An effective validation of analytical method for determination of a polar complexing agent: the illustrative case of cytotoxic bleomycin.

The effectiveness of highly polar agents in cancer treatment is well recognized, but their physicochemical properties make their analytical determination a demanding task. Their analysis requires peculiar sample preparation and chromatographic separation, which heavily impacts the precision of such an analytical method. As a case study, we chose a polar cytotoxic bleomycin, which is a mixture of complexing congeners with relatively high molecular mass, a fact that creates an added challenge in regard to its detection via electrospray mass spectrometry. These issues combined lead to a deprived method performance, so the aim of this study is manifold, i.e., to optimize, validate, and establish quality performance measures for determination of bleomycin in pharmaceutical and biological specimens. Quantification of bleomycin is done at diametrically different concentration levels: at the concentrations relevant for analysis of pharmaceutical dosage forms it is based on a direct reversed-phase HPLC-UV detection, involving minimum sample pretreatment. On the contrary, analysis of bleomycin in biological specimens requires phospholipid removal and protein precipitation followed by HILIC chromatography with MS/MS detection of bleomycin A2 and B2 copper complexes being the predominant species. This study further attempts to solve the traceability issue in the absence of certified reference standards, determines measurement uncertainty, investigates BLM stability and method performance characteristics, and, last but not least, provides an explanatory example of how a method quality assurance procedure should be established in case of an exceedingly complex analytical method.

Aerobic degradation of tetramethyl bisphenol F (TMBPF) with activated sludge: Kinetics and biotransformation products.

This study investigated the bio-degradation kinetics of tetramethyl bisphenol F (TMBPF), a non-estrogenic alternative to bisphenol A (BPA). Batch biotransformation experiments were performed whereby samples were inoculated with activated sludge and analysed using liquid chromatography-Orbitrap-tandem mass spectrometry (LC-Orbitrap-MS) utilising two non-targeted workflows (commercial and freely available online) for biotransformation products (BTP) identification. The degradation of TMBPF followed single first-order reaction kinetics and depended on the initial concentration (ci) with faster degradation -kt = 0.16, (half-life = 4.4 days) at lower concentrations ci = 0.1 mg L-1, compared with -kt = 0.02 (half-live = 36.4 days) at ci = 10.0 mg L-1. After 18 days, only 8% of the original TMBPF remained at the lowest tested concentration (0.1 mg L-1). Twelve BTPs were identified, three of which were workflow and one condition-specific. The highest relative quantities of BTPs were observed in nutrient-mineral and mineral media after ten days, while after 14 days, 36 and 31% of TMBPF (ci = 1 mg L-1) remained in the nutrient-mineral and mineral media, respectively. Also, the kinetics of TMBPF and its BTPs were the same with and without an additional carbon source. A newly proposed biodegradation pathway for TMBPF involves cleavage of the methylene bridge, hydroxylation with further oxidation, sulphation, nitrification, nitro reduction with further oxidation, acetylation, and glycine conjugation, providing a deeper insight into the fate of TMBPF during biological wastewater treatment.

Stability, biological treatment and UV photolysis of 18 bisphenols under laboratory conditions.

The limited knowledge on the stability, removal, and the fate of bisphenol A analogues in the aqueous environment led us to assess the removal by hydrolysis, adsorption, biological treatment and UV photolysis of eighteen common bisphenol compounds (BPs). Hydrolysis of BPs does not occur. The main factor affecting their stability in wastewater samples is storage time, and safe storage conditions were found to be -20 °C or 4 °C for up to four weeks. The results also revealed no significant reduction in the levels of BPs standards when stored in either methanol or ultrapure water. BPE was found to be the most stable, followed by BPF isomers, BPS and BPF, while BP26DM was the least stable and BPM, BPPH, BPP, BPBP and BPFL were quickly adsorbed. For most BPs, the removal efficiency of biological treatment was >85%, and there was no difference between the suspended activated sludge and moving bed bioreactors. Different adsorption affinities of the BPs to biomass were observed and reflect the differences in their Kow. In terms of degradability, direct UV photolysis in water produced three groups of BPs: (A) highly removable (RE > 94%), (B) moderately removable (RE 50-80%) and (C) poorly removable (RE 25-45%). In nearly all cases degradation followed pseudo-first-order kinetics.

