The Toxin in Rice- Arsenic in Our Food: Developing a Novel Testing

The Toxin in Rice- Arsenic in
Our Food: Developing a Novel
Testing Method and Analyzing
Arsenic Levels in Rice
By:
Anuush Vejalla
Major Sources of Assistance-For the most part this project was completed without
much assistance and at home. However, in the beginning Mr.Menton, the head of the
Science Department, was essential in helping me obtain a test kit that tested for arsenic
levels in water, which I later modified to test for arsenic levels in any type of food. Later
Mr. Dougherty, the AP Pyschology teacher, taught one how to keep a systematic
notebook for science fair and emphasized to me the importance of taking the pictures of
one’s results. Mrs. Langley, an AP Statistical Analysis teacher, worked with me to
analyze my data with the use of statistics. However, most of the statistical analysis
presented was researched and completed alone without the aid of anyone else. Finally
Erin Zhang, a Senior at Detroit Country Day, helped one analyze some of the chemical
reactions that took place during the experiment.
Table of Contents
Introduction:………………………………………………………………………..2-5
Background…………………………………………………………...2-4
Working Hypothesis and Research Objective………………….....5
Materials, Methods, and Challenges…………………………………………....5-8
Data………………………………………………………………………………....9-11
Data Analysis……………………………………………………………………....12-14
Conclusion………………………………………………………………………….14-15
Limitations…………………………………………………………………….........15
Challenges………………………………………………………………………….16-17
Extension……………………………………………………………………………17
Novelty of This Experiment………………………………………………….........18
Works Cited…………………………………………………………………………19
Introduction
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Recently arsenic in rice, juices, and vegetables has caused concern. In fact South
Korea has halted the import of rice from the U.S. due to these concerns (“Lawmakers
Seek Limits on Arsenic”). Billions of people around the world rely on rice as their staple
food. If there really is arsenic in rice then the sustainability and health of much of the
world’s population would be greatly affected (“Arsenic in Your Food”).
Due to the implications of arsenic contamination in rice, the purpose of the experiment
is, “How the Type of Rice Affects the Amount of Inorganic Arsenic Present”.
Health Affects of Arsenic Poisoning
Arsenic is a heavy metal that, when exposed to in large amounts in a short
amount of time, can lead to irritation of the gastrointestinal tract, nausea, vomiting, and
diarrhea which eventually can lead to death. There are also many long-term affects of
arsenic poisoning. Arsenic can get into the blood through rice due to the fact that
arsenic is very soluble. It can dissolve in the water that the rice seed draws up when
cooked and so when eaten, gets into the blood stream and causes many problems with
the organs that pump or filter the blood. Continuous exposure to arsenic in the blood
can lead to high blood pressure and cardiovascular disease (“Arsenic”). Also, it may
cause the cancers of the bladder, lung, and skin. It may also cause the degeneration of
the kidneys and liver (“Arsenic in Your Food”). Finally it may cause hyperpigmentation,
where dark spots form on the skin are formed, or hyperkeratois, which is the thickening
of the skin. Exposure to arsenic for 10 years or longer can cause many of these
problems ("Health Affects of Arsenic").
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Types of Arsenic
Two main types of arsenic exist, inorganic and organic. Inorganic arsenic has two
forms Arsenic (III)-As2O3, also known as Arsenious Acid, and Arsenic (V)-H3AsO4, also
known as Arsenate or Arsenic Acid. Inorganic Arsenic is more toxic to humans than
organic arsenic due to the fact that the body is not able to excrete it as easily. In this
study only levels of inorganic arsenic were tested. Inorganic arsenic can exist naturally.
Such as when Arsenopyrite, a naturally occurring arsenic compound, is oxidized, it turns
into Arsenic V. Inorganic arsenic can also be manmade for it is used as an insecticide
in pesticides and fertilizers (“Arsenic in Your Food”).
Arsenic is found in the Earth’s crust as Arsenopyrite: FeAsS. When exposed to
oxygen, Arsenopyrite turns into H3AsO3 or Arsenic (V).
2FeAsS + 11 O2 + 6 H2O --> 4Fe2+ + 4 H3AsO3 + 4 SO42-
Inorganic Arsenic can also be found naturally as Arsenic Trisulfide-As2S3. When
Arsenic Trisulfide reacts with water it forms Hydrogen Sulfide and H3AsO3. The rice
plant may thus pick up sulfur, a nutrient it needs, along with the Arsenic.
As2S3+ 6H2O 3H2S + 2H3AsO3
Inorganic Arsenic is also man made. It is used as an insecticide in many
pesticides and fertilizers. The feed, that farmers feed the livestock that make the
manure that makes the fertilizer, contains high levels of arsenic (Arsenic in Your Food).
In terms of organic rice, USDA Organic means that only organic pesticides or
fertilizers are used. Organic fertilizer is a fertilizer that is made from all natural sources,
like from cow manure. But this cow manure may have arsenic from the feed that the
cow is fed (“Organic Standards”).
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The Rice Plant and Arsenic in the United States
Rice contains greater amounts of arsenic than other plants because it absorbs
water better than most plant, due to the fact that it is grown in annually flooding areas.
