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FLOW CYTOMETRY

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  • This metadata record contains the results from 3 bioassays conducted with the Antarctic marine microalgae Cryothecomonas armigera (incertae sedis). These tests assessed the toxicity of copper, cadmium, lead, zinc and nickel. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (60-90 micromol/m2/s, cool white 36W/840 globes), in 80 mL natural filtered (0.22 microns) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). Filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Tests 1 and 2 consisted of metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Test 3 consisted of metal treatments in an increasing series (no replicates) alongside 3 replicate controls. Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets as dissolved metal concentrations measured on day 0, and day 24. The average of the dissolved metal concentrations were used for further statistical analysis. The age of C.armigera at test commencement was 25-30 days. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x10^3 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The flow cytometer was also used to simultaneously measure changes in the following cellular parameters: chlorophyll a autofluorescence intensity (FL3), cell size (FSC) and cell complexity (SSC). The molecular stain BODIPY 493/503, was used to measure neutral lipid concentrations. Changes in cellular parameters were measured by applying a gate that captured greater than 95% of control cells in a region, R2. Changes in cellular parameters were observed in metal treatments as a shift of the cell population from the R2 region to R1 (for relative decreases) or to R3 (for relative increases). The proportion of cells in each region is expressed as a percentage of the total cell population. The pH was measured on the first and last day of the test. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on 24. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-microns membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). Metal concentrations were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn. Detection limits for Cu, Cd, Pb, Ni and Zn were 1.0, 0.3, 3.2, 1.4, and 1.0 micrograms per litre, respectively. Calculations of effect concentrations (EC 10 and 50) were made using the 'Dose Response Curve' package of R statistical analysis software. Concentration-response curves had several models applied to them, and were tested for best fit by comparing residual standard errors and Akaike's 'An Information Criterion' function . Generally, log-logistic models with 3 parameters provided the best fit. Data for each toxicity test is combined in a single excel spreadsheet, "Cryothecomonas armigera single metal toxicity". The first worksheet is titled "Test Conditions" which provides information on the toxicity test, e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and measured metal concentrations. The third worksheet contains the measured physiological parameters: Neutral lipid concentrations (BODIPY 493/503), chlorophyll a autofluorescence (FL3), cell complexity (SSC), and cell size (FSC). The final worksheet contains the output of statistical analysis; dose-response curves for each metal with applied log-logistic model and 95% confidence interval, a table summarising the effect concentrations (EC10 and EC50), and No Effect Concentration (NEC) is also provided. The file "C. armigera combined.csv" contains rows representing individual exposures with columns for the metal treatment (Metal), averaged dissolved metal concentration for each exposure (Conc), growth rate (Growth), and growth rate as a percent of the control (Pcon). This data was used for data analysis in R statistics. Note that this contains data from all bioassays conducted with C. armigera, including those conducted by Francesca Gissi (doi:10.4225/15/551B2B65A73F3) The script used for data analysis is provided in the document "R statistics script for C. armigera single metal.docx"

  • This metadata record contains observed and predicted toxicity data from bioassays with two species of Antarctic marine microalgae: Phaeocystis antarctica (Prymnesiophyceae) and Cryothecomonas armigera (Cercoza). Bioassay exposures were of mixtures of 5 metals at two ratios, an Environmental (ENV) and Equitoxic (EC) mixture. The measured dissolved metal concentrations were used in two mixture reference models, Independent Action (IA) and Concentration Addition (CA), to predict toxicity as population growth rate inhibition. A Flow Cytometer (BD-FACSVerse) was used to measure the density of microalgae over time, which was then converted to a growth rate. An inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES), was used to measure metal concentrations. Data for each microalga is provided in individual excel spreadsheets, identified by the species tested. A word document is provided that contains the R code used to predict toxicity to the two microalgae by the reference models Independent Action and Concentration Addition. The R code also includes the steps required to extend the models to include a deviation parameter “a” that allows for departure from model additivity. A nested F-test then tests for significance between the fit of each test to observed toxicities. This R code has been adapted to use EC10 as parameter estimates, rather than EC50s. The code was adapted from the approach outlined in Hochmuth, J. D.; Asselman, J.; De, S. Are Interactive Effects of Harmful Algal Blooms and Copper Pollution a Concern for Water Quality Management? Water Res. 2014, 60, 41–53. DOI: 10.1016/j.watres.2014.03.041. Single-metal toxicity data and experimental protocols for P. antarctica from the following paper: and C. armigera used in this study can be found in the following papers: A robust bioassay to assess the toxicity of metals to the Antarctic marine microalga Phaeocyctis antarctica. Francesca Gissi, Merrin S. Adams, Catherine K. King, Dianne F. Jolley (2015). Environmental Toxicology and Chemistry. 2015 Feb 20. doi: 10.1002/etc.2949. Chronic toxicity of five metals to the polar marine microalga Cryothecomonas armigera – Application of a new bioassay. Darren J. Koppel, Francesca Gissi, Merrin S. Adams, Catherine K. King, and Dianne F. Jolley, (2017). Environmental Pollution, Volume 228, 2017, Pages 211-221, doi.org/10.1016/j.envpol.2017.05.034.

