The Dual Effect of Hematite-Amended Constructed Wetlands: Reducing the Toxicity of SMX Degradation Products and Increasing the Dissemination of Antibiotic Resistance Genes

Highlights

What are the main findings?

• Hematite enhanced the SMX removal while promoting the ARGs dissemination.
• Hematite increases the SMX degradation pathway and reduced intermediate toxicity.

What is the implication of the main finding?

• Hematite promotes the SMX removal by altering the microbial community structure, while simultaneously enhancing cell-cell adhesion, thereby facilitating the ARGs dissemination.
• While hematite may alleviate the SMX toxicity, the long-term ecological risks associated with its presence warrant further attention.

Abstract

Iron ore may enhance the treatment performance of antibiotics within constructed wetlands (CWs), but its effects on the toxicity of degradation products and antibiotic resistance genes require further investigation. This study investigated the sulfamethoxazole (SMX) removal efficiency, SMX degradation pathway, and dissemination of antibiotic resistance genes (sul1 and sul2) linked to SMX in hematite-amended CW microcosms. Hematite, due to its large specific surface area and formation of high redox potential, promoted SMX removal (99.05–99.26%) by adsorption, thus enhancing microbial biodegradation. The addition of hematite increased SMX degradation pathways and simultaneously attenuated the ecotoxicity of intermediate products. However, hematite also stimulated the production of extracellular polymeric substances by microorganisms, enhancing cell–cell adhesion and increasing membrane permeability, ultimately leading to a rise in the abundance of sul1 and sul2. Therefore, although iron ore provides benefits in practical applications, the potential environmental risks it poses deserve serious consideration.

1. Introduction

Sulfonamides (SAs) are among the most commonly utilized antibiotics in medicine, being widely prescribed for the treatment and prevention of various diseases [1]. Among these, sulfamethoxazole (SMX), frequently employed in the treatment of infections, is favored for its superior antibacterial properties [2,3]. Nevertheless, SMX is not fully metabolized and has been extensively detected in aquatic ecosystems [4]. Wastewater treatment plants (WWTPs) cannot completely remove SMX, resulting in the presence of 0.22–2.20 μg/LSMX in effluent [5,6] and even reaching as high as 1.34 mg/L in pharmaceutical effluent [7]. Its prolonged persistence presents a significant risk to aquatic and neighboring ecosystems, potentially facilitating the dissemination of antibiotic resistance genes (ARGs) [8].

Constructed wetlands (CWs) have been widely utilized due to their effective treatment performance, low energy consumption, and low maintenance costs. Previous studies have demonstrated that vertical-flow CWs (VFCWs) generally exhibit superior antibiotic removal performance compared to other CW configurations [9]. Nevertheless, enhancing the average removal efficacy of CWs remains a critical priority due to the considerable variability in the SMX removal efficiency (54.1% [10]–99.6% [11]) of VFCWs. CWs primarily rely on plant uptake, substrate adsorption, and biodegradation for pollutant removal [12]. Previous studies have indicated that plant uptake and substrate adsorption contribute significantly less than biodegradation [10,12]. However, it is important to note that the substrate not only serves as the medium for plant growth, but also provides attachment sites for microorganisms [13]. Therefore, optimizing substrate types is a key strategy for enhancing the pollutant removal efficiency of CWs. Many studies have reported that iron ore can enhance SMX removal by substrate optimization [10,14,15]. Iron ore with a larger surface area could enhance antibiotic adsorption and facilitate microbial attachment [16]. Furthermore, iron ore, characterized by its high redox potential and semi-conductive properties, could enhance SMX degradation processes related to bacterial growth and iron cycling, and mediate direct interspecies electron transfer (DIET) [14,17]. Moreover, the iron ions released from iron ore form an “iron film” (oxidized iron) on the surface of the plant root, enhancing antibiotic adsorption [18]. However, the “iron film” may also attenuate radial oxygen loss, potentially limiting oxygen diffusion from roots [18,19,20], which may be unfavorable for the aerobic degradation of antibiotics. Furthermore, root-surface iron film may reduce the absorption of antibiotics by plants [21,22]. While existing studies have primarily investigated the efficiency and mechanisms of pollutant degradation by iron ore, research on the transformation pathways and associated environmental risks in antibiotic conversion remains insufficient.

