<figure><img src="image_1.png" /></figure>

HAL
open science

Novel stable isotope concepts to track antibiotics in
wetland systems

Xiao Liu, Junya Zhang, Hans Hermann Richnow, Gwenaël Imfeld

► To cite this version:

Xiao Liu, Junya Zhang, Hans Hermann Richnow, Gwenaël Imfeld. Novel stable isotope concepts to track antibiotics in wetland systems. Journal of Environmental Sciences, 2024, 10.1016/j.jes.2024.02.005. hal-04544636

HAL Id: hal-04544636

https://hal.science/hal-04544636

Submitted on 12 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
# Novel stable isotope concepts to track antibiotics in wetland systems

Xiao Liu, Junya Zhang, Hans Hermann Richnow, Gwenaël Imfeld

<figure><img src="image_1.png" /></figure>

## To cite this version:

Xiao Liu, Junya Zhang, Hans Hermann Richnow, Gwenaël Imfeld. Novel stable isotope concepts to track antibiotics in wetland systems. Journal of Environmental Sciences, 2024, 146, pp.298-303. 10.1016/j.jes.2024.02.005. hal-04544636

HAL Id: hal-04544636

https://hal.science/hal-04544636v1

Submitted on 12 Apr 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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

ELSEVIER

Available online at www.sciencedirect.com

ScienceDirect

www.elsevier.com/locate/jes

JES

JOURNAL OF
ENVIRONMENTAL
SCIENCES

www.jesc.ac.cn

Perspective

# Novel stable isotope concepts to track antibiotics in wetland systems

Xiao Liu<fnref n="1" />, Junya Zhang<fnref n="2" />, Hans Hermann Richnow<fnref n="3" />,<fnref n="4" />, Gwenaël Imfeld<fnref n="1" />,*

ARTICLE INFO

Article history:

Received 29 November 2023

Revised 8 February 2024

Accepted 8 February 2024

Available online xxx

Keywords:

Constructed wetlands

Micropolluants

Antibiotics

Degradation, CSIA

ABSTRACT

Antibiotics, their transformation products, and the translocation of antibiotic-resistant genes in the environment pose significant health risks to humans, animals, and ecosystems, aligning with the One Health concept. Constructed wetlands hold substantial yet underutilized potential for treating wastewater from agricultural, domestic sewage, or contaminated effluents from wastewater treatment plants, with the goal of eliminating antibiotics. However, the comprehensive understanding of the distribution, persistence, and dissipation processes of antibiotics within constructed wetlands remains largely unexplored. In this context, we provide an overview of the current application of stable isotope analysis at natural abundance to antibiotics. We explore the opportunities of an advanced multiple stable isotope approach, where isotope concepts could be effectively applied to examine the fate of antibiotics in wetlands. The development of a conceptual framework to study antibiotics in wetlands using multi-element stable isotopes introduces a new paradigm, offering enhanced insights into the identification and quantification of natural attenuation of antibiotics within wetland systems. This perspective has the potential to inspire the general public, governmental bodies, and the broader research community, fostering an emphasis on the utilization of stable isotope analysis for studying antibiotics and other emerging micropollutants in wetland systems.

© 2024 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/)

Antibiotics play an essential role in enhancing livestock and aquaculture production as well as promoting human

health. Nevertheless, their excessive use and improper ap-
plication can facilitate the evolution and dissemination
of antibiotic resistance genes (ARGs), thereby diminishing
their effectiveness against human and animal pathogens.
This represents a significant threat to both ecosystems and

<footnote n="1">¹Institut Terre et Environnement de Strasbourg (ITES), Université de Strasbourg/EOST/ENGEES, CNRS UMR 7063, F-67084 Strasbourg, France</footnote>

<footnote n="2">²State Key Joint Laboratory of Environmental Simulation and Pollution Control, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China</footnote>

<footnote n="3">³Department of Technical Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany</footnote>

<footnote n="4">⁴Isodetect GmbH, Deutscher Platz 5b, 04103 Leipzig, Germany</footnote>

<footnote>* Corresponding author.
E-mail: imfeld@unistra.fr (G. Imfeld).</footnote>
<figure><img src="image_3.png" /></figure>

ELSEVIER

Available online at www.sciencedirect.com

ScienceDirect

www.elsevier.com/locate/jes

JES

JOURNAL OF
ENVIRONMENTAL
SCIENCES

www.jesc.ac.cn

Perspective

Novel stable isotope concepts to track antibiotics in
wetland systems

Xiao Liu<fnref n="1" />, Junya Zhang<fnref n="2" />, Hans Hermann Richnow<fnref n="3" />,<fnref n="4" />, Gwenaël Imfeld<fnref n="1" />,*

ARTICLE INFO

Article history:

Received 29 November 2023

Revised 8 February 2024

Accepted 8 February 2024

Available online xxx

Keywords:

Constructed wetlands

Micropolluants

Antibiotics

Degradation, CSIA

A B S T R A C T

Antibiotics, their transformation products, and the translocation of antibiotic-resistant genes in the environment pose significant health risks to humans, animals, and ecosystems, aligning with the One Health concept. Constructed wetlands hold substantial yet underutilized potential for treating wastewater from agricultural, domestic sewage, or contaminated effluents from wastewater treatment plants, with the goal of eliminating antibiotics. However, the comprehensive understanding of the distribution, persistence, and dissipation processes of antibiotics within constructed wetlands remains largely unexplored. In this context, we provide an overview of the current application of stable isotope analysis at natural abundance to antibiotics. We explore the opportunities of an advanced multiple stable isotope approach, where isotope concepts could be effectively applied to examine the fate of antibiotics in wetlands. The development of a conceptual framework to study antibiotics in wetlands using multi-element stable isotopes introduces a new paradigm, offering enhanced insights into the identification and quantification of natural attenuation of antibiotics within wetland systems. This perspective has the potential to inspire the general public, governmental bodies, and the broader research community, fostering an emphasis on the utilization of stable isotope analysis for studying antibiotics and other emerging micropollutants in wetland systems.

