Daphnia Bioassay Essay Research Paper Plan The

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Daphnia Bioassay Essay Research Paper Plan The

Daphnia Bioassay Essay, Research Paper

Plan The initial variable to be considered will be the consequence that caffeine and temperature has on the bosom round and if it has a direct or indirect consequence on the water flea & # 8217 ; s lifetime. If there is adequate clip to see a 2nd variable nevertheless, the effects of a temperature or intoxicant on the water flea & # 8217 ; s organic structure system will besides be considered alongside the initial variable of caffeine. These will so be slotted into the same graph which will demo us how the figure correspond to the consequences.

In order to carry on a true experiment the undermentioned inquiries must be considered and answered and merely so can acurate decision be drawn.

& # 183 ; How variable is the bosom rate in an single Daphnia?

& # 183 ; How does the drug caffeine affect the water flea & # 8217 ; s bosom round?

& # 183 ; At what concentration is the bosom rate slowest? The fastest?

& # 183 ; What indicant in the graph is the optimal concentration for bosom rate of Daphnia?

& # 183 ; What is the significance of the form of the graph line when the information is plotted?

& # 183 ; & # 183 ; What would go on if you increased the temperature in 5C increases ( if clip is available ) ?

& # 183 ; What would go on if intoxicant was added into the solution and why would this impact the water flea & # 8217 ; s bosom rate and general behaviour. To to the full understand the manner the water flea works one must analyze its organic structure and the utilizations for the different parts of it: – ( fig.1 ) [ 1 ] & # 8211 ; Second aerial, used for swimming and feeling the environment [ 2 ] & # 8211 ; oculus controlled by musculuss with nervus connexions to the encephalon [ 3 ] & # 8211 ; legs used for garnering nutrient stabilising the animate being as it easy sinks [ 4 ] & # 8211 ; An bowel where land up nutrient atoms are digested [ 5 ] & # 8211 ; A brood pouch for incubating immature that hatch from big yolk filled eggs [ 6 ] & # 8211 ; A protective outer shell [ 7 ] & # 8211 ; A heartthat pushes clear circulatory fluid around the organic structure [ 8 ] & # 8211 ; Undigested stuff is eliminated out the anus.

As it is now seen that the water flea incorporates its nutrient through the usage of its legs it could explicate why the drugs such as intoxicant or caffeine take slightly longer to come in its encephalon and change its bodily functions-also its organic structure acts as a signifier of a semi-permeable barrier of which little molecules ( such as caffeine & # 8216 ; C8H10O2N4H20 & # 8242 ; ) can perforate the walls of its internal variety meats, this is why it penetrates the whole of the water flea & # 8217 ; s organic structure at one time ( see & # 8216 ; anticipation & # 8217 ; for information relating to this fact ) . If temperature is to be used as a chosen variable, it must be considered that water flea are inhuman animate beings, they do non thermoregulate. This means that their organic structure temperature is the same as the H2O they are drifting in. The chemical reactions that occur in the cells of Daphnia are dependent on certain enzymes, or proteins, to assist the reactions proceed. As you increased the temperature of the H2O, the metamorphosis of the Daphnia increased every bit good, because chemical reactions occur faster at higher temperatures. This means that the bosom rate will rush up in order to supply O to the cells as the metamorphosis additions. However, at 40 grades Celsius, the enzymes break down, and the chemical reactions can no longer happen, so metamorphosis Michigans and the Daphnia dies. In its natural environment, Daphnia does non of all time experience temperatures of 40 grades, so this is improbable to happen. However, they do see alterations in temperature in their natural environment, and their metamorphosis does increase or diminish as the temperature alterations.

