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Article

Acidified Cow Dung-Assisted Phytoextraction of Heavy Metals by Ryegrass from Contaminated Soil as an Eco-Efficient Technique

by
Sana Ashraf
1,*,
Sajid Rashid Ahmad
1,
Qasim Ali
2,
Sobia Ashraf
3,
Muzaffar Majid
1 and
Zahir Ahmad Zahir
4
1
College of Earth and Environmental Sciences, Quaid-e-Azam Campus, University of the Punjab, Lahore 54590, Pakistan
2
Department of Soil Science, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
3
Department of Environmental Sciences, Government College University Faisalabad, Faisalabad 38000, Pakistan
4
Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040, Pakistan
*
Author to whom correspondence should be addressed.
Sustainability 2022, 14(23), 15879; https://doi.org/10.3390/su142315879
Submission received: 4 November 2022 / Revised: 13 November 2022 / Accepted: 19 November 2022 / Published: 29 November 2022
(This article belongs to the Special Issue Sustainable Ecological Environment Restoration)
Browse Figures
Versions Notes

Abstract

:
Heavy metal contamination of soil is an alarming environmental dilemma all over the world. With increasing industrialization, timely development of low-cost and eco-friendly remedial techniques for heavy metal-contaminated soil is needed. Phytoremediation is an emerging technique to remove heavy metals from contaminated soil for environmental sustainability. In the present study, ryegrass was used for phytoextraction of lead and cadmium from contaminated soil in a pot experiment. To enhance the bioavailability of heavy metals, cow dung was acidified by amending with elemental sulfur and molasses and also bioaugmented with an SS-16 sulfur-oxidizing strain to boost biological sulfur oxidation and, hence, four chemically different organic products were prepared. The pot experiment was conducted for a period of 60 days under Pb- and Cd-spiked soil for growing ryegrass with the application of a 10% slurry of each acidified organic product. A significant increase in root and shoot fresh mass as well as Pb and Cd accumulation in the root and shoot of the ryegrass was recorded. As compared to the control and the acidified organic product, P4 was the most effective product overall. Bioconcentration and translocation factors of ryegrass for Pb and Cd were also calculated. At the same time, acidified cow dung slurry (10%) also improved the antioxidative defense mechanism of ryegrass. The results suggest that acidified organic products could be effective for phytoextraction of lead and cadmium from contaminated soil, and in the future acidified cow dung slurry can be used to restore heavy metal-polluted soils in an environmentally sustainable way.

