Blevins – Hagan 1

Introduction

All liquids have a pH level; from the soda you drink to the water in your shower. Rain

water from the sky also has a pH level. When the pH level of rain becomes too low, or acidic, it

becomes known as what is acid rain. Acid rain can harm plants that have roots that sit in the soil

that has absorbed acid rain.

Acid rain often negatively effects the germination rates and growth of different plants

because in the cell membrane of a plant, normally only hydrogen and oxygen are allowed in,

which help to determine the pH gradient of the plant. However, when acid rain is added to this

mix, it can often disturb the pH gradient of the plant, which causes the growth and germination

issues. The remnants of sulfate left inside the plant can suck out important nutrients (Seefeldt).

Acid rain can sometimes have an direct effect itself, but more often than not, the problem lies

within the soil and the plant absorbing the precipitation. Since acid rain has to do with the pH of

the plant, the experiment in a sense simulates seeds soaking in acid rain on a smaller scale, with

absorbent paper towel acting as the soil, and seeing how their germination rate is affected.

When radish seeds are grown in higher temperatures, they tend to germinate faster than

seeds grown in lower temperatures. Growing seeds in very high temperatures reduces the

efficiency of the enzymes in the seeds, this causes the cellular protein within the seed to

be denatured, which kills the seed (Hatting). Due to this, the temperatures 36 degree Celsius, 37

degree Celsius, and 38 degree Celsius were chosen for the seeds to be germinated

in. Temperatures over 38 degree Celsius would be to harmful to the seeds.

The objective of the experiment was to germinate using different pH levels of solutions

and varying temperatures. They were three different solutions each with a different pH level.

Twenty radish seeds were placed in between paper towel, and placed in a plastic sandwich size
 Blevins – Hagan 2

bag. The seeds were then watered with 20 mL of one of the three solutions. The bags of seeds

were then placed into one of three incubators, each with a different temperature. The seeds

stayed in the incubators for four days, and then taken out and examined to see what percentage of

the twenty seeds had germinated in their conditions.

The data was recorded by counting the number of seeds that had germinated out of

twenty. This number was then turned into a percent in order to find the average germination

percentage for the seeds. This method of recording the data was the most efficient and produced

the easiest numbers to work with and interpret.

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Problem Statement

Problem:

How will different pH levels and temperature affect the germination rate of

ten Raphanus sativus seedlings over a 4-day growing sequence?

Hypothesis:

If the seedlings are treated with the low solution (a pH of 6) and the high level

temperature (38 degrees Celsius), then the lowest percentage of seeds will germinate.

Data Measured:

The independent variables in the experiment are the temperature the seeds are kept at and

the pH of the solution poured on the seeds. The temperature will be measured in

degrees Celsius and the solution will be measured in pH. The high, standard, and low levels for

the pH were chosen by basing it off of the fact that water has a neutral pH of 7. Therefore, in the

experiment the pH of 6 will be used as our low, which will be the acidic solution, the pH of 7

will be used as our standard, which is the neutral solution of water, and the pH of 8 will be used

as our high, which is the basic solution. The high, standard, and low levels for the temperature in

degrees Celsius was based off of the average human body temperature, which is 37 degrees

Celsius. To get our low and high level are simply one degree above and below 37 degrees

Celsius, making our low level 36 degrees Celsius and our high level 38 degrees Celsius. The

dependent variable of the experiment is the germination rate of the seeds, as the pH levels and

temperature will affect the germination rates. To measure the experiment, the number of seeds

that have germinated will be counted.

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Experimental Design

Materials:
(6) 500 mL Water Bottles (1) Paper Towel Roll
½ teaspoon of Citric Acid ½ teaspoon of Baking Soda
(105) Ziploc Sandwich Bags (2100) Radish Seeds
(1) Permanent Marker (1) 36 Degree Celsius Incubator
(1) 38 Degree Celsius Incubator (1) 37 Degree Celsius Incubator
(1) 20 mL Graduated Cylinder (1) Bottle of pH Test Strips
(1) ¼ teaspoon Measuring Spoons

Procedure:

