Ecological Engineering 69 (2014) 93–105

Contents lists available at ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Removal and fate of arsenic in the rhizosphere of Juncus effusus

treating artificial wastewater in laboratory-scale constructed
wetlands
Khaja Zillur Rahman a,∗ , Arndt Wiessner b , Peter Kuschk b , Manfred van Afferden a ,
Jürgen Mattusch c , Roland Arno Müller a
a
Centre for Environmental Biotechnology, UFZ−Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany
b
Department of Environmental Biotechnology, UFZ−Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany
c
Department of Analytical Chemistry, UFZ−Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany

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

Article history: The deposition, fate and distribution of arsenic (As) under dynamic redox conditions within the rhi-
Received 24 October 2013 zosphere of helophytes in treatment wetlands are still poorly understood. For this purpose, long-term
Received in revised form 30 January 2014 experiments were carried out in specially designed laboratory-scale constructed wetland reactors treat-
Accepted 29 March 2014
ing artificial domestic wastewater containing As (200 ␮g As l−1 ) in order to investigate the key aspects
of As immobilization, to identify the main As removal pathway by using a mass balance approach and
Keywords:
to assess the role of different sulfate (SO4 2− ) concentrations on As mass retention. The results with a
Arsenic removal
highly efficient As mass retention (>92%) indicated a better performance under C-deficient and oxidized
Artificial wastewater
Constructed wetland
conditions (Eh ∼324–795 mV) regardless to the SO4 2− concentration in the inflow wastewater. An ele-
Juncus effusus vated SO4 2− concentration (25 mg S l−1 in the inflow) facilitated high As-retention (>90%) under C-surplus
Mass balance and microbial dissimilatory SO4 2− reducing condition (Eh ∼−225–−149 mV) within the root-near envi-
Rhizosphere ronment of the rhizosphere in constructed wetlands. Mean pH in a range of 6.6–7.7 might be favoring
the immobilization of As but a comparatively low pH (3.9–5.9) within the root vicinity might enhance
plant uptake. In general, higher As concentrations were exhibited by the plant roots (90–315 mg As kg−1
dry wt) as compared to the shoots (3.5–3.8 mg As kg−1 dry wt). Nearly 3.5-fold higher As concentrations
within the roots from the experimental reactor as compared to the roots collected from control reactor
clearly indicated that a higher amount of As was retained, accumulated, adsorbed, metabolized to other
forms on root surface and/or translocated into the roots of Juncus effusus, where organic C and SO4 2− were
abundant. Based on As mass balance calculation, the reactor with the highest SO4 2− loading was found
to be retained nearly 85% of the total As mass input. Out of which only <1% of the total inflow As mass
was sequestered or translocated into the plant shoots, 42.2% was accumulated/recovered within the plant
roots, 17.2% was entrapped or deposited within the sediments of the gravel bed, 16.2% was recovered in
the pore water and 15.3% was flushed out as outflow. The remaining 9% was considered as unaccountable,
which might be released due to volatilizations or lost due to various unknown reasons. A 5-fold higher
SO4 2− concentration within the reactor might facilitate lower pH (3.9–5.9) and consequent remobiliza-
tion caused a higher amount of free or exchangeable As in the pore water (16.2%), that probably resulted
in a higher As uptake (42.2%) by the plant roots as compared to the roots from the control reactor (only
13%). The findings demonstrate the deposition and fate of As within the rhizosphere, which are of high
importance for an efficient treatment of wastewater containing As under constructed wetland conditions.

© 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +49 341 235 1019/179 940 9506; fax: +49 341 235 1830.
E-mail address: khaja.rahman@ufz.de (K.Z. Rahman).

