Straw Incorporation in Contaminated Soil Enhances Drought Tolerance but Simultaneously Increases the Accumulation of Heavy Metals in Rice

Abstract

Heavy metals (HMs) and drought stress are worldwide issues of concern because of their adverse effects on the growth and productivity of rice. Straw burning causes air pollution via greenhouse gas (GHG) emissions and it requires sustainable management. The introduction of HMs into the food chain poses a major health risk to humans. In this regard, straw incorporation into the soil could reduce air pollution and drought stress. However, its simultaneous impact on HMs’ uptake and drought stress tolerance in crops is unknown. Therefore, the present study aimed to investigate the impact of rice straw incorporation in soil on HMs (Cd, Cu, Pb, and Fe) availability, accumulation, and drought stress tolerance in rice (Oryza sativa L.) grown in pots under glasshouse conditions. The soil samples were collected from a non-contaminated agricultural field (control) and the contaminated field, irrigated with industrial effluent and treated with straw. Straw (1% w/w) was mixed in soils and control plants without straw application were grown under both contaminated and normal soil conditions. The results showed that straw incorporation in soils significantly enhanced the accumulation of HMs in rice grain and other vegetative parts of rice as compared to control. Moreover, straw application harmed chlorophyll and carotenoids. Straw application significantly increased proline in leaves (274.0 µg mL⁻¹) as compared to the control (166.8 µg mL⁻¹). Relative water contents were higher in straw-treated plants, thereby increasing drought stress tolerance. Straw application increased the accumulation of HMs and consequently reduced the biomass of the plant. In conclusion, straw incorporation enhanced drought stress tolerance but simultaneously elevated the accumulation of HMs under contaminated soil in Oryza sativa L.

1. Introduction

Rice (Oryza sativa L.) is the world’s most significant food crop, which is cultivated in over 9% of the cultivatable area on earth. It provides high-quality food to more than half of the world’s population and meets 21 and 15% of the world’s energy and protein needs, respectively [1]. It is grown under various climatic conditions with different temperature, soil, and water conditions. Adverse environmental conditions severely threaten rice productivity. Abiotic stresses, such as drought, heavy metals (HMs), and salinity, are a serious risk to sustainable rice production in many regions of the world [2,3]. Rice (Oryza sativa), being a paddy field crop, is more susceptible to drought stress. It has been estimated that approximately 50% of the production of rice is affected due to drought in the world [4]. Drought stress conditions make changes in the chlorophyll contents by affecting the chlorophyll contents and damaging the photosynthetic apparatus to inhibit photosynthesis. The reduction in chlorophyll contents under drought stress is due to damage to chloroplasts caused by reactive oxygen species [5].

Human exposure to HMs has intensely increased due to exponential growth in various industries [6,7,8,9,10]. The solid waste or liquid effluent contaminates water and surface soil, entering the food chain through plants or aquatic organisms and resulting in adverse effects on human health [11]. Cadmium (Cd) and lead (Pb) are among the most commonly found metals in contaminated soils as cited in recent literature [6,7]. Moreover, the inadequate supply of micronutrients such as Fe and Cu also disrupts various physiological and biochemical attributes of plants as well as humans [3]. HMs imbalance the ongoing metabolic processes, hormones, and enzymes in the human body through redox reactions, which cause oxidative damage to DNA and protein [12]. Some HMs such as arsenic, nickel, chromium, and cobalt are carcinogenic. An excessive amount of lead damages the nervous, circulatory, skeletal, endocrine, and immune systems. Cadmium causes kidney dysfunction, bone fracture, lung cancer, prostatic proliferative lesions, and pulmonary adenocarcinomas [13].

