Capillary Electrophoresis as a Complementary Analytical Tool for the Separation and Detection of Nanoplastic Particles.

Capillary electrophoresis (CE) is presented as a technique for the separation of polystyrene nanoparticles (NPs, particle diameters ranging from 30 to 300 nm) through a bare fused silica capillary and ultraviolet detection. The proposed strategy was also assessed for other types of nanoplastics, finding that stronger alkaline conditions, with an ammonium hydroxide buffer (7.5%, pH = 11.9), enabled the separation of poly(methyl methacrylate), polypropylene, and polyethylene NP for the first time by means of CE for particle diameters below 200 nm. Particle behavior has been investigated in terms of its effective electrophoretic mobility, showing an increasing absolute value of effective electrophoretic mobility from the smaller to the larger sizes. On the other hand, the absolute value of surface charge density decreased with increasing size of NPs. It was demonstrated and quantified that the separation mechanism was a combination of linear and nonlinear electrophoretic effects. This work is the first report on the quantification of nonlinear electrophoretic effects on nanoplastic particles in a CE system.

Recent developments in mass spectrometry for the characterization of micro- and nanoscale plastic debris in the environment.

Development of analytical methods for the characterization (particle size determination, identification, and quantification) of the micro- and nanoscale plastic debris in the environment is a quickly emerging field and has gained considerable attention, not only within the scientific community, but also on the part of policy makers and the general public. In this Trends paper, the importance of developing and further improving analytical methodologies for the detection and characterization of sub-20-µm-range microplastics and especially nanoplastics is highlighted. A short overview of analytical methodologies showing considerable promise for the detection and characterization of such micro- and nanoscale plastic debris is provided, with emphasis on recent developments in mass spectrometry (MS)-based analytical methods. Novel hyphenated techniques combining the strengths of different analytical methods, such as field flow fractionation and MS-based detection, may be a way to adequately address the smallest fractions in plastic debris analysis, making such approaches worthwhile to be further explored.

Highly Selective Removal of Perfluorinated Contaminants by Adsorption on All-Silica Zeolite Beta.

Perfluorinated alkylated substances (PFASs) are widely used in industrial and commercial applications, leading to a widespread occurrence of these persistent and harmful contaminants in our environment. Removal of these compounds from surface and waste waters is being mandated by European and U.S. governments. Currently, there are no treatment techniques available that lower the concentrations of these compounds for large water bodies in a cost- and energy-efficient way. We hereby propose a hydrophobic, all-silica zeolite Beta material that is a highly selective and high-capacity adsorbent for PFASs, even in the presence of organic competitors. Advanced characterization data demonstrate that the adsorption process is driven by a very negative adsorption enthalpy and favorable steric factors.

Mothers and children are related, even in exposure to chemicals present in common consumer products.

BACKGROUND: Phthalates, bisphenol A (BPA) and triclosan (TCS) are detectable in the vast majority of people. Most humans are continuously exposed to these chemicals due to their presence in food or in everyday consumer products. The measurement of these compounds in family members may help to explore the impact of major lifestyle factors on exposure. Mothers and (young) children are especially interesting to study, as they mostly share considerable parts of daily life together. MATERIALS AND METHODS: Phthalate metabolites, bisphenol A (BPA) and triclosan (TCS) were measured in first morning void urine, collected in mother-child pairs (n = 129) on the same day. The mothers (27-45y) and their children (6-11y) were recruited in the Brussels agglomeration and rural areas of Belgium in the context of the European COPHES-DEMOCOPHES human biomonitoring project. Face-to-face questionnaires gathered information on major exposure sources and lifestyle factors. Exposure determinants were assessed by multiple linear regression analysis. RESULTS: The investigated compounds were detectable in nearly all mothers (92.8-100%) and all children (95.2-100%). The range (P90 vs. P10) of differences in urinary concentrations within each age group was for most compounds around 10-20 fold, and was very high for TCS up to 35 and 350-fold in children and mothers respectively. Some participants exceeded the tolerable daily intake guidelines as far as they were available from the European Food Safety Authority (EFSA). Overall, for BPA, the urinary concentrations were similar among both age groups. Most urinary phthalate metabolites were higher in children compared to the mothers, except for monoethyl phthalate (MEP). TCS levels were generally higher in the mothers. Despite the difference in mothers' and children's urinary concentrations, the creatinine-corrected levels were correlated for all biomarkers (Spearman rank r = 0.32 to 0.66, p < 0.001). Furthermore, for phthalates, similar home and lifestyle factors were associated with the urinary concentrations in both age groups: home renovation during last two years or redecoration during the last year for di-ethyl phthalate (DEP); PVC in home for di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP) and butyl benzyl phthalate (BBzP), and personal care products use for DiBP and DnBP. Based on questionnaire information on general food type consumption patterns, the exposure variability could not be explained. However, comparing the phthalate intake from the current study with earlier assessed Belgian food intake calculations for both ages, food in general was estimated to be the major intake source for di-ethyl hexyl phthalate (DEHP), with diminishing importance for BBzP, DiBP and DnBP. CONCLUSION: Our results confirm, that children and their mothers, sharing diets and home environments, also share exposure in common consumer products related chemicals. By collecting morning urine levels on the same day, and using basic questionnaires, suspected exposure routes could be unraveled.

