Response of toxigenic Pseudo-nitzschia sp. to oil and dispersant exposure over 6 days
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Gulf of Mexico Research Initiative
Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers 2 (ADDOMEx-2)
Mount Allison University / Environmental Science
phytoplankton, Pseudo-nitzschia, domoic acid, photosynthesis, diatoms, oil spills, Corexit, water accommodated fraction (WAF), chemically enhanced WAF (CEWAF), diluted CEWAF (DCEWAF), polycyclic aromatic hydrocarbon (PAH)
Pseudo-nitzschia is a common component of phytoplankton communities in the Gulf of Mexico, and some species can produce domoic acid, a potent neurotoxin. Some data suggests that Pseudo-nitzschia is fairly tolerant of oil exposure, but little to no work exists on how oil and dispersants impact its physiology and toxin production. This dataset is for a culture experiment done on a Pseudo-nitzschia sp. colony recently isolated from the Gulf of Mexico. Pseudo-nitzschia sp. was exposed to oil using the water accommodated fraction (WAF), and the dispersant Corexit by using a chemically enhanced WAF (CEWAF) and a diluted CEWAF (DCEWAF). Growth dynamics and photosynthetic performance was measured each day through the experiment, and measurements for domoic acid production and pigment content were done on the final day. Total oil concentration, as well as the alkane and polycyclic aromatic hydrocarbon (PAH) concentration, were characterized for each treatment.
Bretherton L, Hillhouse J, Bacosa H, Setta S, Genzer J, Kamalanathan M, Finkel Z, Quigg A. 2019. Response of toxigenic Pseudo-nitzschia sp. to oil and dispersant exposure over 6 days. Distributed by: Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC), Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-qpqz-hn88
Bretherton, L., Hillhouse, J., Bacosa, H., Setta, S., Genzer, J., Kamalanathan, M., … Quigg, A. (2019). Growth dynamics and domoic acid production of Pseudo-nitzschia sp. in response to oil and dispersant exposure. Harmful Algae, 86, 55–63. doi:10.1016/j.hal.2019.05.008
This data was collected to better understand how oil and dispersant exposure impact toxigenic microalgae that are common to the Gulf of Mexico and to better understand the wider impacts that oil spills and dispersant application can have on the microbial ecology of the Gulf of Mexico.
Data Parameters and Units:
Time (days), Treatment (C = Control, O = WAF, M = CEWAF, DM = DCEWAF), Replicate (no units), EOE (estimated oil equivalents, mg/L), cell count (cells/mL), Fv/Fm (quantum yield, dimensionless), Sigma (photosystem II cross-section, quanta/A^2), p (PSII connectivity factor, dimensionless), Tau1 (plastoquinone reoxidation time, microseconds), Tau2 (PSII reoxidation time, microseconds), ETR (electron transfer rate, mol electrons/m^2/s), Growth rate (d-1), Lag time (days), Chl a (chlorophyll a, pg/cell), PCS (carotenoids, pg/cell), Total DA (total domoic acid, fg/cell), Int DA (intracellular domoic acid, fg/cell), Ext DA (extracellular domoic acid, fg/cell), Alkanes (ppb), PAHs (polycyclic aromatic hydrocarbons, ppb). Note: The empty columns/cells in an excel file indicates that data was not collected for those days.
