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Labile dissolved organic carbon supply limits
hyporheic denitrification
Jay P. Zarnetske,
1,2
Roy Haggerty,
3
Steven M. Wondzell,
4
and Michelle A. Baker
5
Received 4 April 2011; revised 11 October 2011; accepted 22 October 2011; published 31 December 2011.
[1]We used an in situ steady state
15
N-labeled nitrate (
15
NO
3
) and acetate (AcO
)
well-to-wells injection experiment to determine how the availability of labile dissolved
organic carbon (DOC) as AcO
influences microbial denitrification in the hyporheic zone
of an upland (third-order) agricultural stream. The experimental wells receiving
conservative (Cl
and Br) and reactive (
15
NO
3
) solute tracers had hyporheic median
residence times of 7.0 to 13.1 h, nominal flowpath lengths of 0.7 to 3.7 m, and hypoxic
conditions (<1.5 mg O
2
L
1
). All receiving wells demonstrated
15
N
2
production during
ambient conditions, indicating that the hyporheic zone was an environment with active
denitrification. The subsequent addition of AcO
stimulated more denitrification as
evidenced by significant d
15
N
2
increases by factors of 2.7 to 26.1 in receiving wells and
significant decreases of NO
3
and DO in the two wells most hydrologically connected to the
injection. The rate of nitrate removal in the hyporheic zone increased from 218 kg ha
1
yr
1
to 521 kg ha
1
yr
1
under elevated AcO
conditions. In all receiving wells, increases
of bromide and
15
N
2
occurred without concurrent increases in AcO
, indicating that
100% of AcO
was retained or lost in the hyporheic zone. These results support the
hypothesis that denitrification in anaerobic portions of the hyporheic zone is limited by
labile DOC supply.
Citation: Zarnetske, J. P., R. Haggerty, S. M. Wondzell, and M. A. Baker (2011), Labile dissolved organic carbon supply limits
hyporheic denitrification, J. Geophys. Res.,116, G04036, doi:10.1029/2011JG001730.
1. Introduction
[2] There are many environments where denitrification
occurs, but studies have shown that stream systems are par-
ticularly efficient at removing and retaining excess nitrogen
(N) [Seitzinger et al., 2006] with headwater and midnetwork
streams being the most effective in regulation of downstream
N exports [Peterson et al., 2001; Alexander et al., 2000;
Mulholland et al., 2008]. Characteristic of these headwater
and midnetwork streams is the prominence of stream water–
groundwater (hyporheic) exchange flux relative to surface
water flux [Anderson et al., 2005], especially during periods
of low discharge [Wondzell, 2011]. Hyporheic exchange is
also known to strongly influence N transformations and
cycling in streams by increasing solute residence times and
solute contact with reactive biofilms [Duff and Triska, 1990;
Holmes et al., 1994; Jones, 1995; Wondzell and Swanson,
1996; Valett et al., 1996; Hedin et al., 1998; Hill et al.,
1998]. Therefore, it follows that hyporheic exchange can
exert a primary hydrologic control on the export of N
from small to midnetwork watersheds.
[3] A key factor in the fate of N traveling through a
hyporheic zone (Figure 1) is the organic carbon (C) conditions
(substrate quality and quantity) in the stream and hyporheic
zone [Baker et al.,1999;Kaplan and Newbold, 2000; Sobczak
and Findlay, 2002]. In streams, the predominant form of C
is dissolved organic carbon (DOC) [Fisher and Likens,
1973] and only a fraction of that DOC is readily labile
(i.e., bioavailable) [Swank and Caskey, 1982]. For hyporheic
systems, either surface or groundwaters enriched with DOC
are advected into hyporheic systems and fuel aerobic and
anaerobic hyporheic metabolism [Findlay, 1995; Jones,
1995; Baker et al., 2000].
[4] As reviewed by Duff and Triska [2000, and references
therein], the occurrence of denitrification in the hyporheic
zone is complex and is not just related to DOC and NO
3
availability. Denitrification along hyporheic flowpaths is also
a function of (1) the concentration of DO across the hyporheic
zone which is controlled by biochemical oxygen demand and
advected supply; (2) hyporheic water temperature because it
controls microbial activity and DO saturation in water; and
(3) the hydraulics that drive the physical transport and resi-
dence time of water such as the head gradient, hydraulic
conductivity, advection, and dispersion. Of these factors,
1
Department of Geosciences and Water Resources Graduate Program,
Oregon State University, Corvallis, Oregon, USA.
2
Now at School of Forestry and Environmental Studies, Yale University,
New Haven, Connecticut, USA.
3
Department of Geosciences and Institute for Water and Watersheds,
Oregon State University, Corvallis, Oregon, USA.
4
Pacific Northwest Research Station, Olympia Forestry Sciences
Laboratory, Olympia, Washington, USA.
5
Department of Biology and the Ecology Center, Utah State University,
Logan, Utah, USA.
Copyright 2011 by the American Geophysical Union.
0148-0227/11/2011JG001730
JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 116, G04036, doi:10.1029/2011JG001730, 2011
G04036 1of13
the biogeochemical conditions controlling hyporheic deni-
trification primarily vary by the amount of NO
3
and quality
and quantity of DOC present in the system [Findlay, 1995;
Kaplan and Newbold, 2000]. If both NO
3
and labile DOC
are abundant then denitrification rates can be large [e.g.,
Holmes et al., 1996, Storey et al., 2004]. Spatial patterning of
DOC availability along hyporheic flowpaths adds further to
the complexity. DOC and DO are expected to decline at the
head of the hyporheic flowpaths as microbial processes
preferentially utilize DO and labile fractions of DOC [Vervier
and Naiman, 1992; Sobczak et al., 2003; Zarnetske et al.,
2011]. Further along the hyporheic flowpaths the labile DOC
availability can become depleted leading to DOC quality
limitations on denitrification [Sobczak et al., 2003; Zarnetske
et al., 2011]. Also, hyporheic DOC sources from stream and
groundwater vary with discharge and season [Vervier and
Naiman, 1992; Baker and Vervier, 2004] which, in turn,
vary hyporheic metabolism and denitrification.
