Citation: Younas, F.; Bibi, I.; Afzal,

M.; Niazi, N.K.; Aslam, Z.

Elucidating the Potential of Vertical

Flow-Constructed Wetlands

Vegetated with Different Wetland

Plant Species for the Remediation of

Chromium-Contaminated Water.

Sustainability 2022, 14, 5230.

https://doi.org/10.3390/su14095230

Academic Editor: Agostina Chiavola

Received: 25 March 2022

Accepted: 23 April 2022

Published: 26 April 2022

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sustainability

Article

Elucidating the Potential of Vertical Flow-Constructed WetlandsVegetated with Different Wetland Plant Species for theRemediation of Chromium-Contaminated WaterFazila Younas 1, Irshad Bibi 1,* , Muhammad Afzal 2 , Nabeel Khan Niazi 1 and Zubair Aslam 3

1 Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Faisalabad 38040, Pakistan;2015ag441@uaf.edu.pk or fazila.younas@gmail.com (F.Y.); nabeelkniazi@gmail.com ornabeel.niazi@uaf.edu.pk (N.K.N.)

2 Soil and Environmental Biotechnology Division, National Institute for Biotechnology and GeneticEngineering (NIBGE), Faisalabad 38000, Pakistan; afzla@nibge.org

3 Department of Agronomy, University of Agriculture Faisalabad, Faisalabad 38040, Pakistan;zubair.aslam@uaf.edu.pk

* Correspondence: irshad.niazi81@gmail.com or irshad.niazi@uaf.edu.pk

Abstract: Water scarcity is one of the key global challenges affecting food safety, food security, andhuman health. Constructed wetlands (CWs) provide a sustainable tool to remediate wastewater. Herewe explored the potential of vertical flow-CWs (VF-CWs) vegetated with ten indigenous wetlandplant species to treat chromium (Cr)-contaminated water. The wetland plants were vegetated todevelop VF-CWs to treat Cr-contaminated water in a batch mode. Results revealed that the Crremoval potential of VF-CWs vegetated with different wetland plants ranged from 47% to 92% at low(15 mg L−1) Cr levels and 36% to 92% at high (30 mg L−1) Cr levels, with the maximum (92%) Crremoval exhibited by VF-CWs vegetated with Leptochloa fusca. Hexavalent Cr (Cr(VI)) was reducedto trivalent Cr (Cr(III)) in treated water (96–99 %) of all VF-CWs. All the wetland plants accumulatedCr in the shoot (1.9–34 mg kg−1 dry weight (DW)), although Cr content was higher in the roots(74–698 mg kg−1 DW) than in the shoots. Brachiaria mutica showed the highest Cr accumulation inthe roots and shoots (698 and 45 mg kg−1 DW, respectively), followed by Leptochloa fusca. The high Crlevel significantly (p < 0.05) decreased the stress tolerance index (STI) percentage of the plant species.Our data provide strong evidence to support the application of VF-CWs vegetated with differentindigenous wetland plants as a sustainable Cr-contaminated water treatment technology such astannery wastewater.

Keywords: chromium; constructed wetlands; indigenous wetland plants; environmental risk; reuseof Cr-contaminated water

1. Introduction

The freshwater resources are diminishing globally due to uncontrolled human con-sumption of clean water and untreated wastewater being discharged into water reservoirsand waterways [1,2]. As a result, the world population faces water security, food security,and food safety challenges. The industrial sector is one of the main water contaminationsources that generate copious amounts of contaminants in the environment [3,4]. Amongthe various contaminants, chromium (Cr) is of huge concern due to its mutagenic, carcino-genic, and teratogenic effects on human health [5]. In the aquatic and terrestrial systems,Cr exists in trivalent Cr (Cr(III)) and hexavalent Cr ((Cr(VI)), of which Cr(VI) is highly toxicand mobile [6,7]. Chromium(VI) toxicity is well-reported; it can impair plant growth, causeultrastructural modifications of the cell membrane and chloroplast, persuade chlorosis inleaves, damage root cells, reduce pigment contents, disturb water relations and mineralnutrition, and alter enzymatic activities [6]. Various methods have been employed to reme-diate Cr-contaminated wastewater, including chemical reduction, membrane separation,

Sustainability 2022, 14, 5230. https://doi.org/10.3390/su14095230 https://www.mdpi.com/journal/sustainability

Sustainability 2022, 14, 5230 2 of 18

ion exchange, and adsorption [8,9]. However, all these Cr removal technologies havesome disadvantages in terms of their high application and maintenance cost, secondarycontamination, and difficult operational procedure [10–12]. Therefore, it is imperativeto deploy low-cost, sustainable, eco-friendly, and effective remediation technologies totreat Cr-contaminated water such as tannery wastewater. Constructed wetlands (CWs)can provide low-cost, less energy-consuming, environmentally-friendly, and sustainablesolutions for the removal and detoxification of Cr from water, and also fulfill the criteria ofUN-Sustainable Development Goals 2030 [7,13,14].

Vertical flow-CW (VF-CW) is one of the types of CWs that involves the vertical flow ofwastewater in bedding media. This type of CW became popular after understanding thedisadvantages of other subsurface flow CW systems in terms of nitrification capacity ofwastewater. Despite its denitrification efficiency, the VF-CW system is characterized by ahigh oxygen transfer rate and degree of removal and nitrification of organic substances [15].Constructed wetland with vertical flow is considered an efficient treatment technology thatcan withstand faults and variable quality of influence and temperature changes [16]. InCWs, many mechanisms such as biosorption, uptake by microbes and plants, adsorptionon bedding media, biodegradation, co-precipitation, and sedimentation occur [17–19].However, it remains a challenge to select suitable wetland plant species which are usefulfor removing specific toxic metal ions from water [20].

A wide range of wetland plant species, such as Brachiaria decumbens, Canna indica,Iris pseudacorus, Pennisetum purpureum, Phragmites australis Juncus effusus, Schoenoplectusamericanus, Typha latifolia, Cyperus rotundus, have been used in CWs to treat industrialwastewater [21]. In this regard, the most used wetland plant was P. australis for tannerywastewater treatment [22]. Various wetland plant species have different capacities fortaking up various metals [23]. Therefore, the selection of wetland plant species is the mostpromising aspect of CWs to remove a specific metal from contaminated water [24].

Schück and Greger [25] tested thirty-four wetland plant species in CWs for their tol-erance to Cd, Cu, Zn, and Pb. Their data showed a large variation in capacity and metalremoval rate between the investigated plant species. The authors found that C. pseudocype-rus and C. riparia were the most promising plants to remove all the four metals studiedfrom wastewater. Both metal type and levels in wastewater affect the performance andmetal removal potential of wetland plant species. For example, Zn is generally accumu-lated to a higher extent than Cu, followed by Cd and Pb [23]. Therefore, the selection ofwetland plant species is the most promising part of CWs to remove any specific metal fromcontaminated water because of variation in specificity and capacity between species formetals accumulation. Hence, appropriate wetland plants may enhance the removal abilityof specific metals from wastewater [24].

The objective of this study was to evaluate the performance of VF-CWs vegetated withten indigenous wetland plant species (Phragmites australis, Leptochloa fusca, Brachiaria mutica,Typha domingensis, Canna indica, Cyperus laevigatus, Cymbopogon citratus, Cynodon dactylon,Pennisetum purpureum, and Paspalum dilatatum) for the remediation of Cr-contaminatedwater. The survival and growth of wetland plants exposed to Cr-contaminated water weredetermined. The Cr removal efficiency of VF-CWs vegetated with ten wetland plant specieswas evaluated, and speciation of Cr in water and plants tissues was studied. Moreover, thebioaccumulation of Cr in plants and their stress tolerance index (%) to different levels of Crwas measured.

