2. Materials and
Methods
2.1 Ocean Station Papa
This study is part of the first EXPORTS field campaign, which took place
in late summer 2018 (August 14 to September 9) at Ocean Station Papa
(‘Station P’). Station P is a time-series site located in the Northeast
Pacific Ocean (nominally 50°N 145°W) that has been regularly monitored
since 1949 (e.g., Tabata 1965). It is a high-nutrient and
low-chlorophyll system with a ‘muted’ spring bloom owing to persistently
low concentrations of iron (Martin and Fitzwater, 1988; Boyd et al.,
1996). During our study, Station P was characterized by a shallow mixed
layer (average: 29 \(\pm\ \)4 m), and strong vertical and weak
horizontal gradients in hydrographic properties (Siegel et al., 2021).
Mixed layer macronutrient concentrations were elevated and chlorophylla concentrations were low with a mean of 0.21 μg
L-1, which was less than typical August values
(2000–2017) for Station P (Siegel et al., 2021). This condition
resulted in an isolume depths of the 1% photosynthetically available
radiation (PAR) between 70 to 90 m (mean: 78 \(\pm\) 6 m) (Siegel et
al., 2021), which was slightly deeper than climatological conditions
(Siegel et al., 2021). The site was characterized by a highly recycled
food web in the mixed layer (Meyer et al., 2020; McNair et al., 2023),
with low POC fluxes (Buesseler et al., 2020; Estapa et al.,2021), which
were in line with previous observations performed at Station P (Charette
et al., 1999; Wong et al., 2002; Kawakami et al., 2010; Timothy et al.,
2013; Mackinson et al., 2015). An in-depth discussion of how POC fluxes
measured during EXPORTS compare to previous POC flux measurements is
provided in Buesseler et al., (2020).
The observations presented here were conducted from the R/V Roger
Revelle , which sampled ecological and biogeochemical properties
following an instrumented Lagrangian Float drugged at
~100 m depth. A detailed operational description of the
EXPORTS North Pacific field campaign can be found in Siegel et al.,
(2021).
2.2 Sample collection with the Marine Snow
Catchers
Profiles of suspended and sinking particles were collected using three
Marine Snow Catchers (MSC; Lampitt et al., 1993, OSIL, UK). The MSC is a
large volume (volume: VMSC \(=\ \)89.8 L, height:
hMSC\(\ =\) 1.5 m) water sampler with a removable base
section (~ 8 L) that enables the partitioning of
particles according to a predefined settling time (Riley et al., 2012;
Giering et al., 2016). The MSCs were deployed at two to three depths
ranging between 20 and 500 m at 13 stations (Table S1 ).
Deployment depths were chosen to include (1) just below the mixed layer
depth (20–65 m), (2) at ~100 m (Lagrangian float
depth), and (3) between 100–500 m. Depths were adjusted to match where
particle maxima were detected immediately before the MSC deployments,
either via chlorophyll fluorescence profile, or particle counts from the
Underwater Vision Profiler-5 (Picheral et al., 2010, Hydroptic, FR). MSC
casts were conducted in the afternoons on days 1, 3, and 6 of each of
the three 8-day sampling epochs, with additional two-depths casts on
days 5 of epochs 2 and 3 (Table S1 ).
A full description of the MSC and its sampling methodology has been
published by Riley at al. (2012) and Giering et al. (2016). We modified
the protocol slightly as follows: During deployment, the terminal
apertures of the top and base sections of the MSC were kept open and
were closed at the target depth through a wire-guided messenger trigger
mechanism. A tray (height: htray = 4.4 cm, area:
Atray \(=\) 0.028 m2, approximate
volume: ~ 1 L) was placed at the bottom of the MSC base
section prior to each deployment. Immediately following deployment and
retrieval, the MSC was secured in an upright position, the initial bulk
population of particles was sampled from the MSC’s central tap (time
zero, T0 ), and particles were allowed to settle
for 2 hours. We operationally defined three particle fractions,top (t ), base (b ) and tray(tr ) according to their sampling location within the MSC after
the 2-hour settling period. The top fraction was collected from
the central tap in the top section of the MSC and represents the
suspended particle pool, i.e., particles that did not sink during the 2
hours settling period. Subsequently, the water contained in the upper
part of the MSC was gently drained, and the upper part of the MSC
removed. The water in the base section overlying the tray was siphoned
off, constituting the base fraction (volume:
Vbase; 4.5–5.5 L). Water in the tray was considered as
the tray fraction (volume: Vtray 0.33–0.98 L).