Urinary bisphenol A in children, mothers and fathers from Slovenia: Overall results and determinants of exposure.

In the present study, urinary bisphenol A (BPA) levels were reported for the first time in the Slovenian general population and were evaluated with regard to dietary and non-dietary exposure sources, and compared according to age, gender and area of residence. First morning urine was collected from children (6-11 years), their mothers (30-52 years) and fathers (30-53 years), living in urban and rural areas of Slovenia. Besides basic questionnaire data on general population characteristics, socio-economic status and dietary habits, BPA-specific data was also collected, including consumption of food and beverages from plastic and canned containers, presence of white dental fillings, the use of specific consumer products and hormonal treatments. Urine samples were analysed for both free and conjugated BPA using GC-MS/MS. The urinary levels of total BPA in children, mothers and fathers were low, with geometric means of 1.51, 0.79, and 0.20 µg/g creatinine, respectively. The levels were comparable with the levels reported for other European countries and were all below the current health-based guidance values. In line with large-scale surveys, the data revealed age-dependant BPA urinary levels, with the highest levels in the youngest age group. In mothers, urinary levels of BPA were determined by hormonal interactions more than dietary sources, while a positive association between urinary BPA and diet was apparent in children (canned food/drink and food from plastic material) and fathers (canned food/drink). The study clearly shows that physiological and behavioural differences account for differences in levels of urinary BPA among study groups, a finding that sets the priorities for future research.

Risk characterization of bisphenol-A in the Slovenian population starting from human biomonitoring data.

The current study aims to characterize exposure and risk associated to bisphenol-A (BPA) exposure in Slovenia, starting from biomonitoring data. Based on the urinary data, daily intake for the individuals was back-calculated using a physiology based biokinetic (PBBK) model properly parameterized for BPA, coupled with an exposure reconstruction algorithm. Re-running the PBBK model in forward mode allowed the estimation of biologically effective dose (free plasma BPA) and the respective daily area under the curve (AUC). Finally, risk characterization ratio was derived using both external and internal dose metrics. The urinary BPA levels were found low, with GM of 0.79, 1.51 and 0.20 µg/g creatinine for mothers, children and fathers respectively, similar to the levels of other European countries. Based on the above and accounting for the dynamics of exposure and biokinetics, daily intake was estimated, median exposure levels have been estimated equal to 0.019, 0.035 and 0.005 µg/kg_bw/d for mothers, fathers and children respectively. The highest estimated intake level was found in a child, equal to 0.87 µg/kg_bw/d, while the maximum intake for mothers and fathers were 0.7 and 0.8 µg/kg_bw/d respectively. The respective RCR levels using the EFSA t-TDI of 4 µg/kg_bw/d were 2 magnitudes of order lower below 1, independently of the selected method. It has to be noted that had daily intake been estimated solely based on the urinary concentrations mass balance, the estimated intake would be lower, as a result of the oversimplification on exposure and elimination time dynamics. This highlights the importance for using PBBK modelling based exposure reconstruction schemes for rapidly metabolized and excreted compounds such as BPA, as well as the study design of efficient sampling for rapidly metabolized compounds.

Biomarkers of exposure in environment-wide association studies - Opportunities to decode the exposome using human biomonitoring data.