2. Materials and Methods2.1. Chemicals

Chemicals such as hydrogen peroxide (30% w/v) and nitric acid (assay 68–70% w/v)were used to digest plants. A solution of potassium dichromate (K2Cr2O7) was used toprepare artificial Cr-contaminated water (15 and 30 mg L−1). These two Cr levels wereselected because of the average concentration of Cr in the tannery wastewater of Kasur

Sustainability 2022, 14, 5230 3 of 18

in Punjab, Pakistan, which ranged from 15–49 mg L−1 in the tannery wastewater. All thechemicals were of analytical grade.

2.2. Experimental Setup

The wetland plant species used to develop VF-CWs were obtained from the nurseryof the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad,Pakistan. These wetland plant species were selected based on their adaptability to localclimate and flooded conditions, easy accessibility, rapid growth, and tolerance to the harshenvironment (e.g., extreme heat and cold). These plants are perennial grasses that pose noother environmental issues, such as invasive plants [21,26]. These ten wetland plant species(P. australis, L. fusca, B. mutica, T. domingensis, C. indica, C. laevigatus, C. citratus, C. dactylon,P. purpureum, and P. dilatatum) can grow in almost all areas of the country [7].

Cobbles, small and medium-size gravels, and sand were used to fill the VF-CWs thathelp vegetate the local wetland plants upon it [27]. The VF-CWs system was establishedin the greenhouse of the NIBGE for the treatment of Cr-contaminated water. A total ofninety similar VF-CWs mesocosms, each with a length of 3 m, width of 2.5 m, and heightof 3 m, were constructed to evaluate the Cr-removal efficiency of wetland plant species(Figure 1). The total water storage capacity of each VF-CWs unit was 3 L. Each VF-CWunit was filled with a 7.62 cm layer of cobbles, a 5.08 cm layer of medium-sized gravels,and a 2.54 cm layer of small gravels and sand (from bottom to top in the same order)(Figure 1). Cobbles were used at the bottom of the CWs system near the outlet, whichprovides support to avoid clogging and facilitate water distribution. The total period ofplant growth in CWs was from April–September 2021. Before the experiment, the wetlandplants were grown and acclimatized in tap water from April–August and then exposedto Cr stress from August–September. In Pakistan, summer is long and harsh compared towinter, so we selected this duration for our study.

Sustainability 2022, 14, x FOR PEER REVIEW 3 of 19

2. Materials and Methods

2.1. Chemicals

Chemicals such as hydrogen peroxide (30% w/v) and nitric acid (assay 68–70% w/v) were

used to digest plants. A solution of potassium dichromate (K2Cr2O7) was used to prepare

artificial Cr-contaminated water (15 and 30 mg L−1). These two Cr levels were selected

because of the average concentration of Cr in the tannery wastewater of Kasur in Punjab,

Pakistan, which ranged from 15–49 mg L−1 in the tannery wastewater. All the chemicals

were of analytical grade.

2.2. Experimental Setup

The wetland plant species used to develop VF-CWs were obtained from the nursery

of the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisala-

bad, Pakistan. These wetland plant species were selected based on their adaptability to

local climate and flooded conditions, easy accessibility, rapid growth, and tolerance to

the harsh environment (e.g., extreme heat and cold). These plants are perennial grasses

that pose no other environmental issues, such as invasive plants [21,26]. These ten wet-

land plant species (P. australis, L. fusca, B. mutica, T. domingensis, C. indica, C. laevigatus,

C. citratus, C. dactylon, P. purpureum, and P. dilatatum) can grow in almost all areas of the

country [7].

Cobbles, small and medium-size gravels, and sand were used to fill the VF-CWs

that help vegetate the local wetland plants upon it [27]. The VF-CWs system was estab-

lished in the greenhouse of the NIBGE for the treatment of Cr-contaminated water. A

total of ninety similar VF-CWs mesocosms, each with a length of 3 m, width of 2.5 m,

and height of 3 m, were constructed to evaluate the Cr-removal efficiency of wetland

plant species (Figure 1). The total water storage capacity of each VF-CWs unit was 3 L.

Each VF-CW unit was filled with a 7.62 cm layer of cobbles, a 5.08 cm layer of medium-

sized gravels, and a 2.54 cm layer of small gravels and sand (from bottom to top in the

same order) (Figure 1). Cobbles were used at the bottom of the CWs system near the

outlet, which provides support to avoid clogging and facilitate water distribution. The

total period of plant growth in CWs was from April–September 2021. Before the experi-

ment, the wetland plants were grown and acclimatized in tap water from April–August

and then exposed to Cr stress from August–September. In Pakistan, summer is long and

harsh compared to winter, so we selected this duration for our study.

Figure 1. Layout of vertical flow constructed wetlands used in this study.In the VF-CWs, the plan-

tation process was performed based on the United States Environmental Protection Agency

Sub

stra

tes

Wate

r leve

l

Sand: 2.54 cm Small gravels: 2.54 cm

Medium gravels: 5.08 cmCobbles: 7.62

Width: 2.5 m

Length: 3m

Height: 3m

Water direction

Initial planting spacing 0.5–0.10cm

Inlet

outlet

Shape

Rhizome/root cutting

Figure 1. Layout of vertical flow constructed wetlands used in this study.

In the VF-CWs, the plantation process was performed based on the United StatesEnvironmental Protection Agency (USEPA) recommendation (USEPA, 1993). Wetlandplants were planted with a hand-keeping initial spacing of 0.50 to 0.10 cm, and rhizome/rootor stem cuttings material was placed in the porous media at a depth equal to the operationalwater level (Figure 1). An individual rhizome/root or stem cuttings material with a growingshoot at least 0.2 m in length was planted (Figure 2).

Sustainability 2022, 14, 5230 4 of 18

Sustainability 2022, 14, x FOR PEER REVIEW 4 of 19

(USEPA) recommendation (USEPA, 1993). Wetland plants were planted with a hand-keeping ini-

tial spacing of 0.50 to 0.10 cm, and rhizome/root or stem cuttings material was placed in the porous

media at a depth equal to the operational water level (Figure 1). An individual rhizome/root or

stem cuttings material with a growing shoot at least 0.2 m in length was planted (Figure 2).

Figure 2. Schematic diagram of VF-CWs and illustration of mechanisms involved in removal of

chromium (Cr) from wastewater (modified from Younas et al. [7]).

A few dead wetland plants were replaced with new ones during adaptation peri-

ods. The wetland plant species were planted on the VF-CWs media in triplicate and fed

with tap water until it was adapted to VF-CWs. When all selected wetland plants were

completely adapted to the VF-CWs, the units were irrigated with tap water (control; Cr

0 mg L−1) and Cr-contaminated water (15 and 30 mg L−1) from low to high Cr concentra-

tions to improve the plants’ resilience under Cr stress. The system was operated in batch

mode.

