Application Of Common Duckweed (Lemna Minor) In Phytoremediation Of Chemicals In The Environment State And Future Perspective

Accepted Manuscript
Application of Common Duckweed ( ) in Phytoremediation of
Lemna minor
Chemicals in the Environment: State and Future Perspective
Abraham O. Ekperusi, Francis D. Sikoki, Eunice O. Nwachukwu
PII: S0045-6535(19)30241-3
DOI: 10.1016/j.chemosphere.2019.02.025
Reference: CHEM 23137
To appear in: Chemosphere
Received Date: 18 June 2018
Accepted Date: 06 February 2019
Please cite this article as: Abraham O. Ekperusi, Francis D. Sikoki, Eunice O. Nwachukwu,
Application of Common Duckweed ( ) in Phytoremediation of Chemicals in the
Lemna minor
Environment: State and Future Perspective, (2019), doi: 10.1016/j.chemosphere.
Chemosphere
2019.02.025
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1 Application of Common Duckweed (Lemna minor) in Phytoremediation of
2 Chemicals in the Environment: State and Future Perspective
3
4 Abraham O. *Ekperusi12, Francis D. Sikoki3 and Eunice O. Nwachukwu4
5 1 World Bank Africa Centre of Excellence, Centre for Oilfield Chemicals Research, Institute
6 of Petroleum Studies, University of Port Harcourt, Choba, Rivers State, Nigeria
7 2 Department of Marine Environment & Pollution Control, Faculty of Marine Environmental
8 Management, Nigeria Maritime University, Okerenkoko, Delta State, Nigeria
9 3 Department of Animal & Environmental Biology, Faculty of Science, University of Port
10 Harcourt, Choba, Rivers State, Nigeria
11 4 Department of Plant Science & Biotechnology, Faculty of Science, University of Port
12 Harcourt, Choba, Rivers State, Nigeria
13
14 *Correspondence author: +2348033851884, ekperusiab@gmail.com,
15 abraham.ekperusi@aceuniport.org
16
17 ABSTRACT
18 Over the past 50 years, different strategies have been developed for the remediation of
19 polluted air, land and water. Driven by public opinion and regulatory bottlenecks, ecological
20 based strategies are preferable than conventional methods in the treatments of chemical
21 effluents. Ecological systems with the application of microbes, fungi, earthworms, plants,
22 enzymes, electrode and nanoparticles have been applied to varying degrees in different media
23 for the remediation of various categories of pollutants. Aquatic macrophytes have been used
24 extensively for the remediation of pollutants in wastewater effluents and aquatic environment
25 over the past 30 years with the common duckweed (L. minor) as one of the most effective

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26 macrophytes that have been applied for remediation studies. Duckweed has shown strong
27 potentials for the phytoremediation of organic pollutants, heavy metals, agrochemicals,
28 pharmaceuticals and personal care products, radioactive waste, nanomaterials, petroleum
29 hydrocarbons, dyes, toxins, and related pollutants. This review covers the state of duckweed
30 application for the remediation of diverse aquatic pollutants and identifies gaps that are
31 necessary for further studies as we find pragmatic and sound ecological solutions for the
32 remediation of polluted environment for sustainable development.
33
34 Keywords: phytoremediation, chemical pollutants, macrophytes, duckweed, constructed
35 wetlands, petroleum hydrocarbons
36
37 Contents
38 Abstract
39 1.0 Introduction
40 2.0 Phytoremediation
41 2.1 Phytoremediation in Air
42 2.2 Phytoremediation in Soil
43 2.3 Phytoremediation in Water: Application of Macrophytes
44 3.0 Role of Constructed Wetlands in Macrophytes Phytoremediation
45 3.1 Classifications of Wetlands
46 3.1.1 Surface Flow Constructed Wetlands
47 3.1.2 Subsurface Flow Constructed Wetlands
48 4.0 The Common Duckweed (Lemna minor): An Invasive Floating Macrophytes
49 4.1 Biology of Lemna minor
50 4.2 Distribution of Lemna minor

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51 4.3 Taxonomy of Lemna minor
52 4.4 Economic Importance of Lemna minor
53 4.5 Control of Lemna minor
54 4.6 Cultivation of Lemna minor
55 4.7 Uses of Lemna minor
56 5.0 Application of Lemna minor in Phytoremediation
57 5.1 Phytoremediation of Organic Pollutants
58 5.2 Phytoremediation of Heavy Metals
59 5.3 Phytoremediation of Agricultural Chemicals
60 5.4 Phytoremediation of Pharmaceuticals and Personal Care Products
61 5.5 Phytoremediation of Radioactive Wastes
62 5.6 Phytoremediation of Nanomaterials
63 5.7 Phytoremediation of Petroleum Hydrocarbons
64 5.8 Phytoremediation of Toxins, Dyes, Bacteria, Algae
65 6.0 Future Perspective
66 Conclusion
67 References
68
69 1.0 Introduction
70 Pollution is one of the critical existential problems affecting modern society. The industrial
71 revolution of the last century and the dramatic increased in human population over time has
72 resulted in the generation of an unprecedented amount of waste materials and pollutants into
73 the environment. According to the Lancet Commission on Pollution and Health, air, water
74 and soil pollution were responsible for 16% of deaths worldwide in 2015. About 92% of such
75 deaths occurred in developing countries with children being at high risk (Landrigan et al.,

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76 2017). The report further indicated that global pollution cost $4.6 trillion per year, which is
77 equivalent to 6.2% of economic output worldwide (Landrigan et al., 2017). An earlier report
78 from the World Bank and the Institute for Health Metrics and Evaluation put the cost at US
79 $5 trillion worldwide in 2013 (World Bank/IHME, 2016). Over the past 50 years, different
80 strategies have been introduced for the remediation of polluted air, land and water. Many of
81 these strategies, driven by government policies and public opinion, favours ecological based
82 treatment as against conventional methods. Ecological based strategies such as the
83 application of microbes, fungi, earthworms, plants, electrode, and nanoparticles have been
84 applied to varying degrees in different media for remediation of different categories of
85 pollutants (Gadd, 2001; Gregory et al., 2004; Nwachukwu and Osuji, 2007; Mmom and
86 Deekor, 2010; Omokeyeke et al., 2013; Ichor et al., 2014; Makky et al., 2014; Ekperusi et
87 al., 2015; Yan and Reible 2015). This paper evaluates the major concept in phytoremediation
88 and the ecological role of aquatic macrophytes. It also reviewed the application of L. minor
89 for the removal of pollutants in aquatic environment, and future areas of interest in the
90 application of the plant for phytoremediation studies.
91
92 2.0 Phytoremediation
93 Phytoremediation is an aspect of bioremediation that deals with the application of plants for
94 the remediation of polluted environment. It is the potentials of plant species to remove
95 pollutants from polluted media. Phytoremediation deals with the application of certain plants
96 species to accumulate pollutants in terrestrial and aquatic environment. Plant species selected
97 for phytoremediation have the potentials to accumulate specific or wide range of pollutants
98 (Nwachukwu and Osuji, 2007; Omokeyeke et al., 2013; Udeh et al., 2013). In some cases,
99 plants known as hyperaccumulators have the potentials to bioaccumulate pollutants several
100 times above the plant biomass (Van Epps, 2006). Several species of plants have the potentials

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101 to take up, bioaccumulates, immobilize and degrade pollutants in their tissues.
102 Phytoremediation has been applied in the three matrices of the environment such as air, land
103 and water pollution (Van Epps, 2006).
104
105 2.1 Phytoremediation in Air
106 Air pollution is one of the greatest challenges of modern society, due to the relative ease
107 of the movement of pollutants in the atmosphere. In the presence of wind, air pollutants
108 such as soot, carbon monoxide, sulphur dioxide and volatile organic hydrocarbons can
109 travel a considerable distance within a short time in the atmosphere, moving across
110 national boundaries within hours or days. Pollutants in the air can be in the form of foul
111 smell or particles generally termed as particulate matter. Foul or repugnant pollutants in the
112 air, are quickly avoided by humans by moving to areas devoid of such pollutants. Except in
113 cases of smog, humans may be unaware of the presence of particulate matter in the
114 atmosphere. Particulate matter (PM) may be in the air for short or long period as aerosols
115 suspension where it can cause significant harm to human health (Dockery and Pope, 1994; Le
116 Tertre et al., 2002). Breathable PM in the air get into human respiratory tract and the
117 bloodstream via the trachea, lungs and alveoli causing increased difficulty in breathing,
118 especially for those with respiratory conditions such as bronchitis and asthma (Kaupp et al.,
119 2000; Morawska and Zhang, 2000; Nemmar et al., 2002; Zanobetti et al., 2003; Silva et al.,
120 2013). Although, there are many technologies to treat pollutants in the atmosphere, one of the
121 safest and cost-effective is the use of trees for the phytoremediation of air pollutants. Certain
122 plant acts as bio-filters to filter pollutants from the atmosphere via leaf surfaces and shoot of
123 plants (Popek et al., 2013; Nowak et al., 2006). Several species of trees such as Acer
124 platanoides, Artocarpus heterophyllus, Bauhinia variegate, Betula pendula, Ficus spp,
125 Hedera helix, Lagerstroemia speciosa, Mangifera indica, Pinus spp, Psidium guajava,

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126 Stephanandra incisa, Taxus spp and Tilia cordata have been exploited to remove pollutants
127 from the air (Popek et al., 2013; Nawrot et al., 2011; Dzierżanowski et al., 2011; Przybysz et
128 al., 2014a and b; Sæbø et al., 2012; Rai and Panda, 2014; Popek et al., 2017) with varying
129 efficiencies. This is one of the hallmarks of designing sustainable cities as we move into the
130 future. It has been indicated that duckweed can remove about 100 million tonnes of carbon
131 dioxide from the atmosphere (Zhao et al., 2012).
132
133 2.2 Phytoremediation in Soil
134 Land pollution is prevalent across different regions of the world. Pollutants in soil have
135 consequences for soil biota, groundwater and public health (Schaefer and Filser, 2007).
136 Pollutants such as pesticides, diesel, bitumen, can alter soil chemistry and render soil useless
137 for agricultural and other purposes (Okpokwasili and Odokuma, 1990; Anderson and
138 LaBelle, 2000; Obot et al., 2006). Pollutants especially metals in soil could be
139 bioaccumulated in plants tissues and then transferred to humans that consumed plant directly
140 or indirectly. Pollutants can also leach downward and affects groundwater or reach the water
141 table (UNEP, 2011). Abstraction of such polluted water will also result in a direct impact on
142 human health resulting in damage to vital organs such as liver, kidney, lungs, with children
143 and elderly being at greater risk (Lewander and Aleguas, 2007; Landrigan et al., 2017).
144 Phytoremediation on land has gained so much attention within the last three decades as an
145 ecological based solution for the removal of pollutants in soil. Phytoremediation of pollutant
146 in contaminated soil is one of the most extensive applications of plant-based remediation.
147 Over the years, several species of plants have been identified, and screened for the removal of
148 pollutants such as organic pollutants, heavy metals, pesticides, hydrocarbons and munitions
149 from polluted soil (Ndimele, 2010; Nwaichi et al., 2011; Udeh et al., 2013; Nwaichi et al.,
150 2015). Several researchers have recorded varying degree of efficiency with the application of

