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Effect of pH, temperature, and oxygenation on arsenic phytofiltration by

aquatic moss ( Warnstorfia fluitans )

Article  in   Journal of Environmental Chemical Engineering · May 2018

DOI: 10.1016/j.jece.2018.05.044

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 Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx

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Effect of pH, temperature, and oxygenation on arsenic phytofiltration by aquatic moss
(Warnstorfia fluitans)
Arifin Sandhia⁠ ,⁠ b⁠ ,⁠ ⁎⁠ , Tommy Landberga⁠ , Maria Gregera⁠

PR
a
Department of Ecology, Environment and Plant Sciences, Stockholm University, Svante Arrhenius väg 20A, SE-106 91 Stockholm, Sweden
b
Land and Water Resources Engineering Division, Department of Sustainable Development, Environmental Science and Engineering, KTH Royal Institute of Technology, Teknikringen 76, SE -100 44
Stockholm, Sweden

ARTICLE INFO ABSTRACT

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Keywords: Phytofiltration of arsenic (As)-contaminated water could reduce As in irrigation and surface water. In a previous
Arsenic removal study, we found that the aquatic moss Warnstorfia fluitans efficiently removes arsenic from water contaminated
Aquatic moss with arsenate and arsenite. This work investigates how factors such as pH, temperature, and oxygenation influ-
pH ence As removal, since these factors vary in the environment. Plants were grown in a medium with 5 or 10 μM
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Phytofiltration
arsenite or arsenate and: 1) a pH of 2.5, 6.5, or 9.5; 2) a temperature of 12, 20, or 30 °C; and 3) oxygenation
Oxygenation
Temperature of <2 or 13 mg O2⁠ L− ⁠ 1. Removal of As was monitored over 48–96 h, and the content and speciation of As were

analysed in moss plants at the termination of the experiments. Results indicate that As removal was faster in
arsenite than arsenate solutions. Arsenic removal from arsenite solution was the fastest, i.e., 80–90% within 2 h,
at pH 6.5 and 9.5 and at 20 and 30 °C. At pH 2.5, plants were stressed and the net removal was low throughout
the treatment period. Arsenic removal was more efficient at low than high oxygenation levels. Besides this, no As
EC

net efflux process was seen in the water system except after 48 h in arsenate-treated medium in high-temperature
(30 °C) regimes. Regardless of As species added, usually only arsenite was found in the plants after treatment.
Most internal As, i.e., 95% in the arsenate and 85% in the arsenite treatments, was firmly bound to the tissue.
The study found that at 20 °C, neutral pH, and low oxygenation, this aquatic moss has great potential for As
phytofiltration.
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water fern Micranthemum umbrosum [4]. The present work investigates

1. Introduction
Understanding the effects of arsenic (As) contamination of the wa-
ter system as well as how to reduce this contamination are important
global tasks. There is a huge problem of drinking and crop-irrigation
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aquatic plant collected from As-contaminated mining sites in Northern
Sweden part, the aquatic moss Warnstorfia fluitans, which efficiently re-
moves As from As-contaminated water in laboratory tests [5].
Abiotic characteristics in water system such as pH, temperature, and
oxygenation vary between waters and show influence towards As accu-

water around the world containing excessive levels of As [1], a met-
alloid causing serious human health problems such as skin lesions and
lung and bladder cancer [2]. For reduction of As content in the ground-
water system, a relatively large and economic solution required com-
pared to As problem in drinking water system. By using phytofiltra-
tion technique with help of high As accumulating aquatic plant species
could be an promising option, in where the submerged and the emer-
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mulation in plants [6]. These factors may also influence the As specia-
tion in water and thus regulate As uptake in the plant system. Arsenic
exists in both organic and inorganic forms, but water contains in most
cases only inorganic species, i.e., arsenate and arsenite [7].
Arsenic is found in different oxidative states due to the variation
in pH and redox levels: arsenite occurs mainly in reducing and hy-
drothermal groundwater, whereas arsenate is more available in oxidiz-

gent plants species are used to remove As from the water resources. ing and surface groundwater [8]. The pH plays an important role in the
Most relevant studies have tested plants from warmer climates for As availability of As species in different kinds of groundwater [8]. Arsen-
phytofiltration, such as the macrophytes Hydrilla verticillata, Myriophyl- ite (AsO3⁠ 3⁠ –) occurs in uncharged form (H3⁠ AsO3⁠ ) in neutral-pH water,
lum spicatum, Potamogeton crispus, and Vallisneria natans [3] and the