Supercritical CO2 Extraction of Nannochloropsis sp.: A Lipidomic Study on the Influence of Pretreatment on Yield and Composition.

Algal lipids have gained wide interest in various applications ranging from biofuels to nutraceuticals. Given their complex nature composed of different lipid classes, a deep knowledge between extraction conditions and lipid characteristics is essential. In this paper, we investigated the influence of different pretreatments on lipid extraction with supercritical CO2 by a lipidomic approach. Pretreatment was found to double the total extraction yield, thereby reaching 23.1 wt.% comparable to the 26.9 wt.% obtained with chloroform/methanol. An increase in acylglycerides was concurrently observed, together with a nearly doubling of free fatty acids indicative of partial hydrolysis. Moreover, an alteration in the distribution of glyco- and phospholipids was noted, especially promoting digalactosyldiglycerides and phosphatidylcholine as compared to monogalactosyldiglycerides and phosphatidylglycerol. At optimized conditions, supercritical CO2 extraction provided a lipid extract richer in neutral lipids and poorer in phospholipids as compared to chloroform/methanol, though with a very similar fatty acid distribution within each lipid class.

Case Study on Screening Emerging Pollutants in Urine and Nails.

Alternative plasticizers and flame retardants (FRs) have been introduced as replacements for banned or restricted chemicals, but much is still unknown about their metabolism and occurrence in humans. We identified the metabolites formed in vitro for four alternative plasticizers (acetyltributyl citrate (ATBC), bis(2-propylheptyl) phthalate (DPHP), bis(2-ethylhexyl) terephthalate (DEHTP), bis(2-ethylhexyl) adipate (DEHA)), and one FR (2,2-bis (chloromethyl)-propane-1,3-diyltetrakis(2-chloroethyl) bisphosphate (V6)). Further, these compounds and their metabolites were investigated by LC/ESI-Orbitrap-MS in urine and finger nails collected from a Norwegian cohort. Primary and secondary ATBC metabolites had detection frequencies (% DF) in finger nails ranging from 46 to 95%. V6 was identified for the first time in finger nails, suggesting that this matrix may also indicate past exposure to FRs as well as alternative plasticizers. Two isomeric forms of DEHTP primary metabolite were highly detected in urine (97% DF) and identified in finger nails, while no DPHP metabolites were detected in vivo. Primary and secondary DEHA metabolites were identified in both matrices, and the relative proportion of the secondary metabolites was higher in urine than in finger nails; the opposite was observed for the primary metabolites. As many of the metabolites present in in vitro extracts were further identified in vivo in urine and finger nail samples, this suggests that in vitro assays can reliably mimic the in vivo processes. Finger nails may be a useful noninvasive matrix for human biomonitoring of specific organic contaminants, but further validation is needed.

Ultrasound assisted extraction combined with dispersive liquid-liquid microextraction (US-DLLME)-a fast new approach to measure phthalate metabolites in nails.

