Extrapolation of cytotoxic masked effects in planar in vitro assays.

The masking of specific effects in in vitro assays by cytotoxicity is a commonly known phenomenon. This may result in a partial or complete loss of effect signals. For common in vitro assays, approaches for identifying and quantifying cytotoxic masking are partly available. However, a quantification of cytotoxicity-affected signals is not possible. As an alternative, planar bioassays that combine high-performance thin layer chromatography with in vitro assays, such as the planar yeast estrogen screen (p-YES), might allow for a quantification of cytotoxically affected signals. Affected signals form a typical ring structure with a supressed or completely lacking centre that results in a double peak chromatogram. This study investigates whether these double peaks can be used for fitting a peak function to extrapolate the theoretical, unaffected signals. The precision of the modelling was evaluated for four individual peak functions, using 42 ideal, undistorted peaks from estrogenic model compounds in the p-YES. Modelled ED50-values from bisphenol A (BPA) experiments with cytotoxically disturbed signals were 13 times higher than for the apparent data without compensation for cytotoxicity (320 ± 63 ng versus 24 ± 17 ng). This finding has a high relevance for the modelling of mixture effects according to concentration addition that requires unaffected, complete dose-response relationships. Finally, we applied the approach to results of a p-YES assay on leachate samples of an elastomer material used in water engineering. In summary, the fitting approach enables the quantitative evaluation of cytotoxically affected signals in planar in vitro assays and also has applications for other fields of chemical analysis like distorted chromatography signals.

Multi-element analysis of unfiltered samples in river water monitoring-digestion and single-run analyses of 67 elements.

Routine analysis of inorganic analytes in whole water samples from rivers (unfiltered river water) is rarely reported in scientific publications. However, this sample type is valuable and often used in long-term monitoring, regulation, and catchment element budgets, as it includes the dissolved, colloidal, and particulate fraction in one sample type. Preservation measures are not needed and solid-liquid partitioning can be disregarded, which simplifies automated sampling and storage procedures. In this study, we provide several digestion protocols for whole water samples from rivers and the subsequent multi-element analysis of 67 major, minor, and trace elements: Li, Be, B, Na, Mg, Al, Si, P, S, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, Ru, Ag, Cd, In, Sn, Sb, Te, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Ir, Pt, Au, Hg, Tl, Pb, Bi, Th, U. In the absence of whole water reference materials for inorganic analytes, we introduce simulated whole water samples by suspending sediment reference materials as quality control measures. The applicability for improved routine water quality monitoring was successfully tested on samples from different rivers revealing variations of the element fingerprints over time.

Metabolites are overlooked in environmental risk assessments and monitoring of pharmaceuticals: The case study of pantoprazole.

The proton-pump inhibitor pantoprazole (PPZ) is one of the most consumed pharmaceuticals worldwide. Despite its high usage, reported PPZ concentrations in environmental water samples are comparatively low, which can be explained by the extensive metabolism of PPZ in the human body. Since most previous studies did not consider human PPZ metabolites it can be assumed that the current environmental exposure associated with the application of PPZ is substantially underestimated. In our study, 4'-O-demethyl-PPZ sulfide (M1) was identified as the predominant PPZ metabolite by analyzing urine of a PPZ consumer as well as the influent and effluent of a wastewater treatment plant (WWTP) using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). M1 was found to be ubiquitously present in WWTP effluents (max. concentration: 3 000 ng/L) and surface waters in Germany. On average, the surface water concentrations of M1 were approximately 30 times higher than those of the parent compound PPZ. Laboratory scale experiments demonstrated that activated carbon can considerably adsorb M1 und thus improve its removal during wastewater and drinking water treatment. Laboratory ozonation experiments showed a fast oxidation of M1, accompanied by the formation of several ozonation products. Certain ozonation products (identities confirmed via synthesized reference standards) were also detected in water samples collected after ozonation in a full-scale WWTP. Overall lower signal intensities were observed in the effluents of a sand filter and biologically active granular activated carbon filter, suggesting that the compounds were significantly removed during these post-ozonation treatment stages.

