Food Additives & Contaminants: Part B

Surveillance

ISSN: 1939-3210 (Print) 1939-3229 (Online) Journal homepage: http://www.tandfonline.com/loi/tfab20

Mycotoxins, pesticides and toxic metals in

commercial spices and herbs

Ingars Reinholds, Iveta Pugajeva, Konstantins Bavrins, Galina Kuckovska &

Vadims Bartkevics

To cite this article: Ingars Reinholds, Iveta Pugajeva, Konstantins Bavrins, Galina Kuckovska &
Vadims Bartkevics (2016): Mycotoxins, pesticides and toxic metals in commercial spices and
herbs, Food Additives & Contaminants: Part B

To link to this article: http://dx.doi.org/10.1080/19393210.2016.1210244

Accepted author version posted online: 09

Jul 2016.
Published online: 09 Jul 2016.

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 Publisher: Taylor & Francis

Journal: Food Additives & Contaminants: Part B

DOI: 10.1080/19393210.2016.1210244
Mycotoxins, pesticides and toxic metals in commercial spices and herbs

Ingars Reinholds1*, Iveta Pugajeva1, Konstantins Bavrins1, Galina Kuckovska1, Vadims Bartkevics1

1
Institute of Food Safety, Animal Health and Environment “BIOR” (Latvia).
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Abstract
A total of 300 samples representing six condiments (black pepper, basil, oregano, nutmeg,
paprika, and thyme) were analysed for 11 mycotoxins, 134 pesticides, and 4 heavy metals by
ultra-high performance liquid chromatography – tandem quadrupole mass spectrometry and
inductively coupled plasma mass spectrometry. Mycotoxins were detected in 4%, 10%, and
30% of all nutmeg, basil, and thyme samples, respectively. The residues of 24 pesticides
were detected in 59% of the analysed condiments. The maximum residue levels of pesticides
were exceeded in 10% of oregano and 46% of thyme samples. A risk assessment of heavy
metals was performed, indicating daily intake levels far below the tolerable intake levels.

Keywords: spices; herbs; mycotoxins; pesticide residues; toxic metals; HPLC-QqQ-MS/MS;

ICP-MS; maximum residue limits; regression analysis.

INTRODUCTION
Spices and herbs are widely used in culinary to improve the aroma, taste, and functionality of processed
foods and also applied in medicine, pharmacology, and cosmetology due to their antioxidant properties and
nutritional benefits (Peter 2012). The growing consumption rate emphasizes the issue of safety and quality
control of these products (Datta et al. 2015). Recent reports of the Rapid Alert System of Food and Feed of
the European Commission (RASFF 2014), the Codex Committee of the Food and Agriculture Organisation
and the World Health Organisation (FAO 2014; WHO 2007) have raised concerns about environmental
contamination and adulteration of commercial condiments (Balbus et al. 2013). The progress in analytical
chemistry instrumentation has led to improved selectivity and sensitivity of mass spectrometric methods

