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Uptake of oxytetracycline, sulfamethoxazole and ketoconazole from fertilised

soils by plants

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Uptake of oxytetracycline, sulfamethoxazole and

ketoconazole from fertilised soils by plants
a a b
Carmen Lidia Chitescu , Anca Ioana Nicolau & Alida Adriana Maria Stolker
a
Faculty of Food Science and Engineering, University Dunarea de Jos Galaţi, Galaţi,
Romania
b
RIKILT – Wageningen University and Research, Wageningen, The Netherlands

Accepted author version posted online: 29 Aug 2012.Version of record first published: 21
Sep 2012.

To cite this article: Carmen Lidia Chitescu, Anca Ioana Nicolau & Alida Adriana Maria Stolker (2012): Uptake of
oxytetracycline, sulfamethoxazole and ketoconazole from fertilised soils by plants, Food Additives & Contaminants: Part A,
DOI:10.1080/19440049.2012.725479

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 Food Additives & Contaminants: Part A
2012, 1–9, iFirst

Uptake of oxytetracycline, sulfamethoxazole and ketoconazole from fertilised soils by plants

Carmen Lidia Chitescua*, Anca Ioana Nicolaua and Alida Adriana Maria Stolkerb
a
Faculty of Food Science and Engineering, University Dunarea de Jos Galat¸i, Galat¸i, Romania; bRIKILT – Wageningen
University and Research, Wageningen, The Netherlands
(Received 29 June 2012; final version received 26 August 2012)

This study was performed to investigate the potential for a set of two antibiotics and one antifungal compound
to be taken up from the soil by plants. Plants are used for animal or human consumption, and so the measured
concentrations in the plant material will be used to model potential human exposure to these compounds.
The uptake by two types of plants (grass and watercress) from two types of soil was studied. The compounds
used for these experiments were sulfamethoxazole, oxytetracycline and ketoconazole at concentrations of 5 and
Downloaded by [Wageningen UR Library] at 02:10 10 October 2012

10 mg kg1 in the soil. The compounds of interest were extracted out of the plant matrix by applying accelerated
solvent extraction. Analyses were carried out by a LC–MS/MS. From the results, it was concluded that the plant
materials used for this study were able to take up sulfamethoxazole and ketoconazole when the soil was
contaminated with these compounds at a concentration ranging from 5 to 10 mg kg1. Sulfamethoxazole was
detected in all samples, at levels ranging from 7 to 21 mg kg1 for grass and 4 to 7.5 mg kg1 for watercress.
For ketoconazole, the results showed low absorption. Oxytetracycline was not detected in any sample.
A partition-limited model approach was applied for the comparison of experimental and estimated data, and the
relationship between physicochemical properties of the compounds and plant uptake was highlighted.
Keywords: plant uptake; antibiotics; antifungal; partition model; passive transport

Introduction detected in soils, surface waters and groundwater

Veterinary medicines are widely used in livestock
treatment and are released in the fields either directly
in faeces or urine or indirectly through the application
of manure as a fertiliser (Kumar, Singh, et al. 2005).
Veterinary and human medicines are increasingly being
monitored in slurry, soils, surface waters and ground
water. In Austria, concentrations up to 46 mg kg1
chlortetracycline, 29 mg kg1 oxytetracycline and
23 mg kg1 tetracycline in pig manure were reported
(Martinez-Carballo et al. 2007). The concentration of
sulphonamides in manure ranged between 10 and
91 mg kg1 (Pfeifer et al. 2002; Jacobsen and Halling-
Sørensen 2006; Martinez-Carballo et al. 2007).
In sludge, sulphonamides were also detected at
concentrations up to 197 mg kg1 for sulfapyridine and
73 mg kg1 for sulfamethoxazole in Swiss wastewater
treatment plants (Gobel et al. 2005). Sukul and
Spiteller (2006) proposed that with manure slurry
being applied in the field as fertiliser at a maximum
dose rate of 50 m3 ha1, sulphonamide residues in soil
could reach 1 kg ha1, which is of the same order of
magnitude as the application rate of modern pesticides.
A large range of veterinary medicines such as
macrolides, sulphonamides, quinolones, penicillin,
tetracyclines and antifungals have already been
(Hirsch et al. 1999; Thiele-Bruhn 2003; Fatta-
Kassinos et al. 2011). Humans may be exposed to
residues of veterinary medicines in the environment
(i.e., soil, water, sediment). One possible exposure
route is the consumption of crops that have accumu-
lated substances from soils as a result of exposure
to contaminated manure and slurry. A second route of
exposure of human to residues of veterinary medicines
is the consumption of meat, eggs, milk, water and so
on contaminated with veterinary medicines through
the food chain (Boxall et al. 2006).
Although according to Council Directive 96/23/EC
(European Commission 1996), veterinary medicines
are routinely monitored in food materials from treated
animals to ensure that concentrations are below the
maximum residue limits, the sum of the exposure via
different routes and the health impact of such exposure
have not been extensively quantified.
At present, statements about the behaviour of
veterinary medicines/pharmaceuticals in soil are highly
speculative. Evaluation of data on biodegradation
is very difficult as kinetics are mostly unknown
(Winker 2009). Therefore, future investigations are
necessary. The uptake of veterinary and human med-
icines by plants, and their effects, is of major interest

