Urinary phthalate/DINCH metabolites associations with kisspeptin and reproductive hormones in teenagers: A cross-sectional study from the HBM4EU aligned studies.

BACKGROUND: Exposure to phthalate/DINCH metabolites can induce human reproductive toxicity, however, their endocrine-disrupting mechanisms are not fully elucidated. OBJECTIVE: To investigate the association between concentrations of phthalate/DINCH metabolites, serum kisspeptin, and reproductive hormones among European teenagers from three of the HBM4EU Aligned Studies. METHODS: In 733 Belgian (FLEHS IV study), Slovak (PCB cohort follow-up), and Spanish (BEA study) teenagers, ten phthalate and two DINCH metabolites were measured in urine by high-performance liquid chromatography-tandem mass spectrometry. Serum kisspeptin (kiss54) protein, follicle-stimulating hormone (FSH), total testosterone (TT), estradiol (E2), and sex hormone-binding globulin (SHBG) levels were measured by immunosorbent assays. Free Androgen Index (FAI) was calculated as a proxy of free testosterone. Adjusted sex-stratified linear regression models for individual studies, mixed effect models (LME) accounting for random effects for pooled studies, and g-computation and Bayesian kernel machine regression (BKMR) models for the phthalate/DINCH mixture were performed. RESULTS: The LME suggested that each IQR increase in ln-transformed levels of several phthalates was associated with lower kisspeptin [MnBP: %change (95%CI): -2.8 (-4.2;-0.4); MEHP: -1.4 (-3.4,0.2)] and higher FSH [∑DINP: 11.8 (-0.6;25.1)] levels in females from pooled studies. G-computation showed that the phthalates/DINCH mixture was associated with lower kisspeptin [-4.28 (-8.07;-0.34)] and higher FSH [22.13 (0.5;48.4)] also in females; BKMR showed similar although non-significant pattern. In males, higher phthalates metabolites [MEHP: -12.22 (-21.09;-1.18); oxo-MEHP: -12.73 (-22.34;-1.93)] were associated with lower TT and FAI, although higher DINCH [OH-MINCH: 16.31 (6.23;27.35), cx-MINCH: 16.80 (7.03;27.46), ∑DINCH: 17.37 (7.26;29.74)] were associated with higher TT levels. No mixture associations were found in males. CONCLUSION: We observed sex-specific associations between urinary concentrations of phthalate/DINCH metabolites and the panel of selected effect biomarkers (kisspeptin and reproductive hormones). This suggests that exposure to phthalates would be associated with changes in kisspeptin levels, which would affect the HPG axis and thus influence reproductive health. However, further research is needed, particularly for phthalate replacements such as DINCH.

Association of exposure to perfluoroalkyl substances (PFAS) and phthalates with thyroid hormones in adolescents from HBM4EU aligned studies.

BACKGROUND: Perfluoroalkyl substances (PFAS) and phthalates are synthetic chemicals widely used in various types of consumer products. There is epidemiological and experimental evidence that PFAS and phthalates may alter thyroid hormone levels; however, studies in children and adolescents are limited. AIM: To investigate the association of exposure to PFAS and phthalate with serum levels of thyroid hormones in European adolescents. METHODS: A cross-sectional study was conducted in 406 female and 327 male adolescents (14-17 years) from Belgium, Slovakia, and Spain participating in the Aligned Studies of the HBM4EU Project (FLEHS IV, PCB cohort, and BEA, respectively). Concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), free thyroxine (FT4), free triiodothyronine (FT3), and thyroid-stimulating hormone (TSH) were measured in sera from study participants, and urinary metabolites of six phthalates (DEP, DiBP, DnBP, BBzP, DEHP, and DiNP) and the non-phthalate plasticizer DINCH® were quantified in spot urine samples. Associations were assessed with linear regression and g-computational models for mixtures. Effect modification by sex was examined. RESULTS: In females, serum PFOA and the PFAS mixture concentrations were associated with lower FT4 and higher FT3 levels; MEP and the sums of DEHP, DiNP, and DINCH® metabolites (∑DEHP, ∑DiNP, and ∑DINCH) were associated with higher FT4; ∑DEHP with lower FT3; and the phthalate/DINCH® metabolite mixture with higher FT4 and lower FT3. In males, PFOA was associated with lower FT4 and the PFAS mixture with higher TSH levels and lower FT4/TSH ratio; MEP and ∑DiNP were associated with higher FT4; and MBzP, ∑DEHP, and the phthalate/DINCH® metabolite mixture with lower TSH and higher FT4/TSH. PFOA, mono-(2-ethyl-5-hydroxyhexyl) phthalate (OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (oxo-MEHP), and monocarboxyoctyl phthalate (MCOP) made the greatest contribution to the mixture effect. CONCLUSIONS: Results suggest that exposure to PFAS and phthalates is associated with sex-specific differences in thyroid hormone levels in adolescents.

