Skip to main content
PMC home page
Environmental Health Perspectives logoLink to Environmental Health Perspectives
. 2015 Mar 17;123(7):705–711. doi: 10.1289/ehp.1408325

Measurement of Total and Free Urinary Phenol and Paraben Concentrations over the Course of Pregnancy: Assessing Reliability and Contamination of Specimens in the Norwegian Mother and Child Cohort Study

Virginia T Guidry 1,✉, Matthew P Longnecker 2, Heidi Aase 3, Merete Eggesbø 4, Pål Zeiner 5, Ted Reichborn-Kjennerud 6, Gun P Knudsen 6, Randi J Bertelsen 7, Xiaoyun Ye 8, Antonia M Calafat 8, Stephanie M Engel 1
PMCID: PMC4492266  PMID: 25782115

Abstract

Background

Exposures to environmental phenols and parabens may be harmful, especially in utero. Prior studies have demonstrated high within-person variability of urinary concentrations across pregnancy.

Objectives

We sought to measure phenol and paraben biomarker concentrations for the Norwegian Mother and Child Cohort (MoBa) study, assess within-person variability, and investigate any possible external phenol or paraben contamination of specimens.

Methods

We collected three spot urine samples at approximately 17, 23, and 29 weeks gestation in a hospital setting and added a preservative containing ethyl paraben. We measured urinary concentrations and within-person variability for phenols and parabens in a MoBa sample (n = 45), including a subgroup of 15 participants previously randomly selected for a bisphenol A (BPA) exposure study who had unusually high total BPA concentrations. Additionally, we compared reliability results for total, conjugated, and free concentrations of phenolic compounds.

Results

We detected total and free BPA, butyl paraben, propyl paraben, and methyl paraben in 100% of samples, total benzophenone-3 in 95% of samples, and infrequently detected free benzophenone-3 and total and free 2,4-dichlorophenol and 2,5-dichlorophenol. Intraclass correlation coefficients (ICCs) for total, conjugated, and free concentrations ranged from relatively low for BPA to moderate for propyl paraben. ICCs were generally similar overall and by subgroup.

Conclusions

Using conjugated concentrations improved reliability estimates only for BPA. Measuring total and free concentrations, an approach that may be useful for future studies, allowed us to identify likely BPA and butyl paraben contamination of archived MoBa urine specimens.

Citation

Guidry VT, Longnecker MP, Aase H, Eggesbø M, Zeiner P, Reichborn-Kjennerud T, Knudsen GP, Bertelsen RJ, Ye X, Calafat AM, Engel SM. 2015. Measurement of total and free urinary phenol and paraben concentrations over the course of pregnancy: assessing reliability and contamination of specimens in the Norwegian Mother and Child Cohort Study. Environ Health Perspect 123:705–711; http://dx.doi.org/10.1289/ehp.1408325

Introduction

Phenols, including bisphenol A (BPA) and benzophenone-3, and parabens, which also contain a phenolic ring, are found in a number of common consumer products [Centers for Disease Control and Prevention (CDC) 2009, 2010]. The ubiquity of human exposure to phenolic compounds has sparked questions regarding toxicity, especially from exposures during sensitive periods, for example, in utero (Bushnik et al. 2010; Calafat et al. 2008, 2010; Mahalingaiah et al. 2008; Vandenberg et al. 2010; Wolff et al. 2008; Woodruff et al. 2011). Prenatal exposures to phenols have been associated with decreased gestational age at birth (BPA and benzophenone-3) (Tang et al. 2013), decreased birth weight (2,4-dichlorophenol and 2,5-dichlorophenol) (Philippat et al. 2013; Wolff et al. 2008), and child behavior problems, with some evidence of sex differences (BPA) (Braun et al. 2009, 2011b; Harley et al. 2013; Perera et al. 2012). Concentrations of phenol and paraben biomarkers in spot urine samples, which provide exposure measurements at one point in time, are frequently used to estimate recent exposures. However, many phenolic compounds are rapidly excreted (Völkel et al. 2002), and the degree to which a single measure can be used to represent longer-term or cumulative exposure depends partly on population-specific factors such as geographic location, demographic characteristics, and lifestyle (Koch et al. 2014; Meeker et al. 2013; Ye et al. 2009).

Phenolic compounds in humans can be metabolized via conjugation [Matthews et al. 2001; U.S. Environmental Protection Agency (EPA) 2002]; these conjugated species are readily excreted via urine (Völkel et al. 2002). Generally, conjugated phenolic compounds comprise the bulk (≥ 90%) of the total biomarker concentration in urine (Koch et al. 2012; Liao and Kannan 2012; U.S. EPA 2002; Völkel et al. 2002; Ye et al. 2005b). The remainder of the total urinary phenolic biomarker concentration consists of unconjugated (free) species that can arise from several sources: a) free phenolic compounds that passed through the body without conjugation, representing a biologically active form (Koch et al. 2012; U.S. EPA 2002; Völkel et al. 2002); b) free phenolic compounds that entered the sample via contamination during collection or processing (Koch et al. 2012; U.S. EPA 2002; Völkel et al. 2002); c) conjugated phenolic compounds hydrolyzed to the free form by enzymes in local tissues (Waechter et al. 2007); or d) conjugated phenolic compounds hydrolyzed to the free form following improper storage or handling (Ye et al. 2007).

In field studies, exposure assessment is complicated by the ubiquitous presence of phenolic compounds in the environment. This has implications for population exposure levels as well as protocols for sample collection, processing, and analysis, which must be designed to minimize the potential for external sample contamination (Calafat and Needham 2009). For example, the use of BPA in laboratory supplies necessitates fastidious handling to ensure sample integrity (Ye et al. 2013). Both collection conditions and preservatives to prevent bacterial growth in urine can contribute to contamination of urine samples with BPA (Longnecker et al. 2013). Similarly, personal care products used by laboratory technicians that contain benzophenone-3 and parabens have contaminated biological samples (Ye et al. 2013).

Accurate exposure assessment is critical to establishing reliable estimates of associations between background phenolic exposures and health outcomes. Exposures to select phenols (Meeker et al. 2013; Woodruff et al. 2011; Ye et al. 2009), and the reliability of a single spot urine sample to estimate exposure to phenolic compounds over the course of pregnancy can vary substantially across populations (Braun et al. 2011a, 2012; Jusko et al. 2014; Meeker et al. 2013; Philippat et al. 2013; Quirós-Alcalá et al. 2013). These findings suggest that reliability estimates for these compounds may not be generalizable across populations, particularly when patterns of product usage or population exposures may be different.

The purpose of our study was 3-fold. First, we wanted to measure the concentrations of phenols and parabens in archived urine collected from women in the Norwegian Mother and Child Cohort Study (MoBa). Second, we sought to characterize the reliability of a single spot urine sample to estimate environmental exposure to these compounds over the course of pregnancy in the MoBa cohort. Third, given the recently reported potential for contamination of MoBa specimens (Longnecker et al. 2013), we investigated whether using the free and conjugated concentrations of phenolic biomarkers might improve reliability estimates.

