Effectiveness of N Fertilizers with Nitrification Inhibitors on Winter Barley Nutrition and Yield

Abstract

Excessive N loss through leaching and volatilization is a major concern in modern agriculture, reducing N use efficiency, groundwater contamination, and environmental degradation. To address these issues, this research evaluates the impact of N fertilizers containing nitrification inhibitors (NIs), which restrict the conversion of ammonium (NH₄⁺) into nitrate (NO₃⁻), thereby enhancing N retention in the soil. This study examines the effects of different N fertilizer applications on the yield and nutrient dynamics of winter barley (Hordeum vulgare L.) Field experiments were conducted to compare the effects of a one-time and split application of granular N fertilizers ENSIN (with NIs) and DASA (without NIs) on winter barley yield and N dynamics. The highest grain yield was observed with a single ENSIN application (8.35 Mg.hm⁻²), followed by a divided DASA application (7.97 Mg.hm⁻²), both significantly outperforming the control (no N). The most efficient N use was recorded for the single ENSIN application, yielding 27.4 kg of grain per kg of applied N. Agrochemical analyses were conducted to assess soil NH₄⁺ and NO₃⁻ content throughout the vegetation period, and lysimetric methods were used to determine leaching losses. The results highlight the potential of NIs to improve nutrient uptake efficiency, reduce N loss, and enhance sustainable barley production. Through optimizing fertilizer application strategies, this study contributes to the development of more sustainable agricultural practices that improve crop yield while minimizing environmental impacts, particularly in reducing N runoff and groundwater contamination.

1. Introduction

N is an essential macronutrient for plant growth and development, directly affecting crop yield and environmental sustainability [1]. Efficient N management increases plant productivity while reducing nutrient leaching and groundwater contamination [2,3].

The widespread use of N-based mineral fertilizers has significantly contributed to global agricultural productivity. However, intensified agricultural practices exacerbate N losses through leaching and gaseous emissions, with up to 50% of anthropogenic N inputs being lost to the environment [4]. If effective control and regulatory measures are not implemented, N availability may become a limiting factor for crop production [5].

A major challenge in N fertilization is its rapid transformation in the soil. Nitrification, the microbial conversion of ammonium (NH₄⁺) into nitrate (NO₃⁻), accelerates N loss through leaching, increasing the risk of groundwater contamination [6]. Addressing these losses through improved N management strategies is essential for enhancing nutrient use efficiency and reducing environmental impacts.

One of the primary drivers of N₂O emissions from agricultural soils is the application of inorganic fertilizers and/or manure when crops cannot absorb all the available N, resulting in its microbial conversion into nitrous oxide [7]. Moreover, higher N₂O emissions from organic fertilizers than synthetic N fertilizers have been observed in sandy soils under a cool temperate climate [8]. Roche et al. (2016) reported that N₂O emissions varied depending on the formulation of N fertilizers and stabilizer use. In a temperate spring barley system, emissions were significantly lower than the 1% default emission factor established by the Intergovernmental Panel on Climate Change (IPCC) across all fertilizer types, highlighting the importance of N formulation and stabilizers in mitigating emissions [9]. Consequently, balanced N fertilization is crucial for optimizing N retention and minimizing environmental impact.

Strategies to influence ammonium availability include synchronizing fertilization with plant uptake, using slow-release fertilizers or inhibitors, maintaining continuous plant growth to enhance N assimilation, and promoting internal N cycling through immobilization [10]. NIs, which block or slow the conversion of NH₄⁺ into NO₃⁻, have emerged as a promising way to mitigate the environmental risks associated with N loss while simultaneously maintaining or enhancing crop productivity. An alternative approach involves directly inhibiting nitrifiers using commercial (synthetic) or biological nitrification inhibitors. While commercial inhibitors demonstrate effectiveness, their application is often constrained by climate variability and regulations governing organic farming. The commercial inhibitor Instinct^® II has been shown to reduce NO₃⁻-N and total mineral N (TMN) levels, whereas Agrotain^® Ultra has exhibited limited efficacy in inhibiting nitrification in irrigated wheat fields [11]. For instance, ammonium sulfate treated with the nitrification inhibitor (NI) dicyandiamide has demonstrated potential not only in reducing NO₃⁻-N production in loamy and clayey soils but also in decreasing the overall soil nitrification rate [12]. The effectiveness of N fertilizers, particularly when fortified with nitrification inhibitors, is crucial for optimizing winter barley nutrition and yield while minimizing environmental impact.

