Vermicompost Amendment in Soil Affects Growth and Physiology of Zea mays Plants and Decreases Pb Accumulation in Tissues

Abstract

Minimization of the possible harmful effects of soil pollution on agricultural production and food safety are the major challenges in modern agriculture. There is great scientific interest in the detailed understanding of the physiology of lead uptake and toxicity in Zea mays, together with the search for approaches to minimizing Pb accumulation in tissues. The aim of the present study was to explore the possibility of reducing Pb accumulation in Z. mays plants cultivated in Pb-contaminated soil, by means of vermicompost amendment. Z. mays plants were cultivated at three soil vermicompost amendment rates (10, 20, and 30%), with the addition of 1000 mg L⁻¹ of Pb in the form of Pb(NO₃)₂ or an equivalent amount of nitrogen in the form of NH₄NO₃. Additional nitrogen had a significant stimulatory effect on plant growth and physiology, but only for control plants, and at a low vermicompost amendment rate. Independently, Pb had an insignificant negative effect on plant growth and biomass partitioning, but significantly negatively affected the mineral nutrition of Z. mays plants. At a 10 and 20% soil vermicompost amendment rate, the Pb concentration in plant leaves and roots decreased by 65%, while plant biomass increased four to five times in comparison to soil-grown control plants, together with accelerated flowering. It was concluded that vermicompost is one of the most promising soil amendments for reducing heavy metal uptake and accumulation in crop plants, while also being an efficient organic fertilizer.

1. Introduction

Agricultural practices leading to increased soil sustainability and food safety are gaining more interest from both scientists and farmers [1,2]. Minimization of the possible harmful effects of soil pollution on agricultural production and food safety are the major challenges in this respect. While the problem of the negative effect of soil heavy metal contamination on yields can be solved by developing metal-tolerant crop varieties [3], the presence of toxic metal levels in agricultural crop products still represents an insufficiently addressed problem [4].

Lead is one of the environmental contaminants that is mostly associated with anthropogenic impacts [5]. Lead bioavailability in soils is controlled by complex interactions, as Pb is readily complexed with both inorganic constituents as well as organic ligands, or adsorbed on the surface of different types of particles [6]. Therefore, different soil amendments have been explored for their ability to adsorb Pb and other heavy metals and, thus, decrease their bioavailability. Vermicompost is an especially promising product for soil stabilization of heavy metals, as it also acts as a valuable organic fertilizer. The primary positive effect of vermicompost on plant growth is related to the high content of plant-available mineral nutrients as well as plant-growth-promoting biologically active substances—as reported in a recent review [7].

The majority of adsorption studies with vermicompost and heavy metals have been performed in laboratory conditions without plants [8,9,10,11]. Adsorption studies with vermicompost using plants in controlled conditions are relatively scarce [12,13]. However, there have been some field experiments on the effect of organic amendments on heavy metal accumulation [14,15].

Zea mays L. is a crop species with a relatively high potential for use in phytoremediation [16]. There is still a great scientific interest in the detailed understanding of the physiology of lead uptake and toxicity in Z. mays, as evidenced by a recent review [17]. It appears that studies aiming at understanding the amendment methods and functional mechanisms leading to reduced Pb uptake and accumulation are critically relevant.

Direct deleterious effects of heavy metals in plants, including Pb, often have not been distinguished from controlled physiological responses. Even non-biogenous heavy metals can induce changes in gene expression patterns due to chemical similarity with essential metals [18]. The downregulation of photosynthesis and resource allocation to defense due to heavy metal treatment are typical cases of induced responses associated with physiological alterations [19]. Alternatively, endogenous oxidative stress through the enhanced formation of reactive oxygen species can lead to the inhibition of photosynthesis in heavy-metal-stressed plants [20]. Therefore, non-destructive methods of physiological measurement, such as chlorophyll analysis and chlorophyll a fluorescence assays, can be used as indicators of the physiological status of plants [21], including in studies associated with mineral nutrient availability [22,23].

One of problems in studies aiming at assessing the effects of Pb on plants is related to the fact that lead nitrate is very often used for treatments, without an additional control in the form of a balancing nitrogen fertilizer [13]. As a result, any growth stimulation or other physiological changes by lead nitrate can be erroneously interpreted as the effect of Pb itself [24,25].

The aim of the present study was to explore the possibility of reducing Pb accumulation in Z. mays plants cultivated in Pb-contaminated soil, by means of vermicompost amendment. In addition, the effect of vermicompost for growth improvement during Z. mays cultivation was assessed. Special care was taken to evaluate the possible effect of increased soil nitrogen content due to treatment with high doses of Pb nitrate.

2. Materials and Methods

2.1. Plant Material and Substrates

Seeds of Zea mays L. var. saccharata cv. ‘Zlota Karlova SNF’ (Toraf, Kujakowice Górne, Poland) were used for the experiment. The variety is a very early, dwarf-type sugar corn.

Agricultural soil (loamy sand, 2% organic matter) collected in October from a field (Valmiera Municipality, Latvia) employed for cereal production was used as a substrate for plant cultivation. Analysis of plant-available mineral nutrient concentration in soil was performed in a certified agrochemical laboratory (Table 1). According to the established criteria [26], the soil was relatively rich in plant-available nutrients. The soil Pb concentration was <2.3 mg kg⁻¹.

Vermicompost (Eko Zeme, Bauska District, Latvia) was purchased from a local supplier. The vermicompost was produced from composted cow manure and grass biomass, and was certified for organic agriculture. The analysis of plant-available mineral nutrient concentration in vermicompost was performed in a certified agrochemical laboratory (Table 1). The used vermicompost was a very good source of plant-available N, P, K, Mg, Mn, and Zn. The vermicompost Pb concentration was <2.3 mg kg⁻¹.

2.2. Plant Cultivation and Treatments

The experiments were performed in winter in an experimental greenhouse with an automatic control system (HortiMaX, Maasdijk, The Netherlands). Additional light was supplemented by Master SON-TPIA Green Power CG T 400 W (Philips, Amsterdam, The Netherlands) and Powerstar HQI-BT 400 W/D PRO (Osram, Munich, Germany) lamps (380 μmol m^–2 s⁻¹ at the plant level) for a 16 h photoperiod, with a day/night temperature 25/16 °C and relative air humidity of 60 to 70%.

