D4.3 – Recommendations on EAOP-CW based treatment system for water reuse applications
Abstract
The increasing presence of industrial persistent, mobile and toxic compounds (iPM(T)s) and per- and polyfluoroalkyl substances (PFAS), presents a critical barrier to advancing sustainable water management and the circular economy. To address these challenges, the PROMISCES project, part of the Horizon 2020 Green Deal initiative, aims to develop and demonstrate advanced water treatment technologies integrating electrochemical advanced oxidation processes (EAOP) with constructed wetlands (CW) focuses on reducing the concentration of potentially harmful substances in reclaimed water for agricultural irrigation.This study contributes to the European Union's goals outlined in the Green Deal, the Zero Pollution Action Plan, and the Circular Economy Action Plan by addressing the removal of persistent contaminants from wastewater to enable safe agricultural irrigation. This initiative is particularly significant for water scarce regions such as Catalonia, Spain. The Besòs case study focused on the Montornès del Vallès Wastewater Treatment Plant (WWTP), where approximately 60% of the incoming wastewater flow comes from industrial sources. The PROMISCES project complies with stringent European and Spanish regulations for wastewater treatment and reuse, addressing the demand for advanced systems to adhere to evolving directives, such as the new Urban Wastewater Treatment Directive 2024/3019 and Spanish Royal Decree 1085/2024, approving the EU Water Reuse Regulation (2020/741) in Spain.This hybrid system combines electrochemical oxidation and ozonation (e-Peroxone or EAOP), generating hydroxyl radicals for the efficient degradation of iPM(T)s and PFAS, while minimizing the energy consumption and reducing the toxic by-product formation. The system also includes a constructed wetland (CW) system, which mimic natural wetlands for pollutant filtration and nutrient absorption. Using plant species such as Phragmites australis and Iris pseudacorus, CWs serve as a post-treatment stage, enhancing effluent quality and providing additional environmental benefits, including promoting biodiversity.Based on the results, this technological integration tackles the water scarcity challenges commonly faced by Mediterranean regions, positioning reclaimed water as a sustainable and viable solution for agricultural irrigation. Ongoing research and investment in such innovations are essential for driving the advancement of circular water economies.
Full text
Project ID N°: 101036449 Call: H2020-LC-GD-2020-3 Topic: LC-GD-8-1-2020 - Innovative, systemic zero-pollution solutions to protect health, environment, and natural resources from persistent and mobile chemicals Preventing Recalcitrant Organic Mobile Industrial chemicalS for Circular Economy in the soil-sediment-water System Start date of the project: 1st November 2021 Duration: 42 months Main authors: Jessica Meijide Fernández, Jofre Herrero, Leónidas Pérez-Estrada, Carme Bosch (EURECAT), Dana Pierina Orlando, Rocío Inés Bonansea, Miren López de Alda, Marta Llorca, Marinella Farré, Alicia Cano, Víctor Matamoros (CSIC), Toni Mas, Begoña Martínez, Josep Pascual (CBT), Sandra Valero (CCB), Peter Behnisch, Harrie Besselink (BDS) Lead Beneficiary: EURECAT Type of delivery: Report Dissemination Level: PU Filename and version: PROMISCES_D4-3_Recommendation-EAOP-CW (version 1) Website: https://promisces.eu/ Due date: M40 (28 February 2025) D4.3 – Recommendations on EAOP-CW based treatment system for water reuse applications
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 2 © European Union, 2025 No third-party textual or artistic material included on the publication without the copyright holder’s prior consent to further dissemination by other third parties. Reproduction is authorized provided the source is acknowledged Disclaimer The information and views set out in this report are those of the author(s) and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 3 Document History This document has been through the following revisions: Authorisation Distribution This document has been distributed to: Version date Author/Reviewer Description V0.1 02/12/2024 Jessica Meijide, Jofre Herrero, Sandra Valero, Toni Mas, Josep Pascual, Marta Llorca, Dana Pierina Orlando, Rocío Inés Bonansea, Miren López de Alda, Alicia Cano, Peter Behnisch Initial version V0.2 17/01/2025 Jessica Meijide, Carme Bosch, Miren López de Alda, Begoña Martínez, Toni Mas, Sandra Valero, Victor Matamoros Initial check V0.3 10/02/2025 Jessica Meijide, Carme Bosch, Dana Pierina Orlando, Rocío Inés Bonansea, Miren López de Alda, Sandra Valero, Alicia Cano, Victor Matamoros Document updated after review V0.4 18/02/2025 Veronika Zhiteneva Validation V0.5 26/02/2025 Jessica Meijide, Dana Pierina Orlando, Alicia Cano, Victor Matamoros, Sandra Valero Final version after validation V0.6 26/02/2025 Floriane Sermondadaz Quality control V1.0 27/02/2022 Julie Lions Version for submission Authorisation Name Status Date Review M. Sgroi WP4 participant 22/01/2025 Validation U. Miehe WP4 Leader 21/02/2025 Quality Control F. Sermondadaz Administrative and financial manager 26/02/2025 Approval J. Lions Project coordinator 27/02/2025 Name Title Version issued Date of issue EURECAT, CSIC, CBT, BDS, KWB WP4.2 Partners & WP Leader Version 1 03/03/2025
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 4 Executive Summary The increasing presence of industrial persistent, mobile and toxic compounds (iPM(T)s) and perand polyfluoroalkyl substances (PFAS), presents a critical barrier to advancing sustainable water management and the circular economy. To address these challenges, the PROMISCES project, part of the Horizon 2020 Green Deal initiative, aims to develop and demonstrate advanced water treatment technologies integrating electrochemical advanced oxidation processes (EAOP) with constructed wetlands (CW) focuses on reducing the concentration of potentially harmful substances in reclaimed water for agricultural irrigation. This study contributes to the European Union's goals outlined in the Green Deal, the Zero Pollution Action Plan, and the Circular Economy Action Plan by addressing the removal of persistent contaminants from wastewater to enable safe agricultural irrigation. This initiative is particularly significant for water scarce regions such as Catalonia, Spain. The Besòs case study (CS#3) focused on the Montornès del Vallès Wastewater Treatment Plant (WWTP), where approximately 60% of the incoming wastewater flow comes from industrial sources. The PROMISCES project complies with stringent European and Spanish regulations for wastewater treatment and reuse, addressing the demand for advanced systems to adhere to evolving directives, such as the new Urban Wastewater Treatment Directive 2024/3019 and Spanish Royal Decree 1085/2024, approving the EU Water Reuse Regulation (2020/741) in Spain. This hybrid system combines electrochemical oxidation and ozonation (e-Peroxone or EAOP), generating hydroxyl radicals for the efficient degradation of iPM(T)s and PFAS, while minimizing the energy consumption and reducing the toxic by-product formation. The system also includes a constructed wetland (CW) system, which mimic natural wetlands for pollutant filtration and nutrient absorption. Using plant species such as Phragmites australis and Iris pseudacorus, CWs serve as a post-treatment stage, enhancing effluent quality and providing additional environmental benefits, including promoting biodiversity. The CS#3 followed a multi-phase approach, beginning with contaminant screening through nontarget and targeted analytical methods, to identify priority contaminants, such as industrial chemicals and pharmaceuticals, in the effluent streams of the WWTP. Next, lab scale testing was conducted to assess the efficiency of ozonation, electrooxidation, and e-Peroxone in removing contaminants from synthetic wastewater. Pilot-scale testing was then conducted at the Montornès del Vallès WWTP to assess the performance of the e-Peroxone process and a wetland system, validating their efficiency under real-world conditions. The final phase involved a crop irrigation study, analyzing the feasibility of using reclaimed water on the growth of Lactuca sativa (lettuce) and assessing the potential contaminant uptake. Key findings include the high performance of the EAOP, which achieved excellent removal efficiencies exceeding 90% for most target iPM(T)s, whereas PFAS removal was negligible, with e-Peroxone outperforming standalone ozonation or electrooxidation. Additionally, the CW system served as an effective post-treatment polishing stage, reducing nutrient levels and improving effluent quality. Reclaimed water also met stringent microbiological and chemical standards for agricultural use, with lettuce metabolomics indicating no significant health risks. Based on the CS#3 results, this technological integration tackles the water scarcity challenges commonly faced by Mediterranean regions, positioning reclaimed water as a sustainable and viable solution for agricultural irrigation. Ongoing research and investment in such innovations are essential for driving the advancement of circular water economies.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 5 Table of contents 1 Introduction ................................................................................................................................. 10 1.1 e-Peroxone treatment: An overview ................................................................................... 10 1.2 Nature based treatment: A constructed wetland ............................................................... 11 1.3 The case for combining EAOP and CW ................................................................................ 11 1.4 Objectives............................................................................................................................. 12 2 Site description ............................................................................................................................ 13 2.1 General overview of the WWTP Montornès del Vallès ....................................................... 13 2.2 Infrastructure and layout ..................................................................................................... 13 2.3 Infrastructure Overview ....................................................................................................... 14 2.4 Secondary effluent composition .......................................................................................... 14 2.5 Environmental and regulation context ................................................................................ 15 3 Pilot plant configuration .............................................................................................................. 16 3.1 EAOP prototype ................................................................................................................... 16 3.2 Wetland system ................................................................................................................... 18 4 Methodology ................................................................................................................................ 19 4.1 Non-target screening of secondary WWTP effluent and target contaminant selection (Subtask 4.2.1) ................................................................................................................................. 19 4.2 Testing of technologies at lab-scale (TRL 4/5) (Subtask 4.2.2) ............................................ 20 4.3 Pilot-scale technology testing (TRL 6) (Subtask 4.2.3) ......................................................... 20 4.4 Analytical methods for pilot monitoring ............................................................................. 21 4.4.1 Physicochemical parameters ....................................................................................... 21 4.4.2 Nutrients ...................................................................................................................... 21 4.4.3 iPM(T)s ......................................................................................................................... 21 4.4.4 PFAS ............................................................................................................................. 23 4.4.5 Inorganic ions ............................................................................................................... 24 4.4.6 Toxicological assays ..................................................................................................... 24 4.4.7 Microbiological analysis ............................................................................................... 25 4.5 Study of the uptake and distribution of contaminants in vegetable crops irrigated with reclaimed water (Subtask 4.2.4) ...................................................................................................... 26 4.6 Criteria for evaluating the use of reclaimed water in irrigation .......................................... 26 5 Results .......................................................................................................................................... 28 5.1 Non-target screening of secondary WWTP effluent and target contaminant selection .... 28 5.2 Testing of technologies at lab-scale (TRL 4/5) ..................................................................... 30 5.2.1 Synthetic secondary effluent tests .............................................................................. 30 5.2.2 Real secondary effluent trials ...................................................................................... 33
