Simulation of the Fate of Triclosan in a Paddy Soil Co-Contaminated with Graphene Nanomaterials: Enhanced Formation of Bound Residues and Potential Long-Term Risks

Abstract

The co-occurrence of graphene-based nanomaterials such as reduced graphene oxide (RGO) and triclosan in agricultural soils is an emerging concern. This study investigates the impact of RGO on the formation and characteristics of bound residues (BRs) of triclosan in paddy soil using ¹⁴C-isotope tracing and LC-QTOF-MS. Results demonstrate that RGO significantly enhances the accumulation of triclosan BR in a dose-dependent manner, with the highest concentration (1.19 mg kg^–1; 57.0%) observed at 500 mg kg^–1 RGO. While the BR is primarily associated with the humin fraction (>63.8%), RGO shifts the distribution of ¹⁴C-triclosan, enhancing its retention in humin by 1.89–7.59% and in humic acid by 20.7–52.1%. RGO may increase the sequestered BR (8.8–24.7%), and it enhances the covalent BR of triclosan by increasing the proportions of both ether- (3.78–4.58%) and ester-bound (22.8–39.5%) forms. Metabolite analysis reveals limited transformation of triclosan (0.057–0.082 mg kg^–1) in BRs, with carboxylated derivatives identified as minor products. The findings indicate that RGO enhances the persistence of triclosan BRs, which may be attributed to strong adsorption and microbial inhibition, raising concerns about their potential future remobilization and entry into the food chain. This underscores the need to assess the ecological risks of nanomaterial co-contamination for soil health and sustainable agriculture.

1. Introduction

Graphene nanomaterials (GNs), such as reduced graphene oxide (RGO), are increasingly used in industrial and consumer applications due to their exceptional physicochemical properties [1]. Their widespread use results in these materials inevitably entering agricultural systems, potentially through pathways such as wastewater irrigation, sludge amendment, or the use of nano-enabled agrochemicals [2]. Owing to their high specific surface area, adsorption capacity, and variety of functional groups, GNs can strongly adsorb organic pollutants—including pesticides and pharmaceuticals—through hydrophobic and π–π interactions, thereby influencing the pollutants’ fate and persistence in soil [3,4,5]. For instance, graphene oxides were shown to act as carriers, facilitating the transport of contaminants like dimethyl phthalate [6]. In another study, RGO improved the debromination extent of 2,2′,4,4′,5-pentabromodiphenyl ether and the transformation rate of 3,4-DCA in percogenic paddy soil [7]. These studies demonstrate that GNs can significantly alter the fate of organic pollutants in terrestrial environments, either by facilitating their transport or by inhibiting their degradation, depending on the specific conditions and properties of both the GNs and the pollutants.

Triclosan—a broad-spectrum antimicrobial agent widely used in pharmaceutical and personal care products [8]—represents a pollutant of particular concern in agricultural settings. Its frequent detection in agricultural soil is linked to irrigation with reclaimed water and the application of biosolids [9,10]. Known for its persistence and adsorption to soil particles, triclosan may pose ecological and health risks, including potential uptake by crops and disruption of soil microbial communities [10,11]. Research on the effects of graphene on triclosan in aquatic environments shows that RGO has a strong adsorption affinity for triclosan [12]. The adsorption is primarily attributed to the attachment of extracellular polymeric substances to microbially reduced RGO, which enhances its capacity [13], and is further facilitated by minimal electrostatic repulsion of triclosan from the negatively charged RGO surface [14], along with significant contributions from hydrophobic and π–π interactions [15]. In addition, one study has found that RGO affects the occurrence and degradation of triclosan in soils by its strong adsorption of triclosan and inhibition of triclosan-degrading bacteria [16]. Although previous research has demonstrated RGO’s strong adsorption affinity for triclosan in aqueous environments, its impact on triclosan’s fate in soil—particularly regarding the formation of bound residues (BRs)—remains poorly understood. Elucidating this process is essential for scientific evaluation of the environmental impact of such co-contamination in agricultural soil.

