Occurrence of Glucocorticoids, Mineralocorticoids, and Progestogens in Various Treated Wastewater, Rivers, and Streams.

In the current study a high sensitive analytical method was developed for the determination of 60 steroids including glucocorticoids (GC), mineralocorticoids (MC), and progestogens (PG) in WWTP effluents and surface water using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). The limits of quantification (LOQ) ranged between 0.02 ng/L (cortisone) to 0.5 ng/L (drospirenone) in surface water and from 0.05 ng/L (betamethasone) to 5 ng/L (chlormadinone) in treated wastewater. After optimization, the developed method was applied to WWTP effluents, rivers, and streams around Germany. Numerous steroids have been detected during the sampling campaign and predominant analytes from all steroid types were determined. Moreover, the occurrence of dienogest, mometasone furoate, flumethasone pivalate, and the metabolites 6ß-hydroxy dienogest, 6ß-hydroxy triamcinolone acetonide, 7α-thiomethyl spironolactone, and 11α-hydroxy canrenone is reported for the first time. In addition, this study revealed the ubiquitous presence of topically applied GC monoesters betamethasone propionate, betamethasone valerate, and 6α-methylprednisolone propionate in WWTP effluents and surface water.

Quaternary Triphenylphosphonium Compounds: A New Class of Environmental Pollutants.

A nontarget screening using high-resolution mass spectrometry (HRMS) was established to identify industrial emerging contaminants in the Rhine River. With this approach, quaternary triphenylphosphonium compounds (R-Ph3P(+)) were identified as new emerging contaminants in the aquatic environment. The suggested chemical structures were elucidated by MS fragmentation and chemical databank searches and eventually confirmed via authentic standards. R-Ph3P(+) are used worldwide by the chemical industry to synthesize alkenes via the Wittig reaction. In total, five compounds [R = butyl (Bu), R = ethyl (Et), R = methoxymethyl (MeOMe), R = methyl (Me), and R = phenyl (Ph)] were found in German rivers and streams. R-Ph3P(+) were detected only in those rivers and streams that received an appreciable portion of wastewater from the chemical industry. Up to 2.5 µg/L Et-Ph3P(+) was quantified in a small stream from the Hessian Ried, and in the Rhine, up to 0.56 µg/L Me-Ph3P(+) was detected. R-Ph3P(+) were also identified in suspended particulate matter and sediments in the Rhine catchment, with MeOMe-Ph3P(+) concentrations of up to 0.75 mg/kg and up to 0.21 mg/kg, respectively. Because of the lack of ecotoxicological studies, the environmental risks caused by R-Ph3P(+) can be assessed for neither pelagic nor benthic organisms.

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.

Analysis of the aerobic biodegradation of glucocorticoids: Elucidation of the kinetics and transformation reactions.

Glucocorticoids (GCs) are one of the most prescribed pharmaceutical classes worldwide. They have reached the focus as environmental pollutants in the current scientific research, due to their potential risks to aquatic organisms even in the lower ng L-1 range. The objective of this study was to determine the kinetic behavior of selected GCs and to identify their main transformation products (TPs) in lab scaled biodegradation experiments. Therefore, we analyzed the removal of 13 GCs in aerated incubation experiments with activated sludge taken from a German municipal wastewater treatment plant (WWTP) as inoculum. For all steroids, an exponential decrease of the concentrations was observed, which was modelled by pseudo-first order kinetics. Overall, the rate constants kbiol. ranged from 0.07 L gss-1 d-1 (triamcinolone acetonide) to 250 L gss-1 d-1 (prednisolone). These results emphasize the broad variation in the biodegradability and recalcitrance of certain GCs. The selection of the studied GCs enabled a deduction of microbiological stability related to functional groups. Based on the identified TPs, a variety of enzymatically mediated reactions were postulated. Moreover, the identified TPs are characterized by an intact steroid core structure. Thus residual endocrine activity cannot be ruled out. The main observed reactions were regioselective hydrogenation of carbon double-bonds, degradation of the steroid C17 side-chain, ester hydrolysis and oxidative hydroxylation. In total, 41 TPs were tentatively identified and 22 of them were unambiguously confirmed via reference standards. Additionally, 12 TPs were detected in the effluents of municipal WWTPs and, to the best of our knowledge, the occurrence of eight of these TPs has been shown for the first time. These TPs might significantly contribute to the detected residual endocrine activities in the aquatic environments. Therefore, there is a strong need for efficient removal strategies, in particular for persistent steroid hormones with elevated potencies.

Spatial distribution and temporal trends of pharmaceuticals sorbed to suspended particulate matter of German rivers.