Arsenic is very soluble so the rice plant absorbs it
with the water. (“Arsenic in Your Food”). This is
exemplified in Bangladesh where the rice has the
same amount of arsenic as the water used to
irrigate it. In fact the rice in Bangladesh is thought
to be a greater source of arsenic poisoning than
water (“Arsenic in Bangladesh”). The rice plant
takes up arsenic so well due to the fact that it
mistakes arsenic for silicon, one of its nutrients.
(Plos Blogs).
In the last 100 years about 1.6 million tons of Arsenic have been used for farming
in the U.S.
The southern states of Arkansas, Louisiana, Missouri, and Texas are
infamous for using arsenic containing pesticides and fertilizers. Thus places with less
pesticides and fertilizers, like developing nations, may have rice containing less
amounts of arsenic.
There are three main types of rice black, brown, and white. Black and brown rice
are manufactured by only removing the husk of the rice. While, white rice is
manufactured by removing the husk, the bran, and germ layers of the rice leaving only
the endosperm (Dr.BenKim). The bran and germ layers retain many toxins and
nutrients. So when this outer hull is removed, when rice is made white, much of the
Vejalla 5
arsenic contamination is also removed (“Arsenic in Your Food”). Black rice, contains
high levels of antioxidants in the bran and the hull, preventing arsenic from binding in
the bran and hull (L.).
Working Hypothesis and Research Objective
Based on research, I hypothesize that 1) Brown rice will contain higher levels of
arsenic than white rice due to the fact that it contains two extra layers, the germ and the
bran, that white rice does not contain. The arsenic would concentrate in the outer
layers, so if these outer layers are removed a lot of the contamination may be removed.
2) That rice grown domestically would contain higher levels of arsenic than in
developing nations due to the fact that farmers in the United States use more arsenic
containing pesticides and fertilizers than in developing nations.
Materials, Methods, and Challenges
Due to the fact that no lab would accept me because of my young age I had to test the
level of arsenic in rice using a test kit. The test kit only measured the amount of arsenic
in aqueous solutions due to the fact that its purpose was to measure arsenic in food and
soil. Due to this problem a way to make the rice aqueous had to be found. I first cooked
the rice and tested the water, which remained, for arsenic. But this turned out to be an
inaccurate way to measure the arsenic level for a great of percentage of arsenic would
still remain in rice after cooking. So I then decided to grind up 1 cup of rice and mix it
into 1500 mL of distilled water heated at 95 degrees Celsius for about 10 minutes. This
rice solution was then taken and filtered with coffee paper so that only the truly aqueous
particles came through.
Vejalla 6
40 mL of this filtered solution and 10 mL of ethyl
alcohol were then mixed together. Ethyl alcohol was
added to this solution due to the fact that before when
this alcohol was not added the bubbles that contained
the arsine gas would come to the top of the rice
Reaction Vessel
solution and sit there, so there showed to be no arsenic
in the solution many times. I did not notice that this was
the problem. So a Standard was ordered to see if the Test Kit actually worked. What
was exemplified from this Standard was that the bubbles that contained the arsenic in
the solution came to the top and popped. This exemplified that the rice solution had too
much surface tension, so ethyl alcohol was found to reduce surface tension and thus
was added to the solution. This new solution of rice solution
Arsine Gas Not Released
Arsine Gas Released
and ethyl alcohol was added to a reaction vessel. Ethyl alcohol, 90%, acts a surfactant
to reduce the surface tension of the solution. The surfactant reduces the liquid-gas
Vejalla 7
interface present, allowing for the Arsine Gas to escape the solution, for clear
boundaries of the states of matter are now defined.
Now
Sodium
Phosphate
(Na3PO4)
and
Oxone
Monopersulfate
(2KHSO5·KHSO4·K2SO4) were added to the solution to oxidize sulfide into sulfate. Due
to the fact that sulfide is a big interferer. It turns into a gas alongside with the arsenic
and reacts with the Mercury Bromide test strip to make it look like there is more arsenic
than there actually is.
Na3PO4 + 2KHSO5·KHSO4·K2SO4 + S2-  SO42- + K+ + H3PO4 + 2KHO-·KHSO4·K2SO4
Potassium Peroxymonopersulfate or Oxone Monopersulfate, a triple salt, was then
neutralized due to the fact that it is also an interferer in the process. To neutralize
Oxone, Disodium EDTA (C10H12 N2Na2O6) was added.
2KHSO5·KHSO4·K2SO4 + C10H12 N2Na2O6  H2SO5 ·KHSO4·K2SO4 + 2K+ + C10H11
N2Na2O6
Finally the arsenic could be extracted. Sulfamic Acid (NH2SO3H) and Zinc (Zn)
were added to the solution to turn both Arsenic III (As2O3) and Arsenic V (H3AsO4,) into
Arsine Gas (AsH3).
