A comprehensive dataset for assessing the impact of ammonium salts and zeolite on anaerobic digestion performance, microbial dynamics, and metabolomic profiles.

This article presents comprehensive data derived from lab-scale batch anaerobic digesters that were subjected to inhibition by various sources of ammonia. To counter this inhibition, zeolite was introduced into selected digesters. The provided dataset offers a detailed depiction of degradation performance dynamics over time, as well as insights into both microbial and metabolic changes during the inhibition. In detail, 10 conditions were tested in triplicate. In a first series of 15 bioreactors ammonia was introduced to achieve a TAN concentration of 8 g/L, utilizing NH3 solution, NH4Cl salt, (NH4)2CO3 salt, or (NH4)2PO4 salt as inhibitors. A control condition without ammonia was also set up. A second series of 15 bioreactors was set up exactly as the first one, with the addition of zeolite at a concentration of 15 g/L. The data provided includes information on operational conditions, degradation performance measurements throughout the entire process (using biogas production and composition, dissolved organic and inorganic carbon, volatile fatty acids, pH, free and total ammonia nitrogen, apparent isotopic fractionation of biogas as indicators), microbial community analysis using 16S rRNA gene sequencing (50 samples analysed), and metabolomic analysis through liquid chromatography-mass spectrometry (LC-MS) (108 samples analysed). Sequencing data were generated by using IonTorrent PGM sequencer. The sequencing data have been deposited with links to project PRJEB52324, in ENA database from EBI (https://www.ebi.ac.uk/ena/browser/view/PRJEB52324). Sample accession numbers go from SAMEA14277573 to SAMEA14277621. The metabolomic data were generated using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, MA, US). The metabolomic data have been deposited to the EMBL-EBI MetaboLights database with the identifier MTBLS7859 (https://www.ebi.ac.uk/metabolights/MTBLS7859). This data can be used as a source for comparisons with other studies focusing on the inhibition of anaerobic digestion by ammonia, particularly in the context of exploring microbial or metabolomic dynamics during inhibition. Additionally it provides a multi-omic dataset (metataxonomic and metabolomic) with detailed associated metadata describing anaerobic digesters. The dataset is directly is associated to the research article titled "Inhibition of anaerobic digestion by various ammonia sources resulted in subtle differences in metabolite dynamics." [1].

Generic role of zeolite in enhancing anaerobic digestion and mitigating diverse inhibitions: Insights from degradation performance and microbial characteristics.

Zeolite was shown to mitigate anaerobic digestion (AD) inhibition caused by several inhibitors such as long-chain fatty acids, ammonia, and phenolic compounds. In this paper, we verified the genericity of zeolite's mitigating effect against other types of inhibitors found in AD such as salts, antibiotics, and pesticides. The impacts of inhibitors and zeolite were assessed on AD performance and microbial dynamics. While sodium chloride and erythromycin reduced methane production rates by 34% and 32%, zeolite mitigated the inhibition and increased methane production rates by 72% and 75%, respectively, compared to conditions without zeolite in the presence of these two inhibitors. Noticeably, zeolite also enhanced methane production rate by 51% in the uninhibited control condition. Microbial community structure was analyzed at two representative dates corresponding to the hydrolysis/fermentation and methanogenesis stages through 16S rRNA gene sequencing. The microbial characteristics were further evidenced with common components analysis. Results revealed that sodium chloride and erythromycin inhibited AD by targeting distinct microbial populations, with more pronounced inhibitory effects during hydrolysis and VFAs degradation phases, respectively. Zeolite exhibited a generic effect on microbial populations in different degradation stages across all experimental conditions, ultimately contributing to the enhanced AD performance and mitigation of different inhibitions. Typically, hydrolytic and fermentative bacteria such as Cellulosilyticum, Sedimentibacter, and Clostridium sensu stricto 17, VFAs degraders such as Mesotoga, Syntrophomonas, and Syntrophobacter, and methanogens including Methanobacterium, Methanoculleus, and Methanosarcina were strongly favored by the presence of zeolite. These findings highlighted the promising use of zeolite in AD processes for inhibition mitigation in general.

Inhibition of anaerobic digestion by various ammonia sources resulted in subtle differences in metabolite dynamics.

