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NWP Global Registry of Apprentice Ecologists - Backyard, Tulsa, Oklahoma, USA

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Backyard, Tulsa, Oklahoma, USA
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Registered: August 2011
City/Town/Province: Tulsa
Posts: 1
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Red Diesel fuel is widely used in agricultural machines and heavy machine equipment used in farming. The government assesses heavy fines to users of Red Diesel fuel. Red Diesel fuel has been found to be toxic to a number of aquatic organisms as it creates a film over the surface of water, and possibly alters oxygen production in plants. I strongly believe that high concentrations of Red Diesel fuel will create significant damage to the cellular function of Elodea densa. Red Diesel fuel is a hydrocarbon molecule that will more than likely be taken up readily by the cell plasma membrane. However, in large quantities this organic compound can be absorbed through the plasma membrane of the plant cell since it is similar in composition (hydrophobic). Once again I am hypothesizing that since red diesel is a hydrocarbon that it will mix into the plasma membrane since it is similar in composition of the fatty acid tail of the phospholipids. The purpose of this experiment was to examine the effects of red diesel fuel on Elodea densa. The Elodea densa will serve as a model for other plant life exposed to petroleum like products in the environment. I wanted to do this project to study the effects of red diesel fuel cellular damage on the Elodea densa in an aquatic environment. This study is important because it mimics an oil spill like condition and the experiment will be useful in studying the effects of flora damage in oceanic aquatic environments.

What are the effects of red diesel on Elodea densa’s: stolon growth, stem growth, condition of plasma membrane and chloroplast population?

To determine the effects of red diesel and examine the cellular and physiological changes in the Elodea densa population within an aquatic environment. This study is important because it mimics an oil spill like condition and the experiment will be useful in studying the environmental effects of flora damage in oceanic environments.

The purpose of this experiment was to study the effects of red diesel on cellular changes in the Elodea densa population in an aquatic environment. Elodea densa was adversely affected by the different concentrations of red diesel. The red diesel concentrations tested for Elodea densa were as follows: 200ml of red diesel (40%), 100 ml of red diesel (20%), 50 ml of red diesel (10%), 25 ml of red diesel (5%) and 0 ml of red diesel (0%) respectively. Elodea densa was exposed to UV light for approximately 4 weeks. Chloroplast population decreased each week. Furthermore, based on the data the Elodea densa which was subjected to 200 ml of red diesel (40%) experienced approximately 83.8% loss. Red diesel concentrations of 20% and 10% experienced a 77.4% to 54.0% significant loss when compared to the control. Elodea densa cells, when observed underneath the microscope were plasmolyzed. Furthermore, chloroplast organelles were clustered within the plasmolyzed membrane of the cells. The survival limit, for Elodea densa was observed at 5% red diesel concentration, which experienced a 20% chloroplast loss when compared to the control. Growth of stolon was not significantly different when compared against the control. Furthermore, data obtained from measuring the stem length for various concentrations revealed no significant difference. In conclusion, red diesel was found to be toxic to the Elodea densa cells by readily entering their plasma and chloroplast membranes, thereby, reducing the chloroplast population within the epidermis of the leaf.

- clean gravel
- water
- 40 Elodea densa plants
- sharp knife
- ruler
- fluorescent lamp
- microscope
- masking tape
- marking pen
- note book
- 500ml beaker
- Red Diesel
- boxes
- beaker
- camera
- razor
- tweezers

1. To each of the fishbowls add enough gravel to cover the bottom, and add 2,125 milliliters of tap water.
2. Plants were cut with a sharp razor to up to 10cm in length.
3. Plants were separated into five groups; eight Elodea densa stems were placed in each group in each bowl.
4. Label the fishbowls according to the amount of red diesel they will receive: 200ml, 100ml, 50ml, 25ml, and 0m (control). Measure the appropriate amounts of red diesel with a beaker.
5. The concentrations were prepared in the following manner: (40%) combined 300ml of water with 200ml of red diesel, (20%) 400ml of water with 100ml red diesel, (10%) 450ml of water combined with 50ml of red diesel, and (5%) 475ml of water and 25 ml of red diesel. All total volume for each bowl was 500ml. The control contained approximately 500ml water and no red diesel.
6. The plant groups were placed into their designated bowls to ensure adaptation to their environment.
7. Elodea densa contained in the bowls were subjected to fluorescent light. Fluorescent light was consistently applied to all of the bowls containing Elodea densa. Plants were allowed to adjust to its environment for approximately one week.
8. Average number of chloroplast was determined using a given field view of 3 cells. All three cells were counted for total chloroplast and divided by 3.
9. Stem length was determined by placing one plant from each group on a ruler and measuring its average growth in length per week in centimeters.
10. Stolons were cut completely off to approximately 0 cm. Stolon growth was measured to determine the amount growth per week in centimeters.
11. The plasma membranes of Elodea densa cells were observed from leaf tissue underneath the microscope, using a 40x objective lens.
12. The average number of chloroplast, condition of plasma membrane, and length of stem and stolon were all recorded in the notebook.


