Daphnia Bioassays Using Salt
In conducting a bioassay, populations of organisms are exposed
to various concentrations of a chemical to determine its toxicity. A specific
response or endpoint is selected for measurement. Example endpoints for Daphnia
include death, increased heart rate, or decreased appetite. In this experiment,
we will measure Daphnia death rates at various concentrations of salt.
It is best to use Daphnia that are the same age, preferably
newborns, to minimize biological variability among the test organisms. At each
concentration, the number dead are recorded after a specified period of time.
Once the general range of sensitivity is determined, further experiments can
be carried out using smaller differences between the solution concentrations.
- dissecting microscope
- analytical balance
- culture vessel approximately 10 gallons or greater
- water chemistry
kits (pH, hardness, dissolved oxygen)
- transparent cups or beakers
- 10 mL pipette
- pipette with 5 mm diameter opening
water or unpolluted stream water
- Daphnia magna culture,
- Chem Wipes or other tissues
- food: possibilities include
Roti-Rich (Ward Scientific), dried yeast, or unicellular algae such as Selenastrum
Making the Solutions
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 in Table 1:
Table 1. Solution Concentrations
0.2 M NaCl (mL)
Distilled Water (mL)
Carrying Out the Bioassay
- Prior to conducting the bioassay, check the Daphnia
to ensure the culture is healthy.
- Working in groups of 2-4 students,
select your test organisms. Although you may use mixed age populations for bioassays,
it is better to use only young individuals in order to minimize biological differences
among the test organisms. Because the appearance of resting eggs indicates a poor
culture environment, do not use Daphnia with resting eggs. To obtain a
good supply of young Daphnia, begin 24 hours in advance by removing females
bearing embryos from the stock culture and placing them in 400-mL beakers containing
300 mL of spring or stream water and the appropriate amount of food. Five beakers,
each containing 10 adults, usually will supply enough young individuals for one
toxicity test. When you are ready to begin your bioassay, choose young (small)
Daphnia from these cultures.
- Introduce the same number of
neonates (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 young introduced into each labeled vessel.
- 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
- Ideally it is best to check again in 4 hours
and again after 24 hours. If schedules do not permit, just check at approximately
one and 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. Steps 4-6 will take about 15 minutes of two consecutive class periods.
- 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.