The impact of ammonia on anaerobic digestion performance and microbial dynamics has been extensively studied, but the concurrent effect of anions brought by ammonium salt should not be neglected. This paper studied this effect using metabolomics and a time-course statistical framework. Metabolomics provides novel perspectives to study microbial processes and facilitates a more profound understanding at the metabolic level. The advanced statistical framework enables deciphering the complexity of large metabolomics data sets. More specifically, a series of lab-scale batch reactors were set up with different ammonia sources added. Samples of nine time points over the degradation were analyzed with liquid chromatography-mass spectrometry. A filtering procedure was applied to select the promising metabolomic peaks from 1262 peaks, followed by modeling their intensities across time. The metabolomic peaks with similar time profiles were clustered, evidencing the correlation of different biological processes. Differential analysis was performed to seek the differences in metabolite dynamics caused by different anions. Finally, tandem mass spectrometry and metabolite annotation provided further information on the molecular structure and possible metabolic pathways. For example, the consumption of 5-aminovaleric acid, a short-chain fatty acid obtained from l-lysine degradation, was slowed down by phosphates. Overall, by investigating the effect of anions on anaerobic digestion, our study demonstrated the effectiveness of metabolomics in providing detailed information in a set of samples from different experimental conditions. With the statistical framework, the approach enables capturing subtle differences in metabolite dynamics between samples while accounting for the differences caused by time variations.

Monitoring of cellulose-rich biowaste co-digestion with 3D fluorescence spectroscopy and mass spectrometry-based metabolomics.

Anaerobic digestion (AD) is a promising waste management strategy that reduces landfilling while generating biogas. Anaerobic co-digestion involves mixing two or more substrates to enhance the nutrient balance required for microorganism growth and thus improve the degradation. Monitoring AD is crucial for comprehending the biological process, optimizing process stability, and achieving efficient biogas production. In this work, we have used three dimensional excitation emission fluorescence spectroscopy and mass spectrometry metabolomics, two complementary techniques, to monitor the anaerobic co-digestion (AcoD) of cellulose, ash wood or oak wood with food waste. The two approaches were compared together and to the biogas production records. Results of this experiment demonstrated the complementarity of both analytical techniques with the measurement of the biogas production since 3D fluorescence spectroscopy and MS metabolomics revealed the earlier molecular changes occurring in the bioreactors, mainly associated with the hydrolysis step, whereas the biogas production data reflected the biological activity in the last step of the digestion. Moreover, in all cases, the three data sets effectively delineated the differences among the substrates. While the two wood substrates were poorly degradable as they were richer in aromatic compounds, cellulose was highly degradable and was characterized by the production of several glycolipids. Then, the three tested AcoDs resulted in a similar 3D EEM fluorescence and metabolomics profiles, close to the one observed for the AD of food waste alone, indicating that the incorporation of the food waste drove the molecular degradation events in the AcoDs. Substrate-specific differences were appreciated from the biogas production data. The overall results of this research are expected to provide insight into the design of guidelines for monitoring AcoD.

Screening potential toxicity of currently used herbicides in the freshwater amphipod Gammarus fossarum based on multi-level biomarker responses to field-realistic exposures.

Herbicides are widely used to control weeds and maximize crop growth. Because of agricultural runoff, these chemicals are potentially hazardous to aquatic wildlife. However, their ecotoxicity and resulting disturbance in individual performance remain scarcely documented in freshwater crustaceans. This study aimed to screen the potential toxicity of currently used herbicides in the ecosystem engineer Gammarus fossarum using multi-level biomarkers. In microcosms, gammarids were exposed for 72 h to 12 herbicides individually (quinmerac, mesotrione, bentazone, isoproturon, chlortoluron, metazachlor, chloridazone, diflufenican, flufenacet, aclonifen, prosulfocarb and metolachlor) at a field-realistic concentration (i.e. 10 µg/L). The sublethal effects were assessed by monitoring several biochemical, physiological and behavioural traits. In exposed gammarids, alterations in behavioural activities were observed, i.e. increased locomotion and respiration as a general trend. Moreover, biochemical biomarkers suggested herbicide-dependent disruptions in moulting, antioxidant responses and cell integrity. Integrating multi-metric variations through statistical analyses allowed us to identify herbicide clusters likely to trigger common sets of biological responses. Depressed antioxidant defence at the cell level and impaired respiration at the individual level were the predominant toxic effects of herbicides, related to their hydrophobic feature. Furthermore, establishing relationships between sublethal alterations in gammarids and acute lethality or chronic toxicity values defined for regulatory purposes supports the relevance of these alterations as early warnings of toxicity. Our findings demonstrate that currently used herbicides have unexpected toxicological effects in a non-target wild animal, with possible long-term alterations in population dynamics and associated ecological functions, which constitute promising diagnostic tools for risk assessment in agricultural areas.