[5] The use of stable isotope (
15
N) tracers has greatly
advanced understanding of aquatic N cycling by allowing the
tracing of N through ecosystem pathways experiencing dif-
ferent physical and biological conditions. For example,
Böhlke et al. [2004] and Mulholland et al. [2004, 2008]
demonstrated the usefulness of the
15
N tracer approach for
estimating denitrification rates of streams at the reach scale.
More recently, subsurface
15
N tracing has been successfully
used to study hyporheic processes associated with reactive
N transport [Clilverd et al., 2008; Böhlke et al., 2009;
Zarnetske et al., 2011]. Direct hyporheic
15
N addition enables
the distinction between microbial assimilation and retention
pathways and the respiratory denitrification pathway (i.e.,
microbial N
2
production), which was not possible in previous
hyporheic coupled C-N studies that relied on C and N mass
balance approaches [e.g., Hedin et al., 1998; Baker et al.,
1999; Sobczak et al., 2003]. On the other hand, previous
studies have directly measured hyporheic denitrification (via
acetylene block, N
2
:Ar ratios, or
15
NO
3
additions) across
spatial and temporal ranges of DOC and NO
3
conditions and
found that a positive correlation exists between the quantity
of labile DOC and N
2
O production [Baker and Vervier, 2004;
Smith et al., 2006; Arango et al., 2007; Böhlke et al., 2009].
Though the previous studies on coupled hyporheic DOC and
N dynamics consistently indicate that the type and quality of
DOC in a hyporheic system influences denitrification rates,
none have both isolated the role of labile DOC supply on
denitrification and made direct measurements in a hyporheic
environment.
[6] We evaluate the role of labile DOC on denitrification
along a redox gradient that forms along hyporheic flowpaths
in a gravel bar. We combined
15
NO
3
tracing techniques with
an addition of labile DOC to examine the role of DOC in
controlling denitrification. The previous work at this study
site demonstrated that denitrification and DOC dynamics
were closely coupled [Zarnetske et al., 2011]. Under base
flow conditions, DOC persisted across all residence times,
but denitrification rates and DOC uptake decreased beyond
threshold flowpath lengths and residence times (solid curves
in Figure 2). Further, labile DOC was preferentially lost at
the heads of hyporheic flowpaths, leaving the less labile
fraction to be transported to more distal portions of the
Figure 1. Illustration showing a simplified longitudinal cross-sectional view of a stream hyporheic (HZ)
environment with commonly observed microbially mediated pathways for nitrate (NO
3
) transformations.
These pathways were observed in the present study’s hyporheic system [Zarnetske et al., 2011]. Surface
waters supply NO
3
and dissolved organic nitrogen (DON) and carbon (DOC) to the hyporheic zone.
In aerobic regions of the hyporheic zone, DON can be mineralized to NH
4
+
, which can be transformed
via nitrification to create additional NO
3
. The DOC and NO
3
also can be retained in the hyporheic zone
via microbial assimilation processes. DOC and NO
3
entering anaerobic portions of the hyporheic zone
can be utilized for denitrification, which produces dinitrogen oxide (N
2
O) and dinitrogen (N
2
). The
N
2
O and N
2
can degas out of the stream system and return to the atmosphere (ATM).
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
2of13
flowpath [Zarnetske et al., 2011]. Based on our previous
results [Zarnetske et al., 2011], we hypothesized that deni-
trification in this hyporheic zone is limited by the amount of
labile DOC supplied to the hypoxic-anoxic portions of the
hyporheic zone. We expected that adding labile DOC to the
middle of these hyporheic flowpaths would increase meta-
bolic processing rates downgradient of the addition such
that DO will decrease, N
2
production would increase, and
overall NO
3
mass export would decrease (dashed curves in
Figure 2).
2. Methods
2.1. Study Site
[7] The study site is a gravel bar in Drift Creek
(Figure 3a), a third-order stream in the Willamette River
basin in western Oregon, USA (44.9753°N, 122.8259°W).
The drainage basin is 6517 ha, and has mixed land use
dominated by agriculture (lower catchment) and forestry
(upper catchment). The basin’s population is primarily rural
and residences are serviced by septic systems, another poten-
tial source of N in the study stream. Annual precipitation is
1190 mm and comes predominantly during the winter as rain.
Base flow discharge gradually decreases to an annual mini-
mum (<50 L s
1
) in early September. The study reach was
modified by channelization in the past, as were many of the
streams in this agricultural region. The channelized stream is
incised into competent bedrock (andesite flow breccias) and
is now separated from its floodplain. The active channel is
5–20 m wide and is bounded by steep banks 3–5 m high.
The alluvial thickness above bedrock (as depth to refusal)
varies from 0 to ≥1.5 m, which constrains the extent of the
hyporheic zone. The study reach has a slope of 0.007 m m
1
and the morphology is primarily a planebed channel with
occasional pool-riffle sequences (see Montgomery and
Buffington [1997] for definitions of channel types). The
streambed and gravel bar consists of poorly sorted sand,
gravel, cobbles, and boulders.
[8] The hyporheic zone study site is a lateral gravel bar
approximately 6.1 m by 4 m (Figure 3b) with a riffle on one
side and connected to the bedrock channel bank on the other
side. The gravel bar separates two pools and spans a head
loss across the riffle of 0.135 m. The alluvium comprising
this gravel bar was uniformly 1.2 m thick. This gravel bar
was instrumented with a well network (n= 11) of 3.8 cm I.D.
schedule 40 PVC wells screened 0.2–0.4 m below ground
surface. The observed and modeled subsurface exchange
across this gravel bar primarily occurs along lateral flowpaths
from the head to the tail of the bar (Figure 3b). A previous
investigation demonstrated that all wells are connected to
stream water and that well waters come from the stream and
not the local groundwater aquifer [Zarnetske et al., 2011].
Based upon multiple tracer tests at the study site, the
hydraulic conductivity of the gravel bar is 4.03 to 6.63 m d
1
.