http://dx.doi.org/10.1016/j.ecoleng.2014.03.050
0925-8574/© 2014 Elsevier B.V. All rights reserved.
94 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105
1. Introduction
Arsenic (As) is a toxic metalloid which can pollute water, soil,
crops and the environment at large, ultimately affecting human
health (Zhao et al., 2010). More than 245 minerals contain As, and
the principal source of As is geological. However, human activities
such as mining, pesticide application, and burning of fossil fuels
also cause As pollution (Sharma and Sohn, 2009). In recent years,
there has been an increasing contamination of water, soil and crops
by this metalloid in many regions of the world (Tripathi et al., 2007),
particularly in some countries of southern Asia (Meharg, 2004). It is
therefore very important to choose appropriate methods to control
As in the environment. Several treatment technologies have been
applied for the removal of As from contaminated waters, such as
coagulation/filtration, ion exchange, lime softening, adsorption on
iron oxides or activated alumina, reverse osmosis etc. (Zouboulis
and Katsoyiannis, 2002).
Constructed wetlands are low-energy based ‘green’ technolo-
gies that have been increasingly applied in wastewater treatment
since the mid-1980s (Sun and Saeed, 2009) and have consid-
erable potential to remove metals and metalloids, including As
(Buddhawong et al., 2005; Rahman et al., 2008a; Ye et al., 2003).
Wetland plants have been shown to play important roles in con-
structed wetlands to remove As from wastewater (Rahman et al.,
2008a, 2011; Singhakant et al., 2009). Complex interactions of
As under redox gradient (both micro- and macro) conditions
have already been investigated in different laboratory-scale hor-
izontal subsurface-flow constructed wetlands treating an artificial
wastewater (Rahman et al., 2008a).
The rhizosphere of constructed wetlands offers specific macro-
and micro gradients of redox conditions enabling the development
of highly diverse microbial consortia capable of different beneficial
redox reactions (Bezbaruah and Zhang, 2004; Liesack et al., 2000;
Wiessner et al., 2005b). Particularly due to the release of oxygen
and organic carbon at the same time by the roots of helophytes
into the rhizosphere, spatial and temporal micro-scale gradients
of oxygen concentrations and redox states are established close
to the root surfaces. These conditions enable the development of
microbial mats and layers of functionally different microorgan-
isms which simultaneously realize multiple interactive processes
like nitrification, denitrification, mineralization of organic carbon,
methanogenesis, reduction and oxidation of several sulfur and
As compounds on a small spatial scale (Bezbaruah and Zhang,
2004; Darrah et al., 2006; Rahman et al., 2008b; Wiessner et al.,
2005b). Recently, the application of a specially designed laboratory-
scale constructed wetland (Kappelmeyer et al., 2002) in order to
evaluate micro-gradient processes within the near-root environ-
ment of the rhizosphere was shown to be useful (Wiessner et al.,
2005a,b).
The behavior of metals in aquatic systems is complex and may
include interactions among or between the major wetland com-
partments, above-ground plant parts, roots, litter, biofilms, soil,
and water (Kadlec and Knight, 1996). Volatilization of metals into
a gaseous phase occurs with mercury, selenium, and arsenic to a
lesser degree. Dissolved metals can adsorb onto particles, or exist
complexes to inorganic and organic ligands, or be present in solu-
tion in the free-ion state. The adsorption and co-precipitation of
As on hydrous oxides of Fe, Mn or Al oxides and Fe sulfides is an
important sink for As immobilization (Jacks et al., 2002). Moreover,
dissimilatory reduction caused by iron- and sulfate-reducing bacte-
ria is widely considered the primary mechanism responsible for the
rapid As reduction and release observed in anaerobic environments
(Islam et al., 2004; Kirk et al., 2004).
Based on the characteristic of metal hyperaccumulation in
plants, suitable and sustainable remediation strategies could be
developed (Rai et al., 1995). Fitz and Wenzel (2002) proposed
that hyperaccumulators may enhance metal solubility in the rhi-
zosphere via root exudation, consequently increasing plant metal
uptake. Larios et al. (2012) showed that the plants accumulated
extremely high amounts of total As in their tissues which varied
depending on the part of the plant, with roots accumulating the
most As in all the studied plants (up to 1400 mg kg−1 dry wt). In the
context of constructed wetlands, García et al. (2010) reported that
the direct uptake and accumulation of As in plants appears to play a
very minor role in As removal. The same conclusion was drawn by
Singhakant et al. (2009), who reported that only 0.5–1% of the total
As input was accumulated in plant tissues. However, there are also
studies indicating that wetland plants have a remarkable effect on
As retention (Rahman et al., 2011; Sasmaza and Obek, 2009).
First results of laboratory-scale investigations showed the trans-
formation processes and redox dynamics of As-species particularly
in the near-root environment of the Juncus effusus in model con-
structed wetlands. Changes in dynamic redox conditions and
re-oxidation of reduced sulfur into other S species (e.g. S0 , SO4 2– )
caused a total sulfur enrichment and a consequent As remobiliza-
tion within the rhizosphere in this study (Rahman et al., 2008b).
But it is necessary to investigate the role of organic C, S and pH
on As retention within the root vicinity and enhanced plant uptake
under different redox conditions. Differences between correspond-
ing inflow and outflow data of total As indicated remarkable
amounts of As immobilization within the rhizosphere. Therefore, in
addition to the investigations of As-removal efficiency and dynam-
ics of As-species (Rahman et al., 2008b), it is necessary to deepen
the understanding of the deposition, fate and mass balance of
As within the micro-scale root zone environment of the rhizo-
sphere in treatment wetlands. However, Rahman et al. (2011) in
another study showed the fate and distribution of As along the
flow path of a laboratory-scale horizontal subsurface-flow con-
structed wetland. But the knowledge regarding the accumulation
and mass balance of As under the micro-scale gradient of redox
conditions and the role of C, S and pH within the rhizosphere
of helophytes is still insufficient. In the past, little attention has
been paid and virtually no information is available until now that
directly addresses the removal, fate and plant uptake of As under
dynamic redox conditions within the rhizosphere of constructed
wetland.
In the context of our research work in this field, several inves-
tigations like fundamental aspects and mechanisms of As fixation,
influences of dynamic redox conditions on As biotransformation
processes, stability of As within the planted and unplanted wet-
lands, bioaccumulation and uptake of As in plant biomass, sludge
sediment analysis, mass balance of As and S, etc., were carried out
in different laboratory-scale constructed wetlands (Rahman et al.,
2008a,b; Rahman et al., 2011). In principle, the major objectives
of this study were i) to investigate the fate, accumulation and dis-
tribution of As under redox dynamic conditions on a micro-scale
gradient within the rhizosphere; ii) to use a mass balance approach
to identify the main As removal pathway under “ideal flow” con-
ditions; and iii) to assess the role of organic C, pH and different
SO4 2− concentrations on As mass retention within the rhizo-
sphere of helophytes in treatment wetlands. By using Juncus effusus,
long-term experiments in a specially designed laboratory-scale
constructed wetland treating an artificial wastewater containing
As (200 ␮g l−1 ) were performed to evaluate all these processes. The
results of this study may help to better understand the key aspects
of As immobilization within the plant root-zone of the rhizosphere
under constructed wetland conditions and to optimize manage-
ment practices for maximum As retention in different wetland
compartments (shoots, roots, sediments etc.) through a complete
mass balance analysis.
 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105
Fig. 1. Schematic diagram of a specially designed laboratory-scale constructed wet-
land (planted fixed bed reactor–PFBR): (1) feeding storage tank; (2) pump; (3) glass
vessel; (4) distribution chamber; (5) suction cylinder; (6) gravel bed; (7) recir-
culation pump; (8) magnetic valve; (9) on-line measurement; (10) outflow; (11)
plants.
(Adapted from Kappelmeyer et al., 2002).
2. Materials and methods
2.1. Experimental design: Planted fixed-bed reactors
The experiments were carried out in three laboratory-scale
planted-fixed bed reactors (R1, R2 and R3), which were estab-
lished under conditions of complete mixing of the pore water by
a permanent circulation. Since the internal flow conditions were
comparable to an ideal mixed vessel, and therefore, macro-scale
gradients of concentrations were equalized and the effects of the
micro-gradient changes could be determined. The design and the
principles of operation of the reactor (PFR – planted fixed bed reac-
tor) have been described previously in detail (Kappelmeyer et al.,
2002; Wiessner et al., 2005a,b).
Briefly, the rhizosphere of the reactors was represented by a
rooted gravel bed (particle size 2–4 mm) in a glass vessel of 28 cm
diameter and a height of 30 cm. Each reactor was planted with five
rush plants (Juncus effusus) with an initial total shoot number of
72, 84 and 80 in the reactors R1, R2 and R3, respectively. The reac-
tors were closed tightly with a Teflon lid containing five circular
openings through which the plants were grown in the gravel bed
(Fig. 1). The glass reactors were covered to prevent algal growth.
The free pore water volume in the planted beds amounted to 10
l and the hydraulic retention time was adjusted to 5 days. The
reactors were placed in a greenhouse and operated under defined
environmental conditions to simulate an average summer day in a
moderate climate (Kappelmeyer et al., 2002; Wiessner et al., 2005b,
2008). The temperature was set to 22 ◦ C from 6 a.m. to 9 p.m. to
simulate daytime and to 16 ◦ C at night. One lamp (Master SON-
PIA 400 W, Phillips, Belgium) was switched on during daytime as
an additional artificial light source whenever the natural light fell
below 1110 ␮mol m−2 s−1 (Wiessner et al., 2013).
The main reason to use three model reactors operating in par-
allel was to investigate the influences of simultaneously increasing
inflow feeding SO4 2− concentrations on the As mass retention,
both under C-deficient and C-surplus conditions in the respective
reactors. Obtained results from the reactors R2 and R3 were then
95
compared with the control reactor R1, which was constantly fed
with limited or very low inflow SO4 2− concentration.
2.2. Experimental conditions
An artificial wastewater (Wiessner et al., 2005a) simulated a
typical secondary effluent of domestic wastewater was used in
this investigation. The inflow concentrations of the used ingre-
dients were (in mg l−1 ): 204.9 CH3 COONa, 107.1 C6 H5 COONa,
117.8 NH4 Cl, 28 K2 HPO4 , 7 NaCl, 3.4 MgCl2 ·6H2 O, 4 CaCl2 ·2H2 O,
0.89/22.2/44.4/110.9 Na2 SO4 , 0.2 As(V) (Titrisol® As2 O5 in H2 O,
Merck, Germany) and 1 ml l−1 of trace mineral solution which
was adapted from Buddhawong et al. (2005). The resulting con-
centrations of the parameters were (in mg l−1 ): 340 chemical
oxygen demand (COD), 122.3 total organic carbon (TOC), 30.8
ammonia-N, 5 phosphate-P, 0.2/5/10/25 sulfate-S and 0.2 As(V).
These compounds were dissolved in deionized water and fed to
the corresponding reactors in different experimental phases.
Artificial wastewater was freshly prepared in every 3 days
to prevent microbial degradation during storage and operation
(Fig. 1). N2 gas was vigorously purged through and bubbled out of
the liquid phase of the wastewater for approximately 20−25 min
after each preparation of fresh feeding solution in order to remove
any traces of dissolved oxygen. Each reactor was fed separately
from a feeding glass bottle with a volume of 10 l. In order to keep
an anoxic environment inside the feeding glass bottle contain-
ing the artificial wastewater, a continuous purging of nitrogen gas
(N2 ) through the headspace of the feeding bottles was maintained
throughout the whole operation period (Fig. 1).
The reactors were operated for several months (nearly 4–5
months) using similar artificial wastewater (Wiessner et al., 2005a)
but without any SO4 2− or As to establish biofilms and microbiolog-
ical activity. Within these 4–5 months of normal operation, new
shoots were grown from those five plants in each reactor. Prior to
start of our experiment by using simulated wastewater with As and
SO4 2− , the total numbers of healthy green shoots were counted as
231, 245 and 241 in R1, R2 and R3, respectively. The duration of
the main experimental period was 340 days for all three reactors
in this study. At the end of the experimental period of 340 days, the
total shoot numbers were decreased down to a minimum of 180,
211 and 198 in R1, R2 and R3, respectively.
The reactors were fed and run under five different experimental
phases (phase I, II, III, IV and V) realized by varying SO4 2− concen-
trations in the simulated wastewater inflow solution. The operation
conditions in all the phases within the reactors R1, R2 and R3
are listed in Table 1. The hypothesis behind different experimen-
tal phases was to investigate how the redox dynamics of As was
influenced under C-deficient and oxidized condition with simulta-
neous increasing of SO4 2− concentrations in each reactors (phase I),
under C-surplus and reducing condition with both constant (in R1)
and simultaneous increasing and/or decreasing of SO4 2− concen-
trations (phase II, III, IV and V) in the reactors R2 and R3. Each phase
had a sufficient duration to guarantee a representative number of
samples that could be taken from each reactor.
In experimental phase I (duration of first 83 days), the reac-
tors were fed by a continuous inflow of As-contaminated artificial
wastewater (200 ␮g As l−1 ) associated with SO4 2− concentrations
of 0.2, 5 and 25 mg S l−1 but without organic C in the reactor R1,
R2 and R3, respectively. The resulted molecular ratio of SO4 2− -S to
As(V) varied as 1:1, 25:1 and 125:1 in the inflow wastewater of the
reactors R1, R2 and R3, respectively.
In experimental phase II (from day 83 to day144), the reactors
were fed and operated under conditions of surplus C by adding
organic C-sources (resulting a COD of ∼340 mg l−1 ) along with
As(V) and only traces of SO4 2− (0.2 mg S l−1 ) in the simulated
96 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105

Table 1
Operation conditions within the model reactors R1, R2 and R3 accomplished by defined arsenic, organic carbon and sulfate inflow concentrations of the artificial wastewater
(phase I–V).

