Methods:
CROSERF method Singer et al., (2000; 2001) was followed for CROSERF method. In summary, 2L of fresh water (DI) was added to the 2L aspirator bottle and stir bar was set at 300 rpm to make sure there is no vortex. Headspace was less than 5% of total volume and the stoppers were either Teflon or glass made. Then, 0.023, 0.07, 0.23 and 0.84 ml of Maconde surrogate oil (density 0.86 g/ml) were added with the help of a gas tight Hamilton syringe for 10, 30, 100 and 360 mg/L oil loadings respectively. Total 72 hours was allowed to mix the oil with water. Singer, M.M., Aurand, D., Bragin, G.E., Clark, J.R., Coelho, G.M., Sowby, M.L., & Tjeerderma, R.S. (2000). Standardization of the Preparation and Quantitation of Water-accommodated Fractions of Petroleum for Toxicity Testing. Mar. Pollut. Bull. 40(11): 1007-1016. doi: 10.1016/S0025-326X(00)00045-X Singer, M.M., Jacobson, S., Tjeerdema, R.S., & Sowby, M. (2001). Acute effects of fresh versus weathered oil to marine organisms: California findings. In: Proceedings, 2001 International Oil Spill Conference, American Petroleum Institute, Washington, DC. pp. 1263-1268. Silicone Tube Method WAF through silicone tube (Fig. 1c) was prepared following Redman et al., (2017). In summary, a medical grade silicone tube (6 inch-1.5 ft) from A-M System Inc. WA with 0.058-inch X 0.077-inch X 0.0095-inch dimension was used (Fig. 1d). The predetermined amount of oil (e.g. 0.84 ml/2L for oil loading of 360 mg/L) was introduced into the silicone tube by gas tight Hamilton syringe and both ends were closed tightly. The silicone tube was then put into the 2L aspirator bonded with a stir bar (Fig 1c). Similar as CROSERF method, it was then stirred for 72 hours at 300 rpm before collection of samples. Redman, A.D., Butler, J.D., Letinski, D.J., & Parkerton, T.F. (2017). Investigating the role of dissolved and droplet oil in aquatic toxicity using dispersed and passive dosing systems. Environ. Toxicol. Chem. 36(4): 1020-1028. doi: 10.1002/etc.3624 O-ring method Oil absorbed by O-ring Polydimethyl siloxane (PDMS) standard O-ring were bought from O-ring West (Part number: SF70 212 and 002; http://www.oringswest.com/). O-rings were soaked in ethyl acetate for 24 hours followed by methanol (3x each 24 hours) and distilled water (3x each 24 hours). They were then oven dried at 110º C for 1 hour. Total 26 O-rings were weighted (Toledo Model) and average weight was taken. Then total 16 O-rings were soaked in Maconde Surrogate Oil (MC252, Slick A and B) for total 100 hours. Loaded 3-4 O-rings were taken out at 20, 50, and 100 hours and cleaned with DI and kimwipes and the weights were taken. The amount of absorbed oil was calculated from pre and post weight of O-rings. This experiment gave insight of the oil loading kinetics for PDMS O-rings with MC 252 and weathered oil such as Slick A and B. Hydrocarbons from oil partition to the O-rings according to their hydrophobicity and this is very similar to the way hydrocarbons partition to lipid of biological tissues (Letinski et al., 2014). Letinski, D., Parkerton, T., Redman, A., Manning, R., Bragin, G., Febbo, E., Palandro, D., & Nedwed, T. (2014). Use of passive samplers for improving oil toxicity and spill effects assessment. Mar. Pollut. Bull. 86(1-2): 274-282. doi: 10.1016/j.marpolbul.2014.07.006 WAF using loaded O-rings Hydrocarbons from loaded O-rings desorb to the exposure media following the Raoult’s Law. This method is similar in concept of Silicone tube method but very useful with viscous weathered oils (like Slick A and B). Appropriate numbers of O-rings were loaded for each oil loading treatments. Similar to CROSERF and Si tube method, treatments were stirred for 72 hours at 300 rpm. All experiments were done at room temperature (22˚ C) and under the artificial lights.
Instruments:
The analytical procedure used for the measurement of polycyclic aromatic and aliphatic hydrocarbons (PAHs and ALHs, respectively) were based on established GERG SOPs. Briefly, exactly 10 mL of water samples were extracted in duplicate by solvent partition against small volumes (i.e., 2 mL in triplicate) of dichloromethane (DCM) after the appropriate additions of surrogate standards (e.g., d8-naphthalene, d10-acenaphthene, d10-phenanthrene, d12-chrysene, and d12-perylene and d26-nC12, d42-nC20, d50-nC24, and d62-nC30 for PAHs and ALHs, respectively). After extraction, duplicate sample extracts were reduced to approximately 1 and 0.1 mL and spiked with appropriate amounts of deuterated compounds as internal standards (e.g., d10-Fluorene and d12-Benzo(a)pyrene and d54-nC26 for PAHs and ALHs, respectively). PAHs and ALHs were quantitatively analyzed by gas chromatography with mass spectrometric detection (Agilent 6890N GC/5975C inert MSD) in the selected ion mode (SIM) using a 30 m x 0.25 mm i.d. (0.25 µm film thickness) DB-5 fused silica capillary column (J&W Scientific, Inc.). The oven temperature was programed from an initial temperature of 60°C to 300°C at 12°C min-1 and held at this temperature for 6 min. The molecular ions for each target PAH were used for quantification. The oven temperature for aliphatic hydrocarbon analyses was programed from 40°C and ramped to 150°C at 15°C min-1, then to 220°C at 5°C min-1, and finally to 300°C at 10°C min-1 with a final hold of 10 min. The mass spectral data were acquired, and the molecular ions for each of the PAH analytes were used for quantification. The gas chromatograph/mass spectrometer (GC/MS) was calibrated by the injection of standards at five different concentrations. Analyte identification was based on the retention time of the quantitation ion for each compound and a series of confirmation ions.
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