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Daphnia Bioassay
By: Rachel Hughes

Time: 8 class periods (can be shortened if necessary)
Prep Time: 1 hour
Materials:

Daphnia Culture, beakers, depression slides, microscopes, Copper Sulfate, safety goggles, aprons, gloves, beakers, boiled egg, pipettes, distilled water,
Students will keep a lab notebook during this lesson.

Daphnia anatomy sheet unlabelled
Daphnia anatomy sheet - labelled - for the teacher
Investigation Development Sheet
Student Instruction Sheet
Daphnia Worksheet - data collection and response graphs

Abstract
Environmental bioassays are one method of assessing the presence of potentially harmful compounds. Bioassays use organisms that show particular sensitivity to possible pollutants.

In this activity students explore the dose response of the freshwater crustacean, Daphnia, and then use this information to discuss the use of Daphnia as an organism for environmental bioassay. The lesson is written to provide an opportunity for students to develop their own inquiry questions and an experimental design. However, it may be shortened dependent upon time restrictions.

This is part of the explore and explain portion of the learning cycle. Students should have a grasp of the phrase ‘parts per million’ or ppm. The math lesson, Calculating Parts Per Million, supports development of this concept.

Objectives
Students will:
1. Articulate a research question and design and conduct an experiment that addresses the question and subsequent hypothesis that students develop.
2. Describe the dose response of Daphnia to a number of substances in a laboratory report
3. Identify bioassays as a tool for toxicity within their laboratory report.

National Science Education Standards
Content Standard A: Science as Inquiry

IDENTIFY QUESTIONS AND CONCEPTS THAT GUIDE SCIENTIFIC INVESTIGATIONS. Students should formulate a testable hypothesis and demonstrate the logical connections between the scientific concepts guiding a hypothesis and the design of an experiment. They should demonstrate appropriate procedures, a knowledge base, and conceptual understanding of scientific investigations.

DESIGN AND CONDUCT SCIENTIFIC INVESTIGATIONS.Designing and conducting a scientific investigation requires introduction to the major concepts in the area being investigated, proper equipment, safety precautions, assistance with methodological problems, recommendations for use of technologies, clarification of ideas that guide the inquiry, and scientific knowledge obtained from sources other than the actual investigation. The investigation may also require student clarification of the question, method, controls, and variables; student organization and display of data; student revision of methods and explanations; and a public presentation of the results with a critical response from peers. Regardless of the scientific investigation performed, students must use evidence, apply logic, and construct an argument for their proposed explanations.

Content Area C: Life Science

THE INTERDEPENDENCE OF ORGANISMS
Human beings live within the world's ecosystems. Increasingly, humans modify ecosystems as a result of population growth, technology, and consumption. Human destruction of habitats through direct harvesting, pollution, atmospheric changes, and other factors is threatening current global stability, and if not addressed, ecosystems will be irreversibly affected.

THE BEHAVIOR OF ORGANISMS
Organisms have behavioral responses to internal changes and to external stimuli. Responses to external stimuli can result from interactions with the organism's own species and others, as well as environmental changes; these responses either can be innate or learned. The broad patterns of behavior exhibited by animals have evolved to ensure reproductive success. Animals often live in unpredictable environments, and so their behavior must be flexible enough to deal with uncertainty and change. Plants also respond to stimuli.

Content Standard F: Science in Personal and Social Perspectives

ENVIRONMENTAL QUALITY
Natural ecosystems provide an array of basic processes that affect humans. Those processes include maintenance of the quality of the atmosphere, generation of soils, control of the hydrologic cycle, disposal of wastes, and recycling of nutrients. Humans are changing many of these basic processes, and the changes may be detrimental to humans. [See Content Standard C (grades 9-12) ]
Materials from human societies affect both physical and chemical cycles of the earth.
Many factors influence environmental quality. Factors that students might investigate include population growth, resource use, population distribution, overconsumption, the capacity of technology to solve problems, poverty, the role of economic, political, and religious views, and different ways humans view the earth.

Teacher Background

Bioassay is a shorthand commonly used term for biological assay and is a type of scientific experiment.

