Soil Enzymatic Response to Nicosulfuron: A Preliminary Study in a Chernozem Typical to the Banat Plain, Western Romania

Abstract

Nicosulfuron, despite being a post-emergence herbicide commonly used in corn crops to combat weeds, there is still little information on nicosulfuron toxicity for soil microbiota. Little information exists on the impact of nicosulfuron on the enzymatic activities of soil dehydrogenases (Deh), urease (Ure), catalase (Cat), and alkaline phosphatase (Alp). We used a multiple dose- and time point (7, 14, 21, and 28 days) study design to determine the effect of nicosulfuron on these parameters during the first 28 days post-application. The soil pH, electrical conductivity (EC), organic matter content (OM), water content, ammonium-nitrogen (NH₄-N), nitrate-nitrogen (NO₃-N), and available phosphate were also monitored. Ure was the most responsive enzyme to nicosulfuron. This herbicide exerted a transitory dose- and time-dependent inhibitory effect on Deh activity; maximum inhibition occurred at 14 days at doses from the normal recommended dose onward. For Ure, the maximal inhibitory effect started at 7 days of exposure to half the normal recommended dose and continued for another 14 days. The effect on Cat occurred later, whereas Alp activity was affected by nicosulfuron between 7 and 21 days but only for triple the normal recommended dose. OM showed the most consistent relationships with these parameters, being strongly positively correlated with Deh, Cat, and Alp activities. These results indicate that nicosulfuron may alter the soil metabolic activity, thus affecting its fertility.

1. Introduction

Long-term, intensive use of agrochemicals (e.g., herbicides, fungicides, insecticides, nematicides, chemical fertilizers) in agriculture exerts adverse effects on soil biodiversity, agricultural sustainability, and food safety [1,2]. It is estimated that about a third of the Earth’s land surface is affected by some form of soil degradation related to agricultural activities, including 500 million hectares of land impacted by extensive use of different. Importantly, more than 40% of global land area is now used for agriculture, and degradation processes affect over 50% of humanity, contributing to poverty, food insecurity, and environmental instability [2,3]. An important facet of these deleterious repercussions is their adverse impact on the diversity and activity of soil microbial com-munities, thereby perturbing the biochemical processes occurring within this key environmental matrix [3,4,5,6]. These effects can be both direct and indirect, and they may depend on the type of agrochemical, its concentration, application frequency, and other factors [5,6]. Of particular concern is the fact that such changes may reduce (or even totally deplete) the population of certain beneficial microorganisms (e.g., mycorrhizal fungi, nitrogen-fixing bacteria) or drastically reduce their enzymatic activities, thus impacting nutrient cycling and plant health [7].

Nicosulfuron is a high-profile sulfonylurea herbicide that is commonly used for post-emergence control of both annual weeds and perennial weeds in corn (Zea mays ssp.) cultures [8]. Its mechanism of action relies on absorption into the weed leaves and translocation via the xylem/phloem towards the meristematic zone, where it blocks cell division and plant growth by inhibiting the activity of acetolactate synthase [9]. This herbicide has a broad spectrum of activity, as well as high efficiency and selectivity even at relatively low application doses [8]. As a result, it has gained large public approval, being extensively used in large corn-planted areas in Europe, the United States, China, and South America [10]. Extensively used by corn growers since its launch in the early 1990s, the global market of nicosulfuron-based products has increased from $370 million (USD) in 2017 to $425 million (USD) in 2022, with the Asia-Pacific (APAC) region accounting for the largest consumption share (>75%) [11].

As the frequency of nicosulfuron use in intensive agriculture increased, the problems related to the presence and persistence of its residues in the soil gradually became evident [8,12]. It was thus found that these residues can persist on soil surfaces and in groundwater, potentially causing different degrees of phytotoxicity to later crops [13,14]. Studies have demonstrated that this herbicide dissipates relatively quickly in the soil; half-lives ranged between 13.64 days and 20 days, depending on the soil type and environmental conditions [15,16]. However, its mobility into subsurface layers supports that residues could persist longer in deeper soil layers. Thus, the dissipation dynamics in different regions, such as Beijing and Changchun, showed a 90% reduction in soil residues within 21 days, further supporting the herbicide’s relatively short persistence [3]. Despite this, the herbicide’s presence in water bodies across various countries, including the United States, Canada, Germany, France, and Brazil, with detection frequencies of 15% and concentrations ranging from 0.016 to 1.5 micrograms per liter (μg L⁻¹), indicates potential environmental mobility and persistence [17]. Ecotoxicological studies also showed that this herbicide can exert toxic effects on aquatic plants and soil microbial populations [9]. In addition, sulfonylurea herbicides, including nicosulfuron, were shown to affect human health, e.g., by inducing hypoglycemia and increasing the risk of cardiovascular diseases [9]. Therefore, this herbicide can pose serious, unignorable risks to the environmental health.

As key players in terrestrial ecosystems, soil microorganisms are involved in the transformation of organic matter, biogeochemical cycling of nutrients, and degradation of xenobiotics [6,18,19,20,21]. Their enzymatic activity is responsive to natural or anthropogenic changes [4,22,23,24,25] and central to biological processes occurring at soil level, ensuring the decomposition and mineralization of organic matter [26,27]. Measuring the enzymatic activity of soil microbiota can hence be considered an important indicator of soil quality and health [19,20,21,22]. In this context, several studies have investigated the effect of nicosulfuron on soil microbiome enzymatic activity [28,29,30,31]. Other investigations have addressed the soil degradation pathways of nicosulfuron-based products [32,33,34,35]. However, the impact of nicosulfuron and other sulfonylureas on key enzymatic activities of soil microbiota, e.g., dehydrogenases, urease, alkaline phosphatase, and catalase, is still not clear, although these processes are pivotal for organic matter cycling in the soil [18,19,35]. In particular, the temporal dynamics of nicosulfuron effects on these metabolic processes remain to be clarified. The relationships between the soil physicochemical parameters and enzymatic activities are also still poorly understood. Furthermore, no such data are available for the Banat Plain; located in western Romania, it is one of the most important regions for cereal cultivation in both Romania and Europe. This area, part of the larger Pannonian Plain, is characterized by its fertile chernozem soils, favorable climate, and well-developed agricultural infrastructure, making it highly suitable for intensive cereal production [3,6].

