Scientific Publications

Under evolutionary pressure, the kinematic and energetic characteristics of animal locomotion have been optimized for survival. We investigated the kinematics and energetic performance of zebrafish eleutheroembryo escape swims triggered by electrical stimuli in fluids of increasing viscosity. Eleutheroembryos exhibited a decrease in both tail movement frequency and swimming velocity in more viscous environments, while the amplitude of body curvature remains constant. We then combined experimental imaging of freely swimming eleutheroembryos with Navier-Stokes numerical simulations. The results showed that the mechanical power output was initially maximal and remained essentially stable with increasing viscosity, while the cost of transport was linearly correlated with viscosity. Eleutheroembryos maximize neuromuscular power output during the fast-start escape response, enabling them to potentially escape predators under all circumstances in a natural environment. This model may be used to identify genetic and toxicological factors that reduce the mechanical power developed by the neuromuscular system or induce a loss of efficiency in its use.

MLinvitroTox is an automated Python pipeline developed for high-throughput hazard-driven prioritization of toxicologically relevant signals detected in complex environmental samples through high-resolution tandem mass spectrometry (HRMS/MS). MLinvitroTox is a machine learning (ML) framework comprising 490 independent XGBoost classifiers trained on molecular fingerprints from chemical structures and target-specific endpoints from the ToxCast/ Tox21 invitroDBv4.1 database. For each analyzed HRMS feature, MLinvitroTox generates a 490-bit bioactivity fingerprint used as a basis for prioritization, focusing the time-consuming molecular identification efforts on features most likely to cause adverse effects. The practical advantages of MLinvitroTox are demonstrated for groundwater HRMS data. Among the 874 features for which molecular fingerprints were derived from spectra, including 630 nontargets, 185 spectral matches, and 59 targets, around 4% of the feature/endpoint relationship pairs were predicted to be active. Cross-checking the predictions for targets and spectral matches with invitroDB data confirmed the bioactivity of 120 active and 6791 nonactive pairs while mislabeling 88 active and 56 non-active relationships. By filtering according to bioactivity probability, endpoint scores, and similarity to the training data, the number of potentially toxic features was reduced by at least one order of magnitude. This refinement makes the analytical confirmation of the toxicologically most relevant features feasible, offering significant benefits for cost-efficient chemical risk assessment.

Scientific Contribution:
In contrast to the classical ML-based approaches for toxicity prediction, MLinvitroTox predicts bioactivity for HRMS features (i.e., distinct m/z signals) based on MS2 fragmentation spectra rather than the chemical structures from the identified features. While the original proof of concept study was accompanied by the release of a MLinvitroTox v1 KNIME workflow, in this study, we release a Python MLinvitroTox v2 package, which, in addition to automation, expands functionality to include predicting toxicity from structures, cleaning up and generating chemical fingerprints, customizing models, and retraining on custom data. Furthermore, as a result of improvements in bioactivity data processing, realized in the concurrently released pytcpl Python package for the custom processing of invitroDBv4.1 input data used for training MLinvitroTox, the current release introduces enhancements in model accuracy, coverage of biological mechanistic targets, and overall interpretability.

Access to information about chemicals in products and articles is critical for supporting enforcement of chemical regulations, assessing risks from chemicals, allowing informed consumer choices, and enabling product circularity. In this work, we identified and evaluated available databases (DBs) on chemicals in products and articles from the literature using a defined protocol and from European national market surveillance authorities, nongovernmental agencies, and industrial sector groups using questionnaires. This is the first comprehensive review of DBs that provide information about chemicals in products and articles. A majority of these DBs are heterogeneous in terms of scope, ontologies, and data structures. Among the 57 identified DBs, 49 identified specific substances and only 30 reported their concentration in their products. In addition, 35 DBs included hazard information and 27 DBs provided safety information about products or chemicals. The analysis highlights the lack of comprehensive or accessible data on chemicals in products and articles for most categories of products/articles and jurisdictions. The limitations of existing DBs were attributed to scattered regulatory information requirements, a lack of data for unregulated substances, the complexity of supply chain communication, and confidentiality issues. In response to these challenges, we identified opportunities for improving existing information transfer structures and exploring alternative data sources to promote product and article safety and circularity.

Introduction
The European Partnership for the Assessment of Risks from Chemicals (PARC) is a 7-year multinational partnership aimed at consolidating and strengthening European Union’s (EU) research and innovation capacity for chemical risk assessment (RA) to protect human health and the environment. It consists of nine work packages (WP) involving more than 200 participating organisations from 29 countries. PARC is currently mapping the most relevant needs in the field of European chemical RA to steer PARC’s future activities in the coming years. The present study aims to gather the perspectives of WP/Task/Project Leaders of PARC to understand their experience during the first prioritisation round of PARC activities and to identify potential points of improvement for future rounds.

Methods
Three online 90-min focus group discussion (FGD) sessions were conducted between the 3rd and 9th of May 2023. Each session was attended by 4-5 participants with at least one representative from each PARC WPs 4, 5 and 6 (n = 13). The sessions were recorded and transcribed, then analysed in NVivo 12 software using thematic analysis.

