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The zebrafish embryo toxicity and teratogenicity assay

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THE PHILIPPINE BIOTA
Volume XLIV
October 2011

THE ZEBRAFISH EMBRYO TOXICITY AND TERATOGENICITY ASSAY

Jordan Ferdin A. Halili and Jonas P. Quilang

Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon
City 1101

For correspondence:

Dr. Jonas P. Quilang

Institute of Biology, College of Science, University of the Philippines 1101 Diliman,
Quezon City. email: jpquilang@gmail.com; telefax no. 632-9205471

ABSTRACT

The zebrafish, Danio rerio (Hamilton, 1822), has recently emerged as a model
organism for genetic research and for the study of vertebrate development. It is also
extensively used to test for toxicity and teratogenicity of drugs, chemicals, and other
substances. It offers a number of advantages as a model organism, such as its small
size, high fecundity, ease of rearing, and external development which allows for direct
observation. Being a vertebrate, the embryotoxic and teratogenic effects of substances
may be extrapolated to higher vertebrates such as humans. A few studies that have
used zebrafish as a model as well the endpoints used are given. The materials needed,
the general procedure for this assay, analysis, and presentation of data are also given.

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INTRODUCTION

The zebrafish, Danio rerio (Hamilton, 1822), is used widely in studying vertebrate
biology. Both developmental and genetic analyses can easily be done in zebrafish
(Teraoka et al., 2002).This species is relatively small in size and thrives in freshwater
habitats, and thus can easily be reared and maintained in aquarium. It is sexually
mature in three months and spawns a large number of eggs. Its spawning is triggered
by light and each female may produce 100 large eggs per day (Herrmann, 1993;
Samson & Shenker, 2000). The embryos are transparent and develop externally, which
allows easy observation (Hill et al., 2005). Most organs develop within one day at 26 C
and the embryos hatch after three to four days. The larvae are also small which allow
them to be placed in small labware like Petri dishes and cell-culture plates. These
features make the species an excellent model for studying embryotoxic and teratogenic
effects of drugs, pollutants or toxic substances (Samson & Shenker, 2000; Hallare et al.,
2004).

A number of studies have been done to examine the embryotoxicity and

teratogenicity of substances on zebrafish. Samson and Shenker (2000) identified the
stages of embryonic development in Danio rerio that were most sensitive to
methylmercury exposure. Results proved that zebrafish is an excellent organism in
assessing the toxicity of methylmercury. The results also identified which stages were
most vulnerable to finfold abnormalities and tail flexures upon exposure to
methylmercury. Samson and Shenker (2000) found that these periods coincided with
the time when the tail and median fin develops. Another study by Tiedeken et al. (2005)
examined the developmental toxicity of domoic acid on zebrafish. Reduced hatching
and spinal deformities were observed. Other prominent effects of domoic acid included
involuntary movements in the zebrafish larva such as uncontrolled pectoral fin motions
and convulsions. This study showed that zebrafish could be at par with other bioassay
organisms such as rodents.

Zebrafish assay has also been used in assessing pollutants present in the
environment and in testing ecotoxicity of substances (Nagel, 2002). A Campagna et al.
(2007) determined the effects of organochlorine pesticides (aldrin and heptachlor) on
the survival, growth, and gill morphology of zebrafish. Even though survival was not
affected, exposure of zebrafish embryos to low concentrations of these pesticides
showed sublethal effects such as reduced body lengths and presence of gill lesions.
Rampant use of such pesticides may pose risks to the survival of aquatic species and
may compromise ecological balance. In the Philippines, Hallare et al. (2005) used a
sediment contact assay on zebrafish to determine the contamination levels of Laguna
Lake. Developmental defects were observed in embryos exposed to whole sediment
treatments. Moreover, severe teratogenic effects were seen in embryos exposed to
organic extracts of the sediments obtained from the lake. This study provided proof that
zebrafish assay is highly sensitive in assessing sediment toxicity. Quilang et al. (2008)
found deformities and significant reductions in average length and number of
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melanocytes of zebrafish exposed to high concentrations of Polychlorinated biphenyls

(PCBs) which are common environmental pollutants.

