Urinary metabolites of phthalates and di-iso-nonyl cyclohexane-1,2-dicarboxylate (DINCH)-Czech mothers' and newborns' exposure biomarkers.

To assess human exposure to hazardous diesters of phthalic acid and their substitute di-iso-nonyl cyclohexane-1,2-dicarboxylate (DINCH), concentrations of their metabolites in urine should be determined. For the purpose of this biomonitoring study, a quick and easy sample preparation procedure for the simultaneous determination of eight phthalate and four DINCH metabolites in urine has been implemented and validated. Following the enzymatic hydrolysis and dilution with methanol, the sample is ready for the analysis by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The limits of quantification of this method ranged from 0.15 to 0.4 ng/mL urine with recoveries of 60-126% and repeatability in the range of 1-11%. The validated method was subsequently used for the analysis of urine samples collected from mothers and their newborn children living in two localities of the Czech Republic (Karvina and Ceske Budejovice, 2013-2014). Median concentrations of all measured metabolites (∑metabolites) were slightly lower in the urine samples collected from children (77.7 ng/mL urine) compared to their mothers (115.3 ng/mL urine), but no correlation was found between the concentrations of target compounds in children's and mothers' urine samples. The analyte with the highest concentration was monobutyl phthalate (MBP), with the median concentration of 32.1 ng/mL urine in the urine samples collected from mothers and 17.2 ng/mL urine in the samples collected from their children. This compound was also found in almost all of the measured samples. On the other hand, mono-isononyl-cyclohexane-1,2-dicarboxylate (MINCH) was not found in any urine sample. The most contaminated samples were collected from children living in the Karvina locality (median ∑metabolites 103.2 ng/mL urine), where the mono (2-ethyl-5-carboxypentyl) phthalate (cx-MEHP) compound contributed 43% to the total content of phthalate metabolites in newborns' urine. The results from our study are comparable with concentrations of the target compounds from Norway and Germany and lower compared to the results concluded in Sweden.

Integration of five groups of POPs into one multi-analyte method for human blood serum analysis: An innovative approach within biomonitoring studies.

Within this study, a new analytical strategy was developed and validated for the simultaneous determination of 78 organohalogenated contaminants in human blood serum, namely 40 flame retardants (FRs) including 7 "novel" brominated and chlorinated FRs (novel FRs), 19 perfluoroalkylated substances (PFASs), 11 organochlorine pesticides (OCPs) and 8 polychlorinated biphenyls (PCBs). The integral sample preparation procedure was implemented for the isolation of non-polar compounds, based on three-step solvent extraction using a mixture of n-hexane:diethylether (9:1, v/v), followed by purification using a solid-phase extraction (SPE) on a Florisil® column. For isolation of more polar and lipophobic analytes, the remaining fraction from the first extraction step was further processed, using a modified QuEChERS method. Depending on the polarity and volatility of target compounds, either gas chromatography coupled to (tandem) mass spectrometry (GC-MS/(MS)), or ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC-MS/MS), was employed for their identification/quantification. Within the subsequent pilot study, the new validated procedure was successfully applied to the monitoring of organohalogenated contaminants in 38 samples of human blood serum obtained from Prague, Czech Republic. From 78 targeted analytes, 10 PFASs, 10 OCPs, 8 PCBs and 6 BFRs were detected in serum at concentrations above method quantification limits (MQLs). In the serum samples, the amounts of determined PFASs were in the range<0.01-8.97ngmL-1 (mean 0.631ngmL-1), OCPs and PCBs ranged from <0.1-1626ngg-1 lw (mean 40.0ngg-1 lw) and<0.1-481ngg-1 lw (mean 63.3ngg-1 lw), respectively.

Determinants of prenatal exposure to perfluoroalkyl substances in the Slovak birth cohort.

