Investigating the Effect of Varying pH Environments (pH 5, 6, 7, 8,

9) on the Germination Rate and Initial Growth (measured by radicle

length) of Mung Beans (Vigna Radiata) Over 5 Days.

Research Question
How does altering the pH of the growing environment by changing the pH of water (pH 5, 6,
7, 8, 9) affect the germination rate and initial growth (measured by radicle length in cm ) of
mung beans (Vigna Radiata) over a period of 5 days?
Introduction
Mung beans (Vigna Radiata ) are a staple food in many Asian cultures, being used in soups,
stir fry and many other popular dishes. By investigating how different pH environments affect
the germination and growth of Vigna Radiata, it can be understood which conditions lead to
the optimum growth of the plants.

Background
Mung beans (known scientifically as Vigna radiata)
are small, spherical legumes that have been
cultivated and grown in many cultures for centuries.
In addition to being used in culinary dishes, the
beans are also often used in scientific experiments
measuring germination and growth thanks to their
fast growth rates when grown in water (seed to
mature shoot in around 1 week). Thanks to their
popularity and fast growth rates, Vigna Radiata
were selected as the plant to be measured for this
investigation. By investigating the effects of pH
changes on the bean's growth and germination it
can be better understood which conditions are
optimal for the growth of the seeds to maximize Fig.1: Germinated Vigna Radiata with
growth. exposed radicles

Vigna Radiata are traditionally grown in two ways: it can be planted in soil to produce a new
plant that yields small beans, or it can be soaked in water to sprout tender shoots. In this
experiment, the seeds were soaked in water, and the length of the resulting radicle (shoot)
was used to measure the initial growth. Opting for water germination was based on two
reasons. Firstly, it ensured fairness in the experiment methodology by eliminating external
factors present in the soil, such as additional nutrients or microbial and animal life, all of
which could impact growth. Secondly, water germination significantly accelerated the seed
growth rate, enabling the experiment to be completed within a relatively short timeframe (5
days), compared to the 2-3 weeks required for a full-sized plant to develop when grown in
soil.

One way to measure the impact of varying pH on Vigna Radiata is to measure the
germination rate. Seed germination is the process by which a dormant seed transitions to an
actively growing seedling when in contact with water. The water acts as a catalyst, activating
enzymes within the seed and triggering the mobilization of nutrients ultimately leading to the
initial growth of the seed, evident by the sprouting of a radicle. The germination rate is the
percentage of seeds that have successfully sprouted or germinated over the total number of

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seeds within a given time frame. The germination rate is a vital measurement within the
investigation, providing insight into the effectiveness of the germination process.

Another method of measuring the impact of varying pH environments is to measure the initial
growth of the plant by measuring the length of the radicle. The radicle is the embryonic root
of the seedling which serves to anchor the plant in soil and absorb water and nutrients for
growth. Radicle length is often a clear indicator of the health of a plant, with shorter radicles
indicating a slower rate of growth and longer radicles indicating a higher rate of growth (note
that this is about the same plant). By averaging out the initial growth rates for the seeds in
each pH environment after 5 days of growth, the overall health and condition of the plant can
be determined.

As stated before, one of the key factors affecting the growth and health of a plant is the pH
environment it is growing in. pH, also known as the potential of hydrogen, is a measure of
the acidity or alkalinity of a given solution. pH is measured by measuring the concentration of
+¿¿
hydronium ions ( H 3 O ) in an aqueous solution. pH varies from a value of 1 to 14 with 1
being very acidic, 14 being very basic and 7 being neutral. pH is a critical environmental
factor affecting all plants, impacting a plant's ability to uptake nutrients, function on a cellular
level, and overall growth.

This can be explained by the effect pH has on enzymes within the seed. Enzymes are
biological molecules, typically proteins, that act as catalysts in biochemical reactions.
Enzymes facilitate reactions by lowering the activation energy required for the reaction to
occur, thus accelerating the conversion of reactants into products. Enzymes are vital in all
forms of life as they allow for a larger number of reactions to occur without requiring
excessive amounts of energy or time. Much like most living matter, enzymes can be found in
Vigna Radiata seeds and play an extremely important role. The enzymes are responsible for
the breaking down of stored proteins, carbohydrates, and lipids in the seed which are then
used to provide energy and the material required for the initial stages of growth. A key
aspect of enzymes is their reaction to varying pH environments. If an enzyme is exposed to
a substance with a pH too far out of its optimum range, said enzyme will begin to denature.
Denaturation occurs when the three-dimensional structure of an enzyme is altered, resulting
in the loss of its biological activity. pH affects the structure of enzymes by disrupting the ionic
interactions and hydrogen bonding, leading to a loss of function. If the enzymes in the Vigna
Radiata seeds were to denature, the seed would begin to lose a large portion of its biological
ability, resulting in reduced growth and even a failure to germinate.