BACKGROUND: The European Union's 7th Framework Programme (EU's FP7) project HEALS - Health and Environment-wide Associations based on Large Population Surveys - aims a refinement of the methodology to elucidate the human exposome. Human biomonitoring (HBM) provides a valuable tool for understanding the magnitude of human exposure from all pathways and sources. However, availability of specific biomarkers of exposure (BoE) is limited. OBJECTIVES: The objective was to summarize the availability of BoEs for a broad range of environmental stressors and exposure determinants and corresponding reference and exposure limit values and biomonitoring equivalents useful for unraveling the exposome using the framework of environment-wide association studies (EWAS). METHODS: In a face-to-face group discussion, scope, content, and structure of the HEALS deliverable "Guidelines for appropriate BoE selection for EWAS studies" were determined. An expert-driven, distributed, narrative review process involving around 30 individuals of the HEALS consortium made it possible to include extensive information targeted towards the specific characteristics of various environmental stressors and exposure determinants. From the resulting 265 page report, targeted information about BoE, corresponding reference values (e.g., 95th percentile or measures of central tendency), exposure limit values (e.g., the German HBM I and II values) and biomonitoring equivalents (BEs) were summarized and updated. RESULTS: 64 individual biological, chemical, physical, psychological and social environmental stressors or exposure determinants were included to fulfil the requirements of EWAS. The list of available BoEs is extensive with a number of 135; however, 12 of the stressors and exposure determinants considered do not leave any measurable specific substance in accessible body specimens. Opportunities to estimate the internal exposure stressors not (yet) detectable in human specimens were discussed. CONCLUSIONS: Data about internal exposures are useful to decode the exposome. The paper provides extensive information for EWAS. Information included serves as a guideline - snapshot in time without any claim to comprehensiveness - to interpret HBM data and offers opportunities to collect information about the internal exposure of stressors if no specific BoE is available.

In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol.

The aim of the present study was to identify the in vitro Phase I and Phase II metabolites of three new psychoactive substances: α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV), and methedrone, using human liver microsomes and human liver cytosol. Accurate-mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry. Six Phase I metabolites of α-PVP were identified, which were formed involving reduction, hydroxylation, and pyrrolidine ring opening reactions. The lactam compound was the major metabolite observed for α-PVP. Two glucuronidated metabolites of α-PVP, not reported in previous in vitro studies, were further identified. MDPV was transformed into 10 Phase I metabolites involving reduction, hydroxylation, and loss of the pyrrolidine ring. Also, six glucuronidated and two sulphated metabolites were detected. The major metabolite of MDPV was the catechol metabolite. Methedrone was transformed into five Phase I metabolites, involving N- and O-demethylation, hydroxylation, and reduction of the ketone group. Three metabolites of methedrone are reported for the first time. In addition, the contribution of individual human CYP enzymes in the formation of the detected metabolites was investigated.

Urinary BPA measurements in children and mothers from six European member states: Overall results and determinants of exposure.

For the first time in Europe, both European-wide and country-specific levels of urinary Bisphenol A (BPA) were obtained through a harmonized protocol for participant recruitment, sampling and quality controlled biomarker analysis in the frame of the twin projects COPHES and DEMOCOPHES. 674 child-mother pairs were recruited through schools or population registers from six European member states (Belgium, Denmark, Luxembourg, Slovenia, Spain and Sweden). Children (5-12 y) and mothers donated a urine sample. Information on socio-demographic characteristics, life style, dietary habits, and educational level of the parents was provided by mothers. After exclusion of urine samples with creatinine values below 300 mg/L or above 3000 mg/L, 653 children and 639 mothers remained for which BPA was measured. The geometric mean (with 95% confidence intervals) and 90th percentile were calculated for BPA separately in children and in mothers and were named "European reference values". After adjustment for confounders (age and creatinine), average exposure values in each country were compared with the mean of the "European reference values" by means of a weighted analysis of variance. Overall geometric means of all countries (95% CI) adjusted for urinary creatinine, age and gender were 2.04 (1.87-2.24) µg/L and 1.88 (1.71-2.07) µg/L for children (n=653) and mothers (n=639), respectively. Multiple regression analysis was used to identify significant environmental, geographical, personal or life style related determinants. Consumption of canned food and social class (represented by the highest educational level of the family) were the most important predictors for the urinary levels of BPA in mothers and children. The individual BPA levels in children were significantly correlated with the levels in their mothers (r=0.265, p<0.001), which may suggest a possible common environmental/dietary factor that influences the biomarker level in each pair. Exposure of the general European population was well below the current health-based guidance values and no participant had BPA values higher than the health-based guidance values.

