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Macro and Trace Elements in Hemp
(Cannabis sativa L.) Cultivated in
Greece: Risk Assessment of Toxic
Elements
Effrosyni Zafeiraki
1
*, Konstantinos M. Kasiotis
1
, Paul Nisianakis
2
and Kyriaki Machera
1
*
1
Laboratory of Pesticides’Toxicology, Department of Pesticides Control and Phytopharmacy, Benaki Phytopathological Institute,
Athens, Greece,
2
Chemical Laboratory, Athens Analysis Laboratories, Athens, Greece
The accumulation of hazardous contaminants in Cannabis sativa L. raises warning signs
regarding possible adverse effects on human health due to the consumption of herbal
medicines and/or other herbal edible products made from cannabis. Thus, there is an urge
to investigate the levels of hazardous contaminants, such as heavy metals, in cannabis
plant. In the present study, 29 macro and trace elements, including both beneficial and
toxic elements (heavy metals and metalloids), were investigated in 90 samples of Cannabis
sativa L.collected from Greece. According to the results, the detected concentrations of
macro elements in the leaves/flowers of cannabis ranged between 28 and 138,378 ppm,
and of trace elements between 0.002 and 1352.904 ppm. Although the concentrations of
elements varied among the samples, their accumulation pattern was found to be similar,
with the contribution of toxic elements to the total concentration of trace elements being
below 1%. The detected levels of the most toxic elements were below the prescribed limits
established by the WHO, while the calculated THQ and CR values showed no risk (non-
carcinogenic and carcinogenic) for the population exposed to the current cannabis
samples. Positive correlation between the concentration of elements and cannabis
geographical origin and variety was observed. Cannabis leaves/flowers were more
contaminated with trace and macro elements than seeds.
Keywords: Cannabis sativa L., trace and macro elements, heavy metals, ICP-MS, THQ
INTRODUCTION
Cannabis sativa L.is one of the earliest and widely cultivated herbaceous plants. It contains more
than 113 cannabinoids, among which cannabidiol (CBD) and tetrahydrocannabinol (Δ9-THC) are
well known for their healing properties and medicinal use (Russo et al., 2007). Apart from its long-
term use for the treatment of pain, spasms, asthma, insomnia, depression, and loss of appetite,
nowadays, Cannabis sativa L. contributes also to the treatment of nausea and vomiting associated
with cancer chemotherapy, spasticity in multiple sclerosis, and anorexia in HIV/AIDS (Colorado
Department of Public Health and Environment (CDPHE), 2016). In addition, there is substantial
evidence that cannabinoids are also effective in movement disorders and neuropathic pain
(Grotenhermen and Muller-Vahl, 2012).
Besides its medicinal use, Cannabis sativa L. finds application in more than 25.000 products
globally, including industrial cannabis (hemp) and numerous edible products (Salentijn et al., 2015).
Edited by:
Alberto Salomone,
University of Turin, Italy
Reviewed by:
Alice Ameline,
Hˆopitaux Universitaires de
Strasbourg, France
Przemyslaw Niedzielski,
Adam Mickiewicz University, Poland
*Correspondence:
Effrosyni Zafeiraki
e.zafeiraki@bpi.gr
Kyriaki Machera
k.machera@bpi.gr
Specialty section:
This article was submitted to
Analytical Chemistry,
a section of the journal
Frontiers in Chemistry
Received: 15 January 2021
Accepted: 16 February 2021
Published: 22 April 2021
Citation:
Zafeiraki E, Kasiotis KM, Nisianakis P
and Machera K (2021) Macro and
Trace Elements in Hemp (Cannabis
sativa L.) Cultivated in Greece: Risk
Assessment of Toxic Elements.
Front. Chem. 9:654308.
doi: 10.3389/fchem.2021.654308
Frontiers in Chemistry | www.frontiersin.org April 2021 | Volume 9 | Article 6543081
ORIGINAL RESEARCH
published: 22 April 2021
doi: 10.3389/fchem.2021.654308
Cannabis seeds consist of 20–35% oil, which makes them
appropriate for the production of cooking/seasonal oil, dietary
supplements, plant-based superfoods, beverages, and also body
care products, fuel, paint, etc., while the fiber of cannabis (stalk
part) is useful for the production of textiles, insulators, ropes,
paper, and biomaterials. Meanwhile, the increasing demand and
use of its medicinal and food products has led to the harvest of
mainly leaves and seeds in approximately 30 countries. Another
viewpoint not to disregard is that the consumption of raw
unprocessed cannabis is gaining ground as well, since it is
believed that raw leaves and buds are also rich in nutrients
(
˙
Zuk-Gołaszewska and Gołaszewski, 2018).
As cannabis can accumulate both natural and anthropogenic
contaminants of high concern during its growth, it is considered a
potential source of risk for human health (Fu et al., 2018;Craven
et al., 2019;Atapattu and Johnson, 2020). In particular, in
cannabis, trace and macro elements can build up, including
also toxic ones, mainly via the soil and water in which it
grows, or through the deposition of fertilizers, pesticides, and
fungicides that are commonly applied to crops and contain such
elements (Galic et al., 2019). The variety of the plant, harvesting
time, geographical origin, topography, and duration of the
exposure to the contaminants are factors playing an essential
role in the accumulation of elements in the cannabis plant
(Arpadjan et al., 2008;Nagajyoti et al., 2010).
