N-Fertilizer (Urea) Enhances the Phytoextraction of Cadmium through Solanum nigrum L.

Heavy metal contamination is currently a major environmental concern, as most agricultural land is being polluted from municipal discharge. Among various other pollutants, cadmium (Cd), one of the most harmful heavy metals, enters into the food chain through the irrigation of crops with an industrial effluent. In the present study, a pot experiment was designed to assess the effect of different nitrogen (N)-fertilizer forms in the phytoremediation of Cd through Solanum nigrum L. Two types of N fertilizers (NH4NO3 and urea) were applied to the soil in different ratios (0:0, 100:0, 0:100, and 50:50 of NH4NO3 and urea, individually) along with different Cd levels (0, 25, and 50 mg kg−1). The plants were harvested 70 days after sowing the seeds in pots. Cadmium contamination significantly inhibited the growth of leaves and roots of S. nigrum plants. Cadmium contamination also induced oxidative stress; however, the application of N-fertilizers increased the plant biomass by inhibiting oxidative stress and enhancing antioxidants’ enzymatic activities. The greatest plant growth was observed in the urea-treated plants compared with the NH4NO3-treated plants. In addition, urea-fed plants also accumulated higher Cd concentrations than NH4NO3-fed plants. It is concluded that urea is helpful for better growth of S. nigrum under Cd stress. Thus, an optimum concentration of N-fertilizers might be effective in the phytoremediation of heavy metals through S. nigrum.


Introduction
Anthropogenic activities have resulted in environmental degradation and a significant reduction in soil productivity [1]. Abiotic stress, such as heavy metals, extreme temperature, and salinity, affect crop production [2]. Industrial discharge, sewage sludge, mining, and agriculture have become major precursors of heavy metal discharge into water bodies and soil [3]. Compared with all other heavy metals, cadmium (Cd) is considered lethal once it becomes part of the food cycle.
Around 70% of the dietary intake of Cd occurs from consuming the vegetables and grain crops grown in Cd-contaminated soils [4]. Cadmium is highly toxic for plant growth and seed germination, in addition to the antioxidant, photosynthetic, and enzyme activities [5]. It also causes visual toxicity

Experimental Design
A pot experiment was conducted in a botanical garden located in Government College University Faisalabad (Faisalabad, Pakistan; 31 • 25 0" N, 73 • 5 28" E). The soil texture was measured by the method of Bouyoucos [27]; the pH and electrical conductivity (EC) with pH and EC meters, respectively; organic carbon was measured using the Walkley-Black method [28]; and the sodium absorption ratio (SAR) and soluble ions were measured using the method described by Page et al. [29]. The soil physicochemical characteristics are shown in Table 1. The soil was spiked and entirely mixed with different Cd (CdCl 2 2.5H 2 O) levels (0, 25, and 50 mg kg −1 ). Nitrogen fertilizers of two different kinds (NH 4 NO 3 and urea) with varying ratios (0-0, 100-0, 0-100, and 50-50 mg kg −1 ) were added into the soil. It is noteworthy that the contents for Cd, Pb, Cu, and Zn were fundamentally low and unnoticeable in these fertilizers. In table, EC stands for electrical conductivity and SAR for sodium absorption ratio.

Soil Pot Experiments
In this study, 5% sodium hypochlorite (NaClO) was applied for 10 min in order to sterilize the S. nigrum seeds, which were then washed four times with deionized water. The washed and blot-dried seeds were planted in a plastic tray filled with sand, and half-strength Hoagland solution was applied. After three weeks of germination, the uniform seedlings were transferred into pots (four seedlings in each pot). Every single pot was filled with 5 kg of soil. All of the pots were set following complete randomized design (CRD) with four repeats for each treatment, and the water was topped up with tap water in order to maintain the 70% soil water-holding limit.

Plant Harvesting
The plants were harvested 70 days after sowing the seeds and were sectioned into shoots and roots. The length of the roots and shoots, numbers of leaves, leaf area, and fresh weight of plants were measured. The root and shoot samples were further dried in an oven for 72 h at 70 • C, and the dry weights were measured.