Schedule-dependent interaction between vinblastine and irradiation in experimental sarcoma.

BACKGROUND AND PURPOSE: Prolonged vinblastine (VLB) infusion and irradiation (IR) lead to favourable results in certain tumours types; however the underlying biological mechanisms of interaction are not well known. The aim of our study was to evaluate the dose- and time-dependent interactions between split-dose VLB treatment (mimicking prolonged infusion) and IR of sarcoma SA-1 tumours in A/J mice. METHODS: Antitumor effectiveness of different VLB-IR schedules was determined by a tumour growth delay assay, the VLB amount in the tumours by liquid chromatography coupled to mass spectrometry and DNA cell cycle analysis. RESULTS: A positive antitumor effect was obtained when tumours were irradiated immediately after the first (0 h) or second (4 h) injection of VLB treatment, despite the lower amount of VLB in the tumours as well as decreased number of cells in the IR-sensitive G2M phase at these times points as opposed to the second half of VLB split-dose scheduling. CONCLUSION: Preferential binding of VLB to microtubules (with consequent lack of available VLB to bind to DNA where it acts as a radioprotector) and the absence of radiobiologically relevant hypoxia are presumably leading to the observed therapeutic benefit of applying IR at the beginning of the prolonged VLB infusion.

Trace analysis of benzophenone-derived compounds in surface waters and sediments using solid-phase extraction and microwave-assisted extraction followed by gas chromatography-mass spectrometry.

This study describes a procedure for determining eight benzophenone-derived compounds in surface waters and sediments. These include the pharmaceutical ketoprofen, its phototransformation products 3-ethylbenzophenone and 3-acetylbenzophenone, and five benzophenone-type ultraviolet (UV) filters. The proposed analytical method involves the pre-concentration of water samples by solid-phase extraction (SPE) and microwave-assisted extraction (MAE) of sediment samples followed by derivatization and analysis by gas chromatography-mass spectrometry. Different parameters were investigated to achieve optimal method performance. Recoveries of 91 to 96 % from water samples were obtained using HLB Oasis SPE cartridges, whereas MAE of sediments (30 min at 150 °C) gave recoveries of 80 to 99 %. Limits of detection were between 0.1 and 1.9 ng L(-1) for water samples and from 0.1 to 1.4 ng g(-1) for sediment samples. The developed method was applied to environmental samples and revealed the presence of UV filters in the majority of the surface waters with up to 690 ng L(-1) of 2-hydroxy-4-methoxybenzophenone. By contrast, ketoprofen (≤2,900 ng L(-1)) and its degradation products (≤320 ng L(-1)) were found in only two rivers, both receiving wastewater treatment plant effluents. Sediment analysis revealed benzophenone to be present in concentrations up to 650 ng g(-1), whereas concentrations of other compounds were considerably lower (≤32 ng L(-1)). For the first time, quantifiable amounts of two ketoprofen transformation products in the aqueous environment are reported.

Serum Cortisol and Blood Glucose Concentrations in Anesthetized Dogs Administered Levobupivacaine and Low-Dose Dexmedetomidine for Regional Anesthesia of the Oral Cavity.

The effects on the stress response, postanesthetic sedation, and altered behavior were evaluated following regional anesthesia and dental treatment in 40 dogs. Serum cortisol and blood glucose concentrations were measured following the administration of levobupivacaine (LBUP) 0.5% and dexmedetomidine (DEX) (0.5 µg/kg) or a placebo. The dogs were randomly assigned to 4 groups of 10 dogs each. All dogs received a regional nerve block using LBUP 0.5%. Group 1 (LBUP + DEX IV) also received DEX intravenously (IV); group 2 (LBUP + PLC IV) also received a placebo IV; group 3 (LBUP + DEX IO) also received DEX in one infraorbital (IO) block; and group 4 (LBUP + DEX IA) also received DEX in one inferior alveolar (IA) block. Serum cortisol and blood glucose concentrations were determined before the administration of oral blocks and at the end of the procedure. Sedation and behavior scores were assessed before premedication and hourly for 6 h after the end of anesthesia. Cortisol concentration did not change in any group at either evaluation time. The glucose concentration was higher (P < .05) only in the LBUP + DEX IA group at the end of the procedure. The sedation score was higher until the end of the observation period only in the LBUP + DEX IV and LBUP + PLC IV groups. No change in behavior score was observed in any of the groups. The reduction of perioperative stress response in all groups was due to the use of LBUP and not DEX.

