Quantification and stability assessment of urinary phenolic and acidic biomarkers of non-persistent chemicals using the SPE-GC/MS/MS method.

Nowadays, people are exposed to numerous man-made chemicals, many of which are ubiquitously present in our daily lives, and some of which can be hazardous to human health. Human biomonitoring plays an important role in exposure assessment, but complex exposure evaluation requires suitable tools. Therefore, routine analytical methods are needed to determine several biomarkers simultaneously. The aim of this study was to develop an analytical method for quantification and stability testing of 26 phenolic and acidic biomarkers of selected environmental pollutants (e.g., bisphenols, parabens, pesticide metabolites) in human urine. For this purpose, a solid-phase extraction coupled with gas chromatography and tandem mass spectrometry (SPE-GC/MS/MS) method was developed and validated. After enzymatic hydrolysis, urine samples were extracted using Bond Elut Plexa sorbent, and prior to GC, the analytes were derivatized with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA). Matrix-matched calibration curves were linear in the range of 0.1-1000 ng mL-1 with R > 0.985. Satisfactory accuracy (78-118%), precision (< 17%), and limits of quantification (0.1-0.5 ng mL-1) were obtained for 22 biomarkers. The stability of the biomarkers in urine was assayed under different temperature and time conditions that included freezing and thawing cycles. All tested biomarkers were stable at room temperature for 24 h, at 4 °C for 7 days, and at -20 °C for 18 months. The total concentration of 1-naphthol decreased by 25% after the first freeze-thaw cycle. The method was successfully used for the quantification of target biomarkers in 38 urine samples.

Reviewing the variability in urinary concentrations of non-persistent organic chemicals: evaluation across classes, sampling strategies and dilution corrections.

Various biomonitoring studies have been carried out to investigate the exposure of populations by measuring non-persistent organic chemicals in urine. To accurately assess the exposure, study designs should be carefully developed to maximise reproducibility and achieve good characterization of the temporal variability. To test these parameters, the intraclass correlation coefficients (ICCs) are calculated from repeated measurements and range from poor (<0.4) to excellent (≥0.75). Several studies have reported ICCs based on diverse study designs, but an overview, including recommendations for future studies, was lacking. Therefore, this review aimed to collect studies describing ICCs of non-persistent organic chemicals, discuss variations due to study design and formulate recommendations for future studies. More than 60 studies were selected, considering various chemical classes: bisphenols, pyrethroids, parabens, phthalates, alternative plasticizers and phosphate flame retardants. The variation in ICCs for an individual chemical was high (e.g. ICC of propyl paraben = 0.28-0.91), showing the large impact of the study design and of the specific exposure sources. The highest ICCs were reported for parabens (median = 0.52), while lowest ICCs were for 3-phenoxybenzoic acid (median = 0.08) and bisphenol A (median = 0.20). Overall, chemicals that had an exposure source with high variation, such as the diet, showed lower ICCs than those with more stable exposure sources, such as indoor materials. Urine correction by specific gravity had an overall positive effect on reducing the variability of ICCs. However, this effect was mostly seen in the adult population, while specific compounds showed less variation with creatinine correction. Single samples might not accurately capture the exposure to most non-persistent organic chemicals, especially when small populations are sampled. Future studies that examine compounds with low ICCs should take adequate measures to improve accuracy, such as correcting dilution with specific gravity or collecting multiple samples for one participant.

Parameters of ovarian reserve in relation to urinary concentrations of parabens.

