Evaluation of Phytoremediation Potential of an Aquatic Macrophyte (Eichhornia crassipes) in Wastewater Treatment

Abstract

Wastewater generation is a major concern, as most of it goes untreated. Industries, urban areas, and agriculture are the major contributors to wastewater. Phytoremediation is an effective method of wastewater treatment. However, the potential of local aquatic species for hyper-accumulation of heavy metals remains elusive. This study focuses on evaluating the native macrophyte Eichhornia crassipes for phytoremediation potential in different source-based water environments: freshwater (FW), industrial (IW), and urban wastewater (UW). Physico-chemical analysis was conducted on water samples (five samples from each source) along with the corresponding E. crassipes plants for assessing physiological, nutritional, and heavy metal parameters. The results showed distinct characteristics among the water sources. The FW had a high pH, and the IW exhibited elevated levels of electrical conductivity (EC: 1746 μS cm⁻¹), total dissolved solids (TDS: 864 mg L⁻¹), chloride (Cl⁻: 557.83 mg L⁻¹), sulfate (SO₄⁻: 137.27 mg L⁻¹), and calcium (Ca⁺⁺: 77.83 mg L⁻¹) ions. The UW exhibited high bicarbonate (HCO₃⁻: 123.38 mg L⁻¹), sodium (Na⁺: 154 mg L⁻¹), and potassium (K⁺: 37.12 mg L⁻¹) ions. The Cd contamination exceeded World Health Organization (WHO) limits (0.003 mg L⁻¹) in the FW (0.05 mg L⁻¹ in FW-5) and UW (0.05 mg L⁻¹ in UW-3); Cr contamination was higher than the permissible limits of the WHO, National Environmental Quality Standards (NEQS), and the European Union (EU) (0.05 mg L⁻¹) in FW, IW, and UW; arsenic (As) in IW exceeded the WHO, United States Environmental Protection Agency (USEPA), and EU limits of 10 μg L⁻¹, and Pb in UW exceeded the WHO (0.01 mg L⁻¹), NEQS (0.05 mg L⁻¹), and EU (0.01 mg L⁻¹) limits. E. crassipes displayed different traits depending on the water sources. FW-grown plants had a higher biomass and chlorophyll-b content, while UW-grown plants had higher photosynthesis rates and chlorophyll-a content. Shoots accumulated more Na⁺, K⁺, and Ca⁺⁺ ions than roots. Metal translocation from roots to shoots followed specific patterns for each source: the TFs of Zn = 3.62 in FW > Cd = 2.34 in UW > Cr = 1.61 and Pb = 1.29 in IW and BCFs were found in ascending order: Zn > Ni > Cd > As > Pb in FW, Cd > Zn > Ni > Cr > Pb > As in IW, and Cd > Ni > Pb > Cr > Zn > As in UW. The bioconcentration factor was higher in the roots than in the shoots. These findings suggest that E. crassipes shows promise as a phytoremediation option for heavy metal-contaminated wastewater due to its ability to thrive in harsh wastewater conditions with a higher TF > 1 and BCF > 1. Therefore, the utilization of these macrophytes holds potential for wastewater treatment.

1. Introduction

Water is the most significant resource to sustain life on the planet Earth. Available freshwater resources make up about 2.5% of the global water reserves [1]. Anthropogenic activities have boosted the rate of urbanization, industrialization, and agriculture [2], creating a huge pressure on freshwater availability across the globe that may cause water insecurity in the future [3]. Industries generate millions of tons of wastewater with solvents, toxic materials, and heavy metals and discharge them into water bodies untreated [4]. Wastewater constituents, especially heavy metals, and metalloids are of great concern and have destructive effects on the environment and human health [4,5]. Small quantities of certain heavy metals, i.e., copper, iron, zinc, and manganese are essential for normal growth and body functions, but higher concentrations of them are highly toxic [6]. Some of the heavy metals (mercury, lead, and cadmium), even at low concentrations, are injurious to human health and the environment [5].

Various physical (membrane filtration, physical adsorption), chemical (precipitation, ion exchange, coagulation, and flocculation), and biological (biosorption) approaches are being introduced to detoxify heavy metals’ impact from wastewater and promote the reuse of water to mitigate water shortages [3,4,7]. Nevertheless, these approaches are not only economically non-sustainable and non-eco-friendly [8], but their efficiency (50%) is still questionable [9]. In recent years, researchers have been growing interest in exploring alternative, cost-effective, and environmentally friendly approaches, such as phytoremediation [8,9,10], for wastewater treatment.

Phytoremediation utilizes the natural abilities of plants to remove, degrade, or immobilize pollutants from various environmental media, including water [11]. Phytoremediation relies on plants’ ability to tolerate and accumulate pollutants from the environment [10] and offers cost-effective, sustainable, and versatile waste treatment with minimal environmental impact [11]. Various phytoremediation technologies are phytostabilization, phytovolatilization, phytoextraction, phytostimulation, phytofiltration, phytotransformation, and phytodegradation [12]. These plants (phytoremediator) can reduce the hazardous effect of various organic and inorganic contaminants by being reduced, converted, and catabolized by plants [13]. Inorganic contaminants are chemically non-degradable, but some plant species treat them either by immobilization in the root zone (rhizosphere) and accumulation in the root (phytostablization) or shoots (phytoextraction) [14]. Phytofiltration (rhizofiltration and caulofiltration in roots and shoots, respectively) is the only remediation technology for wastewater [15] in which pollutants are accumulated and precipitated [16]. The mechanism of adsorption and absorption is followed by phytofiltration [17].

Phytoremediation efficiently removes contaminants from water, including heavy metals, using diverse plant species. Plants that are native and have high growth rates, high biomass production, adaptability to the environment, an extensive root system, and high accumulation potential should be considered for phytoremediation [18]. Aquatic macrophytes have demonstrated promising potential in wastewater treatment due to their ability to absorb and metabolize contaminants [19].

Water hyacinth (Eichhornia crassipes), a highly productive and free-floating aquatic plant from the Pontedriaceae family, is renowned for its exceptional nutrient absorption, pollution resistance, and massive growth, making it the most toxic weed [18]. Among the hyper-accumulators, E. crssipes is an idealistic aquatic macrophyte for significant bioaccumulation of toxic metals like Cd, Co, Ni, Hg, and Pb and metalloids like arsenic [9,10]. This species exhibits high adsorption efficiency for heavy metals like Cr, Pb, Cu, etc., effectively removing up to 90% of these metals from water. Additionally, it can be reused multiple times in the adsorption process [20].

