Bioresource Technology 99 (2008) 84368440

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Bioresource Technology
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Arsenic removal from waters by bioremediation with the aquatic plants Water
Hyacinth (Eichhornia crassipes) and Lesser Duckweed (Lemna minor)
Sandra Alvarado a, Magdiel Gudez a, Marc P. Lu-Mer b,*, Graterol Nelson a,b, Anzalone Alvaro b,
Arroyo C. Jess c, Zray Gyula d,e
a

Universidad Nacional Experimental Politcnica Antonio Jos de Sucre Vice-rectorado de Barquisimeto, Dpto. de Ingeniera Qumica, Barquisimeto, Edo. Lara, Venezuela
Universidad Centro Occidental Lisandro Alvarado, Decanato de Agronoma, Dpto. Qumica y Suelos, Ncleo Tarabana, Cabudare, Edo. Lara, Venezuela
c
Universidad Centro Occidental Lisandro Alvarado, Decanato de Ingeniera Civil, Dpto. de Construccin, Barquisimeto, Edo. Lara, Venezuela
d
Department of Chemical Technology and Environmental Chemistry, University Eotvoes Lrand, Budapest, Hungary
e
Hungarian Satellite Centre of Trace Elements Institute to UNESCO, Budapest, Hungary
b

a r t i c l e

i n f o

Article history:
Received 13 July 2007
Received in revised form 20 February 2008
Accepted 21 February 2008
Available online 28 April 2008
Keywords:
Bioremediation
Arsenic
Water Hyacinth (Eichhornia crassipes)
Lesser Duckweed (Lemna minor)

a b s t r a c t
In this study the removal of arsenic by the Water Hyacinth (Eichhornia crassipes) and Lesser Duckweed
(Lemna minor) was monitored under a concentration of 0.15 mg L1 of the element. Plant densities were
1 kg/m2 for Lesser Duckweed and 4 kg/m2 for Water Hyacinth on a wet basis. The arsenic was determined
in foliar tissue and water samples by hydride generation atomic absorption spectroscopy. The element
was monitored as a function of time during 21 days. No signicant differences were found in the bioaccumulation capability of both species. The removal rate for L. minor was 140 mg As/ha d with a removal
recovery of 5%. The Water Hyacinth had a removal rate of 600 mg As/ha d and a removal recovery of 18%,
under the conditions of the assay. The removal efciency of Water Hyacinth was higher due to the biomass production and the more favorable climatic conditions. This specie represents a reliable alternative
for arsenic bioremediation in waters.
2008 Elsevier Ltd. All rights reserved.

1. Introduction
The arsenic is one of the most toxic elements that could be found
in waters (Tseng et al., 2002; Centeno et al., 2002; Katsoyiannis and
Zouboulis, 2004; Jain and Ali, 2000); it is considered as carcinogenic
(USEPA, 2003; Hughes, 2002) in the A group by The United States
Environmental Protection Agency. The presence of the element over
the maximum allowed limit of 0.05 mg/L is affecting several countries (USEPA, 2003; Das et al., 2004). The arsenic could be found in
water as the result of the dissolution of minerals from volcanic or
sedimentary rocks as well from the dilution of geothermal waters
(Schmger et al., 2000). This element is also employed in the manufacture of lasers, semiconductors, in the glass industry, pharmaceutical products, and pigments among other uses (Watts, 1997).
In the agriculture the element is used as herbicide and plaguicide
in the form of the compounds sodium methyl arsenate, disodium
methyl arsenate and dimethyl arsenic acid. It could be considered
that the main reason of the water contamination by arsenic is the
agriculture and industrial efuents discharge (Schmger et al.,
2000; Watts, 1997; Pickering et al., 2000).
* Corresponding author.
E-mail address: mparra@ucla.edu.ve (M.P. Lu-Mer).
0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.02.051