In this context, the primary objective of this study was to determine the specific doses and exposure durations at which nicosulfuron significantly impacts soil enzymatic activities in a chernozem typical to the Banat Plain, Western Romania. We hypothesized that nicosulfuron may cause dose- and time-dependent alterations in the activities of key soil enzymes—dehydrogenases (Deh), urease (Ure), alkaline phosphatase (Alp), and catalase (Cat). Selected soil physicochemical parameters, which are the pH, electrical conductivity (EC), organic matter content (OM), water content, ammonium-nitrogen (NH₄-N), nitrate-nitrogen (NO₃-N), and available phosphate, were also monitored and correlated with the aforementioned enzymatic activities. Such associations are relevant for precisely assessing and managing the environmental impact of nicosulfuron, as these interactions can influence the herbicide’s behavior, persistence, and effects on soil health [9,10,12]. The results of this study are important since they provide us with a comprehensive and integrative assessment of nicosulfuron’s impact on multiple soil enzymatic activities across various doses and time points in a chernozem typical to a high-profile area for the European agriculture. By correlating these enzymatic activities with soil physicochemical properties, our outcomes also provide a deeper understanding of how nicosulfuron interacts with soil ecosystems. Moreover, the present investigation was conducted at field-realistic concentrations, thus ensuring practical relevance, and it addresses significant gaps in the existing literature by focusing on the globally important herbicide, nicosulfuron. The findings of the present study are hence critical for advancing knowledge on herbicide effects on soil health, with implications for environmental risk assessment and sustainable agricultural practices.

2. Materials and Methods

2.1. Herbicide

The herbicide used in this study was the sulfonyl urea herbicide nicosulfuron, i.e., 2-[(4,6-dimethoxypyrimidin-2-yl)carbamoylsulfamoyl]-N,N-dimethylpyridine-3-carboxamide (C₁₅H₁₈N₆O₆S) [36]. Its 2-dimensional (2D) structural formula is given in Figure 1. Experiments were conducted using the product sold on the local market as “MILAGRO 40OD” (Syngenta UK Limited, Glissando, Timisoara, Romania), a suspension containing 40 grams per liter (g L⁻¹) nicosulfuron. MILAGRO 40OD exerts strong action against monocotyledonous weeds that are common and difficult to fight pests of corn crops [37].

2.2. Soil Sampling and Treatment

Chernozem soil samples were obtained from an unpolluted experimental field (no known use of pesticides or chemical fertilizers) located in the village of Ghiroda (Timis county; latitude 45°55′13.44 N, longitude 21°14′16.16 E). Chernozem, known for its high fertility and rich organic matter content, is prevalent in the Banat Plain, Western Romania, the region where Ghiroda is located. This region’s climate and topography contribute to the development of deep, humus-rich soils, which are ideal for agriculture. The chernozem in Ghiroda shares these features, making it an excellent reference point for studying the broader chernozem soil type in this part of the country. Moreover, the area has been extensively studied, providing a wealth of data on soil properties, agricultural potential, and environmental impact, which is applicable to the entire region [3,6].

These samples were collected from the top soil layer (0–25 cm) at five different locations, in quantities varying between 25 and 30 kilograms (kg). All these samples were mixed in order to obtain a representative sample of soil, which was next ground and sieved to 2 mm. After that, triplicate jars of 5 kg each were randomly sampled for each. The five nominal nicosulfuron treatments were 0, 0.2, 0.4, 0.8, and 1.2 micrograms per gram of soil (μg g⁻¹ soil)—abbreviated as M, V1, V2, V3, and V4. We chose to include the higher doses (double and triple the recommended dose) to simulate potential real-world scenarios of over-application and to better understand the threshold at which nicosulfuron exerts significant toxic effects on soil enzymatic activities. This approach allowed us to construct a comprehensive dose–response relationship and assess the potential environmental risks associated with excessive herbicide use. The correspondence between these treatments and the doses recommended by the manufacturer is given in

Table 1. Nicosulfuron (as MILAGRO 40OD) treatments used.

Treatment Abbreviation	Dose	Explanation
M	0	0 μg nicosulfuron g−1 soil
V1	D/2	0.2 μg nicosulfuron g−1 soil
V2	D	0.4 μg nicosulfuron g−1 soil
V3	2xD	0.8 μg nicosulfuron g−1 soil
V4	3xD	1.2 μg nicosulfuron g−1 soil

M, reference group; D/2, half the normal recommended dose; D, the normal recommended dose (based on the product label); 2xD, double the normal recommended dose; 3xD, triple the normal recommended dose.

.

For each replicate jar of each treatment group, the experiments were conducted in polyethylene boxes (1 m × 1 m × 0.2 m). The concentration of nicosulfuron to be applied on soil samples was calculated based on the normal dose of herbicide recommended by the manufacturer (1 L per hectare), the concentration of nicosulfuron in the applied product (40 g L⁻¹), and the amount of soil on which the herbicide was applied. After being transferred into the polyethylene boxes, the soil (5 kg per box) was sprayed with nicosulfuron (according to the aforementioned table) and maintained under laboratory conditions (temperature: 22.3–23.1 °C) for 28 days. Moisture was adjusted every seven days, with 50 mL of water being sprayed onto the soil samples for each adjustment in order to restore moisture levels to a constant value, thus ensuring optimal conditions for the experiment.

Soil sampling was performed at 7, 14, 21, and 28 days. The surface layer of soil was carefully removed, and samples were collected from the top 5 centimeters (cm) of the soil profile. Triplicate samples were randomly obtained for each triplicate jar of each treatment using a soil auger. The triplicate soil samples corresponding to each jar were then mixed to provide us with a homogeneous sample, which was further used for enzymatic analyses. After each sampling, the humidity was adjusted and the soil was re-homogenized. The experiment was carried out in laboratory conditions in 2022.

