Article

pubs.acs.org/JAFC

Cadmium and Lead in Chocolates Commercialized in Brazil

Javier E. L. Villa, Rafaella R. A. Peixoto, and Solange Cadore*
Institute of Chemistry, University of Campinas, Post Oce Box 6154, 13083-970 Campinas, Sao Paulo, Brazil

ABSTRACT: Cadmium (Cd) and lead (Pb) concentrations and their relationship to the cocoa content of chocolates
commercialized in Brazil were evaluated by graphite furnace atomic absorption spectrometry (GF AAS) after microwave-assisted
acid digestion. Several chemical modiers were tested during method development, and analytical parameters, including the
limits of detection and quantication as well as the accuracy and precision of the overall procedure, were assessed. The study
examined 30 chocolate samples, and the concentrations of Cd and Pb were in the range of <1.7107.6 and <21138.4 ng/g,
respectively. The results indicated that dark chocolates have higher concentrations of Cd and Pb than milk and white chocolates.
Furthermore, samples with ve dierent cocoa contents (ranging from 34 to 85%) from the same brand were analyzed, and linear
correlations between the cocoa content and the concentrations of Cd (R2 = 0.907) and Pb (R2 = 0.955) were observed. The
results showed that chocolate might be a signicant source of Cd and Pb ingestion, particularly for children.
KEYWORDS: chocolate, cadmium, lead, cocoa, GF AAS

INTRODUCTION
Chocolates are a type of food widely consumed around the
world. The large consumption of chocolate is attributable
mainly to its pleasant avor and the feeling of well-being
promoted by its ingestion,1 which make it a very popular food
among people of all ages, although children are often the major
consumers.
The primary base of chocolate products is cocoa, the seed of
the Theobroma cacao tree.13 To be considered as chocolate, a
food product must contain at least 25% cocoa solids or 20%
cocoa butter.4 According to the International Cocoa Organ-
ization, American production of cocoa beans accounts for
approximately 16% of the worldwide production, and Brazil and
Ecuador are the largest American producers.5 In addition to
being a large producer, Brazil is also an important market for
cocoa derivatives. Dark, milk, and white are the main types of
chocolates available on the Brazilian market, and the
compositions of these varieties dier in terms of their cocoa
solids, milk fat, and cocoa butter contents.6
Numerous studies have demonstrated that the ingestion of
cocoa and its derivatives can benet health, mainly because of
the high levels of avonoids and antioxidants present in cocoa-
based foods.2,7,8 This type of food may prevent harmful eects
caused by free radicals in the human body, contributing to the
reduction of cardiovascular disease and cancer risk.9,10 In
addition, cocoa and its derivatives are also an important source
of essential inorganic constituents in the diet, such as Ca, P, Fe,
Mg, Cu, Zn, K, and Mn.2,11,12 However, the presence of
potentially toxic elements has also been reported,1317
particularly lead and cadmium.18,19
The presence of lead in cocoa and its derivatives has been
discussed for many years.20 Although lead is naturally found in
the environment, this element has no recognized function in
the human body and is harmful even at trace levels. Once in the
human body, lead can aect the metabolism of calcium and
other essential nutrients by competing for binding sites in
biological systems.21,22 In adults, the primary symptoms of lead
poisoning are abdominal pain, arthralgia, hypertension, head-
ache, increased intracranial pressure, central nervous system
dysfunction, anemia, and renal dysfunction. In children, the
eects are more severe and are associated with long-term
eects, such as growth failure, behavioral changes, language
delay, and anemia.23 Some studies have also noted a
relationship between childhood exposure to lead and delayed
intellectual development.2428
Cadmium, another element that is potentially toxic at trace
levels, is found in certain food crops grown in soils containing
high levels of this element and is the major cause of non-
workplace-related exposure to this element.29 The presence of
cadmium in the human body results in damage to several
organs and can, thus, cause hepatic damage or kidney
failure.30,31 In addition, it may cause pneumonitis, destruction
of mucous membranes, glomerular and tubular damage,
testicular necrosis, estrogen-like eects, disruption of steroid-
hormone synthesis, and numerous other harmful eects.32
Thus, controlling the cadmium and lead contents in
chocolate products is a public health concern, especially
because children are more susceptible to lead and cadmium
poisoning through the consumption of chocolate products.
However, the assessment of cadmium and lead concentrations
in chocolate products remains an analytical challenge because
of the complexity of the matrix. A successful approach must
combine adequate sample treatment with a sensitive analytical
technique.1
In this study, the concentrations of cadmium and lead found
in dierent brands and types of chocolate bars available in the
Brazilian market are presented. The relationship between lead
and cadmium and the cocoa content of chocolates
commercialized in Brazil were also evaluated.
Received: June 3, 2014
Revised: August 12, 2014
Accepted: August 15, 2014
Published: August 15, 2014

