Chapter 18

Evaluation of Embryotoxicity Using the Zebrafish Model

Lisa Truong and Robert L. Tanguay

Abstract
The embryonic zebrafish model offers the power of whole-animal investigations (e.g., intact organism,
functional homeostatic feedback mechanisms, and intercellular signaling) with the convenience of cell
culture (e.g., cost- and time-efficient, minimal infrastructure, small quantities of solutions required). The
model system overcomes many of the current limitations in rapid to high-throughput screening of drugs/
compounds and casts a broad net to rapidly evaluate integrated system effects. Additionally, it is an ideal
platform to follow up with targeted studies aimed at the mechanisms of toxic action. Exposures are carried
out in multi-well plates so minimal solution volumes are required for the assessments. Numerous morpho-
logical, developmental, and behavioral endpoints can be evaluated noninvasively due to the transparent
nature of the embryos.

Key words Zebrafish, Development, Embryos, In vivo, Vertebrate, Rapid screening, High-throughput
screening

1 Introduction

Numerous biological models can be employed for toxicity evalua-

tions. In vitro techniques, such as cell culture systems, are often
preferred because they are both cost- and time-efficient. While
these studies are useful, direct translation to whole organisms and
human health is often difficult to infer. In vivo studies can provide
improved prediction of biological response in intact systems but
often require extensive facilities and infrastructure [1–3]. Zebrafish
(Danio rerio) offer a number of practical advantages as a model
organism that overcome these limitations, making these verte-
brates highly amenable for toxicologically relevant research.
Zebrafish can be employed as a powerful in vivo model system to
assess biological interactions and are an outstanding platform to
detail the mechanisms by which substances elicit specific biological
responses. A remarkable similarity in cellular structure, signaling
processes, anatomy, and physiology exists among zebrafish and
other high-order vertebrates, particularly early in development
[4–8]. Current estimates indicate that over the zebrafish and

Jean-Charles Gautier (ed.), Drug Safety Evaluation: Methods and Protocols, Methods in Molecular Biology, vol. 1641,
DOI 10.1007/978-1-4939-7172-5_18, © Springer Science+Business Media LLC 2017

325
326 Lisa Truong and Robert L. Tanguay

human genomes 79% are similar and over 80% of the genes associ-
ated with human diseases are present in zebrafish [9]. Thus, inves-
tigations using this model system can reveal chemical and genome
interactions that are likely to be conserved across species.
Features of the zebrafish’s biology are favorable for adapting this
model system to high-throughput assays. Female zebrafish are able to
produce hundreds of eggs weekly, so large sample sizes are easily
achieved, allowing for statistically powerful dose-response studies. This
abundant supply of embryos also makes it possible to simultaneously
assess the toxicity of a large number of substances in a short period.
The vertebrate’s rapid developmental progression compared to other
mammals makes it an ideal model for high-­throughput screening [10].
For example, neuronal plate formation occurs at 10 hours post fertil-
ization (hpf), followed by organogenesis at 24 hpf, which compared to
a rat occurs at 9.5 days and 5–6 days respectively. The first heartbeat
occurs at 30 hpf for the zebrafish and 10.2 days for rats [11].
Zebrafish embryos can be individually exposed in wells of a
multi-well plate so the required volume needed for the model is
small; thus, only limited amounts of materials are needed to assess
an entire suite of biological interactions and responses. Early devel-
opmental life stages are often uniquely sensitive to environmental
insult, due in part to the enormous changes in cellular differentia-
tion, proliferation, and migration required to form multiple cell
types, tissues, and organs [4, 7, 8, 12]. Since development is highly
coordinated requiring specific cell-to-cell communications, if
exposure to a substance during that critical period perturbed these
interactions, development would be expected to be disrupted.
Embryos are waterborne–exposed to a chemical using a continu-
ous method in which 24 embryos are exposed per concentration in
individual wells of a multi-well plate from 8 to 120 hpf. Exposure
until 120 hpf is the ideal duration for a developmental toxicity test-
ing; primarily due to the vertebrate model’s ability to obtain its
nutrients from its yolk sac until 5 days, which will not introduce
additional external confounding factors. Perturbed development
can manifest as morphological malformations, behavioral abnor-
malities, or death of the embryos. Zebrafish embryos develop
externally and are optically transparent so it is possible to resolve
individual cells in vivo throughout the duration of an exposure
using simple microscopic techniques and numerous effects can be
assessed noninvasively over the course of development.

