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Water quality: new insights thanks to a more efficient integrated approach

August 9, 2018 | Mirella Wepf

The development of novel, less costly analytical methods is of crucial importance in addressing the issue of micropollutants in surface waters. This was one of the main goals of EDA-EMERGE, an EU-funded programme in which Eawag also participated.

While increasing numbers of chemicals are entering surface waters, routine monitoring in Switzerland and the EU focuses on a relatively small number of compounds. These priority substances were originally defined as being of particular environmental concern. However, recent studies have shown that non-priority or even unidentified substances can also pose significant risks to aquatic life and human health. This was confirmed by a European research programme in which Eawag scientists also participated. The aim of EDA-EMERGE was to develop new effect-directed analysis (EDA) techniques, which were applied in a Europe-wide demonstration programme.

Combining biotesting and chemical analysis

In EDA, biotesting is combined with chemical analysis: while bioassays (e.g. involving human cells or zebrafish embryos) can detect toxicity in water samples, it often remains unclear what substances are causing the effects – hence the need for chemical analysis.

In the EDA-EMERGE programme, this approach was further optimised and simplified. Key components are selected bioassays and a newly developed device for the enrichment of micropollutants from large-volume water samples.

Unexplained adverse effects on organisms

Using the new methods, 37 scientists from 15 institutions investigated water quality near wastewater treatment plants (WWTPs) on four European rivers. One of the sites studied was the Urtene river (in the Bernese Midland) near the Moossee-Urtenenbach WWTP.

In samples from all four rivers, bioassays detected toxic effects on algae and fish embryos and endocrine effects which could not be explained solely in terms of the standard list of target compounds. The effects were observed, in particular, downstream of WWTP outlets.

Chemical analysis revealed that the samples also contained numerous substances not included on the target list. Frequently occurring compounds included, for example, gabapentin (an anticonvulsant), TMDD (a surfactant) and phthalimide (a fungicide transformation product). (For more information see: Tousova et al. 2017 / https://doi.org/10.1016/j.scitotenv.2017.06.032)

Outlet of the Moossee-Urtenenbach WWTP. (Photo: Jennifer Schollée, Eawag)

Outlet of the Moossee-Urtenenbach WWTP.
(Photo: Jennifer Schollée, Eawag)

A closer look at the Urtenenbach

After the initial study phase, more detailed investigations were carried out on the Urtenenbach by a smaller (8-strong) team, comprising scientists from Eawag and INERIS (the French National Institute for Industrial Environment and Risks).

Marc Suter, Head of the Bioanalytics Group in Eawag’s Environmental Toxicology department, explains the background: “In the first phase of the study, the project team investigating all four rivers used familiar standard tests and divided the samples into four fractions.” In the second phase, new bioassays were used to study the mode of action of substances and mixtures in more detail. “We also collected far more samples, which were separated on the basis of physicochemical properties into 40 fractions – an extremely high resolution.” The more finely water samples are fractionated, the more clearly can the biological activity observed be attributed to individual compounds.

Precise results thanks to fine fractionation

This method was used to validate the initial study results and to exclude the possibility of laboratory findings being distorted by antagonists. Suter gives an example: “In the second series of tests, estrogenic activity was detected in 20 fractions. In the more crudely separated fractions used in the first test series, it would have been possible for antiestrogens (estrogen antagonists) to mask the effects of estrogens occurring in the same fraction, leading to false‑negative results.” High-resolution fractionation provides a better picture of reality.

Higher glucocorticoid activity

Overall, according to Suter, the results of the first and the second study differed less than had originally been expected. There were, however, some notable differences: “For example, with the aid of finer fractionation, we detected much higher glucocorticoid activity.” Glucocorticoids can have adverse effects on the immune system in humans and animals. This was shown in a zebrafish study carried out by Eawag doctoral student Anita Hidasi as part of the EDA-EMERGE programme (Hidasi et al. 2017  / https://doi.org/10.1016/j.ecoenv.2016.11.024).

Although the second phase of the project required major efforts, it yielded valuable insights: thanks to the combination of existing EDA techniques with new bioassays, Suter says, we now have a better understanding of various adverse effects observed in the laboratory. This is an important step towards improved risk assessment of surface water contaminants – particularly in WWTP effluents. In their conclusions, the scientists also emphasize the need for more research on the effects of glucocorticoids on aquatic organisms.

Financing

EDA-EMERGE is a 4-year Marie Curie ITN project, funded by the European Commission. It aims to train a new generation of young scientists in interdisciplinary techniques.

Publications

An integrative approach combining passive sampling, bioassays and effect-directed analysis to assess the impact of wastewater effluent

