phytoaccumulation of heavy metals from municipal...
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Phytoaccumulation of Heavy Metals from Municipal Solid Waste
Leachate Using Constructed Wetland
By
Ammara Batool
Institute of Environmental Sciences and Engineering (IESE)
School of Civil and Environmental Engineering (SCEE)
National University of Sciences and Technology (NUST)
Islamabad, Pakistan
2018
Dedicated to My Daughter
Aman
ii
Phytoaccumulation of Heavy Metals from Municipal Solid Waste
Leachate Using Constructed Wetland
By
Ammara Batool
(NUST201290035PSCEE1812F)
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Environmental Science
Institute of Environmental Sciences and Engineering (IESE)
School of Civil and Environmental Engineering (SCEE)
National University of Sciences and Technology (NUST)
Islamabad, Pakistan
2018
iii
Acknowledgements
My all praises and gratitude are for Almighty Allah Who is Most gracious and merciful to give me strength to walk through thick and thin of my life and to undertake this research task and enabling me to its completion. After that, I would like to express my sincere gratitude to my advisor Dr. Zeshan for the continuous support of my Ph.D study and related research, for his patience, and motivation. His guidance helped me in the time of research and writing of this thesis.
Besides my advisor, I would like to thank the guidance and examination committee members: Dr. Muhammad Arshad, Dr Sher Jamal Khan, and Dr. Syed Ali Musstajab, for their insightful comments and encouragement, but also for the hard questions which incented me to widen my research from various perspectives.
My sincere thanks also goes to Dr. Imran Hashmi, Associate Dean who provided me support to avail accommodation at university. Without his support it would not be possible to complete my research work.
Laboratory staff of the Institute have been very kind enough to extend their help at various phases of this research, whenever I approached them, and I do hereby acknowledge all of them. I thank Mr Basharat and Mr Mamoon for their cooperation in sample analysis at laboratories of Institute of Environmental Sciences and Engineering. I specially, thank my friends their constant support and encouragement during grey hours.
My gratefulness to Higher Education Commission of Pakistan to award me fully sponsored PhD fellowship which allowed me to undertake this research.
I am very much indebted to my parents and siblings, who encouraged and helped me at every stage of my personal and academic life, and longed to see this achievement come true. At last but surely not least love of my life my daughter Aman, who geared me up to face all obstacles and to continue my work. My work is dedicated to you My Aman, you are pride and joy of my life.
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Table of Contents
Abstract xvii Chapter 1 Introduction 1 1.1 Background 1 1.2 Objectives of the Study 4 1.3 Scope of the Study 4 Chapter 2 Literature Review 6 2.1 Reasons for leachate treatment 6 2.2 Leachate Composition 6 2.2.1 Organic Loading 7 2.2.2 Metals 7 2.3 Leachate Biodegradation 7 2.3.1 Aerobic Degradation and Hydrolysis – Phase I 7 2.3.2 Fermentation and Hydrolysis – Phase II 8 2.3.3 Acetogenesis – Phase III 8 2.3.4 Methanogenesis – Phase IV 8 2.3.5 Oxidation 8 2.4 Leachate Treatment Methods 9 2.5 Types of Constructed Wetlands 13 2.5.1 Free Water Surface System 14 2.5.2 Subsurface Flow System 15 2.5.3 Horizontal Flow system 15 2.5.4 Vertical Flow system 16 2.5.5 Hybrid Systems 16 2.6 Mechanism of pollutant removal in Constructed Wetland 16 2.6.1 Suspended Solids 18 2.6.2 Organic Matter 18 2.6.3 Inorganic Matter 19 2.7 Role of Plants in constructed wetland 20 2.8 Role of substrates in constructed wetland 27 2.9 Operational Parameters of constructed wetland 30 2.10 Research Need for the Dissertation 33 Chapter 3 Materials and Methods 36 3.1 Growth behavior comparison of different wetland species in 36
x
mesocosm constructed wetland 3.2 Effect of substrates on phytoremediation of trace metals from solid
waste leachate 37
3.3 Effect of chelators and substrates on phytoremediation of trace metals from synthetic leachate
38
3.4 Kinetic study for removal of metals and COD by substrates and plants
40
3.5 Pilot Scale Constructed Wetland Experiment 42 3.5.1 Site Selection 42 3.5.2 Design and construction of pilot scale constructed wetland 43 3.5.3 Filling of wetland 43 3.5.4 Plantation in pilot scale constructed wetland 43 3.5.5 Leachate collection, simulation and application 44 3.6 Operational conditions of pilot scale constructed wetland 44 3.6.1 Batch mode 44 3.6.2 Continuous mode 44 3.6.3 Details of chambers (Plants and substrates) 47 3.6.4 Sampling and analysis 48 3.6.4.1 Metal and COD analysis 49 3.6.5 Quality control and quality assurance 50 3.6.6 Metal accumulation factors 51 3.6.7 Statistical analysis 53 Chapter 4 Results and Discussion 54
4.1 Growth behavior comparison of three species exposed to municipal solid waste leachate in microcosm constructed wetland
54
4.2 Effect of substrate on phytoremediation of trace metals from solid waste leachate.
57
4.3 Effect of chelators and substrates on phytoremediation of trace metals from synthetic leachate
69
4.4 Kinetics study of metal and COD removal by substrates and plants 76 Summary of Phase I – Laboratory scale experiments 85 4.5 Pilot scale vertical flow constructed wetland 86 4.5.1 Removal of Cu, Zn, Pb and COD in pilot scale constructed wetland at
HRT of 21 and 14 days in batch mode 86
4.5.2 Removal of Cu, Zn, Pb and COD in pilot scale constructed wetland at HRT of 35 and 5 days in continuous mode
93
xi
4.5.3 Accumulation of Cu, Zn and Pb in plants and substrates 100 4.5.4 Translocation and bioaccumulation of metals in plants of different
chambers of pilot scale vertical flow constructed wetland 104
Summary Phase II – Pilot scale experiment 110 Chapter 5 Conclusions 113 Recommendations 114 References 116
xii
List of Figures
Figure 2.1 Different processes in engineered landfill site(Garcia et al., 2006)
9
Figure 2.2 Different types of constructed wetlands based on direction of flow
14
Figure 2.3 Types of constructed wetland A: Free waster surface, B: Horizontal, C: Vertical and D: Hybrid constructed wetlands
17
Figure 2.4 Interrelated factors in constructed wetland
20
Figure 2.5 Hyperaccumulators of Cu, Zn and Pb; A: P. australis, B: T. latifolia, C: V. zizanioides, D: C. gyana, E: E. globulus and F: C. indica
25
Figure 2.6 Different factors supported by substrates in constructed wetlands
29
Figure 3.1 Design and experimental conditions of experiment; growth behavior comparison of wetland species in mesocosm constructed wetland
37
Figure 3.2 Design and experimental conditions of experiment; effect of substrate on phytoremediation of trace metal from solid waste leachate
38
Figure 3.3 Design and experimental conditions of experiment; effect of chelators and substrates on phytoremediation of trace metals from synthetic leachate
40
Figure 3.4 Kinetic removal study of Cu and Zn from plants, substrates and combination of plants and substrates
42
Figure 3.5 Batch mode of operation in chamber A, B, C and D of pilot scale constructed wetland
45
Figure 3.6 Continuous mode of operation in chamber A, B, C, D and E in pilot scale constructed wetland
46
Figure 3.8 Substrates used in constructed wetland; A:crushed brick, B:steel slag, C: sand
49
Figure 3.9 Materials used in pilot scale constructed wetland; A: Guaze; B: Pipes; C: Valves
49
Figure 4.1 Initial and final height of T. latifolia, P. australis and V. zizanioides in different leachate concentrations
55
Figure 4.2 Chlorophyll level in T. latifolia, P. australis and V. zizanioides
55
Figure 4.3 Accumulation of Cu in roots and shoots of a)P. australis, b)T. latifolia and c) V.zizanioides
56
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Figure 4.4 Percentage removal of trace metals in leachate planted with a) P. australis b) T. latifolia
60
Figure 4.5 Trace metal accumulation in shoot, root and substrates a) P. australis
62
Figure 4.6 Translocation of trace metals by plants in substrates a) P. australis
63
Figure 4.7 Biotransferrable factor of trace metals by plants in different substrates
68
Figure 4.8 Percentage removal of Cu and Zn a) Adsorbents b) Chelators (T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b: Typha CA)
70
Figure 4.9 Accumulation of a) Cu and b) Zn in shoots, roots and substrates (T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick)
71
Figure 4.10 Shoots and roots accumulation of a) Cu and b) Zn in treatment system with chelators (EDTA, CA) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b: Typha CA)
72
Figure 4.11 Translocation factor in treatment systems with a) adsorbents and b) chelators(T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b: Typha CA)
74
Figure 4.12 Metal Tolerance Index of T.latifolia substrates and chelators(T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b: Typha CA)
76
Figure 4.13 Percentage removal of Cu by P. australis and T. latifolia
79
Figure 4.14 Percentage removal of Cu by different substrates
79
Figure 4.15 Percentage removal of Cu by P. australis and T. latifolia in different substrates
80
Figure 4.16 Percentage removal of Zn by P. australis and T. latifolia
81
Figure 4.17 Percentage removal of Zn by substrates
81
Figure 4.18 Percentage removal of Zn by P. australis and T. latifolia in different substrates
81
Figure 4.19 COD removal by plants 82
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Figure 4.20 COD removal by substrates
83
Figure 4.21 COD removal by P. australis and T. latifolia in different substrates
84
Figure 4.22 Removal of a) Cu, b) Zn and c) Pb at HRT of 21 days in Pilot scale Constructed Wetland in Batch mode
89
Figure 4.23 Removal of COD in chamber A and B at HRT of 21 days in Batch mode
90
Figure 4.24 Removal of a) Cu, b) Zn and c) Pb at HRT of 14 days in Pilot scale Constructed Wetland in Batch mode
91
Figure 4.25 Removal of COD at HRT of 14 days in Pilot scale Constructed Wetland in Batch mode
92
Figure 4.26 Removal of a) Cu, b) Zn and c) Pb with HRT of 35 days in Continuous mode in Pilot scale Constructed Wetland
94
Figure 4.27 Percentage removal of COD at HRT of 35 day in Continuous mode in Pilot scale Constructed Wetland
95
Figure 4.28 Percentage removal of Cu, Zn and Pb at HRT of 5 days in Pilot scale Constructed Wetland in Continuous mode
96
Figure 4.29 Loading rate of COD in Pilot Scale Constructed Wetland at HRT of 5 days in Continuous mode
98
Figure 4.30 Accumulation of a) Cu, b) Zn and c) Pb in shoots and roots of plants in chamber A, B, C, D and E of constructed wetland
105
Figure 4.31 Accumulation of a) Cu, b) Zn and c) Pb in substrates of chambers A, B, C, D and E.
106
Figure 4.32 Translocation of Cu, Zn and Pb in plants in different chambers of pilot scale constructed wetland
108
Figure 4.33 Bioconcentration of Cu, Zn and Pb in different plant species. 108
xv
List of Tables
Table 2.1 Leachate composition in acidic and methanogenic phase 10 Table 2.2 Studies of different advance leachate treatment methods 12 Table 2.3 Metal removal studies in constructed wetlands 22 Table 2.4 Metal accumulation by different plant species in constructed wetland 26 Table 2.5 Safe limits for irrigation, toxic concentration and hyperaccumulation
capacity 28
Table 2.6 Studies of different substrates in constructed wetlands 31 Table 2.7 Operational parameters affecting the performance of constructed wetland 33 Table 3.1 Leachate characteristics, retention time and sampling interval of
experiment 4; kinetic study of removal of metals and COD by substrates and plants
41
Table 3.2 Specifications of pilot scale constructed wetland 47 Table 3.3 Material used for construction 48 Table 3.4 Substrates used in pilot scale constructed wetland 48 Table 3.5 Composition of leachate used in pilot scale constructed wetland 48 Table 3.6 Loading rate of Cu in pilot scale constructed wetland 51 Table 3.7 Loading rate of Zn and Pb in pilot scale constructed wetland 52 Table 4.1 Porosity, density and SEM images of crushed brick and steel slag 61 Table 4.2 Leachate composition and Pakistan Environment Protection Department
guidelines 61
Table 4.3 Wet and Dry weight of T. latifolia and P. australis 64 Table 4.4 Shoot and root length of T. latifolia and P. australis 64 Table 4.5 Pseudo second order kinetic model for removal of Cu in different
treatments 78
Table 4.6 Pseudo second order kinetic model for removal of Zn in different treatment
78
Table 4.7 Removal of Cu at HRT of 35 and 5 days in continuous mode of operation 99 Table 4.8 Removal of a) Zn, b) Pb and c) COD at HRT of 35 and 5 days in
continuous mode of operation 101
xvi
List of Abbreviations
Al Aluminum
BCF Bioconcentration factor
BDL Below detection limit
BOD Biological oxygen demand
BR Batch reactor
C. alternifolius Cyperus alternifolius
C. dactylon Cynodon dactylon
C. esculenta Colocasia esculenta
C. gyana Chloris gyana
C. indica Canna indica
CA Citric Acid
Cd Cadmium
Co Cobalt
COD Chemical oxygen demand
Cr Chromium
Cu Copper
CW Constructed Wetland
DO Dissolved oxygen
E. globulous Eucalyptus globulus
EDTA Ethylene diamine tetra acetic acid
Fe Iron
FWS Free water surface
G. sagittatum Gynerum sagittatum
g L-1 gram per liter
g m-2 day-1 gram per meter square per day
H. psittacorum Heliconia psittacorum
xvii
HFCW Horizontal flow constructed wetland
HLR Hydraulic loading rate
HRT Hydraulic retention time
HSSF Horizontal subsurface flow
J. acutus Juncus acutus
L. monopetalum Limoniastrum monopetalum
Mg Magnesium
mg kg-1 milligram per kilogram
mg L-1 milligram per liter
mm mili meter
Mn Manganese
NH4-N Ammonium nitrogen
Ni Nickel
P. australis Phragmites australis
P. pusillus Potamogeton pusillus
Pb Lead
PO4-P Orthophosphate
S. perrenis, Sarcocornia perrenis,
SF Surface flow
T. latifolia Typha latifolia
T. parviflora Tamarix parviflora
TF Translocation factor
TN Total nitrogen
TSS Total suspended solid
V. zizanioides Vetiveria zizanioides
VFCW Vertical flow constructed wetland
VSSF Vertical subsurface flow
Zn Zinc
xviii
Abstract
Improper solid waste management is a growing issue in both developed and developing countries. One of the chronic environmental hazards of solid waste is the leachate generation. Safe disposal of leachate, generated either through engineered or un-engineered landfill sites, has become a major environmental problem. Leachate can be highly toxic in nature due to the presence of heavy metals and other toxic pollutants, therefore it needs efficient treatment before disposal. The domains of municipal solid waste leachate treatment have been unattended in developing countries like Pakistan, creating increasingly concerning situation. To that end, the aim of the present research was to study phytoaccumulation of heavy metal of leachate using potential of different hyperaccumulator species the laboratory and in constructed wetland. To enhance the efficiency of metal removal different adsorbents were used also substrates in the constructed wetland. Moreover, wetland was operated in batch and continuous flow modes at various retention times and loading rates. The experiments of this study were carried out in two phases. The first phase consisted of four lab-scale experiments whereas the second phase constituted the pilot scale investigations. The first laboratory scale experiment was carried out to investigate growth potential of plants (P. australis, T. latifolia and V. zizanioides) in different dilutions of leachate. In the following experiment, the effect of two substrates: crushed brick and steel slag on removal of Cu and Zn was investigated in a lab-scale wetland planted with P. australis and T. latifolia. The percentage removal of Zn and Cu by T. latifolia in crushed brick was 71 and 95%, respectively whereas in the case of steel slag it was 72 and 94%, respectively. P. australis in presence of crushed brick removed 78 and 99% of Zn and Cu, respectively whereas in presence of crushed brick and 73 and 80% in presence of steel slag, respectively. Comparison of percentage removal by plants P. australis and T. latifolia in substrates namely crushed brick and steel slag and chelators namely EDTA and citric acid was also investigated during third lab-scale experiment. At 15 mg L-1 dose of Cu, approximately 90% removal of Cu was observed for both chelators and both plants while at dose of 5 mg L-1, the observed removal was 99% for both the plants spiked with both chelators. Kinetics study for COD and metal removal by plants P. australis and T. latifolia, and the substrates crushed brick, steel slag and limestone as well as by their combination was conducted in fourth lab-scale experiment. The results showed that T. latifolia in presence of brick (r2 = 0.85) and slag (r2 = 0.90) removed Cu significantly. Whereas, Cu removal by P. australis was also efficient (r2 = 0.98) in presence of crushed brick. The pilot scale constructed wetland during the second phase of this study was operated in batch and continuous mode at different COD and metal (Cu, Zn and Pb) loading rates and varying HRTs (21, 14, 35 and 5 days). Wetland comprised of five chambers with an area of 2.15 m2 for each chamber. In batch mode, each chamber of multi-chambered wetland acted as discrete chamber and removal of metal and COD exceeded 90% of the applied concentration in each chamber. Besides, in the case of continuous operation mode, all chambers were inter-connected and removal of more than 90% of applied concentration was achieved in first chamber alone while only 10% of the applied concentration or lesser than that got removed in the following four chambers. The result suggests that in continuous mode, all chambers after first chamber were lightly loaded as more than 90% of applied loads were removed in first chamber.
1
Chapter 1 Introduction
1.1 Background
Municipal and industrial solid waste is commonly disposed in sanitary landfills . Total of 96%
world wide collected municipal solid waste (MSW) is disposed in landfills (Akinbile et al.,
2012). Large quantity of severely polluted leachate is generated from sanitary landfill inducing
potential hazard for the ecosystem including fauna and flora. Strength and composition of
leachate varies widely depending upon type of waste. Report of other researchers is that
concentration of metal toxicants depend on age of landfill site (EPA, 1998). The fresh leachate
produced in landfill sites and composting facilities depend on the waste composition, contains
high concentrations of metals and organic pollutants hence, it is a major environmental hazard
(Aziz et al., 2011; Kjeldsen et al., 2002). It may escape from dumpsite and percolate into ground
water aquifers which in turn may cause health threats. Metals in leachate may transform
physically and chemicaly. The heavy metals possible can cause toxicity, carcinogenecity, and
impairment to the ecosystem.
Open dumpsites have no lining and may allow percolation of leachate to groundwater aquifers.
Release of trace metals through leachate contamination in surface, ground and run off water may
affect human health. In engineered landfills lining not only prevents percolation but also routes
leachate to on site treatment units (Kadlec and Knight, 1996). In situ leachate treatment is cost
effective approach instead of ex-situ hauling. During active years and for more additional years
leachate may need treatment even after dumpsite is closed. Treatment technologies for metal
removal from leachate and wastewater treatment are not satisfactory due to economic constraints
(Trang et al., 2010). There are several options for treatment of leachate like leachate biological
treatment (Xie et al., 2014), advance oxidation biological process (Chemlal et al., 2014),
coagulation or fenton process (Moradi and Ghanbari, 2014), ozone per sulfate advanced
oxidation (Abu Amr et al., 2013) and sequencing batch biofilm reactor (Zhang et al., 2014).
Developing countries cannot afford high technologies with high capital cost. Alternatively,
natural treatment systems are suitable option for developing countries to treat leachate
(Qasaimeh et al., 2015). Natural systems are based on biological system for purification of waste
2
water without any external source of energy except solar energy. Process of purification is very
slow because of higher retention time as compared to conventional treatment systems. Natural
systems remain reliable in extreme weather and high loading rates of COD content with easy and
cheap maintenance (Bakhshoodeh et al., 2016). Natural systems include constructed wetlands
that can be designed as subsurface flow system or free water surface system or horizontal flow or
vertical flow typically consists of impermeable basin with substrates, emergent or submerged
vegetation, and low water depths. Flow direction of constructed is important for removal of
organic and inorganic pollutants (Vymazal, 2010). Vertical flow constructed wetland is
considered more effective for removal of COD and BOD as compared to horizontal flow
constructed wetland (Abou et al., 2013). Combination of two horizontal subsurface flow pilot-
scale constructed wetland and two vertical flow pilot-scale constructed wetlands were used for
enhanced removal of chromium (Vassiliki et al., 2017). Whereas efficient removal of COD,
phosphorus and ammonia was achieved in VFCW planted with (T. parviflora, J. acutus, S.
perrenis and L. monopetalum) (Fountoulakis et al., 2017). Removal of toxicity (metals and
inorganics) and chemical processing are common functions of vegetation (Vymazal, 2011).
Sustained growth of plants ensures the efficient working of wetland (Kadlec and Wallace, 2008)
and growth habits of plants are categorized by surface of water; emergent woody plant, floating
mats, floating plants and submerged aquatic plants. Common reed (P. australis), cattail (T.
latifolia), bulrush (Scirpus) are considered hyperaccumulators for Cu, Zn, Pb, Ni and Mn. Other
plant species like C. indica and V. zizanioides are also planted in wetlands for removal of toxicity
(Calheiros et al., 2007). The defining criteria hyperaccumulators of Co, Cu, Ni and Pb is 1,000
mg kg-1, whereas the threshold level of Mn and Zn is 10,000 mg kg-1. Hyperaccumulators
species subsidize the constructed wetland treatment process chiefly through the rhizosphere and
roots, resulting in improved sedimentation and filtering, prevention of clogging and flow velocity
reduction (Vymazal, 2011). The rhizosphere, provides favorable conditions for the proliferation
of microbes; accumulation in plants by utilizing metals and nutrients (Weis and Weis, 2004).
Usually, single plant species is vegetated in constructed wetlands. However, recent studies report
that the polyculture of plant species can enhance the performance of constructed wetland
(Fountoulakis et al., 2017; Papaevangelou et al., 2017; Kumari and Tripathi, 2015). For extended
performance of constructed wetland good hydraulic conductivity and adsorption capacity of
substrates must be ensured. Substrates play role of filtration, adsorption, sedimentation,
3
flocculation, precipitation and ion exchange. Hydraulic permeability and adsorption capacity are
main characteristics of substrates. They also provide foundation for plants to growth and
microbial activity. Substrates with poor infiltration capacity reduce efficiency of system.
Substrates with Al and Fe ions may help in reducing phosphate from waste water and leachate
(Ryan, 2014). COD and metal loading rate directly influence the efficiency of constructed
wetland which can be controlled by appropriate selection of substrates and plants (Korkusuz et
al., 2005). Palm tree (Melián et al., 2017), rice husk (Tee et al., 2009), palm tree mulch,
limestone coco peat mixture (Allende et al., 2014), slag and gravels (Ge et al., 2015), crushed
brick and steel slag (Batool and Zeshan, 2017) were recently used as substrates for removal of
COD and trace metals in constructed wetlands. Sorption is the main function of these substrates,
which involves transformation of ions to a solid phase from soluble phase. It may result in
variation in process of stabilization. Sorption is explained by cluster of mechanisms, including
adsorption i.e., physical mechanism with chemisorption, fragile bindings, absorption is
biochemical mechanism explaining entrance of compound in vacuoles of plants from external
source and mechanism of precipitation. Metals are adsorbed by process of exchange of ions
depending upon metal and other elements contending for active sites.
Other important parameters affecting the removal efficiency of trace metals include retention
time, loading rate and plant development (García et al., 2015). Provision of long retention time
for high loading (COD and metals) is considered vital for maximum toxicity removal (Abou et
al., 2013). Maintenance and design factors of different kinds of constructed wetland are
important for hydrology including hydraulic loading rate, hydraulic retention time, infiltrative
capacity, hydraulic conductivity, evapotranspiration (W.P.C.F, 1990). Recent studies emphasized
the importance of hydraulic retention time in constructed wetland (Solís et al., 2015; Sultana et
al., 2015; Liu et al., 2016). It has also been demonstrated that constructed wetland can tolerate
high variation in loading rates in terms of influent quality (Dan et al., 2011). Similarly, redox
potential, pH, vegetation development and sedimentation are justified by retention time in
constructed wetland. Tao et al. (2006) reported positive correlation between long retention time
and removal of tinnin, lignin and COD. At laboratory scale, researchers (Aluko and Sridhar,
2014; Xu et al., 2017; Madera-Parra et al., 2013) investigated removal of nutrients and metals in
horizontal and vertical flow constructed wetland. Whereas authors suggested that series of
4
constructed wetland cells at pilot scale with long hydraulic retention time may reduce large land
requirement. Moreover, multistage constructed wetland may improve the quality of effluent
(Auvinena et al., 2017; Vymazal and Kröpfelová, 2015).
Therefore, this research was intended to remove metals and COD efficiently from municipal
solid waste leachate in multi-chamber pilot-scale vertical flow constructed. Moreover, different
hydraulic retention time were provided to enhance performance of constructed wetland. Poly
culture of plants; P. australis, T. latifolia, V. zizanioides, C. Gayna, E. globulus and C. indica
grown in presence of crushed brick, steel slag, sand and gravels also exploited the research gap
of metal removal from municipal solid waste leachate in pilot scale vertical flow constructed
wetland.
1.2 Objectives of the Study Overall, the objectives of the present research were to evaluate the performance of laboratory
experiments and of a pilot scale constructed wetland for treating municipal solid waste leachate.
More specifically, we aimed to:
1. Investigate the different substrates and selected plant species for trace metal
accumulation.
• Growth behavior comparison of three species exposed to municipal solid waste
leachate in microcosm constructed wetland
• Effect of substrates on phytoremediation of heavy metal from MSW leachate
• Effect of chelators and substrates on phytoremediation of metals from MSW leachate
• Kinetic study of metal removal from leachate by plants and substrates
2. Operate and evaluate the performance evaluation of a pilot scale vertical flow constructed
wetland.
• Removal of metal and COD at different HRTs and loading rate in Batch mode
• Removal of metals and COD at different HRTs and loading rate in Continuous mode
1.3 Scope of the Study
With the aim to accomplish the above objectives, the scope of study is given as follows:
5
1. The growth performance of P. australis, T. latifolia and V. zizanioides in different concentration
of real municipal solid waste leachate was evaluated in labscale constructed wetland.
2. The removal of metals from municipal solid waste leachate by P. australis and T. latifolia in
presence of crushed brick and steel slag in labscale constructed wetland was investigated.
3. The effect of chelators: ethylene diamine acetic acid, citric acid and substrates; crushed brick and
steel slag for metal removal by P. australis and T. latifolia from synthetic was studied in labscale
constructed wetland
4. The removal of metals and COD from synthetic leachate with time by P. australis and T. latifolia
in presence of crushed brick, steel slag and limestone was investigated.
5. The removal of metals and COD from municipal solid waste leachate was studied in pilot scale
vertical flow constructed wetland with five chambers at various retention time in batch and
continuous mode.
Journal and Conference Publications
1. International Conference on Advances in Agricultural, Biological and Environmental Sciences
(AABES), London, United Kingdom. 22nd and 23rd July, 2015 “Growth Behavior comparison of
three wetland species in municipal solid waste leachate using constructed wetland” – Paper
published in conference proceedings
2. Comparison of chelators and substrates for phytoremediation of trace metals in synthetic
leachate. 2017. Soil and Sediment Contamination. 26 (2): 220-233
3. Effect of substrate on phytoremediation of trace metals in leachate. 2018. Toxicological and
Environmental Chemistry – Under Review
4. Bio approaches for sustainability Conference at Institute of Environmental Sciences and
Engineering, NUST, Islamabad. 22nd and 23rd Feb, 2017. Effect of substrate on phytoremediation
of trace metals in leachate - Poster Presentation
6
Chapter 2 Literature Review
Proper waste management can be a solution to different environmental hazards. Waste should be
properly collected, segregated, treated and disposed otherwise environmental health is at stake
(El-Fadel, 1997). Designed landfill play important role in managing solid waste (Fig 2.1). Open
dumping without segregation and pre treatment is a common practice in developing countries,
which causes different environmental hazards. It deteriorates environmental quality by leaching
contaminants to ground water aquifers (Kanmani, 2013). Leachate starts producing after
dumping of waste as waste decomposes (Kjeldsen et al., 2002). Strength and composition of
Leachate varies widely depending upon type of waste. Report of other researchers is that
concentration of metal toxicants depend on age of Landfill site (EPA, 1998).
2.1 Reasons for leachate treatment Leachate generated from open dump sites can be more hazardous than landfill leachate. It easily
flows to nearby ditches , drains and water courses causing environmental hazard, therefore it
needs to be controlled. It may escape from dump site and percolate in ground water aquifers
which in turn may pose health threats (HRB, 2003). High concentration of metals, COD, BOD,
hazardous organic chemicals, metals, pesticides, pharmaceutical residues; all need to be removed
before discharging it in nearby natural water system. Composition of leachate depends on type of
waste, transfer station, degree of compaction, moisture content and climate. General
characterization of leachate are high COD values, nitrogen, ammonia, strong odour, color.
Whereas characterisitics may vary by its volume, composition, putrescible organic content
making leachate treatment a great challenge (Safaa et al., 2013).
2.2 Leachate Composition Composition of leachate depends on biological processes within body of waste. Type and
amount of waste along with environmental conditions determine the composition of MSW
leachate (Ward et al., 2005). Leachates are defined as the high strength wastewater produced as a
result of percolation of rainwater through dumped wastes, different biological and chemical
mechanism in deposited waste and the intrinsic content of water present in it. Large amount of
biodegradable organic matter is present in leachates, other important constituents are heavy
7
metals, ammonia-nitrogen, chlorinated organic, phosphorous, and inorganic salts (Renou et al.,
2008). Parameters of significant importance are:
2.2.1 Organic Loading Putrescible material and organic compounds present in leachate is termed as organic loading. It
is commonly measured as chemical oxygen demand (COD), biological oxygen demand (BOD).
Significance of different treatments systems are determined by removal of COD from leachate.
Sormunen et al. (2008) found COD in the range of 642 – 8037 mg L-1 in methanogenic phase.
2.2.2 Metals Leachate is chemically aggressive in nature and contains high concentration of metals (Mg, Cu,
Pb, Fe) depending on nature of waste. Heavy metals remain insoluble in methanogenic phase and
later render to be high. Cu, Zn, Pb, Cr, nickel are found in leachate ranging from moderate to
high concentration (Mor et al., 2006). With the passage of time, the mobility of metals is
amplified because pH of waste becomes more acidic and oxidizing circumstances increase
(Kjeldsen et al., 2002). Dominating concentrations of metals; Hg, Cd, Cu, Ni, Zn, Mg and Pb
have been allied with leachates and transport ability of these metals enhance with DOC and
organic compounds; fulvic, hydrophilic and humic acid (Asadi, 2008).
2.3 Leachate Biodegradation During breakdown processes principal organic matter is formed. It is usually measured in terms
of COD or BOD. Quality of leachate varies with time as it continues to degrade inside itself.
Composition of leachate varied extensively depending mainly upon their degree of stabilization.
Different climatic conditions influence seasonal production of leachate (Table 2.1).
2.3.1 Aerobic Degradation and Hydrolysis – Phase I At initial stage oxygen is available to decompose organic waste. Aerobic microbiota which
facilitates the metabolization of waste to simple carbondioxide, water and simple hydrocarbons
by exothermic process. As a result carbonic acid is produced which lowers pH. This process
takes few days till available oxygen is exhausted. Precipitation and release of moisture by
compaction of waste generate most of leachate produced through the dumped refuse (Kjeldsen et
al., 2002). Once waste is covered, oxygen is not replenished as life of aerobic phase is only few
days (Sarubbi and Sarmiento, 2009). In this phase COD ranges from 6000- 60,000 mg L-1 with
8
pH from 4 – 7. Leachate is considered young at this stage with high organic values (Kjeldsen et
al., 2002). Whereas Aziz et al. (2010) reported COD in the range of 935 – 2345 mg L-1 in
aerobic phase.
2.3.2 Fermentation and Hydrolysis – Phase II After depletion of oxygen, activities of facultative organism are started. These microorganism
ferment putrescible materials and cellulose converting them into volatile fatty acids which rise
the level of COD with acidic level of (pH 5 – 6) with high concentration of ammonia (1000 mg
L-1) and unpleasant odor. This phase may last for decades (Kjeldsen et al., 2002).
2.3.3 Acetogenesis – Phase III Organic acids formed in phase II are converted into acetic acid, carbondioxide and hydrogen by
specific microorganisms. At the end of this stage carbondioxide and hydrogen tends to fade
away. Lower hydrogen concentration promotes generation of methane by methanogens (methane
generating microorganism) (Renou et al., 2008). Characteristics of leachate in acidic phase are
shown in Table 2.1.
2.3.4 Methanogenesis – Phase IV Main processes in this phase Pb to production of methane. Chemical processes are slow which
may take years for completion. Anaerobic conditions with depleted oxygen remain same as of
previous two phases. Methane is generated from conversion of carbondioxide and hydrogen to
methane and water. pH of leachate raise due to low levels of hydrogen. In this phase chemical
oxygen demand is far less than acidic phase ranging from 500 mg L-1 to 4500 mg L-1.
Concentration of metals increased at this stage after leaching from the collected waste thus
increasing toxicity of leachate (Kjeldsen et al., 2002).
2.3.5 Oxidation Final stage of biodegradation of waste involve oxidation processes. Processes of biodegradation
are dynamic depending on suitable conditions. Leachate of phase III has high COD level. After
transition to methane formation phase IV, level of COD decreases with increase of pH value.
Inorganic contaminant like phosphorous, ammonia and chloride are not altered. Similarly, heavy
metals also remain undegraded (Whittleton, 2004).