Reactor Parameter Unit Experimental phases

I n II n III n IV n V n

R1 As (V) ␮g l−1 202 ± 8 12 205 ± 6 10 196 ± 4 13 202 ± 5 15 203 ± 6 6

SO4 2− -S mg l−1 0.2 ± 0.1 12 0.2 ± 0.1 10 0.2 ± 0.1 13 0.2 ± 0.1 15 0.2 ± 0.1 6
TOC mg l−1 bdl 12 120 ± 4 10 118 ± 10 13 120 ± 9 15 128 ± 4 6
R2 As (V) ␮g l−1 204 ± 5 12 201 ± 5 10 199 ± 8 13 201 ± 6 15 201 ± 4 6
SO4 2− -S mg l−1 5±1 12 0.2 ± 0.1 10 5 ± 1 13 5 ± 1 15 bdl 6
TOC mg l−1 bdl 12 124 ± 5 10 120 ± 9 13 125 ± 8 15 125 ± 5 6
R3 As (V) ␮g l−1 198 ± 10 12 199 ± 10 10 201 ± 7 13 199 ± 7 15 204 ± 7 6
SO4 2− -S mg l−1 25 ± 3 12 0.2 ± 0.1 10 25 ± 5 13 10 ± 2 15 bdl 6
TOC mg l−1 bdl 12 126 ± 7 10 118 ± 11 13 123 ± 6 15 126 ± 6 6

bdl: below the detection limit (<1 mg TOC l−1 ; <0.1 mg SO4 2− -S l−1 ).

wastewater inflow solution of all three reactors. Similar running
conditions were prevailing at the end of this phase and the resulted
molecular ratio of SO4 2− -S to As(V) was 1:1 in the reactors.
In phase III (from day 144 to 235), the same C-dosage and SO4 2−
concentration as like in phase II was maintained in the control reac-
tor R1 and only the SO4 2− concentration was increased to 5 and
25 mg S l−1 in the inflow feeding solutions of the reactors R2 and
R3, respectively. During experimental phase IV (corresponding to
day 235 to 315), conditions in all three reactors were similar to
the inflow feeding solution of phase III operation, but only excep-
tion was the concentration of SO4 2− in reactor R3. Instead of a
SO4 2− concentration of 25 mg S l−1 (as in phase III), the amount was
decreased to 10 mg S l−1 in this particular phase IV in the reactor R3
(Table 1). The newly resulting molecular ratio of SO4 2− -S to As(V)
was established as 50:1 under this experimental phase in reactor
R3. Maintaining the same operating conditions as like in Phase IV,
the supply of the inflow SO4 2− was completely stopped in reactor
R2 and R3 (phase V) until the termination of the experiment on day
340. A comparative analysis was carried out in between the reac-
tors under each circumstance and also within different operation
phases.
Plant transpiration represents 98% of the total water loss
(Wiessner et al., 2005a) and was measured by balancing the inflow
and the outflow amounts of water once a week. The flow balances
were also used to control and adjust the inflow rate. All green shoots
from a length of at least approximately 2 cm were counted once a
month.
2.3. Sample collection and analysis
Samples for total As, total organic carbon (TOC) and sulfate
(SO4 2− ) analysis were taken once a week. The preservation tech-
nique of the collected samples for measuring the total As content
and the analytical procedure of hydride generation atomic adsorp-
tion spectrometry (HG-AAS) have already been described by Daus
et al. (2002) and Schmidt et al. (2004).
The volatile As species were analyzed by gas chromatog-
raphy/mass spectrometry (GC–MS, Shimadzu-GC-17A-Shimadzu
GC-MS-Qp5000) with electron ionization and quadrupole analyzer,
using the method described by Pantsar-Kallio and Korpela (2000).
The analyses were performed isothermally at 50 ◦ C, and helium was
used as a carrier gas.
Due to the reactor design, the circulation flow represents the
actual concentration of the pore water inside of the reactor and
thus the pH and redox potential (Eh ) were controlled continuously
in the circulation flow and the data were recorded online twice per
hour (Fig. 1).
SO4 2− was analyzed by ion chromatography (DIONEX 100,
columns AS4A-SC/AG4A-SC and CS12A/CG12A; Idstein, Germany)
and a conductivity detector. TOC was analyzed by using a TOC ana-
lyzer (Shimadzu, TOC 600, Duisburg, Germany).
After the termination of the experiment, plant biomass sam-
ples (shoots and roots) and sludge sediments were collected from
each reactor in order to investigate the potential for As removal
efficiency. Plant samples were sectioned into their shoot and root
components after collecting them from each reactor. The roots
were first thoroughly washed with tap water and then with deion-
ized water to remove any gravel aggregate or sludge sediment. The
plant shoots and roots collected from each reactor were freshly
weighed, dried at 105–108 ◦ C for 3 days, allowed to cool, and then
the dry weights were determined and the water content was cal-
culated. These dry weights are used throughout the text unless
otherwise specified. The dried samples were ground to a fine pow-
der using a mortar and pestle under liquid nitrogen in order to
obtain a homogeneous sample and then preserved in sealed plastic
bottles for analysis.
For the analysis of the total As concentration in plant
biomass (shoots and roots), the homogenized powdered sam-
ples were digested by microwave extraction (PE Anton Paar
GmbH, Graz, Austria). For digestion, 2.0 ml of digestion mixture
(HNO3 :HCl = 4:1) were added to 0.5 g powder in a Teflon pres-
sure bomb and heated to 260 ◦ C for 1 h. After the digest cooled
down, it was filled with deionized water to a total volume of 10 ml,
mixed and filtered using a 0.45 ␮m syringe filter (Satorius AG,
Goettingen, Germany). The filtrate solution was analyzed for total
As by using hydride generation atomic absorption spectrometry
(HG-AAS) with a detection limit of 0.3 ␮g As l−1 . Acid blanks were
analyzed in order to assess possible contamination. All analyses
were performed in duplicate.
Analysis of the sludge sediment samples was performed by the
energy dispersive X-ray fluorescence (EDXRF) spectrometer XLAB
2000 (SPECTRO Instruments) running with the software package
XLAB Pro 2.2. Collected sludge sediments from each reactor were
dried at 105 ◦ C for 24 h using oven MA4O (Satorius, Germany) and
were ground by means of an agate ball mill (Retsch). Well-ground
sample material (1 g) was mixed with stearine wax (Hoechst,
Germany) for XRF analysis as a binder in a ratio of 80:20 (w/w)
and subsequently pressed at 150 MPa to pellets (with an internal
diameter of 32 mm) and analyzed by means of energy dispersive X-
ray fluorescence analysis (EDXRF). The mean value of two replicates
was calculated. The relative error of the method was 2–3%.
Experimental results were evaluated statistically (e.g. mean,
standard deviation, bar graphs error bar) by using spread-
sheet program Microsoft Excel (Microsoft Corporation) in this
study.
 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105 97

2.4. As mass balance calculation

After 340 days of the experiment, a complete mass balance of As

was investigated in each reactor by considering the total As mass
input, the total As mass output, and the total As retained in the plant
biomass (the shoots and the roots), in the pore water and in the
sludge sediments. The remaining (loss or gain) of the As mass from
the mass balance calculation was considered to be unaccountable.
The total As mass input and output in each reactor was calculated
from the cumulative total As mass inflow and outflow during the
whole operation time period (Fig. 2). A simple As mass balance
was computed according to the following Eq. (1) (Singhakant et al.,
2009):

Asin = Asout + Asplant + Assed. + Aspw + Asunaccount (1)

Where Asin is total As mass in the inflow (g); Asout is total As

mass in the outflow (g); Asplant is total As mass in the plant biomass
(g), including plant roots and shoots; Assed. is total As mass retained
in the sludge sediment (g); Aspw is total As mass retained in the
pore water or standing water (g); Asunaccount. is the total As mass
that was unaccountable (g), including the loss or gain of As from
the mass balance calculation.