Major advances in zebrafish studies have been done not only in developmental
biology and ecotoxicology but also in the field of medicine specially on screening for
drugs and on the understanding of diseases (Dooley & Zon, 2000; Rubinstein 2006).
Macrae & Peterson (2003) studied the potential of zebrafish assay in the discovery of
small molecules. Berghmans et al. (2008) investigated the effects of 16 pharmacological
compounds on cardiac, visual, and gut contraction functions in zebrafish. They found
good correlation between their results using zebrafish and known clinical adverse
effects of these compounds to other in vivo models.

A number of endpoints are used in evaluating the toxicity and teratogenicity of

chemicals on zebrafish embryo. Among these are egg and embryo mortality,
pigmentation, heart rate, hatching success, larval viability, larval length, malformations
(Black et al. 1988; Sinha & Kanamadi, 2000; Cheng et al. 2000; Todd & Van Leeuwen,
2002; Hallare et al. 2004; Tiedeken et al. 2005). Most recently used endpoints include
malformations in somite, otoliths, eyes, spinal cord, heart, yolk, tail, fin, heart, facial
structure, brain, jaw, and pharyngeal arch and other organs such as swimbladder,
stomach, intestine and liver (Busquet et al., 2008; Selderslaghs et al., 2009; Brannen et
al., 2010).

Objectives

(1) To determine whether a drug, chemical, pollutant, or a test substance is

embryotoxic or teratogenic and at which concentration;

(2) Other than testing for toxicity or teratogenicity of substances, the materials and
methods that follow may also be used for a simple class activity to visualize the
stages in the development of a vertebrate.

Materials

50 - 100 zebrafish
30-L aquarium
2, 20-L aquaria
24-well culture plates (96-well or 384-well plates if small amounts of test solutions are
used)
Petri dish
fluorescent light
green plastic mesh
spawning tray
stereomicroscope
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blue pipette tips for transferring eggs (around 5 mm from the tip is cut to widen the
orifice and allow egg to pass through; the other end is fitted with an aspirator from a
dropper)
embryo medium (294 mg CaCl2, 123.25 mg MgSO4, 64.75 mg NaHCO3, and 5.75 mg
KCl dissolved in 1 L of distilled water)
pipettor and tips (if small amounts of test solutions are used)
different concentrations of the test substance
positive control (2% ethanol)
negative control (embryo medium)
fish flakes
blood worms

Methodology

1. Acclimatize 25 – 50 adult zebrafish separately for each sex for 2 to 3 weeks in a

20-L aquarium. Zebrafish may be obtained from pet shops. Tap water to be used
in rearing the fish should be aged by storing in a bucket for about 15 days to
remove chlorine. Before use, the tap water should be filtered and aerated and
analyzed for some physico-chemical parameters like dissolved oxygen, pH,
temperature, and salinity.

2. The zebrafish are fed with commercial fish flakes two times a day and with blood
worm two to three days before spawning. The fish are kept in a 12-h/12-light/dark
cycle.

3. For spawning, male and female zebrafish are reared together in a 30-L
aquarium. The fish are placed inside a plastic mesh shaped like a box (Figure 1);
this is to prevent the parents from eating their eggs. The aggs are caught in a
spawning tray placed below the plastic mesh. Spawning is triggered once the
light is turned on and it usually lasts from 30 minutes up to one hour (Hermann
1993; Hallare et al. 2004).

4. One hour after switching on the light, the eggs are collected and rinsed with tap
water. Unfertilized eggs (opaque or with an irregularly shaped first blastomere)
are identified and discarded (Figure 2). All the eggs to be used in the experiment
should be laid by the same group of adults.

5. At around 2 - 6 h postfertilization, only the fertilized eggs (blastula stage) are

selected and transferred to Petri dishes containing embryo medium.

6. One fertilized egg that is in blastula stage, at 50 % epiboly (Figure 2), is placed
on each well of a 24-well culture plate containing 2 ml of test solutions or
controls. Twenty-four (24) fertilized eggs are used per concentration of the test
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substance and for each of the controls (positive, negative, and carrier solvent). At
least three trials should be done. Alternatively, if there is a constraint on the
volume or availability of test substance, 96- or 384-well culture plates may be
used. Peterson et al. (2000) used 50-μl and 200-μl in each well of a 384-well and
96-well plate, respectively. However, embryos placed in 96-well plates tended to
have skeletal abnormalities (Selderslaghs et al. 2009). The test solution in each
well may be replaced daily or replenished.