Although several studies confirmed a wide distribution of pharmaceuticals in rivers and streams, a limited knowledge is available about the partitioning of pharmaceuticals between the water phase and suspended particulate matter (SPM). To close this gap of knowledge, we developed and validated a sensitive and high throughput analytical method for the analysis of 57 pharmaceuticals, 42 metabolites and transformation products (TP) as well as the artificial sweetener acesulfame sorbed to SPM. The method was based on pressurized liquid extraction (PLE) followed by a clean-up via solvent exchange and detection via direct injection-reversed phase LC-MS/MS and freeze-drying-HILIC-MS/MS. Freundlich isotherms were determined for 90 analytes. All showed a linear sorption behavior. Distribution coefficients (Kd) ranged from 0.64 L/kg to 9300 L/kg. For 18 pharmaceuticals, Kd values were found to be above 100 L/kg. SPM of annual composite samples were analyzed to determine the pharmaceutical concentrations between 2005 and 2015 at four sites of the river Rhine: Weil, Iffezheim, Koblenz and Bimmen as well as between 2006 and 2015 at one site of the river Saar, at Rehlingen. In these SPM samples, up to 61 of the 100 analytes were detected with concentrations up to 190 ng/g d.w. (dry weight) for guanylurea, a transformation product of the antidiabetic metformin. For most analytes, increasing concentrations were found along the length of the Rhine and higher concentrations were measured in Rehlingen/Saar. Normalization of the data with the antiepileptic drug carbamazepine as an intrinsic tracer for municipal wastewater indicated possible industrial discharges for four analytes. For most pharmaceuticals, the annual concentrations exhibited a good correlation with the consumption volumes in Germany.

The fate of selected micropollutants in a single-house MBR.

Membrane bioreactor (MBR) technology is an interesting option for single-house wastewater treatment or small communities. Because typically a very high effluent quality is achieved with respect to pathogens, suspended solids, organics and nitrogen, the permeate is well suited for reuse. Little is known about the fate of micropollutants in such small systems. The differences between centralized and decentralized biological wastewater treatment with respect to micropollutants are manifold: besides the operational parameters like hydraulic and sludge retention time, the main difference is in the load variation. While the influent load is expected to be more or less constant in large catchments, it varies strongly in small MBRs due to irregular consumption (e.g. of medication by individuals). Concentrations of micropollutants are higher by a factor 50-1000 than in centralized treatment. It is also unknown how reliable degradation of micropollutants is in case of irregular exposure. In this study, two experiments were conducted in a small MBR treating the wastewater of a three-person household. During normal operation of the treatment plant, 25 pharmaceuticals (antibiotics, antiphlogistics, lipid regulators, iodinated contrast media and hormones) that had not been used by members of the household were added in concentrations typical for municipal wastewater. The removal of most substances was in the same range as for centralized wastewater treatment. It was shown that biological transformation was the main elimination process while adsorption to the activated sludge was negligible for most substances due to the low sludge production at high sludge retention time. No appreciable lag for inducing biological degradation was observed. The high hydraulic and sludge residence time had a positive effect on the elimination of slowly degradable substances, but this was partly compensated by the lower biological activity. An experiment with antibiotics concentrations typical for decentralized treatment (between 500 and 1000 microg l(-1); sulfamethoxazole, sulfapyridine, trimethoprim, clarithromycin, roxithromycin) did not show an inhibitory effect on either nitrification or denitrification.

Quaternary (triphenyl-) phosphonium compounds: Environmental behavior and toxicity.

An analytical method based on high resolution mass spectrometry coupled with liquid chromatography (LC-HRMS) for 25 quaternary phosphonium compounds (QPCs) and derived phosphine oxides (POs) was developed and validated. To investigate the occurrence and fate of QPCs in the aquatic environment, water, suspended solids and sediments from the rivers Rhine and Elbe (upper and middle Elbe as well as tidal Elbe) were analyzed, as well as samples from tributaries bearing significant loads of QPCs. For the first time, the quaternary phosphonium compound tetrabutylphosphonium (Bu4P+) was detected. In the river Elbe concentrations were determined of up to 4700 ng/L (surface water) and 1000 µg/kg (sediment), respectively. Analysis of a time series of suspended solids (2005-2015) showed that QPCs have been present in the Elbe and Rhine catchment for at least one decade, with partly rising tendency. A degradation experiment with Rhine sediment revealed that triphenylphosphonium compounds (R-Ph3P+) and Bu4P+ are persistent in contact with sediment and suspended solids and tend to sorb onto sediment particles. Toxicological studies (reactive oxygen species (ROS) after substance exposure, Ames test, Micronucleus test, determination of cytotoxicity) with selected QPCs confirmed that all of them exhibit cytotoxicity and some even genotoxic potential at elevated concentrations, which emphasizes the need for an emission regulation of these compounds.

