Journal of Environmental Protection, 2011, 2, 1360-1363
doi :1 0.4236/ jep.2011. 210157 Published Online December 2011 (http://www.SciRP.org/journal/jep)
Improving Production of Zebra Fish Embryos in
the Lab
Robert Ohene Adu, Jens Peter Thomsen
Eco-Toxicology Research Group, Esbjerg Institute of Technology (EIT), Aalborg University, Esbjerg, Denmark.
Received August 21 st, 2011; revised September 27th, 2011; accepted October 29th, 2011.
ABSTRACT
The utilization of fish embryos in toxicity testing of hazardous chemicals has recently been adopted in order to satisfy
stricter rules and regulations related to using adult animals in toxicity testing. This paper presents optimising steps
towards improving zebra fish embryo production in the laboratory. Culture conditions were maintained in the aquaria
as stipulated in the OECD draft proposal for a new guideline on fish embryo tests. Furthermore, a sequence of steps
were adopted and followed to improve upon previous work done in the lab in 2006. About 200 eggs were produced in
one spawn trap within an hour of onset of light, an improvement over the 50 - 60 eggs produced in the previous work.
This result demonstrates that with the right culture conditions and proper optimisation of procedure the required num-
ber of embryos needed for toxicity testing can be obtained.
Keywords: Hazardous Chemicals, Zebra Fish Embryo, Toxicity Testing, Spawn Trap, Culture Conditio ns
1. Introduction
The use of juvenile and adult fish in toxicity testing of
chemicals and effluents has been a conventional practice
in eco-toxicology worldwide.
A number of factors render the use of fish suitable and
important in eco-toxicity and environmental risk assess-
ment. Some of these factors are:
 accidental fish kills are visible when they occur and
raise public concern about t he need to protect natural
waters from pollutants;
 fish are considered sentinels for the drinking quality
of waters used by man;
 the aquatic environment is a sink for many chemi-
cals and this is evidenced by the occasionally high
pollution levels and frequent che mical spills;
 fish play a critical role in aquatic food webs by re-
gulating top-down and bottom-up flow of nutrient
and energy;
 fish are an important source of food for humans;
 fishing has a large recreatio n value in many cultures.
In Europe toxicity testing with fish is done in order to
regulate amounts of chemical substances in line with the
goals of REACH (Registration, Evaluation, Authorization
and restriction of Chemicals), under the EU Chemicals
Agency, (ECHA) [1]. The increased production of che-
micals on the market means that more and more fish have
to be killed or poisoned for the purpose of establishing
lethal and sub lethal concentrations.
As of 2005, experts predicted an additional 1.5 million
animal tests needed to be carried out on about 30,000
existing chemicals in ord er to fully implement the obj ect-
tives of REACH, [2]. The establishment of REACH in
2007 has eliminated the distinction between existing sub-
stances and new substances in the EU market and has
replaced some 40 EU Directives and Regulations [3,4].
This has meant that these 30,000 existing substances with
annual market volumes of more than 1 ton, especially
those marketed above 100 tons per year, will need to be
tested under new toxicity testing schemes and their risk
to human health and the environment will be assessed
[3 , 5, 6] . Ou t of these, some 14 0 chemicals have bee n given
special attention by EU member countries and are to un-
derg o comprehensive risk assessment [7].
At the same time, the continuous use of juvenile and
adult fish in ecotoxicology work has come under attack
from animal welfare groups for ethical, economic and
scientific reasons. It is argued that in these bio -assays the
primary endpoint is mortality and fish usually suffer pain
or distress [7]. This, coupled with Directive 86/609/EEC
regulating the use of animals in scientific experiments
has provided the impetus for a reverse in the trend in fish
use for toxicity testing. Thus an alternative testing pro-

Improving Production of Zebra Fish Embryos in the Lab1361
cedure using the embryos of fish instead in toxicity test-
ing, called the fish embryo test (FET) has emerged. The
embryonic develop ment in rel ation to to xic conce ntratio n
is registered during a test period of 48 - 96 hours. In
Ge r ma ny, fo r e xa mp l e, FE T ha s replaced the conventio nal
fish test since 2005 as an obligatory requirement for routine
whole effluent testing (WET) and the test protocol is ame-
nable to changes to make it suitable for chemical testing
as well [8].