*Corresponding author. Email: chitescucarmenlidia@yahoo.com

ISSN 1944–0049 print/ISSN 1944–0057 online

ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/19440049.2012.725479
http://www.tandfonline.com
 2 C.L. Chitescu et al.
for agriculture areas, with regard to fertilisation and
the problem of antimicrobial resistance. Several studies
have recently demonstrated that plants can take up
pharmaceutical compounds from the growth media
via their roots (Kumar, Gupta, et al. 2005; Boxall et al.
2006; Dolliver et al. 2007; Herklotz et al. 2010).
The objective of this research was to evaluate
the plant uptake of three medicines (human and
veterinary): oxytetracycline, sulfamethoxazole and
ketoconazole. The substances were selected (Table 1)
to cover different pharmaceutical classes (sulphona-
mides, tetracyclines and antifungals) and different
environmental properties (hydrophobicity, soil absorp-
tion potential and soil persistence). Oxytetracycline
and sulfamethoxazole are widely used in veterinary
and human practices, and both have a high potential
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to be released into the environment. Tetracylines are
the most frequently used antibiotic within the porcine
and chicken farms; sulphonamides are used for cattle
and swine (Kumar, Singh, et al. 2005). Ketoconazole is
a synthetic antifungal drug used to prevent and treat
skin and fungal infections. Ketoconazole is licensed as
a human medicine but – like many of those antifungals
available for the treatment of mycoses in humans – is
Table 1. Selected physicochemical properties of target compounds.
Molecular
weight
Compound (g/mol) pK1a log K2ow K3ch
Oxytetracycline 460.434 pKa(1) ¼ 3.3 0.95 0.1 27792–93317
pKa(2) ¼ 7.3
pKa(3) ¼ 9.1
Sulfamethoxazole 253.279 pKa(1) ¼ 1.85 0.89 0.2
pKa(2) ¼ 5.6
Ketoconazole 531.431 pKa(1) ¼ 6.51 4.34/3.84 3
pKa(2) ¼ 2.94
Notes: aChen and Huang (2009); bTrapp (2000); cTOXNET (2005); dLi et al. (2005); eJones et al. (2005); fQiang and Adams
(2004); gTeira-Esmatges et al. (2010); hHansch et al. (1995); iSangster (1997); jDoucette (2000); kDoucette (2003); lGerstl (1990).
used by veterinary practitioners and prescribed by drug
producers for poultry (Rochette et al. 2003).
While the plant uptake of tetracyclines or sulpho-
namides has been investigated in several studies
(Boxall et al. 2006; Dolliver et al. 2007), ketoconazole
has never been evaluated from this point of view,
although the tendency of azole to persist in soil is well
known (Huang et al. 2010). While many studies were
mainly focused on different plant species designed for
human consumption such as potatoes (Dolliver et al.
2007), cucumbers (Shenker et al. 2011), lettuce
(Dolliver et al. 2007), carrots (Boxall et al. 2006) or
cereals (Dolliver et al. 2007; Wu et al. 2010), this
experiment also considers grass, a plant for animal
consumption, regarding a future evaluation on the
indirect contamination of animal food.
Materials and methods
Concentrations of pharmaceuticals
Considering the available data (Martinez-Carballo
et al. 2007) on tetracycline and sulphonamides’
contamination levels in sludge or soil, a 5 mg kg1
K4oc (l/kg) Structure Reference
1(a, b, c)
2(c)
3(d)
4(e, c)
1(f, c)
30–500
2(g, c, h)
3(d)
4(g)
1(c)
4897–12,882
2(i)
3(d)
4(j, k, l)