9
Figure 2.1 Different processes in engineered landfill site (Garcia et al., 2006)
Piles of solid waste either openly dumped or in engineering landfills (Fig 2.1) are collected over
years. One-time deposited waste is later covered by more waste extending it over wide areas. In
developing countries waste is dumped without segregation which affects the characteristics of
leachate in each phase. Working with samples of 20-year-old refuse excavated from a landfill,
they showed that the concentrations of Zn, Cd, Cr, S roughly doubled when the refuse was
decomposed under aerobic conditions relative to anaerobic conditions in reactors. They
suggested that aeration of the decomposed refuse resulted in the production of chelating agents
that enhance metals mobility (Kjeldsen et al., 2002). They showed an increase in the fraction of
Cu, Zn, Pb, Cr, Ni, and Cd present as carbonates and sulfides in anoxic refuse relative to aerobic
refuse. This observation confirms the widely held expectation that metals are largely
immobilized by precipitation under anaerobic conditions (Renou et al., 2008). Prudent et al.
(1996) reported on the concentrations of metals in 14 components of MSW. Authors showed that
much of the total metal content was present in forms that were not likely to be reactive in
landfills. For example, plastics were the major contributors of Cd and scrap metal and rubber
were major contributors of Zn.
2.4 Leachate Treatment Methods
Leachate treatment and disposal is one of the difficult problem. Open dumpsites have no lining
which may allow percolation of leachate to groundwater aquifers. In engineered landfills lining
not only prevent percolation but also route leachate to on site treatment units (Kadlec and
Knight, 1996). In situ leachate treatment is cost effective approach in spite of ex-situ hauling.
10
During active years and for more additional years leachate may need treatment even after
dumpsite is closed.
Table 2.1 Leachate composition in acidic and methanogenic phase
S.No. Parameters Unit Acid Phase Methanogenic
Phase
1 pH - 4-7 7-9.5
2 COD mg L-1 6000-60,000 500-4500
3 Zn mg L-1 1,000 0.03 -4
4 Cu mg L-1 9.9 0.065
5 Pb mg L-1 14.2 0.09
6 Cr mg L-1 22.5 0.28
Kjeldsen et al. (2002)
There are several options for treatment of leachate like leachate recirculation, biological
treatment (aerobic and anaerobic), physical/chemical treatment and natural treatment systems
(Table 2.2). Onsite treatment facilities must be established. It may be located behind the dump
site so that leachate may be pumped into closely located pond. Settling of leachate is mandatory
so that chemical compounds and solid particle may be collected at the bottom before its goes for
second treatment. Chemical precipitation, coagulation, flocculation, ammonium removal,
activated carbon adsorption, and advance oxidation process may be applied. Removal of organic
compound which non-biodegradable in nature and heavy metals is done by adding coagulant
shown by Moradi et al. (2014). Whereas precipitation of organic matter with heavy metals after
addition of precipitating reagents, later filtration may remove the remnants from leachate.
Chemical oxidation is a widely studied method for the treatment of effluents containing
refractory compounds such as landfill leachate. Growing interest has been recently focused on
advanced oxidation processes. Most of them, use a combination of strong oxidants, e.g. O3 and
H2O2, irradiation, e.g. ultrasound, ultraviolet, or electron beam, and catalysts, e.g. photocatalyst
or transition metal ions (Renou et al., 2008). Although many of the previous researchers using
ozonation have demonstrated the effectiveness in eliminating COD (reduction is about 50–70%
in most cases) (Hilles et al., 2016), most of them only used this process as tertiary treatment prior
to discharge in the environment. Jung et al. (2017) reported comparison of two chemical
11
processes for treatment of landfill leachate; fenton process and ozonation for removal of
dissolved organic. Results revealed that fenton process exhibited efficient removal of organic
matter. Whereas, organic matter was reduced from 1840 to 112 mg L-1 from biologically treated
leachate using ozonation (Chys et al., 2015). Work by Amor et al. (2015) reported combination
of flocculation and solar photo fenton process. Results showed that 80% turbidity, 63% COD,
and 74% total polyphenols removed by flocculation and remaining load is treated by solar fenton
process.
Anaerobic membrane bioreactor was also used to treat leachate by treating 62% COD (Xie et al.,
2014). Recent study of reducing xenobiotic organic carbon from landfill leachate using
sequential thermophilic-mesophilic anaerobic was carried out by Pathak et al. (2018). Use of
granular activated carbon or powdered activated carbon to adsorb particles to its surface with its
attraction forces is also an effective way to remove suspended matter from leachate. In recent
work activated carbon produced from banana stem was used to absorb color and remove COD
from leachate (Ghani et al., 2017). Combination of coagulation/flocculation, fenton process
following by activated carbon adsorption. Subsequent activated carbon adsorption of ozonated,
coagulated and untreated leachate resulted in 77% removal of COD (Chys et al., 2015). It
showed that combination of more than one treatment process improved removal of COD.
Removal of ammonia is commonly done by ammonia stripping at pH 7 or below. At pH 11
ammonia gas formed and released from wastewater. Another process is exchange of ions without
inducing any change in structure. This ion exchange process is operated in batch and continuous
mode in which resins are stirred in leachate and removed after it settled down (Liu, 2013).
Electrochemical methods include electrocoagulation, electroxidation, and electrofloatation.
These methods are affective in recovering nutrients and metals from solution (Kabuk et al.,
2014). solar PEF and photo-electro-Fenton was assessed for the treatment of a sanitary landfill
leachate previously subjected to coagulation and biological processes (Moreira et al., 2016). It
can be observed that combination of methods facilitate the efficient removal of pollutants i-e
COD from landfill leachate. These technologies are liable to remove single pollutant from
leachate despite of high installation cost. Comparatively, natural treatment systems can remove
organic and inorganic pollutants from landfill leachate at low cost.
12
Table 2.2 Studies of different advance leachate treatment methods
Natural systems are based on biological system for purification of waste water without any
external source of energy except solar energy. Process of purification is very slow because of
higher retention time as compared to conventional treatment systems. Significance of natural
treatment systems are:
S. No. Method Organic and
Inorganic
Removal (%) Reference
1 Anaerobic membrane
bioreactor COD 65
Xie et al. (2014)
2 Coagulation, Fenton BOD
COD
60
72
Moradi and
Ghanbari (2014)
3 Advanced oxidation
biological process
BOD
COD
75
90
Chemlal et al.
(2014)
4 Sodium persulfate/H2O2
based oxidation process
COD
NH3-N
63
50
Hilles et al.
(2016)
5 Ozone per sulfate
advanced oxidation
COD
NH4
80
68 Abu et al. (2013)
6 Electrocoagulation
TOC
TSS
N
NH3-N
82
85
70
75
Kabuk et al.
(2014)
7 Sequencing batch biofilm
BOD
COD
TOC
78
84
80
Zhang et al.
(2014)
8 Micro-aerobic bioreactor COD
TN
58
74 He et al. (2015)
13
1. Natural systems remain reliable in extreme weather and high loading rates of organic
content.
2. Aesthically good and provides habitat to wildlife
3. Easily maintained without any specific skills
4. Cost effective.
Developing countries cannot afford high technologies with high capital cost. Comparatively,
natural treatment systems are suitable option for developing countries to treat waste water and
leachate (Mara, 1976).
2.5 Types of Constructed Wetlands
Constructed wetlands are engineered systems that have been constructed and designed to utilize
the natural processes involving soils, wetland vegetation, and the associated microbial
communities to assist in treating wastewaters. Constructed wetlands are designed to take benefit
of many of the same processes that occur in natural wetlands, but in controlled environment. The
first experiments aimed at the possibility of wastewater treatment by wetland plants were
undertaken by Käthe Seidel in Germany in the early 1950s at the Max Planck Institute (Seidel et
al., 1955). Seidel then carried out numerous experiments aimed at the use of wetland plants for
treatment of various types of wastewater, including phenol wastewaters (Seidel et al., 1966).
Most of experiments were carried out in constructed wetlands with either horizontal or vertical
subsurface flow, but the first fully constructed wetland was built with free water surface in the
Netherlands in 1967 (De Jong et al., 1976). Various types of constructed wetlands may be
combined in order to achieve higher treatment effect, especially for nitrogen. Hybrid systems
comprise most frequently VF and HF systems arranged in a staged manner but, in general, all
types of constructed wetlands could be combined in order to achieve more complex treatment
efficiency.
Flow pattern of leachate/waste water classify the type of wetland. Two main categories are
surface flow and subsurface flow constructed wetland (Fig 2.2). Removal of toxicity (metals and
inorganics) and chemical processing are common functions of vegetation. Good growth of plants
ensures the efficient working of wetland (Kadlec and Wallace, 2008) and growth habits of plants
are categorized by surface of water.
Emergent woody plants
14
Emergent soft tissue plant
Floating mats
Floating plants
Submerged aquatic plants
Dominating macrophytes are emergent soft tissue plants as they have rhizome system and
extensive root. Common reed (P. australis), cattail (T. latifolia), bulrush (Scirpus).
Figure 2.2 Different types of constructed wetlands based on direction of flow
2.5.1 Free Water Surface System Modified natural lagoons of 0.3 to 0.4 meter depth are free water surface system. Waste water/
leachate flows through stems and leaves of plants. Microbes also play an important role in
rhizomes of plants. Coverage of plants in free water surface system may not be uniform and
homogenous (Fig 2.3). Burgoon et al. (1999) employed a series of free surface wetlands
followed by a vertical flow wetland and denitrifying free surface wetlands to provide treatment
of potato processing wastewater. The system was subjected to 0.5 kg m-3d-1 COD loading.
Overall NH4-N, COD, organic N, and TN removals ranged between 72, 92 and 94%, and 84%,
Types of constructed wetland
Free water surface system
Horizontal
Vertical flow
Sub surface Flow system
Horizontal
Vertical flow
Hybrid System Hybrid System
15
66 and 63%, and 42 and 60%, respectively. The performance of FWS CW system was also
studied for domestic wastewater treatment with theoretical hydraulic retention times of 7, 5 and
10 days. Important parameters, such as NH4-N, BOD5, COD, TSS, PO4-P, DO, pH and fecal
coliforms in both raw and treated wastewaters were monitored during a macrophytes life cycle.
Based on the studies, it is concluded that minimum 5 days HRT is necessary for the treatment of
wastewater in FWSCW using C. Lilies or T. latifolia (Shrikhande et al., 2014). Compared to
other intensive (high-rate) anaerobic and aerobic treatment options (e.g. activated
sludge), constructed wetlands are natural systems, which work efficiently and extensively.
However, treatment may require land and time, but it is cost effective and require lower
operation with no electricity input.
2.5.2 Subsurface Flow System Water flow in contact with roots and substrates and wetland depth is 0.3 to 0.9 meters. This
system provides environment for proliferation of biofilm and for removal of pollutants (García et
al., 2015). Common reed and bulrush are commonly used in subsurface flow system. Study by
Mustapha et al. (2018) evaluated the performance of pilot-scale vertical subsurface flow
constructed wetlands planted with three plants species, i.e. C. alternifolius, T. latifolia, and C.
dactylon, in removing heavy metals from secondary treated refinery wastewater. Whereas
removal of ibuprofen, acet-aminophen, diclofenac, tonalide, oxybenzone, triclosan,
ethinylestradiol, bisphenol A was also studied in subsurface vertical flow constructed wetland
(Avila et al., 2014). Sand media provided a larger available surface area for microbial growth, as
well as higher oxygen availability which worked efficiently for removal of pollutants. Zheng et
al. (2016) investigated removal of nutrient by combining free surface and horizontal subsurface
wetland in presence of macrophytes. The effect of interspecific competition was notable for P.
australis, whereby it showed the highest growth performance in both FWS and SSF wetland
2.5.3 Horizontal Flow system Through granular bed water flows horizontally with depth of 0.3 to 0.9 meters with 0.05 – 0.1
cubic meter flow rate (García et al., 2015). Horizontal flow system have good distribution
network of pipes and varies with feasibility (Fig 2.3). Coarse gravels are filled inlet and outlet for
filtration purposes. P. australis are commonly planted in HSF. Papaevangelou et al. (2017)
16
reported removal of chromium in horizontal and vertical flow constructed wetland planted with
macrophytes. Results showed that pilot-scale vertical flow units exhibited lower performance in
comparison to horizontal flow units, with lower removal capacity and higher effluent
concentrations. Whereas Sultana et al. (2015) investigated removal of chromium in two units of
horizontal flow wetland. Approximately 88% removal of chromium was attained in horizontal
flow constructed wetland.
2.5.4 Vertical Flow system Water flow vertically downward through bed of substrates and not flooded permanently (Fig
2.3). As compared to horizontal flow treatment capacity of vertical flow system is high with
same organic loading rate. At the same time underground piping system can be clogged as pipes
are 0.05 -0.01 buried under soil (Moshiri, 1993). Yalcuk and Ugurlu (2009) investigated removal
of COD, ammonia and metals in presence of two vertical flow units in combination with one
horizontal flow unit. Results showed that ammonia was efficiently removed in vertical flow
system. Efficient removal of nutrients, metals in vertical flow constructed was investigated by
several authors (Saeed and Sun, 2011; Abou-Elela and Hellal, 2012; Bohórquez et al., 2016).
2.5.5 Hybrid Systems Hybrid systems are combination of horizontal and vertical SSFs for removal of nitrogen and
nitrates (Fig 2.3). Similarly different other combinations are also possible using different types of
wetland vertical SSFs with FWS etc. Recently multistage hybrid system was used by Vymazal
and Kröpfelová (2015) for removal of nitrogen and COD. System achieved 83% and 79%
removal of COD and nitrogen. He et al. (2017) investigated two horizontal subsurface flow
constructed wetlands (HSSF CWs, down-flow (F1) and up-flow for removal of COD, ammonia
and metals. Results showed significant removal of ammonia as compared to other parameters.
Removal of nutrient and metals using hybrid systems was investigated by various researchers
(Ye and Li, 2009; Wojciech et al., 2017; Hussain et al., 2015; Auvinen et al., 2017).
17
Figure 2.3 Types of constructed wetland A: Free waster surface, B: Horizontal, C: Vertical and D:
Hybrid constructed wetlands (Garcia et al., 2006)
A
B
C
D
18
2.6 Mechanism of pollutant removal in Constructed Wetland Removal mechanism of toxic compounds, metals, organic and inorganic pollutant rely on
physical, biological, chemical processes. Death, decay and growth of plants play role in
biogeochemical cycle.
During growth of plants maximum pollutants are removed by macrophytes and also providing
suitable environment to microbes for proliferation. Macrophytes assimilate metals in their tissues
of roots and leaves (Moshiri, 1993). Constructed wetland is used to treat industrial, municipal,
acid mine drainage and agricultural waste water. Mechanism for removal of pollutants through
constructed wetland are described below.
2.6.1 Suspended Solids Solids retained on filter paper of glass fiber with pore size of 1.2 µm are termed as total
suspended solid including settable solids and dissolved solids. TSS are removed naturally in CW
by interception and sedimentation. Chemical precipitation of pollutants combined with internally
generated SS including plant’s detritus and microbes (Fig 2.4).
Denser and larger particles are removed in CW by settling theory. Filtration is not significant
process in FWS wetland but particles adhere themselves to plant surfaces. Usually plant surfaces
are coated with periphyton biofilm (USEPA, 2000). In horizontal subsurface flow wetland
removal of TSS takes place near inlet. As waste water/ leachate flows through gravel bed
suspended solids are exponentially decreased, removal was achieved up to 90% with 20 mg L-1
hydraulic loading rate (García, 2008). At the same time high HLRs may clog the filter media by
reducing the removal efficiency.
2.6.2 Organic Matter Main constituent of raw waste water is organic matter (W.P.C.F, 1990). Removal of organic
matter involves biological, chemical and physical processes. There is a diverse array of organic
matter characterizing volatile solids and total carbon gives measurement of total organic matter,
COD is organic matter which is chemically oxidized and biologically degradable is BOD
(USEPA, 2000). In free water surface wetland, dissolved organic matter is degraded by
photolysis, sorption, oxidation and biodegradation. Macrophytes also help in supplying small
amount of oxygen. Oxygen is depleted with increase of depth in CW with dense population of
macrophytes. Whereas aerobic conditions have been observed in CW with submerged plants.
19
Surface has aerobic conditions in horizontal SSF and biological processes are anaerobic in
nature. In vertical constructed wetlands organic matter is degraded aerobically. Filtration in
horizontal SSF retain particulate matter by inlet. Abiotic fragmentation convert particulate matter
to smaller particles and extra cellular enzymes hydrolyze them. Fermentative bacteria or aerobic
heterotrophs excrete these enzymes. Aerobic heterotrophs oxidize organic compounds of low
molecular weight generated by hydrolysis (García, 2008).
2.6.3 Inorganic Matter Nitrogen, phosphorous and metals are inorganic in nature. Cycle of nitrogen is complex in
wetlands and its control is challenging. It is found in organic nitrogen and ammonia form
constituting peptides, proteins, urea and nucleic acid. Depending upon pH and temperature NH4-
N is found in ionized form NH4 which is predominant (USEPA, 2002). Eutrophication is caused
by excess of nitrogen by discharging waste water and leachate to ground and surface water. In
constructed wetland, organic ammonia is removed in same way as of TSS by physical processes
(filtration, adsorption). Through microbial mechanism and depletion of oxygen, nitrification is
converted to denitrification. Carbon and nitrogen cycles are coupled through process of
denitrification. Autotrophs bacteria in presence of oxygen convert organic ammonia to nitrate
and nitrite. While denitrification is carried out by heterotrophic bacteria (Garcia, 2008).
2.6.4 Metals
Removal of metals in constructed wetland is complicated process including plant uptake, abiotic
and biotic reaction (flocculation, sedimentation, precipitation, exchanges of cations and anions,
reduction and oxidation) (Kosopolov, 2004). Total removal of metals is not possible but their
physical and chemical properties are changed (Ujang, 2005). Work on removal of metals in CW
by different researchers is shown in Table 2.3. Vegetation play an important role in removal of
metals either by process of phytostabalization, phytoaccumulation, phytovolatization. Mustapha
et al. (2018) reported efficient removal of chromium and iron by T. latifolia. The metals were
taken up into the (stem, leaves and roots) parts of the plants, with the roots being the most
significant. Recently various studies revealed importance of plants for removal of metals (Khan
et al., 2015; Bonanno et al., 2017; Pan et al., 2016). At the same time, bioavailability of metals
20
also influence their removal by plants (Qasaimeh et al., 2015) in constructed wetland (Vymazal
and Brezinová, 2016). Detail of metal removal by plants and substrates is given in the following
section.
Figure 2.4 Interrelated factors in constructed wetland
2.7 Role of Plants in constructed wetland
Vegetation play fundamental role in wetland treatment system by transferring oxygen through
their roots to the bottom of treatment wetlands, and by providing a medium beneath the water
surface for the attachment of micro- organisms that perform the biological treatment (Fig 2.4).
The plants used frequently in constructed wetlands include water hyacinth, cattails, reeds,
duckweed and rushes (Qasaimeh et al., 2015).
Reeds grow along the shoreline and in water up to 1.5 m but are poor competitors in shallow
waters; they are selected for SFS systems because the depth of rhizome penetration allows for
the use of deeper basins (Vymazal and Brezinová, 2016). Water Hyacinth (E. crassipes) is an
Constructed Wetland
Design of CW
Plant Species
Microbes
Loading rate (COD and metal)
Retention Time
Season
Availability of sunlight and
Substrates
21
aquatic plant that grows very vigorously and uses highly the nutrients in the environment. The
growth rate of water hyacinth is affected by the water quality, nutrient content, harvesting
interval, and solar radiation. Aquatic plants have ability to uptake trace metals; this phenomenon
has brought wetlands to new scale of treatment. Usually native plants are chosen for removal of
pollutants. Plants should be readily available in case of severe plant damage or harvesting.
Perennial plants should be selected to guarantee continuity of treatment and operation with
proper growth of two seasons. P. australis and T. latifolia have good growth with properties of
hyperaccumulation for different heavy metals (Table 2.3). Zheng et al. (2016) reported
interspecific competition was notable for P. australis, whereby it showed the highest growth
performance in both SSF and FWS wetland. In a mixed culture, P. australis demonstrates
superiority in terms of competitive interactions for space between plants. Exposure to metals
may affect growth of plant as reported by various researchers (Mahmood et al., 2007; Kamran et
al., 2015; Kamran, 2016). Other plant species like C. indica, C. zizanioides are also planted in
wetlands for removal of metals. Variety of different emergent, macrophytes have been
experimented in constructed wetland for removal of metals shown in Table 2.4. T. latifolia and
P. australis are most important among all hyperaccumulators. Vymazal and Brezinová (2016)
found properties of P. arundinacea similar to P. australis. Organics, inorganics and metals are
efficiently removed in presence of these plants (Scholz and Hedmark, 2010) by absorption and
storage in roots. Lee et al. (2007) found that less than 2% of metal is accumulated in plants.
Moreover, less amount of metals are translocated to shoots of plant. Long distance translocation
of metals between roots and shoots is described by Lu et al. (2013). Meager translocation of
metals by roots of plants may be due to sequestration in vacuoles of roots thus natural action of
defense to remediate potential toxic effects of metals (Shanker et al., 2005). Acetate, oxalate,
malonate, oxalate and citrate are ions excreted as root exudates performing as chelators for
metallic ions (Ryan et al., 2001). Substrates help plants in mechanism of rhizodeposition of
metals and perform role of catalyst involving organic acids (Kidd et al., 2009).
Detoxification mechanism towards metals stress is multigenic adaptability of plant (Pal and Rai,
2010). Biomineralization leading towards precipitation of metals is resistance mechanism of
plants toward excessive exposure. Another mechanism is complex formation with enzyme
glutathione (GSH) and transportation to vacuoles of roots. Thirdly plant produces organic
22
ligands enriched in non protein (NP- SH) thiols such as metallothioneins and phytochelatins and
cysteine (Verbruggen et al., 2009). Phytochelatins help in chelating complexes and metals, later
transporting them to vacuoles (Pal and Rai, 2010).
Table 2.3 Metal removal studies in constructed wetlands
Trace Metals Location Type of
CW Study
Duration Number of chambers
Removal (%) References
Cu
Zn
Pb
USA SF 120 2
78
81
73
Crites et al.
(1997)
Zn
Pb
Cd
Asia HSSF - 1
80
87
66
Lim et al.
(2003)
Fe
Zn
Cu
Hg
USA,
Europe SF,HSSF 504
3
99.8
76.7
33.9
41.6
Kamal et al.
(2004)
Cr
Zn
Ni
USA SF 168 11
82
55
69
Hadad et al.
(2006)
Cu
Pb
Zn
USA SF 48 1
85
71
88
Nelson et al.
(2006)
Cr
Cu
Pb
Asia BR 360 6
75
83
69
Zhang et al.
(2007)
Cr,
Ni
Fe
Zn
USA SF 168-288 3
66
72
61
70
Maine et al.
(2009)
Zn
Cr Asia BR 48-360 8
76
61
Mishra and
Tripathi, (2009)
Cu Asia SF 40 11 48 Khan et al. (2009)
23
Metallothioneins protect against oxidative stress and help to bind metals and maintain
homeostasis (Palmer and Guerinot, 2009). Monferrán et al. (2012) found that short exposure of
Cu to P. pusillus responded by producing antioxidant enzymes named peroxidase, glutathione
reductase and glutathione peroxidase. Constitutive feature of wetland species is metal tolerance
for example T. latifolia and P. australis have constitutive tolerance towards Zn (Vymazal and
Březinová, 2016).
Bioconcentration and translocation factors (TF) are the best way to measure the efficiency of
plants for trace element accumulation and translocation from contaminated environments. Work
by Madera-Parra et al. (2013) investigated translocation and bioconcentration of C. esculenta, G.
sagittatum and H. psittacorum in constructed wetlands. Results showed the important difference
in levels of metals found in plant tissues may imply low mobility of metals from the root to
shoots, a condition that was equally validated against the low TF values that in general terms
remained <1. In spite of the good bioaccumulation of the metals, revealed a decreasing tendency
in the order of Pb > Cd > Cr (VI) > Hg; and in general terms accumulation decreased in leaves.
Rai et al. (2015) reported bioconcentration and translocation of three macrophytes in summer
Pb
Cr
Ni
50
89
74
Cu
Pb
Zn
Cr
Asia HSSF 90 2
39
29
35
52
Bakhshoodeh et
al. (2016)
Zn
Cu UK HSSF 360 2
89
81
Šíma et al.
(2017)
Cr
As
Ni
Cd
Pb
Asia HSSF - 2
85
38
83
90
97
He et al. (2017)
24
and winter season which showed that translocation factor was low (<1) in all the studied
macrophytes, T. latifolia, P. australis and C. esculenta in winter season except for Mn (1.01) in
T. latifolia and As (1.25) in C. esculenta.
Low TF indicates immobilization of trace elements from root to shoot or vacuole as in agreement
with the findings of various authors (Weis and Weis, 2004; Bonanno et al., 2017; Maine a et al.,
2009; Maine et al., 2017). In order to enhance bioconcentration chemical amendment like
addition of chelators is being used to improve the phytoextraction process (Ebrahimi, 2014).
However, chelator assisted phytoremediation has widely been used to decontaminated soil.
Use of DPTA by Rashid et al. (2014) to increase bioconcentration and translocation of Cd and
Pb in lettuce was investigated. Study identified increasing trend of bioconcentration and
translocation of metals with decrease in growth biomass of plants. Similar work by Muhammad
et al. (2009) to remove Cd, Pb, Cu and chromium from soil by T. ausgustifolia by adding
chelators; EDTA and CA. High accumulation of metals was observed in shoots of plants with 0.5
mM application of EDTA. Results showed less growth of plant as compared to control.
Therefore, chelator may improve bioavailability of metals by compromising plant growth.
Hyperaccumulator species are naturally capable to accumulate metals at high concentration
without using chelators (Kumari and Tripathi, 2015). Hyperaccumulation criteria of plants is
shown in Table 2.5. Plants are categorized as hyperaccumulator based on their accumulation
capacity of metals. Accumulation capacity of Cu and Pb is 1000 mg kg-1 and 10,000 mg kg-1 Zn
(Anning et al., 2013). Whereas different environmental factors may affect their tolerance
towards metals like salinity, temperature and geography (Marchand et al., 2010). Accumulation
of metals in macrophytes has been shown in Table 2.3. Application of macrophytes like P.
australis, T. latifolia, S. littoralis in constructed wetland is a promising solution for removal of
pollutants from different types of effluent.
Macrophytes with higher biomass production accumulate significant amount of metals in roots,
shoots and flowers. Marchand et al. (2010) reported that removal rates of metals are higher than
70% in constructed wetlands planted with common macrophytes like P. australis, T. latifolia
(Table 2.4).
25
Figure 2.5 Hyperaccumulators of Cu, Zn and Pb; A: P. australis, B: T. latifolia, C: V. zizanioides, D: C. gyana, E: E. globulus and F: C. indica
A B
C D
E F
26
Table 2.4 Metal accumulation by different plant species in constructed wetland
Species
Name
Common
name Metals
Type of
CW
Influent
mg L-1
Shoots
mg kg-1
Roots
mg kg-1 Location Reference
Phragmites
australis Reed
Cu
Pb HSSF
28.5
99.9
4.38
0.14
14.4
1.62 USA
Peverly et al.
(1995)
Phragmites
australis Reed Cr HSSF 150 1.26 4.20 Portugal
Calheiros et
al. (2008)
Typha
latifolia
Cattail Cu
Zn FWSF
4.1
8.4
55
470 Estonia
Maddison et
al. (2009)
Phragmites
australis Reed
Cu
Zn HSSF
6800,
173000
11.2
111.1
17
66.2 Italy
Galletti et al.
(2010)
Typha
latifolia Cattail
Cu
Zn
Pb
SSF
0.31
0.42
0.19
65
30
4
30
38
5.8
Gahna Anning et al.
(2013)
Typha
latifolia Cattail
Cu
Cr
As
Integrated
CW
2.60
<1
<1.5
38.30
9.00
4.00
France Salem et al.
(2014)
Vetiveria
zizanioides
Vetiver
grass Cu Mesocosm Pakistan
Batool and
Baig (2015)
Phragmites
Karka Reed
Zn
Pb
Cr
Fe
VSB
3.32
0.04
29.22
0.06
- - Nigeria
Badejo et al.
(2015)
Vetiveria
zizanioides
Vetiver
grass
Zn
Mg
Ni
Labscale
63.7
31.3
51.4
- - Zimbabwe Mudhiriza et
al. (2015)
Vetiveria
Zizanioides
Vetiver
grass
Cr
Ni HF
0.38
2.60
3.52
9.54
16.50
21.50 Iran
Bakhshoodeh
et al. (2016)
27
P. australis is well known hyperaccumulator of Cu, Cd, Ni, Cr and Pb (Kumari and Tripathi,
2015; Vymazal and Březinová, 2016). T. Latifolia has thick mass of roots which are efficient
accumulator of metals especially Cu, Zn, Pb and Cd (Sasmaz et al., 2008; Bonanno and Cirelli,
2017). Vetiver is a grass species strong affiliation for metals in soil and waste. V. zizanioides is
known for hyperaccumulation of Cu and Pb. It has thick mass of roots commercially utilize in
pharmacy, cosmetics and perfumes (Danh et al., 2014; Suelee, 2015). Rhodes grass has tolerance
of Pb. Exposure to higher level of Pb may Pb to bending and swelling of roots (Mahdavian et al.,
2016). E. globulus is hyperaccumulator of Pb and Cu. It grows in all seasons. Pulp of E.globulus
is also used for metal adsorbption from biodiesel (Squissato et al., 2017). Bark of E. globulus is
used for adsorption of chromium (Sarin and Pant, 2006). C. indica is used for removal of
nutrients in constructed wetland (Calheiros et al., 2007). Their easy growth and wide availability
also make them as appropriate choice. Selection of appropriate plant species along with efficient
substrates may significantly affect the performance of constructed wetland. Besides techniques
like biostimulation, bioaugmentation, genetically modified microbes or plants; progressive
approach toward enhancing the accumulation property of macrophytes can be employed by
introducing novel substrates which may improve mechanism of phytostabilization of metals in
roots.
2.8 Role of substrates in constructed wetland Substrates play role of filtration, adsorption, sedimentation, flocculation, precipitation and ion
exchange. Hydraulic permeability and adsorption capacity are main characteristics of substrates.
They also provide foundation for plant’s growth and microbial activity (Fig 2.6). Substrates with
Pb
Zn
1.76
37.92
3.93
7.51
20.50
17.52
Scripus
littoralis
Mn
Ni
Cu
Zn
Pb
494.9
56.37
144.9
207.9
93.08
India Bhattacharya
et al. (2006)
28
poor infiltration capacity reduce efficiency of system. Chemical characteristics of substrates will
determine its adsorption capacity. Substrates with Al and Fe ions may help in reducing
phosphate from waste water or leachate (Ryan, 2014). Commonly used substrates in constructed
wetland are gravel and sand. Washed gravels increase filtration of wetlands and minimize
clogging. For reed bed sandstone (Fluvio-glacial) are ideal. Gravel size for reed bed range
between (3 – 12 mm). Gravels increase nitrification process and higher denitrification occur in
reed systems with soil as substrate (Hogain, 2004). Plant growth can be supported by gravels,
sand and soil. Crushed stone may also provide support to plant’s growth and also provide surface
for microbial growth and ion exchange. Research on removal of pollutants from leachate by
different substrates is shown in Table 2.6. Different waste materials like alum sludge, oyster
shell, organic wood mulch, gravel wood mulch, zeolite, quartz, sand, gravels, slag and crushed
brick are used as adsorbent in different studies shown in Table 2.6. These waste materials were
used as insitu immobilized sorbents for heavy metal and inorganics. Surface precipitation and
specific adsorption by minerals present on the surface of these waste material determine the
efficiency of these sorbents.
Table 2.5 Safe limits for irrigation, toxic concentration and hyperaccumulation capacity
S.No Parameter Cu Zn Pb
1 Water (mg L-1) 0.31 0.42 0.19
2 Soil (mg kg-1) 34 6.7 1.75
3 Safe limit (mg L-1) 0.2 2.0 5.0
4 Toxic concentration (mg kg-1) 20-100 100-400 30-300
5 Hyperaccumulation limit (mg
kg-1)
1000 10000 1000
Pescod, (1992); Massa et al. (2010)
Similarly, functional group (carboxyl, hydroxyl, amine) in organic molecules like lignin, humic
substance, chitin, react with heavy metals explain the sorption capacity of heavy metals.
With surface ligands, anions and cations of heavy metals are exchanged to form partial covalent
bonds with ions on the charged surface areas of adsorbent. Studies in Table 2.6 explained that
surface structure also played an important role as crystalline and amorphous surfaces have Mn,
Fe, Al oxides with alumino-silicates. Blast furnace slag consist of calcium alumino-silicates with
29
Al and Fe oxides whereas steel slag analysis showed presence of Fe oxides and calcium – iron
oxides. Crushed brick (red mud) contains oxides of iron along with oxides of aluminum. Fly ash
contains ash particles with amorphous ferro-aluminosilicates (Zhou and Haynes, 2010). Outer
and inner sphere complexes are formed in adsorption reactions. Complexes in out sphere are
formed with one molecule of aqueous solution interposed between bound ions and functional
group involving electrostatic bonding between them. In inner sphere complexes no solvent
molecule is involved instead direct covalent bond is formed with functional group of surface.
Proximity of distribution of ions of charged surface can be termed as “electrical double layer”.
The process of adsorption can be visualized in three planes i) charged adsorbent surface ii) plane
of adsorbent iii) thirdly in near surface water layer balancing of different ions in outside plane.
Combination of adsorption of ions in inner sphere and outer sphere ions makes a charge on
adsorbent surface. Charged surface maintain its electro-neutrality by countering indifferent ions
with equal and opposite in magnitude and charge to surface charge.
Figure 2.6 Different factors supported by substrates in constructed wetlands
Whereas pH of aqueous solutions also play important role in these adsorption reaction in both
inner and outer spheres. In particular steel slag has highly reactive charged surface and therefore
known for efficient removal of Ni, Cu and Zn.
