The Daphnia LC50
Toxicity Test
Earth and Environmental Science
Lab 4
Daphnia
2
The Daphnia LC50 Toxicity Test
INTRODUCTION:
In 1977, Congress passed the Clean Water Act that prohibits the discharge of pollutants in toxic
levels into our oceans, lakes, streams and other waterways. With thousands of chemical
compounds and elements being discharged into the environment daily from industrial,
agricultural, and residential sources, there was only limited, or in many cases nonexistent
information regarding the levels at which such waste products became toxic when introduced
into various ecosystems. What information that was available was based on data from toxicity
studies of a limited number of chemical compounds or elements. Such studies also failed to
account for the potential threat from effluents that contain a number of seemingly harmless waste
products that could have an antagonistic or synergistic effect upon one another when mixed.
Today it would be cost prohibitive to conduct toxicity tests on the thousands of chemical
compounds in daily use, or to analyze effluents to determine their chemical composition and/or
potential threats to the environment. For these reasons the most economical and effective
method to gauge effluent toxicity is to perform an effluent toxicity test that uses aquatic
organisms as indicators of toxicity. A small, developing organism is an ideal model system for
testing effluent toxicity. The developing organism exhibits rapid growth and sensitivity to the
toxic material that is exhibited by arrested development, abnormal growth or death. We will use
Daphnia, an organism that is low on the aquatic food chain, to test the level of effluent that is
harmful to this organism and to the ecosystem as a whole. Such tests are used in commercial and
government laboratories to ascertain the concentration of an effluent, usually expressed as a
percent volume, that is lethal to 50% of the test organisms that are subjected to the effluent for a
predetermined time period. This value is expressed as the Lethal Concentration 50 or the LC50.
There are two basic types of toxicity tests used to determine the LC50 of an effluent; flowthrough and static. First is a flow-through test, where various concentrations of fresh effluent are
pumped through test chambers for a predetermined length of time. This Aquatic Toxicity Lab
incorporates the second method, a static test in which the assay organisms are exposed to the
same test samples for the duration of the test (here, 1 hour and 24 hours). Performing this activity
will result in a better understanding of the concept and basic methods of effluent toxicity testing
which is an invaluable tool used to protect one of our most precious resources—clean water.
OBJECTIVE:
The purpose of this laboratory exercise is to study the effects of different concentrations of an
“effluent” on aquatic organisms in order to find out when these concentrations reach dangerous
levels. This effluent was obtained from a local spill, and officials are concerned about
environmental impacts.
DEFINING THE PROBLEM:
The problem you are faced with is this: What is the level of this effluent that is harmful to
Daphnia, a representative aquatic organism?
Daphnia
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HYPOTHESIS (to be filled in by the student):
METHODS:
The effluent solutions have been prepared for you. They range in concentration from a 100%
stock solution to a 0%, or control, solution. The list below shows the specific concentrations of
“effluent” you will use.
A. 0% (Daphnia Water Control)
B. 12.5%
C. 25%
D. 50%
E. 100%
MATERIALS NEEDED PER GROUP:
Labeling tape
Goggles
Metric Ruler
Culture of Daphnia
5 (30 ml) test tubes
Test tube rack
1 ml disposable pipette and delivery syringe
Beaker containing rinse water (Daphnia water) for pipette
Marking pen
parafilm
1 container of control solution (Daphnia water)
4 containers of prepared concentrations of the toxin/pollutant to be used in the test
PROCEDURE:
PRELIMINARY:
Choose a recorder (writes down observations and data), transfer specialist (transfers
Daphnia from culture to various solutions after pouring solutions into test tubes),
timer (times observations at 10 minute intervals and aids observer), and observer/data
specialist (observes Daphnia behavior and counts living and dead Daphnia at 10
minute intervals).
1. Label each test tube with the toxicity level corresponding to the solutions you will be testing
in this experiment.
