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PII: S0889-1575(17)30290-9
DOI: https://doi.org/10.1016/j.jfca.2017.12.012
Reference: YJFCA 3000
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Please cite this article as: Kruszewski, Bartosz., Obiedziński, Mieczysław Wiesław.,
& Kowalska, Jolanta., Nickel, cadmium and lead levels in raw cocoa and processed
chocolate mass materials from three different manufacturers.Journal of Food
Composition and Analysis https://doi.org/10.1016/j.jfca.2017.12.012
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Original Research Article
Nickel, cadmium and lead levels in raw cocoa and processed chocolate mass materials from
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Department of Fruit and Vegetable Product Technology, Prof. Wacław Dąbrowski Institute
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of Agricultural and Food Biotechnology, Rakowiecka 36, 02-532 Warsaw, Poland
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Faculty of Food Sciences, Department of Food Technology, Warsaw University of Life
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Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland
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*Corresponding author: bartosz.kruszewski@ibprs.pl (B. Kruszewski)
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Lomza State University of Applied Sciences (PWSIiP), Akademicka 14, 18-400 Łomża,
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Poland.
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Graphical abstract
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Highlights
Nickel was metal with the highest levels in cocoa raw materials and chocolate.
Good selection of the raw materials for chocolate production is highly important.
Cocoa bean winnowing and conching are process steps causing metals content decrease.
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Lead level in chocolate was low and did not pose a threat to human health.
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Abstract
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This study is a comprehensive approach to contamination from heavy metals (cadmium Cd,
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lead Pb, nickel Ni) in raw cocoa and the masses resulting from the various steps of the
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chocolate manufacturing process in three different companies. This study provide new and
reliable data for food safety authorities, stakeholders and consumers. It also broadens
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knowledge of the contribution made by particular raw materials, production processes and/or
machines in the overall level of these metals in the final product. The total reduction observed
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in the levels of metals varied according to the production line (10.5 – 33% Cd, 0 – 100% Pb,
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11 – 42% Ni). It was noted that the two steps which have the greatest impact on decreasing
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the concentration of theses metals are winnowing of cocoa bean shell and conching. Nickel
was the most abundant toxic metal (max. 12.1 mg/kg in raw cocoa and 4.5 mg/kg in
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chocolate) and its presence creates a serious risk to children’s health. The chocolate obtained
from one of the producers posed a similar danger in terms of its cadmium content (0.43
mg/kg). Therefore, chocolate products should be monitored constantly and an absolute limit
1. Introduction
Over the past decade, research into the contamination of cocoa raw materials and chocolate
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products with elements such as nickel, cadmium, lead, arsenic, and mercury has been widely
undertaken (FAO/WHO CX/CF 15/9/6, 2014; European Commission, 2004). This threat
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increases in line with the low age and body weight of the potential consumer. The
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International Agency for Research on Cancer (IARC) has classified cadmium as carcinogenic
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to humans (group 1), while lead and nickel as possibly carcinogenic to humans (group 2B)
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(IARC, 2016).
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In the past five years, RASFF (The Rapid Alert System for Food and Feed) notifications
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relating to heavy metals in cocoa-based foods have concerned only cocoa powders (RASFF
Portal, 2016). Elevated concentrations of nickel were confirmed only in cocoa fat and palm
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oil samples (Güldaş et al., 2008). In the case of chocolate products, the highest amounts of
cadmium, lead and nickel were determined in chocolates with a very high cocoa content (≥
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70%) (Sager, 2012). Some studies have shown that children, who consume a lot of chocolate
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may be at risk of heavy metal poisoning, especially from lead, if they exceed the daily intake
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limit (Villa et al., 2014; Yanus et al., 2014; Peixoto et al., 2016).In 2012, the European Food
Safety Authority (EFSA) published a report on cadmium dietary exposure in the European
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population. According to this document, high levels of cadmium were found in chocolate
products, which were among the food categories that contributed the most to dietary exposure
to this metal across all age groups. As a result of this report, Commission Regulation No
1881/2006 was amended by Commission Regulation No 488/2014 in May 2014 which stated
the maximum permissible levels of cadmium in specific cocoa and chocolate products such as
cocoa powders and chocolates with various total dry cocoa solids content. Restrictions of 0.1
up to 0.8 mg/kg of wet weight will apply from 1 January 2019. To date it is the European
Union’s only restriction in terms of levels of heavy metals in cocoa and chocolate products.
Nickel, cadmium and lead are naturally occurring chemical compounds, detected in the
human organism at trace levels. Nickel exhibits biological activity even at very low
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concentrations, and short-term exposure is causing allergic reactions in an increasing number
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of people. Higher concentrations can accumulate in the organs, such as the lungs and kidneys,
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resulting in their impairment. Throat, stomach, lung, sinus and nose cancer have been
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attributed to long-term exposure to nickel (Duda-Chodak & Blaszczyk, 2008). Excessive
ingestion or absorption of lead causes poisoning known as saturnism. The most characteristic
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symptoms include painful contraction of the intestinal smooth muscle (lead colic), anemia,
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weight loss, oxidative damage to the liver and/or kidney and, in certain patients, mental
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depression or cognitive impairment (Ahamed & Siddiqui, 2007). Whereas the presence of
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cadmium, in the form of free ions, interferes with many of the various metabolic cycles, such
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as the transformation of thiamine, proteins, and in the case of chronic poisoning also the
Research teams dealing with the micro- and macroelements present in raw cocoa
materials and chocolate products agree that their contamination with harmful elements such as
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nickel, lead and cadmium could have many sources. It could be due to from the environment
in which the cocoa beans undergo various processes such as fermentation, drying and
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crushing. The source of contamination could also be due to the metal devices where the
grinding, mixing and storage are carried out (Taylor, 2005; Rankin et al., 2005; Rehman &
Husnain, 2012; Ščančar et al., 2013). Heavy metals are introduced into the chocolate products
via cocoa powder and cocoa fat, which are obtained by pressing the cocoa mass, as well as
hydrogenated plant oils (Mounicou et al., 2003; Rankin et al., 2005; Pedro et al., 2006). The
level of these elements also depends on the cocoa plant’s geographical origin, the
environment in which they are cultivated (use of fertilizers, application of pesticides, metals
naturally occurring in the soil and air), local pollution, the use of leaded fuel, and the transport
and storage conditions of the cocoa beans (Taylor, 2005; Dahiya et al., 2005; Rehman &
Husnain, 2012; Bertoldi et al., 2016). Furthermore, the products from various batches can
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differ significantly, as each batch could be contaminated due to the influence of various
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factors and concentrations of raw materials (Dahiya et al., 2005; Pedro et al., 2006).