3. Results

3.1. As removal efficiency

Arsenic removal efficiencies in terms of total amount of the As
mass inflow and the total As retention in all three corresponding
reactors during the whole study period of 340 days is shown in
Fig. 2.
A total inflow mass of 140.0, 135.1 and 123.3 mg As was fed and a
total of 72.8, 27.7 and 18.9 mg As mass was flushed out through the
outlet of reactors R1, R2 and R3, respectively. Therefore, a cumula-
tive total mass of 67.2, 107.4 and 104.4 mg As was retained, which
resulted in nearly 48%, 80% and 85% of the total As mass retention
in the corresponding reactors R1, R2 and R3, respectively (Fig. 3).
During the stoppage period of SO4 2− -S supply (in phase V), no
remobilization or loss of the total As mass was observed in the
reactors, R2 and R3.
Fig. 2. Cumulative total As mass inflow and retention in the three laboratory-scale
reactors (R1, R2 and R3) during the whole experimental period of 340 days.
Under highly oxidized conditions in phase I within the first 83
days of operation, the corresponding mean removal rate was cal-
culated as 0.388 ± 0.012, 0.384 ± 0.015 and 0.347 ± 0.06 mg As per
day and thus contributed to a mean As retention of 94%, 94% and
92%, in the reactors R1, R2 and R3, respectively (Table 2). Addition
of organic C-sources in phase II (day 83–144) showed an immedi-
ate effect with a considerably high As concentration in the outflow,
which resulted in a lower As retention of only 15, 28 and 61% in
the reactors R1, R2 and R3, respectively. During the experimen-
tal phase III (from day 144 to day 235) under anaerobic condition

Table 2
Summary of the treatment performance in the reactors R1, R2 and R3 during the whole operational period of 340 days (phase I–V).

Phase Duration (day) Parameter Unit Reactor

R1 R2 R3

I 0–83 Inflow rate mg/day 0.411 ± 0.002 0.408 ± 0.002 0.379 ± 0.065
Outflow rate mg/day 0.023 ± 0.012 0.023 ± 0.016 0.032 ± 0.014
Removal % 94 94 92
n – 12 12 12
II 83–144 Inflow rate mg/day 0.415 ± 0.001 0.408 ± 0.001 0.364 ± 0.056
Outflow rate mg/day 0.352 ± 0.093 0.294 ± 0.091 0.146 ± 0.066
Removal % 15 28 61
n – 10 10 10
III 144–235 Inflow rate mg/day 0.411 ± 0.009 0.404 ± 0.009 0.326 ± 0.052
Outflow rate mg/day 0.278 ± 0.028 0.045 ± 0.025 0.033 ± 0.010
Removal % 32 89 90
n – 13 13 13
IV 235–315 Inflow rate mg/day 0.410 ± 0.002 0.404 ± 0.002 0.393 ± 0.009
Outflow rate mg/day 0.243 ± 0.021 0.017 ± 0.011 0.021 ± 0.013
Removal % 41 96 95
n – 15 15 15
V 297–340 Inflow rate mg/day 0.412 ± 0.001 0.403 ± 0.001 0.397 ± 0.001
Outflow rate mg/day 0.276 ± 0.038 0.037 ± 0.020 0.019 ± 0.003
Removal % 33 91 95
n – 6 6 6

n: number of samples.
98 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105

All three reactors showed an efficient TOC removal (>74%) dur-

ing the experimental phases with the addition of organic C-sources.
Interestingly, a highly efficient TOC removal in the range of 86–94%
was observed in the reactors R2 and R3, as compared to control
reactor R1 (with a range of 74–86%).

3.3. Redox (Eh ) and pH

Table 4 shows the summary of the mean pH and redox val-

ues (also the ranges) in different experimental phases within the
reactors R1, R2 and R3. No addition of organic C-sources ensured a
Fig. 3. As mass retention capacity of the model reactors R1, R2 and R3 calculated persistent aerobic condition (with an Eh ∼ 324–795 mV) inside the
as a percentage of the inflow total As mass that retained inside the reactors during reactors during experimental phase I (first 83 days).
the whole experimental period of 340 days.
Reducing conditions were established immediately after the
addition of organic C-sources (122.3 mg TOC l−1 ) with a drop
down of Eh value from an aerobic (721–795 mV) to anaerobic
Table 3 (−217–−144 mV) condition within all three corresponding reac-
Removal efficiency of SO4 2− -S and TOC within the model reactors R1, R2 and R3 in
different experimental phases (phase I–V).
tors in experimental phase II (Table 4). But a simultaneous addition
of SO4 2− with constant C-dosage resulted in a low but relatively
Reactor Parameter Removal efficiency (%) stable redox potential value (Eh ∼ −225–−149 mV) within all three
Phase I Phase II Phase III Phase IV Phase IV reactors in phase III–V.
R1 SO4 2− -S − − − − −
Mean pH value of 3.1 ± 1.2, 3.6 ± 0.6 and 3.2 ± 0.4 was observed
TOC − 75 86 74 75 at the beginning (in phase I) and after addition of C-dosage in phase
R2 SO4 2− -S − − 73 68 − II, the pH remarkably raised to a mean value 4.3 ± 0.4, 6.6 ± 0.7
TOC − 86 94 93 92 and 5.9 ± 0.5 in the pore water of the corresponding reactors R1,
R3 SO4 2− -S − − 84 71 −
R2 and R3, respectively (Table 4). Afterwards, a relatively steady
TOC − 89 88 89 93
pH-value was observed in the reactors during the remaining exper-
imental phases. Interestingly, a decrease of nearly 2–3 pH units was
recorded in R3 than in the reactor R2 during the phases III–V.
along with varying SO4 2− concentration, the data demonstrated a
comparatively much higher As retention than the previous phase II
3.4. Concentration of total As in plant biomass (shoots and roots)
with a mean value 89% and 90% in R2 and R3, respectively and only
32% was retained in the control reactor R1 (Table 2). Traces (2–3 ␮g
After the termination of all experimental phases, the sampling
As l−1 ) of volatile As compound [gaseous arsine (AsH3 )] was found
and measurement of plant biomass (shoots and roots) in terms of
in this experimental phase but only in the reactor R3. Data in Table 2
fresh and dry weight, water content (%) and thereby the dry weight
also shows that at least 5% more As was retained within reactor R3
(in kg) of total shoots and roots in each reactor were carried out and
when SO4 2− concentration was 10 mg S l−1 (in phase IV) instead of
all these measurements are shown in Table 5. We measured a rela-
25 mg S l−1 (in phase III).
tively lower total root biomass of 0.93 kg (fresh wt) collected from
reactor R3, as compared to the other two reactors R1 and R2 with
3.2. SO4 2− -S and TOC removal efficiency 1.10 and 1.16 kg (fresh wt), respectively. Based on the water con-
tent analysis data from the plant biomass samples collected from
Table 3 shows the removal efficiency of SO4 2− -S and TOC in the reactors R1, R2 and R3, the dry weight of the total shoots were
different experimental phases within the reactors R1, R2 and R3. 0.16, 0.18, and 0.14 kg, and the corresponding dry weights of the
Overall, a comparatively higher SO4 2− -S removal was observed in total roots were calculated as 0.20, 0.21, and 0.16 kg, respectively
R3 (71–84%) in comparison to the reactor R2 (68–73%) under C- (Table 5). The analytical results of the total As concentrations in
surplus conditions (phase II–V). As expected, no SO4 2− reduction plant biomass are presented in Fig. 4. The mean total As concentra-
was observed under C-deficient and oxidized conditions in exper- tion was measured as 3.88 ± 0.13, 3.62 ± 0.07 and 3.57 ± 0.08 5 mg
imental phase I. As kg−1 (dry wt) in the shoots and was considerably very high in

Table 4
Summary of the average pH, redox (Eh ) values (mean ± SD) and ranges of the values (from minimum to maximum) in each reactor during the whole operational period
(phase I–V).

