Development of an analytical method to quantify N-acyl-homoserine lactones in bacterial cultures, river water, and treated wastewater.

N-Acyl-homoserine lactones (AHL) play a major role in the communication of Gram-negative bacteria. They influence processes such as biofilm formation, swarming motility, and bioluminescence in the aquatic environment. A comprehensive analytical method was developed to elucidate the "chemical communication" in pure bacterial cultures as well as in the aquatic environment and engineered environments with biofilms. Due to the high diversity of AHLs and their low concentrations in water, a sensitive and selective LC-ESI-MS/MS method combined with solid-phase extraction was developed for 34 AHLs, optimized and validated to quantify AHLs in bacterial conditioned medium, river water, and treated wastewater. Furthermore, the developed method was optimized in terms of enrichment volume, internal standards, limits of detection, and limits of quantification in several matrices. An unanticipated variety of AHLs was detected in the culture media of Pseudomonas aeruginosa (in total 8 AHLs), Phaeobacter gallaeciensis (in total 6 AHLs), and Methylobacterium mesophilicum (in total 15 AHLs), which to our knowledge have not been described for these bacterial cultures so far. Furthermore, AHLs were detected in river water (in total 5 AHLs) and treated wastewater (in total 3 AHLs). Several detected AHLs were quantified (in total 24) using a standard addition method up to 7.3±1.0 µg/L 3-Oxo-C12-AHL (culture media of P. aeruginosa).

Bromination of Quorum Sensing Molecules: Vanadium Bromoperoxidase and Cerium Dioxide Nanocrystals via Free Active Bromine Transform Bacterial Communication.

The halogenation of quorum sensing molecules (QSMs) is known to be catalyzed by enzymes such as haloperoxidase (HPO) as well as cerium dioxide nanocrystals (NC), which mimic enzymes. Those enzymes and mimics can influence biological processes such as biofilm formation, where bacteria use QSMs for the "chemical" communication between each other and the coordination of surface colonization. However, not much is known about the degradation behavior of a broad spectrum of QSMs, especially for HPO and its mimics. Therefore, in this study, the degradation of three QSMs with different molecule moieties was elucidated. For this purpose, different batch experiments were carried out with HPOs, NCs and free active bromine (FAB). For N-ß-ketocaproyl-homoserine lactone (3-Oxo-C6-AHL), N-cis-tetradec-9Z-enoyl-homoserine lactone (C14:1-AHL) and 2-heptyl-4-quinolone (HHQ) a fast degradation and moiety-specific transformations were observed. The HPO vanadium bromoperoxidase as well as cerium dioxide NCs catalyzed the formation of the same brominated transformation products (TPs). Since the same TPs are formed in batch experiments with FAB it is very likely that FAB is playing a major role in the catalytical reaction mechanism leading to the transformation of QSMs. In this study in total 17 TPs could be identified in different levels of confidence and the catalytic degradation processes for two QS groups (unsaturated AHLs and alkyl quinolones) with cerium dioxide NCs and vanadium bromoperoxidase were expanded.

Elucidation of removal processes in sequential biofiltration (SBF) and soil aquifer treatment (SAT) by analysis of a broad range of trace organic chemicals (TOrCs) and their transformation products (TPs).

Many chemicals with different physico-chemical properties are present in municipal wastewater. In this study, the removal of a broad range of trace organic chemicals (TOrCs) was determined in two biological treatment processes differing in hydraulic retention time: sequential biofiltration (SBF) and soil-aquifer treatment (SAT), operated in Germany and Spain. Occurrence and the degree of removal of more than 150 TOrCs with different physico-chemical properties were analysed, including precursors as well as human metabolites and environmental transformation products (TPs). Ninety TOrCs were detected in the feed water of the SBF system, 40% of these showed removal efficiencies of higher than 30% during biological treatment. In SAT, 70 TOrCs were detected in the feed water, 60% of these could be reduced by more than 30% after approximately 3 days of subsurface treatment. For uncharged and negatively charged TOrCs biological degradation was mainly responsible for the removal, while positively charged TOrCs were most likely also removed by ionic interactions. The detections of TPs confirmed that biodegradation was a major removal process in both systems. The analysis of positively and negatively charged, neutral and zwitterionic TOrCs and the simultaneous analysis of precursors and their biologically formed TPs enabled a detailed understanding of underlying mechanisms of their removal in the two systems. On this basis, criteria for site-specific indicator selection were proposed.

