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Funding Information: This work was supported by the European Union's Horizon 2020 research and innovation program projects HBM4EU [grant number 733032 ]. Authors acknowledge the editorial assistance of Richard Davies. Publisher Copyright: © 2023 The Author(s) Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2′-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals. publishersversion published

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Publicly available chemical assessments of hydraulic fracturing related waters are generally based on shale gas practices in the U.S. There is a lack of information on hydraulic fracturing related gas development from EU countries and more generally on other types of extractions. This research fills this knowledge gap by presenting chemical and bioassay assessments of hydraulic fracturing related waters from a tight gas development in the Netherlands. Fracturing fluid, flowback water and groundwater from surrounding aquifers before and after the actual fracturing were analysed by means of high resolution liquid chromatography tandem mass spectrometry, the Ames test and three chemical activated luciferase gene expression bioassays aimed at determining genotoxicity, oxidative stress response and polyaromatic hydrocarbon contamination.After sample enrichment a higher number of peaks can be found in both fracturing fluid and flowback samples. No clear differences in chemical composition were shown in the groundwater samples before and after hydraulic fracturing. Preliminary environmental fate data of the tentatively identified chemicals points towards persistence in water. Clear genotoxic and oxidative stress responses were found in the fracturing fluid and flowback samples. A preliminary suspect screening resulted in 25 and 36 matches in positive and negative ionisation respectively with the 338 possible suspect candidates on the list.Extensive measures relating to the handling, transport and treatment of hydraulic fracturing related waters are currently in place within the Dutch context. The results of the present study provide a scientific justification for such measures taken to avoid adverse environmental and human health impacts.

The document provides an overview of the development of a research plan to undertake the analyses of effect biomarkers in connection with exposure to the first set of priority substances in some of the aligned studies. A subset of the aligned studies was also identified to undertake analyses for exposure biomarkers for the 2nd set of priority chemicals.

This report provides an update on the activities throughout the first 3 years of Task 8.1 in the HBM4EU project. The report provides a description of the sampling frame developed for the aligned studies including a definition of the sampling age groups to be included. The substances to be analysed within the age groups are also stated.

Arsenic (As) is a highly toxic element which naturally occurs in drinking water. In spite of substantial evidence on the association between many illnesses and chronic consumption of As, there is still a considerable uncertainty about the health risks due to low As concentrations in drinking water. In the Netherlands, drinking water companies aim to supply water with As concentration of

Toxicological risk assessment of contaminants of emerging concern (CEC) in (sources of) drinking water is required to identify potential health risks and prioritize chemicals for abatement or monitoring. In such assessments, concentrations of chemicals in drinking water or sources are compared to either (i) health-based (statutory) drinking water guideline values, (ii) provisional guideline values based on recent toxicity data in absence of drinking water guidelines, or (iii) generic drinking water target values in absence of toxicity data. Here, we performed a toxicological risk assessment for 163 CEC that were selected as relevant for drinking water. This relevance was based on their presence in drinking water and/or groundwater and surface water sources in downstream parts of the Rhine and Meuse, in combination with concentration levels and physicochemical properties. Statutory and provisional drinking water guideline values could be derived from publically available toxicological information for 142 of the CEC. Based on measured concentrations it was concluded that the majority of substances do not occur in concentrations which individually pose an appreciable human health risk. A health concern could however not be excluded for vinylchloride, trichloroethene, bromodichloromethane, aniline, phenol, 2-chlorobenzenamine, mevinphos, 1,4-dioxane, and nitrolotriacetic acid. For part of the selected substances, toxicological risk assessment for drinking water could not be performed since either toxicity data (hazard) or drinking water concentrations (exposure) were lacking. In absence of toxicity data, the Threshold of Toxicological Concern (TTC) approach can be applied for screening level risk assessment. The toxicological information on the selected substances was used to evaluate whether drinking water target values based on existing TTC levels are sufficiently protective for drinking water relevant CEC. Generic drinking water target levels of 37 μg/L for Cramer class I substances and 4 μg/L for Cramer class III substances in drinking water were derived based on these CEC. These levels are in line with previously reported generic drinking water target levels based on original TTC values and are shown to be protective for health effects of the majority of contaminants of emerging concern evaluated in the present study. Since the human health impact of many chemicals appearing in the water cycle has been studied insufficiently, generic drinking water target levels are useful for early warning and prioritization of CEC with unknown toxicity in drinking water and its sources for future monitoring. Keywords: Contaminants of emerging concern, Chemical water quality screening, Human health risk assessment, (Provisional) drinking water guideline values, Threshold of Toxicological Concern