A new, fast, and environmentally friendly method based on ultrasound assisted extraction combined with dispersive liquid-liquid microextraction (US-DLLME) was developed and optimized for assessing the levels of seven phthalate metabolites (including the mono(ethyl hexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-oxo-MEHP), mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP), and mono-benzyl phthalate (MBzP)) in human nails by UPLC-MS/MS. The optimization of the US-DLLME method was performed using a Taguchi combinatorial design (L9 array). Several parameters such as extraction solvent, solvent volume, extraction time, acid, acid concentration, and vortex time were studied. The optimal extraction conditions achieved were 180 µL of trichloroethylene (extraction solvent), 2 mL trifluoroacetic acid in methanol (2 M), 2 h extraction and 3 min vortex time. The optimized method had a good precision (6-17 %). The accuracy ranged from 79 to 108 % and the limit of method quantification (LOQm) was below 14 ng/g for all compounds. The developed US-DLLME method was applied to determine the target metabolites in 10 Belgian individuals. Levels of the analytes measured in nails ranged between <12 and 7982 ng/g. The MEHP, MBP isomers, and MEP were the major metabolites and detected in every sample. Miniaturization (low volumes of organic solvents used), low costs, speed, and simplicity are the main advantages of this US-DLLME based method. Graphical Abstract Extraction and phase separation of the US-DLLME procedure.

Are nails a valuable non-invasive alternative for estimating human exposure to phthalate esters?

Most human biomonitoring studies conducted in the past year for assessing the human exposure to phthalate esters (PEs) employed measurements of PE metabolites in urine. Although urine is recognized as a valuable non-invasive matrix, it has also limitations regarding the short time window for exposure. Therefore, in this pilot feasibility study we aimed to assess the human exposure to seven PE metabolites (including mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-oxo-MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP) and mono-benzyl phthalate (MBzP)) using human nails. Paired nails and urine samples from the same individuals were used for comparison. Median levels of specific PE metabolites measured in nails and in spot urine of twenty Belgian individuals ranged from

Evaluation of exposure to phthalate esters and DINCH in urine and nails from a Norwegian study population.

Phthalate esters (PEs) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) used as additives in numerous consumer products are continuously released into the environment, leading to subsequent human exposure which might cause adverse health effects. The human biomonitoring approach allows the detection of PEs and DINCH in specific populations, by taking into account all possible routes of exposure (e.g. inhalation, transdermal and oral) and all relevant sources (e.g. air, dust, personal care products, diet). We have investigated the presence of nine PE and two DINCH metabolites and their exposure determinants in 61 adult residents of the Oslo area (Norway). Three urine spots and fingernails were collected from each participant according to established sampling protocols. Metabolite analysis was performed by LC-MS/MS. Metabolite levels in urine were used to back-calculate the total exposure to their corresponding parent compound. The primary monoesters, such as monomethyl phthalate (MMP, geometric mean 89.7ng/g), monoethyl phthalate (MEP, 104.8ng/g) and mono-n-butyl phthalate (MnBP, 89.3ng/g) were observed in higher levels in nails, whereas the secondary bis(2-ethylhexyl) phthalate (DEHP) and DINCH oxidative metabolites were more abundant in urine (detection frequency 84-100%). The estimated daily intakes of PEs and DINCH for this Norwegian population did not exceed the established tolerable daily intake and reference doses, and the cumulative risk assessment for combined exposure to plasticizers with similar toxic endpoints indicated no health concerns for the selected population. We found a moderate positive correlation between MEP levels in 3 urine spots and nails (range: 0.56-0.68). Higher frequency of personal care products use was associated with greater MEP concentrations in both urine and nail samples. Increased age, smoking, wearing plastic gloves during house cleaning, consuming food with plastic packaging and eating with hands were associated with higher levels in urine and nails for some of the metabolites. In contrast, frequent hair and hand washing was associated with lower urinary levels of monoisobutyl phthalate (MiBP) and mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), respectively.

A First Step in the Quest for the Active Constituents in Filipendula ulmaria (Meadowsweet): Comprehensive Phytochemical Identification by Liquid Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry.

Filipendula ulmaria (meadowsweet) is traditionally used for the treatment of inflammatory diseases and as a diuretic and antirheumatic. Extracts of Filipendulae herba are on the market in the European Union as food supplements. Nevertheless, its active constituents remain to be revealed. During this study, the phytochemical composition of Filipendulae Ulmariae Herba was comprehensively characterised for the first time with two complementary generic ultrahigh-performance liquid chromatography-photodiode array-accurate mass mass spectrometry methods. Selective ion fragmentation experiments with a hybrid quadrupole-orbital trap mass spectrometer significantly contributed to compound identification: a total of 119 compounds were tentatively identified, 69 new to F. ulmaria. A rich diversity of phenolic constituents was detected and only a few non-phenolic phytochemicals were observed. Metabolisation and pharmacological studies should be conducted to investigate which of these constituents or metabolites there of contribute to the activity of F. ulmaria after oral intake.