HBM4EU-MOM: Prenatal methylmercury-exposure control in five countries through suitable dietary advice for pregnancy - Study design and characteristics of participants.

BACKGROUND: Seafood is a major source of vital nutrients for optimal fetal growth, but at the same time is the main source of exposure to methylmercury (MeHg), an established neurodevelopmental toxicant. Pregnant women must be provided with dietary advice so as to include safely fish in their diet for nutrition and mercury control. The aim of this work is to present the design of a multicentre randomized control trial (RCT), which combines human biomonitoring (HBM) with dietary interventions using seafood consumption advice to pregnant women for MeHg control, and to collect information about other possible sources of exposure to mercury. It also presents the materials developed for the implementation of the study and the characteristics of the study participants, which were self-reported in the first trimester of pregnancy. METHODS: The "HBM4EU-MOM" RCT was performed in the frame of the European Human Biomonitoring Initiative (HBM4EU) in five coastal, high fish-consuming European countries (Cyprus, Greece, Spain, Portugal and Iceland). According to the study design, pregnant women (≥120/country, ≤20 weeks gestational age) provided a hair sample for total mercury assessment (THg) and personal information relevant to the study (e.g., lifestyle, pregnancy status, diet before and during the pregnancy, information on seafood and factors related to possible non-dietary exposures to mercury) during the first trimester of pregnancy. After sampling, participants were randomly assigned to "control" (habitual practices) or "intervention" (received the harmonized HBM4EU-MOM dietary advice for fish consumption during the pregnancy and were encouraged to follow it). Around child delivery, participants provided a second hair sample and completed another tailored questionnaire. RESULTS: A total of 654 women aged 18-45 years were recruited in 2021 in the five countries, primarily through their health-care providers. The pre-pregnancy BMI of the participants ranged from underweight to obese, but was on average within the healthy range. For 73% of the women, the pregnancy was planned. 26% of the women were active smokers before the pregnancy and 8% continued to smoke during the pregnancy, while 33% were passive smokers before pregnancy and 23% remained passively exposed during the pregnancy. 53% of the women self-reported making dietary changes for their pregnancy, with 74% of these women reporting making the changes upon learning of their pregnancy. Of the 43% who did not change their diet for the pregnancy, 74% reported that their diet was already balanced, 6% found it difficult to make changes and 2% were unsure of what changes to make. Seafood consumption did not change significantly before and during the first trimester of pregnancy (overall average ∼8 times per month), with the highest frequency reported in Portugal (≥15 times per month), followed by Spain (≥7 times per month). During the first-trimester of pregnancy, 89% of the Portuguese women, 85% of the Spanish women and <50% of Greek, Cypriot and Icelandic women reported that they had consumed big oily fish. Relevant to non-dietary exposure sources, most participants (>90%) were unaware of safe procedures for handling spillage from broken thermometers and energy-saving lamps, though >22% experienced such an incident (>1 year ago). 26% of the women had dental amalgams. ∼1% had amalgams placed and ∼2% had amalgams removed during peri-pregnancy. 28% had their hair dyed in the past 3 months and 40% had body tattoos. 8% engaged with gardening involving fertilizers/pesticides and 19% with hobbies involving paints/pigments/dyes. CONCLUSIONS: The study design materials were fit for the purposes of harmonization and quality-assurance. The harmonized information collected from pregnant women suggests that it is important to raise the awareness of women of reproductive age and pregnant women about how to safely include fish in their diet and to empower them to make proper decisions for nutrition and control of MeHg, as well as other chemical exposures.

PFAS association with kisspeptin and sex hormones in teenagers of the HBM4EU aligned studies.

Exposure to Perfluoroalkyl acids (PFAS) can impair human reproductive function, e.g., by delaying or advancing puberty, although their mechanisms of action are not fully understood. We therefore set out to evaluate the relationship between serum PFAS levels, both individually and as a mixture, on the Hypothalamic-Pituitary-Gonadal (HPG) axis by analyzing serum levels of reproductive hormones and also kisspeptin in European teenagers participating in three of the HBM4EU Aligned Studies. For this purpose, PFAS compounds were measured in 733 teenagers from Belgium (FLEHS IV study), Slovakia (PCB cohort follow-up), and Spain (BEA study) by high performance liquid chromatography-tandem mass spectrometry (HPLC/MS) in laboratories under the HBM4EU quality assurance quality control (QA/QC) program. In the same serum samples, kisspeptin 54 (kiss-54) protein, follicle-stimulating hormone (FSH), total testosterone (TT), estradiol (E2), and sex hormone-binding globulin (SHBG) levels were also measured using immunosorbent assays. Sex-stratified single pollutant linear regression models for separate studies, mixed single pollutant models accounting for random effects for pooled studies, and g-computation and Bayesian kernel machine regression (BKMR) models for the mixture of the three most available (PFNA, PFOA, and PFOS) were fit. PFAS associations with reproductive markers differed according to sex. Each natural log-unit increase of PFOA, PFNA, and PFOS were associated with higher TT [18.41 (6.18; 32.31), 15.60 (7.25; 24.61), 14.68 (6.18; 24.61), respectively] in girls, in the pooled analysis (all studies together). In males, G-computation showed that PFAS mixture was associated with lower FSH levels [-10.51 (-18.81;-1.36)]. The BKMR showed the same patterns observed in G-computation, including a significant increase on male Kiss-54 and SHBG levels. Overall, effect biomarkers may enhance the current epidemiological knowledge regarding the adverse effect of PFAS in human HPG axis, although further research is warranted.

