Arsenic Concentration in Single-Source Origin Cacao Products
A Thesis Presented
By
Jeff Astor
To the Joint Science Department
Of The Claremont Colleges
In partial fulfillment of
The degree of Bachelor of Arts
Senior Thesis in Biology
Spring 2011
Astor 1
Table of Contents
Abstract ..........................................................................................Error! Bookmark not defined.
Introduction ....................................................................................Error! Bookmark not defined.
Materials and Methods .................................................................................................................. 17
Results ........................................................................................................................................... 21
Discussion ..................................................................................................................................... 29
Conclusion .................................................................................................................................... 36
Acknowledgements ....................................................................................................................... 37
References ..................................................................................................................................... 38
Appendix ...................................................................................................................................... 43
Astor 2
Abstract
Recently a heavy emphasis has been put on diminishing the amount of arsenic
(As) present in food products and water sources in developing countries. Chocolate bars
have been identified as source of arsenic that may pose a threat to the average
consumer in recent studies, and this fact calls for a more thorough examination. Using
analysis of of single-source origin chocolate bars from different manufacturers coupled
with previously dry-ashed samples of bars from the same region, high levels of arsenic
were identified and scrutinized by means of ICP-MS. Arsenic was found at an average
concentration of 429 ng/g (ppb), though results varied greatly amongst various regions
and producers. Although some outliers had levels of arsenic bordering the PTWI,
bioavailability in the bloodstream and the human body’s own metabolic processes most
likely limits the amount of toxic arsenic present post-consumption. No correlation was
found between arsenic uptake from soil and other toxic contaminants discovered in the
shells of cacao pods, though raw cacao nibs and powder had highly elevated
contaminant levels in comparison to the finished chocolate product. Without direct
knowledge of the mineral composition of the soil local to the Theobroma cacao trees, it is
difficult to attribute the presence of these toxic chemicals to anything other than
external environmental contaminants with close proximity to the site of cacao growth.
Astor 3
Introduction
Recently there has been a large deal of controversy concerning allowable arsenic
levels in our air and drinking water according to EPA tolerable standards. The Clean Air Act
of 1990 worked to reduce toxic output by many industries, yet left out power plants. The
National Defense Resource Council (NDRC) filed a suit against the EPA, and by 1994 they
settled on the EPA reviewing their standards on power plants and complete the investigation.
Following extensive delays, the EPA was forced to review its report and released it by 2000
and the EPA Administrator at the time, Carol Browner, decreed that toxic air pollutants
generated by fossil fuel-fired power plants must be reduced using protective Maximum
Achievable Control Technology (MACT) standards promoted by the law. MACT standards
were developed in an effort to reduce hazardous air pollution emissions to an optimal
amount, while still ensuring that reduction costs don’t outweigh the benefits. However, in
2005 the NRDC stated that “in 2004, the Bush administration EPA issued rulemaking
proposals that made clear it had no intention of following the law to require MACT standards
that would reduce all toxic air pollutants from power plants.”
Following this occurrence, the administration issued what was known as a “rescission
rule” and decided that the EPA would not follow up on its prior decision to enforce the
MACT standards set up for power plant pollution. After brief deliberation, the U.S. Court of
Appeals for the D.C. Circuit made a ruling that the EPA had intentionally and illegally
avoided the decree of the Clean Air Act. Now multiple organizations are teaming up to
ensure that the EPA follows these standards and reduces the arsenic in our air.
Arsenic is not just found in our air and water sources, however. It is becoming clearer
by the year and from recent current events that these are not the only elevated points of
Astor 4
arsenic supply. Knowing this, food sources grown on arsenic contaminated soil are a clear
threat to the well-being of those who ingest them for various reasons, and examining cacao
plants will provide a case study of this.
Arsenic
Arsenic (As) is a metalloid widely distributed in the earth’s crust and present at an
average concentration of 2 mg/kg. It occurs in trace quantities in all rock, soil, water and air,
though it is generally found combined with elements like sulfur, oxygen, and chlorine. This
form is known as inorganic arsenic, while organic arsenic refers to the combination of
arsenic with carbon and hydrogen. Arsenic can exist in four valence states: –3, 0, +3 and +5.
Under reducing conditions, arsenite (As(III)) is the dominant form, yet arsenate (As(V)) is
generally the stable form in oxygenated environments. Elemental arsenic is not soluble in
water, while most inorganic and organic arsenic compounds are white or colorless powders
that do not evaporate. They have no smell, and most have no special taste. Thus, you
usually cannot tell if arsenic is present in your food, water, or air. For this reason, it is
important to examine the food we eat and analyze levels of potentially dangerous substances,
such as arsenic. Many organizations world-wide have taken to this mission, and produced
varying results, especially with arsenic.
Very little literature exists dealing with both arsenic and its source and cacao – which
can be somewhat attributed to the Total Diet Studies (TDS) conducted by the FDA that
breaks down the components of the majority of food products consumed by the people. Over
the last decade, no chocolate products have been found with levels of arsenic exceeding
0.001 mg. This would deter most studies from addressing this issue directly with any
Astor 5
consistency. Only one study by Lee and Low from 1985 has been published showing levels
of arsenic in raw cacao and finished chocolate products, with the result showing arsenic
levels just bordering below the minimal risk level (MRL). However, the dark and especially
the cooking chocolates were shown to contain higher metal contents in their study. This
indicates that the higher the fraction of cocoa mass in the finished product, the higher its
metal contents. A goal of this project is to present a modern analysis of cacao and chocolate
products and determine if their arsenic concentrations need to be dealt with in the future.
Arsenic has been identified by The Department of Health and Human Services, the
EPA, and The International Agency for Research on Cancer (IARC) as an element that is
carcinogenic to humans even in small quantities when consumed in its inorganic form
(Gomez-Caminero et al. 2001). The IARC classifies arsenic as a Group 1 carcinogen. Just
0.005 mg/kg/day in the gastrointestinal tract is enough to be considered above the MRL by
the Agency for Toxic Substances and Disease Registry for acute symptoms, and barely
0.0003 mg/kg/day ingested orally can cause chronic damage to an individual (ATSDR 2009).
To put that into perspective, an 180 lb man is about 81 kg, meaning that quantities of
ingested arsenic >0.405 mg are considered toxic in acute periods (within 14 days), as is
>0.0243 mg daily over the course of a year. Chronic arsenic poisoning has the capacity to
induce negative health effects such as gastrointestinal symptoms, disturbances of cardiovascular and nervous system functions, and eventually death.
Inorganic arsenic in the blood stream undergoes metabolism through a specific
pathway. The first involves converting arsenate to arsenite. The second involves methylating
arsenite, or adding a single carbon grouped with three hydrogens, to remove it from the body
by excretion of urine. Both of these are directly dependent on an enzyme catalyzing the
Astor 6
reaction. The capacity of individuals to rapidly remove arsenic depends on the enzyme rate
and concentration. Arsenic interferes with cellular longevity by allosteric inhibition of an
essential metabolic enzyme pyruvate dehydrogenase (PDH) complex, which catalyzes the
oxidation of pyruvate to acetyl-CoA by NAD+. Inhibiting this enzyme results in elevated
levels of p53, and the energy system of the cell is disrupted resulting in a cellular apoptosis
episode. In terms of biochemistry, high levels of arsenic can prevent utilization of thiamine
resulting in prognosis similar to cretin’s disease associated with thiamine deficiencies.
Arsenic poisoning can raise blood lactate levels and eventually lead to lactic acidosis. When
blood potassium levels are low, there is an increase in the risk for experiencing a lifethreatening heart rhythm problem from arsenic trioxide As2O3. Also, arsenic in cells can
promote the production of hydrogen peroxide (H2O2), and when H2O2 reacts with certain
metals such as iron or manganese it produces a highly reactive hydroxyl radical which can
wreak havoc. Arsenic trioxide, in its inorganic form, can affect cell function by dismantling
voltage-gated potassium channels, resulting in central nervous system malfunctioning and
possibly death. Even drinking water found in the United States can have decent
concentrations of arsenic trioxide. Certain oncogenetic promoters can be turned on through
massive choromosomal rearrangement involving methylation at the histone level. Riechard
and Puga (2010) suggest that the exact process has not been identified, but it may involve
methyltransferase competition for S-adenosylmethionine (SAM). SAM depletion by arsenic
metabolism may impose cofactor restriction on DNA methyltransferase (DNMT) enzymes.
However, Mass and Wang (1997) say that “arsenic alters cytosine methylation patterns of the
promoter of the tumor suppressor gene p53 in human lung cells,” which would decrease
cancer cell apoptosis, or self-assisted cell suicide.