Methods

Study design. MoBa is a prospective population-based pregnancy cohort conducted by the Norwegian Institute of Public Health (Magnus et al. 2006). MoBa was initiated for the study of exposures and diseases related to pregnancy and child development, including potential health effects associated with exposures to environmental chemicals (Magnus et al. 2006). From 1999 through 2008, pregnant women across Norway were recruited at their first ultrasound visit (approximately 17–18 weeks gestation), and 38.5% of invited women consented to participate. The final cohort includes 90,700 mothers and 108,000 pregnancies (Norwegian Institute of Public Health 2013).

Beginning in 2002, as part of the standard data collection protocol, MoBa participants provided a spot urine specimen in a collection cup at the ultrasound visit. Because of evidence of bacterial growth upon receipt at the central processing facility, MoBa revised the protocol to include the addition of a preservative to urine specimens. Laboratory staff transferred 8 mL of urine from each specimen to a Vacutainer tube (Urinalysis Preservative Plus Urine Tube; BD Diagnostics) with a mixture of three preservatives (sodium propionate 94%, ethyl paraben 5.6%, and chlorhexidine 0.4%) and shipped the specimen, unrefrigerated, to a central processing facility (Hoppin et al. 2006; Rønningen et al. 2006). At the processing facility, urine samples were partitioned into 930-μL aliquots and stored at –80°C until use (Rønningen et al. 2006).

From November 2007 through December 2008, a subset of MoBa participants (n = 671) donated a spot urine sample at approximately 17 weeks of gestation plus additional urine specimens at approximately 23 and 29 weeks of gestation for a reliability substudy. These participants were recruited from hospitals representing four regions of Norway [northwest (Sunnmøre Hospital HF Ålesund), southwest (Stavanger University Hospital HF), central (St Olavs Hospital HF), and east (Østfold Hospital HF Fredrikstad)] and specimens were collected using the protocol described above.

From the subset of 671, we randomly selected 30 participants for this reliability substudy. Another 15 participants from the same subset of 671 that were part of a previous random selection for an unpublished pilot study were also included. The pilot study showed that these 15 women had the highest measured total BPA concentrations (range, 22.9–52.1 μg/g creatinine); we included them to determine whether the high concentrations could possibly be attributed to contamination. The final study sample thus included 135 urine samples from 45 women.

For quality control (QC), in 2011 we collected spot urine specimens from 10 Norwegian women using the same collection protocol and materials used for MoBa participants, except that the specimens did not require shipping because they were collected from processing facility staff. QC samples were pooled before transfer to the tube with preservative and aliquotted before analysis.

Informed consent was obtained from each MoBa participant on recruitment. The MoBa study was approved by The Regional Committee for Medical Research Ethics in South-Eastern Norway (#2011/1386). The reliability substudy was also approved by The Regional Committee for Medical Research Ethics in South-Eastern Norway and the University of North Carolina Institutional Review Board. The involvement of the CDC laboratory did not constitute engagement in human subjects research.

Measurement of phenol and paraben concentrations. In 2012, we analyzed urine specimens for free and total (conjugated plus free) concentrations of nine compounds: BPA, butyl paraben, ethyl paraben, methyl paraben, propyl paraben, benzophenone-3, triclosan, 2,4-dichlorophenol, and 2,5-dichlorophenol. Triclosan results are presented elsewhere (Bertelsen et al. 2014). Analyses were conducted using online solid phase extraction–isotope dilution–high performance liquid chromatography–tandem mass spectrometry (online SPE-HPLC-isotope dilution-MS/MS) (Ye et al. 2005a, 2008) at the CDC (Atlanta, GA, USA) in three analytical batches. All urine specimens from a participant were analyzed in the same analytical batch in random order. The pooled urine for QC was distributed into 15 aliquots, and we included 5 aliquots in each of the three analytical batches. Phenol and paraben concentrations are reported in micrograms per liter and micrograms per gram of creatinine to account for urine dilution.

Statistical analyses. All statistical analyses were conducted separately for the complete sample (n = 45 participants, 135 samples; hereafter “complete sample”) as well as for the two subgroups: randomly selected participants with no previously measured BPA concentrations (n = 30 participants, 90 samples; hereafter “random sample”) and participants with high total BPA concentrations in a previous random selection (n = 15 participants, 45 samples; hereafter “high-BPA subgroup”).

We summarized characteristics of the study population using data from version 7 of the quality-assured MoBa data files released for research in June 2012. We also computed coefficients of variation (CVs) for all QC specimens and between the three QC batches. Between-batch CVs based on the QC samples were generally low (< 15%) except when mean concentrations were near the limit of detection (LOD) (see Supplemental Material, Table S1).

For the study sample, we calculated conjugated concentrations as the difference between total and free concentrations, when both total and free concentrations were detectable. Negative conjugated values occurred when free concentrations exceeded total concentrations at levels close to the LOD, which are subject to higher measurement error. We computed geometric means for total, free, and conjugated concentrations with all time periods combined as well as for each of the three time points. We used the instrumental values for total phenol and paraben concentrations below the LOD; for free phenol and paraben concentrations, instrumental values below the LOD were not provided (i.e., missing) because the majority of free concentrations were so low that the instrument software recorded them as “no peak” or “< 0.” We analyzed data only for compounds with > 50% detection frequency; analytes fulfilling this criterion all had 95–100% detection frequency, so we did not employ any additional correction (e.g., imputation) for these values. To examine potential contamination of specimens, we also compared the distributions of free phenol and paraben concentrations as a percentage of the total concentration for the five compounds with sufficient detection: BPA, butyl paraben, methyl paraben, propyl paraben, and benzophenone-3.

We assessed the proportion of variance attributed to between-person variability across the three time points in pregnancy using intraclass correlation coefficients (ICCs). ICCs typically range from 0 to 1 with a value close to 1 indicating high temporal reliability of measurements, with most of the variance attributable to differences between subjects rather than within-person differences between time points. We calculated ICCs for total, free, and conjugated concentrations in micrograms per liter and micrograms per gram creatinine. ICCs and 95% confidence intervals (CIs) were computed with natural logarithm–transformed data using sums of squares from generalized linear models generated by the intracc macro for SAS (Hamer 1995). For comparison, ICCs were also computed using random effects models to estimate within- and between-person variance, assuming an unstructured covariance matrix to allow each covariance to be uniquely estimated and provide maximum model flexibility. The ICCs computed with random-effects models produced results similar to those shown and thus are not presented.

All statistical analyses were conducted using SAS software version 9.3 (SAS Institute Inc., Cary, NC, USA).

Results

Most MoBa participants were married or cohabitating, had at least some college education, and had a moderate income (Table 1). Although nearly half were ever-smokers, most did not smoke during the current pregnancy. The median age of participants at delivery was 30 years with a range of 20–41 years (random sample: median = 31, range 20–41 years; high-BPA subgroup: median = 29, range 21–37 years). The majority of specimens (79.2%) spent 1 day in transit with the remainder spending 2–5 days.

Table 1.

