Science of the Total Environment 601–602 (2017) 812–820

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Science of the Total Environment

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

Bioaccumulation of pharmaceutically active compounds and endocrine

disrupting chemicals in aquatic macrophytes: Results of hydroponic
experiments with Echinodorus horemanii and Eichhornia crassipes
N. Pi, J.Z. Ng, B.C. Kelly ⁎,1
Department of Civil and Environmental Engineering, National University of Singapore, 5A Engineering Drive 1, 117411, Singapore

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• PhACs and EDCs were readily taken up

E. crassipes and E. horemanii.
• Diphenhydramine and triclosan exhibit-
ed the highest degree of bioaccumula-
tion.
• BPA, E1, E2 and warfarin exhibited rela-
tively low bioaccumulation potential.
• No clear relationship was observed be-
tween physicochemical properties and
bioaccumulation behaviour.

a r t i c l e i n f o a b s t r a c t

Article history: Information regarding the bioaccumulation behaviour of pharmaceutically active compounds (PhACs) and endo-
Received 7 March 2017 crine disrupting chemicals (EDCs) in aquatic plants is limited. The present study involved controlled hydroponic ex-
Received in revised form 14 May 2017 periments to assess uptake and elimination rate constants (ku, ke), bioconcentration factors (BCFs) and translocation
Accepted 15 May 2017
factors (TFs) of several PhACs and EDCs in two aquatic macrophyte species, including one submerged species
Available online xxxx
(Echinodorus horemanii) and one free-floating species (Eichhornia crassipes). The results revealed that the studied
Editor: Jay Gan compounds are readily taken up in these aquatic plants. While bioconcentration factors (BCFs) and translocation
factors (TFs) of the test compounds varied substantially, no discernible relationship with physicochemical proper-
Keywords: ties such as octanol-water distribution coefficient (Dow), membrane-water distribution coefficient (Dmw) and or-
Pharmaceutically active compounds ganic carbon-water partition coefficient (Koc). Diphenhydramine and triclosan exhibited the highest degree of
Endocrine disrupting chemicals uptake and bioaccumulation potential. For example, the whole-plant BCF of triclosan in E. horemanii was
Aquatic macrophytes 4390 L/kg, while the whole-plant BCF of diphenhydramine in E. crassipes was 6130 L/kg. BCFs of 17β-estradiol
Bioaccumulation factors (E2), 17α-ethinylestradiol (EE2), estrone (E1) and bisphenol A (BPA) were relatively low (2–150 L/kg). BCFs
Translocation factors
were generally higher in free-floating aquatic macrophyte species compared to the submerged species. For the
free-floating species, E. crassipes, the majority of PhACs and EDCs were more allocated in roots compared to leaves,
with TFs b 1. However, some compounds such as caffeine, atrazine, diphenhydramine, E2 and carbamazepine were
more allocated in leaf tissue (TFs N 1). The study findings may be useful for design and implementation of
phytoremediation systems, as well as aid future modeling and risk assessment initiatives for these emerging organic
contaminants.
© 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author.
E-mail address: bckelly@nus.edu.sg (B.C. Kelly).
1
Department of Civil and Environmental Engineering, National University of Singapore, Block E1A, #07-03, No.1 Engineering Drive 2, 117576, Singapore.

