Materials and Methods
All project protocols were approved by the Portland State University Office of Research Compliance and Institutional Review Board. Dr. Stephanie Farquhar, the project’s Principal Investigator, provided oversight of the study methodology, data collection, laboratory testing, and data analyses.
The ten participants in this study were selected for diversity in age, geography, occupation, ethnicity, and gender. Trained research assistants met with potential subjects to review project goals and methodologies, answer questions, and complete formal consent documents. Each participant was asked to complete an exposure assessment questionnaire and provide information about their residences, occupations, diet, and potential toxic exposures.
Samples were collected in March, April, and May of 2007 using containers and procedures supplied by the analytical laboratories. A nurse collected approximately 50 milliliters of blood from each participant following all necessary safety and sample collection protocols. After clotting, serum was obtained by centrifuging tubes and pouring off or pipetting serum into storage vials. Approximately 10 milliliters of whole blood was maintained for each participant for mercury testing. Samples were processed as necessary, frozen, placed upright in appropriate containers with ice packs, and mailed via overnight courier to the analytical laboratories.
Participants provided first morning void urine samples for phthalate, bisphenol A, organophosphate pesticide, and creatinine clearance testing. Urine samples were collected in the appropriate containers and transferred to storage containers. Urine samples were refrigerated and mailed overnight to the analyzing laboratories. The analytical laboratories provided all the appropriate collection materials and shipping instructions.
All samples were coded to preserve anonymity of the participants. All samples collected were used solely for this project.
Chemical Analysis
Phthalate, Bisphenol A, and PFC Analysis
AXYS Analytical Services, LTD, in Victoria, British Columbia analyzed urine samples for phthalates and bisphenol A and blood serum samples for perfluorinated chemicals (PFCs). Below are the laboratory’s methods in brief.
Phthalates and bisphenol A. Urine samples were analyzed for phthalate mono-esters and total BPA amounts, including both the glucuronidated and free forms of BPA by AXYS Method MLA-059, Analysis of Bisphenol A and Phthalate Metabolites in Urine by LC/MS/MS. This method allows for the combined work up of urine samples for both bisphenol A and phthalate ester metabolites. Accurately measured (approximately 1 milliliter) samples were spiked with isotopically labeled surrogate standards and incubated with an enzyme to release the mono-esters from their glucoronated form. The incubated urine was diluted with high purity water, pH adjusted, and loaded onto SPE cartridges for extraction and clean up. The SPE cartridges were eluted, and the extracts were analyzed on a high performance liquid chromatograph coupled with a triple quadruple mass spectrometer, running manufacturers MassLynx v.4.0 software.
PFCs. Serum was analyzed for PFCs by AXYS Method MLA-042, Analysis of Perfluorinated Organic Compounds (PFC) in Blood Serum by LC/MS/MS. Samples of 0.5 milliliters were spiked with 13C-labeled PFCs and extracted with formic acid. Extracts were loaded onto pre-conditioned Waters Oasis WAX SPE cartridges, which were washed and then eluted with basic methanol. The cleaned-up extracts were spiked with 13C-labeled PFC recovery standards, diluted to final volume with methanol, and analyzed by LC/MS/MS. Analysis was performed on a Micromass Quattro Ultima MS/MS coupled to a Waters 2795 HPLC equipped with a reverse-phase C18 column (7.5cm, 21 mm i.d., 3.5mm particle size). The LC/MS/MS was operated in the MRM mode at unit resolution, using Negative Ion Electrospray ionization. PFC concentrations were determined by isotope dilution or internal standard quantification against the labeled surrogates added at the beginning of the analysis.
Organophosphate Pesticide and PCB Analysis
Pacific Toxicology Labs in Los Angeles, California analyzed urine samples for organophosphate pesticides and serum for PCBs. Below are the laboratory’s methods, in brief.
Organophosphate pesticide metabolites. Urine samples were derivatized with a benzyltoytriazine reagent to produce benzyl derivatives of alkylphosphate metabolites. A saturated salt solution was added to the tubes and the benzyl derivatives were extracted with cyclohexane and analyzed by gas chromatography with flame photometric detection.
PCBs. Serum samples were analyzed using the Webb-McCall method in which PCBs were extracted from de-proteinized serum with 1:1 hexane/ethyl ether. PCBs were separated from by chromatography on silica gel using hexane as eluent. PCB concentrations in the eluent were determined by electron capture gas chromatographic analysis using Webb-McCall mean with percent factors and the internal standard method.
Mercury Analysis
Mercury analysis in whole blood was conducted by Brooks Rand in Seattle, Washington. Samples were analyzed using the EPA 1630 Mod. (BR-0011), Monomethyl Mercury in Blood/Serum - Ultra-low Method. Samples were digested in a KOH/methanol solution. The digestates were then distilled in Teflon distillation vials. Samples were then analyzed by ethylation, Tenax trap pre-concentration, gas chromatography separation, pyrolytic combustion, and atomic fluorescence spectroscopy.
Data Analysis
In order to be consistent with methods used by the CDC, for phthalates, bisphenol A, PFCs, and PCBs, medians were calculated setting non-detectable values at the detection limit divided by the square root of two. Medians were not calculated for organophosphate pesticides because of the relatively high number of participants with undetectable levels. To calculate the sum total for phthalates and PFCs, any value reported as non-detected was assigned a value of _ the detection limit.
