Journal of Food Composition and Analysis 59 (2017) 141–144

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Journal of Food Composition and Analysis

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Short communication

Mercury content in whey protein and potential risk for human health
Leticia Fraga Matos Campos de Aquino* , Roberta de Oliveira Resende Ribeiro,
Julia Siqueira Simoes, Sérgio Borges Mano, Eliane Teixeira Mársico,
Carlos Adam Conte Junior
Departamento de Tecnologia de Alimentos, Centro Laboratorial Analítico, Universidade Federal Fluminense (UFF), Vital Brasil Filho 64, Niterói, RJ, CEP: 24230-
340, Brazil

A R T I C L E I N F O A B S T R A C T

Article history:
Received 12 December 2016 This study investigated total Mercury (THg) contamination in whey protein and was performed to
Received in revised form 14 February 2017 monitor the exposure of the Brazilian population to THg contamination in nineteen brands of whey
Accepted 23 February 2017 protein with different formulation and estimate the potential health risks of mercury exposure to
Available online 24 February 2017 humans through whey protein consumption. Results indicated that the highest Hg concentration level
was 9.41  0.295 ng g 1, while the lowest Hg level was 0.548  0.029 ng g 1. These concentrations were
Keywords: below the maximum limit for food products. However, there is no World Community regulation
Food analysis regarding mercury contamination on dairy products. The risk to human health concerning Hg ingestion
Food composition
was calculated based on consumption data in Brazil and has indicated that potential health risks related
Food safety
to exposure to total mercury from whey protein ingestion need more attention from researchers and
Hg content in food
Supplement more studies are needed, especially including specific intake of mercury from other food products that
Toxicological risk are included in a balanced diet.
Dietary intake © 2017 Elsevier Inc. All rights reserved.

1. Introduction These contaminants could potentially cause toxic effects

Whey protein (WP) is a collective term for soluble protein
fractions extracted from cheese manufacture Whey proteins are
excellent functional supplements with minimal fat, carbohydrate
and lactose (Bucci and Unlu, 2000), making them an effective
ingredient in sports nutrition supplements (Senan et al., 2016).
It is known that milk can assimilate and accumulate com-
pounds and nutrients from animal feed. Ruminants are exposed
during grazing and also by soil ingestion from surface layers
(Laurent et al., 2005). Due to growing environment pollution
caused by increase of industrial, agricultural and urban emissions,
milk and dairy products contain differing concentrations of toxic
contaminants (Ataro et al., 2008; Bilandzic et al., 2011; Maas et al.,
2011). There are many factors that could influence metals presence
in milk, and consequently, in whey protein, such as climate,
geographical location and environmental conditions. In addition,
new technologies in food production may increase the chances of
contamination of food with several environmental pollutants,
especially heavy metals (Shahbazi et al., 2016).
* Corresponding author.
E-mail address: leticiaaquino@veterinaria.med.br (L.F.M.C.d. Aquino).
counteracting the advantages of consumed whey protein. Other
concerns are the lack of regulation for mercury in dairy products,
and standardization of products such as whey protein. However,
the risk associated with dairy products consumption depends on
factors like type of ingested products, amount consumed, exposure
route, intake level, solubility, metal oxidation state, retention
percentage, length of exposure, intake frequency, absorption rate
and the efficiency of excretion mechanisms (Gaucheron, 2013).
Among contaminants, mercury (Hg) is an environmental
pollutant due to its high toxicity and persistence, even at low
concentrations (US EPA, 2003; UNEP, 2013). Food consumption has
been known to be a major source of Hg ingestion. Hg is consistently
exposed to the environment because it is present in the food chain
from both natural and anthropogenic sources (Carrasco et al.,
2011). Mercury is a key contaminant, as it can bio-accumulate and
bio-concentrate in an organism's tissues (Coelho et al., 2013).
Hence the World Health Organization (WHO) has considered this
heavy metal as one of the top ten elements of major public health
concern (WHO, 2010). The ingestion of high amounts of organic Hg
has been associated with the occurrence of several disorders, like
autism, neural tube defects and later neurocognitive development
in children, which may persist into adulthood (Al-Saleh et al., 2016;
Choi et al., 2009; Jin et al., 2016; Kern et al., 2016).