Hydro-geomorphic perspectives on microplastic distribution in freshwater river systems: A critical review.

Freshwater river systems are commonly defined as the main transport paths of microplastics (MP) from land into the seas. A shift in research interest from oceans to rivers can be observed, as a large number of i) case studies, ii) review papers and iii) experimental studies in this field have been published recently. Still, studies often lack an in-depth consideration of quantification, as units are mostly based on item numbers. Spatiotemporal aspects are often neglected. Transport paths linking MP sources and sinks in the environment are insufficiently understood and only recently the awareness increased that sustainable management of the MP pollution cannot be addressed without a sound knowledge of water- and sediment-driven MP transport. Within this review paper, we therefore i) reviewed 92 MP case-studies, with a special focus on spatiotemporal aspects and ii) gathered and compared global load-estimation data from these studies. We then outlined the key processes determining MP movement in rivers on the basis of existing laboratory experiments and theoretical approaches. A procedure to effectively compare units of MP in the water column and in riverine sediments was developed on the basis of i) an extensive MP-dataset in German waterways and ii) suspended sediment concentrations (SSC) of nearest monitoring stations of the German water and shipping authority. Our analysis indicates that relating MP in water samples to SSC reduces the often stated large difference between MP concentrations in the water column and bed sediments and therefore relativizes the importance of river beds as a major "MP sink". As for a quantification of MP fluxes, the use of MP masses as unit is crucial, we applied an approach to convert MP items to masses with the help of i) a power-law distribution of MP-particle size, triangular distributions of ii) form-ratios and iii) polymer densities. An evaluation with an own, extensive dataset of MP-particles showed reasonable results. Therefore, we translated global load data from item numbers to mass values for further analysis. Values were within a reasonable range, especially when considering the respective catchment size of each river at the sampling site.

Occurrence and distribution of emerging micropollutants in the central part of the German Bight.

Micropollutants (MPs) are transported via rivers from industrial and urban areas to the German Bight (G.B.). In contrast to the mounting rivers less information is available on the occurrence of MPs and their transformation products (TPs) in the marine environment of the G.B. In this study 83 compounds, including 26 metabolites of pharmaceuticals and environmental TPs were measured in water at 46 sampling sites in estuaries of Ems, Weser, Elbe, and the G.B. 36 MPs were even detected in the open sea areas (salinity > 34 psu) at 0.07-5.1 ng/L and to the best of our knowledge 10 MPs were detected in the marine environment for the first time. Concentrations of 8 MPs exceeded PNEC values suggesting a potential risk for sea life. Spatial distribution and relation of MPs with salinity allowed identifying emission paths for certain compounds and revealed the emissions from the River Elbe and Rhine.

Efficiency of five samplers to trap suspended particulate matter and microplastic particles of different sizes.

Suspended particulate matter (SPM) plays a major role in nutrient cycles and for the transport of pollutants within local and transboundary water catchments. Obtaining representative SPM samples from rivers, lakes, inland and coastal waters is crucial for quantitative and qualitative chemical analyses to correctly describe the chemical status of a water body. However, a representative sampling of SPM over time is challenging due to the heterogeneity of SPM particles sizes, their non-uniform distribution in rivers, and a variety of sampling devices being in use. Therefore, we investigated the efficiencies of five different sampling devices commonly used in national and international monitoring programs to collect representative SPM samples. We tested three passive sedimentation-based samplers (SBSs: sedimentation box, SB; sedimentation tank, ST; Raetz Sampler, RS), and two active separation techniques (continuous flow centrifuge, CFC; vacuum filtration, VF) in an experimental laboratory setup using in-house SPM standard suspensions (mineral, organic, and microplastic particles) with defined particle sizes. The mass-based efficiencies of the three examined SBSs were 0-66% for the mineral and organic particles <75 µm, where the mean particle sizes of collected samples were always shifted to bigger sizes compared to the initial suspensions. The efficiencies of the three SBSs to collect microplastic particles <80 µm were <20% due to the lower densities of microplastic compared to organic and mineral particles. In contrast to the SBSs, VF and CFC units showed excellent efficiencies >86% for all tested materials, with similar particle size distributions of the sampled material compared to those of the inlet suspensions. In conclusion, SPM sampling efficiencies of sampling units have to be carefully considered and compared to the respective aims of the monitoring approaches, especially when statements are derived from quantitative results on SPM.