NIs can reduce the conversion of NH₄⁺ into NO₃⁻ and have become a promising solution for minimizing the environmental risks of nitrogen losses while maintaining or improving crop productivity [13]. The primary hypothesis of this research is that applying nitrogen fertilizer containing NIs, specifically dicyandiamide (DCD) and 1,2,4-triazole (TZ) (ENSIN fertilizer), may improve nitrogen retention in the soil, potentially increase winter barley (Hordeum vulgare L.) yield, and enhance nitrogen use efficiency compared to conventional fertilization methods using nitrogen fertilizers like DASA fertilizer, which do not contain NIs. Additionally, NIs will reduce N leaching and nitrate accumulation, minimizing environmental contamination while sustaining or improving crop productivity.

This study investigates the effects of fertilizer containing a NIs on winter barley cultivation. Through assessing both agronomic parameters, including grain yield and nitrogen use efficiency, and ecological indicators, such as NH₄⁺ and NO₃⁻ concentrations in the substrate and associated leaching losses, we evaluate the efficacy of this fertilization strategy. Our findings are intended to contribute to the development of more sustainable agricultural practices, providing insights into how improved nitrogen management can optimize plant nutrition while mitigating environmental impacts.

2. Materials and Methods

2.1. Plant Object

The Barcelona variety was used in the field experiment with winter barley (Horedum vulgare L.). Barcelona var. is a standard, two-row, medium–late-maturing winter barley cultivar with a plant height of 84 cm. It has moderate lodging resistance and satisfactory winter hardiness, with a survival rate of 88%. Its overall disease resistance is average, showing moderate resistance to powdery mildew (Blumeria graminis), brown spot (Cochliobolus sativus), and barley rust (Puccinia hordei). The grain size is medium, with a thousand kernel weight (TKW) of 45 g, and its grain yield fraction, above 2.5 mm, is considered average.

Barcelona var. does not have specific agronomic requirements. The optimal sowing time depends on the production region, ranging from September 20 to 30 in corn-growing areas, September 15 to 25 in sugar-beet-growing regions, and up to September 20 in higher-altitude potato-growing areas. Under good agronomic conditions, the recommended seeding rate is 4.0–4.5 million viable seeds per hectare [14].

2.2. Establishment and Organization of the Winter Barley Experiment

A small-plot field experiment with winter barley (Hordeum vulgare L.), cultivar Barcelona, was conducted over two consecutive growing seasons at the Central Controlling and Testing Institute in Agriculture, Testing Station Veľké Ripňany. This station is located in the sugar beet–barley production region of western Slovakia, in the Topoľčany district, at an elevation of 188 m. The region has an average growing season temperature of 15.5 °C, an annual mean temperature of 9.7 °C, and an annual precipitation of 582 mm.

The soil at the experimental site is derived from Tertiary marine Neogene sediments, primarily clayey, with overlying Quaternary loess and Holocene alluvial deposits. In the first year of the experiment, the soil had low inorganic nitrogen (Nin_{in}) content (7 mg·kg⁻¹); medium to high levels of phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg); low organic carbon (Cox_{ox}) content (1.12%); and a slightly acidic pH of 6.5. In the second experimental year, the inorganic N content in the soil was higher (14 mg·kg⁻¹), and the other elements had medium to high levels, similar to the first year. The Cox content (1.03%) remained low, and the pH was 6.8.

The experiment followed a strip–split block design. Each treatment was replicated four times, resulting in 20 experimental plots. Each plot measured 8.9 m × 1.125 m, with a harvest area of 10 m², and the plots were separated by 0.5 m wide paths. The total harvest area was 200 m². Barley seeds were sown in October of each year at a seeding rate of 3.5 million viable grains per hectare. In the second growing season, the experiment was repeated on another site after peas (Pisum sativum L.) as the preceding crop.

Five fertilization treatments were tested in four replicates, with fertilizers applied manually. The fertilization scheme and N application rates at different barley growth stages are presented in

Table 1. Nutrient application management scheme for winter Barley, variety Barcelona.

Nutrient Management Variant	Fertilizer	Regenerative Fertilization (First Decade of April, Tillering Phase) BBCH25		Production Fertilization (“First Node Detectable” Stage) BBCH 32		Quality Fertilization (the Flag Leaf Ligule Visible to Flowering Stages) BBCH 49–51
		Nutrient Doses in kg ha−1
		N	S	N	S	N	S
1	Control	-	-	-	-	-	-
2	DASA 26/13 split application	60	30	50	25	30	15
3	ENSIN split application	60	30	50	25	30	15
4	DASA 26/13 one-time application	140	70	-	-	-	-
5	ENSIN one-time application	140	70	-	-	-	-

.

