The Effect of Herbicides and Biostimulant Application on the Seed Yield and Seed Quality of Soybean (Glycine max (L.) Merr.)

Abstract

The profitability of growing soybean (Glycine max (L.) Merr.) is largely determined by the successful elimination of weeds and the weather conditions, the adverse effect of which can be minimized by the use of biostimulants. This study aims to evaluate the effect of several herbicides and biostimulants on the seed yield and contents of protein, fat, amino acids, and fatty acids in soybean seeds. The results demonstrate that the yield and quality of soybean seeds were most beneficially affected by the use of prosulfocarb immediately after sowing in combination with a subsequent foliar application of bentazone and imazamox with an adjuvant containing methyl oleate and fatty alcohol. This treatment ensured the highest seed yield (3.32 t ha⁻¹⁾, the highest contents in seeds of protein (342.4 g kg⁻¹ DM), phenylalanine (15.65 g kg⁻¹ DM), leucine (23.54 g kg⁻¹ DM), and most of the endogenous amino acids. All herbicide treatments increased the contents of tryptophan, serine, and glutamic acid compared to the seeds without herbicide treatment. The study results indicate that soybean responded positively to all biostimulants, as indicated by a higher seed yield (from 5.3% to 11.3%), plant height, and contents of tryptophan, serine, glutamic acid, cysteine, tyrosine, and C18:1n9c + C18:1n9t acids in the seeds.

1. Introduction

Soybean (Glycine max (L.) Merr.) is considered to be the most valuable leguminous crop from the perspective of both human nutrition and animal feeding. It is grown for either green mass or seeds. In the first case, it is cultivated in warm climate areas, where it is used as an invaluable source of high-value and high-protein green or dried fodder, while in the second case, its production focuses on seeds, which is the main purpose of its cultivation from the global economy perspective. Soybean seeds contain about 20% fat rich in unsaturated fatty acids, which makes this crop the second-largest plant oil producer in the world [1]. In addition, the high content of protein (approximating 40% of seed composition), which contains all the essential exogenous amino acids, makes the soybean a key protein crop [1,2,3]. Soybean also exerts a positive effect on soil fertility, as its cultivation leaves plenty of residues rich in nitrogen and, therefore, creates excellent conditions for follow-up plants. This is also due to the coexistence of soybean with Rhizobia bacteria of the genus Bradyrhizobium, which fix atmospheric nitrogen and, thanks to the well-developed root system, are capable of extracting nutrients from the deeper layers of the soil. Therefore, soybean cultivation introduces a significant amount of nitrogen into the soil, which contributes to an increase in the yield of follow-up crops and, at the same time, to a reduction in expenditures related to fertilizer use [3,4,5].

Soybean is, however, poor at competing with weeds. The profitability of its cultivation is, therefore, largely dependent on the effective elimination of weeds, which may be accomplished by the use of properly selected herbicides [6,7,8]. It is recommended to eradicate weeds as soon as possible, i.e., immediately after sowing, by using soil herbicides, because weeds that sprout at the same time as soybean plants grow faster and higher. This may result in lower seed yield and diminished seed quality [9]. However, it is believed that the use of herbicides before seedling emergence can adversely affect the germination capacity of soybean seeds and that application after seedling emergence can cause plant damage, resulting in lower seed yield [10,11,12,13,14]. The herbicides applied may, therefore, negatively affect soy development, root nodulation, and nitrogen fixation [15]. In addition to the influence of herbicides on plant growth and development, and consequently, on their yield, their application may cause differences in the quality of the yield produced, including the contents of protein and fat [16] as well as fatty acids and amino acids in soybean seeds [17,18].

Adverse conditions for plant growth and development linked, i.e., to climate change, can result in significant losses in crop yields. Biostimulants have emerged in response to this challenge. They support the regeneration of plants after the occurrence of stress factors, not only those related to adverse weather conditions but also those triggered by the use of herbicides [19], and thus contribute to the improvement in the size and quality of crop yields [20,21]. Biostimulants can affect the metabolism of plants, improve their biochemical and morphological properties, and stimulate physiological processes in plants [22]. Currently available biostimulants contain various types of organic and inorganic compounds and can be synthetic or natural. Natural biostimulants most often contain humic substances or amino acids, whereas synthetic preparations are usually composed of phenolic compounds, growth regulators, inorganic salts, and nutrients [23,24,25]. The use of biostimulants is particularly justified in the case of species sensitive to adverse climatic conditions, such as soybean. Like all species of the Fabaceae family, this crop is particularly exposed to temperature-related stress, drought stress, and water scarcity, especially in developmental stages such as germination, emergence, flowering, and pod formation. The shortage of precipitation in these developmental stages causes a water deficit in plant tissues, which, by inhibiting the course of various physiological processes, affects the growth and development of plants and, consequently, the yield and chemical composition of their seeds [26,27,28]. Research into the effects of biostimulants on the yield and quality of soybean seeds is fully justified in the face of the growing popularity of this crop and climatic changes adverse to plant growth and development. For the above reasons, an experiment was conducted that attempted to determine the response of soybean plants to selected biostimulants and different herbicides. It was assumed that the use of biostimulants would have a positive effect on the seed yield and contents of protein, lipids, amino acids, and fatty acids in soybean seeds.

2. Materials and Methods

2.1. Location of the Experiment and Soil and Climatic Conditions

The field experiment was conducted in 2020–2022 at the experimental farm in Czesławice belonging to the University of Life Sciences in Lublin (51°18′23″ N, 22°16′1″ E) in Poland. The experiment was established on loess soil with the grain size distribution of silt loam and classified as good wheat soil complex (soil class II). The soil on which the experiment was established had a neutral pH (pH at 1 mol KCl—7.2). It had high contents of phosphorus (P—130.5 mg kg⁻¹ soil) and potassium (K—176.6 mg kg⁻¹ soil) and a very high content of magnesium (Mg—67.6 mg kg⁻¹ soil). The organic matter content was 1.4%.

The total precipitation and air temperature recorded during the growing season of soybean were higher in all study years compared to the multi-year period (

Table 1. Total precipitation and mean monthly air temperature in the growing season of soybean, recorded by the Meteorological Station in Czesławice (Poland).

Months	Years
	2020		2021		2022		LTA * 1963–2010
	mm	°C	mm	°C	mm	°C	mm	°C
May	111.4	11.2	68.0	11.6	39.9	13.4	63.5	13.6
June	170.3	17.4	68.3	18.6	25.1	19.7	72.7	16.5
July	67.8	18.8	82.4	22.0	190.2	19.7	80.0	18.3
August	59.3	20.4	197.8	17.2	44.8	20.9	69.5	17.7
September	128.5	15.7	57.1	13.1	72.5	12.3	59.5	13.1
Sum/Mean (May–September)	537.3	16.7	473.6	16.5	372.5	17.2	345.2	15.8

* LTA—long-term averages.

). The highest total precipitation was recorded in 2020, especially in May and June, and also in the soybean harvest month (September). Among the study years, the growing season of 2022 was characterized by the highest air temperature in May, June, and August.

2.2. Experimental Design and Agronomic Practices

The two-factor experiment was established in a randomized block design, in three replications, on plots with an area of 24 m² (4 m × 6 m). The Lajma cultivar of soybean (early cultivar, growing period 115–125 days) was sown on the post-spring wheat plot.

The experimental factors included:

I. Herbicides

A. Control plot (no herbicides)—mechanical treatment only;

B. Soil herbicide Boxer 800 EC (prosulfocarb—800 g L⁻¹);

C. Foliar herbicide Corum 502.4 SL (bentazone—480 g L⁻¹, imazamox—22.4 g L⁻¹) + Dash HC adjuvant (methyl oleate—348.75 g L⁻¹, fatty alcohol—209.25 g L⁻¹);

D. Boxer 800 EC soil herbicide and Corum 502.4 SL foliar herbicide + Dash HC adjuvant.

II. Biostimulant type

a. Control plot (no biostimulant);

b. Asahi SL (sodium para-nitrophenolate—3 g L⁻¹, sodium ortho-nitrophenolate—2 g L⁻¹, sodium 5-nitroguaiacolate—1 g L⁻¹);

c. Aminoplant—a biostimulant containing free amino acids and short peptide chains (organic nitrogen (Norg)—8.7%, ammonium nitrogen (N-NH4)—0.4%, free amino acids (FAAS)—10.0%, and organic carbon (Corg)—24.0%);

d. Kelpak SL—an extract of Ecklonia maxima algae (auxins—11 mg L⁻¹ and cytokinins—0.031 mg L⁻¹).