Plants uptake HMs that are present in soil solution and use some HMs in a minute amount for optimum growth. However, an excessive amount of these metals is toxic for plant growth and productivity [14]. The effects of heavy metal toxicity are different according to a particular heavy metal. HMs such as Pb, Cd, Hg, and As do not have any beneficial role in plant growth but pose negative effects even at very low concentrations in plants [15]. Shoot and root growth were significantly reduced in wheat grown under contaminated soil with 5 mg Cd kg⁻¹ [16]. Heavy metal toxicity severely affects photosynthetic activities and plant mineral nutrition, with enhanced enzyme activities involved in reactive oxygen species (ROS) scavenging [17]. Abiotic stress causes plants to produce ROS in chloroplasts, mitochondria, and peroxisomes, which are highly reactive and harm biological processes. Abiotic stress causes the generation of ROS such as superoxide (O₂•), hydroxyl radicals (•OH), hydrogen peroxide (H₂O₂), and singlet oxygen (•O). Plants generate ROS as a primary defense response against different environmental stresses. Long-term exposure results in the overproduction of ROS, which disturbs the normal redox state of cells. ROS can cause the oxidation of proteins and lipids in membranes, damage DNA structure, and inhibit enzymes to activate programmed cell death [18].

Proline is the most significant indicator of abiotic stresses. It plays a key role in osmotic adjustment and improving the capability of plants to bear dehydration at the cellular level, which is caused due to water shortage [19]. Normally, proline storage occurs in the cytoplasm and chloroplast of plants [20]. Even small variations in the level of proline in the cytosolic region had a significant impact on osmotic adjustment at the cellular level [21].

Straw is produced in large amounts during different farming activities annually. The straw produced is mostly burnt in most countries, which has become a threatening air pollutant in the form of smog and other greenhouse gases (GHGs) in different countries, especially in Asia [22]. It is the need of the hour to determine certain strategies, which help reduce the production of air pollutants and offer sustainable ways to manage this waste. The incorporation of organic amendments such as straw into the soil is one such strategy, which requires fewer management costs with no air pollutants. Straw is used in the soil to help minimize pollutants and enrich the soil and this approach is being encouraged in many countries. The incorporation of straw into the soil has a significant effect on soil and the atmosphere [23]. Straw is composed of different organic contents, which are not easily transformed into the soil via microbial decomposition as compared to other organic matter, humic and fulvic acids [24,25].

Straw incorporation increases the soil nutrients via carbon cycling through microbial activities [26]. Two different contradictory results were found, largely unexplained that the straw amendment either increases or decreases the mobility, bioavailability, bioaccumulation, and immobilization of HMs [27,28,29,30]. The addition of straw might decrease the availability of HMs with an increase in pH. Cui [27] found that rice straw added to artificial Cd-contaminated soil @ 6% (w/w), after 6 months of incubation significantly decreased total soluble concentration of Cd from 20 to 15 nmol L⁻¹ and free Cd concentration from 16 to 12 nmol L⁻¹. However, various studies have reported an increase in dissolved organic carbon (DOC) and HMs bioavailability with the addition of crop residues [22,23,24,25,26]. In comparison to the control treatment, straw added to Cu smelter-contaminated soil at a rate of 1% (w/w) dramatically increased Cd in plant stems from 197 to 365 mg kg⁻¹ [28]. Wheat straw 0.25 to 1% added to artificial Cd-contaminated soil increased the soluble Cd concentration by 10–33% and 120% in plant tissues [31]. Similarly, the addition of straw returns organic C, N, P, and Si but it also increases the mobility and bioavailability of metals. For example, MeHg concentration was increased (40–70%) in the plants grown under two mining-impacted soils after the straw amendment [32]. Despite the role of straw incorporation into the soil to mitigate pollution, the behavior of HMs and drought stress tolerance in crops with straw incorporation is still unclear. To clarify the real situation, the present study was conducted to investigate: (1) the impact of rice straw on physio-biochemical attributes and the accumulation of HMs in the root, stem, leave and grains, and (2) to study the impact of rice straw on drought and heavy metal stress tolerance in rice.

2. Materials and Methods

2.1. Soil and Straw Collection

The contaminated soil was collected from the industrially contaminated area and the normal soil from a non-contaminated agricultural field, which was used as reference soil. The HMs contaminated soil was collected from fields near the steel mill in Sakhakot and these fields have been irrigated with steel mill effluent for crop production. Soil samples were air-dried, ground, and sieved (2 mm). The rice straw was collected from the rice-harvested field in Chakdara. The straw was taken to the Laboratory of Biotechnology, which was cut into small pieces and then crushed into small pieces using a commercial grinder so that the small pieces of straw could easily be decomposed in soil.