A longitudinal study of the effect of temperature modification in full-scale anaerobic digesters - dataset combining 16S rDNA gene sequencing, metagenomics, and metabolomics data.

Data in this article provides detailed information on the microbial dynamics and degradation performances in two full-scale anaerobic digesters operated in parallel for 476 days. One of them was kept at 35 °C for the whole experiment, while the other was submitted to sub-mesophilic (25 °C) conditions between days 123 and 373. Sludge samples were collected from both digesters at days 0, 80, 177, 218, 281, 353, and 462. The provided data include the operational conditions of the digesters and the characterization of the sludge samples at the physicochemical level, indicative of the digesters' degradation performance. It also includes the characterization of the sludge samples at the multiomics level (16S rRNA gene sequencing, metagenomics, and metabolomics profiling), to decipher the changes in the microbial structure and molecular activity. The 16S rDNA gene sequencing, metagenomics, and metabolomics data were generated using an IonTorrent PGM sequencer, an Illumina NextSeq 500 sequencer, and LTQ-Orbitrap XL mass spectrometer respectively. The 16S rDNA gene raw data and the metagenomics data have been deposited in the BioProject PRJEB49115, in the ENA database (https://www.ebi.ac.uk/ena/browser/view/PRJEB49115). The metabolomics data has been deposited at the Metabolomics Workbench, with study id ST002004 (DOI: 10.21228/M8JM6B). The data can be used as a source for comparisons with other studies working with data from full-scale anaerobic digesters, especially for those investigating the effect of the temperature modification. The data is associated with the research article "Metataxonomics, metagenomics, and metabolomics analysis of the influence of temperature modification in full-scale anaerobic digesters" (Puig-Castellví et al [1]).

Metataxonomics, metagenomics and metabolomics analysis of the influence of temperature modification in full-scale anaerobic digesters.

Full-scale anaerobic digesters' performance is regulated by modifying their operational conditions, but little is known about how these modifications affect their microbiome. In this work, we monitored two originally mesophilic (35 °C) full-scale anaerobic digesters during 476 days. One digester was submitted to sub-mesophilic (25 °C) conditions between days 123 and 373. We characterized the effect of temperature modification using a multi-omics (metataxonomics, metagenomics, and metabolomics) approach. The metataxonomics and metagenomics results revealed that the lower temperature allowed a substantial increase of the sub-dominant bacterial population, destabilizing the microbial community equilibrium and reducing the biogas production. After restoring the initial mesophilic temperature, the bacterial community manifested resilience in terms of microbial structure and functional activity. The metabolomic signature of the sub-mesophilic acclimation was characterized by a rise of amino acids and short peptides, suggesting a protein degradation activity not directed towards biogas production.

Gradual development of ammonia-induced syntrophic acetate-oxidizing activities under mesophilic and thermophilic conditions quantitatively tracked using multiple isotopic approaches.

Insights into microbiota adaptation to increased ammonia stress, and identification of indicator microorganisms can help to optimize the operation of anaerobic digesters. To identify microbial indicators and investigate their metabolic contribution to acetoclastic methanogenesis (AM), syntrophic acetate oxidation (SAO) or hydrogenotrophic methanogenesis (HM), 40 anaerobic batch reactors fed with acetate of 110 mmol/L were set up at NH4+-N concentrations of 0.14 g/L, 5.00 g/L or 7.00 g/L, inoculated with thermophilic or mesophilic microbiota with or without pre-exposure to ammonia stress. Four stable carbon isotope probing approaches were applied in parallel, with [1,2-13C]-CH3COOH, [2-13C]-CH3COOH, [13C]NaHCO3 or non-labeled CH3COOH used individually. The last three approaches were used to quantify the methanogenic pathways by tracking labeled 13C or natural 13C signatures in the resulting CH4 and CO2, and consistently detected the dynamic transition of dominant pathways from AM to SAO-HM under ammonia stress. Results of quantitative PCR and fluorescence in-situ hybridization illustrated the procedure, acetotrophic methanogens being outcompeted by acetate-oxidizing syntrophs. The first and last isotope-labeling approaches were designed to probe the active acetate-mineralizing microbes with DNA-SIP. Known acetate-oxidizing bacteria like Syntrophaceticus and Tepidanaerobacter, as well as novel members of Pseudomonas, Bacillus and Symbiobacteraceae were detected, with Methanoculleus as the predominant H2/CO2-utilizing partner. Using NanoSIMS, some bacterial cells were observed to be fixing CO2 from [13C]NaHCO3. In this study, Methanosaeta was only active with ammonia < 200 mg-N/L; the syntrophs catalyzing SAO-HM started to compete with AM-conducting Methanosarcina at intermediate concentrations of ammonia, i.e. 200-500 mg-N/L, and outcompeted the acetotrophic methanogens with ammonia > 500 mg-N/L. Under ammonia stress, diverse known and novel microbial taxa were involved in acetate mineralization, comparable with those identified in previous studies.