The scheme of the field experiment is shown in Scheme 1.

Herbicides and biostimulant doses were determined based on the recommendations of the manufacturer of a given chemical, in accordance with the product label. Mechanical treatment consisting of harrowing at the stage of the third trifoliate leaf on the second node (BBCH 12—Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) [29] was performed on all experimental plots. On plots B and D, prosulfocarb soil herbicide was used immediately after sowing soybean seeds at a dose of 3.5 L ha⁻¹. On plots C and D, bentazone and imazamox herbicide were used at a dose of 1.25 L ha⁻¹ in combination with the Dash HC adjuvant at a dose of 1 L ha⁻¹ at the stage of successive leaf development (BBCH 19) [29].

During the growing season of soybean, all biostimulants were used twice: at the stage of the trifoliate leaf developed on the third node (BBCH 13) [29] and at the beginning of flowering (BBCH 61) [29]. The following biostimulant doses were applied on both dates: Asahi SL—0.5 L ha⁻¹, Aminoplant—1.5 L ha⁻¹, and Kelpak SL—2.5 L ha⁻¹.

Soil cultivation under soybean consisted of the following treatments: shallow plowing + harrowing, harrowing, pre-winter plowing; and in the spring, harrowing, NPK fertilization, cultivation with harrowing, seed sowing, and harrowing.

Soybean seeds were sown in the first ten days of May to a depth of 3 cm, in a row spacing of 22.5 cm, in a planned plant density of 70 plants per 1 m². Prior to sowing, soybean seeds were inoculated with Bradyrhizobium japonicum bacteria and dressed with Maxim 025 FS (fludioxonil—25 g L⁻¹) in a dose of 400 mL 100 kg⁻¹ seeds with the addition of water in a ratio of 1:1.

NPK mineral fertilization was applied before sowing in the following doses: N—30 kg ha⁻¹ (34.5% ammonium sulfate), P—40 kg ha⁻¹ (40% superphosphate), and K—80 kg ha⁻¹ (60% potassium salt).

Soybean seeds were harvested annually in the first ten days of September, at the stage of full maturity (89 on the BBCH scale) [29].

2.3. Scope of Study and Statistical Analysis

The characteristics of the yield and crop growth parameters (plant height, first pod height, pod number per plant, number and weight of seeds per plant) were determined based on a sample of 30 plants randomly collected from each plot. The assessment of plant density after emergence and before harvest was carried out in two rows across a length of 2.5 m. The 1000-seed weight was determined in accordance with the Polish Standard PN-68/R-74017 [30].

Seed yield was weighed separately from each plot, i.e., from an area of 24 m², and the results obtained were converted per hectare.

The protein content of the seeds was determined with the Kjeldahl method (PN-75-/a-04018) [30]. The protein amino acid content was determined by ion exchange chromatography (INGOS amino acid analyzer). The crude fat content was determined via the Soxhlet extraction-gravimetric method (PN-64/a-74039) [30], and the fatty acid content by the gas chromatography (GC/FID method). The above analyses were carried out at the Central Research Laboratory of the University of Life Sciences in Lublin (Poland).

The study results collected over the period of 2020–2022 were processed using the analysis of variance (ANOVA), and the significance of differences was estimated using Tukey’s test at a significance level of p ≤ 0.05. The influence of herbicides, type of biostimulant, study years, and their interactions on seed yield, plant density, plant height, first pod height, number of pods and seeds per plant, seed weight per plant, 1000-seed weight, and the content of protein, fat, amino acids, and fatty acids in seeds was determined. A total of 144 results were used for individual statistical calculations (4 herbicide treatments × 4 types of biostimulants × 3 years × 3 replications). Standard deviation (SD) was additionally calculated for the protein and fat contents of the seeds. Calculations were made using Statistica 14.0.0 software (TIBCO Software Inc., Tulsa, OK, USA).

3. Results

3.1. Yield and Yield Components

The analysis of variance demonstrated that herbicides significantly modified soybean yield (

Table 2. Yield of seed, yield, and crop growth parameters of soybean depending on herbicides, type of biostimulant (mean for 2020–2022), and experimental year.

Specification	Seeds Yield (t ha−1)	Plant Density after Emergence (pcs. m−2)	Plant Density before Harvest (pcs. m−2)	Plant Height (cm)	First Pod Height (cm)	Number of Pods per Plant (pcs.)	Number of Seeds per Plant (pcs.)	Seed Weight per Plant (g)	1000-Seed Weight (g)
Herbicides
A	3.09 b	57.2 ab	52.6 a	78.4 b	7.9 b	17.2 b	33.9 c	5.95 c	176.5 a
B	3.28 a	59.6 ab	55.0 a	81.5 ab	8.1 b	18.5 a	36.8 ab	6.49 ab	175.1 a
C	3.11 b	56.5 b	52.0 a	80.9 ab	8.2 ab	17.7 b	36.4 b	6.42 b	174.5 a
D	3.32 a	59.7 a	54.0 a	83.0 a	8.6 a	19.2 a	37.7 a	6.70 a	175.3 a
p-Value	***	*	ns	**	**	***	***	***	ns
Biostimulants
No biostimulant	3.01 c	58.4 a	51.7 a	76.4 b	7.9 b	17.9 bc	36.0 b	6.20 c	173.2 a
Asahi SL	3.27 ab	58.4 a	54.3 a	84.3 a	8.2 ab	18.4 ab	36.0 b	6.45 b	176.7 a
Aminoplant	3.17 b	59.4 a	55.1 a	82.2 a	8.2 ab	17.4 c	34.1 c	6.05 c	177.7 a
Kelpak SL	3.35 a	56.9 a	52.5 a	81.0 a	8.5 a	19.0 a	38.7 a	6.85 a	173.8 a
p-Value	***	ns	ns	***	*	***	***	***	ns
Years
2020	2.77 c	53.2 c	48.6 c	70.8 c	6.5 b	15.1 b	32.6 b	6.03 b	184.7 a
2021	3.28 b	57.7 b	54.0 b	92.5 a	9.0 a	19.5 a	38.4 a	6.18 b	160.7 c
2022	3.55 a	63.9 a	57.6 a	79.6 b	9.1 a	19.9 a	37.6 a	6.96 a	180.7 b
p-Value	***	***	***	***	***	***	***	***	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone, and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ns—no significant difference between treatments at p ≤ 0.05, * significance level at p ≤ 0.05, ** significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

). The mean seed yield and pod number per plant recorded for the three study years on plots B and D with the treatments performed using prosulfocarb as well as prosulfocarb and bentazone + imazamox with the adjuvant were significantly higher than on the plots without herbicides (A) and on those where only the foliar active ingredients bentazone and imazamox with the adjuvant (C) was applied. The highest seed yield was obtained on plot D, which was 7.4% and 6.8% higher compared to that obtained on the control plot (A) and the plot where the crop was protected only with the foliar herbicide (C), respectively. The treatment with the soil and foliar herbicide (plot D) also produced the highest plant density after emergence, plant height, and the first pod height. The use of herbicides (plots B, C, and D) had a positive effect on the number and weight of seeds per plant compared to the treatment without chemical protection against weeds (A). In turn, herbicides caused no significant differences in plant density before harvest and in the 1000-seed weight.

The soybean seed yield was substantially higher in the treatments with all biostimulants tested compared to the no-biostimulant treatment (Table 2). A similar correlation was also found in the case of plant height. The highest seed yield was produced after using the biostimulant with an extract from Ecklonia maxima algae—it was significantly higher than on the plot without biostimulant and on that with Aminoplant treatment, i.e., by 11.3 and 5.7%, respectively. Among the tested biostimulants, only Kelpak SL had a positive effect on the first pod height compared to the no-biostimulant treatment. The values of this characteristic did not differ significantly among the treatments with all studied preparations. The lowest number of pods and the number and weight of seeds per plant were obtained using Aminoplant preparation containing free amino acids. The biostimulants caused no significant differences in plant density after emergence and before harvest, and also in the 1000-seed weight.

