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Organic Geochemistry 36 (2005) 385–397
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Structural characterisation of groundwater hydrophobic
acids isolated from the Tomago Sand Beds, Australia
Cameron McIntyre
a
a,1
, Christopher McRae
Alessandro Piccolo b
a,*
, Barry D. Batts a,
Division of Environmental and Life Sciences, Department of Chemistry, Macquarie University, Sydney, NSW 2109, Australia
b
Dipartimento di Scienze del Suolo, della Pianta e dellÕAmbiente (DISSPA) Sezione di Scienze Chimico-Agrarie,
Università di Napoli ‘‘Federico II’’, Via Università 100, 80055 Portici, Italy
Received 22 March 2004; accepted 7 October 2004
(returned to author for revision 28 June 2004)
Available online 8 December 2004
Abstract
Groundwater hydrophobic acids isolated from the Tomago Sand Beds, Australia have been structurally characterised using multiple analytical techniques. Temporal variability was observed during the study period (1996–2003) and
data for two representative samples isolated in 1996 and 2002 are presented. The sample isolated in 2002 was a highly
aromatic fulvic acid with branched aliphatic functional structures and high oxygen content. It differed from structural
concepts which suggest that groundwater hydrophobic acids isolated from organically lean aquifers are predominantly
aliphatic in nature, with a low oxygen content resulting from extended degradation processes. Data are consistent with
the hydrophobic acids having inputs from both surface recharge and mineralised sedimentary organic carbon. The sample isolated in 1996 was significantly more aliphatic, indicating that a change in microbial activity/community has
occurred at the site.
2004 Elsevier Ltd. All rights reserved.
1. Introduction
Groundwater is an important source of potable water
worldwide and its contamination with organic and inorganic compounds is a major concern. The dissolved organic carbon (DOC) in groundwater contains a
significant amount of humic substances (HSs) derived
from the breakdown of biogenic matter (Thurman,
1985a,b). HSs bind and transport beneficial and pollut*
Corresponding author. Tel.: +612 9850 8288; fax: +612
9850 8313.
E-mail address: [email protected] (C. McRae).
1
Current address: CSIRO Petroleum, P.O. Box 136, North
Ryde, NSW 1670, Australia.
ant species such as metal ions and organic compounds in
a range of aquatic and terrestrial environments (Koopal
et al., 2001), and in groundwaters they have been shown
to increase the solubility of pesticides and form mutagenic compounds during the chlorination of drinking
water (Johnson-Logan et al., 1992; Koivusalo and Vartiaanen, 1997). They have a significant geochemical role
and understanding the structural features of HSs has
been identified as a basic requirement for comparison
of samples, understanding their chemistry and the determination of their environmental and ecological importance (Frimmel and Abbt-Braun, 1999; Nikolaou and
Lekkas, 2001). As an example, Leenheer et al. (2001) recently characterised the dissolved organic matter
(DOM) and its reactivity towards chlorine in reclaimed
0146-6380/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.orggeochem.2004.10.004
386
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
and receiving groundwater of Los Angeles County, CA
to assess its safety for use as drinking water.
The structural features of HSs in groundwater have
received relatively little attention compared with samples from other aquatic and terrestrial environments.
Thurman (1985a) provided a review of the first major
study of groundwater HSs which established their chemical, physical and spectroscopic properties, and several
unique characteristics were found compared to surface
water HSs. The content of DOC in groundwater was
generally low (>5 mg L1) and the isolated HSs contained low amounts of humic acid, aromatics and oxygenated functional groups. The exception was when
the source was located in organic rich sediments containing kerogen, where DOC, humic acid and aromaticity
were exceptionally high. Several other studies have been
conducted on the specific characterisation of groundwater HSs or have included groundwater samples in
broader aquatic HS characterisations (Wassenaar
et al., 1990; Kim et al., 1990; Gron et al., 1996; Artinger
et al., 2000; Alberts et al., 1992; Pettersson et al., 1989,
1994). These confirm that groundwater DOC is structurally unique as a result of extended exposure to adsorption and degradation processes. Adsorption during
infiltration and movement tends to remove the humic
acids, lowering DOC and aromaticity, while degradation
results in the loss of oxygenated functional groups.
Groundwater HSs have also been characterised as part
of studies on landfill leachates, contaminated sites, the
mineralisation of sedimentary organic carbon (SOC),
drinking water studies and DOC budgets (Zwiener
et al., 1999; Wong et al., 2002; Wassenaar et al., 1991;
Thorn and Aiken, 1998; Cronan and Aiken, 1985; Leinweber et al., 2001; Kumke et al., 1999; Christensen et al.,
1998; Calace et al., 2001; Backau et al., 2000; Nanny and
Ratasuk, 2002). Given that the much of the literature
does not deal specifically with background structural
characterisations or Australian sites, data for these areas
remain somewhat limited and inconsistent. In particular,
the variability in the chemical, physical and spectroscopic techniques applied makes inter-study comparisons difficult.
The Tomago Sand Beds near Sydney, Australia are a
remnant dune system and shallow sandy aquifer and
have been mined for mineral sands and used to supply
drinking water. The groundwater geochemistry was radically altered during mining activities, leading to the
potential contamination of water supplies for nearby
populations with species such as iron and arsenic.
DOC levels were also observed to increase with mining
activities and it was thought that organic acids may be
involved in the release and transportation of the inorganic contaminants. The objective of this study was to
conduct a comprehensive background structural characterisation of the groundwater hydrophobic acids isolated
from groundwater at the Tomago Sand Beds and com-
pare the findings with other studies. The hypothesis
was that the hydrophobic acids at this Australian site
are structurally unique and have a significant role in
the groundwater geochemistry. Structural changes in
the hydrophobic acids were observed as the study progressed from 1996 to 2003 and thus data are presented
for two representative samples isolated in 1996
(GW96) and 2002 (GW02).
2. Site description
A detailed description of the study area can be found
in Prosser and Roseby (1995) and a brief summary is given here. The Tomago sand beds are located 16 km
north of Newcastle, Australia. They are characterised
by a dune system of reworked Pleistocene marine sands
that have developed podzol soil profiles since stabilisation (12,000 BP). Vegetation is dominated by dry sclerophyll forests with low lying areas containing swamps.
The sands are dominated by quartz and an extensive
aquifer underlies the beds, with the water table depth
ranging from 1 to 5 m. The podzol soils are predominantly humic in nature at low relief and iron/iron-humic
at high relief. The depth of the B horizon varies with the
water table and ranges from 0.5 to 3 m. The sands contain economically viable quantities of heavy minerals
and these were mined for 30 years (production has
ceased). The groundwater is used a source of potable
water by the local water corporation for nearby populations and potential contamination resulting from mining
has been an ongoing issue of concern. A monitoring programme for the groundwater is ongoing.
3. Experimental
3.1. Background data
Background data for the study site were obtained by
personal communication with Peter R. Wieland,
Department of Chemistry, Macquarie University, Sydney. The data were extracted from the records of the
groundwater monitoring programme at RZ Mines Pty
Ltd, Newcastle. Additional data obtained from Peter
R. Wieland were part of a separate research study of
the site.
