CSIRO PUBLISHING
www.publish.csiro.au/journals/ajsr
Australian Journal of Soil Research, 2005, 43, 239–249
Organic compounds at different depths in a sandy soil and their role
in water repellency
C. P. MorleyA,D , K. A. MainwaringA , S. H. DoerrB , P. DouglasA , C. T. LlewellynA , and L. W. DekkerC
A Department
of Chemistry, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK.
of Geography, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK.
C Alterra, Land Use and Soil Processes Team, PO Box 47, 6700 AA Wageningen, The Netherlands.
D Corresponding author. Email: [email protected]
B Department
Abstract. The causes of soil water repellency are still only poorly understood. It is generally assumed that
hydrophobic organic compounds are responsible, but those concerned have not previously been identified by
comparison between samples taken from a water repellent topsoil and the wettable subsoil. In this study we separated,
characterised, and compared the organic compounds present at 4 different depths in a sandy soil under permanent
grass cover that is water repellent in the upper 0.30 m but wettable below this.
Soil samples were extracted using a mixture of isopropanol and aqueous ammonia (7 : 3 v : v). Samples
were wettable after extraction and re-application of the extract from each sample onto wettable sand induced
water repellency. The chloroform-soluble portions of the extracts were analysed by gas chromatography
and gas chromatography-mass spectrometry. The compounds identified at all soil depths included longchain carboxylic acids (C16 –C24 ), amides (C14 –C24 ), alkanes (C25 –C31 ), aldehydes or ketones (C25 –C29 ),
and more complex ring-containing structures. 1 H and 13 C nuclear magnetic resonance spectroscopy, and the
carbon/hydrogen ratio as determined by microanalysis, confirmed the predominantly aliphatic character of
the extracts.
Both wettable and water repellent samples contained hydrophobic compounds. The 3 water repellent samples
contained far more organic material, although the amount extracted was not related to the degree of water repellency.
Perhaps more importantly, they contained polar compounds of high relative molecular mass, which were almost
absent from the wettable subsoil. It may be speculated that these are the compounds in this soil whose presence in
significant amounts is necessary for water repellency to be exhibited.
Additional keywords: hydrophobicity, gas chromatography.
Introduction
It is widely accepted that soil water repellency is caused by
hydrophobic organic compounds deposited on soil mineral
or aggregate surfaces or present as interstitial matter (Wallis
and Horne 1992; Doerr et al. 2000). The organic matter
from which these compounds are derived may originate from
decomposing soil organic matter (McGhie and Posner 1981)
or, more specifically, from plant root exudates (Dekker and
Ritsema 1996; Doerr et al. 1998), certain fungal or microbial
species (Bond and Harris 1964; Jex et al. 1985; Hallett et al.
2001), and surface waxes from plant leaves (McIntosh and
Horne 1994). The types of organic compounds suggested
in previous studies to cause repellency include plant and
cuticular waxes (McIntosh and Horne 1994), alkanes (Savage
et al. 1972; Ma’shum et al. 1988; Roy et al. 1999; Horne
and McIntosh 2000), fatty acids and their salts and esters
(Schnitzer and Preston 1987; Ma’shum et al. 1988; Franco
et al. 2000; Hudson et al. 1994; Roy et al. 1999; Horne and
© CSIRO 2005
McIntosh 2000), and phytanes, phytols, and sterols (Franco
et al. 1994).
Recent publications on water repellency by Roy et al.
(1999) and Doerr et al. (2000) have, however, argued that
despite the significant advances made in previous studies,
sufficient separation and exact chemical characterisation
of these compounds have yet to be achieved and that
consequently the molecular basis of water repellency is still
only poorly understood. Our ongoing research is focussed
on this gap in current knowledge. Its overall objective is the
establishment of a reliable and widely applicable procedure
to separate and identify organic compounds present in water
repellent soils.
Achieving a fundamental understanding of the
(bio)chemical origin of water repellency is critical not
only in the amelioration of repellency by, for example,
developing more effective and environmentally friendly
wetting agents, it is also important in allowing mankind
10.1071/SR04094
0004-9573/05/030239
240
Australian Journal of Soil Research
C. P. Morley et al.
to balance the detrimental impacts of repellency with its
beneficial effects such as the enhanced stability of soil
organic carbon (Piccolo et al. 1999), reduced evaporative
water losses, or increased aggregate stability (DeBano 2000;
Doerr et al. 2000).
