Phthalate and Bisphenol A Leaching from Food Packaging:GC–MS Profiling and Hepatotoxicity Assessment in Zebrafish and HepG2 Cells

Abstract

Background: The migration of endocrine disrupting chemicals (EDCs), particularly phthalates and bisphenol A (BPA), from plastic food packaging into food matrices represents a major global public health concern. In Pakistan, where plastic packaging is pervasive across both formal retail and informal street food sectors with minimal regulatory oversight, exposure risks remain poorly characterized.

Methods: Forty plastic food packaging samples (PET, PVC, LDPE, PP, PS) were collected from urban markets and street vendors across Lahore, Multan, and Islamabad. Chemical migration was simulated under food contact conditions (60–80°C, 24–48 h) using methanol/hexane extraction. Migrated compounds were identified and quantified by GC–MS (Agilent 7890B/5977A). Hepatotoxicity was evaluated using a dual model: zebrafish (Danio rerio) embryo/larval exposure (96 h, 4 concentration levels) and HepG2 human hepatocellular carcinoma cells (24 h/48 h exposure). Cell viability was measured by MTT assay and oxidative stress assessed by DCFH DA fluorescence (ROS assay).

Results: GC–MS analysis identified 11 distinct compounds, including di(2 ethylhexyl) phthalate (DEHP, mean 342.7 ± 28.4 µg/L), dibutyl phthalate (DBP, 184.3 ± 19.6 µg/L), benzyl butyl phthalate (BBP, 97.6 ± 12.1 µg/L), and BPA (211.4 ± 23.8 µg/L) as dominant leachates. PVC and PS samples exhibited significantly higher migration (p < 0.001). Zebrafish mortality reached 68% at the highest concentration (500 µg/L combined exposure) with pronounced hepatic vacuolation on histology. HepG2 cell viability declined to 31.4 ± 4.2% at 200 µg/L (48 h), yielding an IC50 of 98.6 µg/L. ROS levels increased 4.7 fold above baseline at 200 µg/L. All results were statistically significant (p < 0.05).

Conclusion: Plastic food packaging in Pakistani markets leaches phthalates and BPA at concentrations sufficient to induce measurable hepatotoxicity in both in vivo and in vitro models. These findings provide quantitative evidence to support urgent regulatory intervention for food contact materials in Pakistan.