Food Chemistry 324 (2020) 126867

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Food Chemistry
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Short communication

Doxycycline transfer from substrate to white button mushroom (Agaricus T

bisporus) and assessment of the potential consumer exposure
Małgorzata Gbylik-Sikorska , Anna Gajda, Ewelina Nowacka-Kozak, Andrzej Posyniak
⁎

Department of Pharmacology and Toxicology, National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Puławy, Poland

ARTICLE INFO ABSTRACT

Keywords: The presence of antibiotic residues in the food chain may pose a serious risk to human health. Locating and
Doxycycline evaluating new sources of consumer exposure to antibiotic residues in food is a very important element of health
Button mushroom protection. The possibility of doxycycline uptake from the substrate for mushroom cultivation by the white
Transfer button mushroom (Agaricus bisporus) fruit body was investigated. Mushrooms were experimentally cultivated on
ADI
substrate contaminated with 8 different doxycycline concentrations in substrate and analyte concentrations in
mushroom samples were measured using ultra-high performance liquid chromatography – triple quadrupole
tandem mass spectrometry (UHPLC–MS/MS) The obtained results clearly indicated that doxycycline transfers
from contaminated substrate to mushrooms at concentrations ranging from 0.87 to 72.3 µg/kg, depending on
substrate contamination concentration level and order of harvesting.

1. Introduction be used as a component of selenium (selenium-enriched mushroom

The button mushroom (Agaricus bisporus) is one of the most widely
cultivated mushroom species in the world. The popularity of this
mushroom owes all to its taste, aroma, low calorie, high fibre and good
quality protein content, which qualifies it to the healthy food, super-
food and functional food groups. A. bisporus contains a wide range of
substances with proven biological activity (Aida, Shuhaim, Yazid, &
Maaruf, 2009, Wani, Bodha, & Wani, 2010, Colmenares-Cruz, Sanchez,
& Valle-Mora, 2017; Siwulski et al., 2020). The substances in A. bisporus
are a valuable resource for the proper functioning of the human body
(Hess, Wang, Gould, & Slavin, 2018; Sławińska et al., 2016; Sari,
Prange, Lelley, & Hambitzer, 2017). It includes vitamins, poly-
saccharides, indole, phenolic and terpene compounds as well as mi-
nerals and bioelements like iron, magnesium, potassium, selenium and
zinc (Muszyńska et al., 2015; Ramos et al., 2019; Siwulski et al., 2020).
White button mushrooms contain a high level of ergosterol (a pro-vi-
tamin form of vitamin D2), exposure of mushrooms to UV radiation can
require the production of vitamin D2. Sławińska et al. (2016) de-
termined the stability of ergocalciferol in dried mushrooms (A. bis-
porus), reporting that drying as a method of processing and preservation
of mushrooms is suitable to assure products with a high content of vi-
tamin D2. Commercially cultivated A. bisporus were analysed for their
beta-glucan content with very remarkable results (Sari et al., 2017).
White button mushrooms watered with selenium during cultivation can
aqueous enzymatic extracts or selenoamino acids) which may help in
prevention of diseases resulting from low selenium levels (Cremades
et al., 2012; Maseko et al., 2013). White button mushrooms are also
very popular as study models among scientists from various fields,
especially in medicine and food science (Cavalieri, Bolzoni, & Bandini,
2010; Kanaya et al., 2011; Al-Habib, Holliday, & Aladahmy, 2018;
Bhushan & Kulshreshtha, 2018; Siwulski et al., 2020). The data pro-
vided by Adams, Chen, Phung, Wu, and Ki (2008) illustrate the antic-
ancer potential of phytochemicals in mushroom extract and promote
the recommendation of white button mushroom as a dietary component
that may help in the prevention of prostate cancer in men. Also, Jeong,
Koyyalamudi, Jeong, Song, and Pang (2012) reported a beneficial
therapeutic - antitumor effect of A. bisporus. Francisco et al. (2018),
reported that the usage of alcoholic extracts (microencapsulated) of A.
bisporus as a functional ingredients in yogurt resulted with promising
bioactive properties. Their relatively easy and fast cultivation is an
important rationale for such widespread interest in both the food and
scientific industries. Many studies have shown that A. bisporus have a
high potential as a source of valuable compounds. However, it is still
necessary to carry out scientific research into their application, quality
of products and safety for peoples.
Veterinary antibacterial drugs are administered to animals to treat
diseases and protect their health. The need for antibiotic treatment of
farmed animals on large-scale operations is often unavoidable, because