3.2. Samples, sampling methodology extraction
procedures
Samples were taken from a control piezometer located at UTM 56H 372940 1371020 (RZ Mines Pty
Ltd identification: location 3502, piezometer number
2). The sampling site was a control one unaffected by
mining activities, with piezometers set at depths of 3.6,
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
6.5, 10.4 and 13.9 m below ground level. The piezometer
used was at 6.5 m and was selected because of its high
DOC and hydrogen sulfide content. It samples at least
4 m below the groundwater level (GWL) and below
the B horizon of the soil. Standard Suwannee River fulvic acid (SRFA) was obtained from the International
Humic Substances Society (IHSS; St. Paul, MN,
USA). Standard SRFA was chosen as it has been well
characterised (Averett et al., 1994) and was considered
to be a suitable reference material for the characterisation of the groundwater hydrophobic acids in this study.
Elemental data for SRFA were supplied by the IHSS
with the sample.
Representative samples were collected on 12th October 1996 (3 · 10 L) and 18th March 2002 (150 L). These
samples highlight the structural changes observed over
the study period (1996–2003) and data for other samples
are not included for brevity. Samples were pumped
mechanically and pressure-filtered with nitrogen through
a 0.45 lm cellulose acetate membrane (Advantech MFS,
USA) into glass (1996) and HDPE (2002) containers.
The samples were acidified to pH 2 with concentrated
HCl and transported to the laboratory. The hydrophobic
acid fraction was isolated by adsorption on to a column
of XAD-7 resin using a procedure based on that of Thurman and Malcolm (1981) and detailed in McIntyre et al.
(1997). Elution of the acids was with 0.01 M KOH and
cation exchange was effected with Amberlite IR-120 resin. The resulting solution was freeze dried without separation of the humic acids in order to obtain the whole
hydrophobic acid fraction.
3.3. Dissolved organic carbon, elemental analysis and
isotopic composition
Non-purgeable organic carbon (NPOC) was determined on filtered and acidified samples using a Shimadzu TOC 5000A total organic carbon analyzer.
Samples were sparged with CO2-free oxygen for 2 min
prior to analysis. The instrument was calibrated over
an appropriate range using potassium hydrogen phthalate; % DOC as hydrophobic acids was calculated as
the mass of isolated hydrophobic acids L1 of groundwater · % C/DOC content of the groundwater. Humic
acid content was determined gravimetrically by dissolving a known weight of hydrophobic acids in water and
precipitating the humic acids at pH 1 with concentrated
HCl. The humic acids were filtered off, dried, weighed
and % wt determined.
Elemental microanalysis was performed by The
Campbell Microanalytical Laboratory (Department of
Chemistry, University of Otago, NZ). C, H, N, S, O
(dry ash free) were determined for GW02. C, H, N, S
(dry ash free) were determined for GW96. Samples were
dried for 24 h at 60 C under vacuum prior to elemental
microanalysis. Reported elemental compositions of 75
387
groundwater fulvic acids were averaged for comparison
(Wassenaar et al., 1990; Thurman, 1985a; Kim et al.,
1990; Frimmel and Abbt-Braun, 1999; Artinger et al.,
2000; Alberts et al., 1992; Pettersson et al., 1994).
Stable isotope analysis for sulfur and carbon were
performed at the Stable Isotope Science Laboratory,
Department of Physics and Astronomy, University of
Calgary, Canada.
3.4. Carboxyl group content
Carboxyl group content was determined by titration
to pH 8 using the method of Christensen et al. (1998).
Titrations were conducted using a Radiometer Copenhagen TTA80 titration assembly, PHM82 standard pH
meter and a GK2401C pH electrode. Carboxylic acid
content was not determined for GW96 due to limited
sample quantity. Phenol content was not determined
as titration has been shown to give variable results above
pH 8 due to hydrolysis reactions (Averett et al., 1994).
3.5. Infra-red spectroscopy
Infra-red (IR) spectra were recorded on a Perkin–
Elmer Paragon 1000PC FT-IR Spectrometer. Samples
were dried at 60 C under vacuum for 24 h and analyzed
as 1% w/w KBr discs. Spectra were recorded over the
range 4000–400 cm1 using 16 scans at a resolution of
2 cm1 and were smoothed after acquisition.
3.6. 1H and
13
C NMR spectroscopy
1
H Spectra were recorded on a Bruker DPX400–400
MHz spectrometer using a WATERGATE pulse sequence for suppression of the water resonance (Liu
et al., 1998). Samples were dissolved in D2O and 10,240
transients were collected with a spectral width of 5,995
Hz, an acquisition time of 2.73 s and pulse delay of
2 s. The solvent peak at 4.6 ppm was irradiated so protons in this region were not observed. Peak assignments
were based on the literature (Wilson, 1987; Malcolm,
1990; Leenheer et al., 1998).
Conditions used for Solid-State Cross Polarisation
Magic Angle Spinning (CPMAS) 13C NMR spectroscopy are detailed in Leenheer et al. (2003). Insufficient
sample prevented a spectrum for GW96 being recorded.
Peak assignments were based on the literature (Malcolm, 1990; Nanny and Ratasuk, 2002).
3.7. High-pressure size exclusion chromatography
High-pressure size exclusion chromatography
(HPSEC) is an established method in the analysis of
HSs for determination of molecular weight and size
(Cozzolino et al., 2001; Conte and Piccolo, 1999). Analysis was performed using a Shimadzu LC10AD pump, a
388
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
Perking–Elmer LC295 UV–Vis detector and a 600 · 7.8
mm Polysep GFC P 3000 column with a Polysep P 3000
guard column (Phenomenex). The eluent was 0.05 M
phosphate buffer at pH 7 and the analytes were dissolved
in the eluent at a concentration of 0.2 g L1. Standards
used for calibration were polystyrenesulfonates (LabService Analytica s.r.l., Italy) with molecular weights of
6.78, 16.8, 32 and 130 kDa. A calibration curve relating
molecular weight to elution time was obtained by plotting the natural logarithm of the molecular weights
[ln (Mw)] of the polystyrenesulfonate standards versus
time. Linear regression was used to obtained the relationship between ln (Mw) and time. The injection volume
was 100 lL and spectra were recorded at 250 nm for
standards and 280 nm for samples. The different wavelengths have been shown to have a minimal effect
(OÕLoughlin and Chin, 2001). Spectra were acquired
using Perking–Elmer PE Turbochrom 4.0.1 software.
Data were processed manually by exporting the chromatograms as time versus signal ASCII data files into
Microsoft Excel and only time and signal data corresponding to the major peak of interest were retained.
For each time slice a corresponding molecular weight,
Mi, was calculated using the linear regression equation
fitted to the calibration data. The weight fraction, wi,
of analyte in any time slice was calculated by dividing
the detector voltage response (baseline subtracted) by
the sum of detector voltage
P responses for each analyte
peak (i.e., wi = intensityi/ intensityi). M w was obtained
from the
P sum of the products of wi and Mi (i.e.,
M w ¼ wi M i ). 1=M n was obtained
P from the sum of
the ratios of wi and Mi (i.e., M n ¼ X i M i , where Xi is
the mole fraction of
Pmolecules of molecular weight mass
Mi, thus M n ¼ 1= ðwi =M i Þ).