Pharmacokinetics of single dose levobupivacaine after peri-incisional subcutaneous infiltration in anaesthetized domestic pigs.

Increasing use of pigs as models in translational research, and growing focus on animal welfare are leading to better use of effective analgesics and anaesthetics when painful procedures are performed. However, there is a gap in basic knowledge such as pharmacokinetics of different anaesthetics in these species. The main objective of our study was to determine the pharmacokinetics of levobupivacaine in domestic pigs. Twelve female grower pigs weighing 31.17 ± 4.6 kg were subjected to general anaesthesia and experimental surgery, at the end of which they received 1 mg/kg levobupivacaine via peri-incisional subcutaneous infiltration. Plasma samples were collected before administration of levobupivacaine and at 0.5, 1, 2, 4, 8, 12, 24 and 48 h thereafter. Concentrations of levobupivacaine were determined by liquid chromatography coupled with tandem mass spectrometry. Following single dose of levobupivacaine, all animals had measurable plasma concentrations 0.5 h after drug administration, with most peak concentrations observed at the 1-h time point. In all 12 animals, levobupivacaine was below the limit of quantification 48 h after drug administration. The mean maximum plasma concentration, area under the curve and half-life were determined to be 809.98 µg/l, 6552.46 µg/l h and 6.25 h, respectively. Plasma clearance, volume of distribution and weight-normalized volume of distribution were 4.41 l/h, 35.57 l and 1.23 l/kg, respectively. Peak plasma concentrations in our study were well below concentrations that were found to produce toxicity in pigs.

Associations between Urinary Phthalate Metabolites with BDNF and Behavioral Function among European Children from Five HBM4EU Aligned Studies.

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.

Innovative analytical methodologies for characterizing chemical exposure with a view to next-generation risk assessment.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

Exposure to bisphenol A in European women from 2007 to 2014 using human biomonitoring data - The European Joint Programme HBM4EU.

BACKGROUND: Bisphenol A (BPA; or 4,4'-isopropylidenediphenol) is an endocrine disrupting chemical. It was widely used in a variety of plastic-based manufactured products for several years. The European Food Safety Authority (EFSA) recently reduced the Tolerable Daily Intake (TDI) for BPA by 20,000 times due to concerns about immune-toxicity. OBJECTIVE: We used human biomonitoring (HBM) data to investigate the general level of BPA exposure from 2007 to 2014 of European women aged 18-73 years (n = 4,226) and its determinants. METHODS: Fifteen studies from 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Israel, Luxembourg, Slovenia, Spain, Sweden, and the United Kingdom) were included in the BPA Study protocol developed within the European Joint Programme HBM4EU. Seventy variables related to the BPA exposure were collected through a rigorous post-harmonization process. Linear mixed regression models were used to investigate the determinants of total urine BPA in the combined population. RESULTS: Total BPA was quantified in 85-100 % of women in 14 out of 15 contributing studies. Only the Austrian PBAT study (Western Europe), which had a limit of quantification 2.5 to 25-fold higher than the other studies (LOQ=2.5 µg/L), found total BPA in less than 5 % of the urine samples analyzed. The geometric mean (GM) of total urine BPA ranged from 0.77 to 2.47 µg/L among the contributing studies. The lowest GM of total BPA was observed in France (Western Europe) from the ELFE subset (GM=0.77 µg/L (0.98 µg/g creatinine), n = 1741), and the highest levels were found in Belgium (Western Europe) and Greece (Southern Europe), from DEMOCOPHES (GM=2.47 µg/L (2.26 µg/g creatinine), n = 129) and HELIX-RHEA (GM=2.47 µg/L (2.44 µg/g creatinine), n = 194) subsets, respectively. One hundred percent of women in 14 out of 15 data collections in this study exceeded the health-based human biomonitoring guidance value for the general population (HBM-GVGenPop) of 0.0115 µg total BPA/L urine derived from the updated EFSA's BPA TDI. Variables related to the measurement of total urine BPA and those related to the main socio-demographic characteristics (age, height, weight, education, smoking status) were collected in almost all studies, while several variables related to BPA exposure factors were not gathered in most of the original studies (consumption of beverages contained in plastic bottles, consumption of canned food or beverages, consumption of food in contact with plastic packaging, use of plastic film or plastic containers for food, having a plastic floor covering in the house, use of thermal paper…). No clear determinants of total urine BPA concentrations among European women were found. A broader range of data planned for collection in the original questionnaires of the contributing studies would have resulted in a more thorough investigation of the determinants of BPA exposure in European women. CONCLUSION: This study highlights the urgent need for action to further reduce exposure to BPA to protect the population, as is already the case in the European Union. The study also underscores the importance of pre-harmonizing HBM design and data for producing comparable data and interpretable results at a European-wide level, and to increase HBM uptake by regulatory agencies.