concentration was selected as the worst-case scenario
for the range of veterinary medicines measured in soils.
As a comparison, the concentration of 10 mg kg1 was
also used.
There is no available data on ketoconazole con-
tamination of sludge or soil. According to the VetPec
model approach (Equations (1) and (2)) (Montforts
2006), the following estimated concentrations were
obtained:
Qexcreted ¼ Qc Ttrat Fexcr Manim Ncyclus ð1Þ
Qexcr kdeg manure Tstorage
Cmanure ¼ e ð2Þ
Pmanure
where Qexcreted is the quantity of active substance
excreted in faeces and urine, Qc is the dosage of the
active substance used, Ttrat is the duration of treat-
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ment, Fexcr is the fraction of active substance excreted
in faeces and urine, Manim is the animal-averaged body
weight, Ncyclus is the number of cycles per storage
period, Pmanure is the total quantity of manure per
storage period, kdeg manure is the degradation rate
constant for manure, and Tstorage is the storage time
of manure. For oral dosage, a 38% rate removal as
parent drug was considered (McEvoy 2006). The
manure production by an animal in stable was 10 kg
place1 year1 (Teira-Esmatges et al. 2010). The
degradation rate constant for manure used in this
prediction was calculated by using Equation (3)
(Boxall et al. 2006).
lnð2Þ
kdeg manure ð3Þ
DT50
where DT50 is the half-life of the active drug in manure
(in days). Thirty days was considered the DT50 for
ketoconazole (no available data).
For a treatment dosage of 30 mg day1 (Rochette
et al. 2003), for 7 days for chicken, for two storage
periods (winter and summer) of manure, a maxi-
mum concentration of 11.7 mg kg1 in manure and
5 mg kg1 in soil can be achieved. The spiking concen-
trations of pharmaceuticals used in this experiment are
consistent with these estimated values.
Tested soil
Two sandy soils were used in the study. The soil,
characteristic sandy arable land in the agricultural area
of the Netherlands, was highly homogeneous, with
a relatively high organic carbon content because of
organic fertilisation and crop residues. The soil was
collected in the summer of 2011 from two farms
located close to Harskamp and Wageningen, The
Netherlands. The pH of the soils was 4.5 and 5.5,
respectively. According to RIVM report 2008 on
‘‘Soil ecosystem profiling in The Netherlands’’
(Rutgers et al. 2008), an average value of 5.2% soil
Food Additives & Contaminants: Part A 3
organic matter (SOM) was considered for both sandy
soils. After collection, the soil was air dried and
passed through a 3-mm sieve and mixed to ensure
homogeneity.
Tested plants
A commercially available mixture of grass species seeds
(75% English Ray Grass, Lolium perenne, 25% Field
Meadow Grass, Poa pratensis, Poa trivialis) and
watercress (Nasturtium officinale) seeds were selected
for the study. The plants were planted from the seeds.
Tested chemicals and soil spiking
The sources of the test pharmaceuticals were as
follows: Sigma-Aldrich Chemie B.V. (Zwijndrecht,
The Netherlands) for oxytetracycline and sulfameth-
oxazole and EDQM (Strasbourg, France) for ketoco-
nazole. Individual stock solutions of 10 mg ml1 of the
test substance were prepared in distilled water for each
compound. Proper quantities of each stock solution
were mixed, diluted to 50 ml in pure water and added
to 2 kg of air-dried soil to give a nominal substance
concentration of 5 and 10 mg kg1 of dried weight.
Additional distilled water was added to the soil to give
a moisture content of 20% w/w.
Uptake studies
For this study, plastic containers filled with soil were
used. To each 2.5-L plastic container (Ø ¼ 18.5 cm),
2 kg of soil was added. The experiment was designed in
duplicate. For each soil sample-plant combination,
three pots were prepared (two spiked and one control
soil), which resulted in a total number of 24 pots.
Separate pots for watercress and grass were used.
To prevent salt damage to germinating seeds, a thin
layer of fresh soils was added to the surface of each
pot. Finally, 50 ml of demineralised water was added to
enhance the germination of seeds. Approximately 1.5 g
of grass seeds and 1 g of watercress seeds were sown on
the soil surface, according to the recommended quan-
tities. A polyvinylchloride tube with a diameter of 2 cm
and a length of 20 cm was vertically introduced into the
centre of each pot. The first 6 cm of the tube below
the soil surface had small holes for watering the soil.
The pots were irrigated to maintain a water content
of about 50% to 60% of the pot’s water-holding
capacity.
In general, germination occurred very fast, and
visible shoots appeared after 2 days in the case of
watercress and after a week for grass.
The experiment was conducted in a laboratory of
RIKILT, Wageningen University and Research, from
June to August. Temperature was kept constant at
20 C, and humidity was maintained at 70%. Pots were
 4 C.L. Chitescu et al.
watered every 2 days with demineralised water and
rotated to equalise exposure to light.
After 8 weeks for grass and 6 weeks for watercress,