Determination of Photosynthetic, Chlorophyll, and Carotenoid Content Parameters
Fresh leaf samples (0.5 g) were soaked in acetone (85%, v/v, Sigma) and placed in the dark. The soaked samples were centrifuged (4000× g for 10 min, 4 • C) and the supernatant was collected. Data were recorded on a spectrophotometer at wavelengths of 470, 647, and 664.5 nm, separately. The chlorophyll content, i.e., chlorophyll a, chlorophyll b, and total chlorophyll, and carotenoids were recorded [30]. On a sunny day (10:00 a.m. to 12:00 p.m.), an infrared gas analyzer (IRGA) was used for the assessment of the conductance of the stomata water-use efficiency, rate of transpiration, and photosynthetic rate in the plants' leaves.

Determination of EL, MDA, H 2 O 2 , and Antioxidant Enzyme Concentration
The measurement of both the oxidative stress markers and the activities of antioxidant enzymes were done 70 days after seed sowing. The shoot and root samples were placed in glass tubes vertically and heated at 32 • C for 2 h in distilled water of a known volume in order to measure the electrolyte leakage. This solution was termed EC 1 . Then, the same solution was heated at 121 • C for 20 min, and EC of this second solution was recorded and named EC 2 . The Dionisio-Sese and Tobita [31] equation was used to estimate the EL content.
For the estimation of the H 2 O 2 content, a phosphate buffer solution (3.0 mL) was added to the sample (50 mg) and centrifuged at 6000× g for 30 min, keeping the temperature at 4 • C. Then, 1 mL of titanium sulfate (0.1%) was mixed in a supernatant and centrifuged at 6000× g for 20 min at 4 • C. Absorption was determined at a wavelength of 410 nm and a coefficient of extinction for H 2 O 2 of 0.28 µmol −1 cm −1 . The peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) enzyme activities were determined following the recommendations of Zhang [32] and Aebi [33]. The samples were prepared in a phosphate buffer (0.05 mmol) and the supernatant was gained through centrifuging for 10 min at 12,000× g and 4 • C.

Cadmium Determination in Plants, Translocation Factor and Bioaccumulation Factor
Crushed root and shoot samples were digested using HNO 3 -HClO 4 (3:1, v/v). The samples were kept in 65% HNO 3 -HClO 4 (3:1, v/v) for one night and then put on a hot plate after adding HNO 3 (5.0 mL). The clear solution obtained after digestion was filtered and the Cd concentration was measured using an atomic absorption spectrophotometer. Translocation factor (TF) was calculated by using Equation (1): and the bioaccumulation factor (BCF) was calculated using Equation (2): TF = Cd concentration in root/Total Cd concentration in soil (2)

Statistical Analysis
A two-way analysis of variance test (ANOVA) was applied in order to test the significance of the N fertilizers and Cd concentrations. Tukey's posthoc test was applied for the multiple means comparison technique. The statistical analysis was performed with SPSS for Windows Software v. 19 (IBM, Armonk, NY, USA).

Assessment of N-Fertilizer on Growth and Biomass
Stunted growth was observed in the control group, while no such signs were observed in the N-fertilizer-treated group ( Figure 1). The roots' and shoots' dry weight, the number of leaves per plant shoot length and root length, and the leaf area of the S. nigrum plants significantly increased in the N-fertilizer treatments ( Figure 1). A maximum plant growth was observed in the urea-treated plants at a Cd level (0 mg kg −1 ), with respect to the NH 4 NO 3 -treated and control plants. In addition, more leaves were observed in the urea-treated plants compared with the NH 4 NO 3 -treated plants ( Figure 1). titanium sulfate (0.1%) was mixed in a supernatant and centrifuged at 6000 g for 20 min at 4 °C.
Absorption was determined at a wavelength of 410 nm and a coefficient of extinction for H2O2 of 0.28 µmol −1 cm −1 . The peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) enzyme activities were determined following the recommendations of Zhang [32] and Aebi [33]. The samples were prepared in a phosphate buffer (0.05 mmol) and the supernatant was gained through centrifuging for 10 min at 12,000 g and 4 °C.