⁎ Corresponding author at: Department of Ecology, Environment and Plant Sciences, Stockholm University, Svante Arrhenius väg 20A, SE-106 91 Stockholm, Sweden.
Email address: asandhi@kth.se (A. Sandhi)

https://doi.org/10.1016/j.jece.2018.05.044
Received 27 March 2018; Received in revised form 25 May 2018; Accepted 26 May 2018
Available online xxx
2213-3437/ © 2018.
A. Sandhi et al.
whereas arsenate (AsO4⁠ 3⁠ –) becomes negatively charged when the pH de-
clines below 2.3. Uncharged arsenate, H3⁠ AsO4⁠ , predominates in the pH
range 1–2, negatively charged H2⁠ AsO4⁠ −⁠ in pH 3–6, and the bivalent neg-
atively charged HAsO4⁠ in pH 7–10 (Fig. 1). Not only As species chem-
2
⁠ –
istry but also pH plays an important role in As uptake in plants. Arsenite
accumulation in the aquatic macrophyte coontail (Ceratophyllum demer-
sum L.) decreased when the pH increased in the surrounding medium
[9]. Besides this, pH also plays an important role in As binding inside
plant cells. The binding affinity for arsenate transport was found to peak
at pH 3–5, and lowering the pH from levels in that range could influ-
ence leakage of accumulated As [10].
The temperature of the medium is another important factor that may
influence As accumulation in aquatic plants. Arsenic accumulation in
Fucus spiralis doubled when the seaweed was exposed to 30 °C compared
with 16 °C water [11]. The same was found for Cd, Zn, Cu and Pb ac-
cumulation in Elodea canadensis [12]. Arsenic accumulation in aquatic
plants was found to be higher in the summer, with higher temperatures,
than the spring [13], however, the results could depend on the plants
growth stage.
Another important factor is oxygen availability, which may differ
between flowing and still waters. It is well documented that the spe-
ciation of inorganic As is greatly dependent on the redox condition of
the medium [14], with arsenite changing to arsenate at high redox po-
tentials. Regarding oxygen content and the ability of aquatic plants to
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optimize their physiological conditions, wetland plant species have de-
veloped aerenchyma, a lacunar system in the tissue that enhances the
oxygen concentration under waterlogged conditions and causes lowered
resistance to internal oxygen movement [15]. Furthermore, the oxidiz-
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ing ability of plants can change the As species present on the root sur-
face when plants grow under waterlogged conditions [16]. Oxygenation
therefore plays a role in As species transformation and As accumulation
in aquatic plants.
In the aquatic system, macrophyte species play an important role
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in phosphorus mobilization, trace element transfer, sediment dynamics,
and freshwater ecosystem hydrology [17]. Of macrophyte species, the
bryophytes (mosses) have displayed great potential to accumulate var-
ious contaminants from water systems due to their rapid exchange and
passive sorption process [18–20]. Many aquatic moss species are found
in the arctic region due to their adaptability and growth ability under
low-nutrient, –temperature, and –light conditions [21]. In previous ex-
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periments, we found that the native aquatic moss Warnstorfia fluitans
(Hedw.) Loeske from northern Sweden accumulated high levels of As
from the aquatic system, especially under low-nutrient conditions [5].
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Fig. 1. Predicted arsenate speciation as a function of pH (Medusa software).
2
Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx
However, the effects of the common environmental parameters pH,
temperature, and oxygenation on As removal by this aquatic moss have
not yet been investigated. For potential use of this kind aquatic plant
based phytofiltration facility in the water system of the mining areas, it
is important to get in depth information about major effects of environ-
mental parameters towards As accumulation in W. fluitans. This work
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accordingly investigates how these three factors influence the accumu-
lation and removal efficiency of As from the water system by W. fluitans.
Since water may contain both arsenite and arsenate treated medium,
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both these As species were considered here. The objectives of this study
were to investigate: (1) the effect of different pH levels on As accumu-
lation and speciation in the moss, (2) the effect of temperatures in the
on As accumulation and speciation, and (3) the effect of high and low
oxygenation on As uptake.
2. Materials and methods
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2.1. Plant culture
The aquatic “floating hook-moss” Warnstorfia fluitans (Headw.)
Loeske, gathered from the Skellefteå field in Lapland (N65°20.943′
E18°35.587′), Sweden, was used in this work. Plants were collected
from a stream, outside a mine tailings impoundment containing ele-
vated arsenic levels in the water. Prior to the experiments, plants were
grown for at least a month in greenhouses under submerged conditions
in sediment with adequate aeration provided to ensure both oxygen sup-
ply and turbulence in the water system (see [5]. Before the experiments,
fresh, newly grown moss plants were collected, rinsed five times with
deionized water, washed of all debris and sediment particles, and placed
in 1% Hoagland nutrient solution [22] in buckets for 24–36 h for ac-
climatization to the specific pH, temperature, and oxygenation condi-
tions of each experiment.
Plants were grown and experiments performed under greenhouse
conditions as follows: 16 h light/8 h dark at 23 °C/19 °C. Mosses are
slowly growing organisms and during the uptake experiments of up to
96 h they did not show any growth increase. No sign of toxic effects on
the mosses was shown by As up to 100 μM [5].
2.2. Arsenic chemicals
Natural water contains in most cases only inorganic As species, es-
pecially arsenate but possibly also arsenite. Arsenite (sodium meta-ar
A. Sandhi et al.
senite, NaAsO2⁠ ; Merck, Kenilworth, NJ, USA) and arsenate (sodium ar-
senate dibasic heptahydrate, Na2⁠ HAsO4⁠ ·7H2⁠ O; Sigma-Aldrich, St. Louis,
MO, USA) were used in all experiments. The above-mentioned chemi-
cals were also used as standards in the analysis of As speciation. An As
standard solution of 1000 mg L−⁠ 1
As (Merck) was used when analysing
total As content of water and moss samples.
2.3. Effect of pH on As accumulation and speciation
This experiment was performed to investigate how pH influences
the As accumulation rate and As speciation in the aquatic moss W. flui-