As2O3 + 12NH2SO3H + 6Zn  2AsH3 + 6Zn(NH2SO3)2 + 3H2O
H3AsO4 + 12NH2SO3H + 4Zn  AsH3 + 2Zn(NH2SO3)2 + 4H2O
The Arsine Gas then reacted with the Mercury Bromide Test Strip (HgBr2) to form
a certain color. Due to the fact that the amount of Arsine Gas varies, the types of
products made also varies.
xAsH3 + yHgBr2  AsH2HgBr + As(HgBr2)3 + other variants
After 30 minutes the test strip was compared to a color grading scale, which told
the level of inorganic arsenic present. This level was then converted to the level of
Vejalla 8
arsenic per 1000 mL using the formula C1 x V1 = C2 x V2. ; where C1 is the
concentration (ppb) of arsenic found in 1500mL, V1 is 1500 mL, V2 is 1000 mL, and C2
is the unknown concentration of arsenic (ppb). This was done so that the level of
arsenic in rice could be compared to the amount of arsenic on different limits on arsenic
in water. Once the trial was complete the level of arsenic per 1000 mL was recorded.
Six trials were completed of each rice sample and 11 different samples of rice were
tested.
Flow Chart of Procedure
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Data: The results showed to be as follows
Figure 1
Origin
Missouri
Rice
Yoga Organic
Long Grain
Brown
Foothills of the
Himalayas
83
75
90
90
105
90
11
90
83
90
98
90
83
89
6
Della Organic
Long Grain
Brown
68
60
83
75
60
75
70
9
Basmathi
Brown
83
68
68
75
55
68
69
9
75
60
68
68
68
75
60
53
68
60
68
75
68
65
5
9
68
60
68
53
53
53
68
60
60
45
45
53
60
54
9
6
60
53
53
38
53
60
53
8
38
53
60
30
60
45
48
12
30
45
30
38
30
53
38
9
Mississippi
Uncle Ben's
Delta, Arkansas White
China
Hinode Black
Missouri
China
Mountain
Highlands of
Thailand
Arkansas
Foothills of the
Himalayas
Standard
Deviation
98
Mississippi
Uncle Ben's
Delta, Arkansas Brown
Missouri
Amount of Inorganic Arsenic per 1000 mL (ppb)
Trial Trial Trial Trial
Trial 1 Trial 2
Average
3
4
5
6
Yoga Organic
Long Grain
White
Hinode White
Jasmine
Mahatma White
Della Jasmine
White
Basmathi White
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Figure 2:
The Affect of Rice Type on the Amount of
Inorganic Arsenic
Average Amount of Inorganic Arsenic per 1000 mL (ppb)
100
90
80
70
60
90
89
70
69
68
65
50
40
30
60
54
53
48
38
20
10
0
Uncle
Yoga
Della Basmathi Uncle
Organic Ben's Organic Brown Ben's
Brown
White
Long
Long
Grain
Grain
Brown
Brown
Hinode Yoga Hinode Jasmine Della Basmathi
Black Organic White Mahatma Jasmine White
White White
Long
Grain
White
Types of Rice
Vejalla 11
Data Analysis
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1) The first hypothesis that brown rice would contain higher levels of arsenic than
white rice was proven because of the extra outer two layers that brown rice
contains.
1a) Based on the Student’s t-test, brown and white rice had a significantly different
levels of inorganic arsenic. The p value was 0.017, which was below 0.05, disproving
the Null Hypothesis and suggesting that there is a significant difference between the
levels of arsenic in brown and white rice. The Anova Test analyzed with the Post Hoch
Tukey Test gives a certain number, in this case 18. If the difference in the average
arsenic levels of any two samples is greater than this number then they are
significantly different. This test exemplified that every brown rice had significantly
higher levels of arsenic than its white counterpart. The Yoga Brown had 90 ppb of
arsenic, while its white counterpart Yoga White only had 60 ppb. Uncle Ben’s brown had
89 ppb of arsenic, while its counterpart only had 68 ppb. Basmathi Brown had 69 ppb of
arsenic and Basmathi White had 38 ppb of arsenic. The difference between all of the
brown rice and its counterpart white rice had means greater than 18. Thus proving that
brown rice contains higher levels of arsenic than white rice, due to the extra outer two
layers that brown rice contains.
1b) Only Hinode Black and Hinode White did not have significantly different levels of
arsenic according to the Anova Post Hoc Tukey Test, even though black rice contains
the two extra outer layers that brown rice does. This may be due to the fact that black
rice contains significantly higher levels of antioxidants than brown rice. These
antioxidants concentrate in the outer layers, giving the black rice its “black” color. They
Vejalla 13
prevent arsenic from binding, causing the arsenic either to be excreted or to concentrate
in the endosperm. Thus making the white and black rice have the statistically same
amounts of arsenic.
2) The second hypothesis was disproven. The place where the rice was grown did
not have a significant correlation with the level of arsenic.
2a) A Students t-test was used to compare domestic and foreign rice The p value
showed to be 0.117 when testing all the rice (domestic vs. foreign) and 0.251 when
testing only the white rice (domestic vs. foreign). Both these values were greater then
0.05 so the null the hypothesis was disproven so domestic rice and foreign rice
statistically had no difference.
2b) Though certain rice like Yoga and Uncle Ben’s rice, grown in the Mississippi Delta,
Arkansas, or Missouri, had significantly higher levels of arsenic than Jasmine Mahatma
Rice, grown in Thailand, or Basmathi White, grown in India, on average where the rice
was grown did not have a significant impact on the level of arsenic, as proven by the
ANOVA Test analyzed by the Post Hoc Tukey Test.
