Thomas Andreas Ternes

We report a freeze-dried ready-to-use yeast thyroid screen (YTS), preserving the general dose–response characteristics of the freshly prepared counterpart. This field-deployable method reduces the assay time of the overall procedure from several days to 5 h with no requirement for sterile conditions, thus fulfilling key requirements for on-site implementation in a biosensor array. The effects of cell density and concentration of the cryoprotectant trehalose on median effective concentrations (EC₅₀), limit of detection (LOD) and biosensor induction (IF) were determined and monitored over a storage period of 5 months. In addition, the impact of these parameters was monitored on the biosensor survival rate during freeze-drying and the subsequent storage process. Throughout the 5-month study, the freeze-dried recombinant yeast assay retained comparable dose–response characteristics to those of the freshly prepared counterpart, displaying median values of EC₅₀ in the range of 350 nM to 550 nM and LODs in the range of 20 nM to 45 nM of the reference compound thyroxine (T4). Long-term stabilization is demonstrated using spiked (T4, 2 µM) river water and extracted wastewater effluent. After 5 months of storage, the T4-equivalent activities were 96 ± 38% and 112 ± 15% for river water and wastewater, respectively. In summary, we have successfully demonstrated a proof of principle of a field-deployable yeast thyroid screen (YTS) by using freeze-dried cells and trehalose as a cryoprotectant to achieve storability for up to 5 months at 4 °C.

We report a freeze-dried ready-to-use yeast thyroid screen (YTS), displaying detection sensitivity and performance comparable to that of the freshly prepared counterpart. This field-deployable method reduces the duration of the worktime from several days to 5 hours with no requirement for sterile conditions, fulfilling key requirements for implementation in a biosensor array with on-site application. The effects of cell density and concentration of the cryoprotectant trehalose on median effective concentrations (EC₅₀), limit of detection (LOD) and biosensor induction (IF) were determined and monitored over a storage period of 5 months. In addition, the impact of these parameters was monitored on the biosensor survival rate during freeze-drying and the subsequent storage process. Throughout the 5-month study, the freeze-dried recombinant yeast-assay retained comparable dose-response characteristics to those of the freshly prepared counterpart, displaying median values of EC₅₀ in the range of 350 nM to 550 nM and LODs in the range of 20 nM to 45 nM of the reference compound thyroxine (T4). A proof of principle of the long-term stabilization is demonstrated using spiked (T4, 2 µM) river water and extracted wastewater effluent. After 5 months of storage, the T4 recoveries were 96 ± 38% and 112 ± 15% for river water and wastewater, respectively.

The objective of this study was to characterize monitoring sites with regard to their contaminating sources and to identify potential pollution hotspots as well as previously unknown compounds. Several evaluation methodologies were combined via a category approach consisting of site-specific detections using a combination of an extended target analysis via GC/MS and LC/MS/MS, NTS using LC-reversed phase high resolution MS, and suspect screening via spectral libraries. Furthermore, we considered the fraction of treated wastewater, the agricultural land use, and industry located in the vicinity. In order to evaluate our concept, more than 520 water samples of rivers from 79 locations throughout Saxony were analyzed by target analysis and NTS. Overall, a very good comparability between the results of target analysis and NTS was observed for pharmaceuticals and pesticides. In a first step, NTS data were used to identify hotspots of pollution with organic micropollutants. By this approach, 13 of 79 sites were characterized as “anomalous” as defined by empirical thresholds. More than 350 compounds could be identified using our spectral data library. In a second step, features of two sampling sites, which were likely contaminated by treated industrial wastewater, were investigated in detail, leading to the identification of four currently unknown compounds. These were cyclohexyldiethanolamine (CDA), 2,4,6-tri-(6-amino-hexanoic acid)-1,3,5-triazine (THAT), 2-(dibutylamino)ethanol, and 3,6,9-trioxaundecanedioic with estimated concentrations of up to 100 µg/L in rivers and streams using a semi-quantitative approach. However, based on the NTS database obtained, the identification of further currently unknown compounds can be easily done. The suggested category approach was also suitable to identify pollution hotspots of organic chemicals. Graphical Abstract

