Introduction
In Europe, many individuals enjoy access to clean and safe water simply by turning on the faucet, yet they often remain unaware of its source and treatment processes The provision of such quality water hinges on high raw water quality, which is becoming a significant global challenge Emerging organic compounds, including pharmaceuticals and industrial chemicals, threaten our water resources, as conventional municipal wastewater treatment plants (WWTPs) typically fail to adequately address these contaminants Consequently, these substances enter aquatic environments, compromising water quality and raising concerns about human health, ecology, and economic implications With an increasing identification of micropollutants in surface and groundwater, there is an urgent need for innovative treatment and management strategies to ensure sustainable and cost-effective solutions throughout Europe.
Recent studies have highlighted the prevalence of organic micropollutants, such as endocrine disrupting compounds (EDCs) and pharmaceuticals, in aquatic environments These pollutants, primarily of anthropogenic origin, are often released through wastewater, making even tertiary treated effluents significant contributors to their presence in water bodies Alarmingly, even trace concentrations of these micropollutants can adversely affect aquatic organisms and limit the use of water resources for human consumption The escalating demand for water, coupled with reduced availability due to climate change, has intensified research into advanced technologies aimed at effectively removing these organic trace pollutants from wastewater.
Biological processes, particularly the conventional activated sludge process, dominate wastewater treatment plants (WWTPs) globally Despite this prevalence, traditional organic sum parameters like Chemical Oxygen Demand (COD) are often used to assess treatment efficiency.
BOD are removed to a high degree, others comprising micropollutants are released into the environment unchanged or metabolized (Krzeminski et al., 2019; Quintana et al.,
2005) To mitigate this release, particular attention has been directed towards advanced treatment technologies
Advanced wastewater treatment technologies utilizing ozone (O3) and granular activated carbon (GAC) effectively reduce various endocrine-disrupting compounds (EDCs) in wastewater treatment plant (WWTP) effluents A multibarrier system incorporating both O3 and GAC shows promise, as ozonation can eliminate adsorbed molecules and restore the adsorption capacity of activated carbon GAC's extensive surface area allows for effective adsorption and reaction with ozone and organic pollutants While the O3-GAC method is a viable option for diminishing or mineralizing organic pollutants, achieving complete mineralization of resistant organic matter requires substantial ozone consumption To enhance the economic viability of ozonation, it is often paired with biological processes in water and wastewater treatment.
Ozone application is an effective technology for the removal of organic micropollutants from urban wastewater, already implemented at full scale in countries like Switzerland, Germany, and Sweden The efficiency of removing various micropollutants depends on their reactivity with ozone and hydroxyl radicals, the ozone dosage, and the wastewater composition To minimize ozone scavenging by organic matter, ozonation is typically performed after biological treatment In biologically treated wastewater, ozone effectively targets electron-rich structures, including olefins, aromatic rings, and amines.
2012) and thus reacts with micropollutants (Lee et al., 2016; Rizzo et al., 2019)
Oxidative transformation of matrix components leads to the formation of both inorganic byproducts, such as bromate, and organic compounds like nitrosamines and aldehydes These byproducts may possess greater toxicological risks than their original substances Therefore, it is essential to minimize the generation of these transformation products during technical operations.
The ozone dose plays a crucial role in the formation of unwanted oxidation byproducts during ozonation, alongside the chemical matrix and precursor substances in raw water Research indicates that when the ozone dose is maintained below 0.5g O3/g DOC, the formation of bromate is minimal due to the rapid decomposition of ozone, resulting in reduced ozone exposure (Lee et al., 2013).
Scope and Structure of the Work
This Ph.D research is an interdisciplinary study encompassing advanced wastewater treatment, toxicology, and water quality The experiments were primarily conducted in the laboratories of the Research Unit Water Quality Management at the Institute for Water Quality and Resource Management, TU Wien.
This Ph.D thesis aims to address the identified needs and facts regarding the application of ozonation in municipal wastewater treatment plants, focusing on its effectiveness and potential benefits.
- Evaluate the correlation between ozone dose to the effective removal of trace organic compounds (TrOCs) and the formation of oxidation byproducts
- Evaluate the impact of ozonation on the biodegradability change of recalcitrant COD in treated urban wastewater
- The toxicological evaluation of the treatment efficiency, general cytotoxicity, and decrease of endocrine activity after ozonation
Hence this Ph.D thesis can be divided into three main aspects
Stage 1 focuses on assessing the removal of trace organic contaminants (TrOCs) and the generation of oxidation byproducts, specifically bromate, during the ozonation process Utilizing effluent from an Austrian wastewater treatment plant (WWTP), the study analyzes nine TrOCs commonly found in municipal wastewater, selected based on EU legislation, human metabolism and excretion, environmental prevalence, treatment persistence, and aquatic toxicity The contaminants under investigation include pharmaceuticals, corrosion inhibitors, and artificial sweeteners, with specific research questions guiding the experimental process.
- How is the decomposition performance of ozonation for TrOCs?
- How is the bromate formation in the investigated wastewater related to the ozone dose?
In order to answer the research questions, batch tests were conducted with different nitrite compensated specific ozone doses (0.2, 0.4, 0.6, 0.8, and 1.0 g O3/g DOC)
Stage 2 focused on assessing how ozonation affects the biodegradability of recalcitrant chemical oxygen demand (COD) in urban wastewater following conventional biological treatment Key parameters analyzed included biochemical oxygen demand (BOD5), COD, and dissolved organic carbon (DOC).
UV absorption at 254 nm (UV254) Additionally, two micropollutants were analyzed to validate the experimental setup for ozonation batch tests Specifically, the study aimed to answer the following research questions:
An increase in specific ozone doses used for micropollutant removal in urban wastewater may influence organic sum parameters that are commonly evaluated in wastewater treatment These parameters serve as quality criteria and thresholds for treatment targets in conventional wastewater processes Understanding this impact is crucial for optimizing treatment efficacy and ensuring compliance with environmental standards.
- Does ozonation result in an increase in biodegradability of substances previously recalcitrant to biological degradation, and is there a correlation with the specific ozone dose?
