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Toxicity of metal mixtures to two Antarctic marine microalgae
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.
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Toxicity of metals to Antarctic marine microalgae
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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
Single metal toxicity to the Antarctic marine microalga Cryothecomonas armigera
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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
Toxicity of single metals and metal mixtures to Antarctic Marine Copepods
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This metadata record contains the results of two bioassays testing the response of Antarctic marine copepods to both individual and combined metals via 14 day toxicity tests. The tests were conducted during the 2012-2013 season at Davis Station, East Antarctica. Three metals (cadmium, copper and zinc) were tested singularly and in metal mixture combinations. The concentrations used in the two tests are outlined in the excel spreadsheet (AAS4100_12-13_MixedMetalTox.xlsx). Tests were carried out in 70 mL plastic vials (exposure vials) that contained 50 mL of test solutions. Test solutions were prepared by mixing stock solutions with filtered (0.45 microns) sea water and were stored in a constant temperature cabinet at 0 plus or minus 1 degree C for at least 2 hours prior to the start of tests in order to get to the required test temperature. Each treatment included four replicates and each test included eight controls. Within each replicate vial, 9-12 copepods were carefully added. No additional air, food or water was provided over the test period. At five days a water change was completed by removing the old test solution and replacing it with freshly prepared test solution at the same concentration. The tests were carried out in a constant temperature cabinet set at 0 plus or minus 1 degree C on a 16:8 light:dark photoperiod over 14 days. The number of surviving copepods were counted in each test container, at the same time each day, for 10 days and then a final count was completed on day 14. Mortality was determined by observing the copepods over 20 seconds and if there was no movement they were considered dead. Test solutions were sampled four times during the tests for measurement of metal concentrations. Samples were collected at day 0, day 5 pre-water change, day 5 post-water change and at day 10. Concentrations of the three test metals were determined in theses samples using Inductively-Coupled Plasma Optical Emission Spectrometer (ICP-OES) with appropriate matrix matched standards and blanks to ensure quality control. For all analyses, measured metal concentrations (as opposed to nominal concentrations) were used. Point estimates, including LC10 and LC50 values, were determined using the maximum likelihood-probit method using the software ToxCalc (version 5.0.26 Tidepool Scientific Software). Point estimates were calculated at 4, 7, 10 and 14 days of exposure. Whenever the assumptions for the maximum likelihood-probit method were not met then the Trimmed Spearman-Karber Analysis was used. Data are provided in an Excel workbook (filename: AAS4100_12-13_MixedMetalTox.xlsx). The first worksheet ("/Test Conditions") provides descriptive details for the tests and a key to abbreviations and units. Each worksheet includes a "This worksheet provides..." statement to assist interpretation of the data. A second data file is provided (filename: AAS4100_12-13_ToxCalc.xlsx) containing relevant test data from AAS4100_12-13_MixedMetalTox.xlsx, for input to ToxCalc software for analysis. This file also contains subsequent ToxCalc outputs, with key data (LC10 and LC50 values) provided in a summary worksheet. Other support files provided are seven images of the test species (images by Frances Alexander) and two figures showing copepod response to test solutions (% survival) over the exposure period of the two tests. Copepod samples were collected from the nearshore environment of Prydz Bay, offshore from Davis Station, on two days: 20 December 2012 and 9 January 2013. The 20 December collection was composed of Tisbe sp., collected from benthic habitats and the 9 January collection was composed of Paralabidocera Antarctica, collected from surface waters. Two 14-day laboratory-based toxicity tests were conducted in the Davis laboratories. The test dates were: 2 - 16 January 2013 (test 01; using Tisbe sp., collected 20 December 2012) and 10 - 24 January 2013 (test 02; using P. Antarctica, collected 9 January 2013).
