What you extract is what you see: Optimising the preparation of water and wastewater samples for in vitro bioassays.

The assessment of water quality is crucial for safeguarding drinking water resources and ecosystem integrity. To this end, sample preparation and extraction is critically important, especially when investigating emerging contaminants and the toxicity of water samples. As extraction methods are rarely optimised for bioassays but rather adopted from chemical analysis, this may result in a misrepresentation of the actual toxicity. In this study, surface water, groundwater, hospital and municipal wastewater were used to characterise the impacts of common sample preparation techniques (acidification, filtration and solid phase extraction (SPE)) on the outcomes of eleven in vitro bioassays. The latter covered endocrine activity (reporter gene assays for estrogen, androgen, aryl-hydrocarbon, retinoic acid, retinoid X, vitamin D, thyroid receptor), mutagenicity (Ames fluctuation test), genotoxicity (umu test) and cytotoxicity. Water samples extracted using different SPE sorbents (Oasis HLB, Supelco ENVI-Carb+, Telos C18/ENV) at acidic and neutral pH were compared for their performance in recovering biological effects. Acidification, commonly used for stabilisation, significantly altered the endocrine activity and toxicity of most (waste)water samples. Sample filtration did not affect the majority of endpoints but in certain cases affected the (anti-)estrogenic and dioxin-like activities. SPE extracts (10.4 × final concentration), including WWTP effluents, induced significant endocrine effects that were not detected in aqueous samples (0.63 × final concentration), such as estrogenic, (anti-)androgenic and dioxin-like activities. When ranking the SPE methods using multivariate Pareto optimisation an extraction with Telos C18/ENV at pH 7 was most effective in recovering toxicity. At the same time, these extracts were highly cytotoxic masking the endpoint under investigation. Compared to that, extraction at pH 2.5 enriched less cytotoxicity. In summary, our study demonstrates that sample preparation and extraction critically affect the outcome of bioassays when assessing the toxicity of water samples. Depending on the water matrix and the bioassay, these methods need to be optimised to accurately assess water quality.

Extended anaerobic conditions in the biological wastewater treatment: Higher reduction of toxicity compared to target organic micropollutants.

Extended anaerobic conditions during biological wastewater treatment may enhance the biodegradation of micropollutants. To explore this, we combined iron-reducing or substrate-limited anaerobic conditions and aerobic pilot-scale reactors directly at a wastewater treatment plant. To investigate the detoxification by these processes, we applied two in vitro bioassays for baseline toxicity (Microtox) and reactive toxicity (AREc32) as well as in vivo bioassays with aquatic model species in two laboratory experiments (Desmodesmus subspicatus, Daphnia magna) and two on-site, flow-through experiments (Potamopyrgus antipodarum, Lumbriculus variegatus). Moreover, we analyzed 31 commonly occurring micropollutants and 10 metabolites. The baseline toxicity of raw wastewater was effectively removed in full-scale and reactor scale activated sludge treatment (>85%), while the oxidative stress response was only partially removed (>61%). A combination of an anaerobic pre-treatment under iron reducing conditions and an aerobic nitrification significantly further reduced the residual in vitro toxicities by 46-60% and outperformed the second combination consisting of an aerobic pre-treatment and an anaerobic post-treatment under substrate-limiting conditions (27-43%). Exposure to effluents of the activated sludge treatment did not induce adverse in vivo effects in aquatic invertebrates. Accordingly, no further improvement in water quality could be observed. Compared to that, the removal of persistent micropollutants was increased. However, this observation was restricted to a limited number of compounds and the removal of the sum concentration of all target micropollutants was relative low (14-17%). In conclusion, combinations of strictly anaerobic and aerobic processes significantly enhanced the removal of specific and non-specific in vitro toxicities. Thus, an optimization of biological wastewater treatment can lead to a substantially improved detoxification. These otherwise hidden capacities of a treatment technology can only be uncovered by a complementary biological analysis.

Removal of the iodinated X-ray contrast medium diatrizoate by anaerobic transformation.

