A Proud Global Partner Pilot Study for Relative Bioavailability Study of PAHs in Coal Tar Pitch of Clay Target Fragments Brian Magee, PhD, ARCADIS Glenn Hoeger, ARCADIS Million Woudneh, PhD, AXYS Analytical Anita Meyer, PhD, USACE SETAC North America 35th Annual Meeting November 11, 2014 Imagine the result 6 November 2014 1 © 2014 ARCADIS A Proud Global Partner Pilot Study Purpose • Purpose of the Pilot Study is to develop procedures for evaluating the relative bioavailability of PAHs in clay target fragments including: ‒ Preparing soil test articles for feeding study in mice ‒ Preparing test diets with known concentrations of PAHs from soil test articles and extracts ‒ Identifying PAH metabolites having commercially available control standards ‒ Developing analytical methodology for quantifying PAH metabolites in mouse urine ‒ Providing initial relative bioavailability data for PAHs in clay target fragments 2 6 November 2014 © 2014 ARCADIS A Proud Global Partner Issue: PAHs in Soil at Skeet Range Sites 1. Clay targets made of coal tar pitch and limestone 2. Clay targets are fragile and readily broken into small fragments 3. Coal tar pitch source of high-molecular weight PAHs to soil 3 6 November 2014 © 2014 ARCADIS A Proud Global Partner Conceptual Model for PAHs in Soil Target Fragments Soil Particles 4 6 November 2014 © 2014 ARCADIS A Proud Global Partner Relative Bioavailability Approach Sieved soil w/target fragments 0.25 mm Apply extract to diet Apply soil directly to diet extract Feed mice Collect urine Analyze for metabolites 5 6 November 2014 © 2014 ARCADIS A Proud Global Partner Pilot Study 1. Sample Collection / Test Article & Diet Preparation 2. PAH Metabolite Analytical Method Development 3. Urine Sample Analytical Results 6 6 November 2014 © 2014 ARCADIS A Proud Global Partner Sample, Test Article, and Diet Preparation • Air dry samples – oven drying changes PAH composition in soil • Two-stage sieve/homogenization approach to achieve uniform soil test article from dried soil sample: 1. Sieve #10 (2mm), 1st homogenization 2. Sieve #60 (0.25mm), 2nd homogenization • Analyze soil test article in triplicate, RSD ≤ 20 % • Extract using methylene chloride • Diet Preparation – 5%, 10%, and 20% soil test article in diet, match extract PAH mass to 10% diet preparation • Analyze diet in triplicate, RSD ≤ 20 % 7 6 November 2014 © 2014 ARCADIS A Proud Global Partner PAHs Soil, Fragments, Extract, and Diet Soil Test Article (ppm) Diet Preparation 10% Soil (ppm) Diet Preparation Soil Extract (ppm) Fragment Test Article (ppm) Diet Preparation 10% Frag. (ppm) Diet Preparation Frag. Extract (ppm) Benzo(a)pyrene 255 27 25 1,267 123 104 11% 14% 3% 5% 5% 20% Benz(a)anthracene 230 21 18 1,067 106 90 12% 10% 11% 6% 8% 23% Chrysene 249 26 25 1,200 127 118 12% 17% 3% 2% 5% 19% 8 6 November 2014 © 2014 ARCADIS A Proud Global Partner PAH Metabolite Analysis • Method developed to detect PAH metabolites in urine at approximately 10 ng/L derived • Control standards & appropriate surrogate standards available for: 1. 2. 3. 4. 