http://dx.doi.org/10.1016/j.scitotenv.2017.05.137
0048-9697/© 2017 Elsevier B.V. All rights reserved.
 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820
1. Introduction
Emerging organic contaminants (EOCs) such as pharmaceutically
active compounds (PhACs) and endocrine disrupting chemicals
(EDCs) are widely used and produced substances, with annual produc-
tion volumes typically in the kiloton to megaton range (EC, 2011; WHO,
2012; USEPA, 2014). PhACs can enter environment as the parent phar-
maceutical compound or pharmacologically active metabolites
(Halling-Sorensen et al., 1998). Aquatic environments are considered
to be a major sink for PhACs and EDCs, since these chemicals are not
fully removed by conventional wastewater treatment technologies
(Petrie et al., 2013).
Due to high polarity and low volatility, most PhACs and EDCs tend to
be easily transported in aquatic environments (Breton and Boxall,
2003). More than eighty PhACs/EDCs have been detected in aquatic eco-
systems (Crane et al., 2006; Huerta et al., 2016; Jayasiri et al., 2013),
some of which are known to be relatively persistent, such as carbamaz-
epine and ibuprofen (Crane et al., 2006; Monteiro and Boxall, 2009).
PhACs and EDCs and/or their metabolites, upon entering the water en-
vironment, can pose potential ecological and human health risks, even
at trace levels ranging from ng/L to μg/L (Carlsson et al., 2006;
Corcoran et al., 2010; Daughton and Ternes, 1999; la Farre et al.,
2008). Aquatic organisms within wastewater impacted surface waters
are particularly susceptible to PhACs and EDCs (Garcia-Rodriguez
et al., 2014; Morley, 2009).
In recent years, aquatic plant-based phytoremediation systems
such as constructed wetlands have been proposed for the removal
of various waterborne contaminants (Matamoros et al., 2008;
Zhang et al., 2014). Aquatic plants play a significant role in removing
waterborne contaminants, via direct adsorption and/or absorption
(Dordio and Carvalho, 2013; Li et al., 2014; Pi et al., 2011). Water-
borne contaminants can be taken up and translocated (Felizeter
et al., 2012), accumulated (Felizeter et al., 2012; Kannan et al.,
2005; Nakata, 2005) and/or degraded via the metabolic transforma-
tion (Li et al., 2014). Uptake and translocation of organic contami-
nants in the whole plant system are highly dependent on the plant
species (Garcia-Rodriguez et al., 2014; Zarate et al., 2012), contami-
nant concentrations (Dordio and Carvalho, 2013; Garcia-Rodriguez
et al., 2014), and physicochemical properties of the contaminants,
including hydrophobicity (Garcia-Rodriguez et al., 2014), polarity
(Dordio and Carvalho, 2013; Garcia-Rodriguez et al., 2014) and
water solubility (Dordio and Carvalho, 2013; Garcia-Rodriguez
et al., 2014).
Previous studies have demonstrated that aquatic macrophytes can
be effective at accumulating and/or removing a variety of waterborne
contaminants, including nutrients (Pi et al., 2011), heavy metals (Pi
et al., 2011), and organic compounds such as perfluoroalkyl acids
(Felizeter et al., 2012). However, studies of PhACs and EDCs bioaccumu-
lation in aquatic macrophytes are limited (Li et al., 2014). Several stud-
ies have reported that the presence of plants in constructed wetlands
played a positive role in the removal of various PhACs, including
diclofenac, ibuprofen, ketoprofen, naproxen, salicylic acid, amoxicillin,
ampicillin, erythromycin, sulfadiazine, sulfamethazine, sulfamethoxa-
zole, atenolol, clofibric acid, carbamazepine and caffeine
(Hijosa-Valsero et al., 2016; Li et al., 2014; Matamoros et al., 2012;
Zhang et al., 2012a; Zhang et al., 2012b). However, these studies have
mainly focused on assessing contaminant removal efficiencies rather
than chemical bioaccumulation behaviour (Hijosa-Valsero et al., 2016;
Matamoros et al., 2012; Zhang et al., 2012a; Zhang et al., 2012b).
The objective of the present study was to conduct controlled hydro-
ponic experiments to assess uptake and elimination kinetics, transloca-
tion and bioaccumulation behaviour of several PhACs and EDCs in
aquatic macrophytes. Two aquatic macrophytes species were studied,
including a free-floating species (Eichhornia crassipes (Mart.) Solms)
and a submerged species (Echinodorus horemanii Rataj). The target
PhACs and EDCs exhibited a wide range of physicochemical properties.
813
2. Material and methods
2.1. Chemicals
Nineteen PhACs and EDCs were examined in this study (Table 1, and
Table S1, Supporting information), due to their wide distribution in
aquatic environment detected in organisms at different trophic levels
(Bayen et al., 2013; Crane et al., 2006; Huerta et al., 2016; Jayasiri
et al., 2013; Kannan et al., 2005; Nakata, 2005). High purity chemicals
of the native compounds were purchased. Atenolol, caffeine, linuron
and sulfamethoxazole were obtained from Wako Pure Chemical
(Wako Pure Chemical Inc., Japan), atrazine, bisphenol A (BPA),
carbamazapine, diclofenac, dilantin, diphenhydramine, estrone (E1),
17β-estradiol (E2), 17α-ethinylestradiol (EE2), gemfibrozil, ibuprofen,
naproxen, thiabendazole, triclosan and warfarin were supplied by
Sigma-Aldrich (Sigma-Aldrich, Saint Louis Mo, USA). Isotopically la-
beled compounds were purchased and used as the internal standards.
Atenolol-d7 (N99%), atrazine-d5 (N 99%), carbamazepine-d10 (N99%),
EE2-d4 (N 99%), ibuprofen-d3 (N99%), 13C12-triclosan (N98%) and
warfarin-d5 (N99%) were obtained from CDN Isotopes (C/D/N Isotopes
Inc., Canada), bisphenol A-d6 (98%), 13C3-caffeine (99%), E2-d4 (95–
97%), gemfibrozil-d6 (N 98%), 13C6-sulfamethoxazole (N98%) and 13C6-
thiabendazole (N98%) were supplied by Cambridge Isotope Laboratory
(Cambridge Isotope Laboratory, Inc., USA). HPLC-grade solvents were
obtained from Fisher Scientific (UK) and Tedia (USA).
2.2. Hydroponic experiments
The hydroponic experiments were conducted at Singapore's Van
Kleef Aquatic Science Centre, which offers a covered outdoor environ-
ment, allowing good growing conditions avoiding rain-water interfer-
ence. Nine plastic tanks, each had a dimension of 28 × 18 × 18 cm
(total volume was 9072 cm3), were prepared and divided into three
sets, E. crassipes, E. horemanii and control, with triplicates in each set.
Thirty E. crassipes and E. horemanii each (previously grown for
3 months), were bought from Green Park Tropical Fish Farm and
allowed to acclimate in the tanks for about one week, with 10 seedlings
per tank, the remaining tanks engaged as control were no plants pres-
ent. Each tank was fully covered with aluminum foil on the sides to pre-
vent the photo degradation of PhACs and EDCs, and filled with 6 L of tap
water, which pH value was adjusted to 7.0 ± 0.05 using hydrochloric
acid and was previously passed through the column containing activat-
ed carbon to remove chlorine. The water was renewed every 2 days for
another one week, to allow the plants acclimatized to new condition.
After acclimation, the hydroponic experiment was started with ex-
posure and depuration phase, from day 0 to 14 and day 15 to 28, respec-
tively. In exposure phase, the mixture of native PhACs and EDCs listed in
Table S1 were spiked into the tap water, with amount of each com-
pound in each tank at 120 μg and the final concentration was 20 μg/L.
To keep the concentration constant at 20 μg/L over the exposure
phase, the water with PhACs and EDCs was renewed every 2 days.
From day 15 onwards, water was still renewed every 2 days but without
chemicals spiking, to keep the concentration of PhACs and EDCs in each
tank constant at 0.
Before starting the exposure experiment, the possible decay of target
pollutants in water was considered and tested in the present study.
Three plastic tanks, filled with the same medium (a mixture of test com-
pounds with amount of 120 μg each were spiked into the tank contain-
ing 6 L of tap water, giving a final concentration of 20 μg/L for individual
PhACs and EDCs) as that used for the exposure experiment but without
plants, were prepared and covered with aluminum foil on the sides, to
preclude the photodegradation of target compounds. The water concen-
trations of target compounds in each tank were then analyzed immedi-
ately (Cw0) and two days later (Cw2). The difference between Cw0 and
Cw2 for all the target compounds was lower than 5%.
814 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820

Table 1
List of target compounds and corresponding physicochemical properties.

