Enhanced remediation of paddy soils contaminated with butachlor through the application of nitrogen-fixing cyanobacteria as a nitrogen fertilizer substitute

Enhanced remediation of paddy soils contaminated with butachlor through the application of nitrogen-fixing cyanobacteria as a nitrogen fertilizer substitute | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced remediation of paddy soils contaminated with butachlor through the application of nitrogen-fixing cyanobacteria as a nitrogen fertilizer substitute Huiyao Liu, Yu Zhu, Xiao Liang, Kan Wang, Hongjie Qin, Genbao Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6625888/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Butachlor, a widely used herbicide in paddy fields, exhibits persistent environmental residues, posing significant ecological concerns. Nitrogen-fixing cyanobacteria (NFC), as a biofertilizer, not only enhance soil fertility and crop growth but also show potential in influencing the environmental fate of herbicides. However, the specific effects of NFC as a nitrogen fertilizer substitute on butachlor remediation in paddy soils remains unexplored. To address this, a 105-day outdoor pot experiment was conducted to evaluate the impact of NFC on soil properties, microbial community dynamics, and butachlor removal efficiency. The results showed that NFC application increased soil carbon and nitrogen content, enhanced enzyme activities (e.g., urease), and reshaped the microbial community structure compared with conventional fertilization. Co-occurrence network analysis further showed that NFC increased the complexity of soil microbial ecological networks, leading to closer species interactions and favoring the aggregation and proliferation of microorganisms that mediate butachlor degradation (e.g., Sphingomonas ). As a result, the extinction of butachlor was accelerated, and the amount of butachlor residue at harvesting stage was reduced by 35.81–57.06% relative to conventional fertilization. This study provides a theoretical foundation for the application of NFC in paddy soils, highlighting its potential to improve soil health and sustainability in the context of butachlor use. Butachlor Microbial co-occurrence networks Microbial communities Nitrogen-fixing cyanobacteria Paddy soils sustainability Soil health Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Rice is a staple food crop that sustains over 50% of the global population (Yuan et al. 2021 ). Ensuring high rice yields is therefore critical to global food security. To achieve this, the agricultural sector heavily relies on agrochemicals, including pesticides, fungicides, and herbicides, to control pests, diseases, and weeds, thereby minimizing yield losses and maintaining product quality. Among these, herbicides are the most extensively used. In China, commonly applied herbicides include organophosphorus hydrolase (OPH), phenoxy acid (PAA), and chloroacetanilide herbicide (ANH), among others (Tan et al. 2024 ). Butachlor, a prominent ANH herbicides, ranks among the top three most widely used herbicides in China (Janaki et al. 2015 ). As a selective preemergence herbicide, butachlor inhibits protein and lipid biosynthesis pathways (He et al. 2013 ) and is primarily employed to control a number of broadleaf and grassy weeds in paddy fields (Mohanty and Jena 2019 ). It enters the paddy field environment through spray drift, soil leaching, and surface runoff (Agrawal et al. 2014 ). Recent studies have detected butachlor residues in agricultural soils across various regions, including Northeast China, where contents from 0.04 to 396.04 µg/kg, with a detection rate as high as 94.39% (Chiang et al. 2001 ; Li et al. 2024 ). Elevated levels of butachlor have also been found in rivers due to improper paddy water management or rainfall (Chau et al. 2015 ; Zheng et al. 2016 ). However, butachlor is not a natural soil component and is both toxic and environmentally persistent. Its residues pose ecological risks to non-target organisms in paddy ecosystems, damage subsequent crops, and contribute to exceeding pesticide residue standards in agricultural products and environmental pollution (Anbumani and Mohankumar 2015 ; Shuman-Goodier et al. 2017 ; Yu et al. 2023 ). Studies have shown that butachlor significantly affects soil enzyme activities and microbial communities, with soil acid phosphatase activity being notably inhibited at typical application concentrations (Nazima et al. 2014). Additionally, beneficial soil microorganisms are adversely impacted, and high butachlor levels can severely alter microbial community structures (Fang et al. 2009 ; WANG et al. 2009). Therefore, the persistence of butachlor residues in paddy fields and their associated ecological risks warrant urgent attention. Bioremediation techniques are increasingly employed to mitigate the impact of residual agrochemicals on agricultural soils (Ekram et al. 2020 ). These methods include the use of agrochemical-degrading bacteria (Liu et al. 2023 ), livestock manure (Zhang et al. 2013 ), straw return (Zhao et al. 2013 ), and compost (Feng et al. 2023 ) to degrade organic pollutants. Among these, organic fertilizers are considered a promising, eco-friendly, and safe approach to soil remediation. Organic fertilizers not only provide more nutrients than chemical fertilizers but also significantly alter the physicochemical properties of rhizosphere soils, regulate microbial communities, and directly or indirectly influence the degradation and transformation of pollutants (Liang et al. 2024 ; Song et al. 2021 ; Qing et al. 2022 ). However, the extent to which organic fertilizers promote the degradation of herbicides like butachlor remains unclear, and the underlying mechanisms requires further investigation. Nitrogen-fixing cyanobacteria (NFC) are recognized as effective bioorganic fertilizers. These prokaryotic microorganisms possess heterocysts, which serve as nitrogen-fixing sites. Capable of photosynthesis, NFC derive energy to fix atmospheric nitrogen and convert it into biologically active inorganic nitrogen forms that plants can directly utilize (Massey and Davis 2023 ; Singh et al. 2011 ; Stanier 1977 ). Due to their autotrophic photosynthesis and nitrogen-fixing capabilities, NFC thrive in polluted environments and exhibit significant potential for remediating environmental pollutants, including heavy metals (Singh et al. 2016 ), oil (Nejidat et al. 2023 ), xenobiotics (Megharaj 1987), and pesticides (Megharaj et al. 1994 ). Certain NFC, such as Anabaena and Nostoc , have demonstrated the ability to degrade lindane residues and organophosphorus herbicides (El-Bestawy et al. 2007 ; Forlani et al. 2008 ). Extensive research has explored the effects of NFC on grain production and soil properties (Singh et al. 2016 ; Song et al. 2022b ). When used as nitrogen fertilizer substitutes, NFC have been shown to enhance nitrogen use efficiency in rice cultivation while maintaining yields and reducing fertilizer inputs. Additionally, NFC application improves soil chemical properties, microbial composition, community structure, and functionality in paddy soils (Liang et al. 2024 ; Song et al. 2021 ; Zhang et al. 2021 ). However, the potential of NFC as a green bioremediation strategy for butachlor-contaminated paddy soils remains unexplored. Therefore, investigating the effects of NFC as a substitution for nitrogen fertilizer on the removal of butachlor from paddy soils can provide valuable insights for sustainable agricultural practices. This study hypothesizes that replacing nitrogen fertilizer with NFC can enhance soil properties and microbial communities in butachlor-treated paddy soils, thereby accelerating butachlor elimination. To test this hypothesis, a 105-day outdoor rice pot experiment was conducted under conditions simulating natural environment. The study examined the differential effects of NFC on soil properties and microbial communities under varying fertilization and butachlor application regimes. Furthermore, potential correlations between NFC-induced changes in soil properties and microbial communities and butachlor residue levels were analyzed. The findings aim to provide theoretical support for the health and sustainability of herbicide-treated paddy soils and to promote wider application adoption of NFC in agriculture. 2. Materials and methods 2.1. Design of the outdoor rice pot experiment The soil used in the pot experiments was collected from a farm in Fuyuan City, Heilongjiang Province, China (47°73′N, 134°28′E), located within the Mollisol Zone and classified as albic soil. The chemical and biochemical properties of the soil provided in Table S1 . The soil was sieved through a 2 mm mesh to remove impurities, thoroughly mixed, and distributed into 21 pots. Each pot measured 18 cm in height and 17 cm in top diameter, with a total volume of 3.5 L. Rice seedlings were cultivated in nursery trays in mid-June and transplanted into the pots on 20 July. Each pot contained four rice seedlings. Prior to transplantation, butachlor was applied to the pots to treat the soil (Panda and Sahu 2004 ). The water layer was maintained at approximately 3 cm, and rice seedlings were transplanted after natural water infiltration for 3 days. Based on previous studies, replacing 30% of nitrogen fertilizer with NFC was determined to be the optimal rate for maintaining soil fertility and rice yield (Zhang et al. 2021 ). The required NFC biomass was calculated as described in Section 2.2 . The experiment comprised three groups with a total of seven treatments. 1. Group N: Application of chemical fertilizer only. 2. Group NB: Application of chemical fertilizer and three doses of butachlor, including: NB1: Recommended dose (0.1118 g a.i./m²). NB1.5: 1.5 times the recommended dose (0.1677 g a.i./m²). NB2: 2 times the recommended dose (0.2236 g a.i./m²). 3. Group NCB: Application of chemical fertilizer, NFC replacing 30% of nitrogen fertilizer, and three doses of butachlor, including: NCB1: Recommended dose (0.1118 g a.i./m²). NCB1.5: 1.5 times the recommended dose (0.1677 g a.i./m²). NCB2: 2 times the recommended dose (0.2236 g a.i./m²). All treatments were performed in triplicate. NFC was applied as tiller fertilizer 15 days after transplanting (tillering stage) and as panicle fertilizer 60 days after the heading stage. The pot experiment lasted for 105 days, from rice transplanting to harvest. Throughout the experimental, rice was grown at an average ambient temperature of 23°C, with the water layer maintained at approximately 3 cm. Fertilizer and butachlor application details for each treatment are shown in Table 1 . Soil samples were collected from a depth of 0–15 cm at five time points: day 1 (rice transplanting), day 15 (tillering stage), day 45 (heading stage), day 75 (filling stage), and day 105 (harvesting stage). Three parallel samples per treatment were combine into composite samples, air-dried, and sieved through a 2 mm mesh for analysis of physical and chemical properties and the butachlor residues. Fresh soil samples collected at harvesting stage were stored at -80°C for DNA extraction. Table 1 Fertilizer, NFC addition, and butachlor application in the seven treatments. Treatments Butachlor (g·a.i./m 2 ) Basal fertilizer Tiller fertilizer Panicle fertilizer Total fertilizer Urea (g/m 2 ) P 2 O 5 (g/m 2 ) KCl (g/m 2 ) Urea (g/m 2 ) NFC (g dry wt./m 2 ) Urea (g/m 2 ) NFC (g dry wt./m 2 ) KCl (g/m 2 ) Urea (g/m 2 ) NFC (g dry wt./m 2 ) P 2 O 5 (g/m 2 ) KCl (g/m 2 ) Group N 0 9.00 9.00 6.43 8.00 0 3.00 0 4.29 20.00 0 9.00 10.72 Group NB NB1 0.1118 9.00 9.00 6.43 8.00 0 3.00 0 4.29 20.00 0 9.00 10.72 NB1.5 0.1677 9.00 9.00 6.43 8.00 0 3.00 0 4.29 20.00 0 9.00 10.72 NB2 0.2236 9.00 9.00 6.43 8.00 0 3.00 0 4.29 20.00 0 9.00 10.72 Group NCB NCB1 0.1118 9.00 9.00 6.43 3.00 6.27 2.00 1.26 4.29 14.00 7.53 9.00 10.72 NCB1.5 0.1677 9.00 9.00 6.43 3.00 6.27 2.00 1.26 4.29 14.00 7.53 9.00 10.72 NCB2 0.2236 9.00 9.00 6.43 3.00 6.27 2.00 1.26 4.29 14.00 7.53 9.00 10.72 2.2 NFC culture and characterization On the basis of preliminary experiments, a cyanobacterium strain, Nostoc sp. (FACHB-87), which exhibits notable tolerance to butachlor and nitrogen fixation potential, was selected. This strain was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology. The strain was cultivated under laboratory conditions at a large scale, using sterilized nitrogen-free BG110 medium, with temperature maintained at 27 ± 1°C, light intensity set to 56 µmol m⁻² s⁻¹, a 12-hour light/dark cycle, and continuous aeration with filtered air. The culture was harvested by filtration through 300-mesh gauze, yielding fresh algal biomass with 97% water content. The nitrogen content of cultured Nostoc sp. (FACHB-87) was approximately 71.68 g N kg − 1 dry weight (DW). Under paddy field conditions, the biomass increased 5.19-fold within two weeks, indicating that 2680 g DW of NFC could replace at least 1000 g of nitrogen. 