U.S. Environmental Protection Agency Office of Research and Development EPA www.epa.gov Introduction Methods Conclusions Brett Blackwell l blackwell.brett@epa.gov l 218-529-5078 Empirical measurement of PFAS dosing within in vivo aquatic high throughput assays B. Blackwell1, E. Stacy1, K. Bush2, K. Flynn1, J. Hoang2, M. Le2, D. Villeneuve1 1USEPA Great Lakes Toxicology and Ecology Division, Duluth, MN; 2Oak Ridge Institute for Science and Education (ORISE), Duluth, MN • Understanding per- and polyfluoroalkyl substances (PFAS) toxicity is a priority of the US EPA, and high- throughput testing (HTT) has been developed as a rapid, low-cost alternative to traditional in vivo toxicity testing. • The unique physicochemical properties of PFAS preclude accurately modeling free chemical concentrations using current in vitro disposition models. • Characterizing chemical behavior and partitioning in HTT format is critical for comparison of HTT- derived points of departure with those from traditional aquatic toxicity assays. • Here, 22 individual PFAS (Table 1) were screened for toxicity to juvenile Daphnia magna (see poster 4.21.P-Th175) or Pimephales promelas (poster 2.14.P-We058) following 24 h exposure in 96 well format. • Final concentration of each PFAS in exposure media was empirically measured by LC-MS to accurately assess exposure conditions. Chemical Exposures • PFAS (Table 1) were acquired from EvoTec (South San Francisco, CA) solubilized in DMSO at a nominal concentration of 30 mM and 9.5 mM. Subsequent concentrations were made in a log dilution series from these two solutions for a total of 9 concentrations at ½ log steps. Nominal exposure concentrations ranged 0.03 – 100 μM for each PFAS. • Organisms were exposed to 8 concentrations of each chemical (plus a control) in 1mL polypropylene 96-well plates (n=5 individuals per concentration) in a total volume of 700 µL Lake Superior water spiked with working stock (concentration of DMSO<0.33%). • Working stocks were saved in 1:1 acetonitrile:media or 3:1 acetonitrile:media for chemical analysis. • Exposures were performed for 24 hours at 20 °C (D. magna) or 25°C (P. promelas) with a 16:8 light:dark cycle. Following this, media was transfered from exposure plates into a clean well plate, diluted 1:1 or 3:1 with acetonitrile, and stored at -20°C. • Three replicates per concentration were analyzed for each individual PFAS. For P. promelas, individual replicate wells from one exposure plate were measured while for D. magna, 5 replicate wells per concentration were pooled within 3 replicate exposure plates. The lowest concentration of each PFAS was not measured due to concentrations consistently below or near detection limits (n = 14-21 wells per chemical). Effluent Discharge *Contents of this poster neither constitute, nor necessarily reflect, official US EPA policy. Use or mention of trade names trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government* Chemical Verification • Chemical stocks used to prepare exposure plates and media from post-exposure plates were analyzed by LC-MS using a Vanquish LC system coupled to a TSQ Altis tandem MS. • One method based on EPA Method 1633 (method details given below) was used for quantification of most target analytes. Individual methods were developed for 6:1 FTOH, PFPB, and PFTP. • A stable mass labeled internal standard was used where possible to account for potential matrix effects and analyte loss during sample preparation. • For compounds without a matched internal standard, a stable labeled surrogate with the nearest retention time was used for quantification. Thermo Scientific Vanquish LC system • Column: StableBond C18, 2.1 x 50mm, 1.8μm • Column Temp: 40°C • Injection volume: 5 μL • Mobile phase: A = H2O+5mM ammonium acetate B = MeOH Flow rate: 0.4 mL/min • Gradient: 10% B to 55% B at 2.5 min, to 90% B at 9 min, to 100% B at 9.5 min, held for 2 min, return to 10% B. • Total