Wastewater treatment plant (WWTP) effluents are major sources of endocrine disrupting chemicals (EDCs) and of other chemicals of toxicological concern for the aquatic environment. In this study, we used an integrated strategy combining passive sampling (Chemcatcher®), developmental toxicity and mechanism‐based in vitro and in vivo bioassays to monitor the impacts of a WWTP on a river. In vitro screening revealed the WWTP effluent as a source of estrogen (ER), glucocorticoid (GR), aryl hydrocarbon (AhR) receptor mediated activities, impacting the downstream river site where significant activities were also measured, albeit to a lesser extent than in the effluent. Effect‐directed analysis (EDA) of the effluent successfully identified the presence of potent estrogens (estrone, 17α‐ethinylestradiol and 17β‐estradiol) and glucocorticoids (clobetasol propionate and fluticasone propionate) as the major contributors to the observed in vitro activities, even though other unidentified active chemicals were likely present. The impact of the WWTP was also assessed using zebrafish embryo assays, highlighting its ability to induce estrogenic response through up‐regulation of aromatase promoter‐dependent reporter gene in the transgenic (cyp19a1b–GFP) zebrafish assay and to generate teratogenic effects at non‐lethal concentrations in the zebrafish embryo toxicity test. This study argues for the use of such an integrated approach, combining passive sampling, bioassays and EDA, to comprehensively identify endocrine active compounds and associated hazards of WTTP effluents.
Sonavane, M.; Schollée, J. E.; Hidasi, A. O.; Creusot, N.; Brion, F.; Suter, M. J. -F.; Hollender, J.; Aït-Aïssa, S. (2018) An integrative approach combining passive sampling, bioassays and effect-directed analysis to assess the impact of wastewater effluent, Environmental Toxicology and Chemistry, 37(8), 2079-2088, doi:10.1002/etc.4155, Institutional Repository

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.
Tousova, Z.; Oswald, P.; Slobodnik, J.; Blaha, L.; Muz, M.; Hu, M.; Brack, W.; Krauss, M.; Di Paolo, C.; Tarcai, Z.; Seiler, T.-B.; Hollert, H.; Koprivica, S.; Ahel, M.; Schollée, J. E.; Hollender, J.; Suter, M. J. -F.; Hidasi, A. O.; Schirmer, K.; Sonavane, M.; Ait-Aissa, S.; Creusot, N.; Brion, F.; Froment, J.; Almeida, A. C.; Thomas, K.; Tollefsen, K. E.; Tufi, S.; Ouyang, X.; Leonards, P.; Lamoree, M.; Torrens, V. O.; Kolkman, A.; Schriks, M.; Spirhanzlova, P.; Tindall, A.; Schulze, T. (2017) European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters, Science of the Total Environment, 601, 1849-1868, doi:10.1016/j.scitotenv.2017.06.032, Institutional Repository

Clobetasol propionate causes immunosuppression in zebrafish (Danio rerio) at environmentally relevant concentrations

Synthetic glucocorticoids (GCs) are potential endocrine disrupting compounds that have been detected in the aquatic environment around the world in the low ng/L (nanomolar) range. GCs are used as immunosuppressants in medicine. It is of high interest whether clobetasol propionate (CP), a highly potent GC, suppresses the inflammatory response in fish after exposure to environmentally relevant concentrations. Bacterial lipopolysaccharide (LPS) challenge was used to induce inflammation and thus mimic pathogen infection. Zebrafish embryos were exposed to ≤1000 nM CP from ~1 h post fertilization (hpf) to 96 hpf, and CP uptake, survival after LPS challenge, and expression of inflammation-related genes were examined. Our initial experiments were carried out using 0.001% DMSO as a solvent vehicle, but we observed that DMSO interfered with the LPS challenge assay, and thus masked the effects of CP. Therefore, DMSO was not used in the subsequent experiments. The internal CP concentration was quantifiable after exposure to ≥10 nM CP for 96 h. The bioconcentration factor (BCF) of CP was determined to be between 16 and 33 in zebrafish embryos. CP-exposed embryos showed a significantly higher survival rate in the LPS challenge assay after exposure to ≥0.1 nM in a dose dependent manner. This effect is an indication of immunosuppression. Furthermore, the regulation pattern of several genes related to LPS challenge in mammals supported our results, providing evidence that LPS-mediated inflammatory pathways are conserved from mammals to teleost fish. Anxa1b, a GC-action related anti-inflammatory gene, was significantly down-regulated after exposure to ≥0.05 nM CP. Our results show for the first time that synthetic GCs can suppress the innate immune system of fish at environmentally relevant concentrations. This may reduce the chances of fish to survive in the environment, as their defense against pathogens is weakened.
Hidasi, A. O.; Groh, K. J.; Suter, M. J. -F.; Schirmer, K. (2017) Clobetasol propionate causes immunosuppression in zebrafish (Danio rerio) at environmentally relevant concentrations, Ecotoxicology and Environmental Safety, 138, 16-24, doi:10.1016/j.ecoenv.2016.11.024, Institutional Repository

Photos

Jennifer Schollée deploying a passive sampler (Chemcatcher) at the Moossee Urtenenbach WWTP effluent discharge site. As a doctoral student, Schollée, together with Anita Hidasi, was part of the 7-strong Eawag team ... (Photo: Anita Hidasi, Eawag)

Jennifer Schollée deploying a passive sampler (Chemcatcher) at the Moossee‑Urtenenbach WWTP effluent discharge site. As a doctoral student, Schollée, together with Anita Hidasi, was part of the 7-strong Eawag team participating in EDA-EMERGE. The programme was explicitly designed to train early-career scientists. (Photo: Anita Hidasi, Eawag)

In the laboratory, Eawag doctoral student Jennifer Schollée prepares passive samplers for the second sampling campaign on the Urtenenbach. Passive sampling is used to determine concentrations of water pollutants. (Photo: Birgit Beck, Eawag)

In the laboratory, Eawag doctoral student Jennifer Schollée prepares passive samplers for the second sampling campaign on the Urtenenbach. Passive sampling is used to determine concentrations of water pollutants.
(Photo: Birgit Beck, Eawag) 

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