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OECD/OCDE 236
Adopted:
26 July 2013
1
© OECD, (2013)
You are free to use this material for personal, non-commercial purposes without seeking prior consent
from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to
written permission from the OECD.
OECD GUIDELINES FOR THE TESTING OF CHEMICALS
Fish Embryo Acute Toxicity (FET) Test
INTRODUCTION
1. This Test Guideline (TG) 236 describes a Fish Embryo Acute Toxicity (FET) test with the
zebrafish (Danio rerio). This test is designed to determine acute toxicity of chemicals on embryonic
stages of fish. The FET-test is based on studies and validation activities performed on zebrafish
(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The FET-test has been successfully applied to a wide
range of substances exhibiting diverse modes of action, solubilities, volatilities, and hydrophobicities
(reviewed in 15 and 16).
2. Definitions used in this Test Guideline are given in Annex 1.
PRINCIPLE OF THE TEST
3. Newly fertilised zebrafish eggs are exposed to the test chemical for a period of 96 hrs. Every
24 hrs, up to four apical observations are recorded as indicators of lethality (6): (i) coagulation of
fertilised eggs, (ii) lack of somite formation, (iii) lack of detachment of the tail-bud from the yolk sac, and
(iv) lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive
outcome in any of the four apical observations recorded, and the LC50 is calculated.
INITIAL CONSIDERATIONS
4. Useful information about substance-specific properties include the structural formula, molecular
weight, purity, stability in water and light, pKa and Kow, water solubility and vapour pressure as well as
results of a test for ready biodegradability (OECD TG 301 (17) or TG 310 (18)). Solubility and vapour
pressure can be used to calculate Henry's law constant, which will indicate whether losses due to
evaporation of the test chemical may occur. A reliable analytical method for the quantification of the
substance in the test solutions with known and reported accuracy and limit of detection should be
available.
5. If the Test Guideline is used for the testing of a mixture, its composition should, as far as
possible, be characterised, e.g., by the chemical identity of its constituents, their quantitative occurrence
and their substance-specific properties (see paragraph 4). Before use of the Test Guideline for regulatory
testing of a mixture, it should be considered whether it will provide acceptable results for the intended
regulatory purpose.
6. Concerning substances that may be activated via metabolism, there is evidence that zebrafish
embryos do have biotransformation capacities (19)(20)(21)(22). However, the metabolic capacity of
embryonic fish is not always similar to that of juvenile or adult fish. For instance, the protoxicant allyl
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alcohol (9) has been missed in the FET. Therefore, if there are any indications that metabolites or other
transformation products of relevance may be more toxic than the parent compound, it is also
recommended to perform the test with these metabolites / transformation products and to also use these
results when concluding on the toxicity of the test chemical, or alternatively perform another test which
takes metabolism into further account.
7. For substances with a molecular weight ≥ 3kDa, a very bulky molecular structure, and
substances causing delayed hatch which might preclude or reduce the post-hatch exposure, embryos are
not expected to be sensitive because of limited bioavailability of the substance, and other toxicity tests
might be more appropriate.
VALIDITY OF THE TEST
8. For the test results to be valid, the following criteria apply:
1. The overall fertilisation rate of all eggs collected should be ≥ 70% in the batch tested.
2. The water temperature should be maintained at 26 ± 1 °C in test chambers at any time during
the test.
3. Overall survival of embryos in the negative (dilution-water) control, and, where relevant, in the
solvent control should be ≥ 90% until the end of the 96 hrs exposure.
4. Exposure to the positive control (e.g., 4.0 mg/L 3,4-dichloroaniline for zebrafish) should result
in a minimum mortality of 30% at the end of the 96 hrs exposure.
5. Hatching rate in the negative control (and solvent control if appropriate) should be ≥ 80% at the
end of 96 hrs exposure.
6. At the end of the 96 hrs exposure, the dissolved oxygen concentration in the negative control
and highest test concentration should be ≥80% of saturation.
DESCRIPTION OF THE METHOD
9. An overview of recommended maintenance and test conditions is available in Annex 2.
Apparatus
10. The following equipment is needed:
a. Fish tanks made of chemically inert material (e.g., glass) and of a suitable capacity in relation to
the recommended loading (see “Maintenance of brood fish”, paragraph 14);
7. Inverted microscope and/or binocular with a capacity of at least 80-fold magnification. If the
room used for recording observations cannot be adjusted to 26 ± 1 °C, a temperature-controlled
cross movement stage or other methods to maintain temperature are necessary;
8. Test chambers; e.g., standard 24-well plates with a depth of approx. 20 mm. (see "Test
chambers", paragraph 11);
9. e.g., self-adhesive foil to cover the 24-well plates;
10. Incubator or air-conditioned room with controlled temperature, allowing to maintain 26 ±1 °C
in wells (or test chambers);
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11. pH-meter;
12. Oxygen meter;
13. Equipment for determination of hardness of water and conductivity;
14. Spawn trap: instrument trays of glass, stainless steel or other inert materials; wire mesh (grid
size 2 ± 0.5 mm) of stainless steel or other inert material to protect the eggs once laid; spawning
substrate (e.g., plant imitates of inert material) (OECD 229, Annex 4a (23));
15. Pipettes with widened openings to collect eggs;
16. Glass vessels to prepare different test concentrations and dilution water (beakers, graduated
flasks, graduated cylinders and graduated pipettes) or to collect zebrafish eggs (e.g., beakers,
crystallisation dishes);
17. If alternative exposure systems, such as flow-through (24) or passive dosing (25) are used for
the conduct of the test, appropriate facilities and equipment are needed.