Byproducts generated during biodegradation may exhibit greater toxicity than the parent compound [23]. SMX degradation products containing nitro and nitroso groups may demonstrate higher environmental toxicity than the parent substance [24]. Nevertheless, the impacts of iron ore on the toxicity of SMX degradation intermediates in CWs remain unclear. Additionally, previous studies have largely overlooked the potential role of iron ore in ARG dissemination. Iron ore, with its high specific surface area, may provide attachment sites for ARGs [25], which may be subsequently re-released during microbial processes in CWs [26,27], thereby enhancing their mobility and posing potential ecological risks.

This study utilized the richer and more easily obtainable hematite as a representative iron ore. The mechanism of hematite on SMX degradation and ARG dissemination was elucidated by SMX removal performance, material structure analysis, microbial community structure, and ARG abundance. Our findings provide a comprehensive and systematic assessment of pollutant management strategies for iron ore-based CWs.

2. Materials and Methods

2.1. Chemicals and Materials

Hematite particles ranging from 1 to 2 mm in size were sourced from Hebei, China. SMX standards were supplied by Aladdin (Shanghai, China). Chemical reagents (including oxalic acid, sodium citrate, NaCl, and Na₂-EDTA) for SMX extraction and analysis in plants and substrates were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methanol and acetonitrile were purchased from Merck (Darmstadt, Germany).

2.2. Experimental Setup and Operation

In this study, two groups of VFCWs were prepared using polyvinyl chloride material with an inner diameter of 15 cm and a height of 60 cm in a simulated environmental laboratory (maintained at 25 ± 1 °C) at the Qingdao campus of Shandong University. Each group consisted of three microcosms, with an effective volume of 4.0 L per microcosm (Figure 1). The hematite-amended CW (Fe-CW) was filled with a mixture of hematite and quartz sand at a volume ratio of 1:6, while the control group (C-CW) was filled with quartz. A PVC pipe with perforations (55 cm in height and 3 cm in diameter) was positioned at the center of the substrate to facilitate the in situ monitoring of physicochemical parameters. Each continuous-flow microcosm was equipped with a water outlet positioned 10 cm from the top to facilitate water sample collection.

Three Iris pseudacorus samples with comparable growth morphology were implanted into each CW microcosm. Before implantation, the plants were cultured for two weeks in 10% Hoagland nutrient solution to ensure effective growth. The CW microcosms employed a downward vertical flow configuration and operated continuously, maintaining a hydraulic retention time of 2 days. The influent was simulated according to the effluent standards for wastewater treatment plants (Table S1). To assess the impact of hematite on SMX removal more effectively, the influent concentration of SMX was kept at 0.5 mg/L, reflecting the typical concentrations found in domestic and medical wastewater [7,28,29,30,31]. After each microcosm had stabilized for 60 days, water samples were collected and analyzed every six days.

2.3. Characterization of Substrate

To investigate the influence of hematite on the substrate surface, the surface characteristics were analyzed using biological SEM. A Pt-Pd coating was applied to the samples using a Hitachi IM1000 ion sputter coater (Tokyo, Japan). A subsequent morphological analysis was then conducted at an accelerating voltage of 3 kV using a Hitachi SU8010 field emission scanning electron microscope (Text S1) [32].

2.4. Measurement of Extracellular Polymeric Substance (EPS)

Substrate samples (150 g) were collected. The samples were subjected to 10 min of ultrasonication to solubilize the EPS into the aqueous phase. The EPS was extracted from each sample using the hot alkali extraction method [33] and then quantified using a total organic carbon analyzer (TOC-L CPN, Shimadzu, Japan). The detailed operating procedures are provided in Supporting Information Text S2.

