Data Parameters and Units:
EOE: Time (hours), EOE (mg/L), Oil loading (mg/L), Dissolved concentration (ug/L), Standard concentration (mg/L), Emission, Average, Standard deviation, Standard error BE-SPME: BE-SPME (uM/mL PDMS), Oil loading (mg/L), Average, Standard deviation GCGC of MC252: Starting Carbon number, Ending Carbon number, Composition (%), n-P, i-P, n-CC5, n-CC6, i-N, Di-N, n-Olefins, i-Olefins, Poly-N, AIS, MoAr, NMAr, DiAr, NDiAr, PolyAr, ArS Note: GCxGC analysis of oil only provides % composition of each chemical classes to a total of 100%. This % composition of chemical classes are used as input for PETROTOX model. Separate analyses were done on oil to find their PAHs PIANO SHC concentration. These concentrations were used in PETROTOX model again to predict the aqueous concentrations and compared with concentrations measured by GCMS (PTvsmeasured). PAHs PIANO SHC: Class, Abbreviation, Analytes, Result (mg/kg) PTvsMeasured: C#, Analyte, Oil loading (mg/L), Cdis = predicted dissolved concentration (ug/L), Caq Obs = measured concentration (ug/L), Microdroplet concentration (ug/L), Ctotal predicted total concentration (ug/L) Note: Microdroplet concentration is concentration of droplets (mg/L) created during mixing of oil and water. Evo, slow means mixing of oil and water was done slowly at 300 rpm (rotation per min). Samples for GCMS were taken after the experiments were over i.e. 72 hrs. TU vs oil loadings: Oil loading (mg/L), Toxic units (unitless), CTLBB = critical target lipid body burden, TU(GCXGC) = toxic unit calculated from GCXGC composition of oil, TU(VOC) = toxic unit calculated for VOC from measurements of speciated compounds of oil, TU(PAH) = toxic unit calculated for PAH from measurements of speciated compounds of oil Note: Oil Loading is the amount of oil (mg) added to the aqueous phase (L). This is for oil MC252. Toxic Units (TUs) are acute toxicity units and are calculated by PETROTOX model. For calculation, please see Redman and Parkerton (2015). Redman AD, Parkerton TF. 2015. Guidance for improving comparability and relevance of oil toxicity tests. Mar Pollut Bull 98:156-170.
Methods:
Estimated Oil Equivalents (EOE): Estimated total oil equivalents (EOE) measures aromatic hydrocarbons (mainly mono- and di-aromatic) that contain unsaturated bonds in their structure. EOE was calculated from total scanning fluorescence (TSF), which was measured with an Aqualog Horiba Fluorometer (Model Aqualog-UV-800). First, the TSF was measured for MC252 and the optimal wavelengths for maximum intensity (MI) were established (Excitation=257 nm and Emission=357 nm). Depending on the range of sample concentrations, a 5–7 point calibration curve was made using diluted MC252 in dichloromethane with concentrations ranging from 2 mg/L to 0.1 mg/L. Then, 10 mL samples were extracted with dichloromethane (DCM) and the extract was measured for TSF. MI at established optimal wavelengths was used to estimate the EOE from the calibration curve. Polycyclic-aromatic-hydrocarbons (PAHs) and aliphatic hydrocarbons The analytical procedure used for the measurement of PAHs and aliphatic hydrocarbons (ALHs) were based on established standard operating procedures utilized by Geochemical Environmental Research Group (GERG), Texas A & M University. Briefly, 1 L of sample was extracted in by solvent partition against small volumes (i.e., 50 mL in triplicate) of DCM after the appropriate additions of surrogate standards (e.g., d8-naphthalene, d10-acenaphthene, d10-phenanthrene, d12-chrysene for PAHs, and d12-perylene and d26-nC12, d42-nC20, d50-nC24, and d62-nC30 for ALHs). After extraction, duplicate sample extracts were cleaned through Silica-gel column and then the 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 for PAHs and d54-nC26 for ALHs). 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 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 increased 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 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. Biomimetic Extraction (BE) Briefly, 20 mL of samples were transferred to a glass vial (with Teflon faced septum cap) with no headspace. Then, 30 µm polydimethyl siloxane (PDMS) (0.12 µL) solid phase microextraction (SPME) fibers were given 100 minutes to equilibrate in each vial at 30°C. The vials were agitated at a speed of 250 rpm during this period. After the equilibrium period, an automated system retracted the SPME fiber and thermally desorbed it in the GC-FID injection port. FID detector response was then normalized to 2,3 dimethylnaphthalene to estimate molar concentrations on the fiber that serve as a surrogate for target lipid, and hence predicted toxic units of dissolved oil. BE-SPME samples were taken right after the experiments were done i.e. after 72 hours. During analysis of BE-SPME, SPME fibers were kept in