We have examined a range of extraction procedures
used in previous studies and demonstrated that compounds
capable of causing water repellency can be extracted from a
wide range of sandy soils, using a mixture of isopropanol
and aqueous ammonia, and that these extracts induce
hydrophobicity when applied to acid-washed sand (Doerr
et al. 2005, this issue). The present paper outlines the
results obtained on the separation and characterisation by
gas chromatography (GC) and gas chromatography-mass
spectrometry (GC-MS) of compounds present in extracts
obtained in this way from 3 water repellent and 1 wettable
samples taken from a sandy soil under permanent grass cover
that is water repellent in the upper 0.30 m, but wettable below
this depth. This study was designed to establish whether
differences in soil water repellency may be associated with the
occurrence of particular compound types or variations in their
relative abundance.
Materials and methods
Soil samples
The soil studied consists of non-calcareous dune sand material and is
located near Ouddorp in the south-western part of the Netherlands (WGS
coordinates 51◦ 48 51.2 N, 3◦ 54 32.4 E). The area experiences a humidtemperate climate (mean monthly temperatures vary between 1.7◦ C in
January and 17.0◦ C in July) with rainfall occurring throughout the year
(annual precipitation is 765 mm). During the growing season there is a
small precipitation deficit, in autumn and winter a precipitation surplus
(Dekker et al. 2000). The soil is of uniform sand texture (predominantly
220–270 µm) with <0.1% clay to a depth of c. 0.70 m, and has been
classified as mesic Typic Psammaquent (Dekker et al. 1998). It has an
approximately 0.10-m-thick humous surface layer with c. 36% total
organic carbon content, which decreases to <0.1% below 0.30 m depth
(see Table 2). The soil at this site is well drained and is known to exhibit
severe to extreme water repellency when it dries to less than c. 14%
and 3% soil moisture (by volume) in the top 0.10 m and 0.10–0.20 m,
respectively (Dekker et al. 2001). As a result, recurring preferential
flow pathways can develop during rainfall events following prolonged
dry periods (Dekker and Ritsema 1994). The site is grass-covered, is
in use as pasture, and has not been tilled for at least several decades.
Soil samples were taken at 4 depths (0–0.10, 0.10–0.20, 0.20–0.30,
0.30–0.40 m), dried at 20◦ C, passed through a 2-mm sieve, coned and
quartered (see Doerr et al. 2005, this issue), and stored in plastic bags
prior to further analysis. It should be noted that apart from some
larger organic debris, all soil components passed the 2-mm sieve,
resulting in an unaltered textural composition of the sample material.
The 3 samples taken from the top 0.30 m exhibited considerable water
repellency when dry, whereas the sample taken at 0.30–0.40 m depth
was wettable. Sample characteristics are summarised in Table 1. Total
organic carbon (TOC) data were determined on at least 3 subsamples
using a Skalar PrimacsSC TOC Analyzer. Inorganic carbon content
was negligible, so total carbon content was used as a measure of
the TOC content.
Water repellency assessments
Water repellency levels of the soil samples were determined using
the Water Droplet Penetration Time (WDPT) method (Letey 1969)
after equilibrating subsamples to a controlled atmosphere of 20◦ C and
45–55% relative humidity for 24 h in order to avoid any influence of
changing atmospheric conditions on the measurement results (Doerr
et al. 2002). Five droplets of distilled water (∼80 µL) were placed
on the soil surface and the time recorded for droplet penetration.
Eleven WDPT time intervals were distinguished (e.g. Doerr 1998),
which correspond to 5 descriptive ratings of water repellency, based
on the median time needed for the water droplets to penetrate into
the soil: non-repellent (infiltration within 5 s), slightly (>5, 10, 30,
60 s), strongly (>60, 180, 300, 600 s), severely (>600 s, 900 s, 1 h),
and extremely water repellent (>1 h) (Bisdom et al. 1993). Although
this widely used non-parametric method does not necessarily relate
well to the wetting properties of in situ soil, it allows the relatively
rapid and reproducible evaluation of the persistence of the water
repellency of dry soil samples under laboratory conditions as required
in this study. After additional heating at 105◦ C for 24 h and subsequent
equilibration of subsamples as described above, further WDPT
measurements were performed, as previous studies have shown that
samples classified as wettable after drying at 20◦ C may develop
water repellency after heating to 105◦ C (Ma’shum et al. 1988; Dekker
et al. 1998).