With this knowledge of how differing pH levels can affect enzymes in seeds and
subsequently the germination and growth of the plant, a sensible hypothesis was drawn up.

Hypothesis
It is hypothesized that the growth and germination of Vigna Radiata seeds, measured by the
germination rate and the length of the radicle after 5 days, will reach a combined maximum
point at a pH value of 7, with the germination rate and initial growth falling as the pH
environment extends further away from pH 7.

Independent Variable
The experiment's independent variable was the pH value (5, 6, 7, 8, 9) to which each set of
seeds were exposed. Variations in pH simulate environmental factors like acid rain,
facilitating the measurement of Vigna Radiata growth and its response to different pH levels.
Effectiveness was assessed through germination rate and radicle length.

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 Dependant Variable
The dependent variables of the experiment were the germination rate and initial growth rate
of the seeds over a five-day period. The germination rate reflected the percentage of seeds
that successfully germinated, offering insights into seed responses to varying pH levels. The
initial growth rate, measured by radicle length, indicated plant growth effectiveness across
different pH environments. To maintain fairness, the following various control measures were
identified and addressed.

Control Variables
Variable to be Possible Effect on Data How Variable Was Controlled
Controlled
Volume of pH Variations in pH buffer volumes may The volume of pH Buffer solution given to
Solution influence the growth and germination of plants was precisely measured using a
Vigna Radiata seeds, complicating the measuring cylinder (± 0.5 ml ¿ to maintain
determination of whether growth results consistency across all experimental
from the solution's pH or its volume. trials.

pH of Fluctuating pH levels throughout the Before adding the solution to the plants,
Environment experiment could potentially impact the a pH meter was employed to measure
growth of Vigna Radiata seeds, leading to the pH of the growth medium, ensuring
data that may not accurately reflect the consistency between the measured pH
measured pH values. values and the actual pH of the
environment.
Growth Time Differences in the duration of growth trials Plant growth was consistently measured
may result in biased growth data, where at the same time of day over a period of
longer trials could lead to larger growth five days to ensure uniform data
irrespective of the pH value being tested. collection, thereby mitigating the potential
for variations in growth due to the
passage of time.
Number of Varying seed quantities across trials could To maintain consistency across trials,
Seeds introduce inconsistency in the data, 100 Vigna Radiata seeds were counted
potentially skewing results due to uneven for each pH environment. These seeds
sample sizes. were evenly distributed into five separate
beakers for growth, ensuring an equal
number of Vigna Radiata for each pH
value.
Source of Using seeds from multiple sources may Seeds were obtained from a single
seeds compromise data validity due to potential source to ensure uniformity across all
variations in seed properties. This could five pH environments, maintaining
obscure the ability to distinguish whether consistency throughout the experiment.
observed differences are attributable to pH
variations or seed characteristics.

Tools
 100ml Beaker x5
 Camera
 Tray x5
 Paper towel
 100ml measuring cylinder (± 0.5 ml )
 pH meter (+- 0.001)
 String (for measuring growth)
 Ruler 30cm (± 0.05 cm)
Materials

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  Vigna Radiata seeds x500
 pH 5,6,7,8,9 buffer solution (250ml each)
 Tap water (100ml )
Method Fig.2: Vigna Radiata seeds submerged
in pH buffer solution
1. Measured out 50ml of each buffer solution and
transferred into respectively labeled 100ml beakers (5 total for all pH values).

2. Counted 100 Vigna Radiata seeds for each pH environment (500 total) and placed all
seeds into each 100ml beaker.

3. Allowed Vigna Radiata to soak overnight for 8-12 hours in beaker. Kept beakers in a
cool, dark location and covered them with paper towel.

4. After soaking, removed the buffer solution and transferred the Vigna Radiata seeds
onto the sheet tray. (Ensured to label tray with respective pH buffer).

