High-throughput in vitro bioassays to assure water quality

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Bioassays complement instrumental analysis in water quality monitoring to ensure we do not overlook any components in the chemical cocktail. Beate Escher, Peta Neale and Frederic Leusch, authors of a new book from IWA Publishing, explain how advances in high-throughput screening are making their way into water quality assessment.


In vitro bioassays have a long tradition in water quality assessment. They replace animal testing carried out with algae, aquatic invertebrates and fish, and can also target human health-relevant endpoints (values indicating potential health effects) in the case of drinking water. More than one hundred studies have applied comprehensive batteries of tests that relied on in vitro and simple in vivo bioassays targeting general (apical) effects such as cytotoxicity, as well as diverse specific mechanisms, including effects on the hormone system, xenobiotic metabolism, reactive toxicity, and adaptive stress responses (see Figure 1).

Figure 1. Overview of studies published (1988-2020) that have applied test batteries of in vitro and in vivo assays indicative of at least three different endpoints and at least one cell-based assay to a range of water types. Figure reprinted from Bioanalytical tools in water quality assessment (Escher et al., 2021)

Water types that have been assessed range from contaminated sites and wastewater to treated waters and surface water, often looking at changes across treatment trains. These bioassays are so sensitive that effects can even be detected in drinking water with the correct sample preparation. Treatment efficacy can be assessed with bioanalytical tools, and water quality can also be compared with effect-based trigger values (EBT) to determine if the water is suitable for drinking or safe for aquatic organisms.

Many of the bioassays applied for water quality testing have initially been developed for chemical risk assessment and are inspired by mechanistic toxicological studies of how chemicals interact with living organisms to cause toxicity. The US National Research Council’s strategy to modernise toxicity testing with high-throughput pathway-based assays and the associated Tox21 and ToxCAST programmes, as well as the parallel implementation of an integrated testing strategy in the European Chemicals Regulation REACH, has greatly advanced our approach to chemical risk assessment.

“The field of water quality assessment has profited enormously from the progress made in chemical risk assessment”

The field of water quality assessment has profited enormously from the progress made in chemical risk assessment because a large number of high-throughput screening (HTS) in vitro bioassays have been developed and validated. This effort has also produced vast datasets of effect data for single chemicals, which have become publicly available via the CompTox Chemistry Dashboard (US EPA, 2021) and can serve not only to evaluate toxicity and conduct risk assessment, but also for interpreting water testing results, linking bioassay responses to chemical analysis, and even to predict mixture effects of chemicals detected in water samples.

The set-up of the Tox21 HTS bioassay profiling platform: NCATS = National Center for Advancing Translational Sciences, qHTS = quantative high-throughput screening. Figure based on Sakamuru et al. (2020), Profiling the Tox21 Chemical Library for Environmental Hazards: Applications in Prioritisation, Predictive Modelling, and Mechanism of Toxicity Characterisation. In Big Data in Predictive Toxicology, Editors Neagu and Richarz, pp. 242-263. © 2020, The Royal Society of Chemistry

The Tox21 HTS bioassay profiling platform includes carefully selected endpoints, which were optimised for HTS analysis on robotic systems, permitting rapid testing of almost 10,000 chemicals in more than 50 endpoints (see box). In addition, in the ToxCAST initiative, 1000 chemicals have been screened in approximately 1000 assay endpoints. The first generation of HTS assays were mainly receptor binding assays and assays making use of reporter genes, but presently ongoing efforts on developing other high-throughput approaches (using HT transcriptomics and HT phenotyping) also have potential for water quality applications.

“Ultra-HTS, more reliance on imaging techniques, and assay multiplexing would greatly increase our capacity to evaluate water samples”

The progress in applying HTS for risk assessment also paved the way for applications in environmental monitoring (Schroeder et al, 2016) and human biomonitoring (Vinggaard et al, 2021). In vitro assays have long been used for water quality monitoring, but the stronger scientific underpinning of the pathway-based approaches and risk assessment of chemicals will help their acceptance in regulation in the future.

In risk assessment, individual chemicals or very simple mixtures are evaluated, but in water quality monitoring we are faced with a complex cocktail of thousands, potentially hundreds of thousands, of chemicals. How chemicals interact in mixtures can be easily evaluated in HTS assays, especially when using advanced dosing devices, such as inkjet-type dispensers for preparing complex mixtures (Neale et al, 2020). We need an effective interplay of advanced instrumental analysis and in vitro bioassays to identify mixture risk drivers and evaluate water quality. Using the 96-well plate format traditionally used with manual pipetting allows approximately 10 to 20 water samples to be characterised in one experiment, but with 384-well plates and robotic support an experiment can include 100 to 200 samples. With such tools available, true monitoring programmes can be set up, as exemplified in a recent study where seven treatment wetlands, and one municipal wastewater treatment plant, were monitored over one year with five different bioassays, amounting to more than 3000 bioassay runs on a robotic 384-well plate platform (Sossalla et al, 2021).