Influence of environmental parameters and of their interactions on the release of metal(loid)s from a construction material in hydraulic engineering.

Besides the leaching behaviour of a construction material under standardised test-specific conditions with laboratory water, for some construction materials it is advisable to test their environmental behaviour also under close to end use conditions. The envisaged end use combined with the product characteristics (e.g. mineral phases) is decisive for the choice of environmental factors that may change the release of substance that potentially cause adverse environmental effects (e.g. fertilisation or ecotoxicity). At the moment an experimental link is missing between mono-factorial standardised test systems and non standardised complex incubation experiments such as mesocosms which are closer to environmental conditions. Multi-factorial batch experiments may have the potential to close the gap. To verify this, batch experiments with copper slag were performed which is used as armour stones in hydraulic engineering. Design of experiments (DoE) was applied to evaluate the impact of pH, ionic strength, temperature and sediment content on the release of As, Cu, Mo, Ni, Pb, Sb and Zn. The study shows that release and sediment-eluent partitioning of metal(loid)s are impacted by interactions between the studied factors. Under the prevalent test conditions sediment acts as a sink enhancing most strongly the release of elements from the material.

Quality assessment of digested sludges produced by advanced stabilization processes.

The European Union (EU) Project Routes aimed to discover new routes in sludge stabilization treatments leading to high-quality digested sludge, suitable for land application. In order to investigate the impact of different enhanced sludge stabilization processes such as (a) thermophilic digestion integrated with thermal hydrolysis pretreatment (TT), (b) sonication before mesophilic/thermophilic digestion (UMT), and (c) sequential anaerobic/aerobic digestion (AA) on digested sludge quality, a broad class of conventional and emerging organic micropollutants as well as ecotoxicity was analyzed, extending the assessment beyond the parameters typically considered (i.e., stability index and heavy metals). The stability index was improved by adding aerobic posttreatment or by operating dual-stage process but not by pretreatment integration. Filterability was worsened by thermophilic digestion, either alone (TT) or coupled with mesophilic digestion (UMT). The concentrations of heavy metals, present in ranking order Zn > Cu > Pb > Cr ~ Ni > Cd > Hg, were always below the current legal requirements for use on land and were not removed during the processes. Removals of conventional and emerging organic pollutants were greatly enhanced by performing double-stage digestion (UMT and AA treatment) compared to a single-stage process as TT; the same trend was found as regards toxicity reduction. Overall, all the digested sludges exhibited toxicity to the soil bacterium Arthrobacter globiformis at concentrations about factor 100 higher than the usual application rate of sludge to soil in Europe. For earthworms, a safety margin of factor 30 was generally achieved for all the digested samples.

Quantification of long-term wastewater fluxes at the surface water/groundwater-interface: an integrative model perspective using stable isotopes and acesulfame.

The suitability of acesulfame to trace wastewater-related surface water fluxes from streams into the hyporheic and riparian zones over long-term periods was investigated. The transport behavior of acesulfame was compared with the transport of water stable isotopes (δ(18)O or δ(2)H). A calibrated model based on a joint inversion of temperature, acesulfame, and piezometric pressure heads was employed in a model validation using data sets of acesulfame and water stable isotopes collected over 5months in a stream and groundwater. The spatial distribution of fresh water within the groundwater resulting from surface water infiltration was estimated by computing groundwater ages and compared with the predicted acesulfame plume obtained after 153day simulation time. Both, surface water ratios calculated with a mixing equation from water stable isotopes and simulated acesulfame mass fluxes, were investigated for their ability to estimate the contribution of wastewater-related surface water inflow within groundwater. The results of this study point to limitations for the application of acesulfame to trace surface water-groundwater interactions properly. Acesulfame completely missed the wastewater-related surface water volumes that still remained in the hyporheic zone under stream-gaining conditions. In contrast, under stream-losing conditions, which developed after periods of stagnating hydraulic exchange, acesulfame based predictions lead to an overestimation of the surface water volume of up to 25% in the riparian zone. If slow seepage velocities prevail a proportion of acesulfame might be stored in smaller pores, while when released under fast flowing water conditions it will travel further downstream with the groundwater flow direction. Therefore, under such conditions acesulfame can be a less-ideal tracer in the hyporheic and riparian zones and additional monitoring with other environmental tracers such as water stable isotopes is highly recommended.

Electrochemical treatment of iopromide under conditions of reverse osmosis concentrates--elucidation of the degradation pathway.

