Daphnia Bioassays Using Salt (NaCl)
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.

Equipment

• dissecting microscope
• analytical balance
  
• culture vessel approximately 10 gallons or greater
• water chemistry kits (pH, hardness, dissolved oxygen)
  

Materials

• thermometer
• transparent cups or beakers
• microscope slide
• 10 mL pipette
• pipette with 5 mm diameter opening
• spring water or unpolluted stream water
• Daphnia magna culture, mixed age
• Chem Wipes or other tissues
• food: possibilities include Roti-Rich (Ward Scientific), dried yeast, or unicellular algae such as Selenastrum
• NaCl

Procedure

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

Carrying Out the Bioassay

1. Prior to conducting the bioassay, check the Daphnia to ensure the culture is healthy.
2. 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.
   
3. 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.
   
4. 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.
   
5. 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.
   
6. 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.