Assessment of Environmental Pollution and Human Exposure to Pesticides by Wastewater Analysis in a Seven-Year Study in Athens, Greece.

Pesticides have been used in large amounts around the world for decades and are responsible for environmental pollution and various adverse effects on human health. Analysis of untreated wastewater can deliver useful information on pesticides' use in a particular area and allow the assessment of human exposure to certain substances. A wide-scope screening method, based on liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry, was applied, using both target and suspect screening methodologies. Daily composite influent wastewater samples were collected for seven or eight consecutive days in Athens between 2014 and 2020 and analyzed for 756 pesticides, their environmental transformation products and their human metabolites. Forty pesticides were quantified at mean concentrations up to 4.9 µg/L (tralkoxydim). The most abundant class was fungicides followed by herbicides, insect repellents, insecticides and plant growth regulators. In addition, pesticide transformation products and/or metabolites were detected with high frequency, indicating that research should be focused on them. Human exposure was evaluated using the wastewater-based epidemiology (WBE) approach and 3-ethyl-carbamoyl benzoic acid and cis-1,2,3,6-tetrahydrophthalimide were proposed as potential WBE biomarkers. Wastewater analysis revealed the presence of unapproved pesticides and indicated that there is an urgent need to include more transformation products in target databases.

Wide-scope target screening of >2000 emerging contaminants in wastewater samples with UPLC-Q-ToF-HRMS/MS and smart evaluation of its performance through the validation of 195 selected representative analytes.

This study presents the development and validation of a comprehensive quantitative target methodology for the analysis of 2316 emerging pollutants in water based on Ultra-Performance Liquid Chromatography Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-ToF-HRMS/MS). Target compounds include pesticides, pharmaceuticals, drugs of abuse, industrial chemicals, doping compounds, surfactants and transformation products, among others. The method was validated for 195 analytes, chosen to be representative of the chemical space of the target list, enabling the assessment of the performance of the method. The method involves a generic sample preparation based on mixed mode solid phase extraction, a UPLC-QTOF-MS/MS screening method using Data Independent Acquisition (DIA) mode, which provides MS and MS/MS spectra simultaneously and an elaborate strong post-acquisition evaluation of the data. The processing method was optimized to provide a successful identification rate >95 % and to minimize the number of false positive results (< 5 %). Decision limit (CCα) and detection capability (CCß) were also introduced in the validation scheme to provide more realistic metrics on the performance of a HRMS-based wide-scope screening method. A new system of identification points (IPs) based on the one described in the Commission Decision 2002/657/EC was applied to communicate the confidence level in the identification of the analytes. This system considers retention time, mass accuracy, isotopic fit and fragmentation; taking full advantage of the capacities of the HRMS instruments. Finally, 398 contaminants were detected and quantified in real wastewater.

Assessment of the Acute Toxicity, Uptake and Biotransformation Potential of Benzotriazoles in Zebrafish ( Danio rerio) Larvae Combining HILIC- with RPLC-HRMS for High-Throughput Identification.

The current study reports on the toxicity, uptake, and biotransformation potential of zebrafish (embryos and larvae) exposed to benzotriazoles (BTs). Acute toxicity assays were conducted. Cardiac function abnormalities (pericardial edema and poor blood circulation) were observed from the phenotypic analysis of early life zebrafish embryos after BTs exposure. For the uptake and biotransformation experiment, extracts of whole body larvae were analyzed using liquid chromatography-high-resolution tandem mass spectrometry (UPLC-Q-TOF-HRMS/MS). The utility of hydrophilic interaction liquid chromatography (HILIC) as complementary technique to reversed phase liquid chromatography (RPLC) in the identification process was investigated. Through HILIC analyses, additional biotransformation products (bio-TPs) were detected, because of the enhanced sensitivity and better separation efficiency of isomers. Therefore, reduction of false negative results was accomplished. Both oxidative (hydroxylation) and conjugative (glucuronidation, sulfation) metabolic reactions were observed, while direct sulfation proved the dominant biotransformation pathway. Overall, 26 bio-TPs were identified through suspect and nontarget screening workflows, 22 of them reported for the first time. 4-Methyl-1- H-benzotriazole (4-MeBT) demonstrated the highest toxicity potential and was more extensively biotransformed, compared to 1- H-benzotriazole (BT) and 5-methyl-1- H-benzotriazole (5-MeBT). The extent of biotransformation proved particularly informative in the current study, to explain and better understand the different toxicity potentials of BTs.

Amniotic Fluid and Maternal Serum Metabolic Signatures in the Second Trimester Associated with Preterm Delivery.