The base and tray sampled volumes varied across
deployments depending on sampling efficiency (i.e., how much volume was
lost during sampling). In the laboratory, the tray was visually
investigated for the presence of marine-snow-sized particles. In two
instances (settling time experiments, see section 2.6) the trayand base fraction were combined before sampling (referred to asbottom fraction).
All three fractions (top , base , tray ) were
subsampled for various parameters (Table S1 ). Subsamples for
particulate organic carbon and nitrogen (POC and PON) analysis and for
counts and identification (particle imaging via the FlowCam) were
collected at all stations, except on August 17 when no samples were
fixed. Subsamples for the analysis of total particulate carbon (TC),
from which we obtained particulate inorganic carbon estimates (PIC) by
difference (PIC = TC - POC), biogenic and lithogenic silica (bSi and
lSi, respectively) were taken on days 3 and 5 of each epoch. Subsamples
for the determination of TEP were collected on days 1 and 5 of each
epoch, and the abiotic aggregation potential was determined on seawater
collected on day 1 of each epoch.
2.3 Particle imaging and sizing with the
FlowCam
Particle size distribution of suspended and sinking particles was
assessed using particle imaging. Subsamples (50 mL) were fixed with 37%
formalin (hexamethylenetetramine buffered) to a final concentration of
1–2% and stored in dark at 4°C until analysis. Subsamples were
analyzed using a FlowCam 8000 Series (Sieracki et al., 1998, Yokogawa
Fluid Imaging Technologies, US) in auto-image mode (particles imaged at
a pre-defined flow rate) (Table S2 ). To assess relevant
particle sizes, we first analyzed subsamples using a x40 objective
combined to a 300x1500-μm flow cell and then a x200 objective with a
50x300-μm flow cell. Note that 13 out of 30 suspended particle samples
were not analyzed with the x40 objective due to time constraints. The
minimum sizes reliably resolved by the objectives were 30 μm and 3 μm,
respectively. The maximum size was dictated by the flow cell size and
was 300 μm for the x40 objective and 50 μm for the x200 objective.Base and tray fractions were pre-filtered using a 44-μm
mesh filter to avoid clogging the 50x300-μm flow cell when using the
x200 objective.
Based on the size of the particles and the FlowCam resolution, we chose
to restrict our analysis to particles with an Equivalent Spherical
Diameter (ESD) between 4 and 128 μm. We calculated the minimum number of
particles required to estimate a robust particle size spectrum using the
empirical relationship given by Blanco et al. (1994) and assuming that a
size bin is well represented if there are at least 10 particles in it
(Álvarez et al., 2011). Thus, we imaged around 1,300 particles (i.e.,
minimum desired count and lowest number of counted particles) to cover a
size range of 4 to < 32 μm at x200 and a size range of ≥ 32 to
128 μm at x40.
Imaged particles were sized using FlowCam’s VisualSpreadsheet© software
version 4.15.1. and grouped in the five size bins based on their ESD.
The complete method settings and example images are presented inTable S2 . Particle concentrations of top , baseand tray were calculated by dividing the counted number of
particles in each size bin by the sample volume imaged. Suspended and
sinking particle abundances were calculated as explained in section 2.7.
Particle size distributions (PSDs) were estimated as differential number
size distributions N(D) (# particle mL-1μm-1) for top and tray fractions. The
slope of the PSDs was calculated as the slope of a one-degree polynomial
fit to the log10 -transformed PSD andlog10 -transformed arithmetic mean of each size
bin (μm) over the entire measured size range (polyfit function,
Matlab R2021b).
2.4 Biogeochemical analyses
Typically, 1 L of the T0 , top andbase fractions and ~ 0.1 L of the trayfraction were filtered each for analysis of POC and PON, TC, bSi and
lSi, and TEP. For the occasions when we carried out the settling time
methodological test, between 0.5–1.5 L of the top andbottom subsamples were filtered instead.