Figure 2. Steady state denitrification dynamics at the study site hyporheic zone (solid curves, modified
from Zarnetske et al. [2011]) showing the hypothesized response of denitrification (dashed curves) to an
addition of labile dissolved organic carbon (DOC). The shaded regions represent a net change in the abun-
dance of a solute due to the addition of labile DOC. Note that the labile fraction of the total DOC is pre-
sented as the upper shaded region under the DOC curve with the remainder of the mass representing less
bioavailable forms of DOC. Overall, we hypothesize that the addition of labile DOC to the hyporheic zone
will significantly increase dinitrogen (N
2
) production, decrease total nitrate (NO
3
) concentration and mass
transported through the system, and facilitate additional dissolved oxygen (DO) consumption.
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
3of13
2.2. Tracer Experiment
[9] A 48 h steady state well injection of a conservative
tracer, Cl
, and,
15
NO
3
was used to quantify ambient
hyporheic denitrification via
15
N
2
production. Following
ambient plateau measurements of denitrification during the
first 24 h, a second conservative tracer, Br
, and labile DOC
source, acetate (AcO
), were coinjected for an additional
24 h to measure hyporheic denitrification under increased
labile DOC supply. AcO
was selected because (1) it is
naturally produced and consumed in many aquatic systems,
including hyporheic zones [Chapelle and Bradley, 1996;
Baker et al., 1999], (2) it is highly bioavailable to microbes
[Drake, 1994], (3) it is highly soluble (NaAcO solubility at
20°C = 464 g L
1
), and (4) has demonstrated low sorption
potentials in natural stream sediments (e.g., K
d
≈0[Baker
et al., 1999]). Further, AcO
was used as a pure DOC
source so as to avoid the confounding factor of using more
complex natural DOC sources (e.g., leaf leachate) which
contains additional nutrients [Sobczak et al., 2003].
[10] The injection experiment was performed on 1–3
September 2008 when stream discharge and hyporheic head
gradients were stable. The injection consisted of two steady
state experimental periods: “Pre-DOC”and “DOC.”During
the Pre-DOC period, an injection solution of
15
NO
3
(as 99%
enriched K
15
NO
3
) and Cl
(as NaCl
) was released at a
constant rate (3 mL min
1
) using a metering pump (FMI
QG150, Fluid Metering, Inc., Syosset, NY, USA; note that
the use of trade names in this publication is for reader
information and does not imply endorsement by the U.S.
Department of Agriculture of any product or service) into
the upgradient injection well, H1, for 47.75 h starting at
14:45 on 1 September. During the DOC period, an injection
solution of conservative tracer Br
(as KBr) and NaAcO was
released at a constant rate (6 mL min
1
) into H1 with a
second FMI QG150 metering pump for 21.17 h starting at
17:20 on 2 September.
[11] Based upon pilot studies of dilution factors between
the injection site and the downgradient receiving wells, the
injected solutes needed to be substantially more concentrated
than the ambient hyporheic conditions. The
15
NO
3
introduced
at the injection site, H1, had a concentration of 30.7 mg L
1
,
which was calculated to produce a maximum hyporheic d
15
N
enrichment plume of 40,000‰in the hyporheic water NO
3
leaving the injection site. The Cl
injection concentration
was 4,367 mg L
1
based upon an addition target to elevate
the background hyporheic Cl
by 1,260% and generate clearly
detectable specific conductivity (SC) increase in downgradient
hyporheic flowpaths. The AcO
injection concentration was
504 mg L
1
, while the Br
injection concentration was
50 mg L
1
. During the entire injection time, the injection
well water column was continually mixed via closed system
peristaltic pumping to ensure full solute mixing and equal
concentration release across the entire screened well section
(0.2–0.4 m below ground surface). The low injection pump
rates during the experiment did not create a detectable
increase in head elevation in well H1. The injection solution
had an O
2
concentration of 4.4 mg O
2
L
1
during the injec-
tion, which matches the ambient O
2
concentration observed
at H1 prior to the start of the injection. We did not observe a
change in the O
2
concentration at the injection site during the
experiment.
[12] We used specific conductivity to measure the real-
time Cl
transport between the source well and downgradient
receiving wells. The specific conductivity measurements
were taken every 60 s in 10 wells (H1, H2, H3, I1, I3, J1, J2,
J3, K2, K3) and in the stream water at the head and tail of the
gravel bar to detect if stream specific conductivity varied
during the experiment. These specific conductivity mea-
surements were made with 12 multiplexed, in situ, CS547A
conductivity and temperature probes connected to a CR1000
(Campbell Scientific, Logan, Utah, USA). The specific con-
ductivity measurements were used to characterize the con-
servative solute transport dynamics including flow rates, flow
Figure 3. (a) Map of the Drift Creek study site showing
lateral gravel bar hyporheic site. (b) Map of the hyporheic
study site showing locations of wells (dots with cross hairs),
hyporheic injection site, and water potentiometic surface dur-
ing the injection experiment. Note that the stream briefly
bifurcates near the gravel bar (i.e., not a tributary confluence)
and water chemistry is the same across the channel. Dotted
arrow indicates a single representative simulated advective
flowpath from the injection well (H1) to the stream.
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
4of13
paths, and residence times as well as to inform the timing of
the sampling regime described below (for additional detail,
see Zarnetske et al. [2011]).
[13] The water sampling regime consisted of collecting
multiple rounds of hyporheic samples during the three phases
of the experiment: (1) Preinjections, (2) Pre-DOC plateau
(
15
NO
3
and Cl
steady state), and (3) DOC plateau (AcO
and Br
steady state). For each location (10 wells plus
stream water at the gravel bar head), repeated sampling
occurred during the preinjections (n= 3), Pre-DOC plateau
(n= 5), and during DOC amendment (n= 5) periods. The
Pre-DOC plateau sampling period was initiated at 18.5 h after
15
NO
3
and Cl
injection started when all hyporheic wells
demonstrated steady state specific conductivity values. Fol-
lowing the Pre-DOC Cl
transport times, the DOC amend-
ment sampling period was initiated at 18.5 h after AcO
and
Br
injected started. Repeated hyporheic samples were col-
lected approximately every 2 h during each respective
plateau period. All samples were analyzed for solutes rele-
vant to denitrification and tracing injection solutes (d
15
NO
3
,
d
15
N
2
, as well as concentrations of NO
3
, AcO
, total DOC,
DO, Cl
, and Br
). Hydraulic transport parameters (head,
flow rates, flowpaths, and residence times) were also measured
along the instrumented hyporheic zone.