Column experiment conducted at laboratory scale by Kietlińska et al. (2005) to reveal metal
retention capacity of slag. Total 300L was fed to columns and >60% of Cu and Zn and 20% Ni
Substrates
Plants
Microbes Sedimentation
Filtration Biofilms
Absorption Adsorption
30
was achieved, respectively. Prochaska and Zouboulis (2006) experimented sand and dolomite as
substrates in constructed wetland. Analysis of sand by X-ray diffraction revealed presence of
hallousite (Al2Si2O5 (OH)4·2H2O), silicon oxide SiO2, iron oxide (FeO), and silicon phosphate
oxide (SiP2O7), and anorthite (CaAl2Si2O8). Where XRD analysis of dolomite found calcite
(CaCO3) and (CaMg (CO3)2). Authors found that oxide of iron performed efficiently in removal
of phosphorous as compared to oxide of aluminium. Zhao et al. (2011) used alum sludge as
substrates in constructed wetland for removal of phosphorous with high concentration of organic
matter. Different studies in Table 2.6 shows that variety of waste materials have been used for
removal of metals, organics and inorganics from different effluent. Substrates supports plants,
bacteria, biofilms in constructed wetland along with mechanism of adsorption, absorption,
sedimentation and filtration (Fig 2.6). Presence of plants in efficient substrates provide them with
organic matter thus reducing sulphate production with immobilization of metals (Marchand et
al., 2010). Therefore appropriate selection of substrates may improve the removal efficiency of
constructed wetlands. Substrates efficient for metal removal and plant growth are suitable to
apply in constructed wetland. Slag (He et al., 2017; Kamran et al., 2015), crushed brick (Batool
and Zeshan, 2017), in combination with sand (Eleonora et al., 2011; Papaevangelou et al., 2017;
Prochaska and Zouboulis, 2006; Ge et al., 2015; Syranidou et al., 2016) are considered as
efficient substrates which can be applied in constructed wetland for removal of nutrients and
metals.
2.9 Operational Parameters of constructed wetland Maintenance and design factors of different kinds of constructed wetland are important for
hydrology including hydraulic loading rate, hydraulic retention time, infiltrative capacity,
hydraulic conductivity, evapotranspiration (W.P.C.F, 1990). Recent studies emphasized on
importance of hydraulic retention time in constructed wetland. Solís et al. (2015) found that HRT
played an important role in removal of COD, BOD, phosphorous and nitrogen in constructed
wetland (Table 2.7). Authors found that retention time of three days is optimum for efficient
removal of nutrients and organic matter. Without proper optimization of retention time may Pb
to malfunctioning of the system. For operation and empirical design of CW, retention time is
considered an important parameter. Cárdenas et al. (2016) operated constructed wetland at
31
retention time of five days for enhancing removal of organic matter pollutants (OMP’s) and
achieved 50% removal at HRT of three days.
Table 2.6 Studies of different substrates in constructed wetlands
Substrate Type of CW Metal
s
Removal
(%) Inorganics
Removal
(%) Organic
Removal
(%) Reference
Sand
and
dolomite
VF - P - 45
Prochaska
and
Zouboulis
(2006)
Oyster
Shells Integrated -
N
P
TSS
85
98
94
BOD 92
Park and
Polprasert
(2008)
Organic
wood
mulch,
gravel
wood
mulch
VF, HF -
TN
TP
NH4-N
97
60
99
BOD 71 Saeed and
Sun (2011)
Alum
Sludge Integrated
TSS
TP
NH4-N
97
88
87
BOD 96 Wu et al.
(2011)
Zeolite Mesocosm
Cu
Fe
Zn
Mn
60
40
75
58
NH4-N - Zhu et al.
(2011)
Alum
sludge
Pilot scale
CW -
TN
TP
NH4-N
11-78
75-94
49-93
COD,
BOD5
57-84,
36-84
Zhao et al.
(2011)
Sand,
gravel VFCW - TP, TN
TOC,
COD
Zhao et al.
(2011)
Organic VF - TP TKN 97 COD 96 Korboulews
32
In short retention time anaerobic conditions in CW may not prevail for long and helps in better
oxygenation in rhizomes/ roots areas and within the substrates. Short retention time and efficient
removal may also help in optimizing the land requirement of constructed wetland.
At the same time, loading rates must be optimized with short retention time for better
performance of constructed wetland. To sustain bacteria and enzymes for degradation of toxic
organic polymers and recalcitrant may need long exposure to acclimatize. Similarly, redox
potential, pH, vegetation development and sedimentation are justified by retention time in
manure 98 ky et al.
(2012)
Gravels
IVF
-
TN, TP
15
52
COD
62.8
Chang et al.
(2012)
Sand
and
gravel
Mesocosm As,
Zn 65, 80 - -
Arroyo et
al. (2013)
Zeolite,
quartz
sand,
volcanic
rock
TF - NH4 97 - Liu et al.
(2014)
Sand,
gravel HSSF
Cr
Co
Pb
As
81
76
86
82
NH4-N
PO4-P
91
94 BOD
Rai et al.
(2015)
Slag HSSF - TP 23 BOD
COD
Ge et al.
(2015)
Slag,
crushed
brick
Mesocosm Cu
Zn
80
77 -
COD
-
Batool and
Zeshan
(2017)
Gravels mesocosm B
Se
26-45
50-69 - -
Zhu and
Bañuelos
(2017)
33
constructed wetland. Tao et al. (2006) reported positive correlation between long retention time
and removal of tinnin, lignin and COD.Authors suggested that series of constructed wetland cells
with long HRT may reduce large land requirement. Moreover, series of CW cells may improve
the quality of effluent. Apart from organic matter efficient removal of tramadol, carbamazepine
and diclofenac can be achieved at longer HRTs (Auvinena et al., 2017). Çakir et al. (2015)
reported that hydraulic loading rates directly influence the removal rates of BOD, COD, oil and
grease and TSS in constructed wetland. Bojcevska et al. (2007) also found positive correlation
between loading rates and removal rates of total phosphorous and ammonium. High loading rates
with short retention time may reduce the removal rates of pollutant (Chang et al., 2012).
Reducing contact time for denitrifying bacteria and nitrate by increasing loading rates may
decrease performance of constructed wetland. In start up phase with less plant density could limit
the carbon source which is required for denitrification (Lin et al., 2005). For respiration and
increased microbial production higher loading rate is recommended while microbial growth will
be inhibited after prolonged overloading. Tao et al. (2006) also suggested that increased loading
rate increased mircrobial activity for significant increase in removal of lignin and tinnin in
constructed wetland.
It can be summarized that operational parameters have tendency to influence plant growth
(Fountoulakis et al., 2017), development of biofilms (Lin et al., 2005; Solís et al., 2015;
Marchand et al., 2014),adsorption and absorption of metal and nutrients by substrates (Moreira et
al., 2016; Papaevangelou et al., 2017; Batool and Zeshan, 2017), nutrient and metal removal by
plants (Kumar, 2017; Kumari and Tripathi, 2015).
2.10 Research Need for the Dissertation
From this review it can be observed that optimization of operational conditions for removal of
specific pollutant are required.
Removal of Cu, Zn, Pb and COD have been experimented at large scale constructed wetlands
with long hydraulic retention time. Large land area requirement reduces worldwide acceptability
of constructed wetland despite of low cost. Moreover, long retention time induces different
environmental problems like odor, vector borne diseases, ultimately affecting the performance of
constructed wetland.
34
Table 2.7 Operational parameters affecting the performance of constructed wetland
CW Type Organic
Loading
rates g L-1
Metal
Loading
rates g L-1
Retention
time
(days)
Organic
Removal %
Metal
Removal
%
Reference
SF
mesocosm
0.6 5.3 21.3 Tao et al. (2006)
FWS 0.17 3
5
7
37
61
69
Chen et al. (2006)
HSSF Cr
0.000017
6.8 64 Calheiros et al.
(2007)
HSSF 0.44 0.5 42 Konnerup et al.
(2009)
VF1
VF3
HF
0.023
32
21
11.8
8
12.5
27.3
30.6
35.7
Yalcuk and
Ugurlu (2009)
HSSF Cu
0.0000031 -1
5-7 10 Anning et al.
(2013)
VFCW 0.07-0.016 - 3.5 89 - Avila et al. (2014)
HSSCW 0.6 - 2 80 - Merino-Solís et al.
(2015)
VSBCW Zn
0.01
90 70 Badejo et al.
(2015)
HSSF 0.030 - 5 90 - Cárdenas et al.
(2016)
HF 0.259 0.5 - 1
61.3-82
Upadhyay et al.
(2016)
Therefore optimization of hydraulic retention time in series of chambers in vertical flow
constructed wetland at small land area should be investigated.
High loading rates of metal and organic content reduces the removal efficiency of constructed
wetland. Moreover, plants show wilting and signs of toxicity in leafs and shoots at high exposure
35
to metals. Thus optimization of loading rates with retention time need to be investigated to
improve the removal efficiency with sustained plant growth. Furthermore, literature showed that
either plants species as hyperaccumulators or effective adsorbents were used individually in
constructed wetland. The combination of hyperaccumulators with efficient sorbents may not
have been experimented for removal of Cu, Zn, Pb and COD in vertical flow constructed
wetland. Thus there is a need to figure out the combined efficiency of hyperaccumulators with
sorbents as substrates for significant removal of Cu, Zn, Pb and COD at higher loading rates.
36
Chapter 3 Materials and Methods
The research work of this dissertation consists of two phases. In first phase four experiments
were conducted. First experiment investigated growth behavior of T. latifolia, P. australis and V.
zizanioides in different concentration of leachate. Second experiment investigated removal of
metals from leachate by T. latifolia, P. australis in presence of crushed brick and steel slag. In
third experiment, removal of metals by T. latifolia, P. australis in presence of substrates and
chelators were compared. Whereas fourth experiment explored kinetic study of metal removal by
substrates, plants separately as well as combination of substrates and plant from leachate. In
second phase removal of metals and COD was studied in pilot scale vertical flow constructed
wetland in batch and continuous mode.
3.1 Growth behavior comparison of different wetland species in mesocosm constructed wetland
Landfill leachate was collected from dumpsite on I.JP (Inter Junction Principal) road Islamabad.
Leachate was characterized for different physicochemical parameters which include pH,
temperature, EC, nitrate, phosphate and COD following standard methods (APHA, 2012).
Leachate was stored in plastic container and different leachate concentrations were prepared by
dilution of leachate (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%) using distilled water. Control
was distilled water (Fig 3.1). Young plant species (V. zizanioides, T. Latifolia and P. australis)
were obtained from NARC (National Agriculture Research Centre). Experiments were carried
out for three months. Plants were allowed to grow under hydroponic conditions. Three replicates
of each plant species were planted in all different leachate concentrations. Natural environmental
conditions were provided for plant growth. Plant growth (shoot length), chlorophyll content and
wiltage were measured with monthly interval. Chlorophyll content was measured on monthly
basis by CCM-200 Plus. Plants were harvested after three months and dried for laboratory
analysis. Dry weight of harvested plants was measured. Plants were oven dried at 80 oC for 24
hours. Heavy metal digestion was carried out for harvested and oven dried plant samples
(APHA, 2012).
37
Figure 3.1 Design and experimental conditions of experiment; growth behavior comparison of wetland species in mesocosm constructed wetland
3.2 Effect of substrates on phytoremediation of trace metals from solid waste leachate
Plastic tubs with 4 inches height and 12 inches diameter were used as vertical flow lab scale
constructed wetland system. Gravel and sand were filled in constructed wetland as base material.
Recycled waste material including crushed brick and steel slag in two treatment systems, which
made their top layer. Five plants each of P. australis and T. latifolia were planted in two
treatment system. Leachate was obtained from a dump site in Islamabad. Initial characterization
of leachate showed that the concentration of Cu was 24 mg L-1 and Zn was 30 mg L-1,
respectively. Leachate volume of 33.5 L was applied to each wetland system manually (Fig 3.2).
Plants were allowed to grow under hydroponic conditions for 21 days with three replicates of
each system. In control systems plant species were grown under hydroponic conditions without
substrates. Base material and crushed brick material was collected locally and it was sieved to
size of 2-6 mm. Steel slag (2-6 mm) was obtained from a re-rolling mill in Islamabad. After
Leachate Collection and Characterization
Leachate Dilutions Preparations
(10% - 100%) leachate
HRT 21 days
Length 19 inches
Diameter 4 inches
P. australis T. latifolia V. zizanioides
38
retention of 21 days in growth chamber (30 oC) plants (shoots and roots) were harvested and
leachate samples were collected to assess the efficiency of each system (Fig 3.2).
Figure 3.2 Design and experimental conditions of experiment; effect of substrate on phytoremediation of trace metal from solid waste leachate
3.3 Effect of chelators and substrates on phytoremediation of trace metals from synthetic
leachate
Synthetic leachate with COD of 5000 mg L-1 was prepared with different concentration of Cu
and Zn (5, 10 and 15 mg L-1) with pH 8. Plastic pots were prepared with 10 cm diameter and
P. australis and
T. Latifolia
P. australis and
T. Latifolia
Leachate Applied
Cu (24 mg L-1) Zn (30 mg L-1)
COD (5000 mg L-1)
Constructed wetland with crushed brick
Constructed wetland with steel slag
Substrate Characterization (XRD, SEM, XRF)
Length 19 inches Diameter 4 inches Vol applied 33.5 L Base material
Sand and Gravels HRT – 21 days
Treated leachate Treated leachate Sampling and Analysis (Zn, Cu)
39
45 cm height. After sieving, 2 kg of substrates (Crushed brick and Steel slag) was added to pots.
Five plants of each species; P. australis and T. latifolia were planted in two treatment systems
with crushed brick T1 and steel slag T2, respectively. They were allowed to grow under
hydroponic conditions for 21 days with three replicates of each system. Crushed brick material
was collected locally and it was sieved to size of 2-6 mm. Steel slag (2-6 mm) was obtained from
a re rolling mill in Islamabad. Dried samples of crushed brick and steel slag materials were
characterized for trace elements by EDX (energy dispersive X-ray spectroscopy). It revealed the
presence of ions of iron and silicon which might help in adsorption of trace elements. Control
systems were with plant species (T. latifolia and P. australis) and without substrates (Fig 3.3).
Selected dose of EDTA (2.5 mM) and CA (2.5 mM) was applied to coded plastic pots (C1a= P.
australis, C1b= T. Latifolia) and C2a = P. australis, C2b = T. Latifolia) by sprinkling,
respectively (Five plants per species in each pot system). Solutions of EDTA and CA were
prepared from disodium salt and citric acid, respectively. EDTA chelator was stable at pH 8.46
which was pH of synthetic leachate as well. Citric acid also performs efficiently beyond pH 5 for
removal of trace elements. Similarly, plant species (i.e., P. australis and T. latifolia) which are
native to Europe, central and South East Asia (Eflora.org) were selected for this experiment
based on the previous literature. Young and healthy plants were collected from local nursery; P.
australis and T. latifolia were planted individually in both treatment systems with three replicates
of each treatment(s) system. Treatment-1a) was
P. australis in crushed brick, Treatment-1b) was T. latifolia in crushed brick. Treatment-2a) was
P. australis in steel slag and Treatment-2b) was T. latifolia in steel slag. After retention of 21
days in growth chamber (30 oC) plants (shoots and roots) were harvested and leachate samples
were collected from each treatment to assess the efficiency of each treatment. It should be noted
that before applying leachate, plants were acclimatized for two weeks, fed with tap water (1-2 L)
and Hoagland solution (Composition of Hoagland solution was (mg L-1) 505 KNO3, 1200
Ca(NO3), 1.81 MnCl2.4H2O, 0.11 Na2MoO4.2H2O (Madera-Parra et al., 2015). Element
tolerance index in plant was calculated as:
Element tolerance index was calculated Tindex = Ʃ(Gx/Gmax)/n (Mahmood et al., 2007)
Gmax = Maximum value of growth parameter
Gx = Individual growth parameter
40
Figure 3.3 Design and experimental conditions of experiment; effect of chelators and substrates on phytoremediation of trace metals from synthetic leachate
3.4 Kinetic study for removal of metals and COD by substrates and plants
Three treatment systems set up were developed i) combination of plants and substrates, ii)
substrates and iii) plants only. Height and width of plastic were 21 inches and 5 inches,
respectively. Composition of leachate was 10 mg L-1 Cu and 10 mg L-1 Zn with COD 5000 mg
L-1 with pH 8. P. australis and T. latifolia were used as hyperaccumulators with crushed brick,
steel slag and limestone as Treatment 1 with three replicates (Fig 3.4). Substrates (crushed brick,
steel slag, limestone) were serving as Treatment 2 with three replicates. P. australis and T.
latifolia were used in Treatment 3 without any substrates with three replicates. Retention time of
21 days was provided and sampling was carried out on every fourth day for removal of trace
metals. However, COD was measured on daily basis.
Slag CB
Experimental Conditions
Cu (5,10,15 mg L-1)
Zn (5,10,15 mg L-1)
COD (5000 mg L-1)
pH (8.86)
Height 18 inches
Diameter 4 inches
(HRT 21 days)
T. latifolia
Chelators (2.5 mM)
Substrates (2 kg)
C.A EDTA Slag C.B
Chelators (2.5 mM)
Substrates (2 kg)
C.A EDTA
P. australis
41
Efficiency of metal removal by crushed brick and steel slag with plants was investigated in
previous experiments. Limestone was introduced in current experiment to compare metal
removal efficiency of our selected substrates; crushed brick and steel slag.
Based on solid capacity an expression of the pseudo-second-order rate has been presented for the
kinetics of sorption of divalent Cu and Zn ions by plants only, onto substrates and plant plus
substrates (Ho et al., 2006):
qt = qe2kt (1+qekt )-1
k = pseudo-second-order rate constant (g mg-1 day-1),
qe = amount of metal ion sorbed at equilibrium (mg g-1),
qt = amount of metal ion on the substrates or by plant’s roots at any time, t, (mg g-1)
Where:
qe = intercept slope-1
k = Slope2 intercept-1
Table 3.1 Leachate characteristics, retention time and sampling interval of experiment 4; kinetic study of removal of metals and COD by substrates and plants
S.No. Parameters Values Unit
1 pH 8.84
2 EC 347 µs cm-1
3 COD 5000 mg L-1
4 Cu 10 mg L-1
5 Zn 10 mg L-1
6 HRT 21 days
7 Sampling Interval 4, 8, 12, 16, 21 days
42
Figure 3.4 Kinetic removal study of Cu and Zn from plants, substrates and combination of plants and substrates
3.5 Pilot Scale Constructed Wetland Experiment
Pilot scale vertical flow constructed wetland was established and operated in batch and
continuous mode at varying loading rates and retention time for removal of metals and COD
from municipal solid waste leachate. Poly culture of P. australis, T. latifolia, V. zizanioides, C.
gyana, E. globulus and C. indica were planted in presence of crushed brick, steel slag, sand and
gravel. Details are given as follows:
3.5.1 Site Selection
The site was selected within Institute of Environmental Sciences and Engineering (IESE)
because of availability of suitable land and research facilities in labs of IESE. It was designed as
ex situ treatment for solid waste leachate (Fig 3.5). Pilot scale constructed wetland was designed
Treatment 1 Treatment 2 Treatment 3
Plants
P. australis
T. latifolia
Substrates
Limestone
Crushed Brick
Steel Slag
P. australis T. latifolia
Limestone
Crushed Brick
Steel Slag
43
and constructed at research station Institute of Environmental Sciences and Engineering (IESE),
NUST.
3.5.2 Design and construction of pilot scale constructed wetland
Pilot scale constructed wetland was designed and constructed at research station Institute of
Environmental Sciences and Engineering (IESE), NUST. The wetland consisted of five inter-
connected chambers (A, B, C, D and E) each having an area of 2.15 m2 with controlling valve in
each chamber (Fig 1). Batch and continuous mode were applied to influent. Real leachate was
collected from dumpsite near IJP road Islamabad and transported to the laboratory of IESE in
cold conditions. After successful construction and establishment of pilot scale vertical flow
constructed wetland, operation was started and leachate was allowed to flow in different
chambers in definite time. Leachate flowed vertically in chambers with the help of gravity
through substrates and plants (Fig 3.6).
3.5.3 Filling of wetland
After completion of construction beds were filled with substrates. Crushed brick with sand and
gravel was filled in chamber A. Steel slag with 5 cm sand and 5 cm gravel was filled in chamber
B. Chamber C and D were filled with 5 cm sand and 5 cm gravel only. Crushed brick 5cm and
5cm steel slag was filled in chamber E (Fig 3.8).
3.5.4 Plantation in pilot scale constructed wetland
Ten plants each of P. australis and T. latifolia were planted in chamber A and B. Similarly, V.
zizanioides and C. gayna were planted in chamber C. Moreover, E. globulus was planted in
chamber D and C. indica was planted in chamber E. P. australis, T. latifolia and V. zizanioides
were selected on the basis of laboratory scale experiments. However, selection of C. gayna, E.
globulus and C. indica was on basis of their tolerance towards Cu, Zn and Pb. Polyculture is
capable to treat multiple metals simultaneously and this trend is reported by Fountoulakis et al.
(2018).
44
3.5.5 Leachate collection, simulation and application Leachate was collected from dump site at IJP road Islamabad. Collected leachate was
characterized and synthetic leachate was simulated. Leachate application rate was 400 L per
chamber in batch mode. Whereas, 400 L was applied in chamber A which flowed to inter
connected chambers in continuous mode (Table 3.4).
3.6 Operational conditions of pilot scale constructed wetland
3.6.1 Batch mode
In present work, only chamber A and B were activated at initial stage and operated as batch at
retention time of 21 days in each chamber. Under these conditions the wetland was operated for
four runs. Loading rate of metals and COD shown in Table 3.5 and 3.6 were calculated by the
following formula:
Loading rate (g m-2 day -1) = [concentration of metal (g L-1) * Volume of leachate (L)] /
[area of chamber (m2) ] * HRT (days)
Ryan et al. (2014) treated leachate in constructed wetland having various chambers which were
activated individually for better performance. In second set of batch experiments four chambers
(A, B, C and D) of wetland were activated and were operated at retention time of 14 days in each
chamber. Total three runs were conducted under these conditions (Table 3.5 and Table 3.6).
Sampling of effluent from respective chambers was carried out after completion of retention time
with three replicates of each sample.
3.6.2 Continuous mode Pilot scale constructed wetland was operated continuously for two different hydraulic retention
times (35 days and 5 days). In continuous mode of operation chamber, A was fed manually with
freshly simulated leachate and effluent of chamber A served as influent for chamber B and
effluent of chamber B served as influent for chamber C and so on. All chambers (A, B, C, D and
E) during continuous mode of operation were active and four runs were operated with both
HRTs. Operational conditions for batch and continuous flow are mentioned in Table 3.5 and
Table 3.6. Sampling of effluent from all chambers was carried out after completion of retention
time with three replicates of each sample.
45
Chamber A
Typha latifolia
Phragmites australis
Crushed Brick
Influent
Effluent
Chamber B
Typha latifolia
Phragmites australis
Steel Slag
Chamber C
Vetiveria zizanioides
Chloris gyana
Sand & Gravels
Chamber D
Eucalyptus globulus
Sand & Gravels
Influent
Influent Influent
Effluent
Effluent Effluent
Figure 3.5 Batch mode of operation in chamber A, B, C and D of pilot scale constructed wetland
46
Chamber A
Typha latifolia
Phragmites australis
Crushed Brick
Chamber B
Typha latifolia
Phragmites australis
Steel Slag
Chamber C
Vetiveria zizanioides
Chloris gyana
Sand & Gravels
Chamber D
Eucalyptus globulus
Sand & Gravels
Chamber E
Canna indica
Crushed Brick
Steel Slag
Influent
Figure 3.6 Continuous mode of operation in chamber A, B, C, D and E in pilot scale constructed wetland
Effluent
47
3.6.3 Details of chambers (Plants and substrates)
Five chambers were constructed in wetland at different level from each other. Detail
specifications are given in table below. Each chamber has valve linked with porous pipe
encovered by sieving guaze. Chamber A was 0.60 m high from ground (head height 0.91 m).
Manual valve was installed for discharge of effluent. Porous Pipes (1 ft) was connected with
valve for filtration purpose. Substrates in chamber A were crushed brick (0.05 m), sand (0.03 m)
and gravels (0.03 m). P. australis and T. latifolia were planted. Chamber B was 0.30 m high
from ground (head height 0.60 m) with 0.30 m below Chamber A. This difference in level helped
leachate to flow at gravity from chamber A to chamber B. Length was 0.30 and width 1.52 m
with volume capacity of 700 L. Manual valve was installed for discharge of effluent. Porous
Pipes (1 ft) was connected with valve for filtration purpose. Substrates were steel slag (0.05 m),
sand (0.03 m) and gravels (0.03 m). P. australis and T. latifolia were planted (same as of
chamber A). Height of chamber C was 0.30 m and width was 1.52m. Its difference of 0.30 m
height from chamber B. Manual valve was installed for discharge of effluent. Porous Pipes (1 ft)
was connected with valve for filtration purpose. Substrates were sand (0.06 m) and gravels (0.06
m). V. zizanioides and C. gayana were planted in it. Height of Chamber D was 0.30 m with
width of 1.52 m. Difference of height from chamber C was 0.30 m. Manual valve was installed
for discharge of effluent. Porous pipe (1 ft) was connected with valve for filtration purpose.
Substrates were sand and gravels. E. globolus was planted. Chamber E was planted with C.
indica in presence of crushed brick and slag.
Table 3.2 Specifications of pilot scale constructed wetland
Specifications of each chamber Values
1 Width 1.52 m
2 Length 1.42 m
3 Height 0.3 m
4 Surface area 2.15 m2
5 Volume 0.64 m3
48
Table 3.3 Material used for construction
Material Required Quantity Specs
1 Valves 5 Ø 2inches
2 Pipes 5 2ft
3 Sieve Guaze 1 sheet
4 Cement 4 Bags
5 Brick 2 trollys
Table 3.4 Substrates used in pilot scale constructed wetland
*Djeribi and Hamdaoui (2008; Wang et al. (2016) Table 3.5 Composition of leachate used in pilot scale constructed wetland
3.6.4 Sampling and analysis
Samples of treated leachate was collected after particular retention time. Plants shoots and roots
were collected after harvesting. Plants shoots, roots and leachate were digested according to
methods provided by APHA (2012). Atomic absorption spectrophotometer (analytic Jena) was
used for heavy metals analysis (APHA, 2012).
Substrates Porosity (%) Density (g L-1) Size (mm)
1 Crushed brick 1.26 - 1.46 0.60 – 0.80 3-6
2 Steel slag 1.40 - 1.66 0.40 - 0.50 3-6
3 Sand 1.28 1.12 >2
4 Gravels 1.02 2.66 12-15
Leachate Composition Concentration (mg L-1)
1 Copper 3.75
2 Zinc 1000
3 Lead 7
4 Chemical Oxygen Demand 5000 - 10000
49
Figure 3.7 Substrates used in constructed wetland; A:crushed brick, B:steel slag, C: sand
and D: gravels
Figure 3.8 Materials used in pilot scale constructed wetland; A: Guaze; B: Pipes; C: Valves
3.6.4.1 Metal and COD analysis
Acid digestion of plant samples and substrates were carried using wet acid digestion method by
nitric acid and perchloric acid (v/v~2:1) (Madera-Parra et al., 2013) for Cu analysis .For this
purpose analytical grade chemicals purchased from Merck chemicals Germany were used. In 100
mL flask, 0.5 g of sample was transferred and HNO3 and HClO4 (2:1) was added and digested on
hot plate for two hours approximately with gradual increase in temperature up to 150 oC. The
A B
C D
A B C
50
solutes were cooled after digestion and filtered with Whatman filter paper of pore size 2.5 µm.
Volume of filtrate was raised up to 50 mL by adding Milli Q water. Cu analysis were carried out
by atomic absorption spectrophotometer (AAS) Analytic Jena 2000 using acetylene gas as carrier
gas, at wavelength of 324.8 nm. COD analysis was carried out by closed reflux method 5220B
(APHA, 2012). To determine the concentration of COD in the effluent, 2.5 mL of sample was
placed into a 10 mL test tube. Than, 3.5 mL sulfuric acid reagent was added. Tube was
inverted to mix the acid reagent in sample. Afterwards, 1.5 mL of potassium dichromate
digestion solution was added and samples was shaken. After tightly capping, the tubes were
placed in blocked digester at 150 oC and refluxed for 2 hrs. Once digestion is completed, test
tubes were removed and cooled to room temperature. Later, digested sample was transferred to
50 ml flask for titration. 1 -2 drops of ferroin indicator was added and stirred on magnetic
stirrer while tirating with standardized 0.10 M ferrous ammonium sulphate. End point is
detected by sharp change in color from bluish green to reddish brown. Similarly, blank was
refluxed and titrated.
Calculation:
COD as mg L-1 = (A-B) * M * 8000/ sample (mL)
Where:
A= mL FAS used for blank
B = mL FAS used for sample
M = molarity of FAS
8000 = milliequivalent weight of oxygen * 1000 mL L-1
3.6.5 Quality control and quality assurance
Concentrations (mg L-1) of metals were analyzed by atomic absorption spectrophotometer (AAS)
model Analytic Jena 2000. Calibration was performed by using standard solutions of different
concentration. (Kamran et al., 2015). Quality of data was ensured by repeated analysis of
51
standard solutions. For quality control and assurance all analytical procedures were carried out
by following standard methods of analysis (APHA, 2012).
Table 3.6 Loading rate of Cu in pilot scale constructed wetland
3.6.6 Metal accumulation factors
Factors of metal accumulation in different tissues of plants (shoots and roots) and in substrates
were calculated. Accumulation factors scientifically explained the difference of metal uptake by
roots, shoots from leachate and substrates.
Translocation factor reveals the transfer of metal from roots (mg kg-1) to shoots (mg kg-1)
S.
No
Operational
Mode
Individual
Chamber
HRT
(Days)
Overall
HRT
(Days)
No.
of
Runs
Total
Run
Time
(Days)
Chambers
Used
Cu loading
rate (g m-2day-1)
COD
loading rate (g m-2day-1)
1
Batch
21 21
4 84 A 0.0176 44.12
21 84 B 0.0176 44.12
2 14
14
3
42 A 0.0496 66.18
14 42 B 0.0496 66.18
14 42 C 0.0496 66.18
14 42 D 0.0496 66.18
3
Continuous
7 35 4
28 A 0.0992 132
28 B 0.0022 4.57
28 C 0.00079 3.06
28 D 0.00071 1.95
28 E 0.00021 1.21
4
1 5 4
4 A 0.9266 1853
4 B 0.0311 932
4 C 0.0076 640
4 D 0.0016 329
4 E 0.0024 133
52
Translocation factor = Shoots/Roots……………………….. ……………(1)
TF>1 means trace element uptake from root to shoot has been efficiently carried out (Soda et al.,
2012). Bioconcentration factor was calculated by formula given in Gosh and Singh, (2005).
Bioconcentration factor gives the estimates of the capability of plant species to extract trace
elements from substrates.
Table 3.7 Loading rate of Zn and Pb in pilot scale constructed wetland
Bioconcentration factor = Cp/Cs ………………………………. (2)
Where Cp is metal concentration in whole plant species (mg kg-1) and Cs is concentration of
metal in substrates (mg kg-1)
S.
No
Operational
Mode
Individual
Chamber
HRT (Days)
Overall
HRT
(Days)
No. of
Runs
Total
Run
Time
(Days)
Chambers
Used
COD
loading rate
g m-2 day-1
Zn loading
rate
g m-2day-1
Pb loading
rate
g m-2 day-1
1
Batch
21 21
4 84 A 44.12 0.022 0.061
21 84 B 44.12 0.022 0.061
2 14
14
3
42 A 66.18 1.323 0.092
14 42 B 66.18 1.323 0.092
14 42 C 66.18 1.323 0.092
14 42 D 66.18 1.323 0.092
3
Continuous
7 35 4
28 A 132 2.64 0.185
28 B 4.57 0.288 0.0061
28 C 3.06 0.106 0.003
28 D 1.95 0.028 0.001
28 E 1.21 0.011 0.0007
4
1 5 4
4 A 1853 92.66 1.29
4 B 932 10.54 0.198
4 C 640 3.55 0.168
4 D 329 1.58 0.129
4 E 133 0.437 0.106
53
Metal Removal = [(Ci-Ce)/Ci]*100………………………………….(3)
In above equation Ci is applied influent concentration and Ce is effluent concentration. This
equation provides percentage removal of pollutant (elements) from leachate (Elhafez et al.,
2016).
3.6.7 Statistical analysis
Data was collected and entered in MS Excel version 2015. One way ANOVA with Post
Hoc tukey kramer test (p<0.05) was applied for statistical analysis of data. Various formulas
mentioned in above section were applied to critically analyze the element translocation and
bioconcentration in plants.
54
Chapter 4 Results and Discussion
The results and discussion chapter has been divided into six sections. In first section, growth of
macrophytes was discussed in different leachate concentrations. In second section, effect of
substrates on removal of metals by macrophytes; P. australis and T. latifolia was discussed. In
third section, effect of chelators and substrates on phytoremediation of metals in synthetic
leachate were discussed. Fourth section, discussed about kinetic removal study of Cu and Zn
from leachate by plants, substrates and their combination. In fifth section, removal of metals and
COD from leachate in pilot scale vertical flow constructed wetland in batch mode at HRT of 21
and 14 days was discussed. In last section, removal of metals and COD in continuous mode at
HRT of 35 and 5 days in pilot scale constructed wetland has been discussed. Accumulation of
metals in plants and substrates are also discussed with translocation and bioconcentration factors
as part of pilot scale study.
4.1 Growth behavior comparison of three species exposed to municipal solid waste leachate
in microcosm constructed wetland
This study investigated comparison of growth of three species; T. latifolia, P. australis and V.
zizanioides in different leachate concentrations. Control was distill water (without leachate).
