Aquatic Toxicity

    1. ECVAM validated test methods

    2. Test methods under validation by ECVAM

    3. Development and optimisation of alternative methods

    4. Workshop Reports

    5. Publications




Aquatic toxicity refers to the effects of a compound to organisms living in the water and is usually determined on organisms representing the three trophic levels, i.e. vertebrates (fish), invertebrates (crustaceans as Daphnia spp.) and plants (algae).

Our work focuses on methods which could replace, reduce or refine the use of fish.


The two endpoints using fish are:

  • acute fish toxicity (short-term exposure; determines the concentration which is lethal to 50% of the fish, LC50)
  • chronic fish toxicity (long-term exposure; covers life-cycle of the fish; sublethal effects, determines No Observed Effect Concentration (NOEC), Lowest Observed Effect Concentration (LOEC), ECx


Regulatory Framework

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

  • Chemicals (REACH; acute fish toxicity from 10t/year and chronic fish toxicity test from 100 t/year onwards) pdf icon 
  •  Plant protection products pdf icon
  •  Biocides - until end of August 2013 pdf icon Directive 98/8/EC and from 1st September 2013 pdf icon Regulation 528/2012
  •  Pharmaceuticals (acute fish toxicity only for veterinary pharmaceuticals)
    • Human pharmaceuticals: pdf icon
    • Veterinary pharmaceuticals: pdf icon 
  • Feed additives pdf icon
  • others


List of EU/OECD test guidelines


OECD test guideline/ guidance document

EU Test method

Fish acute toxicity *



Threshold Approach for Acute Fish Toxicity

GD 126

Fish embryo acute toxicity test



Fish early-life stage toxicity


Fish short-term toxicity test on embryo and sac-fry stages



Fish juvenile growth



* It should be noted that 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.



 Alternative test methods and approaches


1. ECVAM validated test methods


1.1 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.

This concept was first described for pharmaceuticals by Hutchinson et al (2003) and further developed for chemicals by EURL ECVAM and the former European Chemicals Bureau taking into consideration the requirements of the limit test as described in OECD TG 203 (Jeram et al, 2005; Hoeger et al, 2006).



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 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 threshold concentration, it demonstrates that that the 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.


ESAC statement

  • pdf icon ESAC statement
    Note: the ESAC statement refers to the Threshold Approach as "Upper Threshold Concentration (UTC) approach"


Regulatory acceptance

  • OECD Guidance Document 126 "Short Guidance on the Threshold Approach for Acute Fish Toxicity" (published in 2010) pdf icon 
  • Included in REACH Guidance on "Information Requirements and Chemical Safety Assessment" pdf icon
  • Recommended for acute fish toxicity testing in the new Biocides Regulation (Regulation (EU) no. 528/2012)



2. Test methods under validation by ECVAM


2.1  Zebrafish embryo toxicity test

On behalf of the OECD, the EURL ECVAM has coordinated the validation (transferability, intra- and interlaboratory reproducibility) of the zebrafish embryo toxicity test (ZFET). The validation project is linked to the ongoing development of an OECD test guideline "New test guideline Fish Embryo Toxicity [FET] Test" (OECD project 2.7). 

The zebrafish embryo toxicity test (ZFET) is a potential alternative to the use of juvenile/adult fish for acute toxicity testing. It is designed to determine acute lethal effects of compounds on embryonic stages of the zebrafish (Danio rerio). Newly fertilised zebrafish embryos (20/concentration and control) are exposed for 96h to at least five concentrations of the test compound and appropriate controls. The following observations indicate lethal effects to the embryos: coagulation of the egg/embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat.
The numbers of dead embryos per concentration are counted and used for the calculation of the LC50 value. 

Validation study

The validation study was steered by a validation management group established in accordance with OECD. The study aimed to evaluate the transferability, intra- and interlaboratory reproducibility of the zebrafish embryo toxicity test. 20 chemicals were tested at 5 different concentrations in 3 independent runs in at least 3 laboratories with appropriate controls. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. 