2.3. Biochemical Analyses

The enzymatic activities chosen for the assay were dehydrogenases (EC 1.1.1.1), urease (EC 3.5.1.5), catalase (EC 1.11.1.6), and alkaline phosphatase (EC 3.1.3.1). These numbers refer to the Enzyme Commission (EC) numbers, which are part of the standard nomenclature for classifying enzymes based on the reactions they catalyze. The activities of Deh, Ure, and Alp were determined with a T90 UV/Vis spectrophotometer (PG Instruments, England), whereas the titration method was applied to determine the catalase activity. Deh activity was quantified using 2,3,5-triphenyltetrazolium chloride (TTC) as substrate, monitoring the reaction product (triphenylformazane, TPF) at 485 nm. This enzymatic marker was expressed as mg TPF g⁻¹ soil at 48 h [6,38]. Ure activity estimates the rate at which urea decomposes in ammonia (NH₃) and carbon dioxide (CO₂). The absorbance was measured at 445 nm, with Ure activity being expressed as mg NH₃-N g⁻¹ h⁻¹ soil at 24 h [39,40].

Alp activity was assessed via hydrolytic separation of phenyl phosphate by phosphomonoesterases; the final products were disodic phosphate and phenol. The latter compound reacts with the Gibbs reactive (2,6-dibromchinon-chloramide) resulting in a blue precipitate. Alp activity was determined at 597 nanometers (nm) and was expressed as mg phenol g⁻¹ soil at 48 h [15,41]. Cat activity was determined using the permanganometric method, being expressed as mg H₂O₂ undecomposed g⁻¹ soil [42,43].

The analytical protocol for each soil sample was conducted in triplicate under controlled laboratory conditions, with all analyses performed by the same researcher on the same day. Enzymatic activity was measured at four time points—7, 14, 21, and 28 days—ensuring consistency and accuracy across the different stages of the experiment.

2.4. Physicochemical Properties of the Soil

The following physicochemical parameters of soil were monitored during this study: temperature, pH, electrical conductivity (EC), organic matter content (OM), water content, ammonium nitrogen content (NH₄-N), nitrate nitrogen content (NH₄-NO₃), and available phosphate content. Temperature, pH, and EC were measured by using a handheld multimeter Multi 340i/SET WTW (Weilheim, Germany), fitted with a specific sensor for each parameter. Prior to the experiment, we measured the soil physicochemical parameters using a composite sample from the entire batch of soil. This approach was chosen to ensure a representative baseline for the entire experimental setup, as the soil was thoroughly mixed to create a uniform sample before being divided into the portions allocated to each treatment group (3 portions × 5 kg soil per treatment). By measuring the parameters from this composite sample, we ensured consistency in the initial conditions across all treatment groups, minimizing variability and allowing us to accurately assess the effects of nicosulfuron on soil enzymatic activities under controlled conditions.

Soil samples were extracted with double-distilled water (1:1 water to soil suspension) to determine the soil pH. A suspension of 5:1 double-distilled water to soil was used for assessing soil EC. In order to determine the soil–water content, thermogravimetric analysis of samples was performed using the thermo-gravimetric method [44].

OM levels were determined by the calcination method, with weight loss by combustion being assessed by direct weighing. The soil samples were calcinated in a muffle furnace; the temperature increased gradually up to 550 °C and was then maintained constant for 4 h. OM corresponding to each soil sample was calculated taking into account the weight of the soil sample at room temperature and the weight of the ash sample [45].

The determination of NH₄-N content was performed by measuring the absorbance using a spectrophotometer T90 UV/Vis (PG Instruments, Lutterworth, UK) at a wavelength of 630 nm [43]. The same method was employed to assess the available phosphate content and NO₃-N content using, respectively, a wavelength of 882 nm [46] and 543 nm [47].

All the methods used for the enzymatic analyses and the physicochemical parameters are presented in detail in other studies carried out by the research team [37,38].

2.5. In Silico Characterization of Nicosulfuron Stability and Toxicity

Nowadays, nicosulfuron has found extensive application in corn fields owing to its high efficacy and minimal toxicity, leading to significant accumulation of residues in both soil and water. These determine undesirable effects not only on the environment but also on human health. Understanding the nicosulfuron structure in terms of its reactivity and toxicity to humans is critical: (i) firstly, it allows researchers and regulators to predict and assess potential risks associated with exposure to the pesticide; (ii) secondly, knowledge of the structure helps in the development of safer alternatives with reduced toxicity. To predict the stability of the nicosulfuron molecule, the Jaguar tool [48] of the Schrödinger suite (Schrödinger Release 2022-4, Schrödinger; LLC: New York, NY, USA, 2022) was engaged. The density functional theory (DFT) using B3LYP/6-31G (d,p) basic set was used for geometry optimization. The toxicity parameters for nicosulfuron were computed using pKCSM [49], STopTox [50], and BeeToxAI [51] online tools.

2.6. Statistical Analysis

For each enzymatic activity investigated (Deh activity, Ure activity, Cat activity, and Alp activity), the log-transformed data sets (decimal logarithmation) corresponding to each combination of the groups of the two independent variables (nicosulfuron dose, exposure time) were tested for normality using Kolmogorov–Smirnov tests, and for homoskedasticity (homogeneity of variance) via Bartlett’s tests. By transforming the data through logarithms, we aimed to reduce the impact of outliers and minimize the impact of potential skewed and heteroskedastic data. For enzymatic activities meeting both these conditions, two-way ANOVAs were conducted, with dose and time being used as factors (independent variables) [24]. Post hoc testing was run using the Neuman–Keuls methodology for each significant main effect. All comparisons for dose and time were conducted versus controls, when dose was considered the main effect; and versus the earliest time point, when time was regarded as the main effect. In the case of significant interactions between nicosulfuron dose and exposure duration, these comparisons were run per time point using the corresponding controls as reference groups. Finally, Pearson’s correlations were applied to pooled means of enzymatic activities and the measured values for pH, temperature, moisture, organic matter content, nitrogen nitrate level, nitrogen ammonia level, and available phosphorus [52]. All statistical analyses were performed using the Statistica version 7 software (StatSoft Inc., Tulsa, OK, USA), with statistical significance being defined as p < 0.05 [53,54].

