Environmental Technology & Innovation 24 (2021) 101928

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Environmental Technology & Innovation

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

Phytoremediation of partially treated MSW leachate by

selected free floating and emergent macrophytes in
subsurface vertical flow constructed wetlands
∗
Perera, K.R.S a , Mangala Yatawara b ,
a
Postgraduate Institute of Science, University of Peradeniya, Peradeniya, 20 400, Sri Lanka
b
Department of Zoology and Environmental Management, Faculty of Science, University of Kelaniya, Kelaniya, 11 600, Sri Lanka

article info a b s t r a c t

Article history: Phytoremediation of partially treated municipal solid waste (MSW) leachate with se-
Received 14 June 2021 lected free floating (Eichhornia crassipes and Pistia stratiotes) and emergent macrophytes
Received in revised form 1 September 2021 (Typha angustifolia and Chrysopogon zizanioides) was assessed at three steps in order
Accepted 6 September 2021
to improve the quality of leachate prior to discharge to the environment. At the 1st
Available online 10 September 2021
step, growth performance of macrophytes was assessed at leachate dilutions of 0%,
Keywords: 25%, 50% and 75%. Results revealed that all leachate dilutions were detrimental to P.
Constructed wetlands stratiotes. Nevertheless, leachate greater than 50% dilutions and all leachate dilutions
Emergent macrophytes favored the growth of E. crassipes and emergent macrophytes respectively. At the 2nd
Free floating macrophytes and 3rd steps, phytoremediation with E. crassipes and emergent macrophytes at 50% and
Phytoremediation phytoremediation with emergent macrophytes at 0% leachate dilution were assessed
MSW leachate respectively. Results revealed that the percentage reductions of measured parameters
(pH, EC, turbidity, color, TSS, BOD, COD, phosphate, ammonium nitrogen, nitrate, sulfate
and color) increased in remediated leachate. Of the emergent macrophytes, C. zizanioides
significantly reduced EC, COD, TSS, nitrates, ammonium nitrogen, sulphates and color
(P<0.05: one way ANOVA) when compared with their controls and T. angustifolia. More-
over, C. zizanioides proved its higher ability in accumulating nitrogen and phosphorous
from leachate than that of T. angustifolia. Nevertheless, accumulation of nitrogen was
higher than phosphorous in shoots of both plants. Although both species performed
better in harsh conditions, C. zizanioides could be recommended as the best plant for
the remediation of partially treated MSW leachate.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction

Landfill leachate is characterized by high chemical and biological oxygen demand (COD, BOD) and often consists of
high concentrations of organic contaminants, heavy metals, toxic materials, ammonia and inorganic materials as well as
refractory compounds, such as humic substances (Chávez et al., 2019). Treatment of landfill leachate consists of biological,
physical, chemical and physico-chemical techniques which carries its own merits and demerits (Kamaruddin et al., 2015).
The most cost-effective form of treatment for high levels of BOD, COD and ammonia is intense biological oxidation that
can achieve from sequence batch reactor system (SBR). The SBR is a form of activated sludge treatment.

∗ Corresponding author.
E-mail address: mmkyat@kln.ac.lk (M. Yatawara).

https://doi.org/10.1016/j.eti.2021.101928
2352-1864/© 2021 Elsevier B.V. All rights reserved.
P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

However, unexpected significantly high leachate generations during rainy seasons in small size SBR systems may lead
discharges of partially treated leachate to the environment. This may eventually cause public health and environmental
damage. Use of additional economically feasible methods for leachate treatment is therefore necessary in order to avoid
such discharges of partially treated leachate to the environment.
Phytoremediation using constructed wetlands (CWs) is an environmentally friendly low-cost method practiced in many
areas of the world for the removal of pollutants in leachate. Over the last few decades, CW system has been recognized as
an appropriate and practical alternative for landfill leachate treatment, making it safe to discharge into the surrounding
environment (Ogata et al., 2015; Justin et al., 2010). Although these systems are classified as free water surface system
(FWS) and subsurface flow system (SSFS) based on the flowing of leachate, SSFS is highly recommended for landfill
leachate treatment since it may act as fixed-film bioreactors (Lavrova and Koumanova, 2010). A FWS has an exposed water
surface and oxygen is prevalently introduced into the wetland via photosynthesis of algae. Many free floating macrophyte
such as Pistia stratiotes, Eichhornia crassipes and Salvinia natans species are recommended for FWSs.
In addition, SSFS is of two types as horizontal and vertical. Although the geometrical configuration and physical layout
of the vertical flow (VF) systems is very similar to the horizontal flow system (HF) (Gorgoglione and Torretta, 2018), the
main difference between two systems is how the wastewater flows through the inert medium. In horizontal flow system
(HF), the leachate is fed into the inlet and continues its way under the surface of the bed in a horizontal path until it reaches
the outlet zone (Fig. 1a). This creates a predominantly anoxic environment, rich in aerobic micro-sites in close proximity
to plant roots, which operate as oxygen transfer systems from the atmosphere to the inside of the filter bed. In addition,
creation of alternating aerobic, anoxic, and anaerobic zones in this system would allow the development of several families
of specific microorganisms for a better degradation of pollutants. Nevertheless, the limited oxygen availability in some
zones decreases nitrification rates (Gorgoglione and Torretta, 2018); thus, careful calculation of hydraulics is necessary
for optimal O2 -supply. On the contrary, in the vertical flow (VF) systems, landfill leachate is fed on the whole surface
area through distribution system and passes the filter in a vertical path (Fig. 1b). Therefore, vertical flow systems can
achieve high outcomes in eliminating organic material and to improve the nitrification since they have a much higher O2
transmission ability than the horizontal flow systems from the beginning (Herath and Vithanage, 2015). Although reed
species are ideal for VF systems, Kumar and Singh (2019) stated that different floating macrophytes such as Eichhornia
crassipes, Hydrilla verticillata, and Salvinia natans could be used to treat the municipal wastewater in a vertical flow CW.
The present study was conducted to assess the potential of selected commonly found free floating (Eichhornia crassipes
and Pistia stratiotes) and emergent macrophytes (Typha angustifolia and Chrysopogon zizanioides) in improving the quality
of partially treated MSW landfill leachate discharged from a SBR system using VF constructed wetland system. Since the
landfill leachate used in the present study consists of leachate from a compost plant and non-hazardous materials, it was
assumed that the leachate is free of heavy metals. Frequent monitoring also confirmed the absence of heavy metals in
MSW leachate selected for the present study.

