LOCATION DESCRIPTION
We have initiated a long-term effort to monitor soil nitrate (NO3-)
and ammonium (NH4+) concentrations, microbial biomass carbon (C) and
nitrogen (N) content, microbial respiration, potential nitrification
and N mineralization rates, and denitrification potential in the
experimental watersheds at Hubbard Brook. In 1994 we began sampling in
the Bear Brook Watershed (west of Watershed 6). In 1998, we added
Watershed 1 to our sampling regime in an effort to monitor and
quantify microbial response to a whole-watershed calcium addition.
In the Bear Brook Watershed we used the “west of watershed 6 litter
trap transects” described by Hughes and Fahey (1994). These are four
100 m transects, 50 m apart with five traps per transect located at
low, mid, upper and high elevations - 20 traps per elevation. We
sampled within 1.5 m of five traps at each elevation. Litter quality,
quantity and composition have been monitored on these transects since
1984. In 1997 we sampled at the upper and high elevations and also at
four locations just above and to the west of Rain Gauge 9 in a mixed
stand dominated by red spruce and balsam fir. In 1998 we began
regularly sampling more extensively in the spruce/fir zone, and
discontinued our sampling at the "upper" site. We sampled
within 1.5 m of the center of each of 5 randomly chosen sites in this
spruce/fir area. Vegetation in the Bear Brook Watershed is roughly
equivalent to that in Watershed 6, which is an approximately 80 year
old “reference” watershed dominated by northern hardwoods (American
beech, sugar maple, yellow birch) at the lower elevations with
moderate amounts of red spruce, balsam fir and white birch at the
“high” elevation sites.
In Watershed 1 we sampled near a subset of the lysimeter sites
established for the calcium addition study. Our sites included the
"spruce/fir" Lysimeter Site 2 at the top of Watershed 1, the
"high" Lysimeter Site 3, the "mid" Lysimeter Site
4, and the "low" Lysimeter Site 6. These site types and
elevations correspond to those in the Bear Brook Watershed. At each
lysimeter site we chose 5 replicate plots that were all within ~30 m
of the center lysimeter stake. We revisited the same approximate plot
during each sampling date. In 1999 we marked out 2 X 3 m plots in
these areas, covered them during the helicopter wollastonite addition,
then uncovered them and hand-applied wollastonite. We have sampled
entirely within these marked plots since the application.
SAMPLING DESIGN
Samples were collected three times each year (note the above
exceptions) to correspond with key plant phenological stages:
pre-green, peak green, and senescence. Sampling dates were generally
in May, July, and October. We also sampled once in December 1999, two
months after the wollastonite addition, to determine if there were
immediate responses to the calcium. In 1994, May and July 1995, May
and July 1996, and from July 1998 to July 1999 we sampled soils using
a bulb corer method. In October 1995 and 1996, all of 1997, and in May
1998 we used a pin block method. From May 2000 to 2010 we sampled
using a split-PVC corer method. From May 2000 on we have sampled using
a split-PVC corer method.
In the pin block method, long (13.2 cm) nails are driven through holes
along the edge of a 15 X 15 cm square of ~1 cm plywood that has been
placed on the forest floor. There are 4 holes on each side of the pin
block. These nails firmly attach the block to the soil, and enclose a
"box" of soil for sampling. We use a small saw to cut away
roots and soil from the edge of the nails and from underneath the
block. We then remove the soil contained by the block and the nails
(15 X 15 X 13.2 cm). In 1997 Oe and Oa were collected as a single
horizon, and mineral soil was discarded. In other years we separated
the organic soil into two layers (Oi/Oe and Oa/A horizons), discarded
the mineral soil from the pin block, and used a bulb corer to take a
10 cm mineral core directly under the (removed) soil block.
In the bulb corer method, a typical flower planting and gardening bulb
corer is used. These corers are metal, have a handle on top, and have
a diameter that gets slightly narrower from top to bottom. The
diameter at the bottom of the corer we used is 6.5 cm. Typically,
anywhere from 2-8 cores are taken at a given site, depending on
horizon depth and density of soils. The corer is inserted 10 cm into
the soil and removed with an intact core. The core is pushed out
through the top of the corer onto a plastic sheet, where horizon
depths are noted and horizons are separated. The mineral soil is
discarded and the remaining soil is split into two layers (Oi/Oe and
Oa/A horizons). Each layer is measured and placed into a sample bag,
with all cores composited by horizon. To obtain a mineral sample we
dig down to the first sign of mineral soil (E or B, depending on the
site) and attempt to take a full 10 cm core. If it is not possible to
obtain a full 10 cm mineral core we take 2-3 partial mineral cores and
combine them.
In the split-PVC corer method, a 5 cm diameter split PVC corer is used
to take all samples. A split PVC corer consists of a piece of 2 inch
(5 cm) PVC pipe, about 15-20 cm long, split lengthwise on both sides.
The corer is actually in two pieces. We put the corer together along
the cuts, and duct-tape one side -- the "hinge" side.
