Effects of antimony on the soil enzymes, microbial, and rhizobial activity of legumes

ABSTRACT

Leguminous farmland in China is severely polluted by antimony. Here, to explore the effects of antimony stress on rhizobia and soil microorganisms in leguminous plants, mung beans, kidney beans, and peanuts were grown in soil with different concentrations of Sb³⁺, Sb⁵⁺, and Sb³⁺+Sb⁵⁺. Antimony had a promotional effect on soil sucrase and catalase activities and an inhibitory effect on urease activity. The effects of Sb³⁺ on sucrase, catalase, and urease activities were 3.3–11.1, 1.27–1.29, and 1.24–3.05 times those of Sb⁵⁺, respectively. Antimony reduced the rhizobial activity by 20.63–57.84%, with the inhibitory effect positively related to the antimony content. Additionally, the inhibitory effect of Sb³⁺ was 1.10–2.22 times that of Sb⁵⁺. This inhibition decreased the total nitrogen and ammonia nitrogen content in the soil. However, the decrease in Sb³⁺ was 0.46–1.54 times that of Sb⁵⁺. Sb³⁺ significantly altered the microbial activity.

KEYWORDS:

• Antimony
• soil enzyme activities
• nitrogen content
• rhizobia
• microbial

1. Introduction

China currently holds 70–80% of the world’s antimony stockpile [1], with 3.28 million tons of antimony ore reserves identified [2]. With China’s rapid economic development, large amounts of antimony have entered the environment [3,4]. Antimony concentrations in the Taipu River basin in China have exceeded the standard on several occasions, affecting the normal operation of the Taipu River water source to varying degrees and even leading to the closure of the water intake at the downstream water source [5]. Antimony levels in the contaminated Taipu River basin range from 21.00 to 115.50 mg·kg⁻¹. Southwest China is a severely affected area of antimony pollution [6]. Antimony concentrations in soil contaminated with heavy metals have been found to reach 36.793 mg·kg⁻¹, which seriously increases the risk of cancer [7]. A previous study found that the average antimony contents in the sediments of the Hunan tin mine and Yatta gold mine were 1,163 and 149 mg·kg⁻¹, respectively, ranging from 57.17 to 7,315 mg·kg⁻¹ and from 111 to 223 mg·kg⁻¹, respectively [8]. The detrimental effects of antimony on biological growth have been widely documented [9]. Antimony is extremely mobile and readily enters plants [10]. Farmland located near antimony smelters and chemical plants that contain antimony is seriously contaminated with antimony [11]. The roots of some plants have a strong ability to absorb antimony, and can accumulate a large amount of trivalent and pentavalent antimony [12]. In another study, researchers tested 34 plants and found that antimony concentrations ranged from 3.92 to 143.69 mg∙kg⁻¹. These researchers found that plants in the Sargassaceae family had the highest antimony content (98.23 mg·kg⁻¹), while plants in the Dryoptera family had the lowest antimony content (6.43 mg·kg⁻¹) [13]. The roots, stems, and leaves of Ficus plants commonly found in mining areas exhibit a strong antimony enrichment ability, and the highest antimony concentrations at the subcellular level are found in the cell wall (72.4%–87.5%) and cytoplasm (8.2%–18.6%) [14]. The World Health Organization stipulates that the daily antimony intake of humans should not exceed 0.86 mg·kg⁻¹ [15], but in polluted areas, this value is easily exceeded. Antimony pollution increases the risk of human cancer and can also damage soil quality.

Antimony in the soil changes from Sb³⁺ to Sb⁵⁺, and the extent to which antimony changes influences its toxic effects in the soil. Most previous studies focused on the toxicity of trivalent antimony to the soil environment [16]. Researchers have investigated the effects of Sb³⁺ on soil and microorganisms, including the distribution of microorganisms in antimony-contaminated soil [17] and the potential applications of microorganisms in improving antimony-contaminated soil [18], while fewer studies have explored the differences in soil toxicity between Sb³⁺, Sb⁵⁺, and mixed antimony. In this study, soils contaminated with different concentrations of Sb³⁺, and Sb⁵⁺, and mixed antimony were set up. Mung bean (Vigna radiata), kidney bean (Phaseolus vulgaris), and peanut (Arachis hypogaea Linn.) were planted into each group of soils to investigate the effects of antimony in different shelf states on the soil enzyme activity, soil nitrogen content, rhizobial activity, and microbial diversity; to analyze the differences in the effects of three types of antimony pollution on soil and microorganisms; and to explore the differences in toxicity between Sb³⁺ and Sb⁵⁺.

