Biology Experiment Report

Name: Lee Shun Ming

Class: 9M9

SID Number: 2008203008

Experiment Number: 4

Title: Investigation of Effect of Concentration of Enzyme on Rate

of
Reaction

Date of Experiment: 9/10/2009

Date of Submission of Report:

Objective of Experiment:
 The structural formula of a The space-filling model of a
caffeine molecule caffeine molecule

In 1819, caffeine (IUPAC nomenclature: 1,3,7-trimethyl- 1H-purine- 2,6(3H,7H)-

dione) was discovered by a German chemist, Friedrich Ferdinand Runge. It is a bitter
white crystalline xanthine that acts as a psychoactive stimulant drug and a mild diuretic
in humans and other animals. Caffeine, an odourless and slightly bitter alkaloid, is found
in coffee, tea, kola nuts, ilex plants, and, in small amounts, in cocoa, where it acts as a
natural pesticide that paralyzes and kills certain insects feeding on the plants. It is also
known as guaranine when found in guarana, mateine when found in mate, and theine
when found in tea; all of these names are synonyms for the same chemical compound. It
is most commonly consumed by humans in infusions extracted from the cherries of the
coffee plant and the leaves of the tea bush, as well as from various foods and drinks
containing products derived from the kola nut. Other sources include yerba mate, guarana
berries, and the Yaupon Holly.

Caffeine is absorbed rapidly into the bloodstream from the gastro-intestinal tract
in human whenever consumed. It reaches maximum concentration in circulatory system
within about an hour. The blood distributes it throughout the body. It even manages to
pass through the blood-brain barrier.

Early experiments showed that low concentrations of caffeine may produce small
decreases in heart rate in human, whereas higher concentrations may make the heart beat
abnormally fast. In the brain it constricts the cerebral blood vessels. If one is used to
drinking several cups of coffee a day but quit drinking later, those blood vessels will
dilate, maybe enough to give that person a powerful headache. It is one of the best known
withdrawal symptoms.

In humans, caffeine is a central nervous system (CNS) stimulant, having the effect
of temporarily warding off drowsiness and restoring alertness. It is the most commonly
used mind-altering drug in the world. When used in moderation, caffeine acts as a mild
stimulant to the nervous system, blocking the neurotransmitter adenosine and resulting in
a feeling of well-being and alertness. Also, it is found that caffeine binds to receptors on 391 words
the surface of heart muscle cells which leads to an increase in the level of cAMP inside
the cells (by blocking the enzyme that degrades cAMP), mimicking the effects of
epinephrine (which binds to receptors on the cell that activate cAMP production). cAMP
acts as a "second messenger," and activates a large number of protein kinase A (PKA;
cAMP-dependent protein kinase). This has the overall effect of increasing the rate of
glycolysis and increases the amount of ATP available for muscle contraction and
relaxation. According to one study, caffeine in the form of coffee, significantly reduces
the risk of heart disease in epidemiological studies. However, the protective effect was
found only in participants who were not severely hypertensive (i.e. patients that are not
suffering from a very high blood pressure). Furthermore, no significant protective effect
was found in participants aged less than 65 years or in cerebrovascular disease mortality
for those aged equal or more than 65 years.

Beverages containing caffeine, such as coffee, tea, soft drinks and energy drinks
enjoy great popularity. Caffeine is the world's most widely consumed psychoactive
substance, but unlike many other psychoactive substances it is legal and unregulated in
nearly all jurisdictions. In North America, 90% of adults consume caffeine daily. The
U.S. Food and Drug Administration lists caffeine as a "Multiple Purpose Generally
Recognized as Safe Food Substance".

Excessive intake of caffeine can result in restlessness, insomnia, and heart

irregularities. The effects of caffeine vary from person to person, as people excrete it at
different rates. Physical dependence and unpleasant symptoms upon withdrawal
(headache, fatigue and depression) are common in regular caffeine users.

A study of effect of caffeine in human heart rate will be very valuable. However,
there are many difficulties in conducting an experiment that involves human as “guinea
pig”. Thus, an alternative is to use a model organism to represent human. The results of
the experiment can then be used as a reference to the effect of caffeine on human.
Daphnia are selected as the model as they can be easily handled in many ways in the
investigation.