3. Results

3.1. Assessment of the Enzymatic Activities

Average values for the measured enzymatic activities at 7, 14, 21, and 28 days of incubation are given in

Table 2. Enzymatic activities in soil samples treated with various doses of nicosulfuron.

Time	Dose	Deh Activity (mg TPF g−1 Soil)		Ure Activity (mg NH3-N g−1 h−1 Soil)		Cat Activity (mg H2O2 Undecomposed g−1 Soil)		Alp Activity (mg Phenol g−1 Soil)
7 days	M	3.82 ±	0.26	1.26 ±	0.09	6.23 ±	0.30	2.63 ±	0.04
7 days	V1	3.35 ±	0.27	0.68 ±	0.11 ***	6.15 ±	0.52	2.33 ±	0.41
7 days	V2	2.47 ±	0.36 **	0.65 ±	0.17 ***	5.74 ±	0.08	2.44 ±	0.39
7 days	V3	2.40 ±	0.29 ***	0.62 ±	0.16 ***	4.45 ±	0.15	2.06 ±	0.35
7 days	V4	1.24 ±	0.08 ***	0.59 ±	0.18 ***	4.41 ±	0.40	1.82 ±	0.25 **
14 days	M	3.17 ±	0.23	1.18 ±	0.12	5.36 ±	0.47	2.72 ±	0.13
14 days	V1	3.22 ±	0.23	1.07 ±	0.04	5.78 ±	0.39	2.54 ±	0.29
14 days	V2	1.61 ±	0.22 ***	0.81 ±	0.06 **	4.91 ±	0.24	2.10 ±	0.20
14 days	V3	1.04 ±	0.17 ***	0.87 ±	0.07 *	4.67 ±	0.14	2.16 ±	0.04
14 days	V4	0.31 ±	0.07 ***	0.78 ±	0.10 **	3.93 ±	0.06	1.96 ±	0.05 *
21 days	M	2.44 ±	0.13	2.33 ±	0.11	3.52 ±	0.06	2.48 ±	0.03
21 days	V1	2.60 ±	0.44	1.39 ±	0.11 ***	3.22 ±	0.05	2.30 ±	0.06
21 days	V2	2.44 ±	0.23	1.03 ±	0.10 ***	3.08 ±	0.03	2.26 ±	0.12
21 days	V3	2.52 ±	0.09	0.85 ±	0.03 ***	2.93 ±	0.05	1.52 ±	0.33 ***
21 days	V4	2.30 ±	0.09	0.79 ±	0.03 ***	2.91 ±	0.01	1.33 ±	0.23 ***
28 days	M	2.62 ±	0.07	2.14 ±	0.01	2.90 ±	0.06	2.14 ±	0.01
28 days	V1	2.70 ±	0.05	1.94 ±	0.03	2.71 ±	0.05	2.11 ±	0.05
28 days	V2	2.23 ±	0.08	1.69 ±	0.47	2.32 ±	0.02	1.96 ±	0.02
28 days	V3	2.18 ±	0.08	1.88 ±	0.01	2.29 ±	0.01	1.91 ±	0.01
28 days	V4	2.13 ±	0.01	0.98 ±	0.00 ***	2.17 ±	0.04	1.82 ±	0.02

Deh, dehydrogenases; Ure, urease; Cat, catalase; Alp, alkaline phosphatase; M, reference group (0 μg nicosulfuron g⁻¹ soil); V1, half the normal recommended dose (D/2)-0.2 μg nicosulfuron g⁻¹ soil; V2, the normal recommended dose (D)-0.4 μg nicosulfuron g⁻¹ soil; V3, double the normal recommended dose (2xD)-0.8 μg nicosulfuron g⁻¹ soil; V4, triple the normal recommended dose (3xD)-1.2 μg nicosulfuron g⁻¹ soil. Data (absolute values) are expressed as mean with one standard deviation. For significant time x dose interactions, comparisons were conducted at each time point using the corresponding controls (M treatment) as reference groups. Marked values (*) indicate significant differences as compared to the reference group (Neuman–Keuls test, *** p < 0.001, ** p < 0.01, * p < 0.05).

. The measured levels ranged: (i) for Deh activity, between 0.30 mg TPF g⁻¹ soil for the V4 variant at 14 days and 3.81 mg TPF g⁻¹ soil for control (M) at 7 days; (ii) for Ure activity, between 0.59 mg NH₃-N g⁻¹ h⁻¹ soil for the V4 variant at 7 days and 2.32 mg NH₃-N g⁻¹ h⁻¹ soil for control at 21 days; (iii) for Cat activity, between 2.16 mg H₂O₂ undecomposed g⁻¹ soil for the V4 variant at 28 days and 6.23 mg H₂O₂ undecomposed g⁻¹ soil for control at 7 days; (iv) for Alp activity, between 1.32 mg phenol g⁻¹ soil for the V4 variant at 21 days and 2.571 mg phenol g⁻¹ soil for control at 14 days. Data sets for these variables were normally distributed (p ≥ 0.123). Homogeneity analysis revealed that variance was similar between groups irrespective of enzymatic activity analyzed (p ≥ 0.064). The assumptions underlying the application of the two-way ANOVA were therefore fulfilled for both factors (nicosulfuron dose, exposure time).

Figure 2 and Figure 3 reveal the changes in Deh, Ure, Cat, and Alp activities depending on nicosulfuron dose and exposure time, respectively.