Characteristics of complete study sample and subgroups [n (%)].a

Characteristic Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15)
Civil status
Married 20 (44.4) 14 (46.7) 6 (40.0)
Cohabitating 21 (46.7) 13 (43.3) 8 (53.3)
Single 2 (4.4) 1 (3.3) 1 (3.3)
Missing 2 (4.4) 2 (6.7) 0 (0.0)
Completed maternal education
< College/university 12 (26.7) 9 (30.0) 3 (20.0)
College/university (up to and including 4 years) 24 (53.3) 16 (53.3) 8 (53.3)
> College/university 6 (13.3) 2 (6.7) 4 (26.7)
Missing 3 (6.7) 3 (10.0) 0 (0.0)
Gross income ($US)
< 34,800 8 (17.8) 5 (16.7) 3 (20.0)
34,800–69,600 27 (60.0) 18 (60.0) 9 (60.0)
> 69,600 7 (15.6) 4 (13.3) 3 (20.0)
Missing 3 (6.7) 3 (10.0) 0 (0.0)
Ever smoked
No 23 (51.1) 16 (53.3) 7 (46.7)
Yes 21 (46.7) 13 (43.3) 8 (53.3)
Missing 1 (2.2) 1 (3.3) 0 (0.0)
Smoked during this pregnancy
No 38 (84.4) 26 (86.7) 12 (80.0)
Sometimes or daily 4 (8.8) 2 (6.7) 2 (13.3)
Missing 3 (6.7) 2 (6.7) 1 (6.7)
aComplete sample (n = 45 participants, 135 samples); random sample: randomly selected participants with no previously measured BPA concentrations (n = 30 participants, 90 samples); high-BPA subgroup: participants with high BPA concentrations in a previous random selection (n = 15 participants, 45 samples).
Open in a new tab

Percent detection was comparable among subgroups but varied by analyte, with most compounds detected in the majority of samples (Tables 2 and 3). We detected free and total concentrations in 100% of samples for four analytes (BPA, butyl paraben, methyl paraben, propyl paraben). We detected total benzophenone-3 in the majority (> 92%) of samples and free benzophenone-3 in approximately 10%. Total concentrations of 2,4-dichlorophenol and 2,5-dichlorophenol were detected in < 50% of samples, with negligible detection of free concentrations for these analytes. Ethyl paraben could not be reliably quantitated because it was a component of the urine preservative added to prevent bacterial growth. Additionally, two samples that did not pass the laboratory QA (quality assurance)/QC criteria for free phenolic compounds concentrations were not reported.

Table 2.

Limit of detection, percent detectable results, and geometric means for complete study sample and subgroups (μg/L).a

Analyte and speciesb Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15)
n (%) > LOD GM all time periods GM 17, 23, 29 weeks n (%) > LOD GM all time periods GM 17, 23, 29 weeks n (%) > LOD GM all time periods GM 17, 23, 29 weeks
Bisphenol A (LOD = 0.4 μg/L)
Total 135 (100) 7.7 8.9, 7.5, 6.7 90 (100) 7.5 7.8, 7.4, 7.2 45 (100) 8.1 11.6, 7.8, 5.9
Free 133 (100) 5.4 7.2, 5.2, 4.3 89 (100) 5.3 6.0, 5.6, 4.5 44 (100) 5.6 10.5, 4.6, 3.8
Conjugated 133 (100) 1.3 1.1, 1.3, 1.6 89 (100) 1.2 1.0, 1.1, 1.4 44 (100) 1.7 1.5, 1.8, 1.9
Butyl paraben (LOD = 0.2 μg/L)
Total 135 (100) 6.3 6.7, 6.1, 5.9 90 (100) 6.4 7.6, 6.0, 5.7 45 (100) 6.1 5.3, 6.4, 6.5
Free 133 (100) 1.8 1.8, 1.8, 1.8 89 (100) 1.8 1.8, 1.8, 1.9 44 (100) 1.8 1.8, 1.9, 1.8
Conjugated 133 (100) 3.1 3.6, 3.1, 2.8 89 (100) 3.5 4.8, 3.8, 2.5 44 (100) 2.5 2.0, 2.2, 3.3
Methyl paraben (LOD = 1.0 μg/L)
Total 135 (100) 1235.7 1,332, 1,249, 1,134 90 (100) 1213.7 1,297, 1,256, 1,097 45 (100) 1280.9 1,405, 1,234, 1,212
Free 133 (100) 25.7 24.1, 26.4, 26.7 89 (100) 25.7 24.7, 25.2, 27.3 44 (100) 25.7 22.9, 28.9, 25.5
Conjugated 133 (100) 1204.5 1,308, 1,212, 1,104 89 (100) 1181.0 1,271, 1,218, 1,066 44 (100) 1253.5 1,392, 1,202, 1,186
Propyl paraben (LOD = 0.2 μg/L)
Total 135 (100) 32.3 29.5, 37.8, 30.2 90 (100) 36.4 40.1, 38.3, 31.4 45 (100) 25.4 16.1, 36.9, 27.8
Free 133 (100) 1.6 1.4, 1.7, 1.6 89 (100) 1.7 1.6, 1.6, 1.7 44 (100) 1.4 1.1, 1.9, 1.3
Conjugated 133 (100) 28.9 25.5, 35.1, 27.3 89 (100) 33.1 35.7, 36.2, 28.2 44 (100) 22.1 12.3, 33.0, 25.6
Benzophenone‑3 (LOD = 0.4 μg/L)
Total 128 (95) 6.1 4.4, 7.0, 7.3 86 (96) 6.7 5.3, 6.5, 8.7 42 (93) 5.0 3.0, 7.9, 5.2
Free 13 (10) NC NC 9 (10) NC NC 4 (9) NC NC
Conjugated 13 (10) NC NC 9 (10) NC NC 4 (9) NC NC
2,4-DCP (LOD = 0.2 μg/L)
Total 60 (44) NC NC 42 (47) NC NC 18 (40) NC NC
Free 2 (2) NC NC 1 (1) NC NC 1 (2) NC NC
Conjugated 2 (2) NC NC 1 (1) NC NC 1 (2) NC NC
2,5-DCP (LOD = 0.2 μg/L)
Total 19 (14) NC NC 11 (12) NC NC 8 (18) NC NC
Free 0 (0) NC NC 0 (0) NC NC 0 (0) NC NC
Conjugated 0 (0) NC NC 0 (0) NC NC 0 (0) NC NC
Abbreviations: 2,4-DCP, 2,4-dichlorophenol; 2,5-DCP, 2,5-dichlorophenol; GM, geometric mean; LOD, limit of detection; NC, not calculated due to < 50% detection. aComplete sample (n = 45 participants, 135 samples); random sample: randomly selected participants with no previously measured BPA concentrations (n = 30 participants, 90 samples); high-BPA subgroup: participants with high BPA concentrations in a previous random selection (n = 15 participants, 45 samples). bInstrumental readings used for total concentrations; values > LOD used for free concentrations. Conjugated concentrations, which are the difference between measured total and free concentrations, were calculated if both total and free were > LOD. Two specimens lacked reportable free phenol concentrations due to instrument error.
Open in a new tab

Table 3.

