Determination of the Effect of Organic Matter Addition to Mineral Soil on Nickel Detoxification in Radish, Its Yield, Nitrogen Metabolism and Chloroplast Pigments

Abstract

Understanding soil properties that govern physicochemical and biological processes is essential for achieving high crop quality and yield. Organic matter is an important element of soil fertility and fertility in vegetable cultivation. In the process of decomposition of organic matter in the soil, humus of various quality is formed. The quality of humus depends on the content of individual acids (fulvic, humic and hymatomalanic acids) in it, which can affect the binding–chelation of heavy metals, limiting their availability to plants. The conducted studies determined the effect of adding organic matter (high peat, brown coal and wheat straw) to mineral soil on nickel detoxification in radish, its yield, nitrogen management and chloroplast pigments. The studies were conducted for three years in a greenhouse in a container system. The tested substrates were contaminated with nickel in the amount of 50, 75 and 100 mg dm⁻³. It was found that introducing organic matter into mineral soil can affect the reduction as well as the increase in nickel content in edible parts of radish. The type of organic material introduced into mineral soil as a source of organic matter has a significant impact on nickel content in radish. It was shown that nitrate reductase activity (NR) depends to a large extent on the substrate in which the plants are grown as well as on the applied dose of nickel. A similar relationship was demonstrated in the case of changes in the level of chloroplast pigments (chlorophyll a, chlorophyll b and carotenoids).

1. Introduction

In 2008, nickel was voted ‘Allergen of the Year’ by the American Contact Dermatitis Society [1]. Nickel can cause a number of adverse human health effects. The element enters the human body through the inhalation of fumes and dusts and through drinking water and food. The nickel content in the diet is strongly related to its concentration in the soil. As plant tissues generally contain more nickel than animal tissues, dietary intake depends largely on the consumption of plant-derived foods [2].

Contact with this heavy metal can most commonly cause allergies (contact dermatitis), as well as cardiovascular and renal diseases, pulmonary fibrosis and lung and nasal cancers. Nickel is believed to cause mitochondrial dysfunction, mainly by inducing oxidative stress and the production of reactive oxygen species [3]. Nickel allergy is categorized as a so-called disease of modern society. The source of nickel for humans is vegetables. In a study by Onianwa et al. [4], an analysis of 78 food items from a Nigerian market was conducted. They found that nickel levels ranged from 0.05 to 9.22 mg kg⁻¹ on a dry weight basis. The highest nickel content in vegetables was found in carrots (9.2 mg kg⁻¹), beans (7.0 mg kg⁻¹), soybeans (5.07 mg kg⁻¹), tomatoes (3.83 mg kg⁻¹), cabbage (3.53 mg kg⁻¹) and onions (3.13 mg kg⁻¹). A similarly high content of this element was reported in cauliflower, cabbage, onion and potatoes [5]. It is known that Ni at low concentrations is an essential nutrient for plant growth and development [6]. In contrast, when Ni contamination in the environment increases, it becomes hazardous. Therefore, it is very important to understand its functional roles and toxic effects on plants [7]. Nickel in soil occurs in inorganic and organic forms, including crystalline minerals and sediments. It is absorbed on the surface of organic cations, as well as dissolved in water and in soil solution as free ions and chelated complexes [8]. In agricultural soils, nickel content ranges widely from 3 to 1000 mg Ni kg⁻¹ soil, but near-significant sources of the element, e.g., metallurgical industries, levels can increase from 24,000 to 53,000 mg Ni kg⁻¹ [9]. According to Kabata-Pendias and Szteke [10], worldwide, the average content of this element in soil ranges from 13 to 37 mg kg⁻¹. In vegetable production, sources of nickel can be mineral fertilizers, pesticides and herbicides [11,12]. The availability of Ni in the soil increases with decreasing pH, so a lower pH value facilitates the transport of this element from the soil solution to the plant roots [13].

Many literature reports indicate that plant uptake of nickel is influenced by the soil organic carbon content [14,15,16,17,18]. The source of organic carbon in the soil is organic matter, which in vegetable cultivation is introduced in various types of organic material. The type of organic material used has a significant effect on the amount of fulvic, humic and hymatomelanic acids formed during the humification of organic matter in the soil. The content of each type of acid influences the formation of complex combinations of nickel with organic functional groups (for example, carboxyl and hydroxyl). Depending on the quality of the humus formed, this can affect nickel detoxification [18] or increased accumulation in plants [19,20].

Data in the literature provide information on the role of this metal in various metabolic processes of the plant. For example, nickel has been shown to influence, among other things, RFT levels, osmoprotectant synthesis, the activation of stress tolerance pathways and the maintenance of intracellular pH and ionic balance in plant cells through interactions with other metal ions, especially iron ions. Additionally, this element modulates nitrogen metabolism by regulating the activity of enzymes involved in nitrogen assimilation [21].

The aim of this study was to determine the effect of the addition of organic matter to mineral soil on nickel detoxification in storage roots of radish, their yield, nitrogen management and chloroplast pigments.

Vegetables whose edible part is the root thickening and the leaves are most susceptible to heavy metal accumulation [22,23]. Vegetables accumulating a high amount of heavy metals in the edible part include radishes, which were selected for the study. Various organic materials (high peat, brown coal and wheat straw) were used as a source of organic carbon in the soil in which radishes were grown, adding them at a rate of 30% by volume to the mineral soil (loamy sand).

2. Materials and Methods

The vegetation studies were conducted in an unheated greenhouse of the Department of Plant Physiology, Poznań University of Life Sciences, during the spring–summer season, 2018–2020. Radish (Raphanus sativus var. sativus) cv. ‘Crispar F1’, a species classified as a plant prone to the accumulation of significant amounts of heavy metals and nitrates in its storage of roots, was selected for this study.

The experimental factors were four substrates: mineral soil (100% control object), mineral soil representing 70% to which high peat was added at 30% by volume, mineral soil 70% to which brown coal 30% was added and mineral soil 70% to which wheat straw 30% was added. These substrates were contaminated with nickel in increasing doses (0, 50, 75 and 100 mg Ni dm⁻³ of substrate). The study was conducted for three years.

Three vegetation experiments were conducted over the three years of the study. The experiments included 48 combinations and each combination consisted of 5 replications. One replication consisted of 5 radish plants growing in one 1 dm³ polyethylene container.

Using a mixture of acids (HNO₃ and HClO₄) at a volume ratio of 3:1, the total nickel content (mg kg⁻¹ dry weight) of the organic materials used in the study was found to be 0.82 (high peat), 0.39 (brown coal) and 0.82 (wheat straw).

In the first week of April, the first year of the study, the growing substrates for all years of the experiments were prepared. Drainless containers with a capacity of 1 dm³ were filled with mineral soil (control) and the same soil to which organic matter was introduced in the amount of 30% by volume in the form of high peat, brown coal and wheat straw. The mineral soil (loamy sand) accounted for 70% of the volume of the substrate, and its granulometric composition was tested using the Cassagrande method modified by Prószynski [24] (

Table 1. Granulometric composition of mineral soil.

Granulometric Composition of Mineral Soil (%)
Send	69
Dust	23
Clay parts	8
Corg content	1.14

).

In the experimental substrates prepared in this way, using the “Universal” method (extraction solutionin 0.03 M CH₃COOH, substrates-to-solution ratios 1:10) [25] for the determination of macronutrients and the Lindsay method (substrates-to-solution ratios 1:5, solution containing 5 g ethylenediaminetetraacetic acid; 9 cm³ of a 25% NH₄OH solution; 2 g Ca(CH₃COO)₂ 2H₂O; 4 g of citric acid in 1 dm³) [26] for the determination of micronutrients, the following chemical composition was found:

• Mineral soil (loamy sand): pH (H₂O) 7.15; EC (mS cm⁻¹) 0.193 and in mg dm⁻³ N-NH₄ 7; N-NO₃ 14; P 16, K 52; Ca 6255; Mg 98; Na 14; S-SO₄ 12; Cl 7; Fe 34.9; Mn 8.2; Cu 2.5; Zn 11.1 and Ni 1.32;
• Mineral soil (loamy sand) + high peat: pH (H₂O) 7.03; EC (mS cm⁻¹) 0.190 and in mg dm⁻³ N-NH₄ 11; N-NO₃ 4; P 34, K 82; Ca 6188; Mg 52; Na 10; S-SO₄ 8; Cl 14; Fe 50.7; Mn 11.5; Cu 3.4; Zn 6.7 and Ni 1.47;
• Mineral soil (loamy sand) + brown coal: pH (H₂O) 7.20; EC (mS cm⁻¹) 0.211 and in mg dm⁻³ N-NH₄ traces; N-NO₃ 5; P 28, K 78; Ca 6311; Mg 178; Na 28; S-SO₄ 17; Cl 9; Fe 48.6; Mn 9.1; Cu 2.8 Zn 12.1 and Ni 1.37;
• Mineral soil (loamy sand) + wheat straw: pH (H₂O) 7.09; EC (mS cm⁻¹) 0.197 and in mg dm⁻³ N-NH₄ 2; N-NO₃ 9; P 33, K 89; Ca 6198; Mg 87; Na 16; S-SO₄ 15; Cl 8; Fe 38.7; Mn 8.8; Cu 2.6; Zn 10.1 and Ni 1.28.