The high applicability of Cannabis sativa L. in medicines and
food products, combined with the ability of the plant to
accumulate contaminants, has raised concerns regarding the
safety of the product (Ware and Tawfik, 2005). According to
the WHO, “This risk can be confined by ensuring that herbal
medicines possessing harmful contaminants and residues do not
reach the public, by evaluating the quality of the medicinal plants,
herbal materials, and final herbal products before they reach the
market”(World Health Organization, 2007).
To this end, the aim of the current study was to investigate the
occurrence of trace and macro elements, including both beneficial
and toxic elements (heavy metals and metalloids), in the leaves/
flowers of 90 hemp samples collected from Greece (2018–2019), a
representative Mediterranean country. Although the specific
samples were intended for industrial purpose, their mineral
composition can be used as a surrogate to potentially reflect
the respective content of cannabis intended for human
consumption. It is noteworthy that the main difference
between hemp and other cannabis focuses on the
differentiation of THC and CBD levels. In addition, the
provisions for the production of medical cannabis in Greece
are governed by the Greek Law 4523/2018 (OGG, 2018) that is in
place from March 2018; hence, such samples were not relevant to
this study. Consequently, the human health risk assessment of the
most toxic elements is attempted and presented. Considering
Greece’s geology and the diversity of the composition of the soil
around the country, potential differentiations between elements’
accumulation and sampling location of cannabis are also
investigated. In the same context, the variety of the plants is
also investigated as another potential factor influencing the
accumulation of elements in cannabis. In order to further
examine the distribution of the elements among the different
parts of cannabis, the leaves/flowers and the seeds of 21 samples
are separated and further analyzed. For the analysis of all the
cannabis samples and the detection of trace and macro elements
in them, an inductively coupled plasma mass spectrometry (ICP-
MS) is used.
Overall, the current study was conducted in an effort to fill
knowledge and literature gaps of the particular scientificfield
being explored. It is important to stress that the novelty of the
study lies both in terms of addressing the problem of metals’
occurrence in cannabis, which although of local character
informs one of the very few relevant studies in the field, and
in terms of establishing a multi-analyte method for the
quantification of several minerals. What is more? To our
knowledge, this is the first study presenting information on
several trace and macro elements in such a large number of
cannabis samples cultivated in Greece, by using an ICP-MS.
MATERIALS AND METHODS
Sample Collection
Sampling was carried out during a 2-year period, that is,
2018–2019. Ninety samples of 9 different varieties of cannabis
plants cultivated in 13 regions in Greece were collected by several
producers and delivered to the Benaki Phytopathological Institute
(Figure 1 and Supplementary Table S1). Upon arrival of the
samples at the lab, 5 or more individual branches collected from
each crop were pooled and further treated for their analysis. The
varieties of cannabis collected were the following: Finola, Futura
75, Fedora 17, Gamagnola, Fellina 32, Dora, CS, Fibror 79, and
Compolti.
Chemicals
The analytical method applied in the current study is suitable for
the quantification of 29 chemical elements: boron (B), sodium
(Na), magnesium (Mg), aluminum (Al), phosphorus (P),
potassium (K), calcium (Ca), titanium (Ti), vanadium (V),
chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel
(Ni), copper (Cu), zinc(Zn), arsenic (As), selenium (Se), strontium
(Sr), molybdenum (Mo), silver (Ag), cadmium (Cd), tin (Sn),
antimony (Sb), barium (Ba), mercury (Hg), thallium (Tl), lead
(Pb), and uranium (U). For the quantification of the elements,
inductively coupled plasma mass spectrometry (ICP-MS) and
internal standards (IS) were used. The standard solution/
mixture of 25 components (Al, Ba, B, Cu, Fe, Sr, Zn, Be, Cr,
Co, Li, Mn, Mo, Ni, Ti, V, Sb, As, Cd, Pb, Se, Ag, Tl, U, andSn) and
the standard solutions of individual elements (Hg, Na, Mg, Ca, P,
and K) used for the preparation of two calibration curves, and the
solution of elements (lithium (
6
Li), scandium (Sc), germanium
(Ge), ytrium (Y), indium (In), terbium (Tb), and iridium (Ir)) used
as internal standard, were all provided by CPAchem (Stara Zagora,
Bulgaria). The standard solution/mixture of 25 components (Al,
Be, Co, Li, SeL, Sn, Zn, Sb, B, Cu, Mn, Ag, Ti, As, Cd, Fe, Mo, Sr, U,
Ba, Cr, Pb, Ni, Tl, and V) used for the preparation of quality
assurance (QC) standard was also purchased from CPAchem
(Stara Zagora, Bulgaria). The two CRMs, BCR-191 brown bread
and BCR-679 white cabbage, were provided by the European
Frontiers in Chemistry | www.frontiersin.org April 2021 | Volume 9 | Article 6543082
Zafeiraki et al. Macro and Trace Elements in Hemp
Commission. Nitric acid (HNO
3
)67–69%, hydrochloric acid 37%
(HCl), and hydrogen peroxide (H
2
O
2
) for trace elements analysis/
trace metal grade were all purchased from Seastar Chemicals Inc.