http://dx.doi.org/10.1016/j.jfca.2017.02.014
0889-1575/© 2017 Elsevier Inc. All rights reserved.
142 L.F.M.C. Aquino et al. / Journal of Food Composition and Analysis 59 (2017) 141–144
Whey protein is the most consumed supplement worldwide,
sometimes even indiscriminately. However, no information is
available regarding Hg concentrations in this matrix and exposure
level. There are two aims in this study: one is to determine the Hg
content in 19 different brands of whey protein and the other is to
assess the exposure level of populations by using the Hazard
Quotient (HQ) for Hg, on the basis of the provisional tolerable
weekly intake of Hg by a human, according to the FAO/WHO
recommendations.
2. Material and methods
2.1. Samples
Whey protein samples from 19 different brands were purchased
and kept under the conditions specified by each manufacturer. At
the laboratory, samples were weighed (weight ranged from 0.09 to
0.100 g) immediately before analysis to avoid any external
interference. It was not necessary for any previous preparation
of samples.
2.2. Analytical determination
Analytical determinations of total Hg in whey protein products
were performed by direct mercury analyzer (DMA-80; Milestone,
Sorisole, Italy). This equipment typically contains an automatic
sampler, a quartz furnace, a cobalt-manganese oxide catalyst, a
gold-coated sand amalgamator and an atomic absorption detection
cell with three different path lengths (165, 120 and 4 mm). For Hg
measurement in whey protein, all parameters of DMA-80TM are
shown in Table 1. Quartz sample boats (Milestone, DMA 8347)
were used for development. An internal thermocouple (ATC)
sensor controlled drying/decomposition temperature. For Hg
reduction, a catalyst system was employed (Milestone, DMA
8333) and for Hg vapor trapping a gold amalgamator system was
used (Milestone, DMA 8134) with an infrared temperature sensor.
A gold amalgamator was used to selectively trap and pre-
concentrate the Hg from the flow of decomposition products. An
optical cell for detection via atomic absorption spectrometry at
253.65 nm was used to quantify elemental mercury, which was
thermally desorbed. This system has a dual cell spectrophotometer
that affords an extremely wide detection range. The system
detection contains an Hg-specific lamp, a low-pressure Hg vapor
lamp, which emits light at a wavelength of 253.65 nm and a silicon
UV diode detector that was used to quantify Hg. Peak height was
used for signal evaluation.
Table 1
Parameters for measurement of mercury (Hg) in animal tissues by direct mercury
analyser (DMA-80TM).
parameter
maximum start temperature ( C)
drying temperature ( C)
drying time (s)
decomposition temperature ( C)
decomposition time (s)
amalgamator heating time (s)
signal recording time (s)
purge time (s)
cuvettes temperature ( C)
cuvette type
oxygen flow rate (mL min 1)
oxygen pressure (bar)
frequency (Hz)
room temperature ( C)
relative humidity (%)
raw sample mass (mg)
2.3. Analytical procedure
2.3.1. Optimization of analytical conditions
Starting from recommended conditions by manufacturer for
determination of Hg in milk powder, optimization of analytical
parameters for determination of Hg in whey protein was carried
out initially. A set of experiments was carried out to obtain the
