In 2007, 200 adults were recruited from the C8 Health Project to participate in an ongoing study of PFOA toxicokinetics. The C8 Health Project, conducted in 2005–2006, was a cross-sectional study of approximately 69,000 individuals who had lived in at least one of six affected water districts near the Washington Works plant; participants provided a blood sample and answered a questionnaire regarding residential and occupational history, water use, health history, and demographic information (Frisbee et al. 2009
; Steenland et al. 2009
). The C8 Health Project was initiated as a result of the settlement agreement in a class action lawsuit filed in West Virginia (Leach v. E.I. du Pont de Nemours & Co. 2002
). The settlement also appointed a group of epidemiologists, the C8 Science Panel, to assess whether a link between PFOA exposure and disease in the community exists (C8 Science Panel 2009
Using phone numbers reported during the C8 Health Project, we attempted to contact eligible individuals for our study of PFOA toxicokinetics by telephone in May 2007 to ask additional eligibility screening questions. The random sample was stratified by sex, water service provider, and primary drinking water source (bottled vs. public). By design, approximately 75% of our participants were recruited from Lubeck to allocate most study resources to observing short-term elimination patterns because filtration began there soon after our baseline interviews. The remaining 25% were recruited from Little Hocking, which did not begin filtration until about 5 months after our baseline interviews. These participants served as a comparison group to assess short-term serum PFOA patterns in the absence of filtration. We recruited approximately equal numbers of males and females to maximize statistical power for detecting any sex differences. Although most of our participants were selected on the basis of consuming public drinking water, our recruitment targets included a small number of bottled water drinkers (20 participants in Lubeck and 10 in Little Hocking) to investigate the rate of decline in their PFOA serum concentrations. Up to six attempts were made to contact each randomly selected individual, including calls on weekdays and weekends and during daytime and early evenings, until the individual was reached or refused to speak with us, or until the recruitment goal had been reached for that individual’s stratum.
We attempted to contact 1,092 individuals before reaching our recruitment goals. Of these, 94 had no working phone number reported to the C8 Health Project, 435 had working phone numbers but could not be reached within six attempts, and 46 refused to answer our questions or were not able to complete the telephone interview. Of the 517 individuals who completed our screening interview, 304 were ineligible based on the additional eligibility screening questions, and 7 were unavailable during the first round of blood sampling or refused to participate in the full study. An additional 6 individuals were initially retained as backup participants but were not followed after our 200 primary study participants completed their first blood draw.
Based on 2005–2006 C8 Health Project data, we used the following eligibility criteria for the study of PFOA toxicokinetics: serum PFOA concentration of at least 50 ng/mL, residential water service provided by Lubeck Public Service District or Little Hocking Water Association, not growing their own vegetables at home, never employed by the DuPont Company, and a signed consent form allowing C8 Health Project data to be shared with the C8 Science Panel. Two groups were recruited: those reporting primary use of public water for water consumption at home, and a smaller group consisting of those primarily using bottled water for residential water consumption. To be eligible, participants had to have reported primary use of public water for water consumption at home in 2005–2006 and in 2007; primary use of public water for cooking, showering, and bathing in 2007; and no use of carbon water filters at home. Eligible bottled water drinkers had to have reported primary use of bottled water for residential water consumption both in 2005–2006 and in 2007. Those who were not served by the same water district in 2007 as in 2005–2006 or those with any history of DuPont employment or likely occupational PFOA exposures since 2000 were excluded from enrollment for both groups. A total of 172 eligible public water drinkers and 28 eligible bottled water drinkers consented to enroll in the study. The ages of the participant ranged from 18 to > 89 years. lists baseline characteristics of enrolled participants.
Baseline characteristics of enrolled participants in May–June 2007, by water district and primary source of drinking water (public water source or bottled water).
