Figure 1: Conceptual diagram describing the hypothesized
effects of changing oxygen conditions on coupled Fe-OC interactions in a
lake or reservoir. Under oxic conditions (top), complexation of Fe and
OC (both through co-precipitation and adsorption) leads to increased
concentrations of Fe-OC in sediments (increased Fe-OC protection),
though oxic conditions may also lead to increased OC respiration rates.
Under hypoxic conditions, reductive dissolution of Fe(III) in Fe-OC
complexes increases dissolved concentrations of Fe(II) and OC in the
water column while decreasing the amount of Fe-OC in sediment
(decreasing Fe-OC protection), though hypoxia may also decrease OC
respiration rates. The net effect of these processes on OC sequestration
remains unknown, motivating this study. This figure is a simplification
of complex interactions happening on a whole-ecosystem scale, and
focuses on hypothesized dominant processes operating on the time scale
of days to years.
2. Methods
2.1 Study Sites: Falling Creek
and Beaverdam Reservoirs
Falling Creek Reservoir (FCR; 37.30ºN, 79.84ºW) and Beaverdam Reservoir
(BVR; 37.31ºN, 79.81ºW) are small (FCR: 0.12 km2, 9.3
m deep; BVR: 0.39 km2, 11 m deep), eutrophic drinking
water reservoirs located in southwestern Virginia, USA (Hounshell et
al., 2021). Both reservoirs are located in forested catchments and both
are dimictic, with summer stratified periods that typically last from
May to October. BVR is located 3 km upstream of FCR and serves as the
primary inflow source for FCR. Fe levels are high in in these
reservoirs’ catchments as a result of weathering and erosion of Fe-rich
metamorphic rocks (Chapman et al., 2013; Woodward, 1932); the bedrock
underlying both reservoirs is layered pyroxene granulite (Virginia
Division of Mineral Resources, 2003). Both reservoirs have been owned
and operated for drinking water provision by the Western Virginia Water
Authority (WVWA) since their construction (FCR: 1898, BVR: 1872; Gerling
et al., 2016; Hamre et al., 2018).
A suite of variables are routinely sampled in FCR and BVR as part of a
long-term monitoring program; all data analyzed in this manuscript are
available in the Environmental Data Initiative (EDI) repository with
detailed metadata (Carey, Lewis, McClure, et al., 2022; Carey, Wander,
Howard, et al., 2022; Carey, Wander, McClure, et al., 2022; Lewis et
al., 2022; Lewis, Schreiber, et al., 2022; Schreiber et al., 2022)
2.2 Whole-ecosystem oxygenation
experiments
In 2012, FCR was equipped with a side-stream supersaturation
hypolimnetic oxygenation (HOx) system to improve water quality in the
reservoir (Gerling et al., 2014). This type of HOx system functions by
withdrawing water from the bottom of the reservoir, adding concentrated,
pressurized oxygen gas to supersaturate the water with dissolved oxygen
(DO), and then returning the oxygenated water at the same depth and
temperature. Previous work in FCR has shown that the HOx system
effectively increases DO concentrations throughout the hypolimnion
without altering temperature or decreasing thermal stability (Gerling et
al., 2014). From 2013–2019, the HOx system in FCR was operated at
variable rates, maintaining an oxygenated hypolimnion for at least part
of the summer (Carey, Thomas, et al., 2022). Conversely, oxygenation was
reduced in 2020 and 2021, maintaining primarily hypoxic conditions
throughout the summer stratified period. To assess the effects of
multiannual changes in oxygen availability on OC and Fe-OC in sediment,
we compared sediment core and sedimentation trap data from summer 2019
(which had a history of high-oxygen conditions during the preceding six
years) to summer 2021 (which followed a summer of hypoxic conditions in
2020; Figure S2). Sediment data were not collected in 2020 due to the
Covid-19 pandemic.
To assess how short-term changes in hypolimnetic DO concentrations
impact Fe-OC on a whole-ecosystem scale, we operated the HOx in FCR on a
variable schedule throughout the summer of 2019 (Carey, Thomas, et al.,
2022). Oxygen was added in approximately two-week intervals at a rate of
25 kg O2 day-1 to the whole
hypolimnion. Between oxygenation periods, we allowed the hypolimnion to
become hypoxic over periods of at least two weeks without oxygenation.
