Supplementary Materials
Site Description
General Soils
Since the site was mined, soil information obtained from National Resources Conservation
Service (NRCS) Soil Surveys is not applicable. Prior to mining, the mitigation site was
composed of soils from the Dekalb-Marowbone-Lantham-Cloverlick-Shelocata-Cutshin,
Shelocta-Gilpin-Hazelton, and Shelocta-Gilpin-Kimper complex mapping units (Hayes 1991).
Soils in the Dekalb-Marowbone-Lantham complex are moderately deep, well-drained, and
formed from sandstone, shale, and siltstone. These soils are located on the upper 1/3 section of
the hill-slopes that are typically xeric, and contain rock outcroppings. Soils in the CloverlickShelocata-Cutshin complex are very deep, well-drained soils formed in colluvium on steep
slopes. They occupy the north and east facing coves and cool slopes. The soils in the SheloctaGilpin-Hazleton complex consist of steep, well-drained, soils formed in colluvium and residuum.
These soils occur on the warm slopes with south and east aspects.
Currently, only a sparse amount of ridge-top soils from the Dekalb-Marowbone-Lantham
complex remain, primarily from the Marowbone soil series. The remainder of the site is
composed of shallow to very deep spoils with varying degrees of weathering and soil
development. A soil pit was dug on the site in 2004 and described by NRCS Soil Survey staff.
As with most surface mines in the area, the soil was classified as an Udorthent in the Fairpoint
soil series (loamy-skeletal, mixed, active, nonacid, mesic Typic Udorthents).
Climate and Geology
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The study was conducted at the University of Kentucky’s Robinson Forest (37º 27’ north latitude
and 83º 08’ west longitude) which is located in the Cumberland Plateau region of eastern
Kentucky. Robinson Forest is a 5,983 ha teaching, research and extension experimental forest
composed of eight discontinuous properties, with the main block comprising approximately
4,200 ha. During the mid-1990s, a portion of Robinson Forest (approximately 600 ha of the 900
ha Laurel Fork watershed) was surface mined for coal. Topographically, Robinson Forest is
characterized by steep slopes with well-drained residuum or colluvial soils formed from
sandstone, shale, and siltstone. The sandstone, shale, siltstone and coal geologic stratigraphy are
horizontally interbedded and are classified as part of the Breathitt Formation (McDowell 1985).
The well-drained soils and geologic layers of minimal permeability result in rapid streamflow
responses to storm events via sub-surface flow (Coltharp and Springer 1980). Elevations on the
forest range from 268 to 475 m.
Although the main block of Robinson Forest has not been surface mined, nearly all of the
adjacent properties have been surface mined for coal. The forest was last harvested by the
Mowbray-Robinson Lumber Company between 1890 and 1920 (Overstreet 1984). The
regenerated forest is classified as mixed-mesophytic with oak (Quercus sp.), hickory (Carya sp.),
and yellow-poplar (Liriodendron tulipifera), and American beech (Fagus grandifolia) as
dominant overstory species, with eastern hemlock (Tsuga canadensis) common in riparian zones.
The climate of Robinson Forest is classified as temperate-humid-continental with warm summers
and cool winters. The average annual temperature for Breathitt County is 12.8 °C, The average
annual precipitation for southeastern Kentucky is 116.4 cm while the 26-year average for three
precipitation collectors at Robinson Forest is 117.5 cm (Cherry 2006). Average monthly
precipitation is 9.79 cm and March tends to be wetter than average and October tends to be drier.
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Analytical Methods
Sediment Oxygen Demand (SOD), denitrification enzyme activity (DEA), Basal Respiration
(BR), Organic Matter (OM) and soil moisture measurement
SOD was measured in the perennial reaches in the field by the method of Hill et al.
(1998). Well-aerated, ambient stream water was collected in a clean plastic beaker from an area
with strong flow. Depending on sediment availability and expected organic matter levels, about
10 to 70 g (dry weight) aliquots of homogenized sediment were dispensed into each of five 250ml Nalgene bottles, which were then completely filled with ambient stream water so that no air
bubbles were visible. Two separate 250-ml Nalgene bottles were filled with stream water only
so that oxygen demand in the water could be accounted for. All seven bottles were incubated
together in the dark for about four hours in a small cooler filled with ambient stream water.
Following incubation, dissolved oxygen (DO) and temperature were measured in each bottle
with a WTW Multi 197i meter and WTW StirOx G stirring DO probe. The mean DO in the
water-only bottles was subtracted from the DO in each sediment-plus-water bottle and the mean
SOD was calculated. BR was calculated from SOD as described by Doering et al. (2011).