Method I have ab initio decided to see how the drug caffeine affects the pulse and lifetime of a water flea, A water flea will be extracted from a pool nearby, so the status of the water flea will non be altered excessively drastically during conveyance. These water flea will be allowed to set to their new milieus and so be taken out through the usage of a pipette and placed in a petri dish incorporating the relevant sum of caffeine/alcohol dressed ore solution ( aq ) . This water flea will be left in the solution for a minimal clip of one minute before any kind of consequences are taken ( for this experiment one and a half proceedingss were used ) this is to let the water flea to set to its new milieus and its semi-permeable barrier to in-secrete the environing solution, although when making this, it must be remembered that if left excessively long, the water flea will hold adjusted itself and the pulse will get down to modulate once more. Once the period of one minute 30 seconds has elapsed, a & # 8216 ; clicker counter & # 8217 ; will be used to number the figure of beats of the water flea & # 8217 ; s bosom in one minute ( evidently a right focussed microscope will be necessary to see the water flea & # 8217 ; s bosom and how it beats ) for jobs associating to this method please see & # 8216 ; rating & # 8217 ; . The concentration of alcohol/caffeine will so be varied to see how this affects the ordinance the water flea & # 8217 ; s pulse. ( the water flea & # 8217 ; s bosom is illustrated in the undermentioned diagram fig.2 ) . In an ideal lab status the program would be such as this, although the clip period and equipment are limited so this method will be followed every bit close as is possible: –

1. Prior to carry oning the bio-assay, look into the Daphnia to guarantee the civilization is healthy. 2.select trial being. Although you may utilize assorted age populations for bio-assaies, it is better to utilize merely immature persons in order to minimise biological differences among the trial being. Because the visual aspect of resting eggs indicates a hapless civilization environment, do non utilize Daphnia with resting eggs. To obtain a good supply of immature Daphnia, get down 24 hours in progress by taking females bearing embryos from the stock civilization and puting them in 400-mL beakers incorporating 300 milliliter of spring or watercourse H2O and the appropriate sum of nutrient. Five beakers, each incorporating 10 grownups, normally will provide adequate immature persons for one toxicity trial. When you are ready to get down the bio-assay, choose immature ( little ) Daphnia from these civilizations.

3. Introduce the same figure of newborns ( at least 10 ) into each trial vas and control utilizing a plastic, disposable pipette with a 5-mm diameter. Be certain to let go of the immature below the surface to avoid killing them by pin downing air under their shells.

4. Fix a tabular array that records the per centum concentration, the clip, and the entire figure of dead at each clip interval. As near to one hr as possible, but prior to the terminal of the period, record the figure dead at each concentration. Remember Daphnia moult to turn, so newborns will shed as they develop. Therefore, do non number the moult castings, which appear as clear shells of the Daphnia on the underside of each cup. Remove dead Daphnia and molt castings at each monitoring interval.

5. Ideally it is best to look into once more in 4 hours and once more after 24 hours. If agendas do non allow ( such as ours holding to be completed in a one hr 45 minute clip period ) , Must non feed animate beings during trials. Steps 4-6 will take about 15 proceedingss of two back-to-back category periods.

6. At the terminal of the bio-assay, prove the H2O to find the pH, hardness, and dissolved O content. Count and enter how many Daphnia in each dish have died, so analyze the data.. Apparatus Equipment

dissecting microscope

H2O chemical science trial kits ( pH, hardness, dissolved O )

Light ( from lamp )

petri dishes

salt solution ( NaCl )

clicker counters Materials

5mm diameter pipette or oculus dropper



microscope slide

1 milliliters pipette with.01 divisions


spring H2O or uncontaminated watercourse H2O

Daphnia magna civilization, assorted age

Rubs or other tissues

nutrient: possibilities include Roti-Rich dried barm, or unicellular algae such as Selenastrum Diagram