2. Material and methods

The study was carried out at three steps.

2.1. Step 1 - Growth performance of selected plants at different leachate concentrations

Young plants of E. crassipes, P. stratiotes, C. zizanioides and T. angustifolia collected from unpolluted water bodies were
acclimatized for 07 days in de-chlorinated tap in a pre-arranged set-up. Plant growths were studied in a dilution series
of leachate (0%, 25%, 50% and 75%) using partially treated leachate discharged from the SBR system and aged tap water.
For this, batch type wetlands of 20 L capacity of diluted leachate were used. Acclimatized plant species (fresh weight:
180.0 g) were introduced and exposed for 28 days in above dilutions. The experiment was triplicated. Wet weights of
plants were obtained at the end of 28th day and the relative growth rates of plants in respective dilution were calculated
following Eq. (1) as described by (Hunt, 1978).
ln w2 − ln w1
Relative growth rate (RGR) = (1)
t2 − t1
where W1 and W2 are initial fresh weight and final fresh weight, respectively, and (t2 –t1 ) is the duration of the experiment
in days.
Based on the results of the step 1, step 2 was arranged. E. crassipes showed positive growth at 50% and 75% leachate
dilutions. Therefore, leachate quality improvement was assessed at 50% dilution considering the amount of water used
for dilution. P. stratiotes was not used at step 2 since it showed negative growth at all dilutions.

2.2. Step 2 – Phytoremediation with E. crassipes, C. zizanioides and T. angustifolia at 50% dilution

Wetlands were constructed as VF system (Fig. 1b) in plastic barrels of 248 L capacity. The hydraulic conductivity (K)
is a key factor that is closely correlated with the efficiency of landfill leachate treatment. According to Duggan (2005)
sandy and gravelly soils possess high K values, whereas clay soils have low K values. Hence, the materials for wetlands
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Fig. 1. Subsurface flow constructed wetland (a) Horizontal (b) Vertical.

consisted of gravel, sand and soil (laterite soil) and the other assembling materials for the preparation of filter media in
wetland models were procured from local market. Filter media in each wetland model, consisted of two layers starting
from the bottom layer of 25 cm height with gravel of 20–30 mm size; top layer of 30 cm height with sand of 2–3 mm
size and a soil mixture.
Gravel and sand were pre-washed with de-chlorinated tap water to remove dust and debris to avoid blockage of
the drainage outlet. Each layer of the filter media was prepared by adding calculated quantity of each material using a
graduated stick for uniform distribution in barrels. Each wetland consisted of a polyvinyl chloride (PVC) inlet pipe at its
top and an outlet drain pipe at the bottom. A plastic tank having the capacity of 1000 L were used as a major reservoir
to store and distribute leachate (50% and 0% dilution).
Young plants of E. crassipes, C. zizanioides and T. angustifolia (fresh weight: 800 g) collected from unpolluted water
bodies were acclimatized for 07 days in de-chlorinated tap in a pre-arranged set-up. The subsurface vertical flow CWs
for (i) E. crassipes (T50 1 ) (ii) T. angustifolia (T50 2 ) and C. zizanioides (T50 3 ) at 50% leachate dilutions were arranged. Control
(without plants) wetlands (C50 ) were also arranged. All the CWs were triplicated. After planting, each CW was fed with
fresh water in 8 mL/min flow rate for one week and then adjusted to 27.7 mL/min with partially treated leachate to satisfy
the required hydraulic retention time (5 days).
Leachate samples from the reservoir and outlet pipes of the wetlands were collected once in five days for thirty-
five days respectively into sterile labeled 1 L polyethylene terephthalate (PET) bottles. Samples were analyzed for
pH, electrical conductivity (EC), turbidity, biochemical oxygen demand (BOD), chemical oxygen demand (COD), total
suspended solids (TSS), phosphate (PO4 3− ), ammonium nitrogen (NH4 + -N), nitrate (NO3 − ), sulfate (SO4 2− ), and color
according to the Standard Methods for Water and Wastewater Examination (APHA, 1999). The quality of remediated
leachate was compared with national standards.

