Sampling and analysis
Lateral subsurface flow begins mid to late April at the start of
snowmelt, increases to the peak in early to mid-May, then declines and
halts by mid to late June (Fig 1a, Troendle & Reuss, 1997). We
collected hillslope export samples as lateral subsurface flow at the
base of each hillslope (i.e., at the piped trenches) weekly in 2016-2018
and analyzed for nutrients, dissolved organic carbon (DOC), total
dissolved nitrogen (TDN), and DOM chemical characteristics. These
samples were collected in pre-combusted (heated for 3 hours at 500 ⁰C)
amber glass bottles then filtered through 0.7 µm pore-size glass fiber
filters (Millipore Corp, Burlington, MA) within 24 hours of collection
and analyzed for DOC, TDN, and DOM fluorescence spectroscopy. We
collected DOM reactivity samples in 250 mL pre-combusted amber glass
bottles, then filtered through (GF/C Whatman®, 1.2 µm effective pore
size) filters within 3 hours of collection to remove bacterial grazers.
DOC and TDN were determined using a Shimadzu TOC-VCPNtotal organic carbon analyzer, with 2M HCl addition before analysis to
remove mineral C (Shimadzu Corporation Columbia, MD). Detection limits
for DOC and TDN were 50 µg L-1.
The chemical character of DOM exported from each hillslope (i.e.,
lateral subsurface flow) was analyzed using UV-Visible fluorescence
spectroscopy on a Horiba Scientific Aqualog (Horiba-Jobin Yvone
Scientific Edison, New Jersey, US). Sample C concentration was
standardized to 5 mg C L-1 using deionized water
(>18 mΩ). Ultraviolet absorbance was analyzed at the 254 nm
wavelength. Excitation emission matrices (EEMs) fluorescence scans were
completed from 240 nm-600 nm excitation and emission wavelengths, with 3
nm band-pass, 3 nm increments, and 3 second integrations. Scans were
blank corrected using deionized water and corrected for inner filter
effects (Kubista, Sjoback, Eriksson, & Albinsson, 1994). First- and
second-order Rayleigh scattering were masked (10 nm width masking), and
samples were normalized by the area of the deionized water Raman
scattering peak (Lawaetz & Stedmon, 2009).
SUVA254 (L mg C-1m-1) was calculated by dividing ultraviolet absorbance
at 254 nm by DOC concentration (Weishaar et al., 2003). Humification
index (HIX) was calculated as the area of spectra collected at 254 nm
emission under 435-480 nm excitation divided by the area under the peak
300-345 nm emission (Zsolnay, Baigar, Jimenez, Steinweg, & Saccomandi,
1999). Fluorescence Index (FI) was calculated as the ratio between
emission at 470 nm and 520 nm at 370 nm excitation (McKnight et al.,
2001; Cory & McKnight, 2005). Fluorescence regional integration
modelling (FRI) was applied to EEMs data to account for areas of
increased fluorescence not identified by standard EEMs indices (Chen,
Westerhoff, Leenheer, & Booksh, 2003). Briefly, EEMs scans were
separated into five regions associated with distinct DOM chemical
components. Total fluorescence intensity for each region was divided by
total intensity of the entire scan, resulting in percentages of total
FDOM derived from each DOM chemical component region.
