Groundwater samples were collected in June 2018 from a background (reference) well located 200 m upgradient from the source and five wells along a flowline in the plume at 39, 68, 102, 125, and 254 m downgradient from the source. Before sampling, at least three times the water volume in the well casing was purged and field parameters (temperature, dissolved oxygen, specific conductance, and pH) were stable. Two samples from each well were collected into unpreserved 1 L amber bottles and shipped on ice overnight to a commercial lab. The two samples were extracted using dichloromethane (DCM; EPA Method 3510). One sample extract was treated with silica gel cleanup (SGC) column (USEPA method 3630C). Aliquots of the above two extracts were used for the high-throughput bioassays (Attagene Inc. Morrisville, NC).To ensure compatibility with the bioassays, DCM-total and DCM-SGC extracts were dried under nitrogen gas and, once dry, reconstituted in 1 mL of dimethysulfoxide (DMSO) resulting in 1000x concentration. A third water sample was collected from each well to perform Attagene bioassays on the organics obtained with the HLB solid phase extraction. These samples were kept on dry ice in the field and stored at 20C. Samples were processed before Aug 17, 2018 (within 22 days). They were filtered using a GF/F filter (1.0 μm); 250 mL of each filtrate was concentrated using OASIS hydrophilic-lipophilic balance (HLB) 5 cm3 200 mg cartridges (Waters, Milford, MA). The cartridges were eluted with 6 mL of methanol, followed by 6 mL of a 50:50 mixture of methanol and DCM, and brought to dryness under nitrogen gas at 20°C. The extracts were reconstituted with 0.5 mL dimethysulfoxide (DMSO) resulting in 500x concentration. The preparation method removes the volatile fraction. As a result, the toxicity assays performed in this study did not assess the effects of the volatile components in the plume as measured by the TPHg analyses. Extracts generated using DCM-total, DCM-SGC and HLB were tested in Attagene assays at 1x concentration relative to the groundwater (i.e. 1 µL of 1000x extract or 2 µL of 500x extract were added to 1 mL of growth media). Bioassays that evaluate activation of 46 molecular targets (CIS-FACTORIAL) were performed on these three extracts in duplicate (Attagene Inc. Morrisville, NC). The assay method was described by Romanov et al.38 and deployed for identification of molecular targets of interest in oil-contaminated groundwater samples 20 and a variety of surface waters39. Briefly, human hepatoma (HepG2) cells transfected with reporter constructs activated by transcription factors (TF) were used. The reporter transcript abundance was measured by isolating the produced RNA, reverse transcription, amplification, labeling and capillary electrophoresis. Abundance data are reported as the induction by sample of interest relative to abundance induced by a DMSO solvent control (abundance in environmental sample was divided by abundance in solvent control). Positive control assays were performed for a subset of molecular targets (Table S2) including AhR (6-Formylindolo [3,2-b] Carbazole) and PXR (Rifampicin).