https://doi.org/10.52973/rcfcv-e33222
Received: 07/12/2022 Accepted: 09/01/2023 Published: 21/02/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33222, 1 – 7
ABSTRACT
The current study was undertaken to determine the 17-β Estradiol
(E2) and Progesterone (P4) levels in different milk products and
some baby follow-on milk samples collected from retail markets
in Türkiye. For this purpose, a total of 50 samples from 8 different
product groups with different fat levels, production technologies,
and production series were analyzed for natural P4 and E2 levels
using a commercial ELISA kit. The highest E2 level was determined in
UHT whole milk (3%) (39.20 ± 6.73 pmol·L
-1
) while the lowest level was
found in buttermilk (26.50 ± 1.85 pmol·L
-1
) samples. For P4 levels, the
highest concentration were found in baby follow-on milk (<1 year old)
(45.83 ± 2.95 nmol·L
-1
) and the lowest values were found in pasteurized
milk samples (23.00 ± 6.66 nmol·L
-1
). Also, in this study, differences
among the product groups for E2 and P4 were not found statistically
(P>0.05). In conclusion, the natural P4 and E2 were detected in all milk
and baby follow-on milks at various levels and generally their levels
associated with the product fat level.
Key words: Progesterone; estradiol 17-β; milk, baby follow-on milk;
ELISA
RESUMEN
El estudio actual se llevó a cabo para determinar los niveles de 17-β
estradiol (E2) y progesterona (P4) en diferentes productos lácteos y en
algunas muestras de leche de seguimiento para bebés recolectadas
en mercados minoristas de Turquía. Para ello, se analizaron un total
de 50 muestras de 8 grupos de productos diferentes con diferentes
niveles de grasa, tecnologías de producción y series de producción
para determinar los niveles naturales de P4 y E2 utilizando un kit ELISA
comercial. El nivel más alto de E2 se determinó en leche entera UHT
(3%) (39,20 ± 6,73 pmol·L
-1
), mientras que el nivel más bajo se encontró
en muestras de suero de leche (26,50 ± 1,85 pmol·L
-1
). Para los niveles
de P4, la concentración más alta se encontró en leche de continuación
para bebés (<1 año) (45,83 ± 2,95 nmol·L
-1
) y los valores más bajos se
encontraron en muestras de leche pasteurizada (23,00 ± 6,66 nmol·L
-1
).
Además, en este estudio, no se encontraron estadísticamente diferencias
entre los grupos de productos para E2 y P4 (P>0.05). En conclusión, los
niveles naturales de P4 y E2 se detectaron en todas las leches y leches
de continuación para bebés en varios niveles y, en general, sus niveles
se asociaron con el nivel de grasa del producto.
Palabras clave: Progesterona; estradiol 17-β; leche; leche de
continuación para bebés; ELISA
Determination of the levels of 17-β Estradiol and Progesterone in Cow milk
and Baby Follow-on milk by ELISA
Determinación de los niveles de 17-β estradiol y progesterona en leche de vaca y leche de
continuación para bebés mediante ELISA
Pınar Şeker
1
, Ali Rişvanlı
2,3
, İbrahim Şeker
4
* and Mehmet Çalıcıoğlu
5
1
Elazig Provincial Directorate of Agriculture and Forestry, Food and Feed Branch, Elazığ, Türkiye.
2
Kyrgyz-Turkish Manas University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology. Bishkek, Kyrgyzstan.
3
Firat University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology. Elazığ, Türkiye.
4
Fırat University, Faculty of Veterinary Medicine, Department of Zootechny, Elazığ, Türkiye.
5
Firat University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology. Elazig, Türkiye.
*Corresponding author: iseker52@gmail.com
17-β Estradiol and Progesterone Levels in Milk and Baby Follow-On Milk / Şeker et al. _____________________________________________
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INTRODUCTION
Cow milk is a food that looks simple but quite a complex food in
composition. Some of the chemicals that it contains are hormones.
The presence of 18 different hormones has been identified in
cow milk thus far. Hormones such as Progesterone (P4), α and β
Estradiol, Estrone, α and β Testosterone, Dehydroepiandrosterone
and Androstenedione can be given as examples for steroid hormones
[12]. The steroid hormones, P4, and Estradiol (E2) are quite stable
and preserve their structure during the processing stages of milk.