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According to the literature, cocoa beans can contain smaller amounts of heavy metals
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than processed cocoa bean products and also some chocolate confectionary (Mounicou et al.,
2003; Taylor, 2005; Yanus et al., 2014). Studies have shown an even lower concentration of
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heavy metals in the cocoa bean core (Rankin et al., 2005; Yanus et al., 2014). This could be
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due to the natural defense provided by the shell which protects the core from external physical
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and chemical threats, and which is removed during cocoa bean processing. The maximum
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shell content recommended by the Food and Drug Administration (FDA) is 1.75% in non-
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alkalized products, while the current Codex Standard 141-1983 imposes a maximum of 4.5%
shell content in non-alkalized cocoa mass (FDA, 2016; FAO/WHO, Codex Stan 141-1983,
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2014). Cocoa shells have a high absorption capacity, mainly of lead, but also of other heavy
metals, from their surroundings. This is the subject of on-going research and this
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characteristic is currently being used to eliminate metal pollution from various fluids, soils,
and, in the future, also from other materials (Rankin et al., 2005; Meunier et al., 2003, 2004).
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Apart from the heavy element content in cocoa and chocolate products, their
bioavailability is also very important. A team led by Mounicou (2003) investigated lead and
cadmium concentrations and their bioavailability in cocoa powders, beans, fat, and masses.
The potential bioavailability ranged from 10–50% and below 10% for cadmium and lead,
respectively. In the published research, both content and bioavailability differed significantly
in terms of the geographical origin of the raw material (Mounicou et al., 2003).
At present there are no reports on the internal factors which influence the heavy metal
content such as machines and the recipes used by the manufacturers. The aim of this study
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was to monitor the amount of cadmium, lead and nickel present during all the steps in the
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chocolate manufacturing process on three different production lines (at three manufacturing
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sites). These three metals were chosen taking account of the most problematic contamination
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in cocoa products and chocolate in the past decade. The contribution of each component to the
overall content of these elements in the final product was also investigated.
Concentrated nitric acid and perhydrol of analytical grade were supplied by Avantor
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Performance Materials Poland S.A. (Gliwice, Poland). The deionised water used in sample
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preparation was purified using a Milli-Q Plus high purity water device of resistivity of 18.0
MΩ cm (Millipore,Bedford, Ma., USA). All measurements were carried out using argon with
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The samples were gathered from the production lines of three different chocolate factories
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located in central Poland. The samples consisted of raw cocoa materials (cocoa beans,
powder, mass and fat), raw non-cocoa materials (cocoa butter equivalents, lecithin, sugar,
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polyglycerol polyricinoleate (PGPR), vanillin aroma), and masses from each stage of the
production process including the final product – dark chocolates (Figure 1 & 2). The cocoa
beans were imported from Ecuador (subspecies Nacional) and the Dominican Republic
(subspecies Trinitario) in an already fermented and dried form- processes carried out in the
country of origin. Immediately after being placed in polyethylene containers, the samples
Materials such as lecithin, PGPR, and sugar from all the producers (if used) exhibited a very
similar composition and physical properties (data not shown), so the results of their analysis
were presented as an average of all the samples collected. The addition of vanillin aroma was
in the order of hundredths of a percent, so it was presumed it could not significantly affect the
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composition of the samples, therefore aroma was eliminated from the analysis.
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Because of the production method: on manufacturing scale (factory Z) or on a mass scale
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(factories X and Y), the producers used different machine parks and purchased their raw
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materials from different suppliers (Figure 1 & 2). The technology and production process
parameters also varied, as well as did the length of the producers presence on the food market
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and the size of the assortment offered. Because the producers wanted to remain anonymous,
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their names were coded using the letters X, Y and Z.