Cultivation of Pseudo-nitzschia sp.: Colonies of Pseudo-nitzschia sp. were isolated from the GOM off the coast of Louisiana (28°86 N, 90°49 W) by M. Parsons (Florida Gulf Coast University) on 8 April 2017. The in situ salinity at the isolation site was 32 psu. Prior to experimentation, the colony of Pseudo-nitzschia sp. was cultivated in natural seawater collected from the GOM off Galveston, TX and enriched with f/2 nutrients, metals and vitamins (Guillard 1975). Cultures were monitored daily by counting cells with a haemocytometer, and diluted with fresh f/2 media to maintain a density of ~100,000 cells mL-1 and ensure cells were exponentially growing. The cultures were maintained at a temperature of 19° C, under light:dark (L:D) cycle of 12:12 h and a light intensity of 150 µmol m-2 s-1. Experimental set up: WAF, CEWAF and DCEWAF were prepared using a modified version of the CROSERF method (Singer et al. 2001). Pre-prepared f/2 seawater media was transferred into 1 L glass aspirator bottles with bottom spigots. To each aspirator, 400 µL of either surrogate Macondo Louisiana crude oil (for WAF) or a mixture of the dispersant Corexit and oil in a 20:1 ratio (for CEWAF) was added. Each aspirator was then stirred at such a speed that a vortex occupied the upper ~25% of the volume, and left for 24h in the dark at room temperature. The WAF and CEWAF mixtures were each pooled into a larger 9 L glass aspirator, forming the stock solutions. When decanting from the 1 L glass aspirators, the media was passed through a 20 µm nylon mesh sieve to remove large particles and droplets. The surface slick was not allowed to pass through the spigots of the 1 L aspirator bottles. In order to make the DCEWAF stock solution, a volume of the CEWAF was diluted with fresh f/2 media by a factor of 10. Stock solutions (850 mL) were transferred into sterile 1 L glass Duran bottles and inoculated with 150 mL of exponentially growing Pseudo-nitzschia sp. culture. Controls were prepared in the same manner, with 850 mL of fresh f/2 media instead. All treatments were prepared in triplicate. Additionally, 500 mL of WAF, CEWAF and DCEWAF stock culture were each transferred to a 1 L Duran bottle, with no phytoplankton added. These formed non-biological controls and served to correct for background fluorescence from both the oil and dispersant for many of the measurements taken (described below). All experimental bottles were maintained at 19° C, with a L:D cycle of 12:12 h and light intensity of 150 µmol m-2 s-1 for 6 days. EOE: The oil concentration was monitored daily in each culture vessel by measuring the estimated oil equivalents (EOE; mg L-1). To determine EOE, aliquots (10 mL) were taken from each experimental bottle, as well as initial stock solutions of WAF, CEWAF and DCEWAF, and were extracted into 10 mL dichloromethane (DCM) in 20 mL scintillation vials. DCM allows for detection of oil as low as 0.7 µg L-1 (Wade et al. 2011). Approximately 3 mL of the DCM fraction was transferred into a quartz cuvette, and the maximum intensity was measured at an excitation wavelength of 322 nm and an emission wavelength of 376 nm in a Shimadzu spectrofluorophotometer (RF-5301PC, Shimadzu, Houston, TX, USA). A calibration curve was made using dilutions of crude oil in DCM in order to calculate the EOE in each sample. This method is able to measure hydrophilic hydrocarbons and does not capture fractions such as n-alkanes. Alkanes, PAHs: The concentration of alkanes and polycyclic aromatic hydrocarbons (PAHs) in the WAF, CEWAF and DCEWAF stocks was determined at the start and at the end of the experiment. 500 mL of each stock was decanted into a glass bottle and immediately stored at -20° C until analysis. On the final day, samples were collected from each bottle again to measure alkane and PAH concentration. Hydrocarbon analysis was performed according to a previously established protocol (Bacosa et al. 2015, 2016; Liu et al. 2017) with some modifications (Bacosa et al. 2018). Briefly, the samples were spiked with a mixture of deuterated standards, extracted three times with 20 mL dichloromethane in a separatory funnel, and filtered through a chromatographic column with anhydrous sodium sulfate. The extracts were then concentrated by rotary evaporator and nitrogen gas to 500 µL. Alkanes and PAHs were analyzed using HP-6890 Series GC (Hewlett Packard) interfaced with an Agilent 5973 inert mass selective detector (MSD). The column was an Agilent DB-5MS column. The running conditions are mentioned elsewhere (Bacosa et al. 2018). The hydrocarbons were quantified using the added deuterated standards and were recovery-corrected. Cell counts, Growth rate, Lag time: A sample for cell counts were taken from each culture bottle every day of the experiment. 