# Temporal dynamic of soil microbial communities and antibiotic resistance markers exposed to increasing concentrations of sulfamethoxazole

Oriane Della-Negra, Marilia Camotti Bastos, Maria Vittoria Barbieri, Valérie Bru-Adan, Gaëlle Santa-Catalina, Nassim Ait-Mouheb, Serge Chiron, Dominique Patureau

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Oriane Della-Negra, Marilia Camotti Bastos, Maria Vittoria Barbieri, Valérie Bru-Adan, Gaëlle Santa-Catalina, et al.. Temporal dynamic of soil microbial communities and antibiotic resistance markers exposed to increasing concentrations of sulfamethoxazole. Environmental Pollution, 2025, 364 (2), pp.125306. 10.1016/j.envpol.2024.125306. hal-04781481

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Temporal dynamic of soil microbial communities and antibiotic resistance markers
exposed to increasing concentrations of sulfamethoxazole

Oriane Della-Negra, Marilia Camotti Bastos, Maria Vittoria Barbieri, Valérie Bru-Adan, Gaëlle Santa-Catalina, Nassim Ait-Mouheb, Serge Chiron, Dominique Patureau

<figure><img src="image_2.png" /></figure>

PII: S0269-7491(24)02023-2

DOI: https://doi.org/10.1016/j.envpol.2024.125306

Reference: ENPO 125306

To appear in: *Environmental Pollution*

Received Date: 15 July 2024

Revised Date: 18 October 2024

Accepted Date: 10 November 2024

Please cite this article as: Della-Negra, O., Bastos, M.C., Barbieri, M.V., Bru-Adan, V., Santa-Catalina, G., Ait-Mouheb, N., Chiron, S., Patureau, D., Temporal dynamic of soil microbial communities and antibiotic resistance markers exposed to increasing concentrations of sulfamethoxazole, *Environmental Pollution*, https://doi.org/10.1016/j.envpol.2024.125306.

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© 2024 Published by Elsevier Ltd.
Temporal dynamic of soil microbial communities and antibiotic
resistance markers exposed to increasing concentrations of
sulfamethoxazole

4

5 Oriane Della-Negra<sup>a,b</sup>, Marilia Camotti Bastos<sup>a,b</sup>, Maria Vittoria Barbieri<sup>b</sup>, Valérie Bru-Adan<sup>a</sup>, Gaëlle

6 Santa-Catalina<sup>a</sup>, Nassim Ait-Mouheb<sup>c</sup>, Serge Chiron<sup>b</sup>, Dominique Patureau<sup>a*</sup>

7

8 <sup>a</sup> INRAE, University of Montpellier, LBE, Av. des Étangs, 11100 Narbonne, France

9 <sup>b</sup> UMR HydroSciences Montpellier, University of Montpellier, IRD, CNRS, 15 Av. Charles Flahault
10 34093 Montpellier cedex 5, France

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11 <sup>c</sup> INRAE, University of Montpellier, UMR GEAU, 361 rue Jean-François Breton, 34196 Montpellier,
12 France

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13 *Corresponding author, email address: dominique.patureau@inrae.fr

14

15 Abstract

16 The reuse of treated wastewater (TWW) for irrigation is widely applied to alleviate pressure on
17 freshwater resources. However, WW contains antibiotics that once in soils, can exert selective
18 pressure, promoting the emergence and spread of antimicrobial resistance (AMR) in the environment.
19 Current environmental risk assessments for antibiotic residues rely on indicators such as Predicted No
20 Effect Concentrations (PNECs), usually determined in liquid media. These PNECs aim to predict
21 antibiotic concentrations that may promote resistance in the environment. Given the complexity of soil
22 matrices, few studies have established PNEC values for soil, which likely differ significantly from
23 aquatic environments.

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To address this gap, we developed a simplified experimental model using soil microcosms irrigated with TWW and the antibiotic sulfamethoxazole (SMX) to estimate threshold concentrations favouring resistance transfer or/and emergence within the soil microbiome. We identified SMX concentrations between 0.01 and 0.1 mg/kg<sub>dry soil</sub> that likely increased the abundance of sulfonamide resistance genes in soil. A time window of 1 to 7 days post-exposure showed a temporary rise in *sull* and *intl* gene abundance (over 1 log/soil 16S rDNA), the appearance of SMX transformation products, and an increase in some Rhodocyclaceae. After 1.5 months of incubation and complete SMX transformation, the relative abundance of *sull* and *intl* remained about 0.5 log higher than in SMX-free controls and soils with
SMX levels below 0.1 mg/kg dry soil. A persistent transformation product, 4-N-glucuronide-SMX, was also observed.

Here, the estimated PNEC for SMX in soil, between 0.01 and 0.1 mg/kg, exceeds typical SMX concentrations found in soils exposed to TWW. This may suggest low impact on resistance selection for this compound in the context of TWW exposure. However further studies on other soils, water, and antibiotics need to be conducted to expand our knowledge on soil PNECs.

**Keywords:**

Sulfamethoxazole, transformation products, reclaimed wastewater, antimicrobial resistance, PNEC

## 1.Introduction

Reuse of treated wastewater (TWW) for irrigation is already applied in many countries as a strategy to address water scarcity (Ungureanu et al., 2020). In this way, farmers have access to a perennial source of water and can meet the growing demands associated with population growth and manage the intensifying droughts caused by global warming (Leiva et al., 2021). However, TWW coming from urban effluents is known to contain micropollutants, including chemical pollutants (salts, trace metals, detergents, microplastics, pharmaceuticals, hydrocarbons, hormones, nanoparticles, etc.) and biological contaminants (pathogens, antibiotic resistant bacteria and antibiotic resistance genes, viruses, etc., Rizzo et al., 2013). Once released into agro-ecosystems, TWW can alter soil composition and impact soil endogenous microbial community structure linked to high input of carbon, nutrients, ions and minerals and low input of chemical and biological contaminants. It may also promote the spread of antimicrobial resistance (AMR) via the settlement of TWW exogenous bacteria carrying antibiotic resistance genes (ARGs), facilitating horizontal ARG transfer to soil endogenous bacteria or exerting selective pressure through the input of residual antibiotics (Grenni et al., 2024; Gullberg et al., 2011; Martin-Laurent et al., 2019).

Most of the processes used for wastewater treatments are not sufficient to completely remove antibiotic residues (Leiva et al., 2021). Even very low concentrations of antibiotics (below the minimum inhibitory concentration) can trigger genetic changes in bacterial community, enhance ARG transfer, and select resistant strains, impacting microbial functions such as carbon mineralization and nitrogen cycling, leading to temporary or perennial shifts in soil microbial structure (Leiva et al., 2021; Rizzo et al., 2013). And in the context of agricultural crops, the risk is of particular concern as antibiotics, ARGs and
antibiotic resistant bacteria (ARB) can be transferred from soil to plants, thus directly entering the food chain (Xiao et al., 2023).

Currently, European regulations on TWW reuse only set thresholds for BOD (biochemical oxygen demand), SS (suspended solids), turbidity and the presence of the fecal indicator *E. coli* (European Parliament, Council of the European Union, 2020). However, no limit is set on antibiotic content, which can be found at concentrations close to µg/L in TWW, depending on their nature (Rodriguez-Mozaz et al., 2020). In the context of environmental risk assessment for antimicrobials, various methodologies and indicators have been developed and continue to evolve to evaluate the selective and persistent potential of antibiotics and their environmental impact (Joint Research Centre et al., 2015; Le Page et al., 2017; Murray et al., 2021). This includes defining concentration thresholds such as Predicted No-Effect Concentrations (PNECs) for resistance or persistence selection (Bengtsson-Palme and Larsson, 2016; Murray et al., 2021). While most of these indicators have been developed for single species in liquid media or complex communities in aquatic environments (Bengtsson-Palme and Larsson, 2016; Murray et al., 2021; Stanton et al., 2020), few if any PNECs have been determined for antibiotics in soil. Typically, soil PNECs are estimated based on water PNECs and adjusted using factors like Koc (organic carbon to water partition coefficient) and foc (fraction of organic carbon) (Castaño-Trias et al., 2024).