The substrate for cultivation was prepared from air-dried samples of soil and vermicompost at three vermicompost amendment rates: 10, 20, and 30% (v/v). The control treatment contained only soil. Deionized water was added to achieve 50% substrate moisture, measured with a HH2 moisture meter equipped with a WET-2 sensor (Delta-T Devices, Burwell, Cambridge, UK). For each of four substrate vermicompost amendment rates, there were three subtreatments: control, Pb(NO₃)₂, and NH₄NO₃ (Table 2). The amount of Pb(NO₃)₂ applied per 1 L of substrate (1598 mg) gave a content of 1000 mg L⁻¹ of Pb. Accordingly, 388 mg of NH₄NO₃ per 1 L of substrate had the amount of N equivalent to that in the Pb(NO₃)₂ treatment. Necessary amounts of both salts were dissolved in deionized water and applied to the respective substrate in the form of a 10% solution. Prepared substrates were placed in 1.2 L plastic containers, 1 L per container.

Seeds were surface-disinfected with 1% KMnO₄ solution, rinsed 10 times with deionized water, and imbibed for 6 h in water. Seeds were placed on filter paper in Petri dishes and germinated in the dark for 5 days at 20 °C. Well-developed germinated seeds were individually sown in containers with the prepared substrate at a 1 cm depth, 10 seeds per treatment. Containers were randomly placed in the greenhouse. After one week, typical uniform seedlings, five per treatment, were selected for further cultivation. Plants were watered with deionized water to maintain substrate moisture at the 50–60% level. Once a week, individual containers were randomly repositioned on a greenhouse bench.

2.3. Measurements and Termination

To monitor plant growth, starting from week 3, the height of individual plants was measured weekly, from the base of the stem to the tip of the longest leaf.

Starting from week 4, nondestructive physiological measurements were performed weekly. For each individual plant, three of the upper photosynthetically most active leaves were selected for measurements. Leaf chlorophyll concentration was measured using a chlorophyll meter CCM-300 (Opti-Sciences, Hudson, NH, USA). Chlorophyll a fluorescence was measured in leaves dark-adapted for at least 20 min by a Handy PEA fluorometer (Hansatech Instruments, Pentney, King’s Lynn, UK). Fluorescence data analysis was performed by PEA Plus software (Hansatech Instruments, Pentney, King’s Lynn, UK). The photochemical efficiency of photosynthesis was estimated by the multiparametric fluorescence indicator, Performance Index Total, combining information on the status of both photosystems, as well as the electron flow between the two systems on an absorption basis [27].

The experiment was terminated after nine weeks of cultivation, when plants in all treatments started to develop male inflorescences. Stem height up to the inflorescence base was measured. Individual plants were cut at the substrate level and separated into individual parts: dry leaves, live leaves, inflorescence, and stem. Roots were separated from the substrate and washed under running tap water to remove any adhered particles, rinsed with deionized water, and blotted dry with paper towels. The fresh mass of individual parts was measured and tissues were dried in a oven at 60 °C until no change in biomass occurred; then, dry mass was measured. Water content in the plant tissues was calculated as g H₂O per g dry mass.

2.4. Analytical Measurements

Dried plant material was used for measurement of soluble K⁺ and NO₃⁻ concentration in the flag leaf, base leaf, stem base, and roots. Plant material (about 5 g) was crushed in pieces and homogenized, and a sample of 0.2 g was randomly taken from the plant material. Tissues were ground with a mortar and pestle to a fine powder and 10 mL of deionized water was added. After filtration through a nylon mesh cloth (No. 80), homogenate was used for measurement of the K⁺ concentration by a LAQUAtwin compact meter B-731 and NO₃⁻ concentration by a LAQUAtwin compact meter NO3-11 (Horiba, Kyoto, Japan). For nitrate measurement, a nitrate interference suppressor solution (Mettler-Toledo, Schwerzenbach, Switzerland) was used according to the manufacturer’s instructions. For each treatment, three samples from individual plants were measured in at least three analytical replicates and the average value was calculated.

Pb analysis was performed in a certified analytical laboratory. Briefly, H₂O₂ and HNO₃ were added to 0.3 g of homogenized plant material. The samples were digested using a microwave digestion system Mars 6 (CEM Corporation, Matthews, NC, USA). Pb was measured using an Agilent 7700 Series ICP-MS (Agilent Technologies, Santa Clara, CA, USA). Five replicate samples for leaves and three replicate samples for roots were analyzed for each treatment.

2.5. Data Analysis

The results were analyzed by KaleidaGraph (v. 5.0, Synergy Software, Reading, PA, USA). The statistical significance of differences was evaluated by one-way ANOVA using post hoc analysis (Tukey’s HSD).

3. Results

3.1. Effect on Growth

Changes in the growth of Z. mays plants due to vermicompost amendment and nitrogen and Pb treatment were estimated by weekly measurements of plant height. The treatment of control plants with NH₄NO₃ resulted only in a temporary increase in plant height (Figure 1A). The greatest positive effect of vermicompost amendment on Z. mays growth was observed for the 10% vermicompost (Figure 1B) and 20% vermicompost treatments (Figure 1C). However, the growth of plants amended with 30% vermicompost was initially suppressed in comparison to control plants (Figure 1D). The nitrogen and Pb nitrate treatments did not result in a growth response in comparison to the control. However, the final height of plants clearly showed that the nitrogen treatment of Z. mays plants amended with 30% vermicompost resulted in a statistically significant negative effect (Figure 2). A similar effect was observed for changes in shoot dry biomass (Figure 3).

Biomass partitioning in Z. mays plants indicated that the growth of all plant parts was stimulated by vermicompost amendment, but to a different extent (Figure 4). The highest degree of stimulation at 10 and 20% vermicompost amendment rates occurred for flowers (Figure 4A) followed by stems (Figure 4B), but also the biomass of leaves (Figure 4C) and roots (Figure 4D) significantly increased. At the 10% vermicompost amendment rate, the biomass of all parts of the plants treated with nitrogen and Pb nitrate tended to be higher in comparison to the control plants, but the differences were not statistically significant. In general, plants amended with 30% vermicompost had a lower biomass of their parts, but significant growth reduction was evident for the leaves, stems, and roots of Z. mays plants treated with nitrogen and the roots of plants treated with Pb nitrate.

The total number of leaves was not significantly affected by vermicompost amendment and nitrogen and Pb nitrate treatments (data not shown). However, the number of dry leaves significantly decreased (Figure 5A) and that of live leaves significantly increased (Figure 5B) for Z. mays plants growing in soil amended with 30% vermicompost.

