In response to the Deepwater Horizon oil spill, the Natural Resource Trustees implemented a toxicity testing program that included 4 different Deepwater Horizon oils that ranged from fresh to weathered, and 3 different oil-in-water preparation methods (including one that used the chemical dispersant Corexit 9500) to prepare a total of 12 chemically unique water accommodated fractions (WAFs). We determined how the different WAF preparation methods, WAF concentrations, and oil types influenced the chemical composition and concentration of polycyclic aromatic hydrocarbons (PAHs) in the dissolved and particulate phases over time periods used in standard toxicity tests. In WAFs prepared with the same starting oil and oil-to-water ratio, the composition and concentration of the dissolved fractions were similar across all preparation methods. However, these similarities diverged when dilutions of the 3 WAF methods were compared. In WAFs containing oil droplets, we found that the dissolved phase was a small fraction of the total PAH concentration for the high-concentration stock WAFs; however, the dissolved phase became the dominant fraction when it was diluted to lower concentrations. Furthermore, decreases in concentration over time were mainly related to surfacing of the larger oil droplets. The initial mean diameters of the droplets were approximately 5 to 10 μm, with a few droplets larger than 30 μm. After 96 h, the mean droplet size decreased to 3 to 5 μm, with generally all droplets larger than 10 μm resurfacing. These data provide a detailed assessment of the concentration and form (dissolved vs particulate) of the PAHs in our WAF exposures, measurements that are important for determining the effects of oil on aquatic species. Environ Toxicol Chem 2017;36:1460-1472. © 2017 SETAC.