1. Introduction

Industrial revolution and anthropogenic activities have aggravated the problem of environmental contamination. Heavy metals (HMs) concentrations in the ecosystem have increased considerably in recent decades. These HMs are incredibly dangerous environmental pollutants owing to their persistence, toxicity, and bioaccumulation within living bodies [1,2]. Use of sewage water to irrigate agricultural lands serves as one of the key reasons for the accumulation of HMs in soil [3]. Their transfer and subsequent biomagnification via food chains threaten all life forms [4,5]. They have raised severe issues at the local, regional, and national levels [6]. Biomagnification of different HMs—such as cadmium (Cd), arsenic (As), lead (Pb), cobalt (Co), mercury (Hg), nickel (Ni), chromium (Cr), and copper (Cu)—have substantially impacted various living organisms as well as the marine ecosystem [7,8]. Organisms at higher trophic levels are more vulnerable to biomagnification, and the presence of toxic HMs can endanger their lives [5]. That HMs induced toxic impacts on the environment and terrestrial organisms can be demonstrated by significant changes in the physicochemical and biological properties of soil [9,10]. Over the years, the HMs accumulate in various plant parts and result in decreased plant growth and development [11,12]. HMs contamination leads to severe morphological, biochemical, physiological, and ultra-structural alterations in plants [13,14].
Among HMs, Pb and Cd are the most hazardous elements; they are enormously poisonous to human health even at minute concentrations and are added to the ecological system on account of various environmental and anthropogenic activities [15]. Different sources of Pb include mining, Pb-acid batteries, fuels, insecticides, and automobile exhausts [16]. Pb and its compounds impart adverse impacts on the digestive, cardiovascular [17], hematopoietic, and endocrine systems etc. [18]. Upon their accumulation, Pb and Cd severely damage the kidneys and nerves [19]. In addition, these toxic elements create free radicals, which can impair proteins, DNA, and lipid molecules [20]. Cd accumulation in humans takes place via food chains and results in various disorders, i.e., cancer, neurotoxicity, nephrotoxicity, and Itai-Itai disease [21]. In plants, Cd stress results in chlorosis and interferes with the absorption of essential elements like phosphorus, potassium, calcium, and magnesium and consequently plant growth is suppressed. Furthermore, Cd accumulation inside plants affects the activity of antioxidant enzymes as well as carbohydrate metabolism [3,22,23]. Plants under Pb stress also suffer from mineral deficiencies, water imbalance, and reduced membrane permeability as well as enzyme activities [12]. Moreover, higher Pb concentrations also affect chlorophyll synthesis [24].
Numerous in-situ and ex-situ techniques have emerged for the management of polluted sites, such as soil washing, vitrification, and encapsulation [25]. Although these management techniques are efficient in soil treatment, they are costly, time consuming, and often not recommended for treating larger volumes of soil [4]. Phytoremediation, on the contrary, is a more worthwhile alternative owing to great applicability, easier execution, and low cost. Phytoremediation reflects using plants alongside amendments/microorganisms for improving soil quality via different processes, such as pollutant degradation, stabilization, or extraction [26]. Phytoextraction refers to the accumulation of toxic substances in the plant body after their extraction from the soil and is also known as phytoaccumulation. It is effectively used for removing toxic contaminants from soil, beginning with their uptake from contaminated sites, followed by transport from roots towards above-ground plant parts, where they are ultimately accumulated in these tissues [27]. The phytoextraction process is highly dependent upon the absorption, translocation, sequestration, and accumulation of high levels of toxic contaminants in above-ground plant parts, and all these factors characterize hyperaccumulating plants. The phytoextraction process occurs in the rootzone of the plants, and mobility as well as bioavailability of the contaminant are the key factors in the smooth running of the process. Increased mobility of HMs can be achieved by supplementing certain chelating agents that have the potential to make complexes with HMs in the soil solution [28]. Moreover, several organic and inorganic amendments have been used in recent decades to accelerate the restoration of contaminated sites [29].
Phytoextraction efficiency is sometimes halted by low solubility of HMs in soil solution as well as less availability for plant uptake [30]. Generally, these issues can be addressed by adopting some common practices, such as using synthetic chelates [31] or applying different acids or acidic fertilizers [32] for reducing the soil pH and improving the solubility of HMs. But they negatively affect plants and soil properties, as well as cause groundwater pollution via leaching [33]. Elemental sulfur (S0) application to soil reduces pH and enhances the solubility and bioavailability of HMs [34]. S0 is also required for the normal growth and functioning of plants [35,36], but the high cost of S0 input and its poor oxidation due to less population of SOB in soil limits its application. The application of organic amendments has resulted in a tremendous acceleration in the activities of these bacteria, as well as S oxidation in the soil [37]. Ryegrass (Lolium perenne L.) is widely distributed throughout the world. It is often used as a turf grass on account of its high tolerance and adaptability, fast growth rate, and regeneration ability, all of which reflect its strong tendency to grow at polluted sites. It has been reported that ryegrass has tremendous growth adaptability in contaminated sites and possesses high contaminant degradation rates [38]. Thus, elemental sulfur can be applied in cow dung and bioaugmented with SOB to acidulate the cow dung and to enhance the efficiency of phytoextraction process. Hence, keeping in consideration the significance of agricultural soil and associated detrimental consequences of heavy metals, the present research was conducted to evaluate the potential of ryegrass for lead and cadmium uptake and accumulation from the soil amended with acidulated cow dung slurry.

2. Materials and Methods

2.1. Preparation of Acidulated Cow Dung Slurry

The current study was performed to evaluate the effect of four different type of products on the phytoextraction potential of ryegrass against Pb and Cd from contaminated soil. The four products were prepared by combining cow dung (CD) with elemental sulfur (S°) @ 7.5 g kg−1 of CD, molasses, and sulfur-oxidizing bacteria (SOB). Product one (P1) comprised of only CD integrated with S° @ 7.5 g kg−1 of CD, whereas product two (P2) consisted of CD along with S° @ 7.5 g kg−1 of CD and molasses. Similarly, product three (P3) contained the combined application of CD, S° @ 7.5 g kg−1 of CD and SOB. The fourth product (P4) used a combination of all of these. These products were tested under normal as well as contaminated soil conditions (Pb- and Cd-spiked soil), and their comparative phytoextraction efficacy was investigated by using ryegrass as a test crop. The optimization experiment for S° indicated 7.5 g kg−1 CD as the best level. Similarly, the optimization experiment for molasses depicted 0.05% as the best rate on the basis of the pH reduction test. The SOB in the current trial were isolated and characterized in a previous study [39]. A brief description includes sampling from various ecologies—i.e., sewage water and sludge, industrial waste sludge, tannery effluent, sulfur-contaminated soil, cow dung and normal soil—and bacteria were isolated by following the dilution plate technique on thiosulfate medium. The bacterial colonies obtained from the sewage sludge indicated maximum efficiency, and these bacteria were screened and characterized for different activities to obtain the most efficient bacterial strains using methods such as pH reduction, the sulfate ion production test, minimum inhibitory concentration (MIC), P-solubilization potential, the catalase and oxidase test, and siderophore production by following suitable protocols. Later, 16S rRNA gene sequencing was used for identifying the successful bacterial isolates. Based on the results of the principal component analysis (PCA), gram positive strain (SS-16) proved to be more efficient among different strains in all the measured attributes, which was used in the current experiment. At the end of the experiment, four different products P1, P2, P3, and P4 having different pH 1.67, 1.46, 1.34, and 1.25, respectively, were prepared. The 10% slurry of each product was prepared to be applied in the pot trial.

