1

Lingering Heavy Metal and Metalloid Toxins in the

Allium cepa Root Tip and Bulb from the Animas and
San Juan Riverbeds, after the 2015 Gold King Mine
Spill

Ms. Sunny Nez

University of New Mexico, Albuquerque New Mexico

Copyright @ 2017
Ms. Yolanda Flores, Advisor
Daniel Winarski, Mentor
2017
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Lingering Heavy Metal and Metalloid Toxins in the Allium cepa Root Tip and Bulb from the
Animas and San Juan Riverbeds, due to the 2015 Gold King Mine Spill
Sunny Nez
1st year
University of New Mexico
Albuquerque, NM
Mentor: Dr. Daniel Winarski

ABSTRACT

Finding potential heavy metal and metalloid toxins in the Animas and San Juan riverbeds
and onion bulb after the 2015 Gold King Mine Spill are crucial to the effects of the mitotic index
of Allium cepa root tip. Initial tests on the mitotic index from the Animas and San Juan Rivers
were conducted in 2015-2016. The hypothesis states that if the both Riverbeds, and onion bulb
samples have potential heavy metals and metalloids, then the samples from both rivers will contain
abbreviated cells, and a lower mitotic index.

Water and riverbed samples were collected after the 2015 Gold King Mine Spill. Green
onions were planted in each water and riverbed sample for one month. The onion root tips were
then analyzed using a digital microscope at 400X for mitotic phases and cellular aberrations. Non-
dividing and dividing cells were counted to measure the mitotic index. Onion bulbs exposed to the
riverbed were tested for metals using Oxford ED-2000 Geology Majors + Traces XRF.

The hypothesis was accepted; both riverbeds affected the mitotic index (0%) of the root tip with
cellular aberrations. Onion bulbs in the XRF study had higher ppm concentrations of Zinc
indicating cytotoxic effects on the mitotic index. The Animas Riverbed had high weight
percentages of specific metal oxides, thus reducing the weight percent of common sand. The XRF
Machine also found that the Animas River having higher concentrations of metalloids, while the
San Juan Riverbed had higher weight percentages of sand because it had less specific metal oxides.
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Table of Contents
List of Tables ……………………………………………………………………………… 4
List of Figures …………………………………………………………………………….. 4
Abstract …………………………………………………………………………………… 1
Introduction………………………………………………………………………………... 3
Materials and Methods…………………………………………………………………….4-5
Results…………………………………………………………………………………....9-15
Discussion and Conclusions…………………...………………………………………. 12-14
References.............................................................................................................................15
Key Words
 Allium cepa- Onion plant
 NDC- Non-Dividing cells; cells that are not undergoing mitosis
 DC- Dividing-Cells; cells that undergo mitosis
 Mitotic Index- a ratio between the numbers of cells that undergo mitosis to the overall
population of cells that do not undergo mitosis.
List of Tables
Table 1. Mitotic Index of Allium cepa Root Tip …………………………………………....6
Table 2. RAW DATA - Table of Chemical Elements found via Oxford Geology Majors + Traces
XRF………………………………………………………………………………………......12
Table 3. RAW DATA - Table of Chemical Elements found via Oxford Geology Majors + Traces
XRF………………………………………………………………………………………......12
List of Figures
Figure 1. Four Riverbed Sample Locations: 2015 Gold King Mine Spill ……………………9
Figure 2. Mitotic Index of Allium cepa Root Tip ………….…………………………….…16
Figure 3. Mitotic Index of Allium cepa Root Tip…………………………………………….16
Figure 4. Significant Changes in Major Chemical Elements between San Juan and Animas
Riverbeds, in Average Weight Percent………………………………………………………17
Figure 5. Significant Changes in Heavy Metals and Arsenic between San Juan and Animas
Riverbeds, in Average PPM…………………………………………………………………18
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INTRODUCTION
Introduction/Background Research
Mitosis is a part of a cell division that plays a small role in the cell cycle where a parent
cell divides into two or more daughter cells. The cell cycle is mostly focused on mitosis and
interphase, which is a longer process and is not really part of Mitosis. Interphase has its own stages
which are known as G1, S (time of replication), and G2 of the cell cycle. G1, and 2 are gap phases
where the cells are trying to grow. There is a process in mitosis where there is DNA replications
in eukaryotes that separates chromosomes in its cell nucleus into two different nuclei.
The process of Mitosis is divided into four stages: Prophase, Metaphase, Anaphase and
Telophase. During Prophase, the chromosomes super coil, condense and are visible for first time
during the cell cycle. The spindle fibers start forming, and the nuclear membrane starts
disappearing. During Metaphase, the spindle fibers reach and attach to centromere of each sister
chromatids. The chromosomes align along the center plane of the cell, and the nuclear membrane
is all gone. During Anaphase, the centromeres start to split and the sister chromatids start to move
towards the opposite sides of the cell.
The onion has 8 chromosomes and the cell division happen at a fast rate in growing root
tips in seed or bulbs, which is a round part of an ordinary onion. The onion root tip is a part of the
onion that grows rapidly and as a result there will be many cells that are visible that undergo
mitosis. The mitotic index is the percentage of cells that undergo mitosis, and help us quantify the
cell division
Water contaminations can play a role of affecting the Allium cepa onion root tip. The
mitotic index is analyzed with the onion root tip and has showed there have been decreases in the
mitotic index as well as cell aberrations due to poor water quality in urban streams, such as a recent
study in South Brazil in 2015. There has also been recent research on the San Juan and Animas
Rivers water quality in 2015.
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Materials and Methods