[14] Hyporheic well samples were collected with a field
peristaltic pump (Masterflex L/S, Vernon Hills, Illinois, USA)
[Woessner, 2007]. All water samples were immediately fil-
tered through ashed Whatman GF/F glass fiber filters (0.7 mm
pore size) into acid washed HDPE bottles (60 mL for nutrient
chemistry and 1 L for d
15
N isotope samples). Following fil-
tering, nutrient chemistry samples and isotope samples were
stored on ice in the field and later refrigerated at 4°C or frozen
in the laboratory until processed and analyzed. DO con-
centrations were measured in situ with a calibrated YSI DO
Meter (Model 52) at all locations prior to collecting each
round of samples. Samples were also collected for d
15
N
2
O(g),
but could not to be analyzed due to technical problems at
the stable isotope laboratory. Nonetheless, denitrification in
freshwater and nearshore marine system sediments consists
almost entirely of N
2
production with N
2
O/N
2
production
ratios generally between <0.001 and <0.05 [Seitzinger, 1988;
Mulholland et al., 2004], so d
15
N
2
by itself is capable of
characterizing the majority of the denitrification dynamics.
[15] The d
15
N gas collection for each sample occurred in
the field and followed procedures adapted from Hamilton
and Ostrom [2007]. A low-flow peristaltic pump was used
to collect 80 mL water samples into a 140 mL plastic syringe
(Becton-Dickinson, Franklin Lakes, NJ, USA) fitted with
stopcocks. All detectable bubbles were expelled to create a
zero headspace. Sample syringes were submerged under
water in a processing tub kept at stream temperature to avoid
atmospheric N contamination. An underwater transfer of
40 mL high-purity He was added to each sample syringe.
Equilibration of the N
2
(g) into the He headspace of each
sample was achieved by gently agitating the syringes for
10 min. After equilibration, 14 mL of headspace gas was
injected into preevacuated 12 mL exetainers (Labco Ltd.,
Wycombe, UK). Preevacuation of exetainers was achieved
by pumping them down to a pressure of <50 mTorr using
a Welch vacuum pump (Model DirectTorr 8905, Skokie,
Illinois, USA). All exetainers were stored underwater in He
purged DI water-filled centrifuge tubes until sample collec-
tion. All sample-filled exetainers were returned to their zero
headspace He purged DI water-filled centrifuge tubes for
storage until analysis.
[16] We conducted a detailed survey of surface water
elevations and channel topography around the instrumented
gravel bar using a Topcon total station (Model GTS-226,
Livermore, California, USA) during the tracer experiment.
The applied standard surveying methods had a spatial reso-
lution of x≤0.1 m, y≤0.1 m, z≤0.005 m for the instru-
mented gravel bar.
2.3. Laboratory Procedures
[17] Stream and hyporheic samples were analyzed for
NO
3
-N, DOC-C, AcO
(CH
3
COO
), Cl
, and Br
at the
Oregon State University Institute for Water and Watersheds
Collaboratory (Corvallis, USA). The NO
3
-N measurements
were made by a Technicon Auto-Analyzer II using standard
colorimetric methodology with detection limits of 0.001 mg
L
1
. The concentration of total DOC was determined with
a Shimadzu TOC-VCSH Combustion Analyzer (Tokyo,
Japan; detection limit = 0.05 mg L
1
). The AcO
,Cl
, and
Br
were determined by ion chromatography (Dionex 1500,
Sunnyvale, California, USA; detection limit = 0.01 mg L
1
).
[18] The
15
N content of the stream and hyporheic water
NO
3
was determined by methods adapted from Sigman et al.
[1997] and Mulholland et al. [2004]. These methods are
briefly summarized below. Prior to
15
N analysis,
15
NO
3
samples with blanks and standards were processed as follows:
(1) A volume of each sample (0.25–1 L; processing volume
is dependent on N content of each sample) was stripped of
its dissolved NH
4
+
and had its NO
3
concentrated; (2) the
concentrated sample NO
3
was captured on a prepared filter
via a reduction/diffusion/sorption procedure (full reduction
of NO
3
to NH
4
+
, which is then converted to NH
3
that dif-
fuses into the headspace and ultimately gets captured on the
acidified sorption filter); and (3) after complete transfer of
NO
3
to the sample filter, the samples were sealed and sent
for
15
NO
3
analysis. All
15
NO
3
and
15
N-gas samples were
analyzed by the Marine Biological Laboratory Stable Isotope
Facility (MBL, Woods Hole, Massachusetts, USA). Data are
reported using delta notation, where
d15N¼1000 Rsample
Rstandard
1
ð1Þ
and Ris the ratio of
15
N:
14
N in the sample or standard
(atmospheric gas). Replicate analyses of the water and gas
samples show the precision of d
15
NO
3
and d
15
N
2
isotope
measurements is 80.0‰and 0.2‰, respectively.
2.4. Parameter and Statistical Calculations
[19] The specific conductivity breakthrough curves (as a
measure of Cl
transport) were used to calculate the median
residence time of the hyporheic water flowpaths for all
downgradient wells receiving a detectable specific conduc-
tivity increase from the injection site. The median residence
time was calculated as the time required to raise the specific
conductivity in the well to one half the plateau specific con-
ductivity [Zarnetske et al., 2011]. The nominal flowpath
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
5of13
length was measured as linear distance between the injection
site and each downgradient well [Zarnetske et al., 2011].