2.2. Pot Trial

The pot trial was conducted at the wire house of the Institute of Soil and Environmental Sciences (ISES), University of Agriculture Faisalabad (UAF), to examine the Pb and Cd accumulation potential of ryegrass from the soil amended with a 10% slurry of each product. The experiment was conducted under normal, Pb-, and Cd-spiked soil, and 5 kg soil per pot was used. The spiking levels of Pb and Cd were kept at 600 mg Pb/kg soil and 80 mg Cd/kg soil, and Pb(NO3)2 and Cd(NO3)2 salts were used for spiking. The wetting-drying mixing technique was continued for two months to equilibrate adsorption of added Pb and Cd salts. Twenty seeds of ryegrass (Lolium perenne) were sown in each pot and thinned to 12 seedlings per pot 1 week after germination. Basal fertilizers were applied to the soil at the rates of 75 mg N/kg (CH4N2O), 80 mg P/kg ((NH4)2HPO4), and 100 mg K/kg (K2SO4). A 10% slurry of each product at field capacity level was applied after every 15 days and the grass was grown for 60 days. There were three replications of each treatment and the CRD factorial design was used. The parameters related to plant physiology (SPAD, chlorophyll ‘a’, ‘b’, and carotenoid contents) and different gas exchanges were also recorded. Upon harvesting, different parameters related to plant growth were recorded and the degree of HMs accumulation was assessed in terms of the bioconcentration factor (BCF) and translocation factor (TF). Soil for determining the Pb and Cd concentrations was collected from 6 inches of depth. Moreover, the soil used in the current experiment was pre-characterized for different physiochemical attributes—i.e., textural analysis (sandy clay loam), volumetric water contents (39%), organic matter contents (0.71%), pH (7.96), EC (1.43 dS m−1), total nitrogen (N; 0.05%), available phosphorous (P; 6.13 mg kg−1), and extractable potassium (K; 115 mg kg−1)—and the total concentration of Pb and Cd in the soil was calculated by following the methods described by [40].

2.3. Plant Analyses

Upon completion of the experiment, all the plant samples were rinsed with de-ionized water and oven-dried for 48 h at 70 °C following their grinding by agate mill. Later, these samples were digested to determine the Pb and Cd contents.

2.3.1. Digestion

The method of [41] was followed for digesting the plant samples prior to their elemental analyses. For the purpose, a di-acid mixture comprising conc. HNO3 and HClO4 in 9:4 ratio was used. Colorless material obtained upon completion of the digestion process was filtered and further diluted using DI water.

2.3.2. Pb and Cd Determination

The Pb and Cd concentrations in all of the plant samples were determined by using atomic absorption spectrophotometry (AAS), where PbSO4 and CdSO4 were used for preparing the stock solution and working standards.

2.3.3. Chlorophyll Contents

The SPAD value was recorded during 10–11 a.m. by using the chlorophyll meter (SPAD-502). Each leaf sample was monitored at three different areas and the average of three readings was taken. Similarly, chlorophyll ‘a’, ‘b’ and carotenoid contents were measured spectrophotometrically at 663, 645, and 480 nm, respectively [42], by making a leaf extract of each plant sample in acetone (80%).

2.3.4. Photosynthesis System

The combined infrared analyzing system (CIRAS-3, USA) was used as a portable photosynthesis measurement system to record the photosynthetic attributes of flag leaves during 8:00 to 10:00 a.m. [43].

2.3.5. Antioxidant Determination in Leaves

Ascorbate Peroxidase (APX)

The APX activity in the leaf samples was determined by measuring the decrease in the absorbance of ascorbate at 290 nm due to H2O2 activity. For the purpose, phosphate buffer (50 mM; pH 7.0) was prepared and enzyme extract (20 µL) was added to 660 µL of buffer with exactly the same volume of H2O2 and ascorbic acid solution. The addition of H2O2 started the reaction, so it was added last. It was expressed as nmol ascorbate min−1 mg−1 protein [44].

Catalase (CAT) Assay

The reaction mixture (3 mL) comprised of 2 mL of crude enzyme extract and 1 mL of 10 mM H2O2 and was based on the decline in absorbance due to H2O2 extinction at 240 nm. It was expressed in nmol H2O2 min−1 mg−1 protein [45].

Superoxide Dismutase (SOD) Assay

The SOD activity was measured by method of [46], where 50 mM of sodium phosphate (pH 7.8) buffer, 20 mM pyrogallol, 20 mM of enzyme extract, and 10 mM EDTA made up 1 mL of the reaction mixture, which was spectrophotometrically monitored at 420 nm for 120 s and expressed as nmol mint−1 g−1 protein.

Lipid Peroxidation

The malondialdehyde (MDA) contents were measured to reflect the lipid peroxidation under HMs stress, where 0.5 mL of crude leaf (made in 0.1% trichloroacetic acid) extract was heated with thiobarbituric acid (0.5%) and TCA (20%) for a half hour in a fume hood at 95 °C and cooled using an ice bath. The absorbance of the reaction mixture was first observed at 532 nm and then at 600 nm, and the activity was calculated on the basis of difference between wavelengths (A532-A600) and was measured as nmol MDA mg−1 protein [47].

Peroxidase (POD)

The colorimetric determination of the POD activities in the ryegrass leaves was performed on the basis of the method of [48]. The assay consisted of 4-aminoantipyrine, phenol and H2O2 as coloring agent. The rate of color generation was directly proportional to the rate of H2O2 consumption, which reflected the POD activities at 510 nm wavelength. The PD activity was expressed as µ mol H2O2 min−1.