(Sydney, Canada). Finally, ultrapure Milli-Q water was also used
for the dilution of all the aforementioned solutions, when needed.
Sample Preparation
All samples were dried overnight in the oven at 60°C, for the
reduction of moisture content. To separate the parts of the plant,
the dried cannabis samples were sieved, and the leaves/flowers
were further selected. The seeds of 21 samples were further
separated and subjected to the same treatment as leaves. The
selected part of cannabis samples was then grounded by an
electronic blender, and the final powder was kept into
polyethylene plastic bags for the avoidance of contamination/
adsorption of elements until their digestion.
The digestion of all the samples was performed in a microwave
oven (MARS 5, CEM). In particular, 0.25 g of each sample were
weighted and placed into a vessel. 5 ml HNO
3
, 3 ml ultrapure
water, 2 ml HCl, and 2 ml H
2
O
2
were then added in each vessel,
and the samples were further digested into the microwave
digestion system. The duration of the digestion process for
each sample was 40 min, with a ramp time of 25 min. The
maximum microwave power and temperature applied were
800 W and 180°C, respectively. After the end of the digestion
process, the vessels were cooled to less than 40°C into a clean
hood, while excess pressure was vented slowly. The digestion
solution was quantitatively transferred to a clean container, and
ultrapure water was added until a final volume of 100 ml was
reached.
Instrumental Analysis
All the cannabis samples were analyzed by using inductively
coupled plasma mass spectrometry (ICP-MS)-Thermo iCAP-RQ,
equipped with an ASX-280 autosampler. Analysis was performed
by applying collision cell mode (kinetic energy discrimination
(KED)), using He (collision gas flow: approximately 4.78 ml/min
(autotune dependent)), to selectively attenuate all polyatomic
interferences based on their size. The instrument used Ni sample
and skimmer cones, MicroMist U-Series Nebulizer (0.4 ml/min
with PEEK connector), and a Quartz cyclonic spray chamber.
Plasma power was equal to 1550 W, while the flow of the
nebulizer gas and the cool gas was approximately 1 L/min
(autotune dependent) and 14 L/min, respectively. The
preferred isotopes and the corresponding internal standard for
each isotope used are presented in Supplementary Table S2.
Prior to the analysis of the samples, the ICP-MS system was
allowed to equilibrate for 30 min, and then, the sensitivity and the
stability of the instrument were checked in KED mode by using
tune solution containing 1 μg/L (each) of Ba, Bi, Ce, Co, In, Li,
FIGURE 1 | Map indicating the locations where the samples of Cannabis sativa L. were collected during 2018 and 2019.
Frontiers in Chemistry | www.frontiersin.org April 2021 | Volume 9 | Article 6543083
Zafeiraki et al. Macro and Trace Elements in Hemp
and U in 2% HNO
3
and 0.5% HCl. Then, a performance test in
KED mode was performed using the same tune solution. When it
was necessary, autotune and calibration mass tests were also
performed, in order for the equipment to be optimized. To this
end, the analysis of the samples was further conducted with high
sensitivity, stability of signal, and low levels of doubly charged
ions and cluster ions.
Quantification and Quality Assurance
Calibration curves covering concentrations from 0.1 to
1,000 μg/kg and from 0.1 to 200 mg/kg were prepared to be
matched with the expected concentration ranges of trace
elements (B, Al, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr,
Mo, Ag, Cd, Sn, Sb, Ba, Hg, Tl, Pb, and U) and macro elements
(Na, Mg, P, K, and Ca) in the samples, respectively. The
coefficient of determination (r
2
) was greater than 0.999 for all
the calibration curves. Gold (Au) was added in all the calibration
standards for the stabilization of mercury, while internal
standards (
6
Li, Sc, Ge, Y, In, Tb, and Ir) were added at a
constant rate and concentration to all unknown samples and
calibration standards.
The limit of detection (LOD) was the concentration value
corresponding to three times the standard deviation obtained
from the consecutive measurements of 10 reagent blanks, while
the limit of quantification (LOQ) was equal to ten times the
standard deviation of the latter. Verification of LOQ values was
also made by the analysis of 12 replicates of spiked aqueous
solutions. The final LOQ values and the estimated uncertainty
(u’) for each element are presented in Supplementary Table S2.
BCR-CRM 191 brown bread and BCR-CRM 679 white
cabbage were measured in the same batch with the unknown
samples in order to monitor and assure the accuracy of the
measurement. Bread and cabbage were chosen as reference
materials due to their similarity to the matrix of cannabis
plant. For further verification of the accuracy of the method,
the certified reference materials and two samples were analyzed
by two different laboratories, both applying microwave digestion
and ICP-MS for the analysis of the samples. No significant
differences were observed between the obtained results of the
two labs. In addition, two QC standard solutions of macro and
trace elements, respectively, were measured in every sequence.
