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The Effects of Creatine on

Lumbriculus variegatus

A Biological Study
Conducted by: Dustin Woods, Josh
Morgan, and Julian Nwoko
HCOP PE 2008

• Lumbriculus variegatus:
o They are organisms, also called California Black
Worms, that live in freshwater biomes.
o One of their most well-known characteristics is
their ability to regenerate body segments after
being seperated.
o They have been used repeatedly as effective
species for testing the toxicity of certain

• Creatine:
o Creatine is used as a dietary supplement to
increase endurance and muscle efficiency.
o It functions, mainly, by optimizing the re-
phosporylation of ADP in the body.
o This may result in more active vessel tissue.
Perhaps, resulting in stronger, more frequent
Hypothesis for Pulsation Rate
If the California blackworms are significantly
sensitive to creatine solutions, then their
pulsation rates will increase in a direct
relationship with the solution concentration and
the exposure time.
Materials and Methods
Pulsation Rate Experiment
o Applied 40 mg/100mL and 60 mg/100mL
creatine solutions and spring water conrol
o Divided worms into groups based on
treatment time (10 or 20 minutes) and type
o Put six worms in each group for a total of
o Observed pulsation rate in pulses per
minute by using a microscope
Hypothesis for Regeneration
If the California blackworms are significantly
sensitive to creatine solutions, then they
will grow longer appendages, when cut and left
to regenerate for a period of time, than if they
are left in spring water for the same amount of
Materials and Methods
Regeneration Experiment
o Used 20 mg/100mL and 40 mg/100mL
creatine solutions and a spring water
control group
o Divided worms into anterior and posterior
segments and cut them up into smaller
o Placed segmented worms in individual
wells based on the section of the original
worm that they represented and the type
of solution they were placed in
Pulsation Experiment Layout
Materials and Methods
Regeneration Experiment
o Put small pieces of a paper bag in the bottom of
the wells and stored the wells in a dark closet to
stimulate the original environment of the worms
o Counted the regrowth of the worms after waiting
one week
Pulsation Experiment Layout
Discussion 1
Our experiment was blocked for two different result-
determining factors: Time duration, and Creatine
concentration. The two time durations were 10
minutes and 20 minutes, and the concentrations,
freshwater, 20 mg/100mL, and 40 mg/100mL.
Following the testing, hypotheses tests on our
data suggested that, after twenty minutes, there
was a significant difference between the pulsation
rates of California Black Worms in ordinary
freshwater and those in a creatine solution of 40
µg/100mL or 60 µg/100mL. Oddly, the same amount
of significance wasn't observed after 10 minutes.
Discussion 1 cont.

Therefore, the data failed to reject our initial

hypothesis. Creatine could, very well, increase the
pulsation rates of California Black Worms, given that
enough time is allowed.

Another interesting observation is that we did not

note a high mortality due to over-dosage,
Which brought up another point. Another test we
could have conducted was an LD-50 test to see their
resilience to the drug.
Discussion 2
The second part of the experiment was blocked for
two different result-determining factors: the side of
the worm that is cut, and the concentration of
Creatine solutions. The worms were immersed in
freshwater, 20 mg/100mL, and 40 mg/100mL
creatine solutions.
Then, they were cut into posterior and anterior
halves. 12 worms were immersed each treatment.
After a week of isolation to allow regeneration to
occur, the number of newly regenerated segments
were counted and recorded.
Discussion 2 cont.

Upon analysis, a significant difference was not

noticed between the number of regenerated
segments in either of the creatine solutions and the
control, spring water. Therefore, we must reject our
hypothesis that Creatine has a direct effect on
regeneration rate.
Statistical Analysis

o Performed using ANOVA

o Used as variables treatment,time, and a
cross between treatment and time
o Made mean comparisons using Student's T-
o Reported data as means ±  standard
Video Samples

1. Clip One - a worm under the microscope.

2. Clip Two - a worm seen from outside of the
microscope view.
3. Clip Three - another worm seen from under the
view of a dissecting microscope
4. Clip Four - another worm seen from under the view
of a dissecting microscope
Martinez VG, Zoran MJ, Reddy PK. Hydrobiologia (2006).
EBSCO Host. 1 July 2008.

Sardo, Soares, Gerhardt. Human and Ecological Risk

Assessment, 13: 519-526,2007.

Walter AA, Smith AE, Herda TJ, Ryan ED, Moon JR,
Cramer JT,Stout JR. "International Journal of Sport
Nutrition and Exercise Metabolism 18 (2008): 142-151.
EBSCO Host. 3 July 2008.