To investigate the effects of different concentrations

The H2O fleaDaphniaPulex( see figure 1 ) is a little ( 1.1-3.5mm long ) crustacean normally found in pools in the United Kingdom. The full taxonomy ofDaphnia puluxis as follows:

Kingdom – Branchiopoda

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Phylum – Diplostraca

Class – Cladocera

Order – Anomopoda

Family – Daphniidae

Genus – Daphnia

Subgenus – Pulex

Figure 1. Daphnia Pulex, demoing the clear exoskeleton and inside the organic structure ( Cladoceran website 2005 )

Whilst being crustaceans and holding an exoskeletonDaphniaare easy to analyze as their exoskeleton is really clear so it is possible to see through it and analyze the working of the interior of their organic structure. In peculiar it is possible to see the flow of the circulatory system and the bosom whipping. Therefore they are utile to prove the effects of specific drugs on pulse, as it is easy to see the alterations via an obvious alteration in pulse.

Daphniahave been used in the present experiment as they are readily available and it is simple to analyze the effects of drugs on their systems. They are besides robust to a assortment of chemicals being present in their environment so can be used to measure the effects of these chemicals without fright that the chemicals will kill them immediately.

Aspirin ( acetylsalicylic acid ) , intoxicant ( ethyl alcohol ) , and caffeine are drugs that all normally used by people. All 3 drugs have an consequence on the human bosom, largely through indirect effects on the circulation and factors that affect circulation – eg thrombocyte coagulating. Alcohol causes a lowering of bosom round due to spread outing the venas through vasodilatation. Aspirin besides lowers the bosom rate via its effects on thrombocytes and the manner that they cause venas to compress. Caffeine, nevertheless, acts to increase bosom round.

It is unluckily non practical to prove the consequence of any of the drugs on the pulse of people as the drugs have a batch of other effects within the organic structure every bit good. AlternativelyDaphniaare used as they have a little organic structure so the drug effects are less widespread and the consequence on the bosom is easy to measure. The effects are besides apparent really rapidly so it is possible to transport out a big figure of probes in a comparatively short infinite of clip.

Hypothesis

The experimental hypotheses for each of the three drugs are as follows:

Aspirin will do the bosom rate ofDaphniato take down in a concentration dependent mode.

Alcohol will do the bosom rate ofDaphniato take down in a concentration dependent mode.

Caffeine will do bosom rate ofDaphniato increase in a concentration dependent mode.

The void hypothesis in all instances is that each of the drugs will hold no consequence on the concentration rate ofDaphnia.

Variables

In order to see whether it is really the drugs that are doing the alteration in the bosom rate of Daphnia the merely variable that can alter is the concentration of the drug. Equally good as this each drug will hold to be tested individually to do certain that it is merely the consequence of that drug which is doing the alteration, if any. A 4th set of consequences besides needs to be taken, which is the bosom rate of water flea without any of the drugs added at all.

For this experiment the independent variable will be the drug concentration. An independent variable is one that is chosen and set specifically by the experimentor.

For this experiment the dependant variable – the 1 that changes as a consequence of the independent variable altering – will be the bosom rate of theDaphnia.

Changeless variables will be:

The figure ofDaphnia( approx 100 )

The volume of H2O ( 100ml )

The temperature ( same room temperature of approx 21 i‚°C in all instances )

The species ofDaphnia(Daphnia pulux)

The sum of H2O transferred to microscope slide for each trial ( 1ml )

The same length of clip theDaphniawould be left in the chemical ( 1 hr )

Method

Daphniawill be collected from a pool, utilizing a all right mesh sive and maintain in a contained with pool H2O until they can be counted and the right figure can be transferred to a petri dish incorporating 100ml of distilled H2O for each experiment. Distilled H2O will be used so that there are no other chemicals present which might hold an consequence on bosom rate.

Approximately 100Daphniawill be used for each experiment, although it would non be possible to number precisely that figure so an approximative figure will be used in each instance.

Aspirin will come in a readily available over the counter soluble signifier ( Boots 300mg soluble acetylsalicylic acid )

Caffeine will besides come in a readily available over the antagonistic signifier of ProPlus ( 50 mg Caffeine )

Alcohol will come from vodka ( Smirnoff Blue label 45 % vol ) .