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151 plants (Njoku et al., 2009; Ndimele, 2010; Vaziri et al., 2013) while in some cases, the
152 addition of nutrients was reported to enhance and increase the efficacy of the
153 phytoremediation process by plants (Mâşu et al. 2014).
154
155 2.3 Phytoremediation in Water: Application of Macrophytes
156 Water pollution is one of the major challenges facing emerging economies. Over a billion
157 people are affected by water pollution issues especially in developing countries (Landrigan et
158 al., 2017). Since the beginning of the Anthropocene, humans have treated water with levity.
159 Freshwater resources were largely seen as a receptacle for domestic and industrial waste.
160 Today, water pollution and the growing share of wastewater released into water bodies is a
161 concern for the sustainable development of water resources for society. Phytoremediation in
162 water deals with the application of aquatic plants for the remediation of pollutants in waters
163 resources or aquatic ecosystems. Certain aquatic macrophytes or invasive aquatic plants are
164 well adapted for the remediation of pollutants in water. Over the years, there has been
165 extensive literature on the application of aquatic plants for the treatment of wastewater and
166 the removal of various pollutants in polluted water resources. Several reviews have been
167 published within the last 35 years on the application of aquatic macrophytes in
168 phytoremediation of a wide range of pollutants in waters (Gupta, 1980; Van Epps, 2006;
169 Sasikumar and Papinazath, 2003; Saier and Trevors, 2010; Zhang et al., 2010; Healy and
170 O’Flynn, 2011; Rahman and Hasegawa, 2011; Sood et al., 2011; Gupta et al., 2012; Sood et
171 al., 2012; Vithanage et al., 2012; Luqman et al., 2013; Mahmood et al., 2013; Vymazal,
172 2013; Zhang et al., 2013; Barznji, 2014; Cui and Cheng, 2014; Halaimi et al., 2014; Khan et
173 al., 2014; Sharma et al., 2014; Verlicchi and Zambello, 2014; Usharani and Vasudevan,
174 2014; Rezania et al., 2015; Shafi et al., 2015; Newete and Byrne, 2016; Machado et al.,
175 2016; Rezania et al., 2016; Mishra and Maiti, 2017; Ekperusi et al., 2018). Aquatic plants

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176 have been applied in the laboratory, field trials and in aquatic ecosystems for the remediation
177 of a wide range of pollutants. Macrophytes have been applied in the remediation of organic
178 pollutants (Ali et al., 1999; Chang et al., 2006; Maine et al., 2007; Dhote and Dixit, 2008;
179 Geurts et al., 2009; Chlot et al., 2011; Shilton et al., 2012; Zhao et al., 2013; Wang et al.,
180 2014; Moore et al., 2016; Lopes et al., 2017), heavy metals (Robinson et al., 2003;
181 Yaowakhan et al., 2005; Bragato et al., 2006; Kumar et al., 2008; Yeh and Wu, 2009; Fawzy
182 et al., 2011; Chattopadhyay et al., 2012; Delmail et al., 2013; Sun et al., 2013; Verma et al.,
183 2014; Łojko et al. 2015; Cajamarca et al. 2016; Gwenzi et al., 2017), agrochemicals (Grollier
184 et al., 1997; Hand et al., 2001; Brogan and Relyea, 2013; Mercado-Borrayo et al., 2015; Lv
185 et al., 2016; Zhang et al. 2017), pharmaceuticals and personal care products (Chen et al.,
186 2009; Reyes-Contreras et al., 2012; Zhang et al., 2013; Cardinal et al., 2014; Hijosa-Valsero
187 et al., 2016) and petrochemicals (Larue et al., 2010; Wang et al., 2011; Akpor et al., 2014;
188 Yan et al., 2015; Al-Baldawi et al., 2016; Pi et al., 2017). Macrophytes have also been used
189 effectively for the removal of radioactive wastes (Sharma et al., 2014; He et al., 2015),
190 munitions (Nepovim et al, 2005), plastic (Zhang et al., 2017) and river remediation (Wang et
191 al., 2011; Li et al., 2011). Macrophytes have been reported to reduce or suppress enteric
192 pathogen (Shingare et al., 2017) and algal population (Zuo et al., 2014) in polluted waters. In
193 some cases, macrophytes have been combined with other organisms such as algae (Thomas
194 and Hand 2011; He et al., 2015) and microbes for the phytoremediation of pollutants in
195 waters (Al-Baldawi et al., 2016; Zhang et al., 2017). Biochar generated from macrophytes
196 (Zeng et al., 2013) have also been applied effectively for phytoremediation of polluted
197 waters. The level of macrophytes application keeps expanding across different geographical
198 regions. Macrophytes have been used to remove pollutants from distillery effluent (Kumar
199 and Chandra, 2004), saline conditions (Klomjek et al., 2005), industrial effluents and
200 wastewater (Schröder et al., 2007; Zhang, 2012; Sukumaran, 2013), domestic effluents and

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201 sewage (Chen et al., 2009; Basílico et al., 2017; Shingare et al., 2017), mine tailing and acids
202 (Saha et al., 2016; Gwenzi et al., 2017), eutrophic lake (Zhao et al., 2013), electroplating
203 (Sun et al., 2013), stormwater (Wang et al., 2014), agricultural run-offs (Mercado-Borrayo et
204 al., 2015), textile dyes (Khandare and Govindwar, 2015), pulp and paper (Arivoli et al.,
205 2015), diary (Lopes et al., 2017) and several other categories of wastewater effluents.
206 Three classes of macrophytes are recognized based on their structural habitat or niche they
207 occupy in aquatic ecosystems for the remediation of pollutants in aquatic environment. They
208 include floating, emergent and submerged macrophytes.
209 Floating macrophytes: These are macrophytes that are exclusively found on the surface of
210 water bodies. They are usually found in standing or slow moving waters. Examples include
211 Lemna spp, Pistia stratiotes, Salvinia minima, Landoltia punctata, Spirodela polyrhiza,
212 Riccia fluitans, Wolffiella gladiata, Marsilea mutica, Hottonia inflata, Azolla spp., Nelumbo
213 lutea and Eichhornia crassipes (USDA, 2018).
214
215 Emergent macrophytes: These macrophytes have roots that are attached to the substrate at
216 the bottom of water bodies while the leaves grow to or above the surface of the water.
217 Examples include Butomus umbellatus, Cyperus spp., Diodia virginiana, Distichlis spicata,
218 Hydrochloa caroliniensis, Imperata cylindrica, Iris virginica, Juncus spp., Justicia
219 americana, Nasturtium officinale, Nuphar lutea, Nymphaea spp., Phragmites australis and
220 Typha spp (USDA, 2018).
221 Submerged macrophytes: They are permanently submerged in water. Their root system is
222 attached to the substrate but their leaves do not reach the surface of the water, unlike
223 emergent macrophytes. They are perpetually submerged throughout the year or lifecycle.
224 Examples include Ceratophyllum demersum, Egeria densa, Hydrilla verticillata, Hygrophila
225 corymbosa, Myriophyllum aquaticum, Najas marina, Potamogeton natans, Ruppia maritima,

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226 Stuckenia pectinata, Vallisneria americana and Elodea canadiensis (Mahmood et al., 2013;
227 USDA, 2018). The nature of macrophytes applied in phytoremediation of water pollution
228 depends on the behaviour and distribution of the pollutants in water.
229
230 3.0 Role of Constructed Wetlands in Macrophytes Phytoremediation
231 Natural wetlands are aquatic ecosystems that provide rich ecological and economic resources
232 to communities and local people. Wetlands are largely dominated by aquatic macrophytes as
233 the predominant vegetation. Wetlands regulate pollutants, recharge groundwater, provides
234 habitat for biodiversity and provides water and fishery resources to local people. The
235 structural niches of macrophytes in natural wetlands were conceptualized to design and
236 construct wetlands for the remediation of aquatic pollution. Constructed wetlands also known
237 as man-made or engineered wetlands mimic the natural conditions of wetlands with the
238 selection of appropriate macrophytes for the treatment of polluted waters. The low to zero
239 cost of construction, operation and maintenance of constructed wetlands and low ecological
240 footprint make it attractive to investors, city planners and developers (Kongroy et al., 2012).
241 The first constructed wetland was built in Germany by Seidel at the Max Planck Institute
242 with the application of bulrushes for wastewater treatment. Her research work led to the
243 construction of the first operational constructed wetlands in Liebenburg-Othfresen, Germany
244 in 1974 (Verhoeven et al., 2006).
245
246 3.1 Classifications of Wetlands
247 Constructed wetlands are classified generally into Surface Flow and Subsurface Flow
248 Wetlands (Tousignant et al., 1999). Surface flow wetlands are common in North America,
249 especially in the United States (USEPA, 1993) than subsurface flow wetlands which are
250 common in Europe (Mueller and Goswami, 2003). In general, surface flow wetlands require

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251 more land than subsurface wetlands for the same pollution reduction but are easier and
252 cheaper to design and build. In some cases, subsurface systems are often more efficient but
253 can cost significantly more than surface flow systems (Mueller and Goswami, 2003).
254
255 3.1.1 Surface Flow Constructed Wetlands
256 Any wetland where the flowing water is open to air is termed surface flow systems. Reed and
257 Brown (1992) characterize surface flow wetlands as most closely mimicking natural marshes.
258 Additionally, surface flow wetlands have greater aesthetic appeal, wildlife habitat
259 availability, and recreational opportunities, which foster public support (Mueller and
260 Goswami 2003). Surface wetlands are sub-divided into three categories such as floating,
261 emergent and submerged wetlands (Tousignant et al., 1999) depending on the macrophytes
262 applied.
263 Floating Macrophytes Constructed Wetlands: These categories of wetlands make use of
264 floating plants such as duckweeds, water lettuce and water hyacinth. The wetland is design to
265 treat pollutants found in surface waters (Tousignant et al., 1999). Such pollutants are less
266 dense than water.
267 Emergent Macrophytes Constructed Wetlands: These wetlands are design based on the
268 types of emergent plants applied. Emergent wetlands may be applied for the treatment of
269 surface and below surface pollutants (Tousignant et al., 1999).
270 Submerged Macrophytes Constructed Wetlands: These categories of wetlands apply
271 macrophytes that are submerged in aquatic ecosystems. They are mostly applied in the
272 tertiary treatment of wastewater system (Brix, 1994; Tousignant et al., 1999).
273
274 3.1.2 Subsurface Flow Constructed Wetlands

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275 The advantages of subsurface systems include increased treatment efficiencies, fewer pest
276 problems, reduced risk of exposing humans or wildlife to toxic substances and increased
277 accessibility for upkeep. Saving land area is important at many installations and translates
278 into reduced capital cost for projects requiring a land purchase (Mueller and Goswami, 2003).
279 Subsurface flow constructed wetlands are sub-divided according to their flow pattern into
280 horizontal and vertical flow constructed wetlands (Tousignant et al., 1999). Vertical flow
281 systems have removal mechanisms similar to that of horizontal flow systems but completely
282 different flow mechanisms (Mueller and Goswami 2003). Both allow water to flow through
283 permeable, root-laced media, but some vertical flow systems combine an organic substrate
284 with the permeable media (Mueller and Goswami, 2003).
285 Horizontal Flow Constructed Wetlands: In horizontal flow wetlands, the medium is kept
286 saturated under a continuous wastewater flow with effluents moving horizontally via gravity
287 (Tousignant et al., 1999). Horizontal systems are more prevalent and thereby have a
288 considerably larger knowledge base (Mueller and Goswami, 2003).
289 Vertical Flow Constructed Wetlands: Vertical flow wetlands are operated as a batch
290 process rather than in continuous flow mode. The effluents move vertically from the planted
291 layer down through the substrate. The system is aerated using pumps. Vertical flow wetlands
292 are less common and not as well documented as horizontal flow systems (Tousignant et al.,
293 1999). Vertical systems are more efficient and are common in mining applications (Mueller
294 and Goswami, 2003).
295 Over the years, constructed wetlands have undergone critical modifications and wide
296 acceptance for the treatment of various types of effluents and polluted waters in different
297 parts of the world (Hadad et al., 2006; Maine et al., 2007; Zhang et al., 2010; Idris et al.,
298 2011; Kongroy et al., 2012; Vymazal, 2013; Arivoli et al., 2015; Al-Baldawi et al., 2016; Pi
299 et al., 2017; Zhang et al., 2017). Zhang (2012) reported that constructed wetlands removed