3.8. Vapour pressure osmometry
Vapour pressure osmometry (VPO) was performed
using a Westcor, Vapro 5520 vapor pressure osmometer.
Samples were analyzed using the method of Aiken and
Malcolm (1987) and benzoin was the calibration
standard.
3.9. UV–Vis spectroscopy
UV absorbance was determined on a Varian, Cary
300 Bio UV–Vis Spectrophotometer using the procedure
of Chen et al. (1977). Absorbance values were recorded
at 365 and 250 nm for calculation of E2/E3 ratios and at
280 nm for calculation of molar absorbance.
3.10. Thermochemolysis with TMAH
Offline tetramethylammonium hydroxide (TMAH)
thermochemolysis was performed using the method of
del Rio et al. (1998). The internal standard was eicosane
at a concentration of 25 ng L1. Analysis was performed
using a Fisons Instruments GC8000 gas chromatograph
coupled to a MD800 mass selective detector. The oven
was held at 40 C for 2 min, then ramped at 10 C min1
to 100 C, then ramped at 4 C min1 to 300 C and held
for 2 min. Mass spectra were obtained by scanning from
35 to 500 Da in 1 s. The capillary column was a 50 m
BP1 one (SGE, Australia) with a 0.5 lm film thickness.
Compounds were identified by comparison of the spectra with a NIST 92 library.
3.11. Electrospray ionisation mass spectrometry
Electrospray ionisation mass spectrometry (ESI-MS)
was performed using the methods of McIntyre et al.
(1997, 2002). Samples were dissolved in 75:25 methanol:water at a concentration of 1 mg mL1. Conditions
for the acquisition of spectra are given in McIntyre et
al. (2002). Neutral loss spectra were obtained with the
same parameters used for product ion spectra but with
a mass range of 100–700 Da, a scan time of 3 s and
quadrupole resolution settings to achieve unit mass
resolution.
4. Results and discussion
4.1. Background data
The background data for the piezometer are summarised in Table 1. The pH of the groundwater is acidic
and is controlled by the oxidation of sulfur and/or sulfide to sulfate in the oxic zone, with the low content of
clay minerals minimising buffering of the system.
Increasing sulfate and decreasing iron and sulfide levels
with time indicate a change in microbial activity/composition associated with dissolution of pyrite and reduction
of sulfate (Massmann et al., 2003). The pH has decreased with time, consistent with a reduction in bicarbonate
production
associated
with
microbial
mineralisation of SOC during sulfate reduction and acid
production from pyrite dissolution. DOC concentration
has declined with time, indicating a reduction in the
microbial mineralisation of SOC or reduced DOC recharge from surface environments. The decrease in concentration of iron and sulfur is consistent with the
former (Backau et al., 2000). Oxidation/reduction potential (ORP) values indicate a reducing environment,
although the periodic low levels of dissolved oxygen
(DO) reflect the sampling depth being proximal to the
water table. Conductivity (Cond.) is constant and indicates that the spear has not been affected by mining
activities.
Table 1 is incomplete as data were derived from several sources. Trends at this piezometer were observed in
adjacent control piezometers set at different depths (data
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
389
Table 1
Background geochemical data for groundwater located at the Tomago Sand Beds, Australia
Date
GWL
(m)
pH
Temperature
(C)
Total Fe
(mg/L)
Sulfate
(mg/L)
Sulfide
(mg/L)
Total S
(mg/L)
NPOC
(mg/L)
ORP
(mV)
DO
(mg/L)
Cond.
(mS/cm)
09/04/96
08/08/97
01/14/98
01/17/98
05/05/98
06/17/98
08/19/98
01/14/99
05/24/99
06/01/99
06/09/99
03/23/00
05/04/00
01/04/01
05/23/01
07/25/01
11/12/01
06/06/02
07/09/02
1.76
5.3
5.5
5.4
5.3
5.1
5.3
5.3
5.3
5.4
5.1
5.1
5.5
4.8
5.2
5.0
4.9
5.3
4.9
4.7
–
–
–
–
19.6
19.2
–
–
–
20.6
20.2
–
21.2
–
18.9
15.4
–
19.3
17.1
2.1
1.4
2.6
2.2
1.8
2.2
2.0
1.4
1.5
1.7
0.5
1.4
1.9
1.0
0.7
–
0.8
0.8
0.6
–
9.0
6.9
1.7
–
–
1.3
3.0
4.2
–
–
7.6
–
9.6
80
32
11
30
25
–
–
–
–
5.7
5.5
–
–
–
3.5
3.7
–
3.9
–
4.1
0.6
–
0.3
0.6
–
–
–
–
340
128
–
–
–
68
132
–
11
–
40
–
–
27
10
–
–
–
–
8.2
7.0
–
–
–
–
–
–
–
–
–
–
–
4.5
2.2
–
–
–
–
36
26
–
–
–
25
35
–
109
–
47
220
–
43
75
–
–
–
–
0.16
0.27
–
–
–
0.08
0.11
–
0.13
–
0.18
0.24
–
0.40
0.22
–
0.16
0.20
0.19
0.16
0.15
0.19
0.15
0.13
0.10
0.10
0.17
0.16
0.20
0.16
0.11
0.19
0.15
0.14
1.31
0.80
1.82
1.75
0.10
0.32
0.23
0.89
0.30
1.16
1.43
1.18
1.60
0.80
0.31
0.45
not shown), confirmed their validity. All values were
within normal variation for the site which has been monitored for 30 years. Variation in the parameters could
not be correlated with rainfall and season but a major
influence on the system is the high lateral flow rate of
groundwater (1 m week1) which results in the rapid dispersal of ground water and surface recharge. The background data indicate this is a typical groundwater
system.
4.2. DOC composition
The concentration of DOC in the groundwater in
2002 (Table 2) is consistent with previous studies that
have reported concentrations in soil solutions which
range from 20 to 50 mg L1 in the O/A horizons,
decreasing to >3 mg L1 in groundwater as infiltration
occurs (Wassenaar et al., 1991; Thurman, 1985a,b; Pettersson et al., 1994). This reduction is generally attributed to extended exposure to adsorption and
degradation processes. The DOC concentration in
1996 was considerably higher (Table 2). The background
data indicates a higher level of microbial sulfate reduction in 1996 and are consistent with an elevated DOC
being the result of microbial dissolution of SOC.
Previous studies have reported the hydrophobic acid
content of groundwater in organically lean aquifers (%
DOC as hydrophobic acids) as 5–58% with a predominance of fulvic acids (Wassenaar et al., 1990; Thurman,
1985a; Gron et al., 1996; Artinger et al., 2000; Pettersson
et al., 1994). Data for GW96 are consistent with this;
however, for GW02 the hydrophobic acid content is
above the upper end of the range. Higher levels of
microbial sulfate reduction in an organically lean aquifer
have been shown to produce a greater proportion of
hydrophilic acids in the DOC (Wassenaar et al., 1991);
this explains the lower value for GW96 and suggests that
inputs of surface DOC may be more significant for
GW02. The hydrophobic acid content as related to
DOC content is not consistent with the findings of Wassenaar et al. (1990) and Thurman (1985a) who showed
that humate content increases with DOC content in
groundwater. This is also attributed to a change in
microbial activity/community which has altered the total
DOC inputs over the study period. The low humic acid
content of both samples is consistent with removal by
adsorption and degradation processes as water infiltrates and migrates through the ground and also reflects
the organically lean composition of the aquifer.
