Battery of In Vitro Bioassays: A Case Study for the Cost-Effective and Effect-Based Evaluation of Wastewater Effluent Quality
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sampling
2.2. Effect-Based Monitoring Battery of Bioassays
3. Results and Discussion
3.1. Wastewater Monitoring with Battery of Bioassays
3.2. Risk Assessment Based on Effect-Based Trigger Values
3.3. Wider Application of the Battery in Wastewater Quality Control
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Troger, R.; Kohler, S.J.; Franke, V.; Bergstedt, O.; Wiberg, K. A case study of organic micropollutants in a major Swedish water source—Removal efficiency in seven drinking water treatment plants and influence of operational age of granulated active carbon filters. Sci. Total Environ. 2020, 706, 135680. [Google Scholar] [CrossRef] [PubMed]
- Gasparotti, C. The main factors of water pollution in Danube River basin. EuroEconomica 2014, 33, 91–106. [Google Scholar]
- Ng, K.; Alygizakis, N.; Androulakakis, A.; Galani, A.; Aalizadeh, R.; Thomaidis, N.S.; Slobodnik, J. Target and suspect screening of 4777 per- and polyfluoroalkyl substances (PFAS) in river water, wastewater, groundwater and biota samples in the Danube River Basin. J. Hazard. Mater. 2022, 436, 129276. [Google Scholar] [CrossRef] [PubMed]
- Skrbic, B.D.; Kadokami, K.; Antic, I.; Jovanovic, G. Micro-pollutants in sediment samples in the middle Danube region, Serbia: Occurrence and risk assessment. Environ. Sci. Pollut. Res. Int. 2018, 25, 260–273. [Google Scholar] [CrossRef]
- Alygizakis, N.A.; Samanipour, S.; Hollender, J.; Ibanez, M.; Kaserzon, S.; Kokkali, V.; van Leerdam, J.A.; Mueller, J.F.; Pijnappels, M.; Reid, M.J.; et al. Exploring the Potential of a Global Emerging Contaminant Early Warning Network through the Use of Retrospective Suspect Screening with High-Resolution Mass Spectrometry. Env. Sci. Technol. 2018, 52, 5135–5144. [Google Scholar] [CrossRef] [PubMed]
- Diamanti, K.S.; Alygizakis, N.A.; Nika, M.C.; Oswaldova, M.; Oswald, P.; Thomaidis, N.S.; Slobodnik, J. Assessment of the chemical pollution status of the Dniester River Basin by wide-scope target and suspect screening using mass spectrometric techniques. Anal. Bioanal. Chem. 2020, 412, 4893–4907. [Google Scholar] [CrossRef]
- Movalli, P.; Duke, G.; Ramello, G.; Dekker, R.; Vrezec, A.; Shore, R.F.; Garcia-Fernandez, A.; Wernham, C.; Krone, O.; Alygizakis, N.; et al. Progress on bringing together raptor collections in Europe for contaminant research and monitoring in relation to chemicals regulation. Environ. Sci. Pollut. Res. Int. 2019, 26, 20132–20136. [Google Scholar] [CrossRef]
- Brack, W.; Ait-Aissa, S.; Burgess, R.M.; Busch, W.; Creusot, N.; Di Paolo, C.; Escher, B.I.; Mark Hewitt, L.; Hilscherova, K.; Hollender, J.; et al. Effect-directed analysis supporting monitoring of aquatic environments--An in-depth overview. Sci. Total Environ. 2016, 544, 1073–1118. [Google Scholar] [CrossRef]
- Vasquez, M.I.; Lambrianides, A.; Schneider, M.; Kummerer, K.; Fatta-Kassinos, D. Environmental side effects of pharmaceutical cocktails: What we know and what we should know. J. Hazard. Mater. 2014, 279, 169–189. [Google Scholar] [CrossRef]
- Jarosova, B.; Ersekova, A.; Hilscherova, K.; Loos, R.; Gawlik, B.M.; Giesy, J.P.; Blaha, L. Europe-wide survey of estrogenicity in wastewater treatment plant effluents: The need for the effect-based monitoring. Environ. Sci. Pollut. Res. Int. 2014, 21, 10970–10982. [Google Scholar] [CrossRef]
- Joint NORMAN and Water Europe Position Paper: Contaminants of Emerging Concern in Urban Wastewater. 2019. Available online: https://www.normandata.eu/sites/default/files/files/Publications/Position%20paper_CECs%20UWW_NORMAN_WE_2019_Final_20190910_public.pdf (accessed on 14 November 2022).
