Ecotoxicological assessment of the wastewater treatment plant Aachen-Soers and the receiving stream before and after implementation of a large-scale ozonation plant

  • Ökotoxikologische Bewertung der Kläranlage Aachen-Soers und ihres Vorflutes vor und nach Implementierung einer großtechnischen Ozonungsanlage

Wolf, Yvonne; Hollert, Henner (Thesis advisor); Schäffer, Andreas (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Around two-thirds of the world is covered by water; only 2.5 % is classified as freshwater, of which the majority is locked in glaciers and ice caps. Furthermore, half of the freely accessible water is already being used by mankind. This aquatic environment does not only play an important role as habitats for different aquatic species, it also provides ecosystem functions important for humans and as a resource for drinking water. Thus, the protection of the aquatic environment is of high importance. Yet, the pressure on this environment is increasing. The growing human population and increasing industrial development leads to higher disposal of anthropogenic chemicals and waste into the environment. Therefore, the European Water Framework Directive (EU-WFD) was set into operation in 2000. Its main goal is to achieve a "good biological" and a "good chemical" status of all surface waters until 2027. Among other things, this should be achieved by reducing the number of hazardous compounds introduced into the aquatic environment. For this purpose, 45 priority substances were selected that gradually should be reduced in surface waters below their so-called environmental quality standards (EQS). To support the future inclusion of new compounds into the list of priority substances, the EU watch list was published; it included ten substances or substance classes. One class consists of estrogenic compounds, including the natural estrogen 17β-estradiol as well as the synthetic compound 17α-ethinylestradiol. While these micropollutants (MPs) occur often at very low concentrations (pg · L-1 and ng · L-1 range), they are also designed to exert their biological effect at these low concentrations. However, standard chemical analytical methods are often not sensitive enough to detect them at concentrations around their EQS values. Furthermore, chemical analytical methods are not able to detect mixture effects of substances. Recent studies have shown that effect-based methods (EBMs) can detect effects at lower concentrations. Hence, they might be suitable to monitor the effects of the previously mentioned MPs. To implement EBM into the revised version of the EU-WFD as a supporting tool for chemical analysis has already been proposed. It could be shown that wastewater treatment plants (WWTPs) are one major point source of MPs into the aquatic environment, since most conventional treatment processes often fail to sufficiently reduce MPs (like benzotriazole, diclofenac, or carbamazepine). The present thesis was part of the interdisciplinary project DemO3AC. The main goal of this project was to improve the quality of the released water by the WWTP Aachen-Soers. Its recipient is the Wurm River that has shown a dissatisfying or poor biological and chemical status in the EU-WFD screenings. Downstream of the WWTP Aachen-Soers the Wurm River consists of at least 70 % of treated wastewater. As a measure to improve the quality of the Wurm River a large-scale ozonation plant was built. The present thesis aimed to ecotoxicologically assess the impact of the large-scale ozonation plant by analyzing its cleaning efficiency before (phase 1) and after (phase 2) ozonation. As a result of the treatment with ozone, an improvement of the water quality downstream of the WWTP Aachen-Soers was expected. The testing strategy was mainly based on the extended triad approach by Chapman and Hollert (2006) covering different lines of evidence (LOE; in vitro and in vivo EBMs, in situ experiments, residue analysis). The chosen EBMs were previously applied within the SOLUTIONS and NORMAN project. The in vivo methods employed belong to the classical bioassay battery (algae, daphnids, fish, luminescent bacteria) that is proposed for the assessment of wastewater by German authorities. It could be shown, that the influent of the WWTP Aachen-Soers included significant effects in the mentioned assays. Yet, the toxic potential was already reduced/eliminated by the conventional treatment processes during the first project phase. However, it has to be considered that only apical endpoints can be detected by these bioassays; mechanism-based assays may still detect effects at lower levels of biological organization. For this reason, a bigger focus was set on endocrine disrupting compounds (EDCs). These substances are continuously being released into the environment by WWTP effluents since they are not fully eliminated by conventional treatment methods. EDCs can lead to adverse effects at very low concentrations by interacting with receptors, such as the estrogenic (ER) or androgenic (AR) receptor. However, ozonation for example has shown the potential to reduce or even eliminate these effects. To investigate those effects, a bioassay battery assessing different levels of biological organization (from cell to organism) was chosen. To detect receptor-mediated agonistic (estrogenic and androgenic) as well as antagonistic (anti-estrogenic and anti-androgenic) potentials, a panel of CALUX (Chemical Activated Luciferase Expression) assays was conducted. An endocrine potential was detected before and