Overall, the end points were more variable at the later time point, while the variability of the extractable mass was unchanged between time points. This difference suggested that while the amount of extractable material did not appear to change, there was a change in its composition during this period, which is supported by reported changes in the PAH content of the burnt plastic within this timeframe. 9 The bioactivities of the extracts from the pyroplastic were species-specific. The AquaTox FACTORIAL bioassay revealed differences in nuclear receptor activation among fish species and between fish and human receptors. End points included the induction of speciesspecific estrogen receptors (ER and ER), androgen receptors (AR), peroxisome proliferatoractivated receptors (PPAR and PPAR), and retinoid X receptors (RXR) for humans (HU), Danio rerio (zebrafish; ZF), Oryzias latipes (Japanese medaka; JM), and Oncorhynchus mykiss (rainbow trout; RT) that were expressed as GAL4-NR hybrid proteins in human HepG2 cells. Only end points for human receptors (ER and RXR) were activated by extracts from the white nurdles. Fish ER were largely unresponsive to the extracts from the spilled plastic (Figure ); only medaka ER displayed activity above the 1.5-fold induction criteria in response to the combustion remnant extract (3.09  0.06, n=3). ER was the most sensitive to the pyroplastic extracts, and fish ER were more responsive to the pyroplastic extracts than human ER (Figure ). Medaka and rainbow trout ER were activated in response to the burnt plastic and combustion remnant extracts. Human ER and zebrafish ERb expressed activity in response to the combustion remnant extract. None of the extracts elicited AR activity (Figure ). Fish PPAR and PPAR were not activated by the plastic extracts, while the human PPARs were activated (Figure ). Human and medaka RXR showed activity in response to the pyroplastic extracts (Figure ). As with the TF-and NR-FACTORIAL bioassay results, the activity of the nurdles and pyroplastics collected eight months after the spill trended toward lower values (Figure , Tables ). In conjunction with these semi-quantitative results made at a single concentration, dose-activity measurements (Figure , Table ) suggest the potential for the plastic-associated chemicals to disrupt fish estrogen signaling via different pathways depending on the fish species. In contrast, their ability to disrupt fish androgen and lipid metabolism via direct ligand-activated pathways is unlikely. ) except for their ER activation, which was more akin to BaP and BaA than BbF. Notably, none of the single PAH compounds mentioned above activated PXRE/PXR and RXR and few activated PPRE/PPAR while the pyroplastics robustly activated these end points (Figures ). These results suggest that other compounds in the complex chemical mixture were the cause of their activity.