© 2024 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
This is an open access article under the CC BY license
([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/))

Antibiotics play an essential role in enhancing livestock
and aquaculture production as well as promoting human

health. Nevertheless, their excessive use and improper ap-
plication can facilitate the evolution and dissemination
of antibiotic resistance genes (ARGs), thereby diminishing
their effectiveness against human and animal pathogens.
This represents a significant threat to both ecosystems and

<footnote n="1">¹Institut Terre et Environnement de Strasbourg (ITES), Université de Strasbourg/EOST/ENGEES, CNRS UMR 7063, F-67084 Strasbourg, France</footnote>

<footnote n="2">²State Key Joint Laboratory of Environmental Simulation and Pollution Control, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China</footnote>

<footnote n="3">³Department of Technical Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany</footnote>

<footnote n="4">⁴Isodetect GmbH, Deutscher Platz 5b, 04103 Leipzig, Germany</footnote>

<footnote>* Corresponding author.
E-mail: imfeld@unistra.fr (G. Imfeld).</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1 – Dissipation and bioaccumulation processes of antibiotics and related stable isotope fractionation in constructed wetlands using sulfamethoxazole (SMX) as model antibiotic. TPs: transformation products.</figcaption></figure>

global health, in accordance with the One Health concept (Hernando-Amado et al., 2019). The application of manure in agriculture can result in antibiotic accumulation and persistence in the soil, establishing it as a reservoir (Chen et al., 2022). Subsequently, agricultural runoff transports antibiotics into aquatic ecosystems, including wetlands, where they can sorb, accumulate and degrade in water, sediment, and organisms, including the vegetation. Constructed wetlands (CW) are recognized as ecologically friendly, cost-effective and highly efficient natural-based solutions for water treatment (Wu et al., 2023). CW find extensive application in the treatment of wastewater from agriculture, domestic sewage, and contaminated water effluents from wastewater treatment plants (WWTP) with the primary goal of removing micropollutants (Wu et al., 2023), including antibiotics (He et al., 2021; Lv et al., 2022). The removal of antibiotics in CW relies on a myriad of co-occurring removal processes, encompassing sorption, hydrolysis, photodegradation, aerobic/anaerobic microbial degradation, as well as phytoremediation.

Here we use sulfamethoxazole as a model compound to illustrate the fate of antibiotics in CW (Fig. 1). In CW, SMX photodegradation in the water column includes both direct and indirect processes. Direct photodegradation induces several bond cleavages, resulting in the formation of dominant transformation products (TPs) such as sulfanilic acid (Boreen et al., 2004). In contrast, indirect photodegradation, occurring in the presence of dissolved organic matter or nitrate, may be particularly relevant in wetlands, leading to the hydroxylation of SMX as dominant TPs (Boreen et al., 2004). In addition, mi

crobial degradation can occur under both aerobic and anaerobic conditions, and include transformation reactions such as acetylation (Achermann et al., 2018), hydroxylation (Liang and Hu, 2019), and ipso-hydroxylation (Kim et al., 2019) of SMX. Plant degradation may result in methylation and several bond cleavages of SMX (Ruan et al., 2022). Nevertheless, the comprehensive understanding of SMX distribution and persistence, and the contribution of diverse processes and mechanisms to the overall antibiotic dissipation in CW compartments often remains elusive with classical monitoring approaches.

Traditionally, monitoring of changes in the concentration of parent compounds and the generation of TPs in CW systems to establish inlet-outlet balances has conventionally served to evaluate the antibiotics dissipation. While this approach may in some cases aid in distinguishing dissipation processes, non-degradative processes such as sorption, evaporation, and dilution also affect antibiotic concentrations in CW and remain challenging to distinguish from degradative processes. Moreover, relying solely on TPs to differentiate degradation processes, especially in field conditions, poses challenges. Indeed, TPs may also degrade and the potential observation of similar transformation products in various and possibly co-occurring degradation pathways may complicate their identification (Fig. 1). Despite the challenges involved, differentiating between the various processes contributing to overall antibiotic dissipation in CW is crucial for informing risk assessments, quantifying the extent of degradation in CW, and estimating potential antibiotic exposure to higher organisms. This distinction can be instrumental in guiding remediation strategies by identifying the dominant dissipation pro-
<figure><img src="image_4.png" /><figcaption>Fig. 1 – Dissipation and bioaccumulation processes of antibiotics and related stable isotope fractionation in constructed wetlands using sulfamethoxazole (SMX) as model antibiotic. TPs: transformation products.</figcaption></figure>

global health, in accordance with the One Health concept (Hernando-Amado et al., 2019). The application of manure in agriculture can result in antibiotic accumulation and persistence in the soil, establishing it as a reservoir (Chen et al., 2022). Subsequently, agricultural runoff transports antibiotics into aquatic ecosystems, including wetlands, where they can sorb, accumulate and degrade in water, sediment, and organisms, including the vegetation. Constructed wetlands (CW) are recognized as ecologically friendly, cost-effective and highly efficient natural-based solutions for water treatment (Wu et al., 2023). CW find extensive application in the treatment of wastewater from agriculture, domestic sewage, and contaminated water effluents from wastewater treatment plants (WWTP) with the primary goal of removing micropollutants (Wu et al., 2023), including antibiotics (He et al., 2021; Lv et al., 2022). The removal of antibiotics in CW relies on a myriad of co-occurring removal processes, encompassing sorption, hydrolysis, photodegradation, aerobic/anaerobic microbial degradation, as well as phytoremediation.

Here we use sulfamethoxazole as a model compound to illustrate the fate of antibiotics in CW (Fig. 1). In CW, SMX photodegradation in the water column includes both direct and indirect processes. Direct photodegradation induces several bond cleavages, resulting in the formation of dominant transformation products (TPs) such as sulfanilic acid (Boreen et al., 2004). In contrast, indirect photodegradation, occurring in the presence of dissolved organic matter or nitrate, may be particularly relevant in wetlands, leading to the hydroxylation of SMX as dominant TPs (Boreen et al., 2004). In addition, mi-

crobial degradation can occur under both aerobic and anaerobic conditions, and include transformation reactions such as acetylation (Achermann et al., 2018), hydroxylation (Liang and Hu, 2019), and ipso-hydroxylation (Kim et al., 2019) of SMX. Plant degradation may result in methylation and several bond cleavages of SMX (Ruan et al., 2022). Nevertheless, the comprehensive understanding of SMX distribution and persistence, and the contribution of diverse processes and mechanisms to the overall antibiotic dissipation in CW compartments often remains elusive with classical monitoring approaches.

Traditionally, monitoring of changes in the concentration of parent compounds and the generation of TPs in CW systems to establish inlet-outlet balances has conventionally served to evaluate the antibiotics dissipation. While this approach may in some cases aid in distinguishing dissipation processes, non-degradative processes such as sorption, evaporation, and dilution also affect antibiotic concentrations in CW and remain challenging to distinguish from degradative processes. Moreover, relying solely on TPs to differentiate degradation processes, especially in field conditions, poses challenges. Indeed, TPs may also degrade and the potential observation of similar transformation products in various and possibly co-occurring degradation pathways may complicate their identification (Fig. 1). Despite the challenges involved, differentiating between the various processes contributing to overall antibiotic dissipation in CW is crucial for informing risk assessments, quantifying the extent of degradation in CW, and estimating potential antibiotic exposure to higher organisms. This distinction can be instrumental in guiding remediation strategies by identifying the dominant dissipation pro-
cess of antibiotics and monitoring their overall dissipation in
various CW compartments over time.

