Chlorophyll:

Collection and wet Lab:

Paired 1000ml samples of seawater were collected at the same site using a single Niskin bottle every half an hour, one at 2m and the other at 25-28m over a 7 hour period. A 60ml syringe was then cleaned with 60ml of the respective seawater sample under testing and the water discarded overboard. 50ml of the seawater sample was then taken from the original sample bottle and a 0.2μm filter of 25mm diameter attached to the syringe to retain the organic matter from the extracted subsample. The filter paper was then removed, transferred and sealed in a test tube containing 90% acetone to form a lysate solution. These tubes were then later analysed in the lab using a fluorometer.

Zooplankton from MTS Terramare being identified and counted under a microscope

The start of the time series, Station 13, records a higher zooplankton count (cells/m3) in the collected water samples at this time. The value decreases in the water sample collected later in the day. There is a significantly higher cell count in the shallow water samples than in the deep water samples for both time series stations.

Figure 10. Total zooplankton count (cells/m3) in water samples collected on board Terramare on 6th July 2017, for both 100 µm and 200 µm plankton nets. Station 13, sampled at 09:58:16 at 50° 05.420’ N  004° 52.379' W. Station 17, sampled at 14:33:38 at 50° 05.740’ N  004° 52.012' W. Shallow water sample, collected at a depth of 2m. Deep water sample, collected at the predicted DCM (20m at Stn 13, 28m at Stn 17).

An opposite relationship to the time series occurs with the phytoplankton cell count (cells/L) (fig.11). Where zooplankton count is the highest, at Station 13 (fig.10), the phytoplankton count is the lowest (fig.11). There is a strong relationship between the two populations retained in the same ecosystem, where the main taxa of zooplankton fed on phytoplankton (Gołdyn & Kowalczewska-Madura, 2008), causing this contrast in cell count.

Station 17 presents a higher phytoplankton count in the deep water sample than in the shallow water Niskin bottle water sample (fig.11).

Figure 11. Total phytoplankton count (cells/L) in water samples collected on board Terramare on 6th July 2017. Station 13, sampled at 09:58:16 at 50° 05.420’ N  004° 52.379' W. Station 17, sampled at 14:33:38 at 50° 05.740’ N  004° 52.012' W. Shallow water sample, collected from a Niskin Bottle at a depth of 2m. Deep water sample, collected at the predicted DCM (20m at Stn 13, 28m at Stn 17).

Lab analysis:

Chlorophyll samples taken from the previous day on the MTS Terramare using a niskin bottle were placed into a glass tube and measured for fluorescence using a 10AU-fluorometer. Samples had been stored in a refrigerator overnight to help preserve them. The glass tube was placed in the fluorometer and a black cap was placed over the top to prevent light affecting the results. The measurement was taken by emitting light into the sample and measuring the light re-emitted back. This was done for all the samples (3 replicates per site) in a random order and the fluorescence measurement was used to calculate the chlorophyll concentration. This was done by multiplying the fluorescence measurement with 6/50 - 6ml of acetone in the sample collected divided by the 50ml of seawater that was filtered into the sample using MF200 glass microfiber paper. Chlorophyll concentration could then be calculated in µg/l.

Phytoplankton:

Collection & Wet Lab:

A single Niskin Bottle was deployed into the water column using a hand winch to collect samples from different depths. The device was open at both ends to take a pure sample at the depth of interest before being sealed off at both ends by a weighted messenger from the surface. The depth at which the samples were collected were determined from the prediction of the deep chlorophyll maximum (DCM), measured by the CTD readings deployed previously at the same time stations. A surface water sample at 2 meters was consistently measured between each station, giving a water sample for both surface and depth. Maintaining the same geographical position throughout the day provided a data time series.

After triggering the water capture and returning the Niskin Bottle to deck using the hand winch, containers used to store the water samples were rinsed to avoid contamination and transferred into glass bottles containing 1ml of Lugols for preservation and staining purposes (Williams et al., 2016) to be stored in a dark cool box to be transported to the dry lab.