tans. Before the experiment, plants were acclimatized for 24 h in 1%
Hoagland nutrient solution adjusted to pH 2.5, 6.5, and 9.5 with 0.1 M
HNO3⁠ and 0.1 M NaOH. In addition to serving as a nutrient source, the
nutrients helped stabilize the pH level in the medium.
Each acclimatized plant (12 g) was gently wiped and weighed before
being put in a pot and provided with adequate aeration. Plants were
grown in 1-L pots containing 1% Hoagland nutrient solution and 0 (con-
trol) and 10 μM As in the form of arsenate or arsenite. The solution pH
was adjusted to 2.5, 6.5, and 9.5 using 0.1 M HNO3⁠ or 0.1 M NaOH at the
beginning of the experiment and pH data in the water medium was mea-
sured in every 24 h and re-calibrated according to the initial pH level
with the help of 0.1 M HNO3⁠ or 0.1 M NaOH (data not shown). These
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pH levels were chosen based on the following considerations: 1) arsen-
ite is mainly uncharged at pH 6.5; 2) arsenate exists in different charged
forms at different pH levels (Fig. 1) [23]; and 3) the acid mine-drainage
sites where this plant was collected have low pH levels around 2.5 [8].
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The pH level of the treatment was measured and adjusted to the initial
pH every 24 h using 0.1 M HNO3⁠ or 0.1 M NaOH.
Water samples of 5.0 mL were collected from the medium in each
pot 0, 2, 8, 12, 24, 26, 32, 36, and 48 h after the plants had been added
to the medium. The samples were kept refrigerated at 8 °C before total
EC
As analysis. After 48 h of treatment, the moss plants were removed from
each pot and rinsed with deionized water; excess water was gently re-
moved from the plants using paper towels. Mosses were then weighed
and divided into two portions: one for analysing total As concentration
and another for analysing As speciation. The experiment was performed
in three replicates using a randomized block design.
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2.4. Temperature influence on As uptake
This experiment determined the influence of temperature on As up-
take in the mosses. Plants were acclimatized in 1% Hoagland nutrient
solution at the various temperatures for 24 h before the experiment. The
temperature of the medium was set to 12, 20, and 30 °C, maintained by
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placing the cultivation pots in three water baths. The temperature of the
12 °C bath was maintained using an immersion cooler (FT401; Julabo,
Seelbach, Germany) while the temperatures of the 20 and 30 °C baths
were maintained using thermostat heaters (Aqua Thermostat 300; Hy-
dor, Bassano del Grappa, Italy). During the experiment, 0 and 10 μM As
was supplied as either arsenate or arsenite to each 1-L pot containing
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aerated 1% Hoagland nutrient solution. To each pot, 12 g (fresh weight)
of moss sample was added. Five-mL samples of solution were taken from
each pot 0, 12, 24, 48, and 72 h after the plants were placed in the so-
lution. These water samples were kept in a cold room at 10 °C before
analysis of total As. After 72 h, a moss sample was taken from each pot,
rinsed with deionized water, and gently dried with paper towels. The
moss samples were then weighed and dried. The experiment was orga-
nized in a randomized block design, and each treatment was performed
in three replicates.
3
Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx
2.5. Effect of oxygenation on As uptake
An experiment was performed to study how the oxygen level in
the water influenced As mobility in the water–moss system and the As
speciation in the aquatic moss. E-flasks (350 mL) were filled with 1%
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Hoagland nutrient solution under high and low oxygenation, and a 6-g
(fresh weight) moss sample was placed in each of them for 36 h for ac-
climatization before the experiment. Low oxygenation ( < 2 mg O2⁠ L− ⁠ 1)
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was maintained by bubbling N2⁠ gas (AGA, Stockholm, Sweden), 8 h per
day, through aeration tubes in cultivation pots sealed with parafilm.
High oxygenation (∼13 mg O2⁠ L− ⁠ 1
) was maintained by bubbling air
from the aeration system through the aeration tubes. With bubbling
means a higher speed than the adequate aeration mentioned earlier. Af-
ter 36 h, these two oxygen levels were divided into three subgroups:
no added As and 5 μM As added as arsenate or arsenite. The experi-
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ment lasted for 96 h, but for the last 24 h, the N2⁠ supply was kept run-
ning continuously until moss samples were collected from the low-oxy-
gen-treatment experiment. Water samples were taken from each pot 0
and 96 h after the mosses had been placed in the containers, and the
samples were kept at 10 °C until total As analysis. At the end of the ex-
periment, after 96 h, the moss samples were collected from all the pots,
rinsed with deionized water, wiped with paper towels. Mosses were then
weighed and divided into two portions: one for analysing total As con-
centration and another for analysing As speciation. The experiment was
run in three replicates.
2.6. Total As analysis
Moss samples were oven dried at 80 °C for 48 h and then weighed. To
determine the actual dry weight of the moss samples, a supplementary
amount of moss biomass was dried at 105 °C for 24 h. The 80 °C-dried
moss samples were then wet digested in HNO3⁠ :HCIO4⁠ (7:3,V/V) for 19 h
in a heating programme in which the temperature increased stepwise up
to 225 °C. Transformation of As-species were very slow, however, sam-
ples were analysed directly after the experiment termination.
The total As concentrations in moss and water samples were de-
termined using an atomic absorption spectrophotometer (AAS) (Varian
SpectrAA 55B; Varian, Palo Alto, CA, USA) equipped with a vapour gen-
eration accessory (VGA-77; Varian). Sodium borohydride (3%; Merck),
sodium hydroxide (2.5%; EKA Chemicals, Marietta, GA, USA), and hy-
drochloric acid (6 M; VWR International, Radnor, PA, USA) were used
for the hydride generation. Since filtering of water samples did not give
different results from non-filtered water, all data shown are from non
non-filtered water samples The detection limit for analysis was 7 μg As
L−
⁠ 1
. Certified reference material (GBW07604 GSV-3, poplar leaves) was
used each batch of 40 samples to ensure the quality of the analysis with
a recovery of 87.3% ± 2.4. To compensate the interaction effects of the
sample matrix, standard As addition method was applied for all samples
analysed.
2.7. Arsenic speciation
Moss samples were stored at −80 °C before analysis of inorganic
As species. The As extraction procedure was based on the As species