BACKGROUND: Parabens are synthetic chemicals commonly used in cosmetics, pharmaceuticals, food and beverage processing as antimicrobial preservatives. In experimental animals, parabens exposure was associated with adverse effects on female reproduction. Despite the widespread use of parabens little is known about their effect on female fecundity. The objective of the current analysis was to evaluate the associations of urinary parabens concentrations with parameters of ovarian reserve among women undergoing treatment in a fertility clinic. METHODS: Five hundred eleven female aged 25-39 years who attended the infertility clinic in central region of Poland for diagnostic purposes were recruited between September 2014 and February 2019. Urinary concentrations of parabens were measured by a validated gas chromatograohy ion-tap mass spectrometry method. Parameters of ovarian reserve were: antral follicle count (AFC), anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and estradiol (E2) levels. RESULTS: The geometric mean of specific gravity adjusted urinary concentrations of methyl (MP), ethyl (EP), propyl (PP), butyl (BP) and izobutyl paraben (iBuP) were 107.93 µg/L, 12.9 µg/L, 18.67 µg/L, 5.02 µg/L and 2.80 µg/L. Urinary concentrations of PP in the third quartile of exposure ((50-75] percentyl) were inversely associated with antral follicle count (p = 0.048), estradiol level (p = 0.03) and positively with FSH concentration (p = 0.026). MP, EP, BP and iBuP parabens were not associated any with parameters of ovarian reserve. CONCLUSIONS: Chronic exposure to PP may potentially contributing to reduced fecundity and impair fertility. As this is one of the first study to investigate the potential effect of parabens on ovarian reserve further epidemiological studies with longer duration of observation are needed.

Environmental exposure to parabens and sperm chromosome disomy.

Parabens are widely used as antimicrobial preservatives in cosmetics, pharmaceuticals, food and beverage processing due to their board spectrum of activity, inertness, and low cost. The study population consisted of 156 men under 45 years of age who attended the infertility clinic for diagnostic purposes with normal semen concentration of 15-300 mln/ml. Participants were interviewed and provided a semen sample. The parabens concentrations: ethyl paraben (EP), butyl paraben (BP), methyl paraben (MP), and iso-butyl paraben (iBuP) were analyzed in the urine using a validated gas chromatography ion-tap mass spectrometry method. The positive association was found between urinary level of BP and XY18 disomy (p = 0.045) and PP and disomy of chromosome 13 (p = 0.007). This is the first study to examine these relationships, and replication of our findings is needed before the association between parabens concentration in urine and aneuploidy can be fully defined. These findings may be of concern due to increased parabens use.

Personal environmental exposure to plasticizers and organophosphate flame retardants using silicone wristbands and urine: Patterns, comparisons, and correlations.

Plasticizers (PLs) and organophosphate flame retardants (OPFRs) are ubiquitous in the environment due to their widespread use and potential for leaching from consumer products. Environmental exposure is a critical aspect of the human exposome, revealing complex interactions between environmental contaminants and potential health effects. Silicone wristbands (SWBs) have emerged as a novel and non-invasive sampling device for assessing personal external exposure. In this study, SWBs were used as a proxy to estimate personal dermal adsorption (EDdermal) to PLs and OPFRs in Belgian participants for one week; four morning urine samples were also collected and analyzed for estimated daily intake (EDI). The results of the SWBs samples showed that all the participants were exposed to these chemicals, and the exposure was found to be highest for the legacy and alternative plasticizers (LP and AP), followed by the legacy and emerging OPFRs (LOPFR and EOPFR). In urine samples, the highest levels were observed for metabolites of diethyl phthalate (DEP), di-isobutyl phthalate (DiBP) and di-n-butyl phthalate (DnBP) among LPs and di(2-ethylhexyl) terephthalate (DEHT) for APs. Outliers among the participants indicated that there were other sources of exposure that were not identified. Results showed a significant correlation between EDdermal and EDI for DiBP, tris (2-butoxyethyl) phosphate (TBOEP) and triphenyl phosphate (TPhP). These correlations indicated their suitability for predicting exposure via SWB monitoring for total chemical exposure. The results of this pilot study advance our understanding of SWB sampling and its relevance for predicting aggregate environmental chemical exposures, while highlighting the potential of SWBs as low-cost, non-invasive personal samplers for future research. This innovative approach has the potential to advance the assessment of environmental exposures and their impact on public health.

Investigation of in vitro biotransformation of tris (1-chloro-2-propyl) phosphate and confirmation in human urine.

