Sertraline Hydrochloride Exposure Leads to Reactive Oxygen Species Burst in Model Microalgae Species (Raphidocelis subcapitata)

Microalgae play important ecological roles and serve as useful models for assessing impacts of environmental contaminants on lower trophic aquatic organisms. Sertraline, a commonly prescribed selective serotonin reuptake inhibitor (SSRI) found in urban waters, has been observed to induce chlorosis in Raphidocelis subcapitata close to environmentally relevant concentrations. This study estimated potentially hazardous concentrations of sertraline hydrochloride by deriving transcriptomic and metabolomic points of departure (tPODs and mPODs) below which chronic adverse effects would not be expected. Additionally, an Adverse Outcome Pathway describing the mechanism by which sertraline hydrochloride impacted R. subcapitata was proposed using a multi-omics approach. The mPOD derived from nontargeted metabolomics was comparable to the tPOD (455 µg/L vs 690 µg/L) and annotated dysregulated metabolites along with reduced photosynthetic capability indicated that uncoupling of the cyclic electron flow required for photosynthesis led to a reactive oxygen species (ROS) burst. This probable ROS burst altered cell membrane composition and downregulated genes associated with protoporphyrinogen IX and heme biosynthesis pathways. This research highlights the molecular mechanisms underlying pharmaceutical-induced chlorosis in a model microalgae species and demonstrates the utility of integrating metabolomics and transcriptomics for assessing the potential ecological risks of SSRIs. R. subcapitata (5 × 106 cells/mL) were exposed to nominal concentrations of 50, 125, 314, 784, 1,960, 4,900, 12,250, and 30,620 µg sertraline hydrochloride/L (CAS #: 79559-97-0; Purity: ≥ 98.0 %, Millipore-Sigma, MA, USA) or solvent control (0.01% DMSO) for 24 h in triplicate plates in 96-deep-well (2.0 mL) plates using 800 µL of solution per well (Catalog # 267007; Beckman Coulter, CA, USA) sealed with gas-permeable, optically clear adhesive covers (BEM-1, Diversified Biotech, MA, USA). There were five replicate wells per treatment (one replicate well per concentration for atrazine), per plate (n = 3), with treatments distributed such that there were equal numbers of interior and exterior (edge) wells for each treatment. Throughout the 24 h exposure period, R. subcapitata were held under constant light and kept in suspension by gentle shaking using a temperature controlled orbital shaker (Titramax 1000, Heidolph Instruments GmbH, Germany). RNA was extracted from pooled homogenates (n = 3) using Plant/Fungi Total RNA Purification kits (Catalog #31900, Norgen Biotek, Ontario, Canada) with a protocol slightly modified from the manufacturer’s instructions. Total RNA was shipped to the Michigan State University Research Technology Support Facility Genomics Core for library preparation using QuantSeq 3’ mRNA-Seq V2 Library Prep Kit (Lexogen GmbH, Austria) and sequenced on NovaSeq SP flow cells (Illumina, Inc, CA, USA).