2. Materials and methods

2.1. Experimental materials

2.1.1 Test seeds

Mung bean, kidney bean, and peanut seeds were purchased from the Jing Yue seed market of Changchun City, Jilin Province, China

2.1.2 Soil configuration

The soil was sampled from the green area of Dong Hua University. The soil type and the estimated key chemical attributes and composition of the soil are listed below in Table 1.

Table 1. Basic physicochemical properties of test soil.

Physicochemical properties	Results
Soil type	Loam
pH	7.7
Sand	7%
Clay	40%
Silt	53%
Cation exchange capacity	8 cmol·kg^−1
Total organic content	19.20 g·kg^−1
Electrical conductivity	269 μs·cm^−1
Available phosphorus	56.7 mg·kg^−1
Total K	175.45 mg·kg^−1
Total Ca	6.04 g·kg^−1
Total Mg	1.01 g·kg^−1
Total Zn	11.57 mg·kg^−1
Total Fe	5.25 g·kg^−1
Total Cu	0.78 mg·kg^−1

2.2. Experimental method

Soil was placed into basins, with 1.5 kg per basin. L-antimony potassium tartrate (C₈H₄K₂O₁₂Sb₂) and SbCl₅ were used to prepare antimony-bearing soils with concentration gradients of 0, 100, 200, and 400 mg·kg⁻¹. KCl was added to compensate for the concentration of K⁺ in the control samples. After evenly mixing the solution with the soil and letting it stand for a week, the seeds of three leguminous plants (mung bean, kidney bean, and peanut) were sown in the soil. The experiment used three parallel replicas, with the only differences being in the concentration of the heavy metal antimony; otherwise, each pot had similar conditions. Distilled water was applied periodically to compensate for water evaporation during incubation [19]. After 2 months of growth, the rhizobia and nearby soil were collected as samples. The rhizobia of the three leguminous plants were tested synchronously after being extracted from the roots. The samples were stored in a − 5°C freezer.

2.3. Determination method

2.3.1 Enzyme activity

Soil sucrase activity was determined using the colorimetric method [20]. Soil catalase activity was determined using the UV spectrophotometric method [21]. Soil urease activity was determined using colorimetric method [10].

2.3.2 Rhizobial activity

Determination of rhizobia activity used the acetylene method. Acetylene could be reduced to ethylene by rhizobia. Acetylene gas was injected into a closed vessel containing an ammonia solution of a monovalent copper salt, and the interaction produced acetylene copper, which made the solution appear purplish red. The obtained color was determined by a spectrophotometer [22].

2.3.3 Ammonia nitrogen and total nitrogen content

Ammonia nitrogen content was determined using the spectrophotometric method [23]. The total nitrogen content was determined using the Kjeldahl method [24].

