Introduction

Increasing urbanization, broad-scale industrialization, and rapid population emergence since the last few decades are considered as dominant factors in deteriorating the quality of the environment and leading to heavy metal contamination in all spheres of the environment (Qu et al. 2011; Ali et al. 2013a). Discharge of heavy metal-enriched industrial effluents to exposed water channels is posing a highly deleterious impact on flora and fauna which is grabbing the scientists’ attention worldwide (Sood et al. 2012; Júnior et al. 2015). Among all heavy metals, chromium (Cr) is highly toxic, carcinogenic, and persistent in nature (Knasmüller et al. 1998; Sharma and Pandey 2014; Adrees et al. 2015a). Sources of Cr contamination in the environment mainly include metal finishing, leather processing, leachate from sanitary landfills, tobacco emissions, oxidative dyeing, cement plants, timber processing, chromic acid manufacturing, metal plating, and paper and pulp production (Nagajyoti et al. 2010; Afshan et al. 2015; Ali et al. 2015b). Trivalent Cr and hexavalent Cr are the most stable forms of Cr among the variety of valence states ranging from −2 to +6. Furthermore, hexavalent Cr is identified as the more toxic form of Cr compared to trivalent Cr (Becquer et al. 2003). Hexavalent Cr possesses highly mutagenic and carcinogenic impacts on humans and animals (Gerber et al. 1980; Sun et al. 2008; Mondol et al. 2011). Hexavalent Cr toxicity interferes in various physiological processes of plants and ultimately leads to reduction in seed germination, root growth, dry weights, number of leaves, photosynthesis, and induced changes in leaf protein profiles and root microRNA expression (Ali et al. 2011; Bukhari et al. 2015, 2016a, b; Zaheer et al. 2015). Therefore, scientists are more concerned about the remediation techniques to reduce the Cr pollution in a sustainable way to protect the agricultural land for food crops. Phytoextraction is the extraction of heavy metals from soil and water by plants’ natural mechanism of uptake and can be used to decontaminate the polluted soil and water bodies (Adrees et al. 2015a, 2015b; Rizwan et al. 2016a). Phytoremediation is a simple, cost-effective, environment-friendly, economically suitable, and self-sustaining substitute of traditional remediation methods (Rizwan et al. 2016b, 2017). The degree of metal translocation in plants depends upon several factors including heavy metal type, plant species, and surrounding conditions (Jadia and Fulekar 2008; Farooq et al. 2013). Duckweed plant species due to their capability to hyperaccumulate heavy metals are gaining attention around the globe (Radić et al. 2010). Lemna minor, Lemna gibba, and Spirodela polyrhiza are the potential candidates for this technique (Axtell et al. 2003; Charles et al. 2006; Zhang et al. 2011). Lemna minor (duckweed) can be useful in phytoextraction of heavy metals because of its high metal accumulation capability, natural occurrence, rapid growth, tolerance toward cold, and ease of harvesting (Sharma and Gaur 1995; Radić et al. 2010). The average lifespan of L. minor is about 5–6 weeks with a production rate of 0.45 fronds per day and doubling its mass in 2–3 days (Isaksson et al. 2007). Its rapid growth rate can make it a potential candidate for phytoremediation. For the bioavailability of heavy metals in the medium, their mobility and solubility must be ensured. Addition of synthetic chelators like diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacitic acid (EDTA) and organic chelators like citric acid can solve this problem by solubilizing the heavy metals in the growth medium (Sinhal et al. 2010; Szczygłowska et al. 2011; Farid et al. 2015). In addition, chelators also enhance nutrient uptake by plants, which are supportive to plants and help them maintain their normal physiological activities under heavy metal stress (Freitas et al. 2014). Application of synthetic chelators is not so eco-friendly as they can contaminate the ground water because of their persistence in the environment due to their non-biodegradable nature (Anwer et al. 2012; Bareen 2012; Rizwan et al. 2016b). Citric acid (CA), a highly biodegradable low atomic weight organic acid, can serve as an alternate to EDTA in improving metal solubility in the growth medium and their uptake by plants (Ding et al. 2005; Farid et al. 2013; Shakoor et al. 2014). Previous studies have identified the capability of CA for metal uptake and accumulation in plants (Luo et al. 2005; Sinhal et al. 2010). However, scarce information is available regarding the use of CA for phytoextraction of Cr with L. minor.

