Daphnia Ecotoxicology 

• Set up a properly designed environmental toxicology experiment using Daphnia  as a model organism 

• Create a column graph to display the influence of NaCl concentration on  survival of Daphnia magna

• Use ANOVA and Tukey's post-hoc tests to analyze data from your Daphnia environmental toxicology experiment

• Write the Lab Title on a new page on the right-hand side of your notebook. *Remember to include the Lab Date!*

• Write the Background, Aim and your Hypothesis for the lab

• Draw Data Table 2 (See Procedures)

• Add an entry for this lab in your Table of Contents

• Model Organism:  Daphnia magna

• Experimental Questions: 

  • What concentration(s) of salt (NaCl) are lethal to Daphnia?

  • At what concentration(s) of salt (NaCl) is Daphnia survival significantly different from the control?

• Independent Variable: NaCl concentration

  • Control Group: 0 g/L NaCl      

  • Experimental groups: 2 g/L NaCl, 5 g/L NaCl, 10 g/L NaCl, 15 g/L NaCl, 20 g/L NaCl

• Dependent Variable: Survival (out of 5 Daphnia)

• I hypothesize that NaCl influences survival in Daphnia, and predict that as NaCl concentration increases, survival will significantly __increase / decrease.

  • I predict that survival of Daphnia will  significantly differ from the control (0M NaCl) at the following NaCl concentration(s) : _______*See Table 1 for concentrations*

OR

I hypothesize that NaCl exposure will not significantly influence survival in Daphnia

What is a Model Organism?

Scientists use "model organisms" to answer biological questions. We can then apply what we learned to other organisms that may be more challenging to study in the lab, due to practical or ethical constraints. Model organisms are studied extensively, so a substantial amount of data has been accumulated on these organisms. Typically, model organisms are inexpensive and easy to maintain in the laboratory. They tend to have short life cycles and produce a large number of offspring. Today, many model organisms also have had their entire genomes sequenced and made publicly available for research.

Daphnia

Daphnia are commonly called "water fleas." They are actually not insects, but belong to a closely related class of organisms. Daphnia is a genus of planktonic  crustaceans (the class that includes lobsters, crabs, shrimp, etc.). They live in fresh water like lakes, ponds and streams. Because Daphnia bodies are transparent, they are ideal for observing internal organs and systems and physiological responses to their environment.  Daphnia are excellent model organisms to use for bioassays, because they are sensitive to changes in water chemistry. This also makes them good environmental indicators.

What is a Bioassay?

A bioassay involves exposing a biological organism to various concentrations of a chemical to determine its toxicity.   These methods are often used to test water or soil quality, especially near hazardous waste sites. Bioassays are also used test the effects of compounds being considered for use in medicines or skin care products.

In a bioassay, 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 survival at various concentrations of salt.

It is best to use Daphnia that are about the same age to minimize biological variability among the test organisms. At each concentration, the number surviving 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 (or using different toxicants).

Daphnia Bioassay Using Salt (NaCl)

Table 1.  Preparation of Salt (NaCl) Solutions

1.  Prepare solutions with different salt concentrations.

a.  Use tape to label 6 small "exposure" beakers with the concentrations shown in column 1.

b.  Refer to column 2 and add the indicated amounts of 2% salt solution (20g NaCl / L) to each beaker.

c.   Refer to column 3 and add the indicated amount of spring water to each beaker.

d.  Stir the salt solutions to mix thoroughly. 

2.  Use a wide-mouth pipette to carefully place 5 Daphnia into each exposure beaker. 

3.  Observe and note the Daphnia behavior.  (What are they doing?)

4.  At 0, 5, 10, 15, and 20 minutes, count the total number of Daphnia that are surviving and observe the behavior of the living Daphnia.  Note the area of the beaker where they are located, amount of movement, etc.  Record this information in your data table (see  example below).

5. At the end of the experiment (20 minutes) calculate the percent of Daphnia (#/5 *100) that died in each beaker.

Table 2.  The Effect of Salt Concentration on Daphnia Survival

Daphnia Survival over time in varying NaCl concentrations

You will use Excel to create a multi-series scatterplot of Daphnia survival (Y-axis) over time (X-axis). Your graph will display different lines for each concentration of NaCl.

Use the survival values collected by lab bench to create a properly formatted multi-series scatterplot graph in Excel

Column Graph of Mean ± SD

You will use Excel to create a column graph to display mean mortality of Daphnia  at each concentration according to the class data. 

Use the mean and SD values calculated in your Excel file (second tab in spreadsheet) to create a properly formatted column graph.

ANOVA

You will use an online calculator to perform ANOVA tests to examine significant differences in survival among your NaCl concentrations. The ANOVA test will tell you  if any significant differences exist and (2) the Post-Hoc tests will tell you what specific pairs of treatments  significantly differ from each other.

See detailed instructions for the ANOVA test and for how to incorporate ANOVA results in your figures and results paragraphs