Stage 3 of the study focused on the effluent from a wastewater treatment plant (WWTP) treated using a multibarrier system, specifically ozone and granular activated carbon (GAC), at a pilot-scale facility within a full-scale WWTP The primary objective was to conduct long-term toxicological monitoring of this advanced wastewater treatment, utilizing a mode of action (MOA)-based in vitro bioassay battery to evaluate key toxicological endpoints Following the installation and training of WWTP operators to ensure robust operation, the integration of the plant's operation into their daily routine was prioritized Over the course of one year, monthly routine monitoring samples were collected to assess the performance and suitability of the technologies for broader implementation The study aimed to address critical research questions regarding the effectiveness of the multibarrier treatment approach.
- How is the suitability of the multibarrier system with O3 and GAC for advanced wastewater treatment with regard to toxicity?
- How is the toxicity abatement of the two treatment technologies in real-life conditions?
In order to answer the research questions, two approaches were employed:
- The biological equivalent concentrations (BEQs) decrease was determined for the various steps of the multibarrier system
- The BEQs were compared to currently discussed MOA-specific effect-based trigger values (EBTs)
Base on the research questions, this PhD thesis consists of six chapters The outline of these chapters can be given as
Chapter 1 presents the necessary background information and motivations to perform this thesis
Chapter 2 describes the scope and the structure of this thesis
Chapter 3 provides extensive background on micropollutants in wastewater, advanced wastewater treatment technology, and ozonation
Chapter 4 starts with materials and methods It describes lab-scale and pilot scale experimental setup used in this thesis
Chapter 5 reports and discusses the results obtained in experimental investigation
Chapter 6 provides a summary and conclusion of this Ph.D thesis
Background
Micropollutants in wastewater
Water is an essential resource for all living beings and is integral to human activities However, contaminants of emerging concern (CECs), or micropollutants, are present in drinking water, surface waters, and groundwater at concentrations ranging from a few ng/L to several µg/L These trace organic compounds (TrOCs) include everyday products such as pharmaceuticals, industrial chemicals, personal care items, and pesticides, which can harm human health, the environment, and aquatic life The pollution caused by these micropollutants poses significant risks to marine organisms and can have detrimental effects on human health within the ecosystem.
Between 2002 and 2019, the Chemical Abstract Service Registry expanded from 20 million to 156 million chemicals (Escher et al., 2020) In response to the need for chemical regulation, the European Chemicals Agency (ECHA) was created to oversee the registration of chemicals under the EU-wide REACH regulation (EG 1907/2006), which currently lists approximately 22,614 compounds as of June 2021 However, there are no existing regulations or discharge standards for many trace substances in urban wastewater within the European Union To safeguard water resources, the EU Water Framework Directive 2000/06/CE identifies 45 priority compounds or groups, including pesticides, heavy metals, PAHs, phthalates, and endocrine-disrupting chemicals (EDCs) Additionally, Decision 2015/495/EU established a trace list for monitoring a variety of synthetic and natural chemicals across the EU (Barbosa et al., 2016).
Micropollutants in aquatic environments have emerged as a significant global issue, primarily stemming from various sources such as industrial wastewater, agricultural runoff, effluents from medical facilities, and waste generated by concentrated livestock operations.
Runoff from agricultural and livestock areas significantly contributes to the presence of trace organic contaminants (TrOCs), particularly through the use of pesticides and hormonal steroids (Song et al., 2007) The reuse of wastewater for irrigation exacerbates this issue, introducing various TrOCs and their transformation products into field water systems (Barbosa et al., 2016) Additional sources of TrOCs include wastewater treatment plants, landfill leaks, industrial waste, and septic systems (Matthiessen et al., 2006) Domestic wastewater is a prominent source of pharmaceutical residues (such as lipid modifiers, anticonvulsants, and antibiotics), personal care products (including perfumes and disinfectants), and steroid hormones like estrogen (Luo et al., 2014) Moreover, everyday domestic usage of these compounds contributes to their prevalence in the environment (Kanaujiya et al., 2019) An overview of the primary sources of micropollutants in aquatic ecosystems is provided in Table 3.1, with detailed descriptions in the subsequent sections.
Table 3.1 Micropollutants categories and their major sources (according to Luo et al (2014), modified)
Category Important subclasses Major sources Examples
Antibiotics, antidiabetics, analgesics, anticonvulsants, lipid regulators, anticonvulsants, antibiotics, - blockers, and stimulants
Urban wastewater (excretion) Hospital effluents
Acetaminophen, diclofenac, ibuprofen, ketoprofen, mefenamic acid, naproxen, carbamazepine, bezafibrate, sulfamethoxazole, metoprolol, caffeine, atenolol, etc
Personal care products Fragrances, disinfectants, UV filters, and insect repellents
Urban wastewater (bathing, shaving, spraying, swimming and etc.)
Benzophenone, diltiazem, chloroprene, triclosan, methyl benzylidene, chloroprene, tonalite, etc
Urban wastewater (excretion) Hospital effluents
Estradiol, estrone, progesterone, testosterone, etc
Urban wastewater (bathing, laundry, dishwashing and etc.)
Industrial wastewater (industrial cleaning discharges)
Alkylphenol ethoxylates, alkylphenols (nonylphenol and octyl-phenol), perfluorooctanesulfonates acid, perfluorooctanoic acid
Industrial chemicals Plasticizers, fire retardants Urban wastewater (by leaching out of the material)
Benzotriazole, phthalates, polybrominated compounds, dioxin and furans, polycyclic hydrocarbons, trichloroethylene, benzene, toluene, etc
Pesticides Insecticides, herbicides and fungicides
Urban wastewater (improper cleaning, run-off from gardens, lawns and roadways and etc.) Agricultural runoff
The global use of pharmaceutical products is rapidly increasing, with over 200 different medicinal compounds detected in river water (Hughes et al., 2013) Commonly studied pharmaceuticals include anti-depressants, beta-blockers, non-steroidal anti-inflammatory drugs, and the antiepileptic carbamazepine (Petrie et al., 2015) Antibiotics and anti-inflammatory medications are among the most frequently used pharmaceuticals, and their residues in water pose chronic toxicity risks to both humans and animals (Waleng et al., 2022) The environmental prevalence of antibiotics is particularly concerning, as they are extensively used to treat bacterial infections in humans and animals, as well as in livestock production, with more than 250 different antibiotics and 63,151 tons utilized in medical applications (Ashfaq et al., 2016).