BR formation is a critical process controlling the long-term bioavailability and potential remobilization of organic pollutants [17,18,19]. On one hand, the immobilization of pollutants through BR formation is often viewed as a natural detoxification mechanism—primarily due to the marked decrease in bioavailability [20,21]. On the other, studies on compounds including bisphenol S, pendimethalin, and 4-n-dodecylphenol have indicated that BRs may not remain permanently stable and can be remobilized over time, posing a potential long-term environmental risk in agricultural soil [17,22,23]. However, the behavior and stability of triclosan-derived BRs in agricultural soils amended with RGO remain largely uncharacterized. Conventional analytical methods often fall short in reliably discriminating between free and bound pollutant states. A more robust strategy involves coupling isotope tracing techniques (e.g., ¹⁴C labeling) with advanced high-resolution mass spectrometry (such as LC-QTOF-MS), which together provide a powerful tool for elucidating the formation, transformation, and potential release of BRs in agricultural soil [17,23].

Based on the known adsorptive properties of RGO and its potential inhibitory effects on soil microbes [7,24,25], we hypothesized that RGO promotes the formation of triclosan BRs in soil by strong adsorption and suppression of microbial degradation. To test this hypothesis, this study systematically investigated the impact of RGO on the formation and characteristics of triclosan BRs in paddy soil. The distribution of triclosan across humus, sequestered/covalent BR, and ether-/ester-linked BR was quantified using ¹⁴C-isotope tracing analysis. The chemical compositions of the major binding products were identified using LC-QTOF-MS. A mechanism for the effect of RGO on triclosan BR formation is proposed. Our findings contribute to a better understanding of nanomaterial–pollutant interactions in agricultural systems, supporting more accurate risk assessment and sustainable soil management practices.

2. Materials and Methods

2.1. Reagents

[Dichlorophenyl-U-¹⁴C]-triclosan was synthesized according to the method described by Nie et al. [26]. ¹⁴C-triclosan had a specific activity of 10.0 mCi mmol^–1 and a radiochemical purity of >98.0%. The RGO powder, purchased from Suzhou Carbon Graphene Technology Co., Ltd. (Suzhou, China), had a chemical purity of >98% with the following characteristics: thickness, 0.5–3 nm; diameter, 0.5–5 μm; BET surface area, 1000–1217 m²g^–1; zeta potential, −35.1 mV; and C/O ratio, 5.7. High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other solvents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The radioactivity of the samples was measured using a TriCarb-2910 liquid scintillation counter (LSC, PerkinElmer, Waltham, MA, USA).

2.2. Soil Culture and Sampling

The soil was collected from the surface layer (5–20 cm) of a paddy field in Hangzhou, Zhejiang Province, China. The precise geographical coordinates of the sampling site are 30°26′94″ N and 120°19′55″ E. The field had been under intensive rice (Oryza sativa L.) cultivation in the seasons immediately prior to sampling. The soil was classified as a silt loam. It was air-dried and sieved through a 2 mm mesh. The basic physicochemical properties of the soil were as follows: pH, 6.80; sand, silt, and clay contents, 12.1%, 20.6%, and 67.3%; cation exchange capacity, 9.8 cmol kg^–1; total organic carbon, 5.0%; organic matter content, 2.9%; electrical conductivity, 1.0 dS m⁻¹; bulk density, 1.2 g cm⁻³; and soil microbial activity (Chao1 index), 2695.1. The air-dried soil was moistened with distilled water to achieve 30% of the maximum field water holding capacity (WHC) and was further adjusted to 60% WHC. Portions of the prepared soil (300 g) were transferred into 1000 mL glass jars and incubated at 25 ± 1 °C. ¹⁴C-triclosan (1.5 mg kg^–1 soil and 2.5 × 10⁵ Bq kg^–1 soil, dry weight) and RGO (50 mg kg–1: RGO-50; 100 mg kg^–1: RGO-100; 500 mg kg^–1: RGO-500) were added to the tested soils, with three replicates per treatment. Samples were collected at 7, 14, 35, 63, and 98 days after treatment initiation and stored at −20 °C for subsequent analysis.