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laboratory grinder (IKA, Staufen im Breisgau, Germany). 0.25 g from each homogenized
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sample was weighed and placed in HPS-100 TFM teflon vessels (Spectro-Lab, Warsaw,
Poland) able to withstand pressure of up to 11 x 106 Pa. Digestion was conducted with 5 mL
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of nitric acid (65%, v/v) and 2 mL of a 35% (v/v) perhydrol solution. After digestion, the
samples were submitted to heating in a microwave unit in an MLS 1200 Mega mineralizer
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(Milestone, Shelton, Ct., USA). The high-pressure mineralization treatment for all samples
and certified reference materials (CRMs) was as follows: 10 min 250 W / 8 min 500 W / 4min
400 W / 2 min 300 W. After microwave processing the teflon vessels were placed in an ice
bath and cooled for 30 min. Each vessels content was diluted in up to 25 mL with deionised
water in polypropylene volumetric flasks. All laboratory containers which had contact with
the sample solutions were cleaned before use with 20% (v/v) nitric acid and rinsed with
deionised water, then dried in a dust free environment. For all tested materials was made three
2.3.2. Instrumentation
Nickel quantification was performed using the inductively coupled argon plasma atomic
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emission spectroscopy method (ICP AES) with a Thermo iCAP 6500 DUO (Thermo
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Scientific, USA) device. Measurements were carried out using the axial plasma observation
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mode. The wavelength of the studied element were 231.60 nm,. The other program
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parameters were as follows: generator power 1200 W, plasma gas flow rate 14 L/min,
auxiliary gas flow rate 1.5 L/min, micro-concentric nebulizer flow rate 1 mL/min, cyclonic
Japan). The Zeeman background correction method (application of a constant magnetic field)
was applied for measurements carried out with a hollow cathode lamp adequate for each
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2.4. Validation
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GFAAS and ICP AES methods were calibrated using multi-point calibration curve at
concentrations 0 – 0.01 and 0 - 10 mg/L, respectively. For ICP AES method multi-element
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standard solutions (Sigma-Aldrich, Darmstadt, Germany) while for GFAAS method single-
element solutions (Sigma-Aldrich, Darmstadt, Germany) were used. Three calibration curves
were generated for Cd, Pb, Ni, and the obtained results were used to fit the equation by the
least-squares regression. An adequate linear fit for all cases was found (R2 = 0.9980, 0.9990,
The limit of detection (LOD) and quantification (LOQ) were calculated as three and ten times
the standard deviation of the blanks, respectively. LOD and LOQ for Ni were 0.005 and 0.016
mg/kg, respectively. In Table 1 it can be found LOD, LOQ and characteristic mass (mo) for
Cd and Pb. The characteristic mass was calculated from the slope (a) of calibration curve
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using the equation mo = 0.0044 x 20/a, for a sample volume of 20 µL. The element
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concentrations analysis in the blank samples after every tenth measurement was done. The
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average blank measurements for Cd and Pb was below the limit of detection, while for Ni it
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was 0.009 mg/mL.
The accuracy of methods was determinated against CRMs: Skimmed Milk Powder ERM®-
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BD 150 (Joint Research Centre, Geel, Belgium) and Soya Bean Flour INCT-SBF-4 (Institute
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of Nuclear Chemistry and Technology, Warsaw, Poland). Table 2 shows the results of CRMs
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All data was presented as a mean with standard deviations for two repetitions of three samples
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taken from each raw material and the processed masses. The effects of the various steps in the
production process on the heavy elements content were analysed using one way analysis of
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variance (ANOVA). All the results obtained were checked for normal distribution according
values for data that was normally distributed, the Tukey HSD test was used with a
significance level of P ≤ 0.05. If the tested data did not come from normal distribution, the
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Kruskal–Wallis test was used instead. All statistical analyses were performed using Statistica
12.0 software.
for cadmium and lead. A soya bean flour (INCT-SBF-4) was used to check the nickel analysis
because there is no certified value for this element in skimmed milk powder. Table 2 shows
the data. Recoveries were 98.5%, 100% and 95% for Cd, Pb and Ni, respectively. The Student
t-test was used to evaluate the results. All three computed t-values were lower than the
tabulated one. Moreover, the computed p-values were greater than α=0.05 (data not shown).
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This indicates no significant statistical difference between the obtained and the certified
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values.
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3.2. The nickel (Ni) content
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The concentration of nickel in the cocoa powders used by factories X and Y was relatively
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high, 12.10 and 11.70 mg/kg respectively (Table 3). An even higher content was determined
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by Sager (2012) in three different cocoa brands (a range of 10.77 – 17.12 mg/kg) and
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Grembecka & Szefer (2012) in twenty-four cocoa samples (a range of 8.2 – 17.0 mg/kg). The
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report on special monitoring research carried out in Holland found the nickel content in cocoa
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powders to be in the range of 9 – 16 mg/kg (Dolman & Houwen-Claassen, 1999). The cocoa
masses used by factories X and Y were characterized by approximately half the nickel content
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in cocoa powders – 5.72 and 5.87 mg/kg, respectively. The lowest nickel concentration
among all the raw materials containing cocoa was detected in the cocoa beans used by factory
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Z – beans from Ecuador (2.32 mg/kg). The nickel content in cocoa beans originating from the
Dominican Republic (7.62 mg/kg) was more than three times higher than in beans from
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Ecuador. This discrepancy indicates that the geographical location of the cocoa trees has an
great impact on the nickel content of cocoa beans, as can clearly be seen in the research
results of Bertoldi et al. (2016). Both the natural composition of the soil and environmental
contamination are of great importance in cocoa cultivation. All these factors have a direct
impact on the heavy metal content in the cocoa-type ingredients used in chocolate production.
Also the wide range of nickel concentration reported in publications concerning raw cocoa
The cocoa fats used on the X and Y production lines, as well as the cocoa butter equivalent
and PGPR were distinguished by a very low (below 90 μg/kg) nickel concentration (Table 3)
which points to the soft refinement of these ingredients, or even lack of processing through
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hydrogenation, where hydrogen is incorporated into the unsaturated fatty acids in the presence
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of a nickel catalyst, resulting in the contamination of the product by this metal as a residue
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(Güldaş et al., 2008; Dohnalova et al., 2017). In the cocoa fats analysed by Güldaş et al.
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(2008), nickel amounts lower than in the current study (an average of 54 μg/kg) were
determined. Dutch monitoring research on white chocolate, in which cocoa butter was the
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main ingredient, showed a nickel content of from 60 up to 120 μg/kg (Dolman & Houwen-
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Claassen, 1999).
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In the case of all the production lines under discussion, the nickel concentrations decreased
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during the processing of the masses (Tables 4 & 5). At factories Y and X, the total reduction
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in relation to mass after mixing was 11.4 and 16%, respectively. During chocolate production
straight from the cocoa beans by factory Z, the total reduction was about 11% in the case of
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beans from Ecuador, and about 42% if beans from the Dominican Republic were used.