Nicosulfuron dose exerted a significant effect on Deh activity (F(4, 59) = 170.49, p < 0.001, η² = 0.393). Post hoc analysis using the Neuman–Keuls procedure revealed a significant decrease in the measured values. This decrease was dose-dependent (Figure 2a), starting from the second lowest dose onward (the recommended dose). The effect of exposure duration on Deh activity was not only significant (F(3, 59) = 115.32, p < 0.001, η² = 0.200) (Figure 3a), but its impact was also two-fold lower versus that of nicosulfuron dose. Post hoc analysis revealed a significant decrease in Deh values at 14 days (Figure 3a). However, the measured values increased again at 21 days at levels below those seen at 7 days and remained at the same levels at 28 days (Figure 3a). The meaning of this effect was qualified by a significant outcome of the dose-time interaction (F(12, 59) = 55.05, p < 0.001, η² = 0.381). This indicates that the main effect of exposure time depends on the level of nicosulfuron applied. Taken together with data from Table 2, these results indicate a transitory dose- and time-dependent inhibitory effect of nicosulfuron on Deh activity, with maximum inhibition being observed at 14 days post-application for doses from the normal recommended dose onward.

We also identified a significant main effect of nicosulfuron dose on Ure activity (F(4, 59) = 63.60, p < 0.001, η² = 0.342). Application of post hoc comparisons for this factor revealed a significant dose-dependent decrease in Ure activity (Figure 2b). There was also a significant main effect of exposure time on levels of on Ure activity (F(3, 59) = 121.87, p < 0.001, η² = 0.497), with the measured values increasing in a time-dependent manner (Figure 3b). The relative impact of this variable was by 132% higher than that seen for the nicosulfuron dose. The interaction between dose and time was statistically significant (F(12, 59) = 6.21, p < 0.001, η² = 0.098). The measured levels for Ure activity at 7 days (Table 2) decreased with herbicide dose, being significantly lower versus controls for all treatment groups. The same tendency was detected at 14 days and 21 days (Table 2). In contrast, at 28 days only the values measured for the highest dose treatment (V4) showed significant differences compared to control (M). These findings support that nicosulfuron has a stronger inhibitory effect on Ure activity compared to Deh activity. However, this effect, which seemed again to be temporary, occurred starting at 7 days of exposure to half the normal recommended dose and continued for all treatments for two weeks, being observed at the last time point only for triple the normal recommended dose.

However, herbicide dose had no effect on Cat activity (F(4, 59) = 2.05, p = 0.384), despite an evident decrease observed at 21 days (Figure 2c). In contrast, exposure duration exerted a significant main effect on this enzymatic marker (F(3, 59) = 15.42, p < 0.001,η² = 0.428). Post hoc testing disclosed significantly reduced values for Cat activity at 21 days compared to 7 days, but not at 14 days (Figure 3c). The measured levels at 28 days were comparable to those seen at the previous time point (Figure 3b). Overall, these results suggest that nicosulfuron may reduce soil Cat activity, but this effect occurs later compared to Deh activity and Ure activity.

There was also a significant main effect of nicosulfuron dose on Alp activity (F(4, 59) = 25.38, p < 0.001, η² = 0.488). The measured values decreased in a dose-dependent manner from the second lowest dose onwards (the recommended dose) (Figure 2d). Exposure duration exerted a significant effect on this enzymatic parameter (F(3, 59) = 9.96, p < 0.001, η² = 0.139), and its relative impact on Alp activity was three-fold lower relative to the herbicide dose. The measured values at 14 days were similar to those observed at 7 days; they decreased significantly at 21 days but increased at 28 days, although the measured values were still significantly lower than those seen at 7 days (Figure 3d). A significant interaction between nicosulfuron dose and exposure duration was detected (F(12, 59) = 3.14, p = 0.003, η² = 0.180). The measured values at 7 days and 14 days were significantly lower versus controls for the highest dose treatment (Table 2). At 21 days, Alp activities for the V3 and V4 treatment groups showed a similar tendency (Table 2). In contrast, no effect was observed at 28 days. These data show that Alp activity is less and later impacted by nicosulfuron compared to Deh activity and Ure activity; and this effect is transitory, being most evident in the 7–21 day period for a dose of triple the normal recommended dose.

The values obtained for the four enzymatic activities at the level of the soil samples from the experimental lots were compared with those obtained in the control soil sample. Correlation coefficients depending on the time variable are shown in Supplementary Table S2, the correlation coefficients depending on the dose variable are shown in Supplementary Table S3, and the correlation coefficients according to the two variables, time and dose, are presented in the additional Supplementary Tables S4–S6.

3.2. Correlations between the Enzymatic Activities and Physicochemical Properties of the Soil

The soil physicochemical parameters prior to the experiment were: pH: 7.40; temperature: 22.5 °C; soil conductivity (EC): 286.52 µS; water content: 8.87 mg g⁻¹ soil; organic matter content (OM): 21.54 mg g⁻¹ soil; nitrogen nitrate content (N-NO₃): 2.70 mg g⁻¹ soil; ammonium nitrogen content (N-NH₄): 22.33 mg g⁻¹ soil; phosphate content: 60.95 mg g⁻¹ soil. Correlation analysis revealed significant associations between the enzymatic activities investigated and the soil physicochemical parameters (

Table 3. Correlations between soil enzymatic activities and physicochemical parameters.

Physicochemical Parameters/ Enzymatic Activity	pH	EC (µS cm−1)	Water Content (mg g−1)	OM (mg g−1)	N-NH4 (mg kg−1)	N-NO3 (mg g−1)	Phosphate (mg kg−1)
Deh	0.37 *	−0.02	0.01	0.69 ***	0.37 *	0.24	0.45 **
Ure	0.03	0.20	0.03	−0.03	−0.28	0.07	0.08
Cat	0.59 ***	0.20	−0.08	0.84 ***	0.54 ***	0.30 *	0.57 ***
Alp	0.61 ***	0.37 *	−0.02	0.69 ***	0.18	0.51 ***	0.60 ***

Deh, dehydrogenases; Ure, urease; Cat, catalase; Alp, alkaline phosphatase; EC, electric conductivity; OM, organic matter content; N-NH₄, ammonium nitrogen content; N-NO₃, nitrate nitrogen content. Marked boxes (*) indicate significant differences as compared to the reference group (*** p < 0.001, ** p < 0.01, * p < 0.05).