In the third week of April, each year of the study, nutrients were added to the substrates in the form of solutions bringing them to recommended contents (mg dm⁻³): 150 N (KNO₃); 150 P (KH₂PO₄); 250 K (KH₂PO₄); 130 Mg (Mg(NO₃)₂ 6H₂O); 75 Fe (FeSO₄ 7H₂O); 15 Mn (MnSO₄ H₂O); 10 Cu (CuSO₄ 5H₂O) and 20 Zn (ZnSO₄ 7H₂O). Magnesium was not added to the brown coal substrate because the brown coal substrate contained a 178 mg dm⁻³.

Increasing doses of nickel were added to the test substrates in nickel sulphate solution (NiSO₄ 6H₂O) at 0 (control); 50; 75 and 100 mg dm⁻³ of substrate.

Seeds of radish variety ‘Crispar F1’ came from Syngenta Polska Sp. z o.o. This variety is intended for spring, summer and autumn cultivation under covers, it has a strong, erect leaf, ball-storage, and uniformly red skin color, is compact, has white flesh and shows IR resistance: Fusarium oxysporum sp. raphani.

At the end of April, each year of the study, the radish was sown directly into the growing containers. Eight radish seeds were sown per container. For the germination of the seeds, the containers were covered with transparent foil for better and even germination. After germination, the plants were interrupted and subjected to selection. Five undamaged, evenly sized plants were selected for further stages of the experiments.

Harvesting was done at 6 weeks: 8 days germination and 42 days growth after sowing the seeds. After harvesting, the radish storage roots were cleaned, cut off from the leaves and weighed. The plant material was then crushed and dried at 105 °C. The plant material was thus prepared and then mineralized in HNO₃ and HClO₄ acids (3/1 v/v) [27]. Nickel was determined after mineralization using the FAAS (flame atomic absorption) method, with a ZEISS AAS-5 spectrophotometer (Ni—lamp current 5.0 mA, wavelength 232.0 nm, slit 0.20 mm and detectability 0.055 ppm). Physiological and biochemical analyses were carried out on fresh plant material. The starting material for the determination of nitrate levels and nitrate reductase activity was the storage of radish roots. In turn, chloroplast pigment levels were determined in the leaves.

2.1. Nitrate Content

The content of nitrates in radish roots was determined colorimetrically by the nitration of salicylic acid [28]. The plant material (250 mg) was placed in glass tubes, poured with 10 cm³ of deionized water and boiled in a water bath (20 min). The extracts were transferred to centrifuge tubes and centrifuged at 4000 g for 15 min. The supernatant (0.1 cm³) was taken, and 0.4 cm³ of 5% salicylic acid (dissolved in concentrated H₂SO₄) was added. At the same time, a reference sample was prepared. After 20 min, 9.5 cm³ of 2 M NaOH was added to all the tubes and cooled. The absorbance of the sample was measured at a wavelength of 410 nm relative to the reference sample. The content of nitrates was read from the standard curve prepared for KNO₃ and was expressed as milligrams of nitrites per gram of fresh weight [mg NO₃⁻ × g⁻¹ FW].

2.2. Nitrate Reductase Activity

The in vivo determination of nitrate reductase (NR) activity was based on Jaworski [29]. Fragments of radish storage roots (250 mg) were incubated in 50 cm³ glass flasks in 5 cm³ of the incubation mixture (0.1 M phosphate buffer, pH 7.5; 0.1 M KNO₃ and 2% propanol) into which the enzyme diffused. Enzyme activity was measured by the number of nitrites formed due to the reduction in nitrates in this mixture. In parallel, flasks with an incubation mixture without nitrates were prepared. After one hour of incubation at 30 °C, one cm³ of the solution was pipetted into glass tubes, then 1 cm³ of SAA (1% sulfanilamide in 1 M HCl) and 1 cm³ of NED (0.01% N-[1-naphthyl]-ethylenediamine dihydrochloride) were added. After 15 min of incubation (room temperature), absorbance at 540 nm was measured. Based on the standard curve prepared for nitrites (NO₂⁻), the number of nmoles of nitrites formed was calculated. The NR activity was given as the number of nmoles of the product [NO₂⁻] formed in 1 h per gram of fresh weight (nmoles NO₂⁻ × h⁻¹ × g⁻¹ FW).

2.3. Photosynthetic Pigments

Photosynthetic pigments: chlorophyll a, chlorophyll b and carotenoids, were estimated according to the method of Hiscox and Israelstam [30]. Leaf tissue (50 mg) was cut into pieces, poured with 10 mL dimethyl sulfoxide (DMSO) and incubated for 60 min in a water bath at 65 °C. The absorbance of extracts was measured spectrophotometrically at 663, 645 and 480 nm. The pigment content was calculated using modified Arnon formulas [31] and expressed in micrograms per gram of fresh weight.

2.4. Organic Carbon Content

The organic carbon content of C_org. was performed using 1 g of dry soil sifted through a 1 mm mesh sieve, weighed and transferred into an Erlenmeyer flask. To the flask, 50 cm³ of 0.1 N KMnO₄, 10 cm³ of H₂SO₄ (1:3) and 100 cm³ of distilled water were also added. The flask was then placed on a hotplate and heated to boiling, which was maintained for 1 h. If the solution discolored during this time, then another 50 cm³ of 0.1 N KMnO₄ was added. Then, 50 cm³ of 0.1 N oxalic acid (H₂C₂O₂) was added. After the solution was decolorized, it was titrated with 0.1 N KMnO₄ until the color changed to violet.

2.5. The Index of Toxic Effect of Increasing Doses of Nickel (Ti)

This study also determined the index of toxic effect of increasing doses of nickel (Ti) on storage of radish roots yield. The tolerance index value obtained was calculated from the ratio of the storage of radish roots yield obtained in the medium with the appropriate dose of nickel to the yield of plants growing in the control substrate (without nickel addition). A negative effect of nickel on radish yield is indicated by a Ti value below 1, while a value above 1 indicates a positive effect of this metal on the storage of radish roots yield. In the case of no effect of nickel on the storage of radish roots yield, the coefficient Ti = 1.

2.6. Statistical Analysis

Trivariate statistical analysis was performed for five replications for one combination, representing a total of 48 combinations for the entire study. STAT BAT (for orthogonal factorial experiments) was used for statistical calculations, using the DUNCAN test, and differences between mean values were assessed at α = 0.05. The statistical evaluation involved an analysis of variance, three-factorial, for radish storage root yield; nickel content in radish edible parts; organic C content in substrates after radish cultivation; nitrate content and nitrate reductase activity in radish storage root after cultivation; and chloroplast pigment content in radish leaves after cultivation.

3. Results and Discussion

The availability of nickel to plants depends on the chemical, biological and physical properties of the soil [32]. Organic matter introduced into a mineral soil undergoes two processes: mineralization and humification. Humification produces soil humus, the quality of which depends on the type of organic material used. Sources of organic matter in the soil can be crop residues, green manures and other organic materials. The most common and versatile organic fertilizer is manure, but as a result of the introduction of barn-free animal husbandry, its availability is steadily decreasing. Sources with which to supplement soil organic matter can be straw, brown coal and peat [18,33].

3.1. Organic Carbon Content of Substrates After Radish Cultivation

In this study, three organic materials were used as a source of C_org.: high peat, brown coal and wheat straw, differing in the degree of decomposition in the soil. After a single application of these organic materials to the soil, the time required for complete mineralization is estimated to be 4–13 years for high peat [34], 12 years for brown coal [35] and 1.4 years for wheat straw [34].

The lowest C_org content (0.98%) was found in the mineral soil (

Table 2. The content of C_org (%) of substrates after radish cultivation.