3-Hydroxy benzo(a)pyrene (3OH-BaP) 9-Hydroxy benzo(a)pyrene (9OH-BaP) 3-Hydroxy benz(a)anthracene (3OH-BaA) 3-Hydroxy chrysene (3OH-Chry) • Accuracy evaluation: control standard recovery 77-101% for five replicates; MS/MSD recoveries 84-132% • Precision evaluation: spike replicate recoveries RPDs < 5% 9 6 November 2014 © 2014 ARCADIS A Proud Global Partner Analytical Procedure Analysis Flow Chart 4 mL of Urine Spike antioxidant to control analyte oxidation Spike standards: - Quantification standards - De-conjugation standards - ẞ-glucuronidase enzyme Incubate sample at 37oC for approximately 17 hours Extract 3x with 4 mL pentane Spike with derivatization control and recovery standards and derivatize with MSTFA at 60oC Analyze with HRGC-HRMS 10 6 November 2014 © 2014 ARCADIS A Proud Global Partner Pilot Test Results 1. Laboratory Animal Data • • • Body weight gain (grams/mouse) Food consumption (grams/day) Urine production (mL/mouse) 2. PAH ingested dose (µg/day) 3. PAH metabolites in urine (µg/day) 4. Relative Bioavailability (RBA) Factor (Preliminary) 11 6 November 2014 © 2014 ARCADIS A Proud Global Partner Biological Data Body Weight Gain (g)1 Daily Food Ingestion Rate (g/mouse/day) Urine Production (mL/mouse/day) Control Soil Test Article 5% Soil Test Article 10% Soil Test Article 20% Extract (based on 10% Soil) 3.88 3.25 1.83 1.08 1.9 4.3 4.0 3.7 3.9 3.2 ±1.5 ±1.5 ±1.5 ±1.6 ±0.8 1.37 1.55 0.92 1.17 1.47 ±0.45 ±0.35 ±0.4 ±0.4 ±0.87 1 – Weight gain represents difference in average body weight on 14th day less average body weight on 1st day. 12 6 November 2014 © 2014 ARCADIS A Proud Global Partner PAH Doses 5% Soil 10% Soil 20% Soil 10% Extract Benzo(a)pyrene 194 448 849 314 Benz(a)anthracene 152 353 668 233 Chrysene 191 442 838 327 Benzo(a)pyrene 172 368 785 305 Benz(a)anthracene 136 290 618 225 Chrysene 170 363 775 318 Day 7 Day 14 All Doses in units of micrograms per day (µg/day) 13 6 November 2014 © 2014 ARCADIS A Proud Global Partner PAH Metabolite Excretion 5% Soil 10% Soil 20% Soil 10% Extract Benzo(a)pyrene* 0.84 2.40 3.30 5.37 Benz(a)anthracene 0.06 0.13 0.19 0.22 Chrysene 0.29 0.40 0.54 0.65 Benzo(a)pyrene* 0.96 2.21 3.54 5.86 Benz(a)anthracene 0.05 0.11 0.23 0.25 Chrysene 0.31 0.41 0.55 0.64 Day 7 Day 14 All excretions in units of micrograms per day (µg/day) * Benzo(a)pyrene based on sum of 3OH-BaP and 9OH-BaP 14 6 November 2014 © 2014 ARCADIS A Proud Global Partner Calculated Relative Bioavailability Relative Bioavailability Factor (7-day exposure) Relative Bioavailability Factor (14-day exposure) 3OH-BaP 30% 31% 9OH-BaP 28% 34% 3OH-BaA 38% 35% 3OH-CHR 42% 57% PAH Metabolites 15 6 November 2014 © 2014 ARCADIS A Proud Global Partner Main Study Protocol Revisions • Air-dry soil samples • Two-stage sieving/homogenization • Target Relative Standard Deviation of 20% • Extract preparation using methylene chloride rather than acetone • Diet preparation at 5% Soil Test Article in Diet • Urine volume of 1 mL required for analysis • Anti-oxidant added to urine preparation to control autooxidation of PAH metabolites 16 6 November 2014 © 2014 ARCADIS Imagine the result Brian Magee, Ph.D. ARCADIS U.S., Inc. Two Executive Drive, Suite 303, Chelmsford, MA 01824 Tel: 978.937.9999 x319 [email protected] 17 6 November 2014 © 2014 ARCADIS Glenn Hoeger ARCADIS/Malcolm Pirnie One South Church Ave, Suite 1120, Tucson, AZ, 85701 Tel: 520-629-8282 [email protected]