Results
Some important aspects for the prioritisation of activities that were mentioned include: (1) having a transparent prioritisation process even though each WP might need different prioritisation criteria, (2) balancing the fulfilment of short-term regulatory needs and anticipating long-term needs in chemical RA, (3) maintaining alignment and synergy between the WPs and with other relevant EU initiatives to avoid duplication and to ensure continuity of work and (4) making sure that PARC can effectively respond to requests from different PARC stakeholders.

Conclusions
The next round of PARC research activity steering process will provide an opportunity to implement the various improvements identified. PARC should utilise the advantage of having stakeholders from different backgrounds (e.g., risk assessors, policymakers, regulatory bodies, academia, etc.) within its consortium and its advising bodies to prioritise projects and activities that will support its overall objectives. These recommendations could also be of interest outside PARC in the context of prioritising research and innovation needs related to chemical RA.

The acoustic startle response (ASR) is leaded by a sudden and intense acoustic stimulus. ASR has several forms of plasticity, including habituation and sensorimotor gating. Although ASR and its plasticity have been intensively studied in zebrafish (Danio rerio) larvae, information in adult zebrafish is still very scarce. In this manuscript we present Zebra_K, a new automated high-content kinematic analysis platform for assessing ASR, its habituation and prepulse inhibition (PPI), a quantitative measure of sensorimotor gating, in adult zebrafish. The analysis of the kinematic parameters of ASR in adult zebrafish has shown a single response wave consistent with the short-latency C-bend described in zebrafish larvae. Moreover, protocols have been designed and validated in Zebra_K for the analysis of sensitivity, habituation and PPI of this response. Then, the effect of the time of day and the gender on zebrafish ASR plasticity has been analyzed for the first time. Females exhibited higher responsiveness and a lower habituation and PPI than males, a result consistent with the gender effect described in other animal models and in humans. This platform has also been used to determine the effect of a pharmacological modulators of ASR plasticity, the NMDA-receptor antagonist ketamine. As described in other animal models, ketamine increased the responsiveness to the acoustic stimuli, decreasing habituation and leading to complete abolition of PPI. These results enhance the interest of using adult zebrafish to assess the potential effect of environmental pollutants on ASR plasticity.

The main objective of the ZebraCool programme was to create a positive attitude and curiosity towards science by bringing experimental activities within schools using an introductory cognitive and sensory approach. This innovative programme was offered at all levels of primary and secondary education including vocational high schools. Thematic workshops can be carried out on various themes such as comparative anatomy and embryology, molecular biology and evolution, or toxicology and endocrine disruptors. They were on an ad hoc basis or as part of an annual school project using zebrafish as a model. This animal was a very attractive entry point for the educator to motivate students to appreciate biology, in particular in the field of molecular biology and evolution. For each practical workshop, the student was an actor in his/her learning, which was intended to arouse the curiosity and desire to understand and learn. The programme was based on close collaboration between class teachers and programme educators to adapt workshops’ content to the school curriculum. Students conducted their own experiments, formulated and tested hypotheses, learned laboratory techniques, collected and analysed data. ZebraCool scientific activities fell within a conceptual framework of evolutionary biology through which participants perceived their own inner fish through the comparison of biological processes between humans and zebrafish.

Background
Asthma and allergic diseases are among the common causes of morbidity and mortality globally. Various environmental pollutants are linked to the development of asthma and allergic diseases. Evidence on the role of oxidative stress and immune markers in the association of environmental pollutants with asthma and allergy is scant. We examined cross-sectional associations between environmental pollutants and asthma and allergy, investigated mixture effects and possible mediation by oxidative stress or immune markers.

Methods
We used data from the Flemish Environment and Health Study 2016–2020 (FLEHS IV), including 409 adolescents aged 13–16 years. Fifty-four pollutants, including metals, phthalates, Di(isononyl) cyclohexane-1,2-dicarboxylate (DINCH), bisphenols, currently used and legacy pesticides, flame retardants, per- and polyfluoroalkyl substances (PFAS), polyaromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) were analyzed. Outcomes were self-reported asthma, rhinitis, eczema, allergies, respiratory infection, and airway inflammation, measured through fractional exhaled nitric oxide (FeNO). Single pollutant models using multiple regression analysis and multipollutant models using Bayesian Kernel Machine Regression (BKMR) were fitted. As sensitivity analysis, Bayesian model averaging (BMA) and elastic net (ENET) models were also performed. For Bayesian models, posterior inclusion probabilities (PIP) were used to identify the most important chemicals. Mediation analysis was performed to investigate the role of oxidative stress, measured by urinary 8-hydroxy-2' -deoxyguanosine (8-OHdG), and immune markers (eosinophils, basophils, InterLeukin 8, InterLeukin 6, and Interferon-ᵧ in blood).