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	0.96 *a–c	1.02 a–f	0.95 ab	0.98 a	0.98 a
		50	0.95 ab	0.99 a–d	1.05 c–g	1.00 a
		75	0.93 a	0.99 a–d	0.98 a–c	0.97 a
		100	0.95 ab	1.02 a–f	1.01 a–e	0.99 a
Mean B·A			0.95 a	1.01 bc	0.99 b
Loamy sand with high peat		0	1.95 ij	2.01 j–l	2.01 j–l	1.99 c	1.99 c
		50	1.95 ij	1.95 ij	2.01 j–l	1.97 c
		75	1.95 ij	2.01 j–l	2.01 j–l	1.99 c
		100	1.89 i	2.04 j–m	2.06 k–n	2.00 c
Mean B·A			1.94 f	2.00 g	2.02 g
Loamy sand with brown coal		0	2.15 no	2.15 no	2.04 j–m	2.11 e	2.10 d
		50	2.25 p	2.13 m–o	1.95 ij	2.11 e
		75	2.10 l–n	2.12 mn	1.95 ij	2.06 d
		100	2.10 l–n	2.22 op	2.00 ij	2.11 e
Mean B·A			2.15 h	2.15 h	1.98 g
Loamy sand with wheat straw		0	1.05 c–g	1.17 h	1.04 b–f	1.09 b	1.08 b
		50	1.08 d–h	1.14 gh	1.01 a–e	1.08 b
		75	1.05 c–g	1.11 f–h	1.04 b–f	1.07 b
		100	1.04 b–f	1.14 gh	1.10 e–h	1.09 b
Mean B·A			1.05 d	1.14 e	1.04 cd
Mean A			1.52 a	1.58 b	1.51 a
Mean C		dose 0 Ni (I–III)	dose 50 Ni (I–III)		dose 75 Ni (I–III)		dose 100 Ni (I–III)
		1.54 a	1.54 a		1.52 a		1.55 a
Mean A·C	year	dose 0 Ni	dose 50 Ni		dose 75 Ni		dose 100 Ni
	I	1.53 a–c	1.56 cd		1.51 ab		1.49 a
	II	1.59 de	1.55 b–d		1.56 cd		1.61 e
	III	1.51 ab	1.50 a		1.49 a		1.54 a–c

A—year, B—substrate and C—dose. * Homogeneous groups were identified using the Duncan test; data followed by the same letters do not differ significantly at α = 0.05.

) and compared to this content, more C_org was found by 103.06% in the substrate with high peat, by 114.29% in the substrate with brown coal and by 10.20% in the substrate with wheat straw. The highest C_org (2.10%) was found in the substrate with brown coal.

In mineral soil, the lowest C_org content (0.95%) was found in the first year of this study, while the highest (1.01%) was found in the second year. In the substrate with high peat, the lowest C_org content (1.94%) was found in the first year and the highest in the second (2.00%) and third (2.02%) years of this study. In the substrate with brown coal, the lowest C_org content (1.98%) was obtained in the third year of cultivation and significantly higher in the first (2.15%) and second (2.15%) years of this study. In the substrate with wheat straw, the lowest C_org content (1.04%) was obtained in the third year of cultivation and it did not differ significantly from the amount in the first year. In the second year of this study, the highest C_org content of 1.14% was obtained in this substrate.

Analyzing the average for the individual years of this study, the highest C_org of 1.58% was obtained in the second year of cultivation and the lowest in the first (1.52%) and third (1.51%) year.

No effect of increasing nickel concentrations, regardless of the substrate and years, was found on significant changes in the amount of C_org.

Soil organic matter consists of sedimentary organic rocks—bioliths, dead plant and animal remains, living organisms: fungi, bacteria, cyanobacteria and insects and earthworms. The basic component of organic matter is humus substances. Humus is a naturally produced mixture of polymolecular and heterogeneous organic matter, dark in color, representing the various stages of decomposition of plant and animal remains [36]. Humus compounds are mainly composed of heterogeneous groups of acids (fulvic and humic acids) and their salts (humates). Humic acids are amorphous, polymeric organic acids composed of carbon, hydrogen, oxygen and, in smaller amounts, sulfur and nitrogen. The largest quantities are hydrogen and carbon, which are incorporated in these acids in aliphatic chains, ring connections and functional groups.

A major role in humic acids is played by functional groups, which have properties such as hydrophilicity, ion exchange capacity, acidic character and the reversible nature of chemical reactions. The most important functional groups are the carboxyl (-COOH), hydroxyl (-OH), alkoxy (-OCH₃) and carbonyl (>C=O) groups. The functional groups have ion-exchange abilities towards heavy metals [37,38,39]. Humus substances, due to their widespread occurrence, have a great impact on the environment and especially on the transport and mobility of heavy metals in the soil [40]. According to Pandey et al. [41], the stability of humic acid–metal bonds is ranked in order: Cu > Fe > Pb > Ni > Co > Ca > Cd > Zn > Mn > Mg. Such a bond can be chelated [37,38,39]. The carboxyl group (-COOH) increases its amount in the soil as humification of organic matter progresses [42]. The effect of organic matter on heavy metal detoxification in plants has been found by many researchers [12,13,28,36]. Studies are also available that found an increase in heavy metal uptake by plants under the influence of soil organic matter [19,20,43].

The toxicity of nickel, in a study by [44], was mitigated in the presence of fulvic acids. The authors concluded that this may be an effect of the formation of Ni–fulvic acid complexes. In contrast, Alashty et al. [20] found an increase in nickel in radish and lettuce under the influence of increased soil organic matter. The proportion of suitable acids in the humus that is formed from different organic materials has a significant effect on the nickel content of plants. Many authors have argued that humus in which fulvic acids predominate may influence a higher uptake of nickel by plants compared to humus with more humic acids [37,45]. This is motivated by the different structures of the acids and their solubility. Fulvic acids have a lower molecular weight compared to humic acids. Humic acids contain functional groups, including carboxyl and hydroxyl groups responsible for the binding of nickel. A study by Lalas et al. [46] showed that the maximum heavy metal binding capacity in water was demonstrated by fulvic acids in the order Cr > Pb > Ni > Cu > Cd (97.8; 96.5; 95.4; 95.1 and 83.3%, respectively), while humic acids demonstrated Pb > Ni > Cr > Cu > Cd (81.3; 70.7; 68.6; 67.0 and 66.8%, respectively). As can be seen from the above studies, the type of organic material in soil organic fertilization has a significant impact on the chemical structure of humus and its quality. Soil humus containing more humic acids will affect the permanent immobilization of nickel, as exemplified by high peat fertilization. In contrast, humus whose chemical composition is dominated by fulvic acids will affect nickel mobilization and transport to the plant, which can be used in soil phytoremediation techniques. Authors assume that fertilization with wheat straw influences the formation of humus with a higher composition of fulvic acids.

Using fertilization with composted manure, a reduction in nickel uptake by rapeseed was found by Naveed et al. [47]. On the other hand, Zhang et al. [17] found the best efficiency of organic fertilization in detoxifying nickel in winter wheat crops after applying brown coal, followed by biocarbon and chicken manure. The application of brown coal affected the reduction in nickel in lettuce leaves in a study by Misiak and Bosiacki [18]. Rahman et al. [48] found a reduction in nickel in corn after the application of cattle manure.

3.2. Nickel Content of Radish Storage Roots

Radishes grown in high peat substrate exhibited the lowest nickel content (11.61 mg kg⁻¹), whereas levels reached 14.30 mg kg⁻¹ in other treatments (

Table 3. Nickel content in storage roots of radish (mg kg⁻¹).

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C		Mean B
			I	II	III
Loamy sand (mineral soil)		0	7.55 *e	7.68 e	5.70 b–e	6.97 b		12.78 b
		50	9.81 f	11.87 f–i	13.42 h–l	11.70 c
		75	14.11 i–n	15.72 m–p	15.82 m–p	15.22 e
		100	18.38 qr	14.88 k–o	18.41 qr	17.22 f
Mean B·A			18.38 qr	12.54 b	13.33 bc
Loamy sand with high peat		0	3.08 a	7.51e	4.86 a–c	5.15 a		11.61 a
		50	13.74 i–n	11.24 f–h	11.25 f–h	12.07 c
		75	16.00 n–p	12.13 g–j	12.97 g–k	13.70 d
		100	16.82 o–q	14.03 i–n	15.74 m–p	15.53 e
Mean B·A			12.41b	11.23 a	11.20 a
Loamy sand with brown coal		0	7.05 de	6.59 c–e	4.53 a–c	6.06 ab		12.39 b
		50	13.32 g–l	11.11 fg	9.91 f	11.44 c
		75	14.77 k–o	12.19 g–j	14.23 j–n	13.73 d
		100	20.06 rs	13.63 i–m	21.32 st	18.33 f
Mean B·A			13.80 cd	10.88 a	12,50 b
Loamy sand with wheat straw		0	4.00 ab	7.53 e	5,01 a–d	5.51 a		14.30 c
		50	14.84 k–o	12.93 g–k	15,71 m–p	14.49 de
		75	20.09 rs	15.44 l–p	17,41 pq	17.64 f
		100	18.70 qr	16.95 o–q	23,07 t	19.57 g
Mean B·A			14.41 de	13.21 bc	15.30 e
Mean A			13.27 b	11.96 a	13.08 b
Mean C		dose 0 Ni (I–III)	dose 50 Ni (I–III)		dose 75 Ni (I–III)		dose 100 Ni (I–III)
		5.92 a	12.43 b		15.07 c		17.66 d
Mean A·C	year	dose 0 Ni	dose 50 Ni		dose 75 Ni		dose 100 Ni
	I	5.42 a	12.93 de		16.24 g		18.49 h
	II	7.33 b	11.79 c		13.87 e		14.87 f
	II	5.02 a	12.57 cd		15.11 f		19.63 i

A—year, B—substrate and C—dose. * Homogeneous groups were identified using the Duncan test; data followed by the same letters do not differ significantly at α = 0.05.

