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ECOLOGICAL BIOASSAY OF COPPER SULFATE IN DAPHNIA MAGNA

Donald M. Serva
1993 Woodrow Wilson Biological Institute


INTRODUCTION

This laboratory can follow the introduction in the classroom of ecosystems and productivity levels, bioassays and what and how they measure, and Daphnia magna as an index organism, i.e., a species whose health within an ecosystem will indicate the over-all ecological health of that ecosystem. In addition the concepts of LC50, i.e., the "lethal concentration" of a toxicant that will kill 50% of the index organism, and the IQ Toxicity Test, both developed by Aqua Survey, Inc., are presented.

This ecological laboratory uses the biotechnological tool of the bioassay to demonstrate the effects of toxic substances on living organisms. The bioassay is detected through fluorescent stimulation of a metabolized marker on galactose ingested by Daphnia magna that have been exposed to varying concentrations of a chemical that may be a possible environmental contaminant. After exposure to the toxic chemical the Daphnia's enzymes systems will be rendered nonfunctional. Ordinarily death would follow in a few hours or several days. But with the bioassay kit provided by Aqua Survey, Inc., the damaged enzyme systems of Daphnia can be detected by their inability to metabolize a fluorescently marked sugar. Healthy organisms with unimpaired enzyme systems will glow under longwave ultraviolet light ("black light"). Damaged organisms (that will eventually die) will not glow. The ratio of glow to non-glow organisms is used to determine graphically the LC50 value.

TARGET AGE/ABILITY GROUP

The IQ ECOLOGY LABORATORY is geared to university students, but it can be easily adapted to high school students.

STUDENT/CLASS TIME REQUIRED

The IQ ECOLOGY LABORATORY ecotoxicology assessment test can be performed for 25 students and the data analyzed within a two hour laboratory period. However, adapted for high school laboratories of one hour, the procedure can be run and the data procured in less than one hour. Later, in class or for homework the data can be graphically presented and analyzed.


MATERIALS

The kit from Aqua Survey, Inc., contains the following material:

Item                                              Quantity

IQ Exposure Chamber                                  25
IQ Additive (the tagged sugar)                        1
IQ Solvent (water for dilution)                       1
Plastic Cup                                          75
Wide Bore Organism Pipette                           25
Large Capacity Pipette                               50
Calibrated IQ Additive Pipette                        5
Student Directions (master copy)                      1
Instructor Notes                                      1
Toxicant Solution Concentrate (copper sulfate)        1
Longwave U.V. Lights                                  2

The kit sells for $175, but a special introductory price of $150 is available to any school that places an order before September 1, 1993. As a favor to Woodrow Wilson Biology Institute participants, this offer has been extended to September 1, 1994; simply identify yourself as a former participant when ordering. Notice, however, that the price is a one-time investment. The chambers can be used from class to class. Additional tagged sugar can be ordered from Sigma (#M 1633 at $8.20 for 250 mg). The pipettes can be cleaned and saved or ordered again inexpensively. The copper sulfate solution is easily made. The U.V. lights are the most expensive part of the kit. If the instructor can secure more lights ("black lights") from school or other sources, he or she could make do probably with only one kit.

Additional items to be provided are:

  • 8 "AA" batteries for the U.V. lights

  • protective safety glasses for those using the U.V. lights (recommended)

  • 1 clean graduated glass container which holds 1L (for mixing solutions)

  • 1 lightbox (not required, but facilitates the handling of organisms)

  • Daphnia magna (about 1000 are given free with the kit; see instructions in Teacher Guide)

  • SAFETY PRECAUTIONS

    Safety glasses are recommended for viewing the U.V. light. All solutions can be discarded down the drain and the plastic items thrown into the trash. A Material Safety Data Sheet for the IQ Additive and the Toxicant Solution will be provided from Aqua Survey, Inc. upon request.

    Teacher Guide For Preparation Of Materials

    One Week Before Lab:

    1. One week before the laboratory exercise order Daphnia magna. You will need about 1,000 organisms for a class of 25 students. As part of the price of the kit, a thousand organisms with 2 liters of dilution water can be ordered from Aqua Survey, Inc., at 800/654-4684 one week before the lab in order to ensure unstressed organisms.

      On Laboratory Day Itself:

    2. To a 1L beaker add the vial of Toxicant Solution Concentration from the kit to 1L of dilution water, provided with the organisms. Mix thoroughly; the final concentration of this solution will be 0.5 ppm copper. This will be the 100% test solution in the Exposure Chamber. Aliquot 40 mL of this solution to each student in a labeled plastic cup provided with the kit.

    3. To a second labeled plastic cup from the kit aliquot 60 mL of dilution water for each student.

    4. To a third plastic cup allot 40 test organisms in a mixture of dilution water and shipping water to about 50 mL for each student. (The organisms should be allowed to acclimate in these plastic cups for a period of at least half an hour.)