Compound Formula MW Group Class/Use Ionizable functional pKab % ionized Water Log Log Log Log
group molecules solubility Kow,Nb Kocb Dowc Dmwd
(pH 7) (mg/L)b (pH 7) (pH 7)

Atenolol C14H22N2O3 266.3 PhACs Beta blocker Basic 9.6 99.7 9.54 × 105 0.2 1.8 −2.3 −0.2
Atrazine C8H14ClN5 215.7 EDCs Pesticide Basic 1.6 0.00 214.0 2.6 2.4 2.6 2.7
BPA C15H16O2 228.3 EDCs Plasticizer Acidic 9.5 0.05 172.7 3.3 4.6 3.3 3.5
Caffeine C8H10N4O2 194.2 PhACs Stimulant Acidic 0.6 86.3 11,308 −0.1 1.0 -1.0 1.2
Carbamazapine C15H12N2O 236.3 PhACs Antiepileptic Neutral 15.4 0.00 112.0 2.4 3.1 2.4 2.5
Diclofenac C14H11Cl2NO2 296.2 PhACs NSAIDa Acidic 4.0 99.9 4.52 4.5 2.7 1.8 2.7
Dilantin C15H12N2O2 252.3 PhACs Antiepileptic Acidic 8.3 4.77 2.15 2.5 3.1 2.5 2.6
Diphenhydramine C17H21NO 255.4 PhACs Antihistamine Basic 9.0 99.0 362.7 3.3 3.9 1.3 2.2
E1 C18H22O2 270.4 EDCs Steroid estrogen Acidic 10.8 0.02 19.9 3.1 4.4 3.1 3.3
E2 C18H24O2 272.4 EDCs Steroid estrogen Acidic 10.8 0.02 50.1 4.0 4.2 4.0 4.2
EE2 C20H24O2 296.4 EDCs Synthetic Acidic 10.4 0.04 13.2 3.7 4.7 3.7 3.9
estrogen
Gemfibrozil C15H22O3 250.3 PhACs Lipid regulator Acidic 4.7 99.7 8.42 4.8 2.6 2.4 3.1
Ibuprofen C13H18O2 206.3 PhACs NSAIDa Acidic 4.4 98.4 41.1 4.0 2.6 2.2 2.5
Linuron C9H10Cl2N2O2 249.1 EDCs Pesticide Neutral 12.1 0.00 44.3 3.2 2.5 3.2 3.4
Naproxen C14H14O3 230.3 PhACs NSAIDa Acidic 4.2 99.9 44.1 3.2 2.5 0.5 1.4
Sulfamethoxazole C10H11N3O3S 253.3 PhACs Antibiotic Acidic 6.1 88.8 869.5 0.9 2.4 −0.05 0.1
Thiabendazole C10H7N3S 201.3 EDCs Pesticide Basic 4.7 0.50 482.1 2.5 3.6 2.5 2.6
Triclosan C12H7Cl3O2 289.5 PhACs Antibacterial Acidic 7.8 13.7 9.29 4.8 4.4 4.7 4.9
Warfarin C19H16O4 308.3 PhACs Anticoagulant Acidic 5.0 99.0 4.09 2.7 2.6 0.7 2.1
a
NSAID: nonsteroidal anti-inflammatory drugs.
b
Compiled from US EPA's Estimation Programs Interface (EPI) Suite™.
c
Octanol-water distribution coefficient (Dow) at pH 7 was determined as Dow (pH 7) = fN × Kow, N + fI × Kow, I, where fN and fI are fraction of neutral and ionic species at pH 7, re-
spectively, as predicted from the Henderson-Hasselbach equation; Kow, N and Kow, I are the octanol-water partition coefficients (Kow) of the neutral and ionic species, respectively. Kow, I was
determined by assuming Kow, N was approximately 3 log units higher (i.e., Δow = 3.1).
d
Membrane-water distribution coefficient (Dmw) at pH 7 was determined from liposome-water partition coefficients following Armitage et al. (2013). Specifically, the liposome-water
partition coefficient for neutral species was determined as Log Kmw, N = 1.01 × Log Kow + 0.12, the liposome-water partition coefficient for ionic species was determined as Log Kmw, I
= Log Kmw,N − Δmw. Subsequently, Dmw was determined as, Dmw (pH 7) = fN × Kmw, N + fI × Kmw, I where fN is the fraction of chemical in neutral form and fI is the fraction of chemical
in charged form at pH 7, as predicted by the Henderson-Hasselbalch equation.

