Name: ___________________________
General Biology II Lab
Lab Topic: Ecotoxicology
Spring 2024-25
Lab Activity Handout
Lab #11: Ecotoxicology
Objectives:
•
Explore the impacts of various types of pollutants on Daphnia as an indicator species.
•
Demonstrate how to determine the LD₅₀ for a specific pollutant affecting Daphnia.
•
Describe the effects of acid and organic pollution on producers and consumers in aquatic ecosystems.
Activities List
•
Activity # 1- Ecotoxicology Bioassay
•
Activity # 2- Aquatic Microecosystems
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Activity # 1 - Bioassay
In this activity, you will perform a bioassay for an assigned pollutant to determine the LD₅₀ for Daphnia. You
will then collect class data for the pollutants being tested in lab to determine the LD₅₀ for Daphnia for each
pollutant.
Materials
•
•
•
•
•
•
•
•
Glass-marking pens
5 scintillation vials (to incubate the Daphnia)
Cultures of Daphnia
Hot water baths and thermometers (only if you are assigned thermal pollution)
Ice (only if you are assigned thermal pollution)
Transfer pipettes modified to transfer Daphnia.
Pond water
Pond water containing various pollutants.
Methods
1. Each group in lab will be assigned a pollutant to test: thermal, acid, organic, or salt.
2. Determine your experimental setup by completing the experimental design section below.
•
A few restrictions:
i. Each group may use only four Daphnia per vial.
ii. Each group must test five concentrations of their assigned pollutant. Only four
concentrations may be provided—consider the best option for your fifth concentration.
Hint: This will most likely be your negative control.
iii. You must incubate the Daphnia for at least 60 minutes.
3. Once you have determined the concentrations for each vial, fill the vial about 2/3 full with each
concentration.
• Note: If you are testing thermal pollution, you will transfer regular pond water to all five vials
and select different temperatures.
4. Add four Daphnia to each vial using a plastic dropper pipette with the tip cut off. Record the total
number of Daphnia added to the vials in Table #1.
5. Allow the Daphnia to incubate for at least 60 minutes.
6. After 60 minutes, determine the number of Daphnia that have died in each vial. Record this number in
Table #1.
7. Calculate the percent (%) mortality of the Daphnia (percentage of the total that died). Record this in
Table #1.
8. Graph your results in Figure #1.
9. Collect class data for each pollutant and record the data on Tables #2 - #5.
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10. Graph the class data for the various pollutants in Figures #2 - #5.
11. Determine the LD₅₀ for each pollutant based on your graphs. The LD₅₀ is the concentration of the
pollutant at which 50% of the population dies.
Experimental Design:
Use the spaces below to design your experiment and form your hypothesis. For the experimental setup,
describe your experiment step by step in enough detail that someone could recreate it. This must be checked
by your instructor before beginning experimentation.
1. Pollutant assigned: _________________________________
2. Hypothesis:
___________________________________________________________________________________
___________________________________________________________________________________
_______________________________________________________________________
3. Variables:
i.
Independent variable: _____________________________
ii.
Dependent variable: ______________________________
4. Controls:
i.
Positive control: ________________________________
ii.
Negative control: ________________________________
5. Constants (Note: You may use only four Daphnia per vial):
___________________________________________________________________________
6. Concentrations/Temperature/pH
Vial #1 _______ | Vial #2 _______ | Vial #3 _______ | Vial #4 _______ | Vial #5 _______
7. Incubation time: 60 minutes
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8. Outline your experimentation step by step:
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
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Data:
Your Group’s Data:
9. Record your group’s bioassay data in Table #1.
Table # 1: Daphnia mortality after incubation with __________________(assigned pollutant)
Vial
(include
concentration)
# of living
Daphnia at
the start of
incubation
# of dead
Daphnia to
the end of
incubation
Vial #1
Vial #2
Vial #3
Vial #4
Vial #5
% mortality
of Daphnia
10. Graph your results:
100
90
% mortality of Daphnia
80
70
60
50
40
30
20
10
0
Figure # 1: Dose-response Curve for Daphnia exposed to ________________ pollution.
11.Estimate the estimated LD50 for your pollutant based on your group’s data: ___________
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Class Data:
12. Class Data for acid pollution:
Table # 2: Daphnia mortality after incubation for acid pollution
Vial
(include pH)
Vial #1
Vial #2
Vial #3
Vial #4
Vial #5
# of living
Daphnia at
the start of
incubation
# of dead
Daphnia to
the end of
incubation
% mortality
of Daphnia
13.Graph of class data for acid pollution
100
90
% mortality of Daphnia
80
70
60
50
40
30
20
10
0
Figure # 2: Dose-response curve for Daphnia exposed to acid pollution.
14. LD50 for Acid Pollution: _______________________________
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15. Class data for organic, pesticide pollution (glyphosate):
Table # 3: Daphnia mortality after incubation for organic,
pesticide pollution (Glyphosate)
Vial
(include
concentration)
Vial #1
Vial #2
Vial #3
Vial #4
Vial #5
# of living
Daphnia at the
start of
incubation
# of dead
Daphnia at the
end of
incubation
% mortality of
Daphnia
16. Graph of class data for organic, pesticide pollution (glyphosate):
100
90
% mortality of Daphnia
80
70
60
50
40
30
20
10
0
Figure # 3: Dose-response curve for Daphnia exposed to organic, pesticide pollution (glyphosate).