1. Prepare the acidic Solution (Low Solution)

a. Pour ¼ teaspoon of Citric Acid into a 500 ml water bottle

b. Shake the water bottle until the Citric Acid is completely combined

2. Repeat steps 1 through 1b, with another 500 ml water bottle.

3. Prepare the basic Solution (High Solution)

a. Pour ¼ teaspoon of Baking Soda into a 500 ml water bottle

b. Shake the water bottle until the Baking Soda is completely combined

5. Repeat steps 3 through 3b, with another 500 ml water bottle.

5. Take a piece of paper towel and place twenty radish seeds inside

6. Fold the paper towel over so the radish seeds are securely tucked inside

7. Pour 20 ml of the acidic solution on the paper towel and place the towel inside the Ziploc bag

8. Make sure the bag is sealed well

9. Label the bag "acidic Solution #1" in permanent marker

10. Repeat steps 2-6 with the basic solution, making sure to label the plastic bag with "basic

Solution #1"

11. Repeat steps 2-7 twice more, this time changing the labels to say "acidic Solution #2" "basic

Solution #2" so on and so forth

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12. Repeat steps 2-6 with the standard solution (water)

13. Place "basic Solution #1" and "acidic Solution #1" in an incubator set at 36 degrees Celsius

14. Place "basic Solution #2" and "acidic Solution #2" in another incubator set at 38 degrees

Celsius

15. Place the bag with the standard solution in an incubator set at 37 degrees Celsius

16. Leave the seeds for four days

17. Take all of the bags out of the incubators and see how many out of 20 have germinated

Diagram:

Figure 1. Seeds and Solutions

Once the solutions have been made and poured onto the seeds, the result should
look like this.
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Figure 2. Seeds Inside Incubators

This is how the plastic bags with the seeds should look once inside the incubators.
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Data and Observations

Data:

Table 1
Design of Experiment Values
Incubation Temperature ( C ) pH of Solution
(-) Standard (+) (-) Standard (+)
36 37 38 6 7 8

Table 1 above shows low, high, and standard values for each variable. The standard for

the incubation temperature is 37 degrees Celsius. This value was chosen because 37 degrees

Celsius is the temperature of the human body. In addition, there are only three different

incubators were available for use. The three incubators available were 36 degrees Celsius, 37

degrees Celsius, and 38 degrees Celsius. The standard value for the pH of the solution is 7 pH.

This was chosen based on that water has a natural pH level of 7.

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Table 2
Rate of Seed Germination in Each DOE
Percentage of Seeds that Germinated (out of 20)
DOE pH Level, Temperature in Degree Celsius
(+,+) (+,-) (-,+) (-,-) Standard Standard Standard
1 45 10 70 20 45 65 65
2 55 20 40 15 45 65 70
3 70 20 30 5 90 85 90
4 50 20 30 10 75 95 65
5 45 20 30 10 75 50 85
6 50 20 50 10 60 60 50
7 30 30 20 30 75 60 65
8 25 30 40 15 50 70 80
9 40 35 20 20 70 65 75
10 30 35 36 15 75 90 95
11 35 20 25 15 65 65 60
12 30 25 35 15 65 80 75
Average 42 23.33 40.42 15.83 65.833 70.833 68.75
Table 2 above shows all twelve runs and the order in which they were conducted. It also

shows the three standards of each run, in the order they were conducted. It also shows the overall

averages for the (+,+), (+,-), (-,+), (-,-), and all three standards.

Observations

Table 3
Observations
Date Observations
The seeds that had been kept inside of the highest incubator (at 38
Throughout The degrees Celsius) have lots of yellowing on the paper towel around
Experiment them. (see Figure 3.)
The seeds were omitting a foul odor after being removed from the
Throughout The incubators. This was the case for all treatments except for the
Experiment standards
In the (minus, plus) bag, which is the seeds treated with a 6 pH
and set in the 38 degree incubator, had a large circle patch of what
3/28/17 appeared to be mold.
In the (plus, minus) bag, the paper towel covering the seeds had
3/28/17 turned a yellow-brownish color. (see Figure 5.)
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Figure 3. Seeds Inside Highest Incubator

Figure 3 shows an example of the yellowed paper towel from the experiment. The

yellowing occurred for the seeds treated with the high pH, and also the seeds treated with the low

pH.
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Figure 4. Seeds that Developed Mold

Figure 4. shows the seeds that were treated with the low pH and set in the high incubator.

