a Daphnia Culture
You can obtain a culture of Daphnia from several of
the scientific supply companies such as Carolina Biological
or Flinn Scientific.
You can maintain your own culture
of Daphnia relatively easily. See the various websites
above for more information. While not necessarily ideal,
the author has previously used a bucket to maintain Daphnia,
an aquarium is recommended. Prior to receiving the Daphnia
fill the aquarium with tap water and allow the water
to age. Add a crushed boiled egg yolk to the container
two days prior to receiving the Daphnia. The egg yolk
will encourage bacterial growth that the Daphnia will
be able to feed on. Daphnia food can also be bought,
but is not necessary. The water should be a little cloudy
as a result of the bacterial growth. Maintaining a little
bacterial growth jar with water and egg yolk so that
you can continue to feed the Daphnia is advisable. Some
groups recommend using distilled water and adding a variety
of ingredients to it, this maybe advisable if your tap
water has an overabundance of minerals.
Day 1 - Before Class
1. Prepare a small beaker for each group of students
that includes about 10 Daphnia per beaker
2. Prepare Stock solutions of copper sulfate
in 1000, 500, 100, 10 ppm. The preparation
of these solutions will
depend on the type of copper sulfate that is purchased.
The following example used a 20% copper sulfate solution
available from Carolina Biological Supply Company.
I. To calculate the ppm contained in 1 ounce of material
first solve for B:
A x 75 = B
A = the % active ingredient (CuSo4)
B = ppm contained in 1 ounce of the material in 100
gallons of water
To calculate the number of ounces of material
required to make up a desired ppm concentration
solve for C:
C = Desired ppm conc. / B
B = ppm contained in 1 ounce of the material
in 100 gallons of water (from above).
C = number of ounces of material to add
to 100 gallons of water to achieve the
III. To calculate the number of ounces needed for
1 gallon of water
D = C / 100
D = number of ounces of material to add to
1 gallon of water to achieve the desired
IV. 1 ounce is equal to 29.6mL.
3. For the 1000ppm solution – 0.7 ounce (20.7mL)
copper sulfate to a gallon of water, for the 500ppm solution – 0.3
ounce (8.9mL) copper sulfate to a gallon of water, for
the 100ppm solution – 0.07 ounce (2.1mL) copper sulfate
to a gallon of water, and for the 10ppm solution – 0.007
ounce (0.2mL) copper sulfate to a gallon of water. .
Bioassays to Students
1. At the beginning of class ask students to respond
to the following questions in their notebooks,
is in your water?
it just water?
could you tell whether water in a stream was good enough
discussion will be a review if
students have done the lessons earlier in this cycle.
students have written their responses, ask them to
share them with the class. Discuss with students
that even drinking water that comes
out of their tap has salts
and minerals in it; tap water isn’t just H2O.
utility companies constantly monitor their water to make
sure that it is fit to drink
and meets the US Environmental
Protection Agency standards. Bodies of water that are
the source for drinking water
are also monitored. Ask
students “How can the quality
of water be assessed?” Again, this
is a review if they have completed the earlier
lessons. If you have
not done these lessons you may want to review
them for information to share.
2. Explain that one way
people who monitor environmental
assess water quality
(along with the
tests mentioned previously)
is by performing bioassays.
that an environmental
one way that scientists
whether there may be
the presence of potentially
harmful compounds. Bioassays
use organisms that show
to possible pollutants.
they will be introduced
to an organism that is
They will be
an experiment using Daphnia
the impact of a change
in environmental conditions
3. Share that Daphnia
are a freshwater crustacean.
To be able to use
the Daphnia as a bioassay
tool, they have
to be familiar with
normal Daphnia behavior and
with Daphnia anatomy, see the Daphnia
anatomy sheet labelled.
4. In groups have students
look at the Daphnia
within the beakers.
would they describe
behavior of the Daphnia?
How do the
Daphnia move? They
down in their lab notebooks.
5. Show students
how to pipette
had some moderate success using Petri dishes
to observe the Daphnia if depression slides
are not available. Remind
students to make sure that there is sufficient
water around the Daphnia. Review microscope
skills if necessary.
If you have the capability to link a microscope
to some form of projection this can be very
with the students
the various parts of the Daphnia anatomy.
them to identify structures.
they distinguish the intestinal
the Daphnia gravid (pregnant)?
parts of Daphnia on the Daphnia anatomy sheet
6. Make sure that
the heart beating.
to take the heart
beat of the Daphnia.
as you observe
through a microscope,
by just dotting
a piece of paper
you see the heart
beat for 10 seconds
of the Daphnia.
Have the students
a class chart.
Are the heart
a range that appears
students that toxins
not just circulation,
see the heart
of the Daphnia
they want to take
a response to a possible
2 & 3 Dose Response Guided Experience
1. Students should be wearing appropriate safety
equipment, safety goggles, lab apron and gloves.