The iodinated X-ray contrast medium diatrizoate is known to be very persistent in current wastewater treatment as well as in environmental compartments. In this study, the potential of anaerobic processes in soils, sediments, and during wastewater treatment to remove and transform diatrizoate was investigated. In anaerobic batch experiments with soil and sediment seven biologically formed transformation products (TPs) as well as the corresponding transformation pathway were identified. The TPs resulted from successive deiodinations and deacetylations. The final TP 3,5-diaminobenzoic acid (DABA) was stable under anaerobic conditions. However, DABA was further transformed under air atmosphere, indicating the potential for the mineralization of diatrizoate by combining anaerobic and aerobic conditions. With the development of a methodology using complementary liquid chromatography-electrospray ionization-tandem mass spectrometry and liquid chromatography-inductively coupled plasma-mass spectrometry techniques, all identified TPs were quantified and the mass balance could be closed without having authentic standards for four of the TPs available. The detection and quantification of diatrizoate TPs in groundwater, in technical wetlands with anaerobic zones, and in a pilot wastewater treatment plant established for anaerobic treatment highlights the transferability and up-scaling of the results attained by laboratory experiments to environmental conditions.

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.

Transformation of the X-ray contrast medium iopromide in soil and biological wastewater treatment.

In water/soil systems, the iodinated contrast medium iopromide was quantitatively biotransformed into several transformation products (TPs). Twelve TPs were identified via HPLC-UV and LC tandem MS. The chemical structures of the TPs were elucidated via fragmentation in MS2 and MS3 of LC tandem MS with a linear ion trap and 1H and 13C NMR analyses. All TPs exhibited transformations at the side chains containing either carboxylic moieties and/or primary and secondary amide moieties, while the triiodoisophthalic acid structure remained unaltered. A transformation pathway was proposed based on the sequence of TP formation in aerobic batch experiments. Additionally, the occurrence of iopromide TPs was investigated in native water samples. All TPs identified were found in municipal WWTP effluents because of their formation during biological wastewater treatment with maximum concentrations of up to 3.7 +/- 0.9 microg/L (TP 819). Predominantly, those TPs were present at higher concentrations in WWTP effluents which were formed at the beginning of the transformation pathway. Furthermore, four TPs formed at the end of the transformation pathway (TP 759, 701A/B, and 643) were also found in bank filtrate up to 0.050 microg/L and in groundwater of an wastewater irrigation area up to 4.6 microg/L.

Combined in situ and in vitro assessment of the estrogenic activity of sewage and surface water samples.

In order to investigate the estrogenic activities of two municipal sewage treatment plant (STP; sites A and B) effluents and of Rhine water sampled at Worms (site C; Rhine-Neckar triangle, Germany), data from in situ experiments measuring hepatic vitellogenin expression from caged rainbow trout (Oncorhynchus mykiss) were compared with data from in vitro bioassays (yeast estrogen screen [YES], ER luciferase assay with HEK 293 cells [HEK], primary rainbow trout hepatocytes [PH]) and chemical analysis. Three sampling campaigns were carried out at each site between November 2000 and September 2001. Vitellogenin (VTG)-mRNA expression in male rainbow trout exposed for two weeks ranged from 3 +/- 5 to 619 +/- 188 and from 226 +/- 38 to 3373 +/- 1958 pg/microg total RNA at sites A and B, respectively. E2-equivalents obtained from the in vitro bioassays gave values up to 0.21 +/- 0.04 nM (57.3 +/- 10.2 ng/l, PH), 0.07 +/- 0.03 nM (20.2 +/- 6.9 ng/l; YES) and 0.008 +/- 0.002 nM (2.1 +/- 0.7 ng/l; HEK). In contrast, in one-year-old rainbow trout exposed at site C, no VTG-mRNA induction could be observed after two weeks of exposure. In vitro bioassays (YES, HEK, PH) indicated estrogenic activity at site C, which, however, was lower than at the investigated STP effluents. Chemical analysis of representative water samples from site A identified steroidal estrogens up to 5.6 ng/l 17beta-estradiol (E2), 19 ng/l estrone as well as 1.5 ng/l 17alpha-ethinylestradiol. Furthermore, the sum of fecal- and phytosteroids, resorcyclic lactones, and flavonoid concentrations were 280 (A) and 1.200 ng/l (B). In addition, site C (river Rhine) contained 3.9 ng/l E2 and 250 ng/l of fecal- and phytosteroids, respectively. Thus, STP effluents and Rhine water contain biologically relevant concentrations of estrogenic compounds, the activity of which can be detected by means of various bioassays.

Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase.

BACKGROUND: Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells. METHODS AND RESULTS: Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation). CONCLUSIONS: In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.