Soils, with their rich microbial diversity, present a complex environment where microbial interactions (competition, protection) can significantly influence PNEC values, as observed in aquatic environments (Murray et al., 2018; Stanton et al., 2020). Antibiotics in soil can adsorb to particles, limiting their bioavailability and thereby reducing antimicrobial efficiency (Menz et al., 2018); and can also be degraded generating transformation products with varying antimicrobial properties (Löffler et al., 2023). To address knowledge gaps regarding antibiotic concentrations that promote resistance selection in soils, we propose in this study an experimental soil microcosm approach, focusing on the model antibiotic sulfamethoxazole (SMX).

SMX is of particular interest, due to its global use, its relative persistence in soils, degradability (Chen and Xie, 2018; Martin-Laurent et al., 2019; P. J. M. Reis et al., 2018; Wang and Wang, 2018), frequent detection in TWW (with a concentration that could reach hundreds of ng/L, Cacace et al., 2019), and presence in European soils at concentrations below 0.1 mg/kg dry soil due to TWW irrigation (Chen et al., 2014; Cycoń et al., 2019). SMX inhibits bacterial dihydrofolic acid synthesis and several bacteria exhibit resistance via the acquisition of plasmid-borne *sul* genes (*sul123*) coding for the enzyme dehydropteroate synthase involved in the bacterial folic acid pathway (Chen et al., 2022a; Collado et al., 2013; Hu et al., 2022). For this molecule, PNEC was assessed at 0.016 mg/L in liquid medium, for single species (Bengtsson-Palme and Larsson, 2016), and estimated at 0.02 mg/kg in soils (Bourdat-Deschamps et al., 2017).
Here, the described model study aimed to experimentally determine a soil PNEC for SMX, in order to compare it with estimated values, while monitoring the temporal dynamic of SMX and its transformation products, soil microbial community, ARGs and some N-cycling related genes. The purpose here was to identify the key factors (SMX concentration, endogenous bacteria, transformation products, specific time windows) influencing sulfonamide resistance selection and dissemination in soils exposed to TWW.

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## 2. Material and Methods

### 2.1. Sampling

#### 2.1.1. Treated wastewater sampling

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Treated wastewater (TWW) came from a wastewater treatment plant located at Murviel-les Montpellier (France). The wastewater treatment plant included the usual pre-treatment (screening and grit removal) followed by a biological treatment with constructed wetland (CW: forced aeration filters with reeds), supplemented by phosphorus treatment with ferric chloride and extensive treatment in a lagoon (Moulia et al., 2023). This wastewater treatment plant has a nominal capacity of 3000 Inhabitant Equivalent (Ait-Mouheb et al., 2022). The experimental site is used to irrigate a 0.5 ha plot in Murviel-lès-Montpellier using a drip system since 2017, where vineyards, alfalfa and fruit trees are grown. The quality of the TWW meets the criteria for class C reclaimed water (European Parliament, Council of the European Union, 2020).

The samples were collected at the outlet of the TWW plant with hermetically protected bottles.

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#### 2.1.2. Soil samples

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Soils were sampled at the experimental platform of Murviel-lès-Montpellier, on an agricultural plot that was irrigated with TWW (treated by constructed wetlands) for two years (Figure S1). Irrigation was activated six times per day (30 minutes on and 90 minutes off) from 10 am to 8 pm to avoid long periods without irrigation during the high summer temperatures. Globally, the plot received 6000 L/m² the first year and 6500 L/m² the second year. These are the volumes needed for olive and apricot trees during one irrigation season and for vines during two irrigation seasons. This soil and the crops grown on it are representative of the Mediterranean climate. In recent years, the region has suffered increasingly from drought and is turning to the use of reclaimed wastewater for irrigation (Ait-Mouheb et al., 2018). This matrix is therefore a model of Mediterranean agricultural soil regularly irrigated with TWW.

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Three soil samples were taken on the field site, each separated by a few metres. In these three spots, the surface layer was removed (first 10 cm), then holes of 50x50 cm and 30 cm deep were dug and sampled with a shovel. The samples were then homogenized, dried for 48 hours at 35°C and sieved < 2 mm. Soil samples were stored at 4°C before use.

## 2.2. Experimental set-up

The experiments were conducted in microcosms. 25 g of dry soil were added to 250 ml amber glass flasks topped with carded cotton plugs to allow basal respiration. A part of the flasks was autoclaved (20 mins at 121°C), incubated for 24 hours at room temperature, then autoclaved again to reduce bacterial richness and diversity. All the flasks were then acclimated at room temperature for 3 days to activate the native soil microorganisms. They were then irrigated in one shot with 20 mL of either TWW or milliQ water spiked with sulfamethoxazole (SMX, Fluka, 97%) to obtain final SMX concentrations of 0, 0.01, 0.1, 0.5 or 1 mg/kg dry soil. Flasks were incubated at 35 °C for 42 days in the dark to prevent sulfamethoxazole photodegradation. Soil moisture content was adjusted and maintained with milliQ water, at 80% of the field capacity during the whole experiment. Each condition was tested in triplicate, sacrificial mode was adopted and included 7 sampling times (0.4, 1, 3, 7, 14, 28, and 42 days after the one-shot irrigation, Figure 1).

<figure><img src="image_3.png" /></figure>

Soil treatment

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Irrigation type

TWW

<figure><img src="image_6.png" /></figure>

TWW

<figure><img src="image_7.png" /></figure>

milliQ water

<figure><img src="image_8.png" /></figure>

SMX concentration (mg/kg dry soil)

<figure><img src="image_9.png" /></figure>

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Sampling time (days)

0.4, 1, 3, 7, 14, 28, 42

0.4, 1, 3, 7, 14, 28, 42

0.4, 42

<caption>Figure 1. Schematic representation of the experimental set-up (TWW = treated wastewater)</caption>

## 2.3. Molecular biology analyses

### 2.3.1. DNA extraction and analyses
DNA extraction was performed using the DNeasy® PowerWater® kit (Qiagen) for waters and the FastDNA™ SPIN Kit for Soil (MP Biomedical) for soil samples. The kits were used according to the protocol provided by the manufacturer. The quality of the extracted DNA (260/280 ratio), was measured using the Spark® multimode microplate reader. This extraction method was already described and used previously (Della-Negra et al., 2024; Moulia et al., 2023).

### 2.3.2. Quantitative PCR (qPCR)

Amplification reactions were performed in reaction mixtures (12 µL) containing 6 µL of Sybr Super Mix (Biorad) and primers at concentrations indicated in Table 1. Total bacterial quantification was performed by qPCR targeting the V3 region from 16S rDNA. Genes related to antibiotic resistance (sulfonamide resistance: *sul1*, mobile genetic element: *intl*), sulfonamide degradation (sulfonamide mono-oxygenase: *sadA*, arylamine N-acetyltransferase: *nat*) and nitrogen cycle (nitrite reductase: *nirS*, nitrate reductase, *narG*, ammonia mono-oxygenase: *amoA* and nitrogenase *nifH*) were quantified. For 16S rDNA, *sul1*, *intl*, *sadA*, *nat*, *narG*, *amoA-B* and *nifH*, the qPCR programs consisted in initial denaturing at 95°C (2 mins), followed by 40 cycles at 95° (15 s or 10s for 16S rDNA) for denaturation and Ta (Table 1) (60 s or 20s for 16S rDNA, main manuscript) for annealing and amplification. For *nirsS*, the 40 cycles started at 95° (15 s), then Ta (Table 1) (30 s) and 72 °C for 30s. The fluorescence data was acquired at the annealing temperature or at 72°C for *nirS*, and the final melting curve was constructed with temperature ramping up from 72°C to 95°C.

Primers targeting *nat* gene were designed on the basis of sequence alignment of the *nat* gene found in nine species of *Pseudomonas* and *Burkholderia* (details and accession numbers available in Table S1). Their specificity was then checked using Primer-BLAST (Ye et al., 2012).

