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    Effects of Pollution on the Reproduction and Development of Fish (Guppy)
    Elementary School Lab Experiments & Background Information
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    This experiment is courtesy of 

    An Experiment with Endocrine Disruption in Fish

    Developers:

    Patricia A. Schleeweiss
    St. Agnes-Sacred Heart School
    Hilltown, Pennsylvania

    Vincent J. Kramer, Ph.D.
    Toxicology Department
    Rohm and Haas Co.
    Spring House, Pennsylvania

     

    Grade Level:

    4 through 6 (Can be adapted for older or younger students.)

     

    Discipline:

    Life science, earth science, environmental science, chemistry, mathematics, health science.

     

    Goals:

    1. To develop the working skills of the scientific method by:
      1. formulating and testing a hypothesis
      2. predicting an outcome
      3. identifying and observing similarities and differences
      4. constructing a simple chart, table, or graph
      5. using test results to form a conclusion
    2. To develop good safety practices in everyday classroom procedures.
    3. To use cooperative attitudes in developing our scientific activities.

     

    Objectives:

    1. To learn about a specific species of fish.
    2. To study the effects of outside substances on the reproductive development of a fish.
    3. To understand the process of a wastewater treatment facility.

     

    Background:

    1. Cooperative learning is the practice of students working with their peers to attain a common goal. Classroom science lends itself to this method of teaching. Cooperative learning teams of four students are suggested. Job descriptions are included to provide individual accountability of all tasks.
    Material Organizer: - gathers and organizes materials and equipment needed
    - manipulates materials
    - directs cleanup.
       
    Writer/Recorder: - writes down the team's observations and answers to questions
    - makes sketches
       
    Reader/Reporter: - reads directions to the team
    -shares team's results and conclusions with the class
       
    Manager/Supporter: - keeps time
    - makes sure safety procedures are followed
    - encourages group
    - makes sure everyone is constructively participating
    1. Water pollution is the addition of effluents to water in quantities that will degrade the quality of that water. There are several sources of water pollution:
      1. Sewage treatment plants provide primary and secondary phases of wastewater treatment before it is reintroduced into the waterways. These treatments however, do not remove all the nutrients such as phosphorus and nitrogen.
      2. Paper mills use huge amounts of water to strip the bark off the logs, to soak, cook, wash, and cool the paper during the manufacturing process. The waste water from this process is released into streams and rivers.
      3. Agricultural pollution is caused by liquid and solid wastes from all types of farming. This includes runoff from animal waste disposal areas and the land used for livestock and crop production; erosion and dust from plowing; and crop residues and debris.
    2. A stream's ecosystem is the interaction of living organisms in their non-living environment. Algae, zooplankton, macroinvertebrates, and fish interrelate and are effected by effluents that are discharged into their habitat.
    3. Reproduction and the development of fish are necessary processes for the survival of the fish species in a stream. Hormones control these processes. Pollution (specific pollutant chemicals) can disrupt these hormones. This is called endocrine disruption. Endocrine disruption is the alteration of the endocrine system resulting in temporary and/or permanent alterations in the functioning of the endocrine system, usually focusing on the reproductive endocrine system. These alterations can include changes in reproductive behavior, secondary sex characteristics, and reproductive output.
    4. We decided to study guppies because they are live bearers, reproduce readily (supposed to!), and one can easily distinguish the male from the female. They grow quickly, and they are cute, likeable, and energetic little fish. Three natural materials will be tested for endocrine disrupting effects on the reproduction of guppies to demonstrate that endocrine disrupting materials are present in our everyday world and that human activities can cause the release of these materials to rivers and streams. The three natural materials to be tested are: pregnant mare's urine, soybean extract, and pine wood chip extract.

     

    Activity 1: Noticing Male-Female Differences

     

    Objectives:

    1. To learn about a specific species of fish.
    2. To observe the life cycle of a guppy.
    3. To observe similarities and differences of the guppies.
    4. To develop an appreciation for humane care of a living thing.

     

    Background:

    The guppy (Poecilia reticulata) is a small, pretty, live bearing fish. Like many species found in nature the male is more colorful than the female. He is smaller in size. The male uses his colorful tail and fins to attract and court the female. It is fascinating to watch as the males display their colors. The males' sexual organ is a gonopodium. Sperm packets are transferred to the females' genital pore. These sperm packets are stored for three or four broods. The gestation period is 4 to 6 weeks depending on food, light, and temperature. The female will deliver live babies (fry); they will drop an inch or two and swim off. The adult fish should be separated from the fry, as the adults will eat the young. The use of a breeding chamber or providing several small-leafed plants will provide a hiding place and help the fry survival rate.

     

    Materials:

    10 gallon tanks
    Large corner cartridge filters
    Medium size fish nets
    Bottle of Stress Coat
    Small brine shrimp net
    Air pump
    Fish food
    Covers for fish tanks

     

    Suggested Time:

    2 periods

     

    Procedure:

    Set up as many tanks as needed.