BACKGROUND: Perfluoroalkyl substances (PFASs) are man-made fluorinated compounds with endocrine-disrupting properties, detected in 99% of serum samples worldwide and associated with adverse childhood health outcomes. The aim of this study was to describe determinants of prenatal exposure to PFASs in Slovakia. METHODS: This study was based on Slovak multicentric prospective mother-child cohort PRENATAL (N = 796). Cord blood samples were collected within 2010-2012 and PFASs were analyzed in a subpopulation of 322 newborns. Concentrations of perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) were measured in the samples of cord blood using an ultrahigh-performance liquid chromatography- mass spectrometry (U-HPLC-MS) method. From questionnaires, we obtained information on medical history of mother, socio-demographic factors, nutrition and environmental factors. Association between maternal characteristics and PFASs exposure was analyzed using multivariable linear regression models. RESULTS: The highest cord blood concentration (geometric mean ±â€¯SD) was observed for PFOA (0.79 ±â€¯2.21 ng/ml) followed by PFOS (0.36 ±â€¯2.56 ng/ml), PFNA (0.20 ±â€¯2.44 ng/ml) and PFHxS (0.07 ±â€¯2.36 ng/ml). Primiparity was associated with higher levels of all four PFAS: PFOS (exp. ß = 1.25; 95%CI[1.03; 1.53]), PFOA (exp. ß = 1.49; 95%CI[1.18; 1.89]), PFNA (exp. ß = 1.30; 95%CI[1.05; 1.60]) and PFHxS (exp. ß = 1.49; 95%CI [1.20; 1.86]). In addition, maternal age category 29 years and more was associated with higher PFNA and PFHxS levels (exp. ß = 1.27; 95%CI[1.04; 1.55] and exp. ß = 1.30; 95%CI[1.06; 1.60], respectively) and higher educational level of mother was associated with higher PFNA levels (exp. ß = 1.32; 95%CI[1.04; 1.68]). Higher fish consumption was associated with lower PFNA levels (exp. ß = 0.49; 95%CI[0.26; 0.92]). CONCLUSIONS: We observed that PFASs cord blood concentrations were comparable or lower than those measured in western or northern European countries. We identified parity as the main determinant of PFASs exposure in our population and maternal age and education as factors that might be associated with exposure to certain PFASs.

Multiclass analytical method for the determination of natural/synthetic steroid hormones, phytoestrogens, and mycoestrogens in milk and yogurt.

Within this study, a new method enabling monitoring of various estrogenic substances potentially occurring in milk and dairy products was proposed. Groups of compounds fairly differing in physico-chemical properties and biological activity were analyzed: four natural estrogens, four synthetic estrogens, five mycoestrogens, and nine phytoestrogens. Since they may pass into milk mainly in glucuronated and sulfated forms, an enzymatic hydrolysis was involved prior to the extraction based on the QuEChERS methodology. For the purification of the organic extract, a dispersive solid-phase extraction (d-SPE) with sorbent C18 was applied. The final analysis was performed by ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS). Method recovery ranged from 70 to 120% with a relative standard deviation (RSD) value lower than 20% and limits of quantification (LOQs) in the range of 0.02-0.60 µg/L (0.2-6.0 µg/kg dry weight) and 0.02-0.90 µg/kg (0.2-6.0 µg/kg dry weight) for milk and yogurt, respectively. The new procedure was applied for the investigation of estrogenic compounds in 11 milk samples and 13 yogurt samples from a Czech retail market. Mainly phytoestrogens were found in the studied samples. The most abundant compounds were equol and enterolactone representing 40-90% of all estrogens. The total content of phytoestrogens (free and bound) was in the range of 149-3870 µg/kg dry weight. This amount is approximately 20 times higher compared to non-bound estrogens.

Evaluation of 11 polycyclic aromatic hydrocarbon metabolites in urine of Czech mothers and newborns.