Metal and organic contaminants in marine invertebrates from Antarctica
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Metadata record for data from ASAC Project 1005 Metal and organic contaminants in marine invertebrates from Antarctica, field study of their concentrations, laboratory study of their toxicities. See the link below for public details on this project. Data from this project are now unrecoverable. Several publications arising from the work are attached to this metadata record, and are available to AAD staff only. Taken from the referenced publications: Bioaccumulation of Cd, Pb, Cu and Zn in the Antarctic gammaridean amphipod Paramoera walkeri was investigated at Casey station. The main goals were to provide information on accumulation strategies of the organisms tested and to verify toxicokinetic models as a predictive tool. The organisms accumulated metals upon exposure and it was possible to estimate significant model parameters of two compartment and hyperbolic models. These models were successfully verified in a second toxicokinetic study. However, the application of hyperbolic models appears to be more promising as a predictive tool for metals in amphipods compared to compartment models, which have failed to adequately predict metal accumulation in experiments with increasing external exposures in previous studies. The following kinetic bioconcentration factors (BCFs) for the theoretical equilibrium were determined: 150-630 (Cd), 1600-7000 (Pb), 1700-3800 (Cu) and 670-2400 (Zn). We find decreasing BCFs with increasing external metal dosing but similar results for treatments with and without natural UV radiation and for the combined effect of different exposure regimes (single versus multiple metal exposure) and/or the amphipod collective involved (Beall versus Denison Island). A tentative estimation showed the following sequence if sensitivity of P. walkeri to an increase of soluble metal exposure: 0.2-3.0 micrograms Cd per litre, 0.12-0.25 micrograms Pb per litre, 0.9-3.0 micrograms Cu per litre and 9-26 micrograms Zn per litre. Thus, the amphipod investigated proved to be more sensitive as biomonitor compared to gammarids from German coastal waters (with the exception of Cd) and to copepods from the Weddell Sea inferred from literature data. This study provides information on LC50 toxicity tests and bioaccumulation of heavy metals in the nearshore Antarctic gammarid, Paramoera walkeri. The 4 day LC50 values were 970 micrograms per litre for copper and 670 micrograms per litre for cadmium. Net uptake rates and bioconcentration factors of these elements were determined under laboratory conditions. After 12 days of exposure to 30 micrograms per litre, the net uptake rates were 5.2 and 0.78 micrograms per gram per day and the bioconcentration factors were 2080 and 311 for copper and cadmium respectively. The body concentrations of copper were significantly correlated with the concentrations of this element in the water. Accumulation of copper and cadmium continued for the entire exposure suggesting that heavy metals concentrations were not regulated to constant concentrations in the body. Using literature data about two compartments (water-animal) first-order kinetic models, a very good agreement was found between body concentrations observed after exposure and model predicted. Exposure of P. walkeri to mixtures of copper and cadmium showed that accumulation of these elements can be assessed by addition of results obtained from single exposure, with only a small degree of uncertainty. The study provides information on the sensitivity of one Antarctic species towards contaminants, and the results were compared with data of similar species from lower latitudes. An important finding is that sensitivity to toxic chemicals and toxicokinetic parameters in the species investigated are comparable with those of non-polar species. The characteristics of bioaccumulation demonstrate that P. walkeri is a circumpolar species with the potential to be a standard biological indicator for use in monitoring programmes of Antarctic nearshore
Toxicity of physically and chemically dispersed fuels to the Antarctic microgastropod, Skenella palludinoides