Irrespective of the production line used, conching was a crucial processing step which
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lowered the concentration of nickel. It is the obvious effect of adding non-cocoa ingredients in
this step, such as sugar, CBE, PGPR, and cocoa fat, which contain significantly lower
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amounts of nickel. During this step, the nickel content in the masses produced by factories Y
and X showed a significant statistical drop – of 5.8% and 7.8%, respectively. In the samples
taken from factory Z, the decrease was insignificant statistically (6.0%) in the case of beans
from Ecuador, and significant (25.2%) if beans from the Dominican Republic were used. A
second important step in the processing, which influences the nickel concentration, and which
occurs only in factory Z, was winnowing of crushed cocoa beans. The concentration of nickel
decreased slightly (6.1%) in the case of Ecuadorian beans but significantly (19.5%) in beans
from the Dominican Republic as a result of removing nearly all the cocoa shell. As mentioned
earlier, cocoa shells absorb certain metals (Rankin et al., 2005; Meunier et al., 2003, 2004).
The greater reduction noted in the beans from the Dominican Republic can be explained by
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their greater nickel contamination.
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The highest nickel content was determined in the chocolate produced by factories Y and Z (in
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the case of cocoa beans originating from the Dominican Republic), 4.50 and 4.43 mg/kg
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respectively. The chocolate from factory X (3.60 mg/kg) contained less nickel. The most
favorable nickel concentration was found in the chocolate obtained from factory Z, based on
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cocoa beans originating from Ecuador (2.07 mg/kg). Compared with the chocolate produced
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by factories Y and Z (Dominican beans), almost the same nickel content was found in
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chocolate samples of Polish, European and American origin, Grembecka & Szefer (2012)
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obtained a nickel level in the range of 1.9 – 7.9 mg/kg. In twenty-three dark chocolate
samples, Dahiya et al. (2005) reported a nickel content of between 0.049 and 8.29 mg/kg. The
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authors observed a high variability in two batches of individual brands. The differences
between the batches were explained as the lack of repeatability in chocolate production due to
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the different elemental composition of the raw materials. A wide range of nickel
concentration was also observed in this study depending on the cocoa-based materials used.
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Nickel content can be linked to the cocoa content in chocolate products. This statement was
supported by Sager’s (2012) studies of chocolate with 50% and 70% cocoa content. The
median and maximum determined nickel concentrations were higher in chocolate with 70%
cocoa content. Similar results were found by the team led by Güldaş et al. (2008) which show
that of all the types of chocolate, statistically it is dark chocolate which has the highest nickel
content (about 4.5 mg/kg). Moreover Ščančar et al. (2013) proved that the nickel content
increases (0.93 – 5.75 mg/kg) in line with the cocoa content in chocolate (12 – 90%).
The high concentration of nickel in final cocoa products may be due to its rich natural
presence in the environment in which the cocoa beans are cultivated, such as the cocoa
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3.3. The lead (Pb) content
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The highest amount of lead was determined in cocoa mass and cocoa powder produced by
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factory X, 0.585 and 0.575 mg/kg, respectively. Compared to cocoa raw materials produced
by factory Y, the concentration was at least four times higher (Table 3). The cocoa mass used
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by factory Y was very low (below 0.0008 mg/kg) in lead. In the cocoa beans imported by
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factory Z, the lead content was about four times lower than in the cocoa raw materials used by
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All the non-cocoa materials included in the chocolate recipes were characterized by a less
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than 0.0008 mg/kg concentration of lead (Table 3). These results were better than those
At present the European Union or Codex Alimentarius Commission have not imposed any
restrictions regarding the level of lead in cocoa beans, cocoa bean processing products and
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chocolates However, the levels of this metal in all the materials mentioned are constantly
The results published by Sager (2012) for three brands of cocoa (0.002 – 0.196 mg/kg) are
similar to those described in this paper for raw cocoa materials . Also the lead concentration
level in cocoa powders of varying geographical origin were not much higher in the results
obtained by the research teams lead by Mounicou et al. (2003) (an average of 0.011 mg/kg),
Rankin et al. (2005) (0.147 – 0.188 mg/kg) and Yanus et al. (2014) (an average of 0.103
mg/kg). Grembecka & Szefer (2012) did not detect any lead in twenty-four cocoa and six
instant cocoa samples. Both Israeli and French researchers have found the presence of lead in
cocoa fats to be in the range of 0.015 – 0.067 mg/kg (Mounicou et al., 2003; Yanus et al.,
2014). There are two explanations for this phenomenon. The first is the possible
hydrophobicity of some lead compounds present in the cocoa beans (Mounicou et al., 2003).
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Another reason is the possibility of the deshelled cocoa beans being contaminated by small
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quantities of shell (Manton, 2010).
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In the case of cocoa beans, an equal concentration of lead was observed (Table 3) irrespective
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of geographical origin (Ecuador, Dominican Republic). According to Aikpokpodion et al.
(2013), a wide range of lead content (0.40 - 3.45 mg/kg) was noticed in cocoa beans grown on
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adjacent cocoa plantations in the same region of Nigeria. However, in some cocoa cultivating
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countries the soil is naturally- or as a result of pollution- rich in heavy metals, as evidenced by
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monitoring analyses (Huamani-Yupanqui et al., 2012; Kouakou et al., 2013). Comparing our
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results to those obtained by Bertoldi et al. (2016) on cocoa beans from South and Central
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America, there is a suspicion that the study materials from Ecuador and the Dominican
Republic may be contaminated. Likewise other authors determined the lead in cocoa beans of
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different geographical origin (including in Ecuador, among others) to be at a much lower level
than those obtained in this work: 0.051 mg/kg (Mounicou et al., 2003), 0.018 – 0.094 mg/kg
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On all the production lines, with the exception of factory X, the lead content in the chocolate
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mass decreased significantly as a result of the manufacturing process, close to the limit of
quantification or below that limit (Tables 4 & 5). In the final product produced by factory X,
the lead concentration was 0.279 mg/kg and differed slightly compared to the initial
concentration of 0.298 mg/kg. Even the addition of raw non-cocoa like materials, which
contain barely any lead, did not affect the content by weight. It may have become
contaminated with metal from the machines used during the various stages of production,
especially conching, when the highest decrease should occur. Factory X does indeed have a
On production line Z, a drastic decrease in the concentration of lead was noticed after the
winnowing and grinding stage (Table 5). This corroborates an earlier observation mentioned
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in the literature (Mounicou et al., 2003; Rankin et al., 2005; Yanus et al., 2014). Of all the
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microelements and toxic metals, lead is the best absorbed by the shell of cocoa beans. There
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are many applications and attempts to future use that property for reclaiming land with
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contaminated soil and the purification of different types of waste (Meunier et al., 2003, 2004).