Determination of H2O2 Contents

Leaf tissue (0.2 g) was homogenized with 3 cm3 of trichloroacetic acid (0.1% m/v) in an ice bath followed by centrifugation for 15 min at 12,000× g. For this purpose, 0.5 cm3 aliquot of the supernatant was added to 1 cm3 of 1M potassium iodide and 0.5 cm3 of buffer (pH 7.0). Absorbance was read at 390 nm and H2O2 contents were determined using the coefficient of absorbance (ε) 0.28 μM−1 cm−1 [49].

2.3.6. Statistical Analyses

For the statistical analyses of the data, Statistix® software (version 9.1,) was used, means were computed, and graphs were constructed by using Microsoft Excel 2019®. Similarly, the Least Significant Difference (LSD) test was used as a post-hoc test for comparing the significant means [50].

3. Results

In the current pot trial, the heavy metal accumulation potential of ryegrass was evaluated by preparing four different products comprising acidified cow dung slurry amended with molasses, SOB, and a combination of both under normal, Pb-, and Cd-spiked soils. The contaminated soil conditions led to a substantial decline in all the plant growth and physiological attributes, whereas application of cow dung slurry ameliorated the toxic impacts of these hazardous contaminants on all the measured attributes. Details of all the findings is given below.

3.1. Growth Attributes

Under normal soil conditions, maximum increment in plant height (17%), root length (23%), shoot fresh (17%) and dry weights (72%), root fresh (50%) and dry weights (50%) was observed under the application of P4 followed by P3, which increased the plant height (14%), root length (17%), shoot fresh (15%) and dry weights (55%), root fresh (29%) and dry weights (19%) (Table 1 and Table 2). Similarly, under Pb contamination, the P4 application resulted in a tremendous improvement in all the growth attributes—i.e., plant height (26%), root length (1.1 fold), shoot fresh (72%) and shoot dry weights (1.38 fold), root fresh (96%) and dry weights (2.05 fold)—in comparison with the corresponding control treatment, followed by P3, which increased the growth attributes, i.e., plant height (20%), root length (91%), shoot fresh (51%) and dry weights (1.16 fold), root fresh (80%) and dry weights (1.26 fold). In the same way, under Cd-contaminated soil conditions, the application of P4 led to significant increment in all the measured growth traits—such as plant height (24%), root length (43%), shoot fresh (52%) and dry weights (1.35 fold), root fresh (87%) and dry weights (1.97 fold)—in comparison with its corresponding control treatment, followed by P3, which increased the growth attributes, i.e., plant height (18%), root length (27%), shoot fresh (47%) and dry weights (89%), root fresh (56%) and dry weights (1.55 fold). So, the application of P4 (cow dung + elemental sulfur + molasses + SOB) proved to be the most efficient in terms of mitigating the adverse effects of Pb and Cd contamination on the growth attributes of ryegrass, followed by P3 (cow dung + elemental sulfur + SOB), in comparison with the control treatment.

3.2. Physiological Attributes

The outcomes regarding the effect of Pb and Cd stress on the physiological attributes of ryegrass (Table 3) depicted a negative correlation between heavy metal contamination and plant physiology, i.e., SPAD value, chlorophyll ‘a’, chlorophyll ‘b’, and carotenoid contents. It was revealed from the results that the application of acidified cow dung slurry alone and in combination with molasses and SOB significantly alleviated the heavy metal-mediated toxic effects on the physiology of ryegrass. In the absence of heavy metal contamination, the P4 application caused a significant improvement in all the measured plant physiological attributes, such as SPAD value (1.19 fold), chlorophyll ‘a’ (17%), chlorophyll ‘b’ (22%), and carotenoid contents (40%), as compared to the control treatment.
The P4 application under Cd contamination increased the SPAD value (65%), chlorophyll ‘a’ (57%), chlorophyll ‘b’ (62%), and carotenoid contents (33%) more than the control treatment, followed by P3, which also significantly increased the SPAD value (55%), chlorophyll ‘a’ (41%), chlorophyll ‘b’ (33%), and carotenoid contents (33%). A similar trend was observed under Pb stress, where the application of P4 improved the plant physiology, as evidenced by the tremendous increase in the SPAD value (79%), chlorophyll ‘a’ (19%), chlorophyll ‘b’ (35%), and carotenoid contents (75%) in comparison to their respective control treatment, followed by P3, which increased the SPAD value (55%), chlorophyll ‘a’ (11%), chlorophyll ‘b’ (22%), and carotenoid contents (52%) over control. It was concluded that the P4 application yielded the most significant outcomes in terms of improving the plant physiology under heavy metal stress.