Detailed methodology is provided in section 3.1. Height and chlorophyll level of plants were
measured. T. latifolia has shown good growth without any signs of wilting and necrosis in
different leachate dilutions. Final height of T. latifolia was higher than initial height as compared
to other plant species in 100% leachate (Fig 4.1). Slight increase in height of P. australis was
observed as compared to initial height. Final height of V. zizanioides was increased in lower
concentration of leachate 10% and 20% as compared to higher leachate concentration. T. latifolia
and P. australis are hyperaccumulators of metals (Cu, Cd and Cr) with significant growth rate
under harsh conditions (Zheng a et al., 2016). Therefore, growth of macrophytes have shown
significantly different results as compared to V. zizanioides. Chlorophyll level was significantly
high in P. australis in 60% leachate (Fig 4.2). On the other side chlorophyll level gradually
decreased with increase in leachate concentration. No significant difference was observed in
chlorophyll level of T. latifolia and V. zizanioides (p<0.05).
55
Accumulation of Cu was highest in roots of T. latifolia in 30% leachate with gradual decrease to
70% leachate (Fig 4.3). Increase in accumulation of Cu in roots of T. latifolia was observed in
80% concentration of leachate revealing that T. latifolia has acclimatized itself with increasing
harsh conditions. On the other side, T. latifolia showed decreasing trend in accumulation of Cu in
roots at highest leachate concentration. Work by Sasmaz et al. (2008) showed that roots of T.
latifolia accumulated high amount of metals and this plant could be used as bioindicator in
polluted sites. Dense growth of T. latifolia indicate high concentration of nutrients or pollutants.
Significantly high accumulation of Cu by roots of P. australis in 70% leachate was observed (Fig
4.3). Accumulation of Cu was gradually decreased with increase in leachate concentration. On
the other side increase in root accumulation of Cu was observed in 100% leachate concentration.
Work by Badejo et al. (2015) showed that P. karka has ability to accumulate higher
concentration of different metals with increase in plant growth.
Figure 4.1 Initial and final height of T. latifolia, P. australis and V. zizanioides in different leachate concentrations
Figure 4.2 Chlorophyll level in T. latifolia, P. australis and V. zizanioides
0
10
20
30
40
100 90 80 60 40 30 20 10 Control
Plan
t hei
ght(
inch
es)
Leachate concentration (%)
T.Initial
T.Final
P.Initial
P.Final
V.Initial
V.Final
02468
1012
100 90 80 70 60 50 40 30 20 10 Control
Chlo
roph
yll C
onte
nt
Inde
x
Leachate (%)
Typha Phragmites Vetiver
56
Figure 4.3 Accumulation of Cu in roots and shoots of a)P. australis, b)T. latifolia and c) V.zizanioides
In case of V. zizanioides significantly high accumulation of Cu was noticed in roots in 90%
applied leachate concentration (Fig 4.3). V. zizanioides has accumulated 15 mg kg-1 Cu in shoots
02468
1012141618
100 90 80 70 60 50 40 30 20 10
Cu (m
g kg
-1)
Leachate concentration (%)
Phragmites.roots Phragmites.shoots
0
5
10
15
20
25
100 90 80 70 60 50 40 30 20 10
Cu (m
g kg
-1)
Leachate concentration (%)
Typha.roots Typha.shoots
b)
0
5
10
15
20
25
100 90 80 70 60 50 40 30 20 10
Cu (m
g kg
-1)
Leachate concentration (%)
Vetiver roots Vetiver.shoots
c)
a)
57
in 20% leachate concentration. Badejo et al. (2015) concluded that V. zizanioides accumulate
high amount of Cr with good growth. In present study, growth of V. zizanioides was stunted with
increase in leachate concentration (Cu concentration) revealed by plant height. P. australis and
T. latifolia were strong weedy species and V. zizanioides was a grass species. It showed that
biomass of plant affect the efficacy of plants for metal removal. Jarecki et al. (2005) investigated
growth of tomato and marigold in leachate generated from two different compost material.
According to authors nutrients and metal availability was high in runoff leachates. Mahmood et
al. (2007) reported effect of Cu on growth; root and shoot length of rice, barley and wheat.
Recently Mor et al. (2013) reported growth behavior of wheat exposed to different concentration
of municipal solid waste leachate. Their results showed that high exposure of leachate inhibited
growth and chlorophyll level in plants. Leachate has high concentration of metals which reduce
plants growth by production of oxidants in shoot and roots. Therefore, toxicity of metals should
be taken into account before applying leachate on basis of its richness in nutrient.
It can be summarized that efficient and sustained growth in terms of plant height (without any
visible signs of wilting) was observed in P. australis in higher concentration of leachate (100%)
with significant high accumulation of Cu in its roots. Chlorophyll level was high in P. australis
in 60% leachate concentration. Chlorophyll level decreased in all plants as exposed to high
concentration of leachate. Cu accumulation was more in roots than shoots of P. australis, T.
latifolia and V. zizanioides at 100, 80 and 90% leachate concentration, respectively.
4.2 Effect of substrate on phytoremediation of trace metals from solid waste leachate.
Present study aims at evaluating the effect of steel slag and crushed brick for removal of Cu and
Zn from municipal solid waste leachate by P. australis and T. latifolia in constructed wetlands.
In control, P. australis and T. latifolia were planted without substrates in hydroponic condition.
Detailed methodology is provided in section 3.2. Substrates were characterized by Scanning
electron microscopy and energy dispersive X-ray spectroscopy. Scanning electron microscope
images were examined to characterize the structure of substrates as given in Table 4.1. The SEM
images of substrates at 1 mm and 1µm showed amorphous and porous surface of crushed brick
and edgy structure of steel slag with particle size of 6mm which might help in intra particle
58
diffusion of leachate in these substrates. Density of crushed brick and steel slag materials was
0.6- 0.8 and 0.4-0.5 g mL-1, respectively (Table 4.1). Energy dispersive X-ray spectrometry
analysis of dried samples of crushed brick and steel slag was carried out to detect the presence of
trace metals. It revealed presence of silicon, aluminum, titanium and calcium ions in crushed
brick and steel slag as shown in supplementary figure 2 and 3. High peak of silicon was observed
at energy of 1.8 kev whereas aluminum and titanium were showing peaks at energy of 1.6 and
0.5 kev in steel slag, respectively. In EDX analysis of crushed brick high peak of iron was
observed at energy of 6.5 kev which was higher than that of titanium 0.5 kev. Small peaks of
magnesium and calcium were also observed at energy of 1.4 and 4 kev, respectively in crushed
brick. No presence of Cu and Zn was observed in both substrates.
At the end of experiments, plants were harvested and weight with root and shoot length was
measured shown in Table 4.3 and 4.4, respectively. Fresh weight of plants was measured and
the plants were oven dried at 80 oC for 12 hours to determine dry weight. Shoots and roots length
is presented in Table 4. Fresh weight of shoots and roots of T. latifolia was 23.31 and 38.71 g in
presence of crushed brick and 21.83 and 32.22 mg kg-1 in presence of steel slag, respectively.
Fresh and dry weight of shoot and root for both the plants in presence of crushed brick was
higher than that of steel slag and control as shown in Table 4.3.
It can be observed in Table 4.3 that weight of shoots and roots T. latifolia and P. australis in
presence of crushed brick is significantly higher (p<0.05) than that of control. No significant
difference was observed in weight of shoots and roots of T. latifolia and P. australis in presence
of steel slag as compared to that of control plants. Crushed brick has high content of iron oxides
which is growth promoter in plants. Colombo et al. (2014) investigated interaction of iron
minerals with plants and found that iron help plants to release siderophores, flavonoid and
organic acids supporting plant growth and metabolism. Gogoi et al. (2018) found that iron oxides
present in adsorbent helped in removal of Zn and Cu from industrial wastewater.
Length of shoot and root for both the plants in presence of crushed brick was more than that of
steel slag and control as shown in Table 4.4. Shoot and root length of T. latifolia was 14.21 and
4.95 inches in presence of crushed brick and 13.29 and 3.23 inches in presence of steel slag,
respectively. Whereas shoot and root length of P. australis was 7.88 and 3.66 inches in presence,
of crushed brick and 6.71 and 1.76 inches in presence of steel slag. In control, shoot and root
59
length of T. latifolia was 12.0 and 3.2 inches and 6.0 and 2.3 inches of P. australis, respectively.
It is worthy to note that shoot and root length of both T. latifolia and P. australis in presence of
crushed brick was significantly higher (p<0.05) as compared to that of control plants. However,
no significant change was observed in the length of both plants in the presence of steel slag as
compared to control. Roots of T. latifolia was heavier and longer while shoots of P. australis was
heavier and longer in presence of crushed brick.
Zn accumulation by T. latifolia was 10.83 and 6.04 mg kg-1 in shoot and roots, respectively in
presence of crushed brick which itself absorbed 4.5 mg kg-1 (Fig 4.5). Similarly, steel slag
absorbed 3.94 mg kg-1 Zn, whereas its accumulation in shoots and roots of T. latifolia was 13.04
and 5.92 mg kg-1, respectively. It was found that T. latifolia has accumulated significantly more
Zn in shoots (p<0.05) in presence of steel slag as compared to that in crushed brick. On the other
hand, roots of T. latifolia in crushed brick accumulated slightly more Zn than in steel slag.
Similarly, comparison of substrate showed that crushed brick absorbed slightly more Zn than
steel slag with no significant difference (p<0.05). Zn accumulation in shoots and roots of control
T. latifolia was 0.59 mg kg-1 and 0.70 mg kg-1 and control P. australis was 0.59 mg kg-1 and 0.26
mg kg-1, respectively.
Crushed brick absorbed 0.573 mg kg-1 Cu, whereas its accumulation in shoots and roots of T.
latifolia was 2.91 and 19.28 mg kg-1 , respectively. Similarly, steel slag absorbed 0.346 mg kg-1
Cu whereas its accumulation in shoots and roots of T. latifolia was 0.30 and 22 mg kg-1,
respectively. It was observed that T. latifolia has accumulated significantly more Cu (p<0.05) in
roots in presence of steel slag as compared to that in crushed brick. On the other hand, substrates
comparison showed no significant difference (p<0.05) in Cu absorption by crushed brick and
steel slag. Cu accumulation in shoots and roots of control T. latifolia was 0.22 mg kg-1 and 0.18
mg kg-1 and control P.australis was 0.55 mg kg-1 and 0.74 mg kg-1, respectively. Zn
accumulation by P. australis was 18.27 and 3.57 mg kg-1 in shoots and roots, respectively in
presence of crushed brick which itself absorbed 1.56 mg/g. Similarly, steel slag absorbed 1.46
mg kg-1 whereas it’s accumulation in shoots and roots of P. australis was 15.83 and 4.63 mg kg-
1, respectively (Fig 4.5). It showed that shoots of P. australis accumulated significantly more Zn
(p<0.05) in presence of crushed brick as compared to steel slag. Comparison of substrates
showed no significant difference (p<0.05) in sorption of Zn. Cu accumulation by P. australis
60
was 5.32 and 12.16 mg kg-1 in shoot and roots, respectively in presence of crushed brick which
itself absorbed 6.27 mg kg-1. Whereas, steel slag has absorbed 0.466 mg kg-1 of Cu and its
accumulation in shoots and roots of P. australis was 0.535 and 18.23 mg kg-1, respectively.
Figure 4.4 Percentage removal of trace metals in leachate planted with a) P. australis b) T. latifolia
0
10
20
30
40
50
60
70
80
90
100
Crushed brick Steel slag Control
Perc
enta
ge re
mov
al -
P. a
ustr
alis
(%)
Zn Cu
0
10
20
30
40
50
60
70
80
90
100
Crushed brick Steel slag Control
Perc
enta
ge re
mov
al -
T. L
atifo
lia (%
)
Zn Cub)
a)
61
Table 4.1 Porosity, density and SEM images of crushed brick and steel slag
*Djeribi and Hamdaoui (2008); Wang et al. (2016)
Table 4.2 Leachate composition and Pakistan Environment Protection Department guidelines
*BDL: Below detection limit
Therefore, it was found that roots of P. australis has accumulated significantly more Cu (p<0.05)
in steel slag as compared to that in crushed brick. On the other hand, crushed brick has absorbed
significantly more Cu (p<0.05) than steel slag (Fig 4.5). It can also be noted from Figure 4.5 that
Zn accumulation was more in shoots as compared to roots by both plants whereas Cu
accumulation was more in roots as compared to shoots by both plants in presence of both
substrates.
Substrates Density
(g mL-1)
Pore
volume
SEM Images (1mm) SEM Images (1µm)
Crushed Brick 0.6 - 0.8 1.26
Steel Slag
0.4 - 0.5 1.40
S.No Parameters Leachate
Composition
Units Pak National
Environmental
Quality Standards
1 pH 8.76 ± 0.45 - 6 - 9
2 EC 616 ± 1.32 µs/cm -
3 COD 5000 ± 0.5 mg L-1 150
4 Ammonia 80 mg L-1 40
5 Nitrogen 150 mg L-1 -
6 Pb BDL mg L-1 0.5
7 Se BDL mg L-1 0.5
8 Cu 24 ± 0.71 mg L-1 1
9 Zn 30 ± 0.53 mg L-1 5
62
Figure 4.5 Trace metal accumulation in shoot, root and substrates a) P. australis b) T. latifolia
Translocation factor of Zn by T. latifolia was 1.79 and 2.20 in presence of crushed brick and
steel slag, respectively. On the other hand, translocation factor of Cu by T. latifolia was 0.15 and
0.01 in crushed brick and steel slag, respectively. Translocation of Zn by P. australis was 5.11
and 3.41 in crushed brick and steel slag. Whereas translocation of Cu by P. australis in crushed
brick and steel slag was 0.43 and 0.02, respectively (Fig 4.6). It was found that translocation of
Zn was significantly higher (<1- greater than 1) than Cu by both T. latifolia and P. australis in
presence of both substrates.
0
5
10
15
20
25
Shoot Root Substrates Shoot Root Substrates
Zn Cu
P. a
ustr
alis
(mg
kg-1
)
Brick Steel slag
* *
0
5
10
15
20
25
Shoot Root Substrates Shoot Root Substrates
Zn Cu
T. la
tifol
ia (m
g kg
-1)
Brick Steel slag
*
*
b)
a)
63
Figure 4.6 Translocation of trace metals by plants in substrates a) P. australis b) T. latifolia
Whereas translocation of Cu was >1 by both plants in presence of both substrates.
Bioconcentration of Zn by T. latifolia was 3.74 and 4.81 in presence of crushed brick and steel
slag, respectively (Fig 4.7). Whereas bioconcentration of Cu by T. latifolia was 38.70 and 64.32
in the presence of crushed brick and steel slag, respectively. It was found that bioconcentration
of Zn to T. latifolia was not significantly higher (p<0.05) in crushed brick as compared to that in
steel slag. On the other hand, bioconcentration of Cu by T. latifolia was significantly more in
steel slag (p<0.05) than that in crushed brick.
0
1
2
3
4
5
6
Crushed brick Steel slag Control
TF in
Phr
agm
ites a
ustr
alis
Zn Cua)
0
0.5
1
1.5
2
2.5
Crushed brick Steel slag Control
TF -
T. L
atifo
lia
Zn Cu
*
*
b)
64
Table 4.3 Wet and Dry weight of T. latifolia and P. australis
Wet weight (g) Dry Weight (g)
T. latifolia P. australis T. latifolia P. australis
Substrates Shoot Root Shoot Root Shoot Root Shoot Root
Crushed
Brick
43.19*
± 0.37
52.81*
± 0.69
48.51*
± 0.36
34.63*
± 0.21
22.78
±0.42
37.01
±0.57
33..09
±0.6
13.25
±0.31
Steel Slag 41.51
±0.82
48.76
± 0.44
46.44
±0.27
31.66
±0.18
19.89
±0.45
30.16
±0.28
30.69
±0.75
9.81
±0.59
Control 39.88
±0.61
46.27
±0.73
45.19
±0.91
32.11
±0.25
20.03
±0.42
34.88
±0.53
31.63
±0.88
9.75
±0.11
Table 4.4 Shoot and root length of T. latifolia and P. australis
T. Latifolia P. australis
Substrates Shoot length
(inches)
Root Length
(inches)
Shoot length
(inches)
Root length
(inches)
Crushed Brick 14.2* 4.9* 7.8* 3.6*
Steel Slag 13.2 3.2 6.7 1.7
Control 12.0 3.2 6.0 2.3
Bioconcentration of Zn by P. australis in presence of crushed brick and steel slag was 14.97 and
13.98, respectively. Bioconcentration of Cu to P. australis was 2.78 and 40.21 in crushed brick
and steel slag, respectively (Fig 4.7). There was no significant difference in bioconcentration of
Zn by P. australis in presence of crushed brick and steel slag. On the other side, statistically
significant difference was found in bioconcentration of Cu by P. australis planted in steel slag
(p<0.05) and crushed brick.
Solid waste leads to production of leachate which is toxic and harmful in environment. The
effective removal of trace metals from solid waste leachate is one of most important
environmental issues for many industrialized countries. T. latifolia and P. australis have been
used widely for uptake of trace metals (Ait et al., 2004) in constructed wetland since many
65
decades. Trace metals can be phytoaccumulated in shoots and roots or can be phytostabilized by
rhizomes. In the present study, T. latifolia and P. australis were used in constructed wetland for
removal of trace metals from leachate in combination with different substrates. The selected
emergent species can phytoaccumulate Zn and Cu present in leachate with sustained growth
(weight and height). Similarly, translocation and bioconcentration of trace metals in wetland
species varied according to the provided substrates. The current approach used to clean up the
metals from solid waste leachate which involves the use of metal-accumulating plants to remove,
transfer, stabilized the metals from leachate. Plants growing on substrates; crushed brick and
steel slag have accumulated the metals and finally reduced the appreciable amount of metal
concentration in leachate. P. australis and T. latifolia produce good biomass so they were
assumed to uptake considerable amount of metals from real leachate in current study. Young
plants uptake nutrients with their growth from provided substrates (Yadav et al., 2009) so their
growth performance can be correlated with phytoremediation ability.
Removal of Cu in CW of crushed brick and steel slag, planted with T. latifolia showed an
increased removal efficiency (>95%), which might be due to assimilation capacity of plants, high
photosynthetic rate, and characteristics of substrate. Removal percentage of Zn was less efficient
by both macrophytes in crushed brick and steel slag as compared to Cu. P. australis showed the
highest removal of Cu (>95%) in crushed brick and low removal >75% in steel slag without any
visible signs of wilting and necrosis. This might be due to the increased antioxidative
metabolism in P. australis, under conditions of metal stress (Kumari and Tripathi, 2015).
Whereas, Batool and Zeshan (2017) showed that P. australis and T. latifolia performed
efficiently in crushed brick as compared to steel slag for removal of metals (Zn and Cu) without
use of chelators. Young plants especially wetland species (P. australis and T. latifolia) are
known to have dense roots with ability to phytostabilize trace metals (Vymazal and Brezinová,
2016).
After harvesting of plants, the maximum biomass achieved by shoots of P. australis and roots of
T. latifolia was recorded in presence of crushed brick. Overall an increasing growth trend of both
macrophytes was observed in crushed brick as compared to steel slag and control. Crushed brick
contained major concentration of iron which is an essential micronutrient and highly required for
plant metabolism. These findings revealed that variations in metal uptake by these plants might
66
be due to presence of micronutrients (Fe, Mg) in substrate (Kumari and Tripathi, 2015). High
biomass of the plant played a prominent role in the extraction of metals in solid waste leachate.
The results of our study showed that the growth and metal accumulation capabilities of P.
australis was enhanced within period of 21 days and these findings were also found similar as
reported by Madera-Parra et al. (2015). Both trace metals served as plant’s nutrients and helped
in metabolic activity of plant but higher concentrations can be toxic (Vymazal and Březinová,
2016). Higher concentration of Cu and Zn may affect the osmosis, transport, uptake and
regulation of vital ions disrupting the plant’s metabolic activity (Ait et al., 2004). Elongation in
root and shoots length of T. latifolia was increased in presence of crushed brick under metal
stress. EDX spectrometry showed that crushed brick contained major concentration of iron,
which is an essential micronutrient and highly required for plant metabolism. P. australis has
also shown increased shoot and root length in presence of crushed brick. Steel slag inhibited the
root elongation of T. latifolia and P. australis as compared to crushed brick and control. Results
suggested that both macrophytes can tolerate stress of Cu and Zn in presence of crushed brick
compared to steel slag.
The amount stored in the P. australis shoots represented a large proportion of the removed Zn
and less in the both substrates. While retained Cu was stored mainly in the roots biomass in the
same macrophyte in both substrates. The process of partitioning is a common strategy of
macrophytes to accumulate toxic ions in the roots preventing any adverse effect on shoots and
leaves which act as site of metabolic activities and photosynthesis (Rai et al., 1995). P. australis
is used in CWs for the treatment of wastewater containing metals (Bragato et al., 2006; Kumari
and Tripathi, 2015). P. australis, T. latifolia, and C. esculenta used to treat sewage in constructed
wetland grew well without visual appearance of toxicity and any impact on their growth (Rai et
al., 2015). The below ground biomass of T. latifolia indicated greater Cu accumulation potential
compared to the aboveground biomass. Generally, the rhizosphere is the zone where
physicochemical and biological processes occur through interactions between plants, substrate,
microorganisms and pollutants. Zn was mainly stored in above ground biomass of T. latifolia.
The removal of metals retained in the aboveground biomass can be removed through harvesting
and proper disposal in landfills (Vassiliki et al., 2017).
67
Crushed brick showed the highest Cu and Zn removal performances in presence of P. australis
and T. latifolia, respectively. The difference in the metal removal due to the substrates is
probably related to the fine character that favors physical conditions as adsorption, absorption,
and biofilm growth surface (Hamdaoui, 2006). Crushed brick is a highly alkaline in nature with a
pH of 8–13 due to presence of the sodium hydroxide. Crushed brick is mainly composed of fine
particles containing ions of aluminium, iron, silicon, titanium oxides and hydroxides shown in
results of EDX spectrometry. The red color is caused by the oxidized iron present, which can
make up to 60% of the mass of it (Bhatnagar et al., 2011). Maximum adsorption of Cu was
found at pH 5.5 in the case of crushed brick. Longer contact time played important role and
enhanced the interaction between the metal and the substrate (Djeribi and Hamdaoui, 2008).
Translocation factor of Cu was less than 1 in T. latifolia and P. australis in presence of both
substrates. Low TF indicates immobilization of trace elements in vacuole or from root to shoot as
in agreement with the findings of various authors (Maine et al., 2017; Madera-Parra et al., 2015)
. Low translocation from roots to aerial parts is an advantage because metals are not available for
herbivorous animals (Maine et al., 2017). Cu remained immobilized in the roots or substrates.
However, T. latifolia showed high translocation (>1) of Zn in both, crushed brick and steel slag.
TF of Zn by P. australis was found to be higher in crushed brick than steel slag, greater than 1 in
both substrates. Zn is required for production of tryptophan which is an originator of indole-3-
acetic acid (IAA hormone) and accomplishes its function in stems (Hopkins and Huner, 2009).
Because of these functions, Zn is often found rather in the aboveground than belowground
biomass of wetland plants. However, efficient translocation signifies a key trait of
hyperaccumulation and efficient trace element transportation from roots to shoots (Zhao et al.,
2016).
Bioconcentration factor is the capacity of metal accumulation in relation with plant biomass from
soil and reported for many plants (Yadav et al., 2009). T. latifolia showed lower BCF value of
Zn in comparison to P. australis in presence of crushed brick. Higher metal removal amounts in
the P. australis might also be related to its high endurance ability towards toxic metals in
leachate (Maine et al., 2017). The BCF value for Cu was higher in steel slag than crushed brick
by both macrophytes. It suggested that both substrates helped plants to retain Cu in roots as an
action of filtration to protect shoots and leaves for trace metal induced impairments (Klink et al.,
68
2013). High BCF values for wetland plants is also reported by (Soda et al., 2012). On the other
side, such a high metal concentration factor shown by macrophytes resulted into lowering the
metal content of solid waste leachate and improvement in quality of treated leachate. A large
value of BCF implies a better phytoextraction and phytoaccumulation capability of plants
(Madera-Parra et al., 2013). Difference in BCF values depends upon substrate conditions,
exposure time, concentrations in the environment, metal chelating compounds released from the
roots, affinity of trace elements for the adsorption sites, type of absorption mechanism, and
sampling period.
Figure 4.7 Biotransferrable factor of trace metals by plants in different substrates
a) P. australis b) T. latifolia
0
5
10
15
20
25
30
35
40
45
Crushed brick Steel slag
BCF
in P
. aus
tral
is
Zn Cu
*
*
a)
0
10
20
30
40
50
60
70
Crushed brick Steel slag
BCF
in T
. Lat
ifolia
Zn Cu
*
*
b)
69
Overall, both plants showed a differential potential for bioconcentration factor depending on the
substrates. Synergistic relation of T. latifolia and P. australis with steel slag and crushed brick
has improved their phytoaccumulation without any impairment to growth. It can be summarized
that performance of T. latifolia and P. australis was proficient for removal of Cu and Zn in steel
slag and crushed brick, respectively. Substrates supported the process of phytostabilization by
restricting translocation of Cu to shoots thus sustaining better growth of plants leaving plants
viable for herbivorous purposes. Zn might also played an important role in growth as
micronutrient and increased defense mechanism of plants against Cu. Phytoremediation in
presence of crushed brick and steel slag in constructed wetlands clearly provides an efficient and
cost-effective means for achieving goal of metal removal from leachate. The treatment system is
easy to construct and can be replicated at open dump sites in developing countries
4.3 Effect of chelators and substrates on phytoremediation of trace metals from synthetic leachate
The present study aimed to recycle waste materials, crushed brick and slag, and compare their
efficiency with chelators (EDTA and C.A) for the removal of trace elements to develop a cost-
effective and environment-friendly approach. Chelators mobilize metals for easy and quick
accumulation by plants. At the same time, chelators have adverse effects on plant growth and
expensive to use. On the other side, substrates are cost effective with no harmful effects on plant
growth. Control was plants without substrates and chelators. Detailed methodology is provided
in section 3.3. P. australis and T. latifolia have shown highest percentage removal of Cu in
crushed brick as compare to control in dose of 5 mg L-1 in leachate (Fig 4.8). In higher applied
concentration of Cu in T. latifolia set up removal of Cu was less in both substrates; slag and
crushed brick as compare to control. Percentage removal of Zn was higher in lower applied range
(5 mg L-1 >15 mg L-1 >10 mg L-1 ) with significant difference at (p < 0.05). Performance of P.
australis in slag and T. latifolia in crushed brick was better than other treatments systems. Zn
might have played the role of micronutrient which helped in removal of Cu along with better
performance of plants in substrates. Accumulation Zn in treatment system of T. latifolia was
higher than P. australis in citric acid treatment. Removal of Zn by chelating treatment with
70
EDTA was >90% in lower applied range of Cu and >80% in 15 mg L-1 >10 mg L-1 Cu in
synthetic leachate (Fig 4.8). Removal percentage of Cu was approximately 99% in both chelating
treatment systems as compare to control in 5 mg L-1 >10 mg L-1>15 mg L-1. Efficient removal
percentage can be observed in substrates treatment systems and chelating treatment systems. It
revealed that crushed brick (2 kg) and slag (2 kg) have shown efficiency equivalent to optimum
(2.5 mM) dose of EDTA and C.A for removal of Zn and Cu.
Figure 4.8 Percentage removal of Cu and Zn a) Adsorbents b) Chelators (T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b:
Typha CA)
0102030405060708090
100
T1a T1b T2a T2b ContPhrg
Cont Typ T1a T1b T2a T2b ContPhrg
Cont Typ
Copper Zinc
Rem
oval
with
ads
orbe
nts (
%)
5 mg/L 10 mg/L 15 mg/L
0102030405060708090
100
C1a C1b C2a C2b ContPhrg
Cont Typ C1a C1b C2a C2b ContPhrg
Cont Typ
Copper Zinc
Rem
oval
with
che
lato
rs (%
)
5mg/L 10mg/L 15mg/L
a)
b)
71
Efficient sorption capacity for different elements by crushed brick (Djeribi and Hamdaoui, 2008)
and slag (Wang et al., 2016) was due to active sites on particle surface. Cu and Zn content in
different parts of plant in experimental set up with adsorbents are shown in Fig 4.10. Different
parts of plants (P. australis, T. latifolia) have different affinity for Cu and Zn.
Figure 4.9 Accumulation of a) Cu and b) Zn in shoots, roots and substrates (T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick)
Cu was accumulated more in roots in applied concentration of 5 mg L-1 by P. australis in slag
(T2a) as compared to crushed brick (T1a). More accumulation of Cu was observed in shoots than
in roots in higher applied concentration of Cu (15 mg L-1 >10 mg L-1) by P. australis in slag than
in crushed brick with significant difference (p<0.05). The reason could be the composition of
steel slag with heterogenous oxides comprising different functional groups (oxide groups of Al+3
0123456789
T1a T1b T2a T2b ContPhrg
ContTyp
T1a T1b T2a T2b ContPhrg
ContTyp
T1a T1b T2a T2b ContSlag
ContBrick
Shoots Roots Substrates
Cu (m
g kg
-1)
5mg/L 10mg/L 15mg/L
*
* *
*
*
a)
0123456789
T1a T1b T2a T2b ContPhrg
ContTyp
T1a T1b T2a T2b ContPhrg
ContTyp
T1a T1b T2a T2b ContSlag
ContBrick
Shoots Roots Substrates
Zn (m
g kg
-1 )
5mg/L 10mg/L 15mg/L
*
*
*
* *
b)
72
and Fe+3) (Wang et al., 2016) which not only promoted good growth of P. australis but enhanced
translocation of Cu.
Figure 4.10 Shoots and roots accumulation of a) Cu and b) Zn in treatment system with chelators (EDTA, CA) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b: Typha CA)
Level of Cu was high in roots as compared to shoots in T. latifolia (T1b>T2b) lower applied
range of 10 mg L-1. Concentration of Cu increased in shoots as compared to roots (T1b>T2b)
with increase in given concentration of Cu in synthetic leachate 15 mg L-1. Previously, P.
australis and T. latifolia have been extensively used for uptake of trace elements with sand or
gravel as substrates in constructed wetland (Kumari and Tripathi, 2015). Kumari and Tripathi
0123456789
10
C1a
C1b
C2a
C2b
Cont
.Phr
g
Cont
.Typ C1
a
C1b
C2a
C2b
Cont
.Phr
g
Cont
.Typ
Shoots Roots
Cu (m
g kg
-1)
5mg/L 10mg/L 15mg/L
*
a)
*
0123456789
10
C1a-
Sh
C1b-
Sh
C2a-
Sh
C2b-
Sh
Cont
-Phr
g
Cont
-Typ
C1a-
R
C1b-
R
C2a-
R
C2b-
R
Cont
-Phr
g
Cont
-Typ
Shoots Roots
Zn (m
g kg
-1 )
5mg/L 10mg/L 15mg/L
* *
b)
*
73
(2015) have studied removal of Cu, Zn, Pb, Fe, Ni, Cd and Cr by T. latifolia and P. australis at
HRT of 14 days. Bernardini et al. (2016) mentioned that P. australis has higher accumulation
capacity for Cu, Cd, Cr, Ni and Fe. In present work P. australis as compared to T. latifolia also
performed significantly well (p<0.05) in crushed brick and slag for removal of Cu and Zn. Zhao
et al. (2011) used turf grass with EDTA application for removal of Cu, Zn and Pb. With
increasing dose of EDTA (5 mM to 10 mM) accumulation of elements increased in plants. At the
same time leaching of metals in leachate from compost increased with time. Leaching of metals
by EDTA was also explained by Ebrahimi (2013) as prospective and potential hazard of this
remediation technique. According to findings of researchers, growth of plants were retarded by
application of 0.5 g kg-1 EDTA because it has significant adverse effect on plant’s health.
In present study accumulation of Cu was higher in shoots than roots in plants spiked by EDTA as
compared to CA. Whereas roots has accumulated more Cu in plants spiked by Citric acid.
Present study results revealed that trace elements were significantly removed by recycling waste
materials (crushed brick and steel slag) as substrates with efficiency compatible to chelators like
EDTA (ethylene diamine tetra acetic acid), C.A (Citric acid) for selected range of Cu and Zn (5
mg L-1 , 10 mg L-1 , 15 mg L-1). Trend of Cu (5 mg L-1) accumulation with chelators was high
in roots than shoots by P. australis as compared to T. latifolia spiked with EDTA (C1a>C1b)
(Fig 4.10). Accumulation of Cu (5 mg L-1) was high in roots of P. australis spiked with C.A was
greater than EDTA (C2a>C1a). Higher concentration of Cu was removed by roots in 10 mg L-1
as compared to 15 mg L-1 from synthetic leachates spiked with EDTA. Trend of translocation
factor of Cu (5 mg L-1) by P. australis in steel slag (T1a>T2b>T2a>T1b) was lower than T.
latifolia in slag (Fig 4.11). If translocation is greater than one it depicts more accumulation of
trace element in shoots than roots. Trend of translocation was T1b>T2b>T1a>T2a in (10 mg L-1
>15 mg L-1) applied concentration of Cu in synthetic leachate. Translocation of Cu may be
depending on amount of organic complexes of Cu formed in solution and consistency of this
organically complexed Cu in provided pH (Liao, 2000). Translocation of Zn was higher in 5 mg
L-1 as compared to higher doses in T1b>T2a>T1a>T2b. Zn was more translocated by T. latifolia
in steel slag (C1b>C2b>C1a>C2a) in 5 mg L-1 applied concentration of Zn in synthetic leachate.
Ait et al. (2004) reported that higher amount of Zn (above 50 mg L-1) induced toxicity to plants
by chlorosis and young shoot’s rolling and curling. Effect of higher doses of trace elements was
74
noticed by visual growth sign of plants. Chlorophyll level of plants may also indicates the
response of plants towards toxicity of Cu and Zn.
