Title: The effect of concentration of lead ions in the soil on the production of garlic

crops

Research question: “To what extent does the concentration of lead in polluted soils affect

the mitotic index in the meristem of Allium sativum?”

Subject: Biology

Candidate number: gtt042

Word count: 3,824.

Acknowledgements:

I would like to express my appreciation to my extended essay supervisor, Mr Abdelghany

Zaghal, for his guidance and assistance throughout the making of this essay.

I would also like to thank Mrs Nibal Hamdan for her constant help throughout this experience.

Finally, I would like to take this chance to express my deep gratitude to my family, for their

constant support and encouragement.

Thank you for making this an unforgettable experience.

 Table of contents

1. Introduction ............................................................................................................................. 1

1. The concentration of lead ions in polluted soil ....................................................................... 2

2. The growth of Allium sativum in light of its mitotic index ..................................................... 3

3. Investigating the effect of different lead concentrations on the mitotic index of Allium

sativum: ........................................................................................................................................... 5

4.1 Variables: .............................................................................................................................. 5

4.2 Experimental predictions and hypothesis:............................................................................. 6

4.3 Experimental materials and protocol .............................................................................. 6

4.3.1 Preparation of metallic solutions: ................................................................................... 6

4.3.2 Harvest roots of garlic cloves to be used for the experiment:......................................... 6

4.3.3 Procedure for preparing slides: ....................................................................................... 7

5. Experimental findings: ............................................................................................................ 8

6. Experimental calculation ........................................................................................................ 9

7. Analysis and evaluation: ....................................................................................................... 11

8. Conclusion: ........................................................................................................................... 14

Improvements and further studies: ............................................................................................ 16

9. Bibliography: ........................................................................................................................ 17
 1. Introduction

Pollution is a widely spread issue that negatively impacts the environment on a global scale.

Pollution affects the water we drink and the food we eat. Lebanon is one of the countries

suffering from this global issue, both its water and soils are polluted (Masri, 1995). Sources of

pollution such as sewage, industry effluence, solid wastes, and agricultural remains containing

heavy metals are released in most rivers, lakes, and seas which in turn are used for watering

plants (Masri, 1995). The waters in Lebanon are polluted to the extent that one can see dead fish

floating on their surfaces. According to an article written by Nadine Mazloum, 36% of the water

in the Litani River is now turned into a “brownish muck”, making it not suitable for irrigation

and swimming (Mazloum, 2016). Furthermore, the metallic ions present in the above-mentioned

pollutants can pass into the soil, resulting in its contamination. The clay-rich nature of the soil in

Lebanon aggravates this problem by absorbing most of these heavy metals (Lebanon State of the

Environment Report, n.d). One of the main heavy ions found in high concentrations in the soil is

lead (Wuana & Okieimen, 2011). Lead has affected not only crops but also human health. Lead

in soils can enter the human body through vegetables or fruits grown in the lead-polluted soil.

Once lead enters the body, it can proceed to act as a carcinogen, and it can affect bone and

muscle growth (“Protect Your Family from Lead in Your Home”, 2013). After further exploring

this issue and its adverse effects on agriculture, I have decided to investigate how the

contamination of soils by heavy metallic ions, lead in this case, affects crop production.

In this investigation, the crop that will be used is Allium sativum. Allium sativum is widely

needed for its medicinal uses. It is known to regulate blood pressure, decrease cholesterol level,

prevent diseases and tumors, cure earaches, and stomach aches (“GARLIC: Uses, Side Effects,

Interactions and Warnings.”, n.d). It also decreases the glucose level in the blood, which makes it

1
a possible antidiabetic agent (Sher, Fakhar-ul-Mahmood, Shah, Bukhsh, & Murtaza, 2012).

Moreover, the presence of germanium in Allium sativum enhances the immune system

(Germanium, 2017). On another note, the distinguished taste of Allium sativum makes it a

commonly used ingredient for several dishes such as garlic bread, spinach fatayer, and several

types of pasta. Given all these benefits and uses of Allium sativum, a decrease in its production as

a result of soil pollution may not allow the human population to make use of its many medicinal

benefits along with its distinct taste. For that reason, in this investigation, the effect of increasing

lead concentrations on the crop production of Allium sativum will be tested.

