Transformation of organic micropollutants along hyporheic flow in bedforms of river-simulating flumes.

Urban streams receive increasing loads of organic micropollutants from treated wastewaters. A comprehensive understanding of the in-stream fate of micropollutants is thus of high interest for water quality management. Bedforms induce pumping effects considerably contributing to whole stream hyporheic exchange and are hotspots of biogeochemical turnover processes. However, little is known about the transformation of micropollutants in such structures. In the present study, we set up recirculating flumes to examine the transformation of a set of micropollutants along single flowpaths in two triangular bedforms. We sampled porewater from four locations in the bedforms over 78 days and analysed the resulting concentration curves using the results of a hydrodynamic model in combination with a reactive transport model accounting for advection, dispersion, first-order removal and retardation. The four porewater sampling locations were positioned on individual flowpaths with median solute travel times ranging from 11.5 to 43.3 h as shown in a hydrodynamic model previously. Highest stability was estimated for hydrochlorothiazide on all flowpaths. Lowest detectable half-lives were estimated for sotalol (0.7 h) and sitagliptin (0.2 h) along the shortest flowpath. Also, venlafaxine, acesulfame, bezafibrate, irbesartan, valsartan, ibuprofen and naproxen displayed lower half-lives at shorter flowpaths in the first bedform. However, the behavior of many compounds in the second bedform deviated from expectations, where particularly transformation products, e.g. valsartan acid, showed high concentrations. Flowpath-specific behavior as observed for metformin or flume-specific behavior as observed for metoprolol acid, for instance, was attributed to potential small-scale or flume-scale heterogeneity of microbial community compositions, respectively. The results of the study indicate that the shallow hyporheic flow field and the small-scale heterogeneity of the microbial community are major controlling factors for the transformation of relevant micropollutants in river sediments.

Enantiomeric Fractionation during Biotransformation of Chiral Pharmaceuticals in Recirculating Water-Sediment Test Flumes.

Many organic contaminants entering the aquatic environment feature stereogenic structural elements that give rise to enantiomerism. While abiotic processes usually act identical on enantiomers, biotic processes, such as biodegradation often result in enantiomeric fractionation (EFr), i.e., the change of the relative abundance of enantiomers. Therefore, EFr offers the opportunity to differentiate biodegradation in complex environmental systems from abiotic processes. In this study, an achiral-chiral two-dimensional liquid chromatographic method for the enantioseparation of selected pharmaceuticals was developed. This method was then applied to determine the enantiomeric compositions of eight chiral pharmaceuticals in 20 water-sediment test flumes and test EFr as an indicator of biodegradation. While all eight substances were attenuated by at least 60%, five (atenolol, metoprolol, celiprolol, propranolol, and flecainide) displayed EFr. No EFr was observed for citalopram, fluoxetine, and venlafaxine despite almost complete attenuation (80 to 100%). Celiprolol, a barely studied ß-blocker, revealed the most distinct EFr among all investigated substances; however, EFr varied considerably with biodiversity. Celiprolol-H2 was identified as a biological transformation product possibly formed by reduction of the celiprolol keto group through a highly regio- and enantioselective carbonyl reductase. While celiprolol-H2 was observed across all flumes, as expected, its formation was faster in flumes with high bacterial diversity where also EFr was highest. Overall, EFr and transformation product formation together served as good indicators of biological processes; however, the strong dependence of EFr on biodiversity limits its usefulness in complex environmental systems.

Bacterial Diversity Controls Transformation of Wastewater-Derived Organic Contaminants in River-Simulating Flumes.

Hyporheic zones are the water-saturated flow-through subsurfaces of rivers which are characterized by the simultaneous occurrence of multiple physical, biological, and chemical processes. Two factors playing a role in the hyporheic attenuation of organic contaminants are sediment bedforms (a major driver of hyporheic exchange) and the composition of the sediment microbial community. How these factors act on the diverse range of organic contaminants encountered downstream from wastewater treatment plants is not well understood. To address this knowledge gap, we investigated dissipation half-lives (DT50s) of 31 substances (mainly pharmaceuticals) under different combinations of bacterial diversity and bedform-induced hyporheic flow using 20 recirculating flumes in a central composite face factorial design. By combining small-volume pore water sampling, targeted analysis, and suspect screening, along with quantitative real-time PCR and time-resolved amplicon Illumina MiSeq sequencing, we determined a comprehensive set of DT50s, associated bacterial communities, and microbial transformation products. The resulting DT50s of parent compounds ranged from 0.5 (fluoxetine) to 306 days (carbamazepine), with 20 substances responding significantly to bacterial diversity and four to both diversity and hyporheic flow. Bacterial taxa that were associated with biodegradation included Acidobacteria (groups 6, 17, and 22), Actinobacteria (Nocardioides and Illumatobacter), Bacteroidetes (Terrimonas and Flavobacterium) and diverse Proteobacteria (Pseudomonadaceae, Sphingomonadaceae, and Xanthomonadaceae). Notable were the formation of valsartan acid from irbesartan and valsartan, the persistence of N-desmethylvenlafaxine across all treatments, and the identification of biuret as a novel transformation product of metformin. Twelve additional target transformation products were identified, which were persistent in either pore or surface water of at least one treatment, indicating their environmental relevance.