When consumed, they are absorbed and enter to the blood stream
of the consumer and may affect their cells. However, hormones
in protein structure can lose their function through denaturation
by pasteurization, sterilization, or other processing methods [38].
Hormones like Parathyroid hormone are reported to be found in similar
rates in fresh and pasteurized milk. The effects of pasteurization and
Ultra High Temperature (UHT) techniques were variable on hormones
depending on their structure [25].
The most prevalent source of endogenous steroid hormones are
pregnant cows. In an economically sustainable dairy farming, it is
recommended that the cows become pregnant at the postpartum 2
nd
and 3
rd
months and continue producing milk for human consumption
until 2 months before delivery. This means that the milk is obtained
for approximately 7 months during the cows’ pregnancy. This is
the conventional type of modern dairy cow farming in the world.
Progesterone, known as the pregnancy – hormone, found in the plasma
of cows, and passes into milk at levels of 2–33 nanograms·microliters
-1
(ng·µL
-1
) (3.3–42.27 nanomol·liters
-1
(nmol·L
-1
)) [16]. Its level decreases
1–2 weeks (wk) before delivery. During this time, colostrogenesis,
production of colostrum, occurs. The level of estrogen hormone begins
to increase at the second trimester of pregnancy or at 4–5 months of
pregnancy and prepares the genital organs for delivery. Consequently,
during 10–months of lactation, the cow milk includes high levels of P4
during 7 months and high levels of both P4 and estrogen hormones
during 4 months. Pape–Zambito et al. [35] reported that 17β–estradiol
(E2) levels in the milk samples taken from 1
st
, 2
nd
, and 3
rd
third from the
same cows were 5.4 ng·mL
-1
(6.92 nmol·L
-1
), 16.2 ng·mL
-1
(20.75 nmol·L
-1
),
and 39.4 ng·mL
-1
(50.47 nmol·L
-1
), respectively.
P4 is secreted from the corpus luteum (CL), placenta and the
adrenal glands. The blood concentration varies depending on species,
pregnancy, sexual cycle, and various disorders [26, 49]. Regal et al.
[39] determined the steroid hormone levels in raw milk obtained
from pregnant and non-pregnant animals. While the level of P4 was
2.86 nmol·L
-1
in pregnant ones, level of P4 was reported to be 0.31
nmol·L
-1
in the milk of non-pregnant cows. It has been reported that
the amount of steroids such as P4, which has lipophilic properties,
varies according to the fat level of milk and dairy products [21, 29].
A limited number of studies is available investigating the effects
of steroid hormones of milk origin. The available studies are mostly
about E2. The reason for this is that E2 plays a role in hormone-
dependent cancers, mainly breast cancer. E2 is in the list of Group 1
carcinogens prepared by the International Agency for Research on
Cancer (WHO-IARC).
Concerns about the effects of steroid hormones in milk are divided
into 2 groups as cancer and reproductive system-related. It has been
reported that increased consumption of animal-derived food may have
adverse effects on the development of hormone-dependent cancer
in humans. It has been claimed that drinking the milk of pregnant
cows, known to contain signicantly high P4 levels, or eating products
derived from this milk may increase the hormone-related health risks
[18]. Farlow et al. [13] reported that the risk of ovarian cancer in women
consuming more than 4 glasses of milk or equivalent dairy products
per day was 100% higher than those who consumed 2 glasses. Gao
et al. [19], on the other hand, reported that consumption of 3 glasses
per day (600 mL milk = yogurt, = 130 grams (g) cheese, 15 g = butter)
increased the risk of prostate cancer in men by 39%.
There are data available reporting that early puberty, acne and
especially breast cancer can be seen in humans who consume foods
obtained from synthetic or natural P4-treated animals, and that
the rate of such diseases may increase in men [2, 5, 13, 27, 28, 32].
However, there are also publications reporting that P4-containing
milk does not cause such effects [41]. The possible side effects on
human health have recently been mentioned, especially since milk and
dairy products obtained from pregnant cows contain high natural P4.