Limit of detection, percent detectable results, and geometric means for complete study sample and subgroups (μg/g creatinine).a

Analyte and speciesb Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15)
n (%) > LOD GM all time periods GM 17, 23, 29 weeks n (%) > LOD GM all time periods GM 17, 23, 29 weeks n (%) > LOD GM all time periods GM 17, 23, 29 weeks
Bisphenol A (LOD = 0.4 μg/L)
Total 135 (100) 10.9 17.9, 9.3, 7.8 90 (100) 9.6 12.3, 9.2, 7.8 45 (100) 14.1 37.8, 9.4, 7.8
Free 133 (100) 7.7 14.3, 6.5, 5.0 89 (100) 6.9 9.5, 7.1, 4.9 44 (100) 9.6 34.1, 5.5, 5.2
Conjugated 133 (100) 1.8 2.1, 1.6, 1.8 89 (100) 1.4 1.5, 1.3, 1.5 44 (100) 2.8 4.5, 2.2, 2.5
Butyl paraben (LOD = 0.2 μg/L)
Total 135 (100) 8.9 13.5, 7.5, 6.9 90 (100) 8.2 12.0, 7.5, 6.1 45 (100) 10.5 17.3, 7.6, 8.9
Free 133 (100) 2.6 3.6, 2.3, 2.1 89 (100) 2.4 2.9, 2.3, 2.0 44 (100) 3.1 5.8, 2.2, 2.4
Conjugated 133 (100) 4.3 6.9, 3.6, 3.3 89 (100) 4.4 7.1, 4.3, 2.7 44 (100) 4.2 6.5, 2.7, 4.5
Methyl paraben (LOD = 1.0 μg/L)
Total 135 (100) 1758.9 2,676, 1,539, 1,322 90 (100) 1563.0 2,051, 1,572, 1,185 45 (100) 2227.5 4,559, 1,474, 1,645
Free 133 (100) 36.6 48.0, 33.0, 31.1 89 (100) 33.3 39.0, 32.2, 29.4 44 (100) 44.2 74.8, 34.5, 34.6
Conjugated 133 (100) 1713.4 2,604, 1,512, 1,286 89 (100) 1530.8 2,010, 1,553, 1,150 44 (100) 2152.1 4,536, 1,435, 1,609
Propyl paraben (LOD = 0.2 μg/L)
Total 135 (100) 46.0 59.3, 46.6, 35.1 90 (100) 46.8 63.3, 47.9, 33.9 45 (100) 46.2 52.1, 44.0, 37.8
Free 133 (100) 2.2 2.8, 2.1, 1.9 89 (100) 2.1 2.5, 2.1, 1.9 44 (100) 2.4 3.6, 2.2, 1.8
Conjugated 133 (100) 41.0 50.7, 43.1, 31.8 89 (100) 42.6 56.4, 45.2, 30.4 44 (100) 38.0 40.2, 39.5, 34.8
Benzophenone‑3 (LOD = 0.4 μg/L)
Total 128 (95) 8.5 8.8, 8.3, 8.5 86 (96) 8.5 8.4, 7.7, 9.4 42 (93) 8.6 9.6, 9.5, 7.0
Free 13 (10) NC NC 9 (10) NC NC 4 (9) NC NC
Conjugated 13 (10) NC NC 9 (10) NC NC 4 (9) NC NC
2,4-DCP (LOD = 0.2 μg/L)
Total 60 (44) NC NC 42 (47) NC NC 18 (40) NC NC
Free 2 (2) NC NC 1 (1) NC NC 1 (2) NC NC
Conjugated 2 (2) NC NC 1 (1) NC NC 1 (2) NC NC
2,5-DCP (LOD = 0.2 μg/L)
Total 19 (14) NC NC 11 (12) NC NC 8 (18) NC NC
Free 0 (0) NC NC 0 (0) NC NC 0 (0) NC NC
Conjugated 0 (0) NC NC 0 (0) NC NC 0 (0) NC NC
Abbreviations: 2,4-DCP, 2,4-dichlorophenol; 2,5-DCP, 2,5-dichlorophenol; GM, geometric mean; LOD, limit of detection; NC, not calculated due to < 50% detection. aComplete sample (n = 45 participants, 135 samples); random sample: randomly selected participants with no previously measured BPA concentrations (n = 30 participants, 90 samples); high-BPA subgroup: participants with high BPA concentrations in a previous random selection (n = 15 participants, 45 samples). bInstrumental readings used for total concentrations; values > LOD used for free concentrations. Conjugated concentrations, which are the difference between measured total and free concentrations, were calculated if both total and free were > LOD. Two specimens lacked reportable free phenol concentrations due to instrument error.
Open in a new tab

The geometric mean concentration of analytes for the random sample and high-BPA subgroup were generally comparable when expressed on a micrograms per liter basis (Table 2). When expressed on a micrograms per gram creatinine basis, however, we observed a higher mean free BPA concentration at week 17 for the high-BPA subgroup (Table 3). In turn, we observed a difference in mean total BPA concentrations between subgroups, as confirmed with a t-test comparing log-normally distributed means (data not shown). We also observed a higher mean conjugated methyl paraben for the high-BPA subgroup, which led to a significant difference in mean total methyl paraben concentrations between subgroups (data not shown).

For all phenols and parabens, the percentage of free species in the total concentration was similar for the random sample and high-BPA subgroup (Table 4). The majority of BPA and butyl paraben was in its free form. Free concentration comprised over 20% of the total biomarker concentration in approximately 96% of the samples analyzed for BPA and approximately 65% of the samples analyzed for butyl paraben (Table 4). By contrast, free propyl paraben represented > 20% of the total concentration in approximately 11% of samples, and most of the detected methyl paraben and benzophenone-3 was conjugated.

Table 4.

Comparison of free concentrations as a percent of total concentration (μg/L) for select phenols and parabens within complete study sample and subgroups [n (%)].a

