Kayce Cook Lab Report

Effects of Rock Salt (Calcium Magnesium Acetate) On Lumbriculus variegatus

Abstract

Furthering the study of how L. variegates react to different substances, our

group chose Calcium magnesium acetate to test. The group created four solutions
to test: 0.5% CMA, 1% CMA, 3% CMA, and 5% CMA. During the course of our tests,
the 3% and 5% solutions were found to be lethal doses while the other two solutions
were below the tolerance levels of the specimens. Our worms were subjected to a
pulsations test, a locomotion test, and a chemotatic test to discover how the
solutions affected their behavior. The results of these tests confirmed two of our
three hypotheses.

Introduction/Background

The purpose of this study was to discover how calcium magnesium acetate
affects aquatic freshwater invertebrates, the surrounding environments, and
humans that come into contact with the substance. To measure the extent of the
effects of the substance, L. variegates, or the California black worm was used. The
first objective of the experiment was to measure the tolerance levels of the worms
by using different concentrations of the same solution, and from there, determine
how the different concentrations affected behavior, locomotion, and health of the
worms and to use that data in application to human and vegetative studies.

To further our knowledge of the specimen and our substance, the group
researched other studies performed. The group found that humans can be
susceptible to the CMA in large quantities and concentrations, and that it could
compromise the hypersensitivity of the skin and cause rashes and irritation. i Other
studies showed that our substance could have a negative effect on the environment
by introducing a higher salt content to the soil and water ways surrounding roads
and highways. ii These studies laid the groundwork for the experiments to follow.

Hypotheses

If a freshwater aquatic organism (L. variegates) is added to a saltwater

solution, and the hypertonic solution is pushed towards equilibrium, then L.
variegates will experience significant changes in their pulsation rates, which will rise
above that of the control group which are in spring water.

If a freshwater aquatic organism (L. variegates) is subjected to a hypertonic

solution of Calcium magnesium acetate, then the occurring equilibrium will slow the
locomotory motions of the worms by a significant rate.
 If a freshwater aquatic organism (L. variegates) is subjected to a hypertonic
solution in a chemotatic test, being a solution adverse to its own habitat, then the
worm will react to the solution as a repellent and will try to escape it.

Methodology

The first experiment that was conducted was the Pulsation Experiment. This
test was performed at the Life Sciences building, the Histology Lab on June 25. The
test required the following materials: three petri dishes, 30 worms, the control
solution of spring water, the two prepared solutions of 1% CMA and 5% CMA, three
pipettes for each solution, three sets of wells for the exposure to the solutions, a
timer for the allotted time period of exposure, and a microscope to count the
pulsations.

The process for this experiment includes separating the 30 worms into three
groups. Each group is placed in a set of wells for the five to fifteen time period of
exposure to each solution. After the exposure time is finished, each worm is
removed and the pulsations are counted for 10 seconds. The data collected is then
multiplied by six to get the number of pulsations per minute for each worm.

That data is then entered into the AN OVA (JMP v. 7 SAS) program, and using
the Student’s t-Test function, the mean and standard deviations for the data set is
calculated.

The second experiment, the Locomotion Experiment, involved measuring the

distance covered by the worms while under the influence of the solution. This test
was also performed at the Histology Lab at the Life Sciences building on July 2. The
following materials are needed: three petri dishes, 30 worms separated into the
three test groups, a petri dish track to measure the distance of each worm, three
sets of wells for the exposure periods, a timer for the exposure time periods and for
the intervals of measuring the worms’ distances, and the 1% CMA and 3% CMA
solutions and the control solution of spring water.

To make the petri dish track, one must first take a petri dish and measure
around the circumference of the outside, marking in grease pencil each centimeter.
Then, take a piece of stiff paper that is measured to be as tall as the petri dish and
fold it into a circle, taping the ends together. Place this circle of paper in the middle
of the petri dish, fixing it in place with two rubber bands. The purpose of this circle
of paper is so that the worms will have a fixed track to move in and the distance can
be accurately measured without the worm moving across the middle of the petri
dish, skewing the results of the test.

The procedure for this experiment includes putting the separated groups of
10 worms into their designated wells filled with each solution, including the control
solution of spring water. Then set the timer for the five to fifteen minute exposure
periods for each group of worms. After the time periods are completed, extract the
worms and place them in the petri dish track. Set the timer for 30 seconds and
monitor the worms. After the time period is complete, record the data for each
worm in the three test groups. After all the worms have been in the track, take the
data collected and multiply each value by two to get the distance traveled per
minute. Then entering the data into the ANOVA program and calculating the mean,
standard deviation, and the t-test.

This third experiment was also performed at the Life Sciences building on July
9. For the final experiment, the Chemotatic Experiment, the subject to be measured
is if the worms react to the solution as an attractant or a repellent by how the
worms move in relation to their tails. A positive response is described by the worm
moving towards its tail, where the solution will be placed. A negative response is
described by the worm moving away from its tail and the solution.

