VARIABLES
As well as pH levels, there are other variables which can change unintentionally, and thus affect the outcome of the experiment. These include:
- Volumes and concentrations of solutions
- Temperature
- Light availability
- Wind/Movement
The concentrations will be varied intentionally, and therefore do not need to be controlled. The volumes of solution that each sample receives will be kept constant amongst all the samples. I will attempt to control the remaining variables by keeping all of the samples in the same room, in the same area, with closed windows and doors. In the event of any of these variables (bar volume and concentration) changing beyond my control, I do not believe that it will be detrimental to the experiment as a whole, as all of the samples will be subject to identical conditions, whatever the change.
After completing the experiment, it was clear that the only samples which were successful in any germination were those of both acids with either 0% concentration or 20% concentration i.e. those with the lowest concentrations. I took the pH values of each solution using universal indicator, and discovered that all of the solutions (bar the distilled water) had a pH value of either 1 or 2. I had intended for the solutions to have a more diverse range of pH values, as I had already suspected that such acidic conditions would not have resulted in any germination. As expected, the samples with only distilled water showed great success in germination, and those with only 20% concentration showed very little, but some signs of germination.
RESULTS
After evaluating the results of the pilot experiment, I decided to make certain modifications to the experiment:
- I would alter the volumes used when diluting the acid so that there would be a more diverse range of pH values amongst the solutions.
- As the sample with the lowest concentration of Sulphuric Acid germinated more successfully than the sample with the lowest concentration of Hydrochloric Acid, I decided to use only diluted concentrations of Sulphuric Acid, but with repeat tests.
- For greater accuracy in my results, I would use 50 seeds for each sample rather than 40.
- I would no longer use mass as a measure of germination, and focus on length of stem and general physical appearance.
In light of the necessary modifications, I will produce the following solutions:
I will produce one repeat sample for each solution, making a total of 14 samples.
I would use the apparatus as used in the pilot with the following exceptions:
- No Hydrochloric Acid
- No Scales
- No 10cm³ Measuring Cylinders
- 3 less Bottles (total 5)
- 1 additional 100cm³ Measuring Cylinder (total 4)
- Additional 720cm³ Distilled Water (total 1320cm³)
- Additional 380cm³ Sulphuric Acid (total 380cm³)
- 220 additional Cress Seeds (total 700)
- 2 additional Petri Dishes (total 14)
- 5 x 10cm³ Syringes
As acid rain is a common occurrence today due to profuse emissions of gases such as Sulphur Dioxide and Nitric Oxide, I carried out an experiment based on the hypothesis that when grown in a variety of acidic pH values, germination success will decrease as acidity increases. I attempted to germinate several samples of cress seeds in petri dishes lined with cotton wool, using solutions diluted with different concentrations of Sulphuric Acid. I chose Sulphuric Acid, as Sulphur Dioxide is one of the constituents of acid rain. The pH values of the solutions ranged from 1 to 7 (there was also a sample given only distilled water). I made two samples for each solution in order to make an average for more accurate data analysis. Everyday for five days, I would measure the length of any sprouts or stems, and take note of significant changes in physical appearance, as well as adding more of each solution to the corresponding samples.
The samples with the two highest concentrations (10% and 100%) of Sulphuric Acid did not germinate at all, whilst the remaining samples germinated at different rates. The remaining samples all reached 100% germination by day two, although the samples with the lowest concentrations of Sulphuric Acid had almost doubled the % germination of others after day one. There was also significant difference in physical appearance between the samples. Once again, the two samples with the lowest concentration (0% and 0.1%) of Sulphuric Acid continued to grow to up to 58mm, whilst the remaining samples (of 0.3%, 0.5%, and 1% concentrations) reached between 3mm and 7mm at day three, and then stopped. To prove/disprove my hypothesis, I tested my results using the Chi-squared goodness of fit test. The results of this statistical analysis proved that there was a significant difference of germination success amongst the different acidic conditions which did not occur by chance.
At the end of the experiment, with the supporting evidence of the statistical analysis, I concluded that my hypothesis was correct in that the samples which were only given the least concentrated solution of Sulphuric Acid germinated more successfully than the others. To further support my hypothesis, I also noticed that although the remaining samples did germinate, the success of germination decreased as the level of acidity increased (with the exception of the samples given only distilled water), indicating that pH level does affect the germination of seeds.
A seed is a fertilised and ripened ovule, consisting of the plant embryo, varying amounts of stored food material (endosperm), and a protective outer seed coat (aleurone layer) (1). The embryo consists of the cells that will develop into the structures of the adult plant (root, bud, stalk, and leaf), and secretes hormones such as cytokinin, and indole acetic acid, which promote cell division (2). The endosperm contains insoluble food stores including starch, proteins, and lipids, all of which are used in the initial germination stages. The aleurone layer contains proteins, and under the influence of gibberellic acid (manufactured and secreted by the embryo) synthesises amino acids into hydrolytic enzymes such as -amylase and a protease. These enzymes are then used to hydrolyse starch glucose, and proteins amino acids for further growth and development (1).