Astor 7
Flora that develop near anthropogenic regions or around active geothermal areas
show much higher arsenic levels than other biota, which may be directly linked to
contamination of the soil by human interference. The plants then aggregate arsenic by means
of soil uptake by the roots of the plants. Theobroma cacao (cacao trees) is native to the
Amazon, and has been distributed throughout Central America, Mesoamerica, before many
African countries and hotter nations took up the crop as well. According to the Food and
Agriculture Organization (FAO) in 2011, Côte d'Ivoire, Ghana, Indonesia, Nigeria, and
Brazil are the top producers of cacao beans with the producing being done by both large
industrial plantation operations and small scale local growers. The production of cacao beans
is still increasing also, mainly due to production expansion rather than yield efficiency.
However, recent completion of the cacao genome (Matina 1-6 genotype) sequencing in
September of 2010 opens new doors for cacao efficiency in breeding and cultivation. The
three varieties of Theobroma cacao are Forastero, Criollo, and Trinitario, with the last one
comprising at least 80% of the world’s cacao and the most simple taste. The Criollo form has
been said to produce the least bitter taste, and is coveted by many, though the Criollo variety
is also more susceptible to disease, making it somewhat impractical. The Trinitario is a
hybrid of the two, containing elements of both, and may hold the answers to future hybrid
cacao trees.
In the cacao harvesting and chocolate producing process, the large pods are initially
severed from their respective tree where they are cracked open and the beans are removed
from the pods and milky pulp inside. The beans are then placed in heaps, crates, or on grates
under sunlight where they are meticulously dried and fermented for upwards of two weeks.
Astor 8
Illustration 1 World production of cocoa beans, in thousand tonnes, over a course of 15 years (data
collected by UNCTAD from International Cocoa Organization).
Certain operations just place the piles directly on the ground, a main theorized contact point
at which arsenic is absorbed. On occasion, large batches are covered with leaves to allow
optimal fermentation. In the case of large scale operations, the beans, after having been
packed into jute sacks, weighed and classified, vast quantities of the tropical fruit are loaded
into the copious holds of ocean-going freighters to begin the journey across the oceans to the
ports of Europe and North America. For local farmers, the dried bean product is sold to a
director who organizes massive amounts of cocoa beans and ships them to the same
manufacturers. The recipients of the beans will decide the combination of beans needed to
produce the appropriate chocolate, sort them appropriately, and then deshell the beans,
cleansing them of their soil contaminants using winnowing machines. They are refined to
their roasted nib, and heated to a liquor. The liquor is blended with various ingredients and
conching machines (among others) are utilized to create the proper amount of cacao butter,
powder, and chocolate.
This long, arduous process is filled with many steps that convolute the sources, and
make it difficult to determine the exact location(s) that arsenic is introduced into the cacao
Astor 9
product. With beans being mixed and matched according to the chocolate requested, regional
data is hard to obtain with most commercial chocolate products. One avenue that is available
to overcome this problem is the rise of small artisan chocolatiers that create high-quality
chocolate bars from single-source origin beans. This new wave of “bean to bar” chocolate
producers offer a commodity which can be analyzed with respect to geographical regions.
Thus, purchasing chocolates from a particular high-end chocolate distributer known to make
single-source origin chocolate bars from different global sectors and analyzing them could
prove beneficial in determining areas with higher As concentration in the soil.
Exposure to arsenic that doesn’t involve job activity happens almost exclusively by
ingesting contaminated food or water. While data may be demonstrated in terms of total
arsenic amount, arsenic exists in inorganic forms and organoarsenic forms. The most
common of the organoarsenicals, arsenobetaine, is found in various sea foods. The actual
total arsenic concentrations in foodstuffs from various countries will vary widely depending
on the food type, growing conditions (soil type, vicinity of contaminated regions, water,
geochemical activity, use of arsenical pesticides) and processing techniques. A few studies
have been done determining when heavy metals are introduced into the product, and there is
a general consensus, especially in regards to Lee and Lowe (1985) and Manton et al (2010).,
that compounds such as arsenic are present before the industrial processing.
In terms of speciation, the organic species predominates in fruits, vegetables, and
seafood, with between 0-10% being inorganic arsenic for each type. According to
preliminary data and studies done before, estimations show that almost a quarter of an
average person’s daily arsenic intake can be of the inorganic species (US EPA, 1988, Yost et
al., 1998). Looking at chemical literature has shown that the bond between arsenic and
Astor 10
carbon is extraordinarily strong, almost impossible for any mammal to break this via
metabolic pathways. Subsequently, the breakdown of organic arsenicals during metabolism
does not necessarily create inorganic arsenic. In most species, including humans, ingested
organic components such as MMA(V) or monomethylarsine and DMA(V) or trimethylarsine
undergo limited metabolism, do not readily enter the cell, and are primarily excreted
unchanged in the urine. Thomas et al. (2001) contests that methylated and dimethylated
arsenicals that contain arsenic in the trivalent oxidation state are more cytotoxic, more
genotoxic, and more potent inhibitors of the activities of some enzymes than are inorganic
arsenicals that contain arsenic in the trivalent oxidation state. Hence, it is reasonable to
describe the methylation of arsenic as a pathway for its activation, not as a mode of
detoxification. Williams et al. (2005) discusses arsenic levels in rice and describes the main
As species detected in the rice extract were AsIII, DMAV, and AsV. In European,
Bangladeshi, and Indian rice 64.1% (n=7), 80.3% (n=11), and 81.4% (n=15), respectively, of
the recovered arsenic was found to be inorganic.
Two studies show how species of arsenic can be introduced into edible regions of
commercial food crops solely by absorbing the contents from the arsenic-laden air and soil
(Helgesen and Larsen, 1998; Woolson 1973). Helgesen and Larsen (1998) worked on the
bioavailability of arsenic pentoxide during its uptake from contaminated soil for a wood
preservative plant to carrots and established that it was 0.47 ± 0.06% of total soil arsenic
content. The same study demonstrated that although only arsenite and arsenate initially
existed in the soil, both previous species along with MMA and DMA were found in the final
product. With regard to soils introduced with 0-500 µg/g of arsenate, various species of green
bean, lima bean, spinach, cabbage, tomato, and radish grown there aggregated high levels of
Astor 11
arsenic in the edible regions. Radish, spinach, and green beans showed values of 76 µg/g, 10
µg/g, and 4.2 µg/g, respectively. The range of arsenic accumulation in the other three
vegetable crops ranged from 0.7–1.5 µg/g. All of these studies point to the potential of
movement of multiple arsenic species from soil into agronomic crops. This is emphasized by
a 2008 study by Kim et al., which demonstrates that concentrations of arsenic, lead,
cadmium, and others in horticultural soils remain phytoavailable. According to the FDA’s
arsenic profile, they determined that site-specific risk assessments must be implemented with
vegetable garden care-takers adhering to soil guidelines. A study conducted by Zhang et al.
in 2002 uses information from previous studies to identify that arsenic predominates in its
inorganic form in throughout most terrestrial and aquatic biota (Helgesen and Larsen, 1998).
In cacao plants, it has been proposed that the drying process of the beans gives them time to
absorb contaminants in the soil such as these.
A study of Chinese Tea plants and the accumulation and distribution of arsenic and
cadmium is addressed in a study by Shi et al. (2008) showing a large discrepancy in
concentrations of arsenic and cadmium amidst random cultivators, indicating a direct
correlation to different growing conditions. Arsenic was acquired by means of roots and
stems in the tea plants and was the central segment of aggregation. Arsenic extractability
increases with elevated levels of arsenic in the soil and heavy metal concentrations in biota
increases linearly with extractable levels. This correlation may explain why certain regions
produce higher levels of arsenic in their produce than others. Arsenic does not generally
accumulate enough in uncontaminated soil to be a potential threat to humans in terms of
toxicity, yet in arsenic contaminated soil, the uptake of arsenic by the plant tissue is
significantly elevated, particularly in vegetables and edible crops. Therefore, there is concern
Astor 12
regarding accumulation of arsenic in agricultural crops and vegetables grown in arsenicaffected areas. With more relaxed environmental standards in other nations, there is
understandable caution that soil from contaminated regions will go overlooked and find its
way beneath cacao trees.
Dealing with the metabolism of large doses of arsenic in the gastrointestinal tract and
excretion of arsenic in both inorganic and organic forms requires insight into the valence
state and speciation of the ingested arsenic. Of the various forms arsenic appears in, humans
are more likely to frequently encounter the trivalent (arsenite) and pentavalent (arsenate)
species. As long as oxidative and reductive factors are minimal (low Eh) and arsenite is
present at physiological pH (around 7.4), it exists in its non-ionized form.