Daphnia Size & # 8211 ; There is a large size difference in the Daphniidae,

depending on the species. Newly hatched Moina are somewhat

larger than freshly hatched seawater runt, and twice every bit large as

mean grownup rotifers, but freshly hatched Daphnia are twice as

large as Moina, and may non be suited for some of the smaller

fish Fry because of their size. Life rhythm of Daphnia & # 8211 ; The water flea has both sexual and

nonsexual stages. In most environments, the population consists

wholly of females that reproduce asexually. Under optimum

conditions, a female may bring forth more than 100 eggs per

brood, reiterating every 3 yearss. A female may hold every bit many as

25 broods in its life-time, but the norm is about 6. The

female will get down to reproduce at approximately 4 yearss old with a brood

size of 4 to 22 eggs. Under inauspicious conditions, males are

produced, and sexual reproduction begins. The consequence is the

laying of resting eggs, merely like the seawater runt. Factors that

can trip this are a deficiency of nutrient, low O supply, a high

population denseness, or low temperatures. Nutritional Value & # 8211 ; The nutritionary content of Daphnia

varies with age, and what its been eating. The protein content

is normally about 50 % of dry weight. Quite the antonym from

Artemia, grownups usually have a higher fat content than

juveniles, about 20-27 % for grownups, and 4-6 % for juveniles.

Some species have been reported to hold protein contents

transcending 70 % . Live Moina are about 95 % H2O, 4 % protein,

0.54 % fat, 0.67 % saccharides, and 0.15 % Ash.

The fatty acerb composing of nutrient is of import to the endurance

and growing of fish Fry. Omega-3 extremely saturated fatty acids

are indispensable for many species of fish. Moina cultured on

bakers yeast are high in monoenoic fatty acids. By utilizing what

is called w-yeast ( yeast enriched with cuttlefish oil ) , Moina

will incorporate really high degrees of Omega-3 fatty acids. Moina can

take up lipoids really easy from the emulsion, but there is a

side consequence to this, seemingly it besides slows productiveness, so

this emulsion should merely be fed to a batch offprint from the

chief growout settlement. Commercial expressions are available in pet

supply houses for the enrichment of Artemia, Rotifer, and

Daphnia civilizations. Physical Requirements & # 8211 ; Salinity & # 8211 ; Daphnia are typically freshwater beings, but,

some are found in somewhat brackish H2O. Some species have

been observed in salts up to 4 ppt, and salts of 1.5 to

3.0 ppt are common in pool civilizations in the East. Oxygen & # 8211 ; Daphnia are by and large tolerant of hapless H2O

quality, and dissolved O varies from about zero to

supersaturation. Like the Brine Shrimp, their ability to last

in an O hapless environment is in their ability to synthesise

haemoglobin. The production of haemoglobin may be promoted by

high temperatures, and a high population. Besides, like seawater

runt, Daphnia are non tolerant of all right air bubbles. A slow

aeration is needed with Daphnia as a big bubble column will

deprive the Daphnia out and kill them. pH and ammonium hydroxide & # 8211 ; A pH between 6.5 and 9.5 is acceptable.

High ammonium hydroxide degrees, with high pH will drastically cut down

reproduction, but will non impact the existent wellness of the

animate beings themselves. So it seems that on the little graduated table that

we require, monitoring of pH and ammonium hydroxide is non critical to

success. Dissolved minerals & # 8211 ; In contrast to their tolerance of low

O, Daphnia are really sensitive to perturbations of the Attic

composing of their environment. They become immobile and

finally die with the add-on of salts like Na,

K, Mg, and Ca. Low concentrations of

P ( less than 0.5 ppm ) will excite reproduction,

but concentrations higher than 1.0 are lethal to the immature.

Daphnia magna are rather immune to phosphorus and can

withstand concentrations every bit high as 5-7 ppm. Daphnia are non

affected by the add-on of N in fertilisers for the

publicity of algae growing. As with any aquarium venture, the

H2O used should be treated with aeration or de-chlor to

take Cl before the civilization is started. Concentrations

of merely 0.01 ppm Cu will ensue in decreased motion in

Daphnia. They are highly sensitive to metal ions like

Cu and Zn, pesticides, detergents, bleaches and other

dissolved toxins. Municipal and good H2O may be

contaminated adequate to kill the civilization. The best beginning of

H2O is filtered watercourse or lake H2O, rain H2O collected

for low air polluted countries, or, use the H2O from your

fish tank H2O alterations. Temperature & # 8211 ; Daphnia have a broad tolerance to

temperature. The optimal temperature for Daphnia Magna is

18-22 deg C ( 64-72 F ) Moina withstand extremes even more,

defying day-to-day fluctuations of 5-31 deg C ( 41-88 F ) ; their

optimal being 24-31 deg C ( 75-88 F ) . The higher temperature

tolerance of Moina make this species a better pick where

temperatures may lift above the comfort degrees for Magna at

certain times of the twelvemonth.

