PFΔScreen - an open-source tool for automated PFAS feature prioritization in non-target HRMS data.

Per- and polyfluoroalkyl substances (PFAS) are a huge group of anthropogenic chemicals with unique properties that are used in countless products and applications. Due to the high stability of their C-F bonds, PFAS or their transformation products (TPs) are persistent in the environment, leading to ubiquitous detection in various samples worldwide. Since PFAS are industrial chemicals, the availability of authentic PFAS reference standards is limited, making non-target screening (NTS) approaches based on high-resolution mass spectrometry (HRMS) necessary for a more comprehensive characterization. NTS usually is a time-consuming process, since only a small fraction of the detected chemicals can be identified. Therefore, efficient prioritization of relevant HRMS signals is one of the most crucial steps. We developed PFΔScreen, a Python-based open-source tool with a simple graphical user interface (GUI) to perform efficient feature prioritization using several PFAS-specific techniques such as the highly promising MD/C-m/C approach, Kendrick mass defect analysis, diagnostic fragments (MS2), fragment mass differences (MS2), and suspect screening. Feature detection from vendor-independent MS raw data (mzML, data-dependent acquisition) is performed via pyOpenMS (or custom feature lists) with subsequent calculations for prioritization and identification of PFAS in both HPLC- and GC-HRMS data. The PFΔScreen workflow is presented on four PFAS-contaminated agricultural soil samples from south-western Germany. Over 15 classes of PFAS (more than 80 single compounds with several isomers) could be identified, including four novel classes, potentially TPs of the precursors fluorotelomer mercapto alkyl phosphates (FTMAPs). PFΔScreen can be used within the Python environment and is easily automatically installable and executable on Windows. Its source code is freely available on GitHub ( https://github.com/JonZwe/PFAScreen ).

Non-extractable PFAS in functional textiles - characterization by complementary methods: oxidation, hydrolysis, and fluorine sum parameters.

Per- and polyfluoroalkyl substances (PFAS) are widely used for durable water-repellent finishing of different fabrics and textiles such as outdoor clothing, carpets, medical textiles and more. Existing PFAS extraction techniques followed by target analysis are often insufficient for detecting widely used side-chain fluorinated polymers (SFPs) that are barely or non-extractable. SFPs are typically copolymers consisting of a non-fluorinated backbone with perfluoroalkyl side-chains to obtain desired properties. We compared the accessible analytical information and performance of complementary techniques based on oxidation (dTOP and PhotoTOP assays), hydrolysis (THP assay), standard extraction, extractable organic fluorine (EOF), and total fluorine (TF) with five functional textiles and characterized 7 further textiles only by PhotoTOP oxidation. The results show that when applied directly to textile samples, dTOP and PhotoTOP oxidation and also hydrolysis (THP) are able to capture large fractions of TF in the form of perfluoroalkyl side-chains present in the textiles while methods relying on extracts (EOF, target and non-target analysis) yield much lower fractions of TF (e.g., factor ∼25-50 lower). The conversion of large fractions of the measured TF into PFCAs or FTOHs from fluorinated side chains is in contrast to previous studies. Concentrations ranged from

Practical application guide for the discovery of novel PFAS in environmental samples using high resolution mass spectrometry.

BACKGROUND: The intersection of the topics of high-resolution mass spectrometry (HRMS) and per- and polyfluoroalkyl substances (PFAS) bring together two disparate and complex subjects. Recently non-targeted analysis (NTA) for the discovery of novel PFAS in environmental and biological media has been shown to be valuable in multiple applications. Classical targeted analysis for PFAS using LC-MS/MS, though growing in compound coverage, is still unable to inform a holistic understanding of the PFAS burden in most samples. NTA fills at least a portion of this data gap. OBJECTIVES: Entrance into the study of novel PFAS discovery requires identification techniques such as HRMS (e.g., QTOF and Orbitrap) instrumentation. This requires practical knowledge of best approaches depending on the purpose of the analyses. The utility of HRMS applications for PFAS discovery is unquestioned and will likely play a significant role in many future environmental and human exposure studies. METHODS/RESULTS: PFAS have some characteristics that make them standout from most other chemicals present in samples. Through a series of tell-tale PFAS characteristics (e.g., characteristic mass defect range, homologous series and characteristic fragmentation patterns), and case studies different approaches and remaining challenges are demonstrated. IMPACT STATEMENT: The identification of novel PFAS via non-targeted analysis using high resolution mass spectrometry is an important and difficult endeavor. This synopsis document will hopefully make current and future efforts on this topic easier to perform for novice and experienced alike. The typical time devoted to NTA PFAS investigations (weeks to months or more) may benefit from these practical steps employed.

