The Effects of Creatine on Physiological Characteristics of Lumbriculus variegatus

By Joshua Morgan

Abstract

This study examined the effects that creatine has on the pulsation rate and
regeneration ability of Lumbriculus variegatus with respect to concentration of
solution and exposure time. Creatine is a staple for athletes as it is believed to
stimulate muscle productivity, and it has been subjected to adequate prior
experimentation as a result. Essentially, this experiment was designed to expose
precisely how widespread the drug’s benefits are; its introduction into a species in
which it is not biologically synthesized presented the potential for valid and
meaningful results. In a pulsation rate experiment, it was hypothesized that the
presence of creatine would increase the species’ pulsation rate. In a regeneration
experiment, it was hypothesized that the presence of the drug would result in
increased regeneration. In addition, it was hypothesized that these effects would
occur in direct relationships with time and concentration. In the pulsation rate
experiment, our hypothesis could not be rejected due to the calculated T-test
values, but the T-test values in the regeneration experiment negated the second
hypothesis. Therefore, it could only be concluded that the pulsation rate of
Lumbriculus variegatus can possibly increase with exposure to creatine.

Introduction/Background

California blackworms developed into a basis for numerous scientific inquiries

in the closing years of the twentieth century, essentially as a result of the newfound
appreciation the scientific community adopted for the species’ accessibility and
economic justification. 1 Another trend was the widespread use of creatine as an
enhancer for muscle strength and energy sustenance. 2 Due to widespread debate
of whether or not creatine’s effects are of a high extent, much information about the
topic is unclear in the scientific community. The basis behind this experiment was
to examine the results of creatine’s introduction into an organism in which it is not
naturally synthesized, or any invertebrate. This should have presented insight
into how universally creatine can alter organisms’ physiological processes.
This experiment was preceded by a multitude of related ones, some of which
involved the introduction of new substances into the environment of Lumbriculus
variegatus and some that tested creatine in organisms. It has been determined in a
scientific study that the alteration of sediment types in the environment of
Lumbriculus variegatus produces effects in areas such as pulsation rate and
regeneration. 3 It has been found in an experiment that introducing the worms to
boric acid (H₃B) decreases their regrowth.4 On the subject of experimentation
involving creatine and its effects on organisms and their biological functions, one
test found that creatine loading on young men did not have any significant effect on
their fatigue threshold levels.5 These and other experiments helped to justify the
two major subjects as plausible to use in this experiment, as it has been proven that
creatine’s effects are testable in laboratory conditions and that Lumbriculus
variegatus represents a reliable test species in toxicology experiments. In this
manner, these sources upheld these tests as worthwhile experiments that could
support or reject a given hypothesis.

Hypotheses

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.

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 time.

Methodology

The pulsation rate experiment used two creatine solutions, one with .4 mg of
creatine per 100 mL of water and one with .6 mg of creatine per 100 mL of water,
and a control of the spring water that is found in the species’ environment. Overall,
36 worms were divided into six groups of six worms, two control groups with
different exposure times, and two groups from each of the experimental groups.
After a worm was placed in a group and in the petri dish containing the solution it
would be tested with, it was timed individually for 10 or 20 minutes. After a worm’s
exposure time was complete, it was placed on a piece of filter paper and observed
through a microscope. The worm’s ratio of pulsations per minute was determined
by observing how many times a rush of blood passed through one of its segments in
one minute. This process was carried out for each individual worm and the data
was recorded for all of them. Finally, the statistical data was calculated and the t-
test was performed to determine the validity of the hypothesis.
The regeneration experiment also used two creatine solutions, but a .2 mg/
100 mL solution replaced the .6 mg/ 100 mL solution. Each worm was divided into
its anterior and posterior sections to determine if biological differences exist among
them. The segments were placed in wells based on the body section they represent
and the control or experimental group they will be a part of. In this experiment, 12
worms and 24 segments were used in each of the three test groups, for a total of 36
worms and 72 segments to be tested. Small pieces of brown paper were placed at
the bottom of the wells to stimulate the original environment of the worm species,
and the wells were placed in a dark area to achieve the same effect. One week
later, the wells were taken out of the dark place and the regrowth of the segments
was counted from under the microscope. This was relatively easy to do as the
regenerated segments of the worms were much lighter than the original segments.
After the results were recorded, the same statistical data from the first experiment
were recorded.

Results
 Figure 1

Figure 2

Figure 1 demonstrates that the experimental groups reported a mean

pulsation rate that was higher than the mean for the spring water control group.
The graph implies that the difference between the control and experimental groups
was especially significant after the 20 minute time period. However, it also shows
little difference between the means for the 0.4 mg/mL group and the 0.6 mg/mL.
No clear correlation is shown regarding the time of exposure for the worms that is
consistent with the three test groups.
Figure 2 implies that the addition of creatine to the environment of
Lumbriculus variegatus did not affect the regeneration rate of the worms in any
major way. It also shows the mean for the experimental group with the more
concentrated solution to be lower than the mean for the group with the lower
concentrated solution. The figure shows that the posterior segments generally had
slightly more regrowth than the anterior ones. It also demonstrates the high level of
variance in the data through the y-error bars, a factor that may induce some
unavoidable error in our predictions.

Conclusions/Future Implications

Through an examination of the p-values collected from Analysis of Variance

(ANOVA) testing, it was concluded that the exposure of Lumbriculus variegatus to
creatine increased the species’ pulsation rate as predicted in the hypothesis, but
the difference only became significant after the worms are exposed for a time t in
which 10<t≤20. Though the data supported the hypothesis for this test, more
experiments must be performed before this hypothesis can be labeled as theory.
This test confirmed the hypothesis is reasonable and therefore should be opened up
for further experimentation in order for it to be made even more viable.
The ANOVA tests rejected the hypothesis given for the regeneration
experiment. The p-values for tests involving treatment, body section, and the cross
between the two, were all above 0.05, and were therefore interpreted as indication
of insignificant differences in the data. This indicated that the hypothesis could not
be accepted in the form of scientific logic. Note that this did not prove the
hypothesis incorrect and it remained possible that the conclusion of this experiment
could have been skewed by factors such as misleading data or significant
procedural errors. In speculation, these results could be questioned by another test
that produces contradictory results.
Due to uncontrollable factors, the experiments performed in this study were
not entirely flawless. The tests were performed under a set time period that was
organized by a party that was not directly involved in carrying out the experiments.
The experiments were constrained to a block of time in a classroom setting that was
held only once a week. The study was held in its duration over a six week time
period. Had this study been performed under a flexible schedule set to the needs of
its performers, it probably would have been more characteristic of a profession
scientific study. For example, the test groups would have been much larger,
minimizing the possibility of outliers misconstruing the data collected and the
conclusions that were drawn from it. In addition, some factors, such as
temperature, that were intended to remain constant were assumed to have little
variation but were not actually measured.
1
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