Comparative study of Zn-phytoextraction potential in guar (Cyamopsis tetragonoloba L.) and sesame (Sesamum indicum L.): tolerance and accumulation

Abstract

Phytoextraction is a plant-based technique for removing heavy metals from polluted soil. The experiment reported in this paper was undertaken to study the Zn phytoextraction potential of Cyamopsis tetragonoloba in comparison with Sesamum indicum in the framework of a pot experiment. Plants were subjected to six Zn concentrations (0, 50, 100, 200, 300, 400 mg kg⁻¹ soil) for 90 days to investigate Zn tolerance and accumulation. Results demonstrated that, at higher Zn levels, root, shoot lengths, biomass and chlorophyll content were all significantly reduced (p < 0.05). A steady increase in Zn accumulation with increasing concentration in soil was observed for all treatments. Both plant species had relatively high Zn tolerance and accumulation capacity, with C. tetragonoloba being more tolerant and having higher Zn accumulation than S. indicum. At 400 mg Zn kg kg⁻¹, accumulation of Zn in C. tetragonoloba was highest in the root (439.33 mg kg⁻¹) followed by stem (436.00 mg kg⁻¹), leaf (40.67 mg kg⁻¹) and pod (11.33 mg kg⁻¹). Considering the rapid growth, high biomass, tolerance, accumulation efficiency, bioconcentration factor (BCF), bioaccumulation coefficient (BAC) and translocation factor (TF) (all greater than 1) established C. tetragonoloba as a potential candidate plant for Zn phytoextraction.

Keywords:

• Soil pollution
• zinc
• phytoextraction
• accumulation
• translocation

1. Introduction

Soil pollution by heavy metals released from anthropogenic activities is a worldwide environmental problem. Toxic metals have made their entry into agricultural soils primarily because of rapid industrialization, inappropriate utilization and disposal of toxic metal containing wastes, excessive use of fertilizers and pesticides (Amel et al., 2016; Dmitry, Alexander, Naser, Ahmad, & Eduarda, 2015). The most common heavy metal contaminants frequently released in the environment are Cd, Hg, Pb, Cr, Ni, Co, Cu and Zn (Subhashini, Swamy, & Hema Krishna, 2013). Their presence in the soil may leads to harmful effects on both the ecosystem and living organisms (Demim et al., 2014).

Among heavy metals, zinc (Zn) is an essential trace element belongs to the list of transition metals and stands 24th among the most abundant elements on the earth’s crust. In soil, Zn mainly occurs as sulphide (Neha, Hari, & Balwinder, 2016; Pratap et al., 2014). Agricultural soil is contaminated with zinc by natural and anthropogenic activities including mining and industrial processes and agricultural practices. The use of commercial fertilizers, pesticides, liming materials, or manures, being added to Zn-deficient agricultural soils to achieve enhanced plant growth and productivity, has become major factors contributing elevated levels of Zn in world agricultural soils (Alloway, 2013; Douglas et al., 2017; Kabata-Pendias, 2011).

Like other heavy metals, Zn is non-biodegradable contaminant persist in the environment, inevitably accumulate and eventually get into the food chain (Sveta et al., 2016). The toxicological impact of Zn depends on their concentration in the environmental context. At low levels, Zn plays an important role in several metabolic processes of plant; activate enzymes, involved in protein synthesis and in carbohydrate, nucleic acid and lipid metabolism (Demim, Drouiche, Aouabed, & Semsari, 2013; Fässler, Robinson, Stauffer, & Gupta, 2010) but at high doses Zn adversely affects plants and cause alterations in various morphological and physiological processes. Considering known toxic effects of Zn in plants such as inhibited plant growth and development, elevated oxidative stress, and impaired cellular metabolism, their eventual impact on photosynthesis and biosynthesis of chlorophyll as well as plant productivity (Pratap et al., 2014), root growth and mitotic efficiency, chromosomal aberrations and nutrient accumulation abnormalities (Radha, Srivastava, Solomon, Shrivastava, & Chandra, 2010), sustainable minimization of Zn in agricultural soils is imperative.

Soil remediation by various physicochemical methods has been well documented in literature. However, remediation of heavy metal polluted soils by traditional physical and chemical methods are not suitable for agricultural lands, required large investments and technological resources (Oh, Li, Cheng, Xie, & Yonemochi, 2013). Thus, a plant-based technology has emerged as a cure for the sustainable control of elevated levels of various toxic metals in soils and received great attention in recent years. Phytoextraction refers to the efficient use of metal-accumulating plants to transport and concentrate metals from soil into the harvestable parts of roots and shoot biomass, (i.e. root and shoot) and appears to be a promising, cost-effective technology for the remediation of metal-polluted soils (Amanullah et al., 2016).