Application of reverse osmosis for the reuse of treated wastewater on the one hand offers a way to provide high quality effluent waters. On the other hand reverse osmosis concentrates exhibiting highly concentrated contaminants are produced simultaneously. Electrochemical treatment of those concentrates is regarded as one possible answer to the problem of their disposal into surface waters. Nevertheless, due to the diversity of direct and indirect degradation processes during electrolysis, special care has to be taken about the formation of toxic transformation products (TPs). In this study the electrochemical transformation of the X-ray contrast medium iopromide was investigated as a representative of biologically persistent compounds. For this purpose, anodic oxidation at boron doped diamond as well as cathodic reduction using a platinum electrode were considered. Kinetic analyses revealed a transformation of 100 µM iopromide with first order kinetic constants between 0.6 and 1.6 × 10(-4) s(-1) at the beginning and a subsequent increase of the reaction order due to the influence of secondary oxidants formed during electrolysis. Mineralization up to 96% was achieved after about 7.5 h. At shorter treatment times several oxidatively and reductively formed transformation products were detected, whereas deiodinated iopromide represented the major fraction. Nevertheless, the latter exhibited negligible toxicological relevance according to tests on vibrio fisheri. Additional experiments utilizing a divided cell setup enabled the elucidation of the transformation pathway, whereas emerging TPs could be identified by means of high resolution mass spectrometry and MS(n)-fragmentations. During electrolysis the iodine released from Iopromide was found to 90% as iodide and to 10% as iodate even in the open cell experiments, limiting the potential formation of toxic iodo-disinfection by-products. Chlorinated TPs were not found.

Determination of the long-term release of metal(loid)s from construction materials using DGTs.

Long-term leaching experiments are crucial to estimate the potential release of dangerous substances from construction materials. The application of Diffuse Gradients in Thin film (DGT) in static-batch experiments was tested to study the long-term release of metal(loid)s from construction materials for hydraulic engineering, for half a year. Long-term release experiments are essential to improve calculations of the life-time release for this materials. DGTs in batch experiments were found to be a space and labour efficient application, which enabled (i) to study, in a non-invasive manner, the total release of nine metal(loid)s for half a year, (ii) to differentiate between release mechanisms and (iii) to study mechanisms which were contrary to the release or caused experimental artefacts in the batch experiments. For copper slag (test material) it was found that eight metal(loid)s were released over the whole time period of 184 d. Cu, Ni and Pb were found to be released, predominantly caused by (the) weathering of sulphide minerals. Only for Zn a surface depletion mechanism was identified. The results from the long-term batch experiments deliver new information on the release of metal(loid)s during the life cycle of construction materials with regard to river basin management objectives.

Photodegradation kinetics and transformation products of ketoprofen, diclofenac and atenolol in pure water and treated wastewater.

Pharmaceutical compounds such as ketoprofen, diclofenac and atenolol are frequently detected at relatively high concentrations in secondary effluents from wastewater treatment plants. Therefore, it is important to assess their transformation kinetics and intermediates in subsequent disinfection processes, such as direct ultraviolet (UV) irradiation. The photodegradation kinetics of these compounds using a medium pressure (MP) lamp was assessed in pure water, as well as in filtered and unfiltered treated wastewater. Ketoprofen had the highest time- and fluence-based rate constants in all experiments, whereas atenolol had the lowest values, which is consistent with the corresponding decadic molar absorption coefficient and quantum yield. The fluence-based rate constants of all compounds were evaluated in filtered and unfiltered wastewater matrices as well as in pure water. Furthermore, transformation products of ketoprofen, diclofenac and atenolol were identified and monitored throughout the irradiation experiments, and photodegradation pathways were proposed for each compound. This enabled the identification of persistent transformation products, which are potentially discharged from WWTP disinfection works employing UV photolysis.

Leaching of metal(loid)s from a construction material: influence of the particle size, specific surface area and ionic strength.

Construction materials are tested worldwide for a potential release of dangerous substances to prevent adverse effects on humans and biota. It is crucial to identify and understand the processes which are decisive for the release of hazardous substances. The current study compares the results of different test methods. Taking copper slag as model material, the influence of material particle size, eluant composition and ionic strength was tested. Ionic strength and salinity significantly influenced the release of metal(loid)s in the water phase. Furthermore, it was elucidated that colloids can cause methodological artefacts. The available specific surface area exhibited a positive correlation with the release of hazardous substances. The specific surface areas of materials were determined by the Brunauer, Emmett and Teller model (BET) and four other methods. The aluminium foil method showed the best results with regard to the statistical uncertainty, compared to a 3D laser scanning method. With help of the roughness factor λ it is possible to compare the results from surface area measurements with different material particle sizes (0-250 mm). This comparability offers the potential to match the release of metal(loid)s from laboratory studies with field applications and catchment area calculations/modelling, based on the release per m(2).