<table><caption>Table 1. Information about qPCR primers and conditions used in quantification of target genes.</caption><thead><tr><th>Gene</th><th>Primers</th><th>Sequence (5'-3')</th><th>Temperature of annealing</th><th>Concentration of primers</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">16S rRNA</td><td>F</td><td>ACGGTCCAGACTCCTACGGG</td><td rowspan="2">61 °C</td><td>200 nM</td><td rowspan="2">(Wéry et al., 2008)</td></tr><tr><td>R</td><td>TTACCGCGGCTGCTGGCAC</td><td>200 nM</td></tr><tr><td rowspan="2">Sul1</td><td>F</td><td>CGCACCGGAAACATCGCTGCAC</td><td rowspan="2">60 °C</td><td>250 nM</td><td rowspan="2">(Cerqueira et al., 2019)</td></tr><tr><td>R</td><td>TGAAGTTCCGCCGCAAGGCTCG</td><td>250 nM</td></tr><tr><td rowspan="2">Intl</td><td>F</td><td>GATCGGTCGAATGCGTGT</td><td rowspan="2">60 °C</td><td>200 nM</td><td rowspan="2">(Cerqueira et al., 2019)</td></tr><tr><td>R</td><td>GCCTTGATGTTACCCGAGAG</td><td>200 nM</td></tr><tr><td rowspan="2">sadA</td><td>F</td><td>GAACGCGATTGACACTGCAC</td><td rowspan="2">63 °C</td><td>900 nM</td><td rowspan="2">(A. C. Reis et al., 2018)</td></tr><tr><td>R</td><td>GATGGACTCTCGACATAGCAC</td><td>900 nM</td></tr><tr><td rowspan="2">nat</td><td>F</td><td>GTGCTCGAAGCAGTAACCGC</td><td rowspan="2">63.5 °C</td><td>250 nM</td><td rowspan="2">This study</td></tr><tr><td>R</td><td>TTCGAGAACCTCGACGTG</td><td>250 nM</td></tr><tr><td rowspan="2">nirS</td><td>F</td><td>GA(C/G)TTC GG(A/G)TG(C/G)GTC TTGA</td><td rowspan="2">58 °C</td><td>250 nM</td><td rowspan="2">(Throbäck et al., 2004)</td></tr><tr><td>R</td><td>GT(C/G)AACGT(C/G)AAGGA(A/G) AC(C/G)GG</td><td>250 nM</td></tr><tr><td>narG</td><td>F</td><td>TCGCCSATYCCGGCSATGTC</td><td>60 °C</td><td>200 nM</td><td></td></tr></tbody></table>
<table><tr><td></td><td>R</td><td>GAGTTGTACCAGTCRGCSGAYTCSG</td><td>200 nM</td><td>(Kandeler et al., 2006)</td></tr><tr><td>amoA-B</td><td>F</td><td>GGGGTTTCTACTGGTGGT</td><td>150 nM</td><td>(Rotthauwe et al., 1997)</td></tr><tr><td></td><td>R</td><td>CCCCTCGGGAAAGCCTTCTTTC</td><td>150 nM</td><td>(Poly et al., 2001)</td></tr><tr><td>nifH</td><td>F</td><td>TGCGAYCCSAARGCBGACTC</td><td>500 nM</td><td>(Poly et al., 2001)</td></tr><tr><td></td><td>R</td><td>AGSGCCATCATYTCRCCKGA</td><td>500 nM</td><td>(Poly et al., 2001)</td></tr></table>

### 2.3.3. Illumina-Based 16S rDNA sequencing and sequence data analysis

Sequencing analyses of the DNA extracted from the soil and water were carried out within the Genotoul Lifescience Network Genome and Transcriptome Core Facility platform in Toulouse. The protocol is described in SI (section 1.2).

## 2.4. Chemical analyses

### 2.4.1. Chemicals

Acetonitrile (> 99.9%) and methanol (> 99.9%) were purchased from Fischer Scientific. EDTA-Mcllvaine solution was prepared from 1.5 g of disodium hydrogen phosphate dihydrate salt (> 99%, Merck), 1.3 g of citric acid monohydrate (> 99%, Merck) and 0.372 g of EDTA (> 99%, Merck), dissolved in 100 mL of milliQ water.

Sulfamethoxazole (97%) was purchased from Fluka, d₄-sulfamethoxazole (98%) from AlsaChim, nitro-sulfamethoxazole, acetyl-sulfamethoxazole and sulfanilamide were purchased from Merck. sulfamethoxazole-β-D-glucuronide was obtained from Toronto Research Chemicals.

### 2.4.2. General analyses

Analyses of pH, conductivity, nitrite, nitrate, ammonium, sulfate, and phosphate were performed through the sequential analyzer (Gallery Plus Thermo Fischer, https://doi.org/10.15454/1.557234103446854E12). Liquid samples were directly injected after filtration on 0.45 µm filters. Solid samples were freeze-dried and 3 g of dry soils were then diluted in 9 mL of milliQ water, vortexed for 5 minutes, centrifuged for 5 minutes at 4000 rpm, filtered on 0.45 µm filters and injected.

### 2.4.3. Soil extraction

Soil samples were freeze-dried and extracted using the QuEChem extraction method described elsewhere (Manasfi et al., 2021). The complete protocol is described in SI (section 1.3).
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## 2.4.4. Sulfamethoxazole quantification and identification of transformation products

Sulfamethoxazole extraction and detection and quantification methods are described in SI (section 1.4).

For TPs identification, a literature review of the most common and already identified SMX TPs was constituted and used as a list for their subsequent screening in soil samples (P. J. M. Reis et al., 2018; Wang and Wang, 2018). Based on this list, their molecular ions [M + H]⁺ were extracted from the (+)ESI-HRMS chromatograms and [M - H]⁻ from the (-)ESI-HRMS chromatograms with an isolation width of 5 ppm. If available, structures of some TPs were compared and confirmed with their reference standards (Table S2).

For the identification of 4-N glucuronide SMX TP, full scan data dependent MS/MS fragmentation (dd-MS2) was set to acquire TP fragment spectra. Collision energies of 10, 20, 30 and 40 eV were tested.

## 2.5. Statistical analyses

Statistical analysis and graphical representations were performed using R software (R Core Team, 2022). Community richness (Observed index, representing the number of species in a sample) and diversity (Shannon index) and PCoA (Principal Coordinate Analysis) analyses were assessed and performed by ggplot2 (Wickham, 2009) and phyloseq packages (McMurdie and Holmes, 2013). Linear discriminant analysis effect size (LEfSe) were performed using microbiomeMarker package (Cao et al., 2022) to identify biomarkers between SMX concentrations, with an absolute linear discriminant analysis (LDA) score > 3.0 as cut-off value. Significant differences were assessed performing ANOVA followed by post hoc tests.

## 3. Results and Discussion

The experimental setup presented here was designed to simulate the introduction of residual antibiotic concentrations into soils via TWW. The following results aim to describe and understand the behavior of the model antibiotic sulfamethoxazole once introduced into the soil, to assess its impact on the soil microbiome and specific resistance genes. The goal was to identify the concentration levels at which sulfamethoxazole poses a risk for promoting AMR in the soil.
239 3.1.Sulfamethoxazole degradation in soil samples

240 3.1.1.Effect of SMX concentration on degradation rate

241 SMX in TWW was quantified at 9.7 ± 2.7 µg/L, which is included in the common range of SMX concentration found in TWW and aquatic environment (from ng/L to a dozen of µg/L). In historical soil samples, it was quantified at 6.9 ± 4.7 µg/kg dry soil, which is included in the common range of SMX concentration found in soils irrigated with TWW (0-55 µg/kg dry soil, Chen et al., 2014).

246 LC-HRMS analysis and the use of d₄-SMX as internal standard enabled the monitoring and quantification of SMX in soil samples. In both reuse_soil and low_div_soil irrigated with TWW a decrease of SMX concentration was observed across time (Figure 2a-b). After 42 incubation days, SMX was not detected anymore in reuse_soil whereas it was still quantified around 0.01-0.03 µg/kg dry soil in low_div_soil for the three highest SMX concentrations. In low_div_soil irrigated with milliQ water, no SMX degradation was observed as the same concentrations were recovered after 42 days of incubation. It could be explained by the absence of SMX (photo)degradation (microcosms incubated in the dark, absence of co-substrates) and the reduction of bacterial activity by autoclaved treatment.