Polycyclic aromatic hydrocarbons (PAHs) represent a large group of ubiquitous contaminants of the environment, including food chain where they are released as by-products of incomplete combustion of an organic matter. Epidemiological studies have shown that exposure to PAHs correlated with increased incidence of cancer. Carcinogenicity is associated mainly with metabolites that are formed during metabolic degradation of these substances in exposed organism. In this study monohydroxylated PAHs (OH-PAHs), the major metabolites excreted into urine, were determined in 531 urine samples collected from mothers and their newborns from two localities of the Czech Republic - heavily air polluted Karvina and control locality of Ceske Budejovice and in two sampling rounds - August-October 2013 (summer, less air polluted season) and January-April 2014 (winter, more air polluted season). From all targeted analytes, naphthalene-2-ol was the most abundant compound present in 100% of the samples and it represented also the analyte with the highest concentration. Median concentration of ΣOH-PAHs in the urine of children was on average 1.6 times lower compared to the respective mother which correlates with higher intake of PAHs by mothers. ΣOH-PAHs concentrations determined in mothers' urine collected in the summer were comparable in both localities. No significant increase occurred in Ceske Budejovice in winter, while in samples from the Karvina region a statistically significant difference (α=0.05) in the amount of ΣOH-PAHs was observed. The median concentrations of ΣOH-PAHs in mothers' urine samples in the winter were 1.5 times higher than in the summer in the same locality. The amounts of ΣOH-PAHs in newborns' urine from Karvina in the winter season were 1.5 times higher than in the summer collected in the same locality and 3.3 times higher when compared with the less polluted locality of Ceske Budejovice.

A novel strategy for the determination of polycyclic aromatic hydrocarbon monohydroxylated metabolites in urine using ultra-high-performance liquid chromatography with tandem mass spectrometry.

In this study, a novel analytical approach for the determination of 11 monohydroxylated polycyclic aromatic hydrocarbon metabolites (OH-PAHs) in urine was developed and validated. The rapid, simple and high-throughput sample preparation procedure based on ethyl acetate extraction and subsequent purification by dispersive solid-phase extraction (d-SPE) employing a Z-Sep sorbent is used for the first time. For the identification/quantification of target compounds, ultra-high-performance liquid chromatography (U-HPLC) interfaced with tandem mass spectrometry (MS/MS) was applied. The results of validation experiments performed on the Standard Reference Material (SRM) 3673 (organic contaminants in non-smokers' urine) were in accordance with the certified values. The method recoveries ranged from 77 to 114 % with the relative standard deviation lower than 20 % and the quantification limits in the range of 0.010-0.025 ng mL(-1) (except for benzo[a]pyren-3-ol with 0.9 ng mL(-1)). Within the pilot study, the new method was used for the analysis of OH-PAHs in 50 urine samples. The concentrations of ΣOH-PAHs were in the range of 0.87-63 ng mL(-1) (1600-33,000 ng g(-1) creatinine), with naphthalen-2-ol (2-OH-NAP) and phenanthren-1-ol (1-OH-PHEN) being the most abundant exposure biomarkers detected in all samples.

Perfluoroalkyl substances (PFASs) and other halogenated compounds in fish from the upper Labe River basin.

This study summarizes results on levels of 25 perfluoroalkyl substances (PFASs), three hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols and four hydroxylated derivates of polybrominated diphenyl ethers (OH-PBDEs) in 59fish samples collected in nine localities on two major rivers from the Czech Republic. To identify potential sources of these chemicals, several sampling sites located close to highly industrialized areas were also involved. The major PFAS representatives, perfluorooctane sulfonate (PFOS), C9-C14 perfluoroalkyl carboxylic acids (PFCAs) and perfluorooctane sulfonamide (FOSA) were detected in 100% fish samples. The concentration ranges of individual substances in the respective groups of PFASs were as follows: 0.572-61.3ngg(-)(1) wet weight (ww) for Tot-PFOS, 0.007-0.121ngg(-)(1)ww for perfluoroalkane sulfonates (PFSAs) (without PFOS isomers), 0.007-22.0ngg(-)(1)ww for PFCAs and 0.026-7.76ngg(-)(1)ww for FOSA. The highest contents of ∑PFASs (51.9ngg(-)(1)ww and 47.8ngg(-)(1)ww) were measured in fish muscle tissue from the locality Trmice situated on the Bílina River and Verdek on the Labe River, where chemical and/or textile industry is located. From 11 targeted BFRs, five compounds (α-HBCD, ß-HBCD, γ-HBCD, TBBPA and 2,4,6-tribromophenol (TBP)) were determined in analyzed samples. The concentration ranged as follows: 3.15-1211ngg(-)(1) lipid weight (lw) for ∑HBCD, 4.99-203ngg(-)(1)lw for TBBPA and 1.76-107ngg(-)(1)lw for 2,4,6-TBP.