2.5. Microbial Analysis

Approximately 10 g of each substrate sample was used for DNA extraction. Genomic DNA was extracted from the substrates using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). To determine the concentration and quality of the extracted DNA, spectrophotometry analysis (NanoDrop ND-1000, Thermo Scientific, Waltham, MA, USA) and 1.0% agarose gel electrophoresis was used. The V3-V4 region of the 16S rRNA gene was amplified using the primer 338 F (5′-ACTCCTACGGGAGGCAGA-3′), and then analyzed on the Illumina MiSeq platform (Majorbio, Shanghai, China).

2.6. Determination of Antibiotics

Water samples were directly extracted using solid-phase extraction. For solid samples (substrate and plant), ultrasonic extraction was performed with a mixture of acetonitrile and citrate buffer (1:1), followed by solid-phase extraction with a SAX-HLB column. The full procedures for antibiotic extraction can be found in Text S3.

The HPLC system, equipped with a C18 column (Acclaim™; 4.6 mm × 250 mm, 5 μm), was used to quantify the SMX content in the sample. SMX degradation products were identified by high-resolution mass spectrometry (Agilent 6520, Agilent Technologies, Santa Clara, CA, USA), with the specific HPLC and mass spectrometry conditions outlined in Text S3.

2.7. ARG Quantification

High-throughput qPCR analysis was performed using the Wafergen SmartChip real-time PCR system (Wafergen, Fremont, CA, USA), which allows for large-scale analysis by processing up to 5184 micro-well reactions in a single run. In this study, ARGs associated with SMX (sul1 and sul2), intl-1, and 16S rRNA were analyzed. All samples were sent to China Yuanzai Biotechnology Co., Ltd. (Wuhu, China) for testing. A threshold cycle (CT) of 31 was used as the detection limit, and only ARGs with amplification in all replicates were regarded as positive.

$$\mathrm{Relative} \mathrm{copy} \mathrm{number}={10}^{(31-\mathrm{Ct})/(10/3)}$$

(1)

2.8. Statistical Analysis

Differences in SMX removal, α and β diversity, and microbial community abundance among the groups were analyzed using one-way analysis of variance (ANOVA). Principal coordinate analysis (PCoA), based on Bray–Curtis distances, was conducted to assess microbial community composition, utilizing the R package (R4.5.1) “vegan”. Principal component analysis (PCA) was used to explore the relationship between hematite, microbial communities, and ARGs. All statistical analyses were performed in R (v4.3.3), with graphical visualization created using Origin 2021.

3. Results and Discussion

3.1. Hematite-Amended CWs Improved SMX Removal

By comparing two groups of CWs with different substrates, we found that the addition of hematite significantly improved SMX removal performance (p < 0.05). The effluent SMX concentration of C-CW was 14.03–14.89 μg/L, whereas the effluent SMX concentration of Fe-CW reduced by approximately two-thirds compared to that of C-CW (p < 0.05) with an effluent concentration of 3.68–4.99 μg/L (Figure 2a), which aligned with findings from previous studies [10,34]. Mass balance analysis revealed that “Others” (mainly microbial degradation [35,36]) represented the primary removal pathway for SMX in both CW microcosms, with its relative contribution significantly enhanced by 6.29% in Fe-CW (Figure 2b). To further elucidate these phenomena, the effect of hematite on the microbial community was investigated.

The microbial communities were distinctly categorized into two groups: one with iron oxide addition and the other without (Figure 3a). The microbial richness and diversity in Fe-CW were significantly higher than those in C-CW (Figure 3b,c, p < 0.05). Furthermore, PCoA clearly revealed distinct differences in microbial community structure between C-CW and Fe-CW (Figure 3d). PCoA indicated that SMX-degrading bacteria seemed to exhibit a stronger association with Fe-CW, suggesting that hematite addition significantly altered the microbial community structure within the CW microcosms (Figure 3e).