In turn, the seed yield was significantly modified by weather conditions in the individual study years (Table 2). The highest seed yield was obtained during the growing season of 2022, characterized by the highest average air temperature, among others, in the month of sowing soybean seeds (May), which probably had a positive effect on plant emergence and, consequently, on crop yield. In the third year of the study (2022), the values of the following yield traits were also higher than in the other study years: plant density after emergence and before harvest and seed weight per plant. The first year of the study (2020) proved to be the least beneficial for soybean yield, as the recorded seed yield was 22.0 and 7.6% lower than in 2022 and 2021, respectively. The air temperature recorded in 2020 in the months of seed sowing and initial growth of soybean (May, June) was the lowest among the study years, which probably adversely affected the emergence of this plant and its initial growth and, consequently, caused the lowest seed yield in this year. In addition, very heavy rainfall in September 2020 made it difficult to harvest soybeans. In 2020, the lowest values were determined for the following yield traits: plant density after emergence and before harvest, plant height, first pod height, as well as pod number, seed number, and seed weight per plant. In this year, only the 1000-seed weight was higher than in the remaining study years.

The interaction of herbicides and biostimulant type did not significantly differentiate seed yield but still affected some traits of the soybean yield and crop growth parameters, as presented in

Table 3. Crop growth parameters of soybean depending on the interaction of herbicides and the type of biostimulator (mean for 2020–2022).

Herbicides	Biostimulants	Plant Height (cm)	First Pod Height (cm)	Number of Pods per Plant (pcs.)	Number of Seeds per Plant (pcs.)	Seed Weight per Plant (g)
A	No biostimulant	72.1 e	7.3 c	16.4 e	32.8 d	5.52 e
	Asahi SL	83.5 abcd	7.9 bc	17.6 cde	33.8 d	5.99 cde
	Aminoplant	75.7 de	7.6 bc	17.3 de	34.5 d	6.02 cde
	Kelpak SL	82.3 abcd	8.7 ab	17.4 de	34.4 d	6.28 bcde
B	No biostimulant	77.5 bcde	7.6 bc	19.4 abc	40.2 a	6.97 ab
	Asahi SL	81.8 abcd	8.4 abc	18.5 bcd	35.9 bcd	6.55 abcd
	Aminoplant	86.4 ab	7.6 bc	16.7 de	33.0 d	5.92 cde
	Kelpak SL	80.2 abcde	8.7 ab	19.5 abc	38.1 abc	6.51 abcd
C	No biostimulant	76.7 cde	8.2 abc	17.1 de	35.0 cd	5.81 de
	Asahi SL	86.5 ab	8.3 abc	17.0 de	35.0 cd	6.45 abcde
	Aminoplant	78.9 abcde	8.1 bc	18.2 cde	34.6 cd	6.15 bcde
	Kelpak SL	81.6 abcd	8.3 abc	18.6 bcd	41.0 a	7.26 a
D	No biostimulant	79.3 abcde	8.4 abc	18.6 bcd	35.9 bcd	6.51 abcd
	Asahi SL	85.3 abc	8.3 abc	20.4 ab	39.2 ab	6.80 abc
	Aminoplant	87.7 a	9.5 a	17.3 de	34.2 d	6.12 bcde
	Kelpak SL	79.8 abcde	8.3 abc	20.6 a	41.3 a	7.35 a
p-Value		*	***	***	***	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). * Significance level at p ≤ 0.05, *** significance level at p ≤ 0.001.

. In the treatment with the Aminoplant biostimulant, the highest plants and the highest pod heights were noted on the plot where the crop was treated with both the soil herbicide and the foliar herbicide (plot D). The use of Kelpak SL biostimulant combined with complex herbicides (D) turned out to exert the most beneficial effect on pod number, seed number, and weight per plant. The lowest values of these traits of the yield and crop growth parameters were determined on the plots not treated with biostimulants or herbicides.

The interaction of herbicides and weather conditions, in particular growing seasons of soybean, significantly affected the plant density after emergence, plant height, pod number per plant, seed weight per plant, and 1000-seed weight (

Table 4. Crop growth parameters of soybean depending on the interaction of herbicides and experimental year.

Years	Herbicides	Plant Density after Emergence (pcs. m−2)	Plant Height (cm)	Number of Pods per Plant (pcs.)	Seed Weight per Plant (g)	1000-Seed Weight (g)
2020	A	47.4 d	69.2 f	13.4 e	5.38 g	184.3 ab
	B	55.3 bcd	73.3 e	15.7 cd	6.27 cde	183.8 ab
	C	51.8 cd	67.4 f	14.6 de	5.92 ef	181.8 ab
	D	58.2 bc	73.2 ef	16.6 c	6.56 abcd	188.9 a
2021	A	57.7 bc	91.5 ab	18.7 b	5.71 fg	158.4 c
	B	57.3 bc	88.9 bc	20.2 ab	6.22 def	159.0 c
	C	57.7 bc	91.9 ab	18.9 b	6.31 cde	164.9 c
	D	58.2 bc	97.6 a	20.3 ab	6.47 bcd	160.5 c
2022	A	66.5 a	74.4 ef	19.4 ab	6.77 abc	186.8 ab
	B	66.3 a	82.3 cd	19.7 ab	6.97 ab	182.5 ab
	C	60.0 ab	83.5 cd	19.7 ab	7.02 a	176.9 b
	D	62.8 ab	78.3 de	20.8 a	7.07 a	176.4 b
p-Value		**	***	*	**	**

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). * Significance level at p ≤ 0.05, ** significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

). In the first year of the experiment (2020), the 1000-seed weight was similar on all plots (Table 4). The values of the other characteristics were the lowest on the plots without the herbicide treatment (A) or with only the foliar active ingredients bentazone + imazamox with the adjuvant (C). In the growing season of 2021, the plant density after emergence, pod number per plant, and 1000-seed weight were not significantly differentiated by the herbicides applied. However, the 1000-seed weight was significantly lower than that determined in the other study years, regardless of the herbicide treatment. In contrast, the height of the plants was the highest on plot D and similar on plots without herbicide (A) and those with bentazone and imazamox with the adjuvant (C). The value of this feature recorded on all plots in 2021 was significantly higher than in the other experimental years, regardless of the herbicides applied. In 2022, plant density after emergence, pod number per plant, seed weight per plant, and 1000-seed weight were similar on plots, whereas the highest plants were found on the plots with only foliar herbicide (C) or only soil herbicide (B) treatment. The lowest seed weight per plant was determined in the treatment with no herbicide (plot A) in 2020 and 2021.

The yield of soybean seeds and the traits of the yield and crop growth parameters listed in

Table 5. Yield of seed, yield, and crop growth parameters of soybean depending on the interaction of the type of biostimulant and experimental year.

Years	Biostimulants	Seeds Yield (t ha−1)	Plant Height (cm)	First Pod Height (cm)	Number of Pods per Plant (pcs.)	Number of Seeds per Plant (pcs.)	Seed Weight per Plant (g)	1000-Seed Weight (g)
2020	No biostimulant	2.42 f	60.2 d	6.2 b	13.9 e	30.6 e	5.63 e	186.7 a
	Asahi SL	2.91 de	78.8 c	6.9 b	15.5 de	32.2 de	6.02 de	182.6 a
	Aminoplant	2.75 e	78.1 c	6.4 b	14.0 e	30.4 e	5.70 e	187.4 a
	Kelpak SL	2.98 cde	66.0 d	6.4 b	17.0 cd	36.9 bc	6.78 bc	182.0 a
2021	No biostimulant	3.09 cd	91.4 a	8.7 a	19.7 ab	39.1 ab	6.01 de	153.3 c
	Asahi SL	3.37 abc	93.0 a	8.9 a	19.8 ab	39.1 ab	6.41 cd	165.0 b
	Aminoplant	3.22 bcd	88.9 b	8.7 a	19.7 ab	37.1 bc	6.03 de	162.3 bc
	Kelpak SL	3.45 ab	96.6 a	9.6 a	18.9 b	38.4 ab	6.27 cd	162.3 bc
2022	No biostimulant	3.51 ab	77.7 c	8.7 a	20.1 ab	38.2 ab	6.97 ab	179.5 a
	Asahi SL	3.54 a	81.0 c	8.8 a	19.8 ab	36.6 bc	6.92 bc	182.6 a
	Aminoplant	3.53 ab	79.5 c	9.5 a	18.5 bc	34.8 cd	6.43 cd	183.4 a
	Kelpak SL	3.61 a	80.3 c	9.5 a	21.2 a	40.7 a	7.50 a	177.1 a
p-Value		*	***	*	***	***	***	*

Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). * Significance level at p ≤ 0.05, *** significance level at p ≤ 0.001.

differed significantly in the individual study years as affected by the biostimulant used. In 2020, the no-biostimulant treatment produced significantly lower seed yield, i.e., by 18.8, 16.8, and 12.0%, compared to the seed yields recorded after treatments with Kelpak SL, Asahi SL, and Aminoplant preparations, respectively. In contrast, the pod number per plant, as well as seed number and weight per plant reached the highest values after the Kelpak SL biostimulant, whereas plant height after treatments with Asahi SL and Aminoplant. In the first growing season of soybean, the first pod height and 1000-seed weight were similar on all plots (without and with the biostimulant treatment). However, the height of the plants was significantly lower than in the other study years, regardless of the biostimulant applied. In 2021, the seed yield produced on the plot without biostimulant treatment was significantly lower (by 10.4%) only after the application of Kelpak SL. In contrast, the plant height was the lowest after the Aminoplant biostimulant application and the 1000-seed weight on the plots without the biostimulant treatment. In the second growing season of soybean (2021), the remaining traits of the crop and yield structure included in the table reached similar values regardless of the biostimulant treatment. In the last year of the experiment (2022), the number of pods per plant and the number and weight of seeds per plant were the lowest after Aminoplant treatment, while the remaining traits of the crop and yield structure and seed yield reached similar values on all plots.

3.2. Seed Quality Parameters

The experimental factors significantly affected the protein content of soybean seeds (Figure 1a,b). The highest protein content was determined in the seeds from the treatment with prosulfocarb and bentazone + imazamox (plot D). Compared to this treatment, the protein content was 1.4% lower in the seeds harvested from the plots that were not treated with herbicides (Figure 1a). The highest protein content was determined in the seeds of soybean treated with Aminoplant biostimulant and a similar one in those from the crop treated with Kelpak SL preparation (Figure 1b). In soybean seeds harvested from the plots without the biostimulant treatment, the content of protein was significantly lower (by 1.7%) only compared to those from the Aminoplant treatment.

The highest fat content was determined in the seeds of soybean harvested from the lots without herbicides (A), whereas similar fat contents were obtained in the seeds from plots B and C (Figure 2a). After application of both soil and foliar herbicides (plot D), the fat content of the seeds was significantly lower compared to the herbicide-free plot (A) and to the plot with the bentazone and imazamox active ingredients treatment (C), i.e., by 2.5 g kg⁻¹ and 3.0 g kg⁻¹, respectively. The highest fat content was determined in soybean seeds harvested from the plot without biostimulant application; however, it was significantly higher only compared to the seeds from the Aminoplant treatment (by 2.2%) (Figure 2b). The fat content of soybean seeds was similar in the use of all biostimulants tested.

The protein and fat contents of soybean seeds differed significantly depending on the weather conditions in the individual study years (Figure 3a,b). The highest protein content was determined in 2022, which was characterized by the highest average air temperature and the lowest precipitation during the growing season of soybean. Compared to the third year of the experiment (2022), the protein content of the seeds harvested in 2020 was lower by 2.8 g kg⁻¹ and that of the seeds harvested in 2021 by 5.8 g kg⁻¹. In turn, the fat content of the seeds harvested in 2020 was significantly lower compared to those produced in 2021 and 2022, by 3.5 and 2.7 g kg⁻¹, respectively.

The protein and fat contents were also significantly differentiated by the interaction of herbicides and biostimulant types (Figure 4 and Figure 5). In contrast, the statistical analysis did not confirm any significant effect of the interaction between the experimental factors and study years on the contents of these components.

On the plot where only soil herbicide (B) was applied and on the plots without herbicides (A), the tested biostimulant caused no significant differences in the protein content of soybean seeds (Figure 4). On the plots with the foliar herbicide (plot C), the application of Aminoplant biostimulant increased its content by 3.2 and 2.9% compared to the treatments with Asahi SL and no-biostimulant, respectively. Also, on the plot with complex herbicides (D), the protein content of the seeds was most beneficially affected by the Aminoplant preparation containing free amino acids. Comparing all plots, the highest protein content was determined in the seeds from plot D after Aminoplant treatment and the lowest one in the seeds from plot C after Asahi SL application, i.e., a preparation from the group of nitrophenols.

The biostimulants significantly differentiated the fat content of soybean seeds only on plot D, where both the soil herbicide prosulfocarb and the foliar herbicide bentazone and imazamox were used (Figure 5). On this plot, a significantly higher (by 4.0%) fat content was determined in the seeds from the treatment with no biostimulant compared to the treatment with Aminoplant.

The experimental factors affected the content of protein amino acids to a different extent (

Table 6. Content of exogenous amino acids in soybean seeds (g kg⁻¹ DM) depending on herbicides, type of biostimulator (mean for 2020–2022), and experimental year.

Specification	Lys	Met	Phe	Thr	Leu	Ile	Val	His	Trp
Herbicides
A	18.63 a	6.66 a	15.22 ab	11.84 a	22.54 b	12.79 a	13.29 a	7.94 a	5.87 b
B	18.71 a	6.71 a	15.09 b	11.99 a	22.99 ab	12.78 a	13.46 a	7.68 a	6.89 a
C	18.60 a	6.44 a	15.13 b	12.13 a	22.54 b	12.54 a	13.38 a	7.72 a	7.23 a
D	19.07 a	6.65 a	15.65 a	12.21 a	23.54 a	12.98 a	13.84 a	7.91 a	7.01 a
p-Value	ns	ns	**	ns	*	ns	ns	ns	***
Biostimulants
No biostimulant	18.31 b	6.33 b	15.24 a	11.90 ab	22.90 a	12.59 a	13.29 a	7.58 a	5.24 d
Asahi SL	18.60 ab	6.47 ab	15.21 a	11.60 b	22.84 a	12.87 a	13.46 a	7.87 a	7.16 b
Aminoplant	19.08 a	6.82 a	15.17 a	12.32 ab	22.59 a	12.80 a	13.57 a	7.90 a	7.99 a
Kelpak SL	19.02 ab	6.85 a	15.46 a	12.36 a	23.26 a	12.83 a	13.64 a	7.90 a	6.61 c
p-Value	*	**	ns	*	ns	ns	ns	ns	***
Years
2020	18.69 b	6.53 b	15.19 b	12.02 b	22.79 b	12.76 b	13.42 b	7.73 b	6.85 a
2021	18.47 b	6.51 b	14.93 b	11.75 b	22.45 b	12.32 b	13.13 b	7.51 b	6.32 b
2022	19.09 a	6.82 a	15.69 a	12.36 a	23.46 a	13.24 a	13.92 a	8.20 a	7.07 a
p-Value	*	*	***	*	**	*	*	***	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ns—no significant difference between treatments at p ≤ 0.05, * significance level at p ≤ 0.05, ** significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

and

Table 7. Content of endogenous amino acids in soybean seeds (g kg⁻¹ DM) depending on herbicides, type of biostimulator (mean for 2020–2022), and experimental year.