2.3. Determination of Bound Residues

The BRs were extracted following the method of Nie et al. [27]. Briefly, soil samples (10 g wet weight) were placed in 100 mL Teflon centrifuge tubes. Then, 50 mL of acetonitrile, 50 mL of methanol, and 50 mL of acetone were added sequentially. The mixtures were shaken on an orbital shaker at 200 rpm for 2 h and then centrifuged at 6000× g for 5 min. The resulting soil pellets were combusted using an OX-501 biological oxidizer (RJ Harvey Instruments, Bridgewater, NJ, USA). The released ¹⁴CO₂ was trapped with 10 mL of scintillation cocktail I (capture efficiency > 90%), and its radioactivity was quantified by LSC. This radioactivity was defined as the BR of triclosan.

2.4. Fractionation of Bound Residues in Soil Humus

The BRs of triclosan in humus were fractionated according to the following procedure [28]. First, 0.3 g of air-dried soil samples were mixed with 1.2 mL of 0.1 M NaOH solution (degassed). The mixture was agitated at 200 rpm for 16 h to fully dissolve the humic substances and then centrifuged at 10,000× g for 15 min. This step separated the alkaline-soluble humus (supernatant) from the insoluble humin pellet. The humin pellet was dried in an oven at 50 °C prior to combustion analysis. Next, the supernatant (containing the alkaline-soluble humus) was transferred to a 5 mL conical-bottom centrifuge tube. It was acidified to pH 1.0 by adding a predetermined volume of 6 M HCl and then centrifuged again at 10,000× g for 30 min. This step separated the acid-insoluble humic acid (HA, precipitate) from the acid-soluble fulvic acid (FA, supernatant). Finally, the radioactivity of the FA fraction was determined by taking a 1.0 mL aliquot, adding it to a scintillation cocktail, and measuring it by LSC. The radioactivity associated with the HA and humin fractions was determined by oxidative combustion using a biological oxidizer, followed by LSC counting of the trapped ¹⁴CO₂.

2.5. Distribution of Sequestered and Covalently Bound Residues of Triclosan in Soil

The distribution of sequestered and covalent BR of triclosan in soil was determined using a sequential extraction and derivatization protocol based on the method of Cao et al. [23]. Trimethylchlorosilane (TMCS) was used as a silylation agent to target and protect hydroxyl (-OH) and carboxyl (-COOH) functional groups in the BRs, thereby enhancing their stability and volatility for subsequent analysis. First, 1 g of soil (that had been previously extracted to remove soluble fractions) was weighed into a centrifuge tube. Then, 10 mL of acetone and 1 mL of TMCS were added to the tube. The mixture was shaken for 16 h (overnight) to facilitate derivatization. After shaking, the sample was centrifuged at 10,000× g for 5 min to separate the supernatant from the soil pellet. The supernatant was carefully transferred to a vial. The solvent in the vial was evaporated to dryness under a fume hood. The dry residue was redissolved in 1 mL of methanol, and its radioactivity was quantified by LSC. This fraction was operationally defined as the sequestered BR. Simultaneously, the remaining soil pellet was transferred to an oven and dried at 50 °C. The radioactivity associated with this pellet was determined by oxidative combustion followed by LSC and was operationally defined as the covalently bound fraction.

2.6. Distribution of Ether- and Ester-Linked Bound Residues of Triclosan in Soil

The distribution of triclosan bound via ester and ether linkages was determined through a sequential alkaline hydrolysis and extraction procedure. First, 1 g of soil was reacted with 4 mL of 1 M NaOH solution at 90 °C for 4 h with continuous stirring to hydrolyze ester linkages. After hydrolysis, the mixture was centrifuged at 5000× g for 15 min. The supernatant (the first hydrolysate) was collected, and the residual soil pellet was washed once with distilled water. The washed pellet was then subjected to a second, more severe hydrolysis with 4 mL of 1 M NaOH at 120 °C for 15 min to cleave ether linkages. After centrifugation, this second supernatant (hydrolysate 2) was collected. The hydrolysate from the second step (120 °C) was acidified to pH 1–2 and extracted with dichloromethane (DCM) to isolate the organic phase. This DCM extract was concentrated by rotary evaporation and reconstituted in methanol, and its radioactivity was quantified by LSC. This fraction was designated as the ester-linked BR. The supernatants from the first and second hydrolyses (hydrolysate 1 and 2) were combined. The combined alkaline solution was acidified to pH 1–2 and extracted with DCM. The radioactivity in this DCM extract was quantified by LSC, and the extract was designated as the ether-linked BR. Finally, the remaining soil residue after the two hydrolyses was considered to contain other, more recalcitrant bound forms. Its radioactivity was determined by oxidative combustion using a biological oxidizer, followed by LSC.

