Methodology:

4 cruises: JR16006 / HH180101 / HH230418 / JR17006

Seawater was collected from six depths from a standard environmental CTD cast as close to midday as possible. The CTD was positioned in full sunlight and not in the shadow of the ship. Sampling depths were selected based on the PAR irradiance readings from the CTD at the surface of the water (approx. 2m) after being initially stabilised at 10m and bought back to the surface. Set percentages of light 100%, 50%, 25%,15%,3% and 1% were calculated from the surface PAR and the depths chosen accordingly. For phytoplankton samples three depths were selected - Surface, Chlorophyll maximum and deep (exception was on HH180101 where only surface waters were taken due to it being wintertime). 10L acid washed carboys and acid washed tubing with 200 µm mesh to pre-screen the water were used to collect the water samples. The carboys were stored in black bags either in the cold room or on deck in a low light area. Location of the collected water for storage until processing was dependent on the temperature of the surface water at the point of collection. 4mls of Lugol's solution, 1% (final concentration) was dispensed into 500ml glass amber bottles. 400ml of water sample was added, and samples stored in the cold room at 4-5 °C.

Laboratory analysis:

Before observation through a light microscope, samples fixed with 1% Lugol's iodine were settled using a Hydro-Bios glass plate chamber and 50ml tower. The inverted samples were poured into the tower, so it was just over full. A glass coverslip was placed over the top of the tower, ensuring there were no bubbles in the column. Vacuum pressure holds the chamber set up together. The base plate and tower were then carefully lifted into a tray and left to settle for a minimum of 20 hours.

After adequate settling time, the chamber was removed from the tray and distilled water was dropped either side of the tower base. The tower was removed by gently pushing it across the base plate, followed by a square coverslip, ensuring there was no gap to minimise bubbles. The baseplate and tower were then lifted carefully, and the remaining contents were emptied into a waste beaker. The base plate was dried, ready for microscopy. The waste beaker, tower and tower cover slip were washed in a sink with hot water.

The prepared base plates were transferred to an inverted light microscope, the light source was set to a preferred intensity and viewed at a x20 objective, using Phase2 contrast conditions.

Where cells were in high abundance, a minimum of 100 cells were counted for statistical reasons, which was achieved by determining a set number of fields of view. Where cells are rare or in low abundance, the whole of the chamber was counted.

A calibrated eyepiece graticule was used to size phytoplankton cells from the chlorophyll maximum samples only. Imaging software confirmed eyepiece sizing on a select number of cells. Conversion of cell sizes to biomass volumes was achieved using geometric calculations - specific for each cell type. Calculations take into consideration the shrinkage effect of the fixative on the cells to give a live volume. From this live volume C:N ratios were calculated.

Cell enumeration results for each species at each station are provided as - cell numbers/litre.

Cell sizes for biomass calculations are provided as - Length in µm, Breadth in µm.

Biomass volumes are provided as - pgC per cell and Total µgC for each station.

Data collection:

Standard CTD and associated software used to determine ocean parameters and define collection depths on board the ship.

Sample analysis conducted on a Zeiss S100 inverted microscope by Elaine Mitchell. Axio Imager software version 4.1 was used to take images and confirm eyepiece graticule sizing.

Data from microscope analysis was entered into Comma Separated Value (csv) files for cell enumerations, biomass and C:N ratio calculations.

Data quality:

Sizing of cells for biomass calculations were done using an eye piece graticule on the microscope. A small number of images were taken and the manual eyepiece measurements were compared to the measurements taken using the Axio imager software and in each case they were almost the same if you rounded up the imaging sizes to the nearest micron.