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FIMOI 2WdlESV39161112~378522ESE~0399W6.R1.3d Journal ot Env~ronrnenlalSclence and Heallh P a n A IESEI

JOURNAL O F ENVIRONMENTAL SCIENCE AND HEALTH

Part A--Toxic/Hazardous Substances & Environmental Engineering
Vol. A39, N O . 6, pp. 1535-1 545, 2004

Identification of Recalcitrant Hydrocarbons

Present in a Drilling Waste-Polluted Soil

J. M. ~ r c e - ~ r t e g a N.
,' G. ~ojas-~veliza~a,'
and R. ~ o d r i ~ u e z - ~ i z ~ u e z " *

' ~ e p t ode
. Biokcnologia y Bioingenieria, Cenlro de Investigacion y de
Estudios Avanzados del Instituto Politecnico Nacional, Mexico
'~rograrnade Biotecnologia del Petroleo.
Instituto Mexicano del Petroleo. Mexico

ABSTRACT

During spills of hydrocarbons in soil, it has been observed that aliphatic and the
slightly aromatic hydrocarbons are first to be removed. however, branched
aliphatic and aromatic compounds, polycyclic aromatic hydrocarbons (PAHs)
and their similar heteroatoms with sulfur (PAS) remain strongly absorbed to
soil particles. It is important to point out that studies of biodegradation of
alkyl-substituted PAHs and PAS are scarce and most of them have been carried
out using only available standard compounds. The aim of this investigation was
to identify and to quantify the aliphatic. alkyl polycyclic aromatic, and sulfured
recalcitrant fractions present in a contaminated soil with drilling wastes. A
modified method of shaking-centrifu~atio~~ extraction was implemented for the
extraction of compounds from contaminated soil. The organic extract obtained

*Correspondence: Dr. R. Rodriguez-Vizquez, Depto. de Biotecnologia y Bioingenieria,

Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacion;il
Av. Instititto Politecnico Nacional 2508, A.P. 14-740. Col. San Pedro Zacatenco, C.P
07360, Mexico, D.F: Fax: 52-55-50613313; E-mail: rerovaz(a~yahoo.com.mx;rrodrig(i+nail.
cinvestav.mx.

D01: 10.108lI ESE- 120037851 1093-4529 (Print); 1532-41 17 (Onlme)

Copyr~ghtQ 3-004 by Marcel Dekker, Inc www.dekker.com
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1536 Arce-Ortega, Rojas-Avelizapa, and Rodriguez-Vazquez

was purified and fractionated using aluminum oxide. Gas Chromatograph with
flame ionization detector (GC-FID) and Gas Chromatograph with mass
spectrometer detector (GC-MS) identified the aliphatic, PAHs and PAS
fractions. Hydrocarbon composition in the soil contaminated with 140,000mg
TPHs,'Kg soil, consisted in 80% of branched aliphatic compounds of C10 to
C22. 15% of alkyl PAHs, and 5% of PAS compounds. Lineal, lineal branched.
and cyclic branched aliphatic hydrocarbons, as well as their alkyl naphthalene,
anthracene and phenantrene, methyldibenzothiophene. dimethyldibenzothio-
phene, and diniethylnaphto[2,3-blthiophene compounds were identified by
CG-MS. The identification of compounds in soil P31, allowed us to speculate
on the origin of the contamination and the natural attenuation that had
occurred at this site.

Key Words: PAHs-Polycyclic aromatic hydrocarbons; PAS-Polycyclic

aromatic hydrocarbons, heteroatoms with sulfur; PANs-Polycyclic aromatic
hydrocarbons, heteroatoms with nitrogen; HTPs-Hydrocarbons total
petroleum; Hydrocarbon extraction; Fractionation hydrocarbons; Aliphatic
hydrocarbons.