The recovery of the two CRMSs, the two QCs, and the internal
standards ranged between 80 and 120% in all the samples,
verifying the sufficient ionization of the elements and the
absence of matrix effect. For the avoidance of spectral
interferences, one additional isotope was measured when
possible, while reagent blanks were prepared under the same
conditions as the samples and measured in every batch.
Statistical Analysis
Statistical analysis was performed by using the SPSS software
(IBM SPSS statistics for Windows). The Shapiro–Wilk test was
performed in order to investigate the normality of variance
between the element concentration and the sampling location,
and between the former and the variety of cannabis samples. The
nonparametric Kruskal–Wallis test was also applied in order to
examine statistically significant differences between the
concentration of each individual element and the sampling
locations or the variety of the samples. The statistical
significance level was acceptable at p<0.05. Possible
correlation of each element concentration between the leaves
and the seeds of the same cannabis plants was also investigated by
applying the Pearson correlation coefficient test.
Human Health Risk Assessment
Although there are several pathways through which humans can
be exposed to heavy metals and trace elements, the main route is
the dietary consumption (ingestion), representing a 90% of the
overall health risk (Doabi et al., 2018;Guo et al., 2020). Hence, in
the presented work, calculations for risk assessment were
regarded only by this pathway.
In the current study, the estimated daily intake (EDI) of the
most toxic elements was calculated for both non-carcinogenic
and carcinogenic risks, based on the following equation (Eq. 1):
EDI FIR ×C×EF ×ED ×CF
BW ×AT ,(1)
where C is the concentration of the element (mg/kg), FIR (food
intake rate): 3 g per person per day, EF (exposure frequency):
365 days/year, ED (exposure duration): 27 and 70 years for non-
carcinogenic and carcinogenic risk, respectively, AT (average
exposure time (crop)) for noncarcinogens: 365 days*ED, BW
(average body weight): 70 kg for adults, and CF: unit
conversion factor.
Non-carcinogenic Risk
The target hazard quotient (THQ) was further estimated, for the
investigation of the potential non-carcinogenic risk of cannabis
samples. The calculation of THQ was based on the following
equation (Eq. 2):
THQ EDI
RfD,(2)
where RfD is the oral reference dose (mg/kg/day).
Cumulative health risk was assessed by calculating the hazard
index (HI). The latter was derived from the summation of all
separate THQs (k number of heavy metals regarded),
considering the ingestion pathway using the equation below
(Eq. 3):
HI
n
k1
THQk.(3)
Carcinogenic Risk
The carcinogenic risk accounts for the cumulative probability of
developing cancer in an individual’s lifetime as a result of the
exposure to a potential carcinogen. Consequently, two endpoints
were considered: the EDI of each toxic element and the oral
cancer potency (or slope) factor (CPF
o
). To determine the EDI for
carcinogenic risk, the same equation was used (Eq. 1), projecting
AT to the average lifetime (365 days * 70 years 25,550 days).
CPF
o
represents a metric of cancer risk, defined as the justifiable
upper-bound estimate of the probability that an individual will
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Zafeiraki et al. Macro and Trace Elements in Hemp
develop cancer if exposed to a chemical for a lifetime of 70 years
(Farris and Ray, 2014). Values were retrieved mainly from the US
Environmental Protection Agency (US EPA, 2020), the Office of
Environmental Health Hazards Assessment (OEHHA, 2020),
and the Risk Assessment Information System (RAIS, 2020). In
this regard, the cancer risk (CR) was assessed using the following
equations (Eqs. 4,5)
CRkEDI ×CPFo,(4)
CR
n
k1
CRk.(5)
CR was considered negligible when it was below 1 ×10
–6
, and
likely harmful when above 1 ×10
–4
. Values within the range 1 ×
10
–6
to 1 ×10
–4
signify an acceptable or tolerable risk.
RESULTS AND DISCUSSION
Levels of Trace and Macro elements in the
Leaves/Flowers of Cannabis Sativa L
In the current study, 29 elements were quantified in 90 samples of
Cannabis sativa L. leaves/flowers. The distribution of the detected
concentrations of the analyzed elements and the skewness are
shown through displaying the data quartile and averages (Figures
2,3), while the concentrations of individual elements for each of
the cannabis sample are illustrated in Supplementary Table S3.
The elements quantified can be divided into two main
categories based on their toxicity: macro elements (Mg, Na, K,
P, and Ca) and trace elements (B, Al, Ti, V, Cr, Mn, Fe, Co, Ni,
Cu, Zn, As, Se, Sr, Mo, Ag, Sn, Sb, Ba, Tl, and U), which are
mainly essential for the body and thus for human health, and
FIGURE 2 | Box plot of macro elements’concentration in cannabis samples.
FIGURE 3 | Box plot of trace elements’concentration in cannabis samples.
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Zafeiraki et al. Macro and Trace Elements in Hemp
toxic heavy metals and metalloids (Pb, Cd, Hg, and As) that are
trace elements characterized by high toxicity and also accused for
the cause of severe health effects.