Scientific evidence strongly suggests tha t toxicity test -
ing of chemicals with fish embryos is neither better nor
worse than testing with adult fish. Results from a com-
parative re-evaluation of both fish embryo and adult fish
toxicity data carried out on 143 substances showed a
strong correlation between the two [7]. Indeed, using the
zebra fish as an example, a comparison of 21 data pairs
of lethal concentrations that kill 50% of the population
(LC50s) carried out on the adult fish and the embryo
showed a regression with a correlation of 0.81 and a
slope of 1.12 [7].
The Zebra fish (Danio rerio sp.), a tropical freshwater
fish has become a model in ecotoxicology and environ-
mental sciences with a wide range of future applications
for integrative risks assessment of chemicals [9]. T he spe-
cies is native to the streams of Southeastern Asia, es-
pecially Nepal, India, Pakistan, Bangladesh among others. It
has been noted to possess sterling qualities such as small
size of the species, its cheap and easy husbandry, superb
fertility, pr oductio n of tra nspa rent embr yos, rapid in-vitro
development of the embryos and simple, accurate che-
mical delivery [10].
This study, as a prelude to the embryo assay, looked at
steps towards enhancing the production of embryos of
the Zebra fish (Danio rerio sp.) for use in the fish em-
bryo toxicity testing. It is aimed at developing an appro-
priate methodology towards maximising the production
of embryos of the zebra fish in the lab. This test has been
proposed for testing the toxicity of a number of che-
micals such as saponins, speculated to be responsible for
recent fish kills in a small stream in Denmark [11]. Sapo-
nins are naturally-occurring glycosides considered to be
present in horse chestnuts (Aesculus hippocastanum sp.).
They are known to cause hemolysis of red blood cells in
fish [10].
Previous work done in the laboratory by [12] saw ire-
gular egg production and therefore inadequate embryos for
a thorough test. When eggs were produced they were usua-
lly between 50 and 60, less than the number required for a
duplicate test. As a result there is need for a standardized
procedure to maximize egg production for the series of
tests required. According to the procedure outlined in the
OECD Draft guideline for Fish Embryo Toxicity test [13],
the test should be performed in 2 - 3 replicates, each hav-
ing at least 5 concentrations of the test substance and a
control group in 24-well plates. This requires a minimum
of 120 embryos to meet the statistical requirements. To
achieve this, a number of optimising steps were needed to
ensure this production .
2. Materials and Methods
2.1. Experimental Set-Up
The set-up involved 2 aquaria (Waterhome) each of 40 L
capacity and a maintenance tank to maintain an optimum
water level. A breeding stock of mature zebra fish (Danio
rerio sp.) of about 6 months were used. Each aquarium
had 20 fishes (~13 males and 7 females) for adequate
spawning and fertilization. To maintain a suitable tempe-
rature range of 26˚C ± 1˚C for the zebra fishes, two 50 W
heaters (Elite 50 W) were installed on the aquaria. Two
lamps (Sun-Glo A1590) were fitted inside the aquaria
and connected to a clock timer to regulate a light-dark
regime of 14 hours light and 9 hours darkness. As aqua-
rium water, suitable dilution water with oxygen saturation
at 26˚C ± 1˚C not less than 80% was used for breeding
the zebra fish [13]. Oxygen was supplied through pumps
installed inside the two aquaria. The dissolved oxygen
concentration was measured by means of an OxyTop Dis-
solved Oxygen (D.O) Probe. The pH of the aq uari um wa-
ter was measured with a standard pH Meter (Meter Lab
pH M210) and ensured to be between 6.8 and 8.4 [13].
Dry flaky food was supplied about 3 times a day to the
fishes through a programmable fish-feeding timer (Nutra-
fin ProFeed Plus) installed on each aquarium whilst live
Artemia species were manually administered once a day
to promote breeding. The fishes require a combination of
both flakes and live foods to ensure food variety. In their
natural waters zebra fish feed on smaller organisms such as
zooplanktons. Care was how- ever taken not to over feed the
fish.