Co-authors: Glória Isidro, Henriqueta Louro, Maria João Silva (INSA) The current deliverable describes the process of revision of the first list of indicators (published in June 2017 as D6.1.) and presents the indicator leaflets ((see attachment 1) that link key results of HBM4EU to the objectives of the project as laid down in the description of action. These indicator leaflets already contain a lot of results on the revised list of indicators, and give very valuable information on the progress of HBM4EU in relation to the specific goals of the project. Combining the information from these leaflets in the frame of expected impacts will allow us to put forward conclusions towards impact and sustainability of HBM4EU. As such, this restructured list answers to the main comments on the first list of indicators from the task 6.5 partners, the Management Board, the Governing Board and the EU Policy Board, in concretu to: • Drastically reduce the number of indicators from 48 indicators (including 9 internal indicators) on the first list to 28 indicators on the revised list without losing essential information. Moreover by bundling related indicators we now have 22 indicator leaflets (and 1 overview leaflet); • Make the relationship between the indicators and the goals of HBM4EU more clear by structuring the list of indicators according to the overarching objectives and specific goals; • Use the indicators to say something about the impact of the HBM4EU project: the indicator leaflets were used to give input for the impact section of the periodic technical reporting 2018 to describe the progress made for the 5 expected impacts of HBM4EU. This exercise will be continued and ameliorated in the 2019 periodic technical reporting as more indicator leaflets will be available compared to 2018; • Link the indicators with the work on sustainability of HBM in Europe: the indicators were presented at the sustainability workshop in Paris. Participants indicated that they think the leaflets will be useful for institutional discussions, national hub meetings, meetings with policy makers and other meetings and that they would like to use them as soon as they are available. The added value of having indicators of success, is to monitor the implementation and achieved impact of HBM4EU. This will allow for a more efficient tracking of achieved goals. This deliverable will help to further optimize and revise the first set of indicators to monitor the implementation of the HBM4EU and the achieved impact. The indicators of success are written in a clear language, they are concise and capture the main achievements in the list of indicators that has been agreed amongst the partners. Therefore, they can be easily used by all partners across the consortium, the EU Policy Board and our HBM4EU ambassador Thomas Jackl. HORIZON2020 Programme Contract No. 733032 HBM4EU info:eu-repo/semantics/publishedVersion

A plethora of in vitro bioassays is developed in the context of chemical risk assessment and clinical diagnostics to test effects on different biological processes. Such assays can also be implemented in effect-based monitoring (EBM) of (drinking) water quality in parallel to chemical analyses. EBM can provide insight in the risks for environmental and human health associated with exposure to (unknown) complex low-level mixtures of micropollutants, which fits in the risk-based approach that was recently introduced in the European Drinking Water Directive. Some challenges remain, in particular related to selection and interpretation of bioassays. For water quality assessment, carcinogenesis, adverse effects on reproduction and development, effects on xenobiotic metabolism, modulation of hormone systems, DNA reactivity and adaptive stress responses are considered as the most relevant toxicological endpoints. An evaluation procedure of the applicability and performance of in vitro bioassays for water quality monitoring based on existing information has been developed, which can be expanded with guidelines for experimental evaluations. Besides, a methodology for the interpretation of in vitro monitoring data is required, since the sensitivity of specific in vitro bioassays in combination with sample concentration may lead to responses of chemicals (far) below exposure concentrations that are relevant for human health effects. Different approaches are proposed to derive effect-based trigger values (EBTs), including EBTs based on 1) relative ecotoxicity potency, 2) health-based threshold values for chronic exposure in humans and kinetics of reference chemicals, and 3) read-across from (drinking) water guideline values. EBTs need to be chosen carefully in order to be sufficiently but not over-conservative to indicate potential health effects. Consensus on the crucial steps in the selection and interpretation of in vitro bioassay data will facilitate implementation, as well as legal embedding in the context of water quality monitoring of such assays in EBM strategies. This article is protected by copyright. All rights reserved.