Changes in SMX-degrading bacterial communities were observed in both CWs (Figure 4). No significant alterations were observed in the dominant genera (Delftia, Microbacterium, and Thauera) at the genus level. Notably, the relative abundance of Microbacterium declined from 73.84% in C-CW to 61.22% in Fe-CW, potentially due to its potential reliance on SMX as the sole carbon and energy source [37,38]. With the exception of Acinetobacter, Arthrobacter, Microbacterium, and Rhodococcus, the relative abundances of other bacterial genera in Fe-CW increased by 0.16–6.53% compared to those in C-CW. Previous research has demonstrated that Brevundimonas may utilize Fe(III) as an electron acceptor in anaerobic respiration [39], while Thauera may utilize Fe as an electron donor for iron cycling [40]. Additionally, Paenarthrobacter exhibit enhanced siderophore production and population growth under heavy metal stress conditions [41]. This may be attributed to the fact that low concentrations of hematite may enhance the proliferation of some microbial communities [42], where iron–mineral–microbe interactions facilitate microbial energy acquisition and microbial abundance [38].

3.2. Potential Degradation Pathways of SMX and Toxicity Assessment of Intermediate Byproducts

To elucidate the degradation intermediates and pathways of SMX, UPLC-qTOF/MS was employed to identify 16 intermediate compounds, thereby proposing five potential major degradation pathways of SMX (Figure 5a). It is noteworthy that the S-N hydrolysis process and partial mechanisms of electrophilic substitution were exclusively observed in Fe-CW. SMX undergoes hydrolysis of the S-N bond in the sulfonyl group, leading to the formation of TP174 as the primary biodegradation product (Figure 5a), followed by the subsequent generation of TP158 by further biodegradation processes [43]. Previous studies have proposed that iron may activate specific or non-specific enzymes responsible for hydrolyzing S-N bonds in sulfonamide groups [34]. Additionally, iron may induce the generation of hydroxyl radicals, which can attack the isoxazole ring, resulting in the formation of TP158 [44]. As for the electrophilic response, TP270 was found in both groups of artificial wetlands. The formation of TP270 was attributed to the electrophilic substitution of a hydrogen atom on the SMX benzene ring, a process also observed in water/sediment studies on the biodegradation of SMX transformation products [45]. However, TP288 and TP274 were only found in Fe-CW. Additionally, TP288 can form either through TP270 hydroxylation [34] or via the electrophilic attack and substitution of the C=C bond in the isoxazole ring of SMX [46]. Subsequently, the aromatic ring of TP288 undergoes hydroxylation to form TP304, which may be attributed to the interaction of ·OH radicals with aniline [46]. The aniline radical hydrolyzes to yield hydroxylated radical intermediates, which then react with O₂ to produce hydroxylated aniline derivatives [47]. The formation of TP274 could be attributed to the reductive cleavage of the N-O bond on the isoxazole ring of TP288. Additionally, the formation of TP274 could be attributed to the electrochemical reduction and addition reaction of the N-O bond of the isoxazole ring of TP288.

In addition, both CWs can degrade SMX through the ammonia oxidation process (Pathway 1), hydroxylation (Pathway 2), acetylation (Pathway 3), and partial mechanisms of electrophilic substitution (Pathway 4).

