Daphnia Bioassay Essay, Research Paper

Plan The initial variable to be considered will be the effect that caffeine and temperature has on the heart beat and if it has a direct or indirect effect on the daphnia’s lifespan. If there is enough time to consider a second variable however, the effects of a temperature or alcohol on the daphnia’s body system will also be considered alongside the initial variable of caffeine. These will then be slotted into the same graph which will show us how the figure correspond to the results.

In order to conduct a true experiment the following questions must be considered and answered and only then can acurate conclusion be drawn.

· How variable is the heart rate in an individual Daphnia?

· How does the drug caffeine affect the daphnia’s heart beat?

· At what concentration is the heart rate slowest? The fastest?

· What indication in the graph is the optimum concentration for heart rate of Daphnia?

· What is the significance of the shape of the graph line when the data is plotted?

· ·What would happen if you increased the temperature in 5C increments (if time is available)?

· What would happen if alcohol was added into the solution and why would this affect the daphnia’s heart rate and general behavior. To fully understand the way the daphnia works one must examine its body and the uses for the different parts of it:- (fig.1) [1] – Second antenna, used for swimming and sensing the environment[2] – eye controlled by muscles with nerve connections to the brain[3] – legs used for gathering food stabilizing the animal as it slowly sinks[4] – An intestine where ground up food particles are digested[5] – A brood pouch for incubating young that hatch from large yolk filled eggs [6] – A protective outer shell [7] – A heartthat pushes clear circulatory fluid around the body[8] – Undigested material is eliminated out the anus.

As it is now seen that the daphnia incorporates its food through the use of its legs it could explain why the drugs such as alcohol or caffeine take somewhat longer to enter its brain and alter its bodily functions-also its body acts as a form of a semi-permeable barrier of which small molecules (such as caffeine ‘C8H10O2N4H20′) can penetrate the walls of its internal organs, this is why it penetrates the whole of the daphnia’s body at once (see ‘prediction’ for information relating to this fact). If temperature is to be used as a chosen variable, it must be considered that daphnia are cold-blooded animals, they do not thermoregulate. This means that their body temperature is the same as the water they are floating in. The chemical reactions that occur in the cells of Daphnia are dependent on certain enzymes, or proteins, to help the reactions proceed. As you increased the temperature of the water, the metabolism of the Daphnia increased as well, because chemical reactions occur faster at higher temperatures. This means that the heart rate will speed up in order to provide oxygen to the cells as the metabolism increases. However, at 40 degrees Celsius, the enzymes break down, and the chemical reactions can no longer occur, so metabolism stops and the Daphnia dies. In its natural environment, Daphnia does not ever experience temperatures of 40 degrees, so this is unlikely to occur. However, they do experience changes in temperature in their natural environment, and their metabolism does increase or decrease as the temperature changes.

Method I have initially decided to see how the drug caffeine affects the heartbeat and lifespan of a daphnia, A daphnia will be extracted from a pond nearby, so the condition of the daphnia will not be altered too drastically during transport. These daphnia will be allowed to adjust to their new surroundings and then be taken out through the use of a pipette and placed in a petri dish containing the relevant amount of caffeine/alcohol concentrate solution (aq). This daphnia will be left in the solution for a minimum time of one minute before any sort of results are taken (for this experiment one and a half minutes were used) this is to allow the daphnia to adjust to its new surroundings and its semi-permeable barrier to in-secrete the surrounding solution, although when doing this, it must be remembered that if left too long, the daphnia will have adjusted itself and the heartbeat will begin to regulate again. Once the period of one minute thirty seconds has elapsed, a ‘clicker counter’ will be used to count the number of beats of the daphnia’s heart in one minute (obviously a correctly focussed microscope will be necessary to see the daphnia’s heart and how it beats) for problems relating to this method please see ‘evaluation’. The concentration of alcohol/caffeine will then be varied to see how this affects the regulation the daphnia’s heartbeat. (the daphnia’s heart is illustrated in the following diagram fig.2). In an ideal lab condition the plan would be such as this, although the time period and equipment are limited so this method will be followed as close as is possible:-

1. Prior to conducting the bioassay, check the Daphnia to ensure the culture is healthy. 2.select test organisms. Although you may use mixed age populations for bioassays, it is better to use only young individuals in order to minimize biological differences among the test organisms. Because the appearance of resting eggs indicates a poor culture environment, do not use Daphnia with resting eggs. To obtain a good supply of young Daphnia, begin 24 hours in advance by removing females bearing embryos from the stock culture and placing them in 400-mL beakers containing 300 ml of spring or stream water and the appropriate amount of food. Five beakers, each containing 10 adults, usually will supply enough young individuals for one toxicity test. When you are ready to begin the bioassay, choose young (small) Daphnia from these cultures.

