The Daphnia LC50 Toxicity Test

Earth and Environmental Science Lab 4

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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 resourcesclean 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?

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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.

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5. 6. 7. 8. 9.

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. 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. After 1 hour, place a square of parafilm over each test tube containing any live Daphnia and place labeled test tubes in rack. Transcribe your group data for 60 minutes of exposure to the board and copy the class data onto Data Table 2. After 24 hours, record the number of dead Daphnia in each test tube in Data Table 1. 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: ________

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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)

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More report info at: http://lasi.lynchburg.edu/perault_d/public/classes/research/report.htm