This study successfully used effect-directed analysis to identify a transformation product of N,N'-substituted p-phenylenediamines (PPD) rubber-antiozonants as the main driver of estrogenicity in aqueous leachates from elastomer membranes. First, two signals with estrogenic activity were detected via high-performance thin-layer chromatography (HPTLC) in a planar yeast estrogen screen (p-YES). After the active fractions were extracted from the HPTLC plate, they were analyzed by nontarget screening using liquid chromatography coupled to high-resolution mass-spectrometry. Via a prioritization process, the elucidation of the MS²-spectra, and a library research, 4-hydroxydiphenylamine (4HDPA) and N-phenyl-p-benzoquinone monoimine (QMI) were identified (m/z 186.0915 and 184.0757, respectively, in positive mode). 4HDPA is a hydrolysis product of PPD antiozonants, whereas QMI is formed by the oxidation of 4HDPA. By measuring the leachate within the YES medium, we found that QMI was transformed into 97 ± 1% of the estrogenic active compound 4HDPA. Therefore, YES results of redox couple compounds need to be carefully assessed due to potential transformations occurring in the YES medium itself. Finally, the estrogenic activity of 4HDPA was identified for the first time, and we confirmed that the overall estrogenicity observed in the elastomer leachates was predominantly caused by the elution and formation of 4HDPA by 120 ± 30%. Furthermore, 4HDPA was found in river and stream water at concentrations ranging between 7 and 20 ng/L.

There is an urgent need to improve the knowledge about occurrence and behavior of microplastics in the aquatic environment. The aim of this study was to determine the temporal variability and spatial distribution of poly(vinyl chloride) (PVC) in the larger German rivers Rhine and Moselle. Monitoring campaigns using a continuous flow centrifuge (sampling of suspended matter in a particle size range of 1 mm-1 μm) revealed a strong dependence of PVC concentrations of up to 3.3 μg/L on the discharge. It could be shown that PVC microplastic concentrations have strong correlations with suspended matter (correlation coefficients >0.94 with p-values <0.05). PVC microplastics and suspended matter were thus evidenced to show an identical temporal behavior in the Rhine River and Moselle. Furthermore, our results indicate that the pollution with PVC microplastic is mainly originated from diffuse rather than point sources. Elevated PVC loads were mainly observed during floods. Our study confirmed that approximately 80% of the PVC loads can be exclusively attributed to these high discharge events. Calculated annual PVC loads (2019-2022) ranged from 2-17 t/a (Moselle) to 10-38 t/a (Rhine). In addition, long-term trend analysis of loads (2006-2022; particle size range: <2 mm) at the Rhine for Weil, Iffezheim, Koblenz, and Bimmen showed significant decreasing trends (ranging from 0.41 to 1.72 t/a) for all these sites with an overall decrease ranging between 38 and 58%.

The focus in microplastic research has shifted from marine ecosystems towards freshwater ecosystems. Still, most studies are based on small sample numbers, both spatially and temporally. Little is known about the spatiotemporal variability of microplastics (MPs) in large river systems such as the Rhine River, Germany. Within our study, we performed four cross-sectional sampling campaigns at two sites in the Rhine River, at Koblenz and Emmerich, involving depth-distributed sampling over a particle size range from 10 µm to 25 mm. For plastic particle analysis, we used both optical and thermoanalytical approaches to determine mass-based polymer concentrations. Our results show that MP variability within the water column is complex, but mostly follows the particles density: the ratio between superficial MPs concentration and mean concentration of the verticals was >1 for lighter polymers with a density below 1.04 g/cm³ and 3 among all size classes with only a few exceptions, even though the Rouse theory would indicate a more homogeneous distribution for small particle sizes. Large sampling volumes are essential, particularly for larger MP particles, as the coefficient of variation rises with particle size. At our study sites, no significant lateral variation was apparent, while during a flood event, MP concentrations were significantly higher than during low and mean water stages. This study is the first to (i) gain insights into cross-sectional MPs distribution in the Rhine River and (ii) account for particle mass concentrations, and thus lays the foundation for potential future MPs flux monitoring.