Daphnia sp. are small, planktonic crustaceans. They, between 0.2 to 5mm in
length, are common freshwater cladocerans, often classified with other
tiny crustaceans as “microcrustaceans.” Cladocerans are commonly
known as water fleas not only due to their salutatory swimming style, but
also because of their resemblance to real fleas, though real fleas are insects and share
only an extremely distant common ancestry with Daphnia, since both crustaceans and
insects are arthropods. Most species in the Order Cladocera are freshwater
species, although there are some marine species. The classification of
Cladocera is as an order within the Subclass Diplostraca within the Class
Branchiopoda within the Subphylum Crustacea. All species of Daphnia occur in
different strains - sometimes the same species can look completely different, both in
terms of size and shape, depending on its origin, and environmental factors at that
location. There are approximately 150 known species in North America, and a similar
number in Europe (many of these species are found on both continents, either through
accidental introduction by man, or nature). Many foreign species have been introduced to
America and Europe from Asia and Africa (the most notorious of which is Daphnia
lumholtzi, which is native to Africa). It is not uncommon to collect 20 or more species in
one small area of lake bottom! Daphnia sp. live in various aquatic environments
ranging from acidic swamps to freshwater lakes, ponds, streams and rivers.
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The figure below shows the anatomy of a typical Daphnia sp.
 In terms of nutritional information, Daphnia have a protein content of around 50%
dry weight and a fat content of 20-27% for adults (4-6% for juveniles), nevertheless some
species have been reported to have a higher protein content. Daphnia tend to be almost
kidney shaped, possessing only a single compound eye (though they have an ocellus, a
simple eye), two doubly-branched antennae (frequently half the length of the body or
more), and leaf-like limbs inside the carapace that produce a current of water which
carries food and oxygen to the mouth and gills. The carapace covers the body, including
the 4 to 6 pairs of thoracic appendages, and is used as a brood chamber. The abdomen
and post-abdomen (distal to the anus) is generally bent forward under the thorax. The
post-abdomen bears two large claws used primarily for cleaning debris out of the
carapace. Swimming is accomplished mainly by downward strokes of the large second
set of antennae. The action of this second set of antennae is responsible for the jumping
motion too. Their bodies are almost transparent and with a microscope the heart beating
can be observed easily, and sometimes even their last meal (the gut may appear green if
the individual has been feeding on algae). As a result they make excellent subjects for the
microscope as one can observe the beating heart. The heart is at the top of the back, just
behind the head, and the average heart rate is approximately 180 bpm under normal
conditions.

Males are distinguished from females by their smaller size, larger antennules, 286 words
modified post-abdomen, and first legs, which are armed with a hook used in clasping.

A few Daphnia prey on tiny crustaceans and rotifers, but most are filter feeders,
ingesting mainly unicellular algae (e.g. Chlamydomonas sp.) and various sorts of organic
detritus including protists and bacteria. In the summer months, they can often be seen
"blooming" in ponds and lakes as the concentration of algae builds up. They also eat
forms of yeast, but mostly in laboratories or controlled environments.

The life span of Daphnia, from the release of the egg into the brood chamber until
the death of the adult, is highly variable depending on the species and environmental
conditions (Pennak, 1978). Typically, the lifespan of a Daphnia does not exceed one year
and is largely temperature dependent. It is discovered that the life span increases as
temperature decreases, due to lowered metabolic activity.

Daphnia reproduce by parthenogenicity (the ability to self-replicate without

fertilisation of any form), during the late spring, summer and early autumn (depending on
temperature, food availability and presence of waste products of their metabolism). The
entire race is made up of females during this period. Developing embryos are often
visible in the mother's body without the aid of a microscope. However, when food is
scarce, some eggs develop into males and the females produce eggs that must be
fertilised. These eggs develop into small embryos which then go into suspended
animation, and are shed with the carapace as dark brown/black saddle-shaped cases
known as ephippia. These can survive harsh conditions. When conditions improve again,
the egg producing generations begin producing live young once again (all females), and
the male sex dies out completely until it is needed when conditions worsen once again.

Once thought of as an animal of polluted waters, Daphnia have been proven to be

very sensitive to poor water conditions and a number of research and industrial groups
use Daphnia to test water quality. For example, they are very sensitive to halide
concentration, like the chloride or fluoride in tap water, which are extremely toxic to
Daphnia. They are also sensitive to metal ion concentration, like sodium, potassium,
magnesium and calcium, which in increased concentrations can cause immobility and
death, and Daphnia are extremely sensitive to copper, zinc and most dissolved toxins
(e.g. dichromate ions). They are often used to monitor water quality so that only safe
water is released into the environment by industry and water treatment plants.