The drug will be added to the distilled H2O in the petri dish before adding the water flea and stirred with a clean glass rod to do certain that it is to the full dissolved and spread throughout the liquid. Then the water flea will be transferred to the petri dish utilizing a pipette.

Drugs will be in the measures shown in tabular arraies 1-3

Table 1. A table demoing the figure of caffeine tablets used in each experiment and the ensuing caffeine concentration

Number of tablets

Sum of caffeine ( milligram )

Sum of H2O ( cubic decimeter )

Caffeine concentration ( mg / cubic decimeter )

0

0

0

0.1

1

50

0.1

5

2

100

0.1

10

3

150

0.1

15

4

200

0.1

20

Table 2. A table demoing the figure of aspirin tablets used in each experiment and the ensuing aspirin concentration

Number of tablets

Sum of acetylsalicylic acid ( milligram )

Sum of H2O ( cubic decimeter )

Caffeine concentration ( mg / cubic decimeter )

0

0

0.1

0

1

300

0.1

30

2

600

0.1

60

3

900

0.1

90

4

1200

0.1

120

Table 3. A table demoing the sum of vodka used in each experiment and the ensuing intoxicant concentration

Sum of vodka ( milliliter )

Sum of intoxicant ( milligram )

Sum of H2O ( cubic decimeter )

Alcohol concentration ( mg / cubic decimeter )

0

0

0.1

0

50

22.5

0.1

2.25

100

45

0.1

4.5

150

67.5

0.1

6.75

200

90

0.1

9

There would really be merely 1 control experiment, to look into for differences caused by the drug, so the top row of each tabular array really corresponds to the same experiment, where there is merely H2O in the petri dish and no added chemicals.

As the water flea are little beings with a comparatively permeable exoskeleton they would be able to absorb the caffeine nowadays in the H2O and it could hold its consequence upon the organic structure.

In each instance the Daphnia would be left in the H2O and drug for a clip of 1 hr to guarantee that the drug has had any consequence. As the Daphnia are merely traveling to be used for a short period of clip they would have no nutrient as it would non be necessary.

Whilst wholeDaphniaare seeable to the bare oculus, a microscope is necessary to see the really bosom beats. Therefore a clean pipette will be used to reassign a droplet of the treated H2O and water flea onto a microscope slide. This would so be covered utilizing a screen faux pas to forestall the liquid from traveling. This will so be looked at under the microscope and the figure of times the Daphnia’s bosom beats per minute recorded. Each clip that the bosom round is counted it will be repeated 3 times to obtain an norm.

Different microscope slides would be used in each instance to avoid cross taint by different drugs or drug concentrations.

Equipment list

Fine mesh screen

Plastic container to hive away water flea in

Distilled H2O ( 13 x 100 milliliters )

13 clean plastic pipettes

13 clean petri dishes

13 microscope slides and cover faux pass

Light microscope

Caffeine tablets ( ProPlus )

Aspirin tablets ( Boots soluble acetylsalicylic acid )

Alcohol ( Smirnoff blue label vodka )

Measuring cylinder

Hazard appraisal

There are a figure of specific countries which involve hazard within this experiment. These are the initial aggregation of water flea, the handling of water flea and the handling of chemicals. Specific hazard appraisal are as follows:

If alcohol / drug is ingested so rinse out oral cavity instantly and seek medical advice.

If alcohol / drug solution is spilt on apparels rinse affected country and take apparels if necessary.

If alcohol / drug solution gets into eyes so rinse out instantly and seek medical advice.

An grownup ( over 18 ) must buy the vodka and be present during its usage.

Care should be taken when obtaining the water flea from a pool and an grownup should ideally make the collection.

If pond H2O is ingested so rinse out oral cavity instantly and seek medical advice.

Consequences

It was found that, as Daphnia bosom round was really fast it was easier and more dependable to number in 15 2nd subdivisions and so change over this to crush per minute. So this was done in all instances.

The bosom rate of water flea under control conditions was found to be an norm of 350 beats / minute. The consequences for each of the 3 drugs are considered individually below.

Caffeine

The consequences from the experiment to look into the consequence of caffeine upon the bosom rate of Daphnia are shown in figure 2 below.

