Potential of Duckweed ( Lemna minor ) for the Phytoremediation of Landfill Leachate

Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou 310058, China Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000, Pakistan Department of Environmental Sciences and Engineering, Government College University, Allama Iqbal Road, Faisalabad 38000, Pakistan Sustainable Development Study Center, Government College University, Lahore, Pakistan Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad, Pakistan


Introduction
Ever-increasing population and expansion of industrial activities along with changing lifestyle gave rise to exponential generation of solid waste over the last few years [1].
erefore, management of municipal solid waste is of utmost importance under current scenario [2].Feasible and ideal options for the management of municipal solid waste (MSW) at low cost are hard to decide because of diverse considerations [3].Landfilling is the most acceptable management option for waste disposal in ird World countries.Technical feasibility, low operational cost, less supervision, and simplicity make landfilling the most preferred method for the management of MSW [4].Degradation of solid waste takes place due to physicochemical changes occurring in the solid waste and matrix of the landfill soil.Landfill leachate, assisted by rainwater and biochemical, physical, and chemical reactions, percolates all the way through the matrix of solid waste.Quality and quantity of leachate depends upon the weather discrepancies, age of landfill, precipitation, and amount/type of solid waste composition [5].While passing through the waste mass, leachate is polluted with toxic substances and heavy metals [6].In leachate, the major sources of toxic metals are electronic waste, dyes, pesticides, batteries, and fluorescent lamps [7].
Heavy metal solubility and mobility is highly dependent on the age of landfill, pH, and ratio of organic and inorganic substances [8].Acid formation at low pH is considered as the most deliberate phase which boosts up the occurrence of high concentration of toxic metals in landfill leachate [9].Heavy metals are among the major environmental contaminants because of their toxic effects, ability to accumulate in the aquatic system, and nonbiodegradable nature [10].Landfill leachate is thus regarded as a major environmental hazard which pollutes the surrounding environment affecting local biota, groundwater, and other aquatic systems [5].Similarly presence of organic parameters like BOD, COD, and pH are also among complicated parameters in landfill leachate.Higher fractions of these organics are primarily more challenging as they are less biodegradable in nature and have selective toxicity for biological process [11].Hence, selection of suitable methods for the treatment of the landfill leachate, before its final disposal into any water body, is an important step to avoid the environmental degradation.
Traditional heavy metals remediation techniques like ion exchange, filtration, and adsorption are not cost effective and may adversely affect the aquatic ecosystem [12].Use of plants in purification process is called phytoremediation, and it has gained attention as a suitable option for the treatment of landfill leachate [6,13].In case of heavy metals, phytoremediation is a self-sustaining and economical alternative treatment technology [14].During the last two decades, phytoremediation has attained substantial significance and the discovery of hyperaccumulator plants made it more promising because of their ability to accumulate high amount of heavy metals in aerial parts of the body [15,16].Ideally, the plants used for phytoremediation should have the ability to produce high biomass, survive in extremely toxic environment, and accumulate the contaminants in high concentration.Degradation of different contaminants depends upon the selection of phytoremediation technique and type of the particular contaminants [17].Plants act as host for endophytic bacteria by providing nutrients and offering protection against physical environment.ese symbiotic relations promote the growth and competitiveness of plants to respond to the external stresses such as nutrients and heavy metals [18].
Recent studies have described some aquatic plant species best suited for the remediation of heavy metals like Cu, Zn, Fe, Cd, Pb, Cr, Hg, and Ni [19,20].Aquatic plants like Eichhornia crassipes, Azolla filiculoides, Pistia stratiotes, Hydrilla verticillata, Typha domingensis, Salvinia Cucullata, Azolla caroliniana, Azolla pinnata, Lemna minor, Lemna aequinoctialis, Lemna gibba, and Spirodela polyrhiza are suitable aquatic plants for the removal of heavy metals as reported by several researchers [21][22][23][24][25][26][27].Aquatic plants play a vital role in harmonizing the water bodies.ey naturally have the tendency to treat different wastewater streams including landfill leachate [28].Duckweed (L.minor) is an aquatic plant that belongs to the family Lemnaceae.Owing to rapid growth rate, cold tolerance, ease of harvesting, and cost effectiveness, Lemna minor is a much better candidate than other aquatic plants for phytoextraction of heavy metals [29].Duckweed has been reported to be very effective in the phytoextraction of organic matter, suspended solids, heavy metals, and soluble salts from wastewater [30].In wastewater treatment studies, L. minor is used for the monitoring of heavy metals [31].In this study, phytoremediation potential of duckweed (L.minor), for the treatment of landfill leachate, was evaluated for 15 days.Heavy metal content and physiochemical parameters of landfill leachate were investigated.Plants were carefully washed with water to remove the insect larvae and epiphytes.Plant samples were put into the plastic jar, filled with water, for one week to acclimatize with the existing environment.After that, plants of the same size were collected for the research experimentation.Samples of landfill leachate and Lemna minor were collected in the month of July 2016.