During the whole experiment, one seedling of each plant species
was randomly collected from each tank every 4 days, washed and proc-
essed into leaves and roots. After being weighed, the leaves and roots
samples were stored at − 20 °C before chemical analysis. Part of the
leave and root samples were dried in an oven at 70 °C for 48 to 72 h
and the oven-dried weight of plant samples were measured, to obtain
the plant dry biomass. Water samples were collected on day 0, 2, 4, 14
(the day 0 of the depuration phase), 16 and 18, just before and immedi-
ately after water renewal, to determine chemical concentrations in
water.
2.3. Extraction and cleanup of water and macrophyte samples
Water samples were processed based on the previous studies
(Bayen et al., 2013; Togola and Budzinski, 2008; Wu et al., 2010b) and
USEPA method 1694 (USEPA, 2007), with some modifications in pres-
ent study and the actual method used was described below in detail.
Water samples with volume of 500 mL each were filtered through a
glass fiber filter (diameter 47 mm, particle retention 1 μm) and the pH
was adjusted to 7.0 ± 0.05 using 1 M of hydrochloric acid. Prior to SPE
extraction, the mixture of labeled PhACs and EDCs was spiked, with
amount of each compound listed in Table S2, and equilibrated in the
dark at 4 °C for about 14–18 h. Samples collected during exposure
phase (day 0, 2 and 4) were then directly injected into LC/MS/MS for
chemical analysis. Samples taken during depuration phase (day 14, 16
and 18) were further extracted using Hydrophilic Lipophilic Balance
(HLB) cartridges (60 mg, 3 mL, Phenomenex), pre-conditioned with
5 mL of methanol, 5 mL of Milli-Q water and 5 mL of Milli-Q water
with pH at 7.0. After the samples were loaded, the cartridges were
washed with 5 mL of MilliQ water, and vacuum-dried for 15 min.
Analytes were eluted with 12 mL of methanol followed by 6 mL of ace-
tone and methanol (1:1 v/v). Combined elution was concentrated to a
final volume of 0.5 mL under nitrogen stream, transferred to 2 mL of
GC vial, and spiked with 50 ng of 13C6-TCPAA and Atrazine-d5. The pre-
pared samples were stored at −20 °C before LC/MS/MS analysis.
For macrophytes, extraction and cleanup were based on previous
methods for PhACs and EDCs in biota (Bayen et al., 2013; Holling
et al., 2012; Metcalfe et al., 2010; Vanderford and Snyder, 2006),
USEPA method 1694 (USEPA, 2007)), with some modifications and
the actual method used in present study was described below. Specifi-
cally, 5 g of wet plant sample were weighed, grinded to dryness with an-
hydrous sodium sulfate, and transferred to 50 mL polypropylene tube.
20 mL of methanol (HPLC grade, fisher, UK) was added to each sample
tube and the mixture of labeled PhACs and EDCs was spiked, with
amount of each compound listed in Table S2. The contents of each
tube were then mixed with a vortex for 20 s, prior to the ultrasonic-
extraction operated in an ultrasonic cleaner (Elma, S 60 H, Alphasonics
Pte., Ltd.) at room temperature for 15 min. The extract was centrifuged
at 1100 gravity for 5 min and the supernatant was then decanted to a
round bottom flask. The procedures were repeated for three times. In
order to increase the extraction efficiency of triclosan, acetone was
used at the second round instead of methanol, as acetone is more effec-
tive than methanol to extract triclosan from biota samples (USEPA,
2007). The extract was combined and the final 60 mL of extract in
each flask was then concentrated to approximately 2 mL using a rotary
evaporator at 120 rpm and a temperature of 40 °C. Then 50 mL of Milli-Q
water was added to each flask, mixed and prepared for the clean-up.
The pH of the mixture was adjusted to 7.0 ± 0.05 using diluted hydro-
chloric acid at concentration of 1 M.
Solid Phase Extraction (SPE) clean-up was performed using HLB car-
tridges (200 mg, 6 mL, Phenomenex). Cartridges were pre-conditioned
by passage of 5 mL of methanol, followed by 5 mL of Milli-Q water and
5 mL of Milli-Q water with pH of 7.0. Then samples obtained above were
passed through the pre-conditioned cartridges at a rate of 1 drop/s.
After all the samples were loaded, the cartridge was washed with
5 mL of Milli-Q water and vacuum-dried for approximately 15 min.
The cartridges were then eluted with 12 mL of methanol, followed by
6 mL of acetone and methanol (1:1 v/v). The combined elution was con-
centrated under a gentle stream of nitrogen at 5 bar and 35 °C, to a final
volume of 0.5 mL. The resulting solution was transferred to 2 mL of GC
 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820
vial and 50 ng of instrument internal standards (13C6-TCPAA and
Atrazine-d5) were spiked to each vial. The vials were then stored at
− 20 °C before sending to liquid-chromatography electrospray
ionization-tandem mass spectrometry (LC-ESI-MS/MS) for the chemical
analysis.
2.4. Instrumental analysis using LC-ESI-MS/MS
A Shimadzu LCMS-8030 Tandem Quadruple Liquid Chromatograph
Mass Spectrometer (LC/MS/MS) was used for the identification and
quantification of target analytes. A Poroshell 120 Stable bond SB-C18
column (2.1 × 150 mm, 2.7-micron) preceded by a Poroshell 120 Stable
bond SB-C18 guard column (2.1 × 5 mm, 2.7-micron) was selected for
analysis. The mobile phase comprised 5 mM of ammonium acetate in
Milli-Q water (A) and ACN/MeOH (1:1 v/v, B) and was delivered at a
flow rate of 300 μL/min. The injection volume was 10 μL. The mass spec-
trometer was operated in multiple reaction monitoring (MRM) mode,
with some compounds in positive ion and the others in negative ion
(Table S1, Supporting Information). The linear gradient program for
positive ion mode was started with mobile phase B at 10%, and in-
creased to 40% at 4 min, to 100% at 10 min, and kept at 100% until
19 min, then decreased to 10% at 20 min and kept until 22 min. The lin-
ear gradient program for negative ion mode was started with B at 10%
and kept until 0.5 min, increased to 50% at 2 min, to 100% at 6 min,
kept at 100% until 10 min, then decreased to 10% at 10.5 min and kept
till 14 min. The transitions (Q1 and Q3) for each compound were listed
in Table S1.
2.5. Quality assurance/quality control
All samples were divided into several batches for the treatment and
analysis. Each batch consisted of 1 procedural blank and 10–12 samples.
Analytes were identified and quantified based on the following criteria:
1) two MRM transitions of each analyte were detected during the entire
chromatographic run; 2) retention time (RT) of the peak of analyte was
within 3 s of the RT obtained from analysis of authentic compounds in
the calibration standards; 3) the difference of the observed ratio of the
two monitored ions for each compound was within 20% between the
analytes and calibration standards; and 4) the signal-to-noise (S/N)
ratio resulted from the peak response of the two corresponding ions
was ≥ 10 for proper quantification of the analyte. Internal standard
isotope-dilution method was used for the calculation of concentrations
of analytes, using mean relative response factors (RRF) derived from 7
calibration standards with relative standard deviation (RSD) below
20%. Method detection limits (MDLs) were set equal to the instrument
detection limit considering the matrix effects of each sample, deter-
mined from each analyte peak response with a S/N ratio of 5. The
MDLs and absolute recoveries for target PhACs and EDCs are provided
in Table S3 and Table S4 respectively. Isotope labeled surrogate com-
pounds and the isotope dilution quantification approach were utilized,
which inherently corrected for exact losses in every sample. Thus, all
samples were recovery corrected. Compounds with blank levels lower
than 10% of extracted sample matrix were further calculated and statis-
tically analyzed without blank correction.
2.6. Data analysis
The approach for data treatment and analysis were similar as that
described in Pi et al. (2017). In brief, plant growth rate constant (kg)
was determined using the measured dry biomass during exposure
phase:
Mt ¼ M 0 ekg t
where Mt. and M0 are the total plant dry biomass at time t and 0
respectively.
815
Uptake and elimination rate constants (ku, ke) of target compounds
were determined using their observed concentrations in plant tissues
(Cp) during exposure and depuration phase, respectively:
C P ¼ C 0 e−ke t d ð2Þ
" #
ku h i
CP ¼ Cw
 1−e−ðke þkg Þt u ð3Þ
ke þ kg
where Co is the initial chemical concentration in plant at the start of
depuration, Cw is the chemical concentrations in water, td is the time
measured from the start of depuration phase, and tu is the time mea-
sured from the start of the experiment until the end of exposure phase.
Kinetically-derived bioconcentration factors (BCFk) values were de-
termined as ku/(ke + kg). Apparent steady-state bioconcentration fac-
tors (BCFss) were determined as the ratio of the chemical
concentration observed in plants (Cp) and water (Cw) at the end of
the exposure phase (i.e., Cp/Cw). Translocation factors (TFs) were deter-
mined as the ratio of the chemical concentration (ng/g dry weight) ob-
served in leaf and roots. Separate TF values were determined for the
exposure and depuration phase on day 14 and 28, respectively. Physico-
chemical properties of individual PhACs and EDCs were used to evaluate
uptake, translocation and bioaccumulation behaviour patterns.
Physicochemical properties of the target PhACs and EDCs are shown
in Table1. Octanol-water partition coefficients of the neutral com-
pounds (Kow,N) and organic carbon-water partition coefficients (Koc)
were compiled the US EPA's Estimation Programs Interface (EPI)
Suite™. Octanol-water distribution coefficient (Dow), which is an esti-
mate of the ratio of the sum of the concentrations of all chemical species
(ionized + un-ionized) in octanol and water, was determined from the
acid dissociation constant (pKa) at pH 7. Membrane-water distribution
coefficient (Dmw) values were estimated from observations of
liposome-water partition coefficients, following the approach used by
Armitage et al. (2013). The compiled information was used to better as-
sess the influence of physicochemical properties on transport and
partitioning behaviour of these ionizable organic compounds in neutral
lipids, phospholipids and non-lipid organic matter of aquatic
macrophytes.
3. Results and discussions
3.1. PhAC and EDC concentrations in water
Concentrations of test compounds in experimental and control tank
water ranged between 19.6 and 20.3 μg/L and 19.7 to 20.4 μg/L on day 0,
between 18.0 and 18.6 μg/L and 19.0 to 20.5 μg/L on day 2, and between
18.1 and 18.9 μg/L and 19.1 to 20.6 μg/L on day 4. The test compounds in
water were below MDLs during the depuration phase (day 14, 16 and
18). The mass balance of chemicals in water was mostly between
90.1% and 103.1%, indicating that fluctuations were negligible. There-
fore, it can be assumed that the 2-day refilling of water with PhACs
and EDCs was able to maintain a constant water concentration of
chemicals and no further collection of water samples was needed.
3.2. Uptake and elimination of PhACs and EDCs
Uptake of PhACs and EDCs in plants was observed for both species,
with amount and rate specific in individual parts, plant species and
compounds. Observed concentrations of individual PhACs and EDCs in
plant root and leaf tissues during the experimental period are shown
in Figs. 1 and 2, and Figs. S1 and S2 (see Supporting information). During
ð1Þ the exposure phase, concentrations of tested chemicals in plant roots
and leaves of both species typically increased rapidly between day 0
and 8, continuing to increase until an apparent steady state was
reached, typically between day 8 and 14. During the depuration phase,
816 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820