In the field experiment, the following N-sulfur fertilizers were used: DASA 26/13 (ammonium nitrate + ammonium sulfate) (without NIs) and ENSIN (with NIs). ENSIN is a N–sulfur fertilizer that typically contains ammonium sulfate (NH₄)₂ SO₄) along with additional sulfur content. The exact composition can vary slightly by manufacturer, but generally, ENSIN contains around 26% N and 13% sulfur.

A total N dose of 140 kg.ha⁻¹ was applied to variants 4 and 5 as a one-time dose at the beginning of barley vegetation during regenerative fertilization in April or March of each experimental year. Additionally, the total N dose was divided into three parts (60 + 50 + 30 kg.ha⁻¹) and applied to variants 2 and 3 during regenerative fertilization in April or March; production fertilization in May or March; and qualitative fertilization in May or April. Through the DASA 26/13 and ENSIN fertilizers, sulfur (S) was applied to variants 2 through 5 at a total dose of 70 kg.ha⁻¹.

2.3. Fertilizer Characteristics in the Evaluated Experiments

DASA 26/13 is a granular N fertilizer containing sulfur. N was in ammoniacal and nitrate forms, and sulfur was in a water-soluble sulfate form. The granules were pink to brown. This fertilizer does not contain any nitrification inhibitors.

• Technical Specifications:
• Total N: 26% by weight
• Ammoniacal N: 18.5% by weight
• Nitrate N: 7.5% by weight
• Water-soluble sulfur: 13% by weight.

ENSIN is a granular N fertilizer with sulfur and nitrification inhibitors (dicyandiamide—DCD and 1,2,4-triazol—TZ) with the same nutrient content as DASA 26/13.

2.4. Agrochemical Soil Analyses Before Establishing the Trials

Laboratory soil analyses were conducted at the Department of Agrochemistry and Plant Nutrition, Faculty of Agrobiology and Food Resources of Slovak University of Agriculture in Nitra (Slovakia). The N content in the soil and lysimetric water was determined using the following methods:

Ammonium N content was colorimetrically determined in the soil (mg N-NH₄⁺kg⁻¹ soil) in a 1% K₂SO₄ leachate with Nessler’s reagent and in lysimetric water (mg N-NH₄⁺ L⁻¹) using the same lysimetric method.

Nitrate N content in the soil (mg N-NO₃⁻.kg⁻¹ soil) was colorimetrically determined in a 1% K₂SO₄ leachate with 2,4-phenol disulfonic acid and in lysimetric water (mg N-NO₃⁻ L⁻¹) using the same method.

2.5. Agrochemical Analyses of Soil Samples for Ammonium and Nitrate N Content in the Soil During the Vegetation of Winter Barley

Soil samples for agrochemical analyses taken from individual variants and repetitions of the field trial for nitrate and ammonium N content were collected from a soil profile depth of 0.0–0.3 m and 0.3–0.6 m using a soil probe. During the vegetation of winter barley, four soil sample collections were carried out at 3- to 4-week intervals from the tillering of barley to the full botanical maturity of barley grains (

Table 2. Soil sample collection dates to determine ammonium and nitrate N content in the soil during the winter barley growing season.

Soil Sampling	Sampling Date
	First Experimental Year	Second Experimental Year
Sampling	2.5	29.3
2. Sampling	24.5	20.4
3. Sampling	10.6	12.5
4. Sampling	29.6	2.6

). The ammonium N content and nitrate N content in the soil were determined using the methods outlined above.

As an indicator of the NIs’ effectiveness in the ENSIN fertilizer, the ratio of nitrate N and ammonium N from the total inorganic N content in the soil was evaluated in all treatments/variants. It was then compared with the values achieved in variants with fertilizers not containing nitrification inhibitors. The ratio of nitrate and ammonium N from the total inorganic N content in the soil was calculated using the following formula:

P = A/B × 100

where

• P is the ratio of nitrate or ammonium N from the total inorganic N content in the soil (%);
• A is the nitrate or ammonium N content in the soil (mg·kg⁻¹);
• B is the inorganic N content in the soil (mg·kg⁻¹).

The inorganic N (Nin) content in the soil was calculated as the sum of ammonium and nitrate N in the soil (mg·kg⁻¹): Nin = N-NH₄ ⁺ + N-NO₃⁻.

Winter barley was harvested with a small-plot combine during the first and second vegetative years, at the time of botanical maturity.

The effectiveness of the fertilizers on grain yield was calculated with the N use efficiency (NUE) coefficient. The NUE indicates a grain yield increment per 1 kg of applied N:

NUE = ΔY/140 (kg.kg⁻¹),

where

• ΔY is an increase in grain yield compared with the control treatment (kg.ha⁻¹);
• 140 = total rate of N (kg.ha⁻¹).