A simple method for direct mercury analysis in dried blood spots (DBS) samples for human biomonitoring studies.

Human exposure to mercury can have serious health effects, especially in vulnerable groups such as children and fetuses. The use of dried blood spot (DBS) samples to collect capillary blood greatly facilitates sample collection and fieldwork, being a less invasive alternative to blood collection by venipuncture, needing a small volume of sample, and does not require specialized medical staff. Moreover, DBS sampling reduces logistical and financial barriers related to transport and storage of blood samples. We propose here a novel method to analyze total mercury in DBS samples in a Direct Mercury Analyzer (DMA) that allow the control of the volume of the DBS samples. This method has shown good results in terms of precision (<6% error), accuracy (<10% coefficient of variation) and recovery (75-106%). The applicability of the method in human biomonitoring (HBM) was demonstrated in a pilot study involving 41 adults aged 18-65. Mercury concentrations of DBS samples from capillary blood collected by finger prick (real DBS samples) were determined in the DMA and compared with those determined in whole blood (venous blood) by ICP-MS, the method usually used in HBM. The sampling procedure was also validated by comparison of real DBS samples and DBS generated artificially in the laboratory by depositing venous samples in cellulose cards (laboratory DBS). There were no statistically significant differences in the results obtained using both methodologies (DMA: Geometric Mean (confidence interval 95%) = 3.87 (3.12-4.79) µg/L; ICP-MS: Geometric Mean (confidence interval 95%) = 3.46 (2.80-4.27) µg/L). The proposed method is an excellent alternative to be applied in clinical settings as screening methodology for assessing mercury exposure in vulnerable groups, such us pregnant woman, babies and children.

Coordination of chemical analyses under the European Human Biomonitoring Initiative (HBM4EU): Concepts, procedures and lessons learnt.

The European Human Biomonitoring Initiative (HBM4EU) ran from 2017 to 2022 with the aim of advancing and harmonizing human biomonitoring in Europe. More than 40,000 analyses were performed on human samples in different human biomonitoring studies in HBM4EU, addressing the chemical exposure of the general population, temporal developments, occupational exposure and a public health intervention on mercury in populations with high fish consumption. The analyses covered 15 priority groups of organic chemicals and metals and were carried out by a network of laboratories meeting the requirements of a comprehensive quality assurance and control system. The coordination of the chemical analyses included establishing contacts between sample owners and qualified laboratories and monitoring the progress of the chemical analyses during the analytical phase, also addressing status and consequences of Covid-19 measures. Other challenges were related to the novelty and complexity of HBM4EU, including administrative and financial matters and implementation of standardized procedures. Many individual contacts were necessary in the initial phase of HBM4EU. However, there is a potential to develop more streamlined and standardized communication and coordination in the analytical phase of a consolidated European HBM programme.

Application of human biomonitoring data to support policy development, raise awareness and environmental public health protection among countries within the HBM4EU project.

Most countries have acknowledged the importance of assessing and quantifying their population's internal exposure from chemicals in air, water, soil, food and other consumer products due to the potential health and economic impact. Human biomonitoring (HBM) is a valuable tool which can be used to quantify such exposures and effects. Results from HBM studies can also contribute to improving public health by providing evidence of individuals' internal chemical exposure as well as data to understand the burden of disease and associated costs thereby stimulating the development and implementation of evidence-based policy. To have a holistic view on HBM data utilisation, a multi-case research approach was used to explore the use of HBM data to support national chemical regulations, protect public health and raise awareness among countries participating in the HBM4EU project. The Human Biomonitoring for Europe (HBM4EU) Initiative (https://www.hbm4eu.eu/) is a collaborative effort involving 30 countries, the European Environment Agency (EEA) and the European Commission (contracting authority) to harmonise procedures across Europe and advance research into the understanding of the health impacts of environmental chemical exposure. One of the aims of the project was to use HBM data to support evidence based chemical policy and make this information timely and directly available for policy makers and all partners. The main data source for this article was the narratives collected from 27 countries within the HBM4EU project. The countries (self-selection) were grouped into 3 categories in terms of HBM data usage either for public awareness, policy support or for the establishment HBM programme. Narratives were analysed/summarised using guidelines and templates that focused on ministries involved in or advocating for HBM; steps required to engage policy makers; barriers, drivers and opportunities in developing a HBM programme. The narratives reported the use of HBM data either for raising awareness or addressing environmental/public health issues and policy development. The ministries of Health and Environment were reported to be the most prominent entities advocating for HBM, the involvement of several authorities/institutions in the national hubs was also cited to create an avenue to interact, discuss and gain the attention of policy makers. Participating in European projects and the general population interest in HBM studies were seen as drivers and opportunities in developing HBM programmes. A key barrier that was cited by countries for establishing and sustaining national HBM programmes was funding which is mainly due to the high costs associated with the collection and chemical analysis of human samples. Although challenges and barriers still exist, most countries within Europe were already conversant with the benefits and opportunities of HBM. This article offers important insights into factors associated with the utilisation of HBM data for policy support and public awareness.

