Measurement of Total and Free Urinary Phenol and Paraben Concentration over the Course of Pregnancy : Assessing Reliability and Contamination of Specimens in the Norwegian Mother and Child Cohort Study Guidry et al, 2015 Chorong Shin July. 22, 2015

Introduction 2019-10-21 hEX-Tox paper review

Introduction Background of this study Phenols (including BPA, Benzophenone-3, Parabens) → found in a number of common consumer products. Prenatal exposure to phenols : endocrine disruption (e.g. decrease gestational age at birth or birth weight), child behavior problems 'Biomarkers' of phenol and paraben : Spot urine sample, which provide exposure measurement at one point in time, are frequently used to estimate recent exposure. However, many phenolic compounds are rapidly excreted (t1/2=1~4.5hr). "Can 'A spot urine' represent longer-term or cumulative exposure?" 2019-10-21 hEX-Tox paper review

Introduction Metabolism of phenolic compounds Phenols are conjugated with sulfate and glucuronic acid after hydrolysis of the esters and excreted primarily in the urine. U.S. EPA, 2002 2019-10-21 hEX-Tox paper review

Introduction Increase unconjugated (free) biomarker conc. Source 1. : free phenolic compounds that passed through the body without conjugation. Source 2. : free phenolic compounds that entered the sample via contamination during collection or processing Source 3. : conjugated phenolic compounds hydrolyzed to the free form by enzymes in local tissues Source 4. : conjugated phenolic compounds hydrolyzed to the free form following improper storage or handling (Contamination). 2019-10-21 hEX-Tox paper review

1 ppb in unpolluted ground water 0.01 - 1 ppb in unpolluted rivers Introduction Environmental exposure to phenolic compounds Introduction 0.03 ppb from U.S. air (1982) 0.07 - 0.7 mg/kg from U.S. sediment ample (2006) 1 ppb in unpolluted ground water from U.S. (1985) 0.01 - 1 ppb in unpolluted rivers (ASTDR, 2008) 2019-10-21 hEX-Tox paper review

Introduction Environmental exposure to phenolic compounds Phenol enters the air, water, and soil as a result of its manufacture and use. Phenol has a short half-life in air, less than 1 day. In air, it reacts with photochemically-produced hydroxyl radicals. Phenol generally remains in soil only about 2-5 days. In soil, phenol biodegrades under both aerobic and anaerobic conditions. Phenol is rapidly degraded in water, but it can remain in water for a week or more if present in high conc. Phenol does not accumulate in fish, other animals, or plants. (ASTDR, 2008) 2019-10-21 hEX-Tox paper review

Introduction Aim of study To measure the conc. of urinary phenols and parabens in the MoBa. To characterize the reliability of a single spot urine sample to estimate environmental exposure to these compounds To identify potential contaminations of MoBa specimens. 2019-10-21 hEX-Tox paper review

Methods Study design [N=671] MoBa cohort study (1999-2008) at 17-18 week gestation The final cohort : 90,700 mothers + 108,000 pregnancies From 2002, MoBa participants provided their spot urine specimen. Because of bacterial growth upon receipt, add a preservative to urine specimens. ; 8mL of urine + a mixture of 3 preservatives (sodium propionate 94%, ethyl paraben 5.6%, chlorhexidine 0.4%) Nov. 2007 - Dec. 2008 : a subset of MoBa participants donated a spot urine sample at 17, 23, 29 weeks of gestation for a reliability substudy. [N=671] For quality control (QC), in 2011, newly collected spot urine specimens from 10 MoBa mothers. 2019-10-21 hEX-Tox paper review

Methods Study design [N=30] [N=671] [N=15] 'Random Sample' MoBa-sub-study (for reliability) participants randomly selected by author [N=30] 'Random Sample' through pilot study high-BPA subgroup [N=15] 22.9 - 52.1 μg/g creatinine ** 3 urine samples for each participant. 2019-10-21 hEX-Tox paper review

Methods Measurement free and total (conjugated + free form) Conc. of 9 compounds BPA, MP, EP, BP, PP, TCS, BP-3, 2,4-dichlorophenol, 2,5-dichlorophenol online SPE-HPLC-isotope dilution-MS/MS analysis Total 3 analytical batches : (45 urine samples + 5 QC samples) / 1 batch 2019-10-21 hEX-Tox paper review

Methods Statistical analyses ICC is a measure of the reliability of measurements. - 반복성, 재현성을 평가하는데 주로 사용되는 신뢰도 지표로서 0 (전혀 일치하지 않음) ~ 1 (완벽하게 일치함) 사이의 값을 갖는다. -계산법 : 𝐈𝐂𝐂= 𝐁𝐞𝐭𝐰𝐞𝐞𝐧 𝐯𝐚𝐫. 𝐭𝐨𝐭𝐚𝐥 𝐯𝐚𝐫. 관찰값들의 전체 분산(total variance = between-pair var. + within var.) 중 쌍들 간 분산(between-pair var.)이 차지하는 비율에 해당한다. - 해석 : 값이 1에 가까울수록 → Most of the variance between time points attributable to differences between subjects rather than within-person difference. 2019-10-21 hEX-Tox paper review