4.3. Elemental and isotopic composition
The average values of elemental composition from
previous studies (cf. 3.3, 1 s.d. given in brackets) for carbon content, hydrogen content and H/C ratio were
53.9% (2.1), 4.95% (0.54) and 1.11 (0.12), respectively
(n = 75). Oxygen content and the O/C ratio were
37.6% (2.0) and 0.53 (0.5) (n = 72). Nitrogen and sulfur
contents were 1.07% (0.35) (n = 73) and 2.97% (0.68)
(n = 48).
Compared to reported values the oxygen content of
GW02 is high. The lower oxygen content of reported
samples has been attributed to the removal of carboxyl
groups from fulvic acids during microbial and chemical
degradation processes (Pettersson et al., 1994). A lower
hydrogen content and H/C ratio indicate that GW02
390
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
Table 2
Sample information and analysis results for the hydrophobic acids isolated from groundwater (GW02 and GW96) and standard
Suwannee River fulvic acid (SRFA)
Parameter
1996/GW96
2002/GW02
SRFA
Sampling date
Volume collected (L)
DOC (mg C L1)
Hydrophobic acids isolated (mg)
% of DOC as hydrophobic acids
Humic acid content (% wt)
12/10/96
10 · 3
10.1
226
36
<5
18/03/02
150
2.2
410
64
<5
–
–
–
–
C (%)
H (%)
O (%)
N (%)
S (%)
Ash (%)
H2O (%)
H/C
O/C
d13C (&)
d34S (&)
48.2
4.21
–
1.32
–
8.42
–
1.04
–
27.6
9.9
52.3
4.10
41.5
1.20
2.58
2.87
6.62
0.93
0.56
27.6
1.6
52.4
4.31
42.2
0.72
0.44
0.46
8.8
0.97
0.60
27.6
–
Carboxyl content (meq g1)
Molecular weight (Da) by VPO
Mw (Da) by HPSEC
Mn (Da) by HPSEC
Polydispersity (Mw/Mn) by HPSEC
E2/E3 ratio
Absorbance at 280 nm (L mol OC1 cm1)
–
–
1773
1356
1.31
4.5
310
5.5
953
1810
1499
1.21
4.3
530
5.7
859
2438
1972
1.24
4.4
420
has a less aliphatic character. Groundwater fulvic acids
are suggested to have a lower aromatic content due to
removal of humic acids by adsorption and degradation.
In surface environments where these processes are less
extensive, elemental compositions of fulvic acids are
similar to that presented for SRFA. The C, H, O data
for GW02 is closer to that of SRFA and suggests that
the sample is relatively immature and/or may still receive
some allochthonous DOC. If the DOC is derived in situ,
this suggests that it has been oxidised, resulting in a
higher oxygen content, or that the character of the sedimentary organic matter is unique. Dating (14C) of the
samples and characterisation of the sediments at the site
have not been done and thus conclusions with regard to
DOC age/origin are not yet possible. The higher sulfur
content of GW02 compared to SRFA is characteristic
of groundwater environments and is indicative of hydrogen sulfide/colloidal sulfur produced by microbes during
sulfate reduction being incorporated into the hydrophobic acids. Average N and S values of reported values are
comparable to GW02 and typical for groundwater HSs.
The elemental composition of GW96 is significantly
different from that of GW02 and the reported values given above. Limited data were obtained due to the limited amount of sample available, but a lower carbon
content and a higher H/C ratio suggest that GW96 contains more aliphatic (possibly oxygenated) compounds
–
than GW02. This is consistent with an addition of low
molecular weight organic acids produced during microbial action on aquifer SOC during sulfate reduction, as
proposed by Wassenaar et al. (1991), and the background data presented above.
The carbon isotope ratio of 27.6& for all three
samples (Table 2) indicates that they are most likely derived from higher plant matter (Smith and Epstein,
1971). This is consistent with published data (Wassenaar
et al., 1990; Thurman, 1985a). The organic sulfur isotope ratios are consistent with literature values and are
consistent with a change in biological activity at the site
(Nissenbaum and Kaplan, 1972). GW96 is isotopically
heavier, indicating greater depletion in 32S through sulfate reduction by microbes. This is supported by higher
sulfide levels observed in the background data prior to
2002. The d 34S values of the dissolved sulfate and sulfide
at the site (data not shown) also support this finding and
indicate that the microbial community has changed or
that microbial activity was occurring at shallower depths
and closer to the piezometer, prior to 2002.
4.4. Carboxyl content
GW02 has a carboxylic acid content which lies between values reported by Thurman (1985b) for fulvic
acids in ground water (5.1–5.5 meq g1) and surface
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
391
waters (5.5–6.2 meq g1). It is higher than a calculated
mean value of 5.2 meq g1 for groundwater fulvic acids
reported by Pettersson et al. (1994) and similar to the 5.7
meq g1 determined for SRFA (Table 2). These data are
consistent with the elevated oxygen content determined
by elemental analysis. The high carboxyl group content
and oxygen content when compared to literature values
again suggest that GW02 is immature and has been exposed to a lesser degree of decomposition or to greater
oxidation.
4.5. Infrared spectroscopy
Infrared spectra are similar to those reported by Kim
et al. (1990) and Alberts et al. (1992) for groundwater
fulvic acid (Fig. 1) and show a close resemblance to
those of hydrophobic acids isolated from streams, lakes
and forest floor solutions (Guggenberger et al., 1994;
David and Vance, 1991). The data are typical for terrestrially derived aquatic hydrophobic acids, but also highlight the fairly non-specific nature of infrared
spectroscopy. The IR spectra indicate the hydrophobic
acids are primarily carboxylic (1720, 3400 cm1; C@O,
OH stretching) with a low content of humic acids and
complexed metal ions (1620 and 1400 cm1).
Fig. 2. 1H NMR spectra of hydrophobic acids isolated from
groundwater (GW02 and GW96) and the relative distribution
of hydrogen atoms (U = phenyl).
4.6. 1H NMR spectroscopy
GW02 has the majority of the observable protons
occurring between 1.75 and 3 ppm (Fig. 2), indicating
a high content of protons on carbon atoms adjacent to
carboxyl groups. The peak at 2.5 ppm indicates a significant proportion of these are in branched aliphatic structures. In unbranched aliphatic structures, protons in this
spectral region give a peak at around 2.2 ppm. Although
Fig. 1. Infrared spectra of hydrophobic acids isolated from
groundwater (GW02 and GW96).
protons of methyl groups attached to aromatic rings
may also appear in this region, 13C NMR spectra presented in the next section confirm that branched aliphatic structures are dominant in GW02. The
spectrum is substantially different from that for hydrophobic acids isolated from a control groundwater, presented by Thorn and Aiken (1998), Well 310), which
gave a major peak at 1.1 ppm corresponding to aliphatic
methylene groups. It also differs substantially from marine and terrestrial HSs which have been shown to have
major peaks in the aliphatic (1 ppm) and carbohydrate
(4 ppm) regions (Hatcher et al., 1979; Malcolm, 1990).