[20] The connectivity of the receiving wells to the injec-
tion well was based on the conservative tracer (Cl
and Br
)
transport during the experiment. The hydrologic connectivity
(D) to the injection well at steady state, i.e., the fraction of
water arriving at the receiving well that originated at the
injection, was calculated as:
Dx;t¼Cx;tCx;t¼0
Cinj Cx;t¼0
ð2Þ
where cis the concentration of the conservative tracer Cl
,x
is the well location, tis the time of sample, and inj is the
injection site. In the absence of biological or chemical
removal pathways, conservative and reactive tracer transport
should be identical. Based upon this assumption, we used the
hydrologic connectivity, D, to calculate the predicted reac-
tive solute concentrations for NO
3
, DOC, and AcO
in each
of the receiving wells before and during the AcO
augmen-
tation period as:
Spred;x;t¼SinjDx;tþSx;t¼01Dx;t
ð3Þ
where S
pred
is the predicted concentration of the solute of
interest at steady state. We then calculated the difference
between the measured and predicted reactive solute con-
centrations for each well during the AcO
augmentation
plateau conditions. NO
3
, DOC, and AcO
removal occurs
when the observed concentration is less than the predicted
concentration and production occurs when the observed con-
centration is greater than the predicted concentration [Hedin
et al., 1998; Baker et al., 1999]. Further, if the difference
between predicted and observed NO
3
concentration increases
following AcO
augmentation, it indicates increased rates of
NO
3
removal along the hyporheic flowpath when AcO
was
added to the system.
[21] We compared changes in concentrations of solutes for
the Pre-DOC and DOC treatment periods using a paired ttest
(significance at p= 0.05, df = 4). We treated repeated samples
within a well as independent replicates. We ran paired ttests
to determine how strongly means of Pre-DOC and means of
DOC differed in each well. We excluded wells H3, I1, I3, J1,
and J3 because they did not receive measureable concentra-
tions of the added solutes.
3. Results
3.1. Background Stream and Hyporheic
Biogeochemical Conditions
[22] Streamflow conditions were relatively stable over the
experiment with a mean flow of 32 L s
1
and a variance of
3.2 L s
1
. We did not observe any detectable change in
head across the gravel bar during the experiment. Stream
and hyporheic water temperature ranged between 12.8 and
15.8°C during the injection experiment. Measured stream
surface water nutrient and chemistry conditions were stable
during the experiment with mean and 1 standard deviation
(n= 13) values of NO
3
(0.61 0.01 mg N L
1
), AcO
(<0.01 0.005 mg L
1
), DOC (1.94 0.36 mg C L
1
),
DO (10.62 0.98 mg O
2
L
1
), Cl
(3.60 0.25 mg L
1
),
Br
(0.010 0.005 mg L
1
) and pH (6.8 0.2).
[23] Background hyporheic conditions collected imme-
diately prior to the start of the injection experiment showed
flow and biogeochemical conditions consistent with the pre-
vious study by Zarnetske et al. [2011]. Hyporheic flowpaths
originated at the head of the gravel bar and are discharged to
the stream at the tail of the gravel bar (Figure 3b). The bio-
geochemical conditions showed that the proximal ends of
hyporheic flowpaths were characterized as oxic while distal
ends were hypoxic and indicated net NO
3
removal (Table 1).
The supply of DOC and DO to the hyporheic zone was pri-
marily from stream water advecting into the sediment at the
head of the gravel bar (Figures 4e and 4g, respectively). The
DO and DOC removal rates were greatest in the first 2 m of
the hyporheic flowpaths, but removal persisted across the
entire gravel bar. While the patterns were similar along the
hyporheic flowpaths of the earlier study (solid curves in
Figure 2) [Zarnetske et al., 2011], there were differences.
No AcO
was detected in the stream or hyporheic water
above the detection limit of 0.01 mg L
1
. Both Cl
and Br
were present before the experiment, but did not vary between
stream and hyporheic waters or among wells (Table 1). Rel-
ative to the previous study, the background DOC conditions
Table 1. Ambient Stream and Hyporheic Temperature and Biogeochemical Parameters and Nominal Transport Distance From the H1
Injection Site to Each Well
a
Location
Temperature
(°C)
DO
(mg O
2
L
1
)
NO
3
(mg N L
1
)
DOC
(mg C L
1
)
Acetate
(mg AcO
L
1
)
Cl
(mg L
1
)
Br
(mg L
1
)
Nominal Distance
From Injection
b
(m)
Stream 12.8 10.62 0.612 1.94 <0.01 3.596 0.011 0.5
G1 13.1 4.24 0.433 1.54 <0.01 3.497 0.011 1.54
H1 (injection site) 13.2 4.31 0.453 1.14 <0.01 3.485 0.010 0
H2 13.3 2.5 0.447 1.15 <0.01 3.528 0.014 +0.67
H3 13.8 0.83 0.37 0.85 <0.01 3.818 0.016 +1.5
I1 13.2 2.95 0.477 1.15 <0.01 3.537 0.013 +1.02
I3 13.8 0.88 0.394 0.91 <0.01 3.471 0.021 +1.75
J1 14.1 0.76 0.317 0.8 <0.01 3.421 0.018 +1.97
J2 14 0.88 0.413 0.84 <0.01 3.461 0.012 +2.18
J3 13.5 1.62 0.45 0.81 <0.01 3.500 0.012 +2.83
K2 14 0.7 0.348 0.76 <0.01 3.440 0.011 +3.12
K3 13.7 1.31 0.43 0.86 <0.01 3.479 0.010 +3.66
a
Reported as means, n=3.
b
Plus and minus symbols denote upgradient and downgradient of H1, respectively.
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
6of13
Figure 4. Spatial biogeochemical and d
15
N signatures during steady state Pre-DOC and DOC augmen-
tation periods of the experiment. Reported values are the means for each period (n= 5).
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
7of13
Figure 5. Change in solute concentrations for the Pre-DOC and DOC treatment periods in receiving
wells. K2, K3, and J3 demonstrate steady state conditions and are sorted from left to right by increasing
levels of connectivity to injection. Note that asterisks denote ttest significant difference of means at
p< 0.05 (one asterisk), p< 0.01 (two asterisks), and p< 0.001 (three asterisks) with df = 4.
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
8of13
were lower in stream and hyporheic waters and a strong
nitrification region at the head of the hyporheic flowpaths
was not present.