2.3 Determination of soil chemical and biochemical properties Soil pH was measured using a pH meter (METTLER TOLEDO 220, Switzerland) in a 1:2.5 (w / v ) soil-water suspension (Abollino et al. 2002 ). Soil total phosphorus (TP) was determined via the alkali fusion method (HJ632-2011, China). Soil total nitrogen (TN) and total organic carbon (TOC) were measured using an elemental analyzer (Elementar Vario TOC, Germany). Ammonium nitrogen (NH 4 + -N) and nitrate nitrogen (NO 3 − -N) were extracted with KCl and quantified using a continuous flow analyzer (Skalar San++, The Netherlands) (Zhang et al. 2020 ). Soil enzyme activities, including catalase (S-CAT), acid phosphatase (S-ACP), urease (S-UE), and sucrase (S-SC), were measured using spectrophotometric kits (Solarbio, China). 2.4 Determination of butachlor residues in soil Butachlor was extracted from soil using acetonitrile, purified via dispersive solid-phase extraction, and quantified using gas chromatography-tandem mass spectrometry (GC-MS/MS) (Agilent 7890B-7000C, USA). Briefly, 5.0 g of soil was mixed with 10 mL of ultrapure water in a 50 mL centrifuge tube and left to stand for 30 minutes before 10 mL of acetonitrile and 4 g of NaCl were added for vortex extraction for 2 minutes. Then, centrifugation was performed at 3500 r/min for 5 minutes before the supernatant was transferred. 5 mL of the supernatant was transferred to a 10 mL plastic centrifuge tube containing 300 mg of MgSO 4 , 100 mg of primary secondary amine (PSA), 100 mg of C18, and 50 mg of graphitized carbon black (GCB). After centrifugation at 10,000 r/min for 5 minutes, 2 mL of the supernatant was blown dry under nitrogen and dissolved in 1 ml of hexane. The extract was filtered through a 0.22 µm membrane and analyzed using GC-MS/MS with a 5% phenyl methylpolysiloxane capillary column (30 m × 0.25 mm × 0.25 µm) (Agilent 122–5532 DB-5 ms). Helium was used as the carrier gas at 2.25 mL/min. The injection port temperature was 280°C, and the column temperature program was as follows: 60°C for 1 minute, increased to 170°C at 40°C/min, and then to 310°C at 10°C/min for 3 minutes. The dissipation kinetics of butachlor in soil were described using the first-order kinetic equation: C t = C 0 e −kt , where C t is the butachlor content time t (d) after application (µg kg − 1 ), C 0 is the initial content (µg kg − 1 ), and k is the first-order rate constant (d − 1 ). 2.5 Metagenomic data processing and annotation Total genomic DNA was extracted from soil samples collected at the rice harvesting stage, fragmented, and used to construct paired-end libraries for sequencing. Data processing, gene annotation, and related methodologies are detailed in Text S1. 2.6 Statistical analysis Statistical analyses were performed using SPSS 26.0 (SPSS, Inc., USA) with one-way ANOVA ( P < 0.05). Prokaryotic microbial functions were annotated and predicted using FAPROTAX software (version 1.2.6). Redundancy analysis (RDA) of environmental variables and butachlor residues was conducted using the vegan package in R 4.3.3 (Oksanen 2011 ). Microbial co-occurrence networks were constructed at the genus level using the Hmisc, igraph, and microeco packages (Liu et al. 2021 ). Similarity matrices were generated using Pearson correlation analysis, and networks were constructed based on a Random Matrix Theory (RMT) threshold, with Spearman correlation coefficients > 0.70 and P < 0.05 considered significant. The networks were visualized using the Fruchterman–Reingold layout in Gephi software (version 0.10.1). Nodes within the networks were classified into four categories based on intra-module connectivity (Zi) and inter-module connectivity (Pi): (1) network hubs (Zi > 2.5, Pi > 0.62), (2) connectors (Zi ≤ 2.5, Pi > 0.62), (3) peripheral nodes (Zi ≤ 2.5, Pi ≤ 0.62), and (4) module hubs (Zi > 2.5, Pi ≤ 0.62). To explore the complex interaction between NFC and butachlor residues in soil, partial least squares path modeling (PLS‒PM) was employed. The model incorporated three sets of endogenous variables: (1) soil properties, including pH, TOC, TN and NH 4 + -N; (2) soil enzyme activities, comprising S-CAT, S-UE and S-SC; and (3) community diversity and richness, represented by the Chao1 and Shannon indices. All PLS-PM analyses were performed using the plspm package. 3. Results 3.1 Butachlor residues in paddy soils Table 2 Parameters of the first-order kinetic equation for butachlor elimination in soil under groups NB and NCB. Treatments First order kinetic equation k (d − 1 ) Determination coefficient (R 2 ) Half-life (d) NB1 C t = 543.07e − 0.0120t 0.0120 0.922 57.76 NCB1 C t = 565.13e − 0.0188t 0.0188 0.964 36.87 NB1.5 C t = 786.12e − 0.0127t 0.0127 0.960 54.58 NCB1.5 C t = 801.40e − 0.0175t 0.0175 0.967 39.61 NB2 C t = 1067.52e − 0.0091t 0.0091 0.926 76.17 NCB2 C t = 1148.34e − 0.0179t 0.0179 0.978 38.72 The residual levels of butachlor in paddy soils were determined at the initial application of NFC and at four key stages of rice growth: tillering, heading, filling and harvesting stages (Fig. 1 ). In all six treatments with three doses of butachlor, the elimination curves followed first-order kinetics, as indicated by determination coefficients (R 2 ) exceeding 0.9. After the 105-day experiment, the butachlor content in the three sub-treatments of the group NB (without NFC) decreased from initial values of 538.15, 729.67, and 1012.83 µg kg − 1 to 110.83, 150.67, and 246.60 µg kg − 1 , respectively. In contrast, the butachlor content in the three sub-treatments of the group NCB (with NFC) decreased from initial values of 526.19, 716.13, and 1044.42 µg kg − 1 to 47.59, 96.71, and 135.94 µg kg − 1 , respectively. Significant differences were detected between NB1 and NCB1, as well as between NB2 and NCB2 ( P < 0.05). The residual butachlor levels in the group NCB were 35.81–57.06% lower than those in the group NB. The elimination rates of butachlor in the three sub-treatments of the group NB were 0.0120, 0.0127, and 0.0091 d − 1 , which were lower than those in the group NCB (0.0188, 0.0175, and 0.0179 d − 1 , respectively). The half-life of butachlor elimination in the group NB ranged from 54.58 to 76.71 days, while the group NCB exhibited a shorter half-life of 36.87–39.61 days (Table 2 ). 3.2 Chemical properties and enzyme activities of paddy soils The results of the chemical analysis of the soil at the rice harvesting stage are presented in Fig. 2 . The data indicate that the different treatments led to variations in several key indicators. Specifically, the NB1 and NB1.5 treatments showed a slight decrease in soil pH, TOC, S-ACP, S-UE, and S-SC compared to the N treatment. However, these differences were not statistically significant. In contrast, the NB2 treatment significantly reduced the S-ACP, S-UE, and S-SC activities compared to the N treatment ( P < 0.05). When comparing the three sub-treatments of the group NB with those of the group NCB, the latter exhibited higher values of pH, TOC, TN, NH 4 + -N, S-ACP, S-UE and S-SC. For instance, the TN levels in the NCB1, NCB1.5, and NCB2 treatments were higher than those in the NB1, NB1.5, and NB2 treatments, respectively. Similarly, the NH 4 + -N levels in the NCB1 and NCB1.5 treatments were significantly greater than those in the NB1 and NB1.5 treatments ( P < 0.05). Additionally, the S-UE and S-SC levels in the NCB1.5 and NCB2 treatments were significantly higher than those in the NB1.5 and NB2 treatments, respectively ( P < 0.05). Interestingly, the group NB demonstrated increased S-CAT activity, whereas the group NCB showed the opposite trend. 3.3 Composition, structure and function of microbial communities in paddy soils Based on metagenomic sequencing results, the composition of soil microbial communities was analyzed, with a focus on the 15 most abundant bacterial phyla in each treatment. The microbial communities across most treatment groups were predominantly composed of Proteobacteria, Acidobacteria, Chloroflexi, Actinobacteria, Verrucomicrobia, Bacteroidota, and Gemmatimonadetes (Fig. 3 A). The highest relative abundance of Proteobacteria was observed in the group NB, ranging from 23.46 to 26.74%. Replacing nitrogen fertilizer with NFC in the group NCB further increased the relative abundance of Proteobacteria to 25.43–30.74%. The relative abundance of Verrucomicrobia in the group NB (6.39–6.88%) was lower than that in the group N (8.31%), while the addition of NFC in the group NCB increased its abundance to 7.58–8.64%. The relative abundance of Bacteroidota was higher in the group NCB compared to the groups N and NB, whereas the relative abundance of Actinobacteria was lower in the group NCB than in the groups N and NB. At the genus level, the use of different doses of butachlor in the group NB reduced the relative abundance of unclassified _p_Verrucomicrobia compared to the group N. In contrast, the addition of NFC in the group NCB increased its relative abundance, with the order N > NCB > NB across all three groups (Fig. 3 B). The relative abundance of Ramlibacter in the group NCB (1.51–1.81%) was also higher than that in the group NB (1.35–1.43%). Additionally, the relative abundance of unclassified _o_Anaerolineales in the group NCB (1.19–2.47%) was substantially greater than that in the groups N (0.60%) and NB (0.63–1.28%). Analysis of cyanobacteria composition at the genus level revealed that the dominant genus in the groups N and NB were more complex and variable, whereas Nostoc was clearly dominant in the group NCB, with a relative abundance of 6.17–22.92% (Fig. S1 ). Furthermore, the α-diversity indices (Chao1, Shannon, and Simpson) of the soil microbial communities were calculated based on metagenomic sequencing results (Fig. 3 C). The Chao1 index in the three sub-treatments of the group NB was significantly higher than that in the group N ( P < 0.05). Although no significant difference was observed between the NCB1 and NCB2 sub-treatments and the NB1 and NB2 sub-treatments, the group NCB exhibited higher values, indicating greater community richness. The Shannon index, which characterizes community diversity, followed a similar trend, with NCB > NB > N. The Simpson index also reflected this pattern, with the group NCB showing higher values than the groups N and NB, indicating greater soil microbial community diversity. NMDS analysis revealed distinct clustering patterns of microbial communities across the different treatment groups (Fig. 4 A). PERMANOVA further confirmed significant differences in microbial community β-diversity among the groups N, NB, and NCB (F = 6.594, P = 0.001; Table S2). Functional annotation of soil microbial bacterial communities using FAPROTAX identified 63 functional microbial groups (Fig. 4 B). The most abundant groups included chemoheterotrophic, aerobic chemoheterotrophic, phototrophic, and photoautotrophic microbiota. Compared to the group N, the abundance of sulfate-respiring microbiota in the group NB was significantly lower ( P < 0.01), while the addition of NFC in the group NCB significantly increased their abundance by 2.45–4.32 times compared to the group NB, with no significant difference from the group N. Additionally, the abundance of nitrogen-fixing microbes was significantly higher in the group NCB than in the groups N and NB ( P < 0.01). Although not statistically significant, an increase in the abundance of aromatic-degrading microbiota was observed in the group NCB. 3.4 Relationships between butachlor residues, microbial communities and environmental factors in paddy soils Redundancy analysis (RDA) identified key drivers of variations in soil microbial communities across different fertilization treatment. The first and second sorting axes explained 34.26% and 11.19% of the variation, respectively, cumulatively accounting for 45.45% (Fig. 4 C). NFC was identified as the primary driver of community composition variation (r² = 0.896, P = 0.002), with S-CAT, pH and NH 4 + -N being the largest contributors among the environmental factors (r 2 = 0.531, P = 0.003; r 2 = 0.513, P = 0.003; and r 2 = 0.465, P = 0.009, respectively; Table S3). Pearson correlation analysis revealed significant positive correlations between NFC and pH, TOC, TP, NH 4 + -N, S-UE, and S-SC ( P < 0.05) (Fig. 4 D). In contrast, NFC was significantly negatively correlated with S-CAT and butachlor residues ( P < 0.05) and significantly positively correlated with Chao1 and Shannon indices of microbial community α-diversity ( P < 0.01). Butachlor residues were significantly negatively correlated with pH, NO 3 − -N, S-ACP, S-UE, and S-SC ( P < 0.01), as well as with the Chao1 and Shannon indices ( P < 0.05). 