Even though estimates of total arsenic uptake of all species can be determined by
arsenic levels in the urine according to the World Health Organization’s 2001 Arsenic report,
it is difficult to interpret what the actual negative effects of arsenic might be without knowing
the amount of bioavailable arsenic. Bioavailability refers to the component of the ingested
portion that is taken into the bloodstream. Xu and Thornton (1985) measured mean total
arsenic levels of 300 mg/kg in the soil of gardens that had been contaminated by previous
mining in close proximity. However, over 98% of the arsenic present in this soil was neither
water-soluble, nor extractable. In a rat model, the absolute bioavailability of these
contaminated soils relative to arsenite and arsenate ranged from 1.02 to 9.87% and 0.26 to
2.98% respectively. Freeman et al. (1993) determined both the absolute and comparative
bioavailability of arsenic in soil from a smelter site using male rabbits and monkeys. When
compared to the intravenous administration of sodium arsenate, the absolute bioavailability
was reported as 25.9% in rabbits and 24.2% in monkeys. When compared to an oral dose of
Astor 13
sodium arsenate, the comparative bioavailabilities were 67.8% in rabbits and 43.6% in
monkeys, in general agreement to findings of Ng et al. (1998) in rats. Such data on
availability of arsenic in soil needs to be considered in assessing human uptake of arsenic
from soil. It should be noted, however, that the bioavailability of ingested inorganic arsenic
will vary depending on the vector in which it is ingested (food, water, soil), the solubility of
the arsenical compound itself and the presence of other food constituents and nutrients in the
gastrointestinal tract. Chocolate would only be consumed via an oral route, so arsenic would
be most potent in this sense, in its inorganic form.
In common with studies in experimental animals, controlled ingestion studies in
humans indicate that trivalent and pentavalent arsenic are both well absorbed from the
gastrointestinal tract. For example, FDA’s Arsenic profile indicates that Pomroy et al. (1980)
reported that healthy male human volunteers excreted 62.3 ± 4.0% of a 0.06-ng dose of 74.
As-arsenic acid (As(V)) in urine over a period of 7 days, whereas only 6.1 ± 2.8% of the
dose was excreted in the feces. Few other controlled human ingestion studies have actually
reported data on both urine and fecal elimination of arsenic. However, between 45% and
75% of the dose of various trivalent forms of arsenic is excreted in the urine within a few
days, which suggests that gastrointestinal absorption is both relatively rapid and extensive.
Yamauchi and Yamamura (1983) showed by HGAAS that arsenic accumulates in the tissues
of the body with age, a finding consistent with laboratory studies done on animals.
In his thesis conducted over the 2009-2010 year, Stjernholm found that heavy metals
available in soils can find ways into cacao plants in varying forms, and eventually to the
consumer, in the form of finished chocolate products. One of his results showed this:
“Arsenic was not a main focus of this study, due to the inability to determine the speciation
present in chocolate bars and the lack of previous literature to assist in the analysis. Thus, the data
for arsenic will be presented in Appendix A for reference. The range of arsenic concentrations in
Astor 14
chocolate bars was found to be 23–1200 ng/g with an average of 520 ng/g [or 0.5 mg/kg]. Although
these values are much higher than both lead and cadmium, analysis of the As species present in
chocolate would need to be performed before focusing on the possible toxicities.”
He also determined that cadmium occurred in greater concentrations than did lead,
and that environmental factors played a part in the uptake of these dangerous metals. As
different soils will contain varying elements contributed by their surroundings, various
geographic regions’ chocolates will be assessed to shed light on arsenic uptake and the
dangers of high concentrations of its inorganic form. Though analyzing arsenic species may
be out of the scope of this project, current publications may be able to offer insight on the
causes and sources of the toxic heavy metal. Understanding how arsenic affects the body
would also require a determination of the transformational processes that arsenic goes
through in the metabolic pathway.
This current study on arsenic in chocolate will deal with the assessment of different
collection of chocolates that were previously ashed with unanalyzed arsenic levels from
numerous manufacturers, and from different regions within each manufacturer. Production
values will be put against recently ashed chocolates from various regions that can help
produce evidence that contamination of chocolate is soil-dependent and that various factors
impact the results. Total arsenic concentrations of each chocolate type will be assessed, and
an attempt to determine the relative toxicities of the various chocolates will be undertaken. If
the values are above the recommended intake, the amount of chocolate ingested needs to be
considered to analyze what dangerous amounts of specific chocolate intake have to be to
invoke serious side effects. The results will be compared and contrasted with the only other
available study that discusses arsenic levels in chocolate – Lee and Lowe’s 1985 study. Also,
the correlation between cadmium, arsenic, and lead uptake will be focused on to establish
whether or not consumption of arsenic containing chocolates will also bring potentially toxic
Astor 15
levels of other heavy metals analyzed in previous literature, and to control for the values of
arsenic and ensure that the company that produces the chocolate does not have a significant
impact on the arsenic levels. Furthermore, raw cacao nibs and cacao powder from Ecuador, a
nation rife with environmental turmoil that may cause contamination of soil in the areas of
Theobroma cacao growth, will be analyzed and evaluated.
Astor 16
Materials and Methods
Chocolate Samples
Ghirardelli bars with cacao percentages between 60%-100% were used in the study
and include Ghirardelli1 60% cacao milk chocolate bar, Ghiradelli, 60% cacao Evening
DreamTM, Ghirardelli 86% cacao Midnight ReverieTM, and Ghirardelli 100% cacao baking
bar. Single origin chocolate bars were purchased from Burdick Chocolates2 and previous
samples with unanalyzed data from Escazu Artisan Chocolates3, Black Mountain
Chocoloate4, and Theo Chocolates5 were used. Single-origin bars include: Escazu GuapilesCosta Rica 65% cocoa chocolate bar, Escazu Carenero-Venezuela 81% cacoa chocolate bar,
Black Mountain Chocolate 70% cocoa Carenero-Superior Venezuela, Black Mountain
Chocolate 70% cocoa Matiguas-Nicaragua, Black Mountain Chocolate 70% cocoa La RedDomincan Republic, Theo 84% cocoa Ghana, Theo 91% cocoa Costa Rica, and Theo 74%
cocoa Madagascar (Stjernholm 2010) were used. The Single-Source Burdick Chocolate Bars
include: Burdick Antonanarivo, Madagascar 66% cacao, Burdick Venezuela-Caracas 71%
cacao, Burdick Quito, Ecuador 74%, Burdick La Paz, Bolivia 68%, and Burdick St. George,
Grenada 75%. Cacao nibs and cacao powder from Earth Shift® were also purchased and
analyzed. Each individual chocolate bar was dry-ashed to produce two samples.
Dry Ashing Procedure
Cervera and Montoro (1994) suggested that dry ashing is the correct method of
delivering the food product to an ICP-MS machine, and as such it was the method of delivery
1
http://www.ghirardelli.com
http://www.burdickchocolate.com/
3
http://www.escazuchocolates.com/
4
http://www.blackmountainchocolate.com/
5
http://www.theochocolate.com/
2
Astor 17
for this procedure. The previous study’s methodology to dry ashing included temperatures of
600 °C and around 6 hours of total ashing. With some prior testing of yield percentage, and
researched knowledge about arsenic, a temperature of 450 °C was determined to be the most
efficient quantity to complete the task. It has been reported that a temperature of 450 °C can
be sufficient for remove metal ions from its surrounding organic complexes for elemental
analysis (Lee, 1985) and he reported a 90-97% recovery of metal from chocolate samples
with spiked metal concentrations when dry ashing at 450°C. Stjernholm had developed a way
for dry-ashing techniques by sampling different methods, and from the information he
presented 450 °C was the temperature settled on to effectively ash chocolates of different
origins, weights, and cacao contents.
Initially, the masses of chocolate that fit in pre-prepared porcelain crucibles were
weighed out. The pre-ashed masses were recorded for reference, and the product was placed
into the crucibles which were then placed into a Barnstead Thermolyne 1300 Furnace with
porcelain lids covering the three areas containing the chocolate product. This is to minimize
the amount of dry ash lost during the period of organic volatilization. The furnace was turned
on and allowed to heat up to 450 °C over a period of about 10-15 minutes, before the
chocolate was left to sit for about 3 hours. The furnace was then turned off, and the porcelain
lids were carefully removed from the crucibles. The ashed products were allowed to cool
before they were weighed and the masses recorded. The furnace was once again turned on
and allowed to heat to 450 °C, before the product was placed back into the furnace. The
chocolate ash was then left to sit for 5 hours.