2.3. Step 3 – Phytoremediation with C. zizanioides and T. angustifolia at 0% dilution

The procedure done at 2.2 was repeated with T. angustifolia (T0 1 ) and C. zizanioides (T0 2 ) and Control (without plants)
wetlands (C0 ) at 0% leachate dilution (Fig. 2).
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Fig. 2. Experimental set up of (T0 1), (T0 2) and (C0 ).

The percentage reductions of each parameter were calculated as described by Sanchez et al. (2018).
Nutrient (Nitrogen and phosphorous) accumulation in macrophytes was carried out at the beginning and at the end of
the experiment (after 40 days) in each wetland. Plants were separated into roots and shoots. Separated roots and shoots
parts were thoroughly washed with water. Then, these samples were washed with 0.2% detergent solution to remove
the waxy coating of leaf surface. Finally, they were washed with 0.1 M HCl followed by thorough washing with distilled
water. The samples were soaked to dry with tissue paper. The samples were air dried on a perfectly clean surface at room
temperature for 3 days in a dust-free atmosphere. The plant samples were oven dried at 70 ◦ C for 48 h. The samples
were ground in an electric stainless-steel mill. The cup and blades of the grinding mill were cleaned before each sample.
Then, the samples were put in an oven, and dried for constant weight. Nitrogen (NH3 -N and organic N) and phosphorous
analyses of shoots and roots were carried out in accordance with Motsara and Roy (2008).
Determination of nitrogen (NH3 -N and organic N)
The dried plant sample (0.5 g) was weighed to the nearest 0.001 g and transferred into a digestion tube of Kjeldatherm
Digestion System. Copper (ll) sulfate pentahydrate (0.45 g) and Potassium sulfate (4.5 g) were then added to the digestion
apparatus. After adding sulfuric acid (50% diluted, 3.0 mL) the mixture was digested. It was maintained at 400 ± 2 ◦ C for
2 h and cooled.
Boric acid (25.0 mL) and Methyl red–bromocresol green mixture solution (0.1 mL) were added to the Erlenmeyer
flask. Both the digestion tube and Erlenmeyer flask were placed in Gerhardt Kjeldahl Distilling apparatus Vapodest 20
and distillation was accomplished. The distillation conditions are summarized in Table 1.
The distillate was titrated against the sulfuric acid (0.25 mol/dm3 ) till the color change to light red. The total nitrogen
content present by mass of the sample was calculated using Eq. (2).
Vs × C × 28014
%TN = (2)
m
where,
% TN = Total Kjeldahl nitrogen content present by mass (dry weight basis)
C = the concentration of standard sulfuric acid
Vs = the volume in milliliters of standardized sulfuric acid solutions used for the sample
m = the mass in grams of the sample used
Table 1
Distillation conditions.
Distillation conditions
Distillation time 240 s
NaOH concentration 35%
NaOH 7 s

Determination of phosphorous
The dried plant sample (0.5 g) was heated in a muffle furnace at 550 ± 25 ◦ C for 4 h. Ash was transferred in to a flask.
Conc. HNO3 acid (5.0 mL) and distilled water (5.0 mL) were added to the flask. The contents were mixed by swirling.
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Table 2
Relative growth rates of E. crassipes and P. stratiotes {Data shows the mean ± standard
error of the mean (SEM) in each leachate dilutions. Different superscript letters in each
column denote significant differences (P < 0.05) indicated by Tukey’s pairwise significant
difference test}.
Leachate dilution Relative growth rate (RGR) (gg−1 d−1 ) of
plant species
E. crassipes P. stratiotes
75% 3.45 × 10−3 ± 6.95× 10−4a −0.46 ± 0.06a
50% 1.44 × 10−3 ± 2.15× 10−4a −1.14 ± 0.10b
25% −2.68 × 10−2 ± 7.36 × 10−4b −1.03 ± 0.05b
0% −4.68 × 10−2 ± 1.14× 10−3c −1.52 ± 0.11c