Tris (1-chloro-2-propyl) phosphate (TCIPP) is one of the major organophosphate flame retardants present in the indoor and outdoor environment. Knowledge of biotransformation pathways is important to elucidate potential bioavailability and toxicity of TCIPP and to identify relevant biomarkers. This study aimed to identify TCIPP metabolites through in vitro human metabolism assays and finally to confirm these findings in urine samples from an occupationally exposed population to propose new biomarkers to accurately monitor exposure to TCIPP. TCIPP was incubated with human liver microsomes and human liver cytosol to identify Phase I and Phase II metabolites, by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Using a suspect-screening approach, the established biomarkers bis (1-chloro-2-propyl) hydrogen phosphate (BCIPP) and 1-hydroxy-2-propyl bis (1-chloro-2-propyl) phosphate (BCIPHIPP) were identified. In addition, carboxyethyl bis (1-chloro-2-propyl) phosphate (TCIPP-M1), bis (1-chloropropan-2-yl) (-oxopropan-2-yl) phosphate (TCIPP-M2) and 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3) were identified. TCIPP-M2, an intermediate product, was not reported before in literature. In urine samples, apart from BCIPP and BCIPHIPP, TCIPP-M1 and TCIPP-M3 were identified for the first time. Interestingly, BCIPP showed the lowest detection frequency, likely due to the poor sensitivity for this compound. Therefore, TCIPP-M1 and TCIPP-M3 could serve as potential additional biomarkers to more efficiently monitor TCIPP exposure in humans.

Urinary Bisphenol A Concentrations and Parameters of Ovarian Reserve among Women from a Fertility Clinic.

BACKGROUND: Human exposure to environmentally widespread endocrine disruptors, especially bisphenol A (BPA), has been suggested to affect reproductive health. Animal studies indicate that BPA may play a role in the process of reproduction and impact on maturing oocytes, meiotic cell division or fertilization rate. Nevertheless, data regarding the effects of exposure to BPA on women's ovarian function are still limited. Therefore, the aim of the current study is to assess the effects of environmental exposure to BPA on ovarian reserve. METHODS: The study participants consisted of 511 women in reproductive age (25-39 years) who attended an infertility clinic for diagnosis, due to the couples' infertility. BPA urinary concentrations were assessed by the validated gas chromatography ion-trap mass spectrometry method. The ovarian reserve was assessed using ovarian reserve parameters: Hormones concentrations: E2 (estradiol), FSH (follicle stimulating hormone), AMH (anti-Müllerian hormone), and AFC (antral follicle count). RESULTS: In the present study, the negative association between BPA urinary concentrations and AMH (p = 0.02) and AFC (p = 0.03) levels was found. Exposure to BPA was not related to other examined parameters of ovarian reserve (FSH, E2). CONCLUSIONS: Our results suggest that BPA exposure may affect women ovarian reserve parameters and reduce ovarian reserve. As this is one of the first studies of its kind, the findings need confirmation in a further investigation.

Concentrations of urinary biomarkers and predictors of exposure to pyrethroid insecticides in young, Polish, urban-dwelling men.

Pyrethroid insecticides are a class of pesticides with multiple agricultural and residential applications. However, widespread use of these chemicals may pose a threat to human health. Biomarkers of pyrethroid exposure are frequently detected in populations around the world, but some groups may be underrepresented. Moreover, there is an ongoing debate on factors contributing to pyrethroid burden in humans. To address these problems, we measured urinary biomarkers of pyrethroid exposure in urine samples from 306 young men living in urban area of Lódz, Poland, and gathered questionnaire data to identify predictors of exposure. Limit of detection (LOD) of gas chromatography-mass spectrometry (GC-MS) method was 0.1 ng/mL for all quantified pyrethroid metabolites, namely cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (trans-DCCA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), and 3-phenoxybenzoic acid (3-PBA). Detection rate ranged from 32% (cis-DBCA) to 76% (trans-DCCA). Concentrations of urinary biomarkers in studied sample were in lower range of these observed in similar studies, with unadjusted geometric means (GMs) of most prevalent biomarkers, trans-DCCA and 3-PBA, equal to 0.268 and 0.228 ng/mL, respectively. As for questionnaire data, the statistical analysis revealed that non-dietary factors, especially dog ownership and pesticide use on household pets, contribute significantly to urinary trans-DCCA and 3-PBA concentrations (p ≤ 0.009). Moreover, a few dietary sources of exposure were identified, such as seeds and nuts consumption for 3-PBA (p < 0.001) and vegetable juice intake for trans-DCCA (p = 0.015). Multivariate analyses further highlighted the importance of non-dietary factors in pyrethroid exposure. Compared to other works, our results confirm widespread exposure to pyrethroids observed in other studies and stress the role of residential pyrethroid use in pyrethroid burden in humans.