the plants were harvested from approximately 1.5 cm
above the soil’s surface. After chopping and homo-
genising, the fresh plant material from each pot was
stored in the freezer at 20 C in plastic tubes until the
analyses were performed.
Compounds analysis
Sample preparation
Extraction was performed on an accelerated solvent
extractor, ASE 350 system equipped with a solvent
selector (Dionex, ASE 350, USA). The optimised
operating conditions were as follows: extraction tem-
perature, 50 C; extraction pressure, 1500 psi; two
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cycles of 5 min each, static extraction; 50% flush
volume; and a 60-s purge with nitrogen. The extraction
solvent used was methanol/citric acid (0.2 M, 50:50, pH
adjustment at 4.5 with sodium hydroxide). After the
extraction, 100 ml Na2EDTA (1 M) was added to each
sample extract and Milli-Q water was added until a
final volume of 50 ml was reached. The sample was
centrifuged for 15 min at 2800 g (Falcom 6/300 MSE
Refrigerated Centrifuge, London, UK). An aliquot of
the final extract, corresponding to 1 g of the sample
(16.6 ml), was diluted with Milli-Q water to a final
concentration of 10% organic solvent (85 ml).
A solid phase extraction (SPE) procedure was
applied in order to clean and concentrate the extract by
using Strata X, 200 mg/6 ml, reverse-phase, SPE car-
tridge. The cartridge was previously preconditioned
with 6 ml methanol followed by 6 ml water. Before
loading the cartridge, the sample pH was adjusted to 3
with acetic acid. After sample application, the cartridge
was rinsed with 6 ml water, followed by 6 ml methanol/
water (30% v/v), and vacuum dried for 1 min. The
analytes were eluted with 6 ml methanol. The eluate
was concentrated by evaporation under flow of high-
purity nitrogen in a water bath at 42 C (Turbo Vap LV
Evaporator, Zymark, Hopkinton, MA) and dissolved
in 25 ml methanol and 225 ml Milli-Q water.
The validated procedure is described in a method
development study (Chitescu et al. 2012) performed
at RIKILT Institute, Wageningen, The Netherlands,
for the same group of compounds on soil and plant
material matrix. Recovery of the three compounds
investigated by spiking plant material was 53% for
oxytetracycline, 59% for ketoconazole and 60% for
sulfamethoxazole.
Full-scan analysis
One LC–full-scan MS configuration was used:
exactive high performance benchtop LC–MS mass
spectrometer powered by Orbitrap Technology from
Thermo Fisher Scientific (Breda, The Netherlands)
coupled to an ultrahigh-pressure liquid chromatogra-
phy chromatograph (Accela, Thermo Fisher Scientific)
system. The resolution was set at 50,000 full width
at half maximum. Full-scan acquisition of m/z ranged
from 100 to 1000; the scan rate used was 2 scans/s;
heated electrospray ion source was operated in the
positive mode.
For separation, an Acquity ultrahigh-pressure
liquid chromatography column C18 (100  2.1 mm,
1.8 mm) (Waters, Etten-Leur, The Netherlands) was
used. A flow rate of 0.4 ml min1 was set for the
separation of the selected compounds. The mobile
phase consisted of eluent A (100% water containing
2 mM ammonium formate and 160 ml formic acid) and
eluent B (100% methanol containing 2 mM ammonium
formate and 160 ml formic acid) (pH 3.5). The column
temperature was set at 40 C. The step gradient was as
follows: 0 to 1 min, 100% A; 1 to 2.5 min, linear
increase to 40% B; 2.5 to 10 min, linear increase to
100% B and hold 3 min; 13 to 13.2 min, decreasing
to 0% B; and 13.2 to 15 min, 100% A.
Detection was based on the calculated exact mass
and on the retention time of target compounds. Data
were evaluated by the Quan Browser Xcalibur 0606
(Thermo Fisher) and Thermo ToxID (Thermo Fisher).
Confirmatory analysis
The sample extracts were (re)analysed by LC–MS/MS
for the confirmation of the proposed identity. Triple
quadrupole-based precursor scans were performed
on a Micromass Quattro Ultima MS/MS (Waters,
Milford, MA) equipped with an electrospray interface
coupled to an LC-20AD (Shimadzu, USA). The same
analytical column and gradient solvents were used for
separation. For the confirmation of the identity,
the criteria described in EU decision 2002/657/EC
(European Commission 2002) were applied, including
the detection of two fragment ions with the appropri-
ate ion ratio. The linearity of the analytical method
was good (R 4 0.99) within 0 to 50 mg kg1. The
detection limits for sulfamethoxazole, oxytetracycline
and ketoconazole, based on calibration curves, were
4.4, 2.6 and 1.1 mg kg1, respectively.
Results
Experimental uptake by plants
The uptake of selected test pharmaceuticals by plants
was evaluated by extraction and analysis of fresh
plant material. Both grass and watercress took up
sulfamethoxazole and ketoconazole (Figure 1).
Sulfamethoxazole was detected in all samples, at
levels ranging from 7 to 21 mg kg1 for grass and
from 4 to 7.5 mg kg1 for watercress. No major
differences were observed between the uptake at the
two spiking levels. Watercress had a lower absorption.
 Food Additives & Contaminants: Part A 5