The flask was placed on a hotplate and boiled for 5 min. Distilled water (5 mL) was added and boiling was continued for
another 10 min. The contents were cooled and filtered through Whatman No. 1 filter paper. The volume was made up
with the distilled water in 25 mL volumetric flask. Each batch of samples for digestion was contained one reagent blank.
The digested plant sample (5.0 mL) was put in a 50.0 mL volumetric flask, and vanadomolybdaye reagent (10.0 mL)
was added. The volume was made up with distilled water. It was shaken thoroughly and kept for 30 min. The absorbance
for the developed yellow color was read at 420 nm on spectrophotometer. The respective phosphorus content was then
obtained from the phosphorus standard curve. The standard curve was prepared by measuring the absorbances for series
of known standard phosphate solutions.
The total phosphorus content of the sample taken for the test was calculated using Eq. (3).
y × 250
%TP = (3)
m
where,
% TP = Total Phosphorus content present in mg/kg (dry weight basis)
y = Phosphorus concentration (µg/mL) from graph
m = the mass in grams of the sample used
Percentage accumulation of nitrogen and phosphorus in each species was calculated using Eq. (4).
(Cf − Ci)
Percentage accumulation (%) = × 100% (4)
Ci
where, Cf = concentration of nutrient level after 40 days and Ci = concentrations of nutrients at initial.
Translocation factor for nutrient accumulation was calculated by Eq. (5) (Singh et al., 2010).

Concentration of nutrient accumulated in shoot

Translocation factor = (5)
Concentration of nutrient accumulated in roots
Data were analyzed using MINITAB (version 17) and presented as the mean ± standard error (SEM). The means
were compared statistically using One-Way Analysis of Variance (ANOVA) followed by Tukey’s Test after the Arc sign
transformation when analyzing emergent plants and controls. The Results were considered significant at p > 0.05. In
addition, two sample t-test at 95% confidence interval was applied as appropriate.

3. Results

3.1. Growth performance of selected plants at different leachate concentrations

E. crassipes plants in 25% and 0% leachate dilutions, showed signs of stress such as wilting (loss of rigidity), altering
color and necrosis (the death of most or all of the cells in an organ or tissue) and showed negative growth rates. E.
crassipes plants in 75% and 50% leachate dilutions appeared healthy (Fig. 3) and showed the highest growth rate at the
75% leachate dilution (Table 2). There was no significant difference between the growth rate of E. crassipes plants treated
with 75% and 50% leachate dilutions (P > 0.05, one way ANOVA). Nevertheless, negative growth rates were observed in
wetlands contained P. stratiotes for all leachate dilutions (Table 2). Chlorosis (loss of the normal green coloration) and
necrosis were observed on leaves. Roots were lost and darkened (Fig. 3).
On the contrary, T. angustifolia and C. zizanioides plants showed positive growth rates in all the dilutions of leachate.
No significant morphological degradation was observed in each leachate dilution (Fig. 4). Both plants showed the highest
growth rate at 75% leachate dilution, followed by 50% leachate dilution and the lowest growth rate at 0% leachate dilution
(Table 3). Similarly, relative growth rates decreased with increased concentration of leachate. There was no significant
difference between the growth rate of T. angustifolia and C. zizanioides plants treated with 75% and 50% leachate dilutions
(P > 0.05, one way ANOVA). Also, there was no significant difference between the growth rate of T. angustifolia plants
and C. zizanioides treated with 25% and 0% leachate dilutions (P > 0.05, one way ANOVA).
5
P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Fig. 3. Growth of E. crassipes (above) and P. stratiotes (below) plants at different Leachate dilutions after 28 days (a - 75% dilution, b - 50% dilution,
c - 25% dilution and d - 0% dilution).. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)

3.2. Phytoremediation with E. crassipes, C. zizanioides and T. angustifolia at 50% dilution in sub-surface vertical flow CWs

All remediated leachate quality parameters analyzed for the present study showed more or less similar variation at
each week (results not shown). The pooled percentage reductions of EC, turbidity, BOD, COD, TSS, phosphates, nitrates,
6
P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Table 3
Relative growth rates of T. angustifolia and C. zizanioides {Data shows the mean ± standard
error of the mean (SEM) in each leachate dilutions. Different superscript letters in each
column denote significant differences (P < 0.05) indicated by Tukey’s pairwise significant
difference test}.
Leachate dilution Relative growth rate (RGR) (gg−1 d−1 ) of
plant species
T. angustifolia C. zizanioides
75% 2.20 × 10−3 ± 1.45 × 10−4a 3.10 × 10−3 ± 1.02 × 10−4a
50% 1.70 × 10−3 ± 1.68 × 10−4ab 2.63 × 10−3 ± 6.5 × 10−5b
25% 1.24 × 10−3 ± 1.33 × 10−4bc 1.63 × 10−3 ± 1.27 × 10−4c
0% 7.64 × 10−4 ± 7.01 × 10−5c 1.03 × 10−3 ± 9.5 × 10−5d

Table 4
The pooled percentage reductions of parameters by T50 1 and Controls (C50 ). Different
superscript letters in each row denote significant differences (P < 0.05) indicated by
Student’s t-test at 95% confidence level.
Parameter Units T50 1 (E. crassipes) Control (C50 )
EC µS/cm 42.76 ± 0.77a 37.18 ± 0.69b
Turbidity NTU 89.76 ± 0.28a 88.41 ± 0.34a
BOD mg/L 91.54 ± 0.45a 90.00 ± 0.20a
COD mg/L 88.60 ± 0.34a 87.06 ± 0.32a
TSS mg/L 58.16 ± 2.16a 53.21 ± 2.07b
PO4 3 mg/L 98.27 ± 0.14a 97.67 ± 0.13a
NO3 − mg/L 62.76 ± 0.87a 55.81 ± 1.40b
NH4 + -N mg/L 76.04 ± 0.62a 70.51 ± 1.56b
SO4 2− mg/L 68.21 ± 1.14a 59.70 ± 1.64b
Color Platinum Cobalt Units 74.13 ± 0.76a 60.13 ± 3.74b