Triclosan exposure and in vitro fertilization treatment outcomes in women undergoing in vitro fertilization.

Triclosan (TCS) is a widespread environmental endocrine-disrupting chemical. Animal and in vitro studies suggested that triclosan may affect homesostasis of sex and thyroid hormones and impact on reproduction. Due to limited data derived from human epidemiological studies, this study was performed to examine the association between urinary concentration of triclosan and in vitro reproductive outcomes (methaphase II (MII) oocyte yield, top quality embryo, fertilization rate, implantation rate, and clinical pregnancy) among women from infertility clinic. The study participants were enrolled in an Infertility Center in Poland. A total of 450 women aged 25-45 (n = 674 IVF cycles) provided urine samples. The urinary concentrations of triclosan were evaluated using validated gas chromatography ion-tap mass spectrometry method. Clinical outcomes of IVF treatment were abstracted from patients electronic chart records. Triclosan was detected in urine of 82% of women with geometric mean 2.56 ± 6.13 ng/mL. Urinary concentrations of triclosan were associated with decrease implantation rate (p = 0.03). There were no association between other examined IVF outcomes: MII oocytes, embryo quality, fertilization rate, and exposure to triclosan. As this is one of the first study on this topic, studies among larger and more diverse population are needed to confirm the results.

Urinary bisphenol A concentrations and in vitro fertilization outcomes among women from a fertility clinic.

Bisphenol A (BPA) is a widespread environmental endocrine disrupting chemical. Although many animals and in vitro studies reported that BPA may affect female fertility through the effect on maturing oocytes and meiotic cell division, but the data from human studies are limited and inconclusive. The study was conducted to examine the association between urinary BPA concentration and in vitro reproductive outcomes (metaphase II (MII) oocyte yield, top quality embryo, fertilization rate, implantation rate and clinical pregnancy) among women from an infertility clinic. The study participants were enrolled in the Infertility Center in Poland. 450 women aged 24-44 (n = 674 IVF cycles) provided urine samples. The urinary concentrations of BPA were evaluated using validated gas chromatography ion-tap mass spectrometry method. Clinical outcomes of IVF treatment were abstracted from patients electronic chart records. To assess the relationship between urinary BPA concentrations early examined reproductive outcomes generalized linear mixed models were used. The detection rate of BPA in urine samples was 98% and the geometric mean 1.59 ± 2.15 ng/ml. A significant decrease was observed between urinary concentration of BPA and implantation (p = 0.04) and decreased MII oocyte count (p = 0.03). There was no association between other examined IVF outcomes: embryo quality, fertilization rate and clinical pregnancy and BPA exposure. Exposure to BPA may have a negative effect during the early stages of human development. The studies among the larger and more diverse population are needed to confirm the results.

Evaluation of 1-year urinary excretion of eight metabolites of synthetic pyrethroids, chlorpyrifos, and neonicotinoids.