Advancements in high-resolution mass spectrometry based methods have enabled a shift from pure target analysis to target, suspect and non-target screening analyses to detect chemicals in water samples. The multitude of suspect chemicals thereby detected needs to be prioritized for further identification, prior to health risk assessment and potential inclusion into monitoring programs. Here, we compare prioritization of chemicals in Dutch water samples based on relative intensities only to prioritization including hazard information based on high-throughput in vitro toxicity data. Over 1000 suspects detected in sewage treatment plant effluent, surface water, groundwater and drinking water samples were ranked based on their relative intensities. Toxicity data availability and density in the ToxCast database were determined and visualized for these suspects, also in regard to water relevant mechanisms of toxicity. More than 500 suspects could be ranked using occurrence/hazard ratios based on more than 1000 different assay endpoints. The comparison showed that different prioritization strategies resulted in significantly different ranking, with only 2 suspects prioritized based on occurrence among the top 20 in the hazard ranking. We therefore propose a novel scheme that integrates both exposure and hazard data, and efficiently prioritizes which features need to be confidently identified first.

The present study explores the ToxCast/Tox21 database to select candidate bioassays as bioanalytical tools for measuring groups of chemicals in water. To this aim, the ToxCast/Tox21 database was explored for bioassays that detect polycyclic aromatic hydrocarbons (PAHs), aromatic amines (AAs), (chloro)phenols ((C)Ps) and halogenated aliphatic hydrocarbons (HAliHs), which are included in the European and/or Dutch Drinking Water Directives. Based on the analysis of the availability and performance of bioassays included in the database, we concluded that several bioassays are suitable as bioanalytical tools for assessing the presence of PAHs and (C)Ps in drinking water sources. No bioassays were identified for AAs and HAliHs, due to the limited activity of these chemicals and/or the limited amount of data on these chemicals in the database. A series of bioassays was selected that measure molecular or cellular effects that are covered by bioassays currently in use for chemical water quality monitoring. Interestingly, also bioassays were selected that represent molecular or cellular effects that are not covered by bioassays currently applied. The usefulness of these newly identified bioassays as bioanalytical tools should be further evaluated in follow-up studies. Altogether, this study shows how exploration of the ToxCast/Tox21 database provides a series of candidate bioassays as bioanalytical tools for measuring groups of chemicals in water. This assessment can be performed for any group of chemicals of interest (if represented in the database), and may provide candidate bioassays that can be used to complement the currently applied bioassays for chemical water quality assessment.

Het doel van dit onderzoek was het beantwoorden van de vraag in hoeverre de kwaliteit van geloosd spuiwater met gewasbeschermingsmiddelen verbetert door het toepassen van (geavanceerde) oxidatietechnieken, en om zicht te krijgen op mogelijke negatieve bijwerkingen van deze technieken op het ecosysteem. Hiervoor zijn verschillende technieken vergeleken: Waterstofperoxide, Waterstofperoxide + Lage druk UV, Waterstofperoxide + middendruk UV, Waterstofperoxide + ozon, Ozon + lage druk UV, Waterstofperoxide + ozon + lage druk UV.