The spectrum more closely resembles that of surface
water fulvic acids isolated from streams and lakes, where
resonances near 2 ppm are more prominent (Malcolm,
1990; Ma et al., 2001). In particular, the spectrum shows
a similar down field shift of protons adjacent to carboxyl
groups as observed in the metal binding fraction of
SRFA, which was attributed to clustering of the carboxyl groups (Leenheer et al., 1998).
GW96 has a significantly different spectrum to that of
GW02, highlighting the change in composition of the
hydrophobic acids in the groundwater at the site since
1996 (Fig. 2). More intense peaks at 1.2 and 2.2 ppm
indicate a greater content of aliphatic acids, and this is
consistent with greater microbial mineralisation of
SOC and other data. These may be branched methyl
structures rather than linear aliphatic/alicyclic compounds, as resonances at 1.6–1.8 ppm due to protons
392
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
b to carboxylic acids increase only very slightly compared to corresponding peaks in GW02.
4.7. CPMAS
13
C NMR spectroscopy
The normal spectrum has a high content of aromatic
carbon (130 ppm), which is not consistent with the concept of groundwater humic substances having a low content of aromatic structures due to adsorption of humic
acids (Fig. 3). The major aliphatic peak at 43 ppm is
indicative of branched aliphatic carbons while the peak
at 30 ppm is due to methylene carbons. The low signal
at 15–20 ppm due to methyl carbons and the major resonance occurring at 43 ppm indicate that the peak at 2.4
ppm in the 1H NMR can be attributed to protons on
branched aliphatic groups adjacent to carboxyl groups,
rather than to methyl groups attached to aromatic rings.
The small shoulder at 105 ppm and the peak at 76 ppm
indicate a low content of anomeric carbon of carbohydrates and C–O linkages, respectively. Shoulders at
115 ppm and 155 ppm indicate lignin-derived aromatics
are present in low quantities. Carboxyl carbons (170
ppm) are moderate in content and normal for aquatic
fulvic acids (cf. Malcolm, 1990). The low signal level
at 58 ppm also indicates methoxyl carbons of lignin
Fig. 3. Normal and dipolar-dephased CPMAS 13C NMR
spectra of hydrophobic acids isolated from groundwater
(GW02) and relative distribution of carbon atoms.
and esters have a minimal contribution. The data are
consistent with previous sections and indicate predominantly aryl-branched aliphatic structures with a low content of lignin-derived compounds and C–O linkages
(carbohydrates).
The dipolar dephased spectrum indicates that the
majority of aromatic and aliphatic carbon (0–160
ppm) is non-quaternary (Fig. 3). Terminal methyl at
15 ppm, quaternary methine at 48 ppm and tertiary
alcohols at 80 ppm are evident while a low proportion
of the aromatic carbon is protonated. These structures
are hypothesised to be indicative of degraded terpenoid
structures, as reported by Leenheer et al. (2003). For this
site, selective or incomplete absorption processes in the
vadose zone of the soil during water infiltration may
have resulted in the enrichment of these types of species.
The overlying vegetation (Eucalyptus) at the site is rich
in terpenoids and is a ready source of precursor terpenoid molecules.
4.8. UV–Vis spectroscopic, molecular weight and
molecular size measurements
SRFA has been reported to have a Mn by VPO of 840
Da and a Mn, Mw and polydispersity by HPSEC of 1652
Da, 2290 Da and 1.39 (Aiken and Malcolm, 1987;
OÕLoughlin and Chin, 2001). Chin et al. (1994) determined the molar absorbance at 280 nm to be 389 L cm1
(mol OC)1. The results presented for SRFA in this
study correlate well with the above data (Table 2). The
E2/E3 ratio was measured for comparison of aromaticity
and average molecular weights as it has been shown to
give a stronger correlation than the E4/E6 ratio (Peuravuori and Pihlaja, 1997).
The molecular weight of GW02 determined using
VPO is high compared with values for aquatic fulvic
acids reported by Aiken and Malcolm (1987) of 500–
950 and 859 Da obtained for SRFA (Table 2). Pettersson et al. (1994) published molecular weights for aquatic
fulvic acids determined by HPSEC using polystyrenesulphonate standards. Their results showed that
the fulvic acids in deep groundwater were lower in
molecular weight than in surface waters and this was
attributed to adsorption and degradation processes.
The VPO result for GW02 is not consistent with this
concept and suggests a lesser degree of exposure to these
processes, allowing higher molecular weight compounds
to stay in solution.
Peuravuori and Pihlaja (1997) have shown that
molecular weights determined by HPSEC have a linear
relationship to those determined by VPO. HPSEC data
for GW02 (Table 2) infer that it has a lower molecular
weight than SRFA. However, given the branched, compact nature of the sample as indicated by NMR, this
suggests it is more likely to reflect a smaller hydrodynamic size. The polydispersity of GW02 is slightly lower
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
then that determined for SRFA and indicates that the
sample has a more homogenous composition. This is
consistent with the findings of Pettersson et al. (1994)
which indicated groundwater fulvic acids were less polydisperse than surface water fulvic acids, which again was
attributable to maturity and a greater exposure to
adsorption and degradation processes. Molar absorption at 280 nm and E2/E3 ratio have been shown to be
related to aromaticity and inversely related to average
molecular weight (Peuravuori and Pihlaja, 1997). The
results for these parameters are consistent with 13C
NMR and VPO data and confirm that GW02 is both
more aromatic than and higher in molecular weight than
SRFA.
The molecular weight for GW96 could not be determined using VPO as in higher ash content resulted its
being insoluble in the solvent. Molar absorbance at
280 nm and E2/E3 ratio indicate that the sample is less
aromatic than GW02 and SRFA and that it has a lower
molecular weight. The polydispersity is higher and indicates that the sample has a less homogenous composition. These data are consistent with the suggestion of
greater microbial sulfate reduction and SOC mineralisation at the site in 1996 producing additional low molecular weight aliphatic acids and an increase in DOC. This
also infers that the molecular weight determined by
393
HPSEC for GW96 reflects both the lower molecular
weight acids produced by microbes as well as a smaller
hydrodynamic size.
4.9. Thermochemolysis with TMAH
The total ion current (TIC) chromatogram for GW02
(Fig. 4) is very similar to that for SRFA presented by del
Rio et al. (1998) and indicates that the sample contains
terrestrially derived aquatic DOC. TMAH thermochemolysis products identified in Table 3 are dominated by
lignin derived compounds with guaiacyl and syringyl
structures (Clifford et al., 1995) which are consistent
with the overlying angiosperm vegetation. Products arising from tannin and cutan (peaks 14 and 21) are present
and, even though the technique is not very efficient for
carbohydrates, some markers can be seen as peaks 13
and 38. The total yield of products is <1.0% assuming
a response factor of 1 for all compounds; this is consistent with the low content of these biopolymer classes observed using NMR spectroscopy. The data indicate that
there is incomplete adsorption of DOC from surface
water recharge. Ratios of vanillyl and syringyl acid:aldehyde are used to indicate lignin degradation (del Rio
et al., 1998). Although the presence of vanillyl and syringyl aldehyde could not be confirmed, the methyl esters
Fig. 4. TMAH thermochemolysis GC/MS total ion chromatograms for hydrophobic acids isolated from groundwater (GW02 and
GW96). Peak identities are given in Table 3; * denotes internal standard eicosane.