More specifically, macro elements are nutritionally important
minerals and contribute to the normal growth and function of the
human body. In the current study, Ca was the most predominant
element in terms of detected concentration. The highest Ca
concentration was found in a Fedora 17 sample from Hrakleio,
and it was equal to 138,378 mg/kg. The next element with the highest
concentrations observed was K. According to the results, K
concentration ranged between 13,459 and 54,393 mg/kg. Mg
minimum and highest concentrations detected in the current
cannabis samples were 7,140 and 20,202 mg/kg, respectively. In
the same context, the concentration of two other essential elements
for the body, P and Na, ranged from 2,018 to 15,088 mg/kg, and
between 28 and 1,672 mg/kg, respectively.
Apart from macro elements, there are also essential trace
elements, such as Fe, Zn, Cr, Mn, Co, Cu, Mb, Ni, and Se
(Prashanth et al., 2015). According to the current findings, Fe,
Mn, and Zn had the highest concentrations among the trace
elements quantified. Fe maximum concentration was found in a
Fibror 79 sample from Trikala and was equal to 1,338 mg/kg,
while its average concentration was 401 mg/kg. In cannabis
samples, Zn concentration ranged between 23.1 and
158 mg/kg, with the latter being detected in a sample of CS
variety collected from Messinia. The detected average
concentration of Mn in the leaves of cannabis analyzed was
equal to 195 mg/kg.
Ni is recognized as an essential nutrient for the proper growth
of plants. However, depending on the way of its assumption into
the organism of humans, and to the amount, duration of contact,
and route of exposure, Ni can also be responsible for several
adverse effects, such as asthma, dermatitis, gastrointestinal
manifestations, cardiovascular diseases, lung fibrosis,
respiratory track cancer, and nasal cancer (Genchi et al.,
2020). The International Agency for Research on Cancer
(IARC) has already classified soluble and insoluble Ni
compounds as Group 1 (carcinogenic to humans) (IARC, 2012).
In the same context, although Cu is an essential trace element
for the body and also a nutrient for plants, human exposure via
digestion of high levels of copper, around 70 mg/day, can lead to
serious adverse effects on health, including liver damage and
gastrointestinal symptoms (National Research Council
Committee on Copper in Drinking Water, 2000;World Health
Organization, 2007). Considering that Cu is strongly
bioaccumulated in nature, the likelihood of exposure to copper
is heightened. Thus, the WHO recommends the control of copper
levels in plants, like herbal ones, that are likely to persist Cu
(World Health Organization, 2007).
Similar to Cu and Ni, Cr can be both essential and toxic. The
toxicity of Cr depends on the oxidation state of the metal. In
particular, Cr (VI) has been associated with increased incidents of
lung cancer, DNA damage, chromosomal aberrations, and
alterations in the epigenomic instability (Zhitkovich, 2005),
while Cr (III) is an essential nutrient, playing an important
role in glucose and lipid metabolism. Even though the use of
large doses of Cr (III) supplements contributes to the
improvement of glucose metabolism, there is a growing
concern over the possible genotoxicity of these compounds
(Stearns, 2000).
Cetain heavy metals (Hg, Pb, and Cd) and metalloids (As) are
also bioaccumulated in nature and food commodities, provoking
toxic effects even at low concentrations and regardless of their
oxidation state. Indicatively, mean concentrations of Hg in
several fish species (from United States, Canada) varied from
0.095 to 0.976 mg/kg; hence, in some cases, the maximum
permissible concentration (or action level) of 1 mg/kg (or ppm)
ascribed by the US Food and Drug Administration was
approximated (Rice et al., 2014, and references therein).
Biomonitoring studies showed for Hg a cord blood concentration
of 0.085 mg/L to be linked to early neurodevelopmental effects
(Centers for Disease Control and Prevention (CDC), 2019).
Nevertheless, the reported mean levels of Hg in blood in the US
population from 2003 to 2016 (National Biomonitoring Program)
were lower than the aforementioned concentration (Centers for
Disease Control and Prevention (CDC), 2019).Inthesamecontext,
for Cd, the highest measured urine concentrations (mean levels did
not surpass 0.06 μg/L) were in proximity, but did not supersede the
levels associated with indications of kidney alterations (Centers for
Disease Control and Prevention (CDC), 2019). Human exposure to
Cd mainly occurs due to the consumption of contaminated food,
inhalation of tobacco smoke, and inhalation by workers in a range of
industries (Rahimzadeh et al., 2017). In particular, Cd
predominantly accumulates in the kidney and liver, exerting toxic
effects on these organs. Cd can cause oxidative stress (Cuypers et al.,
2010;Patra et al., 2011), epigenetic changes in DNA expression
(Wang et al., 2012), renal dysfunction, diabetes (Edwards and
Prozialeck, 2009), hypertension (Gallagher and Meiker, 2010),
and impair vitamin D metabolism (Joint FAO/WHO Food
Standards Program, 2011). Regarding Pb, it has been blamed for
a wide range of biological effects, including hematological,
neurological, behavioral, renal, cardiovascular, and reproductive
system effects (Flora et al., 2012). Depending on the level and
duration of exposure, symptoms can vary, while children are
more vulnerable to the effects of Pb than adults. According to
the WHO, “lead exposure can have serious consequences for the
health of children. At high levels of exposure, Pb attacks the brain
and the central nervous system to cause coma, convulsions, and even
death”(World Health Organization, 2019;Joint FAO/WHO Food
Standards Program, 2011). Hg or methylmercury [MeHg]
+
is toxic
for the central and peripheral nervous system. The inhalation of Hg
vapor can cause harmful effects in the nervous system, lungs,
kidneys, and digestive and immune system, or even ends up to
become fatal. Neurological and behavioral disorders, motor
dysfunction, memory loss, and headaches have been already
observed after the inhalation, ingestion, or dermal exposure to
Hg (World Health Organization, 2017). Moreover, long-term
exposure, of minimum five years, to As usually leads to skin
lesions and cancer, while cancer in the bladder and lungs is also
possible. Other adverse effects that maybe related to the long-term
ingestion of As include diabetes, cardiovascular disease,
developmental effects, adverse pregnancy outcomes, and infant
mortality. On the other hand, the immediate symptoms of acute
As poisoning, that is, the exposure to As occurring over a short
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Zafeiraki et al. Macro and Trace Elements in Hemp
period of time (often less than a day), include vomiting, abdominal
pain, and diarrhea, followed by numbness of the extremities and
muscle cramping, and maybe death (World Health Organization,
2018).