2.3.4 Soil microbial diversity

The microbial diversity was tested by Hangzhou Yanqu Information Technology Co., Ltd. to complete the composition spectrum analysis of microbial community diversity (limited by experimental funding, the samples with the most pronounced data were sent for sampling: the control (CK), Sb⁵⁺ (200 mg·kg⁻¹), and Sb³⁺ (200 mg·kg⁻¹) treatments). Briefly, the polymerase chain reaction (PCR) for the bacterial V4–V5 region of the 16s rDNA gene was amplified using the Novaseq-PE250 (GTGCCAGCMGCCGCGGTAA CCGTCAATTCMTTTRAGTTT) with Q5 high-fidelity DNA polymerases. The quality of DNA extraction was checked using 1.2% agarose gel electrophoresis. PCR amplification was performed using Pfu high-fidelity DNA polymerase purchased from Alltech Gold, and the number of amplification cycles was strictly controlled [25–27]. The PCR amplification products were quantified based on fluorescence. The fluorescence reagent used was the Quant-iT PicoGreen dsDNA Assay Kit, and the quantification instrument was a microplate reader (BioTek, FLx800) [28]. Sequencing libraries were prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit [29,30]. A base was added at the 3’ end of the DNA sequence to prevent the self-association of the DNA fragment and to ensure that the target sequence could be attached to the sequencing junction (there was a prominent T base at the 3’ end of the sequencing junction) [26]. A sequencing junction containing a library-specific tag (i.e. an index sequence) was added at the 5’ end of the sequence to enable the DNA molecule to be immobilized on the Flow Cell;. BECKMAN AMPure XP Beads were used to purify the library system after the addition of the junction by removing the self-linked fragments through magnetic bead screening [31,32]. PCR amplification was performed on the above-linked DNA fragments to enrich the sequencing library template, and BECKMAN AMPure XP Beads were employed to purify the library enrichment product again. Final fragment selection and purification of the library were performed using 2% agarose gel electrophoresis. The final selection and purification of the library were performed via 2% agarose gel electrophoresis.

Each of the high-quality denoised sequences was called an amplicon sequence variant (ASV), and their sequences were clustered into operational taxonomic units (OTUs) at a sequence similarity level of > 97%. The Green Genes database was used to compare the 16S rRNA genes of bacteria, construct a phylogenetic tree, and calculate the abundance of each taxon [33,34].

2.4. Statistical analyses

The average and standard deviation for the results of each experiment are displayed (M ± SD, n = 3). The statistical analysis was conducted using MS Excel 2016, whereas the supporting charts, plots, and curves were created using Origin 2021.

3. Results

3.1. Effect of antimony on soil enzyme activity

Figure 1(a) depicts the effects of different valence states of antimony on sucrase in soil. The results indicated that sucrase activity in the soil increased with increasing antimony content. When the concentrations of Sb³⁺ were 100, 200, and 400 mg·kg⁻¹, sucrase activity increased by 32.05%, 34.26%, and 42.73%, respectively. When the concentrations of Sb⁵⁺ were 100, 200, and 400 mg·kg⁻¹, sucrase activity increased by 2.90%, 9.25%, and 12.92%, respectively. When the concentrations of a mixture of both Sb³⁺ and Sb⁵⁺ were 100, 200, and 400 mg·kg⁻¹ (50% each), sucrase activity increased by 26.52%, 29.77%, and 37.43%, respectively. The amount of increase in sucrase activity in Sb³⁺-containing soils was 3.31–11.06 times higher than that of Sb⁵⁺-containing soils (Figure 1(a)).

Figure 1. Effects of Sb³⁺ on soil enzyme activity. Note a sucrase activity sucrase activity was expressed as milligrams of 1 g of soil glucose after 24 h); b Catalase promotes (Catalase activity was expressed as milliliters of 0.02 mol/L potassium permanganate consumed by lg soil); c urease activity (urease activity was expressed as milligrams of NH₃-N in lg soil after 24 h).

Figure 1(b) depicts the effects of different valence states of antimony on catalase in soil. The results indicated that catalase activity in the soil increased with increasing antimony content. When the concentrations of Sb³⁺ were 100, 200, and 400 mg·kg⁻¹, catalase activity increased by 8.34%, 12.45%, and 23.05%, respectively. When the concentrations of Sb⁵⁺ were 100, 200, and 400 mg·kg⁻¹, catalase activity increased by 6.49%, 9.80%, and 17.88%, respectively. When the concentrations of a mixture of both Sb³⁺ and Sb⁵⁺ were 100, 200, and 400 mg·kg⁻¹ (50% each), catalase activity increased by 10.07%, 11.50%, and 22.19%, respectively. The amount of increase in catalase activity in Sb³⁺-containing soils was 1.27–1.29 times higher than that of Sb⁵⁺-containing soils (Figure 1(b)).