G_0815_20
U1_110826_028 MRM of six Channels ES+
4.94 531.14 > 488.93
1.06e4
6.52
4.18
%
3.95 5.21 6.59
2.62 3.10 3.22 3.30 3.89 4.38 7.41
4.65 5.34 5.67 5.83 6.28 7.72
1
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
U1_110826_028 MRM of six Channels ES+
6.50 531.14 > 82.09
3.59
4.57e3
%

3.12 3.23 4.75 4.96 5.04 5.58 5.71

3.77 4.08 4.43 6.13 6.39 6.83 7.06 7.38 7.57 7.98
2.78
3
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
U1_110826_028 MRM of six Channels ES+
4.28 461.1 > 337
3.21 4.23 5.28 6.12 6.67e3
3.61 4.41 4.80
3.19 3.67 5.60 6.00 6.20 7.58
5.11 7.08
Downloaded by [Wageningen UR Library] at 02:10 10 October 2012

3.53
5.81 6.46 6.75 7.56 7.98
7.13 7.70
2.88
3
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
U1_110826_028 MRM of six Channels ES+
3.23 461.1 > 201
6.97 2.07e4
4.01 4.43
3.19 5.74 5.79 6.63
4.41 7.02
%

3.52 3.74 4.71 4.82 5.01 6.38

5.48 7.53 7.83 7.95
2.98 6.75
1
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
U1_110826_028 MRM of six Channels ES+
3.58 254 > 108
9.64e4
%