Preterm delivery (PTD) represents a major health problem that occurs in 1 in 10 births. The hypothesis of the present study was that the metabolic profile of different biological fluids, obtained from pregnant women during the second trimester of gestation, could allow useful correlations with pregnancy outcome. Holistic and targeted metabolomics approaches were applied for the complementary assessment of the metabolic content of prospectively collected amniotic fluid (AF) and paired maternal blood serum samples from 35 women who delivered preterm (between 29 weeks + 0 days and 36 weeks +5 days gestation) and 35 women delivered at term. The results revealed trends relating the metabolic content of the analyzed samples with preterm delivery. Untargeted and targeted profiling showed differentiations in certain key metabolites in the biological fluids of the two study groups. In AF, intermediate metabolites involved in energy metabolism (pyruvic acid, glutamic acid, and glutamine) were found to contribute to the classification of the two groups. In maternal serum, increased levels of lipids and alterations of key end-point metabolites were observed in cases of preterm delivery. Overall, the metabolic content of second-trimester AF and maternal blood serum shows potential for the identification of biomarkers related to fetal growth and preterm delivery.

Chlorination of benzothiazoles and benzotriazoles and transformation products identification by LC-HR-MS/MS.

The fate of four benzotriazoles [1-H-benzotriazole (1-H-BTRi), tolyltriazole (TTRi), xylyltriazole (XTRi) and 1-hydroxy-benzotriazole (1-OH-BTRi)] and three benzothiazoles [benzothiazole (BTH), 2-hydroxy-benzothiazole (2-OH-BTH) and 2-amino-benzothiazole (2-amino-BTH)], during chlorination batch experiments was investigated. In the first step, their degradation under different experimental conditions (applied molar ratio of NaOCl and the target contaminant (m.r.), reaction's contact time, pH value of the reaction's solution and the influence of total suspended solids (TSS) presence) was investigated and their removal kinetics parameters (kobs and t1/2) were determined. In the second step, LC-QTOFMS/MS was used for the detection and identification of transformation products (TPs) formed during chlorination, through the application of suspect and non-target screening approaches. Four and five TPs of XTRi and 2-amino-BTH, respectively, were detected and tentatively identified, while 1-H-BTRi was proven to be formed by the chlorination of 1-OH-BTRi. Moreover, since the identified TPs were also detected in spiked wastewater samples, after lab-scale chlorination experiments, toxicity assessment was carried out by ECOSAR calculations for the environmental relevance of their occurrence. The proposed chlorinated TPs were proven to be more toxic than their parent compounds.

Liquid chromatography-tandem mass spectrometric methods for the determination of spinosad, thiacloprid and pyridalyl in spring onions and estimation of their pre-harvest interval values.

Two liquid chromatography-tandem mass spectrometric methods were developed and validated to determine spinosyn A and D, thiacloprid and pyridalyl in spring onions cultivated under Egyptian field conditions. The degradation rates, the pre-harvest interval (PHI) values and the half-life values of the three pesticides were estimated. QuEChERS was used for sample preparation and the separation was performed on an X-Bridge C18 column with ACN-formic acid 0.1% as the mobile phase. Linear range, method detection limits (MDLs), precision, recovery and matrix effects were estimated. The multi-residue MDLs ranged from 0.02µg/kg (spinosyn A & D) to 0.05µg/kg for pyridalyl. All the investigated pesticides showed high degradation rates. For spinosad the half-life value was 1.2days, for thiacloprid it reached 2.2days and for pyridalyl 4.4days. Furthermore, the calculated PHI values, according to the maximum residue levels set by the EU, were 0days for spinosad, 9.8days for thiacloprid and 39.4days for pyridalyl.

Extended Suspect and Non-Target Strategies to Characterize Emerging Polar Organic Contaminants in Raw Wastewater with LC-HRMS/MS.

An integrated workflow based on liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) was developed and applied to detect and identify suspect and unknown contaminants in Greek wastewater. Tentative identifications were initially based on mass accuracy, isotopic pattern, plausibility of the chromatographic retention time and MS/MS spectral interpretation (comparison with spectral libraries, in silico fragmentation). Moreover, new specific strategies for the identification of metabolites were applied to obtain extra confidence including the comparison of diurnal and/or weekly concentration trends of the metabolite and parent compounds and the complementary use of HILIC. Thirteen of 284 predicted and literature metabolites of selected pharmaceuticals and nicotine were tentatively identified in influent samples from Athens and seven were finally confirmed with reference standards. Thirty four nontarget compounds were tentatively identified, four were also confirmed. The sulfonated surfactant diglycol ether sulfate was identified along with others in the homologous series (SO4C2H4(OC2H4)xOH), which have not been previously reported in wastewater. As many surfactants were originally found as nontargets, these compounds were studied in detail through retrospective analysis.