In this context, an emerging approach for distinguishing
dissipation processes and monitoring the degradation of an-
tibiotics in complex environmental systems is multi-element
compound-specific isotope analysis (ME-CSIA) (Nijenhuis and
Richnow, 2016; Vogt et al., 2016). ME-CSIA entails the eval-
uation of several stable isotope ratios in individual organic
compounds at natural isotopic abundances (see Box 1 for
definitions). Principally, CSIA can be employed to distin-
guish between degradative and non-degradative processes.
Non-degradative, mostly physical processes generally do
not alter the isotopic signature of contaminants or have a
negligible effect compared to degradative processes. Degrada-
tive processes, involving bond cleavage, typically result in
kinetic stable isotope fractionation. Therefore, the applica-
tion of isotopic concepts allows for differentiation between
non-degradative and degradative processes. In the context
of distinguishing various antibiotic degradation processes
through ME-CSIA, this goal can be achieved as distinct
degradation processes exhibit specific isotope pattern. These
include different directions of isotope fractionation, which
can manifest as normal or inverse isotope fractionation, and
variation in the extent of isotope fractionation denoted by the
isotope fractionation factor (ε), along with the relationship of
stable isotope fractionation of different elements represented
by dual-element isotope values (δ) (Fig. 2). For example, an
inverse isotope fractionation and mass-independent frac-
tionation of sulfur isotopes has been commonly observed
in photodegradation of several pollutants. Furthermore, the
quantification of the antibiotic transformation presents an-
other advantage of ME-CSIA in tracking the fate of antibiotics
in CW compared to traditional concepts. Antibiotic ME-CSIA
holds the potential to facilitate the assessment of in situ an-
tibiotic transformation in CW, and may enable the estimation
of the degradation extent using the Rayleigh concept and ε
derived from laboratory reference experiments (Fig. 2).

Nevertheless, the main challenge in implementing ME-
CSIA for antibiotics in CW lies in the low concentration and
high polarity of antibiotics, along with the limited availabil-
ity of reference laboratory degradation studies to infer trans-
formation processes in wetland systems. The high polarity of
antibiotics poses challenges in GC measurements. Mass en-
richment of sufficient analyte, typically a few mg for mul-
tiple injections, for isotope analysis involves the extraction
from large amounts of water (Elsner and Imfeld, 2016). Solid
phase extraction (SPE) is commonly employed for the extrac-
tion and enrichment of antibiotics from water. However, dur-
ing SPE, non-volatile matrix components are also enriched
along with antibiotics. This co-enrichment of matrix com-
ponents can potentially hinder chromatographic separation
and the accurate identification of target antibiotics, a phe-
nomenon referred to as the "matrix effect" (Elsner and Im-
feld, 2016; Höhener et al., 2022). In addition, the polarity of
antibiotics poses challenges for direct GC measurement, of-
ten necessitating a derivatization step before GC separation
(Blessing and Baran, 2022). Hence, the upcoming tasks in-
volve the development of reliable, non-fractionating extrac-
tion and separation methods, as well as adapted instrumental
approaches. Currently, the existing literature on isotope anal-

ysis of antibiotics predominantly centers on carbon isotope
fractionation, utilizing Liquid Chromatography Isotope Ratio
Mass Spectrometry (LC-IRMS) in photodegradation and mi-
crobial degradation (Birkigt et al., 2015; Willach et al., 2017;
Willach et al., 2018) (Table 1). Nevertheless, there are lim-
itations in utilizing LC-IRMS for the separation of analytes
in environmental extracts, and this technique is restricted
to carbon isotope analysis. LC-IRMS systems utilize online
wet-chemical oxidation to convert all carbon within the com-
pounds to CO2. This CO2 is then extracted from the mobile
phase and directed to the mass spectrometer for carbon iso-
tope ratio analysis (Perini and Bontempo, 2022). However, LC-
IRMS is currently limited to the measurement of carbon iso-
tope ratios. The current separation unit is designed to work
exclusively with CO2, making it challenging to strip other el-
ements (Perini and Bontempo, 2022). Recent advancements
have successfully extended antibiotics CSIA to carbon and
hydrogen stable isotope analysis using Gas Chromatography-
IRMS (GC-IRMS) (Ouyang et al., 2023), thereby shedding new
light on the applicability of ME-CSIA. Furthermore, previous
studies have harnessed dual-element isotope analysis (e.g.,
carbon and chlorine) to differentiate between various degra-
dation pathways of chlorinated organic pollutants in soil-
plant and food web systems (Liu et al., 2021, 2020). In the fu-
ture, with the establishment of suitable derivatization proce-
dures for various types of antibiotics, it may become feasible
to measure nitrogen, chlorine, and sulfur isotopes by GC-IRMS
and Gas Ghromatograph Coupled with a Multiple-collector
Inductively Coupled Plasma Mass Spectrometer (GC-MC-ICP-
MS). For instance, a derivatization method of antibiotics is cur-
rently developed for SMX using trimethylsilyldiazomethane
(TMSD), involving methylation on the nitrogen atom in the
S-N bond (data not published). Additionally, there have been
recent developments in universal chlorine and sulfur isotope
analysis (Horst et al., 2017; Kümmel et al., 2020), presenting
an opportunity to develop and validate these methods for an-
tibiotics, with the perspective to follow-up their stable isotope
composition in CW.

Given the widespread use of antibiotics and the poten-
tial health risks they pose, it is also imperative to develop in-
novative frameworks for assessing the extent of degradation
and elucidating the pathways of antibiotics within CW. These
ubiquitous artificial ecosystems, renowned for their adapt-
ability in bioremediation of micropollutant contaminated wa-
ter, can serve as a focal point for this endeavor. Newly devel-
oped isotope concepts offer significant potential to address
multiple challenges. Primarily, these concepts can facilitate
the distinction between dissipation processes, encompassing
both non-degradative and degradative pathways, as well as
among various transformation pathways in CW. This capabil-
ity can aid in prioritizing degradation pathways within CW,
thereby improving risk assessment and the implementation
of remediation strategies. These advancements can provide
guidance for formulating risk management approaches by im-
proving predictions of transformation processes within CW.
Furthermore, integrating isotope concepts into the toolkit for
evaluating the kinetics and processes of antibiotic removal in
wetland systems will enhance the establishment of a more
universal framework. This framework can generate standard-
ized concepts that go beyond the realm of CW research, uti-
cess of antibiotics and monitoring their overall dissipation in
various CW compartments over time.