Therefore, in this study, it was hypothesized whether E. crassipes had the ability to perform phytoremediation of inorganic nutrients (heavy metals) from freshwater, urban, and industrial wastewater. The aim of this study is to evaluate the phytoremediation potential of E. crassipes in wastewater treatment. By investigating its performance in removing specific pollutants from wastewater, we seek to contribute to the understanding of its efficiency and effectiveness as a natural treatment option. This research will provide valuable insights into the suitability of E. crassipes for wastewater treatment systems and help in designing and optimizing phytoremediation strategies for water pollution control.

2. Materials and Methods

2.1. Study Site

Burewala is located at 30°9′33″ N and 72°40′54″ E in District Vehari, Punjab, Pakistan (https://mcburewala.lgpunjab.org.pk/history.html (accessed on 24 May 2023) (Figure 1). Water hyacinth (E. crassipes) and water samples were collected from five sites of each source, i.e., freshwater (FW) (Canal System, Burewala), urban (UW) (Katchi Abadi, Burewala), and industrial (IW) (Budh Byaas, Burewala) wastewater.

2.2. Sample Collection

Water samples were collected as described by Khan et al. [21]. From each site (Figure 1), the water samples were collected by using a clean polythene bottle filled with no air bubbles. For heavy metal(loids) analysis, the samples were filtered through Whatman No. 42 filter paper and digested using nitric acid (HNO₃) (99%, Sigma-Aldrich, St. Louis, MO, USA). From each site, plant (E. crassipes) samples were collected, washed to remove foreign particles, and air-dried for one hour to measure the fresh weight of roots and shoots. Then, the samples were oven dried for 48 h to measure the dry weight of roots and shoots separately.

2.3. Water Analysis

Electrical conductivity (EC), pH, and total dissolved solids (TDS) were measured by an EC meter, HANNA HI9811-5 (HANNA Instruments, Cecili, Italy) pH meter, and TDS meter, respectively. Carbonates, bicarbonates, and chlorides were estimated by the titration method described by Estefan et al. [22] and APHA [23]. For carbonates, 10 mL of each sample was taken in a beaker, and a few drops of phenolphthalein indicator (Sigma-Aldrich, St. Louis, MO, USA) were added; if pink colour appeared, then titrated against 0.01 N H₂SO₄ (99.998%, Sigma-Aldrich, St. Louis, MO, USA) to colourless endpoint, and if no pink colour appeared, then methyl orange indicator (Fisher Scientific Company, Waltham, MA, USA), (M-21676166 LOT 726294; 99% purity) was added to the same sample and titrated against 0.01 N H₂SO₄ showing dark red orange endpoint. For chlorides, 10 mL of each water sample was taken, and a few drops of potassium chromate indicator (Fisher Scientific Company, USA, for Chloride, Certified, 5% (w/v), LabChem™, Singapore) were added and titrated against 0.01 N AgNO₃ with the brick red endpoint. Sulfates were calculated according to equation SO₄²⁻ = TDS − (CO₃²⁻ + HCO₃⁻ + Cl⁻) described by Khalid et al. [24]. Heavy metal(loid)s (Pb, Ni, Zn, As, Cr, Cd) were analysed through atomic absorption spectroscopy (AAS) (Solaar S-100, CiSA, Thermo Fisher Scientific, Warrington, UK) as described by Ahmad et al. [25]. Potassium, calcium, and sodium were estimated by flame photometer (JENWAY, PFP7) by following the methods of Estefan et al. [22].

2.4. Plant (E. crassipes) Analysis

Chlorophyll contents were measured by preparing plant samples in 80% acetone and analysing them through a spectrophotometer at 645 nm and 663 nm for chlorophyll-a and chlorophyll-b, respectively, as described by Arnon [26]. For heavy metal(loids), 200 mg of powdered dry plant material was digested in 8 mL of concentrated HNO₃ and left overnight, then digested by adding 2 mL hydrogen peroxide (Solvay Interox, Deer Park, TX, USA) and heated to obtain a transparent residue that was filtered and diluted to 50 mL in a volumetric flask with distilled water (Milli-Q^® (Darmstadt, Germany); resistivity 18.2 megohm-cm; MilliporeSigma, Darmstadt, Germany) and then analysed through AAS as explained by Ahmad et al. [25]. Sodium, potassium, and calcium ions were estimated with flame photometer by using the method of Estefan et al. [22]. Physiological parameters, i.e., net photosynthesis, stomatal conductance, transpiration rate, and intercellular carbon dioxide concentrations, were measured through an infra-red gas analyser (IRGA, LI-COR6200, Lincoln, NE, USA). The intact leaf of E. crassipes plants was directly placed in an IRGA chamber to determine above mentioned parameters.

2.5. Bioconcentration Factor (BCF)

Bioconcentration factor (BCF) is an index value that provides the heavy metal(loids) accumulation in plants. BCF was calculated by Equation (1) [27].

$$\mathrm{BCF}=\frac{\mathrm{Heavy}\mathrm{metal}\left(\mathrm{loids}\right)\mathrm{concentration}\mathrm{in}\mathrm{plant}\mathrm{tissue}}{\mathrm{Heavy}\mathrm{metal}\left(\mathrm{loids}\right)\mathrm{concentration}\mathrm{in}\mathrm{water}}$$

(1)

2.6. Translocation Factor (TF)

The translocation factor (TF) is a ratio of heavy metal(loids) accumulated in shoots to heavy metal(loids) accumulated in roots (Equation (2)). Higher values indicate greater translocation of respective heavy metal(loids) [25,28].

$$\mathrm{TF}=\frac{\mathrm{Heavy}\mathrm{metal}\left(\mathrm{loids}\right)\mathrm{accumulation}\mathrm{in}\mathrm{plant}\mathrm{shoot}}{\mathrm{Heavy}\mathrm{metal}\left(\mathrm{loids}\right)\mathrm{accumulation}\mathrm{in}\mathrm{plant}\mathrm{root}}$$

(2)

2.7. Statistical Analysis

Data were analysed by using statistical software (Statistix v8.1). Descriptive statistics were applied to compute mean, minimum, maximum, and standard deviation on water and plant samples. The values were compared with the standard values described by international environmental agencies like National Environment Quality Standard (NEQS), Pakistan, World Health Organization (WHO), the United States Environmental Protection Agency (USEPA), and European Union (EU) standards.