The furnace was once again turned off, allowed to cool, and the chocolate product
was taken from inside. Once the porcelain crucibles had cooled enough to be handled, the
Astor 18
dry-ash was placed in a tared weigh-boat where the final mass was recorded. Weighing the
products twice was necessary to ensure that all of the organic matter had been sufficiently
lost. At the beginning of the trials, each sample was then heated again for one more hour and
then weighed again to ensure that the 8 hours total was necessary to completely remove the
organic material from the chocolate. If the mass decreased, then clearly more time was
needed. No samples showed significant loss past the 8-hour mark, and this process was
considered successful. Each sample of the chocolate bars weighed a little over 4 grams, with
the sample of nibs weighing about 3 grams, as they are harder to pack into crucibles. At the
end of the ashing, about 0.09 grams of product was obtained from the 4-gram samples, and
about 0.10 grams was obtained from the nibs. This 2.5% yield of dry-ash for chocolates is
consistent with previous literature, and the 3.5% yield for nibs can be attributed to their much
higher cacao content, which will yield higher quantities of final ash product (Stjernholm
2010).
ICP-MS Analyzing
Once a total of 0.5 grams of ash was collected for each sample, the ash was collected
in vials and sent to Activation Labs6 for Inductively Coupled Plasma Mass Spectroscopy
(ICP-MS) data analysis using the Perkin Elmer Sciex Elan 9000s. The methodology used is
discussed by Afthan et al. (1992). 6M HNO3 was used to cleanse the porcelain crucibles for a
half hour, followed by a half hour of boiling. This pre-wash process is crucial to ensure that
contamination is minimized throughout the procedure. Any glassware that would eventually
come into contact with any product or materials was also thoroughly rinsed with 6M HNO3.
The 0.5 g of dry ashed material collected was transferred into the clean porcelain crucibles
6
www.actlabs.com
Astor 19
and 5 mL of 6M HCl was volumetrically added to each crucible, ensuring that the acid came
in contact with all of the ashed material. Preparing 6M HCl requires taking 0.5 L of
concentrated HCl and adding upwards of 1 L of H20 to it. The diluted acid was placed on a
hot plate with a mild heat setting to evaporate the acid, but careful emphasis was placed on
not bringing it to a boil. The residues were then dissolved in 10.0 mL of 0.1M HNO 3 and
swirled to ensure that all ash had come in contact with the acid. The nitric acid solution was
prepared by diluting 7 mL of concentrated HNO3 with H2O up to 1000 mL. Each crucible
was covered with a watchglass and stirred with a glass stir rod periodically for about 2 hours.
Statistical analysis and calculations was performed using GraphPad Prism 5©.
Using ICP-MS, concentrations of the ashed samples were collected and numerical
calculations were run to convert the amount in the ash to the amount in a single chocolate
bar. To do this, the ashed arsenic concentration was multiplied by the ratio of the arsenic
concentration to the mass of ash. This was then multiplied by the entire mass of the chocolate
bar.
Astor 20
Results
Using 16 types of chocolate bars, cacao nibs, and cacao powder as samples, arsenic
and heavy metal concentrations were determined by Activation Labs using an ICP-MS
machine. Though the results seem to be indicative of region, variability within specific
countries points to the fact that in the future large amounts of artisan chocolates from the
same area might help to verify the validity of the data. A main concern with this approach is
that single-source origin chocolates is a relatively novel idea, and as such, few companies
have products from the same city. Regardless, this analysis focuses on the samples available
and will take the number of samples and large deviation into consideration.
Arsenic levels varied widely with a mean of 366 ng/g and a standard deviation of 307
ng/g. With a high standard deviation, it is interesting to note the variability even within the
same regions, as shown by Figure 1. Black Mountain Venezuela (70% cacao content)
chocolate bar shows the highest concentration of arsenic with a total of 1199 ng/g according
to Table 1. Venezuela would seem to produce arsenic-heavy chocolate as the next highest
amount comes from Escazu Venezuela (81% cacao content) with 1170 ng/g. These are
insignificantly different, and point to substantial arsenic uptake from Venezuelan soil.
Unsurprisingly, these single-source origin chocolate bars also have some of the highest
cadmium and lead concentrations as Lee and Lowe (1985) show arsenic having high
correlation coefficients for both cadmium and lead contents. On the other hand, the Burdick
Venezuela bar shows a value of only 19.5 ng/g, substantially lower than either of the other
two bars, and lower than most by a great deal. This value is lower than any other non-Theo
single-source origin bar.
Astor 21
Other regions show signs of intense regional variability as well. The Theo chocolate
bar from Madagascar (90% cacao content) showed only 4 ng/g of arsenic compared to its
Burdick Madagascar (66% cacao content) counterpart’s 430 ng/g. Theo Madagascar also
happens to have the minimum amount of arsenic in all the samples selected. To add support
to this, Theo and Escazu Costa Rica bars have values of 11 ng/g and 200 ng/g, respectively.
As Figures S2a and S2b point out, the arsenic levels in the previously ashed samples have a
much higher average value than the Burdick chocolate bars do, with only four values in S2b
being below the mean of the Burdick bars. The standard deviation for both groups is still a
very high number, however, as Table S2 shows.
Though the highest arsenic concentrations occur in the two Venezuelan chocolates,
the Ghiradelli 100% and Ghiradelli 86% chocolates both have very high levels of arsenic
with 1020 ng/g and 600 ng/g, respectively. On the other hand, Ghiradelli 60% shows a value
of only 12 ng/g. This points to an increase in heavy metal concentration as cacao percentage
rises, which is in correspondence to Lee and Lowe’s article discussing baking chocolates and
manufactured milk chocolate bars. Figure 2 shows the direct relationship between rising
cacao portions and rising arsenic levels in this study. Ghiradelli is a large commercial
company, however, and it is interesting to note that the levels in these chocolate bars were
much higher than the consistent zero values that the TDS reports show from 1991-2008. It is
impossible to specify where the beans came from directly, as Ghiradelli states that they “use
a proprietary blend of bean varieties that has been refined over the company's 150-year
history to provide the company's distinct and intense chocolate taste. The exact source of the
beans is a closely held family secret.” This does not provide direct information on location-
Astor 22
specific arsenic levels, but helps understand how cacao percentages relate to chocolate bar
heavy metal concentrations.
Figure 1. Arsenic levels in single-source origin chocolate bars from varying regions and cacao content.
*Actual numerical results listed in Appendix. (D.R. = Dominican Republic)
1500
Arsenic
1000
500
0
B
la
ck
E
sc
a
M Thzu
ou e Co
o
E nta C sta
sc in o R
az V sta ic
u en R a
V e ic 65
B
e z a %
la
ck
G nez uel 91
hi u a %
M
ra el 70
ou
G
d a %
B n
la ta Ghir ell 81
ck in h ad i 1 %
M N ira ell 00
ou ic d i 8 %
a e
T
B h T nta rag lli 0%
ur eo h in u 6
di M eo D a 0%
c
7
B k Mada Gh .R. 0%
ur a g an 7
B di da as a 0%
ur ck g c 8
d G a ar 4%
B Bu ick re sca 90
ur r E n r %
di di c ad 66
c k c k ua a %
V Bo do 75
en l r %
ez ivi 74
ue a 6 %
l a 8%
71
%
Arsenic Levels in ng/g (ppb)
Arsenic Concentrations
Chocolate Bar Type
Table 1. Comparison of the summary of arsenic concentration results illustrated in Figure 1 and arsenic
concentrations in Lee and Lowe (1985) study.
[Arsenic] in this study (ng/g)
[Arsenic] in Lee and Lowe (ng/g)
1199
1280
Maximum
(Black Mountain Venezuela 70% cacao)
4
830
Minimum
(Theo Madagascar 90%)
Mean
366.4
980.6
Standard Deviation
307.3
147.6
The local Malaysian chocolates used by Lee and Lowe have a much higher average
arsenic concentration, but only a slightly higher maximum value. The standard deviation is
much lower, and the consistency may be indicative of the chocolates coming from similar
soil conditions. This may correlate to the decades of peninsular mining that Malaysia has
Astor 23
experienced, which have taken a heavy toll on the environment in terms of river pollution
and siltation. Agriculture has suffered as a result, and the chocolate industry must have felt
the repercussions. These results are offered more validity by the fact that Knezevic found an
average arsenic concentration of 770 ng/g in Malaysian chocolate-containing foods in studies
produced in 1980, 1981, and 1982.
Figure 2. Direct relationship between a rise in cacao percentage in chocolate and a rise in arsenic
levels.