As shown in the image, there is a large patch of what appears to be mold growing on the paper

towel in which the seeds were left to germinate in. This did not occur during any of the other

trials, or any of the other treatments, so it can likely be concluded that some kind of error

occurred during the prepping of this trial.

 Blevins – Hagan 11

Figure 5. Yellowish Browning in Bag

Figure 5. shows the color difference between the paper towel for the seeds placed in the

low incubator with the high pH compared to the other bags with different treatments from that

run. The whole paper towel has turned a dark yellow-brown shade, while most of the others had

remained their original white color.

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Data Analysis and Interpretation

A two-factor design of experiment (DOE) was used to calculate the affects that different

pH solutions and incubation temperatures had on Raphanus sativus seed germination. The data

from the experiment was collected by placing twenty radish seeds inside of a folded paper towel,

watering them with either a high pH of eight or a low pH of 6, and then placing them inside an

incubator at either thirty-six or thirty-eight degrees Celsius. In this experiment, each trial was

conducted twelve times. There were thirty-six standard trials, each treated with a neutral pH

solution and placed in a thirty-seven degrees Celsius incubator.

Table 4
Table of Values
pH Level Temperature in Degrees Celsius
- Standard + - Standard +
6 7 8 36 37 38

Table 4 shows the low, standard, and high levels of pH, along with the low standard and

high levels for temperature. The values for pH were chosen because a pH of 7 (the standard) is a

neutral solution of water, so the high and low values are one number to change the pH, but not so

much that it will kill the plants.

Table 5
Averages
(+,+) (-,-) (+,-) (-,+)
Average (%) 42.08 15.83 23.33 40.42
Grand Average(%) 30.415

Table 5 above shows the averages for each treatment value and the grand average. The

data represents the percentage of seeds that germinated (out of 20 seeds). The grand average was

calculated by averaging the four averages together. To do this, add the four averages

together then divide the sum by four.

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Table 6
Effect of pH Level
Effect of pH Level 45.00%

PERCENTAGE OF SEEDS GERMINATED

pH Level (Percent Germinated) 40.00%
(-) 6 (+) 8 35.00%
15.83 42.08 30.00%
25.00%
40.42 23.33
20.00%
Avg = 28.125 Avg = 32.705
15.00%
10.00%
5.00%
0.00%
-1 1
Figure 1. Effect of pHPHLevel
LEVEL

Figure 6. Effect of pH Level

Table 6 shows the trial outcomes from when the seeds were treated with a low pH and a

high pH. The seeds treated with the low pH solution had an average germination percentage of

28.125. The average germination percentage for the seeds treated with the high pH solution was

32.205. The difference between the two averages for pH is not that large, and comes out to be

4.58. This means that on average, as the pH level that the seeds were treated with increases, the

percentage of the seeds that germinated also increases. As determined but the test of

significance in Figure 5, the effect value of 4.58 was not statistically significant.
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Table 7
Effect of Temperature Effect of
Temperature C° (Percent Germinated)
(-) 36 (+) 38 Temperature
45.00%
15.83 42.08

PERCENTAGE OF SEEDS GERMINATED

40.00%
23.33 40.42
Avg = 19.58 Avg = 41.25 35.00%
30.00%
25.00%
20.00%
15.00%
10.00%
5.00%
0.00%
-1 1
TEMPERATURE OF INCUBATOR
Figure 7. Effect of Temperature (Degrees Celsius)

Table 7 above shows the numbers recorded for the seeds placed in the high temperature

incubator (38 degrees Celsius), and the low temperature incubator (36 degrees Celsius). The

average germination percentage for the seeds that were placed in the incubator set at 36 degrees

Celsius was 19.58, while the average germination temperature for the seeds that were placed in

the incubator set at 38 degrees Celsius was 41.25. The difference between these two numbers is

21.67. This means that on average, as the temperature of the incubator the seeds are placed

within increases, the percentage of the seeds that germinate increases as well. As determined by

the test of significance in Figure 10, the effect value of 21.67 was found to not be statistically

significant.
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Table 8
Interaction Effect Effect of Temperature
pH Level (Percent and pH Level
Germinated)
45.00%
(-) 6 (+) 8