Because hypolimnetic volume varied throughout the summer (generally
decreasing throughout the summer as the thermocline deepened), the mean
concentration of oxygen added to the whole hypolimnion throughout an
oxygenation period in 2019 ranged from 0.80 mg L-1day-1 to 0.90 mg L-1day-1.
BVR does not have a HOx system and experiences seasonal hypoxia from May
through November (Hounshell et al., 2021). Consequently, BVR serves as a
reference ecosystem to analyze the effects of oxygenation in FCR.
2.2.1 Oxygen
We monitored DO concentrations throughout the full water column
approximately two times per week in FCR and one time per week in BVR
(Carey, Lewis, McClure, et al., 2022). High-resolution
(~1 cm) depth profiles were taken using a conductivity,
temperature, and depth profiler (CTD; Sea-Bird, Bellevue, Washington,
USA) equipped with a DO sensor (SBE 43; Carey, Lewis, McClure, et al.,
2022) from the reservoir’s surface to the sediments. We also measured
dissolved oxygen using a YSI ProODO DO probe when the CTD was not
available due to maintenance (YSI Inc. Yellow Springs, Ohio, USA; Carey,
Wander, McClure, et al., 2022). YSI measurements were taken at discrete
1 m depth intervals. For a comparison of YSI and CTD measurements, see
Carey et al. (2022).
2.2.2 Hypolimnetic Fe and
DOC
We collected water samples for DOC and Fe analysis at the deepest site
in each reservoir with a 4-L Van Dorn sampler (Wildlife Supply Company,
Yulee, FL, USA). Samples were collected once per week at seven depths in
FCR (0.1, 1.6, 3.8, 5.0, 6.2, 8.0, and 9.0 m), which corresponded to the
reservoir’s extraction depths, and five depths in BVR (0.1, 3.0, 6.0,
9.0, and 11.0 m). In 2019, we conducted a limited amount of additional
sampling in FCR on a second day each week, and these measurements
included DOC from 0.1, 1.6, 5.0, and 9.0 m depths.
We analyzed DOC by filtering water samples through a 0.7-µm glass fiber
filter into an acid-washed bottle, which was rinsed with the filtered
water three times before sample collection. The filtered samples were
frozen for less than six months before analysis on an OC analyzer
(Elementar Vario TOC cube, following APHA standard method 5310B;
American Public Health Association, 2018b).
We collected both total and dissolved (filtered through 0.45-µm filters)
samples for Fe. Samples were preserved in the field using trace metal
grade nitric acid and analyzed using ICP-MS (Thermo Electron X-Series,
Waltham, MA, USA) following APHA Standard Method 3125-B (American Public
Health Association, 2018a; Krueger et al., 2020; Munger et al., 2019;
Schreiber et al., 2022).
2.2.3 Fe-OC in sediments
We analyzed the concentration of Fe-OC in surficial sediments from both
FCR and BVR on multiple dates throughout the summer stratified periods
of 2019 and 2021. In 2019, sediment cores in FCR were collected
immediately before the HOx system was turned on or off, resulting in the
most oxic or hypoxic conditions during that SSS activation or
deactivation interval, respectively. Sediment cores at BVR were taken
once in the middle of summer and once approximately two weeks before
fall turnover in 2019. In 2021, sediment core samples were taken from
both reservoirs on the same dates, approximately once per month.
Additional sediment core samples were collected in March 2021, when both
reservoirs were unstratified and had oxic hypolimnia.
On each sampling date, we collected four replicate hypolimnetic sediment
cores using a K-B gravity sediment corer (Wildlife Supply Company,
Yulee, FL, USA). Cores were collected in the deepest part of each
reservoir, approximately 20 m from where water samples were taken. In
2019, each core was capped and kept on ice while transported back to the
lab, where the top 1 centimeter of sediment from each core was
immediately extruded, collected, and frozen in scintillation vials for
future analysis. In 2021, cores were extruded in the field, and the
samples were kept on ice while being transported back to the lab.