Photocatalytical transformation of fluorotelomer- and perfluorosulfonamide-based PFAS on mineral surfaces and soils in aqueous suspensions.

Per- and polyfluorinated substances (PFAS) are widely used industrial and household chemicals and occur on various contaminated field sites. To better understand their behavior on soils, spike experiments were performed with 6:2 diPAP (6:2 polyfluoroalkyl phosphate diester) on pure mineral phases (titanium dioxide, goethite and silicon dioxide) in aqueous suspensions under artificial sunlight. Further experiments were performed with uncontaminated soil and four precursor PFAS. Titanium dioxide (referenced as 100 %) showed the highest reactiveness to transform 6:2 diPAP to its primary metabolite 6:2 fluorotelomer carboxylic acid, followed by goethite with the addition of oxalate (4.7 %), silicon dioxide (1.7 %) and soil (0.0024 %). Experiments with four precursors [6:2 diPAP, 6:2 fluorotelomer mercapto alkyl phosphate (FTMAP), N-ethyl perfluorooctane sulfonamide ethanol-based phosphate diester (diSAmPAP), N-ethyl perfluorooctane sulfonamidoacetic acid (EtFOSAA)] on natural soils showed a transformation of all four precursors by simulated sunlight. The production of the primary intermediate from 6:2 FTMAP (6:2 FTSA, rate constant k = 2.7∗10-3h-1) was approximately 13-times faster than from 6:2 diPAP (6:2 FTCA, rate constant k = 1.9∗10-4h-1). EtFOSAA was completely decomposed within 48 h whereas only ~7 % diSAmPAP was transformed in the same time. The primary photochemical transformation product of diSAmPAP and EtFOSAA was PFOA, PFOS was not detected. The production rate constant of PFOA varied significantly between EtFOSAA (k = 0.01h-1) and diSAmPAP (k = 1.3∗10-3h-1). Photochemically produced PFOA consisted of branched and linear isomers and can therefore be used in source tracking. Experiments with different soils suggest that the oxidation of EtFOSAA to PFOA is expected to primarily be driven by hydroxyl radicals, whereas for the oxidation of EtFOSAA to further intermediates, another mechanism instead or in addition to the oxidation by hydroxyl radicals is responsible.

PFAS-Contaminated Soil Site in Germany: Nontarget Screening before and after Direct TOP Assay by Kendrick Mass Defect and FindPFΔS.

Soil contaminations with per- and polyfluoroalkyl substances (PFAS) are of great concern due to their persistence, leading to continuous, long-term groundwater contamination. A composite sample from contaminated agricultural soil from northwestern Germany (Brilon-Scharfenberg, North Rhine-Westphalia) was investigated in depth with nontarget screening (NTS) (Kendrick mass defect and MS2 fragment mass differences with FindPFΔS). Several years ago, selected PFCAs and PFSAs were identified on this site by detection in nearby surface and drinking water. We identified 10 further PFAS classes and 7 C8-based PFAS (73 single PFAS) previously unknown in this soil including some novel PFAS. All PFAS classes except for one class comprised sulfonic acid groups and were semi-quantified with PFSA standards from which ∼97% were perfluorinated and are not expected to be degradable. New identifications made up >75% of the prior known PFAS concentration, which was estimated to >30 µg/g. Pentafluorosulfanyl (-SF5) PFSAs are the dominant class (∼40%). Finally, the soil was oxidized with the direct TOP (dTOP) assay, revealing PFAA precursors that were covered to a large extent by identified H-containing PFAS and additional TPs (perfluoroalkyl diacids) were detected after dTOP. In this soil, however, dTOP + target analysis covers <23% of the occurring PFAS, highlighting the importance of NTS to characterize PFAS contaminations more comprehensively.