254 To assess the SMX degradation rate in the microcosms, a 1st order kinetic was assumed:

255 (1) v = d[SMX] / dt = -k × [SMX]

256 (2) ln[SMX] = ln[SMX]₀ - k × t

257 with [SMX] corresponding to SMX concentration, [SMX]₀, the SMX concentration after spiking at T0, t the incubation time in days and k the kinetic constant expressed in day⁻¹. After plotting ln[SMX] against time, linear regressions yielded Pearson coefficients R² between 0.85 and 0.96 for reuse_soil and low_div_soil spiked at 0.5 and 1 mg/kg dry soil (Table S3), thus validating the 1st order kinetic hypothesis. 258 259 260 Assays carried out on the biodegradation of sulfonamides in soils showed that this class of antibiotic 261 262 followed a pseudo 1st order kinetic, thus corroborating the results here (Li et al., 2020).

263 To compare with literature, kinetic constants k were transformed into DT₅₀ using the following formulae 264 265

265 (3) DT₅₀ = ln(2) / k

266 Along with the kinetic constants, DT₅₀ depended on initial SMX concentrations (Figure 2c). For SMX 267 concentrations of 0.5 and 1 mg/kg dry soil, DT₅₀ of 1.4 and 1.7 days were obtained respectively in 268 reuse_soil. Similar values around 1.3-3 days were found in literature under aerobic condition (Chen and 269 Xie, 2018). The DT₅₀ increased by a factor 10 in low_div_soil, just as in another study comparing
historical and sterile soils (from 2.2 to 32.2 days at 2.5 mg/kg dry soil, Li et al., 2020) confirming the ability of autoclaved soil irrigated with TWW to degrade SMX. Generally, the two main mechanisms explaining SMX degradation in sterile soils are photodegradation or hydrolysis (Li et al., 2020). Here microcosms were incubated in the dark, thus limiting photodegradation. For lower SMX concentrations (0.01 and 0.1 mg/kg dry soil), DT₅₀ were found to be higher: at 3.2 et 8.8 days respectively in reuse_soil. For this concentration range in soils, no comparable data was available in literature. Between 0.01 and 0.1 mg/kg dry soil the increase of DT₅₀ could be attributed to the alteration of degrading community structure or function, but it could also be associated with an overestimation of the DT₅₀ for SMX concentration of 0.01 mg/kg dry soil due to its proximity with the LOQ. However, for SMX concentration > 0.1 mg/kg dry soil, it seems that SMX degradation was enhanced indicating changes in microbial community structure and/or function via the possible selection of SMX degrading bacteria or the over-expression of genes encoding for molecule degradation (Fang et al., 2023).

From these results it meant that (i) soil exposed to TWW had the ability to degrade SMX, (ii) once the soil bacterial diversity is reduced (low_div_soil), remaining soil endogenous bacteria (exposed to milliQ) do not degrade SMX and no abiotic transformation was highlighted here, (iii) bacteria brought by TWW could settle in low_div_soil or TWW addition could favour the growth of remaining bacteria, able to degrade SMX but at a lower rate, (iv) there is a key SMX concentration in between 0.01 and 0.5 mg/kg dry soil that might impact microbial community with a switch from sensitivity to selectivity with stimulation of its degradation.
<figure><img src="image_22.png" /><figcaption>Figure 2. (a) SMX concentration dynamic in reuse_soil. (b) SMX concentration dynamic in low_div_soil. (c) Degradation rate assessed by k, the kinetic constant of a 1st order degradation kinetic and their respective half-life DT₅₀.</figcaption></figure>

### 3.1.2. Monitoring of SMX transformation products

Along with SMX degradation, known transformation products (TP) were searched (Wang and Wang, 2018). In reuse_soil, several TPs whose structures are depicted in Figure 3 were identified on the basis of their exact mass and/or by comparison with their commercial reference (m/z and retention time, black stars in Figure 3e). Overall in reuse_soil, the majority of TPs were observed between 0.4 and 3 days of incubation (Figure 3). In particular, desamino-SMX and nitro-SMX appeared as early as 0.4 days and then tended to disappear over time. The area of the peaks seemed to indicate that these TPs were more abundant with higher SMX concentrations (Figure 3a). Desamino-SMX and nitro-SMX, corresponding respectively to desamination and nitrification, are usually generated via co-metabolism by nitrifying and denitrifying bacteria (Kassotaki et al., 2016).

Physicochemical analysis showed a decrease of nitrate concentrations in reuse_soil (13.1 ± 0.5 mg/kg dry soil, 8.2 ± 1.4 mg/kg dry soil, 0.9 ± 0.1 mg/kg dry soil, at 0, 0.4 and 1 day respectively, Figure S2), while nitrite concentration increased between 0 and 0.4 days, and then decreased (1.4 ± 0.1 mg/kg dry soil, 3.0 ±
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0.3 mg/kg *dry soil*, 0.2 ± 0.1mg/kg *dry soil*, at 0, 0.4 and 1 day respectively, Figure S2). It suggested that denitrifying conditions were established in the soil system. In these conditions, it is known that SMX can undergo abiotic transformation via the formation of unstable diazonium cations which then can react with NO₂⁻ or nitrous acid to give desamino-SMX and nitro-SMX (Nödler et al., 2012), which supported their measurement in the reuse_soil.

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Hypothetical 4-hydroxy-SMX also appeared from 0.4 day; the peak area increased up to 3 incubation days and then disappeared (Figure 3b). This putative TP has already been identified in SMX biodegradation assay with some *Rhodococcus* species (Gauthier et al., 2010) and was detected in activated sludge in the presence of autotroph nitrifying bacteria (Chen et al., 2022a; Müller et al., 2013). Acetyl-SMX was also identified in the first three days of incubation and only at 1 mg/kg *dry soil* of SMX. This transformation is usually performed via N-acetyl transferase and at high SMX concentrations (<10 mg/L), (Wang et al., 2021) which is consistent with what is observed here. *nat* gene encoding for the arylamine N-acetyl transferase and associated with this transformation was quantified in the soil microcosms and TWW (Table S4) but no SMX concentration effect was detected. This transformation was probably a minor pathway and it is likely that the TP was not observed for the lower SMX conditions due to the limit of detection.

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Additionally, no TP associated with the ipso-hydroxylation of SMX (involving 2 mono-oxygenase and FMN reductase encoding by *sadABC* genes, A. C. Reis et al., 2018) was observed here. It suggested that in these conditions and at environmental concentrations, this transformation pathway was not involved (assumption reinforced by the no-quantification of *sadA* gene, below the LOD).

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<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

Figure 3. (a) Dynamic of desamino-SMX peak area over time. (b) Dynamic of OH-SMX peak area over time. (c)

Dynamic of 4-N-glucuronide-SMX peak area over time. (d) Dynamic of sulfanilamide peak area over time. (e)

Putative structures of SMX transformation products detected in soil microcosms (star = TP whose commercial

standard was available).
334 3.1.3.Identification of a new SMX transformation pathway in soils

335 Interestingly an additional SMX-TP was detected at the beginning and at the end of the experiment (Figure 3c) with an exact mass corresponding to the SMX-β-D-glucuronide molecule (m/z: observed 336 428.0762 [M-H⁻], calculated: 428.0758 for C₁₆H₁₈N₃O₉S⁻, Table S2). Compared to the reference, the 337 exact mass matched (428.0762), but the retention time was different (7.7 min for sulfamethoxazole, 4.9 338 min for SMX-β-D-glucuronide and 7.4 min for the unknown TP, Figure S3). MS2 fragmentation in 339 negative mode of SMX gave the following fragments: m/z 156 (NH₂-C₆H₄-SO₂⁻), m/z 92 (H₂N-C₆H₄⁻) 340 and m/z 64 (C₅H₄⁻) with a typical cleavage of the SO₂-NH bond (Niessen, 2012). As expected, the 341 fragmentation of SMX-β-D-glucuronide gave m/z 252 corresponding to (SMX-H⁻) due to the cleavage 342 of N-glucuronide bond, and fragments observed with SMX due to the SO₂-NH bond cleavage. 343 Additional fragments characteristic of glucuronide fragmentation were also detected (m/z 175, 113, see 344 Figure S4) (Karlsson et al., 2010). For the TP found in this study, m/z 252 corresponding to (SMX-H⁻) 345 was also detected, validating the bond between the two molecules. Fragments attributed to m/z 332 346 (glucuronide-NH-C₆H₄-SO₂⁻), m/z 288 (glucuronide[-CO₂]-NH-C₆H₄-SO₂⁻) corresponding to the loss of 347 glucuronide CO₂, m/z 268 (glucuronide-NH-C₆H₄⁻) and m/z 176 (C₆H₈O₆⁻) were also observed, thus 348 following the same pattern as SMX fragmentation (Figure 4). It seems here that 4-N-glucuronide-SMX 349 TP was formed in the soils instead of SMX-β-D-glucuronide. 350