The highest concentration of lead was determined in the chocolate produced by factory X
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(0.279 mg/kg), while other chocolate produced by factories Y and Z showed lead
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concentrations below the limit of quantification(Tables 4 & 5). These results were similar to
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the level of ranges published by Yanus et al. (2014) (0.062 – 0.145 mg/kg), Villa et al. (2014)
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(0.021 – 0.138 mg/kg), Sager (2012) (0.014 – 0.114 mg/kg), Güldaş et al. (2008) (an average
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of 0.033 mg/kg) and Wojciechowska-Mazurek et al. (2008) (an average of 0.056 mg/kg).
Other authors obtained higher levels of lead in chocolate, such as Rehman & Husnain (2012)
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(0.060 – 1.4 mg/kg) and much higher levels such as Dahiya et al. (2005) (0.236 – 8.04
mg/kg), Iwegbue (2011) (0.4 – 2.3 mg/kg) and Jalbani et al (2009) (an average of 2.48
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mg/kg). Grembecka & Szefer (2012) did not detect any lead in forty-six dark chocolate
samples.
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In this paper it was hard to find a correlation between the cocoa content in chocolate and the
content of individual heavy metals because the cocoa content in the analysed products were
quite similar: 60, 64 and 70%. Nevertheless, in the literature, it has been observed that this
relationship has linear characteristics. The designated correlation for lead in chocolates
achieved a coefficient of even 0.955 (Yanus et al., 2014; Villa et al., 2014). However, such
correlation generally occurs in chocolate with varying cocoa content, manufactured in the
same factory. On the other hand, the differences in metal concentration between
miscellaneous brands of chocolates with the same cocoa content can be very high, due to the
origin and composition of the raw materials and various production processes (Yanus et al.,
2014; Villa et al., 2014). Isotopic studies have confirmed that the high lead content often
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encountered in cocoa beans is not only due to pollution of the cultivation site, but is
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frequently the effect of transport and/or the processing of cocoa beans and then the production
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of chocolate (Rankin et al., 2005; Sager, 2012).
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3.4. The cadmium (Cd) content
Cocoa beans imported from Ecuador showed an elevated cadmium content (0.629 mg/kg)
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indicating a higher level of contamination. Cocoa beans originating from the Dominican
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Republic contained about five times less cadmium (0.128 mg/kg). Similar amounts of
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cadmium were determined in cocoa powders used by factories X and Y (Table 3). The lowest
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cadmium level was found in the cocoa masses produced by factories X and Y: 0.058 mg/kg
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and 0.098 mg/kg respectively. The report of the tenth session of the Codex Committee on
Contaminants in Foods (CCCF) dated 2016 suggested a limit of cadmium content in cocoa
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powder of 4.0 mg/kg [FAO/WHO, CX/CF 16/10/9 – Add.1., 2016]. The cocoa powders
chocolate will be limited to the level of 0.60 mg/kg fresh weight. The cocoa powders analysed
The results obtained by Sager (2012) (0.112 – 0.171 mg/kg), Yanus et al. (2014) (an average
of 0.125 mg/kg) and Güldaş et al. (2008) (an average of 0.181 mg/kg) were close to those
mentioned above for cocoa powders. Much higher amounts of cadmium, in the range of 0.094
– 1.833 mg/kg, were reported by the team led by Mounicou (2003). Grembecka & Szefer
(2012) did not detect any cadmium in twenty-four cocoa and six instant cocoa samples. In the
case of cocoa beans, various authors have recorded different cadmium contents. A relatively
low concentration, of about 0.072 mg/kg, was determined by Yanus et al. (2014). A slight
presence of cadmium (below the limit of detection, max. 0.080 mg/kg) in cocoa beans from
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three different regions of Nigeria was found by Aikpokpodion et al. (2013). Quite similar
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findings (an average of 0.372 mg/kg) to the results shown in this study were reported by
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Mounicou et al. (2003).
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All the above studies and data collected by COABISCO (Chocolate, Biscuits and
The highest amounts of cadmium in cocoa beans is observed in Latin America and the
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Caribbean, while the least contaminated crops come from West Africa (Bertoldi et al., 2016).
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Cadmium was detected in non-cocoa tested materials, both in the fats and emulgators, at a
level of below 2 pbb (Table 3). Slightly higher amounts, of from 11 up to 14 ppb, were
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During the production process, irrespective of the type of production line, the cadmium
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content in the chocolate mass decreased (Tables 4 & 5). This was contrary to the observations
of some researchers (Yanus et al., 2014), and in accordance with others results (Mounicou et
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al., 2003). In factory X, it decreased by 33% in relation to the cocoa mass after mixing step,
whereas in factory Y it decreased by only 10.5%. In factory Z, chocolate prepared using raw
materials from Ecuador contained 32% less cadmium than the cocoa beans from which it was
produced. But in the chocolate made from raw materials of Dominican origin, the reduction in
cadmium was 16.4%. As in the case of the previously discussed heavy metals, the reduction
in the concentration of cadmium on any type of production line was due to adding raw
materials such as sugar and cocoa fats which contain low traces of these metals. Moreover,
The highest concentration of cadmium was determined in the chocolate produced by factory Z
from raw material originating from Ecuador (0.429 mg/kg). Four times less cadmium was
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found in chocolate produced by the same manufacturer but made from cocoa beans imported
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from the Dominican Republic (Table 5). Chocolates manufactured by factories X and Y
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exhibited even lower contents of cadmium, 0.029 and 0.068 mg/kg, respectively. The results
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of analyses of chocolate with various cocoa masses suggest a relationship between the amount
of cocoa in chocolate products and the quantity of cadmium (Jalbani et al., 2009; Yanus et al.,
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2014). Therefore, the ninth session of the Codex Committee on Contaminants in Foods has
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proposed establishing different limits for cadmium, depending on the type of chocolate
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[FAO/WHO, CX/CF 15/9/6, 2014]. According to the proposed limit of 2.00 mg/kg for
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chocolate containing cocoa equal to or greater than 50%; all the above types of chocolate
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2019 the limit of cadmium content in chocolate (≥ 50% cocoa) of 0.80 mg/kg fresh weight
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will be obligatory for all European Union Member States. Each of the types of chocolate
produced by X, Y and Z factories which were analysed would not exceed the specified limits.