2.2. Soil Analysis

Different physicochemical parameters such as pH, electrical conductivity (EC), texture, and water-holding capacity (WHC) of the collected soil samples were measured by using standard methods [33]. A pH meter (InoLab-WTB GmbH; Weilheim, Germany), an electrical conductivity meter (WTW–330i), and a hydrometer technique were used to determine pH, EC, and soil texture. The soil samples were digested with concentrated nitric acid (HNO₃), and perchloric acid (HClO₄) using a block digester. After digestion, the concentration of HMs was determined using an atomic absorption spectrophotometer (Perkin-Elmer Model No. 2380, Oak Brook, IL, USA).

2.3. Seeds Germination

Mature and well-developed rice seeds (cv. Fakhari Malakand) purchased from the Chakdara grain market were separated for germination purpose by dipping the seeds in a water beaker. The seeds were surface sterilized by dipping in 0.1% HgCl₂ for 20 min in a beaker and then washed (2–3 times) with sterilized distilled water. A plastic pot filled with river sand was taken. Then, rice seeds spread over the surface and were properly kept moist until seedlings emerged.

2.4. Transplantation of Rice Plants

Seedlings of rice having equal size were selected and one seedling was transplanted in each plastic pot (3 × 3 × 5 inches in length, width, and height, respectively). The pots were filled with 1 kg uniformly mixed soil. There were four treatments in total viz. C = Normal soil from the non-contaminated field only, T₁ = Normal soil with the addition of straw, T₂ = Contaminated soil without the addition of straw, and T₃ = Contaminated soil with the addition of straw. The treatments with straw addition received straw at the rate of 1% (w/w) which was added and mixed uniformly. The treatments were arranged in a completely randomized design (CRD) in triplicate. The pots were irrigated with tap water twice a week; the temperature was kept at 25 ± 2 °C in the 14/10 h (light/dark) photoperiod in the greenhouse. After four months of plant transplantation, plants in each treatment were harvested carefully and washed with tap water three times and then three times with sterile distilled water to eliminate the dust particle adhered to the plant surface. Different plant parts were separated and properly labeled in paper envelopes and stored in the refrigerator for further analysis. After harvesting plants, the root, shoot, and leaves biomass in each treatment was determined by using a digital balance. After harvesting the plants, leaf samples were used for the determination of different biochemical parameters.

2.5. Estimation of Chlorophyll Contents

The amount of photosynthetic pigments such as chlorophyll a and chlorophyll b was analyzed by using the procedure of Arnon [34]. The green leaf samples from the rice plants in both the control and treated pots were collected. Fresh leaves (200 mg) were taken in the mortar and homogenized with a pestle by adding 2 mL acetone (80%). The extract was poured into the centrifuge tube, which was centrifuged at 1000 rpm for 5 min. The supernatant was taken and transferred into a clean test tube. The volume was made at 6 mL using acetone. Then, the extract was analyzed for chlorophyll contents using a UV–visible spectrophotometer (UV-1700 Shimadzu, Kyoto, Japan). Acetone (80%) was used as blank. Three replicates from each treatment were used for the estimation of chlorophyll contents. Chlorophyll “a” was measured at 663 nm and chlorophyll “b” at 645 nm wavelength. The following formula was used for the calculation of chlorophyll “a”, “b” and “total chlorophyll contents”:

$$\mathrm{Chlorophyll} \mathrm{a} ({\mathsf{\mu }\mathrm{g} \mathrm{mL}}^{-1})=12.7\times {\mathrm{A}}_{663}-2.69\times {\mathrm{A}}_{645}$$

(1)

$$\mathrm{Chlorophyll} \mathrm{b} ({\mathsf{\mu }\mathrm{g} \mathrm{mL}}^{-1})=22.9\times {\mathrm{A}}_{645}-4.68\times {\mathrm{A}}_{663}$$

(2)

$$\mathrm{Total} \mathrm{chlorophyll} \mathrm{contents} ({\mathsf{\mu }\mathrm{g} \mathrm{mL}}^{-1})=8.02\times {\mathrm{A}}_{663}+20.20\times {\mathrm{A}}_{645}$$