Compound extraction, separation, and identification
Extraction procedure
Soil samples (240 g) were extracted in a Soxhlet apparatus for
24 h using a 2.4-L isopropanol : aqueous ammonia (35%, 0.88 SG)
(7 : 3 v : v) solvent mixture (Doerr et al. 2005, this issue). It was
necessary to pre-wet the samples with the solvent mixture for 15 min
prior to refluxing, as ammonia is lost during the extraction procedure.
The extracted material dissolved in the solvent was then filtered,
concentrated under reduced pressure on a rotary evaporator at 45◦ C,
transferred to a pre-weighed porcelain evaporating dish, and taken to
dryness on a hot water bath. A blank consisting of a clean, empty
cellulose thimble was also extracted to provide a correction factor for the
presence of residual organics in the solvents and/or Soxhlet apparatus.
Table 1. Sample codes and water repellency levels of samples before extraction
Water Droplet Penetration Time classes (WDPT, s) and associated repellency ratings are detailed
in the Materials and methods
Sample
code
NL1
NL2
NL3
NLC
Sampled depth
(m)
WDPT class
(dried at 20◦ C)
Repellency
rating
WDPT class (s)
(dried at 105◦ C)
Repellency
rating
0–0.10
0.10–0.20
0.20–0.30
0.30–0.40
180
3600
18 000
<5
Strong
Severe
Extreme
Non-repellent
18 000
18 000
3600
<5
Extreme
Extreme
Severe
Non-repellent
Organic compounds and water repellency
Extract re-applications
The procedures of Ma’shum et al. (1988) were followed. A portion of
the dried extract was re-dissolved in chloroform, filtered, and re-applied
to 5 g of wettable (WDPT <5 s) acid-washed sand (AWS; quartz sand
washed with hydrochloric acid, supplied by Riedel de Haën). The ratio
of sand to extract was chosen such that it was reapplied at the same
mass ratio as it was extracted. Based on laser particle size analysis
(Malvern Mastersizer), the AWS used has a mean diameter of 270 µm
and distribution width of ±70 µm, which is very similar to the particle
size of the soil samples used in this study.
Separation of extracted material
The dried extract was dissolved in 200 mL of chloroform and
water mixture (1 : 1 v : v). The phases were separated and washed with
2 × 100 mL aliquots of the other solvent. The aqueous phase contains
amphipathic and polar material and the chloroform phase the lipid
compounds (Horne and McIntosh 2000). Both phases were taken to
dryness on a hot water bath (although only the chloroform phase was
used in this study).
Gas chromatography and gas chromatography-mass
spectrometry analysis
The dried chloroform phase was dissolved in tetrahydrofuran (THF,
chosen in preference to chloroform because it dissolved more material),
filtered through tightly packed glass wool in order to remove insoluble
particles, and analysed by GC and GC-MS. Chromatograms were
obtained using a Hewlett Packard 5890 series II gas chromatograph
equipped with a flame ionisation detector (FID) and a ZB5 5% phenyl
polysiloxane coated capillary column (30 m, 0.32 mm i.d., 1.0 µm
diameter film). Samples (2 µL) were injected splitlessly (0.6 min)
and helium was used as the carrier gas. The oven temperature was
programmed from 210◦ C to 280◦ C at 2◦ C/min and held there for 2.5 min
followed by a second program of 15◦ C/min to a final temperature
of 310◦ C (held for 80 min). The injection port was set at 250◦ C.
A Fisons GC8000 gas chromatograph interfaced directly with a Fisons
Masslab MD800 low-resolution GC-MS instrument was used to obtain
electron impact spectra. The Fisons GC8000 gas chromatograph also
contained a ZB5 5% phenyl polysiloxane capillary column (30 m,
0.32 mm i.d., 0.25 µm diameter film). Splitless, 1-µL injections with
hydrogen carrier gas and a temperature program of 40◦ C isothermal for
2 min, then ramped at 10◦ C/min to 300◦ C and held for 30 min were used.
The injection port was set at 250◦ C. Compounds were identified based
on retention times, mass spectral interpretation, use of the NIST mass
spectral search program and NIST/EPA/NIH mass spectral library v.2.0,
and comparison with authentic compounds where possible.
Microanalysis
Microanalysis of the extracts was performed by Butterworth
Laboratories Ltd at Teddington in Middlesex using a Leeman Labs Inc.
Model 44 CHN Elemental Analyser.