Experimental phases

Reactor Parameter I II III IV V

R1 pH 3.1 ± 1.2 4.3 ± 0.4 4.9 ± 1.4 6.7 ± 0.1 6.9 ± 0.1
(1.1–5.0) (3.4–4.8) (3.5–6.6) (6.6–6.8) (6.8–7.1)
Eh (mV) 499 ± 203 28 ± 162 −187 ± 6 −182 ± 6 −191 ± 4
(324–740) (−168–307) (−196–−179) (−189–−169) (−196–−188)
R2 pH 3.6 ± 0.6 6.6 ± 0.7 7.4 ± 0.1 7.6 ± 0.1 7.7 ± 0.1
(3.2–5.1) (5.1–7.4) (7.2–7.5) (7.4–7.8) (7.6–7.8)
Eh (mV) 597 ± 88 −97 ± 176 −217 ± 4 −219 ± 5 −216 ± 10
(396–721) (−217–277) (−225–−213) (−225–−210) (−224–−202)
R3 pH 3.2 ± 0.4 5.9 ± 0.5 5.8 ± 0.2 5.2 ± 0.5 3.9 ± 0.1
(2.8–4.1) (5.1–6.5) (5.5–6.1) (4.4–5.6) (3.8–4.1)
Eh (mV) 670 ± 88 −52 ± 139 −195 ± 17 −201 ± 13 −199 ± 23
(495–795) (−144–259) (−211–−149) (−215–−172) (−220–−166)
 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105 99

Table 5
Plant analysis data in terms of fresh and dry weight of shoots and roots samples, water content and total dry weight of collected shoots and roots biomass (in kg) from each
reactor after the termination of the experiment.

Reactor Shoots Roots

Sample Sample dry Water Total fresh Total dry Sample Sample dry Water Total fresh Total dry
fresh wt (g) wt (g) content (%) wt (kg) wt (kg) fresh wt (g) wt (g) content (%) wt (kg) wt (kg)

R1 1.393 0.512 63 0.421 0.156 11.440 2.029 82 1.100 0.195

R2 2.130 0.689 68 0.575 0.184 7.620 1.352 82 1.160 0.207
R3 2.236 0.860 62 0.361 0.137 9.817 1.728 82 0.930 0.164

Table 6
Distribution of total As in each reactor, which includes the total inflow and outflow mass of As and the distribution in different compartments (shoots, roots, sediment, etc.)
over the whole operation period of 340 days. Data are given in mg As, and the values in parentheses are percentile amounts of total inflow As.

Reactor Total inflow (mg As) Shoots (mg As) Roots (mg As) Sediment (mg As) Pore water (mg As) Unaccountable (mg As) Outflow (mg As)

R1 140.0 (100%) 0.6 (0.4%) 17.6 (12.6%) 6.2 (4.4%) 6.5 (4.6%) 36.5 (26.1%) 72.8 (52.0%)
R2 135.1 (100%) 0.5 (0.4%) 22.9 (17.0%) 30.9 (22.9%) 13.9 (10.3%) 39.1 (28.9%) 27.7 (20.5%)
R3 123.3 (100%) 0.4 (0.3%) 51.6 (41.9%) 21.2 (17.2%) 20.0 (16.2%) 11.1 (9.0%) 18.9 (15.3%)

the roots as 90 ± 2.83, 110.5 ± 3.54, 315 ± 4.24 mg As kg−1 (dry wt)
within the reactors R1, R2 and R3, respectively. On the contrary,
the mean As concentration was found to be only 0.5 ± 0.14 and
2.25 ± 0.2 mg As kg−1 (dry wt) in the control shoot and root sam-
ples of J. effusus (collected from natural, non-contaminated source),
respectively.
3.5. Concentration of the total As in the sediment
The results from the collected sediments showed a mean total As
concentration of 206 ± 5, 806 ± 9 and 941 ± 4 mg As kg−1 (dry wt) in
the corresponding three reactors R1, R2 and R3, respectively (Fig. 4).
In comparison, a mean As concentration of 3.2 ± 0.4 mg As kg−1 (dry
wt) in the control sediment sample (collected from a natural, non-
contaminated source) was shown to be nearly 251–294 times lower
than the concentration found in the sediments collected from the
reactors R2 and R3.
3.6. Data for As mass balance
Table 6 represents a summary of the total As mass deposition
and distribution in the different wetland compartments (shoots,
roots, sediments etc.) and a complete mass balance calculation
within the reactors over the whole operation period of 340 days.
Considering the inflowing As mass within the reactors as 100%, the
percentile value of the outflowing As mass and retention was esti-
mated. For instance, a total mass of 140.0 mg As (100%) was fed
as an inflow, and a total mass of 72.8 mg As (52%) was flushed out
of the reactor R1 as outflow. The recovered As masses in the pore
water, within the shoots, the roots and in the sediments collected
from this reactor (R1) were measured as 6.5, 0.6, 17.6 and 6.2 mg
As, which resulted in a nearly 4.6%, 0.4%, 12.6% and 4.4% of the total
inflowing As mass retention in these compartments, respectively.
The remaining 36.5 mg As (nearly 26% of the total inflow As mass)
was considered to be unaccountable or retained in uncounted sinks.
Similarly, after calculating the total As mass in the plant shoots,
roots and sediments of other two reactors, it was observed that
nearly 17% and 42% of the total inflowing As mass were accumu-
lated/recovered/concentrated within the roots and nearly 23% and
17% were entrapped or deposited within the gravel bed (as sedi-
ments) of the reactors R2 and R3, respectively (Fig. 5). Only 9% of the
total inflow As mass was calculated as unaccountable within R3 in
comparison with the reactor R2, where nearly 29% was considered
to be retained in uncounted sink or termed as unaccountable. In all
three reactors, only a very little amount (<1% of the inflow As mass)
was translocated into the shoots of J. effusus in this experiment.