We sampled the organic (O horizon) and mineral (A horizon; 10 cm depth)
soil layers at ten locations distributed across the old-growth and
second-growth hillslopes (n=20). O horizon material was sampled within a
30 cm by 30 cm quadrat and total O-horizon depth was measured in each
quadrat corner. O horizons were separated into litter and duff layers as
follows (FIA 2019): needles and recognizable plant material (<
6 mm diameter) were classified as litter, while unrecognizable,
fragmented material between the litter and mineral soil layers was
considered duff. There was no measurable duff in the second-growth
forest. Mineral soil (0-10 cm depth) was collected using a 7.5 cm
diameter corer after the O horizon was removed. Mineral soils had rocks,
mosses, and lichens removed and were then sieved to 2 mm. We determined
gravimetric soil moisture after drying a subsample at 105 ⁰C for 48
hours. Organic horizon samples were well mixed and moss, lichen, and
rocks were removed by hand. Organic horizon samples were dried for 6
days at 60 ⁰C and the total mass was recorded. Total bulk density for
mineral and organic soils was calculated by dividing the dry weight of
the total soil (including > 2 mm fraction in mineral soils)
by the sample volume. A subset of each sample was dried for 48 hours at
60 ⁰C, then ground and analyzed for total C and N by dry combustion
(LECO 1000 CHN analyzer, LECO Corporation, St. Joseph, MI, USA). Total C
and N pools were calculated by multiplying C and N concentrations by the
associated O- and A-horizon masses. Water extractable soil organic
matter (WEOM) samples were created by leaching a litter layer sample
from each hillslope’s O-horizon to replicate DOM inputs (Sparling,
Vojvodic-Vukovic, & Schipper, 1998). Litter samples were air dried for
3 days then 10 g subsamples were steeped in 50 mL of DI
(>18 mΩ) at 70 ⁰C for 18 hours, shaken and filtered through
0.7 µm pore size glass fiber filters.
Biological oxygen demand assays were performed to assess the reactivity
of DOM inputs (WEOM litter leachates) and DOM exports (lateral
subsurface flow). DOM input incubations were diluted and standardized to
30 mg C L-1 to mirror C concentrations of DOM export
incubations. Each DOM input sample was inoculated with 88 mL of water
from an adjacent stream (Sparling et al. 1998), and export DOM was
incubated with microbes that occurred naturally in the subsurface flow.
Relationships in reactivity between input and output DOM should be
considered tentative correlations, as different microbial cultures were
used in litter and subsurface leachate incubations. Experimental
controls, which only contained the stream water microbial culture with
no additional DOM and analytical controls which only contained DI
(>18 mΩ) were incubated simultaneously with the oxygen
demand assays. Microbial dissolved oxygen consumption (mg
O2 L-1) was continuously measured (15
second intervals) using Oxy-4 probes (PreSens, Precision Sensing GmbH,
Regensburg, Germany). Input and export samples were incubated in the
dark at 20 ⁰C and the hourly oxygen consumption rate (mg
O2 L-1 hr-1) was
averaged for each sample over the 24-hour incubation period. No samples
approached hypoxic conditions (<4 mg O2L-1). Oxygen consumption rates were normalized per
gram of C to remove any bias of C quantity on consumption rate.
Post-incubation samples were filtered to remove microbial biomass
(Nucleopore®, polycarbonate filter, 0.2 µm). Concentration of C
mineralized (Δ mg C L-1) was calculated as the
difference between measured pre- and post- incubation DOC
concentrations, then converted to loss as CO2(ΔCO2, in mg CO2 L-1),
under the assumption all CO2 produced by incubations was
removed from the sample with HCl addition during C measurement. The
respiratory quotient (RQ), was calculated by dividing C lost to
respiration (ΔCO2, in mg CO2L-1) by the change in O during the incubation
(ΔO2 in mg O2 L-1).
DOC and TDN concentrations, and FDOM indices in subsurface flow exports
were compared between old-growth and second-growth land cover types
using Welch-Satterthwaite unequal variance t-test with significance
assigned at p < 0.01 (R Core Team, 2019). Regional percentages
for FDOM components (FRI modelling) in export samples were logit
transformed before statistical analyses to remove biases inherent in
proportional data. We compared input nutrient concentrations and pools
in organic and mineral horizon soils, along with comparisons of oxygen
consumption and respiratory quotients in DOM input and export samples
using the Student’s t-tests for parametric data, with significance
assigned at p < 0.01 (t.test function, R Core Team, 2019). The
data that support the findings of this study are available from the
corresponding author upon reasonable request.