Arsenic Levels in ng/g (ppb)
Arsenic concentrations as a function of rising cacao
portions in Ghiradelli Dark Chocolate Bars
1500
1000
y = 24.917x - 1498.6
R2 = 0.9941
500
0
50
60
70
80
90
Cacao Percentage
100
110
Though a small sample size was used to examine the link between these two factors,
the very high r2 indicates a significant relationship. The slope of the line was significantly a
non-zero value with a p-value of 0.0490 indicating a correlation, and the linear-fit line falls
well between the error marks.
Figure 3 shows the values of heavy metal concentrations for each chocolate bar type
from varying regions. Arsenic levels are higher in the majority of samples studied, with
cadmium being the next largest average values. Burdick chocolates have a high amount of
heavy metal content across the board, with Burdick Ecuador having the highest cadmium
concentration at 1159 ng/g, more than double the closest value – Burdick Grenada with 570
ng/g. Burdick Ecuador also contains at least 300% more lead then the next highest bar with
Astor 24
429 ng/g. For both of these chocolate bars, arsenic levels are below the mean, yet Burdick
Madagascar has an arsenic concentration of 430 ng/g. Lead values are all relatively low,
except for the Burdick Ecuador chocolate bar which shows a concentration of 429 ng/g.
Figure S3 and S4 clarify that previously ashed samples have much higher arsenic values, but
much lower lead and cadmium values than the Burdick chocolates.
Figure 3. Heavy metal and arsenic concentrations in single source origin chocolate bars from different
locations.
Burdick Venezuela 71%
Burdick Bolivia 68%
Burdick Ecuador 74%
Burdick Grenada 75%
Burdick Madagascar 66%
Theo Madagascar 90%
Theo Ghana 84%
Black Mountain D.R. 70%
Black Mountain Nicaragua 70%
Ghiradelli 60%
Ghiradelli 86%
Ghiradelli 100%
Escazu Venezuela 81%
Black Mountain Venezuela 70%
Theo Costa Rica 91%
Escazu Costa Rica 65%
Cadmium
00
15
10
50
0
00
Arsenic
Lead
0
Contaminant Levels in ng/g (ppb)
Contaminant Concentrations
Chocolate Bar Type
Table 2. Summary heavy metal and arsenic concentrations in single-source origin chocolate bars
Cadmium
(ng/g)
Arsenic
(ng/g)
Lead
(ng/g)
7-
Range
Mean
Std. Deviation
55-570
285.9
171.5
4-1199
366.4
307.3
429
73.1
103.4
Table 2 shows that while the cadmium mean value is lower than the mean of
arsenic, the lowest amount is much higher than the lowest of arsenic. Lead appears to be
lower than both across the board, though it has a large outlier in Burdick Ecuador that may
Astor 25
have skewed the results. Looking at Figure 4 seems to make it more clear that sorting the
values out by the companies that produced each bar shows more consistent high contaminant
content in the Burdick Chocolates, and shows that the Theo chocolate bars have the
consistently lowest contaminant contents. Escazu chocolates have a severe outlier in the
Venezuelan chocolate’s arsenic level, though this company’s chocolate has a generally lower
contaminant concentration.
Figure 4. Sorting out the levels of the various heavy metals into groups of companies that
produced the chocolate bars.
di
c
B kM
u
B rdi ada
ur ck g
Contaminant Levels in ng/g (ppb)
di G as
B
B u ck re ca
ur r E n r
di dic c ad 66
u a
c
Es k V k B ado 75%
en ol r %
B
Escaz
ez ivia 74
la
u
c
ue 6 %
c
az C
B k
l 8
la M
o
ck o u V sta a 7 %
1%
en R
M un
ez ic
ou ta
a
B n in
la ta V uel 65
a %
ck in e
M N nez 81%
ou ic u
nt ara ela
ai g 7
Th
n ua 0%
D 7
eo Th
.R 0
Th M eo
. %
eo ad Gh 70%
C ag an
os as a
ta ca 84
R r9 %
ic 0
a %
91
%
Grouped by Company
1500
Cadmium
Arsenic
Lead
1000
500
B
ur
0
Bar Type
Black Mountain chocolate bars show high arsenic levels for each one of their
products and Venezuela is also an outlier here. Nevertheless, none of these groupings
produced a significant result distinctly differentiating the chocolates based on company,
Astor 26
supporting the theory that contamination occurs from soil uptake rather than industrial effects
of the chocolate producing process. This identification is in stark contrast to results produced
by Rankin et al. that showed heavy metals being introduced in chocolate from sources other
than the cacao beans or shells, though Manton et al. (2010) vilifies the study as unscientific
and biased.
The correlation between uptake of separate heavy metals varies amidst each metal as
is shown in Table 3 and Figure 5. Cadmium and lead uptake both correspond very
significantly to each other with a p-value of 0.0002175. However, arsenic does not have a
direct correlation to either of the other two heavy metals, and in fact, is very far from it. This
result will be analyzed in greater detail in the discussion, but Lee and Lowe’s 1985 study
shows a more direct correlation between other heavy metals and arsenic in particular with a
correlation coefficient of 0.95 for cadmium and 0.93 for lead as is shown in Table 3.
Figure 5. Correlation between contaminant concentrations in single source origin chocolate bars from
different locations.
Correlation of Heavy Metal Concentrations
1.0
Cadmium
Arsenic
Lead
0.6
0.4
0.2
Le
ad
ic
rs
en
A
ad
m
-0.2
iu
m
0.0
C
P-Values
0.8
Heavy Metal Type
Astor 27
Table 3. Matrix of correlation coefficients for Cd, As, and Pb contents in chocolate bars from Lee and
Lowe (1985) study and from this study.
This
Study
Cadmium Arsenic Lead
Cadmium
0.004
0.898
Arsenic
0.004
0.009
Lead
0.898
0.009
Lee and
Lowe
Cadmium Arsenic Lead
Cadmium
0.948 0.889
Arsenic
0.948
0.927
Lead
0.889
0.927
The last comparison done involves looking at Earth Shift Ecuadoran Nibs and Earth
Shift Ecuadoran Cacao Powder heavy metal values, and examining the difference between
chocolate bar values. The nibs expressed a 480 ng/g concentration of arsenic, 46 ng/g of lead,
and 1470 ng/g of cadmium. The cadmium level is very high – 5 times as much cadmium as
the mean value listed in Table 2. This outpaces even results such as the ones Mounicou et al.
found with the range of cadmium levels of cacao nibs from across the globe between 375450. The cacao powder contained almost double the cadmium level of the nibs with 2462
ng/g, arsenic levels of 358 ng/g, and lead levels of 280 ng/g. Previous studies have not found
levels of cadmium this high, though the arsenic levels are below, or on par, with other
literature values. The overall trend of arsenic levels shows that the TDS values are far lower
than the literature results and those observed in this study.
Astor 28
Discussion
Chocolate bars used in this study hold a mass of about 100 ± 20 g, though for
the sake of the study, 100 grams will be used as a barometer. Taking these arsenic values into
consideration, a range of 4-1199 ng/g of arsenic gives an individual ingesting one full bar of
chocolate a total consumption of between 0.4-120 µg of arsenic, or an average of 36 µg. As
previously mentioned, bioavailability to the bloodstream has to be taken into consideration,
and when looking at the literature, appears in values between 10-60% though a more
appropriate level of around 30% is more consistent in animal studies. The amount of
bioavailable arsenic in an individual’s circulatory system following consumption of one
chocolate bar would be 10 µg on average. Utilizing the PTWI of arsenic (15 µg/kg) and
converting that to daily intake of a child weighing 65 kg, 10 µg results in only 9% of
maximal intake. However, looking at the high end of arsenic results and bioavailability data
gives 60 µg of bioavailable arsenic, which makes up 45% of a 65 kg child’s maximal daily
intake.
A very small child weighing only 30 kg would have a bigger issue as one chocolate
bar of Black Mountain Venezuela 70% cacao would be enough to almost equal the maximum
tolerable daily intake. It must be emphasized for accuracy that PTWI is expressed in terms of
a weekly basis to underscore the importance of long-term exposure for accumulating
contaminants, which arsenic is. In 2005, the average chocolate consumption per person in the
United States is estimated at 5.2 lbs per year or 0.014247 lbs per day. This amounts to 6.4
grams/day and not enough to evoke any kind of toxic reaction even over the long-term,
though it may contribute somewhat when coupled with arsenic available in other terrestrial
crops and seafood.