5. On tray, Counted the number of seeds that had successfully germinated. Additionally,
measured the lengths of any radicles that had developed by measuring from base of
radicles to tip using measuring string.

6. After collecting data, placed the Vigna Radiata

back into the beaker and refilled with 50 ml of
the respective pH solution.

7. Repeated steps 4 to 6 until all data had been

collected for all pH trials.

8. Once data had been collected, placed all trials

back into a cool dark place and checked again
after 24 hours.

9. Repeated steps 4-7 for every day that the

experiment was performed. (For this
experiment, the trials were observed for 5
days). Fig.3: Vigna Radiata seeds in the process of being
measured for germination and radicle growth

Ethical, Environmental and Safety Issues

Issue Description of Issue Method of Minimizing
pH Solution pH solution could be spilled resulting in Appropriate PPE will be always
Burns skin burns. worn when handling the experiment
to prevent burns from occurring.

Glassware Glassware could be dropped resulting in A sharps disposable bin will be

Breakages sharp projectiles being produced which always present in the lab when the
could cause harm to individuals in the experiment is being performed and
lab. appropriate PPE will be worn while
handling glassware to prevent injury.

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Plant As Vigna Radiata are often consumed in All beakers will be clearly labeled
Consumption food, someone who is not familiar with with "do not eat" and the experiment
the experiment may try and consume the will be clearly labeled to prevent
plants which could cause harm as they individuals from eating the Vigna
are gown in extreme pH water values Radiata shoots.

Environmental Contaminated pH buffer solution could All pH buffer waste was placed into
Contaminatio cause harm to plants and animal life if waste beakers for appropriate
n incorrectly disposed of disposal
pH Buffer Potentially dangerous chemicals could The chemicals used for pH buffer in
Chemicals be used to make a pH buffer solution, the school lab and subsequent
which could cause harm if handled experiment were considered of little
incorrectly to no risk, thus eliminating the
possibility of chemical-related
injuries due to pH buffer

Analysis
Over the course of the experiment, both the germination rate and the average radicle growth
for all five pH value trials were collected and tabulated. This data was collected to
understand how the Vigna Radiata reacted to differing pH conditions and to determine at
which pH the seeds grew most effectively.
The results from the 5 trials were all compiled into two separate graphs and tables displaying
germination rate and radicle growth respectively.

Raw Data Table 1: Table displaying the number of germinated Vigna Radiata seeds
in each pH environment (5, 6, 7, 8, 9)over 5 days
pH Environment
Value Day 1 Day 2 Day 3 Day 4 Day 5
pH5 87 88 88 89 89
pH6 81 85 90 91 91
pH7 92 97 99 99 99
pH8 74 75 77 77 77
pH9 66 72 74 74 74

Raw Data Table 2: Table displaying the average Radicle Length (± 0.05 cm¿ of Vigna
Radiata seeds in each pH environment (5, 6, 7, 8, 9) over 5 Days
pH Environment Day 1 ( Day 2 Day 3 ( Day 4 ( Day 5 (
Value ± 0.05 cm¿ (± 0.05 cm¿ ± 0.05 cm¿ ± 0.05 cm¿ ± 0.05 cm¿
pH5 0 0.2 0.3 0.4 0.4
pH6 0 0.3 0.7 1.2 1.8
pH7 0 0 2 3.7 5.2
pH8 0 0 0.3 0.5 0.5
pH9 0 0 0 0.1 0.1

Qualitative Observations

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 - pH8 and pH9 buffer solutions turned a strong green colour after having Vigna Radiata
soak
- Lower pH trials (pH5,6) had more germinated seeds when compared to higher pH
trials (pH8,9) after 1 day of growth
- Ph5 and pH6 trials displayed first signs of growth after 2 days of growth
- By day 3 all pH trials had seeds growing
- pH9 trial was last to start growing, taking 4 days to do so
- Germination appeared to stagnate after 3 days of growth for all pH environments

Processed Data
The raw data collected from each trial at each specific pH increment was collected and
processed to produce a specific average figure for the average germination rate and
average radicle growth for each pH trial.