Identification of Real-Life Mixtures Using Human Biomonitoring Data: A Proof of Concept Study.

Human health risk assessment of chemical mixtures is complex due to the almost infinite number of possible combinations of chemicals to which people are exposed to on a daily basis. Human biomonitoring (HBM) approaches can provide inter alia information on the chemicals that are in our body at one point in time. Network analysis applied to such data may provide insight into real-life mixtures by visualizing chemical exposure patterns. The identification of groups of more densely correlated biomarkers, so-called "communities", within these networks highlights which combination of substances should be considered in terms of real-life mixtures to which a population is exposed. We applied network analyses to HBM datasets from Belgium, Czech Republic, Germany, and Spain, with the aim to explore its added value for exposure and risk assessment. The datasets varied in study population, study design, and chemicals analysed. Sensitivity analysis was performed to address the influence of different approaches to standardise for creatinine content of urine. Our approach demonstrates that network analysis applied to HBM data of highly varying origin provides useful information with regards to the existence of groups of biomarkers that are densely correlated. This information is relevant for regulatory risk assessment, as well as for the design of relevant mixture exposure experiments.

A step towards harmonising human biomonitoring study setup on European level: Materials provided and lessons learnt in HBM4EU.

Internal exposure of the human body to potentially harmful chemical substances can be assessed by Human Biomonitoring (HBM). HBM can be used to generate conclusive data that may provide an overview of exposure levels in entire or specific population groups. This knowledge can promote the understanding of potential risks of the substances of interest or help monitoring the success of regulatory measures taken on the political level. Study planning and design are key elements of any epidemiologic study to generate reliable data. In the field of HBM, this has been done using differing approaches on various levels of population coverage so far. Comparison and combined usage of the resulting data would contribute to understanding exposure and its factors on a larger scale, however, the differences between studies make this a challenging and somewhat limited endeavour. This article presents templates for documents that are required to set up an HBM study, thus facilitating the generation of harmonised HBM data as a step towards standardisation of HBM in Europe. They are designed to be modular and adaptable to the specific needs of a single study while emphasising minimum requirements to ensure comparability. It further elaborates on the challenges encountered during the process of creating these documents during the runtime of the European Joint Programme HBM4EU in a multi-national expert team and draws up lessons learnt in the context of knowledge management.

Current exposure to phthalates and DINCH in European children and adolescents - Results from the HBM4EU Aligned Studies 2014 to 2021.