Approximately 70% of antibiotics are not absorbed or metabolized by humans or animals, resulting in their excretion into the environment via feces (Ahmad et al., 2019b) The extensive use of antibiotics in both human and veterinary medicine contributes to elevated concentrations in aquatic ecosystems These pharmaceuticals can enter the environment through various channels, including manufacturing facilities, wastewater from individuals and patients, sewer systems, and improper disposal methods Additionally, antibiotics may contaminate groundwater through agricultural practices such as fertilization and leaching (Ahmad et al., 2019a; Kümmerer, 2009).
Antibiotics target prokaryotic cells by disrupting the synthesis of the cell envelope, proteins, and nucleic acids (DNA/RNA), leading to potential resistance in bacteria Concentrations of antibiotics in soil and water can range from a few nanograms to hundreds of nanograms per kilogram or liter, particularly near hospitals and animal production farms While some antibiotics, like penicillin, can degrade over time, others, such as tetracycline, persist longer in the environment, resulting in significant ecological impacts The continuous release of pharmaceutical products contributes to their increasing prevalence in the environment, raising concerns about their long-term effects.
Personal care products (PCPs) encompass a wide range of items, including perfumes, cosmetics, shampoos, skincare products, oral care products, soaps, and sunscreens, which are utilized globally in large quantities A subset of PCP ingredients includes fragrances like nitro and polycyclic musk, as well as sunscreens, disinfectants, antiseptics, repellents, and preservatives Many of these products serve as additives, with cosmetic ingredients often comprising lipids or oils, such as those found in sunscreens Consequently, there is a significant diversity in the ingredients used in personal care products (Liu et al., 2013).
Wastewater treatment plants are significant contributors to the introduction of pharmaceuticals and personal care products (PCPs) into aquatic environments, as certain PCPs remain und degraded during the treatment process (Blair et al., 2015; Liu et al., 2013; Meador et al., 2016).
Water contamination by personal care products (PCPs) poses significant risks to aquatic ecosystems, humans, and animals due to their toxic nature Research indicates that PCPs are persistent, bioactive, bioaccumulative, and can disrupt endocrine functions (Niemuth et al., 2015).
Yu et al (2013) highlight that the release of certain compounds is influenced by various factors, including waste stream flow and the usage patterns of pharmaceutical and personal care products (PCPs), which can differ by region and season Montes-Grajales et al (2017) further emphasize that these variations significantly affect the environmental fate and concentration of these substances.
The extensive use of surfactants in both industrial and household applications has led to significant environmental accumulation, with annual synthetic surfactant production surpassing 12.5 million tons (Ahmad et al., 2019a; Edser, 2006) Surfactants are categorized into four types—cationic, anionic, nonionic, and amphoteric—based on the charge of their head groups, which influences their physicochemical properties and various applications Unfortunately, these surfactants and their residues can infiltrate surface water and groundwater through wastewater systems, resulting in detrimental environmental impacts (Ivanković et al.).
2010) The presence of surfactants can cause physiological, pathological, and biochemical effects on humans, animals, and aquatic life (Ahmad et al., 2019a)
Pesticides are substances utilized to safeguard crops from pests, encompassing various types such as insecticides, herbicides, fungicides, molluscicides, rodenticides, and nematicides, along with their use as plant growth regulators (USEPA, 2014) The World Health Organization (WHO) reports approximately 3 million cases of pesticide poisoning and 220,000 fatalities annually, highlighting the serious health risks associated with their use This extensive application of pesticides has led to elevated levels of pesticide residues in global water bodies, with organochlorine and organophosphorus pesticides identified as particularly concerning groups (Ahmad et al., 2019a).
Pesticides pose significant risks to non-target species, leading to detrimental effects on humans, animals, and terrestrial ecosystems Studies indicate that 80–90% of applied pesticides volatilize, harming plants and other organisms (Sonal et al., 2019) These chemicals can infiltrate the environment through various channels, including agricultural runoff, industrial waste, and leaks from landfills and sewage systems Highly toxic to both humans and animals, pesticides disrupt endocrine functions and reproductive health, earning them the label of xenohormones Moreover, the overuse of herbicides, fungicides, and pesticides contributes to deforestation by diminishing tree and shrub populations (Ahmad et al., 2019a; Sonal et al., 2019).
Industrial chemicals, including corrosion inhibitors, dishwasher detergents, and antifreeze, are significant micropollutants found at concentrations of 22.1 àg/L and 24.3 àg/L, respectively (Deeb et al., 2017; Rogowska et al., 2020).
Advanced wastewater treatment
Table 3.2 illustrates that various trace organic contaminants (TrOCs), including endocrine-disrupting compounds (EDCs), pharmaceutical and personal care products (PPCPs), pesticides, and certain household chemicals, are inadequately removed during biodegradation (Margot et al., 2015; Yang et al., 2017) The presence of these compounds in biological treatment systems highlights the necessity for supplementary processes to effectively eliminate them from wastewater Therefore, enhancing existing wastewater treatment plants (WWTPs) with advanced treatment technologies presents a viable solution for improving wastewater quality.
Recent efforts have focused on identifying advanced wastewater treatment methods that effectively reduce micropollutants beyond conventional biological treatments Techniques such as powdered activated carbon (PAC) adsorption, membrane filtration, and oxidation are being explored for their technical and economic viability in wastewater treatment plants.
The effectiveness of activated carbon in removing micropollutants is influenced by factors such as the type and dosage of carbon, operating conditions, and the composition of the water matrix Its high specific surface area and well-developed porous network contribute to its ability to adsorb a wide range of contaminants While granular activated carbon (GAC) has been utilized in various treatment plants, its efficiency can diminish based on the specific compound and the frequency of regeneration or replacement In contrast, powdered activated carbon (PAC), due to its smaller particle size, typically exhibits superior adsorption kinetics and may outperform GAC in terms of efficiency.