2c) As proven by the ANOVA Test analyzed by the Post Hoc Tukey Test, Della Organic
Long Grain Brown, Uncle Ben’s White, and Yoga White rice, all grown domestically, had
statistically the same amount of arsenic as Basmathi Brown, Hinode Black, Hinode
White, and Jasmine Mahatma White, which were all grown in foreign countries, for all of
these different types of rice had an average amount of arsenic within 18 ppb of each
other. Where the rice was grown did not impact the level of arsenic due to many things,
such as: the contamination of ground water or the soil, whether a company was
Vejalla 14
polluting the water with arsenic, whether for some reason the farmer used more or less
pesticides or fertilizers, if the farmers in developing nations sent only the best rice to the
United States, or even if rice was grown in different locations and combined. So there
are many other factors that influence the correlation between where the rice is
grown and the amount of inorganic arsenic.
3) Organic and non-organic rice did not have a significant difference according to
the student’s t-test. When comparing organic vs. non-organic rice with the Student’s Ttest, the p-value was 0.3546, which was higher than 0.05; proving that organic and nonorganic rice had statistically the same amount of inorganic arsenic. This may because
the fertilizers used still have arsenic in them or the water used is contaminated.
Conclusion
In the end arsenic in rice is a real problem and will pose a problem to the sustainability
of the human race. The federal limit for arsenic in water is 10 ppb per liter. The amount
of arsenic in rice, in these finding, range from 4 to 9 times that limit.
 Federal limits on the amount of arsenic in rice should be laid down at least the old
limit for arsenic in water, 50 ppb. So that companies will have time to adjust to these
new standards, for there has never been a standard on arsenic in rice before.
 Limits on the amount of arsenic should also be put on pesticides that farmer’s use
and the feed that farmers feed the animals that make their fertilizer, for pesticides
and fertilizers are the root cause of the problem. This should be a limit enforced by
the World Health Organization.
Vejalla 15
 Limits should be put on the water that is used to irrigate the rice fields, for this is also
a major factor. This should also be a limit enforced by the World Health
Organization.
 For the time being it is safer to eat white rice instead of brown rice.
 Finally there is no difference in the level of arsenic in organic rice compared to nonorganic.
Challenges
1. Formation of Rice Solution- Making the rice solution in the beginning was a
challenge in itself. The Arsenic Test Kit only measured levels of arsenic in water and
soil, not in rice; so I had to find a way to make the rice solution aqueous.
2. Unable to Extract Arsine Gas- In the beginning, when I first started testing there
showed to be no arsenic in the rice. But when I tested the Standard Solution to see if
the Arsenic Test Kit from Hach actually worked, I realized that the arsenic containing
bubbles that came up burst right away in the Standard, but they accumulated at the top
of the solution when I tested for arsenic in rice. I decided that it was an issue with
surface tension. I found that I could reduce the surface tension by adding ethyl alcohol.
When I added the ethyl alcohol, the arsine gas-containing bubbles popped when at first
I tested for arsenic in Uncle Ben’s Brown Rice.
3. Not enough Ethyl Alcohol-When I tested for arsenic in Yoga Organic Long Grain
Brown Rice and Hinode Black Rice the bubbles did not pop once again. I decided to
increase the amount of ethyl alcohol-continously added to the rice solution from 4 mL to
10 mL and this problem was then resolved.
Vejalla 16
Limitations
1. The technique that I followed may not have extracted a 100% of Arsenic from rice in
the water, due to the fact that my Test Kit was designed to test for the arsenic level in
the water. So if I had access to better equipment at the Michigan State Environmental
Lab my results would be more accurate.
2. If more trials were conducted then the results would be more accurate.
3. In this experiment I tested Arsenic III and Arsenic
V. Furtherer research is required to find if any
Color Grading Scale
other arsenic compounds are present.
4. If I had a more accurate way of measuring the
amount of inorganic arsenic present in the rice;
like a Gas Chromatograph, a Mass Spectrometer
or a PID controller, the experiment would have
been more accurate. The Arsenic Test Kit from
Hach only had 8 numbers ranging from 0-500 to
which one could compare the color of the test strip. This made it so that I would have
to estimate the amount of arsenic present because I only had 8 colors to match to the
test strip to, so it could have more accurate. But the same margin of error existed for
each trial. So even thought the numbers may not be correct, the trends are correct.
The numbers may not be exact but there still are still significant amounts of arsenic in
rice.
5. The rice was ground up, so little rice particles existed in the aqueous solution of rice,
resulting in only the arsenic from the surface of the particle escaping, while the arsenic
inside of the particle did not escape. This would result in the findings to be lower than
Vejalla 17
the actual arsenic level. However the arsenic that remained inside of the rice particles
would be so minute that a major change in results would not occur.
Extension:
Continuation of this Project:
•
Testing the amount of arsenic in the urine of people who eat rice and the urine of
people who do not eat rice.
•
Studying the correlation between certain types of deaths or diseases with rice
consumption based on ethnicities.
•
Testing the amount of arsenic in the different soils where rice is grown to see if
there is any correlation between the amount of arsenic in the rice and soil.
•
Checking the amount of organic arsenic in rice.
•
Testing the level of arsenic in different pesticides or fertilizers and the water that
is used to irrigate the rice.
•
Testing levels of arsenic in rice from different lots buy buying the rice at different
times in the year.