In this context, an emerging approach for distinguishing
dissipation processes and monitoring the degradation of an-
tibiotics in complex environmental systems is multi-element
compound-specific isotope analysis (ME-CSIA) (Nijenhuis and
Richnow, 2016; Vogt et al., 2016). ME-CSIA entails the eval-
uation of several stable isotope ratios in individual organic
compounds at natural isotopic abundances (see Box 1 for
definitions). Principally, CSIA can be employed to distin-
guish between degradative and non-degradative processes.
Non-degradative, mostly physical processes generally do
not alter the isotopic signature of contaminants or have a
negligible effect compared to degradative processes. Degrada-
tive processes, involving bond cleavage, typically result in
kinetic stable isotope fractionation. Therefore, the applica-
tion of isotopic concepts allows for differentiation between
non-degradative and degradative processes. In the context
of distinguishing various antibiotic degradation processes
through ME-CSIA, this goal can be achieved as distinct
degradation processes exhibit specific isotope pattern. These
include different directions of isotope fractionation, which
can manifest as normal or inverse isotope fractionation, and
variation in the extent of isotope fractionation denoted by the
isotope fractionation factor (ε), along with the relationship of
stable isotope fractionation of different elements represented
by dual-element isotope values (δ) (Fig. 2). For example, an
inverse isotope fractionation and mass-independent frac-
tionation of sulfur isotopes has been commonly observed
in photodegradation of several pollutants. Furthermore, the
quantification of the antibiotic transformation presents an-
other advantage of ME-CSIA in tracking the fate of antibiotics
in CW compared to traditional concepts. Antibiotic ME-CSIA
holds the potential to facilitate the assessment of in situ an-
tibiotic transformation in CW, and may enable the estimation
of the degradation extent using the Rayleigh concept and ε
derived from laboratory reference experiments (Fig. 2).

Nevertheless, the main challenge in implementing ME-
CSIA for antibiotics in CW lies in the low concentration and
high polarity of antibiotics, along with the limited availabil-
ity of reference laboratory degradation studies to infer trans-
formation processes in wetland systems. The high polarity of
antibiotics poses challenges in GC measurements. Mass en-
richment of sufficient analyte, typically a few mg for mul-
tiple injections, for isotope analysis involves the extraction
from large amounts of water (Elsner and Imfeld, 2016). Solid
phase extraction (SPE) is commonly employed for the extrac-
tion and enrichment of antibiotics from water. However, dur-
ing SPE, non-volatile matrix components are also enriched
along with antibiotics. This co-enrichment of matrix com-
ponents can potentially hinder chromatographic separation
and the accurate identification of target antibiotics, a phe-
nomenon referred to as the “matrix effect” (Elsner and Im-
feld, 2016; Höhener et al., 2022). In addition, the polarity of
antibiotics poses challenges for direct GC measurement, of-
ten necessitating a derivatization step before GC separation
(Blessing and Baran, 2022). Hence, the upcoming tasks in-
volve the development of reliable, non-fractionating extrac-
tion and separation methods, as well as adapted instrumental
approaches. Currently, the existing literature on isotope anal-

ysis of antibiotics predominantly centers on carbon isotope
fractionation, utilizing Liquid Chromatography Isotope Ratio
Mass Spectrometry (LC-IRMS) in photodegradation and mi-
crobial degradation (Birkigt et al., 2015; Willach et al., 2017;
Willach et al., 2018) (Table 1). Nevertheless, there are lim-
itations in utilizing LC-IRMS for the separation of analytes
in environmental extracts, and this technique is restricted
to carbon isotope analysis. LC-IRMS systems utilize online
wet-chemical oxidation to convert all carbon within the com-
pounds to CO2. This CO2 is then extracted from the mobile
phase and directed to the mass spectrometer for carbon iso-
tope ratio analysis (Perini and Bontempo, 2022). However, LC-
IRMS is currently limited to the measurement of carbon iso-
tope ratios. The current separation unit is designed to work
exclusively with CO2, making it challenging to strip other el-
ements (Perini and Bontempo, 2022). Recent advancements
have successfully extended antibiotics CSIA to carbon and
hydrogen stable isotope analysis using Gas Chromatography-
IRMS (GC-IRMS) (Ouyang et al., 2023), thereby shedding new
light on the applicability of ME-CSIA. Furthermore, previous
studies have harnessed dual-element isotope analysis (e.g.,
carbon and chlorine) to differentiate between various degra-
dation pathways of chlorinated organic pollutants in soil-
plant and food web systems (Liu et al., 2021, 2020). In the fu-
ture, with the establishment of suitable derivatization proce-
dures for various types of antibiotics, it may become feasible
to measure nitrogen, chlorine, and sulfur isotopes by GC-IRMS
and Gas Ghromatograph Coupled with a Multiple-collector
Inductively Coupled Plasma Mass Spectrometer (GC-MC-ICP-
MS). For instance, a derivatization method of antibiotics is cur-
rently developed for SMX using trimethylsilyldiazomethane
(TMSD), involving methylation on the nitrogen atom in the
S-N bond (data not published). Additionally, there have been
recent developments in universal chlorine and sulfur isotope
analysis (Horst et al., 2017; Kümmel et al., 2020), presenting
an opportunity to develop and validate these methods for an-
tibiotics, with the perspective to follow-up their stable isotope
composition in CW.

Given the widespread use of antibiotics and the poten-
tial health risks they pose, it is also imperative to develop in-
novative frameworks for assessing the extent of degradation
and elucidating the pathways of antibiotics within CW. These
ubiquitous artificial ecosystems, renowned for their adapt-
ability in bioremediation of micropollutant contaminated wa-
ter, can serve as a focal point for this endeavor. Newly devel-
oped isotope concepts offer significant potential to address
multiple challenges. Primarily, these concepts can facilitate
the distinction between dissipation processes, encompassing
both non-degradative and degradative pathways, as well as
among various transformation pathways in CW. This capabil-
ity can aid in prioritizing degradation pathways within CW,
thereby improving risk assessment and the implementation
of remediation strategies. These advancements can provide
guidance for formulating risk management approaches by im-
proving predictions of transformation processes within CW.
Furthermore, integrating isotope concepts into the toolkit for
evaluating the kinetics and processes of antibiotic removal in
wetland systems will enhance the establishment of a more
universal framework. This framework can generate standard-
ized concepts that go beyond the realm of CW research, uti-
<figure><img src="image_4.png" /><figcaption>Fig. 2 – Conceptual framework of Multiple Element Compound-Specific Stable Isotope Analysis (ME-CSIA) applied to antibiotics in constructed wetlands (CW). (A) The footprint of antibiotic degradation in CW: Stable isotope composition of antibiotics in CW may vary due to kinetic isotope effects when antibiotics undergo degradation processes such as photo-, phyto-, and microbial degradation, as shown for Element A, B, and C. (B) Degradation mechanisms and pathway identification from reference laboratory experiments: The direction and magnitude of isotope fractionation may indicate the underlying reaction chemistry. Stable isotope fractionation values (ε) for individual elements and dual isotope element values (Λ) (represented by δ¹⁵N versus δ¹³C values) are retrieved, serving for the identification and differentiation of pathways within CW. (C) Quantitation of the extent of antibiotic degradation in field samples: The degradation extent can be quantified using the Rayleigh equation, employing reference stable isotope fractionation values (Vogt et al., 2016).</figcaption></figure>