2.6. Establishment of 2-Year Pot Experiment with Barley

Along with the field trial, a 2-year pot experiment with barley was established, aimed at assessing the effect of fertilizers containing nitrification inhibitors on nitrate N leaching. The barley experiment was set up in plastic containers with a diameter of 290 mm and a height of 260 mm with perforated bottoms. Each container was filled with 15 kg of soil, which was first thoroughly homogenized by mixing. The containers were filled with soil 10 days before sowing to allow the soil to settle properly. The individual fertilization variants (the same as in the field experiment) were repeated four times. Barley seeds were sown on October 5 (first year) and October 2 (second year), and 20 barley seeds were sown to a depth of 35 mm in each container. Fertilizer application was performed manually before sowing barley seeds to a depth of 70 mm in the soil profile, with the second part (for the split application) applied at the first node detectable (BBCH 32) stage on the surface of the soil. The total N dose was 9 g per container, and for the split application, it was 4.5 g + 4.5 g per container.

During the winter, the pots were maintained in a greenhouse, where soil moisture was controlled by watering the soil profile as needed. Each pot received 150 mL of water every day until the first sampling for the lysimetric solution. As plant growth advanced, water volumes increased to 300 mL per pot. In early April, the pots were transferred to an outdoor vegetation cage. After the pots were moved outdoors, the water supply was adjusted according to plant growth and weather conditions to maintain a soil water-holding capacity of 50–60%. During storms or heavy rainfall, the pots were temporarily covered with a protective plastic roof. At the time of sampling, each pot was watered with 950–1200 mL of water (according to the actual moisture of soil in the pots) to facilitate leaching into a container placed below each experimental pot with plants.

During the barley growing season, four lysimetric solution samples were taken—two samples in the autumn and two samples in the spring (sampling dates are listed in

Table 3. Dates of lysimeter solution sampling during the barley-growing season.

Sampling of Lysimetric Solutions	Sampling Date
	First Experimental Year	Second Experimental Year
1. Sampling	5.11	10.11
2. Sampling	25.2	28.2
3. Sampling	20.4	19.4
4. Sampling	22.5	30.5

).

This combined field and pot experiment allowed for a comprehensive assessment of barley growth, soil nutrient dynamics, and environmental influences across different cultivation conditions.

2.7. Statistical Evaluation of the Results

The grain yield of winter barley (Mg.hm⁻²); the nitrate and ammonium N contents in the soil (mg·kg⁻¹); and the nitrate N-leaching contents in the lysimetric solutions (mg.L⁻¹) were evaluated using the STATGRAPHICS Plus program (version 5.1) through an analysis of variance, with differences between the levels of the monitored factors assessed using an LSD test (Least Significant Difference) at a 95% probability level (α = 0.05).

3. Results

3.1. Evaluation of the Effect of Fertilization on the Grain Yield of Winter Barley

The winter barley grain yields achieved in the first year of the experiment are presented in

Table 4. Grain yield of winter barley in the first experimental year season (average of four repetitions).

Nutrient Variant		Grain Yield (Mg.hm−2)	Relative %
1: Control, without N		4.44 a	100.0
2: DASA split application		7.25 bd	163.3	100.0
3: ENSIN split application		5.15 c	116.0	71.0	100.0
4: DASA one-time application		6.68 b	150.5	92.1	129.7	100.0
5: ENSIN one-time application		7.85 d	176.8	108.3	152.4	117.5
LSD	α = 0.05	0.71

The same letters for the average yields of individual variants indicate statistical insignificance.

. These values indicate that both split and one-time fertilizer applications statistically significantly increased the grain yield of barley compared with the unfertilized control.

The significant effect of nitrification inhibitors on the ENSIN fertilizer was confirmed in the field trial with winter oilseed rape, where a one-time application increased the seed yield by 44.2%, relative, compared with the one-time application of the DASA fertilizer at the same N dose [15].

The highest grain yield (7.85 Mg.hm⁻²) was achieved in the variant fertilized with ENSIN fertilizer (with NIs) with a one-time application in the first evaluated year (Table 4). The least effective was the split application of ENSIN fertilizer, which resulted in the lowest grain yield (5.15 Mg.hm⁻²) compared with the other fertilization variants. With ENSIN fertilization, a one-time application was more effective, resulting in a 1.52-fold higher yield compared with its split application. In the DASA fertilizer application variants (without NIs), the split application was the most effective, with a grain yield of 7.25 Mg.hm⁻², which was not significantly different from the one-time application of ENSIN fertilizer; however, the DASA split application increased application costs.