Test chambers
11. Glass or polystyrene test chambers should be used (e.g., 24-well plates with a 2.5 – 5 ml filling
capacity per well). In case adsorption to polystyrene is suspected (e.g., for non-polar, planar compounds
with high KOW), inert materials (glass) should be used to reduce losses due to adsorption (26). Test
chambers should be randomly positioned in the incubator.
Water and test conditions
12. Dilution of the maintenance water is recommended to achieve hardness levels typical of a wide
variety of surface waters. Dilution water should be prepared from reconstituted water (27). The resulting
degree of hardness should be equivalent to 100-300 mg/L CaCO3 in order to prevent excessive
precipitation of calcium carbonate. Other well-characterised surface or well water may be used. The
reconstituted water may be adapted to maintenance water of low hardness by dilution with deionised
water up to a ratio of 1:5 to a minimum hardness of 30-35 mg/L CaCO3. The water is aerated to oxygen
saturation prior to addition of the test chemical. Temperature should be kept at 26 1 C, in the wells,
throughout the test. The pH should be in a range between pH 6.5 and 8.5, and not vary within this range
by more than 1.5 units during the course of the test. If the pH is not expected to remain in this range, then
pH adjustment should be done prior to initiating the test. The pH adjustment should be made in such a
way that the stock solution concentration is not changed to any significant extent and that no chemical
reaction or precipitation of the test chemical is caused. Use of hydrogen chloride (HCl) and sodium
hydroxide (NaOH) to correct pH in the solutions containing the test chemical is recommended.
Test solutions
13. Test solutions of the selected concentrations can be prepared, e.g., by dilution of a stock
solution. The stock solutions should preferably be prepared by simply mixing or agitating the test
chemical in the dilution water by mechanical means (e.g., stirring and / or ultra-sonification). If the test
chemical is difficult to dissolve in water, procedures described in the OECD Guidance Document No. 23
for handling difficult substances should be followed (28). The use of solvents should be avoided, but may
be required in some cases in order to produce a suitably concentrated stock solution. Where a solvent is
used to assist in stock solution preparation, its final concentration should not exceed 100 µl/L and should
be the same in all test vessels. When a solvent is used, an additional solvent control is required.
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Maintenance of brood fish
14. A breeding stock of unexposed, wild-type zebrafish with well-documented fertilisation rate of
eggs is used for egg production. Fish should be free of macroscopically discernible symptoms of infection
and disease and should not have undergone any pharmaceutical (acute or prophylactic) treatment for
2 months before spawning. Breeding fish are maintained in aquaria with a recommended loading capacity
of 1 L water per fish and a fixed 12 – 16 hour photoperiod (29)(30)(31)(32)(33). Optimal filtering rates
should be adjusted; excess filtering rates causing heavy perturbation of the water should be avoided. For
feeding conditions, see Annex 2. Surplus feeding should be avoided, and water quality and cleanness of
the aquaria should be monitored regularly and be reset to the initial state, if necessary.
Proficiency Testing
15. As a reference substance, 3,4-dichloroaniline (used in the validation studies (1)(2)), should be
tested in a full concentration-response range to check the sensitivity of the fish strain used, preferably
twice a year. For any laboratory initially establishing this assay, the reference chemical should be used. A
laboratory can use this chemical to demonstrate their technical competence in performing the assay prior
to submitting data for regulatory purposes.
Egg production
16. Zebrafish eggs may be produced via spawning groups (in individual spawning tanks) or via
mass spawning (in the maintenance tanks). In the case of spawning groups, males and females (e.g., at a
ratio of 2:1) in a breeding group are placed in spawning tanks a few hours before the onset of darkness on
the day prior to the test. Since spawning groups of zebrafish may occasionally fail to spawn, the parallel
use of at least three spawning tanks is recommended. To avoid genetic bias, eggs are collected from a
minimum of three breeding groups, mixed and randomly selected.
17. For the collection of eggs, spawn traps are placed into the spawning tanks or maintenance tanks
before the onset of darkness on the day prior to the test or before the onset of light on the day of the test.
To prevent predation of eggs by adult zebrafish, the spawn traps are covered with inert wire mesh of
appropriate mesh size (approx. 2±0.5 mm). If considered necessary, artificial plants made of inert
material (e.g., plastic or glass) can be fixed to the mesh as spawning stimulus (3)(4)(5)(23)(35).