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 6 5.2.3 Scaling up: real secondary effluent at larger scale ...................................................... 37 5.3 Challenges and adaptations during pilot plant installation ................................................. 41 5.4 Pilot-scale technology operation (TRL 6) ............................................................................. 42 5.4.1 EAOP system ................................................................................................................ 42 5.4.2 CW system ................................................................................................................... 42 5.4.3 Monitoring ................................................................................................................... 45 5.5 Parameters and removal efficiency of the pilot-scale technology testing (TRL 6) .............. 45 5.5.1 Physicochemical parameters ....................................................................................... 45 5.5.2 Nutrients ...................................................................................................................... 46 5.5.3 iPM(T)s removal ........................................................................................................... 47 5.5.4 PFAS removal ............................................................................................................... 51 5.5.5 Formation and fate of inorganic ions .......................................................................... 52 5.5.6 Energy consumption .................................................................................................... 54 5.5.7 Toxicological analysis ................................................................................................... 54 5.5.8 Microbiological and physicochemical analyses analysis .............................................. 55 5.6 Control wetland system - potential for removal of PMTs and PFAS ................................... 55 5.6.1 Physicochemical parameters and nutrients ................................................................ 55 5.6.2 iPM(T)s removal ........................................................................................................... 57 5.6.3 PFAS removal ............................................................................................................... 59 5.7 Compliance with Directive (EU) 2024/3019 on urban wastewater treatment ................... 60 5.8 Challenges and adaptations during pilot plant operation ................................................... 64 6 Use of reclaimed water in vegetable crop irrigation ................................................................... 65 6.1. Characteristics of the water used for irrigation ................................................................... 65 6.2. Uptake and distribution of contaminants in vegetable crops ............................................. 66 6.2.1 iPM(T)s ......................................................................................................................... 66 6.2.2 PFAS ............................................................................................................................. 69 6.3. Impact on crop yield and quality ......................................................................................... 70 6.3.1 Crop productivity and quality ...................................................................................... 70 6.3.2 Crop metabolomics ...................................................................................................... 71 6.4. Environmental and health implications ............................................................................... 72 6.4.1 iPM(T)s ......................................................................................................................... 72 6.4.2 PFAS ............................................................................................................................. 75 7 Conclusions and recommendations ............................................................................................. 76 7.1 Key findings on process operation ....................................................................................... 76 7.2 Recommendations for optimizing the treatment and crop studies .................................... 76 7.3 Future considerations for system scalability ....................................................................... 77 8 References ................................................................................................................................... 79
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 7 List of figures Figure 1. Circular Economy Route for Case Study 3 (Source: KWB) .................................................................. 10 Figure 2. Technological treatment train for water reuse for agricultural irrigation (Source: KWB) ................. 12 Figure 3. Location of Montornès del Vallès WWTP........................................................................................... 13 Figure 4. Schematic representation of the EAOP prototype ............................................................................ 16 Figure 5. EAOP prototype (APRIA Systems, Photobench LED275-8a) ............................................................... 16 Figure 6. P&ID diagram of the EAOP prototype (APRIA Systems, Photobench LED275-8a) ............................. 17 Figure 7. CW system (Montornès del Vallès WWTP) ........................................................................................ 18 Figure 8. Experimental set-up design for the CWs ............................................................................................ 19 Figure 9. Experimental setup design for testing of technologies at lab-scale .................................................. 20 Figure 10. Experimental setup depicting the individual containers for cultivating lettuce ............................. 26 Figure 11. Sum of peak areas for each sample preparation (SP) method evaluated through suspect screening . ........................................................................................................................................................................... 28 Figure 12. iPM(T)s removal by ozonation, electro-oxidation and e-Peroxone using synthetic secondary effluent ........................................................................................................................................................................... 32 Figure 13. iPM(T)s removed by electro-oxidation and e-Peroxone using real secondary effluent .................. 34 Figure 14. PFAS removal from real secondary effluent using electro-oxidation and e-Peroxone .................... 35 Figure 15. PFAS removal in real secondary effluent using electro-oxidation and e-Peroxone ........................ 36 Figure 16. iPM(T)s removal using electro-oxidation and e-Peroxone on real secondary WWTP effluent at larger scale ................................................................................................................................................................... 38 Figure 17. PFAS removal using electro-oxidation and e-Peroxone on real secondary WWTP effluent at larger scale ................................................................................................................................................................... 39 Figure 18. Weekly precipitation (blue bar) and evapotranspiration (orange bar) values (mm) and average weekly temperature (red line) (ºC) calculated using data from Parets del Vallès Meteorological Station. ..... 43 Figure 19. Average residence time (green line), irrigation time (red line) and weekly treated water volume for the post EAOP (blue bar) and control (orange bar) CW systems ...................................................................... 44 Figure 20. Nutrient removal of the post EAOP CW at Montornès del Vallès WWTP ........................................ 47 Figure 21. Pharmaceutical removal using e-Peroxone at Montornès del Vallès WWTP .................................. 48 Figure 22. Pharmaceuticals removal in the post EAOP CW system at Montornès del Vallès WWTP ............... 48 Figure 23. Pesticides/Drugs of abuse/Others removal using e-Peroxone at Montornès del Vallès WWTP .... 49 Figure 24. Pesticides/Drugs of abuse/Others removal by post EAOP CW system at Montornès del Vallés WWTP ........................................................................................................................................................................... 49 Figure 25. Industrial chemicals removal using e-Peroxone at Montornès del Vallès WWTP ........................... 50 Figure 26. Industrial chemical removal in the post EAOPs CW system at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) ...................................................................... 50 Figure 27. Nutrient removal of control CW at Montornès del Vallès WWTP .................................................. 57 Figure 28. Pharmaceutical removals by control CW system at Montornès del Vallès WWTP ......................... 57 Figure 29. Pesticides/Drugs of abuse/Others removals by control CW system at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) ........................................................... 58 Figure 30. Industrial chemical removals by control CW system at Montornès del Vallés WWTP .................... 58 Figure 31. Agronomical parameters of lettuce crops irrigated with different water types (n=10) .................. 70 Figure 32. Score plot with samples normalized using the triphenylamine feature and data scaled via Pareto scaling. ............................................................................................................................................................... 72
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 8 List of tables Table 1. Design values for the Montornès del Vallès WWTP ............................................................................ 14 Table 2. Average composition of the secondary effluent during pilot operation (April 1 to July 31, 2024) .... 15 Table 3. List of target analytes included in the LC-MS/MS-based targeted method developed ...................... 22 Table 4. PFAS quantified by IDAEA-CSIC ........................................................................................................... 23 Table 5. Maximum allowable value established for Quality A.A. standards ..................................................... 25 Table 6. Soil-grown crops monitoring parameters ........................................................................................... 27 Table 7. Results obtained from analysis of target compounds in 85 water samples collected during the pilot and field-scale experiments ............................................................................................................................ 29 Table 8. Target pollutants and their theoretical concentrations in the spiked solution .................................. 30 Table 9. EE/O values for each pollutant involved in the treatment processes using spiked synthetic effluent water ............................................................................................................................................................... 33 Table 10. Formation of chlorinated by-products using electro-oxidation and e-Peroxone on real secondary effluent ............................................................................................................................................................ 36 Table 11. EE/O values for each pollutant involved in the treatment processes for real secondary effluent ... 36 Table 12. Formation of chlorinated by-products using electro-oxidation and e-Peroxone on real secondary WWTP effluent at a larger scale ..................................................................................................................... 39 Table 13. Formation of brominated by-products using electro-oxidation and e-Peroxone on real secondary WWTP effluent at a larger scale ..................................................................................................................... 40 Table 14. EE/O values for each pollutant involved in the treatment processes for real secondary WWTP effluent ........................................................................................................................................................................ 41 Table 15. Management of CW system operations and regulation of water inflows and outflows. ................. 44 Table 16. Average monthly sulfonic PFAS levels before and after e-Peroxone ............................................... 51 Table 17. Average monthly carboxylic PFAS levels before and after e-Peroxone ........................................... 51 Table 18. Average monthly sulfonic PFAS levels before/after post e-AOP CW ............................................... 51 Table 19. Average monthly carboxylic PFAS levels before/after post e-AOP CW ............................................ 52 Table 20. Formation of chlorinated by-products by e-Peroxone at Montornès del Vallès WWTP .................. 52 Table 21. Removal of chlorinated by-products by constructed wetland ......................................................... 53 Table 22. CALUX analysis results of WAX-SPE water sample extracts .............................................................. 54 Table 23. Comparison of physicochemical parameters between CW systems (control vs post-EAOP) ........... 56 Table 24. Average monthly sulfonic PFAS levels before (initial) and after (final) the control CW at Montornès del Vallés ......................................................................................................................................................... 59 Table 25. Average monthly carboxylic PFAS levels (initial) and after (final) the control CW at Montornès del Vallés ............................................................................................................................................................... 59 Table 26. Summary of iPMTs data: EAOP inlet concentration and removal efficiencies .................................. 61 Table 27. Summary of PFAS data: EAOP inlet concentration and removal efficiencies ................................... 63 Table 28. General water quality parameters for each type of irrigation water (n=5) ...................................... 66 Table 29. Concentration of iPM(T)s in irrigation waters (n=3) and soil used for crop studies ......................... 67 Table 30. Overview of the iPM(T)s compounds in lettuce samples irrigated with bottled water (BW), secondary effluent wastewater (WW), and reclaimed water (RW) ................................................................................. 68 Table 31. Overview of the PFAS compounds in lettuce samples irrigated with bottled water (BW), secondary effluent wastewater (WW), and reclaimed water (RW) (n=10) ..................................................................... 69 Table 32. Risk quotient (RQs) values for iPMT compounds detected in untreated wastewater (uWW), secondary effluent wastewater (WW) and reclaimed water (RW) ................................................................ 73 Table 33. Threshold of toxicological concern (TTC) analysis results for detected compounds ........................ 75 Table 34. Weekly intake for PFAS (sum of PFOA, PFOS and PFHxS) ................................................................. 75
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 9 List of abbreviations BOD5: Biochemical Oxygen Demand (5 days) COD: Chemical Oxygen Demand CWs: Constructed Wetlands DO: Dissolved Oxygen DOC: Dissolved Oxygen Concentration DPG: N,N'-diphenylguanidine EAOP: Electrochemical Advanced Oxidation Processes EE/O: Electrical Energy per Order HSSF CWs: Horizontal Subsurface Flow Constructed Wetlands iPM(T): Industrial Persistent, Mobile, and Potentially Toxic Compounds PFAS: Perand Polyfluoroalkyl Substances SHE: Standard Hydrogen Electrode SP: Sample Preparation SS: Suspended Solids TN: Total Nitrogen TP: Total Phosphorus TRL: Technology Readiness Level WWTP: Wastewater Treatment Plant