⁎
Corresponding author.
E-mail address: malgorzata.gbylik@piwet.pulawy.pl (M. Gbylik-Sikorska).

https://doi.org/10.1016/j.foodchem.2020.126867
Received 18 February 2019; Received in revised form 17 April 2020; Accepted 18 April 2020
Available online 20 April 2020
0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
M. Gbylik-Sikorska, et al.
the keeping of a large number of animals (poultry, pigs, or cattle) in a
relatively small area may lead to outbreaks and very fast spread of
bacterial infections. Despite the implementation of all sanitary and
hygienic standards and improvement of animal welfare, the only ef-
fective solution which counteracts infection in most cases is antibiotic
therapy. Unfortunately, after drug administration, up to 90% of an
antibiotic (parent compounds and metabolites) may be excreted via
urine, faeces and manure. It may cause organic fertilisers which contain
animal manure to be contaminated with antibiotics and/or their me-
tabolites (Berendsen et al., 2018; Ho, Zakaria, Latif, & Saari, 2012; Ho,
Zakaria, Latif, & Saari, 2014; Karcı & Balcıoğlu, 2009; Peng et al.,
2016). The use of manure-based fertilisers is very popular and is often
integral to cultivation in all parts of the world. Organic fertilisers in-
cluding manure-based kinds owe their attractiveness to the high con-
tent of nutrients necessary for proper growth such as nitrogen, po-
tassium and phosphorus. Also, the reuse of waste from the animal
husbandry industry is a very important aspect of the eco-economy. At
the moment, many reports in the literature describe soil and crop
contamination with antibiotics caused by the use of organic manure-
based fertilisers (Boxall et al., 2006; Karcı & Balcıoğlu, 2009; Seo, Cho,
Kang, Jeong, & Jung, 2010; Tasho & Cho, 2016; Madikizela, Ncube, &
Chimuka, 2018; Zhang et al., 2016) and their potential accumulation in
crop tissues (Pan & Chu, 2017a, 2017b; Ahmed et al., 2015; Hu, Zhou,
& Luo, 2010; Chung et al., 2017). Therefore, organic fertilisers con-
taminated with antibacterial drug residues may constitute a separate
source of antibiotic contamination in food. Locating new sources of
human exposure to antibiotic residues, drug-resistant bacteria or re-
sistance genes is a very important element of human health protection
(Bengtsson-Palme, 2017; Kümmerer, 2003).
An industry which uses chicken and/or horse manure is commercial
mushroom cultivation. One of the most important elements in white
button mushroom cultivation is a suitable substrate (compost) pre-
paration which generally contains wheat straw, cereals, horse and/or
chicken manure and gypsum (Andrade et al., 2008; Baysal et al., 2007;
Elenwo & Okere, 2007; Isikhuemhen & Mikiashvili, 2009; Stoknes,
Beyer, & Norgaard, 2013). Unfortunately, high-quality horse manure is
much more difficult to get than chicken manure. This is due to limited
access to horse manure and its attendant high price. The desirability of
managing the increasing amount of poultry manure generated by a
constantly growing number of poultry farms is further dissuasion from
using horse manure. Chicken manure may contain antibiotic residues
because of their common use in the poultry industry to treat bacterial
infections, which can appear during the relatively short time in which
birds are raised (Berendsen et al., 2018; Ho et al., 2012; Ho et al., 2014;
Peng et al., 2016). Tetracyclines, antibacterial compounds with a broad
spectrum of activity, are often used in infections occurring in chickens.
Doxycycline (DC), (α-6-deoxy-5-hydroxytetracycline) with broad spec-
trum activity against Gram-positive and Gram-negative bacteria
(Holmes & Charles, 2009), comes in for especially frequent use. Due to
its high lipid solubility, good tissue distribution, long elimination half-