Figure 4.11 Translocation factor in treatment systems with a) adsorbents and b) chelators(T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick) (C1a: Phragmites EDTA; C1b: Typha EDTA;
C2a: Phragmites CA; C2b: Typha CA)
Different trace elements adsorbed by plants in different parts varies with their form in leachate or
waste water, on water transportation and types of plants. Vascular system of plant plays
important role for accumulation of elements by translocation and compartmentalization in roots,
shoots or leaves (Kim et al., 2008). In chelators translocation of Cu was high in 5 mg L-1 >10 mg
L-1 >15 mg L-1 in C2b>C1b>C1a>C2a. It showed that citric acid helped T. latifolia for
translocation Cu from roots to shoots. Trend of translocation of Zn was more as compare to Cu
05
101520253035404550
T1a
T1b
T2a
T2b
Cont
.Phr
g
Cont
.Typ T1
a
T1b
T2a
T2b
Cont
.Phr
g
Cont
.Typ
Copper Zinc
TF w
ith a
dsor
bent
5mg/L 10mg/L 15mg/L
a)
012345678
C1a
C1b
C2a
C2b
Cont
-Phr
g
Cont
Typ C1
a
C1b
C2a
C2b
Cont
.Phr
g
Cont
.Typ
Copper Zinc
TF w
ith c
hela
tors
5mg/L 10mg/L 15mg/L
b)
75
(Fig 4.11). Zn might played the role of micronutrient which benefited plants for removal of Cu
from synthetic leachate. Cu is also micronutrient below 0.2 mg, which is quite lower than applied
dose of Cu (Kumari and Tripathi, 2015) Low concentration is not effective and higher
concentration of chelators induced adverse effects on plants. Work by Ebrahimi (2013) showed
maximum uptake of Pb by shoots of E globolus in higher applied dose of EDTA. There are
various mechanism behind enhanced uptake of trace elements by applying chelaters. This
mechanism is depending on plant species (Chen et al., 2014), chemical and physical nature of
elements (Saifullah et al., 2009) substrates/soil, exposure time and chelator mode of application
(Begonia et al., 2004). Screening plants species for specific pollutants/toxicity for
phytoremediation can be calculated by element tolerance index. It revealed and marked threshold
levels of specific plant’s species tolerance towards elemental toxicity (Cu and Zn) in different
treatment systems (Fig 4.12). P. australis and T. latifolia are known as hyperaccumulator for Cu
in contaminated soils and waste water (Kumari and Tripathi, 2015). In present research these two
wetland species were exposed to different levels of Cu and Zn (low to high) in both treatment
systems (substrates and chelators). In treatments spiked by EDTA, height of plants was stunted
in all ranges of both elements with significant difference as compare to citric acid and these
results are in accordance with different studies (Cay et al., 2015; Ebrahimi, 2016; Zhao et al.,
2011). Height of plants was better in treatments with substrates as compare to treatments with
chelator which determined plant’s tolerance indexing with significant difference (p<0.05).
Figure 4.12 Metal Tolerance Index of T.latifolia substrates and chelators(T1a: Phragmites slag; T1b: Phragmites crush; T2a: Typha slag; T2b: Typha brick) (C1a: Phragmites EDTA; C1b: Typha EDTA; C2a: Phragmites CA; C2b:
Typha CA)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
T1a T1b T2a T2b C1a C1b C2a C2b
Ti
5mg/L 10mg/L 15mg/L
76
Whereas overall element tolerance index of T2b>T2a (T. latifolia in slag >crushed brick) was
high in 5 mg L-1 >15 mg L-1 >10 mg L-1. T. latifolia as compared to P. australis has shown
more elemental tolerance (5 mg L-1 >10 mg L-1 >15 mg L-1 ) in steel slag with significant
difference from crushed brick. Overall, tolerance of plants in substrates was better than in both
chelators which shows that chelators induced toxic effects on plants growth. Reduced and
stunted growth decreased tolerance towards element uptake. Mahmood et al. (2007) revealed
effects of Cu on metal tolerance of crop seedlings and Cu imposed more adverse effects as
compare to Zn. Moreover they found that element induced morphological and structural
properties of plant’s roots which may Pb towards reduced tolerance. In present study substrates
reduced the load on plants species by sorption of trace elements which might have increased
their tolerance index with sustained growth unlike chelators.
In developing countries like Pakistan, solid waste leachate management is still an environmental
issue leading to elemental toxicity in ground water aquifers and surrounding environment.
Applying chelators for remediation of elemental toxicity is costly approach for developing
countries. It can be summarized that significantly high removal of Cu and Zn was achieved by
plants in chelators at dose of 15 mg L-1. Chelator effected growth of plants as wilting in higher
dose of Cu was observed. Treatment with substrates withstand better growth of plants by
restricting translocation of toxicity to shoots which supported better photosynthesis in higher
exposure. This work presents an environment friendly and cost effective alternate of chelators.
Crushed brick is a waste material readily available at brick kilns in Pakistan. Similarly, steel slag
is also a waste material spared by steel re rolling industries in Pakistan. Recycling these waste
products of brick kilns and steel industries for phytoremediation of trace elements with T.
latifolia and P. australis opens environmental friendly and new avenue in field of remediation.
4.4 Kinetics study of metal and COD removal by substrates and plants
The aim of this study was to investigate removal of Cu and Zn by i) removal of metals from
leachate in presence of substrates ii) metal removal from by hyperaccumulators; P. australis and
T. latifolia from leachate iii) metal removal from leachate by combination of both; plants and
substrates with variation in time, respectively. Psuedo second order kinetics was applied on
77
results of metal removal by different treatments. Detailed methodology is provided in section
3.4. It was found that T. latifolia in steel slag and P. australis in crushed brick was efficient for
removal of Cu (Table 4.5). Whereas T. latifolia and P. australis both were equally efficient in
crushed brick for removal of Zn (Table 4.6). In treatment 1 removal percentage of Cu was
significant by P. australis as compared to T. latifolia without substrates (Fig 4.13). P. australis
achieved highest Cu removal of 97% in presence of crushed brick at 21st day of experiment as
shown in fig 4.15. Different studies showed that plant accumulate maximum concentration of
metal with maximum retention time (Bouchama et al., 2016; Fountoulakis et al., 2017; Madera-
Parra et al., 2013). In treatment 2 Cu removal by crushed brick was significantly different from
other substrates (limestone, steel slag) without plants (Fig 4.14). In treatment 3, P. australis in
crushed brick removed Cu above 80% at 14th of experiment instead of 21st day (Fig 4.15).
Performance of T. latifolia was prominent in presence of steel slag for 88% removal of Cu at 14th
day instead of 21st day of experiment as shown in Fig 4.15. It is important note that either plants
or substrates have not removed maximum concentration of metal at 14th day of experiment
compared to metal removal by combination of plants and substrates. It depicts that P. australis
and T. latifolia in combination with slag and crushed brick can removal metals from leachate in
short time. Plants were performing role of hyperaccumulators and substrates were providing
active site for metal. sorption developed synergistic combination for efficient metal removal
from leachate. Work by Ye et al. (2003) showed that P. australis wilted in Cu contaminated site.
It can be assumed here that macrophyte hyperaccumulate Cu at the cost of its health and
longevity. Kumari and Tripathi (2015) and Anning et al. (2013) reported P. australis and T.
latifolia are hyperaccumulators of Cu and Zn with maximum accumulation capacity of 1000 and
10,000 mg kg-1 , respectively. However, initial concentration of Cu and Zn was 10 mg L-1 in
leachate of present work providing less exposure to hyperaccumulators compared to their
accumulation capacity. At the same time, crushed brick and steel slag have proven efficiency for
metal adsorption. For this reason, no wilting or necrosis was observed in treatment of plants in
presence of all substrates. Exposure to Cu in synthetic leachate to plants was shared by substrates
in treatment 3 which helped in good growth of plants. Metal ions in synthetic leachate were in
soluble form and can be easily accumulated by plant. But presence of substrates played counter
role by adsorbing metal ions primarily and plants accumulated limited amount of metal from
substrates (Batool and Zeshan, 2017).
78
Table 4.5 Pseudo second order kinetic model for removal of Cu in different treatments
S.No. Treatments - Cu Slope k r2
1 T1.Phrg 1.0 0.001 0.89
2 T1.Typha 0.9 0.001 0.95
3 T2.Lime 1.1 0.003 0.87
4 T2.Brick 1.9 0.014 0.74
5 T2.slag 1.7 0.014 0.77
6 T3.Phragmites.Lime 4.0 0.10 0.78
7 T3.Phragmites.Brick 1.2 1.16 0.98
8 T3.Phragmites.Slag 3.9 0.10 0.86
9 T3.Typha.Lime 2.5 0.09 0.81
10 T3.Typha.Brick 7.0 0.01 0.85
11 T3.Typha.Slag 4.3 0.12 0.90
Table 4.6 Pseudo second order kinetic model for removal of Zn in different treatment
S.No. Treatments - Zn Slope k r2
1 T1.Phragmites 0.69 1.4 0.86
2 T1.Typha 1.84 12 0.80
3 T2.Lime 2.01 16 0.87
4 T2.Brick 2.05 16 0.88
5 T2.slag 1.10 4 0.82
6 T3.Phragmites.Lime 1.13 4.8 0.90
7 T3.Phragmites.Brick 1.81 12 0.92
8 T3.Phragmites.Slag 0.53 1.12 0.56
9 T3.Typha.Lime 2.10 16 0.82
10 T3.Typha.Brick 0.39 0.36 0.95
11 T3.Typha.Slag 0.75 1.96 0.60
79
Figure 4.13 Percentage removal of Cu by P. australis and T. latifolia
Figure 4.14 Percentage removal of Cu by different substrates
Proficient removal of Zn was observed by T. latifolia as compared to P. australis in treatment 1
at 14th day achieving 97 and 85% removal, respectively (plants only) (Fig 4.16).
Removal of Zn by crushed brick was better than steel slag and limestone at 4th, 8th, 11th and 17th
day of experiment attaining 31, 52, 78 and 98% in treatment 2, respectively. (Fig 4.17).
Performance of crushed brick and limetstone for removal of Zn was 98% at 21st day,
respectively. Removal of Zn was prominent by T. latifolia at 17th in crushed brick achieving 93%
removal. Significant removal of Zn was 98% by T. latifolia in limetstone at 14th, 17th and 21st
day, respectively (Fig 4.18). Main reason of quick removal of Zn by treatment of plants was
affinity of T. latifolia and P. australis for Zn. Hyperaccumulation capacity of both are 10,000 mg
0
10
20
30
40
50
60
70
80
4 8 11 14 17 21
Cu re
mov
al (%
)
Time (Days)
P. australis T. latifolia
0
20
40
60
80
100
4 8 11 14 17 21
Cu re
mov
al (%
)
Time (Days)
Lime Brick Slag
80
L-1 (Anning et al., 2013) that is significantly higher than initial concentration provided in this
experiment. Moreover, initial concentration of Zn might have played role of micronutrient which
helped in plant growth and significant assimilation.
Figure 4.15 Percentage removal of Cu by P. australis and T. latifolia in different substrates
Significant difference was observed in removal of chemical oxygen demand by P. australis
achieving 766 mg L-1 as compared to 715 mg L-1 by T. latifolia at 21st day of experiment (Fig
4.19). In treatment 2 limestone proficiently removed up to 474 mg L-1 COD as compared to 662
mg L-1 in crushed brick and 881 mg L-1 in steel slag at 21st day of experiment (Zhang et al.,
2013). P. australis in steel slag removed COD more efficiently as compared to other substrates
(Fig 4.20). It should be noted here that COD as organic matter served as plant manure which
helped in growth of plants. Most importantly respiration of plant’s roots helped in degrading of
COD. Steel slag alone can help in removal of COD by Beh et al. (2012) than combination with
plant can improve the removal of COD. P. australis achieved COD removal up to 433, 332, and
87 mg L-1 in presence of lime, crushed brick and steel slag. Whereas T. latifolia attained 449,
112 and 581 mg L-1 COD in presence of lime, crushed brick and steel slag at 21st day of
experiment. Significant decline in COD values was observed in treatment of P. australis in
presence of steel slag starting from 17th day of experiment to 21st day attaining 988, 733, 616,
433, 298, 167, 113 and 87 mg L-1, respectively. Whereas P. australis in presence of lime
removed COD in the range of 1298 mg L-1 and 433 mg L-1 from 17th to 21st day of experiment.
0102030405060708090
100
4 8 11 14 17 21
Cu -
Rem
oval
(%)
Time (Days)
Phragmite.Lime Phragmite.Brick Phragmite.Slag Typha.Lime Typha.Brick Typha.Slag
81
Figure 4.16 Percentage removal of Zn by P. australis and T. latifolia
Figure 4.17 Percentage removal of Zn by substrates
Figure 4.18 Percentage removal of Zn by P. australis and T. latifolia in different substrates
0
20
40
60
80
100
4 8 11 14 17 21
Zn re
mov
al (%
)
Time (Days)
P. australis T. latifolia
0
20
40
60
80
100
4 8 11 14 17 21
Zn re
mov
al (%
)
Time (Days)
Lime Brick Slag
0
20
40
60
80
100
4 8 11 14 17 21
Zn -
Rem
oval
(%)
Time (Days) Phragmite.Lime Phragmite.Brick Phragmite.Slag Typha.Lime Typha.Brick Typha.Slag
82
T. latifolia in presence of lime removed COD from 1322 to 449 mg L-1 from 17th to 21st day of
experiment.
On other side, T. latifolia in crushed brick removed COD efficiently (Jia et al., 2014) than in
presence of lime stone and steel slag, respectively. Respiration of plants and ions present in
crushed brick assisted in rapid degradation of COD.
Based on solid capacity an expression of the pseudo-second-order rate has been presented for the
kinetics of sorption of divalent Cu and Zn ions by plants only, onto substrates and plant plus
substrates (Ho et al., 2006):
qt = qe2kt /1+qekt
k = pseudo-second-order rate constant (g/mg days),
qe = amount of metal ion sorbed at equilibrium (mg/ g),
qt = amount of metal ion on the substrates or by plant’s roots at any time, t, (mg/g)
Where:
qe = intercept/slope
k = Slope2/intercept
Figure 4.19 COD removal by plants
0500
100015002000250030003500400045005000
1 3 5 7 9 11 13 15 17 19 21
COD
(mg
L-1 )
Time (Days)
Phragmites Typha
83
Figure 4.20 COD removal by substrates
Best fitted model was achieved by plotting graph between 1/qt and 1/t for Cu and Zn. In order to
determine absorption capacity of substrates or plants from concentrated this second-order rate
equation has been applied termed as a pseudo-second-order. It can be determined by plotting 1/qt
against 1/t. Other factors influencing the sorption capacity, includes pH, temperature,
conductivity and initial sorbate concentration (Ho et al., 2004). Pseudo second order kinetics has
been linearized to determine the sorption of Cu and Zn with time. Table 4.1 and 4.2 shows the
data obtained by using linear method for sorption of Cu and Zn in different treatment systems.
Values of slopes and r2 have been listed in Tables. In this study, the coefficient of determination,
r2, was used to test the best-fitting of the kinetic model to the experimental data. Pseudo second
order kinetics model favorably explained chemical sorption of substrates. R square values close
to 0.80 indicates positive evidence of metal sorption in different treatment systems. Results
showed that T. latifolia in presence of brick (r2 = 0.85) and slag (r2 = 0.9) removed Cu
significantly. On the other side, P. australis removed Cu (r2 = 0.98) in presence of crushed brick.
In case of Zn, T. latifolia and P. australis showed better performance in presence of crushed
brick (r2 = 0.95) and (r2 = 0.92) and lime (r2 = 0.90). Similar sorption results of crushed brick and
lime were obtained by different researchers (Djeribi and Hamdaoui, 2008; Hamdaoui 2006; Aziz
et al., 2008). Crushed brick comprised of high content of iron oxides and traces of titanium and
aluminium oxide revealed by XRD analysis (supplementary data) which provide active sites for
metal sorption. Pepper et al. (2017) found that iron, aluminium and titanium in red mud (crushed
brick) develop a synergistic interaction to provide active sites for metal sorption. At the same
time, iron present in crushed brick might also played role of micronutrient (Colombo et al.,
0
1000
2000
3000
4000
5000
1 3 5 7 9 11 13 15 17 19 21
COD
(mg
L-1 )
Time (Days)
Lime Brick Slag
84
2014) thus providing healthy substrates for growth of plant roots and no signs of wilting were
observed during the experiment.
Figure 4.21 COD removal by P. australis and T. latifolia in different substrates
P. australis has performed efficiently in presence of brick crushed (r2 =0.92). Data showed the
sorption equilibrium capacity, the sorption rate constant of Cu and Zn are function of the time. It
can be summarized that P. australis and T. latifolia in presence of crushed brick and steel slag
showed efficient removal of Cu and Zn. High coefficients of determinants in pseudo second
order kinetics were obtained in treatment of P. australis in presence of crushed brick and T.
latifolia in steel slag for Cu removal. Thus combination of plants with substrates was performing
significantly better than individual plants and substrates for removal of Cu and Zn from leachate.
0500
100015002000250030003500400045005000
1 3 5 7 9 11 13 15 17 19 21
COD
(mg
L-1 )
Time (Days)
Phragmite.Lime Phragmite.Brick Phragmite.Slag
0500
100015002000250030003500400045005000
1 3 5 7 9 11 13 15 17 19 21
COD
(mg
L-1 )
Time (Days)
Typha.Lime Typha.Brick Typha.Slag
85
Summary of Phase I – Laboratory scale experiments
In phase I, growth of P. australis, T. latifolia and V. zizanioides was investiagted in ten
concentration of leachate. Both plant species have performed significantly well revealing their
sustainability in different ranges of leachate. Significantly high accumulation of Cu by roots of
P. australis in 70% leachate was observed (Fig 4.3). Accumulation of Cu was gradually
decreased with increase in leachate concentration. On the other side increase in root
accumulation of Cu was observed in 100% leachate concentration. In case of V. zizanioides high
accumulation of Cu was noticed in roots in 90% leachate concentration (Fig 4.3). P. australis
and T. latifolia were strong weedy species and V. zizanioides was a grass species. It showed that
type and biomass of plant affect the efficacy of plants for metal removal.
In second experiment, growth and uptake of metals was analyzed in presence of new substrates;
crushed brick and steel slag. It was found that P. australis and T. latifolia have shown good
growth with significant removal of metals from MSW leachate. Percentage removal of Zn and
Cu by T. latifolia in crushed brick was 71 and 95% whereas in case of steel slag it was 72 and
94%, respectively. P. australis removed 78 and 99% of Zn and Cu in presence of crushed brick
and 73 and 80% in steel slag, respectively. It showed that removal of Cu was significantly high
by both macrophytes in both substrates thus proving that both species can growth in novel
substrates; crushed brick and steel without compromising their quality of metal
hyperaccumulation from MSW leachate.
After this finding, third experiment was conducted to compare metal removal efficiency of
substrates against chelators. The results showed that trace elements were significantly removed
by recycling waste materials (crushed brick and steel slag) as substrates with efficiency
compatible to chelators like EDTA and C.A for selected range of Cu and Zn (5 mg L-1, 10 mg L-1
and 15 mg L-1). P. australis as compared to T. latifolia has performed significantly well (p=0.05)
in crushed brick and slag for removal of Cu and Zn. Whereas trend of Cu (5 mg L-1)
accumulation with chelators was high in roots than shoots by P. australis as compared to T.
latifolia spiked with EDTA (C1a>C1b) (Fig 4.11). Accumulation of Cu (5 mg L-1) was high in
roots of P. australis spiked with C.A was greater than EDTA (C2a>C1a). Higher concentration
86
of Cu was removed by roots in 10 mg L-1 as compared to 15 mg L-1 from synthetic leachates
spiked with EDTA. It revealed that crushed brick (2 kg) and slag (2 kg) have shown efficiency
equivalent to optimum (2.5 mM) dose of EDTA and C.A for removal of Zn and Cu. Efficient
sorption capacity for different elements by crushed brick (Djeribi and Hamdaoui, 2008) and slag
(Wang et al., 2016) was due to active sites on particle surface.
In fourth experiment, kinetic study of Cu and Zn removal from MSW leachate was conducted to
analyze metal removal with time variation and results showed that P. australis achieved highest
removal of Cu at 21st day of experiment. In treatment 2, Cu removal of crushed brick was
significantly different from other substrates (limestone, steel slag) without plants. In treatment 3
P. australis in crushed brick removed 80% and 95% Cu at 14th and 21st day of experiment,
respectively. Performance of T. latifolia was prominent in steel slag for removal of Cu at 11th,
14th, 17th and 21st day of experiment.
Based on the results of Phase I, P. australis and T. latifolia were selected for plantation in pilot
scale vertical flow constructed wetland. Crushed brick and steel slag were chosen as efficient
substrates for above mentioned macrophytes in pilot scale CW.
4.5 Pilot scale vertical flow constructed wetland
Performance of pilot scale vertical flow constructed wetland in treatment of municipal solid
waste leachate at different retention time in multi chambers was investigated. The batch and
continuous mode of treatment were used in constructed wetland. Detailed methodology is
provided in section 3.5. The reason behind batch mode ascribed to high performance efficiency
of constructed wetland as long time is required for better interaction between plants (Rai et al.,
2015) and substrates.
4.5.1 Removal of Cu, Zn, Pb and COD in pilot scale constructed wetland at HRT of 21 and 14 days in batch mode
In present work chamber A and B were activated at initial stage as shown in section of
methodology. Loading rate of Cu was 0.017 g m-2 day-1 with percentage removal of 96 and 98%
87
with HRT of 21 days in chamber A and B, respectively (Fig 4.22). Chamber A was planted with
two hyperaccumulator species (P. australis and T. latifolia) in presence of crushed brick and
sand and gravel. At initiation stage maximum retention time was provided for plants and
substrates to acclimatize and develop synergistic ecological relation. Madera-Parra et al. (2013)
provided retention time of 21 days to hyperaccumulator species in order to maximize metal
removal from synthetic leachate.
Loading rate of Zn was 0.22 g m-2 day-1 with percentage removal of 98 and 97 % in chamber A
and B at HRT of 21 days, respectively (Fig 4.22). On the other side loading rate of Pb in
chamber A and B was 0.061 g m-2day-1 with removal percentage of 90 and 94% at same HRT,
respectively. Chamber A and B were planted with two hyperaccumulator species (P. australis
and T. latifolia) in presence of crushed brick and steel slag, respectively.
Despite of high loading rate of Zn 0.22 g m-2 day-1, both chambers A and B showed stable and
efficient removal at HRT of 21 days as compared to that of Pb. Chamber B has shown better
removal percentage of Pb 94% as compared to chamber A 90% at HRT of 21 days (Fig 4.21).
Loading rate of COD was 44 g m-2day-1 with removal percentage of 93 and 94% in chamber A
and B at HRT of 21 days, respectively (Fig 4.23). Loading rate of COD was higher (44 g m-2 day-
1) as compared to Zn (0.22 g m-2 day-1) and Pb (0.061 g m-2 day-1). Both chambers have shown
efficient removal of COD at HRT of 21 days. The Zn removal rates were also higher (98%) in
chamber A and (97%) in chamber B. Crushed brick and steel slag were performing the role of
substrates in chamber A and B, respectively. Energy dispersive x-ray (supplementary data)
revealed presence of different ions including Fe and Al ions which might helped in absorption of
Zn and Pb from leachate. Whereas two macrophytes species in chamber A and B have affinity
for metals like Cu, Zn, Pb and Cr (Kumari and Tripathi, 2015) and played role in improving
effluent quality. It is worthy to note that substrates and macrophytes may play their role if
maximum retention is provided. Therefore parameter of retention time is key factor affecting
performance of plants, substrates, microbes (Ghosh and Gopal, 2010). High residence time of
leachate in chamber A removed metals COD significantly. Respiration of plants, mixing of
oxygen at surface of chamber area played important role in aerobic degradation of COD. Fan et
al. (2013) revealed efficient removal of COD with aeration in constructed wetland in presence of
88
macrophytes. Bisone et al. (2016) reported intermittent feeding strategy improved performance
of CW as it supported higher loads with longer feeding which favored infiltration and promoting
accumulation in plants (Madera-Parra et al., 2013).
Four chamber (A, B, C and D) were activated at second stage as shown in Table 2. In this set of
run, hydraulic retention time in chambers was reduced to 14 days and loads of Cu and COD were
increased. Loading rate of Cu was 0.049 g m-2 day-1in chamber A, B, C and D with percentage
removal 93, 95, 98 and 98%, respectively (Fig 4.24). It should be noted at HRT of 14 days that
trend of percentage removal of Cu decreased in chamber A and B as compared to that in 21 days.
Plants and substrates stabilized in chamber A and B by developing synergistic relation at HRT of
21 days and it supported plants and substrates to remove metal at HRT of 14 days. Percentage
removal in chamber C (98%) and D (98%) was higher as compared to chamber A (93%) and B
(95%). Plants and substrates in chamber C and D responded significantly well.
Loading rate of Zn was 1.32 gm-2day-1with removal rate of 99, 98, 99 and 99% in chamber A, B,
C and D, respectively (Fig 4.24). Whereas loading rate of Pb was 0.092 gm-2day-1with removal
efficiency of 95, 95, 97 and 95% in chamber A, B, C and D at HRT of 14 days, respectively.
COD loading rate was 66.18 g m-2 day-1with removal percentage of 92, 93, 93 and 93% in
chamber A, B, C and D at HRT of 14 days, respectively. It is worthy to note that loading rates of
Zn (1.32 g m-2 day-1), Pb (0.092 g m-2 day-1) and COD (66.18 g m-2 day-1) increased by
decreasing HRT from 21 days to 14 days. Minor fluctuations in removal percentage of Zn in
chamber A (99%) and chamber B (98%) at HRT of 14 days were observed as compared to
chamber A (98%) and chamber B (97%) at HRT of 21 days, respectively. Removal percentage of
Zn in chamber C (99%) and D (99%) was slightly higher than chamber A (99%) and B (98%).
Removal percentage of Pb in chamber A and B was 95% and 95% at HRT of 14 days (Fig 4.23),
higher as compared to removal percentage in chamber A (90%) and B (94%) at HRT of 21 days,
respectively. Removal rate of Pb in chamber C and chamber D was 97.83% and 95.89% which
was slightly higher than removal rate of chamber A (95%) and chamber B (95%) at HRT of 14
days, respectively. Papaevangelou et al. (2017) investigated removal of chromium in batch and
continuous experiments in parallel operation of pilot scale horizontal subsurface flow and
vertical flow constructed wetland planted with T. latifolia. Planted units of both CW have
performed better for removal of chromium at HRT of 6, 8 and 20 days. Present work was in
89
accordance to performance results of T. latifolia for removal of Zn and Pb in pilot scale VFCW
at HRT of 21 and 14 days reported by Papaevangelou et al. (2017).
Figure 4.22 Removal of a) Cu, b) Zn and c) Pb at HRT of 21 days in Pilot scale Constructed Wetland in Batch mode (Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis,
T. latifolia, steel slag)
94
95
96
97
98
99
100
0
0.006
0.012
0.018
0.024
21 21
Cu -
Rem
oval
(%)
Cu -
Load
ing
Rate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate %Removal
A B
96
97
98
99
100
0
0.06
0.12
0.18
0.24
0.3
21 21
Zn -
Rem
oval
(%)
Zn L
oadi
ng R
ate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B
b)
8486889092949698100
0
0.02
0.04
0.06
0.08
21 21
Pb -
Rem
oval
(%)
Pb L
oadi
ng R
ate
(g m
-2 d
ay-1
)
Time (Days) Loading rate Removal (%)
A B
c)
a)
90
Figure 4.23 Removal of COD in chamber A and B at HRT of 21 days in Batch mode
(Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag)
Performance of P. australis and T. latifolia in CW was studied by Kumari and Tripathi (2015)
for removal of Cu, Zn, Pb and Cr within 14 days in batch experiment. They found that mixture of
both species enhanced the removal of metals significantly as compared to alone culture. On the
other side, crushed brick and steel slag were playing key role in metal sorption from MSW
leachate in chamber A and B, respectively. Barca et al. (2013) experimented steel slag in
subsurface horizontal flow constructed wetland for removal of phosphorous in batch mode at
HRT of 3 and 1 day, respectively. Authors found that increase in temperature increases pH with
excessive CaO dissolution which help in precipitation of phosphate. Carbonates and oxides
present in slag in chamber B probably helped in removal of metals from MSW leachate. Another
work by Hussain et al. (2015) slag was used as substrate in constructed wetland for removal of
phosphorous, nitrogen and COD. Slag was also used as sorbent for removal of metals for
removal of Cu, Cd and Zn (Wang et al., 2016) in column studies. Aziz et al. (2008) studied
removal of Cu, Zn, Pb, Ni and Cr in batch series at various pH using crushed brick and gravels.
Presence of carbonates in crushed brick was reasons for metals removal. Jia et al. (2014) found
COD decreased with increase in dosage of crushed brick in batch study. Crushed brick and steel
slag were used in lab scale constructed wetland for removal of Cu and Zn by Batool and Zeshan
(2017). In present work crushed brick and steel slag also performed efficiently for removal of
Cu, Zn, Pb and COD in pilot scale vertical flow constructed wetland at HRT of 21 and 14 days.
86
88
90
92
94
96
98
100
44
44.03
44.06
44.09
44.12
21 21
COD
- Rem
oval
(%)
COD-
Loa
ding
Rat
e (g
m-2
day
-1)
Time (Days)
Loading Rate %Removal
A B
91
Figure 4.24 Removal of a) Cu, b) Zn and c) Pb at HRT of 14 days in Pilot scale Constructed
Wetland in Batch mode (Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus,
sand and gravel)
88
90
92
94
96
98
100
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
14 14 14 14
Cu -
Rem
oval
(%)
Cu -
Loa
ding
Rat
e
(g
m-2
day
-1)
A B C D a)
98
99
100
00.20.40.60.8
11.21.41.6
14 14 14 14
Zn -
Rem
oval
(%)
Zn L
oadi
ng R
ate
(g
m-2
day
-1)
A B C D b)
919293949596979899100
0
0.02
0.04
0.06
0.08
0.1
0.12
14 14 14 14
Pb -
Rem
oval
(%)
Pb L
oadi
ng R
ate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B C D c)
92
Figure 4.25 Removal of COD at HRT of 14 days in Pilot scale Constructed Wetland in Batch mode
(Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus, sand and gravel)
Two different grasses (V. zizanioides, C. gyana) were planted in chamber C in presence of sand
and gravels. Efficient removal of Zn and Pb revealed the good performance of these grass
species. Suelee, (2015) found V. zizanioides is efficient in removal of Cu, Pb, Mn, Zn and Fe
without showing any signs of necrosis and wilting. E. globulus planted in chamber D in presence
of sand and gravels efficiently removed Zn and Pb from landfill leachate. Bakhshoodeh et al.
(2017) reported efficient removal of metals and COD from leachate using three stage pilot scale
horizontal flow constructed wetland planted with V. zizanioides at HRT of 15 days. Quality of
treated leachate was unable meet the Iranian effluent standards thus concluding that alone V.
zizanioides in multi stage HFCW cannot be used to treat leachate. In present work poly culture of
V. zizanioides and C. gyana in presence of sand and gravel have performed efficiently for
removal of metals and COD in batch mode at HRT of 14 days.
Minor fluctuation were observed in percentage removal of COD in chamber A (92%) and B
(93%) at HRT of 14 days (Fig 4.25) as compared to removal percentage in chamber A (93.03%)
and B (94%) at HRT of 21 days, respectively. No significant difference was observed in removal
percentage of COD in chamber C (93%) and chamber D (93%) to that of in chamber A (92%)
and B (93%) at HRT of 14 days. Akratos and Tsihrintzis (2007) reported that retention time
above eight days is optimum for removal of COD achieving removal percentage of (91%) in
horizontal subsurface flow constructed wetland. In present study retention time was reduced to 7
days after attaining maximum removal of Zn, Pb and COD at HRT of 14 days.
86
88
90
92
94
96
98
100
10
20
30
40
50
60
70
80
14 14 14 14
COD
- Rem
oval
(%)
COD
- Loa
ding
Rat
e
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B C D
93
4.5.2 Removal of Cu, Zn, Pb and COD in pilot scale constructed wetland at HRT of 35 and 5 days in continuous mode
All chambers were active in this stage (A, B, C, D and E) with residence time of 7 days in each
chamber mentioned in section of methodology. Leachate flowed continuously from chamber A
to E in four runs. In this phase all chambers (A, B, C, D and E) were active and wetland was
operated for a set of four runs in continuous flow mode at HRT of 7 days (overall HRT of 35
days). As stated in methodology section, effluent of preceding chamber served as influent for the
following chamber. As no significant difference in removal was found at HRT of 21 and 14 days
therefore it was operated in continuous mode. It was also assumed here that capacity of plants
and substrates have been build up for efficient removal based on results of previous HRT’s.
Significant removal of Cu was achieved in chamber A at HRT of 7 days and remaining Cu was
removed in the following chambers. For instance 49% of remaining Cu in effluent of chamber B
was removed at same HRT. Removal trend of Cu was 22, 40 and 67% increased as the leachate
moved through the following chambers C to E. This may be due to the minute amount that was
available for different plants and substrate. Loading rate of Zn was 2.64, 0.288, 0.1069, 0.0289
and 0.0111 g m-2 day-1in chamber A, B, C, D and E at HRT of 7 days in continuous mode of
operation, respectively (Fig 4.26). Removal rates of Zn was 83, 51, 52, 46 and 50% in chamber
A, B, C, D and E at same HRT, respectively. Removal of Zn was significantly high in chamber
A as compared to following chambers. Loading rates of Zn decreased subsequently from
chamber A followed by chamber E (2.64, 0.288, 0.1069, 0.0289 and 0.0111 g m-2 day-1) at HRT
of 35 days (Fig 4.26) due to continuous mode of operation. Ghosh and Gopal, (2010) found that
high retention time implies lower loading rates and provide more contact time thus enhancing the
stability of constructed wetland. Loading rate of Pb was 0.1853, 0.00612, 0.0033, 0.00169 and
0.000767 g m-2 day-1with removal percentage of 95, 27, 38, 48 and 42% in chamber A, B, C, D
and E at HRT of 35 days (Figure 4.26) in continuous mode of operation, respectively (Fig 4.26).