Time-course analysis of metabolomic and microbial responses in anaerobic digesters exposed to ammonia.

Omics longitudinal studies are effective experimental designs to inform on the stability and dynamics of microbial communities in response to perturbations, but time-course analytical frameworks are required to fully exploit the temporal information acquired in this context. In this study we investigate the influence of ammonia on the stability of anaerobic digestion (AD) microbiome with a new statistical framework. Ammonia can severely reduce AD performance. Understanding how it affects microbial communities development and the degradation progress is a key operational issue to propose more stable processes. Thirty batch digesters were set-up with different levels of ammonia. Microbial community structure and metabolomic profiles were monitored with 16 S-metabarcoding and GCMS (gas-chromatography-mass-spectrometry). Digesters were first grouped according to similar degradation performances. Within each group, time profiles of OTUs and metabolites were modelled, then clustered into similar time trajectories, evidencing for example a syntrophic interaction between Syntrophomonas and Methanoculleus that was maintained up to 387 mg FAN/L. Metabolites resulting from organic matter fermentation, such as dehydroabietic or phytanic acid, decreased with increasing ammonia levels. Our analytical framework enabled to fully account for time variability and integrate this parameter in data analysis.

Single and combined effects of insecticides on multi-level biomarkers in the non-target amphipod Gammarus fossarum exposed to environmentally realistic levels.

Aquatic media are ultimate recipients of various contaminants including pesticides pervasively applied in agrosystems. Characterizing the ecotoxicity of pesticides and their mixtures to aquatic wildlife at field-realistic levels is thus crucial for environmental risk assessment. This study aims at assessing the effects of two current-use insecticides, imidacloprid and chlorpyrifos, on Gammarus fossarum using multi-level biomarkers. In microcosms, gammarids were exposed for 72 h to insecticides tested individually or in mixture at 0.01, 0.1 and 1 µg/L of each chemical. Multi-metric responses were assessed at the individual level (behavioural traits: locomotion, respiration and amplexus formation) and the cellular level (enzymes involved in growth, moulting, digestion and cell stress). The results showed insecticide-elicited behavioural and biochemical responses from the lowest concentration of 0.01 µg/L. Overall, single exposures stimulated behavioural traits and inhibited enzymatic activities, highlighting subtle impacts at different organizational levels but these were not dose related. For binary mixtures, antagonistic effects (i.e. less-than-additive) on biomarkers were mainly observed when compared with single exposures. Multi-variable analyses indicated the complementarity of behavioural and biochemical biomarkers in identifying sublethal biological alterations and dose-dependent multiple action sites of insecticides. Besides, the mortality observed only for the mixture at 1 µg/L demonstrated a high lethal potential of insecticides in a simple binary combination. To conclude, this study demonstrates disturbances in individual performances and cellular impairments occurring at environmentally realistic exposure levels in a non-target wild species. Since the sublethal effects, such as those identified with this multi-biomarker approach, could lead to long-term alterations in population dynamics of agricultural areas, they constitute promising early endpoints for risk assessment of insecticides.

Co-digestion of wastewater sludge: Choosing the optimal blend.

Anaerobic co-digestion (AcoD) is a promising strategy to increase the methane production of anaerobic digestion plants treating wastewater sludge (WAS). In this work the degradability of six different mixtures of WAS with fish waste (FW) or garden-grass (GG) was evaluated and compared to the three mono-digestions. Degradation performances and methanogenic pathways, determined with the isotopic signatures of biogas, were compared across time. Fish and grass mono-digestion provided a higher final methane production than WAS mono-digestion. In co-digestion the addition of 25% of fish was enough to increase the final methane production from WAS while 50% of grass was necessary. To determine the optimal blend of WAS co-digestion two indicators were specifically designed, representing the maximum potential production (ODI) and the expected production in mono-digestion conditions (MDI). The comparison between these indicators and the experimental results showed that the most productive blend was composed of 75% of co-substrate, fish or grass, with WAS. Indeed, the final methane production was increased by 1.9 times with fish and by 1.7 times with grass associated to an increase of the methane production rate by 1.5 times. Even if the same succession of methanogenic pathways across time was observed for the different mixtures, their relative proportions were different. Sewage sludge degradation was mostly achieved through hydrogenotrophic pathway while acetoclastic pathway was dominant for fish and grass degradation. These results were confirmed by the identification of Archaea with 16S sequencing.

