Effects of Long-Term Multi-Treatment Experiments on Organic Matter and Enzymatic Activity in Sandy Soil

Abstract

This study shows an evaluation of the condition of organic matter against enzymatic activity in soil. Long-term static field experiments with fertilisation with manure (FYM), different minerals, and mineral–manure were used for the research. Assays were obtained of the content of total organic carbon (TOC), dissolved fraction (DOC), susceptibility to oxidation (CL1) and (CL), total nitrogen (TN), dissolved nitrogen fraction (DTNT), and available forms of potassium, phosphorus, and magnesium. The activity of enzymes dehydrogenases, catalase, β-glucosidase, proteases, alkaline, and acid phosphatase was determined. We calculated the enzymatic indices and those evaluating the labile organic carbon management (CMI and CPI) in soil. An increase in TOC, up to 8.85 g kg⁻¹ and 8.56 g kg⁻¹ (FYM, FYM + KN), respectively, as compared with the control (5.67 g kg⁻¹), did not have a significant effect on the content of labile carbon fraction CL for the fertilisation treatments. Only a higher CL content was found in the soil with the FYM + PN and FYM + NPK + Mg treatments (2.07 g kg⁻¹ and 2.05 g kg⁻¹). All the fertilisation treatments under study demonstrated a decrease in the value of the carbon management index (CMI). Similar DOC values (on average, 75.14 mg kg⁻¹) were noted. The average percentage share of the DOC fraction accounted for 1.163% TOC, and it was lower as compared with the control variant (1.33% TOC). The mineral fertilisation treatments decreased soil enzyme activities. Multiparametric enzymatic soil fertility indices differed due to soil properties, depending on the fertilisation applied.

1. Introduction

The organic matter of soil is subject to transformations in mineralization and humification processes. Agrotechnical practices affect the course and the rate of its decomposition [1,2]. The long-term dynamic use of soils and frequently unbalanced fertilisation result in many processes leading to, e.g., a decrease in biological activity, acidification, and lowering the content of humus [3,4]. The assessment of these processes should be based not only on determining the current organic carbon content but also on the share of fractions of organic matters related to mineralization processes.

The method that offers such possibilities is the analysis of susceptibility to the chemical oxidation of soil organic matter, with the use of potassium manganate (VII) solution [5,6]. The content of the soil organic matter fraction, which is water soluble or soluble in salt solutions of a pH~7 (DOM), also facilitates evaluating the quality of the organic matter of soils [7]. DOM in soil is expressed as the content of dissolved organic carbon (DOC). That fraction includes mostly low-molecular organic compounds, which can act as growth stimulants and inhibitors in plants. DOC is also an easily available energy material for microorganisms [8]. Manure (FYM) and mineral fertilisers are common fertilisers applied to manage nutrients, enhancing soil fertility and quality, and thus increasing the yields [9,10]. However, an intensive use of mineral fertilisers can lead to soil degradation. For that reason, using FYM as an alternative for mineral fertilisers can be an effective fertilisation strategy to maintain soil health and biodiversity [9]. Nutrients found in FYM are gradually released, which depends on the origin [11] and the ratio of TOC/TN. Fertilisers change the environmental parameters of soil, e.g., TOC and pH; they can change the composition and diversity of soil microorganisms and, as a result, affect biochemical processes [2,12]. The processes are a parameter, showing the biological conditions of soils. The level of the enzymatic activity of soils involves a sensitive soil fertility index and soil productivity index as well as information about the ecological changes in the soil environment [13,14]. Soil enzymes are released by microorganisms and plant roots [15]. They take part in the formation and decomposition of soil organic matter (SOM) and the biogeochemistry of C, N, P, and S [16]. As reported by Li et al. [17], fertilisation with mineral nitrogen has an inconsiderable effect on the biomass of microorganisms and thus on the enzymatic activity of soil. The application of enzymatic soil quality indices, which facilitate specifying the anthropogenic changes, allows the identification of the trends that occur in soil for a longer period [18].

The research hypothesis assumes that the manure and mineral fertilisation are factors that significantly modify the quality of organic matter and differentiate the activity of soil enzymes. With that in mind, the aim of this study was to determine the quantitative and qualitative changes in the organic matter of light soil (Luvisol) under the influence of long-term, varied manure and mineral fertilisation. We also investigated the activity of redox (dehydrogenases and catalase) and hydrolytic enzymes responsible for C (β-glucosidase), N (proteases), and P (alkaline and acid phosphatase) transformations. The values of indices evaluating the labile organic carbon management in soil and the enzymatic soil fertility indices have been demonstrated.

2. Materials and Methods

2.1. Material

The experiment was set up in 1948 in Mochełek, in close vicinity to Bydgoszcz, the Kujawsko-Pomorskie province, Poland (53°13′ N, 17°51′ E, 95 m above sea level) in Luvisol. The grain size composition of topsoil (0–20 cm) was classified as sand 61.1%, silt 33.6%, and clay 5.3%. The experiment was established in the region of the impact of a cold climate with dry seasons and a warm summer; Dfb [19]. The annual mean precipitation is 485 mm, and the temperature—8.1 °C. The aim of the experiment has been an evaluation of the effects of the mineral and natural forms of manure (FYM) fertilisation variants on soil properties and crop productivity. For 76 years, some changes were made in experimental treatments and crop rotation [20]. In 2002, reclamation liming was carried out, and since 2011, for the control, until then, there was no fertilisation; mineral fertilisation was introduced equal to half an NPK rate (nitrogen, phosphorus, and potassium). The crop rotation included pea, winter wheat, winter rapeseed, corn, and spring barley. Soil was sampled in 2022, after the corn harvest, and prior to spring barley sowing, from 12 fertilisation treatments, each treatment had four replications: T0—control (without fertilisation); T1—NPK; T2—FYM (FYM every 4 years); T3—FYM + PK; T4—FYM + KN; T5—FYM + KN + Mg (+magnesium); T6—FYM + PN; T7—FYM + PN + Mg; T8—FYM + NPK; T9—FYM + NPK + Mg; T10—FYM + NPK + Ca (liming once every 4 years); T11—FYM + NPK + Ca + Mg. Over the last two decades the annual mean fertilisation was as follows: nitrogen—96 kg N ha⁻¹, phosphorus—88 kg P₂O₅ ha⁻¹, potassium—120 kg K₂O ha⁻¹, magnesium—20 kg MgO ha⁻¹, FYM 8t ha⁻¹, and liming (CaO) 1.2 t ha⁻¹.