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300 about 87% of pollutants in wastewater treatment. An appraisal of about forty constructed
301 wetlands in Brazil by Machado et al. (2016) concluded that constructed wetlands are highly
302 efficient in the treatment of a wide range of wastewaters. They advocated for the expansion
303 and continued use of constructed wetlands in Brazil. Zhang et al. (2015) reviewed records of
304 constructed wetlands application in the phytoremediation of wastewater within a fifteen years
305 period (2000 and 2015). They concluded that all forms of constructed wetlands are very
306 efficient in the removal of organic pollutants in wastewater.
307
308 4.0 The Common Duckweed (Lemna minor): An Invasive Floating Macrophyte
309 The common duckweed (L. minor) is an invasive floating aquatic macrophyte with ecological
310 and economic implication wherever the colony of the plant exist. To understand the ability of
311 the plant to play a vital role in the phytoremediation of a wide range of pollutants, there is
312 need to briefly review the ecological role of the plant as a vital component of the aquatic
313 ecosystem.
314
315 4.1 Biology of Lemna minor
316 L. minor is the smallest of all angiosperm or flowering plants in the plant kingdom.
317 Duckweed is a small floating monocotyledons plant that forms a thick blanket in nutrient-rich
318 fresh and brackish waters. The plant is composed of one or few leaves called fronds and a
319 single root or rootlet with no stem. It reproduces vegetatively by simply dividing to form
320 separate individual plants (Correll and Correll, 1972). L. minor is about 2 to 4 mm across. It
321 aggregates together forming colonies on surface waters (Rusoff et al., 1980). Frick (1985)
322 reported that the frond doubling time for L. minor was about 1.4 days. Duckweed cultured in
323 the laboratory can grow indefinitely if nutrients, light and water are provided, thus producing
324 unlimited duckweed specimens for use at any moment. It produces a considerable number of

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325 daughter fronds during its lifetime, but mother frond usually dies after production of six
326 generations (Ziegler et al., 2015). Kutschera and Niklas (2014) dubbed the duckweed family
327 as ‘Darwinian Demons’ due to their ubiquitous reproductive capacity, sporadic development
328 and ability to almost 'live forever'.
329
330 4.2 Distribution of Lemna minor
331 L. minor is widely distributed across different geographical regions from the tropics to
332 temperate zones, from freshwater to brackish water (Hillman and Culley, 1978). It is native to
333 Africa, Asia, Europe and North America but present in Australia and South America
334 (Appenroth et al., 2015). Birds are important in dispersing duckweeds to new sites. The
335 sticky root enables the plant to adhere to the plumage or feet of water birds, aiding the spread
336 to different aquatic ecosystems (Mbagwu and Adeniji, 1988).
337
338
339 Figure 1: Global distribution of duckweed, (Landolt, 1986)

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340
341 4.3 Taxonomy of Lemna minor
342 L. minor belongs to the family Lemnaceae, which is monophyletic to the Araceae family
343 (Klaus et al., 2013). There are about 40 species in five genera: Lemna, Landoltia, Spirodela,
344 Wolffia and Wolffiella. Duckweeds are extremely reduced in morphology, present a
345 developmental hybrid of leaf, and stem origin (Lemon and Posluszny, 2000). The extreme
346 reduction in its morphological characteristics, global distribution and high phenotypic
347 plasticity to environmental conditions (Vaughan and Baker 1994; Bog et al., 2010), have
348 made the taxonomy of the group very difficult for scientists over the past 200 years. The
349 phylogeny of the family still has an ongoing dispute. The simplified morphology of
350 duckweeds has made it particularly difficult to reconcile their inter-specific relationships (Les
351 et al., 2002; Bog et al., 2010). Systematics approach relying solely on morphological and
352 biochemical markers (Crawford et al., 1996, 2005; Les et al., 1997) is insufficient for
353 classification of the group. This has lead to the application of advanced identification
354 techniques such as molecular studies for the elucidation of the group (Yamamoto et al., 2001;
355 Les et al., 2002; Mardanov et al., 2008; Xue et al., 2011). The classification for duckweed
356 includes;
357 Domain: Eukaryota
358 Kingdom: Plantae
359 Phylum: Spermatophyta
360 Subphylum: Angiospermae
361 Class: Monocotyledonae
362 Order: Araceae (Arales)
363 Family: Lemnaceae
364 Genera: Lemna

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365 Species: Lemna minor
366
367 4.4 Economic Importance of Lemna minor
368 The sporadic growth of duckweed inflicts serious damage to aquatic resources with several
369 economic implications. The dense and extensive mat created by the plant on surface waters
370 block water channels and makes activities such as water flow, navigation, boating, swimming
371 and fishing impossible. It also affects irrigation, flood canals, clog hydroelectric turbines
372 (Sculthorpe, 1967; Bruner, 1982; Sharma, 1984; Dray and Center, 2002) and disrupt rice
373 fields (Holm et al., 1977; Waterhouse, 1993). A dense cover of duckweed shuts out and
374 inhibits competing submerged aquatic plants including algae from sunlight (Sculthorpe,
375 1967; Sharma, 1984).
376
377 4.5 Control of Lemna minor
378 Control of duckweed can be by physical (mechanical), chemical or biological. In some cases,
379 integrated control involving the combination of two methods for the control of duckweed is
380 required. Physical or mechanical control includes physical removal by hand or machines.
381 Special floating harvesters are used in lakes and rivers, and harvested plants are transported
382 to the shore for proper disposal. Chemical control is carried out by the application of
383 herbicides by spraying duckweed infested waters. Effective herbicides include diquat,
384 triclopyr, glyphosate, chlorsulfuron, and endothall (Langeland and Smith, 1993; Rivers,
385 2002). The application of herbicides is highly discouraged due to environmental and public
386 health issues. Several species of herbivores are known as natural enemies of duckweeds.
387 Species such as Anas sp, Aphis sambuci, Elophila sp, Hydrellia williamsi, Lemnaphila
388 neotropica, Lemnaphila scotlandae, Mesovelia mulsanti, Neohydronomus affinis,
389 Rhopalosiphum nymphaeae and Tanysphyrus lemnae are known for the biological control of

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390 duckweed. Many of the species rely on duckweed as food and for egg deposition. Others bore
391 into the Lemna thalli (Scotland, 1940; Buckingham, 1984). Also, almost all species of duck
392 rely on duckweeds as source of food.
393
394 4.6 Cultivation of Lemna minor
395 Duckweed culture requires a regular supply of water and nutrient from organic manure or
396 fertilizer. A single frond can produce as many as 10 generations of daughter plants over a
397 period of 10 days to several weeks before dying (Adesina et al., 2005). The plant doubles its
398 mass in less than 2 days under ideal conditions of nutrients availability, sunlight, and
399 temperature faster than any other higher plants (Adesina et al., 2005). Daily attention and
400 frequent harvesting are required throughout the year to ensure the productivity and health of
401 duckweed colonies (Adesina et al., 2005). For culture purposes, nutrients sources include
402 animal manure, kitchen wastes, waste from food processing plants and abattoir wastes. Under
403 culture conditions, duckweed should be harvested frequently, preferably daily. About 10-35%
404 could be harvested daily leaving the remaining plants in the pond for further growth (Hasan
405 et al., 2009). Duckweed requires a pH of 5 to 9, a temperature of 6 to 33 °C and pond depth
406 of 0.5 metres (Leng et al., 1995; Hasan et al., 2009). It also requires about 60 mg L-1 of
407 nitrogen and a minimum of 1 mg L-1 of phosphorous for growth. Under optimal conditions, a
408 duckweed farm can produce 10 to 30 tonnes of dried duckweed per hectare per year (Leng et
409 al., 1995). Under laboratory conditions duckweed requires a pH of 6 to 7.5 and an
410 appropriate amount of nitrogen, phosphorous and potassium as well as other essential
411 nutrients like sulphur and sodium.
412
413 4.7 Uses of Lemna minor

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414 Growing interest in duckweed has lead to series of international conferences in various parts
415 of the world giving researchers opportunity to interact and advance the study and application
416 of the plant for human progress (Zhao et al., 2012; Lam et al., 2014). L. minor has a long
417 history of application in aquaculture, livestock production, poultry, pharmaceuticals, biofuels,
418 toxicity testing, environmental monitoring and for the remediation of polluted wastewater. In
419 some cases, duckweed has been reported as human food (Boyd, 1968; Chang et al.,
420 1977; Culley et al., 1973; Rusoff et al., 1978; Adesina et al., 2005; Appenroth et al., 2015).
421 In many parts of the world, farmers have used duckweed as a feed source for animal
422 production especially for pigs, ducks and rabbits (Khan et al., 2014). Duckweed protein is
423 reported to have a high concentration of essential amino acids. The plant also has a high
424 concentration of trace elements, carotene and xanthophylls, which makes duckweed a
425 valuable supplement for poultry and animal feeds (Haustein et al., 1988). Fish species such as
426 grass carp (Ctenopharyngodon idella), silver barb (Puntius gonionotus) and tilapia
427 (Oreochromis sp.) are readily fed with duckweed (Iqbal, 1999). There is a passive interest in
428 the use of duckweed as human food supplement. In few reports, it was indicated that
429 duckweed has been used as food by humans (Culley et al., 1973; Rusoff et al., 1977) but no
430 concrete information regarding the application is available. On the nutritional properties of
431 duckweed as a potential food source for humans, Appenroth et al. (2017) reported that
432 protein contents and amino acids in duckweeds are almost the same for WHO recommended
433 value for humans. L. minor has been manipulated to produce monoclonal antibodies for the
434 treatment of human diseases such as tissue inflammation, autoimmune condition and
435 cancerous cells (Cox et al., 2006). Naik et al. (2012) reported a pure and high form of
436 antibody extracted from transgenic Lemna similar to those already available in the market for
437 medical application. Cantó-Pastor et al. (2015) advance the application of genetic
438 engineering with a transgenic L. minor by successfully orchestrating the artificial slicing of

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439 precursor gene for the production of microRNA. A review of previous research by Cui and
440 Cheng (2014) indicated that Lemna could be utilized for the production of butanol, alcohol
441 and biogas. Xu et al. (2011) reported high starch content in duckweed needed for the
442 conversion into ethanol. Zhao et al. (2012) estimated that about 17 million tonnes of fuel
443 annually, which is about 25% of the annual volume of fuel consumed in China, could be
444 produced from duckweed. Research is ongoing to genetically manipulate duckweed to
445 increase lipid contents for increased oil production for biofuels generation (Zhao et al., 2012).
446 Duckweed has been used as model plants for biological monitoring. The plant have been
447 applied to study the ecotoxicological profile of several categories of pollutants in aquatic
448 environment, to have a better understanding of the effect of such pollutants to biological
449 resources and public health (Sinha et al., 2005; Tkalec et al., 2005; Tkalec, et al., 2007;
450 Mufarrege et al., 2009; Senavirathna et al., 2013). National and international standards have
451 been developed using duckweed for toxicity studies (Brain and Solomon, 2007; Bog et al.,
452 2010; Brain et al., 2012). In some cases, toxicity studies have been reported to be more
453 effective using duckweed than animals (Hughes et al., 1988). L. minor is an efficient
454 bioaccumulator of pollutants (organic pollutants, heavy metals, agrochemicals, PPCPs,
455 radioactive waste, nanomaterials and hydrocarbons) in surface waters (Hasan et al., 2009;
456 Reinhold et al. 2011; Mohedano et al. 2012, Basile et al. 2012; Megateli et al. 2013;
457 Shuvaeva et al., 2013; Bokhari et al. 2015; Van Hoeck et al. 2015; Iatrou et al. 2017; Amare
458 et al., 2018; Ergen and Tunca 2018; Ohlbaum et al. 2018). The sporadic distribution and
459 invasive nature of the plant and the ability to thrive in diverse habitats increased the
460 potentials of the plant to withstand harsh environmental conditions including polluted or
461 degraded waters (Sukumaran, 2013; Das et al., 2014).
462
463 5.0 Application of Lemna minor in Phytoremediation

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464 The rapid expansion and increasing sophistication of the global chemical industry for the past
465 century have resulted in increasing levels of complex toxic effluents released into the
466 environment, especially aquatic ecosystem. More than 140,000 new chemicals and pesticides
467 have been synthesised since 1950, with over 5000 produced in large quantity becoming
468 widely dispersed in the environment and are responsible for universal human exposure
469 (Landrigan et al., 2017). L. minor has been applied extensively for the remediation of diverse
470 chemical pollutants. The plant is used separately or in combination with other aquatic
471 macrophytes as an ecological based pollution treatment technology. The various treatment
472 technologies will be considered based on the categories of the pollutant below (Figure 2).
473
474

