Methods:
Experimental The experimental design followed methods described in Rahsepar et al. (2017) and van Eenennaam et al. (2018). Twenty-one, completely glass microcosms (25 cm square) were filled with a 5 cm layer of sediment, and 15 cm of natural seawater (0.45 μm filtered Eastern Scheldtwater). Sediment was collected at low tide from the top 10 cm of an intertidal mudflat in the Dutch Wadden Sea (approximate location: 52° 56.112 N, 004° 59.976 E) and transported directly to the Wageningen Marine Research Laboratory, Den Helder, The Netherlands. Sediment was sieved at 1 mm to remove large organisms and particles, and thoroughly mixed. The sediment settled for one day prior to the start of the experiment. The mesocosms remained at 14 °C, with a light schedule of 16 hours of light and 8 hours of dark for the duration of the experiment. The top 5 cm of the water column was aerated continuously. After allowing the sediment settle for one day, 40 Corophium volutator (amphipod), and 20 Hydrobia ulvae (gastropod) were added to each microcosm. These organisms were collected with the sediment. The foraminifera used in this study were already present in the sediment. The organisms were given four days to adjust to the new environment before treatment started. For this (artificial) marine snow and kaolin clay with varying concentrations of oil were gradually added to the mesocosms over a period of four days. Each day, 25% of the total amount of treatment material was suspended in the water column and allowed to settle on the sediment surface. The formulation of the treatment materials included marine snow and clay, with or without oil. The biodegradation and weathering of oil compounds is described in detail in Rahsepar et al. (2016), Rahsepar et al. (2017) and van Eenennaam et al. (2018). There were seven treatments in total: 1)a control (no sedimentation, no oil), 2)marine snow (which included kaolin clay) additions with three varying concentrations of oil (0 g m-2, 3 g m-2, 10 g m-2) and 3)kaolin clay additions with three varying concentrations of oil (0 g m-2, 3 g m-2, 10 g m-2). All treatments were performed in triplicate (reported as “A, B and C”). The oil used to mimic the physical, biological, and chemical cycling of Macondo Oil during the experiment was a light-sweet crude oil (provided by British Petroleum), which was chemically similar to the oil associated with the DWH event. Rahsepar, S., Langenhoff, A.A.M., Smit, M.P.J., van Eenennaam, J.S., Murk, A.J., & Rijnaarts, H.H.M. (2017). Oil biodegradation: Interactions of artificial marine snow, clay particles, oil and Corexit. Marine Pollution Bulletin, 125(1-2): 186-191. doi: 10.1016/j.marpolbul.2017.08.021 van Eenennaam, J.S., Rahsepar, S., Radović, J.R., Oldenburg, T.B.P., Wonink, J., Langenhoff, A.A.M., Murk, A.J., & Foekema, E.M. (2018). Marine snow increases the adverse effects of oil on benthic invertebrates. Marine Pollution Bulletin, 126: 339-348. doi: 10.1016/j.marpolbul.2017.11.028 Rahsepar, S., Smit, M.P.J., Murk, A.J., Rijnaarts, H.H.M., & Langenhoff, A.A.M. (2016). Chemical dispersants: Oil biodegradation friend or foe? Marine Pollution Bulletin, 108(1-2): 113-119. doi: 10.1016/j.marpolbul.2016.04.044 Benthic Foraminfera Methods After 42 days, one sediment sub-sample was taken from the surface 1 cm of each microcosm using a 2.5 cm diameter syringe. These sub-samples were stained with 2.5 mL of 3.3 mg L-1 rose Bengal (purity 95%, Sigma-Aldrich, CAS Number: 632-69-9) and preserved in 4% formaldehyde and sent to the University of South Florida, College of Marine Science for further analyses. There, the sub-samples were weighed and washed with a sodium hexametaphosphate solution through a 63-μm sieve to disaggregate detrital particles from foraminiferal tests (Osterman, 2003). The fraction remaining on the sieve (>63-μm) was dried in an oven at 32 oC for 12 hours, weighed again, and stored at room temperature (Osterman, 2003). Stained, unstained, and total (stained+unstained) counts were reported for greater than 300 individuals per sample and identified to species. Taxonomic references included: Linnaeus (1758), d'Orbigny (1839), and Hofker (1977) L.E. Osterman (2003). Benthic foraminifers from the continental shelf and slope of the Gulf of Mexico: an indicator of shelf hypoxia. Estuarine, Coastal and Shelf Science, 58(1): 17-35 doi: 10.1016/S0272-7714(02)00352-9 C. Linnaeus (1758). Systema Naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Editio decima, reformata. Laurentius Salvius: Holmiae. ii, 824 pp. A.D. Orbigny d' (1839). Foraminifères, in de la Sagra R., Histoire physique, politique et naturelle de l'ile de Cuba. pap. J. Hofker (1933). Foraminifera of the Malay Archipelago. Papers from Dr. Th. Mortensen's Pacific Expedition, 1914-16, Pacific Expedition, vol. 22. Videnskabelige