Synthetic pyrethroids, chlorpyrifos, and neonicotinoids are representatives of non-persistent insecticides ubiquitously used against insects all over the world. Their widespread use causes prevalent exposure to these compounds, which may be hazardous to human health. The insecticides have short biological half-lives and are mostly excreted in urine within 24 h after entering the human body; thus, the urinary concentration of their metabolites is highly dependent on the time elapsed between exposure and sample collection. Considering the within-day fluctuations in urinary concentration, one randomly collected sample may cause misclassification of long-term exposure. We evaluated the variability of excretion of eight insecticide metabolites in 24-h urine samples collected from 14 volunteers once or twice per month over 12 consecutive months. High detection frequency above 70% for non-specific metabolites of pyrethroid, chlorpyrifos, and neonicotinoids confirmed widespread exposure to these insecticides in the studied population. A long-term variability of exposure was assessed based on intraclass correlation coefficient (ICC). We found relatively low variability of excretion for non-specific pyrethroid metabolites and 3,5,6-trichloro-2-pyridinol (ICC > 0.75), but poor repeatability for 6-chloronicotinic acid. Constantly higher ICCs were observed for daily excretion than for unadjusted concentrations. Seasonal differences were observed for 3,5,6-trichloro-2-pyridinol and 6-chloronicotinic acid, with the highest and the lowest median concentration, respectively, in the summer. Due to high ICC values and lack of seasonal variations, one 24-h urine sample was considered sufficient to characterize long-term excretion of non-specific pyrethroid metabolites in non-occupationally exposed population. In addition, we calculated the daily intake (DI) for cypermethrin, permethrin, deltamethrin, and chlorpyrifos. The estimated DI values were mostly below the acceptable daily intake, which indicates that the evaluated exposure is non-hazardous to the population.

Triclosan exposure and ovarian reserve.

The objective of the current analysis was to investigate the associations of urinary triclosan concentrations with parameters of ovarian reserve. Five hundred eleven female aged 25-39 years who attended the infertility clinic for diagnostic purposes were recruited. Urinary concentrations of triclosan were measured by a validated gas chromatograohy ion-tap mass spectrometry method. Parameters of ovarian reserve were: antral follicle count (AFC), anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and estradiol (E2) levels. Urinary concentrations of triclosan decrease antral follicle count. There were no statistically significant associations between other parameters of ovarian reserve (estradiol, FSH and AMH levels) and triclosan concentrations. Triclosan exposure may negatively affect antral follicle count, a marker of ovarian reserve. As the data on triclosan exposure and ovarian reserve are scarce additional study is needed to confirm the results.

Human Semen Quality, Sperm DNA Damage, and the Level of Urinary Concentrations of 1N and TCPY, the Biomarkers of Nonpersistent Insecticides.

The aim of the study was to evaluate the association between environmental exposure to nonpersistent insecticides and semen quality (concentration, motility, morphology, computer-aided semen analysis [CASA] parameters, and sperm DNA damage). Urine samples ( n = 315) collected from men who attended the infertility clinic with normal semen concentration of 15 to 300 mln/ml and age under 45 years were analyzed for two metabolites (1-naphthol [1N] and 3,5,6-trichloro-2-pyridinol [TCPY]) of nonpersistent insecticides. Participants provided semen, blood, and saliva samples; additionally, men filled a detailed questionnaire. The results identified that urinary TCPY concentration was significantly associated with a decrease in motility; also there was a positive association between TCPY and DNA fragmentation index (DFI). 1N concentration was negatively associated with a percentage of sperm with normal morphology and positively with one of the CASA parameters (curvilinear velocity [VCL]). The results suggest that environmental exposure to nonpersistent insecticides may have an impact on semen quality parameters and sperm DNA damage.

Off-line microextraction by packed sorbent combined with on solid support derivatization and GC-MS: Application for the analysis of five pyrethroid metabolites in urine samples.