Urinary concentrations of phthalate/DINCH metabolites and body mass index among European children and adolescents in the HBM4EU Aligned Studies: A cross-sectional multi-country study.

BACKGROUND: Phthalates are ubiquitous in the environment. Despite short half-lives, chronic exposure can lead to endocrine disruption. The safety of phthalate substitute DINCH is unclear. OBJECTIVE: To evaluate associations between urinary concentrations of phthalate/DINCH metabolites and body mass index (BMI) z-score among children and adolescents. METHOD: We used Human Biomonitoring for Europe Aligned Studies data from 2876 children (12 studies, 6-12 years, 2014-2021) and 2499 adolescents (10 studies, 12-18 years, 2014-2021) with up to 14 phthalate/DINCH urinary metabolites. We used multilevel linear regression to assess associations between phthalate/DINCH concentrations and BMI z-scores, testing effect modification by sex. In a subset, Bayesian kernel machine regression (BKMR) and quantile-based g-computation assessed important predictors and mixture effects. RESULTS: In children, we found few associations in single pollutant models and no interactions by sex (p-interaction > 0.1). BKMR detected no relevant exposures (posterior inclusion probabilities, PIPs < 0.25), nor joint mixture effect. In adolescent single pollutant analysis, mono-ethyl phthalate (MEP) concentrations were associated with higher BMI z-score in males (ß = 0.08, 95 % CI: 0.001,0.15, per interquartile range increase in ln-transformed concentrations, p-interaction = 0.06). Conversely, mono-isobutyl phthalate (MiBP) was associated with a lower BMI z-score in both sexes (ß = -0.13, 95 % CI: -0.19, -0.07, p-interaction = 0.74), as was sum of di(2-ethylhexyl) phthalate (∑DEHP) metabolites in females only (ß = -0.08, 95 % CI: -0.14, -0.02, p-interaction = 0.01). In BKMR, higher BMI z-scores were predicted by MEP (PIP=0.90) and MBzP (PIP=0.84) in males. Lower BMI z-scores were predicted by MiBP (PIP=0.999), OH-MIDP (PIP=0.88) and OH-MINCH (PIP=0.72) in both sexes, less robustly by DEHP (PIP=0.61) in females. In quantile g-computation, the overall mixture effect was null for males, and trended negative for females (ß = -0.11, 95 % CI: -0.25, 0.03, per joint exposure quantile). CONCLUSION: In this large Europe-wide study, we found age/sex-specific differences between phthalate metabolites and BMI z-score, stronger in adolescents. Longitudinal studies with repeated phthalate measurements are needed.

GC-EI-MS datasets of trimethylsilyl (TMS) and tert-butyl dimethyl silyl (TBDMS) derivatives for development of machine learning-based compound identification approaches.