Early Ultrastructural Changes in Biopsies From Patients With Symptomatic CKD of Uncertain Etiology.

Introduction: Although the investigation of chronic kidney disease of uncertain etiology (CKDu) has identified many possible influencing factors in recent years, the exact pathomechanism of this disease remains unclear. Methods: In this study, we collected 13 renal biopsies from patients with symptomatic CKDu (Sym-CKDu) from Sri Lanka with well-documented clinical and socioeconomic factors. We performed light microscopy and electron microscopic evaluation for ultrastructural analysis, which was compared with 100 biopsies from German patients with 20 different kidney diseases. Results: Of the 13 Sri Lankan patients, 12 were men (92.3%), frequently employed in agriculture (50%), and experienced symptoms such as feeling feverish (83.3%), dysuria (83.3%), and arthralgia (66.6%). Light microscopic evaluation using activity and chronicity score revealed that cases represented early stages of CKDu except for 2 biopsies, which showed additional signs of diabetes. Most glomeruli showed only mild changes, such as podocyte foot process effacement on electron microscopy. We found a spectrum of early tubulointerstitial changes including partial loss of brush border in proximal tubules, detachment of tubular cells, enlarged vacuoles, and mitochondrial swelling associated with loss of cristae and dysmorphic lysosomes with electron-dense aggregates. None of these changes occurred exclusively in Sym-CKDu; however, they were significantly more frequent in these cases than in the control cohort. Conclusion: In conclusion, our findings confirm the predominant and early alterations of tubular structure in CKDu that can occur without significant glomerular changes. The ultrastructural changes do not provide concrete evidence of the cause of CKDu but were significantly more frequent in Sym-CKDu than in the controls.

Efficient PFAS prioritization in non-target HRMS data: systematic evaluation of the novel MD/C-m/C approach.

Non-target screening (NTS) based on high-resolution mass spectrometry (HRMS) is necessary to comprehensively characterize per- and polyfluoroalkyl substances (PFAS) in environmental, biological, and technical samples due to the very limited availability of authentic PFAS reference standards. Since in trace analysis, MS/MS information is not always achievable and only selected PFAS are present in homologous series, further techniques to prioritize measured HRMS data (features) according to their likelihood of being PFAS are highly desired due to the importance of efficient data reduction during NTS. Kaufmann et al. (J AOAC Int, 2022) presented a very promising approach to separate selected PFAS from sample matrix features by plotting the mass defect (MD) normalized to the number of carbons (MD/C) vs. mass normalized to the number of C (m/C). We systematically evaluated the advantages and limitations of this approach by using ~ 490,000 chemical formulas of organic chemicals (~ 210,000 PFAS, ~ 160,000 organic contaminants, and 125,000 natural organic matter compounds) and calculating how efficiently, and especially which, PFAS can be prioritized. While PFAS with high fluorine content (approximately: F/C > 0.8, H/F < 0.8, mass percent of fluorine > 55%) can be separated well, partially fluorinated PFAS with a high hydrogen content are more difficult to prioritize, which we discuss for selected PFAS. In the MD/C-m/C approach, even compounds with highly positive MDs above 0.5 Da and hence incorrectly assigned to negative MDs can still be separated from true negative mass defect features by the normalized mass (m/C). Furthermore, based on the position in the MD/C-m/C plot, we propose the estimation of the fluorine fraction in molecules for selected PFAS classes. The promising MD/C-m/C approach can be widely used in PFAS research and routine analysis. The concept is also applicable to other compound classes like iodinated compounds.

Identification of disinfection by-products (DBP) in thermal water swimming pools applying non-target screening by LC-/GC-HRMS.

