General description: The associated file contains sediment pigment data from the antFOCE project 4127. Units: all pigment data in ug/g, 0 = below detection limit of HPLC. Sample collection details: At the start and end of the antFOCE experiment, four sediment core samples were taken from inside and outside each chamber or open plot by divers. The top 1 cm of the cores was then removed and placed in the dark, first at -20ºC for 2 hours, then at -80ºC until analysis at the Australian Antarctic division. Pigment analysis Frozen samples were transported under liquid N2 to a freeze drier (Dynavac, model FD-5), in pre-chilled flasks with a small amount of liquid N2 added. Custom made plumbing fitted to the freeze drier enabled samples to be purged with N2 to prevent photo-oxidation up until solvent extraction. Prior to pigment extraction five 2 g stainless steel ball bearings were added to homogenise the freeze dried sediment. The samples were bead beaten for 1 minute (Biospec products). Subsamples (~0.05 g) were immediately transferred to cryotubes with 700 µl of dimethylformamide (DMF) for two hours. Samples were kept at -80ºC and under a safe light (IFORD 902) at all times. All pigment concentrations are standardised to sediment weight. Pigments were extracted with dimethylformamide (DMF 700 µl) over a two hour period at -20ºC. Zirconia beads, and 100 µl of Apo 8 and an internal standard were added to each sub-sample. After a two hour extraction, sub-samples were bead beaten for 20 seconds and then placed in a centrifuge with filter cartridge inserts for 14 minutes at 2500 rpm at -9ºC to separate the solvent from the sediment. The supernatant was transferred into to a vial and placed in a precooled rpHPLC autosampler. The rpHPLC system used is described in Hodgson et al. (1997). Pigment detection was at 435, 470 and 665 nm for all chlorophylls and carotenoids, with spectra from 300–700 nm being collected every 0.2 seconds. Pigment identification was carried out using a combination of rpHPLC and normal phase HPLC retention times, light absorbance spectra and reference standards (see Hodgson et al., 1997). These techniques assisted in the accurate identification of pigments and their derivatives to a molecular level and enabled several pigment derivatives to be analysed. The HPLC was previously calibrated with authentic standards and protocols outlined in SCOR (1988). Data set headers: (A)Treatment: Example code 4127_SOP7_6-1-15_PlotB_R1, = prodject code_Standard Operating Procedure(SOP) used to collect samples(see antFOCE parent file)_ Date_Chamber/plot(A,B,C,D)_replicate core within Chamber/plot(1,2,3) (B) BB carot= BB caroten, type of pigment detected by HPLC. See Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more details. (C) Chl c1 = Chlorophyll derivatives see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (D) Chl c2 = Chlorophyll derivatives see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (E) Chl c3 = Chlorophyll derivative see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (F) Chla = Chlorophyll a see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (G) Ddx =Diadinoxanthin see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information (H) dtx = Diatoxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information (I) epi = Chlorophyll epimer pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (j) Fuc = Fucoxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (k) Gyro2 = Gyroxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (L) Pras = Prasanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (m) Zea = Zeaxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (n) Date = Samples taken at the start of antFOCE experiment or at the end (o) chamber = The antFOCE chamber (A,B,C,D) (p) Treatment = The associated pH level in chambers (Acidified ~7.8, Control ~8.2) (Q) Position = Samples were taken within chambers and outside chambers (outside, inside) (r) rep= Subsamples were taken within each chamber/position (R1=replicate one, R1-R4) Spatial coordinates: 66.311500 S, 110.514216 E Dates: between 1/12/2014 and 1/3/2015 Timezone:UTC+11