2.3. Maintenance and Operations of VF-CW Treatment Systems

The VF-CWs treatment systems were checked daily for maintenance to ensure

proper functioning. The main reason for these inspections was to attend to the circulation

and distribution of water from the outlet to the inlet zone. Daily, distilled water was

supplied to all the VF-CW units to replace the reduction of water volume due to evapo-

transpiration [21]. Distilled water was maintaining the water level in CWs without af-

fecting Cr levels.

Schematic Diagram of Wetland Mechanism

Sorption

Phytoremediation

Bioremediation

Coagulation/Flocculation/adsorption

Figure 2. Schematic diagram of VF-CWs and illustration of mechanisms involved in removal ofchromium (Cr) from wastewater (modified from Younas et al. [7]).

A few dead wetland plants were replaced with new ones during adaptation periods.The wetland plant species were planted on the VF-CWs media in triplicate and fed with tapwater until it was adapted to VF-CWs. When all selected wetland plants were completelyadapted to the VF-CWs, the units were irrigated with tap water (control; Cr 0 mg L−1) andCr-contaminated water (15 and 30 mg L−1) from low to high Cr concentrations to improvethe plants’ resilience under Cr stress. The system was operated in batch mode.

2.3. Maintenance and Operations of VF-CW Treatment Systems

The VF-CWs treatment systems were checked daily for maintenance to ensure properfunctioning. The main reason for these inspections was to attend to the circulation anddistribution of water from the outlet to the inlet zone. Daily, distilled water was supplied toall the VF-CW units to replace the reduction of water volume due to evapotranspiration [21].Distilled water was maintaining the water level in CWs without affecting Cr levels.

2.4. Water Sampling and Analysis

Water sampling and analysis were conducted at various intervals (1, 15, and 30 days)during the experimental period (30 days). This time interval was selected on the base ofplants growth. The first interval was selected after one day of Cr exposure, the second atthe plants’ maximum growth stage, and the last interval before harvesting when plantsstarted to die. The pH and redox potential (Eh) of the water were measured at the samplingsite using pH (ST 300, Ohaus, Parsippany, NJ, USA) and redox meters (Model 8424, Hanna-USA), respectively. Water samples were collected from the outlet of CWs at various intervals(1, 15, and 30 days) during the experiment to determine the total Cr, Cr(VI), and Cr(III).

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2.5. Morphological and Chemical Parameters of Wetland Plant Species

After one month of Cr exposure, wetland plants were carefully separated from thebedding medium of VF-CWs to observe the effect of Cr-contaminated water on the growthand biomass of all the wetland plant species. After harvesting, the wetland plants wereseparated into roots and shoots. The root and shoot length of all wetland plant species wasmeasured with a measuring tape. The dry weight of root and shoot was measured afterdrying in an oven at 65 ◦C. The oven-dried wetland plant samples were powdered (<1 mm)and digested in a mixture of HNO3 and H2O2 (1:1 ratio) at 120 ◦C [28]. Digested sampleswere then filtered and stored in a refrigerator at 4 ◦C

2.6. Estimation of Chromium and Nutrient Elements in Roots and Shoots

Total Cr concentration in the root and shoot was determined using a flame atomicabsorption spectrometer (F-AAS, Thermo-AA®, Solar Series, Waltham, MA, USA). TheCr(VI) concentration in wetland plants tissues was determined using 1,5-diphenylcarbazidemethod at 540 nm on a UV-Visible spectrophotometer (NovAA® 800 series, Analytik Jena,Germany) (APHA 2005). The concentration of Cr(III) was calculated by the differencebetween total Cr and Cr(VI) in plant tissues. The Mn, Fe, and Zn concentrations were ana-lyzed using an F-AAS. The concentrations of Ca, Na, and K in wetland plant samples wereanalyzed using a flame photometer (BWB Model BWB-XP, 5 Channel Flame Photometer,Newbury, England. All the metals and nutrient elements analyses were replicated thrice,and reagent blanks were included for quality assurance.

2.7. Bioaccumulation and Translocation Factors

The wetland plant species efficiency for Cr remediation was calculated by translocationfactor (TF) and bioaccumulation factor (BAF) [26].

The translocation factor provides an index of the wetland plants’ ability to transfer Crfrom root to shoot (Equation (1)):

TF = CA/CU (1)

where CA is the concentration of Cr in the shoot (mg kg−1 dry weight (DW)) and CU is theconcentration of Cr in the root (mg kg−1 DW). The TF > 1 indicates an efficient translocationof metals such as Cr from root to shoot.

The bioaccumulation factor (BAF) of Cr was determined by Equation (2):

BAF = CP/CW (2)

whereas CP is the concentration of Cr in wetland plant species shoot (mg kg−1 DW), andCW is the concentration of Cr in the water (mg L−1). Wetland plants with BAF > 1 areclassified as bioaccumulators.

2.8. Stress Tolerance Index (STI)

The stress tolerance index is an important parameter for measuring the high biomassproduction and stress tolerance potential of plant species. Stress tolerance indexes forvarious growth parameters were calculated using the following formulae [29].

Shoot length STI (SLSTI) = (shoot length of stressed plant/shoot length ofcontrol plant) × 100

Root length STI (RLSTI) = (root length of stressed plant/root length ofcontrol plant) × 100

Shoot fresh weight STI (SFSTI) = (shoot fresh weight of stressed plant/shootfresh weight of control plant) × 100

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Root fresh weight STI (RFSTI) = (fresh root weight of stressed plant/rootfresh weight of control plant) × 100

Shoot dry weight STI (SDSTI) = (shoot dry weight of stressed plant/shootdry weight of control plant) × 100

Root dry weight STI (RDSTI) = (root dry weight of stressed plant/root dryweight of control plant) × 100

2.9. Quality Assurance and Quality Control of Chromium Analysis

Appropriate quality assurance precautions and procedures were followed to ensuredata reliability and accuracy. The F-AAS was calibrated after every five readings usingdrift and blank reagents. After every 12 samples, a reference sample with known Cr(VI))concentration was run on a spectrophotometer for quality control and analytical accuracy.Three reagent blanks were also included with each batch of plant samples during the aciddigestion to assure quality.

2.10. Statistical Analysis

Data collected from the screening experiment was analyzed using Sigma Plot (ver-sion10). Significant statistical differences (p < 0.05) among wetland plant species for Crremoval were determined by two-way analysis of variance (ANOVA). Variation in Crreduction efficacy among tested wetland plant species was determined by Tukey’s HSDtest at p < 0.05. For Pearson correlation and principal component analysis (PCA), XLSTAT2018 software was used [30].

3. Results and Discussion3.1. Redox Potential and pH of the VF-CWs Medium

During the experiment, a change over time in the redox potential (Eh) and pH ofthe VF-CWs medium was observed depending on wetland plants species in the VF-CWs(Table S1, Supplementary Information). The CWs were mainly operating under reducingconditions with Eh values spanning in the negative region (−62 to −122 mV) in all the VF-CWs having various wetland plant species. Water pH in the CWs fed with Cr-contaminatedand un-contaminated water showed some fluctuation with a trend for a slight increase (pH7.8–8.9) at various intervals of water sampling (1, 15, and 30 days). However, a decrease(9.1 to 8.0) in the pH of the water treated by VF-CWs vegetated with B. mutica was observedat both levels (15 and 30 mg L−1) of Cr-contamination and control. It may be becausethe roots of these wetland plants release organic acids, which can decrease the pH of themedia [31].