  • This data set was collected from a ocean acidification minicosm experiment performed at Davis Station, Antarctica during the 2014/15 summer season. It includes: - description of methods for all data collection and analyses. - flow cytometry counts; autotrophic cells, heterotrophic nanoflagellates, and prokaryotes

  • These data come from a set of experiments conducted on the coastal waters near Davis Station in January 2017. The first set of data are from a transect near the Sorsdal glacier and out to sea, to characterise DMSP-mediated phytoplankton bacteria interactions along a salinity gradient. The second data set are from a series of incubation experiments to gain deeper insight into the role of various infochemicals in Antarctic phytoplankton-bacteria relationships. Specifically, DMSP, VitB12, Tryptophan and Methionine. The last data set is derived from two incubation experiments: a short term DMSP addition experiment to look at its uptake and utilisation by the microbial community; and a longer-term (5 day) stable isotope probing experiment to track DMSP through the lower trophic food web.

  • This data set was collected from two minicosm experiments conducted at Davis Station, Antarctica. 1. Variance experiment - 2013/14 summer season 2. Ocean acidification experiment - 2014/15 summer season It includes: - description of methods for all data collection and analyses. - environmental data logged throughout the experiment; nutrients, temperature, light climate. - flow cytometry counts; autotrophic cells, heterotrophic nanoflagellates, and prokaryotes. - FlowCam counts; individual phytoplankton species data. - microscopy counts; individual phytoplankton species data.

  • This metadata record contains the results from 11 bioassays conducted with 2 species of Antarctic marine microalgae. Seven tests were conducted with Phaeocystis antarctica (Prymnesiophyceae), assessing the toxicity of copper, cadmium, lead, zinc and nickel. Four tests were conducted with Cryothecomonas armigera (Incertae sedis), assessing the toxicity of copper only. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (150-200 micro mol/m2/s, cool white 36W/840 globes), in natural filtered (0.45 microns for P.antarctica and 0.22 microns filtered for C. armigera) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). For both species, filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Test volumes for P.antartica and C.armigera were 50 mL and 80 mL, respectively. All tests consisted of 3-5 metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets. The following replicate toxicity tests were completed for P. antarctica: - 5 tests with copper (1-20 micro g/L) - 4 tests with lead (10-500 micro g/L) - 3 tests with cadmium (100-2000 micro g/L) - 3 tests with zinc (100-2000 micro g/L) - 3 tests with nickel (200-1000 micro g/L) For C. armigera, 1 rangefinder test was carried out testing 6 concentrations (1-100 micro g/L), and 3 definitive test, with 5 concentrations (15-100 micro g/L). The age of P. antarctica and C.armigera at test commencement was 8-12 days, and 25-30 days, respectively. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x103 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. The flow cytometer was also used to simultaneously measure change sin chlorophyll a fluorescence intensity, cell size and internal cell granularity. Toxicity tests were continued until cell densities in the control treatments had increased 16-fold. Toxicity tests with P. antarctica were carried out over 10 days, with cell densities in each replicate flask measured every 2 days. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The pH in all treatments was measured on the first and last day of the test, as well as on day 6 for P. antarctica tests and an additional two times per week for C. armigera tests. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on days 0, 6 and 10 for P. antarctica tests, and on days 0, 7, 14, 21, and 24 for C. armigera tests. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-micron membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). All toxicity test results were calculated using measured dissolved metal concentrations, which were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn and using inductively coupled plasma-mass spectrometry (ICP-MS; Agilent 7500CE) for lowest concentration Cu samples (nominal concentration 1 micro g/L). Detection limits for Cu, Cd, Pb, Ni and Zn were 1, 0.12, 1.7, 1.2 and 0.1 micro g/L, respectively (ICP-AES) and 0.05 micro g/L (ICP-MS) for low concentration Cu samples. The specific growth rates (u) and corresponding measured metal concentrations were used to calculate toxicity test values using Toxcalc (Version 5.0.23, TidePool Scientific Software, San Francisco, CA, USA). Data were tested for normal distribution using Shapiro-Wilk's test (p greater than 0.01); and equal variances using Bartlett's test (p = 0.09). The inhibitory concentration which reduced population growth rate by x% (ICx) compared to controls was calculated using linear interpolation. The Dunnett's multiple comparison test was used to determine which treatments were significantly different to the control (2 tailed, p less than or equal to 0.05), and to calculate the no observable effect concentration (NOEC) and the lowest observable effect concentration (LOEC). Data for each toxicity test are provided in individual excel spreadsheets, identified by the species tested, the test number for that species and the date the test started. A summary table of details for the 11 tests is provided in the file: Summary table.xlsx. The first worksheet for each test file is titled "Test Conditions". This sheet provides information on the toxicity test e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and the measured pH and metal concentrations. For C. armigera data sheets, there is an additional worksheet, "Measured Cu and pH" which includes all measured pH values and metal concentrations across the 24-day period. Following the growth rate sheets are the statistical outputs for each metal, which were all generated using Toxcalc. Finally, if additional cellular parameters were measured (Chlorophyll a fluorescence, cell size and internal cell granularity), the raw data for each parameter is include in a worksheet, "Metal cellular parameters". Data were collected in an Australian laboratory (CSIRO Land and Water, Centre for Environmental Contaminants Research, Lucas Heights, 2234, NSW) during May 2013 - April 2014. The tests used microalgal strains that had been previously collected from the Southern Ocean and are cultured within the microalgal collection at the Australian Antarctic Division (AAD). Daughter daughter cultures were transferred to CSIRO, where they were cultured for this work.