2.2. Methods

2.2.1. Chemical Analysis of the Included Soil

In the air-dried and sieved (<2 mm) soils, the following were assayed:

-

pH in 1 M KCl and H₂O by potentiometric method;

-

total organic carbon (TOC) and total nitrogen (TN) were assayed with the Vario Max CN analyser (Elementar, Langenselbold, Germany);

-

the amount of dissolved organic carbon (DOC) and dissolved total nitrogen (DTN) were measured in the solutions following the extraction with 0.004 M CaCl₂. DOC and DTN were assayed with the Muli N/C 3100 Analityk Jena analyzer (Jena, Germany, assay sensitivity of 1 µg L⁻¹) and expressed in mg kg⁻¹ d.w. of the soil sample as well as the percentage share in the pool of TOC and TN, respectively;

-

humus fractions susceptible to oxidation [5,6]. The method is based on assaying the fractions of organic carbon susceptible to oxidation by acting on the soil sample with a 0.333 M solution (CL labile carbon) and (CNL non-labile carbon) as well as a 0.0333 M KMnO₄ solution (CL1 labile carbon) in a neutral environment.

The results were used to calculate soil organic carbon management indices:

-

carbon pool size index CPI:

$$CPI={\displaystyle \frac{TOC sample}{TOC reference}}$$

(1)

-

carbon management index CMI:

$$CMI=CPI\times LI\times 100$$

(2)

where the lability index

$$LI={\displaystyle \frac{Lsample}{Lreference}},$$

and L is calculated as follows:

$$L={\displaystyle \frac{CL}{CNL}},$$

where the L–lability of C (C in fraction oxidised by KMnO₄; C remaining unoxidized by KMnO₄).

In the soil, the content of available macroelements was also assayed:

-

the content of available phosphorus (P) PN-R-04023 [21] and potassium (K) PN-R-04022 [22] were assayed with the Egner–Riehm method (DL) [23], and the content of available magnesium (Mg) PN-R-04020 [24] was assayed, according to the Schachtschabel method [25].

2.2.2. The Activity of Enzymes in Soil

The activity of selected enzymes representing the oxidoreductases (dehydrogenases and catalase) and hydrolases (alkaline and acid phosphatase, β-glucosidase, and proteases) was measured for fresh sieved (<2 mm) soils. The soils were stored at 4 °C (for two weeks). The activity of the following enzymes in soil was investigated:

-

the activity of dehydrogenases (DEH) was assayed with the Thalmann [26] method after sample incubation with 2,3,5-triphenyltetrazolium chloride, and the measurement of absorbance of triphenylformazan (TPF) at 546 nm was expressed in mg TPF kg⁻¹ 24 h⁻¹.

-

the activity of catalase (CAT) was assayed with the Johnson and Temple [27] method with a 0.3% solution of hydrogen peroxide as a substrate. The other H₂O₂ was determined with a titration of 0.02 M KMnO₄ in acid conditions.

-

the activities of alkaline phosphatase (AlP) and acid phosphatase (AcP) were measured from the detection of p-nitrophenol (pNP) released after incubation (37 °C, 1 h) for a pH~6.5 for acid phosphatase and for a pH~11.0 for alkaline phosphatase [28].

-

the activity of β-glucosidase (BG) was measured with the Eivazi and Tabatabai [29] method, applying p-nitrophenyl-β-D-glucopyranoside as a substrate. The concentrations of p-nitrophenol were assayed with an immediate readout of the sample at 400 nm after alkalization with the buffer Tris/NaOH (pH 10.0) and CaCl₂.

-

the activity of proteases (PRO) was assayed with the Ladd and Butlera [30] method, where the concentration of the amino acid tyrosine (Tyr) was assayed in the soil samples after incubation with sodium caseinate. Absorbance was measured with the spectrophotometer at a rem λ = 680.

The results of the activity of the enzymes under study were used to calculate the soil indices:

-

enzymatic index of the soil pH from the activity of alkaline (AlP) and acid (AcP) phosphatase [31]:

$$AlP/AcP$$

(3)

-

the geometric mean GMea [32]:

$$GMea=\sqrt[6]{DEH\times CAT\times AlP\times AcP\times BG\times PRO}$$

(4)

-

to evaluate the total activity of soil enzymes (TEI) (total enzyme activity index), the following was calculated [33]:

$$TEI=\sum {\displaystyle \frac{Xi}{\overline{Xi}}}$$

(5)

where Xi is the activity of soil enzyme i, and $\overline{Xi}$ is the mean activity of enzyme i in all the samples.

-

the results of the metabolic activity index (MAI) [34] for the total soil activity are also presented:

$$MAI=\sum {\displaystyle \frac{Pij}{Pcij}}$$

(6)

where $P_{ij}={\displaystyle \frac{A_{ij}}{{Ref}_j}}$, ${Pc}_{ij}={\displaystyle \frac{{Ac}_{ij}}{{Refc}_j}}$ and A_ij is the value of activity of each enzyme; Ref_j is the reference parameter—TOC; Ac_ij is the value of the activity of each enzyme in the control soil; Ref_cj is the reference parameter in the control soil.

2.3. Statistical Analyses

The data received were exposed to the statistical analysis performed in MS Excel, Statistica 13.3. The normality of the distribution of the parameters was verified with the Shapiro–Wilk test of normality. To determine the dependence among the basic soil properties, total organic carbon (TOC), total nitrogen (TN), dissolved organic carbon and dissolved nitrogen (DOC, DTN), fractions of labile carbon (CL1), content of available P, K, and Mg forms, the activity of selected soil enzymes, and the coefficients of correlation were calculated using PAST 4.13 [35]. Statistically significant values of correlation coefficients are presented as the correlogram. For the results, a single-factor analysis of variance was performed with the post hoc HSD Tukey test for a level of significance of p = 0.05. The results were expressed as the arithmetic mean. To account for the diversification of soil in terms of physical, chemical, and biochemical parameters, we used a multidimensional exploration technique, principal component analysis (PCA), for the first two components.

