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Study Methodology

Polluted Pets: Study Methodology

April 17, 2008

Introduction. For 20 dogs and 37 cats, a trip to the vet in December and January included a special procedure – the donation of a small amount of blood or urine needed for an exploratory study of industrial pollutants in pets. In the most comprehensive tests ever conducted on companion animals, we analyzed samples for a broad battery of industrial chemicals and pollutants – 70 chemicals in total, from 5 chemical classes. To our knowledge this work includes the first reported biomonitoring tests for 23 of the targeted chemicals in pets. Information below describes the components of this new study, detailing the sample collection procedures, sample preparation and analysis methods, and the quality assurance and quality control provisions included in the study design.

Blood and urine sample acquisition and storage. Samples were collected from dogs and cats visiting the Hanover Animal Hospital in Mechanicsville, Virginia. Each pet owner was informed of the nature of the study, and signed a consent form in order to participate. A dog typically can safely provide 3 to 4 mL of blood and 1 mL of urine, while due to smaller size, a cat typically can safely provide only 1 to 2 mL of blood and 1 mL of urine.

Number of pets sampled, sample medium, and sample storage container varied depending on the class of chemicals targeted for analysis:

  • 5 dogs and 10 cats provided blood samples stored and processed in plastic vacutainers and vials for perfluorochemicals analysis;
  • 5 dogs and 5 cats provided urine samples for phthalates and BPA analysis;
  • 10 dogs and 17 cats provided blood samples stored and processed in glass vacutainers and vials for PBDEs analysis;
  • 5 dogs and 10 cats provided blood samples stored in EDTA-treated plastic vacutainers for metals analysis.
Blood samples collected for perfluorochemical and PBDE analyses were allowed to clot for 30 minutes, then centrifuged. The serum was extracted and stored for analysis. Blood samples collected for metals analysis were used whole. Serum, whole blood, and urine samples were frozen and shipped at the end of the collection period to Axys Analytical Services in British Columbia, Canada. There, individual samples were combined to create a single dog and a single cat serum sample for perfluorochemicals analysis, a single dog and a single cat urine sample for phthalates and BPA analysis, and a single dog and a single cat serum sample for PBDEs analysis. Whole blood samples were shipped to Brooks Rand in Seattle, Washington, where they were composited to create a single dog sample and a single cat sample for metals analysis. Samples were stored at -20 degrees C until analyzed.

Analysis of Perfluorochemicals (PFCs). Analysis for perfluorochemicals was conducted on approximately 0.5 mL of serum. The sample was first spiked with three 13C-labeled perfluorochemical surrogate standards prior to extraction using formic acid on a solid phase extraction cartridge. The extract was spiked with labeled recovery standards and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) using a Micromass Quattro Ultima MS/MS coupled with a Waters 2690 high performance liquid chromatographic (HPLC) system. Target compounds are quantified using the internal standard method, comparing the area of the quantification ion to that of the 13C-labeled standard and correcting for response factors. Final sample concentrations were determined by isotope dilution/internal standard quantification against matrix-matched calibration standards carried through the analysis procedure alongside the samples. Measurements are reported in nanograms per milliliter (wet weight) of blood serum.

Analysis of Phthalate Metabolites and Bisphenol A (BPA). Phthalate metabolites and BPA were co-extracted from 1 mL urine samples. Urine samples were first buffered with ammonium acetate, and spiked with 13C-labeled phthalate monoesters, d6-bisphenol A, 13C4-4-methylumbelliferone, and 4-methylumbelliferyl glucuronide. Urine samples were also spiked with beta-glucuronidase enzyme (for deconjugation of glucuronidated forms of the target analytes). The treated samples were incubated to hydrolyze the glucuronides (the completeness of hydrolysis was monitored by the recovery of 4-methylumbelliferone).

Samples were extracted and cleaned up using solid phase extraction (SPE) procedures. Extracts were spiked with labeled recovery standards for both phthalate metabolites and BPA, split into 2 portions, and each portion was analyzed separately by LC/MS/MS using a Micromass Quattro Ultima MS/MS coupled with a Waters 2695 HPLC system. The method determined the total of the free and the glucuronidated phthalate metabolites and bisphenol A. Analyte concentrations were determined using isotope dilution quantification.

Values are reported in units of micrograms per gram creatinine, a urine protein, to account for variation in the dilution of the urine samples due to different levels of fluid intake by the participating animals.

Analysis of Polybrominated Diphenyl Ethers (PBDEs). Analyses for PBDEs were achieved using a 6 gram serum sample. Samples were spiked with a suite of 13C-labeled surrogate standards, extracted and cleaned up using solid phase extraction (SPE) procedures, followed by acid silica clean up. Extracts were spiked with labeled recovery standards and analyzed by gas chromatography with high-resolution mass spectrometric detection (GC/HRMS) in accordance with EPA method 1614. GC/HRMS analyses were performed using a Micromass Ultima high-resolution mass spectrometer equipped with a Hewlett-Packard 6890 gas chromatograph. Quantification of target analytes was achieved by isotope dilution quantification using the 13C-labeled surrogate standards.

Values are reported in units of nanograms per gram of lipid in the blood serum, as PBDEs accumulate in the lipid fraction of blood.

Analysis of Metals. Analysis for metals was conducted by Brooks Rand LLC (Seattle, WA). To measure methylmercury, 0.1 gram whole blood samples were subjected to alkaline digestion, followed by ethylation, then purging and trapping of the resulting ethylated mercury derivatives. The mercury compounds were thermally desorbed and transferred to a gas chromatography column, which separated the species by mass. Ethylated mercury compounds were heated to form elemental mercury, which was measured using cold vapor atomic fluorescence spectroscopy (CVAFS). Values are reported in units of micrograms per liter (wet weight) of whole blood.

To measure total mercury, 1 gram whole blood samples were subjected to acid digestion and oxidation. Mercury ions in the sample were reduced to elemental mercury, then purged and trapped. The concentrated mercury was measured using CVAFS. Values are reported in units of micrograms per liter (wet weight) of whole blood.

To measure lead, 200 microliter whole blood samples were first diluted by a factor of 50 in a 1% nitric acid solution and filtered. Analysis was conducted using inductively couple plasma – mass spectrometry (ICP-MS).

Values are reported in units of micrograms per deciliter (wet weight) of whole blood.

Procedures for quality assurance and quality control (QA/QC). All organic analyses were conducted in accordance with AXYS' accredited QA/QC program including regular analysis of QC samples and participation in international inter-laboratory comparison programs. Each analysis batch included a procedural blank to demonstrate cleanliness and a spiked laboratory control sample to monitor precision and recovery. The sample results were reviewed and evaluated in relation to the QA/QC samples worked up at the same time. The sample surrogate standard recoveries and detection limits, procedural blank data and the laboratory control sample data were evaluated against method criteria to ensure acceptable data quality.

All metal analyses were conducted in accordance with Brooks Rand's accredited QA/QC program. Samples were analyzed in conjunction with NIST 966 standard-spiked blanks, and 4 method blanks.