Development of a broad spectrum method for measuring flame retardants - overcoming the challenges of non-invasive human biomonitoring studies.

Flame retardants (FRs), such as polybrominated diphenyl ethers (PBDEs) and phosphate flame retardants (PFRs), are a diverse group of compounds that are used to improve fire safety in many consumer products, such as furniture, textiles, electronics, etc. As these compounds are potentially harmful for human health, there is a need to better understand human exposure. Exposure to environmental contaminants can be monitored by the measurement of external sources of exposures and also by the determination of contaminant levels in human samples. For ethical and practical reasons, noninvasive matrices, such as hair, are preferred but, unfortunately, not widely used due to methodological limitations. A major challenge is sample availability: only small amounts can be sampled per individual. Multi-residue methods are therefore essential in order to determine multiple compounds in low sample amounts. In the framework of the FP7 project (INFLAME), an analytical method for the simultaneous determination of PBDEs and PFRs in human hair has been optimized and validated. Before extraction, hair samples (200 mg) were denaturated in nitric acid (HNO3) for 25 min at 25 °C. Consecutively, the samples were extracted using a mixture of hexane:dichloromethane, and extracts were further fractionated on Florisil. Fraction A which contained PBDEs was additionally cleaned on acidified silica gel and measured by gas chromatography coupled with electron capture negative ionization mass spectrometry (GC-ECNI-MS), while fraction B containing PFRs was directly analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). This approach resulted in recoveries between 81-120% for PBDEs and 75-113% for PFRs (relative standard deviation (RSD) < 16%, n = 9). The optimized multi-residue method has been applied to 20 human hair samples. The obtained results indicated that the levels of PBDEs in hair samples were very low (0.2-12 ng/g) in relation to PBDE levels in human hair samples from other studies and most of the time below the method limit of quantification (LOQm). On the contrary, the PFR levels were relatively high as they were in the range of the levels previously found in dust samples (2-5,032 ng/g hair). We would like to highlight that the contribution of air and dust cannot be neglected (especially in the case of PFRs); therefore, we suggest that hair might be a good indicator of retrospective and integral exposure (which includes atmospheric deposition as well as endogenous mechanisms). Moreover, the aim of our study is focused on exposure assessment and levels detected in hair (independently of whether they come from internal or external exposure) and will significantly contribute to the exposure assessment.

Human biomonitoring of emerging pollutants through non-invasive matrices: state of the art and future potential.

Human biomonitoring (HBM) is a scientific technique that allows us to assess whether and to what extent environmental pollutants enter humans. We review here the current HBM efforts for organophosphate esters, emerging flame retardants, perfluoroalkyl substances, and phthalate esters. Use of some of these chemicals has already been banned or restricted; they are regularly detected in the environment, wildlife, and human matrices. Traditionally, blood and urine collection have been widely used as sampling methods. New non-invasive approaches (e.g., saliva, hair, nails) are emerging as valid alternatives since they offer advantages with respect to sampling, handling, and ethical aspects, while ensuring similar reliability and sensitivity. Nevertheless, the identification of biomarkers of exposure is often difficult because chemicals may be metabolized in the human body. For many of the above-mentioned compounds, the mechanisms of the favorable metabolization pathways have not been unraveled, but research on important metabolites that could be used as biomarkers of exposure is growing. This review summarizes the state of the art regarding human exposure to, (non-invasive) HBM of, and metabolism of major organophosphate esters, emerging flame retardants, perfluoroalkyl substances, and phthalate esters currently detected in the environment.

Detection of flavonoids in microalgae from different evolutionary lineages.