Thus, by knowing the importance of Cr in plants, the present study aimed to investigate the effects of exogenous CA on L. minor growth under Cr stress. Furthermore, physiological and biochemical approaches were used to identify the CA-induced Cr tolerance mechanisms in L. minor by evaluating the roles of key components such as (i) plant growth and biomass, (ii) photosynthesis, (iii) Cr accumulation, and (iv) oxidative stress and activities of antioxidant enzymes.

Material and methods

Experimental growth conditions and treatments

Already grown healthy and uniform plants of L. minor were collected from the domestic wastewater pond and thoroughly rinsed with distilled water and transferred to glass platters of 1 L capacity with complete randomized design (CRD). The physico-chemical properties of wastewater and Cr concentration in L. minor collected from pond are given in Table 1. Aeration was continuously provided through pumping. An experimental setup was placed in a laboratory with sunlight and air and at normal room temperature varying between 20 and 25 °C. After transplanting, plants were supplied with Cr as K2CrO4 and citric acid and sprayed with 10% of nutrient solution prepared by the recipe of Hoagland and Arnon (1950) consisting of (in μM) KNO3 3000; KH2(PO4) 100; Ca(NO3)2 2000; MgSO4 1000; H3BO3 50; ZnSO4·7H2O 0.8; MnCl2·4H2O 0.05; CuSO4·H2Mo4·H2O 0.10; 5H2O 0.3; and FeNa-EDTA 12.5. The treatments were as follows: T1, control; T2, CA (2.5 mM); T3, Cr (10 μM); T4, Cr (100 μM); T5, Cr (200 μM); T6, Cr (10 μM) + CA (2.5 mM); T7, Cr (100 μM) + CA (2.5 mM); and T8, Cr (200 μM) + CA (2.5 mM) with three replications. The control system was provided with neither K2CrO4 nor citric acid. The solutions were renewed after every 3 days by draining the previous solution completely. The abovementioned treatments were maintained for 6 weeks. The solutions were stirred up with the help of spatula after every 10 h.

Table 1 Wastewater physico-chemical parameter analysis
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Physiological assessment

After 6 weeks of treatment application, the plants were sampled for biomass (fresh and dry weight), leaf color, and leaf area. The whole plant samples were kept at 70 °C for at least 72 h for the measurement of dry biomass. For biochemical analysis, fresh whole plant samples were used.

Biochemical assessment

Determination of chlorophyll content

After continuous application of treatments for 6 weeks, carotenoid and chlorophyll contents were measured in fully expanded fresh plants by following the method of Metzner et al. (1965) with certain amendments. The fresh leaves were incubated till discoloration in aqueous acetone (85%, v/v) at 4 °C under darkness with continuous shaking for pigment extracts. Further, the extract was centrifuged at 4 °C and 4000 rpm for the next 10 min and the supernatant was collected from the surface for the measurement of light absorbance at 452.5, 644, and 663 nm by using a Halo DB-20/DB-20S (Dynamica Company, London, UK) spectrophotometer. Finally, calculations were made by using an equation and adjusted extinction coefficients given by Lichtenthaler (1987).

Equation for measuring chlorophyll and carotenoid contents:

$$ \begin{array}{l}\begin{array}{l}\mathrm{Chlorophyll}\ \mathrm{a}\ \left(\upmu \mathrm{g}/\mathrm{mL}\right)={10.3}^{\ast }-{\mathrm{E}}_{663}-0.98\ast {\mathrm{E}}_{644}\hfill \\ {}\mathrm{Chlorophyll}\ \mathrm{b}\ \left(\upmu \mathrm{g}/\mathrm{mL}\right)=19.7\ast {\mathrm{E}}_{644}-3:87\ast {\mathrm{E}}_{663}\hfill \\ {}\mathrm{Total}\ \mathrm{chl}\mathrm{orophyll}=\mathrm{chlorophyll}\ \mathrm{a}+\mathrm{chlorophyll}\ \mathrm{b}\hfill \end{array}\\ {}\mathrm{Total}\ \mathrm{carotenoids}\left(\upmu \mathrm{g}/\mathrm{mL}\right)=4.2\ast {\mathrm{E}}_{4552.5}-\Big\{\left({0.0264}^{\ast}\mathrm{chl}\ \mathrm{a}\right)+\left(0:{426}^{\ast}\mathrm{chl}\ \mathrm{b}\right)\end{array} $$