meddelelser fra Dansk naturhistorisk forening i Kjøbenhavn. vol. 93. Statistical Methods Multiple indices were applied to each assemblage including Shannon, Evenness and Fisher’s Alpha (Hammer and Harper, 2006). Total foraminifera abundance was reported as individuals per unit mass (indiv./g). Univariate analysis of variance (ANOVA) was performed using the PAST paleo-statistics suite (Hammer and Harper, 2006) to test for differences between oil concentration (3 g m-2, 10 g m-2 treatments vs. control), sediment drivers (clay, snow treatments vs. control) and combined sediment and oil (Snow: 3 g m-2, 10 g m-2, Clay: 3 g m-2, 10 g m-2 vs. control) treatments for the following dependent variables: stained abundance (indiv./g), Fisher's Alpha, Evenness and Shannon. To assure that any variability between treatments was not simply due to natural variability between aquaria, ANOVA was also performed using replicate aquaria as a potential driver. Multivariate analyses were performed on the benthic foraminifera assemblages using Primer-e (Clarke and Gorley, 2015). The individual counts were square root transformed and a Bray Curtis similarity matrix was created. A Non Metric MDS (nMDS) with 100 restarts and a minimum stress of 0.09 was derived from the similarity matrix and a cluster analysis was performed to determine the degree of similarity between samples. Hammer, Ø. & Harper D. (2006). Paleontological Data Analysis. – Blackwell Publishing, Oxford. 351pp. ISBN 1–4051–1544–0. Clarke, K.R. & Gorley R.N. (2015). Getting started with PRIMER v7. PRIMER-E: Plymout, Plymouth Marine Laboratory, 20 pp. Stable Isotopes Stained Ammonia beccarii tests were picked from the “A” replicate of each treatment to obtain greater than 150 micrograms of CaCO3. Translucent tests were preferentially selected (Wycech et al., 2016) to avoid any authigenic overgrowth influence on the stable isotope measurements. The Ammonia beccarii tests were lightly cracked between two sterile glass plates and homogenized. They were then cleaned ultrasonically in reagent-grade methanol to disaggregate any clay and authigenic carbonate material (Hill et al., 2004). delta13C and delta18O (3 replicates of 50 micrograms CaCO3) were measured with a ThermoFisher MAT253 stable isotope ratio mass spectrometer coupled to a GasBench-II peripheral in continuous-flow mode located at the University of South Florida College of Marine Science Stable Isotope Biogeochemistry Lab. Measurement followed established procedures (Révész and Landwehr, 2002; Spötl and Vennemann, 2003; Duhr and Hilkert, 2004; Burman et al, 2005; Spotl, 2011). Secondary reference materials (TSF-1 delta13C = 1.95±0.05 ‰, delta18O =-2.20±0.06 ‰; Borba delta13C = 2.87±0.05 ‰, delta18O =-6.15±0.09 ‰; LECO-carb delta13C =-15.45±0.16 ‰, delta18O =-20.68±0.16 ‰, all calibrated with NBS19, NBS18 and LSVEC certified reference materials) were used to normalize measurements to the VPDB scale. Measurement uncertainty, expressed as ±1 standard deviation of n=42 measurements of the TSF-1 laboratory reference material was 0.064 ‰ and 0.107 ‰ for delta13C and delta18O, respectively. Wycech, J., Kelly, D.C., & Marcott, S. (2016). Effects of seafloor diagenesis on planktic foraminiferal radiocarbon ages. Geology, 44(7): 551-554. doi: 10.1130/G37864.1 Hill, T.M., Kennett, J.P., & Valentine, D.L. (2004). Isotopic evidence for the incorporation of methane-derived carbon into foraminifera from modern methane seeps, Hydrate Ridge, Northeast Pacific. Geochemica et Cosmochimica Acta, 68(22): 4619-4627. doi: 10.1016/j.gca.2004.07.012 Révész, K.M. & J.M. Landwehr. (2002). δ13C and δ18O isotopic composition of CaCO3 measured by continuous flow isotope ratio mass spectrometry: statistical evaluation and verification by application to Devils Hole core DH‐11 calcite. Rapid Communications in Mass Spectrometry, 16(22): 2102-2114. doi: 10.1002/rcm.883 Spötl, C. & T.W. Vennemann. (2003). Continuous-flow isotope ratio mass spectrometric analysis of carbonate minerals. Rapid Communications in Mass Spectrometry, 17(9): 1004-1006. doi: 10.1002/rcm.1010 Duhr, A. & Hilkert, A.W. (2004). Application note 30049: Automated H2/H2O equilibration for dD determination on aqueous samples using thermo scientific GasBench II. Bremen, Germany: Thermo Fisher Scientific. Burman, J., Gustafsson, O., Segl, M., & Schmitz, B. (2005). A simplified method of preparing phosphoric acid for stable isotope analyses of carbonates. Rapid Communications in Mass Spectrometry, 19(21): 3086-3088. doi: 10.1002/rcm.2159 C. Spötl (2011). Long-term performance of the Gasbench isotope ratio mass spectrometry system for the stable isotope analysis of carbonate microsamples. Rapid Communications in Mass Spectrometry, 25(11): 1683-1685. doi: 10.1002/rcm.5037
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