Développement d'une méthode d'extraction en phase solide et chromatographie liquide-spectrométrie de masse en tandem pour l'analyse des antibiotiques dans le fumier

International audiencePharmaceuticals are now well known micro-pollutants of aquatic systems. They are studied due to sanitary and environmental risks they could represent and especially the antibiotics because of the antibiotic resistance phenomenon. We have focused our research on that group of compounds. Antibiotics mainly enter into the environment either via sewage treatment plant (SWTP) effluent (human origin) or via runoff of land fertilized with sludge from SWTP or manure from livestock (veterinary origin). We have worked on that second origin and studied 2 different types of process before the manure spreading: i) the simple storage of the manure during few months in huge tank ; ii) the treatment process with solid/liquid separation system and 2 different ways of treatment for each phase. Whatever the origin of the contamination, it appears to be a chronic contamination and it is necessary to quantify compounds at trace level (ng/L). For this purpose, we have developed a method for the simultaneous quantification of 23 antibiotics belonging to 9 families. The first step is an extraction of molecules by SPE on Oasis HLB cartridges and the second one is the analysis by Rapid Resolution Liquid Chromatography-tandem mass spectrometry (RRLC/MS/MS) with a 5 cm C18 column and an electrospray ionisation source. The preliminary results show an important contamination of stored pig manure by lincomyin, tetracycline, oxytetracycline and sulfadiazine. The results are discussed according to various ways of manure treatment and uses

Evidence for a Complex Relationship between Antibiotics and Antibiotic-Resistant Escherichia Coli: From Medical Center Patients to a Receiving Environment

International audienceThe aim of this study was to investigate the relationship between antibiotics and antibiotic-resistant fecal bacteria (E. coli) in water along a medical center−wastewater treatment plant−river continuum (4 km). A multiresidue chemical analysis methodology, using solid phase extraction coupled with liquid chromatography tandem mass spectrometry, was performed to detect whether low levels of contamination by 34 antibiotics were related to antibiotic resistance of E. coli and antibiotic use. The contamination of water by antibiotics and antibiotic-resistant E. coli decreased along the continuum. Although amoxicillin was predominantly prescribed, only ofloxacin (1 ng*L−1) and sulfamethoxazole (4 ng*L−1) persisted in the river. At the retirement home, in the medical center, even though no tetracycline and sulfamethoxazole were consumed, the highest occurrences of antibiotic resistance were in classes of quinolones (42.0%), sulfonamides (24.0%), tetracyclines (38.0%), and penicillins (38.0%), mainly due to the presenceof multiple antibiotic-resistance genes on class 1 integrons. Along the continuum, the occurrence of E. coli resistant to antibiotics and those carrying class 1 integrons decreased in water samples (p-value <0.001). Interestingly, in the river, only persistent antibiotic compounds (ofloxacin, sulfamethoxazole) were found, but they did not correspond to the major resistances (tetracycline, amoxicillin) of E. coli

Utilisation d'échantillonneurs passifs (POCIS et SPMD) pour évaluer l'efficacité des traitements tertiaires des eaux usées