3.3. Gas Exchange Attributes

Data regarding the effect of heavy metal contamination on the gas exchanges attributes of the ryegrass leaves and the effect of acidified cow dung application in mitigating their adverse impacts (Figure 1) revealed that heavy metal contamination (Pb and Cd) imparted adverse effects on the gas exchange traits of ryegrass. However, the acidified cow dung amendment to the soil solely or in combination with molasses and SOB improved the gas exchange in the leaves of ryegrass under normal and contaminated soil conditions. The maximum increment under the stressed conditions in the measured gas exchange traits—i.e., photosynthetic rate (1.19 fold), stomatal conductance (46%), transpiration rate (58%), and Water use efficiency (38%)—were pragmatic due to application of P4, followed by P3, as compared to the uncontaminated soil conditions. Similarly, the P4 application under Cd stress uplifted the gas exchanges attributes of ryegrass leaves—for example, transpiration rate (37%), photosynthetic rate (3.08 fold), water use efficiency (1.97 fold), and stomatal conductance (72%)—were prominent due to application of P4 followed by P3, which increased the photosynthetic rate (1.49 fold), transpiration rate (23%), stomatal conductance (47%), and Water use efficiency (1.01 fold) in comparison to the corresponding control treatment. Similarly, under Pb-contaminated soil conditions, the exogenous application of P4 caused maximum increment in the gas exchange properties of ryegrass leaves—i.e., photosynthetic rate (3.86 fold), transpiration rate (42%), stomatal conductance (66%), and Water use efficiency (2.41 fold)—followed by P3 application, which improved the photosynthetic (2.71 fold) and transpiration rates (29%), stomatal conductance (34%), and Water use efficiency (1.89 fold) in comparison to the corresponding control treatment. Therefore, the P4 product comprising the acidulated cow dung slurry along with molasses and SOB displayed the most promising results in terms of increasing the gas exchange attributes under heavy metal contamination, followed by product P3, which consisted of acidulated cow dung slurry in combination with SOB (Figure 1).

3.4. Antioxidant Activities

The application of acidulated cow dung slurry individually as well as in combination with SOB or molasses mitigated the effects of heavy metal-mediated oxidative stress (Figure 2) on the ryegrass plant as evidenced by improved antioxidant activities in all of the treatments in comparison to the control. Under normal soil conditions, maximum decrement in the antioxidant activities—i.e., H2O2 contents (13%), MDA contents (4%), APX (6%), CAT (4%), SOD (0.04%) and POD (6%) activities—was observed under the application of P4, followed by P3, which decreased the H2O2 contents (11%), MDA contents (3%), APX (6%), CAT (19%), SOD (0.01%) and POD (4%) activities.
Similarly, under Pb contamination, the P4 application resulted in a tremendous improvement in all of the antioxidant activities—i.e., H2O2 contents (60%), APX (1.71 fold), MDA (46%), SOD (81%), POD (66%) and CAT (50%) activities—in comparison to the corresponding control treatment, followed by P3, which decreased antioxidant activities, such as H2O2 contents (53%), APX (1.29 fold), MDA (40%), SOD (51%), POD (55%), and CAT (34%). Moreover, under Cd-contaminated soil conditions, the application of P4 led to significant improvement in all of the measured antioxidant activities—such as H2O2 contents (57%), APX (1.26 fold), MDA (24%), SOD (32%), POD (36%), and CAT (45%)—in comparison to the corresponding control treatment, followed by P3, which reduced the antioxidant activities, i.e., H2O2 contents (36%), APX (98%), MDA (19%), SOD (20%), POD (26%), and CAT (30%). Thus, the application of P4 (cow dung + elemental sulfur + molasses + SOB) proved to be the most efficient in terms of mitigating the adverse effects of Pb- and Cd-induced oxidative stress on the ryegrass, followed by P3 (cow dung + elemental sulfur + SOB), in comparison with the control treatment (Figure 2).

3.5. Heavy Metal (Pb and Cd) Concentration in Plant Parts and Soil after Harvest of Ryegrass

The data regarding the effect of acidulated CD slurry on the HMs uptake by the ryegrass shoots and roots (Figure 3) indicated that the application of P4 substantially increased the solubility of heavy metals and improved the Pb (52%) and Cd (56%) uptake by ryegrass shoots, followed by P3, which increased the concentration of Pb (35%) and Cd (43%) by ryegrass shoots in comparison to the control treatment. Similarly, P4 application maximally increased the Pb (39%) and Cd (26%) uptake by ryegrass roots as compared to the control treatment, followed by P3, which increased the Pb (24%) and Cd (23%) uptake by ryegrass roots over the control treatment. Similarly, the impact of acidulated CD slurry alone and along with molasses and SOB on the residual Pb and Cd concentration in soil after the harvest of ryegrass (Figure 3) depicted that the maximum available heavy metal concentration was observed in the control treatment, whereas the P4 application led to the minimum percentage in the remaining Pb (57%) and Cd (61%) concentrations in the soil, followed by P3, which also decreased the residual Pb and Cd concentration by 75% and 95%, respectively, under contaminated soil conditions and in comparison to the control treatment (Figure 3).