3. Results and Discussion

3.1. Physicochemical Properties of the Soil

The soils studied in the surface horizon demonstrated a decrease in the active acidity of pH of H₂O (by 5.96 on average), and exchangeable acidity in the pH of KCl (by 4.68 on average), as compared with the T0 (

Table 1. Values of pH and the organic matter properties.

Fertilisation	pH KCl	pH H2O	TOC g kg−1	TN g kg−1	TOC/ TN	DOC mg kg−1	DTN mg kg−1	DOC %TOC	DTN %TN
T0	5.3	6.3	5.67	0.53	10.7	75.65	7.28	1.33	1.37
			±0.005	±0.005		±0.10	±0.005
T1	4.5	6.0	5.07	0.94	5.4	64.55	6.30	1.27	0.70
			±0.005	±0.005		±0.15	±0.005
T2	4.4	5.8	8.85	0.68	13.0	67.95	6.41	0.80	0.94
			±0.005	±0.005		±0.05	±0.01
T3	4.2	5.6	6.75	0.55	12.3	78.20	6.60	1.16	1.20
			±0.005	±0.005		±0.00	±0.005
T4	4.2	5.6	8.56	0.50	17.1	80.05	11.41	0.92	2.28
			±0.005	±0.005		±0.05	±0.01
T5	4.4	5.6	5.57	0.46	12.1	79.50	8.02	1.43	1.74
			±0.005	±0.005		±0.10	±0.005
T6			6.52	0.57	11.4	78.40	7.58	1.20	1.33
	4.4	5.7	±0.01	±0.005		±0.20	±0.005
T7			6.16	0.54	11.4	80.20	8.03	1.30	1.50
	4.5	5.9	±0.005	±0.005		±0.10	±0.005
T8			6.82	0.58	11.7	82.10	10.87	1.20	1.88
	4.6	5.9	±0.01	±0.005		±0.10	±0.005
T9			6.36	0.56	11.3	70.25	7.59	1.10	1.35
	4.9	6.2	±0.01	±0.005		±0.05	±0.02
T10			6.72	0.58	11.6	67.10	8.08	1.00	1.39
	5.4	6.5	±0.005	±0.000		±0.00	±0.005
T11			6.23	0.53	11.7	77.70	7.70	1.25	1.45
	5.4	6.5	±0.005	±0.005		±0.10	±0.005
Mean			6.607	0.585	11.642	75.137	7.993	1.163	1.428
HSD			0.039	0.028	n.s.	0.516	0.063	0.062	n.s.

Abbreviations: T0—control (without fertilisation); T1—NPK; T2—FYM (FYM every 4 years); T3—FYM + PK; T4—FYM + KN; T5—FYM + KN + Mg (+magnesium); T6—FYM + PN; T7—FYM + PN + Mg; T8—FYM + NPK; T9—FYM + NPK + Mg; T10—FYM + NPK + Ca (liming once every 4 years); T11—FYM + NPK + Ca + Mg. TOC—total organic carbon; TN—nitrogen total; DOC—dissolved organic carbon; DTN—dissolved total nitrogen; DOC%—percentage share in the pool TOC, DTN%—percentage share in the pool TN; HSD LSD—Honestly Significant Difference± Standard Deviation; n.s.—not statistically significant.

). The fertilisation variants which included liming were the only ones for which the values of those parameters corresponded to the T0 level (5.3; 6.3). Research by Jaskulska et al. [2] showed that over 70 years of applying manure and mineral fertilisation caused strong acidification of the soil. The soil reaction affects the solubility of the minerals and the content of their forms that is available to plants [36]. It is also the basic factor regulating many biological processes in soil [37]. Soil acidity has an unfavourable effect on soil conditions, and it has already become a serious problem in intensifying agricultural systems all across the world. Unfortunately, such changes were observed in long-term field experiments, especially in light soils. In extreme cases, soil degradation was noted [38].

Soil organic carbon (SOC) determines the persistence of ecosystems and environmental processes, including the quality of soil. An inadequate long-term use of agrotechnical practices usually leads to SOC exhaustion, and it affects the content of soil organic carbon. The average TOC in the soils analysed was 6.607 g kg⁻¹, and it was higher than the T0 (5.67 g kg⁻¹). The treatment with only mineral fertilisation was the only one with a decrease in TOC to 5.07 g kg⁻¹ in soil, which is also reported by Nardi et al. [3]. The content of the labile fractions of organic carbon and the value of indices evaluating the management of that parameter in soil are equally important [39,40]. The analysis of the condition of organic matter in the soils under study involved the use of the organic carbon fraction susceptible to oxidation with KMnO₄ solution with a concentration of 0.0333 M dm⁻³, namely the fraction most susceptible to oxidation (CL1), and a concentration of 0.3333 M dm⁻³ (CL). The content of CL1 for the control variant was 0.321 g kg⁻¹, whereas the average content for the treatments studied was 0.264 g kg⁻¹ (

Table 2. The impact of different fertilisations on labile and non-labile organic carbon fractions.