Figure 1(c) depicts the effects of different valence states of antimony on urease in soil. The results indicated that urease activity in the soil decreased with increasing antimony content. When the concentrations of Sb³⁺ were 100, 200, and 400 mg·kg⁻¹, urease activity decreased by 42.28%, 51.96%, and 45.50%, respectively. When the concentrations of Sb⁵⁺ were 100, 200, and 400 mg·kg⁻¹, urease activity decreased by 13.88%, 30.66%, and 36.79%, respectively. When the concentrations of a mixture of both Sb³⁺ and Sb⁵⁺ were 100, 200, and 400 mg·kg⁻¹ (50% each), urease activity decreased by 38.40%, 46.79%, and 49.70%, respectively. The amount of increase in urease activity in Sb³⁺-containing soils was 1.24–3.05 times higher than that of Sb⁵⁺-containing soils (Figure 1(c)).

3.2. Effect of antimony on soil nitrogen content

Soil nitrogen content is an important indicator of farmland soil fertility. Because the soil nitrogen content after planting legumes is closely related to rhizobial activity, the soil nitrogen content can also reflect the degree of antimony toxicity to plant roots and soil.

As shown in Figure 2, the total nitrogen content and ammonia nitrogen content of the soil under antimony stress were lower than those of the blank control group. The total nitrogen content of the soil decreased by 8.37–18.28% and the ammonia nitrogen content of the soil decreased by 12.58–24.71% under Sb³⁺ stress. Both the total nitrogen content and ammonia nitrogen content reached their lowest values of 1942.55 ± 15.56 mg·kg⁻¹ and 18.73 ± 0.77 mg·kg⁻¹, respectively, at the Sb³⁺ content of 100 mg·kg⁻¹. Under Sb⁵⁺ stress, the total nitrogen content in soil decreased by 11.91–18.09% and the ammonia nitrogen content in soil decreased by 5.29–13.43%, both of which reached their lowest values of 1946.50 ± 7.78 mg·kg⁻¹ and 21.53 ± 0.36 mg·kg⁻¹, respectively, at 400 mg·kg⁻¹ of Sb⁵⁺. Under mixed antimony stress, the total nitrogen content in soil was reduced by 12.54–17.69% and the ammonia nitrogen content in soil was reduced by 13.38–20.14%, both of which reached their lowest values of 1956.11 ± 3.25 mg·kg⁻¹ and 19.87 ± 0.44 mg·kg⁻¹, respectively, at the antimony content of 100 + 100 mg·kg⁻¹. The variation of ammonia nitrogen content in soil contaminated with Sb³⁺ was 0.94–4.67 times that in soil contaminated with Sb⁵⁺. The change in total nitrogen content in soil contaminated with Sb³⁺ was 0.46–1.54 times that in Sb⁵⁺-contaminated soil.

Figure 2. Effects of Sb³⁺ on soil nitrogen content. Note. a total nitrogen content in soil (mg·kg⁻¹); b ammonia nitrogen content in soil (mg·kg⁻¹).

3.3. Effect of antimony on rhizobia

Antimony had a significant effect on the physical indexes of the rhizobia of mung beans (Table 2), kidney beans (Table 3) and peanuts (Table 4). With the increase in the concentration of antimony, the average mass of rhizobia was reduced by 15.87–26.98%, and the average diameters of rhizobia were reduced by 5.2–10.53%. The rhizobia were incapable of normal development under the toxic effect of antimony. Using the spread plate method to culture the rhizobia samples, it was found that the number of bacterial colonies in rhizobia under antimony stress was also significantly lower than that of the blank control group, and there was a slight difference in the types of colonies cultured.

Table 2. Physical indicators of mung bean root nodules.

	Soil Sb concentration (mg·kg^−1)	Average mass (g)	Average diameter (mm)	Bacterial count of plate coating culture
Sb^3+	0	0.0551 ± 0.0037	1.56 ± 0.071	16
	100	0.0478 ± 0.0067	1.53 ± 0.057	14
	200	0.0428 ± 0.0068	1.49 ± 0.069	12
	400	0.0434 ± 0.0092	1.49 ± 0.083	11
Sb^5+	0	0.0551 ± 0.0037	1.56 ± 0.071	16
	100	0.0466 ± 0.0034	1.52 ± 0.074	14
	200	0.0438 ± 0.0046	1.42 ± 0.058	14
	400	0.0442 ± 0.0084	1.40 ± 0.048	12
Sb^3++ Sb^5+	0	0.0551 ± 0.0037	1.56 ± 0.071	16
	50 + 50	0.0459 ± 0.0055	1.52 ± 0.067	15
	100 + 100	0.0471 ± 0.0039	1.44 ± 0.064	13
	200 + 200	0.0433 ± 0.0066	1.42 ± 0.049	12

Table 3. Physical indicators of kidney bean root nodules.

