Acute Aquatic Toxicity

One of the most frequently used aquatic toxicity tests is the acute fish toxicity test – Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 203 – which uses lethality as an endpoint. Replacement, reduction, and refinement approaches are available that can instead be used to protect environmental health.

Rainbow Trout Gill Cell Line Cytotoxicity Assay

An in vitro assay for cytotoxicity using the rainbow trout gill cell line, RTgill-W1 (OECD TG 249), was adopted by the OECD in 2021 and has the potential to play an essential role in replacing the acute fish toxicity test. The assay predicts fish acute toxicity for a broad range of substances in many areas, including product development, screening, and as an element of testing strategies. In contrast to the acute fish toxicity test, this method provides mechanistic information by quantifying cell viability using fluorescent measurements for metabolic activity and integrity of cell and lysosomal membranes. Moreover, with a shorter exposure time compared to the acute fish toxicity test, the cell line assay allows for higher throughput.

The Fish Embryo Acute Toxicity Test

The Fish Embryo Acute Toxicity Test (FET; OECD TG 236) is a refinement of the acute fish toxicity test, because embryos are tested instead of adult fish. The FET is not regulated under Directive 2010/63/EU, as the study is completed before the animals have become independently feeding larval forms. The European Chemicals Agency (ECHA) has acknowledged that the FET can be used within weight-of-evidence approaches together with other information to address the acute fish toxicity data requirement under the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation.

The Threshold Approach for Acute Fish Toxicity

Applying the Threshold Approach set out in OECD Guidance Document (GD) 126 can substantially reduce the number of adult fish used to assess acute fish toxicity while protecting environmental health. In this approach, an initial fish test is conducted at one concentration derived from test responses in Daphnia and algae and continued testing is triggered only if death is observed at this threshold concentration.

In Silico Models

Quantitative Structure–Activity Relationship (QSAR) models help predict toxicity based on analogue substances and chemical categories. Available QSAR models for acute fish toxicity include literature models, free data repositories for pre-calculated QSAR predictions (e.g. the Danish (Q)SAR database and an element of the OECD QSAR Toolbox), free computerised models (e.g. US Environmental Protection Agency (EPA) ECOSAR), commercial computerised models (e.g. MultiCASE EcoTox Models), and review papers. QSAR models can be used in a weight-of-evidence approach to predict efficiently the potential environmental effects of chemicals.

Integrated Approaches to Testing and Assessment for Acute Fish Toxicity

As a member of the International Council on Animal Protection in OECD Programs (ICAPO), the Science Consortium is co-leading an OECD project together with Austria (project no 2.54 on the OECD Work Plan for the Test Guidelines Programme), to replace, reduce, and refine the use of adult fish in acute fish toxicity testing by developing a GD with integrated approaches to testing and assessment (IATA) for acute fish toxicity. The IATAs under development include a broad range of information, such as the physicochemical properties of the test chemical and data generated using QSARs, in vitro methods such as the RTgill-W1 cytotoxicity test, and/or the fish embryo acute toxicity test. The IATAs aim to estimate whether algae, aquatic invertebrates, or fish are the most sensitive species as surrogates for all aquatic organisms according to the EU Regulation 1272/2008 on classification, labelling and packaging. It has been shown that the application of environmental extrapolation models combined with data from alternative methods, including mechanistic indicators of toxicity, may provide at least the same level of environmental protection as the acute fish toxicity test. A presentation on the development of IATAs was given at the World Congress in 2021 and a poster on a draft IATA was presented at the SETAC Europe meeting in 2019.

A Cefic Long-Range Research Initiative–funded project titled “Strengthening Weight of Evidence for FET Data to Replace Acute Fish Toxicity (SWiFT)” is centred around a probabilistic Bayesian network approach to enhancing the prediction of acute fish toxicity. The Science Consortium is represented in the project’s monitoring team, and the outcomes of this project will be taken into account in the OECD IATA GD.

Another activity in the area of reducing fish testing is a retrospective analysis of acute fish toxicity tests being conducted by the US EPA Office of Pesticide Programs to investigate the relative sensitivity among species to help determine whether there is a basis for reducing the number of species used in acute fish toxicity testing. A recording of a presentation about this subject is available in the Science Consortium and EPA webinar series on the use of new approach methodologies (NAMs) in risk assessment.

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