Abstract:
A surface water survey and corresponding bioassay experiment was conducted to examine the impact of nutrients on microbial responses to the Texas City “Y” oil spill on March 22, 2015 in Galveston Bay, Texas. On March 25th and March 28th, 2014 surface (1m depth) water samples were collected from 8 stations. Complementary measurements of water temperature and salinity were also taken at each station. Aliquots of collected water were preserved in glass at -20°C for subsequent estimated oil equivalence (EOE), total polycyclic hydrocarbon (PAH), relative PAH, and nutrients, including nitrate (NO3-1), nitrite (NO2-), ammonium (NH4+) and phosphate (Pi), concentration measurements. Nano- and pico-plankton (2-20 um) were enumerated by flow cytometry. Surface water from stations 2 and 3 was used as the initial substrate for bioassay incubations. Water samples were distributed to triplicate pre-autoclaved polycarbonate bottles (1L) and incubated for a 7-day period. Nitrogen (as nitrate) and Pi were added corresponding to standard f/2 medium (https://ncma.bigelow.org/) for nutrient enriched treatments. Incubated water was sampled from each bottle at 0, 1, 2, 3, 4, 5, 6 and 7 days for flow cytometric analysis. Additionally, water was preserved at the end of the 7-day incubation for EOE, total PAH, relative PAH and nutrient measurements as described above. In addition, a meta-analysis documenting historical anthropogenic releases of petrochemicals into Galveston Bay, Texas was conducted. This included a compilation of the total number of spills from 1998-2014 and the total volumes of petrochemicals released by each of the spills.
Data Parameters and Units:
Historical_Data.csv: Year, Spill volume (L) TexasCityYOilSpill_Data.csv: Date (MM.DD.YYYY), Time point (hrs), Station, Treatment, Replicate, Total PAH (mg L-1), Relative PAH (%), Heterotrophic Abundance (cells mL-1), Autotrophic Abundance (cells mL-1), Nutrient Concentration (umol L-1) Values defined as BDL indicate that the measurements were below the detection limits of the instrumentation. The extreme value (-999) was used to indicate a parameter not measured. Station, latitude (decimal degrees), longitude (decimal degrees) Station 1, 29.37 N, 94.82 W Station 2, 29.36 N, 94.75 W Station 3, 29.34 N, 94.77 W Station 4, 29.34 N, 94.78 W Station 5, 29.32 N, 94.78 W Station 6, 29.32 N, 94.79 W Station 7, 29.31 N, 94.81 W Station 8, 29.32 N, 94.83 W
Methods:
Sampling: Surface (1m depth) water samples were collected on cruises of opportunity onboard the R/V Flying Fish Too and the R/V Bateau in Galveston Bay. Complementary measurements of water temperature (degrees C) and salinity (unit-less practical salinity scale) were taken. Aliquots of collected water were preserved in glass at -20°C for subsequent EOE, total PAH, relative PAH, and nutrient concentration measurements. Additionally, triplicate samples of water (1 mL) were gravimetrically passed through a 20 um mesh-size sieve into sterile 1.5 mL microcentrifuge tubes containing 0.2 um filtered paraformaldehyde and molecular biology grade gluteraldehyde at final concentrations of 1% and 0.01% respectively (Kamiya et al. 2007). All samples were stored at -20 deg C until processing for flow cytometry. The 20 um mesh was chosen because it allowed us to focus on the pico (less than 2 um) and nano (2-20 um) plankton size fractions. Estimated Oil Equivalence (EOE): Water samples were extracted into dichloromethane and the maximum intensity (MI) from the extracted sample was measured at an excitation wavelength of 260 nm and an emission wavelength of 370 nm corresponding to the MI from the Texas City “Y” oil following procedures described in Wade et al. (2011). Total and Relative Polycyclic Aromatic Hydrocarbons (PAH): Concentrations of PAH’s including C0-C4 naphthalenes, biphenyl, acenaphthylene, acenphthene, C0-C3 fluorenes, phenanthrene, C0-C4 anthracenes, C0-C3 dibenzothiophenes, C0-C3 fluoranthenes, pyrene, benzo(a)anthracene, C0-C4 crysenes, benzo(b)fluoranthene, benzo(k) fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene, indeno(1,2,3-c,d)pyrene, dibenzo(a,h)anthracene, and benzo(g,h,i)perylene were determined by gas chromotagraphy mass spectrometric detection in selected ion mode as described in Kirman et al. 2016. Nutrient Concentrations: Determination of dissolved nutrient concentrations followed a standard operating procedure established by The NELAC Institute, which does not enrich nutrients during filtration. Water (50 mL) was filtered through a 0.7 μm glass fiber filter (Whatman, Kent, UK), and the filtrate was stored in sterile centrifuge tubes at -20°C until processing. The Texas A&M Univer- sity Geochemical and Environmental Research Group determined concentrations of nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), and phosphate (Pi-inorganic pool) from each water sample using an auto-analyzer according to Koroleff (1999). The ratio of dissolved inorganic nitrogen (DIN) to Pi was calculated after summing the individual dissolved nitrogen inputs (DIN = NO3-1 + NO2- + NH4+). Flow Cytometry: Heterotrophic and autotrophic microbial plankton groups were resolved using SYBR Green I staining procedures modified from (Marie et al. ) on a Gallios 3-laser flow cytometer (Beckman Coul- ter, Brea, CA). The operational definition of microbial plankton cells used herein is based on a pre-filtration targeting cells less than 20 um in size and the frame of reference and volume used during flow cytometric processing. This method can capture individuals within both the pico- and nano-plankton size fractions (0.2–2 μm and 2–20 μm respectively) but most of the particles counted were smaller than internal standard beads used (less than 10 μm), indicating that the majority of these are pico-plankton (0.2–2 μm) cells. Further, because in situ nano-plankton (2–20 μm) abundance is expected to be less than 1000 cells mL-1 the volume of sample processed herein would not capture enough nano-plankton cells to significantly alter the total and relative abundances described. Aliquots of preserved sample were stained with 1/1000 diluted 10000X concentrated SYBR Green I (Invitrogen, Carlsbad, CA). SYBR Green staining and persistent fluorescence was enhanced by the addition of potassium citrate (30 mmol L-1 final concentration) to each sample. Samples were incubated in the dark at room temperature for 15 min.This incubation procedure did not cause variation in naturally occurring pigments prohibitive to group isolation. Internal size (10 μm) and enumeration (973 beads μL-1) standard flow count fluorophores were added to each sample tube post incubation (Beckman Coulter, Brea, CA.). Particles were isolated within IsoFlow sheath fluid (Beckman Coulter, Brea, CA) and were exposed to 488 nm and 638 nm excitation by lasers and fluorescence was evaluated. Chlorophyll a emission was collected through a 695 nm band-pass filter ± 15 nm targeting its emission maximum of 667 nm. SYBR Green I emission was collected through a 525 nm band-pass filter ± 15 nm targeting its emission maximum of 522 nm. Phycoerythrin emission was collected through a 575 band-pass filter ± 15 nm targeting its emission maximum of 576 nm. Phycocyanin emission was collected through a 660 nm band- pass filter ± 15 nm targeting its emission maximum of 642 nm. Samples were analyzed for 5 min. at a flow rate of 4–8 μL min-1 discriminating on SYBR Green I fluorescence. Data analysis was conducted using Kaluza Cytometry Analysis software (Version 1.2 Beckman-Coulter, Brea, CA). Autotrophic and heterotrophic cells were discriminated using Boolean gating on a combination of bivariate scatter plots (cytograms) or histograms with parameters including SYBR Green I, orange, and red fluorescence. Individual cells were grouped by the similarity in their physiological characteristics on the basis of previously reported observations of cultured and environmental samples. Kimaya E., S. Izumiyama, M. Nushimura, J. G. Mitchell, K. Kogure. (2007). Effects of fixation and storage on flow cytometric analysis of marine bacteria. Journal of Oceanography. 63: 101-112. Wade, T. L., S. T. Sweet, J. L. Sericano, N. L. Guinasso Jr., A. R., Diercks, R. C. Highsmith, V. L. Asper, D. Joung, A. M. Shiller, S. E. Lohrenz, S. B. Joye. 2011. Analyses of water samples from the Deepwater Horizon oil spill: Documentation of the subsurface plume. In Monitoring and Modeling the Deepwater Horizon Oil Spill: A Record-Breaking Enterprise Book Series: Geophysical Monograph Series. 195: 77-82. Kirman, Z., J. Sericano, T. Wade, T. Bianchi, F. Marcantonio, A. Kolker. (2016). Composition and depth distribution of hydrocarbons in Barataria Bay marsh sediments after the Deepwater Horizon Oil Spill. Environmental Pollution. 214: 101-113. Koroloff, H. (1999). Determination of Nutrients. In: Methods of Seawater Analysis. Grasshoff, K., editor. Weinheim, Germany. Wiley-VCH Verlag GmbH. Pp. 159-227. Marie D., F. Partensky, S. Jacquet, D. Vaulot. (1997). Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Applied and Environmental Microbiology. 63: 186-193.
Instruments:
Temperature and salinity were measured using either a Datasonde 5 (Hydrolab Corporation, Austin, TX, USA) or a hand-held CTD (Castaway-CTD, SonTek, San Diego, CA, USA). In some cases, to protect equipment from oil fouling, a refractometer calibrated to the hydrolab was used to measure salinity. Estimated oil equivalents were measured on a Aqualog Fluorometer (Horiba Scientific, Kyoto, Japan). Total and relative PAH concentrations were measured on a gas chromatograph mass spectrometer 6890N GC/5975C inert MSD, (Agilent, Santa Clara, CA, USA). Concentrations of nutrients were measured on an auto-analyzer (Astoria-Pacific, Clackamas, OR, USA). Abundance of microbial groups was measured on a Gallios 3-Laser Flow Cytometer (Beckman Coulter, Irving, TX, USA).
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