Literature has reported about 500 vascular plant species for metal absorption from contaminated soil. The heavy metal uptake, accumulation, mechanism of metal concentration, exclusion and compartmentation vary among different plant species and also between various plant parts (Sharma, Singh, & Manchanda, 2014; Singh & Agrawal, 2010). However, the use of field crop plants for the management of long-term pollutant dispersion has been given much emphasis in this perspective to crop plants from Brassicaceae followed by Fabaceae, Asteraceae and Poaceae (Zaidi, Wani, & Khan, 2012).

Guar (Cyamopsis tetragonoloba L.) is one of important annual legume crops, belonging to the family Fabaceae. The endosperm of guar seeds contains gum, which is gaining importance as non-food item (Ashraf, Akhtar, Sarwar, & Ashraf, 2002). Recently, the oil industry has started using guar gum in hydraulic fracturing in which high pressure is used to crack rock. Guar gum in the fracking fluid increases its viscosity and improves the efficiency of natural gas extraction. The increased use of guar gum in oil fracking has boosted the demand of guar worldwide (Abidi et al., 2015; Deepak, Sheweta, & Bhupendar, 2014). A key feature of legumes as a resource for phytoremediation is their role in providing additional N-compounds to the soil, thus improving its fertility and ability to support biological growth (Xiuli et al., 2013).

On the other hand, sesame (Sesamum indicum L.) is an economically important oil seed crop of family Pedaliaceae, planted in arid and semi-arid regions of the world (Elleuch, Besbes, Roiseux, Blecker, & Attia, 2007). Sesame is widely used in pharmaceutical industry in many countries because of its high oil, protein and antioxidant contents (Saydut, Duz, Kaya, Kafadar, & Hamamci, 2008). Sesame seed has one of the highest oil contents of any seed and is considered to be the oldest oilseed crop known to man, highly resistant to drought and has the ability of growing where most crops fail (Dawodu, Ayodele, & Bolanle-Ojo, 2014). Sesame seeds have been used as an alternative feedstock for the production of a biodiesel fuel. The methyl ester of the sesame plant can successfully be used as petrodiesel (Ahmad et al., 2011). Viscosity and density of methyl esters of sesame seed oil are found to be very close to that of diesel. The heating value (7.5%), calorific value (5.4%) of biodiesel is found to be slightly lower than that of diesel (Saydut et al., 2008).

This experiment was set up to identify a plant species that could tolerate concentrations of Zn in soil. C. tetragonoloba and S. indicum are plant species with rapid growth and good biomass production. It was hypothesized that plant species with high tolerance could then be tested for their phytoextraction potential. Thus, the aims of the present investigation are (i) to study the effect of Zn on plant growth and physiology and, (ii) to evaluate the phytoextraction potential of C. tetragonoloba and S. indicum, with respect to Zn concentrations.

2. Material and methods

2.1. Seed collection and sterilization

Seeds of the guar variety BR-99 were collected from the Institute of Fodder Research Program and sesame variety TH-6, from the Institute of Oilseeds Research Program, National Agricultural Research Centre (NARC), Islamabad (Table 1). In order to avoid any microbial contamination, seeds were surface sterilized with 0.1% HgCl₂ for 10 min and washed 7 times with sterilized water (Pourakbar, Khayami, Khara, & Farbidina, 2007).

Table 1. Plant species selected for pot experiment.

No.	Botanical name	Vernacular name	Family
1	Cyamopsis tetragonoloba L.	Guar	Fabaceae
2	Sesamum indicum L.	Sesame	Pedaliaceae

2.2. Soil collection and preparation

Soil samples of sandy-loam soil were collected from uncontaminated agricultural fields located in Jamshoro, Sindh, Pakistan, at depth of 0–15 cm using hand spade. Equidistant (2 m) collected samples were homogenized to prepare one bulk sample. For the greenhouse experiment, soil was air dried (at room temperature for 15 days) and ground to a final particle size of 2 mm. To enhance soil porosity, sand was mixed in 3:1 ratio with sandy loam soil.

2.3. Soil measurements

For soil characterization, the soil samples were air dried at room temperature, ground in a ceramic mortal to pass through a 2-mm mesh sieve, homogenized, and stored in polyethylene bags for subsequent analysis (Table 2). Soil pH was measured with a pH-meter (InoLab-WTB GmbH; Weilheim, Germany) using glass electrode at the 1:2 (w/v) ratio of soil to water suspension (Rachit, Verma, Meena, Yashveer, & Shreya, 2016). The electrical conductivity (EC) was measured with an electrical conductivity meter (WTW – 330i) at the 1:2 (w/v) ratio of soil to water suspension (Rachit et al., 2016). Organic matter (OM) and organic carbon (%) were measured according to Walkley and Black (chromic acid titration) method (Fanrong et al., 2011).