The water content increased in photosynthetically active leaves of Z. mays control plants treated with nitrogen and Pb nitrate, in Pb nitrate-treated plants that received 20% vermicompost amendment, and for all plants in the 30% vermicompost treatment (Figure 6A). Moreover, the water content in stems significantly increased in Pb-nitrate-treated plants for the 20% vermicompost amendment, and in all plants for the 30% vermicompost amendment, and this effect was especially pronounced for nitrogen-treated plants (Figure 6B).

3.2. Effect on Physiological Parameters

For soil-grown plants Z. mays, chlorophyll concentration in the main photosynthesizing leaves of the plants treated with nitrogen and Pb nitrate started to increase over control values on Week 6 (Figure 7A). Soil amendment with vermicompost at a 10% rate resulted in a significant increase in leaf chlorophyll concentration, with an additional increase due to treatment with nitrogen and Pb nitrate (Figure 7B). However, at higher vermicompost amendment rates, differences between control plants and those treated with nitrogen and Pb nitrate levelled off (Figure 7C) and completely disappeared (Figure 7D).

There was a temporary increase in chlorophyll a fluorescence parameter Performance Index Total in soil-grown plants in the nitrogen and Pb nitrate treatment (Figure 8A). The increase in Performance Index Total became more pronounced and continuous with increased vermicompost amendment rate (Figure 8B–D). Treatment of Z. mays plants with nitrogen and Pb nitrate at the 10 and 20% vermicompost substitution rate resulted in an additional increase in Performance Index Total, but this effect was no longer evident at the 30% vermicompost amendment rate.

3.3. Effect on Accumulation of Ions and Pb

Nitrate concentration in different plant parts was measured as a possible indicator of nitrogen status. Nitrate concentration in flag leaf tissue was not affected by the treatments (Figure 9A). Surplus nitrate accumulated in the base leaf of plants cultivated at the 30% vermicompost amendment rate, and especially for plants treated with additional nitrogen and Pb nitrate (Figure 9B). Similarly, nitrate accumulated in the stem base of Z. mays plants treated with additional nitrate, and this effect increased with increasing soil vermicompost amendment rate, but Pb drastically reduced the nitrogen-dependent increase in nitrate accumulation (Figure 9C). Root nitrate concentration significantly increased due to the increase in soil vermicompost amendment rate, and it was stimulated by additional treatment with nitrogen at the 30% amendment rate (Figure 9D). However, Pb treatment resulted in a significant reduction in nitrate accumulation in roots.

The effect of vermicompost amendment and addition treatments on general plant nutrition was evaluated by tissue K⁺ concentration in different plant parts (Figure 10). The effect of additional plant-available K⁺ through soil amendment with vermicompost was clearly observed for all tested plant parts, and particularly in the stem base and base leaf. A stimulative effect of additional nitrogen availability on K⁺ accumulation was the most pronounced in flag leaf tissues, together with a striking negative effect of Pb (Figure 10A), which was evident also in stem base tissue (Figure 10C).

The concentration of Pb was 1.34 and 3.01 mg kg⁻¹ in the leaves and roots of plants grown in uncontaminated soil, respectively. In plants treated with 1 g L⁻¹ of Pb in the form of nitrate, these levels reached 100 and 500 mg kg⁻¹, respectively (Figure 11). However, soil amendment with vermicompost significantly decreased Pb accumulation approximately by 80%. This decrease in Pb accumulation with increased vermicompost amendment rate occurred in both leaves and roots (Figure 11, inset).

4. Discussion

Lead tolerance mechanisms in plants are largely related to limiting its intake in roots and restricting its transport to above-ground parts. Root-released uronic-acid-containing exudates bind lead ions to their carboxyl groups, thus inhibiting their uptake [28]. The majority of lead taken up in roots is stored in cell walls in the form of relatively stable complexes with galacturonic and glucuronic acids [29]. A layer of thickened cells in endoderm tissue, called a Casparian strip, acts as a physical barrier for apoplastic transport of lead, which further limits its translocation to above-ground parts [30]. As a result, lead predominantly accumulates in the root tissues of plants. However, lead transport and accumulation in above-ground organs can be stimulated by chelating substances. In a classical study of Huang and Cunningham (1996) [31], Z. mays plants exhibited the highest shoot Pb accumulation potential among 11 plant species grown in Pb-contaminated soil, reaching 225 mg kg⁻¹. By use of chelating agents, it is possible to achieve a many-fold higher Pb accumulation in the shoots of Z. mays, reaching 771 mg kg⁻¹ [32]. As an extreme, 10-day-old Z. mays seedlings cultivated in soil with 2500 mg kg⁻¹ of Pb for 7 days accumulated more than 10,000 mg kg⁻¹ of Pb in the shoots under the effect of synthetic chelate [31]. Due to this high accumulation potential and relatively high tolerance against Pb, Z. mays has been used as a model in phytoremediation studies [16].

In addition to genetic and physiological mechanisms, soil properties can significantly affect Pb availability for plants. Therefore, in the context of food security, agricultural practices leading to decreased heavy metal accumulation in Z. mays plants need to be considered. One such approach involves increasing the stabilization of heavy metals in soil, while providing sufficient mineral nutrient supply in plant-available forms in soil, as well as increasing soil sustainability. In this respect, organic fertilizers have drawn the greatest attention of researchers. Vermicompost is an organic fertilizer with a relatively high amount of plant-available nutrients and plant-growth-promoting substances, and has high microbiological activity, which make it a promising choice for soil remediation [7].

Usually, raw organic materials have less heavy metal absorption capacity in comparison to processed ones. Thus, the adsorption capacity for Pb of cow manure was observed to be 103 mg g⁻¹, compared to 171 mg g⁻¹ of cow manure vermicompost [9]. In contrast, both sheep manure and vermicompost were ineffective as stabilizing materials of Pb, but biochars produced from the two materials were effective [10]. Highly variable effects of different organic materials on Pb accumulation in Z. mays and other plant tissues have been reported. From the opposite side, some studies used vermicompost as a material for the possibly stimulated accumulation of heavy metals in plant tissues [33,34]. In particular, a study with Avena strigosa indicated that the high rate of vermicompost addition to soil (>50%) increased the bioavailability of Cr and Pb, resulting in an enhanced accumulation of the metals in plant tissues [33]. However, this effect should not be confused with similar effects resulting from plant cultivation in the presence of sewage-sludge-derived vermicompost, as in the case of Z. mays, where vermicompost samples contained elevated concentrations of various heavy metals [14], and in the case of experiments with tannery sludge vermicompost [35].

