Sampling the Sea Surface Microlayer (SML) and Sub-Surface Water

A total of 1479 samples were collected from 120 stations: 67 on AMT24 and 53 on AMT25 (Table A.2). The surface microlayer (SML) was sampled first, in triplicate twice daily, during pre-dawn (05.00 - 06.00 GMT) and solar noon (13.00 - 14.00 GMT) hydrocasts. A custom-made Garrett screen (Garrett, 1965) (width:60 cm, breadth: 60 cm, mesh:16, wire diameter:

0.36 mm, effective surface area: 2025 cm²) was used, following a standard protocol (Cunliffe and Wurl, 2014).

Sampling took place from the deck of the ship, over the bow. The approximate deployment position was 8.3 metres above the sea surface. Sampling was only carried out when the ship was stationary, as recommended by Cunliffe and Wurl (2014), and following a visual inspection to establish that there were no ship-derived effluents that would contaminate the samples. No effluent was discharged from the ship 1 hour before arriving at the next sampling station so as not to disturb the surface and contaminate samples. This procedure was developed and evaluated by Salter et al. (2011) and used successfully in an investigation of air-sea gas exchange mediated by an artificial surfactant slick.

The need to sample from the ship’s deck was dictated by cruise logistics and is the principal reason why the Garret Screen (Figure 2.5 A) was selected over alternative samplers such as the glass plate (see section 1.1.2), which cannot be deployed in this way. The Garret Screen has a number of other advantages, including ease of cleaning with 10% HCl or by baking at 450^oC for 6 hours, and the ability to accumulate relatively large sample volumes rapidly (15 ml/dip).

The screen was deployed horizontally, pre-rinsed in seawater and submerged just under the water surface for 5 seconds before being lifted back up through the SML. Based on multiple screen deployments, the Garrett screen used typically samples the uppermost 65-80 µm of the

sea surface (Pereira et al., 2016). However, this may vary depending on both the oceanographic and meteorological conditions at the time of sampling (Cunliffe and Wurl, 2014).

The adhering small rectangular cells of seawater samples captured in the spaces of the wire mesh by means of surface tension were drained along one of the screen corners into pre-rinsed 15 ml high-density polyethylene (HDPE) tubes (Schneider-Zapp et al., 2014) (Figure 2.5 B) and stored in a cool box to minimize the possibility of photochemical degradation and/or biological modification prior to analysis. The HDPE tubes were previously shown to be inert to surfactant activity (Schneider-Zapp et al., 2014; Pereira et al., 2016).

(A)

(B)

Figure 2.5. Garrett screen orientation during sampling on board RRS James Clark Ross: (A) Garrett Screen at the sea surface during AMT24: (B) Method of draining sample water from the Garrett Screen.

At high sea states the screen was deployed within wave troughs and in the case of any splash back, the samples were excluded.

Sub-Surface water (SSW) samples were routinely collected simultaneously via a hull-mounted

“non-toxic” seawater inlet 7 m below the waterline and during each 500 m hydrocast (a total of 357 “non-toxic” SSW samples).

To investigate the vertical profile of CDOM in more detail (see section 3.6), at 14 stations on AMT24 and 13 stations on AMT25, 9 samples were collected: at the 5 selected light penetration depths (see section 2.4), at the base of the mixed layer, at the deep chlorophyll maximum (DCM) and at the oxygen (O2) maximum and minimum depths (as indicated by an oxygen sensor on the CTD rosette). Sample allocations for the analysis of surfactant activity (SA) and CDOM absorbance (see sections 2.5.3 and 2.5.6) are shown in Figure 2.6.

Figure 2.6. Sample collection and sample processing protocols for surfactant and CDOM absorbance on AMT24 and AMT25

In general, 7 samples were collected from each cast including 5 samples from various selected light penetration depths (97%; 55%; 33%, 14% and 1%). Samples were collected from the Niskin bottles using acid-washed silicon tubing that was pre-rinsed with excess sample.

Samples for SA determination were unfiltered because filtration can modify SA by removing insoluble and most absorbable compounds and/or releasing intracellular matter due to cell rupture (Ẑutić et al., 1981; Ćosović et al., 1985; Williams, 1986; Ćosović and Vojvodić, 1998;

Schneider-Zapp et al., 2014; Pereira et al., 2016; Sabbaghzadeh et al., 2017). Reduction in total

SA by ~13% in seawater samples (i.e. north Adriatic Sea) and ~70% in estuarine samples following filtration was reported previously (Ćosović and Vojvodić, 1998).

All samples for SA determination were analysed immediately following sample collection.

Typically there was a maximum of 3 hours between sample collection and the final analysis for each cast. Samples were kept in the dark in a cool box after Gašparović et al. (2011), Salter et al. (2011) and Pereira et al. (2016). Schneider-Zapp et al. (2013) showed that untreated SA samples can be stored at 4°C in the dark for up to two weeks with little overall change.

Samples for CDOM determination were collected in 250 ml borosilicate volumetric flasks (analytical grade glassware, SCHOTT, GL 45, 250 ml, Fisher brand) pre-rinsed with excess sample. All glassware was pre-combusted at 450°C for 8 hours to remove any accumulated organic matter (Stubbins et al., 2006). To minimize contamination during filtration, syringes and all containers were pre-rinsed with pure laboratory water (18.2 Ohm Milli-Q, Millipore System Inc., USA), hereinafter referred to as MilliQ, and a small aliquot of sample (~ 3 ml) prior to use. This procedure was repeated prior to each subsequent filtration. Powder free disposable gloves were worn during all sampling and sample processing.

After sample collection, sub-samples (20 ml) were transferred to a ground glass syringe (20 ml, SAMCO) and filtered (10 ml min^-1) through 0.22 µm surfactant-free single use Millipore syringe filters (MILLEX GP, Millipore, PES membrane).

CDOM samples were filtered based on previous protocols (Nelson et al., 1998; Helms et al., 2008; Nelson and Siegel, 2013). Unfiltered samples include particulate components such as picophytoplankton, bacteria, other organics and mineral grains, in addition to dissolved species that contribute to light attenuation and hence modify sample absorbance characteristics (Michaels and Knap, 1996; Nelson and Siegel, 2013). Particulate absorbance tends to become dominant at wavelengths > 360 nm, which importantly affects the calculation of spectral slope due to changed absorption characteristics (Stubbins et al., 2006). For example, Nelson et al.

(1998) reported 50% of total particulate absorption at 440 nm by detritus.