Phthalates are mainly used as plasticizers for polyvinyl chloride (PVC). Exposure to several phthalates is associated with different adverse effects most prominently on the development of reproductive functions. The HBM4EU Aligned Studies (2014-2021) have investigated current European exposure to ten phthalates (DEP, BBzP, DiBP, DnBP, DCHP, DnPeP, DEHP, DiNP, DiDP, DnOP) and the substitute DINCH to answer the open policy relevant questions which were defined by HBM4EU partner countries and EU institutions as the starting point of the programme. The exposure dataset includes ∼5,600 children (6-11 years) and adolescents (12-18 years) from up to 12 countries per age group and covering the North, East, South and West European regions. Study data from participating studies were harmonised with respect to sample size and selection of participants, selection of biomarkers, and quality and comparability of analytical results to provide a comparable perspective of European exposure. Phthalate and DINCH exposure were deduced from urinary excretions of metabolites, where concentrations were expressed as their key descriptor geometric mean (GM) and 95th percentile (P95). This study aims at reporting current exposure levels and differences in these between European studies and regions, as well as comparisons to human biomonitoring guidance values (HBM-GVs). GMs for children were highest for ∑DEHP metabolites (33.6 µg/L), MiBP (26.6 µg/L), and MEP (24.4 µg/L) and lowest for∑DiDP metabolites (1.91 µg/L) and ∑DINCH metabolites (3.57 µg/L). In adolescents highest GMs were found for MEP (43.3 µg/L), ∑DEHP metabolites (28.8 µg/L), and MiBP (25.6 µg/L) and lowest for ∑DiDP metabolites (= 2.02 µg/L) and ∑DINCH metabolites (2.51 µg/L). In addition, GMs and P95 stratified by European region, sex, household education level, and degree of urbanization are presented. Differences in average biomarker concentrations between sampling sites (data collections) ranged from factor 2 to 9. Compared to the European average, children in the sampling sites OCC (Denmark), InAirQ (Hungary), and SPECIMEn (The Netherlands) had the lowest concentrations across all metabolites and ESTEBAN (France), NAC II (Italy), and CROME (Greece) the highest. For adolescents, comparably higher metabolite concentrations were found in NEB II (Norway), PCB cohort (Slovakia), and ESTEBAN (France), and lower concentrations in POLAES (Poland), FLEHS IV (Belgium), and GerES V-sub (Germany). Multivariate analyses (Survey Generalized Linear Models) indicate compound-specific differences in average metabolite concentrations between the four European regions. Comparison of individual levels with HBM-GVs revealed highest rates of exceedances for DnBP and DiBP, with up to 3 and 5%, respectively, in children and adolescents. No exceedances were observed for DEP and DINCH. With our results we provide current, detailed, and comparable data on exposure to phthalates in children and - for the first time - in adolescents, and - for the first time - on DINCH in children and adolescents of all four regions of Europe which are particularly suited to inform exposure and risk assessment and answer open policy relevant questions.

Human urinary arsenic species, associated exposure determinants and potential health risks assessed in the HBM4EU Aligned Studies.

The European Joint Programme HBM4EU coordinated and advanced human biomonitoring (HBM) in Europe in order to provide science-based evidence for chemical policy development and improve chemical management. Arsenic (As) was selected as a priority substance under the HBM4EU initiative for which open, policy relevant questions like the status of exposure had to be answered. Internal exposure to inorganic arsenic (iAs), measured as Toxic Relevant Arsenic (TRA) (the sum of As(III), As(V), MMA, DMA) in urine samples of teenagers differed among the sampling sites (BEA (Spain) > Riksmaten adolescents (Sweden), ESTEBAN (France) > FLEHS IV (Belgium), SLO CRP (Slovenia)) with geometric means between 3.84 and 8.47 µg/L. The ratio TRA to TRA + arsenobetaine or the ratio TRA to total arsenic varied between 0.22 and 0.49. Main exposure determinants for TRA were the consumption of rice and seafood. When all studies were combined, Pearson correlation analysis showed significant associations between all considered As species. Higher concentrations of DMA, quantitatively a major constituent of TRA, were found with increasing arsenobetaine concentrations, a marker for organic As intake, e.g. through seafood, indicating that other sources of DMA than metabolism of inorganic As exist, e.g. direct intake of DMA or via the intake of arsenosugars or -lipids. Given the lower toxicity of DMA(V) versus iAs, estimating the amount of DMA not originating from iAs, or normalizing TRA for arsenobetaine intake could be useful for estimating iAs exposure and risk. Comparing urinary TRA concentrations with formerly derived biomonitoring equivalent (BE) for non-carcinogenic effects (6.4 µg/L) clearly shows that all 95th percentile exposure values in the different studies exceeded this BE. This together with the fact that cancer risk may not be excluded even at lower iAs levels, suggests a possible health concern for the general population of Europe.

Assessment of exposure to pesticide mixtures in five European countries by a harmonized urinary suspect screening approach.

Humans are exposed to a mixture of pesticides through diet as well as through the environment. We conducted a suspect-screening based study to describe the probability of (concomitant) exposure to a set of pesticide profiles in five European countries (Latvia, Hungary, Czech Republic, Spain and the Netherlands). We explored whether living in an agricultural area (compared to living in a peri-urban area), being a a child (compared to being an adult), and the season in which the urine sample was collected had an impact on the probability of detection of pesticides (-metabolites). In total 2088 urine samples were collected from 1050 participants (525 parent-child pairs) and analyzed through harmonized suspect screening by five different laboratories. Fourty pesticide biomarkers (either pesticide metabolites or the parent pesticides as such) relating to 29 pesticides were identified at high levels of confidence in samples across all study sites. Most frequently detected were biomarkers related to the parent pesticides acetamiprid and chlorpropham. Other biomarkers with high detection rates in at least four countries related to the parent pesticides boscalid, fludioxonil, pirimiphos-methyl, pyrimethanil, clothianidin, fluazifop and propamocarb. In 84% of the samples at least two different pesticides were detected. The median number of detected pesticides in the urine samples was 3, and the maximum was 13 pesticides detected in a single sample. The most frequently co-occurring substances were acetamiprid with chlorpropham (in 62 urine samples), and acetamiprid with tebuconazole (30 samples). Some variation in the probability of detection of pesticides (-metabolites) was observed with living in an agricultural area or season of urine sampling, though no consistent patterns were observed. We did observe differences in the probability of detection of a pesticide (metabolite) among children compared to adults, suggesting a different exposure and/or elimination patterns between adults and children. This survey demonstrates the feasibility of conducting a harmonized pan-European sample collection, combined with suspect screening to provide insight in the presence of exposure to pesticide mixtures in the European population, including agricultural areas. Future improvements could come from improved (harmonized) quantification of pesticide levels.