). The amount of nickel in radish storage roots increased under the influence of increasing doses of this metal. The obtained statistical average for the years of the study indicates that the lowest Ni content (5.92 mg·kg⁻¹) was found in radishes growing in substrates to which no nickel was added. An increase in nickel content in radish storage root by 109.97% was observed at the dose of 50 mg dm⁻³, by 154.56% at the dose of 75 mg Ni dm⁻³ and by 189.31% at 100 mg Ni dm⁻³ in comparison to the content in plants growing in nickel-free substrate.

In the substrates to which no nickel was added (average from three years), the highest content of this metal (6.97 mg kg⁻¹) was found in the roots of plants growing in mineral soil, whereas the lower content of this metal was found in plants growing in the substrate with high peat and straw. In the substrates with a dose of 50 mg Ni dm⁻³, the lowest amount of Ni was obtained in the storage roots in the substrate with brown coal (11.44 mg kg⁻¹) and it did not differ significantly from the content obtained in plants in mineral soil without the addition of organic matter and in the peat substrate. In the substrate with the addition of straw, the highest amount of Ni was obtained, amounting to 14.49 mg kg⁻¹. After applying a dose of 75 mg Ni dm⁻³, it was found that the lowest Ni content was characteristic of radish storage root growing in peat and brown coal substrates, while 17.64 mg kg⁻¹, which was the highest amount, was obtained in storage root growing in the substrate with straw. After applying a dose of 100 mg Ni dm⁻³, the lowest nickel content of 15.53 mg kg⁻¹ was obtained when growing radish in mineral soil with the addition of high peat, and the highest (19.57 mg kg⁻¹) when growing it in a straw substrate.

The research by Singh et al. [5] shows that 86% of nickel accumulates in radish roots, and the remaining 14% in leaves. In tomato plants, 8% of nickel is accumulated in the fruit, 17% in the roots, 70% in the stem and 25% in the leaves. Bosiacki and Roszyk [49] studied various vegetables whose edible parts are leaves, roots and fruits and found that the highest nickel content was found in the group of vegetables with edible leaves (cabbage, lettuce, leek and parsley), while the lowest was found in the group of vegetables whose edible parts are fruits (cucumber and tomato). In the study by Grembecka et al. [50] on the Ni content in 22 different vegetables, it was found that the highest nickel content was found in parsley root, and the lowest in tomato fruit. Moreover, lettuce leaves and storage roots of radish contained similar amounts of nickel. The amount of the discussed metal in the tested vegetables ranged from 0.0003 to 0.05 mg 100 g⁻¹ of product, with an average of 0.01 mg 100 g⁻¹. In the studies by Curyło [51] on the content of Ni in edible parts of eight vegetables, the highest amount of the tested metal was obtained in lettuce leaves.

For example, the nickel content (µg g⁻¹) in edible parts of vegetables according to Singh et al. [5] was as follows: radishes, 10; carrots, 12; onion, 28; spinach, 11; cauliflower, 40; cabbage, 29; tomato, 17 and peas, 15. Onianwa et al. [4] obtained a nickel content in leafy and fruit vegetables in the range of 0.80–9.22 mg kg⁻¹. Bosiacki and Roszyk [49] found, using the technique of mineralization of plant material in acids (wet mineralization), the following nickel contents (mg kg⁻¹): lettuce from 0.07 to 10.12; cabbage, 0.21–30.30; parsley, 0.26–10.31; leek, 0.01–0.70; carrot, 0.003–0.08; tomato, 0.003–0.57 and cucumber, 0.003–0.30.

In the experiments conducted in substrates to which nickel was not introduced, the Ni content in radish storage root ranged on average for the three years of this study from 5.15 (radish growing in a substrate with high peat) to 6.97 (radish growing in mineral soil). The radish storage root contains 95% water. After converting these contents to wet weight (content of 0.26 to 0.35 mg kg⁻¹ wet weight), they were compared with the Commission Regulation (EU) 2024/1987 [52], concerning the maximum levels of nickel in root vegetables of 0.9 mg kg⁻¹ wet weight. There was no exceedance of nickel content in the storage root of radishes growing in substrates without nickel. In Commission Regulation (EU) 2024/1987 of 30 July 2024 [52] amending Regulation (EU) 2023/915 [53], as regards maximum levels of nickel in certain foodstuffs including vegetables, the maximum level of Ni (mg kg⁻¹ wet weight) is as follows: root and tuber vegetables and bulb vegetables, 0.9; fruiting vegetables, 0.40; brassica vegetables, 0.50 leafy vegetables, 0.50; fresh herbs, 1.20 and stem vegetables 0.40.

Due to insufficient data, WHO [54] has not established a daily dose requirement for the tested metal for humans. However, the daily dose of nickel for animals is 0.2 mg Ni per day, while the daily maximum dose should not exceed 0.6 mg, as allergic reactions on the skin may occur.

3.3. Fresh Mass Yield of Storage Roots of Radish

In the first year of this study, the average yield of radish storage roots differed significantly (

Table 4. Average yield of fresh mass of radish storage roots (g pot ⁻¹).

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	38.2 *b–j	18.2 a	29.2 a–e	28.5 a	33.9 a
		50	40.6 c–j	29.4 a–e	31.0 a–f	33.7 ab
		75	34.6 b–i	34.0 b–g	34.0 b–g	34.2 ab
		100	49.0 g–j	28.2 a–d	40.4 c–j	39.2 bc
Mean B·A			40.6 de	27.5 a	33.7 abc
Loamy sand with high peat		0	49.2 g–j	27.0 a–c	45.2 f–j	40.5 bc	41.7 c
		50	44.0 e–j	34.8 b–i	49.4 h–j	42.7 c
		75	47.2 g–j	39.4 c–j	43.6 e–j	43.4 c
		100	38.4 b–j	29.2 a–e	53.0 j	40.2 bc
Mean B·A			44.7 ef	32.6 ab	47.8 f
Loamy sand with brown coal		0	39.0 b–j	29.8 a–e	40.8 c–j	36.5 bc	36.2 ab
		50	34.2 b–h	29.4 a–e	48.0 g–j	37.2 bc
		75	34.0 b–g	24.0 ab	48.0 g–j	35.3 a–c
		100	37.6 b–i	29.8 a–e	39.2 b–j	35.5 a–c
Mean B·A			36.2 bcd	28.3 a	44.0 ef
Loamy sand with wheat straw		0	49.6 ij	27.4 a–d	42.4 d–j	39.8 bc	38.5 bc
		50	48.6 g–j	36.4 b–i	35.6 b–i	40.2 bc
		75	43.8 e–j	30.6 a–f	40.6 c–j	38.3 bc
		100	41.2 c–j	26.6 a–c	38.6 b–j	35.5 a–c
Mean B·A			45.8 ef	30.3 ab	39.3 c–e
Mean A			41.8 b	29.6 a	41.2 b
Mean C		dose 0 Ni (I–III)	dose 50 Ni (I–III)		dose 75 Ni (I–III)		dose 100 Ni (I–III)
		36.3 a	38.5 a		37.8 a		37.6 a
Mean A·C	year	dose 0 Ni	dose 50 Ni		dose 75 Ni		dose 100 Ni
	I	44.0 c	41.9 c		39.9 c		41.6 c
	II	25.6 a	32.5 b		32.0 b		28.5 ab
	III	39.4 c	41.0 c		41.6 c		42.8 c

A—year, B—substrate and C—dose. * Homogeneous groups were identified using the Duncan test; data followed by the same letters do not differ significantly at α = 0.05.