Using recirculating flumes and a response surface model to investigate the role of hyporheic exchange and bacterial diversity on micropollutant half-lives.

Enhancing the understanding of the fate of wastewater-derived organic micropollutants in rivers is crucial to improve risk assessment, regulatory decision making and river management. Hyporheic exchange and sediment bacterial diversity are two factors gaining increasing importance as drivers for micropollutant degradation, but are complex to study in field experiments and usually ignored in laboratory tests aimed to estimate environmental half-lives. Flume mesocosms are useful to investigate micropollutant degradation processes, bridging the gap between the field and batch experiments. However, few studies have used flumes in this context. We present a novel experimental setup using 20 recirculating flumes and a response surface model to study the influence of hyporheic exchange and sediment bacterial diversity on half-lives of the anti-epileptic drug carbamazepine (CBZ) and the artificial sweetener acesulfame (ACS). The effect of bedform-induced hyporheic exchange was tested by three treatment levels differing in number of bedforms (0, 3 and 6). Three levels of sediment bacterial diversity were obtained by diluting sediment from the River Erpe in Berlin, Germany, with sand (1 : 10, 1 : 1000 and 1 : 100 000). Our results show that ACS half-lives were significantly influenced by sediment dilution and number of bedforms. Half-lives of CBZ were higher than ACS, and were significantly affected only by the sediment dilution variable, and thus by bacterial diversity. Our results show that (1) the flume-setup is a useful tool to study the fate of micropollutants in rivers, and that (2) higher hyporheic exchange and bacterial diversity in the sediment can increase the degradation of micropollutants in rivers.

Passive sampling of organic contaminants across the water-sediment interface of an urban stream.

Passive sampling is a well-established tool for monitoring time-weighted average concentrations of polar and semi-polar organic contaminants in streams at flow velocities between 0.1 and 0.4 m s-1. However, its application under low-flow conditions (10-5 to 0.01 m s-1) - as encountered in hyporheic zones - has been scarcely reported. In this study, 3 novel passive sampler configurations were developed for the monitoring of (semi-)polar organic pollutants and related transformation products across the water-sediment interface and thus across varying hydrodynamic conditions. Their design was inspired by Chemcatcher and diffusive gradients in thin films for organics. To determine the most optimal sampler design, an uptake experiment was completed involving the 3 novel passive sampler configurations and a reference Chemcatcher in polar configuration. The experiments consisted of a circular flume that simulated the main channel of a stream and an aquarium with stagnant water that represented the underlying hyporheic zone. The systems were exposed to 192 organic pollutants at environmental concentrations, and the samplers were then collected, extracted and analyzed using liquid chromatography high-resolution mass spectrometry after 2, 6 and 14 days. The configuration that was most insensitive to different hydrodynamic conditions consisted of a reversed-phase sulfonated styrenedivinylbenzene disk as the receiving phase that was covered by an agarose diffusion gel and topped with a polyethersulfone membrane filter. To further evaluate its environmental application, samplers were installed downstream of a sewage treatment plant located at an urban stream in Berlin, Germany (Erpe). The samplers were mounted on custom-made holders which were subsequently embedded in the stream bed to position samplers above (0.30 m) and within the sediment (-0.15/-0.30/-0.45 m) for 11 days. Target and suspect screening workflows were then applied to identify common concentration patterns and link parent attenuation to transformation product formation. A total of 104 concentration profiles were determined, suggesting the efficiency of the proposed sampling strategy in the water-sediment interface. Valsartan acid was the only known transformation product indicative of hyporheic zone-driven attenuation as its concentration in porewater by far exceeded its concentration in surface water. Similar patterns were observed for a larger list of suspected transformation products, of which a sotalol transformation product was tentatively identified. Overall, the established sampling methodology can be effectively used to quantify organic contaminants during low-flow conditions and is suitable for the characterization of attenuation patterns of organic pollutants in hyporheic zones.