3.2. Solute Transport and Retention: Pre-DOC
Addition
[24] Four of the nine downgradient wells were hydrolog-
ically connected to the H1 well injection site as determined
by a significant increase (p< 0.05; ttest; df = 4) in Cl
during the first Pre-DOC injection plateau period (H2, J2,
K2, K3; Figures 4 and 5). The steady state hydrologic con-
nectivity (D) to the injection was 0.013 at J2, 0.0020 at K2,
and 0.0090 at K3 (Table 2). (Note that the tracer injection
rate was very low and was diluted into a large volume of
hyporheic water flowing through the gravel bar. Still, the
relatively high concentration of conservative and
15
N tracers
resulted in distinct breakthroughs, despite the apparent low
connectivity). The H2 well was connected to the injection
well, but the level of connectivity varied between Pre-DOC
and DOC periods thereby failing the experimental assump-
tion of steady state flow dynamics between the experimental
injection periods as shown in section 3.3. The specific con-
ductivity did not vary significantly between Pre-DOC and
DOC periods in the other wells (data not shown but see Cl
in Figures 4a and 4b). Nominal flowpath lengths from the
injection site to the receiving wells ranged from 2.18 m (J2)
to 3.66 m (K3). The median hyporheic transport times from
the injection site to J2, K2, and K3 were 9.8, 11.3, and 13.3 h,
respectively (Table 2).
[25]TheCl
connectivity consistently corresponded with the
amount of
15
NO
3
reaching the receiving wells (d
15
NO
3
‰=
721.8 * [Cl
mg L
1
]2248, r
2
= 0.999, n= 30), so the
15
NO
3
tracer spatial plume is the same as the Cl
plume
(Figure 4). Nitrate concentrations were differentially altered
by the K
15
NO
3
addition. The two most connected wells
showed increased NO
3
concentration (J2 increased from
0.41 to 0.50 mg N L
1
and K3 increased from 0.43 to
0.49 mg N L
1
) and there was no change in the least
connected well K2 (Table 2). The DOC in three connected
wells was lower than background conditions. During this
period no AcO
was detected in any of the repeated sampling
rounds (Table 2).
15
NO
3
tracer enrichment and
15
N
2
pro-
duction via denitrification were also detected at significant
but varying levels in each of the receiving wells (Figures 4
and 5).
3.3. Solute Transport and Retention:
Post-DOC Addition
[26] The steady state conditions of
15
NO
3
and Cl
tracer
addition was confirmed as we detected no significant dif-
ferences (p> 0.05; ttest, df = 4; Figure 5) between the Pre-
DOC and DOC Cl
concentrations at 3 of the 4 connected
wells (J2, K2, K3). Well H2 had a significantly higher Cl
concentration and
15
NO
3
enrichment during the DOC period
compared to the Pre-DOC period (see auxiliary material
Figure S1).
1
Therefore, H2 is not used for comparison
between Pre-DOC and DOC treatment periods of the experi-
ment. Of the connected wells, H2 is located adjacent and
closest to the injection site (nominal flowpath length = 0.67 m;
Figure 4). The second conservative tracer, Br
, injected with
the AcO
showed the same dilution and downgradient tracer
plume behavior as the Cl
tracer. The injected AcO
during
the DOC addition period formed a plume from the injection
site that behaved similar to the conservative tracers (Figure 4).
However, the elevated total DOC concentrations did not per-
sist along the flowpaths and were near Pre-DOC conditions at
the most distal downgradient receiving well, K2. Accounting
for the AcO
injected into the hyporheic zone and dilution
along the flowpaths to each well showed that all of the AcO
was retained in the hyporheic zone, with as much as 6.36 mg
AcO
L
1
being retained along flowpath between wells H1
and J2 (Table 2).
[27] The DO, NO
3
, and d
15
N
2
conditions changed signif-
icantly during the DOC addition. The DO in the receiving
wells all showed significantly decreased concentrations
under elevated labile DOC conditions creating more anoxic
conditions in the three receiving wells ( p< 0.05; ttest; df = 4;
Figure 5). The mean NO
3
concentration decreased in all
three receiving wells, with highly significant decreases in
NO
3
seen in the two most connected wells ( p< 0.001 for
K3 and J2; Figure 5). This decrease in NO
3
resulted in
overall NO
3
conditions lower than the background condi-
tions, and after accounting for dilution showed that NO
3
was
retained along the flowpaths at levels between 0.05 and
0.37 mg N L
1
. The d
15
NO
3
enrichment did not vary sig-
nificantly following the addition of AcO
, but the d
15
N
2
signature increased significantly in all receiving wells with
Table 2. Steady State DOC Augmentation Period Transport
Times, Well Hydrologic Connectivity, and Solute Responses for
the Three Wells Hydrologically Connected to H1
a
Hyporheic Condition
Location
K2 K3 J2
Connectivity to injection, D() 0.002 0.009 0.013
Median transport time from
injection (h)
11.3 13.3 9.8
NO
3
(mg N L
1
)
Background 0.35 0.43 0.41
Pre-DOC 0.35 0.49 0.50
With DOC
Predicted, S0.40 0.73 0.76
Observed 0.35 0.38 0.39
Retention 0.05 0.35 0.37
DO (mg O
2
L
1
)
Background 0.70 1.31 0.88
Pre-DOC 0.93 1.38 1.06
With DOC
Predicted, S0.80 1.34 0.92
Observed 0.79 1.18 0.79
Retention 0.01 0.16 0.13
Acetate (mg AcO
L
1
)
Background <0.01 <0.01 <0.01
Pre-DOC <0.01 <0.01 <0.01
With DOC
Predicted, S0.80 4.77 6.36
Observed <0.01 <0.01 <0.01
Retention 0.80 4.77 6.36
a
Solute background values are means (n= 3); Pre-DOC and DOC period
values are means (n= 5).
1
Auxiliary material is available in the HTML. doi:10.1029/
2011JG001730.
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
9of13
d
15
N
2
enrichment increasing up to 26.1 times that of the
Pre-DOC levels (p< 0.001; ttest; df = 4; Figure 5).