(3)

2.6. Estimation of Proline in Leaves

For extraction and biochemical estimation of proline, the method proposed by Bates et al. [35] was used. Fresh leaf tissue (100 mg) was placed in 2 mL Eppendorf tubes, along with 1.5 mL sulfosalicylic (3%), and homogenized. The homogenate was centrifuged for 5 min at 13,000 rpm. After centrifugation, 300 mL supernatant, 2 mL of each glacial acetic acid, and acid ninhydrin were added to the test tube and left for 1 h at a boiling temperature in the water bath. Then, these tubes were transferred to an ice bath to stop the reaction immediately. After this, 1 mL toluene was mixed with the extract and mixed vigorously for 10–30 s. The toluene-containing chromophore layer was withdrawn from the top aqueous phase and pipetted to room temperature, after which the absorbance was measured using a UV–visible spectrophotometer (UV-1700 Shimadzu, Kyoto, Japan) at 520 nm. As a blank, toluene was employed. The proline contents were determined from different samples using a standard curve. For each sample, three replicates were used for proline estimation.

2.7. Estimation of Carotenoids Contents in Leaves

The carotenoids contents in leaf samples were estimated by using acetonic (90%) extract of leaves and analyzed at 480 nm wavelength using a UV–visible spectrophotometer (UV-1700 Shimadzu, Kyoto, Japan). Acetone (90%) was used as blank [36]. Specific wavelengths were used to measure the carotenoids contents. The following formula was used for the calculation of the carotenoids contents:

$$\mathrm{Carotenoid} \mathrm{contents} ({\mathsf{\mu }\mathrm{g} \mathrm{g}}^{-1} \mathrm{FW})={\mathrm{A}}_{480}+\left(0.114\times {\mathrm{A}}_{663}\right)-\left(0.638\times {\mathrm{A}}_{645}\right)$$

(4)

2.8. Relative Water Content

The assessment of drought resistance under different treatments was carried out by full leaf assays [37]. Distilled water with different test concentrations of polyethylene glycol (PEG 6000) 0, 5, 10, and 15% denoted by T₀, T₁, T₂, and T₃, respectively was prepared and sterilized. The sterilized solution was poured into a sterilized falcon tube under aseptic conditions in a laminar cabinet and properly labeled. Leaf samples from all the treatments having equal size were collected and surface sterilized with ethanol (70%) for a few seconds and with bleach (10%) for 10 min, followed by three rinses with sterilized water and transferred to properly labeled falcon tubes. Then, all tubes were kept overnight at room temperature to become turgid. The following day, the turgid weight (TW) of each leaf was recorded, using 5 decimal place analytical balance. After measuring TW, the tubes with leaf samples were incubated for three days. Then, the fresh weight of each leaf sample was measured and denoted as FW. The dry weight was determined after drying leaf samples from each treatment and denoted as DW. All the measurements were taken in triplicates. The relative water contents (RWC) for leaf samples in each treatment were calculated using the following formula:

$$\mathrm{Relative} \mathrm{water} \mathrm{contents} (\%)=\frac{\mathrm{FW}-\mathrm{DW}}{\mathrm{TW}-\mathrm{DW}}\times 100$$

(5)

2.9. Heavy Metals (HMs)

For HMs analysis, plant tissues were kept at 80 °C in an oven for 48 h and completely dried. The dried plant tissues were crushed in an electrical blender and converted into powder, properly labeled, and properly packed for further analysis. Then, the powder (0.5 g) from each root, stem, and leaf sample was taken in a 50 mL flask and a 6.5 mL mixture of concentrated nitric acid (0.1 N), concentrated sulfuric acid (0.2 N), and concentrated perchloric acid (0.01 N) in a 5:1:0.5 ratio was added. Then, these flasks were kept on the electronic hot plate in the fume hood and heated until a clear supernatant appeared [38]. The samples were allowed to cool at room temperature before raising the volume to 50 mL by adding deionized distilled water. The extract was filtered using Whatman filter paper, and the liquid was kept until it was evaluated for distinct HMs using an atomic absorption spectrophotometer (Perkin-Elmer Model No. 2380, Oak Brook, IL, USA).