3. Introduce the same number of neonates (at least 10) into each test vessel and control using a plastic, disposable pipette with a 5-mm diameter. Be sure to release the young below the surface to avoid killing them by trapping air under their carapaces.

4. Prepare a table that records the percent concentration, the time, and the total number of dead at each time interval. As close to one hour as possible, but prior to the end of the period, record the number dead at each concentration. Remember Daphnia molt to grow, so neonates will molt as they develop. Therefore, do not count the molt castings, which appear as clear shells of the Daphnia on the bottom of each cup. Remove dead Daphnia and molt castings at each monitoring interval.

5. Ideally it is best to check again in 4 hours and again after 24 hours. If schedules do not permit (such as ours having to be completed in a one hour 45 minute time period), Must not feed animals during tests. Steps 4-6 will take about 15 minutes of two consecutive class periods.

6. At the end of the bioassay, test the water to determine the pH, hardness, and dissolved oxygen content. Count and record how many Daphnia in each dish have died, then analyze the data.. Apparatus Equipment

dissecting microscope

water chemistry test kits (pH, hardness, dissolved oxygen)

Light (from lamp)

petri dishes

salt solution (NaCl)

clicker counters Materials

5mm diameter pipette or eye dropper

Alcohol

Caffeine

microscope slide

1 ml pipette with .01 divisions

thermometer

spring water or unpolluted stream water

Daphnia magna culture, mixed age

Wipes or other tissues

food: possibilities include Roti-Rich dried yeast, or unicellular algae such as Selenastrum Diagram

Daphnia Size – There is a big size difference in the Daphniidae,

depending on the species. Newly hatched Moina are slightly

larger than newly hatched brine shrimp, and twice as big as

average adult rotifers, but newly hatched Daphnia are twice as

big as Moina, and may not be suitable for some of the smaller

fish fry because of their size. Life cycle of Daphnia – The daphnia has both sexual and

asexual phases. In most environments, the population consists

entirely of females that reproduce asexually. Under optimum

conditions, a female may produce more than 100 eggs per

brood, repeating every 3 days. A female may have as many as

25 broods in its lifetime, but the average is about 6. The

female will start to reproduce at about 4 days old with a brood

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

produced, and sexual reproduction begins. The result is the

laying of resting eggs, just like the brine shrimp. Factors that

can trigger this are a lack of food, low oxygen supply, a high

population density, or low temperatures. Nutritional Value – The nutritional content of Daphnia

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

is usually around 50% of dry weight. Quite the opposite from

Artemia, adults normally have a higher fat content than

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

Some species have been reported to have protein contents

exceeding 70%. Live Moina are about 95% water, 4% protein,

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

The fatty acid composition of food is important to the survival

and growth of fish fry. Omega-3 highly saturated fatty acids

are essential for many species of fish. Moina cultured on

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

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

will contain very high levels of Omega-3 fatty acids. Moina can

take up lipids very easily from the emulsion, but there is a

side effect to this, apparently it also slows productivity, so

this emulsion should only be fed to a batch separate from the

main growout colony. Commercial formulas are available in pet

supply houses for the enrichment of Artemia, Rotifer, and

Daphnia cultures. Physical Requirements – Salinity – Daphnia are typically freshwater organisms, but,

some are found in slightly brackish water. Some species have

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

3.0 ppt are common in pond cultures in the orient. Oxygen – Daphnia are generally tolerant of poor water

quality, and dissolved oxygen varies from almost zero to

supersaturation. Like the Brine Shrimp, their ability to survive

in an oxygen poor environment is in their ability to synthesize

hemoglobin. The production of hemoglobin may be promoted by

high temperatures, and a high population. Also, like brine

shrimp, Daphnia are not tolerant of fine air bubbles. A slow

aeration is needed with Daphnia as a large bubble column will

strip the Daphnia out and kill them. pH and ammonia – A pH between 6.5 and 9.5 is acceptable.

High ammonia levels, with high pH will drastically reduce

reproduction, but will not affect the actual health of the

animals themselves. So it seems that on the small scale that

we require, monitoring of pH and ammonia is not critical to

success. Dissolved minerals – In contrast to their tolerance of low

oxygen, Daphnia are very sensitive to disturbances of the ionic

composition of their environment. They become immobile and

eventually die with the addition of salts like sodium,

potassium, magnesium, and calcium. Low concentrations of

phosphorus (less than 0.5 ppm) will stimulate reproduction,

but concentrations higher than 1.0 are lethal to the young.