2 Materials

2.1 Zebrafish 1. Fish water: 0.3 g/L Instant Ocean salts (Aquatic Ecosystems,
Husbandry Apopka, FL) in reverse osmosis (RO) water.
2. Incubator set at 28 ± 0.1 °C.
3. Fish food: Gemma Micro (Skretting Inc, Tooele France).
 Evaluation of Embryotoxicity Using the Zebrafish Model 327

2.2 Dechorination 1. Compound stereo microscope for viewing embryos.

2. 60 mm glass petri dish.
3. 22.24 U/mL of total activity pronase per 100 μL aliquot
(Roche, Catalog No: 10165921001) in ultra pure water.
Assuming the activity level of the powder pronase is ≥7 U/
mg, weigh 317.77 mg of pronase into 50 mL glass beaker
and fill it with 5 mL of ultra pure water. Gently vortex the 50
mL pronase: ultra pure water mixture with stir bar in refrig-
erator for 30 min. Remove beaker and keep on ice. Aliquot
100 μL into 1.5 mL microcentrifuge tube and place them
into a freezer box, then immediately place into the box into
the freezer. This will make 50 1.5 mL microcentrifuge tubes
that can be stored for up to 4 months. Aliquots can be thawed
just prior to use.
4. Timer.

2.3 Exposure 1. Multi-well plates.

2. 8 or 12 multichannel pipette.
3. 50 mL reagent reservoir.
4. Wide-bore Pasteur pipette.

2.4 Assessment 1. Anesthesia: 4 mg/mL of 3-aminobenzoate ethyl ester meth-

anesulfonate salt (tricaine, Sigma-Aldrich) in RO water, pH
adjusted to 7.0 with 0.1 M Tris–HCl, pH 9.0.
2. Methyl cellulose: 10 mg/mL of methyl cellulose (Sigma-­
Aldrich, see Note 1).

3 Methods

3.1 Zebrafish 1. Rear adult zebrafish Danio rerio in standard laboratory con-
Husbandry ditions of 28 °C with a pH of 7 ± 0.2 on a 14 h light/10 h
dark photoperiod [13].
2. House zebrafish in 2.0-L polycarbonate tanks with recirculat-
ing water system. Keep adult zebrafish in groups to allow for
large quantities of embryos to be collected. Group spawning
also helps to increase genetic diversity.
3. Feed the fish twice daily with Gemma Micro feed.
4. Spawning: place male and female zebrafish into spawning bas-
kets in polycarbonate tanks the afternoon before the embryos
are needed. Zebrafish will typically spawn when the lights come
on after the 10 h dark period.
5. The following morning, newly fertilized eggs are collected,
rinsed several times in system water, and placed into fresh fish
water in a 150 mm plastic petri dish.
328 Lisa Truong and Robert L. Tanguay

Fig. 1 Six hours post fertilization embryos. (a) Six hpf embryo with its chorion. (b) Six hpf embryo after using
pronase to enzymatically remove its chorion

6. Remove embryos that are unfertilized or necrotic prior to plac-

ing the petri dish into the incubator to keep warm until the
embryos reach 6 hours post fertilization (hpf) (Fig. 1) [10].
7. Remove embryos that are not the same stage as the majority
prior to experimental use (see Note 2).

3.2 Dechorination 1. To avoid barrier effects potentially posed by the chorion, all
embryos should be dechorionated at 6 hours post fertilization
(hpf) using a protocol for pronase enzyme degradation
[14–16].
2. Place 6 hpf embryos into a 60 mm glass petri dish with 25 mL
fish water (see Note 3). Up to 1000 embryos can be processed
in a single dish using this method [17].
3. Add 83 μL of 31.77 mg/mL pronase (with ≥7 U/mg pronase
activity level) to the center of the dish and continuously swirl
gently to mix the solution.
4. Set a timer for 6 min, and continuously swirl the embryos
while occasionally observing the petri dish under the micro-
scope to check for embryos without chorions, chorion pieces
in the solution, and “deflated” chorions.
5. When 7 min have passed, or when the above are observed,
remove the pronase solution by diluting the solution with fresh
fish water, slowly decanting over the edge of the petri dish
continuously for 1 min, then repeat this procedure for a total
of 10 min (see Note 4).
6. After the rinse, allow the embryos to recover in the petri
dish in an incubator (or a room at 28 °C) for 30 min (see
Note 5).
 Evaluation of Embryotoxicity Using the Zebrafish Model 329