Specification	Arg	Asp	Ser	Glu	Pro	Gly	Ala	Cys	Tyr
Herbicides
A	20.66 a	35.13 a	15.07 b	53.54 b	15.70 b	12.45 a	13.09 a	6.54 b	10.06 b
B	20.90 a	35.39 a	16.28 a	56.98 a	15.90 b	12.51 a	13.15 a	6.70 ab	10.01 b
C	21.01 a	35.60 a	16.30 a	57.09 a	16.47 ab	12.64 a	12.96 a	6.65 ab	10.00 b
D	21.15 a	35.85 a	16.50 a	57.35 a	17.23 a	12.64 a	13.43 a	6.84 a	10.36 a
p-Value	ns	ns	**	***	**	ns	ns	**	***
Biostimulants
No biostimulant	20.28 b	34.78 a	14.81 b	53.14 b	16.14 a	12.09 a	12.76 a	6.38 b	9.84 b
Asahi SL	20.77 ab	35.78 a	16.53 a	57.39 a	16.25 a	12.59 a	12.93 a	6.80 a	10.17 a
Aminoplant	21.31 a	35.59 a	16.68 a	57.76 a	16.67 a	12.82 a	13.41 a	6.76 a	10.15 a
Kelpak SL	21.35 a	35.82 a	16.14 a	56.68 a	16.25 a	12.74 a	13.53 a	6.78 a	10.28 a
p-Value	**	ns	***	***	ns	ns	ns	***	***
Years
2020	20.99 b	35.51 b	16.03 b	56.08 b	16.42 b	12.57 a	13.20 b	6.73 b	10.12 b
2021	20.30 c	34.71 c	15.50 c	53.63 c	15.74 c	12.20 a	12.67 c	6.36 c	9.89 c
2022	21.49 a	36.25 a	16.60 a	59.01 a	16.82 a	12.91 a	13.60 a	6.95 a	10.32 a
p-Value	***	**	*	***	*	ns	*	***	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ns—no significant difference between treatments at p ≤ 0.05, * significance level at p ≤ 0.05, ** significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

). Among the exogenous amino acids, herbicides significantly differentiated contents of phenylalanine (Phe), leucine (Leu), and tryptophan (Trp), and among the endogenous ones, contents of serine (Ser), glutamic acid (Glu), proline (Pro), cysteine (Cys), and tyrosine (Tyr). The seeds harvested from the plot where both soil and foliar herbicide were used (D) had the highest contents of phenylalanine, leucine, and most endogenous amino acids, including serine, glutamic acid, proline, cysteine, and tyrosine. In the treatments with only soil herbicide (plot B) or only foliar herbicide (plot C), contents of phenylalanine, leucine, proline, cysteine, and tyrosine in soybean seeds were similar to these determined in the seeds from the plot without herbicide treatment (A). All treatments of herbicides (B, C, D) significantly increased the contents of tryptophan, serine, and glutamic acid compared to those determined in the seeds harvested from the plot without herbicides (A).

Among the exogenous amino acids, the use of biostimulants significantly modified the content of lysine (Lys), methionine (Met), threonine (Thr), and tryptophan (Trp), and among the endogenous amino acids, the content of arginine (Arg), serine (Ser), glutamic acid (Glu), cysteine (Cys), and tyrosine (Tyr) (Table 6 and Table 7). The use of the Aminoplant biostimulant had the most beneficial effect on the accumulation of lysine, but its content in the seeds from this treatment was significantly higher only compared to the seeds harvested from the plot without biostimulant application (by 4.2%). All tested biostimulants caused an increase in the content of tryptophan in soybean seeds compared to the seeds from the plot without the biostimulant, with the greatest increase observed upon the use of Aminoplant (by 52.5%) and the least one upon the use of Kelpak SL (by 26.1%). Crop treatment with Kelpak SL had a more beneficial effect on the threonine content compared to Asahi SL (Table 6). All tested biostimulants caused a significant increase in the contents of the following endogenous amino acids in soybean seeds: serine, glutamic acid, cysteine, and tyrosine (Table 7). The content of arginine and methionine (a representative of exogenous amino acids) was also higher upon the use of biostimulants, but it differed significantly compared to the seeds from the plot without biostimulant application only in the case of treatments with Aminoplant and Kelpak SL.

Weather conditions also significantly differentiated the contents of protein amino acids in soybean seeds (Table 6 and Table 7). The content of tryptophan turned out to be similar in the seeds harvested in 2020 and 2022 but significantly lower in those from the 2021 harvest, i.e., by 7.7% compared to the first year of the experiment (2020) and by 10.6% compared to 2022 (Table 6). The contents of the other exogenous amino acids were the highest in the seeds from 2022, which was characterized by the highest temperature and the lowest precipitation. Contents of exogenous amino acids determined in the seeds in the other study years were similar and significantly lower than in those harvested in 2022. Among the endogenous amino acids, only the content of glycine did not differ significantly between the individual years of the experiment (Table 7). The contents of the other endogenous amino acids were the highest in seeds from 2022 and the lowest in those from 2021 when the air temperature recorded in the month of soybean ripening (August) turned out to be the lowest among the study years and compared to the multi-year period.

The interaction of herbicides and biostimulant types significantly differentiated the contents of amino acids listed in

Table 8. Content of exogenous amino acids in soybean seeds (g kg⁻¹ DM) depending on the interaction of herbicides and the type of biostimulator (mean for 2020–2022).

Herbicides	Biostimulants	Lys	Phe	Leu	His	Trp
A	No biostimulant	17.40 b	14.30 cd	21.92 abc	6.81 e	3.76 h
	Asahi SL	18.98 ab	16.35 a	24.18 a	8.59 ab	8.63 ab
	Aminoplant	18.42 ab	14.17 d	20.51 c	7.39 cde	7.58 abcd
	Kelpak SL	19.71 a	16.05 ab	23.53 ab	8.95 a	3.50 h
B	No biostimulant	18.03 ab	15.53 abc	23.13 abc	7.59 bcde	5.23 g
	Asahi SL	18.87 ab	15.03 bcd	22.80 abc	7.55 bcde	7.81 abc
	Aminoplant	19.08 ab	14.98 bcd	23.44 ab	8.06 abcd	6.71 cdef
	Kelpak SL	18.85 ab	14.80 bcd	22.59 abc	7.53 bcde	7.82 abc
C	No biostimulant	18.92 ab	15.31 abcd	22.73 abc	8.35 abcd	6.11 efg
	Asahi SL	17.59 b	14.46 cd	21.48 bc	7.23 de	6.26 defg
	Aminoplant	18.92 ab	15.70 ab	23.08 abc	7.68 bcde	8.81 a
	Kelpak SL	18.95 ab	15.04 bcd	22.86 abc	7.63 bcde	7.74 abc
D	No biostimulant	18.88 ab	15.82 ab	23.83 ab	7.57 bcde	5.87 fg
	Asahi SL	18.95 ab	15.01 bcd	22.91 abc	8.11 abcd	5.95 fg
	Aminoplant	19.88 a	15.84 ab	23.33 ab	8.47 abc	8.85 a
	Kelpak SL	18.58 ab	15.93 ab	24.07 ab	7.49 bcde	7.36 bcde
p-Value		*	***	***	***	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). * Significance level at p ≤ 0.05, *** significance level at p ≤ 0.001.

and

Table 9. Content of endogenous amino acids in soybean seeds (g kg⁻¹ DM) depending on the interaction of herbicides and the type of biostimulator (mean for 2020–2022).

Herbicides	Biostimulants	Arg	Ser	Glu	Pro	Cys	Tyr
A	No biostimulant	19.00 b	12.74 c	47.01 d	15.97 abc	5.75 e	9.02 f
	Asahi SL	21.47 a	16.58 ab	56.78 abc	16.44 abc	7.00 ab	10.55 bc
	Aminoplant	20.43 ab	15.33 abc	54.72 bc	16.54 abc	6.72 abcd	9.58 e
	Kelpak SL	21.74 a	15.63 abc	55.65 abc	13.86 c	6.70 abcd	11.10 a
B	No biostimulant	20.01 ab	14.63 bc	53.17 c	16.04 abc	6.35 cd	9.77 de
	Asahi SL	20.76 ab	17.68 a	60.30 a	15.75 abc	7.07 a	9.92 de
	Aminoplant	21.04 ab	15.84 ab	56.03 abc	15.61 abc	6.46 bcd	10.30 bcd
	Kelpak SL	21.78 a	16.97 ab	58.40 ab	16.21 abc	6.92 ab	10.05 cde
C	No biostimulant	21.11 ab	16.43 ab	56.59 abc	16.74 ab	6.71 abcd	10.56 bc
	Asahi SL	19.98 ab	15.11 abc	54.83 bc	14.78 b	6.26 de	9.52 ef
	Aminoplant	21.69 a	17.61 a	60.10 a	16.70 abc	6.86 abc	9.90 de
	Kelpak SL	21.24 ab	16.06 ab	56.85 abc	17.64 ab	6.76 abcd	10.02 de
D	No biostimulant	21.01 ab	15.43 abc	55.80 abc	15.79 abc	6.70 abcd	10.00 de
	Asahi SL	20.88 ab	16.74 ab	57.63 abc	18.02 a	6.88 abc	10.70 ab
	Aminoplant	22.09 a	17.93 a	60.17 a	17.83 a	7.01 ab	10.80 ab
	Kelpak SL	20.62 ab	15.90 ab	55.81 abc	17.29 ab	6.76 abcd	9.94 de
p-Value		**	***	***	***	***	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ** Significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

. Contents of lysine, histidine, tryptophan, arginine, serine, glutamic acid, cysteine, and tyrosine were the lowest in the seeds from the plot without herbicide (A) and biostimulant treatment. The use of the Aminoplant biostimulant on the plot with soil and foliar herbicide (D) had the most beneficial effect on the contents of lysine, tryptophan, arginine, and serine. In turn, the seeds harvested from the plot without herbicide treatment (A) but with Kelpak SL application had the highest contents of histidine and tyrosine. Furthermore, the highest contents of phenylalanine and leucine were determined in the seeds after Asahi SL application on plots without chemical protection (A), and that of proline in the seeds after this biostimulant application on the plot with both prosulfocarb and bentazone + imazamox herbicides (D). The use of only the soil herbicide (B) and the Asahi SL preparation had the most beneficial effect on the content of glutamic acid and cysteine in soybean seeds.