Results
In single pollutant models, FeNO was significantly higher by 20% (95% CI: 6, 36%) and 13% (95% CI: 2, 25%) per interquartile range (IQR) fold in mono-n-butyl phthalate (MnBP) and mono-benzyl phthalate (MBzP), respectively. In BKMR analysis, the group PIPs indicated phthalates and DINCH as the most important group (group PIP = 0.509), with MnBP being the most important pollutant within that group (conditional PIP = 0.564; %change = 28%; 95%CI: 6, 54%). Similar patterns were observed in all multipollutant models. Eosinophil count mediated 37.8% (p = 0.018) and 27.9% (p = 0.045) of the association between MBzP and FeNO, and the association between MnBP and FeNO, respectively. 8-OHdG plays a significant mediating role in the association of 2,4-Dichlorophenoxyacetic acid (2,4-D) (55.4%), 3,5,6-Trichloro-2-pyridinol (TCPY) (48.1%), and 1-Naphthylamine (1-NAP) (32.7%) with rhinitis, while the total effects of these chemicals on rhinitis were not statistically significant.

Conclusions
This study found associations between phthalates, MnBP and MBzP, and elevated FeNO, which appeared to be mediated by eosinophil count. 8-OHdG plays a significant mediating role in the association between 2,4-D, TCPY, and 1-NAP with rhinitis, while their direct effects remain non-significant. Use of inflammatory and oxidative stress markers can enhance the understanding of inflammatory processes in asthma and allergic diseases due to environmental pollutants.

Context: In vitro toxicology studies are increasingly being included as evidence in systematic reviews and chemical risk assessments. INVITES-IN, a tool for assessing the internal validity of in vitro studies, is currently under development. The first step in developing INVITES-IN involves the creation of an “item bank”, an overview of study assessment concepts that may be relevant to evaluating the internal validity of in vitro toxicology studies. The item bank and methodology for its creation presented in this manuscript are intended to be a general resource for supporting the development of appraisal tools for in vitro toxicology studies and potentially other study designs.

Methods: We derived the item bank from seven literature sources (one existing item bank created from a systematic review of assessment criteria for in vitro studies, and six purposively sampled study appraisal tools) and the transcripts of three focus groups. Assessment criteria plausibly relating to internal validity were abstracted from the literature sources and focus group transcripts, disaggregated into individual criteria, then normalised to express in the simplest achievable language the core issue in each criterion – an “item bank” of assessment concepts. The items were then mapped onto a set of bias domains. We conducted simple descriptive statistical analyses and visualisations to describe patterns in the dataset and developed recommendations for the use and development of the item bank.

Results: The item bank contains 405 items of potential relevance to evaluating the internal validity of in vitro toxicology studies.

Discussion: To our knowledge, this is the second item bank of any kind to have been created for toxicology studies, and the first to use focus groups as a data source alongside literature analysis. The large number of items contributed by focus group discussions suggests this is an efficient method for capturing internal validity issues that are not easily identifiable in the literature. We believe our item bank and methodology for its creation will be a useful resource for supporting the development of appraisal tools. Due to the broad applicability of many items in the item bank, it may be informative for study designs beyond the in vitro domain.

Bisphenol A (BPA), a synthetic chemical widely used in the production of polycarbonate plastic and epoxy resins, has been associated with a variety of adverse effects in humans including metabolic, immunological, reproductive, and neurodevelopmental effects, raising concern about its health impact. In the EU, it has been classified as toxic to reproduction and as an endocrine disruptor and was thus included in the candidate list of substances of very high concern (SVHC). On this basis, its use has 
been banned or restricted in some products. As a consequence, industries turned to bisphenol alternatives, such as bisphenol S (BPS) and bisphenol F (BPF), which are now found in various consumer products, as well as in human matrices at a global scale. However, due to their toxicity, these two bisphenols are in the process of being regulated. Other BPA alternatives, whose potential toxicity remains largely unknown due to a knowledge gap, have also started to be used in manufacturing processes. The gradual restriction of the use of BPA underscores the importance of understanding the potential risks associated with its alternatives to avoid regrettable substitutions. This review aims to summarize the current knowledge on the potential hazards related to BPA alternatives prioritized by European Regulatory Agencies based on their regulatory relevance and selected to be studied under the European Partnership for the Assessment of Risks from Chemicals (PARC): BPE, BPAP, BPP, BPZ, BPS-MAE, and TCBPA. The focus is on data related to toxicokinetic, endocrine disruption, immunotoxicity, developmental neurotoxicity, and genotoxicity/carcinogenicity, which were considered the most relevant endpoints to assess the hazard related to those substances. The goal here is to identify the data gaps in BPA alternatives toxicology and hence formulate the future directions that will be taken in the frame of the PARC project, which seeks also to enhance chemical risk assessment methodologies using new approach methodologies (NAMs).

Background

With the high influx of low-cost plastic toys on the market, there is growing concern about the safety of such toys. Some of these plastic toys contains hazardous chemicals like polybrominated diphenyl ethers (PBDEs) due to the use of recycled plastics in new toy manufacturing. Here, we investigated if toys marketed in Europe are compliant with EU directives to assess the safety of currently used children's toys and identify implications of PBDE content in toys.

