Journal of Food Composition and Analysis 104 (2021) 104159

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Health risk assessment of toxic metals and toxigenic fungi in commercial

herbal tea samples from Belgrade, Serbia
Marko Krstić a, Miloš Stupar b, Danijela Đukić-Ćosić c, *, Katarina Baralić c,
Svetlana Đogo Mračević a
a
Department of Analytical Chemistry, University of Belgrade — Faculty of Pharmacy, Vojvode Stepe 450, 11221, Belgrade, Serbia
b
Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade, Faculty of Biology, Studentski trg 16, 11000, Belgrade, Serbia
c
Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade — Faculty of Pharmacy, Vojvode Stepe 450, 11221, Belgrade, Serbia

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

Keywords: This study aimed to: (i) determine the content of toxic metals and toxigenic fungi in 14 commercial herbal tea
food analysis samples from the market of Belgrade, Serbia; and (ii) assess their risk to human health. After the microwave
food composition digestion (HNO3/H2O2, 7:1,v/v), toxic metal content was determined by graphite furnace atomic absorption
toxic metals
spectroscopy (GF-AAS). The health risk as a result of Cd, Pb, As, Mn, Ni, and Cr exposure via herbal tea intake
toxigenic molds
risk assessment
was assessed through estimated daily intake (EDI), target hazard quotient (THQ), and hazard index (HI). Fungi
isolated from the tested herbal tea samples were identified on the basis of colony morphology and microscopic
characteristics of reproductive structures. Toxic metals concentration in herbal tea samples for Cd, Pb, As, Mn, Ni
and Cr ranged 0.04-0.93, 0.09-2.54, 0.03-0.77, 1.82-651.04, 0.97-9.01, 0.49-3.47 mg/kg of dried plant material,
respectively. Values of THQ for investigated toxic metals and HI were below 1, indicating the absence of human
health risk. All 14 samples of the tested herbal teas were contaminated with fungi, while total count of cultivable
fungi in each sample did not exceed national standards (105 CFU/g). Presence of 23 morphologically different
isolates suggested moderate fungal diversity of the samples, while the highest isolation frequency (IF) of 100%
was documented for Penicillium spp. and Aspergillus spp. The obtained risk assessment results suggest that all the
tested samples are safe for human health regarding the toxic metal and fungal content and provide an important
basis for understanding the potential risks of toxic metals and toxigenic fungi intake via commercially available
herbal teas.

1. Introduction Tibetan, African or Amazonian) (Kosalec et al., 2009). The so-called

“real” tea (Camellia sinensis), as well as the herbal infusions of medical
It is believed that healthy eating and drinking habits protect from a plants, are generally rich in biologically active compounds, such as
variety of diseases. In that sense, phytotherapy, based on a very long polyphenols, flavonoids, alkaloids, enzymes, minerals, proteins, amino
tradition and common in the history of mankind, plays a significant role acids, aroma-forming substance, vitamins, fibers, volatile oils, purines,
(Kalny et al., 2007). Nowadays, herbal teas are considered very popular carbohydrates, etc. (Aboulwafa et al., 2019) and contain a variety of
refreshing drinks, which are also consumed because of their healing minerals and trace elements (Popović et al., 2017). Furthermore, many
properties (Poswal et al., 2019). Hot water infusions of medical plants beneficial properties of tea and other herbal infusions of medical plants,
have been used as drinks for centuries in the Far East and Mediterranean such as antioxidative, anticarcinogenic, antimicrobial and antidiabetic
countries, mostly because of their taste and flavors (Kosalec et al., 2009; potential, have been proven in vitro (Farzaneh and Carvalho, 2015).
Ravikumar, 2018; Poswal et al., 2019). Considering that many of them However, in spite of the widespread usage and accepted opinion that
are believed to have medical properties, they are also used as natural natural herbal products are not harmful, their inappropriate use can lead
remedies to treat minor disturbances and illnesses. Thus, many of them to intoxication (Ernst, 2004, 2006). Pesticides, radioactive particles,
were a part of traditional medicines (e.g. Ayurveda, Unani, Chinese, toxic metals and mycotoxins can be found among the numerous

* Corresponding author at: University of Belgrade - Faculty of Pharmacy, Vojvode Stepe 450, 11221, Belgrade, Serbia.
E-mail addresses: [email protected] (M. Krstić), [email protected] (M. Stupar), [email protected] (D. Đukić-Ćosić),
[email protected] (K. Baralić), [email protected] (S.Đ. Mračević).

https://doi.org/10.1016/j.jfca.2021.104159
Received 16 May 2021; Received in revised form 31 August 2021; Accepted 1 September 2021
Available online 3 September 2021
0889-1575/© 2021 Elsevier Inc. All rights reserved.
M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

