3.1. Effect of EAF Slag on Nutrient Status in Soil and Bean Plant Organs
The soil enriched with the EAF slag had higher pH-value and conductivity in comparison to the control soil (
Table 1). The effect of the EAF slag on the soil pH-value and conductivity can be ascribed to its high content of oxides, primarily free calcium oxide. These compounds can be dissolved in the aqueous solution of the soil, resulting in the release of hydroxide ions and thus higher alkalinity [
16,
18,
28,
29].
The contents of the assessed nutrients elevated in the soils supplemented with the EAF slag (
Table 1). Several studies have reported that concentrations and availability of Mg, Ca and K cations rise with time in soil enriched with steel slag [
15,
18,
28,
29]. Khan et al. [
29] concluded that increase of soil pH-value due to the addition of EAF slag improves the ratio of Ca:Mg ions and thus ensures the greater availability of Ca ions that are generally unavailable in acid soils. In this study, elevated concentrations of Mg, Ca and K in the soils enriched with the EAF slag, and even in the soil enriched with combined 2% EAF slag and NPK, were detected. Interestingly, the contents of Mg and K in the soil increased with an increase of the level of EAF slag, while the content of Ca was the highest in the soil enriched with 1% EAF slag.
The significant uptake of Mg and K by plant organs coincided with increased concentrations of those nutrients in the slag enriched soil (
Table 2). The EAF slag supplementation markedly increased leaf and seed Mg content, showing even better performance than NPK fertilizer (
Table 2). The lower level of the EAF slag significantly increased the leaf and husk K content. Enhanced nutrient uptake by plants in the EAF slag enriched soils might be connected with the beneficial effects of slag on soil bacterial communities, which in turn increased nutrient availability [
17,
18].
The major determinants in N availability in a soil solution are the content of the soil organic matter and conditions for mineralization (moisture, temperature, aeration), while the soil pH has minimal effect on the turnover of N in alkaline soils [
30]. Higher plant-available N was recorded in the soils supplemented either by the EAF slag or NPK fertilizer compared to the control soil (
Table 1). This might be related to the higher degree of nitrification in soils at a pH greater than 6.0, since the optimal pH for nitrification is established to be around 8.5 [
31]. Interestingly, the increase of leaf and seed N content was higher in plants cultivated in soils supplemented by a lower level of the EAF slag than in those supplemented by a higher level of it. A moderate-to-strong positive relationship was found between N and K content in different plant organs (r from 0.68 to 0.84), suggesting the possible effect of K on the N level (
Table 3). This could be the case in our study, since it was demonstrated that a higher amount of K in the slag, when released into the soil, can increase the availability of NH
4+ ions in the process of cation exchange [
32,
33]. In this study, a significant induction of leaf NR activity was observed in all amended soils (
Table 4), which corroborates the assumption of a higher nitrification rate in tested soils. In addition, NR activity and content of leaf N were closely related, as evidenced by a very strong positive correlation (r = 0.89;
Table 3). In a study by Das et al. [
17], it was determined that steel slag amendment improved the N uptake of rice straw. The authors suggested that increased N uptake could be connected with the stimulation of nitrogen fixation in soil amended with steel slag.
Both total and plant available P was higher in the soils supplemented either by the EAF slag or NPK fertilizer than in the control soil (
Table 1). Available P in alkaline soils is generally low; it may, however, increase depending on the amount of soluble organic matter, as P tends to be less stable at higher pH [
34]. Kristen and Erstad [
35] found that the increase of P in soil could be related to the presence of Si in slag. Specifically, Si can be replaced with plant-available soil P. In this study, the increase of plant-available P coincided with the increase of Si in the EAF slag amended soils, especially in those amended with 1% EAF slag (
Table 1). Accordingly, the leaf and seed P content significantly increased in plants cultivated in slag supplemented soils (
Table 2).