Soil amendment with date palm leaf waste biochar (0.5–3%) decreased the soil availability of heavy metals and did not result in a change in Pb concentration in leaves of Z. mays plants, and there was a tendency of decreased root Pb accumulation by 23% at the highest amendment rate [36]. Organic formulation panchakavya decreased Pb accumulation in the shoots and roots of Z. mays by 32 and 37%, respectively [37]. The addition of chicken manure decreased Pb accumulation in Z. mays by 53% [38]. Vermicompost application (at 10% amendment rate) decreased Pb concentration in the shoots of Brassica chinensis by 67% [13]. Vermicompost decreased the soil plant-available concentration of Pb by 43% [12]. When maize straw biochar and maize straw compost was compared with respect to their ability to reduce heavy metal accumulation in Z. mays plants, the greatest effect was shown by biochar [39]. In contrast, while biochar had high Cd retention capacity, it also increased the Cd bioavailability and accumulation potential in plants [40].

The ability of organic materials to adsorb heavy metals, including Pb, has been mostly associated with the presence of humic substances [41,42]. However, the existence of other types of interaction cannot be ruled out, as humin was shown to be inefficient in decreasing the availability of Cd, in comparison to vermicompost and vermicompost solid residue [40]. The high concentration of Ca in the vermicompost sample used in the current study (Table 2) might have reduced the root uptake of lead, due to the inhibition of ion pumps in root cell membranes [43]. In addition, the high concentration of Fe²⁺ in the vermicompost might have negatively affected Pb uptake due to the antagonistic interaction between the two metals [44].

It has also been documented that the improvement of mineral supply promotes heavy metal accumulation in plants. The increased concentration of soil-available nitrogen stimulated Pb accumulation in Z. mays and Spinacia oleraceae plants [45]. This clearly was not the case in the present study, where the plant-available mineral nutrient pool in soil was enhanced by vermicompost amendment on the background of an increasing concentration of organic matter.

Differences in experimental conditions seem to greatly affect the results in this type of studies. One crucial aspect is related to the concentration of Pb (or any other heavy metal of interest) in soil samples used for plant cultivation in metal uptake and accumulation experiments. In some studies, the Pb concentration range was only several tens of mg kg⁻¹ [39,46], while other studies used extremely high Pb loads (1000–2500 mg kg⁻¹) [31,34].

One of the possible reasons for Pb toxicity on plant physiological processes is associated with its negative effect on mineral nutrition, especially at the level of mineral element uptake [47]. The Pb treatment of Z. mays plants resulted in a significant reduction in K⁺ concentration in the roots but not in the shoots [48]. The hypothesis that the shoot growth inhibition of Z. mays seedlings is due to its negative effect on the K⁺ pool through K⁺ leakage from the root cells has been tested but not confirmed [48]. Nitrogen metabolism at the level of nitrate represents another nutritional target of Pb toxicity [6]. This effect was clearly seen in the present study, as there was a decrease in nitrate accumulation in Z. mays even on the background of the additional nutrient supply through vermicompost amendment.

Leaf chlorophyll concentration and chlorophyll a fluorescence parameter Performance Index Total were not negatively affected by Pb treatment (Figure 7 and Figure 8), while it is generally accepted that the decrease in chlorophyll concentration is one of the reasons for the diminished photosynthetic activity together with the negative effects on photosynthetic electron transport [49,50]. On the other hand, the increasing rate of vermicompost amendment resulted in a typical response of increased leaf chlorophyll concentration and Performance Index Total, showing an optimization of the physiological status of plants under the effect of vermicompost. A similar effect was observed previously in studies with different crops [51,52], and it is evident that leaf chlorophyll concentration positively responds to vermicompost amendment in a concentration-dependent manner. Most likely, this effect is associated with a prolonged growth period of leaves due to the better mineral supply of vermicompost-treated plants. However, the positive response of Performance Index Total to vermicompost amendment seems to be associated with the stimulation of activity of a water-splitting complex or other photochemical reactions at the donor side of photosystem II [52]. These responses are characteristic of plants at optimal mineral nutrient availability [22,23], reflecting the highest possible physiological performance of the plant.

In contrast to the effects on plant mineral nutrition, there were no significant negative morphological effects of Pb treatment on Z. mays plants, even for plants growing in soil without vermicompost amendment, while the total biomass of Pb-treated plants insignificantly decreased by 10% (Figure 2). It is possible that any negative effect of Pb was masked by the presence of an increased concentration of N in the substrate due to treatment with PbNO₃. When exposure to Pb nitrate and Pb acetate were compared using other model plants, it appeared that the two salts indeed had different effects [53,54,55]. When Pb acetate was used as a treatment in an experimental small plot field study with Z. mays plants, it significantly decreased the growth, morphological parameters, and grain yield [56].

When using high doses of Pb in the form of nitrate, as in the present study, it is impossible to dissect the effects of Pb from those of elevated nitrogen availability, without the use of an appropriate control. Especially when Pb-tolerant plants are used as model species, a seemingly positive effect of Pb on plant growth and physiological status can be due to the effect of surplus nitrogen [24,25]. In the present study, by using an additional control with the same amount of nitrogen as received in the Pb nitrate treatment, it was possible to dissociate Pb-specific effects from those of nitrate, both at morphological and physiological levels. Most importantly, Pb had a clear negative effect on NO₃ concentration in the base leaf, stem base, and roots, in comparison to the stimulative effect of surplus nitrogen (Figure 9), and a negative effect on K⁺ concentration in the flag leaf, stem base, and roots (Figure 10). Interestingly, the negative effects of Pb treatment on plant height and biomass accumulation were efficiently prevented for plants grown in vermicompost-amended soil, due to a significant decrease in Pb accumulation capacity in plant tissues, but the above-mentioned negative effects on mineral nutrition were only diminished but not fully lost. The relationship between nitrogen and Pb was also affected not only by the presence of vermicompost, but also by the vermicompost amendment rate, as nitrogen treatment tended to give a higher stem and root biomass in comparison to Pb nitrate treatment at the 20% amendment rate, but the dry mass of leaves, stems, and roots was significantly lower in nitrogen-treated plants in comparison to the Pb-nitrate-treated plants at the 30% amendment rate (Figure 4). The total biomass of ammonium-nitrate-treated plants cultivated at the 30% vermicompost amendment rate was significantly decreased in comparison to that of the control and Pb-nitrate-treated plants (Figure 3). This more likely indicates the appearance of ammonium toxicity in conditions of high nutrient availability [57]. Recently, transcriptional signatures in roots of Z. mays have been compared for nitrate and ammonium, and it was shown that both overlapping and distinct pathways indeed are regulated [58].