*
Corresponding author: Email: Ingars.Reinholds@bior.lv.
 combined with gas chromatography (GC) and high or ultra-high performance liquid chromatography (HPLC,
UHPLC). The use of fluorescence (FLD), triple quadrupole tandem mass spectrometry (QqQ-MS/MS) and
time-of-flight or Orbitrap high resolution mass spectrometry (TOF- and Orbitrap-HRMS) detectors has
enabled reliable quantification of contaminants (pesticides, mycotoxins) at concentrations far below the
maximum limits (MLs) set by the authorities (Maghrabi 2014; Turner et al. 2015; Tosun et al. 2016).
Mycotoxins are highly toxic food contaminants produced by Aspergillus, Fusarium, Penicillium,
and other species of fungi during improper post-harvest processing, storage, or manufacturing of food
products (Do et al. 2015). Aflatoxins and ochratoxin A are considered extremely toxic and carcinogenic to
humans, and their presence has been detected in spices (nutmeg, paprika, turmeric) cultivated in subtropical
climate zones (Gallo et al. 2015). The MLs in the European Union (EU) for aflatoxin B1 and the total
aflatoxins in dried spices/herbs are set to 5 µg kg−1 and 10 µg kg−1 (EC 2010). The ML for OTA in dried
spices, herbs, and their mixtures is equal to 15 µg kg−1 (EC 2012). In 2014, Yogendrarajah et al. reported
aflatoxin AFB1 content elevated above the ML in 65% of commercial chilli samples originating from paprika
cultivated in Sri Lanka. Recently, Özkan et al. (2015) determined AFB1 exceeding the maximum level in
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27% of the samples of ground red chilli pepper harvested in Gaziantep, Turkey. Fumonisins (FB1, FB2),
trichothecenes (deoxynivalenol), and zearalenone, commonly found in unprocessed and processed cereals,
have been found in spices and herbs (Santos et al. 2010). In 2013, Waskiewicz et al. reported elevated
ochratoxin A levels in samples of cayenne pepper and black pepper, while high concentrations of fumonisins
were found in several herbs available on the market in Poland.
Pesticides, insecticides, and herbicides are frequently used in agriculture to protect plants from
insects, pests, pathogens that may produce mycotoxins, and to improve the amount and quality of harvest
(Attallah et al. 2012). The accumulation of pesticide residues in plants may cause toxic and allergenic effects
on human health due to the consumption of contaminated spices and herbs (Akoto et al. 2015). The
maximum residue limits (MRLs) for pesticides have been set in Commission Regulation 396/2005, the latest
information on the MRLs and toxicity of pesticide residues is included in the Pesticide database of the
European Union (EC 2005; 2016). The analysis of pesticide residues in condiments requires the use of
sensitive multi-component methods, which are based mainly on GC-MS or HPLC-QqQ-MS/MS and HPLC-
Orbitrap-HRMS techniques (Masiá et al. 2014).
Metal ions of high or moderate toxicity are common contaminants in agricultural plants arising from
improper waste management, rapid industrialisation and urbanisation of agricultural regions, causing the
accumulation of metal ions in cultivated spices and herbs through water, soil, and air (Tripathy et al. 2015).
The most toxic elements are heavy metals (especially arsenic (As), cadmium (Cd), mercury (Hg), and lead
(Pb)), which have no biological roles in any living species, and may induce a wide range of toxic effects and
developmental abnormalities, especially in children and embryos, have been linked to Alzheimer's and
Parkinson's diseases, autism, and cancer. For these reasons, the presence of heavy metals in agricultural
products, including dried spices and herbs, should be strictly controlled (Donggyu et al. 2015). Among the
heavy metals, chromium has also been recently investigated in spices and herbs due to the acute toxicity and
proved mutagenic effects of chromium(VI) in animal tests. While trace levels of chromium(III) are essential
for the regulation of metabolic reactions in human body, elevated concentrations of chromium may lead to
the formation of chromium(VI) compounds having toxic effects on human health (Haney 2015). The
regulations of tolerable concentration levels for those contaminants may vary depending on the country. The
 majority of regulations are attributed to the European Food Safety Authority, while the limits set by WHO for
the maximum concentrations of As, Pb, Cd, Cr, and Hg in herbal medicines are 5.0 µg g-1, 10.0 µg g-1,
0.3 µg g-1, 2.0 µg g-1, and 0.2 µg g-1, respectively (WHO 2007). MLs of 0.3 µg g-1 and 0.2 µg g-1 have been
set for Pb and Cd in condiments (WHO 2013). Inductively coupled plasma techniques with mass
spectrometric, optical emission, and atomic emission spectrometric detectors (ICP-MS, ICP-OES, and ICP-
AES) are the major analytical approaches used for the assessment of heavy metal contamination of spices and
herbs. It has been reported that ICP-MS ensures a better sensitivity and accuracy for reliable multi-element
screening compared to other methods, while atomic absorption spectroscopy technique is commonly used for
single element assessment (Reinholds et al. 2015; Bua et al. 2016). In 2009, Mubeen et al. reported high
levels of Pb and Cr (up to 70 µg g-1 and 368 µg g-1) found in commercial chilli spices from Pakistan markets,
with the contamination significantly exceeding MLs set by WHO recommendations. Kulhari et al. (2013)
reported Cr levels above the limits recommended by WHO that were found in several medicinal herbs, where
the Cr levels in leaf samples of those products were up to 5 times higher compared to stem samples (Kulhari
et al. 2013). Recent studies by several authors have indicated a common occurrence of heavy metal
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contamination in commercial spices and herbs, indicating a need for a proper multi-contaminant control to
mitigate the risks to human health (Seddigi et al. 2016).
In the context of safety of commercial condiments, the present research deals with a combined
occurrence analysis of multi-class pesticide residues, conventional mycotoxins and the most common toxic
metallic elements (Cd, Pb, As, Cr), which have been assessed in various samples of commercial spices and
herbs distributed in the European market. For the analysis, two highly sensitive mass spectrometric methods
based on HPLC-QqQ-MS/MS and ICP-MS were used in order to evaluate the occurrence of contaminants in
those condiments. Statistical data analysis was used in order to evaluate the interference from various types
of contaminants. Regarding the high quality requirements of commercial foodstuffs, the levels of
contaminants in the samples were compared to the tolerance levels set by the EC Regulations and the
recommendations established by WHO for heavy metals.

MATERIALS AND METHODS

Sample collection

Sample sets of six commercial condiments (three types of ground herbs: basil from India, oregano from
Turkey, and thyme from Poland, and three types of ground spices: blends of black pepper originating from
Brazil and Vietnam, nutmeg from Indonesia, and sweet paprika / chilli blends originating from Brazil and
China) were provided in 2014 by the collaborating company FUCHS Gewürze GmbH (Germany). From all 6
commodity types 50 samples of 100 g were taken, making up 300 samples in total. The samples were
packaged in separate air-tight plastic bags and stored in the dark at room temperature until processing.
Description of analysed samples is given in the excel file of the database.
 Chemicals, reagent, and standards