Fertilisation	CL1 g kg−1	CL1 %TOC	CL g kg−1	CNL g kg−1
T0 *	0.321 ± 0.001	5.7	1.07 ± 0.005	4.60
T1	0.191 ± 0.000	3.8	0.64 ± 0.005	4.43
T2	0.200 ± 0.000	2.3	0.61 ± 0.000	8.24
T3	0.266 ± 0.000	3.9	0.64 ± 0.005	6.11
T4	0.211 ± 0.000	2.5	0.56 ± 0.005	8.00
T5	0.253 ± 0.003	4.5	0.65 ± 0.010	4.92
T6	0.243 ± 0.003	3.7	2.07 ± 0.010	4.45
T7	0.267 ± 0.003	4.3	0.64 ± 0.010	5.52
T8	0.274 ± 0.004	4.0	0.80 ± 0.010	6.02
T9	0.311 ± 0.001	4.9	2.05 ± 0.005	4.31
T10	0.302 ± 0.002	4.5	1.05 ± 0.005	5.67
T11	0.323 ± 0.003	5.2	1.07 ± 0.010	5.16
Mean	0.264	4.108	0.998	5.428
HSD	n.s.	n.s.	n.s.	3.791

Abbreviations: *; ±—see Table 1. CL1—labile carbon (fraction oxidised 0.0333 M KMnO₄), CL—labile carbon (fraction oxidised 0.333 M KMnO₄), CNL—non-labile carbon (C remaining unoxidized by 0.333 M KMnO₄), n.s.—not statistically significant

). Important information is provided by the percentage share of that fraction in the TOC pool. However, also in that case, the CL1 values (% TOC) for all the fertilisation variants were lower (from 2.3% TOC for FYM to 5.2% TOC for FYM + NPK + Ca + Mg), as compared with the control: 5.7% TOC. The indices of carbon management (CMI) and carbon level (CPI) facilitate evaluating the effects of varied mineral and FYM fertilisation on the quality of organic matter (Figure 1). The results of this study demonstrate that an increase in TOC, up to 8.85 g kg⁻¹ and 8.56 g kg⁻¹ (FYM, FYM + KN), respectively, as compared with the control (5.67 g kg⁻¹), did not have a significant impact on the content of the labile fraction (CL) in respective fertilisation treatments. We noted only a higher content of that fraction for FYM + PN and FYM + NPK + Mg treatments (2.07 g kg⁻¹ and 2.05 g kg⁻¹). Unfortunately, all the fertilisation treatments demonstrated a decrease in the value of the carbon management index (CMI) (Table 2).

The lowest decrease in the CMI value (down to 78.4% and to 65.3% of the reference sample) was recorded for the treatments with the highest CL fraction content. According to Blair et al. [41], introducing FYM and mineral fertilisation increases the content of total organic carbon and labile fractions. Unfortunately, the results of the analyses of soils in respective fertilisation treatments do not confirm the working hypothesis presented by the authors. Only the application of FYM + PN and FYM + NPK + Mg increased the labile fraction of organic carbon, as compared with the control. The soil carbon accumulation index points to an unfavourable condition of soil organic matter. It is, therefore, worth stressing that the agrotechnical treatments in the present field experiment did not enhance the quality of organic matter despite higher contents of organic carbon in the fertilisation treatments [42]. It was found that the average content of total nitrogen (TN) in the treatments analysed was 0.583 g kg⁻¹ (Table 1). As compared with the TN (0.53 g kg⁻¹) in the control soil, a decrease in the content of that parameter was recorded for FYM + KN (0.50 g kg⁻¹) and FYM + KN + Mg (0.46 g kg⁻¹) treatments. However, the highest content of TN was recorded for the treatment with mineral fertilisation only; NPK (0.94 g kg⁻¹). The TOC and TN results were used to calculate the values of the ratio of TOC/TN, which is an indicator of the degree of soil organic matter decomposition. In general, it is assumed that FYM fertilisation results in a decrease in the value of that ratio, which is due to a greater accumulation of the content of nitrogen than carbon [43]. The TOC/TN value for the control was 10.7, and the average value for the fertilisation treatments analysed was 11.6 (Table 1). Importantly, higher TOC/TN values, from 11.3 to 17.1, were recorded for almost all the fertilisation treatments, except for the NPK variant (5.4), which is due to the lowest TOC (5.07 g kg⁻¹) and the highest TN (0.94 g kg⁻¹). A high value of the ratio of TOC/TN (17.10) for the FYM + KN variant, on the other hand, can point to a mineralization process slowdown [42].

A mobile fraction of soil organic matter (DOM) is the portion that is soluble in water or in salt solutions with a pH~7. The DOM in soil is expressed as the content of dissolved organic carbon (DOC). It is the most mobile and the most active soil component, acting as an easily accessible source of nutrients and energy for microorganisms and other living organisms. It determines the processes of soil structure formation, and it is responsible for nutrient transport [8]. Both the agrotechnical practices and the organic material introduced into soil have an unquestionable effect on the content of dissolved organic matter in soils under agricultural use [7]. In the soil under study, for the fertilisation variants analysed, there were noted similar DOC contents (on average, 75.14 mg kg⁻¹) (Table 1). The significantly highest DOC content, as compared with the control variant (75.65 mg kg⁻¹), was identified in the soil from FYM + NPK (82.10 mg kg⁻¹) and FYM + PN + Mg (80.20 mg kg⁻¹) treatments. One should also note a decrease in the content of the fraction of organic carbon in such fertilisation variants such as NPK, FYM, FYM + NPK + Mg, and FYM + NPK + Ca. However, the DOC contents in the soils studied did not affect the percentage share of that carbon fraction in soil humus. The average percentage share of the DOC fraction in the total organic carbon accounted for 1.16% TOC, and it was lower than in the control variant (1.33% TOC). A lack of significant differences in the DOC expressed in % TOC can suggest, according to Gonet et al. [7], that it depends on the absolute amount of soil organic matter. For the dissolved total nitrogen (DTN), the average content was 7.99 mg kg⁻¹, and it was higher than the contents in the control variant (7.28 mg kg⁻¹). Interestingly, however, for the NPK, FYM, and FYM + PK fertilisation variants, we recorded a decrease in the content of that fraction (6.30; 6.41 and 6.60 mg kg⁻¹, respectively). The fertilisation variants applied in the long-term field experiment did not have a significant effect on the percentage share of that fraction in the total nitrogen.

3.2. The Content of Available Macronutrients

The ANOVA demonstrated a significant effect of fertilisation on changes in the content of available P, K, and Mg (

Table 3. The content of available phosphorus, potassium, and magnesium.