2.7. Analysis of Triclosan Metabolites in BR by LC-MS/MS

Analysis of triclosan metabolites within the ether- and ester-linked BR fraction was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Agilent 1260 HPLC system (Santa Clara, CA, USA) coupled to an Agilent 6530 Q-TOF mass spectrometer. Chromatographic separation was performed on a Zorbax SB-C18 column (4.0 mm × 250 mm, 5 μm particle size) maintained at 40 °C. The mobile phase consisted of (A) water and (B) acetonitrile. The gradient elution program was applied at a flow rate of 1.0 mL min^–1: 0–20 min, 40% B; 20–35 min, 40% to 80% B; 35–40 min, 80% to 95% B; 40–47 min, 95% to 40% B; and 47–50 min, 40% B. The injection volume was 40 μL.

Mass spectrometric detection was performed in negative electrospray ionization mode. The ion source parameters were set as follows: source temperature, 250 °C; capillary voltage, 3000 V; nebulizer pressure, 35 psi. Data were acquired with a collision energy ramp from 5 to 50 eV. All data were processed using Agilent MassHunter Workstation software (version B.06.00).

2.8. Quality Assurance and Quality Control

Method blanks were processed with each batch of samples (one blank per three samples) to monitor potential contamination. The recovery of ¹⁴C-triclosan from spiked control soil samples exceeded 97%. The mass balance of ¹⁴C was calculated for all samples, with recovery rates ranging from 90% to 105%. For LC-QTOF-MS analysis, the limits of detection (LOD) and quantification (LOQ) were determined to be 0.01 and 0.05 mg kg⁻¹, respectively, based on signal-to-noise ratios of 3 and 10. For LSC, the LOD and LOQ were 1 Bq and 2 Bq (equivalent to 0.006 and 0.012 μg kg⁻¹), respectively. Instrument calibration was verified daily, and a procedural standard was analyzed after every 10 samples to ensure reproducibility. All procedures involving ¹⁴C-triclosan were conducted in accordance with institutional radiation safety protocols.

2.9. Data Analysis

All data were statistically analyzed using one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test for multiple comparisons. The analysis was performed using SPSS software (version 19.0; IBM SPSS Statistics, Armonk, NY, USA). Differences were considered statistically significant at p < 0.05. Results are presented as the mean ± standard error of the mean from three independent replicates (n = 3).

3. Results and Discussion

3.1. Effect of RGO on Total BRs of Triclosan

Figure 1 illustrates the concentrations of triclosan BR coexisting with RGO at various time intervals in a paddy soil system. Initially, the concentration of total BR increased, followed by stabilization with prolonged incubation time. By day 98, the concentrations of BR in the control and treatment groups (RGO-50, RGO-100, and RGO-500) reached 0.74–1.19 mg kg^–1. This represented a substantial portion of the initial compound (1.5 mg kg^–1), which is consistent with the high BR formation (50–60% of the initial amount) previously reported for ¹⁴C-labeled sulfadiazine, sulfamethoxazole, and triclosan in soil [29,30]. One study has indicated that triclosan readily incorporates into soil and forms BR [16]. The hydroxyl groups present in triclosan may facilitate the rapid formation of BRs in soils. Notably, at this time point, the trend of BR concentrations observed was RGO-500 (1.19 mg kg^–1) > RGO-100 (0.88 mg kg^–1) > RGO-50 (0.83 mg kg^–1) > control (0.74 mg kg^–1). Throughout all incubation periods, the application of 500 mg kg^–1 of RGO significantly enhanced the formation of triclosan BR in soil (F = 25.7, p = 0.007). Similarly, multi-walled carbon nanotubes (20–2000 mg kg^–1) have been reported to promote the formation of BRs of 2,4-dichlorophenol in soil [31].