Similar to many animals, Daphnia sp. are prone to alcohol intoxication, and make
excellent subjects for studying the effects of the depressant on the nervous system – due
to the translucent exoskeleton, and the visibly altered heart rate. They are tolerant of
being observed live under a cover slip and appear to suffer no harm when returned to
open water. In this experiment, it is intended to study the effect of different concentration
of caffeine solutions on the heart rate of Daphnia.

Problem Statement:
What is the effect of caffeine on heart rate of Daphnia?

Hypothesis:
There is a correlation between concentration of caffeine solution and the heart rate of
Daphnia. In this experiment, when the concentration of caffeine solution increases, the 518 words
heart rate of Daphnia increases.

Aim:
The aim of this experiment is to study the effect of caffeine on heart rate of Daphnia

List of Materials and Apparatus:

The materials and apparatus needed for this experiment are culture of Daphnia, petri
dishes, cavity slide, dropping pipettes, distilled water or pond water, caffeine powder,
cotton wool or gauze or muslin cloth, beakers, spatula, stopwatch, tissue paper or filter
paper, electronic mass balance, scientific calculator and light microscope.

Variables:
1. Manipulated variable: Concentration of caffeine solution
1.1) Manipulating the variable: Using different amount of caffeine powder to prepare
five caffeine solutions of different concentration, that is 0.1%, 0.2%, 0.3%, 0.4% and
0.5% concentration of caffeine solution.
2. Dependant variable: The heart rate of Daphnia
2.2) Recording the variable: The beating of the legs of Daphnia is actually proportional
to the heart rate of Daphnia. Observing the Daphnia under the light microscope, the heart
rate of Daphnia is recorded by counting the beatings of the legs of Daphnia in 15
seconds.
3. Constant variable: Temperature of the environment of Daphnia, time of recording
heart beat, type of Daphnia used, pH of the environment of Daphnia
3.3) Controlling the variable: Throughout the experiment, the experiment is conducted in
an air-conditioned room of temperature 28oC. Every recording of heart beat of Daphnia is
done in 15 seconds. Only Daphnia (sp.) is used throughout the experiment. Throughout
the experiment, the Daphnia are kept only in pond water or mixture of pond water and
caffeine solution.

Technique:
In this experiment, five techniques are applied to conduct this experiment. The five
techniques are as follow:
1. Transferring of Daphnia from a container to another
2. Placing Daphnia on a cavity slide and immobilising it by using cotton wool or
gauze or muslin cloth.
3. Counting the beat of the leg of Daphnia to identify the heart beat rate
4. Use of microscope to observe Daphnia
5. Programming a scientific calculator to count heart beat of Daphnia.

Precautionary Steps:
Before handling the experiment, the safety precautions must be prioritized. The
precautionary steps that should be taken in this experiment are as follow:
1. During the experiment, lab coat should be worn to prevent chemicals from
spoiling clothes.
2. Eye protection/goggles and gloves should be worn to protect the eyes and skin.
3. The compound microscope should be handled with great care for they are
precision instruments and it is expensive to replace. After using it, make sure it is
stored under cover or in a box to protect it from dust.
4. When carrying the compound microscope, always use two hands, holding it by 417 words
the limb and under the “feet” (base); put it down the bench workstation without
jarring. (Never carry microscope by the microscope tube or stage)
5. Keep the lenses and working surface (the stage) clean and dry, uncontaminated by
reagents, stains and fixatives. Take special care to avoid contact between the
lenses and the fingers of the experimenter (and later, with any of the solvents,
stains and mounting fluids in use).
 6. Lenses and the mirror should be wiped only with lens tissue.
7. Do not use direct sunlight to illuminate the mirror or condenser lens of the
microscope, since sunlight when focused on to the retina may damage the light-
sensitive cells there.
8. Take care when using electrical equipment like microscope light near to water to
prevent from being electrocuted.
9. Take care when clipping the cavity slide to the stage of the microscope to prevent
breakage.
10. If a stroboscope is used to show the Daphnia’s heart rate and the experimenter
knows he/she suffer from photosensitive epilepsy, the lecturer should be informed
of this and appropriate precautions be taken.
11. The caffeine solution is ensured not too concentrated so that the Daphnia does not
die of it.
12. Each experiment of different concentration of caffeine solution ought to be
conducted as fast as possible for there is a risk for the Daphnia to die in a new
environment.
13. The Daphnia should be place back into the petri dish which contains pond water
after each experiment of different concentration of caffeine solution so that it
restores to its normal heart rate before it being used for another experiment.
14. The Daphnia used should be handled with care to avoid it from injury and thus
affect the results of the experiment.
15. Glassware like beaker and cavity slide must be handled carefully to avoid
breakage.