2.2.4 Sediment traps
To determine the amount of Fe-OC and total OC in samples of material
settling from the water column (i.e., not estimate deposition rates), we
deployed 19-L buckets approximately 1 m above the sediments at the
deepest point of each reservoir (8 m at FCR and 10 m at BVR). These
sediment traps were deployed from June–December 2021 and sampled every
two weeks by slowly bringing the bucket to the surface, decanting and
discarding water from the bucket, collecting up to 5 L of the remaining
water and particulate matter, and transporting this material back to the
lab on ice. Upon arriving at the lab, we allowed the particulates to
settle for approximately 5 minutes before decanting and discarding as
much water as possible and filling four 50-mL centrifuge tubes with the
remaining material. The samples were centrifuged for 10 minutes at 3100
rpm, then combined into one vial and frozen for later analysis. No
sediment traps were deployed for Fe-OC analysis in 2019.
2.3 Microcosm incubations
To isolate the effects of oxygen from other interacting factors that
affect Fe and OC on a whole-ecosystem scale, we conducted six-week
microcosm incubations using hypolimnetic sediment and water from FCR.
Incubations were conducted in 177-mL glass jars (Figure S1), after
extensive pilot testing revealed that these jars were highly effective
at maintaining hypoxic conditions when sealed and oxic conditions when
uncapped. We started the experiment with 102 microcosms split evenly
into oxic (uncapped) and hypoxic (capped) treatments. After two weeks
(similar to the 2019 whole-ecosystem HOx manipulation), we switched the
treatment of approximately half of the remaining microcosms, generating
two additional oxygen regimes: hypoxic-to-oxic and oxic-to-hypoxic.
Starting on week two, there were consequently a total of four oxygen
regimes: hypoxic, oxic, hypoxic-to-oxic, and oxic-to-hypoxic.
To set up the experiment, we collected sediment and water from the
deepest site in FCR on 30 June 2021, when the hypolimnion was hypoxic.
Water was collected from 9 m depth using a Van Dorn sampler, and
sediment was collected from the same location using an Ekman sampler.
Samples were transported on ice back to the lab, then homogenized by
stirring and shaking. We used a syringe to add the sediment slurry (20
mL) to each jar, then slowly added 150 mL of hypolimnetic water, making
an effort to minimize sediment disturbance. We stored the microcosms in
an unlit incubation chamber at 15 ºC for the duration of the experiment,
which corresponded to warm, end-of-summer conditions in the hypolimnion
of FCR (Carey, Lewis, McClure, et al., 2022).
2.3.1 Microcosm sampling
Microcosms were sampled destructively for DO, total and dissolved Fe,
total and dissolved OC, pH, sediment OC, and sediment Fe-OC. For the
continuous oxic and hypoxic treatments, we sampled 3–6 replicates two
times per week for four weeks (6 replicates: days 2, 6, 9, 13; 3
replicates: days 16, 20, 23). We added additional sampling for the
hypoxic-to-oxic and oxic-to-hypoxic treatments: these treatments were
sampled for the first three days after switching the oxygen regime (days
14, 15, 16), twice the following week (days 20, 23), and one more time a
total of four weeks from when treatments were switched (day 34), with
three replicates analyzed per sampling event. All microcosms under a
hypoxic treatment were sampled in an anaerobic chamber which maintained
mean ambient oxygen conditions <200 ppm (Coy Laboratory, Grass
Lake, MI, USA) to reduce oxygen exposure during sampling.
To begin sampling a microcosm, DO was measured using a YSI DO probe.
While measuring DO, we used the probe to gently swirl the water in the
microcosm, homogenizing the water sample while minimizing sediment
disturbance. Next, we used an acid-washed syringe to withdraw 30 mL of
water for total OC (TOC), 13 mL for total Fe, 30 mL of water for DOC,
and 13 mL for dissolved Fe analyses. DOC samples were filtered through a
0.7-µm glass fiber filter, and dissolved Fe samples were filtered
through 0.45-µm filters. After taking samples for Fe and DOC, we
withdrew as much water as possible without disturbing the sediment and
measured pH from this sample in a separate container using an Ohaus
Starter 300 pH probe (Parsippany, NJ, USA). Finally, we swirled the
sediment with remaining water (approximately 1–5 mL) and poured this
mixture into a 20 mL glass EPA vial, which we then froze for Fe-OC
analysis. Hypoxic microcosms were stored in the anaerobic chamber for
approximately two hours before analysis to ensure oxygen concentrations
in the chamber were sufficiently low before opening the jars. Oxic
microcosms were sampled immediately after removal from the incubator.