3.6. Bioconcentration and Translocation Factors of Ryegrass for Lead and Cadmium

The bioconcentration factor (BCF) values for heavy metal concentrations revealed that the application of P4 significantly increased the BCF values for Cd and Pb by 26% and 39%, respectively, followed by P3, which increased the BCF values for Pb and Cd by 24% and 23%, respectively, over the control treatment (Figure 4). Moreover, the data regarding the translocation factor (TF) for Pb and Cd indicated that P4 application increased their TF by 22% and 40%, respectively, over the control, followed by P3, which increased the TF for Pb and Cd by 14% and 26%, respectively (Figure 4).
Thus, in all of the results, it was found that the P4 (Cow dung + sulfur + molasses + SOB) application was the most efficient in ameliorating the toxic effects of Pb and Cd on all of the measured attributes of ryegrass and increasing its phytoextraction potential. Furthermore, the BCF and TF values indicated that the ryegrass could be ideally used as a potential accumulator to decrease Pb- and Cd-contaminated soils.

4. Discussion

Heavy metal (HM) pollution has created alarming concern with respect to human health risks, hence the need to explore various efficient and effective remediation strategies has become inevitable. In recent times, phytoremediation has emerged as an extremely promising approach owing to its inexpensive, environment-friendly, and aesthetically more pleasing nature than other remediation techniques. In the current investigation, the phytoremediation ability of ryegrass was evaluated due to ryegrass’ relatively fast growth rate, high biomass, deep massive and fibrous root system, adaptable nature under severe environmental circumstances, and most importantly, high tolerance of heavy metal toxicities. For improving the bioavailability of HMs, cow dung was acidified by amending it with elemental sulfur and also bioaugmented with SOB. Similarly, a C source for heterotrophic SOB was added to the acidified cow dung in the form of molasses.
The effect of HMs on growth attributes depicted a negative correlation between heavy metal stress and plant growth traits. These findings are in line with the results of earlier studies [3,51,52]. But the application of acidified cow dung slurry alone and amended with molasses and SOB mitigated the adverse effects of HMs on the growth attributes of ryegrass. The findings of this study are somewhat similar to those observed by Jacob et al. [53], who checked the effect of cow dung application in ameliorating the hazardous impacts of Pb, Cd, and Cr on the growth of maize plants grown in contaminated soil. Their results indicated that the cow dung application remediated the heavy metal-induced toxic effects on the maize plant and improved its growth. Similar results were obtained by Baghaie [54], who stated that application of sulfur and cow manure amended with Thiobacilus bacteria resulted in a considerable increase in the Cd concentration of the plant. Improvement in plant growth under contaminated soil might be due to the beneficial role of sulfur in decreasing soil pH, which resultantly led to a considerable increase in nutrient uptake by the plant and, hence, plant growth was improved. Besharati et al. [55] also reported that the application of sulfur in the presence of Thiobacillus resulted in significant improvement in the plant biomass.
In the current study, a significant correlation was observed between the applied acidified cow dung slurry (alone and in combination with molasses and SOB) and the photosynthetic attributes of the ryegrass plant, where the maximum improvement in the plant physiology was observed in the product P4, which was comprised of sulfur-amended CD slurry, SOB, and molasses (as a C source). The harmful impacts of heavy metals on plant physiology might be ascribed to a disturbance in mineral uptake under stressed conditions, which may have disturbed enzyme activities and subsequently decreased the biosynthesis of photosynthetic pigments [56,57]. Moreover, Bashir et al. [57] reported that the combined application of biochar and elemental sulfur in the presence of SOB mitigated the adverse impacts of Cr on the maize physiology and improved the chlorophyll contents and the efficiency of the photosynthetic apparatus. A negative correlation was observed between the HMs contamination and gas exchange traits of ryegrass leaves, and at the same time, a substantial increase in the gaseous exchange attributes upon the sole application of acidified cow dung slurry and its combination with molasses and SOB under Pb and Cd stress was also found in the current study. The heavy metals decrease the size and number of leaves, the size of stomata, and also the cell membranes of stomatal guard cells [58], which lead to turgor loss in the leaves and, as a result, the gaseous exchange, i.e., the transpiration rate, is affected [59]. Similarly, water use efficiency is related to the stomatal ability to efficiently utilize water for producing their biomass. Under heavy metal stress, the stomatal closure also resulted in a considerable decline in the water use efficiency of the plant. However, the application of acidified cow dung slurry reduced the soil pH and increased the nutrient uptake by the plant, and in this way, might have improved the efficiency of the photosynthetic apparatus and, hence, the overall gas exchange traits.
Furthermore, a significant relation was found between the applied acidified cow dung slurry and the antioxidant defense system of the plant under heavy metal contamination. Under heavy metal stress, the plants are exposed to the production of numerous reactive oxygen species (ROS), which results in lipid peroxidation and damage to the structural and functional integrity of cellular membranes [60,61]. However, the application of acidified cow dung slurry along with SOB and molasses led to a tremendous decline in antioxidant enzymes activities, and this might be due to the greater improvement in nutrient uptake under low pH conditions, which might have diluted the heavy metal stress to the plants, and relatively less activity of antioxidant enzymes was observed. The lower production of antioxidant enzymes is an index of less oxidative stress to the plant.
We also observed a considerable increase in the heavy metal solubility as well as uptake by the plant under the application of acidified cow dung slurry along with SOB and molasses as a carbon source, as evidenced by the obtained BCF and TF values, which indicated the phytoextraction potential of ryegrass. An increase in the solubility and availability of heavy metals in the current experiment is due to the activity of SOB, which oxidized the elemental sulfur present in the cow dung and converted it into sulfuric acid (H2SO4) and, resultantly, decreased the soil pH. The prevailing acidic conditions increased the availability, hence, uptake of the heavy metals by the plants and the phytoextraction capacity of the ryegrass plant was established.