A novel, fast and eco-friendly analytical method using microextraction by packed sorbent coupled to large volume injection-gas chromatography-mass spectrometry (MEPS-LVI-GC-MS) was developed for the determination of five pyrethroid metabolites (cis-2,2-dimethyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-cyclopropanecarboxylic acid, cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acids, cis-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzoic acid) in human urine. MEPS was performed off-line using a manually-operated semiautomatic syringe (eVol), and several parameters including the sample pH, extraction sorbent, washing solvent, volume and type of elution solvent and number of draw-eject cycles were optimized. Analytes were extracted from enzymatically hydrolyzed urine using a C18 solid phase with subsequent simultaneous derivatization and elution with a mixture of 1,1,1,3,3,3-hexafluoroisopropanol and diisopropylcarbodiimide in n-hexane (on-line derivatization). The optimized method was validated, with linearity established from 0.05 to 25ngmL-1 and R values > 0.99. Obtained quantification limits were in the range of 0.06-0.08ngmL-1, and the precision expressed as relative standard deviation (RSD) was below 14% for all of the analytes. The method was cross-validated with a reference approach based on liquid-liquid extraction-gas chromatography-mass spectrometry (LLE-GC-MS) by analyzing 21 urine samples.

Urinary Bisphenol A Levels and Male Fertility.

Bisphenol A (BPA) is a high-production volume industrial chemical found in many consumer products. BPA is a suspected potent endocrine disruptor, with endocrine-disrupting properties demonstrated in animal studies. Few human studies have examined bisphenol A exposure in relation to male fertility and, results are divergent. The aim of the study is to examine the associations between urinary BPA concentration and male fertility. Bisphenol A urinary concentrations were measured using gas chromatography coupled with tandem mass spectrometry in 315 men under 45 years of age with normal sperm concentration (⩾15 mln/ml) recruited from a male reproductive health clinic. Participants were interviewed and provided a semen sample. BPA was detected in 98.10% of urine samples, with a median concentration of 1.87 µg/l (1.63 µg/ g creatinine). A multiple linear regression analysis identified a positive association between the urinary concentrations of bisphenol A 25th-50th percentile and total sperm sex chromosome disomy ( p = .004). Also when modeled as continuous variable urinary BPA concentration increased total sperm sex chromosome disomy ( p = .01). Urinary concentration of BPA also increase the percentage of immature sperm (HDS) ( p = .018) and decrease motility ( p = .03). The study provides evidence that exposure to BPA is associated with poorer semen quality. Future studies are needed to confirm these findings.

Environmental levels of triclosan and male fertility.

Triclosan is a synthetic chemical with broad antimicrobial activity that has been used extensively in consumer products, including personal care products, textiles, and plastic kitchenware, although the exposure which is widespread evidence from human studies is scarce. Our study aims to investigate the relationship between triclosan exposure and male fertility. Triclosan (TCS) urinary concentrations were measured using gas chromatography coupled with tandem mass spectrometry in 315 men recruited from a male reproductive health clinic with normal sperm concentration (≥ 15 mln/ml) (WHO 2010) under 45 years of age. Participants were interviewed and provided a semen sample. TCS was detected in 84.13% of urine samples, with a median concentration of 2.83 µg/l (2.57 µg/g creatinine). A multiple linear regression analysis showed a positive association between the urinary concentrations of triclosan 50th-75th percentile and ≥ 50 percentile and percentage of sperm with abnormal morphology (p = 0.016 and p = 0.002, respectively). The study provides evidence that exposure to triclosan is associated with poorer semen quality. Future studies are needed to confirm these findings.

Human Semen Quality, Sperm DNA Damage, and the Level of Reproductive Hormones in Relation to Urinary Concentrations of Parabens.

OBJECTIVE: The aim of this study was to evaluate the association between environmental exposure to parabens and semen quality parameters [main semen parameters, computer-aided semen analysis (CASA parameters], sperm chromatin structure, and the level of reproductive hormones in men [follicle-stimulating hormone (FSH), testosterone, estradiol]. METHODS: Urine samples collected from 315 men who attended the infertility clinic for diagnostic purposes with normal semen concentration of 15 to 300 mln/mL were analyzed for five parabens concentrations using a validated gas chromatography ion-tap mass spectrometry method. Participants were interviewed and also provided a semen, saliva, and blood samples. RESULTS: Urinary parabens concentrations were significantly associated with an increase in the percentage of sperm with abnormal morphology, in sperm with high DNA stainability and a decrease in the percentage of motility and testosterone level. CONCLUSIONS: This is one of the first study on this topic, so the observation of the relationship between parabens and semen quality warrants further investigation.