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The occurrence of polar pesticides in sediments has not been extensively investigated because of their relatively poor hydrophobicity and apparently less persistence in the environment. However, their continuous release into the aquatic systems calls for the evaluation of their potential accumulation in sediments and the role of this matrix as a potential source of these compounds. Considering this, a method based on pressurized liquid extraction (PLE), extract clean-up by solid phase extraction (SPE), and analyte determination by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated to analyze 50 relevant (frequently used and/or regulated or found in water) medium to highly polar pesticides in sediments. The method showed good performance regarding accuracy (relative recoveries between 76 and 124%), precision (relative standard deviation values < 20%), sensitivity (LODs in the low nanogram per gram for most compounds), linearity (coefficients of determination > 0.99), and matrix effects (negligible for all analytes). The use of an isotope dilution approach for quantification ensures result reliability. As a part of the validation process, the method was applied to the analysis of the target pesticides in sediments from the Llobregat River (NE Spain) showing the presence of five of them, namely, terbutryn, dichlorvos, terbuthylazine, diazinon, and irgarol. All 5 pesticides, due to both the concentrations found and their physical-chemical characteristics, demonstrate high potential for bioaccumulation and risk to aquatic organisms. Additional multi-disciplinary studies that investigate pesticide occurrence in different aquatic compartments and evaluate the potential risks for aquatic ecosystems are required to assess the environmental impact and significance of the presence of pesticides in sediments.

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

The identification of pollutants is crucial to protect water resources and ensure food safety. The available analytical methodologies allow reliable detection of organic pollutants such as pesticides; however, there is the need for faster, direct and continuous methodologies for real‐time monitoring of pesticides. Fluorescent‐based biosensors have been recently proposed as a valid alternative due to their advantage of being easy, cheap and specific. In this context, the aim of the present EU‐FORA fellowship programme was to develop and apply a fluorescence‐based biosensing device for the detection of organophosphate (OP) pesticides in water samples and drinkable food. The study was addressed using a mutant of the thermostable esterase‐2 from Alicyclobacillus acidocaldarius (EST2‐S35C) as a bioreceptor for OP pesticides. The use of EST2 involves some significant advantages including specificity and affinity towards OPs, and high stability over time in a different range of temperatures and pH. The protein was labelled to the fluorescent probe IAEDANS and fluorescence measurements of quenching in solution and in immobilised form were performed. The results showed good stability and sensitivity, reaching low limits of detection and quantification and a constant signal intensity over time. The addition of paraoxon quenched the fluorescence of the complex, reaching a plateau at 100 pmol paraoxon. The decrease of enzymatic activity of EST2‐S35C‐IAEDANS in the presence of paraoxon correlated the inhibition of the labelled enzyme with the decrease in fluorescence. The results from the application of the biosensor with real samples showed a decrease in fluorescence in surface water samples, contaminated by OPs. The use of the developed fluorescence‐based biosensor demonstrated its applicability for real samples monitoring and could ensure the production of large amounts of data in a short period of time which can be used to address environmental and food safety risk assessment.

Faster, sensitive and real‐time methods for detecting organophosphate (OP) pesticides are urged for in situ monitoring of these widely spread contaminants. For this reason, several efforts have been addressed for the development of performant biosensors. The thermostable enzyme esterase‐2 from Alicyclobacillus acidocaldarius (EST2), with a lipase‐like Ser‐His‐Asp catalytic triad with a high affinity to OPs, is a promising candidate as a bioreceptor for biosensor development. Within this EU‐FORA fellowship project, two different components of the biosensor were evaluated: (i) the use of the enzymatic bioreceptor in solution or immobilised in a solid membrane; (ii) the measurement of fluorescence quenching by direct measurement of the fluorescence probe intensity signal or by fluorescence resonance energy transfer (FRET) from the tryptophans located in the catalytic site of the enzyme to a binded fluorescence probe. Fluorescence spectroscopy is among the most used techniques in analytical chemistry laboratories, mainly due to its high sensitivity and simplicity. To this aim, the developed IAEDANS‐labelled EST2‐S35C mutant has been used. Fluorometric measurements with both methods showed linearity with increased EST2‐S35C concentrations. No significant interference on FRET measurements was observed due to changes in medium pH or due to the addition of other organic components (glucose, ascorbic acid, yeast extract). Both methods presented similar sensitivity towards detecting OPs, with fluorescence quenching due to the presence of paraoxon at environmentally relevant concentrations from 0.09 µM. The obtained results are of high relevance to further development of biosensors for the pesticide monitoring that: (i) decrease the expenses of the analysis; (ii) simplify the procedures for pesticide detection; (iii) reduce the time of response. Furthermore, the use of biosensors for pesticides real‐time and in situ detection of pesticides promises to increase the number of samples analysed, providing a larger amount of data for food safety risk assessment.