Synchrotron based FTIR macromolecule profiles of 5 diatom species from the AAS_4026 ocean acidification project. Data represent the peak areas for wavenumbers related to key macromolecules. For details on methods see Duncan et al. (2021) New Phytologist. Experimental design and mesocosm set up Mesocosm set up and conditions were as described previously (Deppeler et al., 2018; Hancock et al., 2018). Briefly, a near-shore, natural Antarctic microbial community was collected from an ice-free area among broken fast ice approximately 1km offshore from Davis Station, Antarctica (68° 35ʹ S, 77° 58ʹ E) on 19 November 2014. This community was incubated in 6 x 650L polyurethane tanks (mesocosms) across a gradient of fCO2 levels (343, 506, 634, 953, 1140 and 1641 μatm; denoted M1 – M6). These fCO2 levels corresponded to pH values ranging from 8.17 to 7.57. Temperature was maintained at 0.0 °C ± 0.5 °C and the mesocosms were stirred continuously by a central auger (15 r.p.m.) for gentle mixing and covered with an air-tight lid. Irradiance was initially kept low (0.8 ± 0.2 μmol photons m-2s-1), while cell physiology was left to acclimate to increasing fCO2 levels (over 5 days). When target fCO2 levels were reached in all six mesocosms, light was gradually increased (days 5-8) to 89 ± 16 μmol photons m-2s-1 on a 19 h:5 h light:dark cycle, to mimic current natural conditions. To generate the gradient in carbonate chemistry, filtered seawater saturated with CO2 was added to five of the mesocosms. Daily measurements were taken to monitor pH and dissolved inorganic carbon (DIC). For details of fCO2 manipulations, analytical procedures and calculations see Deppeler et al., (2018). Samples for physiological and macromolecular measurements in this study were taken on day 18, at the end of the incubation period (Deppeler et al., 2018). Cell volume Cell volume was determined for selected taxa from M1 and M6 via light microscopy. Cells were imaged on a calibrated microscope (Nikon Eclipse Ci-L, Japan) and length, width and height (24-77 cells per taxa) determined using ImageJ software (Schneider et al., 2012). Biovolume was then calculated according to the cell morphology and corresponding equations described by Hillebrand et al (1999). Macromolecular content by FTIR The macromolecular composition of the selected diatom taxa sampled from all six mesocosms on day 18 was determined using Synchrotron based FTIR microspectroscopy on formalin-fixed (2% v/v final concentration) cells. Measurements were made on hydrated cells and processed according to previous studies (Sackett et al. 2103; 2014; Sheehan et al. 2020). Briefly, fixed cells were loaded directly onto a micro-compression cell with a 0.3 mm thick CaF2 window. Spectral data of individual cells (between 15-49 cells per taxon per mesocosm) were collected in transmission mode, using the Infrared Microspectroscopy Beamline at the Australian Synchrotron, Melbourne, in November 2015. Spectra were acquired over the measurement range 4000− 800 cm−1 with a Vertex 80v FTIR spectrometer (Bruker Optics) in conjunction with an IR microscope (Hyperion 2000, Bruker) fitted with a mercury cadmium telluride detector cooled with liquid nitrogen. Co-added interferograms (n = 64) were collected at a wavenumber resolution of 6 cm−1s. To allow for measurements of individual cells, all measurements were made in transmission mode, using a measuring area aperture size of 5 × 5 µm. Spectral acquisition and instrument control were achieved using Opus 6.5 software (Bruker). Normalised spectra of biologically relevant regions revealed absorbance bands representative of key macromolecules were selected. Specifically, the amide II (~1540 cm-1), Free Amino Acid (~1452 cm-1), Carboxylates (~1375 cm-1), Ester carbonyl from lipids (~1745 cm-1) and Saturated Fatty Acids (~2920 cm-1) bands were selected. Infra-red spectral data were analysed using custom made scripts in R (R Development Core Team 2018). The regions of 3050-2800, 1770-1100 cm-1, which contain the major biological were selected for analysis. Spectral data were smoothed (4 pts either side) and second derivative (3rd order polynomial) transformed using the Savitzky-Golay algorithm from the prospectr package in R (Stevens and Ramirez-Lopez, 2014) and then normalised using the method of Single Normal Variate (SNV). Macromolecular content for individual taxon was estimated based on integrating the area under each assigned peak, providing metabolite content according to the Beer-Lambert Law, which assumes a direct relationship between absorbance and relative analyte concentration (Wagner et al., 2010). Integrated peak areas provide relative changes in macromolecular content between samples. Because of the differences in absorption properties of macromolecules, peak areas can only be used as relative measure within compounds.

Long-term experiment on increased CO2 level on krill physiology. Krill were exposed to a range of CO2 conditions 400-4000ppm over a year, and their growth, mortality, and physiology were monitored. -List of files- Ericson Krill Ocean Acidification Study Raw Data_for data centre.xlsx: This file contains data on krill growth, mortality, physiology, and biochemistry, as well as information on water chemistry throughout 1 year period of the experiment. Ericson et al. Adult krill OA MS final submission.pdf: Unpublished manuscript of the experiment including all methods of the experiment.

Refer to antFOCE report section 4.5.1 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 The download file contains an Excel workbook with one data spreadsheet and one of notes relevant to the data. The data are the total number of each motile organism collected from 2 recruitment tiles deployed in chambers or open plots during the antFOCE experiment. The 2 tiles were deployed together in a metal stand in either a horizontal or vertical orientation. Background The antFOCE experimental system was deployed in O’Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – “antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis”. This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

Refer to antFOCE report section 4.5.1 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 The download file contains an Excel workbook with one data spreadsheet and one of notes relevant to the data. The data are the total number of each sessile organism collected per tile as per the census methods detailed in the Notes spreadsheet. Tiles were deployed in chambers or open plots during the antFOCE experiment on a metal stand in either a horizontal or vertical orientation. Background The antFOCE experimental system was deployed in O’Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – “antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis”. This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