Research Question
Will the contaminated water and riverbed samples from the Animas and San Juan Rivers on the
Navajo Reservation affect the mitotic index of Allium cepa root tip?
Hypothesis
If the Animas and San Juan water and riverbed samples have medium to poor quality, then
samples will contain cellular aberrations, and a lower mitotic index in the Allium cepa root tip
and bulbs.
Variables
The independent variables for this experiment is the effects of the Animas River and San Juan
Riverbeds. The dependent variables are the number of dividing and non-dividing cells indicating
the mitotic index of onion root tip. The heavy metals and metalloids present in the riverbeds and
onion bulbs using Oxford Geology Majors + Traces XRF
Materials
The materials needed for this experiment were green onions, 10 250 mL beakers,
riverbed samples from 2 sites for 2 rivers, containers to collect samples, IBM’s Oxford ED-2000
XRF Machine
Procedures
1. Collect riverbed and water samples from two locations; Penny Lane in Aztec, NM and Berg
Park from the Animas River in Farmington, NM. As well as Kirtland and Shiprock for the San
Juan River 1 year and 3 months after the 2015 Gold King Mine Spill.
2. Take pictures of all four sites for the Animas and San Juan Rivers
3. Obtain a small amount of the riverbed soil and secure it safely in double Ziploc bags for each
site.
4. This soil was sent off for laboratory work for the IBM’s Oxford ED-2000 X-Ray Fluorescence
Machine for heavy metal contaminates.
5. Set up 8 250 mL beakers and label the river, site, and trial number.
6. Insert 5 green onions into each beaker, make sure the onion root tip is embedded into the soil.
7. The first part of the experiment will be observed for physical changes for one month for the
mitotic index of normal and abnormal cells with well water and riverbed samples.
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8. Make sure the onions stay watered and do not dry out.
9. When the one month is up, take out a green onion and cut the tip of the root with a scalpel.
10. Make sure to slit the root tip so the whole root tip lays flat on a slide.
11. Squash the root tip lightly with a slide cover onto the slide and insert onto a microscope
under 400X. Take one picture of each cell for each trial for both the Animas and San Juan River.
12. Record the number of dividing cells and non-dividing cells.
13. Calculate the mitotic index of the non-dividing cells.
14. Observe for abnormal cells as well.
15. When the onion roots are finished with the biological testing portion of this experiment, three
onion roots will be analyzed for heavy metal and metalloid contamination.
16. Cut the onion approximately an inch from the bulb with a scalpel for each of the onions.
17. Place the onion bulbs with its root tips still attached in glass bowls and dry them.
18. Place each onion bulb in double plastic Ziploc bags. These plastic bags will undergo the
same X-Ray Fluorescence laboratory work from IBM as did the riverbed samples.

Figure 1. Four Riverbed Sample Locations: 2015 Gold King Mine Spill
Source of Map: https://summitvoice.files.wordpress.com/2015/08/goldkingminespill.jpg
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Results
The results are summarized in Table 1 and Figure 2, on the effects of the riverbed samples on the
mitotic index of Allium cepa root tip.