3. Results

3.1. Water Analysis

3.1.1. Physico-Chemical Parameters of Water

Freshwater (FW) showed a high pH as compared to industrial (IW) and urban (UW) wastewater sources (FW > IW > UW). The average pH values for FW, IW, and UW were 8.54, 8.32, and 7.94, respectively (

Table 1. Descriptive analysis of physicochemical parameters of freshwater (FW), industrial (IW), and urban (UW) wastewater samples.

Water Sources		pH	EC	TDS	HCO3−	CO32−	Cl−	SO42−	Na+	K+	Ca2+
			(μS cm−1)	(mg L−1)
FW	Min.	8.40	420.00	200.00	71.22	N/A	74.80	14.03	12.60	3.10	32.30
	Max.	8.70	470.00	230.00	109.83	N/A	82.24	61.98	13.80	7.50	43.10
	Ave.	8.54	436.00	212.00	93.57	N/A	79.33	19.10	13.38	4.03	35.75
	SD	0.13	19.49	10.95	19.66	N/A	3.00	21.11	0.48	0.70	3.81
IW	Min.	7.90	1420.00	700.00	54.92	12.00	425.54	34.25	34.50	17.10	71.30
	Max.	8.50	2430.00	1200.00	146.44	91.53	744.50	273.05	38.10	37.80	89.50
	Ave.	8.32	1746.00	864.00	107.38	61.52	557.83	137.27	37.03	30.37	77.33
	SD	0.24	402.53	198.82	34.13	35.41	133.47	92.91	1.31	8.54	6.69
UW	Min.	7.80	1230.00	610.00	91.53	12.04	425.40	12.95	38.20	17.80	45.80
	Max.	8.00	1520.00	740.00	170.84	18.01	602.65	62.73	302.00	94.00	74.40
	Ave.	7.94	1366.00	672.00	123.38	14.43	496.20	38.00	154.10	37.12	58.65
	SD	0.09	123.00	58.05	29.66	3.27	70.82	20.37	101.68	28.42	11.03
Permissible Limits
NEQS (2008)		6.5 to 8.5	N/A *	<1000	N/A	N/A	<250	N/A	N/A	N/A	N/A
WHO (2008)		6.5 to 9.5	N/A	1000	N/A	N/A	250	250	200	N/A	N/A
USEPA		N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
EU		≥6.5 to ≤9.5	2500 at 20 °C	N/A	N/A	N/A	250	250	200	N/A	N/A

* Not available.

). The highest pH value was observed for FW, which was 8.70. FW showed a little bit higher values than NEQS but within the defined limits (6.5–9.5) of the WHO. The EC values for the water samples increased in the following order IW > UW > FW. The mean concentrations of EC values were 436 μS cm⁻¹, 1746 μS cm⁻¹, and 1366 μS cm¹ for FW, IW, and UW, respectively (Table 1).

The results showed that a lower value of TDS was observed in FW than in IW and UW. The TDS of water increased in the following order IW > UW > FW. The average concentration of TDS was 212 mg L⁻¹, 864 mg L⁻¹, and 672 mg L⁻¹ for FW, IW, and UW, respectively. The TDS level was higher in IW-5 than the defined permissible limits (Table 1).

A lower value of chlorides was observed in FW as compared to IW and UW. The chloride of the water increased in the following order UW > IW > FW. The average concentration of chloride was 79.33 mg L⁻¹, 557.83 mg L⁻¹ and 496.20 mg L⁻¹ for FW, IW, and UW, respectively (Table 1). Chloride was higher than the permissible limit in both IW and UW, while the FW value was within the permissible limit. Carbonates were not detected in FW sources. The average bicarbonate concentration in FW, IW, and UW was 93.57 mg L⁻¹, 107.38 mg L⁻¹, and 123.38 mg L⁻¹, respectively. Higher bicarbonates were detected in UW water followed by IW and FW sources, i.e., 170.84 mg L⁻¹. The average concentrations of carbonates detected in IW and UW were 61.52 mg L⁻¹ and 14.43 mg L⁻¹, respectively (Table 1).

The average concentrations of sulfate ions reported in FW, IW, and UW were 19.10 mg L⁻¹, 137.27 mg L⁻¹, and 38 mg L⁻¹, respectively. Only the IW-5 displayed a greater concentration than the permissible limit set by the WHO and EU (250 mg L⁻¹) (Table 1). Potassium analysis showed a lower concentration of K⁺ ions in FW than in IW and UW. The order of K⁺ concentration increase was UW > IW > FW. The results showed a lower value of sodium (Na⁺) ions in FW than in IW and UW. The Na⁺ concentration of water increased in the following order UW > IW > FW. Na⁺ concentration was higher at UW-3 and UW-5 compared to the permissible limit for Na⁺ (200 mg L⁻¹) set by the WHO and EU (Table 1). Calcium analysis presented a lower concentration of Ca²⁺ in FW than in IW and UW. The Ca²⁺ concentration in water increased in the following order IW > UW > FW (Table 1).

3.1.2. Heavy Metal(loids) Analysis of Water

A descriptive analysis of the heavy metal(loids) in the water from all three sources is given in

Table 2. Descriptive analysis of heavy metal(loid)s detected in FW, IW, and UW resources.

Water Sources		Cd	Cr	Zn	Pb	Ni	As
		mg L−1					μg L−1
FW	Min.	0.001	0.05	0.02	0.01	0.01	0.61
	Max.	0.005	0.15	0.03	0.22	0.02	2.71
	Ave.	0.003	0.09	0.03	0.08	0.01	1.92
	SD	0.001	0.05	0.00	0.09	0.01	0.83
IW	Min.	0.001	0.02	0.03	0.03	0.01	17.30
	Max.	0.004	0.08	0.05	0.06	0.03	33.89
	Ave.	0.002	0.05	0.04	0.04	0.02	28.45
	SD	0.001	0.03	0.01	0.01	0.01	7.62
UW	Min.	0.002	0.01	0.06	0.01	0.01	12.52
	Max.	0.005	0.07	0.18	0.09	0.06	25.69
	Ave.	0.003	0.05	0.10	0.05	0.03	19.62
	SD	0.001	0.02	0.05	0.03	0.02	5.44
Permissible Limits
NEQS		0.01	0.05	5	0.05	0.02	50
WHO		0.003	0.05	3	0.01	0.07	10
USEPA		0.005	0.1		0.015		10
EU		0.005	0.05		0.01	0.02	10

. The observed average cadmium (Cd) concentrations in the FW, IW, and UW were 0.003 mg L⁻¹, 0.002 mg L⁻¹, and 0.003 mg L⁻¹, respectively. Cd was detected in all water samples; the order was FW > UW > IW. In FW and UW, it was 0.005 mg L⁻¹, higher than the WHO permissible limit (0.003 mg L⁻¹). The average chromium (Cr) concentration was 0.09 mg L⁻¹ for FW, 0.05 mg L⁻¹ for IW, and 0.05 mg L⁻¹ for UW. Cr metal values were higher in FW and marginal for UW and IW in comparison to permissible limits (Table 2).