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 16 3 Pilot plant configuration 3.1 EAOP prototype The electrochemical advanced oxidation processes (EAOP) prototype (APRIA Systems, Photobench LED275-8a) (Figure 4, Figure 5 and Figure 6) has been designed to integrate three advanced oxidation technologies, each engineered to operate independently or in combination with the others: (i) an ozone generation system equipped with an ozoniser and a Venturi tube; (ii) an annular UV-C LED photoreactor (not used in PROMISCES project); and (iii) an electrochemical cell equipped with BBD (boron-doped diamond) electrodes. This flexible configuration enables for tailored operation depending on the treatment requirements optimizing performance and efficiency in the pollutant degradation. Figure 4. Schematic representation of the EAOP prototype (APRIA Systems, Photobench LED275-8a) Figure 5. EAOP prototype (APRIA Systems, Photobench LED275-8a)
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 17 Ozonation unit The treatment process initiates by channelling the water into a Venturi tube. As the water flows through this tube, ozone is introduced into the stream. This ozone is generated by the ozoniser at a maximum rate of approximately 7 g/h, using atmospheric air as the feed gas. Subsequently, the ozonated water enters a contact reactor, specifically engineered to promote optical mixing and maximize the interaction between the ozone and water. Within this chamber, contact time is carefully extended, facilitating complete ozone dissolution and enhancing the overall effectiveness of the treatment process. Any remaining ozone in the gas phase is efficiently removed using a thermocatalytic ozone destructor, preventing any excess ozone from being released to the environment. Upon completion of the treatment process, the ozonated water can be either recirculated back to the feed tank for further processing or directed to the following treatment unit for additional treatment steps. Photochemical unit In the photochemical treatment, the water undergoes processing within a glass annular photoreactor that consists of two concentric tubes. Water flows through the outer tube while a powerful LED lamp is housed within the inner tube. This light source is composed of 80 UV-C LEDs (=275 nm) distributed across four strips, providing adjustable irradiated power. To achieve the desired treatment dose, either the flow rate of the water or the irradiated power of the lamp must be modified accordingly. Additionally, the photoreactor features an air-cooling system and a temperature probe, ensuring effective temperature monitoring and control throughout the process. Figure 6. P&ID diagram of the EAOP prototype (APRIA Systems, Photobench LED275-8a) Electrooxidation unit The electrochemical cell featured a boron-doped diamond (BDD) anode and a stainless steel (SS) cathode, designed with optimized active surface areas. Each BDD electrode, fabricated with a niobium substrate and double-sided coating, and each SS electrode measured 5 × 15 cm, providing a total surface area of 150 cm²/electrode. The electrodes were assembled in an alternating stack with
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 18 a 3 mm gap between them, forming a set of 5 BDD and 6 SS electrodes for an active area of 1500 cm² (excluding the outermost SS electrodes' external surfaces). Additionally, the system is outfitted with a safety pressure switch which guarantees operations stay within safe limits, with a maximum operating pressure established at 4.5 bar. Temperature, pH, redox potential, dissolved ozone and conductivity values of the treated water are monitored via the PLC screen located on the front of the electrical and control enclosure. Furthermore, the installation features three visual flowmeters and three pressure gauges, each linked to the respective unit, providing real-time flow and pressure readings. 3.2 Wetland system Two CW channels were designed to maximize their natural filtration capabilities, each measuring 74 cm in width, 47 cm in depth, and 3 meters in length, and employing a horizontal subsurface flow configuration (Figure 7). These artificial ecosystems incorporate a layered gravel system (1.5 cm diameter), enhancing water flow and filtration, along with the introduction of two macrophyte species: Iris pseudacorus and Phragmites australis. Figure 7. CW system (Montornès del Vallès WWTP) One of the flumes receives water directly from the outlet of the Montornès del Vallès WWTP, utilizing secondary effluent without prior filtration and the second channel is supplied with water from the EAOP prototype outlet buffer tank, which undergoes both filtration and e-Peroxone process (Figure 8). Both constructed wetlands are fed from the lower section of the flume and function as subsurface horizontal flow systems, with water flowing through a granular medium at a depth influenced by the size and spread of the plant roots. Both CWs operated under the same hydraulic conditions, defined by the volume of water treated by the EAOP. The water produced by the EAOP was stored in multiple buffer tanks to maintain a steady flow. The system operated on a daily irrigation schedule, running for 7 hours from Monday to Friday during working hours, with a flow rate of 0.35 L/min. From Friday to Sunday, the flow rate was reduced to 0.20 L/min for a duration of 66 hours. The average inlet flow rate over the entire period was 0.24 L/min, calculated by dividing the total liters consumed by the total watering hours.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 19 The CWs have an effective porosity of 25% (determined through granulometric analysis, according to Sanders (1998)). The CWs have a maximum capacity of 250 liters and operate under normal conditions with the water filled to one-third of their total depth, which allows them to hold an estimated volume of 80-90 liters. The average hydraulic residence time, calculated based on the typical inlet flow rate and total volume treated, ranges from 5 to 6 hours. However, on sample days, the hydraulic residence time increases to approximately 6 to 7 hours, as the system operates in batch mode to optimize water contact with the rhizomes, processing an average daily volume of 210 liters. This estimation may vary due to multiples factors such as changes in the inflow rates, irrigation durations, evapotranspiration, rainfall, and sampling frequency, all of which influence fluctuations in outflow during the pilot operation. Figure 8. Experimental set-up design for the CWs 4 Methodology 4.1 Non-target screening of secondary WWTP effluent and target contaminant selection (Subtask 4.2.1) A non-target screening approach of secondary WWTP effluent was conducted using three different sample preparation methods to achieve a comprehensive identification of iPM(T), as detailed in Deliverable D1.4. The identified compounds were then prioritized based on their persistence, bioaccumulation potential, mobility, and toxicity. Subsequently, the 25 most relevant compounds from this study were selected to develop a target method using liquid chromatography-tandem mass spectrometry with a hybrid quadrupole time-offlight analyzer (SPE-LC-QToF-MS/MS) in positive ionization mode. This selection was expanded to approximately 40 compounds by including additional compounds of interest identified in previous studies at a wastewater regeneration station.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 20 4.2 Testing of technologies at lab-scale (TRL 4/5) (Subtask 4.2.2) The technology underwent rigorous testing at the lab-scale, utilizing the batch experimental setup illustrated in Figure 9. The ozone generated from compressed oxygen is directed into a specially designed 5L contact tank with an integrated cooling system. This setup optimizes the interaction between ozone and wastewater, keeping temperatures within an ideal range to maximize the efficiency of the oxidation process. The cooling system ensures stable conditions, preventing thermal degradation and sustaining the optimal environment for effective treatment. Figure 9. Experimental setup design for testing of technologies at lab-scale In addition, the system integrates an electrochemical cell that complements the ozone treatment by generating reactive species, enhancing the degradation of these contaminants in the wastewater. This combination establishes a robust, multi-faceted treatment process, enabling a thorough assessment of the technology's performance within a controlled laboratory environment. The system’s precise design allows for meticulous monitoring throughout the 240-min treatment period and fine-tuning of operational parameters, yielding valuable insights into the efficiency and overall effectiveness of the treatment approach under evaluation. 4.3 Pilot-scale technology testing (TRL 6) (Subtask 4.2.3) The pilot scale testing was conducted using the pilot plant configuration, as detailed in chapter 3. Pilot plant configuration. This set-up was specifically designed to evaluate the efficiency and effectiveness of the treatment processes under realistic operational conditions. The operational conditions establish a thorough framework for assessing the technology, including factors such as flow rates, temperature, and specific characteristics of the wastewater. Throughout the pilot plant operation, the samples were collected from several key points including the outlet of the secondary treatment (which serves as the input to the EAOP prototype), the outlet of the EAOP prototype, the buffer tank supplying treated water from the EAOP prototype to the post EAOP wetland, the buffer tank feeding the control wetland with water from the secondary treatment outlet, and the wetland outlet.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 21 After collecting the samples, sodium thiosulfate (1M) was added to quench the oxidation reaction, using a ratio of 1 mL of sodium thiosulfate per 50 mL of sample. The samples were then refrigerated for 24 hours before being sent to the CSIC facilities. There, they were prepared and conditioned appropriately for subsequent injection. 4.4 Analytical methods for pilot monitoring 4.4.1 Physicochemical parameters The physicochemical parameters were continuously monitored via the front panel of the electrical cabinet of the EAOP prototype. This interface displayed real-time readings of pH, conductivity, operating temperature and redox potential, measured through dedicated probes. The system automatically recorded the values of all variables over time, ensuring precise tracking of operational conditions. As part of quality control, pH, conductivity, dissolved oxygen, and temperature were measured during each periodic sampling using the Hanna Instruments HI98494 multiparameter device. These manual measurements complemented the automated monitoring system, ensuring the accuracy and reliability of the data collected. In addition, chemical oxygen demand (COD) was determined using Hach LCK314 cuvette test kits (range 15–150 mg/l) with a TR420 Thermoreactor and DR2800 spectrophotometer (Hach Lange, UK). 4.4.2 Nutrients Ammonium was measured using Hach LCK304 (0.015–2 mg/L) and LCK305 (1–12 mg/L) cuvette test kits, with analysis performed on a DR2800 spectrophotometer (Hach Lange, UK). Nitrate levels were quantified using the Hach LCK339 kit (0.23–13.5 mg/L) on the same spectrophotometer. For total nitrogen and total phosphorus, Hach LCK138 (1–16 mg/L) and LCK349 (0.05–1.5 mg/L) cuvette test kits were used, analyzed with TR420 Thermoreactor and DR2800 spectrophotometer (Hach Lange, UK). 4.4.3 iPM(T)s Water sample analysis was conducted using an Elute UHPLC system paired with an Impact II Q-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Detailed chromatographic and mass spectrometric conditions can be found in Deliverable D1.4, sections 2.4.4 and 2.4.5. The method demonstrated robust validation at two concentration levels (0.2 and 2 μg/L), delivering reliable results in terms of sensitivity (LOD ranging from 0.04 to 166 ng/L), reproducibility (RSD <15% for most compounds), and recoveries (absolute values generally between 60% and 140%). Table 3 provides an overview of the compounds included in the target method.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 22 Table 3. List of target analytes included in the LC-MS/MS-based targeted method developed by CSIC for waters Nº Compound CAS No. Substance category 1 1,2,3-Benzotriazole 95-14-7 Industrial Chemicals 2 1,8-Diazabicyclo [5.4.0]undec-7-ene 6674-22-2 Industrial Chemicals 3 10,11Dihydroxycarbamazepine 35079-97-1 Pharmaceuticals (metabolite) 4 2,4-Diaminotoluene 95-80-7 Industrial Chemicals 5 2-Amino-4-cresol + 6-Methyl-2pyridinemethanol 95-84-1 / 1122-71-0 Industrial Chemicals 6 2-Aminophenol 95-55-6 Personal Care Products 7 2-Ethyl-1,5-dimethyl-3,3diphenylpyrrolinium 21409-27-8 Pharmaceuticals (metabolite) 8 2-Ethylhexyl diphenyl phosphate 1241-94-7 Industrial Chemicals 9 2-Methoxy-5-methylaniline 120-71-8 Industrial Chemicals 10 3,5-di-tert-Butyl-4-hydroxybenzoic acid 1421-49-4 Industrial Chemicals 11 (4+5)-Methylbenzotriazole 29878-31-7 Industrial Chemicals 12 6-Methoxyquinoline 5263-87-6 Industrial Chemicals 13 Bis(2-ethylhexyl)amine 106-20-7 Industrial Chemicals 14 Caffeine 58-08-2 Others 15 Caprolactam 105-60-2 Industrial Chemicals 16 Carbamazepine 298-46-4 Pharmaceuticals 17 Carbendazim 10605-21-7 Pesticides 18 Dibutyl adipate 105-99-7 Personal Care Products 19 Dibutyl hydrogen phosphate 107-66-4 Industrial Chemicals 20 Dibutyl phthalate 84-74-2 Industrial Chemicals 21 Diethyl phthalate 84-66-2 Personal Care Products 22 Diuron 330-54-1 Pesticides 23 Flecainide 54143-55-4 Pharmaceuticals 24 Galaxolidone 507442-49-1 Personal Care Products 25 MDMA 42542-10-9 Drugs of abuse 26 Melamine 108-78-1 Industrial Chemicals 27 N,N'-Diphenylguanidine 20277-92-3 Industrial Chemicals 28 N-Phenyl-1-naphthylamine 90-30-2 Industrial chemicals 29 O-Desmethyl Venlafaxine 93413-62-8 Pharmaceuticals (metabolite) 30 Ofloxacin 82419-36-1 Pharmaceuticals 31 Secbumeton 26259-45-0 Pesticides 32 Sitagliptin 486460-32-6 Pharmaceuticals 33 Sulpiride 15676-16-1 Pharmaceuticals 34 Temazepam 846-50-4 Pharmaceuticals 35 Terbutryn 886-50-0 Pesticides 36 Theophylline 58-55-9 Others (metabolite) 37 Tributyl phosphate 126-73-8 Industrial Chemicals 38 Tributylamine 102-82-9 Industrial Chemicals 39 Triethyl phosphate 78-40-0 Industrial Chemicals 40 Tris(2-butoxyethyl) phosphate 78-51-3 Industrial Chemicals 41 Venlafaxine 93413-69-5 Pharmaceuticals