The growth of garlic root tips will be measured using the mitotic index, which in turn, would

give an idea about how the production of Allium sativum is affected. The mitotic index is an

indirect way to calculate cell proliferation (through a micrograph). In other words, it measures

the ratio of the number of cells undergoing mitosis to the number of cells not undergoing mitosis

(Cornell, n.d). Prior understanding of mitosis and its sub-phases will be intensively needed to

discuss, analyze and interpret the micrograph readings, allowing the deduction of the mitotic

indexes and conclusions.

The results of this investigation are environmentally worthy given that they shed light on the

issue of pollution and how it is affecting the production of a medically and domestically

important crop such as Allium sativum This investigation with the hope that this will raise the

awareness of people and urge them to take action and put an end to this alarming issue.

1. The concentration of lead ions in polluted soil

High concentrations of lead in soils can have drastic effects on crops and can also affect

human health. According to an article by Pennsylvania State University, lead is a naturally

occurring element in soil. Unpolluted soil naturally contains low concentrations of lead that

2
range from 10 to 50 mg/L (“Lead in Residential Soils: Sources, Testing, and Reducing

Exposure”, 2017). Polluted soils contain concentrations of lead that range from 50 to 1,500 mg/L

(“Lead in Residential Soils: Sources, Testing, and Reducing Exposure”, 2017). Research shows

that high levels of lead concentrations can kill the decomposers present in the soil (Greene, n.d);

hence, preventing the recycling of nutrients that are essential for plant growth. Moreover,

different concentrations of lead cause a bias in the organisms present in the soil, affecting the

type of plants that can grow in this area. Other than the fact that lead-contaminated soil can affect

crop production and quality, it has been identified as an important contributor to high blood lead

levels (Madhavan, Rosenman, & Shehata, 1989) and therefore, a risk factor of different diseases

such as cancer.

Given that lead is naturally occurring in soils in concentrations between 10 to 50 mg/L and

polluted soils contain up to 1,500 mg/L, the concentrations of lead solutions to be used in this

investigation as representative samples of polluted soil will be 220mg/L, 420mg/L, 1000mg/L

and 1500mg/L. A lead solution of concentration 20mg/L will be used as a control that represents

the unpolluted soil.

2. The growth of Allium sativum in light of its mitotic index

Several ways can be used to measure the effect of different lead concentrations on the

production of garlic, including measuring root growth at different concentrations and measuring

the mitotic index. I chose to complete my investigation by measuring the mitotic index since it is

more accurate and more indicative about the growth rate because it can show at which phase the

growth was inhibited, allowing more detailed and accurate analysis. A thorough understanding of

the process and phases of cell division is a prerequisite to measuring the mitotic index.

The process of cell division can be summarized as follows (Cornell, n.d):

3
 Cell division has three steps: Interphase, mitosis (the division of the nucleus), and

cytokinesis (the division of the cytoplasm). Each of these steps has their subphases.

Interphase:

- Gap 1: the cell starts getting larger, and the organelles duplicate.

- Synthesis phase: the chromosomes replicate.

- Gap 2 phase: the cell continues to grow larger, and the organelles continue to duplicate.

Mitosis:

- Prophase: chromosomes condense and super coil, the nuclear membrane disappears, the

centrioles start moving to opposite sides of the cell, and spindle fibers start to form.

- Metaphase: The centrosomes are at opposite poles of the cell, spindle fibers attach to the

centromeres of the chromosomes, aligning them on the equatorial plate.

- Anaphase: the spindle fibers contract, causing the two sister chromatids to separate and

move to opposite poles of the cell.

- Telophase: The sister chromatids are at opposite poles of the cell, the nuclear membrane

starts forming, spindle fibers start to disappear, and the chromosomes start to uncoil.

Cytokinesis is the division of the cytoplasm of the cell. Plant cells separate by forming a cell

plate that extends and fuses with the cell wall, splitting the cell into two daughter cells. On the

other hand, in animal cells, a cleavage furrow is formed. The cleavage furrow deepens until it

meets, and the cell becomes pinched off, and it separates to form two daughter cells.

Measuring the mitotic index necessitates the identification of the phase of the cell followed by

further calculations using a formula. To identify the cell phase, a photograph of the cells can be

taken and then analyzed as follows:

- A cell with an intact nucleus is in interphase

4
 - A cell with unorganized chromosomes with no nucleus is in prophase.