Processed Data Table 1: Table displaying the final Vigna Radiata germination rate
(day 5) for each pH environment (5, 6, 7, 8, 9)
pH Environment Value Germination Rate (%)
pH5 89%
pH6 91%
pH7 99%
pH8 77%
pH9 74%

Processed Data Table 2: Table displaying the Average radicle Growth (± 0.05 cm¿
for Vigna Radiata over 5 days for each pH environment (5, 6, 7, 8, 9)
Average Radicle Length (5th
pH Environment Value day) (± 0.05 cm¿ Standard Deviation
pH5 0.4 0.206398
pH6 1.7 0.600016
pH7 5.1 0.636651
pH8 0.5 0.303808
pH9 0.1 0.107313

Calculations
To calculate the Germination Rate for each pH trial, the following formula was used.

Number of Germinated Seeds

Germination rate= × 100
Total Number of Seeds
Sample Calculation
84
Germination Rate= × 100
100
Germination Rate=8 4 %

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To calculate the average Radicle length for each pH trial, the following formula was used.

Average Radicle Length=

∑ of measured Radicle Lengths
Total Number of Seeds
Sample Calculation

0.5+0.7+0.6 +0.5+0.3
Average Radicle Length=
5
Average Radicle Length=0.52 cm

Graphs

Processed Data Graph 1: Graph displaying the average germination rates of Vigna
Radiata seeds in differing pH environments (5, 6, 7, 8, 9)

120%

100%

R² = 0.853599516031458
Germination Rate (%)

80%

60%

40%

20%

0%
4 5 6 7 8 9 10

pH Environment

Processed Data Graph 2: Graph displaying the average radicle growth length of
Vigna Radiata seeds in differing pH environments (5, 6, 7, 8,9)

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 6

Length (±𝟎.𝟎𝟓𝒄𝒎)
4

0
5 6 7 8 9

pH Environment Value

Analysis 1
The results from the experiment were processed and respectively placed on processed
data graph 1 and 2. By analysing the data presented in the processed data table and graph,
it could be confidently stated that the pH environment in which Vigna Radiata seeds had the
highest average germination rate was a pH environment of pH7. The average germination
rate at this specific pH value was 99%, which was 8% greater than the next closest pH
value, this being pH6 (91% avg, germination rate) and 33% greater than the lowest
germination rate (pH 9 with an avg. of 66%). This result was concurrent with external
research performed which stated that the optimum pH environment for Vigna Radiata growth
was “between pH 6.2 and 7.2”. In order to check that the trendline was displaying a
significant correlation, the R2 value was calculated. R2 is simply a measure of how well a
model “fits” a set of data, with a value closer to 1 indicating a higher degree of fit and thus
accuracy. The R2 value was 0.8536 indicating a strong correlation between the data and the
trendline (the R2 value can be seen on the processed data graph 1). As a result of the final
germination rates being calculated from the 5th day of growth, there was no standard
deviation present in the final value thus explaining the absence of error bars on processed
data graph 1.

Observing processed data graph 2 displaying average radicle growth over 5 days, it was
observed that the average length of the pH7 value radicles was much higher than the other
pH trials with an average of approximately 5.1 ±0.05 cm. However, it was also observed that
there were large error bars present on the graph indicating high standard deviation values.
The presence of large and overlapping error bars (as seen in the pH8 and pH9 trials) made it
impossible to confidently determine whether the change in radicle length was due to the
independent variable or random chance. To determine whether a significant statistical
difference was present in the data collected for the average radicle length, a single factor
ANOVA test was performed.

ANOVA Test
The use of the ANOVA test allowed for the determination of whether there was a significant
statistical in the collected data set

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Null Hypothesis ( H 0)
There is no statistical difference/relationship between the pH of the surrounding environment
and the average radicle length of Vigna Radiata seeds

Alternative Hypothesis ( H 1)
There is a statistical difference/relationship between the pH of the surrounding environment
and the Germination rate of Vigna Radiata seeds

The ANOVA Test was performed with a significant value of 0.05

As the F value (2354.888) was greater than the F Critical value (2.389948) at a p-value of 0,
the null hypothesis could be rejected as the test was deemed as significant. Since the null
hypothesis was rejected, the alternative is accepted, which stated that there was a
significant difference between the pH environments and the average radicle length of the
Vigna Radiata seeds.

