The significance of the new method lies in the sampling matrix itself. Historically, urine has been considered the gold standard for assessing phthalate exposure in humans, because metabolism renders these compounds hydrophilic and readily excreted. After ingestion, inhalation or dermal absorption, phthalate diesters are hydrolyzed in the liver and small intestine into monoester metabolites, then conjugated into water-soluble glucuronides that are flushed from the body. But collecting urine from a wild dolphin requires catheterization during a catch-and-release health assessment, a logistically demanding and stressful procedure. Blubber, by contrast, can be obtained from free-swimming animals using remote biopsy dart sampling, a technique already used worldwide to measure persistent organic pollutants and trace metals in cetaceans.

Blubber is a biologically compelling target for contaminant surveillance. Composed of 80 to 90 percent lipid, it serves roles in buoyancy, thermal insulation and energy storage, and acts as a reservoir for lipophilic anthropogenic chemicals. High-molecular-weight phthalates such as DEHP, DINP and DIDP carry high octanol-water partition coefficients, meaning they preferentially partition into fatty tissues. Moreover, evidence from human breast milk, which shares lipid-rich characteristics with blubber, has long shown that monoester metabolites can be retained in such matrices. The research team reasoned that blubber might capture metabolites too hydrophobic to be efficiently excreted in urine, potentially extending the detectable window of exposure in a way urine cannot.

To build the protocol, the researchers adapted existing methods that had extracted phthalate metabolites from roach muscle tissue and harbor porpoise liver. They validated the technique using a single full-depth blubber sample collected during necropsy from a bottlenose dolphin stranded near Charleston, South Carolina. The finalized workflow is technically precise: approximately 200 milligrams of blubber is placed in methanol-rinsed amber glass vials, submerged in ammonium acetate buffer, and spiked with isotopically labeled internal standards. The mixture undergoes two 20-minute rounds of sonication, followed by an enzymatic deglucuronidation step using beta-glucuronidase from E. coli K12, incubated at 37 degrees Celsius for 90 minutes.

The extracts are then cleaned up and concentrated using solid-phase extraction on Bond Elut NEXUS cartridges, conditioned with acetonitrile and phosphate buffer, rinsed with formic acid and HPLC-grade water, and eluted with ethyl acetate followed by acetonitrile. After evaporation to dryness under nitrogen at 50 degrees Celsius, samples are reconstituted in water and analyzed by high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry with negative electrospray ionization. Separation is achieved in just 11 minutes using a water-acetonitrile gradient. Twelve phthalate metabolites, spanning both low- and high-molecular-weight compounds, were targeted, with calibration curves required to achieve correlation coefficients of at least 0.995 for every analytical batch.

Quality assurance was rigorous. Reagent blanks, field blanks, reagent spikes and matrix spikes accompanied every batch of six samples, and blank-derived contamination was subtracted from sample values. Mean recoveries for most analytes fell comfortably within the 70 to 130 percent range accepted by the U.S. Environmental Protection Agency, and solid-phase extraction recoveries in validation matrix spikes exceeded 90 percent for all compounds. MEHP proved the exception, with a lower but consistent mean recovery of roughly 60 to 90 percent, a pattern the authors attribute to possible binding to matrix components, incomplete cartridge elution, ion suppression, and pervasive background contamination from DEHP in laboratory materials, a well-documented problem given DEHP’s ubiquity.

Once validated, the method was applied to blubber samples from 27 bottlenose dolphins captured and released during health assessments in Sarasota Bay, Florida, in 2016, 2017, 2022 and 2023. The Sarasota Bay community of roughly 170 dolphins has been studied continuously for more than 50 years, making it one of the best-characterized marine mammal populations in the world and an ideal proving ground for a new exposure biomarker. The sampled animals included 13 males and 14 females aged 2 to 34 years, with 67 percent classified as sexually immature.

The results revealed detectable concentrations of three metabolites. MEHP, the monoester of the plasticizer DEHP, was found in six dolphins at 5.2 to 12.4 nanograms per gram wet weight. MEP, the metabolite of diethyl phthalate used in personal care products, appeared in two animals at 39.7 to 50.1 nanograms per gram. MIDP, the metabolite of the industrial plasticizer diisodecyl phthalate, was detected in three dolphins at 26.0 to 27.4 nanograms per gram. Overall, at least one metabolite was detectable in 37 percent of sampled dolphins, and a single two-year-old female carried detectable levels of two metabolites simultaneously. The oldest sampled dolphin, a 34-year-old female, had the highest concentration of any analyte.

Compared with earlier urine-based studies in the same population, which found multiple phthalate compounds in roughly 75 percent of dolphins tested, the blubber detection rate was lower, and the authors interpret this through the lens of chemical partitioning. Low-molecular-weight phthalates and their metabolites are more hydrophilic and are preferentially excreted in urine rather than stored in lipid, whereas high-molecular-weight parent compounds may partition into blubber before or after metabolism. Elevated limits of detection, driven by the deliberately small 200-milligram sample mass needed to avoid clogging extraction cartridges with co-extracted lipids, also likely suppressed detection rates. Larger samples introduced excessive lipid material that overwhelmed the cleanup process, forcing a trade-off between extraction practicality and analytical sensitivity.

The study’s authors emphasize that this is the first analysis of phthalate metabolites in blubber from live, healthy dolphins, filling a critical gap in marine mammal health surveillance. Remote biopsy darts typically yield full-thickness blubber samples of 0.05 to 0.8 grams, comparable to the tissue mass used here, suggesting the method could be deployed on remotely collected samples in future work. That capability would allow phthalate exposure assessments in at-risk or remote populations where catch-and-release assessments are infeasible, increase sampling efficiency, and reduce stress on protected animals. While blubber remains a less reliable indicator of total exposure than urine, particularly for low-molecular-weight compounds from personal care products, it offers a powerful complementary tool for high-molecular-weight compounds from plastics. As researchers begin to trace the trophic transfer of phthalates from microplastics through prey to dolphins, this new blubber-based protocol provides a standardized foundation for expanding chemical surveillance across the world’s coastal oceans, with direct implications for marine conservation and, given dolphins’ role as sentinels for ecosystem and human health, for people who share the same contaminated waters.

Subject of Research: Development of a blubber-based detection protocol for phthalate ester metabolites in bottlenose dolphins

Article Title: Development and application of a blubber-based detection protocol for phthalate ester metabolites in bottlenose dolphins (Tursiops truncatus)

Article References: Development and application of a blubber-based detection protocol for phthalate ester metabolites in bottlenose dolphins (Tursiops truncatus). (n.d.). https://doi.org/10.1007/s11356-026-38215-8

Image Credits: AI Generated

DOI: 10.1007/s11356-026-38215-8

Keywords: bottlenose dolphins, phthalates, blubber, marine mammals, endocrine disruptors, plastic pollution, LC-MS/MS, Sarasota Bay, environmental monitoring, contaminant exposure, biopsy sampling, ecotoxicology