Nuclear magnetic resonance (NMR) spectroscopy
1
H and 13 C NMR spectra of the chloroform-soluble portions of the
extracts were recorded in deuterated chloroform at 400 MHz on a Bruker
Avance 400 Spectrometer.
Results and discussion
The total organic carbon content and the amount of material
extracted were found to decrease with sampling depth
(Table 2). The wettable subsoil yielded the least extract.
A similar difference in extracted amounts between water
repellent and wettable soils was observed by Hudson (1992)
Australian Journal of Soil Research
241
Table 2. WDPT classes and associated repellency ratings resulting
from re-application of extracts to wettable acid-washed sand
Water Droplet Penetration Time classes (WDPT, s) and associated
repellency ratings are detailed in the Materials and methods
Extract
from
NL1
NL2
NL3
NLC
WDPT
class
(dried at
20◦ C)
Repellency
rating
WDPT
class
(dried at
105◦ C)
Repellency
rating
3600
18 000
18 000
3600
Severe
Extreme
Extreme
Severe
>18 000
>18 000
>18 000
18 000
Extreme
Extreme
Extreme
Extreme
and Hudson et al. (1994). It should be noted, however, that
there is no clear general relationship between either the total
organic carbon content, or the amount of organic matter that
can be extracted, and the degree of soil water repellency
(Doerr et al. 2005, this issue).
For the water repellent soil samples dried at 20◦ C, the
severity of water repellency increased with depth. The reverse
was observed, however, for the soil samples that had been
dried at 105◦ C (see Table 1), a trend which had been noted
previously by Dekker et al. (2000). We have not attempted
to interpret this behaviour, as the most recent work by
us and others (Doerr et al. 2005, this issue; Ziogas et al.
2005, this issue) has shown that the effects of drying water
repellent soil samples at 105◦ C are not consistent across
all soil types.
The re-application of each extract to wettable acid-washed
sand induced water repellency, confirming the presence in all
the extracts of compounds that can cause water repellency
(Table 3). Extraction of empty Soxhlet thimbles was carried
out in order to demonstrate that there was no significant
contribution to the extract from the apparatus used. Only trace
quantities of material (<3 mg per extraction) were obtained
in this way.
Each extract was partitioned between chloroform and
water, following the procedures of Ma’shum et al. (1988).
The GC-MS chromatograms of the THF-soluble portion of
the dried chloroform phases are shown in Fig. 1. Similarities
Table 3. Mass of material extracted from each sample and total
organic carbon content (TOC) before and after extraction (g/kg)
Sample
code
NL1
NL2
NL3
NLC
TOC
Pre-extractionA Post-extractionA
36.2 (± 2.9)
5.9 (± 0.3)
0.8 (± 0.2)
n.d.
25.6 (± 1.0)
3.5 (± 0.2)
n.d.
n.d.
Mass
extractedB
9.76 ± 1.49
2.64 ± 0.33
1.10 ± 0.59
0.55 ± 0.23
n.d., Not detected.
A
Based on at least 3 subsamples.
B
Based on at least 4 determinations. Masses are corrected for the
contribution of the apparatus (2 ± 1 mg per sample).
242
Australian Journal of Soil Research
C. P. Morley et al.
15–17 18–20
21–23
9–11
100
12–14
(a)
24–26
27–28
6–8
1 2
31–35
36
37
3–5
50
29
30
0
12–14
9–11
100
(b)
21–23
15–17
18–20
24–26
1
6–8
3–5
27–28
2
50
31–35
36
37
29
30
(%)
0
21–23 18–20
100
(c)
12–14
6–7
50
9–11
4
3
27
24–26
28
15–17
5
29
31–35
1
8
2
30
36
37
0
9–11
100
12–14
(d )
3–5
1
50
15–17
18–20
6–8
21–23
24–26
27–30
2
31–35
36
37
0
10.00
15.00
20.00
25.00
30.00
35.00
Time (min)
Fig. 1. GC-MS chromatograms of tetrahydrofuran-soluble extracts from (a) NL1, (b) NL2, (c) NL3, and (d) NLC.
in the chromatograms were expected, as the 4 samples
were obtained from the same soil at 4 different depths
as summarised in Table 1, and hence would have been
exposed to very similar sources of organic material. In
order to compare the results the main peaks in each
chromatogram have been numbered 1–37 (Fig. 1). The
assignments of these peaks are listed in Table 4. In some
cases it was also possible to confirm the identities of the
compounds by comparing the retention times and mass
spectra obtained with those of purchased authentic standards.