The findings demonstrate that RGO enhances the formation of triclosan BR through several primary mechanisms: First is direct facilitation: RGO itself has a strong adsorption capacity for triclosan. It may also alter soil particle surface properties, further promoting triclosan adsorption and ultimately leading to its sequestration into BRs. Second is indirect inhibition: The strong adsorption of triclosan onto RGO reduces its bioavailability to soil microorganisms, thereby inhibiting its degradation and indirectly promoting BR formation. BR derived from compounds and their major metabolites can be categorized as type-I BR (sequestered within soil organic matter and clay minerals) or type-II BR (covalently bound to soil organic matter) [32]. Given that type-I BR involves sequestration within soil organic matter and clay minerals, the strong adsorption capacity of RGO for triclosan may specifically enhance the formation of this type of BR in soil [27]. Furthermore, high concentrations of RGO can adversely affect soil microorganisms through physical damage, such as membrane disruption and cell entrapment [33], thereby affecting the fate of pollutants like 2,2′,4,4′,5-pentabromodiphenyl ether and 3,4-dichloroaniline [7]. This inhibitory effect extends to key triclosan-degrading bacteria (e.g., Sphingomonas and Pseudomonas), reducing triclosan dissipation and ultimately promoting the formation of BRs [16]. The enhancement of BR by RGO may have implications for long-term soil health and contaminant persistence. This underscores the need to evaluate the ecological risks of nanomaterial co-exposure in agroecosystems, particularly when assessing the fate of antimicrobial agents such as triclosan.

3.2. Effect of RGO on Bound Residues of Triclosan in Humus Fractions

The presence of RGO greatly influenced the distribution of BRs derived from triclosan among humus fractions in agricultural soil, as depicted in Figure 2. Overall, the percentage of ¹⁴C-triclosan across humus fractions followed the order: humin > FA > HA, with the proportion in humin ranging from 63.8% to 85.1%. The results indicate that ¹⁴C-triclosan preferentially binds to humin in soil. Similarly, previous studies have indicated that ¹⁴C-pyraoxystrobin and prosulfocarbin are predominantly associated with humin [34,35]. This distribution may be attributed to the high organic carbon content in humin—including black carbon and kerogen—which promotes stronger sorption of hydrophobic pollutants compared to HA or FA [35].

RGO greatly altered the distribution of ¹⁴C-triclosan among humus fractions. Specifically, it increased the proportion of ¹⁴C-triclosan in humin and HA while decreasing it in FA. For instance, the percentages of ¹⁴C-triclosan in humin/FA/HA were 68.8%/20.8%/10.3% (control), 72.3%/17.0%/10.7% (RGO-50), 76.4%/12.2%/11.4% (RGO-100), and 78.1%/6.8%/15.2% (RGO-500). Furthermore, compared with the control, the magnitude of change in treatment groups showed dose-dependence on RGO concentration (Figure S1, r ≥ 0.86). This observation aligns with previous findings that GNs can induce higher ¹⁴C accumulation in humin and HA [34]. This phenomenon may be attributed to the extensive aromatic domains of RGO, which can interact with the inherent aromatic structures in humin via π–π stacking [36]. This interaction likely creates a more condensed, hydrophobic environment that favors the partition and entrapment of hydrophobic compounds like triclosan [12]. The chlorophenolic ring of triclosan can further participate in π–π interactions and halogen bonding with this augmented aromatic network, leading to the observed increases in humin BRs. Given that residues bound to humin exhibit the highest stability, the shift in distribution toward this fraction suggests that RGO can reduce the short-term mobility and bioavailability of triclosan, potentially lowering the immediate risks of plant uptake or leaching. However, the long-term implications warrant careful consideration. As humin is a key component of soil organic matter and contributes to soil structure and nutrient cycling, the incorporation of triclosan residues into this pool may represent a lingering source of contamination. Future assessments of soil health and organic matter management in nano-amended agricultural systems should consider the potential for such interactions to influence the persistence and long-term release potential of organic pollutants.