Results

Eighty-four toys purchased from international toy retailers were screened for bromine using X-ray fluorescence (XRF), and 11 of those with bromine content higher than 500 µg/g were analyzed for ten PBDEs using GC–HRMS. PBDEs were detected in all 11 toys. Ʃ₁₀PBDE concentrations ranged up to 23.5 mg/g (with a median concentration of 8.61 mg/g), with BDE-209 being the most abundant compound (4.40 mg/g). Eight samples exceeded the EU’s Low POP Content Limit (LPCL) of 500 µg/g for the Ʃ₁₀PBDEs by 6–47 times and the Unintentional Trace Contaminant (UTC) limits of 10 µg/g for Deca-BDE by 12–800 times.

Conclusions

PBDEs were up to percent levels, suggesting direct recycling of flame retarded plastic, e.g., e-waste plastics, into toy components. This is a call for concern and requires intervention from all stakeholders involved in the toy market. Overall, the occurrence of non-compliant toys in the EU market, as indicated in this study is primarily attributed to gaps in regulations, inadequate legislation for recycled plastics, the rise of online sales, complexities in global and national supply chains, and economic challenges. Failure to address these issues will hinder the efforts of the plastics industry to transition into a circular economy. This suggests that more actions are needed to address gaps in cross-border enforcement, and stricter sanctions are required for toy manufacturers who fail to adhere to regulations and safety standards.

Innovative tools suitable for chemical risk assessment are being developed in numerous domains, such as non-target chemical analysis, omics, and computational approaches. These methods will also be critical components in an efficient early warning system (EWS) for the identification of potentially hazardous chemicals. Much knowledge is missing for current use chemicals and thus computational methodologies complemented with fast screening techniques will be critical. This paper reviews current computational tools, emphasizing those that are accessible and suitable for the screening of new and emerging risk chemicals (NERCs). The initial step in a computational EWS is an automatic and systematic search for NERCs in literature and database sources including grey literature, patents, experimental data, and various inventories. This step aims at reaching curated molecular structure data along with existing exposure and hazard data. Next, a parallel assessment of exposure and effects will be performed, which will input information into the weighting of an overall hazard score and, finally, the identification of a potential NERC. Several challenges are identified and discussed, such as the integration and scoring of several types of hazard data, ranging from chemical fate and distribution to subtle impacts in specific species and tissues. To conclude, there are many computational systems, and these can be used as a basis for an integrated computational EWS workflow that identifies NERCs automatically.

While pesticide use is subject to strict regulatory oversight worldwide, it remains a main concern for environmental protection, including biodiversity conservation. This is partly due to the current regulatory approach that relies on separate assessments for each single pesticide, crop use, and non-target organism group at local scales. Such assessments tend to overlook the combined effects of overall pesticide usage at larger spatial scales. Integrative landscape-based approaches are emerging, enabling the consideration of agricultural management, the environmental characteristics, and the combined effects of pesticides applied in a same or in different crops within an area. These developments offer the opportunity to deliver informative risk predictions relevant for different decision contexts including their connection to larger spatial scales and to combine environmental risks of pesticides, with those from other environmental stressors. We discuss the needs, challenges, opportunities and available tools for implementing landscape-based approaches for prospective and retrospective pesticide Environmental Risk Assessments (ERA). A set of “building blocks” that emerged from the discussions have been integrated into a conceptual framework. The framework includes elements to facilitate its implementation, in particular: flexibility to address the needs of relevant users and stakeholders; means to address the inherent complexity of environmental systems; connections to make use of and integrate data derived from monitoring programs; and options for validation and approaches to facilitate future use in a regulatory context. The conceptual model can be applied to existing ERA methodologies, facilitating its comparability, and highlighting interoperability drivers at landscape level. The benefits of landscape-based pesticide ERA extend beyond regulation. Linking and validating risk predictions with relevant environmental impacts under a solid science-based approach will support the setting of protection goals and the formulation of sustainable agricultural strategies. Moreover, landscape ERA offers a communication tool on realistic pesticide impacts in a multistressors environment for stakeholders and citizens.