Cadmium Determination in Plants, Translocation Factor and Bioaccumulation Factor
Crushed root and shoot samples were digested using HNO3-HClO4 (3:1, v/v). The samples were kept in 65% HNO3-HClO4 (3:1, v/v) for one night and then put on a hot plate after adding HNO3 (5.0 mL). The clear solution obtained after digestion was filtered and the Cd concentration was measured using an atomic absorption spectrophotometer. Translocation factor (TF) was calculated by using Equation (1): and the bioaccumulation factor (BCF) was calculated using Equation (2): TF = Cd concentration in root / Total Cd concentration in soil (2)

Statistical Analysis
A two-way analysis of variance test (ANOVA) was applied in order to test the significance of the N fertilizers and Cd concentrations. Tukey's posthoc test was applied for the multiple means comparison technique. The statistical analysis was performed with SPSS for Windows Software v. 19 (IBM, Armonk, NY, USA).

Assessment of N-Fertilizer on Growth and Biomass
Stunted growth was observed in the control group, while no such signs were observed in the Nfertilizer-treated group ( Figure 1). The roots' and shoots' dry weight, the number of leaves per plant shoot length and root length, and the leaf area of the S. nigrum plants significantly increased in the Nfertilizer treatments ( Figure 1). A maximum plant growth was observed in the urea-treated plants at a Cd level (0 mg kg −1 ), with respect to the NH4NO3-treated and control plants. In addition, more leaves were observed in the urea-treated plants compared with the NH4NO3-treated plants ( Figure 1).

Assessment of Gas Exchange, Chlorophyll, and Carotenoid Content Attributes
Differential responses of chlorophyll and gas exchange attributes were recorded in urea-treated and NH4NO3-treated plants. Significant changes with the maximum values of Chl a, Chl b, total Chl, and carotenoid contents were observed in the urea-fed plants ( Figure 2). A nonsignificant increase in chlorophyll content was observed in the urea-treated plants at 25 mg kg −1 Cd concentrations, in contrast to the NH4NO3-treated and control plants. In addition, a higher expression of gas attributes was recorded in the urea-treated plants than that of the NH4NO3-fed plants ( Figure 3).

Assessment of Gas Exchange, Chlorophyll, and Carotenoid Content Attributes
Differential responses of chlorophyll and gas exchange attributes were recorded in urea-treated and NH 4 NO 3 -treated plants. Significant changes with the maximum values of Chl a, Chl b, total Chl, and carotenoid contents were observed in the urea-fed plants ( Figure 2). A nonsignificant increase in chlorophyll content was observed in the urea-treated plants at 25 mg kg −1 Cd concentrations, in contrast to the NH 4 NO 3 -treated and control plants. In addition, a higher expression of gas attributes was recorded in the urea-treated plants than that of the NH 4 NO 3 -fed plants ( Figure 3).

Assessment of Gas Exchange, Chlorophyll, and Carotenoid Content Attributes
Differential responses of chlorophyll and gas exchange attributes were recorded in urea-treated and NH4NO3-treated plants. Significant changes with the maximum values of Chl a, Chl b, total Chl, and carotenoid contents were observed in the urea-fed plants ( Figure 2). A nonsignificant increase in chlorophyll content was observed in the urea-treated plants at 25 mg kg −1 Cd concentrations, in contrast to the NH4NO3-treated and control plants. In addition, a higher expression of gas attributes was recorded in the urea-treated plants than that of the NH4NO3-fed plants ( Figure 3).