394
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
Table 3
Peak identification table for Fig. 4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Butanedioic acid, dimethyl ester
Methylbutanedioic acid, dimethyl ester
C7-Alkenone
2-Methoxy phenol
Pentanedioic acid, dimethyl ester
1,2-Dimethoxybenzene
1,3-Dimethoxybenzene
Hexanedioic acid, dimethyl ester
Dimethoxytoluene
1,2,3-Trimethoxybenzene
3-Methoxybenzoic acid, methyl ester
4-Methoxyacetophenone
1,2,4-Trimethoxybenzene
1,3,5-Trimethoxybenzene
4-Methoxybenzeneacetic acid, methyl ester
1,2,3-Trimethoxytoluene
3,4-Methylenedioxybenzoic acid, methyl ester
4-Methoxypropanoic acid, methyl ester+
Nonanedoic acid, dimethyl ester
3,4-Dimethoxyacetophenone
3,5-Dimethoxybenzoic acid, dimethyl ester
3,4-Dimethoxybenzoic acid, dimethyl ester
3,4-Dimethoxybenzeneacetic acid, methyl ester
1,2,4-Trimethoxyphenylpropene
3,4,5-Trimethoxyacetophenone
Hexadecene
3,4,5-Trimethoxybenzoic acid, methyl ester
3,4-Dimethoxyphenylpropanoic acid, methyl ester
Tetradecanoic acid, methyl ester
3,4,5-Trimethoxyphenylacetic acid, methyl ester
Pentadecanoic acid, methyl ester
Hexadecenoic acid, methyl ester
Hexadecanoic acid, methyl ester
Hexadecanoic acid
Octadecenoic acid, methyl ester
Octadecanoic acid, methyl ester
Nonadecanoic acid, methyl ester
Methylated glucose compound
Eicosanoic acid, methyl ester
Docosenoic acid, methyl ester
Docosanoic acid, methyl ester
of vanillyl and syringyl benzoic acids (peaks 21, 22, 27)
are present in significant amounts. This indicates that
the lignin components are highly degraded and this is
consistent with the high oxygen content. Nanny and
Ratasuk (2002) have suggested that C4–6 n-alkanedioic
acids are products of catechol degradation and microbial fermentation processes. These compounds are present (peaks 1, 2, 5, 8) and indicate some microbial activity
is still occurring at the site.
GW96 has a similar TIC chromatogram to GW02
and to SRFA, indicating predominantly terrestrially derived aquatic DOC (del Rio et al., 1998), but with two
significant differences. The C4–6 n-alkanedioic acids are
more intense and peaks corresponding to the methyl es-
ters of fatty acids (peaks 29, 31–38, 40–41) are prominent. The increased quantity of n-alkanedioic acids is
consistent with higher levels of microbial degradation
of aromatic compounds (Nanny and Ratasuk, 2002)
while the fatty acids are consistent with the mineralisation of sedimentary organic carbon and higher microbial
input. Grasset and Amblès (1998) demonstrated that lipids with an even carbon number preference are released
after the enzymatic degradation of humin. These findings are consistent with other data suggesting a higher
level of microbial sulfate reduction and/or SOC mineralisation in 1996. The similar distribution of ligninderived compounds in both samples suggests they are
derived from the same origin.
4.10. Electrospray ionisation mass spectrometry
Negative ion ESI-MS mass spectra for GW02, GW96
[Fig. 5(a) and (b)] and SRFA (not shown) are typical for
aquatic fulvic acids and there are no major differences,
reflecting the non-specific nature of the technique at this
level. The spectra show a distribution of ions at even
mass from m/z 100–700, with groups of ions spaced
every 14 Da. Ions spaced at 2 and 14 Da have been
shown for SRFA, using high resolution mass spectrometry, to result from structural differences of two hydrogen atoms and CH2 units such as those found in
aliphatic, alicyclic and olefinic structures (Stenson et al.,
2002). High-resolution mass data obtained for a sample
obtained in 1998 at this site found similar mass differences (unpublished data) and from this finding it is inferred that similar structural entities exist in the
samples used in this study. The spectrum for GW02 differs slightly from those of GW96 and SRFA. GW02
contains ions that were less well resolved and with a
more uniform intensity, showing that GW02 does contain a different assemblage of compounds, although
the exact nature of the compounds the ESI-MS spectrum represents is still unclear (Stenson et al., 2002;
McIntyre et al., 2001). The greater resolution between
the groups of ions spaced at 14 Da in GW96 is consistent with a higher content of aliphatic acids, as indicated
by 1H NMR spectra and elemental analysis.
Tandem mass spectrometric experiments have been
used to reveal more specific structural features of fulvic
acids (McIntyre et al., 1997, 2002). The product ion
spectra for both samples indicate the presence of aliphatic polycarboxylic acids [Fig. 5(c) and (d)]. Major
fragments spaced at 44 and 18 Da result from loss of
CO2 and H2O from carboxylic acids. Lower mass ions
such as 59 Da (acetate) and 73 Da (propanoate) are suggestive of fragmentation of aliphatic structures (Leenheer et al., 2003). The product ion spectrum for GW96
contains low mass ions of greater intensity than
GW02, consistent with greater aliphaticity. Neutral loss
of 88 Da mass spectra (loss of two CO2 molecules) was
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
(a)
(c)
(e)
(b)
(d)
(f)
395
Fig. 5. Negative ion electrospray ionisation spectra for hydrophobic acids isolated from groundwater: (a) GW02 and (b) GW96, and
product ion spectra of m/z 351 for: (c) GW02 and (d) GW96, and neutral loss of 88 Da spectra for: (e) GW02 and (f) GW96.
used to indicate compounds containing at least two carboxylic acid groups [Fig. 5(e) and (f)]. Based on the
product ion spectra of individual ions (data not shown)
the neutral loss spectrum shows aromatic polycarboxylic
acids, consistent with the products of lignin degradation,
at m/z 171, 181, 197, 209 and aliphatic polycarboxylic
acids predominantly at m/z 200–400. These data indicate
that the samples contain a mixture of aryl aliphatic polycarboxylic acids and lignin-derived compounds.
5. Conclusions
Groundwater hydrophobic acids isolated in 2002
from the Tomago Sand Beds contained predominantly
fulvic acids with a high content of aromatic and
branched aliphatic polycarboxylic structures. The
molecular weight and oxygen content are high and similarities to the surface water fulvic acid SRFA were
found, suggesting some input from surface DOC still occurs. Low polydispersity and a low content of carbohydrates and lignin-derived compounds indicated that
adsorption and degradation processes are a major control on the DOC composition. Although the origin of
the hydrophobic acids could not be conclusively determined, it is hypothesised that compounds derived from
precursor terpenoid molecules, selectively degraded
and adsorbed during infiltration of surface water recharge, may contribute to this fraction. The high lateral
flow rate of the system is likely to have minimised the
time of exposure of recharge DOC to natural processes.
