Skip to main content
EU Science Hub

The assessment of aquatic toxicity is an important component of the environmental hazard and risk assessment of all types of chemicals, and is therefore included in several pieces of EU chemicals legislation.

Aquatic toxicity in general refers to the effects of a chemical on organisms living in water and is determined with organisms representing the three trophic levels:

  • Algae or plants, representing "primary producers"
  • Invertebrates (e.g. crustaceans such as Daphniaspp.), representing "primary consumers/secondary producers"
  • Vertebrates (usually fish), representing "secondary consumers"

EURL ECVAM's work focuses on methods which could replace, reduce or refine the use of fish.

In general, there are acute and chronic endpoints in aquatic toxicity. Acute toxicity is usually determined with short-term exposure of fish to a series of concentrations of a chemical.
The concentration that is lethal to 50% of the test fish is calculated and expressed as LC50 value.

Chronic toxicity is about longer-term exposure. It covers effects on hatching, growth and survival and is used for the determination of NOEC (No Observed Effect Concentration) values, LOEC (Lowest Observed Effect Concentration) or ECx values where x is a % (e.g. 10%) and is concentration of a chemical where 10% of the population show some sort of effect.

Whereas acute aquatic toxicity testing is a basic requirement in most pieces of EU chemicals legislation, chronic aquatic toxicity testing may be required when the outcome of the acute testing indicates a risk, or in the case that long term exposure is expected.

The EURL ECVAM Strategy to replace, reduce and refine the use of fish in aquatic toxicity and bioaccumulation testing (published in 2014) provides an overview of ongoing projects.

Regulatory framework


Aquatic toxicity testing is stipulated for environmental hazard and risk assessment by the regulatory frameworks on:

EU/OECD test guidelines

Acute fish toxicity

  1. Fish acute toxicity*: OECD test guideline 203 / EU test method C.1
  2. Threshold approach for acute fish toxicity: OECD guidance document GD 126
  3. Fish embryo acute toxicity test**: OECD test guideline 236

* OECD TG203/C.1 allows conducting a limit test, where fish are exposed to a single concentration (100 mg/L). If no mortality is observed at this concentration it is concluded that the LC50 is greater than 100 mg/L and in consequence the substance not toxic to fish.

** ECHA published in 2016 a report on the suitability of OECD TG 236 for fish toxicity testing and recommendations for its use. The report of a workshop on the use of TG 236 under REACH has been published by UBA and ECHA in 2018.

Chronic fish toxicity

  1. Fish early-life stage toxicity: OECD test guideline 210
  2. Fish short-term toxicity test on embryo and sac-fry stages*: OECD test guideline 212 / EU test method C.15
  3. Fish juvenile growth*: OECD test guideline 215 / EU test method C.14

*It should be noted that OECD TG 212 and 215 cover only embryonic or juvenile life stages and may not be appropriate for testing chronic fish toxicity.


EURL ECVAM validated test methods


1. Threshold approach for acute fish toxicity testing


The Threshold Approach for Acute Fish Toxicity testing is a tiered testing strategy which has the potential to significantly reduce the number of fish used for acute aquatic toxicity testing.

It is based on the fact that the LC50/EC50 value of the most sensitive of the three test species (fish, algae and invertebrates) is commonly used for hazard and risk assessment and that fish is often not the most sensitive test species.


When fish acute toxicity data need to be generated, the fish limit test as described in OECD TG 203 / C.1 should be carried out at a threshold concentration using far less fish than the full LC50 test.

The threshold concentration (TC) corresponds to the lowest LC50/EC50 derived from reliable acute invertebrate (e.g. Daphnia, OECD TG202) and algae (e.g. OECD TG 201) data.

If no fish mortality is observed at the TC, it demonstrates that fish is not the most sensitive test species. No further testing is required and the TC value can be used as LC50 fish (LC50 fish is greater than TC with 99% of confidence).

If mortality occurs at the threshold concentration, a full LC50 test should be conducted.

Validation study documents

Available on request.

Regulatory acceptance

2. Zebrafish Embryo Acute Toxicity Test (ZFET)

This method can be used to identify concentrations of chemicals that cause acute toxicity in fish in aquatic environments.