environmental pollutants potentially present in dry plant material of the present multidisciplinary research were to: (i) determine the
(Kosalec et al., 2009). Many metals are minerals/trace elements, concentrations of toxic metals (Cd, Pb, As, Mn, Ni, and Cr) in commer­
essential for human health due to their role in metabolic processes cial herbal tea samples; (ii) assess the potential human health risks of
(Razic and Kuntic, 2013). However, it must not be neglected that some these 6 metals in herbal tea infusions; (iii) identify fungi in commercial
of them could also be toxic. Lead (Pb), mercury (Hg) and cadmium (Cd) tea samples, and (iv) estimate the presence of toxigenic moulds in the
are the most important toxic metal pollutants which can be detected in herbal tea samples and their adverse effect on human health.
medical plants. It has also been shown that toxic metals are prone to
bioaccumulation, and, thus, might provoke a variety of negative effects 2. Material and methods
on the immune, nervous, and reproductive system (Razic and Kuntic,
2013; Popović et al., 2017). Toxic metals are naturally present in the 2.1. Samples
stone substrata and, carried by the water flow, they might reach into the
soil. Moreover, anthropogenic activities such as industry, mining and All tested tea samples (Camellia sinensis and herbal infusions of the
increased application of fertilizers can be viewed as a cause of the higher selected medical plants) used in this study were commercially available
content of toxic metals in the air, water and soil. Additionally, plants and collected from markets, healthy food stores and supermarkets in
mainly absorb toxic metals through root system from soil, and via foliar Belgrade, Republic of Serbia. Samples were bought either as filter or
organs from atmosphere, and store them in the plant tissue (Caldas and bulk products. Three samples of each commercial herbal tea sample
Machado, 2004; Azevedo and Lea, 2005). were obtained and analyzed, taking into account the possible biological
Apart from toxic metals, herbal teas can also be contaminated, pri­ variability, geographical origin, climatic conditions, time and year of
mary or secondary, with microbes (Nogueira Carogio et al., 2015; harvest, etc. A total of 14 tested herbal teas, along with their English and
Dayananda et al., 2017). binomial names are listed in the Table 1.
Water, air and soil are the sources for primary microbial contami­
nations of plants in the field. Improper production processes (drying,
2.2. Toxic metal analysis
packaging and storage), as well as inadequate distribution of herbal
products increase the possibility of secondary microbial contaminations
2.2.1. Chemicals and reagents
(Nogueira Carogio et al., 2015). According to Dayananda et al. (2017),
All solutions were prepared using the analytical grade reagents and
during the tea production process, microbial contaminations can be
deionized water with resistivity of 18.2 MΩ cm− 1 obtained by a Milli-Q
much higher if the processing steps are conducted with unwashed fresh
system (Millipore, Bedford, Massachusetts, USA). Nitric acid (65%)
tea leaves. Among all of the microorganisms that could colonize aerial
suprapure quality (HNO3, G.R., Lach-ners.r.o., Naratovice, Czech Re­
plant parts, presence of the filamentous fungi, potential mycotoxin
public) and H2O2 30% solution (Macron Fine Chemicals, Avantor Per­
producers, must not be overlooked. In the literature, these fungi are
formance Materials, Gliwice, Poland) were used for sample digestion.
often referred to as “toxigenic moulds”. They are ubiquitously distrib­
The accuracy of analyses was validated with standard reference material
uted in the nature. Their spores are always present in the indoor and
Apple Leaves 1515 standard reference material (Sigma-Aldrich, St.
outdoor air, while their concentration depends on the environmental
Louis, Missouri, United States). Quantification of Cd, Pb, As, Mn, Ni, and
factors (Kasprzyk, 2008). Under the appropriate growth conditions
Cr was carried out using an external standard method. Calibration was
(temperature, humidity, nutrients), spores and other viable fungal
performed using multielement standard solution 1 g/L (Merck, Darm­
propagules could germinate and colonize available substrata,
stadt, Germany) in diluted nitric acid.
comprising of not only living plants in the fields, but also commodities,
including cereals, oil seeds, nuts, herbs, and spices (Škrinjar et al.,
2.2.2. Sample preparation
2011). During the secondary metabolism of fungi, mycotoxines are
Microwave digestion method (CEM, Matt hews, North Carolina,
biosynthesized and extracted from mycelium into the sub­
USA) was used for the preparation of the herbal tea samples for toxic
strata–extrolites (Richard, 2007). Many mycotoxines are mutagenic,
metal determination. Each tea sample (0.25 g) was put into quartz
teratogenic, cytotoxic, while some of them, such as aflatoxines (Group I
vessels with the addition of 7 mL HNO3 and 1 mL of H2O2. The condi­
carcinogens) and ochratoxines (Group 2B carcinogens), have been
tions for sample mineralization were set by adjusting the digestion
classified as carcinogens by the International Agency for Research on
program parameter as follows: (I) heating until the temperature reaches
Cancer (IARC). Hence, their presence, along with the presence of toxi­
genic moulds in tea and herbal infusions, represents a real hazard to
Table 1
human health. Additionally, many moulds are opportunistic pathogens,
Commercial herbal tea samples for toxic metals and toxigenic fungi analysis.
which, if found in herbal beverages, can cause infection and pose a
threat to immunosuppressed patients (Kosalec et al., 2009). Therefore, it Names
Sample Family
is essential that these products are microbiologically safe. In that English/Common Binomial
context, the microbiological control of tea and other herbal products 1. Peppermintf,* Mentha piperitaL.
must be highlighted, with the main purpose to analyze the contamina­ 2. Basilb,* Ocimumbasilicum L.
tion by filamentous fungi (Nogueira Carogio et al., 2015). World Health 3. Thymef,* Thymus vulgaris L. Lamiaceae
Organization (WHO) contaminants guidelines propose that contamina­ 4. Sagef,* Salvia officinalis L.
5. Winter savoryf,* SaturejamontanaL.
tion should be avoided and controlled by implementing quality assur­
6. Chamomile f,* MatricariachamomillaL.
ance measures, such as good agricultural and collection practices 7. Yarrowf,* Achileamillefolium L.
Asteraceae
(GACP) (in the case of herbal plants), as well as good manufacturing 8. Green teaf,** Camellia sinensis(L.) Kuntze Theaceae
practices (GMP) (in the case of herbal medicines). Additionally, 9. St John’s wortf,* HypericumperforatumL. Hypericaceae
10. Dog Rosef,* Rosa canina L. Rosaceae
considering that some of the contaminants present in the plants have
11. Rosellef,* Hibiscus sabdariffa L. Malvacea
well-known toxic effects and that some even have accumulative abili­ 12. Nettleb,* UrticadioicaL. Urticaceae
ties, it is necessary to assess their risk to human health (Khan et al., 13. Bearberryb,* Arctostaphylosuva-ursi (L.) Spreng Ericaceae
2009; Asgari Lajayer et al., 2017). 14. Black elderb,* SambucusnigraL. Adoxaceae
Considering that, to the best of our knowledge, in the literature f
Filter.
published so far, health risk assessment of commercial herbal tea sam­ b
Bulk.
ples available in the Serbian market in the light of the presence of both *
Cultivated in Serbia.
toxic metals and toxigenic fungi has not been conducted, the objectives **
Foreigner origin.

2
M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

200 ◦ C (15 min), (II) maintaining the high temperature (200 ◦ C) for
EDIn
another 15 min and (III) ventilation and cooling of the system for 20 THQn = ; n = 1, 2, 3, …, 6 (2)
RfDn
minutes. After the mineralization, samples were transferred into the
volumetric flask (25 mL) and filled with deionized water to the final For each metal (n), RfDn (mg/kg/day) represents the oral reference
volume of 25 mL. A mixture prepared only with oxidizing agents (HNO3 dose regulated by the U.S. Environmental Protection Agency (US EPA),
and H2O2 - 7: 1; v/v) was used as a blanc. EDIn is the daily average exposure dose (mg/kg/day), while THQn is the
target hazard quotient of the toxic metal. The RfD values for Cd, Pb, As,
2.2.3. Toxic metal content determination Mn, Cr, and Ni were 5.0 × 10− 4, 1.5 × 10− 3, 3.0 × 10− 4, 1.4 × 10− 1,
The toxic metal concentration was determined by electrothermal 0.02 mg, and 1.5 kg/day, respectively (Cai et al., 2019) (US EPA, 2019)
atomization using a graphite furnace atomic absorption spectroscope
(GF-AAS) (Agilent Technologies, Santa Clara, California, USA). Experi­ 2.2.7. Combined risk of multiple toxic metals through intake of herbal teas
mental conditions for Cd, Pb, As, Mn, Ni and Cr determination are The overall human health risk effects may result from the exposure to
presented in the Table 2. The accuracy and precision, LOD (limits of more than one contaminant. Thus, the hazard index (HI) was used to
detection) and LOQ (limits of quantification) of the method were tested estimate the total non-carcinogenic health hazards caused by the
by the above mentioned standard reference material, Apple Leaves exposure to multiple toxic metals (US EPA, 2007). Given the potential
1515. The obtained recovery values were in the range from 85.2% to non-carcinogenic risk posed by the different toxic metals, it is assumed
109.7%. Concentrations of metals in the investigated samples were that adverse effects of toxic metals in herbal teas are additive rather than
expressed in mg/kg of dried plant material. The experiment was carried synergistic or antagonistic. Therefore, HI is equal to the sum of the target
out in triplicate, while the results are expressed as mean ± standard hazard quotients of all the studied contaminants. It can be estimated by
deviation. the following equation (3) (US EPA, 2007):
∑
Hazard Index(HI) = THQn
2.2.4. Risk assessment of toxic metals intake through herbal tea infusions
The health risk to herbal tea consumers as a result of exposure to Cd, = THQ1 + THQ2 + THQ3 + ⋅⋅⋅ + THQn; n
Pb, As, Mn, Ni, and Cr through herbal tea intake was assessed based on = 1, 2, 3, ...6 (3)
the estimated daily intake (EDI), target hazard quotient (THQ), and
hazard index (HI) (Zhang et al., 2018). In this equation, HI represents the total health risk related to the
toxic metals (N = 6). THQn represents the THQ value of an individual
2.2.5. Estimated daily intake toxic metal, while HI is the hazard index of multiple metals. In the case
The following equation (1) of EDI was used to calculate the exposure of HI value which is less than 1, the exposure dose is lower than the
assessment to toxic metals from consumption of herbal teas: adverse reaction threshold (for non-carcinogens). On the other hand, if
the HI value is higher than 1, the exposure dose is higher than the
EDIn =
(Cn × IR × TRn)
; n = 1, 2, 3, ..., 6 (1) adverse reaction threshold, and, in that case, it is very likely that toxic
(BW × 1000) metals will have negative effects on human health (Zhang et al., 2018).
In this equation, Cn represents toxic metal concentration in herbal
tea samples (mg/kg), n is toxic metal type (1,2,…,6), IR is the ingestion 2.3. Fungal isolation and identification
rate of herbal tea for adults (11.4 g/person/day) (Peng et al., 2018;
Zhang et al., 2018), TRn is the transfer rate of toxic metal from herbal A small amount of each herbal tea sample (0.2 g) was suspended in
tea sample into the infusion (Zhang et al., 2018), BW is body weight (60 sterile distilled water (1.8 mL) and vigorously shaken for 5 minutes. Two
kg for adults) (de Oliveira et al., 2018; Zhang et al., 2018). The TR values temperatures of the sterile distilled water were chosen for the experi­
used in this study were 14.18% for Cd, 7.11% for Pb, 23.83% for As, ment: control (25 ± 2 ◦ C) and heat treatment (80 ± 3 ◦ C). Heat treat­
22.5% for Mn, 67.71% for Ni, and 11.45% for Cr (Zhang et al., 2018). ment was set to 15 minutes. Prepared suspensions were inoculated on
the following nutrient media: Potato Dextrose agar (PDA) and Malt
2.2.6. Health risk of individual toxic metals through intake herbal teas Yeast 40% Sucrose agar (MY40). PDA was selected as the suitable me­
Target hazard quotient (THQ) was used to quantitatively evaluate dium for isolation of mesophilic fungi, while MY40 was used for isola­
the potential non-carcinogenic effects of individual toxic metals (Zhang tion of xerophiles and xerotolerants. Both media were prepared
et al., 2018). A THQ value less than 1 indicates that there is no signi­ according to the instructions given by Samson (2010). Petri plates (Ø 90
ficant risk of non-carcinogenic effects for the exposed population. THQ mm) with inoculated media were incubated in thermostat (UE 500,
can be calculated by the equation (2): Memmert) on 25 ± 2 ◦ C, while fungal colony formation was monitored
over a week. After the incubation period, colonies formed on each media
were counted and converted to CFU/g (Colony Forming Unites per gram

