Abstract:
Culture experiments were conducted on fifteen phytoplankton species to examine their biological and physiological responses during exposure to oil and a combination of oil and dispersant at Texas A&M University at Galveston. The species tested included Species 1: Thalassiosira pseudonana CCMP 1335, Species 2: Synechococcus elongatus CCMP 1334, Species 3: Skeletonema grethae CCMP 776, Species 4: Dunaliella tertiolecta UTEX LB999, Species 5: Skeletonema grethae CCMP 775, Species 6: Chaetoceros diversum CCMP 179, Species 7: Odontella mobiliensis CCMP 597, Species 8: Phaeodactylum tricornutum UTEX 646, Species 9: Lithodesmium undulatum CCMP 472, Species 10: Emiliania huxleyi CCMP 371, Species 11: Odontella aurita CCMP 816, Species 12: Emiliania huxleyi CCMP 1280, Species 13: Chaetoceros affinis CCMP 159, Species 14: Navicula sp. UTEX BSP11, and Species 15: Skeletonema costatum UTEX LB2308. Cultures (300 mL initial volume) grown in standard f/2 media were maintained at 19°C under 12-hour diurnal light cycle conditions for a 14-day period in triplicate for controls and each experimental treatment. Treatments including WAF (water accommodated fraction of oil), a combination of WAF and Corexit (CEWAF), and a combination of WAF and Corexit diluted (DCEWAF), such that the concentration of oil (mg L-1) was the same as the WAF-only addition, were applied to each species. Further, non-phytoplankton controls (i.e. f/2 media) were also exposed to each treatment type in order to account for the photodegradation of the oil when exposed to light. At two-day intervals the cultures were sampled to determine the concentration of oil (EOE, estimated oil equivalence), chlorophyll a relative fluorescence intensity and chlorophyll a fluorescence induction and relaxation (FIRe) curve parameters (Fv/FM, σPSII, P, and t1).
Suggested Citation:
Quigg, Antonietta; Williams, Alicia . 2016. Biological and physiological responses during 14 day incubations of 15 phytoplankton species in culture exposed to oil and combinations of oil and dispersant. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N79P2ZPD
Publications:
Bretherton, L., Williams, A., Genzer, J., Hillhouse, J., Kamalanathan, M., Finkel, Z. V., & Quigg, A. (2018). Physiological response of 10 phytoplankton species exposed to macondo oil and the dispersant, Corexit. Journal of Phycology. doi:10.1111/jpy.12625
Purpose:
The purpose of the ADDOMEX Task 1 Phytoplankton Screening experiment was to determine the measurable biological and/or physiological responses to oil and a combination of oil and dispersants of several phytoplankton species native to the Gulf of Mexico. This baseline information was intended to help make insightful choices into which phytoplankton species would be ideal candidates for continued research objectives associated with Task 1 goals.
Data Parameters and Units:
Date (MM.DD.YYYY); Time point (days); Culture (Non-Phytoplankton Control, Blank, Species 1, Species 2, Species 3, Species 4, Species 5, Species 6, Species 7, Species 8, Species 9, Species 10, Species 11, Species 12, Species 13, Species 14, Species 15); Treatment (WAF, CEWAF, DCEWAF, Blank, Control); Replicate (A, B, C, Blank); Estimated Oil Equivalence (mg L-1) (Wade et al. 2011). All fluorescence parameters are based on relative fluorescence intensities (unitless) unless otherwise specified. Chlorophyll a relative fluorescence intensity (Turner Designs, n.d.), Fv/FM (maximum quantum yield of Photosystem II (PSII) photochemistry), σPSII (functional absorption cross section of PSII, Å2 quanta−1), p (connectivity factor defining the excitation energy transfer between individual photosynthetic units), and t1 (time constant of the relaxation kinetics of the fluorescence yield following the single turnover flash, microsecond) (Satlantic, 2012). The extreme value (-999) was used to indicate a parameter not measured.