Based on toxicological evidence, children’s exposure to phthalates may contribute to altered neurodevelopment and abnormal regulation of brain-derived neurotrophic factor (BDNF). We analyzed data from five aligned studies of the Human Biomonitoring for Europe (HBM4EU) project. Ten phthalate metabolites and protein BDNF levels were measured in the urine samples of 1148 children aged 6–12 years from Italy (NACII-IT cohort), Slovakia (PCB-SK cohort), Hungary (InAirQ-HU cohort) and Norway (NEBII-NO). Serum BDNF was also available in 124 Slovenian children (CRP-SLO cohort). Children’s total, externalizing and internalizing behavioral problems were assessed using the Child Behavior Checklist at 7 years of age (only available in the NACII-IT cohort). Adjusted linear and negative binomial regression models were fitted, together with weighted quantile sum (WQS) regression models to assess phthalate mixture associations. Results showed that, in boys but not girls of the NACII-IT cohort, each natural-log-unit increase in mono-n-butyl phthalate (MnBP) and Mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) was cross-sectionally associated with higher externalizing problems [incidence rate ratio (IRR): 1.20; 95% CI: 1.02, 1.42 and 1.26; 95% CI: 1.03, 1.55, respectively]. A suggestive mixture association with externalizing problems was also observed per each tertile mixture increase in the whole population (WQS—IRR = 1.15; 95% CI: 0.97, 1.36) and boys (IRR = 1.20; 95% CI: 0.96, 1.49). In NACII-IT, PCB-SK, InAirQ-HU and NEBII-NO cohorts together, urinary phthalate metabolites were strongly associated with higher urinary BDNF levels, with WQS regression confirming a mixture association in the whole population (percent change (PC) = 25.9%; 95% CI: 17.6, 34.7), in girls (PC = 18.6%; 95% CI: 7.92, 30.5) and mainly among boys (PC = 36.0%; 95% CI: 24.3, 48.9). Among CRP-SLO boys, each natural-log-unit increase in ∑DINCH concentration was associated with lower serum BDNF levels (PC: −8.8%; 95% CI: −16.7, −0.3). In the NACII-IT cohort, each natural-log-unit increase in urinary BDNF levels predicted worse internalizing scores among all children (IRR: 1.15; 95% CI: 1.00, 1.32). Results suggest that (1) children’s exposure to di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) metabolites is associated with more externalizing problems in boys, (2) higher exposure to DINCH may associate with lower systemic BDNF levels in boys, (3) higher phthalate exposure is associated with higher urinary BDNF concentrations (although caution is needed since the possibility of a “urine concentration bias” that could also explain these associations in noncausal terms was identified) and (4) higher urinary BDNF concentrations may predict internalizing problems. Given this is the first study to examine the relationship between phthalate metabolite exposure and BDNF biomarkers, future studies are needed to validate the observed associations.

Harmful algal blooms and the toxins produced during these events are a human and environmental health concern worldwide. Saxitoxin and its derivatives are potent natural aquatic neurotoxins produced by certain freshwater cyanobacteria and marine algae species during these bloom events. Saxitoxins effects on human health are well studied, however its effects on aquatic biota are still largely unexplored. This work aims at evaluating the effects of a pulse acute exposure (24 h) of the model cladoceran Daphnia magna to 30 μg saxitoxin L⁻¹, which corresponds to the safety guideline established by the World Health Organization (WHO) for these toxins in recreational freshwaters. Saxitoxin effects were assessed through a comprehensive array of biochemical (antioxidant enzymes activity and lipid peroxidation), genotoxicity (alkaline comet assay), neurotoxicity (total cholinesterases activity), behavioral (swimming patterns), physiological (feeding rate and heart rate), and epigenetic (total 5-mC DNA methylation) biomarkers. Exposure resulted in decreased feeding rate, heart rate, total cholinesterases activity and catalase activity. Contrarily, other antioxidant enzymes, namely glutathione-S-transferases and selenium-dependent Glutathione peroxidase had their activity increased, together with lipid peroxidation levels. The enhancement of the antioxidant enzymes was not sufficient to prevent oxidative damage, as underpinned by lipid peroxidation enhancement. Accordingly, average DNA damage level was significantly increased in STX-exposed daphnids. Total DNA 5-mC level was significantly decreased in exposed organisms. Results showed that even a short-term exposure to saxitoxin causes significant effects on critical molecular and cellular pathways and modulates swimming patterns in D. magna individuals. This study highlights sub-lethal effects caused by saxitoxin in D. magna, suggesting that these toxins may represent a marked challenge to their thriving even at a concentration deemed safe for humans by the WHO.

Existing regulatory frameworks often prove inadequate in identifying contaminants of emerging concern (CECs) and determining their impacts on biological systems at an early stage. The establishment of Early Warning Systems (EWSs) for CECs is becoming increasingly relevant for policy-making, aiming to proactively detect chemical hazards and implement effective mitigation measures. Effect-based methodologies, including bioassays and effect-directed analysis (EDA), offer valuable input to EWSs with a view to pinpointing the relevant toxicity drivers and prioritizing the associated risks. This review evaluates the analytical techniques currently available to assess biological effects, and provides a structured plan for their systematic integration into an EWS for hazardous chemicals in the environment. Key scientific advancements in effect-based approaches and EDA are discussed, underscoring their potential for early detection and management of chemical hazards. Additionally, critical challenges such as data integration and regulatory alignment are addressed, emphasizing the need for continuous improvement of the EWS and the incorporation of analytical advancements to safeguard environmental and public health from emerging chemical threats.