Flavonoids are important secondary plant metabolites believed to be present mainly in land plants. As phenolics were detected previously in microalgae using photometric assays, we wanted to investigate the nature of these phenolics and verify whether flavonoids are present. Therefore, in this study, we used state-of-the-art ultra-high performance liquid chromatography-two-dimensional mass spectrometry (UHPLC-MS/MS) technology to investigate whether microalgae also contain flavonoids. For this, representative microalgal biomass samples from divergent evolutionary lineages (Cyanobacteria, Rhodophyta, Chlorophyta, Haptophyta, Ochrophyta) were screened for a set of carefully selected precursors, intermediates, and end products of the flavonoid biosynthesis pathways. Our data unequivocally showed that microalgae contain a wide range of flavonoids and thus must possess the enzyme pool required for their biosynthesis. Further, some of the microalgae displayed an intricate flavonoid pattern that is compatible with the established basic flavonoid pathway as observed in higher plants. This implies that the flavonoid biosynthesis pathway arose much earlier in evolution compared to what is generally accepted.

Prenatal exposure to mixtures of per- and polyfluoroalkyl substances and organochlorines affects cognition in adolescence independent of postnatal exposure.

BACKGROUND: Studies on cognitive and neurodevelopmental outcomes have shown inconsistent results regarding the association with prenatal exposure to perfluoroalkyl substance (PFAS) and organochlorines. Assessment of mixture effects of correlated chemical exposures that persist in later life may contribute to the unbiased evaluation and understanding of dose-response associations in real-life exposures. METHODS: For a subset of the 4th Flemish Environment and Health Study (FLEHS), concentrations of four PFAS and six organochlorines were measured in respectively 99 and 153-160 cord plasma samples and 15 years later in adolescents' peripheral serum by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Sustained and selective attention were measured at 14-15 years with the Continuous Performance Test (CPT) and Stroop Test as indicators of potential neurodevelopmental deficits. Quantile g-computation was applied to assess the joint associations between prenatal exposure to separate and combined groups of PFAS and organochlorines and performance in the CPT and Stroop Test at adolescence. Subsequently, individual effects of each chemical compound were analyzed in mixed effects models with two sets of covariates. Analytical data at birth and at the time of cognitive assessment allowed for off-setting postnatal exposure. RESULTS: In mixtures analysis, a simultaneous one-quantile increase in the natural log-transformed values of PFAS and organochlorines combined was associated with a decrease in the mean reaction time (RT) and the reaction time variability (RTV) in the CPT (ß = -15.54, 95% CI:-29.64, -1.45, and ß = -7.82, 95% CI: -14.97, -0.67 respectively) and for the mixture of PFAS alone with RT (ß = -11.94, 95% CI: -23.29, -0.60). In the single pollutant models, these results were confirmed for the association between perfluorohexanesulfonate (PFHxS) with RT (ß = -17.95, 95% CI = -33.35, -2.69) and hexachlorobenzene with RTV in the CPT (ß = -5.78, 95% CI: -10.39, -0.76). Furthermore, the participants with prenatal exposure above the limit of quantification for perfluorononanoic acid (PFNA) had a significantly shorter RT and RTV in the CPT (ß = -23.38, 95% CI: -41.55, -5.94, and ß = -9.54, 95% CI: -19.75, -0.43, respectively). CONCLUSION: Higher prenatal exposure to a PFAS mixture and a mixture of PFAS and organochlorines combined was associated with better sustained and selective attention during adolescence. The associations seemed to be driven by PFHxS and were not linked to exposure levels at the time of assessment.

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69 % of spiked compounds using LC-HRMS and 20-60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants' own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from -9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

Exposure to Phthalates in European Children, Adolescents and Adults since 2005: A Harmonized Approach Based on Existing HBM Data in the HBM4EU Initiative.