HPLC grade acetonitrile and methanol were obtained from Merck-Millipore (Darmstadt, Germany). ACS
grade formic acid (≥96.0%), ammonium formate (99%), and nitric acid (≥69.0%) of trace analysis grade were
purchased from Sigma–Aldrich (St. Louis, MO, USA). Hydrogen peroxide (30%) of extra purity was
obtained from Scharlau (Spain). Ultrapure water was generated by a Millipore Milli-Q™ system (Billerica,
MA, USA). A QuEChERS ("Quick, Easy, Cheap, Effective, Rugged, and Safe") buffer-salt mixture was
obtained from Phenomenex (Torrance, CA, USA) and each portion consisted of 1 g trisodium citrate
dihydrate, 1 g sodium chloride, 0.5 g disodium hydrogen citrate sesquihydrate, and 4 g of anhydrous
magnesium sulphate. Disposable Ultrafree® PVDF membrane filters (0.22 µm) were purchased from Merck
Millipore (Darmstadt, Germany). Standards of mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, fumonisin
B1, fusarenon X, HT-2 toxin, nivalenol, ochratoxin A, sterigmatocystin, T-2 toxin, and zearalenone) were
purchased from Romer Labs Biopure (Tulln, Austria). Standards of the 134 following pesticides were
obtained from AccuStandard (New Haven, CT, USA) and Dr. Ehrenstorfer (Germany): acephate,
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acetamiprid, aldicarb, aldicarbsulfone, aldicarbsulfoxide, azinphos-methyl, azoxystrobin, bitertanol, boscalid,

bromuconazole, bupirimate, buprofezin, carbaryl, carbendazim, carbofuran, chlorantaniliprole,
chlorfenvinphos, clofentezine, clothianidin, cymoxanyl, cyproconazole, cyprodinil, diazinon, dichlorvos,
dicrotophos, diethofencarb, difenoconazole, dimethoate, dimethomorph, diniconazole, dodine,
epoxiconazole, ethion, ethirimol, ethoprophos, famoxadone, fenamidone, fenamiphos, fenamiphossulfone,
fenamiphossulfoxide, fenarimol, fenazaquin, fenbuconazole, fenhexamid, fenoxycarb, fenpropimorph,
fenpyroximate, fenthion, fenthionsulfone, fenthionsulfoxide, fluopyram, fluquinconazole, flusilazole,
flutriafol, formetanate, formothion, fosthiazate, hexaconazole, hexythiazox, hydroxycarbofuran, imazalil,
imidacloprid, iprovalicarb, isocarbophos, isofenphos-methyl, isoprocarb, kresoxim-methyl, linuron,
malaoxon, malathion, mandipropamide, mepanipyrim, metalaxyl, metconazole, methamidophos,
methidathion, methiocarb, methiocarbsulfone, methiocarbsulfoxide, methomyl, methoxyfenozide,
metobromuron, monocrotophos, myclobutanil, nitenpyram, omethoate, oxadixyl, oxamyl, oxydemeton-
methyl, paclobutrazole, penconazole, pencycuron, pendimethalin, phenthoate, phosalone, phosmet, phoxim,
pirimicarb, pirimiphos-methyl, profenofos, propamocarb, propiconazole, propoxur, propyzamide,
pymetrozine, pyraclostrobin, pyridaben, pyrimethanil, pyriproxyfen, quinoxyfen, rotenone, spinosynA,
spinosynD, spirodiclofen, spiromesifen, spiroxamine, tebuconazole, tebufenozide, tebufenpyrad,
terbuthylazine, tetraconazole, tetramethrin, thiabendazole, thiacloprid, thiamethoxam, thiodicarb,
thiophanate-methyl, triadimefon, triadimenol, triazophos, trichlorfon, trifloxystrobin, triflumuron,
triticonazole. The multi-element standard solution “5” for ICP (TraceCERT®) in 10% nitric acid was
obtained from Sigma-Aldrich (France).

Mycotoxins and pesticides analysis

Standard solutions and sample preparation

Stock solutions of pesticides and mycotoxins were prepared by dissolving solid standards (approximately
10 mg of each) in appropriate solvents (10 mL). The mix of working standard solutions was prepared at
10 µg mL-1 concentration for mycotoxins and at 4 µg mL-1 concentration for pesticides. Mixed working
 standards were stored at −20°C for no longer than 1 month. Working standard solutions of mycotoxins and
pesticides were used to fortify blank samples at the appropriate concentrations: 10, 50, 100, 250 and
500 µg kg-1. Samples of roughly ground herbs (basil, oregano, and thyme) were pre-ground by using a
porcelain mortar. Samples of spices that were already in ground form (particle size ≤ 150 µm) were used for
analysis without any pre-treatment.

Extraction and sample preparation

A buffered QuEChERS solid-phase extraction (SPE) procedure (EN 15662 2008) was used to obtain the
organic extracts for mass spectrometric analysis. In the cases of calibration and quality control, standard
solutions were added at the appropriate spiking level. Samples (aliquots of 2 g) were weighed into 50 mL
centrifuge tubes and eluted with deionised water (10 mL) and acetonitrile (10 mL). Portions of QuEChERS
buffer-salt mixture were then added to each tube. The samples were vortexed for 1 min and shaken rapidly
for 10 min using an automatic shaker. The sample tubes were centrifuged for 5 min at 4500 rpm using a
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Heraeus Multifuge 3L-R centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). An aliquot of the
organic phase from each sample (8 mL of supernatant) was transferred into a 15 mL PP centrifuge tube and
frozen out at -80°C for 30 min, using the Thermo Fisher Scientific Heto Ultra freeze (Waltham, MA, USA),
followed by centrifugation of the obtained organic sample fraction for 5 min at 4500 rpm. Then the samples
(3 mL) were evaporated to dryness using a TurboVap LV evaporator (Biotage, Sweden) and reconstituted in
500 µL of water/methanol (50:50, v/v). The extracts were filtered through 0.22 µm PVDF centrifuge filters
and transferred to autosampler vials for the analysis.