Considering that toxic metals are abundant on nature, they
are likely to be present in many foods, and thus the ensurance
of the safety of herbal products is of major importance. To this
end, the WHO has already established guidelines in order to
assess the quality of herbal medicines and products and
prescribed maximum concentration limits for the toxic
elements (As, Pb, Cd, Cr, and Hg) (World Health
Organization, 2007). Recommendation levels for Cu and Ni
in raw herbal materials are not established yet, and thus these
elements are not included in Table 1.
By comparing the obtained levels of key toxic elements with the
limits prescribed for raw herbal materials (intended for herbal
medicines use) by the WHO, it was found that none of them
exceeded the prescribed limits (Table 1). In particular, the
concentrations of Hg, Pb, Cd, and As were found to be lower
than the standards in all the samples, except for one sample where
Cd concentration was 1.4 times higher than the recommended level.
The obtained levels of Hg, Pb, Cd, and As are not believed to
comprise a risk for human health, based on the mentioned
established limits.
Apart from the elements above, Cr concentrations were also
compared to the limits prescribed by the WHO, and it was
observed that the levels of Cr exceeded the limit in
approximately 25% of the analyzed samples. To this end,
further investigation on the quality assurance of cannabis
plants before their use in medicinal and edible products is
strongly recommended, in order for humans to avoid chronic
exposure to toxic elements, such as Cr.
The current results are in agreement with a previous study
investigating heavy metals in medicinal plants, including
Cannabis sativa L., in which Pb and Cr concentrations
exceeded the limits set by the WHO, raising warning signs
regarding the ingestion of herbal medicines (Kumar et al., 2018).
However, both in the present study and in Kumar’s study, Cr
levels were higher than the standards of the WHO, and Cr current
concentrations (range: 0.337–7.89 mg/kg) were lower than the
ones detected in the Kumar’s study (range: 17.6–58.6 mg/kg). In
the same context, other studies focusing on metals in Cannabis
sativa L. reported higher levels of heavy metals than the ones of
the present study (Zerihum et al., 2015). On the other hand, in
another study, quantifying toxic elements in medicinal food
homologous plants, the detected levels of As, Cd, Hg, and Pb
in the fruit of Cannabis sativa L. were at the same range with the
current ones (Fu et al., 2018). Similarly, in a previous study
examining the levels of macro and microelements in various
herbs, Cd detected concentration was close to the current findings
(Moghaddam et al., 2020). In contrast, the mean Pb
concentration in Moghaddam’s study was equal to 6.32 mg/kg,
while in the present study, it was 0.430 mg/kg. Regarding the rest
of the common elements analyzed in the two aforementioned
studies, their concentration was in most of the cases lower than
that in the present study.
TABLE 1 | Concentration ranges of the most toxic elements compared with the maximum limits set by the WHO.
Element Minimum concentration (ppm) Maximum concentration (ppm) Average of C (ppm) WHO 2007 recommendation
levels (ppm)
a
Hg 0.006 0.107 0.020 0.2
Cd 0.007 0.431 0.049 0.3
Pb 0.095 1.752 0.433 10
As 0.031 0.742 0.159 5
Cr 0.337 7.886 1.686 2
a
Canadian values
FIGURE 4 | Average contribution (%) of macro and trace elements
among all the cannabis samples.
Frontiers in Chemistry | www.frontiersin.org April 2021 | Volume 9 | Article 6543087
Zafeiraki et al. Macro and Trace Elements in Hemp
Finally, it is worth mentioning that a more extended
comparison of cannabis plants’contamination with elements is
hampered due to the insufficient available data. Thus, the
collection and analysis of more cannabis samples would be
necessary in order to reach a more solid conclusion.
Element Pattern
Despite the difference in levels, the pattern of the elements was
found to be similar among all the cannabis samples, with Ca and
Fe being the most predominant elements in macro and trace
elements, respectively. The contribution of each macro and trace
element to the total concentration of elements was calculated
based on the average concentration of each element and is
presented in the following figures (Figure 4).