Additional experiments in field conditions using natural contaminated soil and different forms of organic amendment at various rates are necessary for obtaining practically useful results, as the experimental system used had typical limitations characteristic for vegetation pot studies [59].

5. Conclusions

The main conclusion from this study was that, in addition to the pronounced positive effects of vermicompost soil amendment on the growth and physiology of Z. mays plants, it also significantly decreased Pb accumulation in plant leaves and roots. The most favorable effect was evident at 10 and 20% vermicompost amendment rates, resulting in a 65% decrease in Pb concentration in tissues of Pb-treated plants, while plant biomass increased four to five times in comparison to soil-grown control plants, together with accelerated flowering. Thus, vermicompost is one of most favorable and sustainable organic products for reducing heavy metal uptake and accumulation in crop plants, while also being an efficient organic fertilizer.

Author Contributions

Conceptualization, M.V. and G.I.; methodology, M.V.; investigation, Z.L.-S.; writing—original draft preparation, G.I. and Z.L.-S.; writing—review and editing, M.V. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data reported here are available from the authors upon request.

Acknowledgments

We thank Jānis Freibergs (Eko Zeme, Latvia) for providing the vermicompost sample used in the present study. We thank Guntis Brūmelis (University of Latvia) for English correction.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Effect of rate of soil vermicompost (V) amendment, NH4NO3, and Pb(NO3)2 treatment on relative growth of Zea mays plants grown in soil (A), and at soil vermicompost amendment rates of 10% (B), 20% (C), and 30% (D). Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE. V 0: no vermicompost amendment; V 10%, V 20%, and V 30%: vermicompost amendment by 10, 20, and 30% (v/v), respectively.

Figure 2. Effect of rate of soil vermicompost (V) amendment, NH4NO3 (N), and Pb(NO3)2 (Pb) treatment on final height of Zea mays plants. Amendment rate is given as v/v% added to soil. Data are means from 5 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05).

Figure 3. Effect of rate of soil vermicompost amendment, NH4NO3, and Pb(NO3)2 treatment on shoot dry mass of Zea mays plants. Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05).

Figure 4. Effect of rate of soil vermicompost (V) amendment, NH4NO3 (N), and Pb(NO3)2 (Pb) treatment on dry mass of leaves (A), dry mass of stem (B), dry mass of flowers (C), and dry mass of roots (D) of Zea mays plants. Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05).

Figure 5. Effect of rate of soil vermicompost (V) amendment, NH4NO3 (N), and Pb(NO3)2 (Pb) treatment on number of dry leaves (A) and number of live leaves (B) of Zea mays plants. Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05).

Figure 6. Effect of rate of soil vermicompost (V) amendment, NH4NO3 (N), and Pb(NO3)2 (Pb) treatment on water content in leaves (A) and water content in stems (B) of Zea mays plants. Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05).

Figure 7. Effect of NH4NO3 (N) and Pb(NO3)2 (Pb) treatment on relative time course of leaf chlorophyll concentration of Zea mays plants grown in soil (A), and at soil vermicompost amendment rates of 10% (B), 20% (C), and 30% (D). Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE, with 3 separate measurements each. V 0: no vermicompost amendment; V 10%, V 20%, and V 30%: vermicompost amendment by 10, 20, and 30% (v/v), respectively.

Figure 8. Effect of NH4NO3 (N) and Pb(NO3)2 (Pb) treatment on relative time course of chlorophyll a fluorescence parameter Performance Index Total of Zea mays plants grown in soil (A), and at soil vermicompost amendment rates of 10% (B), 20% (C), and 30% (D). Amendment rate is given as v/v % added to soil. Data are means from 5 replicates ± SE, with 3 separate measurements each. V 0: no vermicompost amendment; V 10%, V 20%, V 30%: vermicompost amendment by 10, 20, and 30% (v/v), respectively.

Figure 9. Effect of rate of soil vermicompost amendment, NH4NO3, and Pb(NO3)2 treatment on NO3− concentration in flag leaf (A), base leaf (B), stem base (C), and roots (D) of Zea mays plants. Amendment rate is given as v/v % added to soil. Data are means from 3 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05). DM, dry mass.

Figure 10. Effect of rate of soil vermicompost amendment, NH4NO3, and Pb(NO3)2 treatment on K+ concentration in flag leaf (A), base leaf (B), stem base (C), and roots (D) of Zea mays plants. Amendment rate is given as v/v % added to soil. Data are means from 3 replicates ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05). DM, dry mass.

Figure 11. Effect of rate of soil vermicompost amendment on Pb accumulation in roots and leaves of Pb(NO3)2-treated Zea mays plants. Inset shows relative changes in Pb concentration. Amendment rate is given as v/v % added to soil. Data are means from 5 replicates for leaves and 3 replicates for roots ± SE. Different letters indicate statistically significant differences between the treatments according to Tukey’s HSD test (p < 0.05). DM, dry mass. Control level of Pb was 1.34 and 3.01 mg kg−1 in leaves and roots, respectively.

Table 1

Properties of agricultural soil and vermicompost used in the present study.

Nutrient or Property (Unit)	Soil	Vermicompost	Optimum for Cultivated Plants
N (mg L−1)	90	730	120
P (mg L−1)	316	4251	60
K (mg L−1)	560	16,500	150
Ca (mg L−1)	1700	25,000	800
Mg (mg L−1)	320	4500	50
S (mg L−1)	23	925	50
Fe (mg L−1)	925	420	30
Mn (mg L−1)	145	165	1.5
Zn (mg L−1)	11	80	1.0
Cu (mg L−1)	2.75	6.00	0.50
Mo (mg L−1)	0.09	0.04	0.02
B (mg L−1)	1.1	3.0	0.2
Na (mg L−1)	32	780	n.a.
pHKCl (pH units)	5.87	7.29	n.a.
Electrical Conductivity (mS m−1)	1.84	36.7	n.a.

Plant-available concentrations are indicated, measured in 1 M HCl extract. Electrical conductivity was measured at 1:5 extraction ratio.

Table 2

Treatments used in the present study.