The average zinc (Zn) concentrations detected were 0.03 mg L⁻¹ for FW, 0.04 mg L⁻¹ for IW, and 0.10 mg L⁻¹ for UW. The Zn concentration found in all studied water sources was within the permissible limit (Table 2). The average concentrations of lead (Pb) detected in FW, IW, and UW were 0.08 mg L⁻¹, 0.04 mg L⁻¹, and 0.05 mg L⁻¹, respectively. The average Pb concentrations in all studied water sources were higher according to the WHO, the US-EPA, and EU standards but within the permissible limit (0.05 mg L⁻¹) of NEQS except for FW (Table 2). FW was highly contaminated with Pb as compared to IW and UW.

The average concentrations of nickel (Ni) detected were 0.01 mg L⁻¹ in FW, 0.02 mg L⁻¹ in IW, and 0.03 mg L⁻¹ in UW. Ni concentrations found in all water sources were within the permissible limits (0.07 mg L⁻¹) of WHO standards. The average arsenic (As) concentrations analysed were 1.92 μg L⁻¹ for FW, 28.45 μg L⁻¹ for IW, and 19.62 μg L⁻¹ for UW. The observed average As values were within the permissible limits defined by NEQS (50 μg L⁻¹) but higher than WHO (10 μg L⁻¹), USEPA (10 μg L⁻¹), and EU (10 μg L⁻¹) standards. Only the FW As concentration (1.92 μg L⁻¹) was according to the standards (Table 2). IW showed the highest As concentrations followed by UW. In IW, the highest As concentration was detected in IW-2 (33.89 μg L⁻¹) followed by IW-1 (33.88 mg L⁻¹). In UW, the highest As concentration was observed in UW-1 (25.69 μg L⁻¹) followed by UW-2 (23.97μg L⁻¹) (Table 2).

3.2. Plant Analysis

3.2.1. Plant (E. crassipes) Physiological Analysis

A descriptive analysis of various plant physiological parameters is given in (

Table 3. Descriptive analysis of growth and physiology of E. crassipes grown in FW, IW, and UW. Shoot fresh weight (SFWt), shoot dry weight (SDWt), root fresh weight (RFWt), root dry weight (RDWt), transpiration rate (TR), stomatal conductance (SC), photosynthesis rate (AR), intercellular CO₂ concentration (ICC), chlorophyll-a and b contents (Chl-a and Chl-b).

Water Sources		TR	SC	AR	ICC	Chl-a	Chl-b		SFWt	SDWt	RFWt	RDWt
		mmol m2 s−1		μmol m2 s−1	μmol mol−1	mg g−1			g
FW	Min.	1.01	397.70	0.40	22.00	0.01	0.64		24	1.59	16	1.01
	Max.	3.76	999.90	2.40	31.30	0.12	1.31		56	4.31	30	3.76
	Ave.	2.67	755.56	1.26	25.60	0.52	0.98		40	2.99	22	2.67
	SD	1.14	267.79	0.76	3.84	0.05	0.30		13	1.23	7	1.14
IW	Min.	1.27	257.15	0.25	10.50	0.01	0.78		24	1.24	12	1.27
	Max.	3.63	968.00	1.87	40.30	0.08	1.29		86	6.21	40	3.63
	Ave.	2.69	536.57	0.82	23.16	0.03	1.05		42	2.72	19	2.69
	SD	0.96	283.38	0.66	12.32	0.03	0.23		25	2.04	12	0.96
UW	Min.	1.10	463.00	0.30	18.00	0.02	0.47		20	0.84	10	1.10
	Max.	3.37	999.90	3.40	33.70	0.11	1.67		66	4.71	32	3.37
	Ave.	2.47	755.16	1.66	24.14	0.06	1.21		41	2.80	19	2.47
	SD	0.93	261.39	1.30	7.19	0.04	0.44		22	1.81	10	0.93

). The average fresh biomass of E. crassipes shoots was 40, 42, and 40.8 g for FW, IW, and UW, respectively. A greater fresh shoot biomass was observed for IW. The average dry biomass of the shoots was 2.99, 2.72, and 2.78 g for FW, IW, and UW, respectively. The average fresh biomass of E. crassipes roots collected was 22, 19.20, and 18.80 g for FW, IW, and UW, respectively. A greater fresh root biomass was observed for IW. The average dry biomass of roots was 1.39, 0.85, and 1.03 g for FW, IW, and UW, respectively (Table 3). The highest fresh and dry biomass yields were displayed by FW plant samples.

The chlorophyll contents were higher in FW plant samples, i.e., 0.52 mg g⁻¹ of the leaf, while IW and UW plants displayed 0.04 mg g⁻¹ and 0.06 mg g⁻¹ of the leaf. Similarly, chlorophyll-b contents were higher in UW plant samples followed by IW plant samples, i.e., 1.21 mg g⁻¹ and 1.05 mg g⁻¹, respectively (Table 3). Chlorophyll-a analysis showed that FW plant samples had higher chlorophyll-a and lower chlorophyll-b contents, while IW and UW plant samples displayed lower chlorophyll-a contents while higher chlorophyll-b contents.