Daphnia magna are quite resistant to phosphorus and can

withstand concentrations as high as 5-7 ppm. Daphnia are not

affected by the addition of nitrogen in fertilizers for the

promotion of algae growth. As with any aquarium venture, the

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

remove chlorine before the culture is started. Concentrations

of only 0.01 ppm copper will result in reduced movement in

Daphnia. They are extremely sensitive to metal ions like

copper and zinc, pesticides, detergents, bleaches and other

dissolved toxins. Municipal and well water may be

contaminated enough to kill the culture. The best source of

water is filtered stream or lake water, rain water collected

for low air polluted areas, or, use the water from your

aquarium water changes. Temperature – Daphnia have a wide tolerance to

temperature. The optimum temperature for Daphnia Magna is

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

resisting daily variations of 5-31 deg C (41-88 F); their

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

tolerance of Moina make this species a better choice where

temperatures may rise above the comfort levels for Magna at

certain times of the year.

Prediction To create a reasonable prediction, it must first be known how daphnia react in their surroundings this background knowledge will undoubtedly help when attempting to make conclusions and/or predictions relating to the daphnia’s bodily systems and reactions. Daphnia typically live 40-56 days. This varies according to species and environmental conditions. Each brood typically holds 6-10 eggs, which turn into embryos and are released within a few days. Juveniles reach sexual maturity in 6 to 10 days.

Daphnia is a small Crustacean – barely visible with the naked eye. It lives in water and has large antennae in comparison with the rest of its body. These are used to make (jumpy) movements – hence the name “water flea”.

Daphnia’s seem to serve two purposes:

(1) they are very popular fish foodespecially because it is quite easy to keep them in culture, they reproduce at high rates and they keep the aquarium water clean

(2) aschanges in the heart rate might suggest a chemical compound has some physiological effect, and – more importantly – Daphnia magna is used to measure the toxicity of a chemical compound in water (LD50 measurements).

From a sciencepoint of view, Daphnia is not intensively studied. In essence, a few ATPase subunits and ribosomal RNA genes are sequenced.

Also under investigation is the nature of this species unusual pigmentation. daphnia are translucent and are usually amber or devoid of color. These dune daphnia, when found in their natural environment, possess a striking melanic pigmentation. It has been hypothesized that the melanism serves as a photoprotection device, but it is possible that it also serves as a defensive protection from the predatory copeopods which also inhabit these ponds. In a study in which melanic forms of daphnia were found in arctic ponds a strong correlation was found between whether or not the pond was surrounded by vegetation and the extent to which melanic forms were present. It was found that in all cases, melanic forms were present in ponds which were devoid of surrounding vegetation. It was hypothesized that organic nutrient runoff from local fauna filters out harmful ultraviolet radiation within the first few inches of water. Ponds lacking this natural UV protection thus harbored the pigmented forms. Indeed the ponds found near Florence are not only devoid of any vegetation, but they are also shallow, reinforcing the need for photoprotection

These ponds don’t resurface in the same spot every year, because the sands shift considerably throughout the summer after the ponds dry up. If there is a uniform amount of genetic variation between all ponds, it is likely that the winds blow the diapausing embryos around, mixing up the gene pools each year. If the amount of variation within a pond is low, it is likely that it was founded by one or a few hatched epphipia. If there is a high amount of variation, what maintains it? It is a well known fact that variation within populations is absolutely necessary for species to adapt in a changing environment. Because of the inherent instability of dune ecosystems, and the apparent uniqueness of this species of daphnia, could actually be dealing with an endangered species. Caffeine is a drug stimulant which acts directly upon the CNS (central nervous system) and brain which due mainly to the daphnia’s mass and size, a less amount of each substance (caffeine or alcohol) would effect the daphnia’s body than the human body, this could show the relation, ‘the greater the mass the more of the drug needed to affect the body system’. Therefore I predict that as the concentration of caffeine increases the heartbeat will increase as it does. Drinks such as ‘coffee’ or ‘coca cola’ contain a lot of caffeine which scientist report are linked to high BP (blood pressure or hypertension) rates. Limited research has stated that there is sucha link with heavy coffee consumption with high BP, all these can be linked back to the daphnia who’s bodily systems are based upon the same basic principles of survival.