Concentrations of POC and PON were determined by filtering subsamples
onto two replicate pre-combusted (450 °C, 30 minutes) GF/F filters (25
mm, Whatmann, UK). The filters were dried at 60°C and stored at room
temperature until analysis. Filters were analyzed using a CEC 44OHA
elemental analyzer (Control Equipment, US) after treatment with 10%
HCl. Replicates were averaged (see average of the relative standard
deviations of the filter replicates in Table S3 ). Detection
limit of POC and PON ranged between 0.8–16 μg and 0.2–3.9 μg,
respectively. All POC values were above the detection limit of the
instrument. However, for PON, 24 of 237 values were below detection,
seven of which were negative and therefore substituted with zero (μg
L-1). Values were corrected to account for non-target
carbon on the filter using the average POC and PON mass (12.6 μg for
POC; 2.1 μg for PON) of 28 blanks obtained using a multiple volume (0.5,
1 and ~ 2L) regression approach (Moran et al., 1999) of
water collected with Niskin Bottles from the CTD-rosette system within
the mixed layer (campaign-wide correction). A detailed explanation of
the blank correction method and a reconciliation of all the POC and PON
measurements obtained using different methodologies during the EXPORTS
field campaign is presented by Graff et al., (in review ).
Total particulate carbon (TC) was measured following procedures similar
to POC but without prior acidification. Only one filter per fraction was
generated due to volume constraints. No measured values fell below the
instrument detection limit (1.2 to 4.5 μg). Particulate inorganic carbon
(PIC) was calculated by subtracting the uncorrected POC from TC.
Biogenic and lithogenic silica concentrations were determined by
filtering samples onto 0.6-μm pore size polycarbonate membrane filters
(47 mm diameter, Isopore, Millipore), which were dried at 60°C and
stored at room temperature until analysis. Filters were digested in
Teflon tubes by adding 4 mL of 0.2 N (normal) NaOH (95°C, 40 minutes)
and cooled immediately afterwards. The resulting solutions were
neutralized by adding 1 mL of 1 M (molar) HCl and centrifuged (10
minutes, 2500 rpms) to separate lSi from bSi. 4 mL of the solution was
diluted with 6 mL of Milli-Q water and assessed via the molybdosilicic
acid spectrophotographic method to measure bSi (Strickland and Parsons,
1968). The remaining 1 mL of solution, which was left at the bottom of
the Teflon tube together with the filter, was rinsed using Milli-Q
water, left to dry, and cooled. To extract lSi, 0.25 mL of 2.5 M (molar)
hydrofluoric acid were added. After 48 hours 9.75 mL 1 M (molar)
saturated boric acid solution were added, and filters were centrifuged.
8 mL of the resulting solution was added to 2 mL 1 M (molar) saturated
boric acid solution and assessed spectrophotometrically as
aforementioned.
Concentrations of TEP were determined colorimetrically on triplicate
samples. Subsamples were filtered onto 0.4-μm pore size polycarbonate
filters (25 mm, Whatmann, UK), stained with Alcian blue, and stored
frozen until analysis. Filter blanks, prepared by staining and rinsing
wet filters, were processed like the samples. Stained filters were
soaked for at least 2 hours in 80% sulfuric acid (Fisher Scientific;
95% w/w), and the absorption at 787 nm was measured
spectrophotometrically (Thermo Scientific GENESYS 10S UV- VIS; Passow
and Alldredge, 1995). The stained filters were compared to a standard
curve developed using Gum Xanthan (Sigma-Aldrich) and hence TEP
determinations were expressed as standardized Gum Xanthan equivalents
(GXeq) (Bittar et al., 2018). TEP determinations were considered above
detection, if the absorbance value of a sample at 787 nm was at least
twice the absorbance value of the blank at 787 nm (Passow and Alldredge,
1995). A total of 28 of 112 TEP determinations measured from the MSC
were below this detection limit. TEP associated with sinking particles
between 300 and 500 m was always at or below detection limit. In
addition to the MSC samples, we also determined water column TEP
concentration on water collected using Niskin bottles fitted to a CTD
rosette (depth range: 5–500 m; n = 153). A total of 22 of 153 TEP
measured on the water column sampled with the Niskin bottles were at or
below the detection limit. We did not remove TEP values at or below the
limit of detection but consider these observations an upper bound
estimate of the real in situ concentrations. We expressed TEP in carbon
units (TEP-C) using a conversion factor of 0.75 μg C µg
L-1/ GXeq L-1 (Engel and Passow,
2001).