3.3. Effect of RGO on the Distribution of Sequestered and Covalently Bound Residues of Triclosan in Soil

The ¹⁴C concentrations (mg kg^–1) associated with sequestered and covalently BRs in a paddy soil system under control and RGO treatments (RGO-50, RGO-100, and RGO-500) at different sampling time points (7, 14, 35, 63, and 98 d) are presented in Figure 3. In all treatment groups, the ¹⁴C concentration in covalent BR (0.1–0.9 mg kg^–1) was significantly higher than that in sequestered BR (0.07–0.4 mg kg^–1) (F = 382.3, p = 0.001). For instance, at the end of incubation (98 d), the ¹⁴C concentrations reached 0.9 mg kg^–1 in covalent BR versus 0.3 mg kg^–1 in sequestered BR. This distribution pattern was similar to that reported for sequestered and covalent BR of 4-n-dodecylphenol in soil [22]. The temporal patterns differed between the two fractions: sequestered BR concentrations initially increased, then decreased, whereas covalent BR concentrations increased progressively before plateauing. This decrease in sequestered BR indicates a more rapid release compared to covalent BR, as the former is primarily held by relatively reversible physical forces (e.g., adsorption, entrapment) [37,38]. In contrast, the progressive accumulation and stability of covalent BR underscore the formation of stronger, irreversible chemical bonds (e.g., ester, ether linkages) with soil organic matter. This interpretation is consistent with the observed predominance of ester-linked residues in Section 3.4.

While RGO (100 mg kg^–1 or 500 mg kg^–1) did not alter the fundamental pattern of sequestered BR being more readily released than covalent BR, it significantly increased the absolute amount of both residue types (F = 31.3, p = 0.005). These results demonstrate that RGO enhances the accumulation of ¹⁴C in both sequestered and covalently BRs. This dose-dependent enhancement (Figure S1, r ≥ 0.90) can be mechanistically interpreted as follows: RGO, with its high specific surface area and aromatic structure, acts as a powerful adsorbent via π–π interactions and hydrophobic effects with the chlorophenolic rings of triclosan [12,15]. By concentrating triclosan molecules at the RGO–soil interface, this intense adsorption not only directly increases the pool of sequestered BR but, more importantly, by concentrating triclosan molecules at the RGO–soil interface, prolongs their residence time and increases the probability of nucleophilic attack from functional groups (e.g., -OH, -COOH) in soil organic matter, thereby catalyzing the formation of stable covalent bonds [32]. Furthermore, the physical encapsulation of triclosan within RGO aggregates provides an additional pathway for sequestration, shielding the compound from microbial degradation and contributing to the observed long-term persistence. Consequently, the RGO-induced increase in sequestered BR indicates a reduction in the immediate mobility and bioavailability of triclosan. However, this larger reservoir of sequestered residues also represents a pool of contaminants that may be remobilized over time [23]. The simultaneous and substantial increase in the more stable covalent BR pool suggests a dual role of RGO: It temporarily immobilizes triclosan while concurrently promoting its integration into the soil matrix through stable chemical bonds. This shift toward covalent bonding has profound implications for long-term fate, potentially leading to protracted but low-level release, which underscores the need to assess the long-term bioavailability and environmental risk of triclosan residues in RGO-amended soils.

3.4. Effect of RGO on the Distribution of Ether- and Ester-Linked BR of Triclosan in Soil

Figure 4 shows the proportional distribution of ¹⁴C among ether-linked, ester-linked, and other BR forms in both control and RGO-treated soils over time. Generally, the proportion of ¹⁴C in ester-linked BR (20.7–67.8%) was consistently higher than that associated with ether-linked BR (9.3–30.7%) across all treatments and time points. These findings are consistent with a previous report showing a higher prevalence of ester-linked BR of tetrabromobisphenol A compared to ether-linked forms in soil systems [39]. Cao et al. [23] also reported that ester-linked BR accounted for 31.5 ± 3.0% of the total BR (28 d), and the contribution of ester-linked BR was much larger than that of ether-linked BR bisphenol S (BPS) to the total BR of BPS in soil systems.