Percentage of free/total Bisphenol A Butyl paraben Methyl paraben Propyl paraben Benzophenone-3
Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15) Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15) Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15) Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15) Complete sample (n = 45) Random sample (n = 30) High-BPA subgroup (n = 15)
Missing 2 (1.5) 1 (1.1) 1 (2.2) 2 (1.5) 1 (1.1) 1 (2.2) 2 (1.5) 1 (1.1) 1 (2.2) 2 (1.5) 1 (1.1) 1 (2.2) 2 (1.5) 1 (1.1) 1 (2.2)
Free < LOD 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 120 (88.9) 80 (88.9) 40 (88.9)
0–5% 0 (0.0) 0 (0.0) 0 (0.0) 6 (4.4) 5 (5.6) 1 (2.2) 130 (96.3) 87 (96.7) 43 (95.6) 71 (52.6) 51 (56.7) 20 (44.4) 9 (6.7) 6 (6.7) 3 (6.7)
> 5–10% 1 (0.7) 1 (1.1) 0 (0.0) 18 (13.3) 10 (11.1) 8 (17.8) 2 (1.5) 1 (1.1) 1 (2.2) 31 (23.0) 17 (18.9) 14 (31.1) 2 (1.5) 2 (2.2) 0 (0.0)
> 10–20% 2 (1.5) 1 (1.1) 1 (2.2) 21 (15.6) 16 (17.8) 5 (11.1) 1 (0.7) 1 (1.1) 0 (0.0) 16 (11.9) 12 (13.3) 4 (8.9) 1 (0.7) 1 (1.1) 0 (0.0)
> 20–50% 16 (11.9) 13 (14.4) 3 (6.7) 38 (28.2) 28 (31.1) 10 (22.2) 0 (0.0) 0 (0.0) 0 (0.0) 7 (5.2) 3 (3.3) 4 (8.9) 1 (0.7) 0 (0.0) 1 (2.2)
> 50% 114 (84.4) 74 (82.2) 40 (88.9) 50 (37.0) 30 (33.3) 20 (44.4) 0 (0.0) 0 (0.0) 0 (0.0) 8 (5.9) 6 (6.7) 2 (4.4) 0 (0.0) 0 (0.0) 0 (0.0)
LOD, limit of detection. aComplete sample (n = 45 participants, 135 samples); random sample: randomly selected participants with no previously measured BPA concentrations (n = 30 participants, 90 samples); high-BPA subgroup: participants with high BPA concentrations in a previous random selection (n = 15 participants, 45 samples).
Open in a new tab

For the conjugated concentrations of BPA, butyl paraben, and propyl paraben (Table 5), the ICCs ranged from low for BPA (~ 0.25) to moderate for butyl paraben (~ 0.40) and propyl paraben (~ 0.60); this was the case regardless of subgroup or method of expressing concentration. For conjugated methyl paraben, the ICCs varied both by subgroup and method of expressing concentration, with values ranging from moderate to low—for example, 0.34 in micrograms per gram creatinine for the random sample and 0.06 in micrograms per gram creatinine for the high-BPA subgroup.

Table 5.

ICCs (95% CIs) for total, conjugated, and free concentrations of phenols and parabens for complete study sample and subgroups.a

Analyte Unadjusted concentration (μg/L) Adjusted concentration (μg/g creatinine)
Total Conjugated Free Total Conjugated Free
Complete sample (n = 45)
BPA 0.24 (0.09, 0.39) 0.29 (0.13, 0.44) 0.15 (0.01, 0.29) 0.04 (–0.07, 0.17) 0.24 (0.09, 0.39) 0.02 (–0.09, 0.14)
Butyl paraben 0.34 (0.18, 0.49) 0.36 (0.20, 0.50) 0.10 (–0.04, 0.26) 0.38 (0.23, 0.53) 0.41 (0.26, 0.55) 0.12 (–0.01, 0.26)
Methyl paraben 0.48 (0.33, 0.61) 0.47 (0.33, 0.61) 0.34 (0.18, 0.48) 0.24 (0.10, 0.40) 0.24 (0.09, 0.39) 0.24 (0.10, 0.39)
Propyl paraben 0.55 (0.41, 0.67) 0.57 (0.44, 0.69) 0.56 (0.42, 0.68) 0.62 (0.49, 0.72) 0.64 (0.51, 0.74) 0.61 (0.47, 0.72)
Benzophenone-3 0.43 (0.28, 0.57) NC NC 0.50 (0.35, 0.63) NC NC
Random sample (n = 30)
BPA 0.36 (0.20, 0.50) 0.28 (0.13, 0.43) 0.24 (0.09, 0.40) 0.13 (–0.01, 0.29) 0.15 (0.00, 0.31) 0.11 (–0.02, 0.26)
Butyl paraben 0.27 (0.12, 0.43) 0.34 (0.19, 0.49) 0.11 (–0.04, 0.26) 0.30 (0.16, 0.46) 0.36 (0.21, 0.51) 0.22 (0.08, 0.38)
Methyl paraben 0.53 (0.39, 0.66) 0.52 (0.39, 0.65) 0.34 (0.18, 0.48) 0.35 (0.21, 0.50) 0.34 (0.20, 0.50) 0.24 (0.10, 0.40)
Propyl paraben 0.51 (0.37, 0.64) 0.53 (0.39, 0.65) 0.57 (0.43, 0.69) 0.58 (0.45, 0.70) 0.59 (0.47, 0.71) 0.71 (0.61, 0.80)
Benzophenone-3 0.38 (0.23, 0.52) NC NC 0.38 (0.23, 0.53) NC NC
High BPA sample (n = 15)
BPAb 0.01 (–0.11, 0.14) 0.22 (0.07, 0.37) 0.00 (–0.10, 0.13) –0.12 (–0.25, –0.12) 0.18 (0.05, 0.35) –0.11 (–0.23, –0.07)
Butyl parabenb 0.50 (0.34, 0.62) 0.42 (0.26, 0.55) 0.11 (–0.03, 0.27) 0.54 (0.44, 0.69) 0.54 (0.42, 0.67) –0.05 (–0.16, 0.05)
Methyl paraben 0.36 (0.21, 0.51) 0.35 (0.20, 0.50) 0.37 (0.23, 0.52) 0.06 (–0.03, 0.22) 0.06 (–0.04, 0.21) 0.21 (0.10, 0.39)
Propyl paraben 0.66 (0.56, 0.77) 0.67 (0.58, 0.78) 0.54 (0.43, 0.68) 0.73 (0.62, 0.81) 0.75 (0.65, 0.82) 0.47 (0.35, 0.63)
Benzophenone-3 0.59 (0.50, 0.74) NC NC 0.80 (0.71, 0.86) NC NC
NC, not calculated due to < 50% detection. aComplete sample (n = 45 participants, 135 samples); random sample: randomly selected participants with no previously measured BPA concentrations (n = 30 participants, 90 samples); high-BPA subgroup: participants with high BPA concentrations in a previous random selection (n = 15 participants, 45 samples). bWhen the variance within time points exceeds the variance between subjects, a negative ICC is mathematically possible and can be interpreted as approximately 0 (Shrout and Fleiss 1979).
Open in a new tab

For the free concentrations of BPA and butyl paraben, the ICCs were lower than for the conjugated concentrations, even approaching 0 for free BPA. This was true regardless of method of expressing concentration or subgroup. The ICC for free methyl paraben was approximately 0.35 across subgroups in micrograms per liter and approximately 0.2 in micrograms per gram creatinine. For creatinine-adjusted free propyl paraben, the ICC was higher in the random sample (0.71) than the high-BPA subgroup (0.47), whereas unadjusted values were more consistent across subgroups (~ 0.55).

The ICC for the total concentrations of these phenols and parabens can be thought of as a weighted average of the values for the conjugated and free compounds. Thus, for BPA and butyl paraben, with a relatively greater proportion of free compound, the ICCs for total concentrations were generally low. For methyl paraben and propyl paraben, with relatively little free compound, the ICCs for total concentrations largely reflected the ICCs of conjugated concentrations. For BP-3, where a lack of detectable free concentrations prevented the computation of conjugated concentrations, the ICC was 0.38 in the random sample and 0.80 in the high-BPA subgroup.