Table 2
Experimental conditions for Cd, Pb, As, Mn, Ni and Cr determination by graphite furnace atomic absorption spectrometry (GF-AAS)
Element Wavelength Argon Chemical Drying 1 (◦ C) Drying 2 (◦ C) Drying 3 (◦ C) Pyrolysis (◦ C) Atomization Clean temperature
(nm) flow l/ modifier (ramp time (ramp time (ramp time (ramp time (s), temperature (◦ C) (◦ C) (ramp time (s),
min (s), hold time (s), hold time (s), hold time hold time (s)) (ramp time (s), hold hold time (s))
(s)) (s)) (s)) time (s))

Pb 283.3 3 10% Triton X 85 (5, 20) 95 (3, 20) 120 (2, 10) 400 (10, 20) 2100 (0, 3) 2400 (1, 5)
NH4H2PO4
Cd 228.8 3 10% Triton X 85 (5, 20) 95 (3, 20) 120 (2, 10) 250 (10, 20) 1800 (0, 3) 2600 (1, 5)
NH4H2PO4
Cr 357.9 3 Pd(NO3)2 85 (5, 20) 95 (3, 20) 120 (2, 10) 1000 (10, 20) 2100 (0, 3) 2400 (1, 5)
As 193.7 3 Pd(NO3)2 Mg 85 (5, 20) 95 (3, 20) 120 (2, 10) 1400 (10, 20) 2100 (0, 3) 2600 (1, 5)
(NO3)2
Ni 232.0 3 Pd(NO3)2 Mg 85 (5, 20) 95 (3, 20) 120 (2, 10) 800 (10, 20) 2100 (0, 3) 2400 (1, 5)
(NO3)2
Mn 279.5 3 Pd(NO3)2 85 (5, 20) 95 (3, 20) 120 (2, 10) 700 (10, 20) 2100 (0, 3) 2400 (1, 5)

3
M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

of herbal sample), which represents a total fungal count. 2009; Zhang et al., 2020a). Furthermore, different metals accumulate in
Identification of fungi was performed according to the colony plants at different rates depending on the aforementioned factors (Zhang
morphology and microscopic characteristics of reproductive structures et al., 2020a).
(mostly conidiogenous apparatus’ and ascocarps, detected via micro­
scope Nicon Eclipse E200 (Nicon, Tokyo, Japan), equipped with camera 3.1.1. Cadmium
BresserMikroCam PROHDMI (Bresser, Rhede, Germany)) using identi­ In the current research, the lowest Cd concentration (0.04 mg/kg)
fication keys: Raper and Fennell (1965), Pitt (1979), Watanabe (2002) was detected in Urticadioica L., while its highest concentration was
and Samson (2010). All filamentous fungi were identified to genus, observed in Hypericum perforatum L. In that sense, it could be assumed
section or species level. Filamentous fungi (which remain sterile) and that the obtained Cd concentration in St John’s wort (0.93 mg/kg) is
yeast were not identified, but their colonies were considered in the total significantly higher than permitted values according to the WHO and
fungal count. European Pharmacopeia.
Isolation frequency (IF) of the specific fungal taxa was determined Hypericum perforatum L. is recognized as Cd hyperaccumulator, and
according to Esquivel (2003): other studies have also revealed the high Cd concentration in its dried
leaf samples. Mamani et al. (2005) reported that Cd concentration in St
number of times a specific fungal taxa is detected
IF(%) = × 100 John’s worth samples from China ranged from 0.56-1.08 mg/kg
total number of samplings realized
(Mamani et al., 2005), while, according to Chizzola et al. (2003), con­
For all the identified fungi recovered on PDA and MY40 after the heat centration of Cd in tested St John’s wort tea samples from Austria was
treatment survival rate was determined and expressed in percentage. even higher, 1.3 mg/kg (Chizzola et al., 2003). In Serbia, Popović et al.
(2017) have also examined various tea samples and reported elevated
2.4. Statistical analysis Cd concentration in St John’s wort (0.97 mg/kg) and yarrow (0.72
mg/kg) compared to the other samples (Popović et al., 2017). On the
Statistical analysis of the metal content in herbal teas, as well as risk other hand, Djukić-Ćosić et al. (2011) have demonstrated that Cd con­
assessment, were performed by Microsoft Excel 2016 (Microsoft, Red­ tent significantly varies in Hypericum perforatum samples collected from
mond, WA, USA). the different locations in Serbia. The lowest Cd level was found in the
samples obtained from the one of the Rtanj localities (0.25 mg Cd/kg),
3. Results and Discussion while the highest was observed in a sample from Ozren (1.24 mg/kg).
Furthermore, these authors have observed that the levels of Cd in three
3.1. Levels of toxic metals in commercial herbal tea samples of four investigated localities were higher than those proposed by the
WHO (0.3 mg/kg dried herb material) (Djukić-Ćosić et al., 2011). They
The total concentrations of Cd, Pb, As, Mn, Ni in the tested tea concluded that discrepancies in Cd concentration among Hypericum
samples are presented in the Table 3. The obtained results showed that plant species from different localities depend not only on the different
concentrations of Cd, Pb, As, Mn, Ni and Cr in herbal tea samples ranged soil mineral composition, more intensive metal absorption in plants at
0.04-0.93, 0.09-2.54, 0.03-0.77, 1.82- 651.04, 0.97-9.01, 0.49-3.47 mg/ low pH conditions, low ion exchange capacity, but also scarce organic
kg dried plant material, respectively. matter and phosphate content. Additionally, all of these differences can
It is important to mention that toxic metals might accumulate in further be influenced by anthropogenic factors (Djukić-Ćosić et al.,
certain plants, and, therefore, their concentrations depend primarily on 2011).
the physiological properties and metal absorption mechanisms of the
investigated plants, chemical speciation of metals in soil, as well as soil 3.1.2. Lead
physicochemical properties (e.g. pH) (Zhang et al., 2020a). For example, In the case of Pb, the lowest concentration was observed in chamo­
tea plants cultivated in soils which originate from mineralized rocks can mile (0.09 mg/kg), and the highest in common nettle (2.54 mg/kg).
lead to high concentrations of toxic metals (Yaylali-Abanuz and Tüysüz, None of the tested samples exceeded neither of the permitted values.