4. Discussion
[28] This study demonstrates that labile DOC limits deni-
trification along hyporheic flowpaths and that NO
3
losses
can be attributed in part to denitrification in this hyporheic
zone. The tracing of the denitrification pathway of
15
NO
3
to
15
N
2
under ambient and elevated AcO
(as labile DOC)
conditions confirm earlier theoretical- and observation-based
conclusions regarding the role of hyporheic DOC quantity
and quality on denitrification dynamics in hyporheic systems
[Hedin et al., 1998; Baker et al., 1999; Hill et al., 2000;
Sobczak and Findlay, 2002].
[29] The hyporheic conditions and steady state experimental
design allowed us to evaluate our hypotheses about the role
of labile DOC in hyporheic denitrification (Figure 2). The
post-DOC conditions clearly show that the addition of labile
DOC to this hyporheic zone increased metabolic processing
rates downgradient of the addition such that the additional
labile DOC was utilized and removed rapidly (Figure 4).
Utilization of labile DOC stimulated additional aerobic res-
piration as seen in the increased DO deficit conditions
(Figure 5). Consequently, the increased anaerobic conditions
and elevated supply of carbon substrate, lead to significant
increases in denitrification rates. Overall, the addition of
labile DOC resulted in a significant increase in the total NO
3
mass removed by this gravel bar hyporheic zone (Figure 5).
4.1. Hyporheic Zone as a Stream DOC Sink
[30] This study shows that the hyporheic zone is a sink for
stream DOC and that within the hyporheic zone, the avail-
ability of labile DOC is a critical factor controlling denitri-
fication. We observed a DOC gradient across the gravel bar
flowpaths. At the head of the gravel bar, the hyporheic water
DOC concentrations are similar to the DOC of surface waters,
but hyporheic DOC concentration consistently declined along
the flowpaths (Figure 4). Spatial declines of hyporheic DOC
along flowpaths are consistently seen in gravel bar hyporheic
investigations where the major source of hyporheic DOC is
stream water [e.g., Vervier and Naiman, 1992; Findlay et al.,
1993]. We also know that, in this gravel bar, the lability of
DOC declined along the flowpath and at a rate greater than the
decline of total DOC [Zarnetske et al., 2011]. Declines in
labile DOC along hyporheic flowpaths have been previously
documented in natural and experimental hyporheic systems
where DOC quality was measured [e.g., Sobczak and Findlay,
2002; Sobczak et al., 2003]. For example, when bioavailable
nitrogen is available, labile DOC will decline along riparian
and hyporheic flowpaths due to microbial productivity and
assimilation [Sobczak et al., 1998; Sobczak and Findlay,
2002] and increased metabolism [Baker et al., 2000].
[31] A portion of the DOC advected through the gravel bar
is used for microbial aerobic and anaerobic respiration, as
shown by the rapid depletion of DO and the ambient levels
of denitrification occurring along the flowpaths (Figures 4
and 5). We also know that despite the large addition of
AcO
to this gravel bar, no above-background AcO
con-
centrations were detected at any downgradient points before
or during the AcO
addition. The 100% retention of the
AcO
across a 24 h plateau injection period (i.e., the DOC,
as AcO
, uptake capacity was not achieved) combined with
the known low sorption potential and high solubility of
AcO
[e.g., Baker et al., 1999] indicates that in this hypor-
heic zone, metabolism is strongly limited by DOC supply
even under the elevated DOC conditions of the experiment.
The controlled experimental observations of DOC limitation
on hyporheic metabolism in this study mirror the findings of
Jones [1995] and Baker et al. [2000], who observed that
natural variations in stream-sourced DOC supply correlated
with hyporheic metabolism and N transformation rates.
4.2. Labile DOC Supply Controls Hyporheic
NO
3
Dynamics
[32] The hyporheic denitrification rates are DOC substrate
limited in this gravel bar as documented along the extended
flowpaths leading to wells J2, K2, and K3, where all other
conditions for denitrification are present except for a suffi-
cient source of labile DOC to serve as an electron donor. This
was proven under controlled, steady state conditions where
just a change in the labile DOC availability resulted in more
extensive anaerobic conditions and increased denitrification
rates (both NO
3
concentrations decreased and d
15
N
2
signa-
ture increased significantly (Figures 4 and 5)).
[33] The hyporheic DOC and NO
3
dynamics fit the con-
ceptual model of Findlay [1995], which hypothesizes that
the influence of the hyporheic zone at the reach scale is a
function of two variables: the rate of hydrologic exchange in
and out of the hyporheic zone and the rate of biogeochemical
processes in the hyporheic zone. In this system, we see that
denitrification is limited by the amount of labile DOC sup-
plied via hydrologic exchange from the DOC-rich surface
waters. No lateral hillslope or groundwater inflow has been
detected at this gravel bar site. Thus, surface water is the
primary source of DOC to this hyporheic zone at summer
low flow. Consequently, denitrification in this system will
vary primarily as a function of the quantity and quality of the
DOC in the surface water and the hydrologic conditions
promoting hyporheic exchange across the gravel bar. During
a prior investigation on the same gravel bar [Zarnetske et al.,
2011], the hyporheic zone had a much greater NO
3
removal
efficiency with approximately 99% of the NO
3
removed
along flowpaths traversing the gravel bar versus only 57% in
this study. During the previous study, hydrologic exchange
conditions were similar (i.e., head gradient, hydraulic con-
ductivity, and residence time) as were the ambient stream
NO
3
concentrations (0.54 mg L
1
and 0.61 mg L
1
,
respectively), but a key difference was that stream DOC
concentration entering the hyporheic zone was much higher:
3.01 mg L
1
in the previous study versus 1.94 mg L
1
in the
current study. The larger flux of DOC to the head of the
gravel bar during the earlier study also stimulated greater
DO consumption rates. These higher DO consumption rates
lead to the development of anoxic conditions occurring over
shorter flowpath lengths than the present study. Therefore, a
larger portion of the hyporheic zone had reducing conditions
conducive to denitrification in the study by Zarnetske et al.
[2011]. However, after adding the labile DOC source to
the hyporheic zone in this study we see that the gravel bar
becomes more anoxic and NO
3
removal efficiency almost
ZARNETSKE ET AL.: DOC CONTROLS HYPORHEIC DENITRIFICATION G04036G04036
10 of 13
doubled from the Pre-DOC to the DOC periods (Table 2 and
Figure 5).