Fertilisation	P	K	Mg
	mg kg−1
T0 *	73.52 ± 1.26	59.80 ± 3.58	10.32 ± 1.98
T1	118.4 ± 3.59	168.2 ± 9.12	11.91 ± 2.59
T2	128.7 ± 7.25	172.9 ± 7.56	12.11 ± 2.14
T3	154.2 ± 9.37	195.4 ± 8.23	13.29 ± 1.89
T4	89.31 ± 2.53	141.2 ± 6.12	15.04 ± 2.56
T5	92.70 ± 3.57	175.9 ± 12.11	22.98 ± 3.45
T6	142.7 ± 8.21	78.50 ± 5.73	19.63 ± 4.11
T7	149.7 ± 7.11	67.90 ± 6.32	23.17 ± 4.96
T8	163.8 ± 11.89	172.4 ± 9.63	21.75 ± 4.87
T9	159.7 ± 10.46	179.2 ± 10.45	24.89 ± 3.28
T10	168.9 ± 8.26	183.7 ± 9.58	17.56 ± 3.56
T11	142.9 ± 7.63	211.5 ± 13.44	26.97 ± 2.31
Mean	132.0	150.6	18.30
HSD0.05	4.170	11.62	3.151

Abbreviations: *, ±—see Table 1. P—available phosphorus; K—available potassium; Mg—available magnesium.

). The significantly highest content of available P (168.9 mg kg⁻¹) was recorded in the soil T10. There was 130% more compared with the control (73.52 mg kg⁻¹). The lowest P content was recorded in the soil T4 (89.3 mg kg⁻¹) and T5 (92.7 mg kg⁻¹). Menšík et al. [44] reported the highest content of N, P, and K in the topsoil for the NPK, FYM, cattle slurry and straw, and cattle slurry fertilisation treatments and the lowest for the control. FYM is an important source of organic and mineral P; it is mostly mineral P which is uptaken by the plants and, in general, it accounts for 45% to 90% of P in FYM. The soil from all the variants, except for the control, according to PN-R-04023 [21], can be considered class I with a very high P content. In such a case, phosphorus fertilisation is considered redundant. P is an element that is hardly mobile, available to plants only in the immediate vicinity of roots, and P uptake depends a lot on soil reaction and temperature. An excess of phosphorus is not harmful to plants; however, too much of that element in soil can impair the absorption of some microelements, e.g., potassium, copper, iron, and zinc. An intensive phosphorus fertilisation to increase the yield and its quality poses a greater risk of dispersion of that nutrient to the environment and penetration into waters. Filipek and Skowron [45] showed that 20–30% of the excessive phosphorus introduced into soil with fertiliser, as compared with phosphorus uptake with the yield, is accumulated in soil in an available form of phosphorus extractable with the Egner–Riehm solution. Such an amount is a potential source of phosphorus loss due to leaching.

The significantly highest content of available K (211.5 mg kg⁻¹) was recorded for T11, which accounted for 254% more as compared with the T0 (59.8 mg kg⁻¹) (Table 3). According to PN-R-04022 [22], soil from that treatment is considered class I with a very high K content. We found no significant differences in the K content across T1, T2, T5, T8, and T9 (168.2 mg kg⁻¹, 172.9 mg kg⁻¹, 175.9 mg kg⁻¹, 172.4 mg kg⁻¹, and 179.2 mg kg⁻¹, respectively). Reports by Arbačauskas et al. [46] demonstrated that the application of only potassium resulted in an increase in available P, contrary to nitrogen and phosphorus fertilisation. The T6 and T7 (without K) fertilisation variants had a significantly decreased K content in soil (78.5 mg kg⁻¹ and 67.9 mg kg⁻¹, respectively). According to PN-R-04022 [22], those soils are considered class IV with a low content of that macronutrient. A long-term field experiment carried out by Balik et al. [47] showed that the content of available K decreased over 21 years in non-fertilised plots, which must have been due to a release of potassium from the unexchangeable form. Those changes, however, do not always reflect the K equilibrium.

The content of available Mg was significantly modified by long-term mineral, natural, and natural and mineral fertilisation (Table 3). The highest content (26.97 mg kg⁻¹, 24.89 mg kg⁻¹, and 23.17 mg kg⁻¹) was noted in the soil samples with Mg fertilisation (T11, T9, and T7, respectively). However, we recorded no significant differences in the Mg content in the soils sampled from those treatments. These soils, according to PN-R-04020 [24], can be qualified as soils with a low content (class IV) of available Mg. The significantly lowest Mg content was found in soil T0 (10.32 mg kg⁻¹), T1 (11.91 mg kg⁻¹), and T2 (12.11 mg kg⁻¹). Introducing combined organic and mineral fertilisation into the soil resulted in a significant increase in the content of that macroelement. Similar results were recorded by Sienkiewicz et al. [48]. They found that FYM with mineral fertilisers increases the Mg content as compared with only mineral fertilisation. FYM enhances the sorption of the soil complex, which retains cations due to physicochemical sorption. It limits the process of element leaching, including Mg.

3.3. The Activity of Enzymes

The activity of the enzymes is presented in

Table 4. The activity of enzymes in soil.