life and low cost it is often used with good results in the treatment of
poultry.
The European “One Health” concept assumes that human health is
tightly connected to animal health and the environment. (Robinson
et al., 2016). The need of prevention against the constantly rising an-
timicrobial resistance, increases year by year. This approach makes it
necessary to identify unexplored sources of antibiotic residues and re-
servoirs of antimicrobial resistance on food chain. So, searching and
identification of a potential antibiotics residue sources is of great im-
portance for human health. Animal by-products such as soil improvers
(manure) or organic manure-based fertilisers can be the source/re-
servoire of antibiotics resistance bacteria. Considering the fact that
antibiotics present in manure, organic fertilizer or poultry litter could
transfer to crops also in high concentration, there is a high possibility of
contamination of substrates for mushrooms cultivation with that com-
pounds. Due to the possibility of contamination with DC of button
2
Food Chemistry 324 (2020) 126867
mushroom during cultivation, which could cause a potential threat to
consumer, it was necessary to conduct an experiment allowed to in-
vestigate the possibility of DC transfer from experimentally con-
taminated substrate to the mushroom fruit body, its accumulation and
potential consumer exposure. To the best of our knowledge, this study
is the first to explore the possibility of antibacterial drug uptake by
white button mushrooms.
2. Material and methods
2.1. Chemicals and reagents
DC was selected because of its frequent use in the treatment of
bacterial infections in poultry therapy and its solubility in water.
Analytical grade DC (97% purity) and demeclocycline (DMC) (91.5%)
internal standards (IS) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Acetonitrile, methanol and formic acid were obtained from
J.T. Baker (Deventer, the Netherlands) and trichloroacetic acid was
sourced from Sigma-Aldrich. Water was deionised (> 18 MΩ cm−1) by
the Millipore system. Polyvinylidene fluoride (PVDF) syringe filters in
0.22 μm size were from Restek (Bellefonte, PA, USA).
2.2. Experimental design
Commercially available indoor-growing mushroom (A. bisporus) kits
were purchased from a local producer. Each kit contained inoculated
mushroom substrate (5 kg) packed in a plastic bag and cardboard box.
The kits included also a separate bag of wet peat soil (a casing) con-
taining calcium carbonate. Before the experiment substrate and soil
were checked for freedom from antibiotic residues by a modified pre-
viously described analytical method (Gbylik-Sikorska et al., 2014).
Then, composts were weighed and fortified with DC at 8 different
concentration levels (10, 50, 100, 250, 500, 1000, 2000 and 5000 µg/
kg of substrate) in 3 replicates. The solutions of DC in water were added
to the substrate with a spray (2 mL), which ensured even distribution
(Fig. 1). Fortified substrates were incubated in a closed bag at 18 °C for
24 h. Thereafter, peat soil was applied to the substrate surface and
watered.
2.3. Conditions of mushroom cultivation
Experiments were conducted under the same controlled laboratory
conditions (80–90% relative air humidity and temperature of 12–18 °C
in a darkened room). For the first 10 days of mushroom cultivation, the
room temperature was kept between 16 and 18 °C, and on the 11th day
it was lowered to 12–14 °C (this was necessary for the suppression of
mycelium growing through the casing surface); the air humidity was
constant throughout the duration of the experiment. In order to main-
tain adequate humidity of the cultivation soil, water was sprayed onto
the casing surface once a day. Temperature and humidity were mon-
itored twice a day.
2.4. Analytical determination of DC in button mushrooms