In continuous mode of operation effluent of preceding chamber was serving as influent of
following chamber therefore loading rate of Pb decreased subsequently from chamber A
followed by chamber E. Significant removal percentage in chamber A showed that maximum
removal of Pb occurred initially leaving less concentration of Pb for uptake by following
chambers. Loading rate of COD was 132, 4.57, 3.06, 1.95 and 1.21 g m-2 day-1 with removal
94
percentage of 96, 30, 34, 36 and 39% in chamber A, B, C, D and E at HRT of 35 days,
respectively (Fig 4.27).
Figure 4.26 Removal of a) Cu, b) Zn and c) Pb with HRT of 35 days in Continuous mode in Pilot
scale Constructed Wetland (Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E.
globulus, sand and gravel, Chamber E; C. indica, crushed brick and steel slag)
94
95
96
97
98
99
100
0
0.04
0.08
0.12
7 14 21 28 35
Cu -
Rem
oval
(%)
Cu -
Load
ing
Rate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B C D E
75
80
85
90
95
100
0
0.2
0.4
0.6
0.8
1
1.2
7 14 21 28 35
Zn -
Rem
oval
(%)
Zn L
oadi
ng R
ate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
b)
90
92
94
96
98
100
0
0.05
0.1
0.15
0.2
0.25
7 14 21 28 35
Pb -
Rem
oval
(%)
Pb L
oadi
ng R
ate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B C D E c)
a)
95
Figure 4.27 Percentage removal of COD at HRT of 35 day in Continuous mode in Pilot scale Constructed Wetland (Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T.
latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus, sand and gravel, Chamber E; C. indica, crushed brick and steel slag)
Significant removal of COD was observed in chamber A. Konnerup et al. (2009) also found that
heavy loads of domestic wastewater achieved higher removal percentage of TSS (>88%) and
COD (42 – 83%) in the beds of horizontal subsurface flow constructed wetland with nominal
detention time varying from 12 hrs to 4 days. All chambers were active with residence time of 1
day in each chamber and wetland was operated for four set of runs in continuous flow mode.
Effluent of preceding chamber served as influent for the following chamber. Loading rate of Cu
increased up to 0.92 g m-2 day-1 by lowering residence time to one day and significant removal of
96% Cu was achieved in chamber A. In the present study experiments with crushed brick and
macrophytes showed highest removal of metals. Aziz et al. (2008) found significant removal of
Cd, Pb, Zn, Ni, Cu and Cr in presence of crushed brick. Removal percentage of Cu was 41, 24,
14 and 56% with loading rate 0.03107, 0.00768, 0.00165, and 0.0024 g m-2 day-1 in chamber B,
C, D and E, respectively (Fig 4.28). Removal percentage was high in chamber B, D and E at
HRT of 35 days due to low loading rates as compared to that at HRT of 1 day. With decrease in
HRT from 35 to 5 days, Cu removal percentage decreased, however difference was significant in
chamber A and C.
Çakir et al. (2015) showed that increase in loading rates decrease the removal rate of COD.
Similarly, removal rate decreased with reduction in HRT and increase in HLR.
93
94
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99
100
0
20
40
60
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100
120
140
7 14 21 28 35
COD
- Rem
oval
(%)
COD
- Loa
ding
Rat
e (g
m-2
day
-1)
Time (Days)
Loading Rate Removal (%)
A B C D E
96
Figure 4.28 Percentage removal of Cu, Zn and Pb at HRT of 5 days in Pilot scale Constructed Wetland in Continuous mode (Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P.
australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus, sand and gravel, Chamber E; C. indica, crushed brick and steel slag)
Whereas, series of the same experiments in CW built the capacity of plants and substrates with
synergistic ecological interaction with each other to achieve maximum removal of metal at
lowest retention time in whole wetland. Work done by Xu et al. (2014) showed that removal
9293949596979899100
0
0.4
0.8
1.2
1 2 3 4 5
Cu -
Rem
oval
(%)
Cu -
Load
ing
Rate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B C D E aa)
75
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90
95
100
0
20
40
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100
1 2 3 4 5
Zn -
Rem
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(%)
Zn L
oadi
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ate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
b) A B C D E
75
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00.20.40.60.8
11.21.41.6
1 2 3 4 5
Pb -
Rem
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(%)
Pb L
oadi
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ate
(g m
-2 d
ay-1
)
Time (Days) Loading Rate Removal (%)
A B C D E c)
97
efficiency of total phosphorous, total suspended solids, Kjeldahl nitrogen increased with decease
in loading rates in constructed wetland. Loading rate of Zn was significantly high in chamber A
after lowering retention time to 1 day achieving 85% removal. Remaining concentration of Zn in
effluent of chamber A was treated in chamber B significantly achieving 94% removal with HRT
of one day. Trend of percentage removal of Zn increased from chamber B to chamber E and
attained 99% removal (Fig 4.28). Loading rate of Pb was significantly high in chamber A with
82% removal. Trend of percentage removal increased from chamber B onwards and attained
89% removal in chamber E. With high COD loading rate percentage removal of COD was 50%
in chamber A (Fig 4.29). 30% of remnant COD was removed in chamber B with significant
removal in chamber C, D and E (98%). Removal of chemical oxygen demand differed
significantly in all chambers influenced by design of constructed wetland, characteristics of
substrates, retention time and importantly oxygen concentration in beds/substrates (Vymazal,
2010).
Maximum removal of Zn, Pb and COD was achieved at HRT of 35 days therefore it was reduced
to 5 days in pilot scale vertical flow constructed wetland. Loading rate of Zn was 92.66, 10.54,
3.55, 1.58 and 0.437 g m-2 day-1 in chamber A, B, C, D and E with removal percentage of 87.03,
61.64, 53.83, 57.75 and 59.08%, respectively at HRT of 5 days (Fig 4.26). Loading rate of Pb
was increased with decrease in retention time from 7 days to 1 day in chamber A (1.29 g m-2 day-
1) with removal percentage of 82.86%. Mass loading of Pb was 0.198, 0.168, 0.129 and 0.106 g
m-2 day-1in chamber B, C, D and E with removal rate of 13.56, 0.79, 5.28 and 12.63%,
respectively at HRT of 5 day (Fig 4.27). Reduction in HRT from 35 days to 5 days increased
mass loading of COD (1853 g m-2day-1) in chamber A. Increased loading rate of COD decreased
the removal percentage in chamber A (49%) at HRT of 1 day (Fig 4.29) as compared to removal
percentage (96%) of COD at HRT of 7 days, respectively. Removal percentage decreased in
chamber B (31%) as compared to that in chamber A (49%) at HRT of 1 day in continuous mode
of operation. Significant increase in removal percentage of COD was observed from chamber C
to chamber E (47, 59 and 96%) at HRT of 1 day per chamber (Fig 4.29). Jia et al. (2014) found
COD decreased with increase in dosage of crushed brick in batch study. Furthermore, presence
of oxides and carbonates in steel slag also help in oxidation of COD. Substrate in chamber E was
composed of crushed brick and steel slag thus merging the benefits of both substrates enhanced
98
removal of COD from MSW leachate at HRT of 1 day. Stefanakis and Tsihrintzis (2012) studied
ten units of pilot scale vertical flow constructed wetland in continuous mode for three years.
Figure 4.29 Loading rate of COD in Pilot Scale Constructed Wetland at HRT of 5 days in
Continuous mode Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus, sand
and gravel, Chamber E; C. indica, crushed brick and steel slag)
Authors planted two macrophytes P. australis and T. latifolia in presence of porous media
substrate composed of zeolite, carbonate, igneous, and bauxite. Under high loads of 200 g m-2
day-1 significant COD removal was achieved by authors. Role of macrophytes in presence of
porous media has not shown significantly high removal of metals and is therefore not
recommended by authors. However, in present work crushed brick and steel slag has improved
performance of both macrophytes for significantly high removal of metals and COD. Matamoros
(2016) investigated difference between batch and continuous feeding mode for removal of
pesticides by micro algae and found higher removal efficiency in continuous mode of operation
at HRT of 2, 4 and 8 days. Papaevangelou et al. (2017) reported difference of batch and
continuous mode of operation in VFCW and HFCW. Authors found that hydraulic conditions
and feeding regime significantly affected the removal efficiency of chromium in both CWs.
Furthermore, mechanism for chromium removal involved higher retention in substrates as
compared to accumulation in plants. Results of present study also revealed significant
accumulation of Zn in substrate of chamber A and Cu in chamber B. Whereas Pb was
significantly accumulated in substrate chamber E as compared to preceding chambers. Energy
dispersive x-ray spectroscopy revealed presence of silicon and aluminum ions, carbonates and
0
20
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0
500
1000
1500
2000
2500
1 2 3 4 5
COD
- Rem
oval
(%)
COD
- Loa
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Rat
e (g
m-2
day
-1)
Time (Days) Loading Rate Removal (%)
A B C D E
99
oxides making it an efficient adsorbent for Cu, Zn and Pb. Akratos and Tsihrintzis (2007) have
investigated effect of HRT, vegetation, porous media in five pilot scale units of horizontal
subsurface wetlands operated in continuous mode for two years. Operating with four different
HRT’s (6, 8, 14 and 20 days) maximum removal of COD, P-PO4-3 and TKN was attained at HRT
of 8 days. Whereas present work showed maximum COD removal achieved at HRT of 5 days
VFCW in continuous mode. Results showed that plants and substrates in five chambers were
performing key role for removal of COD, Cu, Zn and Pb at different HRTs. Accumulation of
metals in plant species and substrates are discussed in the following section. It is safe to assume
that vertical flow pilot scale constructed wetland has performed efficiently for removal of metals
and COD at high loading rate with least retention time of one day. Five chambered VFCW with
poly culture of plant species in presence of different substrates provided a full leachate treatment
with effluent quality meeting national wastewater guidelines.
It can be summarized that statistically high removal percentage of Zn, Pb and COD at HRT 35
and HRT of 5 days was observed. In continuous mode, maximum removal of metals was
achieved in first chamber thus secondary treated effluent was irrigating other chambers which
enhanced the overall performance of VFCW at lower HRT. In case of COD maximum removal
was achieved in chamber E exhibiting performance of tertiary treatment in presence of crushed
brick and steel slag planted with C. indica.
Table 4.7 Removal of Cu at HRT of 35 and 5 days in continuous mode of operation
*significant removal at p < 0.05
HRT 35 days HRT 5 days
Chambers Cu Loading
Rate
(g m-2 day-1 )
Removal (%) Cu Loading
Rate
( g m-2day-1 )
Removal (%)
A 0.0992 96* 0.926 96*
B 0.0022 49 0.0311 41
C 0.00079 22 0.0076 24
D 0.00071 40 0.0016 14
E 0.00021 67 0.0024 56
100
Less retention of leachate in chambers provide less contact time with substrates and plants.
Difference in removal percentage of COD (49, 31, 47, 59 and 96%) in Table 4.8 was observed at
HRT of five days as compared to that of at HRT of 35 days (96, 30, 34, 36 and 39%) (Table 4.8).
Results showed that maximum removal of COD, Zn and Pb was achieved in chamber A reducing
the efforts of preceding chambers. On the other side, COD with loading rate of 132 g m-2 day-1
supported plant growth and removal efficiency of Pb and Zn simultaneously.
The summary of pilot scale constructed wetland in continuous mode has been shown in Table
4.3. Loading rate of Zn decreased (2.64, 0.288, 0.106, 0.0289, 0.0111 g m-2 day-1) from chamber
A through B, C, D to chamber E in continuous mode at HRT of 35 days at individual chamber
(Table 4.8). The trend of percentage removal was high in chamber A (83%) as compared to the
following chambers which is in line with other study Vassiliki et al. (2017). Percentage removal
of Pb in continuous mode of operation was efficient in chamber A (95%) and it decreased in
preceding chambers B, C, D and E (27, 38, 48 and 42%). Decrease in retention time increased
loading rate of COD in chamber A (132 g m-2 day-1) with gradual decrease (4.57, 3.06, 1.95 and
1.21 g m-2 day-1) in the following chambers (B, C, D and E) at HRT of 35 days (Table 4.8).
Despite of increase in loading rate with decrease in retention time, removal efficiency remained
stable at HRT of 35 days. Loading rates significantly increased with sharp decline in retention
time at HRT of 5 days. Detail discussion of metal accumulation in plants and substrates are given
in the following sections.
4.5.3 Accumulation of Cu, Zn and Pb in plants and substrates
Accumulation of Cu, Zn, and Pb was studied in shoots and roots of P. australis, T. latifolia in
chamber A and B, V. zizanioides, C. Gyana in chamber C, E. globulus in chamber D and C.
indica in chamber E, respectively. Accumulation of Cu in P. australis in chamber A was
significantly higher in roots (0.51 mg kg-1) as compared to shoots (0.21 mg kg-1). Whereas high
Cu accumulation was measured in shoots (0.27 mg kg-1) of T. latifolia as compared to roots (0.21
mg kg-1) (Fig 4.30). Results by Maddison et al. (2009) showed high accumulation of Cu in
shoots of cattail which supports the result of present study. Absorption of Cu in substrate of
chamber A was 4.6 mg kg-1.
101
Table 4.8 Removal of a) Zn, b) Pb and c) COD at HRT of 35 and 5 days in continuous mode of operation
*significant removal at p < 0.05
*significant removal at p < 0.05
a) HRT 35 days HRT 5 days
Chambers Zn Loading
Rate
( g m-2 day-1)
Removal (%) Zn Loading Rate
( g m-2 day-1)
Removal (%)
A 2.64 83.68* 92.66 87.03*
B 0.28 51.79 10.54 61.64
C 0.10 52.40 3.55 53.83
D 0.02 46.42 1.58 57.75
E 0.01 50.26 0.437 59.08
b) HRT 35 days HRT 5 days
Chambers Pb Loading
Rate
( g m-2day-1)
Removal (%) Pb Loading Rate
( g m-2day-1)
Removal (%)
A 0.185 95.01* 1.29 82.86*
B 0.006 27.47 0.198 13.56
C 0.003 38.92 0.168 0.790
D 0.001 48.04 0.129 5.28
E 0.0007 42.21 0.106 12.63
c) HRT 35 days HRT 5 days
Chambers COD Loading
Rate
(g m-2 day-1)
Removal (%) COD Loading
Rate
(g m-2day-1)
Removal (%)
A 132 96* 1853 49*
B 4.57 30 932 31
C 3.06 34 640 47
D 1.95 36 329 59**
E 1.21 39 133 96***
102
No significant difference was observed (p<0.05) in Cu accumulation by shoots (0.08 mg kg-1)
and roots (0.04 mg kg-1) of P. australis in chamber B (Fig 4.30). On the other side significant
amount of Cu (p<0.05) was measured in roots (0.49 mg kg-1) of T. latifolia in chamber B as
compared to shoots (0.11 mg kg-1). High metal sequestration in roots reveals metal tolerance
strategy of plants to prevent above ground biomass (shoots and flowers) from metal induced
toxicity and injuries (Klink et al., 2013).
V. zizanioides accumulated significantly (p<0.05) high concentration of Cu in shoots (0.44 mg
kg-1) as compared to roots (0.07 mg kg-1). Badejo et al. (2015) found V. zizanioides and P.
australis were able to remove 97% of Cr with sustained growth in constructed wetland.
Moreover he found that 75% of heavy metals were bonded with roots and substrates in
constructed wetland. As the plants and substrates assimilate trace metals therefore these plants
must be dumped in engineered landfill sites for safe disposal (Salt et al., 1995).
No significant difference was observed in shoot (0.24 mg kg-1) and root (0.13 mg kg-1) of C.
gyana in chamber C (Fig 4.30). Significant amount (p<0.05) of Cu was accumulated in shoots
(0.24 mg kg-1) of E. globulus as compared to roots (0.05 mg kg-1). Slightly higher concentration
of Cu was measured in shoots (0.29 mg kg-1) of C. indica as compared to roots (0.12 mg kg-1).
Work by Konnerup et al. (2009) found that C. indica increased the aesthetic value of
constructed wetland along with good growth after irrigation with high nutrient wastewater. In
present work polyculture in different chambers of wetland supported the removal of Cu with
better growth performance in substrates. According to Bragato et al. (2006) P. australis and T.
latifolia were not considered as hyperaccumulator species, besides they have high biomass, fast
growth, dense root system and possesses quality to accumulate metals in their aerial tissues
which highlighted them as hyperaccumlator. Plants of chamber B have accumulated less amount
of Cu as compared to plants of chamber A supported by significantly high absorption of Cu by
steel slag (Fig 4.31). Despite of Cu impurities present in steel slag, absorption of Cu was
prominent in substrate of chamber B. Korkusuz et al. (2005) studied removal of phosphorus in
constructed wetland filled with steel slag and planted with P. australis. They observed higher
removal of phosphorous in wetlands with slag beds as compared to gravel filled wetland. Work
by Abou-Elela et al. (2013) suggested that combinations of different plant species in constructed
wetland may improve efficiency and show variation in metal accumulation in different tissues of
plant.
103
In present study COD facilitated plant growth in all chambers of constructed wetland. Better
growth of plant supported efficient removal of metals as plants convey oxygen to roots and
rhizosphere and creates microsite aerobically facilitating Nitrosomonas bacteria to convert nitrite
to nitrate (Ye and Li, 2009).
Accumulation of Zn, and Pb was studied in shoots and roots of P. australis, T. latifolia in
chamber A and B, V. zizanioides, C. Gyana in chamber C, E. globulus in chamber D and C.
indica in chamber E, respectively. Significantly higher accumulation of Zn was observed in roots
of P. australis (148 mg kg-1as compared to shoots (11.72 mg kg-1) in presence of crushed brick
(chamber A). Similar trend was observed in roots of T. latifolia (48 mg kg-1) as compared to
shoots (11.30 mg kg-1) in chamber A (Fig 4.30). High metal sequestration in roots reveals metal
tolerance strategy of plants to prevent above ground biomass (shoots and flowers) from metal
induced toxicity and injuries (Klink et al., 2013). Accumulation of Zn in roots of P. australis (29
mg kg-1) was significantly high (p<0.05) than shoots (6.7 mg kg-1) and shoots of T. latifolia (7.8
mg kg-1) has significantly higher Zn than roots (5.02 mg kg-1) in chamber B, respectively (Fig
4.30). Zn is required for production of tryptophan which is an originator of indole-3-acetic acid
(IAA hormone) and accomplishes its function in stems (Hopkins and Huner, 2009). Because of
these functions, Zn is often found rather in the aboveground than belowground biomass of
wetland plants. However, efficient translocation signifies a key trait of hyperaccumulation and
efficient trace element transportation from roots to shoots (Zhao et al., 2016).
Roots of V. zizanioides (7.8 mg kg-1) and C. gayana (21.4 mg kg-1) accumulated more Zn than
shoots of both plants (4.6 mg kg-1) and (4.7 mg kg-1), respectively (Fig 4.30). Similar trend was
observed in roots of E. globulus (8.9 mg kg-1) and C. indica (3.9 mg kg-1) as compared to shoots
(5.4 and 5.1 mg kg-1) in sand, gravels and crushed brick, slag, respectively. It can be summarized
that significant difference was observed in sorption of Zn by different substrates in multi
chambered VFCW (Fig 4.31). Absorption of Zn was significantly (p<0.05) prominent in crushed
brick (614 mg kg-1) and steel slag (347 mg kg-1), sand and gravels in chamber C (184 mg kg-1),
and D (160 mg kg-1), respectively (Fig 4.31). Pb accumulation was significantly high in roots of
P. australis (12.5 mg kg-1) and T. latifolia (6.9 mg kg-1) as compared to shoots (0.5 and 1.45 mg
kg-1) in crushed brick. Comparatively less accumulation of Pb in roots of both species (6.1 and
6.4 mg kg-1) was observed as compared to shoots (0.74 and 0.81 mg kg-1) in steel slag in
chamber B, respectively (Fig 4.31). High accumulation of Pb was noticed in roots of V.
104
zizanioides (5.6 mg kg-1) and C. gayana (9.2 mg kg-1) as compared to shoots (0.42 and 0.61 mg
kg-1). E. globulus and C. indica accumulated significantly high concentration of Pb in roots (4.1
and 4.4 mg kg-1) in chamber D and E than shoots (0.47 and 0.45 mg kg-1), respectively. Work by
Ebrahimi (2014) showed that E. globulus has potential to removal Zn and Pb from metal
contaminated sites. Bouchama et al. (2016) found that GPOX enzyme helps in the elimination of
excess H2O2 produced in the roots, which could be an adaptation mechanism in response to
heavy metals effects, especially in plants who accumulate heavy metals in roots more than in
leaves like P. australis. C. indica has accumulated less amount of Pb as compared to all other
plant species because significant amount of Pb was absorbed by substrates (20.7 mg kg-1) of
chamber E (Fig 4.31). Absorption of Pb was significantly high in chamber B consisted of steel
slag (7.13 mg kg-1) and chamber E consisted of crushed brick and steel slag (20.72 mg kg-1) as
compared to chamber A consisted of crushed brick (11.5 mg kg-1), sand and gravels in C (8.38
mg kg-1) and D (7.39 mg kg-1), respectively (Fig 4.31). Whereas significantly high amount of
metals inhibited translocation of metals to shoots. Substrates were not only providing medium
for plant growth but also acts as a primary sink of metals. In present work it was found that
crushed brick and steel slag are mainly composed of fine particles containing ions of aluminium,
iron, silicon, titanium oxides and hydroxides shown in results of EDX spectrometry
(supplementary data). Different sorption studies (Ebbs et al., 2016; Wang et al., 2016;
Nehrenheim et al., 2008) showed that presence of silicon, aluminium ions, oxides and
hydroxides help in sorption of different metals like Cu, Zn, Pb, Cr and Cd. Additionally, the
removal of metals retained in the aboveground biomass can be removed through harvesting and
proper disposal. The fact that the VF chambers operated more than a year highlights the
contribution of the root zone to the continuous entrapment of Cu, Zn and Pb.
4.5.4 Translocation and bioaccumulation of metals in plants of different chambers of pilot scale vertical flow constructed wetland
Translocation is metal accumulation in shoots as compared to roots of plants. If accumulation is
higher in shoots than translocation will be considered efficient and vice versa (Fig 4.32). In
present study plants were harvested at the end of experiments in pilot scale constructed wetland.
P. australis and T. latifolia were at maximum exposure and shown translocation of Cu 0.23 and
1.26 in chamber A and 2.06 and 0.22 in chamber B, respectively.
105
Figure 4.30 Accumulation of a) Cu, b) Zn and c) Pb in shoots and roots of plants in chamber A, B, C, D and E of constructed wetland Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E.
globulus, sand and gravel, Chamber E; C. indica, crushed brick and steel slag)
0
0.1
0.2
0.3
0.4
0.5
0.6
Phragmites Typha Phragmites Typha Vetiver Chloris Eucalyptus CannaCu a
ccum
ulat
ion
(mg
kg-1
)
Plant species
Shoots Roots
C B A D E
a
b
c
d
b
a
b
c
d
c
020406080
100120140160180
Phragmites Typha Phragmites Typha Vetiver Chloris Eucalyptus Canna
Zn a
ccum
ulat
ion
(mg
kg-1
)
Plant species Shoots Roots
A B C D E a
b c
a b d
b)
0
2
4
6
8
10
12
14
Phragmites Typha Phragmites Typha Vetiver Chloris Eucalyptus Canna
Pb a
ccum
ulat
ion
(mg
kg-1
)
Plant species
Shoots Roots
A B C D E a
b
c
d
b
d
c)
a)
106
Figure 4.31 Accumulation of a) Cu, b) Zn and c) Pb in substrates of chambers A, B, C, D and E. Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus, sand and gravel,
Chamber E; C. indica, crushed brick and steel slag)
0
50
100
150
200
250
300
350
A B C D ECu A
ccum
ulat
ion
(mg
kg-1
)
Chambers
a b
0
100
200
300
400
500
600
700
A B C D E
Zn a
ccum
ulat
ion
(mg
kg-1
)
Chambers
a
b
c
d
c
b)
0
5
10
15
20
25
A B C D E
Pb a
ccum
ulat
ion
(mg
kg-1
)
Chambers
a
b
c c
d
a)
c)
107
Bragato et al. (2006) have worked with P. australis and found Cu higher in shoots than roots. T.
latifolia and P. australis in presence of crushed brick and steel slag has bioaccumulated
significantly high amount of Cu, Zn and Pb as compared to other plant species (Fig 3a). Both
macrophytes are known as hyperaccumulators (Vymazal and Březinová, 2016; Kumari and
Tripathi, 2015). A hyperaccumulator plant can be distinguished from a non-hyperaccumulator by
its capability to absorb and accumulate exceptionally high (50–100 times than non-
accumulators) concentrations (Table 2.1) of metals in their leaves without severe damage to vital
physiological processes and plant growth. Translocation of Zn and Pb in T. latifolia and P.
australis was poor despite of higher accumulation of metals in biomass. In this scenario
substrates played substantial role in retaining metals by absorption, thus also increasing
accumulation in roots of macrophytes. Badejo et al. (2015) found V. zizanioides and P. australis
were able to remove 97% of chromium with good growth in constructed wetland. Moreover he
found that 75% of heavy metals were bonded with roots and substrates in constructed wetland.
Generally, the rhizosphere is the zone where biological and physicochemical processes occur
through interactions between microorganisms, plants substrate and pollutants (Philippe et al.,
2015; Sultana et al., 2015). Phytoremediation processes regarding the overall removal of Cu, Zn
and Pb are considered effective when both metals entrapped in the rhizome system (belowground
biomass) (Calheiros et al., 2008; Rai et al., 2015).
Drzewiecka et al. (2011) has found better accumulation of Cu in leaves of P. australis and T.
augustifolia in two different seasons. Results of present work were similar to Bragato et al.
(2006) as substrates accumulated significantly high Cu in chamber B. On the other side
significantly high (p<0.05) translocation of Cu was observed in V. zizanioides (TF<7) in
chamber C as compared to other plant species. V. zizanioides was planted in sand and gravel
instead of crushed brick and steel slag. Results showed that sand and gravel as substrates may
not accumulated significant amount of Cu in chamber C (3.4 mg kg-1) and in chamber D (1.9 mg
kg-1) which facilitated its translocation to shoots of V. zizanioides (Suelee, 2015). E. globulus has
shown significantly high (p<0.05)translocation (4.74) with low bioconcentration of Cu and these
findings are similar to result obtained by Shukla et al. (2011). C. indica has shown low
translocation of Cu (2.42) in chamber E as compared to V. zizanioides and E. globulus (Fig
4.32). Chamber E has combination of substrates (crushed brick and steel slag) has high Cu
accumulation thus acting as filter by retaining maximum Cu (303 mg kg-1) in substrates. It must
108
be noted here that P. australis and T. latifolia in crushed brick and steel slag, respectively has not
shown significant translocation of Cu as compared to C. indica because biomass of C. indica was
less than that of macrophytes (Sultana et al., 2015). High translocation of Zn 1.55 was shown by
T. latifolia in steel slag (chamber B) and 1.28 by C. indica in presence of steel slag and crushed
brick in chamber E (Fig 4.32).
Figure 4.32 Translocation of Cu, Zn and Pb in plants in different chambers of pilot scale constructed wetland Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T.
latifolia, steel slag, Chamber C; V. zizanioides, C. gayna, sand and gravels, Chamber D; E. globulus, sand and gravel, Chamber E; C. indica, crushed brick and steel slag)
Figure 4.33 Bioconcentration of Cu, Zn and Pb in different plant species. Chamber A; P. australis, T. latifolia, crushed brick, Chamber B; P. australis, T. latifolia, steel slag, Chamber C; V. zizanioides, C.
gayna, sand and gravels, Chamber D; E. globulus, sand and gravel, Chamber E; C. indica, crushed brick and steel slag)
0
1
2
3
4
5
6
7
Phragmitesaustralis
Typhalatifolia
Phragmitesaustralis
Typhalatifolia
Vetiverzizanioides
Chloris gayna Eucalyptusglobulus
Canna indica
Tra
nslo
catio
n Fa
ctor
Plant Species Cu Zn Pb
A B C D E
0
5
10
15
20
25
30
35
Phragmitesaustralis
Typhalatifolia
Phragmitesaustralis
Typhalatifolia
Vetiverzizanioides
Chlorisgayna
Eucalyptusglobulus
Canna indica
Bio
conc
entr
atio
n Fa
ctor
Plant Species
Cu Zn Pb
A B C D E
109
P. australis (0.078), T. latifolia (0.232) in chamber A, and P. australis (0.22) in chamber B and
V. zizanioides (0.59), Chloris gyana (0.22) in chamber C and E. globulus (0.612) in chamber D
have shown minimal translocation of Zn as compared to other metals. Ait et al. (2004) reported
high accumulation of Zn in roots which decreased its translocation to shoots (Fig 4.32). In case
of Pb no significant translocation was observed in P. australis (0.046), T. latifolia (0.21) in
chamber A, P. australis (0.12), T. latifolia (0.124) in chamber B, V. zizanioides (0.074), C.
gyana (0.065) in chamber C, E. globulus (0.11) in chamber D and C. indica (0.11) in chamber E,
respectively. Pb was significantly absorbed (p<0.05) by roots of plants therefore inhibiting its
translocation to shoots as action of defensive mechanism (Fig 4.32).
Ability of phytoextraction is depicted by bioconcentration of metals in different tissues and
biomass of plants. According to Reeves, (2003), normal capacity of hyperaccumulator is to
accumulate 1000 ppm Cu, 10,000 ppm Zn and Pb (Anning et al., 2013) therefore showing high
bioconcentration in them. In present study plants were harvested at the end of experiment to
calculate total accumulation of metals. Pilot scale constructed wetland was working in batch and
continuous mode from chamber A followed by chamber E. Bioconcentration of Cu was
calculated on basis of metal accumulation in plant and metal present in substrates.
Bioconcentration of Cu in P. australis and T. latifolia was 2.77 and 2.15 in chamber A and 0.009
and 0.047 in chamber B, respectively (Fig 4.33). Significantly high (p<0.05) sorption of Cu in
chamber B (296 mg kg-1) revealed that substrates trapped maximum amount of Cu as action of
filtration to prevent plants from toxicity. Therefore bioconcentration of Cu was less in plants of
chamber B as compared to chamber A.
Bioconcentration of Cu by V. zizanioides and C. gyana in chamber C was 6.38 and 1.73,
respectively. Sand and gravels in chamber C has shown less sorption of Cu (3.4 mg kg-1) thus
allowing plants to accumulate maximum amount of metal. Similarly bioconcentration of E.
globulus was 1.50 in presence of sand and gravels (1.9 mg kg-1). C. indica in presence of crushed
brick and steel has shown less bioconcentration of Cu (0.034) as compared to plant species of
chamber A (Fig 4.33). Though Konnerup et al. (2009) showed that C. indica have ability to
remove nutrients at high loading rates. Crushed brick and steel slag as substrates in chamber E
has played important role in retaining Cu (303 mg kg-1) in them. Thus high bioconcentration
110
factor of P. australis and T. latifolia showed their role for remediation of Cu from MSW leachate
in vertical flow constructed wetland.
Bioconcentration of Zn in P. australis and T. latifolia was 5.23 and 1.95 in chamber A and 2.40
and 0.84 in chamber B, respectively. V. zizanioides and C. gyana showed 0.81 and 1.7
bioconcentration of Zn in chamber C (Fig 4.33). Bioconcentration of Zn in E. globulus was
0.897 in chamber D and 0.56 in C. indica in chamber E, respectively. It can be observed that P.
australis and T. latifolia have shown significantly high bioconcentration in chamber A as
compared to other plants. Whereas C. gyana has also shown high BCF of Zn in chamber C. High
absorption of metals by substrates may help plants to accumulate them in roots or shoots (Deng
et al., 2004) which directly affects the translocation and bioconcentration factor. High
bioconcentration of Pb by P. australis and T. latifolia was 22.69 and 14.50 was observed in
chamber A as compared to chamber B (9.31 and 9.67), respectively. V. zizanioides and C. gyana
revealed 8.7 and 14 bioconcentration of Zn in chamber C and 6.13 by E. globulus in chamber D
and 5.93 by C. indica in chamber E, respectively (Fig 4.33). Results showed that Pb was readily
available to plants for accumulation. Phytoavailability depends on exposure and nature of metals
(Xiong et al., 2014). Phytoavailability depends on exposure and nature of metals (Xiong et al.,
2014). Moreover, indigenous microbial community in roots of plants managed to cope with the
contaminants (Syranidou et al., 2016) and enhance their ability of phytoremediation. It can be
summarized that translocation and bioaccumulation of metals varied in six plants species
depending upon substrates. Substrates were playing key role in determining the behavior of
plants toward metal stress.
Summary Phase II – Pilot scale experiment
Pilot scale vertical flow constructed wetland was operated with four different retention time of
21, 14, 35 and 5 day in batch and continuous mode. Results showed that high retention time
provided more margin for removal of Cu, Zn and Pb from VFCW. Four runs were operated at
HRT of 21 days in batch mode achieving approximately 95% removal of Cu, Zn, Pb and COD.
Three runs were operated at HRT of 14 days in batch mode attaining high removal in chamber C
111
and D as compared to chamber A and B. Overall removal was approximately 90% in all
chambers at HRT of 14 days.