All microcosm samples were analyzed following standard methods. We
stored TOC and DOC samples in bottles that had been acid-washed and
rinsed three times with the sample water. All DOC and TOC samples were
frozen for <6 months prior to analysis on an OC analyzer
(Elementar Vario TOC cube, following Standard Method 5310B; American
Public Health Association, 2018b) Fe samples were preserved using trace
metal grade nitric acid and analyzed using the ferrozine method (Gibbs,
1979). We also analyzed Fe samples from days 16 and 23 using inductively
coupled plasma mass spectrometry (ICP-MS). All microcosm data are
published with complete metadata in the Environmental Data Initiative
repository (Lewis et al., 2022).
2.4 Fe-OC analysis
We analyzed the amount of Fe-OC in both the whole-ecosystem and
microcosm sediment samples using the citrate bicarbonate dithionite
(CBD) method. This method was first described for marine systems by
Lalonde et al. (2012) and has since been adapted for freshwater lakes by
Peter and Sobek (2018). It is important to note that our measurement of
Fe-OC as the percentage of OC that is extractable using the CBD method
is an operational definition (Fisher et al., 2021). We used this method
to enable comparisons both between oxygen treatments and with other
published work that used the same general approach (e.g., Lalonde et
al., 2012; Peter & Sobek, 2018).
Following the CBD method, each sediment sample was freeze-dried and
divided into three treatments: initial, reduction, and control.
“Initial” samples received no treatment and were used to measure the
OC content of the sediment. “Reduction” samples were treated with a
metal-complexing agent (trisodium citrate) and reducing agent (sodium
dithionite) in a buffered solution (sodium bicarbonate) to measure how
much Fe and OC were released as a result of Fe reduction. Control
samples were used to account for the release of Fe and OC in the
reduction treatment that resulted from processes other than Fe
reduction. They were treated with the same buffer (sodium bicarbonate)
and sodium chloride in the same ionic strength as the trisodium citrate
and sodium dithionite of the reduction treatment.
For both the control and reduction treatments, we measured 100 mg of
homogenized, freeze-dried sediment into 15-mL polypropylene centrifuge
tubes (Falcon Blue, Corning Inc., Corning, NY, USA). We then added 6 mL
of either control or reduction buffer solution (0.11 M sodium
bicarbonate) to each tube. The reduction buffer contained 0.27 M
trisodium citrate, while the control buffer contained 1.6 M sodium
chloride. After heating samples to 80ºC in an oven, 0.1 g sodium
dithionite was added to the reduction samples and 0.088 g sodium
chloride was added to control samples, and samples were kept at 80ºC for
an additional 15 min. Samples were centrifuged for 10 min at 3100 RPM,
and the supernatant was collected in a 50-mL centrifuge tube. This
extraction process was repeated two more times for both treatments
(Peter and Sobek, 2018). Finally, samples were rinsed three times using
artificial lake water, which was prepared by diluting Artificial Hard
Water from Marking and Dawson (1973) to 12.5% with Type I reagent grade
water.
After extraction, all sediment samples (including those in the initial
treatment) were dried and acid-fumigated for 48 hours to remove
remaining citrate and bicarbonate (Harris et al., 2001). Samples were
then run on a CN analyzer (Elementar VarioMax, Ronkonkoma, NY, USA) to
determine the amount of OC per unit mass of sediment. We adjusted
sediment mass to account for Fe loss during control and reduction
treatments. The amount of OC removed with Fe reduction (CBD-extractable
OC) was calculated as the difference between the OC content of the
control and reduction samples and expressed as a percentage of the
initial OC content of the sediment.
2.5 Data analysis
All analyses were performed in R (version 4.0.3; R core team 2020) using
packages tidyverse (Wickham et al., 2019), lubridate (Grolemund &
Wickham, 2011), ggpubr (Kassambara, 2020), egg (Auguie, 2019), rstatix
(Kassambara, 2021), akima (Akima et al., 2022), colorRamps (Keitt,
2022), rLakeAnalyzer (Winslow et al., 2019), and tseries (Trapletti et
al., 2022). All novel analysis code is archived as a Zenodo repository
(Lewis, 2022).