The highest grain yield (8.85 Mg.hm⁻²) was achieved in the second experimental year, just like the first year, with a one-time application of ENSIN fertilizer (with NIs). The most effective DASA fertilization was the split application with a grain yield of 8.69 Mg.hm⁻². The lowest grain yield (8.33 Mg.hm⁻²) was found with a one-time DASA application (without NIs), but the difference in yield compared with the split application was not statistically significant (

Table 5. Grain yield of winter barley in the second experimental year season (average of four repetitions).

Nutrient Variant		Grain Yield (Mg.hm−2)	Relative, %
1: Control, without N		4.58 a	100
2: DASA split application		8.69 bc	189.7	100
3: ENSIN split application		8.70 bd	190.0	100.1	100
4: DASA one-time application		8.33 c	181.9	95.9	95.8	100
5: ENSIN one-time application		8.85 d	193.2	101.8	101.7	106.2
LSD	α = 0.05	0.37

The same letters for the average yields of the individual variants indicate statistical insignificance.

).

The average grain yield of winter barley over two growing seasons (

Table 6. Grain yield of winter barley (average of the two-year experiment and four replications).

Nutrient Variant		Grain Yield (Mg.hm−2)	Relative %
1: Control, without N		4.51 a	100.0
2: DASA split application		7.97 bd	176.7	100
3: ENSIN split application		6.92 c	153.4	86.8	100
4: DASA one-time application		7.50 bc	166.3	94.1	108.4	100
5: ENSIN one-time application		8.35 d	185.1	104.8	120.7	111.3
First experimental year		6.27 a
Second experimental year		7.83 b
LSD variants	α = 0.05	0.69
LSD years	α = 0.05	0.37

The same letters for the yield averages of individual variants indicate statistical insignificance.

) confirms the results observed in individual years, with the highest yield achieved through a one-time application of ENSIN fertilizer (8.35 Mg·hm⁻²). The split application of ENSIN fertilizer resulted in a statistically significantly lower yield compared with its one-time application. The split application of DASA fertilizer produced a statistically non-significantly lower yield (7.97 Mg·hm⁻²) compared with the one-time application of ENSIN fertilizer while also yielding a slightly higher but statistically insignificant yield compared with the one-time application of DASA fertilizer. These findings suggest that nitrification inhibitors in the ENSIN fertilizer did not reduce winter barley grain yield when applied as a one-time dose compared with DASA fertilizer (without inhibitors), whether applied in a one-time or split dose. However, the split application of ENSIN fertilizer resulted in lower grain yield compared with fertilizers that do not contain nitrification inhibitors.

The first growing season had deficit precipitation levels but was exceptionally warmer than the long-term average. In contrast, the second growing season was normal in terms of precipitation and had average temperatures higher than the long-term mean. The second growing season provided more favorable conditions for winter barley grain yield. The average grain yield during this season (7.83 Mg·hm⁻²) was statistically significantly higher by 1.56 Mg·hm⁻² than the average grain yield in the first season (6.27 Mg·hm⁻²).

3.2. Evaluation of the Fertilization Efficiency of Winter Barley

The highest increase in barley grain yield per 1 kg of applied N (NUE) was achieved in both individual years and the multi-year average with a one-time application of ENSIN fertilizer (with NIs) (

Table 7. N use efficiency of winter barley (kg.kg⁻¹) (average of 4 replications).

Treatment	First Year		Second Year		Average
	ΔY	NUE	ΔY	NUE	ΔY	NUE
1: Control, without N	-		-		-
2: DASA split application	2810	20.1	4110	29.4	3460	24.7 ad
3: ENSIN split application	710	5.1	4120	29.4	2410	17.2 b
4: DASA one-time application	2240	16.0	3750	26.8	3000	21.4 c
5: ENSIN one-time application	3410	24.4	4270	30.5	3840	27.4 d

ΔY is an increase in yield compared with the control treatment (kg.ha⁻¹); LSD = 3.22; α = 0,05; NUE is N use efficiency; NUE = ΔY/140 (kg.kg⁻¹), which means an increase in grain yield per 1 kg of applied N compared with the control (unfertilized) treatment; 140 = total rate of N (kg.ha⁻¹). Different letters (a, b, c, d) indicate statistical differences between treatments. The same letter shared between treatments means those treatments are not significantly different from each other.

). The split application of DASA fertilizer resulted in an average yield increase of 24.7 kg.kg⁻¹ per 1 kg of applied N over the years. The lowest yield increases were observed in the split applications of ENSIN (17.2 kg.kg⁻¹) and DASA (21.4 kg.kg⁻¹). These results indicate that the N dose in the one-time application of ENSIN fertilizer contributed the most to increasing barley grain yield.

3.3. Assessment of Ammonium and Nitrate N Content in the Soil Under Winter Barley

Table 8. Ammonium and nitrate N content in the soil under winter barley (average of four sampling events and four replications).