3.5 Co-occurrence network of soil microbial communities Co-occurrence networks were constructed to examine potential interactions within soil microbial communities under varying doses of butachlor, with or without NFC addition. The group NB network consisted of 237 nodes and 1524 links (Fig. 5 A), while the NCB group network had 268 nodes and 2103 links (Fig. 5 D; Table 3 ). The modularity indices of the two networks were 0.620 and 0.549, respectively, with nodes clustered into 11 and 10 modules. The average degree (avgK), average path length (GD), and average clustering coefficient (avgCC) were higher in the group NCB (15.694, 4.117, and 0.549, respectively) than in the group NB (12.861, 4.101, and 0.620, respectively). Modules 2, 3, 5, and 8 were dominant in the group NB, while modules 1, 2, 7, and 8 were dominant in the group NCB. The network density of the group NCB (0.058) was also higher than that of the group NB (0.054). Table 3 Topological characterization of co-occurrence networks within microbial communities of the groups NB and NCB. Network Indices Groups RMT threshold Total nodes Total links R 2 of power law Positive links (ratio) Negative links (ratio) Average degree (avgK) Average network Distance (GD) Network density Average clustering coefficient (avgCC) Betweenness centrality Network diameter Modularity index NB 0.70 237 1524 0.43 1270 (83.33%) 254 (16.67%) 12.861 4.101 0.054 0.620 0.097 9.783 0.578 NCB 0.70 268 2103 0.684 1206 (57.35%) 897 (42.65%) 15.694 4.117 0.058 0.549 0.127 15.167 0.540 These two networks were divided into different modules, which can be used to analyze the relationships between important genera in different modules. Correlation analysis revealed that modules 1, 2, 4, 5, 6, 7 and 8 in the group NB were negatively correlated with butachlor residues, while modules 3 and 9 showed positive correlations, though not significant (Fig. 5 B). In the group NCB, module 1 exhibited a significant negative correlation with butachlor residues ( P < 0.05), while modules 2, 4, 5, 6 and 7 were negatively correlated, and modules 3 and 8 were positively correlated (Fig. 5 E). Key connectors in the NB network included Novosphingobium , unclassified_ p_Candidatus_Moranbacteria , Nitrosopumilus , and unclassified_ o_Phycisphaerales (Fig. 5 C). In the NCB network, unclassified_ o_Myxococale was identified as a connector, and unclassified_ o_Rhodocyclales and unclassified_o_ Pirellulales were identified as module hubs (Fig. 5 F). PLS‒PM was employed to assess the direct and indirect influences of NFC, soil properties, soil enzyme activities, and soil microbial community diversity and richness on butachlor residues. The model revealed that butachlor residues were significantly negatively influenced by soil enzyme activities and soil microbial community diversity and richness ( P < 0.01) (Fig. 6 A). NFC significantly positively regulated soil properties (r = 0.811, P < 0.001), enzyme activities (r = 0.722, P < 0.001), and soil microbial community diversity and richness (r = 0.706, P < 0.01). Soil properties negatively regulate butachlor residues and positively regulate microbial community diversity and richness. The standardized total effects indicated that NFC, soil properties, soil enzyme activities, and microbial community diversity and richness were negatively correlated with butachlor residues, with standardized total effect values of -0.565, -0.350, -0.670 and − 0.593, respectively (Fig. 6 B). 4. Discussion 4.1 Changes in soil properties and enzyme activities in paddy soils due to NFC addition In this experiment, the effect of three doses of butachlor on soil properties and residual butachlor levels were evaluated under different fertilization conditions at the harvesting stage. The results revealed that soils treated with butachlor exhibited lower pH and TOC compare to soil under normal fertilization without butachlor (Fig. 2 ). In contrast, the addition of NFC to butachlor-treated soils led to increased pH, TOC, TN and NH 4 + -N. Butachlor, an amide herbicide, primarily degrades in soil through microbial transformation, with additional pathways including leaching and plant uptake (Yu et al. 2003 ). The adsorption of butachlor in soil is closely linked to soil properties, which influence its environmental behavior (Xu et al. 2005 ). For instance, soils with higher TOC content exhibit adsorption capacity for butachlor (Liu et al. 2008 ). During butachlor degradation, N -chloramide, a byproduct of chlorination, can oxidize inorganic and organic substances, including sulfite and organic sulfur (Ding et al. 2018 ). However, amide chlorination is directly influenced by soil pH (Zhang and Gunten 2023 ). The favorable conditions for NFC growth in paddy soils, including optimal temperature, humidity, and nutrients, enable NFC to play a critical role in nitrogen fixation and utilization. NFC converts atmospheric nitrogen into compound nitrogen, releasing a potion into the soil during its growth, and the remainder can be retained in the soil through mineralization (Prasanna and Nayak 2006 ). This process resulted in higher contents of TN and NH 4 + -N in NFC-treated soils (Fig. 2 ), consistent with previous findings (Song et al. 2022a ). The increased TOC content following NFC addition may be attributable to the photosynthesis of NFC, which facilitates organic carbon input into the soil (Kaushik 2014 ). Furthermore, the growth and decay of NFC release extracellular polymeric substances (EPS) and the accumulation of biomass, significantly contributing to soil organic carbon accumulation (Dron et al. 2012 ). Soil enzymes, sensitive to exogenous pollutants, serve as crucial indicators of soil contamination (Margesin et al. 2000 ). In this study, the activities of S-ACP, S-UE, and S-SC were lower in the group NB compared to the group N (Fig. 2 ), suggesting that higher butachlor doses inhibit these enzyme activities. This finding aligns with previous research (Wang et al. 2006 ). However, NFC addition enhanced the activities of S-ACP, S-UE and S-SC in butachlor-treated soils (Fig. 2 ). S-UE is integral to nutrient cycling, particularly nitrogen metabolism (Ni and Pacholski 2022 ). NFC improves soil nutrient availability, thereby positively influencing enzyme activity. Certain Nostoc species produce extracellular enzymes capable of decomposing organic residues, while their high biomass and EPS secretion increases polysaccharide content and microbial activity, further enhancing soil quality in butachlor-treated soils. 4.2 Changes in the structure and function of soil microbial communities in paddy soils due to NFC addition Soil microorganisms, as the most active component of soil ecosystems, drive plant productivity, biogeochemical cycling, and organic pollutant degradation, playing a fundamental role in maintaining soil sustainability (Mühlbachová 2008 ). In this study, NFC addition increased the abundance of Proteobacteria and Bacteroidota in butachlor-treated soils (Fig. 3 A). These phyla are key players in biogeochemical cycling, with Proteobacteria involved organic matter decomposition (Dang and Lovell 2016 ) in soil ecosystems (Delmont et al. 2018 ) and Bacteroidota contributed to polysaccharide metabolism (Shi et al. 2020 ). The improved soil properties and increased the TN and TOC content following NFC addition likely favored the growth of copiotrophic taxa such as Proteobacteria and Bacteroidota, which thrive one labile carbon sources (Fig. 3 A). Additionally, Verrucomicrobia, which play roles in nitrogen fixation and methane emission reduction (Chiang et al. 2018 ; Dunfield et al. 2007 ), were positively influenced by NFC-mediated changes in soil carbon and nitrogen dynamics. The carbon and nitrogen contents in soil represent the main environmental variables affecting taxa such as Verrucomicrobia (Wang et al. 2021 ). Adding NFC directly affects the biogeochemical cycling of carbon and nitrogen in butachlor-treated soils. Consequently, this resulted in a notable effect on Verrucomicrobia, which play important roles in carbon and nitrogen cycling. At the genus level, butachlor application reduced the relative abundance of Ramlibacter , a genus within Proteobacteria (Fig. 3 B). However, NFC addition counteracted this decline. We hypothesized that this alteration may be largely related to the input of NFC. Ramlibacter , a genus of Proteobacteria, is involved in several important pathways in the soil nitrogen cycle, including nitrogen fixation and intracellular ammonium accumulation and transport (Hu et al. 2023 ). These nitrogen-cycling processes mediated by Ramlibacter promote the production of ammonium in cells. In this study, the positive correlation between NFC and NH₄⁺-N content (Fig. 4 D), together with the results of our measurements of the NH₄⁺-N content of the soil (Fig. 2 ), may further support this hypothesis. NFC addition also increased the α-diversity of soil microbial communities, with no negative effects observed from butachlor application (Fig. 3 C). However, significant differences in β-diversity among treatment groups (Fig. 4 A, Table S2) suggest that NFC addition altered the original ecological balance, altering microbial community structure. Environmental factors such as pH, TOC, TP, NH₄⁺-N, S-UE, S-SC, and S-CAT, along with residual butachlor, likely contributed to these changes (Fig. 4 C). Functional annotation using FAPROTAX revealed that NFC addition increased the abundance of sulfate-respiring microbiota (Fig. 4 B). Sulfate-reducing bacteria capable of sulfate respiration can induce the conjugation of some sulfur-containing compounds with chloroacetamide herbicides like butachlor, thereby dechlorinating chloroacetamide and accelerating its degradation (Liu et al. 2022 ; Torabi et al. 2020 ). Additionally, the higher abundance of aromatic-degrading microbiota in the group NCB may also contribute to butachlor elimination (Fig. 4 B). Co-occurrence network analysis shows that the addition of NFC increases the network nodes and links, making the network more complex (Fig. 5 D). Some modules in the group NB are negatively correlated with butachlor residues, but none of the correlations are significant. In contrast, network module 1 in the group NCB shows a significant negative correlation with butachlor residues ( P < 0.05) (Fig. 5 E). The core genus in Module 1 was Sphingomonas , which is capable of nitrogen fixation and phytohormone production, enabling it to promote plant growth and improve stress tolerance (Asaf et al. 2020 ). More importantly, Sphingomonas exhibits a distinct advantage in degrading chloroacetamide herbicides. Certain Sphingomonas strains can utilize butachlor as the sole carbon source and achieve its complete mineralization through synergistic metabolism (Kim et al. 2013 ; Hou et al. 2014 ). Additionally, Sphingomonas can produce the amide hydrolase CmeH , which hydrolyzes the butachlor intermediate 2-chloro- N -(2,6-dimethylphenyl) acetamide to 2,6-diethylaniline (DEA). DEA is further mineralized into aniline and catechol under the action of Sphingomonas (Li et al. 2013 ). All of the above intermediates can ultimately be converted into carbon dioxide and water. The unclassified_ o_Rhodocyclales and unclassified_ o_Pirellulales are the module hubs and key nodes with relatively high abundances in the group NCB (Fig. 5 F), and they are significantly negatively correlated with butachlor residues ( P < 0.05) (Fig. S2). Some members of the denitrifying bacteria Rhodocyclales have the ability to degrade hydrocarbons (Abdalrhman et al. 2020 ). Studies indicated that Pirellulales in plant rhizosphere soils may play a pivotal role in nitrogen fixation and ammonium retention processes (Hu et al. 2024 ). Therefore, the continuous growth and death of NFC in the soil provide nutritional conditions for the growth and metabolism of other microorganisms, creating a favorable environment for the proliferation of microorganisms capable of degrading butachlor or promoting its degradation in the soil. These changes the assembly pattern of the microbial community, making the soil microbial ecological network more complex and the interactions between species closer. 