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The previously mentioned results for chocolate were in line with those obtained by other
research teams: Villa et al. (2014) (0.032 – 0.107 mg/kg), Yanus et al. (2014) (0.065 – 0.141
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mg/kg), Sager (2012) (0.025 – 0.510 mg/kg) and Güldaş et al. (2008) (an average of 0.034
mg/kg). The team led by Wojciechowska-Mazurek (2008) surveyed 266 chocolate products
which had a lower concentration of cadmium, averaging 0.013 mg/kg; 90% of the results did
not exceed 0.033 mg/kg. By contrast, higher levels of cadmium, within a wide range of from
0.010 – 2.73 mg/kg, were found by researchers in different brands of chocolate on the Indian
market (Dahiya et al., 2005). Grembecka & Szefer (2012) did not detect any cadmium in
All the chocolate samples analysed fulfilled the maximum permissible concentration
requirements which had been established in the past (1 mg/kg for Pb by FAO, withdrawn in
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2003) and mandatory in the immediate future (0.8 mg/kg for Cd by Commission Regulation
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No 488/2014, as of 1 January 2019). The guidance for industry document prepared by the
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FDA recommended a maximum lead level of 0.1 mg/kg in candy products which are likely to
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be consumed frequently by small children. Our study samples of chocolate from factory X
(60% of cocoa solids) exceeded this limit almost three times, so it is recognized as posing the
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greatest risk for young consumers among all the chocolate tested.
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The tolerable weekly intake (TWI) for Cd and Ni recommended by the EFSA is 2.5 and 19.6
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µg/kg body weight (b.w.), respectively. The provisional tolerable weekly intake (PTWI)
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proposed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) are higher:
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5.8 and 35.0 µg/kg b.w., respectively. Taking into account an average body weight of 70 kg
for adults and a consumption of 70 g of chocolate per week, the analysed chocolate did not
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Assuming a child weighs of 20 kg, the maximum contribution of cadmium was 60% of the
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EFSA TWI and 26% of the JECFA PTWI, while the median for all chocolate samples was
12% and 5%, respectively. Therefore, only the consumption of chocolate from factory Z (ZE -
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made of Ecuador cocoa beans) was a significant source of Cd for children. In the case of Ni,
represent a serious risk to children’s health because of the 64%, 80%, 79% and 37%
be considered health protective. The EFSA found the previous PTWI established as 25 µg/kg
b.w. to no longer be appropriate, as a result of the insightful assessment. However, EFSA has
not proposed any recommendations. But referring to this limit, all analysed chocolate was
considered as safe either for adults and children, contributed maximum 4% of the tolerable
intake.
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4. Conclusions
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The chocolate production described in this study, which took place in three different factories
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in central Poland shows the mechanism behind the contents of three heavy metals in cocoa
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masses. It was noted that the two steps in the process which decrease their concentration the
most, are conching, when the addition of non-cocoa ingredients induces a dilution effect, and
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winnowing, when the shell of the cocoa bean, saturated with these metals is removed. The
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analysis of the experimental data indicates that the best way to control the contamination of
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chocolate from lead, nickel and cadmium is to carefully select the raw materials and have an
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in-depth understanding of the manufacturing process within the context of the presence of
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these metals. Based on the results, it seems that factory Z has the best chocolate production
methodology. During all the steps in the manufacturing process, the heavy element content in
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the mass remained the same or fell, but it was never raised. The results of the present study
contribute new andreliable data to the food safety authorities and broaden knowledge of the
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contribution made by particular raw materials and production processes on the overall level of
markets should be continued. This study shows that contamination with heavy metals may be
the result of the manufacturing process, as was the case on the production line of factory X,
which uses old machines. Judging from this work it can be concluded that the contamination
of cocoa beans also takes place before the manufacturing process, such as during their
cultivation, pre-treatment and transportation. Furthermore, some of the cultivation areas are
More than 10 years ago it was pointed out that the concentration of lead in chocolates
is the highest among those found in food products. However, the vast majority of recent
research, as well as that undertaken in this study, indicates that the level of lead in products
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containing cocoa has decreased significantly to a relatively safe level. The data obtained in
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this study confirm this situation. However, this does not mean that a definitive solution has
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been found to the problem of lead in cocoa products. According to the results in this paper,
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nickel was the most prominent toxic metal, with the highest levels in raw cocoa materials and
chocolate of various origins. The concentration of this element in the chocolate samples of
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factories X, Y and Z creates a serious risk to the health of children who consume this type of
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sweet regularly. ZE chocolate poses a similar threat in terms of its cadmium content. Bearing
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in mind the risk assessment conducted in our study, we would like to give our support to the
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suggestion put forward by other researchers (Rehman & Husnain, 2012; Wojciechowska-
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Mazurek et al., 2008) regarding the establishment of an absolute limit for the level of
cadmium, lead and nickel concentrations in cocoa powder, chocolate and products containing
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chocolate. Such limits have already been established in the European Union for cadmium.