•
Measuring the level of arsenic in rice grown completely without pesticides or
fertilizers to see for sure if pesticides or fertilizers affect the level of arsenic
present.
•
Analyzing correlation between rate of certain disease and consumption of rice in
countries that eat high levels of rice, for arsenic poisoning may be the cause of
their diseases.
•
I finally want to repeat my experiment in a lab with better equipment and more
samples of rice. This can be accomplished by using the processes of Gas
Chromatography and Multiple Ion Detection Mass Spectrometry.
Vejalla 18
Novelty of this Experiment- No known technique or published procedure to directly
measure the amount of arsenic in rice was available to me. Sophisticated equipment to
measure arsenic, like the Gas Chromatograph Mass Spectrometer, Multiple Ion
Detection
Mass
Spectrometry,
or
the
Graphite
Furnace
Atomic
Absorption
Spectrometry, were not within my reach. The arsenic test kit procured could only test for
the arsenic in water. I had to then find a way to make it so that the test kit would
measure the level of arsenic in rice. Then, along the way, problems, such as the high
surface tension of the rice solution, came up that I had to solve with perseverance. In
the end, I developed a cheap effective procedure that could test for arsenic in any type
of food at a relatively low price of around 1 dollar per test, which the World Health
Organization could greatly utilize.
Works Cited
Vejalla 19
"Arsenic in Your food Our Findings Show a Real Need for Federal Standards for This Toxin."
Consumer Reports Nov. 2012: n. pag. Consumer Reports. Web. 12 Nov. 2012.
<http://www.consumerreports.org>.
"Arsenic, Organic Foods, and Brown Rice Syrup." Environmental Health Perspectives. N.p.:
n.p., n.d. N. pag. Gale Science in Context. Web. 12 Nov. 2012.
<http://ic.galegroup.com>.
"Health Affects of Arsenic." Civil and Environmental Engineering. U of Maine, n.d. Web. 12
May 2013. <http://www.civil.umaine.edu>.
L., Patrick. "Toxic Metals and Antioxidants: Part II. The Role of Antioxidants in Arsenic and
Cadmium Toxicity." PubMed.gov. US National Library of Medicine, n.d. Web. 12 May
2013. <http://www.ncbi.nlm.nih.gov>.
"Lawmakers Seek Limits in Arsenic in Rice." Reuters Health Medical News 22 Sept. 2012: n.
pag. Gale Science in Context. Web. 12 Nov. 2012. <http://ic.galegroup.com>.
"Organic Standards." United States Department of Agriculture. N.p., n.d. Web. 12 May 2013.
<http://www.ams.usda.gov>.
Purpose and Motivation
• How Does the Type of Rice Affect the Amount
of Inorganic Arsenic Present?
• Inspiration
– South Korea halted the import of rice from the
United States
– Rice is staple food for billions
Background on Arsenic
• Two major types of inorganic arsenic
– Arsenic III: As2O3
– Arsenic V- H3AsO4
• Sources of Arsenic Contamination
– Natural
• Exists as Arsenopyrite and Arsenic Trisulfide in the Earth’s crust
• Arsenic very soluble, rich plant siphons abnormally high
amounts of water, and mistakes the arsenic for silicon
– Manmade
• Used as insecticides in pesticides and fertilizers
• EPA limit 10 ppb per liter
– Ingestion detrimental to health
Background (Rice)
• Rice
– White, brown, black
• Black and brown only
remove husk
• White remove husk
and endosperm
• Black contains high
levels of antioxidants
Hypotheses
1) Rice grown domestically will contain higher
levels of arsenic than rice grown in developing
nations, because farmers in the U.S. use more
arsenic- containing pesticides and fertilizers.
1) brown and black rice will contain higher levels of
arsenic than white rice, because brown and black
rice have an extra outer shell that retains arsenic
that white rice does not have.
Flow Chart of Procedure
TESTING –Arsine Gas Released
Bubbles can
be seen
TESTING – Arsine Gas Not Released
Key Chemical Reactions
Oxidation:
Na2HPO4 + 2KHSO5·KHSO4·K2SO4 + H2S  SO42- + H3PO4 + 2KHSO-·KHSO4·K2SO4 + 2Na+
Oxone®
Neutralization:
2KHSO5·KHSO4·K2SO4 + C10H12 N2Na2O6  H2SO5·KHSO4·K2SO4 +2K+ + C10H11 N2Na2O6
EDTA2Arsine Gas Formation:
Arsenic III and Arsenic V are turned into Arsine Gas by adding Sulfamic Acid and Zinc
As2O3 + 12NH2SO3H + 6Zn  2AsH3 + 6Zn(NH2SO3)2 + 3H2O
Sulfamic Acid
H3AsO4 + 4NH2SO3H + 2Zn  AsH3 + 2 Zn(NH2SO3)2+ 4H2O
Arsine Gas Reacts with the Mercury Bromide Test Strip:
AsH3 + HgBr2  AsH2HgBr + As(HgBr)3 + other variants
Amount of Inorganic Arsenic
vs. Type of Rice
Average Amount of Inorganic Arsenic per 1000 mL
(ppb)
100
90
90
89
80
70
70
69
68
65
60
60
50
54
53
48
38
40
30
20
10
0
Yoga
Organic
Long
Grain
Brown
Uncle
Ben's
Brown
Della Basmathi Uncle
Organic Brown
Ben's
Long
White
Grain
Brown
Hinode
Black
Type of Rice
Yoga
Organic
Long
Grain
White
Hinode Jasmine Della Basmathi
White Mahatma Jasmine White
White
White
Data Analysis
1) Brown rice contains higher levels of arsenic than
white rice because of the extra outer two layers
that brown rice contains.