Prediction To make a sensible anticipation, it must foremost be known how daphnia react in their milieus this background cognition will doubtless assist when trying to do decisions and/or anticipations associating to the water flea & # 8217 ; s bodily systems and reactions. Daphnia typically live 40-56 yearss. This varies harmonizing to species and environmental conditions. Each brood typically holds 6-10 eggs, which turn into embryos and are released within a few yearss. Juveniles reach sexual adulthood in 6 to 10 yearss.

Daphnia is a little Crustacean & # 8211 ; hardly seeable with the bare oculus. It lives in H2O and has big aerials in comparing with the remainder of its organic structure. These are used to do ( jumpy ) movements & # 8211 ; hence the name & # 8220 ; H2O flea & # 8221 ; .

Daphnia & # 8217 ; s seem to function two intents:

( 1 ) they are really popular fish foodespecially because it is rather easy to maintain them in civilization, they reproduce at high rates and they keep the fish tank H2O clean

( 2 ) aschanges in the bosom rate might propose a chemical compound has some physiological consequence, and & # 8211 ; more significantly & # 8211 ; Daphnia magna is used to mensurate the toxicity of a chemical compound in H2O ( LD50 measurings ) .

From a sciencepoint of position, Daphnia is non intensively studied. In kernel, a few ATPase fractional monetary units and ribosomal RNA cistrons are sequenced.

Besides under probe is the nature of this species unusual pigmentation. water flea are semitransparent and are normally amber or devoid of colour. These dune water flea, when found in their natural environment, possess a dramatic melanic pigmentation. It has been hypothesized that the melanosis serves as a pho

toprotection device, but it is possible that it besides serves as a defensive protection from the predatory copeopods which besides inhabit these pools. In a survey in which melanic signifiers of water flea were found in north-polar pools a strong correlativity was found between whether or non the pool was surrounded by flora and the extent to which melanic signifiers were present. It was found that in all instances, melanic signifiers were present in pools which were devoid of environing flora. It was hypothesized that organic alimentary overflow from local zoology filters out harmful UV radiation within the first few inches of H2O. Ponds missing this natural UV protection therefore harbored the pigmented signifiers. Indeed the pools found near Florence are non merely devoid of any flora, but they are besides shallow, reenforcing the demand for photoprotection

These pools don & # 8217 ; t resurface in the same topographic point every twelvemonth, because the littorals displacement well throughout the summer after the pools dry up. If there is a unvarying sum of familial fluctuation between all pools, it is likely that the air currents blow the diapausing embryos around, blending up the cistron pools each twelvemonth. If the sum of fluctuation within a pool is low, it is likely that it was founded by one or a few hatched epphipia. If there is a high sum of fluctuation, what maintains it? It is a good known fact that fluctuation within populations is perfectly necessary for species to accommodate in a changing environment. Because of the built-in instability of dune ecosystems, and the evident singularity of this species of water flea, could really be covering with an endangered species. Caffeine is a drug stimulation which acts straight upon the CNS ( cardinal nervous system ) and encephalon which due chiefly to the water flea & # 8217 ; s mass and size, a less sum of each substance ( caffeine or intoxicant ) would consequence the water flea & # 8217 ; s organic structure than the human organic structure, this could demo the relation, & # 8216 ; the greater the mass the more of the drug needed to impact the organic structure system & # 8217 ; . Therefore I predict that as the concentration of caffeine increases the pulse will increase as it does. Drink such as & # 8216 ; java & # 8217 ; or & # 8216 ; coca Cola & # 8217 ; incorporate a batch of caffeine which scientist study are linked to high BP ( blood force per unit area or high blood pressure ) rates. Limited research has stated that there is sucha nexus with heavy java ingestion with high BP, all these can be linked back to the water flea who & # 8217 ; s bodily systems are based upon the same basic rules of endurance.