Figure 2. The consequence of caffeine on the bosom rate of Daphnia Pulex ( norm of 3 counts )

It can be seen that there is a positive correlativity between caffeine concentration and Daphnia bosom rate. The per centum addition in bosom rate, when compared to command, is shown in table 4 below.

Table 4. A table demoing the consequence of increasing caffeine concentration on daphnia bosom rate

Caffeine concentration ( mg/l )

Heart rate addition ( beats/minute )

Percentage addition

50

13 beats per minute

3.5 %

100

15 beats per minute

4.3 %

150

17 beats per minute

4.9 %

200

20 beats per minute

5.8 %

Aspirin

Unfortunately the concentrations of acetylsalicylic acid used really killed the water flea whilst they were in the solution for the needed hr. Therefore it was necessary to change the concentrations and reiterate the experiment. As the water flea died at all concentrations of acetylsalicylic acid even 30 milligrams / liter must hold been excessively concentrated for them. Therefore the concentration was reduced to 10 % of original values, and the experiment repeated. The new concentrations were obtained by thining the dissolved tablets in more distilled H2O, eg 10 times the sum to obtain concentrations of 1/10. unluckily, due to clip restraints, the counts could non be repeated 3 times so the figure are for a individual count at each concentration.

The consequences from these new concentrations are shown in figure 3 below.

Figure 3. The consequence of acetylsalicylic acid on the bosom rate of Daphnia

It can be seen from figure 3 that there is no peculiar form to the consequences, other than the fact that acetylsalicylic acid is linked to a decrease in bosom rate. However this decrease does non look to be concentration dependant, or at least non at the concentrations used in this experiment.

Alcohol

The consequence of increasing intoxicant concentration is shown in figure 4.

Figure 4. The consequence of intoxicant on the bosom rate of Daphnia ( norm of 3 counts )

It can be seen that intoxicant had rather a big consequence on the bosom rate of water flea and there was an reverse correlativity between intoxicant concentration and bosom rate. The per centum lessening in bosom rate, when compared to command, is shown in table 5 below.

Table 5. A table demoing the consequence of increasing intoxicant concentration on daphnia bosom rate

Alcohol concentration ( mg/l )

Heart rate lessening ( beats/minute )

Percentage lessening

2.25

104 beats per minute

29.8 %

4.5

120 beats per minute

34.3 %

6.75

134 beats per minute

38.3 %

9

148 beats per minute

42.3 %

Statistical analysis of consequences

It was non possible to set about accurate statistical analysis of the aspirin consequences as merely 1 count was taken at each concentration degree on the perennial experiment. However it was possible to set about a t trial analysis on the intoxicant and caffeine consequences, utilizing the original informations as opposed to the average consequences shown in the graphs. Statistical analysis was carried out utilizing the package available from Graphpad quickcalcs. These statistically analysed consequences are shown in table 6 and 7 below.

Table 6. A tabular array to demo the comparative statistical significance of changing concentrations of caffeine upon the bosom rate of water flea

Concentration compared with control ( mg/l )

2 tailed P value

Statistical significance

5 mg/l

0.2567

None

10 mg/l

0.0301

Yes

15 mg/l

0.0575

None

20 mg/l

0.0579

None

Table 7. A tabular array to demo the comparative statistical significance of changing concentrations of intoxicant upon the bosom rate of water flea

Concentration compared with control ( mg/l )

2 tailed P value

Statistical significance

2.5 mg/l

0.0001

highly

4.5 mg/l

0.0012

really

6.75 mg/l

& lt ; 0.0001

highly

9 mg/l

0.0019

really

Discussion

The consequences from this experiment indicate that both caffeine and intoxicant affect bosom rate of water flea. Aspirin may impact bosom rate but the consequences obtained here do non supply plenty grounds to back up this position to the full.

Why does caffeine affect bosom rate?

The consequences from this experiment show that caffeine causes bosom rate to increase by around 0.05 % per milligram of caffeine. Whilst it can non be known precisely why this happens there are assorted possible grounds.

