Sample preparation

PAHs were analyzed using a method modified from one reported previously [20]. The collected sediment was thawed, mixed, and sieved through a 1-mm mesh. Triplicate wet sediment samples from each site (approximately 10–20 g per sample) were extracted twice with acetone after spiking with a deuterium-labeled PAH surrogate standard mixture (phenanthrene-d₁₀, anthracene-d₁₀, fluoranthene-d₁₀, pyrene-d₁₀, chrysene-d₁₀, benzo[a]pyrne-d₁₂, and perylene-d₁₂). Thereafter, the acetone extract was concentrated to approximately 20 mL by use of a rotary evaporator. The concentrated extract was added to 50 mL of 1 M KOH/EtOH solution and shaken for 15 hours at room temperature (ca. 25°C) in the dark for alkaline decomposition. Following decomposition, the solution was extracted twice with hexane. The extracted hexane solution was washed with water, dehydrated with anhydrous sodium sulfate, filtered, and concentrated to about 1 mL by a rotary evaporator and N₂ gas. The concentrated solution was subjected to column chromatography with 5% H₂O-deactivated silica gel and 100 mL of 1% acetone/hexane solution. The eluate was concentrated and diluted to 1 mL with hexane after the addition of p-terphenyl-d₁₄ to the sample as an internal standard, and was analyzed by gas chromatography–mass spectrometry (GC–MS).

Bivalve samples were prepared using the same procedure used for the sediment samples, with some minor adjustments [20]. The soft parts of bivalves were thawed and then homogenized using a food processor. About 6 g of the homogenized sample (n = 3) was decomposed with 50 mL of 1 M KOH/EtOH solution for 15 hours using a magnetic stirrer at room temperature after adding the deuterium-labeled PAH surrogate standard mixture. The samples were then shaken vigorously for 2 hours for complete decomposition. The remaining procedure was the same as that used for sediment samples.