The steps mentioned above are the precautionary steps to be taken. Otherwise, good
laboratory practice is sufficient to take account of any hazards and avoid significant risks.

Risk Factors:
There are some risk factors in this experiment. They are as follow:
1. Breakage of cavity slide
2. Suffering from photosensitive epilepsy (only if stroboscope is used in the
experiment)

The risk of mentioned above can be minimised by handling the materials and apparatus
with extra care.

Procedure:
1. Caffeine solutions of concentrations of 0.1%, 0.2%, 0.3%, 0.4% and 0.5% are
prepared using caffeine powder and distilled water. To prepare 0.1% caffeine
solution, 0.1g of caffeine powder is dissolved in 100ml of distilled water. The
amount of caffeine powder is altered accordingly to obtain the caffeine solutions
of respective concentration.
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2. A light microscope with microscope light is set up. A low power objective lens of
40x magnification is selected.
3. One Daphnia is selected and scooped cautiously from the container using a petri
dish along with moderate amount of water.
4. The Daphnia is transferred from the petri dish onto a cavity slide, by using a
spatula, along with a small amount of pond water.
 5. A few strands of muslin cloth are placed on the Daphnia, so that to make sure that
it is held in position and does not move while it is being observed under the
microscope. Some water is inevitablely absorbed. One or two extra drops of water
is be added to prevent the Daphnia from dying.
6. The cavity slide is placed onto the microscope stage and held in position using
stage clips.
7. The microscope is focused by adjusting the coarse focusing knob and fine
focusing knob until a clear image of Daphnia is observed. The position of cavity
slide is adjusted until the heart of Daphnia can be observed clearly. In case of
unable to locate the heart, the legs of the Daphnia are located. The beating of the
legs of Daphnia is known to be proportional to the heart beat.
8. One student from the group is assigned to observe the Daphnia using a
microscope and count the number of heart beat or leg beat made by the Daphnia
within 15 seconds. A scientific calculator is programmed so that every successive
tapping of the “=” button records one heart beat or beating of legs. Another
student is assigned to record the time by using a stopwatch. This step is repeated
again to obtain a second reading. The average of value of the readings are worked
out.
9. The Daphnia is returned to the petri dish which contains fresh pond water for one
minute.
10. Steps 4 to 9 are repeated for five times by adding caffeine solution of
concentrations 0.1%, 0.2%, 0.3%, 0.4% and 0.5% to the cavity slide respectively.
11. Each average value of heart beat of Daphnia is multiplied by four to obtain the
value of heart beat of Daphnia in unit of beats per minute. The results of the
experiment are tabulated and a graph of the heartbeat of Daphnia per minute
against the concentration of caffeine solution is drawn.

Observation:
1) Table:
The table below shows the different concentration of caffeine solutions (%) and the 407 words
respective heart rate of Daphnia

Concentration of caffeine The Number of Heart Beats of Heart rate of

solution (%) Daphnia in Daphnia
15 seconds ( Beats/minute)
Reading 1 Reading 2 Average
Control (pond water of 45 44 44.5 178
original habitat)
0.1 54 56 55 220
0.2 74 73 73.5 294
0.3 80 77 78.5 314
0.4 75 76 75.5 302
0.5 60 59 59.5 238

2) Graphs:
The graph below shows the Graph of Heart Rate of Daphnia Against Concentration of
Caffeine Solution.