PERCENTAGE OF GERMINATED SEEDS

40.00%
Solid
(+) 38 40.42 42.08 35.00%
Temperature Segment 30.00%
(in degrees
Dotted 25.00%
C) (-) 36 15.83 23.33
Segment 20.00%
15.00%
10.00%
5.00%
0.00%
-1 1
PH LEVEL

Figure 8. Interaction Effect

Table 8 above shows the interaction effect between the temperature and the pH

level. The interaction effect between the two variables was calculated by taking 40.42 and

subtracting it from 42.08 and dividing it by two, then taking 23.33 and subtracting 15.83, and

also dividing that answer by two. From there, the solution from the first subtraction equation

(42.08/2 - 40.42/2, which equals 0.83) and subtracting the solution from the second subtraction

equation (23.33/2 - 15.83/2, which equals 3.75). This gives a final answer of –2.92 as the

interaction effect. The interaction effect represents how the two variables affected each

other. Since the slopes of the two line segments are both positive, it can be concluded that there

was little to no interaction effect between the two variables. In addition, based on the test of

significance in Figure 10. It was found that the interaction effect pf -2.92 was not statistically

significant.
 Blevins – Hagan 16

Scatter Plot of Standards

100%
Percentage of Germinated Seeds

80%

60%

40%

20%

0%
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Standard Number

Figure 9. Scatter Plot of Standards

Figure 9. above shows the thirty-six standards that were conducted throughout

the experiment. From standards one to seven, the numbers seem to have a slightly upward trend,

and after standard seven, the numbers appear to fluctuate up and down slightly, with no dramatic

increases. The standards of the experiment act as a control, because the rest of the data taken

should always be compared to the standards. If the highs and lows of the experiment produce

results that are very close to the standards, then there is likely no data that is statistically

significant. Since the standards do not remain constant throughout the experiment, there is a

likely possibility that all the data taken could have happened by chance alone, and not actually

been effected by the pH level or the incubation temperature. This also means that there could

have been lurking variables when we actually carried out the experiment, such as how long the

solutions had sat or things of that sort.

 Blevins – Hagan 17

Dot Plot of Effects

4.58

-2.92 21.67

-100-95-90-85-80-75-70-65-60-55-50-45-40-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95100
Figure 10. Dot Plot of Effects

Figure 10. above shows the three effect values graphed on a number line. The

pH effect value is 4.58 (green dot). The temperature of the incubator effect value is 21.67 (blue

dot). Lastly, the interaction effect is -2.92 (red dot). The range of standard was found to be 50%.

To find if anything was statistically significant, the range of standards in multiplied by 2. This

will be our fences. 50% multiplied by 2 is 100%. This means the fences are 100 and -100 (black

lines). If any of the effects fall between these fences, that means it is not statistically

significant. Since the fences are 100, this means that nothing could have been statistically

significant. From this it can be concluded that the values of the experiment might have been too

close in range. To fix this, the values in Table 4 would need to be changed. All three of the effect

values are not statistically significant. However, the interaction effect value of 21.67 is the

farthest away from 0 so that means, that the temperature of the incubators, while it did not pass

the test for statistical significance, had the biggest effect on the percentage of seeds germinated.

Parsimonious Prediction:
Y = 30.395 + “noise”

Figure 11. Parsimonious Prediction

The parsimonious prediction equation is simply equal to the range of standards, because

nothing in the experiment was determined to be one of the "vital few". The parsimonious

prediction equation is to determine what the outcome of the experiment would be if it were to

be performed again with different predictor variables, to further improve the research. However,
 Blevins – Hagan 18

in this example, there are no statistically significant factors to tweak to redo the experiment once

again. This makes it very difficult to infer what could happen if this experiment was done with

interpolated numbers. However, since the temperature factor was practically significant, the

degrees in Celsius could be interpolated so that they were a father distance a part, to see how the

temperature really had an effect on the data. If this experiment was ran again, it could be

expected that the results would show that on average, 30.395% of the seeds would have

germinated.

Interpretation:

In summary, nothing in this experiment was deemed statistically significant. This means

that there is a very likely chance that all the data that has been collected could have simply

occurred by purely chance alone. However, even though there were no statistically significant

variables in this experiment, the temperature was a variable that was practically significant. This

is because there is quite a large difference in the averages of the seeds grown in the high

temperature versus the seeds grown in the low temperatures.