Degradation of emerging contaminants from water under natural sunlight: The effect of season, pH, humic acids and nitrate and identification of photodegradation by-products.

Both photodegradation and hydrolysis of non-steroidal anti-inflammatory drugs (NSAIDs) and endocrine disrupting chemicals (EDCs) were investigated in order to evaluate their photochemical fate in aquatic environment and to assess the effect of season and specific characteristics of water (pH, humic acids and nitrate concentration) on the removal of target EDCs and NSAIDs through photodegradation. An additional objective was the identification of the photodegradation by-products of specific NSAIDs and their dependence on irradiation time. Selected compounds' transformation was investigated under natural sunlight radiation while control experiments were conducted in the dark. As expected, most of compounds' degradation rate decreased with decreasing light intensity between two different experimental periods. Most of the tested compounds exhibited different rates of degradation during direct and indirect photolysis. The degradation rate of the selected compounds increased in the presence of NO3(-) and the photodegradation rate was higher for some compounds in alkaline than in acidic solution. The effect of humic acids' presence in the water depends on the absorbance spectrum of the compound and the produced photosensitizers. More specifically, humic acids act as inner filter toward most of the selected NSAIDs and as photosensitizers toward most of the EDCs. The results of the irradiation experiments in the presence of both humic acids and NO3(-), indicate that the direct photolysis is much more efficient than indirect photochemical processes. Finally, several degradation by-products of ketoprofen and diclofenac were identified in the samples, exposed to sunlight. The dependence of these by-products on radiation time is also demonstrated.

Qualitative Multiresidue Screening Method for 143 Veterinary Drugs and Pharmaceuticals in Milk and Fish Tissue Using Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry.

A wide-scope screening methodology has been developed for the identification of veterinary drugs and pharmaceuticals in fish tissue and milk using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). The method was validated using a qualitative approach at two concentration levels. The detection of the residues was accomplished by retention time, accurate mass, and the isotopic fit using an in-house database. Product-ion spectra were used for unequivocal identification of the compounds. Generic sample treatment was applied. The majority of the compounds were successfully detected and identified at concentration levels of 150 ng mL(-1) in milk and 200 µg kg(-1) in fish (>80% of the compounds in both matrices), whereas satisfactory results were also obtained at concentration levels of 15 ng mL(-1) in milk and 20 µg kg(-1) in fish (>60% of the compounds detected and identified).

Benzosulfonamides in wastewater: method development, occurrence and removal efficiencies.

Benzosulfonamides (BeSAs) are a family of compounds with a broad application as industrial chemicals: plasticizers, intermediates for pesticides and drugs or used in the production of artificial sweeteners, among others. BeSAs constitute a class of organic compounds of emerging environmental concern. However, analytical methodologies for their determination in the environment are scarce as well as there is a lack of environmental occurrence data for these substances. An analytical methodology based on pseudo-SRM liquid chromatography-tandem mass spectrometry (LC-(ESI-)MS/MS) was developed and optimized for the determination of three BeSAs (benzenesulfonamide (BSA), o-toluenesulfonamide (o-TSA) and p-toluenesulfonamide (p-TSA)) in wastewater. The solid phase extraction protocol was also carefully optimized. The method provided recoveries in the range 83-118% (three different fortification levels). Good precision (<10.2% (intra-day) and <13.6% (inter-day)) and limits of detection achieved (11-89 ng L(-1)) allow a reliable quantification of these compounds in both effluent and influent wastewaters. The presence of BeSAs has been evaluated during seven consecutive days in influent and effluent samples from the wastewater treatment plant of Athens, Greece. o-TSA and p-TSA were detected in 100% of the samples, while BSA was detected in all the influents and in 43% of the effluents. All three compounds were determined in relevant concentrations (up to 1.4 µg L(-1) in the case of p-TSA), constituting the first evidence of the presence of these compounds in Greece, and contributing to the scarce occurrence data. Removal efficiencies of BeSAs during wastewater treatment was also assessed and discussed. In this regard, different behaviors were observed: while BSA and p-TSA were removed in different rates, o-TSA was formed during wastewater treatment. A daily load of 0.48 kg of o-TSA and 0.47 kg of p-TSA was discarded in the receiving Saronikos Gulf.

Removal of endocrine disruptors and non-steroidal anti-inflammatory drugs through wastewater chlorination: the effect of pH, total suspended solids and humic acids and identification of degradation by-products.