17. LD50 for glyphosate (organic, pesticide) pollution: ______________________
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18. Class data for salt pollution:
Table #4: Daphnia mortality after incubation for salt pollution
Vial
(include
concentration)
# of living
Daphnia at the
start of
incubation
# of dead
Daphnia at the
end of
incubation
Vial #1
Vial #2
Vial #3
Vial #4
Vial #5
% mortality of
Daphnia
19. Graph of class data for salt pollution:
100
90
% mortality of Daphnia
80
70
60
50
40
30
20
10
0
Figure #4: Dose-response curve for Daphnia exposed to salt pollution.
20. LD50 for salt pollution: _______________________________
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21. Class data for thermal pollution:
Table #5: Daphnia mortality after incubation for thermal pollution
Vial
(include
temperature)
# of living
Daphnia at the
start of
incubation
# of dead
Daphnia at the
end of
incubation
Vial #1
Vial #2
Vial #3
Vial #4
Vial #5
% mortality of
Daphnia
22. Graph of class data for thermal pollution:
100
90
% mortality of Daphnia
80
70
60
50
40
30
20
10
0
Figure #5: Dose-response curve for Daphnia exposed to thermal pollution.
23. LD50 for thermal pollution: _______________________________
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Thought Questions:
24. What is an indicator species? Explain why Daphnia are used as an indicator species?
25. What is the difference between an acute impact of a pollutant and a chronic impact of a pollutant?
26. Based on the results, which of the pollutants appeared to have an acute impact on Daphnia?
27. Were there any pollutants with 0% mortality across the incubation period? If yes, propose a hypothesis
as to why.
28. Even if a pollutant doesn’t cause acute effects, it might still impact organisms higher in the food chain.
What is this phenomenon called? Explain how it works.
29. If Daphnia populations are reduced by pollution, predict the effects on the following organisms:
a. Microscopic algae: ____________________________
b. Small fish: _________________________________
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Activity # 2- Aquatic Micro-ecosystems
In this section, you will analyze aquatic micro-ecosystems set up in the laboratory to simulate three lake
environments:
1. Normal
2. Polluted with acid (i.e., acid rain)
3. Polluted with organic, nutrient pollution (i.e., fertilizer)
Your task is to compare these ecosystems to determine the effects of acid and organic pollution on the
diversity and density of organisms.
Materials
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Aquatic ecosystems in 500-ml beakers simulating:
o Normal
o Acid Pollution
o Organic pollution
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Note: There is one set for the entire week of labs, please treat with care!
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Keys for identifying pond life
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Dropper pipets
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Microscope slides and cover glasses
Methods
1. Observe the three micro-ecosystems macroscopically (with the naked eye) to determine
the relative density of autotrophs.
o
This is easily done by noting the degree of clarity and greenish color of the
water.
2. Observe the three micro-ecosystems macroscopically to see if any visible heterotrophs
are present.
3. Prepare a wet-mount slide of each of the three micro-ecosystems to determine the
relative abundance of autotrophs and heterotrophs in each system.
o
To prepare a wet-mount slide:
•
Use a dropper pipet to place 1–2 drops of pond water on a clean
microscope slide. Place a clean cover slip on its edge at a 45° angle and
slowly lower it onto the sample to avoid bubbles.
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4. Draw or take a photo of your prepared slides. Identify at least one autotroph and one
heterotroph in each image.
(If you cannot find these organisms, make a note of that on your drawings.)
5. Record your observations and answer the questions below.
Thought Questions:
1. Based on macroscopic (naked eye) observations, which microecosystem seems to have:
a. The greatest density of autotrophs?
b. The lowest density of autotrophs?
c. The greatest density of heterotrophs?
d. The lowest density of heterotrophs?
2. Based on examination of your prepared slides microscopically, record the presence of autotrophs in the
microecosystems by placing an +, ++, or +++ depending on the relative amount in the appropriate
spaces in the table.
Table # 6: Presence of various organisms in the aquatic micro-ecosystems
Organism
Acid Pollution
Normal
Nutrient Pollution
Green Algae
Cyanobacteria
Rotifers
Protozoa
Crustacea
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3. Microscopic observations of microecosystems. Identify and label at least one autotrophic and one
heterotrophic organism for each slide.
Sample: “Normal” pond water micro-ecosystem
Total Magnification: __________
Notes: ______________________________
____________________________________
Figure #6: Microscopic sample
from the “Normal” microecosystem
Sample: Nutrient pollution micro-ecosystem
Total Magnification: __________
Notes: ______________________________
____________________________________
Figure #7: Microscopic sample
from the nutrient polluted microecosystem
Sample: Acid pollution micro-ecosystem
Total Magnification: __________
Notes: ______________________________
____________________________________
Figure #8: Microscopic sample
from the acid polluted microecosystem.
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4. True or False: Pollutants typically only have localized (nearby) impacts for a short period of time.
5. What is one source of nutrient pollution?
6. How could nutrient pollution lead to hypoxia (decreased oxygen availability)?
7. What is one source of acid pollution?
8. You are hiking in the Adirondack Mountains you come across a lake with extremely clear water. What
might this clarity indicate about the overall health of the lake? What type of pollution might be
impacting this lake? Refer to food chains or food webs in your explanation.
9. Pesticide pollution and nutrient pollution are both considered types of organic pollution. Describe the
different impacts these two types of organic pollution could have on ecosystems.
10. After a pollutant enters the environment, ecotoxicologists assess impacts at the organism, population,
and ecosystem levels.
Based on your understanding of both ecosystem dynamics and ecotoxicology, at which level is it more
challenging to “deal with” a pollutant, and why? Support your answer with an example.
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