[34] The addition of the labile DOC along midflowpaths
also changed the transport of advected stream DOC through
the gravel bar. There were higher total DOC concentrations
in receiving wells J2 and K3 during the acetate addition
period (Figure 5). The acetate addition did not directly add to
the measured total DOC concentrations as no acetate was
recovered in the down gradient wells. However, the acetate
may have indirectly increased downgradient total DOC
concentrations in two ways. First, the acetate is preferentially
consumed over many other stream-sourced DOC compounds
[Hall and Meyer, 1998], which could decrease the uptake rate
of stream-sourced DOC. Second, the physical adsorption of
hyporheic DOC is concentration dependent [Findlay and
Sobczak, 1996] and leads to DOC immobilization [Fiebig
and Lock, 1991]. The subsurface acetate addition may have
filled many of the DOC sorption sites with acetate. Thus
decreasing the number of available DOC sorption sites along
the flowpaths, and decreasing the uptake rate of stream-
sourced DOC. Together the preferential uptake of acetate and
decrease in available DOC sorption sites, in effect, would
shunt the stream-sourced DOC further down the flowpaths
resulting in the higher total DOC concentrations in J2 and K3
during the acetate injection period.
[35] We conclude that, with sufficient upgradient supply of
labile DOC entering hyporheic environments, the hyporheic
zone can play a very significant role in regulating down-
stream NO
3
export in Drift Creek. To demonstrate this point,
we compare ambient NO
3
removal rates to the DOC aug-
mented removal rates at the gravel bar site. A calibrated
groundwater flow model of the hyporheic zone site shows
a minimum of 226 L m
2
d
1
of hyporheic exchange
flow occurs under the summer low-flow conditions of the
DOC experiment [Zarnetske, 2011]. Given this hydrologic
exchange flow and the background observed NO
3
retention
rates, the NO
3
mass removal rate at this gravel bar at this
time is 218 kg ha
1
yr
1
. This removal rate is within the
range of previous surface water–groundwater exchange
studies (e.g., 10–39 kg ha
1
yr
1
[Lowrance et al., 1997];
up to 6600 kg ha
1
yr
1
[Hedin et al., 1998]). In com-
parison to the NO
3
and DOC values seen in these previous
studies, the ambient NO
3
and DOC in our system was an
order of magnitude lower for both N and C constituents.
The decreased availability of NO
3
and DOC alone can limit
the potential for total NO
3
removal via denitrification com-
pared to the previous investigations. However, by artificially
increasing only the supply of labile hyporheic DOC to our
receiving wells (i.e., not the entire gravel bar), we were able
to increase the NO
3
removal rate by 303 kg ha
1
yr
1
,toa
total of 521 kg ha
1
yr
1
. This is an increase of 139% above
background removal rates.
[36] The injection of labile DOC in this experimental
gravel bar is akin to groundwater–surface water environ-
ments where a DOC-rich groundwater or riparian flowpath
converges with other hyporheic flowpaths. Hedin et al.
[1998] and Hill et al. [2000] observed high denitrification
rates where hydrologic conditions promoted mixing of DOC-
rich groundwaters with DOC-poor waters that contained
NO
3
. Therefore, the formation of these denitrification hot
spots is governed by the complex groundwater hydraulics
that mix waters containing DOC and NO
3
[McClain et al.,
2003]. In this gravel bar hyporheic zone, the ambient DOC
gradient is the result of an imbalance between the hydrologic
transport and biogeochemical reaction kinetics: the advected
supply rate is less than the biochemical demand rate for labile
DOC. By experimentally manipulating the labile DOC gra-
dient with an acetate addition we altered the balance between
transport and reaction kinetics and created a denitrification
hot spot and altered total DOC transport in this hyporheic
zone.
[37] This experiment also indicates that anthropogenic
(intentional and unintentional) or natural additions of labile
DOC to a hyporheic system will facilitate greater denitrifi-
cation if NO
3
supply is not limiting. Similar to groundwater
remediation practices (e.g., denitrification walls, in the sense
of Schipper and Vojvodic-Vukovic [1998]), strategic addi-
tions of a labile DOC source to the near stream environment
could mitigate NO
3
flux into a stream system [Pfenning and
McMahon, 1997; Hedin et al., 1998], but that would require
extensive understanding of the complex hydrologic flow-
paths and flow rates leading to the denitrification sites.
5. Conclusions
[38] We showed that hyporheic environments are an
important sink for both DOC and nitrate in freshwater eco-
systems and that the fate of nitrate, in this case denitrification,
is tightly coupled to C dynamics. The in situ steady state
labile DOC and
15
NO
3
addition experiment definitively
showed that denitrification is occurring in the hyporheic zone
of this upland agricultural stream and that the availability of
labile C strongly limits the overall denitrification potential.
We echo Gruber and Galloway [2008]: C and N cycles are
coupled in river systems and they should be evaluated and
modeled as coupled processes. The use of total DOC in
hyporheic denitrification models is likely to overestimate
denitrification potentials, especially if all the DOC is con-
sidered available for microbial processes. This study indi-
cates that future hyporheic denitrification investigations
should measure and account for the labile fraction of DOC
and not just the total DOC.
[39]Acknowledgments. Support for this project was primarily
provided by a NSF Ecosystem Informatics IGERT fellowship (grant
DGE-0333257) to J.P.Z.; research grants from the OSU Institute for Water
and Watersheds and a North American Benthological Society Endowment
Fund to J.P.Z.; and NSF grants EAR-0409534 and EAR-0409591 to R.H.,
S.M.W., and M.A.B. Additional support was provided by the Hollis M. Dole
Environmental Geology Fund at OSU. Any opinions, findings, and con-
clusions or recommendations expressed in this material are those of the
authors and do not necessarily reflect the views of NSF. We thank the
Harry Klopfenstein Farm for generously granting access to the Drift Creek
research sites. Special thanks to V. Adams, P. Zarnetske, and J. Yin for
field assistance and C. Jones and K. Motter of CCAL and OSU IWW Col-
laboratory for help with analyzing general water chemistry.
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