Discussion:
1. As the concentration of the caffeine solution increases from 0.0% (control) to 100 words
0.3%, the graph shows an upward trend. After concentration of 0.3%, further
increase in concentration of caffeine solution, that is 0.4% and 0.5% of caffeine
solution, results in a decrease in heart rate of the Daphnia.
2. As the concentration of caffeine increases, heart rate of Daphnia increases for
caffeine concentration up to 0.3% because caffeine is a stimulant which raises the
heart rate of animals. Assumption that a similar effect would result in human can
be made.
3. However, there is a drop in the heart rate for 0.4% and 0.5% of caffeine solution.
This is an anomaly shown in the experiment. Reference to other research papers
and the results from other students doing the same experiment as well as
consultation to experienced lecturers reveal that the heart rate of the Daphnia
should increase from 0.0% up to 0.5% of caffeine solution. This is because 0.0 –
0.5% of caffeine solution is still within the concentration range which a typical
Daphnia can tolerate. The probable explanation to this occurrence is that the
Daphnia selected for this experiment is a weak one and thus has a narrower
concentration range which it can tolerate, most probably from 0.0-0.3%. Another
explanation to this anomaly is that throughout the experiment, the same Daphnia
is used instead of using fresh Daphnia for each different caffeine solution of
different concentration. Due to the fact that caffeine is primarily an antagonist of
the central nervous system's receptors for the neurotransmitter adenosine, the
body of Daphnia which regularly consume caffeine adapt to the continual
presence of the drug by substantially increasing the number of adenosine
receptors in the central nervous system. This increase in the number of the
adenosine receptors makes the body much more sensitive to adenosine.
Consequently, the stimulatory effects of caffeine are substantially reduced, a
phenomenon known as a tolerance adaptation, thus the heart rate reduces even
though the concentration of caffeine solution increases.
4. The experiment for each concentration of caffeine solution is repeated twice in
order to obtain higher accuracy. Random errors could be minimised in this way.
5. The anatomy of Daphnia is revised before carrying out the experiment to ensure a
higher efficiency while carrying out the experiment.
6. The Daphnia should be handled with extra care as it is a very delicate creature.
Muslin cloth is used in holding the Daphnia in position. This makes the Daphnia
unable to move when observing it under the microscope, thus make the counting
of heart beat or the beating of the legs easier.
7. Excess water is removed by using filter paper from the cavity slide as too much
solution will result in vigorous movement of Daphnia. Counting of heart beat or
beatings of the legs would then be more difficult.
8. There is an alternative in counting the heart beat or beating of legs of Daphnia, if
no scientific calculator is available. It is by tapping a pencil on a piece of paper
and count up the pencil marks at the end of the time period. Obviously, this
method is more primitive and errors are more likely to occur because some pencil
marks may be too close together and cause confusion in counting the number 505 words
pencil mark. However, in the absence of a scientific calculator, this method can be
applied as it is better than counting the heart beat or beating of legs of Daphnia
mentally.
9. The limited caffeine concentration for Daphnia is about 1%. That means high
concentration of caffeine solution should not used for Daphnia may die of it.
Hence, the highest concentration of caffeine solution used in the experiment is
only 0.5%.

10. There are a few unavoidable limitations in this experiment which may affect the
accuracy of the results. The following are the limitations and their respective
improvements:
• Due to the scarce availability of Daphnia in the laboratory, the same
Daphnia is used throughout the experiment. As mentioned above, this may
cause the Daphnia to develop a tolerance towards caffeine and thus affects
the results in the experiment. To improve this, sufficient supply of
Daphnia must be prepared to be used for the experiment.
• Temperature of the environment of the Daphnia is one of the factors that
affects the heart rate of it. Prolong exposure of the Daphnia to the
microscope light will overheat the Daphnia. To prevent the Daphnia from
overheating, the microscope light should be turned off between
observations and a heat sink should be used.
• Heart beat or beating of legs of the Daphnia can be very fast when it is in
0.3-0.5% of caffeine solution. Missing a few beats when counting the
heart beat or beating of legs is almost inevitable. An improvement to this
is to set a video camera above the eyepiece so that the heart beat of
Daphnia is recorded. Later, the video can be played in slow motion with
the timeline of the video displaying on the screen. This helps to obtain a
more accurate heart beat rate. Alternatively, an ICam can be placed above
the eyepiece of the microscope to project the image of the slide onto a
large screen to help with counting. In the Journal of Biological Education
(1997) 31, pp. 253-255 by Foster, a method of using a stroboscope to
freeze the motion is suggested. The use of stroboscope may overcome the
problems of counting faster heart rates. When the frequency of the
stroboscope is in phase with the heart beat, it looks as if the motion of
 heart is frozen. However positioning the light sources and strobe is tricky.
It is very difficult to freeze the motion and viewing with a strobe light can
cause eye strain and dizziness. This increases the risk of the experiment.
Therefore, this method is not used in this experiment.
• Besides the use of muslin cloths to restrict the movement of the Daphnia,
there is an alternative way of restricting the movement of the Daphnia.
Some plasticine can be used to form a barrier around the depression of the
cavity slide so that the Daphnia is confined to the space in the depression
of the cavity slide.
• The Daphnia may be hurted and injured while it was being transferred
from one container to the other. This may affect the experimental results.
The only step can be taken is to handle the Daphnia carefully to avoid
injuring it.
• Since the Daphnia is not observed under its natural habitat, its stressed 531 words
level in the experiment may be higher than usual. This might cause
fluctuations in its heart rate or an undesired change in the heart rate. The
only thing to be done is to count the heart beat or beating of legs of the
Daphnia as soon as it is ready to be observed.
• The results of the experiment would be more accurate and valid if a “blind
study” is done. This is because the experimenter who is counting the heart
beat of the Daphnia is unaware of the concentration of caffeine in the
solution it is in. This can prevent bias due to the observer’s expectations.
Studies have shown that observer’s expectations can significantly
influence the results.