Development of an analytical method to determine avermectins in water, sediments and soils using liquid chromatography-tandem mass spectrometry.

A comprehensive analytical multi-residue method has been developed for the determination of seven avermectins (abamectin, doramectin, ivermectin, emamectin benzoate, eprinomectin, moxidectin and selamectin) in surface water, sediment and soil samples. Solid samples were extracted applying pressurised liquid extraction followed by a solid-phase extraction (SPE) clean-up step. For aqueous samples, extraction was done utilising only SPE. All compounds were measured using liquid chromatography coupled to tandem mass spectrometry using atmospheric pressure chemical ionisation. The recoveries were 38-67% (relative standard deviation: 9-26%), 63-88% (16-23%) and 63-80% (9-15%) for spiked Rhine water, spiked sediment and spiked soil samples, respectively, and limit of quantifications were 2.5-14 ngl(-1) in water and 0.5-2.5 ngg(-1) in soil and sediment.

First interlaboratory exercise on non-steroidal anti-inflammatory drugs analysis in environmental samples.

Comparability of monitoring data are essential for any meaningful assessment and for the management of environmental risks of emerging pollutants. The reliability and comparability of data at European level is often limited, because analytical methods for emerging pollutants are often not fully validated, not harmonized or not suitable for all relevant matrices. This paper describes a collaborative interlaboratory exercise for the analysis of non-steroidal anti-inflammatory drugs (NSAIDs) residues in freshwater and wastewater, held in the framework of the EU project "Network of reference laboratories for monitoring of emerging environmental pollutants" (NORMAN). The NSAID compounds selected in this study were ketoprofen, naproxen, ibuprofen and diclofenac. Thirteen laboratories distributed along nine European Countries (Austria, France, Germany, Greece, Italy, Slovak Republic, Slovenia, Spain, and Switzerland) took part in this exercise, 126 samples were analyzed and a total number of 473 values in duplicate were collected. Samples selected in this study include environmental water (river water and waste water) and artificial water (fortified environmental and distilled water) with different ranges of complexity. Two analytical methods were proposed by the organiser; one is based on the use of solid phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the second one is based on SPE followed by gas-chromatography-mass spectrometry (GC-MS), however, in the first round some different approaches were also admitted. The main goals of this interlaboratory comparison were to evaluate the available analytical schemes for NSAID analysis in natural waters, to evaluate the repeatability (r) and reproducibility (R) between participating laboratories, and to evaluate the influence of the analytical method and sample matrices on the results.

Are we about to upgrade wastewater treatment for removing organic micropollutants?

Activated sludge treatment allows only for a partial removal of micropollutants, mainly via sorption and biological degradation. Ozonation and activated carbon filtration are processes bearing the potential to drastically reduce the micropollutant load discharged to the environment after (centralized) biological treatment. The estimated total costs between 0.05 and 0.20 euro per m3 treated water (depending on plant size and effluent DOC content) represent only a small fraction of the total costs for urban wastewater management and are therefore considered feasible. Full scale testing is currently planned or under way with the aim to a) confirm this cost estimation and b) to demonstrate the benefit by quantification of the effect of removal and by documenting the impact on the ecology of receiving waters. Ozonation would have the additional advantage of achieving partial disinfection. Another issue currently being intensively studied is the byproducts formed during ozonation and their toxicity. Evidence is needed that the formed ozonation byproducts are either harmless or easily degradable. Since a 5% to 20% loss of sewage is occurring due to sewer leakage and combined sewer overflow an improved reduction of micropollutant input to the aquatic environment requires that advanced centralized treatment is complemented with measures taken before discharge into the sewer. Options hereto may be waste design, labeling of compounds according to environmental friendliness or separate treatment of quantitatively significant point sources (e.g. hospital wastewater, nursery homes, industrial wastewater).

Distribution of estrogens, 17beta-estradiol and estrone, in Canadian municipal wastewater treatment plants.