Determination of protein and antioxidant enzymes

After continuous application of treatments for 6 weeks, the content of soluble protein was measured by following the protocol of Bradford (1976) using a standard (bovine serum albumin) and dye (Coomassie Brilliant Blue G-250). In detail, fresh whole plants were grounded with mortar and pestle under chilled conditions and then mixed in 10 mL of buffer solution made by 50 mM of sodium phosphate containing polyvinylpyrolidine 40 (2%,w/v) and 1 mM EDTA. Further, the supernatant was collected after centrifuging the mixture at 4 °C and 11,000 rpm for 15 min and was used for the measurement of antioxidant enzymes (POD and SOD) and proteins.

To estimate the entire protein concentration, 100 μL of sample extract was further homogenized with 1 mL of Bradford solution and 595 nm wavelength of the spectrophotometer was selected to measure light absorbance.

The protocol described by Aebi (1984) was followed to measure the activity/concentration of catalases (CAT, EC 1.11.1.6). For this, 3 mL of assay mixture consisted of 2.8 mL solution of phosphate buffer (50 mM with 2 mM citric acid, pH 7.0), 100 μL of hydrogen peroxide (300 mM), and 100 μL of enzyme extract. The concentration of CAT was estimated by recording the changes in absorbance at 240 nm which occurred due to the disappearance of hydrogen peroxide (ε = 39.4 mM−1 cm−1).

The activity/concentration of ascorbate peroxidase (APX, EC 1.11.1.11) was estimated by following the protocol presented by Nakano and Asada (1981). An assay mixture of 3 mL contained 2.7 mL phosphate buffer solution (50 mM with 2 mM citric acid, pH 7.0), 100 μL ascorbate (7.5 mM), 100 μL of enzyme extract, and 100 μL of hydrogen peroxide (300 mM). The concentration of APX was estimated by recording the changes in wavelength at 290 nm occurred due to its oxidation (ε = 2.8 mM−1 cm−1).

Hydrogen peroxide contents

To estimate the content of H2O2, the assay mixture was prepared by homogenizing the fresh whole plant (50 mg) with 3 mL of phosphate buffer solution (pH 6.5, 50 mM) and centrifuging at 6000×g and 4 °C for 25 min. Further, 3 mL of extracted supernatant was mixed with 1 mL of sulfuric acid (20%, v/v) and titanium sulfate (0.1%), then again the mixture was centrifuged at 6000×g and 4 °C for 15 min. The concentration of H2O2 was estimated at 410 nm by recording changes in the intensity of the supernatant’s yellow color and calculated by the following extinction coefficient of 0.28 μmol−1 cm−1.

Malondialdehyde content

Malondialdehyde concentration (a product of lipid peroxidation) in whole plant tissues was estimated by following the reaction method of thiobarbituric acid (TBA) as documented by Heath and Packer (1968) along with some modifications added by Zhang and Kirham (1994) and Dhindsa et al. (1981).

Assessment of electrolyte leakage

After completing the 6 weeks of treatments, whole plants were put in test tubes having 8 mL of distilled water. The initial electrical conductivity (EC1) was recorded after incubating the test tubes for 2 h at 32 °C in a water bath. For the measurement of subsequent electric conductivity (EC2), the sample test tubes were autoclaved for 20 min at 121 °C to discharge maximum electrolytes and then were cooled at room temperature, and electrolyte leakage (EL) was determined by following the method described by Dionisio-Sese and Tobita (1998).

Electrolyte leakage was computed by the following formula:

$$ \mathrm{EL}=\left({\mathrm{EC}}_1/{\mathrm{EC}}_2\right)\times 100 $$

Chromium concentration

Plants were dried at 90 °C and then turned to ash in a muffle furnace at 600 °C for 6 h. The ash samples were dissolved in concentrated solution of HNO3 and HCl (3 mL each) and filtered in a volumetric flask, then distilled water was added to make a final volume of 50 mL. The Perkin Elmer AAnalyst 100 (USA) method was adopted to determine the Cr concentration. Data was analyzed according to Analytical Software, Tallahassee, USA.