2.10. Statistical Analysis

The collected data were expressed as the means ± standard deviations. For statistical analysis, the analysis of variance (ANOVA) technique was employed using GraphPad Prism (version 8.0 GraphPad Software LLC, CA, USA) and MS Excel (version 2016, Microsoft, Washington, DC, USA). The treatment means were compared using the least significant design test at α = 0.05.

3. Results

3.1. Physicochemical Properties of Soil

Soil samples from the industrially contaminated area and the normal soil from the non-contaminated field soil were tested regarding various physicochemical parameters (

Table 1. Physicochemical properties and heavy metal contents of the soil samples.

	Normal	Contaminated
EC (dS m−1)	3.04 ± 0.19 b	5.28 ± 0.80 a
pH	8.18 ± 0.09 a	7.91 ± 0.19 b
Texture	Loam	Loam
WHC (mL)	331.70 ± 4.4 b	350.0 ± 2.9 a
Cd (mg kg−1)	0.026 ± 0.002 b	0.248 ± 0.001 a
Pb (mg kg−1)	0.038 ± 0.002 b	0.503 ± 0.003 a
Cu (mg kg−1)	0.1810 ± 0.0057 b	1.252 ± 0.0034 a
Fe (mg kg−1)	40.03 ± 1.14 b	94.69 ± 0.57 a

The values following the mean values represent the standard deviation where n = 3. Different letters indicate a significant difference in treatments at p ≤ 0.05.

). The electrical conductivity of contaminated soil was higher than normal soil. The pH of the non-contaminated (as reference) soil sample was 8.18 while that of the industrial contaminated soil sample was 7.91. The result revealed that non-contaminated soil pH was higher than 7.0, which shows that both the soils were alkaline. The water-holding capacity of the contaminated soil sample was also higher than the normal soil sample. The HMs (Cd, Pb, Cu, and Fe) contents in contaminated soil were higher than in normal soil.

3.2. Fresh and Dry Biomass

Fresh and dry biomass were significantly decreased with the application of straw compared to control without straw application (Figure 1). Lower fresh and dry biomass, i.e., 1.142 and 0.173 g were recorded with the addition of straw (T₃) under contaminated soil while higher fresh and dry biomass, i.e., 1.606 and 0.273 g were recorded under normal soil without straw application (C).

3.3. Physiological Attributes

3.3.1. Chlorophyll Contents

The effect of straw and heavy metal on chlorophyll a, b, and total chlorophyll (a + b) contents are presented in Figure 2. The chlorophyll contents were significantly reduced in straw-treated plants when compared with control without straw application (Figure 2). The lowest amount of chlorophyll “a” (7.93 µg mL⁻¹) and “b” (19.11 µg mL⁻¹) and total chlorophyll (27.04 µg mL⁻¹) was found in the leaves of Oryza sativa L. grown under contaminated soil with the application of straw (T₃). Similarly, the highest chlorophyll “a” (13.94 µg mL⁻¹) chlorophyll “b” (29.88 µg mL⁻¹), and total chlorophyll (42.94 µg mL⁻¹) were recorded in plants grown under normal soil without straw (C).

3.3.2. Carotenoids Contents

The effect of straw application under normal and HMs contaminated soils is presented in Figure 3. With straw application, carotenoids contents were significantly decreased in comparison to the control without straw application. The lowest carotenoids contents, i.e., 0.025 µg mL⁻¹ were recorded in the rice plants treated with straw and grown under contaminated soil whereas the highest carotenoids contents, i.e., 0.042 µg mL⁻¹ were recorded in the rice plants without straw application and grown under normal soil.

3.3.3. Proline Contents

The impact of straw application on proline accumulation in rice plants is presented in Figure 4. The results showed that proline contents were significantly increased in straw-treated plants when compared to the plants without straw treatment (control). Contaminated soil showed higher concertation of proline as compared to normal soil with straw application. Lower proline contents (166.8 µg mL⁻¹) were observed in the plants grown under normal soil without the straw addition (C) to the rice plants while the maximum proline contents (274.0 µg mL⁻¹) were recorded in the plants grown under contaminated soil with straw application (T₃).