Our study provides the most comprehensive dataset for high-precision radiogenic isotopes of lead (Pb) in blood for the western European population. It investigates their potential for elucidating the contribution of soil Pb to blood Pb using a human biomonitoring survey involving 81 adults and 4 children living in the urban area of Liège (Belgium). Soils in the area show moderate (median of 360 mg/kg) to high (95th percentile of 1000 mg/kg) Pb concentrations, due to former metal processing activities. Blood lead levels (BLL) measured in the study population are, on average, quantitatively consistent with a ∼ 20 % increase due to the exposure to Pb from soils, as estimated by a single-compartment biokinetic model. Consistently, its isotopic composition does not represent an endmember that fully accounts for the variability of Blood lead isotope (BLI) compositions measured in the study population. While some individuals show more thorogenic BLI ratios (relatively more enriched in ²⁰⁸Pb), which could be consistent with a greater exposure to local soils and/or by their country of birth, the BLI data mostly follow a trend roughly parallel to the European Standard Lead Pollution (ESLP) line, within the European leaded gasoline field, even two decades after the withdrawal of this source. Differences in BLI are probably associated with factors related to the presence of Pb in dwellings (pipes, paint) and drinking water distribution system, suggesting that the anthropogenic Pb in use, relevant to human exposure, may contain ore components of different origins, including the Australian Pb ore signature.

The focus on implementation of systematic review (SR) principles in chemical risk assessments (CRAs) is growing as it has the potential to advance the rigour and transparency of the CRAs. However, the SR and CRA communities use their own specific terminologies. Understanding the meaning of core SR and CRA terms and where they overlap is critical for application of SR methods and principles in CRAs. Moreover, it will increase the possibility for cross-sectorial collaboration, avoid misunderstandings, and improve communication among risk assessors, researchers, and policy makers.

We present a process for the cross-mapping of core CRA terms and core SR terms. Core terms for study appraisal, evidence synthesis and integration used in the SR and CRA communities will be included. The outcome will be an overview of how core SR terms map onto core CRA terms and vice versa, and a description of the relationship and conceptual overlap between the terms.

The cross-mapping is divided in three phases, where in the first phase the core SR and CRA terms will be identified. In the second phase, existing SR and CRA definitions will be mapped. In the third phase, descriptions of the relationship and conceptual overlap between the terms will be derived. The third phase will include weekly one-hour online meetings for SR and CRA experts.

We investigated the level of protection of reproductive and developmental toxicity offered through occupational exposure limits (OELs) and Derived No-Effect Levels for workers’ inhalation exposure (wDNELs). We compared coverage of substances that have a harmonised classification as reproductive toxicant 1 A or 1B (Repr.1 A/B), numerical values and scientific basis of 12 lists of OELs and wDNELs from REACH Registrants’ and the Committee for Risk Assessment. Across the 14 sources of OELs and wDNELs, 53 % of the Repr1A/B-substances had at least one exposure limit (counting groups of metals as one entry). Registrants’ wDNELs covered the largest share, 40 %. The numerical values could be highly variable for the same substance across the lists. How often reproductive toxicity is identified as the critical effect varies between the examined lists, both due to different assessments of the same substance and different substance coverage. Reviewing the margin of safety to reproductive toxicity cited in the documents, we found that 15 % of safety margins were lower to reproductive toxicity than the critical effect. To conclude, neither the REACH nor work environment legislation supply wDNELs or OELs for a substantial share of known reproductive toxicants. EU OELs cover among the fewest substances in the range, and in many cases national OELs or wDNELs are set at more conservative levels.

Increased wildfire activity increases the demands onfire rescue services and firefighters’ contact with harmful chemicals.This study aimed to determine firefighters’ exposure to toxicmetal(loid)s and its association with the lipid profile. CELSPAC-FIREexpo study participants (including 110 firefighters) providedurine and blood samples to quantify urinary levels of metal(loid)s (arsenic, cadmium (Cd), mercury and lead (Pb)) and serum lipidbiomarkers (cholesterol (CHOL), low-density lipoprotein choles-terol (LDL), high-density lipoprotein cholesterol (HDL), and triglycerides (TG)). The associations were investigated by usingmultiple linear regression and Bayesian weighted quantile sum(BWQS) regression. Higher levels of Pb were observed infirefighters. Pb was positively associated with CHOL and TG.Cd was negatively associated with HDL. In the BWQS model, the mixture of metal(loid)s was associated positively with CHOL (β =14.75, 95% CrI = 2.45−29.08), LDL (β = 15.14, 95% CrI = 3.39−29.35) and TG (β = 14.79, 95% CrI = 0.73−30.42), whilenegatively with HDL (β = −14.96, 95% CrI = −25.78 to −1.8). Pb emerged as a key component in a metal(loid) mixture. Theresults suggest that higher exposure to lead and the mixture of metal(loid)s is associated with the alteration of the lipid profile, whichcan result in an unfavorable cardiometabolic profile, especially in occupationally exposed firefighters.