The compounds whose identities have been confirmed by the
injection of purchased standards are indicated in bold text
in Table 4.
There were 5 main types of compounds identified by
GC-MS in all 4 samples, long chain fatty acids (C16 –C24 ),
amides (C14 –C24 ) of similar chain lengths to the acids,
slightly higher chain length alkanes (C25 –C31 ), aldehydes
or ketones (C25 –C29 ), and more complex ring-containing
structures. An unsaturated acid and its amide (C18 ), and
a phthalate (1,2-benzene dicarboxylate), were also present.
There were indications of the presence of alkenes, alcohols,
esters, and diols, but further work is needed (e.g. using
chemical ionisation mass spectrometry) before unequivocal
identifications of these compounds can be made. The
chemical structures of the compounds found to be present
in the soil extracts are shown in Fig. 2. Preliminary estimates
of relative abundances can be obtained on the basis of
peak heights. More precise quantitative determination of
extract composition using internal standards is already
in progress, but these initial measurements allow gross
differences between the chromatograms to be identified
and discussed.
GC-MS analysis indicated the presence of long-chain
fatty acids with 16–24 carbon atoms (C16 , C18 , C20 , C21 , C22 ,
C23 , C24 ). Even-chain acids predominated. A similar
distribution of mostly even-numbered long chain fatty acids,
but with 16–32 carbon atoms, was noted by Ma’shum
et al. (1988) in organic fractions responsible for water
repellency in some Australian soils. Research carried out
on water repellent soils by Franco et al. (1994) and
Organic compounds and water repellency
Australian Journal of Soil Research
243
Table 4. Assignments of selected peaks present in gas chromatography-mass spectrometry of the
extracts (see Fig. 1)
Compounds were identified based on retention times, mass spectral interpretation, use of the NIST mass
spectral search program and NIST/EPA/NIH mass spectral library v.2.0
Peak
no.
Retn time
(min)
1
2
3
4
5
6
7aE
7bE
8
9aE
9bE
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33aE
33bE
34
35
36
37
20.0
20.1
21.7
21.9
22.1
23.5
23.6
23.6
23.8
24.6
24.6
24.7
24.8
25.2
25.3
25.5
26.1
26.3
26.4
26.9
27.1
27.2
27.8
27.9
28.2
28.5
28.7
28.9
29.3
29.4
29.5
29.8
31.3
31.6
31.9
31.9
32.3
33.2
33.5
34.8
IdentificationA
Formula
n-Hexadecanoic acid
Tetradecanamide
Oleic acid (cis-9-octadecenoic acid)B
Octadecanoic acid
Hexadecanamide
4,8,12,16-tetramethylheptadecan-4-olide
9-octadecenamide
Eicosanoic acid (NL2 & 3 only)
Octadecanamide
Benzamide type compoundD
Heneicosanoic acid (NL1 only)
Pentacosane
Currently unidentified
Phthalate (1,2-benzene dicarboxylate)D
Docosanoic acid
Hexacosane
Tricosanoic acid
Heptacosane
Aldehyde/ketoneC
Tetracosanoic acid
Octacosane
Docosanamide
Nonacosane
Aldehyde/ketoneC
Currently unidentified
Triacontane
Tetracosanamide
Currently unidentified
Hentriacontane
Stigmasterol type compoundD
Aldehyde/ketoneC
Currently unidentified
AlkaneC
Aldehyde/ketoneC
d-Friedoolean-14-en-3-one
22, 23-Dihydrostigmasterol
Taraxerol
Stigmasta-3,5-dien-7-one
d-Friedoolean-14-en-3-ol, acetate (3β)
Currently unidentified
C16 H32 O2
C14 H29 NO
C18 H34 O2
C18 H36 O2
C16 H33 NO
C21 H40 O2
C18 H35 NO
C20 H40 O2
C18 H37 NO
Not known
C21 H42 O2
C25 H52
Not known
Not known
C22 H44 O2
C26 H54
C23 H46 O2
C27 H56
C25 H50 O
C24 H48 O2
C28 H58
C22 H45 NO
C29 H60
C27 H54 O
Not known
C30 H62
C24 H49 NO
Not known
C31 H64
Not known
C29 H58 O
Not known
Not known
Not known
C30 H48 O
C29 H50 O
C30 H50 O
C29 H46 O
C32 H52 O2
Not known
A
The identification of compounds in bold type has been confirmed by comparison of their spectra with
those of authentic samples.