4.3 Potential role of NFC as a driver of butachlor degradation in soil NFC addition enhanced butachlor degradation in paddy soils, as evidenced by reduced residual butachlor levels (Fig. 1 ). The physical and chemical properties of soil significantly influence the degradation rate of butachlor and its environmental fate (Liu et al. 2008 ). PLS-PM showed that the improvement of soil properties and enzyme activity mediated by NFC directly or indirectly promoted the degradation of butachlor (Fig. 6 ). NFC also directly had a positive impact on the diversity and richness of the soil microbial community, altering and optimizing the soil microbial community and thus enhancing the stability of the molecular ecological network. Therefore, the addition of NFC to soil improved soil nutrient conditions, promoted enzyme activity, stimulated and altered the metabolic activities and aggregation of microorganisms, and further promoted the soil adsorption behavior of butachlor and the microbially-mediated biodegradation pathway. In other words, it promoted the biodegradation of butachlor. In conclusion, replacing nitrogen fertilizers with NFC is an effective strategy to improve soil fertility and reduce butachlor residues, which can lower the potential risks to crops and non-target microorganisms. 5. Conclusion This study investigated the effects of replacing nitrogen fertilizers with NFC on the properties and community structure of paddy soils treated with different doses of the herbicide butachlor, as well as its impact on butachlor elimination. The results indicated that substituting nitrogen fertilizers with NFC increased the soil carbon and nitrogen contents, as well as the activities of enzymes such as S-UE, compared with those in soils treated with various doses of butachlor under normal fertilization conditions. In conclusion, NFC improved the chemical properties and soil enzyme activities of paddy soils with butachlor and remodeled the composition and structure of microbial communities, favoring the aggregation and growth of microbial taxa potentially associated with mediating the biodegradation of butachlor, thus indirectly contributing to the elimination of butachlor from the soil. These results suggest that replacing a portion of nitrogen fertilizer with NFC is a promising management strategy for paddy soils treated with butachlor. This approach not only improves soil fertility but also reduces the environmental risks associated with residual herbicides. However, further investigation is needed to elucidate the underlying mechanisms by which NFC promotes the microbial-mediated degradation of butachlor. Declarations Competing Interest The authors have no competing interests to declare that are relevant to the content of this article. Funding This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28030102) and Special Fund for Scientific Innovation Strategy-construction of High Level Academy of Agriculture Science (R2021YJ-QG003, R2023PY-JG024). Author Contribution Conceptualization: Dunhai Li; Methodology: Huiyao Liu, Xiao Liang, Kan Wang, Hongjie Qin, Dunhai Li; Formal analysis and investigation: Huiyao Liu, Yu Zhu; Writing-original draft: Huiyao Liu; Writing - review and editing: Chengrong Peng, Dunhai Li; Funding acquisition: Hongjie Qin, Chengrong Peng, Dunhai Li; Supervision: Genbao Li, Chengrong Peng, Dunhai Li. Data availability Data will be made available on request. References Abdalrhman AS, Zhang Y, Arslan M, Gamal El-Din M (2020) Low-current electro-oxidation enhanced the biodegradation of the recalcitrant naphthenic acids in oil sands process water. J Hazard Mater 398:122807. doi: 10.1016/j.jhazmat.2020.122807 Abollino O, Aceto M, Malandrino M, Mentasti E, Sarzanini C, Petrella F (2002) Heavy metals in agricultural soils from Piedmont, Italy. Distribution, speciation and chemometric data treatment. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6625888","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459847740,"identity":"ca261200-6f73-4529-95b2-873a19639f2c","order_by":0,"name":"Huiyao Liu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Huiyao","middleName":"","lastName":"Liu","suffix":""},{"id":459847741,"identity":"ef26f8b1-9642-4b8d-8afe-7cf03bc88200","order_by":1,"name":"Yu Zhu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhu","suffix":""},{"id":459847742,"identity":"a93f637f-4a88-4cbd-b04e-8283679996cf","order_by":2,"name":"Xiao Liang","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Liang","suffix":""},{"id":459847743,"identity":"5f75bd5b-3227-48db-920b-db8dfbec4b94","order_by":3,"name":"Kan Wang","email":"","orcid":"","institution":"Central-Southern Safety \u0026 Environment Technology Institute Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kan","middleName":"","lastName":"Wang","suffix":""},{"id":459847744,"identity":"9982d319-85cd-4d9e-ad18-ecc7e6ac1b6e","order_by":4,"name":"Hongjie Qin","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongjie","middleName":"","lastName":"Qin","suffix":""},{"id":459847745,"identity":"fe4a2c71-3b15-4252-ac2c-aa7c9e160eaf","order_by":5,"name":"Genbao Li","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Genbao","middleName":"","lastName":"Li","suffix":""},{"id":459847746,"identity":"bf6bb4e5-31f5-42d0-8e2e-b6032dcd61e5","order_by":6,"name":"Chengrong Peng","email":"","orcid":"","institution":"China Three Gorges Corporation","correspondingAuthor":false,"prefix":"","firstName":"Chengrong","middleName":"","lastName":"Peng","suffix":""},{"id":459847747,"identity":"45a7802c-1316-4391-abd4-623e8ed4df4c","order_by":7,"name":"Dunhai Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAn0lEQVRIiWNgGAWjYBACPmYGM4YPBgcSiNfCBtTCOIM0LQwMZsw8DCRpYWfe9tim4E4eA/vhBww/dxDlMLZy4xyDZ8UMPGkGjL1niNLCYyadY3A4sYEhh4GZsY1YLRYgLfxvSNHCANIiQbwtbGWSPQaHi9kknhkc7CVGCz//4W0SP/4czuPnT3744CcxWhDWAfEBUjSMglEwCkbBKMADAKpfK43TQZ30AAAAAElFTkSuQmCC","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Dunhai","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-09 07:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6625888/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6625888/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83307536,"identity":"db05d592-d7cf-4b50-81b7-99594f0d0a6e","added_by":"auto","created_at":"2025-05-22 17:04:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":463519,"visible":true,"origin":"","legend":"\u003cp\u003eElimination of butachlor in paddy soils at three doses under different fertilization treatments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/79f55b7249f8a4a71db08f99.png"},{"id":83307535,"identity":"5dbc8a0b-ef69-42bf-af3c-c7f680ec9989","added_by":"auto","created_at":"2025-05-22 17:04:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":890651,"visible":true,"origin":"","legend":"\u003cp\u003eChemical properties and enzyme activities of soils at the rice harvesting stage. Different lowercase letters represent significant differences between treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/9774b5718da89da608233812.png"},{"id":83307539,"identity":"ddfa6447-f1b8-4798-9f2a-115e6c88e336","added_by":"auto","created_at":"2025-05-22 17:04:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1078543,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of soil microbial communities at the (A) phylum and (B) genus levels under different treatments at the rice harvesting stage. (C) Soil microbial community α-diversity indices under different treatments at the rice harvesting stage.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/fd494a8d2ba6a08de1b96457.png"},{"id":83307537,"identity":"1785a17e-132b-4ed1-9258-ebfa6e8c56d8","added_by":"auto","created_at":"2025-05-22 17:04:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1452792,"visible":true,"origin":"","legend":"\u003cp\u003e(A) NMDS analysis of soil microbial communities under different treatment at the rice harvesting stage. (B) Relative abundance of soil bacterial functional microbiota predicted using FAPROTAX. (C) RDA analysis between soil microbial community, butachlor residue and environmental factors under different treatments at the rice harvesting stage. (D) Pearson correlation analysis between butachlor residues, microbial communities, and multiple environmental factors in the soil at the rice harvesting stage (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/93e9445e183c69d7d4de0e84.png"},{"id":83308722,"identity":"363aea10-3f17-4815-8116-200f6b2c9065","added_by":"auto","created_at":"2025-05-22 17:36:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1921065,"visible":true,"origin":"","legend":"\u003cp\u003eCo-occurrence network analysis of soil microbial communities at the rice harvesting stage in the groups NB (A) and NCB (D). Correlations between module-characterized genus and butachlor residues in the NB (B) and NCB (E) networks (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Key genus identified in the microbial networks of the groups NB (C) and NCB (F).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/7ed0bb0270721ab27048c67e.png"},{"id":83308166,"identity":"f94a50ed-e91c-4b00-a68b-73f09bbc515b","added_by":"auto","created_at":"2025-05-22 17:20:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":522360,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Analysis of the effects of NFC, soil properties, soil enzyme activities, and soil microbial community diversity and richness on butachlor residues using PLS-PM and (B) the standardized total effect of PLS-PM on butachlor residues. Red and blue arrows indicate positive and negative causal flows, respectively. Solid arrows representsignificant paths (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/9bc45dddbd956e514a675f1c.png"},{"id":83307538,"identity":"31be7990-f285-404d-9adc-c99cf92053ab","added_by":"auto","created_at":"2025-05-22 17:04:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":861842,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6625888/v1/80a57def983f3bd56f0160d2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced remediation of paddy soils contaminated with butachlor through the application of nitrogen-fixing cyanobacteria as a nitrogen fertilizer substitute","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRice is a staple food crop that sustains over 50% of the global population (Yuan et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ensuring high rice yields is therefore critical to global food security. To achieve this, the agricultural sector heavily relies on agrochemicals, including pesticides, fungicides, and herbicides, to control pests, diseases, and weeds, thereby minimizing yield losses and maintaining product quality. Among these, herbicides are the most extensively used. In China, commonly applied herbicides include organophosphorus hydrolase (OPH), phenoxy acid (PAA), and chloroacetanilide herbicide (ANH), among others (Tan et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eButachlor, a prominent ANH herbicides, ranks among the top three most widely used herbicides in China (Janaki et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As a selective preemergence herbicide, butachlor inhibits protein and lipid biosynthesis pathways (He et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and is primarily employed to control a number of broadleaf and grassy weeds in paddy fields (Mohanty and Jena \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It enters the paddy field environment through spray drift, soil leaching, and surface runoff (Agrawal et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Recent studies have detected butachlor residues in agricultural soils across various regions, including Northeast China, where contents from 0.04 to 396.04 \u0026micro;g/kg, with a detection rate as high as 94.39% (Chiang et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Elevated levels of butachlor have also been found in rivers due to improper paddy water management or rainfall (Chau et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, butachlor is not a natural soil component and is both toxic and environmentally persistent. Its residues pose ecological risks to non-target organisms in paddy ecosystems, damage subsequent crops, and contribute to exceeding pesticide residue standards in agricultural products and environmental pollution (Anbumani and Mohankumar \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shuman-Goodier et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies have shown that butachlor significantly affects soil enzyme activities and microbial communities, with soil acid phosphatase activity being notably inhibited at typical application concentrations (Nazima et al. 2014). Additionally, beneficial soil microorganisms are adversely impacted, and high butachlor levels can severely alter microbial community structures (Fang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; WANG et al. 2009). Therefore, the persistence of butachlor residues in paddy fields and their associated ecological risks warrant urgent attention.