Progress, applications, and challenges in high-throughput effect-directed analysis for toxicity driver identification - is it time for HT-EDA?

The rapid increase in the production and global use of chemicals and their mixtures has raised concerns about their potential impact on human and environmental health. With advances in analytical techniques, in particular, high-resolution mass spectrometry (HRMS), thousands of compounds and transformation products with potential adverse effects can now be detected in environmental samples. However, identifying and prioritizing the toxicity drivers among these compounds remain a significant challenge.

Identification of antimicrobial and glucocorticoid compounds in wastewater effluents with effect-directed analysis.

Pharmaceuticals, such as glucocorticoids and antibiotics, are inadequately removed from wastewater and may cause unwanted toxic effects in the receiving environment. This study aimed to identify contaminants of emerging concern in wastewater effluent with antimicrobial or glucocorticoid activity by applying effect-directed analysis (EDA). Effluent samples from six wastewater treatment plants (WWTPs) in the Netherlands were collected and analyzed with unfractionated and fractionated bioassay testing.

Identifying antimicrobials and their metabolites in wastewater and surface water with effect-directed analysis.

This study aimed to identify antimicrobial contaminants in the aquatic environment with effect-directed analysis. Wastewater influent, effluent, and surface water (up- and downstream of the discharge location) were sampled at two study sites. The samples were enriched, subjected to high-resolution fractionation, and the resulting 80 fractions were tested in an antibiotics bioassay.

Characterisation of (anti-)progestogenic and (anti-)androgenic activities in surface and wastewater using high resolution effectdirected analysis.

The quality of surface waters is threatened by pollution with low concentrations of bioactive chemicals, among which those interfering with steroid hormone systems. Induced by reports of anti-progestogenic activity in surface waters, a two-year four-weekly survey of (anti-)progestogenic activity was performed at three surface water locations in the Netherlands that serve as abstraction points for the production of drinking water. As certain endogenous and synthetic progestogenic compounds are also potent (anti-)androgens, these activities were also investigated.

Development of a framework to derive effect-based trigger values to interpret CALUX data for drinking water quality.

Bioassays are increasingly being implemented for water quality monitoring as targeted chemical analyses are not always sufficient for the detection of all emerging chemicals or transformation products. However, the interpretation of bioassay results remains challenging, in particular because a positive response does not necessarily indicate that there may be an increased risk. For this purpose, effect-based trigger (EBT) values have been introduced as thresholds above which action needs to be undertaken to determine the cause of the response.

High resolution effect-directed analysis of steroid hormone (ant)agonists in surface and wastewater quality monitoring.

Monitoring of chemical water quality is extremely challenging due to the large variety of compounds and the presence of biologically active compounds with unknown chemical identity. Previously, we developed a high resolution Effect-Directed Analysis (EDA) platform that combines liquid chromatography with high resolution mass spectrometry and parallel bioassay detection. In this study, the platform is combined with CALUX bioassays for (anti)androgenic, estrogenic and glucocorticoid activities, and the performance of the platform is evaluated.

Identification of mutagenic and endocrine disrupting compounds in surface water and wastewater treatment plant effluents using high-resolution effect-directed analysis.

Effect-directed analysis (EDA) has shown its added value for the detection and identification of compounds with varying toxicological properties in water quality research. However, for routine toxicity assessment of multiple toxicological endpoints, current EDA is considered labor intensive and time consuming. To achieve faster EDA and identification, a high-throughput (HT) EDA platform, coupling a downscaled luminescent Ames and cell-based reporter gene assays with a high-resolution fraction collector and UPLC-QTOF MS, was developed.

Statistical analysis of a large set of semi-quantitative GC-MS screening data to evaluate and prioritize organic contaminants in surface and drinking water of the Netherlands.

Due to anthropogenic activities in the catchments, surface waters are contaminated with a large variety of chemical compounds. Drinking water companies in the Netherlands use surface water from the rivers Rhine, and Meuse, Lake IJssel and water from a reclaimed land area as sources for the production of drinking water. Samples from the abstraction points and the produced drinking waters were investigated using chemical screening with gas chromatography coupled to mass spectrometry to detect an as wide as possible range of organic contaminants, generating enormous data sets.

Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve.

We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1-30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator.

Steroid hormonal bioactivities, culprit natural and synthetic hormones and other emerging contaminants in waste water measured using bioassays and UPLC-tQ-MS.

Emission of compounds with biological activities from waste water treatment plant (WWTP) effluents into surface waters is a topic of concern for ecology and drinking water quality. We investigated the occurrence of hormone-like activities in waste water sample extracts from four Dutch WWTPs and pursued to identify compounds responsible for them. To this aim, in vitro reporter gene bioassays for androgenic, anti-androgenic, estrogenic, glucocorticoid and progestogenic activity and a UPLC-tQ-MS target analysis method for 25 steroid hormones used in high volumes in pharmacy were applied.