). Regardless of the applied nickel doses, the highest radish mass (45.8 and 44.7 g pot⁻¹) was obtained in plants growing in a substrate consisting of a mixture of mineral soil with the addition of straw and high peat. The lowest mass of storage roots (36.2 g pot⁻¹) was found in mineral soil with brown coal. In the second year of cultivation, no significant differences were obtained in the yield of radish storage roots. In the third year of the study, the highest yield was found in plants growing in a substrate with the addition of high peat, which amounted to 47.8 g pot⁻¹, and it was the highest yield obtained in all the years studied. The lowest yield (33.7 g pot⁻¹) in the third year of this study was observed in plants growing in a substrate consisting of mineral soil.

The average yield of radish storage roots from the three years of the study, obtained in plants growing in the tested substrates, regardless of the nickel dose, was significantly the highest in the mineral soil with the addition of peat (41.7 g pot⁻¹), and it was 23% higher than the lowest yield (33.9 g pot⁻¹) obtained in the mineral soil without the addition of organic matter.

Comparing the average radish yield in subsequent years of the study, regardless of the type of substrate and nickel dose, the highest was obtained in the first and third year of cultivation (41.8 and 41.2 g pot⁻¹, respectively), while it was significantly lower in the second year of this study. Regardless of the type of substrate and years of the study, no significant effect of increasing nickel doses on radish yield was found.

However, when comparing the effect of increasing nickel doses and subsequent years of research, regardless of the type of substrate on radish yielding, it was found that the lowest radish storage roots yields were obtained in the second year of research at all the applied doses.

Significant differences in radish yield were found under the influence of the tested substrates and nickel doses, regardless of the year of study. The lowest yield (28.5 g pot⁻¹) was obtained in plants growing in mineral soil to which no nickel was introduced, and the highest yield (42.7 and 43.4 g pot⁻¹) was obtained in mineral soil with high peat, to which 50 and 75 mg Ni dm⁻³ was introduced.

Latif [55] conducted research on the cultivation of two radish varieties treated with nickel sulphate and found that increasing nickel doses of 50, 100, 150 and 200 mg kg⁻¹ caused a decrease in the fresh and dry mass of radish. In our own research, the reason for the radish variety tolerance was the soil pH buffering ranging from 7.03 to 7.20. Lower yields of other vegetable species under the influence of increasing nickel concentrations were also found by Balaguer et al. [56] and Kumar et al. [57], while in the research conducted by Palacios et al. [58], a decrease in yield was found only at a dose of 240 mg kg⁻¹. Lower nickel doses (60, 120 mg kg⁻¹) did not significantly affect tomato yield. Similar results were obtained by Poulik [59], who did not observe any negative effect of nickel dose on the growth, development and yield of lettuce and tomatoes.

In our own studies, the applied nickel doses from 50 to 100 mg kg⁻¹ had a generally positive effect on radish yield in selected experimental combinations. In view of the above, it is important to continue research on determining the limit values of nickel in vegetable crops in various horticultural technologies.

3.4. Tolerance Index (Ti) of Radish to Increasing Doses of Nickel

A tolerance index above 1, indicating no toxic effect on yield when growing plants in mineral soil, was obtained in all the years of this study for the Ni dose of 50 and 100 mg dm⁻³ and in the second and third year for the dose of 75 mg dm⁻³ (

Table 5. Tolerance index for storage root of radish to increasing doses of nickel.

Substrate	Dose of Ni (mg dm−3)	Year of Research
		I	II	III
Loamy sand (mineral soil)	50	1.06	1.62	1.06
	75	0.91	1.87	1.16
	100	1.28	1.55	1.38
Loamy sand with high peat	50	0.89	1.29	1.09
	75	0.96	1.46	0.96
	100	0.78	1.08	1.17
Loamy sand with brown coal	50	0.88	0.99	1.18
	75	0.87	0.81	1.18
	100	0.96	1.00	0.96
Loamy sand with wheat straw	50	0.98	1.33	0.84
	75	0.88	1.12	0.96
	100	0.83	0.97	0.91

).

Radish growing in peat substrate achieved a tolerance index above 1 in the second year of cultivation at all applied doses of Ni and in the third year using doses of 50 and 100 mg dm⁻³ (Table 5). When growing radish in brown coal substrate a positive effect of this metal on yield was found in the third year of the study at the Ni dose of 50 and 75 mg dm⁻³ (Table 5). Radish growing in straw substrate achieved a tolerance index above 1 in the second year of cultivation at the applied Ni dose of 50 and 75 mg dm⁻³ (Table 5).

3.5. Nitrate Content and Nitrate Reductase (NR) Activity in Storage Roots of Radish

Nickel plays important roles in various metabolic processes of the plant, including affecting, among others, the level of RFT, the synthesis of osmoprotectants, the activation of stress tolerance pathways, and the maintenance of intracellular pH and ionic balance in plant cells through mutual contact with other metal ions, especially iron ions. Additionally, this element has been shown to modulate nitrogen metabolism by regulating the activity of enzymes involved in nitrogen assimilation [21].

Data in the literature indicate that among all food consumed by humans, vegetables provide more than 70% of the total amount of nitrates [60]. The values regarding the norms of nitrate content (mg NO₃⁻ kg⁻¹) in individual vegetable groups are included in the EU Regulation No. 1258/2011 [61]. The presence of nitrates in plants is, among other things, a result of the natural nitrogen cycle in nature, which is a normal phenomenon. Importantly, nitrates are not evenly distributed throughout the plant. In the case of vegetables, plant organs can be arranged with decreasing nitrate content in the following order: petiole > leaf > shoot > root > inflorescence > tuber > fruit > seed [62]. It should also be emphasized that the content of nitrates and nitrites may change under the influence of environmental factors (including solar radiation, temperature and rainfall) and agrotechnical factors (cultivation date, harvest date, vegetation period, soil type and fertilizer application). However, the most important factor is the plant species and variety [63,64].

Organic matter, including the direct input of straw to the field, can increase the source and supply of soil carbon and nitrogen, change the soil microbial biomass and enzyme activity and affect the soil organic carbon sequestration, which in turn affects soil fertility and quality [65]. A lot of available nitrogen means more intensive uptake by plants, which means greater activity of enzymes responsible for nitrogen assimilation. In plants, nitrates are responsible for regulating the activity of nitrate reductase—if they are present, the activity of the enzyme increases. A lot of ammonium nitrogen has the opposite effect—it often inhibits the formation of reductase, and as a result, the use of nitrates to build protein. Due to containing an abundance of essential nutrients, straw has significant potential to mitigate carbon (C), nitrogen (N), phosphorus (P) and potassium (K) deficits in soil [66]. Rehman et al. [48] suggest that the effect of adding organic matter to the soil on Ni stabilization may result from the high capacity for cation exchange, an increase in soil pH and an increase in the uptake of microelements by plants. As is known, heavy metals can cause disturbances in various physiological, biochemical and metabolic processes, including nitrogen (N) uptake and assimilation. A metal-induced decrease in the N assimilation process can also reveal a disturbance in the overall homeostasis and metabolic activity in plants. It has been shown that the most effective NO₃⁻ inhibitor among heavy metals is Cd, which inhibits NO₃⁻ absorption even at very low concentrations [67]. Disturbances in N metabolism cause a decrease in NO₃⁻ uptake and change enzyme activity [68]. Nitrate reductase (NR) is a very important enzyme and controls the initiation of the NO₃⁻ assimilation process in plants. Studies indicate that the effect of heavy metals on N assimilation enzymes depends on the level of enzyme sensitivity and their location in cells/organs, the concentration of metals in the soil, their mobility and the time of plant exposure to metal toxicity. One of the mechanisms of heavy metals action on the activity of nitrogen metabolism enzymes is their ability to bind with essential sulfhydryl (SH) groups [69]. The above is confirmed by the results of studies on the effect of Al and Cu on NR activity. The cause of the decrease in enzyme activity was the direct interaction of metals with functional SH groups present in the active sites of NR [70,71]. It is also worth noting that the activity of leaf nitrate reductase in the circadian cycle often decreases as the day progresses, which may reflect its degradation and/or synthesis blockage. Thanks to NR, inorganic forms of nitrogen are effectively incorporated into organic compounds [72,73].

Undoubtedly, in recent years, there has been an increase in the number of reports on Ni tolerance and toxicity in plants. However, little is still known about the effect of this element on nitrogen metabolism, especially in plants grown on organic substrates. In view of the above, it was extremely important to investigate the effect of increasing nickel doses (0, 50, 75 and 100 mg Ni dm⁻³ of substrate) on the nitrate content in radish (a vegetable classified as susceptible to nitrate accumulation), which was grown in substrates consisting of mineral soil with the addition of high peat, brown coal and wheat straw.