- Hinnenkamp, V.; Balsaa, P.; Schmidt, T.C. Target, suspect and non-target screening analysis from wastewater treatment plant effluents to drinking water using collision cross section values as additional identification criterion. Anal. Bioanal. Chem. 2022, 414, 425–438. [Google Scholar] [CrossRef]
- Lopez, F.J.; Pitarch, E.; Botero-Coy, A.M.; Fabregat-Safont, D.; Ibanez, M.; Marin, J.M.; Peruga, A.; Ontanon, N.; Martinez-Morcillo, S.; Olalla, A.; et al. Removal efficiency for emerging contaminants in a WWTP from Madrid (Spain) after secondary and tertiary treatment and environmental impact on the Manzanares River. Sci. Total Environ. 2022, 812, 152567. [Google Scholar] [CrossRef]
- Liška, I.; Wagner, F.; Sengl, M.; Deutsch, K.; Slobodník, J. Joint Danube Survey 4: A Comprehensive Analysis of Danube Water Quality; ICPDR—International Commission for the Protection of the Danube River: Vienna, Austria, 2021; ISBN 978-3-200-07450-7. [Google Scholar]
- Letzel, T.; Bayer, A.; Schulz, W.; Heermann, A.; Lucke, T.; Greco, G.; Grosse, S.; Schussler, W.; Sengl, M.; Letzel, M. LC-MS screening techniques for wastewater analysis and analytical data handling strategies: Sartans and their transformation products as an example. Chemosphere 2015, 137, 198–206. [Google Scholar] [CrossRef]
- Liu, Q.; Zhao, Z.; Li, H.; Su, M.; Liang, S.X. Occurrence and removal of organic pollutants by a combined analysis using GC-MS with spectral analysis and acute toxicity. Ecotoxicol. Env. Saf. 2021, 207, 111237. [Google Scholar] [CrossRef]
- Mehinto, A.C.; Jayasinghe, B.S.; Vandervort, D.R.; Denslow, N.D.; Maruya, K.A. Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays. J. Vis. Exp. 2016, 118, 54725. [Google Scholar] [CrossRef]
- Lundqvist, J.; Mandava, G.; Lungu-Mitea, S.; Lai, F.Y.; Ahrens, L. In vitro bioanalytical evaluation of removal efficiency for bioactive chemicals in Swedish wastewater treatment plants. Sci. Rep. 2019, 9, 7166. [Google Scholar] [CrossRef] [PubMed]
- Coors, A.; Vollmar, P.; Sacher, F.; Polleichtner, C.; Hassold, E.; Gildemeister, D.; Kuhnen, U. Prospective environmental risk assessment of mixtures in wastewater treatment plant effluents—Theoretical considerations and experimental verification. Water Res. 2018, 140, 56–66. [Google Scholar] [CrossRef] [PubMed]
- Kienzler, A.; Bopp, S.K.; van der Linden, S.; Berggren, E.; Worth, A. Regulatory assessment of chemical mixtures: Requirements, current approaches and future perspectives. Regul. Toxicol. Pharm. 2016, 80, 321–334. [Google Scholar] [CrossRef] [PubMed]