analysis program for determination of Hg in whey protein. The
optimization of analysis procedure was performed with three
parameters: limit of quantification (LOQ), limit of detection (LOD)
and accuracy (Miller and Miller, 2000). Quantification and
detection limits determined for blank analysis was recommended
by 2011/836/EU Regulation. The limits were calculated using the
equations: “LOD = 3 Sa/b” (0.025 ng g 1) and “LOQ = 10 Sa/b”
(0.089 ng g 1), from calibration curve data presented in a general
form as “y = a + bx”, where “Sa” is the standard deviation of the
response “y” and “b” is the slope of calibration curve.
Method accuracy was verified by analyzing standard reference
material for whey powder (IAEA-155TM), as shown in Table 2.
Analyses were performed in triplicate. Before each weighing, boats
were washed with soap and rinsed with distilled water, dried, and
heated at 650  C for 3 min in the DMA-80. Before running each
sample in triplicate, the blind value was verified (<0.001 Hg [ng]).
2.3.2. Calibration curve
Standard Hg solutions were prepared (0.5, 1, 2, 3, 5, 10, 15, 25, 50,
150, 400 and 1000 ng g 1) by stepwise dilutions from 1000 mg L 1
of stock solution purchased from Sigma-Aldrich Brazil Ltda (São
Paulo, Brazil). These solutions were used to built two calibration
curves, one ten-point curve (0.0–15 ng g 1; y = 22.085x 0.3217;
r2 = 0.9992) and the other a five-point curve (25–1000 ng g 1;
r2 = 0.9999). In addition, confirmation tests for calibration curves
were carried out during each run, with two concentrations from
curve 1 and one concentration from curve 2.
2.4. Potential risk for human health
The hazard quotient (HQ) was calculated in order to assess the
threat of ingesting Hg through whey protein consumption, which
reflects the potential risk related to regular ingestion of
contaminants (EPA, 2000). This quotient indicates the risk level
of a certain population to a contaminant exposure. HQ is obtained
from the ratio between contaminants exposure (whey protein
ingestion rate, in this case) and the reference oral dose (RfD). If HQ
<1, there is no evident risk; if HQ >1, there may be a possibility of
harmful effects to human health. The HQ can be calculated from
the following equation (EPA, 2000):
“HQ = EF  ED  WPIR  CHg/RfD  WAB  TA”
where “EF” is the exposure frequency (260 days/year); “ED” is the
exposure duration corresponding to average lifetime (55 years);
setting “WPIR” is whey protein ingestion rate (25–65 g/person/day,
200  10 depending on the producer recommendation); “CHg” is whey
200  10 protein Hg concentration (mg g 1); “RfD” is an estimate of a safe
60
daily oral exposure (Hg = 0.1 mg kg 1 day 1; EPA, 2000); “WAB” is
650  10
120 the average body weight (70 kg), and “TA” is the average exposure
12 time for non-carcinogens (260 days/year  “ED”).
24
60 Table 2
12 The results of mercury (Hg) determination in Certified Reference Materials (CRMs).
Quartz
3–3.5 certified reference certified value found value recovery
60–80 material (ng g 1) (ng g 1)a %
60 IAEA-155 2.60  0.14 2.78  0.31 106.02
25 whey powder
60–85
0.090–0.100 a
Average of six determinations at 95% level of confidence.
 L.F.M.C. Aquino et al. / Journal of Food Composition and Analysis 59 (2017) 141–144 143