The major fatty acids of soybean oil were saturated acids, including palmitic (C16:0) and stearic (C18:0), monounsaturated acids: oleic + elaidic (C18:1n9c + C18:1n9t), and polyunsaturated acids, linoleic + linolelaidonic (C18:2n6c + C18:2n6t) and α-linolenic (C18:3n3 alpha). The remaining acids accounted for only 3.58% of total FAs (

Table 10. Fatty acid profile (%) in soybean oil depending on herbicides, type of biostimulant (mean for 2020–2022), and experimental year.

Specification	C16:0	C18:0	C18:1n9c + C18:1n9t	C18:2n6c + C18:2n6t	C18:3n3 (Alpha)	Others
Herbicides
A	10.76 a	4.06 a	20.02 a	53.79 a	8.26 a	3.11 b
B	10.88 a	3.98 a	20.35 a	53.34 a	8.09 a	3.36 b
C	11.00 a	4.03 a	19.08 b	53.01 a	8.28 a	4.60 a
D	10.77 a	3.92 a	20.23 a	53.71 a	8.12 a	3.25 b
p-Value	ns	ns	***	ns	ns	***
Biostimulants
No biostimulant	11.00 a	4.02 a	19.15 c	52.68 b	8.33 ab	4.82 a
Asahi SL	10.70 b	3.95 a	19.98 b	54.15 a	8.08 bc	3.14 b
Aminoplant	11.16 a	4.05 a	19.89 b	53.36 ab	8.47 a	3.07 b
Kelpak SL	10.54 b	3.98 a	20.65 a	53.66 ab	7.88 c	3.29 b
p-Value	***	ns	***	*	***	***
Years
2020	10.30 b	3.87 b	20.86 a	53.42 a	7.80 b	3.75 a
2021	11.04 a	4.08 a	19.38 b	53.77 a	8.31 a	3.42 b
2022	11.21 a	4.05 ab	19.51 b	53.20 a	8.45 a	3.58 ab
p-Value	***	*	***	ns	***	**

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ns—no significant difference between treatments at p ≤ 0.05, * significance level at p ≤ 0.05, ** significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

).

Herbicides significantly differentiated the percentage of monounsaturated fatty acids, C18:1n9c + C18:1n9t, in the fatty acid profile. The significantly lowest content of these acids was shown in the soybean oil from the seeds harvested from the plot protected exclusively with the foliar herbicide bentazone and imazamox (C) (Table 10). The highest total saturated fatty acid (SFA) content and the lowest monounsaturated fatty acid (MUFA) content were determined in the oil from the seeds harvested from plot C (

Table 11. Fatty acid profile (%) in soybean oil depending on herbicides, type of biostimulant (mean for 2020–2022), and experimental year—continuation.

Specification	SFA	MUFA	PUFA	OMEGA 3	OMEGA 6	OMEGA 9
Herbicides
A	15.99 b	20.50 b	63.51 a	8.61 a	54.82 a	20.45 a
B	16.14 ab	21.02 a	62.84 a	8.42 a	54.43 a	20.89 a
C	16.43 a	19.88 c	63.68 a	8.84 a	54.85 a	19.76 b
D	15.93 b	20.83 ab	63.24 a	8.45 a	54.79 a	20.71 a
p-Value	**	***	ns	ns	ns	***
Biostimulants
No biostimulant	16.44 a	20.02 c	63.54 a	8.90 a	54.64 ab	19.89 c
Asahi SL	15.87 b	20.53 b	63.61 a	8.40 ab	55.20 a	20.44 b
Aminoplant	16.34 a	20.41 bc	63.26 a	8.86 ab	54.32 b	20.31 bc
Kelpak SL	15.85 b	21.28 a	62.87 a	8.14 b	54.72 ab	21.17 a
p-Value	***	***	ns	*	*	***
Years
2020	15.55 b	21.56 a	62.89 a	8.02 b	54.84 ab	21.46 a
2021	16.33 a	19.89 b	63.78 a	8.76 a	55.02 a	19.78 b
2022	16.49 a	20.23 b	63.29 a	8.95 a	54.31 b	20.12 b
p-Value	***	***	ns	**	*	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ns—no significant difference between treatments at p ≤ 0.05, * significance level at p ≤ 0.05, ** significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

). The use of foliar herbicide only (plot C) significantly reduced the content of Omega 9 acids compared to plots A, B, and D.

In soybean oil from the seeds harvested from the plot without biostimulants, the C16:0 content and the sum of SFA were similar to those achieved after Aminoplant treatment and significantly higher than after Asahi SL and Kelapk SL application (Table 10 and Table 11). The use of all tested biostimulants had a beneficial effect on the content of C18:1n9c + C18:1n9t acids. On the other hand, the total content of monounsaturated fatty acids (MUFA) in the oil was significantly higher only after the application of Asahi SL and Kelpak SL preparations compared to the treatment without biostimulant. The total content of polyunsaturated fatty acids (PUFA) did not differ significantly depending on the biostimulant. However, the use of Kelpak SL reduced the content of polyunsaturated acid C18:3n3 (alpha) compared to the treatment without biostimulant and to that with Aminoplant. A significant increase in the content of C18:2n6c + C18:2n6t compared to the treatment without the biostimulant was noted only after Asahi SL. The seeds harvested from the plot without the biostimulant treatment had the highest sum of Omega 3 acids and the lowest sum of Omega 9 acids. However, the sum of Omega 3 was significantly higher only compared to treatmentsntly from that obtained in the treatment with Aminoplant.

Heavy rainfalls during the soybean growing season of 2020 promoted the accumulation of monounsaturated fatty acids (MUFA), especially oleic + elaidic (C18:1n9c + C18:1n9t), as well as Omega 9 acids, and decreased the contents of palmitic and α-linolenic acid, the sum of saturated fatty acids (SFA) and Omega 3 acids in the seeds (Table 10 and Table 11). In contrast, in 2022, characterized by the highest temperature and the lowest precipitation, the seeds had the highest contents of palmitic and α-linolenic acid and the sum of saturated fatty acids (SFA) and Omega 3 acids, which differed significantly from those determined in the seeds harvested in 2020.

The interaction of herbicides and the type of biostimulant significantly differentiated the contents of fatty acids listed in

Table 12. Fatty acid profile (%) in soybean oil depending on the interaction of herbicides and the type of biostimulant (mean for 2020–2022).