B
Comparison with an authentic sample of the isomeric elaidic acid supports this assignment.
C
The molecular ion for this species was not detected.
D
Although the molecular formula and the principal functional groups present have been established, the
structures of these compounds could not be determined on the basis of their electron impact mass
spectra alone.
E
Only one peak was observed in the gas chromatograph, as 2 compounds co-elute.
Horne and McIntosh (2000) also showed the presence of
long-chain fatty acids. The presence of long-chain fatty
acids is not surprising, particularly in samples from an
undisturbed sandy soil as investigated here, since these
substances are particularly difficult to degrade (Hayes
and Graham 2000). They may originate from a range of
sources including plant cuticles (Hayes 1998). Fatty acids
are thought to contribute to the hydrophobic behaviour
that humic substances display in some circumstances
(Clapp et al. 1993).
244
Australian Journal of Soil Research
C. P. Morley et al.
CH 3 (CH 2 )nCO2 H
CH 3 (CH 2 )nCONH 2
CH 3 (CH 2 )nCH 3
(1)
(4)
(7b)
(9b)
(13)
(15)
(18)
(2)
(5)
(8)
(20)
(25)
(10)
(14)
(16)
(19)
(21)
(24)
(27)
n = 14
n = 16
n = 18
n = 19
n = 20
n = 21
n = 22
CH 3 (CH 2 )nCH =CH(CH2)nCO2 H
(3) n = 7
n = 12
n = 14
n = 16
n = 20
n = 22
n = 23
n = 24
n = 25
n = 26
n = 27
n = 28
n = 29
CH 3 (CH2 ) nCH=CH(CH2) nCONH2
(7a) n = 7
R
O
O
O
N
H
O
(6) C21 H40 O 2
(9a)
O
R
O
O
R
(12)
OH
O
HO
(33a) C30 H48 O
(34) C30 H50 O
(33b) C 29 H50 O
O
O
O
(35) C29 H46O
(36) C 32 H52O 2
Fig. 2. Structures of the compounds identified in the extracts.
A similar chain-length distribution of amides was
observed. For many of the long-chain acids, there appeared to
be an amide of the same chain length (e.g. C16 , C18 , C22, C24 ),
which makes it doubtful whether the amides were originally
present in the soils or were formed as a result of heating for
long periods in the presence of ammonia.
Alkanes were present with a slightly higher chain-length
distribution (C25−31 ) than that of the acids and amides.
Edlington et al. (1962) reported that most plant hydrocarbons
are straight chain, saturated compounds with an odd number
of carbon atoms. The chain length distribution of the alkanes
observed is shown in Fig. 3. There is a predominance of
compounds having an odd number of carbon atoms. Each
chain length between 25 and 31 carbon atoms was, however,
detected, which could suggest that not all the alkanes were
of plant origin. Alternatively, they may have been subjected
to decay or microbial/fungal attack. Franco et al. (1994)
and Horne and McIntosh (2000) also observed the presence
of alkanes.
Aldehydes or ketones having 25, 27, and 29 carbon atoms
were detected. The precise structures of these compounds
could not, however, be determined on the basis of the mass
spectra obtained. The ring-containing compounds observed
were predominantly stigmasterol derivatives.
An unsaturated acid was found to be present in the
NL1 and NL2 soil extracts. This is 9-octadecenoic acid,
which is oleic or elaidic acid depending on the cis- or
trans-orientation of the double bond. The mass spectrum
obtained appeared to be a better match with that of oleic acid.
The corresponding unsaturated amide was also identified,
9-octadecenamide, C18 H35 NO. The phthalate (benzene 1,2dicarboxylate) detected (peak 12) is most likely due to
contamination from the plastic bags that were used for storing
the soil samples.
All 4 soil sample extracts contained alkanes, in similar
relative abundances (see Fig. 3). The observed difference
in behaviour between NLC (the wettable sample) and the
others cannot therefore be ascribed to the presence or absence
Organic compounds and water repellency
Australian Journal of Soil Research
245
50
(a)
40
30
20
10
0
50
(b)
40
30
Relative abundance
20
10
0
50
(c)
40
30
20
10
0
50
(d )
40
30
20
10
0
10 (C25)
14 (C26)
16 (C27)
19 (C28)
21 (C29)
24 (C30)
27 (C31)
31 (?)
