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Popularized on social media, hand-moldable plastics are formed by consumers into tools, trinkets, and dental prosthetics. Despite the anticipated dermal and oral contact, manufacturers share little information with consumers about these materials, which are typically sold as microplastic-sized resin pellets. Inherent to their function, moldable plastics pose a risk of dermal and oral exposure to unknown leachable substances. We analyzed 12 moldable plastics advertised for modeling and dental applications and determined them to be polycaprolactone (PCL) or thermoplastic polyurethane (TPU). The bioactivities of the most popular brands advertised for modeling applications of each type of polymer were evaluated using a zebrafish embryo bioassay. While water-borne exposure to the TPU pellets did not affect the targeted developmental end points at any concentration tested, the PCL pellets were acutely toxic above 1 pellet/mL. The aqueous leachates of the PCL pellets demonstrated similar toxicity. Methanolic extracts from the PCL pellets were assayed for their bioactivity using the Attagene FACTORIAL platform. Of the 69 measured end points, the extracts activated nuclear receptors and transcription factors for xenobiotic metabolism (pregnane X receptor, PXR), lipid metabolism (peroxisome proliferator-activated receptor γ, PPARγ), and oxidative stress (nuclear factor erythroid 2-related factor 2, NRF2). By nontargeted high-resolution comprehensive two-dimensional gas chromatography (GC × GC-HRT), we tentatively identified several compounds in the methanolic extracts, including PCL oligomers, a phenolic antioxidant, and residues of suspected antihydrolysis and cross-linking additives. In a follow-up zebrafish embryo bioassay, because of its stated high purity, biomedical grade PCL was tested to mitigate any confounding effects due to chemical additives in the PCL pellets; it elicited comparable acute toxicity. From these orthogonal and complementary experiments, we suggest that the toxicity was due to oligomers and nanoplastics released from the PCL rather than chemical additives. These results challenge the perceived and assumed inertness of plastics and highlight their multiple sources of toxicity.

Environmental pollution is a threat to humans and wildlife species. Of particular concern are endocrine disrupting chemicals (EDCs). An important target of EDCs is nuclear receptors (NRs) that control endocrine and metabolic responses through transcriptional regulation. Owing in part to structural differences of NRs, adverse effects of EDCs vary significantly among species. Here, we describe a multiplexed reporter assay (the Ecotox FACTORIAL) enabling parallel assessment of compounds' effects on estrogen, androgen, thyroid, and PPARγ receptors of representative mammals, birds, reptiles, amphibians, and fish. The Ecotox FACTORIAL is a single-well assay comprising a set of species-specific, one-hybrid GAL4-NR reporter constructs transiently transfected into test cells. To harmonize cross-species assessments, we used a combination of two approaches. First, we used the same type of test cells for all reporters; second, we implemented a parallel detection of reporter RNAs. The assay demonstrated excellent quality, reproducibility, and insignificant intra-assay variability. Importantly, the EC50 values for NR ligands were consistent with those reported for conventional assays. Using the assay allowed ranking the hazard potential of environmental pollutants (e.g., bisphenols, polycyclic aromatic hydrocarbons, and synthetic progestins) across species. Furthermore, the assay permitted detecting taxa-specific effects of surface water samples. Therefore, the Ecotox FACTORIAL enables harmonized assessment of the endocrine and metabolic disrupting activity of chemicals and surface water in humans as well as in wildlife species.

While chemical analysis of contaminant mixtures remains an essential component of environmental monitoring, bioactivity-based assessments using in vitro systems increasingly are used in the detection of biological effects. Historically, in vitro assessments focused on a few biological pathways, for example, aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) activities. High-throughput screening (HTS) technologies have greatly increased the number of biological targets and processes that can be rapidly assessed. Here we screened extracts of surface waters from a nationwide survey of United States streams for bioactivities associated with 69 different end points using two multiplexed HTS assays. Bioactivity of extracts from 38 streams was evaluated and compared with concentrations of over 700 analytes to identify chemicals contributing to observed effects. Eleven primary biological end points were detected. Pregnane X receptor (PXR) and AhR-mediated activities were the most commonly detected. Measured chemicals did not completely account for AhR and PXR responses. Surface waters with AhR and PXR effects were associated with low intensity, developed land cover. Likewise, elevated bioactivities frequently associated with wastewater discharges included endocrine-related end points ER and glucocorticoid receptor. These results underscore the value of bioassay-based monitoring of environmental mixtures for detecting biological effects that could not be ascertained solely through chemical analyses.