Lab analysis:

The top 90ml of the settled mixture was removed using a vacuum pump. The remaining 10ml of the solution was analysed, which theoretically contained all the phytoplankton cells from the 100ml sample.

A transfer pipette was used to place a 1ml into a Sedgwick Rafter cell, as a representative volume of the 100ml sample. Each square in the cell contained a volume of 1μl. For each sample, the phytoplankton contents of 100 out of the 1000 squares were identified and recorded under a 10x magnification with a 10- AU Light Microscope (Turner Designes, CA).

Coastal Plankton: Photo Guide for European Seas (Larink & Westheide, 2011) was used to ID the observed species on the cell. Multiplying the abundance of each species in the Sedgwick Rafter cell by 1000, created a value of abundance per litre.

Zooplankton:

Collection and wet Lab:

Direct samples of living zooplankton were collected using bongo plankton nets (60cm diameter) with respective filtering sizes of 100μm and 200μm, which were lowered to 2m below surface with a davit crane and towed for 10 minutes. After, the paired nets were rinsed overboard with seawater and retrieved for collection, filtering and transfer into two 1000ml plastic sample bottles with respective labelling. The samples were then fixed and preserved with 50 ml of 10% formaldehyde solution and two new sample bottles fixed, straight away, onto the nets and the procedure repeated at a new depth of 28m. This process was carried out over hourly intervals at the same site.

Lab analysis:

To analyse the 1L water sample collected for zooplankton was inverted to mix up the settled cells to a homogenous mixture. A 10ml sample was removed using a 10ml measuring cylinder. From this sample, 2ml at a time was moved into a Bogorov Chamber using a 3ml transfer pipette to clearly identify and record the present phytoplankton species for the 10ml sample.

The chamber contents were viewed under a 10x magnification with a WILD M3Z light microscope (Serial No.: GY746). Coastal Phytoplankton: Photo Guide for Northern European Seas (Kraberg et al., 2011) was used to ID the observed species on the cell.

A calculation was completed for the species abundance to present the data as abundance per m-3 of seawater.

The volume of sea water sampled in m3 (V) is calculated by:

V = Π r2 L

Where r2 is the radius of plankton net opening (m), squared and L is the towing distance (m).

From this the number of zooplankton per m3 (N) is calculated by:

               N = (n x 100)/ V

Where n is the number of zooplankton in 10ml of a 1L sample, V is the volume of seawater sampled (m3).

Figure 12. Phytoplankton count (cells/L) in water samples collected on board Terramare on 6th July 2017. Total cells count from Station 13 to Station 17, from 09:58:16 to 14:33:38. Start location of time series at 50° 05.420’ N  004° 52.379' W. Shallow water sample, collected from a Niskin Bottle at a depth of 2m.

Figure 13. Phytoplankton count (cells/L) in water samples collected on board Terramare on 6th July 2017. Total cells count from Station 13 to Station 17, from 09:58:16 to 14:33:38. Start location of time series at 50° 05.420’ N  004° 52.379' W. Deep water samples collected from a Niskin bottle at the predicted DCM (between 20 -28m ).

Disclaimer: The views and opinions expressed are those of the contributors and do not reflect the views and opinions of the University of Southampton

Gołdyn, R, Kowalczewska-Madura, K, Interactions between phytoplankton and zooplankton in the hypertrophic Swarzędzkie Lake in western Poland, J Plankton Res 2008; 30 (1): 33-42.

Kraberg, A, Bauman, M, Dürselen, C-D, 2010, Coastal Phytoplankton: Photo Guide for Northern European Seas, 2nd, München: Dr Arrow.

Williams, O, Beckett, R, & Maxwell, D, 2016, ‘Marine phytoplankton preservation with Lugol’s: a comparison of solutions’, Journal of Applied Phycology, 28, 1705.

Lanrink, O, Westheide, W, 2011, Coastal Plankton: Photo Guide for European Seas, 2nd, München: Pfeil Verlag.