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This metadata record contains the results from bioassays conducted to show the response of the Antarctic gastropod, Skenella palludinoides to contamination from combinations of Special Antarctic Blend (SAB) diesel, chemically dispersed with fuel dispersant Ardrox 6120. Fuel only water accommodated fractions (WAF), chemically enhanced water accommodated fractions (CEWAF) and dispersant only treatments were prepared following the methods in Singer et al. (2000) with adaptations from Barron and Ka’aihue (2003). WAF was made using the ratio of 1: 25 (v/v), fuel to filtered seawater (FSW) following the methods of Brown et al. (2017). Ratios for chemically dispersed treatments were 1: 100 (v/v), fuel to FSW and 1: 20 (v/v) dispersant to fuel. Dispersant only treatments were made using ratios for CEWAF, substituting the fuel component with FSW. Mixes were made in 5 L or 10 L glass aspirator bottles using a magnetic stirrer to achieve a vortex of approximately 20% in the FSW before the addition of test media. The same mixing energy was used to prepare all WAFs for enhanced reproducibility and comparability of results (Barron and Ka’aihue, 2003). Mixes were stirred in darkness to prevent bacterial growth for 18 h with an additional settling time of 6 h at 0 plus or minus 1 oC. A dilution series of four concentrations were made from the full strength aqueous phase of each mix using serial dilution. WAF test concentrations were 100%, 50%, 20% and 10% while CEWAF concentrations were 10%, 5%, 1% and 0.1%. These concentrations were chosen in order to quantify the mortality curve and allow statistical calculation of LC50 values. To facilitate comparisons of dispersant toxicity in the presence and absence of fuel, dispersant only test concentrations reflected those of CEWAF treatments. WAF was sealed in airtight glass bottles stored at 0 plus or minus 1 oC for a maximum of 3 h before use. Fresh test solutions were prepared every four days to ensure consistent water quality and replace hydrocarbons that adsorbed or evaporated into the atmosphere. Each test concentration was represented by five replicates with five FSW control beakers, with approximately 10 S.palludinoides individuals per replicate. The healthiest and most active individuals were chosen. Beakers were filled to 200 ml and were left open to allow the natural evaporation of lighter monoaromatic hydrocarbon components that would occur during a real spill. Animals were not fed during experiments to prevent hydrocarbons being ingested, thereby introducing an additional exposure pathway. Experiments ran for a total of 35 d exposure duration for WAF and CEWAF experiments and 15 d for dispersant only experiments. Experiments were run in cold temperature-controlled cabinets set at a temperature of 0 plus or minus 1 oC, fluorescent lights in the cabinets were set to a light regime of 18 h light, 6 h darkness, following the methods in Brown et al. (2017) to reflect Antarctic summer environmental conditions. Lethal and sublethal observations were made at test times of: 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d, 14 d, 16 d, 20 d, 21 d, 28 d and 35 d for SAB + Ardrox 6120 experiments and 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d, 14 d, 15 d for Ardrox 6120 only experiments. The health status of each individual was classified as per the criteria listed below: Attached to the vial with horns in or out Unattached (often upside down), horns out, will reattach if flipped over Not attached but if touched, will retract Closed but attached and out of water Operculum closed Dead, operculum open a little (muscles no longer working), if touched, operculum will not move and tissues might disintegrate Dead animals were removed and preserved in 80% ethanol at each observation period. In order to simulate a repeated pulse pollutant, 90 to 100% of the test solution volume of each beaker was renewed with freshly made test concentrations every four days to replenish hydrocarbons lost through evaporation and
Responses of Antarctic marine biota to contaminants - Brown Ostracod toxicity tests, Kingston 2007
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Metadata record for data from AAS (ASAC) Project 2933. While it is generally thought that Antarctic organisms are highly sensitive to pollution, there is little data to support or disprove this. Such data is essential if realistic environmental guidelines, which take into account unique physical, biological and chemical characteristics of the Antarctic environment, are to be developed. Factors that modify bioavailability, and the effects of common contaminants on a range of Antarctic organisms from micro-algae to macro-invertebrates will be examined. Risk assessment techniques developed will provide the scientific basis for prioritising contaminated site