The concentrations of DC in A. bisporus were determined using a
modified previously reported analytical procedure for the determina-
tion of antibacterial compounds in button mushrooms (Gbylik-Sikorska,
Gajda, Nowacka-Kozak, & Posyniak, 2019)
2.4.1. Sample preparation
First, mushroom samples were sliced and blended at room tem-
perature and frozen with liquid nitrogen, and then they were blended
for a second time. Five grams of blended sample was weighed into a
50 mL plastic tube, then 20 µL of DMC IS (2 µg mL−1) was added,
mixed and stored in a dark place at 5 °C for 30 min. After incubation,
8 mL of 5% trichloroacetic acid was added, the samples were vortexed
M. Gbylik-Sikorska, et al.
Fig. 1. Experiment diagram of button mushrooms cultivation.
for 1 min, put into an ultrasonic bath for 1 h and mixed with a rotary
stirrer for 30 min at room temperature. Thereafter samples were cen-
trifuged at 2930 × rcf for 15 min at 5 °C. Finally, 1 mL of supernatant
was filtered by 0.22 µm PVDF membrane syringe filters into an ana-
lytical vial.
2.4.2. Ultra-high-performance liquid chromatography–mass spectrometry
(UHPLC–MS/MS)
The DC concentration was determined using a Nexera X2 ultra high
performance liquid chromatography–tandem mass spectrometry
(UHPLC–MS/MS)(Shimadzu, Japan) system connected to a QTRAP®
4500 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA,
USA). Analyst 1.6.3 software (AB Sciex) controlled the UHPLC–MS/MS
system and processed the data. The chromatography separation was
performed on a Luna C18(2) 100A column (50 × 3.0 mm, particle size
3 µm) (Phenomenex, Torrance, CA, USA) integrated with a guard
column of the same type (Fig. 2). The mobile phase for analysis con-
sisted of acetonitrile:methanol (80:20 v/v) (A) and 0.1% formic acid
(B). Gradient elution was performed as follows: 0.00–1.00 min (15% A),
2.30–3.30 min (95% A) and 3.31–4.00 min (15% A) at a flow rate of
600 µL/min. Detection was performed in the negative ionization mode
in the selected reaction monitoring mode (SRM). Monitoring was of m/z
445.0 > 428.0 and 445.0 > 154.0 for DC and 465.0 > 448.0 for IS
transitions.
3
Food Chemistry 324 (2020) 126867
2.5. Method validation
Linearity, selectivity, repeatability, reproducibility and recovery
(European Decision 2002/657/EC), limit of quantification (LOQ), and
limit of detection (LOD) of the method were evaluated. The linearity
was determined by a matrix-matched calibration curve which was
prepared by fortifying blank mushroom samples at 6 concentration le-
vels (0.5–500 µg/kg). The repeatability was calculated as the coefficient
of variation (CV, %) of results obtained after fortifying six blank
mushroom samples at three concentration levels (1, 10 and 100 µg/kg).
Preparation and analysis of the spiked samples took place on the same
day with the same instrument utilized and the same operators executing
the tasks. The within-laboratory reproducibility was calculated as the
CV (%) of the results obtained after fortifying another two sets of blank
samples at the same concentration levels of analysed compound as for
the repeatability and analysing them on two days with the same in-
strument and different operators. Estimation of the LOQ was at ten
times the signal-to-noise ratio (S/N) of DC in fortified samples, at the
minimum detectable concentration level samples. The LOD was de-
termined as a signal-to-noise of three. The average recovery was eval-
uated in the same experiment as repeatability by comparing the mean
measured concentration with the fortified concentration of the samples
in relation to the matrix-matched calibration curve. Specificity was
checked by analysis of 20 blank mushroom samples to verify the ap-
pearance of possible interfering substances at the retention times of the
DC.
M. Gbylik-Sikorska, et al. Food Chemistry 324 (2020) 126867

