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Acute aquatic toxicity: 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.

Determination of the lethal concentration (50%; LC50) is a standardised approach to compare the acute toxicity of chemicals or other substances in a particular context (in this case water).

The method aims to reduce tests that are carried out in juvenile or adult fish.

The OECD (with the support of EURL ECVAM) ran a validation study to ensure that the method is reproducible within and across laboratories.

The results and full reports will be soon available on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.

Aquatic (fish) toxicity


Aquatic toxicity in general refers to the effects of a chemical or substance on organisms living in water and is determined with organisms representing various levels of the food chain in the water:

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

In general, there are acute and chronic endpoints in aquatic toxicity.

Acute fish 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 fish 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.
In this case, test method and validation focuses on fish and specifically zebrafish (Danio rerio).


Zebrafish Embryo Acute Toxicity Test Method (ZFET)

[collapsed]ZFET is carried out with newly fertilised eggs from zebrafish (Danio rerio).

It is an acute test that uses short-term exposure (96 hours) to a substance and determines the concentration that is lethal to 50% of zebrafish embryos (LC50) as an indicator of acute fish toxicity.

Indications of death of an embryo include: coagulation of the embryo, lack of somite formation (a somite is an early stage division of a body part of an embryo that eventually go on to form vertebrate, skeletal muscle, cartilage, tendons and skin of the back), non-detachment of the tail and/or lack of heartbeat. All are easily detectable using light microscopy.


Animal testing replacement?

[collapsed]The use of the test will likely result in an overall reduction in the number of juvenile and adult (zebra)fish used in aquatic toxicity testing.

Directive 2010/63/EU on the protection of animals used for scientific purposes covers larval forms of non-human vertebrate animals once they are independently feeding.

This means the following: the ZFET in the form validated is outside the scope of the Directive. This is because zebrafish embryos do not start to independently feed before five days and the method only uses zebrafish embryos up to four days (i.e. 96 hours) post-fertilisation.


Validation study

[collapsed]The ZFET validation study was coordinated by EURL ECVAM on behalf of the OECD.

The study ran between 2008 and 2012 and was designed to evaluate the reproducibility of the ZFET to ultimately support the development of an OECD test guideline (OECD test guideline (TG) No 236 Fish embryo acute toxicity (FET) test was published in 2013).

In parallel to the validation study, Belanger et al. (2012, 2013) evaluated the predictive capacity of (zebrafish) fish embryo acute toxicity tests for acute fish toxicity testing.

Belanger et al. directly compared data from acute fish toxicity tests on juvenile and adult fish (i.e. the 'standard' method) and acute fish embryo toxicity tests (i.e. the 'alternative' method).

Once both studies were completed, we requested that ESAC peer review both the prospective study that the OECD performed and the retrospective analysis of Belanger et al. (2012).

ESAC finalised its review in March 2013. After fully endorsing ESAC's opinion, EURL ECVAM published its recommendations on 25/07/2014.


Validation study outcomes

[collapsed]The validation study demonstrated that the ZFET method is transferable and reproducible within and across laboratories. The retrospective study meanwhile demonstrated that there was a strong correlation (r=0.9) between fish acute toxicity data and fish embryo acute toxicity data (i.e. the alternative method).

This analysis was based on data from 144 chemicals that covered a broad range of physico-chemical properties, modes of action and functional use. This included chemicals with industrial uses, plant protection uses, surfactants, pharmaceuticals and biocides. On that basis, we concluded that ZFET has a wide applicability domain.

Overall, ZFET can provide information on acute fish toxicity that is comparable to the information that can be derived from standard tests.[/collapse]

EURL ECVAM recommendations

[collapsed]As a result of the validation study and other published reports, we recommended the following:

  • When appropriate, ZFET should be used for generating information on acute fish toxicity. Prospective users should consult EURL-ECVAM's Database for Alternative Methods (DB-ALM protocol no. 140) for the detailed ZFET protocol.
  • Prior to use a number of limitations should be considered. The following limitations are highlighted:
    • It is not fully understood how embryonic metabolism compares to that of juvenille or adult fish. This means there is a possibility that chemicals (and any transformed metabolites) may have effects on embryos that differ from juvenile or adult fish.
    • There is some evidence that certain chemicals with a high molecular weight may not pass the chorion (the outermost membrane that surrounds an embryo). This reduced bioavailability may therefore result in artificially lower apparent toxicity.
  • To support the use of ZFET, it should be included in respective regulations and associated guidance documents.
  • An OECD guidance document on the use of OECD TG236 (i.e. fish embryo acute toxicity test (FET)) should be developed and it should include the use ZFET to generate information relating to acute fish toxicity (this is now available, please see below).
  • The use of ZFET will result in an overall reduction in the numbers of juvenile and adult fish required for acute aquatic toxicity testing.
  • The database containing fish embryo acute toxicity data and acute fish toxicity data (i.e. Belanger et al 2012, 2013) should be maintained and updated.
  • Further efforts should be made in terms of development and validation of methods that avoid the use of fish in environmental hazard and risk assessment.[/collapse]