Penta- and 2,4,6-tri-chlorophenol biodegradation during municipal solid waste anaerobic digestion.

In this study isotopic tracing using (13)C labelled pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP) is proposed as a tool to distinguish the loss of PCP and 2,4,6-TCP due to biodegradation from other physical processes. This isotopic approach was applied to accurately assess in situ PCP and 2,4,6-TCP degradation under methanogenic conditions in several microcosms made up of household waste. These microcosms were incubated in anaerobic conditions at 35°C (mesophilic) and 55°C (thermophilic) without agitation. The volume of biogas produced (CH4 and CO2), was followed for a period of 130 days. At this stage of stable methanogenesis, (13)C6-PCP and (13)C6-2,4,6-TCP were introduced anaerobically in microcosms and its monitoring at mesophilic and thermophilic conditions was performed in parallel by gas chromatography mass spectrometry (GC-MS) and gas chromatography isotope-ratio mass spectrometry (GC-IRMS). This study proved the almost total dechlorination of bioavailable PCP and 2,4,6-TCP into 4-CP at 35°C. Nevertheless, high rate adsorption in particular materials of the two compounds was observed. Furthermore, Carbon-13 Nuclear Magnetic Resonance ((13)C-NMR) Spectroscopy analysis of (13)C labelled 2,4,6-TCP mesophilic incubations showed the partial mineralization of 4-CP at 35°C to acetate and then to HCO(3-). Consequently, NMR results confirm the biogas isotopic results indicating the mineralization of (13)C labelled 2,4,6-TCP into (13)C (CH4 and CO2). Concerning (13)C labelled PCP mesophilic incubations, the isotopic composition of the biogas still natural until the day 262. In contrast, no dechlorination was observed at 55°C. Thus PCP and 2,4,6-TCP were persistent in thermophilic conditions.

Stable isotope probing of acetate fed anaerobic batch incubations shows a partial resistance of acetoclastic methanogenesis catalyzed by Methanosarcina to sudden increase of ammonia level.

Ammonia inhibition represents a major operational issue for anaerobic digestion. In order to refine our understanding of the terminal catabolic steps in thermophilic anaerobic digestion under ammonia stress, we studied batch thermophilic acetate fed experiments at low (0.26 g L(-1)) and high (7.00 g L(-1)) Total Ammonia Nitrogen concentrations (TAN). Although methane production started immediately for all incubations and resulted in methane yields close to stoichiometric expectations, a 62-72% decrease of methanogenic rate was observed throughout the incubation at 7.00 g L(-1) of TAN compared to 0.26 g L(-1). Stable Isotope Probing analysis of active microbial communities in (13)C-acetate fed experiments coupled to automated ribosomal intergenic spacer analysis and 16S rDNA pyrotag sequencing confirmed that microbial communities were similar for both TAN conditions. At both TAN levels, the (13)C-labeled bacterial community was mainly affiliated to Clostridia-relatives, with OPB54 bacteria being the most abundant sequence in the heavy DNA 16S rDNA pyrotag library. Sequences closely related to Methanosarcina thermophila were also abundantly retrieved in the heavy DNA fractions, showing that this methanogen was still actively assimilating labeled carbon from acetate at free ammonia nitrogen concentrations up to 916 mg L(-1). Stable isotopic signature analysis of biogas, measured in unlabeled acetate fed experiments that were conducted in parallel, confirmed that acetoclastic methanogenic pathway was dominant at both ammonia concentrations. Our work demonstrates that, besides the syntrophic acetate oxidation pathway, acetoclastic methanogenesis catalyzed by Methanosarcina can also play a major role in methane production at high ammonia levels.

Elucidation of the thermophilic phenol biodegradation pathway via benzoate during the anaerobic digestion of municipal solid waste.