The discovery of new disinfection by-products (DBPs) is still a rarely investigated research area in past studies. In particular, compared to freshwater pools, therapeutic pools with their unique chemical composition have rarely been investigated for novel DBPs. Here we have developed a semi-automated workflow that combines data from target and non-target screening, calculated and measured toxicities into a heat map using hierarchical clustering to assess the pool's overall potential chemical risk. In addition, we used complementary analytical techniques such as positive and negative chemical ionization to demonstrate how novel DBPs can be better identified in future studies. We identified two representatives of the haloketones (pentachloroacetone, and pentabromoacetone) and tribromo furoic acid detected for the first time in swimming pools. Non-target screening combined with target analysis and toxicity assessment may help to define risk-based monitoring strategies in the future, as required by regulatory frameworks for swimming pool operations worldwide.

Effects of the Antidepressant Amitriptyline on Juvenile Brown Trout and Their Modulation by Microplastics.

Pharmaceuticals such as antidepressants are designed to be bioactive at low concentrations. According to their mode of action, they can also influence non-target organisms due to the phylogenetic conservation of molecular targets. In addition to the pollution by environmental chemicals, the topic of microplastics (MP) in the aquatic environment came into the focus of scientific and public interest. The aim of the present study was to investigate the influence of the antidepressant amitriptyline in the presence and absence of irregularly shaped polystyrene MP as well as the effects of MP alone on juvenile brown trout (Salmo trutta f. fario). Fish were exposed to different concentrations of amitriptyline (nominal concentrations between 1 and 1000 µg/L) and two concentrations of MP (104 and 105 particles/L; <50 µm) for three weeks. Tissue cortisol concentration, oxidative stress, and the activity of two carboxylesterases and of acetylcholinesterase were assessed. Furthermore, the swimming behavior was analyzed in situations with different stress levels. Exposure to amitriptyline altered the behavior and increased the activity of acetylcholinesterase. Moreover, nominal amitriptyline concentrations above 300 µg/L caused severe acute adverse effects in fish. MP alone did not affect any of the investigated endpoints. Co-exposure caused largely similar effects such as the exposure to solely amitriptyline. However, the effect of amitriptyline on the swimming behavior during the experiment was alleviated by the higher MP concentration.

PhotoTOP: PFAS Precursor Characterization by UV/TiO2 Photocatalysis.

To unravel the complexity of per- and polyfluoroalkyl substances (PFAS) in products and environmental samples, sum parameters that provide relevant information on chemical characteristics are necessary since not all PFAS can be captured by target analysis in case of missing reference standards or if they are not extractable or amenable to the analytical method. Therefore, we evaluated photocatalysis (UV/TiO2) as a further total oxidizable precursor approach (PhotoTOP) to characterize perfluoroalkyl acid precursors via their conversion to perfluoroalkyl carboxylic acids (PFCAs). Photocatalysis has the advantage that no salts are needed, allowing direct injection with liquid chromatography-mass spectrometry without time-consuming and potentially discriminating sample cleanup. OH radicals were monitored with OH probes to determine the reactivity. For eight different precursors (diPAPs, FTSAs, FTCAs, N-EtFOSAA, PFOSA), mass balance was achieved within 4 h of oxidation, and also, in the presence of matrix, complete conversion was possible. The PhotoTOP was able to predict the precursor chain length of known and here newly identified precursors qualitatively when applied to two PFAS-coated paper samples and technical PFAS mixtures. The length of the perfluorinated carbon chain (n) was mostly conserved in the form of PFCAs (n-1) with only minor fractions of shorter-chain PFCAs. Finally, an unknown fabric sample and a polymer mixture (no PFAS detectable in extracts) were oxidized, and the generated PFCAs indicated the occurrence of side-chain fluorinated polymers.

FindPFΔS: Non-Target Screening for PFAS─Comprehensive Data Mining for MS2 Fragment Mass Differences.

