Source Materials for IHSS Samples

Standard Samples

At the time of its inception, the IHSS decided to collect, distribute, and maintain standard samples that were isolated from soil, peat, leonardite, and river water. By definition, an IHSS standard sample must satisfy all four of the following criteria:

  1. the sample must have come from a site specifically designated by the IHSS for this purpose;
  2. the sample must have been prepared according to a specific procedure designated by the IHSS;
  3. the operations involved in (1) and (2) must have been conducted under the direct supervision of the IHSS;
  4. the sample must be designated as a standard by the IHSS.

Upon depletion of the existing supply of a standard humic or fulvic acid, another batch of that standard will be prepared from the existing stock of bulk soil, peat, or leonardite, or from the Suwannee River. Successive batches of a standard material are expected to be very similar but not necessarily identical. The sources of the four standard samples are:

Suwannee River – The Suwannee River rises in the Okefenokee Swamp in south Georgia and flows southwest to the Gulf of Mexico. The Okefenokee Swamp contains extensive peat deposits; however, decomposing vegetation is believed to provide most of the dissolved organic carbon (DOC) to its waters. At its headwaters in the Okefenokee Swamp, the Suwannee River is a blackwater river, with DOC concentrations ranging from 25-75 mg/L and pH values of less than pH 4.0. At the time of collection of the IHSS standard aquatic samples, the water level in the Okefenokee Swamp was regulated by a series of dams in a sill along the western edge of the swamp. The IHSS samples were collected at the southernmost dam on this sill.

Additional information about the Suwannee River and the Okefenokee Swamp is found in: Humic Substances in the Suwannee River, Georgia: Interactions, Properties, and Proposed Structures, R. C. Averett J. A. Leenheer, D. M. McKnight, and K. A. Thorn, Editors, USGS Water-Supply Paper 2373 (1994).

Elliott Soil – The Elliott soil is typical of the fertile prairie soils of the U.S. states of Indiana, Illinois, and Iowa. The IHSS sample was obtained from an undisturbed area on the grounds of the Joliet Army Ammunition Plant near Joliet, Illinois. The Elliott series consists of very deep, somewhat poorly drained soils on moraines and till plains. They formed in as much as 20 inches of loess or silty material and silty clay loam glacial till. Permeability is moderately slow in the solum and moderately slow or slow in the underlying material. Slope gradients range from 0 to 7 percent. Mean annual precipitation is about 33 inches, and mean annual air temperature is about 50 degrees F.

Taxonomic Class: Fine, illitic, mesic Aquic Arguidolls.

Typical Pedon: Elliott silt loam ñ on a west-facing slope with less than a 1 percent gradient in a cultivated field (Colors are for moist soil unless otherwise stated).

Ap – 0 to 6 inches; black (10YR 2/1) silt loam, dark gray (10YR 4/1) dry; moderate fine granular structure; friable; common fine roots; moderately acid; abrupt smooth boundary.

A – 6 to 11 inches; black (10YR 2/1) silty clay loam, dark gray (10YR 4/1) dry; moderate fine granular structure; friable; common fine roots; slightly acid; clear smooth boundary. (Combined thickness of the A horizons is 10 to 20 inches.)

For more information see the USDA-NRCS soil series description for the Elliott series.

Pahokee Peat – The Pahokee peat is a typical agricultural peat soil of the Florida Everglades. The IHSS sample was obtained from the University of Florida Belle Glade Research Station. The Pahokee series consists of very poorly drained soils that are 36 to 51 inches thick over limestone. Pahokee soils formed in organic deposits of freshwater marshes.

Taxonomic Class: Euic, hyperthermic Lithic Medisaprists

Typical Pedon: Pahokee muck – cultivated

Oap – 0 to 10 inches; black (10YR 2/1) muck; less than 5 percent unrubbed fiber; moderate coarse subangular blocky structure; very friable; 10 percent mineral content; neutral; abrupt smooth boundary. (6 to 12 inches thick)

Oa1 – 10 to 28 inches; black (5YR 2/1) muck; 65 percent unrubbed fiber, 10 percent rubbed fiber; massive; 10 percent mineral content; medium acid; gradual smooth boundary.