Table 1. Mitotic Index of Allium cepa Root Tip

Non-Dividing and Dividing Cells on the Mitotic Index of Allium Cepa Root Tip
Trial 1 Trial 2 Average Mitotic Index
NDC DC NDC DC NDC DC Percentage (%)
Control (prepared slide) 64 19 62 5 126 24 16.0
Animas River (Penny Lane, NM) 80.0 0.00 69.0 0.00 149 0.00 0.00
Animas River (Berg Park, NM) 56.0 0.00 55.0 0.00 105 0.00 0.00
San Juan River (Kirtland, NM) 51.0 0.00 64.0 0.00 115 0.00 0.00
San Juan River (Shiprock, NM) 12.0 0.00 7.00 0.00 19.0 0.00 0.00
*NDC- Non-dividing cells
*DC- Dividing cells
Figure 2. Mitotic Index of Allium cepa Root Tip
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Figure 3. Oxford ED-2000 X-Ray Fluorescence, using their Geology Majors + Traces Software

http://www.oxford-instruments.com
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Table 1. Riverbed DATA - Table of Chemical Elements found via Oxford Geology Majors +
Traces XRF

(Chemical elements with little or no weight percent have been deleted from this list)
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Table 2. Riverbed DATA - Table of Chemical Elements found via Oxford Geology Majors +
Traces XRF

(Chemical elements with little or no weight percent have been deleted from this list)
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Table 4. Discussion of Riverbed DATA – Significant Changes in Chemical Elements between

Animas and San Juan Riverbeds

(Chemical elements with little or no weight percent have been deleted from this list)
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Figure 4. Significant Changes in Major Chemical Elements between San Juan and Animas
Riverbeds, in Average Weight Percent

80
Average weight percent

70
60
50
40
(Wt%)

30
20
10 San Juan…
0 Animas River
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Figure 5. Significant Changes in Heavy Metals and Arsenic between San Juan and Animas
Riverbeds, in Average PPM

600
500
Average PPM

400
300
200 San Juan River
Animas River
100
0
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Table 5: Onion Bulb XRF: bulbs absorbed Chemical Elements from the Riverbed Samples,
showing the Elements can enter the Food Chain
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DISCUSSION AND CONCLUSIONS

Discussion
The number of dividing cells for all two trials for the Animas and San Juan River were
zero. The cells in trial 2 for Site 1 in San Juan, Shiprock NM were positive with no visibility of
mitosis. Cells were elongated, longer and had thicker cell walls. The other trials however did show
that the cells had already went through different mitotic phases. There was also an exception of
the Control, there were numbers rating average. It makes sense that the San Juan River samples
had higher weight percentages for sand (SiO4), per Table 4, as the Animas river samples had
significantly higher weight percentages for Aluminum Oxide, Calcium Oxide, and Iron Oxide. The
higher concentration of Lead and Arsenic in the Animas River samples indicates that the San Juan
River Water has less of these toxic elements upstream, prior to the confluence of the two rivers.
In Figure 2, the Animas Riverbed has higher weight percent of Aluminum, Calcium, and
Iron oxides, which reduces the weight percent of common sand. The San Juan Riverbed also has
a higher weight percentage of sand because it has less Aluminum, Calcium, and Iron oxides. In
Figure 3, the Animas Riverbed has higher Lead, Arsenic, Zinc, Cobalt, and Nickel. The upstream
San Juan Riverbed was also not affected by the Gold King mine spill and its cleaner water must
dilute the toxins flowing in from the Animas River, at the confluence of the two.
Zinc (found both in the riverbed samples and absorbed by the onion bulbs) can be toxic to
plants. “Zinc toxicity and problems with regard to tolerance and ecological significance are briefly
discussed. Differential tolerance of plant genotypes exposed to zinc toxicity is a promising
approach to enrich our understanding of zinc tolerance in plants.” Knowledge concerning the
composition and biochemistry with regard to phytotoxicity, acceptance and transport of zinc and
tolerance and its characterization are also discussed. “The cytotoxic effects of zinc on plants are
elucidated. The major change was seen in the nucleus of the root tip cells due to zinc toxicity.”
“The chromatin material was highly condensed and some of the cortical cells showed disruption
and dilation of nuclear membrane in presence of 7.5 mM zinc.” The cytoplasm was beginning to
have less structure, “disintegration of cell organelles and the development of vacuoles were also
observed. The number of nucleoli also increased in response to zinc resulting in the synthesis of
new protein involved in heavy metal tolerance.”
http://link.springer.com/chapter/10.1007/978-90-481-2666-8_53
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These results may be useful to our local agencies, showing that there are other ways to
monitor our water, such as using mitotic index, heavy metal, and metalloid toxin tests to determine
this. With these results, they may also be helpful to a national government agency. Considering
rivers, streams, and other water select communities, using the mitotic index and metal tests will
help determine if there is progress or remaining constant.
In reference to the experiment in 2015/2016 on the mitotic index from the Animas and San
Juan Rivers, the mitotic index was fairly low. With the exception of the control which was rating
average. This year’s study, with the inclusion of the water and riverbed, that the two rivers have
not healed themselves. It is true the once heavily polluted water has moved on, however the effects
of the Gold King Mine Spill still linger in the respective riverbeds. As for IBM's Oxford ED-2000
X-Ray Fluorescence (XRF) machine, it empirically excites materials with X-Rays, to move
electrons from their normal shells to outer shells. When those excited electrons fall back into their
normal shells, they emit characteristic X-Ray spectra, spectra which are unique to each chemical
element. These unique X-Ray spectra become a form of fingerprint which the Geology + Majors
software uses to identify the chemical elements found in the riverbeds and onion bulbs.