The scale used to measure the acidity or alkalinity of a solution is the pH scale, which generally ranges in values from 0-14. Acidic solutions have pH values below pH 7, which is the value of a solution considered to be neither acid nor alkaline i.e. neutral. In pure water, the concentration of hydrogen ions is equal to 10ˉ ; when an acid is added to pure water, the hydrogen ion concentration increases above this level (3). The pH of a solution is a measure of the hydrogen ion concentration in that solution. The pH scale is logarithmic, so a small change in pH represents a large change in hydrogen ion concentration. For example, the hydrogen ion concentration of gastric juices (pH 1) is nearly 400 times greater than that of pure water (pH 7) (2).
The pH level of the solution in which the seeds are germinating affects the availability of elements. Extreme acid/alkaline conditions may adversely affect plant growth by altering selected nutrient availability (3). Proteins have specifically charged groups such as carboxyl groups and amino groups. Since the charge of these groups depends on pH, a protein molecule can have different charges according to pH. In general, positive charges and negative charges on the surface of protein are well-balanced around neutral pH. Because of electrostatic attraction, the shape of the protein is compact and stable. However, for example, at extremely low pH levels, the carboxyl group is broken and negative charges are decreased. Thus, proteins will lose the stability which comes from electrostatic attraction, and gain more electrostatic repulsion between the increased positive charges. This is how proteins are denatured at extreme pH (4).
Rain water is naturally slightly acidic due to the presence of carbon dioxide in the atmosphere which forms weak carbonic acid in water. Acid rain is a form of air pollution formed when oxides of Sulphur and Nitrogen combine with atmospheric moisture to create sulphuric and nitric acids. The emissions of these gases come mainly from traffic exhaust and smoke from factories, which has increased since the industrial revolution. Acid rain can significantly affect the growth of plants by damaging root systems, and rendering leaves unable to carry out sufficient photosynthesis (2).
I began the experiment by labelling the bottles with the concentration of solution that they would hold: 0.1%, 0.3%, 0.5%, 1%, and 10%. The 0% and 100% concentrations did not need bottles as I used the H2SO4 and H2O directly from the containers in which I received them (which were already labelled). I also labelled 2 of the 100cm³ measuring cylinders with ‘Sulphuric Acid’ and ‘Water’ to prevent any accidental contamination. Similarly, I labelled the 10cm³ syringes with the concentration that each would be used for throughout the experiment (0.1%, 0.3%, 0.5%, 1%, and 10%). The next step was measuring out all of the solutions of H2SO4 and H2O to produce the respective concentrations. I began by making the 10% concentrated solution; using the 100cm³ measuring cylinder labelled ‘Water’, I measured 180cm³ of H2O into the corresponding bottle labelled ‘10%’. I then used the second 100cm³ measuring cylinder labelled ‘Sulphuric Acid’ to measure 20cm³ of H2SO4 and added this to the bottle, and shook the bottle gently to mix the solution. I then made the 1% concentrated solution; I first measured 360cm³ of H2O using the 100cm³ measuring cylinder labelled ‘Water’, and put this into the corresponding bottle labelled ‘1%’. Using one of the remaining two 100cm³ measuring cylinders I measured 40cm³ of the 10% concentrated solution which I added to the bottle, and shook gently to mix the solution. I then made the 0.5% concentrated solution; I measured 100cm³ of H2O using the 100cm³ measuring cylinder labelled ‘Water’ and put this into the corresponding bottle labelled ‘0.5%’. Using the second of the remaining two 100cm³ measuring cylinders, I measured 100 cm³ of the 1% concentrated solution which I added to the bottle and shook gently to mix the solution. I then made the 0.3% concentrated solution; I first measured 140cm³ of H2O using the 100cm³ measuring cylinder labelled ‘Water’ and put this in the corresponding bottle labelled ‘0.3%’. Once again I used the second of the remaining two 100cm³ measuring cylinders to measure 60cm³ of the 1% concentrated solution which I added to the bottle and shook gently to mix the solution. I then made the 0.1% concentrated solution; I measured 180cm³ of H2O using the 100cm³ measuring cylinder labelled ‘Water’ and put it in the corresponding bottle labelled ‘0.1%’. Using the second of the remaining two 100cm³measuring cylinders I then measured 20cm³ of the 1% concentrated solution which I added to the bottle and shook gently to mix the solution. After all the solutions had been made, I used Universal Indicator to measure the pH value of all the solutions (including the Distilled Water and undiluted Sulphuric Acid), and recorded the results. I then cut out fourteen circles of cotton wool to fit in all of the petri dishes, and labelled two dishes for each solution (0.1%, 0.3%, 0.5%, 1%, and 10%). Because there were two samples for each concentration, I also labelled one sample ‘A’ and the other ‘B’ (for all concentrations). I then used the forceps to count fifty cress seeds for each sample, and sprinkled them into the petri dishes by hand in a random fashion. Using five separate 10cm³ syringes (which were all labelled ‘0.1%’, ‘0.3%’, ‘0.5%’, ‘1%’, and ‘10%’ according to the samples for which they were going to be used), I then added 30cm³ of each solution to the corresponding sample petri dishes, and placed all of them on the same shelf, side by side, in a straight line. The shelf was opposite a window, with nothing to obstruct the light. 24 hours later, I measured the longest stems in each sample with a ruler, and recorded the length in mm of the longest one. I also took note of any change in physical appearance of each sample as a whole. Using the same five labelled 10cm³ syringes, I added a further 10cm³ of each solution to the corresponding sample petri dishes. I repeated these daily tasks every 24 hours for a further 4 days. Throughout the experiment, I did as much as I could to control all variables to make the experiment more accurate and fair. The concentrations were varied intentionally, and therefore did not need to be controlled. The volumes of solution that each sample received were kept constant amongst all the samples. I attempted to control the remaining variables of temperature, light availability, and wind/movement by keeping all of the samples in the same room, in the same area, with closed windows and doors.