Chemical and toxicological assessment of leachates from UV-degraded plastic materials using in-vitro bioassays.

The broad use of plastics and the persistence of the material results in plastic residues being found practically everywhere in the environment. If plastics remain in the (aquatic) environment, natural weathering leads to degradation processes and compounds may leach from plastic into the environment. To investigate the impact of degradation process on toxicity of leachates, different types of UV irradiation (UV-C, UV-A/B) were used to simulate weathering processes of different plastic material containing virgin as well as recyclate material and biodegradable polymers. The leached substances were investigated toxicologically using in-vitro bioassays. Cytotoxicity was determined by the MTT-assay, genotoxicity by using the p53-CALUX and Umu-assay, and estrogenic effects by the ERα-CALUX. Genotoxic as well as estrogenic effects were detected in different samples depending on the material and the irradiation type. In four leachates of 12 plastic species estrogenic effects were detected above the recommended safety level of 0.4 ng 17ß-estradiol equivalents/L for surface water samples. In the p53-CALUX and in the Umu-assay leachates from three and two, respectively, of 12 plastic species were found to be genotoxic. The results of the chemical analysis show that plastic material releases a variety of known and unknown substances especially under UV radiation, leading to a complex mixture with potentially harmful effects. In order to investigate these aspects further and to be able to give recommendations for the use of additives in plastics, further effect-related investigations are advisable.

Quantification of Poly(vinyl chloride) Microplastics via Pressurized Liquid Extraction and Combustion Ion Chromatography.

A reliable analytical method has been developed to quantify poly(vinyl chloride) (PVC) in environmental samples. Quantification was conducted via combustion ion chromatography (C-IC). Hydrogen chloride (HCl) was quantitatively released from PVC during thermal decomposition and trapped in an absorption solution. Selectivity of the marker HCl in complex environmental samples was ensured using cleanup via pressurized liquid extraction (PLE) with methanol at 100 °C (discarded) and tetrahydrofuran at 185 °C (collected). Using this method, recoveries of 85.5 ± 11.5% and a limit of quantification down to 8.3 µg/g were achieved. A variety of hard and soft PVC products could be successfully analyzed via C-IC with recoveries exceeding >95%. Furthermore, no measurable overdetermination was found for various organic and inorganic matrix ingredients, such as sodium chloride, sucralose, hydroxychloroquine, diclofenac, chloramphenicol, triclosan, or polychlorinated biphenyls. In addition, sediments and suspended particular matter showed PVC concentrations ranging up to 16.0 and 220 µg/g, respectively. However, the gap between determined polymer mass and particle masses could be significant since soft PVC products contain plasticizers up to 50 wt %. Hence, the results of the described method represent a sum of all chlorine-containing polymers, which are extractable under the chosen conditions.

Quantification of 68 elements in river water monitoring samples in single-run measurements.