Phthalates are mainly used as plasticizers and are associated inter alia with adverse effects on reproductive functions. While more and more national programs in Europe have started monitoring internal exposure to phthalates and its substitute 1,2-Cyclohexanedicarboxylic acid (DINCH), the comparability of results from such existing human biomonitoring (HBM) studies across Europe is challenging. They differ widely in time periods, study samples, degree of geographical coverage, design, analytical methodology, biomarker selection, and analytical quality assurance level. The HBM4EU initiative has gathered existing HBM data of 29 studies from participating countries, covering all European regions and Israel. The data were prepared and aggregated by a harmonized procedure with the aim to describe-as comparably as possible-the EU-wide general population's internal exposure to phthalates from the years 2005 to 2019. Most data were available from Northern (up to 6 studies and up to 13 time points), Western (11; 19), and Eastern Europe (9; 12), e.g., allowing for the investigation of time patterns. While the bandwidth of exposure was generally similar, we still observed regional differences for Butyl benzyl phthalate (BBzP), Di(2-ethylhexyl) phthalate (DEHP), Di-isononyl phthalate (DiNP), and Di-isobutyl phthalate (DiBP) with pronounced decreases over time in Northern and Western Europe, and to a lesser degree in Eastern Europe. Differences between age groups were visible for Di-n-butyl phthalate (DnBP), where children (3 to 5-year olds and 6 to 11-year olds) had lower urinary concentrations than adolescents (12 to 19-year-olds), who in turn had lower urinary concentrations than adults (20 to 39-year-olds). This study is a step towards making internal exposures to phthalates comparable across countries, although standardized data were not available, targeting European data sets harmonized with respect to data formatting and calculation of aggregated data (such as developed within HBM4EU), and highlights further suggestions for improved harmonization in future studies.

Concurrent Assessment of Phthalates/HEXAMOLL® DINCH Exposure and Wechsler Intelligence Scale for Children Performance in Three European Cohorts of the HBM4EU Aligned Studies.

Information about the effects of phthalates and non-phthalate substitute cyclohexane-1,2-dicarboxylic acid diisononyl ester (HEXAMOLL® DINCH) on children's neurodevelopment is limited. The aim of the present research is to evaluate the association between phthalate/HEXAMOLL® DINCH exposure and child neurodevelopment in three European cohorts involved in HBM4EU Aligned Studies. Participating subjects were school-aged children belonging to the Northern Adriatic cohort II (NAC-II), Italy, Odense Child Cohort (OCC), Denmark, and PCB cohort, Slovakia. In each cohort, children's neurodevelopment was assessed through the Full-Scale Intelligence Quotient score (FSIQ) of the Wechsler Intelligence Scale of Children test using three different editions. The children's urine samples, collected for one point in time concurrently with the neurodevelopmental evaluation, were analyzed for several phthalates/HEXAMOLL® DINCH biomarkers. The relation between phthalates/HEXAMOLL® DINCH and FSIQ was explored by applying separate multiple linear regressions in each cohort. The means and standard deviations of FSIQ were 109 ± 11 (NAC-II), 98 ± 12 (OCC), and 81 ± 15 (PCB cohort). In NAC-II, direct associations between FSIQ and DEHP's biomarkers were found: 5OH-MEHP+5oxo-MEHP (ß = 2.56; 95% CI 0.58-4.55; N = 270), 5OH-MEHP+5cx-MEPP (ß = 2.48; 95% CI 0.47-4.49; N = 270) and 5OH-MEHP (ß = 2.58; 95% CI 0.65-4.51; N = 270). On the contrary, in the OCC the relation between DEHP's biomarkers and FSIQ tended to be inverse but imprecise (p-value ≥ 0.10). No associations were found in the PCB cohort. FSIQ was not associated with HEXAMOLL® DINCH in any cohort. In conclusion, these results do not provide evidence of an association between concurrent phthalate/DINCHHEXAMOLLR DINCH exposure and IQ in children.

Supercritical CO2 Extraction of Bioactive Compounds from Mango (Mangifera indica L.) Peel and Pulp.

The potential of supercritical CO2 (SC-CO2) for the extraction of bioactive compounds from mango by-products was assessed. Carotenoid extraction was optimized using a design of experiments based on temperature (35, 55 and 70 °C), pressure (10 and 35 MPa) and co-solvent addition (0%, 10% and 20% of ethanol or acetone). Moreover, the co-extraction of phenolic acids, flavonoids and xanthonoids was evaluated in a subset of parameters. Finally, a comparison was made between SC-CO2 and a two-step organic solvent extraction of the bioactive compounds from the pulp and peel fractions of two Ecuadorian varieties. The optimal extraction temperature was found to be dependent on the bioactive type, with phenolics requiring higher temperature than carotenoids. The optimal overall conditions, focused on maximal carotenoids recovery, were found to be 55 °C, 35 MPa and 20% of ethanol. The main carotenoid was ß-carotene, while phenolics differed among the varieties. The bioactive content of the peel was up to 4.1-fold higher than in the pulp fraction. Higher antioxidant activity was found in the extracts obtained with organic solvents. SC-CO2 is a promising technology for the isolation of valuable compounds from mango by-products.