Instrumentation used for the HPLC-QqQ-MS/MS method

The determination of mycotoxins and pesticides was performed by a previously developed analytical method,
proven to be sensitive for analysis of these contaminants in paprika. The detection parameters (retention
times, quantitative and confirmation ionic transitions and the respective collision energies), as well as the
performance characteristics of both mycotoxins and pesticide residues in condiments are given in Reinholds
et al. (2016). In this survey the method was applied to 5 other sample types: nutmeg, black pepper and 3
different herbs. It is based on a Waters Acquity HPLC system (Milford, MA, USA) coupled to an AB SCIEX
QTrap 5500 QqQ-MS/MS detector (Framingham, MA, USA). Ionisation was achieved by an electrospray
ionisation (ESI) source in positive and negative ion modes. The separation of analytes was performed on a
Phenomenex 100 mm × 2.1 mm i.d., 2.6 µm Kinetex C18 analytical column (Torrance, CA, USA). For
mycotoxin analysis, the mobile phase consisted of (A) water containing 0.1 % formic acid and (B) 100%
methanol with a flow rate of 0.3 mL min-1. The percentage of organic solvent in the mobile phase varied as
follows: 0 min, 5%; 6 min, 5%; 10 min, 95%; 11 min, 95%, 11.1 min 5%, and 15 min, 5%. For pesticide
analysis, the mobile phase of (A) 5 mM ammonium formate in 0.1% formic acid and (B) 100% acetonitrile
was used with the flow rate of 0.4 mL min-1. For pesticide analysis, the percentage of organic solvent varied
as follows: 0 min, 20%; 1 min, 20%; 10 min, 80%; 11 min, 80%; 11.1 min, 20%, and 15 min, 20%. Injection
volume of 10 μL was used both for the analysis of mycotoxins and pesticides. Sample and column
temperatures were 10°C and 40°C, respectively.
 Quality assurance and quality control

Control samples with known standard concentrations (100 µg kg-1) were analysed in each sample batch. The
method performance was estimated from the results obtained by validation studies developed in accordance
to the EC guidance document SANCO/12571/2013 (SANCO, 2013). The following validation parameters
were checked: linearity, accuracy, precision, and sensitivity (Table 2).

Heavy metals analysis

Standard solutions and sample preparation

Each sample (a 0.3 g replicate) was accurately weighed on an analytical balance (±0.0001 g) and transferred
to a PTFE digestion vessel. Deionised water (2 mL) was added, followed by concentrated nitric acid (8 mL)
and concentrated hydrogen peroxide (2 mL). The vessels were kept at room temperature for 2 to 8 h until the
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formation of bubbles ceased. This duration was dependent of the type of matrix, where the longer time was
necessary for herbal samples due to the high content of oils in these matrices. Then the vessels were closed
and placed inside a MARS 6 microwave oven (CEM Corporation, Matthews, NC, USA) for digestion. The
temperature was controlled by the following microwave program: 0 min, 23°C (ambient temperature);
20 min, 150°C; 30 min, 150°C; 50 min, 200°C; 80 min, 200°C; and 120 min, ambient temperature. The
prepared solutions of digested samples were filtered through a filter with 8 – 10 μm pore size, quantitatively
transferred to graduated polypropylene volumetric flasks (only plastic flasks were used), and diluted to 50
mL volume with deionised water. Blank samples of standard solutions were prepared by the same procedure
as used for the analysed samples. The standard solutions used in the construction of calibration curves for
quantification studies, were prepared at concentration levels of 5, 10, 100, 500 and 1,000 μg L-1 using a
standard solution containing 10 mg L-1 of each target element per 100 mL of the mixed standard solution.

The ICP-MS method

Determination of heavy metals was performed on an Agilent 7700x ICP-MS instrument (Tokyo, Japan). The
optimised instrumental parameters are summarised in Table 1. This method was used for the determination of
ICP-MS signals of Cr-52, As-75, Cd-111, and Pb-208 ions. The concentrations of the metallic elements (As,
Cd, Cr, and Pb) were calculated from the obtained external calibration lines, with each line constructed from
five calibration points of elemental standards at 10-1,000 µg L-1 concentration levels and an additional blank
sample measured under the same conditions and adjusted by the least-squares regression analysis.

Quality assurance and quality control

The six-point calibration curves (five standards and one blank) were constructed for all the metal ions. The
calibration curve correlation coefficients were checked to be better than 0.999 before the start of the sample
analysis (Table 1). For quality assurance and quality control purposes, daily analyses of quality control
materials (with the concentrations of 2 µg L-1 and 50 µg L-1) were done to monitor the performance
parameters (accuracy, repeatability) of the ICP-MS method and the cleanliness of vessels (background
 samples). The results of method validation are summarised in Table 3.