concentrations of chemicals absorbed in root and leaf decreased rapidly
between day 14 and 21.
The uptake and elimination rate constants (ku, ke) for the various
PhACs and EDCs are summarized in Table 2 and Table S5 (see
Supporting Information). On a whole-plant basis (leaf + root), ku values
in some PhACs and EDCs, including atenolol, BPA, caffeine,
carbamazapine, dilantin, diphenhydramine and thiabendazole, were
found much higher in E. crassipes, ku values of sulfamethoxazole and
warfarin were comparable between the two plant species, while ku
values of the remaining compounds were observed higher in
E. horemanii. ku values ranged from 0.9 L/kg·d for warfarin to
843 L/kg·d for triclosan in E. horemanii, and ranged from 0.4 L/kg·d
for E1 to 1550 L/kg·d for diphenhydramine in E. crassipes. Between
the two plant species, whole-plant ke values for E. horemanii ranged ap-
proximately from 0.07 d− 1 (atenolol and BPA) to 0.47 d− 1
(carbamazapine). ke values observed for E. crassipes were between
0.10 d−1 (E1) and 0.42 d−1 (dilantin).
3.3. Bioconcentration factors (BCFs)
The BCFs of individual PhACs and EDCs in E. horemanii and
E. crassipes, varied substantially (Table 3). On a whole-plant basis,
PhACs exhibited BCF values ranging from 10.1 (warfarin) to 4390 L/kg
(triclosan) and from 9.4 (warfarin) to 6130 L/kg (diphenhydramine)
in E. horemanii and E. crassipes respectively. BCFs of EDCs in
E. horemanii and E. crassipes ranged from 5.8 (BPA) to 291 L/kg (thiaben-
dazole) and from 2.2 (E2) to 763 L/kg (thiabendazole). BCFs of individ-
ual PhACs and EDCs were different between the two plant species.
Atrazine, diclofenac, E1, E2, EE2, gemfibrozil, ibuprofen, naproxen, sulfa-
methoxazole and triclosan exhibited higher BCFs in the submerged spe-
cies, E. horemanii. Atenolol, BPA, caffeine, carbamazapine, dilantin,
diphenhydramine, linuron and thiabendazole exhibited higher BCFs in
the free-floating species, E. crassipes.
BCFs based on leaf and root tissue concentrations were clearly differ-
ent between the two macrophyte species E. crassipes, the floating