Triple-quadrupole inductively coupled plasma mass spectrometry (ICP-QQQ-MS) is a unique analytical technique which is, next to speciation analyses, applied for the determination of total element concentrations in several matrices. Due to its wide linear range, short analysis times, and the collision-reaction gas technology, it is capable of addressing a high number of analytes in a single run with sufficient low limits of quantification for river water monitoring. Over the last decades, the focus of the environmental monitoring changed from "traditional" and regulated analytes to elements of possibly rising concern from new applications such as the so-called technology-critical elements (TCE). By widening the analytical window of this method for applications in networks of future river water monitoring, a better understanding of natural transport processes and global biogeochemical element cycles will be established and the total number of methods can be reduced. During method development and validation, certified reference materials, calibration check solutions, and spiked river water samples from 12 major German rivers covering different catchment areas were measured and evaluated with the three cell gases He, H2 and O2. The method delivers a best as possible undisturbed simultaneous determination for 68 out of 71 target analytes with recoveries in an accepted range of 80-120% for river water samples (dissolved fraction; <0.45 µm). After comprehensive evaluation, we offer a novel best-practice multi-element method for river water monitoring with the goal of fostering the exchange and discussion between practitioners in long-term river monitoring. It enables the readers to create their own methods based on the scientific needs to monitor elemental "fingerprints" of rivers and their catchments.

Protein fractionation and shotgun proteomics analysis of enriched bacterial cultures shed new light on the enzymatically catalyzed degradation of acesulfame.

The removal of organic micropollutants in municipal wastewater treatment is an extensively studied field of research, but the underlying enzymatic processes have only been elucidated to a small extent so far. In order to shed more light on the enzymatic degradation of the artificial sweetener acesulfame (ACE) in this context, we enriched two bacterial taxa which were not yet described to be involved in the degradation of ACE, an unknown Chelatococcus species and Ensifer adhaerens, by incubating activated sludge in chemically defined media containing ACE as sole carbon source. Cell-free lysates were extracted, spiked with ACE and analyzed via target LC-MS/MS, demonstrating for the first time enzymatically catalyzed ACE degradation outside of living cells. Fractionation of the lysate via two-dimensional fast protein liquid chromatography (FPLC) succeeded in a partial separation of the enzymes catalyzing the initial transformation reaction of ACE from those catalyzing the further transformation pathway. Thereby, an accumulation of the intermediate transformation product acetoacetamide-n-sulfonic acid (ANSA) in the ACE-degrading fractions was achieved, providing first quantitative evidence that the cleavage of the sulfuric ester moiety of ACE is the initial transformation step. The metaproteome of the enrichments was analyzed in the FPLC fractions and in the unfractionated lysate, using shotgun proteomics via UHPLC-HRMS/MS and label-free quantification. The comparison of protein abundances in the FPLC fractions to the corresponding ACE degradation rates revealed a metallo-ß-lactamase fold metallo-hydrolase as most probable candidate for the enzyme catalyzing the initial transformation from ACE to ANSA. This enzyme was by far the most abundant of all detected proteins and amounted to a relative protein abundance of 91% in the most active fraction after the second fractionation step. Moreover, the analysis of the unfractionated lysate resulted in a list of further proteins possibly involved in the transformation of ACE, most striking a highly abundant amidase likely catalyzing the further transformation of ANSA, and an ABC transporter substrate-binding protein that may be involved in the uptake of ACE into the cell.

Identification and trend analysis of organic cationic contaminants via non-target screening in suspended particulate matter of the German rivers Rhine and Saar.