\u003c/p\u003e \u003cp\u003eBioremediation techniques are increasingly employed to mitigate the impact of residual agrochemicals on agricultural soils (Ekram et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These methods include the use of agrochemical-degrading bacteria (Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), livestock manure (Zhang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), straw return (Zhao et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and compost (Feng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) to degrade organic pollutants. Among these, organic fertilizers are considered a promising, eco-friendly, and safe approach to soil remediation. Organic fertilizers not only provide more nutrients than chemical fertilizers but also significantly alter the physicochemical properties of rhizosphere soils, regulate microbial communities, and directly or indirectly influence the degradation and transformation of pollutants (Liang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qing et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the extent to which organic fertilizers promote the degradation of herbicides like butachlor remains unclear, and the underlying mechanisms requires further investigation.\u003c/p\u003e \u003cp\u003eNitrogen-fixing cyanobacteria (NFC) are recognized as effective bioorganic fertilizers. These prokaryotic microorganisms possess heterocysts, which serve as nitrogen-fixing sites. Capable of photosynthesis, NFC derive energy to fix atmospheric nitrogen and convert it into biologically active inorganic nitrogen forms that plants can directly utilize (Massey and Davis \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stanier \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Due to their autotrophic photosynthesis and nitrogen-fixing capabilities, NFC thrive in polluted environments and exhibit significant potential for remediating environmental pollutants, including heavy metals (Singh et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), oil (Nejidat et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), xenobiotics (Megharaj 1987), and pesticides (Megharaj et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Certain NFC, such as \u003cem\u003eAnabaena\u003c/em\u003e and \u003cem\u003eNostoc\u003c/em\u003e, have demonstrated the ability to degrade lindane residues and organophosphorus herbicides (El-Bestawy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Forlani et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Extensive research has explored the effects of NFC on grain production and soil properties (Singh et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). When used as nitrogen fertilizer substitutes, NFC have been shown to enhance nitrogen use efficiency in rice cultivation while maintaining yields and reducing fertilizer inputs. Additionally, NFC application improves soil chemical properties, microbial composition, community structure, and functionality in paddy soils (Liang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the potential of NFC as a green bioremediation strategy for butachlor-contaminated paddy soils remains unexplored. Therefore, investigating the effects of NFC as a substitution for nitrogen fertilizer on the removal of butachlor from paddy soils can provide valuable insights for sustainable agricultural practices.\u003c/p\u003e \u003cp\u003eThis study hypothesizes that replacing nitrogen fertilizer with NFC can enhance soil properties and microbial communities in butachlor-treated paddy soils, thereby accelerating butachlor elimination. To test this hypothesis, a 105-day outdoor rice pot experiment was conducted under conditions simulating natural environment. The study examined the differential effects of NFC on soil properties and microbial communities under varying fertilization and butachlor application regimes. Furthermore, potential correlations between NFC-induced changes in soil properties and microbial communities and butachlor residue levels were analyzed. The findings aim to provide theoretical support for the health and sustainability of herbicide-treated paddy soils and to promote wider application adoption of NFC in agriculture.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Design of the outdoor rice pot experiment\u003c/h2\u003e \u003cp\u003eThe soil used in the pot experiments was collected from a farm in Fuyuan City, Heilongjiang Province, China (47\u0026deg;73\u0026prime;N, 134\u0026deg;28\u0026prime;E), located within the Mollisol Zone and classified as albic soil. The chemical and biochemical properties of the soil provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The soil was sieved through a 2 mm mesh to remove impurities, thoroughly mixed, and distributed into 21 pots. Each pot measured 18 cm in height and 17 cm in top diameter, with a total volume of 3.5 L. Rice seedlings were cultivated in nursery trays in mid-June and transplanted into the pots on 20 July. Each pot contained four rice seedlings. Prior to transplantation, butachlor was applied to the pots to treat the soil (Panda and Sahu \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The water layer was maintained at approximately 3 cm, and rice seedlings were transplanted after natural water infiltration for 3 days. Based on previous studies, replacing 30% of nitrogen fertilizer with NFC was determined to be the optimal rate for maintaining soil fertility and rice yield (Zhang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The required NFC biomass was calculated as described in Section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe experiment comprised three groups with a total of seven treatments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1. Group N: Application of chemical fertilizer only.\u003c/h3\u003e\n\n\u003ch3\u003e2. Group NB: Application of chemical fertilizer and three doses of butachlor, including:\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNB1: Recommended dose (0.1118 g a.i./m\u0026sup2;).\u003c/p\u003e \u003cp\u003eNB1.5: 1.5 times the recommended dose (0.1677 g a.i./m\u0026sup2;).\u003c/p\u003e \u003cp\u003eNB2: 2 times the recommended dose (0.2236 g a.i./m\u0026sup2;).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e3. Group NCB: Application of chemical fertilizer, NFC replacing 30% of nitrogen fertilizer, and three doses of butachlor, including:\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNCB1: Recommended dose (0.1118 g a.i./m\u0026sup2;).\u003c/p\u003e \u003cp\u003eNCB1.5: 1.5 times the recommended dose (0.1677 g a.i./m\u0026sup2;).\u003c/p\u003e \u003cp\u003eNCB2: 2 times the recommended dose (0.2236 g a.i./m\u0026sup2;).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll treatments were performed in triplicate. NFC was applied as tiller fertilizer 15 days after transplanting (tillering stage) and as panicle fertilizer 60 days after the heading stage. The pot experiment lasted for 105 days, from rice transplanting to harvest. Throughout the experimental, rice was grown at an average ambient temperature of 23\u0026deg;C, with the water layer maintained at approximately 3 cm. Fertilizer and butachlor application details for each treatment are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eSoil samples were collected from a depth of 0\u0026ndash;15 cm at five time points: day 1 (rice transplanting), day 15 (tillering stage), day 45 (heading stage), day 75 (filling stage), and day 105 (harvesting stage). Three parallel samples per treatment were combine into composite samples, air-dried, and sieved through a 2 mm mesh for analysis of physical and chemical properties and the butachlor residues. Fresh soil samples collected at harvesting stage were stored at -80\u0026deg;C for DNA extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFertilizer, NFC addition, and butachlor application in the seven treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"15\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eButachlor\u003c/p\u003e \u003cp\u003e(g\u0026middot;a.i./m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eBasal fertilizer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eTiller fertilizer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003ePanicle fertilizer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c15\" namest=\"c12\"\u003e \u003cp\u003eTotal fertilizer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKCl\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNFC\u003c/p\u003e \u003cp\u003e(g dry wt./m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNFC\u003c/p\u003e \u003cp\u003e(g dry wt./m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eKCl\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eNFC\u003c/p\u003e \u003cp\u003e(g dry wt./m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eKCl\u003c/p\u003e \u003cp\u003e(g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eGroup N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eNB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNB1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eNCB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCB1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 NFC culture and characterization\u003c/h2\u003e \u003cp\u003eOn the basis of preliminary experiments, a cyanobacterium strain, \u003cem\u003eNostoc\u003c/em\u003e sp. (FACHB-87), which exhibits notable tolerance to butachlor and nitrogen fixation potential, was selected. This strain was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology. The strain was cultivated under laboratory conditions at a large scale, using sterilized nitrogen-free BG110 medium, with temperature maintained at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, light intensity set to 56 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;, a 12-hour light/dark cycle, and continuous aeration with filtered air. The culture was harvested by filtration through 300-mesh gauze, yielding fresh algal biomass with 97% water content. The nitrogen content of cultured \u003cem\u003eNostoc\u003c/em\u003e sp. (FACHB-87) was approximately 71.68 g N kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry weight (DW). Under paddy field conditions, the biomass increased 5.19-fold within two weeks, indicating that 2680 g DW of NFC could replace at least 1000 g of nitrogen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of soil chemical and biochemical properties\u003c/h2\u003e \u003cp\u003eSoil pH was measured using a pH meter (METTLER TOLEDO 220, Switzerland) in a 1:2.5 \u003cem\u003e(w\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) soil-water suspension (Abollino et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Soil total phosphorus (TP) was determined via the alkali fusion method (HJ632-2011, China). Soil total nitrogen (TN) and total organic carbon (TOC) were measured using an elemental analyzer (Elementar Vario TOC, Germany). Ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) and nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) were extracted with KCl and quantified using a continuous flow analyzer (Skalar San++, The Netherlands) (Zhang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Soil enzyme activities, including catalase (S-CAT), acid phosphatase (S-ACP), urease (S-UE), and sucrase (S-SC), were measured using spectrophotometric kits (Solarbio, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of butachlor residues in soil\u003c/h2\u003e \u003cp\u003eButachlor was extracted from soil using acetonitrile, purified via dispersive solid-phase extraction, and quantified using gas chromatography-tandem mass spectrometry (GC-MS/MS) (Agilent 7890B-7000C, USA). Briefly, 5.0 g of soil was mixed with 10 mL of ultrapure water in a 50 mL centrifuge tube and left to stand for 30 minutes before 10 mL of acetonitrile and 4 g of NaCl were added for vortex extraction for 2 minutes. Then, centrifugation was performed at 3500 r/min for 5 minutes before the supernatant was transferred. 5 mL of the supernatant was transferred to a 10 mL plastic centrifuge tube containing 300 mg of MgSO\u003csub\u003e4\u003c/sub\u003e, 100 mg of primary secondary amine (PSA), 100 mg of C18, and 50 mg of graphitized carbon black (GCB). After centrifugation at 10,000 r/min for 5 minutes, 2 mL of the supernatant was blown dry under nitrogen and dissolved in 1 ml of hexane. The extract was filtered through a 0.22 \u0026micro;m membrane and analyzed using GC-MS/MS with a 5% phenyl methylpolysiloxane capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m) (Agilent 122\u0026ndash;5532 DB-5 ms). Helium was used as the carrier gas at 2.25 mL/min. The injection port temperature was 280\u0026deg;C, and the column temperature program was as follows: 60\u0026deg;C for 1 minute, increased to 170\u0026deg;C at 40\u0026deg;C/min, and then to 310\u0026deg;C at 10\u0026deg;C/min for 3 minutes.