International audienceOur objective is to study some advanced processes, such as ozonation (O3) or Granular Activated Carbon (GAC) as example, used in tertiary stage of wastewater treatment plants (WWTP). The challenges when studying such processes is to be able to measure organic micropollutants at very low concentrations (ng/L). To achieve this goal, passive samplers (PS) like POCIS (Polar Organic Chemical Integrative Sampler) and SPMD (Semi-Permeable Membrane Device) were used. The results of chemical analyses on PS were compared with those on grab water samples. Four one-month-long sampling campaigns were carried out in 3 different WWTP: 1) O3 (full-scale) followed by GAC (pilot) in WWTP A, 2) O3 only (full-scale) in the same WWTP A, 3) GAC (pilot) in WWTP B, 4) polishing pond (full-scale) in WWTP C. In all campaigns, POCIS were exposed in water during 14 days and SPMD during 28 days. To be able to compare these 4 campaigns and the molecule concentrations upstream and downstream the tertiary treatment, POCIS and SPMD were always immersed in an aquarium in the same controlled experimental conditions of flow (280 mL/min), temperature (20° C) and light. To insure quality of data, POCIS and SPMD were exposed in triplicate and field blanks were realized (i.e. PS exposed to the ambient air). In parallel, 2h composite water samples were collected at D0, D14 and D28. Targeted chemical analyses were performed on POCIS extracts for 117 hydrophilic compounds belonging to pharmaceutical, hormone, pesticide and alkylphenol classes and on SPMD extracts for 46 lipophilic compounds belonging to chlorine pesticides, Polycyclic Aromatic Hydrocarbon, PolyChloroBiphenyl, PolyBrominated DiphenylEthers classes. Hydrophilic compounds and PAH were also analysed in water samples. We will present results obtained for SPMD and POCIS. As an example, compared to grab sampling, POCIS allowed detecting 7 more pharmaceuticals in influent of tertiary treatments and 4 more in effluents in the 2 first sampling campaigns. Nevertheless, information on process efficiency is the same since the decrease of pharmaceutical concentrations after advanced water treatment is in the same order of magnitude with grab and passive sampling in the 2 first sampling campaigns. Hence, POCIS appears as an interesting tool to characterize tertiary stages of WWTPs with analysis of organic micropollutants at very low concentrations

Étude de l'efficacité des traitements avancés des eaux usées et du traitement des boues de stations d'épuration au travers d'analyses de 168 molécules ciblées et d'analyses de molécules non-ciblées

International audienceThe general objective of the ECHIBIOTEB research program (“Innovating tools for sampling, chemical and biological analyses for the diagnosis of wastewater advanced tertiary treatments and sludge treatments”, 2011-2014, financed by the French National Research Agency, coordinated by Irstea) is to use innovative and complementary tools to characterize the efficiency of water advanced treatments and sludge treatments in wastewater treatment plant. To achieve this goal, various strategies were combined: chemical analysis of a large panel of selected priority and emerging contaminants, non-target chemical analysis, in vitro and in vivo biological analysis and innovating sampling tools such as integrative samplers (e.g. POCIS (Polar Organic Chemical Integrative Sampler) and SPMD (Semi-Permeable Membrane Device)). Our presentation will focus on results on the chemical analyses of selected molecules and non-target analysis. Until today, 11 sampling campaigns, 8 for water and 3 for sludge, have been conducted. Among the potential treatments used for advanced (or tertiary) water treatments, ozone (O3), Granular Activated Carbon (GAC), O3 + GAC, ozone peroxide (H2O2) and UV/H2O2 were studied in full scale or large scale pilot design, located on the outlet of secondary biological treatments. For sludge treatments, solar dryer, compost and reed-bed filter were tested. A total of 138 contaminants belonging to pharmaceuticals, oestrogenic hormones, alkylphenols (AkP), Endocrine Disruptor Compounds (EDC), Polycyclic Aromatic Hydrocarbons (PAH) and pesticides were measured in the dissolved phase of water samples; and 113 contaminants, belonging to pharmaceuticals, oestrogenic hormones, AkP, EDC, organochlorine pesticides, PAH, PCB (PolyChloroBiphenyl) and PBDE (PolyBrominated DiphenylEthers) were measured in sludge samples. After extraction by adapted methods such as Liquid-Liquid Extraction, Solid Phase Extraction or Solid Phase MicroExtraction for waters, and Accelerated Solvent Extraction or microwave extraction for sludge, molecules were analysed by gas (GC) or liquid phase chromatography coupled with single or tandem mass spectrometry. Non-target analysis were realised with two-dimensional GC coupled to Time Of Flight mass spectrometer. For most of the compounds, targeted analysis showed a significant reduction in contaminant concentration in the dissolved phase of water after all tested treatments. Indeed, pharmaceuticals and AkP were efficiently eliminated with concentrations in effluents respectively about 100 and 10 times lower than in influents. In contrast, aminomethylphosphonic acid (AMPA) and glyphosate were not eliminated at all from secondary wastewaters. Many of the selected compounds are refractory to conventional secondary treatments; nonetheless, we demonstrated that they can be efficiently removed thanks to selected advanced tertiary treatments. In the presentation, we will discuss which treatment seems to be the most efficient to eliminate the selected compounds. Depending on the process and the contaminant class, concentrations in raw and treated sludge varied widely. Non-target analyses of 3 different sludge samples allowed to obtain a list of 190 molecules of interest, among which some nitrogen heterocyclic compounds as potential emerging contaminants. Further investigations are needed, especially combining chemical with biological analysis, to determine which compounds among these 190 would be the most relevant to be considered in environmental studies