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Authors wish to confirm that there are no known conflicts of interest associated with
this publication and there has been no significant financial support for this work that could
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1. Adewole, E., Ogunmodede, O. T., Talabi, J., Ajayi, O. O., Oso, O. A., Lajide, L. (2011).
Physico-chemical properties of cocoa (Theobroma cacao L.) farm soil in Ikota, Ifedore,
2. Ahamed, M., & Siddiqui, M. K. J. (2007). Low level lead exposure and oxidative stress:
T
3. Aikpokpodion, P. E., Atewolara-Odule, O. C., Osobamiro, T., Oduwole, O. O., Ademola,
IP
S. M. (2013). A survey of copper, lead, cadmium and zinc residues in cocoa beans
R
obtained from selected plantations in Nigeria. Journal of Chemical and Pharmaceutical
SC
Research, 5(6), 88-98.
4. Bernard, A. (2008). Cadmium & its adverse effects on human health. The Indian Journal
fingerprinting and geographic traceability of Theobroma cacao beans and cocoa products.
M
levels for certain contaminants in foodstuffs. Official Journal of the European Union, L
PT
364, 5-24.
8. Dahiya, S., Karpe, R., Hedge, A. G., Sharma R. M. (2005). Lead, cadmium and nickel in
chocolates and candies from suburban areas of Mumbai, India. Journal of Food
hydrogenated fats and selected chocolate bars in Czech Republic. Food Chemistry, 217,
456-460.
10. Dolman, H. A., & Houwen-Claassen, A. A. M. (1999). Nickel content of carobe and
11. Duda-Chodak, A., & Błaszczyk, U. (2008). The impact of nickel on human health.
T
Journal of Elementology, 13, 685-696.
IP
12. EFSA. (2012). Cadmium dietary exposure in the European population, EFSA Journal,
R
10(1): 2551, 37 pp.
SC
13. European Commission (2004). Assessment of the dietary exposure to arsenic, cadmium,
lead and mercury of the population of the EU Member States: SCOOP, Scientific Co-
U
operation on Questions Relating to Food: task 3.2.11: final report 2004.
N
14. FAO/WHO. (2014). Proposed draft maximum levels for cadmium in chocolate and
A
Foods, New Dehli, 16-20 March 2015. CX/CF 15/9/6. Agenda Item 6.
ED
15. FAO/WHO. (2014). Standard for cocoa (cacao) mass (cocoa/ chocolate liquor) and
16. FAO/WHO. (2016). Proposed draft maximum levels for cadmium in cocoa and cocoa-
derived products. The 10th session of the Codex Committee on Contaminants in Foods,
E
CC
17. FDA. (2016). Electronic Code of Federal Regulations, Title 21, Chapter 1, Part 163-
A
Cacao Products.
18. Grembecka, M., & Szefer, P. (2012). Differentiation of confectionery products based on
some trace elements in different chocolate types produced in Turkey. Journal of Food
T
21. IARC. (2016). Agents classified by the IARC Monographs, volumes 1-117. updated 24
IP
October 2016.
R
22. Iwegbue, C. M. A. (2011). Concentrations of selected metals in candies and chocolates
SC
consumed in southern Nigeria. Food Additives and Contaminants Part B, 1(4), 22-27.
23. Jalbani, N., Kazi, T. G., Afridi, H. I., Arain, M. B. (2009). Determination of toxic metals
U
in different brand of chocolates and cadies, marketed in Pakistan. Pakistan Journal of
N
Analytical and Environmental Chemistry, 10, 48-52.
A
24. Kouakou, Y. K. N., Yao, G. F., Nangah, K. Y., Guety, T. P., Yao-Kouame, A. (2013).
M
25. Manton, W. I. (2010). Determination of the provenance of cocoa by soil protolith ages
and assessment of anthropogenic lead contamination by pb/nd and lead isotope ratios.
E
CC
26. Meunier, N., Blais, J. F., Tyagi, R. D. (2004). Removal of heavy metals from acidic soil
A
73, 225-235.
27. Meunier, N., Laroulandie, J., Blais, J. F., Tyagi, R. D. (2003). Cocoa shells for heavy
and bioavailability of cadmium and lead in cocoa powder and related products. Food
29. Pedro, N. A. R., de Oliveira, E., Cadore, S. (2006). Study of the mineral content of
30. Peixoto, R. R. A., Devesa, V., Vélez, D., Cervera, M. L., Cadore, S. (2016). Study of the
T
factors influencing the bioaccessibility of 10 elements from chocolate drink powder.
IP
Journal of Food Composition and Analysis, 48, 41-47.
R
31. Rankin, C. W., Nriagu, J. O., Aggarwal, J. K., Arowolo, T. A., Adebayo, K., Flegal, A.
SC
R. (2005). Lead Contamination in cocoa and cocoa products: isotopic evidence of global
33. Rehman, S., & Husnain, S. M. (2012). Assessment of trace metal contents in chocolate
M
11.
34. Sager, M. (2012). Chocolate and Cocoa Products as A Source of Essential Elements in
PT
35. Ščančar, J., Zuliani, T., Milačič, R. (2013). Study of nickel content in Ni-rich food
E
CC
36. Taylor, D. A. (2005). Lead in cocoa products: where does contamination come from?
A
37. Villa, J. E. L., Peixoto, R. R. A., Cadore, S. (2014). Cadmium and lead in chocolates
8763.
38. Wojciechowska-Mazurek, M., Starska, K., Brulińska-Ostrowska, E., Plewa, M., Biernat,
noxious to human health Part I. Wheat cereal products, vegetable products, confectionery
and products for infants and small children (2004 year) (in Polish). Roczniki PZH, 59(3),
251-266.
39. Yanus, R .L., Sela, H., Borojovich, E. J. C., Zakon, Y., Saphier, M., Nikolski, A.,
T
Gutflais, E., Lorber, A., Karpas, Z. (2014). Trace elements in cocoa solids and chocolate:
IP
an ICPMS study. Talanta, 119, 1-4.