\u003c/p\u003e \u003cp\u003eThe dissipation kinetics of butachlor in soil were described using the first-order kinetic equation:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = C\u003csub\u003e0\u003c/sub\u003ee\u003csup\u003e\u0026minus;kt\u003c/sup\u003e,\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere C\u003csub\u003et\u003c/sub\u003e is the butachlor content time t (d) after application (\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), C\u003csub\u003e0\u003c/sub\u003e is the initial content (\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and k is the first-order rate constant (d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Metagenomic data processing and annotation\u003c/h2\u003e \u003cp\u003eTotal genomic DNA was extracted from soil samples collected at the rice harvesting stage, fragmented, and used to construct paired-end libraries for sequencing. Data processing, gene annotation, and related methodologies are detailed in Text S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS 26.0 (SPSS, Inc., USA) with one-way ANOVA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Prokaryotic microbial functions were annotated and predicted using FAPROTAX software (version 1.2.6). Redundancy analysis (RDA) of environmental variables and butachlor residues was conducted using the vegan package in R 4.3.3 (Oksanen \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Microbial co-occurrence networks were constructed at the genus level using the Hmisc, igraph, and microeco packages (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarity matrices were generated using Pearson correlation analysis, and networks were constructed based on a Random Matrix Theory (RMT) threshold, with Spearman correlation coefficients\u0026thinsp;\u0026gt;\u0026thinsp;0.70 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant. The networks were visualized using the Fruchterman\u0026ndash;Reingold layout in Gephi software (version 0.10.1). Nodes within the networks were classified into four categories based on intra-module connectivity (Zi) and inter-module connectivity (Pi): (1) network hubs (Zi\u0026thinsp;\u0026gt;\u0026thinsp;2.5, Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.62), (2) connectors (Zi\u0026thinsp;\u0026le;\u0026thinsp;2.5, Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.62), (3) peripheral nodes (Zi\u0026thinsp;\u0026le;\u0026thinsp;2.5, Pi\u0026thinsp;\u0026le;\u0026thinsp;0.62), and (4) module hubs (Zi\u0026thinsp;\u0026gt;\u0026thinsp;2.5, Pi\u0026thinsp;\u0026le;\u0026thinsp;0.62).\u003c/p\u003e \u003cp\u003eTo explore the complex interaction between NFC and butachlor residues in soil, partial least squares path modeling (PLS‒PM) was employed. The model incorporated three sets of endogenous variables: (1) soil properties, including pH, TOC, TN and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N; (2) soil enzyme activities, comprising S-CAT, S-UE and S-SC; and (3) community diversity and richness, represented by the Chao1 and Shannon indices. All PLS-PM analyses were performed using the plspm package.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Butachlor residues in paddy soils\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters of the first-order kinetic equation for butachlor elimination in soil under groups NB and NCB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst order kinetic equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ek (d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDetermination coefficient (R\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHalf-life (d)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = 543.07e\u003csup\u003e\u0026minus;\u0026thinsp;0.0120t\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e57.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = 565.13e\u003csup\u003e\u0026minus;\u0026thinsp;0.0188t\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNB1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = 786.12e\u003csup\u003e\u0026minus;\u0026thinsp;0.0127t\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCB1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = 801.40e\u003csup\u003e\u0026minus;\u0026thinsp;0.0175t\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = 1067.52e\u003csup\u003e\u0026minus;\u0026thinsp;0.0091t\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.926\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e76.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003et\u003c/sub\u003e = 1148.34e\u003csup\u003e\u0026minus;\u0026thinsp;0.0179t\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.978\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e38.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe residual levels of butachlor in paddy soils were determined at the initial application of NFC and at four key stages of rice growth: tillering, heading, filling and harvesting stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In all six treatments with three doses of butachlor, the elimination curves followed first-order kinetics, as indicated by determination coefficients (R\u003csup\u003e2\u003c/sup\u003e) exceeding 0.9. After the 105-day experiment, the butachlor content in the three sub-treatments of the group NB (without NFC) decreased from initial values of 538.15, 729.67, and 1012.83 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 110.83, 150.67, and 246.60 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. In contrast, the butachlor content in the three sub-treatments of the group NCB (with NFC) decreased from initial values of 526.19, 716.13, and 1044.42 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 47.59, 96.71, and 135.94 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Significant differences were detected between NB1 and NCB1, as well as between NB2 and NCB2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The residual butachlor levels in the group NCB were 35.81\u0026ndash;57.06% lower than those in the group NB. The elimination rates of butachlor in the three sub-treatments of the group NB were 0.0120, 0.0127, and 0.0091 d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which were lower than those in the group NCB (0.0188, 0.0175, and 0.0179 d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). The half-life of butachlor elimination in the group NB ranged from 54.58 to 76.71 days, while the group NCB exhibited a shorter half-life of 36.87\u0026ndash;39.61 days (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Chemical properties and enzyme activities of paddy soils\u003c/h2\u003e \u003cp\u003eThe results of the chemical analysis of the soil at the rice harvesting stage are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The data indicate that the different treatments led to variations in several key indicators. Specifically, the NB1 and NB1.5 treatments showed a slight decrease in soil pH, TOC, S-ACP, S-UE, and S-SC compared to the N treatment. However, these differences were not statistically significant. In contrast, the NB2 treatment significantly reduced the S-ACP, S-UE, and S-SC activities compared to the N treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eWhen comparing the three sub-treatments of the group NB with those of the group NCB, the latter exhibited higher values of pH, TOC, TN, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, S-ACP, S-UE and S-SC. For instance, the TN levels in the NCB1, NCB1.5, and NCB2 treatments were higher than those in the NB1, NB1.5, and NB2 treatments, respectively. Similarly, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N levels in the NCB1 and NCB1.5 treatments were significantly greater than those in the NB1 and NB1.5 treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the S-UE and S-SC levels in the NCB1.5 and NCB2 treatments were significantly higher than those in the NB1.5 and NB2 treatments, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, the group NB demonstrated increased S-CAT activity, whereas the group NCB showed the opposite trend.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Composition, structure and function of microbial communities in paddy soils\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on metagenomic sequencing results, the composition of soil microbial communities was analyzed, with a focus on the 15 most abundant bacterial phyla in each treatment. The microbial communities across most treatment groups were predominantly composed of Proteobacteria, Acidobacteria, Chloroflexi, Actinobacteria, Verrucomicrobia, Bacteroidota, and Gemmatimonadetes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The highest relative abundance of Proteobacteria was observed in the group NB, ranging from 23.46 to 26.74%. Replacing nitrogen fertilizer with NFC in the group NCB further increased the relative abundance of Proteobacteria to 25.43\u0026ndash;30.74%. The relative abundance of Verrucomicrobia in the group NB (6.39\u0026ndash;6.88%) was lower than that in the group N (8.31%), while the addition of NFC in the group NCB increased its abundance to 7.58\u0026ndash;8.64%. The relative abundance of Bacteroidota was higher in the group NCB compared to the groups N and NB, whereas the relative abundance of Actinobacteria was lower in the group NCB than in the groups N and NB. At the genus level, the use of different doses of butachlor in the group NB reduced the relative abundance of unclassified\u003cem\u003e_p_Verrucomicrobia\u003c/em\u003e compared to the group N. In contrast, the addition of NFC in the group NCB increased its relative abundance, with the order N\u0026thinsp;\u0026gt;\u0026thinsp;NCB\u0026thinsp;\u0026gt;\u0026thinsp;NB across all three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The relative abundance of \u003cem\u003eRamlibacter\u003c/em\u003e in the group NCB (1.51\u0026ndash;1.81%) was also higher than that in the group NB (1.35\u0026ndash;1.43%). Additionally, the relative abundance of unclassified\u003cem\u003e_o_Anaerolineales\u003c/em\u003e in the group NCB (1.19\u0026ndash;2.47%) was substantially greater than that in the groups N (0.60%) and NB (0.63\u0026ndash;1.28%). Analysis of cyanobacteria composition at the genus level revealed that the dominant genus in the groups N and NB were more complex and variable, whereas \u003cem\u003eNostoc\u003c/em\u003e was clearly dominant in the group NCB, with a relative abundance of 6.17\u0026ndash;22.92% (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the α-diversity indices (Chao1, Shannon, and Simpson) of the soil microbial communities were calculated based on metagenomic sequencing results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The Chao1 index in the three sub-treatments of the group NB was significantly higher than that in the group N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although no significant difference was observed between the NCB1 and NCB2 sub-treatments and the NB1 and NB2 sub-treatments, the group NCB exhibited higher values, indicating greater community richness. The Shannon index, which characterizes community diversity, followed a similar trend, with NCB\u0026thinsp;\u0026gt;\u0026thinsp;NB\u0026thinsp;\u0026gt;\u0026thinsp;N. The Simpson index also reflected this pattern, with the group NCB showing higher values than the groups N and NB, indicating greater soil microbial community diversity.\u003c/p\u003e \u003cp\u003eNMDS analysis revealed distinct clustering patterns of microbial communities across the different treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). PERMANOVA further confirmed significant differences in microbial community β-diversity among the groups N, NB, and NCB (F\u0026thinsp;=\u0026thinsp;6.594, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Table S2).