). The values obtained for the physicochemical parameters in the soil samples from the experiment are shown in Supplementary Table S1.

Low and moderate positive correlations were identified between the Deh and the soil pH, organic matter, nitrogen ammonium content, and available phosphorus content. In contrast, the Ure showed no significant associations with the soil physicochemical parameters. For Cat, a strong positive relationship with the soil organic matter content was found, as well as moderate positive associations with the soil pH, nitrogen (as ammonium) level, and available phosphorus. This enzymatic activity also revealed a weak association with nitrogen (as nitrate) concentration. Alp also displayed direct, moderate correlations with pH, soil organic matter, nitrogen (as nitrate), and available phosphorus, and a weak correlation with soil electric conductivity.

3.3. The Results of in Silico Characterization of Nicosulfuron Stability and Toxicity

The quantum chemical reactivity descriptors estimated on predicted HOMO (Highest Occupied Molecule Orbital) and LUMO (Lowest Un-occupied Molecular Orbital) values, and the molecular electrostatic potential (MESP) give significant information about nicosulfuron (Figure 4).

The energy gap calculated as the difference between the energy of HOMO and LUMO orbitals, refers to the amount of energy required to remove an electron from a molecule or atom in its ground state. A high gap energy suggests that the electrons within the molecule are tightly bound, making it more difficult to remove them. The value of −4.385 eV of the gap energy of nicosulfuron indicates that it is a less reactive compound and more resistant to chemical changes.

The results of in silico nicosulfuron characterization are detailed in

Table 4. Acute toxicity prediction with pKCSM, STOPTox, and BeeToxAI online tools.

	pKCSM
Parameters	Predicted	Descriptions
AMES toxicity	No	AMES mutagenicity test indicates that it may act as a carcinogen
Max. human tolerated dose	0.602	Dose (log mg/kg/day); Toxic effect > 0.477 log mg kg−1 day−1
hERG I inhibitor	No	hERG I/II inhibitors could cause the development of the acquired long QT syndrome, which leads to fatal ventricular arrhythmia
hERG II inhibitor	No
Oral Rat Acute Toxicity	2.272	LD50 (mol kg−1)
Hepatotoxicity	Yes	Categorical (Yes/No)
Skin Sensitisation	No	Categorical (Yes/No)
T.Pyriformis toxicity	0.279	log μg L−1; If value is >−0.5 log μg L−1 is considered to be toxic
Minnow toxicity	3.690	log mM; If values is <−0.3 indicate high acute toxicity
	STOPTox
Endpoint	Prediction/Confidence	Predicted fragment contribution
Acute Inhalation Toxicity (* RF: MACCS fingerprints)	Non-Toxic (−) 90.0%
Acute Oral Toxicity (* RF: MACCS fingerprints)	Non-Toxic (−) 95.0%
Acute Dermal Toxicity (* RF: MACCS fingerprints)	Non-Toxic (−) 90.0%
Eye Irritation and Corrosion (* RF: MACCS fingerprints)	Toxic (+) 88.0%
Skin Sensitization (* RF: Morgan EFCP4)	Non-Sensitizer (−) 70.0%
Skin Irritation and Corrosion (* RF: Morgan EFCP4)	Negative (−) 90.0%
	BeeTOX
Endpoint	Prediction/Confidence
Acute oral Toxicity (* RF: MACCS fingerprints) # Honey bee (Apis mellifera)	Non-Toxic (−) 54.0%
Acute Contact Toxicity (* SVM: FeatMorgan FCFP2) # Honey bee (Apis mellifera)	Non-Toxic (−) 95.0%

. From them, the hepatoxicity parameter suggests that nicosulfuron has a significant likelihood of causing hepatotoxicity. The Assessment of Acute Eye Irritation/Corrosion parameter marked as Toxic (+) with a confidence score of 88.0% provides details regarding the potential health risks associated with exposure to nicosulfuron. If the effects are irreversible (after monitoring for a minimum of 21 days), the chemical may be flagged as corrosive. Moreover, Table 4 reveals the forecasted contribution of fragments, visually represented by a red area, signifying a heightened likelihood of increased toxicity, while the green regions indicate that the fragment is predicted to decrease toxicity. Anyway, further research studies are required to compare the results with similar compounds/pesticides from the same class.

4. Discussion

The application of pesticides to soil can disturb the metabolism of local microbiota and their enzymatic activities. The negative impact of various pesticides on the activities of enzymes found in soil is registered for hydrolases (especially dehydrogenase) and oxidoreductases [7,55]. Concerning the herbicides, the literature data reveal their effects on the activities of the enzymes found in soil that highlight the stimulation or inhibition of the enzymes activities depending on the dose of the applied herbicide, the incubation, application interval, inorganic and organic soil content, soil type, soil maintenance work, and other environmental factors [56,57,58]. The decrease in enzyme activities in soils where sulfonylurea herbicides were applied can be attributed to the depletion of available nutrients in the soil, the degradation compounds of nicosulfuron, the persistence of nicosulfuron in the soil, but also to the inhibition of the metabolic activity of the community of microorganisms.

Enzymatic activities: dehydrogenase and urease are the most frequently determined in the case of testing the effects of pesticides on soils, the effect being one of inhibition of the two activities [18,19,25,59].