2. Add ~5 ml of each solution to the appropriate beaker, then to each test tube.
3. Transfer specialist--don goggles.
Daphnia
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4. Place 5 Daphnia of the same size in each tube while minimizing the transfer of culture water
and placing Daphnia under the liquid to prevent air being trapped under the carapace
(causing the daphnia to die). Transfer Daphnia to effluent tubes in increasing order of
toxicity. To avoid contamination, rinse the pipette between uses. Also limit the amount
of liquid being transferred.
5. Record the numbers of Daphnia dead at 10 minute intervals in each solution you are testing
in Data Table 1. At the same time note any changes in swimming habits over time.
6. After 1 hour, place a square of parafilm over each test tube containing any live Daphnia and
place labeled test tubes in rack.
7. Transcribe your group data for 60 minutes of exposure to the board and copy the class data
onto Data Table 2.
8. After 24 hours, record the number of dead Daphnia in each test tube in Data Table 1.
9. Dump tube contents down the drain, remove labels, and rinse tubes.
DATA TABLE 1---Group Data Showing the Number of Dead Daphnia at Specific Times
Start Time:________________________________________________________
SOLUTION
Control (0%)
12.5%
25%
50%
100%
0________________________________________________________
10________________________________________________________
20________________________________________________________
TIME
30________________________________________________________
(MIN.)
40________________________________________________________
50________________________________________________________
60________________________________________________________
24 HOURS________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
DATA TABLE 2---Pooled Class Data Showing the Number of Dead Daphnia at 60 Minutes
GROUP 
1
2
3
4
TOTAL
Control (0%)_________________________________________________________
12.5% _________________________________________________________
SOLUTION 25% _________________________________________________________
50% _________________________________________________________
100% _________________________________________________________
______________________________________________________________________________
10. Use the SoftTox software to calculate the LC50 for your group data at 60 minutes, the class
data at 60 minutes, and your group data at 24 hours.
Start  All Programs  WindowsChem  SoftTox
LC50 for Group Data at 60 minutes: ________
LC50 for Class Data at 60 minutes: ________
LC50 for Group Data at 24 hours: ________
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Assessment
QUESTIONS
(Due 1 week following lab, questions and answers typed)
10 Points
1. Provide the 3 LC50 results from the previous page.
2 points
2. Based on your 60 minute group data, plot with Excel the number of dead Daphnia at each 10
minute time point for each solution. You should have one graph (with time on the X axis; #
of dead daphnia on the Y axis) and five lines on the graph, labeled with the proper
identification. (Use the Excel file provided to make a line graph)
2 points
3. Based on the pooled class data, plot with Excel the total number of dead Daphnia at the 60
minute time point for each solution. % toxicity should be on the x axis. (Column graph)
2 points
4. Based on your 24 hour group data, plot with Excel the number of dead Daphnia at the 24
hour time point for each solution. % toxicity should be on the x axis. (Column graph)
2 points
5. List at least 3 potential or observed errors associated with this lab.
1 point
6. How could this test aid environmental scientists trying to prevent pollution? What
suggestions can you offer in using the LC50 to possibly treat an effluent so that it
may be discharged into the environment? (remember to include this point in your paper)
1 point
LAB REPORT
(due 2 weeks following lab, typed)
20 Points
1. Title (1 point)
- should be fewer than twelve words and should reflect the factual content of the paper.
- a good title is straight-forward and uses key words
2. Introduction (3 points)
- outline the scientific purpose(s) or objective(s) of the research performed and give the
reader sufficient background to understand the rest of the report
- also include background information on the biology of the study organism
3. Hypothesis (1 point)
- your prediction(s)
4. Materials and Methods (3 points)
- a concise description of the materials, procedures, and equipment used (not as a list)
5. Results (4 points)
- a written summary of the results from the research WITHOUT discussing implications
- includes tables, figures, and graphs (should be redone from above assignment) and
included as supplements
6. Discussion (4 points)
- data are interpreted in relation to the hypotheses or purposes
- errors and improvements are reviewed
- relevance of the research is discussed
7. Literature Cited (1 point)
- need at least 3 citations (primary literature preferred); should also be cited in text
Overall writing such as clarity, grammar, use of headings and subheadings, etc. (3 points)
More report info at: http://lasi.lynchburg.edu/perault_d/public/classes/research/report.htm