Herbicides	Biostimulants	C16:0	C18:2n6c + C18:2n6t	C18:3n3 (Alpha)	SFA	MUFA	OMEGA 6	OMEGA 9
A	No biostimulant	11.02 bcd	53.19 ab	8.54 abc	16.21 bc	20.58 bcd	54.30 ab	20.38 cde
	Asahi SL	11.49 ab	54.72 a	8.64 ab	16.69 ab	18.59 e	55.53 a	18.59 f
	Aminoplant	10.70 bcde	53.54 ab	8.16 bcd	15.94 bcd	20.73 bcd	54.52 ab	20.83 bcd
	Kelpak SL	9.84 e	53.70 ab	7.69 bcd	15.12 d	22.10 ab	54.92 ab	22.00 ab
B	No biostimulant	11.13 abc	52.91 abc	8.31 bcd	16.41 ab	20.86 bcd	54.05 ab	20.69 bcd
	Asahi SL	11.30 ab	54.01 ab	8.39 bcd	16.41 ab	19.79 cde	54.98 ab	19.68 def
	Aminoplant	10.80 bcde	53.99 ab	8.17 bcd	15.92 bcd	20.50 bcd	55.06 ab	20.44 cde
	Kelpak SL	10.29 cde	52.45 bc	7.50 d	15.83 bcd	22.92 a	53.61 ab	22.74 a
C	No biostimulant	10.68 bcde	50.98 c	7.95 bcd	16.78 ab	18.66 e	55.48 ab	18.63 f
	Asahi SL	10.04 d	54.35 ab	7.60 cd	15.23 cd	21.56 abc	55.46 ab	21.45 abc
	Aminoplant	12.04 a	52.66 bc	9.38 a	17.25 a	19.40 de	53.44 b	19.16 ef
	Kelpak SL	11.22 abc	54.06 ab	8.20 bcd	16.47 ab	19.90 cde	55.01 ab	19.80 def
D	No biostimulant	11.18 abc	53.65 ab	8.50 abc	16.35 ab	19.98 cde	54.74 ab	19.86 def
	Asahi SL	9.97 e	53.52 ab	7.69 bcd	15.14 d	22.16 ab	54.83 ab	22.05 ab
	Aminoplant	11.10 abc	53.23 ab	8.16 bcd	16.25 ab	20.99 bcd	54.25 ab	20.80 bcd
	Kelpak SL	10.82 bcde	54.43 ab	8.12 bcd	15.98 bcd	20.20 cde	55.35 ab	20.14 cde
p-Value		***	***	***	***	***	**	***

A—Control plot (no herbicides), B—prosulfocarb, C—bentazone and imazamox, D—prosulfocarb, bentazone and imazamox. Different letters denote significant differences (p ≤ 0.05). The same letter means not significantly different values (p ≤ 0.05). ** Significance level at p ≤ 0.01, *** significance level at p ≤ 0.001.

. The highest content of C18:2n6c + C18:2n6t acids and the sum of Omega 6 acids was determined in soybean oil from the seeds harvested from the plot without herbicides (A) after the Asahi SL biostimulant application. The use of only the soil herbicide (plot B) and the Kelpak biostimulant promoted the accumulation of monounsaturated fatty acids and Omega 9 acids. The treatment with only the foliar herbicide (C) and the Aminoplant preparation resulted in the highest contents of palmitic and α-linolenic acids and the sum of saturated fatty acids (SFA). The use of complex herbicides (plot D) in combination with the foliar Asahi SL biostimulant caused the greatest reduction in the content of saturated fatty acids (especially C16:0 acid), the excessive consumption of which adversely affects human health.

The fatty acid content of soybean seed oil was not significantly differentiated by the experimental factors in the individual years of the study.

4. Discussion

Soybean development and, consequently, its yield are influenced by various biotic and abiotic stress factors. Among them, weed infestation of the crop is an important biotic constraint reducing soybean yield [16]. This is due to the fact that the initial growth of soybean is slow, and the first 30 days after sowing are considered critical in terms of its competition with weeds. Heavy weed infestation leads to reduced yield and adversely affects seed quality [31,32]. Currently, the available herbicides may be applied both directly after sowing and after soybean emergence, and their earliest possible use is recommended, given the high competitiveness of weeds in the initial period of soybean development [33]. In the present study, prosulfocarb applied immediately after seed sowing, both when used alone and in combination with follow-up application of a foliar herbicide (bentazone, imazamox), proved better in crop protection and had a more beneficial effect on the quality of soybean seeds than the use of a foliar herbicide (bentazone, imazamox) alone. In the study by Chaudhari et al. [32], the pre-emergence application of herbicides (imazethapyr) ensured higher soybean productivity compared to the post-emergence treatment. Likewise, in the present study, Nainwal and Saxena [18] showed that omitting the herbicide treatment reduced seed yield and yield parameters, such as, among others, pod number per plant. However, these authors determined a higher seed yield after applying the herbicide before plant emergence (diclosulam) than after soybean sowing, followed by haloxyfop application post-emergence. Young et al. [34] obtained different results than those presented in our study. They demonstrated reduced soybean yields in the case of acifluorfen and imazethapyr treatments compared to those obtained on plots without herbicides, by 1.5 and 2.1%, respectively. In the study by Poston et al. [10], the reduction in seed yield after herbicide application depended on the type of active substance, as pendimethalin reduced the yield by 4% and S-metolachlor by 3%. The yield reduction resulted from herbicide-induced plant damage, the severity of which was largely dependent on the soybean cultivar and weather conditions and reached even up to 50%. In the present study, we did not observe any phytotoxic effect of prosulfocarb, bentazone, and imazamox on the crop plant, which was also indicated by the similar density of soybean plants determined on the control and herbicide-protected plots.

The present study results demonstrated that among the tested herbicides, the one with prosulfocarb applied directly after sowing in combination with a follow-up application of a foliar herbicide (bentazone, imazamox) significantly increased the protein content and reduced the fat content of soybean seeds. According to Guo et al. [35], the content of protein is a quantitative feature usually negatively correlated with the oil content. This was also confirmed in our experiment. In contrast, Movahedpour et al. [9] obtained higher protein and oil contents in the seeds from plots treated with trifluralin herbicide (application before sowing), which, according to these authors, was associated with greater competitiveness of weeds on the plot not protected with the herbicide. Similarly, Nainwal and Saxena [18] demonstrated that all herbicides analyzed in their experiment, including fluchloralin, pendimethalin, and diclosulam, increased the protein and fat contents of soybean seeds by about 12 and 15% compared to the seeds from the plot with no herbicides. Also, the total content of unsaturated fatty acids (oleic, linoleic, linolenic) was higher in the treatments with herbicides. Conversely, the present study showed that the applied herbicides did not differentiate the total content of polyunsaturated fatty acids (PUFA), including that of linoleic and linolenic acids. On the other hand, the lowest total content of monounsaturated fatty acids (MUFA) was obtained after the application of only the foliar herbicide bentazone and imazamox. In the treatment with prosulfocarb, the total content of MUFA in the seeds was higher than in those from the plot without chemical protection against weeds. According to Nainwal and Saxena [18], a lower total saturated fatty acid content of the seeds is achieved upon the use of certain herbicides (fluchloralin, pendimethalin, haloxyfop), which may result from a modification or inhibition of the rate of lipid metabolism by these chemicals. However, these authors did not observe such an effect after the application of diclosulam. Also, the herbicides used in our study did not cause a significant decrease in the SFA content, and the herbicide bentazone and imazamox used alone actually increased the total content of saturated fatty acids compared to the treatment without chemical protection.

Amino acids are essential to maintain proper body functions, as they are involved in metabolic processes, transport, and storage of all nutrients, including carbohydrates, proteins, vitamins, minerals, water, and fats [36,37]. Soybean seeds represent a high-value source of protein because they contain all essential amino acids that must be supplied with food [38]. Investigations into the effect of herbicides on the content of amino acids in seeds of crops indicate that this effect depends on the type of active substance [36,39]. Free amino acids accumulate in plants mostly as a result of natural environmental stress but also under the influence of herbicides containing diuron [17,40]. The present study results showed that weed control with the prosulfocarb immediately after sowing, followed by the application of a foliar herbicide bentazone and imazamox, caused the greatest increase in the content of phenylalanine and leucine and most endogenous amino acids. In turn, a study by El-Sobki et al. [37] showed that the use of foliar herbicides (pinoxaden, clodinafop-propargyl, and pyroxsulam) led to a significant decrease in the content of essential and endogenous amino acids and protein in seeds. However, the active substance tribenuron-methyl increased the levels of proline, glycine, arginine, and histidine but had no effect on cystine and threonine contents compared to the control treatment. In turn, in the present study, among the amino acids mentioned, the herbicides used differentiated only the contents of proline and cysteine, which were the highest when prosulfocarb was applied immediately after seed sowing and followed by the application of a foliar herbicide (bentazone, imazamox). These results are similar to those obtained by other researchers, who found that the accumulation of proline in seeds increased significantly along with an increase in the dose of active substances [41,42]. This accumulation of proline can be explained as a defense mechanism against stress triggered by the use of chemical substances [42]. According to Fayez et al. [42], the effect of herbicides on the content of amino acids in plants may vary depending on their dose because treatments with a lower herbicide dose caused an increase in the content of free amino acids, whereas higher doses of fusilade and basagran were observed to decrease it.