3.20 3.45 4.00 4.23 4.52 4.78 4.86 5.26 6.26 6.55 7.79 7.92
5.71 6.15 7.19 7.26
0
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
U1_110826_028 MRM of six Channels ES+
3.58 254 > 92.2
1.59e5
%

3.29 3.88 4.17 4.58 4.82 5.03 5.78 5.87 6.17 6.78 7.04 7.47 7.82
0 Time
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

Figure 1. Chromatogram of a grass sample grown in contaminated soil (5 mg kg1). From the top to the bottom: ketoconazole:
m/z 531.14 ! 82.09, RT ¼ 6.5, m/z 531.14 ! 488.93, RT ¼ 6.5; oxytetracycline: m/z 461.1 ! 337, RT ¼ 3.6, m/z 461.1 ! 201,
RT ¼ 3.6; sulfamethoxazole: m/z 254.10 ! 92.20, RT ¼ 3.5, m/z 254.10 ! 108, RT ¼ 3.5. For LC–MS–MS conditions, see
Compounds analysis. RT, reaction temperature.

For ketoconazole, the results were less homogeneous.
For two samples of grass and five samples of water-
cress, the compound was not detected. The rest of the
samples showed low absorption. Oxytetracycline was
not detected in any sample. Antibiotics were not
detected in any of the unexposed (blank) samples.
Detailed uptake data for plants exposed to 5 and
10 mg kg1 antibiotics are presented in Table 2.
Comparison of experimental results with predicted
values of uptake concentration
The partition-limited model considers the passive
transport of small molecules, such as pesticides,
fungicides, antibiotics and pharmaceuticals, via soil/
pore water plants (Chiou et al. 2001; Montforts
2006). The uptake process is considered as a series of
partitions of small molecules (contaminant) between
plant water, carbohydrates and lipids fraction.
The model is expressed as follows:
Csoil
Cw ¼ ð4Þ
Foc soil Koc
where Cw is the contaminant’s concentration in exter-
nal water, Csoil is the contaminant’s concentration in
the soil, Foc soil is the fraction of organic matter in the
soil (SOM) and Koc is the contaminant’s partition
 6 C.L. Chitescu et al.

Table 2. Experimental values of plant uptake for selected compounds.

Compounds’ concentration (mg kg1a)

Sample Sulfamethoxazole Oxytetracycline Ketoconazole

Grass in soil 1_spiked at 5 mg kg1 21.5 54 51

Grass in soil 1_spiked at 5 mg kg1 10.5 54 1.7
Grass in soil 1_spiked at 10 mg kg1 17.5 54.4 2.9
Grass in soil 1_spiked at 10 mg kg1 12.9 54.4 1.7
Grass in soil 2_spiked at 5 mg kg1 7.1 54.4 51
Grass in soil 2_spiked at 5 mg kg1 6.8 54.4 2.0
Grass in soil 2_spiked at 10 mg kg1 14.5 54.4 1.7
Grass in soil 2_spiked at 10 mg kg1 5.0 54.4 1.9
Watercress in soil 1_spiked at 5 mg kg1 4.9 54.4 2.4
Watercress in soil 1_spiked at 5 mg kg1 4.1 54.4 51
Watercress in soil 1_spiked at 10 mg kg1 7.0 54.4 51
Watercress in soil 1_spiked at 10 mg kg1 4.2 54.4 51
Watercress in soil 2_spiked at 5 mg kg1 7.6 54.4 7.0
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Watercress in soil 2_spiked at 5 mg kg1 – 54.4 51

Watercress in soil 2_spiked at 10 mg kg1 4.5 54.4 51
Watercress in soil 2_spiked at 10 mg kg1 7.5 54.4 7.1

Note: aThe concentrations for contaminants are corrected for the recovery values.

