Comparisons of analytical chemistry and biological activities of extracts from North Pacific gyre plastics with UV-treated and untreated plastics using in vitro and in vivo models

Plastic debris is an emerging worldwide threat to marine biota. Marine species may face unique challenges in low-flow estuarine systems with a high abundance of "macro-sized" (> 4.75 mm) plastic due to the leaching of constituents and adsorbed contaminants. To simulate this leaching process, plastic samples recovered from the North Pacific Gyre along with corresponding UV-irradiated virgin plastic and non-irradiated virgin plastic counterparts were incubated in saltwater for 30 days at ambient temperatures ranging from 17 to 25 degrees C. Following solid-phase extraction, water samples were fractionated with sequential methanol elution from 10 to 100% and evaluated using in vitro assays assessing estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) activities. In vivo responses (vitellogenin [vtg] and cytochrome p450 1A [cyp 1a] mRNA) were measured following 5-day exposures in Japanese medaka (Oryzias latipes) larvae (3 days post hatch). Estrogenic plasticizers, co-planar PCBs and PAHs were quantified in the extracts using targeted GC-MS/MS and UPLC-MS/MS. In vitro estrogenicity showed highest activity in the 70% methanol fraction for all plastic leachate exposures. Whole extract in vitro estradiol equivalent (EEQ) values were 4.34 +/- 2.65, 8.79 +/- 2.09 and 13.78 +/- 3.64 ng/L, for virgin plastic, UV-irradiated virgin plastic and North Pacific Gyre-recovered plastic, respectively (mean +/- SD). Significant vtg induction was observed in medaka larvae exposed to leachate extracts from North Pacific Gyre recovered plastic and UV-irradiated virgin plastic (9.9-fold, p = 0.039 and 10.1-fold, p = 0.042, respectively). Chemically-determined EEQ values were also localized in the 70% methanol fraction. Whole leachate extract chemical EEQ values were 0.33 +/- 0.07, 1.64 +/- 0.62 and 11.4 +/- 2.13 ng/L, for virgin plastic, UV-irradiated virgin plastic and North Pacific Gyre-recovered plastic, respectively. In-vitro AhR activity was highest in the 70% methanol elution with greater activity in North Pacific Gyre-recovered plastic than in virgin plastic and UV-irradiated virgin plastic (toxic equivalency [TEQ] = 1.06 +/- 0.54, 0.38 +/- 0.07 and 0.71 +/- 0.47 ng/L, respectively). CYP1A mRNA was significantly induced in larval medaka exposed to North Pacific Gyre-recovered plastic leachates (17.8-fold, p = 0.02) while exposure to virgin plastic and UV-irradiated virgin plastic leachates caused no significant change. Chemically-determined TEQ analysis for AhR indicated highest activity in the 90% methanol fraction for all leachates, with whole extract in vitro TEQs being 1.47 +/- 0.87, 0.03 +/- 0.05 and 0.42 +/- 0.38 ng/L for North Pacific Gyre-recovered plastic, virgin plastic and UV-irradiated virgin plastic, respectively. These results indicate that weathering and UV radiation release estrogenic plasticizers and demonstrate the ability for plastics to transport adsorbed persistent organic pollutants at eco-toxicologically relevant concentrations.