Conclusions
In conclusion, the hypothesis was accepted. The mitotic index in each cell of all two trials
for the Animas and San Juan River water was 0%. Control had a mitotic index of 16.0%, indicating
that the factor was heavy metal and metalloid toxin contamination. There were no phases of mitosis
seen in the San Juan River, Animas River. However, there were observations showing that the
cells in all of the trails from the Animas River and site 2 of the San Juan River in Shiprock went
through different phases of mitosis. There were also no visible signs of mitotic aberrations,
however cell abnormalities were observed. The cells were elongated, longer and had thicker cell
walls for trial 2 in Shiprock, NM.
There were also significant changes in major chemical elements between San Juan and
Animas riverbed samples in average weight percent. The Animas Riverbed has higher weight
percentages of Aluminum, Calcium, and iron oxides, which reduces the weight percent in common
sand. The San Juan Riverbed has a higher weight percentage of sand because it has less of the
chemical elements found in the Animas Riverbed.
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This means that heavy meatal contamination of water in the Animas and San Juan River from the
2015 Gold King Mine Spill is still affecting life in the area. The Animas and San Juan River water
affected the mitotic index of Allium cepa root tip.

Future Research
In the future, I would like to use proper micro technique procedures that must be followed
in order to produce the best microscopic slides of onion root tip. This means that I would like to
cut the root tip properly so the cells do not overlap each other while observing the cells in the
microscope. Another component that I would like to add is to test sample water in Church Rock
New Mexico for Metalloid Toxins and Heavy Metal and determine which site is more toxic, The
Animas and San Juan River or Church Rock Well Water. I would also like to test for biological
contaminants that may contaminate the roots, affecting to the contribution of the root tip growth
along with heavy metal and metalloid toxins. Lastly, I would gather riverbed samples in the San
Juan River, upstream of the confluence with the Animas River, to help confirm that the San Juan
River is initially cleaner and then contaminated by the Animas River at the confluence of the two
rivers.
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References
[1] vlab.amrita.edu. Mitosis in Onion Root Tips. 24 Feb. 2016. Web. 2011.
http://vlab.amrita.edu/?sub=3&brch=188&sim=1102&cnt=1
Adekunle A. Bakare, Okunola A. Alabi, Adeyinka M. Gbadebo, Olusegun I. Ogunsuyi and
Chibuisi G. Alimba. In Vivo Cytogenotoxicity and Oxidative Stress Induced by Electronic Waste
Leachate and Contaminated Well Water. www.mdpi.com/2078-1547/4/2/169/htm
[2] The Biology Project, Cell Biology. The Cell Cycle & Mitosis Tutorial. 24 Feb. 2016. Web
.1997 http://www.biology.arizona.edu/cell_bio/tutorials/cell_cycle/cells3.html
Prabhakar Bhatta and S.R. Sakya. Study of Mitotic Activity and Chromosomal Behaviour in
Root Meristem of Allium cepa l. Treated with Magnesium Sulphate. 24 Feb. 2016. Web.
file:///E:/Mitotic%20Index%20Study.pdf
[3] Andrea Menéndez-Yuffá, Rafael Fernandez Da Silva, Liliana Rios, Nereida Xena de Enrech.
Mitotic aberrations in coffee (Coffea arabica cv. 'Catimor') leaf explants and their derived
embryogenic calli. 24 Feb. 2016. Web. file:///E:/Mitotic%20aberrations.pdf
[4] Camila Gonçalves Athanásio, Daniel Prá, and Alexandre Rieger. Hindawi, Scientific World
Journal. Water Quality of Urban Streams: The Allium cepa Seeds/Seedlings Test as a Tool for
Surface Water Monitoring. 24 Feb. 2016. Web. 2014.
http://www.hindawi.com/journals/tswj/2014/391367/
[5] Reichman, S.M. The Responses of Plants to Metal Toxicity: A review focusing on Copper,
Manganese and Zinc. AUSTRALIAN MINERALS & ENERGY ENVIRONMENT
FOUNDATION. 2002. 24 Feb 2016. Web.
http://plantstress.com/Articles/toxicity_i/Metal_toxicity.pdf
[6] Oxford ED-2000 XRF Machine from IBM, Tucson Arizona