3.4. Drought Stress Tolerance

The effect of straw application on drought stress tolerance in rice plants was determined using rice leaves under different concentrations of polyethylene glycol (PEG 6000) stress (Figure 5). The higher RWC was recorded in straw-treated plants grown under contaminated soil compared to without straw-treated plants grown under normal soil in all concentrations of PEG (0, 5, 10, and 15%).

3.5. Bioaccumulation of Heavy Metals in Root, Stem, and Leaf Samples of Oryza sativa

Heavy metals (HMs) accumulation in root, stem, and leaf samples of Oryza sativa parts is presented in Figure 6. Comparatively highest accumulation Cd, Cu. Fe and Pb were found in the root and the lowest in leaves of Oryza sativa. The highest Cd (0.050 ± 0.002 mg kg⁻¹ DW), Cu (0.365 ± 0.002 mg kg⁻¹ DW), Fe (36.08 ±1.01 mg kg⁻¹ DW), and Pb (0.224 ± 0.006 mg kg⁻¹ DW) contents were recorded in roots of Oryza sativa grown under contaminated soil with the addition of straw. Stem and leaves also showed higher accumulation under contaminated soil with the addition of straw. The accumulation of HMs in stem was: Cd (0.040 ± 0.002 mg kg⁻¹ DW), Cu (0.104 ± 0.003 mg kg⁻¹ DW), Fe (5.55 ± 0.50 mg kg⁻¹ DW) and Pb (0.072 ± 0.004 mg kg⁻¹ DW). In leaves, Cd, Cu, Fe, and Pb contents were 0.035 ± 0.001, 0.097 ± 0.002, 3.30 ± 0.100 and 0.033 ± 0.004 mg kg⁻¹ DW, respectively. HMs contents (Cd, Cu, Fe, and Pb) were higher in plants grown under contaminated soils and treated with straw as compared to other treatments. The accumulation of HMs in different plant parts was in order, i.e., root > stem > leaves. The lowest accumulation of HMs (Cd, Cu, Fe, and Pb) was found in control C (normal soil) without straw as compared to all other treatments. Among these Cd, Cu, Fe, and Pb metals, Fe contents were the highest while the Cd contents were the lowest in different plant parts.

3.6. Bioaccumulation of Heavy Metals in Grain Samples of Oryza sativa

The effect of straw application on heavy metal accumulation under normal and contaminated soils was also determined in rice grains (Figure 7). The results showed that the accumulation of HMs was significantly increased in straw-treated plants when compared to those without straw-treated plants (control). The maximum accumulation of Cd, Cu, Fe, and Pb was found in contaminated soil with the addition of straw (T₃). The accumulation of different metals was as Cd (0.0167 ± 0.00066 mg kg⁻¹ DW), Cu (0.0121 ± 0.00066 mg kg⁻¹ DW), Fe (0.0746 ± 0.002 mg kg⁻¹ DW), and Pb (12.03 ± 0.3367 mg kg⁻¹ DW). The minimum accumulation of Cd (0.0093 ± 0.00066 mg kg⁻¹ DW), Cu (0.0787 ± 0.001 mg kg⁻¹ DW), Fe (0.0356 ± 0.0026 mg kg⁻¹ DW), and Pb (8.237 ± 0.06 mg kg⁻¹ DW) in grain samples collected from the plants grown under normal soil conditions without straw application.

3.7. Pearson Correlation

The proline and relative water content had a positive and significant correlation in leaves, while chlorophyll and carotenoids showed a negative correlation with Cd, Cu, Fe, and Pb (

Table 2. Correlation among heavy metal contents, different growth, yield, and biochemical parameters.