Chromium concentration was calculated for the plant as a whole by the following formula:

Metal (μg g−1) in plant = metal reading of digested sample (mg L−1) × dilution factor

where

$$ \mathrm{Dilution}\ \mathrm{factor}=\frac{\mathrm{Total}\ \mathrm{volume}\ \mathrm{of}\ \mathrm{sample}\ \left(\mathrm{mL}\right)}{\mathrm{Weight}\ \mathrm{of}\ \mathrm{plant}\ \mathrm{material}\ \left(\mathrm{g}\right)} $$

The metal uptake in whole plant was calculated using the following formula: (Zayed et al. 1998)

Metal uptake = metal concentration in plant (μg g−1) × plant’s whole dry weight (g)

which was used to calculate the bio-concentration factor.

The actual Cr concentration in plant tissues (Cra) = Cr conc. in plant organ (Crb) is subtracted by the already available concentration in plant (Crc) (0.098 ± 0.001):

$$ {Cr}_a={Cr}_b\hbox{--} {Cr}_c $$

Statistical analysis

Data presented in this paper are the average of three replicates ± S.D. Analysis of variance (ANOVA) and graphical representation were performed with software Statistix 10.0, further followed by Tukey’s test to find significant differences among mean values of treatments.

Results

Agronomic traits

The variations in L. minor growth parameters including fresh weight and dry weight per hundred plants as a whole are given in Table 2. Chromium application reduced both fresh and dry biomass of plants as compared to the control treatment. The weight was gradually decreased with increasing Cr concentrations in culture medium. Application of CA (2.5 mM) along with Cr 10, 100, and 200 μM significantly increased the fresh and dry biomass of plants compared to those plants treated with respective Cr treatments alone. A similar trend was found in the leaf area of L. minor under Cr and CA application (Table 2). Addition of CA increased the fresh weight by 20.2, 21.8, 22.2, and 24.9% at 0, 10, 100, and 200 μM Cr treatments compared to the respective Cr treatments alone. The dry weight was dramatically decreased under CA by 11.53 and 16.66% at Cr 10 and 200 μM, respectively.

Table 2 Effect of chromium and citric acid on fresh weight, dry weights, leaf area, and Cr concentration and accumulation (mg plant−100) of L. minor grown in solution culture with increasing Cr concentrations (10, 100, and 200 μM) treated or not with 2.5 mM citric acid
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Leaf greenness of plants was also reduced with increasing concentrations of Cr in the nutrient solution (Fig. 1). Plants applied with 200 μM Cr showed the lowest greenness while the highest leaf greenness was observed for the plants treated with CA compared to the control plants. Application of CA in combination with Cr enhanced the leaf greenness compared to the plants treated with Cr only.

Fig. 1
figure 1

. Effect of Cr and citric acid on leaf color of L. minor grown in solution culture with increasing Cr concentrations (10, 100, and 200 μM) treated or not with 2.5 mM citric acid

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Photosynthetic pigments

Chlorophyll (Chl a, Chl b, and total Chl) and carotenoid contents significantly decreased with the addition of Cr in the solution compared to the control (Fig. 2). The maximum total chlorophyll and carotenoid contents were observed in plants treated with CA alone while the highest reduction in photosynthetic pigments was observed in plants treated with 200 μM Cr. Exogenous application of CA along with Cr significantly enhanced the Chl a, Chl b, total Chl, and carotenoid contents in plants as compared to Cr only-treated plants. The addition of CA increased the total chlorophyll contents by 2.14, 14.65, 14.54, and 24.86% at 0, 10, 100, and 200 μM Cr treatments compared to the respective Cr treatments without CA application.

Fig. 2
figure 2

Effect of Cr and citric acid on chlorophyll a, b, total chlorophylls, and total carotenoids in L. minor grown in solution culture with increasing Cr concentrations (10, 100, and 200 μM) treated or not with 2.5 mM citric acid. Values are demonstrated as means of three replicates along with standard deviation. Different normal or italicized small and capital letters indicate that values are significantly different at P < 0.05

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Antioxidant enzyme activity

Four key antioxidant enzymes, APX, SOD, POD, and CAT, were analyzed to investigate the impact of Cr and CA amendments on antioxidant capacity of L. minor (Fig. 3). Chromium stress alone significantly inhibited the antioxidant enzyme activities in a dose-additive manner except in 10 μM Cr where antioxidant enzyme activities were increased compared to the control. Exogenous CA + Cr addition further enhanced the antioxidant enzyme activities and showed synergetic effects compared to the respective Cr treatments alone. The antioxidant enzyme activities were boosted up under CA at initial concentrations of Cr, i.e., 10 and 100 μM, while at the highest concentration of 200 μM, a reduction of the activities of antioxidant enzymes was observed.

