Cu and Pb Accumulation and Removal From Aqueous Medium by Enydra Fluctuans DC. (Asteraceae) - A Medicinal Plant With Potential for Phytoremediation

Enydra uctuans DC. (Asteraceae) is an edible semi-aquatic oating or trailing herbaceous plant widely distributed in tropical Africa, South and South East Asia, and Australia. Its leaves, which are consumed as a vegetable, are also used in traditional medicine to treat several diseases. The ecacy of this plant in removal of copper and lead from aqueous medium was tested in the present study. Accumulation of both Cu and Pb was signicantly higher in root than that in leaf and stem. Though all the bioconcentration factor (BCF) values were greater than unity, none of the translocation factor (TF) values was greater than unity, indicating that this plant could not be considered a hyperaccumulator of these metals. Nevertheless, E. uctuans could remove Cu from aqueous medium at rates ranging from 98.8–99.7 %, with a mean reduction of 99.2 % after 96 h exposure at various concentrations. The removal of Pb ranged from 97.1–99.1 %, with a mean reduction of 98.2 %. Thus, E. uctuans showed high potential in removal of Cu and Pb from aqueous medium, and has the prospect of being used in phytoremediation of these metals.


Introduction
Various human activities such as industrialization and urbanization accompanied by rapid population growth have added different types of pollutants into the freshwater ecosystems (CPCB 2008). Among these pollutants, heavy metals are of worldwide concern because of their toxic properties, tendency to accumulate in biota, and persistent nature (Rai et al. 1981 . Under these circumstances, it is necessary to identify broad-spectrum, simple and costeffective technologies for removal of toxic metals (Zhang et al. 2020). The idea of using plants to cleanup pollutants from the environment was introduced in 1983, though this method was being applied for the last 300 years (Ali et al. 2020). These technologies are also named as green remediation, botanoremediation, vegetative remediation, and agro remediation (Sarwar et (Singh et al. 1996;Miretzky et al. 2004;Putra et al. 2015). Plants that are used in the process of phytoremediation take up heavy metals through their roots and later transfer these contaminants to the above-ground parts of their body (Ashraf et al. 2018).
Aquatic macrophytes are of structural and functional signi cance in aquatic ecosystems because they provide stable habitats, and are a source of food and oxygen to the macroinvertebrate and sh fauna.
They contribute to nutrient cycling, improve water quality by regulating oxygen balance and also play an important role in heavy metal accumulation (Srivastava et al. 2008;Dhote and Dixit 2009). Their high biomass yield, fast growth rate, high tolerance, ability to accumulate heavy metals, and direct exposure to contaminated water, facilitate their remediation ability, which enables them to act as natural water ltration systems (Sood et al. 2012). The roots, shoots, and leaves of aquatic macrophytes have the capacity to absorb heavy metals from aquatic environments, while their roots have the potential to analgesic (Rahman et al. 2002); and hypotensive (Kuri et al. 2014). Furthermore, it is packed with vitamins and other nutrients (Dua et al. 2015). The plant grows abundantly in rice elds, ditches, drains, natural channels and edges of sh ponds, where they propagate by fragmentation and often choke the water courses (Ali et al. 2013). However, its potential for removal of heavy metals from aqueous medium has not been assessed to any great extent, though it is known to have the ability to remove the metalloids arsenic and boron from water (Shaheen et al. 2006(Shaheen et al. , 2007. The two heavy metals copper and lead were selected for several reasons. Copper (Cu) is an essential micronutrient for the growth of plants and a constituent trace nutrient of the protein component of several enzymes, most of which are involved in catalyzing redox reactions, electron ow, etc. However, Cu can be toxic at high concentrations that are extremely harmful to both plants and animals (Devi and Prasad 1998; Wang and Dei 2001; Khumanleima Chanu and Gupta 2014). Due to direct exposure to its toxic effects, many aquatic plants are more sensitive to copper than their terrestrial counterparts (Fernandes and Henriques 1991). Cu is commonly used as a fungicide in tea plantations and different agricultural lands in the study area (Khumanleima Chanu and Gupta 2014). Lead at concentrations of more than 0.03 mg/100 gm (FAO/WHO 2001) can cause blood diseases and affect nervous system, teeth and gum, liver, pancreas, and bones in humans (Khan 2015). Lead is dense, soft, durable, and corrosionresistant with a relatively low melting point, making it a major constituent of paint, ammunition, leaded glass, solder, storage batteries, etc. (Abadin et al. 2007). The principal consumption of lead (i.e., ~ 80% of the total use of lead) is for the lead-acid storage battery, and untreated wastewater containing lead used in recycling of batteries is released into the water bodies in the study area (Bedabati Chanu and Gupta 2016). In developing countries, there is a rapid increase in the demand for lead batteries due to an increase in the number of motorized vehicles, solar panels, telecommunication and computers (OK International 2021). A review of the available scienti c literature reveals that the Cu and Pb accumulation and removal by E. uctuans are not documented. Considering the above facts, the present paper is an attempt to critically evaluate the potential of this plant to accumulate Cu and Pb in its different tissues, and to remove these heavy metals from aqueous medium.