Statistical analysis

Descriptive statistics of the determined results were calculated by using MS Office Excel software, and the
Excel add-in “Data Analysis suite” was applied for the linear regression analysis (LRA).

Health risk assessment

Human health risk assessment was performed by estimating the consumer daily intake values (ADI) in order
to describe the “safe” levels of intake expressed on either a daily basis (the tolerable daily intake – TDI), or
on a weekly basis (TWI – the tolerable weekly intake). We used the following formula (ATSDR, 2015) to
indicate the exposure doses according to the mean concentrations determined in the study samples, the
average consumer body weight of 60 kg and the daily human intake of condiments equal to 10 g (1):
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D = C × 10/1000 × T/60 (1)

-1 -1
where: D – exposure dose in µg g per day or µg kg per week. When multiplied by the time factor
(T = 7 days per week), the determined concentrations of mycotoxins and heavy metals were expressed in
µg g-1. For the evaluation of risks to the health of consumers, the obtained exposure values were compared to
the published toxicological data – the provisional maximum tolerable daily or weekly intake values
summarised by the Codex Committee on Food Additives and Contaminants in their recent report about heavy
metals (As, Cd, Pb) and major mycotoxins (FAO, 2014).

RESULTS AND DISCUSSION

Method validation

During the initial stage of this study, once sample preparation, separation and mass spectrometric
detection parameters were optimised, a full validation was performed for both the HPLC-QqQ-MS/MS and
ICP-MS methods. The validation results regarding the quantification of 11 mycotoxins and 134 pesticides
showed an adequate method performance with the following parameters determined for spices (black pepper,
nutmeg) and three herbs: the regression coefficients (R2) of method linearity ranged from 0.90 to 0.99; the
accuracy determined as mean recoveries (calculated for spiked blank samples at 10, 50, 100, 250, and
500 µg kg-1) was in the range 80-118% (except for 200% in the case of fusarenon X) in spices and in the
range 70-97% for the analysis of organic contaminants in the samples of herbs; the relative standard
deviations (RSDs) indicating method repeatability for mycotoxins and pesticides were in the range 8-15% for
spices and 14-20% for herbs, where the higher values for herbal samples could be attributed to the complex
matrices of herbs with a relatively high content of oils. The calculated limits of detection (LODs) and limits
of quantification (LOQs) for most of the components are summarised in Table 2, indicating that this method
is sufficiently sensitive. The LOD and LOQ values were higher for herbal samples due to matrix complexity,
however, the sensitivity and other parameters for the determination of mycotoxins and pesticide residues in
 all analysed matrices were in accordance with the requirements defined by EU Regulations (2006, 2009).
The results of ICP-MS method validation summarised in Table 3 indicate good linearity, sufficient
accuracy and precision, and sensitivity for the detection of toxic metals at trace levels. The assessment of
validation results indicated that the validated methods were fully appropriate for the quantification of toxic
compounds in samples of spices and herbs in accordance to the requirements of EU legislation and the
current state of the art in contaminant analysis.

Mycotoxins in spices and herbs

The previous study of mycotoxin contamination in paprika samples (Reinholds et al. 2016) indicated minor
traces of ochratoxin A, sterigmatocystin and fumonisin B1 (FB1) in sixteen of the fifty analysed samples,
where the less common fumonisin was found at the range of 11-33 µg kg–1 in four of the thirty six sweet
paprika samples and in ten of fourteen hot paprika samples, with the concentration of fumonisin B1 varying
from 26 µg kg–1 to 124 µg kg–1 (Reinholds et al. 2016). The analysis of two spices (black pepper, nutmeg)
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and three herbs (basil, oregano, and thyme) indicated a rather significant difference in mycotoxin
contamination in these condiments. Black pepper and basil were the only matrices in which we did not find
any traces of mycotoxins above the LOD. Only one sample of nutmeg was determined to contain ochratoxin
A at a level close to ML (14 µg kg–1) and one sample contained 25 µg kg–1 of FB1. The rather low
contamination of spices with mycotoxins may be attributed to the stringent selection procedure for raw
materials used by the supplier (Fuchs Gewürze).
In the three analysed herbal matrices, traces of three Fusarium mycotoxins (sterigmatocystin,
zearalenone, and deoxynivalenol) of the 11 analysed mycotoxins were detected only in five oregano samples
(10%) and in fifteen thyme samples (30%). Sterigmatocystin was the only compound determined at trace
levels (15-28 µg kg-1) in the oregano samples, and was also present in two thyme samples (4%) at
concentrations of 10 µg kg-1 and 14 µg kg-1, respectively. Zearalenone and deoxynivalenol were found in
nine (18%) and seven (14%) of the analysed thyme samples, respectively, and the levels of both mycotoxins
were distributed over the ranges of 40-209 µg kg-1 and 10-142 µg kg-1, respectively. The results for thyme
samples may be attributed to agricultural practices in the country of origin (Poland) causing variable
contamination levels depending on the growth and storage conditions.