The systems for water treatments to reduce the arsenic content

in waters are complex. Some methods are the adsorptioncoprecipitation using aluminum and iron salts (Song et al., 2006),
adsorption onto activated alumina (Lin and Wu, 2001), activated
coal or bauxite (Daus et al., 2004), inverse osmosis, ionic interchange (Kim and Benjamin, 2004), nanoltration, among others
(Pena et al., 2005; Maity et al., 2005). These methods have high
costs and difcult implementation and maintenance. It is necessary to evaluate non-expensive simple effective and efcient systems for contaminated water remediation.
Some aquatic plants have a high capability to accumulate heavy
elements or toxic ones by different mechanisms, and then allow for
the purication of high contaminated waters, due to industrial or
agrochemical discharges (Maine et al., 2001; Chua, 1998; So
et al., 2003). The species Water Hyacinth Eichhornia crassipes and
Lesser Duckweed Lemna minor were previously used for decontamination or reduction of contaminant levels in water.
Kiran et al. (1991) tested four aquatic plants (E. crassipes, Pistia
stratiotes, Salvinia rotundifolia and L. minor) and evaluated the removal of nitrogen and phosphorus. They observed that E. crassipes
has the highest capacity for nitrogen extraction during summer
and rainy season, while the phosphorus was more efciently extracted during summer with the order: Eichhornia, Pistia, and Lemna

S. Alvarado et al. / Bioresource Technology 99 (2008) 84368440

and Salvinia. During winter L. minor has the best removal efciency
for phosphorus. The removal capacity of the plants rose with the
increment of the nitrogen and phosphorus in the water. Removal
of N as nitrate was between 42% and 96.2%, and for phosphate was
between 36.3% and 70.2%. The high potential of these aquatic plants
for the removal of N and P, reduction of BOD, QOD, is demonstrated.
De Souza et al. (1999) studied the phytoaccumulation of trace elements Cd(II), Cr(VI), Cu(II), Ni(II), Se(VI) with the specie (E. crassipes)
under hydroponic conditions and concentrations ranging from 0.1 to
10 mg/L in a period of 14 days. For a concentration level of 0.10 mg/L
the bioaccumulation capabilities were: 1.85 mg Cd/kg d; 1.15 mg
Cu/kg d; 0.53 mg Cr/kg d; 0.58 mg Se/kg d and 1.04 mg Ni/kg d. Shaban et al. (2005) demonstrated that the non-living roots of the Water
Hyacinth can rapidly remove arsenic from water.
In this work the Water Hyacinth and Lesser Duckweed are evaluated and compared for the removal rate of arsenic from water.
The performance of these two species for arsenic removal is not
well documented in the literature. The arsenic concentration was
determined as a time function in water and foliar tissue under controlled conditions at the level of contamination of 0.15 mg/L of the
element and in control groups. The agronomical behavior was also
evaluated by means of dry weight percentage, growth rate and
plant density to determine the reliability of the species for the phytoremediation of waters contaminated with the element.
2. Experimental
2.1. Assay location
The experiments were carry out in a greenhouse at the Estacin
Experimental Miguel Luna Lugo of the Decanato de Agronoma,
Universidad Centroccidental Lisandro Alvarado, Tarabana,
Municipio Palavecino, Edo. Lara, Venezuela. The area is characterized as a very dry tropical forest, with a height of 510 m.o.s.l, with
a mean of 658.3 mm of rain, potential evapotranspiration of
2048.1 mm per year, mean temperature of 25.1 C, a mean photoperiod of 7.9 h and relative humidity of 70%. The latitude is
1010 2500 N and the longitude of 69170 W.
2.2. Experimental design
The experimental design was random with ve treatments and
ve repetitions: two experimental groups, two control groups and
an additional control of water with arsenic, without plants. The
treatments are described in Table 1. The experimental units were
identical plastic vessels MANAPLAST of 20 L capacity and water
lamina of 46.5 cm, surface area 0.7854 m2, surface area of water
lamina 0.6793, deep of 13.3 cm, with plants and treatments (as
shown in Table 1).
2.3. Plants
Young plants of Water Hyacinth (E. crassipes) and Lesser Duckweed (L. minor), collected in eld, were taken in big plastic bags to

Table 1
Treatments used for the experiment
Treatment

Description

T1
T2
T3

Water with out As, Eichhornia crassipes plants (1000 g of fresh weight)
Control: water without As, Lemna minor plants (670 g of fresh weight)
Water with arsenic (0.15 mg/L), Lemna minor plants (670 g of fresh
weight)
Control: water with arsenic (0.15 mg/L), Eichhornia crassipes plants
(1000 g of fresh weight)
Control: water with arsenic (0.15 mg/L), without plants