<figure><img src="image_28.png" /><figcaption>Figure 4. (a)MS² spectrum of SMX in HCD mode and a collision energy of 10 eV. (b) MS2 spectrum of SMX-β-D-glucuronide in HCD mode and a collision energy of 10 eV, (c) MS2 spectrum of 4-N-glucuronide-SMX in HCD mode and a collision energy of 20 eV, (d) MS2 spectrum of 4-N-glucuronide-SMX in HCD mode and a collision energy of 30 eV.</figcaption></figure>

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SMX-β-D-glucuronide, is generally obtained from SMX glucuronidation via UDP glucurotransferase in liver to facilitate xenobiotic transport via transporters and efflux pump (Radke et al., 2009). If it can be found in soils as SMX-derivative contaminant, it is generally retransformed in SMX by glucuronide bond cleavage and glucuronidase. However, it can persist in soils up to 82 days, which could explain why its isomer was detected until the end in our experiment (Löffler et al., 2023). One study reported the glucosylation and glucuronidation of SMX in *Arabidopsis thaliana* cells and cucumber seedlings (Dudley et al., 2018). Even if it is not yet well studied and reported, glucuronidation was also found in bacteria, mediated via glucuronosyltransferase and identified in *Xanthomonas campestris*, *Pasteurella multocida*, *Escherichia coli*, *Sphingomonas paucimobilis*, and *Streptococci* (Zhao et al., 2016). To our knowledge, this was the first time that this 4-N-glucuronide-SMX TP was identified in soils. The structure differing from SMX-β-D-glucuronide suggests that other enzymes or mechanisms might be at work here.

In low_div_soil, SMX TPs were mainly found in the late stage (14-42 days), probably due to a slower degradation compared to reuse_soil. Putative 4-hydroxy-SMX was detected between 14 and 28 incubation days, 4-N-glucuronide-SMX between 14 and 42 incubation days and sulfanilamide between 7 and 28 days (Figure 3d). This last one observed once at 28 incubation days in reuse_soil could be formed by amihydrolase, an activity performed by some *Pseudomonas* and sulfate-reducing bacteria or via abiotic hydrolysis (Wang and Wang, 2018).

None of the TPs mentioned before were detected neither in soils at T0 nor in TWW, confirming that transformations occurred during the experiments, in the microcosms.

Among these TPs, desamino-SMX, nitro-SMX, 4-hydroxy-SMX and N-acetyl SMX were found to exhibit higher antimicrobial activity compared to SMX (determined by bacterial inhibition tests, Majewsky et al., 2014; Osorio et al., 2016).

These first results showed that SMX concentration influenced DT₅₀, with a key concentration around 0.1 mg/kg dry soil, inducing an increase in biodegradation rate in reuse_soil. Although it was slower, a partial degradation of SMX was also observed in low_div_soil irrigated with TWW, suggesting a role of TWW-exogenous bacteria or autoclaved-resistant bacteria in SMX biodegradation. Several TPs with varying structures were also detected, mainly in the first 3 days for reuse_soil and at the end of the experiment (14-42 days) for low_div_soil. For the first time, a derivative of SMX glucuronide TP also appeared in these soil samples, as a proper biotransformation pathway.
388 3.2.Impact of SMX concentration on reuse_soil bacterial diversity

389 3.2.1.Temporal dynamic in the reuse_soil irrigated with TWW

390

391 Irrigation of reuse_soil with TWW (mostly containing Proteobacteria and Bacteroidetes, Figure S5) did not disturb soil diversity or richness (around 1800-1900, Table S5), but a temporal dynamic of Observed and Shannon indices was seen with a decrease from 0.4 to 28 incubation days (from 1788 to 1372 for Observed index and 6.1 to 5.0 for Shannon index) and an increase between 28 and 42 incubation days (up to 1722 for Observed index and 5.7 for Shannon index, Figure S6, Table S5). In the PCoA graph shown in Figure 5a, a temporal dynamic was also observed after exposure to TWW. At the phylum level, Firmicutes, represented by *Gracilibacter*, unclassified Family XVIII (Clostridia), *Lutispora* and *Anaerosolibacter* were the most impacted: it increased just after TWW irrigation and then decreased across the time from 7-14 incubation days (Figures S7-8). The abundance of these bacteria generally increase in soils irrigated with TWW (associated with organic matter amendment, and nutrients supply, Ibekwe et al., 2018). Less variation was observed among the other phyla although a temporal dynamic was seen at the genus level (Figures S7-8).

403 Overall it seems that TWW exposure brought or make some soil-already-present-components more available (such as nutrients, ions, minerals) which stimulated/modified the community structure of reuse_soil (Della-Negra et al., 2024; Frenk et al., 2015; Moulia et al., 2023).

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407 3.2.2.Identification of early stage biomarkers of the response to SMX concentration

408

409 The PCoA graph depicted in Figure 5a showed that SMX concentration disturbed reuse_soil endogenous bacteria as distinct bacterial groups could be observed between 1 and 7 incubation days.

411 Linear discriminant analysis Effect Size (LEfSe analysis) was thus performed on sampling times 1, 3, 7 (time points grouped together for the analysis). This kind of analysis is generally used to determine biomarkers of conditions and was performed here to reveal the bacterial genera statistically differing between SMX concentrations (LEfSe analysis combines standard tests for statistical significance and additional tests encoding biological consistency and effect relevance, Segata et al., 2011).

416 Thus several biomarkers associated with the initial SMX concentration gradient were identified (Table 2) and corresponded to:

418 (i) inhibition effect for Clostridiaceae family XVIII (Firmicutes), representing up to 10% of the bacterial relative abundance for SMX concentration < 0.01 mg/kg *dry soil* and less than 0.5 % for SMX concentrations > 0.1 mg/kg *dry soil*, *Flavisolibacter* (Bacteroidetes), representing up to 7% of the bacterial

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relative abundance for SMX concentration < 0.01 mg/kg dry soil and less than 3 % for SMX concentrations > 0.1 mg/kg dry soil and less abundant bacteria including spirillum shape bacteria from α- et β-Proteobacteria (Noviherbaspirillum, Azospirillum, Magnetospirillum) and some unclassified γ-Proteobacteria;

(ii) selection effect for some unclassified Rhodocyclaceae (β-Proteobacteria) for which a positive gradient in relative abundance was correlated with SMX concentration, ranging from 0.7% to 5% and finally S0134 terrestrial group of Gemmatimonadetes phylum.

Among the inhibited bacteria Clostridiaceae family XVIII, Azospirillum and Magnetospirillum are known to be N-fixing bacteria (Bazylinski et al., 2000; Steenhoudt and Vanderleyden, 2000), Flavisolibacter a denitrifying bacteria (Bourgeois et al., 2015), and Noviherbaspirillum involved in denitrifying processes (Ishii et al., 2017). Clostridia, Proteobacteria and Bacteroidetes were already identified to be inhibited in soils (Archundia et al., 2021; Garbini et al., 2022) and wetland sediments (Chen et al., 2022a) in the presence of SMX.