Compared to the control, RGO greatly altered the proportional distribution of ¹⁴C among BR forms. At 98 d, the proportions of ester BR decreased in the following order: RGO-500 (67.8%) > RGO-100 (56.5%) > RGO-50 (51.2%) > control (28.4%), with a similar trend observed for ether-linked BR. These results indicate that RGO enhances the retention of ester-linked BR forms in a dose-dependent manner (Figure S1, r ≥ 0.98). This enhancement can be attributed to a combination of physical and chemical processes. The proposed mechanism involves two key aspects: First, the hydroxyl and carboxyl groups on RGO [40] are not merely mentioned but are proposed to act as direct binding sites for esterification with carboxylic acid metabolites (like carboxy-triclosan) or for etherification with phenolic hydroxyl groups. Second, and more important, the powerful adsorption capacity of RGO for triclosan and its metabolites (via π–π stacking and hydrophobic interactions) concentrates these reactants at the RGO–soil organic matter interface [16]. This localized increase in concentration considerably enhances the probability of collisions between reactive functional groups, effectively catalyzing the nucleophilic substitution reactions that lead to ester bond formation. However, the increased formation of covalent bonds introduces a long-term environmental risk. The stability of these bonds differs markedly: ester-linked residues are relatively labile and susceptible to hydrolysis, resulting in potential slow release, whereas ether-linked residues are more stable and represent a persistent sink. Consequently, environmental perturbations or agricultural practices such as tillage and changes in soil pH could potentially remobilize the less stable ester-linked fraction over time. Therefore, future research should prioritize the long-term bioavailability and remobilization potential of these specific BR forms under realistic conditions.

3.5. Effect of RGO on Metabolites of Triclosan in Bound Residues

HPLC−¹⁴C-LSC analysis of the soil extracts from BRs showed that triclosan was transformed, with one major metabolite detected alongside the parent compound (Figure 5a). Two radioactive peaks were detected in the control and treatments, corresponding to the parent compound triclosan and M1. These signals of metabolites M1 and triclosan exhibited the characteristic isotopic patterns of trichlorinated compounds, with an intensity ratio of m/z:m/z + 2:m/z + 4 = 3:3:1 (Figure 5b,c).

The parent compound M0 was observed at m/z 286.9425 ([M-H]⁻). Its MS/MS fragmentation produced three primary daughter ions at m/z 250.9672, 142.9905, and 34.9694 (Figure 6a). The measured molecular mass of the compound was 286.9453 Da, which is consistent with the theoretical value for triclosan (C₁₂H₇Cl₃O₂; 286.9439 Da). The metabolite M1 was identified at m/z 330.9343 ([M-H]⁻). High-resolution mass analysis determined its molecular formula to be C₁₃H₇C₃O₄ (theoretical m/z 330.9348, error −1.5 ppm), which corresponds to the addition of a carboxyl group (-COOH) to the parent triclosan molecule (C₁₂H₇Cl₃O₂). Fragmentation of M1 yielded several key product ions. The predominant ion was at m/z 286.9497, resulting from the neutral loss of 43.9904 Da (CO₂), which is a characteristic fragmentation of carboxylic acids, which implies the presence of the -COOH group. This fragment corresponds to the [M-H]⁻ ion of triclosan itself. Other major fragments included ions at m/z 250.9643 ([triclosan-HCl]^–), m/z 153.0174 ([triclosan-C₆H₄Cl₂-HCl]^–), and m/z 34.9682 (Cl^–). Based on the exact mass, isotopic pattern, and characteristic fragmentation—especially the definitive loss of CO₂—M1 was preliminarily identified as carboxy-triclosan. Furthermore, the mass accuracy observed for the predominant ions was consistently high, ranging from −1.5 to +4.9 ppm. This high level of precision confirms the reliability of the MS data, as mass errors within this range (~10 ppm) are crucial for accurate compound assignment and robust metabolite identification in complex biological samples [41]. The presence of such acidic metabolites is consistent with findings for other halogenated organic compounds in soil, such as 4,4′-dichlorodiphenylacetic acid from dichlorodiphenyltrichloroethane [42]. The proposed metabolic pathways of triclosan in soil are summarized in Figure 7a. Carboxylation was identified as the primary transformation process for triclosan in bound residues. This finding is supported by Luks et al. [17], who also detected 4-carboxy-pendimethalin as a major metabolite of pendimethalin in soil BRs [17]. To our knowledge, this study provides the first evidence of carboxylated triclosan in soil, based on high-resolution mass spectrometry; however, full structural confirmation with a synthesized standard is a necessary step for future work to assess its environmental risks.