4. Discussion

4.1. As mass retention capacities of the model reactors

Overall, the total As mass retention capacities of the reactors

Fig. 4. Concentrations of total As in plant shoots (A), roots (B) and sediments (C)
collected from the reactors R1, R2 and R3 (the values are the mean of two replicates
and the error bars are standard deviations).
R2 and R3 were much higher (80% and 85%, respectively) than
the control reactor R1 (only 48%). In general, SO4 2− loading was
much higher in the reactors R2 and R3, as compared to the reac-
tor R1. Therefore, amount of SO4 2− is playing an important role in
case of As mass retention within the root-near environment of the
rhizosphere in treatment wetlands.
A highly efficient As mass retention (>92%) indicated a
better performance under C-deficient and oxidized conditions
(phase I) regardless to the concentration of SO4 2− in the inflow
100 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105
Fig. 5. Mass balance of As estimated as a percentage of inflow total As mass that
retained in different wetland compartments investigated in the model reactors R1,
R2 and R3.
artificial wastewater of all three reactors (see Fig. 2 and Table 2).
Highly oxidized conditions (Eh ∼ 324–795 mV) were not favor-
able for microbial dissimilatory SO4 2− reduction and therefore no
SO4 2− reductions or removal were found in the corresponding
reactors (see phase I; Table 3). Hence, it can be concluded that
the immobilization of As within the rhizosphere of helophytes
was accomplished by mechanisms other than arsenic-sulfide
precipitation (likely as As2 S3 ). Adsorption and/or concomitant co-
precipitation of As, specifically with Fe(III) oxyhydroxides was the
most probable reasons for As immobilization under such condi-
tions (Bednar et al., 2005; Rahman et al., 2008b). Highly oxidized
conditions due to plant root-mediated O2 release and re-oxidation
of reduced As-species probably influenced higher sorption and pre-
cipitation reactions within the rhizosphere of Juncus effusus.
Addition of organic C-sources probably caused an immediate
mobilization of As within the reactors under limited SO4 2− and
reducing conditions (Rahman et al., 2008b), and hence a compara-
tively low As removal efficiency was observed during experimental
phase II in all three reactors (Table 2). Under such conditions, there
was probably a shift in terminal electron acceptors from O2 to
Fe(III) oxyhydroxides. Thereby, reductive dissolution of Fe(III) oxy-
hydroxides mediated by microbial activities probably contributed
to a release of immobilized As fraction in the aqueous phase of
all the corresponding reactors (Smedley and Kinniburgh, 2002).
This is also in agreement with the fact that under reducing condi-
tions, Fe(III) is reduced to Fe(II) and resulting in the mobilization of
some of the adsorbed As, particularly from sediments and the plant
root-zone (Kneebone et al., 2002). Moreover, reduction of As(V)
to more mobile As(III) and subsequent As(III) enrichment due to
microbial activity might also facilitated As remobilization (Rahman
et al., 2008b). Therefore, it was evident in this study that the addi-
tion of electron donor has a huge impact on As mobility within
the rhizosphere of helophytes in treatment wetlands. Presumably
there were several competing reactions including dissolution or
desorption, precipitation or adsorption and remobilization occur-
ring simultaneously under such conditions.
The occurrence of sulfide (S2– ) under anoxic conditions may
immobilize As due to its high affinity to sulfide minerals or the
formation of As-sulfide minerals (Bostick and Fendorf, 2003). This
might be the reason of highly efficient As mass retention (>89%)
under anaerobic condition by varying SO4 2− concentrations in R2
and R3 (phase III; Table 2). Other authors also suggested that under
reducing environments and in the presence of S and Fe, As can form
insoluble sulfide compounds (Buddhawong et al., 2005; Singhakant
et al., 2009a), such as orpiment As2 S3 , in which As is present as
As(III) and arsenopyrite (FeAsS). Dissimilatory reduction caused by
iron- and sulfate-reducing bacteria is widely considered as the pri-
mary mechanism responsible for the rapid As reduction and release
observed in anaerobic environments (Islam et al., 2004). Recently,
Mattes et al. (2010) provided more details of the wetland system
described in Duncan et al. (2004), highlighting that not only sulfate-
reducing bacteria played a role in As removal, but iron-oxidizing
bacteria also making a significant contribution.
Addition of relatively lower SO4 2− concentration (10 mg S l−1 in
phase IV) contributed to 5% more As retention within the reactor R3
as compared to the SO4 2− concentration in phase III (25 mg S l−1 ).
From this, it can also be concluded that a very high SO4 2− loading
does not necessarily improve As removal performances within the
rhizosphere of constructed wetlands.
4.2. Correlation of Redox (Eh ), pH and As mass retention
The mobility and plant availability of many trace and toxic
metals and metalloids in wetland soils are often governed by
oxidation-reduction (redox) potential and associated pH in the
rhizosphere (Gambrell, 1994), while the oxygen released from
the roots probably influences the redox and pH of rhizosphere
(Yang et al., 2010). Flooding conditions induce an enrichment of
metals in soils surrounding the roots of wetland plants (Wright
and Otte, 1999). Under C-deficient condition in this study, a very
high redox potential (with an Eh ∼ 324–795 mV) were obtained
inside the reactors, which clearly indicated an aerobic condition in
the root-near environment of the rhizosphere. Aerobic conditions
were unfavorable for SO4 2− reducing bacteria and therefore it can
be concluded that adsorption and co-precipitation of As, specifi-
cally predominant and thermodynamically more stable As(V) with
iron(oxy)hydroxide favored As retention under such oxic condi-
tions.
Reducing conditions play an important role for SO4 2− reduc-
tion, which requires a reducing environment and an electron donor.
Under C-surplus condition along with available SO4 2− (in R2 and
R3), the Eh values varied in between −149 and −225 that resulted
in a higher SO4 2− removal (68–84%) and a highly efficient As mass
retention (>89%) within the reactors in this study (phase III–IV;
Tables 2–4). Different authors have reported different Eh values
required by sulfate-reducing bacteria to thrive: less than −200 mV
(Cabrera et al., 2006), less than −100 mV (Willow and Cohen,
2003), or between −150 and −200 mV (Tuttle, 1969). Rahman
et al. (2008a) noted that microbial SO4 2− reduction was greater
 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105
under redox potential values between −160 and −190 mV, and
that higher SO4 2− removal contributed to higher removal of As.
However, these conclusions were drawn based on their measured
water quality parameters (Eh , TOC), without monitoring microbial
community composition or function directly. Microorganisms can
enhance the removal of As by mediating redox and precipitation
processes (Lizama et al., 2011). Talukder et al. (2012) showed that
under aerobic water management (Eh ∼ 135–138 mV), As uptake
by the rice plant parts was significantly less as compared to anaer-
obic environment (Eh ∼ −41–−76 mV). The study has confirmed
that anaerobic water management is the main reason for the high
enhanced As uptake in rice. This was also in agreement in this study
with a remarkably higher As uptake in the plant biomass (mainly
roots) under a persistent anaerobic environment, even in the roots
from the control reactor R1 with a concentration of 90 ± 2.83 mg
As kg−1 (dry wt) (Tables 4 and 6; Fig. 4B).
Several studies have shown that many factors affect the bioac-
cumulation (biosorption) of metals and metalloids in aquatic
ecosystems. Among the physico-chemical factors, pH is possibly
the most important (Gadd, 2009; Lizama et al., 2011) and plays
a vital role for As mass retention within the rhizosphere. Persis-
tent low pH value in all corresponding reactors might referred to
as a rhizosphere acidification under oxidized conditions, which
could have resulted from the effect of K+ uptake and release of
H+ under conditions of low redox buffer capacity (Rahman et al.,
2008b). Arsenic-induced root exudation might also a probable rea-
son for a reduced pH in the rhizosphere. One study showed that
the reduction in rhizosphere pH increases the chemical activity of
most metals, thereby increasing As uptake by the ferns (Tu and Ma,
2004). Nevertheless, As removal is strongly dependent on the pH
and these low pH in the reactors remarkably favored As removal
(>90%, predominantly as arsenate) under this C-deficient oxic con-
dition. In general, sulfate-reducing microorganisms do not grow
well at pH values below 5.5 and prefer higher levels of alkalin-
ity, with 6.6 being optimal (Govind et al., 1999). Therefore, rapid
As removal processes took place within the rhizosphere by other
processes than dissimilatory SO4 2− reduction.
Addition of organic C-sources caused simultaneously a reducing
condition inside the reactors and an increment of rhizosphere pH
level due to buffering effect (phase II; Table 4). Rapid SO4 2− reduc-
tion by dissimilatory sulfate-reducers utilizing already enriched
S-pool might produce alkalinity and triggered the increment of
rhizosphere pH under such conditions. Better As mass retention
in these experimental phases were observed in a highly consistent
pH with an average pH value of 7.0 ± 0.5 and 5.7 ± 0.5 in R2 and
R3, respectively (phase III–V, Tables 2 and 4). Changes in alkalinity
can indicate changes in the speciation of As and sulfate-reducing
bacteria activity, since these bacteria can also provide alkalinity to
the water and affect its pH (Cohen, 2006). Lizama et al. (2011) in
