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:

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 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

The structural formula of a caffeine molecule

The space-filling model of a caffeine molecule

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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.

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, modified post-abdomen, and first legs, which are armed with a hook used in clasping.

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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 heart rate of Daphnia increases.

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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 Daphnia2.2) 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 Daphnia3.3) 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 another2. 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 rate4. Use of microscope to observe Daphnia5. 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.

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4. When carrying the compound microscope, always use two hands, holding it by 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 slide2. 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

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amount of caffeine powder is altered accordingly to obtain the caffeine solutions of respective concentration.

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.

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Observation:1) Table:

The table below shows the different concentration of caffeine solutions (%) and the respective heart rate of Daphnia

Concentration of caffeine solution (%)

The Number of Heart Beats of Daphnia in 15 seconds

Heart rate of Daphnia ( Beats/minute)

Reading 1 Reading 2 AverageControl (pond water of original habitat)

45 44 44.5 178

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.

0 0.1 0.2 0.3 0.4 0.50

50

100

150

200

250

300

350

Graph of Heart Rate of Daphnia Against Concentration of Caffeine Solution

Concentration of Caffeine Solution/%

Hear

t Rat

e of

Dap

hnia

/bpm

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Discussion:1. As the concentration of the caffeine solution increases from 0.0% (control) to

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

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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 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.

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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 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.

11. 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.

12. 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.

13. 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.

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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 of tolerance of Daphnia towards caffeine.

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Benjamin Cummings, 20055. Advanced Biology Principles & Applications Study Guide, CJ Clegg with DG

Mackean, PH Openshaw and RC Reynolds, John Murray (Publishers) Ltd, 19966. 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.shtml10. http://en.wikipedia.org/wiki/Daphnia11. http://en.wikipedia.org/wiki/Caffeine12. http://www.caudata.org/daphnia/

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