In the study of Maruyama et al. [31] men, women and children
were given 600 mL of milk from cows known to be at an advanced
stage of pregnancy, and urine and blood samples were obtained at
15, 30, 45, 60, 90 and 120 minutes (min) and it was observed that the
estrone and P4 levels increased and the luteinizing hormone (LH),
follicle stimulating hormone (FSH) and Testosterone concentrations
decreased in males. In the same study, it was revealed that the
Estrone, E2, Estriol and Pregnandiol concentrations increased in
the urine samples taken from men, women, and children.
Aksglaede et al. [4] stated that due to the fact that the amount of
endogenous steroid hormone in children is very low, children may
be very sensitive even to steroids taken with food. As a result, it has
been reported that girls have an increased risk of early puberty and
developing breast cancer at an advanced age, and that boys have a
risk of a decrease in Sertoli cells and semen quality in advanced age
as a result of FSH suppression, especially by E2, in addition to a risk
of diabetes and obesity in all children.
In a study conducted to determine the daily milk and dairy products
consumption habits of Iranian people and the levels of steroid hormones
they take into their bodies using the ELISA method, the mean E2 and P4
levels in milk samples were reported to be 330.5 ± 190.2 picograms·mL
-1
(pg·mL
-1
) (1.953 ± 1.12 nmol·L
-1
) and 3.57 ± 2.47 ng·mL
-1
(0.12 ± 0.09 nmol·L
-1
),
respectively. However, it was stated that milk consumption was within the
reported range (175–240 mL·day
-1
), and the content of steroid hormones
detected in milk samples could be considered safe in children and adults.
Due to the effects of steroid hormones, especially E2, regular monitoring
of these hormones in milk and dairy products has been recommended
in Iran due to its effect in the etiology of various cancers [33].
Some researchers reported their concerns about that E2 exposure
could lead to health risks due to the facts that most of the dairy
products are made from cow milk and E2 is a quite stable molecule.
In addition, compared to the past, milk, baby follow-on milks and
dairy food consumption is higher today. Therefore, it is important to
carry out surveys for the levels of steroid hormones in dairy products
[33]. In the light of the information provided above, the objective of
the present study was to determine the natural E2 and P4 hormone
levels in milk and infant follow-up milk samples in Türkiye.
MATERIALS AND METHODS
Sample collection
In this study, a total of 50 samples of cow's milk (semi-skimmed UHT
milk), 3% fat UHT milk, organic UHT milk, pasteurized milk, lactose-free
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UHT milk, buttermilk) and baby follow-on milk (baby follow-on milk (under
1 year old), infant follow-on milk (1 year old)), with different fat levels and
properties belonging to 8 product groups, from 2 different production
series produced by various companies in Türkiye and collected from
retail points were used. Information on the samples collected within
the scope of the research has been presented in TABLE I.
The research was approved by Fırat University Animal Experiments
Local Ethics Committee (case number: 2017/02-20).
The preparation of samples of baby follow-on milks for ELISA was
carried out according to the manual containing sample preparation
information for ELISA in solid samples (samples preparation
procedure for ELISA of SunRed).
ELISA
Natural P4 and E2 levels in the collected samples were determined
in the ELISA reader (Bio Tek Instruments, USA) at 450 nanometers
(nm) wavelength using the commercial ELISA kit. In the analysis, a
plate (plate of 96 samples for each hormone) produced and veried
by the relevant company in accordance with the standards for P4 and
estrogen analysis was used (Sundred E2 kit catalogue no: 201–12–1009,
progesterone kit catalogue no: 20112–1008 Shanghai, China) [34, 44].
Hormone levels in milk and milk products were analyzed according
to the procedures by the kit manufacturer (SunRed).
Statistical analyses
In the study, rst, the descriptive statistics of natural E2 and P4
hormones were calculated in the product groups. The Kruskall Wallis
analysis of variance was then used for comparisons between product
groups in terms of natural estradiol E2 and P4 hormone levels. The
Mann Whitney U test was then used in the follow-up for the parameters
that were found to be important as a result of these analyses [3, 10,
46]. These analyses were carried out using the SPSS program [45].
RESULTS AND DISCUSSION
The descriptive statistics of natural E2 and P4 hormone levels in
cow's milk and infant follow-on milk have been presented in TABLE II.