- Alygizakis, N.A.; Besselink, H.; Paulus, G.K.; Oswald, P.; Hornstra, L.M.; Oswaldova, M.; Medema, G.; Thomaidis, N.S.; Behnisch, P.A.; Slobodnik, J. Characterization of wastewater effluents in the Danube River Basin with chemical screening, in vitro bioassays and antibiotic resistant genes analysis. Env. Int. 2019, 127, 420–429. [Google Scholar] [CrossRef]
- Brand, W.; de Jongh, C.M.; van der Linden, S.C.; Mennes, W.; Puijker, L.M.; van Leeuwen, C.J.; van Wezel, A.P.; Schriks, M.; Heringa, M.B. Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays. Env. Int. 2013, 55, 109–118. [Google Scholar] [CrossRef]
- Xu, J.; Wei, D.; Wang, F.; Bai, C.; Du, Y. Bioassay: A useful tool for evaluating reclaimed water safety. J Env. Sci. 2020, 88, 165–176. [Google Scholar] [CrossRef] [PubMed]
- Neale, P.A.; O’Brien, J.W.; Glauch, L.; Konig, M.; Krauss, M.; Mueller, J.F.; Tscharke, B.; Escher, B.I. Wastewater treatment efficacy evaluated with in vitro bioassays. Water Res. X 2020, 9, 100072. [Google Scholar] [CrossRef] [PubMed]
- Escher, B.I.; Asmall yi, U.-A.y.U.S.; Behnisch, P.A.; Brack, W.; Brion, F.; Brouwer, A.; Buchinger, S.; Crawford, S.E.; Du Pasquier, D.; Hamers, T.; et al. Effect-based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European Water Framework Directive. Sci. Total Environ. 2018, 628, 748–765. [Google Scholar] [CrossRef] [PubMed]
- Van der Oost, R.; Sileno, G.; Janse, T.; Nguyen, M.T.; Besselink, H.; Brouwer, A. SIMONI (Smart Integrated Monitoring) as a novel bioanalytical strategy for water quality assessment: Part II-field feasibility survey. Env. Toxicol. Chem. 2017, 36, 2400–2416. [Google Scholar] [CrossRef]
- De Baat, M.L.; Kraak, M.H.S.; Van der Oost, R.; De Voogt, P.; Verdonschot, P.F.M. Effect-based nationwide surface water quality assessment to identify ecotoxicological risks. Water Res. 2019, 159, 434–443. [Google Scholar] [CrossRef]
- Volker, J.; Stapf, M.; Miehe, U.; Wagner, M. Systematic Review of Toxicity Removal by Advanced Wastewater Treatment Technologies via Ozonation and Activated Carbon. Env. Sci. Technol. 2019, 53, 7215–7233. [Google Scholar] [CrossRef]
- Escher, B.I.; Neale, P.A. Effect-Based Trigger Values for Mixtures of Chemicals in Surface Water Detected with In Vitro Bioassays. Env. Toxicol. Chem. 2021, 40, 487–499. [Google Scholar] [CrossRef]
- Black, G.P.; He, G.; Denison, M.S.; Young, T.M. Using Estrogenic Activity and Nontargeted Chemical Analysis to Identify Contaminants in Sewage Sludge. Env. Sci. Technol. 2021, 55, 6729–6739. [Google Scholar] [CrossRef]
- U.S. Environmental Protection Agency. US EPA: Guidance on Cumulative Risk Assessment of Pesticide Chemicals That Have a Common Mechanism of Toxicity. 2002. Available online: https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/guidance-cumulative-risk-assessment-pesticide (accessed on 8 November 2022).