Persistence of antibiotics such as macrolides, tiamulin and salinomycin in soil.

The extensive use of veterinary drugs in agriculture leads to contamination of manure. If this manure is used as fertiliser, soil may be exposed to the respective drugs. Additionally soil exposure may stem from contaminated sewage sludge that is used on some agricultural land as fertiliser. This study focuses on the fate of antibiotics in soil. We present a 120-day degradation experiment of six commonly used antibiotics: erythromycin, roxithromycin oleandomycin, tylosin, salinomycin and tiamulin in soil as well as calculating the resulting half-lives. The half-lives were 20 days for erythromycin, 27 days for oleandomycin, 8 days for tylosin, 16 days for tiamulin and 5 days for salinomycin; all according to 1st order kinetics. The concentration of roxithromycin remained nearly unchanged during the whole experiment.

Occurrence of venlafaxine, other antidepressants and selected metabolites in the Rhine catchment in the face of climate change.

Fate and occurrence of 4 selective serotonin reuptake inhibitors, one serotonin-noradrenergic reuptake inhibitor and one noradrenergic-dopamineric reuptake inhibitor and their human metabolites were determined in a German municipal wastewater treatment plant as well as in the Rhine River and selected tributaries. The enantiomeric fractions of venlafaxine and its metabolites were not altered during wastewater treatment and were similar in all river samples underlining that no appreciable biodegradation occurs. In the Rhine catchment area highest concentrations were detected for venlafaxine, citalopram and their human metabolites. Projected future climate change would lead to an increased portion of treated wastewater in rivers due to reduced discharges during low flow situations by the end of the 21st century. However, the effect of climate change on the pattern and concentrations of antidepressants is predicted to be of minor importance in comparison to altered consumption quantities caused by demographic developments and changes in life styles.

Determination of antibiotics from soil by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry.

A method for the analysis of several macrolide and ionophore antibiotics, as well as tiamulin, from soil was developed using pressurized liquid extraction (PLE), reversed-phase liquid chromatography and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (LC-APCI+-MS-MS). The analytes were extracted from soil by PLE in 30 min and the extracts were cleaned up by solid-phase extraction (SPE) on a diol SPE cartridge. Liquid chromatographic (LC) separation of the antibiotics was achieved in 35 min. Recovery experiments were performed using spiked soil and concentrations varying from 1 to 2000 microg/kg. By using a macrolide internal standard the recovery rates for the macrolides erythromycin and roxithromycin ranged from 43 to 94% (RSD 20-23%), for the ionophore salinomycin the recovery rate was 76% (RSD 29%), while the pleuromutilin tiamulin was completely recovered. The limits of detection ranged from 0.2 to 1.6 microg/kg. In soil samples a maximum concentration of 0.7 microg/kg tiamulin was found.

Identification of putative steroid receptor antagonists in bottled water: combining bioassays and high-resolution mass spectrometry.

Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MS(n) experiments elucidated the molecule's structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MS(n) data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably, dioctyl fumarates and maleates have been overlooked by science and regulation to date. This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.

Removal of antibiotics from urban wastewater by constructed wetland optimization.

Seven mesocosm-scale constructed wetlands (CWs), differing in their design characteristics, were set up in the open air to assess their efficiency to remove antibiotics from urban raw wastewater. A conventional wastewater treatment plant (WWTP) was simultaneously monitored. The experiment took place in autumn. An analytical methodology including HPLC-MS/MS was developed to measure antibiotic concentrations in the soluble water fraction, in the suspended solids fraction and in the WWTP sludge. Considering the soluble water fraction, the only easily eliminated antibiotics in the WWTP were doxycycline (61±38%) and sulfamethoxazole (60±26%). All the studied types of CWs were efficient for the removal of sulfamethoxazole (59±30-87±41%), as found in the WWTP, and, in addition, they removed trimethoprim (65±21-96±29%). The elimination of other antibiotics in CWs was limited by the specific system-configuration: amoxicillin (45±15%) was only eliminated by a free-water (FW) subsurface flow (SSF) CW planted with Typha angustifolia; doxycycline was removed in FW systems planted with T. angustifolia (65±34-75±40%), in a Phragmites australis-floating macrophytes system (62±31%) and in conventional horizontal SSF-systems (71±39%); clarithromycin was partially eliminated by an unplanted FW-SSF system (50±18%); erythromycin could only be removed by a P. australis-horizontal SSF system (64±30%); and ampicillin was eliminated by a T. angustifolia-floating macrophytes system (29±4%). Lincomycin was not removed by any of the systems (WWTP or CWs). The presence or absence of plants, the vegetal species (T. angustifolia or P. australis), the flow type and the CW design characteristics regulated the specific removal mechanisms. Therefore, CWs are not an overall solution to remove antibiotics from urban wastewater during cold seasons. However, more studies are needed to assess their ability in warmer periods and to determine the behaviour of full-scale systems.

