[Which contribution of wastewater treatment plants in the fight against antimicrobial resistance?] / La filière d'assainissement, quel rôle dans la lutte contre l'antibiorésistance ?

Due to the massive use of antibiotics, antimicrobial resistance (AMR) continues to spread, endangering global disease control and environmental quality. The sources of bacteria or antimicrobial resistance genes are linked to human activities: urban, hospital and industrial discharges, livestock farms). The role of sanitation systems-sewerage, wastewater treatment and sludge treatment (WWTP)-in the problem of AMR has not yet been clearly established by the scientific community. The data available to date show that they eliminate part of the bacteria, genes and antibiotics, although this is not their primary vocation. WWTPs thus play an important filtering role to limit dissemination in the environment. On the other hand, some authors warn against their potential involvement in the selection of new resistant germs, given the conditions conducive to the exchange of genetic material between microbial strains of various types and exposed to selective agents. Today, knowledge of the mechanisms involved in the selection of antibiotic resistance and the fate of bacteria and resistance genes within sanitation systems remains limited. Research is needed to better characterize the contribution of wastewater systems and the performance of wastewater, recycled water, stormwater and sludge treatment processes.

Du fait de l'utilisation massive des antibiotiques, l'antibiorésistance (ABR) ne cesse de se répandre, mettant en danger le contrôle des maladies dans le monde et la qualité de l'environnement. Les sources d'émissions de bactéries ou de gènes de résistance aux antimicrobiens sont liées aux activités humaines : rejets urbains, hospitaliers, industriels, élevages. Le rôle que jouent les systèmes d'assainissement ­ réseau, filières de traitement des eaux usées (STEU) et traitement des boues ­ dans la problématique de l'ABR n'a pas encore été clairement établi par la communauté scientifique. Les données disponibles à ce jour montrent que les STEU éliminent une partie des bactéries, gènes et antibiotiques, bien que ce ne soit pas leur vocation première. Elles jouent ainsi un rôle de filtre important pour limiter la dissémination dans l'environnement. Mais à l'inverse, certains auteurs mettent en garde contre leur implication potentielle dans la sélection de nouveaux germes résistants, compte tenu des conditions propices aux échanges de matériel génétique entre souches microbiennes de natures diverses et exposées à des agents sélectifs. Aujourd'hui, les connaissances sur les mécanismes impliqués dans la sélection de l'ABR et le devenir des bactéries et gènes de résistance au sein des systèmes d'assainissement restent limitées. Des travaux de recherche sont nécessaires pour mieux caractériser leur contribution et évaluer les performances des procédés de traitement des eaux usées, recyclées, pluviales et des boues.

Assessment of flow cytometry for microbial water quality monitoring in cooling tower water and oxidizing biocide treatment efficiency.

The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared. Flow cytometry and plate counts methods showed a significant correlation for changes in concentrations despite a 1 to 2-log difference regarding absolute quantification. Concerning intracellular activity, the use of two different flow cytometers (FACSCanto™ II and Accuri™ C6) showed no statistical difference while a difference was observed between flow cytometry and usual methods (culture and ATP measurement). The standard culture and flow cytometry methods were also compared for in vitro bacteria inactivation measurements in the presence of 3 different types of oxidizing biocides commonly used for cooling tower disinfection. Reductions observed ranged between 1 and 2 log depending on (1) the detection method, (2) the bacterial population origin and/or (3) the active biocide molecule used. In conclusion, flow cytometry represents an efficient, accurate and fast approach to monitor water contamination and biocide treatment efficiency in cooling towers.

Enantiomeric fractionation as a tool for quantitative assessment of biodegradation: The case of metoprolol.

An efficient chiral liquid chromatography high resolution mass spectrometry method has been developed for the determination of metoprolol (MTP) and three of its major metabolites, namely O-desmethylmetoprolol (O-DMTP), α-hydroxymetoprolol (α-HMTP) and metoprolol acid (MTPA) in wastewater treatment plant (WWTP) influents and effluents. The optimized analytical method has been validated with good quality parameters including resolution >1.3 and method quantification limits down to the ng/L range except for MTPA. On the basis of this newly developed analytical method, the stereochemistry of MTP and its metabolites was studied over time in effluent/sediment biotic and sterile microcosms under dark and light conditions and in influents and effluents of 5 different WWTPs. MTP stereoselective degradation was exclusively observed under biotic conditions, confirming the specificity of enantiomeric fraction variations to biodegradation processes. MTP was always biotransformed into MTPA with a (S)-enantiomer enrichment. The results of enantiomeric enrichment pointed the way for a quantitative assessment of in situ biodegradation processes due to a good fit (R(2) > 0.98) of the aerobic MTP biodegradation to the Rayleigh dependency in all the biotic microcosms and in WWTPs because both MTP enantiomers followed the same biodegradation kinetic profiles. These results demonstrate that enantiomeric fractionation constitutes a very interesting quantitative indicator of MTP biodegradation in WWTPs and probably in the environment.