  • Metadata record for data from ASAC Project 2300 See the link below for public details on this project. ---- Public Summary from Project---- Antarctic reefs, like their tropical counterparts, harbour a high diversity of animal life. For the first time we will determine how global warming will affect food availability to the animals which comprise the structural components of the reefs. Ultimately, we wish to predict the cascading effect through the community as one component changes. With the confirmation that sponges in Antarctic waters graze on ultraplankton there is now a global overview that sponges are the primary benthic organism that is responsible for linking the pelagic microbial food web to the benthos. Like other shallow water demosponges, sponges in Antarctica are omnivorous sponges that graze nonselectively, consuming both heterotrophic and phototrophic organisms. Retention efficiencies of ultraplankton are similar to other sponges measured using similar techniques from shallow water to the deep sea, the tropics to boreal waters. The large amounts of water processed by these benthic suspension feeders and their diet places these sponges squarely within the functional group of organisms that link the pelagic microbial food web to the benthos. The number of macroinvertebrates that have been shown to side- step the microbial loop and directly utilize the base of the microbial food web as a primary food source is ever growing and currently includes demosponges, ascidians, soft corals, and bivalves. Dense macroinvertebrate communities dominated by demosponges and corals in shallow water have been shown to remove as much as 90% of the ultraplankton from the water that passes over them. The daily fluxes of ultraplankton to these communities ranges from 9 to 1970 mg C day-1 m-2. We conservatively estimate that this single species of sponge, which comprises only a portion of the benthos, mediates a flux of 444 mg mg C day-1 m-2 from the water column, which places it in the range of shallow-water temperate and boreal systems. Furthermore, we found that physical disturbance results in changes in community structure. The subtidal rocky coasts near Casey are similar to many of the exposed rocky coasts of the world that support extensive stands of macroalgae that form a strong positive association with understorey encrusting coralline algae. Loss of canopies of algae on temperate coasts often triggers large and predictable changes to the assemblage of understorey taxa. We observed large negative effects of removing canopies of H. grandifolius on encrusting corallines growing beneath, with such effects consistent with predictions of previous research on tropical and temperate coasts. However, elevating concentrations of nutrients did not greatly reduce the magnitude of the negative effects of canopy removal. Nevertheless, our results suggest that disturbance (removal) to canopies of H. grandifolius has large consequences for those organisms associated with this widely distributed (circumpolar) species of canopy-forming algae. See the full copy of the final report (available for download from the URL given below) for more information. Also included in the download file, are five Excel spreadsheets. The spreadsheets contain the data collected from the transects, quadrats, etc (see the final report for more information). Where possible the spreadsheets have been converted to csv files. The fields in this dataset are: Location depth Species Transect Quadrat Irradiance PAR