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475
476
477 Figure 2: Categories of pollutants remediated by L. minor
478
479 5.1 Phytoremediation of Organic Pollutants
480 The development and advancement in the phytoremediation of aquatic pollutant started with
481 the desire to treat municipal and industrial wastewater effluents. The removal of organic load,
482 odour and colour in order to improve water quality before release into stream, rivers or
483 groundwater spur the diverse research from wastewater treatment plant to natural and man-
484 made or engineered wetlands. Either in single or combined application of macrophytes, L.
485 minor has been reported as a very successful floating macrophyte for the phytoremediation of

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486 organic pollutants (Mohedano et al., 2012). In some cases, L. minor was more effective than
487 normal wastewater treatment plant for remediation of municipal effluents. Primary and
488 secondary treatment only resulted in about 50% reduction in biochemical oxygen demand and
489 phosphate whereas duckweed resulted in 94.45 and 79.39% reduction (Priya et al., 2011).
490 There was 98.0 and 98.8% removal for total nitrogen and phosphorous with an increased
491 level of dissolved oxygen due to an improvement of nutrients load by duckweed (Mohedano
492 et al., 2012). The excellent result reported by both authors indicated that duckweed was
493 highly efficient in the removal of organic pollutant in aquatic ecosystems, especially for the
494 treatment of effluent from industrial and agricultural production plants. Irrespective of the
495 effluents type, duckweed has shown the resilience to remove or reduce organic pollutants in
496 wastewater effluents. The removal of organic pollutant is also reflected in the accumulation
497 of biomass and protein contents in duckweed (Mohedano et al., 2012; Saha et al., 2014). This
498 is an indication that the plant utilizes nutrients from the effluents for growth and
499 development. Mohedano et al. (2012) reported an increased level of plant biomass and 35%
500 increase in protein content at the end of the study whereas Saha et al. (2014) reported
501 duckweed increase biomass by 30% within 21 days. Other workers reported poor
502 performance (Saha et al., 2014; Zhang et al., 2014) and inhibition (Wang et al., 2014;
503 Grijalbo et al., 2016; Wang et al., 2016) of duckweed depending on the concentration of the
504 pollutants and the toxicity of the effluents type. In a study with steel effluents, L. minor was
505 only able to remove chloride (30%), sulphate (16%) and total dissolved solids (14%) after 21
506 days (Saha et al., 2014). The removal rate was quite low compared to other reports. Several
507 factors could be responsible for this. The high concentration of the pollutants in the effluents
508 could influence the low result. There is the possibility that the remediation of steel effluents
509 could be difficult for duckweed considering other inhibitory substances that could be present
510 in the effluents which may not be of concern to the authors. Duckweed performs fairly in the

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511 removal of ammonia under lab conditions (Zhang et al., 2014). The plant grew at a
512 concentration ranging from 7 to 84 mg L-1 and optimally at 28 mg L-1 under ammonium
513 nitrate pollution (Wang et al., 2014). At 840 mg L-1, ammonium nitrate inhibited growth rate,
514 carbon contents, carbon-nitrogen ratio, photochemical cells and induced reactive oxygen
515 stress (ROS) that resulted in cell mortality (Wang et al., 2016). Increased ROS in aquatic
516 plant is an indication of environmental stress, compromising the ability or potentials of such
517 plants to carry out its regular ecological function of regulating nutrients in aquatic
518 environment. In a spent metal effluent, L. minor showed slight inhibition at a concentration of
519 2300 mg L-1 for chemical oxygen demand within 5 days interval (Grijalbo et al., 2016). The
520 low inhibition reported indicates the potency of the plant to withstand high level of pollutants
521 in the environment. It also reinforces the resilience of the plant to pollutants, but the short
522 duration of the study could also be a factor for the slight inhibition. Extending the study
523 duration may have indicated otherwise. The combination of microbes may aid or burden the
524 plant in the phytoremediation of pollutants. The inoculation of a bacterial consortium did not
525 have any effect on plant inhibition or growth; rather it significantly increased the reduction of
526 chemical oxygen demand to 41%, compared to only duckweed application which yielded
527 about 16% in mine effluents (Grijalbo et al., 2016). This result indicated the synergy between
528 duckweed and bacterial interaction for the removal of pollutants. Interestingly, the
529 application of bacterial consortium alone did not yield any positive outcome. Papadopoulos
530 and Tsihrintzis (2011) evaluated the phytoremediation potentials of L. minor for the removal
531 of organic pollutant from sewage effluent over a year period. They reported that duckweed
532 was highly efficient in the removal of biochemical oxygen demand, ammonia, and total
533 suspended solids by 94, 72, and 63% respectively, but increased phosphate in effluent by
534 1.1%. As one of the longest study for organic pollutants using duckweed, it is expected that
535 the selected organic pollutants will be completely removed from the sewage effluents, but

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536 this is not the case. The removal rate for the pollutants especially ammonia, total suspended
537 solids and the slight increased in phosphate may indicate that other factors present in the
538 sewage effluents may be acting to reduce the efficiency of duckweed for the remediation of
539 organic pollutants in the study. Tufaner (2018) reported over 82% removal rates for chemical
540 oxygen demand, biochemical oxygen demand, total nitrogen, ammonium nitrate, and
541 phosphate using L. minor. The combination of different species of duckweeds and other
542 macrophytes revealed varying remediation potentials. This could be attributed to the adaptive
543 capacity of each species when exposed to pollutants in the environment. The combination of
544 two or more duckweed species was reported to be highly effective for the remediation of
545 organic pollutants (Yilmaz and Akbulut, 2011; Van Echelpoel et al., 2016; Zhao et al., 2014).
546 The duckweeds L. minor and L. gibba lowered the biochemical and chemical oxygen demand
547 of wastewater effluents ranging from 85 to 88% and 79 to 83% respectively below the
548 USEPA guidelines (Yilmaz and Akbulut, 2011). The combination of three species of
549 duckweeds such as L. minor, Landoltia punctata and Spirodela polyrhiza in the remediation
550 of nitrate and phosphate in swine effluents was more effective than a single application of
551 duckweeds (Zhao et al., 2014). Comparative assessment of the individual plant potentials
552 indicated that L. minor was slightly more effective than L. gibba in wastewater effluents
553 (Yilmaz and Akbulut, 2011), while in swine effluents, L. minor was the most effective than L.
554 punctata and S. polyrhiza. Although duckweeds share a striking and characteristic closeness
555 in the family Lemnaceae, the various species have different potentials to adapt to a wide
556 range of pollutants or stress in the environment. L. minor may well have a better adaptive
557 potential confers on it along its evolutionary pathway to withstand, accumulates and degrade
558 pollutants in the immediate environment compared to the other species of duckweed. Van
559 Echelpoel et al. (2016) conducted a study to understand the invasive mechanisms of L.
560 minuta with a native population of L. minor in the presence of nutrients. In all cases, L. minor

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561 outperforms L. minuta for nutrients removal and biomass. They concluded that the introduced
562 L. minuta did not show any competitive advantage over the native species of L. minor. The
563 result in this study agrees with previous report where L. minor outperformed L. punctata and
564 S. polyrhiza. Azolla filiculoides was more effective than L. minor in the reduction of electrical
565 conductivity, total dissolved solids, turbidity, chemical oxygen demand, phosphate, total
566 nitrogen, sulphate except for biochemical oxygen demand, where the reverse is the case
567 within 28 days (Amare et al., 2018). The increased selective uptake of biochemical oxygen
568 demand by duckweed deserved further studies. It could be related to competition for
569 resources among both species of macrophytes. There is need for further studies especially
570 combining duckweed and other macrophytes to understand the relationship and capacity for
571 each macrophyte in a mixed system for a comprehensive remediation application. Farid et al.
572 (2013) investigated the removal of organic pollutants by six macrophytes in urban wastewater
573 effluent. Macrophytes such as Pistia stratiotes, Eichhornia crassipess, Hydrocotyleum
574 bellatta, L. minor, Tyhpa latifolia and Scirpus acutus showed potentials of phytoremediation
575 of pollutants, although at varying efficiency. The plants removed about 33.7% of electrical
576 conductivity, 93.1% turbidity, 35.2% total dissolved solids, 61% chloride, 45.7% hardness,
577 32.3% calcium, 55.9% magnesium and 77.6% nitrate. They concluded that a combination of
578 macrophytes in effluent treatment was more effective than the use of individual plant
579 treatment. It is the general opinion that the combination of several macrophytes will increase
580 the removal rate of pollutants since synergistic effects of the various macrophytes will come
581 to bear on the level of the pollutants present, but the moderate removal rate for organic
582 pollutants in this study do not reflect that. The combination of the various macrophytes did
583 not provide a better result compared to only duckweed application. Duckweed has shown
584 great potentials in the removal of vital organic pollutants present in wastewater effluents, but
585 there is a still limitation on the categories of effluents types from the report so far. Future

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586 studies should focus on the potentials of the plant for the removal of organic pollutants in
587 other categories of effluents such as industrial wastewater, acid mines, pulp and paper and
588 diary effluents. The effectiveness of duckweed for other categories of effluents will give a
589 broader understanding of the suitability of the plant as a viable and cost-effective alternative
590 for the ecological remediation of organic pollutants in the environment.
591
592 Table 1: Summary of organic pollutants associated with L. minor remediation
Pollutants Duration Conc. Removal Rate References
EC, TDS,
Turbidity, COD,
BOD, P, TN, SO4
-2
28 days 4.52-2737 mg
L-1
68, 68, 97, 92,
92, 97, 94.6,
77.9%
Amare et al. 2018
EC, turbidity, TDS,
Cl, hardness, Ca,
Mg, Nitrate,
Sulphate
30 days 0.83 dS/m,
89.6 NTU,
1.7-525 mg L-1
33.7, 93.1, 35.2,
61, 45.7, 32.3,
55.9, 77.6%
Farid et al. 2013
COD 5 days 2300 mg L-1 16% Grijalbo et al. 2016
pH, EC, TSS, TDS,
DO, BOD, COD,
Chloride, Nitrite,
Nitrate, PO4, SO4,
K
20 days 8.83, 1800
mho/cm, 1018,
2091.67, 1.67,
520.33, 1196,
597.33, 2.80,
3.10, 0.60,
216.85, 68.63
mg L-1
20.26, 92.10,
86.89, 92.82,
90.88, 92.25,
93.91, 92.76,
91.31, 91.36,
92.12, 91.94,
91.32%
Mishra et al. 2012
TN, N-NH3, TP, 1 yr 264.5, 202.1, 98.3, 98.8, Mohedano et al. 2012

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30.1 kg L-1 94.5%
BOD, NH4
+, TSS,
PO4
+
12
months
343, 81, 193,
1.73 mg L-1
94, 72, 63, -1.1% Papadopoulos and
Tsihrintzis 2011
BOD, phosphate 22 days 414, 3.14 mg
L-1
94.45, 79.39% Priya et al. 2011
chloride, sulphate,
TDS
30 days 250-1000 mg
L-1
30, 16, 14% Saha et al. 2014
COD, BOD, TKN,
NH3-N,TP, PO4-P
25 days 1025, 167, 76,
55, 4.8, 2.4 mg
L-1
88, 83, 94, 96,
97, 95%
Tufaner 2018
N, P 4 days 0-80, 0-25 mg
L-1
92, 72% Van Echelpoel et al.
2016
NH4
+ 7 days 28-840 mg L-1 - Wang et al. 2014
BOD, COD 10 days 224-227, 372-
377 mg L-1
88, 83 % Yilmaz and Akbulut,
2011
NH4
+ 2 weeks 0.5 -14 mg L-1 0.082 mg g-1 Zhang et al. 2014
N, P, 9 days 1020, 224 mg
L-1
60-67.84 % Zhao et al. 2014
593
594 5.2 Phytoremediation of Heavy Metals
595 The application of macrophytes in the uptake and remediation of heavy metals from the
596 environment is one of the most investigated areas in the application of macrophytes for
597 removal of pollutants in aquatic media. Heavy metals pose a serious risk to the environment
598 and all life forms because they are indestructible, easily transported across media and can
599 lead to poisoning of tissues and organs (Adesiyan et al., 2018; Enegide and Chukwuma,