- A cell with chromosomes that are clearly aligned at the equatorial plate is in metaphase.

- A cell whose chromosomes are getting pulled to opposite poles is in anaphase.

- A cell getting pinched in the middle and starting to separate is in telophase.

3. Investigating the effect of different lead concentrations on the mitotic index of

Allium sativum:

4.1 Variables:

In an attempt to respond to the research question, the mitotic index of Allium sativum was

measured under different concentrations of lead ions. Therefore, the concentration of lead

solutions (20mg/L, 220mg/L, 420mg/L, 1000mg/L, and 1500mg/L) in which the garlic cloves

were grown represents the independent variable of this study. The mitotic index of the Allium

sativum root tips represents the dependent variable. The mitotic index will be able to give an idea

about the growth of the Allium sativum in increasing concentrations of lead solution which in

turn, would provide an idea about the effect of different lead concentrations on the crop

production of Allium sativum. All the cups containing the garlic cloves were kept outdoors to

mimic the environment's conditions. The garlic used in this experiment was purchased from the

same place. The garlic cloves put in the cups were all of approximately the same size and shape.

Furthermore, the same type, size, color, and shape of cups were used. The cups containing the

garlic cloves were kept in the same temperature, pressure, light, and humidity conditions.

Moreover, all the prepared lead solutions were of the same volume. Before the experiment, all

the garlic cloves were given three days to grow root tips in the volume and type of water.

5
4.2 Experimental predictions and hypothesis:

As the concentration of the lead solution that the garlic cloves are being grown in increases

the mitotic index will decrease. A low mitotic index indicates less growth of garlic, which in

turn, indicates less production of garlic.

Null hypothesis (H0): The mitotic index will remain the same in all the root tips that were

exposed to different concentrations of lead.

Alternative hypothesis (H1): The mitotic index will be different in the root tips that were

exposed to different concentrations of lead.

4.3 Experimental materials and protocol

4.3.1 Preparation of metallic solutions:

In this step, three solutions of lead were prepared. To prepare the first concentration,

20mg/L, 0.02g of lead was measured. Then, the measured mass was transferred into a 250ml

volumetric flask with the help of a funnel. Then, 50ml of water was added. The mixture was

homogenized using a magnetic stirrer.. Finally, the solution was diluted with water until the line

mark. This step was repeated five times to obtain four different concentrations of lead. The

materials to be used in this preparation are:

 Volumetric flask (250±0.15ml)  Powder lead acetate

 Distilled water  Magnetic Stirrer

 Digital balance (±0.01g)

4.3.2 Harvest roots of garlic cloves to be used for the experiment:

For this step, the garlic cloves were allowed to grow roots. That was done by getting 20

garlic cloves and inserting a toothpick in the center of each garlic clove. The toothpicks were

used to hold the garlic cloves above the water. The toothpicks holding the garlic cloves are then

6
adjusted in different cups containing 100ml of water. After three days, when the garlic cloves

would have already grown roots, they were transferred into different cups containing 100ml of

each concentration of the three metallic solutions. A timer was used to follow the evolution of

the mitotic index in each solution as a function of time. Three days after the immersion of the

cloves in each solution, 5×1cm long root tips were cut by scissors to be used for the preparation

of the microscopic slides. The materials that will be used throughout this preparation are:

 Garlic cloves.

 Plastic cups.  Lead acetate solutions (aq) of

 Gloves concentrations: 20mg/L, 220mg/L,

 Toothpicks 420mg/L, and 1000mg/L

 Tap water.

4.3.3 Procedure for preparing slides:

For this step, slides were prepared and viewed under the microscopes. Each of the cut root

tips was put on a different watch glass. 4 drops of 1M HCl were added to the root tips on the

watch glasses using a dropper and left for 2 minutes. Using another dropper, three drops of the

Orcein pink stain were also added to the root tips. Later, the mixture of root tips, HCl, and

Orcein pink stains was gently heated on a hot plate for five minutes. After heating it, most of the

liquid had evaporated, and the stain had penetrated the root tip. The root tips were then

transferred to a clean microscopic slide using tweezers. One drop on the orcein stain is then

added to the root tips on the microscopic slide. The microscopic slide is then covered with a

clean coverslip. Then, a paper towel was placed on the coverslip and using the side of your hand;

7
the root tip was squashed. That way, any excess stain is dried off, and more importantly, the root

tip is squashed. This will allow the microscope to show a clearer picture of the cell. Finally, the

slide was observed under a 45X microscope. This step is repeated for each of the trials of each

concentration of lead. The materials used for this part of the procedure are:

 Plastic pipettes  Microscope slides

 1M hydrochloric acid  Paper towels

 1% Orcein pink stain.  Scissors

 Watch glass.  Hot plate

 Cover slips.