Insights into reductive dechlorination of triclocarban in river sediments: Field measurements and in vitro mechanism investigations.

Triclocarban (TCC) reductive dechlorination was investigated using a combination of field and laboratory experiments. Field monitoring revealed that TCC reductive dechlorination in river sediments leads to formation of two isomers of its lesser chlorinated congener namely 3,4'-dichlorocarbanilide and 4,4'-dichlorocarbanilide. Monochlorocarbanilide was not detected in sediments supporting that transformation of dichlorocarbanilide into monochlorocarbanilide is the rate limiting step of TCC dechlorination. In vitro experiments were conducted to study TCC potential reductive dechlorination mechanisms. These experiments demonstrated that 4,4'-dichlorocarbanilide was specifically formed upon a nucleophilic addition reaction under anaerobic conditions. The 3,4'-diclorocarbanilide was formed upon a two steps radical mechanism under aerobic conditions which includes TCC activation by one electron oxidation followed by a nucleophilic substitution reaction with glutathione. In vitro results suggested that strict anaerobic conditions might not be required for TCC reductive dechlorination in the environment. Moreover, in vitro reactions were performed using biomimetic or enzymatic systems supposing that TCC dechlorination might occur through microbial action in situ. Measured dichlorocarbanilide isomers/triclocarban ratios were used to evaluate the relative significance of both dechlorination pathways and the pathway leading to 3,4'-dichlorocarbanilide was found significant in all investigated river sediment samples.

Health risk assessment related to waterborne pathogens from the river to the tap.

A two-year monitoring program of Cryptosporidium parvum oocysts, Giardia duodenalis cysts, Escherichia coli, Clostridium perfringens spores and adenovirus was conducted in three large rivers in France used for recreational activities and as a resource for drinking water production. Fifty-liter river water and one thousand-liter tap water samples were concentrated using hollow-fiber ultrafiltration and analyzed by molecular biology or laser-scanning cytometry. In order to evaluate watershed land use influence on microorganism concentration changes, occurrence and seasonality of microorganisms were studied. The highest concentrations of protozoan parasites and C. perfringens were found for one of the three sites, showing a high proportion of agricultural territories, forests and semi-natural environments, which may be partly attributable to soil leaching due to rainfall events. On the contrary, the highest concentrations of adenoviruses were found at the two other sites, probably due to strong urban activities. Health risk assessment was evaluated for each waterborne pathogen regarding exposure during recreational activities (for a single or five bathing events during the summer). The calculated risk was lower than 0.5% for parasites and varied from 1% to 42% for adenovirus. A theoretical assessment of microorganism removal during the drinking water treatment process was also performed, and it showed that an absence of microorganisms could be expected in finished drinking water. This hypothesis was confirmed since all tested tap water samples were negative for each studied microorganism, resulting in a risk for drinking water consumption lower than 0.01% for parasites and lower than 0.5% for adenovirus.

Chlorination of bisphenol A: non-targeted screening for the identification of transformation products and assessment of estrogenicity in generated water.

Besides the performance of water treatments on the removal of micropollutants, concern about the generation of potential biologically active transformation products has been growing. Thus, the detection and structural elucidation of micropollutants transformation products have turned out to be major issues to evaluate comprehensively the efficiency of the processes implemented for drinking water treatment. However, most of existing water treatment studies are carried out at the bench scale with high concentrations and simplified conditions and thus do not reflect realistic conditions. Conversely, this study describes a non-targeted profiling approach borrowed from metabolomic science, using liquid chromatography coupled to high-resolution mass spectrometry, in order to reveal potential chlorination products of bisphenol A (BPA) in real water samples spiked at 50µgL(-1). Targeted measurements first evidenced a fast removal of BPA (>99%) by chlorination with sodium hypochlorite (0.8mgL(-1)) within 10min. Then, the developed differential global profiling approach enabled to reveal 21 chlorination products of BPA. Among them, 17 were brominated compounds, described for the first time, demonstrating the potential interest of this innovative methodology applied to environmental sciences. In parallel to the significant removal of BPA, the estrogenic activity of water samples, evaluated by ER-CALUX assay, was found to significantly decrease after 10min of chlorination. These results confirm that chlorination is effective at removing BPA in drinking water and they may indicate that the generated compounds have significantly lower estrogenic activity.

Differential chemical profiling to identify ozonation by-products of estrone-sulfate and first characterization of estrogenicity in generated drinking water.