	Soil Sb concentration (mg·kg^−1)	Average mass (g)	Average diameter (mm)	Bacterial count of plate coating culture
Sb^3+	0	0.0511 ± 0.0052	1.67 ± 0.088	14
	100	0.0405 ± 0.0065	1.59 ± 0.047	14
	200	0.0396 ± 0.0058	1.52 ± 0.038	12
	400	0.0338 ± 0.0073	1.51 ± 0.054	10
Sb^5+	0	0.0511 ± 0.0052	1.67 ± 0.088	14
	100	0.0408 ± 0.0081	1.62 ± 0.066	13
	200	0.0395 ± 0.0053	1.58 ± 0.074	10
	400	0.0366 ± 0.0034	1.55 ± 0.051	10
Sb^3++ Sb^5+	0	0.0511 ± 0.0052	1.67 ± 0.088	14
	50 + 50	0.0432 ± 0.0077	1.62 ± 0.094	13
	100 + 100	0.0407 ± 0.0038	1.58 ± 0.038	13
	200 + 200	0.0353 ± 0.0054	1.53 ± 0.067	11

Table 4. Physical indicators of peanut root nodules.

	Soil Sb concentration (mg·kg^−1)	Average mass (g)	Average diameter (mm)	Bacterial count of plate coating culture
Sb^3+	0	0.0651 ± 0.0044	1.92 ± 0.053	18
	100	0.0604 ± 0.0022	1.85 ± 0.077	15
	200	0.0573 ± 0.0065	1.81 ± 0.039	12
	400	0.0411 ± 0.0084	1.79 ± 0.082	11
Sb^5+	0	0.0651 ± 0.0044	1.92 ± 0.053	18
	100	0.0618 ± 0.0088	1.83 ± 0.042	16
	200	0.0568 ± 0.0047	1.77 ± 0.088	13
	400	0.0427 ± 0.0063	1.75 ± 0.091	12
Sb^3++ Sb^5+	0	0.0651 ± 0.0044	1.92 ± 0.053	18
	50 + 50	0.0528 ± 0.0074	1.85 ± 0.067	15
	100 + 100	0.0469 ± 0.0031	1.79 ± 0.057	12
	200 + 200	0.0375 ± 0.0056	1.76 ± 0.085	10

As shown in Figure 3, antimony inhibited rhizobial activity. When the concentration of Sb³⁺ was 100 mg·kg⁻¹, the rhizobial activity was the lowest; the mung bean rhizobial activity was reduced by 20.63%, the kidney bean rhizobial activity was reduced by 34.86%, and the peanut rhizobial activity was reduced by 57.84% compared to that of the blank control group. The rhizobial activity under Sb⁵⁺ stress decreased with the increase of the Sb⁵⁺ concentration. When the Sb⁵⁺ concentration reached 400 mg·kg⁻¹, mung bean rhizobial activity decreased by 15.01%, kidney bean rhizobial activity decreased by 13.99%, and peanut rhizobial activity decreased by 21.32%. When legumes were under mixed antimony stress, the magnitude of changes in rhizobial activity depended on the Sb³⁺ content, and the rhizobial activity was the lowest when the Sb³⁺ concentration was 100 mg·kg⁻¹ and the total mixed antimony concentration was 200 mg·kg⁻¹.

Figure 3. Effects of Sb³⁺ on rhizobium activity. Note a mung bean’s rhizobium activity (rhizobium activity was calculated as C₂H₄ content consumed per minute, **: p < 0.01); b kidney bean’s rhizobium activity; c peanut’s rhizobium activity.