Fertilisation	DEH	CAT	AlP	AcP	BG	PRO
T0 *	0.271 ± 0.08	0.063 ± 0.01	0.200 ± 0.09	0.649 ± 0.05	0.529 ± 0.08	16.23 ± 1.23
T1	0.289 ± 0.09	0.089 ± 0.02	0.237 ± 0.07	0.709 ± 0.07	0.612 ± 0.07	37.57 ± 2.35
T2	0.489 ± 0.08	0.265 ± 0.09	0.351 ± 0.07	0.868 ± 0.01	1.351 ± 0.11	22.43 ± 1.98
T3	0.358 ± 0.04	0.183 ± 0.08	0.362 ± 0.08	0.875 ± 0.10	1.107 ± 0.09	18.56 ± 1.23
T4	0.471 ± 0.04	0.251 ± 0.08	0.291 ± 0.06	0.826 ± 0.05	1.309 ± 0.12	19.65 ± 1.58
T5	0.321 ± 0.05	0.138 ± 0.07	0.252 ± 0.07	0.753 ± 0.07	0.531 ± 0.11	15.22 ± 1.66
T6	0.334 ± 0.04	0.169 ± 0.06	0.286 ± 0.09	0.806 ± 0.03	1.122 ± 0.10	19.56 ± 2.08
T7	0.317 ± 0.03	0.156 ± 0.05	0.267 ± 0.11	0.767 ± 0.08	0.961 ± 0.09	17.72 ± 1.83
T8	0.365 ± 0.05	0.198 ± 0.06	0.309 ± 0.11	0.861 ± 0.08	1.128 ± 0.11	20.08 ± 1.98
T9	0.343 ± 0.06	0.172 ± 0.06	0.271 ± 0.09	0.784 ± 0.12	1.139 ± 0.12	19.05 ± 1.76
T10	0.361 ± 0.04	0.181 ± 0.07	0.648 ± 0.12	1.203 ± 0.11	1.186 ± 0.11	20.19 ± 2.08
T11	0.328 ± 0.03	0.161 ± 0.05	0.499 ± 0.11	1.099 ± 0.09	0.984 ± 0.09	17.96 ± 1.81
Mean	0.354	0.169	0.331	0.850	0.997	20.35
HSD0.05	0.022	0.015	0.044	0.029	0.131	2.161

Abbreviations: *; ±—see Table 1. DEH—dehydrogenases (mg TPF kg⁻¹ 24 h⁻¹); CAT—catalase (mg H₂O₂ kg⁻¹ h⁻¹); AlP—alkaline phosphatase (mM pNP kg⁻¹ h⁻¹); AcP—acid phosphatase (mM pNP kg⁻¹ h⁻¹); BG—β-glukosidase (mM pNP kg⁻¹ h⁻¹); PRO—protease (mg TYR kg⁻¹ h⁻¹).

. The results demonstrate that FYM and mineral fertilisers significantly changed the enzymatic activity of soil. T2 and T4 fertilisation increased the activity of dehydrogenases (0.489 mg TPF kg⁻¹ 24 h⁻¹ and 0.471 mg TPF kg⁻¹ 24 h⁻¹, respectively) and catalase (0.265 g H₂O₂ kg⁻¹ h⁻¹ 0.251 g H₂O₂ kg⁻¹ h⁻¹), respectively. For DEH, it was about 73%, and for CAT, it was around 300% higher activity, as compared with the control. The application of NPK (T1) also increased the activity of redox enzymes: DEH—6% only; CAT—41%. Some mineral ions, e.g., Ca, Mg, and Fe, are cofactors that activate enzymes [49]. Their availability in soil determines the level of activity of the enzyme. The activity of dehydrogenases is considered an indicator of oxidative metabolism in soil. Catalase, on the other hand, is an antioxidating enzyme that protects against oxidative stress and catalyses the decomposition of hydrogen peroxide to water and oxygen. The activity of those two enzymes is used to acquire information on the microbiological activity in soil. The AlP and AcP activities were significantly highest in T10 (0.648 mM pNP kg⁻¹ h⁻¹ and 1.203 mM pNP kg⁻¹ h⁻¹, respectively) and T11 (0.499 mM pNP kg⁻¹ h⁻¹ and 1.099 mM pNP kg⁻¹ h⁻¹, respectively) (Table 4). Applying only FYM (T2) significantly increased the activity of AlP (0.351 mM pNP kg⁻¹ h⁻¹) and AcP (0.868 mM pNP kg⁻¹ h⁻¹) (by 75.5% and 34%), as compared with the control (0.200 mM pNP kg⁻¹ h⁻¹ and 0.649 mM pNP kg⁻¹ h⁻¹), respectively. The NPK (T1) application also resulted in a significant increase in the AlP activity (0.237 mM pNP kg⁻¹ h⁻¹) and AcP (0.709 mM pNP kg⁻¹ h⁻¹), however, only by 18.5% and 9%.

We recorded the significantly highest increase in BG activity after the application of FYM (T2) (1.351 mM pNP kg⁻¹ h⁻¹) and FYM + KN (T4) (1.309 mM pNP kg⁻¹ h⁻¹) (Table 4). BG activity was significantly higher for T2 and T4 (155% and 147%, respectively) than in the control (0.529 mM pNP kg⁻¹ h⁻¹). We found no significant differences in BG activity between the T0 and T1 applications (0.612 mM pNP kg⁻¹ h⁻¹). As for the negative effects of chemical fertilisation, Dincă et al. [50] noted a decrease in the enzymatic activity. It was noticeable especially in the soil after applying higher rates of mineral fertilisers, which coincides with organic matter losses. Mineral fertilisation only (T1) increased the PRO activity significantly (37.57 mg TYR kg⁻¹ h⁻¹) (Table 4). It was 131% more than for the control (16.23 mg TYR kg⁻¹ h⁻¹). Also, T2 resulted in a significant PRO increase (by 38%). The significantly lowest PRO activity (15.22 mg TYR kg⁻¹ h⁻¹) was recorded with T5. We found no significant differences in the PRO activity due to the application of T3, T4, T6, T8, T9, and T10. A higher SOC content can be a possible reason for a higher activity of enzymes for FYM as compared with mineral fertilisers [9]. Yang et al. [51] demonstrated a significant impact of magnesium fertilisation in the form of MgSO₄ on hydrolytic enzymes (urease, phosphatase, invertase, and protease). Many enzymes depend on Mg, which can bond the magnesium substrate complex with a poor interaction with Mg or Mg, which can bond directly with the enzyme and change its structure, serving a catalytic function [52]. According to Keeler et al. [53], N added into soil increased the activity of acid phosphatase by an average of 13%, cellobiohydrolase by 17%, and a β-1,4-N-acetylglucosaminidase by 18%. The authors suggest that adding N enhances the count of soil microorganisms and increases the demand for P and C. It leads to an increase in the activity of enzymes taking part in the biogeochemistry of those macroelements. We found, however, no effect of N on the activity of redox enzymes degrading lignin. To evaluate the global model of AlP and AcP activity in soil due to the application of N and/or P, Zheng et al. [54] performed a meta-analysis. It demonstrated that the N application activated AcP by 10.1% (±2.9%); however, it showed a minimal effect on AlP, whereas FYM P fertiliser decreased the AcP activity by 7.7% ± 2.6%, yet it increased the AlP activity slightly. The report by Wang et al. [55] identified that the activities of β-glucosidase and alkaline phosphatase were highest after the application of organic fertilisation and after combined NPK with organic fertilisation (NPKMg). That activity was 161–171% and 75–91% higher, respectively, as compared with the control (without fertilisation). A study of 21 years by Choudhary et al. [56] demonstrated that the activity of enzymes (dehydrogenases, β-glucosidase, invertase, alkaline and acid phosphatase, arylsulfatase, and urease) depended significantly on the FYM + NPK fertilisation, as compared with the other treatments (control, N 120, NPK, FYM, and FYM + N). The application of inorganic fertilisers diversifies the activity of soil enzymes. The activity of some enzymes increases with the concentration of inorganic nutrients. Mineral nutrients applied for fertilisation are an immediate food for soil microorganisms. Their count increases and, as a result, the enzymatic activity increases. Yang and Norton [57] report on the application of organic fertilisers (compost) increasing the activity of soil enzymes (protease, chitinase, urease, and arginase), as compared with the inorganic N fertiliser (ammonium sulphate). Usually, organic additives essentially increase the activity of enzymes [58], which is probably due to a stimulation of the growth of microorganisms and a correlated increase in the activity of extracellular enzymatic complexes.