extraction protocol of Bergqvist and Greger [24]. Moss samples of ap-
proximately 1.4–5.0 g were placed in 15-mL Falcon tubes. Ten mL of
MeOH:H2⁠ O (1:1)solution was added to each tube followed by ultra-mix-
ing (Polytron PT2000 homogenizer; Kinematica AG, Luzern, Switzer-
land) at maximum speed for 10–15 s. The tube was then sub
A. Sandhi et al.
jected to heavy shaking for 5 min followed by sonication in an ultra-
sonic cleaner (Transsonic Digital S; Elma Schmidbauer, Singen, Ger-
many) for 20 min. Finally, the tube was centrifuged at 1700g for 10 min,
after which the supernatant was transferred to a 50-mL Falcon tube.
This method, except for the ultra-mixing, was repeated two additional
times with the same moss pellet. The entire procedure, not cluding the
ultra-mixing, was then repeated three more times, with 0.1 M HCl solu-
tion. The HCl-extracted samples were stored at −20 °C until As species
analysis. The final solution after MeOH:H2⁠ O (1:1) extraction was evap-
orated and reduced at 60 °C, and then resuspended in 3 mL of deionized
H2⁠ O and stored at −20 °C until As species analysis.
Arsenic species in the moss samples were detected using AAS (Spec-
trAA 55B; Varian) with vapour generation (VGA-77; Varian) and sepa-
rated using coupled high-pressure liquid chromatography (HPLC) with a
Hamilton PRP X-100 (250 × 4.6 mm) anion exchange column (Metrohm,
Herisau, Switzerland). The HPLC eluent was a 20 mM ammonium-phos-
phate buffer (pH 5.8) with a flow rate of 2 mL min− ⁠ 1 and was running
for 10 min plus 10 min recovery. Standard solutions of arsenite and ar-
senate (see Section 2.2) were used to identify (Supplementary Fig. 1)
and quantify the concentrations of the As species present in the moss
samples. The samples were analysed 2 times, without and with standard
added. The standard concentration added to samples was 370 μg L−
and 740 μg L −
⁠ 1
for arsenite and arsenate, respectively. The detection
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limit was 1.5 μg L− ⁠ 1 for arsenite and 9 μg L−
⁠ 1 for arsenate. Data from the
AAS detector were collected and analysed using SpectrAA Worksheet
Oriented AA Software, Version 5.1 (Varian). Certified reference mater-
ial (GBW07604 GSV-3, poplar leaves) was used. To compensate the in-
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teraction effects of the sample matrix, standard As addition method was
applied for all samples analysed.
2.8. Statistical analysis and additional information
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Statistical analysis used one-way ANOVA, LSD post hoc testing,
Tukey-Kramer testing, and Student’s t-testing performed with SPSS soft-
ware, version 22 (IBM, Armonk, NY, USA) and JMP, version 11 (SAS In-
stitute, Cary, NC, USA) to analyse statistical significance for As concen-
trations in water and moss samples and As speciation in moss. The sig-
nificant differences between treatments were compared using the LSD
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post hoc test and Tukey-Kramer test. The significance level was set to
P < 0.05.
In the water, the default temperature was approximately 20 °C (day/
night), oxygenation approximately 5 mg O2⁠ L− ⁠ 1, and pH approximately
6.5 if nothing else is indicated regarding the various experiments.
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Fig. 2. Time course of As remaining in growth medium at various pH levels over 48 h of treatment with moss. The medium initially contained nutrients and 10 μM arsenate (left) or
arsenite (right); pH was adjusted 24–26 h after the start. Mean of three replicates, ±SE.
Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx
3. Results
In all experiments, plants took up As from the solution and the As
content of the water declined with time. The concentration of As in-
creased in the plant tissue. The As concentration in the controls was
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high, up to 300 μg As gDW− ⁠ 1, because the plants were collected from
As-contaminated sites.
The solution pH influenced the As removal from the solution by the
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aquatic moss (Fig. 2). At the lowest solution pH of 2.5, the removal of
both arsenate and arsenite was low, being 40% at most, compared with
the other two tested solution pH levels, at which the removal was up
to almost 100%. In both the arsenite and arsenate solutions, the As re-
moval was fastest at pH 6.5. After 2 h, 90% of the As had already been
removed from the arsenite solution, while 50% was removed from the
arsenate medium after 2 h. The biggest difference between arsenite and
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arsenate removal appeared at pH 9.5, at which the removal was slower
in the arsenate than the arsenite solution. In general, As removal was
faster in the arsenite than the arsenate solution.
There was a more or less distinct, but insignificant, fluctuation in the
As removal with time, especially at pH 2.5, with the removal curve dip-
ping 24–26 h from the start of the experiment (Fig. 2). This was likely
⁠ 1
because the pH had been analysed and adjusted at that time. The pH
decreased over 24 h to 5.0 ± 0.2 (SD) in both the pH 6.5 and 9.5 treat-
ments in both the arsenate and arsenite solutions (not shown); however,
the pH did not change in the pH 2.5 solutions (not shown).
The moss plants took up As from the solution during the 48-h treat-
ment period, shown by the higher As concentration in the treated plants
than in the controls (Fig. 3). The As concentration in the control plants
at pH 9.5 tended to be half of that in plants grown at the two lower pH
levels. The As concentration in the As-treated aquatic moss was lower at
pH 2.5 than at pH 6.5 or 9.5 when the moss was growing in the pres-
ence of either arsenite or arsenate. The internal As concentration tended
to be higher in the arsenate- than arsenite-treated moss samples, when
comparing each pH level separately.
Comparing the control and As-treated plants, the As concentration
increased with increasing pH as follows: 2.7 and 1.3 times at pH 2.5,
4.7 and 3.7 times at pH 6.5, and 6.0 and 4.5 times at pH 9.5 in arsen-
ate and arsenite solutions, respectively (Fig. 3). The As concentration in
the mosses grown at the neutral pH of 6.5 was 1400 and 1100 mg of As
kgDW− ⁠ 1 in the forms of arsenate and arsenite, respectively, after 48 h of
treatment. The biomass also increased with increasing pH, although not
significantly, but did not change with As treatment (not shown).
A larger proportion of the internal As concentration in the moss was
firmly bound, as indicated by the low extraction efficiency (Table 1).
4
A. Sandhi et al. Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx

arsenite than the arsenate solution. As removal was almost 100% in the
arsenite solution and up to 80% in the arsenate solution after 48 h of
treatment. After 48 h, the removal stopped and the As level increased
in the arsenate solution, insignificantly at the lowest (12 °C) and signif-
icantly at the highest (30 °C) temperature. In the arsenite solution, 80%
and 90% of the As was removed in 12 h at 20 °C and 30 °C, respectively,

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while it took 24 h to achieve the same removal in the 12 °C medium; the
As level then stayed at this low level. The removal rate was the slowest
at the lowest temperature, 12 °C, in both arsenite and arsenate solutions.

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After 72 h, the As concentrations in moss plants at room temperature
(20 °C) were 1500 and 1750 mg kgDW− ⁠ 1 for arsenate and arsenite, re-

Fig. 3. As concentration in moss treated 48 h with 10 μM arsenite or arsenate at vari-
ous pH levels. Different letters (a-e) indicate significant differences among pH treatments
(p < 0.05). Mean of three replicates, ±SE.
spectively (Fig. 5), higher than when treated for 48 h (Fig. 3). There was
no significant difference in As concentration between As-treated plants
attributable to the temperature or added As species. The difference in
As concentration between the control and As-treated plants was great-
est at room temperature, i.e., 16 and 17.5 times at 20 °C, 7.5 and 6.5

When treated with arsenate, the extraction efficiency was as low as
3.3–5.4% of the total As content. When treated with arsenite, this value
was higher at 13.5–24.7%. At pH 2.5, the extraction efficiency tended
to be higher than at the higher pH levels.
Arsenite was found in the tissue, independently of whether arsenite
or arsenate was added, while only in the arsenate treatment at pH 6.5
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times at 12 °C, and 7.6 and 5.2 times at 30 °C in the arsenate and arsen-
ite solutions, respectively, though the concentration difference between
temperatures was not significant.
Arsenic removal was somewhat influenced by the oxygenation of the
medium. Moss tended to remove more As when growing under low than
high oxygenation (Fig. 6). Under high- and low-O2⁠ conditions, As was
more easily removed from the arsenate than from the arsenite solution,