coefficient between SOM and water. Table 3. Comparison of experimental results with estimated
values of plan uptake.
Cpt ¼ pt Cw ½ fpw þ fch Kch þ flip Klip  ð5Þ
Experimental Estimated
where Cpt is the concentration of the contaminant concentration concentration
in the plant on a fresh-weight base; fpw, fch and flip are Compound (mg kg1) (mg kg1) log Kow
the weight fraction of water, lipids and the sum of Oxytetracycline 54.4 0.10–0.15 0.95
carbohydrates, cellulose and proteins in the plant that Sulfamethoxazole 7–21.5 15–50 0.89
are assumed to have approximately the same partition Ketoconazole 1.7–4 50–73 3.84
coefficient (Kch); Klip is the partition coefficient for
the lipids fraction of the plant, which is assumed to be
equal to Kow, octanol-water partition coefficient; pt is transport model is not applicable for a quasi-
the quasi-equilibrium factor, defined as the ratio of the equilibrium factor of 1.
respective concentrations in plant water and external
water, with pt ¼ 1 denoting an equilibrium state. Discussion
Basically, with passive transport, if the concentrations The behaviour of veterinary medicines in the environ-
in whole plant and external water are at equilibrium, ment is related to a range of factors including the
all parts of the plant must be at equilibrium (Chiou H-bonding potential, cation exchange, cation bridging
et al. 2001). Approximate values for Kch are available at clay surfaces and complexation (Tolls 2001).
according to Kow (Hung et al. 2010). Previous studies have shown that plants take up
Weight composition of grass and watercress shoots less than 2% of the pharmaceuticals applied to soil
was supposed to be comparable with that of ryegrass (Kumar, Gupta, et al. 2005; Dolliver et al. 2007). In
shoots: 88.8% water, 0.97% lipids and 10.2% carbo- this study, two test compounds were able to transfer
hydrates (Li et al. 2005). into plant tissues from soils but their uptake behaviour
Estimated plant uptake values obtained for plant was compound specific. The root uptake of non-ionic
contamination in the case of 5 mg kg1 concentration organic chemicals from soil solution is considered to be
of test pharmaceuticals in soil following the partition- a partition-related process and generally increases with
limited model ranged from 15 to 50 mg kg1 for the increase in a compound’s hydrophobicity (Wu et al.
sulfamethoxazole, from 0.09 to 0.15 mg kg1 for oxy- 2010). The uptake of ionisable compounds can be
tetracycline and from 50 to 75 mg kg1 for ketoconazole affected by hydrophobicity as well as pKa and substrate
(using the estimated value of Kow ¼ 3.84). pH conditions (Trapp 2000). With soils’ pH ranging
On comparing the estimated concentration with between 4.5 and 5.5, sulfamethoxazol was predomi-
the experimental concentration (Table 3), for highly nantly in neutral form, oxytetracycline was 50% in
lipophilic compounds, it was found that the passive neutral form and ketoconazole was 70% to 80% in