The limited availability of analytical reference standards makes non-target screening approaches based on high-resolution mass spectrometry increasingly important for the efficient identification of unknown PFAS (per- and polyfluoroalkyl substances) and their TPs. We developed and optimized a vendor-independent open-source Python-based algorithm (FindPFΔS = FindPolyFluoroDeltas) to search for distinct fragment mass differences in MS/MS raw data (.ms2-files). Optimization with PFAS standards, two pre-characterized paper and soil samples (iterative data-dependent acquisition), revealed Δ(CF2)n, ΔHF, ΔCnH3F2n-3, ΔCnH2F2n-4, ΔCnHF2n-5, ΔCnF2nSO3, ΔCF3, and ΔCF2O as relevant and selective fragment differences depending on applied collision energies. In a PFAS standard mix, 94% (36 of 38 compounds from 10 compound classes) could be found by FindPFΔS. The use of fragment differences was applicable to a wide range of PFAS classes and appears as a promising new approach for PFAS identification. The influence of mass tolerance and intensity threshold on the identification efficiency and on the detection of false positives was systematically evaluated with the use of selected HR-MS2-spectra (20,998) from MassBank. To this end, with the use of FindPFΔS, we could identify different unknown PFAS homologues in the paper extracts. FindPFΔS is freely available as both Python source code on GitHub (https://github.com/JonZwe/FindPFAS) and as an executable windows application (https://doi.org/10.5281/zenodo.6797353) with a graphical user interface on Zenodo.

Occurrence of brominated disinfection by-products in thermal spas.

Thermal spas are gaining more and more popularity among the population because they are used for recreational purposes. Disinfecting these baths without losing the health benefits poses a challenge for swimming pool operators. Previous studies have mainly focused on regulated chlorinated DBPs in freshwater pools with no bromide or seawater pools with very high bromide content. Thermal water pools have a low bromide content and in combination with chlorine can lead to chlorinated, brominated and mixed halogenated DBP species. The occurrence of brominated and mixed halogenated DBPs in these types of pools is largely unexplored, with very few or limited studies published on regulated DBPs and even fewer on emerging DBP classes. In the field of swimming pool water disinfection, apart from extensive studies in the field of drinking water disinfection, only a few studies are known in which >39 halogenated and 16 non-halogenated disinfection by-products, including regulated trihalomethanes (THM) and haloacetic acids (HAA), were investigated in swimming pool water. Calculated bromine incorporation factor (BIF) demonstrated that even small amounts of bromide in swimming pool water can lead to a large shift in DBP species towards brominated and mixed halogenated DBPs. Dihaloacetonitriles (DHANs) accounted for >50% of the calculated cytotoxicity and genotoxicity on average. Comparison of the target analysis with the TOX showed that a major part of the measured TOX (69% on average) could be explained by the regulated classes THMs, HAAs, and the unregulated class of HANs. This study aims to help operators of swimming pools with bromide-containing water to gain a better understanding of DBP formation in future monitoring and to fill the knowledge gap that has existed so far on the occurrence of DBPs in thermal water pools.

LC-HRMS screening of per- and polyfluorinated alkyl substances (PFAS) in impregnated paper samples and contaminated soils.

High per- and polyfluorinated alkyl substance (PFAS) concentrations have been detected in agricultural soils in Southwest Germany. Discharges of PFAS-contaminated paper sludge and compost are suspected to be the cause of the contamination. Perfluorinated carboxylic acids (PFCAs) have been detected also in groundwater, drinking water, and plants in this area. Recently, previously unknown compounds have been identified by high-resolution mass spectrometry (HRMS). Major contaminants were polyfluorinated dialkylated phosphate esters (diPAPs) and N-ethyl perfluorooctane sulfonamide ethanol-based phosphate diester (diSAmPAP). In this study, HRMS screening for PFAS was applied to 14 soil samples from the contaminated area and 14 impregnated paper samples which were from a similar period than the contamination. The paper samples were characterized by diPAPs (from 4:2/6:2 to 12:2/12:2), fluorotelomer mercapto alkyl phosphates (FTMAPs; 6:2/6:2 to 10:2/10:2), and diSAmPAP. In soil samples, diPAPs and their transformation products (TPs) were the major contaminants, but also FTMAPs, diSAmPAP, and their TPs occurred. The distribution patterns of the carbon chain lengths of the precursor PFAS in soil samples were shown to resemble those in paper samples. This supports the hypothesis that paper sludge is a major source of contamination. The presence of major degradation products like PFCAs, FTSAs, or PFOS and their distribution of carbon chain lengths indicate the activity of biotic or abiotic degradation processes and selective leaching processes from the upper soil horizons.