A significant correlation was recorded between TOC and DEH activity (r = 0.98), CAT (r = 0.98), and BG (r = 0.85) (Figure 2). The activity of those enzymes depended on TOC, 96%, 96%, and 72%, respectively. Dehydrogenases are respiratory enzymes that oxidize organic compounds, allocating their two atoms of hydrogen to the acceptors of electrons, producing energy [59]. Those enzymes play an essential role in the biological oxidation of the organic matter of soil by transferring hydrogen from organic substrates to inorganic acceptors [60]. β-glucosidase is an enzyme catalysing the final stage of cellulose hydrolysis, decomposing disaccharides and releasing the glucose available to soil microorganisms [18]. A higher content of organic carbon increases the rate of mineralization by microorganisms in soil. It results in an increase in enzymatic activity. SOC determines an increase in the concentration of the substrate available in soil. An increased C content in soil can often lead to increased water retention, which can increase substrate and enzyme diffusion [61]. Also, Gautam et al. [9] found that the long-term use of organic fertilisers (16 years) enhanced the content of SOC, which is the main substrate of the activity of enzymes in soil.

3.4. Enzymatic Indicators of Soil Quality

Chemical compounds present in the soil environment can activate or inhibit the effect of a single enzyme. For that reason, many multiparametric indicators differentiating soil due to the effect of, e.g., the use or type of vegetation have been developed [62]. According to Paz-Ferreiro and Fu [63], effective indicators should provide early warning of the upcoming changes in the soil environment due to the effect of biotic and abiotic factors. From the activity of soil enzymes determined with varied organic and mineral fertilisation, we calculated the enzymatic indicators of the soil quality (AlP/AcP, GMea, TEI, MAI) (Figure 3). The enzymatic pH indicator (AlP/AcP) was calculated with the results of AlP and AcP activity for varied fertilisation. The AlP/AcP value ranged from 0.31 to 0.54, depending on the fertilisation applied. According to Dick et al. [31], for optimal plant growth and development, such a soil pH value can be considered where AlP/AcP is about 0.50. In the present experiment, the AlP/AcP value exceeded 0.5 only for the T10 application (0.54). The results have been confirmed with excessive soil pH in H₂O and 1 M KCl (Table 1). The highest value of the GMea index was observed in the soil following T2 and T10 applications. According to Jat et al. [14], the GMea of the enzymes studied is related to the physicochemical and biological soil properties. For that reason, it serves as a soil quality indicator. Higher GMea values stand for a higher soil quality, and they can describe qualitative changes in soil, disregarding the physicochemical properties [18]. The MAI value ranged from 7.91 (T5) to 10.82 (T11). According to Picariello et al. [34], the MAI values can range from +1 to +∞; however, the values increase with an increase in the metabolic activity of soil. MAI calculated from the enzymatic activity also provides information on the functional stability of microbial communities. Analysing the soils with varied long-term mineral and organic fertilisation, no differences were observed in the MAI values across T2, T3, T4, T6, T7, T8, and T9. To evaluate the total enzymatic activity of soil, the total enzyme activity index (TEI) was used. Its value varied depending on the fertilisation applied. The highest TEI value was recorded following the application of FYM (T2) (13.04) as well as FYM + NPK + Ca (T10) (12.78). Changes in the TEI value depending on fertilisation were the same as for GMea. Similar results were reported by Zhang et al. [64], who suggest that, indeed, those two indicators reflect changes in the soil environment most and that they are reliable for a comprehensive evaluation of the enzymes’ reactions to biotic and abiotic factors. Wojewódzki et al. [65], on the other hand, demonstrated that MAI and TEI effectively differentiated the soil properties depending on the biocarbon applied. The analysis of correlation between the physical and chemical parameters and enzymatic soil indicators showed a significant positive correlation between TOC and GMea (r = 0.79) and TEI (r = 0.83). These indicators accounted for 62% and 69%, respectively, depending on TOC. The indicator AlP/AcP and MAI depended significantly on the pH of H₂O (r = 0.66 and r = 0.87, respectively) and the pH of KCl (r = 0.68 and r = 0.84, respectively) (Figure 2).