Vacuum-assisted evaporative concentration combined with LC-HRMS/MS for ultra-trace-level screening of organic micropollutants in environmental water samples.

Vacuum-assisted evaporative concentration (VEC) was successfully applied and validated for the enrichment of 590 organic substances from river water and wastewater. Different volumes of water samples (6 mL wastewater influent, 15 mL wastewater effluent, and 60 mL river water) were evaporated to 0.3 mL and finally adjusted to 0.4 mL. 0.1 mL of the concentrate were injected into a polar reversed-phase C18 liquid chromatography column coupled with electrospray ionization to high-resolution tandem mass spectrometry. Analyte recoveries were determined for VEC and compared against a mixed-bed multilayer solid-phase extraction (SPE). Both approaches performed equally well (≥ 70% recovery) for a vast number of analytes (n = 327), whereas certain substances were especially amenable to enrichment by either SPE (e.g., 4-chlorobenzophenone, logDow,pH7 4) or VEC (e.g., TRIS, logDow,pH7 - 4.6). Overall, VEC was more suitable for the enrichment of polar analytes, albeit considerable signal suppression (up to 74% in river water) was observed for the VEC-enriched sample matrix. Nevertheless, VEC allowed for accurate and precise quantification down to the sub-nanogram per liter level and required no more than 60 mL of the sample, as demonstrated by its application to several environmental water matrices. By contrast, SPE is typically constrained by high sample volumes ranging from 100 mL (wastewater influent) to 1000 mL (river water). The developed VEC workflow not only requires low labor cost and minimum supervision but is also a rapid, convenient, and environmentally safe alternative to SPE and highly suitable for target and non-target analysis.

Spatial and Temporal Variability in Attenuation of Polar Organic Micropollutants in an Urban Lowland Stream.

Contamination of rivers by trace organic compounds (TrOCs) poses a risk for aquatic ecosystems and drinking water quality. Spatially- and temporally varying environmental conditions are expected to play a major role in controlling in-stream attenuation of TrOCs. This variability is rarely captured by in situ studies of TrOC attenuation. Instead, snap-shots or time-weighted average conditions and corresponding attenuation rates are reported. The present work sought to investigate this variability and factors controlling it by analysis of 24 TrOCs over a 4.7 km reach of the River Erpe (Berlin, Germany). The factors investigated included sunlight and water temperature as well as the presence of macrophytes. Attenuation rate constants in 48 consecutive hourly water parcels were tracked along two contiguous river sections of different characteristics. Section 1 was less shaded and more densely covered with submerged macrophytes compared to section 2. The sampling campaign was repeated after macrophyte removal from section 1. The findings show, that section 1 generally provided more favorable conditions for both photo- and biodegradation. Macrophyte removal enhanced photolysis of some compounds (e.g., hydrochlorothiazide and diclofenac) while reducing the biodegradation of metoprolol. The transformation products metoprolol acid and valsartan acid were formed along the reach under all conditions.

Microvolume trace environmental analysis using peak-focusing online solid-phase extraction-nano-liquid chromatography-high-resolution mass spectrometry.

Online solid-phase extraction was combined with nano-liquid chromatography coupled to high-resolution mass spectrometry (HRMS) for the analysis of micropollutants in environmental samples from small volumes. The method was validated in surface water, Microcystis aeruginosa cell lysate, and spent Microcystis growth medium. For 41 analytes, quantification limits of 0.1-28 ng/L (surface water) and 0.1-32 ng/L (growth medium) were obtained from only 88 µL of sample. In cell lysate, quantification limits ranged from 0.1-143 ng/L or 0.33-476 ng/g dry weight from a sample of 88 µL, or 26 µg dry weight, respectively. The method matches the sensitivity of established online and offline solid-phase extraction-liquid chromatography-mass spectrometry methods but requires only a fraction of the sample used by those techniques, and is among the first applications of nano-LC-MS for environmental analysis. The method was applied to the determination of bioconcentration in Microcystis aeruginosa in a laboratory experiment, and the benefit of coupling to HRMS was demonstrated in a transformation product screening.

Combined mutagenesis and kinetics characterization of the bilin-binding GAF domain of the protein Slr1393 from the Cyanobacterium Synechocystis PCC6803.

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (λmax =649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 µs, 390 µs, and 1.5 ms) for the red-to-green conversion, and 1.2 µs, 340 µs, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the green-absorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.