2014 American Chemical Society 8759 dx.doi.org/10.1021/jf5026604 | J. Agric. Food Chem. 2014, 62, 87598763
Journal of Agricultural and Food Chemistry

Article

MATERIALS AND METHODS
Chemicals. Deionized water (Milli-Q system, 18.2 M cm,
Millipore, Bedford, MA) and analytical-grade reagents were used to
prepare all solutions and standards. Nitric acid (65%) and hidrogen
peroxide (30%, v/v) were purchased from Merck (Darmstadt,
Germany). Pure argon (99.999%) purchased from White Martins
(Sao Paulo, SP, Brazil) was used as purge and protective gas.
The palladium working solution (10 g/L) was purchased from
PerkinElmer (Norwalk, CT). Magnesium nitrate was purchased from
Merck (Darmstadt, Germany), and the corresponding working
solution (10 g/L) was prepared by diluting an appropriate amount
of the salt in ultrapure water. Ammonium dihydrogen phosphate was
obtained from Fluka (Buch, Switzerland), and its working solution
(100 g/L) was prepared by diluting the appropriate amount of the salt
in deionized water.
The analyte solutions were prepared from 1000 mg/L standard
solutions (Merck Darmstadt, Germany) in 0.2% (v/v) HNO3.
Appropriate dilutions were made from the standard solutions and
solutions were left to cool, transferred to individual 25.0 mL
volumetric asks, and diluted to the mark with deionized water.
A total organic carbon analyzer (TOC-VCPN, Shimadzu, Kyoto,
Japan) was used to determine the residual carbon content (RCC).
Acidbase titration of the digests was performed to determine the nal
acidities of the samples. The titrations were performed with a standard
solution of sodium hydroxide (0.0989 mol/L) and phenolphthalein
[1.0% (m/v) in ethanol].
Validation Studies. The method was validated by determining
various gures of merit, such as the linearity, precision, accuracy,
characteristic mass, and detection limit, as recommended by Welz and
Sperling.33 The accuracy of the method was evaluated using the
following CRMs: corn bran (NIST 8433), wheat our (NIST 1567a),
and whole milk powder (NIST 8435). Furthermore, analyte addition
and recovery experiments were performed for three dierent chocolate
types at two concentration levels (0.2 and 0.5 /L for Cd and 1 and 4
/L for Pb) and in triplicate for each spiked level. The limits of
detection (LOD) and quantitation (LOQ) of each analyte were also
calculated.

stored in polyethylene asks under refrigeration. All glass and quartz
materials were treated with a 10% (v/v) HNO3 solution for 24 h and
then rinsed with deionized water prior to use. RESULTS AND DISCUSSION
Instrumentation. All measurements were carried out using an Sample Digestion. The study of sample digestion
AAnalyst 600 atomic absorption spectrometer (PerkinElmer, Norwalk, procedures is an important topic for the improvement of
CT) with Zeeman background correction that was equipped with a
transversely heated graphite tube atomizer and an AS-800
autosampler. Electrodeless discharge lamps for Cd and Pb were
operated at 230 and 440 mA, respectively. The selected wavelengths
were 228.8 nm for Cd and 283.3 nm for Pb. Aliquots of 20 L of the
samples and calibration solutions were injected with 5 L of chemical
modier directly into the graphite tube. Pure argon (99.999%) was
used as the purge and protective gas. The optimum operating
parameters for GF AAS are shown in Table 1.