Electrochemical Oxidation of 6:2 Polyfluoroalkyl Phosphate Diester-Simulation of Transformation Pathways and Reaction Kinetics with Hydroxyl Radicals.

Polyfluoroalkyl phosphate diesters (diPAPs) are widely used for paper and cardboard impregnation and discharged via waste streams from production processes and consumer products. To improve the knowledge about the environmental fate of diPAPs, electrochemical oxidation (EO) was used to characterize the transformation pathways and reaction kinetics. 6:2 diPAP was transformed electrochemically to perfluorocarboxylic acids (C5-C7 PFCAs) and two intermediates (6:2 fluorotelomer carboxylic acid, FTCA, and 6:2 fluorotelomer unsaturated carboxylic acid, FTUCA). EO of potential intermediates 6:2 monoPAP and 6:2 fluorotelomer alcohol (FTOH) showed similar transformation products but with different ratios. We show that 6:2 diPAP is initiated by OH radical (•OH) reactions, as evidenced by the measured steady-state concentrations of •OH with the probe molecule terephthalic acid, quenching experiments, and pH dependency of the reaction. PFHpA was the main product of 6:2 diPAP oxidation, and it was formed in a pseudo-first-order reaction for which a bimolecular rate constant was estimated to be kO•H,diPAPformPFHpA = 9.4(±1.4) × 107 M-1 s-1 by an initial rate approach. This can be utilized to estimate the environmental half-life of 6:2 diPAP for the reaction with •OH and the formation kinetics of persistent PFCAs.

Separation of Photochemical and Non-Photochemical Diurnal In-Stream Attenuation of Micropollutants.

For a better process understanding of in-stream attenuation of trace organic contaminants (TrOCs), quantitative comparisons between field studies under different environmental conditions and controlled laboratory experiments are important to separate different processes. However, this is hampered by the challenge to transfer kinetics from the laboratory to different field conditions due to the lack of good quantitative measures to account for different boundary conditions. For phototransformation, in situ light conditions in a river are difficult to determine because light is reduced, for instance, by absorption, scattering on suspended particles, and shading effects. In this study, we present an approach to separate photochemical from non-photochemical diurnal in-stream attenuation based on rate constants relative to diclofenac, as a reference compound, to account for the difference in the in situ light conditions combined with laboratory experiments. 12 out of 45 detected target TrOCs showed a diurnal attenuation at a selected river stretch. A non-photochemical process, potentially biotransformation, was responsible for the diurnal attenuation of bisoprolol, metoprolol, O-desmethylvenlafaxine, tramadol, and venlafaxine. Attenuation of amisulpride, flufenamic acid, hydrochlorothiazide, naproxen, and xipamide can be quantitatively explained by phototransformation, partially for sotalol. Attenuation rate constants of hydrochlorothiazide at different field sites from this study and from published data range over 2 orders of magnitude. Differences can be quantitatively explained by different light exposures but not by water chemical parameters.

Storm Event-Driven Occurrence and Transport of Dissolved and Sorbed Organic Micropollutants and Associated Effects in the Ammer River, Southwestern Germany.

