William Paul  Kent Place School  42 Norwood Ave.  Summit, NJ 07901	Jaime Zung  Rye High School  Parsons Street  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 26-30^oC) 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.
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    Students will be able to :

• design an experiment exploring the effects of ethanol and heavy metal pollutants on the development of zebrafish embryos;
• culture and care for zebrafish and their embryos;
• discuss the differences between point and non-point pollution and their effects on embryological development;
• relate the effects of ethanol and heavy metal pollutants to their own lives;
• synthesize strategies, through group discussions, to deal with alcohol abuse and environmental pollutants.

•     High School Biology Students,  Grades 9-12

Notes to the Teacher: 

• Students can work in groups of  4.
• Breeding tanks need to be set up at least 2 weeks prior to beginning the investigations.
• Preparation time needed: zebrafish embryos are readily obtained by regulating the photoperiod of the aquaria.
• Class time needed: 1 period for prelab discussion, 1 period to set up lab,  1 week  to observe embryos,  and 1-3 days for discussion and interpretation of results.  This can be an ongoing experiment depending on students' interest in follow-up activities and experiments.
• Students need to be cautious in the use and disposal of heavy metal chemicals.

        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 26-30^oC.
    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
    for experimentation.

        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 26-30^oC.

    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
    own experiments:

Ethanol experiment
    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.

Lead
    Suggested concentrations 0-0.5ppm for various lengths of time depending on student design.

Cadmium
    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.
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• Formal Lab Write-up

• Alternative Assessment - creative short story, poem, play based on student's reaction to   the laboratory activity

FishScope

    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.
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