Bioassays are typically conducted to measure the effects of a substance on a living organism. Bioassays may be qualitative or quantitative. Qualitative bioassays are used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity. Quantitative bioassays involve estimation of the concentration or potency of a substance by measurement of the biological response that it produces. Quantitative bioassays are typically analyzed using the methods of biostatistics. Bioassays are essential in the development of new drugs, and monitoring pollutants in the environment. Environmental bioassays are generally a broad-range survey of toxicity, and a toxicity identification evaluation is conducted to determine what the relevant toxicants are.

The use of bioassays include:

1. Measurement of the pharmacological activity of new or chemically undefined substances
2. Investigation of the function of endogenous mediators
3. Determination of the side-effect profile, including the degree of drug toxicity
4. Measurement of the concentration of known substances (alternatives to the use of whole animals have made this use obsolete)
5. Assessing the amount of pollutants being released by a particular source, such as wastewater or urban runoff.
[taken from Wikipedia]

Related and Resource Websites

Environmental Bioassays
Environmental Inquiry - Bioassays: Background Information for High School Students
http://ei.cornell.edu/toxicology/bioassays/Uses.asp
Bioassay on Wikipedia: http://en.wikipedia.org/wiki/Bioassay


Daphnia
Daphnia on Wikipedia http://en.wikipedia.org/wiki/Daphnia
Daphnia Genomics Consortium http://daphnia.cgb.indiana.edu/
MBL Aquaculture on Daphnids http://mblaquaculture.com/content/organisms/daphnids.php

Dose Response Curves
Introducing dose-response curves http://www.curvefit.com/logec50_or_ec50_.htm
What is an LD50 and LC50 http://www.ccohs.ca/oshanswers/chemicals/ld50.html

Graphing Guidelines http://misterguch.brinkster.net/graph.html

Activity
Maintaining a Daphnia Culture
You can obtain a culture of Daphnia from several of the scientific supply companies such as Carolina Biological or Flinn Scientific.

You can maintain your own culture of Daphnia relatively easily. See the various websites above for more information. While not necessarily ideal, the author has previously used a bucket to maintain Daphnia, an aquarium is recommended. Prior to receiving the Daphnia fill the aquarium with tap water and allow the water to age. Add a crushed boiled egg yolk to the container two days prior to receiving the Daphnia. The egg yolk will encourage bacterial growth that the Daphnia will be able to feed on. Daphnia food can also be bought, but is not necessary. The water should be a little cloudy as a result of the bacterial growth. Maintaining a little bacterial growth jar with water and egg yolk so that you can continue to feed the Daphnia is advisable. Some groups recommend using distilled water and adding a variety of ingredients to it, this maybe advisable if your tap water has an overabundance of minerals.

Day 1 - Before Class
1. Prepare a small beaker for each group of students that includes about 10 Daphnia per beaker


2. Prepare Stock solutions of copper sulfate in 1000, 500, 100, 10 ppm. The preparation of these solutions will depend on the type of copper sulfate that is purchased. The following example used a 20% copper sulfate solution available from Carolina Biological Supply Company.

Example calculation:
I. To calculate the ppm contained in 1 ounce of material first solve for B:

A x 75 = B

Where:
A = the % active ingredient (CuSo4)
B = ppm contained in 1 ounce of the material in 100 gallons of water

II. To calculate the number of ounces of material required to make up a desired ppm concentration solve for C:

C = Desired ppm conc. / B

Where:
B = ppm contained in 1 ounce of the material in 100 gallons of water (from above).
C = number of ounces of material to add to 100 gallons of water to achieve the desired concentration.

III. To calculate the number of ounces needed for 1 gallon of water

D = C / 100

Where:
D = number of ounces of material to add to 1 gallon of water to achieve the desired concentration

IV. 1 ounce is equal to 29.6mL.

3. For the 1000ppm solution – 0.7 ounce (20.7mL) copper sulfate to a gallon of water, for the 500ppm solution – 0.3 ounce (8.9mL) copper sulfate to a gallon of water, for the 100ppm solution – 0.07 ounce (2.1mL) copper sulfate to a gallon of water, and for the 10ppm solution – 0.007 ounce (0.2mL) copper sulfate to a gallon of water. .

Introducing Bioassays to Students
1. At the beginning of class ask students to respond to the following questions in their notebooks,

  • What is in your water?
  • Is it just water?
  • How could you tell whether water in a stream was good enough to drink?

This discussion will be a review if students have done the lessons earlier in this cycle.