ICCs computed with random effects models produced similar results (data not shown). The Spearman correlation coefficients among serial pairs of concentrations (conjugated, creatinine-adjusted) were generally comparable across pairing and subgroups, consistent with the results in Table 5 (see Supplemental Material, Table S2).

Discussion

The main purpose of our study was to examine the reliability of phenolic concentrations in a single spot urine sample collected during pregnancy to estimate exposure over the course of pregnancy in the MoBa cohort. In general, ICCs were poor to moderate, with the highest ICCs found consistently for propyl paraben (~ 0.60). We expect ICCs for concentrations that have been adjusted for creatinine to more accurately describe exposure variability, rather than variability in urine dilution. Results were generally similar overall and by subgroup. The small sample size (n = 45) may also have contributed to imprecision in the estimates of average concentration and of the ICCs.

Contamination likely accounts for the greater geometric mean total BPA concentrations in this study sample compared with other studies. When free BPA is excluded from the total analyte concentration, the geometric mean for conjugated BPA (1.3 μg/L) is similar to other population estimates for total BPA (Braun et al. 2009; Bushnik et al. 2010; CDC 2013; Hoepner et al. 2013), including those for pregnant women (Quirós-Alcalá et al. 2013), although some previously reported total BPA means for pregnant women have been higher than these conjugated values (Meeker et al. 2013; Woodruff et al. 2011). Quantifying free BPA only provides an estimate of potential contamination. For butyl paraben, 88 (65%) samples had free concentrations greater than 20% of total concentrations, also indicating likely contamination. There was some evidence of contamination with propyl paraben as well; for propyl paraben, 15 (11%) samples had free concentrations > 20% of total, but reliability estimates were consistent for total, conjugated, and free concentrations. There was no evidence of contamination for methyl paraben, benzophenone-3, 2,4-dichlorophenol, or 2,5-dichlorophenol, given either the limited proportion of free concentrations in the total concentration (methyl paraben, benzophenone-3) or the limited detection of any free concentrations among those samples with detectable total concentrations (2,4-dichlorophenol, 2,5-dichlorophenol).

In the case of MoBa, the likely sources of BPA contamination were the urinary preservative (sodium propionate 94%, ethyl paraben 5.6%, and chlorhexidine 0.4%) and the collection conditions, that is, the hospital setting, plastic materials, and handling procedures (Longnecker et al. 2013), despite employing procedures to limit possible contamination. Population sources of BPA exposure are diverse. BPA is employed in the manufacture of polycarbonate plastics (e.g., compact discs, plastic dinnerware, toys), epoxy resins (e.g., can linings, dental composites), and thermal paper (e.g., some receipts) (CDC 2009). Our results suggest that the magnitude of contamination varied, perhaps due to collection conditions or changes in preservative composition, and thus measurement of free concentrations would be required to isolate the possible contaminant from the conjugated fraction. Many investigators take steps to reduce contamination from known sources; however, contamination may also arise in unexpected ways. It is difficult to compare possible sources of contamination resulting from sample collection procedures between studies due to limited detail provided in the peer-reviewed literature. Although most investigators choose to measure only total phenol and paraben concentrations (conjugated and free combined), measuring both species in at least a subgroup of study samples allows for the identification of samples in which the free:total proportion is out of the expected range (Koch et al. 2012; Völkel et al. 2002; Waechter et al. 2007), which may indicate contamination.

We also investigated whether isolating the conjugated concentrations would produce improved ICCs compared with those for total concentrations. Separating the conjugated from the free phenol did not substantially alter reliability estimates for most analytes. For both BPA and butyl paraben; however, the majority of samples had a substantial proportion of these compounds present as free species and the conjugated analyte concentrations appeared more reliable, whereas ICCs for free concentrations were especially low. This supports four conclusions: a) Reliability estimates may be affected by external sources of contamination, as indicated by higher concentrations than typically reported in other studies; b) in this population, the extent of the specimen contamination varied, possibly due to conditions at multiple collection locations or variation in collection tube manufacturing (Table 2); c) these data may also reflect daily variability in individual exposures that make characterization of average longer-term exposure challenging; and d) the reliability of a single specimen’s concentration to categorize BPA exposure is rather poor, as has been reported in other studies (Braun et al. 2011a, 2012; Jusko et al. 2014; Meeker et al. 2013; Philippat et al. 2013). For other phenolic compounds, our results were comparable to previous estimates for methyl paraben, but lower than prior reports for benzophenone-3, butyl paraben, and 2,4-dichlorophenol, and higher than prior reports for propyl paraben (Meeker et al. 2013; Philippat et al. 2013; Smith et al. 2012).

Although the source of butyl paraben contamination in the MoBa samples could not be definitively established with the available information, it appears that the urinary preservative containing 5.6% ethyl paraben was the source of the detected ethyl paraben. The analytic consequences of this preservative are of primary importance to investigators analyzing affected specimens. Online SPE-HPLC-isotope dilution-MS/MS includes three steps: an extraction step in which target analytes are preconcentrated and separated from unwanted matrix components, a separation step in which target compounds are separated from each other and residual matrix biomolecules, and a detection step in which compounds are quantified according to molecular mass. Generally, environmental chemicals, including phenols and parabens, are present in urine at trace concentrations—at or below parts per billion. In the MoBA samples, ethyl paraben concentrations were in the parts per thousand range. Therefore, the detector was oversaturated, and the analytic performance and sensitivity of the mass spectrometer for all phenols and parabens measured was negatively affected, as evidenced by the QC CVs being in some cases twice as large as annual CVs reported by the CDC for these analytes (CDC, National Center for Environmental Health 2011).

Given the extent of interference, exposure assessment for most urinary phenols and parabens may be difficult in the MoBa cohort using the currently available assays. It is unknown to what degree additional analytes might be impacted. We hypothesize that the extraction, separation, or quantification of compounds with physicochemical properties comparable to those of ethyl paraben may also be negatively impacted. Further detailed quality control studies would have to be conducted in order to test this hypothesis.

Conclusions

We demonstrated moderate reliability of a single spot urinary concentration to estimate exposure over an 18-week period for benzophenone-3 and propyl paraben, with lower reliability for the other measured analytes. Studies interested in measuring environmental chemicals should avoid, if at all possible, the use of preservatives that may interfere with analytic procedures, and should provide collection protocols to the analytic laboratory in advance so potential interferences can be anticipated. We document the utility of measuring the total and free phenol and paraben biomarker concentrations as a method for detecting the contamination of specimens from these ubiquitous compounds.

Supplemental Material

(176 KB) PDF
Click here for additional data file. (171.4KB, pdf)

Acknowledgments

We are grateful to all the participating families in Norway who take part in this ongoing cohort study. We acknowledge X. Zhao, J. Kramer, and T. Jia for measuring analyte concentrations.