Weathered plastic materials which do not leach (e.g., phthalates) should be used. Mating, spawning and
fertilisation take place within 30 min after the onset of light and the spawn traps with the collected eggs
can be carefully removed. Rinsing eggs with reconstituted water after collection from spawning traps is
recommended.
Egg differentiation
18. At 26C, fertilised eggs undergo the first cleavage after about 15 min and the consecutive
synchronous cleavages form 4, 8, 16 and 32 cell blastomers (see Annex 3)(35). At these stages, fertilised
eggs can be clearly identified by the development of a blastula.
PROCEDURE
Conditions of exposure
19. Twenty embryos per concentration (one embryo per well) are exposed to the test chemical.
Exposure should be such that ± 20% of the nominal chemical concentration are maintained throughout the
test. If this is not possible in a static system, a manageable semi-static renewal interval should be applied
(e.g., renewal every 24 hrs). In these cases exposure concentrations need to be verified as a minimum in
the highest and lowest test concentrations at the beginning and the end of each exposure interval (see
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paragraph 36). If an exposure concentration of ± 20% of the nominal concentrations cannot be
maintained, all concentrations need to be measured at the beginning and the end of each exposure interval
(see paragraph 36). Upon renewal, care should be taken that embryos remain covered by a small amount
of old test solutions to avoid drying. The test design can be adapted to meet the testing requirements of
specific substances (e.g,. flow-through (24) or passive dosing systems (25) for easily degradable or highly
adsorptive substances (29), or others for volatile substances (36)(37)). In any case, care should be taken to
minimise any stress to the embryos. Test chambers should be conditioned at least for 24 hrs with the test
solutions prior to test initiation. Test conditions are summarised in Annex 2.
Test concentrations
20. Normally, five concentrations of the test chemical spaced by a constant factor not exceeding 2.2
are required to meet statistical requirements. Justification should be provided, if fewer than five
concentrations are used. The highest concentration tested should preferably result in 100% lethality, and
the lowest concentration tested should preferably give no observable effect, as defined in paragraph 28. A
range-finding test before the definitive test allows selection of the appropriate concentration range. The
range-finding is typically performed using ten embryos per concentration. The following instructions
refer to performing the test in 24-well plates. If different test chambers (e.g., small Petri dishes) are used
or more concentrations are tested, instructions have to be adjusted accordingly.
21. Details and visual instructions for allocation of concentrations across 24-well plates are
available in paragraph 27 and Annex 4, Figure 1.
Controls
22. Dilution water controls are required both as negative control and as internal plate controls. If
more than 1 dead embryo is observed in the internal plate control, the plate is rejected, thus reducing the
number of concentrations used to derive the LC50. If an entire plate is rejected the ability to evaluate and
discern observed effects may become more difficult, especially if the rejected plate is the solvent control
plate or a plate in which treated embryos are also affected. In the first case the test must be repeated. In
the second one the loss of an entire treatment group(s) due to internal control mortality may limit the
ability to evaluate effects and determine LC50 values.
23. A positive control at a fixed concentration of 4 mg/L 3,4-dichloroaniline is performed with each
egg batch used for testing.
24. In case a solvent is used, an additional group of 20 embryos is exposed to the solvent on a
separate 24-well plate, thus serving as a solvent control. To consider the test acceptable, the solvent
should be demonstrated to have no significant effects on time to hatch, survival, nor produce any other
adverse effects on the embryos (cf. paragraph 7c).
Start of exposure and duration of test
25. The test is initiated as soon as possible after fertilisation of the eggs and terminated after 96 hrs
of exposure. The embryos should be immersed in the test solutions before cleavage of the blastodisc
commences, or, at latest, by the 16 cell-stage. To start exposure with minimum delay, at least twice the
number of eggs needed per treatment group are randomly selected and transferred into the respective
concentrations and controls (e.g., in 100 ml crystallisation dishes; eggs should be fully covered) not later
than 90 minutes post fertilisation.
26. Viable fertilised eggs should be separated from unfertilised eggs and be transferred to 24-well
plates pre-conditioned for 24 hrs and refilled with 2 ml/well freshly prepared test solutions within 180
minutes post fertilisation. By means of stereomicroscopy (preferably ≥30-fold magnification), fertilised
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eggs undergoing cleavage and showing no obvious irregularities during cleavage (e.g., asymmetry,
vesicle formation) or injuries of the chorion are selected. For egg collection and separation, see Annex 3,
Fig. 1 and 3 and Annex 4, Fig. 2.
Distribution of eggs over the 24-well plates
27. Eggs are distributed to well plates in the following numbers (see also Annex 4, Fig. 1)
20 eggs on one plate for each test concentration;
20 eggs as solvent control on one plate (if necessary);
20 eggs as positive control on one plate;
4 eggs in dilution water as internal plate control on each of the above plates;
24 eggs in dilution water as negative control on one plate.
Observations
28. Apical observations performed on each tested embryo include: coagulation of embryos, lack of
somite formation, non-detachment of the tail, and lack of heartbeat (Table 1). These observations are used
for the determination of lethality: Any positive outcome in one of these observations means that the
zebrafish embryo is dead. Additionally, hatching is recorded in treatment and control groups on a daily
basis starting from 48 hrs. Observations are recorded every 24 hrs, until the end of the test.