Once students have written their responses, ask them to share them with the class. Discuss with students that even drinking water that comes out of their tap has salts and minerals in it; tap water isn’t just H2O.

Water utility companies constantly monitor their water to make sure that it is fit to drink and meets the US Environmental Protection Agency standards. Bodies of water that are the source for drinking water are also monitored. Ask students “How can the quality of water be assessed?” Again, this is a review if they have completed the earlier lessons. If you have not done these lessons you may want to review them for information to share.


2. Explain that one way people who monitor environmental quality also assess water quality (along with the tests mentioned previously) is by performing bioassays. Explain that an environmental bioassay is one way that scientists assess whether there may be the presence of potentially harmful compounds. Bioassays use organisms that show particular sensitivity to possible pollutants. Explain that today they will be introduced to an organism that is used in bioassays, Daphnia. They will be designing an experiment using Daphnia to assess the impact of a change in environmental conditions on health.


3. Share that Daphnia are a freshwater crustacean. To be able to use the Daphnia as a bioassay tool, they have to be familiar with normal Daphnia behavior and be somewhat familiar with Daphnia anatomy, see the Daphnia anatomy sheet labelled.


4. In groups have students look at the Daphnia within the beakers. How would they describe the behavior of the Daphnia? How do the Daphnia move? They should write these observations down in their lab notebooks.


5. Show students how to pipette a Daphnia into a depression slide for observation under a microscope. I’ve had some moderate success using Petri dishes to observe the Daphnia if depression slides are not available. Remind students to make sure that there is sufficient water around the Daphnia. Review microscope skills if necessary. If you have the capability to link a microscope to some form of projection this can be very helpful.

  • Discuss with the students the various parts of the Daphnia anatomy.
  • Ask them to identify structures.
    • Can they distinguish the intestinal tract?
    • The eyes?
    • The heart?
    • Is the Daphnia gravid (pregnant)?
    • Have students identify parts of Daphnia on the Daphnia anatomy sheet - unlabelled.


6. Make sure that students see the heart beating. Show students how to take the heart beat of the Daphnia. This is easily done, as you observe through a microscope, by just dotting a piece of paper with a pencil each time you see the heart beat for 10 seconds and then multiplying by 6.

Have students practice taking the heart rate of the Daphnia. Have the students place their results on a class chart. Are the heart beat counts similar? Can you identify a range that appears to be normal?

7. Clarify with the students that toxins might affect many different systems, not just circulation, but being able to see the heart beat of the Daphnia might be something they want to take account of when they are investigating a response to a possible toxin.

Days 2 & 3 Dose Response Guided Experience
1. Students should be wearing appropriate safety equipment, safety goggles, lab apron and gloves.


2. Each group should be given a beaker with the stock solution of copper sulfate (see above for the preparation of the stock solution.)

For the teacher’s information only, Daphnia has an LC50 in excess of 320 ug/liter. Inform students that copper sulfate is known to affect Daphnia very adversely. They are to look at the impact of different dilutions of this substance on the Daphnia.


3. Provide students with the following instructions - on the Student Instruction Sheet. They are to set up the dose response investigation today and collect data on the subsequent 3 days. Data collection will not take the entire class period and so the following lesson can be conducted on Days 2 & 3.
(The data can be collected on the Daphnia Data excell workbook can be used if computers are available)

Student Instructions: Student Instruction Sheet
You are going to assess the response of Daphnia to copper sulfate. Copper sulfate is routinely added to ponds and lakes to control weeds.
Make sure to take appropriate safety measures. Wear safety goggles, apron and gloves.
When you are finished with the solutions dispose of them where your teacher instructs you. Do NOT pour them down the drain.


1. Label each of the four beakers with the concentration to be created in that beaker; 1000ppm, 500ppm, 100ppm, 10ppm.
2. Using the stock solution of copper sulfate that you have been provided. Make sure you place the dilution in properly labeled beakers.
3. Add 10 Daphnia to each beaker. Be sure to make sure that none of the Daphnia are gravid (pregnant) before placing them in the beakers.
4. Place the beakers on the side and observe at 24, 48, and 72 hours. Count how many survive and record in a data table.
5. Graph the results on a graph. Make sure to identify the dependent (survivors) and independent variables (concentration / hours) and place them appropriately on the graph.