Astor 29
Again, the bioavailable arsenic does not necessarily have as significant an impact on
the overall toxicity level as speciation of arsenic may be necessary to further comment on the
damage consumption of this toxin can do. Unfortunately, speciation of the total arsenic
concentration was difficult to achieve without access to hydride generation atomic
fluorescence spectrometry (HPLC–HG–AFS) involving a PS Analytical Millennium
excalibur system which would involve more funding and resources that are currently out of
the scope of this project. However, it is much easier to speculate on the speciation of arsenic
using case studies that have been presented about other biota that were found containing
large quantities of phytoavailable arsenic of many species. Han et al. (2005) clearly
demonstrated that Chinese Tea plants can show a large range of arsenic concentrations,
including values of up to 4430 ng/g and that a wide spectrum of speciation does not
necessarily mean that it is harmless. If the arsenic is in its trivalent form, it can be up to 70
times more toxic than the methylated pentavalent form, and inorganic arsenic is 100 times
more toxic than organic forms. The methylation process that occurs in the body to excrete the
toxin may not diminish the bioavailable arsenic by as much as previously believed as well.
The correlation that is seen with great significance in the Lee and Lowe’s study
between arsenic, cadmium, and lead is not seen in this study, though cadmium and lead are
significantly correlated. This is not necessarily unexpected; the 1985 study took chocolate
bars manufactured locally from Malaysia and has the advantage of comparable
environmental factors for consistency. Future researchers may find it easier to organize their
experiment with this approach in mind and investigate discrepancies within the same region.
With such a wide variability in the choices of chocolates in this study, controlling for the
large number of impacting factors is both difficult and unmanageable. Extremely large
Astor 30
standard deviations are noted in arsenic concentrations that may be indicative of poor data.
One must take into consideration the fact that due to each chocolate bar being from a
different geographic region, multiple environmental factors come into play which can
significantly affect the data. This is especially noticeable when grouping the concentrations
of heavy metals into categories by the company (Figure 5) that produces them. Even
chocolate bars from the same company have a large variability in which heavy metal is
expressed most significantly, and if certain heavy metals are even present at all.
Previously ashed samples show much higher values of arsenic, yet generally lower
amounts of cadmium and lead. Initially, one might conclude that Burdick chocolates have
been contaminated in the process of manufacturing the chocolate, but further analysis of the
data suggests otherwise. Also, the fact that low values of lead and arsenic are clearly seen in
almost all of the Burdick chocolate samples negates that assessment.
Looking at Figure 2, the Ghiradelli chocolate bars analyzed showed a significant
linear increase in arsenic concentration as cacao percentage of the bars increase. While it is
true that these are not single-source origin chocolates, the blend of beans is most likely
similar, and this data corresponds directly with what Lee and Lowe discovered when they
compared cooking and dark chocolate bars to milk chocolate bars produced by the local
manufacturers. As higher levels of cacao in chocolate points to higher arsenic levels, hotter
environments may promote more effective cacao tree development, leading to pods with
more concentrated contaminate contents. Theobroma cacao is indigenous to the Amazon and
conditions that are optimal for blooming may assist in the uptake of heavy metals as well as
help the trees flourish. Cacao cultivation is mainly efficient in the hot region around 15
degrees both north and south of the equator as is shown in Illustration 3. However, when
Astor 31
attempting to group the chocolate bars based solely on their cacao percentages, no significant
trend is seen in increases in either arsenic, cadmium, or lead. This finding points to the soil
indicative of the geographic region as a more appropriate measure of heavy metal
concentration. Without soil samples from the areas of cultivation, it is difficult to pinpoint
how much arsenic is accrued in the pods themselves, and thus transferred to the beans and
refined product.
Illustration 3. Regions of optimal Theobroma cacao growth7
An interesting note is that the most significant indicator of arsenic concentration in
previously ashed samples, and potentially other heavy metal levels, is whether or not the
chocolate bar has beans that originate in Venezuela. This suggests that these results can be
tied directly to the recent encroachment on Venezuelan rainforests and other flora by mining
and logging companies within the last 15 years. Old mining facilities can contaminate the
surrounding environment by means of airborne toxin particles settling on the leaves, shoots,
and stems of chocolate trees and being absorbed into their edible regions. This can happen in
the same way to the soil, which has been shown to take up arsenic from external sources and
transfer it to the flora that take habitat in the soil. Environmental problems in Venezuela
7
http://exhibits.mannlib.cornell.edu/chocolate/chocolatelands.php
Astor 32
include sewage pollution into Valencia Lake, located not far to the west of Caracas, where
the Burdick Venezuela chocolate originates from. Current concerns also include irresponsible
mining operations that endanger the rain-forest ecosystem and indigenous peoples.
Interestingly enough, Caracas has a bordering town called Caranero where both previously
ashed Venezuelan chocolate bar beans originate from. If Valencia Lake is being used for any
sort of irrigation system in those local chocolate manufacturing industries, then it could be a
source of heavy metal and arsenic contamination. Burdick Venezuela-Caracas has very low
values of both arsenic and lead, yet it comes from a city bordering Caranero where the
highest arsenic levels have been identified. The fact that three very closely geographically
located chocolate manufacturers produced results that hinge mostly on the exact location of
the manufacturers underscores the emphasis on soil contamination from a source of extreme
proximity, rather than the city the chocolate is produced in.
The Ecuadoran nibs and cacao powder analyzed in this study showed extraordinarily
high levels of cadmium and a high arsenic concentration as well. Earth Shift® states that their
cacao beans are hand peeled and never machine processed, which may complicate the
winnowing process of removing the unwanted matter from the nibs and beans. According to
their website, Arriba Nacional-Fino de Aroma, the variety of cacao that Earth Shift uses has
the finest flavor profile and the highest amount of bioavailable nutrients and minerals. With
this statement comes the possibility that along with the nutrients and minerals that are
bioavailable in the chocolate, heavy metals are transferred as well. This can offer a better
explanation for such a dangerously high level of cadmium present in the raw nibs and cacao
powder and a relatively high level of arsenic. Though there is an FDA mandated 1.75% of
shell allowed in the finished chocolate product, a small amount of shell that takes up heavy
Astor 33
metals in much higher concentrations may have been enough to contaminate the nibs or
chocolate with external species, yet would have a hard time explaining values as high as the
cadmium levels shown in both forms of cacao beans.
Other companies’ Ecuadoran products show varied signs of excessive heavy metal
uptake, however, and one is forced to conclude that the nibs and powder must have naturally
had a much higher heavy metal concentration before the beans are refined. This makes sense,
as the powder is more concentrated, and thus contains a larger amount of the contaminated
cacao than any finished chocolate product or other form of cacao bean could. Even still,
Ecuador has the distinction of having the highest deforestation rate and worst environmental
record in South America. Oil exploration, logging, and road building have had a disastrous
impact on Ecuador's primary rainforests and agricultural community. Perhaps in a future
study, an examination of chocolate bars from specific regions across the country would offer
more insight into the environmental factors surrounding Ecuador. The Earth Shift products
website did not specify where the powder and nibs originate, and the fact that the Burdick
chocolate shows high levels of cadmium, arsenic, and especially lead points towards its
origin, the city of Quito, Ecuador, being an area known for contaminated effects. It is one of
the most industrialized cities in Ecuador, and the environmental impact assessment reported
by Carlos Paez (1998) addresses how wastewater discharges have become a main target for
the environment department of the Ecuadoran government.
Clearly, while the countries of bean origin have a slight impact, it is easier to apply
blame to the local regions where the chocolate beans were grown and developed, then to
assess an entire country. An idea for perfecting a study of this type would be to compare and
Astor 34
contrast two local sites within a country and look at the environment directly surrounding the
cacao trees or other external factors that could have an impact on the make-up of the soil.
While the range of arsenic concentrations was extremely large, certain high values
cast some doubt on the safety of eating very large quantities of chocolate with beans that
originate in countries notorious for environmental standards problems. However, this study
concludes that an adult or large child will have little problem consuming large portions of
chocolate, though small children should be wary when consuming vast quantities of
chocolate that come from contaminated regions.
Astor 35
Conclusion
The concentrations of arsenic in analyzed chocolate bars in this study were
found to mostly be below the ranges of previously reported values. Only a few outliers
produced comparable results, though this can be mainly attributed to those studies taking
samples from regions with previously identified areas of environmental contamination. The
results showed much higher values than any of the TDS reports indicated, though those
studies use commercially manufactured chocolates that are submitted to much stricter health
standards. Artisan chocolatiers focus more on the unique blend that each chocolate brings to
the palate, and less on the composition of its mineral makeup. Even so, dangerous levels of
arsenic would only result from consuming chocolate in large quantities over a prolonged
period of time or by supplementing the chocolate with another source of potentially lethal
arsenic, such as seafood or contaminated water.