a review paper stated that the changes in the speciation of As can
affect pH, as the oxidation of As(III) to As(V) decreases the pH value,
whereas the precipitation of arseno-sulfides increases it. This was
also demonstrated within the rhizosphere of Juncus effusus in this
study.
From a very high to a relatively lower or even limited SO4 2−
loading might also be resulting in a decreasing tendency of rhizo-
sphere pH, even though under C-surplus conditions (phase III–V in
R3; Tables 1 and 4). In fact, it was very interesting to observe a com-
paratively lower mean pH in all thorough the experimental phases
(I–V) of R3, specifically a decrease of nearly 2–3 pH units during
the phases III–V as compared to the reactor R2, This lower pH in
the rhizosphere might be a convincing reason for higher amount of
re-mobilized As in the pore water (16.2%) as well as higher As con-
centration in the roots collected from the reactor R3 than the roots
from R1 and R2 (Figs. 4 and 5). So, an increase of rhizosphere pH
101
might be favoring the immobilization of liable and exchangeable As
fractions in the rhizosphere but a lower pH within the root vicinity
of constructed wetlands might consequently enhance plant uptake.
Therefore, a pH change may affect the bioavailability of As and may
also play a vital role for plant As uptake within the rhizosphere.
For example, Wells and Richardson (1985) reported a decrease in
arsenate uptake in the moss Hylocomium splendens with increas-
ing pH. In this moss, arsenate uptake was optimal at pH 5, where
H2 AsO4 − was the dominant form in solution. As the pH increased
to pH 8, where HAsO4 2− was the dominant anion, arsenate uptake
decreased. Mukherjee and Kumar (2005) observed in the aquatic
plant Pistia stratiotes that the maximum As uptake rates occurred
at pH 6.5.
4.3. Accumulation of As in plant biomass
Terrestrial plants are able to accumulate As to a substantial
extent (Visoottiviseth et al., 2002) but survive the stress to dif-
fering degrees of vitality. During the whole experimental period
(341 days), As was accumulated in different compartments of the
plant biomass, mainly into plant shoots and roots. No remarkable
differences can be found in terms of As translocation into the plant
shoots of the corresponding reactors. In general, the mean As con-
centrations (dry wt) within the plant shoots of the reactors were
7-fold higher than the As concentration in the control shoot sam-
ples (Fig. 4A).
The concentrations of total As in the plant roots were extremely
higher than in the shoots in this study (Fig. 4). In general, total As
concentrations exhibited in the roots were 23–88 times higher than
to their shoots in all reactors. A vast majority of As were found to be
fixed in/on the roots and only a very limited amount was translo-
cated to the shoots. Buddhawong (2005) and other authors also
reported similar facts when dealing with As and heavy metals in
constructed wetlands. Roots continuously remained under direct
exposure to As in both oxic and anoxic environment and transloca-
tion rate of As into shoots presumably depends on other factors and
varies within different plant species. Carbonell et al. (1998) studied
the As content in Spartina alterniflora and found As in the range of
0.80–1.77 mg kg−1 in shoots and 6.87–86.60 mg kg−1 in the roots.
The much higher accumulation of As in the plant roots compared
to the above-ground biomass (shoots) corresponded to the studies
for Typha latifolia, Equisetum fluviatile, Triglochin palustre, and Spar-
ganium sp. (Dushenko et al., 1995). However, Rahman et al. (2011)
showed a translocation with a range of 12–20 times from the roots
to the shoots when investigating the fate of As in a laboratory-
scale horizontal subsurface-flow constructed wetland. The low As
translocation from roots to shoots is probably due to the fact that
arsenate is rapidly reduced to arsenite in the roots, followed by
complexation with thiols and sequestration in the root vacuoles
(Zhao et al., 2009). Iron plaque on root surfaces has been shown
to control the uptake and transfer of As by rice (Liu et al., 2005).
Several other studies have shown that roots accumulate more As
than do shoots (Adhikari et al., 2011; Hozhina et al., 2001). Dif-
ferent reasons may explain why As remains mostly in plant roots,
such as limited translocation of As from roots to shoots (Wang et al.,
2002), the presence of Fe and S (Zhao et al., 2010), the effect of As
speciation in the mechanism of translocation and its relationship
to the phosphate transporter (Dhankher, 2005), and the formation
of As(III)-phytochelation (PC) complexes in roots and subsequent
sequestration in root vacuoles (Xue and Yan, 2011). However, the
form of As that is translocated to shoots or how this transloca-
tion occurs is not known (Dhankher, 2005; Lizama et al., 2011).
Further studies on these As species are therefore needed to investi-
gate the mechanism of As uptake, translocation and accumulation,
102 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105
considering both the water and the sediment and taking into
account the relationship with Fe, S and phosphate.
Our results with a mean concentration of 90 ± 2.8, 110.5 ± 3.5
and 315 ± 4.3 mg As kg−1 within the roots of Juncus effusus col-
lected from the three corresponding reactors were definitely not
surprising under an ideal-flow condition, also depending on var-
ious loading conditions and exposure time. Nearly 3-fold higher
As concentrations as compared to the roots from the reactor R2
and 3.5-fold higher from the control reactor R1 clearly indicated
a higher amount of As retention, accumulation, adsorbed, metab-
olized to other forms and/or translocated into the root vicinity of
Juncus effusus in the reactor R3, where organic carbon and SO4 2−
loading were abundant. Nearly 5-fold lower C/S ratio (TOC: SO4 2− -
S) within the reactor R3 might facilitate lower pH and consequent
remobilization caused free or exchangeable As in the pore water
and hence a higher concentration of As in roots as compared to
the reactor R2 was found. Higher enrichment of total sulfur due
to the re-oxidation of reduced S2– to oxidized S-species like ele-
mental sulfur (S0 ), SO4 2− etc. might also cause remobilization of As
(Rahman et al., 2008b) and hence a higher As-concentration in the
roots of the reactor R3 was established. But interestingly, the dry
weight of the total root biomass in reactor R3 was comparatively
lower than the dry weight of the roots collected from other two
reactors R1 and R2 (Table 5). Therefore, a higher As accumulation
or uptake by the plant roots might have some effect on plant root
growth under such long-term investigation.
The results of the study by Favas et al. (2012) revealed high
bioaccumulation levels of As in several species at a magnitude
much higher than the concentration in the surrounding water. The
highest concentrations of As were found in the submerged species
Callitriche lusitanica (2346 mg As kg−1 dry wt), but the measured
concentrations in the other emergent plants were significantly
lower, even in the rhizomes/roots, such as T. latifolia (4.17 mg
As kg−1 dry wt) and J. effusus (14.4 mg As kg−1 dry wt). The sorp-
tion and bio-concentration of As were measured in Schoenoplectus
californicus and Typha angustifolia in a pilot-scale constructed wet-
land receiving wastewater inflows containing As at potentially
hazardous levels (Sundberg-Jones and Hassan, 2007). Moreover,
it has already been shown that roots and shoots As concentrations
significantly increased with increasing As application rates to the
rooting medium of a wetland ecosystem (Mkandawire and Dudel,
2005; Sundberg-Jones and Hassan, 2007). The root vicinity of Juncus
effusus was continuously exposed to an artificial wastewater con-
taminated with As under idealized flow conditions, which might
enhance bioaccumulation of As and the uptake by the roots in this
investigation.
The increase of As uptake attributed to iron plaque on root
surfaces has been reported in rice and Spirodela polyrhiza (Liu
et al., 2005; Rahman et al., 2008c). At the root plaque interface,
siderophores or phytosiderophores exuded by microbes or roots
may complex with Fe(III) and mobilize Fe-bound arsenate, taken
up through phosphate co-transporters, which may lead to simulta-
neous uptake of Fe and arsenate (Liu et al., 2005). This phenomenon
has also been observed for other metals such as Cu and Zn (Ye et al.,
2001). Fitz and Wenzel (2002) reported that root-induced changes
in the rhizosphere altered its chemical composition and facilitated
As uptake by Pteris vittata L (Chinese brake fern), the first-known
arsenic hyperaccumulator. Root architecture and physiology, root-
induced changes in water and nutrient availability, root exudates,
and fungal and bacterial associations (Gahoonia and Nielsen, 2003)
are all components of the dynamic rhizosphere system and prob-
ably facilitated higher As retention within the roots in this study.
The formation of iron plaque on the macrophyte roots has a high
affinity for As, tending to have a higher affinity for As(V) than
As(III) (Chen et al., 2005), and might also be a probable reason for
the higher As concentration within the roots. The ability to carry
oxygen from the air down to its stem and discharge it in the rhizo-
sphere through the roots (Brammer and Ravenscroft, 2009) creates
oxidized micro-environments around the roots in which iron is
oxidized and precipitated to form a coating (Liu et al., 2006). This
oxidation in the rhizosphere may also lead to As precipitating on
root surfaces. The presence of available sulfur due to a high SO4 2−
loading might also be a reason for the formation of iron plaque on
the root surfaces of Juncus effusus in this study. Nevertheless, more
investigations on the effects of O2 release through the plant-roots,