Several values of the biogeochemical measurements were considerably
higher than the average concentrations at their collection depth. This
occurred for ~5% of the total number of observations
and these samples are referred to here as outliers . Outliers were
defined as observations that were at least twice the average
concentration for that depth layer. We refer to background
concentrations when these outliers were removed to assess the baseline
biogeochemical composition of suspended and sinking particles. The
nature of the outliers and the implication of their presence is
discussed in section 4.5 below.
2.5 Abiotic aggregation potential
experiments
The potential for the abiotic formation of aggregates was evaluated
using rolling tanks experiments. Four experiments were conducted using
MSC T0 (August 25 at 95 m), top (August 25
at 95 m) or base (August 17 at 55 m and August 19 at 55 and 95 m)
sample fractions. Each experiment was conducted for 36 hours in 1.1-L
rolling tanks at near in situ temperature (13°C or 4°C) in the
dark. Aggregate formation was monitored by visually checking every 6
hours for the appearance of marine snow-sized particles (ESD
> 0.5 mm).
2.6 Settling time experiments for
MSC
Methodological experiments to determine the effect of settling time on
particle fractions partitioning were conducted twice (on August 20 and
September 03, Table S1 ) from MSC deployments at 60 and 80 m
depth, respectively. Each time, all three MSCs were deployed at the same
depth, but settling times after retrieval varied: particles were allowed
to settle for 1, 2 and 4 hours after recovery of the MSCs. Subsamples oftop and bottom fractions were analyzed for POC and PON,
bSi and TEP.
2.7 Calculations of particle concentrations and
fluxes
Following Riley et al. (2012) and Giering et al. (2016), concentrations
of suspended (cNS), slow-sinking (cSS)
and fast-sinking (cFS) particles were calculated as
follows:
cNS = top concentration (2 )
cSS = (base – top concentrations)\(\times\ \)Vbase / VMSC(3 )
cFS = (tray – base concentrations)\(\times\ \) Vtray /
(Atray \(\times\ \)hMSC)
(4 )
Occasionally, after applying the corrections we obtained negative values
i.e., the top concentration exceeded the baseconcentration, or the base concentration exceeded the trayconcentration. We assigned a zero concentration to the specific
observations. The negative values could reflect a lack of sinking
particles, the presence of ascending particles and/or actively moving
zooplankton. In the analyses that follow, little differences were found
between slow- and fast-sinking particle concentrations (see section
3.2). Hence, these two sinking particle fractions are combined. In four
deployments, TEP and TC were measured only in the top andtray fractions. In these cases, sinking TEP and TC concentrations
were estimated following equation (4) but subtracting the topfraction from the tray fraction.
The standard particle sinking velocity for the MSC is assumed to be 18 m
d-1 as determined geometrically by dividing the
sinking distance (height of the MSC; hMSC =
VMSC/ AMSC = 1.5 m) by the settling time
(t = 2 hours). We assumed that the 18 m d-1 sinking
velocity estimate for sinking particles represents a lower bound
estimate because the sinking particles could have reached the bottom of
the MSC much sooner (Giering et al., 2016).
2.8 Statistical analyses
The relative uncertainties in the calculated concentrations and fluxes
were determined by using a Monte Carlo error propagation with mean
values obtained by averaging the replicate filters and the precision
estimated by calculating the average of the relative standard deviations
of the filter replicates (Table S3 ). A 1% uncertainty was
assumed for the measured values (VMSC,
Atray , hMSC,
Vbase , Vtray,
hMSC and sinking time). Final estimates of the averages
and standard deviations of particle concentrations and fluxes were then
calculated from the simulation results. Simple linear regression was
used to test (Student’s t-test, 95% confidence) for statistically
significant temporal and depth trends displayed by the biogeochemical
content of suspended, sinking particles and their partitioning to total.
The linear analyses were performed using the
function fitlm in Matlab (R2021b). The same approach was performed to
test for significant temporal trends displayed by the biogeochemical
content of suspended and sinking particles during the settling tests.
Paired t-test was used to test for differences among particle fractions.
The data presented in this study, and all the data generated during the
first EXPORTS field campaign can be found at NASA SeaBASS data
repository (https://seabass.gsfc.nasa.gov/cruise/EXPORTSNP).