Demographic information and phthalate metabolite concentrations (µg/L) detected in bottlenose dolphins (Tursiops truncatus) urine sampled from Barataria Bay, LA during 2011-2023 and Sarasota Bay, FL during 2010-2019, 2022-2024

Exposure to phthalate esters has previously been documented in bottlenose dolphins (Tursiops truncatus) inhabiting an urban estuary (Sarasota Bay, FL, USA; 2010-2019). Phthalates are chemicals commonly added to plastic products and consumer goods to enhance qualities such as flexibility, fragrance, and stability. These chemicals are known to leach out of plastic products and into the marine environment, leaving wildlife vulnerable to reproductive, developmental, and metabolic health effects. Environmental phthalate exposure has been shown to vary relative to human activity and urbanization. To evaluate potential differences in dolphin exposure risk, urinary phthalate metabolite concentrations were compared between free-ranging bottlenose dolphins from an urban (Sarasota Bay, FL, USA; 2010-2023; n= 71 ) and rural estuary (Barataria Bay, LA, USA; 2011-2023; n = 45). The magnitude of MEHP detection did not differ significantly between sampling sites (p = 0.97); however, MEHP was detected more frequently in Sarasota Bay dolphins (73.24%; n=52; 95% CI: 61.20-82.73) than Barataria Bay dolphins (33.33%; n = 15; 95% CI: 20.00 - 48.95%). Further, dolphins from Sarasota Bay may be exposed to a greater diversity of phthalates compared to Barataria Bay dolphins as indicated by differences in the detected phthalate metabolite profile. Notably, Barataria dolphins were also affected by the Deepwater Horizon oil spill, warranting additional studies of potential phthalate sources and health implications among these dolphins. 

Methods
Each urine sample was screened for seven phthalate metabolites (MEP, MEHP, MEHHP, MEOHP, MBzP, MiBP, and MBP). Urine samples (1 mL) were spiked with isotopically labeled internal standards for each metabolite before proceeding to a glucuronidation step to release monoesters from their conjugated forms (Blount et al., 2000). Following glucuronidation, samples were extracted via SPE (Agilent Bond Elute Nexus) then separated and quantified using high-performance liquid chromatography (HPLC; Agilent 1100; WatersXBridge BEH C18, 2.5 μm, 2.1 x 50 mm analytical column) coupled to a triple quadrupole mass spectrometer (MS; Applied Biosystems Sciex API 4000) with electrospray ionization (ESI negative) interface. Sample integrations were performed using Analyst software (Sciex ver 1.5). Prior to the acquisition of sample data, the instrument was calibrated (standard reference material (SRM) 3060: monoester phthalates in acetonitrile); coefficients of determination (r2) for all metabolites were ≥ 0.995. 

Quality assurance/quality control (QA/QC) samples (reagent blanks, reagent spikes, matrix spikes, SRM 3672 Organic Contaminants in Smokers’ Urine, and field blanks) were processed alongside the urine samples. Reagent and field blank values were subtracted from the determined concentration value to account for any metabolite contamination resulting from laboratory processes. Acceptable QA/QC criteria for spike (reagent and matrix) and SRM recoveries were 70%-130%. The limit of detection (LOD) was determined for each metabolite and is based on the lowest point on the calibration curve that could be detected on the instrument divided by the volume of the sample extracted.