The productivity of plants, including soybean, can be increased by using biostimulants, which alleviate the adverse effects of various stress factors impeding vital processes in plants [25,43]. Biostimulants provide an additional source of amino acids, facilitating the opening of stomata and water retention in plants, thus stimulating photosynthesis and metabolic processes [44]. A study by Franzoni et al. [19] also indicated that the use of biostimulants alleviates the symptoms of stress in plants by increasing the content of chlorophyll, the level of which decreased significantly upon the use of herbicides.

The present study results also show that all tested biostimulants resulted in significantly higher seed yield and plant height. The highest yield increase was observed after the treatment with Kelpak biostimulant, produced from seaweed and containing auxins and cytokinins. Similarly, experiments conducted by other researchers indicated that the use of seaweed extracts had a positive effect on plant productivity [45] and increased the height of soybean plants [46]. Physiological effects induced in plants by auxins include cell elongation, apical dominance, phototropism and geotropism, root and pod growth, and seed formation enhancement, thus playing a fundamental role in plant growth and development [47]. Cytokinins, on the other hand, perform important functions, such as delaying plant aging as well as reducing the formation of free radicals, and, consequently, inhibiting phospholipid degradation, as well as participating in the regulation of many other processes in plants [48].

Similarly to the present study results, the findings reported by other authors also indicate a beneficial effect of biostimulants on plant yield [21,28,49]. Rymuza et al. [25] showed that the use of Asahi (analyzed in our study) and Improver (potassium p-nitrophenolate, potassium o-nitrophenolate, and potassium 5-nitroguaiacolate) biostimulants contributed to a significant increase in yield and the values of other yield traits, including 1000-seed weight and seed number per pod. Morais et al. [50] also proved that the biostimulant increased the 1000-seed weight and final productivity. The 1000-seed weight determined in our study after the use of biostimulants was also higher but did not differ statistically significantly from the other treatments tested. In the case of soybean treated with algae-based biostimulants, Melo et al. [51] observed higher profitability of cultivation, as well as higher seed yield and weight. A study by Kocira et al. [24] showed that the use of the natural biostimulant Fylloton containing 19 amino acids of plant origin had a positive effect on the number of seeds and pods and, similarly to our experiment, on soybean yield and plant height. The positive effect of biostimulants based on amino acids on plant growth, development, and yield is probably due to the fact that they stimulate the plant’s defense response to stress at the molecular level [52]. The amino acids they contain are involved in the synthesis of many organic compounds and also affect the uptake of macro- and micro-elements [53].

The results presented in this work do not confirm the effect of the interaction of herbicides and biostimulant type on soybean seed yield. Similarly, Franzoni et al. [19] demonstrated that the use of biostimulants in soybean cultivation had a positive effect on its yield, but the combined use of herbicides and biostimulants did not increase it.

The results achieved in the present study and those reported by other authors indicate that the use of biostimulants affects the seed quality characteristics of soybean and other crops. The results of the research by Petrova et al. [54] proved that the use of stimulants (except for those dressed with HL 100—Humusil) caused a slight increase in the fat content of seeds compared to the seeds from the control treatment. Kocira et al. [24] found that the application of a biostimulant containing plant-derived amino acids generally caused an increase in the protein content of the seeds, but this increase was dependent on the number of applications, biostimulant concentration, and soybean cultivar. Likewise, in our study, the seeds from the treatment with Aminoplant preparation containing free amino acids had a significantly higher protein content and a lower fat content compared to the seeds from the plot not treated with the biostimulant. The results of the research by other authors also showed a positive effect of a seaweed extract [55,56] and stimulants containing free amino acids [56,57] on the protein content of legume seeds. Investigations on the effect of biostimulants on the content of amino acids in seeds are, however, scarce. Iwaniuk et al. [58] have pointed to a variable effect of the combined use of a humic biostimulant and pesticides on the content of amino acids in wheat grain that was largely dependent on weather conditions. According to these authors, plant protection products or biostimulants may act as stress factors contributing to the breakdown of complex proteins into free amino acids, which are then used for the biosynthesis of defense proteins. Biostimulants may facilitate the uptake of nutrients by supporting metabolic processes in the soil and plants, i.e., by facilitating the development of mycorrhizal fungi that transport nutrients to the plant [59]. The results of our experiment indicate that biostimulants have a beneficial effect on the content of certain amino acids, including lysine, methionine, tryptophan, and some endogenous amino acids in soybean seeds. In turn, Jakiene [44] demonstrated that the use of a biopreparation containing amino acids affected the content of fatty acids in rapeseed seeds. Our research also demonstrated that crop treatment with Aminoplant biostimulant promoted the accumulation of polyunsaturated C18:3n3 (alpha) acid in soybean oil.

The present study showed that the yield and quality of soybean seeds were related to the weather conditions in the individual growing seasons. The highest seed yield, plant density after emergence and before harvest, and seed weight per plant were obtained in 2022, characterized by the highest average air temperature among the study years, including also in the month of soybean sowing (May), which had a positive effect on the emergence of plants and, consequently, on the yield. In 2020, characterized by the lowest temperature in the month of soybean sowing (May) and flowering (July) and by excessive rainfall (May, June), the seed yields were the lowest. According to Ergo et al. [60], unfavorable weather conditions during the formation of generative organs can disrupt photosynthesis and thus disturb plant metabolism, which results in decreased weight and number of seeds, causing lower soybean yields. This is confirmed by the results obtained in the present study. Rymuza et al. [25] and Hwang et al. [61] demonstrated that the main factors limiting the cultivation of this species are temperature and precipitation during germination and flowering. In the 2020 season, we also showed that the use of biostimulants increased the seed yield compared to the no-biostimulant treatment, which indicates a beneficial effect of these preparations on the development and yield of soybean under stressful conditions. The present study also showed that the protein content of seeds depended on the weather conditions in the individual growing seasons of soybean, with the highest protein content recorded in 2022, characterized by the highest average air temperature and the lowest precipitation in the soybean growing season. Similarly, the studies by Wilcox and Shibles [62] and Gawęda et al. [63] demonstrated that soybean seeds usually accumulated more protein and less oil in warm and dry summers.

5. Conclusions

The complex herbicide protection was found to elicit the most beneficial effect on seed yield and protein content of soybean seeds. It also ensures the greatest increases in contents of phenylalanine and leucine as well as most endogenous amino acids, like serine, glutamic acid, proline, cysteine, and tyrosine, while decreasing the fat content of soybean seeds.

The application of a foliar herbicide alone (bentazone, imazamox) decreased the total content of monounsaturated fatty acids (MUFA) and Omega 9 fatty acids and increased the total content of saturated fatty acids (SFA) in soybean seeds.

The results of the present experiment point to the advisability of using biostimulants in soybean cultivation as an element boosting its seed yield and seed quality parameters. Their application is particularly recommended under stress conditions, as indicated by higher soybean seed yields produced after the application of all tested biostimulants in the study year with low temperatures recorded in the months of seed sowing and initial growth.

All tested biostimulants enable achieving a higher seed yield and higher contents of tryptophan, serine, glutamic acid, cysteine, tyrosine, and C18:1n9c + C18:1n9t acids in the seeds, with the greatest seed yield increased noted upon the application of Kelpak SL biostimulant (an extract from Ecklonia maxima algae).

The biostimulant containing free amino acids (Aminoplant) and that based on the Ecklonia maxima algae (Kelpak SL) were found to positively affect the contents of arginine and methionine in soybean seeds. The first ones additionally enabled producing soybean seeds with high contents of protein and lysine but with a reduced fat content.

The use of a biostimulant with an active compound from a group of nitrophenol (Asahi SL) and the one based on marine algae (Kelpak SL) had a positive impact on the total content of Omega 9 acids and decreased the total content of saturated fatty acids (SFA), palmitic acid (C16:0) in particular, in soybean oil.

The study results demonstrate that the complex herbicide protection, involving the use of both soil and foliar herbicides, coupled with the application of biostimulants, may be recommended for soybean cultivation, particularly in growing seasons with adverse weather conditions.