Table 5
The pooled percentage reductions of parameters by T50 2 , T50 3 and Controls (C50 ). Different superscript
letters in each row denote significant differences (P < 0.05) indicated by Tukey’s pairwise significant
difference test.
Parameter Units T50 2 (T. angustifolia) T50 3 (C. zizanioides) Control (C50 )
EC µS/cm 44.63 ± 1.89b 47.92 ± 0.98a 37.18 ± 0.69c
Turbidity NTU 90.12 ± 0.19a 91.25 ± 0.28a 88.41 ± 0.34c
BOD mg/L 92.46 ± 0.21a 93.78 ± 0.32a 90.00 ± 0.20b
COD mg/L 89.40 ± 0.33b 91.26 ± 0.35a 87.06 ± 0.32c
TSS mg/L 59.45 ± 2.24b 67.92 ± 2.06a 53.21 ± 2.07c
PO4 3 mg/L 98.37 ± 0.14a 99.01 ± 0.10a 97.67 ± 0.13b
NO3 − mg/L 63.48 ± 1.19b 67.85 ± 1.08a 55.81 ± 1.40c
NH4 + -N mg/L 77.07 ± 0.71b 81.15 ± 0.78a 70.51 ± 1.56c
SO4 2− mg/L 70.12 ± 1.08b 75.17 ± 0.84a 59.70 ± 1.64c
Color Platinum Cobalt Units 92.44 ± 0.28b 93.76 ± 0.21a 89.62 ± 0.31b

ammonium nitrogen, sulphates and color of partially treated leachate by (i) T50 1 (E. crassipes) and Controls (C50 ), and (ii)
T50 2 (T. angustifolia), T50 3 (C. zizanioides) and Controls (C50 ) are shown in Tables 4 and 5 respectively. The pooled percentage
reductions of almost all parameters with respect to controls, increased in all plant species. Among the wetlands, T50 3 that
contained C. zizanioides showed the highest percentage reductions of all parameters and the percentage reductions of EC,
COD, TSS, nitrates, ammonium nitrogen, sulphates and color by T50 3 were significantly higher than those of the Controls
and T50 1 (P < 0.05: one way ANOVA) (Table 4). Nevertheless, there were no significant differences in pH of the remediated
samples in each treatment and Controls (P > 0.05, one way ANOVA).
Color of leachate samples at the beginning and at the end of the experiment showed significant variations. Partially
treated leachate was turbid and black in color (Fig. 5) with unpleasant odor. Nevertheless, leachate remediated by T.
angustifolia and C. zizanioides were more or less colorless. But pale brown color was observed in leachate remediated
by E. crassipes. On the contrary, color of leachate in controls showed slightly brown color. There was no any odor in the
treated leachate coming out from the plant treatments and Controls. In addition, all the selected water quality parameters
of remediated samples complied with the tolerance limits of general standards for the discharge of industrial effluent into
inland surface waters in schedule 1 of the National Environmental (Protection and Quality) Regulation No 1 of the gazette
No: 1534/18 dated 01.02.2008.
Significant accumulations of nitrogen and phosphorus were observed in shoots than those in roots of both plants
(P < 0.05: Student’s t-test). Among the wetlands, T50 3 (C. zizanioides) performed better in up taking of both nitrogen
and phosphorus in shoots. The translocation factor of nitrogen and phosphorous in T50 3 were 3.7 and 2.47 respectively
(Table 6).
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Fig. 4. Growth of T. angustifolia and C. zizanioides at different leachate dilutions after 28 days (a - 75% dilution, b - 50% dilution, c - 25% dilution
and d - 0% dilution).

3.3. Phytoremediation with C. zizanioides and T. angustifolia at 0% dilution in sub surface vertical flow CWs

The pooled percentage reductions of EC, turbidity, BOD, COD, TSS, phosphates, nitrates, ammonium nitrogen, sulphates
and color by T0 1, T0 2 and Controls (C0 ) are shown in Table 7. The percentage reductions of almost all parameters increased
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Fig. 5. Color of the initial and remediated samples by each plant at 35th day {a - 50% diluted leachate, b - T50 1 (E. Crassipes), c - T50 2 (T. angustifolia),
d - T50 3 (C. zizanioides) and e - C50 (Control)}.. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