Continuous mode was operated at HRT of 35 and 5 days, respectively in VFCW. It was observed
that decrease in retention time increased loading rates of Cu, Zn, Pb and COD with declined
removal percentage. Above 96% removal of Cu was achieved in chamber A at HRT of 7 days
and remaining Cu was removed in the following chambers. For instance 49% of remaining Cu in
effluent of chamber B was removed at same HRT. Removal trend of Cu was 22, 40 and 67%
increased as the leachate moved through the following chambers C to E.
Removal rates of Zn was 83, 51, 52, 46 and 50% in chamber A, B, C, D and E at same HRT,
respectively. Percentage removal of Pb in continuous mode of operation was efficient in chamber
A (95%) and it decreased in preceding chambers B, C, D and E (27, 38, 48 and 42%). After
lowering HRT to one day; removal percentage of Cu was 41, 24, 14 and 56% in chamber A, B,
C, D and E, respectively. Loading rate of Zn was significantly high in chamber A after lowering
retention time to 1 day achieving 85% removal. Remaining concentration of Zn in effluent of
chamber A was treated in chamber B achieving 94% removal with HRT of one day. Trend of
percentage removal of Zn increased from chamber B to chamber E and attained 99% removal
(Fig 4.26). Loading rate of Pb was high in chamber A with 82% removal. Trend of percentage
removal increased from chamber B onwards and attained 89% removal in chamber E. With high
COD loading rate percentage removal of COD was 50% in chamber A (Fig 4.26).
Approximately, 30% of remnant COD was removed in chamber B with significant removal in
chamber C, D and E (98%).
It can be observed that removal rates were high in batch mode at higher retention time of 21 and
14 days. As HRT lowered removal rate decreased in each chamber but overall removal achieved
was 95% which showed at VFCW performed efficiently at high loading rates of metals and COD
with shorter retention time.
In present study plants were harvested at the end of experiments in pilot scale constructed
wetland. P. australis and T. latifolia were at maximum exposure and shown translocation of Cu
112
0.23 and 1.26 in chamber A and 2.06 and 0.22 in chamber B, respectively. On the other side
significantly high (p<0.05) translocation of Cu was observed in V. zizanioides (TF<7) in
chamber C as compared to other plant species. V. zizanioides was planted in sand and gravel
instead of crushed brick and steel slag. C. indica has shown low translocation of Cu (2.42) in
chamber E as compared to V. zizanioides and E. globulus (Fig 4.29). Chamber E has
combination of substrates (crushed brick and steel slag) has high Cu accumulation thus acting as
filter by retaining maximum Cu (303 mg kg-1) in substrates. It must be noted here that P.
australis and T. latifolia in crushed brick and steel slag, respectively has not shown significant
translocation of Cu as compared to C. indica because biomass of C. indica was less than that of
macrophytes. High translocation of Zn 1.55 was shown by T. latifolia in steel slag (chamber B)
and 1.28 by C. indica in presence of steel slag and crushed brick in chamber E (Fig 4.29). Pb has
not shown significant translocation to shoots of plants in different chambers. Bioconcentration of
Cu in P. australis and T. latifolia was 2.77 and 2.15 in chamber A and 0.009 and 0.047 in
chamber B, respectively (Fig 4.30). Significantly high (p<0.05) sorption of Cu in chamber B
(296 mg kg-1) revealed that substrates trapped maximum amount of Cu as action of filtration to
prevent plants from toxicity. Bioconcentration of Zn in P. australis and T. latifolia was 5.23 and
1.95 in chamber A and 2.40 and 0.84 in chamber B, respectively. High bioconcentration of Pb by
P. australis and T. latifolia was 22.69 and 14.50 was observed in chamber A as compared to
chamber B (9.31 and 9.67), respectively. Despite of high exposure of metals and COD to plants
of chamber A and B P. australis and T. latifolia have performed efficiently with good growth
throughout the operation of VFCW. Substrates of chamber A (crushed brick) and chamber B
(steel slag) and chamber E (crushed brick and steel slag) have also significantly absorbed metals
to support plant growth in shorter retention time with high loading rate.
113
Chapter 5 Conclusions
Phase I
1. Experiment 1: Efficient and sustained growth was observed in T. latifolia in
higher concentration of leachate and significant high accumulation of Cu was observed in
roots than shoots. But accumulation decreased with increased metal concentration in
leachate. Chlorophyll level was high in P. australis in 60% leachate concentration.
Chlorophyll level decreased in all plants as exposed to high concentration of leachate.
2. Experiment 2: Performance of T. latifolia and P. australis was proficient for
removal of Cu and Zn in steel slag and crushed brick, respectively. Substrates supported
the process of phytostabilization by restricting translocation of Cu to shoots thus
sustaining better growth of plants. Zn may also played an important role in growth as
micronutrient..
3. Experiment 3: Significantly high removal of Cu and Zn was achieved by plants in
chelators at dose of 15 mg kg-1 of Cu and Zn. Chelator effected growth of plants as plants
showed wilting in higher dose of 15 mg L-1Cu. Treatment with substrates withstand better
growth of plants by restricting translocation of toxicity to shoots which may supported
better photosynthesis in higher exposure.
4. Experiment 4: P. australis and T. latifolia in presence of crushed brick and steel
slag showed efficient removal of Cu and Zn. High coefficients of determination in pseudo
second order kinetics were obtained in treatment of P. australis in presence of crushed
brick and T. latifolia in steel slag for Cu removal. In other words, copper was efficiently
removed by combination of P. australis in presence of crushed brick and T. latifolia in
steel slag.
Phase II
1. In batch mode, each chamber of multi-chamber wetland acted as discrete chamber,
removal of metal and COD was more than 90% of applied concentration. Whereas in
114
case of continuous mode of operation, all chambers were inter-connected and removal of
more than 90% of applied concentration was achieved in first chamber alone while only
10% of applied concentration or less than that was achieved in the following four
chambers. It also means that in continuous mode, all chambers after first chamber were
lightly loaded as more than 90% of applied loads were removed in first chamber.
2. In Batch mode at both HRT of 21 and 14 days no significant difference was observed in
removal efficiency of metals and COD. At HRT of 21 days removal of metals and COD
was above 90% in active chambers; A and B whereas at HRT of 14 days, metal removal
was increased up to 93% with constant removal of COD in active chambers that are A, B,
C and D.
3. In continuous mode at HRT of 35 days subsequent increase in percentage removal of Cu,
Zn and Pb was observed from chamber A followed by chamber E. Significant decrease in
removal percentage of Zn and COD was observed with low HRT of one day in chamber
A while achieving maximum removal in the preceding chambers.
4. Translocation of Cu, Zn, Pb and COD was restricted in P. australis and T. latifolia in
crushed brick and steel slag. High Cu accumulation was found in roots of T. Latifolia in
presence of steel slag. V. zizanioides and E. globulus has shown significant translocation
of Cu in sand and gravel. Zn accumulation was significantly high in roots of P. australis
in presence of crushed brick. High accumulation of Pb was found in roots and shoots of
T. latifolia in crushed brick. Pb and Cu was highly absorbed by substrates of chamber E
and Zn in chamber A, respectively.
Recommendations
1. For critical analysis it is recommended that in pilot scale constructed wetland (same
combinations of plants and substrates) sampling of substrates at different levels should be
done (upper surface, mid layer and benthic layer) which would help in understanding the
exchange of ions and chemistry of sorption in CW. 2. Under same optimized conditions of HRT’s in same facility of CW at IESE, microbial
and biofilm analysis can open an interesting avenue. It will help to understand that apart
115
from plants and substrate microbes are also playing an important role. Therefore it is the
right momentum for researchers. 3. Analyzing one hyperraccumulator species in all chambers of CW in different substrates
can also lead us to new path. As novel substrates introduced in CW in present study in
combination with different plants. Leading species of present work was T. latifolia which
can be tested in different substrates individually in different chambers under same
optimized conditions. 4. E. globulus proved to be a good accumulator of Cu and Pb in present research. Therefore
it can be tested under steel slag and crushed brick to further analyze its efficiency for
metal removal. 5. Bio augmenting the chamber of CW with similar combinations of plants and substrates
can open door to new innovative work. Single bacterial species can be tested in
combination with the hyperaccumulators of present work. Whereas inoculum of bacterial
consortium might also work out well under similar optimized conditions of present work.
116
References
Avila, C., Nivala, J., Olsson, L., Kassa, K., Headley, T., Mueller, R.A., Bayona, J.M., Garci´a, J., 2014. Emerging organic contaminants in vertical subsurface flow constructed wetlands: Influence of media size, loading frequency and use of active aeration. Sci. Total Environ. 494–495, 211–217. https://doi.org/10.1016/j.scitotenv.2014.06.128
Abou-Elela, S.I., Golinielli, G., Abou-Taleb, E.M., Hellal, M.S., 2013. Municipal wastewater treatment in horizontal and vertical flows constructed wetlands. Ecol. Eng. 61, 460–468. https://doi.org/10.1016/j.ecoleng.2013.10.010
Abou-Elela, S.I. and Hellal, M.S., 2012. Municipal wastewater treatment using vertical flow constructed wetlands planted with Canna, Phragmites and Cyprus. Ecol. Eng. 47, 209–213. https://doi.org/10.1016/j.ecoleng.2012.06.044
Abu Amr, S.S., Aziz, H.A., Adlan, M.N., 2013. Optimization of stabilized leachate treatment using ozone/persulfate in the advanced oxidation process. Waste Manag. 33, 1434–1441. https://doi.org/10.1016/j.wasman.2013.01.039
Ait Ali, N., Bernal, M.P., Ater, M., 2004. Tolerance and bioaccumulation of cadmium by Phragmites australis grown in the presence of elevated concentrations of cadmium, copper, and zinc. Aquat. Bot. 80, 163–176. https://doi.org/10.1016/j.aquabot.2004.08.008
Akinbile, C.O., Yusoff, M.S., Ahmad Zuki, A.Z., 2012. Landfill leachate treatment using sub-surface flow constructed wetland by Cyperus haspan. Waste Manag. 32, 1387–1393. https://doi.org/10.1016/j.wasman.2012.03.002
Aluko, O.O. and Sridhar, M., 2014. Evaluation of effluents from bench-scale treatment combinations for landfill leachate in Ibadan, Nigeria. Waste Manag. Res. 32, 70–78. https://doi.org/10.1177/0734242X13514624
Amor, C., Torres-Socías, E. De, Peres, J.A., Maldonado, M.I., Oller, I., Malato, S., Lucas, M.S., 2015. Mature landfill leachate treatment by coagulation/flocculation combined with Fenton and solar photo-Fenton processes. J. Hazard. Mater. 286, 261–268. https://doi.org/10.1016/J.JHAZMAT.2014.12.036
Anning, A.K., Korsah, P.E., Addo-Fordjour, P., 2013. Phytoremediation of wastewater with Limnocharis flava, Thalia geniculata and Typha latifolia in constructed wetlands. Int. J. Phytoremediation 15, 452–64. https://doi.org/10.1080/15226514.2012.716098
Anwarzeb, K., Khan S., Khan, M.A., Qamar, Z., and Waqas, M., 2015. The uptake and bioaccumulation of heavy metals by food plants, their effects on plants nutrients, and associated health risk: a review. Env Sci Pollut Res 22, 13772–13799.
APHA (American Public Health Association), 2012. Standard Methods for the Examination of Water and Wastewater, Standard Methods. https://doi.org/ISBN 9780875532356
Arroyo, P., Ansola, G., Miera, L.E.S. De, 2013. Effects of substrate, vegetation and flow on arsenic and zinc removal efficiency and microbial diversity in constructed wetlands. Ecol. Eng. 51, 95–103. https://doi.org/10.1016/j.ecoleng.
117
Asadi, M., 2008. Investigation of heavy metals concentration in landfill leachate and reduction by different coagulants, in: The 7th International Conference Faculty of Environmental Engineering Vilnius Gediminas Technical University. Vilnius, Lithuania. 484–488.
Aziz, H.A., Adlan, M.N., Ariffin, K.S., 2008. Heavy metals (Cd, Pb, Zn, Ni, Cu and Cr(III)) removal from water in Malaysia: Post treatment by high quality limestone. Bioresour. Technol. 99, 1578–1583. https://doi.org/10.1016/j.biortech.2007.04.007
Aziz, S.Q., Aziz, H.A., Yusoff, M.S., 2011. Optimum Process Parameters for the Treatment of Landfill Leachate Using Powdered Activated Carbon Augmented Sequencing Batch Reactor (SBR) Technology. Sep. Sci. Technol. 46, 2348–2359. https://doi.org/10.1080/01496395.2011.595753
Aziz, S.Q., Aziz, H.A., Yusoff, M.S., Bashir, M.J.K., Umar, M., 2010. Leachate characterization in semi-aerobic and anaerobic sanitary landfills: A comparative study. J. Environ. Manage. 91, 2608–2614. https://doi.org/10.1016/J.JENVMAN.2010.07.042
Badejo, A.A., Sridhar, M.K.C., Coker, A.O., Ndambuki, J.M., Kupolati, W.K., 2015. Phytoremediation of Water Using Phragmites karka and Veteveria nigritana in Constructed Wetland. Int. J. Phytoremediation 17, 847–852. https://doi.org/10.1080/15226514.2014.964849
Bakhshoodeh, R., Alavi, N., Paydary, P., 2017. Composting plant leachate treatment by a pilot-scale, three-stage, horizontal flow constructed wetland in central Iran. Env. Sci Pollut Res 24, 23803–23814. https://doi.org/10.1007/s11356-017-0002-6
Bakhshoodeh, R., Alavi, N., Soltani Mohammadi, A., Ghanavati, H., 2016. Removing heavy metals from Isfahan composting leachate by horizontal subsurface flow constructed wetland. Environ. Sci. Pollut. Res. 23, 12384–12391. https://doi.org/10.1007/s11356-016-6373-2
Barca, C., Troesch, S., Meyer, D., Drissen, P., Andreìs, Y., Chazarenc, F., 2013. Steel slag filters to upgrade phosphorus removal in constructed wetlands: Two years of field experiments. Environ. Sci. Technol. 47, 549–556. https://doi.org/10.1021/es303778t
Batool, A., Baig, M.A., 2015. Growth Behavior Comparison of Three Species Exposed to Municipal Solid Waste Leachate in Microcosm Constructed Wetland. International Conference on advances in biological, agricultural and environmental sciences, 22nd and 23rd July. London, UK
Batool, A., Zeshan, 2017. Effect of Chelators and Substrates on Phytoremediation of Synthetic Leachate for Removal of Trace Elements. Soil Sediment Contam. An Int. J. 26(2): 220-233. https://doi.org/10.1080/15320383.2017.1274958
Begonia, M.T., Begonia, G.B., Miller, G.S., Gilliard, D., 2004. Effects of Chelate Application Time on the Phytoextraction of Lead-Contaminated Soils. Bull. Environ. Contam. Toxicol 73, 1033–1040. https://doi.org/10.1007/s00128-004-0529-3
Beh, C.L., Chuah, T.G., Nourouzi, M.N., Choong, T., 2012. Removal of heavy metals from steel making waste water by using electric arc furnace slag. E-Journal Chem. 9, 2557–2564. https://doi.org/10.1155/2012/128275
118
Bernardini, A., Salvatori, E., Guerrini, V., Fusaro, L., Canepari, S., Manes, F., 2016. Effects of high Zn and Pb concentrations on Phragmites australis (Cav.) Trin. Ex. Steudel: Photosynthetic performance and metal accumulation capacity under controlled conditions. Int. J. Phytoremediation 18, 16–24. https://doi.org/10.1080/15226514.2015.1058327
Bhatnagar, A., Vilar, V.J., Botelho, C.M., Boaventura, R.A., Ltd, F., 2011. A review of the use of red mud as adsorbent for the removal of toxic pollutants from water and wastewater. Environ. Technol. 32, 231–249. https://doi.org/10.1080/09593330.2011.560615
Bhattacharya, T., Banerjee, D.K., Gopal, B., 2006. Heavy metal uptake by scirpus littoralis schrad . from fly ash dosed and metal spiked soils. Environ. Monit. Assess. 121, 363–380. https://doi.org/10.1007/s10661-005-9133-1
Bisone, S., Gautier, M., Masson, M., Forquet, N., 2016. Influence of loading rate and modes on infiltration of treated wastewater in soil-based constructed wetland. Environ. Technol. 3330, 1–12. https://doi.org/10.1080/09593330.2016.1185165
Bohórquez, E., Paredes, D., Arias, C.A., 2016. Vertical flow-constructed wetlands for domestic wastewater treatment under tropical conditions: effect of different design and operational parameters. Environ. Technol. (United Kingdom) 38(2), 199-208. https://doi.org/10.1080/09593330.2016.1230650
Bonanno, G., Borg, J.A., Di Martino, V., 2017a. Levels of heavy metals in wetland and marine vascular plants and their biomonitoring potential: A comparative assessment. Sci. Total Environ. 576, 796–806. https://doi.org/10.1016/j.scitotenv.2016.10.171
Bonanno, G., Cirelli, G.L., 2017b. Comparative analysis of element concentrations and translocation in three wetland congener plants: Typha domingensis, Typha latifolia and Typha angustifolia. Ecotoxicol. Environ. Saf. 143, 92–101. https://doi.org/10.1016/j.ecoenv.2017.05.021
Bouchama, K., Rouabhi, R., Djebar, M.R., 2016. Behavior of Phragmites australis (CAV.) Trin. Ex Steud used in phytoremediation of wastewater contaminated by cadmium. Desalin. Water Treat. 57, 5325–5330. https://doi.org/10.1080/19443994.2015.1022001
Bragato, C., Brix, H., Malagoli, M., 2006. Accumulation of nutrients and heavy metals in Phragmites australis (Cav.) Trin. ex Steudel and Bolboschoenus maritimus (L.) Palla in a constructed wetland of the Venice lagoon watershed. Environ. Pollut. 144, 967–975. https://doi.org/10.1016/j.envpol.2006.01.046
Burgoon, P.S., Kadlec, R.H., Henderson, M., 1999. Treatment of potato processing wastewater with engineered natural systems. Water Sci. Technol. 40, 211–215. https://doi.org/10.1016/S0273-1223(99)00412-6
Byoung-Hwa Lee, M.S., 2007. What is the role of Phragmites australis in experimental constructed wetland filters treating urban runoff? Ecol. Eng. 29, 87–95. https://doi.org/10.1016/J.ECOLENG.2006.08.001
Çakir, R., Gidirislioglu, A., Çebi, U., 2015. A study on the effects of different hydraulic loading rates (HLR) on pollutant removal efficiency of subsurface horizontal-flow constructed wetlands used for treatment of domestic wastewaters. J. Environ. Manage. 164, 121–128.
119
https://doi.org/10.1016/j.jenvman.2015.08.037
Calheiros, C.S.C., Rangel, A.O.S.S., Castro, P.M.L., 2008. The effects of tannery wastewater on the development of different plant species and chromium accumulation in Phragmites australis. Arch. Environ. Contam. Toxicol. 55, 404–414. https://doi.org/10.1007/s00244-007-9087-0
Calheiros, C.S.C., Rangel, A.O.S.S., Castro, P.M.L., 2007. Constructed wetland systems vegetated with different plants applied to the treatment of tannery wastewater. Water Res. 41, 1790–1798. https://doi.org/10.1016/j.watres.2007.01.012
Cay, S., Uyanik, A., Engin, M.S., Kutbay, H.G., 2015. Effect of EDTA and Tannic Acid on the Removal of Cd, Ni, Pb and Cu from Artificially Contaminated Soil by Althaea rosea Cavan. Int. J. Phytoremediation 17, 568–574. https://doi.org/10.1080/15226514.2014.935285
Chang, J., Wu, S., Dai, Y., Liang, W., Wu, Z., 2012. Treatment performance of integrated vertical-flow constructed wetland plots for domestic wastewater. Ecol. Eng. 44, 152–159. https://doi.org/10.1016/j.ecoleng.2012.03.019
Chemlal, R., Azzouz, L., Kernani, R., Abdi, N., Lounici, H., Grib, H., Mameri, N., Drouiche, N., 2014. Combination of advanced oxidation and biological processes for the landfill leachate treatment. Ecol. Eng. 73, 281–289. https://doi.org/10.1016/j.ecoleng.2014.09.043
Chen, T.Y., Kao, C.M., Yeh, T.Y., Chien, H.Y., Chao, A.C., 2006. Application of a constructed wetland for industrial wastewater treatment: A pilot-scale study. Chemosphere. 64, 497-502. https://doi.org/10.1016/j.chemosphere.2005.11.069
Chen, X.-C., Liu, Y.-G., Zeng, G.-M., Duan, G.-F., Hu, X.-J., Hu, X., Xu, W.-H., Zou, M., 2014. The optimal root length for vetiveria zizanioides when transplanted to Cd polluted soil. Int. J. Phytoremediation. 17, 563–567. https://doi.org/10.1080/15226514.2014.922930
Christos S.Akratos and Vassilios A.Tsihrintzis, 2007. Effect of temperature, HRT, vegetation and porous media on removal efficiency of pilot-scale horizontal subsurface flow constructed wetlands. Ecol. Eng. 29, 173–191. https://doi.org/10.1016/J.ECOLENG.2006.06.013
Chys, M., Oloibiri, V.A., Audenaert, W.T.M., Demeestere, K., Van Hulle, S.W.H., 2015. Ozonation of biologically treated landfill leachate: efficiency and insights in organic conversions. Chem. Eng. J. 277, 104–111. https://doi.org/10.1016/J.CEJ.2015.04.099
Colombo, C., Palumbo, G., He, J.-Z., Pinton, R., Cesco, S., 2014. Review on iron availability in soil: interaction of Fe minerals, plants, and microbes. J. Soils Sediments 14, 538–548. https://doi.org/10.1007/s11368-013-0814-z
Crites, R.W., Dombeck, G.D., Watson, R.C., Williams, C.R., 1997. Removal of Metals and Ammonia in Constructed Wetlands. Water Environ. Res. 69, 132–135. https://doi.org/10.2307/25044854
Dan, T.H., Quang, L.N., Chiem, N.H., Brix, H., 2011. Treatment of high-strength wastewater in tropical constructed wetlands planted with Sesbania sesban: Horizontal subsurface flow versus vertical downflow. Ecol. Eng. 37, 711–720.
120
https://doi.org/10.1016/j.ecoleng.2010.07.030
Danh, L.T., Truong, P., Mammucari, R., Tran, T., Foster, N., 2014. Vetiver grass, Vetiveria zizanioides: a choice plant for phytoremediation of heavy metals and organic wastes. Int. J. Phytoremediation 11, 664–91. https://doi.org/10.1080/15226510902787302
Deblina Ghosh and Gopal Brij, 2010. Effect of hydraulic retention time on the treatment of secondary effluent in a subsurface flow constructed wetland. Ecol. Eng. 36, 1044–1051. https://doi.org/10.1016/J.ECOLENG.2010.04.017
Deng, H., Ye, Z.H., Wong, M.H., 2004. Accumulation of lead, zinc, copper and cadmium by 12 wetland plant species thriving in metal-contaminated sites in China. Environ. Pollut. 132, 29–40. https://doi.org/10.1016/j.envpol.2004.03.030
Djeribi, R., Hamdaoui, O., 2008. Sorption of copper(II) from aqueous solutions by cedar sawdust and crushed brick. Desalination 225, 95–112. https://doi.org/10.1016/j.desal.2007.04.091
Drzewiecka, K., Borowiak, K., Mleczek, M., Zawada, I., Goliński, P., 2011. Bioaccumulation of zinc and copper by Phragmites australis (CAV.) Trin ex Steudel and Typha angustifolia (L.) growing in natural water ecosystems. Fresenius Environ. Bull. 20, 325–333.
Ebbs, S.D., Bradfield, S.J., Kumar, P., White, J.C., Musante, C., Ma, X., 2016. Accumulation of zinc, copper, or cerium in carrot (Daucus carota) exposed to metal oxide nanoparticles and metal ions. Environ. Sci. Nano 3, 114–126. https://doi.org/10.1039/C5EN00161G
Ebrahimi, M., 2016. Enhanced Phytoremediation Capacity of Chenopodium album L. Grown on Pb-Contaminated Soils Using EDTA and Reduction of Leaching Risk. Soil Sediment Contam. An Int. J. 25, 652–667. https://doi.org/10.1080/15320383.2016.1190314
Ebrahimi, M., 2014. Effect of EDTA and DTPA on Phytoremediation of Pb-Zn Contaminated Soils by Eucalyptus camaldulensis Dehnh and Effect on Treatment Time. Desert. 19(1), 65–73.
Ebrahimi, M., 2013. The Effect of EDTA Addition on the Phytoremediation Efficiency of Pb and Cr by Echinochloa crus galii (L.) Beave and Associated Potential Leaching Risk. Soil Sediment Contam. An Int. J. 23, 245–256. https://doi.org/10.1080/15320383.2014.815153
Elhafez, S.E.A., Hamad, H.A., Zaatout, A.A., Malash, G.F., 2017. Management of agricultural waste for removal of heavy metals from aqueous solution: adsorption behaviors, adsorption mechanisms, environmental protection, and techno-economic analysis. Environ. Sci. Pollut. 24(2), 1397-1415. Res. https://doi.org/10.1007/s11356-016-7891-7
EPA, 1998. Guidelines for Ecological Risk Assessment. Fed. Regist. 63, 26846–26924.
Fan, J., Liang, S., Zhang, B., Zhang, J., 2013. Enhanced organics and nitrogen removal in batch-operated vertical flow constructed wetlands by combination of intermittent aeration and step feeding strategy. Environ. Sci. Pollut. Res. 20, 2448–2455. https://doi.org/10.1007/s11356-012-1130-7
Fountoulakis, M.S., Daskalakis, G., Papadaki, A., Kalogerakis, N., Manios, T., 2017. Use of halophytes in pilot-scale horizontal flow constructed wetland treating domestic wastewater. Env. Sci Pollut Res 24, 16682–16689. https://doi.org/10.1007/s11356-017-9295-8
121
Galletti, A., Verlicchi, P., Ranieri, E., 2010. Removal and accumulation of Cu, Ni and Zn in horizontal subsurface flow constructed wetlands: Contribution of vegetation and filling medium. Sci. Total Environ. 408, 5097–5105. https://doi.org/10.1016/j.scitotenv.2010.07.045
García-Pérez, A., Harrison, M., Chivers, C., Grant, B., 2015. Recycled Shredded-Tire Chips Used As Support Material in a Constructed Wetland Treating High-Strength Wastewater from a Bakery: Case Study. Recycling 1, 3–13. https://doi.org/10.3390/recycling1010003
Garcia, T., Angarita, S., Rodríguez, M.., 2006. Classical subsurface flow wetland optimization to heavy metal removal. 10th Int. Conf. Wetl. Syst. Water Pollut. 23rd - 29th Sept. Control.Lisbon, Portugal
Ge, Y., Wang, X., Zheng, Y., Dzakpasu, M., Zhao, Y., Xiong, J., 2015. Functions of slags and gravels as substrates in large - scale demonstration constructed wetland systems for polluted river water treatment. Environ. Sci. Pollut. Res. 22, 12982–12991. https://doi.org/10.1007/s11356-015-4573-9
Ghani, Z.A., Yusoff, M.S., Zaman, N.Q., Zamri, M.F.M.A., Andas, J., 2017. Optimization of preparation conditions for activated carbon from banana pseudo-stem using response surface methodology on removal of color and COD from landfill leachate. Waste Manag. 62, 177–187. https://doi.org/10.1016/J.WASMAN.2017.02.026
Gogoi, H., Leiviskä, T., Heiderscheidt, E., Postila, H., Tanskanen, J., 2018. Removal of metals from industrial wastewater and urban runoff by mineral and bio-based sorbents. J. Environ. Manage. 209, 316–327. https://doi.org/10.1016/j.jenvman.2017.12.019
Guittonny-Philippe, A., Masotti, V., Claeys-Bruno, M., Malleret, L., Coulomb, B., Prudent, P., H??hener, P., Petit, M. ??l??onore, Sergent, M., Laffont-Schwob, I., 2015. Impact of organic pollutants on metal and As uptake by helophyte species and consequences for constructed wetlands design and management. Water Res. 68, 328–341. https://doi.org/10.1016/j.watres.2014.10.014
Hadad, H.R., Maine, M.A., Bonetto, C.A., 2006. Macrophyte growth in a pilot-scale constructed wetland for industrial wastewater treatment. Chemosphere 63, 1744–1753. https://doi.org/10.1016/j.chemosphere.2005.09.014
Hamdaoui, O., 2006. Batch study of liquid-phase adsorption of methylene blue using cedar sawdust and crushed brick. J. Hazard. Mater. 135, 264–273. https://doi.org/10.1016/j.jhazmat.2005.11.062
Hannele Auvinena, Iva Havranb, Laurens Hubaua, L.V., Wilhelm Gebhardt, Volker Linnemann, Dion Van Oirschot, G.D.L, Diederik P.L. Rousseaua, 2017. Removal of pharmaceuticals by a pilot aerated sub-surface flow constructed wetland treating municipal and hospital wastewater. Ecol. Eng.100,157-164
He, H., Duan, Z., Wang, Z., Yue, B., 2017. The removal efficiency of constructed wetlands filled with the zeolite-slag hybrid substrate for the rural landfill leachate treatment. Environ. Sci. Pollut. 24(21), 17547-17555. https://doi.org/10.1007/s11356-017-9402
He, R., Wei, X.-M., Tian, B.-H., Su, Y., Lu, Y.-L., 2015. Characterization of a joint recirculation
122
of concentrated leachate and leachate to landfills with a microaerobic bioreactor for leachate treatment. Waste Manag. 46, 380–388. https://doi.org/10.1016/j.wasman.2015.08.006
Herrera-Cárdenas, J., Navarro, A.E., Torres, E., Herrera-C Ardenas, J., 2016. Effects of porous media, macrophyte type and hydraulic retention time on the removal of organic load and micropollutants in constructed wetlands. J. Environ. Sci. Heal. Part A Toxic/Hazardous Subst. Environ. Eng. J. 51(5), 380-388. https://doi.org/10.1080/10934529.2015.1120512
Herrera-Melián, J.A., Guedes-Alonso, R., Borreguero-Fabelo, A., Santana-Rodríguez, J.J., Sosa-Ferrera, Z., 2017. Study on the removal of hormones from domestic wastewaters with lab-scale constructed wetlands with different substrates and flow directions. Environ. Sci. Pollut. Res. 1, 1–11. https://doi.org/10.1007/s11356-017-9307-8
Hilles, A.H., Abu Amr, S.S., Hussein, R.A., El-Sebaie, O.D., Arafa, A.I., 2016. Performance of combined sodium persulfate/H2O2 based advanced oxidation process in stabilized landfill leachate treatment. J. Environ. Manage. 166, 493–498. https://doi.org/10.1016/j.jenvman.2015.10.051
Ho, Y.S., 2006. Isotherms for the sorption of lead onto peat: Comparison of linear and non-linear methods. Polish J. Environ. Stud. 15, 81–86. https://doi.org/10.1016/j.watres.2005.10.040
Ho, Y.S., 2004. Selection of optimum sorption isotherm. Carbon N. Y. 42, 2115–2116. https://doi.org/10.1016/j.carbon.2004.03.019
Hopkins, H., Huner, N., 2009. Introduction to Plant Physiology, The Univirsity of Western Ontario. 4th Edition, John Wiley and Sons, Ontario, Canada. https://doi.org/10.2134/agronj1951.00021962004300010013x
Hristina Bojcevska and Karin Tonderski, 2007. Impact of loads, season, and plant species on the performance of a tropical constructed wetland polishing effluent from sugar factory stabilization ponds. Ecol. Eng. 29, 66–76. https://doi.org/10.1016/J.ECOLENG.2006.07.015
Hussain, S.I., Blowes, D.W., Ptacek, C.J., Jamieson-Hanes, J.H., Wootton, B., Balch, G., Higgins, J., 2015. Mechanisms of Phosphorus Removal in a Pilot-Scale Constructed Wetland/BOF Slag Wastewater Treatment System. Environ. Eng. Sci. 32, 340–352. https://doi.org/10.1089/ees.2014.0376
HRB (Health Research Board), 2003. Health and Environmental Effects of Landfilling and Incineration of Waste – A literature Review. Published by Health Research Board, Dublin(www.hrb.ie)
Jarecki, M.K., Chong, C., Voroney, R.P., 2005. Evaluation of Compost Leachates for Plant Growth in Hydroponic Culture. J. Plant Nutr. 28, 651–667. https://doi.org/10.1081/PLN-200052639
Jia, C., Li, G., Dai, Y., Wang, F., Liang, W., 2014. Remediation effects of used brick powder on nutrient-laden sediment. Desalin. Water Treat.57(10), 1–10. https://doi.org/10.1080/19443994.2014.993722
Jung, C., Deng, Y., Zhao, R., Torrens, K., 2017. Chemical oxidation for mitigation of UV-quenching substances (UVQS) from municipal landfill leachate: Fenton process versus ozonation. Water Res. 108, 260–270. https://doi.org/10.1016/J.WATRES.2016.11.005
123
Kabuk, H.A., İlhan, F., Avsar, Y., Kurt, U., Apaydin, O., Gonullu, M.T., 2014. Investigation of Leachate Treatment with Electrocoagulation and Optimization by Response Surface Methodology. Clean - Soil, Air, Water 42, 571–577. https://doi.org/10.1002/clen.201300086
Kadlec, R.H., Wallace, S.D., 2008. Treatment Wetlands. CRC Press, Boca Raton, USA.
Kadlec, R. H., & Knight, R. L. (1996). Treatment wetlands. Boca Raton, FL: CRC Press.
