Introduction. The general objective of the ECHIBIOTEB research program (“Innovating tools for sampling, chemical and biological analyses for the diagnostic of wastewater advanced treatments and sludge treatments”, 2011-2014, financed by the French National Research Agency, coordinated by Irstea) is to use innovative and complementary tools to better assess the efficiency of wastewater advanced treatments and sludge treatments. 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 integrative samplers. Through various examples, our presentation will focus on the interest and also the limit of these tools to evaluate the efficiency of wastewater advanced treatments and sludge treatments. The chemical and biological tools tested in the program. -Chemical analysis of targeted contaminants: 14 traces metal and 170 organic molecules belonging to pharmaceuticals, oestrogenic hormones, alkylphenols (AkP), Endocrine Disruptor Compounds (EDC), Polycyclic Aromatic Hydrocarbons (PAH), pesticides, PolyChloroBiphenyl (PCB) and PolyBrominated DiphenylEthers (PBDE) were selected. According to their physico-chemical properties, they are measured in the dissolved phase of water and/or in sludge samples. After extractions by methods such as Solid Phase Extraction or Solid Phase MicroExtraction (SPME) for waters, and Accelerated Solvent Extraction or microwave extraction for sludge, molecules are analysed by gas (GC) or liquid phase chromatography (LC) coupled with single or tandem mass spectrometry. In fine, the aim is to select the relevant compounds to characterize such water and sludge treatment processes. -Non-target chemical analysis: Various instrumental strategies such as GC-2D-MS/TOF (two-dimensional gas phase chromatography coupled to Time Of Flight mass spectrometer), SPME-GC/TOF, HRMS/TOF (High Resolution Mass Spectrometry) are applied to identify new organic contaminants or degradation products. -In vitro and in vivo biological analysis: they are used to link the presence of contaminants to their effects on biological receptors (in vitro assays) or on living organisms (in vivo assays). Receptors studied by in vitro bioassays are estrogen, androgen, glucocorticoid, thyroid and dioxin receptors. Cytotoxicity and genotoxicity are also evaluated. In vivo bioassays are performed either in laboratory on effluents, fresh sludge samples and sludge eluates, or in a modified in situ approach with wastewaters deviated and flow controlled. In vivo laboratory bioassays include aquatic and terrestrial tests: bacteria (acute toxicity, Microtox®), micro-algae (growth), rotifers (reproduction), micro-crustaceans (reproduction) and plants (root elongation and early growth). In vivo modified in situ bioassays are realised on gasteropod (survival, growth, reproduction), crustacean (survival, alimentation rate, reproduction), insect (survival, growth) and fish embryos (survival, hatching, developmental abnormalities). -Integratives samplers: POCIS (Polar Organic Chemical Integrative Sampler) and SPMD (Semi-Permeable Membrane Device), dedicated to water sampling of hydrophilic and hydrophobic organic molecules respectively, are used to increase the samples representativeness1. Their use are combined with target and non target chemical analysis and with in vitro biological tests. -Effect directed analysis (EDA): whose principle is based on a combination of biotesting (by in vitro bioassays), fractionation procedure and chemical analytical methods. The toxic response guide the chemical fractionation and identification of compound responsible for the observed toxic effects. -Interaction of Dissolved Organic Matter (DOM) with micropollutants: the influence of the DOM onto toxicity and bioavailability of contaminants is studied through fast and small microplate tests, based on inhibitive competition of fluorescence. Sampling campaigns. From March 2011 to October 2012, 13 sampling campaigns (10 for water and 3 for sludge) were conducted. Water campaigns were organized either on a short (1 day) or on a long period (1 month). During short campaigns, chemical analysis of targeted contaminants, non-target chemical analysis, in vitro bioassays, in vivo bioassays praticed in laboratory and DOM tests were implemented. During long campaigns, every ECHIBIOTEB tools were used, including integrative samplers and in vivo in situ bioassays. Moreover, global parametres like phosphorus, nitrate, pH and suspended particles were controlled in order to give an indication on the operational conditions of the treatment studied. The treatments used for advanced (or tertiary) water treatments were adsorbants like Granular Activated Carbon (GAC), zeolites or expanded clay, and advanced oxidation processes (AOP) like ozone (O3), ozone peroxide (H2O2), UV/H2O2 , O3 + GAC, and polishing pond ; they were studied in full scale or large scale pilot design, located at the outlet of secondary biological treatments. For sludge treatments, solar dryer, compost and reed-bed filter were tested. First results. - Chemical analysis of targeted contaminants: For most of the compounds and whatever the type of treatment, targeted analysis showed a significant reduction in contaminant concentration in the dissolved phase of water after treatment. Pharmaceuticals concentrations, as example, were reduced in effluents about 100 times lower than in influents. The evaluation and discussion on removal rates are presented in a parallel study (ARMISTIQ research program) and detailed in the abstract “Occurrence of selected micropollutants in treated wastewater and removal with optimised tertiary treatments”. In our presentation, we will discuss on the pertinence of these compounds and more precisely on the selection of a shorter list of targeted compounds to characterize water and sludge treatment processes. -Non-target chemical analysis of 3 different sludge samples allowed to obtain a list of 190 molecules of interest, among which some nitrogen heterocyclic compounds as potential new contaminants. Further investigations are needed, especially combining chemical with biological analysis (EDA), to determine which compounds among these 190 would be the most relevant to be considered for water or sludge treatment characterization. -With in vitro biological analysis, an oestrogenic activity was detected in the aqueous influent of various advanced treatments, whereas it was poorly or not detected in the effluent. Compounds responsible for this toxic activity seem to be efficiently removed by GAC. Moreover, low PAH-like activity was detected in aqueous influent and effluent of wastewater tertiary treament in link to the low PAH concentrations measured in water samples. No dioxin-like or thyroid activity was detected in wastewaters. Genotoxic and cytotoxic activities were detected in some aqueous influents. Genotoxicity was no longer detected in effluents whereas cytotoxic activites are still detected at significant level in the ozone effluent. -With in vivo biological tests, low toxicity was measured in situ and in the laboratory before and after the studied treatment (O3+GAC and O3). Nevertheless, the size of the Japanese Medaka larvae and the hatching efficiency were reduced before those advanced treatments. Regarding the reproduction of Ceriodaphnia dubia, atypical dose-response curves were observed before the O3+GAC treatment showing inhibitory effects at low concentrations and reproduction rates significantly higher than the control at high concentrations. No difference was observed on the sludge toxicity before and after the solar dryer. On the contrary, the composting of sludge reduces the toxicity of the samples in terrestrials tests and tests performed on sludge water extracts, Furthermore, the root elongation test on oat in contact with fresh sludge sample seems suitable for routine analyses because of this simplicity and fastness. -The use of POCIS as integrative samplers allowed us to detect and quantify more -blockers, as an example, than with grab sampling. Nevertheless, the decrease of -blockers concentrations after advanced water treatment is in the same order of magnitude with grab and passive sampling. Compared to grab sampling, POCIS allowed to detect 7 more pharmaceuticals in influent of tertiary treatments and 4 more in effluents. The detection of a greater number of compounds thanks to integrative samplers (i.e. concentration effect) should be helpful to explain some toxicity results.