Fig. 2. Chromatograms of a – blank button mushroom sample; b – DC spiked button mushroom sample (1 µg/kg); c, e, g, i– sample of button mushroom obtained in
the 1st harvesting, containing DC at different concentration level; d, f, h, j – sample of button mushroom obtained in the 2nd harvesting, containing DC at different
concentration level.

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M. Gbylik-Sikorska, et al. Food Chemistry 324 (2020) 126867

2.6. Bioconcentration factor and safety assessment

The bioconcentration factor (BCF) enables evaluation of the ability

of mushrooms to accumulate a chemical substance, in this case DC,
from substrate, and was calculated using the following formula:
Cmushroom
BCF =
Csubstrate
C mushroom is the average concentration of chemical substance (DC)
in analysed mushroom samples (µg/kg), and C substrate is the con-
centration of the same chemical substance (DC) in substrate (µg/kg).
The acceptable daily intake (ADI) is defined as the maximum
amount of a chemical that can be ingested daily over a lifetime with no
appreciable health risk. The ADI value for DC is 3 µg/kg body weight
(bw) (EPMAR, 2015). The estimated daily intake (EDI) for DC was
calculated as follows:
C × Cpd
EDI =
bw
where C is the average concentration of chemical substance (DC) in
analysed mushroom samples (µg/kg), and Cpd means daily average
consumption of button mushrooms per capita (both this and bw in kg).
The harmfulness of consumer exposure was assessed by comparison of
EDI and ADI.

2.7. Statistical analysis

All statistics were calculated using Microsoft® Excel 2013 (Microsoft

Corporation, USA) and one-way ANOVA, differences were considered
significant when p < 0.05.

3. Results and discussion

3.1. Mushroom cultivation and harvesting

The white button mushroom cultivation was carried out twice with
there being eight experimental groups (eight different DC concentra-
tions in substrate) and one control group in each experiment, and each
group containing three kits. After 14 days the first small mushrooms
(0.2–0.6 cm) began to grow in different places on the casing surface
(Fig. 3). The first harvests of mushrooms were started on the 19th day
(Fig. 3), yielding from 700 g to 900 g per kit. The second harvest a week
later provided from 500 g to 650 g. There was no DC impact on
mushroom harvest weight. The collected mushrooms were sliced and
frozen at − 18 °C until sample analysis. The second experiment was
started one month later and was carried out under the same protocol.
Fig. 3. Presentation of the various stages of experimental button mushroom
cultivation.
3.2. Validation results

The method developed for the determination of DC in common
mushroom was validated in accordance with Commission Decision
2002/657/EC. The matrix-matched curves showed good linearity (de-
termination coefficient r2 > 0.99) in the concentration range of
0.5–500 µg/kg. For the DC repeatability, the average CV was
8.9 ± 4.2%, and for within-laboratory reproducibility it was
12.4 ± 3.2%. The average percentage recovery was 97 ± 6.3%. The
LOQ of the method was 0.5 µg/kg and the LOD was 0.08 µg/kg. The
specificity study shows that no peak was detected in these samples at
the retention time corresponding to the DC retention time.
3.3. DC concentration in button mushrooms
All mushrooms were analysed for DC concentration, each harvest
from one kit being homogenized separately. Five randomly selected
samples from each kit were taken for analysis in all experimental
groups. The results obtained in the experiments are presented in
Table 1. DC was only detected in the samples collected in groups with
concentration in substrate in a range from 500 to 5000 µg/kg (Fig. 2c-j).
Mushroom DC concentrations ranged from 0.87 to 72.3 µg/kg and were
two and three times higher in the samples collected in the first harvest.
In this concern, the order of harvesting and DC concentration in sub-
strate significantly affects the concentration of DC in mushrooms
(p < 0.001 – 0.02). There was no significant dereferences between
experimental groups at the same concentration levels (p > 0.05). The
results of the experiment indicated that DC concentrations in mush-
rooms depended on its concentrations (the highest concentrations were
determined in mushrooms harvested for substrate with the highest
concentration of DC) in substrate and on the order of harvesting (sig-
nificantly lowet in the mushrooms collected in the second harvest). To
our knowledge, there are no data published in literature reporting the
occurrence of antibacterial drugs (e.g, tetracyclines, DC) in substrate for
button mushroom cultivation and its transfer to the mushroom fruit
body. Most of the studies available in the literature concern residues of