3.2. Chromium Removal Efficacy of VF-CWs

The concentration of total Cr, Cr(VI), and Cr(III) in the water treated by VF-CWs isshown in Table 1. The total Cr removal efficiencies of the VF-CWs spanned 37–92% and47–92% at an initial concentration of 15 and 30 mg Cr L−1, respectively. After 30 days, atlow (15 mg L−1) initial Cr concentration, L. fusca, C. laevigatus, P. australis, B. mutica, C. indica,P. purpureum, C. citratus, C. dactylon, T. domingensis, and P. dilatatum removed 92%, 88%,80%, 75%, 61%, 53%, 53%, 50%, 48% and 47% of total Cr from the water, respectively. Athigh (30 mg L−1) initial Cr level, L. fusca, C. laevigatus, P. australis, B. mutica, T. domingensis,C. dactylon, C. indica, C. citratus, P. purpureum, and P. dilatatum, removed 92%, 87%, 83%,74%, 58%, 50%, 47%, 46%, 44%, and 37% total Cr, respectively, after 30 days.

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3.3. Removal of Chromium from the Water

The analysis of water samples collected from VF-CWs planted with L. fusca showedthat there was the lowest (0.01 mg L−1) Cr(VI) concentration in the water with 15 mg L−1

Cr treatment (Table 1). The highest Cr(VI) concentration (2.13 mg L−1) was observed intreated water collected from VF-CWs vegetated with C. laevigatus having a high level ofCr (30 mg L−1). The water samples analysis indicated there was a significant reduction inCr(VI) to Cr(III) (96% and 91% at low and high levels of Cr contamination, respectively) inVF-CWs planted with T. domingensis.

The Cr(VI) uptake by the plants uses necessary anionic carriers such as sulfates. Inwetland plant roots, Cr(VI) can be reduced to Cr(III), which may bind with the extracellularcells of roots [32]. Also, wetland plant roots release root exudates that can decreaseCr(VI) concentration in water by making chelating compounds with organic acids [7].The minimum Cr(VI) reduction into Cr(III) occurred in VF-CWs planted with C. indica,40–46% at both levels of Cr. Therefore, Cr(VI) significantly (p < 0.05) reduced to Cr(III) inwater samples of all VF-CWs throughout the experiment because negative Eh (reducedconditions) could provide a highly suitable environment for the reduction of Cr(VI) toCr(III) [7].

However, at the end of the experiment, water samples analysis showed some differencein Cr speciation. Data showed maximum Cr(III) concentration (18.9 mg L−1) in VF-CWsplanted with P. dilatatum with Cr at 30 mg L−1. The minimum Cr(III) concentration(1.5 mg L−1) was observed in VF-CWs planted with C. laevigatus with a high level of Cr.The lowest Cr(VI) concentrations (0.01 mg L−1) and total Cr (1.1 and 2.3 mg L−1) wereobserved in VF-CWs planted with L. fusca at both levels of Cr, respectively.

At both levels of Cr (15 and 30 mg L−1), L. fusca showed maximum Cr removalefficiency from contaminated water, which was 92% Cr removal of total Cr. The minimumremoval efficiency, 47%, and 37%, of total Cr was observed in CWs planted with P. dilatatumat Cr 15 and 30 mg L−1, respectively. L. fusca and C. dactylon showed 92% and 50%removalefficiency at both levels of Cr in water, respectively. The Cr removal from water in allVF-CWs throughout the experiment may be due to plant uptake, adsorption with organicmatter or root exudates, and microbial reduction [33–35]. In this study, most of the Cr(VI)present in the contaminated water was reduced to Cr(III). Similar findings were alsoobserved earlier [36], where more than 90% of Cr(VI) was reduced to Cr(III) by wetlandplant species.

3.4. Morphological Parameters of Wetland Plants

Results revealed that Cr-contamination significantly (p < 0.05) reduced the length ofroot and shoot dry and fresh weight of all the wetland plants compared to plants grownin uncontaminated water (control) (Table 2). There were some visible toxicity symptoms,including leaves necrosis, drying, and shedding in C. indica, T. domingensis, C. citratus,C. dactylon, P. purpureum, and P. dilatatum. Among all the wetland plant species, L. fusca,C. laevigatus, P. australis, and B. mutica exhibited the highest survival at both levels of Crwithout any significant growth difference compared to control.

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Table 1. Total chromium (Cr), Cr(VI), and Cr(III) concentration in treated Cr-contaminated water collected from the outlet of VF-CWs planted with 10 differentwetland plant species. ND: not detected. Values are presented as mean ± standard error of three replicates (n = 3).

1 Day 15 Day 30 Day

Wetland PlantSpecies

Cr Treatment(mg L−1) Cr(III) Cr(VI) Total Cr Cr(III) Cr(VI) Total Cr Cr(III) Cr(VI) Total Cr

Paspalum dilatatumCr0 ND ND ND ND ND ND ND ND NDCr15 11.27 ± 0.4 2.36 ± 0.2 13.63 ± 0.3 8.81 ± 0.8 0.18 ± 0.7 9.00 ± 0.4 7.79 ± 0.5 0.09 ± 0.02 7.89 ± 0.8Cr30 22.18 ± 0.4 13.63 ± 0.1 26.09 ± 0.3 19.78 ± 0.5 0.21 ± 0.7 20.00 ± 0.4 18.9 ± 0.5 0.04 ± 0.03 18.9 ± 0.5

Phragmites australisCr0 ND ND ND ND ND ND ND ND NDCr15 10.45 ± 0.9 1.27 ± 0.2 11.72 ± 0.4 2.68 ± 0.5 0.31 ± 0.6 3.00 ± 0.3 2.86 ± 0.5 0.03 ± 0.02 2.9 ± 0.5Cr30 24.82 ± 0.6 1.58 ± 0.2 26.41 ± 0.4 6.19 ± 0.7 0.86 ± 0.8 7.00 ± 0.3 4.78 ± 0.4 0.08 ± 0.06 4.87 ± 0.5

Cyperus laevigatusCr0 ND ND ND ND ND ND ND ND ND

Cr15 10.35 ± 0.4 0.32 ± 0.07 10.67 ± 0.4 1.72 ± 0.8 0.27 ± 0.7 2.00 ± 0.3 1.59 ± 0.4 0.08 ± 0.06 1.68 ± 0.5Cr30 14.78 ± 0.3 5.60 ± 0.2 20.39 ± 0.4 1.52 ± 0.6 3.47 ± 1.8 5.00 ± 0.5 1.50 ± 0.4 2.13 ± 0.1 3.68 ± 0.8

Typha domingensisCr0 ND ND ND ND ND ND ND ND ND

Cr15 13.56 ± 0.3 0.21 ± 0.03 13.78 ± 0.2 8.86 ± 0.4 0.13 ± 0.7 9.00 ± 0.5 7.58 ± 0.6 0.11 ± 0.05 7.7 ± 0.5Cr30 27.58 ± 0.4 0.20 ± 0.04 27.79 ± 0.4 14.86 ± 0.4 0.13 ± 0.7 15.00 ± 0.4 12.2 ± 0.4 0.11 ± 0.02 12.34 ± 0.7

Canna indicaCr0 ND ND ND ND ND ND ND ND ND

Cr15 6.00 ± 0.3 3.49 ± 0.3 9.50 ± 0.3 5.88 ± 0.5 0.12 ± 0.7 6.00 ± 0.5 5.72 ± 0.4 0.06 ± 0.03 5.78 ± 0.6Cr30 14.17 ± 0.3 6.41 ± 0.2 20.58 ± 0.3 16.92 ± 0.4 0.08 ± 0.02 17.00 ± 0.4 15.6 ± 0.4 0.08 ± 0.06 15.76 ± 0.4