Due to the fact that alcohol is a depressant, somewhat of a reverse of the effects that caffeine would so create, I predict that as the concentration of the alcohol increases the heartbeat of the daphnia would become less until it stops – the concentration at which the heart stops and the daphnia die is a question needed to be answered in this bioassay. Due to these predictions, I do so believe that a graph containing both would look like this:-

(fig 3) The heartbeat may become irregular with high dosage due to the fact the daphnia’s small mass and surface area would struggle severely to cope with the change in environment and this irregularity could perhaps result in death

The higher the concentration of caffeine the higher the heart rate will become. After a few minutes the daphnia will become accustomed to the environment and heart beat will likely regulate – this is remembering that it is not excessively high concentration for the daphnia to die. This would be due to the fact that they are cold blooded animals and react accordingly to their surroundings. Safety This is always a concern when working with any kind of toxin so points such as the following must be maintained to keep working safely:-

When heating water ensure all usual precautions are applied to Bunsen burner usage

Do not contaminate daphnia ‘home’ water with any substance.

Fair Test/Failures To create a higher level of accuracy when obtaining results, the clicker counter was used for 20 seconds and multiplied by 3 to get the results for one minute.

This was done three times and an average was taken to create more acurate results

Preliminary results were also taken just to ensure all anomalies will be seen and accounted for.

An attempt is made to use the same daphnia for all experiments as daphnia’s vary in size and metabolic rates therefore this must have every attempt made to be avoided. Although occasionally when using high concentrations, death is unavoidable.

Also the age of the daphnia can have some effect, although some only live a matter of minutes, like any other animals, their metabolic rates vary depending on age, size and characteristics .

With the equipment in use, it is more or less impossible to know whether the daphnia is ill or in a poor state, so if unlucky an ill daphnia could alter the entire bioassay. Further equipment such as extremely acurate thermal probes, or other physical assessment tools would be necessary if conducting an extremely acurate bioassay.

Although the female daphnia population is in excess of the male, if a male daphnia was taken for the experiment it would make results differ due to the fact that It is difficult to distinguish male and female Daphnia. They are basically identical except that males are generally smaller in size, have larger antenules, and the first legs have a stout hook used in clasping the female during mating. Rather than trying to identify males to detect stressful environments, it is much simpler to inspect the brood chambers of females). Ie. If a male daphnia was used, the results would differ greatly.

Daphnia stress levels can also alter results, in as much as if the daphnia had a high blood pressure (hyper tension) caffeine would heighten this and alcohol lower it, although starting from a plane incorrect to the necessary average.

Also 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 alcohol or caffeine this would also affect this bioassay in a negative fashion.

All Daphnia are extremely sensitive to metal ions and many dissolved

toxins in the water. Moina shows the highest resistance levels. these are likely to be apparent dissolved in the oxygen to be used. Analysis Of Results It can be seen from both graph and table, that as the levels of caffeine in the NaCl solution increase, the daphnia’s heart beats per minute increase at a high rate, which seems to peak at about 340 bpm and then levels off until the daphnia die at around 0.8%. With relation to the alcohol, this showed that the BP dropped from 186 BPM and leveled off at around 100 BPM before they die at a concentrarionof close to 0.9%. It seems that the daphnia have a higher tolerance levels to the drug alcohol than they seem to caffeine, this is obtained due to the fact it takes a higher concentration of solution for it to stop the daphnia’s metabolism completely. To find out more and why these affect the heart so much, more detailed research must go into the heart of this crustacean. The crustacean hearts are known to be neurogenic. The cardiac rhythm is originated in cardiac ganglion.

The cardiac ganglion is composed of the pacemaker (small) and follower (large) neurons. The pacemaker neurons exhibit spontaneous bursting activity, and induce firing of follower neurons through excitatory synapses. The follower neurons make excitatory neuromuscular junctions onto the cardiac muscle and cause muscle contraction, the heartbeat.

The cardiac muscles are simply driven by the motor neurons (followers) in the cardiac ganglion just like other skeltal muscles that are driven by their own motor neurons in the CNS.

In crayfish, the cardiac ganglion is often buried under the cardiac muscle fibers. From the knowledge of this it shows how the pacemaker and follower and motor neurons adapt to this new environment and this is why alcohol and caffeine can effect the crustacean heart so quickly and effectively.