5
M. Gbylik-Sikorska, et al. Food Chemistry 324 (2020) 126867

Table 1 The experiments were conducted under non-sterilised conditions and

The doxycycline (DC) mean concentration results obtained in different experi- microorganisms present in substrates may have led to partial DC bio-
ments. degradation. Thus the BCF values reported in this paper are minimum
Kit no DC DC concentration [µg/kg]* values elicited in experiments. The accumulation of DC estimated for
concentration mushrooms obtained in the first and second harvests was approxi-
level in Group I/1st Group I/ Group II/1st Group II/ mately 0.01 and 0.005, respectively, with one exception (0.003) in the
substrate [µg/ harvest 2nd harvest harvest 2nd harvest
second harvest. It means that accumulation of DC in mushrooms de-
kg of
substrate] pends on the order of harvesting. Both Azanu et al. (2016) and Pan and
Chu (2017a) reported BCF values in a range from 0.1 to 0.44, which
1 10 nd nd nd nd means that the BCF values obtained in our study are significantly lower
2 10 nd nd nd nd
than those in studies on tetracycline uptake by crops. The BCF values
3 10 nd nd nd nd
1 50 nd nd nd nd
presented here are lower by factors of ten and approximately one
2 50 nd nd nd nd hundred than those authors’ findings, it may be caused by DC biode-
3 50 nd nd nd nd gradation in substrate, its tendency to form complexes with ions pre-
1 100 nd nd nd nd sented in the substrate and water and/or differences in water and
2 100 nd nd nd nd
compound uptake and transport from substrate to mushroom fruit
3 100 nd nd nd nd
1 250 nd nd nd nd body. Consumer exposure was estimated for a standard adult weighing
2 250 nd nd nd nd 60 kg. The average daily consumption (7.6 g/day) was calculated based
3 250 nd nd nd nd on data of the Central Statistical Office in Poland, which estimates the
1 500 3.93 ± 2.6 0.87 ± 1.4 3.42 ± 3.4 2.20 ± 2.1
average annual consumption of button mushrooms per capita in Poland
2 500 2.93 ± 3.1 1.03 ± 2.1 5.87 ± 3.8 1.25 ± 1.8
3 500 4.51 ± 2.1 2.42 ± 1.6 5.97 ± 4.1 2.63 ± 1.4
at 2.7 kg/year. Using measured average DC concentrations in harvested
1 1000 8.57 ± 4.2 4.42 ± 1.5 12.8 ± 4.5 7.34 ± 2.4 mushrooms and EDI values, the percentage of ADI was calculated and
2 1000 9.84 ± 3.8 2.16 ± 2.3 10.3 ± 3.3 5.27 ± 2.5 shown in Table 2. The estimated EDI values range from 0.0002 to
3 1000 10.2 ± 2.6 3.24 ± 2.1 12.5 ± 3.7 4.38 ± 1.6 0.0081 µg/kg per day, and are much lower than the ADI. In the worst-
1 2000 19.8 ± 5.1 9.52 ± 3.1 23.2 ± 4.1 12.3 ± 2.8
case scenario it was 0.27% of ADI. The obtained results indicate that the
2 2000 20.2 ± 4.2 8.96 ± 2.2 28.1 ± 3.7 12.4 ± 3.2
3 2000 17.8 ± 4.7 12.9 ± 3.5 21.3 ± 2.4 8.56 ± 3.3 potential risk associated with the consumption of mushrooms such as
1 5000 61.3 ± 4.4 19.3 ± 3.4 56.4 ± 3.6 22.8 ± 4.1 were harvested in the experiment is insignificant. The potential con-
2 5000 72.2 ± 5.1 24.8 ± 3.6 72.3 ± 4.2 26.3 ± 2.7 sumer exposure extrapolated from the tetracycline concentrations in
3 5000 53.8 ± 5.2 21.6 ± 3.1 67.2 ± 3.8 18.5 ± 3.5 the edible part of crops was reported in previous studies. Azanu et al.
(2016) estimated EDI values to be in a range from 0.0002 to 0.0003 µg/
nd – not detected; * – average concentration of five results per kit.
kg per day, this means that the risk to consumer health is probably low.
Also, other estimates of potential human exposure in edible crops were
pesticides (Du et al., 2018) or metal content (Rashid, Rahman, Correll,
below the recommended ADI level (Ahmed et al., 2015; Boxall et al.,
& Naidu, 2018; Rzymski, Mleczek, Siwulski, Gąsecka, & Niedzielski,
2006; Pan & Chu, 2017a), and were similar to our study results.
2016; Rzymski et al., 2017; Sobhanardakani & Jahangard, 2017).
However, there are several studies that have shown that tetracyclines
can be taken up from soil by plants (Hussain, Naeem, Chaudhry, &
4. Conclusions
Iqbal, 2016; Pan, Wong, & Chu, 2014). Moreover, some of them have
described the effect of antibiotic uptake by plants from organic ferti-