Non-target screening of suspended particulate matter (SPM), collected from the German rivers Rhine and Saar, was conducted with the goal of identifying organic, permanent cationic contaminants and of estimating their temporal trends over an extended period. Therefore, annual composite samples of SPM, provided by the German Environmental Specimen Bank, were extracted and analyzed with high resolution LC-QToF-MS/MS. To facilitate the identification of substances belonging to the class "permanent cations", prioritization methods were applied utilizing the physicochemical properties of these compounds. These methods include both interactions of the analyte molecules with cation exchange resins and analyzing mass deviations when changing from non-deuterated to deuterated mobile phase solvents during LC-MS analysis. By applying both methods in a combined approach, 123 of the initially detected 2695 features were prioritized, corresponding to a 95% data reduction. This led to the identification of 22 permanent cationic species. The organic dyes Basic Yellow 28 and Fluorescent Brightener 363 as well as two quaternary ammonium compounds (QACs) were detected in environmental samples for the first time to best of or knowledge. The other compounds include additional QACs, as well as quaternary tri-phenylphosphonium compounds (QPC/TPP). In addition to identification, we determined temporal trends of all compounds over a period of 13 years and assessed their ecotoxicological relevance based on estimated concentrations. The two QACs oleyltrimethylammonium and eicosyltrimethylammonium show significant increasing trends in the Rhine SPM and maximum concentrations in the Saar SPM of about 900 and 1400 µg/kg, respectively. In the case of the dyes, constant trends have been observed at the end of the studied period, but also maximum concentrations of 400 µg/kg for Basic Yellow 28 in 2006 and 1000 µg/kg for Fluorescent Brightener 363 in 2015, potentially indicating a strong ecotoxicological risk.

Communication Breakdown: Into the Molecular Mechanism of Biofilm Inhibition by CeO2 Nanocrystal Enzyme Mimics and How It Can Be Exploited.

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C12-AHL a QS signaling compound used by Pseudomonas aeruginosa, was detected in the presence of CeO2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

Close-range remote sensing-based detection and identification of macroplastics on water assisted by artificial intelligence: A review.

Detection and identification of macroplastic debris in aquatic environments is crucial to understand and counter the growing emergence and current developments in distribution and deposition of macroplastics. In this context, close-range remote sensing approaches revealing spatial and spectral properties of macroplastics are very beneficial. To date, field surveys and visual census approaches are broadly acknowledged methods to acquire information, but since 2018 techniques based on remote sensing and artificial intelligence are advancing. Despite their proven efficiency, speed and wide applicability, there are still obstacles to overcome, especially when looking at the availability and accessibility of data. Thus, our review summarizes state-of-the-art research about the visual recognition and identification of different sorts of macroplastics. The focus is on both data acquisition techniques and evaluation methods, including Machine Learning and Deep Learning, but resulting products and published data will also be taken into account. Our aim is to provide a critical overview and outlook in a time where this research direction is thriving fast. This study shows that most Machine Learning and Deep Learning approaches are still in an infancy state regarding accuracy and detail when compared to visual monitoring, even though their results look very promising.

Making waves: Time for chemical surface water quality monitoring to catch up with its technical potential.

A comprehensive real-time evaluation of the chemical status of surface water bodies is still utopian, but in our opinion, it is time to use the momentum delivered by recent advanced technical, infrastructural, and societal developments to get significantly closer. Procedures like inline and online analysis (in situ or in a bypass) with close to real-time analysis and data provision are already available in several industrial sectors. In contrast, atline and offline analysis involving manual sampling and time-decoupled analysis in the laboratory is still common practice in aqueous environmental monitoring. Automated tools for data analysis, verification, and evaluation are changing significantly, becoming more powerful with increasing degrees of automation and the introduction of self-learning systems. In addition, the amount of available data will most likely in near future be increased by societal awareness for water quality and by citizen science. In this analysis, we highlight the significant potential of surface water monitoring techniques, showcase "lighthouse" projects from different sectors, and pin-point gaps we must overcome to strike a path to the future of chemical monitoring of inland surface waters.

Open-source feature detection for non-target LC-MS analytics.

RATIONALE: Non-target screening techniques using high-resolution mass spectrometers become more and more important for environmental sciences. Highly reliable and sophisticated software solutions are required to deal with the large amount of data obtained from such analyses. METHODS: Processing of high-resolution LC-HRMS data was performed upon conversion into an open, XML-based data format followed by an automated assignment of chromatographic peaks using the open-source programming language R. Raw data from three different LC-HRMS systems were processed as a proof of principle. RESULTS: We present a simple and straightforward algorithm to extract chromatographic peaks from previously m/z-centroided data based on the open-source programming language R and C++. The working principle and processing parameters are explained in detail. A ready-to-use script is provided in the supporting information. CONCLUSIONS: The developed algorithm enables a comprehensible automated peak picking of non-target LC-MS data. Application to three completely different HRMS raw data files showed reasonable False Positives and False Negatives detection and moderate calculation times.