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600 2018; Sodango et al., 2018). They can also result in irreversible impairment or damage and
601 deaths in some cases (Sodango et al., 2018; Adesiyan et al., 2018). The application of plant
602 especially macrophytes for heavy metal pollution treatment is due to macrophytes ability to
603 bioaccumulate substantial amount of metals into their tissues. It has been suggested that
604 macrophytes can effectively bioaccumulate heavy metals over 100,000 times higher (Mishra
605 and Tripathi, 2008) than other bioremediation agents into their biomass. The removal or
606 remediation of metals from polluted environment by macrophytes especially duckweed is
607 based on several prevailing factors such as the concentration of the pollutants present, the
608 bioavailability of the metals and the duration of the study. The variation in these factors could
609 determine the efficiency or effectiveness of any remediation efforts by duckweed.
610 Phytoaccumulation of metals by duckweed in many cases may results in no effect on the
611 plant, inhibition and in some cases death. The process can be effective without any effects on
612 the bioremediator. There are cases where the plant can be inhibited in the course of
613 accumulating metals in tissues. Inhibition can be mild or severe, which may or may not affect
614 the remediation capacity of the plant. Severe inhibition results in oxidative stress in plant. It
615 may also result to death of the plant. It has been reported that high accumulation of cadmium
616 by L. minor resulted in the inhibition of the plant (Razinger et al., 2008). Duckweed was able
617 to recover within days after high exposure to copper, nickel and cadmium toxicity (Drost et
618 al., 2007). It is safe to say, that where plant survives a high level of exposure to a toxicant or
619 stress, there is a potential for full recovery. Although there was high removal of zinc and
620 aluminium from effluents, zinc was largely toxic than aluminium at the same concentration
621 (Radic et al., 2010). Toxicity of both metals resulted in oxidative stress with a decline in
622 enzymatic activity, resulting in the build-up of anti-oxidative mechanism against both metals
623 (Radic et al., 2010). This is expected since zinc is more reactive than aluminium in the
624 presence of organic compounds or acids. Exposure of L. minor to varying concentration of

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625 mercury within 6 days period resulted in physiological, biochemical and molecular response
626 (Zhang et al., 2017), whereas the plant was highly effective in the removal of lead and
627 mercury from effluent (Dirilgen, 2011). Mercury is a highly toxic metal to organisms, so
628 duckweed may have deployed all available defence mechanism to withstand and survive the
629 toxic nature of the metal. Toxic metals and stressors in the environment decreased the
630 functional capacity of macrophytes when such stressors exceed the macrophytes tolerance
631 threshold. As an adaptive mechanism for survival, plant usually triggers anti-stress response
632 such as increased production of enzymes, anti-shock proteins in order to protect vital organs
633 and tissues of the plant and counter the stress from the pollutant. Tufaner (2018) reported
634 over 90% removal for heavy metals (chromium, zinc, aluminium, arsenic, cadmium, cobalt,
635 copper, lead and nickel) while 83% for mercury in a mixed wetland using L. minor. From
636 these studies, duckweed could accumulate more mercury in a mixed media with other metals
637 than a single application of mercury with plant. Uptake of selenium by L. minor increased
638 with increased concentration of metals but selenium was highly lethal to duckweed at higher
639 concentrations (Mechora et al., 2015) while Ohlbaum et al. (2018) reported high uptake of
640 selenium by L. minor and Egeria densa. L. minor was effective in the removal of chromium
641 and lead but inefficient in the removal of copper. Chromium significantly inhibited growth
642 rate of plant (Üçüncü et al., 2013). The speciation of copper present in the effluents may be
643 non-bioavailable for uptake by duckweed. The high level of chromium uptake was attributed
644 to the lack of mechanism that acted as barriers to the transport of chromium in L. minor
645 (Üçüncü et al., 2013). High removal of lead and chromium, when compared with copper
646 accumulation in L. minor, was reported in a follow-up study (Üçüncü et al., 2013). Lead and
647 chromium have the same characteristic behaviour in the remediation study in respect to
648 uptake and accumulation in plant. The highest removal efficiency occurred within 48 hours
649 after the initiation of the study (Üçüncü et al., 2013). Cvjetko et al. (2010) reported that metal

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650 accumulation by L. minor increased when metals (copper and cadmium) were combined
651 compared to separate application. Combination of both metals was less toxic than the
652 application of separate metals to the plant (Cvjetko et al., 2010). L. minor was highly
653 efficient in the removal of metals from two effluents media within a 31 days period as
654 removal efficiency increases with increased in time (Bokhari et al., 2015). Significant
655 removal was also reported for chromium and cobalt (Oporto et al., 2006; Sree et al., 2015).
656 Removal efficiencies of heavy metals in industrial and municipal effluents include cadmium
657 (94.7 and 94.3%), copper (94.5 and 92.2%), lead (97.4 and 89%) and nickel (99 and 84.2%)
658 respectively (Bokhari et al., 2015). Duckweed was more effective for industrial than
659 municipal effluents. Highest removal of metals was observed with nickel and lead for
660 industrial effluent, while cadmium and copper for municipal effluent (Bokhari et al., 2015).
661 The increased effectiveness for industrial effluents compared to municipal effluents for heavy
662 metal removal seems odd, as more removal was expected for municipal than industrial
663 effluents due to the high levels of organics expected in municipal effluents. The complex
664 nature of nutrients and pollutants in the industrial effluents could be responsible for the high
665 rate compared to high organics loads present in municipal wastewater. Environmental
666 conditions present in aquatic media may play a vital role in the uptake of metals by L. minor
667 (Tunca et al., 2015). To what extent is not clear. The state of the metals may also play a role
668 in the uptake and bioaccumulation dynamics in phytoremediation setup. A comparison of the
669 effects of two copper treatments on L. minor revealed that among the treatments, copper ions
670 exerted the highest growth impairment on duckweed than copper oxide with more
671 pronounced effects in the roots than the fronds of the plant (Song et al., 2016). The root being
672 the first point of contact is expected to show pronounced levels of impact compared to the
673 fronds. This may not be true for all cases. The ionic form of copper may be more toxic and
674 readily bioavailable than the oxide of copper which could be more stable form in the

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675 environment. Chawla et al. (1991) reported that uptake of cadmium by L. minor varies across
676 time, pH and temperature gradient. Accumulation tends to decrease with a corresponding
677 decrease in temperature. As the temperature falls below the threshold for duckweeds,
678 strategies to survive in frigid conditions may sets in as against accumulation of nutrients and
679 pollutants. The plant may minimize or shut down the active transport or translocation process
680 thereby affecting the uptake and accumulation of metals for survival in the face of declining
681 temperature. Kara (2004) indicated that removal of copper by L. minor peaked after 48 hours
682 with increasing concentration and then decreased as the concentration of copper increased
683 through time. The plant reached its equilibrium uptake capacity within 2 days and further
684 increased in time and concentration only resulted in decreased potency of the plant to
685 accumulate copper. The increased uptake at the early or initial phase of the study may
686 indicate the readiness of the plant to accumulate metals to balance the essential nutrients
687 levels needed. It may also reflect the fact that uptake may be more pronounced at the early
688 stages of exposure to pollutants when the potency of the plant for uptake is high and the
689 pollutant burden has not yet set in. Teixeira et al. (2014) reported that L. minor was able to
690 successfully removed iron from coalmine effluents forty years after of operation. The success
691 of the plant for the removal of iron after four decades in the environment may indicate that
692 plants may have the mechanism to increase the bioavailability rate and uptake of certain
693 elements in the environment. It is also possible that iron in the mine effluent was made
694 available by the presence of acids in the effluents which can keep the metal readily available
695 despite the long period of time. How effective duckweed can successfully remove pollutants
696 especially metals from polluted environment after several decades needs a comprehensive
697 study for the successful application to the preponderance of abandon mines in different parts
698 of the world. There are reports that the addition or presence of complementary nutrients or
699 safeners acts as a buffer to mitigate stress in phytoremediation setup leading to increased

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700 efficiency in the removal, uptake, accumulation or degradation of pollutant by macrophytes.
701 Appenroth et al. (2008) reported that sulphate facilitated the bioaccumulation of chromate by
702 duckweeds (L. minor and S. polyrhiza). In the presence of sulphur nutrient, L. minor and
703 Salvinia minima were effective in the removal of arsenic, although L. minor take up arsenic
704 eleven fold than S. minima (Leão et al., 2014). Also, L. minor reported no stress in tissues
705 while S. minima showed signs of damage (Leão et al., 2014). Sulphate may have acted as a
706 catalyst for the uptake of metals. The intended uptake of sulphur may have lead to the
707 simultaneous uptake of metals. In contrast, the addition of sulphate as a buffering agent or
708 safener did not improve the removal of selenium rather it impaired the ability of L. minor to
709 remove the metal from the effluents (Lo et al., 2015). In another development, the addition of
710 sulphuric acid decreased the removal efficiency of metals by duckweed (Ohlbaum et al.,
711 2018). L. minor was more tolerant of chromate toxicity than S. polyrhiza and the application
712 of sulphate did not enhance the growth rate of duckweeds (Apprenroth et al., 2008). It is
713 possible that the levels of concentration of sulphate did not act as a potential catalyst for the
714 removal of metals by duckweed. There is also a possibility that sulphate may have created an
715 extra burden for the plant to take up metals. The addition of phosphate and nitrate as nutrients
716 enhanced the removal of chromium from constructed wetlands and aid the movement of
717 metal to the leaves of the plant (Di Luca et al., 2014). The addition of graphene oxide ranging
718 from 1 to 5 mg L-1 reduces stress, enhanced phytochemical activities of L. minor and
719 increased the accumulation of copper, boron, manganese, iron, cobalt and zinc (Hu et al.,
720 2017). A comparative assessment of L. minor and Salvinia auriculata for the removal of
721 copper using benoxacor and dichlormid showed that plant with improvement agents was not
722 inhibited by copper irrespective of concentration applied. The improvement agent effectively
723 increased the remediation of copper by both plants from the media, although, L. minor was
724 more effective than S. auriculata in the bioaccumulation of copper (Panﬁli et al., 2017). The

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725 addition of alginate microspheres to the media containing L. minor slightly enhance the
726 removal of cadmium from effluent, and decreased the burden of cadmium toxicity on
727 duckweed in the medium (Tunca et al., 2017). Sallah-Ud-Din et al. (2017) evaluated the use
728 of citric acid to enhance the response and uptake of chromium by L. minor. Although citric
729 acid improves the uptake of chromium, there was inhibition in photosynthetic activities and
730 fronds development in duckweed. Kruatrachue et al. (2002) reported a maximum uptake of
731 lead by L. minor with the addition of about 20 mg L-1 of humic acid. The acid may have
732 increased the mobility of metals and accelerated the uptake by duckweed. Increasing the
733 concentration of the acid (160 mg L-1) resulted in a significant decline in the accumulation of
734 lead by plant. The increased levels of acids may have resulted in higher mobility of lead
735 beyond the accumulation threshold for L. minor, hence the decline in accumulation rate. The
736 addition of EDTA did not significantly increase the uptake of lead by L. minor, but it
737 significantly decreased the uptake of cadmium in the media (Saygideger and Dogan, 2004).
738 The same concentration of lead (50µg mL-1) inhibited C. demersum than L. minor, while
739 cadmium (0.5µg mL-1) inhibited duckweed than C. demersum. The result shows that C.
740 demersum was more effective in the removal of lead than L. minor while the reverse was the
741 case for cadmium (Saygideger and Dogan, 2004). EDTA being an acid may have increased
742 the bioavailability of lead beyond the plant capacity to accumulate in the media. The
743 application of salicylic acid increased the uptake of vital nutrients by L. minor but decreased
744 the uptake of cadmium (Lu et al., 2018). The presence of acids induced the production of
745 heat shock proteins which is a defence mechanism triggered in the presence of environmental
746 stress. Citric, humic, EDTA and salicylic acids all have the potentials to enhance or increase
747 the mobility or labile phase of metals which in turn could increase the initial uptake in some
748 cases or create a sharp increase in stress in other situation for plants as the case may be.
749 Under prevailing acidic condition, the plant defence system may block increased metal