2.5.1 Sediment Fe-OC
characterization
We calculated summary statistics to describe iron-bound organic carbon
and total organic carbon in surficial sediment (2019 and 2021) and
settling particulate material (2021 only) across both reservoirs. We
then pooled data from both reservoirs to analyze the difference between
settling material and surficial sediments using Welch’s t-tests. Because
data were unavailable for settling material in 2019, the comparison of
settling material to surficial sediment was limited to 2021 data only.
2.5.2 Whole-ecosystem
experiments: short-term responses
We used Welch’s t-tests to assess whether sediment properties differed
between the two-week periods of HOx activation compared to HOx
deactivation during summer 2019 in FCR. Sediment time series did not
exhibit significant temporal autocorrelation, justifying this approach
(Lewis, Schreiber, et al., 2022).
To qualitatively assess whether oxygenation experiments led to
differences in water column chemistry, we overlayed plots of DOC and Fe
from the deepest sampling depth in each reservoir with dissolved oxygen
at the same depths throughout the summer stratified period of 2019 .
2.5.3 Whole-ecosystem
experiments: interannual differences
We assessed whether there were significant differences in sediment
properties among the four reservoir-years—BVR in 2019 (hypoxic), BVR
in 2021 (hypoxic), FCR in 2019 (oxic) and FCR in 2021 (hypoxic). First,
we used Levene tests to assess homogeneity of variance among
reservoir-years (Table S1). While Fe-OC (both per unit sediment and as a
percentage of sediment OC) met the ANOVA assumption of homogeneous
variance, total sediment OC did not. Consequently, we used one-way
ANOVAs and Tukey post hoc tests for Fe-OC metrics, but used Welch
one-way ANOVAs and Games-Howell post-hoc tests, both of which account
for unequal variances, for sediment OC (Tables S2 and S3).
2.5.4 Microcosm
incubations
We used one-way ANOVAs and Tukey post-hoc tests to assess whether
sediment properties differed between microcosm treatments, after testing
for homogeneity of variance using Levene tests (Table S4). For this
analysis, we used data from days 20 and 23 (pooled together because
replicates were sampled destructively), as these were the final days
when data were available for all treatments.
Speciation-solubility calculations were conducted for day 23 of the
microcosm experiments using the Spece8 module of Geochemists’ Workbench
(GWB; Aquatic Solutions LLC, Champaign, IL, USA) and the wateqf
thermodynamic database (Ball & Nordstrom, 1991). The goal of the
calculations was to assess the predicted speciation of Fe in the
presence of OC under the environmental conditions of each microcosm
treatment (following Oyewumi & Schreiber, 2017). Environmental
conditions considered in this analysis included pH, DO, temperature,
DOC, major cations (Ca, Na, K), Fe, and major anions (Cl,
SO4; bicarbonate was not measured so we calculated that
via charge balance). We assumed that DOC consisted primarily of humic
acid for the calculations.
3. Results
3.1 Fe-OC levels in surficial
hypolimnetic sediment are high and greater than in settling particulate
matter
A substantial proportion of sediment OC was associated with Fe in both
FCR and BVR. In FCR (averaged across 2019 and 2021), one gram of
surficial sediment contained a mean of 481 µmol Fe-OC (±138, 1 SD),
31±8% of the total sediment OC pool (n=30). BVR had slightly lower
Fe-OC than FCR on average, and one gram of surficial sediment contained
a mean of 418±121 µmol Fe-OC, 24±7% of the total sediment OC pool
(n=20). Total OC comprised 9±3% of sediment mass in FCR and 10±1% of
sediment mass in BVR.
Levels of Fe-OC, both as a fraction of sediment mass and as a fraction
of total sediment OC, were significantly higher in sediment core samples
than in settling material collected in hypolimnetic traps (Figure 2). In
2021, averaged across both reservoirs, one gram of the reservoir
surficial sediments contained a mean of 443±133 µmol of Fe-OC (n=28),
69% higher than settling material collected in the traps, which
contained a mean of 262±143 µmol Fe-OC (n=17; t32=-4.24,
p<0.001; Figure 2a). A mean of 24±6% of the total sediment OC
pool was bound to Fe in sediments (n=28), while only 9±4% of sediment
OC was bound to Fe in settling material (n=17;
t43=-10.44, p<0.001; Figure 2c). Total OC was
60% higher in settling material (µ=16.5±3.3) than in surficial
sediments (µ=10.3±1.6; t20=7.33, p<0.001;
Figure 2b).