T4
T5

8437

reduce the stress. The plants of the two species were then carefully
selected and reproduced under the assay conditions to ensure the
adaptation. During the adaptation period the associated species as
algae and other aquatic plants were eliminated. A commercial
nutrient solution was added to the vessels. After the adaptation
period the plants were again selected for the assay according to
the criteria of leaves number and size and taken to the vessels.
2.4. Experimental design
The vessels were randomly placed in the greenhouse to avoid
the preferential effect of temperature gradient or air ow in the
groups that could allow to different evaporation rates. To each vessel was added the nutrient solution (0.4 g/L of fertilizer SOLUB in
a volume of 20 L) with a stock composition of SOLUB as follows:
8.0% NNH3, 10.0% NNO3, P2O5 18%, K2O 18%, MgO 1%, S 1%, B
0.01%, CuEDTA 0.019%, FeEDTA 0.04%, MnEDTA 0.05%, Mo
0.001%, ZnEDTA 0.019%, carbonate free, potassium sources free
of Cl and Na. The solution was prepared with tap water. The water
for nutrient solution preparation was characterized for the physicochemical parameters hardness, alkalinity, acidity, total solids,
sedimentable solids and pH, in order to verify the quality. The
water volume was kept constant during the whole experiment.
The water sampling procedure was performed always after water
reposition. The arsenic level in the water of experimental groups
as well in the control group of water with arsenic without plants
was 0.15 mg/L (three times the maximum allowed for water level).
The arsenic solution was prepared from a stock 1000 mg/L solution
Tritisol (Merck, Darmstadt, Germany). The plant mass, as shown in
Table 1 was 670 g of fresh weight for Lesser Duckweed and 1000 g
for Water Hyacinth. The plant densities were 1 kg/m2 for Lesser
Duckweed and 4 kg/m2 for Water Hyacinth in wet basis.
2.5. Sampling procedure
2.5.1. Water samples
The water samples were taken as function of time, with plastic
syringes, after the water reposition, to plastic 100 ml closed vessels. The rst milliliters of sample were used for vessel washing.
The water samples were taken with a 20 ml syringe from different
deeps and points of the vessel. The samples were collected interdaily for a period of 21 days. They were preserved at pH less than 2,
following the methodology described in section 1060 C of the Standard Methods for the Examination for Water and Wastewater (Eaton, 1995).
2.5.2. Foliar tissue
The foliar tissue samples were collected taking including all
plant parts (root, leaves, steam in the case of Water Hyacinth,
and the whole plant in the case of Lesser Duckweed). Three samplings were performed: at the beginning of the experiment, at
the 15th day and at the 21th day. Samples were stored in sealed
plastic bags at 20 C.
2.5.3. Arsenic determination
The total arsenic was determined in water and foliar tissue samples by hydride generation ame atomic absorption spectrometry
(HG-FAAS), in a Perkin Elmer Spectrometer 3110, Waltham, Massachusetts, USA, with a hydride generation module with quartz cell
and a single hollow cathode lamp.
2.5.4. Analysis of water samples
Subsamples of 0.1 ml or 0.2 ml were aphorized to 10 ml with
HCl 0.5 M. The analysis was carry out according to the method
3114C of the Standard Methods for Water and Wastewater Examination (Eaton, 1995), recommended by USEPA (United States

8438

S. Alvarado et al. / Bioresource Technology 99 (2008) 84368440

Environmental Protection Agency) and the AWA (American Water

Association) for the arsenic and selenium determination (USEPA,
2003).
2.5.5. Analysis of plant tissue
Subsamples of 0.5 or 1.5 g of dry tissue were digested using the
method 3030F of the Standard Methods for Water and Wastewater
Examination (Eaton, 1995) with a mixture of HCl/HNO3; nal digested sample volume was 60 ml. The analysis by HG-FAAS was
then performed in a similar way as for the water samples. The
accuracy of the arsenic determination in the plant tissue was veried with the NIST SRM 1573 tomato leaves. The standard reference material was digested and analyzed in the same fashion as
the
foliar
samples.
A
concentration
value
of
0.30 lg g1 0.04 lg g1 was found, a 10% of relative deviation respect to the certied value (0.27 lg g1 0.05 lg g1).
2.6. Agronomical variables
The variables fresh and dry weight were determined at the
beginning of the experiment at the day 14th and at the end of
the experiment (day 21st). Samples of the two species were taken,
weighed and dried in oven at 70 C for 48 h. After the drying procedure the samples were weighed again to determine the percent
of dry weight and the relationship between fresh and dry weight,
the estimated total dry weight in each vessel, total biomass, plant
density in dry basis and growth rate.