Table 6
Percentage accumulation (%) of nitrogen and phosphorous* (mean ± SEM) in shoots and
roots of plant species at 50% dilution. Different superscript letters in each row denote
significant differences (P < 0.05) indicated by Student’s t-test at 95% confidence level.
Treatment type Shoots Roots Translocation factor
T50 1 (E. crassipes) 58.93 ± 1.79a 30.00 ± 0.05b 3.50
46.83 ± 0.39a∗ 37.72 ± 0.62b∗ 2.22*
T50 2 (T. angustifolia) 60.71 ± 1.79a 33.33 ± 0.08b 3.60
48.13 ± 1.32a∗ 39.50 ± 1.65b∗ 2.10*
T50 3 (C. zizanioides) 64.44 ± 2.22a 33.33 ± 0.00b 3.70
51.44 ± 1.13a∗ 41.88 ± 3.42b∗ 2.47*

Table 7
The pooled percentage reductions of parameters by T0 1 , T0 2 and Controls (C0 ). Different superscript
letters in each row denote significant differences (P < 0.05) indicated by Tukey’s pairwise significant
difference test.
Parameter Units T0 1 (T. angustifolia) T0 2 (C. zizanioides) Control (C0 )
EC µS/cm 41.53 ± 0.52 b
47.96 ± 0.55a
35.86 ± 0.95c
Turbidity NTU 90.30 ± 0.19a 91.76 ± 0.16a 88.77 ± 0.18b
BOD mg/L 92.36 ± 0.15a 93.60 ± 0.12a 90.21 ± 0.20b
COD mg/L 88.48 ± 0.23b 90.55 ± 0.26a 86.30 ± 0.30c
TSS mg/L 59.96 ± 1.72b 65.10 ± 1.15a 54.13 ± 1.70c
PO4 3 mg/L 98.34 ± 0.15a 98.82 ± 0.93a 97.62 ± 0.11b
NO3 − mg/L 63.97 ± 1.11b 67.42 ± 1.12a 54.69 ± 0.97c
NH4 + -N mg/L 77.52 ± 0.55b 81.60 ± 0.51a 69.08 ± 1.20c
SO4 2− mg/L 69.41 ± 1.05b 74.13 ± 0.76a 60.13 ± 3.74c
Color Platinum Cobalt Units 92.13 ± 0.15b 93.71 ± 0.18a 89.38 ± 0.14c

by both C. zizanioides and T. angustifolia plant species when compared with Controls as observed at 3.2 above. Of the
wetlands, T0 2 that contained C. zizanioides showed the highest percentage reductions of all parameters and the percentage
reductions of EC, COD, TSS, nitrates, ammonium nitrogen, sulphates and color were significantly higher than that of the
Controls and T0 1 (P < 0.05: one way ANOVA).
Color of the leachate remediated by T0 2 were clear and colorless. Slightly brown color was seen in treated leachate
by T0 1 and brown color was seen in Controls (Fig. 6). There was no any odor in the treated leachate coming out from
the plant treatments and Controls. All the selected water quality parameters of remediated samples complied with the
tolerance limits of general standards for the discharge of industrial effluent into inland surface waters in schedule 1 of
the National Environmental (Protection and Quality) Regulation No 1 of the gazette No: 1534/18 dated 01.02.2008.
Significant accumulations of nitrogen and phosphorus were observed in shoots than those in roots of both plants (P
< 0.05: Student’s t-test). (Table 8). Of the wetlands, T0 2 (C. zizanioides) performed better in up taking of both nitrogen
and phosphorus in shoots. The translocation factor of nitrogen and phosphorous in T0 2 were 3.15 and 2.37 respectively
(Table 8).
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

Fig. 6. Color of the initial and remediated samples by each plant at 35th day {a - 0% diluted leachate, b - T0 1 (T. angustifolia), c - T0 2 (C. zizanioides)
and d - C0 (Control)}. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 8
Percentage accumulation (%) of nitrogen and phosphorous* (mean ± SEM) in shoots and
roots of plant species at 0% dilution. Different superscript letters in each row denote
significant differences (P < 0.05) indicated by Student’s t-test at 95% confidence level.
Treatment type Shoots Roots Translocation factor
T0 1 (T. angustifolia) 60.71 ± 3.09a 31.11 ± 0.17b 2.66
48.28 ± 0.63a∗ 39.41 ± 2.33b∗ 2.11*
T0 2 (C. zizanioides) 62.22 ± 3.85a 33.33 ± 0.08b 3.15
50.97 ± 0.90a∗ 41.28 ± 3.66b∗ 2.47*