Table showing average percentage germination (%) and stem length (mm) over 5 days
Other Observations:
Statistics – The Chi-Squared Goodness of Fit Test
Table showing observed and expected results from day 1 of the experiment
Null hypothesis: success of seed germination is not dependent on pH level .˙. the same number of seeds should germinate in each sample.
I tested this null hypothesis by means of a χ² for the results obtained from day 1.
Degrees of freedom = 6 .˙. critical value at 0.05 = 12.59
χ² = 289.1649048 .˙. null hypothesis is incorrect; there is a significant difference which did not occur by chance.
It is clear from the results that there is a distinct link between pH conditions and the germination of seeds. This is mostly indicated by the almost direct correlation between increase in acidity and decrease in germination success, as well as the supporting evidence from the statistical analysis. However, although the results show that germination success was initially proportional to concentration of Sulphuric Acid (proving my hypothesis to be correct), the actual growth of the cress plants was most successful overall with the 0.1% concentrated solution (proving my hypothesis to be incorrect), Although the latter point proves my hypothesis to be incorrect, the principles that pH is directly linked germination success, and that higher acidic conditions are proportionally linked to the adverse effect on germination success remain true. There are of course, however, other factors involved which may have affected the experiment as a whole. Throughout the experiment, I did not endeavor to keep variables such as temperature, wind and light availability constant, simply for the reasons that all of the samples would be subject to the same conditions and would therefore theoretically make the test fair, and also because I felt that there would not be significant changes that would affect the experiment as a whole. The majority of results gained from the experiment were expected, however there were some anomalies. The main anomaly is made more distinct in the graph showing % germination on day 1. In accordance with my hypothesis, it would have been expected that more seeds would have germinated in the 0.5% concentrated sample than in the 1% concentrated sample (but less than in the 0.3% concentrated sample). My initial estimate to explain this anomaly would be the distribution of seeds at the beginning of the experiment. As the distribution was random, this could have easily contributed to the fairness of the experiment, as some seeds may have been in a better position than others in terms of access to the solution, as well as light availability. For example, the coordination of the cotton wool may have been in such a way that the seeds further away from the light (at the back of the dish) may have received less light due to the cotton wool obstructing them. Moreover, when the solutions were added, this was also random and may not have always soaked the entire cotton wool, thus enabling more access to the solution to some seeds than others. In combination with this theory, movement may have also been a problem as before germination had fully developed in some seeds, they would easily be moved about when the solution was added, thus perhaps giving them more of an advantage/disadvantage. Although the germination rates were different amongst the samples, there was a clear distinction between those that did germinate and those that didn’t at all, and all those that did germinate reached 100% germination regardless. The highest concentrated samples (10% and 100%) did not germinate at all, most likely due to the enzymes required for germination being denatured. However, the seeds in the 10% concentrated sample always remained a lighter colour than those the 100% concentrated sample, indicating that although the concentrations were too strong for any germination, the minor difference between them still affected their outcome (although perhaps not significantly).
Although limitations were fairly minor, I believe that the experiment could be improved as a whole if all the variables could be controlled absolutely. For example, if the seeds were grown in the same way, but placed on the cotton wool in a more controlled manner, one by one if necessary. This would help to prevent blocking of light or accessibility to the solutions by the cotton wool. Alternatively they could be grown in another medium similar to cotton wool in that it would not interfere with the germination process such as paper towels. Additionally, if the samples were kept in a room with a controlled temperature and the availability of natural light was controlled, this too would help to make the experiment more accurate as a whole.
In terms of how acid rain affects the growth of plants in general, tests such as this one could be carried out with other acids found in acid rain such as Nitric Acid and Carbonic Acid varying the concentration of each; perhaps one particular acid in the solution of acid rain has more or less affect on plant germination than another. Furthermore, the same tests could be carried out using a variety of other seeds which may be more/less tolerant to the acidity, which could then be developed to research into what makes plants more/less tolerant to acidic conditions.
1. http://www.bartleby.com/65/ph/pH.html
2. Microsoft Encarta Encyclopedia 2002
3. W R Pickering, Advanced Biology Revision Handbook, 1994, p. 50
4.
Raw Data
* = Average
% = seeds germinating (%)
mm = length of sprouts/stems (mm)