Marine sediments often represent an important reservoir of carbonate minerals that will react rapidly to changing seawater chemistry as a result of ocean acidification. Ocean acidification (the reaction of CO2 with seawater) lowers the saturation state with respect to carbonate minerals and may lead to dissolution of these minerals if undersaturation occurs. There are three main carbonate minerals found in marine sediments: 1. aragonite 2. calcite (also referred to as low-magnesium calcite, containing less than 4mol% MgCO3) 3. high-magnesium calcite (greater than 4 mol% MgCO3) Due to the different structure of these minerals, they have different solubilities with high-Mg calcite the most soluble, followed by aragonite and then calcite. As seawater CO2 increases and the saturation state with respect to carbonate minerals decreases, high-Mg calcite will be the first mineral subject to undersaturation and dissolution. By measuring the carbonate mineral composition of sediments, we can determine which areas are most at risk from dissolution. This information forms an important baseline with which we can assess future climate change. The effect of ocean acidification on carbonates in marine sediments will occur around the world, but due to the lower seawater temperatures in Antarctica, solubility is much lower so the impacts will occur here first. This dataset is a compilation of carbonate mineralogy data from surface sediments collected from the East Antarctic margin. The dataset includes sample metadata, bulk carbonate content, %calcite, % aragonite and mol% MgCO3 (i.e. the magnesium content of high-Mg calcite). This dataset was compiled from new (up to 2020) and archived sediment samples that contacted sufficient carbonates (typically greater than 3% CaCO3)/

Refer to antFOCE report section 4.4.5 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 One camera and flash unit was mounted on the top middle section of each chamber to take one photo of the sediment every 30 minutes. The download file contains two folders with the photos taken from the 28th of January to the 23rd of February 2015 – one for Chamber A and one for Chamber C. A video time-lapse compilation of the Chamber A images is also included. Malfunctioning cameras deployed on Chamber A and C and on B and D during this same period and at other times, meant that no useful images were obtained. Background The antFOCE experimental system was deployed in O'Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – "antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis". This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

Carbonate chemistry data sets for the Antarctic Free Ocean Carbon Dioxide Enrichment experiment, Casey Station, East Antarctica, 2014/15. Project Summary: Currently, a quarter of the CO2 we emit is absorbed by the ocean. CO2 absorption in seawater changes its chemistry – reducing ocean pH (raising its acidity) – which has significant impacts on biological processes and serious implications for the resilience of marine ecosystems. As CO2 is more soluble in cold water we expect polar ecosystems to bear the heaviest burden of this 'ocean acidification'. We will perform the first in situ polar CO2 enrichment experiment to determine the likely impacts of ocean acidification on Southern Ocean sea-floor communities under increasing CO2 emissions.

Metadata record AAS_4127_antFOCE_HardSubstrateFauna contains all data sets relating to the fauna sampled from hard substrates during the antFOCE experiment, including recruitment tiles, artificial substrate units and biofilm slides. Refer to antFOCE report section 4.5 for deployment, sampling and on-station analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 Background The antFOCE experimental system was deployed in O’Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – “antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis”. This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

Carbonate chemistry data for the antFOCE seawater samples. The download file contains an Excel spreadsheet with a number of worksheets detailing the samples collected from O'Brien Bay, Casey Station. The dataset includes information on oxygen levels, pH levels, temperature and salinity levels, as well as the concentrations of various elements (dissolved inorganic carbon, phosphate, nitrate, nitrite, silicate). Free-ocean CO2 enrichment (FOCE) experiments have been deployed in marine ecosystems to manipulate carbonate system conditions to those predicted in future oceans. We investigated whether the pH/carbonate chemistry of extremely cold polar waters can be manipulated in an ecologically relevant way, to represent conditions under future atmospheric CO2 levels, in an in-situ FOCE experiment in Antarctica. We examined spatial and temporal variation in local ambient carbonate chemistry at hourly intervals at two sites between December and February and compared these with experimental conditions. We successfully maintained a mean pH offset in acidified benthic chambers of -0.38 (plus or minus 0.07) from ambient for approximately 8 weeks. Local diel and seasonal fluctuations in ambient pH were duplicated in the FOCE system. Large temporal variability in acidified chambers resulted from system stoppages. The mean pH, Ωarag and fCO2 values in the acidified chambers were 7.688 plus or minus 0.079, 0.62 plus or minus 0.13 and 912 plus or minus 150 micro-atm respectively. Variation in ambient pH appeared to be mainly driven by salinity and biological production and ranged from 8.019 to 8.192 with significant spatio-temporal variation. This experiment demonstrates the utility of FOCE systems to create conditions expected in future oceans that represent ecologically relevant variation, even under polar conditions.