Multi-analyte method for the analysis of various organohalogen compounds in house dust.

In the present study, a novel analytical approach for the simultaneous determination of 27 brominated flame retardants (BFRs), namely polybrominated diphenyl ethers (PBDEs), isomers of hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA) and several novel BFRs (NBFRs), together with 18 perfluoroalkyl substances (PFASs) in indoor dust was developed and validated. To achieve integrated isolation of analytes from the sample and their fractionation, a miniaturized method based on matrix solid phase dispersion (MSPD) was employed. Principally, after mixing the dust (<0.1 g) with the Florisil(®), the mixture was applied on the top of a sorbent (Florisil(®)) placed in glass column and then analytes were eluted using solvents with different polarities. For the identification/quantification of target compounds largely differing in polarity, complementary techniques represented by gas and liquid chromatography coupled to tandem mass spectrometry (GC-MS/MS and LC-MS/MS) were used. The results of validation experiments, which were performed on the SRM 2585 material (for PBDEs, HBCDs and TBBPA), were in accordance with the certified/reference values. For other analytes (NBFRs and PFASs), the analysis of an artificially contaminated blank dust sample was realized. The method recoveries for all target compounds ranged from 81 to 122% with relative standard deviations lower than 21%. The quantification limits were in the range of 1-25 ng g(-1) for BFRs and 0.25-1 ng g(-1) for PFASs. Finally, 18 samples (6 households × 3 sampling sites) were analyzed. The high variability between concentrations of PFASs and BFRs in the dust samples from various households as well as collecting sites in a respective house was observed. The total amounts of PFASs and BFRs were in the range of 1.58-236 ng g(-1) (median 10.6 ng g(-1)) and 39.2-2320 ng g(-1) (median 325 ng g(-1)), respectively. It was clearly shown that dust from the indoor environment might be a significant source of human exposure to various organohalogen pollutants.

Brominated flame retardants and perfluoroalkyl substances in sediments from the Czech aquatic ecosystem.

This study reports results of analysis of various groups of halogenated chemicals, including brominated flame retardants (BFRs), such as polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs), tetrabromobisphenol A (TBBPA) and perfluoroalkyl substances (PFASs) in 31 sediment samples collected in different localities of the Czech Republic. In this survey, identification of potential sources of these compounds was also performed; therefore several sampling sites located in highly industrialized areas were involved. Concentrations of target groups of analytes determined in sediments from several Czech rivers examined within this study decreased in the following order: decabromodiphenyl ether (BDE 209) >>> TBBPA~HBCDs~linear perfluorooctane sulfonate (L-PFOS)>other PBDEs~perfluorinated carboxylic acids (PFCAs)~perfluorooctane sulfonamide (FOSA). When compared the contamination by two monitored groups of halogenated compounds, the total content of ∑BFRs was significantly higher, i.e. in the range from the method quantification limit (MQL) to 528 µg/kg dry weight (dw) (median value 5.68 µg/kg dw), than the total concentration of ∑PFASs, that was in the range from MQL to 25.5 µg/kg dw (median value 1.48 µg/kg dw). The extremely highest content of BFR group (265-528 µg/kg dw) was found in sediments collected in sampling sites on the Labe and Luzická Nisa Rivers, which are located in highly chemical industrialized areas and also in the sample from the locality Lampertice obtained from the sedimentation tank close to the factory processing and storing waste. These concentrations were a little bit higher or comparable to those found in similar highly industrialized areas worldwide.