Pesticides in spices and herbs

Twenty four pesticide residues from the analysed 134 compounds were detected in 59% of the samples that
were analysed. The number of detected pesticide residues ranged from none in nutmeg to sixteen compounds
present at trace or elevated levels in thyme. The concentration levels of pesticide residues determined by
HPLC-QqQ-MS/MS in the analysed samples are given in Table 4.
The residues of two pesticides (carbendazim and metalaxyl) both were found at trace levels (4.0-
25 µg kg-1) below EU MRLs in 49 samples (98%) of black pepper. The results are in agreement with the
studies by Amate et al. (2010), who found a similar range (8.1-17 µg kg-1) of carbendazim residue levels as
the only pesticides present in black and white peppers.
The occurrence rate of pesticide residues in the analysed herbs increased in the following order:
basil < oregano << thyme, where only two insecticides and one fungicide (metalaxyl) were found at trace
levels that were 54% of the levels in basil (Table 4). Oregano contained four insecticides and only one
 fungicide (carbendazim), while nearly all of the detected pesticide residues were found below the determined
MRL levels, with the exception of dimethoate (sum of dimethoate and omethoate) concentration reaching 25
- 44 µg kg-1 in four (8%) of the oregano samples.
A notably high variety and number of pesticide residues (16 different compounds) were detected in
the analysed samples of thyme, which contained the residues of 7 insecticides and 9 fungicides. Only nine
(18%) of the analysed fifty thyme samples were determined to be free of pesticide residues (concentrations
below the LOD). The range of pesticide residues detected in the remaining forty one sample of thyme
typically included one to six compounds in each sample. While most of the pesticide residues in thyme were
found below MRLs, three fungicides in several samples were above the MRL. Cymoxanyl was found in
twelve (24%) of the samples, and its content in seven samples (54-78 µg kg-1) exceeded the MRL (50 µg kg-
1
). These samples also contained increased concentrations of dimethoate (32-72 µg kg-1), which exceeded the
maximum level of 20 µg kg-1. It was concluded that the levels of contamination with cymoxanyl and
dimethoate showed a statistically significant correlation, with a regression coefficient of 0.83, indicating the
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possibility that these compounds were applied together as a mixture for the treatment of thyme (Figure 1).
In addition, the concentration of two fungicides exceeded EU MRLs in several samples of thyme.
The residues of tebuconazole significantly (by 4 to 12 times) exceeded the MRL in three (9%) of the
samples, reaching the concentration of 603 µg kg-1. The levels of fungicide tetraconazole also violated the
MRL in fourteen (18%) of the thyme samples, where the levels found in two samples exceeded the MRL by
5.8 to 8.9 times, reaching the concentration of 117-171 µg kg-1, respectively. Those results were in agreement
with previously published studies by Malinowska and Jankowski (2015), who also found elevated
concentrations of those pesticide residues in thyme grown in Poland. The results of this study emphasize the
importance of monitoring pesticide residues in spice and herbal products, especially in the case of thyme,
which was identified as the most contaminated matrix among the herbs, containing mostly fungicide type
pesticide residues in excess of the MRLs set by the EU regulations (EC 2005).

Heavy metals in spices

The results obtained for the six analysed matrices were evaluated according to the maximum limits (MLs)
set by FAO/WHO for the most common toxic elements (WHO 2007). The mean concentrations and the
ranges of heavy metal concentrations in the analysed spices and herbs are given in Table 5. The results of this
study indicated a broad variation of contaminant levels depending on the type of spices and herbs. The lowest
concentrations of toxic metals were found in nutmeg samples.
Arsenic was found in all the analysed samples of condiments. The range was from <0.005 µg g-1 to
0.041 μg g-1 in nutmeg, while the values in black pepper, paprika, and basil ranged from 0.015 to 0.22 μg g-1,
and the highest values were found in samples of thyme and oregano, with the ranges of 0.13-0.43 μg g-1 and
0.20-0.60 μg g-1, which were below the MLs set by WHO (WHO 2007).
Elevated levels of Cr were found in all samples, ranging from 0.4 μg g-1 to 13.8 μg g-1 in the
analysed samples (Table 5). Levels for Cr in condiments have not been set, thus the determined values were
compared to the limits in raw herbal products (2 μg g-1), which were exceeded in 67.7% of all the analysed
condiments (WHO 2007). Chromium was found at concentrations exceeding these levels by 1.5 to 6.7 times
in 98% of all black pepper samples and by 1.8 to 5.4 times in all samples of paprika (Table 5). There were
 only 4 samples (8%) of nutmeg showing slightly exceeded content of Cr compared to the recommended level
of 2 μg g-1. Chromium levels exceeded this value by 1.6 to 6.5 times in 90% of all thyme samples and by 1.3
to 3.4 times in 64% of oregano samples, indicating that thyme and oregano are the most contaminated herbs
with elevated levels of chromium. In comparison, the levels for Cr were reported to be from less than 0.010
µg g-1 to 1 µg g-1 in commercial condiments (Seddigi et al. 2013).
Cadmium was found at levels below 0.2 μg g-1 in all the samples of spices. Cadmium concentration
in basil and oregano ranged from <0.005 µg g-1 to 0.19 μg g-1. A notably high content of cadmium was
detected in thyme (0.016-0.6 μg g-1), with 44 (88%) of the samples containing this element at concentrations
exceeding the ML value of 0.2 μg g-1 set by WHO for condiments (WHO 2013).
The content of Pb, one of the most toxic environmental pollutants, in the analysed samples of spices
and herbs ranged from 0.37 μg g-1 to 1.20 μg g-1. The concentrations in spices were in the range from <0.005
to 0.37 μg g-1 in nutmeg, 0.11-0.44 μg g-1 in black pepper, and 0.20-0.75 μg g-1 in paprika. Two samples
(4%) of nutmeg, 15 samples (30%) of black pepper, and 43 samples (86%) of paprika exceeded the ML of
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0.3 μg g-1 set by WHO for lead in condiments (WHO 2013). The levels of Pb exceeded the level of 0.3 μg g-1
(WHO, 2013) by up to 2.3 times in 48 samples of oregano (96%), up to 3.4 times in 49 samples of basil
(98%), and in all 50 samples (100%) of thyme, thus pointing to basil and thyme as the herbal condiments
most contaminated with Pb. The results of this investigation are in agreement with some recent studies
raising concerns about elevated content of Cd, Cr, and Pb in commercial condiments (Inam et al. 2013;
Dghaim et al. 2015).