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 23 For lettuce analysis, a complementary method was developed, involving sample extraction via QuEChERS and subsequent analysis using the same conditions applied for water samples. This method has also been validated, and the results from its application to lettuce samples in this project are slated for publication. 4.4.4 PFAS The analysis of PFAS and related compounds through suspect screening builds on IDAEA-CSIC's previous experience with other compound groups. The workflow facilitates the tentative identification of PFAS and related compounds with a confidence level of up to 2, as defined by Schymanski et al. (2014) and scale and refine to level 1 for the PFASs for which CSIC possesses the standard (Table 4), as outlined in Deliverable D1.4. Sample purification and pre-concentration. Water samples were centrifuged at 2000 rpm for 10 min at room temperature before extraction. Subsequently, 200 mL of water was transferred to a PET container and spiked with a mixture of surrogate internal standards in methanol, achieving a final concentration of 10 pg/mL in sample. The extraction protocol was adapted from a previously developed method by IDAEA-CSIC (Barbosa et al., 2023). In summary, solid-phase extraction (SPE) cartridges were conditioned sequentially with 2 mL of methanol and 2 mL of ultrapure water under gravity conditions. Samples comprising 200 mL of surface water and 100 mL of wastewater were loaded under vacuum conditions through PEEK capillary tubes at a controlled flow rate of 1 mL/min. The cartridges were then dried under vacuum for 15 min, and PFASs were eluted using 8 mL of methanol (0.1% NH4OH) into polypropylene tubes. The eluates were evaporated nearly to dryness under a gentle nitrogen stream, transferred into LC vials with 250 μL inserts, and reconstituted in 100 μL of ultrapure water/methanol (90:10). To ensure the reliability of the process, an SPE blank sample was processed alongside real samples to monitor potential cross-contamination. Table 4. PFAS quantified by IDAEA-CSIC Carboxylic acids Sulfonic acids & sulfonamides New PFAS PFBA PFBS 6:2 diPAP PFPeA PFPeS 8:2 diPAP PFHxA PFHxS ADONA PFHpA PFHpS EtFOSA PFOA PFOS EtFOSAA PFNA PFNS FOSAA PFDA PFDS MeFOSAA PFUnA PFDoS MeFOSA PFDoA FOSA HFPO-DA (Gen-X) PFTrDA Fluorotelomer sulfonic acids PFMOAA PFTeDA 4:2 FTSA PFMOPrA PFHxDA 6:2 FTSA PFMOBA PFODA 8:2 FTSA PFO2HxA 10:2 FTSA PFO3OA PFO4DA
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 24 Instrumental Analysis: Chromatographic separation is performed using an Acquity LC system (Waters, Milford, MA, USA) coupled with a C18 Hypersil GOLD PFP LC analytical column (50x3 µm) (Themo Fisher Scientific, San Jose, CA). The mobile phase consists of (A) 20 mM aqueous ammonium acetate and (B) 20 mM methanol ammonium acetate. The elution gradient starts at 20% B, which increases to 80% B within 5 minutes. In the following 5 minutes, the gradient rises to 90% B, which is maintained for 2 more minutes. The initial conditions are restored within 1 minute and maintained for another minute, completing a total run time of 12 minutes per injection at a flow rate of 0.2 mL/min. The optimal injection volume is 10 μL. The chromatographic system is integrated with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific), equipped with an electrospray ionization (ESI) source, operating in negative ionization mode. Data acquisition is performed in full scan (FS) mode (90-1500 Da) with a resolution of 70,000 FWHM, alongside data-dependent scans (ddS) targeting the most intense ions, at a resolution of 15,000 FWHM. The entire system is managed through Xcalibur 4.1 software. The total ion chromatograms (TIC), generated from FS acquisitions, are then processed with Xcalibur software for the quantification of CSIC’s available standards (level 1 confidence). 4.4.5 Inorganic ions The quantification of the chlorinated and brominated ions was conducted using a DIONEX ICS-2100 ion chromatography system. This analysis was performed with a high-performance ion-exchange column, using a gradient elution of H2O and KOH at a flow rate of 1 mL/min, ensuring precise separation and quantification of the target ions. To guarantee optimal analysis quality, samples were filtered through 0.45 µm nylon membrane prior to injection, removing any particulate matter, which could interfere with the chromatography process. 4.4.6 Toxicological assays The samples were divided for extraction using two different SPE columns. A general extraction was performed using the HLB-SPE column, while a PFAS specific extraction was carried out with the WAXSPE column. The eluates from both extractions were evaporated under a gentle stream of N2 and further reconstituted in the appropriate volume of DMSO, based on the extraction method used. Serial dilutions of all sample extracts were prepared in DMSO and then analysed for their potential to disrupt with T4-TTR binding (PFAS CALUX), ERα-receptor activation (ERα CALUX), PXR-receptor activation (PXR CALUX), cytotoxicity (Cytotox CALUX), AR-receptor antagonism (anti-AR CALUX), and PPARγ-receptor antagonism (anti-PPARγ CALUX). TTR-Binding Assay. Serial sample dilutions were incubated overnight at 4°C in Tris-buffer (pH 8.0) with TTR (0.058 µM) and a fixed concentration of T4 (0.052 µM) (3.2% of the sample dilution in the incubation mixture). After incubation, TTR-bound and free T4 were separated using a Bio-Gel P-6DG column. The eluate was then transferred into the assay medium, and TRβ CALUX cells were exposed for 24 hours. CALUX Bioassays. CALUX cells were seeded in 96-well plates with assay medium. After exposing the CALUX cells to serial sample dilutions in triplicate (PFAS CALUX: dilutions after TTR-binding assay; other CALUX: dilution series of sample extracts), the induction of luciferase production was measured by luminescence using a Berthold luminometer following the addition of luciferin substrate. On each
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 25 96-well plate, complete calibration curves for each bioassay are also analysed using the relevant reference compounds. Data analysis. The analysis results of sample extracts, presented as induction relative to the standard reference compound, were interpolated into the calibration curves of each respective bioassay for quantitative assessment of disruptive potential. This process was carried out using the statistical software package GraphPad Prism V5.03. Only dilutions without any signs of cytotoxicity were considered for the final evaluation of the analysis. All findings are expressed as the equivalent amount of reference compound per liter of processed water. 4.4.7 Microbiological analysis The treated water was analysed for microbiological parameters, including E. coli, Legionella, and eggs of parasitic helminths (both Taenia and nematodes), to confirm compliance with the requirements set by Royal Decree 1085/2024 This Spanish regulation establishes the Water Reuse Regulation and introduces several amendments to other decrees about water management. The analysis was carried out by an external laboratory to evaluate whether the treated water complies with Quality A.A. standards for irrigation of vegetable crops, particularly those consumed raw, as established by the Spanish Royal Decree 1085/2024. These crops, where the edible part comes into direct contact with the reclaimed water, require a higher level of safety to prevent any health risks. The laboratory tests aim to verify if the water meets the specific microbiological and chemical criteria outlined for this quality level (Table 5), ensuring its safety for agricultural use in food production. Table 5. Maximum allowable value established for Quality A.A. standards Parameter Maximum allowable value E. coli (CFU/100 mL) 10 Turbidity (NTU) 5 SS (mg/L) 10 DBO5 (mg/L) 10 Intestinal nematodes (egg/L) 1 Legionella spp.a (CFU/L) < 1000 T. saginata and T. solium - Contaminants b - a Legionella spp.: Compliance with Royal Decree 487/2022, which established health requirements for the prevention and control of Legionella, will also be required. b The restricted contaminants listed in the wastewater discharge authorization will be monitored to ensure the production of reclaimed water does not lead to deterioration of the receiving environment. This aligns with the requirements set forth in Royal Decree 817/2015, establishes the monitoring and assessment criteria for surface water status and environmental quality standards, and Royal Decree 1514/2009, regulates the protection of groundwater against contamination and degradation.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 32 Figure 12. iPM(T)s removal by ozonation, electro-oxidation and e-Peroxone using synthetic secondary effluent (1,2,3-benzotriazole: blue line, 4-methylbenzotriazole: grey line, 5-methylbenzotriazole: yellow line, melamine: light blue line N,N'-diphenylguanidine (DPG): green line, and tris(2-butoxyethyl) phosphate: navy blue line)
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 33 Table 9. EE/O values for each pollutant involved in the treatment processes using spiked synthetic effluent water Type of contaminant Contaminant Ozonation EE/O (kWh/m3) Electro-oxidation EE/O (kWh/m3) e-Peroxone EE/O (kWh/m3) iPM(T)s 1,4Dioxane n.a. n.a. n.a. 1,2,3-Benzotriazole 10.1-11.3 17.5 62.4-88.6 2,4Diaminotoluene n.a. n.a. n.a. 4-methyl-1H-benzotriazole n.a. 14.6-78.0 12.5-37.5 N,N'-Diphenylguanidine 10.7-19.1 n.a. n.a. Tris(2-butoxyethyl) phosphate n.a. 24.4-170 63.4-81.1 2-Aminophenol n.a. 160-205 n.a. Melamine n.a. 15.2-35.3 7.7-21.2 5-Methyl-1H-benzotriazole n.a. 12.9-162 18.1-36.7 PFAS PFHxA n.a. n.a. n.a. PFBS n.a. n.a. n.a. PFHxS n.a. n.a. n.a. n.a.: not available For ozonation, EE/O values cannot be calculated because the 90% removal threshold was not reached within the operational timeframe (Table 9). Conversely, in the electro-oxidation and e-Peroxone processes, the removal efficiency for some contaminants exceeded 90% well in advance of collecting the first sample. The results offer a general indication of performance but cannot be directly compared due to the absence of precise 90% removal values. This limitation arises from the insufficient number of samples available, which prevents accurate data fitting and interpolation to refine the analysis. 5.2.2 Real secondary effluent trials Following the lab-scale analysis of iPM(T)s and PFAS removal using a synthetic effluent, additional experiments were conducted with real secondary WWTP effluent. This was intended to assess the impact of the liquid matrix on the iPM(T)s and PFAS removal efficiencies. By comparing all the results from synthetic and real effluents, deeper insights into the complexities introduced by several constituents present in secondary effluent can be obtained. This comprehensive approach helps identify potential challenges and optimize treatment strategies for real-world applications. These experiments analysed both doped and undoped scenarios to evaluate how the presence of multiple contaminants influences the oxidation process. In the doped experiments, known iPM(T)s and PFAS concentrations were used to test the system’s ability to effectively remove these compounds under controlled conditions. In contrast, the undoped experiments measured removal efficiencies in natural settings, without the addition of specific contaminants. Additionally, in this stage, the effectiveness of the oxidation processes was thoroughly evaluated, with a specific focus on the removal efficiencies of PFAS—using a combination of three PFAS compounds—and iPM(T)s, which comprised a mix of nine distinct compounds. The details of these compounds, with their respective theoretical concentrations in the spiked solution, are presented in Table 8. Note that at this point, the concentration of PFAS was reduced to 10 ppb by performing a pre-concentration step during the sample analysis. The subsequent monitoring performed by IDAEA-CSIC revealed only five of the nine iPM(T) compounds spiked into the real secondary effluent could be successfully quantified. The method
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 34 successfully enabled the quantification of 1,2,3-benzotriazole, 2-aminophenol, (4+5)- methylbenzotriazole, N,N'-diphenylguanidine (DPG), and tris(2-butoxyethyl) phosphate. Despite being introduced at the same concentrations, four compounds could not be quantified during the analysis. 2,4-diaminotoluene exhibited concentration increases over time, even in unspiked samples where the initial concentration was nearly zero. In contrast, melamine showed no significant differences between the spiked and unspiked samples. The initial concentrations were practically the same, indicating that spiking did not influence the levels of these compounds in the analysed system. Additionally, the detection of 1,4-dioxane was not feasible because its analysis requires positive ionization mode, whereas the rest of the compounds were measured using negative ionization mode. Despite the analytical challenges encountered in these experiments, higher removal percentages were achieved using e-Peroxone process (operating at 30 A and an estimated ozone dosage of 384 mgO3/L) compared to electro-oxidation alone (Figure 13). Furthermore, as anticipated, the oxidative treatment proved more effective at elevated contaminant concentrations. As a result, these findings confirm the performance of the e-Peroxone process to treat real secondary effluent at the laboratory scale. Figure 13. iPM(T)s removed by electro-oxidation and e-Peroxone using real secondary effluent (1,2,3benzotriazole: blue line, 2-aminophenol: orange line, (4+5)-methylbenzotriazole: grey line, N,N'- diphenylguanidine (DPG): green line and tris(2-butoxyethyl) phosphate: navy blue line) Unfortunately, in the described experiments, the target compounds PFBA, PFPeA and PFOA were not detected in either the spiked or non-spiked assays, suggesting their concentrations were below the detection limits of the analytical method. Similarly, 6:2 FTS remained below the quantification limit in both cases, further complicating the assessment of its removal efficiency within the oxidation