\u003c/p\u003e \u003cp\u003eFunctional annotation of soil microbial bacterial communities using FAPROTAX identified 63 functional microbial groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The most abundant groups included chemoheterotrophic, aerobic chemoheterotrophic, phototrophic, and photoautotrophic microbiota. Compared to the group N, the abundance of sulfate-respiring microbiota in the group NB was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the addition of NFC in the group NCB significantly increased their abundance by 2.45\u0026ndash;4.32 times compared to the group NB, with no significant difference from the group N. Additionally, the abundance of nitrogen-fixing microbes was significantly higher in the group NCB than in the groups N and NB (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Although not statistically significant, an increase in the abundance of aromatic-degrading microbiota was observed in the group NCB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Relationships between butachlor residues, microbial communities and environmental factors in paddy soils\u003c/h2\u003e \u003cp\u003eRedundancy analysis (RDA) identified key drivers of variations in soil microbial communities across different fertilization treatment. The first and second sorting axes explained 34.26% and 11.19% of the variation, respectively, cumulatively accounting for 45.45% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). NFC was identified as the primary driver of community composition variation (r\u0026sup2; = 0.896, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), with S-CAT, pH and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N being the largest contributors among the environmental factors (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.531, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.513, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; and r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.465, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, respectively; Table S3). Pearson correlation analysis revealed significant positive correlations between NFC and pH, TOC, TP, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, S-UE, and S-SC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, NFC was significantly negatively correlated with S-CAT and butachlor residues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and significantly positively correlated with Chao1 and Shannon indices of microbial community α-diversity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Butachlor residues were significantly negatively correlated with pH, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, S-ACP, S-UE, and S-SC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as well as with the Chao1 and Shannon indices (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Co-occurrence network of soil microbial communities\u003c/h2\u003e \u003cp\u003eCo-occurrence networks were constructed to examine potential interactions within soil microbial communities under varying doses of butachlor, with or without NFC addition. The group NB network consisted of 237 nodes and 1524 links (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), while the NCB group network had 268 nodes and 2103 links (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The modularity indices of the two networks were 0.620 and 0.549, respectively, with nodes clustered into 11 and 10 modules. The average degree (avgK), average path length (GD), and average clustering coefficient (avgCC) were higher in the group NCB (15.694, 4.117, and 0.549, respectively) than in the group NB (12.861, 4.101, and 0.620, respectively). Modules 2, 3, 5, and 8 were dominant in the group NB, while modules 1, 2, 7, and 8 were dominant in the group NCB. The network density of the group NCB (0.058) was also higher than that of the group NB (0.054).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTopological characterization of co-occurrence networks within microbial communities of the groups NB and NCB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"13\" nameend=\"c14\" namest=\"c2\"\u003e \u003cp\u003eNetwork Indices\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRMT threshold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal nodes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal links\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e of power law\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive links (ratio)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative links (ratio)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAverage degree\u003c/p\u003e \u003cp\u003e(avgK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAverage network\u003c/p\u003e \u003cp\u003eDistance\u003c/p\u003e \u003cp\u003e(GD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNetwork density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAverage clustering coefficient\u003c/p\u003e \u003cp\u003e(avgCC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eBetweenness centrality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eNetwork diameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eModularity\u003c/p\u003e \u003cp\u003eindex\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1270 (83.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e254 (16.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e9.783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.578\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1206 (57.35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e897\u003c/p\u003e \u003cp\u003e(42.65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e15.167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.540\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese two networks were divided into different modules, which can be used to analyze the relationships between important genera in different modules. Correlation analysis revealed that modules 1, 2, 4, 5, 6, 7 and 8 in the group NB were negatively correlated with butachlor residues, while modules 3 and 9 showed positive correlations, though not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the group NCB, module 1 exhibited a significant negative correlation with butachlor residues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while modules 2, 4, 5, 6 and 7 were negatively correlated, and modules 3 and 8 were positively correlated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Key connectors in the NB network included \u003cem\u003eNovosphingobium\u003c/em\u003e, unclassified_\u003cem\u003ep_Candidatus_Moranbacteria\u003c/em\u003e, \u003cem\u003eNitrosopumilus\u003c/em\u003e, and unclassified_\u003cem\u003eo_Phycisphaerales\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In the NCB network, unclassified_\u003cem\u003eo_Myxococale\u003c/em\u003e was identified as a connector, and unclassified_\u003cem\u003eo_Rhodocyclales\u003c/em\u003e and unclassified_o_\u003cem\u003ePirellulales\u003c/em\u003e were identified as module hubs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003ePLS‒PM was employed to assess the direct and indirect influences of NFC, soil properties, soil enzyme activities, and soil microbial community diversity and richness on butachlor residues. The model revealed that butachlor residues were significantly negatively influenced by soil enzyme activities and soil microbial community diversity and richness (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). NFC significantly positively regulated soil properties (r\u0026thinsp;=\u0026thinsp;0.811, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), enzyme activities (r\u0026thinsp;=\u0026thinsp;0.722, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and soil microbial community diversity and richness (r\u0026thinsp;=\u0026thinsp;0.706, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Soil properties negatively regulate butachlor residues and positively regulate microbial community diversity and richness. The standardized total effects indicated that NFC, soil properties, soil enzyme activities, and microbial community diversity and richness were negatively correlated with butachlor residues, with standardized total effect values of -0.565, -0.350, -0.670 and \u0026minus;\u0026thinsp;0.593, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Changes in soil properties and enzyme activities in paddy soils due to NFC addition\u003c/h2\u003e \u003cp\u003eIn this experiment, the effect of three doses of butachlor on soil properties and residual butachlor levels were evaluated under different fertilization conditions at the harvesting stage. The results revealed that soils treated with butachlor exhibited lower pH and TOC compare to soil under normal fertilization without butachlor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, the addition of NFC to butachlor-treated soils led to increased pH, TOC, TN and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N. Butachlor, an amide herbicide, primarily degrades in soil through microbial transformation, with additional pathways including leaching and plant uptake (Yu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The adsorption of butachlor in soil is closely linked to soil properties, which influence its environmental behavior (Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). For instance, soils with higher TOC content exhibit adsorption capacity for butachlor (Liu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). During butachlor degradation, \u003cem\u003eN\u003c/em\u003e-chloramide, a byproduct of chlorination, can oxidize inorganic and organic substances, including sulfite and organic sulfur (Ding et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, amide chlorination is directly influenced by soil pH (Zhang and Gunten \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe favorable conditions for NFC growth in paddy soils, including optimal temperature, humidity, and nutrients, enable NFC to play a critical role in nitrogen fixation and utilization. NFC converts atmospheric nitrogen into compound nitrogen, releasing a potion into the soil during its growth, and the remainder can be retained in the soil through mineralization (Prasanna and Nayak \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This process resulted in higher contents of TN and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N in NFC-treated soils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), consistent with previous findings (Song et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The increased TOC content following NFC addition may be attributable to the photosynthesis of NFC, which facilitates organic carbon input into the soil (Kaushik \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, the growth and decay of NFC release extracellular polymeric substances (EPS) and the accumulation of biomass, significantly contributing to soil organic carbon accumulation (Dron et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil enzymes, sensitive to exogenous pollutants, serve as crucial indicators of soil contamination (Margesin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In this study, the activities of S-ACP, S-UE, and S-SC were lower in the group NB compared to the group N (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that higher butachlor doses inhibit these enzyme activities. This finding aligns with previous research (Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, NFC addition enhanced the activities of S-ACP, S-UE and S-SC in butachlor-treated soils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). S-UE is integral to nutrient cycling, particularly nitrogen metabolism (Ni and Pacholski \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). NFC improves soil nutrient availability, thereby positively influencing enzyme activity. Certain \u003cem\u003eNostoc\u003c/em\u003e species produce extracellular enzymes capable of decomposing organic residues, while their high biomass and EPS secretion increases polysaccharide content and microbial activity, further enhancing soil quality in butachlor-treated soils.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2 Changes in the structure and function of soil microbial communities in paddy soils due to NFC addition\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSoil microorganisms, as the most active component of soil ecosystems, drive plant productivity, biogeochemical cycling, and organic pollutant degradation, playing a fundamental role in maintaining soil sustainability (M\u0026uuml;hlbachov\u0026aacute; \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this study, NFC addition increased the abundance of Proteobacteria and Bacteroidota in butachlor-treated soils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These phyla are key players in biogeochemical cycling, with Proteobacteria involved organic matter decomposition (Dang and Lovell \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in soil ecosystems (Delmont et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Bacteroidota contributed to polysaccharide metabolism (Shi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The improved soil properties and increased the TN and TOC content following NFC addition likely favored the growth of copiotrophic taxa such as Proteobacteria and Bacteroidota, which thrive one labile carbon sources (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, Verrucomicrobia, which play roles in nitrogen fixation and methane emission reduction (Chiang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Dunfield et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), were positively influenced by NFC-mediated changes in soil carbon and nitrogen dynamics. The carbon and nitrogen contents in soil represent the main environmental variables affecting taxa such as Verrucomicrobia (Wang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Adding NFC directly affects the biogeochemical cycling of carbon and nitrogen in butachlor-treated soils. Consequently, this resulted in a notable effect on Verrucomicrobia, which play important roles in carbon and nitrogen cycling.