Caffeine if a methylxanthine which acts by suppressing the enzyme ( phosphodiesterase ) that metabolises camp. The ensuing rise in camp degrees has the same consequence as increasing degrees of adenylate cyclase, which is one of the chief effects of epinephrine. Adrenaline is known to increase bosom rate via its effects on the i??1adrenoceptors so it is likely that caffeine has a similar consequence. Whilst these effects are known about in worlds, and water fleas are non the same beings, it is likely that the effects are mediated for similar grounds – eg the engagement of enzymes and suppression / stimulation of these.

It is possible that the effects of caffeine were non every bit pronounced as might hold occurred due to a automatic reaction. In worlds an addition in bosom rate is frequently quickly followed by a automatic compensatory mechanism to decelerate the bosom rate once more. This compensation can affect the venas distending and a stimulation of enzymes such as phosphodiesterase in order to cut down camp degrees. In the instance of the water flea it might hold been the instance that a more important rise in bosom rate occurred every bit shortly as the caffeine was absorbed by the water flea but so there was a compensatory mechanism to change by reversal this consequence. However as the solution of caffeine was present around the water flea for the whole continuance of the experiment so this is improbable as each clip new caffeine was absorbed it would do bosom rate to increase, and the daphnia’s system would non be able to invariably undergo the automatic compensations.

How does aspirin affect bosom rate

Aspirin has its chief effects on an enzyme called cyclo-oxygenase. Aspirin Acts of the Apostless by adhering to the active site of the enzyme, therefore forestalling it from adhering to the eiocosanoid and holding its needed consequence. The suppression is irreversible as aspirin binds for good to the active site of the enzyme. Cyclo-oxygenase ( COX ) is the chief enzyme involved in the transition of eiocosanoids found in membrane fatty acids, into the inflammatory go-betweens prostaglandins and leuoktrienes. COX acts to change over arachadonic acid into the prostaglandin precursor prostaglandin G2and besides to change over this precursor into prostaglandin H2. It is from PGH2that the chief inflammatory go-betweens are generated. Therefore the bar of COX prevents even the first portion of the inflammatory rhythm from taking topographic point.

Whilst it is non instantly obvious how the bar of inflammatory go-betweens should cut down bosom rate, it becomes obvious when the consequence of the redness on bosom rate is known. Whilst each person prostanoid has somewhat different effects, the overall one if to do vasoconstriction and besides an addition in myocardial contractility. This latter peculiarly consequences in bosom rate being increased as the bosom musculus in undertaking more often.

This means that decreases bosom rate by forestalling this increased bosom musculus contraction, every bit good as forestalling the vasculature from compressing so much.

Again, it is the effects of acetylsalicylic acid in worlds that is known about, but it could be expected that the consequence is similar in water flea.

Why might alcohol affect bosom rate

It is non known precisely how ethanol Acts of the Apostless to diminish bosom rate, but it is believed to be a knock on consequence from the alterations in peripheral vasculature. Ethanol is known to do vadodilatation – eg dilation of blood vass. If blood vass are dilated so there is more infinite therefore a greater volume for blood so the bosom has to crush fewer times to present the same sum of blood.

It is besides possible that ethyl alcohol has its effects on bosom rate via a cardinal nervous system consequence. Ethanol ab initio increase catecholamine release, including Dopastat. This acts to excite bosom rate. However following this initial consequence ethyl alcohol really reduces the concentration of catecholamines, chiefly noradrenaline, by increasing the rate at which it is removed from cells. As the water flea had been left in the ethyl alcohol incorporating H2O for an hr it would be expected that they had reached this 2nd phase by the clip bosom rate was being measured. However it is possible that the struggle between the initial and subsequent effects of intoxicant could be the ground why the consequences did non follow a peculiar form.

Beginnings of mistake

Whilst statistical significance appears to be present in a figure of consequences it is really hard to be certain with such a little population figure. In order to be more certain of consequences, a much larger sample size should be used in each instance. When a consequence has been shown to be important and is really due to opportunity this is known as a type I statistical mistake.