The distribution of female hormones, 17beta-estradiol and estrone, was determined in effluents of 18 selected municipal treatment plants across Canada. Replicate 24-h composite samples were collected from the influent and final effluent of each treatment plant, and the removal efficiency compared to the operational characteristics of the plants. In conventional activated sludge and lagoon treatment systems, the mean concentrations of 17beta-estradiol and estrone in influent were 15.6 ng/l (range 2.4-26 ng/l) and 49 ng/l (19-78 ng/l). In final effluents, the mean concentrations of both 17beta-estradiol and estrone were reduced to 1.8 ng/l (0.2-14.7 ng/l) and 17 ng/l (1-96 ng/l), respectively. 17beta-estradiol was removed effectively, >75% and as high as 98%, in most of the conventional mechanical treatment systems with secondary treatment. The removal of estrone was much more complex with removal varying from 98% to situations where the concentrations in the effluent were elevated above that detected in the influent. The estrogenicity, measured using a transfected estrogen receptor in yeast (YES) assay, was also variable, ranging from high removal to elevations of estrogenicity in final effluent. Although the apparent removals were not statistically correlated with either hydraulic (HRT) or solid (SRT) retention times, plants or lagoons with high SRT were very effective at reducing the levels of hormones. Well-operated plants that achieved nitrification also tended to have higher removal of hormones than those that did not nitrify. Laboratory aerobic reactor experiments confirmed the rapid removal of 17beta-estradiol, estrone, and estrogenicity when exposed to sewage slurries.

Estrogenicity of solid phase-extracted water samples from two municipal sewage treatment plant effluents and river Rhine water using the yeast estrogen screen.

In the present study, the yeast estrogen screen (YES) was used to estimate the estrogenic potential of solid phase-extracted water samples from the effluents of two municipal sewage treatment plants (STPs 1 + 2) and from four lanes (left to right) of the river Rhine at Worms, Germany, i.e. downstream the STPs. Estrogenic activities of extracted water samples were expressed as 17beta-estradiol equivalents (E(2)-EQs). Estrogenic activity was detected in the effluents of both STPs with values of 0.242 +/- 0.038 nM (65.96 +/- 10.4 ng/l) and 0.125 +/- 0.026 nM E(2)-EQs (34.1 +/- 7.18 ng/l) at STP 1 and 2, respectively. In river Rhine water, estrogenic activity was lower, however, displaying significant differences between the left and right bank of the river (0.044 +/- 0.003 nM E(2)-EQs [11.97 +/- 0.7 ng/l] for lanes 1-3; 0.071 +/- 0.01 nM E(2)-EQs [19.42 +/- 2.8 ng/l] for lane 4). Chemical analysis of corresponding water samples resulted in a potential estrogenic response in the YES, expressed as E(2)-EQs for the known estrogens and phytoestrogens in the STP effluents with values up to 0.0662 nM E(2)-EQs (18.04 ng/l). In Rhine water from lane 4, however, total estrogenic activity of steroidal estrogens was equal to 0.014 nM E(2)-EQs (3.8 ng/l). Furthermore, total concentrations of flavonoids, fecal- and phytosteroids and resorcyclic lactones were about 1.2 microg/l at STP 1, 0.62 microg/l at STP 2 and 0.25 microg/l at the river Rhine, lane 4. Results indicate that estrogenic activity can clearly be measured in SPT effluents as well as in river Rhine water using the YES in combination with chemical analysis. Results from the bioassay, however, indicated a higher estrogenic potential (expressed as E(2)-EQs) than that obtained by chemical analysis.

Trace determination of fluoroquinolone antibacterial agents in urban wastewater by solid-phase extraction and liquid chromatography with fluorescence detection.

Fluoroquinolones (FQs) are among the most important antibacterial agents (synthetic antibiotics) used in human and veterinary medicine. An analytical method based on reversed-phase liquid chromatography with fluorescence detection was developed and validated for the simultaneous determination of nine FQs and the quinolone pipemidic acid in urban wastewater. Aqueous samples were extracted using mixed-phase cation-exchange disk cartridges that were subsequently eluted by ammonia solution in methanol. Recoveries were above 80% at an overall precision of better than 10%. Instrumental quantification limits varied between 150 and 450 pg injected. The presented method was successfully applied to quantify FQs in effluents of urban wastewater treatment plants. The two most abundant human-use FQs, ciprofloxacin and norfloxacin, occurred in primary and tertiary waste-water effluents at concentrations between 249 and 405 ng/L and from 45 to 120 ng/L, respectively. The identity of FQs in urban wastewater was confirmed by recording full fluorescence spectra and liquid chromatography directly coupled to tandem mass spectrometry. These results indicate that conventional environmental risk assessment overestimates FQ concentrations in surface waters by 1 to 2 orders of magnitude.