Post Hoc Tukey Test

While the single factor ANOVA test confirmed that there was a significant statistical
difference in the average radicle length of the Vigna Radiata seeds, it failed to pinpoint
exactly where these significant statistical differences were in the data. In order to pinpoint
where these significant differences where, it was decided to perform a post hoc Tukey test
on the data set. A post Hoc Tukey Test is a statistical analysis test that determines whether
there is a significant statistical difference between specific individual groups of data,
opposed to an entire set as a whole. By doing this the test it could be determined whether
differences between singular groups are significant.

The Post Hoc Tukey Test was performed with an adjusted significance value of 0.005

After performing the Post Hoc Tukey Test, it was found that all trials displayed significant
statistical difference except for the trial between pH5 and pH8. It was observed that the p-
value (0.43316) between the trials was far higher than the significance level (0.005). As a
result of this finding the null hypothesis could not be rejected for this specific trial. As for the
other groups since the observed p values where below the significance value, the alternative
hypothesis could be accepted.
Analysis 2
After performing the single factor ANOVA Test and the Post Hoc Tukey Test, it could
confidently be concluded that the pH value at which the radicle length of the Vigna Radiata

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was on average the longest was at pH7, with an average length of 5.1 ±0.05 cm. This
average length was considerably longer than the other pH value trials, being 3.4 ± 0.05 cm
longer than the next longest average length being from trial pH6 (1.7 ± 0.05 cm) and
5 ± 0.05 cm longer than the shortest average length (pH9). The data appeared to fit in with
previously stated literature values which stated that “Vigna Radiata exhibit optimal growth in
pH environments between pH6.3 and pH7.2”. Despite there being a large standard deviation
in the found results, the use of an ANOVA and Post Hoc Test allowed for a significant
statistical difference be confirmed, thus validating the found results.

Discussion
Several conclusions can be drawn from the data collected from the experiment
By analysing the data presented in the processed data graphs and tables it was
determined that the optimal pH conditions for Vigna Radiata to germinate and grow in was a
pH environment of 7. The Vigna Radiata seeds had a final germination rate of 99% with an
average radicle growth length of 5.1 ±0.05 cm after 5 days of growth. It was also noted that
the Vigna Radiata grown in the pH6 environment, although not to the extent of the pH7
sample, significantly outgrew the other trials reaching a germination rate of 91% with a final
average radicle length of 1.7 ± 0.05 cm. These results coincided with external research
conducted on the same experiment which stated that the optimum pH environment for the
seeds to grow in was from a “pH in the range of 6,3 and 7,2.”

The use of the ANOVA and Post Hoc Tukey Test to measure the statistical significance of
the average radicle growth data also ensured that the collected data was statistically valid
and that the difference in values was due to the independent variable instead of random
chance.
The use of graphs to represent the raw data collected additionally served to visualize the
trends displayed by the beans showcasing how they reacted to the different pH
environments. Regarding the radicle growth graph (processed data graph 2), it can be noted
that the growth tended to trend upwards from pH5 to a peak of pH7, and then suddenly
dropping at pH8 and pH9. It can be seen that at the more extreme pH values, these being
pH5 and pH9 showcased the lowest average initial growth, only reaching an average final
length of 0.4 ± 0.05 cm and 0.1 ± 0.05 cm respectively. This limit in growth compared to the
pH6 and Ph7 trials can be attributed to the denaturation of the enzymes within the Vigna
Radiata seed. For a Vigna Radiata to grow, specific enzymes within the seed itself must
function optimally to contribute to essential functions such as the breaking down and
processing of nutrients vital for growth. If these enzymes cannot function, then the plant will
struggle or fail to grow. Thanks to the extreme pH value that the Vigna Radiata were
exposed to, it can be concluded that the enzymes responsible for the initial growth stages of
the seed were denatured to the point where they lost all their function thus causing the seed
to not grow properly. The enzymes most likely denatured due to the extreme pH values
causing the ionic and hydrogen bonds that hold the enzyme together to break. This is
backed up by the fact that the pH7 and to an extent the pH6 trial significantly outgrew the
pH5 and pH9 trials. Since the environment the seeds were exposed to was more suited to
the optimum pH for the enzymes, they were able to function properly and allow for the
growth of the seed.

