This test along with the fish 96-h acute toxicity test is one of the standbys in aquatic toxicology. D. magna and D. pulex are the common test species.
D. magna require relatively hard water for its culture; D. pulex tolerate softer water.
In general, ten neonates that are less than 24-h old are placed in 125 ml beakers containing 100 ml of test solution at five concentrations and a negative control. The tests are usually run in triplicate. Death is difficult to observe so immobility of the daphnia is used as the endpoint.
An organism is considered immobile (nonmotile) if it does not resume swimming after prodding with a pipet or glass rod. Measurements are made at 24-h intervals. No feeding during course of experiment.
Why not?
Parameters - water temp 20 C;
pH range 7.0-8.6;
DO concentation 40-100%;
aeration - not necessary
photoperiod - 16 light 8 h dark.
Procedure.
First, make a 0.2M NaCl solution by mixing 11.69 g NaCl with enough distilled water to make 1 liter. Then label a series of beakers with the following concentrations:0.2M, 0.1M, 0.075M, 0.05M, and 0.025M. Make up these concentrations from the 0.02M solution using the proportions listed.
100%
50%
37.5%
25%
12.5%
control
100
50.0
37.5
25.0
12.5
0
0
50.0
62.5
75.0
87.5
100
1.Introduce the same number of Daphnia (at least 10) into each test vessel and control using a plastic, disposable pipette with a 5-mm diameter. Be sure to release the young below the surface to avoid killing them by trapping air under their carapaces. Record the time and number of Daphnia introduced into each labeled vessel.
2. Prepare a table that records the percent concentration, the time (1 hr, 24 hrs, 48 hrs), and the total number of dead at each time interval. As close to one hour as possible, but prior to the end of the period, record the number dead at each concentration. Remember Daphnia molt to grow, so neonates will molt as they develop. Therefore, do not count the molt castings, which appear as clear shells of the Daphnia on the bottom of each cup. Remove dead Daphnia and molt castings at each monitoring interval.
3. Ideally it is best to check again in 4 hours and again after 24 hours. Continue observations for a minimum of 48 hours or as long as there is not more than 10% death in the control population. Do not feed animals during tests.
4. At the end of the bioassay, test the water to determine the pH, hardness, and dissolved oxygen content. Count and record how many Daphnia in each dish have died, then analyze your data.
Freshwater species:Selenastrum capricornutum; Scenedesmus subspicatus; Chlorella vulgaris;Microcystis aeruginosa, Anabaena flos-aquae
Number of organisms per chamber ( -10%) 2 x 10 4; Sterile Erlenmeyer flasks; water temp 24 C; continuous cool white fluorescent; pH 7.5; endpoint growth or biomass; cell number.