More specifically, Ca and K were found to contribute more
than the other macro elements to the total concentration of the
latter in all the cannabis samples. In particular, Ca contributed
61%, followed by K (21%) and Mg (11%). As far as trace elements
are concerned, Fe and Al were detected in the highest
concentrations, covering 27 and 24% of the total detected
concentrations in all the cannabis samples.
At this point, it is worth mentioning that the content in toxic
heavy metals and metalloids was very low in all the cannabis
samples (below 1%).
Geographical Origin and Variety of
Cannabis Plants
The concentration of macro and trace elements accumulated by
cannabis varies, depending on several factors, such as the type and
the variety of the plant, the geographical origin (soil) where the plant
grew, the application of pesticides and fertilizers, the drying methods
of the hemp, and the storage conditions (Galic et al., 2019).
In the current study, the samples were collected from 13 different
locations around Greece (Aitoloakarnania, Arkadia, Messinia,
Magnisia, Evoia, Voiwtia, Thessaloniki, Hrakleio, Hleia, Trikala,
Karditsa, Larisa, and Korinthia) and consisted of 9 different
varieties of cannabis (Finola, Futura 75, Fedora 17, CS, Dora,
Carmagnola, Compolti, Fibror 79, and Fellina 32)
(Supplementary Table S1).
In order to investigate a possible correlation between the
detected concentration of elements in each sample and the
geographical origin of the samples, cannabis plants were
grouped based on the location in which they grew,
regardless of their variety. A Shapiro–Wilk test (p>0.05)
and a visual inspection of their normal Q–Qplotsshowedthat
element concentrations were not normally distributed among
thesamplinglocations.Tothis end, the nonparametric
Kruskal–Wallis test was further applied. According to the
result, a statistically significant difference was observed
between the concentration of Na, K, Ca, B, Mn, Ni, Cu,
Zn,As,Sr,Mo,Cd,Ba,Tl,andU,andthegeographical
origin of the samples. For the rest of the elements, the pvalue
washigherthan0.05,revealing that the difference between
their detected levels and sampling points was insignificant.
More specifically, the locations among which a statistically
significant difference for the aforementioned elements was
observed are illustrated in the Supplementary data
(Supplementary Figure S1).
The current findings were in agreement with a previous
study, showing that the sampling site influences the
accumulation of most of the analyzed metals in Cannabis
sativa L. (Zerihum et al., 2015). This could be attributed to
the different soil composition of each area, and also to the
pesticides or fertilizers used for the cultivation of Cannabis
sativa L. in each field. However, since soil was not investigated
in the current study, and the lists of pesticides/fertilizers
applied in the fields were not available, further research on
this topic is recommended.
Similar to geographical origin, the variety of cannabis was
found to influence the accumulation of certain elements in the
plant. Normal Q–Q plots and Shapiro–Wilk test were applied for
the evaluation of the normality among the different cannabis
varieties. According to the results, the samples were not normally
distributed, and thus, a nonparametric Kruskal–Wallis test was
further applied, showing statistically significant difference (p<
0.05) between the concentrations of Na, K, Ca, Ni, Cu, As, Sr, Mo,
Cd, Ba, and U, and the varieties of the samples. More specifically,
the varieties among which there was a statistically significant
difference are presented in Supplementary Figure S2, for each
individual element.
The current variation in metals’accumulation among cannabis
varieties could be attributed to different anatomical and chemical
characteristics of the plant, such as soil type, stage of growth, and
metals absorbed (Verma et al., 2007;Olowoyo et al., 2012). The
current findings are in accordance with a previous study, presenting
differentiation of heavy metals concentration among different
medicinal plants, including Cannabis sativa L. (Kumar et al., 2018).
Distribution of Elements Between Leaves/
Flowers and Seeds of Cannabis Plant
In 21 out of the 90 samples, both leaves/flowers and seeds were
separated and further analyzed for the investigation of elements
distribution between the two parts of the plant. Leaves/flowers
were found to accumulate more trace and macro elements than
the seeds of the same sample, regardless of the cannabis variety
and the location where the plant grew (Supplementary Table S4).
The current results were in consistency with a previous study
reporting that the levels of As, Cd, Cr, Fe, Ni, and Hg in leaves
exceeded those of cannabis seeds (Eboh and Thomas, 2005). For
the investigation of a possible correlation of the concentration of
each element between leaves and seeds, Pearson correlation tests
were applied. According to the results, the correlation between
the two groups was found to be significant (p<0.05) for the
following elements: Na, Al, Mn, Co, Ni, Cu, Zn, As, Se, Sr, Mo,
Cd, Ba, Tl, and U (Supplementary Figure S1). At this point, it is
worth mentioning that among the most toxic elements, As and
Cd were the only ones presenting significant correlation between
leaves and seeds. In addition, further investigation on elements’
accumulation in the different parts of Cannabis sativa L. is
strongly recommended since the obtained information could
contribute to the assurance of the safety of edible cannabis
products made of these parts of the plant.