In the field of environment and health studies, recent trends have focused on the identification of contaminants of emerging concern (CEC). This is a complex, challenging task, as resources, such as compound databases (DBs) and mass spectral libraries (MSLs) concerning these compounds are very poor. This is particularly true for semi polar organic contaminants that have to be derivatized prior to gas chromatography-mass spectrometry (GC-MS) analysis with electron impact ionization (EI), for which it is barely possible to find any records. In particular, there is a severe lack of datasets of GC-EI-MS spectra generated and made publicly available for the purpose of development, validation and performance evaluation of cheminformatics-assisted compound structure identification (CSI) approaches, including novel cutting-edge machine learning (ML)-based approaches [1]. We set out to fill this gap and support the machine learning-assisted compound identification, thus aiding cheminformatics-assisted identification of silylated derivatives in GC-MS laboratories working in the field of environment and health. To this end, we have generated 12 datasets of GC-EI-MS spectra, six of which contain GC-EI-MS spectra of trimethylsilyl (TMS) and six GC-EI-MS spectra of tert-butyldimethylsilyl (TBDMS) derivatives. Four of these datasets, named testing datasets, contain mass spectra acquired by the authors. They are available in full, together with corresponding metadata. Eight datasets, named training datasets, were derived from mass spectra in the NIST 17 Mass Spectral Library. For these, we have only made the metadata publicly available, due to licensing reasons. For each type of derivative, two testing datasets are generated by acquiring and processing GC-EI-MS spectra, such that they include raw and processed GC-EI-MS spectra of TMS and TBDMS derivatives of CECs, along with their corresponding metadata. The metadata contains IUPAC name, exact mass, molecular formula, InChI, InChIKey, SMILES and PubChemID, of each CEC and CEC-TMS or CEC-TBDMS derivative, where available. Eight GC-EI-MS training datasets are generated by using the National Institute of Standards and Technology (NIST)/U.S. Environmental Protection Agency (EPA)/National Institute of Health (NIH) 17 Mass Spectral Library. For each derivative type (TMS and TBDMS), four datasets are given, each corresponding to an original dataset obtained from NIST/EPA/NIH 17 and three variants thereof, obtained after each of the filtering steps of the procedure described below. Only the metadata about the training datasets are available, describing the corresponding NIST/EPA/NIH 17 entires: These include the compound name, CAS Registry number, InChIKey, exact mass, Mw, NIST number and ID number. The datasets we present here were used to train and test predictive models for identification of silylated derivatives built with ML approaches [4]. The models were built by using data curated from the NIST Mass Spectral Library 17 [2] and the machine learning approach of CSI:Output Kernel Regression (CSI:OKR) [2]. Data from the NIST Mass Spectral Library 17 are commercially available from the National Institute of Standards and Technology (NIST)/U.S. Environmental Protection Agency (EPA)/National Institute of Health (NIH) and thus cannot be made publicly available. This highlights the need for publicly available GC-EI-MS spectra, which we address by releasing in full the four testing datasets.

Contaminants of emerging concern in urine: a review of analytical methods for determining diisocyanates, benzotriazoles, benzothiazoles, 4-methylbenzylidene camphor, isothiazolinones, fragrances, and non-phthalate plasticizers.

Human biomonitoring (HBM) frameworks assess human exposure to hazardous chemicals. In this review, we discuss and summarize sample preparation procedures and analytical methodology for six groups of chemicals of emerging concern (CECs), namely diisocyanates, benzotriazoles, benzothiazoles, 4-methylbenzylidene camphor, isothiazolinones, fragrances, and non-phthalate plasticizers, which are increasingly detected in urine, however, are not yet widely included in HBM schemes, despite posing a risk to human health. The sample preparation procedures depend largely on the chemical group; however, solid-phase extraction (SPE) is most often used due to the minimized sample handling, lower sample volume, and generally achieving lower limits of quantification (LOQs) compared to other extraction techniques. In terms of sample analysis, LC-based methods generally achieve lower limits of quantification (LOQs) compared to GC-based methods for the selected six groups of chemicals owing to their broader chemical coverage. In conclusion, since these chemicals are expected to be more frequently included in future HBM studies, it becomes evident that there is a pressing need for rigorous quality assurance programs to ensure better comparability of data. These programs should include the reporting of measurement uncertainty and facilitate inter-laboratory comparisons among the reporting laboratories. In addition, high-resolution mass spectrometry should be more commonly employed to enhance the specificity and selectivity of the applied analytical methodology since it is underrepresented in HBM. Furthermore, due to the scarcity of data on the levels of these CECs in urine, large population HBM studies are necessary to gain a deeper understanding of the associated risks.