Experimental Setup.
One set of experimental containers, having three tubs, was arranged.Each experimental container was filled with 20 L landfill leachate and 200 g fresh weight of duckweed.Experiment was conducted in triplicate to attain the average efficiency of the plant, and the study was performed in the month of November.
e mean daily temperature during the study was 23 ± 5 °C, while daily average humidity was 72 ± 15% at the experimental site.Test duration was 0, 3, 6, 9, 12, and 15 days (total 6 observations with pretreatment data).

Heavy Metal Estimation in Leachate and Plant Samples.
Plant samples were washed thoroughly before they were oven dried at 70 °C.After complete drying, plant samples were crushed and sieved to < 1 mm.Plant samples (0.25 g each) were digested with diacid (HNO 3 -HCLO 4 ) by gradually increasing the temperature.After complete digestion, distilled water was added in the sample to make the final volume up to 50 ml.Heavy metal (Zn, Pb, Fe, Cu, and Ni) contents were determined in both plant and leachate samples using the atomic absorption spectrophotometer (AAS) (Z-8230).

2
Journal of Chemistry 2.5.Calculation.Uptake of heavy metals by the plant was calculated using the dilution factor as follows: dilution factor � total volume of sample(ml) weight of the plant(g) . (1) Percentage efficiency was calculated by determining the initial (C0) and final concentration (C1) of metals in the sample as described previously [33]: where C0 and C1 are the initial and final concentration of the metal in the medium (mg • L −1 ).e bioconcentration factor was calculated as described previously [34]: 2.6.Statistical Analysis.Data presented in this paper are the mean of three replicates ± SD. Analysis of variance (ANOVA) and graphical representation were performed with GraphPad prism5 software followed by Tukey's test to get the significant difference between different mean values.

Results and Discussion
3.1.Physiochemical Parameters.Results of the phytoremediation potential study on Lemna minor are given in Table 1.pH of landfill leachate reduced from its initial value (7.9) to the final value of (6.8) by the end of 15-day experiment as depicted in Table 1.Duckweed (L.minor) has the potential to survive under a wide range of pH, i.e., 4.5 to 7.5 [35].Values of TSS and TDS were 63.5 mg  5. Overall, Lemna minor exhibited a great ability to remove all the heavy metals under study from the landfill leachate.e higher potential of L. minor to remove metals from leachate is attributed to huge biomass production and efficient growth in the highly metal-polluted environment [39].

Removal Efficiency.
e present study demonstrates that metal removal efficiency of L. minor from landfill leachate was more than 70 to 90% (Figure 6).e maximum efficiency (91 %) of L. minor was observed for the removal of copper from leachate.Removal efficiency for Pb, Zn, Fe, and Ni was 78, 83, 77, and 76%, respectively.A previous study reported that L. minor removed 76% lead and 82% nickel from the contaminated solution under laboratory conditions [25].Metal removal efficiency of L. minor was in the following order: Cu (91%) > Zn (83%) > Pb (78%) > Fe (77%) > Ni (76%).Similar results for heavy-metal removal efficiency were also reported by other researchers [21,40].Results of a previous study reported that that removal efficiency of L. minor was 58%, 62%, and 68% for copper, lead, and nickel, respectively [41].Similarly, L. minor removes nickel by 74 % and lead by 79% from the industrial wastewater stream [42].[43].Accumulation of zinc was highest on the 6th day, following a gradual decrease with time (Figure 1).Concentration of zinc in L. minor was recorded as 1.15, 1.17, 0.99, 0.95, and 0.93 mg•kg −1 after 3, 6, 9, 12, and 15 days of exposure, respectively.Similarly, in a previous study, L. minor was reported to accumulate higher amount of zinc as compared to L. gibba [44].Zinc is an essential trace element which plays an important role in the growth and development of plants.Zinc is a most commonly found element in several enzymes   Journal of Chemistry such as cytochrome oxides, polyphenol oxides, and ascorbic acid oxides [45,46].Upon absorption by plants, Zn is transformed from insoluble to soluble state (Zn 2+ ) which ultimately enhances the capacity of the aquatic plant to accumulate higher amount of Zn in their body [47].Plants accumulated maximum concentration of lead on the 6th day of the experiment, after which minor changes occurred till the end of the experiment.Highest lead accumulation was 0.68 mg•kg −1 on day 6, while the least 0.39 mg•kg −1 was observed on day 15 (Figure 2).Similar observations for the accumulation of lead by L. minor were also recorded by Singh et al. [19].L. minor also accumulated Pb at the rate of 561 mg•kg −1 dry weight (dw) on day 7 of the experiment at 50 mg•L −1 concentration in the growth medium [48].e results of this study demonstrated that accumulation of Pb increased with the increase in concentration and duration of exposure.Results for accumulation of Pb are in line with the earlier studies on di erent aquatic plants like Wol a arrhiza, [49] Najas indica, [50] C. demersum, [51] Lemna minor, and Lemna gibba [52].Uptake of iron by L. minor was increased gradually till the 12th day of experiment and exhibited a declining trend thereafter.Accumulation of iron on days 3 and 9 of the experiment was 0.87 and 0.74 mg•kg −1 , respectively.It was reduced to 0.64 mg•kg −1 by the end of the treatment (Figure 3).Iron, at the same time, is equally important for the growth and development of plants.Uptake of Fe is crucial for the metabolism of chloroplast and mitochondria.Mostly iron exists in the form of less soluble ferric oxides, which becomes free of oxides at low pH and is converted to readily available form of Fe for the plants to uptake [53].