Materials And Methods
Plant material and culture of plants Enydra uctuans DC. was collected from water bodies of Irongmara area (latitude: 24.689 ºN, longitude: 92.742 ºE) in Cachar district, Assam, India. The plant material was brought to the laboratory in a plastic bag and washed with tap water. The plants were grown in hydroponic culture system till they developed new branches. These new branches were cut and grown in soil ooded with 50% modi ed Hoagland nutrient solution with its pH maintained at 5.8-6.2 (Göthberg et al. 2004

Experimental Methods
Fully grown healthy shoots of similar height (20-25 cm long) were cut from the same mother plant, washed with tap water and placed in 50 % Hoagland nutrient media for 1 week (Temperature: 22-25˚C; dissolved oxygen: 6.28-6.87 mg L 1 ; electrical conductivity: 99.6-107 µS cm 1 ; pH: 5.96-6.5) to produce new roots. After the acclimatization period, plants of similar shoot height were exposed to Cu and Pb in two sets of experiments. Plants were exposed for 96 h to graded concentrations of Cu

Estimation of Cu and Pb
The Cu and Pb content in the plant samples were estimated using standard methods (Gupta 1996; Khumanleima Chanu and Gupta 2014). As soon as the experiments were completed, the leaf, stem and root were separated and cut into small pieces, and after drying to a constant weight at 60 ± 2˚C, were ground and digested with concentrated HNO 3 . The residues were dissolved in deionized water and the concentrations of Cu and Pb were estimated in a graphite furnace atomic absorption spectrophotometer

Bioconcentration and Translocation Factor
The ability of the plant to absorb Cu and Pb from an aqueous medium and its capacity to translocate them from the roots to different tissues of the plant are expressed by the Bioconcentration Factor (BCF) and the Translocation Factor (TF), respectively. BCF and TF were calculated by the following formulae:

Statistical analysis
The data sets were subjected to One-way ANOVA to nd the statistical signi cance of differences among them, with multiple comparisons made by Tukey tests. SPSS 20 software was used for performing all tests and wherever necessary log transformation of data was also done.

Results And Discussion
Accumulation of copper and lead in different tissues of E. uctuans The accumulation of Cu in leaf, stem and root of E. uctuans after 3, 24, 48 and 96 h of exposure to graded concentrations of Cu are shown in Table 1. Cu was not detected in the tissues of the control plants. One-way ANOVA with multiple comparisons by Tukey tests revealed that there were signi cant differences (p < 0.001) in Cu accumulation among the three tissues of Cu-exposed plants at all concentrations and hours of exposure, in the order root > leaf > stem. There was a consistent increase in accumulation of Cu in the root with increasing concentration and duration of exposure, although this trend was not evident in the leaf and stem. The highest Cu concentration of 980.90 ± 20.81 µg g -1 dry weight (DW) was observed in the root of plants exposed to 10.2 mg Cu L -1 at 96 h. On the other hand, the lowest accumulation was 41.04 Cu µg g -1 DW in the stem exposed to 0.55 mg Cu L -1 at 3 h. The highest Cu concentrations recorded in the leaf and stem were 129.01 ± 0.10 and 168.55 ± 0.39 µg g -1 DW, respectively, when exposed to 10.2 mg Cu L -1 at 24 and 3 h, respectively. Values are given as mean ± SD; values with different superscripts in the same column indicate signi cant differences at P < 0.05 among the different tissues of plants exposed to a given Cu concentration; DW: dry weight; ND: not detected Though copper acts as an essential nutrient by playing a prominent role in enhancing photosynthesis, and promoting development, metabolism and growth of plants, it can be highly toxic beyond certain threshold concentrations (Li and Xiong 2004). Copper affects several biochemical and physiological mechanisms in plants, inhibits growth, and imparts toxic effects due to accumulation in the tissues . Cu accumulation was highest in the roots due to its existence as cations i.e., Cu 2+ and Cu + (Krupanidhi et al. 2008) which got attached to negative charges present in the structure of cell wall in the roots (Göthberg 2008). Khumanleima Chanu and Gupta (2014) also found that roots of Ipomoea aquatica accumulated higher amount of copper than the apical parts of the plant.
In the case of Pb accumulation, a pattern similar to that observed for Cu could be observed (Table 2). Pb was not detected in the control plants, and its accumulation was highest in root, followed by that in leaf and stem. One-way ANOVA with multiple comparisons by Tukey tests revealed that there were signi cant differences (p < 0.001) in Pb accumulation among the tissues at all concentrations and hours of exposure, in the order root > leaf > stem. The highest Pb accumulation of 1093.60 ± 4.08 µg g − 1 DW was found in the root after 96 h exposure to 50.

Conclusions
Due to its widespread occurrence in several parts of the world, the plant E. uctuans is a suitable candidate for its possible utility in phytoremediation of metal-contaminated water as well as in the assessment and biomonitoring of metals and metalloids in freshwater habitats. The present investigation revealed that the plant could survive in fairly high Cu and Pb (up to 10.2 Cu and 50.2 Pb mg L − 1 ) contaminated medium. It could also strongly accumulate these metals, especially in the roots, with BCF values for Cu reaching > 400 and those for Pb > 40. Further, the plant could remove Cu and Pb from aqueous medium at an average rate of 99.2 and 98.2 percent, respectively, after a relatively short exposure time of 96 h. These characteristic features, coupled with its ability to asexually propagate by fragmentation, makes it a plant worth exploring for its potential for phytoremediation of water contaminated with toxic metals and metalloids. Declarations