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was arsenate found in the tissue (Table 1). The arsenite concentration
tended to be higher in the arsenite than the arsenate treatments. Only
in the arsenite treatments did the internal arsenite concentrations differ
among the various pH treatments, pH 6.5 giving the highest and pH 2.5
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the lowest arsenite concentration. No As species were detected in the
moss samples from the control treatment.
The temperature effect on the moss removal of As from the growth
medium over time is shown in Fig. 4. The As removal was faster in the
though the difference is not significant. After 96 h of treatment with
aquatic moss, 65 and 60% of the As was removed under high oxygena-
tion and 95 and 80% under low oxygenation, from arsenate and arsen-
ite solutions, respectively. At low-oxygen, removal of arsenic as arsenate
was significantly better than the high-oxygen removal as arsenate or the
high- or low-oxygen removal as arsenite.

Table 1
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Concentrations of arsenite and arsenate in moss after 48 h of treatment with 10 μM arsenite or arsenate at various pH levels. The extraction efficiency, indicating how much As is not tied
up in the tissue, is also shown. Different letters indicate significant differences within each column (p < 0.05). Mean of three replicates, ±SE; nd, not detected.

Treatment Arsenite, mg kg DW−

⁠ 1
Arsenate, mg kg DW−
⁠ 1
Extraction efficiency, %

As specie pH
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Arsenite 2.5 60.5 ± 6.1b⁠ nd 24.7 ± 0.13a⁠

6.5 159.5 ± 35.4a⁠ nd 14.1 ± 0.02b⁠
9.5 112.4 ± 24.3a⁠ b nd 13.5 ± 2.91a⁠ b
Arsenate 2.5 40.4 ± 2.3b⁠ nd 5.4 ± 0.01b⁠
6.5 52.1 ± 16.2b⁠ 17.3 ± 12.7 3.7 ± 0.01b⁠
9.5 35.3 ± 17.9b⁠ nd 3.3 ± 0.01b⁠
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Fig. 4. Time course of As remaining in growth medium at various temperatures over 72 h of treatment with moss. The medium initially contained nutrients and 10 μM arsenate (left) or
arsenite (right). Mean of three replicates, ±SE.

5
A. Sandhi et al. Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx

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Fig. 5. As concentration in moss treated for 72 h with 10 μM arsenite or arsenate at var-
ious temperatures. Different letters (a-b) indicate significant differences among tempera-
ture treatments (p < 0.05). Mean of three replicates, ±SE.

Fig. 7. As concentration in moss treated for 96 h with 5 μM arsenite or arsenate at low

(<2 mg L−⁠ 1) and high (13 mg L−
⁠ 1) oxygen levels. Different letters (a-c) indicate significant

differences among treatments. Mean of three replicates, ±SE.

D
Table 2
Concentrations of arsenite and arsenate in moss after 90 h of treatment with 0 or 5 μM
of arsenite or arsenate at high and low oxygen level. The extraction efficiency, indicating
how much As is not tied up in the tissue, is also shown. Different letters indicate signif-
TE
icant differences within each column (p < 0.05). Mean of three replicates, ±SE; nd, not
detected.

Arsenite,
mg Arsenate, Extraction
Treatment kgDW− ⁠ 1
mg kgDW− ⁠ 1
efficiency, %
EC

As specie O2⁠

Control Low 6 ± 0.4a⁠ 83 ± 21a⁠ 4.0 ± 1.0a⁠

High 11 ± 0.9b⁠ 73 ± 21a⁠ 4.6 ± 2.8a⁠
Arsenite Low 355 ± 49.3c⁠ 1300 ± 146b⁠ 30.3 ± 10.3b⁠ c
High 330 ± 37.2c⁠ 1541 ± 220b⁠ 27.9 ± 3.5b⁠
Arsenate Low 336 ± 63.7c⁠ 1506 ± 390b⁠ 39.0 ± 7.3c⁠
RR

High 253 ± 82.6c⁠ 1178 ± 280b⁠ 41.4 ± 10.6c⁠

Fig. 6. Time course of As remaining in growth medium at low ( < 2 mg L−
⁠ 1)
and high
(13 mg L−
⁠ 1) oxygen levels over 96 h of treatment with moss. The medium initially con-

tained nutrients and 5 μM arsenate or arsenite. Different letters (a-b) indicate significant arsenite or arsenate concentration in the plants due to various oxygen
differences among oxygen treatments (p < 0.05). Mean of three replicates, ±SE. treatments.