Code	Vermicompost (%, v/v)	Pb(NO3)2 (mg L−1)	NH4NO3 (mg L−1)
V0	0	0	0
V0 + N	0	0	388
V0 + Pb	0	1598	0
V10	10	0	0
V10 + N	10	0	388
V10 + Pb	10	1598	0
V20	20	0	0
V20 + N	20	0	388
V20 + Pb	20	1598	0
V30	30	0	0
V30 + N	30	0	388
V30 + Pb	30	1598	0

References

1. Pozza, L.E.; Field, D.J. The science of Soil Security and Food Security. Soil Secur.; 2020; 1, 100002. [DOI: https://dx.doi.org/10.1016/j.soisec.2020.100002]

2. Vidar, M. Soil and agriculture governance and food security. Soil Secur.; 2022; 6, 100027. [DOI: https://dx.doi.org/10.1016/j.soisec.2021.100027]

3. Rai, K.K.; Pandey, N.; Meena, R.P.; Rai, S.P. Biotechnological strategies for enhancing heavy metal tolerance in neglected and underutilized legume crops: A comprehensive review. Ecotoxicol. Environ. Saf.; 2021; 208, 111750. [DOI: https://dx.doi.org/10.1016/j.ecoenv.2020.111750] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33396075]

4. Rai, P.K.; Lee, S.S.; Zhang, M.; Tsang, Y.F.; Kim, K.-H. Heavy metals in food crops: Health risks, fate, mechanisms, and management. Environ. Int.; 2019; 125, pp. 365-385. [DOI: https://dx.doi.org/10.1016/j.envint.2019.01.067] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30743144]

5. Kumar, A.; Kumar, A.; Cabral-Pinto, M.M.S.; Chaturvedi, A.K.; Shabnam, A.A.; Subrahmanyam, G.; Mondal, R.; Gupta, D.K.; Malyan, S.K.; Kumar, S.S. et al. Lead toxicity: Health hazards, influence on food chain, and sustainable remediation approaches. Int. J. Environ. Res. Public Health; 2020; 17, 2179. [DOI: https://dx.doi.org/10.3390/ijerph17072179]

6. Pourrut, B.; Shahid, M.; Dumat, C.; Winterton, P.; Pinelli, E. Lead uptake, toxicity, and detoxification in plants. Rev. Environ. Contam. Toxicol.; 2011; 213, pp. 113-136. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21541849]

7. Ievinsh, G. Review on physiological effects of vermicompost on plants. Biology of Composts; Meghvansi, M.K.; Varma, A. Springer Nature Switzerland AG: Cham, Switzerland, 2020; pp. 63-86.

8. Matos, G.D.; Arruda, M.A.Z. Vermicompost as natural adsorbent for removing metal ions from laboratory effluents. Process Biochem.; 2003; 39, pp. 81-88. [DOI: https://dx.doi.org/10.1016/S0032-9592(02)00315-1]

9. Zhu, W.; Du, W.; Shen, X.; Zhang, H.; Ding, Y. Comparative adsorption of Pb²⁺ and Cd²⁺ by cow manure and its vermicompost. Environ. Pollut.; 2017; 227, pp. 89-97. [DOI: https://dx.doi.org/10.1016/j.envpol.2017.04.048]

10. Boostani, H.R.; Najafi-Ghiri, M.; Hardie, A.G.; Khalili, D. Comparison of Pb stabilization in a contaminated calcareous soil by application of vermicompost and sheep manure and their biochars produced at two temperatures. Appl. Geochem.; 2019; 102, pp. 121-128. [DOI: https://dx.doi.org/10.1016/j.apgeochem.2019.01.013]

11. Zhang, Y.; Tian, Y.; Hu, D.; Fan, J.; Shen, M.; Zeng, G. Is vermicompost the possible in situ sorbent? Immobilization of Pb, Cd and Cr in sediment with sludge derived vermicompost, a column study. J. Hazard. Mater.; 2019; 367, pp. 83-90. [DOI: https://dx.doi.org/10.1016/j.jhazmat.2018.12.085]

12. Moslehi, A.; Feizian, M.; Higueras, P.; Eisvand, H.R. Assessment of EDDS and vermicompost for the phytoextraction of Cd and Pb by sunflower (Helianthus annuus L.). Int. J. Phytoremed.; 2019; 21, pp. 191-199. [DOI: https://dx.doi.org/10.1080/15226514.2018.1501336] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30663886]

13. Wang, F.; Miao, L.; Wang, Y.; Zhang, M.; Zhang, H.; Ding, Y.; Zhu, W. Using cow dung and mineral vermireactors to produce vermicompost for use as a soil amendment to slow Pb²⁺ migration. Appl. Soil Ecol.; 2022; 170, 104299. [DOI: https://dx.doi.org/10.1016/j.apsoil.2021.104299]

14. Zuo, W.; Xu, K.; Zhang, W.; Wang, Y.; Gu, C.; Bai, Y.; Shan, Y.; Dai, Q. Heavy metal distribution and uptake by maize in a mudflat soil amended by vermicompost derived from sewage sludge. Environ. Sci. Pollut. Res.; 2019; 26, pp. 30154-30166. [DOI: https://dx.doi.org/10.1007/s11356-019-06184-w] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31422536]

15. Mwilola, P.N.; Mukumbuta, I.; Shitumbanuma, V.; Chishala, B.H.; Uchida, Y.; Nakata, H.; Nakayama, S.; Ishizuka, M. Lead, zinc and cadmium accumulation, and associated health risks, in maize grown near the Kabawe mine in Zambia in response to organic and inorganic soil amendments. Int. J. Environ. Res. Public Health; 2020; 17, 9038. [DOI: https://dx.doi.org/10.3390/ijerph17239038]

16. Wuana, R.A.; Okieimen, F.E. Phytoremediation potential of maize (Zea mays L.). A review. Afr. J. Gen. Agric.; 2010; 6, pp. 275-287.

17. Abedi, T.; Gavanji, S.; Mojiri, A. Lead and zinc uptake and toxicity in maize and their management. Plants; 2022; 11, 1922. [DOI: https://dx.doi.org/10.3390/plants11151922]

18. Cruz, Y.; Villar, S.; Gutiérrez, K.; Montoya-Ruiz, C.; Gallego, J.L.; Delgado, M.P.; Saldarriaga, J.F. Gene expression and morphological responses of Lolium perenne L. exposed to cadmium (Cd²⁺) and mercury (Hg²⁺). Sci. Rep.; 2021; 11, 11257. [DOI: https://dx.doi.org/10.1038/s41598-021-90826-y] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34045631]

19. Emamverdian, A.; Ding, Y.; Mokhberdoran, F.; Xie, Y. Heavy metal stress and some mechanisms of plant defense responses. Sci. World J.; 2015; 2015, 756120. [DOI: https://dx.doi.org/10.1155/2015/756120]

20. Paunov, M.; Koleva, L.; Vassilev, A.; Vangronsveld, J.; Goltsev, V. Effects of different metals on photosynthesis: Cadmium and zinc affect chlorophyll fluorescence in durum wheat. Int. J. Mol. Sci.; 2018; 19, 787. [DOI: https://dx.doi.org/10.3390/ijms19030787]