Despite the advantages of powdered activated carbon (PAC) in wastewater treatment, its application is hindered by slow reaction rates, challenges in separating PACs from wastewater, and competition from micro-contaminants and low molecular weight compounds (Abegglen et al., 2009; Ruhl et al., 2014; Zietzschmann et al., 2016) Additionally, the potential requirement for extra disinfection to comply with stricter wastewater reuse standards further limits its use (Rizzo et al., 2019) In contrast, micro-granules of granular activated carbon (GAC) have emerged as a promising alternative, offering benefits such as reduced waste solids during treatment and improved efficiency in waste treatment plants.
22 necessary to inject a coagulant such as FeCl3 to prevent leakage of activated carbon and is simpler to operate at a similar cost (Alves et al., 2018; Mailler et al., 2016)
Membrane technology, particularly high-pressure nanofiltration (NF) and reverse osmosis (RO), is gaining attention for its effectiveness in removing trace substances from wastewater, allowing for potential reuse in groundwater and agriculture The compatibility of NF and RO with other systems, such as activated carbon and ozonation, enhances their modularity and integrability However, a significant drawback of NF is the production of a concentrated stream, which can represent 10–20% of the original wastewater volume Additionally, the high energy demands from increased pressure operation, coupled with costs related to clogging and membrane replacement, pose challenges to the sustainability of these filtration techniques.
Ozone is emerging as a promising alternative for advanced municipal wastewater treatment, addressing the limitations of activated carbon and membranes Its versatility allows for significant reductions in micropollutant release into water bodies, while also enhancing wastewater quality for reuse purposes.
Ozone and ozonation process
In 1785, Dutch chemist Martinus van Marum observed an unusual odor while experimenting with electric sparks on water's surface, mistakenly attributing it to an electrical reaction, unaware that he was actually generating ozone.
In 1839, chemist Christian Friedrich Schürnbein identified a distinctive pungent odor associated with lightning, which he named "ozone" based on the Greek word "ozein" meaning to smell The molecular formula for ozone (O3) was established later in 1865 by Jacques-Louis Soret and subsequently verified by Schürnbein in 1867.
Ozone, a reactive molecule made up of three oxygen atoms, is thermodynamically unstable and easily converts back to oxygen Its formation is an endothermic process, and humans can detect its distinct smell at low concentrations.
Ozone is an unstable gas at room temperature, appearing blue when observed in sufficient thickness, with a concentration range of 15 g/m³ to 30-40 g/m³ for clear identification (Baig et al., 2010) The various structures of ozone are illustrated in Figure 3.3.
Figure 3.3 The structure of ozone
Moreover, a summary of the physicochemical and thermodynamic properties of ozone is presented in Table 3.3
(Baig et al., 2010; von Sonntag et al.,
Free molar formation entalpy KJ/mole 142.2
Ozone storage presents significant challenges, leading to the necessity of on-site ozone production (Baig et al., 2010) Modern industrial ozone generators, which have evolved from Werner von Siemens's original design in 1857 (von Sonntag et al., 2012), utilize various generation techniques Among these, only the electric discharge method (Corona) is capable of producing ozone at industrial scales exceeding 2 kg/h, as other methods like electrolysis, high-stress discharge, photolysis, and constant radiation result in rapid conversion of ozone back to oxygen (Baig et al., 2010).
Corona electric discharge occurs when electrical energy flows through a narrow gap filled with air or oxygen, leading to the breakdown of oxygen molecules and the formation of oxygen radicals These radicals then combine with oxygen molecules to create ozone To maintain efficiency, it is essential to remove the residual heat generated during this process using a cooling system (Kreuzinger et al., 2011).
Figure 3.4 Basic principle of an ozone generator
(Adopted from https://www.lenntech.com.pt/library/ozone/generation/ozone-generation.htm)
The concentration of ozone produced is influenced by the type of feed gas used; oxygen-fed ozone systems typically generate ozone concentrations ranging from 6-16%, with an average of 8-12%, while air-fed ozone systems produce lower concentrations, ranging from 1-4% (Rakness, 2011).
3.3.3 Advantages and disadvantages of ozone
Ozone offers several advantages, including its rapid production from air or oxygen through electrical discharge and its ability to react effectively with both organic and inorganic compounds It has a diverse range of applications, such as disinfection, reducing chemical oxygen demand, and improving the color, odor, and turbidity of treated water Additionally, any excess ozone in water decomposes into oxygen without leaving harmful residues As a result, ozone is widely utilized as a chemical reagent in various processes, including the oxidation of biological pollutants and the removal of undesirable tastes, odors, and colors from water, as well as reducing turbidity in water and wastewater treatment.
EU, ozone is used for disinfection and odor absorption in drinking water since 1906 in France, and then in other countries in the region (Ikehata et al., 2018; von Gunten, 2018;
Ozone has notable drawbacks, including challenges in sustaining residual levels after sterilization, which complicates the prevention of microorganism regrowth To ensure water quality, the use of secondary disinfectants, such as chlorine, is often required (Demir et al., 2016) Additionally, ozone can lead to the formation of harmful oxidation byproducts like bromate and aldehydes, and there are difficulties in effectively transferring ozone mass to wastewater.
Recent advancements in ozone technology have expanded its applications in conventional water treatment methods This evolution has revealed the presence of more resilient microorganisms, including Giardia and Cryptosporidium cysts, prompting the implementation of government regulations aimed at safeguarding public health from the risks associated with these pathogens.
3.3.4 Application of ozonation process in wastewater treatment
Ozonation has been extensively evaluated as an advanced wastewater treatment method across laboratory, pilot, and full-scale studies, demonstrating its effectiveness in reducing micropollutants in municipal wastewater Recognized as one of the best available technologies in Switzerland, ozonation helps meet stringent water resource protection requirements by effectively removing an average of 12 indicator substances.
Although German legislation does not mandate the construction of advanced treatment units, many wastewater treatment plants (WWTPs) have been enhanced with ozonation to mitigate micropollutant emissions into water bodies In Austria, pilot plants utilizing ozonation and granular activated carbon (GAC) have been implemented to monitor their application and performance.