Due to the fact that intoxicant is a sedative, slightly of a contrary of the effects that caffeine would so make, I predict that as the concentration of the intoxicant increases the pulse of the water flea would go less until it stops & # 8211 ; the concentration at which the bosom stops and the water flea dice is a inquiry needed to be answered in this bio-assay. Due to these anticipations, I do so believe that a graph incorporating both would look like this: –

( fig 3 ) The pulse may go irregular with high dose due to the fact the water flea & # 8217 ; s little mass and surface country would fight badly to get by with the alteration in environment and this abnormality could possibly ensue in decease

The higher the concentration of caffeine the higher the bosom rate will go. After a few proceedingss the water flea will go accustomed to the environment and bosom round will probably modulate & # 8211 ; this is retrieving that it is non overly high concentration for the water flea to decease. This would be due to the fact that they are cold blooded animate beings and respond consequently to their milieus. Safety This is ever a concern when working with any sort of toxin so points such as the undermentioned must be maintained to maintain working safely: –

When heating H2O guarantee all usual safeguards are applied to Bunsen burner use

Do non pollute water flea & # 8216 ; place & # 8217 ; H2O with any substance.

Fair Test/Failures To make a higher degree of truth when obtaining consequences, the clicker counter was used for 20 seconds and multiplied by 3 to acquire the consequences for one minute.

This was done three times and an norm was taken to make more acurate consequences

Preliminary consequences were besides taken merely to guarantee all anomalousnesss will be seen and accounted for.

An effort is made to utilize the same water flea for all experiments as water flea & # 8217 ; s vary in size and metabolic rates therefore this must hold every effort made to be avoided. Although on occasion when utilizing high concentrations, decease is ineluctable.

Besides the age of the water flea can hold some consequence, although some merely live a affair of proceedingss, like any other animate beings, their metabolic rates vary depending on age, size and features.

With the equipment in usage, it is more or less impossible to cognize whether the water flea is sick or in a hapless province, so if luckless an sick water flea could change the full bio-assay. Further equipment such as highly acurate thermic investigations, or other physical appraisal tools would be necessary if carry oning an highly acurate bio-assay.

Although the female water flea population is in surplus of the male, if a male water flea was taken for the experiment it would do consequences differ due to the fact that It is hard to separate male and female Daphnia. They are fundamentally indistinguishable except that males are by and large smaller in size, have larger antenules, and the first legs have a stout hook used in clasping the female during copulating. Rather than seeking to place males to observe nerve-racking environments, it is much simpler to inspect the brood Chamberss of females ) . Ie. If a male water flea was used, the consequences would differ greatly.

Daphnia emphasis degrees can besides change consequences, in every bit much as if the water flea had a high blood force per unit area ( hyper tenseness ) caffeine would rise this and alcohol lower it, although get downing from a plane incorrect to the necessary norm.

Besides factors such as the heat from the lamp raising the temperature must be considered as it is known that reactions with enzymes occur faster when the temperature is risen.

If a petri dish, or pipette was contaminated with another substance or a high concentration of intoxicant or caffeine this would besides impact this bio-assay in a negative manner.