The sample is also interpreted to have at least a minor
contribution of compounds from microbially mineralised SOC. The high content of aromatics in the sample
is a concern with regards to drinking water supplies as
this could produce higher yields of disinfection byproducts during chlorination. The sample is structurally unique, differing from the current concepts of
groundwater hydrophobic acids which suggest they
should be low molecular weight aliphatic acids depleted
in oxygen content, carboxyl groups and aromaticity.
The findings show the site is unique with regard to the
structure of the hydrophobic acids and that they are
likely to have an important geochemical role with regards to drinking water quality.
Limited data obtained for the hydrophobic acids isolated in 1996 indicate that sample has a significant content of compounds with structural characteristics
different substantially to those of the 2002 sample. Background data and analysis show DOC levels were elevated at that time and suggest that elevated microbial
activity associated with sulfate reduction and mineralisation of SOC led to a greater input of lower molecular
weight (possibly branched) aliphatic acids. The observed
changes in the structural characteristics and composition
of the DOC demonstrate that temporal stability of DOC
in control locations cannot be assumed, even over relatively short periods of time. The dynamic nature of the
system confirms the groundwater DOC has a significant
geochemical role. Dating using 14C, metal binding studies and investigation of SOC at site remain to be done.
Determination of the DOC origin (SOC or recharge)
would greatly assist in the further interpretation of the
structural data.
396
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
Acknowledgments
We thank Jerry A. Leenheer of the U.S.G.S. for
providing the 13C NMR spectrum and assisting with
its interpretation, Dr. Pellegrino Conti at the University of Napoli, Italy for providing the HPSEC data,
Dr. Sinan Ali and Dr. Rita Holland at Macquarie
University, Australia for the use of their vapour pressure osmometer and Dick Holroyde of the Hunter
Water Corporation, Australia for supplying the rainfall data we are also grateful to Simon George, José
C. del Rio, Ian Bull and an anonymous reviewer for
reviews.
Associate Editor—I.D. Bull
References
Aiken, G.R., Malcolm, R.L., 1987. Molecular weight of aquatic
fulvic acids by vapor pressure osmometry. Geochimica et
Cosmochimica Acta 51, 2177–2184.
Alberts, J.J., Filip, Z., Hertkorn, N., 1992. Fulvic and humic
acids isolated from groundwater: compositional characteristics and cation binding. Journal of Contaminant Hydrology 11, 317–330.
Artinger, R., Backau, G., Geyer, S., Fritz, P., Wolf, M., Kim,
J.I., 2000. Characterization of groundwater humic substances: influence of sedimentary organic carbon. Applied
Geochemistry 15, 97–166.
Averett, R.C., Leenheer, J.A., McKnight, D.M., Thorn, K.A.
(Eds.), 1994. Humic substances in the Suwannee River,
Georgia: Interactions, properties and proposed structures.
US Geological Survey Water-Supply Paper 2373, US
Geological Survey, Denver, CO.
Backau, G., Artinger, R., Geyer, S., Wolf, M., Fritz, P., Kim,
J.I., 2000. Groundwater in-situ generation of aquatic humic
and fulvic acids and the mineralization of sedimentary
organic carbon. Applied Geochemistry 15, 819–832.
Calace, N., Liberatori, A., Petronio, B.M., Pietroletti, M., 2001.
Characteristics of different molecular weight fractions of
organic matter in landfill leachate and role in soil sorption
of heavy metals. Environmental Pollution 113, 331–339.
Chen, Y., Senesi, N., Schnitzer, M., 1977. Information provided
on humic substances by E4/E6 ratios. Soil Science Society of
America Journal 41, 352–358.
Chin, Y.-P., Aiken, G.R., OÕLoughlin, E., 1994. Molecular
weight, polydispersity and spectroscopic properties, of
aquatic humic substances. Environmental Science and
Technology 28, 1853–1858.
Christensen, J.B., Jensen, D., Gron, C., Filip, Z., Cristensen,
T.H., 1998. Characterization of the dissolved organic
carbon in landfill leachate-polluted groundwater. Water
Research 32, 125–135.
Clifford, D.J., Carson, D.M., McKinney, D.E., Bortiatynski,
P.G., Hatcher, P.G., 1995. A new rapid technique for the
characterization of lignin in vascular plants: thermochemolysis with tetramethylammonium hydroxide (TMAH).
Organic Geochemistry 23, 169–175.
Conte, P., Piccolo, A., 1999. Conformational arrangement of
dissolved humic substances: influence of solution composition on association of humic molecules. Environmental
Science and Technology 33, 1682–1690.
Cozzolino, A., Conte, P., Piccolo, A., 2001. Conformational
changes of humic substances induced by some hydroxy-,
keto- and sulfonic acids. Soil Biology and Biochemistry 33,
563–571.
Cronan, C.S., Aiken, G.R., 1985. Chemistry and transport of
soluble humic substances in forested watersheds of the
Adirondack Park, New York. Geochimica et Cosmochimica
Acta 49, 1697–1705.
David, M.B., Vance, G.F., 1991. Chemical character and origin
of organic acids in streams and seepage lakes of central
Maine. Biogeochemistry 12, 17–41.
del Rio, J.C., McKinney, D.E., Knicker, H., Nanny, M.A.,
Minard, R.D., Hatcher, P.G., 1998. Structural characterization of bio- and geo-macromolecules by off-line thermochemolysis with tetramethylammonium hydroxide. Journal
of Chromatography A 823, 433–448.
Frimmel, F.H., Abbt-Braun, G., 1999. Basic characterization of
reference NOM from central Europe – similarities and
differences. Environment International 25, 191–207.
Grasset, L., Ambles, A., 1998. Aliphatic lipids released from a
soil humin after enzymatic degradation of cellulose. Organic
Geochemistry 29, 893–897.
Gron, C., Wassenaar, L., Krog, M., 1996. Origin and structures
of groundwater humic substances from three Danish
aquifers. Environment International 22, 519–534.
Guggenberger, G., Zech, W., Schulten, H.R., 1994. Formation
and mobilization pathways of dissolved organic matter:
evidence from chemical structural studies of organic matter
fractions in acid forest floor solutions. Organic Geochemistry 21, 51–66.
Hatcher, P.G., Rowan, R., Mattingly, M.A., 1979. 1H and 13C
NMR of marine humic acids. Organic Geochemistry 2, 77–85.
Koivusalo, M., Vartiaanen, T., 1997. Drinking water chlorination byproducts and cancer. Reviews on Environmental
Health 12, 81–90.
Kim, J.I., Backau, G., Li, G.H., Duschner, H., Psarros, N.,
1990. Characterization of humic acids and fulvic acids from
Gorleben groundwater. FreseniusÕ Journal of Analytical
Chemistry 338, 245–252.
Koopal, K.K., van Riemsdijk, W.H., Kinniburgh, D.G., 2001.
Humic matter and contaminants: general aspects and
modeling metal ion behavior. Pure Applied Chemistry 73,
2005–2016.