Table 3
Levels of toxic metals in commercial herbal tea samples compared to the permitted international values
Toxic metals content (mg/kg)

Tea samples Cd Pb As Mn Ni Cr

1. Mentha piperitaL. 0.08 ± 0.01 0.49 ± 0.02 0.07 ± 0.01 33.0 ± 0.05 1.00 ± 0.06 0.80 ± 0.03
2. Ocimumbasilicum L. 0.08 ± 0.02 0.37 ± 0.03 0.28 ± 0.02 49.40 ± 0.08 1.11 ± 0.07 0.91 ± 0.06
3. Thymus vulgaris L. 0.26 ± 0.01 1.34 ± 0.05 0.37 ± 0.01 58.87 ± 0.09 2.16 ± 0.04 1.92 ± 0.08
4. Salvia officinalis L. 0.22 ± 0.03 1.49 ± 0.02 0.56 ± 0.04 45.21 ± 0.03 1.56 ± 0.05 1.47 ± 0.06
5. Satureja montana L. 0.17 ± 0.02 1.04 ± 0.03 0.17 ± 0.02 12.34 ± 0.07 2.44 ± 0.06 1.74 ± 0.05
6. MatricariachamomillaL. 0.16 ± 0.01 0.09 ± 0.01l 0.34 ± 0.03 1.82 ± 0.09l 2.20 ± 0.06 0.91 ± 0.03
7. Achileamillefolium L. 0.14 ± 0.02 0.27 ± 0.02 0.06 ± 0.01 27.11 ± 0.11 1.17 ± 0.04 0.67 ± 0.01
8. Camellia sinensisL. 0.11 ± 0.01 1.34 ± 0.04 0.77 ± 0.02h 651.04 ± 0.21h 5.59 ± 0.08 0.72 ± 0.05
9. HypericumperforatumL. 0.93 ± 0.03h,* 0.21 ± 0.02 0.03 ± 0.01l 112.34 ± 0.18 9.01 ± 0.21h 1.63 ± 0.06
10. Rosa canina L. 0.20 ± 0.01 0.49 ± 0.05 0.26 ± 0.02 88.49 ± 0.02 2.46 ± 0.05 0.49 ± 0.01l
11. Hibiscus sabdariffa L. 0.19 ± 0.03 0.29 ± 0.02 0.14 ± 0.03 375.26 ± 0.14 3.65 ± 0.04 1.29 ± 0.05
12. UrticadioicaL. 0.04 ± 0.01l 2.54 ± 0.04h 0.73 ± 0.02 19.24 ± 0.03 1.19 ± 0.07 1.54 ± 0.02
13. Arctostaphylosuva-ursi L. 0.12 ± 0.01 1.03 ± 0.01 0.24 ± 0.02 67.21 ± 0.06 1.11 ± 0.03 2.83 ± 0.03
14. SambucusnigraL. 0.14 ± 0.01 0.98 ± 0.03 0.36 ± 0.01 10.24 ± 0.11 0.97 ± 0.02l 3.47 ± 0.04h

Permitted values (mg/kg)

World Health Organization 0.3 10 - - - -
European Pharmacopeia (Ph. Eur. 10.0) 0.5 5 - - - -
European Union 1 3 - - - -
h
The highest level.
l
The lowest level.
*
Value above the recommended/permitted levels.

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M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