5. Conclusions

a.
Phytoextraction is the most efficient ecofriendly soil reclamation technique and application of acidulated cow dung slurry enhanced the efficiency of this technique by increasing the availability of Pb and Cd to ryegrass.
b.
Application of cow dung slurry not only enhanced the availability of Pb and Cd, but also promoted the growth of ryegrass.
c.
Acidulated cow dung slurry also improved the antioxidative defense mechanism of ryegrass.
d.
For future research work, phytoextraction of heavy metals may be done at the field level with the application of acidulated cow dung slurry by growing the same or some other plant species for environmental sustainability.

Author Contributions

Conceptualization, S.A. (Sana Ashraf), S.R.A. and Z.A.Z.; Data curation, Q.A. and S.A. (Sobia Ashraf); Investigation, S.A. (Sobia Ashraf); Methodology, S.A. (Sana Ashraf); Software, S.A. (Sana Ashraf); Supervision, Z.A.Z.; Visualization, S.A. (Sobia Ashraf) and M.M.; Writing—original draft, S.A. (Sana Ashraf); Writing—review & editing, S.R.A., Q.A., M.M. and Z.A.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are thankful to the University of Agriculture, Faisalabad-Pakistan, for providing support and the necessary materials for the successful completion of the experiment.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Effect of acidified cow dung-amended different products on the gas exchange attributes (A) photosynthetic rate, (B) transpiration rate, (C) stomatal conductance, and (D) Water use efficiency of ryegrass under Pb and Cd stress. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB; Means sharing the same letter(s) are statistically similar to each other at the 5% probability level.
Figure 1. Effect of acidified cow dung-amended different products on the gas exchange attributes (A) photosynthetic rate, (B) transpiration rate, (C) stomatal conductance, and (D) Water use efficiency of ryegrass under Pb and Cd stress. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB; Means sharing the same letter(s) are statistically similar to each other at the 5% probability level.
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Figure 2. Effect of acidified cow dung-amended different products on the antioxidant activities (A) hydrogen peroxide, (B) ascorbate peroxidase, (C) malondialdehyde contents (D) superoxide dismutase (E) peroxidase, and (F) catalase activities of ryegrass leaves under Pb and Cd stress. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB; Means sharing the same letter(s) are statistically similar to each other at the 5% probability level.
Figure 2. Effect of acidified cow dung-amended different products on the antioxidant activities (A) hydrogen peroxide, (B) ascorbate peroxidase, (C) malondialdehyde contents (D) superoxide dismutase (E) peroxidase, and (F) catalase activities of ryegrass leaves under Pb and Cd stress. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB; Means sharing the same letter(s) are statistically similar to each other at the 5% probability level.
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Figure 3. Effect of acidified cow dung-amended different products on the (A) Pb concentration in shoot, (B) Pb concentration in roots, (C) Cd concentration on shoots, (D) Cd concentrations in roots, (E) Cd in soil, and (F) Pb in soil for Cd under Pb and Cd stress, respectively. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB. Means sharing the same letter(s) are statistically similar to each other at the 5% probability level.
Figure 3. Effect of acidified cow dung-amended different products on the (A) Pb concentration in shoot, (B) Pb concentration in roots, (C) Cd concentration on shoots, (D) Cd concentrations in roots, (E) Cd in soil, and (F) Pb in soil for Cd under Pb and Cd stress, respectively. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB. Means sharing the same letter(s) are statistically similar to each other at the 5% probability level.
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Figure 4. Effect of acidified cow dung amended different products on the (A) bioconcentration factor for Pb, (B) bioconcentration factor for Cd, (C) Translocation factor for Cd, and (D) Translocation factor for Pb in ryegrass under Pb and Cd stress, respectively. ND; not detected.
Figure 4. Effect of acidified cow dung amended different products on the (A) bioconcentration factor for Pb, (B) bioconcentration factor for Cd, (C) Translocation factor for Cd, and (D) Translocation factor for Pb in ryegrass under Pb and Cd stress, respectively. ND; not detected.