1 mL aliquots were preserved in 10% Lugol’s solution and stored at 4° C until processing. The samples were counted with a haemocytometer slide (Neubauer) using a light microscope. Cell density was used to calculate both growth rate and time spent in the lag phase. Growth rates (µ, d-1) were calculated using the following equation: µ = (lnCt - lnC0) / t where µ is the average specific growth rate between time 0 and time t (d-1), Ct is the cell density at time t, C0 is the cell density at the start of the experiment, and t is the time considered (days). Lag time (λ, days) was calculated by log-transforming the cell densities and plotting them over time using the online applicated GeoGebra (https://www.geogebra.org/graphing). Two lines were then plotted, one where y = C0, and one through the points in the exponential phase. The intercept of these two points was used as an estimate of λ. Chl a, PCS: Daily measurements of chlorophyll a were carried out using a benchtop fluorometer (10AU Turner Designs) on 4 mL aliquots that had been dark-acclimated for 15 minutes. The chlorophyll fluorescence intensity was used to calculate the chlorophyll a concentration (µg/L) in each culture. The fluorescence intensity was also measured in the WAF, CEWAF and DCEWAF non-biological controls, as well as in filtered seawater, to account for background fluorescence. In order to calibrate the fluorometer, a chlorophyll standard from Anacystis nidulans (Sigma-Aldrich) was used to prepare a standard curve. Samples were taken for in-depth characterization of pigment content on the final day of the experiment. 50 mL from each culture was filtered onto a GF filter and stored at -20° C until processing. To extract pigments, the filter was thawed, placed in a 20 mL scintillation vial with 10 mL of 90% acetone and left in the dark for 2 hours at 4° C. Once the pigments had extracted into the acetone, a 2 mL aliquot was placed into a quartz cuvette for analysis on a spectrophotometer (Shimadzu). Measurements were taken at 630, 644 and 750 nm and chlorophyll content was calculated after the equations in Ritchie (2006). Non-biological controls for WAF, CEWAF and DCEWAF were used to correct readings, as well as filtered seawater. Fv/Fm, Sigma, p, Tau1, Tau2, ETR: Daily photophysiology measurements were taken using a Fluorescence Induction and Relaxation (FIRe) and Pulse Amplitude Modulation (PAM) fluorometer. Single turnover (ST) induction curves from the FIRe (Satlantic) were used to measure properties such as photosynthetic efficiency (Fv/Fm), photosystem II (PSII) antenna size (σPSII) and PSII turnover time (τ). The PAM (PhytoPAM, Walz) was used to generate rapid light curves (RLCs) in order to calculate electron transfer rates (ETR). ETR is calculated with the following equation: ETR = Yield ∙PAR ∙0.84 ∙0.5 where ETR is the electron transfer rate (µmol electrons m-2 s-1), Yield is the Fv/Fm measured at a given light intensity (dimensionless), PAR is the photosynthetically active radiation, i.e. light intensity (µmol photons m-2 s-1), 0.84 is the theoretical ratio of photons absorbed by photosynthetic pigments to incident photons, and 0.5 is the theoretical ratio of photons absorbed by PSII relative to photons absorbed by all photosynthetic pigments. The maximum ETR is calculated by plotting ETR over PAR and fitting the Jassby and Platt (1976) model. For all measurements, samples from the WAF, CEWAF and DCEWAF non-biological controls, as well as filtered seawater, were used to account for background fluorescence. Total DA, Int DA, Ext DA: Domoic acid content was measured in all experimental bottles on the final day of the experiment using an enzyme-linked immunosorbent assay (ELISA) kit (Biosense Laboratories). The assay is specific to domoic acid with no cross-reactivity to non-toxic structural analogues such as kainic acid (Garthwaite et al. 2001). The calibrated range of the assay is approximately 10 – 300 pg mL-1. Two 50 mL samples were taken from each culture, one for total domoic acid and one for extracellular domoic acid. The sample for total domoic acid was vortexed for 5 minutes and then filtered through a GF filter (Whatman). The sample for extracellular domoic acid was filtered through a 0.8 um cellulose acetate filter at low pressure. All samples were immediately stored at -20° C until analysis. Intracellular domoic acid was calculated by subtracting the extracellular concentration from the total.