Alhusban et al. (2019) detected Pb in 93% of the analyzed samples of concentration in tea leaves of different types and origin, ranging from
traditional herbs available in Jordanian Market (Alhusban et al., 2019) 457 to 2210 mg/kg, while the highest average concentration of this
and concluded that Pb mean concentration levels in the commonly used metal was observed in black teas (Podwika and Kleszcz, 2018). In the
traditional medicinal herbs can be 4.4 times higher compared to its research by Razic, Kuntic (2013) extremely high Mn concentrations up
mean concentration levels in the pharmaceutical herbal products sold in to 2541.8 mg/kg were reported (Razic and Kuntic, 2013). Furthermore,
pharmacies, which may be attributed to the strict regulations on the these authors also found very high Mn concentrations in tested Roselle
additives and excipients used in the pharmaceutical industry. In their samples (up to 1585.9 mg/kg). The high Mn content in the Roselle
study, Pb concentration levels detected in all the 14 traditional herbs sample is also consistent with the work of Živkov-Baloš et al. (2014) who
were below the Pb permissible limits set by the WHO, apart from the measured Mn content in Hibiscus sample of 453.71 mg/kg (Živkov-Baloš
concentration measured in Persian Thyme (Saturejathymbra) (41.18 et al., 2014). Plants absorb Mn from soil by an active transport mecha­
mg/kg), which exceeded the Pb limits of 3, 5 and 10 mg/kg set by the nism in root’s epidermic cells, and it is easily translocated to the upper
EU, European Pharmacopeia and WHO, respectively (Alhusban et al., plant’s tissues. This phenomenon leads to Mn accumulation in plants
2019). Oliveira et al. (2018) have shown that herbal teas exhibited shoots rather than in root (Kabata-Pendias, 2011; Millaleo et al., 2010).
higher Pb (2.32 mg/kg), as well as As (0.26 mg/kg) and Cd (0.19 mg/kg) High concentrations of Mn in plants could be caused by the cultivation
concentrations than traditional teas, including black, green, and oolong areas being in close proximity to industrial areas or major roads where
(de Oliveira et al., 2018). Comparably to our study, after measuring Pb exhaust fumes caused increased levels of Mn in the soil (Kabata-Pendias,
content in herbal drugs obtained from different local health food stores 2011). In addition to soil contamination, low pH can also contribute to
in Serbia, Kaličanin (2013) detected the lowest overall content of this the metal uptake by plants (Kabata-Pendias, 2011; Ražić,Đogo, 2010).
toxic metal in chamomile (0.73 to 0.77 mg/kg), while the greatest Pb Although the amount of Mn that passes from the plant to the infusion is
content was determined in the samples of the frangula bark (3 mg/kg). not high (about 8%), the rate of Mn uptake by plants could be signifi­
These authors have noted that the overall Pb concentrations in the cantly higher due to the increasing environmental pollution, and could
studied samples were 4-6 times lower compared to the content pre­ consequently lead to health problems during long term tea consumption
scribed by the WHO (Kaličanin and Velimirović, 2012). Comparably to (Mehra, Baker, 2007).
Cd, accumulation of Pb in plants depends on its lifecycle, mobility of the
metal, soil composition, pH values, precipitation, and presence of other 3.1.5. Nickel
ions and molecule types in the soil (Caldas and Machado, 2004; Kali­ Similarly to Cd, highest concentration of Ni was observed in Hyper­
čanin and Velimirović, 2012; Zhong et al., 2016) icum perforatum L. (9.01 mg/kg), while its lowest level was determined
in Sambucusnigra L. (0.97 mg/kg).
3.1.3. Arsenic According to Kastori (1997), average Ni content in plants ranges
Opposite to Cd and Ni, lowest As concentration in the present study from 0.1 to 5 mg/kg (Kastori, 1997). Zhong et al. (2016) have suggested
was determined in Hypericumperforatum L. (0.03 mg/kg), while its that Ni in tea plants mainly originates from foliar and soil applications of
highest concentration was observed in Camellia sinensis L. (0.77 mg/kg). low quality fertilizers and micronutrients. In their study, the highest
Santos et al. (2015) measured 0.02-0.4 mg/kg As in tea leaves level of Ni was reported in white tea (13.41 mg/kg), followed by the
commercialized in Rio De Janeiro, Brazil. In their study, the lowest As yellow tea (12.59 mg/kg) (Zhong et al., 2016). It has been demonstrated
concentration was measured in black and the highest in white tea that high Ni concentration in plant tissue (above 10 mg/kg) could have
(Santos et al., 2015). However, Nkansah et al. (2016) have found even detrimental effect on plants. For example, certain plant disease symp­
higher As level in the selected tea products from Ghanaian market, toms including slow growth, abnormalities in plant photosynthesis,
where As concentrations ranged from 1.40 to 2.00 mg/kg, with a mean chlorosis, and leaf wilting and fading are linked to the high Ni content in
of 1.66 mg/kg (Nkansah et al., 2016). Moreover, out of the 800 analyzed plant tissue (Kabata-Pendias, 2011). As mentioned earlier, in the present
tea samples, Han et al. (2005) detected the highest As concentration of research, the highest Ni concentration was determined in St. John’s wort
4.43 mg/kg, while Yuan et al. (2007) reported maximum As concen­ (9.01 mg/kg), which did not exceed the value of 10 mg/kg. Neverthe­
tration of 4.81 mg/kg in Chinese tea leaves (Han et al., 2005; Yuan et al., less, this could be regarded not only as a potential risk to the plant itself,
2007; Karak and Bhagat, 2010). On the other hand, Zhang et al. (2018) but alsoto tea consumers.
found slightly lower As concentrations (0.073–0.456 mg/kg) in young
tea leaves from Guizhou Province, China (Zhang et al., 2018). It is 3.1.6. Chromium
important to mention that the mobility of As in soil depends on the Lowest Cr concentration was observed in Rosa canina L. (0.49 mg/
properties of soil such as particle size, texture, mineral nutrient content, kg), while the highest concentration was measured in Sambucusnigra L.
pH, presence of other ions, moisture regimes, transformations by mi­ (3.47 mg/kg).
crobes, as well as As chemical form, while As transfer into plants may However, Sungur (2013) measured the highest Cr concentrations in
vary depending on the soil types, cropping pattern, As concentration in chamomile (4.21 mg/kg), licorice (2.80 mg/kg), melissa (2.71 mg/kg),
irrigation water, distance from the water source, depth of water source, marestail (2.66 mg/kg), and anise (1.98 mg/kg) (Sungur et al., 2013).
etc. (Bhattacharya et al., 2012). According to Kastori (1997), the average content of Cr in plants ranges
from 0.01 to 0.5 mg/kg, while values greater than 5 mg/kg of dried plant
3.1.4. Manganese matter can be harmful to the plant and lead to loss of biomass, chlorosis,
Similarly to Pb, lowest concentration of Mn was observed in Cham­ necrosis of leaves and other morphophysiological and metabolic
omile (1.82 mg/kg), while higher concentrations were observed in green changes (Kastori, 1997). Bioaccumulation of Cr within plants depends
tea (651.04 mg/kg), roselle (375.26 mg/kg) and St John’s wort (112.34 upon the plant species, oxidation state of Cr ions, as well as Cr con­
mg/kg). High Mn concentration in C.sinensis samples was also reported centration in the growth medium. However, having in mind that the
in other researches. To name a few, Perić-Grujić et al. (2008) reported mobility of Cr in the plant roots is low, concentration of Cr in roots can
that, in investigated C. sinensis samples, Mn concentrations ranged from sometimes be 100 times higher than in the shoots (Sharma et al., 2020).
149.6 to 199.3 mg/kg and were significantly higher than other detected
heavy metals (Perić-Grujić et al., 2008). Accordingly, in a study con­ 3.1.7. Toxic metal content compared to the permitted values
ducted by Pejin et al. (2012), it was found that, out of the several In the current research, considering that there are currently no reg­
analyzed metals (As, Cd, Cr, Cu, Fe, Pb, Hg, Ni, Zn and Mn) in the moss ulations by Serbian Ministry regarding the officially defined limits of
species R. ontariense, Mn was the most abundant (Pejin et al., 2012). toxic metals concentrations in dried plant material, the obtained con­
Podwika and Kleszcz (2018) also reported relatively high Mn centrations of toxic metals in the investigated herbal teas were

5
M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

compared to available international standards and recommendations. accordance with the results of Zhang et al. (2020), who found that EDI
Additionally, apart from the absence of regulatory requirements at the for the measured metals in tea infusions varies depending on their
national level, international standards are not consistent and do not concentration, which was also heavily related to the plantation tea
provide recommended values for all toxic metals. Thus, WHO and EU leaves were obtained from. The authors concluded that noted variations
provide levels only for Pb and Cd, while the European Pharmacopoeia could be attributed to the natural high background concentration of
provides levels for Cd, Pb and Hg. heavy metals in the soil of certain plantations, as well as the accumu­
Our results have demonstrated that concentrations of these toxic lation capacity of tea leaves (Zhang et al., 2020b). Peng et al. (2018) also
metals in all the investigated tea samples were lower than the corre­ concluded that metal concentration in tea leaves, as well as EDI, may be
sponding thresholds set by the available international standards and influenced by the soil conditions, tea variety, harvest season, and leaf
recommendations, apart from the obtained Cd concentration in St maturity (Peng et al., 2018). As mentioned earlier, other important
John’s wort, which was notably higher than the values permitted by factor which could influence the calculated EDI is the TR of toxic metal
WHO and European Pharmacopeia. On the contrary, in the study by from herbal tea sample into the infusion, which was also concluded by
Zhang et al. (2018), measured Cd concentrations in all the tested sam­ Zhang et al. (2020). Having in mind that these rates might vary in
ples were lower than the EU standards. However, Pb concentration in different studies, this might influence the estimation of the EDI and lead
one sample (3.04 mg/kg) exceeded the EU limit (3 mg/kg) (Zhang et al., to the different results.
2018). On the other hand, de Olivia (2018) found the concentrations of
Cd, Pb, Hg, As, and Cr in all tea samples lower than the corresponding 3.3. Health risk assessment of toxic metals
thresholds set by the WHO and the European Phamacopeia for recom­
mended levels of toxic metals in dried plant materials (de Oliveira et al., The calculated THQs of Cd, Pb, As, Mn, Ni, and Cr and accumulative
2018). HI values for infusions from the 14 herbal tea samples commercially
available in Belgrade market, Republic of Serbia, are presented in the
3.2. Estimated daily intake of toxic metals Table 5.
In accordance with the calculated EDI, the highest THQ was obtained
Transfer of toxic metals from herbal tea into the tea infusions is not for Cd and Ni, which, as mentioned earlier, could be attributed to the
absolute and should be considered (Zhang et al., 2018). Since transfer of ability of this plant to hyperaccumulate these metals (Djukić-Ćosić et al.,
toxic metals from herbal teas into the infusions was not examined in the 2011). Thus, for both Cd and Ni, highest THQ values were calculated for
present study, literature TR values were used to evaluate the EDI. The TR Hypericum perforatum L. On the other hand, for Pb, As, Mn and Cr, the
values were 14.18%, 7.11%, 23.83%, 22.5%, 67.71%, and 11.45% for highest values were obtained for Urtica dioica L, Rosa canina L, Camellia
Cd, Pb, As, Mn, Ni and Cr, respectively (Nookabkaew et al., 2006; Zhang sinensis L. and Sambucus nigra L, respectively. Although the highest THQ
et al., 2018). The calculated EDI values (mg/kg/day) of toxic metals was obtained for As and Mn, it shoud be noted that higher THQ of
through herbal tea infusion intake are listed in the Table 4. certain metals (e.g. As) could be related to the lower RfD values in
For both Cd and Ni, the highest EDI values were obtained for comparison with the other investigated metals. From all the tested
Hypericum perforatum L, as well as the lowest EDI for As. Furthermore, herbal tea samples, the highest HI was noted for Camellia sinensis L, and
for Pb, As and Mn, the highest EDI values were calculated for Camellia the lowest for Achilea millefolium L.
sinensis L, while, for Cr, the highest value was estimated for Sambucus Finally, having in mind that values of both THQ and accumulative HI
nigra L. Contrary to that, EDI for Ni via intake of Sambucus nigra L. was values for Cd, Pb, As, Mn, Ni, and Cr were less than 1, this could suggest
the lowest, as well as the intake of both Pb and Mn via Matricaria cha­ that the risk of adverse effects in adults after the exposure to six tested
momilla L. Lowest EDI values for Cd and Cr were obtained for Urtica metals via herbal tea intake is acceptable Thus, tea beverage made from
dioica L and Rosa canina L, respectively. the commercially available herbal tea samples in Belgrade market, Re­
It should be noted that EDI values are highly dependent on the public of Serbia, should be considered safe for the residents regarding
measured metal concentration, which can be confirmed by drawing a the levels of the investigated toxic metals. Similar results have also been
comparison with the values presented in the Table 3. reported by other authors. For example, in their studies, Peng et al.
Considering that, for Camellia sinensis L. and Hypericum perforatum L, (2018) and Zhang et al. (2018, 2020), have demonstrated that there was
EDI for more than one metal was found to be the highest, it could be no risk to human health from exposure to 8 toxic metals via tea intake
assumed that these herbs could pose the ability to accumulate the said (Peng et al., 2018; Zhang et al., 2018, 2020b). Also, it has been shown
toxic metals (Djukić-Ćosić et al., 2011; Zhang et al., 2020a). This is in that health risks of toxic metals in tea were lower than those in rice and