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Table
Table 1. Effect of acidified cow dung-amended different products on the shoot attributes of ryegrass under Pb and Cd stress.
Table 1. Effect of acidified cow dung-amended different products on the shoot attributes of ryegrass under Pb and Cd stress.
TreatmentsPlant Height (cm)Shoot Fresh Biomass (g)Shoot Dry Biomass (g)
ControlCd
(mg kg−1)		Pb
(mg kg−1)		ControlCd
(mg kg−1)		Pb
(mg kg−1)		ControlCd
(mg kg−1)		Pb
(mg kg−1)
Control57.36 ef 50.57 k50.21 i47.07 d 26.81 h30.23 g3.65 fg2.20 h2.30 h
P161.29 cd52.02 j 52.20 h50.55 c33.33 fg40.62 e5.17 bc3.58 g3.76 fg
P262.45 bc54.38 ij 53.17 gh51.56 bc35.18 f44.28 d5.58 b4.49 de4.15 ef
P365.22 ab59.83 i60.38 fg54.11 ab39.50 e45.51 d5.65 b4.15 ef4.98 cd
P467.06 a62.77 h63.05 de55.08 a40.70 e52.00 a–c6.28 a5.1 bc5.48 bc
HSD value3.47273.11960.54
Means sharing the same letter (s) are statistically similar to each other at 5% probability level.
Table
Table 2. Effect of acidified cow dung-amended different products on the root attributes of ryegrass under Pb and Cd stress.
Table 2. Effect of acidified cow dung-amended different products on the root attributes of ryegrass under Pb and Cd stress.
TreatmentsRoot Length (cm)Root Fresh Biomass (g)Root Dry Biomass (g)
ControlCd
(mg kg−1)		Pb
(mg kg−1)		ControlCd
(mg kg−1)		Pb
(mg kg−1)		ControlCd
(mg kg−1)		Pb
(mg kg−1)
Control47.71 ef28.79 k23.87 l6.72 de 3.37 g4.05 g3.16 ef1.46 i1.53 i
P151.43 cd32.26 j40.49 h8.20 b3.74 g5.25 f3.29 de2.60 h2.80 gh
P253.85 bc34.41 ij42.49 gh8.33 b4.02 g5.36 f3.48 d2.72 h2.98 fg
P355.84 ab36.50 i45.59 fg8.69 b5.26 f 7.28 cd3.77 c3.73 c3.46 d
P458.73 a41.25h50.15 de10.10 a6.3 e7.93 bc4.74 a4.35 b4.65 a
HSD value3.47270.77240.2102
Means sharing the same letter (s) are statistically similar to each other at 5% probability level. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB.
Table
Table 3. Effect of acidified cow dung-amended different products on the physiological attributes of ryegrass under Pb and Cd stress.
Table 3. Effect of acidified cow dung-amended different products on the physiological attributes of ryegrass under Pb and Cd stress.
TreatmentsSPAD ValueChlorophyll ‘a’ (mg g−1 Fresh Weight)
ControlCd
(mg kg−1)		Pb
(mg kg−1)		ControlCd
(mg kg−1)		Pb
(mg kg−1)
Control38.00 de23.60 h21.90 h2.4033 b1.2667 i1.4533 h
P141.83 c30.70 g28.27 g2.5133 b1.6200 e–g1.4600 h
P243.00 bc33.97 f29.30 g2.4767 b1.7033 d–f1.5300 gh
P344.53 b36.51 e33.90 f2.7067 a1.7800 d1.6100 fg
P448.00 a38.97 de39.10 d2.8167 a1.9833 c1.7333 de
HSD value2.47440.0901
TreatmentsChlorophyll ‘b’ (mg g−1 Fresh Weight)Carotenoid Contents (mg g−1 Fresh Weight)
ControlCd
(mg kg−1)		Pb
(mg kg−1)		ControlCd
(mg kg−1)		Pb
(mg kg−1)
Control0.6900 de0.4333 j0.5267 hi0.4200 de0.3000 jk0.2167 m
P10.7300 cd0.5033 i0.5867 fg0.4433 cd0.3200 ij0.2467 lm
P20.7633 bc0.5267 hi0.6100 fg0.4633 cd0.3500 g–i0.2700 kl
P30.8033 ab0.5767 gh0.6400 ef0.5267 b0.3600 gh0.3300 h–j
P40.8433 a0.7000 d0.7100 cd0.5867 a0.4000 ef0.3800 fg
HSD value0.01360.0112
Means sharing the same letter (s) are statistically similar to each other at 5% probability level. Abbreviations: P1: Cow dung + elemental sulfur (7.5 g/kg cow dung), P2: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses, P3: Cow dung + elemental sulfur (7.5 g/kg cow dung) + SOB, P4: Cow dung + elemental sulfur (7.5 g/kg cow dung) + molasses + SOB.
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Ashraf, S.; Ahmad, S.R.; Ali, Q.; Ashraf, S.; Majid, M.; Zahir, Z.A. Acidified Cow Dung-Assisted Phytoextraction of Heavy Metals by Ryegrass from Contaminated Soil as an Eco-Efficient Technique. Sustainability 2022, 14, 15879. https://doi.org/10.3390/su142315879

AMA Style

Ashraf S, Ahmad SR, Ali Q, Ashraf S, Majid M, Zahir ZA. Acidified Cow Dung-Assisted Phytoextraction of Heavy Metals by Ryegrass from Contaminated Soil as an Eco-Efficient Technique. Sustainability. 2022; 14(23):15879. https://doi.org/10.3390/su142315879

Chicago/Turabian Style

Ashraf, Sana, Sajid Rashid Ahmad, Qasim Ali, Sobia Ashraf, Muzaffar Majid, and Zahir Ahmad Zahir. 2022. "Acidified Cow Dung-Assisted Phytoextraction of Heavy Metals by Ryegrass from Contaminated Soil as an Eco-Efficient Technique" Sustainability 14, no. 23: 15879. https://doi.org/10.3390/su142315879

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