Table 1. Apical observations of acute toxicity in zebrafish embryos 24 - 96 hrs post fertilisation.
Exposure times
24 hrs
48 hrs
72 hrs
96 hrs
Coagulated embryos
+
+
+
+
Lack of somite formation
+
+
+
+
Non-detachment of the tail
+
+
+
+
Lack of heartbeat
+
+
+
29. Coagulation of the embryo: Coagulated embryos are milky white and appear dark under the
microscope (see Annex 5, Fig. 1). The number of coagulated embryos is determined after 24, 48, 72 and
96 hrs.
30. Lack of somite formation: At 26±1°C, about 20 somites have formed after 24 hrs (see Annex 5,
Figure 2) in a normally developing zebrafish embryo. A normally developed embryo shows spontaneous
movements (side-to-side contractions). Spontaneous movements indicate the formation of somites. The
absence of somites is recorded after 24, 48, 72 and 96 hrs. Non-formation of somites after 24 hrs might be
due to a general retardation of development. At latest after 48 hrs, the formation of somites should be
developed. If not, the embryos are considered dead.
31. Non-detachment of the tail: In a normally developing zebrafish embryo, detachment of the tail
(see Annex 5, Figure 3) from the yolk is observed following posterior elongation of the embryonic body.
Absence of tail detachment is recorded after 24, 48, 72 and 96 hrs.
32. Lack of heartbeat: In a normally developing zebrafish embryo at 26±1 °C, the heartbeat is
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visible after 48 hrs (see Annex 5, Figure 4). Particular care should be taken when recording this endpoint,
since irregular (erratic) heartbeat should not be recorded as lethal. Moreover, visible heartbeat without
circulation in aorta abdominalis is considered non-lethal. To record this endpoint, embryos showing no
heartbeat should be observed under a minimum magnification of 80x for at least one minute. Absence of
heartbeat is recorded after 48, 72 and 96 hrs.
33. Hatching rates of all treatment and control groups should be recorded from 48 hrs onwards and
reported. Although hatching is not an endpoint used for the calculation of the LC50, hatching ensures
exposure of the embryo without a potential barrier function of the chorion, and as such may help data
interpretation.
34. Detailed descriptions of the normal (35) and examples of abnormal development of zebrafish
embryos are illustrated in Annexes 3 and 5.
Analytical measurements
35. At the beginning and at the end of the test, pH, total hardness and conductivity in the control(s)
and in the highest test chemical concentration are measured. In semi-static renewal systems the pH should
be measured prior to and after water renewal. The dissolved oxygen concentration is measured at the end
of the test in the negative controls and highest test concentration with viable embryos, where it should be
in compliance with the test validity criteria (see paragraph 7f). If there is concern that the temperature
varies across the 24-well plates, temperature is measured in three randomly selected vessels. Temperature
should be recorded preferably continuously during the test or, as a minimum, daily.
36. In a static system, the concentration of the test chemical should be measured, as a minimum, in
the highest and lowest test concentrations, but preferably in all treatments, at the beginning and end of the
test. In semi-static (renewal) tests where the concentration of the test chemical is expected to remain
within ± 20% of the nominal values, it is recommended that, as a minimum, the highest and lowest test
concentrations be analysed when freshly prepared and immediately prior to renewal. For tests where the
concentration of the test chemical is not expected to remain within ± 20% of nominal, all test
concentrations must be analysed when freshly prepared and immediately prior to renewal. In case of
insufficient volume for analysis, merging of test solutions, or use of surrogate chambers being of the same
material and having the same volume to surface area ratios as 24-well plates, may be useful. It is strongly
recommended that results be based on measured concentrations. When the concentrations do not remain
within 80-120% of the nominal concentration, the effect concentrations should be expressed relative to
the geometric mean of the measured concentrations; see Chapter 5 in the OECD Guidance Document
No. 23 for more details (28).
LIMIT TEST
37. Using the procedures described in this guideline, a limit test may be performed at 100 mg/L of
test chemical or at its limit of solubility in the test medium (whichever is the lower) in order to
demonstrate that the LC50 is greater than this concentration. The limit test should be performed using
20 embryos in the treatment, the positive control and – if necessary - in the solvent control and
24 embryos in the negative control. If the percentage of lethality at the concentration tested exceeds the
mortality in the negative control (or solvent control) by 10%, a full study should be conducted. Any
observed effects should be recorded. If mortality exceeds 10% in the negative control (or solvent control),
the test becomes invalid and should be repeated.
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DATA AND REPORTING
Treatment of results
38. In this test, the individual wells are considered independent replicates for statistical analysis.
The percentages of embryos for which at least one of the apical observations is positive at 48 and/or
96 hrs are plotted against test concentrations. For calculation of the slopes of the curve, LC50 values and
the confidence limits (95%), appropriate statistical methods should be applied (38) and the OECD
Guidance Document No. 54 should be consulted (39 ).