A sensitive method for the determination of iodine containing diagnostic agents in aqueous matrices using LC-electrospray-tandem-MS detection.

Iodinated contrast media belong to the most frequently applied compounds in medicine. They exhibit a high polarity and are very persistent against metabolism by the organism and environmental degradation. A sensitive method for the determination of five iodinated contrast media in aqueous matrices is described. Solid phase extraction utilizing Isolute ENV+ material was used for sample enrichment. The contrast media were partially separated on a RP-C18 column, and detection was achieved using electrospray-tandem MS (API III plus and API 365) allowing the sensitive quantitation of these compounds down to the lower ng/L range. The recovery rates generally exceeded 70%, for spiked surface water as well as tap water. The analytes were detected in native samples such as municipal sewage treatment plant (STP) effluent, the river Rhine and even in tap water.

Pharmaceuticals and personal care products in the environment: agents of subtle change?

During the last three decades, the impact of chemical pollution has focused almost exclusively on the conventional "priority" pollutants, especially those acutely toxic/carcinogenic pesticides and industrial intermediates displaying persistence in the environment. This spectrum of chemicals, however, is only one piece of the larger puzzle in "holistic" risk assessment. Another diverse group of bioactive chemicals receiving comparatively little attention as potential environmental pollutants includes the pharmaceuticals and active ingredients in personal care products (in this review collectively termed PPCPs), both human and veterinary, including not just prescription drugs and biologics, but also diagnostic agents, "nutraceuticals," fragrances, sun-screen agents, and numerous others. These compounds and their bioactive metabolites can be continually introduced to the aquatic environment as complex mixtures via a number of routes but primarily by both untreated and treated sewage. Aquatic pollution is particularly troublesome because aquatic organisms are captive to continual life-cycle, multigenerational exposure. The possibility for continual but undetectable or unnoticed effects on aquatic organisms is particularly worrisome because effects could accumulate so slowly that major change goes undetected until the cumulative level of these effects finally cascades to irreversible change--change that would otherwise be attributed to natural adaptation or ecologic succession. As opposed to the conventional, persistent priority pollutants, PPCPs need not be persistent if they are continually introduced to surface waters, even at low parts-per-trillion/parts-per-billion concentrations (ng-microg/L). Even though some PPCPs are extremely persistent and introduced to the environment in very high quantities and perhaps have already gained ubiquity worldwide, others could act as if they were persistent, simply because their continual infusion into the aquatic environment serves to sustain perpetual life-cycle exposures for aquatic organisms. This review attempts to synthesize the literature on environmental origin, distribution/occurrence, and effects and to catalyze a more focused discussion in the environmental science community.

Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge.

Aerobic batch experiments containing a diluted slurry of activated sludge from a real sewage treatment plant (STP) near Frankfurt/Main were undertaken, in order to investigate the persistence of natural estrogens and contraceptives under aerobic conditions. The batch experiments showed that while in contact with activated sludge the natural estrogen 17 beta-estradiol was oxidized to estrone, which was further eliminated in the batch experiments in an approximate linear time dependence. Further degradation products of estrone were not observed. 16 alpha-hydroxyestrone was rapidly eliminated, again without detection of further degradation products. The contraceptive 17 alpha-ethinylestradiol was principally persistent under the selected aerobic conditions, whereas mestranol was rapidly eliminated and small portions of 17 alpha-ethinylestradiol were formed by demethylation. Additionally, two glucuronides of 17 beta-estradiol (17 beta-estradiol-17-glucuronide and 17 beta-estradiol-3-glucuronide) were cleaved in contact with the diluted activated sludge solution and thus 17 beta-estradiol was released. The glucuronidase activity of the activated sludge was further confirmed by the cleavage of 4-methylumbelliferyl-beta-D-glucuronide (MUF-beta-glucuronide) in a solution of a activated sludge slurry and Milli-Q-water (1:100, v/v). The turnover rate obtained was approximately steady state, with a turnover rate of 0.1 mumol/l for the released MUF. Hence, it is very likely that the glucuronic acid moiety of 17 beta-estradiol glucuronides and other estrogen glucuronides become cleaved in a real municipal STP, so that the concentrations of the free estrogens increase.