The average transpiration rate was 2.67 mmol m² s⁻¹ for FW, 2.67 mmol m² s⁻¹ for IW, and 2.47 mmol m² s⁻¹ for UW. The average transpiration rate suggested that a higher transpiration rate was found in IW plant samples followed by FW and UW plant samples (Table 3). The maximum transpiration rate observed was 3.76 mmol m² s⁻¹ for FW-5, 3.63 mmol m² s⁻¹ for IW-3, and 3.37 mmol m² s⁻¹ for UW-5. Similarly, the average stomatal conductance studied was 755.560 mmol m² s⁻¹ for FW, 536.566 mmol m² s⁻¹ for IW, and 755.160 mmol m² s⁻¹ for UW. The stomatal conductance rate was higher in FW plant samples than in UW and IW plant samples. No significant differences were identified in the transpiration rate and stomatal conductance among plant samples from the three water sources.

The average net photosynthesis rate was 1.26 μmol m² s⁻¹ for FW, 0.82 μmol m² s⁻¹ for IW, and 1.67 μmol m² s⁻¹ for UW. The average net photosynthesis rate showed that higher carbon assimilation was achieved in plants with UW reservoirs followed by plants present in FW bodies. The lowest carbon assimilation was achieved in IW plant samples (Table 3). The average intercellular carbon dioxide rate studied was 25.60 μmol mol⁻¹ in FW, 23.16 μmol mol⁻¹ in IW, and 24.14 μmol mol⁻¹ in UW. The average intercellular carbon dioxide rate was higher in FW followed by UW, while the lowest value was identified in IW.

3.2.2. Analysis of Na⁺, K⁺, and Ca²⁺ Ions

The average concentrations of Na⁺ ions in shoots were 101.00 mg L⁻¹ for FW, 120.00 mg L⁻¹ for IW, and 108.20 mg L⁻¹ for UW. The average concentrations of Na⁺ ions in roots were 85.74 mg L⁻¹ for FW, 106.20 mg L⁻¹ for IW, and 114.40 mg L⁻¹ for UW (

Table 4. Descriptive analysis of Na⁺, K⁺, and Ca²⁺ ion concentrations in root and shoot of E. crassipes grown in FW, IW, and UW sources.

Water Sources		E. crassipes Shoots				E. crassipes Roots
		Na+	K+	Ca2+		Na+	K+	Ca2+
		mg L−1
FW	Min.	100.0	152.0	287.0		11.7	62.1	32.3
	Max.	102	312	306		108	160	296
	Mean	101.00	208.0	295.40		85.74	117.20	239.46
	SD	1.00	63.64	7.13		41.52	47.64	115.88
IW	Min.	115	110	318		96	40	309
	Max.	127	318	328		114	153	328
	Mean	120.20	178.60	322.40		106.20	86.00	319.60
	SD	4.66	83.14	3.65		6.72	44.66	8.20
UW	Min.	98	116	293		106	72	302
	Max.	117	312	313		125	174	307
	Mean	108.20	229.00	301.80		114.40	110.80	306.40
	SD	7.26	61.26	7.19		7.40	38.85	3.05

). In freshwater plants, Na⁺ ion concentration was higher in shoots as compared to roots. Similar conditions were observed for the IW wastewater plants. In the case of UW, Na⁺ ion concentration was higher in roots as compared to shoots (Table 4). The maximum Na⁺ ion concentrations in plant shoots of FW, IW, and UW were 102 mg L⁻¹, 127 mg L⁻¹, and 117 mg L⁻¹ respectively while the maximum concentrations in plant roots were 108 mg L⁻¹, 114 mg L⁻¹, and 125 mg L⁻¹, respectively. Na⁺ ion concentration was higher in shoots of IW than in FW and UW plant shoots. On the other hand, the roots of UW plants had higher Na⁺ ion concentrations than FW and IW plants. The average concentrations of K⁺ ions in shoots and roots of FW, IW, and UW were 208.0 mg L⁻¹, 178.6 mg L⁻¹, 229.0 mg L⁻¹, and 117.2 mg L⁻¹, 86.0 mg L⁻¹, and 110.8 mg L⁻¹, respectively (Table 4).

Like Na⁺ ion, K⁺ ion concentration was found higher in shoots as compared to roots. Shoots of UW plants displayed higher K⁺ ion concentration as compared to FW and IW plants and, likewise, roots of FW plants showed higher K⁺ ion concentration than IW and UW wastewater plants. The average concentrations of Ca²⁺ ions found in shoots and roots of FW, IW, and UW were 295.4 mg L⁻¹, 322.4 mg L⁻¹, 301.8 mg L⁻¹, and 239.4 mg L⁻¹, 319.6 mg L⁻¹, and 306.4 mg L⁻¹, respectively. Ca²⁺ concentration was found higher than Na⁺ and K⁺ ions both in roots and shoots. Both the roots and shoots of IW wastewater displayed higher Ca²⁺ ion concentrations than FW and UW plants (Table 4). Results showed that the uptake of Na⁺, K⁺, and Ca²⁺ ions was greater in shoots than in roots of E. crassipes. The orders of Na⁺, K⁺, and Ca²⁺ accumulation in E. crassipes were IW > UW > FW, UW > FW > IW, and IW > UW > FW.

3.2.3. Heavy Metal(loid)s Analysis

In the FW plant samples, the average cadmium (Cd) concentration in the shoots and roots was 0.020 mg L⁻¹ and 0.044 mg L⁻¹, respectively. This suggested that the bioaccumulation of Cd in the roots was higher than in the shoots. Chromium (Cr) showed average concentrations of 0.131 mg L⁻¹ in shoots and 0.299 mg L⁻¹ in roots. The Cr concentration was found higher in roots than in shoots. The average concentrations of lead (Pb) were 0.04 mg L⁻¹ in shoots and 0.14 mg L⁻¹ in roots (

Table 5. Descriptive analysis of heavy metal(loid)s concentrations in shoots and roots of E. crassipes grown in different sites of FW, IW, and UW sources.