Table 1. Heating Programs for Cd and Pb Determination by

GF AAS in Chocolate Samples Using 5 g of Pd + 3 g of
Mg(NO3)2 and 3 g of Mg(NO3)2 + 50 g of NH4H2PO4 as
Chemical Modiers
step temperature (C) ramp (s) hold (s) argon ow rate (mL/min)
1 110 5 20 250
2 130 5 30 250
3 600,a 850b 10 20 250 Figure 1. RCC in samples of each chocolate type digested using
4 1500,a 1600b 0 5 0 dierent nitric acid concentrations.
5 2450 1 3 250
a
Cadmium. bLead. Table 2. Pyrolysis Temperatures and Characteristic Masses
(mo) Obtained for Cd and Pb in a Sample of Dark Chocolate
Certied Materials and Sample Collection. Three certied Using Dierent Chemical Modiers
reference materials (CRMs) from the National Institute of Standards Tpyrolysis mo
and Technology (Gaithersburg, MD), specically, corn bran (NIST analyte chemical modier (C) (pg)
8433), wheat our (NIST 1567a), and whole milk powder (NIST
Cd 10 g of NH4H2PO4 600 1.5
8435), were used to verify the accuracy of the proposed method.
3 g of Mg(NO3)2 + 50 g of NH4H2PO4 700 1.5
A total of 30 chocolate samples of dierent types (labeled by the
manufacturers as white, milk, or dark chocolate) and brands 5 g of Pd + 3 g of Mg(NO3)2 600 1.4
(designated as brands AK) were analyzed in this study. They were Pb 10 g of NH4H2PO4 850 30.8
purchased in the local market of Campinas, State of Sao Paulo, Brazil, 3 g of Mg(NO3)2 + 50 g of NH4H2PO4 850 31
in the period of JanuaryMarch 2014. 5 g of Pd + 3 g of Mg(NO3)2 1000 36.6
Sample Treatment. Sample treatment was performed using a
microwave digestion system (ETHOS1, Milestone, Sorisole, Italy)
equipped with closed polytetrauoroethylene vessels and sensors for analytical methods for the routine determination of trace
temperature and pressure control. Sample masses of 500 mg were elements in food materials. The sample decomposition using
accurately weighed into the closed vessels, and 1.5 mL of 30% (v/v) the minimal amount of reagents can oer advantages as
hydrogen peroxide and 6.5 mL of nitric acid at a concentration of 12, reducing costs and the amounts of residue generated.3436 For
9, 6, or 3 mol/L were subsequently added. The closed vessels were
then placed inside the microwave oven, and the heating program was this, the digestion procedure was evaluated at four dierent
performed over six steps: (1) heat from room temperature to 80 C in HNO3 concentrations (12, 9, 6, and 3 mol/L) by comparing
3 min, (2) hold for 3 min at 80 C, (3) heat from 80 to 140 C in 3 the RCCs and the residual acidities of the digests. The digestion
min, (4) hold for 4 min at 140 C, (5) heat from 140 to 200 C in 8 solutions were free from solid residues when the nitric acid
min, and (6) hold for 16 min at 200 C. After digestion, the resulting concentration was greater than 3 mol/L. The RCC for each
8760 dx.doi.org/10.1021/jf5026604 | J. Agric. Food Chem. 2014, 62, 87598763
Journal of Agricultural and Food Chemistry Article