The CTD on board the Terramare had a broken fluorometer, so no direct chlorophyll measurements could be taken. Because of this, turbidity was used as a proxy for chlorophyll levels, though it should be noted that turbidity in the water column is not solely due to phytoplankton and therefore chlorophyll presence, as other particulate matter and zoo plankton also cause light diffraction. There is a spike in turbidity at mid depths between 20 and 28 m depending on what station is being recorded, which corresponds to the predicted deep chlorophyll max for each station. Any increases seen after the deep chlorophyll max are likely due to raining detritus and sinking particulate matter from dead phytoplankton. There is no clear pattern between time of the sample and the turbidity levels, with turbidity increasing from station 13 to 14, then decreasing to stations 15, 16 and 17.

The phytoplankton cell count time series shows a difference in the variability of the number of phytoplankton between surface and deep, with deep levels being erratic compared to the more stable surface counts (fig. 15). Notable increases/decreases were seen in surface phytoplankton, increasing from 14,000 cell/l to 128,000 cells/l, decreasing back down to 9,000 cells/l then increasing back up to 92,000 cells/l. Surface phytoplankton numbers increase at 13:45 UTC from 22,000 to 51,000 cells/l at 14:33 UTC. This data can be looked at with other nutrient data to see how the presence of phytoplankton effects nutrient concentrations (see chemistry section).

Figure 15. Phytoplankton count (cells/L) in water samples collected on board MTS Terramare on 06.07.2017 from 09:44:31-14:33:38 UTC. Start location ```. Phytoplankton shallow, collected from a Niskin bottle at a depth of 2m. Phytoplankton deep, collected at the predicted DCM ranging between 20 – 28m.  

Figure 16. Chlorophyll concentration time series taken from MTS Terramare between 09:58-14:45 UTC on 06.07.2017 at 50° 05.420’ N 004° 52.379' W. Samples were collected via a Niskin bottle from 2 different depths, a sample from the surface (2m) and a sample from the predicted thermocline (20-28m).

The chlorophyll concentration time series shows that generally deep chlorophyll levels are higher than surface chlorophyll levels, supporting the idea that there is a deep chlorophyll maximum. There are notable increases and decreases in deep chlorophyll (0.6600 µmol/l to 1.7400 µmol/l then back to 0.3707 µmol/l) between 10:00-13:45 UTC and a peak in surface chlorophyll at 13:45 UTC (1.2880 µmol/l).

Phytoplankton and chlorophyll levels both correlate and show a similar pattern, with concentrations and cell numbers being higher at depth than at the surface apart from at station 16, but this may have just been due to water movement as data was collected over a short period of time. The nutrient analysis in the chemistry section correlates with this biological data, supporting that these numbers are an accurate representation of the location at the time. Given deep chlorophyll and phytoplankton values are higher than their surface equivalents, it can be inferred that there is evidence for a deep chlorophyll maximum occurring, although more data over a longer time scale will be needed to make conclusions.

Figure 14. Turbidity depth profile showing turbidity data taken from the CTD on Terramare. Over a Time series.

The dominant phytoplankton species in both the shallow (fig.12) and deep water samples (fig.13) during the offshore time series is Rhizosolenia setigera. The percentage abundance of  R. setigera between the two depths is similar, at 60% (fig.12) and 77.6% (fig.13). Rhizosolenia alata, another cylindrical diatom identified by spine-like features, was the next abundant species for both depths.

However the total phytoplankton cells counts per litre in the deep water sample is much greater, at 308000. The total count for shallow water sample is nearly half the count, at 185000 per litre. Presenting that the R. setigera and R. alata populations in the deep water samples throughout the time series is numerically very large.

Although the total cell count for the deep water sample is larger, the variety of species identified is greater in the shallow water samples. Asterionellopsis sp., Cosconidiscus, Cylindrotheca clasterium, Dinophysis, Guinardia flaccida, Leptocylindrus danicus, Rhizosolenia delicatule and Thalassiosira rotula were identified in the at least one of shallow water samples throughout the time series (fig.12), but lacked in the deep water sample. Whereas Eucampia sp., Karenia mikimotoi and Polykrikos were present in the deep water samples (fig.13) and not the shallow water samples.