The highest concentration for natural E2 hormone levels in the
product groups was determined as 39.20 ± 6.73 pmol·L
-1
in UHT Whole
Milk (3%) and the lowest value was found as 26.50 ± 1.85 pmol·L
-1
in
buttermilk (TABLE II).
The highest concentration for P4 levels in the product groups were
found in Baby Follow-On Milk (<1 year old) (45.83 ± 2.95 pmol·L
-1
) and
the lowest value were found in Pasteurized Milk (23.00 ± 6.66 nmol·L
-1
)
(TABLE II). Also, in this study, differences among the product groups
for E2 and P4 were not found statistically (P>0.05) (TABLE II).
TABLE I
Sample information of the milk and baby follow-on
milk analyzed in the current study
No Dairy Foods
Numbers of
commercial
brands
Numbers
of samples
collected
1
UHT Half-Fat Milk (1.5%) 3 6
2
UHT Whole Milk (3%) 3 6
3
UHT Organic Milk 2 4
4
Pasteurized Milk 3 6
5
UHT Lactose-free milk 3 6
6
Buttermilk 3 6
7
Infant Follow-On Milk (>1 year old) 5 10
8
Baby Follow-On Milk (<1 year old) 3 6
Total
25 50
Sample preparation
The collected samples were stored in deep freeze (Bosch, GSD2111/01
FD6811, Robert Bosch Hausgeröte, Germany) at -18°C until the time of
analysis. When using liquid products such as milk and buttermilk after
they are being thawed and diluted with a standard diluent at a ratio of
1/10. Then, the resulting suspension was centrifugated (Hettich Rotox
32A, Germany) at 1008 Relative Centrifugal Force (RCF) or G-force for
10 min abd the supernatant was removed.
TABLE II
Natural E2 and P4 levels in milk and infant follow-on milk
Dairy Foods n
E2 (pmol·L
-1
) P4 (nmol·L
-1
)
X
̄
± SEM Median X
̄
± SEM Median
UHT Half-Fat Milk(1.5%)
6 36.33 ± 6.12 38.00 25.00 ± 10.28 20.00
UHT Whole Milk (3%)
6 39.20 ± 6.73 37.00 40.80 ± 7.45 50.00
UHT Organic Milk
4 31.67 ± 2.73 30.00 27.00 ± 6.11 31.00
Pasteurized Milk
6 29.50 ± 2.87 27.00 23.00 ± 6.66 19.00
UHT Lactose-free milk
6 37.40 ± 4.85 38.00 37.17 ± 4.89 38.50
Buttermilk
6 26.50 ± 1.85 25.00 24.20 ± 4.51 20.00
Infant Follow-On Milk (>1 year old)
10 32.19 ± 2.29 33.50 39.57 ± 4.52 42.00
Baby Follow-On Milk (<1 year old)
6 32.00 ± 4.62 32.00 45.83 ± 2.95 49.00
ns ns
SEM: Standard error of the mean. ns: Not signicant (
P>0.05)
17-β Estradiol and Progesterone Levels in Milk and Baby Follow-On Milk / Şeker et al. _____________________________________________
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Milk and dairy products have an important role in human nutrition.
Worldwide, cow-derived milk or dairy products are consumed more
frequently than milk or dairy products derived from other species.
Although there are many studies on the components of cow's milk,
the steroid hormones in dairy foods have not been much emphasized.
However, there are previous publications stating that the steroid
hormones carried by milk and dairy products from cows may have
negative effects on human health [14]. There are data showing that
steroid hormones found naturally in milk and dairy products have effects
ranging from accelerating body growth [6] to carcinogenic properties
[47] in humans and animals. There are studies demonstrating that there
is a very strong relationship between high consumption of milk and dairy
products and a high incidence of testicular and prostate cancer [17].