Water Sources		E. crassipes Shoots							E. crassipes Roots
		Cd	Cr	Zn	Pb	Ni	As		Cd	Cr	Zn	Pb	Ni	As
		mg L−1					μg L−1		mg L−1					μg L−1
FW	Min.	0.014	0.07	0.02	0.00	0.02	0.89		0.013	0.09	0.03	0.09	0.06	9.17
	Max.	0.029	0.29	0.35	0.11	0.11	22.81		0.085	0.56	1.53	0.22	0.39	31.26
	Ave.	0.020	0.13	0.20	0.04	0.07	6.47		0.044	0.30	0.67	0.14	0.18	17.96
	SD	0.006	0.09	0.12	0.04	0.03	9.20		0.028	0.18	0.70	0.07	0.13	8.10
IW	Min.	0.015	0.12	0.10	0.04	0.03	4.31		0.021	0.11	0.16	0.05	0.05	24.34
	Max.	0.048	0.33	0.79	0.24	0.07	29.28		0.254	0.19	1.01	0.11	0.11	47.77
	Ave.	0.028	0.22	0.40	0.10	0.05	11.95		0.083	0.14	0.45	0.08	0.08	36.14
	SD	0.013	0.08	0.32	0.08	0.02	10.83		0.096	0.03	0.33	0.02	0.02	8.36
UW	Min.	0.013	0.04	0.03	0.02	0.01	2.51		0.014	0.10	0.13	0.05	0.05	32.45
	Max.	0.080	0.19	0.20	0.14	0.05	36.52		0.029	0.21	0.63	0.17	0.86	48.86
	Ave.	0.042	0.10	0.12	0.08	0.04	18.45		0.020	0.15	0.32	0.10	0.23	39.94
	SD	0.026	0.05	0.08	0.05	0.02	15.12		0.006	0.04	0.21	0.06	0.35	7.01

).

The average concentrations of zinc (Zn) and nickel (Ni) were 0.20 mg L⁻¹ and 0.07 mg L⁻¹ in shoots while 0.67 mg L⁻¹ and 0.18 mg L⁻¹ in roots, respectively. The average concentrations of As were 6.47 mgL⁻¹ in shoots and 17.60 mg L⁻¹ in roots. The highest concentrations of Cd, Cr, Zn, Pb, Ni, and As were 0.03 mg L⁻¹, 0.29 mg L⁻¹, 0.35 mg L⁻¹, 0.11 mg L⁻¹, 0.11 mg L⁻¹, and 22.81 μg L⁻¹ in shoots and 0.09 mg L⁻¹, 0.56 mg L⁻¹, 1.53 mg L⁻¹, 0.22 mg L⁻¹, 0.39 mg L⁻¹, and 31.26 μg L⁻¹ in roots, respectively (Table 5).

The results showed that bioaccumulation of heavy metals and metalloids was higher in shoots than in roots. In IW plant samples, the average concentration of Cd was 0.002 mg L⁻¹ in shoots and 0.028 mg L⁻¹ in root samples. Maximum values of Cd observed in roots and shoots were 0.004 mgL⁻¹ and 0.048 mg L⁻¹, respectively. Cd showed a higher concentration in roots rather than shoots (Table 5). The average concentration of Cr was 0.45 mg L⁻¹ in shoots while it was 0.22 mg L⁻¹ in the roots of the plant samples. Zn, Pb, and Ni had average concentrations of 0.42 mg L⁻¹, 0.43 mg L⁻¹, and 0.02 mg L⁻¹ in shoots and 0.40 mg L⁻¹, 0.11 mg L⁻¹, and 0.05 mg L⁻¹ in roots, respectively.

The results displayed that Zn and Pb concentrations were higher in shoots while Ni had a higher concentration in the roots of E. crassipes. The bioaccumulation in the shoots was higher than in roots except for Ni and Cd. The As was found with average concentrations of 28.45 μg L⁻¹ in shoots and 11.95 μg L⁻¹ in roots of E. crassipes, indicating that the As level was higher in the shoots than in the roots of the studied plants. In UW samples, respective average concentrations of Cd, Cr, Zn, Pb, Ni, and As were 0.042 mg L⁻¹, 0.10 mg L⁻¹, 0.12 mg L⁻¹, 0.08 mg L⁻¹, 0.04 mg L⁻¹, and 18.45 μg L⁻¹ in the shoots of E. crassipes. The roots of E. crassipes displayed an average concentration of 0.020 mg L⁻¹ for Cd, 0.15 mg L⁻¹ for Cr, 0.32 mg L⁻¹ for Zn, 0.10 mg L⁻¹ for Pb, 0.23 mg L⁻¹ for Ni, and 39.94 μg L⁻¹ for As. Results indicated that the shoots of E. crassipes showed lower concentrations of Cr, Zn, Pb, Ni, and As except for Cd (Table 5).

3.2.4. Bioconcentration Factor and Translocation Factor

The average bioconcentration factor (BCF) for Cd in the plant shoots was IW > UW > IW, while for the roots, it was IW > FW > UW. The Cd BCF was higher in the roots than in the shoots of E. crassipes except for UW. The maximum BCF was displayed in shoots by UW-4 (22.94) and in roots by IW-1 (240.32). Likewise, the average BCF for Cr observed in the shoots was IW > UW > FW, while in roots, it was UW > IW > FW. The highest BCF in the shoots was reported in IW-1 (12.48), while in the roots, it was reported in UW-2 (10.19). The average BCF for Ni, Zn, and Pb reported in the shoots were 8.06, 6.95, and 2.26 for FW, 3.78, 10.97, and 2.60 for IW, and 1.79, 1.55, and 3.59 for UW, respectively, showing an order of increase as FW > IW > UW for Ni, IW > FW > UW for Zn, and UW > IW > FW for Pb.

The BCF of Ni in the roots was 24.50, 5.72, and 7.6 for FW, IW, and UW, respectively, and the displayed roots accumulated more Ni than shoots. The average BCF for Zn in the roots was 27.39, 13.05, and 3.04 for FW, IW, and UW, respectively, and the results displayed the roots as having higher Zn accumulators. The average BCF for Pb in the roots was 6.59, 1.86, and 4.91 for FW, IW, and UW, respectively.

The results showed a higher bioaccumulation in FW E. crassipes than in IW and UW. The order of the BCF increases for Pb was FW > UW > IW. The average BCF for As in shoots and roots was 3.16, 0.46, and 0.99 and 11.86, 1.34, and 2.14 for FW, IW, and UW, respectively. Bioaccumulation of As was higher in roots as compared to shoots (

Table 6. Descriptive analysis of bioconcentration factor (BCF) of shoots and roots of E. crassipes in FW, IW, and UW sources.