Table 2. The properties of soil.

Parameter	Values obtained
pH	6.89^b ± 0.04
E.C. (μS cm^−1)	1662^a ± 11
Organic carbon (%)	2.20^b ± 0.04
Organic matter (%)	3.79^b ± 0.02
Zinc total (mg kg^−1)	N.D.*

Notes: Similar letters in same column are statistically non-significant according to Duncan’s Multiple Range Test (p < 0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means; *N.D. = Not detected.

2.4. Preliminary screening

In order to select the Zn concentration for treatments, various doses of ZnSO₄.7H₂O (0, 50, 100, 200, 500, 700, 1000, 1500 and 2000 mg kg⁻¹) were tried in the preliminary screening of the C. tetragonoloba and S. indicum for 20 days. Based on the Zn toxicity symptoms and morphological growth of the seedlings, the following doses (0, 50, 100, 200, 300, 400 mg kg⁻¹) were finally selected (Table 3).

Table 3. Treatment levels selected for pot experiment.

Heavy metal	Salt used	Treatments (mg kg^−1 soil)
		T1	T2	T3	T4	T5	T6
Zinc (Zn)	ZnSO4.7H2O	0	50	100	200	300	400

2.5. Pot experiment

Plastic pots were filled with 5 kg sieved soil, after which soil was artificially spiked with Zn (aqueous solution) using ZnSO₄.7H₂O salt to each pot in increasing concentrations (50, 100, 200, 300, 400 mg kg⁻¹), each with three replicates and kept for 2 weeks to attain equilibrium. The clean soil without Zn spiking was used as control. Pots were arranged in a completely randomized design. After fifteen days of equilibration, 20 surface-sterilized seeds were sown per pot. One week after seed germination, plants were thinned to 5 per pot. A plastic tray was kept below the treatment pot to collect any leachate, which was returned to the pots at next watering. The experiment was conducted in a greenhouse for 90 days. Any symptoms of metal toxicity exhibited by plants were visually noted during the experimental period. Plants were harvested 12 weeks after germination. Soil samples (in triplicate) were also collected for analysis of Zn content by an Atomic Absorption Spectrophotometer (AAnalyst 800, Perkin Elmer, USA). Plant growth and biochemical parameters (chlorophyll) were also measured.

2.6. Germination percentage

The germination percentage, expressed as percentage of germinated seeds to the total number of viable seeds, is calculated by the following equation: (Talebi, Nabavi, & Sohani, 2014)(1)

2.7. Morphological parameters

Plants were taken from each replicate to measure morphological parameters. Root length, shoot length were measured of each replicate with the help of scale. Root and shoot fresh weights were also measured with the help of analytical balance. Plant samples were air dried for one week. Then, they were oven-dried at 80 °C to a constant weight and their dry weights recorded

2.8. Estimation of chlorophyll contents

Photosynthetic pigments, in fully expanded leaves from each treatment, were extracted using 0.5 g of fresh material, ground with 10 mL of 80% aqueous acetone. After filtering, 1 mL of the suspension was diluted with a further 2 mL of acetone, and optical density were determined with a UV-Visible spectrophotometer (Biochrom Libra S22), using two wavelengths (663 and 645 nm) against blank. Chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll contents (mg g⁻¹ f.w) were obtained by calculation, following the method of Arnon (1949).(2) (3) (4)

2.9. Determination tolerance index

Tolerance Index (TI) is expressed as the ratio between the growth parameters (root/shoot length, root/shoot fresh and dry weight) of the plants in contaminated soil in relation to the growth parameters of plants from non-polluted soil calculated by following equation: according to Chen et al., (2011)(5)

2.10. Quality control and quality assurance

All the glassware used during the present experimentation was of high quality, acid resistant Pyrex glass. The analytical grade reagents with a certified purity of 99% and stock metal standard solution (1000 ppm) for AAS analysis were procured from E. Merck (Germany). Working standards were prepared by appropriate dilutions of stock standard solutions with double-distilled water.

2.11. Plant samples preparation, digestion and Zn determination

To determine Zn accumulation in different plant tissues (i.e., root, stem, leaf and pod), harvested plants were washed thoroughly with running tap water, and then deionized water to remove adhered soil particles, oven-dried at 80 °C till constant weight. The oven-dried samples were ground thoroughly using a grinder and passed through a 1.0-mm mesh sieve. The ground plant samples (0.5 g) were digested by HNO₃ and HClO₄ mixed at a ratio of 3:1 (v/v) according to the protocols devised by Altaf et al., (2017). After di-acid digestion, the volume was completed to 50 mL by adding distilled water. The solution was filtered through Whatman’s filter paper.