 Blevins – Hagan 19

Conclusion
It was hypothesized that the low pH solution and the highest temperature would produce

the lowest amount of seed germination. The hypothesis was rejected due to the fact that the low

pH (6) and the low temperature (36°C) produced the lowest amount of seed germination. The

low pH and high temperature preformed the second best out of all of the solutions, contrary to

what was expected to happen.

Acid rain can often have harmful effects on plant germination (Seefeldt). Temperature

can also have negative effects on plant germination (Chen). The intent of this experiment was to

see if crop seeds such as radishes could survive and germinate healthily when exposed to varying

pH levels. The factor of temperature helps to understand where radish seeds can be grown most

successfully if they are simultaneously being exposed to potentially harmful pH levels. To

acquire results, twenty seeds were placed inside a folded paper towel, had 20 mL of either 6 pH

solution or 8 pH solution poured onto them, set inside a Ziploc bag, and then placed inside of an

incubator that was set either at 36, 37, or 38 degrees Celsius. The seeds treated with the low pH

solution (6) had an average germination percentage of 28.125, while the seeds treated with the

high solution (8) had an average germination percentage of 32.705. Temperature, however

played a larger role in effecting how many seeds germinated. The seeds placed in the low

temperature incubator (36℃) had an average germination percentage of about 19.58, and the

seeds that were placed in the high temperature incubator (38℃) had, on average, 41.25 percent of

the seeds germinate. Neither variable played a statistically significant role in effecting the

germination percentage of the seeds, but the temperature did play the largest role in the practical

or scientific significance.

In other similar research studies, seeds were tested with either a variable of temperature

or a variable of pH, but very rarely both. In one example of a pH experiment conducted by the
 Blevins – Hagan 20

Department of Agronomy in Iran, it was found that in low and high variations of pH levels, the

seeds had the most drastic negative changes to the number of seeds that germinated, and the

seeds germination rate (Ghaderi-Far).

There were a few design flaws in the experiment. The first being that the difference

between the pH values were not large enough to have a more significant difference in

the percentage of germination. One procedural issue that occurred was that sometimes when the

seeds were placed in the plastic bags they were not all spaced out evenly. This could have

affected the germination of the seeds.

These flaws can help future research by giving examples on how to improve upon the

experiment. This research can provide as an example to future research about how pH and

temperature effect the germination of radish seeds. The difference between the pH values should

be increased to better see how pH levels affect the percentage of germinated seeds. By making

these changes to the experiment, the way pH and temperature affects the germination of radish

seeds will become clearer. This knowledge can be beneficial to agriculturalists, in order to find

the best growing conditions for radish seeds, or scientists looking to further develop botany

discoveries in these areas.

Important knowledge and experience was gained from conducting this

research. Learning how seed germination is affect by varied factors was enlightening. In

addition, learning how to properly conduct research and lab papers was very important and

beneficial.
 Blevins – Hagan 21

Work Cited
Chen, Lingling, Quanzhu Chen, Lingqi Kong, Fangshan Xia, Huifang Yan, Yanqiao Zhu, and

Peisheng22 Mao. "Proteomic and Physiological Analysis of the Response of Oat

(Avena Sativa) Seeds to Heat Stress under Different Moisture Conditions." Frontiers in

Plant Science. Frontiers Media S.A., 22 June 2016. Web. 14 Mar. 2017.

Ghaderi-Far, F., J. Gherekhloo, and M. Alimagham. "Influence of Environmental Factors on

Seed Germination and Seedling Emergence of Yellow Sweet Clover

(Melilotus Officinalis)." Planta Daninha. Sociedade Brasileira Da Ciência Das Plantas Dani

nhas, 9 Mar. 2010. Web. 14 Mar. 2017.

Hatting, Hesta. "Factors Affecting Wheat Seed Germination." Factors Affecting Wheat Seed

Germination. N.p., June 2012. Web. 14 Mar. 2017.

Seefeldt, Steven. "Re: Why Does Acid Rain Stop Germination of Seeds (water Cress)?" Re: Why

Does Acid Rain Stop Germination of Seeds (water Cress)? n.p., 09 Oct. 1999. Web. 14

Mar. 2017.