Treatment	Depth of Soil Profile	N Content (mg·kg−1 Soil)
		First Experimental Year			Second Experimental Year
		N-NH4+	N-NO3−	Nin	N-NH4+	N-NO3−	Nin
1: Control, without N	0.0–0.3	4.4	3.4	7.8	2.1	3.7	5.8
	0.3–0.6	3.4	3.4	6.8	1.6	3.7	5.3
2: DASA split application	0.0–0.3	17.4	7.6	25.0	8.7	6.1	14.8
	0.3–0.6	10.1	5.2	15.3	4.5	5.6	10.1
3: ENSIN split application	0.0–0.3	32.1	9.5	41.6	35.0	7.7	42.7
	0.3–0.6	20.9	10.6	31.5	11.0	5.7	16.7
4: DASA one-time application	0.0–0.3	19.3	6.2	25.5	11.5	7.8	19.3
	0.3–0.6	10.5	5.0	15.5	6.9	7.8	14.7
5: ENSIN one-time application	0.0–0.3	22.7	7.7	30.4	22.4	7.8	30.2
	0.3–0.6	16.7	5.1	21.8	12.4	8.9	21.3

shows that in the first year, the highest ammonium N (N-NH₄⁺) content (32.1 mg·kg⁻¹ soil) was observed in the split application of ENSIN treatment. Similarly, a high concentration (22.7 mg·kg⁻¹) was recorded with the single application of ENSIN, both exceeding the values found with the DASA fertilizer at a soil depth of up to 0.3 m. A similar trend was observed at a depth of 0.3 to 0.6 m.

The proportion of ammonium N within the total inorganic N content was also higher in the ENSIN-treated plots (with NIs) compared with DASA (without NIs), particularly when ENSIN was applied in a split manner at a depth of up to 0.3 m (

Table 9. Proportion of ammonium N in total inorganic N in the soil (%).

Treatment	Depth (m)	First Year	Second Year
1: Control, without N	0.0–0.3	56.4	36.2
	0.3–0.6	50.0	30.2
2: DASA split application	0.0–0.3	69.6	58.8
	0.3–0.6	66.0	44.6
3: ENSIN split application	0.0–0.3	77.2	82.0
	0.3–0.6	66.3	65.9
4: DASA one-time application	0.0–0.3	75.7	59.6
	0.3–0.6	67.7	46.9
5: ENSIN one-time application	0.0–0.3	74.7	74.2
	0.3–0.6	76.6	58.2

). The proportion of ammonium N in the one-time application of DASA at a depth of up to 0.3 m was nearly identical to that observed for ENSIN, regardless of application method.

In the second year, the highest ammonium N concentrations were again found in the same treatments and at the same soil depths as in the first year, with values of 35 mg·kg⁻¹ in the split ENSIN application and 22.4 mg·kg⁻¹ in its one-time application. These concentrations were higher than those in both DASA application methods (Table 8). At a depth of up to 0.3 m, the proportion of ammonium N relative to total inorganic N content in the soil reached 82% in the split ENSIN application and 74.2% in its one-time application. These values were significantly higher than those recorded in the DASA treatments (without NIs) at both soil depths (Table 9).

These findings indicate that, in both years and at both soil depths, the highest ammonium N concentrations were achieved with ENSIN fertilizer which contains NIs. Additionally, both the absolute values and the proportion of ammonium N relative to total inorganic N were higher in the split ENSIN application compared with DASA fertilizer without NIs. This suggests that the inhibitors in ENSIN slowed the conversion of N-NH₄⁺ into N-NO₃⁻. Furthermore, in both years and at both soil depths, nitrate N (N-NO₃⁻) concentrations were lower than ammonium N levels (except in the control treatment).

In the two-year average, the results are completely analogous, and the inhibitory effect of the inhibitors in the ENSIN fertilizer was evident in both depths, as demonstrated by the increased ammonium N content and its higher proportion of total inorganic N compared with the DASA fertilizer (without NIs). Slightly better results were achieved with the split application of ENSIN compared with its one-time application (

Table 10. Ammonium and nitrate N content in the soil under winter barley (average of 2 years, four samplings, and four repetitions; n = 32).

Treatment	Depth of Soil Profile (m)	N Content (mg·kg−1 Soil)			N-NH4+/Nmin × 100 (%)
		N-NH4+	N-NO3−	Nmin
1: Control, without N	0.0–0.3	3.25	3.55	6.80	47.8
	0.3–0.6	2.50	3.55	6.05	41.3
2: DASA split application	0.0–0.3	13.05	6.85	19.90	65.6
	0.3–0.6	7.30	5.40	12.70	57.5
3: ENSIN split application	0.0–0.3	33.55	8.60	42.15	79.7
	0.3–0.6	15.95	8.15	24.10	66.2
4: DASA one-time application	0.0–0.3	15.40	7.00	22.40	68.8
	0.3–0,6	8.70	6.40	15.10	57.6
5: ENSIN one-time application	0.0–0.3	22.55	7.75	30.30	74.4
	0.3–0.6	14.55	7.00	21.55	67.5

).