Ozonation is increasingly utilized in full-scale treatment plants across France and Sweden, as noted by recent studies (Estman et al., 2019; Penru et al., 2018) Additionally, there has been a notable rise in academic publications focusing on "ozonation and wastewater" over the years, highlighting its growing importance in wastewater treatment.
Figure 3.5 Number of entries searching “ozonation and wastewater” in Science Direct (only Research Articles)
Ozonation involves two key mechanisms for degrading micropollutants: direct reactions with molecular ozone and indirect reactions facilitated by hydroxyl radicals (OH•) produced when ozone interacts with electron-rich organic compounds like phenols and secondary amines Ozone selectively targets compounds with electron-rich elements, including olefins, deprotonated amines, and activated aromatics, demonstrating a wide range of reaction rate constants (kO3) that span from 1 to 10^7 M^-1.
1s -1 (von Sonntag et al., 2012) With relatively low selectivity, OH • is capable of oxidizing many micropollutants species with extremely high reaction rate constants (k
The study highlights that hydroxyl radical (OH•) values range from 10^8 to 10^9 M^-1 s^-1, indicating significant differences that enhance the indirect reaction mechanism for removing ozone-refractory contaminants (Gligorovski et al., 2015; Lee et al., 2013) While dissolved organics and micropollutants are not fully mineralized, they are transformed into smaller, more biodegradable, and less toxic substances (Hỹbner et al., 2015; Vửlker et al., 2019) Effluent organic matter is crucial for ozonation due to its reactive functional groups, which limit the oxidants available for treating trace organic contaminants (TrOCs) (Chys et al., 2017; Rizzo et al., 2019) In the ozonation process of biologically treated municipal wastewater, the specific ozone dose normalized to dissolved organic carbon (g O3/g DOC) is often used to compare effluents with varying DOC levels However, it remains uncertain if the same ozone and OH• exposure is achieved across different substrates at the same g O3/g DOC Assuming similar effluent organic matter characteristics across cities, it can be posited that identical g O3/g DOC levels yield comparable ozone and OH• exposure (Lee et al., 2013) Additionally, nitrite compensation of the specific ozone dose is essential for accurate removal efficiency comparisons, as nitrite rapidly reacts with ozone, consuming 3.43 g O3/g NO2-N (Lee et al., 2013).
2013) To remove micropollutants from WWTPs wastewater, typical ozone dosage ranges from 0.25 to 1.5 g O3/g DOC (Baresel et al., 2016; Lee et al., 2016; Rizzo et al.,
Materials and Methods
Experiment overview
Lab-scale experiment 1 aims to evaluate the elimination of micropollutants and the formation of bromate byproducts during ozonation, utilizing effluent samples from a wastewater treatment plant in Austria Following guidelines established by Swiss experts (Zappatini et al., 2015), the study focuses on assessing the degradation efficiency of micropollutants at varying ozone doses while monitoring bromate (BrO3 -) formation The results are then compared with existing literature to assess the applicability of the findings.
Lab-scale experiment 2 investigated the effects of ozonation on the biodegradability of recalcitrant chemical oxygen demand (COD) in urban wastewater Effluent samples were collected from four Austrian municipal wastewater treatment plants that employed full nitrification and denitrification processes The study utilized three specific ozone doses—low, average, and high—similar to lab-scale experiment 1 The primary objective was to assess the relationship between the ozonation process, the specific ozone dose applied, and the resulting changes in biodegradability, as well as their impact on total organic parameters.
Pilot-scale experiments were carried out at a full-scale wastewater treatment plant (WWTP) utilizing an advanced treatment system that includes ozonation and granular activated carbon The primary objective of this study was to conduct long-term monitoring of wastewater toxicity after it has undergone treatment, using an in vitro biological assay kit designed to assess toxicological endpoints in a "real-life" setting.
30 conditions Routine sampling for chemical contaminants of emerging concern (CEC) analysis and effect-based method testing (EBM) was efficient control and monitoring.
Laboratory experiments
4.2.1 Production of ozone stock solution
The ozone stock solution was produced based on the guideline of Zappatini et al (2015) The experimental setup is illustrated in Figure 4.1
Figure 4.1 The structure of the ozone system
Ozone is inherently unstable and cannot be stored like oxygen; thus, it must be continuously generated using an ozone generator in conjunction with an oxygen tank In this process, oxygen is supplied to the Fischer technology model OZ200/5 ozone generator, operating at 35 W with a flow rate of 10 L/h The generated ozone (O3) is then directed into a 2-liter glass reactor filled with deionized water, which has been refrigerated overnight Gaseous ozone is introduced into the water as fine bubbles through an aeration stone, creating a concentrated O3 stock solution The concentration of this solution can fluctuate significantly with temperature, so it is cooled in an ice bath, following the method outlined by Zappatini et al (2015) The ozone concentration is subsequently measured using the indigo method and photometry, with specific absorbance values referenced from Bader et al (1981) and von Sonntag et al.
To ensure the stability of the stored ozone stock solution, ice is utilized during storage The concentration of ozone in the solution fluctuated between 40 and 55 mg O3/L, depending on the specific experiments conducted It is important to note that not all gaseous ozone dissolves in water, leading to some ozone escaping from the reactor.
A potassium iodide solution bottle is utilized to eliminate residual ozone, while an ozone alarm device offers audible and visual alerts for concentrations as low as 0.1 ppm in the air Given that ozone is a toxic gas with irritating effects, it is crucial to handle it discreetly and prioritize safety measures Figure 4.2 illustrates the ozone system implemented in the laboratory.
Figure 4.2 The ozone system in the laboratory
1 ozone generator, 2 spectrometers, 3 pump, 4 reactor for the O3 stock solution,
5 ice bath, 6 potassium iodide solution, 7 ozone alarm device
4.2.2 Experimental setup for micropollutant abatement
The batch test was conducted to assess the degradation of micropollutants in wastewater by mixing it with an ozone (O3) stock solution in 50 and 100 mL Schott bottles The mixing ratio was determined based on the targeted Dspec, the ozone concentration in the stock solution, and the levels of dissolved organic carbon (DOC) and nitrite in the wastewater The number of Schott bottles utilized varied according to the specific ozone doses being tested For instance, to evaluate specific ozone doses of 0.2, 0.4, 0.6, 0.8, and 1.0 g O3/g DOC, a total of six bottles were employed, with five containing the wastewater and O3 mixture tailored to the specific dose, while the sixth served as a reference containing only the wastewater.