In this study, the antibacterial drug transfer from contaminated
lised soils (Ahmed et al., 2015; Chowdhury, Langenkämper, & Grote,
substrate for mushroom cultivation to the mushroom fruit body was
2016; Dolliver, Kumar, & Gupta, 2007; Kang et al., 2013). Experimental
demonstrated. The experiment allowed the possibility of DC uptake by
studies on the uptake of oxytetracycline into crops from spiked soil
button mushrooms from contaminated substrate to be assessed. Using
(1000 µg/kg) showed that the concentrations in lettuce leaves and
UHPLC–MS/MS analysis, DC concentration in harvested mushrooms
carrots were 7.2 µg/kg and 23 µg/kg, respectively (Boxall et al., 2006).
was determined and was found to depend on the degree of con-
Also, Azanu et al. (2016) applied tetracycline in spiked irrigation water
tamination of the substrate and the order of harvesting. The estimated
(1000 µg/kg) and found it taken up by crops with concentrations from
consumer exposure even in the hypothetical worst-case scenario was
10.0 to 30.0 µg/kg. In both cases the obtained results were similar to
significantly below the ADI value. However, this should not be ignored
ours. Pan and Chu (2017a) found that the concentration of tetracycline
and more research should be carried out, especially with naturally
in crop tissues (lettuce, carrot and tomato) grown in soil exposed to the
contaminated substrate. Moreover, conducting further research in this
antimicrobial in wastewater spiked at the two levels 2 and 20 µg/L were
area, especially the detection of antibacterial drugs in commercially
in a range from 1.56 to 15.6 µg/kg. Our study results showed no DC
available A. bisporus, may contribute to identifying the potential source
accumulation in button mushrooms harvested on substrates with low
of antibiotic residues. Therefore, a detailed knowledge of new sources
DC (10–250 µg/kg) concentration levels. Ahmed et al. (2015) also re-
of antibiotic residues on food is of great importance for human health.
ported that chlortetracycline, oxytetracycline and tetracycline con-
centration in lettuce, cucumber and tomato cultivated on soil spiked
through irrigation to 5000, 10,000 or 25,000 µg/kg were between 2.0
CRediT authorship contribution statement
and 204 µg/kg. The high concentration of tetracyclines in crop tissues
reported in that study could be due to long-term irrigation with spiked
Małgorzata Gbylik-Sikorska: Conceptualization, Data curation,
water, the quadruple doses (one dose per week) and additionally the
Formal analysis, Funding acquisition, Investigation, Methodology,
irrigation of all pots with water containing tetracyclines, in contrast to
Project administration, Supervision, Validation, Visualization, Writing -
our study, in which the substrates were spiked only once.
original draft, Writing - review & editing. Anna Gajda:
Conceptualization, Formal analysis, Investigation, Writing - original
3.4. Bioconcentration factor and potential exposure assessment to DC draft. Ewelina Nowacka-Kozak: Investigation, Methodology,
through mushroom consumption Validation. Andrzej Posyniak: Writing - original draft.

The BCF values for DC in common mushrooms are shown in Table 2.

6
M. Gbylik-Sikorska, et al.
Table 2
Bioconcentration factor and consumer exposure assessment to doxycycline (DC) through mushroom consumption.
Harvest no DC concentration level in substrate [µg/kg of Detected average* DC concentration
substrate] [µg/kg]
1 500 4.40 ± 1.2
1000 10.7 ± 1.6
2000 21.75 ± 3.5
5000 63.8 ± 1.7
2 500 1.72 ± 2.6
1000 4.50 ± 1.6
2000 10.78 ± 2.4
5000 22.5 ± 3.0
Detected average DC concentration for groups I and II experiment results.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was funded by KNOW (Leading National Research
Centre), decision of Ministry of Science and Higher Education No. 05-1/
KNOW2/2015. The author would like to thank Ms. Magdalena Bilecka
for her support in sample preparation for LC-MS/MS analysis.
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