Biology 18b
October 16, 2007
Temperature changes and acetaminophen elicit a response in
daphnia heart rate
J. Gormley, K. Kenning*
Department of Biology, Brandeis University.
Assistant Vincent Mecozzi.
Supervised by Professor Melissa Kosinski-Collins and Teaching
Modifying an organism’s environment can be a useful means of understanding biological
systems. More specifically, how an organism’s heart beat responds to changes in the
environment can be observed and deductions can be made as to why those changes occurred.
Temperature change, for example, or the introduction of food, stimulants, or chemicals, all
represent environmental variation that should elicit some form of response from an organism.
Daphnia are small aquatic crustaceans that are found in fresh water environments (KosinskiCollins, 2007). Since their cytoskeleton is clear and their organs visible, daphnia have been used
to monitor environmental changes and determine how the change affects its heart rate (Pritchard,
2003). In this particular case, monitoring the heart rate of daphnia allowed us to study and
analyze the influences of two specific environmental changes on the biological systems within
daphnia.
First, water at various temperatures was added to daphnia. Daphnia are cold-blooded organisms
and therefore do not thermogregulate (Gerritsen,1982). As a result, introducing water above
room temperature would likely elicit an increased heart rate while water colder than room
temperature would decrease the heart rate. While daphnia do not have blood like humans,
oxygen is still transported via the circulatory system to allow for oxidation reactions that support
the energy needs of cells. Temperature change was expected to affect the availability of oxygen,
either increasing or decreasing its demand and thereby relatively affecting its heart rate.
Daphnia’s response to chemical changes in their environment was also tested. NyQuil is a
pharmaceutical drug often used to treat common cold, aches, or pains. Its main ingredient is
acetaminophen, a pain reliever that, like other antibiotics, inhibits protein synthesis.
Acetaminophen has proven to immobilize daphnia in even low concentrations (Daughton et al.
1999) so its application had to be well monitored. Acetaminophen was expected to lower the
heart rate since reactions involved in the production of oxygen-carrying proteins, such as
hemoglobin, are hindered, affecting the metabolism of the cell and the muscles that contract the
heart.
Environmental changes proved to affect daphnia’s heart rate. The heart rate of daphnia tended to
increase with rising temperature yet decrease at lower temperatures. Furthermore, acetaminophen
in NyQuil influenced the organism’s heart rate, causing it to decrease on average at each
concentration, exhibiting inhibitory characteristics.
* The last author contributed her own report for this experiment
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MATERIALS AND METHODS
Temperature-inducing effect on daphnia heart rate
A single daphnia was prepared as described (Kosinski-Collins, 2007). Water at 23ºC was added
to the daphnia three separate times with a new daphnia each time. The process was repeated for
water temperatures of 4ºC, 15ºC, and 37ºC. The daphnia’s heart rate was recorded before each
temperature was introduced and then after using a hand-counter and dissecting microscope. The
change in heart rate was recorded.
Effect of NyQuil on daphnia heart rate
NyQuil was diluted to 10-, 100-, 1000-, 10000-, and 50000-fold solutions. A single daphnia was
prepared as described (Kosinski-Collins, 2007). The 10-fold solution was added to the daphnia
three separate times with a new daphnia each time. The process was repeated for the 100-fold,
1000-fold, 10,000-fold, and 1:50000 solutions. The daphnia’s heart rate was recorded before each
solution was introduced and then after using a hand-counter and dissecting microscope. The
change in heart rate was recorded.
RESULTS
A single daphnia’s heart rate was recorded under normal conditions (room temperature
water) ten times in order to obtain an average heart rate. The average heart rate was found
by taking the sum of the heart rates from all trials and dividing by ten (the total number of
trials). The average rate was found to be 217 beats per minute (bpm) with a standard
deviation of 15.15 bpm. Standard deviation is given by the formula:
s
 X
i
 X
2
N 1
Once the control heart rate was established, water at different temperatures was added to
the daphnia’s environment in order to determine how its heart rate would be affected.
Table 1: Daphnia heart rate measurements with the addition of different
temperatures of water
Trial 1
23º
Temperature
Water† (bpm)
-6
Trial 2
Change in heart Rate
4ºC Temperature 15ºC Temperature
Water (bpm)
Water† (bpm)
-30
-18
30
12
-36
-36
33
Trial 3
-3
-12
-6
48
Average*
1
-26
-20
37
Stdev**
9.644
12.490
15.100
9.644
*All averages rounded to the nearest whole number
**Standard deviation shown is in +/- beats per minute
†
37ºC Temperature
Water (bpm)
Data retrieved on separate date, September 24, 2007.
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The changes in heart rate were determined by observing the heart rate of daphnia both before and
after each temperature was added. It was found that the heart rate changed 1 bpm ± 9.644
when room temperature water was added. On average, the heart rate decreased 26 bpm
±12.490 when 4ºC water was added. With the addition of 15ºC water, the heart beat also
decreased on average 20 bpm ± 15.100. Lastly with the addition of 37ºC water, it
increased 37 bpm ± 9.644.