4. Discussion

The present study assessed the phytoremediation potential of selected free floating and emergent macrophytes in
sub-surface vertical flow constructed wetlands after confirming their growth at different leachate concentrations. Among
the floating macrophytes, E. crassipes showed its highest growth rate at its optimum dilution of the leachate. Nevertheless,
negative growths of E. crassipes were observed at 25% and 0% leachate dilutions indicating that 25% diluted leachate and
raw leachate are detrimental to E. crassipes. In addition, P. stratiotes showed negative growth rates in all the dilutions
of leachate. Sooknah and Wilkie (2004) stated that higher COD levels are unfavorable for the growth of P. stratiotes. The
higher COD levels of the leachate (360.67–541.19 mg/L) in the present study may be a reason for the negative growth of
P. stratiotes.
Both emergent macrophytes, T. angustifolia and C. zizanioides showed positive growth rates in all leachate dilutions.
Both plants showed the highest growth rate at its maximum dilution (75%) and the lowest growth rate at 0% leachate
dilution. Since the study continued to assess the potential of E. crassipes in improving the leachate quality at 50% leachate
dilution, the same dilution was also selected for the emergent macrophyte species, C. zizanioides and T. angustifolia.
All remediated leachate quality parameters analyzed for the present study showed more or less similar variation at
each week (results not shown). Considering overall results, with respect to controls, percentage reductions of almost all
parameters including EC, turbidity, BOD, COD, TSS, PO4 3− , NO3 − , NH4 + -N, SO4 2− and color increased in wetlands having
either E. crassipes or selected emergent macrophytes. The percentage reduction values of most of the parameters in these
wetlands having plants were significantly higher than those of controls (P < 0.05: Student’s t-test or Tukey’s pair wise
tests). This could be attributed to the collective impact of microbial degradation of pollutants and plant metabolism in
CWs.
Considering percentage reductions of parameter in CWs, Hench et al. (2003) stated that EC reduces because of
evapotranspiration and or movement of substrate by plant roots. With respect to controls, a high EC reduction was
observed in wetlands having plants and reducing abilities varied with plant species. Among different parameters of
leachate, turbidity denotes to particles present within the water column. In general, a portion of incoming carbon
compounds is dissolved within the leachate, whereas the remainder occurs in the form of particulate matter. The solvable
carbon compounds are eliminated by microorganism growth on the medium surfaces and connected to the plant roots
and rhizomes penetrating the substrate (Kadlec, 1995). In constructed wetlands having plants, microbial growth occurs
in plant roots as well as in medium surfaces. Therefore, turbidity reduction in wetlands having plants was comparatively
high.
BOD is a quantifiable measurement of the quantity of oxygen consumed by microbes to oxidize of carbon-based matter.
The decrease in BOD may be due to the consumption of organic matter by the microorganisms in the surrounding area
of plant roots for their growth and multiplication. Mazumder (2013) stated that the organisms involved are principally
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