<table><caption>Table 1 – Overview of antibiotic CSIA studies and associated conditions.</caption><thead><tr><th>Antibiotics</th><th>Method</th><th>Stable isotope</th><th>Conditions</th></tr></thead><tbody><tr><td>Sulfamethoxazole (SMX)</td><td>LC-IRMS<sup>a</sup></td><td>δ<sup>13</sup>C</td><td>Direct photodegradation (Birkigt et al., 2015; Willach et al., 2018)</td></tr><tr><td>SMX</td><td>LC-IRMS<sup>a</sup></td><td>δ<sup>13</sup>C</td><td>Indirect photodegradation with ozone and chlorine dioxide (Willach et al., 2017)</td></tr><tr><td>SMX</td><td>LC-IRMS<sup>a</sup></td><td>δ<sup>13</sup>C</td><td>Aerobic Microbial degradation- Microbacterium sp. strain BR1 (Birkigt et al., 2015)</td></tr><tr><td>SMX</td><td>GC-IRMS<sup>b</sup></td><td>δ<sup>13</sup>C, δ<sup>b</sup>H</td><td>Anaerobic Microbial degradation- Desulfovibrio vulgaris Hildenborough (Ouyang et al., 2023)</td></tr></tbody></table>

lizing them as a prototype for complex and dynamic systems. The potential of such a generic framework may also extend to broader applications, encompassing various micropollutants and a range of environmental scenarios at urban, territorial, or watershed scales (Hofstetter et al., 2024).

To advance the development and implementation of antibiotic CSIA in wetlands, collaborative efforts among research, local and territorial monitoring programs, and government entities are crucial to addressing key milestones. These include the validation and standardization of the antibiotic isotope methodology, encompassing ¹³C, ¹⁵N, ²H, ³⁷Cl, and ³³/³⁴S. This involves ensuring the consistency and reliability

of sampling, extraction, measurements, and interpretation of isotopic data. Moreover, expanding reference antibiotic laboratory studies to encompass a broader range of wetland-specific processes, such as indirect photodegradation or bioaccumulation and biomagnification in food chains, is essential to enhance the applicability of antibiotic CSIA in field studies. This expansion will pave the way for future applications of antibiotic CSIA in CW, including source apportionment, bioaccumulation in trophic levels, biodegradation, and phytoremediation concepts. In turn, such applications will contribute to the development of a framework for risk assessment and regulatory evolution through the application of antibiotic

<footnote>a LC-IRMS: Liquid Chromatography Isotope Ratio Mass Spectrometry.</footnote>

<footnote>b GC-IRMS: Gas Chromatography Isotope Ratio Mass Spectrometry.</footnote>
<figure><img src="image_5.png" /><figcaption>Fig. 2 – Conceptual framework of Multiple Element Compound-Specific Stable Isotope Analysis (ME-CSIA) applied to antibiotics in constructed wetlands (CW). (A) The footprint of antibiotic degradation in CW: Stable isotope composition of antibiotics in CW may vary due to kinetic isotope effects when antibiotics undergo degradation processes such as photo-, phyto-, and microbial degradation, as shown for Element A, B, and C. (B) Degradation mechanisms and pathway identification from reference laboratory experiments: The direction and magnitude of isotope fractionation may indicate the underlying reaction chemistry. Stable isotope fractionation values (ε) for individual elements and dual isotope element values (Λ) (represented by δ¹⁵N versus δ¹³C values) are retrieved, serving for the identification and differentiation of pathways within CW. (C) Quantitation of the extent of antibiotic degradation in field samples: The degradation extent can be quantified using the Rayleigh equation, employing reference stable isotope fractionation values (Vogt et al., 2016).</figcaption></figure>

<table><caption>Table 1 – Overview of antibiotic CSIA studies and associated conditions.</caption><thead><tr><th>Antibiotics</th><th>Method</th><th>Stable isotope</th><th>Conditions</th></tr></thead><tbody><tr><td>Sulfamethoxazole (SMX)</td><td>LC-IRMS<sup>a</sup></td><td>δ<sup>13</sup>C</td><td>Direct photodegradation (Birkigt et al., 2015; Willach et al., 2018)</td></tr><tr><td>SMX</td><td>LC-IRMS<sup>a</sup></td><td>δ<sup>13</sup>C</td><td>Indirect photodegradation with ozone and chlorine dioxide (Willach et al., 2017)</td></tr><tr><td>SMX</td><td>LC-IRMS<sup>a</sup></td><td>δ<sup>13</sup>C</td><td>Aerobic Microbial degradation- Microbacterium sp. strain BR1 (Birkigt et al., 2015)</td></tr><tr><td>SMX</td><td>GC-IRMS<sup>b</sup></td><td>δ<sup>13</sup>C, δ<sup>b</sup>H</td><td>Anaerobic Microbial degradation- Desulfovibrio vulgaris Hildenborough (Ouyang et al., 2023)</td></tr></tbody></table>

lizing them as a prototype for complex and dynamic systems. The potential of such a generic framework may also extend to broader applications, encompassing various micropollutants and a range of environmental scenarios at urban, territorial, or watershed scales (Hofstetter et al., 2024).