TP239 [48] and TP284 [49] were widely recognized as transformation products associated with the co-metabolic oxidation of ammonia-oxidizing bacteria (AOB) and SMX [34]. These compounds were detected in all CW microcosms, which was consistent with previous findings [34]. TP239 is generated from SMX through deamination, which is potentially related to deaminase enzymes encoded in the genomes of the ammonia-oxidizing archaea Nitrososphaera gargensis, AOB Nitrosomonas nitrosa Nm90, and comammox Nitrospira inopinata [48]. Similarly, TP284 has been shown to form through a pathway analogous to that of TP239, involving the co-metabolic oxidation of SMX by AOB, followed by the aminohydrolase-mediated hydrolysis of TP284 to TP217. Additionally, TP290 was shown to form through the hydroxylation of both the -NH₂ group and the isoxazole ring of SMX [50]. Previous studies have demonstrated that the hydroxylation of the isoxazole ring in SMX is primarily catalyzed by CYP450 enzymes [51,52]. TP318 and TP121 were identified as further transformation products of TP290, potentially related to pathways associated with “Xenobiotics biodegradation and metabolism” [34]. Both TP296 and TP300 may originate from SMX acetylation mediated by key enzymes such as POR, FDH, and FTHFS [34]. Moreover, in this study, both CWs were able to degrade SMX through partial mechanisms of electrophilic substitution. TP256 and TP270 were generated through the electrophilic reactions of SMX. TP256 was formed via the electrophilic substitution of the -CH₃ group on the SMX isoxazole ring, followed by ring opening to yield TP225 [43]. These intermediates can ultimately be mineralized to methane, carbon dioxide, and water through microbial metabolism.

Given that distinct metabolites and transformation pathways confer varying ecological risks, we systematically assessed the toxicity of SMX and its degradation products by T.E.S.T, including bioaccumulation potential, developmental toxicity, and mutagenicity (Figure 5b). Compared to SMX, the majority of the intermediates exhibited lower toxicity. Notably, TP239 showed slightly higher bioaccumulation potential than the parent compound. In terms of developmental toxicity, the toxicity of TP256, TP274, and TP318 significantly decreased following CW treatment. Interestingly, most of these toxic intermediates were predominantly detected in C-CW (with the exception of TP274), suggesting that hematite may effectively mitigate the ecological risks associated with SMX removal.

3.3. ARG Dissemination

As illustrated in Figure 6a, the abundance of sul1 (3.38 × 10⁶ and 1.33 × 10⁷) was an order of magnitude higher than that of sul2 (9.75 × 10⁵ and 2.03 × 10⁶) in both CWs. Since Sul1 is easily disseminated, it is frequently utilized as an indicator gene to monitor variations in ARGs within environments [53,54]. Sul1 is typically encoded on the intl-1 gene [55], which plays a pivotal role in shaping the composition and abundance of ARG profiles within the substrate [56], indicating that intl-1 may facilitate the propagation of sul1 through specific mechanisms [56,57,58]. The PCA results further indicate a significant correlation between sul1 and intl-1 (Figure 6b). It was noteworthy that the relative abundances of sul1, sul2, and intl-1 were all increased in Fe-CW compared to C-CW. This may be attributed to the fact that the production of EPS increases the hydrophobicity of the cell surface, which reduces intercellular rejection and increases membrane permeability [42]. SEM analysis (Figure 6c) revealed that the surface of the Fe-CW substrate exhibited a greater accumulation of flocculent substances compared to the C-CW group, with the former being extensively covered by biofilms. Furthermore, quantitative measurements of EPS content confirmed that the EPS levels in the Fe-CW were significantly higher than those in the C-CW (Figure 6d). The elevated production of EPS may enhance cell adhesion, which improves cell–cell communication and facilitates conjugative transfer [42,59]. Additionally, hematite and its derived iron (hydr)oxides possess the ability to adsorb ARGs [60]. However, ARGs may be re-released during microbial-mediated iron reduction, enhancing their mobility [27], thereby ultimately promoting the horizontal gene transfer (HGT) of ARGs.

4. Conclusions

Hematite may enhance SMX removal through surface complexation, hydrogen bonding, and enhancing biodegradation. Furthermore, the presence of iron facilitates an enhanced degradation pathway for SMX, simultaneously reducing the toxicity of the degradation intermediates. However, iron ore also facilitates the HGT dissemination of ARGs by stimulating EPS secretion, enhancing cell surface hydrophobicity, reducing intercellular rejection, and improving membrane permeability. Therefore, while iron ore enhances the effectiveness of water pollution control in practical applications, the potential environmental risks associated with its use must not be overlooked. Additionally, the SMX influent concentration in this study was rather high; thus, the ways in which MP influences SMX removal under low concentrations need further study.