Assessment of Antioxidant Enzyme Activities and EL, MDA, and H2O2
After N-fertilizer supplementation, a significant reduction was noticed in the MDA, H2O2, and electrolyte leakage parameters (Figure 4). Reductions in EL in the leaves of the urea, NH4NO3, and urea + NH4NO3 treated plants were 15%, 33%, and 22%, respectively, compared with the control plants. The catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD) enzyme activities in the S. nigrum leaf were significantly increased after the addition of N-fertilizers ( Figure 5). In contrast to the control group, the increases in the POD, CAT, APX, and

Assessment of Antioxidant Enzyme Activities and EL, MDA, and H2O2
After N-fertilizer supplementation, a significant reduction was noticed in the MDA, H2O2, and electrolyte leakage parameters (Figure 4). Reductions in EL in the leaves of the urea, NH4NO3, and urea + NH4NO3 treated plants were 15%, 33%, and 22%, respectively, compared with the control plants. The catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD) enzyme activities in the S. nigrum leaf were significantly increased after the addition of N-fertilizers ( Figure 5). In contrast to the control group, the increases in the POD, CAT, APX, and

Assessment of Concentration of Cd in Plants
The optimum induction of N-fertilizers remarkably enhanced the Cd concentration in the roots and shoots of S. nigrum compared with the respective controls ( Figure 6). In order to assess the phytoremediation potential of Cd by S. nigrum, the TF and BCF were calculated. Results showed that plants supplemented with urea had higher BCF when compared with other treatments. The optimum induction of N-fertilizers remarkably enhanced the Cd concentration in the roots and shoots of S. nigrum compared with the respective controls ( Figure 6). In order to assess the phytoremediation potential of Cd by S. nigrum, the TF and BCF were calculated. Results showed that plants supplemented with urea had higher BCF when compared with other treatments.

Biomass and Plant Growth
It is an established fact that fertilizer application generally has positive effects on plant growth under heavy metal toxicity [34,35]. In the present study, the highest plant growth was observed in the urea-treated plants at 25 mg kg −1 Cd concentrations, in contrast to the NH4NO3-treated plants (Figure 1). The findings of our study are in accordance with the finding of Lin et al. [36], who found that 2.5 mmol N L −1 was the optimal concentration to boost S. alfredii shoot growth. Moreover, 1.0

Biomass and Plant Growth
It is an established fact that fertilizer application generally has positive effects on plant growth under heavy metal toxicity [34,35]. In the present study, the highest plant growth was observed in the urea-treated plants at 25 mg kg −1 Cd concentrations, in contrast to the NH 4 NO 3 -treated plants (Figure 1). The findings of our study are in accordance with the finding of Lin et al. [36], who found that 2.5 mmol N L −1 was the optimal concentration to boost S. alfredii shoot growth. Moreover, 1.0 mmol N L −1 was found to be an optimal concentration for Cd and Zn storage in the shoots of S. alfredii. Furthermore, stunted plant growth under heavy metal stress was noticed. For example, Rabelo et al. [37] reported that 2.0 mM Cd in Tanzania guinea grass inhibited the production of new tillers and leaves. A reduced nitrate uptake from the nutrient solution was reported by Gouia et al. [38] for Cd-exposed plants. It was investigated that the Cd concentration increased in S. nigrum with a supply of N-fertilizer (NH 4 NO 3 ) without affecting the Cd speciation in plants [39]. Wei et al. [40] confirmed that the accretion of Cd in S. nigrum shoots and roots is augmented by applying urea in the growth medium. In our study, the root and shoot dry weights, shoot length, numbers of leaves per plant, and roots length of S. nigrum plants were significantly enhanced with the N-fertilizer application. In our current study, disparity in the Cd absorption in differently treated plants was observed, which is in line with the results of Ye et al. [41], who described an enhanced absorption of Cd and a phytoextraction efficiency of T. patula after N-fertilizer treatment. Moreover, our results are also supported by Yang et al. [42].