remediation activities in marine environments, and will contribute to the development of guidelines specific to Antarctica. Brown Ostracod toxicity tests, Kingston 2007 Test animals were collected from near shore environments at Casey Station, East Antarctica during Dec 2006 - Jan 2007, and transported to culturing facilities at the Australian Antarctic Division in Tasmania, where tests were conducted during 2007. The test animals were exposed to metals in non-renewable static tests in vials containing 50 mL of the test solution at ambient Antarctic coastal salinity of 34 ppt. Tests were held in temperature controlled cabinets (incubators) at a temperature of 0, 2 or 4 degrees C (+/- approximately 1 degrees C). Ten test animals were introduced into each of 3 to 5 replicate vials per treatment at test commencement, and were exposed for 10 weeks during which periodic observations were made. Test solutions were renewed in weekly water changes. Periodic observations (time since start of test) are given in hr (hours), d (days) or w (weeks). At each observation time, test animals were scored in one of the Endpoint categories described on each worksheet. Each worksheet provides data for a particular test taxa (Brown Ostracods - taxonomy to be verified) for a given test number (T01, T02, T03, T04, T05,) and a given metal contaminant (copper, zinc, cadmium and lead). Test information is provided in the first 14 rows of each worksheet, e.g. Site of collection, Test start date, Endpoint categories etc. ASU = artificial settlement units (plastic scourers used by Sarah Richards, which had been deployed in Newcomb Bay in approximately the year 2000). Concentration micro grams per litre are nominal concentrations. Measured concentrations are provided in the file: Brown Ostracod_chemistry.xls, as described below. Test temperature was 0 degrees C unless otherwise stated. Unit for all temperature data are degrees C. See the readme file in the download for more information.
Responses of Antarctic marine biota to contaminants - Juvenile Gastropod toxicity tests, Kingston 2007
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Metadata record for data from AAS (ASAC) Project 2933. While it is generally thought that Antarctic organisms are highly sensitive to pollution, there is little data to support or disprove this. Such data is essential if realistic environmental guidelines, which take into account unique physical, biological and chemical characteristics of the Antarctic environment, are to be developed. Factors that modify bioavailability, and the effects of common contaminants on a range of Antarctic organisms from micro-algae to macro-invertebrates will be examined. Risk assessment techniques developed will provide the scientific basis for prioritising contaminated site remediation activities in marine environments, and will contribute to the development of guidelines specific to Antarctica. Juvenile Gastropod toxicity tests, Kingston 2007 During 2007 a series of toxicity tests, using Antarctic marine invertebrates, were conducted at Australian Antarctic Division laboratories in Kingston, Tasmania, to test the sensitivity of Antarctic nearshore biota to a range of common metal contaminants. This data record describes two such tests, using juveniles of the microgastropod Skenella paludionoides. The first test (T01) was a 14 day test (start date: 20/08/2007) using juveniles less than 7 days old. The second test (T02) was a 12 week test (start date: 10/09/2007) using juveniles of the same cohort, that were less than 28 days at the commencement of the test. A range of concentrations of three single metals (cadmium, copper and zinc) were applied as test treatments to determine this species sensitivity to these common metal contaminants. T01 included all three metals, T02 used copper and zinc only. Data are provided in the excel file: CaseyKingston0607_Microgastropod.xlsx This file includes descriptive test details, test data and measured metal concentrations of test solutions. Scanned copies of laboratory notebook and test scoresheets are provided in PDF files: - CKing_ Ecotox Kingston 0607.pdf - Kingston07-microgastropod-juv-T01.pdf
Toxicity of physically and chemically dispersed fuels to the common Antarctic amphipod, Paramoera walkeri