Cross-sectional associations between exposure to per- and polyfluoroalkyl substances and body mass index among European teenagers in the HBM4EU aligned studies.

Per- and polyfluoroalkyl substances (PFAS) are widespread pollutants that may impact youth adiposity patterns. We investigated cross-sectional associations between PFAS and body mass index (BMI) in teenagers/adolescents across nine European countries within the Human Biomonitoring for Europe (HBM4EU) initiative. We used data from 1957 teenagers (12-18 yrs) that were part of the HBM4EU aligned studies, consisting of nine HBM studies (NEBII, Norway; Riksmaten Adolescents 2016-17, Sweden; PCB cohort (follow-up), Slovakia; SLO CRP, Slovenia; CROME, Greece; BEA, Spain; ESTEBAN, France; FLEHS IV, Belgium; GerES V-sub, Germany). Twelve PFAS were measured in blood, whilst weight and height were measured by field nurse/physician or self-reported in questionnaires. We assessed associations between PFAS and age- and sex-adjusted BMI z-scores using linear and logistic regression adjusted for potential confounders. Random-effects meta-analysis and mixed effects models were used to pool studies. We assessed mixture effects using molar sums of exposure biomarkers with toxicological/structural similarities and quantile g-computation. In all studies, the highest concentrations of PFAS were PFOS (medians ranging from 1.34 to 2.79 µg/L). There was a tendency for negative associations with BMI z-scores for all PFAS (except for PFHxS and PFHpS), which was borderline significant for the molar sum of [PFOA and PFNA] and significant for single PFOA [ß-coefficient (95% CI) per interquartile range fold change = -0.06 (-0.17, 0.00) and -0.08 (-0.15, -0.01), respectively]. Mixture assessment indicated similar negative associations of the total mixture of [PFOA, PFNA, PFHxS and PFOS] with BMI z-score, but not all compounds showed associations in the same direction: whilst [PFOA, PFNA and PFOS] were negatively associated, [PFHxS] associated positively with BMI z-score. Our results indicated a tendency for associations of relatively low PFAS concentrations with lower BMI in European teenagers. More prospective research is needed to investigate this potential relationship and its implications for health later in life.

Methylmercury Risk Assessment Based on European Human Biomonitoring Data.

A risk assessment (RA) was conducted to estimate the risk associated with methylmercury (MeHg) exposure of vulnerable European populations, using Human Biomonitoring (HBM) data. This RA was performed integrating published data from European HBM surveys and earlier EFSA approaches (EFSA 2012). Children/adolescents (3 to 17 years old) and women of childbearing age (18 to 50 years old) were selected as relevant study population groups for this RA. Two types of HBM datasets were selected: HBM studies (n = 18) with mercury (Hg) levels (blood and hair, total Hg and/or MeHg) in the general population in different EU countries and the DEMOCOPHES harmonized study in child-mother pairs (hair, total Hg) in 17 EU countries as a reference. Two approaches were included in the RA strategy: the first one was based on estimations of the fraction of children/adolescents and women of childbearing age, respectively, from the EU general population exceeding the HBM-I value established by the German Human Biomonitoring Commission, measured as Hazard Quotients (HQ); and the second approach was based on estimations of the fraction of the two population groups exceeding the Tolerable Weekly Intake (TWI) (or their equivalent to Tolerable Daily Intake (TDI)) defined by EFSA in 2012. The HQ approach showed that for both groups, the risk varies across EU countries and that some EU areas are close to or exceeding the exposure guidance values. This is the case of Spain and Portugal, which showed the highest HQ (GM and/or P95), probably due to their higher fish consumption. Results from the EFSA approach show that hair values of children/adolescents and women of childbearing age (both in selected HBM studies and in DEMOCOPHES study) are below the TDI of 1.9 µg/g; therefore, in general, the European population does not exceed the daily average/intake dose for MeHg and/or Hg. A possible risk underestimation was identified in our assessment since for many studies no data on P95 were available, causing loss of relevant information for risk characterization on the upper bound. In addition, data from other European countries also with high seafood consumption, such as France, Greece or Iceland, were not available. For this reason, further RA refinement is needed with harmonized and more widespread HBM data to account for differences in European exposure and associated risks, so that interventions to protect vulnerable citizens, can be applied.