Cymbopogon citratusCr0 ND ND ND ND ND ND ND ND ND

Cr15 7.10 ± 0.3 2.94 ± 0.02 10.0 ± 0.2 7.80 ± 0.4 0.19 ± 0.3 8.00 ± 0.3 6.89 ± 0.7 0.1 ± 0.04 7.00 ± 0.7Cr30 19.84 ± 0.2 4.07 ± 0.1 23.92 ± 0.4 18.72 ± 0.5 0.27 ± 0.4 19.00 ± 0.4 15.8 ± 0.5 0.16 ± 0.1 16.0 ± 0.7

Leptochloa fuscaCr0 ND ND ND ND ND ND ND ND ND

Cr15 9.69 ± 0.2 0.15 ± 0.09 9.84 ± 0.3 2.43 ± 0.7 0.06 ± 0.1 2.50 ± 0.3 1.08 ± 0.8 0.01 ± 0.02 1.1 ± 0.5Cr30 16.19 ± 0.3 2.90 ± 0.2 19.09 ± 0.3 6.73 ± 0.5 0.06 ± 0.1 6.80 ± 0.4 2.2 ± 0.4 0.01 ± 0.05 2.3 ± 0.7

Cynodon dactylonCr0 ND ND ND ND ND ND ND ND ND

Cr15 11.67 ± 0.2 0.16 ± 0.08 11.8 ± 0.5 9.42 ± 0.7 0.08 ± 0.1 9.50 ± 0.3 7.43 ± 0.6 0.06 ± 0.02 7.5 ± 0.8Cr30 25.65 ± 0.2 1.67 ± 0.2 27.32 ± 0.7 16.90 ± 0.5 0.09 ± 0.1 17.00 ± 0.5 14.9 ± 0.4 0.07 ± 0.03 15 ± 0.7

Brachiaria muticaCr0 ND ND ND ND ND ND ND ND ND

Cr15 10.33 ± 0.4 0.33 ± 0.08 10.6 ± 0.3 3.86 ± 0.4 0.13 ± 0.2 4.00 ± 0.2 3.88 ± 0.6 0.09 ± 0.02 3.98 ± 0.7Cr30 21.02 ± 0.3 0.48 ± 0.2 21.51 ± 0.4 9.38 ± 0.4 0.11 ± 0.1 9.50 ± 0.2 7.6 ± 0.7 0.10 ± 0.1 7.78 ± 0.6

Pennisetumpurpureum

Cr0 ND ND ND ND ND ND ND ND NDCr15 9.83 ± 0.2 1.08 ± 0.2 10.9 ± 0.5 8.84 ± 0.5 0.06 ± 0.1 8.90 ± 0.2 6.99 ± 0.5 0.01 ± 0.01 7.00 ± 0.5Cr30 17.42 ± 0.4 5.28 ± 0.2 22.71 ± 0.4 16.92 ± 0.5 0.08 ± 0.1 17.00 ± 0.3 16.6 ± 0.8 0.07 ± 0.01 16.7 ± 0.8

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Table 2. Impact of Cr treatments on morphological parameters of ten wetland plant species planted in vertical flow-constructed wetlands (VF-CWs). Values arepresented as mean ± standard deviation of three replicates (n = 3). Values with different alphabets are significantly different from each other (Tukey’s test at p < 0.05).

Wetland PlantSpecies

Cr Treatments(mg/L) Shoot Length (cm) Root Length (cm) Shoot Fresh Weight (g) Shoot Dry Weight (g) Root Fresh Weight (g) Root Dry Weight (g)

Paspalum dilatatumCr0 27 ± 1 gk 30 ± 1 abc 5.32 ± 0.5 klm 0.56 ± 0.05 jk 15.79 ± 1.6 fg 2.09 ± 0.2 i–n

Cr15 22 ± 1 ijk 25 ± 1 d–g 4.56 ± 0.4 k–n 0.25 ± 0.02 kl 8.61 ± 0.9 i–l 1.82 ± 0.2 j–mCr30 19 ± 1 jk 17 ± 1 h–k 1.14 ± 0.1 n 0.20 ± 0.02 kl 4.07 ± 0.1 m 0.76 ± 0.1 lmn

Phragmites australisCr0 86 ± 3 ab 20 ± 1 f–i 14.45 ± 1.3 def 1.24 ± 0.1 ghi 19.74 ± 2.0 efg 3.52 ± 0.3 f–j

Cr15 78 ± 2 ab 17 ± 1 g–j 11.79 ± 1.0 fgh 0.94 ± 0.06 hij 14.35 ± 1.4 ghi 2.99 ± 0.3 g–kCr30 73 ± 2 bc 17 ± 1 h–k 9.41 ± 1.8 g–j 0.75 ± 0.08 ij 13.33 ± 0.9 hij 2.53 ± 0.2 h–k

Cyperus laevigatusCr0 91 ± 3 a 35 ± 1 ab 9.52 ± 2.5 b 1.09 ± 0.09 ghi 27.27 ± 2.7 abc 16.99 ± 1.0 a

Cr15 81 ± 2 ab 35 ± 1 abc 8.39 ± 1.7 c 1.05 ± 0.1 ghi 24.76 ± 2.5 b–e 14.59 ± 0.4 bCr30 78 ± 2 ab 35 ± 1 bcd 6.16 ± 1.4 cde 0.81 ± 0.1 ij 21.63 ± 2.4 b–e 13.98 ± 1.6 b

Typha domingensisCr0 55 ± 1 de 20 ± 1 f–i 6.46 ± 0.6 i–l 2.54 ± 0.2 ab 22.79 ± 2.3 cde 5.17 ± 0.5 efCr15 47 ± 2 d–g 20 ± 2 g–j 6.27 ± 0.5 i–l 2.40 ± 0.2 abc 22.25 ± 2.2 cde 4.45 ± 0.4 fgCr30 40 ± 2 d–h 17 ± 1 g–j 5.70 ± 0.5 j–m 2.08 ± 0.1 cd 18.17 ± 2.1 def 4.25 ± 0.9 f–i

Canna indicaCr0 58 ± 2 cd 10 ± 1 l–o 39.92 ± 3.5 a 1.39 ± 0.1 fj 26.91 ± 2.7 a–d 2.22 ± 0.2 h–l

Cr15 43 ± 1 d–g 7 ± 1 mno 29.47 ± 2.6 b 1.14 ± 0.1 ghi 19.74 ± 2.0 efg 1.96 ± 0.2 i–nCr30 19 ± 1 f–j 7 ± 1 no 12.12 ± 1.5 cd 1.12 ± 0.1 ghi 8.35 ± 1.4 ghi 1.13 ± 0.2 i–n

Cymbopogon citratusCr0 43 ± 1 d–h 12 ± 1 j–m 8.36 ± 0.7 h–k 2.31 ± 0.2 bc 8.97 ± 0.9 h–k 4.87 ± 0.4 f

Cr15 25 ± 2 h–k 7 ± 1 no 5.32 ± 0.5 klm 1.88 ± 0.1 de 6.46 ± 0.6 j–m 2.19 ± 0.2 h–lCr30 25 ± 1 i–k 5 ± 1 o 2.28 ± 0.2 mn 0.96 ± 0.08 hi 2.87 ± 0.3 lm 1.57 ± 0.4 k–n