ionisation form (MarvinSketch software). For ionisa-
ble organic compounds, their neutral form generally
favours root uptake, whereas ionisation can reduce
their bioaccumulation in plants (Rabølle and Sqliid
2006).
The result obtained for oxytetracycline, consistent
with estimate values, can be explained by its propri-
eties. Oxytetracycline has three pKa values: 3.3, 7.3 and
9.1; hence, it can exist as a cationic, zwitterionic and
anionic species under acidic, moderately acidic to
neutral and alkaline conditions (Thiele-Bruhn 2003;
Kong et al. 2007). The uptake of oxytetracycline is
pH dependent, which is the lowest at pH 5.0 and
the highest at pH 7.0. This trend is related to the
dissociation of oxytetracycline under different pH
conditions and the zwitterion form favouring uptake
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(Kong et al. 2007). The ionisation feature of oxytetra-
cycline can also significantly influence its behaviour,
including hydrolysis in water and sorption onto soil
components (Chen and Huang 2009). Doi and
Stoskopf (2000) found that the half-lives of oxytet-
racycline in water were 46 days at pH 3 and 9 days
at pH 10.
Oxytetracycline can form strong complexes with
metal cations through its multiple O- and N-functional
groups, thus influencing its sorption to the soil
components (minerals and organic matter) (Jones
et al. 2005; Andreu et al. 2009). Once adsorbed
onto the soils, oxytetracycline is hardly desorbed
(suggested by Koc value), with only 0.5% to 2.3%
released from sandy loam and loamy sand soils
(Rabølle and Sqliid 2006).
Sorption coefficients of sulphonamides are very
low in soil (Boxall et al. 2002), which indicates that
sulphonamides are more bioavailable. At soil pH of 4.5
to 5.5, sulfamethoxazole is almost completely in
neutral form, which means that absorption and trans-
port in the plant is a passive transport following the
partitioned model (quasi-equilibrium factor pt ¼ 1).
Within an order of magnitude, the results obtained are
consistent with the estimated value.
The uptake of ketoconazole, in a range of 2 to
5 mg kg1, is not consistent with predicted values,
suggesting that ketoconazole absorption and translo-
cation in the leaves by water mass flow is low. This
statement is supported by studies of translocation
of chemicals into shoots, indicating that uptake is
related to log Kow by a Gaussian curve distribution
(Briggs et al. 1982). Maximum translocation was
observed at a log Kow of 1.8.
Hsu’s studies (Hsu and Kleier 1990) on xenobiotic
absorption and translocation in plants established a
relationship between log Kow and organic matter
content of the soils. In hydroponic conditions, the
maximum absorption of xenobiotics is observed for a
log Kow value of 3 to 3.2. In normal plant growth
conditions, absorption decreases with an increase in
Food Additives & Contaminants: Part A 7
organic matter content, with the maximum absorption
observed for log Kow value of 1 to 1.2.
Compounds with increased polarity are taken up
less well by shoots, and the uptake of highly lipophilic
compounds (log Kow 4 4.5) is low. Considering the
pH of the soil (4.5–5.5), ketoconazole is ionised in
proportion of 70% to 80% and the partition model
is based on passive transport. Ketoconazole’s low
absorption can also be explained by a relatively high
Koc (low availability), and its highly lipophilic charac-
ter, determining strong associations with the organic
matter in soil (Kipopoulou et al. 1999). Based on
experimental results, the calculated value of the quasi-
equilibrium factor derived from Equation (2) is
pt  0.1.
Evaluation of risks for the consumer
The ingested dose due to the plant contamination is sig-
nificantly lower than the acceptable daily intake (ADI)
(0.03 mg kg1 day1 for oxytetracycline; 0.2 mg kg1
day1 for sulfamethoxazole) or the minimum oral
daily dose (3.3 mg kg1 for ketoconazole) and, there-
fore, do not cause an acute risk for humans.
Conclusions
This study demonstrates the ability of plants to uptake
pharmaceutical compounds from fertilised soils.
The effective concentration of the pharmaceuticals
available for plant uptake is considered to be the con-
centration in soil’s interstitial (pore) water, which can
be estimated and calculated. Soil composition influ-
ences by SOM the concentration of the pharmaceutical
in pore water.
The partition-limited model gave satisfactory
results for the passive transport of sulfamethoxazole.
Taking into account the capacity of oxytetracycline to
form strong complexes with the soil mineral or with
hydroxy groups at the surface of the soil particles as
well as the soil pH, no absorption of oxytetracycline
can be explained. In the case of highly lipophilic
compounds such as ketoconazole, there is a reverse
correlation between the quasi-equilibrium factor (pt)
and the Kow value, and, therefore, low absorption
from the soil. The experimental values of the quasi-
equilibrium coefficient, expressing the equilibrium
between pharmaceuticals’ concentration in plant
water and external water, are 1 for oxytetracycline
and sulfamethoxazole and less than or equal to 0.1 for
ketoconazole.
Additional uptake data have to be collected –
additional experiments have to be set up – for the
validation of the model to come to a final conclusion
regarding the applicability of the model to predict the
uptake of pharmaceuticals by plants.
 8 C.L. Chitescu et al.
Based on the uptake and exposure data of phar-
maceuticals by plants collected in this study, it is very
unlikely that there is any direct risk for the consumer
of the plants (human or animal). However, the risk
of allergy, long-term toxicity, exposure to mixtures of
pharmaceutical contaminants and, most importantly,
bacteria resistance development should not be
excluded.
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