Changes in DNA Methylation in Arabidopsis thaliana Plants Exposed Over Multiple Generations to Gamma Radiation.

Previous studies have found indications that exposure to ionising radiation (IR) results in DNA methylation changes in plants. However, this phenomenon is yet to be studied across multiple generations. Furthermore, the exact role of these changes in the IR-induced plant response is still far from understood. Here, we study the effect of gamma radiation on DNA methylation and its effect across generations in young Arabidopsis plants. A multigenerational set-up was used in which three generations (Parent, generation 1, and generation 2) of 7-day old Arabidopsis thaliana plants were exposed to either of the different radiation treatments (30, 60, 110, or 430 mGy/h) or to natural background radiation (control condition) for 14 days. The parental generation consisted of previously non-exposed plants, whereas generation 1 and generation 2 plants had already received a similar irradiation in the previous one or two generations, respectively. Directly after exposure the entire methylomes were analysed with UPLC-MS/MS to measure whole genome methylation levels. Whole genome bisulfite sequencing was used to identify differentially methylated regions (DMRs), including their methylation context in the three generations and this for three different radiation conditions (control, 30 mGy/h, and 110 mGy/h). Both intra- and intergenerational comparisons of the genes and transposable elements associated with the DMRs were made. Taking the methylation context into account, the highest number of changes were found for cytosines followed directly by guanine (CG methylation), whereas only limited changes in CHG methylation occurred and no changes in CHH methylation were observed. A clear increase in IR-induced DMRs was seen over the three generations that were exposed to the lowest dose rate, where generation 2 had a markedly higher number of DMRs than the previous two generations (Parent and generation 1). Counterintuitively, we did not see significant differences in the plants exposed to the highest dose rate. A large number of DMRs associated with transposable elements were found, the majority of them being hypermethylated, likely leading to more genetic stability. Next to that, a significant number of DMRs were associated with genes (either in their promoter-associated region or gene body). A functional analysis of these genes showed an enrichment for genes related to development as well as various stress responses, including DNA repair, RNA splicing, and (a)biotic stress responses. These observations indicate a role of DNA methylation in the regulation of these genes in response to IR exposure and shows a possible role for epigenetics in plant adaptation to IR over multiple generations.

Urinary Polycyclic Aromatic Hydrocarbon Metabolites Are Associated with Biomarkers of Chronic Endocrine Stress, Oxidative Stress, and Inflammation in Adolescents: FLEHS-4 (2016-2020).

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants of public health concern. Multiple biological mechanisms have been hypothesized to contribute to PAHs-associated adverse health effects. Little is known about the impact of PAHs on endocrine stress and inflammation in adolescence. We examined 393 Flemish adolescents (14-15 years) cross-sectionally, measured urinary concentrations of hydroxylated naphthalene, fluorene, phenanthrene and pyrene metabolites, and calculated the sum of all measured metabolites. We determined hair cortisol concentration (HCC) as endocrine stress biomarker, leucocyte counts and neutrophil-lymphocyte ratio (NLR) in peripheral blood as inflammatory biomarkers, and urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) concentration as oxidative stress biomarker. Exposure-response associations were analyzed by multiple regression, adjusted for a priori selected covariates. A doubling of 1-hydroxypyrene concentration was associated with a factor of 1.13 (95% CI: 1.03, 1.24) increase in HCC and a factor of 1.07 (95% CI: 1.02, 1.13) increase in 8-oxodG. Doublings of 2- and 3-hydroxyphenanthrene concentrations were associated with a factor of 1.08 (95% CI: 1.02, 1.14) and 1.06 (95% CI: 1.00, 1.12) increase in 8-oxodG, respectively. Doubling of 2-hydroxyphenanthrene and of the sum of 2- and 3-hydroxyfluorene was associated with, respectively, a factor of 1.08 (95% CI: 1.02, 1.14) and 1.06 (95% CI: 1.01, 1.13) increase in NLR. Our results indicate the glucocorticoid pathway as a potential target for PAH exposure in adolescents and suggest oxidative stress, endocrine stress, and inflammation in adolescence as underlying mechanisms and early markers for PAH-related adverse health effects.