Participants were asked to donate eight blood samples over 4 years, beginning in May 2007. Collection of the first round of blood samples was completed in June 2007. Subsequent blood draws took place approximately 1, 2, 3, 6, and 12 months after the initial donation, with the final sample donated in August 2008. A brief questionnaire regarding recent water use, local fruit/vegetable consumption, and potential occupational exposure was administered by computer-assisted telephone interview before each blood draw. All but 3 of the original 200 participants remained enrolled throughout the first year of the study, completing all six interviews and blood samples. Additional blood draws are planned for mid-2009 and mid-2011.
Blood samples (~ 4 mL) were collected at each visit by trained phlebotomists at a location of the participant’s choosing, generally at home. Samples were transported in coolers to a laboratory on the same day, allowed to clot at room temperature for a minimum of 30 min, and centrifuged for at least 15 min at 2,400 rpm. After centrifuging, serum was transferred to 2 mL polypropylene Nalgene vials (Thermo Fisher Scientific, Waltham, MA) and stored at −30°C for up to 1 week before being shipped on dry ice to the Centers for Disease Control and Prevention (CDC).
At the CDC, all samples were kept at or below −40°C until analysis. Concentrations of PFOA in serum were measured using a modification of online solid-phase extraction (SPE) coupled to reverse-phase high-performance liquid chromatography (HPLC)–isotope dilution tandem mass spectrometry previously described (Kuklenyik et al. 2005
). Briefly, we added 275 μL 0.1 M formic acid and 25 μL 13
PFOA internal standard solution to 100 μL serum. The spiked serum was vortex mixed and sonicated and then injected into a Symbiosis online SPE-HPLC system (Spark Holland, Plainsboro, NJ), allowing for the preconcentration of PFOA on a C18 SPE column. This column was automatically positioned in front of a Betasil C8 analytical HPLC column (2.1 × 50 mm, 5 μm; ThermoHypersil-Keystone, Bellefonte, PA) for the chromatographic identification of PFOA on a Surveyor HPLC system (ThermoFinnigan, San Jose, CA) operated at a 300 μL/min flow rate with 20 mM ammonium acetate (pH 4) in water (mobile phase A) and acetonitrile (mobile phase B). The HPLC gradient program (11 min) started at 10% of mobile phase B and increased from 10% to 55% (0.5–4.0 min), from 55% to 80% (4–7.0 min), and from 80% to 90% (7–9 min), then B content 90% hold (0.5 min), decreased to 10%, and hold 10% (9.6–11 min). Detection and quantification used negativeion heated electrospray ionization (a variant of electrospray ionization) tandem mass spectrometry on a ThermoFinnigan TSQ Quantum Ultra triple-quadrupole mass spectrometer. The isotope-labeled internal standard used for quantification was 13
-PFOA. We used 10 PFOA calibration standards, spiked into calf serum, encompassing the entire linear range of the method (0.1–150 ng/mL), to construct a daily calibration curve for quantification. For samples with PFOA concentrations higher than the highest calibration standard, we used a volume of serum of < 100 μL, so the actual volume of serum used varied between 10 μL and 100 μL, depending on the sample. In addition to the calibration standards, we analyzed blanks and low-concentration (~ 4 ng/mL), medium-concentration (~ 17 ng/mL), and high-concentration (~ 100 ng/mL) quality control (QC) materials, prepared in calf serum, along with each batch of samples to ensure the accuracy and reliability of the data across time (Kuklenyik et al. 2005
). The concentrations of the QC materials, averaged to obtain one measurement each of high-, medium-, and low-concentration QC for each batch, were evaluated using standard statistical probability rules (Caudill et al. 2008
Duplicate 4 mL blood samples were collected from 32 participants during the first round of blood draws in May–June 2007. Spun serum from these samples was shipped to the Southwest Research Institute (San Antonio, TX) as a quality assurance check and analyzed using liquid chromatography–tandem mass spectrometry; the Spearman rank correlation coefficient for paired PFOA concentrations measured by the two laboratories was 0.89. The median ratio between paired concentrations was 1.16, with higher average estimates obtained by the CDC laboratory.