The introduction of organic matter to the mineral soil significantly influenced the change in the nitrate content in storage roots of radish. The lowest nitrate content, regardless of the applied nickel doses and years of study, was found in plants growing in a substrate consisting of mineral soil with the addition of wheat straw and amounted to 1.04 mg g⁻¹ FW (

Table 6. Nitrate content in storage roots of radish (mg NO₃⁻ g⁻¹ FW).

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	3.01 pq	0.68 c–g	0.26 a–c	1.31 cd	1.36 c
		50	2.06 k–m	0.79 e–g	0.83 fg	1.22 bcd
		75	2.05 k–m	1.47 ij	0.33 a–d	1.28 bcd
		100	3.18 q	1.52 ij	0.18 a	1.63 ef
Mean B·A			2.57 h	1.11 d	0.40 b
Loamy sand with high peat		0	3.17 q	0.68 c–g	0.48 a–f	1.44 de	1.55 d
		50	2.62 no	2.21 l–n	0.26 a–c	1.70 f
		75	2.72 op	1.46 ij	0.11 a	1.43 de
		100	3.21 q	1.04 gh	0.62 b–g	1.62 ef
Mean B·A			2.93 i	1.35 e	0.37 ab
Loamy sand with brown coal		0	2.26 mn	0.96 g	0.24 ab	1.15 bc	1.19 b
		50	2.44 m–o	1.36 hi	0.31 a–c	1.37 cd
		75	2.33 m–o	1.56 ij	0.15 a	1.35 cd
		100	1.84 j–l	0.75 d–g	0.06 a	0.88 a
Mean B·A			2.22 g	1.16 d	0.19 a
Loamy sand with wheat straw		0	1.71 i–k	0.74 d–g	0.26 a–c	0.90 a	1.04 a
		50	2.37 m–o	0.42 a–f	0.37 a–e	1.05 ab
		75	2.15 lm	1.57 ij	0.11 a	1.28 b–d
		100	1.82 j–l	0.80 e–g	0.13 a	0.91 a
Mean B·A			2.01 f	0.88 c	0.22 ab
Mean A			2.43 c	1.12 b	0.29 a
Mean C		Dose 0 Ni (I–III)	Dose 50 Ni (I–III)		Dose 75 Ni (I–III)		Dose 100 Ni (I–III)
		1.20 a	1.34 b		1.33 b		1.26 ab
Mean A·C	Year	Dose 0 Ni	Dose 50 Ni		Dose 75 Ni		Dose 100 Ni
	I	2.54 g	2.37 fg		2.31 f		2.51 g
	II	0.77 c	1.20 d		1.51 e		1.02 d
	III	0.31 ab	0.44 b		0.18 a		0.25 a

A—year, B—substrate and C—dose. data followed by the same letters do not differ significantly at α = 0.05.

). The highest nitrate reductase activity was found in plants growing in this substrate (

Table 7. Nitrate reductase activity in storage roots of radish (nmoles NO₂⁻ × h⁻¹ × g⁻¹ FW).

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	26.8 c–k	10.5 ab	14.1 a–f	17.1 a	20.3 a
		50	30.0 f–k	10.3 a	18.8 a–h	19.7 a–c
		75	28.2 d–k	18.6 a–h	15.7 a–g	20.8 a–c
		100	23.7 a–j	26.9 c–k	19.4 a–h	23.4 a–d
Mean B·A			27.2 b	16.6 a	17.0 a
Loamy sand with high peat		0	28.1 d–k	13.2 a–d	14.1 a–e	18.4 ab	22.2 a
		50	26.9 c–k	13.6 a–d	13.8 a–d	18.1 ab
		75	37.3 j–l	16.5 a–h	19.0 a–h	24.3 a–d
		100	52.5 m	17.3 a–h	14.3 a–f	28.0 cd
Mean B·A			36.2 c	15.1 a	15.3 a
Loamy sand with brown coal		0	28.3 d–k	11.6 abc	19.3 a–h	19.7 abc	22.8 a
		50	26.3 b–k	13.8 a–d	35.5 i–l	25.2 a–d
		75	22.1 a–j	17.8 a–h	24.2 a–j	21.4 a–d
		100	32.1 h–l	17.7 a–h	24.1 a–j	24.6 a–d
Mean B·A			27.2 b	15.2 a	25.8 b
Loamy sand with wheat straw		0	29.8 e–k	13.3 a–d	45.7 lm	29.6 d	26.0 b
		50	31.3 g–k	20.7 a–i	26.2 a–k	26.1 b–d
		75	27.8 d–k	10.7 ab	24.9 a–j	21.1 a–d
		100	40.3 k–m	17.3 a–h	23.9 a–j	27.2 cd
Mean B·A			32.3 bc	15.5 a	30.2 bc
Mean A			30.7 c	15.6 a	22.1 b
Mean C		Dose 0 Ni (I–III)	Dose 50 Ni (I–III)		Dose 75 Ni (I–III)		Dose 100 Ni (I–III)
		21.2 a	22.3 ab		21.9 a		25.8 b
Mean A·C	Year	Dose 0 Ni	Dose 50 Ni		Dose 75 Ni		Dose 100 Ni
	I	28.2 d	28.6 d		28.9 d		37.2 e
	II	12.1 a	14.6 ab		15.9 ab		19.8 bc
	III	23.3 cd	23.6 cd		20.9 bc		20.4 bc

A—year, B—substrate and C—dose. data followed by the same letters do not differ significantly at α = 0.05.

). The highest nitrate content of 1.55 mg g⁻¹ FW was found in storage roots of radish growing in mineral soil to which high peat was added.

Analyzing the amounts of nitrates in storage roots of radish obtained in plants growing in substrates in subsequent years of this study, regardless of the applied nickel doses and type of substrate, their lowest content of 0.29 mg g⁻¹ FW was found in the third year of cultivation. The highest content of nitrates was characterized by storage roots of radish grown in the first year of this study.

From analyzing the effect of nickel dose, regardless of the type of substrate and year of research, on the average content of nitrates in storage roots of radish, the lowest amounts were found in substrates to which no nickel was added.

Studies conducted on rice seedlings grown hydroponically with the addition of 50, 100, and 200 μM nickel (NiSO₄ 6H₂O) showed a decrease in the level of nitrates and a decrease in the activity of nitrate reductase (NR) and nitrite reductase (NiR), as well as glutamine synthetase (GS) and glutamate synthase (GOGAT) [74]. A similar relationship was observed in the case of wheat seedlings treated with 50 and 100 μM Ni. In these studies, a reduced content of nitrates, a decrease in the activity of NR, GS and GOGAT were found [75]. Based on the above, it is suggested that the decrease in NR activity may be caused by the disruption between the carbon, sulfur and nitrogen metabolic pathways, leading to a decrease in nitrate content [76]. In addition, studies conducted on tomato plants have shown that the addition of exogenous nickel affects the expression of genes encoding enzymes of nitrogen metabolism and the activity of enzymes, i.e., nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS) and glutamate synthase (GOGAT), and also improves carbohydrate metabolism [77].

The available literature also shows research results indicating the positive participation of nickel in nitrogen metabolism. The above is confirmed by, among others, the results conducted on sugar cane plants (Saccharum officinarum). The use of Ni in the concentration range of 0; 0.25; 0.5; 1; 3 and 9 mg kg⁻¹ increased the activity of urease and nitrate reductase [78]. As is known, nitrate reductase (NR) is the most important enzyme in the nitrate assimilation pathway. An increased NO₃⁻ level affects the increase in nitrate reductase activity. In the conducted studies (Table 7), the activity of nitrate reductase in radish was higher in each year of the study in the substrates to which 100 mg of Ni was added compared to the plants growing in substrates without its addition. Undoubtedly, the above research results contribute new knowledge on the role of nickel in increasing crop tolerance to abiotic stress, which may potentially lead to increased yields [78].

3.6. Chloroplast Pigment Content in Radish Leaves

Nickel can directly and indirectly affect the photosynthesis process. The limitation of photosynthesis may be in particular related to the disruption of the chloroplast structure, blocking of chlorophyll synthesis, disturbed electron transport (including by changing the structure of plastoquinone), and a decrease in the activity of Calvin cycle enzymes [79]. It is believed that the decrease in chlorophyll level under the influence of heavy metals, including nickel, may be caused by a decrease in iron level, inhibition of the activity of enzymes of the chlorophyll biosynthesis pathway and the replacement of central Mg²⁺ molecules in the chlorophyll structure by heavy metals [80].