Biotransformation of pregabalin in surface water matrices and the occurrence of transformation products in the aquatic environment - comparison to the structurally related gabapentin.

The biodegradability of the anticonvulsant pregabalin (PGB) was studied in laboratory incubation experiments in contact with water/sediment systems under different redox conditions. PGB was degraded by biological processes under aerobic conditions reaching half-lives of 8 to 10 d, while inactivated and anaerobic control experiments revealed no significant decrease of PGB concentrations. Within experiments spiked with elevated PGB concentrations, 12 TPs were formed and tentative chemical structures could be proposed by accurate masses and fragmentation pathways detected via measurements with high resolution mass spectrometry (LC-QToF-MS). Four of the proposed TPs were finally confirmed either by authentic reference standards (PGB-Lactam, ISA, TP 157-A (II)) or a self-synthesized standard (NA-PGB). PGB-Lactam was identified as the quantitatively most relevant TP formed via intramolecular cyclization under aerobic conditions, reaching up to 33% of the initial PGB concentration. Incubation experiments spiked with PGB-Lactam revealed three times higher half-lives compared to the parent compound, indicating that PGB-Lactam is more stable than PGB. A comparison with results gained from water/sediment incubation experiments with the structurally related compound gabapentin (GBP) revealed, that the transformation behaviour can be mainly extrapolated to PGB. Most of the observed transformation reactions found for PGB were comparable to the ones found for GBP. The TPs PGB-Lactam and NA-PGB as well as three GBP TPs (GBP-Lactam, NA-GBP and CCHA) have been detected in German wastewater treatment plants (WWTPs) effluents and the river Rhine including some of its tributaries such as Main, Neckar, Moselle and Aare. Moreover, GBP and PGB as well as some of their TPs were detected in German bank filtrates and finished drinking waters up to 260 ng L-1. For that reason these compounds should be monitored in drinking water in the future.

Micropollutant transformation and taxonomic composition in hybrid MBBR - A comparison of carrier-attached biofilm and suspended sludge.

The suspended sludge and carrier-attached biofilms of three different hybrid moving bed biofilm reactor (MBBR) systems were investigated with respect to their transformation potential for a broad range of micropollutants (MPs) as well as their microbial community composition. For this purpose, laboratory-scale batch experiments were conducted with the separated suspended sludge and the carrier-attached biofilm of every system in triplicate. For all batches the removal of 31 MPs as well as the composition of the microbial community were analyzed. The carrier-attached biofilms from two hybrid MBBR systems showed a significant higher overall transformation potential in comparison to the respective suspended sludge. Especially for the MPs trimethoprim, diclofenac, mecoprop, climbazole and the human metabolite 10,11-dihydro-10-hydroxycarbamazepine consistently higher pseudo-first-order transformation rates could be observed in all three systems. The analysis of the taxonomic composition revealed taxa showing higher relative abundances in the carrier-attached biofilms (e. g. Nitrospirae and Chloroflexi) and in the suspended biomasses (e. g. Bacteroidetes and Betaproteobacteria). Correlations of the biodiversity indices and the MP biotransformation rates resulted in significant positive associations for 11 compounds in suspended sludge, but mostly negative associations for the carrier-attached biofilms. The distinct differences in MP removal between suspended sludge and carrier-attached biofilm of the three different MBBR systems were also reflected by a statistically significant link between the occurrence of specific bacterial taxa (Acidibacter, Nitrospira and Rhizomicrobium) and MP transformation rates of certain MPs. Even though the identified correlations might not necessarily be of causal nature, some of the identified taxa might serve as suitable indicators for the transformation potential of suspended sludge or carrier-attached biofilms.