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750 uptake as a survival mechanism and rather increase the uptake of essential nutrients. This
751 could be an indication that plants selectively take up substances from the environment based
752 on their usefulness and capacity for uptake. Liu et al., (2018) reported that L. minor was
753 effective in the removal of boron. The removal rate significantly decline with the introduction
754 of sodium chloride and slightly increased reaching a peak at 100 mM. Sodium chloride
755 inhibited the growth and potentials of the plant to take up boron in the presence of salt. The
756 potentials of duckweed to remove pollutants in saline condition is a precursor to the
757 potentials of the plant to remove pollutants from coastal environment. Although duckweed
758 has been reported to thrive in saline conditions, no comprehensive assessment on the level of
759 salt concentration that will be optimum for pollutant removal under such conditions have
760 been reported. The observed inhibition and limitation in removing boron in saline conditions
761 could explain the behaviour of the plant for the remediation of pollutants outside freshwater
762 effluents. Microbes associated with roots of plant are believed to play a synergistic or
763 mutualistic role in the phytoremediation and degradation of pollutants present in the
764 rhizosphere. Stout et al. (2010) compared the phytoaccumulation of cadmium by L. minor
765 and bacteria isolated from the plant root. They reported that plant removed more cadmium
766 compared to plant and bacteria treatment. The presence of the isolate did not increase the
767 uptake of cadmium by plant rather it inhibited the uptake of cadmium. They concluded that
768 the microbe mounted a defensive role against pollutant entering plant as a mechanism for its
769 survival. Microbes may view the pollutant as a threat to its own survival and hence may play
770 an antagonistic role in the uptake and removal of metals from the polluted environment. This
771 is especially so since metals are non-biodegradable, but the reverse may be the case for
772 organic pollutants which can be degraded and may act as a source of nutrients for microbes.
773 Tang et al. (2015) investigated the effect of a rhizobacterium on the remediation of chromium
774 by L. minor. They observed that without microbe, initial uptake of chromium increased

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775 significantly reaching 70%, but as the concentration of chromium increases from 0.05 to 0.15
776 mM, uptake decreased significantly to 14%. This is expected, since increasing toxicity and
777 stress decrease plant potentials to take up pollutants as the physiological response of the plant
778 is switched towards survival rather than uptake of pollutants. The addition of
779 Exiguobacterium sp to the medium with duckweed at lower chromium concentrations (0.05
780 to 0.10 mM) did not improve the removal rate of chromium, but at 0.15 mM, the removal rate
781 significantly increased to 27.6%. The bacterium appears to enhance the capacity of duckweed
782 to take up chromium within the first 8 days of the study. Strangely, the presence of the
783 bacterium significantly reduces the uptake of chromium for the next 8 days. In essence, the
784 microbe blocks the biosorption of chromium by duckweed towards the second phase of the
785 study. The bacterium may have viewed further uptake of metals by the plant as a potential
786 threat to its own survival, hence it mounted a defence mechanism against further uptake of
787 pollutant by the plant. One earlier suggestion of bacteria potentials to block further uptake of
788 pollutants is by the production of biofilm covering the roots and fronds of duckweeds and
789 protecting the plant from further uptake of pollutants in the media (Stout et al., 2010). The
790 application of L. minor for metals remediation has been successful in many studies and the
791 combination of two or more duckweed species is expected to increase the rate of remediation
792 of pollutants. Even some studies have suggested that L. minor outperformed activated carbon
793 in the removal of metals from effluents (Saygideger et al., 2005). Combining various species
794 of duckweeds is essentials to understand the dynamics and effectiveness as against single
795 application. Lahive et al. (2011) evaluated the phytoaccumulation of zinc by three duckweed
796 species. The three species accumulated zinc preferentially. L. gibba accumulated high level
797 of zinc, L. minor had higher zinc levels in roots compared to fronds while the opposite was
798 the case for L. punctata. In general, L. punctata and L. minor had higher levels of zinc
799 accumulation than L. gibba. This study indicated that when exposed to the same

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800 concentration of pollutant, species of duckweed respond differently. The adaptive mechanism
801 for the different response and accumulation pathway is yet unclear. L. minor and S. polyrhiza
802 were effective in the accumulation of cadmium and showed the same uptake pattern with
803 increased concentration of the metal (Chaudhuri et al., 2013), although, cadmium inhibited
804 growth rate and phytochemical processes in both duckweeds (Chaudhuri et al., 2013). A
805 similar pattern was reported for the removal of boron by L. minor and L. gibba (Gür et al.,
806 2016) with increase inhibition as the concentration increased from 8 to 128 mg L-1. Inhibition
807 was more pronounced in L. minor than L. gibba (Gür et al., 2016). In a separate study, with a
808 combination of four metals L. gibba and L. minor effectively removed copper (57 and 58%),
809 lead (60 and 77%), nickel (60 and 68%) and zinc (62 and 62%) from the treatment (Yilmaz
810 and Akbulut, 2011). In this case, L. minor was slightly more effective than L. gibba. Further
811 studies indicated that despite the effectiveness of both species, L. gibba was more effective
812 than L. minor for the removal of copper, lead, zinc and arsenic from a polluted media
813 (Sasmaz et al., 2015). Zhao et al. (2015) reported the accumulation of copper by two
814 duckweeds (L. punctata and L. minor). They indicated that L. punctata was less tolerant to
815 copper and hence accumulated less copper compared to L. minor. Removal of copper from
816 the medium increased with the combination of both duckweeds compared to single
817 application. Yang et al. (2018) indicated that the three species of duckweeds (L. gibba, L.
818 minor and S. polyrhiza) applied for the monitoring and remediation of mercuric chloride
819 showed significant differences in mercury uptake in the media. The highest was reported with
820 S. polyrhiza and the least was L. gibba. Among the duckweeds, there is still significant
821 variation in the accumulation or uptake of pollutants in various effluents. The concentration
822 of the pollutants in the effluents and the duration of the study may influence the variation
823 observed in the various studies. Among the duckweed applied so far, it is difficult to trace a
824 specific pattern due to the varied application and lack of uniform measurement parameters for

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825 the different study. Aside single or multiple applications of duckweeds, other workers
826 preferred a combination of various macrophytes in their studies, in order to consider a
827 comparative assessment as against single application of the potentials of aquatic plants. The
828 choice is usually dependent on the researcher and perhaps the scope of the study. It has also
829 been argued that different macrophytes are effective in accumulating different pollutants
830 (Wang et al., 2002), especially where different pollutants with different characteristics are
831 present in an effluents. This argument bolster the need for the combination of different
832 macrophytes as each may have tolerance and affinity for certain type of pollutants or metals.
833 Wang et al. (2002) reported that different macrophytes were effective in accumulating
834 different pollutants. L. minor and E. crassipes were effective in the removal of cadmium,
835 Oenanthe javanica was effective with mercury and Lepironia articulata with lead. Although,
836 there is general uptake by all macrophytes, L. minor and Ceratophyllum demersum were
837 observed to be effective in the removal of lead and cadmium, C. demersum was most
838 effective in the removal of lead, while L. minor was more effective in the removal of
839 cadmium (Saygideger and Dogan, 2004). It is safe to argue that the common duckweed may
840 favour the uptake of cadmium compared to other heavy metals. High removal of iron, copper,
841 zinc, manganese, chromium and lead by L. minor, P. stratiotes, and Spirodela intermedia was
842 reported under laboratory setup (Miretzky et al., 2004). Goulet et al. (2005) reported that L.
843 minor showed the best phytoremediation potentials in the presence of aluminium in
844 wastewater while the least was Nuphar sp., but E. crassipes was more effective than L. minor
845 in the removal of arsenic from wastewater at 0.15 mg L-1 of metal and significant differences
846 was reported in the removal of arsenic by both species (Alvarado et al., 2008). Water
847 hyacinth (E. crassipes) and duckweed (L. minor) were more effective in accumulation of
848 metals (iron, chromium, copper, cadmium, zinc and nickel) within 21 days period than S.
849 polyrhhiza (Mishra et al., 2008). Although a general pattern of metal uptake may be the case,

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850 uptake could also be species specific. There is need for further assessment to elucidate the
851 uptake kinetics for different categories of metal pollutants in macrophytes remediation. The
852 idea whether certain plant species have affinity for the uptake of metals still remains a puzzle
853 due to insufficient data to elucidate the kinetics and response to specific metals in place. In a
854 very comprehensive survey, Vardanyan and Ingole (2006) sampled 45 aquatic plants
855 including L. minor for the removal of 14 heavy metals from a lake. They observed that all
856 macrophytes exhibited the same pattern in the accumulation of metals. Metals such as
857 calcium, iron, aluminium, chromium, copper, barium, titanium, cobalt and lead were higher
858 in the roots, manganese, zinc and magnesium were more in the stem or fronds while calcium
859 was more in the leaves. They further observed that accumulation of essential metals (calcium,
860 iron and manganese) was higher than non-essential metals (chromium, cadmium, lead and
861 nickel) in macrophytes. Rai (2010) reported that E. crassipes, L. minor and Azolla pinnata
862 removed about 25% to 71.42% of copper, chromium, iron, manganese, nickel, lead, zinc,
863 mercury and cadmium from power plant, coalmine and alkaline plant effluents within 60
864 days. Bharti and Banerjeen (2012) investigated the removal of heavy metals from a coalmine
865 using A. pinnata and L. minor. They reported that both plants removed heavy metals from the
866 media at varying concentrations. A. pinnata removed manganese (98%) iron (95.4%) zinc
867 (95%) copper (93%) lead (86.9%) cadmium (85%) chromium (77.7%) and nickel (66.2%) in
868 a decreasing trend while L. minor removed manganese (99.5%) copper (98.8%) zinc (96.7%)
869 nickel (94.5%) iron (93.1%) cadmium (86.7%) lead (84%) and chromium (76%) respectively.
870 For both plants, manganese has the highest removal efficiency while nickel was the least for
871 A. pinnata and chromium was the least for L. minor. They concluded that L. minor showed
872 more decrease in chlorophyll, protein and biomass than A. pinnata in the course of the study.
873 Comparison of both plants showed that L. minor was more effective than A. pinnata (Bharti
874 and Banerjee, 2013). All six macrophytes such as E. crassipes, Hydrilla verticillata, Jussiaea

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875 repens, L. minor, P. stratiotes and Trapa natans were effective for the removal of copper and
876 mercury from pulp effluent, but L. minor and E. crassipes were the most efficient (Mishra et
877 al., 2012). Similar outcome was also reported by Vaseem and Banerjee (2015) for A. pinnata
878 and L. minor with manganese, zinc, copper and iron. Azolla caroliniana and L. minor
879 removed arsenic, copper and silicon from a constructed wetlands (Rofkara et al., 2013). L.
880 minor removed higher amount of metals and silicon compared to A. caroliniana (Rofkara et
881 al., 2013). L. minor was more effective, followed by Raphanus sativus while the least was
882 Festuca ovina for the removal of arsenic, cadmium and mercury from single and mixed metal
883 exposure (Charlier et al., 2005). Basile et al. (2012) indicated that aquatic plants such as L.
884 minor, Elodea canadensis and Leptodictyum riparium accumulated metals differently. L.
885 riparium was the most effective in accumulation of copper, zinc and lead, whereas L. minor
886 was the most effective for cadmium. Cadmium was the most toxic metal, followed by lead,
887 copper and zinc. L. minor could withstand higher concentration of cadmium compared to E.
888 canadensis and L. riparium. The physiological and biochemical pathway that confers better
889 ability to withstand environmental stress for L. minor compared to the other aquatic plants is
890 unclear. Othman et al. (2015) investigated the potentials of L. minor and Salvinia natans to
891 remove heavy metals at various concentrations (1, 2 and 5 mg L-1) from a wastewater system.
892 Although, both plants were highly efficient in the removal of heavy metals from the media, S.
893 natans was more effective in the resistance and removal of metals from the system compared
894 to L. minor, while in the presence of a third species such as E. canadensis, L. minor was more
895 effective than both species in the removal of cadmium, copper and zinc (Török et al., 2015).
896 In another study with various concentration (1, 3 and 5 mg L-1) of iron in a Ramsar wetland
897 with four species of macrophytes, E. crassipes was the most effective followed by P.
898 stratiotes, L. minor and the least was Salvinia cucullata within 4, 8 and 12 days interval
899 (Singh and Rai, 2016). There was a high mortality with the phytoaccumulation of iron,