32 sample) All experiments were carried out in duplicates The experimental setup is shown in Figure 4.3 and Table 4.1
Figure 4.3 The experimental set up, including analyzed parameters
Table 4.1 Schematic ratio of wastewater and ozone stock solution in the ozonation batch tests
Applied volumes / sample D Spec (g O 3 /g DOC)
Volume of ozone stock solution
V_WW Volume of the investigated wastewater sample (V_WW) V_WW
4.2.3 Experimental setup for the biodegradability study
The experiments in this study were similar to Chapter 4.2.2 and with three targeted Dspec
In the study, wastewater was treated with varying concentrations of ozone (0.4, 0.6, and 0.8 g O3/g DOC) by mixing it with an ozone stock solution in 0.5 L-Schott bottles The mixing ratio was determined based on the desired specific dose (Dspec), the ozone concentration, and the dissolved organic carbon (DOC) and nitrite levels in the wastewater To maintain consistency across experiments, the total volume of wastewater, ozone stock solution, and deionized water was kept constant, typically comprising 450 mL of wastewater and 50 mL of the ozone stock solution and deionized water All experiments were conducted in triplicate, and after a reaction time of about one hour, samples were aerated for 15 minutes using a fine ceramic aerator to eliminate any residual ozone.
Figure 4.4 The experiment setup, including analyzed parameters
Carbamazepine (CBZ) and benzotriazole (BZT) were evaluated as process control parameters to assess the effectiveness of ozonation experiments By comparing the observed abatement rates with those documented in literature and previous experiments, both substances have been identified as recommended indicators for ozonation, as noted by Jekel et al (2015) CBZ serves as an indicator for highly reactive compounds, while BZT represents moderately reactive compounds, with second-order rate constants of kO3 = 3 x 10^5 M^-1 s^-1 for CBZ and kO3 = 230 M^-1 s^-1 for BZT (Huber et al., 2003) Due to CBZ's high reactivity, an abatement rate of 80-90% is anticipated.
34 expected for Dspec above 0.4 g O3/g DOC A lower abatement can be considered an indication for methodological or experimental shortcomings
Table 4.2 Schematic ratio of deionized water, wastewater and ozone stock solution in the ozonation batch tests
Volume of deionized water (V_DW)
Volume of ozone stock solution (V_O 3 ) V_O 3
Volume of the investigated wastewater sample
Table 4.3 Nitrite compensated specific ozone doses (D spec ) and applied volumes in the ozonation experiments
Pilot plant experimental setup
The pilot plant's flow scheme, illustrated in Figure 4.5, employs a multibarrier approach that integrates ozonation and granular activated carbon filtration, featuring key sampling points for the study Three ozone reactors (O3-R), operating in series with a total volume of 12 m³, exhibited hydraulic retention times ranging from 9 to 40 minutes, influenced by wastewater inflow dynamics The activated carbon filter, containing 1.8 m³ of Epibon A granular activated carbon (GAC) from Donau Carbon, treated a side stream of 8 m³/h, achieving a hydraulic retention time of 13.5 minutes The automated process control system aimed for a specific nitrite-compensated ozone dose of 0.55 g O3/g DOC, with routine operations showing a range of 0.4 to 0.7 g O3/g DOC, and research campaigns varying from 0.2 to 0.9 g O3/g DOC The sampled bed volumes of the granular activated carbon filter increased from approximately 1,000 at the start of monitoring to 33,100 at the final sampling campaign, indicating biological activation of the filter after around 2,000 bed volumes.
Figure 4.5 Flow scheme of the advanced treatment demonstrator plant with the sampling points (O 3 -R…ozone reactor, N…feed tank for GAC-filter,
Analyzed parameters
4.4.1 Sampling and investigating wastewater characteristics
In a lab-scale experiment conducted in Austria, effluents from a wastewater treatment plant (WWTP) were collected using a 20-liter polyethylene tank The samples were promptly refrigerated and then allowed to acclimate at room temperature (23 ± 2 °C) for a minimum of three hours prior to experimentation The average parameters of the wastewater are detailed in Table 4.4.
Table 4.4 Average wastewater parameters of the effluent samples
Parameters Unit Average values ± standard deviation
In a lab-scale experiment, effluent samples from four Austrian municipal wastewater treatment plants (WWTPs) were analyzed, focusing on those operating under full nitrification and denitrification conditions Samples were gathered in a 20-liter polyethylene tank and filtered using 0.45 µm glass fiber filters to ensure measurement reproducibility at low concentrations The study aimed to assess the impact of ozone on the biodegradability of the water matrix, and thus, representative daily composite samples were not collected for every experiment, instead utilizing 24-hour volume proportional composite or grab samples Relevant parameter values are detailed in Table 4.5.
Table 4.5 Average wastewater parameters of the four investigated WWTPs
WWTP1a Grab 15.22 ± 0.00 0.65 ± 0.07 5.29 ± 0.06 0.02 ± 0.00 WWTP1b Composite 18.32 ± 0.63 0.68 ± 0.12 6.45 ± 0.10 0.05 ± 0.00 WWTP2a Grab 15.56 ± 0.00 1.42 ± 0.38 5.85 ± 0.06 0.05 ± 0.00 WWTP2b Composite 14.81 ± 0.64 1.38 ± 0.14 5.81 ± 0.06 0.05 ± 0.00 WWTP2c Grab 15.56 ± 1.92 1.33 ± 0.10 5.22 ± 0.00 0.03 ± 0.00 WWTP3 Grab 17.24 ± 0.00 1.99 ± 0.14 6.28 ± 0.12 0.2 ± 0.00 WWTP4 Grab 18.18 ± 0.00 1.91 ± 0.04 6.82 ± 0.10 0.1 ± 0.00
In pilot-scale experiment 3, a monthly routine monitoring was performed between May
Between 2018 and May 2019, a total of 16 grab samples, each collected in 1.5 L aluminum bottles as per BioDetection Systems BV recommendations, were extracted over a 13-month period Not all bioassays were conducted on every sample, and a comprehensive summary of the dates and operational data is provided in Table 4.6.