INTRODUCTION

For a long time, the main industrial activity in southeastern Mexico has been the
exploitation of petroleum, with high amounts of residuals generated by these oil
activities. and it is not until recent decadcs that attention has been placed on disposal
and treatment during the extraction and refinement of the petroleum. It is known
that the presence of the polycyclic aromatic hydrocarbons (PAHs) and similar
heteroatoms with sulfur (PAS) in contaminated soils implies a high degree of risk,
since at least 16 PAHs are considered carcinogenic and PAS are also
considered carcinogenic and mutagenic due to their similarity with PAHS.[~] There is
also a large quantity of methyl-substituted compounds of PAHs and PAS but
information on their toxicity is scarce.[41
On the other hand, remediation of contaminated soils with hydrocarbons,
has lower operation costs with biological processes than with physico-chemical
methodologies.[" However. PAHs and heterocyclic PAS remain in soil,['] especially
in weathered soils."' In model systems of contaminated soils using standards of
these types of compounds, it has been obscrvcd that PAS inhibit PAHs degradation
and vice versa.'" This situation has emphasized the importance of characterizing
the alkyl-substituted aliphatic and aromatic hydrocarbons[" with the aim of
choosing conditions to improve the biological treatment processes to remove a
larger quantity of recalcitrant and toxic hydrocarbons.[101There are few works
using real hydrocarbon-contaminated soil; most studies have uscd soils with model
compoui~ds.[~'~
In this work, recalcitrant hydrocarbons presents in a real contaminated soil were
identified, fundamentals step during the bioremediation efforts of hydrocarbon-
contaminated soil. The identification of toxic compounds is also of great importance
to dcterminc the risk of a contaminated soil.
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Identification of Recalcitrant Hydrocarbons

MATERIALS AND METHODS

Thc uscd reagents wcre anhydrous sodium sulfate (Fermont), aluminum oxidc
mesh 60-300 activity I (SAI). dichloromethane HPLC degree (Burding & Jackson),
hexane HPLC degree (Burding & Jackson). toluene HPLC degree (Burding &
Jackson), chloroform HPLC degree (Burding & Jackson).
Soil P31 used for this work was takcn from a cesspit of drilling wastes
located in southcastcrn Mexico. Physicochemical tests were determined: texturc,
pH, humidity, C, N, and P concentrations, c o n d u ~ t i v i t ~ , ~
and
" ~ microbiologic
characterization as total and hydrocarboclastic bacteria.[13]

Hydrocarbon Extraction

To obtain thc hydrocarbons from soil P31, modifications to the method of

shaking-centrifugation extraction by Schwab et al.'"] was implemented, which
consisted in the type of shaking, time. and solvent. This method was performed using
dry or wet soil (0.5 to 2 g), 3 g of Na2S04, the mixture was shaken in a vortex until
homogenizcd, then added with 5 mL of dichloromcthane, shakcn again for 45 s, and
the sample centrifuged at 6000 rpm during 15 min. The organic phase was collected.
This procedure was repeated two more times from the addition of dichloromethane
to collect 15 mL of organic phase. The organic phase was concentrated at 740 mbar
and 40'C until dryness, then suspended with 21nL of hexanc (for asphaltenc
separation). Gas Chromatograph with mass spectrometer detector (GC-FID) and
Gas Chromatograph with mass spcctromctcr detector (GC-MS) were used to
analyze conccntrated samples. Results obtained wcre cornparcd with those from a
Soxhlet extraction method according to the D5369-93 ASTM~''] method, and the
3540 EPA method.['b1

Hydrocarbon Separation

Characterization of the extracted hydrocarbons required a methodology that

allowed to separate the aliphatic, PAHs, PAS, and heteroatoms with nitrogen
(PANs) fractions, as reported by Later et al.,[171
and the 3611 EPA method,['" which
describe the separation of cach fraction. However, some of the solvents arc now of
restricted use, for this reason the methodology by Later was modified, replacing
benzene with toluene for elution of the second fraction, in addition to a change in the
eluyent volume. To determine the efficiency of the fraction separation, a standard
mixture labeled STDFA containing aliphatic hydrocarbons C10, C12. C14, C l 6
at 221.4, 233.3, 233.8, 233.9 g/L respectively was used; the PAHs anthracene,
phenanthrene, chrysene, and dibenzo(a,h)anthracene at 113.9, 59.7, 81.6, 90.7 g/L
respcctively; dibcnzothiophenc at 220 g/L as representative compound of the PAS
and carbazol at 133 g/L as reprcsentativc conlpound of PANs.
Since hydrocarbon fractions from soil P31 were difficult to obtain with the
method reported by Later et al..L'71in the prescnt study thc mcthod was modified
until conditions allowed to separatc and obtain a high average percentage of
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1538 Arce-Ortega, Rojas-Avelizapa, and Rodriguez-Vazquez

Table I . Separation of standard STDFA components.