21. Matisons, R.; Krišāns, O.; Jansons, Ā.; Kondratovičs, T.; Elferts, D.; Ievinsh, G. Norway spruce seedlings from an Eastern Baltic provenance show tolerance to simulated drought. Forests; 2021; 12, 82. [DOI: https://dx.doi.org/10.3390/f12010082]

22. Wang, H.; Liu, R.L.; Jin, J.Y. Effects of zinc and soil moisture on photosynthetic rate and chlorophyll fluorescence parameters of maize. Biol. Plant.; 2009; 53, pp. 191-194. [DOI: https://dx.doi.org/10.1007/s10535-009-0033-z]

23. Horaczek, T.; Dąbrowski, P.; Kalaji, H.M.; Baczewska-Dąbrowska, A.H.; Pietkiewicz, S.; Stępień, W.; Gozdowski, D. JIP-test as a tool for early detection of the macronutrients deficiency in Miscanthus plants. Phtosynthetica; 2020; 58, pp. 507-517. [DOI: https://dx.doi.org/10.32615/ps.2019.177]

24. Kovačević, B.; Miladinović, D.; Orlović, S.; Katanić, M.; Kebert, M.; Kovinčić, J. Lead tolerance and accumulation in white poplar cultivated in vitro. South-east Eur. For. SEEFOR; 2013; 4, pp. 3-12. [DOI: https://dx.doi.org/10.15177/seefor.13-01]

25. Parys, E.; Wasilewska, W.; Siedlecka, M.; Zienkiewicz, M.; Drożak, A.; Romanowska, E. Metabolic responses to lead of metallicolous and nonmetallicolous populations of Armeria maritima. Arch. Environ. Contam. Toxicol.; 2014; 67, pp. 565-577. [DOI: https://dx.doi.org/10.1007/s00244-014-0057-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25070267]

26. Osvalde, A. Optimization of plant mineral nutrition revisited: The roles of plant requirements, nutrient interactions, and soil properties in fertilization management. Environ. Exp. Biol.; 2011; 9, pp. 1-8.

27. Banks, J.M. Continuous excitation chlorophyll fluorescence parameters: A review for practicioners. Tree Physiol.; 2017; 37, pp. 1128-1136. [DOI: https://dx.doi.org/10.1093/treephys/tpx059]

28. Sharma, P.; Shanker, R. Lead toxicity in plants. Braz. J. Plant Physiol.; 2005; 17, pp. 35-52. [DOI: https://dx.doi.org/10.1590/S1677-04202005000100004]

29. Inoue, H.; Fukuoka, D.; Tatai, Y.; Kamachi, H.; Hayatsu, M.; Ono, M.; Suzuki, S. Properties of lead deposits in cell walls of radish (Raphanus sativus) roots. J. Plant Res.; 2013; 126, pp. 51-61. [DOI: https://dx.doi.org/10.1007/s10265-012-0494-6]

30. Seregin, I.V.; Shpigun, L.K.; Ivanov, V.B. Distribution and toxic effects of cadmium and lead on maize roots. Russ. J. Plant Physiol.; 2004; 51, pp. 525-533. [DOI: https://dx.doi.org/10.1023/B:RUPP.0000035747.42399.84]

31. Huang, J.W.; Cunningham, D. Lead phytoextraction: Species variation in lead uptake and translocation. New Phytol.; 1996; 134, pp. 75-84. [DOI: https://dx.doi.org/10.1111/j.1469-8137.1996.tb01147.x]

32. Li, H.; Wang, Q.; Cui, Y.; Dong, Y.; Christie, P. Slow release chelate enhancement of lead phytoextraction by corn (Zea mays L.) for contaminated soil—a preliminary study. Sci. Total Environ.; 2005; 339, pp. 179-187. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2004.07.020] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15740768]

33. Hoehne, L.; de Lima, C.V.S.; Martini, M.C.; Altmayer, T.; Brietzke, D.T.; Finatto, J.; Gonçalves, T.E.; Granada, C.E. Addition of vermicompost to heavy metal-contaminated soil increases the ability of black oat (Avena strigosa Schreb) plants to remove Cd, Cr, and Pb. Water Air Soil Pollut.; 2016; 227, 443. [DOI: https://dx.doi.org/10.1007/s11270-016-3142-2]

34. Kouhi, S.M.M.; Ardakani, M.R.S.; Beykkhormizi, A. Irrigation of Helianthus annuus with Pb-polluted water: Improvement of phytoremediation using vermicompost. J. Plant Process Funct.; 2019; 8, pp. 77-83.

35. Gondek, K.; Filipek-Maazur, B. Biomass yields of shoots and roots of plants cultivated in soil amended by vermicomposts based on tannery sludge and content of heavy metals in plant tissues. Plant Soil Environ.; 2003; 49, pp. 402-409. [DOI: https://dx.doi.org/10.17221/4144-PSE]

36. Ahmad, M.; Usman, A.R.A.; Al-Faraj, A.S.; Ahmad, M.; Sallam, A.; Al-Wabel, M.I. Phosphorus-loaded biochar changes soil heavy metals availability and uptake potential of maize (Zea mays L.) plants. Chemosphere; 2018; 194, pp. 327-339. [DOI: https://dx.doi.org/10.1016/j.chemosphere.2017.11.156] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29220749]

37. Praburaman, L.; Park, J.-H.; Park, Y.-J.; He, Z.; Kamala-Kannan, S.; Oh, B.-T. Effect of panchakavya (organic formulation) on phytoremediation of lead and zinc using Zea mays. Chemosphere; 2020; 246, 125810. [DOI: https://dx.doi.org/10.1016/j.chemosphere.2019.125810]

38. Guo, X.; Han, W.; Zhang, G.; Yang, W.; Wei, Z.; He, Q.; Wu, Q. Effect of inorganic and organic amendments on maize biomass, heavy metals uptake and their availability in calcareous and acid washed soil. Environ. Technol. Innov.; 2020; 19, 101038. [DOI: https://dx.doi.org/10.1016/j.eti.2020.101038]

39. Irfan, M.; Mudassir, M.; Khan, M.J.; Dawar, K.M.; Muhammad, D.; Mian, I.A.; Ali, W.; Fahad, S.; Saud, S.; Hayat, Z. et al. Heavy metals immobilization and improvement of maize (Zea mays L.) growth amended with biochar and compost. Sci. Rep.; 2021; 11, 18416. [DOI: https://dx.doi.org/10.1038/s41598-021-97525-8]

40. Pinto, T.O.; García, A.C.; Guedes, J.N.; Sobrinho, N.M.B.A.; Tavares, O.C.H.; Berbara, R.L.L. Assessment of the use of natural materials for the remediation of cadmium soil contamination. PLoS ONE; 2016; 11, e0157547.