	Fresh Biomass	Dry Biomass	Carotenoids	Proline	Chlorophyll a	Chlorophyll b	Total Chlorophyll	Cd	Cu	Fe	Pb	RWC
Fresh biomass	1
Dry biomass	0.9142	1
Carotenoids	0.9692 *	0.8029	1
Proline	−0.8882	−0.9946 **	−0.7807	1
Chlorophyll a	0.9460	0.7334	0.9827 *	−0.6937	1
Chlorophyll b	0.9840 *	0.8412	0.9975 **	−0.8184	0.9772 *	1
Total chlorophyll	0.9739 *	0.8015	0.9970 **	−0.7717	0.9920 **	0.9962 **	1
Cd	−0.9510 *	−0.7486	−0.9940 **	0.7177	−0.9959 **	−0.9874 *	−0.9964 **	1
Cu	−0.9099	−0.6725	−0.9806 *	0.6430	−0.9876 *	−0.9659 *	−0.9802 *	0.9934 **	1
Fe	−0.9881 *	−0.9456	−0.9207	0.9144	−0.9013	−0.9454	−0.9326	0.8992	0.8429	1
Pb	−0.8898	−0.6514	−0.9744 *	0.6298	−0.9704 *	−0.9562 *	−0.9673 *	0.9834 *	0.9960 **	0.8125	1
RWC	−0.8151	−0.9715 *	−0.6545	0.9617 *	−0.5907	−0.7055	−0.6622	0.5960	0.5017	0.8811	0.4680	1

Asterisks indicate a significant difference at * = p ≤ 0.05, ** = p ≤ 0.01.

). Fresh and dry biomass also showed a negative correlation with these HMs. Straw-treated plants showed a higher accumulation of Cd, Cu, Fe, and Pb.

4. Discussion

Soil pollution through heavy metals (HMs) contamination has become a serious environmental issue throughout the world and this problem is growing with each day passing due to rapid industrialization and other anthropogenic activities [39,40,41,42]. Soil samples from the industrially polluted area and normal soil from the non-contaminated agricultural field as reference soil were collected and analyzed regarding various physicochemical parameters. The electrical conductivity (EC), pH, water-holding capacity, and HMs contents (Cd, Pb, Cu, and Fe) were higher than in normal soil. The results are in line with other researchers who also found higher values of pH (7.5), EC (2.49 dS m⁻¹), Cd (9.9 mg kg⁻¹), Cu (40.83 mg kg⁻¹), Fe (143 mg kg⁻¹), and Pb (30.81 mg kg⁻¹) in the distillery, tannery and mixed effluent irrigated soil than control soil [43]. Similarly, the pH of Cd-contaminated soil (7.8) was higher as compared to control soil without Cd spiking (6.9). However, the EC of the both soils was the same, i.e., 0.3 dS m⁻¹ [44]. A similar result was reported that the pH, EC, and HMs contents were higher, and water-holding capacity was lower in industrially contaminated soil as compared to control, i.e., reference soil [45].

Straw recycling in soils is a good agricultural practice to enhance soil fertility [46]. However, the effect of straw application on the accumulation of HMs in plants was still unclear. In the current study, the effect of straw on chlorophyll “a” chlorophyll “b”, carotenoids, proline, and relative water content under polyethylene glycol (PEG) in rice (Oryza sativa L.) was investigated under normal as well as HMs contaminated soil with industrial effluent. Similarly, carotenoids are non-enzymatic antioxidants that play a critical role in alleviating the negative effects of reactive oxygen species produced under abiotic stress conditions such as HMs [47]. In the present study, straw application under HMs contaminated soil resulted in a significantly negative impact on chlorophyll a, b, total chlorophyll, and carotenoids contents as compared to control under normal soil without straw application. Earlier, it was found that HMs, particularly Cd, inhibit the biosynthesis of chlorophyll by stopping the production of photoactive protochlorophyllide reductase enzyme complex and inhibiting the synthesis of aminolevulinic acid. In addition, Cd made complexation with acid active thiol group and blocked sulfhydryl group to synthesize chlorophyll [48]. Similarly, the application of straw from four plants, i.e., Stellaria media L., Cardamine hirsute, Cerastium glomeratum, and Galium aparine L. significantly reduced the chlorophyll a, b, and total chlorophyll contents in Brassica chinensis L. as compared to the control [49]. Straw from Eclipta prostrata reduced the photosynthesis pigments in C. betea seedlings [50]. Similarly, straw from Tagetes erecta and Solanum photeinocarpum L. caused a significant reduction in chlorophyll a, and b in Galinsoga parviflora and Bidens pilosa L. [51]. In addition, the chlorophyll contents and chlorophyll a/b ratio were decreased under Cd stress [52]. The reduction in chlorophyll content was observed in wheat and mustard when these were irrigated with HMs contaminated effluent for 90 days [43]. With the application of Youngia japonica straw under Cd stress, carotenoids contents were reduced in grape seedlings in comparison to the control without straw application [53]. In the present study, straw application under HMs contaminated soils showed a negative effect on photosynthetic pigments and the fresh and dry biomass of rice. The maximum reduction was observed in straw-treated plants grown under heavy metal contaminated soil. Similar results were recorded with the addition of straw under Cd-contaminated soil, with low biomass in rice as compared to the control without straw application. Bai et al. [31] showed that Cd was responsible for inhibited plant growth.