Recovered (%)
First Second Third
Compound fraction fraction fraction Total

C-I0
C-12
C-14
C- 16
Anthracene
Phenanthrene
Chrysene
Dibenzo(a,h)anthracene
DBT
Carbazol

The first fraction was obtained with 10 niL of hexane, the second with 28 mL of toluene, and
the third with 50 mL o r chloroforni.

recovery (>go% ) of STDFA components. Percentage recoveries of the STDFA are

shown in Table 1. The fractionation procedure was as follows: 0.8 g of hydrocarbon
extract were dissolved in 2 mL of chloroform and adsorbed in 3 g of aluminum oxide
(60-300 mesh), the mixture was then shaken thoroughly and the solvent was
evaporated during 2 h. Afterwards, the mixture (3 g) was added to the top of a glass
column (13 min di x 24 cm length) which was previously packed with 6 g of aluminium
oxide. The first fraction (aliphatic compounds) was eluted with lOmL of hcxane, the
second fraction (containing the PAHs and PAS). with 28 mL of toluene, and the third
fraction (with nitrogen compounds) was eluted with 50mL of chloroform.
TPHs and fractions were quantified and identified by gas chromatography.
using a gas chromatograph (Agilent 6850 series G C System) with flame ionization
detector (FID), and a capillary column DB-I. Helium was used as carrier gas at a
flow rate of 1.5mL/min, the temperature of the injector and detector was set at
250C, the temperature of the oven was programmed at 45C for 3n1in. of 45 at
100'C at 20GC/min.of 100 at 200C at 10C/min, of 200 at 255C at 18"C/min, of
255 at 290C at 10GC/min,remaining at 290 for 6min.
T o identify the compounds in each one of the fractions, a gas chromatograph
H P 6890 coupled to a mass spectrometer GC-MS 5973 was used with an MS-5
capillary helium column, helium was used as a carrier gas at a flow rate of
0.9 mL/min. The temperature of the injector was 250C the temperature of the oven
was programmed at 80'C for 1 mln, of 80 at 240C at 15"C/min, of 240 at 290C at
7"C/min, remaining at 290 for 2.5 min.

Quantification of Hydrocarbons

Hydrocarbons in soil were quantified by 8015 and 8440 EPA methods.['93201

Calibration curves were built to correlate the total chromatographic area under
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Identification of Recalcitrant Hydrocarbons 1539

the curvc of all the peaks detected vs. a known concentration of hydrocarbons, using
a hydrocarbon sample, coming from soil P31 (free of asphaltens). The calibration
curve was preparcd at 200, 100, 50, 25, 12.5 6.25g/L in hexanc. Five replicas of
soil P31 werc analyzed by GC-FID and the results obtained were compared with the
analysis of total petroleum hydrocarbons (TPHs) by infrared spectroscopy in a
Nicolct Nexus spectrophotometer taking as rcference the 8440 EPA method.["'

Ide~itificationof Hydrocarbons

Con~pound identification in each fraction was possible by analysis in the

CG-MS of fivc replicas of soil P31. comparing the fragmentation pattern of each
peak in thc chromatograin with the fragmcntation pattern obtained from the NIST
library. considcred with a probability above 85%.

RESULTS

Soil Characteristics

The soil uscd in this study was slightly alkaline clay loam (pH = 7.94) with 39%
moisture content, native C/N/P ratio of 100/2/0.1I, and conductivity 0.03 mS/cm.
Microbial counts corresponded to 4.2 x 10' and 1.9 x 10' CFU/g dry soil for total
and hydrocarbonoclastic bactcria respectively.