Average Change in Heart Rate
(bpm)
Figure 1: Change in heart rate as a function of water temperature
60
50
40
30
20
10
0
-10 0
-20
5ºC Water
15ºC Water
23ºC Water
10
20
30
40
37ºC Water
-30
-40
-50
Water Temperature (ºC )
The average change in heart rate was graphed as a function of the water temperature. Data was based on
table 1.
After recording the changes in heart rate due to varying water temperatures, NyQuil was
added to daphnia at different concentrations to observe how daphnia’s heart rate would
respond.
Table 2: Changes in heart rate caused by concentrations of NyQuil
Change in Heart Rate
1:1000
1:10000
Concentration of Concentration of
NyQuil (bpm)
NyQuil (bpm)‡
-12
-21
Trial 1
1:10
Concentration of
NyQuil (bpm)
-54
Trial 2
-18
-45
-36
-35
Trial 3
-12
-30
-18
-6
Average*
-28
-29
-25
-22
Stdev**
22.716
16.523
9.644
14.640
*All averages rounded to the nearest whole number
**Standard deviation shown is in +/- beats per minute
‡
Data retrieved on separate date, September 24, 2007.
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1:50000
Concentration of
NyQuil (bpm)
-24
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For each different concentration, heart rates were recorded before the NyQuil was added
and then after it was added. It was found that the heart rate, on average, decreased 28
bpm ± 22.716 with the addition of the 10-fold dilution of NyQuil. With the addition of
the 1000-fold dilution, it decreased 29 bpm ± 16.523 on average. The heart rate
decreased on average 25bpm ± 9.644 with the addition of 10000-fold dilution of NyQuil.
Lastly, with the addition of the 50000-fold dilution, it decreased 22 bpm ± 14.640 on
average.
Figure 2: Change in heart rate as a function of NyQuil Concentration
Average Change in Heart Rate
(beats/min)
20
10
0
-10000
0
10000
20000
30000
40000
-10
50000
60000
10-fold Dilution
1000-fold dilution
-20
10000-fold dilution
50000-fold dilution
0-fold dilution
-30
-40
-50
-60
Concentration of NyQuil (x-fold dilution)
The average change in heart rate was graphed as a function of the concentration of NyQuil (plotted as the
number of folds of dilution). Data was based on table 2 except for the 0-fold dilution which was based on
the addition of 23 ºC temperature from table 1.
Discussion:
A decrease in heart rate suggests that acetaminophen affected metabolic processes, such as ATP
synthesase, depriving the heart of enough oxygen. Furthermore, it likely sped down the
circulatory system as nerves did not receive signals from neighboring cells as quickly, slowing
down impulses to the heart.
In the first part of the experiment, the average heart rate of daphnia was determined.
Using a single daphnia, the heart rate was recorded ten times to reach an average of 216
beats per minute with a standard deviation of 15.15 beats per minute. A low standard
deviation in relation to the average suggests that the average heart rate accurately
represents the heart rate of daphnia in environments similar to that of the daphnia being
tested. Even though the daphnia was kept in its normal environment (23ºC water),
several factors likely influenced a change in the observed heart rate. For one, light from
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the microscope likely heated the daphnia and, if its duration under the light had been
prolonged, an increase in heart rate would have likely been observed. It is for this reason
that the daphnia was removed from the light for approximately three minutes between
trials five and six so that the daphnia’s normal environment could be maintained. Also,
the daphnia, deprived of sufficient water to allow it to swim, was temporarily
immobilized, thus creating stressful conditions which may have spiked the heart rate.
Adding water helped maintain a normal heart rate.
In the second part of the experiment, changes in daphnia’s heart rate due to temperature
modifications were analyzed. Daphnia were treated with several water samples at
different temperatures and the average changes of heart rate were recorded. With the
addition of 100μL of 4ºC water, the average change in heart rate was -26 beats per
minute. For 15ºC water, the average change was -64 beats per minute. Lastly, the
addition of 37ºC water caused the heart rate to increase 37 beats per minute on average.
With each addition, the heart rate did change to some extent. Adding water colder than
room temperature decreased the heart rate on average and warmer water increased it on
average. This relationship was expected because metabolism will increase as the
temperature rises and chemical reactions take place faster as a result. With an increased
metabolism, the heart rate needs to provide more oxygen to the cell and will thus beat
faster.
An unexpected result was that the introduction of 15 ºC water lowered the heart rate more
on average than the introduction of the 4 ºC water. The heart rate should have decreased
linearly with the decreasing temperature. However, one explanation for this may have be
that the 5 ºC water created such extreme conditions that it created stressful situations for
the daphnia, increasing its heart rate as the temperature lowered. Human error may have
also played a role in addition to the factors mentioned in the discussion of the average
heart rate of daphnia above.