Journal of Chemistry
From the leachate, plants accumulated highest concentration of copper at the start of the experiment till day 9, followed by no signi cant increase in its accumulation.Maximum accumulation (0.58 mg•kg −1 ) was recorded on day 6 of the experiment (Figure 4).Copper is an essential micronutrient and plays a vital role in the growth and development of plants [54,55].Plants regulate the intercellular copper level by rectifying its uptake and declining the free intercellular copper concentrations by metallochaperones.
ese are Cu-binding soluble proteins which transport it to the sections of the plant cells where they are needed the most [56].During the study, maximum concentration of nickel accumulated on days 3 and 6 of the experiment.Its concentration was 0.83 and 0.86 and 0.64 mg•kg −1 on days 3, 6, and 9, respectively.Nickel promoted the growth and development of Lemna minor fronds when applied at the rate of 0.5 mg•L −1 [57].Lemna minor absorbed nickel more pro ciently as compared with lead [24].L. minor removed 65, 72 and 87% Ni at di erent initial concentrations during a twenty-two-day experimental study [58].Similar result was also reported where L. Minor accumulated more Ni as compared to L. gibba after 80 days of exposure [59].Results of the present study, for the accumulation of nickel, are in agreement with the earlier reports [60][61][62].
Accumulation of heavy metals by the whole plant is depicted in Figure 7. Results obtained from the present study revealed that the uptake of metal by plants was dependent upon their initial concentration in the wastewater.Findings of this study were in line with those of Axtell et al. [24].Similarly, L. minor showed a signi cant phytoremediation potential by accumulating more than 90% of Fe, Zn, and Cu with di erent concentrations at di erent time periods [63].Analysis of variance showed signi cant uptake (p < 0.05) of all heavy metals from land ll leachate during the experimental study.

Bioconcentration Factor for Heavy Metals.
Bioconcentration factor is expressed as the ratio of the concentration of heavy metals absorbed by plant tissues to that in the medium [34].Bioconcentration factor (BCF) is considered as a blueprint for the determination of metal uptake e ectiveness by aquatic plants [64].Bioconcentration factor values, for the accumulation of heavy metals by L. minor at di erent exposure time periods, are given in Table 2. e maximum BCF value for zinc was 0.78 and 0.81 on the 3rd and 6th day of the experiment, respectively.e bioconcentration value for lead increased progressively and attained the maximum level (0.81) on the 6th day of the experiment.From land ll leachate, BCF value for iron was the highest one on the 3rd and 6th day of experiment.BCF value for copper was 0.76, 0.84, 0.68, 0.65, and 0.42 on day 3, 6, 9, 12, and 15 of the experiment, respectively.Maximum BCF value (0.71) for nickel was observed on 6th day of the experiment.Results indicate that L. minor, grown on the land ll leachate, showed maximum BCF values for copper, lead, and zinc.BCF values of heavy metals by L. minor were in the order of Cu (0.84) > Pb (0.81), Zn (0.81) > Fe (0.74) > Ni (0.71).Similarly, BCF values for Cu, Ni, Pb, and Cd were also found to be less than one by L. minor from two di erent kinds of e uents in a hydroponic experiment for 31 days [61].In another study, L. minor came out as an excellent accumulator for Fe, Zn, and Cu having the BCF value more than 1 from di erent lakes in south Urals region, Russia [65].
According to previous studies, di erent oating aquatic plants demonstrated much higher accumulation for these heavy metals with higher bioconcentration factor.Roots of Eichhornia crassipes and Pistia stratiotes showed much better metal accumulation potential as compared to the upper parts of the plants.In this study, values of bioconcentration factor for Zn, Cu, and Pb were more than 1 in both aquatic plants [66].Physiological demand of plant tissues for certain heavy metals and their accumulation kinetics directly or indirectly a ects their absorption from the growth medium [67].e value of BCF more than 1 indicates the suitability of certain aquatic plants (i.e., hyperaccumulators) for phytoextraction of heavy metals.In the current study, the BCF values of L. minor for all the heavy metals were found to be lower than 1. e results suggest that L. minor is a moderate accumulator for Zn, Cu, Pb, Ni, and Cu under given circumstances of the present study.