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900 copper, zinc, lead and cadmium by L. minor and P. stratiotes (Brăhaița et al., 2015). It is
901 strange that inhibition and lethality do not dissuade macrophytes from the accumulation of
902 metallic pollutants in the environment. Romero-Hernández et al. (2016) assessed the varying
903 responses of Typha latifolia, L. minor, E. crassipes and Miriophyllum aquaticum for the
904 uptake of copper, lead, mercury and zinc. They reported that T. latifolia and L. minor were
905 highly inhibited resulting in death of both plant, while E. crassipes and M. aquaticum were
906 highly tolerant and were able to accumulate metals (Romero-Hernández et al., 2016). It is
907 odd that duckweed and T. latifolia could not survive at 1.0 mg L-1 of copper and 0.5 mg L-1 of
908 lead, zinc and mercury after 3 days. The acidic pH (5.8 to 6) applied in this study may have
909 inhibited both plants since metals are known to be more mobile or reactive under acidic
910 conditions. For E. crassipes, uptake of metals was 99.8, 99.6, 97.9, and 94.37% while for M.
911 aquaticum, it was 98.2, 95.2, 94.3 and 86.5% respectively. It is expected that the more the
912 macrophytes applied the more the effectiveness of the remediation, but this is not the case.
913 Rather, fewer macrophytes may be more effective than a wide combination of different
914 species of macrophytes. It is difficult to truly quantify the effectiveness of the combination of
915 two or more species of macrophytes in the remediation of metals in effluents due to the
916 myriads variation in the scope of different studies, considering the fact that factors such as the
917 concentration of metals present and the state of the metals may also influence the removal of
918 metals from wastewater. Lahive et al. (2013) investigated the relationship between zinc
919 uptake in L. minor and the corresponding accumulation by a crustacean Gammarus pulex
920 feed with duckweed. They reported that as concentration increases in duckweed, there was a
921 corresponding increase in crustaceans resulting in mortality of G. pulex. It is interesting that
922 the subsequent accumulation of zinc by the crustacean resulted in the death while there is no
923 mortality with duckweed. This shows the high tolerance of duckweed to pollutants and as a
924 potential organism for pollutant removal in the environment. Singh et al. (2016) compared

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925 the uptake of arsenic by aquatic macrophytes and algae in a wetland. They reported that both
926 macrophytes and algae were able to accumulate varying concentrations of arsenic in polluted
927 water. Phytoaccumulation of arsenic in aquatic macrophytes showed that E. crassipes, L.
928 minor and P. stratiotes accumulated 597, 735 and 24.5 mg kg-1 of arsenic in shoot, leaves and
929 fronds respectively, while among algae, diatoms and Hydrodictiyon reticulatum accumulated
930 760 and 403 mg kg-1 respectively. L. minor and A. filiculoides were very effective in the
931 accumulation of cobalt, zinc and manganese, but L. minor was significantly more effective
932 than A. filiculoides in the removal of the metals at the end of the study (Amare et al., 2017).
933 A follow up report (Amare et al., 2018) indicated that L. minor was more effective than A.
934 filiculoides in the removal of cobalt, cadmium, zinc, chromium, nickel, copper, iron and
935 manganese within 28 days. Both L. minor and Daphnia magna were successful in the
936 removal of heavy metals separately, but duckweed was more successful than Daphnia
937 (Fikirdeşici-Ergen et al., 2017). A combination of both species did not significantly increase
938 the removal of metals (aluminium, barium and iron) from the medium, but there was an
939 increased removal rate especially by duckweed when metals were combined in the medium
940 (Fikirdeşici-Ergen et al., 2017). Bonanno et al (2018) investigated the removal of ten heavy
941 metals (arsenic, cadmium, chromium, copper, mercury, manganese, nickel, lead and zinc) by
942 twenty different macrophytes in a wetland receiving domestic and industrial effluents. They
943 indicated that uptake or translocation of metals show no specific pattern among the plants.
944 Although, removal rate varies significantly, L. minor recorded the highest removal rate
945 among the twenty species. Little is known about the transport and fate of inorganic pollutants,
946 especially metals in duckweed, but mechanisms such as methylation, chelation, sequestration
947 and binding chemicals are largely responsible for the detoxification of metals and the
948 conversion from toxic to less toxic forms (Chandra, 2015). Further research is needed in this
949 direction to fully understand the pathways and fate of metals in plant tissues. Shedding light

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950 in this area can help expand and increased the knowledge of duckweed for the
951 phytoremediation of metals in the environment. Macrophytes, especially duckweed species
952 present in different geographical locality may show diverse outcome when exposed to the
953 same or different levels of pollutants. The tolerance or uptake levels may indicate the
954 adaptive capacity of the local species and the prevailing environmental factors present in such
955 locality as reflected in the different outcome of the studies reported so far. Also, many of the
956 studies reported so far are largely based on laboratory microcosm, where prevailing
957 environmental conditions are largely regulated by researchers. Future studies should consider
958 field mesocosm which has a strong proximity to real life application in order to have a better
959 understanding on the dynamics of heavy metals remediation by duckweeds.
960
961 Table 2: Summary of heavy metals associated with L. minor remediation
As 21 days 0.15 mg L-1 5% Alvarado et al., 2008
Co, Cd, Zn,
Cr, Ni, Cu,
Fe, Mn
28 days 33.17, 11.33,
74.53, 0,
35.33, 18,
828.13, 126
μg L-1
12.76, 2.17,
424.58, 4.45,
12.15, 16.67,
2877.5, 3078.9 mg
kg-1 DW
Amare et al. 2017
Co, Cd, Zn,
Cr, Ni, Cu,
Fe, Mn
28 days 0-12.39 mg
L-1
72-91% Amare et al. 2018
Cr 7 days 100 µM - Appenroth et al. 2008
Cd, Pb, Cu,
Zn
7 days 10-3-10-7 M 95, 93, 86.5,
63.5%
Basile et al. 2012

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43
Fe, Mn, Cu,
Zn, Ni, Pb,
Cr and Cd
7 days 22.91, 9.61,
2.04, 1.03,
0.86, 0.69,
0.18 and 0.06
mg L-1
93.1, 99.5, 98.8,
96.7, 94.5, 84, 76,
86.7%
Bharti and Banerjee,
2012
Fe, Mn, Cu,
Zn, Ni, Pb,
Cr and Cd
7 days 22.906,
9.606, 2.039,
1.034, 0.856,
0.69, 0.182,
0.0598 mg L-
1
48.35, 63.05,
49.06, 55.76,
51.91, 60.07,
42.86%
Bharti and Banerjee,
2013
Cd, Cu, Pb,
Ni
31 days 0.038, 0.062,
0.608, 0.054
mg L-1
94, 94, 99, 97.4% Bokhari et al. 2015
As, Cd, Cr,
Cu, Hg, Mn,
Ni, Pb, Zn
4 months 0.18-30.2 µg
L-1
0.22 - 265 mg kg-1 Bonanno et al. 2018
Cu, Zn, Fe,
Ni, Cd, Pb
14 days 100-14000
µg L-1
5405.5, 5339,
1427.5, 453.9,
357.25, 110.73 mg
kg-1
Brăhaița et al. 2015
Cd, Hg, As 3 days 44, 25, 67
μM
93, 90, 641 nmol g-
1 FW
Charlier et al. 2005
Cd 22 days 0.5-3.0 mg L-
1
42-78% Chaudhuri et al. 2013

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44
Cd 96 hrs 0-0.5 ppm 90% Chawla et al. 1991
Cu, Cd 7 days 2.5-5, 5 μmol
L-1
480.98, 1055.64
µg g-1 DW
Cvjetko et al. 2010
Pb, Hg 7 days 0.1-10, 0.1-2
mg L-1
1959, 490 µg g-1 Dirilgen, 2011
Zn, Cu, Ni,
Cd
7 days 1.9-56.3 μM 128 nmol mg-1
DW
Drost et al. 2007
Al, Ba and
Fe
48 hrs 3000, 20000,
12000 μg L-1
280.20, 1261.80,
891.40 μg L-1
Fikirdeşici-Ergen et al.
2017
Al 58 days 97 μg L-1 17.20 mg g-1 Goulet et al. 2005
Boron 7 days 4 to 128 mg
L-1
4007mg kg-1 Gür et al. 2016
Cu, 5 days 0.05-1.2 mg
L-1
83.3% Hu et al. 2017
Cu 72 hrs 25-100 μM 200-1700 µg g-1
DW
Kanoun-Boulé et al.
2009
Cu 4 days 1-7, 77.78% Kara, 2004
Pb 12 days 50-200/10-
160 mg L-1
43.52 mg g-1 Kruatrachue et al. 2002
Zn 10 days 3 - 100 mg L-
1
431 µg g-1 FW Lahive et al. 2011
Zn 7 days 0.2-30 mg L-
1
75% Lahive et al. 2013
B 4 days 2 mg L-1 15.5% Liu et al. 2018
Se 7 days 5 - 40 ug L-1 10.25 mg kg-1 Lo et al. 2015

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45
Cd 7 days 10 μM 38% Lu et al. 2018
Se 21 days 0-10 mg L-1 19.5 mg g-1 Mechora et al. 2015
Fe, Cu, Zn,
Mn,
Cr and Pb
15 days 1 mg L-1 78.47, 97.56,
95.20, 90.41,
96.94, 98.55%
Miretzky et al. 2004
Fe, Cr, Cu,
Cd, Zn,Ni
21 days 4.8 - 0.07 mg
L-1
62, 59, 57, 56, 55,
47%
Mishra et al. 2008
Cu, Hg 20 days 0.46, 0.23
mg L-1
71.4, 66.5% Mishra et al. 2012
Se 7 days 100 μg L-1 97.8% Ohlbaum et al. 2018
Cr(VI) 16 days 0.5 and 2.0
mg L-1)
1.02 mg g-1 DW Oporto et al. 2006
Fe, Cu, Zn 4 weeks 1-5 mg L-1 90% Othman et al. 2015
Cu 2 weeks 2 mg L-1 54.2% Panﬁli et al. 2017
Zn, Al 15 days 0.15 and 0.3
mM
2041-49457, 1505-
2554µg g-1 DW
Radic et al. 2010
Cu, Cr,
Fe, Mn,
Ni, Pb, Zn,
Hg, Cd
60 days 0.04-98 mg
L-1
25-77.42% Rai, 2010
Cd 7 days 0 to 500 µM 12,320 µg g-1 Razinger et al. 2008
As, Cu, S 14 days 0-20,
2-78,
0-1.8 µM
141, 452, 6995 mg
kg-1
Rofkar et al. 2013
Cu, Hg, Pb, 7 days 0.5 and 0.25 0% Romero-Hernández et al.