bacteria, however additionally include fungi and protozoa. Percentage reduction of BOD raised in planted systems, as a
result of organic substance degradation and also the activity of photosynthesis of plants that create adequate O2 supply for
microbes, rhizosphere to decompose leachate more effectively. This also causes the growth of aerobic bacteria to degrade
the carbon-based complexes, thereby reducing the COD level. Due to microbial development on the medium surfaces of
the substrate in control wetlands, some organic composites are also removed (Kadlec, 1995). O2 necessity is achieved by
O2 transference through the air water edge, or attained from oxygen-containing compounds such as phosphates, nitrates
and sulfates. This may be the other reason for the reduction of COD in the Controls.
Total suspended solids are particles greater than 2 µm occur in the water path. TSS are removed due to physical
methods such as accumulation, sedimentation and interception (Manios et al., 2003). Solids are deposited by the medium
principally through filtration, which occur in the matrix of the medium. Physical processes occur in controls as well as
wetlands having macrophytes. In wetlands consisting of macrophytes, when suspended solids go through the plant roots,
these particles can retain, aggregate and ultimately settle down due to the power of gravity or could be decomposed by
microbes, meanwhile particulate matter settle down to the bottom (Valipour et al., 2015). In controls, due to microbial
growth on the medium surfaces of the bed, suspended particles may have been metabolized by microbes to show reduced
TSS.
Different forms of N such as nitric oxide (NO), NO3 − , and NH3 are highly soluble in water. Nevertheless, it is well-
established that numerous removal processes including nitrification, denitrification, uptake by plants and microorganisms,
NH3 volatilization, and adsorption onto cation exchange are involved in the removal of N in CW (Yang et al., 2001).
In addition, phosphorus is one of the supreme essential elements that exist in wastewater/leachate predominantly as
phosphate, normally so-called orthophosphate. Due to physical–chemical partition, also as biotic conversion, precipitation,
accretion and biomass storage the elimination of phosphate in constructed wetlands can be accomplished (Mazumder,
2013). Similarly, the significant decrease in phosphate concentration could be due to the mineralization of organic
phosphorus to further constant phosphorus compound such as di-phosphate and triple phosphate because of oxidation
procedure. Phosphorus could also be precipitated with inorganic substances such as aluminum, iron and calcium found
in the root bed substrate. Nevertheless, plants absorb inorganic phosphate (PO4 3− ) from their growth medium (Akinbile
et al., 2012). Reduction of PO4 3− in controls might have resulted from absorption by the algae exist or microbes that were
present near the gravel surface.
In general, the variation of redox condition at root structures, together the accessibility of organic carbon, O2 and SO4 2−
reducing microorganisms principally regulate SO4 2− in the leachate. In wetlands consisting plants, detected advance
decreases of SO4 2− might be contributed to the occurrence of suitable root regions for biotic assimilation and sulphates
precipitation procedure. Nevertheless, the minor reduction of SO4 2− detected in controls may be due to the mineral
precipitation.
Color could be detected as an initial indication for harmful condition in water. Because of the occurrence of colored
organic materials, presence of iron, and pollution of the water basis with contaminants, elevated rate of color might be
identified in the raw leachate. These compounds are eliminated by microorganism growth on the medium surfaces and
connected to the plant roots and rhizomes penetrating the substrate (Kadlec, 1995). Therefore, color reduction in wetlands
having plants was relatively high.
Considering nutrient accumulation in plants, within 35 days of study period, all plant species accumulated more than
88% of N from leachate. Nevertheless, both T. angustifolia and C. zizanioides performed better in nitrogen accumulation
at 50% dilution than 0% dilution. From plant assimilation and nitrification/denitrification processes, the effectiveness of
nitrogen removal may be described sufficiently. Mechanism of protein synthesis, electron conveyance in photosynthesis
phenomena and respiration procedures are possibly occurred with association of accumulated nitrogen. Macrophyte roots
are mostly participated in the assimilation process (Gupta et al., 2012). Once macrophytes exposed to nutrient-high
effluent, these organisms incline to gather additional nutrients than are required for development (Valipour et al., 2015).
The translocation factor for N was higher than 2.5 in all macrophyte species. This indicates that, nitrogen is accumulated
highly in shoots than roots. The decrease of nitrogen in roots occurred, because of the upward movement to assist the
high development rate of new sprouts (Sharma et al., 2006).
Within 35 days of study period, C. zizanioides performed better in phosphorus accumulation followed by T. angustifolia
at both 0% and 50% dilutions. The efficiency of phosphorus elimination can be described sufficiently from plant assimilation
and sorption, biomass storage, precipitation and accretion. Percy and Truong (2003) stated that, C. zizanioides had a speed
and extremely large ability for uptake of nutrients, predominantly phosphorus in leachate. The translocation factor for
P was higher than one but lower than 2.5 in all macrophyte species. N: P ratio in wastewater also affects the uptake of
nutrients in macrophytes. Reddy et al. (1990) found that the top values of N and P uptake were occurred while the N: P
ratio in the wastewater is around 2.3 and 5.
As a whole, it seems that plant roots can significantly influence the reduction of pollutants in MSW leachate. Although
E. crassipes is a free-floating species, it was able to reduce a significant level of pollutants in a subsurface vertical flow
constructed wetland system. According to Global Invasive Species Database (2011), as much as 50% of a single water
hyacinth’s biomass can be roots and these roots are adventitious and fibrous, 10–300 cm in length. In addition, as many
as 70 lateral roots per cm give the roots a feathery appearance. Therefore, such root morphology can be attributed for the
significant reduction of selected parameters even in sub-surface VF system. On the other hand, T. angustifolia are able to
thrive in water depths of 10–75 cm. The plant develops with use of rhizomes and requests a huge amount of sunlight to
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P. K.R.S and M. Yatawara Environmental Technology & Innovation 24 (2021) 101928

withstand. T. angustifolia usually uptakes huge levels of nutrients from the soil and aggregating those in upright herbal
sections by way of biomass. Nevertheless, high attraction of C. zizanioides for both organic and mineral chemicals because
of its high root growth showed that it might be utilized to create a low price and eco-friendly purification for wastewater
(Xia et al., 2000). The roots of C. zizanioides are fibrous and reported to reach depths up to 3 m thus being able to stabilize
the soil (Mickovski et al., 2005). Seroja et al. (2018) stated that C. zizanioides is a relatively strong resistance plant and
has high survival rate. Also, Percy and Truong (2003) stated that, C. zizanioides consists of a very huge-water utilization
and nutrient absorbing rates, and the plant’s ability to grow in harsh environments, it is highly matched to remediate
leachate.

5. Conclusions and recommendations

The experimental results revealed that E. crassipes was successful for the remediation of 50% diluted leachate even
in sub-surface VF constructed wetlands. The selected emergent macrophytes, C. zizanioides and T. angustifolia have the
ability to grow in raw leachate and to enhance the quality of leachate (EC, turbidity, BOD, COD, TSS, phosphate, nitrate,
ammonium nitrogen, sulfate and color). The remediated leachate parameters complied with National Environmental
(Protection and Quality) Regulation No 1 of the gazette No: 1534/18 dated 01.02.2008. Nevertheless, C. zizanioides showed
the highest performance in improving leachate quality than T. angustifolia. In addition, nitrogen accumulation was higher
than that of phosphorous in shoots of these plants. Therefore, among the plant species selected for the present study,
C. zizanioides could be recommended as the best plant for the remediation of partially treated MSW leachate using sub
surface vertical flow constructed wetlands.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Acknowledgment

The authors gratefully thank the Central Environmental Authority (CEA)-Sri Lanka and the Department of Zoology
and Environmental Management, Faculty of Science, University of Kelaniya-Sri Lanka respectively for the experimental
supports.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors.

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