A spawn trap was placed inside each aquarium to co-
llect the eggs spawned. To separate these spawned eggs
from their mother fishes, each spawn trap was covered
with a stainless steel grid of mesh size about 1.25 mm,
big enough to let the eggs drop through. Fastened to the
grid on the spawn trap were artificial plants made of
plastic to stimulate breeding. Each spawn trap was placed
on an artificial green plastic carpet inside the aquarium to
serve as substrate (Figure 1).
Photo-resist curtains were fitted around the aquaria to
ensure that during the 9 hour s when the lamps were timed
off at night the zebra fishes experienced complete dark-
ness.
2.2 Methodology
For the culture conditions in the aquaria, the temperature

Improving Production of Zebra Fish Embryos in the Lab 1362
Figure 1. Aquaria with spawn traps.
was set at 2 6˚C ± 1˚C, whilst a photoperiod of 14 hr-light:
8 hr-dark periods was set (9 am to 11 pm light). To en-
sure this, the photo-resist curtains were drawn to cover
the aquaria before leaving lab to ensure that the fishes
experienced complete darkness from 11 pm when the light
was timed off until the next morning at 9 am. Zebra fish
were bred at a male-to-female ratio of 2:1 (~27 males and
13 females). Since fertilization in zebra fish is in-vitro
there is need for twice as many males as females to en-
sure that enough sperms are spawned to fertilize the spa-
wned eggs.
The two spawn traps were gently removed before 9 am
and emptied of any old eggs. Old eggs were transferred
into Petri dishes and the traps were quickly put back
inside the aquaria and covered with the curtain again
before onset of light. One hour after onset of light (10 am)
the spawn traps were removed again and fresh eggs (1-
hour old) were transferred into separate petri dishes. The
live fish food, Artemia, was now introduced. This was
done once a day to promote breeding. When this food had
settled down in the aquaria, the spawn traps were put
back. This was to ensure tha t the spawn traps were al ways
free of Artemia food. Fertilized eggs were identified under
a stereo microscope and counted.
The physico-chemical conditions in the aquaria namely
dissolved oxygen (D.O) level, pH and temperature were
determined using the D.O probe and pH meter respect-
tively.
The dry flakes food was periodically introduced about
3 times a da y by the automatic feeding mac hine installed
with a timer [13].
2.3. pH and Dissolved Oxygen
The pH of the maintenance water inside the two aquaria
were 8.34 and 8.30 whilst dissolved oxygen levels were
7.4 mg/L representing more than 80% saturation which
meet the OECD requirements (Table 1).
3. Results and Discussion
About 200 embryos were obtained in one spawn trap
Table 1. Physicochemical condit ions inside aquaria.
Aquarium 1 Aquarium 2
pH 8.34 8.3
Dissolved Oxygen7.4 mg/L (94%) 7.4 mg/L (94%)
Temperature 27.4˚C 26.5˚C
within one hour of onset of light (Figure 2). This was an
improvement over the previous work which recorded 50
- 60 eggs in an aquarium [12]. Thus newly-formed em-
bryos less than 2 hours old (1-cell stage, Figure 3) were
available for toxicity testing with any chemical or eff-
luent to be used. The sensitivity of embryos to chemical
or effluent exposure has been shown to depend on em-
bryo age at start of exposure. In an experiment testing,
for example, 2,4 dinitrophenol on zebra fish embryos, it
was clearly demonstrated that eggs 8 hours and older sho-
w e d n o mor tality at all af ter 4 8 hou r s of ex posu re [8].
A sensitivity study carried out by Lahnsteiner, 2008 [14]
also confirmed the importance of using early onto-genetic
stages of em b ryo for the test.
4. Conclusions
It is thus possible, from the results obtained, that at least
Figure 2. Newly-fertilized eggs in Petri dish.
Figure 3. Embryo at 1-cell stage under microscope.

Improving Production of Zebra Fish Embryos in the Lab
1363
200 eggs can be obtained in each aquarium within 1 hour
of onset of light, provided that optimal culture conditions
and the right procedural steps as indicated in this study
are followed. Slight modifications in procedure may be
necessary as per the peculiar conditions obtaining in the
lab.
5. Acknowledgements
The authors would like to thank the section leader, Eric
Sogaard, for his approval for this research to be underta-
ken. Especial thanks also go to the lab technician, Linda
Madsen, for her immense support in helping to carry out
the laboratory work.
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