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46
Zn mg L-1 2016
3 days 200 μM 20.2% Sallah-Ud-Din et al.
2017
Cu, Pb, Zn,
and As
3 days 67, 7.5,
7230, and 96
µg L-1
87, 1259, 628,
7070%
Sasmaz et al. 2015
Pb, Cd 7 days 50, 0.5 µg
mL-1
1116, 1136 µg g-1 Saygideger and Dogan,
2004
Cd, Cu and
Ni
3 hrs 100 mg L-1 83, 69 and 59 mg
g-1
Saygideger et al. 2005
Fe 12 days 1-5 mg L-1 21-71% Singh and Rai, 2016
As, Si, Cd,
Pb, Cr, Ni
- 117, 249,
163,79.4,
138, 36.5 mg
L-1
735, 2022, 102,
54.6, 198, 146 mg
kg-1 DW
Singh et al. 2016
Co 7 days 1 - 100 µM 21 mg g-1 DW Sree et al. 2015
Cd 14 days 100 µg L-1 24 µg g-1 Stout et al. 2010
Cr 16 days 0-0.20 mM 27.6% Tang et al. 2015
Fe 21 days 0.03-22.60
mg L-1
19.4 mg g-1 DW Teixeira et al. 2014
Cu, Zn, Cd 6 days 4.10, 4.30,
7.30 mg L-1
0.381, 0.557, 1.251
mg g-1 FW
Török et al., 2015
Hg, Cr, Zn,
Al, As, Cd,
Co, Cu, Pb,
25 days 0.36, 67.33,
49.59, 94.65,
16.31, 1.47,
82.84, 90.25,
98.00, 98.33,
90.95, 97.79,
Tufaner 2018

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47
Ni 24.17, 23.84,
23.37,
346.81 ppb
98.27, 98.46,
99.61, 98.08%
Pb, Cd, Cr,
Cr, Ni, Cu,
Cu, As, Mn,
Ba, Al, Fe,
Zn, Zn
5 days Tunca et al. 2015
Cd 48 hrs 0.01-10 mg
L-1
97.32% Tunca et al. 2017
Cr, Cu and
Pb
7 days 10.946,
4.359, 0.875
mg L-1,
99.97 % Üçüncü et al. 2013
Cu, Cr, Pb 7 days 3000, 10400,
200μg L-1
40, 75, 85% Üçüncü et al. 2013
Ca, Mg, Fe,
Al, Cr, Cu,
Ni, Ba, Mn,
Zn, Pb, Cd,
Ti, Co
- - 127700, 23630,
14320, 4560,
13.48, 23.56, 0.58,
226.1, 3000, 212.5,
7.17, 0.79, 152.3,
16.0 μg g-1 DW
Vardanyan and Ingole
2006
Mn, Cu, Zn,
Fe, Pb, Cr
and Cd
7 days 4.9, 1.432,
0.816, 0.762,
0.655, 0.07,
0.018 mg L-1
94, 86, 62, 74, 84,
63 and 78 %
Vaseem and Banerjee,
2015

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Cd 48 days 0-8 mg L-1 14200 mg kg-1 Wang et al. 2002
Hg 7 days 0.25-8 mg L-
1
2.55 mg g-1 DW Yang et al. 2018
Cu, Pb, Ni,
Mn
10 days 3.24, 6.79,
4.4, 20.2 mg
L-1
58, 62, 68, 77%
(7.62, 5.93, 33.57,
47.12 mg g-1 DW)
Yilmaz and Akbulut,
2011
Hg 6 days 0-30 μM 58.3% Zhang et al. 2017
Cu 7 days 0.01-1 mg L-
1
887.3 mg kg-1 DW Zhao et al. 2015
962
963 5.3 Phytoremediation of Agricultural Chemicals
964 After serving their useful purpose in the production of food, agricultural chemicals add to the
965 burden of environmental pollution. Several metric tonnes of agrochemicals such as fertilizers,
966 pesticides, herbicides and fungicides are produced annually. A considerable amount of these
967 chemicals applied on farmlands and aquaculture ends up in aquatic environment without
968 treatment. Tront and Saunders (2007) evaluated the uptake and accumulation of 2,4-
969 dichlorophenol by L. minor. Analysis of plant indicated that the herbicide and metabolites
970 were found in the plant tissues with some degree of inhibition. The levels of the compound
971 and metabolites increased with time in plant. Less than 10% of the original compound was
972 found in the plant, indicating over 90% sequestration of initial compound accumulated by
973 duckweed. Unlike heavy metals which are non-biodegradable, macrophytes especially
974 duckweeds can accumulate and degrade agrochemicals into useful components for growth
975 and development. Duckweed was severely inhibited by the herbicide norflurazon, but there
976 was a rapid recovery after the plant was removed from the media (Wilson and Koch, 2012).
977 The concentration and reactive potentials of the herbicide may be too high for duckweed. A

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978 comparison of lab and field setup of duckweed to atrazine indicated that both setup did not
979 reflect any difference in atrazine exposure and uptake (Dalton et al., 2013). This is
980 unexpected since the prevailing factors in the field are expected to be different from a
981 simulated setup in a laboratory microcosm. Lactofen did not show any sign of inhibition or
982 toxicity to L. minor during a 5 days period, but the plant selectively take up a non-significant
983 quantity of the pesticide (Wang et al., 2017). This is unusual because, without inhibition,
984 more pollutant is expected to be taken up by duckweed. The selective and low uptake could
985 indicate that lactofen maybe difficult or non-bioavailable for uptake by the common
986 duckweed. Duckweed may respond differently in the presence of two or more agrochemicals
987 in the environment. Megateli et al. (2013) reported different responses and removal rate for
988 copper and dimethomorph at the same concentration (1000 µg L-1) by L. minor. Copper
989 severely inhibited the growth of duckweed while dimethomorph fairly inhibited the plant. L.
990 minor was more effective in the reduction of copper compared to dimethomorph. This is in
991 line with Olette et al. (2008) report where dimethomorp was the most toxic followed by
992 copper sulphate and flazasulfuron when exposed to different macrophytes. This could
993 indicate that where two or more pollutants are present in effluents, macrophytes including
994 duckweed could selectively take up and accumulates pollutants that are less toxic as an
995 adaptive mechanism for survival in such environment. The increased copper accumulation
996 may have weakened the defence system of the plant and exposed it to increased inhibition in
997 the presence of dimethomorph. It may seem that copper could be more readily bioavailable
998 for uptake than dimethomorph. The combination of both fungicides (copper and
999 dimethomorph) resulted in complete inhibition of L. minor resulting in the death of the plant
1000 (Megateli et al., 2013). Both fungicides could have acted synergistically in the mixture to
1001 exert a greater inhibition and impact on the plant resulting in death. The opposite was the
1002 case for copper and chloroacetamide (penthoxamide) herbicide (Obermeier et al., 2015).

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1003 Careful observation of physiological activities shows that the plant was fairly inhibited for
1004 both agrochemicals although at different concentrations. Anti-oxidative processes triggered
1005 as a defence mechanism in plant involves the deposition of more copper sulphate in fronds
1006 while herbicide binds with glutathione (Obermeier et al., 2015). Dosnon-Olette et al. (2011)
1007 reported low uptake for two herbicides isoproturon (25%) and glyphosate (19%) from the
1008 medium with L. minor. Despite the low concentration of both pollutants, the result after four
1009 days was low. Previous reports also indicated a low uptake of isoproturon (Bottcher and
1010 Schroll, 2007) by L. minor, although with a higher concentration than that reported by
1011 Dosnon-Olette et al. (2011). It is probable that duckweed has lower uptake or translocation
1012 potentials for the two herbicides. Other factors such as physical or mechanical stress on the
1013 duckweed prior to application in the study may also affect the growth, functionality and
1014 ability to removed pollutants from the media. Panfili et al. (2019) reported inhibition and
1015 reduce uptake of the herbicide terbuthylazine by duckweed. The addition of safeners or
1016 biostimulants such as Megafol and Benoxacor significantly reduced plant inhibition and
1017 increases the uptake of the herbicide. The combination of different species of macrophytes to
1018 different agrochemicals may also result in different outcomes for different aquatic plants. In a
1019 study of the phytoremediation potentials of three macrophytes (L. minor, E. canadensis and
1020 Cabomba aquatica) exposed to two fungicides (dimethomorp and copper sulphate) and a
1021 herbicide (flazasulfuron), Olette et al. (2008) reported that L. minor was the most successful
1022 in the remediation of pesticides while E. canadensis and C. aquatica have similar
1023 accumulation pattern. Copper sulphate and dimethomorp were readily bioavailable to
1024 macrophytes than flazasulfuron (Olette et al., 2008). Further studies with two fungicides such
1025 as dimethomorph and pyrimethanil showed slight inhibition with the application of five
1026 macrophytes (L. minor, S. polyrhiza, C. aquatica, C. palustris and E. canadensis) (Dosnon-
1027 Olette et al., 2009). Although, there was a poor removal rate for all macrophytes, with

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51
1028 toxicity more pronounced on C. palustris and E. canadensis. L. minor and S. polyrhiza were
1029 more effective for the remediation of dimethomorph and pyrimethanil (about 10-fold) than
1030 the other macrophytes with dimethomorph having a higher removal rate than pyrimethanil
1031 (Dosnon-Olette et al., 2009). For both fungicides, pyrimethanil could be more toxic than the
1032 former. It is also feasible that pyrimethanil may have decreased the toxicity of dimethomorph
1033 in the media resulting in increased uptake of dimethomorph. Dosnon-Olette et al. (2010)
1034 evaluated the phytoremediation of dimethomorph by L. minor and S. polyrhiza in wastewater
1035 effluent. They reported that both macrophytes were effective in fungicide removal. L. minor
1036 removed about 115 μg L-1 compared to 83 μg L-1 removed by S. polyrhiza at 600 μg L-1
1037 dimethomorph. When exposed to the herbicide metazachlor, L. minor performs poorly
1038 compared to S. polyrhiza (Müller et al., 2010), but L. minor was more adapted to atrazine
1039 than M. aquaticum (Teodorovic´ et al., 2012). Following the literature so far, it is safe to say,
1040 that comparative application of macrophytes to the remediation of agrochemicals has shown
1041 that duckweeds (S. polyrhiza and L. minor) have a competitive advantage irrespective of the
1042 agrochemicals applied. The factor responsible for the preferential accumulation of
1043 agrochemical pollutants over other macrophytes is not clear from the above studies since
1044 most macrophytes including duckweeds display a general mechanism for tolerance and
1045 overcoming toxicity when exposed to stress in the environment. Among the duckweeds, L.
1046 minor was more effective than S. polyrhiza. The wider distribution of the common duckweed
1047 compared to the lesser duckweed could have conferred some level of advantage on the
1048 common duckweed. The list of agrochemicals so far reported for duckweed remediation is
1049 limited compared to the myriads of agrochemicals in the market released into the
1050 environment. There is need for more studies on agrochemicals in the environment whose
1051 behaviour and uptake by duckweeds have not been reported. The poor uptake of lactofen,
1052 isoproturon and glyphosate warrant further attention.

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52
1053
1054 Table 3: Summary of agricultural chemicals associated with L. minor remediation
Atrazine 7 days 0-960 μg L-1 - Dalton et al., 2013
dimethomorph and
pyrimethanil
4 days 600 μg L-1 17 and 12 (48
and 33 µg g-1
FW)
Dosnon-Olette et al.
2009
Dimethomorph 4 days 600 μg L-1 41 μg g-1 Dosnon-Olette et al.
2010
Isoproturon,
glyphosate
4 days 0-20, 0-120
μg L-1
25, 19% Dosnon-Olette et al.,
2011
copper,
dimethomorph
7 day 0-30, 0-1000
μg L-1
76, 60% Megateli et al. 2013
Metazachlor 28 days 5 - 500 μg L-1 - Müller et al., 2010
copper and
chloroacetamide
7 days 50-100, 1.25-
2.5 μg L-1
368, 250 μg g-1
DW
Obermeier et al., 2015
copper sulphate,
ﬂazasulfuron,
dimethomorph
7 days 400 µg L-1 2.5 to 50 (30,
27 and 11, μg
g-1 FW)
Olette et al. 2008
Terbuthylazine 2 weeks 0.031-0.500
mg L-1
67% Panfili et al. 2019
Atrazine 12 days 40-640 μg L-1 3.69 µg g-1 Teodorovic´ et al.
2012
4-chloro-2- 77 hrs 26-373 μM 3.51 umol g-1 Tront and Saunders

Application Of Common Duckweed (Lemna Minor) In Phytoremediation Of Chemicals In The Environment State And Future Perspective

Application Of Common Duckweed (Lemna Minor) In Phytoremediation Of Chemicals In The Environment State And Future Perspective

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