Photosynthetic Pigments
Maximum chlorophyll content was observed in the urea-treated plants at a 25 mg kg −1 Cd concentration, compared with the NH 4 NO 3 -treated and control plants ( Figure 2). Our results support the previous reports [43], in which the author claimed that different N forms, e.g., ammonium nitrate (NH 4 NO 3 ), ammonium sulfate ((NH 4 ) 2 SO 4 ), and calcium nitrate (Ca (NO 3 ) 2 ), significantly altered the Chl a, Chl b, total Chl, and carotenoid contents under Cd stress. Cadmium is a phytotoxic metal that causes growth inhibition. A high dose or prolonged exposure to Cd can lead to the death of plants, which is the result of disturbed respiration, reduced photosynthesis, and altered assimilation of N in plants [44]. Cadmium toxicity alters the N metabolism directly or indirectly [45]. It was found that an optimum N dosage may alleviate Cd toxicity to plants by improving the photosynthetic activity, stromal proteins, and the plant growth and biomass [46]. Under stress conditions, plants provoke antioxidant enzymes and certain metabolite activities for their survival in order to negate stress [47]. Jalloh et al. [48] reported that an appropriate dosage of urea and NH 4 + -N under Cd stress stimulated and increased the SOD and POD activities. Significantly increased MDA content in Cd-stressed plants were observed with addition of NH 4 + -N and/or urea in rice plants at the milking stage.

Oxidative Stress, Antioxidant Enzymes, and Cd Concentration
Malmir [49] found that Cr-induced oxidative stress, subsequently increasing the H 2 O 2 and MDA contents. Similar evidence of Cr-induced oxidative stress was observed in Helianthus annuus L. [50] and Brassica. Various studies have shown that different amendments have reduced the Cd toxicity in different plant species, for example, EDTA reduced Cd toxicity in Brassica [51], silicon in Brassica napus L. [52], hydrogen peroxide in Brassica napus L. [53], supermine in Vigna radiate L. [54], and selenium in rapeseed seedlings [55]. The markedly reduced EL and MDA contents in the N-fertilizer-treated, Cd-stressed plants in our study demonstrate the significant role of N fertilizers for Cd stress. Our results revealed that SOD, POD, CAT, and APX activities of S. nigrum were recorded in response to Cd stress. Furthermore, the POD, CAT, SOD, and APX activities significantly increased after the application of N-fertilizers. Similar results were observed previously [43], indicating that N forms had a prominent impact on oxidative stress inflicted by Cr toxicity. Therefore, the Ca(NO 3 ) 2 -fed plants had minimal oxidative stress compared with the (NH 4 ) 2 SO 4 and urea-treated plants.
Similar findings were observed for the N fertilizers for the phytoremediation of Cd, and even with different environmental conditions, soil textures, and experimental durations [56], and only a few analyses, we acknowledged the possible mechanisms for Cd phytoremediation with detailed analysis in our recent study. In addition, urea-treated plants translocated and accumulated a higher concentration of Cd than the NH 4 NO 3 -treated plants. However, the application of N-fertilizers inhibited oxidative damage and enhanced antioxidants' enzyme activities. Our results depicted that at all levels of applied Cd, the both TF and BCF were enhanced with the application of urea alone, as compared with all other treatments ( Figure 6E,F). This shows that among the various treatments, the plants treated with urea alone had greater potential for Cd phytoremediation. In conclusion, the suitable concentration of urea remarkably promoted and assisted the processes of Cd phytoremediation by S. nigrum.

Conclusions
Our study concludes that the application of an appropriate dosage of N fertilizers (NH 4 NO 3 and urea) could be a suitable practice to enhance the remediation of heavy-metal-polluted soils when growing S. nigrum. The plant biomass increased significantly with N fertilizer addition. Maximum plant growth was observed in the urea-treated plants compared with the NH 4 NO 3 -treated plants. In addition, the urea-treated plants also accumulated a higher Cd than the NH 4 NO 3 -treated plants. The application of N fertilizers inhibited oxidative stress and enhanced the antioxidants' enzymatic activities. It is concluded that urea might be helpful for better growth of S. nigrum under Cd stress. Our results indicate that the best N-fertilizer is urea, and that it is recommended for the phytoremediation of Cd. However, the application of urea for heavy metals phytoremediation other than Cd is still unknown, and future studies can be helpful for this investigation.