Closely linked to oxidation–reduction processes, microbial dehydrogenases are pivotal enzymes in the biological oxidation of soil organic matter [20]. These enzymes are sensitive to anthropogenic contamination/pollution (including the application of herbicides), serving as pertinent bioindicators of soil health [60]. Under the present experimental conditions, a 28 day exposure to field-realistic doses of nicosulfuron resulted in a trend towards decreasing soil Deh activity. This general trend is supported by several laboratory studies investigating the effects of nicosulphuron and other sulfonylurea herbicides given at different doses, including the recommended dose and doses above this threshold [3,28,31,61,62,63,64,65]. For example, Radivojević et al. (2012) obtained similar results for the same exposure time frame and nicosulfuron doses of 0.3, 1.5, and 3.0 μg g⁻¹ soil [28]. The investigated soil was a chernozem with a clay loam texture (pH 7.10, organic matter: 3.32%; sand: 21%; silt: 49%: clay: 30%) obtained from Zemun Polje—an urban neighborhood of Belgrade (Serbia), with soil samples being collected at 1, 7, 14, 21, 30, and 60 days post-application [28]. These data provide evidence that nicosulfuron may inhibit soil Deh activity during the first month post-application.

This inhibitory effect was, however, transient, reaching a maximum after 14 days of exposure and being attenuated from 21 days onwards. This concave dose–response relationship is consistent with literature data. Thus, soil Deh activity in the aforementioned investigation showed the lowest value between 1 and 10 days after nicosulfuron application, before increasing from 30 days to 60 days [28]. Since the sampling time points within the 0–30 day period were similar to those used in the present work, it is conceivable that higher exposure doses may have contributed to this earlier inhibition of Deh activity. In general, the application of the herbicides significantly increased the activity of soil Deh and Ure [31]. In this context, it is important to mention that Deh play a critical role in soil microbial metabolism, specifically in the oxidation of organic matter and the transfer of hydrogen to electron acceptors such as NAD⁺, NADP⁺, or FAD [20]. These enzymes are involved in key biochemical processes that are essential for maintaining soil health and fertility [60,61]. Due to their central role in microbial respiration and energy production, Deh activity is often used as a sensitive indicator of soil microbial activity and overall soil health [60,61,62,63]. When soil homeostasis is disturbed—whether due to pollution, the application of agrochemicals like nicosulfuron, or other environmental stressors—Deh activity is typically one of the first parameters to be affected [64]. Its decline can indicate a disruption in microbial communities and a reduction in soil’s biochemical functioning, signaling potential long-term degradation of soil quality [65].

Urease is involved in the soil nitrogen cycle, being produced by microorganisms that catalyze the hydrolysis of urea into carbon dioxide and ammonia [6,7]. Different effects on soil urease activity were reported in the specialty literature in response to herbicide application: no influence [66], slight inhibition effect [67], strong inhibition [16,68,69], or even stimulating effects [69]. Here, nicosulfuron dose exerted an inhibitory impact, whereas exposure duration displayed a stimulatory effect, with the latter factor having a stronger influence on soil urease activity than the former factor. These results may be attributed to several factors. One potential explanation is that while the exposure dose may initially suppress urease activity by harming soil microorganisms, the increased exposure duration may allow the soil microbial community to recover and adapt, leading to an increase in urease activity. This adaptation and recovery could be related to various factors, such as the development of resistant strains of microorganisms, the shift in microbiota towards bacterial strains tolerant to nicosulfuron, the recovery of microbial populations following cessation of abiotic stress, or the decomposition of nicosulfuron by soil microorganisms [9]. It is also plausible that the initial exposure dose of nicosulfuron might reach a threshold beyond which additional doses have a diminishing impact on soil urease activity. This threshold could be reached relatively quickly, and further increases in dose may not significantly affect the already inhibited urease activity [70].

Toxicological literature provides quite discrepant, laboratory-derived data related to the impact of nicosulfuron on Ure activity. Thus, Satric et al. (2018) administered nicosulfuron at rates of 0.3, 0.6, 3.0, and 30.0 μg g⁻¹ soil on two soils with different physicochemical properties (loam and sand), with soil samples being collected at 3, 7, 14, 30, and 45 days post-application. As a general trend, nicosulfuron application yielded a significant increase in soil Ure activity during the first 30 days of exposure (irrespective of soil type). The maximum activity was found at 7 days for the highest treatment group [31]. In contrast, Li et al. (2014) reported inhibition of Ure activity in a loamy sand soil before 21 days of exposure to nicosulfuron doses of 13.5, 27, and 54 μg g⁻¹ soil [71]. Soil samples were collected on days 7, 14, 21, 28, and 56. These findings suggest that the specific effects of nicosulfuron on soil and microbial activity can vary depending on factors like soil type, microbial community composition, herbicide concentration, and application method [72].

We also note that, consistent with our results, the aforementioned study found that even half the normal recommended dose exerts a significant inhibitory effect on Ure activity [71]. Moreover, Ure activity began to recover after 21 days, reaching normal levels in 56 days. This is also in line with our findings derived from the post hoc analysis of the significant time-dose interaction seen in the case of UA [71].

In the context of microbial ecology, Cat activity can be considered a biochemical marker for intracellular biological activity and can provide information about the responses of soil microbiota to exogenous stress, including pesticides [73]. Our results showed that, under the current experimental conditions, soil Cat activity was the least responsive enzymatic parameter to nicosulfuron application. The response of Cat activity to this external insult was relatively weak and delayed in relation to Deh activity and Ure activity, with significant changes being observed only from 21 days onward. The absence of a significant effect of dose on Cat activity suggests that catalase may be less affected by nicosulfuron within a 30 day time frame at the doses typically applied in agricultural settings. Indeed, there is evidence to support this assumption. Thus, Li et al. (2014) have identified a significant (stimulatory) impact of nicosulfuron on Cat activity, but only for doses much higher than those used in the current investigation, which are 13.5, 27, and 54 μg g⁻¹ soil [74], respectively.

Soil Alp activity is important for the transformation of organic phosphorus into inorganic phosphates, which are easily usable by soil microorganisms and provide mineral nutrients for plants. Similar to Cat activity, this enzymatic parameter was less sensitive to nicosulfuron exposure compared to Deh activity and Ure activity. However, we identified an inhibitory effect. Alp activity was found to be less and later affected by nicosulfuron compared to the other Deh and Ure enzymatic activities, and the effect of the herbicide was a transient one, more evident in the first 21 days, at a dose three times the normal recommended dose.