Peak number (chain length)
Fig. 3. Alkane distributions of tetrahydrofuran-soluble extracts from (a) NL1, (b) NL2, (c) NL3, and (d) NLC. Relative
abundance is calculated on the basis of peak height with respect to the sum of the peak heights of all the compounds shown.
of these hydrophobic compounds alone. Figure 4 shows
plots of the relative abundances of the other components
of the extracts. These are generally more polar materials,
all containing oxygen-based functional groups. Under the
conditions employed here for GC, the retention time is
determined by a combination of relative molecular mass
and polarity. Amongst this group of more polar compounds
therefore, smaller molecules elute first. It can be seen
from Fig. 4d that the extract from the wettable sample
contains only small amounts of the larger polar compounds.
In contrast, all 3 of the extracts from the water repellent
samples contain significant amounts of this material. There
are obviously variations in composition between NL1, NL2,
and NL3, and further work will be required to determine
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Australian Journal of Soil Research
14
C. P. Morley et al.
(a)
12
10
8
6
4
2
0
14
12
(b)
10
8
6
Relative abundance
4
2
0
14
(c)
12
10
8
6
4
2
0
14
(d )
12
10
8
6
4
2
0
1
2
3
4
5
6
7
8
9 11 12 13 15 17 18 20 22 23 25 26 28 29 30 32 33 34 35 36 37
Peak number
Fig. 4. Distribution of compounds (no alkanes) for tetrahydrofuran-soluble extracts from (a) NL1, (b) NL2, (c) NL3, and (d)
NLC. Relative abundance is calculated on the basis of peak height with respect to the sum of the peak heights of all the compounds
shown.
whether these can be related to the degree of water repellency,
but a clear distinction can be drawn between the water
repellent and wettable samples.
We do not, at present, have an explanation for the
distribution of high molecular mass polar compounds in
the soil profile. A previous study of soil from the same
site (Ritsema and Dekker 1996) highlighted the role of
preferred flow paths in the leaching of organic substances
from the topsoil, but to our knowledge, compoundspecific studies have not been reported. The mobility
Organic compounds and water repellency
of organic compounds in the soil, the transportive and
chemical effects of microorganisms, and the development
of water repellency as a consequence of changes in
compound distribution with time are areas which warrant
further investigation.
The mechanism whereby the presence of higher molecular
mass polar compounds might induce water repellency in
soils is not known. We can speculate that the inherently
lower solubility of these compounds in water is a key factor.
When water comes into contact with a wettable soil, the
lower molecular mass polar compounds will diffuse relatively
quickly and act in a detergent-like fashion to solubilise other,
inherently more hydrophobic material, such as alkanes. By
contrast, when water comes into contact with a water repellent
soil, the higher molecular mass polar compounds will diffuse
relatively slowly, so that the barrier to water penetration is
maintained for longer. It is also significant that the wettable
sample in this study has a smaller overall organic content
than the water repellent samples, so that the amount of
hydrophobic material that needs to dissolve is much less.
It is important to note, however, that recent work by us and
others (Roy and McGill 2000; Doerr et al. 2005, this issue)
has shown that simple presence or absence of a particular
group of compounds in a soil may not in itself be a sufficient
explanation for wettable or water repellent behaviour, and that
the distribution and molecular arrangement of the organic
material must also be considered. We have already begun
to undertake experiments on model systems, designed to
probe the physico-chemical changes that take place at mineral
surfaces coated with a selection of the organic compounds
identified in this study.
Other analyses performed on the chloroform-soluble
portion of the extracts reinforce the GC-MS results obtained.
The NMR spectra of the extracts confirmed the view that
they consist of primarily long-chain aliphatic compounds,
as the observed signals were predominantly in the expected
regions for aliphatic CH2 groups. The spectra of all the
extracts are very similar, with the major signals due to
aliphatic H and C appearing in the 0–2.5 ppm region in
the 1 H NMR spectra and the 0–45 ppm region in the
13 C NMR spectra. The spectra of the NL1 chloroformsoluble extract are representative and are discussed in more
detail below.