The report of Phase 1 has been approved by the 23rd OECD WNT meeting in 2011 and is available on the OECD website: Phase 1 Report pdf icon and Annexes pdf icon.

The report of Phase 2 has been approved by the 24th OECD WNT meeting (April 2012) and is available on the OECD website: Phase 2 Report pdf icon and Annexes pdf icon.

The ESAC peer review was finalised and the EURL ECVAM Recommendation is under preparation.


Regulatory acceptance

At the 25th WNT (April 2013) of the OECD, the new OECD TG236 Fish embryo acute toxicity (FET) test was adopted and declassified by the OECD Joint Meeting in July 2013. It is available on the OECD website pdf icon .

The OECD TG236 does not indicate whether the fish embryo acute toxicity test can be used as an alternative to the OECD TG203; however, several recently published papers demonstrate that the LC50 values produced with the fish embryo acute toxicity test correlate well with those observed in juvenile or adult fish (Lammer et al, 2009; Knoebel et al 2012; Belanger et al (2013). 

3. Development and optimisation of alternative methods


3.1 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.



EURL ECVAM was scientific advisor to the FP7 Coordinated Action EuroEcoTox (for more information, see EuroEcoTox web site).

The project ended in November 2012 and aimed to promote the cooperation between research centres, industry and other stakeholders in Europe devoted to the R&D and application of alternative methods in environmental toxicity testing



3.3 CEllSens

EURL ECVAM was 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. The project ended in 2011. More information is available on the Ecetoc web site.



4. Workshop reports


  • Castaño, A., Bols, N., Braunbeck, T., Dierickx, P., Halder, M., Isomaa, B., Kawahara, K., Lee, L., Mothersill, C., Pärt, P., Repetto, G., Riego Sintes, J., Rufli, H., Smith, R., Wood, C., Segner, H, 2003. The use of fish cells in Ecotoxicology. The Report and Recommendations of ECVAM Workshop 47. ATLA 31: 317-351.


5. Relevant Publications

  • Busquet, F., Belanger, S., Braunbeck, T., Carr, G., Halder, M., Lillicrap, A., Rawlings, J., Strecker, R., Walter-Rohde, S., Amcoff, P, 2011. Update on the OECD validation study on the transferability, intra- and inter-laboratory reproducibility of the Zebrafish Embryo Toxicity Test. Poster presentation at SETAC Europe 21th Annual Meeting 15-19 May 2011, abstract no TU138.
  • Halder, M., Léonard, M., Iguchi, T., Oris, J.T., Ryder, K., Belanger, S.E., Braunbeck, T.A., Embry, M.R., Whale, G., Norberg-King, T., Lillicrap, A., 2010. Regulatory aspects on the use of fish embryos in environmental toxicology. Integr Environ Assess Manag 6:484-91.
  • Hoeger, B, Jeram, S., Holt, M, Douben, P. Halder, M, 2006. Reduction of animal use in acute aquatic toxicity testing: Further development of the threshold approach and its application to existing chemicals and plant protection products. Poster presentation at SETAC Europe 16th Annual Meeting 7-11 May 2006, abstract no MO1/AM/P05.
  • Hutchinson, T.H., Barrett, S., Buzby, M., Constable, D., Hartmann, A., Hayes, E., Huggett, D., Länge, R., Lillicrap, A.D., Straub, J.O., Thompson, R.S., 2003. A strategy to reduce the numbers of fish used in acute ecotoxicity testing of pharmaceuticals. Environ. Toxicol. Chem. 22, 3031-3036
  • Jeram, S., Riego Sintes, J.M., Halder, M., Baraibar Fentanes, J., Sokull-Klüttgen, B., Hutchinson, T.H. (2005) A strategy to reduce the use of fish in acute ecotoxicity testing of new chemical substances notified in the European Union. Regulatory Toxicology and Pharmacology 42, 218-224.