    1. Obtain materials needed. (Do not purchase fish until tanks are prepared)
    2. Wash out the fish tanks. Do not use soap or detergents.
    3. Remove charcoal from the filters. The charcoal will remove the substances you are testing. Add extra filter fiber to the filters.
    4. Make sure all of the tanks are set up the same. Use the same size tanks, the same type filter, the same pumps, etc. This will allow for a fair test.
    5. Fill tanks with water. Allow the water to circulate for at least 24 hours so that it can dechlorinate. The water temperature should be between 75� and 78�F.
    6. Label all tanks and equipment with their contents. Date and initial each container. Note: Do not set up fish tanks near the window. The sunlight will cause algae to form.
    7. Treat all tanks with Stress Coat.
    8. Add the guppies!

     

    Conclusions:

    Reader/reporter shares results with class

     

    STUDENT LAB SHEET

    TEAM MEMBERS:

    1.______________________ 2.________________________
       
    3.______________________ 4.________________________

    PROCEDURE:

    Spend at least 10 minutes observing your team's guppies. Discuss your observations as your team recorder makes notes. Pay careful attention to the size, color, activity, etc. of your fish.

    Complete the Venn diagram.

    Be sure all team members have looked over this work and are in agreement with the findings.

     

     

     

     

    STUDENT LAB SHEET

    STUDENT NAME

    :__________________________________

     

    PROCEDURE:

    Spend at least 10 minutes observing your guppies. Label the parts of the fish, both male and female.

     

     

     

    Activity 2: Noting Population Differences

    Objectives:

    1. To learn about a specific species of fish.
    2. To formulate and test a hypothesis.
    3. To record results on a graph.

     

    Suggested Time:

    long term (at least 6 weeks)

     

    Procedure:

    1. Use the fish tanks set up in activity 1.
    2. Observe the fish each day
      1. check and record the water temperature
      2. feed your fish
      3. report on the clarity of the water
      4. observe the health of your fish. Record if they are eating, active, etc.

    Continue the above routine until you are able to complete the following lab sheet.

     

    Conclusions:

    This is an example. Draw your own graph on graph paper. Reader/reporter shares results with class.

    This graphing exercise can be completed weekly, since there will most likely be changes in the populations of both the control tank A and the experiment tank B.

     

     

    STUDENT LAB SHEET

    TEAM MEMBERS:

    1.______________________ 2.________________________
       
    3.______________________ 4.________________________

    PROBLEM:

    1. How long does it take to be able to determine if the fry are male or female?
    2. Are there more male fish or female fish in a litter?

     

    HYPOTHESIS:

    1. _________________________________________
    _________________________________________
    _________________________________________
    _________________________________________
    _________________________________________
       
    2. _________________________________________
    _________________________________________
    _________________________________________
    _________________________________________
    _________________________________________

    RESULTS:

    Graph the number of males and females in the original group of fish and in the group of fry once you can identify the sex of the offspring.

     

    CONCLUSIONS:

    What did you learn from this experiment?

     

     

    Activity 3: Effect of a Polluting Substance on the Reproduction and Development of Fish

    Objectives:

    1. To study the effects of outside substances on the reproductive development of a fish.
    2. To understand the process of a wastewater treatment facility.

     

    Background:

    This activity can be repeated three times with each of the different potential endocrine disrupters. An alternative is to assign one of the potential disrupters to each team and have them share results when they have completed the study.

    1. Soybeans, and legumes in general, contain estrogenic materials known as phytoestrogens (meaning plant estrogens). The chemical nature of these phytoestrogens is such that their structure mimics the chemical structure of estrogen. One chemical that is present in soybeans that can be converted to a phytoestrogen by the action of microorganisms is genistin (Chemical Abstracts Service No. 529-59-9). Soybeans or soy flour will be obtained locally.
    2. Mare's urine contains metabolites of estrogen that can be converted by the action of microorganisms back to the original estrogen. One such metabolite of estrogen is estradiol-17b-D-glucuronide (Chemical Abstracts Service No. 15087-02-2). Mare's urine, about 1 L, will be obtained from a local large animal veterinarian. If possible, a pregnant mare's urine will be used because it will contain greater concentrations of estrogen metabolites than a non-pregnant mare.
    3. White pine and many other tree species contain sterols that can be converted by the action of microorganisms to potentially endocrine disrupting substances. One sterol that can be converted to a testosterone mimicking substance is called stigmastanol (Chemical Abstracts Service No. 19466-47-8). White pine wood chips will be obtained from a freshly cut white pine tree and cut into approximately 1 cm x 1 cm chips

     

    Materials:

    10 gallon tanks
    flexible tubing
    gallon glass jugs
    rigid tubing
    large corner cartridge filters
    air pumps
    3-way breeders
    air stones
    filter fiber
    vacuum
    medium size fish nets
    covers for fish tanks
    small brine shrimp net
    yeast
    bottle of Stress Coat
    sugar
    bottle of Ich medicine
    measuring spoons
    Tetramin fish food
    gang valves

     

    Suggested Time:

    long term

     

    Procedure:

    Fish tanks

    Set up tanks as described above. To study all three potential endocrine disrupters you will need at least four tanks a control tank (A), and a tank for each test substance (B, C, D, etc.)