The massive use of pesticides represents one of the main causes of environmental deterioration, as they have adverse effects on non-target organisms. Thus, the development of technologies capable of reducing their release into the environment is urgently needed. This study reports for the first time the white-rot fungus Trametes versicolor as an alternative towards the degradation of medium to highly polar pesticides such as the organophosphate malathion, and the neonicotinoids acetamiprid and imidacloprid. Specifically, T. versicolor could completely remove 1 mg/L of malathion in an Erlenmeyer flask within 48 h, while experiments of acetamiprid and imidacloprid (4 mg/L), conducted in air-pulse fluidized bioreactors, resulted in degradation percentages of 20% and 64.7%, respectively, after 7 days of operation. Enzymatic exploration studies revealed that the cytochrome P450 system, instead of the extracellular enzyme laccase, is involved in the degradation of acetamiprid and imidacloprid. The degradation pathways were proposed based on the main transformation products (TPs) formed in the solutions: seven in the case of malathion, and two and one in the case of imidacloprid and acetamiprid, respectively. Although the TPs identified were predicted to be less toxic than the investigated pesticides, the toxicity of the individual solutions slightly increased throughout the degradation process, according to the Microtox assay. However, the solution toxicity was always below the threshold established in the local regulation. Although additional research is needed to implement this treatment at a pilot plant scale, this work highlights the potential of T. versicolor to bio-remediate pesticide-contaminated waters.

The widespread use of pesticides in the last decades and their accumulation into the environment gave rise to major environmental and human health concerns. To address this topic, the scientific community pointed out the need to develop methodologies to detect and measure the presence of pesticides in different matrices. Biosensors have been recently explored as fast, easy, and sensitive methods for direct organophosphate pesticides monitoring. Thus, the present work aimed at designing and testing a 3D printed adapter useful on different equipment, and a membrane support to immobilize the esterase-2 from Alicyclobacillus acidocaldarius (EST2) bioreceptor. The latter is labelled with the IAEDANS, a bright fluorescent probe. EST2 was selected since it shows a high specificity toward paraoxon. Our results showed good stability and replicability, with an increasing linear fluorescent intensity recorded from 15 to 150 pmol of labelled EST2. Linearity of data was also observed when using the immobilized labelled EST2 to detect increasing amounts of paraoxon, with a limit of detection (LOD) of 0.09 pmol. This LOD value reveals the high sensitivity of our membrane support when mounted on the 3D adapter, comparable to modern methods using robotic workstations. Notably, the use of an independent support significantly simplified the manipulation of the membrane during experimental procedures and enabled it to match the specificities of different systems. In sum, this work emphasizes the advantages of using 3D printed accessories adapted to respond to the newest research needs.

The occurrence of polar pesticides in sediments has not been extensively investigated because of their relatively poor hydrophobicity and apparently less persistence in the environment. However, their continuous release into the aquatic systems calls for the evaluation of their potential accumulation in sediments and the role of this matrix as a potential source of these compounds. Considering this, a method based on pressurized liquid extraction (PLE), extract clean-up by solid phase extraction (SPE) and analyte determination by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated to analyze 50 relevant (frequently used and/or regulated or found in water) medium to highly polar pesticides in sediments. The method showed good performance regarding accuracy (relative recoveries between 76% and 124%), precision (relative standard deviation values <20%), sensitivity (LODs in the low ng/g for most compounds), linearity (coefficients of determination >0.99), and matrix effects (negligible for all analytes). The use of an isotope dilution approach for quantification ensures results reliability. As a part of the validation process, the method was applied to the analysis of the target pesticides in sediments from the Llobregat River (NE Spain) showing the presence of five of them, namely, terbutryn, dichlorvos, terbuthylazine, diazinon, and irgarol. All 5 pesticides, due to both the concentrations found and their physical-chemical characteristics, demonstrate high potential for bioaccumulation and risk to the aquatic organisms. Additional multidisciplinary studies that investigate pesticides occurrence in the different aquatic compartments and evaluate the potential risks for aquatic ecosystems are required to assess the environmental impact and significance of the presence of pesticides in sediments.