A Tiered Approach for Assessing Individual and Combined Risk of Pyrethroids Using Human Biomonitoring Data.

Pyrethroids are a major insecticide class, suitable for biomonitoring in humans. Due to similarities in structure and metabolic pathways, urinary metabolites are common to various active substances. A tiered approach is proposed for risk assessment. Tier I was a conservative screening for overall pyrethroid exposure, based on phenoxybenzoic acid metabolites. Subsequently, probabilistic approaches and more specific metabolites were used for refining the risk estimates. Exposure was based on 95th percentiles from HBM4EU aligned studies (2014-2021) covering children in Belgium, Cyprus, France, Israel, Slovenia, and The Netherlands and adults in France, Germany, Israel, and Switzerland. In all children populations, the 95th percentiles for 3-phenoxybenzoic acid (3-PBA) exceeded the screening value. The probabilistic refinement quantified the risk level of the most exposed population (Belgium) at 2% or between 1-0.1% depending on the assumptions. In the substance specific assessments, the 95th percentiles of urinary concentrations in the aligned studies were well below the respective human biomonitoring guidance values (HBM-GVs). Both information sets were combined for refining the combined risk. Overall, the HBM data suggest a low health concern, at population level, related to pyrethroid exposure for the populations covered by the studies, even though a potential risk for highly exposed children cannot be completely excluded. The proposed tiered approach, including a screening step and several refinement options, seems to be a promising tool of scientific and regulatory value in future.

Improving the Risk Assessment of Pesticides through the Integration of Human Biomonitoring and Food Monitoring Data: A Case Study for Chlorpyrifos.

The risk assessment of pesticide residues in food is a key priority in the area of food safety. Most jurisdictions have implemented pre-marketing authorization processes, which are supported by prospective risk assessments. These prospective assessments estimate the expected residue levels in food combining results from residue trials, resembling the pesticide use patterns, with food consumption patterns, according to internationally agreed procedures. In addition, jurisdictions such as the European Union (EU) have implemented large monitoring programs, measuring actual pesticide residue levels in food, and are supporting large-scale human biomonitoring programs for confirming the actual exposure levels and potential risk for consumers. The organophosphate insecticide chlorpyrifos offers an interesting case study, as in the last decade, its acceptable daily intake (ADI) has been reduced several times following risk assessments by the European Food Safety Authority (EFSA). This process has been linked to significant reductions in the use authorized in the EU, reducing consumers' exposure progressively, until the final ban in 2020, accompanied by setting all EU maximum residue levels (MRL) in food at the default value of 0.01 mg/kg. We present a comparison of estimates of the consumer's internal exposure to chlorpyrifos based on the urinary marker 3,5,6-trichloro-2-pyridinol (TCPy), using two sources of monitoring data: monitoring of the food chain from the EU program and biomonitoring of European citizens from the HB4EU project, supported by a literature search. Both methods confirmed a drastic reduction in exposure levels from 2016 onwards. The margin of exposure approach is then used for conducting retrospective risk assessments at different time points, considering the evolution of our understanding of chlorpyrifos toxicity, as well as of exposure levels in EU consumers following the regulatory decisions. Concerns are presented using a color code, and have been identified for almost all studies, particularly for the highest exposed group, but at different levels, reaching the maximum level, red code, for children in Cyprus and Israel. The assessment uncertainties are highlighted and integrated in the identification of levels of concern.

Harmonization of Human Biomonitoring Studies in Europe: Characteristics of the HBM4EU-Aligned Studies Participants.