Table 3. Determination of the Analytes in Standard Table 6. Concentrations Found (ng/g) of Cd and Pb in
Reference Materials Chocolates (n = 3)
Cd (ng/g) Pb (ng/g) sample type/brand cocoa content (%) Cd Pb
certied certied 1 white/A nia <1.7 <21
CRM value found value found 2 white/B ni <1.7 <21
corn bran 12 5 10 2 140 34 118 12 3 white/C ni <1.7 <21
whole milk nca 110 50 70 9 4 white/D ni <1.7 <21
powder 5 white/E ni <1.7 <21
wheat our 26 2 24.6 0.5 nc 6 white/F ni <1.7 <21
a
nc = not certied. 7 white/G ni <1.7 <21
8 milk/A ni 7.8 1.4 <21
Table 4. Recovery Results (%) for Cd and Pb in Spiked 9 milk/B ni 12.3 0.3 <21
Samples (n = 3) 10 milk/C ni 9.6 1.1 <21
11 milk/D ni 8.7 0.6 <21
Cd Pb
12 milk/E ni 9.9 0.5 <21
chocolate level 1 level 2 level 1 level 2 13 milk/G ni 9.7 0.4 <21
type (0.2 g/L) (0.5 g/L) (1 g/L) (4 g/L)
14 milk/H ni 11.0 0.3 <21
white 93 6 96 4 92 7 95 3 15 dark/A 55 36.7 1.0 138.4 4.4
milk 94 6 92 3 97 5 99 4 16 dark/B 60 38.0 0.8 22.7 2.7
dark 96 4 95 5 102 8 93 3 17 dark/C 43 35.4 1.2 <21
18 dark/E 53 32.9 2.3 <21
Table 5. Figures of Merit for the Determination of Cd and 19 dark/G 70 38.3 0.5 44.8 4.2
Pb in Chocolate 20 dark/I 75 107.6 4.5 44.6 2.7
element linear range (g/L) LOD (ng/g) LOQ (ng/g) RSDrepeat (%) 21 dark/J 40 22.1 0.4 59.5 2.8
22 dark/J 70 40.0 1.9 129.7 6.9
Cd 0.0342 0.5 1.7 1.6
23 ni/A 40 29.8 0.7 <21
Pb 0.4510 6.3 21 2.1
24 ni/B 41 28.2 0.3 39.1 3.4
25 ni/D ni 31.5 0.7 <21
type of chocolate is shown in Figure 1, and the residual acidities 26 milk/K 34 11.6 1.0 27.0 3.8
of the digests prepared using nitric acid at 6, 9, and 12 mol/L 27 milk/K 41 14.4 1.1 28.1 3.1
ranged from 1 to 2.7 mol/L. 28 dark/K 55 37.3 0.9 39.5 3.9
A concentration of 6 mol/L HNO3 was used in subsequent 29 dark/K 70 48.5 0.6 63.6 3.7
experiments to digest 500 mg of chocolate, even though this 30 dark/K 85 89.5 2.3 73.8 4.1
condition presented the highest RCC value, considering that a
ni = not informed.
the technique of GF AAS has a pyrolysis step, which main
function is to eliminate the matrix organic matter prior to the
atomization. As already demonstrated, when using this measurements of the blank and b is the slope of the calibration
technique, it is possible to do the introduction of samples curve. A further multiplication by a factor of 50, considering the
with high contents of organic matter, including slurry and solid mass of the sample (0.5 g) and the nal volume of the solution
samples.14,33 (25 mL), allowed us to obtain the LOD and LOQ in ng/g. The
Optimization of the GF AAS Conditions. Pyrolysis and repeatability was obtained by analyzing a control sample of dark
atomization curves were established using the sample with the chocolate 6 times within a short period of time.
highest RCC (dark chocolate) to guarantee that the heating The gures of merit for the determination of Cd and Pb are
program would be adequate for all of the samples. The provided in Table 5.
pyrolysis temperature and characteristic masses of Cd and Pb Analytical Determinations. Cadmium and lead concen-
using dierent chemical modiers are shown in Table 2. With trations were determined in 30 chocolate samples of dierent
respect to the chemical modiers, the best detectability, types and brands using the proposed method. The results are
analytical signal, and background signal for Cd determination shown in Table 6.
were observed using 3 g of Mg(NO3)2 + 5 g of Pd, whereas The concentrations of Cd and Pb found ranged between
the highest detectability and repeatability for Pb determination <1.7107.6 and <21138.4 ng/g, respectively, and the highest
were observed when 3 g of Mg(NO3)2 + 50 g of NH4H2PO4 concentrations of Cd and Pb were found in dark chocolates.
was used. The results were in agreement with those found by Rehman et
Analytical Features. No chocolate reference material with al.13 (Cd and Pb ranged between 4.3 and 190 and between 29
certied Cd and Pb concentrations is currently available. and 1400 ng/g, respectively) and, in general, lower than those