41. Trevisan, S.; Francioso, O.; Quaggiotti, S.; Nardi, S. Humic substances biological activity at the plant-soil interface. Plant Signal. Behav.; 2010; 5, pp. 635-643. [DOI: https://dx.doi.org/10.4161/psb.5.6.11211]

42. Meng, F.; Yuan, G.; Wei, J.; Bi, D.; Ok, Y.S.; Wang, H. Humic substances as a washing agent for Cd-contaminated soils. Chemosphere; 2017; 181, pp. 461-467. [DOI: https://dx.doi.org/10.1016/j.chemosphere.2017.04.127] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28458221]

43. Kozhevnikova, A.D.; Seregin, I.V.; Bystrova, E.I.; Belyaeva, A.I.; Kataeva, M.N.; Ivanov, V.B. The effects of lead, nickel, and strontium nitrates on cell division and elongation in maize roots. Russ. J. Plant Physiol.; 2009; 56, pp. 242-250. [DOI: https://dx.doi.org/10.1134/S1021443709020137]

44. Dong, Y.; Ma, L.Q.; Rhue, R.D. Relation of enhanced Pb solubility to Fe partitioning in soils. Environ. Pollut.; 1999; 110, pp. 515-522. [DOI: https://dx.doi.org/10.1016/S0269-7491(99)00312-7] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15092830]

45. Peñafiel-Sandoval, Z.B.; Iannacone, J. Effect of urea on lead absorption in corn (Zea mays L.), spinach (Spinacia oleraceae L.) and cabbage (Brassica oleraceae L.). Agron. Columb.; 2020; 38, pp. 105-217.

46. Kacálková, L.; Tlustoš, P.; Száková, J. Chromium, nickel cadmium, and lead accumulation in maze, sunflower, willow, and poplar. Pol. J. Environ. Stud.; 2014; 23, pp. 753-761.

47. Hadi, F.; Aziz, T. A mini review on lead (Pb) toxicity in plants. J. Biol. Life Sci.; 2015; 6, pp. 91-101. [DOI: https://dx.doi.org/10.5296/jbls.v6i2.7152]

48. Małkowski, E.; Kita, A.; Galas, W.; Karcz, W.; Kuperberg, J.M. Lead distribution in corn seedlings (Zea mays L.) and its effect on growth and the concentrations of potassium and calcium. Plant Growth Regul.; 2002; 37, pp. 69-76. [DOI: https://dx.doi.org/10.1023/A:1020305400324]

49. Sengar, R.S.; Pandey, M. Inhibition of chlorophyll biosynthesis by lead in greening Pisum sativum leaf segments. Biol. Plant.; 1996; 38, pp. 459-462. [DOI: https://dx.doi.org/10.1007/BF02896681]

50. Kumar, A.; Prasad, M.N.V. Lead-induced toxicity and interference in chlorophyll fluorescence in Talinum triangulare grown hydroponically. Photosynthetica; 2015; 53, pp. 66-71. [DOI: https://dx.doi.org/10.1007/s11099-015-0091-8]

51. Ievinsh, G.; Andersone-Ozola, U.; Zeipiņa, S. Comparison of the effects of compost and vermicompost soil amendments in organic production of four herb species. Biol. Agric. Hortic.; 2020; 36, pp. 267-282. [DOI: https://dx.doi.org/10.1080/01448765.2020.1812116]

52. Ose, A.; Andersone-Ozola, U.; Ievinsh, G. Substrate-dependent effect of vermicompost on yield and physiological indices of container-grown Dracocephalum moldavica plants. Agriculture; 2021; 11, 1231. [DOI: https://dx.doi.org/10.3390/agriculture11121231]

53. Sharma, S.D.; Chorpa, R.N. Effect of lead nitrate and lead acetate on growth of the moss Semibarbula orientalis (Web.) Wijk. et Marg. grown in vitro. J. Plant Physiol.; 1987; 129, pp. 243-249. [DOI: https://dx.doi.org/10.1016/S0176-1617(87)80083-4]

54. Yang, N.; Zhou, F.-R.; WEang, J.-X. Eco-toxicological effects of two kinds of lead compounds on forest tree seed in alkaline soil. Environ. Monit. Assess.; 2016; 188, 201. [DOI: https://dx.doi.org/10.1007/s10661-016-5198-2] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26927657]

55. Alexandrino, R.C.S.; Lima, F.R.D.; Martins, G.C.; Natal-du-Luz, T.; Sousa, J.P.; Guilherme, L.R.G.; Marques, J.J. Lead acetate toxicity in tropical soils. Ecotoxicology; 2021; 30, pp. 1029-1042. [DOI: https://dx.doi.org/10.1007/s10646-021-02443-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34191243]

56. Sofy, M.R.; Seleiman, M.F.; Alhammad, B.A.; Alharbi, B.M.; Mohamed, H.I. Minimizing adverse effects of Pb on maize plants by combined treatment with jasmonic, salicylic acids and proline. Agronomy; 2020; 10, 699. [DOI: https://dx.doi.org/10.3390/agronomy10050699]

57. Britto, D.T.; Kronzucker, H.J. NH₄ toxicity in higher plants: A critical review. J. Plant Physiol.; 2002; 159, pp. 567-584. [DOI: https://dx.doi.org/10.1078/0176-1617-0774]

58. Ravazollo, L.; Trevisan, S.; Forestan, C.; Varotto, S.; Sut, S.; Dall’Acqua, S.; Malagoli, M.; Quaggiotti, S. Nitrate and ammonium affect the overall maize response to nitrogen availability by triggering specific and common transcriptional signatures in roots. Int. J. Mol. Sci.; 2020; 21, 686. [DOI: https://dx.doi.org/10.3390/ijms21020686]

59. Poorter, H.; Fiorani, F.; Stitt, M.; Schurr, U.; Finck, A.; Gibon, Y.; Usadel, B.; Munns, R.; Atkin, O.K.; Tardieu, F. et al. The art of growing plants for experimental purposes: A practical guide for the plant biologist. Funct. Plant Biol.; 2012; 39, pp. 821-838. [DOI: https://dx.doi.org/10.1071/FP12028]