2. Each group should be given a beaker
with the stock solution of copper
sulfate (see above for
of the stock solution.)
the teacher’s information
only, Daphnia has an LC50 in excess of 320
ug/liter. Inform students that copper
sulfate is known to affect
Daphnia very adversely. They are to look at
the impact of different dilutions of
this substance on the Daphnia.
3. Provide students with the following
instructions - on the Student
Instruction Sheet. They are
to set up the dose response investigation
today and collect data on the subsequent 3
will not take the entire class period and so
the following lesson can be conducted on Days
2 & 3. (The
data can be collected on the Daphnia
Data excell workbook can be used if computers
Instructions: Student Instruction Sheet
You are going to assess the response of Daphnia to copper
sulfate. Copper sulfate is routinely added to ponds and
lakes to control weeds.
Make sure to take appropriate safety measures. Wear safety
goggles, apron and gloves.
When you are finished with the solutions dispose of them
where your teacher instructs you. Do NOT pour them down
1. Label each of the four beakers with the concentration
to be created in that beaker; 1000ppm, 500ppm, 100ppm,
2. Using the stock solution of copper sulfate that you
have been provided. Make
sure you place the dilution in properly labeled beakers.
3. Add 10 Daphnia to each beaker. Be sure to make sure
that none of the Daphnia are gravid (pregnant) before
placing them in the beakers.
4. Place the beakers on the side and observe at 24, 48,
and 72 hours. Count how many survive and record in a
5. Graph the results on a graph. Make sure to identify
the dependent (survivors) and independent
/ hours) and place them appropriately on the graph.
1. What was the response of the Daphnia to copper
2. If copper sulfate is used to clear ponds and
lakes of weeds, what does the information you’ve gained
from this experiment tell you about the use of copper
Once students have their results they should share their
graphs with the rest of the class. (The
graphs can be created in the Daphnia
Data excell workbook can be used if computers are
2. Ask students if the graphs show a similar pattern.
Discuss the use of a dose response graph. (See resources
3. As a group discuss the concept of LD50. Explain
that one way of measuring the toxicity of a substance
it in terms of the amount, given at one time, needed
to kill 50 percent of a test population.
4. Ask students what are some of the problems with
the exercise they performed for assessing LD50 or LC50.
how old, or the health of the Daphnia, they can’t
know whether individuals would have died anyway) Point
conducting multiple trials of the same experiment means
that they may be able to have greater confidence in
than if they just had the results from one trial.
Using Daphnia as a Bioassay Organism
Once students have explored the dose response of Daphnia
to copper sulfate they should develop a procedure using
Daphnia to explore an environmental substance or factor
oxygen, pH etc.) that might affect the health of the Daphnia.
1. Provide students with the Investigation
2. Explain that they are to use Daphnia as a bioassay in
investigating this environmental factor’s impact on
health. You may wish to start by reviewing some substances
and factors that affect our environmental health and substances
that we add to the water cycle, for example cleaning materials,
herbicides and pesticides, etc.
For homework have students
develop 3 potential questions that they could ask using Daphnia
as a bioassay and briefly describe how these questions might
1. Students should share their questions within their
group and then using availability, interest and safety
decide which question they want to pursue as a group.
2. They should then identify how they are going to answer
the question procedurally. Once they have identified
a question and an outline of how this might be investigated
attain teacher approval to continue. When students approach
to receive approval for an investigation this is the
to identify materials that they will have to bring and
those you can provide and also identify any obvious safety
3. Once the question and general outline of the procedure
has been identified, students should research a little
about the factor that they are investigating. Drawing
research and their previous experiences, they should
develop a hypothesis. Review with students what a hypothesis
if necessary. I encourage my students to identify the
dependent and independent variables and develop an “If….then…” statement
using the variable information. They also have to provide
a rationale for their hypothesis based upon their research
and observations. This is described on the Investigation
You may wish to use this time to discuss null and alternative
your students’ readiness.
This is also a good time to remind students that when
discussing the term “hypotheses”, it is more
appropriate to use the terms “supported” or “rejected” rather
4. Students should then flesh out their procedure based
upon their hypothesis; identify materials needed and
who is going
to bring them; identify safety considerations and develop
a data collection method. Students should approach the
teacher for approval to proceed at this point. Make sure
seek approval that their hypothesis is in response to
their identified questions and that the data they will
will address the hypothesis. If their procedures and
data collection methods do not address the question and
they should rewrite them. This approach typically takes
most of a class period, but can be very effective in
focusing students on developing hypothesis and matching
6 – Day 8
Students should conduct their investigations and
write up a lab report. In their lab report they should
state whether their hypothesis was rejected or supported
based on their observations.
If they were unable to
get enough information how might they gain more? If
hypothesis was rejected, do they have a new hypothesis?
Do they have
new questions based upon their investigations? Students
also address how Daphnia respond to the environmental
factor under study and how this might add information
to the environmental
5: Students must come up with 3 questions that they would
like to investigate
Day 6: Students may finish writing up procedures and designing
data collection sheets.
Day 8: Students should work on independent lab reports