Test report
39. The test report should include the following information:
Test chemical:
Mono-constituent substance
physical appearance, water solubility, and additional relevant physicochemical properties;
chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI
code, structural formula, purity, chemical identity of impurities as appropriate and
practically feasible, etc. (including the organic carbon content, if appropriate).
Multi-constituent substance, UVBCs and mixtures:
characterised as far as possible by chemical identity (see above), quantitative occurrence
and relevant physicochemical properties of the constituents.
Test organisms:
scientific name, strain, source and method of collection of the fertilised eggs and subsequent
handling.
Test conditions:
test procedure used (e.g., semi-static renewal);
photoperiod;
test design (e.g., number of test chambers, types of controls);
water quality characteristics in fish maintenance (e.g., pH, hardness, temperature,
conductivity, dissolved oxygen);
dissolved oxygen concentration, pH, total hardness, temperature and conductivity of the test
solutions at the start and after 96 hrs;
method of preparation of stock solutions and test solutions as well as frequency of renewal;
justification for use of solvent and justification for choice of solvent, if other than water;
the nominal test concentrations and the result of all analyses to determine the concentration
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of the test chemical in the test vessels; the recovery efficiency of the method and the limit of
quantification (LoQ) should also be reported;
evidence that controls met the overall survival validity criteria;
fertilisation rate of the eggs;
hatching rate in treatment and control groups.
Results:
maximum concentration causing no mortality within the duration of the test;
minimum concentration causing 100 % mortality within the duration of the test;
cumulative mortality for each concentration at the recommended observation times;
the LC50 values at 96 hrs (and optionally at 48 hrs) for mortality with 95% confidence limits,
if possible;
graph of the concentration-mortality curve at the end of the test;
mortality in the controls (negative controls, internal plate controls, as well as positive
control and any solvent control used);
data on the outcome of each of the four apical observations;
incidence and description of morphological and physiological abnormalities, if any (see
examples provided in Annex 5, Figure 2);
incidents in the course of the test which might have influenced the results;
statistical analysis and treatment of data (probit analysis, logistic regression model and
geometric mean for LC50);
slope and confidence limits of the regression of the (transformed) concentration-response
curve.
Any deviation from the Guideline and relevant explanations.
Discussion and interpretation of results.
LITERATURE
1. OECD (2011) Validation Report (Phase 1) for the Zebrafish Embryo Toxicity Test: Part I
and Part II. Series on Testing and Assessment No. 157, OECD, Paris.
2. OECD (2012) Validation Report (Phase 2) for the Zebrafish Embryo Toxicity Test: Part I
and Part II (Annexes). Series on Testing and Assessment No. 179, OECD, Paris.
3. Braunbeck, T., Böttcher, M., Hollert, H., Kosmehl, T., Lammer, E., Leist, E., Rudolf, M. and
Seitz, N. (2005) Towards an alternative for the acute fish LC50 test in chemical assessment: The
fish embryo toxicity test goes multi-species - an update. ALTEX 22: 87-102.
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4. ISO (2007) International Standard Water quality – Determination of the acute toxicity of waste
water to zebrafish eggs (Danio rerio). ISO 15088:2007(E) International Organization for
Standardization.
5. Nagel, R. (2002) DarT: The embryo test with the zebrafish (Danio rerio) - a general model in
ecotoxicology and toxicology. ALTEX 19: 38-48.
6. Schulte, C. and Nagel, R. (1994) Testing acute toxicity in embryo of zebrafish, Brachydanio rerio
as alternative to the acute fish test - preliminary results. ATLA 22, 12-19.
7. Bachmann, J. (2002) Development and validation of a teratogenicity screening test with embryos
of the zebrafish (Danio rerio). PhD-thesis, Technical University of Dresden, Germany.
8. Lange, M., Gebauer, W., Markl, J. and Nagel, R. (1995) Comparison of testing acute toxicity on
embryo of zebrafish (Brachydanio rerio), and RTG-2 cytotoxicity as possible alternatives to the
acute fish test. Chemosphere 30/11: 2087-2102.
9. Knöbel, M., Busser, F.J.M., Rico-Rico, A., Kramer, N.I., Hermens, J.L.M., Hafner, C.,
Tanneberger, K., Schirmer, K., Scholz, S. (2012). Predicting adult fish acute lethality with the
zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure
concentration analysis. Environ. Sci. Technol. 46, 9690-9700.
10. Kammann, U., Vobach, M. and Wosniok, W. (2006) Toxic effects of brominated indoles and
phenols on zebrafish embryos. Arch. Environ. Contam. Toxicol., 51:97-102.
11. Groth, G., Kronauer, K. and Freundt, K.J. (1994) Effects of N,N-diemethylformamide and its
degradation products in zebrafish embryos. Toxicol. In Vitro 8: 401-406.
12. Groth, G., Schreeb, K., Herdt, V. and Freundt, K.J. (1993) Toxicity studies in fertilized zebrafish
fish eggs treated with N-methylamine, N,N-dimethylamine, 2-aminoethanol, isopropylamine,
aniline, N-methylaniline, N,N-dimethylaniline, quinone, chloroacetaldehyde, or cyclohexanol.