The same trend can be observed for the average values across years, depths, samplings, and repetitions (n = 64), where the fertilizer with NIs (ENSIN) showed approximately twice as high ammonium N concentrations compared with the same fertilizer without an inhibitor (DASA), both with split (greater inhibitor effect) and one-time (smaller inhibitor effect) applications. This is also confirmed by the proportion of ammonium N from the total inorganic N—74.7% for the split application of ENSIN and 71.6% for its one-time application—which is 12 absolute percentage points and 7 absolute percentage points higher, respectively, than the corresponding fertilizers without the inhibitor (

Table 11. Ammonium and nitrate N content in the soil under winter barley (average of 2 years, two depths, four samplings, and four repetitions, n = 64).

Treatment	N Content (mg·kg−1 Soil)			N-NH4+/Nmin × 100 (%)
	N-NH4+	N-NO3−	Nmin
1: Control, without N	2.875 a	3.550	6.425	44.7 a
2: DASA split application	10.175 b	6.125	16.300	62.4 b
3: ENSIN split application	24.750 c	8.375	33.125	74.7 c
4: DASA one-time application	12.050 b	6.700	18.750	64.3 bd
5: ENSIN one-time application	18.550 d	7.375	25.925	71.6 cd

LSD = 5.18; α = 0.05. LSD = 10.2; α = 0.05. Each letter (a–d) indicates statistical differences between treatments, where different letters denote significantly different values according to statistical analysis (e.g., ANOVA with post-hoc test).

).

The container experiment with N leaching indicates that the highest nitrate N content in the leachate was in the variant with a one-time application of the DASA fertilizer (183 mg.L⁻¹). A statistically significant reduction in the amount of leached N-NO₃⁻ was observed with the split application of this fertilizer (

Table 12. Ammonium and nitrate N content (mg.L⁻¹) in the lysimetric solution (average of years, sampling, and repetitions; n = 32).

Treatment	N-NH4+	N-NO3−
1: Control, without N	0.0	4.0 a
2: DASA	0.0	183 b
3: DASA 1/2 + 1/2	2.2	87 c
4: ENSIN	0.2	73 c
5: ENSIN 1/2 + 1/2	1.8	40 d

Each letter (a–d) indicates statistical differences between treatments, where different letters denote significantly different values according to statistical analysis (e.g., ANOVA with post-hoc test).

). The positive effect of NIs in the ENSIN fertilizer was clearly evident. In these variants, the least amount of N-NO₃⁻ was leached, with a more pronounced effect achieved with the split application of ENSIN fertilizer (40 mg.L⁻¹) compared with its one-time application (73 mg.L⁻¹).

4. Discussion

The use of fertilizers containing nitrification inhibitors (NIs) has significant environmental implications, as fertilization and irrigation can increase soil CO₂ and N₂O emissions across various soil types [16]. Li et al. (2016) developed a model to compare N₂O emissions under 20 different climate change scenarios, evaluating the effects of NIs on nitrous oxide (N₂O) emissions [17]. In our barley experiment, the ENSIN fertilizer effectively inhibited the oxidation of ammonium (N-NH₄⁺) into nitrate (N-NO₃⁻), resulting in greater ammonium nitrogen retention within the soil profile compared with treatments with conventional fertilizers.

These findings are consistent with previous studies [18], which highlight the role of NIs in mitigating nitrogen loss through leaching and gaseous emissions while maintaining soil nitrogen availability. Although Hu et al. (2013) reported no significant yield benefits across various crops [18], our results demonstrated a substantial yield increase in winter barley, particularly with a one-time ENSIN application (with NIs). The highest N use efficiency (27.4 kg grain per kg N) was achieved through this approach, suggesting that the agronomic effectiveness of NIs depends on factors, such as crop type, fertilizer composition, and environmental conditions.

Nitrate leaching from the soil was also reduced, as demonstrated by the lysimeter experiment, where nitrate leaching in the ENSIN treatments was 42% lower than in the treatments using fertilizers without nitrification inhibitors. Similar findings were reported by Boy-Roura et al. (2016), who observed a decrease in nitrate leaching using nitrification inhibitors, such as dicyandiamide (DCD) [19]. Further studies conducted in subtropical pastureland have confirmed that while NIs significantly reduce nitrate (NO₃⁻) runoff and leaching, they do not significantly impact pasture biomass yields [20]. Additionally, NIs reduce the availability of nitrate (N-NO₃⁻) as a substrate for conversion into nitrogen oxide (NOx) under anerobic or partially anerobic soil conditions [21]. This process decreases nitrogen losses from fertilizers in gaseous form and mitigates greenhouse gas emissions that contribute to atmospheric warming.