The quantification of zinc (Zn) in respective tissues was carried out using atomic absorption spectrometer equipped with a zinc cathode lamp, under optimum analytical conditions for the estimation of zinc. The optimum conditions for AAS used throughout these studies given in Table 4. The standard calibration method was adopted for the quantification of results and triplicate samples were run to insure the precision of quantitative results.

Table 4. Measurement conditions of F-AAS for zinc (Zn) determination.

Parameters	Values
Wave length (nm)	213.9
Hollow cathode lamp current (mA)	5.0
Flame type	Air-C2H2
Background correction	On
Slit-width (nm)	1.0
Flame condition	Oxidizing
Expansion factor	1

The Zn concentration and accumulation in plant root and shoot were calculated by the following formula: (Muhammad et al., 2014)(6) (7)

2.12. Soil sample preparation, digestion and Zn determination

Soil samples were air dried at room temperature, ground, homogenized, and stored in polyethylene bags for subsequent analysis. Digestions of soil and plant samples were done using aqua regia method (Ogunkunle et al., 2017). To quantify the Zn content in soil, 1 g soil sample was digested using a wet digestion method with HNO₃ and HCl (3:1 ratio v/v) and heated on a hot plate for 2 h at a temperature of 110 °C until the solution becomes clear. After cooling, the volume was completed to 50 mL by adding distilled water. The solution was filtered through Whatman’s filter paper. The filtrate was analysed for Zn content by Atomic Absorption Spectrophotometer.

2.13. Evaluation of phytoextraction efficiency

Phytoextraction potential of plants is influenced by the mobility and availability of contaminants in soil and plants. To evaluate the phytoextraction potential of C. tetragonoloba and S. indicum, the following factors were calculated based on simple ratios of contaminant concentration in plant parts and growth matrix (Rohan, Mayank, João, & Paul, 2013).

2.13.1. Bioconcentration factor (BCF)

The bioconcentration factor (BCF) was calculated as the Zn concentration ratio in plant roots to soil, given in equation (8⁽³⁾(3) )(8)

2.13.2. Bioaccumulation coefficient (BAC)

The bioaccumulation coefficient (BAC) was calculated as a ratio of Zn in shoot to that in soil, given in Equation (9⁽⁴⁾(4) )(9)

2.13.3. Translocation factor (TF)

The translocation factor (TF) was determined as a ratio of heavy metals in plant shoot to that in plant root, given in equation (10⁽⁵⁾(5) )(10)

2.14. Statistical analysis

All experiments were conducted with three replicates and the data collected were analysed statistically using PASW^® Statistics 18 (SPSS Inc., Chicago, IL, USA). To compare the means of the treatments, analysis of variance (ANOVA) was performed followed by Duncan’s multiple range post hoc tests at significance level of p < 0.05 to observe significance difference among means.

3. Results and discussion

3.1. Soil characterization

The soil was sandy loam with slightly acidic to neutral pH (6.89), organic carbon (2.20%), organic matter contents (3.79%) and electrical conductivity (1662 μS/cm). Among soil properties, soil pH was found to play the most important role in determining metal speciation, solubility from mineral surfaces and eventual bioavailability of metals due to its strong effect on solubility and speciation of metals both in the soil as a whole and particularly in the soil solution. The mobility and bioavailability of heavy metals increased with decreased soil pH, whereas organic matter supplies organic chemicals to the soil solution that can serve as chelates and increase metal mobility and availability to plants (Fanrong et al., 2011). Such factors may act individually or in combination with each other and may alter the soil behaviour of the zinc present, as well as the rate of uptake by plants. So, in accordance with soil properties, Zn is more mobile and more bioavailable.

3.2. Zn-induced phytotoxicity

Gradual increase in Zn concentration significantly (p < 0.05) reduced all tested growth and biochemical parameter (chlorophyll content) in two plant species. In the current investigation, the seed germination of C. tetragonoloba and S. indicum was not influence significantly (p > 0.05) by Zn concentrations (Figure 1). Zn is an essential micronutrient that promotes seed germination and growth at optimal concentration but at higher levels inhibits germination and growth (Kabata-Pendias, 2011). The seeds of guar and sesame were able to germinate in the presence of low to moderate concentrations of Zn in soil but germination reduced at 400 mg Zn kg⁻¹ as compared to control. According to Li, Khan, Yamaguchi, and Kamiya (2005), seed is the only stage in the life cycle of plants well protected against the metal stress. The seed coat acts like a barrier between the embryo and the environment; protects the embryo against the heavy metals toxicity.

Figure 1. Effect of Zn stress on germination of C. tertragonoloba and S. indicum seeds after 7 days in soil medium with varying concentrations of Zn.

Notes: Similar letters are statistically non-significant according to Duncan’s Multiple Range Test (p<0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means.