The parent compound triclosan was the dominant radiolabeled component, comprising 85.8% to 92.4% of the extracted ¹⁴C (0.40–0.70 mg kg^–1. This indicates its substantial persistence and limited metabolic turnover within the BRs. The results indicated that the parent compound persisted within the bound residues. This observation is consistent with previous studies; for example, Luks et al. [17] reported that pendimethalin—a parent compound—accounted for 17.8% of BRs, while acidic metabolites constituted 24.0%. The concentration of M1 (carboxy-triclosan) ranged from 0.057 to 0.083 mg kg^–1 (7.6–14.2%). The presence of this acidic metabolite is mechanistically important, as carboxylated compounds readily form ester linkages with soil organic matter [43]. This chemical propensity directly explains the predominance of ester BRs over ether-linked forms observed in this study and suggests a potential pathway for integration into natural humic structures, possibly reducing short-term bioavailability but contributing to a persistent residue pool.

Additionally, the percentage of the parent compound (triclosan) within the BRs increased with higher RGO concentrations in soil. The proportion accounted for by triclosan rose from 85.8% in the control to 87.9% (RGO-50), 88.5% (RGO-100), and 92.4% (RGO-500), while the concentration of M1 concurrently increased from 0.057 mg kg^–1 to between 0.065 and 0.082 mg kg^–1. These results indicate that RGO inhibited the dissipation of triclosan in BRs, which is consistent with previous reports that RGO reduces triclosan degradation through its strong adsorption and suppression of triclosan-degrading bacteria [16,27]. Further research is needed to clarify the interaction mechanisms between RGO and triclosan metabolites at the molecular level, particularly their influence on key bacterial degraders and soil enzyme activity. Furthermore, the use of RGO or similar nanomaterials in agriculture requires careful evaluation of their impact on pesticide degradation kinetics and residue formation, with particular attention to the long-term bioavailability of both parent compounds and their transformation products. Further research should focus on the interaction between nanomaterials and soil microbiomes to elucidate functional impacts on pesticide degradation and the resulting implications for soil health and food safety.

4. Conclusions

This study revealed that RGO significantly influenced the fate and characteristics of triclosan BRs in paddy soil. The presence of RGO, especially at high concentrations (500 mg kg^–1), significantly enhanced the total amount of triclosan-derived BRs in a dose-dependent manner. RGO also profoundly altered the distribution of triclosan residues within the soil matrix, promoting their association with the most stable humus fraction, humin, and increasing the proportion of covalently BRs relative to sequestered forms. A dose-dependent effect of RGO further promoted the formation of ester-linked residues while simultaneously inhibiting triclosan dissipation and increasing the proportion of the parent compound. Notably, a small amount of carboxylated metabolites was detected for the first time in the BR fraction, indicating limited degradation of triclosan in RGO-amended soils. Both acidic metabolites and parent triclosan are likely major contributors to the formation of ester-bound residues. These results suggest that GNs play a prominent role in governing the long-term fate of organic pollutants such as triclosan in paddy soil. By enhancing residue formation, stabilizing them in recalcitrant pools, and inhibiting their degradation, RGO co-contamination could lead to the protracted persistence of triclosan and its transformation products. Although this may temporarily reduce immediate risks, it raises significant concerns regarding long-term soil health, potential gradual remobilization, and the sustainability of agricultural practices involving nanomaterials. Nanomaterial–pollutant interactions must, therefore, be integrated into environmental risk assessment frameworks, with a particular focus on their combined behavior in agricultural systems. Further research should prioritize the bioavailability, plant uptake, and toxicity of BR fractions to fully evaluate their implications for sustainable agriculture and food safety. The study’s findings underscore the need to develop guidelines that account for the combined effects of nanomaterials and organic pollutants to ensure sustainable agricultural practices and prevent long-term ecological risks.