7. The embryos are observed every 24 hours, taking note of mortality, hatching,
and physical abnormalities such as axial curvatures, cardiac and yolk sac edema
and hypopigmentation. Survival rate at 2, 3 and 5 days posfertilization (dpf) are
noted. At 3 dpf, hatching rate and heart beat per minute are recorded. Heart rate
is obtained by counting the number of distinct beats in a span of 15 seconds. At 5
dpf, the body length (from tip of the head to the tip of the tail) of the embryos is
measured. The melanocytes on the left side of the body of each embryo are also
counted under a stereomicroscope at 5 dpf (Figure 3).
Data analysis and presentation
Continuous data such as length, number of melanocytes, heart rate, percent
survival, percent deformities, and hatching rate may be analyzed using Analysis of
Variance (ANOVA) to determine if there is significant difference between the means of
test concentrations and controls. Prior to ANOVA, data are tested if they satisfy the
assumption of normality and homoscedasticity using appropriate statistical software. If
these assumptions are not met, logarithmic, square-root, or arcsin-square-root
transformation may be done on the data. If a statistical sofware is used, the P-value
(Probability or significance value) is given for each F-value in the ANOVA. A P-value of
0.05 or less indicates significant differences in means. A P-value of 0.01 or less
indicates higly significant differences in means. If there are significant differences in the
ANOVA, post-hoc test can be done such as Duncan’s Multiple Range Test (DMRT),
Tukey test, or Dunnet’s test. If after doing each of the suggested data transformations,
the assumptions of ANOVA are not satisfied, Kruskal-Wallis test, a non-parametric test
may be used. Means as well as standard errors or stand deviations of the treatments
and controls are either tabulated (see Quilang et al. 2008 for an example) or presented
as graphs (see Hallare et al. 2005 for an example). Digital images of embryos or larvae
with deformities may also be shown.

ACKNOWLEDGEMENTS

We wish to acknowledge the Institute of Biology, UP Diliman for the use of

facilities, the UP Office of the Vice-President for Academic Affairs for the equipment
grant, and Dr. Sonia D. Jacinto, our mentor, for encouragement and support.
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REFERENCES

Black, D.E., D.K. Phelps, & R.L. Lapan. (1988). The effect of inherited contamination on
egg and larval winter flounder, Pseudopleuronectes americanus. Mar. Environ.
Res. 25: 45-62.
Berghmans, S., P. Butler, P. Goldsmith, G. Waldron, I. Gardner, Z. Golder,
F.M. Richards, G. Kimber, A. Roach, W. Alderton, A. Fleming. (2008). Zebrafish
based assays for the assessment of cardiac, visual and gut function--potential
safety screens for early drug discovery. J. Pharmacol. Toxicol. Methods. 58(1):
59-68.
Brannen, K.C., J.M. Panzica-Kelly, T.L. Danberry, & K.A. Augustine-Rauch. (2010).
Development of a Zebrafish Embryo Teratogenicity Assay and Quantitative
Prediction Model. Birth Defects Res. (Part B) 89: 66-77.
Busquet, F., R. Nagel, F. von Landenberg, S.O. Mueller, N. Huebler, & T. Broschard.
(2008). Development of a new screening assay to identify proteratogenic
substances using zebrafish Danio rerio embryo combined with an exogenous
mammalian metabolic activation system (mDarT). Toxicol. Sci. 104(1): 177-188.
Campagna, A.F., M.N. Eler, R. Fracacio, B.K. Rodrigues, & N.F. Veami. (2007). The
toxic potential of aldrin and heptachlor on Danio rerio juveniles (Cypriniformes,
Cyprinidae). Ecotoxicology 16: 289-298.
Cheng, S.H., A.W.K. Wai, C.H. So, & R.S.S. Wu. (2000). Cellular and molecular basis
of cadmium-induced deformities in zebrafish embryos. Environ. Toxicol. Chem.
19: 3024-3031.
Dooley, K. & L. I Zon. (2000). Zebrafish: a model for the study of human disease. Curr.
Opin. Gen. Dev. 10: 252-256.
Hallare, A.V., H.-R. Kohler, & R. Triebskorn. (2004). Developmental toxicity and stress
protein responses in zebrafish embryos after exposure to diclofenac and its
solvent, DMSO. Chemosphere. 56: 659-666.
Hallare, A.V., T. Kosmehl, T. Schulze, H. Hollert, H.-R. Kohler, R. Triebskorn. (2005).
Assessing contamination levels of Laguna Lake sediments (Philippines) using a
contact assay with zebrafish (Danio rerio) embryos. Sci. Total Environ. 347: 254-
271.
Herrmann, K. (1993). Effects of the anticonvulsant drug valproic acid and related
substances on the early development of the zebrafish (Brachydanio rerio).
Toxicol. in Vitro. 7: 41-54.