Alkanes and PAHs were analyzed using HP-6890 Series GC (Hewlett Packard) interfaced with an Agilent 5973 inert mass selective detector (MSD). The column was an Agilent DB-5MS column. The samples were counted with a haemocytometer slide (Neubauer) using a light microscope. Daily measurements of chlorophyll-a were carried out using a benchtop fluorometer (10AU Turner Designs).
Provenance and Historical References:
Bacosa, H. P., D. L. Erdner, and Z. Liu. 2015. Differentiating the roles of photooxidation and biodegradation in the weathering of Light Louisiana Sweet crude oil in surface water from the Deepwater Horizon site. Marine Pollution Bulletin 95: 265–272. doi:10.1016/j.marpolbul.2015.04.005 Bacosa, H.P., Kamalanathan, M., Chiu, M.H., Tsai, S.M., Sun, L., Labonté, J.M., Schwehr, K.A., Hala, D., Santschi, P.H., Chin, W.C. and Quigg, A. 2018. Extracellular polymeric substances (EPS) producing and oil degrading bacteria isolated from the northern Gulf of Mexico. PLOS ONE 13: e0208406. doi:10.1371/journal.pone.0208406 Bacosa, H. P., K. M. Thyng, S. Plunkett, D. L. Erdner, and Z. Liu. 2016. The tarballs on Texas beaches following the 2014 Texas City “Y” Spill: Modeling, chemical, and microbiological studies. Marine Pollution Bulletin 109: 236–244. doi:10.1016/j.marpolbul.2016.05.076 Y Garthwaite, I., K. M. Ross, C. O. Miles, L. R. Briggs, N. R. Towers, T. Borrell, and P. Busby. 2001. Integrated Enzyme-Linked Immunosorbent Assay Screening System for Amnesic, Neurotoxic, Diarrhetic, and Paralytic Shellfish Poisoning Toxins Found in New Zealand. Journal of AOAC International 84 (5), 1643–1648. Guillard, R. R. L. 1975. Culture of Phytoplankton for Feeding Marine Invertebrates. Culture of Marine Invertebrate Animals, 29–60. doi:10.1007/978-1-4615-8714-9_3 Liu, J., H. P. Bacosa, and Z. Liu. 2017. Potential Environmental Factors Affecting Oil-Degrading Bacterial Populations in Deep and Surface Waters of the Northern Gulf of Mexico. Front. Microbiol. 7. doi:10.3389/fmicb.2016.02131 Ritchie, R. 2006. Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynthesis research 89: 27–68. doi:10.1007/s11120-006-9065-9 Singer, M. M., D. V. Aurand, G. M. Coelho, G. E. Bragin, J. R. Clark, M. Sowby, and R. Tjeerdema. 2001. Making, measuring, and using water-accomodated fractions of petroleum for toxicity testing. International Oil Spill Conference 2001: 1269–1274. doi:10.7901/2169-3358-2001-2-1269 Wade, T.L., Sweet, S.T., Sericano, J.L., Guinasso, N.L., Diercks, A.R., Highsmith, R.C., Asper, V.L., Joung, D., Shiller, A.M., Lohrenz, S.E. and Joye, S.B. 2011. Analyses of Water Samples From the Deepwater Horizon Oil Spill: Documentation of the Subsurface Plume. Geophysical Monograph Series, 77–82. doi:10.1029/2011gm001103