Kent Place School
42 Norwood Ave.
Summit, NJ 07901
Rye High School
Rye, NY 10580
The embryo of the zebrafish is an excellent subject
for use in the science classroom due to several factors: zebrafish can
be easily raised and maintained in the classroom without the need for any
costly or specialized equipment; the fish are small (3 cm) and their generation
time is only 3-4 months; large numbers of eggs, which can be collected
weekly, develop rapidly and in synchrony outside the mother; and the eggs
and embryos are transparent, develop rapidly to hatching (96 hours at 26-30oC)
and can be easily exposed to chemical agents without mechanical interference.
In many vertebrates ethanol is known to cause a wide variety of developmental anomalies including embryonic retardation, craniofacial abnormalities, and neurological disorders. Laale (1971) has shown that zebrafish embryos exposed to ethanol levels between 1.4% and 2.4% for 24 hours also exhibit these abnormalities.
Fish embryos are sensitive to toxins and have been used as teratogenic screens for many years. They have recently been employed in the screening of environmental toxins, as well.
High school students need to be exposed to the dangers of alcohol abuse and the many environmental toxins that pollute our air, land and water. Because of its ease of cultivation and manipulation, the zebrafish embryo can be employed in the classroom to demonstrate the effects of these chemicals on embryonic development.
This laboratory exercise can be used to demonstrate the effects of ethanol, cadmium, and lead on embryological development. Cadmium and lead are two heavy metal contaminants of our environment. In addition, information is included that can be used for extended investigations of the effects on embryonic development of magnetic fields, such as those commonly found at home and in the workplace.
Students will be able to :
Notes to the Teacher:
After reading and participating
in group discussions of zebrafish development and the
danger of heavy metals, students will design experiments that they wish to carry out. Their
designs and procedures need to be approved by the teacher.
The following are suggestions for helping students in setting up this laboratory:
At least two weeks prior
to the beginning of the lab, the breeding tanks should be set up.
Zebrafish breeding is dependent on a constant photoperiod of 12-14 hours/day. The fish
should be exposed to this photoperiod for at least 10-14 days prior to egg collection. A
timer would be most efficient to insure the constant delivery of light. There should be at
least two breeding tanks for the fish: one where eggs will be collected and one tank for rest
and relaxation . (Not for you, but the fish.)
The egg collection tank will have
some differences from the rest tank. A single layer of marbles
will be used to aid in egg collection. As the eggs fall from the female they will land between the marbles
prohibiting the zebra fish from eating them and allowing for easy collection. Also, there should not be
any sort of filtration system set up in the egg collection tank to prevent loss of the eggs in
the filter. The resting tank can be a typical fish tank with a filtration system, gravel, and
any "toys" for the fish. Both tanks should be kept between 26-30oC.
About 25 zebrafish should be placed in each tank and should be rotated from the breeding
tank to rest tank every other day.
The collection of eggs serves
two purposes: the first is to collect eggs; the second is to
clean the tank. Using a large siphon tube in the egg collection tank, start siphoning between
the marbles, but be sure to have a brine shrimp net attached to the opposite end to collect
the eggs. Be thorough, as the eggs are small and may escape visual detection. Once the
eggs have been collected, rinse them with fish water, and place them into individual jars
A third tank will be needed
for students to use in their investigations. This tank will
lack all amenities, except for a submersible heater and aerator. The water level in this tank
needs only be to the top of the tallest jar. The jars are used to contain the developing
embryos and the chemical being tested. In this way, different chemicals at various
concentrations can be juxtaposed in the tank at one time. Once again, the temperature
should range from 26-30oC.
For further questions about setups, breeding, and egg collection click: The Fish Net
The following concentrations should
be used as guidelines for students designing their
Students can expose embryos to 1.4 -2.4% ethanol for 24 hrs. Other concentrations can
be used for longer or shorter time periods depending on student design.
Suggested concentrations 0-0.5ppm for various lengths of time depending on student design.
0.03-4mg/L for various lengths of time depending on student design.
A fourth tank should be used
to house the fry for examination and observations. This
tank can be similar to the resting tank in setup.
Armont, D.R. and Saunders, D.E. 1996. Exposure of embryonic cells to alcohol: Contrasting effects during preimplantation and postimplantation development. Semin. Perinatal 20:127-139.
Cameron, I.L., Hardman, W.E., Winters, W.D., Zimmerman, S., and A.M. Zimmerman. 1993. Environmental magnetic fields influences on early embryogenesis. J. Cell. Biochem. 51:417-425.
Laale, H.W. .1971. Ethanol induced notochord and spinal cord duplications in the embryo of the zebrafish, Brachydanio rerio. J.Exp. Zool. 177:51-64.
Perez-Coll, C.S., Herkovits, J., and A. Saliban. 1985. Effects of cadmium on the development of an amphibian. Arch. Biol. Med. Exp. 18:33-39.
Van Leeuwen, C.J., Grootelaar, E.M., and G. Niebeek. 1990. Fish embryos as teratogenicity screens: A comparison of embryotoxicity between fish and birds. Ecotoxicol Environ. Saf. 20:42-52.
Weber, D.N. 1993. Exposure to sublethal levels of
waterborne lead alters reproductive behavior patterns in fathead minnows,
Pimephales promelas. Neurotoxicology 14:347-358.