the samples for 100 min. Model Analysis The equilibrium dissolved phase concentrations of individual hydrocarbons were calculated by inputting oil loading and composition (Table S2) into the oil solubility model [6]. The difference between total measured concentrations and predicted dissolved concentrations was then used to infer the amount of particulate or colloidal oil present in each WAF treatment. The GCxGC composition (Table S1) was used as input to PETROTOX to predict the loading of fresh Macondo oil that corresponds to predicted acute toxicity (i.e. LL50). PETROTOX simulates the solubility of oil components and predicts toxicity based on additive toxic units from pseudo-constituents, or blocks, based on the target lipid model (TLM ) framework. The predicted sensitivity of different species is determined by the critical target lipid body burden (CTLBB) for the organism/endpoint. To determine the expected range of acute toxicity for the Macondo oil across species, PETROTOX predictions were performed using a CTLBB provided by McGrath and Di Toro corresponding to a sensitive (CTLBB = 24 µg/goctanol), typical (CTLBB = 146.5 µg/goctanol) and insensitive organism (CTLBB = 500 µg/goctanol). To assess the potential for chronic toxicity and acute/chronic ratio (ACR) of 4.5 was applied to acute CTLBB values. CROSERF Methods developed by Singer, et al. [2000], Singer, et al. [2001] were followed for generating WAFs that reflect standard practice. Briefly, 2 L of fresh water (DI) was added to the 2 L aspirator bottle and stir bar was set at 300 rpm to prevent any vortex. Headspace was less than 5% of total volume and the stoppers were either Teflon or glass. Different volumes (0.023, 0.07, 0.23 and 0.84 mL) of MC252 (density 0.86 g/mL) were added using gas tight Hamilton syringes for 10, 30, 100 and 360 mg/L oil loading treatments, respectively. Oil was mixed in water for a total of 72 h, and samples were taken immediately thereafter (no settling period). Singer MM, Aurand D, Bragin GE, Clark JR, Coelho GM, Sowby ML, Tjeerdema RS. 2000. Standardization of the preparation and quantitation of water-accommodated fractions of petroleum for toxicity testing. Mar Pollut Bull 40:1007-1016. Singer MM, Jacobson S, Tjeerdema RS, Sowby M. 2001. Acute effects of fresh versus weathered oil to marine organisms: California findings. International Oil Spill Conference, pp 1263-1268. Silicone tube method for creating Water Accommodating Fractions Water dosed via silicone tubing was prepared following Redman, et al. [2017]. Briefly, medical grade silicone tubing (6 inch–1.5 ft) (A-M Systems Inc., WA) with dimensions of 0.058 X 0.077 X 0.0095-inch was used. The predetermined amount of oil (e.g. 0.84 mL/2 L for oil loading of 360 mg/L) was injected into the silicone tube using a gas tight Hamilton syringe, and both ends were knotted tightly. The silicone tube was attached to the stir bar and submerged in the 2 L aspirator bottle. Analogous to the CROSERF method, the loaded tubing was stirred for 72 h at 300 rpm prior to sample collection. Redman AD, Butler JD, Letinski DJ, Parkerton TF. 2017. Investigating the role of dissolved and droplet oil in aquatic toxicity using dispersed and passive dosing systems. Environ Toxicol Chem 36:1020-1028. O-ring method for creating Water Accommodating Fractions Silicone O-rings were purchased from O-rings West (Part number: SF70 212 and 002; http://www.oringswest.com/). O-rings were cleaned by soaking in ethyl acetate for 24 h followed by methanol (3x each for 24 h) and distilled water (3x each for 24 h). They were then oven dried at 110º C for 1 h. A total of 26 O-rings were weighed using a Toledo Balance (Model MS603TS) and averaged. A total of 16 pre-cleaned O-rings were then soaked in 50 mL of MC252 for 100 h. Loaded O-rings were removed at 20, 50, and 100 h, rinsed with DI and dried with Kimwipes prior to weighing. The amount of absorbed oil was calculated from pre- and post-weight of the O-rings. This experiment provided insights regarding the kinetics of oil absorption into the O-rings for fresh MC252. This method is similar in concept to the silicone tube method, but more practical with viscous weathered oils that cannot be loaded into silicone tubing. Based on the mass of oil that was found to be taken up by O-rings (see above), the appropriate number of O-rings were loaded to provide the oil mass corresponding to the targeted treatment loadings. For example, if O-rings swell to 120% of original mass when soaked in oil, 20% of the mass in the loaded O-rings is attributed to absorbed oil. A typical O-ring weights 1.05 g (n=25), so in this example adding 10 O-rings to 1 L of water is equivalent to an oil loading of 2100 mg/L. Similar to CROSERF and silicone tube method, treatments were stirred for 72 h at 300 rpm.
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