5. Experimental findings:

Table 1 shows the total number of cells undergoing mitosis and the total number of cells in

general, viewed under the microscope.

Concentration
of Pb+ ions 20mg/L 220mg/L 420mg/L 1000mg/L
(mg/L)
Number Number Number Number
Total Total Total Total
of cells of cells of cells of cells
number number number number
in in in in
of cells of cells of cells of cells
mitosis mitosis mitosis mitosis
Trial 1 18 28 9 22 10 29 1 27
Trial 2 14 23 12 25 9 23 0 20
Trial 3 11 22 9 20 8 25 0 23
Trial 4 12 23 13 28 7 22 1 25
Trial 5 14 25 11 24 6 20 0 22

8
 6. Experimental calculation

Table 2 shows the mitotic index of garlic root tips in each different lead concentration in 4

different trials along with the average mitotic index and standard deviation of each concentration.

Concentration of 1,000mg/L
20mg/L 220mg/L 420mg/L
lead solutions
Trial 1 0.64 0.40 0.34 0.04

Trial 2 0.61 0.48 0.39 0.00

Trial 3 0.50 0.45 0.32 0.00

Trial 4 0.52 0.46 0.32 0.04

Trial 5 0.56 0.46 0.30 0.00

Average mitotic
index 0.57 0.45 0.33 0.016

Standard
deviation 0.06 0.03 0.03 0.02

Sample calculation for mitotic index for each trial:

number of cells undergoing mitosis

Mitotic index =
total number of cells

Sample calculation of mitotic index of trial 1 of lead concentration 20mg/L:

18
= 0.64
28

Sample calculation for average mitotic index:

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑚𝑖𝑡𝑜𝑡𝑖𝑐 𝑖𝑛𝑑𝑒𝑥

𝑇𝑟𝑖𝑎𝑙 1𝑀𝐼 + 𝑇𝑟𝑖𝑎𝑙 2𝑀𝐼 + 𝑇𝑟𝑖𝑎𝑙 3𝑀𝐼 + 𝑇𝑟𝑖𝑎𝑙 4𝑀𝐼 + 𝑇𝑟𝑖𝑎𝑙 5𝑀𝐼
=
5

Sample calculation of average mitotic index of lead concentration 20mg/L:

9
 0.64 + 0.61 + 0.50 + 0.52 + 0.56
= 0.57
5.0

Sample calculation of Standard deviation of lead concentration 20mg/L:

Step 1: Subtract the mean from each of values of the trials

 0.64 − 0.57 = 0.07  0.52 − 0.57 = − 0.05

 0.61 − 0.57 = 0.04  0.56 − 0.57 = − 0.01

 0.50 − 0.57 = − 0.07

Step 2: Square each difference

 (0.07)2 = 0.005  (− 0.05)2 = 0.003

 (0.04)2 = 0.002  (− 0.01)2 = 0.0001

 (− 0.07)2 = 0.005

Step 3: Add all the squared values obtained in step 2 and divide them by N-1

0.005 + 0.002 + 0.005 + 0.003 + 0.0001

= 0.004
4

Step 4: Find the square root of the value obtained in step 3.

0.004 = 0.06

Choice of statistical analysis (One-way ANOVA, n.d):

A one-way ANOVA statistical test is used in this investigation to find out whether there are any

statistical differences between 4 different groups of concentrations of lead solutions to find out

whether the mitotic index will be different in the root tips that were exposed to different

concentrations of lead. The choice of a one-way ANOVA test was made in order to know

whether to reject or accept the hypothesis. An ANOVA test was used instead of a t-test due to

the presence of more than two means.