Water Sources		BCF of Shoots							BCF of Roots
		Cd	Cr	Zn	Pb	Ni	As		Cd	Cr	Zn	Pb	Ni	As
FW	Min	4.24	0.47	0.59	0.06	2.95	0.55		4.13	1.74	1.00	0.42	3.55	5.70
	Max	24.10	5.33	10.61	7.64	17.37	8.41		30.75	7.13	59.62	22.41	69.55	26.89
	Ave.	10.57	1.97	6.95	2.26	8.06	3.16		19.41	3.56	27.39	6.59	24.50	11.86
	SD	8.09	1.95	3.77	3.30	5.88	3.27		10.27	2.18	29.45	9.19	26.17	8.99
IW	Min	5.76	2.76	2.23	0.85	2.54	0.13		9.75	1.49	3.61	1.23	2.90	1.02
	Max	22.29	12.48	29.55	5.48	6.77	0.92		240.32	8.29	37.78	2.73	7.95	2.18
	Ave.	13.76	5.81	10.97	2.60	3.78	0.46		61.68	4.17	13.05	1.86	5.72	1.34
	SD	6.64	4.01	11.28	1.92	1.74	0.40		100.16	3.26	14.02	0.73	1.87	0.50
UW	Min	3.79	1.34	0.14	0.26	0.30	0.10		3.71	1.54	1.80	0.64	1.38	1.35
	Max	41.50	2.85	3.14	11.09	4.11	2.27		14.59	10.19	5.63	16.88	21.81	2.69
	Ave.	21.94	2.25	1.55	3.59	1.79	0.99		8.99	4.58	3.04	4.91	7.36	2.14
	SD	16.84	0.64	1.21	4.64	1.53	0.89		3.89	3.65	1.57	6.98	8.38	0.53

).

The translocation factor (TF) for Zn was 3.62, 0.87, and 0.61 for FW, IW, and UW, respectively. The TF of Zn showed a higher accumulation in the UW environment followed by IW. The TF for Pb and Ni was 0.29 and 0.61 for FW, 1.29 and 0.68 for IW, and 0.77 and 0.49 for UW, respectively (

Table 7. Descriptive analysis of the translocation factor (TF) for heavy metal(loid)s in E. crassipes grown in FW, IW, and UW sources.

Water Sources		Cd	Cr	Zn	Pb	Ni	As
FW	Min.	0.24	0.22	0.03	0.03	0.15	0.06
	Max.	1.08	0.90	10.61	0.48	1.44	1.47
	Ave.	0.61	0.52	3.62	0.29	0.61	0.40
	SD	0.37	0.3	4.97	0.17	0.54	0.60
IW	Min.	0.06	0.79	0.43	0.6	0.39	0.09
	Max.	2.29	2.09	1.67	2.12	0.9	0.81
	Ave.	0.79	1.61	0.87	1.29	0.68	0.33
	SD	0.86	0.50	0.47	0.61	0.21	0.29
UW	Min.	0.45	0.28	0.04	0.41	0.01	0.05
	Max.	4.21	1.19	1.38	1.14	1.00	0.91
	Ave.	2.34	0.70	0.61	0.77	0.49	0.47
	SD	1.63	0.38	0.56	0.29	0.36	0.38

). The accumulation rate was found higher in the FW habitat for both Pb and Ni heavy metals. The TF for As was 0.40, 0.33, and 0.47 for FW, IW, and UW, respectively. The As TF showed that bioaccumulation was higher in the IW environment followed by FW. Bioaccumulation of different metals according to the TF in the FW source was Pb > As > Cr > Ni > Cd > Zn, while for IW and UW, it was As > Ni > Cd > Zn > Pb > Cr and As > Ni > Zn > Cr > Pb > Cd, respectively (Table 7).

4. Discussion

Results of this study claimed that some aquatic plants have the potential to remediate water, including FW, IW, and UW, contaminated with heavy metals through anthropogenic activities over time. E. crassipes has shown significant heavy metal removal from the water collected from natural sources consisting of five different sites, i.e., FW (Canal System, Burewala), UW (Katchi Abadi, Burewala), and IW (Budh Byaas, Burewala). To our understanding, we here report, for the first time, about an overlooked or underestimated phenomenon of phytoremediation ongoing under natural conditions as water resources are being strained by both human activities and natural problems [5].

The physicochemical analysis of the water revealed that FW has a high pH as compared to IW and UW sources (FW > IW > UW), while electrical conductivity (EC) shows an opposite trend and increases in the order of IW > UW > FW (Table 1). Freshwater sources often receive inputs from geological formations rich in carbonate minerals, such as limestone. These minerals can dissolve in water, releasing carbonate ions (CO₃⁻²) and bicarbonate ions (HCO₃⁻¹) that act as buffers and maintain a higher pH [29], while the lower pH of IW and UW wastewater might be related to the presence of organic acid usually used in various processes in industry and houses that can contribute to the lower pH of wastewater compared to freshwater [30]. Lower EC of FW compared to IW and UW might be linked with the presence of dissolved ions and contaminants [4]. The TDS and chloride ions contents of water were increased in the order of IW > UW > FW and UW > IW > FW, respectively. The higher levels of TDS and chloride ion content in wastewater compared to FW can be attributed to various factors, including industrial discharges, domestic sewage, and agricultural runoff [30]. Similarly, lower concentrations of bicarbonates, sulfates, potassium, calcium, and sodium were detected in FW when compared to UW and IW. It might be due to natural geological processes, anthropogenic activities, and the addition of chemicals in wastewater. The results are comparable with the previous studies of Abdel-Shafy et al. [31], Gamage and Yapa [32], and Aniyikaiye et al. [33].

It is well-known that heavy metals enter the environment through various anthropogenic activities, such as mining practices, automobile exhaust, paints, batteries, manufacturing, production industries, etc. These substances are toxic to humans, as they easily enter the body and cause neurological disorders and cancer [3,5]. Our results revealed a high degree of heavy metal contamination not only in UW and IW but also in FW, which requires an immediate strategy to mitigate heavy metal contaminations in these resources, so their impact can be minimized on human and plant health. In FW, concentrations of Cd, Pb, As, and Cr were reported higher than the permissible limits, as recorded previously by Kamel et al. [34]. Pb, As, Cd, and Cr were also reported higher than the defined limits in industrial wastewater. In UW, Pb, Cd, Cr, Ni, and As were identified as higher except for Zn. The results are comparable with the findings of Samecka-Cymerman and Kemper [35] and Brankovic [36]. However, our findings have elaborated and emphasized that the roots of E. crassipes acted as a better sink for such heavy metal(loid) accumulation, as previously reported by Lu et al. [37], Hasan et al. [38], and Victor et al. [39], than shoots. The reason for the higher accumulation in roots was due to the absorption capabilities of the root tissue surfaces [40]. This shows that E. crassipes can be used for heavy metal removal from water bodies through the process of rhizofilteration.