\u003c/p\u003e \u003cp\u003eAt the genus level, butachlor application reduced the relative abundance of \u003cem\u003eRamlibacter\u003c/em\u003e, a genus within Proteobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, NFC addition counteracted this decline. We hypothesized that this alteration may be largely related to the input of NFC. \u003cem\u003eRamlibacter\u003c/em\u003e, a genus of Proteobacteria, is involved in several important pathways in the soil nitrogen cycle, including nitrogen fixation and intracellular ammonium accumulation and transport (Hu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These nitrogen-cycling processes mediated by \u003cem\u003eRamlibacter\u003c/em\u003e promote the production of ammonium in cells. In this study, the positive correlation between NFC and NH₄⁺-N content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), together with the results of our measurements of the NH₄⁺-N content of the soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), may further support this hypothesis. NFC addition also increased the α-diversity of soil microbial communities, with no negative effects observed from butachlor application (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, significant differences in β-diversity among treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table S2) suggest that NFC addition altered the original ecological balance, altering microbial community structure. Environmental factors such as pH, TOC, TP, NH₄⁺-N, S-UE, S-SC, and S-CAT, along with residual butachlor, likely contributed to these changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFunctional annotation using FAPROTAX revealed that NFC addition increased the abundance of sulfate-respiring microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Sulfate-reducing bacteria capable of sulfate respiration can induce the conjugation of some sulfur-containing compounds with chloroacetamide herbicides like butachlor, thereby dechlorinating chloroacetamide and accelerating its degradation (Liu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Torabi et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, the higher abundance of aromatic-degrading microbiota in the group NCB may also contribute to butachlor elimination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eCo-occurrence network analysis shows that the addition of NFC increases the network nodes and links, making the network more complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Some modules in the group NB are negatively correlated with butachlor residues, but none of the correlations are significant. In contrast, network module 1 in the group NCB shows a significant negative correlation with butachlor residues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The core genus in Module 1 was \u003cem\u003eSphingomonas\u003c/em\u003e, which is capable of nitrogen fixation and phytohormone production, enabling it to promote plant growth and improve stress tolerance (Asaf et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). More importantly, \u003cem\u003eSphingomonas\u003c/em\u003e exhibits a distinct advantage in degrading chloroacetamide herbicides. Certain \u003cem\u003eSphingomonas\u003c/em\u003e strains can utilize butachlor as the sole carbon source and achieve its complete mineralization through synergistic metabolism (Kim et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, \u003cem\u003eSphingomonas\u003c/em\u003e can produce the amide hydrolase \u003cem\u003eCmeH\u003c/em\u003e, which hydrolyzes the butachlor intermediate 2-chloro-\u003cem\u003eN\u003c/em\u003e-(2,6-dimethylphenyl) acetamide to 2,6-diethylaniline (DEA). DEA is further mineralized into aniline and catechol under the action of \u003cem\u003eSphingomonas\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). All of the above intermediates can ultimately be converted into carbon dioxide and water. The unclassified_\u003cem\u003eo_Rhodocyclales\u003c/em\u003e and unclassified_\u003cem\u003eo_Pirellulales\u003c/em\u003e are the module hubs and key nodes with relatively high abundances in the group NCB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), and they are significantly negatively correlated with butachlor residues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. S2). Some members of the denitrifying bacteria Rhodocyclales have the ability to degrade hydrocarbons (Abdalrhman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies indicated that Pirellulales in plant rhizosphere soils may play a pivotal role in nitrogen fixation and ammonium retention processes (Hu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, the continuous growth and death of NFC in the soil provide nutritional conditions for the growth and metabolism of other microorganisms, creating a favorable environment for the proliferation of microorganisms capable of degrading butachlor or promoting its degradation in the soil. These changes the assembly pattern of the microbial community, making the soil microbial ecological network more complex and the interactions between species closer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Potential role of NFC as a driver of butachlor degradation in soil\u003c/h2\u003e \u003cp\u003eNFC addition enhanced butachlor degradation in paddy soils, as evidenced by reduced residual butachlor levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The physical and chemical properties of soil significantly influence the degradation rate of butachlor and its environmental fate (Liu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). PLS-PM showed that the improvement of soil properties and enzyme activity mediated by NFC directly or indirectly promoted the degradation of butachlor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). NFC also directly had a positive impact on the diversity and richness of the soil microbial community, altering and optimizing the soil microbial community and thus enhancing the stability of the molecular ecological network. Therefore, the addition of NFC to soil improved soil nutrient conditions, promoted enzyme activity, stimulated and altered the metabolic activities and aggregation of microorganisms, and further promoted the soil adsorption behavior of butachlor and the microbially-mediated biodegradation pathway. In other words, it promoted the biodegradation of butachlor. In conclusion, replacing nitrogen fertilizers with NFC is an effective strategy to improve soil fertility and reduce butachlor residues, which can lower the potential risks to crops and non-target microorganisms.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study investigated the effects of replacing nitrogen fertilizers with NFC on the properties and community structure of paddy soils treated with different doses of the herbicide butachlor, as well as its impact on butachlor elimination. The results indicated that substituting nitrogen fertilizers with NFC increased the soil carbon and nitrogen contents, as well as the activities of enzymes such as S-UE, compared with those in soils treated with various doses of butachlor under normal fertilization conditions. In conclusion, NFC improved the chemical properties and soil enzyme activities of paddy soils with butachlor and remodeled the composition and structure of microbial communities, favoring the aggregation and growth of microbial taxa potentially associated with mediating the biodegradation of butachlor, thus indirectly contributing to the elimination of butachlor from the soil. These results suggest that replacing a portion of nitrogen fertilizer with NFC is a promising management strategy for paddy soils treated with butachlor. This approach not only improves soil fertility but also reduces the environmental risks associated with residual herbicides. However, further investigation is needed to elucidate the underlying mechanisms by which NFC promotes the microbial-mediated degradation of butachlor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interest\u003c/h2\u003e \u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28030102) and Special Fund for Scientific Innovation Strategy-construction of High Level Academy of Agriculture Science (R2021YJ-QG003, R2023PY-JG024).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Dunhai Li; Methodology: Huiyao Liu, Xiao Liang, Kan Wang, Hongjie Qin, Dunhai Li; Formal analysis and investigation: Huiyao Liu, Yu Zhu; Writing-original draft: Huiyao Liu; Writing - review and editing: Chengrong Peng, Dunhai Li; Funding acquisition: Hongjie Qin, Chengrong Peng, Dunhai Li; Supervision: Genbao Li, Chengrong Peng, Dunhai Li.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdalrhman AS, Zhang Y, Arslan M, Gamal El-Din M (2020) Low-current electro-oxidation enhanced the biodegradation of the recalcitrant naphthenic acids in oil sands process water. 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Chemosphere 144:1177\u0026ndash;1192. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chemosphere.2015.09.050\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2015.09.050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Butachlor, Microbial co-occurrence networks, Microbial communities, Nitrogen-fixing cyanobacteria, Paddy soils sustainability, Soil health","lastPublishedDoi":"10.21203/rs.3.rs-6625888/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6625888/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eButachlor, a widely used herbicide in paddy fields, exhibits persistent environmental residues, posing significant ecological concerns. Nitrogen-fixing cyanobacteria (NFC), as a biofertilizer, not only enhance soil fertility and crop growth but also show potential in influencing the environmental fate of herbicides. However, the specific effects of NFC as a nitrogen fertilizer substitute on butachlor remediation in paddy soils remains unexplored. To address this, a 105-day outdoor pot experiment was conducted to evaluate the impact of NFC on soil properties, microbial community dynamics, and butachlor removal efficiency. The results showed that NFC application increased soil carbon and nitrogen content, enhanced enzyme activities (e.g., urease), and reshaped the microbial community structure compared with conventional fertilization. Co-occurrence network analysis further showed that NFC increased the complexity of soil microbial ecological networks, leading to closer species interactions and favoring the aggregation and proliferation of microorganisms that mediate butachlor degradation (e.g., \u003cem\u003eSphingomonas\u003c/em\u003e). As a result, the extinction of butachlor was accelerated, and the amount of butachlor residue at harvesting stage was reduced by 35.81\u0026ndash;57.06% relative to conventional fertilization. This study provides a theoretical foundation for the application of NFC in paddy soils, highlighting its potential to improve soil health and sustainability in the context of butachlor use.\u003c/p\u003e","manuscriptTitle":"Enhanced remediation of paddy soils contaminated with butachlor through the application of nitrogen-fixing cyanobacteria as a nitrogen fertilizer substitute","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 17:04:16","doi":"10.21203/rs.3.rs-6625888/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-18T05:46:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T14:52:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110743851077185965683999099326027109995","date":"2025-05-20T14:41:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-20T08:16:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T02:50:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-12T06:10:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2025-05-09T06:55:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0c4972a9-2916-467c-9f5f-96d71e543d71","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-09-19T03:08:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 17:04:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6625888","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6625888","identity":"rs-6625888","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}