All Daphnia are highly sensitive to metal ions and many dissolved

toxins in the H2O. Moina shows the highest opposition degrees. these are likely to be evident dissolved in the O to be used. Analysis Of Consequences It can be seen from both graph and tabular array, that as the degrees of caffeine in the NaCl solution addition, the water flea & # 8217 ; s bosom beats per minute addition at a high rate, which seems to top out at approximately 340 beats per minutes and so degrees off until the water flea dice at around 0.8 % . With relation to the intoxicant, this showed that the BP dropped from 186 BPM and leveled off at around 100 BPM before they die at a concentrarionof near to 0.9 % . It seems that the water flea have a higher tolerance degrees to the drug intoxicant than they seem to caffeine, this is obtained due to the fact it takes a higher concentration of solution for it to halt the water flea & # 8217 ; s metamorphosis wholly. To happen out more and why these affect the bosom so much, more elaborate research must travel into the bosom of this crustacean. The crustaceous Black Marias are known to be neurogenic. The cardiac beat is originated in cardiac ganglion.

The cardiac ganglion is composed of the pacesetter ( little ) and follower ( big ) nerve cells. The pacesetter nerve cells exhibit self-generated bursting activity, and bring on fire of follower nerve cells through excitatory synapses. The follower nerve cells make excitant neuromuscular junctions onto the cardiac musculus and cause musculus contraction, the pulse.

The cardiac musculuss are merely driven by the motor nerve cells ( followings ) in the cardiac ganglion merely like other skeltal musculuss that are driven by their ain motor nerve cells in the CNS.

In crayfish, the cardiac ganglion is frequently buried under the cardiac musculus fibres. From the cognition of this it shows how the pacesetter and follower and motor nerve cells adapt to this new environment and this is why intoxicant and caffeine can consequence the crustaceous bosom so rapidly and efficaciously.

So how does alcohol and caffeine interfere with nervousnesss? All cells have a type of protein embedded in their cell membranes called receptor proteins. These receptors allow communicating between cells to happen. Nervousnesss can have communicating from cells far off or from nerve cells near by. Cells far off secrete endocrines, which find their manner to the mark nervus & # 8217 ; s hormone-receptors. Once these endocrines attach to the receptor, alterations in the nervuss take topographic point. The hormone-receptors, when activated, can alter how rapidly a nervus generates an impulse, or change how fast the nervus passes urges along. Some endocrines stimulate an addition in bosom rate ( such as during exercising ) and other endocrines decrease bosom rate ( such as during slumber ) which shows how an addition in emphasis applied to the cardiac musculus of the bosom, Internet Explorer. From caffeine can do it all in faster, and a lessening such as forced by a sedative such as intoxicant can do it crush more easy.

Other receptors allow communicating between next nervus cells ( such as between the pacesetter nerve cells and the follower nerve cells ) . Alternatively of endocrines, this type of receptor binds to neurotransmitters.

Receptors of any type, nevertheless, are non perfect. Sometimes they by chance bind molecules that they shouldn & # 8217 ; t. This can do an inappropriate addition in nervus activity, or it may do an inappropriate lessening in nervus activity. ( The alteration depends on the type of receptor involved, and the type of adhering the molecule uses. ) The nervus fibres carry oning pulsations to the Black Marias of Daphnia may incorporate receptors that unsuitably bind to ethanol ( or a merchandise of ethyl alcohol after it is broken down ) doing an inappropriate lessening in nervus activity. Which could be another signifier of anomylais consequence.

In relation to my anticipation, my estimated graph was proved to be more or less right and what was stated about caffeine being related to high blood pressure was besides proved to be apparently besides correct in every bit much as the fact that the blood force per unit area greatly rose as the caffeine per centum increased. The rate at which they both increased was similar which shows that caffeine and alcohol addiction can hold and every bit inauspicious consequence on 1s wellness although us as worlds organic structures are more accepting of caffeine than we are alcohol & # 8211 ; the fact that water flea is a cold blooded crustacean shows that crustaceans such as water flea can intake more capacity in comparing to their organic structure mass than we can as worlds and the fact that they are cold blooded makes them more at hazard to the drug caffeine.

It was besides clear that the round did go guerrilla as the dose increased due to the fact they were close to decease or really badly.

Besides although non presented in a tabular array or graph signifier it was shown that the pulse did modulate after three proceedingss of it being in the solution ( at concentrations of 0 % -0.6 % )

My relation anticipations such as & # 8216 ; as the dose increased so will the bosom rate & # 8217 ; nevertheless basic, remain the of import key to the enzymatic reactions taking topographic point inside the water-flea & # 8217 ; s organic structure.