Fe solubility is low in calcareous soils. At pH between 7 and 8.5, this microelement is mainly present in the soil solution in the form of Fe(OH)
2+, Fe(OH)
3 and Fe(OH)
4− [
30]. Despite high soil pH in the slag amended soils, the Fe content was higher in those soils compared to the control soil (
Table 1). This result was expected, as the EAF slag contains a considerable amount of Fe oxides. The Fe uptake in leaves and husks of plants increased either by the EAF slag or NPK fertilizer supplementation compared to the control (
Table 2). These results could be explained by the better solubility of Fe(OH)
4− at pH higher than 8.5 [
36]. It is interesting that a positive moderate-to-strong correlation was established among Fe, Mg, N, P and K contents in different plant organs, indicating their mutual dependency (
Table 3). Similarly, Torkashvand [
16] found that application of steel converter slag at 0.5 and 1% levels significantly increased extractable Fe in calcareous soil, and uptake of Fe, Mn, K, and P in maize shoots, after a two-month growth period. Steel slag also stimulated accumulation of Fe and other nutrients (N, P, K, Mn and Zn) in maize [
14] and radish plants, especially when organic matter was added to calcareous soil [
37]. Additionally, Islam et al. [
18] detected increased uptake of Fe, Mg and Ca in turnips and spinach cultivated in soils amended with steel slag.
Several investigations found a negative correlation between Fe and Mn uptake in shoots, suggesting an antagonistic effect between these micronutrients [
38,
39], however, that was not the case in our study. Here, the EAF slag application significantly increased Mn content in the soil and in the husk of bean plants, while the leaf and seed Mn content was similar to that in the control soil.
The EAF slag supplementation did not cause substantial change in the contents of non-essential, potentially toxic metals (Cd, Pb, Cr) (
Table 1). The content of those metals in the soil amended with 2% EAF slag was slightly higher than in the soils amended with 1% EAF slag and the control soil. In this study, the uptake of non-essential metals in plant organs was not determined, but based on the available data, steel slag at levels up to 2% does not significantly affect the content of those metals in several model plants, including the bean [
15,
40,
41,
42].
3.2. Effect of EAF Slag on Growth and NR Activity of Bean Plant
The application of either EAF slag or NPK fertilizer significantly increased plant height compared to the control soil (16–21% increase compared to the control) (
Figure 1a). EAF slag increased the leaf dry weight (
Figure 1c). However, only amended with 1% EAF slag resulted with a significant increase in the number of husks (
Figure 1b) and in seed dry weight (
Figure 1c). A marked rise in the number of husks was also seen after application of combined 2% EAF slag and NPK fertilizer (
Figure 1c). Such positive impact of the EAF slag on growth parameters may be connected to the increased contents of N, P, K, Mg and Fe in different plant organs (
Table 2).
Specifically, a positive correlation between the leaf dry weight and P, K and Mg content in different plant organs was found (r from 0.55 to 0.71) (
Table 3). In addition, the number of husks was closely related to the husk-N and -K, and Fe, P and K content in different plant organs (r from 0.54 to 0.64) (
Table 3). Previous studies demonstrated that converter steel slag at level 1 and 2% caused an increase of Fe, P and K uptake and concomitant increase of shoot dry matter in maize [
13,
14]. On the other hand, Negim et al. [
15] reported that steel slag-promoted growth of dwarf beans could be related to an increased foliar Ca content. In a study by Chen et al. [
42], molybdenum slag at levels up to 5% improved the growth of pak choi seedlings cultivated in calcareous soil by providing nutrients. Interestingly, in comparison to the performance of NPK amendment, remobilization of N from leaf to seed (which is important for the seed-filling period) was much more effective in plants cultivated in the EAF slag-amended soil that resulted in maximum seed dry weight (
Table 2). This might be due to disturbed N metabolism and higher oxidative damage in plants cultivated in the soil enriched with NPK fertilizer (
Table 5). Indeed, recent research suggests that excessive N application led to considerable changes in N metabolism and to increased lipid peroxidation, which consequently altered grain filling in wheat [
43].
Nitrate reductase (NR) is a crucial enzyme for the acquisition of N in plants and it is a reliable indicator of plant-N status in leaves [
44]. The activity of NR increased in a following order: C < S2 < S1 < F < FS2 (
Table 4). A very strong correlation was found between NR activity and leaf N (r = 0.89), but the activity of that enzyme also correlated with N contents of husk and seed (r from 0.59 to 0.65) (
Table 3).