Exposure of children and adolescents from Northeastern Slovenia to per- and polyfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are of high concern for the environment, wildlife, and human health due to their persistence and potential to cause adverse health effects. Despite political measures to restrict the production and distribution of PFAS and to limit the exposure of populations, PFAS can be measured at commonly high detection frequencies in human samples. Thus, this pilot study aimed to determine the serum concentrations of PFPA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, PFHpS, PFOS, PFHxA, PFDoDA, and PFBS in 113 girls and 112 boys (age 7-10 and 12-15) from Northeastern Slovenia - a rural area characterized by agricultural activities - and to identify potential sources of exposure using questionnaire data. PFAS were analysed by liquid chromatography coupled to mass spectrometry after phospholipid removal. 9 out of 12 analytes were detected at detection frequencies above 30%, with the highest geometric means (GM) being observed for PFOS (GM 1.9 ng/mL) > PFOA (GM 1.0 ng/mL) > PFHxS (GM 0.3 ng/mL) = PFNA (GM 0.3 ng/mL). We identified the participants' socio-economic status, age, sex, sampling region, public water supply, and the consumption of fish and seafood, cereals, and locally produced fruits, vegetables, and mushrooms as the predominant determinants of exposure. Furthermore, we compared our results with the serum and plasma concentrations reported for similar age groups in other studies and concluded that PFAS exposure in this highly agricultural area in Slovenia is notably low. This is the first study systematic HBM study of PFAS exposure in Slovenia, although it was conducted on a limited number of participants representative of rural and agricultural areas, it represents a good basis for upgrading the approach to a nationwide HBM study.

Removal of residues of psychoactive substances during wastewater treatment, their occurrence in receiving river waters and environmental risk assessment.

Continuous consumption combined with incomplete removal during wastewater treatment means residues of psychoactive substances (licit drugs, medications of abuse and illicit drugs) are constantly introduced into the aquatic environment, where they have the potential to affect non-target organisms. In this study, 17 drug residues of psychoactive substances were determined in wastewater influent, effluent and in receiving rivers of six Slovene municipal wastewater treatment plants employing different treatment technologies. Variations in removal efficiencies (REs) during spring, summer and winter were explored, and ecotoxic effects were evaluated using in silico (Ecological Structure-Activity Relationships software-ECOSAR) and in vivo (algal growth inhibition test) methods. Drug residues were detected in influent and effluent in the ng/L to µg/L range. In receiving rivers, biomarkers were in the ng/L range, and there was good agreement between measured and predicted concentrations. On average, REs were highest for nicotine, 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH), cocaine residues, and amphetamine (>90 %) and lowest for methadone residues (<30 %). REs were comparable between treatments involving activated sludge and membrane bioreactors, while the moving biofilm bed reactor (MBBR) removed cotinine, cocaine, and benzoylecgonine to a lesser extent. Accordingly, higher levels of nicotine and cocaine residues were detected in river water receiving MBBR discharge. Although there were seasonal variations in REs and levels of drug residues in receiving rivers, no general pattern could be observed. No significant inhibition of algal growth (Chlamydomonas reinhardtii) was observed for the tested compounds (1 mg/L) during 72 h and 240 h of exposure, although effects on aquatic plants were predicted in silico. In addition, environmental risk assessment revealed that levels of nicotine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), morphine, and 3,4-methylenedioxymethamphetamine (MDMA) pose a risk to aquatic organisms. Since nicotine and EDDP can have acute and chronic effects, the authors support regular monitoring of receiving surface waters, followed up by regulatory actions.