Utilisation des échantillonneurs intégratifs pour évaluer l'efficacité des traitements avancés des eaux usées : cas des molécules pharmaceutiques

International audienceIntroduction. The general objective of the ECHIBIOTEB research program (“Innovating tools for sampling, chemical and biological analyses for the diagnosis of wastewater advanced tertiary treatments and sludge treatments”, 2011-2014, financed by the French National Research Agency) is introduced in a previous presentation. Briefly it consists in developing new tools for the determination of the efficiency of advanced treatments, especially for the sampling. This presentation will describe the potential of Polar Organic Chemical Integrative Sampler (POCIS) for the monitoring of pharmaceuticals in water, compared to grab sampling. Analytical methodology. Until today, two campaigns were performed in order to follow the efficiency of ozone (O3) treatment, alone and coupled with Granular Activated Carbon (GAC) by exposing 20 POCIS before and after the wastewater treatment during 28 days. Each triplicate, associated to a field blank, is dedicated to a kind of analysis (pharmaceuticals, alkylphenols, and biological analysis, for example). In parallel, grab samplings were achieved after 0, 14 and 28 days of POCIS exposition in order to compare these two approaches. 104 molecules belonging to several therapeutic classes such as antibiotics, -blockers, antineoplastics, antivirals, phosphodiesterase type 5 inhibitor (PDE 5), analgesics, broncholidators, non-steroidal anti-inflammatory and psychiatric drugs, lipid regulators and stimulants, were analysed. Therefore, in order to determine the in-situ rate constants of exposed POCIS, 3 Reference Performance Compounds (PRC) were previously added in the adsorbant phase: salbutamol-d3, caffeine-13C and desisopropylatrazin-d5 (DIA d5) and laboratory calibration on pharmaceuticals were performed. Solid Phase Extractions (SPE) were carried out on the different samples depending on compound classes and, then, analyses were performed by liquid phase chromatography coupled with tandem mass spectrometry (LC-MS/MS). Results. Among targeted compounds, 39 molecules were not detected in any samples of wastewater, in the dissolved phase and 32 molecules in any POCIS samplers. As illustrated in Figure 1a, concentrations upstream from the treatments are higher than those downstream; whatever is the nature of wastewater process. Betablockers, macrolides, psychiatric drugs, fluoroquinolones and quinolones are the most abundant compounds upstream these processes and downstream, only two classes (Betablockers and stimulants) remain present as relatively high concentrations. Therefore, it seems that GAC and GAC+O3 treatments involve a drastic reduction of micropollutant concentrations in wastewaters. Regarding concentrations determined in the adsorbant phase of passive samplers (Figure 1b), the tendency between upstream and downstream coincides well with the one of wastewater samples, in terms of pharmaceutical abundances and between samples too. Figure 2 shows that the number of detected targeted compounds depends on the place of sampling (up or downstream) and on the way of sampling (POCIS vs. grab sampling). For the process using granular activated carbon, based on the measurements of 103 molecules, 13 extra compounds are detected in the adsorbant phase of POCIS compared to the dissolved phase, upstream and downstream the process. Until today, regarding the analysis of 93 molecules for ozone+GAC process, the trend is also observed with the detection of 23 and 17 extra compounds, upstream and downstream respectively. These results show the real interest of this sampling mode by the improvement of methodology sensitivity. Laboratory calibration of passive samplers allow to determine rate constants for more than 25 of target compounds and so, to compare directly the results between integrative and grab sampling in terms of concentrations. This methodology of sampling reveals its interest for the quantification of targeted compounds and has been also applied to the analysis of non-targeted compounds in order to complete the knowledge on the efficiency of such process. The presence of pharmaceuticals and non-targeted compounds could explain the potential toxicity of these samples (wastewater and POCIS extracts). Acknowledgements. The authors wish to thank the program ECOTECH-ECHIBIOTEB, the Aquitaine Region and the European Union (CPER A2E project) for financial support. Europe is moving in Aquitaine with the European Regional Development Fund