Results * 50% 미만 detection : 2,4-DCP, 2,5-DCP는 이후 분석 과정에서 제외됨. 2019-10-21 hEX-Tox paper review

Results * 50% 미만 detection : 2,4-DCP, 2,5-DCP는 이후 분석 과정에서 제외됨. 2019-10-21 hEX-Tox paper review

Results 𝐅𝐫𝐞𝐞 𝐓𝐨𝐭𝐚𝐥 (=𝑭𝒓𝒆𝒆+𝒄𝒐𝒏𝒋𝒖𝒈𝒂𝒕𝒆𝒅) ×𝟏𝟎𝟎(%) 𝐅𝐫𝐞𝐞 𝐓𝐨𝐭𝐚𝐥 (=𝑭𝒓𝒆𝒆+𝒄𝒐𝒏𝒋𝒖𝒈𝒂𝒕𝒆𝒅) ×𝟏𝟎𝟎(%) BPA, BP : 전체의 50% 이상이 free form PP : 전체 중 20% 이내가 free form MP, BP-3 : 대부분 conjugated form으로 존재 2019-10-21 hEX-Tox paper review

Results for conjugated conc. : low for BPA (0.18-0.29), moderate for BP(0.36-0.54), PP(0.53-0.75), from low to moderate for MP (varied both by subgroup and method) for free conc. : lower than conjugated conc. for BPA, BP. MP : 0.35 for ug/L, 0.2 for ug/g creatinine. for ug/g creatinine PP : Random sample (0.71) > high-BPA subgroup (0.47) for ug/L PP : consistent across subgroup (0.57, 0.54) 2019-10-21 hEX-Tox paper review

Results for total conc. : can be thought of as a weighted average of the values for the conjugated and free compound. Thus, BPA, BP (free >>> conjugated) : the ICCs for total were generally low. MP, PP (conjugated >>> free) : the ICCs for total largely reflected the ICCs of conjugated 2019-10-21 hEX-Tox paper review

Discussion Reliability of phenolic conc. in a single spot urine Mostly, ICC were poor to moderate level (Table 5) : "Reliability ↓?" cf. The highest ICC : Propyl paraben (among the analyte) ug/L urine vs. ug/g creatinine : 'creatinine adjusted' more accurately describe exposure variability. Results overall and by subgroup (Complete vs. random vs. high-BPA) : similar The small sample size (total N=45) may contributed to imprecision in the estimates of average conc. and ICC. 2019-10-21 hEX-Tox paper review

Discussion Contamination of specimens ; BPA This study : 'total-BPA geometric mean' is greater than other studies. When free-BPA is excluded from the total-BPA conc. (In this study results), the GM (=1.3 ug/L urine) for conjugated BPA is similar to other studies' estimates for total-BPA. Therefore, this study's free-BPA ⇒ from contamination! Contamination Source : urinary preservative(in this study) and the collection conditions like the hospital setting, plastic materials, and handling procedures. 'Quantifying free BPA only' provides an estimate of potential contamination. Also measurement of free conc. would be required to isolate the possible contaminant from the conjugated fraction. 2019-10-21 hEX-Tox paper review

Discussion Contamination of specimens ; Butyl paraben Butyl paraben : 전체 135개(45명*3rep) sample 중 15개, 즉 11% 정도의 sample에서 20% 이상이 Free form인 것으로 나타남. This result is also due to contamination. Because, the reliability estimates (=ICC) of total, conjugated, free conc. : consistent. Contamination Source (in the MoBa samples) : could not be definitively established with the available information. It might be the urinary preservative containing 5.6% ethyl paraben. Generally, environmental phenols are present in urine at or below ppb. [= ng/ml]. But in the MoBa samples, ethyl paraben conc. were in the ppt range. Therefore, the detector was oversaturated, and the analytic performance and sensitivity of the mass spectrometer 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. 2019-10-21 hEX-Tox paper review

Discussion Isolating the conjugated conc. Separating the conjugated from the free phenol did not substantially alter reliability estimates(ICC) for most analytes. However, for BPA & BP : The majority of samples had a substantial proportion of these conjugated conc. appeared more reliable, whereas ICCs for free conc. were especially low. 2019-10-21 hEX-Tox paper review

Discussion Conclusion a) Reliability estimates may be affected by external sources of contamination, as indicated by higher conc. than typically reported in other studies. b) In this population, the extent of the specimen contamination varied, possibly due to conditions at multiple collection location or variation in collection tube manufacturing. c) These data may also reflect daily variability in individual exposures. d) The reliability of a single specimen's conc. to categorize BPA exposure is rather poor, as has been reported in other studies. Exposure assessment for most urinary phenols and parabens may be difficult in the MoBa cohort using the currently available assays. We hypothesize that the online SPE-HPLC-isotope dilution-MS/MS steps to ethyl paraben may be negatively impacted. Further detailed quality control studies would have to be conducted in order to test this hypothesis. 2019-10-21 hEX-Tox paper review

Appendix 'expected' total : free BPA (Liao C et al, 2012) Determination of free and conjugated forms of BPA in human urine and serum by LC-MS/MS

Appendix ICC 비교) Koch et al. 2014 (Koch et al, 2014) Inter- and Intra-individual variation in urinary biomarker concentration over a 6-day sampling period. Part 2: Personal care product ingredients