The method uses zebrafish embryos and determines the concentration at which 50% of the embryos do not survive (i.e. is lethal) after being exposed to a chemical for 96 hours.

More information about this method and its validation study are available here.


Test methods under validation by EURL ECVAM

[collapsed]Currently there are no test methods under validation.[/collapse]

Development and optimisation of alternative methods

[collapsed]EURL ECVAM Strategy

The EURL ECVAM Strategy to replace, reduce and refine the use of fish in aquatic toxicity and bioaccumulation testing (published in 2014) provides an overview of ongoing projects.

OECD projects

EURL ECVAM contributes to several projects on the work plan for OECD test guideline programme (for 2016 see here):

  • Update of OECD Guidance Document 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures
    This project is co-lead by the International Council on Animal Protection in OECD Programmes (ICAPO), the European Commission (JRC - EURL ECVAM) and USA and addresses the use of solvents in aquatic toxicity tests on fish.
    When solvents are used, e.g. for the testing of poorly soluble chemicals, OECD test guidelines require two control groups - a water control and a solvent control.
    Part 1 of the project aims at updating OECD Guidance Document 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000) with advanced methodology for media preparation and exposure systems, and by that minimising the use of solvents.
    Part 2 of the project aims at determining whether it is possible to use only the solvent control. A retrospective review of existing data generated according to OECD test guidelines in the presence of a solvent is ongoing.
  • Revision of OECD Guidance Document 126,the threshold approach for acute fish toxicity.
    The project aims at updating OECD GD126, and integrate the fish embryo acute toxicity test (OECD TG236) into the step-wise approach for determining acute fish toxicity data. The project is co-lead by Austria and ICAPO.
  • Revision of OECD Test Guideline 203 Fish Acute Toxicity. TG 203 determines the concentration of a chemical at which 50% of the fish die (LC50) and is one of the few guidelines still using death as an endpoint.
    The project (led by Switzerland and UK) aims at including the use of non-lethal endpoints (moribund state) to reduce the suffering of the fish.

Scientific Options for Avoiding Chronic Fish Testing on the Basis of Existing Data and Extrapolation Approaches

Acute aquatic toxicity testing is a basic requirement in most pieces of EU chemicals legislation, whereas chronic aquatic toxicity testing may be required on a case by case basis, for example when the outcome of the acute testing indicates a risk, or when a long-term exposure to the chemical is expected.

EURL ECVAM explored whether interspecies extrapolations and acute-to-chronic relationships could be used to scientifically support the waiving of chronic fish tests.
For this purpose, acute and chronic toxicity data for Daphnia and fish were extracted from various databases and analysed to identify possible relationships taking into consideration different mode of actions.

The results of this analysis indicate that several types of aquatic toxicity data can be used to assess the potential for chronic fish toxicity.

In particular, interspecies extrapolations based on invertebrate (Daphnia) data, and acute-to-chronic extrapolations from existing acute fish toxicity data, are recommended as a means of deriving information on chronic fish toxicity without the need to perform additional fish tests.

EURL ECVAM published the report on Scientific options for avoiding chronic fish testing on the basis of existing data and extrapolation approaches in May 2016 (Kienzler et al., 2016).

Collaboration with ILSI HESI

EURL ECVAM is represented on the ILSI HESI project committee on the use of animal alternatives in environmental risk assessment and participates in several projects carried out in the framework of this committee.

- Ecological threshold of toxicological concern

An international collaboration under the ILSI HESI has been established to address challenges relating to the development and application of useful ecological threshold of toxicological concern (eco-TTC) concepts (Belanger et al., 2015).

Use of fish cell lines

EURL ECVAM has been scientific advisor to CEllSens, a project co-sponsored by CEFIC LRi and Defra/UK, which aimed to develop/standardise methods based on the use of fish cell lines and fish embryos for the prediction of acute fish toxicity. More information is available here.

As a follow-up of the CEllSens project, the RTgill-W1 (rainbow trout gill cell line) cytotoxicity assay has been evaluated for its transferability, within- and between-laboratory reproducibility under the umbrella of CEFIC LRI project ECO8.3-NC3Rs-EAWAG.

In addition, the RTgill-W1 cytotoxicity assay had been submitted to EURL ECVAM in early 2014 and the test submitter had been invited to provide a full submission. More details are availabe here. [/collapsed]