2) The place where the rice was grown did not have
a significant correlation with the level of arsenic.
– So there are many other factors that influence the
correlation between where the rice is grown and the
amount of inorganic arsenic.
3) Organic and non-organic rice did not have a
significant difference in arsenic levels.
Conclusion
• In the end arsenic in rice is a real problem and will pose a
problem to the sustainability of the human race. The federal
limit for arsenic in water is 10 ppb per liter. The amount of
arsenic in rice, in these finding, range from 4 to 9 times that
limit.
• Federal limits on the amount of arsenic in rice should be laid
down at least the old limit for arsenic in water, 50 ppb.
• Limits on the amount of arsenic should also be put on
pesticides that farmer’s use and the feed that farmers feed
the animals that make their fertilizer, for pesticides and
fertilizers are the root cause of the problem. This should be
a limit enforced by the World Health Organization.
Limitations
• All of the arsenic might not have
been extracted from the rice in the
water
–laboratory technique not used
• The scale on the Test Kit was not very
good
TESTING - Equipment
Novelty of the Experiment
• No technique or published
procedure to directly measure the
amount of arsenic in rice available
–Arsenic Test kit only tests arsenic in
water
–Had to Figure out way to modify test
kit for testing arsenic in food
Extension- Continuation of Project
• Testing the amount of arsenic in:
– the urine of people who eat rice and the urine of people
who do not eat rice.
– different soils where rice is grown to see if there is any
correlation between the amount of arsenic in the rice
and soil.
– in different pesticides or fertilizers and the water that is
used to irrigate the rice.
– in rice grown completely without pesticides or fertilizers
pesticides or fertilizers affect the level of arsenic present.
• Repeat experiment in a lab with better equipment
and more samples
Acknowledgement
• Mr.Menton (Head of Science Department)-Essential
in helping me obtain a test kit that tested for arsenic
levels in water
• Mr. Dougherty (AP Psychology Teacher)- Taught one
how to keep a systematic notebook for science fair
and emphasized to me the importance of taking the
pictures of one’s results.
• Mrs. Langley (AP Statistics Teacher)- Worked with
me to analyze my data with the use of statistics
• Aaron Zeng and Sid Chand (Seniors at Detroit
Country Day) - helped one analyze some of the
chemical reactions that took place during the
experiment.
The Toxin in Rice – Arsenic in our Food:
Developing a Novel Testing Method and Analyzing Arsenic Levels in Rice
Anuush Vejalla- Southeastern Michigan Symposium
Abstract
Flow Chart of Procedure
The aim of the project is to understand the content of arsenic in rice and to
furnish recommendations based on statistical and experimental results to
both governments and users all over the world. Arsenic is a deadly toxin that
leads to cancer, heart problems, and death. Since there is not any published
method to quantify the levels of inorganic arsenic in rice, a novel testing
method was developed through experimental and theoretical means. The
formation of the rice grain depends on the soil, fertilizer, water, pesticide, and
the location of cultivation, which all need to be considered to understand
arsenic levels in rice. Thus to verify one hypothesis, that rice grown
domestically in the United States would contain higher levels of arsenic than
rice grown in foreign countries, for farmers in the U.S. use more pesticides
and fertilizers while farming, rice from different parts of the world was tested.
Another hypothesis, that brown rice would have higher levels of arsenic than
white rice due to the extra two layers, the germ and the bran that it contains,
was verified by testing brown, black, and white rice. The rice had four to nine
times the EPA limit on arsenic in water, 10ppb. The results show that brown
rice has higher levels of arsenic than white rice, but the correlation between
where the rice is grown and the level of arsenic varies.
Key Chemical Reactions
Oxidation:
Sodium Phosphate Dibasic and Potassium Peroxymonosulfate (Oxone®) are added
to create strong oxidizing conditions, so that Hydrogen Sulfide oxidizes into Sulfate,
for it is an interferer. Sulfide turns into a gas and reacts with the mercury bromide
test strip, making it look like there is more arsenic than actually present.
Na2HPO4 + 2KHSO5·KHSO4·K2SO4 + H2S  SO42- + H3PO4 + 2KHSO·KHSO4·K2SO4 + 2Na+
Oxone®
Neutralization:
Disodium EDTA, a organic Amine acting as an acid, is added to neutralize any
remaining Oxone, acting as a base, for Oxone is also an interferer.
2KHSO5·KHSO4·K2SO4 + C10H12 N2Na2O6  H2SO5·KHSO4·K2SO4 +2K+ +
EDTA2C10H11 N2Na2O6
Arsine Gas Formation:
Arsenic III and Arsenic V are turned into Arsine Gas by adding Sulfamic Acid and
Zinc
Background
Motivation to Conduct This Experiment
After I came back from India and had seen the farming land, I knew I wanted
to do something related to agriculture. I then heard that South Korea had
halted the import of rice from the United States due to concerns of arsenic
in the rice. So I did this experiment to verify South Korea’s concerns for if
there really is arsenic in rice, I would be affected, my family will be affected,
and billions of people around the world would be greatly affected.