Storm events lead to agricultural and urban runoff, to mobilization of contaminated particulate matter, and to input from combined sewer overflows into rivers. We conducted time-resolved sampling during a storm event at the Ammer River, southwest Germany, which is representative of small river systems in densely populated areas with a temperate climate. Suspended particulate matter (SPM) and water from 2 sampling sites were separately analyzed by a multi-analyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for 97 environmentally relevant organic micropollutants and with 2 in vitro bioassays. Oxidative stress response (AREc32) may become activated by various stressors covering a broad range of physicochemical properties and induction of aryl hydrocarbon receptor-chemical-activated luciferase gene expression (AhR-CALUX) by hydrophobic compounds such as dioxins and dioxin-like molecules. Compound numbers, concentrations, their mass fluxes, and associated effect fluxes increased substantially during the storm event. Micropollutants detected in water and on SPM pointed toward inputs from combined sewer overflow (e.g., caffeine, paracetamol), urban runoff (e.g., mecoprop, terbutryn), and agricultural areas (e.g., azoxystrobin, bentazone). Particle-facilitated transport of triphenylphosphate and tris(1-chloro-2-propyl) phosphate accounted for up to 34 and 33% of the total mass flux even though SPM concentrations were <1 g L-1 . Effect fluxes attributed to SPM were similar or higher than in the water phase. The important role of SPM-bound transport emphasizes the need to consider not only concentrations but also mass and effect fluxes for surface water quality assessment and wastewater/stormwater treatment options. Environ Toxicol Chem 2021;40:88-99. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Biodegradation of Pesticides at the Limit: Kinetics and Microbial Substrate Use at Low Concentrations.

The objective of our study was to test whether limited microbial degradation at low pesticide concentrations could explain the discrepancy between overall degradability demonstrated in laboratory tests and their actual persistence in the environment. Studies on pesticide degradation are often performed using unrealistically high application rates seldom found in natural environments. Nevertheless, biodegradation rates determined for higher pesticide doses cannot necessarily be extrapolated to lower concentrations. In this context, we wanted to (i) compare the kinetics of pesticide degradation at different concentrations in arable land and (ii) clarify whether there is a concentration threshold below which the expression of the functional genes involved in the degradation pathway is inhibited without further pesticide degradation taking place. We set up an incubation experiment for four weeks using 14C-ring labeled 2-methyl-4-chlorophenoxyacetic acid (MCPA) as a model compound in concentrations from 30 to 20,000 µg kg-1 soil. To quantify the abundance of putative microorganisms involved in MCPA degradation and their degradation activity, tfdA gene copy numbers (DNA) and transcripts (mRNA) were determined by quantitative real-time PCR. Mineralization dynamics of MCPA derived-C were analyzed by monitoring 14CO2 production and 14C assimilation by soil microorganisms. We identified two different concentration thresholds for growth and activity with respect to MCPA degradation using tfdA gene and mRNA transcript abundance as growth and activity indices, respectively. The tfdA gene expression started to increase between 1,000 and 5,000 µg MCPA kg-1 dry soil, but an actual increase in tfdA sequences could only be determined at a concentration of 20,000 µg. Accordingly, we observed a clear shift from catabolic to anabolic utilization of MCPA-derived C in the concentration range of 1,000 to 5,000 µg kg-1. Concentrations ≥1,000 µg kg-1 were mainly associated with delayed mineralization, while concentrations ≤1,000 µg kg-1 showed rapid absolute dissipation. The persistence of pesticides at low concentrations cannot, therefore, be explained by the absence of functional gene expression. Nevertheless, significant differences in the degradation kinetics of MCPA between low and high pesticide concentrations illustrate the need for studies investigating pesticide degradation at environmentally relevant concentrations.

Temporal and spatial variable in-stream attenuation of selected pharmaceuticals.