Methods:
Cultures (300 mL initial volume) were grown in standard f/2 media (Guillard and Ryther 1962). Samples were collected every 2 days for a period of 14 days. Cultures were obtained from the National Center for Marine Algae (formally CCMP) or the culture collection of Algae at The University of Texas Austin. These included Species 1: Thalassiosira pseudonana CCMP 1335, Species 2: Synechococcus elongatus CCMP 1334, Species 3: Skeletonema grethae CCMP 776, Species 4: Dunaliella tertiolecta UTEX LB999, Species 5: Skeletonema grethae CCMP 775, Species 6: Chaetoceros diversum CCMP 179, Species 7: Odontella mobiliensis CCMP 597, Species 8: Phaeodactylum tricornutum UTEX 646, Species 9: Lithodesmium undulatum CCMP 472, Species 10: Emiliania huxleyi CCMP 371, Species 11: Odontella aurita CCMP 816, Species 12: Emiliania huxleyi CCMP 1280, Species 13: Chaetoceros affinis CCMP 159, Species 14: Navicula sp. UTEX BSP11, and Species 15: Skeletonema costatum UTEX LB2308. All treatments were prepared in glass aspirator bottles that were mixed with a stir bar for 24 hours on stir plates on the lowest setting, such that no visible vortex formed. 200 microL of oil per 1L f/2 media was used to make WAF. A stock solution of oil (10 microL) and corexit (500 microL) was created and 200 microL of this stock per 1L of f/2 media was used to make CEWAF. DCEWAF was prepared by diluting CEWAF with f/2 media until the estimated oil equivalent concentration was similar to that of WAF. Transfer of solutions from aspirator bottles to experimental vessels only included the lower fraction, excluding the visible surface “oil slick” layer and all solutions were passed through a sieve (20 micro m pore size) prior to distribution. A fresh f/2 medium (blank), and WAF, CEWAF and DCEWAF non-phytoplankton containing controls were used to correct for interference from background fluorescence and to account for photodegredation during the 14-day experimental period. Instruments were calibrated according to standard practices described in Turner Designs (n.d.) and Satlantic (2012). To determine estimated oil equivalents (EOE), aliquots of each culture were extracted into dichloromethane in 20 mL scintillation vials. The maximum intensity (MI) from extracted sample was measured at an excitation wavelength of 260 nm and an emission wavelength of 370 nm corresponding to the MI from the Macondo Oil following procedures described in Wade et al. (2011). EOE was calculated according to the following calibration curve equations: Equation 1 (Species 1-8): EOE = 0.0104 * (Spectrofluorometer Fluorescence Intensity) + 0.209 Equation 2 (Species 9-15): EOE = 0.0179 * (Spectrofluorometer Fluorescence Intensity) A second aliquot of each culture was dark acclimated for a minimum of 15 minutes before fluorometric processing. The raw relative fluorescence intensity of Chlorophyll a was collected using an in-vivo method with excitation across the red spectrum (185-870nm) and measuring light emission at 680nm. The concentration range was set at medium and the time constant was set at 2 seconds. The acquisition range was restricted to manual adjustment and was not changed throughout the experiment. The fluorescence induction and relaxation (FIRe) curve parameters were collected from the single turnover (ST) component of the transient using a gain between 400 and 2400 and with 10 acquisitions per sequence, optimizing the signal to noise ratio. Guillard, R. and J. Ryther. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Canadian Journal of Microbiology 8(2): 229-239. 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. Turner Designs. (n.d.). Technical Note: An introduction to Fluorescence Measurements. Revision A. Retrieved from http://www.turnerdesigns.com/t2/doc/appnotes/998-0050.pdf on June 14th, 2016. Satlantic. (2012). Operation manual for the FIRe Fluorometer System. Revision G. Retrieved from http://satlantic.com/sites/default/files/documents/FIRe-RevG-Manual.pdf on June 14th, 2016.
Instruments:
Estimated oil equivalents were measured on a Spectroflurophotometer RF-5301PC (Shimadzu, Houston, TX, USA.) Chlorophyll a relative fluorescence intensity was measured on a Fluorometer 10AUTM (Turner Designs, San Jose, CA, USA). Chlorophyll a fluorescence induction and relaxation curve parameters were measured on a FIRe Fluorometer System (Satlantic, Halifax NS, Canada).
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