The fact that caffeine is a stimulation, and will kill daphnia at high dose has been doubtless proved, and that intoxicant is a sedative and lowers BP, and when In high dose will kill, is similarly proved

The inquiries posed in the program have all been provided an reply to, except, due to the deficiency of clip it was impossible to change the temperature with the truth necessary for this bio-assay. Evaluation My program worked, it proved to be one of the optimal ways to prove the effects of such drugs on a little crustacean with the limited clip, equipment and background research, and for this history, to better this bio-assay farther variables such as temperature combined with one of the drugs, and/or adding nicotine, another sedative that is said to be habit-forming to worlds who smoke coffin nails, over a drawn-out period of clip, it could be seen if the little cold blooded crustacean would besides go mentally dependent on such a drug as nicotine, this could be done, in combination with a per centum of pitch to see how this effects the water flea & # 8217 ; s respiratory system and enzyme reactions.

The evident mistakes that could happen when making an experiment are included under the header & # 8216 ; just test/failures & # 8217 ; .

There was one or two anomylais consequences and these are circled and highlighted on both graph and tabular array, this could be due to any figure of mistakes ( most of which stated under & # 8216 ; just test/failures & # 8217 ; ) these include, sick water flea, ill measured concentration, another substance apparent in petri dish or pipette and so on.

On one or two occasions it was impossible ( with this equipment ) to number the water flea & # 8217 ; s bosom. Neural detectors would be ideal for numbering the BPM & # 8217 ; s of the water flea & # 8217 ; s bosom

The program was besides accurate and my cognition of the water flea & # 8217 ; s background and ideal conditions were important in subtracting, clip, steps and temperatures for the experiment to be carried out at.

The consequences proved to be clear, the dual graph, assisting in seeing how the two sets of figures overlap at room temperature. They were sufficiently good for acurate decisions to be drawn and an efficient bio-assay to happen.

There is Definitive room for farther probe into caffeine and intoxicant playing as stimulations and sedatives severally, but to to the full look into a caffeine molecule ( C8H10O2N4H2O ) fig 5

must be taken apart and seen how variable combinations of C, O, H, consequence the water flea & # 8217 ; s bosom rate. To make this, more advanced equipment and higher degrees of accomplishment and clip must be incorporated to do the bio-assay a success. Daphnia BioassayPreliminary intoxicant exp.

Percentage of intoxicant in H2O solutionTest 1 ( sum of beats in one minute ) Trial 2 ( sum of beats in one minute ) Trial 3 ( sum of beats in one minute )

0 % 186198184

20 % 176178181

40 % 157148150

60 % 142144137

80 % 12011097

100 % deaddead71

Actual Alcohol exp. Percentage of intoxicant in H2O solutionTest 1 ( sum of beats in one minute ) Trial 2 ( sum of beats in one minute ) Trial 3 ( sum of beats in one minute )

0 % 187184189

20 % 179184182

40 % 154157149

50 % 143144139

60 % 137131142

70 % 127125124

80 % 121116101

90 % 1178489

100 % deaddeaddead

Preliminary caffeine exp. Percentage of caffeine in H2O solutionTest 1 ( sum of beats in one minute ) Trial 2 ( sum of beats in one minute ) Trial 3 ( sum of beats in one minute )

0 % 207207207

20 % 244247241

40 % 300309307

60 % 333339345

80 % toomany tocount347349

100 % deaddeaddead

Actual caffeine exp. Percentage of caffeine in H2O solutionTest 1 ( sum of beats in one minute ) Trial 2 ( sum of beats in one minute ) Trial 3 ( sum of beats in one minute )

0 % 204211209

20 % 247249254

40 % 299304311

50 % 310316319

60 % 334339347

70 % 344340339

80 % 341344347

90 % 301deaddead

100 % deaddeaddead