Concentration of P4 and E2 in milk and milk products were determined
in several studies using different analysis methods. These; in a study,
the P4 levels in skimmed milk, buttermilk, skimmed milk powder, and
regular milk were reported to be 1.4, 6, 17, and 12 ng·mL
-1
or ng·g
-1
(1.79,
7.69, 21.77, and 15.37 nmol·L
-1
), respectively [23]. In another study, the
P4 level in milk was determined as 9.81 ng·mL
-1
or ng·g
-1
(12.57 nmol·L
-1
)
[21]. Also, Snoj et al. [42] found that mean P4 concentration in UHT
3.5% milk (10.76 ± 0.43 ng·mL
-1
) were signicantly higher than in UHT
0.5% milk (7.06 ± 0.26 ng·mL
-1
). In addition, previous studies have
shown that P4 is also signicantly associated with the percentage
of fat in milk (r = 0.98) [20]. However, the relationship between milk
fat percentage and P4 is less than that of E2.
Previous studies report that approximately 60-80% of estrogens in
human diets come from milk and dairy products [40]. In a study, the
E2 level in milk was determined as 0.02 ng·mL
-1
[21]. The distribution
of estrogens, especially E2, in the fat or fat-free portions of milk is
still controversial. Studies have shown that there is no difference in
E2 concentrations in whole milk or composite skimmed milk [1, 30].
E2 concentrations in milk correlate with the percentage of milk fat and
are found in higher concentrations in the fat fraction of milk [43, 50].
The level of E2, which is related to the fat ratio of milk, comes out as a
result of the removal of most of the fat during production, causing the
E2 concentrations measured in skimmed milk to differ from estradiol
in whole milk. Pape-Zambito et al. [36] reported that E2 concentration
in raw whole milk averaged 1.4 ± 0.2 pg·mL
-1
of milk (mean ± SEM) and
ranged from non detectable to 22.9 pg·mL
-1
of milk. Snoj et al. [42]
found that mean E2 concentration in UHT 3.5% milk (25.37 ± 1.15 pg·mL
-1
)
were signicantly higher than in UHT 0.5% milk (19.38 ± 0.79 pg·mL
-1
). In
the same study, it was suggested that there were signicant positive
correlations between hormone concentrations and milk fat content,
and that high E2 and P4 concentrations indicated that most of the
milk in the examined commercial milks came from pregnant cows.
Generally, the P4 and E2 levels found in this study are higher than
those reported by Vicini et al. [48] and Pape-Zambito et al. [37].
Potential explanations for these differences may cover the differences
in analysis methods and limited sample size used in the current study as
well as biological factors such as pregnancy period, sexual cycle period,
genetics, mastitis, the animal's diet, and the presence of transport
proteins. For this reason, it has been suggested that the concentration
of E2 in milk and dairy products varies and is not well dened [36].
Judging the level of steroid hormones in food whether it is safe to
consume is complicated. To the present knowledge, there is no toxic
or harmful level, such as Maximum Residue Limits (MRL) for the steroid
hormones determined for any type of food. However, there are the
acceptable daily intake (ADI) level, Non-observed effect level (NOEL),
and lowest-observed effect level established by the Joint Food and
Agriculture Organization of the United Nations (FAO) and World Health
Organization (WHO) Expert Committee on Food Additives (JECFA) [24].
Among those, NOEL is particularly worthy because it is accepted as the
maximum allowed daily intake with no harmful effect for humans. NOEL
values for adults are 3.3 mg· kilograms (kg)
-1
bw per day for P4, and 5
micrograms (µg)·kg
-1
bw per day for βE2 [24]. However, Food and Drug
Administration (FDA) exhibited a different approach on evaluating the
risk from steroid hormones [15]. Simply, daily endogenous production
of P4, Testosterone, and βE2 were calculated and a permitted increase
exposure has been established, which is 1% of the daily production.
For instance, daily production and permitted increase exposure were
reported as 150 µg and 15 µg for P4 and 6 µg and 0,06 µg for βE2.
According to FDA, it can be said that total daily intake of these steroid
hormones should not exceed the permitted increase exposure, which
are too low compared to NOEL values of JECFA [24]. This inconsistency
in the information may make interpreting the results of steroid hormone
levels determined in foods dicult.
Nili-Ahmadabadi et al. [33] carried out a health risk assessment
using the data they obtained on the estimated daily steroid hormone
intake, average body weight of child and adult consumers, the amount
of milk consumed and the level of steroid hormones in milk with the
help of a formula. As a result, it was stated that the levels of steroid
hormones taken with milk and dairy products were close to the levels
found in the human body. However, in the study, it was reported that
due to the hormone levels of some products, attention should be paid
to their intense consumption.