after ozonation at all sampling points of the WWTP. Androgenicity and anti-estrogenicity seemed not to play such an important role in the aquatic environment as estrogenic or anti-androgenic compounds. The estrogenic potential was already reduced by conventional treatment and was further diminished by ozonation, yet, not fully eliminated. Nonetheless, the estrogenic potential was always reduced downstream of the WWTP in comparison to upstream, which indicates a pre-contamination of the Wurm River. Anti-androgenicity showed an increased reduction with higher ozone concentration (> 4 mg · L-1). However, for both endpoints determined equivalent concentrations were higher than the proposed effect-based trigger values indicating a potential risk for wildlife and humans. Additional measurements of single compounds (benzotriazole, bisphenol A, citalopram) showed that mixture effects (additive, antagonistic, synergistic) play a crucial role in the assessment of receptor-related endpoints. The endocrine activity was further assessed by the reproduction assay with Potamopyrgus antipodarum in the field. Results before and after ozonation did not show any effect of the ozonation on reproduction downstream of the WWTP Aachen-Soers after a four-week exposure period. In both cases, the reproduction downstream of the WWTP was significantly increased compared to the sampling sites upstream of the WWTP and the laboratory control. Since this assay was not conducted under control laboratory conditions abiotic factors could also have influenced the results of the experiments. As for the ERα CALUX assay, a pre-contamination was detected upstream of the WWTP at a higher temperature. This temperature was comparable with the temperature downstream of the WWTP for all experiments. Thus, multiple stressors (chemical load, temperature, nutrition) may have led to the increased reproduction downstream of the WWTP Aachen-Soers. Contrary to the ERα CALUX assay, no reduction of the effect was detected downstream of the WWTP Aachen-Soers. Additionally, juvenile rainbow trout (Oncorhynchus mykiss) were exposed for ten days at the Wurm River to assess the impact of the WWTP on different endpoints (body indices, vitellogenin, ethoxyresorufin-O-deethylase activity, glutathione-S-transferase activity, micronuclei, gene expression). For all endpoints assessing different potentials, no significant effect of the released wastewater before and after ozonation was detected, as well as neither a positive nor a negative influence of the installed ozonation plant. Depending on their bioaccumulation potential, some compounds need a longer period to induce adverse effects. Thus, a longer exposure period might result in a more realistic scenario for fish and potential delayed effects could be better determined. This was already shown by other studies. The impact of the WWTP Aachen-Soers was further assessed by the acute feeding assay and by determination of the internal concentration of hazardous compounds in Gammarus pulex. No clear effects in the acute feeding assay were found, likely due to the chemical load of the acute toxic compounds being too low to exert biological effects. The determination of the internal chemical load showed potential chronic effects on Gammarus pulex based on the measured toxic units. However, this effect was detected on all sample sites, and the highest influence was observed downstream of the rain overflow basin and not downstream of the WWTP Aachen-Soers. To conclude, the present thesis elucidated, that the upscaling of the WWTP Aachen-Soers with a large-scale ozonation plant resulted in a reduction of the endocrine potential and chemical load that is released into the Wurm River. Additionally, it was demonstrated that bioassays with different endpoints investigated with different levels of organization (cell and organism), under different conditions (field and laboratory), and with different sample matrices (native water sample and extract) play a crucial role in the assessment of complex environmental samples as surface and wastewaters. They can support chemical analytical methods, without knowledge of the exact sample composition. Yet, chemical analysis is still an important tool in the assessment of complex mixtures. Moreover, not only in vitro test systems should be used, since the complexity of an organism cannot be depicted by cell-based assessment alone. The present thesis was able to show, that different exposure patterns (reduction vs. no impact) were detected by different testing strategies (CALUX vs. reproduction assay). This indicates that abiotic factors can significantly impact the outcome of experiments. Finally, based on the findings of the DemO3AC project the need for implementation of further bioanalytical testing strategies into the assessment of chemicals is even more strengthened. The EU-WFD stated to support further implementation of EBMs into regulation. Other projects also set a basis for further use of those methods (SOLUTIONS, NORMAN). However, more assessment criteria need to be defined. A good start is given by other scientists. Yet, those criteria need to be consistent and find their way into regulation. The present thesis demonstrated that it is possible to make an integrative analysis of complex environmental samples (surface and wastewater) based on an assessment strategy covering different LOE (e.g., EBMs, in situ experiments, chemical analysis).


  • Department of Biology [160000]
  • Chair of Environmental Biology and Chemodynamics [162710]