Fig. 3
figure 3

Antioxidative enzyme activities in L. minor grown in solution culture with increasing Cr concentrations (10, 100, and 200 μM) treated or not with 2.5 mM citric acid. Values are demonstrated as means of three replicates along with standard deviation. Different small normal and italicized letters indicate that values are significantly different at P < 0.05

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Electrolyte leakage, hydrogen peroxide, and malondialdehyde content

Oxidative stress in L. minor was evaluated by measuring the EL and H2O2 and MDA contents in plants (Fig. 4a, b, d). Increasing Cr concentrations significantly enhanced the oxidative damage (MDA and H2O2) and EL. The highest concentrations of EL and H2O2 and MDA were observed in the plants treated with the highest concentration of Cr (200 μM) while the lowest contents were observed in the control plants. Application of CA (2.5 mM) alone and/or in combination with Cr significantly alleviated the oxidative stress as was observed by the reduced production of EL and H2O2 and MDA in L. minor under Cr stress compared to the respective Cr treatments without CA application. With CA application under Cr stress, the maximum reduction in H2O2 and MDA contents was by 12.73 and 23.16% at 10 and 100 μM Cr, respectively.

Fig. 4
figure 4

Effect of Cr and citric acid on electrolyte leakage, H2O2 concentration, protein contents, and MDA contents in L. minor grown in solution culture with increasing Cr concentrations (10, 100, and 200 μM) treated or not with 2.5 mM citric acid. Values are demonstrated as means of three replicates along with standard deviation. Different letters indicate that values are significantly different at P < 0.05

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Soluble protein

Chromium-stressed L. minor showed the reduced soluble protein contents compared to the control (Fig. 4c). The highest reduction in protein contents was observed in the plants treated with 200 μM Cr treatment alone. However, CA application gradually enhanced the protein contents in Cr-stressed plants compared to the plants treated with respective Cr treatments alone. Addition of CA increased the soluble protein content by 5.0, 10, 18, and 11.6% at 0, 10, 100, and 200 μM Cr treatments compared to the respective Cr treatments alone.

Chromium concentration

Chromium concentration in L. minor significantly increased with increasing Cr concentrations in the nutrient solution (Table 2). Application of CA further enhanced the Cr concentrations and uptake by plants compared to the respective treatments without CA application. The highest Cr concentrations were observed in the plants treated with 200 μM Cr along with CA. Addition of CA increased the Cr concentration by 6.1, 26.5, 20.5, and 20.2% at 0, 10, 100, and 200 μM Cr compared to the respective Cr treatments alone. The correlations among all attributes of L. minor studied are given in Table 3. The negative sign showed a significant decrease in the parameter with respect to the other parameter. The correlation among Cr concentrations to all attributes showed significant decrease except antioxidant enzymes, reactive oxygen species, and electrolyte leakage.

Table 3 Correlation among the all physiological and biochemical parameters with Cr concentration and uptake by Lemna minor L.
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Discussion

A possible mechanism for Cr uptake and tolerance in L. minor by the addition of CA during Cr stress was probed in the present research. Chromium stress reduced the plant biomass (Table 2) and leaf color (Fig. 1). It has been reported in previous studies that Cr uptake and accumulation in plant reduced the plant growth and biomass by suppressing mineral uptake which interrupts normal metabolic processes (Ali et al. 2015a, b; Dheeba et al. 2015; Gill et al. 2015). The Cr-induced stress is responsible for the generation of reactive oxygen species (ROS) which deteriorated the growth and development of plants (Singh et al. 2012; Das et al. 2014). The addition of CA significantly improved the growth and biomass of L. minor under Cr treatments, indicating its promotive role in metal stress mitigation (Figs. 1, 2 and 3; Table 2). A similar role of CA in nutrient uptake under metal stress was reported by several researchers (Najeeb et al. 2011; Ehsan et al. 2014; Zaheer et al. 2015).