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This metadata record contains the results from bioassays conducted to show the response of the common Antarctic amphipod, Paramoera walkeri to contamination from combinations of Special Antarctic Blend (SAB) diesel, Marine Gas Oil (MGO) and Intermediate Fuel Oil (IFO 180), chemically dispersed with fuel dispersants Ardrox 6120 and Slickgone NS. Fuel only water accommodated fractions (WAF), chemically enhanced water accommodated fractions (CEWAF) and dispersant only treatments were prepared following the methods in Singer et al. (2000) with adaptations from Barron and Ka’aihue (2003). WAF was made using the ratio of 1: 25 (v/v), fuel to filtered seawater (FSW) following the methods of Brown et al. (in prep). Ratios for chemically dispersed treatments were 1: 100 (v/v), fuel to FSW and 1: 20 (v/v) dispersant to fuel. Dispersant only treatments were made using ratios for CEWAF, substituting the fuel component with FSW. Mixes were made in 5 L or 10 L glass aspirator bottles using a magnetic stirrer to achieve a vortex of 20-25% in the FSW before the addition of test media. The same mixing energy was used to prepare all WAFs for enhanced reproducibility and comparability of results (Barron and Ka’aihue, 2003). Mixes were stirred in darkness to prevent bacterial growth for 42 h with an additional settling time of 6 h at 0 plus or minus 1 oC. Extended stirring times were used following the recommendations determined as part of the hydrocarbon chemistry component of this project (Kotzakoulakis, unpublished data). A dilution series of four concentrations were made from the full strength aqueous phase of each mix using serial dilution. WAF test concentrations were 100%, 50%, 20% and 10% while CEWAF concentrations were 10%, 5%, 1% and 0.1%. These concentrations were chosen in order to quantify the mortality curve and allow statistical calculation of LC50 values. To facilitate comparisons of dispersant toxicity in the presence and absence of fuel, dispersant only test concentrations reflected those of CEWAF treatments. WAF was sealed in airtight glass bottles stored at 0 plus or minus 1 oC for a maximum of 3 h before use. Fresh test solutions were prepared every four days to ensure consistent water quality and replace hydrocarbons that adsorbed or evaporated into the atmosphere. Each test concentration was represented by five replicates with five FSW control beakers, with 10 P. walkeri individuals per replicate. Only healthy and active individuals were chosen with a size range of 7.9 plus or minus 0.7 mm for adults and 2.5 plus or minus 0.2 for juveniles measured from the base of the antennae to the widest part of the dorsal curve. Larger individuals and brooding females were not used to avoid unrelated deaths related to age or reproductive state (Sagar, 1980). Beakers were filled to 200 ml and were left open to allow the natural evaporation of lighter monoaromatic hydrocarbon components that would occur during a real spill. A small square of plankton mesh was placed in each jar to provide a substratum to reduce the stress of laboratory conditions and to help to stem cannibalism. Animals were not fed during experiments to avoid hydrocarbons adsorbed onto food pellets being ingested by the amphipods, thereby introducing an additional exposure pathway. Experiments ran for a total of 12 d exposure duration. Experiments were run in cold temperature-controlled cabinets maintained at a temperature of 0 plus or minus 1 oC, fluorescent lights in the cabinets were set to a light regime of 18 h light, 6 h darkness, following the methods in Brown et al. (2017) to reflect Antarctic summer environmental conditions. Lethal and sublethal observations were made at standard ecotoxicology test times of 24 h, 48 h, 96 h, 7 d, 10 d and 12 d, with an additional observation at 8 d coinciding with one of the 4-day water changes. The health status of each individual was classified on a scale of one to four; one showing no effect up to four being mortality.
Toxicity of Heavy Metals in Seawater to Juvenile Heart Urchins (Abatus spp)
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These data sets describe the toxicity of lead, coppyer, zinc and cadmium spiked seawater to the juveniles of Abatus ingens and Abatus nimrodi. Metals were tested separately over exposure periods of 96 and 240 hours. The experimental endpoint was mortality as defined by cessation of observable movement. The coding system for the data files is J(juvenile)An (Abatus nimrodi) or Ai (Abatus ingens) - Metal name_Dates of the experiment the period of the observation (96 hour or 240 hour). This work falls under the umbrella project ASAC_2201. The fields in this dataset are: Species Toxicant Date Replicate Concentration Moving pH Salinity Dissolved Oxygen