A subgroup of endocrine-disrupting chemicals have the ability to disrupt metabolism. These metabolism-disrupting chemicals (MDCs) can end up in aquatic environments and lead to adverse outcomes in fish. Although molecular and physiological effects of MDCs have been studied in adult fish, few studies have investigated the consequences of metabolic disruption in fish during the earliest life stages. To investigate the processes affected by metabolic disruption, zebrafish embryos were exposed to peroxisome proliferator–activated receptor gamma (PPARγ) agonist rosiglitazone, the PPARγ antagonist T0070907, and the well-known environmentally relevant MDC bisphenol A. Decreased apolipoprotein Ea transcript levels indicated disrupted lipid transport, which was likely related to the observed dose-dependent increases in yolk size across all compounds. Increased yolk size and decreased swimming activity indicate decreased energy usage, which could lead to adverse outcomes because the availability of energy reserves is essential for embryo survival and growth. Exposure to T0070907 resulted in a darkened yolk. This was likely related to reduced transcript levels of genes involved in lipid transport and fatty acid oxidation, a combination of responses that was specific to exposure to this compound, possibly leading to lipid accumulation and cell death in the yolk. Paraoxonase 1 (Pon1) transcript levels were increased by rosiglitazone and T0070907, but this was not reflected in PON1 enzyme activities. The present study shows how exposure to MDCs can influence biochemical and molecular processes involved in early lipid metabolism and may lead to adverse outcomes in the earliest life stages of fish.

Pharmaceuticals and their human metabolites are contaminants of emerging concern in the aquatic environment. Most monitoring studies focus on a limited set of parent compounds and even fewer metabolites. However, more than 50% of the most consumed pharmaceuticals are excreted in higher amounts as metabolites than as parents, as confirmed by a literature analysis within this study. Hence, we applied a wide-scope suspect screening approach to identify human pharmaceutical metabolites in wastewater influent from three Swiss treatment plants. Based on consumption amounts and human metabolism data, a suspect list comprising 268 parent compounds and over 1500 metabolites was compiled. Online solid phase extraction combined with liquid chromatography coupled to high-resolution tandem mass spectrometry was used to analyze the samples. Data processing, annotation, and structure elucidation were achieved with various tools, including molecular networking as well as SIRIUS/CSI:FingerID and MetFrag for MS2 spectra rationalization. We confirmed 37 metabolites with reference standards and 16 by human liver S9 incubation experiments. More than 25 metabolites were detected for the first time in influent wastewater. Semiquantification with MS2Quant showed that metabolite to parent concentration ratios were generally lower compared to literature expectations, probably due to further metabolite transformation in the sewer system or limitations in the metabolite detection. Nonetheless, metabolites pose a large fraction to the total pharmaceutical contribution in wastewater, highlighting the need for metabolite inclusion in chemical risk assessment.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity ↗ has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.

There is indisputable evidence that the environment, humans and wildlife are continuously exposed not to single but to multiple chemicals from diferent sources. Exposure to these mixtures can lead to combined risks not yet suffciently addressed in any of the European chemical legislations. Under the REACH regulation for industrial chemicals, specifc environmental mixture assessments are challenged by a lack of data on toxicity, use and exposures and the communication of data along the supply chain. Within the Chemicals Strategy for Sustainability the European Commission proposed to introduce (a) mixture allocation factor(s) (MAF) as regulatory management tool to reduce exposures, efects and potential risks of unintentional mixtures. The MAF is proposed to be applied as default value within the chemical safety assessments undertaken by companies under REACH. Here, we critically review the relevant literature discussing the conceptual background of the MAF and approaches to derive its magnitude.
The analysis focuses on the environment and key issues for an implementation in regulatory practise together with remaining uncertainties and needs for possible ways forward. At this stage introducing a MAF in REACH Annex I appears the most pragmatic and immediately implementable measure to address risks from unintentional mixtures in the environment. A so-called MAFceiling appears as the preferred option of policy makers, since it would only afect relevant substances close to their respective risk threshold. While the magnitude of a MAF will be decided politically, the choice of methods and assumptions to derive its size should be clear and transparent, build on the available scientifc evidence and take account for uncertainties. A MAF will be most efective reducing environmental releases and exposure levels if risk mitigation measures are implemented in practise. Its socioeconomic impacts and costs need to be assessed in a balanced way together with the benefts for the environment, society, and for companies — also in comparison to the eforts needed for specifc mixture risk assessments. In the future and with the experiences gathered in practise, a discussion is needed on how to assess and regulate unintentional mixtures across diferent pieces of chemicals legislation to consider the true exposure situation and ensure harmonisation.

A tool for evaluation of internal validity of in vitro studies called INVITES-IN is currently under development. The tool is designed specifically for cell culture studies.

This protocol describes the testing of the performance of INVITES-IN. By performance, we mean the extent to which results of using INVITES-IN are the same for different users (consistency), the amount of time and cognitive effort it takes to apply INVITES-IN (assessor workload), the precision and potential for systematic error in results of applying INVITES-IN (accuracy), and how easy it is to use INVITES-IN (user experience).

The participants in the user testing will be representative for the expected end-users of INVITES-IN which are persons preparing literature reviews including in vitro studies (e.g. in the context of chemical hazard and risk assessments or drug development). All end-users are expected to have experience with in vitro methods.