Method
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
XI.
XII.
XIII.
XIV.
XV.
XVI.
Obtain 1 cup or 160 mg of a rice sample
Grind the rice sample to a powder and mix the powder in 1500 mL of
distilled water and heat this solution for 10 minutes at 95oC.
Filter 50mL of the rice solution with filter paper
Mix 40 mL of the filtered rice solution with 10 mL of ethyl alcohol-90%
concentration-to reduce the surface tension
Pour this solution into the reaction bottle from the Arsenic Test Kit
Insert the test strip into the cap of the reaction bottle and close the flap
Begin creating strong oxidizing conditions so sulfide, an interferer in
the process, can be oxidized to sulfate by adding one pillow of Sodium
Phosphate Dibasic: Na2 PO4- to the solution in the bottle and swirling to
mix
Finalize the oxidation of sulfide, an interferer, to sulfate by adding a
second pillow of Potassium Monopersulfate or Potassium
Peroxymonopersulfate, a triple salt: 2KHSO5·KHSO4·K2SO4 into the
solution in the bottle and swirl to mix
Wait 3 minutes
Add a third pillow of a mixture of Disodium and Tetra Sodium
Ethylenediaminetetracetic Acid: C10H12 N2Na2O8 and C10H12 N2Na4O8 - to
remove any remaining Potassium Monopersulfate and swirl to mix
Wait 2 minutes, then swirl again
Add one scoop of Sulfamic Acid- NH2SO3H, to generate AsH3 gas by
reduction and swirl to mix
Add a final pillow of Powdered Zinc, Zn to the solution to also generate
AsH3 by reduction and then immediately attach the cap to the reaction
bottle and wait 30 to 35 minutes so that the solution can react and the
AsH3 gas can react with the HgBr2 test strip, swirl the bottle two times
in this time
Remove the test strip and compare it to the chart on the bottle
containing the test strips and record the number under the color on the
chart that is same as the reacted test strip
Convert the amount of arsenic in 1 cup of rice diluted in 1500 mL of
water to the amount of arsenic in 1 cup of rice diluted in 1000 mL of
water by using the formula C1 x V1 = C2 x V2; where C1 is the
concentration (ppb) of arsenic found in 1500mL, V1 is 1500 mL, V2 is
1000 mL, and C2 is the unknown concentration of arsenic (ppb).
Repeat with other samples of rice
TEMPLATE DESIGN © 2008
www.PosterPresentations.com
Acknowledgements
As2O3 + 12NH2SO3H + 6Zn  2AsH3 + 6Zn(NH2SO3)2 + 3H2O
Sulfamic Acid
H3AsO4 + 4NH2SO3H + 2Zn  AsH3 + 2 Zn(NH2SO3)2+ 4H2O
Arsine Gas Reacts with the Mercury Bromide Test Strip
AsH3 + HgBr2  AsH2HgBr + As(HgBr)3 + other variants
• Brown Rice contains higher levels of arsenic than white rice- Hypothesis
Proven
• Students T-test, black vs. white rice, had a p value of .017, below .05
• Null Hypothesis is disproven and the results are significantly
different
• ANOVA Test analyzed with the Post Hoch Tukey Test gives a certain
number, in this case 18. If the difference in means of any two
samples is greater than this number then they are significantly
different.
• All brown rice significantly different from white rice counterpart
• Reason: Brown rice has an extra shell where arsenic concentrates
• Hinode Black and Hinode White are not statistically different
• From ANOVA Test
• Reason: Black rice has antioxidants in the other layers, which prevent
the arsenic from binding
• Where the rice was grown did not have a significant correlation with the
level of arsenic-Hypothesis Disproven
• Student’s T-test Domestic vs. Foreign: P-value: .117
• ANOVA Test
• Della Organic Long Grain Brown, Uncle Ben’s White, and Yoga
White rice, all grown domestically, had statistically the same amount
of arsenic as Basmathi Brown, Hinode Black, Hinode White, and
Jasmine Mahatma White all grown in foreign countries
• All within 18ppb of each other
• Reason
• When forming the hypothesis only use of pesticides and fertilizers
were taken into account, but other sources of contamination: ground
water or soil contamination, whether a factory was polluting the
water with arsenic, whether for some reason the farmer used more
or less pesticides or fertilizers, if the farmers in developing nations
sent only the best rice to the United States, or even if rice was
grown in different locations and combined, were not taken into
account.
• Organic and Non-Organic Rice have no significant difference
• Proven by Student’s T-test: P-value: .3546
• Reason: FDA organic means that all natural fertilizers and pesticides are
used. Cow manure is a organic fertilizer, but the feed that’s fed the cow’s
that make the manure contains high level of arsenic.
• Final Conclusion
• In the end arsenic in rice is a real problem and will pose a problem
to the sustainability of the human race. The federal limit for arsenic
in water is 10 ppb per liter. The amount of arsenic in rice, in these
finding, range from 4 to 9 times that limit.
