ABSTRACT: Purpose: The goals of this study are to investigate developmental toxicity and hazard potential of a short-chain PFAS homologous series in zebrafish, specifically interrogating the impact of different functional head groups. Developmental toxicity findings were supplemented with investigations into transcriptom-wide effects of body burdens following PFAS exposures. Methods: For developmental toxicity assessments, dechorionated embryonic zebrafish were statically exposed at 8 hours post fertilization (hpf) to either vehicle control (0.5% methanol) or to FBSA, PFBS, PFPeA, or 4:2 FTS across a range of concentrations (1-100 µM, normalized to 0.5% methanol) in 96-well plate format. Morphological and Behavior endpoints were assessed at 24 and 120 hpf. For investigations into transcriptomic effects and body burdens, embryos were exposed to vehicle control or 47 µM of each PFAS and then sampled at 48 hpf, before morphological effects were observable. Transcriptomic effects of FBSA exposure were phenotypically anchored to abnormal morphology at 120 hpf as 47 µM was the concentration at which morphological effects were observed in 80% of the exposure group. At 48 hpf, 10 embryos were pooled per replicate, euthanized via ice bath, and total RNA extracted using a Zymo Research Direct-zol RNA MiniPrep kit and then sequenced on the BGISEQ-500 platform (Beijing Genomics Institute; BGI) for the transcriptomic investigation. Data analysis and visualization was conducted by BGI and their Dr. Tom platform. Briefly, sequencing data was filtered using SOAPnukes to remove adaptor sequences, N content greater than 5%, and low-quality reads. Clean reads were mapped to the reference genome (Danio rerio GRCz11) using HISAT2. Gene fusions and differential splicing were detected using Ericscript and rMATS, clean read alignments using Bowtie2 and expression level calculations using RSEM. Differential expression analysis was performed using DESEq2 (q-value ≤ 0.05), followed by gene ontology enrichment analysis using Phyper and transcription factor enrichment analysis using MetaCore. For body burden measurments, 4 replicates of 40 embryos were pooled per exposure group, euthanized on ice, homogenized, and analyzed by high pressure liquid chromatography and triple quadrupole mass spectrometry. Results: Across the 4-fluorinated carbon short-chain PFAS series, all chemicals induced larval behavior effects, but only FBSA caused morphological, behavioral and large-scale transcriptomic disruptions (specifically at 47 µM). Notably, exposures at concentrations shown to induce only behavior effects resulted in a small number of differentially expressed genes. Biological processes enriched following FBSA exposure at 47 µM were related to lipid metabolism and biosynthesis, cellular homeostasis, brain and nervous system development, and immune processes. The bioaccumulative potential of the series at 47 µM was FBSA > PFBS > 4:2 FTS > PFPeA.