Increased Cr uptake ultimately reduced the Chl a, b, total Chl, and carotenoid contents in L. minor (Fig. 2). Deformation of chloroplast ultrastructure causes altered shape and enlargement of thylakoids which is responsible for lowering the Chl a, b, and carotenoid contents in plant leaves (Parmar et al. 2013). The reduced plant growth might be the consequence of low photosynthetic performance owing to decreased chlorophyll and carotenoid contents. Citric acid alleviated Cr-induced toxicity in L. minor possibly by enhancing the photosynthetic rate and recovering chloroplast ultrastructure damaged by Cr toxicity. The promoting role of CA in enhancing chlorophyll and carotenoid content was observed in many plant species under stress caused by different heavy metals like Brassica napus under Cd stress (Ehsan et al. 2014), Cr stress (Afshan et al. 2015), and Pb stress (Shakoor et al. 2014).

The toxicity caused by Cr and other metals usually induces oxidative stress and negatively affects plant growth and development (Afshan et al. 2015; Habiba et al. 2015). In the present study, Cr toxicity increased oxidative stress which resulted in elevated level of EL, as well as enhanced production of MDA and H2O2 (Fig. 4a, b, d). According to Mittler (2002), plants have the natural ability to combat the toxic impacts of ROS. These results are endorsed with recent studies which identified that many plant species such as wheat, B. napus, barley, and tobacco bear oxidative stress under Cr application (Diwan et al. 2012; Ali et al. 2011; Ali et al. 2015a; Gill et al. 2015; Bukhari et al. 2016a, b). The present results suggested the assisted role of CA which considerably mitigated the toxic effect of Cr and reduced MDA and H2O2 accumulation by decreasing EL. Cr and other metals usually induce oxidative stress and then cause injuries in plants (Ali et al. 2011, 2015a; Habiba et al. 2015).

Production of ROS needs to be controlled by ROS-scavenging mechanisms. Plants are naturally capable to mitigate and repair the ROS-induced damage by activating the antioxidant enzyme defense system (Mittler et al. 2004). In this study, it is identified that low concentration of Cr (10 μM) enhanced POD, SOD, APX, and CAT activities, while their activation was slowed down with the increase in Cr concentrations (Fig. 3a, b). This phenomenon indicates that at higher accumulation of metals in plants, the over-expression of antioxidant enzymes might be an intense mechanism for plants’ survival (Haouari et al. 2012). Similar findings have been reported by Bukhari et al. (2016a, b) about the behavior of the plant enzymatic defense system under Cr stress. However, application of CA helped the plants to survive under Cr stress by increasing activities of antioxidative enzymes in L. minor (Fig. 3a, b).

Chromium stress has been reported for reduced soluble protein in plants (Singh et al. 2012; Das et al. 2014). Our results revealed the same decreasing trend of soluble protein in L. minor under Cr stress (Fig. 4c). It might be the result of increased oxidative damages induced by Cr which suppressed the protein contents (Jabeen et al. 2016). Citric acid application enhanced protein contents in L. minor (Fig. 4c) and decreased H2O2 production and EL which protected the plants from further damage (Fig. 4a, b). The improved plant biomass, leaf greenness, and chlorophyll contents with CA application with Cr stress might be due to enhanced antioxidant enzymatic activity (Figs. 1, 2, and 3; Table 2) and suppression of MDA and H2O2 generation (Fig. 4b, d).

The decline in plant agronomic traits and biomass accumulation might be the result of decreased nutrient uptake and increased metal accumulation by plants under metal stress (Rizwan et al. 2012; Pradas-del-Real et al. 2013; Keller et al. 2015; Khaliq et al. 2016). Addition of CA significantly increased Cr uptake by B. napus (Afshan et al. 2015) and Cd uptake by Sedum alfredii (Lu et al. 2013). According to Shakoor et al. (2014), CA is capable of making heavy metals bioavailable in both soil and aqueous media because of its chelating ability. In this study, CA application to Cr-stressed plants enhanced the Cr uptake as compared to Cr only-treated and control system plants (Table 2). In this work, results are being evaluated for the Cr concentration for the whole plant including its root and two to three fronds. Metal distribution in plants’ tissue is a necessary characteristic as it can be indirectly useful to indicate detoxification mechanism.

The results of the present study are in line with the previous work that the role of L. minor as phytoextraction agent for heavy metals is quite obvious in aqueous media also identified by Bokhari et al. (2016). Improved biomass, metal uptake by plant, and bioavailability of heavy metals in the rhizosphere are the key parameters to be judged for successful phytoextraction plan stated by McGrath and Brooks (1998). However, the efficiency of L. minor to hyperaccumulate Cr as well as the role of CA in enhancing Cr bioavailability in the soil system still bear a question mark, which needs further experimental evidence.