Antiviral drugs in wastewater and surface waters: a new pharmaceutical class of environmental relevance?

An analytical method was developed for the determination of nine antiviral drugs (acyclovir, abacavir, lamivudine, nevirapine oseltamivir, penciclovir, ribavirin, stavudine, zidovudine) and one active metabolite (oseltamivir carboxylate) in raw and treated wastewater as well as in surface water using LC/ESI tandem MS detection. Relative recoveries exceeded 80% and limits of quantification ranged between 0.2 and 10 ng L(-1). Matrix effects were compensated by the use of stable isotope labeled surrogate standards and optimized chromatographic separation on a Hydro-RP column. All antiviral drugs, except ribavirin, were detected in raw wastewater. A significant reduction in concentrations was observed for acyclovir, lamivudine, and abacavir in treated wastewater indicating a substantial removal, while nevirapine, zidovudine and oseltamivir were found in similar concentrations in raw and treated wastewater. Concentrations in river waters were in the lower ng L(-1)-range, with a maximum of 190 and 170 ng L(-1) for acyclovir and zidovudine, respectively in the Hessian Ried. Estimated total compound loads in the Ruhr river ranged between <0.1 and 16.5 g d(-1) while for the Rhine river values between 370 and 1800 g d(-1) were determined. The ratios of oseltamivir to oseltamivir carboxylate were approximately a factor of 10 higher for the Rhine than for the other rivers and streams indicating a significant contribution from other sources such as discharges from manufacturing facilities.

Determination of steroid hormones, hormone conjugates and macrolide antibiotics in influents and effluents of sewage treatment plants utilising high-performance liquid chromatography/tandem mass spectrometry with electrospray and atmospheric pressure chemical ionisation.

In this study we present a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method which has been elaborated to analyse steroid hormones, hormone conjugates, oral contraceptives and macrolide antibiotics unchanged in unfiltered influents and effluents of sewage treatment plants (STPs). HPLC separation of the steroid hormones was achieved in 35 min, as well as those of the antibiotics. The analytes were extracted by solid-phase extraction, followed by clean-up using size exclusion chromatography (SEC). For the final quantification HPLC/MS/MS was used. The two ionisation modes, electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI), in HPLC/MS/MS were compared for the analysis of steroid hormones. For quantitative results drastic matrix effects were observed while using ESI. These effects were less pronounced while using APCI. These pitfalls were additionally reduced by clean-up using SEC as well as isotope dilution. Additionally, two multiple reaction monitoring (MRM) transitions per compound were used to prevent false positive results. Recovery experiments with spiked tap water with concentrations varying from 1 to 1000 ng/L gave constant recovery rates: The recovery rates for the hormones and conjugates ranged from 58 to 107%, those of the contraceptives ranged from 83 to 109%. The relative standard deviation was found to be 7 to 24% and the limits of detection were 0.1 to 4.5 ng/L. The recovery rates of the macrolide antibiotics ranged from 76 to 103%, while the relative standard deviation was found to be 7 to 14% and the limits of detection ranged from 0.6 to 1.8 ng/L. The maximum concentrations found in influents of a STP was 470 ng/L for estriol and 1200 ng/L for erythromycin.

Determination of antibiotics such as macrolides, ionophores and tiamulin in liquid manure by HPLC-MS/MS.

A method for the analysis of several macrolide and ionophore antibiotics as well as tiamulin in liquid manure was developed. Reversed-phase liquid chromatography and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry was used for detection.High-performance liquid chromatographic (HPLC) separation of the antibiotics was achieved in 35 min. The analytes were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on a diol SPE cartridge. Recovery experiments with spiked liquid manure concentrations varying from 6 to 2,000 microg kg(-1) gave constant recovery rates. The recovery rates for the macrolides erythromycin, roxithromycin and oleandomycin were 75-94%, that for the ionophore salinomycin was 119%, while that for the pleuromutilin tiamulin was 123%, when using a macrolide internal standard. The relative standard deviation was found to be 15-36% and the limits of detection were 0.4-11.0 micro g kg(-1). The maximum concentrations found in manure samples were 43 micro g kg(-1) for tiamulin and 11 micro g kg(-1) for salinomycin.