Fig. 1. Observed concentrations of individual PhACs and EDCs in roots and leaf tissue of E. horemanii during the 28-day experiment. Data are represented as mean ± standard deviation (n = 3).
 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820 817

Fig. 2. Observed concentrations of individual PhACs and EDCs in roots and leaf tissue of E. crassipes during the 28-day experiment. Data are represented as mean ± standard deviation (n = 3).

species, generally exhibited higher root BCFs compared to E. horemanii
for most compounds tested. For example, the root BCF for atenolol in
E. crassipes (2660 L/kg) was nearly 20 times higher than that of
E. horemanii (118 L/kg). BCFs in E. horemanii were found to be higher
in leaf, compared to root, for most compounds tested. Conversely,
BCFs for most compounds in E. crassipes were higher in root, compared
to leaf. These results can be attributed to the submerged nature of
E. horemanii, which allows direct chemical exposure and partitioning
between water and leaf tissue.
3.4. Translocation factors (TFs)
The observed differences between the two types of macrophytes
were further shown by the determination of TFs (Table S6, Supporting
information). The difference of TFs was observed among the com-
pounds tested and between species. At the end of exposure phase
(day 14), TFs for the tested compounds were generally higher in
E. horemanii, the submerged species, with exceptions of diphenhydra-
mine and E2. Most tested compounds in E. horemanii were found to al-
locate more in leaf compared to root (i.e., TFs N 1), whereas, most
compounds in E. crassipes were observed to allocate more in root, com-
pared to leaf (i.e., TFs b 1). The observed TFs further highlight that PhACs
and EDCs are more efficiently accumulated in leaf tissue of E. horemanii
due to the submerged nature of this species. For E. crassipes, a free-
floating species with foliage not directly exposed to the water, efficient
internal transport from roots into foliage is required for translocation to
occur.
At the end of depuration phase (day 28), the majority of the PhACs
and EDCs in E. crassipes were allocated more in roots, compared to leaf tis-
sue, which was the same as that in exposure phase (day 14) (i.e., TFs b 1).
However, for E. horemanii, the allocation of some compounds in leaf and
root was observed to be different between exposure and depuration pe-
riod. For example, the TF of caffeine in exposure period was N1 (4.98)
and nearly 5 times higher than that in depuration period (0.91).
818 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820

Table 2
Whole-plant uptake rate constants (ku, L/kg·d) and elimination rate constants (ke, d−1) of
individual PhACs and EDCs in E. horemanii (submerged macrophyte) and E. crassipes (free-
floating macrophyte).

Compound E. horemanii E. crassipes

ku (L/kg·d) ke (d−1) ku (L/kg·d) ke (d−1)

Atenolol 79.1 ± 6.6 0.07 ± 0.01 503 ± 25.5 0.12 ± 0.01

Atrazine 90.0 ± 8.3 0.25 ± 0.02 38.4 ± 2.9 0.25 ± 0.02
BPA 1.1 ± 0.1 0.07 ± 0.01 5.6 ± 0.5 0.12 ± 0.01
Caffeine 17.5 ± 1.3 0.27 ± 0.03 34.7 ± 2.9 0.31 ± 0.02
Carbamazapine 124 ± 10.7 0.47 ± 0.04 176 ± 12.9 0.39 ± 0.04
Diclofenac 20.0 ± 1.7 0.23 ± 0.02 5.7 ± 0.5 0.19 ± 0.02
Dilantin 55.1 ± 4.4 0.32 ± 0.02 102 ± 8.2 0.42 ± 0.03
Diphenhydramine 410 ± 29.9 0.36 ± 0.03 1552 ± 121 0.13 ± 0.01
E1 2.0 ± 0.2 0.11 ± 0.01 0.4 ± 0.0 0.10 ± 0.02
E2 2.2 ± 0.2 0.10 ± 0.01 0.7 ± 0.1 0.15 ± 0.01
EE2 38.2 ± 2.7 0.23 ± 0.02 17.7 ± 1.4 0.15 ± 0.01
Gemfibrozil 62.2 ± 4.2 0.17 ± 0.02 14.5 ± 1.2 0.18 ± 0.02
Ibuprofen 20.3 ± 1.6 0.20 ± 0.02 6.1 ± 0.5 0.19 ± 0.02
Linuron 76.2 ± 5.9 0.35 ± 0.03 129 ± 9.4 0.32 ± 0.02
Naproxen 77.9 ± 6.1 0.13 ± 0.01 62.0 ± 5.1 0.21 ± 0.02
Sulfamethoxazole 3.3 ± 0.3 0.13 ± 0.01 3.0 ± 0.3 0.17 ± 0.02
Thiabendazole 104 ± 7.4 0.32 ± 0.03 259 ± 17.7 0.25 ± 0.02
Triclosan 843 ± 51.6 0.12 ± 0.01 385 ± 28.9 0.24 ± 0.02
Warfarin 0.9 ± 0.1 0.08 ± 0.01 1.1 ± 0.1 0.14 ± 0.01