Limitations
1. The technique that I followed may not have extracted a 100% of Arsenic
from rice in the water, due to the fact that my Hach Arsenic Test Kit was
designed to test for the arsenic level in the water. For example when the
rice is ground up, the rice is in a fine particulate form, so when arsenic
is extracted, it only comes from the surface of the rice grain..
Type of Rice vs Amount of Inorganic Arsenic
Average Amount of Inorganic
Arsenic per 1000 mL (ppb)
Recent studies are showing that toxins in food are causing many health
issues. One of these toxins is inorganic arsenic, arsenic (III)- As2O3,
arsenious acid, and arsenic (V)-H3AsO4, arsenic acid or arsenate. Arsenic
is a carcinogenic substance that if exposed to can lead to death. It is very
soluble in water. For this reason the EPA has put limits on the amount of
arsenic in water, 10 ppb per liter. But there might also be arsenic in rice.
Rice is one of the major crops in the
world. The rice plant siphons abnormally
large amounts water and absorbs most of
the arsenic in that water because it
mistakes it for Silicon, a nutrient for
plants. There are three main types of rice
black, brown, and white. Black and brown
rice are manufactured by only removing
the husk of the rice. While white rice is
manufactured by removing the husk, the bran, and germ layers of the rice
leaving the endosperm. The bran and germ layers retain many toxins and
nutrients. Black rice also has high amounts of antioxidants ,which give it
its black color.
Arsenic Sources:
Arsenic is found in the Earth’s crust as Arsenopyrite: 2FeAsS. When
exposed to oxygen, Arsenopyrite turns into H3AsO3 or Arsenic (V).
2FeAsS + 11 O2 + 6 H2O --> 4Fe2+ + 4 H3AsO3 + 4 SO42Arsenic can also be found naturally as Arsenic Trisulfide-As2S3. When it
reacts with water it forms Hydrogen Sulfide and H3AsO3. The rice plant
may thus pick up the Hydrogen Sulfide present in the water.
As2S3+ 6H2O 3H2S + 2H3AsO3
Arsenic is also man made. It is used as an insecticide in many pesticides
and fertilizers. It is especially used in the United States. In the last 100
years, 1.6 million tons of arsenic have been used for farming. The feed,
that farmers feed the livestock who make the manure that makes the
fertilizer, contains high levels of arsenic. In terms of organic, FDA organic
means that only organic pesticides or fertilizers are used. Organic fertilizer
is a fertilizer that is made from all natural sources, like from cow manure.
Data Analysis
100
90
90
89
80
70
70
69
68
65
60
60
54
53
50
48
38
40
30
20
1. If I had a more accurate way of measuring the amount of inorganic
arsenic present in the rice; like a Gas Chromatograph, a Mass
Spectrometer or a PID controller, the experiment would have been more
accurate. The Arsenic Test Kit from Hach only had 8 numbers ranging
from 0-500 to which one could compare the color of the test strip. This
made it so that I would have to estimate the amount of arsenic present.
10
Challenges
0
Yoga Organic Uncle Ben's Della Organic
Long Grain
Brown
Long Grain
Brown
Brown
Basmathi
Brown
Uncle Ben's Hinode Black Yoga Organic Hinode White
White
Long Grain
White
Type of Rice
Jasmine
Mahatma
White
Della
Jasmine
White
Basmathi
White
During this experiment many hurdles were overcome with the following
innovations:
1. Formation of Rice Solution
Making the rice solution in the beginning was a challenge in itself. The
Arsenic Test Kit only measured levels of arsenic in water and soil, not in
rice; so I had to find to get the arsenic of the rice mixed into the water.
2. Unable to extract arsine gas:
In the beginning, when I first started testing there showed to be no
arsenic in the rice. But when I tested the Standard Solution to see if the
Arsenic Test Kit from Hach actually worked, I realized that the arsenic
containing bubbles that came up burst right away in the Standard, but
they accumulated at the top of the solution when I tested for arsenic in
rice. I decided that it was an issue with surface tension. I found that I
could reduce the surface tension by adding ethyl alcohol. When I added
the ethyl alcohol, the arsine gas-containing bubbles popped when I
tested for inorganic arsenic in Uncle Ben’s Brown Rice. This solved
Pictures of Testing
3. Not enough Ethyl Alcohol:
When I tested for arsenic in Yoga Organic Long Grain Brown Rice and
Hinode Black Rice the bubbles did not pop once again. I decided to
increase the amount of ethyl alcohol added to the rice solution from 4 mL
to 10 mL and this problem was then resolved.
Novelty of the Experiment
No technique or published procedure to directly measure the amount of
arsenic in rice is available to me. Sophisticated equipment to measure
arsenic like the Gas Chromatograph Mass Spectrometer, Multiple Ion
Detection Mass Spectrometry, or the Graphite Furnace Atomic Absorption
Spectrometry were not within my reach. The arsenic test kit procured
could only test the arsenic in water. So I had to mix the rice at a particle
level with water, so that the test kit could measure the level of inorganic
arsenic present. I then also had to heat this solution.
Mr.Dougherty-AP Psychology Teacher, Mr. Menton—AP Chemistry Teacher, Mrs. Langley-AP Statistics Teacher, Aaron Zheng, Siddharth Chand