Data collected from the performance testing will be used for further refinement and development of the release version of INVITES-IN.

In utero and children’s exposure to per- and polyfluoroalkyl substances (PFAS) is a major concern in health risk assessment as early life exposures are suspected to induce adverse health effects. Our work aims to estimate children’s exposure (from birth to 12 years old) to PFOA and PFOS, using a Physiologically-Based Pharmacokinetic (PBPK) modelling approach. A model for PFAS was updated to simulate the internal PFAS exposures during the in utero life and childhood, and including individual characteristics and exposure scenarios (e.g., duration of breastfeeding, weight at birth, etc.). Our approach was applied to the HELIX cohort, involving 1,239 mother–child pairs with measured PFOA and PFOS plasma concentrations at two sampling times: maternal and child plasma concentrations (6 to 12 y.o). Our model predicted an increase in plasma concentrations during fetal development and childhood until 2 y.o when the maximum concentrations were reached. Higher plasma concentrations of PFOA than PFOS were predicted until 2 y.o, and then PFOS concentrations gradually became higher than PFOA concentrations. From 2 to 8 y.o, mean concentrations decreased from 3.1 to 1.88 µg/L or ng/mL (PFOA) and from 4.77 to 3.56 µg/L (PFOS). The concentration–time profiles vary with the age and were mostly influenced by in utero exposure (on the first 4 months after birth), breastfeeding (from 5 months to 2 (PFOA) or 5 (PFOS) y.o of the children), and food intake (after 3 (PFOA) or 6 (PFOS) y.o of the children). Similar measured biomarker levels can correspond to large differences in the simulated internal exposures, highlighting the importance to investigate the children’s exposure over the early life to improve exposure classification. Our approach demonstrates the possibility to simulate individual internal exposures using PBPK models when measured biomarkers are scarce, helping risk assessors in gaining insight into internal exposure during critical windows, such as early life.

Recognition of per- and polyfluoroalkyl substances (PFAS) as widespread environmental pollutants and a consequent risk to human health, has recently made the European Union (EU) adopt several regulatory measures for their management. The coherence of these measures is challenged by the diversity and the ubiquitous occurrence of PFAS, which also complicates the EU’s endeavor to advance justified, harmonized, and transparent approaches in the regulatory assessment of chemical risks. Our study critically reviews the European approach for the risk assessment of PFAS, by applying a comparative analysis of the current and pending regulatory thresholds issued for these chemicals in water bodies, drinking water, and certain foodstuffs. Our study shows that the level of health protection embedded in the studied thresholds may differ by three orders of magnitude, even in similar exposure settings. This is likely to confuse the common understanding of the toxicity and health risks of PFAS and undermine reasonable decision-making and the equal treatment of different stakeholders. Wealso indicate that currently, no consensus exists on the appropriate level of required health protection regarding PFAS and that the recently adopted tolerable intake value in the EU is too cautious. Based on our analysis, we propose some simple solutions on how the studied regulations and their implicit PFAS thresholds or their application could be improved. We further conclude that instead of setting EU-wide PFAS thresholds for all the environmental compartments, providing the member states with the flexibility to consider case-specific factors such as regional background concentrations or food consumption rates, in their national regulatory procedures would likely result in more sustainable management of environmental PFAS without compromising the scientific foundation of risk assessment, the legitimacy of the EU policy framework and public health.

Introduction: Benzotriazoles and benzothiazoles (BTs) are high-production volume chemicals as well as widely distributed
emerging pollutants with potential health risk. However, information about human exposure to BTs and associated health
outcomes is limited.
Objective: We aimed to characterise exposure to BTs among Czech men, including possible occupational exposure among
firefighters, its predictors, and its associations with liver function, serum lipids and oxidative stress.
Methods: 165 participants (including 110 firefighters) provided urine and blood samples that were used to quantify the urinary
levels of 8 BTs (high-performance liquid chromatography-tandem mass spectrometry), and 4 liver enzymes, cholesterol,
low-density lipoprotein, and 8-hydroxy-2’-deoxyguanosine. Linear regression was used to assess associations with population
characteristics and biomarkers of liver function, serum lipids and oxidative stress. Regression models were adjusted for
potential confounding variables and false discovery rate procedure was applied to account for multiplicity.
Results: The BTs ranged from undetected up to 46.8 ng/mL. 2-hydroxy-benzothiazole was the most predominant compound
(detection frequency 83%; median 1.95 ng/mL). 1-methyl-benzotriazole (1M-BTR) was measured in human samples for
the first time, with a detection frequency 77% and median 1.75 ng/mL. Professional firefighters had lower urinary 1M-BTR
compared to non-firefighters. Urinary 1M-BTR was associated with levels of γ-glutamyl transferase (β = − 17.54%; 95%
CI: − 26.127, − 7.962).
Conclusion: This is the first study to investigate BT exposure in Central Europe, including potentially exposed firefighters.
The findings showed a high prevalence of BTs in the study population, the relevance of 1M-BTR as a new biomarker of
exposure, and an urgent need for further research into associated adverse health outcomes.