In the present study, the highest level of E2 was found in UHT Whole
Milk (3%) (39.20 ± 6.73 pmol·L
-1
) and the P4 level was found to be highest
in Baby Follow-On Milk (<1 year old) (45.83±2.95 nmol·L
-1
) (TABLE II).
It was concluded that this situation was due to the fact that the
levels of estradiol and P4 in milk and baby Follow-On Milks changed
depending on different factors other than the fat ratio. In generally,
according to the results of this study, the hypothesis that P4 and
estrogen can be expected in higher frequency and intensity in
high-fat dairy products due to their lipophilic properties has been
proven. However, the differences between the product groups for
E2 concentrations were not signicant (P>0.05).
Although the ELISA method is a successful method in the analysis of
steroid hormones in blood plasma, its use in steroid hormone analysis
in foods is very rare. No study has been found on the determination
of steroid hormones by ELISA method in dairy products, except
milk samples. In this study, ELISA method was used for the rst
time in buttermilk, and Baby Follow-On Milks not milk only, for this
purpose. It is also reported that the ELISA method is useful as it is
inexpensive compared to the advanced methods, does not require a
well-equipped laboratory, yields fast results, and can analyze more
samples at the same time [22]. However, conrmation of results with
more advanced analysis methods could have been better in terms of
the reliability of the results.
Another issue that should be discussed in the current study is the
sample size. It would have been denitely more appropriate to have
a higher sample numbers than it was in the current study. Although
an increased sample size increases the power of the population
proportionally, the increased sample size also increases the time
spent for the research and costs [10, 46]. The most proper sample
size varies according to the objectives of the study and limiting factors
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33222, 1 – 7
5 of 7
(time, cost, number of staff, and others). The sample size used in this
study is considered as a short coming of the study.
Infant formulas have special functions in infant diets as they are the
main and unique source of nutrients during the rst months of life.
These products are within the scope of Commission Directive 2006/141/
EC, in particular, as amended by the Commission Directive 2013/46/EU
[11]. Dairy preparations for infants and children are recognized as an
important source of natural hormones, despite available information
on their concentration in these products being very limited [9, 51].
Hence, it is proposed that more of these hormonal compounds should
be investigated in different products in order to make an estimation
of their consumption and make a comparison with their level in breast
milk [7]. Barreiro et al. [8] reported that there was no reference level or
legal limit for hormones in infant formulas, that there is no obligation
to declare hormone values on product labels, and accordingly, no legal
conclusions could be drawn in terms of food safety.
In the presented study, the concentrations of E2 and P4 in the
follow-on milk of infants over 1 year of age were 32.19 ± 2.29 (pmol·L
-1
)
and 39.57 ± 4.52 (nmol·L
-1
), respectively, and 32.00 ± 4.62 (pmol·L
-1
) and
45.83 ± 2.95 (nmol·L
-1
) in the follow-on milk of infants under 1-year-old
and these rates were found to be within acceptable limits and not
different from other milk and dairy products. Although the importance
of these data for infant health and development is not fully known
today, this study has once again conrmed that formulas are a source
of steroid hormones.
The differences between the E2 and P4 levels in milk, buttermilk,
and Baby Follow-On Milks between the current study and previous
studies can be explained by multiple factors ranging from the
differences in analysis methods and sample numbers to farm factors
including nutrition and care conditions. Also, the breeds of the cows
and lactation periods may cause by these different. The question
of whether these levels of P4 and E2 may pose a health risk to
the consumers does not have a simple answer and requires a risk
assessment based on the exposure level.
CONCLUSIONS
In this study, it was conrmed by the ELISA method that P4 and
E2 are naturally present at various levels in whole milk, buttermilk
and baby follow-on milk, and It was also concluded that, in general,
there appears to be a correlation between the concentrations of
these hormones and the fat content of each product but may also
be affected by other factors. Also, more has been determined that
the levels of these hormones in the products in question are not at
the levels that may be harmful to human health as stated in previous
publications.
Conict of Interest
The authors declare that they have no conicts of interest in the
research.
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