Table 4
Estimated daily intake (EDI) (mg/kg/day) of Cd, Pb, As, Mn, Ni, and Cr for adults associated with the consumption of 14 different herbal tea infusions.
Estimated daily intake (EDI) (mg/kg)

Tea samples Cd Pb As Mn Ni Cr

1. Mentha piperitaL. 2.15E-06 6.62E-06 3.17E-06 1.41E-03 1.29E-04 1.74E-05

2. Ocimumbasilicum L. 2.16E-06 4.99E-06 1.27E-05 2.11E-03 1.43E-04 1.98E-05
3. Thymus vulgaris L. 7.01E-06 1.81E-05 1.68E-05 2.52E-03 2.78E-04 4.18E-05
4. Salvia officinalis L. 5.93E-06 2.01E-05 2.54E-05 1.93E-03 2.01E-04 3.20E-05
5. Satureja Montana L. 4.58E-06 1.40E-05 7.70E-06 5.28E-04 3.14E-04 3.78E-05
6. MatricariachamomillaL. 4.31E-06 1.22E-06l 1.54E-05 7.78E-05l 2.83E-04 1.98E-05
7. Achileamillefolium L. 3.77E-06 3.65E-06 2.72E-06 1.16E-03 1.51E-04 1.46E-05
8. Camellia sinensisL. 2.96E-06 1.81E-05h 3.49E-05h 2.78E-02h 7.19E-04 1.57E-05
9. HypericumperforatumL. 2.51E-05h 2.84E-06 1.36E-06l 4.80E-03 1.16E-03h 3.55E-05
10. Rosa canina L. 5.39E-06 6.62E-06 1.18E-05 3.78E-03 3.16E-04 1.07E-05l
11. Hibiscus sabdariffa L. 5.12E-06 3.92E-06 6.34E-06 1.60E-02 4.70E-04 2.81E-05
12. UrticadioicaL. 1.08E-06l 3.43E-05 3.30E-05 8.23E-04 1.53E-04 3.35E-05
13. Arctostaphylosuva-ursi L. 3.23E-06 1.39E-05 1.09E-05 2.87E-03 1.43E-04 6.16E-05
14. SambucusnigraL. 3.77E-06 1.32E-05 1.63E-05 4.38E-03 1.25E-04l 7.55E-05h
h
The highest level.
l
the lowest level.

6
M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

Table 5
Target hazard quotient (THQ) of Cd, Pb, As, Mn, Ni, and Cr and hazard index (HI) values for adults associated with the consumption of commercial herbal tea samples.
Target hazard quotient (THQ)
Hazard index (HI)
Tea samples Cd Pb As Mn Ni Cr

1. Mentha piperitaL. 4.31E-03 4.41E-03 1.06E-02 1.01E-02 6.43E-03 1.16E-05 0.03613

2. Ocimum basilicum L. 4.31E-03 3.33E-03 4.20E-02 1.51E-02 7.14E-03 1.32E-05 0.07211
3. Thymus vulgaris L. 1.40E-02 1.21E-02 5.58E-02 1.80E-02 1.39E-02 2.78E-05 0.1146
4. Salvia officinalis L. 1.18E-02 1.34E-02 8.45E-02 1.38E-02 1.00E-02 2.13E-05 0.1345
5. SaturejamontanaL. 9.16E-03 9.37E-03 2.56E-02 3.77E-03 1.57E-02 2.52E-05 0.06412
6. MatricariachamomillaL. 8.62E-03 8.11E-04 5.13E-02 5.56E-04 1.42E-02 1.32E-05 0.07510
7. Achileamillefolium L. 7.54E-03 2.43E-03 9.06E-03 8.28E-03 7.53E-03 9.72E-06 0.03514
8. Camellia sinensisL. 5.93E-03 1.21E-02 1.16E-01 1.99E-01# 3.60E-02 1.04E-05 0.369*1
9. HypericumperforatumL. 5.01E-02# 1.89E-03 4.53E-03 3.43E-02 5.80E-02# 2.36E-05 0.1493
10. Rosa canina L. 1.08E-02 4.41E-03 3.92E-01# 2.70E-02 1.58E-02 7.11E-06 0.0977
11. Hibiscus sabdariffa L. 1.02E-02 2.61E-03 2.11E-02 1.15E-01 2.35E-02 1.87E-05 0.1722
12. UrticadioicaL. 2.16E-03 2.29E-02# 1.10E-01 5.88E-03 7.66E-03 2.23E-05 0.1484
13. Arctostaphylosuva-ursi L. 6.47E-03 9.28E-03 3.62E-02 2.05E-02 7.14E-03 4.10E-05 0.0799
14. SambucusnigraL. 7.54E-03 8.83E-03 5.43E-02 3.13E-03 6.24E-03 5.03E-05# 0.0808

Numbers from 1 to 14 indicate the orfer of the calculated HI values, from highest to lowest.
#
(bolded) - the highest THQ for a particular metal.