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 35 process. Notably, these four contaminants were not initially included in the spiked mixture. However, interestingly, PFOS -also absent from the spiked mixturewas detected in both experiments. This result suggests potential cross-contamination or residual presence of PFOS in the liquid matrix or containers used for the experiments. Nevertheless, cross-contamination during chemical analysis has been ruled out by using control blanks in parallel during the entire chemical extraction and analysis to monitor for any possible contamination. In the spiked experiments, four target PFAS contaminants, PFHxA, PFBS, PFHxS, and PFOS were detected; however, their concentrations deviated from the expected theoretical values, hindering an accurate evaluation of the removal efficiency for these compounds. This inconsistency impacts the ability of drawing definitive conclusions about the effectiveness of the treatment process. As can be seen in Figure 14, the fluctuations in the PFAS removal over time did not exhibit a significant trend. Figure 14. PFAS removal from real secondary effluent using electro-oxidation and e-Peroxone (PFHxA: navy blue line, PFBS: blue line, PFHxS: orange line, PFOS: grey line) Furthermore, at this stage, none of the PFAS were detected in the non-spiked experiments, indicating a challenge for scaling up the process at the WWTP Montornès del Vallès. Therefore, the sample volume for PFAS analysis was adjusted. Given that the initial experimental setup involved a 5L reactor with only 50 mL/sample available, the sample volume was increased to 500 mL. This adjustment was conducted to enable pre-concentration before analysis, allowing for more precise PFAS quantification. Looking ahead, future experiments must be conducted without spiking the samples to gain a more accurate understanding of removal efficiencies. To evaluate the feasibility of the e-Peroxone process for effective PFAS removal, Eurecat outsourced the detailed analysis of specific compounds -PFHxA, PFBS and PFHxSto a specialized commercial laboratory. The experiment was conducted under the same operational conditions as previous ones, by spiking these compounds to simulate real-world contamination levels. As can be seen in Figure 15, a notable reduction in long-chain PFHxA and PFHxS was observed over time, while the removal efficiency for short-chain compounds progressively diminished. This decline is attributed to the breakdown of long-chain PFAS, which generates short-chain PFAS as by-products. As a result, the concentration of short-chain PFAS increases, making their removal less efficient over time. This highlights the complex dynamics of PFAS degradation during the e-Peroxone process.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 36 Figure 15. PFAS removal in real secondary effluent using electro-oxidation and e-Peroxone (PFHxA: navy blue line, PFBS: yellow line and PFHxS: orange line) The generation of ions following the oxidative treatment was analysed, revealing the formation of chlorate (Table 10). The analytical method was not sufficiently optimized for quantifying these ions at the observed concentrations. To address this, Eurecat laboratory personnel modified the method to reduce the detection limits by tenfold to 0.5 mg/L and developed a perchlorate method, considering the presence of chlorate could indicate the potential formation of perchlorate as well. Table 10. Formation of chlorinated by-products using electro-oxidation and e-Peroxone on real secondary effluent Treatment Chloride (mg/L) Chlorite (mg/L) Chlorate (mg/L) Initial Final Initial Final Initial Final Electro-oxidation 336 22 < 5 < 5 < 5 69 e-Peroxone 337 26 < 5 < 5 < 5 75 In terms of energy consumption, the electrical energy per order (EE/O) was calculated for each pollutant involved in the treatment process (Table 11). Table 11. EE/O values for each pollutant involved in the treatment processes for real secondary effluent Type of contaminant Contaminant Electrooxidation EE/O (kWh/m3) e-Peroxone EE/O (kWh/m3) iPM(T)s 1,2,3-Benzotriazole n.a. 19.8 2,4Diaminotoluene n.a. 43.4 N, N'-Diphenylguanidine n.a. n.a. Tris(2-butoxyethyl) phosphate 165.2 17.1 2-Aminophenol 88.3 194.9 Melamine n.a. n.a. (4+5)-Methyl-1H-benzotriazole n.a. n.a. PFAS PFHxA n.a. n.a. PFBS n.a. n.a. PFHxS n.a. n.a. n.a.: not available The EE/O values were determined under the specific operational parameters of these assays (Table 11). These values are representative solely of the experimental setup, including the initial concentrations, and may not apply to other scenarios or operational parameters. Similarly to the experiments conducted with synthetic effluent, the results provide a general performance indication
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 37 but lack direct comparability due to the unavailability of precise 90% removal values. This limitation stems from an insufficient sample size, hindering accurate data fitting and interpolation needed for more detailed analysis. 5.2.3 Scaling up: real secondary effluent at larger scale Prior to scaling up the e-Peroxone treatment at the WWTP, Eurecat carried out a series of experiments using the EAOP prototype at their premises in July-August 2023. To evaluate the system’s performance at a larger scale, electro-oxidation and e-Peroxone runs were conducted over a total of 30 hours. The tests were divided into two 8-hour operational sessions, spaced across consecutive days (day 1: 8 hours of operation, 14-hour pause, and day 2: an additional 8 hours). This approach allowed for a comprehensive analysis of the treatment duration and system efficiency under extended operational conditions. Notably, the EAOP prototype is not designed for continuous operation due to the absence of an emergency stop mechanism for handling potential ozone leaks. As a result, some additional safety measures were implemented to ensure secure and controlled operation. During these experiments, the focus was placed exclusively on monitoring iPM(T)s and PFAS removal (Figure 16 and Figure 17), and ion formation (Table 12 and Table 13) over time, as these are critical parameters for the implementation of the EAOP prototype at the WWTP. Given the varying properties and origins of the iPM(T)s analysed by IDAEA-CSIC, these compounds were grouped based on their initial concentration in the influent. Consequently, these target pollutants were classified into five concentration ranges, spanning from values above 1.6 g/L to as low as 0.06 g/L.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 38 Figure 16. iPM(T)s removal using electro-oxidation and e-Peroxone on real secondary WWTP effluent at larger scale As can be seen in Figure 16, the removal efficiency of iPM(T)s fluctuated considerably based on the functional groups within the molecules and their initial concentration. This variation highlights the importance of these factors in optimizing treatment processes for effective contaminant reduction. Although no significant differences were noted between the electro-oxidation process and the ePeroxone system for many compounds, the introduction of ozone resulted in a slight improvement in the removal of specific substances. To determine the ideal treatment duration for practical applications, a compromise was established between pollutant removal efficiency and the energy consumption associated with the treatment time. This detailed analysis identified 360 minutes as the optimal timeframe for reaching effective results.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 39 Unfortunately, no significant removal of PFAS was observed, as depicted in Figure 17, since the initial and the final concentrations at different times are virtually identical. This outcome raises important considerations regarding the efficiency of the treatment process. Figure 17. PFAS removal using electro-oxidation and e-Peroxone on real secondary WWTP effluent at larger scale Several publications reported that oxidation processes involving long-chain PFAS produce short-chain PFAS as by-products. This transformation can result in a relatively stable concentration of short-chain PFAS if the reaction time is inadequate for achieving complete mineralization (Barisci and Suri, 2020; Radjenovic et al., 2020; Mirabediny et al., 2023). The lack of significant changes in PFAS concentrations suggests that either the current treatment parameters are not adequately optimized to effectively target these persistent compounds, or the analytic methods used for quantification are challenged by interferences in the liquid matrix. Considering these insights, the optimal operational parameters for the AOP prototype were defined so that effective removal of iPM(T)s could be reached. Additionally, during the oxidation process, halides (chlorides and bromides) may undergo reactions leading to the generation of chlorinated and brominated by-products (Table 12 and Table 13). This fact not only impacts the overall chemical composition of the effluent but also raises environmental concerns regarding the potential toxicity of these compounds. Furthermore, the formation of such by-products may affect compliance with discharge limits, necessitating careful monitoring to ensure that the treated water meets regulatory standards before being released into the environment. Table 12. Formation of chlorinated by-products using electro-oxidation and e-Peroxone on real secondary WWTP effluent at a larger scale Treatment Chloride (mg/L) Chlorite (mg/L) Chlorate (mg/L) Perchlorate (mg/L) Initial Final Initial Final Initial Final Initial Final Electro-oxidation 367 329 < 0.5 < 0.5 1.3 85 2.8 219 e-Peroxone 386 253 < 0.5 < 0.5 0.5 57 < 0.5 169 The production of chlorinated compounds during the oxidation process was quantified at EURECAT’s laboratory (refer to section 4.6, Quantification of inorganic ions). The analysis revealed a significant increase in chlorate and perchlorate ions following oxidation treatment, attributable to the anodic
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 40 oxidation of chloride ions (Amado-Piña et al., 2022). As illustrated in Table 12, the electro-oxidation process resulted in a greater formation of these ions compared to the e-Peroxone technology. The generation of inorganic oxychlorines during the e-Peroxone process has been explored by Lin et al. (2016). The anodic oxidation plays a crucial role in the conversion from chloride to oxychlorines, particularly during the initial stage of chloride transformation into hypochlorous acid/hypochlorite and the final stage of chlorate conversion into perchlorate. Although ozone and hydroxyl radicals do not directly oxidize chloride, they can oxidize hypochlorite (ClO-) into higher oxychlorines once they are electrochemically generated at the anode (Lin et al., 2016). On the other hand, electro-generated hydrogen peroxide plays a key role in reducing hypochlorite back to chloride and transforming chlorate radicals into chlorate ions, thus helping to minimize the formation of oxychlorines. Table 13. Formation of brominated by-products using electro-oxidation and e-Peroxone on real secondary WWTP effluent at a larger scale Treatment Bromide (mg/L) Bromate (mg/L) Initial Final Initial Final Electro-oxidation < 1 < 0.5 < 0.5 < 0.5 e-Peroxone < 0.5 < 0.5 < 0.5 < 0.5 Bromate formation is practically negligible at low specific ozone doses (< 0.5 mgO3/mg DOC) during the ozonation process. Nevertheless, its formation poses a notable risk when higher ozone doses are applied during ozonation, particularly at or above 1.0 mgO3/mg DOC. Even moderate bromide levels in water (as low as 50 g/L) may trigger significant bromate production, raising both operational and environmental challenges (von Gunten, 2003). The e-Peroxone process effectively reduces bromate formation in bromide-containing water by reducing ozone’s lifetime and neutralizing hypobromous acid (HBrO), which is a key reaction intermediate in bromate production during ozone-based reactions. The formation of brominated compounds was quantified in EURECAT’s lab. As shown in Table 13, the quantification limits of the method were insufficient to accurately measure the levels of bromide and bromate present in both the influent and effluent, respectively. Moving forward, the bromate concentration was analysed in a commercial laboratory with a lower quantification limit of 1 g/L. In terms of energy consumption, the electrical energy per order (EE/O) was calculated for each pollutant involved in the treatment process (Table 14).
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 41 Table 14. EE/O values for each pollutant involved in the treatment processes for real secondary WWTP effluent Type of contaminant Contaminant Electrooxidation EE/O (kWh/m3) e-Peroxone EE/O (kWh/m3) iPM(T)s (4+5)-Methyl-1H-benzotriazole 4.55 4.35 1,2,3-Benzotriazole 5.21 5.42 Galaxolidone n.a. n.a. 2,4Diaminotoluene n.a. n.a. 2-Aminophenol 2.46 2.72 o-desmethyl venlafaxine 1.05 n.a. Sitagliptin 14.10 18.07 10,11-Dihydro-10,11dihydroxycarbamazepine 21.01 n.a. 3,5-di-tert-butyl-4-hydroxybenzoic acid n.a. n.a. Flecainide 12.90 5.69 N, N'-Diphenylguanidine 1.50 4.33 Tris(2-butoxyethyl) phosphate n.a. 15.62 Venlafaxine 3.02 n.a. 6-Methoxyquinoline n.a. n.a. Carbamazepine n.a. n.a. Sulpiride n.a. n.a. Caffeine n.a. n.a. Ofloxacin n.a. n.a. Theophylline n.a. n.a. EDDP/2-ethyl-1,5-dimethyl-3,3diphenylpyrrolinium 3.61 4.85 Diuron n.a. n.a. Temazepam n.a. n.a. Terbutryn n.a. n.a. MDMA n.a. n.a. The primary component of operating costs in oxidation processes stems from the electrical energy required per order. As mentioned above, this metric is defined as the amount of electrical energy (in kWh) needed to achieve a one-order-of-magnitude reduction in the concentration of a specific pollutant within 1 m3 of contaminated water. A lower electrical energy per order indicates a more efficient treatment method, highlighting the efficacy of the process in removing pollutants. This focus on optimizing energy consumption not only improves the economic viability of the treatment system but also aligns with sustainability goals by reducing the overall environmental footprint. 5.3 Challenges and adaptations during pilot plant installation The installation of the pilot plant presented a multifaceted challenge, requiring careful adjustments and innovative solutions to ensure successful execution. From logistical constraints to unforeseen technical difficulties, this section delves into the key challenges encountered during the installation phase into the pilot framework.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 48 Figure 21. Pharmaceutical removal using e-Peroxone at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) Nevertheless, compounds such as sitagliptin, and especially temazepam, had lower removal rates by e-Peroxone process, even at six hours of treatment, suggesting operational limitations in the technology's effectiveness for persistent substances. This fact underscores the potential need for targeted optimization to effectively address specific pharmaceutical contaminants, rather than focusing on improving the removal of all studied compounds, to reduce the treatment time and energy consumption of the e-Peroxone process. The operation of post EAOP CW system depends on the e-Peroxone process. High removal efficiencies in the EAOPs involve minimal further removal of compounds was seen in the CW, as most of the contaminants were already removed in the EAOPs (Figure 22). Nevertheless, compounds as sitagliptin and flecainide, with 60-70% removal during the e-Peroxone process (Figure 21), showed additional removal within the CW systems. These substances were 50-80% removed in the CW, demonstrating that the CW does provide additional compound removal. This revealed the CW’s complementary role in treating certain compounds resistant to complete degradation by the oxidation process. Figure 22. Pharmaceuticals removal in the post EAOP CW system at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) Pesticides/Drugs of abuse/Others As was anticipated, the removal rates varied significantly, with terbutryn (used as a biocide) and MDMA (commonly known as ecstasy) removed >95% after 180 min of treatment (Figure 23). Nevertheless, other compounds such as EDDP (a metabolite of methadone) and carbendazim (a fungicide) demonstrate notable removal differences between the 180-min and 360-min treatment, with removal rates falling below 50% at 180 min.