The increase in arsenic concentration in direct response to an increase in cacao
content points towards the contamination being a result of the cacao beans taking up arsenic
from the soil either in the drying and fermenting process, or during the growth of the pods on
Theobroma cacao trees. Other results added to this conclusion, as there were no significant
differences between chocolate bars from varying manufacturers. Also, a great deal of
variation was discovered even amongst chocolate bars from the same general region,
speaking to the specific composition of the soil as the main origin of arsenic concentrations
in the finished chocolate products. Geographically, South American chocolates produced
generally higher concentrations of arsenic, though Burdick Madagascar chocolates have
levels approaching many of these countries.
A more in-depth look at arsenic levels in chocolate products might involve
narrowing the geographic spread to one or two countries that have been ravaged by industrial
Astor 36
pollution. Then it would be possible to take samples from different regions in the country and
compare the results to those resembling intense contamination. It would also help to take soil
from the site of growth and identify how much of the toxic heavy metals are taken from the
soil to the bean, checking arsenic levels at each step in the refining and manufacturing
process. To fully determine the effect arsenic can have on the body, speciation of the arsenic
present in the finished product would supplement studies of bioavailability and allow a more
thorough breakdown of the toxicity of the chocolate.
Astor 37
Acknowledgements
For all his help and support, I would like to graciously thank Dr. Donald McFarlane as well
as for proposing this project as a senior thesis topic. Together with Dr. Andrew Zanella, they
assisted me through all the experimental processes and aided in data interpretation and
presentation. I am humbled by their expertise and professionalism in handling this project
and showing me the ins and outs of the numerous procedures. I offer my sincerest thank you
for all the time and effort put into this. I would also like to thank Matt Stjernholm for his data
assistance and chocolate-buying guidelines which instructed me along the way.
Astor 38
References
Journal Citations
1. Activation Laboratories Ltd., http://www.actlabs.com. Ontario, Canada.
2. Agency for Toxic Substances and Disease Registry (ATSDR) 1999. Toxicological
Profile for Arsenic. Atlanta, GA:Agency for Toxic Substances and Disease Registry.
Available: http://www.atsdr.cdc.gov/toxprofiles/tp13.html [accessed 20 October
2010].
3. ATSDR (b). 1999. Toxicological Profile for Arsenic (Final Report). NTIS Accession
No. PB99 166621. Atlanta, GA: Agency for Toxic Substances and Disease Registry.
434 pp.
4. Cervera M., and Montoro, R., 1994. Critical Review of the Atomic Spectrometric
Analysis of Arsenic in Foods. Fresenius J. Anal. Chem., 348, 331.
5. Chocolate Bars and Products:
Burdick Chocolates http://www.burdickchocolates.com
Ghiradelli Chocolates http://www.ghirardelli.com
Earth Shift Chocolates http://www.earthshiftproducts.com
6. FDA 1991-2008. Summary of Toxic and Nutritional Elements Found in TDS Foods.
Washington, DC: U.S Food and Drug Administration. Available at
http://www.fda.gov/Food/FoodSafety/
FoodContaminantsAdulteration/TotalDietStudy/ucm184293.htm [accessed 20
November 2010].
7. Freeman, G., Johnson, J., Killinger, J., Liao, S., Davis, A., Ruby, V., Chaney, R.,
Lovre, S., Bergestrom, P., 1993. Bioavailability of Arsenic in Soil Impacted by
Smelter Activities Following Oral Administration in Rabbits. Toxicol. Sci.
(1993) 21 (1): 83-88
8. Han, W., Shi Y., Ma L., and Ruan J., 2005. Arsenic, Cadmium, Chromium, Cobalt,
and Copper in Different Types of Chinese Tea. Bulletin of Environmental
Contamination and Toxicology Volume 75, Number 2, 272-277
9. Helgesen, H., Larsen, E., 1998 Bioavailability and speciation of arsenic in carrots
grown in contaminated soil. May 1998, Vol. 123 (791–796).
10. Kim, N., Gaw, S., Northcott, G., Wilkins, A., and Robinson G., 2008. Uptake of
ΣDDT, Arsenic, Cadmium, Copper, and Lead by Lettuce and Radish Grown in
Contaminated Horticultural Soils. J. Agric. Food Chem., 2008, 56 (15), pp 6584–
6593.
Astor 39
11. Lederer W., Fensterheim R., Wauchope R., 1983. Uptake, translocation and
phytotoxicity of arsenic in plants.. Arsenic: Industrial, Biomedical, Environmental
Perspectives. New York: Van Nostrand Reinhold; 1983. p. 348-375.
12. Lee, C., Low, K., and HOH, R., (1985) Determination of Cadmium, Lead, Copper
and Arsenic in Raw Cocoa, Semifinished and Finished Chocolate Products. Pertanika
8(2) 1985, 243 - 248).
13. Loffredo C., Aposhian H., Cebrian M., Yamauchi H., Silbergeld E., 2003. Variability
in human metabolism of arsenic. Environ Res 92:85–91
14. Mass M., Wang L., 1997. Arsenic alters cytosine methylation patterns of the
promoter of the tumor suppressor gene p53 in human lung cells: a model for a
mechanism of carcinogenesis. Mutat Res 386:263–277
15. Meunier N., Blais J., Tyagi R., 2004. Removal of heavy metals from acidic soil
leachate using cocoa shells in a batch counter-current sorption process. Hydrometall.
2004;73:225–235.
16. Mounicou, S., 2003. Concentrations and Bioavailability of Cadmium and Lead in
Cocoa Powder and Related Products. Food additives and contaminants 20.4 (2003):
343-52.
17. Ng, J., Gomez-Caminero, A., Howe, P., Hughes, M., Kenyon, E., Lewis D.R., Moore,
M., 2001. Environmental Health Criteria for Arsenic and Arsenic Compounds. World
Health Organization, Geneva.
18. Ng, J., Johnson, D., Moore, M., Imray, P., Chiswell, B., 1998. Speciation of arsenic
metabolites in the urine of occupational workers and experimental rats using an
optimized hydride cold-trapping method. Analyst, 1998, Volume 123, pgs. 929-933.
19. OSHA Arsenic. United States Occupational Safety and Health Administration.
http://www.osha.gov/SLTC/arsenic/index.html. [accessed February 15, 2011]
20. Paez, C., 1998. Environmental Auditing: The Case of Ecuadorian Industry.
Environmental Impact Assessment; Quito, Ecuador.
21. Pomroy, C., Charbonneau, S., McCullough, R., and Tam, G., 1980. Human retention
studies with 74Arsenic. Toxicology and Applied Pharmacology, 53:550–556.
22. Rankin, Charles C., 2005. Lead Contamination in Cocoa and Cocoa Products:
Isotopic Evidence of Global Contamination. Environmental health perspectives
113.10 (2005): 1344.
23. Reichard, J., Puga, A., 2010. Effects of arsenic exposure on DNA methylation and
epigenetic gene regulation. Epigenomics 2:87–104
Astor 40
24. Shi, Y., Ruan, J., Ma, L., Han, W., Zhejiang, F., 2008. Accumulation and distribution
of arsenic and cadmium by tea plants. Univ Sci B. 2008 March; 9(3): 265–270.
25. Stjernholm, M., 2010 Lead and Cadmium Contamination in Manufactured Chocolate
Bars. Senior Thesis in Biology-Chemistry; Joint Science Department.
26. Thomas, DJ., Styblo, M., Lin, S., 2001. The cellular metabolism and systemic toxicity
of arsenic. Toxicol Appl Pharmacol. 2001;176:127–14
27. Whitfield, J., Dy, V., McQuilty, R., Zhu, G., Heath, A., 2010. Genetric Effects on
Toxic and Essential Elements in Humans: Arsenic, Cadmium, Copper, Lead,
Mercury, Selenium, and Zinc in Erythrocytes. Environ Health Perspect 118(6)
28. Williams, P., Price, A., Raab, A., Hossain, S., Feldmann, J., Meharg, A., 2005.
Variation in arsenic speciation and concentration in paddy rice related to dietary
exposure. Environ.Sci.Technol. 39(15):5531-5540.
29. Woolson, E., Kearney, P., Environmental Science Technology 1973, 7, 47.
30. Xu, J., and Thornton, L., 1985. Arsenic in garden soils and vegetable crops in
Cornwall, England: Implications for human health. Volume 7, Number 4, 131-133.
31. Yamauchi, H., and Y. Yamamura. 1983. Concentration and chemical species of
arsenic in human tissue. Bull. Environ. Contam. Toxicol. 31:267-270
32. Yost, L., Schoof, R., Aucoin, R., 1998. Intake of inorganic arsenic in the North
American diet. Hum Ecol Risk Assess 4:137–152
33. Zhang, W., Cai, Y., Tu, C., Ma, L., 2002. Arsenic speciation and distribution in
an arsenic hyperaccumulating plant. The Science of the Total Environment, volume
300, issues 1-3, 2 December 2002, pgs. 167-177.