Seedling’s height (root and shoot length) is also among primary determinants of plant growth. Under Zn stress the growth was significantly (p < 0.05) affected in terms of root and shoot lengths (Table 5). In C. tetragonoloba and S. indicum, the longest roots (16.07 cm and 12.38 cm) and shoots (117.70 cm and 120.00 cm) were observed in control treatments with 0 mg Zn kg⁻¹, respectively. At 400 mg Zn kg⁻¹ concentration, root length decreased by 12.24 cm and 9.65 cm while shoot length reduced by 90.88 cm and 86.32 cm in both C. tetragonoloba and S. indicum, respectively. Growth inhibition is a general phenomenon associated with most of the heavy metals. Higher level of Zn in the soil directly influences root growth and specific superficial area, decreasing the capacity of absorption of water and nutrients. Toxic effect of Zn in reduced seedling height is mainly due to the interference with metabolic activities of the plant (Mukhopadhyay et al., 2013) ultimately leading to reduction in growth of the plant species, as reported by Luo et al. (2010).

Table 5. Zn-induced phytotoxic effects on growth parameters of Cyamopsis tetragonoloba L. and Sesamum indicum L.

	Zn applied (mg kg^−1)	Root length (cm)	Shoot length (cm)	Root fresh weight (g plant^−1)	Shoot fresh weight (g plant^−1)	Root dry weight (g plant^−1)	Shoot dry weight (g plant^−1)
C. tetragonoloba	0	16.07^a ± 0.31	117.70^a ± 1.06	13.30^a ± 0.84	38.47^a ± 0.55	8.07^a ± 0.15	21.99^a ± 0.99
	50	15.15^a ± 0.77	116.28^a ± 1.53	11.33^b ± 0.94	37.08^a ± 8.29	7.61^a ± 0.09	17.93^b ± 0.12
	100	14.84^ab ± 0.15	110.31^b ± 2.91	10.22^bc ± 0.38	31.53^ab ± 5.54	7.07^a ± 0.07	16.63^bc ± 0.57
	200	13.62^bc ± 0.15	107.10^b ± 2.46	9.76^c ± 0.34	28.38^b ± 0.68	5.51^b ± 0.88	14.70^bc ± 2.49
	300	13.16^cd ± 0.15	91.80^c ± 2.22	8.59^d ± 0.72	27.07^b ± 2.64	4.51^bc ± 0.88	13.33^c ± 3.05
	400	12.24^d ± 1.53	90.88^c ± 3.06	7.63^d ± 0.43	24.17^b ± 1.05	3.71^c ± 1.22	9.67^d ± 2.08
S. indicum	0	12.38^a ± 0.43	120.00^a ± 1.00	12.07^a ± 0.62	37.33^a ± 0.58	7.07^a ± 0.12	20.00^a ± 1.00
	50	11.38^ab ± 0.29	104.83^b ± 1.44	10.24^ab ± 1.95	30.67^b ± 0.58	6.03^b ± 0.06	14.67^b ± 0.58
	100	10.94^bc ± 1.44	104.40^b ± 1.44	8.66^bc ± 2.19	28.37^c ± 1.58	5.13^c ± 0.23	12.67^c ± 0.58
	200	10.18^bc ± 0.65	97.42^c ± 1.55	7.84^bc ± 1.31	23.67^d ± 1.53	4.17^d ± 0.29	10.67^d ± 0.58
	300	9.94^c ± 0.29	87.11^d ± 2.59	7.51^bc ± 0.63	21.00^e ± 1.00	3.43^e ± 0.40	8.13^e ± 0.12
	400	9.65^c ± 0.14	86.32^d ± 1.51	7.25^c ± 1.48	18.00^f ± 1.00	2.97^f ± 0.06	6.33^f ± 1.24

Notes: Similar letters in same column are statistically non-significant according to Duncan’s Multiple Range Test (p < 0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means.

Zn contamination showed significant (p < 0.05) negative impacts on both fresh and dry biomass of C. tetragonoloba and S. indicum (Table 5). Compared to control treatments, Zn stress at 400 mg kg⁻¹ reduced root fresh weight (7.63 g plant⁻¹ and 7.25 g plant⁻¹) and shoot fresh weight (24.17 g plant⁻¹ and 18.00 g plant⁻¹) in C. tetragonoloba and S. indicum, respectively. The dry biomass follows the same trend as fresh weight. Compared to control treatments, Zn stress at 400 mg kg⁻¹ reduced root dry weight from (3.71 g plant⁻¹ and 2.97 g plant⁻¹) and shoot dry weight (9.67 g plant⁻¹ and 6.33 g plant⁻¹) in C. tetragonoloba and S. indicum, respectively. Plant biomass is a good indicator for characterizing the growth performance of plants in the presence of heavy metal. Decrease in plant biomass may be associated with disturbed metabolic activities due to reduced uptake of essential nutrients when grown under Zn stress (Li et al., 2012). Plants produce high aboveground biomass and possess the ability to accumulate heavy metals. This ability is used for phytoextraction purposes including removal of heavy metals from polluted soil. Our results for Zn phytotoxcity were evident from stunted growth and reduced fresh and dry weights that are in consonance with the same phenomenon observed in Jatropha seedlings under Zn stress (Luo et al., 2010).