Proline is an amino acid, which acts as an antioxidant and accumulates in plants under abiotic stresses, e.g., drought and HMs stresses [54]. In the present study, straw application under HMs contaminated soil significantly enhanced proline accumulation in rice leaves in comparison to control without straw application under heavy metal contaminated soil. Straw enhanced the availability of HMs, which resulted in enhanced accumulation of proline. The results are in line with Rai et al. [55] who reported higher proline concentration in Ocimum tenuiflorum under chromium stress. Similarly, lead stress enhanced proline accumulation in Brassica stem [56].

The relative water contents (RWC) indicate water status in the leaves of plants and are commonly used for the estimation of drought resistance in crops under different degrees of water stress [57,58]. In the present study, the impact of straw application on drought stress tolerance was positive in rice leaves under different polyethylene glycol (PEG 6000) concentrations. RWC was higher with straw application in plants grown under heavy metal contaminated soil as compared to control and other treatment. However, the overall RWC decreased with the increasing concentration of PEG. Various studies reported that RWC was reduced under drought stress, e.g., in safflower [59] and Brassica [60]. Similarly, drought stress decreased water potential in root leaves and pods of soya bean as compared to control [61]. Application of straw from Stellaria media L. Cardamine hirsute, Cerastium glomeratum, and Galium aparine L. reduced the water contents in the roots and shoots of Brassica chinesis L. grown under Cd stress as compared to control [44].

Straw added into the soil had a significant impact on soil nutrients. However, the potential impact of straw on the biogeochemistry of HMs is largely ignored [26]. In the current study, straw application showed a positive impact on the accumulation of HMs in rice roots, stems, leaves, and grains. Maximum heavy metal contents were found in plants grown under heavy metal contaminated soil and treated with straw as compared to control and other treatments. A lower accumulation of HMs was found in control plants grown under normal soil without straw application. The higher accumulation might be due to the presence of higher concentrations of HMs in contaminated soil in comparison to the control soil. This premise is supported by the data presented in Table 1. The incorporation of straw might also enhance the availability of HMs to the plants due to the production of organic acids during decomposition [46]. Heavy metal contents were higher in roots and lower accumulation was found in grains. A similar result was reported by Bai et al. [31] that Cd accumulation was higher in roots and lower in grain as compared to stem and leaves. Similarly, accumulation of chromium was significantly higher in ryegrass shoot and stem when treated with straw and earthworm as compared to control without straw application [62]. Another study also reported a higher accumulation of MeHg in harvested tissues of rice treated with straw [63].

5. Conclusions

Our results indicated that straw application under contaminated soil significantly enhanced the accumulation of HMs (Cd, Cu, Pb, and Fe) in the root, stem, leaves, and grain of Oryza sativa. A higher accumulation of HMs was found in the root. Chlorophyll and carotenoids contents were low while proline was high in straw-treated plants under contaminated soil. Straw application also harmed fresh and dry biomass while it had an opposite impact on percent relative water contents. In conclusion, the results recorded in the present study suggest that the removal of straw from the rice fields is necessary to reduce the accumulation of HMs in rice grain and to decrease associated human health risks. Further research work is needed to investigate the mechanisms behind this accumulation of HMs in rice with straw application.