Barley grain yield was not negatively affected by the ENSIN fertilizer containing NIs. In some crops, comparable yield levels to those achieved with the control fertilizer (without NIs, specifically, 3,4-dimethylpyrazole phosphate) were obtained with one less N application or a reduced N application rate [22]. Over the two-year study, the highest average grain yield (8.35 Mg.hm⁻²) was observed with a one-time ENSIN application (with NIs), followed by the split DASA application (7.97 Mg.hm⁻²). A similar positive effect was observed in winter wheat, where a one-time application of urea with nitrification inhibitors resulted in increased grain yield, while protein content and Zeleny sedimentation values were significantly higher compared with the split N application [23].

The demand for ammonium (NH₄⁺) and nitrate (NO₃⁻) in cereal crops varies across different growth stages [24], with each form of nitrogen playing a specific role in plant development. While NIs can enhance nitrogen retention by limiting the conversion of NH₄⁺ into NO₃⁻, excessive inhibition may interfere with the plant’s ability to utilize both nitrogen forms at critical growth stages [25]. If ammonium accumulation becomes too high or nitrate availability is too low, it could potentially affect crop development and yield [26]. It is important to carefully balance the use of NIs to avoid any adverse effects on plant nutrition.

The results of this study with the Barcelona winter barley variety demonstrate a balanced response to nitrogen nutrition, showing no extreme sensitivity to either ammonium (NH₄⁺) or nitrate (NO₃⁻) within commonly applied nitrogen levels. According to Slamka et al. (2008), this variety maintains stable grain quality and yields up to 70 kg N ha⁻¹, with acceptable malt parameters such as crude protein content (10.8–11.5%), fermentable extract (78.5–79.1%), and the Kolbach index (38.6–40.6%) [27].

While direct comparative studies of Barcelona barley’s specific sensitivity to NH₄⁺ versus NO₃⁻ are limited, its ability to sustain grain quality under moderate N application suggests a balanced uptake and assimilation of both nitrogen forms. Further research could provide a more detailed understanding of its nitrogen sensitivity profile.

Based on these findings, we recommend ENSIN fertilizer (with NIs) for a one-time application during regenerative fertilization, particularly in years with evenly distributed or slightly deficient precipitation, emphasizing the utilization of winter and spring moisture. Our results suggest that ENSIN fertilizer should be applied in a one-time dose rather than split into multiple applications, as this approach reduces application costs compared with split applications of fertilizers without NIs (e.g., DASA 26/13). The split N applications did not significantly impact the agronomic characteristics of arugula (rocket plant) or maize [28,29]. However, the effect of split N applications on yield was more pronounced in winter wheat than in spring wheat. Additionally, split N applications mitigated the negative relationship between yield and grain protein content [30]. However, for the winter barley, a one-time ENSIN fertilizer application (with NIs) during regenerative fertilization potentially enhances both the agronomic and economic efficiency of fertilization, ultimately increasing profitability and providing added value, offsetting the higher cost of the fertilizer.

The N dose applied as a one-time application of ENSIN fertilizer (with NIs) may be used under favorable weather conditions. In contrast, a split application is recommended under less favorable conditions with uneven precipitation distribution during the growing season [31]. Kabir et al. (2021) emphasized that split N applications provide environmental benefits across all weather conditions and offer agronomic advantages under specific weather scenarios [31]. This is particularly important for heavy soil and situations where reduced soil conditions prevail (e.g., soil compaction from heavy machinery; conservation tillage technologies like no-till). In these conditions, ENSIN fertilizer works synergistically with the anerobic environment to prevent N-NO₃⁻ formation in the soil, enhancing its eco-environmental value without negatively impacting production parameters.

5. Conclusions

The one-time ENSIN fertilizer (with NIs) application enhanced winter barley yield (8.35 Mg.hm⁻²) and N use efficiency (27.4 kg grain per kg N) while significantly reducing nitrate leaching (42%) and potential gaseous N losses. By stabilizing soil ammonium N, ENSIN supports sustainable nutrient management without compromising productivity.

The one-time application of ENSIN under favorable weather conditions demonstrated the highest N use efficiency and grain yield. However, further long-term research is required to evaluate the effectiveness of split applications under varying precipitation patterns and to confirm the sustainability of this approach.