vegetables (Huang et al., 2018). Furthermore, the HI values for As, Cd,
Cr, and Pb exposure through three types of teas (green, black, and
oolong) in Taiwan were also less than one, indicating no risk to human
health (Shen and Chen, 2008). Although the human health risk of toxic
metals ingestion via tea intake was relatively small, there are other
pathways that can play an important role in toxic metals exposure to
humans (Hadayat et al., 2018). Hence, for a comprehensive human
health risk assessment from all the sources, the intake of toxic metals
through herbal teasshould also be considered, given the toxicity of
metals to people.
However, the uncertainty of the results of the present study should be
mentioned. Firstly, the tea infusion intake was considered the only route
of exposure for toxic metals. Other sources of toxic metals in the dietary
structure, such as drinking water, vegetables, fruits, and grains, may also
play important roles in forming the probable health risks. A prior study Fig. 1. Total mould count (CFU/g) in tested tea and herbal infusion samples on
indicated that health risks caused by the toxic metals depended on crop Potato Dextrose Agar (PDA) and and Malt Yeast 40% Sucrose agar (MY40):
species, and that grain (e.g., rice, corn, and wheat) posed the higher risk Mentha piperita (Mp), Ocimum basilicum (Ob), Thymus vulgaris (Tv), Salvia offi­
than fruit and vegetables (Zheng et al., 2020). Secondly, toxic metals cinalis (So), Saturejamontana (Sm), Matricaria chamomilla (Mc), Achilea mil­
which enter the human body are not completely taken up by the lefolium (Am), Camellia sinensis (Cs), Hypericum perforatum (Hp), Rosa canina
gastrointestinal tract, while their bioavailability is impacted by many (Rc), Hibiscus sabdariffa (Hs), Urtica dioica (Ud), Arctostaphylosuva-ursi (Ua) and
factors (individual lifestyle, food types, the dose taken up by the Sambucus nigra (Sn).
digestive system, etc.) (Laparra et al., 2005). Additionally, the results of
the present study were based on the contents of toxic metals in unpro­ g). The lowest mould count on MY40 was recorded for Green tea (45.68
cessed dried plant material. It is important to bear in mind that the CFU/g).
after-product of the tea processing may also be contaminated by the Obtained results of the total mould counts from the tested herbal
toxic metals (Zhang et al., 2018). Even though TR of the specified toxic samples were compared with the other available standards and recom­
metals obtained from the literature were used to calculate the EDI for all mendations. Safety parameters regarding the fungal presence in
the investigated herbal tea samples, the actual transfer of metals into the different countries are regulated and standardized by the national leg­
infusion might differ in the real life scenario, since percentage of toxic islations. However, those safety standards vary worldwide. To name a
metal release depends on type of plants as well as content of toxic metal. few, according to the Czech Republic legislations (ČSN ISO 7954) the
Finally, this work included only 6 toxic metals, while others were not total count of cultivable fungi in teas must not exceed 105 CFU/g. Na­
considered. This is significant, especially since the results of Zhang et al. tional legislations in Serbia are the same as those in Czech Republic and
(2018) demonstrated that toxic metal thallium (Tl) had the most sig­ suggested that dried plant materials brewed with hot water must harbor
nificant share in the estimated daily intake, as well as the HI for tea less than 105 CFU/g of total mould count. The similar safety microbio­
infusions (Zhang et al., 2018). logical parameters have been proposed by association Tea, Herbal In­
fusions Europe (THIE), where the total yeast and mould count in C.
sinensis and dried herbal infusions must be lower than 105 CFU/g. Ac­
3.4. Fungi in the tested tea and herbal infusion samples
cording to Technical Regulations of the Customs Union (TR CU 021/
2011) on Food Safety: in mixtures of dried medicinal plants, the total
The total mould count expressed as CFU per g of tested herbal sample
mould count of 103 CFU/g could be tolerated, except if they are intended
is presented in the Fig. 1. All 14 samples of the tested teas and herbal
for children and expectant mothers, where only 50 CFU/g is allowed.
infusions were found to be contaminated with fungi.
According to the WHO, herbal medications, which can be used only after
The highest concentrations of mesophilic fungi were documented in
being brewed with boiling water, can harbor a total count of cultivable
Peppermint (859.26 CFU/g) and Black elder (737 CFU/g) samples. On
fungi less than 104 CFU/g.
the other hand, the lowest concentrations of mesophiles were found in
C.sinensis and herbal infusions tested in this research do not exceed
Sage (90.5 CFU/g) and Basil (120 CFU/g). It has been shown that tested
the proposed above mentioned microbiological standards. The
Dog Rose sample was rich in xerophilesand xerotolerants (990.45 CFU/

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M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

acceptable fungal contaminations in dried tea according to Sri Lanka tea MY40, the low number of fungal colonies formed on this medium cannot
board guidelines range from 102 to 103 CFU/g (Dayananda et al., 2017). be taken into account when product safety is considered. However,
However, US Pharmacopoeia for products that contain raw material of quantitative mycological analyses are not the only parameter which
natural origin established a maximum fungal contamination limit of 2 × should be considered when assessing microbiological safety of tea and
102 CFU/g of the product. When tested tea and herbal infusions are other herbal products. Species richness and physiology of identified
compared with strict parameters given by the US Pharmacopoeia, only fungi must also be taken into account. In the research presented here,
Basil (with 120 CFU/g on PDA), Sage (with 90.5 CFU/g on PDA), Winter fungal isolates which differ in colony morphology and growth rate were
savory (121.41 CFU/g on PDA) and Roselle (110.5 CFU/g on PDA) could identified (Table 6).
be regarded as microbiologically safe. Also, due to the selectivity of the A total of 23 morphologically different isolates suggests moderate

Table 6
List of fungi isolated from tested herbal samples
Fungal isolates IF(%) growth on HT survival rate Human health implications
low aw (%)

Mycoses:
Opportunistichyalohyphomycosis(Fincher et al., 1991)
AcremoniumstrictumW. Gams 7.15 - 0
Toxic extrolites:
-
Mycoses:
- respiratory allergies (Achatz et al., 1995)
AlternariaNeessp. 14.28 - 0 - cutaneous infections (Samson et al., 2002)
Toxic extrolites:
alternariol, altertoxins and tenuazonic acid (Patriarca and Fernández Pinto, 2017)
Mycoses:
Aspergillus P. Micheli spp. Sect. -
85.71 + 83.33
Aspergillus Toxic extrolites:
echinulin, flavoglaucin and auroglaucin(Chen et al., 2017)
Mycoses:
Aspergillus P. Michelispp. Sect. -
71.43 + 30
Circumdati Toxic extrolites:
ochratoxin A, xanthomegnin and viomellein(Visagie et al., 2014)
Mycoses:
invasive aspergilloses, keratitis andcutaneus infections (Rudramurthy et al., 2019)
Aspergillus P. Michelispp. Sect. Flavi 57.14 + 0 Toxic extrolites:
aflatoxins, 3- nitropropionic acid, tenuazonic acid and cyclopiazonic acid (Frisvad et al.,
2019)
Mycoses:
lung infections and otomycosis(Schuster et al., 2002)
Aspergillus P. Michelispp. Sect. Nigri 78.57 + 81.82
Toxic extrolites:
ochratoxin A and fumonisin B2 (Varga et al., 2011)
Mycoses:
Aspergillus P. Michelispp. Sect. opportunistic pathogen (Siqueira et al., 2016)
21.48 + 0
Versicolores Toxic extrolites:
Sterigmatocystin (Siqueira et al., 2016)
Mycoses:
respiratory allergic reaction -’’winegrower’s lung’’ (Samson et al., 2002)
Botrytis cinerea Pers. 14.28 - 0
Toxic extrolites:
-
Mycoses:
Cladosporium cladosporioides (Fresen.) G. nail and skin infections (Samson et al., 2002)
7.15 - 0
A. de Vries Toxic extrolites:
-
Mycoses:
-
EpicoccumnigrumLink 35.71 - 0
Toxic extrolites:
-
Mycoses:
opportunistic onychomycosis and keratomycosis(Howard, 2003)
FusariumLink sp. 7.15 - 0
Toxic extrolites:
fumonisins and trichotecenes(Samson et al., 2002)
Mycoses:
Opportunisticmucormycosis(Cox et al., 2011)
Mucor P. Micheli ex L. sp. 7.15 - 0
Toxic extrolites:
-
Mycoses:
-
PenicilliumLink spp. 100 + 0 Toxic extrolites:
ochratoxin A, patulin, Citreoviridin, Citrinin, Cyclopiazonic acid, Penicillic acid,
Roquefortine C (Perrone and Susca, 2017)
Mycoses:
Opportunisticmucormycosis(Cox et al., 2011)
Rhizopusstolonifer(Ehrenb.) Vuill. 14.28 + 0
Toxic extrolites:
-
Non-sporulating isolates 71.43 +
n/a n/a
Yeasts 64.29 +