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 49 Figure 23. Pesticides/Drugs of abuse/Others removal using e-Peroxone at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) The higher removal observed for diuron and caffeine in June, despite a shorter treatment time, could be due to their concentration dependant behaviour during the treatment process. Besides, seasonal or operational factors could influence pollutant levels and reaction kinetics, adding variability to removal efficiency between months. This suggests that e-Peroxone process may achieve higher removal rates when higher pollutant concentrations are present, even with shorter operational times, while lower concentrations might need extended treatment to reach similar efficiencies. The post EAOP CW system showed lower removal for pesticide and illicit drug compounds (Figure 24) compared to the pharmaceuticals previously discussed. This is due to the high removal of these substances during the oxidation process (Figure 23). For example, carbendazim exhibited high removal in CW system during June, coinciding with the lower removal efficiencies observed in the ePeroxone process for the same compound. In contrast, no removal was detected in April, when the e-Peroxone process effectively eliminated nearly 100% of the carbendazim, leaving a lower concentration in the influent to the CW. Figure 24. Pesticides/Drugs of abuse/Others removal by post EAOP CW system at Montornès del Vallés WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) Industrial chemicals The most prevalent iPM(T)s detected in the secondary effluent of the WWTP include industrial chemicals such as phthalates, aminophenols, benzotriazole, phosphates, and various amines. Phthalates, aminophenol and tributylamine were removed >90% during 180 min of e-Peroxone treatment (Figure 25). This can be likely attributed to their chemical structures, which are more amenable to oxidation processes and can be effectively broken down by the active species generated during treatment.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 50 Organic molecules derived from benzotriazole, more specifically 1,2,3-benzotriazole and (4+5)- methylbenzotriazole) demonstrate relatively low removal percentages. Benzotriazoles possess robust aromatic rings, which make them more resistant to removal by reactive species, leading to only partial removal during the treatment process. According to the literature, compounds like atenolol and benzotriazole, which are more resistant to ozone oxidation, can be progressively eliminated as the ozone dose increases, achieving over 85% removal with a moderate dose (around 0.6 g O₃/g DOC) (Hollender et al., 2009). Figure 25. Industrial chemicals removal using e-Peroxone at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) The operation of the post EAOP CW system revealed that most industrial compounds, except for dibutyl phthalate (72±35%), exhibited removal percentages below 60% (i.e., 56±6% for N,N’-DPG or 14±5% for (4+5)-methylbenzotriazole) (Figure 26). Despite the effective removal in the EAOP, these compounds demonstrated limited additional removal in the CW system, highlighting the challenges of further treating pollutants already efficiently degraded in the initial oxidation stage. Compounds resistant to oxidation, such as 1,2,3-benzotriazole, exhibited relatively higher removal in the CW during April. This performance compensated for the lower elimination efficiencies achieved during the oxidation process. Figure 26. Industrial chemical removal in the post EAOPs CW system at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar)
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 51 5.5.4 PFAS removal The PFAS removal in the e-Peroxone process was difficult to evaluate. The average monthly concentrations of 6:2 FTS revealed unexpected increases, deviating from the anticipated degradation pathway for this compound. Moderate removal rates for PFOS (Table 16) highlight the system’s effectiveness in targeting and degrading this compound. By contrast, PFOA exhibits a lower removal when subjected to the same treatment conditions (Table 17). However, the data fails to show a clear trend in the removal or formation of short-chain PFAS, which complicates the ability to draw definitive conclusions from these results. This uncertainty may arise from various factors, such as the inherent variability of the process and the ever-changing interactions of PFAS within the system. Table 16. Average monthly sulfonic PFAS levels before and after e-Peroxone at Montornès del Vallès Months 6:2 FTS (ng/L) PFOS (ng/L) PFHxS (ng/L) PFBS (ng/L) Initial Final Initial Final Initial Final Initial Final April 41.2 74.1 36.8 6.6 < LOD < LOD 149.7 157.4 May 19.0 40.2 33.3 14.7 < LOD < LOD 132.7 143.4 June < LOD 11.2 14.4 20.9 11.0 12.5 277.9 231.2 July 27.9 28.7 54.2 35.3 6.8 6.7 61.3 52.7 Table 17. Average monthly carboxylic PFAS levels before and after e-Peroxone at Montornès del Vallès Months PFNA (ng/L) PFOA (ng/L) PFHxA (ng/L) PFBA (ng/L) Initial Final Initial Final Initial Final Initial Final April 4.5 1.7 20.4 15.9 < LOD 34.0 < LOD < LOD May 2.1 9.5 18.8 14.3 < LOD 31.5 < LOD < LOD June 3.2 3.5 17.5 16.0 18.4 18.8 17.7 14.2 July 7.5 6.2 15.9 13.4 20.1 18.2 23.1 46.2 The post EAOP CW system demonstrated low efficiency in removing short-chain PFAS compounds, as indicated by the similarity in concentrations between the inlet and outlet (Table 18 and Table 19). This outcome suggests that short-chain PFAS, due to their high solubility and low adsorption potential, are less likely to be retained or degraded within the CW system. Unlike their long-chain counterparts, which may adhere more readily to organic matter or biofilms within the CW system, short-chain PFAS remain largely mobile, passing through the system without significant alteration. Furthermore, the limited removal observed may be attributed to the lack of sufficient biological and/or chemical mechanisms (e.g. adsorption, phytoremediation) capable of effectively removing these persistent compounds.This emphasizes a major challenge in treating short-chain PFAS in CWs and highlights the need for long-term monitoring of full-scale CWs treating real wastewater for collecting additional data to support the development and validation of models (Savvidou et al., 2024). Table 18. Average monthly sulfonic PFAS levels before/after post e-AOP CW at Montornès del Vallès Months 6:2 FTS (ng/L) PFOS (ng/L) PFHxS (ng/L) PFBS (ng/L) Initial Final Initial Final Initial Final Initial Final April 74.1 37.3 6.6 34.93 < LOD < LOD 157.4 166.4 May 40.2 51.8 14.7 36.2 < LOD < LOD 143.4 143.1 June 11.2 < LOD 20.9 14.4 12.5 11.6 231.2 227.5 July 28.7 19.7 35.3 45.5 6.7 7.4 52.7 59.5
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 52 The results also reveal inconsistent removal of long-chain PFAS within the CW system. This irregularity could be attributed to several factors, including the desorption of these compounds from accumulated particulate matter or other wetland matter, or insufficient retention time for thorough degradation. Although the PFAS results are not consistent, PFNA concentrations exhibited an unexpected increase at the wetland outlet compared to the inlet throughout the duration of pilot plant operation, indicating potential contamination occurring within the wetland itself. Table 19. Average monthly carboxylic PFAS levels before/after post e-AOP CW at Montornès del Vallès Months PFNA (ng/L) PFOA (ng/L) PFHxA (ng/L) PFBA (ng/L) Initial Final Initial Final Initial Final Initial Final April 1.7 < LOD 15.9 13.5 34.0 < LOD < LOD < LOD May 9.5 51.5 14.3 21.6 31.5 35.9 < LOD < LOD June 3.5 7.5 16.0 16.3 18.8 17.4 14.2 15.0 July 6.2 227.4 13.4 12.1 18.2 17.2 46.2 15.2 Among the analysed PFAS compounds, only 12 were quantified and included into PFOA-equivalents calculation. This methodology ensures a systematic assessment: however, the inconsistencies in PFAS levels between the influent and effluent across most compounds indicate potential variability in the data, limiting the ability to accurately reflect the overall PFAS burden. 5.5.5 Formation and fate of inorganic ions Chlorinated ions, such as chlorite, chlorate, and perchlorate, were tracked throughout the ePeroxone treatment to assess the monthly average generation of these inorganic by-products alongside chloride removal. As illustrated in Table 20, chloride ions decrease, accompanied by a corresponding rise in chlorate and, most notably, perchlorate ions—the highest oxidation state of chlorine. The significant perchlorate levels observed underscore the strong oxidative conditions in the e-Peroxone process, where reactive oxygen species, mainly hydroxyl radicals, facilitate the stepwise oxidation of the chloride ions to its most stable form, perchlorate. Table 20. Formation of chlorinated by-products by e-Peroxone at Montornès del Vallès WWTP Months Chloride (mg/L) Chlorite (mg/L) Chlorate (mg/L) Perchlorate (mg/L) Initial Final Initial Final Initial Final Initial Final April 216.3 152.7 n.a. n.a. 1.8 8.8 < 2.0 42.8 May 305.8 220.8 n.a. n.a. 3.0 9.9 13.5 39.9 June 129.0 123.0 n.a. n.a. < 0.2 9.6 < 2.0 32.0 July 166.3 138.0 n.a. n.a. < 0.2 4.7 < 2.0 18.0 n.a.: not available
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 53 This generation of oxidized chlorine ions presents operational challenges for discharging the treated effluent. Chlorate and perchlorate are subject to regulation due to their potential impacts on the aquatic ecosystems and human health. These compounds, as final oxidation products, are resistant to further breakdown and thus accumulate, posing potential compliance issues for wastewater discharge. Preliminary results suggest the nature-based post-treatment system effectively reduces concentrations of residual inorganic byproducts from the treated effluent (Table 21). By capturing these persistent ions before discharge, the wetland system slightly mitigates potential environmental impacts, reducing the risk of accumulation in downstream ecosystems. Altogether, this layer enhances water quality protection and strengthens the overall environmental resilience of the treatment train. Table 21. Removal of chlorinated by-products by constructed wetland at Montornès del Vallès WWTP Months Chloride (mg/L) Chlorite (mg/L) Chlorate (mg/L) Perchlorate (mg/L) Initial Final Initial Final Initial Final Initial Final April 153 118 n.a. n.a. 8.8 6.5 42.8 37.2 May 221 166 n.a. n.a. 9.9 5.7 39.9 30.8 June 123 102 n.a. n.a. 9.6 1.7 32.0 24.5 July 138 138 n.a. n.a. 4.7 2.5 18.0 21.0 n.a.: not available At present, no specific regulations or limits exist for chlorate and perchlorate in surface water and reclaimed water. The WHO sets the reference levels for chlorate and perchlorate in drinking water at 700 and 70 μg/L (WHO, 2016a; WHO, 2016b). However, both substances have established residue limits in food products. The Commission Regulation (EU) 2020/749 amends Annex III to Regulation (EC) No 396/2005, setting maximum residue levels for chlorate in certain food items. Similarly, Commission Regulation (EU) 2023/915 defines the maximum permissible levels for contaminants, including perchlorate, in food. These regulations set a maximum residue limit for chlorate and perchlorate, ranging from 0.05 to 0.75 mg/kg of food, depending on the product in question. Therefore, the use of non-chlorinated water for irrigation is recommended. Analogously, brominated ions like bromate were monitored throughout the e-Peroxone treatment to evaluate the monthly average formation of these inorganic by-products alongside bromide reduction. However, unlike chloride, bromide concentrations remained below 1 mg/L, providing a lower baseline for bromate formation. Despite this, careful monitoring remains essential given the potential environmental and health impacts associated with brominated by-products. Bromate ion quantification was done by a commercial laboratory to improve the detection accuracy by lowering the quantification limits for bromate. The results revealed bromate concentrations below the detection threshold of 10 ppb (parts per billion), confirming the absence of bromate formation in the process. Furthermore, as expected, the nitrate ions were generated during the oxidation process, leading to an increase in concentration to average values around 9.8 mg/L. According to Regulation (EU) 2020/741, surface water is considered affected by nitrates if the concentration exceeds 25 mg/l. Therefore, despite the increase in concentration, the level does not exceed the threshold.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 54 5.5.6 Energy consumption In terms of energy consumption, determining the electrical energy per order (EE/O) for the pilotscale experiments at the Montornès del Vallès WWTP was unfeasible. This is primarily due to the absence of time-series data and the unknown time required to achieve a 90% reduction for each compound. As a result, energy consumption estimates are instead based on the total energy demands of the entire treatment. This includes the ozone generator, the electrochemical cell, and the pumping systems used for both filling and emptying the prototype’s feed tank, as well as the pump dedicated to wetland filling. The cell operates at approximately 30 A and 10 V, while the ozone generator has a nominal power rating of 1000 W. During the operation of the pilot, the ozone generator operates at a current of 0.5 A, and consumes an actual power of 110 W, despite having a nominal power rating of 1000 W. The pump in the oxidation prototype consumes 0.6 kW over a 3-hour cycle. With these parameters, the total energy consumption of the oxidation prototype reaches 3 kWh. Water transfer and irrigation were handled by a range of specialized pumps. A stainless-steel submersible pump, with a maximum absorbed power of 0.95 kW and nominal motor power of 0.45 kW, was used to move water from the secondary treatment stage to the oxidation equipment. System drainage was managed by a centrifugal pump with a 0.96 kW power capacity. For wetland irrigation, a high-efficiency circulator pump with a permanent magnet motor, operating at 25 W input power, was employed. In total, the system required 3.81 kWh per cycle, equating to an energy consumption of 12.7 kWh per cubic meter. Considering the EU average price for non-household consumers of 0.2008 €/kWh in the second half of 2023, the energy cost for operating the plant is 2.54 €/m³. 5.5.7 Toxicological analysis Evaluating toxicity of samples was essential, as the removal of certain substances may inadvertently lead to the formation of more toxic by-products. CALUX methods were used to assess the toxicity of the samples, extending beyond the mere quantification of the target contaminants. By focusing on both the removal and the resulting toxicity, the CALUX method contributes to the development of more effective and environmentally treatment strategies. A reduction of approximately 90% PFAS activity was observed throughout the entire treatment process, as determined using the CALUX method (Table 22). This result highlights the effectiveness of the treatment in reducing the presence of these contaminants. Table 22. CALUX analysis results of WAX-SPE water sample extracts Sample PFAS CALUX activity LOQ Inlet EAOPs 620 0.69 Outlet EAOPs 170 0.68 Buffer tank 180 0.68 Outlet PROMISCES wetland 65 0.68 Outlet control wetland 330 0.67