Risk assessment due to heavy metals

The potential weekly exposure to heavy metals from the samples of all six condiments were evaluated by
inserting the calculated concentrations in the equation (1) and comparing the results to the recommended
maximum values according to a recent report by FAO/WHO that indicates 3 µg kg-1 as the daily tolerable
intake of As and Pb. Thus, we multiplied the tolerable day intake values of As and Pb by 7, to evaluate the
maximum weekly consumption of condiments containing these heavy metals (FAO 2014). For a health risk
assessment, the 50-200 µg/day dietary intake limit of chromium, recommended by the National Research
Council, was recalculated for a weekly dose and 60 kg body weight, setting the limit of 5.8-23 µg kg-
1
b.w. week-1 (Seddigi et al. 2015). The calculated potential weekly doses of heavy metals are summarised in
Table 6 and compared to the weekly consumer intake of As, Cd, Pb, and the recalculated levels for Cr (FAO
2014). The results indicate that the determined heavy metal concentrations are far below the levels of concern
and do not approach the maximum tolerable doses, thus do not pose an acute threat to consumer health, even
in case of thyme. However, the influence of pesticide residues at elevated levels and the common occurrence
of such contaminants can present some health concerns in sensitive individuals and should be monitored.

CONCLUSIONS

An occurrence study of pesticide residues, mycotoxins and heavy metals was performed by using ICP-MS
and UHPLC-MS/MS. A total of 300 samples of 3 commercial grade spices and 3 herbs were analysed. The
contamination levels of mycotoxins and pesticide residues in most samples were below the M(R)Ls as set by
 EU regulations. The presence of a wide range of pesticide residues causes concerns regarding the quality of
these food ingredients. The number of detected compounds from in total 132 analysed plant protection agents
varied from none in nutmeg to 2 compounds in 98% of black pepper samples, 3 compounds in basil (54% of
the samples), 5 compounds in oregano (60% of the samples) and 16 compounds detected in 82% of the
thyme samples. The pesticide residues exceeded the EU MRLs in 10% of the oregano samples and 88% of
the thyme samples. In several samples the concentrations of toxic metallic elements exceeded the levels set
for condiments according to (WHO 2013), especially in the case of Pb and Cd in thyme and chromium in
68% of all condiment samples. The results indicate that the analysis of pesticide residues and quantification
of mycotoxins by chromatographic methods, as well as and the assessment of toxic elements is crucial for the
control of the quality and safety of condiments and mitigating the potential risks of consumers.

Conflict of interest statement

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The authors declare that there are no conflicts of interest.

Acknowledgements

This research was funded by the EU-FP7-SEC-2012-1 SPICED project under grant number 312631 and
supported by the National Research Programme “Agricultural Resources for Sustainable Production on
Qualitative and Healthy Foods in Latvia” (AgroBioRes) project No. 4 “Sustainable use of local agricultural
resources for qualitative and healthy food product development”.

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 Figure 1. Correlation between dimethoate and cymoxanyl residues in 12 investigated thyme samples.
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 Table 1. ICP-MS operating conditions for analysis of spices and herbs.
Parameter Value
Plasma mode normal, robust
RF forward power (W) 1,300

Sampling depth (mm) 8.0

Carrier gas flow (L min-1) 0.6
-1
Dilution gas flow (L min ) 0.4
Spray chamber temperature (°C) 2
Extraction lens 1 (V) 0
Kinetic energy discrimination (V) 3

Table 2. LOD and LOQ (µg g-1) for the determination of most investigated mycotoxins and pesticides.
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Black pepper / nutmeg Herbs (basil / oregano / thyme)