    Mini Sewage Treatment Tanks

    1. Obtain materials needed.
    2. Fill jugs with water. Water can be dechlorinated by letting it stand out uncovered overnight. Use as many jugs as tanks you have set up and use similar labels (A, B, etc.)
    3. Aerate water with air stones. One pump can facilitate several jugs.
    4. Prepare substances to be tested.

    Fill jugs with substances to be tested (directions below). Remember to maintain a control jug.

     

    Preparation of Substance to be Tested

    1. Control - Using a gallon jug, fill with dechlorinated water. Add one package of yeast and one teaspoon of sugar.
    2. Soy beans- 3/4 lb. of soybeans purchased at a health food store (organic-- does not contain any additives). They are very hard beans so it is necessary to grind them for several minutes in the Cuisinart. Using a gallon glass jug, fill 3/4 full with dechlorinated water. Add one package of yeast and one teaspoon of sugar. Add ground soybeans (343 grams). Fill jug with more water if needed.

      Suggestion to try: Make a soybean extract with hot water and a coffee filter and put this extract into the reactor instead of the soybeans themselves. Soak beans overnight, heat beans and water that they were soaking in to boiling and pass this through a coffee filter. Use the filtered water and put it in the mini-sewage plant. Discard beans that are left over.

    3. Mare's urine- The mare's urine was collected in a clean bucket by waiting patiently. Many horses will urinate when you bring them into their stalls. This is the habit of this particular mare. However, she enjoyed our company and kept us waiting a lot longer than expected. Add 145 mL of urine from a mare in foal to 1 gallon of dechlorinated water, 1 package of yeast and 1 teaspoon of sugar.

      Suggestions to try:

    4. Urine may be obtained from a large animal veterinarian if you can't find a pregnant mare yourself.
    5. If possible, you may obtain a pharmaceutical for estrogen replacement therapy. Pregnant mare's urine is the source of the estrogens used in some pharmaceuticals.

     

    1. Pine chips- The pine chips were made from a freshly cut branch of a White Pine. Plane enough chips to fill a gallon size plastic bag. Be sure to include the bark of the limb. This should weigh approximately 266 grams. Stuff the chips into a gallon jug, add one package of yeast, one teaspoon of sugar, and enough dechlorinated water to fill the jug.

     

    Testing for the effects of the potential endocrine disrupters

    1. Each day for one month add 40 ml of either the control or test substance to the appropriate tank.
    2. Tank A - Control - use 40 ml of the control liquid from the control jug.

      Tank B - Soybeans - use 40 ml from the soybean jug.

      Tank C - Mare's urine - use 40 ml from the mare's urine jug.

      Tank D - pine chips - use 40 ml from the pine chips jug.

    3. Observe the fish daily and complete the data chart.
    4. When you have enough data, complete your graph.

     

    Conclusions:

    Reader/reporter shares results with class

     

    References:

    Teacher's literature (non-fiction)

    Aquatic Project Wild. U.S.A.: Western Regional Environmental Education Council, 1987.

    Drysdale, D.T. and S.A. Bortone. 1989. Laboratory induction of intersexuality in the mosquitofish, Gambusia affinis, using paper mill effluent. Bull. Environ. Contam. Toxicol. 43:611-617.

    Friswold, Carroll. A Beginner's Guide To Guppies. New Jersey: T.F.H. Publications, Inc., 1986.

    Glass, Spencer. Guppies Today. New Jersey: year BOOKS, Inc.

    Houston, David R. Understanding the Game of the Environment. Washington D.C.: Agriculture Information Bulletin No. 426.

    Howell, W. M., T.E. Denton. 1989. Gonopodial morphgenesis in female mosquitofish, Gambusia affinis affinis, masculinized by exposure to degradation products from plant sterols. Environmental Biology of Fishes 24:43-51.

    Johnson, David W., Johnson, Roger T. Holubec, Edythe Johnson. Circles of Learning. Minnesota: Interaction Book Company, 1990.

    Kramer, V.J., S. Miles-Richardson, S.L. Pierrens and J.P. Giesy. 1998. Reproductive impairment and induction of alkaline labile phosphate, a biomarker of estrogen exposure, in fathead minnows (Pimephales promelas) exposed to waterborne 17b-estradiol. Aquatic Toxicol. 40: 335-360.

    McGavack, John Jr., La Salle, Donald P. Guppies, Bubbles, and Vibrating Objects. New York: The John Day Company, 1969.

    Pelissero, C., B. Bennetau, P. Babin, F. LeMann, J. Dunogues. 1991. The estrogenic activity of certain phytoestrogens in the Siberian Sturgeon (Acipenser baeri). J. Steroid Biochem. Molec. Biol. 38:293-299.

     

    Children's literature (fiction)

    Berger, Melvin. The New Water Book. New York: Thomas Y. Crowell Company, 1973.

    Cleary, Beverly. Henry Huggins. New York: Dell Publishing Co., Inc. 1950

    Coombs, Karen Mueller. Flush! Treating Wastewater. Minneapolis: Carolrhoda Books, Inc. 1995.

    Schmid, Eleonore. The Water's Journey. New York: North-South Books, 1989.

    This experiment is courtesy of 




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