1. Daphnia are selected as the model in this experiment as they are fairly easy to
keep and the transparent body allows easy observation of changes in heart rate
under a microscope without having to dissect it. On the minus side, experimenters
have to be extremely cautious when dealing with them because they are delicate
and vulnerable. In addition, Daphnia is small and delicate as well as belongs to a
different family and phylum from human. It ought not to be assumed that 0.5%
caffeine concentration will induce the same trend of change in human heart rate.
On top of that, researches show that upon consuming a modest amount of
caffeine, human heart rate could be decreased slightly. What is more, Daphnia
have neurogenic hearts, while human have myogenic hearts. Thus, in drawing
parallel the results of this experiment to human, the information mentioned above
should be considered.
2. It should be understood that any experiment involving living organism always
rises up bioethical issues. While performing experiment involving living organism
can bring greater advances in science, it should not be forgotten that the well-
being of the living organisms involved in the experiment should be taken care of,
so that they do not suffer for the experiment or at least their sufferings are
minimized. Therefore, the Daphnia being used in the experiment ought to be
treated with great care to avoid hurting or even killing them. If possible, the
Daphnia should be returned back to their natural habitat after the experiment so
that they can return to their normal lives. This step also avoids any disruption in
the food chain which may affect the ecosystem.
 3. Besides concentration of caffeine solution, factors like surrounding temperature,
amount of alcohol and amount of some drugs such as aspirin can affect the heart
rate of Daphnia. If possible, all these factors can be tested to study how they
affect the heart rate of Daphnia. Daphnia are very sensitive to poor water
conditions too. Further studies can be done by testing how the different pollutants
in the water affects the heart rate of Daphnia.

Conclusion:
Generally, when the concentration of caffeine solution increases, the heart rate of
Daphnia increases. Prolong exposure of Daphnia to caffeine can result in the building up 528 words
of tolerance of Daphnia towards caffeine.

References:
1. Edexcel AS Biology, Students book, Pearson company, 2008
2. Edexcel Biology for AS, CJ Clegg, Hodder Education, 2008
3. Text PRE – U Biology Volume 1, Lee Ching, PEARSON Longman, 2008
4. International Edition Biology Seventh Edition, Campbell, Reece, Pearson
Benjamin Cummings, 2005
5. Advanced Biology Principles & Applications Study Guide, CJ Clegg with DG
Mackean, PH Openshaw and RC Reynolds, John Murray (Publishers) Ltd, 1996
6. Carpenter, SR, & Kitchell, JF (1993) The Trophic Cascade In Lakes. Cambridge
University Press, London, England.
7. Davidson College Department of Biology (1999)
http://www.bio.davidson.edu/index.html.
8. Rith, J (1988) Plant succession on abandoned railways in rural New York State.
Proceedings of the 73rd Annual Meeting of the Ecological Society of America,
Davis, CA.
9. www.sciencebuddies.org/mentoring/project_ideas/Pharm_p009.shtml
10. http://en.wikipedia.org/wiki/Daphnia
11. http://en.wikipedia.org/wiki/Caffeine
12. http://www.caudata.org/daphnia/

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