2.5. Statistical analysis

One-way analysis of variance with repeated measures was used

to identify differences among total Hg concentrations of whey
protein. When a significant F was found, additional post-hoc tests
with Bonferroni adjustment were performed. Pearson correlations
were used to examine the relationship between each physical and
chemical and total Hg concentration in whey protein. Statistical
significance was set at the 0.05 level of confidence. All analysis was
performed with SPSS version 17 (SPSS Inc., Chicago, IL). Multivari-
ate analysis on weighed principal components (PCA) was
performed on all data obtained in the experiment using XLSTAT
software version 2012.6.08 (Addinsoft, Paris, France) to identify
similarities and differences between samples.

3. Results and discussion

3.1. Total mercury in whey protein

Total mercury concentration levels (arithmetic mean, standard

Fig. 1. Principal Component Analysis (PCA) biplot of the whey protein data showing
deviation and range) obtained from DMA in different whey protein
the loading of each variable (fat, protein, carbohydrate and mercury) and the scores
brands, concentration of protein, carbohydrate and fat indicated on of each whey protein (points).
the label for each manufacturer and hazard quotient are given in
Table 3. Overall, the minimum and maximum concentration levels
were 0.548  0.029 and 9.41  0.295 ng g 1. Whey protein samples of total Hg content. Bordajandi et al. (2004) and Anastasio et al.
had protein concentrations that ranged from 67 to 92%. Fat and (2006) found values of Hg in milk between 1.40 and 3.34 ng g 1,
carbohydrate concentrations ranged from 0 to 7% and from 0 to while Pankiewicz (2012) found lower level (0.06 ng g 1). The same
11%, respectively. This substantial difference between whey researchers found levels from 0.2 to 5.6 ng g 1 in milk powder,
protein brands is probably due to different composition (ingre- cream, butter and different kinds of ewe’s cheese samples.
dients), processing methods and conditions employed during However, Hg levels found in whey proteins brands (5.01  2.69 ng
protein separation on manufacturing. When multivariate analysis g 1) were lower than those reported in other foods. For example,
was applied through principal component analysis (PCA), it was average levels for different mushroom species were 554  791 ng
possible to observe a positive correlation between fat, carbohy- g 1 (Falandysz et al., 2003, 2004); for different heat treatments in
drate and Hg, and a negative correlation between protein and Hg samples of tuna were 370  298 ng g 1 (Afonso et al., 2015); for rice
(Fig. 1). from different China regions and for fish of different species were
Mercury concentrations found in different brands used in this 47  52 ng g 1 and 56  64 ng g 1, respectively (Liang et al., 2015).
research are lower than the limit of 0.5 mg Hg/g recommended by These substantial differences in Hg concentration compared to
Brazilian regulations and the International Organization for fish dairy products are probably due to direct contact of the matrix
and seafood (ANVISA, 2013; FAO/WHO, 2010). In previous studies, with the contaminating source (mushroom – soil, rice – water, fish
milk samples and dairy products showed higher or similar values – water and food). In order for mercury to be present in milk, it
must pass not only through the digestive system, but also through
Table 3 the circulatory and secretory systems.
Total mercury (Hg) concentration levels (arithmetic mean and standard deviation)
obtained from direct mercury analyser (DMA-80TM), concentration of protein, 3.2. Potential risk for human health through whey protein ingestion
carbohydrate and fat indicated on the label by each manufacturer and the hazard
quotient (HQ) of 19 whey protein brands.
Considering whey protein consumption average for each brand,
1*
sample [Hg] ng g % protein % carbohydrate % fat HQ HQ showed that the ingestion of whey protein indicates no risk,
1 5.61  0.17a 74 7 7 0.022 with HQ < 1, being a healthy and safe food item in terms of Hg
2 6.44  0.07b 70 16 6 0.030 (Table 3). This indicates that whey proteins marketed in Brazil are
3 4.10  0.04c 79 10 2 0.037 in accordance with safety guidelines. However, having in mind the
4 5.55  0.25a 80 7 3 0.024
5 2.18  0.12d 90 0 0 0.019
high percentage of Hg in other foods and mercury bio-accumula-
6 5.52  0.15a 83 2 1 0.051 tion in the body, this HQ should not be considered in isolation.
7 9.41  0.30e 82 13 7 0.041
8 7.42  0.25f 77 5 3 0.041 4. Conclusions
9 2.02  0.11dg 84 4 0 0.007
10 1.50  0.34gh 79 10 3 0.007
11 5.23  0.03a 73 7 5 0.022 The Hg intakes from whey protein should not endanger human
12 4.46  0.25c 74 15 6 0.022 health. However, manufacturers of formulations based on dairy
13 5.70  0.34a 74 6 4 0.028 products should routinely monitor their products and carry out
14 0.55  0.03i 83 7 0 0.002 rigorous control in order to keep toxic elements at an absolute
15 9.35  0.03e 50 37 0 0.040
minimum. Based on these findings and present knowledge
16 3.85  0.12c 80 4 4 0.014
17 1.18  0.12hi 92 0 0 0.010 regarding Hg toxicity, and taking into account the non-existent
18 8.45  0.32j 67 18 5 0.047 World Community regulations regarding Hg contamination in
19 6.59  0.38b 70 10 8 0.038 dairy products, a maximum guideline concentration should be
*
Average of three determinations with standard deviation. Values with different
established for total Hg, including an additional safety factor for
letters are significantly different (p < 0.05). whey protein.
144 L.F.M.C. Aquino et al. / Journal of Food Composition and Analysis 59 (2017) 141–144
Acknowledgements
The authors are grateful for financial support from the
“Coordenação de Aperfeiçoamento de Pessoal de Nível Superior”
(CAPES) and “Fundação Carlos Chagas Filho de Amparo à Pesquisa
do Estado do Rio de Janeiro” (FAPERJ), process number E-26/
201.185/2014.
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