  • Metadata record for data expected ASAC Project 2382 See the link below for public details on this project. This entry contains: Locations for sampling sites for ASAC project 2382 on voyage 3 of the Aurora Australis in the 2004/5 season, collected between December and February of 2004/5; CTD bottle-derived seawater viscosity data and CTD bottle-derived in vivo fluorescence data. There are four spreadsheet files in this download file. Each spreadsheet file contains several worksheets. 1) I9_Stations.xls: Transect 1 (CLIVAR I9 = 'I9') station and sampling details: CTD stations, CTD profiles, Surface samples. 2) PET_Stations.xls: Transect 2 (Kerguelen Plateau and Princess Elizabeth Trough = 'PET') station and sampling details: CTD stations, CTD profiles. 3) Viscosity.xls: Viscosity data. 4) Fluorescence.xls: In vivo fluorescence data. For all files -999 = missing data A word document details the sampling protocols for viscosity and in vivo fluorescence. Note: ASAC project 2382 operates in direct collaboration with ASAC project 2596 (Three-dimensional microscale distribution and production of plankton populations).

  • This data set contains concentrations of phytoplankton, protozoa, total bacteria and metabolically active bacteria assessed by flow cytometry on transects 12, 1, 3, 5, 7, 9 and 11 of the BROKE-West survey of the Southern Ocean between January and March 2006. Only total bacterial concentrations were assessed for transect 11. Between 4 and 12 depths were sampled for marine microbes and concentrations were assessed using FACScan flowcytometer. Phytoplankton were identified and counted based on the autofluorescense of chlorophyll a when excited by the 488 nm laser of the FACScan. Protozoa were identified and counted after staining with the acid vacuole stain Lysotracker Green. Total bacteria were identified and counted using the cell permeant SYTO 13 nucleic stain. Metabolically active bacteria were identified and counted after staining for intracellular esterases with the esterase stain 6CFDA. The fields in this dataset are: Latitude Longitude Transect Number CTD number, flow file Depth (m) Total bacteria (per millilitre) Active bacteria (per millilitre) Dead bacteria (per millilitre) Protozoa (per millilitre) Phytoplankton (per millilitre) This work was completed as part of ASAC project 40 (ASAC_40).

  • This metadata record is a 'Parent' metadata record for ASAC project 2720. See the link for the related 'Child' metadata records. The overall objective is to characterise Southern Ocean marine ecosystems, their influence on carbon dioxide exchange with the atmosphere and the deep ocean, and their sensitivity to past and future global change including climate warming, ocean stratification, and ocean acidification from anthropogenic CO2 emissions. In particular we plan to take advantage of naturally-occurring, persistent, zonal variations in Southern Ocean primary production and biomass in the Australian Sector to investigate the effects of iron addition from natural sources, and CO2 addition from anthropogenic sources, on Southern Ocean plankton communities of differing initial structure and composition. SAZ-SENSE is a study of the sensitivity of Sub-Antarctic Zone waters to global change. A 32-day oceanographic voyage onboard Australia's ice-breaker Aurora Australis was undertaken in mid-summer (Jan 17 - Feb. 20) 2007 to examine microbial ecosystem structure and biogeochemical processes in SAZ waters west and east of Tasmania, and also in the Polar Frontal Zone south of the SAZ. The voyage brought together research teams from Australasia, Europe, and North America, and was led by the ACE CRC, CSIRO Marine and Atmospheric Research, and the Australian Antarctic Division. The overall goal is to understand the controls on Sub-Antarctic Zone productivity and carbon cycling, and to assess their sensitivity to climate change. The strategy is to compare low productivity waters west of Tasmania (areas with little phytoplankton) with higher productivity waters to the east, with a focus on the role of iron as a limiting micro-nutrient. The study also seeks to examine the effect of rising CO2 levels on phytoplankton - both via regional intercomparisons and incubation experiments. Available for download from this metadata record are various datasets collected from the voyage: - An image showing a map of the cruise track. - An excel document detailing hourly position checks of the ship. - An excel document detailing the event log for the voyage. - A word document detailing prospective papers produced from the voyage. Finally a link is available for users to access the special volume of publications produced as a result of this voyage.