Footnotes

This study was supported by grants from the National Institute of Environmental Health Sciences (NIEHS), National Institute of Health (NIH) (P30ES010126, R01ES021777) and the Intramural Research Program of the NIEHS/NIH. V.T.G. was supported by NIEHS grant ES07018. The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and the Ministry of Education and Research, NIEHS/NIH (contract NO1-ES-75558), National Institute of Neurological Disorders and Stroke, NIH (grants UO1 NS 047537-01 and UO1 NS047537-06A1), and the Norwegian Research Council/Bioinformatics and Functional Genomics (FUGE) (grant 151918/S10).

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

The authors declare they have no actual or potential competing financial interests.

References

  1. Bertelsen RJ, Engel SM, Jusko TA, Calafat AM, Hoppin JA, London SJ, et al. 2014Reliability of triclosan measures in repeated urine samples from Norwegian pregnant women. J Expo Sci Environ Epidemiol 245517–521.; 10.1038/jes.2013.95 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braun JM, Kalkbrenner AE, Calafat AM, Bernert JT, Ye X, Silva MJ, et al. 2011aVariability and predictors of urinary bisphenol A concentrations during pregnancy. Environ Health Perspect 119131–137.; 10.1289/ehp.1002366 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braun JM, Kalkbrenner AE, Calafat AM, Yolton K, Ye X, Dietrich KN, et al. 2011bImpact of early-life bisphenol A exposure on behavior and executive function in children. Pediatrics 1285873–882.; 10.1542/peds.2011-1335 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braun JM, Smith KW, Williams PL, Calafat AM, Berry K, Ehrlich S, et al. 2012Variability of urinary phthalate metabolite and bisphenol A concentrations before and during pregnancy. Environ Health Perspect 120739–745.; 10.1289/ehp.1104139 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braun JM, Yolton K, Dietrich KN, Hornung R, Ye X, Calafat AM, et al. 2009Prenatal bisphenol A exposure and early childhood behavior. Environ Health Perspect 1171945–1952.; 10.1289/ehp.0900979 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bushnik T, Haines D, Levallois P, Levesque J, Van Oostdam J, Viau C. Lead and bisphenol A concentrations in the Canadian population. Health Rep. 2010;21(3):7–18. [PubMed] [Google Scholar]
  7. Calafat AM, Needham LL.2009What additional factors beyond state-of-the-art analytical methods are needed for optimal generation and interpretation of biomonitoring data? Environ Health Perspect 1171481–1485.; 10.1289/ehp.0901108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Calafat AM, Ye X, Wong LY, Bishop AM, Needham LL.2010Urinary concentrations of four parabens in the U.S. population: NHANES 2005–2006. Environ Health Perspect 118679–685.; 10.1289/ehp.0901560 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL.2008Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003–2004. Environ Health Perspect 11639–44.; 10.1289/ehp.10753 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. CDC (Centers for Disease Control and Prevention). Fourth National Report on Human Exposure to Environmental Chemicals. Atlanta, GA:Centers for Disease Control and Prevention. 2009. Available: http://www.cdc.gov/exposurereport/pdf/FourthReport.pdf [accessed 25 June 2013]
  11. CDC. National Biomonitoring Program. Factsheet. Parabens. Atlanta, GA:Centers for Disease Control and Prevention. 2010. Available: http://www.cdc.gov/biomonitoring/Parabens_FactSheet.html [accessed 25 June 2013]
  12. CDC. Fourth National Report on Human Exposure to Environmental Chemicals: Updated Tables, September 2013. Atlanta, GA:Centers for Disease Control and Prevention. 2013. Available: http://www.cdc.gov/exposurereport/pdf/FourthReport_UpdatedTables_Sep2013.pdf [accessed 25 June 2013]
  13. CDC, National Center for Environmental Health. Laboratory Procedure Manual: Bisphenol A and Other Environmental Phenols and Parabens in Urine, NHANES 2009–2010. Method 6301.01. Atlanta, GA:Centers for Disease Control and Prevention. 2011. Available: http://www.cdc.gov/nchs/data/nhanes/nhanes_09_10/EPH_F_met_phenols_parabens.pdf [accessed 20 December 2013]
  14. Hamer RM. Intraclass Correlations. Cary, NC:SAS Institute Inc. 1995. Available: http://support.sas.com/documentation/onlinedoc/stat/ex_code/121/intracc.html [accessed 24 June 2013]
  15. Harley KG, Gunier RB, Kogut K, Johnson C, Bradman A, Calafat AM, et al. 2013Prenatal and early childhood bisphenol A concentrations and behavior in school-aged children. Environ Res 12643–50.; 10.1016/j.envres.2013.06.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoepner LA, Whyatt RM, Just AC, Calafat AM, Perera FP, Rundle AG.2013Urinary concentrations of bisphenol A in an urban minority birth cohort in New York City, prenatal through age 7 years. Environ Res 12238–44.; 10.1016/j.envres.2012.12.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoppin JA, Ulmer R, Calafat AM, Barr DB, Baker SV, Meltzer HM, et al. 2006Impact of urine preservation methods and duration of storage on measured levels of environmental contaminants. J Expo Sci Environ Epidemiol 16139–48.; 10.1038/sj.jea.7500435 [DOI] [PubMed] [Google Scholar]
  18. Jusko TA, Shaw PA, Snijder CA, Pierik FH, Koch HM, Hauser R, et al. 2014Reproducibility of urinary bisphenol A concentrations measured during pregnancy in the generation R study. J Expo Sci Environ Epidemiol 245532–536.; 10.1038/jes.2014.23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koch HM, Aylward LL, Hays SM, Smolders R, Moos RK, Cocker J, et al. 2014Inter- and intra-individual variation in urinary biomarker concentrations over a 6-day sampling period. Part 2: personal care product ingredients. Toxicol Lett 2312261–269.; 10.1016/j.toxlet.2014.06.023 [DOI] [PubMed] [Google Scholar]
  20. Koch HM, Kolossa-Gehring M, Schröter-Kermani C, Angerer J, Brüning T.2012Bisphenol A in 24 h urine and plasma samples of the German Environmental Specimen Bank from 1995 to 2009: a retrospective exposure evaluation. J Expo Sci Environ Epidemiol 226610–616.; 10.1038/jes.2012.39 [DOI] [PubMed] [Google Scholar]
  21. Liao C, Kannan K.2012Determination of free and conjugated forms of bisphenol A in human urine and serum by liquid chromatography-tandem mass spectrometry. Environ Sci Technol 4695003–5009.; 10.1021/es300115a [DOI] [PubMed] [Google Scholar]
  22. Longnecker MP, Harbak K, Kissling GE, Hoppin JA, Eggesbo M, Jusko TA, et al. 2013The concentration of bisphenol A in urine is affected by specimen collection, a preservative, and handling. Environ Res 126211–214.; 10.1016/j.envres.2013.07.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Magnus P, Irgens LM, Haug K, Nystad W, Skjaerven R, Stoltenberg C, et al. 2006Cohort profile: the Norwegian Mother and Child Cohort Study (MoBa). Int J Epidemiol 3551146–1150.; 10.1093/ije/dyl170 [DOI] [PubMed] [Google Scholar]