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Hill, A.J., H. Teraoka, W. Heideman, & R.E. Peterson. (2005). Zebrafish as a Model
Vertebrate for Investigating Chemical Toxicity. Toxicol. Sci. 86(1): 6-19.
Macrae, C.A. & R. T. Peterson. (2003). Zebrafish-Based Small Molecule Discovery.
Chem. Biol. 10:901-908.
Nagel, R. (2002). DarT: The embryo test with the Zebrafish Danio rerio--a general
model in ecotoxicology and toxicology. ALTEX. 19 Suppl 1:38-48.
Peterson, R.T., B.A. Link, J.E. Dowling, & S.L. Schreiber. (2000). Small molecule
developmental screens reveal the logic and timing of vertebrate development.
Proc. Natl. Acad. Sci. USA 97(24): 12965–12969.

Quilang, J.P., M.C. de Guzman, M.H. de Hita-Catalan, R.O. Rubio, S.D. Jacinto, E.C.
Santiago, & E.P. Cao. (2008). Effects of Polychlorinated Biphenyls (PCBs) on
Root Meristem Cells of Common Onion (Allium cepa L.) and on early Life Stages
of Zebrafish (Danio rerio). Phil. J. Sci. 137(2): 141-151.

Rubinstein, A.L. (2006). Zebrafish assays for drug toxicity screening. Expert Opin. Drug
Metab. Toxicol. 2(2): 231-240.
Samson, J.C., & J. Shenker. (2000). The teratogenic effects of methylmercury on early
development of the zebrafish, Danio rerio. Aquat. Toxicol. 48: 343-354.
Sinha, P., & R. Kanamadi. (2000). Effect of mercurial fungicide Emisan®-6 on the
embryonic, developmental stages of zebrafish, Brachydanio (Danio) rerio. J. Adv.
Zool. 21: 12-18.
Selderslaghs, I. W.T., A.R. Van Rompay, W. De Coen, & H.E. Witters. (2009).
Development of a screening assay to identify teratogenic and embryotoxic
chemicals using the zebrafish embryo. Reprod. Toxicol. 28: 308–320.
Teraoka, H., W. Dong, S. Ogawa, S. Tsukiyama, Y. Okuhara, M. Niiyama, N. Ueno,
R.E. Peterson, & T. Hiraga. (2002). 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in
the zebrafish embryo: altered regional blood flow and impaired lower jaw
development. Toxicol. Sci. 65: 192-199.
Tiedeken, J.A., J.S. Ramsdell, & A.F. Ramsdell. (2005). Developmental toxicity of
domoic acid in zebrafish (Danio rerio). Neurotoxicol Teratol. 27: 711-717.
Todd, N. E., & M. Van Leeuwen M. (2002). Effects of Sevin (carbaryl insecticide) on
early life stages of zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 53: 267-272.

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FIGURES

Figure 1. Breeding tank: wire mesh, spawning tray and and light apparatus

A B

C D

Figure 2. Danio rerio eggs. A – 2 hours post fertilization (hpf); B – 6 hpf (50% epiboly); C – 12
hpf; D – unfertilized egg. Scale bar = 0.5 mm

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A B

C D

E F

G H

Figure 3. Normal (A & B) and malformed zebrafish larvae (C-H). A – normal ; B – normal
showing melanocytes; C – tail flexure ; D – spinal bend; E – yolk sac edema; F – cardiac
edema; G – combined cardiac and yolk sac edema; H – combined cardiac and yolk sac edema
with tail deformation. Malformed larvae were exposed to PCBs (C-E), 2% ethanol (F), and coal
tar dyes (G & H). Scale bar = 0.5 mm

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