The studies carried out on the effects of other sulfonylurea herbicides (chlorsulfuron and sulfosulfuron) on enzyme activity in laboratory conditions demonstrated the significant effect of herbicides on enzyme activities. Chlorsufuron caused greater perturbations of enzyme activities than sulfosulfuron, and the changes were predominant, mainly in the first 28 days. The studies indicated that in the first 28 days there were significant changes in enzyme activities; the herbicides changed the activity of cellobiohydrolase, arylsulfatase, dehydrogenases, phosphatases, and FDA hydrolase. Phosphatase activity was decreased in both soils on the 7th and 14th day [74]. By analyzing the effect of nicosulfuron on Alp activity in our study, we conclude that the effect was temporary and significant decreases were identified only when high concentrations of nicosulfuron were used; otherwise, the inhibition effect was minor.

As a general trend, Alp activity tends to decrease in soils treated with herbicides [71]. This response has also been reported after the application of sulfonylurea herbicides [18,19,61,74]. These results are in agreement with our findings. However, the pattern and magnitude of changes induced by this class of herbicides on Alp activity can differ depending on the type of sulfonylurea herbicide [74].

The enzymatic activity in the soil is influenced by the physicochemical properties of the soil. Among the most important parameters, we mention temperature, pH, humidity, organic matter content, ammonium content, nitrates, nitrites, soil type, and texture [37,38,57,73,74,75,76].

The physicochemical parameters of the soil influence the enzymatic activity, establishing correlations between the different activities and the chemical parameters: (i) Deh activity in the soil samples is strongly influenced by the content of organic matter, the increase in the content determines an increase and a stimulation of the dehydrogenase activity [40,41,60,74]; (ii) Ure activity is correlated with the nitrate from the soil, organic matter, and available phosphorus [40,41,64], and other studies show that the organic matter negatively influence this enzymatic marker [41,48,77]; (iii) Cat activity is positively correlated with the organic matter, nitrogen level, nitrogen level and available P from the soil [61]; (iv) Alp activity shows positive correlations with organic matter, nitrogen level, and available P from the soil [40,41,60,78].

The physicochemical parameters of the soil influence the enzymatic activity. The correlations made are very complex because the nicosulfuron present in the soil can influence its physical, chemical, and biological composition, one of its effects being on the enzymatic activities of the soil.

The application of quantum chemical reactivity descriptors provides critical insights into the chemical behavior of nicosulfuron. The energy gap between the HOMO and LUMO indicates that nicosulfuron is a stable molecule with low reactivity, hence resistant to chemical changes in environmental conditions. This stability could account for the persistence of its residues in certain environments, such as underground water bodies [17]. Based on the present findings, one can also expect that this herbicide is relatively safe under controlled use, although its breakdown products or prolonged exposure could lead to significant health risks, especially related to its potential hepatotoxic and ocular effects. This scenario is of particular concern in occupational settings and in agricultural areas where nicosulfuron is heavily used [12,71]. Nonetheless, the fact that nicosulfuron is non-toxic to bees is a positive aspect, considering the importance of these pollinators in agriculture [51].

Several limitations of this preliminary investigation need to be acknowledged. Among these caveats, one limitation is that the present findings may be specific to the Banat Plain and its chernozem soil type, thus limiting their applicability to agricultural areas with different soil types and environmental conditions. On the other hand, the chernozem type investigated is encountered in similar high-fertility agricultural regions in Europe [3,6]. This case study may therefore serve as a potential benchmark for other scenarios with comparable conditions. As this study only lasted 28 days, this timeframe may not be long enough to elucidate the potential long-term effects of this herbicide on soil health. However, our findings can inform short-term agricultural practices and risk management strategies since this time frame is critical for evaluating immediate effects of herbicide application. It is also true that we did not specifically test for pesticide residues before the experiment, which does not entirely rule out the possibility of prior use. Nonetheless, we have carefully selected an experimental field with no known history of agrochemical application, minimizing the likelihood of residual contamination. Moreover, our future studies will incorporate pre-experiment testing for residual pesticides to ensure a more controlled baseline condition.

5. Conclusions

This study provides a comprehensive and integrative assessment of the impact of nicosulfuron on key soil enzymatic activities, revealing dose- and time-dependent inhibitory effects that could compromise soil health and fertility. These findings derived from a chernozem typical of a high-profile area for European agriculture are particularly relevant for sustainable agriculture, highlighting the need for careful herbicide application to mitigate potential risks. This research also contributes to environmental risk assessments and sets the foundation for future studies on the long-term effects of agrochemicals on soil ecosystems. Under the present experimental conditions, the effects of nicosulfuron on selected soil enzymatic activities display variations depending on the dose applied and exposure time. Thus, the effect of nicosulfuron on Deh was transitory, dependent on dose and time, with the highest inhibition rate observed 14 days after application, starting from the normal recommended dose. However, the results obtained highlight a more obvious inhibitory effect of nicosulfuron on Ure activity compared to Deh activity, although this effect was again transient, occurring primarily at higher concentrations compared to the normal dose. The impact of nicosulfuron on Cat activity was weaker and delayed versus the other enzymatic activities. A similar effect was observed in the case of Alp activity, with the outcome being transient and more evident during the time period 7–21 days post-application and for the highest dose treatment. Overall, Ure was the most sensitive soil enzyme to nicosulfuron, followed by Deh, Alp, and Cat. Since most of the herbicides applied to the soil cause imbalances, even transient ones at the soil level, they can affect its fertility over time. It is therefore recommended to apply them in a controlled manner and apply the doses recommended by the producing companies. Based on the results of this study, future research directions will be guided to design and characterize new pesticides that are less toxic to humans and have fewer adverse effects on the environment.