Compound
LOD LOQ LOD LOQ
Aflatoxin B1 0.8 2.7 1.0 3.0

Deoxynivalenol 1.8 5.9 2.5 7.5

Fumonisin B1 3.0 9.0 4.0 12

Ochratoxin A 0.5 1.5 0.8 2.4

Sterigmatocystin 2.2 6.6 4.0 12

Acetamiprid 0.8 2.4 1.2 3.6

Carbendazim 2.8 8.4 1.6 4.8

Cymoxanyl 3.5 10.5 3.9 11.7

Cyproconazole 2.0 6.0 3.0 9.0

Diazinon 0.9 2.7 1.4 4.2

Difenoconazole 2.4 7.2 2.8 8.4

Dimethoate 0.7 2.1 1.0 3.0

Fenthionsulfoxide 2.6 7.8 2.8 8.4

Flusilazole 3.0 9.0 4.0 12

Malathion 1.8 5.4 1.6 4.8

Mandipropamide 2.3 6.9 2.8 8.4

Mepanipyrim 1.0 3.0 1.6 4.8

Metalaxyl 1.0 3.0 1.4 4.2

 Black pepper / nutmeg Herbs (basil / oregano / thyme)
Compound
LOD LOQ LOD LOQ

Methoxyfenozide 3.3 9.9 4.0 12

Monocrotophos 1.8 3.6 1.8 3.6

Omethoate 2.3 6.9 2.7 8.1

Oxadixyl 1.5 4.5 2.1 6.3

Pirimiphos-methyl 3.5 10.5 4 12

Propamocarb 2.5 7.5 2.2 6.6

Tebuconazole 1.5 4.5 1.9 5.7

Tetraconazole 2.2 6.6 2.5 7.5

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Thiacloprid 2.8 8.4 2.3 7.0

Table 3. Validation parameters for heavy metal determination.

M, Recovery LOD LOQ
Element R2 RSD (%) -1
(m/z) (%; n=6) (µg g ) (µg g-1)
As 75 0.9995 99 5 0.005 0.015

Cd 111 0.9994 92 3 0.005 0.015

Cr 52 0.9996 108 3 0.010 0.030

Pb 208 0.9990 97 7 0.010 0.030

 Table 4. Pesticide residues in spices and herbs.
Contaminated Pesticide compound, mean concentration (concentration
Spice / Herb
samples range) and MRL from the EC database (µg kg-1)
Black Pepper 49 carbendazim, 8.0 (4.0-14.9), 100; metalaxyl, 12.2 (5.7-
25.4), 100
Nutmeg 0 No compounds determined
Basil 27 diazinon, 5.7 (1.5-9.4), 20; malathion, 6.4 (1.6-11.2), 20;
metalaxyl, 8.0 (3.3-14.0), 2,000
Oregano 30 carbendazim, 2.2 (1.6-4.0), 100; dimethoate and
omethoate, 14.9 (3.0-51.6), 20; fenthionsulfoxide, 9.4 (2.9-
32.0), 10; monocrotophos, 7.9 (2.1-20.4), 20
Thyme 41 acetamiprid, 7.0 (3.0-11.0), 3,000; cymoxanil, 51.6 (20.0-
78.0), 50; cyproconazole, 18.5 (10.0-27.0), 50;
difenoconazole, 31.0 (31.0), 20,000; dimethoate,
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64.4(35.0-85.0), 20; flusilazole, 16.0 (16.0), 20; metalaxyl,

29.8 (16.0-42.0), 2,000; methoxyfenozide 5.0 (5.0), 4,000;
20; oxadixyl, 10.0 (6.0-14.0), 10; pirimiphos-methyl, 11.8
(5.0-20.0), 50; propamocarb, 15.3 (2.3-50.0), 30,000;
tebuconazole, 85.7 (10.0-603.0), 50; tetraconazole, 56.0
(13.0-171.0), 20; thiacloprid 3.0 (2.0-4.0), 5,000;
thiamethoxam 3.0 (3.0), 1,500
 Table 5. Heavy metals in the investigated samples.
Mean heavy metal content ± RSDs and
Spice/herb concentration range (µg g-1)
As Cr Cd Pb
0.06±0.03 6.34±2.58 0.01±0.005 0.28±0.09
Black pepper
<0.005-0.16 1.76-13.48 <0.005-0.02 0.11-0.44
0.10±0.03 5.71±1.72 0.11±0.04 0.42±0.12
Paprika
0.04-0.22 3.59-10.76 0.03-0.19 0.20-0.75
0.03±0.01 1.19±0.51 0.04±0.02 0.13±0.09
Nutmeg
<0.005-0.04 0.47-2.13 <0.005-0.15 <0.001-0.37
0.30±0.08 7.35±2.88 0.40±0.14 0.79±0.17
Thyme
0.13-0.43 1.20-12.92 0.30±0.16 0.32-1.04
0.13±0.19 2.17±0.93 0.12±0.03 0.48±0.14
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Basil
0.06-0.19 1.31-6.89 0.09±0.16 0.26-1.20
0.37±0.09 2.48±0.94 0.02±0.0.01 0.42±0.09
Oregano
0.20-0.61 1.10-5.55 <0.005-0.07 0.22-0.71

Table 6. Potential weekly intake of heavy metals compared to the tolerable daily intake levels.
Element PWTI The calculated potential weekly intake by consuming 10 g
-1
(µg kg condiments per week (µg kg-1 b.w. week-1)
b.w. week-1) Black Paprika Nutmeg Basil Oregano Thyme
pepper
As 21 0.01 0.02 0.003 0.02 0.06 0.05
Cd 25 0.01 0.13 0.04 0.04 0.02 0.16
Cr 5.80-23.3 7.40 6.66 1.38 2.53 2.89 8.58
Pb 21 0.32 0.49 0.15 0.56 0.49 0.56