The procedure for this experiment requires the following materials: three
petri dishes, 30 worms, the test solutions 0.5% CMA and 1% CMA along with the
control solution of spring water, a timer set for 30 seconds for the monitoring
periods for each worm, a pipette for each solution to be tested, and filter paper to
absorb excess spring water from the worms.

To start the experiment, the worms must be separated into 10 worm groups
for each solution to be tested. First, place each worm into an empty petri dish and
use filter paper to absorb the excess spring water from the worm’s body. This action
prevents the solution being tested from being diluted in the process of monitoring
the worms’ behavior. After the excess spring water has been removed, place a
single drop of the solution being tested on the worm’s tail and set the timer for 30
seconds. After the 30 seconds are complete, record the data for each worm.

This set of data doesn’t need a mean, standard deviation, or t-test. The data
sets can be presented in a positive or negative responses divided by ten for each
test group, where each set of data are in 0%-100% range.

Results

The results of the Pulsations Experiment were that the mean pulsation rate
rose after the worms were exposed to the 1% CMA solution when compared with the
control group. The worms exposed to the 5% CMA solution, however, were
terminated after two minutes in the solution.

The results of the Locomotion Experiment were that the mean distances from
the test group exposed to the 1% CMA solution were less than the control group’s
mean distance. The worms exposed to the 3% CMA solution were also terminated
within two minutes in the solution, so a new solution was created for the conclusion
of the test, 0.5% CMA. The mean distance for this group increased from the first test
group in the 1% CMA solution and the control group in the spring water.

The results of the Chemotatic Experiment were that for both test groups, the
0.5% CMA and 1% CMA solutions, the worms reacted positively for 70% and reacted
negatively for 30% when compared to the 100% positive reactions for the worms in
the control solution of spring water.

Conclusions/Future Implications

The conclusions for the Pulsations experiment are that the solution of 1%
CMA did raise the worms’ pulsation rates, supporting our hypothesis. Improvements
on this experiment could include using the 0.5% CMA in the pulsations test to see
how a solution of less concentration could affect their pulsation rates. This change
could relate to the results of the Locomotion test, showing an increase in pulsation
rates, or a similar decrease in pulsation rates like the 1% CMA solution test group.

The results of the Locomotion experiment show that the solution slowed the
worms’ movements down enough to see a significant change with the 1% CMA
solution group’s mean distance being lower than the control solution’s. However,
the 0.5% CMA solution test group showed an increase in distance when compared
with the control group’s mean distance. This could be because the salt content of
the solution was not great enough to impair the worms’ movements as in the 1%
CMA solution test group. Improvements could be made like lengthening the
exposure time to the 0.5% CMA solution, or creating another CMA solution that is
between 0.5%-1% CMA.

The conclusions for the Chemotatic experiment show that the freshwater
organisms find that salt solutions to be an attractant. An improvement that could be
made would be to repeat this test many more times to calculate the overall mean
responses instead of mainly depending on 10 worm groups for each test solution.
This would minimize the margin of error and confirm the worms’ reactions to the
solutions.

References and Literature Cited

Cushman, J.R.; Duff, V.A.; Butfau, G.H.;Aust, L.B.; Caldwell, N.; Lazer, W.. “Evaluation
of CMA and Road Salt for Contact Hypersensitivity Potential and Dermal Irritancy in
Humans”. Contact Dermatitis (01051873) Apr91, Vol. 24, Issue 4, p.289-292

http://www.ncbi.nlm.nih.gov/pubmed/1831107

McFarland, B.L.; O’Reilly, K.T. “Environmental Impact and Toxicological

Characteristics of Calcium Magnesium Acetate”. Chemical Deicers and the
Environment. Lewis Publishers, Boca Raton, Florida.1992. p 192-227. 9 fig, 6 tab, 50
ref.
http://md1.csa.com/partners/viewrecord.php?requester=gs&collection=ENV&recid=
9306831

Sardo, Soares, Gerhardt. “Behavior, Growth, and Reproduction of Lumbriculus

Variegatus (Oligochaetae) in Different Sediment Types”. Human and Ecological Rich
Assessment, 13:519-526, 2007.

http://www.informaworld.com/smpp/content~content=a778588780~db=all~order=
page
i
Cushman, J.R.; Duff, V.A.; Butfau, G.H.;Aust, L.B.; Caldwell, N.; Lazer, W..
“Evaluation of CMA and Road Salt for Contact Hypersensitivity Potential and
Dermal Irritancy in Humans”. Contact Dermatitis (01051873) Apr91, Vol. 24,
Issue 4, p.289-292

http://www.ncbi.nlm.nih.gov/pubmed/1831107

ii
McFarland, B.L.; O’Reilly, K.T. “Environmental Impact and Toxicological
Characteristics of Calcium Magnesium Acetate”. Chemical Deicers and the
Environment. Lewis Publishers, Boca Raton, Florida.1992. p 192-227. 9 fig, 6
tab, 50 ref.

http://md1.csa.com/partners/viewrecord.php?requester=gs&collection=ENV
&recid=9306831