The As concentrations in the moss after treatment for 96 h under 4. Discussion

CO

low and high oxygenation are shown in Fig. 7. The As concentration in

the treated aquatic moss was about 2.5 times higher than in the con-
trol plants, with a final concentration of approximately 500 mg kgDW− ⁠ 1
and no differences between As-species treatments. Plants growing under
low oxygenation tended to have higher As concentrations than did those
growing under high oxygenation. Between control and treated plants,
the As concentration increased by 2.7 and 2.8 times under low oxy-
UN
In this work we investigated the removal of arsenite or arsenate from
water by the aquatic moss W. fluitans and the influence of pH, temper-
ature, and oxygen on this removal. It is clear that independent of treat-
ment, except at pH 2.5 (Fig. 2), plants were in good condition and re-
moved As from the solutions (Figs. 2, 4, and 6). Removal of As by W.
fluitans was earlier demonstrated to function well, especially at low-nu-

genation versus by 1.6 and 1.7 times under high oxygenation for arse-
nate and arsenite, respectively. The oxygenation in this experiment was
much higher (13 mg O2⁠ L− ⁠ 1) and lower ( < 2 mg O L−
2
⁠
⁠ 1) than in the
other two experiments, in which the oxygen level was approximately
5 mg O2⁠ L−
⁠ 1.
The arsenite and arsenate concentration in moss after oxygen treat-
ment is shown in Table 2. The concentration of both arsenite and ar-
senate was higher after As treatment, compared with the control. The
concentration of arsenate was higher than of arsenite in moss in both
treatments and controls. There is, however, no significant difference in
trient and low-arsenate concentrations [5]. The As removal was much
faster in arsenite than in arsenate solutions (Figs. 2, 4, and 6), and af-
ter 2 h, 93% had already been removed from the arsenite while just
49% had been removed from the arsenate solutions. After 48 h, little
As remained in either type of solution and, except under low oxygena-
tion (Fig. 6), the least As remained in the arsenite solution (Figs. 2 and
4). This does not align with the fact that arsenate is more available
than arsenite in aquatic media [25]. The retention time in water may
be lower for arsenite than arsenate, so that arsenite, in addition to via
plant uptake (Figs. 3, 5, and 7), disappears from the water by sedimen