IF – Isolation frequency; HT –Heat treatment

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M. Krstić et al. Journal of Food Composition and Analysis 104 (2021) 104159

fungal diversity of tested herbal samples. The highest isolation fre­ Bokhary and Aly (2013) showed the dominance of A. parasiticus
quency (IF) of 100% was documented for the members of genera Peni­ (member of Flavi section), A. nigerand Fusarium sp. in tested herbal
cillium and Aspergillus. On the other hand, the lowest IF of 7.15% was samples from various supermarkets and street bazaars in Jeddah (Saudi
recorded for Acremonium strictum, Cladosporium cladosporoides, Fusarium Arabia) (Bokhary and Aly, 2013). Tian et al. (2013) reported the
sp. and Mucor sp. The most dominant group of Aspergilli were the dominance of A. niger and Penicillium sp. in tested Pu’er tea samples
members of Aspergillus section (former Aspergillus glaucus group), found (dried fermented C. sinesis leaves) in Puer City in Yunnan province
in all the tested herbal samples apart from Bearberry and Chamomile. (China) (Tian et al., 2013). Haas et al. (2013) also investigated Pu’er tea
Hence, they are predominatly isolated on MY40, suggesting the sec­ samples in various tea shops in Austria and Germany and reported that
ondary microbial contaminations of the tested herbal samples, most prevalent species were Aspergillus acidus (member of Nigri section),
likely during the storage in dry conditions. Their spores are common in A. fumigatus, Mucoromycotina and Penicillium spp. (Haas et al., 2013).
indoor air and house dust. Thus, fungal growth could occur on substrata These authors detected the presence of OTA in 11.1 % of the tested
with low water activity (aw), such as cereals, or food products with high herbal samples. In research presented by Tournas and Katsoudas (2008),
sugar content, i.e., syrups, jams and jellies, salted meat products, and the most common fungi detected in various herbal infusions were
semi-dry foods and feeds, as well as dried herbal material (Chen et al., Aspergillus niger, Penicillium spp., Eurotium rubrum, E. chevalieri, A. flavus,
2017). Furthermore, Aspergillus spp. sect. Aspergillus showed very high Fusarium spp., Alternaria alternata and yeasts (Tournas and Katsoudas,
heat treatment survival rate (83.33%) suggesting that conidia and as­ 2008). The black Aspergilli were also the most frequently isolated fungi
cospores of these fungi are thermo-tolerant and could stay viable during from green, black and herbal infusions tested in Czech Republic
the tea preparation process. (Řezáčová and Kubátová, 2005). In the study by Cvetnić and Pepeljnjak
According to Škrinjar et al. (2011), members of Aspergillus section (1999), the most prevalent mould isolates in dried medicinal herbs were
were also the dominant group of fungi found in tea samples from various species belonging to the genera Aspergillus, Penicilliumand Mucor. Among
Serbian health food stores and markets. These authors reported the the detected Aspergilli, the most abundant members were Aspergillus,
presence of Eurotium herbariorum and E. awamori, teleomorphic state of Flavi and Nigri section (Cvetnić and Pepeljnjak, 1999).
Aspergillus section in tested Bearberry samples, as well as high heat
treatment survival rate. Řezáčová, Kubátová (2005) reported the high 4. Conclusion
abundance of Eurotium spp. in various types of tea from shops in Prague
(Řezáčová and Kubátová, 2005). Concerning the health issues, Asper­ Taking into account the calculated values of THQ and accumulative
gillus spp. sect. Aspergillus are very seldomly reported as pathogenic HI values for Cd, Pb, As, Mn, Ni, and Cr, results of the health risk
(Siqueira et al., 2016), and are not regarded as toxigenic fungi. However, assessment conducted in this study suggested that the risk of adverse
some extrolites with minor toxic effects, such asechinulin, flavoglaucin effects in adults after the exposure to six tested metals via herbal tea
and auroglaucin are produced by these Aspergilli (Chen et al., 2017). The intake is acceptable. Furthermore, although all tested herbal tea and
high IF was also documented for Aspergillus spp. sect. Nigri (78.57%). infusion samples showed moderate fungal contamination, with the
Black Aspergilli are frequently cited as causative agents of food spoilage highest isolation frequency documented for Penicillium spp. and Asper­
and the main source of these fungi is soil (Varga et al., 2011). Unlike the gillus spp, estimated total fungal count and fungal diversity could be
members of Aspergillus section, the members of Nigri section were iso­ regarded as safe according to the recommended microbiological stan­
lated on both PDA and MY40 media, and could be responsible for both dards. However, in the overall risk assessment process, the presence of
primary and secondary microbial contaminations of plants. Also, it has toxic metal and toxigenic fungi in tea and other herbal products should
been documented that conidia of black Aspergilli could stay viable after not be neglected. Furthermore, risk assessment of toxic metals and
the heat treatment (81.82%), which must not be neglected since they are toxigenic fungi in herbal teas is important not only having in mind their
known as human pathogen and mycotoxin producer (Table 6). High negative effects on human health, but also in order to obtain realistic
isolation frequency (71.43%), ability to grow on low aw, but also mod­ estimates of their intake through these popular beverages.
erate heat treatment survival rate of 30% was recorded for Aspergillus
spp. Sect. Circumdati. Authors’ contributions
Members of both sections, Nigri and Circumdati, as well as some
Penicillia, are producers of ochratoxin A (OTA), one of the most- Marko Krstić: Conceptualization, Methodology, Formal analysis,
abundant food-contaminating mycotoxins (Al-Anati and Petzinger, Investigation, Writing – Original Draft; Miloš Stupar: Methodology,
2006). It is carcinogenic to humans and has been shown to be muta­ Formal analysis, Writing – Original Draft, Danijela Đukić-Ćosić: Meth­
genic, possibly by induction of oxidative DNA damage (Palma et al., odology, Formal analysis, Writing – Review, Editing, Katarina Baralić:
2007). In Balkan countries, the consumption of food with high OTA Writing – Review, Editing, Visualization, Data Curation, Svetlana Đogo
content can be linked to the Balkan endemic nephropathy, chronic renal Mračević: Project Administration, Funding Acquisition, Writing – Re­
disease which often has fatal outcome (Castegnaro et al., 2006). Thus, view, Editing
the presence of OTA producers in tea and other herbal products must not
be neglected. Funding
On the other hand, in research presented here, aflatoxins producers
from Flavi section were less frequntly isolated (57.14%) and showed no This work was partially supported by The Ministry of Education,
survival after heat treatment. Aflatoxins are toxigenic, carcinogenic, Science and Technological Development of the Republic of Serbia (451-
mutagenic and teratogenic. High level of exposure to these fungal 03-9/2021-14/200161 and 451-03-68/2020-14/ 200178).
extrolites could lead to an acute necrosis, cirrhosis, and liver carcinoma
(Groopman et al., 1988).
The fungal diversity documented in this research corresponds with Declaration of Competing Interest
other researches worldwide regarding this topic. To name a few, Bugno
et al. (2006) reported that Aspergillus spp. and Penicillium spp. are major The authors declare that there is no conflict of interest.
fungal contaminants responsible for microbial contamination of herbal
drugs in Brazil (Bugno et al., 2006). The presence of xerophilica fla­ References
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