Anaerobic digestion makes it possible to valorize municipal solid waste (MSW) into biogas and digestate which are, respectively, a renewable energy source and an organic amendment for soil. Phenols are persistent pollutants present in MSW that can inhibit the anaerobic digestion process and have a toxic effect on microbiota if they are applied to soil together with digestate. It is then important to define the operational conditions of anaerobic digestion which allow the complete degradation of phenol. In this context, the fate of phenol during the anaerobic digestion of MSW at 55°C was followed using an isotopic tracing approach ((13)C6-phenol) in experimental microcosms with inoculum from an industrial thermophilic anaerobic digester. With this approach, it was possible to demonstrate the complete phenol biodegradation into methane and carbon dioxide via benzoate. Benzoate is known to be a phenol metabolite under mesophilic conditions, but in this study it was found for the first time to be a phenol degradation product at thermophilic temperature.

Pesticide contamination interception strategy and removal efficiency in forest buffer and artificial wetland in a tile-drained agricultural watershed.

Pesticide pollution is a major threat to aquatic ecosystems that can be mitigated through complementary actions including buffer zones (BZs). This paper discusses the results of 3 yr of field-scale monitoring of the concentration and load transfer of 16 pesticides out of a tile-drained catchment (Bray, France) and their reduction through two BZ: an artificial wetland (AW) and a forest buffer (FB). Typically, the highest concentrations were measured in the first flows following pesticide applications or resuming after periods of low or no flow. An open/close water management strategy was implemented to operate the parallel BZ based on pesticide applications by the farmer. The strategy was efficient in intercepting molecules whose highest concentrations occurred during the first flows following application. Inlet vs. outlet pesticide load reductions ranged from 45% to 96% (AW) and from -32% to 100% (FB) depending on the pesticide molecule and the hydrological year. Partly reversible adsorption was a dominant process explaining pesticide removal; whereas, degradation occurred for sufficiently long water retention time. Apart from the least sorbing molecules (e.g., isoproturon), BZ can partially remove pesticide pollution.

Evaluation of biodegradability of phenol and bisphenol A during mesophilic and thermophilic municipal solid waste anaerobic digestion using 13C-labeled contaminants.

In this paper, the isotopic tracing using (13)C-labeled phenol and bisphenol A was used to study their biodegradation during anaerobic digestion of municipal solid waste. Microcosms were incubated anaerobically at 35 °C (mesophilic conditions) and 55 °C (thermophilic conditions) without steering. A continuous follow-up of the production of biogas (CH(4) and CO(2)), was carried out during 130 d until the establishment of stable methanogenesis. Then (13)C(12)-BPA, and (13)C(6)-phenol were injected in microcosms and the follow-up of their degradation was performed simultaneously by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) and gas chromatography mass spectrometry (GC-MS). Moreover, Carbon-13 Nuclear Magnetic Resonance ((13)C-NMR) Spectroscopy is used in the identification of metabolites. This study proves that the mineralization of phenol to CO(2) and CH(4) occurs during anaerobic digestion both in mesophilic and thermophilic conditions with similar kinetics. In mesophilic condition phenol degradation occurs through the benzoic acid pathway. In thermophilic condition it was not possible to identify the complete metabolic pathway as only acetate was identified as metabolite. Our results suggest that mineralization of phenol under thermophilic condition is instantaneous explaining why metabolites are not observed as they do not accumulate. No biodegradation of BPA was observed.

Design of experiments and detailed uncertainty analysis to develop and validate a solid-phase microextraction/gas chromatography-mass spectrometry method for the simultaneous analysis of 16 pesticides in water.

A solid-phase microextraction (SPME)/gas chromatography (GC)-mass spectrometry (MS) multiresidue analytical method was developed for 16 pesticides presenting different physicochemical properties including diphenyl ether, triazine, ureas, acetamides, benzofuran, thiocarbamate, pyridine carboxamides, chloronitrile, piperedine, and azoles. Optimization was achieved by means of the design of experiments methodology. Extraction temperature, extraction time, desorption temperature, and NaCl addition were the factors exhibiting the most significant effects on pesticide extraction. Validation was carried out through model adequacy and specificity tests, limits of quantification and detection determination, and full uncertainty assessment on the whole analytical method. Good first- and second-order model adequacy was found for pesticide calibration. LOQs were in the 0.05-0.5 microg L(-1) range and specificity recoveries varied from 75% to 140%. These results were considered acceptable for our research purposes on highly concentrated agricultural flows. Uncertainty calculations accounted for several steps: standard preparation, calibration model selection, and use. On average, real sample concentration uncertainties were lower than 10%, indicating that the analytical method performed very well. Its application to 61 real water samples confirmed the presence of some pesticide concentrations in relation to farmer use, whereas other molecules were usually either not detected or not quantified.