3.3. Effect of EAF Slag on Photosynthetic Parameters of Bean Leaves
In this study, the activity of the photosynthetic apparatus was evaluated by estimating maximum quantum yield of PSII (F
v/F
m), net photosynthetic rate of CO
2 assimilation (PS), and content of chlorophylls and carotenoids (
Table 6). Soil amendment with either the EAF slag or NPK fertilizer caused no significant change in F
v/F
m values, nor in the contents of chlorophylls and carotenoids and their ratios in comparison to the control. Since these parameters directly describe the regulation of the processes of absorption and trapping energy fluxes, they are extraordinarily important for maintaining effective primary photochemistry of the PSII [
45,
46]. Based on these data, it can be concluded that PSII was fully functional in all investigated bean plants, which allowed a truthful direct comparison of net photosynthetic rates between investigated plants.
Gas exchange measurements provide a direct measure of the net rate of photosynthetic carbon assimilation [
47]. Photosynthetic parameters obtained by those measurements showed that the EAF slag boosted photosynthesis, as evidenced by increased net photosynthetic rate PS (
Table 6). The highest gain of PS (65% compared to the control) was noted after application of combined 2% EAF slag and NPK fertilizer, whereas a significant increase of PS (35% compared to control) was also observed on application of 1% EAF slag. It seems that the photosynthetic rate is affected by leaf N and NR activity, as the correlation between those parameters was significant and ranged from 0.56 to 0.64 (
Table 3). The relation between PS and leaf-N content could be explained, at least to some extent, by the relatively high investment of N in the proteins of the Calvin cycle and thylakoids, in particular RuBisCO in C3 plants [
48,
49,
50].
Other physiological parameters, such as intercellular CO
2 concentration and stomatal conductance, were improved in plants cultivated in the soil enriched with 1% EAF slag and in the soil enriched with combined 2% EAF slag and NPK fertilizer (
Table 4). On the other hand, the transpiration rate was not affected by either the EAF slag or NPK fertilizer supplementation, in comparison to the control.
3.4. Effect of EAF Slag on Oxidative Stress Parameters of Bean Leaves
Oxidative stress arising due to the imbalance between the surplus production of ROS and immediate inefficiency in their neutralization often occurs in response to a variety of natural and anthropogenic factors. Stress-induced accumulated ROS can harm vital biomolecules, causing protein cross-linking, inhibition of enzyme activity, alterations in membrane fluidity and solute transport, and other detrimental processes, which eventually lead to cell death [
51]. Since a higher oxidation rate occurs in plant leaves, in this study, oxidative stress parameters were assessed only in bean leaves. As markers of oxidative damage to membrane lipids and proteins in the bean leaves, MDA and carbonyl group contents were evaluated. Carbonylation of leaf proteins was not affected by either the EAF slag or NPK fertilizer application, implying that there was no direct oxidation of proteins by ROS (
Table 5). However, the level of MDA significantly increased on application of NPK fertilizer compared to the control. Simultaneously, a significant rise in activity of APX, one of the H
2O
2 detoxifying enzymes, was determined on application of NPK fertilizer. On the other hand, the POX enzyme was obviously not induced in the degradation of the H
2O
2, as evidenced by unchanged POX activity in the plants cultivated in the amended soils. Since a positive correlation (
Table 3) was established between NR activity and MDA (r = 0.65), a higher peroxidation of membrane lipids might be related to a faster rate of N assimilation and higher ROS formation [
43,
52]. Moreover, APX activity correlated with NR activity (r = 0.58) and leaf N content (r = 0.57), corroborating the connection between N metabolism and ROS accumulation. Activity of SOD, one of the major antioxidative enzymes, was unchanged in the plants cultivated in the soils enriched with either the EAF slag and/or NPK fertilizer, which points to another source of H
2O
2 formation (
Table 5). The uncharged and freely diffusible oxygen species can be generated via several enzymatic and non-enzymatic reactions such as the oxidation of glycolate during photorespiration, amine oxidase, xanthine oxidase, NADPH oxidase and so forth [
53].