We identified a significant positive correlation between NT and PRO activity (r = 0.98) (Figure 2). Enzymes are compounds that are rich in N, and so their production is closely regulated with N availability. Proteases take part in nitrogen mineralization in soil. They catalyse the hydrolysis of peptide bonds in proteins and peptides to amino acids [66]. We recorded a positive significant correlation between TOC/TN and DTN (r = 0.61), DEH (r = 0.74), CAT (r = 0.83), and BG (r = 0.63) and a negative one with PRO (r = −0.70) and CL1 (r = −0.83). We also confirmed a significant negative correlation between DOC and CL1 (r = −0.55) and between TOC and CL1 (r = −0.55). As reported by Błońska et al. [67], a high activity of enzymes is closely connected with a higher OM content, with a good rate of decomposition, and with a low TOC/TN ratio. We recorded a significant negative correlation between DOC and the H₂O pH (r = −0.61) and KCl pH (r = 0.50). Cincotta et al. [68] confirm that acidity determines the stability or release of dissolved organic matter.

A significant correlation was also noted between the activity of AlP and H₂O pH (r = 0.71) and KCl pH (r = 0.75) as well as AcP and H₂O pH (r = 0.72) and KCl pH (r = 0.76). According to Dick et al. [31], phosphatases are enzymes that are most sensitive to changes in pH, which is one of the significant factors affecting the rate of chemical reactions [65]. pH affects the level of ionisation of the enzyme and substrate and changes the conditions of an enzyme–substrate complex. Zheng et al. [54] demonstrated that a change in soil pH was the key factor accounting for varied AlP and AcP activities. A significant positive correlation was recorded between the content of available P and the activity of AlP (r = 0.46) and AcP (r = 0.45). Those enzymes were the right parameter for the soils analysed in terms of the content of available phosphorus. However, the activity of AlP and AcP depended only slightly (21% and 20%, respectively) on P. Phosphatases are enzymes that play a key role in the process of the biochemical mineralization of organic phosphorus bonds [69]. To a large extent, the knowledge of the values of those two parameters should facilitate estimating the content of phosphorus available to plants, which can refer to phosphorus assayed with the Egner–Riehm method [70]. A negative significant correlation was identified between the content of CL1 and the activity of CAT (r = −0.68), BG (r = −0.57), and a positive correlation with TN (r = 0.88) and PRO (r = 0.93). We recorded a significant positive correlation between P and pH in H₂O (r = 0.56) and pH in KCl (r = 0.51). Similar dependencies were reported by Lemanowicz et al. [18]. The soil reaction affects the solubility of mineral nutrients and thus their availability to plants. As for the change in the value of soil pH below optimal for a given element, a fast decrease in yield occurs [36]. We identified a significant correlation between TOC and DEH activity (r = 0.98), CAT (r = 0.98), and BG (r = 0.85) (Figure 2). The activity of those enzymes depended on TOC (by 96%, 96%, and 72%, respectively). Dehydrogenases are respiratory enzymes that oxidise organic compounds, allocating two of their hydrogen atoms to the acceptors of electrons and producing energy [59]. These enzymes play an essential role in biological soil organic matter oxidation by transferring hydrogen from organic substrates to inorganic acceptors [60]. β-glucosidase is an enzyme catalysing the final cellulose hydrolysis stage by decomposing disaccharides, releasing glucose available to soil microorganisms [18]. A higher content of organic carbon increases the rate of mineralization by microorganisms in soil. It results in an increase in the enzymatic activity. SOC determines an increase in the concentration of substrate available in soil. An increased C content in soil can often lead to increased water retention, which can increase substrate and enzyme diffusion [61]. Also, Gautam et al. [9] found that the long-term application of organic fertilisers (16 years) enhanced the content of SOC, which is the main substrate of enzyme activity in soil.

The principal component analysis (PCA) was made to identify the potential factors affecting the parameters in soil. We transformed 17 variables into three orthogonal components, which together account for 76.37% of the total variance, two components of which, PC1 and PC2, are presented in Figure 4. The first two components account for 57.94%. The first component (PC 1), accounting for 35.33% of the variance, was most strongly negatively correlated with TOC (−0.873), TOC/TN (−0.876), DON (−0.532), DEH (−0.849), CAT (−0.937), and BG (−0.847) and positively correlated with TN (0.485), CL (0.758), and PRO (0.475). The second component (PC2), accounting for 22.61%, was most strongly negatively correlated with DOC (−0.517), and positively with H₂O pH (0.848), KCl pH (0.863), P (0.475), Mg (0.473), AlP, (0.790), and AcP (0.789). The loading values >0.75, 0.75–0.5, and 0.5–0.3 are referred to as “strong”, “moderate”, and “poor”, respectively [71].

4. Conclusions

The natural fertilisation applied in the fertilisation variants increased the contents of organic carbon in soil. Unfortunately, that state did not determine the values of carbon management (CMI and CPI) significantly. It is evident from a low content of dissolved organic carbon fraction (DOC), as well as from the fractions undergoing oxidation (CL and CL1). The agrotechnical treatments applied in a long-term field experiment did not improve the state of soil organic matter in the fertilisation variants in terms of the DOM availability to microorganisms. It is also worth noting that the soil reaction in the fertilisation variants was slightly acidic, and the FYM + NPK + Ca and FYM + NPK + Ca + Mg treatments were the only ones for which the soil reaction corresponded to that of the control.

A regular use of integrated natural fertilisation in the form of FYM and mineral fertilisation increased the content of the available forms of nutrients.

The lack of manure decreased the activity of catalase, dehydrogenases, alkaline and acid phosphatases, β-glucosidase, and proteases in soil.

The activity of enzymes taking part in C, N, and P biogeochemistry was much higher in soil following the application of manure together with mineral fertilisers and liming as compared with fertilisation with only manure or mineral fertiliser. However, there were observed differences in the activity of the respective enzymes depending on the fertilisation applied. These resulted from their different susceptibility and resistance to environmental factors and from the soil content of substrates specific for enzymatic reactions.

The multiparametric enzymatic soil fertility indices (AlP/AcP, GMea, MAI, and TEI) differentiate the soil properties depending on the fertilisation applied. Therefore, they can be used as indicators of soil fertility. The GMea and TEI indices were mostly correlated with total organic carbon.