For example, Gurpreet et al. [81] showed a decrease in the level of chlorophyll a and b and carotenoids in mung bean (Vigna mungo L.) seedlings under the influence of a 50 and 100 μM nickel concentration. Additionally, the addition of 5 mM nitrogen eliminated the negative effect of the tested heavy metal. A similar relationship was also revealed by Dubey and Pandey [82]. With an increasing nickel dose (20–100 μM), a decrease in the content of photosynthetic pigments was noted due to the induced oxidative stress. Also in maize leaves, the content of chlorophyll decreased with increasing nickel concentration in the same concentration range [83].

In turn, opposite results were obtained in the case of sugar cane (Saccharum officinarum). The use of Ni in the concentration range of 0; 0.25; 0.5; 1; 3 and 9 mg kg⁻¹ increased the level of photosynthetic pigments. The authors suggest that a dose of 0.5 mg Ni kg⁻¹ nickel has a beneficial effect on the plant metabolism (nitrogen and carbohydrate metabolism) and, consequently, increases the yield [78]. Moreover, the results of studies on rapeseed plants showed that the chlorophyll content increased in plants fed with urea as the sole source of nitrogen and treated with nickel. The increase in chlorophyll content indicates a clear improvement in nitrogen assimilation from urea by the addition of nickel, because nitrogen is a component of the chlorophyll molecule [84].

3.6.1. Chlorophyll ‘a’ Content in Radish Leaves After Cultivation

The introduction of organic matter to the mineral soil significantly affected the content of chlorophyll a in radish leaves (

Table 8. Chlorophyll ‘a’ content in radish leaves (mg g⁻¹ F.W.) after cultivation.

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	1.55 *j	1.05 c–i	1.22 e–j	1.27 de	1.22 b
		50	1.07 c–i	1.09 c–i	1.17 c–i	1.11 a–d
		75	1.37 ij	1.17 c–i	1.16 c–i	1.23 b–e
		100	1.29 f–j	1.34 h–j	1.12 c–i	1.25 cde
Mean B·A			1.32 cd	1.16 bc	1.17 bc
Loamy sand with high peat		0	1.26 e–j	1.27 f–j	1.12 c–i	1.22 b–e	1.15 b
		50	1.18 d–i	1.34 h–j	0.98 b–h	1.17 b–e
		75	1.18 c–i	1.09 c–i	1.16 c–i	1.14 b–e
		100	1.18 c–i	0.93 a–f	1.12 c–i	1.08 a–d
Mean B·A			1.20 bcd	1.16 bc	1.10 b
Loamy sand with brown coal		0	1.55 j	1.14 c–i	1.34 h–j	1.34 e	1.15 b
		50	1.20 e–j	1.09 c–i	0.97 b–h	1.09 a–d
		75	1.32 g–j	0.99 b–h	0.80 a–c	1.03 ab
		100	1.29 f–j	1.16 c–i	1.00 b–i	1.15 b–e
Mean B·A			1.34 d	1.09 b	1.03 ab
Loamy sand with wheat straw		0	0.62 a	1.18 c–i	0.95 a–g	0.91 a	0.98 a
		50	0.80 a–d	1.14 c–i	1.17 c–i	1.04 ab
		75	1.19 d–i	1.09 c–i	0.88 a–e	1.05 abc
		100	0.94 a–g	1.15 c–i	0.68 ab	0.92 a
Mean B·A			0.89 a	1.14 b	0.92 a
Mean A			1.19 b	1.14 b	1.05 a
Mean C		Dose 0 Ni (I–III)	Dose 50 Ni (I–III)		Dose 75 Ni (I–III)		Dose 100 Ni (I–III)
		1.19 a	1.10 a		1.12 a		1.10 a
Mean A·C	Year	Dose 0 Ni	Dose 50 Ni		Dose 75 Ni		Dose 100 Ni
	I	1.24 cd	1.06 ab		1.26 d		1.18 b–d
	II	1.16 b–d	1.16 b–d		1.09 a–c		1.14 a–d
	III	1.16 b–d	1.07 a–c		1.00 ab		0.98 a

A—year, B— substrate and C—dose. * Homogeneous groups were identified using the Duncan test; data followed by the same letters do not differ significantly at α = 0.05.

). The lowest content of chlorophyll a, regardless of the applied nickel doses and years of study, was found in plants growing in a substrate consisting of mineral soil with the addition of wheat straw, and it amounted to (0.98 mg g⁻¹ F.W.). When analyzing the amount of chlorophyll a in radish leaves obtained from plants growing in substrates in subsequent years of this study, regardless of the nickel doses used and the type of substrate, its lowest content (1.05 mg g⁻¹ F.W.) was found in the third year of cultivation. The effect of nickel dose, regardless of the type of substrate and year of research, on chlorophyll content was analyzed, and no significant differences in its content were found in radish leaves.

3.6.2. Chlorophyll ‘b’ Content in Radish Leaves After Cultivation

The introduction of organic matter to the mineral soil significantly affected the content of chlorophyll b in radish leaves (

Table 9. Chlorophyll ‘b’ content in radish leaves (mg g⁻¹ F.W.) after cultivation.

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	0.49 jk	0.29 a–g	0.40 g–k	0.39 ef	0.37 b
		50	0.35 d–i	0.29 a–g	0.40 f–k	0.35 a–e
		75	0.42 h–k	0.28 a–g	0.39 d–j	0.36 a–f
		100	0.39 e–j	0.37 a–i	0.38 d–j	0.38 d–f
Mean B·A			0.41 e	0.31 bc	0.39 de
Loamy sand with high peat		0	0.38 d–j	0.33 b–h	0.38 d–j	0.37 c–f	0.35 b
		50	0.39 f–k	0.36 d–i	0.35 c–h	0.37 c–f
		75	0.35 d–i	0.27 a–e	0.39 f–k	0.34 a–e
		100	0.37 d–i	0.24 a–c	0.37 d–i	0.33 a–d
Mean B·A			0.37 de	0.30 bc	0.37 de
Loamy sand with brown coal		0	0.50 k	0.27 a–d	0.47 i–k	0.41 f	0.34 b
		50	0.37 d–i	0.18 a	0.36 d–i	0.30 a–c
		75	0.39 f–k	0.23 ab	0.29 a–g	0.31 a–c
		100	0.38 d–j	0.29 b–g	0.36 d–i	0.35 b–e
Mean B·A			0.41 e	0.24 a	0.37 de
Loamy sand with wheat straw		0	0.17 a	0.30 b–g	0.37 d–i	0.28 a	0.30 a
		50	0.23 ab	0.28 a–f	0.43 h–k	0.31 a–c
		75	0.37 d–i	0.28 a–f	0.32 b–h	0.32 a–d
		100	0.29 a–g	0.33 b–h	0.28 a–e	0.30 ab
Mean B·A			0.27 ab	0.30 b	0.35 cd
Mean A			0.37 b	0.29 a	0.37 b
Mean C		Dose 0 Ni (I–III)	Dose 50 Ni (I–III)		Dose 75 Ni (I–III)		Dose 100 Ni (I–III)
		0.36 b	0.33 a		0.33 a		0.34 ab
Mean A·C	Year	Dose 0 Ni	Dose 50 Ni		Dose 75 Ni		Dose 100 Ni
	I	0.39 de	0.34 b–d		0.38 de		0.36 c–e
	II	0.30 ab	0.28 a		0.27 a		0.31 a–c
	III	0.41 e	0.38 de		0.35 b–d		0.35 b–d

A—year, B—substrate and C—dose. data followed by the same letters do not differ significantly at α = 0.05.

). The lowest content of chlorophyll b, regardless of the applied nickel doses and years of study, was found in plants growing in a substrate consisting of mineral soil with the addition of wheat straw and it amounted to (0.30 mg g⁻¹ F.W.). When analyzing the amount of chlorophyll b in radish leaves obtained from plants growing in substrates in subsequent years of the study, regardless of the nickel doses used and the type of substrate, its lowest content (0.29 mg g⁻¹ F.W.) was found in the third year of cultivation. Analysis was conducted on the effect of nickel dose, regardless of the type of substrate and the year of research, on the content of chlorophyll b in radish leaves. The highest content was found in plants growing in substrates to which no nickel was added, while the lowest in plants growing in substrates with a nickel dose of 50 and 75 mg dm⁻³.

3.6.3. Total Chlorophyll Content in Radish Leaves After Cultivation

The introduction of organic matter to the mineral soil significantly affected the content of total chlorophyll in radish leaves (

Table 10. Total chlorophyll content in radish leaves (mg g⁻¹ F.W.) after cultivation.