10
Anova: single factor.

SUMMARY

Groups Count Sum Average Variance

20mg/L 5 2.83 0.566 0.00348
220mg/L 5 2.25 0.45 0.0009
420mg/L 5 1.67 0.334 0.00118
1,000mg/L 5 0.08 0.016 0.00048

ANOVA
Source of Variation SS df MS F P-value F crit
1.22E-
Between Groups 0.840895 3 0.280298 185.628 12 3.238872
Within Groups 0.02416 16 0.00151

Total 0.865055 19

7. Analysis and evaluation:

The results of the ANOVA statistical analysis that was carried out showed that there is a

significant difference between the groups. The results of the statistical analysis gave a p-value of

1.22×10-12, which is less than 0.05, leading me to reject my null hypothesis and accept my

alternative hypothesis that states that the mitotic index will be different in the root tips that were

exposed to different concentrations of lead. Furthermore, my hypothesis was further proved by

observations and calculations. While viewing the slides under the microscope, there was a clear

decrease in the number of cells undergoing mitosis as the concentration of lead increased.

Moreover, calculations of the mitotic index for each root tip grown in different concentrations of

lead ions proved the hypothesis such that the value of the mitotic index of root tips grown in high

concentrations of lead was very low in comparison to the mitotic index of root tips grown in low

11
concentrations of lead. At the highest concentration, 1000mg/L, mitosis was almost completely

inhibited and the roots had a mitotic index of 0.016, thus, it is suitable to say that lead inhibited

mitosis in garlic and therefore can decrease its crop yield if found in large amounts in the soil.

The results of the trials of each concentration were similar with standard deviations that

varied from 0.02 to 0.061 which increases the reliability of this experiment. The results of this

experiment proves that the increasing lead concentrations in the soil will, most definitely, affect

the yield of the crop production of Allium sativum. This gives us an idea about how the high lead

concentrations in the soil can decrease crop production, especially of crops belonging to the

Allium family, such as onion, shallots and scallions given that they are in the same family as

Allium sativum and are therefore very similar to it.

Although my results showed that increasing concentrations of lead drastically affect the

mitotic index, this can only be applied to real life to a certain extent since it is not possible to

reach maximum accuracy. Factors present in the natural environment that was not present in this

investigation could affect the plant’s absorption of lead. For example, a mineral present in the

soil can inhibit the uptake of lead by plants. Furthermore, the pH of the soils varies, which in

turn can affect the uptake of lead by plants. This is because lead is insoluble at a pH greater than

6.5, and thus, will not be able to enter the roots.

At normal concentrations, since lead is naturally occurring in soils, plants came up with

mechanisms that allow it to avoid the effects of lead. The roots are the first organs that the lead

ions come in contact with. As the first line of defense, the cell walls of the root cells create a

barrier by synthesizing and depositing callose which in turn inhibits the entry of lead ions into

the cells (Fahr et al., 2013). However, plants can’t avoid the effects of lead at high concentrations

since callose is synthesized mainly in the protoderm and in the centre of the root tip and is not

12
deposited regularly in all root cells and tissue (Samardakiewicz, Krzesłowska, Bilski,

Bartosiewicz, & Woźny, 2011). Thus, it would not inhibit the entry of all the lead ions in the

case of a high lead concentration in the soil. Other mechanisms that plants can use to prevent the

effects of lead are to absorb large amounts of lead and isolate them in the vacuoles or translocate

a large amount of lead in root cells to the upper part of the plant (Fahr et al., 2013).

As my experiment proved, high concentrations of lead decrease mitosis. A research

article done by Wusheng Jiang and Donghua Liu in 2010 on the defense system in the root cells

of Allium sativum against lead supports the results of my experiment as it shows that after

several hours of lead treatment, many changes occurred in the cell. Some of these changes

included: the ER and the mitochondria became swollen along with the loss of cristae, the nuclear

envelope of some cells became relatively diluted, cisternae broke down to form vesicles, and the

plasma membrane became injured (Jiang & Lui, 2010). All of these changes lead to the decrease

in mitosis since organelles need to be duplicated in the interphase before allowing the cell to

move into mitosis. Because these cells’ organelles are damaged and swollen, the process of

organelle duplication in interphase will not be complete, inhibiting the cell from moving into

mitosis, therefore, decreasing the mitotic index.