Kumke, M.U., Zwiener, C., Abbt-Braun, G., Frimmel, F.H.,
1999. Spectroscopic characterization of fulvic acid fractions
of a contaminated groundwater. Acta Hydrochimica
Hydrobiologica 27, 409–415.
Leenheer, J.A., Brown, G.K., MacCarthy, P., Cabaniss, S.E.,
1998. Models of metal binding structures in fulvic acid from
the Suwannee River, Georgia. Environmental Science and
Technology 32, 2410–2416.
Leenheer, J.A., Rostad, C.E., Barber, L.B., Schroeder,
R., Anders, R., Davisson, M.L., 2001. Nature and
chlorine reactivity of organic constituents from
reclaimed water in groundwater, Los Angeles County,
California. Environmental Science and Technology 35,
3869–3876.
C. McIntyre et al. / Organic Geochemistry 36 (2005) 385–397
Leenheer, J.A., Nanny, M.A., McIntyre, C., 2003. Terpenoids
as major precursors of dissolved organic matter in landfill
leachates, surface water, and groundwater. Environmental
Science and Technology 37, 2323–2331.
Leinweber, P., Schulten, H.R., Kalbitz, K., Meissner, R.,
Jancke, H., 2001. Fulvic acid composition in degraded
fenlands. Journal of Plant Nutrition and Soil Science 164,
371–379.
Liu, M., Mao, X., Ye, C., Huang, H., Nicholson, J.K., Lindon,
J.C., 1998. Improved WATERGATE pulse sequence for
solvent suppression in NMR spectroscopy. Journal of
Magnetic Resonance 132, 125–129.
Ma, H., Allen, H.E., Yin, Y., 2001. Characterization of isolated
fractions of dissolved organic matter from natural waters
and a wastewater effluent. Water Research 35, 985–996.
Malcolm, R.L., 1990. The uniqueness of humic substances in
each of soil, stream and marine environments. Analytica
Chimica Acta 232, 19–30.
Massmann, G., Tichomirowa, M., Merz, C., Pekdeger, A.,
2003. Sulfide oxidation and sulfate reduction in a shallow
groundwater system (Oderbruch Aquifer, Germany). Journal of Hydrology 278, 231–243.
McIntyre, C., Batts, B.D., Jardine, D.R., 1997. Electrospray
mass spectrometry of groundwater organic acids. Journal of
Mass Spectrometry 32, 328–330.
McIntyre, C., Jardine, D., McRae, C., 2001. Electrospray mass
spectrometry of aquatic fulvic acids. Rapid Communications in Mass Spectrometry 15, 1974–1975.
McIntyre, C., McRae, C., Jardine, D., Batts, B.D., 2002.
Identification of compound classes in soil and peat fulvic
acids as observed by electrospray ionization tandem mass
spectrometry. Rapid Communications in Mass Spectrometry 16, 1604–1609.
Nanny, M.A., Ratasuk, N., 2002. Characterization and comparison of hydrophobic neutral and hydrophobic acid
dissolved organic carbon isolated from three municipal
landfill leachates. Water Research 36, 1572–1584.
Nikolaou, A.D., Lekkas, T.D., 2001. The role of natural
organic matter during formation of chlorination by-products: a review. Acta Hydrochimica Hydrobiologica 29, 63–
77.
Nissenbaum, A., Kaplan, I.R., 1972. Chemical and isotopic
evidence for the in situ origin of marine humic substances.
Limnology and Oceanography 17, 570–582.
OÕLoughlin, E., Chin, Y.-P., 2001. Effect of detector wavelength
on the determination of the molecular weight of humic
substances by high-pressure size exclusion chromatography.
Water Research 35, 333–338.
Pettersson, C., Arsenie, I., Ephraim, J., Boren, H., Allard, B.,
1989. Properties of fulvic acids from deep groundwaters.
The Science of the Total Environment 81/82, 287–296.
Pettersson, C., Ephraim, J., Allard, B., 1994. On the composition and properties of humic substances isolated from deep
groundwater and surface water. Organic Geochemistry 21,
443–451.
397
Peuravuori, J., Pihlaja, K., 1997. Molecular size and spectroscopic properties of aquatic humic substances. Analytica
Chimica Acta 337, 133–149.
Prosser, I.P., Roseby, S.J., 1995. A chronosequence of rapid
leaching of mixed podzol soil materials following sand
mining. Geoderma 64, 297–308.
Smith, B.N., Epstein, S., 1971. Two categories of 13C:12C ratios
for higher plants. Plant Physiology 47, 380–384.
Stenson, A.C., Landing, W.M., Marshall, A.G., Cooper, S.R.,
2002. Ionization and fragmentation of humic substances in
electrospray ionization Fourier transform-ion cyclotron
resonance mass spectrometry. Analytical Chemistry 74,
4397–4409.
Thorn, K.A., Aiken, G.R., 1998. Biodegradation of crude oil
into nonvolatile organic acids in a contaminated aquifer
near Bemidji, Minnesota. Organic Geochemistry 29, 930–
931.
Thurman, E.M., Malcolm, R.L., 1981. Preparative isolation of
aquatic humic substances. Environmental Science and
Technology 15, 463–466.
Thurman, E.M., 1985a. Humic substances in groundwater. In:
Aiken, G.R., McKnight, D.M., Wershaw, R.L., MacCarthy, P. (Eds.), Humic Substances in Soil, Sediment, and
Water: Geochemistry, Isolation, and Characterization.
Wiley, New York, pp. 87–103.
Thurman, E.M., 1985b. Organic geochemistry of natural
waters. Nijhoff and Junk Publishers, Dordrecht, pp. 14–
15, 296.
Johnson-Logan, L.R., Broshears, R.E., Klaine, J., 1992.
Partitioning behavior and the mobility of chlordane in
water. Environmental Science and Technology 26, 2234–
2239.
Wassenaar, L., Aravena, R., Fritz, P., Barker, J.F., 1990.
Isotopic composition (13C, 14C, 2H) and geochemistry of
aquatic humic substances from groundwater. Organic Geochemistry 15, 383–396.
Wassenaar, L.I., Aravena, R., Fritz, P., Barker, J.F., 1991.
Controls on the transport and carbon isotopic composition
of dissolved organic carbon in a shallow groundwater
system, Central Ontario, Canada. Chemical Geology 87,
39–57.
Wilson, M.A., 1987. NMR Techniques and Applications in
Geochemistry and Soil Chemistry. Permagon Press, Oxford.
Wong, S., Hanna, J.A., King, S., Carroll, T.J., Eldridge, R.J.,
Dixon, D.R., Bolto, B.A., Hesse, S., Abbt-Braun, G.,
Frimmel, F.H., 2002. Fractionation of natural organic
matter in drinking water and characterization by 13C crosspolarization magic-angle spinning NMR spectroscopy and
size exclusion chromatography. Environmental Science and
Technology 36, 3497–3503.
Zwiener, C., Kumke, M.U., Abbt-Braun, G., Frimmel, F.H.,
1999. Adsorbed and bound residues in fulvic acid fractions
of a contaminated groundwater: isolation, chromatographic
and spectroscopic characterization. Acta Hydrochimica
Hydrobiologica 27, 208–213.