For a few years, the concern of water treatment companies is not only focused on the removal of target micropollutants but has been extended to the investigation of potential biologically active by-products generated during the treatment processes. Therefore, some methods dedicated to the detection and structural characterization of such by-products have emerged. However, most of these studies are usually carried out under simplified conditions (e.g. high concentration levels of micropollutants, drastic treatment conditions, use of deionized or ultrapure water) and somewhat unrealistic conditions compared to that implemented in water treatment plants. In the present study, a real field water sample was fortified at the part-per-billion level (50 µg L(-1)) with estrone-3-sulfate (E1-3S) before being ozonated (at 1 mg L(-1)) for 10 min. In a first step, targeted measurements evidenced a degradation of the parent compound (>80%) in 10 min. Secondly, a non-targeted chemical profiling approach derived from metabolomic profiling studies allowed to reveal 11 ozonation by-products, among which 4 were found predominant. The estrogenic activity of these water samples spiked with E1-3S before and after treatment was assessed by the ER-CALUX assay and was found to decrease significantly after 10 min of ozonation. Therefore, this innovative methodological strategy demonstrated its suitability and relevancy for revealing unknown compounds generated from water treatment, and permitted to generate new results regarding specifically the impact of ozonation on estrone-3-sulfate. These results confirm that ozonation is effective at removing E1-3S in drinking water and indicate that the by-products generated have significantly lower estrogenic activity.

GC-MS(n) and LC-MS/MS couplings for the identification of degradation products resulting from the ozonation treatment of Acetochlor.

The degradation of the chloracetamide herbicide acetochlor has been studied under simulated ozonation treatment plant conditions. The degradation of acetochlor included the formation of several degradation products that were identified using GC/ion-trap mass spectrometry with EI and CI and HPLC/electrospray-QqTOF mass spectrometry. Thirteen ozonation products of acetochlor have been identified. Ozonation of the deuterated herbicide combined to MS(n) and high-resolution mass measurement allowed effective characterization of the degradation products. At the exception of one of them, the product B (2-chloro-2', ethyl-6', methyl-acetanilide), none of the identified degradation products has been already reported in the literature. Post-ozonation kinetics studies revealed that the concentrations of most degradation products evolved noticeably with time, particularly during the first hours following the ozonation treatment. This raises concerns about the fate of degradation products in the effluents of treatment plants and suggests the need for a better control on these products if their toxicity was demonstrated.

Differential global profiling as a new analytical strategy for revealing micropollutant treatment by-products: application to ethinylestradiol and chlorination water treatment.

The detection and structural elucidation of micropollutants treatment by-products are major issues to estimate efficiencies of the processes employed for drinking water production versus endocrine disruptive compounds contamination. This issue was mainly investigated at the laboratory scale and in high concentration conditions. However, potential by-products generated after chlorination can be influenced by the dilution factor employed in real conditions. The present study proposes a new methodology borrowed to the metabolomic science, using liquid chromatography coupled to high-resolution mass spectrometry, in order to reveal potential chlorination by-products of ethinylestradiol in spiked real water samples at the part-per-billion level (5 µg L(-1)). Conventional targeted measurements first demonstrated that chlorination with sodium hypochlorite (0.8 mg L(-1)) led to removals of ethinylestradiol over 97%. Then, the developed differential global profiling approach permitted to reveal eight chlorination by-products of EE2, six of them being described for the first time. Among these eight halogenated compounds, five have been structurally identified, demonstrating the potential capabilities of this new methodology applied to environmental samples.

Investigation of the dissociation pathways of metolachlor, acetochlor and alachlor under electron ionization - application to the identification of ozonation products.

With the future aim of using gas chromatography coupled with mass spectrometry to characterize the transformation products of ozonated herbicides: metolachlor, acetochlor and alachlor, an interpretation of their electron ionization mass spectra is presented. Fragmentation mechanisms are proposed on the basis of isotopic labelling and multiple-stage mass spectrometry experiments carried out on an ion trap mass spectrometer. We also give examples in order to demonstrate how the elucidation of such fragmentation mechanisms for herbicides may simplify the characterization of their ozonation products.

Analysis of steroid hormones in effluents of wastewater treatment plants by liquid chromatography-tandem mass spectrometry.

This paper presents the development of an analytical procedure for the determination of two sexual steroid hormones: 17beta-estradiol and estrone, and the synthetic contraceptive estrogen, 17alpha-ethynylestradiol in effluents of wastewater treatment plants. Samples are extracted via solid-phase extraction using C18 cartridges. Extracts in ethyl acetate are then purified with a liquid-liquid separation with aqueous sodium chloride, then with a clean-up on a Florisil cartridge. Steroids are analyzed using an LC-MS-MS ion trap system. Internal quantification with the corresponding deuterated steroids leads to limits of quantification at 5 ng/l for estrone and 10 ng/l for estradiol and ethynylestradiol. In mineral spiked water, recoveries are 91% for 17beta-estradiol, 97% for estrone and 87% for 17alpha-ethynylestradiol and RSDs are 15% for 17beta-estradiol, 11% for estrone and 23% for 17alpha-ethynylestradiol.