- Escher, B.I.; Lamoree, M.; Antignac, J.P.; Scholze, M.; Herzler, M.; Hamers, T.; Jensen, T.K.; Audebert, M.; Busquet, F.; Maier, D.; et al. Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project. Int. J. Env. Res. Public Health 2022, 19, 12990. [Google Scholar] [CrossRef]
- Konig, M.; Escher, B.I.; Neale, P.A.; Krauss, M.; Hilscherova, K.; Novak, J.; Teodorovic, I.; Schulze, T.; Seidensticker, S.; Kamal Hashmi, M.A.; et al. Impact of untreated wastewater on a major European river evaluated with a combination of in vitro bioassays and chemical analysis. Env. Pollut. 2017, 220, 1220–1230. [Google Scholar] [CrossRef]
- Neale, P.A.; Ait-Aissa, S.; Brack, W.; Creusot, N.; Denison, M.S.; Deutschmann, B.; Hilscherova, K.; Hollert, H.; Krauss, M.; Novak, J.; et al. Linking in Vitro Effects and Detected Organic Micropollutants in Surface Water Using Mixture-Toxicity Modeling. Env. Sci. Technol. 2015, 49, 14614–14624. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Alameddine, M.; How, Z.T.; Gamal El-Din, M. Advancing the treatment of primary influent and effluent wastewater during wet weather flow by single versus powdered activated carbon-catalyzed ozonation for the removal of trace organic compounds. Sci. Total. Environ. 2021, 770, 144679. [Google Scholar] [CrossRef] [PubMed]
- Enault, J.; Loret, J.-F.; Neale, P.; De Baat, M.; Escher, B.; Belhadj, F.; Kools, S.; Pronk, G.; Leusch, F. How effective are water treatment processes in removing toxic effects of micropollutants? A literature review of effect-based monitoring data. J. Water Health 2023, jwh2023235. [Google Scholar] [CrossRef]
- Phan, L.T.; Schaar, H.; Reif, D.; Weilguni, S.; Saracevic, E.; Krampe, J.; Behnisch, P.A.; Kreuzinger, N. Long-Term Toxicological Monitoring of a Multibarrier Advanced Wastewater Treatment Plant Comprising Ozonation and Granular Activated Carbon with In Vitro Bioassays. Water 2021, 13, 3245. [Google Scholar] [CrossRef]
- Brack, W.; Aissa, S.A.; Backhaus, T.; Dulio, V.; Escher, B.I.; Faust, M.; Hilscherova, K.; Hollender, J.; Hollert, H.; Müller, C.; et al. Effect-based methods are key. The European Collaborative Project SOLUTIONS recommends integrating effect-based methods for diagnosis and monitoring of water quality. Environ. Sci. Eur. 2019, 31, 10. [Google Scholar] [CrossRef]
- Palumbo, M.T.; Russo, S.; Polesello, S.; Guzzella, L.; Roscioli, C.; Marziali, L.; Valsecchi, L.; Cappelli, F.; Pascariello, S.; Tasselli, S.; et al. Integrated Exposure and Algal Ecotoxicological Assessments of Effluents from Secondary and Advanced-Tertiary Wastewater-Treatment Plants. Env. Toxicol. Chem. 2022, 41, 2404–2419. [Google Scholar] [CrossRef]
- Muller, M.E.; Escher, B.I.; Schwientek, M.; Werneburg, M.; Zarfl, C.; Zwiener, C. Combining in vitro reporter gene bioassays with chemical analysis to assess changes in the water quality along the Ammer River, Southwestern Germany. Env. Sci. Eur. 2018, 30, 20. [Google Scholar] [CrossRef]
