EFFECT OF MANURE AND CROP RESIDUES CONTINUOUS APPLICATION ON HUMIC C IN A CULTIVATED SOIL - Soil Ecology Research Developments - page 220

stored under vacuum over silica gel. HAs were extracted from individual replicate plots and
were then combined before analyses.
Total Organic C and δ 13 C
Total organic C (TOC) in soils and HA samples were measured with an elemental
analyzer (Thermo Finnigan mod. EA 1110). The percentage of C in the sample was calculated
using acetanilide as a certified standard containing 71.09 % of carbon. The HA samples were
weighed in tin capsules and analyzed directly, whereas the soil samples were pretreated with
6 M HCl in order to eliminate the carbonate-bound C. About 10 mg of finely ground dry soil
were weighed in silver capsules placed on a cast iron plate. A few drops of acid solution were
added directly to the capsules and the plate was kept at 80 °C until the soil was dry. The
samples were measured using CF-IRMS (Continuous Flow-Isotope Ratio Mass Spectrometry)
by entering the CO 2 from the elemental analyzer into an IRMS (Isotope Ratio Mass
Spectrometer mod. Delta Plus Thermo Electron, Bremen, Germany). The C isotope values
were expressed in delta notation (δ), where: δ(‰) = [(R sample /R standard )-1 x 1000, and R sample
and R standard are 13 C/ 12 C of the sample and standard, respectively. Delta 13 C is referred to the
Vienna Pee Dee Belemnite (VPDB) standard [35]. All measurements were made in triplicate
for each sample. The reproducibility of the delta 13 C values of the samples was, in 90 % of the
cases, better than 0.1 ‰. The variation coefficient was < 0.1%.
Potentiometric titration. Samples were prepared by dissolving 12 mg of the freeze-dried
HA in 20 mL of Milli-Q Millipore water containing 0.05 M NaCl to keep the ionic strength
constant. The pH was adjusted to pH 3 by adding about 1 mL of 0.05 M HCl. The solutions
were titrated to pH 10.5 with 0.05 M NaOH using a VIT 90 Titrator Radiometer (Radiometer,
Copenhagen, DK). The potentiometric titrations were carried out in triplicate at 25 °C, under
N 2 flow and the delivery range was 10 μ L min -1 (± 0.01). The first derivative method was
performed to determine the concentration of COOH groups.
DRIFT Spectroscopy
The spectra were recorded with a Nicolet Impact 400 FT-IR Spectrophotometer
(Madison, WI) equipped with an apparatus for diffuse reflectance (Spectra-Tech. Inc.,
Stamford, CT). Spectra were collected as Kubelka-Munk units using KBr (Aldrich Chemical
Co.) as the background reference. The sample chamber was continuously flushed with CO 2
free dry air. DRIFT spectra were collected from 4000 to 400 cm -1 and averaged over 200
scans (resolution ± 4 cm -1 ). Analyses of spectral data were performed with Grams/386
spectral software (Galactic Industries, Salem, NH). Spectral sections from 1850 to 800 cm -1
were baseline-corrected to an absorbance value of 0.00 at 1850 cm -1 .