Holding the corer firmly together, we hammer it 10-15cm into the
ground. The corer is removed and then opened with the intact soil core
inside. The soil is split into three layers (Oi/Oe, Oa/A, and mineral
horizons). Each horizon is measured and placed into a sample bag. We
typically collect 2-8 cores per site, compositing all cores by
horizon.
DATA DESCRIPTION
In most cases, Oi and Oe horizons were composited into one sample, as
were Oa and A horizons. Mineral samples generally consist of the top
10 cm of mineral soil beginning below the A horizon. In 1994 and May
and July 1995, only Oi/Oe and Oa/A horizons were sampled. In 1997 only
Oe and Oa horizons were collected, and they were composited into one
sample. Samples are either collected once (July) or three times a year
(May, July, and October). Samples were also collected in December
1999.
NOTE ON OUTLIERS IN THE DATA SET
All outliers were left in the data set unless it was evident that
there was a contamination or laboratory procedure problem. In 2005, at
the W1 high site, rep 3 of the Oi/Oe sample had biomass C,
Respiration, Biomass N, Nitrification, Mineralization and DEA that
were values that were, in some cases, 10 times higher than the next
highest value. It was determined that since this pattern was evident
in multiple lab analyses it was not due to laboratory errors, and must
represent a "hot spot" of activity in the soil. These data
were left in the dataset. In 2001, at the Bear Brook high elevation
site, the mineral horizon had extremely high NH4 levels. This mean is
the result of 5 data points, 2 of which were very high. These data
were also left in the dataset.
LABORATORY PROCEDURES
Samples were stored at 4o C between sampling and analysis (from less
than 1 week to up to three weeks). From 1994 to 1996 soils were sieved
(>4 mm). From 1997 to 2010 soils were manually homogenized: all
large rocks, roots, and other non-decomposed organic material were
removed, and samples were thoroughly mixed. No more than three minutes
were spent homogenizing any sample. All samples were held at field
moisture before analysis. Soil water content was determined
gravimetrically.
Microbial biomass C and N content was measured using the chloroform
fumigation-incubation method (Jenkinson and Powlson 1976). Soils were
fumigated to kill and lyse microbial cells in the sample. The
fumigated sample was inoculated with fresh soil and sealed in a jar,
and microorganisms from the fresh soil grew vigorously using the
killed cells as substrate. The flushes of carbon dioxide (CO2) and 2 M
KCl extractable inorganic N (NH4+ and NO3-) released by the actively
growing cells during a 10-day incubation at field moisture content
were assumed to be directly proportional to the amount of C and N in
the microbial biomass of the original sample. A proportionality
constant (0.41) was used to calculate biomass C from the CO2 flush in
the fumigated samples. Biomass N is the total inorganic N flush in the
fumigated samples.
Inorganic N and CO2 production were also measured in
"control" samples. Control samples were prepared in the same
fashion as those listed above, but were not fumigated. These
incubations provided estimates of microbial respiration and potential
net N mineralization and nitrification. Microbial respiration was
quantified from the amount of CO2 evolved over the 10-day incubation.
Potential net N mineralization and nitrification were quantified from
the accumulation of NH4+ plus NO3- and NO3- alone during the 10-day
incubation. We measured 2 M KCl extractable inorganic N in the fresh
soil samples to determine the initial soil NO3- and NH4+
concentrations. Carbon dioxide was measured by thermal conductivity
gas chromatography. Inorganic N was measured colorometerically using
an autoanalyzer.
Denitrification enzyme activity was measured using the short-term
anaerobic assay described by Smith and Tiedje (1979). Sieved soils
were amended with NO3- (100 mg N kg-1), dextrose or glucose (40 mg
kg-1), chloramphenicol (10 mg kg-1) and acetylene (10 kPa) and were
incubated under anaerobic conditions for 90 minutes. Gas samples were
taken at 30 and 90 minutes, stored in evacuated glass tubes and
analyzed for N2O by electron capture gas chromatography. For more
information on any of the methods described above, refer to Robertson
et al. (1999).
Soil pH was measured by weighing 10 g of soil at field moisture into a
small beaker. 20 ml of deionized water was added and swirled to make a
slurry. After 30 minutes the pH of the slurry was measured using a pH
meter calibrated using pH 4 and pH 7 buffers.
CALCULATIONS
All results are expressed on a per gram of dry soil basis. Values can
be converted to a “per area” basis using data on the mass of different
soil horizons found elsewhere on the data page of this website.
REFERENCES
Jenkinson, D. S., and D. S. Powlson. 1976. "The effects of
biocidal treatments on metabolism in soil – V: A method for measuring
soil biomass. Soil Biology & Biochemistry 8:209-213.
Robertson, G.P., C.S. Bledsoe, D.C. Coleman and P. Sollins, editors.
1999. Standard Soil Methods for Long Term Ecological Research. Oxford
University Press, New York.
Smith, M.S., and J. M. Tiedje. 1979. Phases of denitrification
following oxygen depletion in soil. Soil Biology & Biochemistry
11:262-267.