Ozonation products of zidovudine and thymidine in oxidative water treatment.

Ozonation is an advanced treatment technology that is increasingly used for the removal of organic micropollutants from wastewater and drinking water. However, reaction of organic compounds with ozone can also result in the formation of toxic transformation products. In the present study, the degradation of the antiviral drug zidovudine during ozonation was investigated. To obtain further insights into the reaction mechanisms and pathways, results of zidovudine were compared with the transformation of the naturally occurring derivative thymidine. Kinetic experiments were accompanied by elucidation of formed transformation products using lab-scale batch experiments and subsequent liquid chromatography - high resolution mass spectrometry (LC-HRMS) analysis. Degradation rate constants for zidovudine with ozone in the presence of t-BuOH as radical scavenger varied between 2.8 ∙ 104 M-1 s-1 (pH 7) and 3.2 ∙ 104 M-1 s-1 (pH 3). The structural difference of zidovudine to thymidine is the exchange of the OH-moiety by the azide function at position 3'. In contrast to inorganic azide, no reaction with ozone was observed for the organic bound azide. In total, nine transformation products (TPs) were identified for both zidovudine and thymidine. Their formation can be attributed to the attack of ozone at the C-C-double bond of the pyrimidine-base. As a result of rearrangements, the primary ozonide decomposed in three pathways forming two different TPs, including hydroperoxide TPs. Rearrangement reactions followed by hydrolysis and subsequent release of H2O2 further revealed a cascade of TPs containing amide moieties. In addition, a formyl amide riboside and a urea riboside were identified as TPs indicating that oxidations of amide groups occur during ozonation processes.

Fate and behavior of progestogens in activated sludge treatment: Kinetics and transformation products.

Previous studies have shown the high ecotoxicological potential of progestogens (PGs) on the reproductive system of aquatic organisms. Yet the ubiquitous presence of several PGs in wastewater treatment plant (WWTP) effluents indicates an incomplete removal during treatment. To investigate the fate and behavior of PGs during biological wastewater treatment, nine commonly used PGs were incubated in aerobic lab-scale degradation experiments with activated sludge taken from a municipal WWTP. The degradation kinetics revealed a fast removal after 48 h for most of the compounds. Cyproterone acetate and dienogest were the most recalcitrant of the analyzed steroids with half-lives of 8.65 h and 4.55 h, respectively. Thus, only moderate removals of these PGs can be predicted in full-scale WWTPs. Moreover, numerous transformation products (TPs) were detected via high-resolution mass spectrometry. Hydrogenation or dehydrogenation of ring A and non-selective hydroxylations of 17α-hydroxyprogesterone derivatives (medroxyprogesterone acetate, chlormadinone acetate, cyproterone acetate) as well as for 19-nortestosterone derivatives (dienogest, norethisterone acetate, etonogestrel) were observed as major transformation reactions. Seven of the identified TPs were confirmed by reference standards. The biodegradation of cyproterone acetate revealed an almost quantitative transformation to 3α­hydroxy cyproterone acetate which is reported to be genotoxic. In a comparative evaluation of the TPs formed and the steroid structure, it was observed that molecular structure played a role in the inhibition of several transformation reactions, explaining the increased recalcitrance of these compounds. In addition, aromatization of the steroid ring A was identified for the 19-nortestosterone derivatives leading to the formation of estrogen-like TPs. For instance, the degradation of norethisterone acetate led to the formation of 17α-ethinylestradiol, a well-known and very potent synthetic estrogen. The evidence of the conversion of progestogenic to estrogenic compounds and the formation of potentially hazardous TPs indicates the need of a more comprehensive environmental risk assessment for synthetic steroids. Two of the newly identified TPs (3α-hydroxy cyproterone acetate and ∆9,11-dehydro-17α-cyanomethyl estradiol) were detected in WWTP effluents for the first time.