So how does alcohol and caffeine interfere with nerves? All cells have a type of protein embedded in their cell membranes called receptor proteins. These receptors allow communication between cells to occur. Nerves can receive communication from cells far away or from nerve cells near by. Cells far away secrete hormones, which find their way to the target nerve’s hormone-receptors. Once these hormones attach to the receptor, changes in the nerve take place. The hormone-receptors, when activated, can change how quickly a nerve generates an impulse, or alter how fast the nerve passes impulses along. Some hormones stimulate an increase in heart rate (such as during exercise) and other hormones decrease heart rate (such as during sleep) which shows how an increase in stress applied to the cardiac muscle of the heart, ie. From caffeine can make it beat faster, and a decrease such as forced by a depressant such as alcohol can make it beat more slowly .

Other receptors allow communication between adjacent nerve cells (such as between the pacemaker neurons and the follower neurons). Instead of hormones, this type of receptor binds to neurotransmitters.

Receptors of any type, however, are not perfect. Sometimes they accidentally bind molecules that they shouldn’t. This can cause an inappropriate increase in nerve activity, or it may cause an inappropriate decrease in nerve activity. (The change depends on the type of receptor involved, and the type of binding the molecule uses.) The nerve fibers conducting pulses to the hearts of Daphnia may contain receptors that inappropriately bind to ethanol (or a product of ethanol after it is broken down) causing an inappropriate decrease in nerve activity. Which could be another form of anomylais result.

In relation to my prediction, my estimated graph was proved to be more or less correct and what was stated about caffeine being related to hypertension was also proved to be seemingly also correct in as much as the fact that the blood pressure greatly rose as the caffeine percentage increased. The rate at which they both increased was similar which shows that caffeine and alcoholism can have and equally adverse effect on ones health although us as humans bodies are more accepting of caffeine than we are alcohol – the fact that daphnia is a cold blooded crustacean shows that crustaceans such as daphnia can intake more capacity in comparison to their body mass than we can as humans and the fact that they are cold blooded makes them more at risk to the drug caffeine.

It was also clear that the beat did become irregular as the dosage increased due to the fact they were close to death or very ill.

Also although not presented in a table or graph form it was shown that the heartbeat did regulate after three minutes of it being in the solution (at concentrations of 0%-0.6%)

My relation predictions such as ‘as the dosage increased so will the heart rate’ however basic, remain the important key to the enzymatic reactions taking place inside the water-flea’s body.

The fact that caffeine is a stimulant, and will kill daphnia at high dosage has been undoubtedly proved, and that alcohol is a depressant and lowers BP, and when In high dosage will kill, is likewise proved

The questions posed in the plan have all been provided an answer to, except, due to the lack of time it was impossible to vary the temperature with the accuracy necessary for this bioassay. Evaluation My plan worked, it proved to be one of the optimum ways to test the effects of such drugs on a small crustacean with the limited time, equipment and background research, and for this account, to improve this bioassay further variables such as temperature combined with one of the drugs, and/or adding nicotine, another depressant that is said to be addictive to humans who smoke cigarettes, over a prolonged period of time, it could be seen if the small cold blooded crustacean would also become mentally dependant on such a drug as nicotine, this could be done, in combination with a percentage of tar to see how this effects the daphnia’s respiratory system and enzyme reactions.

The apparent faults that could occur when doing an experiment are included under the heading ‘fair test/failures’.

There was one or two anomylais results and these are circled and highlighted on both graph and table, this could be due to any number of faults (most of which stated under ‘fair test/failures’) these include, ill daphnia, poorly 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 count the daphnia’s heart. Neuronic sensors would be ideal for counting the BPM’s of the daphnia’s heart

The plan was also accurate and my knowledge of the daphnia’s background and ideal conditions were crucial in deducting, time, measures and temperatures for the experiment to be carried out at.

The results proved to be clear, the double graph, helping in seeing how the two sets of figures overlap at room temperature. They were sufficiently good for acurate conclusions to be drawn and an efficient bioassay to occur.

There is Definitive room for further investigation into caffeine and alcohol acting as stimulants and depressants respectively, but to fully investigate a caffeine molecule (C8H10O2N4H2O) fig 5

must be taken apart and seen how varying combinations of carbon, oxygen, hydrogen, effect the daphnia’s heart rate. To do this, more advanced equipment and higher levels of skill and time must be incorporated to make the bioassay a success. Daphnia BioassayPreliminary alcohol exp.

Percentage of alcohol in water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount of beats in one minute)

0%186198184

20%176178181

40%157148150

60%142144137

80%12011097

100%deaddead71

Actual Alcohol exp. Percentage of alcohol in water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount 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 water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount 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 water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount of beats in one minute)

0%204211209

20%247249254

40%299304311

50%310316319

60%334339347

70%344340339

80%341344347

90%301deaddead

100%deaddeaddead