To advance the development and implementation of antibiotic CSIA in wetlands, collaborative efforts among research, local and territorial monitoring programs, and government entities are crucial to addressing key milestones. These include the validation and standardization of the antibiotic isotope methodology, encompassing ¹³C, ¹⁵N, ²H, ³⁷Cl, and ³³/³⁴S. This involves ensuring the consistency and reliability

of sampling, extraction, measurements, and interpretation of isotopic data. Moreover, expanding reference antibiotic laboratory studies to encompass a broader range of wetland-specific processes, such as indirect photodegradation or bioaccumulation and biomagnification in food chains, is essential to enhance the applicability of antibiotic CSIA in field studies. This expansion will pave the way for future applications of antibiotic CSIA in CW, including source apportionment, bioaccumulation in trophic levels, biodegradation, and phytoremediation concepts. In turn, such applications will contribute to the development of a framework for risk assessment and regulatory evolution through the application of antibiotic

<footnote>a LC-IRMS: Liquid Chromatography Isotope Ratio Mass Spectrometry.</footnote>

<footnote>b GC-IRMS: Gas Chromatography Isotope Ratio Mass Spectrometry.</footnote>
CSIA, ensuring a robust foundation for addressing environ-
mental concerns associated with antibiotic contamination in
wetlands. Finally, the potential and contribution of antibiotic
CSIA in wetlands may align with the Global Health concept,
promoting a holistic approach to addressing health-related
challenges associated with antibiotics in engineered and
nature-based wastewater treatment solutions worldwide.

Box 1: Classic Definitions

**Isotopes** are naturally occurring variations of elements that have different atomic masses due to a different number of neutrons but identical numbers of protons (e.g. ¹³C, ¹²C)

**Stable isotopes** are non-radioactive naturally occurring forms of elements

**ME-CSIA:** Multi-element Compound-specific Isotope Analysis

**Isotope fractionation/ Isotope effect (IE)** is a change in the isotopic composition due to a biological, chemical or physical process

**Delta value (δ)** describes the difference in the isotopic ratio (e.g. ¹³C/¹²C) of a sample relative to an international standard in %

**Isotope Fractionation factor (ε)** describes the change in the isotopic composition of a substrate over the course of a reaction

**Dual element isotope analysis** is the slope of changes of δ values of two elements (e.g. C–H) compared to the source, which is represented by Lambda value (Λ).

Declaration of Competing Interest

All authors of the article disclose no actual or potential conflict
of interest including any financial, personal or other relation-
ships with other people or organizations within three years
of beginning the submitted work that could inappropriately
influence, or be perceived to influence, their work.

REFERENCES

Achermann, S., Bianco, V., Mansfeldt, C.B., Vogler, B.,
Kolvenbach, B.A., Corvini, P.F.X., et al., 2018. Biotransformation
of sulfonamide antibiotics in activated sludge: the formation
of pterin-conjugates leads to sustained risk. Environ. Sci.
Technol. 52, 6265-6274. doi:10.1021/acs.est.7b06716.

Birkigt, J., Gilevska, T., Ricken, B., Richnow, H.H., Vione, D.,
Corvini, P.F.X., et al., 2015. Carbon stable isotope fractionation
of sulfamethoxazole during biodegradation by
microbacterium sp. strain BR1 and upon direct photolysis.
Environ. Sci. Technol. 49, 6029–6036.
doi:10.1021/acs.est.5b00367.

Blessing, M., Baran, N., 2022. A review on environmental isotope
analysis of aquatic micropollutants: recent advances, pitfalls
and perspectives. TrAC Trend. Anal. Chem. 157, 116730.
doi:10.1016/j.trac.2022.116730.

Boreen, A.L., Arnold, W.A., McNeill, K., 2004. Photochemical fate of
sulfa drugs in then aquatic environment: sulfa drugs
containing five-membered heterocyclic groups. Environ. Sci.
Technol. 38, 3933–3940. doi:10.1021/es0353053.

Chen, J., Yang, Y., Ke, Y., Chen, X., Jiang, X., Chen, C., et al., 2022.
Sulfonamide-metabolizing microorganisms and mechanisms
in antibiotic-contaminated wetland sediments revealed by
stable isotope probing and metagenomics. Environ. Int. 165,
107332. doi:10.1016/j.envint.2022.107332.

Elsner, M., Imfeld, G., 2016. Compound-specific isotope analysis (CSIA) of micropollutants in the environment — Current developments and future challenges. Curr. Opin. Biotechnol. 41, 60–72. doi:10.1016/j.copbio.2016.04.014.

Hernando-Amado, S., Coque, T.M., Baquero, F., Martínez, J.L., 2019.
Defining and combating antibiotic resistance from One Health
and Global Health perspectives. Nat. Microbiol. 4, 1432–1442.
doi:10.1038/s41564-019-0503-9.

He, Y., Zhang, L., Jiang, L., Wagner, T., Sutton, N.B., Ji, R., et al.,
2021. Improving removal of antibiotics in constructed wetland
treatment systems based on key design and operational
parameters: a review. J. Hazard. Mater. 407, 124386.
doi:10.1016/j.jhazmat.2020.124386.

Hofstetter, T.B., Bakkour, R., Buchner, D., Eisenmann, H.,
Fischer, A., Gehere, M., et al., 2024. Perspectives of
compound-specific isotope analysis of organic contaminants
for assessing environmental fate and managing chemical
pollution. Nat. Water 2, 14–30. doi:10.1038/s44221-023-00176-4.

Höhener, P., Guers, D., Malleret, L., Boukaroum, O.,
Martin-Laurent, F., Masbou, J., et al., 2022. Multi-elemental
compound-specific isotope analysis of pesticides for source
identification and monitoring of degradation in soil: a review.
Environ. Chem. Lett. 20, 3927–3942.
doi:10.1007/s10311-022-01489-8.

Horst, A., Renpenning, J., Richnow, H.H., Gehre, M., 2017.
Compound specific stable chlorine isotopic analysis of volatile
aliphatic compounds using gas chromatography hyphenated
with multiple collector inductively coupled plasma mass
spectrometry. Anal. Chem. 89, 9131–9138.
doi:10.1021/acs.analchem.7b01875.

Kim, D.W., Thawng, C.N., Lee, K., Wellington, E.M.H., Cha, C.J.,
2019. A novel sulfonamide resistance mechanism by
two-component flavin-dependent monooxygenase system in
sulfonamide-degrading actinobacteria. Environ. Int. 127,
206–215. doi:10.1016/j.envint.2019.03.046.

Kümmel, S., Horst, A., Gelman, F., Strauss, H., Richnow, H.H.,
Gehre, M., 2020. Simultaneous compound-specific analysis of
δ 33 S and δ 34 S in organic compounds by GC-MC-ICPMS
using medium- and low-mass-resolution modes. Anal. Chem.
doi:10.1021/acs.analchem.0c03253.

Liang, D.hui, Hu, Y., 2019. Simultaneous sulfamethoxazole
biodegradation and nitrogen conversion by *Achromobacter* sp.
JL9 using with different carbon and nitrogen sources.
Bioresour. Technol. 293. doi:10.1016/j.biortech.2019.122061.

