SES 4054 Environmental and Public Health Risk Assessments Lab: Effect of a chemical substance on Daphnia magna A. Preparation of tested chemical substance A dilution series of chemical (potassium dichromate,

K₂Cr2O7) has to be prepared following the steps. 1. Take seven 100 ml calibrated flasks. Label the first flask as "stock 1", the second flask as "stock 2" and the others as C1 - C1 C2 - C3 C4 - C5. 2. Weigh 100 mg potassium dichromate (K₂Cr₂O7) on an analytical balance, transfer it into the "stock 1" flask and fill to the mark with deionized water. 3. Transfer 1 ml "stock 1" solution into "stock 2" flask and fill the latter flask to the mark to make up a 10 mg/l toxicant concentration. 4. Transfer the following volumes of toxicant solution from "stock 2" into the other 100 ml flasks: - 32 ml to flask C1 - 18 ml to flask C2 - 10 ml to flask C3 - 5.6 ml to flask C4 - 3.2 ml to flaskC5 5. Add Standard Freshwater to the mark, stopper the flasks and shake to homogenize the solutions. 6. Fill the multiwell with the toxicant solutions as indicated in section "Filling of the Test Plate". B. Pre-feeding of Daphnia magna 1. Take one vial with Spirulina powder and fill it (with Standard Freshwater. 2. Shake the vial thoroughly to homogenize the contents. N.B. Vortex shaking is advised to obtain a very homogenous suspension of the Spirulina particles. 3. Two hours prior to collecting the neonates for the test, pour the algal suspension into the hatching petri dish and swirl the contents gently to distribute the food evenly. C. Filling of the test plate 1. Transfer 10 ml dilution water into each well of the control row and 10 ml of the respective toxicant concentrations into each well of the corresponding rows, in the sequence of increasing toxicant concentrations. 2. Each test concentration as well as the control have to be assayed in 4 replicates. 3. Each multiwell plate is provided with 4 test wells for the controls and 4 test wells for each toxicant concentration. 4. The test wells in each column are labeled A, B, C and D and the rows are labeled X (controls), 1,2,3,4 and 5 for the five toxicant dilutions. All the wells of each row have to be filled with one toxicant dilution (or with the dilution medium for the control row). X Rinsing wells A B Test wells Put the multiwell plate on the stage of the dissection microscope or on a light table and transfer exactly 5 neonates from the rinsing wells into each of the 4 wells of each column. This transfer shall also be performed in the order of increasing test concentrations. D. Transfer of the neonates to the test wells Transfer of the Daphnids to the multiwell plate is accomplished in two steps: a. transfer of the neonates from the petri dish into the rinsing wells of the multiwell plate (first column to the left). b. transfer of the neonates from the rinsing wells to the 4 test wells of the same rows. 1. Put the Petri dish with the pre-fed neonates on the stage of a dissection microscope or on the transparent stage of the light table with dark light strip. 2. Transfer at least 20 (actively swimming) neonates into each rinsing well in the sequence: row X (control), row 1, row 2, row 3, row 4 and row 5 (i.e. in order of increasing concentrations of toxicant). Try to carry over as little as possible liquid from the petri dish to the wells during this transfer and rinse the micropipette thoroughly after each transfer 3. Put the multiwell plate on the stage of the dissection microscope or on the transparent stage of the light table and transfer exactly 5 neonates from each rinsing well into the 4 wells of each row. This transfer shall also be performed in the sequence of increasing test concentrations. N.B. Count the neonates as they exit the micropipette to be sure of the transfer of exactly 5 test organisms per well. Important remark: Avoid surface floating of test organism Daphnids are quite susceptible to being trapped at the surface of the liquid medium in the test wells, by the phenomenon of "surface tension". Once "floating", some test organisms may not be able to free themselves from the surface and may die. In order to avoid "surface floating" which can seriously jeopardize the outcome of the bioassays, it is of utmost importance, during the transfer of the neonates into the test wells, to put the tip of the micropipette in the medium, and not to drop the organisms onto the surface of the medium. E. Incubation of test organism 1. Put the Parafilm strip on the multiwell plate and put the cover on tightly. 2. Put the multiwell in the incubator at 20°C, in darkness. F. Scoring the results 1. After 24h incubation, put the multiwell plate under the dissection microscope or on the stage of the light table. 2. Record the number of dead and immobilized* neonates, versus that of the actively swimming. test organisms in each well. *The neonates which are not able to swim after gentle agitation of the liquid for 15 seconds shall be considered to be immobilized, even if they can still move their antennae. 3. Record the results on the "Results Sheet". 4. Calculate the total the number of dead and immobile neonates for each toxicant concentration and calculate the mean and the % effect. G. Estimation of the EC50 There are many procedures for calculating 50% effect thresholds. The simplest one, which is outlined hereunder, consists of plotting the calculated effect percentages on a "log concentration/% mortality sheet". A graph paper is provided for such a very simple and rapid EC50 estimation by "graphical interpolation". 1. Indicate the concentrations (or dilutions) used in the toxicant dilution series on the Y-axis. 2. Plot the calculated percent effect on the horizontal line at the height of each concentration or dilution. 3. Connect the plotted points with a straight line 4. Locate the 2 most adjacent points on the graph which are separated by the vertical 50 % effect line and read the EC50 at the intersect of the two lines. Dilution series tested: Concentration 1: Concentration 2: Concentration 3: Concentration 4: Concentration 5: Table 1: The number of dead and immobilized neonates Control Conc. 5 Conc. 4 Exposure (hours) A B с Ꭰ Results Sheet: Total (x/20) % Effect Table 2: Concentration of the chemical Flask Stock solution C1 C2 C3 C4 C5 Conc. 3 Conc. 2 Chemical concentration (mg/L) Conc. 1/n SES4054 Environmental and Public Health Risk Assessments Report guideline for Ecotoxicological Lab: Toxicity Testing using Daphnia Length: 300-500 words Content: A brief report with 1. Objective; 2. Results and 3. Discussion 1. Objective: A brief statement of the objective of the study 2. Results: please fill in the result sheet and report the LC50 (please include the steps of your calculation using the guide). Dilution series tested: Concentration 1: 32ml >10% Concentration 2: 18ml >10% Concentration 3: 10ml >10 Concentration 4: 5.6ml >10' Concentration 5: 3.2ml >10% Table 1: The number of dead and immobilized neonates Control Conc. 5 Exposure (hours) A B с D 24 0 0 0 C1 C2 C3 C4 C5 10 Total (x/20) % Effect 0 0 24 Results Sheet: 20 5555 5555 100 Table 2: Concentration of the chemical Flask Stock solution Conc. 4 24 20 100 Conc. 3 24 10 G जजज 20 100 Conc. 2 24 5 5 5 5 20 100 Chemical concentration (mg/L) 10 Conc. 1 24 0 0 0 5 20 100 3. Discussion: a. Please discuss the major advantages of toxicity testing using Daphnia. b. Please discuss the value of LC50 and EC50 for risk assessment.