Étude de l'efficacité des traitements avancés des eaux usées et du traitement des boues de stations d'épuration au travers d'analyses de 168 molécules ciblées et d'analyses de molécules non-ciblées

International audienceThe general objective of the ECHIBIOTEB research program (“Innovating tools for sampling, chemical and biological analyses for the diagnosis of wastewater advanced tertiary treatments and sludge treatments”, 2011-2014, financed by the French National Research Agency, coordinated by Irstea) is to use innovative and complementary tools to characterize the efficiency of water advanced treatments and sludge treatments in wastewater treatment plant. To achieve this goal, various strategies were combined: chemical analysis of a large panel of selected priority and emerging contaminants, non-target chemical analysis, in vitro and in vivo biological analysis and innovating sampling tools such as integrative samplers (e.g. POCIS (Polar Organic Chemical Integrative Sampler) and SPMD (Semi-Permeable Membrane Device)). Our presentation will focus on results on the chemical analyses of selected molecules and non-target analysis. Until today, 11 sampling campaigns, 8 for water and 3 for sludge, have been conducted. Among the potential treatments used for advanced (or tertiary) water treatments, ozone (O3), Granular Activated Carbon (GAC), O3 + GAC, ozone peroxide (H2O2) and UV/H2O2 were studied in full scale or large scale pilot design, located on the outlet of secondary biological treatments. For sludge treatments, solar dryer, compost and reed-bed filter were tested. A total of 138 contaminants belonging to pharmaceuticals, oestrogenic hormones, alkylphenols (AkP), Endocrine Disruptor Compounds (EDC), Polycyclic Aromatic Hydrocarbons (PAH) and pesticides were measured in the dissolved phase of water samples; and 113 contaminants, belonging to pharmaceuticals, oestrogenic hormones, AkP, EDC, organochlorine pesticides, PAH, PCB (PolyChloroBiphenyl) and PBDE (PolyBrominated DiphenylEthers) were measured in sludge samples. After extraction by adapted methods such as Liquid-Liquid Extraction, Solid Phase Extraction or Solid Phase MicroExtraction for waters, and Accelerated Solvent Extraction or microwave extraction for sludge, molecules were analysed by gas (GC) or liquid phase chromatography coupled with single or tandem mass spectrometry. Non-target analysis were realised with two-dimensional GC coupled to Time Of Flight mass spectrometer. For most of the compounds, targeted analysis showed a significant reduction in contaminant concentration in the dissolved phase of water after all tested treatments. Indeed, pharmaceuticals and AkP were efficiently eliminated with concentrations in effluents respectively about 100 and 10 times lower than in influents. In contrast, aminomethylphosphonic acid (AMPA) and glyphosate were not eliminated at all from secondary wastewaters. Many of the selected compounds are refractory to conventional secondary treatments; nonetheless, we demonstrated that they can be efficiently removed thanks to selected advanced tertiary treatments. In the presentation, we will discuss which treatment seems to be the most efficient to eliminate the selected compounds. Depending on the process and the contaminant class, concentrations in raw and treated sludge varied widely. Non-target analyses of 3 different sludge samples allowed to obtain a list of 190 molecules of interest, among which some nitrogen heterocyclic compounds as potential emerging contaminants. Further investigations are needed, especially combining chemical with biological analysis, to determine which compounds among these 190 would be the most relevant to be considered in environmental studies