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