- Qian, Y.; Wang, X.; Wu, G.; Wang, L.; Geng, J.; Yu, N.; Wei, S. Screening priority indicator pollutants in full-scale wastewater treatment plants by non-target analysis. J. Hazard. Mater. 2021, 414, 125490. [Google Scholar] [CrossRef]
- Houtman, C.J.; Ten Broek, R.; Brouwer, A. Steroid hormonal bioactivities, culprit natural and synthetic hormones and other emerging contaminants in waste water measured using bioassays and UPLC-tQ-MS. Sci. Total Environ. 2018, 630, 1492–1501. [Google Scholar] [CrossRef]
- Farré, M.; Barceló, D. Toxicity testing of wastewater and sewage sludge by biosensors, bioassays and chemical analysis. TrAC Trends Anal. Chem. 2003, 22, 299–310. [Google Scholar] [CrossRef]
- Ahkola, H.; Lindholm-Lehto, P.; Perkola, N.; Valitalo, P.; Merilainen, P.; Maenpaa, K.; Stelzer, J.A.A.; Heiskanen, I.; Jarvisto, J.; Nuutinen, J.; et al. A preliminary study on the ecotoxic potency of wastewater treatment plant sludge combining passive sampling and bioassays. Sci. Total Environ. 2021, 758, 143700. [Google Scholar] [CrossRef] [PubMed]
- Coppens, L.J.; van Gils, J.A.; Ter Laak, T.L.; Raterman, B.W.; van Wezel, A.P. Towards spatially smart abatement of human pharmaceuticals in surface waters: Defining impact of sewage treatment plants on susceptible functions. Water Res. 2015, 81, 356–365. [Google Scholar] [CrossRef] [PubMed]
- Kienle, C.; Werner, I.; Fischer, S.; Luthi, C.; Schifferli, A.; Besselink, H.; Langer, M.; McArdell, C.S.; Vermeirssen, E.L.M. Evaluation of a full-scale wastewater treatment plant with ozonation and different post-treatments using a broad range of in vitro and in vivo bioassays. Water Res. 2022, 212, 118084. [Google Scholar] [CrossRef] [PubMed]
Activity | LOD * | LOQ * | EBT Value |
---|---|---|---|
Estrogenic (ERα) | 0.02 ng 17ß-Estradiol-eq/L | 0.05 ng 17ß-Estradiol-eq/L | 0.1 ng 17ß-Estradiol-eq/L |
Antiandrogenic (anti-AR) | 1.90 μg Flutamide-eq/L | 5.76 μg Flutamide-eq/L | 14 μg Flutamide-eq/L |
Glucocorticoid (GR) | 0.02 ng Dexamethasone-eq/L | 0.05 ng Dexamethasone-eq/L | 100 ng Dexamethasone-eq/L |
PPARγ receptor (PPARγ) | 10.1 ng Rosiglitazone-eq/L | 31 ng Rosiglitazone-eq/L | 10 ng Rosiglitazone-eq/L |
PAH activity (PAH) | 0.32 ng B[a]P-eq/L | 0.96 ng B[a]P-eq/L | 6.2 ng B[a]P-eq/L |
Oxidative stress (Nrf2) | 1.45 μg Curcumine-eq/L | 4.4 μg Curcumine-eq/L | 10 μg Curcumine-eq/L |
Pregnane X receptor (PXR) | 0.74 μg Nicardipine-eq/L | 2.2 μg Nicardipine-eq/L | 3 μg Nicardipine-eq/L |
EoE | Mitigation Plans Proposed for WWTP Operators |
---|---|
Below 1 | -No further action |
Between 1 and 3 | -Perform quality check of data; -Monitor every 3 months for 1 year until EoE is below 1. |
Between 3 and 10 | -All actions of the category above; -Resample and reanalyze immediately to confirm exceedance of EBT; -Quantify toxicity drivers. |
Between 10 and 100 | -All actions of the category above; -Enhance program for source identification; -Monitor the distribution system closer to the point of exposure to confirm attenuation of CECs and to confirm the magnitude of assumed safety factors associated with removal efficiency, dilution, and post treatment. |