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	2.04 jk	1.34 b–i	1.62 e–k	1.67 ef	1.59 b
		50	1.43 b–i	1.37 b–i	1.57 d–i	1.46 b–e
		75	1.79 i–k	1.45 c–i	1.55 d–i	1.60 a–f
		100	1.68 f–k	1.71 g–k	1.50 c–i	1.63 d–f
Mean B·A			1.73 d	1.47 bc	1.56 cd
Loamy sand with high peat		0	1.64 e–k	1.61e–k	1.51 c–i	1.59 c–f	1.50 b
		50	1.58 d–j	1.69 f–k	1.33 b–i	1.53 c–f
		75	1.53 d–i	1.37 b–i	1.55 d–i	1.48 c–e
		100	1.55 d–i	1.17 a–e	1.49 c–i	1.40 a–d
Mean B·A			1.57 cd	1.46 bc	1.47 bc
Loamy sand with brown coal		0	2.05 k	1.41 b–i	1.80 i–k	1.75 f	1.50 b
		50	1.57 d–i	1.27 b–h	1.33 b–i	1.39 a–d
		75	1.71 h–k	1.22 a–g	1.09 a–d	1.34 a–c
		100	1.67 f–k	1.45 c–i	1.36 b–i	1.49 c–e
Mean B·A			1.75 d	1.34 ab	1.40 bc
Loamy sand with wheat straw		0	0.79 a	1.48 c–i	1.32 b–i	1.20 a	1.29 a
		50	1.03 a–c	1.43 b–i	1.60 e–k	1.35 a–c
		75	1.55 d–i	1.37 b–i	1.21 a–f	1.38 a–d
		100	1.23 a–h	1.48 c–i	0.96 ab	1.22 ab
Mean B·A			1.15 a	1.44 bc	1.27 ab
Mean A			1.55 b	1.43 a	1.42 a
Mean C		Dose 0 Ni (I–III)	Dose 50 Ni (I–III)		Dose 75 Ni (I–III)		Dose 100 Ni (I–III)
		1.55 a	1.43 a		1.45 a		1.44 a
Mean A·C	Year	Dose 0 Ni	Dose 50 Ni		Dose 75 Ni		Dose 100 Ni
	I	1.63 c	1.40 ab		1.65 c		1.53 a–c
	II	1.46 a–c	1.44 a–c		1.35 ab		1.45 a–c
	III	1.56 bc	1.46 a–c		1.35 ab		1.33 a

A—year, B—substrate and C—dose. data followed by the same letters do not differ significantly at α = 0.05.

). The lowest content of total chlorophyll, regardless of the applied nickel doses and years of study, was found in plants growing in a substrate consisting of mineral soil with the addition of wheat straw and it amounted to (1.29 mg g⁻¹ F.W.). When analyzing the amount of total chlorophyll in radish leaves obtained from plants growing in substrates in subsequent years of the study, regardless of the nickel doses used and the type of substrate, its highest content (1.55 mg g⁻¹ F.W.) was found in the first year of cultivation. From analyzing the effect of nickel dose, regardless of the type of substrate and years of research, on the content of total chlorophyll in radish leaves, no significant differences in its content were found.

3.6.4. Carotenoid Content in Radish Leaves After Cultivation

The introduction of organic matter to the mineral soil significantly affected the content of carotenoids in radish leaves (

Table 11. Carotenoid content in radish leaves (mg g⁻¹ F.W.) after cultivation.

Substrate		Dose of Ni (mg dm−3)	Year of Research			Mean B·C	Mean B
			I	II	III
Loamy sand (mineral soil)		0	0.45 ij	0.34 b–i	0.37 e–j	0.38 ef	0.35 b
		50	0.31 b–h	0.33 b–h	0.26 a–e	0.30 a–c
		75	0.39 g–j	0.34 c–i	0.34 c–i	0.36 a–f
		100	0.36 d–j	0.40 h–j	0.33 b–h	0.36 d–f
Mean B·A			0.38 de	0.35 cde	0.33 bc
Loamy sand with high peat		0	0.35 d–j	0.38 f–j	0.34 b–i	0.36 c–f	0.34 b
		50	0.35 d–j	0.39 g–j	0.31 b–h	0.35 c–f
		75	0.33 b–h	0.33 b–h	0.35 d–j	0.34 b–e
		100	0.35 d–j	0.28 b–g	0.34 c–i	0.33 a–e
Mean B·A			0.35 cde	0.35 cde	0.34 cd
Loamy sand with brown coal		0	0.46 j	0.33 b–h	0.41 h–j	0.40 f	0.34 b
		50	0.34 c–i	0.32 b–h	0.30 b–h	0.32 a–d
		75	0.37 f–j	0.30 b–h	0.25 a–d	0.31 a–d
		100	0.37 f–j	0.35 d–i	0.31 b–h	0.34 b–f
Mean B·A			0.39 e	0.33 bc	0.32 bc
Loamy sand with wheat straw		0	0.18 a	0.35 d–j	0.30 b–h	0.28 a	0.30 a
		50	0.23 ab	0.33 b–h	0.34 d–i	0.30 a–c
		75	0.35 d–i	0.33 b–h	0.27 a–f	0.32 a–d
		100	0.29 b–g	0.35 d–i	0.23 a–c	0.29 ab
Mean B·A			0.26 a	0.34 c–e	0.29 ab
Mean A			0.34 b	0.34 b	0.32 a
Mean C		Dose 0 Ni (I–III)	Dose 50 Ni (I–III)		Dose 75 Ni (I–III)		Dose 100 Ni (I–III)
		0.35 b	0.32 a		0.33 ab		0.33 ab
Mean A·C	Year	Dose 0 Ni	Dose 50 Ni		Dose 75 Ni		Dose 100 Ni
	I	0.36 c	0.31 a–c		0.36 c		0.34 a–c
	II	0.35 a–c	0.34 a–c		0.33 a–c		0.35 a–c
	III	0.35 bc	0.30 a		0.31 ab		0.31 ab

A—year, B—substrate and C—dose. data followed by the same letters do not differ significantly at α = 0.05.

). The lowest content of carotenoids, regardless of the applied nickel doses and years of study, was found in plants growing in a substrate consisting of mineral soil with the addition of wheat straw and it amounted to (0.33 mg g⁻¹ F.W.). When analyzing the amounts of carotenoids in radish leaves obtained from plants growing in substrates in subsequent years of the study, regardless of the nickel doses used and the type of substrate, its lowest content (0.32 mg g⁻¹ F.W.) was found in the third year of cultivation. The effect of nickel dose, regardless of the type of substrate and the year of research, on the content of carotenoids in radish leaves was analyzed. The highest content (0.35 mg g⁻¹ F.W.) was found in plants growing in substrates to which no nickel was added, while the lowest was found in plants growing in substrates with a nickel dose of 50 mg dm⁻³.

The results obtained in this study showed that changes in the level of chloroplast pigments (chlorophyll a, chlorophyll b and carotenoids) depend on the applied nickel dose. Analyzing the effect of nickel dose, regardless of the type of substrate and years of study, the content of chlorophyll b and carotenoids in radish leaves was the highest in plants growing in substrates to which nickel was not introduced, while the lowest was in plants growing in substrates with a nickel dose of 50 and 75 mg dm⁻³ for chlorophyll b and for carotenoids with a dose of 50 mg dm⁻³.

The obtained results contribute further information in the scope of research on the influence of nickel on nitrogen metabolism, in particular the content of nitrates and the level of chloroplast pigments. Undoubtedly, the wide range of obtained results still indicates the need to continue research on the influence of other organic materials on the content of nickel in edible parts of horticultural plants and to determine the optimal contents of nickel in substrates and soils for the cultivation of horticultural plants.

4. Conclusions

The type of organic material in fertilization has a significant effect on the accumulation of heavy metals in vegetable cultivation. The consequence of applying the right kind of organic material (containing a lot of humic acids in the chemical composition of humus) to soils contaminated with heavy metals is to obtain vegetables, their edible part, with a low content of nickel. Effective organic material application is a critical aspect of soil and crop management. It was found that high peat introduced into mineral soil affected the detoxification of nickel, reducing its content in storage root of radish. In turn, wheat straw introduced into mineral soil increases the nickel content in storage root of radish. All the introduced organic materials into the mineral soil increased the C_org. The fertilization of the mineral soil with high peat and wheat straw influenced the increase in the storage roots of the radish yield. The addition of nickel to the substrate (regardless of the type of substrate and the years of research) caused an increase in the level of nitrates in the storage roots of radish. The activity of nitrate reductase in radish was higher in each year of the study in the substrates to which 100 mg of Ni was added compared to the plants growing in substrates without its addition. It was shown that changes in the level of chloroplast pigments (chlorophyll a, chlorophyll b and carotenoids) depend largely on the substrate in which the plants are grown as well as the applied dose of nickel.