Chlorophyll contents decreased significantly (p < 0.05) with gradual increase in Zn concentration from 0 to 400 mg kg⁻¹ (Figure 2). In C. tetragonoloba and S. indicum, the maximum amount of chlorophyll contents were measured at 50 mg kg⁻¹, while the lowest concentration of chlorophyll a (0.76 mg g⁻¹ and 0.68 mg g⁻¹), chlorophyll b (0.25 mg g⁻¹ and 0.21 mg g⁻¹) and total chlorophyll (1.01 mg g⁻¹ and 0.89 mg g⁻¹) was at 400 mg Zn kg⁻¹, respectively. Reduction in chlorophyll contents may be due to competition of Zn with iron for binding with protoporphyin, a main precursor of chlorophyll synthesis. Production of reactive oxygen species (ROS) upon Zn exposure inhibits chlorophyll production by damaging the pigment–protein complexes located in thylakoid membranes (Sagardoy et al., 2010; Vassilev, Perez-Sanz, Cuypers, & Vangronsveld, 2007).

Figure 2. Effect of Zn stress on photosynthetic pigments chlorophyll-a (a) chlorophyll-b (b) and total chlorophyll (a + b) (c), on C. tertragonoloba and S. indicum after 90 days growth in soil medium with varying concentrations of Zn.

Notes: Similar letters are statistically non-significant according to Duncan’s Multiple Range Test (p < 0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means.

Tolerance indices (TIs) were also affected by Zn toxicity. Both plant species had different tolerance indices (TIs) under Zn stress (Table 6). In this study, C. tetragonoloba was more tolerant to Zn stress than S. indicum. At 400 mg kg⁻¹ Zn treatment, C. tetragonoloba and S. indicum had the TIs for root lengths (76.09% and 88.00%) and shoot lengths (77.23% and 71.93%), root fresh weights (57.68% and 60.15%) and shoot fresh weights (62.88% and 48.20%), root dry weights (46.11% and 42.00%) and shoot dry weights (43.81% and 31.53%), respectively. Metal tolerance of plant is a prerequisite for studying the plant–metal interactions before application for phytoextraction. Plant tolerance to heavy metal stress is estimated based on their root and/or shoot growth inhibition by the metal present in a medium. Growth inhibition is a common response to heavy metal stress and is also one of the most important agricultural indices of heavy metal tolerance. According to Audet and Charest (2007), if TI values less than 1, this indicates that the plant suffered a stress due to metal pollution with a net decrease in biomass. By contrast, if TI values greater than 1 suggest that plants have developed tolerance with a net increase in biomass (hyper accumulator). If TI values equal to 1, the plant is unaffected by metal pollution, indicate no difference relative to control treatments.

Table 6. Effect of Zn stress on the tolerance indices (TIs) of Cyamopsis tetragonoloba L. and Sesamum indicum L.

Plant species	Zn applied (mg kg^−1)	Tolerance indices
		Root length (%)	Shoot length (%)	Root fresh weight (%)	Shoot fresh weight (%)	Root dry weight (%)	Shoot dry weight (%)
C. tetragonoloba	50	94.25^a ± 2.96	98.79^a ± 0.85	85.35^a ± 7.94	96.57^a ± 22.70	94.28^a ± 1.38	81.67^a ± 4.13
	100	92.39^ab ± 0.81	93.72^b ± 1.92	77.15^ab ± 6.60	82.08^ab ± 15.33	87.66^a ± 0.80	75.75^ab ± 4.92
	200	84.79^bc ± 2.57	90.99^b ± 1.50	73.49^ab ± 2.50	73.78^ab ± 0.81	68.41^b ± 12.23	67.06^ab ± 12.99
	300	81.94 ^cd ± 2.51	77.53^c ± 3.14	64.99^bc ± 9.17	70.45^b ± 7.76	56.02^bc ± 12.02	60.57^bc ± 13.17
	400	76.09^d ± 8.08	77.23^c ± 3.17	57.68^c ± 6.65	62.88^b ± 3.57	46.11^c ± 16.11	43.81^c ± 7.97
S. indicum	50	91.99 ^a ± 5.54	87.37^a ± 1.93	84.51^a ± 12.78	82.15^a ± 1.44	85.40^a ± 1.92	73.54^a ± 6.27
	100	88.71^a ± 14.73	87.01^a ± 1.93	71.44^ab ± 16.28	76.03^b ± 5.16	72.67^b ± 3.99	63.52^b ± 5.78
	200	82.36 ^a ± 8.10	81.18^b ± 0.62	64.79^ab ± 8.77	63.37^c ± 3.42	58.99^c ± 4.65	53.43^c ± 4.05
	300	80.35^a ± 5.13	72.61^c ± 2.75	62.21^ab ± 2.06	56.26^d ± 2.84	48.58^d ± 5.71	40.74^d ± 2.15
	400	88.00^a ± 3.89	71.93^c ± 0.98	60.15^b ± 12.04	48.20^e ± 2.06	42.00^d ± 1.49	31.53^e ± 5.01