For more information see the USDA-NRCS soil series description for the Pahokee series.

Leonardite – Leonardite is produced by the natural oxidation of exposed lignite, a low-grade coal. The IHSS sample was obtained from the Gascoyne Mine in Bowman County, North Dakota, U.S.A.

Large quantities of the three solid-phase bulk source materials (Elliott soil, Pahokee peat, and Gascoyne leonardite) were air-dried and then sieved to remove pebbles and gross fibrous matter. After they were homogenized, a sufficient quantity of each source material was set aside for isolation of the standard humic and fulvic acids, and the remainder of each source material was stored. These bulk source materials are available from the IHSS. They are intended for various uses, but are expected to be used primarily to evaluate new isolation procedures.


Reference Samples

The IHSS has also collected and distributed several reference samples that were isolated from soil, peat, and natural waters. By definition, an IHSS reference sample does not satisfy at least one of the criteria for standard humic substances, but does meet both of the following conditions:

  1. the sample must have been prepared from a clearly designated source material, and its preparation must be described in detail;
  2. the material must have been homogenized by the IHSS and must be distributed by the IHSS.

Upon depletion of the existing supply of a reference humic or fulvic acid, that sample might not be replenished. The sources of the reference samples are:

Suwannee River NOM – The same sampling site was used for the standard and reference samples (see previous description).

Pahokee Peat – The same sampling site was used for the standard and reference samples (see previous description).

Hellrudmyra – The reference Nordic humic acid and fulvic acid were collected at this small tarn (700 m2), which is located in a small (8 ha), relatively isolated catchment not far from Oslo, Norway. The tarn has served as reference water for most of the humus research in Norway during the last 30 years. DOC concentrations in Hellrudmyra range from 10-25 mg/L, and pH ranges from 4.0 to 4.9.

Summit Hill Soil – The Summit Hill soil (Orthic Brown Soil (NZ); Typic Dystrudept (USDA)) was taken from a tussock grass area adjacent to the Ahuriri reserve on the southern side of Summit Hill. Summit Hill is in the Port Hills to the south of the city of Christchurch on the South Island of New Zealand in a area with an a annual rainfall of about 1000 – 1100 mm. The soil parent material is loess derived from greywacke rock. The sample was obtained from the Ah horizon (0 – 15 cm) on a moderately steep west facing slope at approximately 350m above sea level. This soil had a carbon content of 4.3 %.

Profile description:
Ah – 0-15 cm; very dark greyish brown (10YR3/2) silt loam; friable; moderately developed fine nut structure; numerous roots; gradual boundary,

Bw – 15-25 cm; yellowish brown (10YR5/4) silt loam; friable; weakly developed fine nut structure; many very dark greyish brown (10YR3/2) worm casts from above; many fine roots; clear boundary

Bw2 – 25-60 cm; yellowish brown (10YR5/4) silt loam (heavier than above); friable; weakly developed fine nut structure; few fine roots; clear boundary,

BC – 60-85 cm; yellowish brown (2.5Y5/4) silt loam; friable; massive; abundant fine and coarse distinct strong brown (7.5YR 5/60 mottles; few small fragments of tuff; very few fine roots.

Waskish Peat – The IHSS Waskish peat was collected in Pine Island Bog in Koochiching County, Minnesota. This is a Sphagnum bog peat typical of northern temperate regions. The Waskish series consists of deep, very poorly drained organic soils that formed mostly in slightly decomposed material from Sphagnum moss on raised bogs on glaciated terrain. Mean annual precipitation is about 56 cm. Mean annual temperature is about 4oC.

Taxonomic Class: Dysic, frigid Typic Sphagnofibrists

Typical Pedon: Waskish peat – on slightly convex slope of less than 1 percent on the crest of a large raised bog on a glacial lake plain under a dense stand of black spruce with ground cover of Sphagnum and Hypnum mosses and Labrador tea.

Oil – 0 to 3 inches; very pale brown (10YR 7/3, broken face) fibric material, very pale brown (10YR 8/4, rubbed and pressed); about 90 percent fiber, about 85 percent rubbed; massive; nonplastic; nonsticky; fibers are primarily Sphagnum; about 10 percent woody fragments; about 7 percent mineral material; extremely acid; clear wavy boundary.