changing of redox and pH for the formation of Fe plaque on root sur-
faces and hence altering the bioavailability of As in the rhizosphere
of constructed wetlands are needed.
4.4. Distribution of As in sediment
A big variety of suspended and biofilm-fixed microorganisms on
plant-root surfaces along with surplus organic C produced sludge
sediments, which were collected from the reactors after the ter-
mination of the experiments. Analytical results of the collected
and dried sediments showed a wide variation with a mean con-
centration of 206 ± 5, 806 ± 9 and 941 ± 4 mg As kg−1 (dry wt) in
R1, R2 and R3, respectively (Fig. 4C). Clear evidence of intensive
dissimilatory SO4 2− reduction, highly efficient TOC removal, high
As adsorption and/or precipitation in the sludge sediment of the
reactor R3 suggested higher microbial activities within the rhizo-
sphere in presence of higher SO4 2− loading as compared to other
two reactors R1 and R2.
4.5. As mass balance
Calculation of total As mass balance was carried out for all the
experimental reactors at the end of our investigations. Since the
reactors were closed tightly enough and well-controlled systems,
we were able to obtain a quantitative mass balance of total As by
expressing its distribution as percentage of the total mass loaded
into the reactors. It was observed that the reactors R2 and R3 were
performing with a much better As deposition than the control reac-
tor R1 with limited SO4 2− loading.
A substantially higher amount of As mass was accumulated in
the roots than sludge sediments in reactor R3 with a higher SO4 2−
loading. Based on the concentration of As in the plant biomass
(shoots and roots) and sludge sediments of the reactor R3, we calcu-
lated a 42% of total inflow As mass were accumulated in the roots
of Juncus effusus and 17.2% were deposited within the sediment
(Fig. 5). A high level of As in the roots of R3 might be related to a
higher SO4 2− loading (i.e. lower C/S ratio), consequently a lower pH
and formation of elemental sulfur (S0 ) within this reactor, hence a
greater remobilization of As in the solution for plant-root uptake.
This was demonstrated by a comparatively higher As retention in
the pore water (16.2%) and lower retention (9%) in unaccountable
sink than the reactors R1 and R2 (Table 4; Fig. 5).
The accumulation or deposition of As in the sediments occurred
probably due to the formation of insoluble precipitates like As2 S3
via abiotic processes or, more probably, driven by the high level of
microbial activity and biotic processes incorporated to the organic
matter content within the rhizosphere. Along with sludge sedi-
ments, plant roots were also therefore considered to be the primary
sink for the sequestration of As in the rhizosphere, which agrees
with an As accumulation range of 44–49% within the roots of
Juncus effusus investigated in the planted horizontal subsurface-
flow constructed wetlands (Rahman et al., 2011) and partially
agrees with many other studies of treatment wetland systems
(Sundaravadivel and Vigneswaran, 2001). Several studies have
identified aquatic plants with high As content: Lagarosiphon major
 K.Z. Rahman et al. / Ecological Engineering 69 (2014) 93–105
(300 mg As kg−1 dry wt) (Brooks and Robinson, 1998), Egeria densa
(>1000 mg As kg−1 dry wt) (Robinson et al., 1995), C. demersum
(>1000 mg As kg−1 dry wt) (Robinson et al., 1995), and Lemna
gibba (1021.7 ± 250.8 mg As kg−1 dry wt) (Mkandawire et al., 2004).
Therefore, the accumulation of As depends on the type of plant
(Lizama et al., 2011; Zhao et al., 2010), and the potential of some
aquatic plants to accumulate As has already been well demon-
strated. This potential of aquatic plants to accumulate As supports
their possible use in phytoremediation of As-contaminated water
(Xue and Yan, 2011). Li et al. (2011) suggested that most of As
uptake was accumulated in 9 wetland species root tissues rather
than on root surfaces or in shoot tissues. The root tissue is there-
fore the main barrier for As transport in wetland plants. These
might be critical considerations for improving the efficiency of
As removal from wastewater in full-scale constructed wetland
systems.
Only <1% of total inflow As mass were accumulated into the
plant shoots (Fig. 5). This result clearly indicated the minor role
by the plant shoots in terms of As uptake and showed a very little
contribution to the overall As mass balance. Standing or pore water
accounted for nearly 5%, 10% and 16% of total inflow As mass in the
three respective reactors with an increasing SO4 2− concentrations.
Enrichment of total sulfur due to the re-oxidation of reduced S2– to
oxidized S-species (S0 , SO4 2− etc.) might cause remobilization of
As and hence a higher percentile As mass can be found in the pore
water of the reactor R3 with a higher SO4 2− loading than the other
two reactors R1 and R2.
Nearly a total of 52%, 21% and 15% of the inflow As mass were
passed through the reactors R1, R2, and R3, respectively and col-
lected at the outlet. The larger amounts of As (52% of the total
inflow As mass) were flushed out through the outlet of the control
reactor R1 in comparison to the reactor R3 (15%), which indicated
a highly stable bonding of As with the attached biofilms within
plant root zone of the rhizosphere associated with higher SO4 2−
loading. Therefore, the higher the SO4 2− loading as well as an
enhanced dissimilatory SO4 2− reduction in the reactor (Rahman
et al., 2008b), the better will be the As binding capacity within
the root-near environment of the rhizosphere in constructed
wetlands.
Only traces (2–3 ␮g l−1 ) of inorganic volatile arsine (AsH3 ) were
measured in this study, which resulted in a very small amount
of As mass that was left out of the systems due to volatilization.
Nearly 26%, 29% and 9% of total As mass in these three reactors were
accumulated into unaccountable sink, which could be due to other
microbial reactions, adsorption/precipitation to other unknown
sink or even lost due to volatilization. Some of the retained or
trapped As might have been bio-transformed by plant-root activity
and associated microbes to other unidentified organic compounds,
which prevented the calculation of a complete As mass balance in
this study.
From the previous results (Buddhawong, 2005), it was found
that the gravel material could not absorb As mass in substantial
amounts from the solution. Therefore, this kind of gravel itself had
no remarkable impact on the mass balance of As in constructed
wetland systems. Fractional analysis of deposited sediment might
also be necessary to investigate different forms of As that were
retaining within the rhizosphere of helophytes in treatment wet-
lands. Singhakant et al. (2009) reported about the forms of the
retained As by using sequential fractionation, which could indi-
cate As complexation with Fe and Mn on the media surface of
31–38% and As trapping into the media of 42–52% of the total
As. However, more attention should be given to the accumulation
of As in different wetland compartments and to avoid poten-
tial toxic effects the accumulated As could pose to the wetland
plants.
103
5. Conclusions
This study reveals that wetland plants possess high As con-
centrations on roots and a very low As translocation from roots
to the shoots. Redox potential and pH within the root-zone are
greatly influencing As deposition and plant uptake. An increase
of rhizosphere pH (alkalinity) might be favoring the immobiliza-
tion of liable and exchangeable As within the root vicinity and
a decrease of pH might cause remobilization of As and enhance
plant (root) uptake. Sulfate loading with an elevated SO4 2− con-
centration is also playing an important role, which facilitates an
efficient SO4 2− reduction and TOC removal as well as a higher
As retention under microbial dissimilatory SO4 2− reducing con-
dition within the rhizosphere. Consequently, high enrichment of
total sulfur due to the re-oxidation might cause remobilization
of As and enhance plant-root mediated As uptake. Nevertheless,
the higher the SO4 2− loading, the better will be the As binding
capacity within the root-near environment of the rhizosphere in
constructed wetlands. Plant shoots (Juncus effusus) are playing a
minor role in terms of As uptake and contributing very little to the
overall As mass balance. Roots and sludge sediments are consid-
ered to be the primary sinks for As retention in the rhizosphere
of constructed wetlands. Accumulation of a small portion of As to
unaccountable sinks and/or a probable loss due to volatilization
or other unknown reasons demands further research under such
strict conditions on a priority basis in order to protect the surround-
ing environment from any toxic effects of volatile As compounds.
Moreover, different physico-chemical properties on the root sur-
face, storage characteristics in root tissues of wetland plants and
precipitation/adsorption/retention of different As species clearly
requires further investigation. Future studies should also intend
to focus on exploring the patterns of microbial ecology and their
interactions, associated As toxicity on plants and microbial biomass
and volatilization of As under dynamic redox conditions within the
rhizosphere of constructed wetland system.
Acknowledgments
The work was funded by a grant from the German Fed-
eral Ministry of Education and Research under the International
Postgraduate Study in Water Technology (BMBF-IPSWaT) pro-
gram and by grants from the Helmholtz-Centre for Environmental
Research–UFZ, Leipzig, Germany. The authors would like to thank
Kerstin Puschendorf, Ines Mäusezahl, Karsten Marien, Jürgen
Steffen, Reinhard Schumann, and Uwe Kappelmeyer for their out-
standing technical and analytical support.
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