Questions
1. What was the response of the Daphnia to copper sulfate?
2. If copper sulfate is used to clear ponds and lakes of weeds, what does the information you’ve gained from this experiment tell you about the use of copper sulfate?

Day 4

1. Once students have their results they should share their graphs with the rest of the class. (The graphs can be created in the Daphnia Data excell workbook can be used if computers are available)

2. Ask students if the graphs show a similar pattern. Discuss the use of a dose response graph. (See resources above)

3. As a group discuss the concept of LD50. Explain that one way of measuring the toxicity of a substance is to describe it in terms of the amount, given at one time, needed to kill 50 percent of a test population.

4. Ask students what are some of the problems with the exercise they performed for assessing LD50 or LC50. (Ex. Without knowing how old, or the health of the Daphnia, they can’t know whether individuals would have died anyway) Point out that conducting multiple trials of the same experiment means that they may be able to have greater confidence in their results than if they just had the results from one trial.

Using Daphnia as a Bioassay Organism
Once students have explored the dose response of Daphnia to copper sulfate they should develop a procedure using Daphnia to explore an environmental substance or factor (available oxygen, pH etc.) that might affect the health of the Daphnia.
1. Provide students with the Investigation Development Sheet.
2. Explain that they are to use Daphnia as a bioassay in investigating this environmental factor’s impact on health. You may wish to start by reviewing some substances and factors that affect our environmental health and substances that we add to the water cycle, for example cleaning materials, herbicides and pesticides, etc.

For homework have students develop 3 potential questions that they could ask using Daphnia as a bioassay and briefly describe how these questions might be explored.

Day 5
1. Students should share their questions within their group and then using availability, interest and safety as criteria, decide which question they want to pursue as a group.

2. They should then identify how they are going to answer the question procedurally. Once they have identified a question and an outline of how this might be investigated groups should attain teacher approval to continue. When students approach to receive approval for an investigation this is the time to identify materials that they will have to bring and those you can provide and also identify any obvious safety risks.

3. Once the question and general outline of the procedure has been identified, students should research a little about the factor that they are investigating. Drawing upon that research and their previous experiences, they should develop a hypothesis. Review with students what a hypothesis is if necessary. I encourage my students to identify the dependent and independent variables and develop an “If….then…” statement using the variable information. They also have to provide a rationale for their hypothesis based upon their research and observations. This is described on the Investigation Development Sheet
. You may wish to use this time to discuss null and alternative hypothesis dependent on your students’ readiness. This is also a good time to remind students that when discussing the term “hypotheses”, it is more appropriate to use the terms “supported” or “rejected” rather than “proven”.

4. Students should then flesh out their procedure based upon their hypothesis; identify materials needed and who is going to bring them; identify safety considerations and develop a data collection method. Students should approach the teacher for approval to proceed at this point. Make sure when students seek approval that their hypothesis is in response to their identified questions and that the data they will collect will address the hypothesis. If their procedures and data collection methods do not address the question and hypothesis they should rewrite them. This approach typically takes most of a class period, but can be very effective in focusing students on developing hypothesis and matching procedures.

Day 6 – Day 8
Students should conduct their investigations and write up a lab report. In their lab report they should explicitly state whether their hypothesis was rejected or supported based on their observations.

If they were unable to get enough information how might they gain more? If their hypothesis was rejected, do they have a new hypothesis? Do they have new questions based upon their investigations? Students should also address how Daphnia respond to the environmental factor under study and how this might add information to the environmental health field.

Homework

Day 5: Students must come up with 3 questions that they would like to investigate
Day 6: Students may finish writing up procedures and designing data collection sheets.
Day 8: Students should work on independent lab reports

Embedded Assessment

Students’ ability to articulate a research question, design and conduct an experiment that addresses the question and subsequent hypothesis can be assessed through the documentation of student ideas on the student inquiry sheet, by participation in the lab and by the final laboratory report.

Students’ grasp of the concept of dose response and the use of bioassays can be assessed during class discussion and in the final laboratory report.



PULSE is a project of the Community Outreach and Education Program of the Southwest Environmental Health Sciences Center and is funded by:


an
NIH/NCRR award #16260-01A1
The Community Outreach and Education Program is part of the Southwest Environmental Health Sciences Center: an NIEHS Award

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Supported by NIEHS grant # ES06694


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Last update: November 10, 2009
  Page Content: Rachel Hughes
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