3.4. Effect of antimony on microorganisms

To demonstrate the species annotation, the identification results were counted as shown in Table 5. The identification results of each sample shown in Table 5 at each taxonomic level were plotted as bar graphs to compare the differences in the number of OTUs and the taxonomic identification results of different samples, as shown in Figure 4.

Figure 4. Statistical chart of the number of microbial taxonomic units at each level. Note. Sb0 represents CK, Sb1 represents soil with added Sb⁵⁺, and Sb2 represents soil with added Sb³⁺.

Table 5. Statistical table of the number of microbial taxonomic units at each level.

ID	phylum	class	order	family	genus	species
A0 (CK)	22	68	144	221	326	50
A1 (200 mg·kg^−1 Sb^5+)	21	66	140	207	283	47
A2 (200 mg·kg^−1 Sb^3+)	24	68	142	207	303	49

The analysis of the experimental results showed that the number of microorganisms and the number of species, families, and classes in the soil under antimony stress were lower than those in the CK group. Under Sb³⁺ stress, the number of microorganisms and the number of species, families, and classes in the soil were similar compared to the CK group.

The abundance rank curves shown in Figure 5 are more abstract, where each fold line represents a soil sample. More fold lines indicated higher homogeneity of the bacterial community, while steeper folds reflected lower homogeneity and greater variation in the OTU abundance [31]. The results showed that soils contaminated with Sb³⁺ had high homogeneity. In contrast, soils contaminated with Sb⁵⁺ had lower homogeneity and greater differences in abundance.

Figure 5. Venn diagram of the sample (group) ASV/OUT.

The ASV/OTU abundance table was used to produce a Venn diagram to analyze which species were common and which were unique among the different samples (Figure 6). In total, 14.2% of microorganisms were not significantly affected, while 23.87% of microorganisms survived only in the blank control and could not survive under antimony stress. Under Sb³⁺ stress, 33.06% of the microorganisms survived. Antimony changed the survival environment of the soil, and the composition of microorganisms in the three soils was significantly different between treatment groups. Microorganisms with higher antimony tolerance that were better at mitigating the toxic effects of antimony replaced most of the native microorganisms in the soil. This demonstrated that the native microorganisms in the soil did not adapt to the environmental changes brought about by antimony contamination.

Figure 6. Abundance grade graph.

As shown in Figures 7 and 8, the activity of microorganisms was analyzed by using heat maps to further compare the differences between samples and to illustrate the species abundance distribution trends for each sample. Under Sb⁵⁺ stress, the composition of active microorganisms was close to that of the blank control group, and the changes in microorganisms were mainly reflected in the changes in the activity of microorganisms of the same species. At this time, the activity of microorganisms declined, but the composition of active microorganisms did not change. However, under Sb³⁺ stress, the composition of active microorganisms changed greatly. The activity of previously active microorganisms in the soil was significantly reduced, while the activity of previously inactive microorganisms increased and dominated.

Figure 7. Heat map of species composition at the genus level for species clustering.

Figure 8. Heat map of species composition at the genus level for biclustering.

Figure 9 shows the visualization of the composition and distribution of each sample at the six classification levels of phyla, class, order, family, genus, and species in the form of a bar chart. Microorganisms in Sb⁵⁺-containing soils were similar to those in CK, but the microbial populations in Sb³⁺-contaminated soils were more variable.

Figure 9. Bar chart of species composition. Note Sb0 represents CK, Sb1 represents soil with added Sb⁵⁺, and Sb2 represents soil with added Sb³⁺.

Figure 9. (Continued).

Figure 9. (Continued).