Notes: Similar letters in same column are statistically non-significant according to Duncan’s Multiple Range Test (p < 0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means.

3.3. Zn concentration in plant tissues

The Zn concentration among the different plant tissues (root, stem leaf and pod) of both plant species are presented in Table 7. In C. tetragonoloba, the highest concentration of Zn accumulated in the root: 439.33 mg kg⁻¹ followed by stem: 436.00 mg kg⁻¹, leaf: 40.67 mg kg⁻¹, and pod: 11.33 mg kg⁻¹ at 400 mg Zn kg⁻¹. However, in S. indicum, Zn accumulated primarily in the leaf: 282.33 mg kg⁻¹, with small amount being transferred to root: 273.67 mg kg⁻¹, stem: 121.33 mg kg⁻¹, and pod: 12.33 mg kg⁻¹ at 400 mg Zn kg⁻¹. The high Zn contents in the plant tissues are clearly related to the concentration of metal in the growing environment. Studies have shown the uptake of metals; their partition and translocation to different plant parts, as well as the degree of tolerance to them are dependent on the metal, its availability, the plant species, and its metabolism (Rohan et al., 2013). The Zn accumulation capacity, based on their availabilities in the soil, varies greatly among different plants species and cultivars, and is also affected by various edaphic conditions (Abioye, Ekundayo, & Aransiola, 2015).

Table 7. Zn concentration, bioconcentration factor (BCF), bioaccumulation factor (BAC) and translocation factor (TF) of Cyamopsis tetragonoloba L. and Sesamum indicum L.

Plant species	Zn applied (mg kg^−1)	Zn concentration				BCF	BAC	TF
		Root (mg kg^−1)	Stem (mg kg^−1)	Leaf (mg kg^−1)	Pod (mg kg^−1)
C. tetragonoloba	50	55.33^e ± 2.52	35.33^de ± 4.16	56.00^a ± 1.00	3.03^d ± 1.95	1.11^a ± 0.05	1.89^a ± 0.05	1.72^a ± 0.07
	100	105.67^d ± 0.58	79.33^d ± 2.08	20.33^d ± 4.04	6.33^c ± 1.15	1.06^a ± 0.01	1.06^c ± 0.03	1.00^b ± 0.03
	200	201.67^c ± 15.89	183.33^c ± 4.93	30.33^c ± 3.22	8.67^b ± 0.58	1.01^a ± 0.08	1.11^c ± 0.02	1.11^b ± 0.11
	300	325.33^b ± 17.89	321.00^b ± 0.00	34.33^c ± 0.58	10.33^ab ± 0.58	1.08^a ± 0.06	1.22^b ± 0.00	1.13^b ± 0.06
	400	439.33^a ± 16.50	436.00 ^a ± 6.93	40.67^b ± 0.58	11.33^a ± 0.58	1.09^a ± 0.04	1.22^b ± 0.02	1.11^b ± 0.06
S. indicum	50	33.33^e ± 4.16	12.33^d ± 0.58	39.33^e ± 4.36	6.33^c ± 1.53	0.67^b ± 0.08	1.16^b ± 0.02	1.75^b± 0.18
	100	72.67^d ± 10.97	60.33^c ± 6.03	72.33^d ± 3.22	7.33^c ± 0.58	0.73^b ± 0.11	1.40^a ± 0.07	1.96^a ± 0.39
	200	189.33^c ± 3.06	82.33^b ± 0.58	203.33^c ± 15.28	10.00^b ± 1.00	0.95^a ± 0.02	1.48^a± 0.08	1.56^b ± 0.06
	300	254.33^b ± 4.04	92.67^b ± 4.62	259.33^b ± 8.62	10.67^ab ± 0.58	0.85^a ± 0.01	1.21^b ± 0.04	1.43^b ± 0.04
	400	273.67^a ± 5.51	121.33^a ± 10.50	282.33^a ± 2.52	12.33^a ± 1.53	0.68^b ± 0.01	1.04^c ± 0.03	1.52^b ± 0.06

Notes: Similar letters in same column are statistically non-significant according to Duncan’s Multiple Range Test (p < 0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means.