Elodea densa plants exposed to red diesel at 200ml (40%) concentration caused almost all the chloroplast to die and become plasmolyzed. The plasmolysis was due in part to the high concentration of red diesel in the water environment. Chloroplast organelles were found to be clustered in red diesel micelle bodies. The average chloroplast count for the 200ml (40%) red diesel for all of the weeks was as follows: week one 16.6, week two 3.6, and week three 7.6. These small numbers represent the low levels of chloroplast found in the cells for the plants exposed to the highest concentration of red diesel. Elodea densa plants subjected between 100ml to 50 ml of red diesel were also found to have low levels of chloroplast organelles. Between these two values of 100ml to 50ml of Red diesel, the lowest level of chloroplasts at three weeks was estimated between an average count between 10.6 and to 21.6 respectively. This data suggests that the higher levels red diesel (200ml, 100ml, and 50 ml) was significantly toxic to the plant. Based on the data, the 25ml concentration of red diesel was least damaging to the life of the plant. Furthermore, the number of chloroplasts (37.6) counted for the third week, when compared to the control demonstrated that Elodea densa had a tolerance for red diesel at this particular concentration.

Condition of Plasma Membrane-
Microscopic observations made of Elodea densa at week one, using a 40X objective lens for the 200ml (40%) concentration of red diesel immediately compromised the integrity of the cell. Observations made using the microscope revealed that chloroplasts were clustered by large droplets of red diesel (micelle bodies). Also, conditions of the plant cell at 100ml (20%) were significantly impaired in that the cell wall appeared damaged and the chloroplast organelles were clustered in red diesel lipid vesicles as compared to the control. At 50ml to 25ml the plasma membrane appeared to be non-plamolyzed with viable green chloroplast and normal Brownian movement.
At week two, the conditions appeared progressively worse for the plasma membrane at both 200ml and 100ml red diesel concentration. The 200ml concentration appeared to have more micelle vesicles than the 100ml red diesel concentration. The plasma membrane appeared plasmolyzed and some red dye was incorporated into the cell. At 50ml and 20ml red diesel concentration, the cell was more intact because the chloroplast appeared to be less damaged than the higher concentrations of red diesel. The 50ml and 25 ml still had some significant damage to the plasma membrane as compared to the control. The control had very green and non-plasmolyzed cells. By the third week, results indicate that all plasma membranes except for the control were severely impaired, by having fragmented plasma membranes. The decreased population of chloroplast at week three also supports the observational findings of the plasma membrane.

Growth of Stolon-
Stolon growth initially was significantly affected by the different concentrations of red diesel at week one. At week one, the control growth for stolon was 5.5cm as compared to .5 cm growth for the 200ml and 1.0 cm growth for the 100ml. However, at week two, stolon growth compared to the control was not significantly different. Also, stolon growth at week three was not significantly different from the control. The ability of stolon to grow under these red diesel conditions may indicate that the plant has an ability to adapt to its environment or may also explain the ability for the cells in this particular region of the plant cell to not be affected by the compounds in red diesel (different components in the cell wall of the stolon cells).

Growth of stem-
Measurements of the stems, for all red diesel concentrations in Elodea densa, found no significant differences compared to the control. For example, at 200ml red diesel concentration for week three, stem length was found to be 11.5cm compared to the control which measured at 12.0 cm. Furthermore, stem growth data was not significantly different for all of the remaining concentrations of red diesel.