3.3 Exposure 1. Chemicals should be dissolved in ultra pure water if possible

(see Note 6). In the case that this is not possible, the solvent of
3.3.1 Waterborne
choice for exposure utilizing the embryonic zebrafish is
Exposure
dimethyl sulfoxide (DMSO) (see Note 7).
2. Pour each test solution into a 50 mL reagent reservoir, which
will fit a multichannel pipette.
3. For each exposure concentration tested, use a multichannel
pipette to fill 16 individual wells in a multi-well plate with 100
μL of chemical solution. Five concentrations and one control
group can be tested using two 96-well plates.
4. At 8 hpf, transfer viable, appropriately developing embryos
into individual wells of a multi-well plate using a wide-bore
glass pipette (see Note 8).
5. Incubate at 28 °C until 24 hpf, then perform assessments.

3.3.2 Microinjection 1. If direct delivery of a chemical is necessary to ensure accurate

Exposure dose delivery, embryos should be microinjected at 8 hpf (see
Note 9).
2. Align 8 hpf embryos in troughs embedded in a 1% agarose
plate filled with fish water as described by The Zebrafish Book
[11, 13].
3. Inject each embryo with 2.3 nL of the desired chemical con-
centration or the appropriate vehicle control directly into the
yolk.
4. Place each embryo into individual wells of a 96-well plate, each
filled with 100 μL of fish water. When directly delivering a
chemical into the yolk sac, any concentration above 0.1%
DMSO caused developmental defects not attributed to the
chemical. If a chemical requires a solvent, two sets of serial
dilutions should be made. The first serial dilution should be
100 times higher than the final concentration desired made
with 100% DMSO (see Note 10) For the second set of serial
dilutions, from the 100% DMSO serial dilution, make a 1:10
dilution from the first serial dilution. Make sure to have an
appropriate control for each chemical, which includes the cor-
rect percentage of solvent used in each solution.
5. Incubate at 28 °C until first assessments at 24 hpf.

3.4 Assessment 1. At 24 hpf, embryos are assessed for viability, developmental

progression, and spontaneous movements (earliest behavior in
zebrafish). Developmental progression is considered perturbed
if zebrafish are more than 12 h delayed compared to control
animals. Spontaneous movements are assessed over a 2 min
period and are considered perturbed if there is a lack of embry-
onic contractions and/or movement.
330 Lisa Truong and Robert L. Tanguay

2. At 120 hpf, larval morphology (body axis, eye, snout, jaw, otic
vesicle, notochord, heart, brain, somite, fin, yolk sac, trunk, cir-
culation, pigment, swim bladder; Fig. 2) is evaluated and
recorded and behavioral endpoints (motility, tactile response)

Control zebrafish 120 hpf

1. 120 hr mortality – dies between 24 and 120 hours post fertilization (hpf)

2. 24 hr mortality – dies before 24 hpf

3. 24 hr sp. Mov – no spontaneous movement at 24 hpf 4

4. 24 hr dev prog - delayed development

5. 24 hr notochord – notochord malformation (wavy notochord) 5

6. axis – curved or bent axis in either direction 6

6, 18
7. brain - brain malformations or necrosis

8. caudal fin – malformed or missing 8

9. circulation – no circulation or blood flow 7, 10

10. eye – eyes malformed, missing or smaller/larger than normal

11. heart – heart malformation, pericardial edema (fluid around the heart)
11, 12, 15, 16, 17, 18, 20, 21
12. jaw – malformed

13. otic – malformed or missing

14. pectoral fin – malformed or missing

15. pigmentation – lack of pigmentation, overpigmentation

12, 13
16. snout – shortened or malformed

17. somite – malformed or disorganized, missing somites

18. swim bladder inflate – failure of swim bladder to inflate

19. touch response – not responsive to touch at 120 hpf

20. trunk – short trunk, malformed or missing

21. YSE – yolk sac edema, swelling around the yolk sac
14

Fig. 2 Visual assessment of zebrafish morphology. Images are given as examples of typical chemical-induced
malformations observed in the zebrafish
 Evaluation of Embryotoxicity Using the Zebrafish Model 331

are thoroughly evaluated in vivo. Test for behavioral endpoints

and then anesthetize animals for thorough morphological anal-
ysis. At the end of the assessments, zebrafish are euthanized
with tricaine.
3. Evaluations are completed in a binary notation (present or not
present) (see Note 11). Control and chemical-exposed groups
are statistically compared using Fisher’s Exact test at p < 0.05
with R software for each endpoint evaluated (Fig. 2).