Table 4.6 Summary of sampling campaigns frequency of sampling for each sampling point, sorted by specific ozone dose
*BV: Bed volumes are only given for routine campaigns
Nine targeted trace organic contaminants (TrOCs) were chosen for analysis based on current and proposed EU legislation, their metabolism and excretion in humans, known environmental presence, persistence in wastewater treatment, and toxicity to aquatic life This selection encompasses pharmaceuticals, corrosion inhibitors, and sweeteners.
Table 4.7 Overview of TrOCs analyzed
Substance Acronym Substance class CAS-Number
Diatrizoic acid dihydrate DTA Iodinated contrast medium 50978-11-5 Diclofenac DCF Analgesic/anti-inflammatory 15307-79-6 Ibuprofen IBP Analgesic/anti-inflammatory 31121-93-4
Metoprolol, a beta-blocker, is primarily prescribed for managing high blood pressure and heart disease Benzotriazole, commonly found in treated municipal wastewater at concentrations of several grams per liter, reacts moderately with ozone but is effectively removed through adsorption on activated carbon Additionally, sulfamethoxazole, an antibiotic belonging to the sulfonamide class, is utilized in the treatment of urinary tract infections.
Carbamazepine, primarily used for epilepsy treatment, is resistant to elimination in activated sludge processes Acesulfame K, a synthetic sweetener found in various foods, serves as an anthropogenic tracer due to its high concentrations in sewage treatment effluent Bezafibrate, a lipid-lowering medication, effectively manages high cholesterol levels While ibuprofen is efficiently degraded in wastewater treatment, diclofenac remains persistent Diatrizoic acid dihydrate is utilized in diagnostic imaging procedures, including urography and computed tomography, particularly for patients with barium allergies (Kreuzinger et al., 2020).
The in vitro bioassay test battery was developed to identify modes of action based on established toxic mechanisms that address key steps in the toxicity pathway, as recommended by Escher et al (2012, 2018) and Neale et al (2017b) While positive signal responses do not directly indicate higher-order effects, they highlight that every adverse outcome starts with a molecular initiating event This connection illustrates how biological responses at the cellular level can lead to higher-order effects at the organ, organism, and ultimately population levels, encapsulated in the concept of adverse outcome pathways, as outlined by Ankley et al (2010).
Figure 4.6 In vitro bioassay panel allocated to the Toxicity Pathway Classifications (according to Neale et al (2017a), modified)
The wastewater extracts were analyzed by BioDetection Systems BV in Amsterdam using nine CALUX® (Chemical Activated Luciferase eXpression) reporter gene bioassays This long-term monitoring identified five modes of action for WWTP effluent monitoring, as recommended in the 2019 NORMAN and Water Europe Position paper by the NEREUS COST Action ES 1403 Additionally, three bioassays were included to assess genotoxicity, cytotoxicity, and anti-estrogenicity, which are important for evaluating water quality (Escher et al., 2021) The bioassay principles are detailed in Alygizakis et al (2019).
Analytical methods
4.5.1 Determination of the ozone concentration using the indigo method
The measurement principle relies on the decolorization of potassium indigotrisulfonate (C16H7K3N2O11S3) by ozone in a stoichiometric reaction By analyzing the decrease in absorbance at a wavelength of 600 nm, the ozone concentration can be accurately calculated, as outlined in DIN 38408-3 (2011) This process utilizes a UV/VIS spectrometer, specifically the Dr Lange-Cadas model.
100) with a quartz cuvette (5 cm) was used to measure the spectrophotometer at 600 nm
The Dissolved Organic Carbon (DOC) was analyzed using the Shimadzu Total Organic Carbon Analyser TOC-L CPH through the direct NPOC (non-purgeable organic carbon) method This process involved acidifying the sample to eliminate Total Inorganic Carbon (TIC) and subsequently measuring the carbon dioxide produced via thermal-catalytic combustion, detected by a non-dispersive infrared (NDIR) cell The concentration of carbon dioxide measured directly correlates to the DOC level in the sample.
A Continuous Flow Analyzer (CFA) - SAN Plus System from Skalar company was used to analyze NO2 - The concentration was determined based photometric principles
COD was analyzed with small tube test (STT) (Hach-Lange DR 2800; Hach-Lange COD Test LCK 314)
BOD was measured after 5 days as BOD5 ATU was added as a nitrification inhibitor to ensure that the consumed oxygen measured as BOD5 was limited to respiration for
Oxygen levels during the oxidation of organic matter were measured using a luminescence-based sensor (SP-PSt3-NAU-D5-YOP, PreSens Precision Sensing GmbH), providing daily results The sensor was affixed to the inner surface of a BOD bottle, enabling signal measurement through electro-optical components without direct contact To validate this method, parallel measurements of residual oxygen after five days were conducted using an oxygen probe (WTW), as detailed in Table 4.8.
Figure 4.7 BOD luminescence-base measurement
Table 4.8 Comparison of BOD 5 determined with two different oxygen sensors
The spectral absorbance coefficient at 254 nm (UV254) was measured with a UV/VIS spectrometer (Dr Lange – Cadas 100)
All measurements were carried out according to the standardized methods listed in Table 4.9
Table 4.9 Overview of the analyzed conventional parameters and the applied methodology
Chemical oxygen demand COD ISO 15705
Biochemical oxygen demand BOD5 ISO 5815-1, EN1899-2
Dissolved organic carbon DOC EN 1484
Spectral absorption coefficient at 254 nm UV245
Nitrate/Nitrite compounds NO3-N / NO2-N ISO 13395
Bromide Br - HPLC MS/MS
Bromate BrO3 HPLC MS/MS
The wastewater samples were filtrated with VWR glass fiber filter diameter 45 mm and pore size 1àm Analytical standard in ethanol concentration of 1mg/mL in ethanol were prepared
In this study, the analysis of micropollutants and the measurement of bromide and bromate concentrations were conducted using an automated online solid-phase extraction (SPE) method combined with LC-MS/MS analysis This innovative approach integrates liquid chromatography (HPLC) with mass spectrometry (MS), allowing for the effective separation of sample components through HPLC, which are then directly characterized using MS For further details, refer to Appendix 1.