Leptochloa fuscaCr0 91 ± 3 a 27 ± 1 cde 13.31 ± 1.2 d–g 1.85 ± 0.07 a 26.91 ± 2.7 a–d 7.16 ± 0.6 cdCr15 88 ± 3 ab 25 ± 1 c–f 13.13 ± 0.7 ghi 1.76 ± 0.09 ef 23.92 ± 2.4 b–e 6.75 ± 0.6 deCr30 86 ± 3 ab 22 ± 1 e–h 12.60 ± 0.9 ijk 1.64 ± 0.03 fgh 22.73 ± 2.3 cde 6.63 ± 0.6 de

Cynodon dactylonCr0 38 ± 1 e–i 17 ± 1 g–j 4.94 ± 0.4 k–n 0.14 ± 0.01 l 14.71 ± 1.5 gh 3.84 ± 0.3 fgh

Cr15 25 ± 1 h–k 15 ± 1 i–l 2.94 ± 0.4 k–n 0.06 ± 0.01 l 4.31 ± 0.4 j–m 2.25 ± 0.2 h–lCr30 25 ± 1 i–k 15 ± 1 i–l 2.66 ± 0.2 lmn 0.08 ± 0.01 l 3.23 ± 0.3 klm 0.55 ± 0.05 lmn

Brachiaria muticaCr0 83 ± 2 ab 35 ± 1 a 16.92 ± 1.5 cd 1.77 ± 0.1 de 32.96 ± 3.3 a 8.62 ± 0.8 c

Cr15 81 ± 2 ab 35 ± 1 ab 16.25 ± 1.2 def 1.38 ± 0.1 fg 30.86 ±3.1 a 8.16 ± 0.7 cdCr30 81 ± 2 ab 33 ± 1 ab 15.73 ± 1.1 efg 1.30 ± 0.1 fgh 28.98 ± 2.9 ab 7.59 ± 0.7 cd

Pennisetum purpureumCr0 45 ± 1 def 20 ± 1 f–i 7.60 ± 0.7 ijk 0.14 ± 0.01 l 2.51 ± 0.3 m 0.64 ± 0.1 lmn

Cr15 20 ± 1 jk 15 ± 1 i–l 5.70 ± 0.5 j–m 0.12 ± 0.01 l 2.15 ± 0.2 m 0.29 ± 0.03 mnCr30 15 ± 1 k 10 ± 1 k–n 2.70 ± 0.5 j–m 0.07 ± 0.01 l 0.97 ± 0.2 m 0.45 ± 0.04 n

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The determination of dry and fresh biomass at the end of the experiment showed thatthe fresh weight of C. indica shoots was higher at 15 mg L−1 Cr level (25.4 g), but a significant(p < 0.05) reduction (12.1 g) occurred at a high Cr level (30 mg L−1). The highest reductionof dry root biomass was observed in C. dactylon (85.6%) and the lowest in L. fusca (7.4%)at a high level of Cr compared to control. The lowest reduction (11.4%) of shoot weightwas observed in L. fusca at 30 mg L−1 of Cr. Some wetland plants under these conditionsshowed a continuous survival at both levels of Cr without significant reduction in growth,which is a crucial parameter of phytoremediation ability. In an earlier study, it was observedthat L. fusca could tolerate a high level of Cr (247 mg L−1) without any significant changein its growth compared to control treatment [27]. Chromium affects various processesin plants, such as seed germination, plant growth, and biomass production, as well asvarious plant physiological processes such as photosynthesis reduction and oxidative andnutrient imbalance [37]. In this study, the growth behavior and morphological parametersof wetland plants revealed differences among plant species exposed to Cr-contaminatedwater. This difference in growth parameters of wetland plant species over time may be dueto their intrinsic nature [33].

In this study, the use of L. fusca, C. laevigatus, B. mutica, and P. australis in VF-CWs totreat Cr-contaminated water proved better in terms of rapid plant growth and Cr removalability than other plant species due to their high Cr removal efficiency [38,39]. P. dilatatum,C. indica, and C. citratus were less active in removing Cr than other plant species because oftheir slow growth and low biomass yield at a high level of Cr-contamination. This studyrevealed that Cr reduction from contaminated water in VF-CWs vegetated with L. fuscaand B. mutica was attributed to their extensive plant root growth, which served as an activezone for inorganic contaminants sequestration by microbial population. Wetland plantspecies having extensive root growth in Cr-contaminated water are more active in removingcontaminants, as observed in earlier studies [40–43]. In the CWs, the extensive root systemof wetland plant species enhances the oxygen transfer efficiency, which increases thecontribution of microbial biomass towards contaminant degradation and treatment [7,44].

3.5. Phytoaccumulation of Cr in Wetland Plants

In this study, there was a significant difference in Cr accumulation in the shoots androots of each wetland plant species (Figure 3). Considering all the wetland plants, B. muticashowed the highest total Cr accumulation in the roots and shoots (698 and 45 mg kg−1

DW, respectively), followed by L. fusca at both levels of Cr (Figure 3b). This may be due totheir rapid growth and extensive root system in the CWs fed with Cr-contaminated water(Table 2). The lowest Cr accumulation was observed in the roots and shoots of C. dactylon(115 and 4.46 mg kg−1 DW, respectively) compared to other plants in VF-CWs.

Among all the wetland plant species, B. mutica showed the highest Cr(III) accumulationin the root (297 and 693 mg kg−1 DW) and shoot (32 and 41 mg kg−1 DW) at 15 and30 mg L−1 Cr levels, respectively, followed by L. fusca (Figure 3c,d; Table 3). The lowestCr(VI) concentration was observed in the roots of P. dilatatum (1.19 mg kg−1 DW) at a Crlevel of 15 mg L−1, and the highest Cr(VI) accumulation was found in the shoot of C. citratus(6.31 mg kg−1 DW) at 30 mg L−1 of Cr (Figure 3). Tadese and Seyoum (2015) reportedthat up to 83% of Cr taken up by wetland plants remained in root cells. In this study, Craccumulation in the roots is consistent with prior research showing that wetland plantsaccumulated various metals mainly in their root tissue followed by the shoot [26,45]. Thisis because of the adsorption of Cr at the extracellular negatively charged sites (e.g., -COO-)of the cell walls of the roots. So, the immobilization of Cr likely occurred in the vacuoles ofroot cells [46].

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(a) (b)

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Figure 3. (a,c,e) Total chromium (Cr), Cr(III) and Cr(VI) concentrations in roots and shoots (T Cr:

(b), Cr(III): (d), Cr(VI): (f)) of ten various wetland plant species grown in vertical flow-constructed

wetlands (VF-CWs) containing Cr-contaminated water (0, 15 and 30 mg L−1). Error bars show ±

standard error of mean of three replicates (n = 3). Similar bars (filled with similar color) labeled with

different alphabets are significantly different from each other (Tukey’s test; p < 0.05).

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Figure 3. (a,c,e) Total chromium (Cr), Cr(III) and Cr(VI) concentrations in roots and shoots (T Cr:(b), Cr(III): (d), Cr(VI): (f)) of ten various wetland plant species grown in vertical flow-constructedwetlands (VF-CWs) containing Cr-contaminated water (0, 15 and 30 mg L−1). Error bars show ±standard error of mean of three replicates (n = 3). Similar bars (filled with similar color) labeled withdifferent alphabets are significantly different from each other (Tukey’s test; p < 0.05).