Liu, X., Li, W., Kümmel, S., Merbach, I., Sood, U., Gupta, V., et al.,
2021. Soil from a hexachlorocyclohexane contaminated field
site inoculates wheat in a pot experiment to facilitate the
microbial transformation of β-hexachlorocyclohexane
examined by compound-specific isotope analysis. Environ.
Sci. Technol. 55, 13812–13821. doi:10.1021/acs.est.1c03322.

Liu, X., Wu, L., Kümmel, S., Merbach, I., Lal, R., Richnow, H.H.,
2020. Compound-specific isotope analysis and enantiomer
fractionation to characterize the transformation of
hexachlorocyclohexane isomers in a soil-wheat pot system.
Environ. Sci. Technol. 54, 8690–8698.
doi:10.1021/acs.est.9b07609.

Lv, M., Zhang, D., Niu, X., Ma, J., Lin, Z., Fu, M., 2022. Insights into
the fate of antibiotics in constructed wetland systems:
removal performance and mechanisms. J. Environ. Manage.
321, 116028. doi:10.1016/j.jenvman.2022.116028.

Nijenhuis, I., Richnow, H.H., 2016. Stable isotope fractionation concepts for characterizing biotransformation of organohalides. Curr. Opin. Biotechnol. 41, 108–113.
doi:10.1016/j.copbio.2016.06.002.

Ouyang, W., Kümmel, S., Adrian, L., Zhu, Y., Richnow, H.H., 2023.
Chemosphere carbon and hydrogen stable isotope
fractionation of sulfamethoxazole during anaerobic
transformation catalyzed by Desulfovibrio vulgaris
CSIA, ensuring a robust foundation for addressing environ-
mental concerns associated with antibiotic contamination in
wetlands. Finally, the potential and contribution of antibiotic
CSIA in wetlands may align with the Global Health concept,
promoting a holistic approach to addressing health-related
challenges associated with antibiotics in engineered and
nature-based wastewater treatment solutions worldwide.

Box 1: Classic Definitions

**Isotopes** are naturally occurring variations of elements that have different atomic masses due to a different number of neutrons but identical numbers of protons (e.g. ¹³C, ¹²C)

**Stable isotopes** are non-radioactive naturally occurring forms of elements

**ME-CSIA:** Multi-element Compound-specific Isotope Analysis

**Isotope fractionation/ Isotope effect (IE)** is a change in the isotopic composition due to a biological, chemical or physical process

**Delta value (δ)** describes the difference in the isotopic ratio (e.g. ¹³C/¹²C) of a sample relative to an international standard in ‰

**Isotope Fractionation factor (ε)** describes the change in the isotopic composition of a substrate over the course of a reaction

**Dual element isotope analysis** is the slope of changes of δ values of two elements (e.g. C–H) compared to the source, which is represented by Lambda value (Λ).

**Declaration of Competing Interest**

All authors of the article disclose no actual or potential conflict
of interest including any financial, personal or other relation-
ships with other people or organizations within three years
of beginning the submitted work that could inappropriately
influence, or be perceived to influence, their work.

REFERENCES

Achermann, S., Bianco, V., Mansfeldt, C.B., Vogler, B.,
Kolvenbach, B.A., Corvini, P.F.X., et al., 2018. Biotransformation
of sulfonamide antibiotics in activated sludge: the formation
of pterin-conjugates leads to sustained risk. Environ. Sci.
Technol. 52, 6265-6274. doi:10.1021/acs.est.7b06716.

Birkigt, J., Gilevska, T., Ricken, B., Richnow, H.H., Vione, D.,
Corvini, P.F.X., et al., 2015. Carbon stable isotope fractionation
of sulfamethoxazole during biodegradation by
microbacterium sp. strain BR1 and upon direct photolysis.
Environ. Sci. Technol. 49, 6029–6036.
doi:10.1021/acs.est.5b00367.

Blessing, M., Baran, N., 2022. A review on environmental isotope
analysis of aquatic micropollutants: recent advances, pitfalls
and perspectives. TrAC Trend. Anal. Chem. 157, 116730.
doi:10.1016/j.trac.2022.116730.

Boreen, A.L., Arnold, W.A., McNeill, K., 2004. Photochemical fate of
sulfa drugs in then aquatic environment: sulfa drugs
containing five-membered heterocyclic groups. Environ. Sci.
Technol. 38, 3933–3940. doi:10.1021/es0353053.

Chen, J., Yang, Y., Ke, Y., Chen, X., Jiang, X., Chen, C., et al., 2022.
Sulfonamide-metabolizing microorganisms and mechanisms
in antibiotic-contaminated wetland sediments revealed by
stable isotope probing and metagenomics. Environ. Int. 165,
107332. doi:10.1016/j.envint.2022.107332.

Elsner, M., Imfeld, G., 2016. Compound-specific isotope analysis (CSIA) of micropollutants in the environment — Current developments and future challenges. Curr. Opin. Biotechnol. 41, 60–72. doi:10.1016/j.copbio.2016.04.014.

Hernando-Amado, S., Coque, T.M., Baquero, F., Martínez, J.L., 2019.
Defining and combating antibiotic resistance from One Health
and Global Health perspectives. Nat. Microbiol. 4, 1432–1442.
doi:10.1038/s41564-019-0503-9.

He, Y., Zhang, L., Jiang, L., Wagner, T., Sutton, N.B., Ji, R., et al.,
2021. Improving removal of antibiotics in constructed wetland
treatment systems based on key design and operational
parameters: a review. J. Hazard. Mater. 407, 124386.
doi:10.1016/j.jhazmat.2020.124386.

Hofstetter, T.B., Bakkour, R., Buchner, D., Eisenmann, H.,
Fischer, A., Gehere, M., et al., 2024. Perspectives of
compound-specific isotope analysis of organic contaminants
for assessing environmental fate and managing chemical
pollution. Nat. Water 2, 14–30. doi:10.1038/s44221-023-00176-4.

Höhener, P., Guers, D., Malleret, L., Boukaroum, O.,
Martin-Laurent, F., Masbou, J., et al., 2022. Multi-elemental
compound-specific isotope analysis of pesticides for source
identification and monitoring of degradation in soil: a review.
Environ. Chem. Lett. 20, 3927–3942.
doi:10.1007/s10311-022-01489-8.

Horst, A., Renpenning, J., Richnow, H.H., Gehre, M., 2017.
Compound specific stable chlorine isotopic analysis of volatile
aliphatic compounds using gas chromatography hyphenated
with multiple collector inductively coupled plasma mass
spectrometry. Anal. Chem. 89, 9131–9138.
doi:10.1021/acs.analchem.7b01875.