3.4. Zn accumulation in root and shoot

Beside concentrations, a total amount of metals accumulated in the shoots is considered as the most important parameter to evaluate the potential of phytoextraction in plants (Hanen et al., 2010). A significant rise in Zn accumulation per plant in root and shoot of both the plant species varied with respect to Zn concentrations in soil (Figure 3). In this study, both C. tetragonoloba and S. indicum accumulated more Zn contents in shoot than root. Root accumulation of Zn in C. tetragonoloba was increased from 50 to 400 mg Zn kg⁻¹; however, in S. indicum, the accumulation was increased from 50 to 200 mg Zn kg⁻¹, while decreased at 300 and 400 mg Zn kg⁻¹. The highest value of Zn accumulation in C. tetragonoloba root (1615.51 μg plant⁻¹) was at 400 mg Zn kg⁻¹, while in S. indicum root accumulation (874 μg plant⁻¹) at 300 mg Zn kg⁻¹. Likewise, shoot accumulation of Zn in C. tetragonoloba was increased from 50 to 300 mg Zn kg⁻¹, while decreased at 400 mg Zn kg⁻¹; however, in S. indicum, increased from 50 to 200 mg Zn kg⁻¹, while decreased at 300 and 400 mg Zn kg⁻¹. The highest value of Zn accumulation in C. tetragonoloba shoot (4873.45 μg plant⁻¹) was at 300 mg Zn kg⁻¹, while in S. indicum the highest shoot accumulation (3155.33 μg plant⁻¹) was at 200 mg Zn kg⁻¹. The decrease in root and shoot accumulation is related with high concentration of Zn in soil that affects the plant biomass production. Thus, in order to evaluate the accumulation, it is necessary to take biomass into consideration. Accumulation of elements in plant biomass always depends on both concentration and biomass (Vymazal, 2016).

Figure 3. Accumulation of Zn in root (a) and shoot (b) of C. tertragonoloba and S. indicum after 90 days growth in soil medium with varying concentrations of Zn.

Notes: Similar letters are statistically non-significant according to Duncan’s Multiple Range Test (p < 0.05), Data are means (n = 3 ± SD), a in superscript represent significantly highest followed by later alphabets for lower means.

3.5. Phytoextraction potential

The BCF, BAC, and TF values (Table 7) help to identify the suitability of plants for phytoextraction or phytostabilzation by explaining the accumulation characteristics and translocation behaviours of metals in plants. Plants with BCF, BAC and TF values > 1 are good phytoextractor, suitable for phytoextraction. Plants with BCF > 1 and TF < 1 are good phytostablisor, suitable for phytostabilziation, whilst plants with BCF < 1 and TF < 1 are not suitable for phytoextraction (Rohan et al., 2013; Varun, Ogunkunle, D’Souza, Favas, & Paul, 2017).

In general, both plant species have potential to be used for phytoextraction purpose. But only the plant species with BCF (bioconcentration factor), BAC (bioaccumulation coefficient) and TF (translocation factor) greater than one have the greater potential to be used for phytoextraction. Between tested plant species, C. tetragonoloba had the BCF, BAC and TF values > 1 at all treatments, than S. indicum, indicating that C. tetragonoloba being a high efficiency plant for Zn translocation from root to the shoot would be a good accumulator and a potential candidate suitable for Zn phytoextraction.

4. Conclusions

From this study, it has been concluded that none of the plant species were identified as metal hyperaccumulators. Considering the rapid growth, biomass, accumulation efficiency, adaptive properties, tolerance, restoration potential towards Zn, and being leguminous and fast-growing crop, C. tetragonoloba can be used as an effective tool to decontaminate Zn polluted soils in quick and successive flushes than S. indicum. With application of these plants in the management and remediation of contaminated environment, the great positive characteristics are that the cost is very low in comparison to other physiochemical methods, and can remove pollutants from soil and reduce their movement towards groundwater, sustains the soil properties, and may improve soil quality and productivity. Moreover, these plants can both remediate brownfields and produce valuable biomass, which can bring income for the owners of the contaminated sites. The harvested biomass could then be incinerated and disposed of or the accumulated metal could also be recovered for commercial uses and thus recycled and reused as biofuel. All in all, phytoextraction is a less invasive and cheaper method than standard techniques, as well as more environment-friendly. It would be a reasonable choice to grow these plants and absorb the hazardous materials from the brownfield and the prevention of future brownfields emerging in industrial sectors. Further work is needed to understand the mechanisms of targeted metal absorption and tolerance in plants.

Disclosure statement

No potential conflict of interest was reported by the authors.