Solvent extracts were prepared in triplicate by incubating ten nurdles or their equivalent mass of plastic in 5 mL (~45 mg/mL) analytical grade dichloromethane (DCM) for 24 h at room temperature in combusted borosilicate glass vials with PTFE/F217 lined caps. DCM was used as a solvent because it would provide parity with previous chemical analyses conducted on the spilled plastic, 4,9 it is commonly used to prepare extracts from combustion-derived plastics and materials for bioassays, and many classes of organic compounds, including hydrocarbons, are readily soluble in it. After extraction, half of the DCM extract (2.5 mL) was blown to dryness under a gentle stream of nitrogen at room temperature and reconstituted in 100 µL of molecular biology grade dimethyl sulfoxide (DMSO). An extraction blank without plastic was also prepared. Specifics of each extract are provided in Table . TF-, NR-, and AquaTox-FACTORIAL bioassays. DMSO-reconstituted DCM extracts were shipped to Attagene, Inc. (Morrisville, NC, USA) for testing by their TF-FACTORIAL (45 TF specific reporters), NR-FACTORIAL (24 human NRs) assays (previously named cis-and trans-FACTORIAL assays, respectively), and AquaTox-FACTORIAL (6 human NRs and 19 fish NRs). The assays use HepG2 cells to assess the activity of endogenous transcription factors (TF assay) or transfected hybrid proteins consisting of a yeast GAL4 DNA binding domain and ligand-binding domain of the human nuclear receptors (NR assay) or fish nuclear receptors (AquaTox assay). These multiplexed assays comprised 89 different measured end points related to cell stress, endocrine activity, growth and differentiation, immunity, and lipid, xenobiotic, and general metabolism. Extracts were tested at a maximum concentration of 3 µL DMSO extract/mL cell culture medium for 24 h. This concentration equates to the extractable content from ~4 mg of spilled plastic (~20% of the mass of a nurdle). Final DMSO concentrations were 0.3% (v/v). Five to six technical replicates of DMSO solvent controls matched to the DMSO concentration of the extracts were run with each sample set. Each extract was run as three technical replicates in Dulbecco's Modified Eagle Medium (DMEM) containing 1% charcoal-stripped fetal bovine serum (FBS). The pyroplastics were evaluated by each FACTORIAL assay twice: the first at the maximum tested concentration for each of three extracts prepared from three independent sets of plastic, and the second as a 6-point serial dilution from the maximum tested concentration for a single representative extract. Each assay format was run once. TF-FACTORIAL assay. HepG2 cells were transfected with TF-FACTORIAL reporter library (46 TF-specific reporter plasmids and seven control reporters) using TransIT-LT1 transfection reagent according to the manufacturer's protocol (Mirus). Transfected cells were plated into 12-well plates (310 5 /well), incubated for 24 h in their growth medium, washed, and treated with samples for 24 h in assay media (DMEM with 1% charcoal-stripped FBS). Cells were collected and processed. NR-FACTORIAL assay. HepG2 cells were transfected with NR-FACTORIAL library (25 GAL4-NR expression vector and corresponding reporter plasmid pairs) using TransIT-LT1 transfection reagent according to the manufacturer's protocol (Mirus). Each pair of GAL4-NR/reporter was transfected separately to avoid cross-reactivity. Transfected cells were pooled together and plated into 12-well plates (310 5 /well), incubated for 24 h in growth media, washed and treated with tested samples for 24 h in assay medium (DMEM with 1% charcoal-stripped FBS). Cells were collected and processed. AquaTox-FACTORIAL assay. HepG2 cells were transfected with AquaTox-FACTORIAL library (25 GAL4-NR expression vector and corresponding reporter plasmid pairs) using TransIT-LT1 transfection reagent according to manufacturer's protocol (Mirus). Each pair of GAL4-NR/reporter was transfected separately to avoid cross-reactivity. Transfected cells were pooled together and plated into 12-well plates (310 5 /well), incubated for 24 h in growth media, washed and treated with tested samples for 24 h in assay medium (DMEM with 1% charcoal-stripped FBS). Cells were collected and processed. Sample processing. Total RNA was isolated using PureLink Pro 96 total RNA Purification Kit (ThermoFisher). Reporter RNA was amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a single pair of common primers. PCR fragments were labeled with fluorescent markers, and cut with HpaI restriction enzyme, generating reporter-specific sizes of labeled DNA fragments that were quantitatively assayed by capillary electrophoresis using a Genetic Analyzer 3500xl. Bioassay responses were expressed as fold-induction relative to the DMSO control by dividing the treated cells' average technical replicate expression by the average technical replicate expression of the appropriate DMSO control. Activation of an end point was operationally-defined as requiring more than 1.5-fold induction across the two independently run assay formats and having a defined dose-response curve. All activities of an extraction blank were below the operationally-defined induction cut-off (Tables ), and all positive control compounds activated receptors as expected (Table ). Statistical analysis. Statistical analyses were conducted using GraphPad Prism 10.2.3 (347). Data are presented as mean ± standard deviation (n = sample size). When appropriate, either parametric or nonparametric tests were used to compare groups. Groups were considered significantly different for a p value less than 0.05. EC50 concentrations and their asymmetrical 95% confidence intervals were calculated by fitting a three-parameter dose-response curve, 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 = 𝑏𝑜𝑡𝑡𝑜𝑚 +