Human biomonitoring has become a pivotal tool for supporting chemicals' policies. It provides information on real-life human exposures and is increasingly used to prioritize chemicals of health concern and to evaluate the success of chemical policies. Europe has launched the ambitious REACH program in 2007 to improve the protection of human health and the environment. In October 2020 the EU commission published its new chemicals strategy for sustainability towards a toxic-free environment. The European Parliament called upon the commission to collect human biomonitoring data to support chemical's risk assessment and risk management. This manuscript describes the organization of the first HBM4EU-aligned studies that obtain comparable human biomonitoring (HBM) data of European citizens to monitor their internal exposure to environmental chemicals. The HBM4EU-aligned studies build on existing HBM capacity in Europe by aligning national or regional HBM studies. The HBM4EU-aligned studies focus on three age groups: children, teenagers, and adults. The participants are recruited between 2014 and 2021 in 11 to 12 primary sampling units that are geographically distributed across Europe. Urine samples are collected in all age groups, and blood samples are collected in children and teenagers. Auxiliary information on socio-demographics, lifestyle, health status, environment, and diet is collected using questionnaires. In total, biological samples from 3137 children aged 6-12 years are collected for the analysis of biomarkers for phthalates, HEXAMOLL® DINCH, and flame retardants. Samples from 2950 teenagers aged 12-18 years are collected for the analysis of biomarkers for phthalates, Hexamoll® DINCH, and per- and polyfluoroalkyl substances (PFASs), and samples from 3522 adults aged 20-39 years are collected for the analysis of cadmium, bisphenols, and metabolites of polyaromatic hydrocarbons (PAHs). The children's group consists of 50.4% boys and 49.5% girls, of which 44.1% live in cities, 29.0% live in towns/suburbs, and 26.8% live in rural areas. The teenagers' group includes 50.6% girls and 49.4% boys, with 37.7% of residents in cities, 31.2% in towns/suburbs, and 30.2% in rural areas. The adult group consists of 52.6% women and 47.4% men, 71.9% live in cities, 14.2% in towns/suburbs, and only 13.4% live in rural areas. The study population approaches the characteristics of the general European population based on age-matched EUROSTAT EU-28, 2017 data; however, individuals who obtained no to lower educational level (ISCED 0-2) are underrepresented. The data on internal human exposure to priority chemicals from this unique cohort will provide a baseline for Europe's strategy towards a non-toxic environment and challenges and recommendations to improve the sampling frame for future EU-wide HBM surveys are discussed.

Selecting the best non-invasive matrix to measure mercury exposure in human biomonitoring surveys.

Exposure to mercury, even at low doses, can affect human health, well-being and life quality at a broad scale. Human biomonitoring is the most straightforward approach to measure and quantify mercury exposure in humans. The objective of the present study is to compare and discuss the relationships between Hg levels in the most used matrices, hair, urine and blood, with the aim to ascertain to what extent mercury exposure and internal mercury levels could be predicted by monitoring non-invasive matrices. The study population (n = 527) is a subsample from Spanish BIOAMBIENT. ES study (18-65 y, both sexes), with data of Hg levels in blood, hair, and urine from the same individuals. We found strong inter-matrix Spearman correlations between blood and hair mercury (r2 = 0.84), while the correlations for urine and blood mercury (r2=0.64) and urine and hair mercury (r2=0.65) were weaker. The geometric mean of the ratios between matrices were (GM, 95%CI): Hair/Blood 280 (271-290), Urine/Blood 0.174 (0.163-0.186) and Hair/Urine 2070 (1953-2194) and Urine/Blood 0.135 (0.128-0.144) for urine corrected by creatinine. High individual variation was observed particularly in those ratios involving urine. Considering the wide range of values observed in the ratios, we do not recommend applying them at individual level. The predictive models indicate that hair Hg was a more accurate predictor than urine. The inclusion of urine values did not increase the predictive accuracy, so, we recommend a cautious interpretation of urine mercury levels. Our study presents clear evidence that in a population highly exposed to food-borne mercury, a large portion of urinary mercury primarily emanates from methylmercury demethylation. We conclude that urine, as a non-invasive matrix, can be used as a reliable qualitative biomarker for Hg exposure when hair measurements not are available. For quantitative individual assessments, still blood measurements are to be preferred.

The European human biomonitoring platform - Design and implementation of a laboratory quality assurance/quality control (QA/QC) programme for selected priority chemicals.

A fundamental objective of the human biomonitoring for Europe initiative (HBM4EU) is to progress toward comparable and robust exposure data for a wide variety of prioritized chemicals in human samples. A programme for Quality Assurance/Quality Control (QA/QC) was designed in HBM4EU with the purpose of creating a network of European laboratories providing comparable analytical data of high quality. Two approaches were chosen for two sets of prioritized chemicals with different timelines: (i) Scheme 1, where interested candidate laboratories participated in multiple rounds of proficiency tests (ii) Scheme 2, where selected expert laboratories participated in three rounds of interlaboratory comparison investigations. In both cases, the results were used to identify laboratories capable of generating consistent and comparable results for sample analysis in the frame of HBM4EU. In total, 84 laboratories from 26 countries were invited to participate in Scheme 1 that covered up to 73 biomarkers from Hexamoll® DINCH, phthalates, bisphenols, per- and polyfluoroalkyl substances, halogenated flame retardants (HFRs), organophosporous flame retardants (OPFRs), polycyclic aromatic hydrocarbons (PAH), cadmium, chromium and aromatic amines. 74 of the participants were successful for at least one biomarker in Scheme 1. Scheme 2 involved 22 biomarkers and successful results were obtained by 2 expert laboratories for arsenic, 5 for acrylamide, 4 for mycotoxins, 2 for pesticides and 2 for UV-filters in skin care products. The QA/QC programme allowed the identification of major difficulties and needs in HBM analysis as well of gaining insight in the analytical capacities of European laboratories. Furthermore, it is the first step towards the establishment of a sustainable European network of HBM laboratories.