Frontiers in Chemistry | www.frontiersin.org April 2021 | Volume 9 | Article 6543088
Zafeiraki et al. Macro and Trace Elements in Hemp
Human Health Risk Assessment
Estimated Daily Intake (EDI)
To our knowledge, there is limited information available on the
consumption of edible cannabis products. Most of the studies
reporting an intake rate of cannabis are mainly focused on the
dosage of medical cannabis. Based on these studies, the daily food
consumption rate of smoked or orally ingested cannabis for
medical purposes was approximately 0.65–3 g of dried
cannabis per person per day (Government of Canada, 2016).
To investigate the estimated daily intake (EDI) at the worst-case
scenario, an assumption was made, and the maximum reported
amount of cannabis (3 g) was used. Based on Eq. 1,theEDIsofAs,
Cd, Hg, Pb, Cr, and Ni were calculated based on the concentration of
each element in each cannabis sample. Children and adolescents
were not regarded in the health risk assessment since they are not
considered major “consumers”of cannabis-containing products
(particularly of raw cannabis). The calculated EDIs of each
element are presented in Supplementary Table S5.
Non-carcinogenic Risk
Based on the previous calculations, the target hazard quotient
(THQ) was next estimated (Eq. 2). The oral RfD values for each
toxic element (As, Cd, Hg, Pb, Cr, and Ni) were retrieved from the
European Food Safety Authority (European Food Safety Authority
(EFSA), 2009;European Food Safety Authority (EFSA), 2012a;
European Food Safety Authority (EFSA), 2012b)andUSEPA
and were equal to 0.3, 1, 0.1, 2, 3, and 20 μg/kg b.w. per day for
As, Cd, Hg, Pb, Cr, and Ni, respectively.
According to the results, all the THQ values were far below 1.
Consequently, the HI values, as a metric of the quantified risk,
were also below 1, indicating that there is no significant risk of
non-carcinogenic effects for the population exposed to the
current cannabis samples, and thus to their products
(Supplementary Table S5). Similar to this outcome, a
previous study displayed that the intake of various plants and
spices could not cause significant health hazard to adults
(Moghaddam et al., 2020).
Carcinogenic Risk
Pb,Cd,Cr,Hg,Ni,andAsareclassified as carcinogenic, while based
on the International Agency for Research on Cancer (IARC, 2012),
Cd, Cr, Ni, and As belong to category 1 of heavy metals inducing
cancer. For Cd and Hg, CPF
o
is not yet assigned. Consequently, the
carcinogenic risk was calculated only for Pb, Cr, Ni, and As, using the
CPF
o
values depicted in Supplementary Table S7.TheresultsofCR
values (Supplementary Table S6) showed a negligible risk, since
these values were far lower than the threshold value of 1 ×10
–6
which US EPA (US EPA, 2020) suggests. Similar conclusion was
derived after summing all individual CR values.
CONCLUSION
To our knowledge, the current study is the first study presenting
insights to the occurrence of macro and trace elements in a
substantial number of Cannabis sativa L. (hemp) samples
collected from different locations around Greece. The analysis of
90 samples of 9 varieties of cannabis showed that all cannabis
samples analyzed contained both macro and trace elements in
their leaves/flowers. Even though the detected concentrations of
elements varied among the samples, the levels of the most toxic
heavy metals and metalloids were below the maximum limits
established by the WHO in all of the analyzed samples,
indicating that no human health risk can be provoked at the first
tier due to the consumption of medicines and edible products based
on the current cannabis samples. Since cannabis is consumed raw
and its products appear on the market for human consumption
either as medicine or as food products, it is important to understand
what is the resulting exposure to elements due to the consumption of
these products, and the subsequent human health risk. In this
context, the EDI, THQ, HI, and CR were further calculated
addressing the most toxic elements. According to the results,
there is no risk (non-carcinogenic and carcinogenic) for the
population exposed to the current cannabis samples, and
consequently to their products. Furthermore, an investigation of
elements’accumulation among the leaves/flowers and seeds of the
same samples was made in order to provide information regarding
the contamination (and its distribution) of the raw material and
further contribute to the safety assurance of the edible cannabis
products made from cannabis leaves and seeds. In addition, the
present study showed positive correlation between elements detected
concentration and cannabis geographical origin and variety.
However, more research is needed to further deepen the
knowledge on elements accumulation in cannabis plants, and as
a consequence to their entrance into the food chain and then to the
human organism.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material; further inquiries can be
directed to the corresponding authors.
AUTHOR CONTRIBUTIONS
EZ: conceptualization, investigation, formal analysis,
visualization, and writing—original draft preparation. KK:
formal analysis and writing—reviewing and editing. PN:
methodology and resources. KM: supervision.
ACKNOWLEDGMENTS
We gratefully acknowledge all the producers who helped us to
carry out this study by providing cannabis samples from their
private cannabis fields in Greece.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fchem.2021.654308/
full#supplementary-material.
Frontiers in Chemistry | www.frontiersin.org April 2021 | Volume 9 | Article 6543089
Zafeiraki et al. Macro and Trace Elements in Hemp
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Zafeiraki, Kasiotis, Nisianakis and Machera. This is an open-
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