3.5. Influence of physicochemical properties

Fig. 3 illustrates plots of log BCFss values of the individual PhACs and
EDCs in whole plant of aquatic macrophyte versus chemical log Dow and
log Dmw. Similarly, Fig. 4 shows plots of the corresponding log TF values
of the studied compounds at the end of exposure phase versus chemical
log Dow and log Dmw. Recent studies of uptake and depuration of
perfluoroalkyl substances (PFASs) in aquatic macrophytes demonstrat-
ed strong relationships between bioconcentration and translocation po-
tential and chemical Dow, Dmw and Koc (Pi et al., 2017). In particular, the
more hydrophobic long-chain PFASs, with higher Dow, Dmw and Koc, ex-
hibited relatively lower TFs, but higher whole-plant BCFs. The data indi-
cate a higher degree of partitioning of those more hydrophobic
compounds into neutral and phospholipids and non-lipid organic mat-
ter of aquatic macrophytes for more hydrophobic compounds. In con-
trast, the results of the present study of PhACs and EDCs do not
indicate any correlation between log BCFs/log TFs and log Dow/log
Dmw values (Fig. 3, Fig. 4). Similarly, there was no clear trend between
Fig. 3. Plots showing the relationship between the whole-plant bioconcentration factors
(log BCFss, L/kg) of test chemicals versus (A) octanol-water distribution coefficient (log
Dow) and (B) membrane-water distribution coefficient (log Dmw).
Log BCFs/Log TFs and the organic carbon-water partition coefficient
(Koc), which is a representation of chemical partitioning in the organic
carbon based constituents (Fig. S3). The lack of a discernible relation-
ship between BCFs and TFs and hydrophobicity may be due, in part, to
variability in degradation of the different compounds. It is also impor-
tant to note that the studied PhACs and EDCs comprise a range of differ-
ent chemical classes, exhibiting different ionizable function groups,
which may also influence uptake and partitioning behaviour.

Table 3
Observed steady-state bioconcentration factors (BCFss, L/kg) of individual PhACs and EDCs in leaf, roots and whole-plant of E. horemanii (submerged macrophyte) and E. crassipes (free-
floating macrophyte).