Among the selected bacteria, Rhodocyclaceae family are denitrifiers known to exhibit sulfonamide resistance (Kanagawa et al., 2000), to be enhanced in the presence of sulfonamide in activated sludge and wetland sediments (Chen et al., 2022b; Collado et al., 2013; Hu et al., 2022) and to possess degradation abilities (Xu et al., 2022; Zhang et al., 2020).
<figure><img src="image_29.png" /><figcaption>Figure 5. (a) PCoA of reuse_soil samples. (b) Temporal dynamic of unclassified Rhodocyclaceae relative abundance (c) Temporal dynamic of *Flavisolibacter* relative abundance in reuse_soil. Unclassified families indicate OTUs for which the genus was no determined.</figcaption></figure>

### 3.2.3.Identification of late stage biomarkers of the response to SMX concentration

LEfSe analysis was also performed on sampling times 14, 28 and 42 incubation days. For these time points, biomarkers of SMX concentrations were also identified and listed in Table 2, and mostly corresponded to inhibition effect of low abundant bacteria (< 1%) of Acidobacteria subgroup 7 (up to 1% for SMX concentrations < 0.01 mg/kg dry soil, and 0.4 % for higher SMX concentrations), some Proteobacteria including unclassified Desulfuromonadaceae, Xanthomonadaceae and Omnitrophicaeota phylum. As shown in Figure S9, the inhibition of these biomarkers seemed to intervene from 14 days of incubation and were still observed after 42 days of incubation for some of them (Figure S9). As SMX
452 was not detected during this period of time, it can be wondered if these inhibition effect was due to a
453 bounce-back effect of SMX use or the residual presence of SMX TPs.

454 Table 2. Mean relative abundance (%) in reuse_soil samples of the biomarkers identified by LEfSe analysis, their
455 LDA score and p_value according to SMX initial concentration. The white background indicates biomarkers
456 identified for the 1-7 days' time period and the grey background indicates biomarkers identified for the 14-42
457 days' time period. The blue color means inhibition phenomena and the red color selection phenomena. Letters in
458 brackets indicate significant differences between SMX concentrations (one-way ANOVA).

<table><thead><tr><th rowspan="2">Taxa (genus level)</th><th rowspan="2">LDA</th><th rowspan="2">p_value</th><th colspan="5">SMX concentration (mg/kg dry soil)</th></tr><tr><th>0</th><th>0.01</th><th>0.1</th><th>0.5</th><th>1</th></tr></thead><tbody><tr><td>Unclassified family XVIII</td><td>4.6</td><td>0.002</td><td>9.1 ± 7.3<sup>(a)</sup></td><td>8.7 ± 8<sup>(a)</sup></td><td>0.1 ± 0.1<sup>(b)</sup></td><td>0.2 ± 0.6<sup>(b)</sup></td><td>0.3 ± 0.6<sup>(b)</sup></td></tr><tr><td>Flavisolibacter</td><td>4.3</td><td>0.003</td><td>5.9 ± 3.6<sup>(a)</sup></td><td>5.2 ± 2.8<sup>(a)</sup></td><td>1.7 ± 0.4<sup>(b)</sup></td><td>2.6 ± 1.1<sup>(b)</sup></td><td>1.8 ± 0.8<sup>(b)</sup></td></tr><tr><td>Noviherbaspirillum</td><td>3.9</td><td>0.002</td><td>1.7 ± 0.8<sup>(a)</sup></td><td>1.3 ± 0.7<sup>(a)</sup></td><td>0.6 ± 0.3<sup>(b)</sup></td><td>0.7 ± 0.4<sup>(b)</sup></td><td>0.1 ± 0.1<sup>(c)</sup></td></tr><tr><td>Azospirillum</td><td>3.6</td><td>0.001</td><td>0.7 ± 0.1<sup>(a)</sup></td><td>0.7 ± 0.2<sup>(a)</sup></td><td>0.1 ± 0.1<sup>(b)</sup></td><td>0.1 ± 0.1<sup>(b)</sup></td><td>0.1 ± 0.1<sup>(b)</sup></td></tr><tr><td>S0134_terrestrial_group</td><td>3.5</td><td>0.01</td><td>0.7 ± 0.1<sup>(b)</sup></td><td>0.7 ± 0.2<sup>(b)</sup></td><td>1.0 ± 0.1<sup>(a)</sup></td><td>1.1 ± 0.3<sup>(a)</sup></td><td>1.1 ± 0.3<sup>(a)</sup></td></tr><tr><td>Unclassified Rhodocyclaceae</td><td>4.3</td><td>0.003</td><td>0.7 ± 0.7<sup>(c)</sup></td><td>0.6 ± 0.4<sup>(c)</sup></td><td>1.4 ± 0.9<sup>(bc)</sup></td><td>1.9 ± 1.2<sup>(b)</sup></td><td>4.4 ± 2.0<sup>(a)</sup></td></tr><tr><td>Acidobacteria subgroup 7</td><td>3.6</td><td>0.008</td><td>0.9 ± 0.1<sup>(a)</sup></td><td>1.0 ± 0.2<sup>(a)</sup></td><td>0.3 ± 0.07<sup>(b)</sup></td><td>0.3 ± 0.1<sup>(b)</sup></td><td>0.2 ± 0.1<sup>(b)</sup></td></tr><tr><td>Unclassified Desulfuromonadaceae</td><td>3.3</td><td>0.008</td><td>0.3 ± 0.1<sup>(a)</sup></td><td>0.1 ± 0.04<sup>(a)</sup></td><td>0 ± 0<sup>(b)</sup></td><td>0 ± 0<sup>(b)</sup></td><td>0.01 ± 0.01<sup>(b)</sup></td></tr><tr><td>Unclassified Xanthomonadaceae</td><td>3.2</td><td>0.008</td><td>0.4 ± 0.06<sup>(a)</sup></td><td>0.3 ± 0.06<sup>(a)</sup></td><td>0.1 ± 0.03<sup>(b)</sup></td><td>0.2 ± 0.01<sup>(b)</sup></td><td>0.2 ± 0.03<sup>(b)</sup></td></tr><tr><td>Omnitrophicaeota genus</td><td>3.2</td><td>0.005</td><td>0.1 ± 0.04<sup>(a)</sup></td><td>0.1 ± 0.02<sup>(a)</sup></td><td>0.01 ± 0<sup>(b)</sup></td><td>0.01 ± 0<sup>(b)</sup></td><td>0 ± 0<sup>(b)</sup></td></tr></tbody></table>

459 Finally, irrigation with TWW induced a temporal dynamic of the endogenous bacterial community of the reuse_soil and mainly of the phylum Firmicutes. SMX concentration effect was mainly observed during the first 7 days of incubation where biomarkers of community response to SMX concentration were identified and corresponded to the inhibition of bacteria involved in the nitrogen cycle and belonging mainly to the phyla Firmicutes and Bacteroidetes; and to the increase of unclassified Rhodocyclaceae whose relative abundance was positively correlated with SMX concentration between 1 and 14 incubation days (Figure S10). Such transitory effect were already observed in water and sediments where community structure and functions were affected by the presence of SMX at environmental concentrations (< 5 µg/L or µg/kg dry sediment) (Kergoat et al., 2021; Pesce et al., 2021).

460 The observed antagonistic effects of selection of Rhodocyclaceae and inhibition of *Flavisolibacter*, for 461 example (present in similar proportions, Figures 5b-c), could be explained in several ways:

462 (i) The pressure exerted by SMX could select the most tolerant and resistant bacteria (such as 463 Rhodocyclaceae bacteria) and the antimicrobial activity of the antibiotic inhibit the most susceptible 464 bacteria (*Flavisolibacter* and some Clostridi). (ii) The selection of tolerant bacteria could also have a 465 protective effect on other more susceptible bacteria by reducing the activity of SMX through its 466 transformation, as observed here (Figures 2 and 3), and/or by maintaining the functional activity of the 467 community (for example in nitrification/denitrification processes, Fang et al., 2023). (iii) Finally, Figure 468 5a clearly shows that the addition of nutrient-rich TWW leads to a shift in microbial communities, 469 certainly linked to competitive effects. The addition of SMX could also constitute a source of carbon 470 and nitrogen that could be incorporated by certain bacteria after transformation. This could therefore 471
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modulate competition phenomena by selecting bacteria most likely to use SMX as substrate, until its
complete transformation.