The determination of perfluoroalkyl substances, brominated flame retardants and their metabolites in human breast milk and infant formula.

In the present study, a novel analytical approach for the simultaneous determination of 18 perfluoroalkyl substances (PFASs) and 11 brominated flame retardants (BFRs) including their hydroxylated metabolites and brominated phenols has been developed and validated for breast milk and infant formula. The sample preparation procedure based on extraction using acetonitrile and subsequent purification by dispersive solid-phase extraction (d-SPE) employing C18 sorbent is rapid, simple and high-throughput. Ultra-high performance liquid chromatography (UHPLC) interfaced with a tandem mass spectrometry (MS/MS) was employed for the identification/quantification of these compounds. The method recoveries of target compounds for both matrices ranged from 80% to 117% with relative standard deviations lower than 28% and quantification limits in the range of 3-200 pg/mL for milk and 5-450 pg/g for infant formula. Within the pilot study, the new method was used for the analysis of PFASs and BFRs in 50 human breast milks and six infant formulas. In the breast milk samples the total contents of PFASs and BFRs were in the range of 38-279 and 45-16,200 pg/mL, respectively. The most abundant PFASs detected in all tested breast milk samples were perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS), the latter contaminant was present not only as a linear form but also as a branched isomers. The incidence of BFRs was lower, the only representatives of this group, tetrabromobiphenol A (TBBPA) and α-hexabromocyclododecane (α-HBCD), were detected in less than 30% of breast milk samples. None of the infant formulas contained BFRs, traces of either PFOS, PFOA or PFNA were found in three samples.

Rapid and simple method for determination of hexabromocyclododecanes and other LC-MS-MS-amenable brominated flame retardants in fish.

In this study, a novel analytical approach for simultaneous determination of hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols, and four hydroxylated derivatives of polybrominated diphenyl ethers (OH-PBDEs) was developed and validated for muscle tissue of both lean and fatty fish. The rapid, simple, and high-throughput sample-preparation procedure was based on acetonitrile extraction then purification by dispersive solid-phase extraction (d-SPE) with a combination of C18 and primary-secondary amine (PSA) sorbents. Ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS-MS) was used for identification and quantification of the analytes. Method recovery for both matrices ranged from 80 to 115% with relative standard deviations (RSDs) <13% for all analytes. Limits of quantification (LOQs) were in the range 0.1-1 µg kg(-1) wet weight. The validated method was used for analysis of brominated compounds in 32 fish and five bivalve samples collected from different European markets within the monitoring survey organized in the framework of the CONffIDENCE project. Of the 12 targeted analytes, only α-HBCD, 2,4-dibromophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP) were quantified in the samples. α-HBCD was found in six fish samples (herring and mackerel) in the range of 0.8-2.5 µg kg(-1) wet weight. 2,4-DBP and 2,4,6-TBP were found in three blue mussel samples in the range of 19.6-43.5 and 2.3-7.5 µg kg(-1) wet weight, respectively.

Occurrence of brominated flame retardants and perfluoroalkyl substances in fish from the Czech aquatic ecosystem.

This study reports results of analysis of various groups of halogenated compounds, including brominated flame retardants (BFRs), such as polybrominated diphenylethers (PBDEs), hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA) and perfluoroalkyl substances (PFASs) in 48 fish samples collected in eight localities from the Czech Republic. In this survey, identification of potential sources of these chemicals was also performed; therefore several sampling sites located in highly industrialized areas were also selected. Perfluorooctanesulfonate (PFOS) was dominating in all tested fish samples. Generally, the content of ∑BFRs was significantly lower, i.e. in the range of 0.21-19.9 µg/kg wet weight, ww (median value 2.37 µg/kg ww) compared to the concentration of ∑PFASs that was in the range of 0.15-877 µg/kg ww (median value 8.5 µg/kg ww). The extremely high content of PFOS (842 µg/kg ww) was found in fish muscle tissue from the locality situated on the Bílina River, where chemical industry is located. This concentration was comparable to those found in similar highly industrialized areas worldwide.