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 55 5.5.8 Microbiological and physicochemical analyses analysis The microbiological analysis of samples was conducted to verify compliance with Quality Standard AA, as specified for irrigation of vegetable crops under Royal Decree 1085/2024. The secondary effluent is currently unsuitable for irrigation due to E. coli concentrations exceeding 250,000 CFU/100 mL, well above the regulatory limit of 100 CFU/100 mL for AA water quality (Table 5). Interestingly, an unexpected increase in E. coli concentrations was observed after the filtration step, just before the e-Peroxone treatment. This increase may be linked to biofilm formation within the filtration system, potentially causing E. coli to be released back into the effluent, or to inadequate filter washing cycles, leading to microbial buildup and breakthrough. Nevertheless, the e-Peroxone process proved effective in eliminating E. coli from the treated water (close to 100%). In contrast, the CW resulted in a slight increase in E. coli concentrations (4 CFU/ 100 ml), though it still complied with the regulatory limits. Regarding Legionella spp., the secondary effluent from Montornès del Vallès WWTP also exceeded the regulatory limits. However, unlike E. coli, complete removal takes place during the filtration step just before the e-Peroxone treatment. As for eggs of parasitic helminths, from both the nematode and Taenia genera, the secondary effluent meets the specifications outlined in Royal Decree 1085/2024. No further changes in concentrations were observed throughout the treatment process in the pilot plant. The key physicochemical parameters for irrigation consideration are turbidity and suspended solids. The secondary effluent had turbidity of approx. 2.3 NTU, which slightly increased during the ePeroxone treatment. This increase can be attributed to the formation of microaggregates and the destabilization of particles during oxidation, which may cause previously settled or colloidal particles to resuspend in the water. Once the water passed through the constructed wetland, turbidity decreased again. This is due to the wetland's role as a natural filtration system, where plants, sediments, and microbial activity remove particles and reduce turbidity. These findings align with the observed trends in suspended solids (SS) concentrations across the samples. The fluctuations in the turbidity correspond closely to the changes in SS, reflecting a similar pattern throughout the treatment process. 5.6 Control wetland system - potential for removal of PMTs and PFAS 5.6.1 Physicochemical parameters and nutrients As described in section 5.4. Pilot-scale technology operation (TRL 6) , both CW systems operated under similar flow conditions, and identical design specifications and climatic factors. The key difference was their inlet water: in the case outlined in section 5.5. Parameters and removal efficiency of the pilot-scale technology testing (TRL 6) , the post-EAOP CW received EAOP effluent, while the control CW was supplied with secondary WWTP effluent (i.e. the same water entering the EAOP). The physicochemical parameters of the inlet water for both CW systems were closely aligned (see pH, electrical conductivity and dissolved oxygen in Table 23). The organic matter, measured as COD, was higher in the CW control (47.4 mg O₂/L) compared to the post EAOP CW system (39.7 mg O₂/L) Despite this, the post EAOP CW demonstrated greater degradation rate, achieving a final COD concentration of 32.2 mg O2/L. The total nitrogen levels in the inlet and outlet were comparable, with nitrogen removal rates ranging from 21% to 24% in both CW systems (Table 23). A notable distinction
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 56 between the two systems is their nitrogen removal mechanisms: the control CW primarily targeted ammonium removal (69%), while the post-EAOP CW focused on nitrate removal (34%). The low concentrations of ammonium in the post-EAOP CW were attributed to its oxidation to nitrate during the e-Peroxone process. Total phosphorous removal was also measured, with the post EAOP CW (36%) outperforming the control (17%). Table 23. Comparison of physicochemical parameters between CW systems (control vs post-EAOP) Control CW Post EAOP CW Inlet channel Outlet channel Inlet channel Outlet channel Av. Min. Max. Av. Min. Max. Av. Min. Max. Av. Min. Max. pH 7.97 7.71 8.20 8.06 7.77 8.21 8.19 7.25 8.74 8.11 7.87 8.30 Electrical Conductivit y µS/cm 2,180 1,756 2,534 2,148 1,645 2,602 2,034 1,544 2,300 2,046 1,502 2,380 Dissolved Oxygen mg/L 9.6 7.9 11.2 9.4 8.3 10.5 9.5 8.2 11.4 9.2 8.0 10.7 COD mg O2/l 47.4 36.8 57.6 41.2 26.7 52.4 39.7 34.9 43.5 32.2 28.0 36.7 COD removal % 13 8 27 19 15 25 N-NH4+ mg N/l 1.18 0.39 3.19 0.34 0.11 1.02 0.17 0.01 0.41 0.26 0.07 0.62 N-NH4+ decline % 69 45 96 0 N-NO3mg N/l 0.56 0.28 0.80 1.01 0.51 1.98 2.29 1.65 2.61 1.51 0.91 1.97 N-NO3decline % 0 34 15 60 N total mg N/l 4.96 3.70 6.60 3.73 2.46 5.95 4.74 4.24 5.72 3.70 3.12 4.01 N decline % 24 9 56 21 11 38 P total mg P/l 0.39 0.18 1.22 0.32 0.09 0.78 0.33 0.22 0.56 0.20 0.07 0.31 P decline % 17 -22 65 36 18 67 In summary, the physicochemical parameters measured in both CW systems showed no significant differences (Table 23, Figure 20 and Figure 27). Further investigation is needed to determine whether variations exist in the water temperature and oxidation reduction potential (ORP) of the EAOPtreated water. A key difference between the two systems was inorganic nitrogen: ammonium dominated in the control CW, whereas nitrate was dominant in the post-EAOP CW. Furthermore, the hydrochemical profile of the EAOP-treated water, including the presence of newly generated ions
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 57 such as chlorate, perchlorate, and bromate, showed no discernible impact on the nutrient removal efficiency of the CW systems. Figure 27. Nutrient removal of control CW at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) 5.6.2 iPM(T)s removal Pharmaceuticals The removal of pharmaceuticals in the control CW revealed that compounds were partially retained by adsorption to the substrate, uptake by vegetation and microbial degradation in the root zone (Figure 28). Venlafaxine and theophylline, both characterized by high hydrophobicity, exhibited a strong affinity for the adsorption to the wetland’s organic matter and substrate particles. This binding mechanism limits their mobility within the control CW, resulting in higher removal. This natural retention process is more pronounced in the control CW compared to the post EAOP CW, where ePeroxone has already degraded a substantial portion of these compounds before they enter the wetland. Figure 28. Pharmaceutical removals by control CW system at Montornès del Vallès WWTP (April: yellow bar, May: blue bar, June: orange bar and July: grey bar) Pesticides/Drugs of abuse/Others Unlike pharmaceuticals, pesticides exhibited poor removal in the control CW system (Figure 29). Despite the moderate/high hydrophobicity of these compounds, their removal can be influenced by multiple factors. Based on the previous studies, carbendazim exhibits different primary species at varying pH levels (Furini et al., 2016). At relatively higher pH values, the solubility of carbendazim strongly decreases, and the predominant species is anionic, leading to weaker soil adsorption. Like
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 64 5.8 Challenges and adaptations during pilot plant operation The operation of the pilot plant at the WWTP Montornès del Vallès represented a complex challenge, requiring careful adjustments to ensure successful operation. From unforeseen technical difficulties to the need for real-time monitoring, this section delves into the key challenges encountered during the operational stage within the PROMISCES project. (i) Varying treatment durations throughout the EAOP operation introduced a notable challenge for result analysis. Since the oxidation equipment was operated at different times during various phases, establishing a consistent baseline for comparison was difficult. The inconsistencies in exposure times impacted the reliability of contaminant removal rates, complicating the assessment of the system's overall performance and effectiveness. (ii) The accumulation of struvite in the EAOP prototype posed significant operational challenges, particularly in the pipes, pumps and on the electrodes of the electrochemical cell. This struvite buildup hindered the overall efficiency of the EAOP prototype, leading to flow obstructions and reduced electrochemical performance. To address these issues, a bi-monthly maintenance routine was established, involving thorough cleaning and inspection of the affected components. This regular maintenance was essential to ensure the continuous performance of the pilot plant, preventing disruptions and maintaining the integrity of the water of the water treatment process. (iii) Ozone quantification in the EAOP prototype was conducted to measure the residual ozone, specifically the fraction not dissolved in the solution. Nevertheless, the lack of non-airtight conditions in the experiment setup prevented an accurate assessment of the ozone dosage within the system. To address this issue, an on-site ozone sensor should be installed to allow real-time monitoring of ozone levels during pilot operation, providing more precise data for optimizing the process. (iv) Evaporation and/or evapotranspiration in the constructed wetlands involved challenges in accurately calculating the contaminant mass balance, hindering the evaluation of contaminant removal efficiency. The imbalance between incoming and outgoing water volumes further complicated accurate assessments. In addition, this variability affected comparison with the control wetland. Thus, the impact of evaporation and/or evapotranspiration on the wetland's treatment performance had to be carefully accounted for in the analysis. (v) Managing the inflow and outflow in wetlands requires precise, manual control of valves to maintain optimal conditions. To prevent overflooding or full drainage, valves are manually adjusted and monitored daily, ensuring the substrate remains sufficiently saturated to support both plant growth and microbial processes. This approach allows for the careful balance of water retention, yet daily oversight is crucial to avoid surface flooding and maintain the desired equilibrium between moisture levels and flow. (vi) The unexpected presence of elevated PFNA concentrations in the treated effluent indicated some construction materials utilized in the wetland may have contained PFAS (Figure 10). This situation complicates the assessment of PFAS removal efficiency, as the specific material composition is rarely provided in detail and often lacks explicit disclosures regarding PFAS content. Experiments to analyse iPMTs and PFAS in the leachates from the materials used in the CWs are currently underway.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 65 6 Use of reclaimed water in vegetable crop irrigation Over the past few decades, water scarcity has intensified into a critical global challenge, driven by the dual forces of climate change and escalating water demand associated with population growth (Ungureanu et al., 2020, Jurado et al., 2012). In response, the reuse of treated wastewater from wastewater treatment plants (WWTPs) for agricultural irrigation has emerged as a viable strategy to reduce reliance on freshwater resources (García Vara et al., 2023). Despite offering considerable advantages, this solution is accompanied by concerns about the environmental safety and potential risks, underscoring the importance of rigorous assessment and the implementation of robust mitigation measures. Recent laboratory research on the effects of PFAS on maize has demonstrated that the uptake and distribution of these compounds within crops are influenced by factors such as chain length, functional groups, and the specific plant tissues involved (Ateia et al., 2019). Similarly, studies on strawberries and lettuce cultivated in both greenhouse and open-field conditions suggest that benzotriazoles can be absorbed, assimilated, and internally regulated by plants (LeFevre, 2017). Despite these findings, the understanding of the mechanisms governing the integration of PMT compounds into the water-soil-plant system and their potential impact on human health remains incomplete. Innovative water reclamation technologies, such as the one explored in this study, hold promise for minimizing crop exposure to PMTs. Nevertheless, evaluating the effects of reclaimed water on crop productivity, quality, and metabolic processes becomes essential. Furthermore, determining whether residual PMTs in the water could accumulate in crop tissues and potentially impact human health warrants focused investigation. 6.1. Characteristics of the water used for irrigation Table 28 presents the physicochemical characteristics of the irrigation waters used in this study, including control water: bottled water (BW), secondary wastewater effluent (WW), and reclaimed water: e-Peroxone + constructed wetland (RW). Four-week-old lettuce seedlings were transplanted into the pots and harvested after 42 days, once they reached commercial size. Daily irrigation frequency was adjusted to maintain soil moisture just below field capacity, effectively preventing leachate production.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 66 Table 28. General water quality parameters for each type of irrigation water (n=5) Units BW RW WW pH - 8.40 ± 0.60 7.90 ± 0.70 7.90 ± 0.90 Redox mV 163.00 ± 58.00 158 ± 57 135.00 ± 73.00 Conductivity S/cm 344.00 ± 92.00 1717.00 ± 111.00 1601.00 ± 213.00 COD mg/L <2.00 24.15 ± 13.40 25.00 ± 20.00 TOC mg/L <1.00 2.00 ± 2.00 14.00 ± 10.00 NH4+ mg/L <0.01 0.30 ± 0.20 1.00 ± 0.80 NO2mg/L <0.01 0.44 ± 0.47 0.37 ± 0.12 NO3mg/L 2.16 ± 0.02 2.41 ± 2.86 1.92 ± 2.39 PO43mg/L <0.10 0.40 ± 0.33 0.47 ± 0.54 SO42mg/L 47.00 ± 15.00 107.00 ± 30.00 113.00 ± 42.00 K+ mg/L 5.00 ± 3.00 25.00 ± 23.00 30.00 ± 19.00 6.2. Uptake and distribution of contaminants in vegetable crops 6.2.1 iPM(T)s The evaluation of contaminant uptake and distribution in lettuce samples irrigated with different water types was conducted using a targeted analysis method that focused on the same iPM(T) compounds identified in the water samples. Table 29 shows the occurrence of iPM(T)s in irrigation waters and soil used for crop cultivation. As expected, some ubiquitous phthalates -though not the main target compounds-, including diethyl phthalate, were detected in all irrigation waters, including bottled water. Plastic bottled water was used to avoid the occurrence of PFAS, the main target group of compounds, from being introduced, rather than using glass bottles. Additionally, both diethyl and dibutyl phthalates were found at notable concentrations in soil, wastewater, and reclaimed water, underscoring the widespread presence of phthalate compounds in the environment (Net et at., 2015). Most of the PMTs studied, such as N,N'-diphenylguanidine (DPG), galaxolidone, and triethyl phosphate, were already present in the soil. However, pharmaceuticals and pesticides appeared to be more closely associated with wastewater sources. No PFAS were detected in bottled water or soil.