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Table 3. Root and shoot uptake of chromium (Cr) by wetland plant species grown in vertical flow-constructed wetlands (VF-CWs).

Wetland Plants Cr Treatment (mg/L) Shoot Cr Uptake(mg/CW)

Root Cr Uptake(mg/CW)

Paspalum dilatatum Cr30 0.002 ± 0.001 0.10 ± 0.01Phragmites australis Cr30 0.023 ± 0.007 1.17 ± 0.04Cyperus laevigatus Cr30 0.010 ± 0.003 2.49 ± 0.09Typha domingensis Cr30 0.026 ± 0.008 0.74 ± 0.02Canna indica Cr30 0.009 ± 0.002 0.19 ± 0.07Cymbopogon citratus Cr30 0.010 ± 0.003 0.29 ± 0.01Leptochloa fusca Cr30 0.056 ± 0.017 4.26 ± 0.15Cynodon dactylon Cr30 0.0003 ± 0.0001 0.06 ± 0.002Brachiaria mutica Cr30 0.058 ± 0.017 5.29 ± 0.19Pennisetum purpureum Cr30 0.0007 ± 0.002 0.06 ± 0.002

CW; Constructed wetland, Cr30: 30 mg L−1.

3.6. Bioaccumulation Factor (BAF) and Translocation Factor (TF)

In this study, the BAF of the wetland plants significantly (p < 0.05) decreased (0.5–41%)with the increase in applied Cr levels (0, 15, and 30 mg L−1) in the water, except for B. muticathat possessed 11% greater BAF at high Cr level (30 mg L−1) compared to control (Table S2,Supplementary Information). L. fusca showed significantly (p < 0.05) higher BAF (29) at alow level of Cr, while B. mutica had significantly (p < 0.05) higher BAF (24) at 30 mg L−1

Cr. At both Cr levels, C. dactylon and P. dilatatum possessed the lowest BAF (3.9–5.1and 1.3–5.2, respectively). Wetland plants with a BAF greater than 1.0 are consideredbioaccumulators [47,48]. Therefore, the wetland plants, L. fusca and B. mutica, having BAFhigher than 20, are the best candidates for accumulating Cr from contaminated water.

At both levels of Cr-contamination, all the wetland plant species had varying TF(0.02–0.11) (Table S2, Supplementary Information). At a high level of Cr, C. indica showedthe highest TF (0.08). Phytoremediation of metals, including Cr, differs between the plantspecies types [49]. Some wetland plants have TF > 1 and show high Cr translocationfrom the root to the shoots of the plants. According to an earlier study [50], high TFof heavy metals, including Cr, indicated metals phytoextraction. The TF of Cr in somewetland plants had values < 1, indicating less translocation from roots to shoots (Table 3).Contaminants translocation from roots to aerial parts of plants is based on the contaminantstypes, wetland plant species, and environmental conditions [51].

The Cr partitioning in plants depends on how plants control and manage contaminantsin the roots to prevent hazardous effects on the site of photosynthesis, leaves, and othermetabolic activities [52]. Moreover, Cr(III) is not an essential element for the plants, sothe plants did not develop any specific mechanisms to translocate Cr(III) from root toshoot [53].

3.7. Chromium Tolerance of the Wetland Plant Species

In this study, L. fusca, C. laevigatus, P. australis, and B. mutica did not show visibletoxicity symptoms compared to other wetland plants. However, in C. indica, T. domingensis,C. citratus, C. dactylon, P. purpureum, and P. dilatatum, leaves and some stem parts died at ahigh concentration of Cr (30 mg L−1). The high Cr level significantly (p < 0.05) decreased thestress tolerance index (STI; %) of wetland plant species (Table S3, Supplementary Information).

The lowest value (33%) of shoot length stress tolerance index (SLSTI) was recordedat 30 mg L−1 for C. indica and the highest value (98%) at 15 mg L−1 for L. fusca. Similarly,the highest RLSTI (98%) was measured for B. mutica at 15 mg L−1 and the lowest (33%)for C. citratus at 30 mg L−1. The SFSTI and RFSTI of all wetland plant species significantly(p < 0.05) decreased as the Cr level increased in VF-CWs. The maximum SDSTI value (97%)was recorded for C. laevigatus at 15 mg L−1 and the minimum (36%) for P. dilatatum at30 mg L−1. Similarly, the RDSTI of all wetland plant species decreased as Cr concentrationincreased in VF-CWs. The excessive metals translocation into old leaves before their

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shedding and detoxification by plant roots may also be considered a mechanism of planttolerance to metals [54]. A study on stress tolerance revealed that the tolerance mechanismhelps plants maintain growth even in the presence of toxic metals concentration [29].

3.8. Mineral Nutrients Content in Root and Shoot of Wetland Plants

Various mineral nutrients (Fe, Zn, Mn, Ca, K and Na) were also analyzed in all wetlandplant species (Figures S1 and S2, Supplementary Information). Across all plant species,shoots nutrient elements data indicated that Fe, Zn, Mn, Ca, K, and Na contents decreasedsignificantly (p < 0.05) (20–100%, 19–100%, 1.4–100%, 5.5–75%, 5.4–55%, and 22–100%,respectively) compared to their controls. In the case of root nutrient concentration of plants,Fe, Zn, Mn, Ca, K, and Na contents decreased significantly (p < 0.05) (31–100%, 20–100%,34–100%, 7.5–67%, 1.4–56%, and 27–100%) at both Cr levels compared to controls.

The nutrient concentration (Fe, Zn, Mn, Ca, K, and Na) in root and shoot of all the wet-land plants decreased with an increase in Cr level from 15 to 30 mg/L (Figures S1 and S2,Supplementary Information). Wetland plants do not have any specific mechanism for Cruptake, so plants uptake Cr using different pathways. The uptake of Cr is through passiveand active mechanisms performed by carriers to uptake various essential elements likesulfate [55]. Due to the structural similarity of Cr(VI) with sulfate and phosphate, its uptakeby roots involves sulfate or phosphate transporters that interfere with the nutrients uptakeand translocation mechanisms of plants [6]. The gradual decrease in the essential nutrientsuptake or translocation could be due to competitive binding Cr potential with carrierchannels and reduced plasma membrane H+ATPase activity [56]. Chromium exposuremay remove the essential nutrients from the binding sites in the plant body. However,Cr is reported to play an antagonistic role in the translocation and uptake of essentialnutrients [57].

Chromium also competes with S, P, and Fe for carrier binding during uptake andreduces the uptake of essential elements [37,58]. Due to the similar ionic form, Cr(VI)also inhibits the absorption of certain essential elements such as Fe, K, Mg, Mn, P, andCa by plants and decreases their uptake [59]. High Cr concentration reduced the uptakeof essential minerals like Fe, Mg, P, and Ca by binding on the sorption sites and forminginsoluble complexes with minerals [37].