4. Discussion

In terms of mechanism, metals may reduce enzyme activity by interacting with enzyme – substrate complexes, denaturing enzyme proteins, and binding to protein-reactive groups, which can essentially be summarized as a biochemical reaction between a protein and a metal. The inhibitory effect of exogenous substances, including heavy metals, on some soil enzymes has been reported in several studies, and the enzyme activity is negatively correlated with the amount of exogenous substances; however, the activity may increase under certain circumstances [35]. Sb³⁺ had a promoting effect on sucrase activity in the present study, similar to the findings of other researchers [36]. Although Sb⁵⁺ also had a promoting effect on sucrase activity, the effect was much lower than that of Sb³⁺. It is hypothesized here that the presence of antimony drastically alters the soil microbial environment, contributing to the elevated microbial demand for organic matter and contributing to the increased sucrase activity. As a metal with strong oxidizing properties, all three antimony pollutants elevated catalase activity to different degrees, similar to other heavy metals. The changes in urease activity in this experiment were similar to the findings of previous researchers, which showed a significant decrease in urease activity under soil Sb content of 200–1000 mg·kg⁻¹ and with the presence of Sb in the soil for 10 days [37]. The three antimony contaminants in the present study showed the same trend of effect on urease, similar to those of Cu and Cd [38,39], and only the degree of the effect differed. Experimental data showed that reduced soil urease activity led to lower soil N content, which was consistent with the results of this experiment. In addition, the lack of nitrogen content led to elevated rhizobial activity. The rhizobia under Sb⁵⁺ and mixed antimony stress exhibited fewer toxic effects than those under Sb³⁺ stress. Sb⁵⁺ had similar effects to Sb³⁺ on both soil enzymes and rhizobial activity, but the degree of effect varied greatly, which was due to the difference in oxidative strength between the two forms of Sb.

Analysis based on the Venn diagram and heat map showed that the activity of the original microorganisms declined under Sb³⁺ stress. The previously active microorganisms were replaced by microorganisms that were more adaptable under Sb³⁺ stress. Unlike the microorganism activity under Sb³⁺ stress, a decrease in microorganism activity was observed under Sb⁵⁺ stress. This confirmed that the change in microbial activity in the soil did not occur because antimony promoted the activity of the original microorganisms in the soil. The microorganism activities changed under Sb³⁺ stress. The activity of native microorganisms almost disappeared, while the activity of microorganisms with extremely low activity prior to the addition of Sb³⁺ significantly increased. This disruption of microbial activity had cascading effects on various soil processes and functions, further exacerbating the adverse effects of antimony on the overall soil fertility and ecosystem functioning. Sb⁵⁺ did not possess the strong oxidizing properties of Sb³⁺, therefore its impact on microbial populations was not as severe as that of Sb³⁺.

Figure 9 Bar chart of species composition. Note Sb0 represents CK, Sb1 represents soil with added Sb⁵⁺, and Sb2 represents soil with added Sb³⁺

5. Conclusion

The conclusions of this paper are as follows:

1. The toxic effect of Sb³⁺ on sucrase activity was 3.31–11.06 times that of Sb⁵⁺. The toxic effect of Sb³⁺ on catalase activity was 1.27–1.29 times that of Sb⁵⁺. The toxic effect of Sb³⁺ on urease activity was 1.24–3.05 times that of Sb⁵⁺. The toxic effect of Sb³⁺ on the rhizobial activity of mung bean was 1.41 times that under Sb⁵⁺ stress, 1.10 times that on the rhizobial activity of kidney bean, and 2.22 times that on the rhizobial activity of peanut.

2. Both valence states of antimony ions had significant toxic effects on microbial populations. Sb³⁺ was more toxic and changed the microbial population categories.

This paper focuses on the toxicity of antimony in different rack states on the soil and rhizobacteria of farmland planted with leguminous plants. As the rhizobacteria of leguminous plants can improve the soil environment, the degree of antimony toxicity in soil planted with other crops will be higher than that found in the results of the present experiments. Limited by the experimental conditions, this experiment failed to analyze the effects of antimony on the rhizobial and microbial activity at the genetic level. The toxic effects of antimony in different rack states on other plants and animals, as well as the effect of the soil in other conditions, need to be further investigated in depth.

Authorship

The article was experimented on and written by Yiran Yang, and modified by Zaifu Yang, with some experimental data provided by Yueyue Tian and Jingyao Xu, and financial support provided by Simeng Kan. Thank you to the authors for their help with the article. All authors agree to proceed with publication.

Availability of data and materials

All relevant data are within the manuscript and its Additional files.

Ethics and consent

The article does not involve any theoretical or moral issues.

Research involving plants

The plants involved in this article are not rare.

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Huadian Jinsha River Upstream Hydropower Development CO., LTD, Chengdu, China [JS-BT-FW/QT-006].