Embryo collection and PAH extraction

On day 4 and day 18, viable embryos (n = 25) were sampled from each incubator and combined with another incubator of the same treatment such that each unique treatment was represented by two sample pools of 50 embryos. Sample pools were immediately frozen at − 80 °C until analysis. Upon analysis, embryos were weighed within 0.01 mg accuracy and the organic compounds were extracted following a method developed by Sørensen and colleagues⁹³. Briefly, surrogate internal standards (same as the water samples) and sodium sulfate were added before the samples were homogenized with n-hexane-dichloromethane using a disperser (IKA 10 basic ULTRA-TURRAX), vortexed, and centrifuged. Supernatants containing the organic extracts were collected and cleaned using solid-phase extraction with silica (500 mg, Agilent Bond Elut SI, Agilent Technologies, USA), and eluted with n-hexane-dichloromethane (1:9, v/v, 6 mL). Purified extracts were concentrated on a heat block (40 °C) under a gentle N₂ steam. Prior to analysis, recovery internal standards (same as the water samples) were added to the purified extracts. A laboratory blank (empty sample vial) was included with each sample set.