Development and implementation of an integrative bioanalytical approach to identify sources of toxicity formed during ozonation of wastewater

Project Information

OzoToxID

Grant agreement ID: 898843

DOI

10.3030/898843

Project closed

EC signature date 20 April 2020

Start date 1 January 2021

End date 31 January 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 203 149,44

EU contribution

€ 203 149,44

Coordinated by

EIDGENOESSISCHE ANSTALT FUER WASSERVERSORGUNG ABWASSERREINIGUNG UND GEWAESSERSCHUTZ
Switzerland

Periodic Reporting for period 1 - OzoToxID (Development and implementation of an integrative bioanalytical approach to identify sources of toxicity formed during ozonation of wastewater)

Reporting period: 2021-01-01 to 2022-12-31

Summary of the context and overall objectives of the project

Ozonation is increasingly integrated into wastewater treatment trains to eliminate organic micropollutants, known to induce adverse effects to aquatic organisms. Additionally, ozonation is sometimes applied in water reuse treatment trains for various treatment goals including disinfection and/or micropollutant abatement. While ozonation has been demonstrated to effectively abate micropollutants in wastewater and reduce toxicity for many endpoints, concerns remain regarding the unintended formation of mutagenic (genotoxic) byproducts. Except for some specific known toxic ozonation byproducts, little is known about the formed mutagenic ozonation byproducts. This knowledge is critical to ensure process optimization by meeting treatment goals while minimizing mutagenicity formation.

OzoToxID aimed at developing and implementing a novel bioanalytical strategy, based on effect-directed analysis (EDA) combining toxicological and advanced chemical analysis, to investigate the formation of toxic ozonation byproducts during wastewater treatment.

An integrative approach combining a mutagenicity assay and advanced chemical analysis as well as mechanistic and kinetic assessments were conducted. Additionally, a non-target screening method for carbonyl compounds, a toxicologically relevant chemical class, has been developed.

Overall, the findings from OzoToxID confirmed the systematic formation of mutagenicity during wastewater ozonation. Subsequent sand filtration allowed to either partially or entirely eliminate the mutagenic byproducts. Nitrite concentration, the applied ozone dose, and the Dissolved Organic Matter (DOM) type played a critical role in the formation of mutagenicity during ozonation. The presence of nitrite in wastewater resulted in a higher formation of mutagenic byproducts during ozonation. This was also accompanied by the formation of nitrocompounds from organic precursors, in a pathway which presumably involves nitrogen dioxide (NO2) as nitrating agent. A unique fingerprint was attributed to effluent DOM in the formation of mutagenicity, in contrast to other DOM types (e.g. lake water, gray water), for which only negligible or very low mutagenicity was formed during ozonation. Furthermore, a novel non-target screening method for the analysis of carbonyl compounds has been validated and applied for the assessment of carbonyl compound formation during ozonation of different water matrices.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

OzoToxID aimed at achieving a better understanding of the formation of mutagenic compounds during advanced wastewater treatment involving an ozonation step. Substantial knowledge was generated on the level of the three main research lines outlined in the following paragraphs:

A systematic assessment has been carried out to evaluate the formation of mutagenicity during ozonation of various water matrices including surface water, greywater, and secondary effluent wastewater originating from different biological processes. This evaluation confirmed the distinct significant formation of mutagenic byproducts during ozonation of wastewater, in contrast to the other water matrices. Mutagenicity formation in secondary effluent wastewater treated with different types of biological treatments (biological oxygen demand removal, nitrification, and denitrification) revealed that the tested biological treatment types did not have a clear influence on mutagenicity formation during ozonation.

In the course of the project, the role of nitrite-ozone reaction in forming nitro compounds during ozonation and the formation of mutagenicity was confirmed. This motivated research focused on the elucidation of the nitration reaction pathway and kinetics. Experiments using ozone-nitrite, peroxynitrite, gamma radiolysis (under conditions forming NO2) were performed. The assessment hinted towards the unexpected role of NO2 as potentially a major reactive nitrogen species involved in of the observed nitration reactions.

The formation of carbonyl compounds is well documented during ozonation of natural organic matter isolates and drinking water. However, there is only limited information on the formation of carbonyl compounds during ozonation of wastewater. Considering the diversity of potential carbonyl precursors in DOM, the currently known carbonyl compounds represent only a small fraction of the universe of this compound class. Since many carbonyl compounds are known to be toxic (including mutagenic and genotoxic effects) due to their nucleophilic properties, a non-target screening method has been developed and applied for the analysis of ozonated water and wastewater samples. The method allowed the identification of several unknown compounds which are formed during ozonation.

Overall, the realization of the above research activities resulted in the achievement of the following milestones and deliverables:
- Development of an effect-directed analysis workflow which was tested and validated by spiking mutagenic compounds into different water matrices (paper in preparation)
- Identification of the role of nitrite in the formation of mutagenicity during ozonation of wastewater and of the fingerprint of effluent DOM in forming mutagenic compounds in contrast to other DOM types (paper in preparation)
- Insights into the mechanisms of nitration of organic compounds during ozonation in the presence of nitrite (paper in preparation)
- Development of a non-target screening method for carbonyl compounds in different water matrices including wastewater, identification of unknown compounds, and determination of formation trends during ozonation (Manasfi et al., 2023 and Houska et al., 2023 published in Water Research)

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The findings regarding the role of effluent DOM, nitrite concentration, and the ozone dose on the formation of mutagenicity during ozonation of wastewater are among the major findings from OzoToxID. In additiona, substantial advances were achieved regarding the formation mechanism of nitrocompounds during ozonation in the presence of nitrite.
Furthermore, a novel analytical method for the non-targeted analysis of carbonyl compounds in water matrices was optimized and validated. The application of the method to ozonated samples elucidated the formation trends of different carbonyl compounds. Overall, OzoToxID provided substantial progress in understanding the formation of (potentially) mutagenic ozonation byproducts during advanced wastewater treatment.

Wastewater ozonation is a timely and relevant issue as increasing public awareness regarding the adverse effects of micropollutants on the environment and potentially human health stimulates the upgrade of conventional WWTPs in Europe. Moreover, ozonation can be applied in potable water reuse treatment trains in water-scarce regions. Therefore, the research carried out in OzoToxID is at the forefront of environmental and human health issues. Conducting research on this topic, with the aim of gaining fundamental understanding which can lead to optimization of treatment in practice, does not only contribute to enhancing European research excellence and competitiveness but also provides progress on a topic with major societal implications in alignment with European strategies for sustainable development and pollution remediation.

OzoToxID Project Overview (created with Biorender.com)