Several hundred grams of the sediment remaining in the sediment collection beaker were bagged
for denitrification enzyme activity (DEA) analyses.
The DEA in sieved soils and sediments was measured by the method of Groffman et al.
(1999), which provides conditions that are non-limiting for denitrification (anaerobic, abundant
nitrate and labile carbon) but which inhibit growth of new organisms. Duplicate ~ 25-g aliquots
of each soil and sediment sample were weighed into small canning jars. Twenty-five ml of a
solution consisting of 100 mg N/L of nitrate, 40 mg/L of dextrose, and 10 mg/L of
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chloramphenicol dissolved in high purity water was added to each jar, which was then sealed
with a lid containing a septum for gas withdrawal. The air headspace was exchanged for N2 by
repeatedly evacuating and adding N2 gas to establish anaerobic conditions. Acetylene, which
blocks the conversion of N2O to N2, was produced by reacting water and calcium carbide.
Acetylene was added to each jar such that it constituted about 10 % by volume of the headspace.
The samples were then incubated at room temperature (22 ± 1 ºC) on a rotary shaker at 100 rpm.
One-ml samples were removed from each jar at incubation times of approximately 30 and 90
minutes. The N2O concentration was immediately analyzed by electron capture gas
chromatography with a Shimadzu GC-14A gas chromatograph (GC) (Shimadzu Scientific
Instruments, Columbia, MD) and a PeakSimple chromatography data system (SRI Instruments,
Torrance, CA). The difference in N2O concentration between the 90-minute sample and the 30minute sample was used to calculate DEA.
The basal respiration (BR) of soil and stream sediments collected from dry ephemeral
and intermittent channels was measured by a method similar to that described by Chantigny et al.
(1999). Duplicate ~ 25-g aliquots of each sieved, field moist sample were weighed into small
canning jars and were allowed to sit open in the lab for several hours to allow equilibration with
room temperature and CO2 concentration. The jars were sealed with lids with a septum for gas
withdrawal and incubated in the dark at room temperature (22 ± 1 ºC) for 16 to 27 hours. Five
jars containing only room air were capped at the beginning of the incubation period to serve as
blanks. At the end of the incubation, headspace CO2 concentration was immediately determined
by thermal conductivity gas chromatography as described by Burke et al. (1997). The difference
in CO2 concentration between the headspace at the end of the incubation and the mean CO2
concentration in the blank jars was used to calculate BR. Three of the intermittent channel
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reaches (FR-I, LM-I, and GC-I) were flooded during the April, 2006 sampling and SOD
measurements were performed. To facilitate the statistical analysis of the effect of mining on
channel sediment BR, these three SOD measurements were converted to a carbon basis assuming
a respiratory quotient of 0.85 (Doering et al. 2011).
Measurement of Concentration and δ13C of DOC and DIC
The concentration and δ13C of DOC and DIC were determined as described by St-Jean (2003)
and Osburn and St-Jean (2007) with an OI Analytical 1030W TIC/TOC analyzer (OI Analytical,
College Station, TX) interfaced to a Thermo Delta V Plus isotope ratio mass spectrometer
(IRMS) (Thermo Fisher Scientific, Waltham, MA). Because of the high DIC concentrations (up
to 60 mg C/L) observed in some of the samples from the mined sites, and very low DOC
concentrations in some samples from the forested streams, samples for δ13C-DOC analysis were
acidified to pH < 2 with phosphoric acid and purged with helium for 10 minutes to remove most
of the DIC before analysis with the IRMS system. This extra DIC removal step, coupled with
the DIC removal step that is part of the normal automatic analysis routine, assured that the δ13CDOC analysis was not contaminated by residual DIC. Stable carbon isotope ratios are expressed
as per mil (‰) in the delta notation versus Vienna Peedee belemnite (VPDB):
δ13C = [[(13C/12C)sample/(13C/12C)standard] – 1 ] x 1000
Standardization of the δ13C-DOC values was accomplished by normalizing the raw isotope
measurements from the IRMS to the Stable Carbon Isotopic Reference Materials (SCIRMs)
IAEA-CH-6 and IAEA-601 with internationally accepted δ13C of -10.45 ‰ and -28.81 ‰ ,
respectively (Coplen et al. 2006). Standardization of δ13C-DIC was accomplished by
normalizing the raw isotope measurements to two in-house sodium bicarbonate standards, kindly
provided by Dr. Pennilyn Higgins of the Univ. of Rochester Stable Isotope Ratios in the
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Environment Laboratory (SIREAL). The SIREAL lab has calibrated these sodium bicarbonate
standards with appropriate SCIRMs and determined δ13C values of -0.84 ‰ and -21.9 ‰,
respectively (Pennilyn Higgins, personal communication). All δ13C analyses were corrected for
instrument drift using the procedure of Mr. Paul Brooks of the UC Berkeley Center for Stable
Isotope Biogeochemistry (http://ib.berkeley.edu/groups/biogeochemistry/downloads.php). Four
vials of a third SCIRM, USGS40 with a δ13C of -26.39 ‰ (Coplen et al. 2006), were run with
every δ13C-DOC set as a check standard. Four vials of an in-house sodium carbonate standard
with a δ13C of 0.15 ‰, which was kindly provided by Professor Howard Spero of UC Davis,
were run with every δ13C-DIC set as a check standard. Mean values of the check standards were
always within 0.3 ‰ of the accepted value, which provided good evidence of the reliability of
our analytical techniques including the normalization and drift correction procedures.