Oi2 – 3 to 14 inches; brown (7.5YR 4/4, broken face) fibric material, dark reddish brown (5YR 3/4, pressed), dark reddish brown (5YR 3/3, rubbed); about 90 percent fiber; about 85 percent rubbed; weak coarse platy structure; nonplastic; nonsticky; fibers are primarily Sphagnum moss; about 15 percent woody fragments; about 6 percent mineral material; few thin layers of sapric material; extremely acid; abrupt smooth boundary.

Oa – 14 to 16 inches; dark brown (7.5YR 3/2, broken face) sapric material, very dark gray (5YR 3/1, rubbed); about 20 percent fiber; about 10 percent rubbed; common medium distinct grayish brown (10YR 5/2) mottles; weak thin platy structure; nonplastic; slightly sticky; mixed Sphagnum moss and herbaceous fiber; about 10 percent woody fragments; about 12 percent mineral material; extremely acid; abrupt smooth boundary.

Oi – 16 to 84 inches; reddish brown (5YR 4/4, broken face) fibric material, brown (7.5YR 5/4, pressed), light yellowish brown (10YR 6/4, rubbed); about 95 percent fiber; about 90 percent rubbed; weak medium and coarse platy structure; nonplastic; nonsticky; fibers are primarily Sphagnum moss; about 5 percent woody fragments; about 3 percent mineral material; about 4 percent thin layers of sapric material; extremely acid.

For more information see the USDA-NRCS soil series description for the Waskish peat.

Nordic Reservoir NOM – This sample was obtained from a drinking water reservoir at Vallsjøen, Skarnes, Norway on October 29 to November 3, 1997. The reservoir is at 225 m above sea level and has a maximum depth of depth of about 14 m. The sample was obtained from the Sør-Odal County Waterworks intake pipe that draws water from at depth of 10m (pH = 5.6, EC = 2.1 mS/m and DOC = 10.7 mg/L). The water temperature was 4°C.

Pony Lake FA reference fulvic acid (1R109F) – It is a purely microbially derived fulvic acid. Pony Lake is a eutrophic, saline coastal pond named after the ponies brought to Antarctica during Ernest Shakelton’s Nimrod expedition in 1907. It was sampled during the austral summer of 2005-2006 by a team consisting of D. McKnight, K. Cawley, C. Jaros, R. Fimmen, R. Cory, P. Miller, Y-P. Chin, C. Foreman from The Ohio State University and the University of Colorado. They collected and processed a FA sample from Pony Lake in Antarctica.

The chemical characteristics of the FA fractions isolated over the time period from 1992 to 2007 show remarkable consistency despite dramatic changes in physical conditions such as extent of ice cover and timing of surface melt. Pony Lake was chosen as the water source for an aquatic microbial end member FA for several reasons: (1) The landscape surrounding the site is devoid of terrestrial plants. Therefore, all of the dissolved organic matter (DOM) in the pond is of microbial origin. (2) The DOC content of that water is very high compared to other Antarctic lakes and ponds. (3) The site was accessible by helicopter, making it possible to bring the water sample back to the laboratory at McMurdo Station for processing. (4) There is extensive background information about the biogeochemistry of the site and it appears to have been a stable ecosystem since the site was occupied by Shackleton in 1907.

In January 2006, 15 m3 of Pony Lake water was collected and filtered through a 100 µm spiral wound pre-filter, a 25 µm Balston glass fiber filter, and finally a 0.5 µm Balston glass fiber filter. Following filtration, the water was pumped into acid washed and DI rinsed 55 gal drums made from high-density polyethelene. The drums were filled on site with filtered water at a rate of one every 30 minutes. The drums were transported via helicopter to Crary Laboratory (McMurdo Station, Antarctica) where the water was acidified within 48 hours to pH 2 (+/- 0.1) and then processed using the method described in Thurman and Malcolm (1981). Briefly, the acidified water was run through a 4 L column packed with XAD-8 resin. The Pony Lake water was applied to the column at a rate of about 900 mL min-1. Once 360 L of Pony Lake water was applied, the column was eluted using 0.1N NaOH. The eluate was immediately acidified and stored in a 4°C environmental room covered in foil to prevent photochemical degradation. The eluate was then reconcentrated on a 2 L column. While on the 2 L column, the sample was rinsed with distilled water to remove chloride. Following the DI rinse, the 2 L column was eluted using 0.1 N NaOH. The eluate was applied to a cation exchange column in order to lower the pH to neutral and hydrogen saturate the FA isolate. Once the sample had been cation exchanged twice, it was lyophilized on site using a tray freeze-dryer. The freeze dried powder was then packaged in acid washed and DI rinsed polyethylene bottles for transport and storage.