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E PT
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Figure 1. Diagrammatic scheme of producer X and Y chocolate manufacturing steps
processing parameters.
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R IP
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U
N
A
M
ED
E PT
CC
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cocoa fat*
cocoa powder cocoa mass sugar
15-20% of
overall addition
MIXING
40-45oC
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ROLLING 30-50oC
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three rollers* ; five rollers#
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DRY CONCHING#
45-55oC U
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A
cocoa fat
75-80% of overall addition* aroma
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80-90oC# CBE*
and PGPR
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TEMPERING
about 30oC
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CHOCOLATE
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processing parameters.
ROASTING
150oC
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CRUSHING
immediately after roasting
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WINNOWING
U
N
A
GRINDING
granite rollers 4h
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CONCHING
sugar
60 h, 50-90oC
E PT
TEMPERING
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about 30oC
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CHOCOLATE
solidification and packing
70% cocoa solids
Figure 2. Diagrammatic scheme of producer Z chocolate manufacturing steps with processing
parameters.
Table 1. Program parameters of Hitachi Z 2700 instrument (GFAAS method)
Cadmium Lead
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wavelength [nm] 228.8 283.3
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slit width [nm] 0.2 0.2
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limit of detection [mg/kg] 0.0007 0.0004
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limit of quantification [mg/kg] 0.002 0.0013
mo [pg ] 15
U 10
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parameter/step drying pyrolysis atomization cleaning drying pyrolysis atomization cleaning
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temperature [oC] 140 550 2400 2700 100 800 2500 2700
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ramp [oC/s] 18 10 0 4 25 10 0 4
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hold [s] 40 18 10 5 60 20 10 5
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#
expressed as mean ± expanded uncertainty (U); U is obtained by multiplying combined standard
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uncertainty by coverage factor k=2 corresponding to 95% confidence level
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*expressed as mean ± standard deviation
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a
Skimmed milk powder ERM®-BD 150
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b
Soya Bean Flour INCT-SBF-4
c
ttab= 2.44 (α=0.05)
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A
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E PT
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Table 3. Element content (mg/kg) of raw materials used in chocolate production
Materials Cd Pb Ni
Cocoa beans with shell Z (E) 0.629 ± 0.067f 0.137 ± 0.030a 2.32 ± 0.45c
Cocoa beans with shell Z (D) 0.128 ± 0.031cd 0.162 ± 0.042a 7.62 ± 0.51e
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Cocoa fat X <0.002 <0.0013 0.086 ± 0.005a
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Cocoa fat Y <0.002 <0.0013 0.070 ± 0.005a
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Cocoa powder X 0.153 ± 0.015d 0.575 ± 0.014b 12.10 ± 0.13g
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Cocoa powder Y 0.174 ± 0.011e 0.155 ± 0.012a 11.70 ± 0.10f
0.002 ± 0.001a
<0.0013
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5.87 ± 0.10d
0.070 ± 0.008a
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Sugar <0.0013
X, Y, Z – producer code; (E,D) – geographical origin of cocoa beans (Ecuador, Dominican Republic)
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Different letters in columns indicate significant differences between element content (P ≤ 0.05)
E PT
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R I
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Table 4. Element content (mg/kg) of masses from production steps in X and Y production lines
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Sample taken Cd Pb Ni
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after: Producer Producer Producer
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X Y X Y X Y
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Mixing 0.043 ± 0.011ab 0.076 ± 0.013a 0.298 ± 0.020a <0.0013 4.28 ± 0.25c 5.08 ± 0.17b
Rolling 0.043 ± 0.012ab 0.074 ± 0.014a 0.318 ± 0.022a <0.0013 4.12 ± 0.15c 4.83 ±0.13b
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Dry conching - 0.068 ± 0.008a - <0.0013 - 4.83 ± 0.08b
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Addition of all
components during wet 0.030 ± 0.005a 0.066 ± 0.007a 0.290 ± 0.010a 0.014 ±0.004a 3.85 ±0.11b 4.36 ± 0.12a
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conching
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End of wet conching 0.033 ± 0.005a 0.068 ± 0.008a 0.285 ± 0.011a <0.0013 3.80 ± 0.05b 4.55 ± 0.06a
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Tempering 0.031 ± 0.006a 0.069 ±0.007a 0.281 ±0.009a <0.0013 3.47 ± 0.08a 4.40 ± 0.06a
Chocolate packaging 0.029 ± 0.005a 0.068 ± 0.007a 0.279 ± 0.010a <0.0013 3.60 ± 0.07a 4.50 ±0.06a
Different letters in columns indicate significant differences between element content (P ≤ 0.05)
Table 5. Element content (mg/kg) of masses from production steps in Z production
line
Sample Cd Pb Ni
taken after:
Cocoa bean origin
Cocoa bean origin country Cocoa bean origin country
country
Dominican Dominican Dominican
Ecuador Ecuador Ecuador
Republic Republic Republic
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0.658 ± 0.130 ± 0.155 ±
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Roasting 0.127 ± 0.028a 2.35 ± 0.39a 7.70 ± 0.61c
0.075b 0.035ab 0.047b
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0.680 ± 0.124 ± 0.152 ±
Crushing 0.123 ±0.034a 2.29 ± 0.42a 7.69 ± 0.55c
b ab
0.066 0.033 0.042b
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Grinding of 0.681 ± 0.127 ±
winnowed <0.0013 <0.0013 2.15 ± 0.30a 6.19 ± 0.27b
0.082b 0.028ab
crumbs
0.476 ± U
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Conching 0.103 ± 0.010a <0.0013 <0.0013 2.02 ± 0.10a 4.63 ± 0.13a
0.024a
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Chocolate 0.429 ± 0.030 ±
0.107 ± 0.012a <0.0013 2.07 ± 0.08a 4.43 ± 0.11a
0.026a 0.010a
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packaging
Different letters in columns indicate significant differences between element content (P ≤ 0.05)
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E PT
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A
32