However, three CRMs (NIST 8433, NIST 1567a, and NIST found by Dahiya et al.18 (Cd and Pb ranged between 1 and
8435) were used. The observed Cd and Pb concentrations were 2730 and between 49 and 8040 ng/g, respectively).
in agreement with the certied values at the 95% condence The Cd and Pb concentrations of all of the samples analized
level (Students t test); the results are shown in Table 3. in this work were below the maximum levels recommended by
Considering the analyte addition and recovery experiments, the Brazilian legislation,37 the European Union,38 and the Food
values ranging from 92 to 102% were obtained, as shown in and Agriculture Organization (FAO)/World Health Organ-
Table 4. ization (WHO).39 According to the U.S. Food and Drug
The LOD and LOQ were calculated as 3Sblank/b and 10Sblank/ Administration (FDA), the levels of Pb in candies must not
b,33 respectively, where Sblank is the standard deviation of 10 exceed 100 ng/g. In this study, two samples of dark chocolates
8761 dx.doi.org/10.1021/jf5026604 | J. Agric. Food Chem. 2014, 62, 87598763
Journal of Agricultural and Food Chemistry
Figure 2. Correlation between the cocoa content (%) and the (A)
cadmium and (B) lead concentrations.
have presented higher concentrations, what is already expected,
considering that the cocoa liquor might be the principal source
of lead.40
The provisional tolerable monthly intake (PTMI) for Cd
recommended by the FAO/WHO is 25 g/kg of body
weight.41 Considering an average weight of 15 kg for children
and 70 kg for adults and a consumption of 10 g of chocolate per
day, the maximum contribution for Cd was 8.6% of the PTMI
for children and 1.8% for adults.
The European Union recommends a tolerable weakly intake
(TWI) of 2.5 g/kg of body weight for Cd,42 and in this case,
the maximum contribution was 20.1% of the TWI for children
and 4.3% for adults. Thus, the dark chocolate consumption
might be a signicative source of Cd, especially for children.
Although the determination of the total concentrations
allowed us to evaluate the maximum levels and tolerable intakes
of Cd and Pb, it is important to point out that the evaluation of
the risk assessment associated with the ingestion of metallic
elements from a food matrix should estimate its bioaccessibility
and bioavailability, considering that only a fraction of the
ingested element is released from the food matrix into the
gastrointestinal tract and is absorbed by humans to nally reach
the bloodstream.21,43,44
With regard to the presence of Cd and Pb in chocolate
samples, the possible sources of contamination include the raw
materials used, the manufacturing processes, and the wrappers
in which the chocolates are stored. As shown in Table 6, the
concentrations of Cd and Pb in chocolates with the same cocoa
content were slightly dierent depending upon the brand,
8762
Article
which may be due to the dierent manufacturing processes
used.
To evaluate the relationship between the cocoa content and
the concentrations of Cd and Pb in chocolate, ve samples with
dierent cocoa contents (ranging from 34 to 85%) from the
same brand were analyzed and the measured concentrations
were plotted against the cocoa content. The results are shown
in Figure 2.
According to the results, a linear correlation exists between
the cocoa content and the concentrations of Cd (R2 = 0.907)
and Pb (R2 = 0.955), which suggests that the main source of
contamination of Cd and Pb in chocolates is the cocoa used in
the manufacturing process.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +55-19-3521-3125. Fax: +55-19-3521-3023. E-
mail: cadore@iqm.unicamp.br.
Funding
The authors gratefully acknowledge the Conselho Nacional de
Desenvolvimento Cientico e Tecnologico (CNPq), the
Fundacao de Amparo a Pesquisa do Estado de Sao Paulo
(FAPESP/Process 2011/07759-7 and Process 2011/21023-3),
and the Instituto Nacional de Ciencias e Tecnologias Analiticas
Avancadas (INCTAA) for their nancial support.
Notes
The authors declare no competing nancial interest.
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