Red diesel fuel is used for tractors and other types of heavy engine equipment. It is a very inexpensive fuel to use for various purposes when operating heavy machinery. According to the EPA (Environmental Protection Agency), red diesel fuel is a primary source of environmental contamination. For example, production of various pollutants such as: VOC’s, NOx, and SOx. These gaseous compounds can react with water to produce acidic compounds which contribute to acid rain and significantly alter the marine life of various different species. Furthermore, Red diesel fuel also contains a significant amount of aromatic compounds that can cause cancer and respiratory problems. The most common chemical formula for red diesel is C12H23. The concentrations tested for the Elodea densa were as follows: 200ml of Red Diesel (40%), 100 ml of Red Diesel (20%), 50 ml of Red Diesel (10%), 25 ml of Red Diesel (5%) and 0 ml of Red Diesel (0%) (control). Chloroplast population decreased each week. Furthermore, based on the data the Elodea that was subjected to 200 ml of Red Diesel (40%) diesel experienced the highest loss of chloroplast organelles, approximately 83.8%. The Elodea which was exposed to Red Diesel fuel at both 100ml and 50 ml also experienced significant loss of chloroplast, 77.4% to 55.3% respectively when compared to the control. Chloroplasts, when viewed underneath the microscope, one could easily observe the plasmolyzed condition of the plasma membrane. Furthermore, the chloroplast where clustered within the plasmolyzed cell. The limit of survival of Elodea densa was observed at 25 ml based on our data. The 25ml red diesel concentration demonstrated a 20% loss in chloroplast population when compared to the control. Stolon growth was examined under each condition and no significant difference was observed when compared to the control. Furthermore, the length of the stems were measured and analyzed for all concentrations of the plant. Data revealed no significant change in stem growth with respect to red diesel concentration. Red diesel is an organic compound that can be readily entered into the plasma membrane of the plant cell and easily entered into the chloroplast membrane. In summary the Red Diesel was found to be toxic to the Elodea plant inner cell structure.
According (Young 1935) oils presumably enter living cells when they pass from living leaves into the tracheae of attached stems. The data obtained in the study by (Young 1935) mentions that the “oil mass theory” believes that petroleum oil touches and mixes with oil miscible substances concentrated in the plasma membrane, forming large mass with an oily continuous phase. Furthermore, according to the study conducted by (Young 1935) Intracellular oil and extra-cellular oil diffuses through the plasma membrane into the cytoplasm of the plant cells. The study conducted by (Young 1935) supports the data obtained in my experiment, because the red diesel entered through the plant cells and caused plasmolysis, and disruption of the chloroplast membrane and ultimately decreasing their population. Although stolon and stem growth were not effected negatively, the damage to the chloroplast carries significant implications to the environment and the ability for marine organism to be able to process and produce photosynthetic oxygen. I strongly believe, alternative and cleaner types of fuel must be produced and utilized for the sake of our environment. Also, there is a definite time effect with respect to damage to the chloroplast. The first week was significantly less damaged that the third week. This implies that when there are oil spills in water that the early the contamination gets cleaned up the better. The longer time the plants species or marine life are exposed to the contaminated red diesel the more devastating effects to the cellular processes of these organisms.

In summary, the Elodea densa plant was able to adapt to its aquatic environment. Examples of adaptation were as follows: (1) the plant was able to demonstrate stolon growth and stem growth under high concentration of red diesel. Measurements taken and recorded showed significant changes when compared to the control for week one. However, week two and three showed that Elodea densa was able to adapt to its aquatic environment. Measurements of stolon growth for week 3 were not significantly different from the control. Data obtained from week 3 provides an explanation that Elodea densa was ultimately able to adapt. In conclusion there were not significant changes in the growth of stem exposed to different concentration of red diesel.
The different concentration levels of red diesel were extremely damaging to the cellular structure of the cell. Damage was easily noticeable at 40X and chloroplast population count was detected at 40x objective. The 200ml concentration was most damaging to the plant in that most of the chloroplast population fell from 47 to 7.6 on average when compared to the control. This is an 83.8% drop in chloroplast population. The function of the chloroplast organelle is to undergo photosynthesis and produce oxygen. One can conclude that the ability for the Elodea to produced oxygen was significantly altered. This finding can potentially be devastating to other aquatic species in the ocean that come across contaminated water with red diesel. Future direction with respect to this project would be to measure the amount of oxygen production in the different concentration levels of red diesel. This would support the damage done to the Elodea densa chloroplast. One of the mistakes noticed with this experiment was that I should have measured everything with a graduated cylinder instead of using a beaker because this would have been more accurate.
In conclusion, stolon growth and stem growth were not significantly affected. This implies that the tolerance level for these variables high. However, long term effect of red diesel concentration can significantly impair the functioning of the plant by affecting photosynthesis.
· Date: August 11, 2011 · Views: 2770 · File size: 12.0kb, 61.3kb · : 567 x 339 ·
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