Using Daphnia for bioassays requires advance planning to make sure that you have a healthy, non-stressed population from which to choose your test organisms. If you order cultures through the mail, be sure to allow sufficient lead time for shipping of replacement cultures in case the original ones arrive in poor condition. After the Daphnia arrive and have stabilized at room temperature, lower the shipping jar into an aquarium or gallon jar containing unchlorinated water (see Culture Water, below).
Plan on maintaining a healthy culture for at least a week or two before using the organisms for bioassay experiments. EPA recommends not using a culture for bioassays if more than 20 percent of the Daphnia die during the two days preceding the test.
Life Cycles of Daphnia
With any of these water sources, you should start by testing the sensitivity of Daphnia to the water. If more than 80% of the individuals living in the water survive for two days or longer, then that source is acceptable for your culture water. In bioassay experiments, dilutions should be made with this same type of water in order to maintain consistency between treatments.
Table 1. Recipe for synthetic culture water for Daphnia
Start by filling the containers with culture water (see discussion above). Although you can buy special food for Daphnia, it is not necessary. Healthy cultures can be maintained using either or both of two simple foods: powdered yeast (the kind used in baking) and unicellular algae such as Selenastrum capricornutum. Simply sprinkle a pinch of yeast on the water surface every couple of days, and/or add several milliliters of concentrated algal solution. Be careful – overfeeding is probably the easiest way to cause a population crash, because excess food will cause oxygen depletion. Aquarium aerators can be used but are not necessary as long as feeding is carried out in moderation. The amount of food needed varies depending on the population density. A general guideline is to feed enough so that the water becomes slightly cloudy but clears again within a day or two. Daphnia are filter feeders, so they gradually clarify the water in which they live.
Using a test-tube sized culture of Selenastrum purchased from a biological supply company, you can create your own never-ending supply of high quality Daphnia food. Simply transfer the algal culture to a larger container such as a liter flask or quart jar, fill with water, and add houseplant fertilizer at the concentration recommended on the packaging. Place the algal culture in a well-lit location, and shake or mix it every few days. Once the solution becomes bright green, it contains billions of algal cells and is ready for use as Daphnia food. Occasionally add more water and fertilizer to replenish the algal solution after feeding your Daphnia cultures.
It is not necessary to periodically clean the Daphnia culture containers. In fact, once organic debris has begun to accumulate it is possible to develop cultures that are relatively self-sustaining because the Daphnia will feed on detritus and decomposer microorganisms. Without supplemental feeding, population growth rates will decline, but the culture may survive weeks or even months unattended.
For optimal culture growth, the following conditions are recommended:
If cultures are maintained under these optimal conditions, a 3-L vessel stocked with 30 Daphnia will produce approximately 300 young per week. You can either let the populations rise and fall in natural cycles, or periodically remove some of the individuals to prevent overcrowding and keep the culture reproducing rapidly. It is a good idea to maintain more than one culture, since even under the best of conditions Daphnia populations occasionally crash for no apparent reason.