Aquatic Toxicity Testing

In 2011, nearly 180,000 fish were used for toxicological and other safety assessments in Europe, and the number used in regulatory tests is likely to rise significantly in response to the 2018 Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) registration deadline.

As assessment of aquatic toxicity is required in various regulatory frameworks, strategies to reduce the number of animals used are urgently needed. PETA Science Consortium International e.V. is working on projects that will replace, reduce, and refine the use of animals in aquatic toxicity tests.

Replacement: In Vitro Methods for Prediction of Fish Toxicity

Although there has been progress towards the development of alternatives to using fish to predict bioaccumulation, metabolism, and acute and chronic toxicity, significantly more resources must be dedicated to finding replacements for the use of animals in aquatic toxicity tests. The Consortium is currently reviewing the state of the science with a view to collaborating with researchers to expedite the development and validation of replacement methods. A number of relevant external publications are listed here.

While we continue to work on a strategy to replace tests on fish, we are also tackling shorter-term goals of reduction and refinement as described below. By supporting the Society of Environmental Toxicology and Chemistry (SETAC) as a Global Affiliate and by attending and presenting at SETAC meetings, the Science Consortium ensures the development and promotion of non-animal methods in ecotoxicology.

Reduction: Eliminating the Water Control When Solvents Are Used in Aquatic Toxicity Tests

In aquatic toxicity tests, the test chemical is usually added to the tank water. To overcome practical issues associated with testing poorly soluble chemicals, a small volume of solvent is sometimes also added. As the solvent can influence the outcome of the study, two controls – one in the presence and one in the absence of solvent – are currently required, doubling the number of fish used in controls and having significant animal-welfare implications.

However, the scientific justification for using two controls is questionable and debated by experts. If only one control is included when solvents are used, the number of fish used in Organisation for Economic Co-operation and Development (OECD) test guidelines (TGs) can be reduced by 14 to 25 per cent.

The aim of the project, which is led by the Consortium and the European Union Reference Laboratory for alternatives to animal testing, is to conduct statistical analyses of existing data and statistical simulations to determine whether one of the controls can be eliminated from fish tests when solvents are used. Further details on the project rationale and statistical simulations are presented on this poster.

With the aim of reducing the number of fish used, we urgently need existing control data in the presence and absence of solvents for the following OECD TGs:

  • OECD TG 203: Fish, Acute Toxicity Test
  • OECD TG 210: Fish, Early-Life Stage Toxicity Test
  • OECD TG 212: Fish, Short-Term Toxicity Test on Embryo and Sac-Fry Stages
  • OECD TG 215: Fish, Juvenile Growth Test
  • OECD TG 229: Fish Short-Term Reproduction Assay
  • OECD TG 230: 21-Day Fish Assay
  • OECD TG 234: Fish Sexual Development Test
  • OECD TG 236: Fish Embryo Acute Toxicity (FET) Test
  • OECD TG 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure
  • OECD Guidance Document (GD) 148: Guidance Document on the Androgenised Female Stickleback Screen

If you can contribute control data for these TGs, please contact Dr Gilly Stoddart at [email protected] for more information and a data template.

Refinement: Incorporating the FET Test Into the Threshold Approach

OECD TG 203, the acute fish toxicity test, is one of the most frequently used aquatic toxicity tests, and as death is the endpoint, animal welfare is a significant concern.

Applying the threshold approach set out in OECD GD 126 can substantially reduce the number of adult fish used in this test. 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.

Furthermore, as embryos are used instead of adult fish, the OECD TG 236 FET test provides a significant refinement to the acute fish toxicity test. In Europe, under Directive 2010/63/EU, this test is considered a replacement, as the study is completed before the animals have become independently feeding larval forms. Therefore, the aim of this project is to reduce and refine the use of adult fish in acute fish toxicity testing by incorporating the FET into the threshold approach for acute fish toxicity.

A strategy is being developed for incorporating the FET into the threshold approach, building on extensive earlier work and three individual efforts:

  1. Development of a new database containing existing acute toxicity data for adult/juvenile fish, fish embryos, Daphnia, and algae to assess how the FET can be incorporated into the threshold approach
  2. Analysis of existing data to clarify the applicability domain, reliability, and relevance of the FET and comparison with the uncertainties of the acute fish toxicity test for the protection of the aquatic ecosystem. This analysis may include assessment of the reproducibility of the FET and the acute fish toxicity test, correlation of the FET to the acute fish toxicity test, and correlation of different forms of the acute fish toxicity test (eg, using different species) to each other.
  3. Consideration of the European Chemicals Agency’s (ECHA) report on the use of the FET for REACH and the outcome of the ECHA and German Federal Environment Agency experts workshop on the potential regulatory application of the FET test under REACH, the Classification, Labelling and Packaging (CLP) Regulation, and the Biocidal Products Regulation (BPR)

On this basis, a concept for defining acceptance criteria for the new approach will be proposed. More information about the project and our collaborators is presented on this poster.