Bull. Environ. Contam. Toxicol. 50: 878-882.
13. Nguyen, L.T. and Janssen, C.R. (2001) Comparative sensitivity of embryo-larval toxicity assays
with African catfish (Clarias gariepinus) and zebra fish (Danio rerio). Environ. Toxicol. 16: 566-
71.
14. Cheng, S.H., Wai, A.W.K., So, C.H. and Wu, R.S.S. (2000) Cellular and molecular basis of
cadmium-induced deformities in zebrafish embryos. Environ. Toxicol. Chem. 19: 3024-3031.
15. Belanger, S. E., Rawlings J. M. and Carr G. J.. Use of Fish Embryo Toxicity Tests for the
Prediction of Acute Fish Toxicity to Chemicals. Environmental Toxicology and Chemistry
(Accepted).
16. Lammer, E., Carr, G. J., Wendler, K., Rawlings, J. M., Belanger, S. E., Braunbeck, T. (2009) Is
the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the
fish acute toxicity test? Comp. Biochem. Physiol. C Toxicol. Pharmacol.: 149 (2), 196−209
17. OECD (1992) Ready Biodegradability, Test Guideline No. 301, Guidelines for the Testing of
Chemicals, OECD, Paris.
18. OECD (2006) Ready Biodegradability, CO2 in sealed vessels, Test Guideline No. 310,
Guidelines for the Testing of Chemicals, OECD, Paris
19. Weigt, S., Huebler, N., Strecker, R., Braunbeck, T., Broschard, T.H. (2011) Zebrafish (Danio
rerio) embryos as a model for testing proteratogens. Toxicology 281: 25-36.
20. Weigt, S., Huebler, N., Strecker, R., Braunbeck, T., Broschard, T.H. (2012) Developmental
effects of coumarin and the anticoagulant coumarin derivative warfarin on zebrafish (Danio
rerio) embryos. Reprod. Toxicol. 33: 133-141.
21. Incardona, J.P, Linbo, T.L., Scholz, N.L. (2011) Cardiac toxicity of 5-ring polycyclic aromatic
hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during
zebrafish development. Toxicol. Appl. Pharmacol. 257: 242-249.
22. Kubota, A., Stegeman, J.J., Woodin, B.R., Iwanaga, T., Harano, R., Peterson, R.E., Hiraga, T.,
Teraoka, H. (2011) Role of zebrafish cytochrome P450 CYP1C genes in the reduced
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mesencephalic vein blood flow caused by activation of AHR2. Toxicol. Appl. Pharmacol. 253:
244-252.
23. OECD (2009) Fish Short Term Reproduction Assay. Test Guideline No. 229, Guidelines for the
Testing of Chemicals, OECD, Paris. See ANNEX 4a.
24. Lammer, E., Kamp, H.G., Hisgen, V., Koch, M., Reinhard, D., Salinas, E.R., Wendlar, K., Zok,
S., Braunbeck, T. (2009) Development of a flow-through system for the fish embryo toxicity test
(FET) with zebrafish (Danio rerio). Toxicol. In Vitro 23: 1436-1442.
25. Brown, R.S., Akhtar, P., Åkerman, J., Hampel, L., Kozin, I.S., Villerius, L.A., Klamer, H.J.C.,
(2001) Partition controlled delivery of hydrophobic substances in toxicity tests using
poly(dimethylsiloxane) (PDMS) films. Environ. Sci. Technol. 35, 4097–4102.
26. Schreiber, R., Altenburger, R., Paschke, A., Küster, E. (2008) How to deal with lipophilic and
volatile organic substances in microtiter plate assays. Environ. Toxicol. Chem. 27, 1676-1682.
27. ISO (1996) International Standards. Water quality - Determination of the acute lethal toxicity of
substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)].
ISO 7346-3: Flow-through method. Available: [http://www.iso.org].
28. OECD (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and
Mixtures. Series on Testing and Assessment No. 23, OECD, Paris.
29. Laale, H.W. (1977) The biology and use of zebrafish, Brachydanio rerio, in fisheries research. A
literature review. J. Fish Biol. 10: 121-173.
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(Brachydanio rerio). 5th edition. Eugene, University of Oregon Press, Institute of Neuroscience,
USA.
31. Canadian Council on Animal Care (2005) Guidelines on: the Care and Use of Fish in
Research, Teaching and Testing, ISBN: 0-919087-43-4
[http://www.ccac.ca/Documents/Standards/Guidelines/Fish.pdf]
32. European Commission (2007) Commission recommendation 2007/526/EC of 18 June 2007 on
guidelines for the accommodation and care of animals used for experimental and other scientific
purposes (notified under document number C(2007) 2525) [http://eur-
lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2007:197:0001:0089:EN:PDF]
33. European Union (2010) Directive 2010/63/EU of the European Parliament and Council of 22
September 2010 on the protection of animals used for scientific purposes. Official Journal of
the European Union, L 276:33-79; 20.10.2010
[http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF]
34. Nagel, R. (1986) Untersuchungen zur Eiproduktion beim Zebrabärbling (Brachydanio rerio,
Ham.-Buch.). J. Appl. Ichthyol. 2: 173-181.
35. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. and Schilling, T.F. (1995) Stages of
embryonic development of the zebrafish. Dev. Dyn. 203: 253-310.
36. OECD (2004) Daphnia sp., Acute Immobilistaion Test. Test Guideline No. 202. Guidelines for
the Testing of Chemicals, OECD, Paris.
37. Weil, M., Scholz, S., Zimmer, M., Sacher, F., Duis, K. (2009) Gene expression analysis in
zebrafish embryos: a potential approach to predict effect conentrations in the fish early life stage
test. Environ. Toxicol. Chem. 28: 1970-1978
38. ISO (2006) International Standard. Water quality - Guidance on statistical interpretation of
ecotoxicity data ISO TS 20281. Available: [http://www.iso.org].
39. OECD (2006) Guidance Document on Current Approaches in the Statistical Analysis of
Ecotoxicity Data: a Guidance to Application. Series on Testing and Assessment No. 54.
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40. Braunbeck, T., Lammer, E., 2006. Detailed review paper “Fish embryo toxicity assays”. UBA
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report under contract no. 20385422 German Federal Environment Agency, Berlin. 298 pp.
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ANNEX 1
DEFINITIONS
Apical endpoint: Causing effect at population level.
Blastula: The blastula is a cellular formation around the animal pole that covers a certain part of the yolk.
Epiboly: is a massive proliferation of predominantly epidermal cells in the gastrulation phase of the
embryo and their movement from the dorsal to the ventral side, by which entodermal cell layers are
internalised in an invagination-like process and the yolk is incorporated into the embryo.
Flow-through test is a test with continued flow of test solutions through the test system during the
duration of exposure.
Internal Plate Control: Internal control consisting of 4 wells filled with dilution water per 24-well plate
to identify potential contamination of the plates by the manufacturer or by the researcher during the
procedure, and any plate effect possibly influencing the outcome of the test (e.g. temperature gradient).
IUPAC: International Union of Pure and Applied Chemistry
Maintenance water: Water in which the husbandry of the adult fish is performed.
Median Lethal Concentration (LC50) is the concentration of a test substance that is estimated to be
lethal to 50% of the test organisms within the test duration.
Semi-static renewal test is a test with regular renewal of the test solutions after defined periods
(e.g., every 24 hrs).
SMILES: Simplified Molecular Input Line Entry Specification
Somite: In the developing vertebrate embryo, somites are masses of mesoderm distributed laterally to the
neural tube, which will eventually develop dermis (dermatome), skeletal muscle (myotome), and
vertebrae (sclerotome).
Static test is a test in which test solutions remain unchanged throughout the duration of the test.
UVCB: substances of unknown or variable composition, complex reaction products or biological
materials
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ANNEX 2
MAINTENANCE, BREEDING AND TYPICAL CONDITIONS FOR ZEBRAFISH EMBRYO
ACUTE TOXICITY TESTS
Zebrafish (Danio rerio)
Origin of species
India, Burma, Malakka, Sumatra
Sexual dimorphism
Females: protruding belly, when carrying eggs
Males: more slender, orange tint between blue longitudinal stripes
(particularly evident at the anal fin)
Feeding regime
Dry flake food (max. 3% fish weight per day) 3 - 5 times daily;
additionally brine shrimp (Artemia spec.) nauplii and / or small
daphnids of appropriate size obtained from an uncontaminated
source. Feeding live food provides a source of environmental
enrichment and therefore live food should be given wherever
possible. To guarantee for optimal water quality, excess food and
feces should be removed approx. one hour after feeding.
Approximate weight of adult
fish
Females: 0.650.13 g
Males: 0.50.1 g
Illumination
Fluorescent bulbs (wide spectrum); 10 - 20 µE/m2/s, 540 - 1080 lux,
or 50 - 100 ft-c (ambient laboratory levels); 12 - 16 hrs photoperiod
Water temperature
26±1 °C
Water quality
O2 ≥80% saturation, hardness: e.g., ~ 30 - 300 mg/L CaCO3, NO3-:
≤48mg/L, NH4+ and NO2-: <0.001 mg/L, residual chlorine <10
µg/L, total organic chlorine <25 ng/L, pH = 6.5 - 8.5
Further water quality
criteria
Particulate matter <20 mg/L, total organic carbon <2 mg/L, total
organophosphorus pesticides <50 ng/L, total organochlorine
pesticides plus polychlorinated biphenyls <50 ng/L
Tank size for
maintenance
e.g., 180 L , 1 fish/L
Water purification
Permanent (charcoal filtered); other possibilities include
combinations with semi-static renewal maintenance or flow-through
system with continuous water renewal
Recommended male to female
ratio for breeding
2:1 (or mass spawning)
Spawning tanks
e.g., 4 L tanks equipped with steel grid bottom and plant dummy as
spawning stimulant; external heating mats, or mass spawning within
the maintenance tanks
Maintenance of parental fish
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Egg structure and appearance
Stable chorion (i.e. highly transparent, non-sticky, diameter ~ 0.8 –
1.5 mm)
Spawning rate
A single mature female spawns at least 50 - 80 eggs per day.