In the third part of the experiment, changes in daphnia’s heart rate due to the addition of
the drug, NyQuil, were analyzed. NyQuil was added in 1:10, 1:1000, and 1:50000
concentrations which resulted in -28, -29, and -22 beats per minute changes in heart rate,
respectively. While the first two concentrations had similar effects, possibly due to error,
the plot of the data shows that decreasing the concentrations decreased the change in
heart rate (made it less negative). For all of the data, however, the drug appeared to act
as a depressant since it lowered the heart rate of the daphnia in all instances. Since it
slowed down the heart rate, the NyQuil likely interfered with the systems and processes
involved in carrying the signal to the cardiac muscle which causes the heart to beat
(Alberts et al. 2004). The circulatory system was likely sped down and, furthermore, the
nerves that generate the impulses did not receive signals from neighboring cells as
quickly. On a molecular level, the metabolism of the daphnia slowed down. With a
decreased heart rate, the blood flow did not provide enough oxygen to the cells. Thus,
specific respiratory processes, such as mitochondria adding oxygen to food to release
energy, decreased (Alberts et al. 2004).
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References:
Alberts, B. Dennis B. Hopkin, K. Johnson, A. Lewis, J. Raff, M. Roberts, K. Walter, P.
2004. Protein structure and function in Essential cell biology, 2nd edition. Garland
Science, New York, NY.
Daughton, C.G., Ternes, T.A. 1999. Pharmaceuticals and Personal Care Products in the
Environment: Agents of Subtle Change? Environmental Health Perspectives. 107: 907 938
Gerritsen, J. 1982. Behavioral response of Daphnia to rate of temperature change:
possible enhancement of vertical migration. Limnology and Oceanography. 27: 254 - 261
Kim, Y., Choi, K., Jung, J., Park, S., Kim, P., Park, J. 2007. Aquatic toxicity of
acetaminophen, carbamazepine, cimetidine, diltiazem and six major sulfonamides, and
their potential ecological risks in Korea. Environmental International. 33: 370-375
Kosinski-Collins, M. 2006. Biology 18b Laboratory Manual. Brandeis University,
Waltham MA.
Pritchard, J.B. 2003. Aquatic Toxicology: Past, Present, and Prospects. Environmental
Health Perspectives. 100: 249-257
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Mitochondria are self-replicating organelles that occur in various numbers, shapes, and
sizes in the cytoplasm of all eukaryotic cells. As mitochondria contain their own genome
that is separate and distinct from the nuclear genome of a cell, they play a critical role in
generating energy in the eukaryotic cell, they give the cell energy by the process of
respiration, adding oxygen to food (typicially pertaining to glucose and ATP) to release
energy
First, it's important to understand how crustacean hearts work. Ethanol must be
interfering somehow with the normal process of heartbeat regulation. So what regulates
crustacean heartbeats? As is the case in most animals, crustacean heartbeats are regulated
primarily by nerve pulses. In the case of crustaceans, these impulses are generated by
pacemaker neurons located in a group of nerve cells called the cardiac ganglion. The
impulses are then transferred to larger follower neurons which carry the signal to the
cardiac muscle, causing it to beat. The University of Calgary has a site describing
crustacean hearts, with a page specifically about the nerve fibers involved.
Somehow ethanol is interfering with the heart rate. The method of action is probably by
interfering with these nerves. (If the quality of the heart beat was affected, we might
consider the cardiac muscle to be the site of action. In this case, however, only the rate is
involved,
so
it's
most
likely
the
nerves
that
are
affected.)
So how does alcohol interfere with nerves? All cells have a type of protein embedded in
their cell membranes called receptor proteins. These receptors allow communication
between cells to occur. Nerves can receive communication from cells far away or from
nerve cells near by. Cells far away secrete hormones, which find their way to the target
nerve's hormone-receptors. Once these hormones attach to the receptor, changes in the
nerve take place. The hormone-receptors, when activated, can change how quickly a
nerve generates an impulse, or alter how fast the nerve passes impulses along. Some
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hormones stimulate an increase in heart rate (such as during exercise) and other
hormones
decrease
heart
rate
(such
as
during
sleep).
Other receptors allow communication between adjacent nerve cells (such as between the
pacemaker neurons and the follower neurons). Instead of hormones, this type of receptor
binds
to
neurotransmitters.
Receptors of any type, however, are not perfect. Sometimes they accidently bind
molecules that they shouldn't. This can cause an inappropriate increase in nerve activity,
or it may cause an inappropriate decrease in nerve activity. (The change depends on the
type of receptor involved, and the type of binding the molecule uses.) The nerve fibers
conducting pulses to the hearts of Daphnia may contain receptors that inappropriately
bind to ethanol (or a product of ethanol after it is broken down) causing an inappropriate
decrease in nerve activity.
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