Physiological parameters of E. crassipes have shown that the plants’ growth order observed in all experiments is FW > UW > IW. However, the E. crassipes plant showed almost similar growth in all three water bodies, i.e., FW, IW, and UW, with little difference in plant biomass (Table 3). Nevertheless, according to Sooknah and Wilkie [41], the growth of E. crassipes decreases as the EC value increases at 4040 μS cm⁻¹, and the growth of E. crassipes is inhibited [42]. In this study, the EC values of IW (1746 μS cm⁻¹) and UW (1366 μS cm⁻¹) might be responsible for the minimum growth and physiology of E. crassipes compared to the FW sources, which have the lowest EC value (436 μS cm⁻¹). Both the roots and shoots of IW displayed higher Ca⁺² and Na⁺ ion concentrations than FW and UW plants (Table 1). Under salt stress, Na⁺ accumulated in the plant tissue triggered Ca⁺² depletion from cytosole, and disequilibrium of Na⁺/K⁺ caused program cell death [43,44]. This might be another reason for the low growth of E. crassipes in IW and UW sources.

In addition, physiological changes due to ion uptake, including shoot and root uptake of Na⁺, K⁺, and Ca²⁺ ions, have shown that the uptake of Na⁺, K⁺, and Ca²⁺ ions is significantly higher in shoots as compared to the roots of E. crassipes. However, it has been observed that the ion uptake in both the root and shoot of the plant are different under different water sources. The order of Na⁺, K⁺, and Ca²⁺ ion accumulation in E. crassipes was observed as IW > UW > FW (Table 1). The significant potential was reported by our studies to accumulate Na⁺, K⁺, and Ca²⁺ ions by E. crassipes. The work of Abdel-Shafy et al. [31] also confirmed that higher Na⁺, K⁺, Ca²⁺, and Mg²⁺ ion concentrations were reported in E. crassipes collected from the Nile River in Egypt. On the contrary, higher concentrations of the studied heavy metal(loid)s were found in the roots of E. crassipes growing under UW, as well as in higher concentrations in all plant samples from all three sources, i.e., FW, IW, and UW. E. crassipes growing in UW showed higher levels of As accumulation as compared to the others (Table 3). E. crassipes is a promising aquatic macrophyte for the remediation of heavy metals and can sustain under moderate concentrations of heavy metals [9,10]. Significant concentrations of Pb, Zn, Ni, Cd, As, and Cr were reported in E. crassipes, confirming its potential as an accumulator for heavy metals [31].

It was previously reported that E. crassipes is an efficient plant for absorption and translocation of Cd, Cu, and Zn in the root or shoot tissues of plants [45]. Our study also claimed higher TF for Pb, Zn, Cd, Ni, and As. Studies by Abdel-Shafy et al. [31] also demonstrated that roots of E. crassipes act as a major sink for Fe, Cd, Pb, Cu, Zn, and Mn comparable with our research work. Sarkar et al. [46] also proved that root powder of E. crassipes can remove 98.83% of Cr and 99.59% of Cu from tannery effluents, while Mokhtar et al. [47] suggested E. crassipes as a hyper-accumulator for Cu (97.3% removal). The Cd uptake was found higher in roots as compared to shoots of E. crassipes except for the plant present in UW. E. crassipes growing in FW showed lower uptake values than IW and UW. The Cd uptake might be affected by slightly alkaline pH as evaluated by [48]. It has been reported earlier that E. crassipes can remove Cd more efficiently than Fe and Cu [49]. Nevertheless, under poor nutrient conditions, ideal Fe removal was reported by Jayaweera et al. [50]. E. crassipes could remove higher concentrations of Pb and Zn more effectively than other metals [51].

In our studies, the BCF demonstrated a higher concentration of Cd followed by Zn and Ni, while a lower one for As. High BCF for E. crassipes make it a powerful aquatic plant to remediate heavy metals from the surrounding environment as recorded by [9,18]. Zhang et al. [52] reported that BCF > 1 indicate a strong enrichment ability of E. crassipes to accumulate heavy metals. Our study inferred greater BCF values in the order: Cd > Zn > Ni > As, which are 10-fold higher than those reported by Zhang et al. 2019 [52]. The average translocation factor reported for Cd was 0.61, 0.79, and 2.34 for FW, IW, and UW, respectively, and displayed that bioextraction was higher in E. crassipes growing in FW sources and lowest in UW plant sources. The TF for Cr was 0.52, 1.61, and 0.70 for FW, IW, and UW. The TF of Cr displayed higher bioextraction in the FW habitat followed by UW. The results of the TF showed that As was more highly accumulated than other heavy metals in IW and UW environments than in FW. The results are comparable with the findings of Jódar-Abellán et al. [53], Azizi et al. [54], and Ibezim-Ezeani and Ihunwo [55]. A TF > 1 indicates good extraction ability of heavy metals by E. crassipes from water (Zhang et al. 2019 [52]). Our study found the TF of Zn = 3.62 in FW, Cd = 2.34 in UW, Cr = 1.61, and Pb = 1.29 in IW, representing good accumulation in E. crassipes shoot.

5. Conclusions

E. crassipes (water hyacinth) is an invasive plant due to massive biomass production. It originated in the tropical zone of America before spreading to tropical countries in other regions of the planet. E. crassipes, on the other hand, is also studied by researchers to remediate various pollutants, including toxic metal(loid)s, under various physical and climatic conditions. Our study confirmed that E. crassipes can adopt diverse environments of FW, IW, and UW due to the presence of various nutrients in these water sources. Most of the physicochemical properties of water (pH, EC, TDS, cations, anions) were found in descending order IW < UW < FW, which reduced E. crassipes biomass and physiology. Researchers considered E. crassipes as an active accumulator of heavy metal(loid)s to treat wastewater, inferred by TFs of Zn = 3.62 in FW, Cd = 2.34 in UW, Cr = 1.61, and Pb = 1.29 in IW and BCFs of Cd > Zn > Ni > As. The least studies were carried out on the phytoremediation potential of E. crassipes under climatic conditions that could be due to the physicochemical nature and complexity of various pollutants. More work is needed to elucidate the heavy metal(loid) phytoremediation potential of E. crassipes under natural climatic conditions.