Temperature is another variable that affects the bosom rate of water flea, apparent by the fact that water flea can be preserved alive at low temperatures due to a deceleration of bosom rate. Therefore it is possible that the heat from the light microscope lamp caused an addition in the bosom rate. However, as the visible radiation was present on each numeration session this variable, whilst perchance impacting bosom rate, would impact all counts by a similar sum. Therefore all counts would be raised from what they should be. This would function to do the consequences from caffeine more important and intoxicant less important. Given that the consequences from intoxicant showed an reverse correlativity so this beginning of mistake could hold merely made the consequences less important. However in the instance of the caffeine it is possible that they were responsible for the alteration in bosom rate observed, instead than the caffeine, as the alterations were instead little anyhow. If the experiment were to be repeated this could be controlled by careful monitoring of temperature and guaranting that this remained changeless throughout the experiment, a variable that was no measured in this experiment.

The fact that intoxicant had to be used from an ab initio diluted signifier, and in a non pure ethanol preparation may hold caused the consequences to be affected. Whilst computations were made to happen the overall concentration of ethyl alcohol into which the water flea were placed, it is non certain whether this was right. The intoxicant could hold contained other chemicals in add-on to ethanol which could hold affected the water flea and their bosom rate.

The preparation of caffeine used was in the signifier of proplus tablets. These are besides non caffeine in a pure format and would incorporate other embroidering chemicals such as sugar and talcum pulverization to do up the tablet. Sugar may good impact the bosom rate of water flea as it is a nutrient material for them, and their bosom rate may alter whilst eating, or following a significant sum of nutrient.

Whilst water flea reproduce asexually they are present in male and female signifiers. It was non possible to determine which gender was being counted on each juncture. It is the females which really give birth to immature. However pregnant females have a lower pulse than non pregnant females and males. Therefore if some of the water flea that were being counted were really pregnant females so this could hold unnaturally lowered the bosom rate consequence. Without analyzing each water flea and guaranting that all were of the same gender and pregnant / non it is non possible to state whether this factor hardship affected the consequences.

Whilst the water flea were held in topographic point slackly on the microscope slide they were non fixed in topographic point. Therefore it was non ever possible to look into that it was the same water flea that was being used for the repeats of counts for each of the drug interventions. It is therefore possible that the fluctuation within each of the 3 counts was due to different water flea being used, instead than a different bosom rate at different times.

It was non possible to determine how old each water flea was which may hold had an consequence on bosom rate as juveniles normally have a higher bosom rate than older beings.

It was assumed that all water flea obtained from the pool were really of the common water flea pulex species. However it is possible that there were a mix of different species within the sample collected. If an being from a different species was used for each of the counts so this could besides give rise to altered consequences.

Evaluation

The consequences from this experiment would look to back up the hypothesis that caffeine increases the bosom rate of water flea. Likewise they besides support the hypothesis that aspirin decreases the bosom rate of water flea.

It was unfortunate that the consequence from intoxicant were inconclusive and can non confirm or confute the hypothesis. Given more clip the consequences would hold been repeated to measure whether the effects were important when more counts were made.

Ideas for future probes

It would hold been utile to see whether the effects which the drugs had on Daphnia bosom rate were reversible. In order to make this the water flea would be transferred to clean distilled H2O and left for an hr before bosom rate was measured once more. It is known that acetylsalicylic acid has an irreversible consequence on COX in worlds but this consequence overall can be reversed when new enzyme is created. A similar consequence could besides happen in water flea.

The effects of different concentrations of drugs were done on different populations of water flea in each case. It would be interesting to measure whether there was any difference in the consequence of bit by bit increasing the drug concentration on the same population. It would hold to be a gradual addition and bosom rate steps be taken from the lowest concentrations in instance a higher concentration had a permanent consequence on the being.

Bibliography

Daphnia image and information from: hypertext transfer protocol: //www.cladocera.uoguelph.ca/taxonomy/daphnia/default.htm accessed 04/05/2005

Human drug information and biochemical information from Rang, H.P. , Dale, M.M. & A ; Ritter, J.M. 1999,Pharmacology, Fourth edn, Churchill Livingstone, Edinburgh.

Information about norm Daphnia bosom rate from Campbell AK, Wann KT, Matthews SB. Lactose causes bosom arrhythmia in the H2O flea Daphnia Pulex. Comparative Biochemistry and Physiology B Biochemistry and Molecular Biology. 2004 Oct ; 139 ( 2 ) :225-34.

Statistical package used – Graphpad Quickcalcs online package. Available at hypertext transfer protocol: //www.graphpad.com/quickcalcs/ttest1.cfm accessed 04/05/2005

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