Depending on the strain, spawning rates may be considerably
higher. The fertilisation rate should be ≥70%. For first time
spawning fish, fertilisation rates of the eggs may be lower in the
first few spawns.
Test type
Static, semi-static renewal, flow-through, 26±1 °C, 24 hrs
conditioned test chambers (e.g., 24-well plates 2.5 - 5 ml per cavity)
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ANNEX 3
NORMAL ZEBRAFISH DEVELOPMENT AT 26OC
Fig. 1: Selected stages of early zebrafish (Danio rerio) development: 0.2 – 1.75 hrs post-fertilisation
(from Kimmel et al., 1995). The time sequence of normal development may be taken to diagnose both
fertilisation and viability of eggs (see paragraph 26: Selection of fertilised eggs).
Fig. 2: Selected stages of
late zebrafish (Danio rerio)
development (de-
chorionated embryo to
optimise visibility): 22 - 48
hrs after fertilisation (from
Kimmel et al., 1995 (35)).
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Fig. 3: Normal development of zebrafish (Danio rerio) embryos: (1) 0.75 hrs, 2-cell stage; (2) 1 hr, 4-
cell stage; (3) 1.2 hrs, 8-cell stage; (4) 1.5 hrs, 16-cell stage; (5) 4.7 hrs, beginning epiboly; (6) 5.3 hrs,
approx. 50 % epiboly (from Braunbeck & Lammer 2006 (40)).
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ANNEX 4
Fig. 1: Layout of 24-well plates
1-5 = five test concentrations / chemical; nC = negative control (dilution water); iC = internal plate control (dilution water);
pC = positive control (3,4-DCA 4mg/L); sC = solvent control
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Fig. 2: Scheme of the zebrafish embryo acute toxicity test procedure (from left to right): production of eggs, collection of the eggs, pre-exposure
immediately after fertilisation in glass vessels, selection of fertilised eggs with an inverted microscope or binocular and distribution of fertilised eggs
into 24-well plates prepared with the respective test concentrations/controls, n = number of eggs required per test concentration/control (here 20), hpf =
hours post-fertilisation.
Spawning unit
Glass vessel with
respective test con-
centrations/controls
at volumes to fully
cover eggs
2n eggs
per conc.
/control
n fertilised eggs
per test concen-
tration/control
100 ml Crystallization dish with
respective concentrations and
controls
10 Fertilized eggs
per concentration
Control of fertili-
zation success
Spawning unit
20 Eggs
per conc.
Waste
Selection of
fertilised eggs
& fertilisation rate
determination
0 hpf
≤ 1.5 hpf
≤ 3 hpf
100 ml Crystallization dish with
respective concentrations and
controls
10 Fertilized eggs
per concentration
Control of fertili-
zation success
Spawning unit
20 Eggs
per conc.
Waste
24 h pre-
conditioning
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ANNEX 5
ATLAS OF LETHAL ENDPOINTS FOR THE ZEBRAFISH EMBRYO ACUTE TOXICITY TEST
The following apical endpoints indicate acute toxicity and, consequently, death of the embryos:
coagulation of the embryo, non-detachment of the tail, lack of somite formation and lack of heartbeat. The
following micrographs have been selected to illustrate these endpoints.
Fig. 1: Coagulation of the embryo: Under bright field
illumination, coagulated zebrafish embryos show a
variety of intransparent inclusions.
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Fig. 2: Lack of somite formation: Although retarded in development by approx. 10 hrs, the 24 hrs old
zebrafish embryo in (a) shows well-developed somites (→), whereas the embryo in (b) does not show any
sign of somite formation (→). Although showing a pronounced yolk sac oedema (*), the 48 hrs old
zebrafish embryo in (c) shows distinct formation of somites (→), whereas the 96 hrs old zebrafish embryo
depicted in (d) does not show any sign of somite formation (→). Note also the spinal curvature (scoliosis)
and the pericardial oedema (*) in the embryo shown in (d).
a
b
a
d
c
*
*
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Fig. 3: Non-detachment of the tail bud in lateral view (a: →; 96 hrs old zebrafish embryo). Note also the
lack of the eye bud (*).
Fig. 4: Lack of heartbeat is, by definition, difficult to illustrate in a micrograph. Lack of heartbeat is
indicated by non-convulsion of the heart (double arrow). Immobility of blood cells in, e.g., the aorta
abdominalis (→ in insert) is not an indicator for lack of heartbeat. Note also the lack of somite formation in
this embryo (*, homogenous rather than segmental appearance of muscular tissues). The observation time
to record an absence of heartbeat should be at least of one minute with a minimum magnification of 80×.
a
*
*
*
*
*