Programmes such as ToxCast and Tox21 have deployed testing platforms that use 1536-well plates and low volume acoustic pipetting robots capable of accurately dispensing volumes as small as 2.5nL. These tools create incredible opportunities for ultra-HTS of environmental samples, but no one has tapped into that potential yet in water quality assessment. Multiplex assays, where multiple effects are monitored simultaneously, would also offer a way to increase testing throughput by creating assays that can measure multiple responses at once. Advances in imaging technology have also made it possible to read out more from existing bioassays: phenotypic profiling, which is already popular for fish embryo toxicity assays, can also be applied to cell-based assays. A study that applied multiparameter phenotypic profiling in a breast cancer cell line (MCF-7) showed how the size and structure of cells is related to biological processes such as cell growth, death, and communication, and applied these tools to testing environmental waters (Wang et al, 2018).

Ultra-HTS, more reliance on imaging techniques, and assay multiplexing would greatly increase our capacity to evaluate water samples – but they also require increasingly complex bioinformatics workflows and matching high throughput in sample preparation.

As of today, we have a broad toolbox of HTS bioassays accessible for many types of water quality monitoring applications (as well as other types of environmental samples, such as sediment, soil, air, biota and food), and cutting-edge research will continue to improve the range, depth and relevance of these in vitro tools. Importantly, HTS in vitro assays can help overcome some of our current regulatory pains, in particular the whack-a-mole game we are faced with in dealing with an increasingly diverse set of contaminants of emerging concern.

It is time for regulators to look into the lessons learned from more than a decade of extensive experience with this effect-based monitoring (EBM) approach and to overhaul our archaic chemical-by-chemical regulatory paradigm to ensure that water is fit-for-purpose and safe for human consumers and aquatic ecosystems.

Bioanalytical Tools in Water Quality Assessment, 2nd edition

Eds: Beate Escher, Peta Neale and Frederic Leusch

Applications for assessing water quality and new water treatment processes are the focus of the new edition of Bioanalytical Tools in Water Quality Assessment. It is aimed at interested parties from science, authorities and management who work in the water sector. After introducing the basics of environmental toxicology and water quality assessment, the authors demonstrate how to develop a bioassay test battery and discuss practical aspects associated with bioassay applications, such as quality control/quality assurance, sample preparation, and data reporting. The book is not only available in print form but also in an ‘open access’ electronic form and with supplementary (training) material on www.ufz.de/bioanalytical-tools


The authors

Beate Escher is internationally recognised for her work on chemical pollution in the environment. She pioneered the field of water quality assessment by addressing complex mixtures of chemical pollutants used in vitro bioassays. Escher is head of the Department of Cell Toxicology at the Helmholtz Centre for Environmental Research in Leipzig, Germany, and professor at the Eberhard Karls University Tübingen, Germany.

Peta Neale is a research fellow at Griffith University and the central theme of her research is to understand the fate and effect of emerging contaminants in the aquatic environment and engineered systems.

Frederic Leusch is a professor and Deputy Head (Research) in the School of Environment and Science at Griffith University, Australia, where he teaches biology and environmental toxicology. He also leads the Toxicology Research Group (ARI-TOX) at the Australian Rivers Institute on the Gold Coast, which focuses on assessing the impact of environmental contaminants on humans and aquatic ecosystems.

More information

Escher, B, et al (2021). Bioanalytical tools in water quality assessment, 2nd edition. IWA Publishing. www.iwapublishing.com/books/9781789061970/bioanalytical-tools-water-quality-assessment-2nd-edition

Neale, PA, et al (2020). Assessing the Mixture Effects in In Vitro Bioassays of Chemicals Occurring in Small Agricultural Streams during Rain Events. Environmental Science & Technology doi.org/10.1021/acs.est.0c02235

Schroeder, AL, et al (2016). Environmental surveillance and monitoring: The next frontiers for high-throughput toxicology. Environmental Toxicology and Chemistry doi.org/10.1002/etc.3309

Sossalla, NA, et al (2021). Removal of micropollutants and biological effects by conventional and intensified constructed wetlands treating municipal wastewater. Water Research doi.org/10.1016/ j.watres.2021.117349

US EPA (2021). CompTox Chemicals Dashboard. comptox.epa.gov/dashboard/ (accessed on 21 March 2021).

Vinggaard, AM, et al (2021). Receptor-based in vitro activities to assess human exposure to chemical mixtures and related health impacts. Environment International doi.org/10.1016/j.envint.2020.106191

Wang, W.L., at al (2018). Multiparameter Phenotypic Profiling in MCF-7 Cells for Assessing the Toxicity and Estrogenic Activity of Whole Environmental Water. Environmental Science & Technology doi.org/10.1021/ acs.est.8b01696