Kim, D.W., Thawng, C.N., Lee, K., Wellington, E.M.H., Cha, C.J.,
2019. A novel sulfonamide resistance mechanism by
two-component flavin-dependent monooxygenase system in
sulfonamide-degrading actinobacteria. Environ. Int. 127,
206–215. doi:10.1016/j.envint.2019.03.046.

Kümmel, S., Horst, A., Gelman, F., Strauss, H., Richnow, H.H.,
Gehre, M., 2020. Simultaneous compound-specific analysis of
δ 33 S and δ 34 S in organic compounds by GC-MC-ICPMS
using medium- and low-mass-resolution modes. Anal. Chem.
doi:10.1021/acs.analchem.0c03253.

Liang, D.hui, Hu, Y., 2019. Simultaneous sulfamethoxazole
biodegradation and nitrogen conversion by *Achromobacter* sp.
JL9 using with different carbon and nitrogen sources.
Bioresour. Technol. 293. doi:10.1016/j.biortech.2019.122061.

Liu, X., Li, W., Kümmel, S., Merbach, I., Sood, U., Gupta, V., et al.,
2021. Soil from a hexachlorocyclohexane contaminated field
site inoculates wheat in a pot experiment to facilitate the
microbial transformation of β-hexachlorocyclohexane
examined by compound-specific isotope analysis. Environ.
Sci. Technol. 55, 13812–13821. doi:10.1021/acs.est.1c03322.

Liu, X., Wu, L., Kümmel, S., Merbach, I., Lal, R., Richnow, H.H.,
2020. Compound-specific isotope analysis and enantiomer
fractionation to characterize the transformation of
hexachlorocyclohexane isomers in a soil-wheat pot system.
Environ. Sci. Technol. 54, 8690–8698.
doi:10.1021/acs.est.9b07609.

Lv, M., Zhang, D., Niu, X., Ma, J., Lin, Z., Fu, M., 2022. Insights into
the fate of antibiotics in constructed wetland systems:
removal performance and mechanisms. J. Environ. Manage.
321, 116028. doi:10.1016/j.jenvman.2022.116028.

Nijenhuis, I., Richnow, H.H., 2016. Stable isotope fractionation concepts for characterizing biotransformation of organohalides. Curr. Opin. Biotechnol. 41, 108–113.
doi:10.1016/j.copbio.2016.06.002.

Ouyang, W., Kümmel, S., Adrian, L., Zhu, Y., Richnow, H.H., 2023.
Chemosphere carbon and hydrogen stable isotope
fractionation of sulfamethoxazole during anaerobic
transformation catalyzed by Desulfovibrio vulgaris
Hildenborough. Chemosphere 311, 136923.

doi:10.1016/j.chemosphere.2022.136923.

Perini, M., Bontempo, L., 2022. Liquid chromatography coupled to isotope ratio mass spectrometry (LC-IRMS): a review. TrAC - Trend. Analyt. Chem. doi:10.1016/j.trac.2021.116515.

Ruan, W., Wang, J., Huang, J., Tai, Y., Wang, R., Zhu, W., et al., 2022.
The in vivo and vitro degradation of sulfonamides in wetland
plants reducing phytotoxicity and environmental pollution.
Environ. Sci. Pollut. Res. 29, 64972-64982.

doi:10.1007/s11356-022-20395-8.

Vogt, C., Dorer, C., Musat, F., Richnow, H., 2016. Multi-element
isotope fractionation concepts to characterize the
biodegradation of hydrocarbons — from enzymes to the
environment. Curr. Opin. Biotechnol. 41, 90–98.

doi:10.1016/j.copbio.2016.04.027.

Willach, S., Lutze, H.V., Eckey, K., Löppenberg, K., Lueling, M.,
Wolbert, B., et al., 2018. Direct photolysis of sulfamethoxazole
using various irradiation sources and wavelength ranges -
insights from degradation product analysis and
compound-specific stable isotope analysis. Environ. Sci.

Technol. doi:10.1021/acs.est.7b04744.

Willach, S., Lutze, H.V., Eckey, K., Löppenberg, K., Lüling, M., Terhalle, J., et al., 2017. Degradation of sulfamethoxazole using ozone and chlorine dioxide - compound-specific stable isotope analysis, transformation product analysis and mechanistic aspects. Water Res. 122, 280-289.

doi:10.1016/j.watres.2017.06.001.

Wu, H., Wang, R., Yan, P., Wu, S., Chen, Z., Zhao, Y., et al., 2023.
Constructed wetlands for pollution control. Nat. Rev. Earth

Environ. 4, 218-234. doi:10.1038/s43017-023-00395-z.
Hildenborough. Chemosphere 311, 136923.

doi:10.1016/j.chemosphere.2022.136923.

Perini, M., Bontempo, L., 2022. Liquid chromatography coupled to isotope ratio mass spectrometry (LC-IRMS): a review. TrAC - Trend. Analyt. Chem. doi:10.1016/j.trac.2021.116515.

Ruan, W., Wang, J., Huang, J., Tai, Y., Wang, R., Zhu, W., et al., 2022.
The in vivo and vitro degradation of sulfonamides in wetland
plants reducing phytotoxicity and environmental pollution.
Environ. Sci. Pollut. Res. 29, 64972-64982.

doi:10.1007/s11356-022-20395-8.

Vogt, C., Dorer, C., Musat, F., Richnow, H., 2016. Multi-element isotope fractionation concepts to characterize the biodegradation of hydrocarbons — from enzymes to the environment. Curr. Opin. Biotechnol. 41, 90–98.

doi:10.1016/j.copbio.2016.04.027.

Willach, S., Lutze, H.V., Eckey, K., Löppenberg, K., Lueling, M.,
Wolbert, B., et al., 2018. Direct photolysis of sulfamethoxazole
using various irradiation sources and wavelength ranges -
insights from degradation product analysis and
compound-specific stable isotope analysis. Environ. Sci.

Technol. doi:10.1021/acs.est.7b04744.

Willach, S., Lutze, H.V., Eckey, K., Löppenberg, K., Lüling, M., Terhalle, J., et al., 2017. Degradation of sulfamethoxazole using ozone and chlorine dioxide - compound-specific stable isotope analysis, transformation product analysis and mechanistic aspects. Water Res. 122, 280-289.

doi:10.1016/j.watres.2017.06.001.

Wu, H., Wang, R., Yan, P., Wu, S., Chen, Z., Zhao, Y., et al., 2023.
Constructed wetlands for pollution control. Nat. Rev. Earth

Environ. 4, 218-234. doi:10.1038/s43017-023-00395-z.