Conclusion
e present study concluded that land ll leachate was loaded with both organic and inorganic pollutants.Phytoremediation experiment of land ll leachate using L. minor was found to be e cient for the reduction of both organic and inorganic pollutants.
e reduction in pH, TSS, TDS, COD, BOD, Zn, Pb, Fe, Cu, and Ni was recorded during 15 days phytoremediation experiment using L. minor.e rate of removal was accelerated from the 3rd to 9th day of the experiment.Accumulation of heavy metals was directly proportional to their initial concentration in land ll leachate.Removal e ciency for all the metals was higher than 70%.Among 5 metals under study, the accumulation of copper in L. minor was the highest one.
e highest BCF values were shown by copper (0.84) and lead (0.81).Plants demonstrate extensive ability to remove heavy metals from land ll leachate.High removal e ciency and accumulation capacity of L. minor for heavy metals indicate its phytoremediation potential.is study provides a deep insight into the potential of duckweed (L.minor) to be used as a convenient and economically feasible method for the phytoremediation of metal-polluted aquatic environment on large-scale basis.

6
Journal of Chemistry

Figure 1 :Figure 2 :Figure 3 :Figure 4 :
Figure 1: Zinc concentration in L. minor at di erent exposure time (days) from leachate.Bars represent standard deviation.Values are mean ± SD of three replicates.

Figure 5 :Figure 6 :
Figure 5: Nickel concentration in L. minor at di erent exposure time (days) from leachate.Bars represent standard deviation.Values are mean ± SD of three replicates.
Collection of Plant Sample.Samples of duckweed (L.minor) were collected from fresh water ponds at the Fisheries Research and Training Institute, Lahore (31.5890 °N, 74.4642 °E).
[38]larly, Zimmo et al.[38]reported a more efficient reduction in BOD of duckweed-based ponds than that of algal-based ones.3.2.Heavy Metal Removal from Leachate.Results on removal of heavy metals (Zn, Pb, Fe, Cu, and Ni) through phytoremediation of landfill leachate by Lemna minor at different time periods of exposure are shown in Figures1-5.Reduction in concentration of heavy metals in landfill leachate depends upon the duration of exposure to L. minor.Zinc concentration of leachate was reduced from 1.47 mg•L −1 to 0.024 mg•L −1 during 15-day experiment.Initial concentration of lead was 0.83 mg•L −1 in the landfill leachate in which significant decrease (p < 0.05) was observed during the first 9 days, following a negligible change thereafter till the end of the experiment.A similar response of iron was observed as its initial concentration (1.17 mg•L −1 ) was significantly reduced to 0.26 mg•L −1 at the end of the experiment.Concentration of copper considerably declined from 0.69 to 0.06 mg•L −1 in landfill leachate (p < 0.01).Concentration of nickel also significantly reduced (p < 0.05) from 1.21 mg•L −1 to 0.29 mg•L −1 after phytoremediation through Lemna minor as described in Figure [37]1 and 1695 mg•L −1 , respectively, in landfill leachate.As demonstrated in Table1, concentration of TSS was reduced with the passage of time, reaching the minimum level of 42 mg•L −1 at the end of the experiment.Results regarding the decrease during the experiment.Results revealed that L. minor successfully reduced COD by 39% (from 1899 to 756 mg•L −1 ) and BOD by 47% (from 889 to 423 mg•L −1 ).In agreement with the results of present research, Azeez and Sabbar[37]also reported a 32.7% and 49.6% decline in COD and BOD, respectively, during a 4-week phytoremediation study on oil refinery by Lemna minor.

Table 1 :
Physicochemical characteristics of land ll leachate before and after phytoremediation experiment.Values are the mean of three replicates.NIQS, National Environment Quality Standards, Pakistan; ND, not detected; ns, nonsigni cant.* , * * signi cantly di erent at p < 0.05 or p < 0.01 level of ANOVA, respectively (mean ± SD, n 3).

Table 2 :
Bioconcentration factor for L. minor for heavy metals at di erent exposure time periods from land ll leachate.Figure 7: Metal concentration in whole plant (mg/tub).Bars represent standard deviation.Values are mean ± SD of three replicates.