Several of the blood samples collected by a single phlebotomist during the month 2 visit appear to have implausible serum PFOA concentrations compared with PFOA measurements for the same individuals at other time points. We strongly suspect that these blood samples were inadvertently mislabeled during collection or processing before shipment to the CDC. Thus, all 19 blood samples collected by this phlebotomist during the month 2 visit were excluded from the analysis.
All statistical analyses were performed using R (R Development Core Team 2008
). Unless otherwise noted, statistics were calculated using all available valid observations; that is, participants who dropped out of the study and those with omitted 2-month measurements were not excluded from the analyses. Baseline comparisons were conducted using unpaired two-sample t
-tests using unequal variances for continuous variables, and chi-square tests with continuity corrections for proportions. All reported p
-values reflect two-tailed hypothesis tests.
Linear mixed effects models were used to assess subject-specific changes in serum PFOA concentrations over time and to estimate serum PFOA half-lives. The following general model form was used to describe the observed data:
is the serum PFOA concentration measured for individual i
at sampling round j
is the subject-specific intercept, λ is the prefiltration slope, sij
is the prefiltration duration (i.e., time elapsed between blood sample collection and the first day of filtration, set to 0 after filtration), ki
is the subject-specific postfiltration slope, tij
is the postfiltration duration (i.e., time elapsed between the first day of filtration and the blood sample collection day, set to 0 before filtration), Xi
is a vector of fixed covariates for individual i
, β is a vector of fixed effects corresponding to the covariates, and ij
is a random error term. The intercept, postfiltration slope, and error terms were all modeled as random effects with normal distributions; other parameters were treated as fixed effects. The prefiltration slope might also be expected to vary across subjects but could not be estimated as a random effect due to a lack of multiple prefiltration blood samples for most of our subjects. This model assumes that log serum PFOA concentrations were changing linearly over time for a few weeks before filtration for the Lubeck measurements, and for several months before filtration for the Little Hocking measurements. It also assumes a linear postfiltration slope in log serum PFOA concentrations over time for each individual. Parameter estimation was accomplished using the lme package in R, with maximization of the restricted log-likelihood.
Our longitudinal analyses using the linear mixed effects models included the following covariates as predictors: water district, public/bottled water drinker, sex, age, local produce consumer, homegrown produce consumer, and public water service at work. These covariates were selected because average serum PFOA concentrations measured by the C8 Health Project were higher in those served by Little Hocking than for Lubeck, and because related factors (i.e., age, tap water drinks per day, servings of homegrown fruit and vegetables) have been reported to be predictors of serum PFOA concentrations among residents served by Little Hocking Water Association (Emmett et al. 2006
). In addition, an interaction term was used between water district and water source (public/bottled water drinker), in order to allow the effect of water source on baseline PFOA to differ by water district. PFOA concentration was the only time-varying measurement entered into the models; baseline values were used for all other covariates. Model assumptions were evaluated using diagnostic plots and by testing the addition of quadratic terms for tij
Notably, our mixed effects model is equivalent to assuming first-order elimination kinetics with one compartment after filtration, if postfiltration PFOA exposure rates are negligible relative to baseline PFOA serum concentrations. The median PFOA serum concentration in the U.S. general population is 4 ng/mL (Calafat et al. 2007
), a value about 15–100 times smaller than the mean baseline serum concentrations in our four study groups. By definition, after exposures cease first-order elimination is described using the following equation:
where Ct is the concentration at time t, and k is the elimination rate constant. The solution to this equation is well known:
which has the same form as the linear mixed effects model used in this study, because sij = 0 for all postfiltration measurements. If postfiltration exposures are negligible, αi + Xiβ represents the baseline log serum PFOA concentration for participant i, − ki represents the subject-specific postfiltration elimination rate constant, and the subject-specific serum half-life is −ln(2)/ki. Because the serum PFOA concentrations appear to be lognormally distributed, the log transformation is appropriate for both the mean model and for the residuals.