Bioactivity varied according to the type of spilled plastic. First, a single extract concentration was tested to semi-quantitatively assess the variability in bioactivity within a sample type (e.g., white nurdle, burnt plastic, and combustion remnant). Subsequently, dose-response relationships were constructed for the bioactivity of the pyroplastics. Results were largely consistent across the three extracts prepared from three independent sets of plastic (Figure , Tables ). The coefficients of variation of the end points for the burnt plastic and combustion remnant ranged from 0.7 to 16.9% with a mean of 5.2% and 0.2% to 15.6% with a mean of 5.3%, respectively. As a result, one extract of each plastic type was used as a representative sample (Tables S6-S7) for evaluating the dose-response activity of the pyroplastics. The variabilities for the activated end points between the two assay formats were within the reported biological variability of the assays. White nurdles. Of the 45 human transcription factor response elements and 24 nuclear receptors tested for activity in the TF-and NR-FACTORIAL bioassays, the white nurdles activated only two end points above the operationally-defined 1.5 fold-induction cut-off. At the concentration tested, only the aryl hydrocarbon receptor response element (AhRE) and the retinoid X receptor  (RXR) nuclear receptor exceeded the cut-off. The extracts induced average fold increases in activity of 1.76  0.48 (n=3) for AhRE and 1.84  0.76 (n=3) for RXR (Table ). This amount of bioactivity was comparable (within the same order of magnitude) to that of polyethylene nurdles collected after the M/V CMA CGM Bianca containership plastic spill that happened along the banks of the Mississippi River in New Orleans, Louisiana, USA, in August 2020. Extracts of these nurdles induced average fold increases in activity of 3.79  3.43 (n=3) for AhRE; all other end points were below the operationally defined cut-off (Table ). This spill was without fire, and so the source of the AhRE activity was attributed to hydrophobic organic contaminants from the Mississippi River that can associate with the nurdles. Thus, the bioactivity of the white nurdles appeared comparable to that of other polyethylene nurdles found in aquatic environments resulting from a containership spill. This finding also agreed with chemical analyses of their PAH content, which did not differ from that of other nurdles collected in the aquatic environment globally. 9 Pyroplastics. The pyroplastic was more bioactive than the white nurdles, and activation trended with the amount of extractable material. The extracts from the burnt plastic and combustion remnant pieces activated several end points related to biotransformation, lipid, endocrine, and cell stress, growth, and differentiation processes (Figure , Tables S6-S7, S9-S10). Specifically, the extracts activated the pregnane X receptor response element (PXRE) and its nuclear receptor (PXR), the estrogen receptor response element (ERE) and its receptor  (ER), the peroxisome proliferator-activated receptor response element (PPRE) and its receptor  (PPAR), RXR, the nuclear erythroid-2 related factor 2-antioxidant response element (NRF2/ARE), the activator protein 1 (AP-1), and AhRE (Figure ). The elevated activity of PXR, ER, and PPAR in the TF and NR assays for pyroplastic extracts suggested that active components of these extracts acted as direct ligands of PXR, ER, and PPAR. Several other end points demonstrated defined dose-response relationships that did not exceed the 1.5-fold induction cut-off operationally-defined for activation or inconsistently exceeded the cutoff between the two independently run assay formats (Figure , Tables S6-S7, S9-S10). These end points included the liver X receptor  (LXR), the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor  (PPAR), the nuclear receptor related 1 (NURR1; also known as the nuclear receptor 4A2), the metal regulatory transcription factor 1 response element (MRE), the hypoxia-inducible factor-1 (HIF1), the vitamin D receptor response element (VDRE), and the retinoic acid receptor-related orphan receptor response element (RORE). The activity of the liver X receptor family (direct repeat 4-binding proteins) response element (DR4/LXR) and the nuclear respiratory factor 1 (NRF1) activity were suppressed with increasing concentration of extractable material (Figure ). The dose-response curves for AhRE, AP-1, CAR, ERE, ER, HIF1, LXR, MRE, NRF2/ARE, NURR1, PPRE, PPAR, PXRE, PXR, and VDRE showed tremendous concordance between the burnt plastic and combustion remnant with only minor deviations (i.e., the curves lined up on top of one another) (Figures and). The dose-response curves for DR4/LXR, PPAR, RORE, and RXR deviated substantially between the two plastic types (Figures and). The deviation was assessed qualitatively as the relative difference between the dose-response curves of the individual datasets and those of a doseresponse curve for their combined dataset. The bioactivity of extracts from the pyroplastic collected eight months after the spill was slightly diminished.