3.9. Principal Component Analysis (PCA)

Recently, multivariate analysis has attained major importance in finding out possiblerelationships and trends among data variables [60]. The principal component analysisconsiders the correlation and variance of various response attributes concerning inputs [61].In this experiment, multivariate analysis divided response variables (wetland plants) intovarious groups for Cr remediation (Figure S3, Supplementary Information). The metalconcentration in wetland plants was grouped to explain a similar trend in response to Crstress. However, total Cr content was grouped with their species, explaining an enhance-ment in their activities with Cr treatment. The other variables’ responses (pH and Eh) werenot grouped in the PCA because of their different responses concerning Cr treatments.However, in roots and shoots, Cr contents clustered together, which may be due to theirsimilar effects under Cr stress. Many previous studies have also reported the PCA correla-tion to trace various metal concentrations in plant parts [62–64]. The relationship betweenvarious wetland plant response attributes and applied Cr contamination was found in someattributes using the Pearson correlation matrix (Table S4, Supplementary Information).

According to multivariate analysis, the attributes which combined provided a mod-erate to a strong linear correlation relationship. This evaluation showed that the overallimpact of various Cr treatments on wetland plant responses varied from each other andfrom control treatment. This also showed that under specific conditions, other simplestatistical analyses might not explain any significant difference between various treatments.Still, the PCA and correlation analysis separates them depending on their overall effect onvarious response attributes. Therefore, this analysis may be preferred over other statistical

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analyses under specific conditions where huge variation in the data set and complicity intreatments do not show clear linear trends.

3.10. Significance in Environmental Risk Reduction of Chromium

The use of Cr-contaminated wastewater has raised health and environmental concerns.Specifically, these concerns relate to treating Cr-contaminated water using CWs because ofits cost-effectiveness and simplicity of operation. The utilization of contaminated water hasincreased because of the global water scarcity, and around half of the world populationis likely to experience water stress by 2030 [65]. More than 70% of water in the world isused for agricultural irrigation purposes. Therefore, the application of treated wastewaterfor irrigation has a huge potential to relieve water resources pressure. Our study showedthat the wetland plant species could reduce the environmental risk of Cr on land andirrigation water in crops by reducing the concentration of contaminated water. Amongthe plant species studied, L. fusca has the highest ability to reduce total Cr concentrationfrom 30 mg L−1 to 2.3 mg L−1 after one month of Cr stress (Table 1). If used for morethan one month, Cr levels would likely reduce and meet the criteria of national andinternational agencies’ recommended limits for the safe reuse of Cr-contaminated water forirrigation purposes.

L. fusca reduced Cr(VI) concentration to 0.01 mg L−1, which is less than the safe limit

of Cr(VI) in wastewater set by WHO, USEPA, and NEQS (0.05, 0.05, and 0.25 mg L−1,respectively) (Table S5, Supplementary Information) [21,66,67]. In this study, L. fuscaremoved 92% of Cr from water within 30 days of Cr exposure. Therefore, the data in thisstudy indicated that the use of the wetland plants, especially L. fusca, for a long period(about 33 days), may remove 100% of Cr and make the Cr-contaminated water safe foragriculture purposes and reduce the environmental risk of Cr accumulation in soil, water,food, and humans.

3.11. The Fate of Wetland Plants Used for Cr Remediation

The challenge of proper management and disposal of wetland plants biomass used inCWs for the Cr remediation is an important aspect that should be considered with cautionbecause its mishandling may cause secondary contamination. As a result, the plant wastematerial with high total metals content will generally need to be disposed of in a confinedand controlled manner [68]. The other alternative is the incineration of used wetland plantsand disposal of the ash safely in specialized dumps or may be used for Cr recovery andreuse in the relevant industrial sector. The incineration is feasible, environmentally sound,and economically acceptable, and can be used to reduce the secondary pollution burdenfrom Cr-containing plant biomass [26].

4. Conclusions

At low Cr levels, the wetland plant species removed 47% to 92% of total Cr from thewater, and at a high Cr level, it spanned 36% to 92%, with the maximum Cr removal (92%)potential of L. fusca. At both levels of Cr, the BAF of B. mutica (22 and 24, respectively) andL. fusca (29 and 22, respectively) were significantly higher than the other wetland plants.The TF of Cr in the studies of wetland plants had values < 1 suggesting lower translocationfrom roots to shoots. High Cr level significantly (p < 0.05) decreased the STI of all thewetland plants, with the maximum STI obtained for L. fusca (97%). Leptochloa fusca showedthe highest Cr removal (92%) coupled reduction of Cr(VI) to Cr(III), followed by Brachiariamutica. The findings of this study show that VF-CWs with suitable plant species (especiallyL. fusca) is a suitable option for the remediation of Cr-contaminated water.

Future research should be focused on understanding the secondary pollution riskof wetland plants after harvesting. There is a need to assess the life cycle of VF-CWs toensure the total time required of VF-CW systems and investigate the life cycle costs ofCWs on a large scale for wastewater treatment. The emission of greenhouse gases duringCr-contaminated wastewater treatment in VF-CWs needs to be monitored, and how to

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overcome this problem for the sustainable use of CWs under changing climate situationsmust be determined. Also, future research on engineering parameters (hydraulic residencetime and flow rate) should be considered when testing the Cr removal efficiency of CWs.

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su14095230/s1. Table S1: Effect of chromium treatments onpH and redox potential of water in constructed wetlands planted with ten wetland plant species.Values are presented as mean ± standard deviation of three replicates (n = 3). Table S2: Chromiumbioaccumulation factor (BAF) and translocation factor (TF) of wetland plant species. Table S3:Stress tolerance index (%) of wetland plant species growing in CWs fed with Cr-contaminatedwater. Table S4: Pearson correlation relation of various attributes. Table S5: Comparison of Crconcentration present in CWs vegetated with ten wetland plant species and its safe limits given byvarious national and international agencies for environmental risk of Cr. Figure S1: Iron, Na, andK concentrations (a, c, e) root, and (b, d, f) shoots of ten wetland plant species grown in VF-CWscontaining Cr-contaminated water (Cr 0, 15, and 30 mg L-1 in three replicates). Error bars show ±S.E of means of three replicates (n = 3). Similar bars (filled with similar color) labeled with differentalphabets are significantly different from each other (Tukey’s test; p < 0.05). Figure S2: Calcium,Mn, and Zn concentrations in (a, c, e) roots, and (b, d, f) shoots of ten wetland plant species grownin VF-CWs containing Cr-contaminated water (Cr 0, 15, and 30 mg/L in three replicates). Errorbars show ± S.E of means of three replicates (n = 3). Similar bars (filled with similar color) labeledwith different alphabet letters are significantly different from each other (Tukey; p < 0.05). Figure S3:Principle component analysis of water and pant samples taken from CWs vegetated with ten wetlandplant species.

Author Contributions: Conceptualization, reading, editing and finalizing the paper: I.B., N.K.N.,M.A. and F.Y.; Experimental work: F.Y. under supervision of I.B., M.A. and N.K.N.; Funds andResources, I.B. and N.K.N.; Z.A. read and improved the paper. All authors have read and agreed tothe published version of the manuscript.

Funding: This research was funded by Higher Education Commission (Project Nos. 6425/Pun-jab/NRPU/R&D/HEC/2016 and 6396/Punjab/NRPU/R&D/HEC/2016), Pakistan.

Data Availability Statement: Not applicable.

Acknowledgments: The authors are thankful to Higher Education Commission (Project Nos. 6425/Pun-jab/NRPU/R&D/HEC/2016 and 6396/Punjab/NRPU/R&D/HEC/2016), Pakistan, for providingfinancial support. Irshad Bibi acknowledges the support from COMSTEQ-TWAS research grant 2018(18-268 RG/EAS/AS_C).

Conflicts of Interest: The authors declare no conflict of interest.

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