Moreover, other research papers that support my experiment state that exposure of cells to a

high concentration of lead affects the process of cell division since lead treatment can cause an

extended cell cycle along with a decrease in the number of cells synthesizing DNA (Wierzbicka,

1998). Other articles state that exposure of crops to lead can also lead to c-mitosis (an unnatural

unsuccessful nuclear division in which the chromosome number is doubled), anaphase bridges

(an event that occurs in which sister chromatids fail to separate), and chromosome stickiness

13
(Liu, Jiang, Wang, Zhao, & Lu, 1992). On another note, lead can also inhibit root growth

(Bharali, Aran, Arangham & Chetry, 2013).

8. Conclusion:

The results of this experiment showed the effect of high lead concentrations on the crop

production of Allium sativum. This can be seen as a real threat that needs to be stopped. To stop

the contamination of soils by lead, people must avoid the main sources that lead to lead

contamination, such as lead-based paint (“Lead in Residential Soils: Sources, Testing, and

Reducing Exposure”, 2017). Lead-based paint may mix with the soil when chips of paint fall

from buildings or when lead-based paint is present in the industry affluence of that is disposed of

in water that enters the soils. According to an investigation published by the Lebanese Company

for Information & Studies in the year 2015 on lead paint in Lebanon, the total average of lead in

paints reaches 537 ppm (“The Lead in paints... disturbing facts ... and immediate actions are

required.”, 2015). This value is much higher than the maximum acceptable average of lead in

paints which is approved in Canada, the United States, and Nepal to be 90 ppm (“The Lead in

paints... disturbing facts ... and immediate actions are required.”, 2015). These numbers that state

the concentrations of lead in paints give an idea about how much the environment, along with the

soils, are affected by these paints. Furthermore, according to an article done by PennState

University in 2017 on lead in residential soils, another source of lead in soils is gasoline which

contained lead compounds and was used up until 1989 (“Lead in Residential Soils: Sources,

Testing, and Reducing Exposure.”, 2017). Even though leaded gasoline is not widely used

anymore, almost 4.5 to 5 million tons of the lead that was present in gasoline remains in the soil

("Lead in Residential Soils", 2017). As for Lebanon in particular, the use of leaded gasoline is

14
not yet banned, and thus, this type of gasoline still poses a threat to the environment and to the

human health.

Not only does the lead present in soils negatively affect plants, but they also harm the human

health. Lead in soils can enter the human body as a result of people eating fruits or vegetables

that were originally grown in lead-contaminated soils. Children are more sensitive to lead than

grown-ups given that their bodies are still developing. Children are exposed to lead entering their

body far more than adults are. This is because they often put their hands on the soil then put it in

their mouths. In children, lead exposure can have several impacts on the body including poor

muscle coordination, decreased muscle and bone growth, hearing damages and learning

disabilities (“Protect Your Family from Lead in Your Home”, 2013). Although adults are less

exposed to lead, once it's in their system, it can lead to muscle and joints pain, fertility problems,

concentration problems, high blood pressures and nerve disorders (“Protect Your Family from

Lead in Your Home”, 2013). As for pregnant women, having lead in their bodies would put the

fetus' at risk since the lead is in her bloodstream which passes through the placenta and into the

babies developing organs (Effects of lead exposure during pregnancy, n.d).

Taking into consideration the drastic effects of lead-contaminated soils on plants, it can be

concluded that coming up with ways to decrease the uptake of lead by plants is necessary and

should be considered a priority. According to an article done by the University of Minnesota,

adding organic matter to the soil decreased the lead uptake by plants. Organic matter decreases

the availability of lead to the plant (Rosen, n.d). The same article also states that increasing the

pH beyond 6.5 also decreases the lead uptake by plants since lead is unavailable to plants beyond

this pH (Rosen, n.d). To avoid the drastic effects of lead on children's health, it is recommended

to test the concentration of lead present in the soil around the house before allowing children to

15
play outside. If that is not possible, one can be extra cautious and take this issue seriously in a

way that they would observe their children while playing.

Improvements and further studies:

In this investigation, the effect of only four concentrations of lead were tested on Allium

sativum root tips. To get a more specific result, a higher number of concentrations with a smaller

range between them can be used. Furthermore, to provide more proof of the effect of lead-

contaminated soil on the crop production, it can be tested on more than one vegetable. Moreover,

soils are contaminated with many ions other than lead; it is also important to test the effect of

these ions on the production of crops.

16
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