More than 100 | -All actions of the category above; -Consult the local environmental authorities immediately to determine the required response action; -Confirm plant corrective actions through additional monitoring to establish an EoE at least below 100. |
Effluent Wastewater Sampling Site | Anti-AR CALUX | ERα CALUX | GR CALUX | PPARγ CALUX | PAH CALUX | PXR CALUX | Nrf2 CALUX |
---|---|---|---|---|---|---|---|
Asten, AT | 2.6 | 26 | 1.5 | 0.5 | 115 | 10 | 9.5 |
Vratsa, BG | 3.0 | 58 | 0.5 | 0.5 | 71 | 0.5 | 0.5 |
Hodonín, CZ | 2.5 | 48 | 0.5 | 0.5 | 96 | 0.5 | 7.6 |
Donauwörth, DE | 3.6 | 30 | 5.0 | 1.0 | 32,292 | 44 | 3.7 |
Županja, HR | 0.5 | 30 | 0.5 | 0.5 | 92 | 27 | 4.4 |
Győr, HU | 1.5 | 17 | 0.5 | 1.3 | 90 | 24 | 2.0 |
Giurgiu, RO | 0.5 | 44 | 2.4 | 0.5 | 36 | 5.9 | 2.2 |
Šabac, RS | 1.3 | 32 | 0.5 | 0.5 | 76 | 3.6 | 3.0 |
Novo Mesto, SI | 0.5 | 5.0 | 0.5 | 0.5 | 58 | 4.4 | 1.9 |
Bratislava, SK | 0.5 | 12 | 0.5 | 0.5 | 177 | 4.7 | 2.5 |
Uzhgorod, UA | 1.3 | 38 | 0.5 | 0.5 | 104 | 5.0 | 2.0 |
Effluent Wastewater Sampling Site | PAH CALUX | ERα CALUX | Nrf2 CALUX | PXR CALUX | Anti-AR CALUX | PPARγ CALUX | GR CALUX |
---|---|---|---|---|---|---|---|
Asten, AT | 17.7 | 13.0 | 6.8 | <LOD | 1.6 | <LOD | 0.4 |
Vratsa, BG | 11.0 | 29.0 | <LOD | <LOD | 1.9 | <LOD | <LOD |
Hodonín, CZ | 14.8 | 24.0 | <LOD | 85.7 | 1.6 | <LOD | <LOD |
Donauwörth, DE | 5000 | 15.0 | 29.0 | 41.7 | 2.2 | 63.0 | 1.2 |
Županja, HR | 14.2 | 15.0 | 18.0 | 49.3 | <LOD | <LOD | <LOD |
Győr, HU | 13.9 | 8.5 | 16.0 | 23.0 | 0.9 | 82.0 | <LOD |
Giurgiu, RO | 5.6 | 22.0 | 3.9 | 25.0 | <LOD | <LOD | 0.6 |
Šabac, RS | 11.8 | 16.0 | 2.4 | 34.3 | 0.8 | <LOD | <LOD |
Novo Mesto, SI | 9.0 | 2.5 | 2.9 | 21.0 | <LOD | <LOD | <LOD |
Bratislava, SK | 27.4 | 6.2 | 3.1 | 28.7 | <LOD | <LOD | <LOD |
Uzhgorod, UA | 16.1 | 19.0 | 3.3 | 22.7 | 0.8 | <LOD | <LOD |
No. of samples with EoE > 1 | 11 | 11 | 9 | 9 | 4 | 2 | 1 |
Mean value | 467 | 15 | 8 | 30 | 1 | 13 | 0.2 |
Standard deviation | 1503 | 8 | 9 | 24 | 1 | 30 | NA |
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Alygizakis, N.; Ng, K.; Maragou, N.; Alirai, S.; Behnisch, P.; Besselink, H.; Oswald, P.; Čirka, Ľ.; Thomaidis, N.S.; Slobodnik, J. Battery of In Vitro Bioassays: A Case Study for the Cost-Effective and Effect-Based Evaluation of Wastewater Effluent Quality. Water 2023, 15, 619. https://doi.org/10.3390/w15040619
Alygizakis N, Ng K, Maragou N, Alirai S, Behnisch P, Besselink H, Oswald P, Čirka Ľ, Thomaidis NS, Slobodnik J. Battery of In Vitro Bioassays: A Case Study for the Cost-Effective and Effect-Based Evaluation of Wastewater Effluent Quality. Water. 2023; 15(4):619. https://doi.org/10.3390/w15040619
Chicago/Turabian StyleAlygizakis, Nikiforos, Kelsey Ng, Niki Maragou, Sylvana Alirai, Peter Behnisch, Harrie Besselink, Peter Oswald, Ľuboš Čirka, Nikolaos S. Thomaidis, and Jaroslav Slobodnik. 2023. "Battery of In Vitro Bioassays: A Case Study for the Cost-Effective and Effect-Based Evaluation of Wastewater Effluent Quality" Water 15, no. 4: 619. https://doi.org/10.3390/w15040619