High-Throughput Effect-Directed Analysis Using Downscaled in Vitro Reporter Gene Assays To Identify Endocrine Disruptors in Surface Water.

Effect-directed analysis (EDA) is a commonly used approach for effect-based identification of endocrine disruptive chemicals in complex (environmental) mixtures. However, for routine toxicity assessment of, for example, water samples, current EDA approaches are considered time-consuming and laborious. We achieved faster EDA and identification by downscaling of sensitive cell-based hormone reporter gene assays and increasing fractionation resolution to allow testing of smaller fractions with reduced complexity.

Development of a luminescent mutagenicity test for high-throughput screening of aquatic samples.

The Salmonella reversion based Ames test is the most widely used method for mutagenicity testing. For rapid toxicity assessment of e.g.

Improved androgen specificity of AR-EcoScreen by CRISPR based glucocorticoid receptor knockout.

The AR-EcoScreen is a widely used reporter assay for the detection of androgens and anti-androgens. Endogenous expression of glucocorticoid receptors and their affinity for the androgen responsive element that drives reporter expression, however, makes the reporter cells sensitive to interference by glucocorticoids and less specific for (anti-)androgens. To create a glucocorticoid insensitive derivative of the AR-EcoScreen, CRISPR/Cas9 genome editing was used to develop glucocorticoid receptor knockout mutants by targeting various sites in the glucocorticoid gene.

Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry.

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was based on its high sensitivity and selectivity, which is required for environmental trace level detection.

Human health risk assessment of the mixture of pharmaceuticals in Dutch drinking water and its sources based on frequent monitoring data.

The presence of pharmaceuticals in drinking water is a topic of concern. Previous risk assessments indicate that their low concentrations are very unlikely to pose risks to human health, however often conclusions had to be based on small datasets and mixture effects were not included. The objectives of this study were to a) investigate if pharmaceuticals in surface and polder water penetrate in drinking water, b) assess the lifelong exposure of consumers to pharmaceuticals via drinking water and c) assess the possible individual and mixture health risks associated with this exposure.

A multicomponent snapshot of pharmaceuticals and pesticides in the river Meuse basin.

The river Meuse serves as a drinking-water source for more than 6 million people in France, Belgium, and The Netherlands. Pharmaceuticals and pesticides, both designed to be biologically active, are important classes of contaminants present in this river. The variation in the presence of pharmaceuticals in time and space in the Dutch part of the Meuse was studied using a multicomponent analytical method for pharmaceuticals combined with univariate and multivariate statistical analyses of the results.

Relating environmental concentrations of pharmaceuticals to consumption: A mass balance approach for the river Rhine.

In this study, pharmaceuticals were frequently monitored in the Rhine delta between the year 2002 and 2008. Average concentrations of several X-ray contrast mediums were above 0.1 microg/L, the average concentration of carbamazepine was about 0.

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays.

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action.

Sample preparation method for the ER-CALUX bioassay screening of (xeno-)estrogenic activity in sediment extracts.

The application of bioassays to assess the occurrence of estrogenic compounds in the environment is increasing in both a scientific and statutory context. The availability of appropriate validated methods for sample pre-treatment and analysis is crucial for the successful implementation of bioassays. Here, we present a sample preparation method for the bioassay screening of estrogenic activity in sediment with the in vitro Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER-CALUX) assay.

Biomonitoring of estrogenic exposure and identification of responsible compounds in bream from Dutch surface waters.

The exposure to and effects of estrogenic compounds in male breams from Dutch freshwater locations were investigated. Ovotestis was observed infrequently (maximum frequency 16%). However, plasma vitellogenin (VTG) concentration was elevated highly at some locations.

Estrogenic and dioxin-like compounds in sediment from Zierikzee harbour identified with CALUX assay-directed fractionation combined with one and two dimensional gas chromatography analyses.

The identity of compounds responsible for estrogenic and dioxin-like activities in sediment from the harbour of the small town Zierikzee in Zeeland, The Netherlands, was investigated using a bioassay directed fractionation approach with the in vitro estrogen and dioxin responsive reporter gene assays ER- and DR-CALUX. For identification of compounds exhibiting activity in the bioassays, either one or two-dimensional GC in combination with quadrupole (MSD), ion trap (ITD) or time-of-flight mass spectrometric detection (ToF-MS) was used, depending on the biological and chemical characteristics and the complexity of the fractions. The natural estrogenic hormone 17-beta-estradiol and its metabolite estrone were identified with GC-ITD as the main contributors to the estrogenic activity.