Apical and transcriptomic points of departure (aPOD and tPOD) of nine PFAS with different carbon chains and functional groups in zebrafish embryos

Per- and poly-fluoroalkyl substances (PFAS) are persistent environmental contaminants prevalent in industrial manufacturing processes and various consumer products. Human health consequences include reduced fertility, liver damage, thyroid disease, kidney disease, cancer, inflammation, hypertension, and obesity. In this study, various approaches were used to determine toxicological points of departure (PODs) for 9 PFAS (PFDS, PFDS, PFOA, PFOS, PFBA, PFBS, PFHxA, PFHxS, PFNA) in Zebrafish embryos exposed for 5 days from 4 to 120 hours post-fertilization. Apical PODs (aPODs) were assessed for developmental toxicity (based on mortality and malformation), behaviour (based in distance swum under light and dark conditions) and metabolism (based on NADH-dependent changes in alamarBlue fluorescence). Transcriptional PODs (tPODs) were determined using TempO-Seq S1500+ Zebrafish Surrogate Assay Panel applied to pools of 10 embryos. Count data were generated using the Omics data analysis framework for regulatory application (R-ODAF) pipeline, available at https://github.com/R-ODAF/R-ODAF_Health_Canada. Transcriptomic BMC modeling was performed using BMDExpress. PFAS toxicity was highest for PFAS with longer carbon chain length, and with a functional group of sulfonic acid compared with carboxylic acid. This toxicity ranking was similar across all aPODs and tPODs. This study demonstrates that for PFAS chemicals, transcriptional PODs can be used to reliably predict other aPOD estimates generated through assessments of metabolism, behaviour, or mortality/malformation. Overall design: Zebrafish embryos were exposed for 5 days (from 4 to 120 hours post-fertilization) to one of nine PFAS chemicals (PFDS, PFDS, PFOA, PFOS, PFBA, PFBS, PFHxA, PFHxS, PFNA) in 0.1% DMSO (final concentration). PFAS concentrations (see concentration in metadata) are in micromolar units, with each PFAS used at 5 different concentrations (plus 0.1% DMSO controls). Following exposure, 10 embryos were pooled to generate a single sample per PFAS/concentration, with typically n=4 sample per group (see replicate in metadata). Samples were processed in two different batches (see processing_batch in metadata). For batch A, some zebrafish were from the Ottawa breeding facility, while others were a mixture of Ottawa and Montreal breeding facilities. These variables determined which samples were used as the control (0.1% DMSO only) group. 'control_mixed' (see chemical in metadata) was the contrast group for PFDS, PFDS, PFOA and PFOS. 'control_ottawa' was the contrast group for PFBA, PFBS, PFHxA and PFHxS. 'control_pfna' was the contrast group for PFNA. 'control_excluded' samples were outliers and not used in any contrast.