The 1 H aliphatic region is dominated by a large signal
at about 1.2 ppm, which is characteristic of CH2 in
polymethylene chains. Four signals downfield from 1.2 ppm
at ∼1.5, 1.9, 2.1, and 2.3 ppm may be attributed to CH2 groups
in polymethylene chains in closer proximity to electronwithdrawing groups, for example the carboxylic acid groups
of long-chain fatty acids, or, particularly in the case of the
signals at 2.1 and 2.3 ppm, perhaps a CH group. Another
common feature is the signal upfield from 1.2 ppm at 0.8 ppm,
which falls into the correct region to be attributed to CH3
groups. It is also worth noting the lack of the carboxylic acid
Australian Journal of Soil Research
247
proton resonance between 11 and 13 ppm, which may be due
to the expected broadness of the signal. Weak signals were
also observed at 5.2–5.3 ppm, which are in the correct region
to be attributed to olefinic protons.
The 13 C NMR spectra of the extracts generally show
the presence of at least 9 carbon environments. For NL1,
resonances at 23.1, 27.6, 30.1, 32.3, and 43.8 ppm may be
attributed to CH2 groups in polymethylene chains, and signals
at 14.5 and 28.4 ppm correspond to a CH3 group and a CH
group, respectively. Again as in the case of the 1 H NMR
spectrum, the carboxylic acid resonance was absent. The
spectra show that the extracts contain predominantly aliphatic
components, hence confirming the GC-MS data.
The compounds identified in the extracts displayed
infrared absorptions characteristic of the groups present. In
particular, a strong band corresponding to C–H stretching
was observed (Doerr et al. 2005, this issue).
Microanalysis was used to determine the elemental
composition of the chloroform-soluble portion of the NL1
extract. The high carbon (68.70 ± 1.30%) and hydrogen
(9.79 ± 0.10%) contents, and the carbon : hydrogen atom
ratio of 1.70, suggest a preponderance of aliphatics in the
material soluble in chloroform and THF, as confirmed by
GC-MS and NMR spectroscopy. The residual content of c.
20% may be assigned primarily to oxygen, indicating the
presence of many oxygen-containing groups (e.g. carboxylic
acid groups); by contrast, the small nitrogen content of
only 1.80 ± 0.24% suggests a small contribution of nitrogencontaining compounds. However, the nitrogen content is
consistent with the presence of amides as suggested
by GC-MS.
Conclusions
The THF-soluble portions of extracts derived from 3 water
repellent samples and one wettable control sample obtained
at consecutive depths from a soil under permanent pasture in
the Netherlands were compared using a range of analytical
techniques. It was found that the composition of the extract
from the wettable sample resembles that of the extracts
from the water repellent samples in most respects. All
4 samples contained long-chain fatty acids (C16 –C24 ), amides
(C14 –C24 ) of similar chain lengths to the acids, slightly
higher chain length alkanes (C25 –C31 ), aldehydes or ketones
(C25 –C29 ), and more complex ring-containing structures.
It is notable that both wettable and water repellent
samples contained hydrophobic compounds (e.g. alkanes),
the presence of which has previously been associated with
the development of water repellency. The major difference
between the wettable and water repellent samples concerns
high molecular mass polar compounds, such as fatty
acids/amides having 23 or 24 carbon atoms and stigmasterols.
These are virtually absent from the wettable subsoil, and it
may be speculated that in this soil their presence in significant
amounts is necessary for water repellency to be exhibited.
248
Australian Journal of Soil Research
Our findings do not allow unequivocal identification of
a specific compound or compound group as the universal
cause of water repellency of soils, nor would we expect
them to, since compounds with hydrophobic properties
are ubiquitous in the environment in general, and soils in
particular exhibit a high spatial variability with respect to
both physical and biochemical parameters. The results of this
study do, however, throw light on the role of common organic
compounds in the water repellency behaviour expressed by
the mineral particles in a soil. Ongoing and future work
will examine other soils to test the wider applicability of
our conclusions.
Acknowledgments
The authors thank K. Oostindie for sample collection and
shipment, I. Matthews for technical help with the GC
(FID) instrument, the staff at the EPSRC National Mass
Spectrometry Service Centre in the Department of Chemistry,
University of Wales Swansea, for advice and the use of
their GC-MS instrument, M. Nettle for running the NMR
spectra, and S. Szajda for technical help. The financial
assistance of the University of Wales Swansea in providing a
postgraduate studentship (KAM) and a postgraduate bursary
(CTL) is acknowledged. We also thank HEFCW for financial
support. This study was supported by EU grant FAIRCT98-4027 and NERC Advanced Fellowship NER/J/S/200200662(SHD). This work does not necessarily reflect the
European Commission’s views and in no way anticipates its
future policy in this area.
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Manuscript received 25 June 2004, accepted 7 January 2005
http://www.publish.csiro.au/journals/ajsr