Organic micropollutants enter rivers mainly with discharges of wastewater treatment plants (WWTP) and pose a risk to aquatic ecosystems and water quality. A considerable knowledge gap exists for disentangling overlapping processes and driving conditions that control the fate of these pollutants. Thus, the aim of this study was to identify the driving parameters for attenuation of selected pharmaceuticals (carbamazepine, diclofenac, tramadol and venlafaxine) under field conditions. The presented study was performed at a small river (Ammer River, mean discharge 0.87 m3 s-1) which is hydrologically complex due to karstification, numerous artificial discharges, and engineered modifications of the channel. We applied a Lagrangian sampling scheme at two sequential river reaches. In general, for the investigated compounds and over the length of the tested reaches, the absolute net attenuation representative for 24 h was low (≤ 23% net attenuation), yet calculated half-lives were comparable to literature. Photodegradation is specifically relevant for the first river reach characterized by a higher net attenuation of the photosensitive compound diclofenac (14.5% ±11.3%) compared to the second section (9.8% ±13.7%). This is likely due to a spatial difference in canopy shading, which is supported by significant correlations (R2 ≥ 0.8) of the temporally changing 'temperature' and 'solar radiation' with time-specific degradation rate constants of photosensitive compounds for consecutive hourly water parcels. In general, the presented spatially and temporally resolved approach is a suitable tool to determine the attenuation of organic micropollutants and to narrow down the interpretation of net attenuation to a few reasonable processes.

Abiotic and biotic transformation of torasemide - Occurrence of degradation products in the aquatic environment.

The pharmaceutical torasemide is an important loop diuretic and was 2017 one of the ten most prescribed drugs in Germany. Despite its detection in different compartments of the urban water cycle including drinking water, no studies were so far performed to elucidate its fate in the environment and the occurrence of transformation products (TPs). Therefore, we investigated the phototransformation, microbial degradation, transformation with human liver microsomes and anodic oxidation of torasemide to obtain good coverage of environmentally relevant degradation products. Overall sixteen products were identified, covering the following reaction mechanisms: aromatic and aliphatic hydroxylation, including further oxidation to carboxylic acids and quinone imines, amide cleavage, N-dealkylation, N-dearylation, and sulfonamide hydrolysis to sulfonic acids. Especially the formation of quinone imines could be of concern as they are highly reactive electrophiles. Torasemide itself was observed in all investigated wastewater treatment plant (WWTP) samples and wastewater-impacted surface waters. The maximum detected concentration was about 350 ng L-1. Only three of the sixteen transformation products were generally observed in at least one of the samples and the most frequently detected TPs were the human metabolites hydroxytorasemide (TP 364a) and carboxytorasemide (TP 378a). The complete removal of TP 364a during wastewater treatment was in agreement with the results of microbial degradation experiments. TP 364a was most likely transformed into TP 378a, which was microbially less degraded in lab experiments. Based on estimated concentrations, TP 378a could reach about 1 µg L-1 in the investigated wastewater matrices.

Influence of Emission Sources and Tributaries on the Spatial and Temporal Patterns of Micropollutant Mixtures and Associated Effects in a Small River.

Organic micropollutants of anthropogenic origin in river waters may impair aquatic ecosystem health and drinking water quality. To evaluate micropollutant fate and turnover on a catchment scale, information on input source characteristics as well as spatial and temporal variability is required. The influence of tributaries from agricultural and urban areas and the input of wastewater were investigated by grab and Lagrangian sampling under base flow conditions within a 7.7-km-long stretch of the Ammer River (southwest Germany) using target screening for 83 organic micropollutants and 4 in vitro bioassays with environmentally relevant modes of action. In total, 9 pesticides and transformation products, 13 pharmaceuticals, and 6 industrial and household chemicals were detected. Further, aryl hydrocarbon receptor induction, peroxisome proliferator-activated receptor activity, estrogenicity, and oxidative stress response were measured in the river. The vast majority of the compounds and mixture effects were introduced by the effluent of a wastewater-treatment plant, which contributed 50% of the total flow rate of the river on the sampling day. The tributaries contributed little to the overall load of organic micropollutants and mixture effects because of their relatively low discharge but showed a different chemical and toxicological pattern from the Ammer River, though a comparison to effect-based trigger values pointed toward unacceptable surface water quality in the main stem and in some of the tributaries. Chemical analysis and in vitro bioassays covered different windows of analyte properties but reflected the same picture. Environ Toxicol Chem 2020;39:1382-1391. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.