Endocrine disrupting chemicals (EDCs) and non-steroidal anti-inflammatory drugs (NSAIDs) are two groups of emerging pollutants the significance of which rests on their persistent detection in the aquatic environment and their possible adverse effects. Wastewater treatment plants are one of the major ways for transporting such chemicals in the aquatic environment. Chlorination is usually the last stage of treatment before wastewater being disposed to the aquatic environment. This work focuses on the evaluation of the effect of chlorine dose and specific wastewater characteristics (pH, total suspended solids and humic acids) on the removal of target EDCs and NSAIDs through chlorination. Another objective of this study is the identification of chlorination by-products of specific EDCs and NSAIDs and their dependence on contact time. Based on the results it is concluded that the effect of chlorine dose and humic acids concentration on the degradation of target compounds during chlorination is minimal. On the contrary, pH is a critical parameter which highly affects process performance. Moreover, it is concluded that not only the free available chlorine species, but also the properties of EDCs and NSAIDs under different pH conditions can affect chlorination process performance. The effect of TSS on the degradation of the target compounds during chlorination is more profound for chemicals with high Kow values and therefore higher affinity to partition to the particulate phase (i.e. nonylphenols, triclosan). Several degradation by-products were identified through chlorination of nonylphenol, bisphenol A and diclofenac. The dependence of these by-products on chlorination contact time is also demonstrated.

Mass loading and fate of linear and cyclic siloxanes in a wastewater treatment plant in Greece.

The occurrence and fate of 5 cyclic (D3 to D7) and 12 linear (L3 to L14) siloxanes were investigated in raw and treated wastewater (both particulate and dissolved phases) as well as in sludge from a wastewater treatment plant (WWTP) in Athens, Greece. Cyclic and linear siloxanes (except for L3) were detected in all influent wastewater and sludge samples at mean concentrations of (sum of 17 siloxanes) 20 µg L(-1) and 75 mg kg(-1), respectively. The predominant compounds in wastewater were L11 (24% of the total siloxane concentration), L10 (16%), and D5 (13%), and in sludge were D5 (20%) and L10 (15%). The distribution of siloxanes between particulate and dissolved phases in influents differed significantly for linear and cyclic siloxanes. Linear siloxanes showed higher solid-liquid distribution coefficients (log K(d)) than did cyclic compounds. For 10 of the 16 compounds detected in influents, the removal efficiency was higher than 80%. Sorption to sludge and biodegradation and/or volatilization losses are important factors that affect the fate of siloxanes in WWTPs. The mean total mass of siloxanes that enter into the WWTP via influent was 15.1 kg per day(-1), and the mean total mass released into the environment via effluent was 2.67 kg per day(-1).

Determination of benzotriazoles and benzothiazoles in human urine by liquid chromatography-tandem mass spectrometry.

Benzotriazole (BTR) and benzothiazole (BTH) derivatives are used in a wide variety of industrial and consumer products and have been reported to occur in the environment. Owing to a lack of analytical methods, human exposure to BTR and BTH is still unknown. In this study, a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI(+)MS/MS) method was developed for simultaneous determination of five 1,2,3-benzotriazoles and five 1,3-benzothiazoles in human urine. The target benzotriazoles were 1H-benzotriazole, 1-hydroxy-benzotriazole, tolyltriazole, xylyltriazole (or 5,6-dimethyl-1H-benzotriazole), and 5-chloro-benzotriazole, and the target benzothiazoles were benzothiazole, 2-hydroxy-benzothiazole, 2-methylthio-benzothiazole, 2-amino-benzothiazole, and 2-thiocyanomethylthio-benzothiazole. Urine specimens were enzymatically deconjugated with ß-glucuronidase and extracted by a solid-phase extraction (SPE) procedure for the measurement of total concentrations (i.e., free + conjugated forms) of BTRs and BTHs. Additionally, a liquid-liquid extraction (LLE) method was developed for comparison of extraction efficiencies between SPE and LLE. The limits of detection (LODs) ranged from 0.07 (2-amino-benzothiazole) to 4.0 ng/mL (benzothiazole) for the SPE method and from 0.04 (tolyltriazole) to 6.4 ng/mL (benzothiazole) for the LLE method. A total of 100 urine specimens, collected from Athens, Greece, were analyzed by enzymatic deconjugation and SPE. Benzothiazole and tolyltriazole were found frequently, and their concentrations were on the order of a few ng/mL. To our knowledge, this is the first study on the occurrence of 10 BTR and BTH compounds in human urine.