Compound E. horemanii E. crassipes

Leaf Root Whole plant Leaf Root Whole plant

Atenolol 463 ± 31.9 118 ± 8.9 402 ± 29.9 1560 ± 112.7 2660 ± 177 2050 ± 133
Atrazine 319 ± 23.3 30.3 ± 2.5 259 ± 18.3 138 ± 12.5 56.9 ± 4.3 106 ± 8.4
BPA 7.9 ± 0.7 16.4 ± 1.1 5.8 ± 0.5 25.6 ± 2.2 71.4 ± 5.9 19.8 ± 1.2
Caffeine 62.5 ± 5.6 8.5 ± 0.7 52.0 ± 5.1 144 ± 12.0 29.7 ± 2.0 98.4 ± 6.3
Carbamazapine 370 ± 31.1 98.3 ± 7.4 330 ± 21.4 458 ± 29.8 313 ± 21.6 402 ± 28.4
Diclofenac 75.9 ± 6.4 96.1 ± 7.0 78.9 ± 7.0 5.2 ± 0.4 42.3 ± 24.3 19.4 ± 1.4
Dilantin 173 ± 14.6 85.8 ± 7.8 161 ± 13.3 203 ± 15.2 266 ± 17.7 233 ± 15.2
Diphenhydramine 1100 ± 97.4 274 ± 19.6 939 ± 57.3 9350 ± 365 1290 ± 102 6130 ± 313
E1 15.4 ± 1.2 5.9 ± 0.6 13.4 ± 1.2 0.1 ± 0.01 9.7 ± 0.9 4.1 ± 0.4
E2 12.3 ± 1.0 1.2 ± 0.1 10.1 ± 0.9 3.3 ± 0.3 0.3 ± 0.04 2.2 ± 0.2
EE2 153 ± 13.8 47.5 ± 3.2 133 ± 11.1 57.0 ± 4.1 140 ± 10.9 57.9 ± 4.4
Gemfibrozil 351 ± 31.1 91.8 ± 7.9 314 ± 23.5 12.9 ± 1.2 126 ± 10.3 46.7 ± 4.7
Ibuprofen 120 ± 10.6 24.3 ± 1.9 105.9 ± 9.1 10.5 ± 0.9 38.5 ± 3.2 21.0 ± 1.7
Linuron 244 ± 19.6 40.9 ± 3.0 214 ± 15.4 222 ± 18.2 432 ± 32.4 307 ± 21.7
Naproxen 532 ± 33.4 95.6 ± 8.1 468 ± 33.6 114 ± 9.2 345 ± 26.3 207 ± 16.3
Sulfamethoxazole 23.7 ± 1.7 21.4 ± 1.9 23.4 ± 1.9 6.7 ± 0.6 15.5 ± 1.2 10.3 ± 0.8
Thiabendazole 287 ± 22.2 315 ± 24.3 291 ± 21.5 284 ± 21.1 1480 ± 117 763 ± 56.5
Triclosan 4570 ± 321 643 ± 43.8 4390 ± 296 809 ± 33.5 1580 ± 125 1050 ± 89.2
Warfarin 7.7 ± 0.6 21.8 ± 1.7 10.1 ± 0.9 2.4 ± 0.2 24.7 ± 2.0 9.4 ± 0.8
 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820
Fig. 4. Plots showing the relationship between the translocation factors (log TFs) of test
chemicals at the end of exposure phase versus (A) octanol-water distribution coefficient
(log Dow) and (B) membrane-water distribution coefficient (log Dmw). The dashed line
represents a TF equal to 1.
Alternatively, the hydrophobicity range investigated (log Dow − 2.3 to
4.7) may be too narrow to observe clear differences.
In the present study, triclosan (log Dow = 4.7) exhibited relatively
high bioaccumulation potential, with whole-plant BCFss values equal
to 1050 and 4390 L/kg in E. crassipes and E. horemanii, respectively
(Table 2, and Table S5, Supporting information). However, less hydro-
phobic compounds such as diphenhydramine (log Dow = 1.3) also ex-
hibited very high bioconcentration potential (BCFss N 6000 L/kg).
Similar to previous studies, carbamazapine (log Dow = 2.4) was found
to exhibit a high degree of bioaccumulation in both E. horemanii (BCFss
of 330 L/kg) and E. crassipes (BCFss of 402 L/kg). Similar results were ob-
served for atenolol (log Dow = −2.3), which exhibited a whole-plant
BCFss of 2050 L/kg in E. crassipes. Surprisingly, more hydrophobic com-
pounds such as BPA, E1, E2 and EE2 exhibited very low bioaccumulation
potential in these aquatic macrophytes.
Regarding translocation, the results show that carbamazapine (log
Dow = 2.4) is effectively transported from root to leaf, with TFs values
in E. horemanii and E. crassipes of 3.76 and 1.46, respectively, which is
consistent with previous studies (Dordio et al., 2011). Caffeine (log
Dow = − 1.0) was also shown to be readily taken up translocated
from root to leaf for both species, with TFs values much higher than 1,
consistent with previous studies (Dettenmaier et al., 2009;
Garcia-Rodriguez et al., 2014; Matamoros et al., 2012; Zhang et al.,
2013a; Zhang et al., 2013b).
In the cell membranes of plant roots, non-specific transporters can
transport PhACs and EDCs into the plants tissues, however, the uptake
819
and translocation of these compounds within plants can be simply driv-
en by diffusion (Dietz and Schnoor, 2001; Dordio et al., 2011; Dordio
and Carvalho, 2013; Li et al., 2014). Previous studies have pointed out
that uptake and translocation of organic contaminants in aquatic sys-
tems caused by the diffusion process can be affected by physicochemical
properties of compounds such as water solubility, hydrophobicity (log
Kow), pKa, as well as pH and exposure concentration in the water
(Dordio and Carvalho, 2013; Garcia-Rodriguez et al., 2014; Hurtado
et al., 2016; Prosser et al., 2014; Tsao, 2003). It has been suggested
that moderately hydrophobic chemicals (log Kow range: 0.5 to 3.5) are
sufficiently lipophilic to move through the lipid bilayer of plant cell
membranes and water soluble enough to travel into the cell fluids of
plants, hence are more likely to be taken up and translocated to leaves
(Dordio and Carvalho, 2013; Li et al., 2014; Pilon-Smits, 2005). For ex-
ample, uptake and translocation of carbamazepine (log Dow = 2.4) in
plants has been attributed to efficient transport across root membranes
and cell fluids (Garcia-Rodriguez et al., 2014; Holling et al., 2012; Paz
et al., 2016; Shenker et al., 2011; Wu et al., 2010a; Zhang et al.,
2013a). Dordio et al. reported that carbamazepine can be readily
taken up by the roots of Typha spp. and then transported from roots to
stems and leaves (Dordio et al., 2011). Similarly, previous studies have
shown caffeine (Log Dow = −1.0) and sulfamethoxazole (Log Dow =
−0.1) could be easily taken up and translocated in cabbage and soybean
plants (Holling et al., 2012; Wu et al., 2010a).
Overall, the results from the present study are consistent with previ-
ous observations of PhAC and EDC bioaccumulation in plants. The re-
sults suggest that bioaccumulation and translocation of PhACs and
EDCs in aquatic plants is not easily predicted by physicochemical prop-
erties. Less hydrophobic compounds such as carbamazapine and di-
phenhydramine are more readily taken up and translocated in leaf
tissue. Triclosan, a weak acid (pKa = 7.8) with a log Dow of 4.7, exhibits
minimal translocation, but a relatively high degree of bioaccumulation
potential on a whole-plant basis. Further empirical and/or modeling
studies are required to better understand the role of physicochemical
properties in the uptake and distribution of these ionizable organic con-
taminants in aquatic macrophytes.
3.6. Implications for phytoremediation and risk assessment
The results from the present study suggest that PhACs and EDCs
can be readily taken up and accumulated in aquatic macrophytes.
The bioaccumulation potential and the distribution between roots
and leaf tissue vary widely among for these compounds. The
bioaccumulation behaviour of PhACs and EDCs in submerged and
free-floating macrophytes is markedly different, as indicated by the
observed ku and BCF values in leaf and root tissues. For submerged
macrophytes such as E. horemanii, PhACs and EDCs are more prefer-
entially allocated in leaf tissue compared to root, due to the fact
leaves are in direct contact with the waterborne compounds. Con-
versely, for free-floating species such E. crassipes, PhACs and EDCs
tend to be more are allocated more in roots compared to leaf tissue,
with the exception of more hydrophilic compounds that can be ef-
fectively translocated.
These findings may be useful for future design and implementation
of phytoremediation systems, as well as environmental risk assessment
initiatives. Phytoremediation involves the use of plant species to re-
move environmental contaminants via uptake, translocation, accumula-
tion and/or degradation. This study provides new information regarding
bioaccumulation behaviour of PhACs and EDCs in aquatic macrophytes,
which can be used for removing waterborne contaminants in wastewa-
ter impacted lagoons or surface waters, as well as in constructed wet-
land systems, through direct sorption and/or translocation. In
addition, the information regarding uptake and elimination kinetics of
PhACs and EDCs in aquatic macrophytes may aid future bioaccumula-
tion modeling and risk assessment initiatives involving PhACs and EDCs.
820 N. Pi et al. / Science of the Total Environment 601–602 (2017) 812–820
Acknowledgements
The work was funded by a Singapore Ministry of Education (MOE)
Academic Research Fund (AcRF) Tier 1 grant to B.C. Kelly. Thanks are al-
so given to Singapore-Delft Water Alliance and NUS Environmental Re-
search Institute (NERI) for facilities support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.05.137.
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