4 Notes

1. Methyl cellulose is unique in that it “melts” when cold and

solidifies when hot. It dissolves best in cold water; however, it
is best to disperse the powder form in warm water and then
continue to mix while chilling. An alternate to the methyl cel-
lulose is Protoslo® (Carolina Biological Supply Company,
Burlington, NC).
2. Eggs can sometimes be laid and fertilized at different times in a
group spawns; therefore always remove embryos that are devel-
oping more rapidly or significantly slower prior to using them
for an experiment. As an alternate, male and female pairs can be
set up in several divided tanks, and the dividers can be removed
at the same time. The resulting stage matched embryos can
then be pooled, prior to random embryo selection.
3. Do not bleach embryos if their chorions are to be removed by
pronase digestion. Bleaching modifies the chorion and pro-
nase treatment is completely ineffective. In addition, when
dechorinating embryos it is essential to use glass petri dishes.
Dechorinated embryos will stick to the bottom of plastic
dishes and will be severely damaged during the procedure.
4. The newly dechorionated embryos are very delicate. Water
should be administered with a gentle flow and not directly
onto the embryos. Some of the embryos will not be out of
their chorion even once the 10 min rinsing period is done.
More will emerge during the recovery period.
5. Once an embryo is dechorinated, do not bleach the embryos.
6. Chemicals or drugs that are thought to be inactive until
metabolized to an active form may be pre-exposed to induce
and active conformation prior to waterborne exposures.
7. The Sinnhuber Aquatic Research Laboratory at Oregon State
University has demonstrated that an embryo elicited no devel-
opmental deformities at 1% DMSO when waterborne-exposed
[3, 18–20].
332 Lisa Truong and Robert L. Tanguay

8. Be sure to allow the embryo to fall to the bottom of the wide-­

bore Pasteur pipette prior to touching the solution in the wells.
If an embryo disintegrates when it reaches the solution, make
sure to replace the solution and place a new embryo in the well.
9. All methods discussed are continuous waterborne exposure,
but if no analytical method is available to determine biological
uptake, an alternative is to directly deliver the chemical into
the animal through microinjection. Because embryos are
transparent, tissue dose and distribution can also be deter-
mined using fluorescently labeled materials and laser scanning
confocal microscopy.
10. Make sure to vortex each microcentrifuge tube prior to the
next dilution to ensure it is a homogenous solution.
11. If more than 20% of the animals in the control group are
adversely affected (any endpoint, including mortality), then
the experiment is not valid and will need to be repeated. Test
chemicals may have specific targets in humans, but this target
may not be completely conserved structurally in other verte-
brate models. The structural differences between vertebrates
and humans can result in either false negatives or false positives.
For example, if a drug is designed to target a human specific
structure that is not well conserved in zebrafish, upon expo-
sure, the drug would not influence the zebrafish target. The
effects observed when this occurs are considered false nega-
tives. Vice versa, a false positive can also occur when effects
observed due to a drug impacting a specific target expressed
only in zebrafish, but this target is not structurally conserved in
humans. Another consideration is that chemical toxicity may
be dependent on metabolic activity. False negatives and false
positives may also occur if the metabolic activity in the zebraf-
ish embryo is distinct from human metabolic activity. It is pos-
sible to use exogenous mammalian metabolic activation system
to reduce false positive and false negatives [21].

Acknowledgments

The authors would like to thank the Sinnhuber Aquatic Research

Laboratory and the Environmental Health Sciences Center at
Oregon State University where much of the protocols were devel-
oped. This work was supported by NIEHS grants P30 ES000210.
 Evaluation of Embryotoxicity Using the Zebrafish Model 333

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