  24. Mahalingaiah S, Meeker JD, Pearson KR, Calafat AM, Ye X, Petrozza J, et al. 2008Temporal variability and predictors of urinary bisphenol A concentrations in men and women. Environ Health Perspect 116173–178.; 10.1289/ehp.10605 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matthews JB, Twomey K, Zacharewski TR.2001In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors α and β. Chem Res Toxicol 142149–157.; 10.1021/tx0001833 [DOI] [PubMed] [Google Scholar]
  26. Meeker JD, Cantonwine DE, Rivera-González LO, Ferguson KK, Mukherjee B, Calafat AM, et al. 2013Distribution, variability, and predictors of urinary concentrations of phenols and parabens among pregnant women in Puerto Rico. Environ Sci Technol 4773439–3447.; 10.1021/es400510g [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Norwegian Institute of Public Health. Publications from the Norwegian Mother and Child Cohort Study. 2013. Available: http://www.fhi.no/artikler/?id=65520 [accessed 31 May 2013]
  28. Perera F, Vishnevetsky J, Herbstman JB, Calafat AM, Xiong W, Rauh V, et al. 2012Prenatal bisphenol A exposure and child behavior in an inner-city cohort. Environ Health Perspect 1201190–1194.; 10.1289/ehp.1104492 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Philippat C, Wolff MS, Calafat AM, Ye X, Bausell R, Meadows M, et al. 2013Prenatal exposure to environmental phenols: concentrations in amniotic fluid and variability in urinary concentrations during pregnancy. Environ Health Perspect 1211225–1231.; 10.1289/ehp.1206335 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Quirós-Alcalá L, Eskenazi B, Bradman A, Ye X, Calafat AM, Harley K.2013Determinants of urinary bisphenol A concentrations in Mexican/Mexican-American pregnant women. Environ Int 59152–160.; 10.1016/j.envint.2013.05.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rønningen KS, Paltiel L, Meltzer HM, Nordhagen R, Lie KK, Hovengen R, et al. 2006The biobank of the Norwegian Mother and Child Cohort Study: a resource for the next 100 years. Eur J Epidemiol 218619–625.; 10.1007/s10654-006-9041-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shrout PE, Fleiss FJ.1979Intraclass correlations: uses in assessing rater reliability. Psychol Bull 862420–428.; 10.1037/0033-2909.86.2.420 [DOI] [PubMed] [Google Scholar]
  33. Smith KW, Braun JM, Williams PL, Ehrlich S, Correia KF, Calafat AM, et al. 2012Predictors and variability of urinary paraben concentrations in men and women, including before and during pregnancy. Environ Health Perspect 1201538–1543.; 10.1289/ehp.1104614 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tang R, Chen MJ, Ding GD, Chen XJ, Han XM, Zhou K, et al. 2013Associations of prenatal exposure to phenols with birth outcomes. Environ Pollut 178115–120.; 10.1016/j.envpol.2013.03.023 [DOI] [PubMed] [Google Scholar]
  35. U.S. EPA (U.S. Environmental Protection Agency). Toxicological Review of Phenol. (CAS No. 108-95-2) In Support of Summary Information on the Integrated Risk Information System (IRIS). EPA/635/R-02/006. Washington, DC:U.S. EPA. 2002. Available: http://www.epa.gov/iris/toxreviews/0088tr.pdf [accessed 5 September 2013]
  36. Vandenberg LN, Chahoud I, Heindel JJ, Padmanabhan V, Paumgartten FJ, Schoenfelder G.2010Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ Health Perspect 1181055–1070.; 10.1289/ehp.0901716 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Völkel W, Colnot T, Csanády GA, Filser JG, Dekant W.2002Metabolism and kinetics of bisphenol A in humans at low doses following oral administration. Chem Res Toxicol 15101281–1287.; 10.1021/tx025548t [DOI] [PubMed] [Google Scholar]
  38. Waechter J, Thornton C, Markham D, Domoradzki J.2007Factors affecting the accuracy of bisphenol A and bisphenol A-monoglucuronide estimates in mammalian tissues and urine samples. Toxicol Mech Methods 17113–24.; 10.1080/15376510600803581 [DOI] [PubMed] [Google Scholar]
  39. Wolff MS, Engel SM, Berkowitz GS, Ye X, Silva MJ, Zhu C, et al. 2008Prenatal phenol and phthalate exposures and birth outcomes. Environ Health Perspect 1161092–1097.; 10.1289/ehp.11007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Woodruff TJ, Zota AR, Schwartz JM.2011Environmental chemicals in pregnant women in the United States: NHANES 2003–2004. Environ Health Perspect 119878–885.; 10.1289/ehp.1002727 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ye X, Bishop AM, Needham LL, Calafat AM.2008Automated on-line column-switching HPLC-MS/MS method with peak focusing for measuring parabens, triclosan, and other environmental phenols in human milk. Anal Chim Acta 6221–2150–156.; 10.1016/j.aca.2008.05.068 [DOI] [PubMed] [Google Scholar]
  42. Ye X, Bishop AM, Reidy JA, Needham LL, Calafat AM.2007Temporal stability of the conjugated species of bisphenol A, parabens, and other environmental phenols in human urine. J Expo Sci Environ Epidemiol 176567–572.; 10.1038/sj.jes.7500566 [DOI] [PubMed] [Google Scholar]
  43. Ye X, Kuklenyik Z, Needham LL, Calafat AM.2005aAutomated on-line column-switching HPLC-MS/MS method with peak focusing for the determination of nine environmental phenols in urine. Anal Chem 77165407–5413.; 10.1021/ac050390d [DOI] [PubMed] [Google Scholar]
  44. Ye X, Kuklenyik Z, Needham LL, Calafat AM.2005bQuantification of urinary conjugates of bisphenol A, 2,5-dichlorophenol, and 2-hydroxy-4-methoxybenzophenone in humans by online solid phase extraction–high performance liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 3834638–644.; 10.1007/s00216-005-0019-4 [DOI] [PubMed] [Google Scholar]
  45. Ye X, Pierik FH, Angerer J, Meltzer HM, Jaddoe VW, Tiemeier H, et al. 2009Levels of metabolites of organophosphate pesticides, phthalates, and bisphenol A in pooled urine specimens from pregnant women participating in the Norwegian Mother and Child Cohort Study (MoBa). Int J Hyg Environ Health 2125481–491.; 10.1016/j.ijheh.2009.03.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ye X, Zhou X, Hennings R, Kramer J, Calafat AM.2013Potential external contamination with bisphenol A and other ubiquitous organic environmental chemicals during biomonitoring analysis: an elusive laboratory challenge. Environ Health Perspect 121283–286.; 10.1289/ehp.1206093 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

(176 KB) PDF
Click here for additional data file. (171.4KB, pdf)

Articles from Environmental Health Perspectives are provided here courtesy of National Institute of Environmental Health Sciences

RESOURCES