Chemical analysis of FA samples isolated from Pony Lake since 1994 and the 2005/06 season show characteristics strongly indicative of microbial origin, such as high nitrogen content and low aromaticity (Table 1 and 2). The 13C NMR data were collected using the methods of Dria et al. (2002). NMR analysis shows that the ratio of aromatic (110-160 ppm) to aliphatic carbon (0-60 ppm) is low, indicating that the FA contains few aromatic functional groups, while it does have abundant carbohydrate-type functional groups from cellular structures and by-products.


Table 1: Elemental analysis for fulvic acid isolates from Pony Lake

Sample date C H N O S Ash
1/1994 48.9 6.2 4.4 40.4 10.1
12/1997 * 49.9 6.09 4.9 35.8 3.22 7.53
1/2005 49.97 5.94 6.09 34.73 3.03 3.07
1/2006 (1R109F) 52.47 5.39 6.51 31.38 3.03 1.25

 * Brown et al., 2004


Table 2: 13C NMR analysis of fulvic acid isolates from Pony Lake.  The chemical shifts correspond to: 0-60 ppm aliphatic and methoxy groups, 60-90 ppm 0-alkyl, 90-110 ppm anomeric carbon, 110-160 ppm aromatic groups, 160-190 ppm amides and esters, and 190-230 ppm ketones.

Sample date 0-60 ppm 60-90 ppm 90-110 ppm 110-160 ppm 160-190 ppm 190-230 ppm
1/1994 42 16.6 6.6 16.5 16.7 2.2
12/1997 53.3 16.3 3.8 11.7 13.8 1.1
1/2005 62.3 10.9 0.9 10.9 17.3 2.2
1/2006(1R109F) 60.8 8.4 0.2 12.1 17.4 1.2


Acknowledgements. Thanks to J. Guerard, A. Roche, M. Dieser, and D. Rosenberger for lab and field work in Antarctica.  Field support: Raytheon Polar Services and Petroleum Helicopters Inc. Funding: The National Science Foundation (OPP-0338299) and The International Humic Substances Society.


(1) Brown, A., McKnight, D.M., Chin, Y.P., Roberts, E.C., and Uhle, M., 2004. Chemical characterization of dissolved organic material in Pony Lake, a saline coastal pond in Antarctica. Marine Chemistry, 89: 327-337.

(2) Cory, R. and McKnight, D.M., 2005. Fluorescence spectroscopy reveals ubiquitous presence of oxidized and reduced quinones in dissolved organic matter. Environmental Science and Technology, 39: 8142-8149.

(3) Dria, K., Sachleben, J., and Hatcher, P., 2002. Solid-state carbon-13 nuclear magnetic resonance of humic acids at high magnetic field strengths. Journal of Environmental Quality, 31: 393-401.

(4) McKnight, D.M., Andrews, E.D., Aiken, G.R., and Spaulding, S.A., 1994. Dissolved humic substances in eutrophic coastal ponds at Cape Royds and Cape Bird, Antarctica. Limnology and Oceanography, 39: 1972-1979.

(5) McKnight, D.M., Boyer, E.W., Westerhoff, P.K., Doran, P.T., Kulbe, T., Anderson, D.T., 2001. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnology and Oceanography, 46: 38-48.

(6) Thurman, E. M. and Malcolm, R. L. 1981. Preparative isolation of aquatic humic substances. Environmental Science and Technology, 15: 463-466.


The IHSS does not maintain or distribute bulk source materials for the reference samples.