Kamal, M., Ghaly, A.E., Mahmoud, N., Côté, R., 2004. Phytoaccumulation of heavy metals by aquatic plants. Environ. Int. 29, 1029–1039. https://doi.org/10.1016/S0160-4120(03)00091-6
Kamran, M.A., Syed, J.H., Eqani, S.A.M.A.S., Munis, M.F.H., Chaudhary, H.J., 2015. Effect of plant growth-promoting rhizobacteria inoculation on cadmium (Cd) uptake by Eruca sativa. Environ. Sci. Pollut. Res. 22, 9275–9283. https://doi.org/10.1007/s11356-015-4074-x
Khan, S., Ahmad, I., Shah, M.T., Rehman, S., Khaliq, A., 2009. Use of constructed wetland for the removal of heavy metals from industrial wastewater. J. Environ. Manage. 90, 3451–3457. https://doi.org/10.1016/j.jenvman.2009.05.026
Kietlińska, A., Renman, G., Jannes, S., Tham, G., 2005. Nitrogen removal from landfill leachate using a compact constructed wetland and the effect of chemical pretreatment. J. Environ. Sci. Health. A. Tox. Hazard. Subst. Environ. Eng. 40, 1493–506.
Kim, D.H., Shin, M.C., Choi, H.D., Seo, C. Il, Baek, K., 2008. Removal mechanisms of copper using steel-making slag: adsorption and precipitation. Desalination 223, 283–289. https://doi.org/10.1016/j.desal.2007.01.226
Kjeldsen, P., Barlaz, M.A., Rooker, A.P., Baun, A., Ledin, A., Christensen, T.H., 2002. Present and Long-Term Composition of MSW Landfill Leachate: A Review. Crit. Rev. Environ. Sci. Technol. 32, 297–336. https://doi.org/10.1080/10643380290813462
Klink, A., Macioł, A., Wisłocka, M., Krawczyk, J., 2013. Metal accumulation and distribution in the organs of Typha latifolia L. (cattail) and their potential use in bioindication. Limnol. - Ecol. Manag. Inl. Waters 43, 164–168. https://doi.org/10.1016/j.limno.2012.08.012
Konnerup, D., Koottatep, T., Brix, H., 2009. Treatment of domestic wastewater in tropical, subsurface flow constructed wetlands planted with Canna and Heliconia. Ecol. Eng. 35, 248–257. https://doi.org/10.1016/j.ecoleng.2008.04.018
Korboulewsky, N., Wang, R., Baldy, V., 2012. Purification processes involved in sludge treatment by a vertical flow wetland system: Focus on the role of the substrate and plants on N and P removal. Bioresour. Technol. 105, 9–14. https://doi.org/10.1016/j.biortech.2011.11.037
Korkusuz, E.A., Beklioǧlu, M., Demirer, G.N., 2005. Comparison of the treatment performances of blast furnace slag-based and gravel-based vertical flow wetlands operated identically for domestic wastewater treatment in Turkey. Ecol. Eng. 24, 187–200. https://doi.org/10.1016/j.ecoleng.2004.10.002
Kumari, M., Tripathi, B.D., 2015. Efficiency of Phragmites australis and Typha latifolia for
124
heavy metal removal from wastewater. Ecotoxicol. Environ. Saf. 112, 80–86. https://doi.org/10.1016/j.ecoenv.2014.10.034
Kumari, M., Tripathi, B.D., 2015. Effect of Phragmites australis and Typha latifolia on biofiltration of heavy metals from secondary treated effluent. Int. J. Environ. Sci. Technol. 12, 1029–1038. https://doi.org/10.1007/s13762-013-0475-x
Liao, M., 2000. Mechanisms of copper absorption. PhD Thesis. Massey University, New Zealand.1-209.
Lim, P.E., Mak, K.Y., Mohamed, N., Noor, A.M., 2003. Removal and speciation of heavy metals along the treatment path of wastewater in subsurface-flow constructed wetlands. Water Sci. Technol. 48, 307–13.
Lin, Y.-F., Jing, S.-R., Lee, D.-Y., Chang, Y.-F., Chen, Y.-M., Shih, K.-C., 2005. Performance of a constructed wetland treating intensive shrimp aquaculture wastewater under high hydraulic loading rate. Environ. Pollut. 134, 411–421. https://doi.org/10.1016/j.envpol.2004.09.015
Liu, H., Hu, Z., Zhang, J., Ngo, H.H., Guo, W., Liang, S., Fan, J., Lu, S., Wu, H., 2016. Optimizations on supply and distribution of dissolved oxygen in constructed wetlands: A review. Bioresour. Technol. 214, 797–805. https://doi.org/10.1016/j.biortech.2016.05.003
Liu, M., Wu, S., Chen, L., Dong, R., 2014. How substrate influences nitrogen transformations in tidal flow constructed wetlands treating high ammonium wastewater? Ecol. Eng. 73, 478–486. https://doi.org/10.1016/j.ecoleng.2014.09.111
Liu, S., 2013. Landfill leachate treatment methods and evaluation of Hedeskoga and Måsalycke landfills. Masters Thesis. Lund University, Sweden. 1–67.
Lizama Allende, K., McCarthy, D.T., Fletcher, T.D., 2014. The influence of media type on removal of arsenic, iron and boron from acidic wastewater in horizontal flow wetland microcosms planted with Phragmites australis. Chem. Eng. J. 246, 217–228. https://doi.org/10.1016/j.cej.2014.02.035
Lu, L., Tian, S., Yang, X., Peng, H., Li, T., 2013. Improved cadmium uptake and accumulation in the hyperaccumulator Sedum alfredii: the impact of citric acid and tartaric acid. J. Zhejiang Univ. Sci. B 14, 106–14. https://doi.org/10.1631/jzus.B1200211
Maine, M.A. H. Hadad, G. Sanchez, C. Bonett, 2009. Influence of vegetation on the removal of heavy metals and nutrients in a constructed wetland. J. Environ. Manag. 90(1), 355-363.
Maddison, M., Soosaar, K., Mauring, T., Mander, Ü., 2009. The biomass and nutrient and heavy metal content of cattails and reeds in wastewater treatment wetlands for the production of construction material in Estonia. Desalination 246, 120–128. https://doi.org/10.1016/j.desal.2008.02.040
Madera-Parra, C., Peña-Salamanca, E.J., Peña, M.R., Rousseau, D.P.L., Lens, P.N.L., 2015. Phytoremediation of Landfill Leachate with Colocasia esculenta, Gynerum sagittatum and Heliconia psittacorum in Constructed Wetlands. Int. J. Phytoremediation 17, 16–24. https://doi.org/10.1080/15226514.2013.828014
125
Mahdavian, K., Ghaderian, S.M., Schat, H., 2016. Pb accumulation, Pb tolerance, antioxidants, thiols, and organic acids in metallicolous and non-metallicolous Peganum harmala L. under Pb exposure. Environ. Exp. Bot. 126, 21–31. https://doi.org/10.1016/j.envexpbot.2016.01.010
Mahmood, T., Islam, K.R., Muhammad, A.S., 2007. Toxic effects of heavy metals on early growth and tolerance of cereal CROPS. Pak. J. Bot 39, 451–462.
Maine, M.A., Hadad, H.R., Sánchez, G.C., Di Luca, G.A., Mufarrege, M.M., Caffaratti, S.E., Pedro, M.C., 2017. Long-term performance of two free-water surface wetlands for metallurgical effluent treatment. Ecol. Eng. 98, 372–377. https://doi.org/10.1016/j.ecoleng.2016.07.005
Manoj Kumar, R.S, 2017. Performance evaluation of semi continuous vertical flow constructed wetlands (SC-VF-CWs) for municipal wastewater treatment. Bioresour. Technol.232; 321-330
Marchand, L., Mench, M., Jacob, D.L., Otte, M.L., 2010. Metal and metalloid removal in constructed wetlands, with emphasis on the importance of plants and standardized measurements: A review. Environ. Pollut. 158, 3447–3461. https://doi.org/10.1016/j.envpol.2010.08.018
Marchand, L., Nsanganwimana, F., Oustrière, N., Grebenshchykova, Z., Lizama-Allende, K., Mench, M., 2014. Copper removal from water using a bio-rack system either unplanted or planted with Phragmites australis, Juncus articulatus and Phalaris arundinacea. Ecol. Eng. 64, 291–300. https://doi.org/10.1016/j.ecoleng.2013.12.017
Massa, N., Andreucci, F., Poli, M., Aceto, M., Barbato, R., Berta, G., 2010. Screening for heavy metal accumulators amongst autochtonous plants in a polluted site in Italy. Ecotoxicol. Environ. Saf. 73, 1988–1997. https://doi.org/10.1016/j.ecoenv.2010.08.032
Merino-Solís, M., Villegas, E., de Anda, J., López-López, A., 2015. The Effect of the Hydraulic Retention Time on the Performance of an Ecological Wastewater Treatment System: An Anaerobic Filter with a Constructed Wetland. Water 7, 1149–1163. https://doi.org/10.3390/w7031149
Mishra, V.K., Tripathi, B.D., 2009. Accumulation of chromium and zinc from aqueous solutions using water hyacinth (Eichhornia crassipes). J. Hazard. Mater. 164, 1059–1063. https://doi.org/10.1016/j.jhazmat.2008.09.020
Monferrán, M. V, Pignata, M.L., Wunderlin, D.A., 2012. Enhanced phytoextraction of chromium by the aquatic macrophyte Potamogeton pusillus in presence of copper. Environ. Pollut. 161, 15–22. https://doi.org/10.1016/j.envpol.2011.09.032
Mor, S., Kaur, K., Khaiwal, R., 2013. Growth behavior studies of bread wheat plant exposed to municipal landfill leachate. J. Environ. Biol. 34, 1083–1087.
Mor, S., Ravindra, K., Dahiya, R.P., Chandra, A., 2006. Leachate Characterization and Assessment of Groundwater Pollution Near Municipal Solid Waste Landfill Site. Environ. Monit. Assess. 118, 435–456. https://doi.org/10.1007/s10661-006-1505-7
Moradi, M., Ghanbari, F., 2014. Application of response surface method for coagulation process
126
in leachate treatment as pretreatment for Fenton process: Biodegradability improvement. J. Water Process Eng. 4, 67–73. https://doi.org/10.1016/j.jwpe.2014.09.002
Moreira, F.C., Soler, J., Fonseca, A., Saraiva, I., Boaventura, R.A.R., Brillas, E., Vilar, V.J.P., 2016. Electrochemical advanced oxidation processes for sanitary landfill leachate remediation: Evaluation of operational variables. Appl. Catal. B Environ. 182, 161–171. https://doi.org/10.1016/J.APCATB.2015.09.014
Mudhiriza, T., Mapanda, F., Mvumi, B., Wuta, M., 2015. Removal of nutrient and heavy metal loads from sewage effluent using vetiver grass,Chrysopogon zizanioides (L.) Roberty. Water SA 41(4), 457-463. https://doi.org/10.4314/wsa.v41i4.04
Kamran,M.A., Eqani, SAMAS., Bibi, S., Amna, R.K.X., Monis, M.F.A., Katsoyiannis, A., Bokhari, H.J.C., 2016. Bioaccumulation of nickel by E. sativa and role of plant growth promoting rhizobacteria (PGPRs) under nickel stress. Ecotoxicol. Environ. Saf. 126, 256–263. https://doi.org/10.1016/J.ECOENV.2016.01.002
Muhammad, D., Chen, F., Zhao, J., Zhang, G., Wu, F., 2009. Comparison of EDTA- and citric acid-enhanced phytoextraction of heavy metals in artificially metal contaminated soil by Typha angustifolia. Int. J. Phytoremediation 11, 558–574. https://doi.org/10.1080/15226510902717580
Mustapha, H.I., Bruggen, J.J.A. Van, Lens, P.N.L., 2018. Fate of heavy metals in vertical subsurface flow constructed wetlands treating secondary treated petroleum refinery wastewater in Kaduna , Nigeria. Int. J. Phytoremediation 20, 44–53. https://doi.org/10.1080/15226514.2017.1337062
Nehrenheim, E., Waara, S., Johansson Westholm, L., 2008. Metal retention on pine bark and blast furnace slag - On-site experiment for treatment of low strength landfill leachate. Bioresour. Technol. 99, 998–1005. https://doi.org/10.1016/j.biortech.2007.03.006
Nelson, E.A., Specht, W.L., Knox, A.S., 2006. Metal Removal from Water Discharges by a Constructed Treatment Wetland. Eng. Life Sci. 6, 26–30. https://doi.org/10.1002/elsc.200620112
Pal, R., Rai, J.P.N., 2010. Phytochelatins: Peptides Involved in Heavy Metal Detoxification. Appl. Biochem. Biotechnol. 160, 945–963. https://doi.org/10.1007/s12010-009-8565-4
Palmer, C.M., Guerinot, M. Lou, 2009. Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nat. Chem. Biol. 5, 333–340. https://doi.org/10.1038/nchembio.166
Pan, X., Wu, P., Jiang, X., 2016. Levels and potential health risk of heavy metals in marketed vegetables in Zhejiang , China, Nature Publishing Group. Nature Publishing Group.Report no; 6, ISSN: 20317. https://doi.org/10.1038/srep20317
Papaevangelou, V.A., Gikas, G.D., Tsihrintzis, V.A., 2017. Chemosphere Chromium removal from wastewater using HSF and VF pilot-scale constructed wetlands : Overall performance , and fate and distribution of this element within the wetland environment. Chemosphere 168, 716–730. https://doi.org/10.1016/j.chemosphere.2016.11.002
Park, W.H., Polprasert, C., 2008. Roles of oyster shells in an integrated constructed wetland system designed for P removal. Ecol. Eng. 34, 50–56.
127
https://doi.org/10.1016/j.ecoleng.2008.05.014
Pathak, A., Pruden, A., Novak, J.T., 2018. Two-stage Anaerobic Membrane Bioreactor (AnMBR) system to reduce UV absorbance in landfill leachates. Bioresour. Technol. 251, 135–142. https://doi.org/10.1016/J.BIORTECH.2017.12.050
Pepper, R.A., Couperthwaite, S.J., Millar, G.J., 2017. Value adding red mud waste: High performance iron oxide adsorbent for removal of fluoride. J. Environ. Chem. Eng. 5, 2200–2206. https://doi.org/10.1016/j.jece.2017.04.031
Pescod, M.B., 1992. Wastewater treatment and use in agriculture - FAO irrigation and drainage paper 47 Food and agriculture organization of the united nations (No. ISBN 92-5-103135-5). Rome.Italy
Petra Kidd,Juan Barceló,M. Pilar Bernal, Flavia Navari-Izzo, Charlotte Poschenrieder, Stefan Shilev, Rafael Clemente, C.M., 2009. Trace element behaviour at the root–soil interface: Implications in phytoremediation. Environ. Exp. Bot. 67, 243–259. https://doi.org/10.1016/J.ENVEXPBOT.2009.06.013
Peverly, J.H., Surface, J.M., Wang, T., 1995. Growth and trace metal absorption by Phragmites australis in wetlands constructed for landfill leachate treatment. Ecol. Eng. 5, 21–35. https://doi.org/10.1016/0925-8574(95)00018-E
Prochaska, C., Zouboulis, I., 2006. Removal of phosphates by pilot vertical-flow constructed wetlands using a mixture of sand and dolomite as substrate. Ecol. Eng. 26, 293–303. https://doi.org/10.1016/j.ecoleng.2005.10.009
Prudent, P., Domeizel, M., Massiani, C., 1996. Chemical sequential extraction as decision-making tool: application to municipal solid waste and its individual constituents. Sci. Total Environ. 178, 55–61. https://doi.org/10.1016/0048-9697(95)04797-2
Qasaimeh, A., Alsharie, H., Masoud, T., 2015. A Review on Constructed Wetlands Components and Heavy Metal Removal from Wastewater. J. Environ. Prot. (Irvine,. Calif). 6, 710–718. https://doi.org/10.4236/jep.2015.67064
Rai, U.N., Sinha, S., Tripathi, R.D., Chandra, P., 1995. Wastewater treatability potential of some aquatic macrophytes: Removal of heavy metals. Ecol. Eng. 5, 5–12. https://doi.org/10.1016/0925-8574(95)00011-7
Rai, U.N., Upadhyay, A.K., Singh, N.K., Dwivedi, S., Tripathi, R.D., 2015. Seasonal applicability of horizontal sub-surface flow constructed wetland for trace elements and nutrient removal from urban wastes to conserve Ganga River water quality at Haridwar, India. Ecol. Eng. 81, 115–122. https://doi.org/10.1016/j.ecoleng.2015.04.039
Rashid, A., Mahmood, T., Mehmood, F., Khalid, A., Saba, B., Batool, A., Riaz, A., 2014. Phytoaccumulation, competitive adsorption and evaluation of chelators-metal interaction in lettuce plant. Environ. Eng. Manag. J. 13, 2583–2592.
Reeves, R.D., 2003. Tropical hyperaccumulators of metals and their potential for phytoextraction. Plant Soil 249, 57–65. https://doi.org/10.1023/A:1022572517197
Renou, S., Givaudan, J.G., Poulain, S., Dirassouyan, F., Moulin, P., 2008. Landfill leachate
128
treatment: Review and opportunity. J. Hazard. Mater. 150, 468–493. https://doi.org/10.1016/j.jhazmat.2007.09.077
Ryan, K., 2014. The treatment of landfill leachate using natural systems. PhD Thesis. University College Cork. Ireland.
Ryan, P., Delhaize, E., Jones, D., 2001. Function and m echanism of organic anion exudation from p lant r oots. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 527–560. https://doi.org/10.1146/annurev.arplant.52.1.527
Saeed, T., Sun, G., 2011. A comparative study on the removal of nutrients and organic matter in wetland reactors employing organic media. Chem. Eng. J. 171, 439–447. https://doi.org/10.1016/j.cej.2011.03.101
Safaa M.Raghab, Ahmed M.Abd El Meguid, H.A.H., 2013. Treatment of leachate from municipal solid waste landfill. HBRC J. 9, 187–192. https://doi.org/10.1016/J.HBRCJ.2013.05.007
Saifullah, Meers, E., Qadir, M., de Caritat, P., Tack, F.M.G., Du Laing, G., Zia, M.H., 2009. EDTA-assisted Pb phytoextraction. Chemosphere 74, 1279–1291. https://doi.org/10.1016/j.chemosphere.2008.11.007
Salem, Z. Ben, Laffray, X., Ashoour, A., Ayadi, H., Aleya, L., 2014. Metal accumulation and distribution in the organs of Reeds and Cattails in a constructed treatment wetland (Etueffont, France). Ecol. Eng. 64, 1–17. https://doi.org/10.1016/j.ecoleng.2013.12.027
Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Dushenkov, V., Ensley, B.D., Chet, I., Raskin, I., 1995. Phytoremediation: A Novel Strategy for the Removal of Toxic Metals from the Environment Using Plants. Nat. Biotechnol. 13, 468–474. https://doi.org/10.1038/nbt0595-468
Yadav, S.K., Juwarkar, A.A., Kumar, G.P., Thawalea, P.R., Singh, S.K., 2009. Bioaccumulation and phyto-translocation of arsenic, chromium and zinc by Jatropha curcas L.: Impact of dairy sludge and biofertilizer. Bioresour. Technol. 100, 4616–4622. https://doi.org/10.1016/J.BIORTECH.2009.04.062
Sarin, V., Pant, K.K., 2006. Removal of chromium from industrial waste by using eucalyptus bark. Bioresour. Technol. 97, 15–20. https://doi.org/10.1016/j.biortech.2005.02.010
Sasmaz, A., Obek, E., Hasar, H., 2008. The accumulation of heavy metals in Typha latifolia L. grown in a stream carrying secondary effluent. Ecol. Eng. 33, 278-284. https://doi.org/10.1016/j.ecoleng.2008.05.006
Scholz, M., Hedmark, Å., 2010. Constructed Wetlands Treating Runoff Contaminated with Nutrients. Water air soil Pollut. 323–332. https://doi.org/10.1007/s11270-009-0076-y
Shanker, A., Cervantes, C., Lozatavera, H., Avudainayagam, S., 2005. Chromium toxicity in plants. Environ. Int. 31, 739–753. https://doi.org/10.1016/j.envint.2005.02.003
Shrikhande, A.N., Nema, P., Mhaisalkar, V.A., 2014. Performance of Free Water Surface Constructed Wetland Using Typhalatifolia and Canna Lilies for the Treatment of Domestic Wastewater. J. Environ. Sci. Eng. 56, 93–104.
129
Shukla, O.P., Juwarkar, A., Singh, S.K., Khan, S., Rai, U.N., 2011. Growth responses and metal accumulation capabilities of woody plants during the phytoremediation of tannery sludge. Waste Manag. 31, 115–123. https://doi.org/10.1016/j.wasman.2010.08.022
Sarubbi, A.J and G. S Sarmiento., 2009. Leachate abatement inside solid waste landfill, Latin American applied research. Asociación Argentina de Investigadores en Ciencias de la Ingeniería Química y Química Aplicada. 39(4), 307-315
Šíma, J., Svoboda, L., Šeda, M., Krejsa, J., Jahodová, J., 2017. Removal of selected risk elements from wastewater in a horizontal subsurface flow constructed wetland. Water Environ. J. 13(4), 582-590. https://doi.org/10.1111/wej.12269
Soda, S., Hamada, T., Yamaoka, Y., Ike, M., Nakazato, H., Saeki, Y., Kasamatsu, T., Sakurai, Y., 2012. Constructed wetlands for advanced treatment of wastewater with a complex matrix from a metal-processing plant: Bioconcentration and translocation factors of various metals in Acorus gramineus and Cyperus alternifolius. Ecol. Eng. 39, 63–70. https://doi.org/10.1016/j.ecoleng.2011.11.014
Sormunen, K., Ettala, M., Rintala, J., 2008. Internal leachate quality in a municipal solid waste landfill: Vertical, horizontal and temporal variation and impacts of leachate recirculation. J. Hazard. Mater. 160, 601–607. https://doi.org/10.1016/J.JHAZMAT.2008.03.081
Squissato, A.L., Lima, A.F., Almeida, E.S., Pasquini, D., Richter, E.M., Munoz, R.A.A., 2017. Eucalyptus pulp as an adsorbent for metal removal from biodiesel. Ind. Crops Prod. 95, 1–5. https://doi.org/10.1016/j.indcrop.2016.10.004
Stefanakis, A.I., Tsihrintzis, V.A., 2012. Effects of loading, resting period, temperature, porous media, vegetation and aeration on performance of pilot-scale vertical flow constructed wetlands. Chem. Eng. J. 181–182, 416–430. https://doi.org/10.1016/J.CEJ.2011.11.108
Suelee, A.L., 2015. Phytoremediation potential of vetiver grass (vetiveria zizanioides) for water contaminated with selected heavy metal A BS Thesis submitted to the Faculty of Environmental Studies,. Universiti Putra Malaysia 1-143
Sultana, M.Y., Chowdhury, A.K.M.M.B., Michailides, M.K., Akratos, C.S., Tekerlekopoulou, A.G., Vayenas, D. V., 2015. Integrated Cr(VI) removal using constructed wetlands and composting. J. Hazard. Mater. 281, 106–113. https://doi.org/10.1016/j.jhazmat.2014.06.046
Syranidou, E., Christofilopoulos, S., Gkavrou, G., Thijs, S., Weyens, N., Vangronsveld, J., Kalogerakis, N., 2016. Exploitation of Endophytic Bacteria to Enhance the Phytoremediation Potential of the Wetland Helophyte Juncus acutus. Front. Microbiol. 07, 1-15.1016. https://doi.org/10.3389/fmicb.2016.01016
Tao, W., Hall, K.J., Duff, S.J.B., 2006. Performance evaluation and effects of hydraulic retention time and mass loading rate on treatment of woodwaste leachate in surface-flow constructed wetlands. Ecol. Eng. 26, 252–265. https://doi.org/10.1016/j.ecoleng.2005.10.006
Tee, H.C., Seng, C.E., Noor, A.M., Lim, P.E., 2009. Performance comparison of constructed wetlands with gravel- and rice husk-based media for phenol and nitrogen removal. Sci. Total Environ. 407, 3563–3571. https://doi.org/10.1016/j.scitotenv.2009.02.017
Trang, N.T.D., Konnerup, D., Schierup, H.-H., Chiem, N.H., Tuan, L.A., Brix, H., 2010.
130
Kinetics of pollutant removal from domestic wastewater in a tropical horizontal subsurface flow constructed wetland system: Effects of hydraulic loading rate. Ecol. Eng. 36, 527–535. https://doi.org/10.1016/j.ecoleng.2009.11.022
Upadhyay, A.K., Bankoti, N.S., Rai, U.N., 2016. Studies on sustainability of simulated constructed wetland system for treatment of urban waste: Design and operation. J. Environ. Manage. 169, 285–292. https://doi.org/10.1016/j.jenvman.2016.01.004
USEPA (United States Environmental Protection Agency), 2002. 2000 National Water Quality Inventory Report to Congress.
Verbruggen, N., Hermans, C., Schat, H., 2009. Mechanisms to cope with arsenic or cadmium excess in plants. Curr. Opin. Plant Biol. 12, 364–372. https://doi.org/10.1016/j.pbi.2009.05.001
Veronica B., Eleonora P. G. M., Brunello C., Sandra M., Ravelo, R.I., 2011. Efficiency assessment of a reed bed pilot plant (Phragmites australis) for sludge stabilisation in Tuscany (Italy). Ecol. Eng. 37, 779–785. https://doi.org/10.1016/J.ECOLENG.2010.05.008
Víctor Matamoros, Y.R., 2016. Batch vs continuous-feeding operational mode for the removal of pesticides from agricultural run-off by microalgae systems: A laboratory scale study. J. Hazard. Mater. 309, 126–132. https://doi.org/10.1016/J.JHAZMAT.2016.01.080
Vymazal, J., 2011. Plants used in constructed wetlands with horizontal subsurface flow: a review.Wetland Restoration, 674: 133-156. https://doi.org/10.1007/s10750-011-0738-9
Vymazal, J., 2010. Constructed Wetlands for Wastewater Treatment. Water 2, 530–549. https://doi.org/10.3390/w2030530
Vymazal, J., Březinová, T., 2016. Accumulation of heavy metals in aboveground biomass of Phragmites australis in horizontal flow constructed wetlands for wastewater treatment: A review. Chem. Eng. J. 290, 232–242. https://doi.org/10.1016/j.cej.2015.12.108
Vymazal, J., Kröpfelová, L., 2015. Multistage hybrid constructed wetland for enhanced removal of nitrogen. Ecol. Eng. 84, 202–208. https://doi.org/10.1016/j.ecoleng.2015.09.017
Wang, Z., Huang, G., An, C., Chen, L., Liu, J., 2016. Removal of copper, zinc and cadmium ions through adsorption on water-quenched blast furnace slag. Desalin. Water Treat. 3994, 1–14. https://doi.org/10.1080/19443994.2015.1135084
Ward, M.L., Bitton, G., Townsend, T., 2005. Heavy metal binding capacity (HMBC) of municipal solid waste landfill leachates. chemosphere.60, 206-215. https://doi.org/10.1016/j.
W.P.C.F (Water Pollution Control Federation). 1990. Natural Systems for Wastewater Treatment. Manual of Practice FD-16. Washington, D.C
Weis, J.S., Weis, P., 2004. Metal uptake, transport and release by wetland plants: Implications for phytoremediation and restoration. Environ. Int. 30, 685–700. https://doi.org/10.1016/j.envint.2003.11.002
131
Wu, S., Austin, D., Liu, L., Dong, R., 2011. Performance of integrated household constructed wetland for domestic wastewater treatment in rural areas. Ecol. Eng. 37, 948–954. https://doi.org/10.1016/j.ecoleng.2011.02.002
Xie, Z., Wang, Z., Wang, Q., Zhu, C., Wu, Z., 2014a. An anaerobic dynamic membrane bioreactor (AnDMBR) for landfill leachate treatment: Performance and microbial community identification. Bioresour. Technol. 161, 29–39. https://doi.org/10.1016/j.biortech.2014.03.014
Xiong, T., Leveque, T., Shahid, M., Foucault, Y., Mombo, S., Dumat, C., 2014. Lead and Cadmium Phytoavailability and Human Bioaccessibility for Vegetables Exposed to Soil or Atmospheric Pollution by Process Ultrafine Particles. J. Environ. Qual. 43(5), 1-9. https://doi.org/10.2134/jeq2013.11.0469
Xu, J., Shi, Y., Zhang, G., Liu, J., Zhu, Y., 2014. Effect of hydraulic loading rate on the efficiency of effluent treatment in a recirculating puffer aquaculture system coupled with constructed wetlands. J. Ocean Univ. China 13, 146–152. https://doi.org/10.1007/s11802-014-2000-3
Xu, J., Zhao, G., Huang, X., Guo, H., Liu, W., 2017. Use of horizontal subsurface flow constructed wetlands to treat reverse osmosis concentrate of rolling wastewater. Int. J. Phytoremediation 19, 262–269. https://doi.org/10.1080/15226514.2016.1217392
Yalcuk, A., Ugurlu, A., 2009. Comparison of horizontal and vertical constructed wetland systems for landfill leachate treatment. Bioresour. Technol. 100, 2521–2526. https://doi.org/10.1016/j.biortech.2008.11.029
Ye, F., Li, Y., 2009. Enhancement of nitrogen removal in towery hybrid constructed wetland to treat domestic wastewater for small rural communities. Ecol. Eng. 35, 1043–1050. https://doi.org/10.1016/j.ecoleng.2009.03.009
Ye, Z.H., Baker, J.M., Wong, M.H., Willis, J., 2003. Copper tolerance, uptake and accumulation by Phragmites australis. Chemosphere 50, 795–800.
Yucong Z, Xiaochang W, Mawuli D, B., Yaqian Z, Huu H N, Wenshan G., Yuan G., Jiaqing X., 2016. Effects of interspecific competition on the growth of macrophytes and nutrient removal in constructed wetlands: A comparative assessment of free water surface and horizontal subsurface flow systems. Bioresour. Technol. 207, 134–141.
Zhang, D.-B., Wu, X.-G., Wang, Y.-S., Zhang, H., 2014. Landfill leachate treatment using the sequencing batch biofilm reactor method integrated with the electro-Fenton process. Chem. Pap. 68, 782–787. https://doi.org/10.2478/s11696-013-0504-8
Zhang, J., Li, R., Li, J., Hu, J., Sun, Q., 2013. [Limestone and pyrite-limestone constructed wetlands for treating river water]. Huan jing ke xue= Huanjing kexue 34, 3445–50.
Zhang, X., Liu, P., Yang, Y., Chen, W., 2007. Phytoremediation of urban wastewater by model wetlands with ornamental hydrophytes. J. Environ. Sci. (China) 19, 902–9.
Zhao, C., Xie, H., Xu, J., Zhang, J., Liang, S., Hao, J., Hao Ngo, H., Guo, W., Xu, X., Wang, Q., Wang, J., 2016. Removal mechanisms and plant species selection by bioaccumulative factors in surface flow constructed wetlands (CWs): In the case of triclosan. Sci. Total
132
Environ. 547, 9–16. https://doi.org/10.1016/j.scitotenv.2015.12.119
Zhao, S., Lian, F., Duo, L., 2011. EDTA-assisted phytoextraction of heavy metals by turfgrass from municipal solid waste compost using permeable barriers and associated potential leaching risk. Bioresour. Technol. 102, 621–626. https://doi.org/10.1016/j.biortech.2010.08.006
Zhao, Y.J., Hui, Z., Chao, X., Nie, E., Li, H.J., He, J., Zheng, Z., 2011. Efficiency of two-stage combinations of subsurface vertical down-flow and up-flow constructed wetland systems for treating variation in influent C/N ratios of domestic wastewater. Ecol. Eng. 37, 1546–1554. https://doi.org/10.1016/j.ecoleng.2011.06.005
Zhao, Y.Q., Babatunde, A.O., Hu, Y.S., Kumar, J.L.G., Zhao, X.H., 2011. Pilot field-scale demonstration of a novel alum sludge-based constructed wetland system for enhanced wastewater treatment. Process Biochem. 46, 278–283. https://doi.org/10.1016/j.procbio.2010.08.023
Zhou, Y.-F., Haynes, R.J., 2010. Sorption of Heavy Metals by Inorganic and Organic Components of Solid Wastes: Significance to Use of Wastes as Low-Cost Adsorbents and Immobilizing Agents. Crit. Rev. Environ. Sci. Technol. 40, 909–977. https://doi.org/10.1080/10643380802586857
Zhu, H., Bañuelos, G., 2017. Evaluation of two hybrid poplar clones as constructed wetland plant species for treating saline water high in boron and selenium, or waters only high in boron. J. Hazard. Mater. 333, 319–328. https://doi.org/10.1016/j.jhazmat.2017.03.041
Zhu, W.-L., Cui, L.-H., Ouyang, Y., Long, C.-F., Tang, X.-D., 2011. Kinetic Adsorption of Ammonium Nitrogen by Substrate Materials for Constructed Wetlands. Pedosphere 21, 454–463. https://doi.org/10.1016/S1002-0160(11)60147-1
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Appendices
Fig 1 Calibration curve of copper in atomic absorption spectrophotometer
Fig 2 EDX analysis of crushed brick
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
0
100
200
300
400
500
600
700
800
900
1000
Coun
ts
CKa
OKa
NaKa
MgK
a AlKa
SiKa
SKa SK
bCl
KaCl
KbKK
a KKb Ca
KaCa
Kb
TiLl
TiLa
TiKa
TiKb
FeLl
FeLa
FeKe
sc
FeKa
FeKb
134
Fig 3 EDX analysis of steel slag
Fig 4 XRD analysis of steel slag
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
0
100
200
300
400
500
600
700
800
900
1000
Coun
ts
CKa
OKa
MgKa
AlKa
ClKa ClK
bKK
a KKb Ca
KaCa
Kb
TiLl T
iLa
TiKa
TiKb
FeLl
FeLa
FeKe
sc
FeKa
FeKb
Ca2SiO4
Ca(OH)2
FeO CaCO3
Al2O3 SiO CuSiO2
135
Fig 5 XRD analysis of steel slag
Fig 6 XRD analysis of sand
Fe2O3
SiO2 Al2O3
SiO2
ZnO
Al2O3