Books Cited
1. Kabata-Pendias, A. Trace Elements in Soils and Plants. CRC Press, Boca Raton,
Florida. 2001, Third Edition. Pgs 27-31, 73-97, 225-230.
Web Documents Cited
1. http://www.epa.gov/iris/subst/0278.htm, 2008. Chronic Health Hazard Assessments
for Noncarcinogenic Effects of Arsenic, Inorganic. United States Environmental
Protection Agency. CASRN 7440-38-2. [accessed December 4, 2010]
2. http://www.fao.org/docrep/006/y5143e/y5143e0x.htm, 2010. Cocoa: Actual and
Projected Production. Food and Agriculture Organization of the United Nations
[accessed February 10, 2011]
Astor 41
3. http://switchboard.nrdc.org/blogs/jwalke/epas_mercury_and_air_toxics_ru.html,
2011. EPA’s Mercury and Air Toxics Rule; John Walke’s Blog. National Resource
Defense Council. [accessed March 20, 2011]
Astor 42
Appendix
Methods of Trace Metal Analysis
ICP-MS:
Inductively Coupled Plasma Mass Spectrometry, or ICP-MS, is a form of mass spectrometry
with a high level of sensitivity, and is able to detect specific elements at concentrations as
small as one part in a trillion. This machine combines an inductively coupled plasma at very
high temperatures with a mass spectrometer. The plasma converts atoms of elements in the
subject at hand, in this case ash, into ions. These ions are susceptible to the mass vs. charge
gradient by which mass spectrometers work. As ions concentrate at specific regions of
mass/charge ratio, the amount of element present is determined and recorded. The device
used in this experiment was Activation Labs located in Ontario, Canada.
Supplemental Data
Figure S1. Residual Values Regression Run on Arsenic Concentrations from Globally Variant Chocolates
showed no correlation or significant slope.
Residual Values of
Arsenic Concentrations
Linear Regression of Chocolate Samples
Organized by Cacao Contents
1000
500
0
-500
50
60
70
80
90
100
110
Cacao Percentage
Astor 43
Es
ca
z
la
ck T u C
M he os
ou o
ta
nt Co R
a
s
Es in ta ica
ca V R 65
zu en ica %
Ve ezu 91
ne ela %
z
G
7
B
hi uel 0%
la
r
ad a 8
ck
G el 1%
M
hi li
ou
ra 10
n
B ta Gh de 0%
la in ir lli
ck
8
N a
M ic del 0%
ou ar li
nt ag 60%
u
Th Th ain a 7
eo eo D. 0%
M G R. 7
ad ha 0
ag na %
as 8
ca 4%
r9
0%
B
Arsenic Levels in ng/g (ppb)
B
ck
di
ur
B
1000
ez
u
Ve
n
el
a
iv
ia
ol
75
%
66
%
%
71
%
68
r7
4%
da
do
ua
B
Ec
ck
di
ur
B
ck
di
ca
r
re
na
G
as
ag
ad
M
di
ck
ur
ur
B
B
ur
di
ck
Arsenic Levels in ng/g (ppb)
Figure S2a. Arsenic levels in Burdick Chocolates
Burdick Arsenic
500
400
Arsenic
300
200
100
0
Chocolate Bar Type
Figure S2b. Arsenic levels in previously ashed unanalyzed arsenic chocolates.
Previously Ashed Arsenic Concentrations
1500
Arsenic
500
0
Chocolate Bar Type
Astor 44
Table S1. Actual Numerical Values for Chocolate Samples utilized in this study
Arsenic (ng/g)
Escazu Costa Rica 65%
200.0
Theo Costa Rica 91%
11.0
Black Mountain Venezuela 70%
1199.0
Escazu Venezuela 81%
1170.0
Ghiradelli 100%
1022.0
Ghiradelli 80%
600.0
Ghiradelli 60%
12.0
Black Mountain Nicaragua 70%
410.0
Black Mountain D.R. 70%
503.0
Theo Ghana 84%
13.0
Theo Madagascar 90%
4.0
Burdick Madagascar 66%
430.0
Burdick Grenada 75%
78.0
Burdick Ecuador 74%
140.0
Burdick Bolivia 68%
50.0
Burdick Venezuela 71%
19.5
Figure S3. Aggregation of Analyzed Contaminant Concentrations (As, Cd, Pb) of All Chocolates Shown with
Error Bars.
Es
ca
M Th zu
ou e Co
o
Es nta C sta Contaminant Mean Levels in ng/g (ppb)
ca in os R
zu Ve ta ica
B
Ve ne Ric 65
la
z a %
ck
G nez uel 91
hi u a %
M
o
G rad ela 70
B un
la ta Ghir ell 81 %
ck in h ad i 1 %
M N ira ell 00
ou ic d i 8 %
a e
T
B h T nta rag lli 6%
ur eo h in u 6
e
di M o D a 0%
c
7
B k Mada Gh .R. 0%
ur a g an 7
B di da as a 0%
ur ck g c 8
d
a a 4
B Bu ick Gre scar 90%
ur r E n r %
di dic c ad 6
ck k ua a 6%
Ve Bo do 75
ne liv r 7 %
zu ia 4%
el 68
a %
71
%
Mean Values of
Contaminant Concentrations
1000
Contaminant Means
800
600
400
200
B
la
ck
0
Chocolate Bar Type
Astor 45
Figure S4. Concentrations of Contaminant Levels in Cacao Products Prior to Chocolate Producing
Process
Contaminat Levels in ng/g (ppb)
Cacao Powder and Nibs
3000
Cadmium
Arsenic
Lead
2000
1000
E
ar
th
S
hi
ft
E
ar
E
cu
th
ad
o
r
S
hi
ft
C
E
cu
ac
ao
ad
or
P
ow
N
ib
de
r
s
0
Chocolate Form
Figure S5a. Levels of Contaminants in Burdick Chocolates Analyzed in this Study
Contaminant Levels in ng/g (ppb)
Burdick Contaminant Levels
1500
Cadmium
Arsenic
Lead
1000
500
%
71
ue
ez
Ve
n
ck
B
ur
di
ur
B
la
ia
ol
iv
B
ck
di
ck
di
ur
B
68
%
74
%
Ec
u
na
G
re
ck
di
ur
B
ad
da
ar
as
c
ag
ad
M
ck
di
B
ur
or
75
66
%
%
0
Chocolate Bar Type
Astor 46
Figure S5b. Concentrations of contaminant levels in previously ashed chocolates.
Es
ca
z
la
ck T u C
Contaminant Levels in ng/g (ppb)
M he os
ou o
ta
nt Co R
Es ain sta ica
ca V
R 6
zu en ica 5%
Ve ezu 91
ne el %
z a
G
B
hi uel 70%
la
r
ad a 8
ck
G
e 1
M
hi lli %
ou
ra 10
B nta Gh de 0%
la in ir lli
ck
8
N a
M ic del 6%
ou ar li
nt ag 60
u %
Th Th ain a
eo eo D. 70%
M G R.
ad ha 70
ag na %
as 8
ca 4%
r9
0%
Previously Ashed Contaminant Concentrations
1500
Cadmium
Arsenic
Lead
1000
500
B
0
Chocolate Bar Type
Table S2. Actual Numerical Values of Contaminant Concentrations from Chocolate Samples Utilized in This
Study.
Cadmium (ng/g) Arsenic (ng/g) Lead (ng/g)
Escazu Costa Rica 65%
150.0
200.0
23.
Theo Costa Rica 91%
215.0
11.0
7.
Black Mountain Venezuela 70%
495.0
1199.0
90.
Escazu Venezuela 81%
265.0
1170.0
111.
Ghiradelli 100%
212.0
1022.0
37.
Ghiradelli 86%
81.0
600.0
18.
Ghiradelli 60%
144.0
12.0
8.
Black Mountain Nicaragua 70%
94.0
410.0
7.
Black Mountain D.R. 70%
153.0
503.0
15.
Theo Ghana 84%
55.0
13.0
50.
Theo Madagascar 90%
170.0
4.0
24.
Burdick Madagascar 66%
293.0
430.0
142.
Burdick Grenada 75%
570.0
78.0
72.
Burdick Ecuador 74%
485.0
140.0
335.
Burdick Bolivia 68%
212.0
50.0
90.
Burdick Venezuela 71%
306.0
20.0
47.
Astor 47