References
Burke, R. A., Zepp, R. G., Tarr, M. A., Miller, W. L., & Stocks, B. J. 1997. Effect of fire on soilatmosphere exchange of methane and carbon dioxide in Canadian boreal forest sites.
Journal of Geophysical Research 102: 29, 289-29, 300.
Chantigny, M. H., Angers, D. A., Prévost, D., Simard, R. R., & Chalifour, F-P. 1999. Dynamics
of soluble organic C and C mineralization in cultivated soils with varying N fertilization.
Soil Biology and Biochemistry, 31, 543-550.
Cherry, M. A. 2006. Hydrochemical characterization of ten headwater catchments in eastern
Kentucky. MS thesis, University of Kentucky, Lexington Kentucky.166 p.
Coltharp, G. B. & Springer, E. P. 1980. Hydrologic characteristics of an undisturbed hardwood
watershed in eastern Kentucky. In: Proceedings of the Central Hardwood Forest
Conferece III, H.E. Garrett and G.S. Cox (Eds.). University of Missouri Press, Columbia
MO, pp 10-20, 465 pp.
Coplen, T. B., Brand, W. A., Gehre, M., Gröning, M. Meijer, H. A., Toman, B., & Verkouteren,
R. M. 2006. New guidelines for δ13C measurements. Analytical Chemistry 78: 24392441.
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Doering, M., Uehlinger, U., Ackermann, T., Woodtli, M., & Tockner, K. 2011. Spatiotemporal
heterogeneity of soil and sediment respiration in a river-floodplain mosaic (Tagliamento,
NE Italy). Freshwater Biology, 56, 1297-1311.
Groffman, P. M., Holland, E. A., Myrold, D. D., Robertson, G. P., & Zou, X. 1999.
Denitrification p. 272-288. In G.P. Robertson et al. (Eds.) Standard Soil Methods for
Long Term Ecological Research (pp. 272-288). New York: Oxford Univ. Press.
Hayes, R. A. 1991. Soil Survey of Breathitt County, Kentucky. USDA-NRCS. U.S. Gov. Print.
Office, Washington, DC.
Hill, B. H., Herlihy, A. T., Kaufmann, P. R., & Sinsabaugh, R. L. 1998. Sediment microbial
respiration in a synoptic survey of mid-Atlantic region streams. Freshwater Biology, 39,
493-501.
McDowell, R. C. 1985. The Geology of Kentucky. Professional Paper, U.S. Geological Survey.
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Osburn, C. L. & St-Jean, G. 2007. The use of wet chemical oxidation with high-amplification
isotope ratio mass spectrometry (WCO-IRMS) to measure stable isotope values of
dissolved organic carbon in seawater. Limnology and Oceanography Methods, 5, 296308.
Overstreet, J. 1984. Robinson Forest Inventory. Department of Forestry, University of
Kentucky.
St.Jean, G. 2003. Automated quantitative and isotopic (13C) analysis of dissolved inorganic
carbon and dissolved organic carbon in continuous-flow using a total organic carbon
analyzer. Rapid Communications in Mass Spectrometry, 17, 419-428.
Map Captions
Map 1. Robinson Forest is an approximately 6,000 ha experimental forest located in portions of
Breathitt, Perry and Knott counties Kentucky. The forest is composed of eight discontinuous
properties, with the main block comprising approximately 4,200 ha.
Map 2. This study was conducted on the main block (outlined in black) and the Laurel Fork tract
(outlined in blue) of Robinson Forest. The main block primarily consisted of second-growth
intact forest that has received little disturbance since it was last logged in the 1920s. The Laurel
Fork tract (now referred to as the Paul Van Booven Wildlife Management Area – PVB WMA)
was surface mined for coal in the late 1990s and early 2000s. The aerial photo shows extensive
active mining (gray area) surrounding the study sites.
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