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At the end of the experiment, even if SMX was not detected anymore, some inhibition effects were still effective on low abundant bacteria belonging to Omnitrophicaeota and some Proteobacteria. Without SMX or at low concentration (0.01 mg/kg *dry soil*), some Acidobacteria subgroup 7, Desulfuromonadaceae, Xanthomonadaceae and Omnitrophicaeota emerged from 14 days, while some others started to decrease (Figure S9). In this case, a change in the local physico-chemical conditions could lead to an increase in these bacteria, better adapted to the new environment. It is also possible that the activity associated with the previous bacteria (*Flavisolibacter*, *Noviherbaspirillum*, *Clostridia* unclassified family XVIII) transformed complex substrates into molecules more easily assimilated by these emerging taxa. With SMX, induced changes in microbial communities might have disturbed metabolic activities and the pool of available substrates/nutrients in the soil preventing from these taxa to emerged. It is also possible that the residual quantity of SMX or the presence of SMX TPs with known bacterial toxicity (Majewsky et al., 2014) inhibits these taxa.

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### 3.3. The effect of SMX on N-cycling bacteria

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During this experiment, phosphate, sulfate, ammonium, nitrate and nitrite concentrations were monitored and the evolution of these chemicals in soils is available in Figure S2 and Figure S11.

A particular attention was given to nitrogen as many nitrifying and denitrifying bacteria are known to be SMX degraders (P. J. M. Reis et al., 2018; Wang and Wang, 2018) or disturbed by high SMX concentrations (Garbini et al., 2022).

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Ammonium concentration increased after TWW irrigation, in reuse_soil, for 3 days (from 1.9 ± 0.1 mg/kg *dry soil* to 2.6 ± 0.2 mg/kg *dry soil*), probably due to ammonification and denitrification processes stimulated by the supply of nutrients (including nitrate and nitrite, Figure S2) and organic matter from TWW. It then decreased across the time via nitrification/denitrification processes to reach 1.5 ± 0.1 mg/kg *dry soil* (Figure S11). Interestingly, lower ammonium concentrations were observed at the beginning of the experiment (0-3 days) for high SMX concentration (> 0.1 mg/kg *dry soil*, Figure S11), suggesting a change in N-cycling bacteria or related activity. This time range matched with the inhibition of N-fixing bacteria (Clostridiaceae family XVIII, *Azospirillum* and *Magnetospirillum*) and denitrifying bacteria (*Flavisolibacter*, *Noviherbaspirillum*). The number of copy of genes related to denitrification (*nirS*, *narG*), nitrification (*amoA-B*) and N-fixation (*nifH*) were not affected by SMX addition (Figure S12, Table S6). However, their expression could be impacted, as SMX is known to inhibit nitrogenase (encoded by *nifH* gene, Garbini et al., 2022) and up-regulate the expression of *amoA* gene associated
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with ammonium oxidizing bacteria (Guo et al., 2022). It would thus explain the difference in ammonium concentration observed for SMX concentration > 0.01 mg/kg dry soil.

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Thus, SMX concentration > [0.01-0.1 mg/kg dry soil] disturbed the activity of N-cycling related bacteria. After the degradation of SMX, this disturbance disappeared and similar level of ammonium was found in each SMX condition suggesting a resilience of the bacterial community after SMX degradation.

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3.3.Effect of SMX concentration on sulfonamide resistance gene *sul1* and mobile genetic element *intl*

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Figure 6 shows the evolution of 16S rDNA, sulfonamide resistance gene *sul1* and mobile genetic element *intl* in reuse_soil according to SMX concentration and over time.

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First of all, the copy number of 16S rDNA in reuse_soil changed relatively little over the course of the experiment. Before irrigation, the copy number/g dry soil was 8.7 x 10⁹ ± 1 x 10⁸. TWW load was more than 2 log lower (1.2 x 10⁷ copy number/g dry soil, Table S4) and did not increase the copy number in soil (as seen in Figure 6, at day 0.4). A slight increase of approximately 0.5 log was observed between 0.4 and 1 day and was attributed to the effect of TWW irrigation and stimulation of endogenous bacteria growth. It then decreased over the time to finally reach the same level at the end of the experiment (day 42, Figure 6).

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In reuse_soil, the temporal evolution of *intl* and *sul1* gene abundance/g dry soil and of their relative abundance/16S rDNA (Figure S13, Figure 6) had a non-linear shape indicating a decorrelation with 16S rDNA abundance. Both gene abundance were correlated which was consistent with their probable co-

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In this study TWW input induced an increase of *intl* gene copy number between 0.4 to 7-14 days, then a steady state was observed between 14 and 42 incubation days. During the increasing period, three levels of *intl* abundance were noticed, according to SMX concentration (i) 0-0.01 mg/kg dry soil, (ii) 0.1-0.5 mg/kg dry soil and (iii) 1 mg/kg dry soil with 1.5 log differences between the extreme levels, at T=3 and 7 days (Figure 6). From 14 incubation days, only 2 levels were still observed for SMX concentration (i) 0-0.01 mg/kg dry soil and (ii) 0.1-1 mg/kg dry soil.

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*sul1* gene copy number followed a similar level trend between 1 and 7 incubation days with (i) a decrease of *sul1* relative abundance of 0.5 log for SMX concentrations < 0.01 mg/kg dry soil, (ii) a less marked decrease/steady state for SMX concentrations between 0.1 and 0.5 mg/kg dry soil and finally (iii) an increase of *sul1* relative abundance (+ 0.5 log) for SMX concentration 1 mg/kg dry soil (Figure 6). A steady state was observed between 14 and 42 incubation days. At the end of the experiment two levels of *sul1*
547 gene copy number were also observed for the SMX concentrations (i) 0-0.01 mg/kg dry soil and (ii) 0.1-1 mg/kg dry soil. Contrary to *intl*, *sull* gene copy number at 42 incubation days for SMX concentration 0-0.01 mg/kg dry soil, was of the same order of magnitude compared to T0.
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<figure><img src="image_30.png" /><figcaption>Figure 6. Evolution of (a) 16S rDNA/g dry soil, (b) *intl*/16S rDNA and (c) *sull*/16S rDNA in reuse_soil across time. Letters indicate significant differences between SMX concentrations (one-way ANOVA).</figcaption></figure>

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3.4.Assessment of Predicted No Effect Concentration (PNEC) for SMX resistance selection in soils

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557 Irrigation with TWW led to an overall increase in the number of copies of the *intl* gene within the
558 microbial community whatever the concentration of SMX. Since TWW contains various contaminants,
559 including pharmaceuticals, we could deduce that it induced selection pressure and favoured the
560 propagation of this integron responsible for horizontal transfer of resistance genes (Gillings et al., 2015).
561 The temporal dynamic of *sull* showed that TWW irrigation disseminate sulfonamide resistance in soils
562 for SMX concentration above 0.01 mg/kg *dry soil*. As observed in a previous study, even if SMX
563 concentration decreased in soil across time, *intl* and *sull* dynamics increased over time (Chen et al.,
564 2019). Maintenance of ARGs could involve fitness costs of the host bacteria and impact their growth
565 rate due to metabolic burden (Fang et al., 2023). However, here it seems that this cost is actually
566 negligible compared to the benefits of resistance. The increase of these genes could be associated with
567 the increase of the most tolerant bacteria, carrying SMX resistant genes and competing with others. This
568 increase could also be due to the presence of SMX TPs still exerting selection pressure, or it could be
569 attributed to resistance gene transfer among the soil bacterial population.

570 After 42 incubation days, the residual but significant increase in both genes abundance for SMX
571 concentration > 0.1 mg/kg dry soil, involved the existence of a threshold between 0.01 and 0.1 mg/kg dry
572 soil, above which the dissemination of sulfonamide resistance was favoured. It seems here that a steady
573 state was reached. If previous statistical analysis (LEfSe analysis on 14-42 days period) did not reveal

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the presence of selected bacteria, the remaining resistance increase could be the result of horizontal
resistance gene transfer within soil bacterial community.

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According to these results, the “No Observed Selection Concentration” for SMX resistance, equivalent to the “Predicted No Effect Concentration” was assessed around 0.01 mg/kg dry soil, whereas the “Lowest Observed Selection Concentration” was determined at 0.1 mg/kg dry soil (Murray et al., 2021). No comparable data were experimentally obtained for soil, however, an equivalent PNEC was calculated from ecotoxicological data and assessment factor (Bourdat-Deschamps et al., 2017) and assessed at 0.02 mg/kg dry soil, in the same order of magnitude to what is observed here.

