Bioassays Using Daphnia
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
Daphnia typically live 40 to 56 days, varying according to species and environmental conditions. Each brood typically holds 6-10 eggs, which turn into embryos and are released within a few days. Juveniles reach sexual maturity in 6 to 10 days. A healthy population of Daphnia consists mostly of females that have been produced asexually. The culture can become stressed if the population density gets too high or if there is a food shortage, poor water quality, or extreme temperatures. Under stressful conditions, Daphnia produce more male embryos and begin to reproduce sexually. The resulting resting eggs will not hatch until they have gone through a certain sequence of environmental changes, including several freeze/thaw cycles. Therefore, if you want to maintain a steady supply of Daphnia in your lab, you will need to avoid the stressful conditions that lead to sexual reproduction.
Daphnia are quite sensitive to the chemistry of the water in which they live. In order to provide standardized culture water, professional scientists start with distilled water and add essential minerals and nutrients (see Table 1). The problem with this approach in schools is that you may not have access to distilled water of sufficient purity. Supermarket-grade distilled or deionized water may contain trace-level contaminants at concentrations high enough to be detrimental to populations of Daphnia. An alternate possibility is to use unchlorinated water such as bottled spring water, or water from a local well, spring, stream, or lake. It also is possible to start with chlorinated tap water and either let it sit long enough for the chlorine to evaporate, or treat it with activated charcoal or one of the products sold for de-chlorination of water to be used in aquaria.
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
Optimal Culture Conditions
Although Daphnia are sensitive to dissolved oxygen, pH, and chemical contaminants, it is not difficult to maintain healthy cultures. Individual students or groups can grow Daphnia in liter flasks or quart jars, or you can maintain larger populations in aquaria or gallon jars. The larger the container, the easier it will be to maintain favorable conditions for long-term survival, but the harder it will be to harvest Daphnia with a pipette for experiments.
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:
|dissolved oxygen||> 6 mg/L|
|hardness||160-180 mg CaCO3/L|
|lighting cycle||16 light/8 dark|
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.
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