Water is an essential resource for all living organisms. The continuous and increasing use of pesticides in agricultural and urban activities results in the pollution of water resources and represents an environmental risk. To control and reduce pesticide pollution, reliable multi-residue methods for the detection of these compounds in water are needed. In this context, the present work aimed at providing an analytical method for the simultaneous determination of trace levels of 51 target pesticides in water and applying it to the investigation of target pesticides in two agriculture-impacted areas of interest. The method developed, based on an isotopic dilution approach and on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry, is fast, simple, and to a large extent automated, and allows the analysis of most of the target compounds in compliance with European regulations. Further application of the method to the analysis of selected water samples collected at the lowest stretches of the two largest river basins of Catalonia (NE Spain), Llobregat and Ter, revealed the presence of a wide suite of pesticides, and some of them at concentrations above the water quality standards (irgarol and dichlorvos) or the acceptable method detection limits (methiocarb, imidacloprid, and thiacloprid), in the Llobregat, and much cleaner waters in the Ter River basin. Risk assessment of the pesticide concentrations measured in the Llobregat indicated high risk due to the presence of irgarol, dichlorvos, methiocarb, azinphos ethyl, imidacloprid, and diflufenican (hazard quotient (HQ) values>10), and an only moderate potential risk in the Ter River associated to the occurrence of bentazone and irgarol (HQ>1).

The occurrence of the extensively used herbicide diuron in the environment poses a severe threat to the ecosystem and human health. Four different ligninolytic fungi were studied as biodegradation candidates for the removal of diuron. Among them, T. versicolor was the most effective species, degrading rapidly not only diuron (83%) but also the major metabolite 3,4-dichloroaniline (100%), after 7-day incubation. During diuron degradation, five transformation products (TPs) were found to be formed and the structures for three of them are tentatively proposed. According to the identified TPs, a hydroxylated intermediate 3-(3,4-dichlorophenyl)-1-hydroxymethyl-1-methylurea (DCPHMU) was further metabolized into the N-dealkylated compounds 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The discovery of DCPHMU suggests a relevant role of hydroxylation for subsequent N-demethylation, helping to better understand the main reaction mechanisms of diuron detoxification. Experiments also evidenced that degradation reactions may occur intracellularly and be catalyzed by the cytochrome P450 system. A response surface method, established by central composite design, assisted in evaluating the effect of operational variables in a trickle-bed bioreactor immobilized with T. versicolor on diuron removal. The best performance was obtained at low recycling ratios and influent flow rates. Furthermore, results indicate that the contact time between the contaminant and immobilized fungi plays a crucial role in diuron removal. This study represents a pioneering step forward amid techniques for bioremediation of pesticides-contaminated waters using fungal reactors at a real scale.

The management of the anthropogenic water cycle must ensure the preservation of the quality and quantity of water resources and their careful allocation to the different uses. Protection of water resources requires the control of pollution sources that may deteriorate them. This is a challenging task in multi-stressed catchments. This work presents an approach that combines pesticide occurrence patterns and stable isotope analyses of nitrogen (delta15N-NO3-, delta15N-NH4+), oxygen (delta18O-NO3-), and boron (delta11B) to discriminate the origin of pesticides and nitrogenpollution to tackle this challenge. The approach has been applied to a Mediterranean subcatchment subject to a variety of natural and anthropogenic pressures. Combining the results from both analytical approaches in selected locations of the basin, the urban/industrial activity was identified as the main pressure on the quality of the surface water resources, and to a large extent also on the groundwater resources, although agriculture may play also an important role, mainly in terms of nitrate and ammonium pollution. Total pesticide concentrations in surface waters were one order of magnitude higher than in groundwaters and believed to originate mainly from soil and/or sediments desorption processes and urban and industrial use, as they were mainly associated with treated wastewaters. These findings are supported by the stable isotope results, that pointed to an organic origin of nitrate in surface waters and most groundwater samples. Ammonium pollution observed in some aquifer locations is probably generated by nitrate reduction. Overall, no significant attenuation processes could be inferred for nitrate pollution. The approach presented here exemplifies the investigative monitoring envisioned in the Water Framework Directive.