6
A. Sandhi et al.
tation. This was not investigated in the present study, which focused on
the plant removal of As from water.
The remaining As in the solution was kept low by the plants for
the rest of the treatment period. However, while no As leaked from the
plants back into the arsenite medium (Figs. 2, 4, and 6), there was clear
As leakage into the arsenate solution after 48 h of removal at high tem-
perature (Fig. 4). Also, Sandhi et al. [5] found such leakage by W. flui-
tans after 48 h of treatment in arsenate solution, though only when no
nutrients were present. This could be because the arsenite taken up is
bound, for example, to cell walls or phytochelatins [26], so leakage is
impossible. According to Moreno-Jiménez et al. [27], non-accumulating
plant species actively release approximately 50–80% of their accumu-
lated As.
When treated with arsenate, most As was strongly bound in the plant
tissue, as indicated by a low extraction factor of approximately 3.5%,
compared with 13.5% for arsenite (Table 1). Sandhi et al. [5] found no
significant difference between arsenite- and arsenate-treated plants in
this regard, and 93–98% of As was in a bound form within the moss
biomass. Sandhi et al. [5] used solution containing no nutrients and, in-
dependent of As treatment form, more arsenate than arsenite was found
in the plant tissue. In contrast, in this work, generally only arsenite
was present in the plant materials, independent of the As species added
(Table 1).
The relatively low As extraction efficiency in W. fluitans can be ex-
D
plained by As complex formation with lipid, cellulose, magnesium pec-
tates, or lignin in the moss [28]. This might be a detoxification mech-
anism attributable to the plants having originally been collected from
As-contaminated sites. This mechanism was earlier discussed by Sandhi
TE
et al. [5].
That arsenite and arsenate were removed from the water by the moss
plants was obvious, since the treated plants contained higher As concen-
trations than did the controls (Figs. 3, 5, and 7). The increase in concen-
tration increased with time, at least up to 72 h of treatment (Figs. 3 and
EC
5). This means that the capacity of the aquatic moss to take up As was
high. It seems as though submerged plants such as aquatic mosses have
a high capacity for As accumulation [24]; Diaz et al., 2012.
The moss plants growing in pH 2.5 solution were clearly influenced
by the low pH, since they removed very little arsenite and arsenate (Fig.
2) and accumulated very little As (Fig. 3). At pH 2.5, the plants were
low in chlorophyll and unhealthy (not shown), so the As net uptake was
RR
likely low for this reason. Most mosses grow very poorly at pH levels
below 4 [29]. In the fern Pteris vittata, growth was negatively affected
by high As at low pH (4.5) but not at high pH (8.5) [30]. In the present
work, the extraction efficiency, i.e., the level of unbound As, is higher
at pH 2.5 than at higher pH levels (Table 1). This may indicate that the
membranes were affected by the low pH, being more permeable and
CO
leaky, so that the net As uptake was low (Fig. 2). This supports the find-
ings of Wells and Richardson [10] that at pH levels below 3, arsenate
leaked from cells of the moss Hylocomium splendens.
The highest As removal from the water was found at the neutral pH
of 6.5 (Fig. 2). A study of arsenite uptake in coontail (Ceratophyllum de-
mersum L.) also suggested that this aquatic macrophyte could accumu-
late more As at neutral than at higher pH levels [9]. The fern Pityro-
UN
gramma calomelanos had the highest As uptake at the neutral pH of 5.9
in a study examining the 3.6–8.9 pH range [31].
Arsenite is a pH-insensitive form of As, so arsenite removal was sim-
ilar at both pH 9.5 and 6.5 (Fig. 2). Arsenate, on the other hand, is
pH sensitive (Fig. 1), so arsenate removal is strongly affected by pH
(Fig. 2). Interpreting the data in relation to the diagram plotted using
Medusa software (Fig. 1) reveals the following information: H2⁠ AsO4⁠ − ⁠
and HAsO4⁠ 2⁠ are found at all pH levels tested; at pH 2.5, the solution
7
Journal of Environmental Chemical Engineering xxx (2018) xxx-xxx
contained neutral H3⁠ AsO4⁠ but not AsO4⁠ 3⁠ –; at pH 6.5, all four As species
were found; and at pH 9.5, AsO4⁠ 3⁠ – but not H3⁠ AsO4⁠ is present. Perhaps
H3⁠ AsO4⁠ is more easily taken up than is AsO4⁠ 3⁠ –, so As uptake is faster in
moss at pH 6.5 than at pH 9.5 (Fig. 2).
Arsenic uptake occurs in three ways in the aquatic macrophyte W.
fluitans: 1) active uptake via phosphate transporters, 2) passive uptake
F
via aquaglyceroporins, and 3) physiochemical adsorption by the plant
surface [32]. The physiochemical forms of arsenate differ at pH 6.5 and
9.5 (Fig. 1) and different forms may vary in uptake efficiency. The arse-
OO
nate form at pH 9.5 is therefore less available to plants than the forms
at pH 6.5.
According to Marin et al. [33], As uptake is higher at low pH in rice,
being up to eight times higher at pH 5.5 than at pH 7.5. However, there
is a relationship between pH and redox potential, and redox potential
has a major impact on As uptake, low redox potential being associated
with up to three times higher uptake.
In the present work, the highest removal of As was found under low
PR
oxygenation in arsenate solution (Fig. 6), while oxygenation had no sig-
nificant effect on the concentration increase of As in the plant biomass
(Fig. 7). Low oxygen may chemically transform arsenate into arsenite,
which is more quickly removed from the solution (Figs. 2 and 4). Ac-
cumulated arsenate can be reduced to arsenite, which can be stored in-
side plant cell vacuoles [27]. However, this does not explain why arse-
nate is removed more quickly than arsenite under low oxygenation. In
addition, plants contained more arsenate than arsenite, independent of
treatment (Table 2), which may be due to that plants adjust the internal
arsenite: arsenate ratio to a certain level. The oxygenation of water in
the natural environment is between 2 and 13 O2⁠ mg L− ⁠ 1, probably more
closed to 13 O2⁠ mg L− ⁠ 1 used in our investigation. At this oxygenation
level, the predominant As form is arsenate. Decreasing the oxygenation
of the water under natural conditions will result in more optimal As re-
moval by the studied plant.
Like pH, temperature did not influence arsenite removal, though
it did influence arsenate removal (Fig. 4). Room temperature, 20 °C,
tended to have the highest and most stable removal time curve com-
pared with the lower and higher temperatures tested (Fig. 4). In arsen-
ate, the most stable removal curve was seen at 20 °C, at which no leak-
age was observed (Fig. 4). At 12 °C, removal occurred very late in the
time course and after 48 h the moss started to leak As, though this leak-
age was not significant at the low temperature. Significant leakage was
seen, however, at the highest temperature, 30 °C (Fig. 4). Since such
leakage was not found in the case of arsenite, it was likely not caused by
the high temperature alone; rather, a synergistic stress reaction of the
membranes, caused by a combination of arsenate and warm tempera-
ture, might account for the leakage [34].
While the As removal time course differed between As species (Fig.
4), there was no difference in As accumulation in plants grown in ar-
senite and arsenate solutions at the various temperatures tested (Fig.
5). This means that it was the initial removal of As that differed; after-
wards, in this case after 72 h of acclimation in the As-containing water,
the plant As removal was about the same independent of temperature
and the As species used (Figs. 4 and 5).
In conclusion, this investigation has found that W. fluitans takes up
As under various environmental and climatic conditions. [13] suggested
that each plant species has a specific set of conditions considered to con-
stitute its “sweet spot” for As accumulation from the environment. From
the present work, we can conclude that the natural conditions of this
plant, i.e., 20 °C and pH 6.5, give its optimal conditions for As uptake.
Although the natural W. fluitans system is stream water, decreasing the
oxygenation of the water under natural conditions will result in even
better As removal by this plant.
A. Sandhi et al.
Acknowledgements
This work was funded by the Environment Foundation of the
Swedish Engineers’ Association, the Knut and Alice Wallenberg Founda-
tions and C. F. Lundström Foundation. Mr Ingvar Granberg and Mr Leif
Granberg are greatly acknowledged for their help with plant material
collection.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.jece.2018.05.044.
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