Automated online solid-phase extraction was conducted using 10 mL sample injections, followed by HPLC separation utilizing a gradient mode with a 0.1% acetic acid solution in deionized water (A) and a 0.1% acetic acid solution in acetonitrile (B) Detailed programs for the online SPE and HPLC separation are provided in Table 4.10.
Table 4.10 The gradient program for online SPE and HPLC separation
Flow Gradient Flow Gradient min mL/min % A % B mL/min % A % B
The study utilized an Agilent high-pressure liquid chromatograph (HPLC) featuring dual binary pumps, a degasser for eluent preparation, a CTC PAL autosampler with a Peltier-Cooler, and a Rheodyne 2-position, 6-port switching valve For mass spectrometry, the research employed an AB Sciex QTrap 3200, a hybrid triple quadrupole linear trap ion trap tandem mass spectrometer.
For automated online solid phase extraction (SPE), a Phenomenex Strata X On-Line extraction cartridge (20 x 2.0 mm; 25µm) was utilized The HPLC separation was performed using a Phenomenex Luna C-18 analytical column (150 x 3.0 mm; 5µm) along with Phenomenex C18-Security guard cartridges (40 x 3.0 mm) For quantitative analysis, MRM Analysis with electrospray ionization mode (MRM ESI) was conducted at 500°C, employing nitrogen as the collision gas.
Table 4.11 Parameter MRM Analysis with electro spray ionization mode
The confirmatory and identifying mass and all other parameters of the MS/MS can be found in Table 4.12
Table 4.12 Mass properties of all analyzed compounds by HPLC MS/MS
Compound Polarity Q1 mass Q3 mass Identifying mass m/z m/z m/z DP CE CXP
The signal to noise ratio (S/N) and lower limit of detection (LOD) are given in Table 4.13
Table 4.13 Analyzed micropollutants and analytical quality criteria
Signal to noise ratio (S/N) and LOD (ng/L) (Standard concentration: 10 ng/L)
All wastewater samples underwent filtration using a glass fiber filter with a pore size of 3 µm, with a maximum sample volume of 1,000 mL The samples were then concentrated through solid-phase extraction (SPE) utilizing Oasis HLB cartridges (500 mg, 6cc, Waters 186000115), following the BDS protocol with minor modifications for the final resuspension of the evaporated sample A detailed outline of the SPE process is illustrated in Figure 4.8.
Cartridges were preconditioned using 6 mL of acetonitrile and 6 mL of deionized water, drawn through under low vacuum to eliminate residual bonding agents Filtered samples were introduced to the cartridges at a controlled flow of a few drops per second, not exceeding 10 mL/min Following sample loading, cartridges were washed with 6 mL of 5% methanol in water and dried under vacuum for 30 minutes to remove excess moisture The analytes were then eluted into a 20 mL culture tube using a mixture of 10 mL methanol and 10 mL acetonitrile at approximately 5 mL/min Finally, the samples were evaporated to about 0.5 mL under a nitrogen stream at room temperature and transferred to a vial, rinsing with 0.5 mL of methanol.
47 and 0.5 mL acetonitrile The final volume of the 1.5 mL extracted sample was kept in the fridge at 7 °C prior to analysis
Figure 4.8 The steps in the SPE process
The CALUX® bioassays yield biological equivalent concentrations (BEQs) per liter, referenced against compounds listed in Table 4.14, allowing for effective quantification of the analyzed effects Each analysis has a specific limit of quantification (LOQ), and genotoxicity is assessed both with and without S9 for metabolic activation Variations in results between the two testing conditions indicate whether metabolization or detoxification of the ingredients has taken place (Escher et al.).
The study conducted in 2012 distinguishes between directly and indirectly acting genotoxic compounds While the hormone-mediated mechanisms of action, specifically the estrogen receptor (ER) and anti-androgen receptor (AR) CALUX®, were assessed in all samples, the remaining six endpoints were evaluated alternately, as indicated in Table 4.14.
When the BEQ fell below the LOQ, half of the LOQ was utilized to ensure that results below the LOQ were still included in the statistical analysis This method accounts for sample-specific LOQs, which may cause the BEQ from results under the LOQ to vary slightly and, in certain instances, suggest an amplified signal during treatment processes.
Table 4.14 Information on the CALUX ® in vitro bioassay panel and frequency of analysis
Bioassay Measured endpoint Reference compound EBT* Frequency of analysis Key reference CAS O 3 GAC
Cytotox Repression of constitutive transcriptional activation / cytotoxic activity Tributyltin acetate - 16 16 7 1
Er * Estrogen receptor -mediated signalling 17 -Estradiol 0.1 ng BEQ/L 16 16 7 2 anti-Er Repression of estrogen receptor -mediated signalling Tamoxifen - 2 2 2 3 anti-AR * Repression androgen receptor activation Flutamide 14 àg BEQ/L 16 16 7 4
The activation of the Nrf2 pathway plays a crucial role in the oxidative stress response, with curcumin exhibiting significant effects at a concentration of 10 µg BEQ/L Additionally, the p53-dependent pathway is activated in response to genotoxicity, both with and without metabolic activation from S9, as seen with cyclophosphamide and actinomycin treatments These findings highlight the importance of p53 in mediating cellular responses to genotoxic stress.
PAH * Aryl-hydrocarbon receptor activation / toxic PAH - xenobiotics metabolism Benzo[a]pyrene 6.2 ng BEQ/L 8 8 4 5
PXR * Activation of pregnane X receptor / xenobiotic metabolism and sensing Nicardipine 3 àg BEQ/L 3 3 3 6
* suggested in the joint NORMAN and Water Europe Position paper (2019);
1 van der Linden et al (2014), 2 Sonneveld et al (2004), 3 van der Burg et al (2010b), 4 van der Burg et al (2010a), 5 Pieterse et al (2013), 6 Escher et al
EBTs associated with MOAs were sourced from literature, including studies by Escher et al (2018) and van der Oost et al (2017) For the endpoints recommended in the joint NORMAN and Water Europe Position paper (2019), the lower EBTs proposed in these publications were utilized.