3.2. Arsenic removal

The arsenic masses in the tissue and water for the Lesser Duckweed and Water Hyacinth are shown in Figs. 1 and 2. The minimal
amount of the element in water for both species is found at the
14th day of the experiment and it is in agreement with the maximum concentration in tissue for both species. After the 14th day
there was a release of the element to water, as consequence of tissue death in both species. A higher accumulation in the tissue of
the Water Hyacinth was observed (see Figs. 1 and 2).
The mass of arsenic removed and the removal rate for the Lesser
Duckweed were low with respect to the initial arsenic level. This
means that the plants should be collected during a longer period
in order to remove the contaminant, to achieve a long residence
time and the management of higher biomasses and residues. The
arsenic absorbed mass and removal rate of the Water Hyacinth
suggest that this plant has a better capability for the removal of
the element during the rst 14 days. The species must be harvested
every 15th days in order to avoid the release of the arsenic to the
water.
Regarding to the bioaccumulation per kilogram of planta higher
value for the Lesser Duckweed was observed, due to the plant characteristics as size and weight. The difference is not signicant at a
condence level p = 0.05. Despite this fact, the higher biomass production of the Water Hyacinths leads to a signicantly higher removal percentage and removal rate of the arsenic from water
(see Table 2). The environmental conditions were more adequate

2.7. Statistical analysis

mg of As

The statistical analysis of the results was performed with the

program STATISTIX 8 (Analytical Software, 2003). The normality
of the analyzed variables (As in water, As in tissue, in treatments,
dry weight %, biomass production and other parameters) was
tested. For variables with normality in error (Wilk Shapiro test),
variance homogenity (Barletts test) a parametric test (ANOVA)
was applied. For those variables that do not accomplish these criteria, the non-parametric test of KruskasWalis was applied.

3. Results
3.1. Plant density and growth rate

0
0

10

15

20

25

Day
Fig. 1. Milligrams of As in water () and foliar tissue (j) as function of time (Lemna
minor). N = 5 for each point.

3.5
3
mg of Arsenic

Plant density (dry basis) for Lesser Duckweed was reduced during the experiment, independently of the treatment due to the climatic conditions of the experiment which also negatively
inuenced the growth rate. A more pronounced decrement was observed in the treatment control (without As). In wet basis non-signicant differences were found between treatments. This could be
the consequence of a major humidity percentage in plants of the
control treatment. Respect to the Water Hyacinth, it was observed
an increment of plant density in dry basis between the rst and
14th days and a decrement between the 15th and 21st days. This
could be a consequence of the reproduction of the plants during
the rst period and the reduction of the available area and nutrients in the vessels during the second period.
Density on a wet basis it was observed as an increment during
the entire experimental period. No signicant differences were
found in the behavior of plants in treatment with As and without
As. The presence of the contaminant had no inuence in the density. The growth rate for Water Hyacinth was positive during the
rst period (day 1 to 14th) and negative during the second period.
In this last period a competence for the area and nutrients was
higher. The presence of the element does not inuence the agronomical behavior of the Water Hyacinth plants.

2.5
2
1.5
1
0.5
0
0

10

12

14

16

18

20

22

24

Day
Fig. 2. Milligrams of As in water () and foliar tissue (j) as function of time (Eichhornia crassipes). N = 5 for each point.