Looking at the germination rate graph (processed data graph 1) further backs up the
findings concluded from the initial growth graph. It can once again be seen that the
germination rate appears to peak at the pH7 environment, with the average germination

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 rates falling as the pH approaches the more extreme values. The implementation of a
trendline serves to further highlight this trend, and its relatively high R2 value of 0.8536
highlights the validity of the line. Despite there being a trend, however, it was noted that the
trend was far less pronounced than what was found regarding the average radicle growth
rate. This smaller variance in average germination rate could suggest that the pH of the
environment in which the Vigna Radiata are grown may not have as much of an effect on the
germination of the seeds in comparison to their growth.

From the data collected and the analysis of results, it can be concluded that the pH
environment in which Vigna Radiata are most likely to grow best is a pH environment of pH
7. This conclusion supports the stated hypothesis that the Vigna Radiata will achieve
optimum growth in a pH environment of Ph7. Thanks to the large amount of accurate data
collected and the statistical analysis performed to ensure it was valid, these conclusions
were able to be drawn confidently.

Strengths
Overall, the experiment conducted accurately represented the effects of altering the pH of
water on the germination and radicle growth of Vigna Radiata seeds. The experimental
process followed allowed for many trials to be performed and the said data from those trials
to be collected and processed in a short time of only 5 days. The data collected from the
trials displayed statistical significance and showed how differing pH environments affect
Vigna Radiata growth and germination. This is backed up by previous research performed
on Vigna Radiata seeds, where the collected data was able to be referenced and compared
to other sources, further backing up the validity of the results found. Although the experiment
performed was relatively accurate, there were several flaws in the experimental process that
could be improved upon.

Weaknesses
Error Impact on Data/Process Improvements
Chemical The chemicals making up the pH A solution could be to utilize a pH buffer that
effects buffer could have had an adverse has the same chemicals and has no effect on
effect on the growth of the Vigna the plant or seed. This would ensure that the
Radiata instead of the pH of the changes in growth and germination are solely
water. For example, the presence due to the different pH
of sodium carbonate in pH9
buffer could affect a plant's ability
to uptake nutrients.
Combination Testing of both germination rate An improvement could be to split the
of Tests and average radicle growth in the experimental process into two parts, where
same experimental process could each dependant variable is measured
provide inconclusive results as independently of one another. For example,
they are affected by different instead of measuring the effect of differing pH
factors. on the average radicle length from
germination, the Vigna Radiata could be
grown in water and then subsequently
exposed to the different pH solution. This
would more accurately test how a change in
pH will affect growth.
Volume of As plants grew, they would Instead of watering plants based on volume a
water consume more water which would designated height could be used to water the
eventually exceed the volume plants. Additionally watering the plants more
being allocate each day. This than once a day could prevent water from

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 meant that the plant may be drying out
restricted from growth due to a
lack of water.

Sample size The sample size for all pH The sample size for each trial could be
environment trials was very large reduced as it was found that the standard
(100 seeds each) which meant a deviation in results was relatively small,
lot of valuable time was wasted meaning that the sample size could be made
counting the seeds for each trial. smaller.

Large pH The large jumps of 1 between the The increment for which the pH is tested
Increments pH values resulted in a large gap could be reduced to 0.5 or even 0.25 jumps,
between data points which could allowing for more precise data on the exact
potentially cause the omission of pH value at which Vigna Radiata grow
more precise data. optimally.

Extensions
An extension to the current experiment could be to apply the same experimental process to
related plants which also grow in a similar pH environment. By doing this it can be
determined whether plants are affected by changes in pH because of their own genetic
makeup or their adaptation to their current environment. By performing tests on similar
plants one can gain a broader understanding of how certain plants may behave similarly or
differently to significant pH changes in their respective environments. Another potential
extending could be to test different chemicals on Vigna Radiata. By using the same
experimental process but instead changing the pH buffer for specific chemicals, one could
determine which chemicals have the greatest effect on the Vigna Radiata germination and
growth. This would allow for the determination of the chemicals which may inhibit or aid in
the growth of Vigna Radiata seeds.

Conclusion
The research question and the hypothesis were both answered and supported respectively
from the data received from the experiment. The pH of water that allowed for the highest
germination rate and initial growth rate was a pH of 7, with this value being supported by the
large amount of experimental data collected as well as being cross referenced with outside
findings. Overall, the experiment performed could be deemed a success as both the
research question and the hypothesis where able to be answered and supported by a large
amount of accurate and precise data.

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