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Furthermore, given the octanol-water partition and adsorption coefficients values for SMX (log(K<sub>ow</sub>) = 0.9 and K<sub>d</sub> in soils = 1-5 L.kg<sup>-1</sup>), (Hu et al., 2019) it could be estimated that in our microcosm system (containing 25 g of dry soil and 20 mL of water) SMX was similarly present in soil and aqueous phase.

From this assumption, the PNEC would be around 0.006 mg/L and the LOSC around 0.06 mg/L. PNEC assessed in this study was relatively close to the PNEC determined in liquid media, obtained from ecotoxicological data (0.0006 mg/L, Bengtsson-Palme and Larsson, 2016). In complex liquid media PNEC values varied a lot and were globally higher compared to this study (Guo et al., 2022; Rauseo et al., 2021; Schuijt et al., 2024; Zhao et al., 2021). The discrepancies observed between the different studies could be due to the diversity of microbial community and functions, whose mechanisms and responses to selection pressure may vary. Soil system generally richer in nutrients, associated with a quite high temperature (35°C) could also maximises numbers of bacterial generations and the probability to observe selection (Stanton et al., 2020). Finally, the observed PNEC could also be the result of the combine effect of SMX and other residual contaminants or TPs.

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3.4.Correlation between resistance, transformation products and microbial communities in reuse_soil

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From all the results obtained previously, a correlation matrix was constructed (Figure 7), linking physicochemical properties, main SMX TPs, resistance genes and reuse_soil bacterial community at the genus level (between 0.4-7 days).

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3.4.1.Correlation between resistance and biomarkers of microbial community answer to SMX concentration

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Int11 gene was positively correlated (p_value < 0.001) with *Gracilibacter* from Clostridia and with β-Proteobacteria including unclassified Rhodocyclaceae, unclassified Burkholderiaceae and *Hydrogenophaga* (Burkhoderiales). *Sull* gene, whose evolution was a little different compared to *intl* gene, was positively correlated with *Hydrogenophaga* and unclassified Rhodocyclaceae (p_value <
608

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0.001). All these bacteria were already identified as resistant to sulfonamides or involved in their degradation (Xiaoting et al., 2021; Zhang et al., 2020) and mostly corresponded to anaerobic organisms. Unclassified Rhodocyclaceae was previously found to be selected with high SMX concentrations (Collado et al., 2013), and correlated with both of these genes. A study conducted in wetlands (using epic-PCR) showed that Burkholderiaceae and Rhodocyclaceae families were the most likely to carry the *intl* gene, followed by members of the Firmicutes. The host of the *sull* gene was most likely to be a member of Burkholderiaceae and Rhodocyclaceae families (Knecht et al., 2023).

615

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Thus, in our study, it seems that the bacteria selected in the presence of the SMX gradient also carry the resistance genes, which supports the hypothesis that the most tolerant bacteria were selected here.

617

<figure><img src="image_31.png" /></figure>

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Figure 7. Correlation matrix between physicochemical properties, SMX concentration, 16S rDNA, *sull* and *intl* genes with reuse_soil microbial communities on 0-7 days’ time period. The red-blue colour gradient is related to the Pearson coefficient measuring linear correlation between chemical and bio-molecular variables and the relative abundance of specific genera (representing more than 0.5% of the total abundance). \* = *p_value* ≤ 0.05; \*\* = *p_value* ≤ 0.01; \*\*\* = *p_value* ≤ 0.001.

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### 3.4.2. SMX biodegradation pathways and SMX resistance

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About SMX TPs, the normalised peak area of the main ones (4-N-glucuronide-SMX, desamino-SMX and 4-hydroxy-SMX) were included in the correlation matrix (Figure 7). As it could be seen, they were
clustered and correlated with distinct factors and bacteria. The desamino-SMX was clustered with nitrate and nitrite concentrations that were positively correlated with nitrifying and denitrifying bacteria ($R^2 > 0.8$), which is consistent with the literature (Kassotaki et al., 2016). Putative 4-hydroxy-SMX and 4-N-glucuronide-SMX were clustered with *sull* gene and associated (low correlation) with the relative abundance of some Clostridia (*Anaerosolibacter*) and β-Proteobacteria (unclassified Rhodocyclaceae, *Hydrogenophaga* and *Azoarcus*) (from Rhodocyclaceae family)). SMX hydroxylation and deamination led to TPs with an apparent antimicrobial effect superior to that of the parent molecule (Majewsky et al., 2014; Osorio et al., 2016). In our case, these TPs could contribute to the inhibitor effect observed within the first incubation week (Figure 5), but the tolerance of the complex soil bacterial community could also be higher than individual strains and therefore not be disturbed by these TPs. Even if hydroxylation via the activity of hydroxylase or cytochrome P450 mono-oxygenase (Ullrich and Hofrichter, 2007) are common pathways for aromatic compound degradation and inactivation, it is highly possible that hydroxylation and deamination were more closely associated with abiotic processes in our cases (Su et al., 2023).

Glucuronyl transferase activity, on the other hand, seems to be correlated with unclassified Rhodocyclaceae and could be a strategy for inactivating SMX and facilitating extracellular release of SMX via efflux pumps (Radke et al., 2009). In this case, this transformation would be related to a resistance mechanism, certainly achieved by a limited group of bacteria in order to resist and/or allow collective tolerance and protection against SMX exposure.

## 4. Conclusions

In the considered agricultural soil, we established threshold concentrations of SMX (ranging from 0.01 to 0.1 mg/kg *dry soil*) that were likely to increase the abundance of sulfonamide resistance genes in the soil. We identified a time window between 1 and 7 days after SMX exposure, during which a transitory increase in the *sull* and *intl* genes (>1 log/soil 16S rDNA) was observed, along with the emergence of SMX transformation products (4-hydroxy-SMX and desamino-SMX) and an increase in specific bacteria (notably, the selection of unclassified Rhodocyclaceae). Even 1.5 months post-exposure and complete transformation of SMX, fingerprints of SMX input were still noticeable, with the relative abundance of ARGs remaining higher (by approximately 0.5 log) compared to the SMX-free control and SMX concentrations < 0.1 mg/kg *dry soil*. The presence of persistent transformation product such as 4-N-glucuronide-SMX was also observed.

This preliminary study allowed us to establish an initial threshold concentration for the selection of sulfonamide resistance, between 0.01 and 0.1 mg/kg of dry soil. This concentration is of the same order
of magnitude as the theoretical estimates and exceeds the levels generally found in soils irrigated with TWW. It also highlights the potential role that TPs may play in maintaining selective pressure and promoting the spread of SMX resistance in the environment.

As the experimental model used here does not fully reflect real-world conditions, future studies should explore various scenarios, including repeated irrigation (or continuous drip irrigation), to evaluate the long-term risks of repeated inputs of antibiotic-contaminated domestic wastewater into soils. The research should also be extended to different soil types to account for the influence of soil physico-chemical properties and endogenous bacterial diversity on SMX degradation, resistome/mobilome dynamics, and the evolution of soil bacterial communities. Indeed, the biomarkers identified in this study may vary between different soil types. Lastly, although the residual concentrations of SMX from TWW may not pose a significant threat for SMX resistance in soils, other antibiotics from different classes (present in higher amounts or with a lower PNEC) may present a greater risk and should be explored the same way.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

This work (2204-003) was funded through Labex AGRO 2011-LABX-002 (under I-Site Muse framework) coordinated by Agropolis Fondation.

## Supporting information

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Highlights

* SMX disturbs soil microbial community and resistome the 1st week after exposure

* SMX PNEC was assessed in soils around 0.01 mg/kg dry soil

* Unclassified Rhodocyclaceae were selected with SMX and correlated with *sul1* gene

* SMX degradation in soil was enhanced from 0.5 mg/kg dry soil

* Soil resistome shows the fingerprint of SMX use despite its degradation after 42 days

<figure><img src="image_32.png" /></figure>
**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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