S. Alvarado et al. / Bioresource Technology 99 (2008) 84368440

Table 2
Bioaccumulation, removal rate and removal % for Water Hyacinth and Lesser
Duckweed

Water Hyacinth
Lesser Duckweed

mg of As/kg n = 5

mg of As/ha d n = 5

Removal % n = 5

1.8 0.5
2.5 0.6

600 140
140 30

18 5
51

for the Water Hyacinth as deduced from the agronomical behavior

and in agreement to the optimal climatic condition. Water Hyacinth can grow up at temperatures till 37 C, while the Lesser
Duckweed was more appropriate for lower than 25 C
temperatures.
The absorption of arsenic by both species is fast till the 14th day
of the assay (see Fig. 3). The reduction of the arsenic mass in the
water without plants during the whole period, specially around
the 14th day and further, is more similar to the reduction in the
case of the Water Hyacinth. This could be explained by the chemical properties of the As(III) solution, that was added to the water
and by the fact of the negative growth rate, the death of the entire
green structure of the Lesser Duckweed with the faster release of
the arsenic to the water. The As(III) or arsenite is slowly oxidized
to As(V) and precipitates (Fox and Doner, 2003; Melndez and Solsona, 2006). The arsenate absorption is related to the phosphate
transport, being a competition between these two chemical species. The arsenite is absorbed in a lesser amount and by a different
mechanism (Mkandawire and Dudel, 2005; Abedin et al., 2002;
Schmger et al., 2000; Santos et al., 2006). The high afnity of
the absorption process for phosphate and arsenate explains the tolerance to the arsenic in some plants as could be Holcus lanatus
(Meharg and Mac Nair, 1992). If considering that the phosphorous
absorption mechanism is highly developed in oating plants, it

3.5

8439

could be explained that the high absorption of the phosphorous

from the natural aquatic systems due to the lack of availability of
this element. In consequence, the high absorption of the arsenic
by both species during the initial phase is due in part to the necessity for the absorption of the phosphate from water. This high
absorption of phosphorous from water has been demonstrated by
Meerhoff et al. (2002) for E. crassipes and by Reddy and De Busk
(1985) for both species. It could be realized that the absorption
by both species is similar during the rst 14 days of the assays,
but it is important to notice that the species did not have a similar
grown up. While E. crassipes had a positive growth rate during the
same period, this parameter was negative for L. minor. L. minor is
supposed to have a higher arsenic absorption efciency. Another
difference is the fact that E. crassipes exhibits in its life cycle, a
gradual senescence of the tissues and necrosis of the older leaves
at rst. The end of the life cycle of the Lesser Duckweed implies
the death of the whole green structure. There is a high release of
the arsenic from the dead material to water since there is not possibility of the re-absorption due to the negative growth rate of the
specie during the assay and the absence of new green biomass. This
situation indicates the necesity of further studies about the dynamic of the arsenic related to the growth cycles of bioaccumulators. The understanding of the relationships allows the
determination of the real possibility of using these plants in the
bioremediation of waters.
4. Conclusions
No signicant differences were found in the bioaccumulation
capability of both species or mg of As/kg of plant. Nevertheless
the removal rate for L. minor was lower (140 mg As/ha d) with a removal recovery of 5%. The Water Hyacinth had a removal rate of
600 mg As/ha d and a removal recovery of 18%, under the conditions of the assay. The removal efciency of Water Hyacinth was
higher due to the biomass production. The climatic conditions of
the experiment area was more adequate for the Water Hyacinth
and inuence the removal capability of the species. The arsenic
contamination at the level of 0.15 mg/L did not inuence the development of the species. The Water Hyacinth represents a reliable
alternative for arsenic bioremediation in waters under the climatic
conditions of very dry tropical forest in Tarabana, Edo. Lara,
Venezuela.
Acknowledgements

mg of Arsenic

2.5

To the FONACIT, Fondo Nacional de Ciencia y Tecnologa, Repblica Bolivariana de Venezuela and to the Hungarian Academy of
Sciences, since this work was performed in the frame of the International project between these two institutions. To the CDCHT
UCLA, Consejo de Desarrollo Cientco Humanstico y Tecnolgico
de la Universidad Centro Occidental Lisandro Alvarado, for the
nancial support to the research by the projects AG 012 2002
and IC 005 2006.

1.5

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0.5

0
0

10

15

20

25

Day
Fig. 3. Milligrams of As in water for Water Hyacinth (), Lesser Duckweed (j) and
water without plants (N) as function of time. N = 5 for each point.

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