Comparison of zebrafish toxicity among different developmental windows of exposure to three environmentally relevant PFAS compounds

The per-and polyfluoroalkyl substances (PFAS) are of significant global concern due to their highly ubiquitous and persistent nature, bioaccumulation in organisms, and potential toxicity. The aquatic environment is known as an important sink for PFAS resulting in high concentrations in aquatic organisms. However, little is known about the developmental windows of sensitivity in which the PFAS chemicals are biologically active, in addition to the toxicity endpoints that best reflect chemical hazard. In this study, zebrafish (Danio rerio) were exposed to a 0.33% DMSO vehicle control, 1uM chlorpyrifos (CAS 2921-88-2) positive control, and eight concentrations (0-100 uM, half-log dilutions) of three environmentally relevant PFAS compounds in concentration-response: PFOS (CAS 1763-21-1), PFOA (CAS 45285-51-6), and PFHxS (CAS 355-46-4). There was also a group of unexposed zebrafish aliquoted from a single pool into all exposure plates to serve as a quality assurance measure. The goal of this study was to generate transcriptomic point of departure (tPOD; a benchmark dose/concentration -based treatment level below which a concerted gene expression response is not observed) estimates for zebrafish exposed to PFAS compounds as a health protective exposure level for risk assessment. Through the use of short-term embryo/larval plate-based high-throughput toxicity tests, tPODs were determined across seven distinct developmental windows (6-24 hours post fertilization (hpf), 6-48 hpf, 24-48 hpf, 6-120 hpf, 24-120 hpf, 48-120 hpf, 96-120 hpf) to assess how common experimental design variables (e.g., different exposure durations, exposure at different developmental stages) affect point of departure estimates. Zebrafish (Danio rerio) embryos were exposed to eight concentrations of three different per- or polyfluoroalkyl substances (PFAS), perfluorooctanesulfonic acid (PFOS; DTXSID3031864); perfluorooctanoic acid (PFOA; DTXSID8031865); and perfluorohexane sulfonate (PFHxS; DTXSID90892476) in concentration-response (0-100 uM, half-log dilutions), and then whole body gene expression was determined using RNA sequencing. Embryos were exposed to each of the three PFAS over seven distinct exposure periods (6 hours post fertilization [hpf]-120 hpf; 6 hpf-48 hpf; 6 hpf-24 hpf; 24 hpf – 120 hpf; 48 hpf-120 hpf; and 96 hpf-120 hpf) with the goal of determining how much the tPOD varies as a function of the developmental window over which the organisms were exposed. Zebrafish were also exposed to 0.33% DMSO (vehicle control) and 1uM chlorpyrifos (transcription positive control). Each PFAS chemical concentration was included on two of the three 96-well plates (n=5 individuals/plate), and DMSO (n=8 individuals/plate) and chlorpyrifos (n=3 individuals/plate) were included on all three plates.