    5. To the IQ Solvent container add the contents of the vial of IQ Additive and shake for 30 seconds. The final volume will be about 50 mL. (According to the kit, it is important to shake the IQ Additive Solution for 5 seconds before the students draw their aliquot from this common stock.)

    6. If you do not have at least an hour and a half laboratory period, the Daphnia can be exposed to the toxicant solution for only half an hour, rather than the recommended one hour. If your laboratory time frame is still constricted, instruct the students that after they have the Daphnia in the pipette, let them swim naturally to the bottom opening in the pipette and then drop them into the Exposure Chamber. This speeds up the time needed to get the Daphnia into the Exposure Chamber. [During the one-half to one hour exposure time, you will have to provide other activities for the students or will have to lecture on such suggested topics as bioassays and how they are used, what is an LC50, what are Daphnia, or the theory behind the IQ Toxicity Test.]


    STUDENT INSTRUCTIONS

    PROCEDURE:

    1. Each student collects from the distribution area: 1 Exposure Chamber; 1 plastic cup with 60 mL of dilution water (DW); 1 plastic cup with 40 Daphia magna in about 50 mL of water; 1 plastic cup with 40 mL of Toxicant Solution (TS); 1 wide bore pipette for the Daphnia; and 2 large capacity pipettes, one each for dilution water and for copper sulfate (or other toxicant).

    2. Using the second large capacity pipette, add toxicant solution up to the "TS" fill lines. The toxicant is copper sulfate (CuS04). The full strength solution is 0.5 parts per million copper.

    3. Use either pipette to stir the toxicant solution and dilution water together. Do not stir 0% or 100%. This procedure produces a series of dilutions of the toxicant solution from 0% (pure dilution water) up to 100% (full strength toxicant solution).

    4. Transfer six Daphnia magna into each cell, starting with the control cell (0% toxicant) and ending with 100% toxicant with the wide bore pipette. During transfer, carry over as little organism water as possible from the organism plastic cup. Your instructor will demonstrate the technique.

    5. When the last animal has been transferred, record the time in your notes. Record the time at the end of exposure to the toxicant.

    6. At the end of one hour, use the calibrated pipette to add 0.25 mL of the IQ Additive sugar solution to each exposure chamber cell. (The pipette is marked in 0.25 mL graduations.) The IQ Additive sugar solution will be centrally located in the laboratory, and you will take turns with other students to accomplish the introduction of the IQ Additive to your test chambers. (If time is limited, one-half hour exposure is sufficient.)

    7. Fifteen minutes after the addition of the sugar solution, go into a darkened room (the darker, the better) and put on protective U.V. glasses (recommended, not required). Illuminate the Daphnia magna by placing the exposure chamber on top of a source of longwave ultraviolet light ("black light"). Two U.V. lights have been provided to your instructor with this kit. You will need to share these lights with other students. The best way to illuminate the test chamber is to hold the chamber on top of the black light. You may need to slowly move the black light up and down the back side of the chamber in order to accurately score your test.

    8. For each cell, count the number of Daphnia magna that are not glowing as brightly as those in the control cell. Use the organisms in the control (0%) cell for comparison. Enter this number in Table 1. Although all the Daphnia may still be alive at this time, a dull glow indicates that the individual is no longer feeding and digesting properly and will soon die. (It is important to use the U.V. light after only fifteen minutes of exposure to the tagged sugar. If more exposure time is given, the fluorescent marker disperses into the solution, making the fluorescent contrast difficult to see.)


      TABLE 1:

      Concentration of toxicant and Daphnia Magna mortality

      Exposure Chamber Cells          Cencentraion of Toxicant
                                      (CuSO4) ppm
      

      0% (control)                          0.00ppm
      6.25%                                 0.03ppm
      12.5%                                 0.06ppm
      25%                                   0.12ppm
      50%                                   0.25ppm
      100%                                  0.50ppm
      

    9. Referring to Table 1, at what concentration of toxicant (in parts per million) did half the Daphnia magna (3 of 6) show lethal effects? Estimate the LC50 by plotting on two cycle semi-log graph paper the toxicant concentration versus the percent adversely affected. Locate the two points on the graph that are separated by the 50% affected line, and connect them with a straight line. If one of these two points is obviously irregular, the data point for the next lower or higher test concentration is used. On the vertical axis, read the value of the point where the diagonal line and the 50% affected line interesect. This value estimates the LC50 for the test, expressed as a toxicant concentration.


    QUESTION FOR THOUGHT

    What concentration of this pollutant should be considered "unacceptable?"

    Why? If all the control organisms do not glow brightly, what might be the causes?


    SOURCES OF MATERIALS

    Aqua Survey, Inc., 499 Point Breeze Road, Flemington, New Jersey 08822.
    Telephone: (908) 788-8700; (800) 654-4684; Fax: (908) 788-9165.


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