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 67 Table 29. Concentration of iPM(T)s in irrigation waters (n=3) and soil used for crop studies Compounds BW (ng/L) RW (ng/L) WW (ng/L) Soil (ng/g) Triethyl phosphate n.d. 61 ± 17 24 ± 10 3.0 ± 0.4 Galaxolidone n.d. 1618 ± 460 2507 ± 306 12 ± 1 Dibutyl adipate 40.38 ± 0.003 n.d. n.d. 14 ± 1 Tris(2-butoxyethyl) phosphate n.d. 299 ± 121 503 ± 81 n.d. N,N'-Diphenylguanidine (DPG) n.d. 208 ± 46 3491 ± 158 462 ± 19 Dibutyl phthalate n.d. 159 ± 130 165 ± 158 49 ± 4 Diethyl phthalate 18419 ± 2 991 ± 614 2086 ± 2846 905 ± 15 Bis(2-ethylhexyl)amine n.d. 20 ± 2 12 ± 3 n.d. 2-Ethylhexyl diphenyl phosphate n.d. n.d. n.d. n.d. MDMA n.d. 186 ± 201 2406 ± 2007 n.d. O-Desmethyl venlafaxine n.d. 19 ± 16 977 ± 315 n.d. Venlafaxine n.d. 34 ± 11 408 ± 95 n.d. 3,5-di-tert-Butyl-4hydroxybenzoic acid n.d. 387 ± 107 378 ± 66 n.d. Carbendazim n.d. 15 ± 16.22 16 ± 18 n.d. (4+5)- Methylbenzotriazole n.d. 510 ± 218 1008 ± 391 n.d. 1,8-Diazabicyclo [5.4.0]undec-7-ene n.d. <LOQ 6 ± 5 n.d. PFBA n.d. 15 ± 1 18 ± 2 n.d. PFOA n.d. 15 ± 5 16 ± 6 n.d. PFHxS n.d. 7 ± 4 7 ± 14 n.d. PFOS n.d. 27 ± 9 27 ± 11 n.d. 6:2 FTS n.d. 30 ± 24 3 ± 6 n.d. n.d.: not detectable As can be seen in Table 29, the concentration of some compounds exhibited an increase after EAOP + CW treatment. Although this outcome may seem inconsistent, the values align with those reported in Table 27, showing negative removal and significant deviation. This could be attributed to analytical variability or matrix effects, which can influence the accuracy of the quantification. Out of the 41 iPM(T)s evaluated, 16 compounds were detected in the lettuce samples (Table 30). The antidepressant venlafaxine and its metabolite O-desmethylvenlafaxine were only found in samples irrigated with effluent wastewater, suggesting residual concentrations in reclaimed water were too low to result in detectable levels in crop leaves. A similar trend was observed for the drug of abuse MDMA and the pesticide carbendazim. This suggests that the EAOP + CW successfully minimized the presence of these pollutants in both reclaimed water and the irrigated crops.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 68 A different trend emerges for certain industrial chemicals, such as 2-ethylhexyl diphenyl phosphate and bis(2-ethylhexyl) amine, which were found at similar concentrations in crops irrigated with all water types, including in the control samples. These results are consistent with the detection of phthalates in all irrigation waters and some PMTs from industrial application found in the soil (Table 29). As a result, the presence of PMTs, including triethyl phosphate, galaxolidone, dibutyl adipate, N,N-diphenylguanidine, dibutyl and diethyl phthalates, in all lettuce samples, including those irrigated with BW, appears to be directly linked to their presence in the soil. Table 30. Overview of the iPM(T)s compounds in lettuce samples irrigated with bottled water (BW), secondary effluent wastewater (WW), and reclaimed water (RW) Average concentration (ng/g) Compounds LOD (ng/g) LOQ (ng/g) BW RW WW Frequency of detection (/30) Triethyl phosphate 0.17 0.57 29 ± 6.5 28 ± 7 22 ± 5.0 30 Galaxolidone 1.1 3.7 7.9 ± 3.1 8.1 ± 3.2 5.5 ± 4.0 29 Dibutyl adipate 0.18 0.61 1.5 ± 0.88 3.3 ± 1.9 3.2 ± 2.6 24 Tris(2-butoxyethyl) phosphate 0.07 0.22 0.24 ± 0.08 0.13 ± 0.08 0.43 ± 0.18 22 N,N'-Diphenylguanidine (DPG) 0.22 0.71 1.1 ± 0.06 0.99 ± 0.01 1.1 ± 0.03 19 Dibutyl phthalate 0.79 2.6 70 ± 32 30 ± 17 51 ± 30 15 Diethyl phthalate 3.4 11 123 ± 88 13 ± 12 741 14 Bis(2-ethylhexyl)amine 0.04 0.15 0.06 ± 0.02 0.01 ± 0.007 n.d. 11 2-Ethylhexyl diphenyl phosphate 0.88 2.9 3.3 ± 2.3 3.5 ± 0.35 4.2 ± 2.7 10 MDMA 0.12 0.39 n.d. n.d. 1.3 ± 0.59 10 O-Desmethyl venlafaxine 0.15 0.51 n.d. n.d. 0.24 ± 0.13 10 Venlafaxine 0.09 0.31 n.d. n.d. 1.2 ± 0.61 10 Carbendazim 0.04 0.13 n.d. n.d. 0.25 ± 0.03 4 (4+5)- Methylbenzotriazole 0.10 0.33 n.d. n.d. 0.05 ± 0.05 3 1,8-Diazabicyclo [5.4.0]undec-7-ene 0.04 0.12 n.d. 0.15 ± 0.04 n.d. 3 n.d.: not detectable
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 69 6.2.2 PFAS The assessment of contaminant uptake and distribution in lettuce samples irrigated with different water types was performed using the analytical procedure detailed in section 4.7. Out of the entire set of PFAS analysed, only five compounds were detected and quantified in at least one sample (Table 31). Table 31. Overview of the PFAS compounds in lettuce samples irrigated with bottled water (BW), secondary effluent wastewater (WW), and reclaimed water (RW) (n=10) Average concentration (ng/g) Compounds LOD (ng/g) LOQ (ng/g) BW RW WW Frequency of detection (/10) PFBA 0.023 0.070 0.44 ± 0.11 1.16 ± 0.7 0.71 ± 0.21 50% BW 100% RW 50% WW PFOA 0.015 0.045 0.05 ± 0.01 0.09 ± 0.07 n.d. 30% BW 40% RW 0% WW PFHxS 0.013 0.039 0.09 ± 0.04 0.1 ± 0.04 0.85 ± 0.79 100% BW 100% RW 100% WW PFOS 0.025 0.074 0.18 ± 0.16 0.14 ± 0.07 7.21 ± 6.95 100% BW 100% RW 100% WW 6:2 FTS 0.043 0.130 0.25 ± 0.05 0.22 ± 0.09 0.22 ± 0.21 100% BW 100% RW 90% WW The results reveal notable differences between the PFAS levels reported in the irrigation waters used in the experiments (detailed in the section 5.5.4.) and the uptake profile observed in the lettuce samples. Only two acids and three sulfonates were detected in the samples. The sulfonates PFHxS, PFOS and 6:2 FTS were the most frequently identified, consistent with their higher log Kow values (2.98, 5.61, and 3.12, respectively), indicating greater potential for uptake and accumulation. The results demonstrated the use of reclaimed water from the combined EAOP + CW technology reduced the concentrations of PFOS and PFHxS in crop leaves. More specifically, the concentration of PFOS in lettuce leaves decreased from 7.21 to 0.14 ng/g when irrigated with WW instead of RW, while PFHxS levels dropped from 0.85 to 0.1 ng/g. Although most PFAS were detected in lettuce leaves irrigated with BW, concentration levels were very close to the method's LOQ, considering the standard deviation values reported in Table 31. Furthermore, the presence of PFAS may stem from other alternative sources, such as the soil used for growing the lettuce crops - a presumably pristine site where no agricultural or industrial activities have occurred in the past-, as previously discussed regarding the other iPM(T)s.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 70 6.3. Impact on crop yield and quality 6.3.1 Crop productivity and quality Figure 31a illustrates that crop productivity, measured by dry weight, was not significantly affected by the quality of the irrigation water. Other agricultural parameters followed a similar trend, with no notable impact on humidity (Figure 31b) or leaf height (Figure 31c). Figure 31. Agronomical parameters of lettuce crops irrigated with different water types (n=10) Regarding the lipid and carbohydrate content (Figure 31e and Figure 31f), the use of WW for irrigation increased lipid levels compared to crops irrigated with BW or RW, with carbohydrate content remaining unaffected. The increase in the plant lipid concentration is not negative outcome and even, it may indicate enhanced production of essential fatty acids or stress-related secondary metabolites. However, if driven by the oxidative stress, contamination, or the buildup of harmful substances, it raises concerns about potential adverse effects. To explore this in greater depth, a metabolomics analysis has been carried out. For chlorophyl content (Figure 31c), no significant differences were observed between treatments. However, the WW treatment resulted in higher chlorophyll levels compared to the other two. Although an increase in chlorophyll can enhance photosynthesis, it may also be induced by stress. Therefore, chlorophyll content should be considered alongside other agronomic parameters. In this case, both lipid content and chlorophyll levels seem to be impacted by the WW treatment. Considering all parameters together, the results suggest that compounds present in WW, such as iPM(T)s and PFAS, may have significantly contributed to the increase in lipid content. Reclaimed water had no notable effect on lettuce agronomics compared to control water, secondary treated wastewater influenced lipid levels. These results suggest that the developed EAOP + CW technology plays a role in mitigating the impact of wastewater constituents on lettuce.
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 71 6.3.2 Crop metabolomics For the metabolomics analysis, intermediate leaves from three lettuce plants per treatment were collected using an 8-mm leaf punch. These samples were immediately frozen in dry ice and transported to the laboratory within two hours, where they were stored at −80 °C until analysis. The metabolomic profiling focused on lettuce grown with the three different irrigation water treatments. Leaf extraction followed a modified version of previously established protocols. Ten mg of homogenized lettuce tissue were placed in an Eppendorf tube, to which 400 μL of methanol was added. The samples were vortexed, sonicated, and centrifuged using a methanol-chloroform-water mixture based on the methodology outlined by Hurtado et al. (2017). The resulting extracts were vacuum-dried, followed by derivatization with methoxyamine in pyridine (20,000 mg/L) and Nmethyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) containing 1 % trimethylchlorosilane (TMCS) according to Jorge et al. (2016). Triphenylamine was added to the samples before analysis. GC-Orbitrap analysis was performed using an instrument equipped with a Zebron ZB-5HT Inferno column (30 m × 0.25 mm × 0.25 μm). The oven temperature program started at 70 °C for 2 minutes, then increased to 100 °C at a rate of 7 °C/min, followed by a rise to 260 °C at 5 °C/min, and finally to 320 °C at 10 °C/min. Data from the GC–MS analysis were processed using Thermo Scientific's TraceFinder 5.1 software. Peak detection, spectral deconvolution, alignment, and library searches were carried out through the TraceFinder plug-in. A minimum total ion chromatogram (TIC) intensity threshold of 1 × 10³ and an ion overlap window of 98% were applied to ensure data accuracy. Feature peak areas of the samples were uploaded to the MetaboAnalyst 5.0 platform (http://www.metaboanalyst.ca) for further analysis. Data normalization was performed using the triphenylamine feature peak, followed by Pareto scaling. Metabolomics data were initially analyzed using univariate analysis (Kruskal-Wallis test) to identify potential significant differences in features. Subsequently, principal component analysis (PCA) was applied to detect distinct patterns and visualize variations in the data (Figure 32).
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 72 Figure 32. Score plot with samples normalized using the triphenylamine feature and data scaled via Pareto scaling. A PCA was then conducted. The shaded area represents the 95% confidence interval. None of the features showed a significant difference in univariate analysis (p-value < 0.05, nonparametric test) across the three treatment groups. Additionally, the PCA of the metabolome did not reveal any noticeable separation between lettuces irrigated with the different water types (Figure 32). The results show no significant metabolomic changes in the lettuce plants, even though varying contaminant concentrations were detected in the samples during this initial growth cycle. This limited impact of irrigation water quality could be attributed to the combined influence of the soil, the lettuce plants, and the diverse microbial communities inhabiting the root zone (rhizosphere). These microorganisms and their associated genes are known for their remarkable diversity and their crucial role in enhancing plant resilience to abiotic stresses caused by soil contaminants (Chaudhry et al., 2005). Additionally, the rhizosphere exhibits a superior capacity for pollutant breakdown compared to unplanted soil (Olson et al., 2003). 6.4. Environmental and health implications 6.4.1 iPM(T)s The ecotoxicological risk of the 38 iPM(T)s identified in influent and effluent water from the WWTP and the e-Peroxone effluent was evaluated through risk quotients (RQs). These RQs were determined by calculating the ratio between the maximum measured concentrations (MECs) and the predicted no-effect concentrations (PNECs) for freshwater, as published by the NORMAN network (https://www.norman-network.com/nds/ecotox/lowestPnecsIndex.php) (Table 32). Based on the established risk categories, considering reclaimed water, fourteen compounds fall under the “no risk” classification for aquatic organisms, with RQ values below 0.1. Fourteen compounds are categorized as “low risk”, exhibiting RQ values between 0.1 and 1. Six compounds
D4.3 – Recommendations on EAOP-CW based treatment system for water reuse 73 show a “moderate risk” potential, with RQ values ranging from 1 to 10, while four compounds are identified as “high risk” with RQ values exceeding 10. Compared to the influent (WW) of the WWTP, a decrease in the number of compounds categorised as “high risk” can be observed, due to a decrease in their concentration, 1) when applying the conventional treatment in the Montornès del Vallès WWTP (secondary effluent wastewater, WW) and 2) after the e-Peroxone (reclaimed water, RW). Table 32. Risk quotient (RQs) values for iPMT compounds detected in untreated wastewater (uWW), secondary effluent wastewater (WW) and reclaimed water (RW) uWW WW RW Compounds PNEC (ng/L) MECs (ng/L) RQ MECs (ng/L) RQ MECs (ng/L) RQ Galaxolidone 100 4.9E+03 49 4.2E+03 42 2.1E+03 21 MDMA 216 1.2E+05 551 1.1E+04 50 2.7E+03 12 O-Desmethyl venlafaxine 6 2.3E+03 370 1.6E+03 267 71 12 Venlafaxine 6 569 93 658 108 70 11 Ofloxacin 26 2.1E+03 80 720 28 223 8.6 Caffeine 100 2.4E+04 240 3.2E+03 32 705 7 Bis(2-ethylhexyl) amine 14 45 3.2 18 1.3 52 3.7 Tris(2-butoxyethyl) phosphate 1.4E+02 1.3E+03 9.0 584 4.2 479 3.4 Theophylline 1.0E+02 2.0E+04 196 1.2E+03 12 203 2.0 3,5-di-tert-Butyl-4hydroxybenzoic acid 1.1E+03 1.1E+03 1.0 574 0.5 1.3E+03 1.1 Flecainide 640 22 0.03 1.7E+03 2.6 536 0.8 (EDDP) / 2-ethyl-1,5dimethyl-3,3diphenylpyrrolinium 85 145 1.7 79 0.9 41 0.5 1,2,3-benzotriazole 7.8E+03 2.7E+03 0.4 4.8E+03 0.6 3.6E+03 0.5 Temazepam 71 345 4.9 42 0.6 32 0.4 2,4-Diaminotoluene 1.2E+04 5.1E+04 4.3 1.6E+04 1.3 5.2E+03 0.4 N,N'-Diphenylguanidine (DPG) 1.1E+03 237 0.2 555 0.5 448 0.4 Dibutyl phthalate 1.0E+04 1.6E+03 0.2 1.2E+03 0.1 4.1E+03 0.4 Diuron 70 28 0.4 26 0.4 22 0.3 Terbutryn 65 45 0.7 83 1.3 20 0.3 10,11-Dihydro-10,11dihydroxycarbamazepine 1.9E+03 2.1E+04 11.0 739 0.4 516 0.3 Carbendazim 150 32 0.2 133 0.9 39 0.3 (4+5)-Methylbenzotriazole 8.0E+03 1.4E+03 0.2 1.5E+03 0.2 869 0.1 Diethyl phthalate 1.6E+04 5.1E+03 0.3 6.3E+03 0.4 1.6E+03 0.1 2-Aminophenol 4.0E+03 2.8E+04 7.0 2.4E+03 0.6 263 0.1 Sulpiride 4.1E+03 407 0.1 887 0.2 114 0.03 Secbumeton 48 n.d. n.d. n.d. n.d. 1 0.02 Carbamazepine 2.0E+03 62 0.03 79 0.04 41 0.02
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