Hydrocarbon Extraction

Hydrocarbons were extracted from five replicas of soil P3 1 and GC hydrocarbon

quantification was pcrformed using the Eq. (1) obtained with the calibration curve

(where A is the area under the curve and C the hydrocarbon concentration).
Corrclation betwcen area undcr curvc and hydrocarbon conccntration was 0.9978.
Results with the shaking-centrifugation method and GC-FID revealed that
hydrocarbon concentration was in the range of 140,909 f23,667-mg TPHs/g soil
and by infrared spectroscopy it was 139,521 f32,841-mg TPHs/g soil. On the other
hand, hydrocarbon extraction by the Soxhlet method and quantified by GC-FID
was 141.438 f40,948 mg TPHs/g soil and by infrared spectroscopy the concentration
was 110.336 k 59,458 mg TPHs/g soil. As shown, the obtaincd concentration by
shaking-centrifugation was similar to the Soxhlet extraction, and presents a lower
standard deviation. The infrared spectroscopy method had a higher standard
deviation than the data obtained with the CG-FID with both extraction methods.
Thus, for hydrocarbon determinations, the shaking-centrifugation method and G C
analysis were preferred. Moreover, this extraction method allowed to process more
soil samples in a short period.
1540 Arce-Ortega, Rojas-Avelizapa, and Rodriguez-Vazquez

Separation of Hydrocarbon Fractions

Results of the hydrocarbon fractionation from soil gave only two important
fractions, the first fraction was composed of aliphatic hydrocarbons to an extent of
80% and the second fraction was the remaining 20% of the total hydrocarbon
extract. N o nitrogen compounds were detected in soil P31.

Identification o f Recalcitrant Hydrocarbons

Thc aliphatic fraction was identified by GC-MS and the chromatographic

profile is shown in Fig. 1. This is similar to the chromatographic profile of crude oil
reported by Reid et al.("] whrrc aliphatic hydrocarbons prevail. This indicates that
the contamination source could have been some type of crude oil. The compounds
(Table 2) were identified by their equivalent carbon numbers in the range of C10
to C21 (Fig. 2) where 44% of them are lineal aliphatic, 37% branched aliphatic
and 12% cyclic aliphatic hydrocarbon compounds.
As mentioned previously, the second fraction containing PAHs and PAS (Fig. 3)
wiis similar to the chromatographic profile of diesel reported by Liang and ~ilotta["]
where PAHs and PAS compounds prevail. The GC-MS analysis showed that PAHs
composed of the second fraction, mainly alkyl substituted naphtalenes, fluorenes.
anthraccnes, and phenanthrenes (Table 3). Twenty-five percent of the second
fraction was composed by PAS where methyl and dimethyl substituted compounds
were the most frequent. Table 4 indicates conlpounds identified by GC-MS.

DISCUSSION

This study showed that thc soil contaminated with 140,000mg of hydrocarbons/
Kg dry soil, contains 112.000mg/Kg of aliphatic fraction (Table 2), 21,0001ng/Kg
are PAHs (Table 3), and 6796 ing/Kg are PAS coinpounds (Table 4). Hydrocarbon
compounds with nitrogen were not detected in this soil.

Abundance

time

Figure I . Chromatographic profile of the first fraction of soil P3I by CG-MS.

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Identification of Recalcitrant Hydrocarbons

Table 2. Concentration and identification of the compounds

found in the first fraction of soil P31 by GC-MS.

(%) mg/Kg of dry soil

C10
C11
C13
C14
CIS
C16
C17
C19
C20
C21
Total aliphatic hydrocarbons

HCR HLR HL
Figure 2. Distribution of aliphatic hydrocarbons in the first fraction, according to the
structure; lineal (HL), branched (HLR). and branched cyclic (HCII). I View rhrs art rn color. at

Abundance
em
:
XI311

Figwe 3. Chromatogram profile of the compounds present in the second fraction of soil P31
by GC-MS.

The selected site had apparently bcen contaminated with drilling wastes based
on diesel. Table 4 shows the sulphur compounds found in the studied sample. Note
the presence of dimethyl and trimethyl benzothiophene, methyl and dimethyl
dibenzothiophene, which are also present in diesel components as reported by
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1542 Arce-Ortegn, Rojns-Avelizapa, and Rodriguez-Vazquez

Table 3. Concentration and identification of the conlpounds

found in the second fraction of soil P31 by GC-MS.

Compound (%) mg/Kg of dry soil

C-Naphthalene
C2-Naphthalene
C3-Naphthalene
Fluorene
C-Fluorene
C2-Fluorene
Anthracene
C-Anthracene
C2-Anthracene
C2-Phenantrene
C3-Phenantrene
Total PAHs

Table 4. Concentration and identification of the compounds

found in the third fraclion of soil P3 I by GC-MS.

Compound ('54 ) mg/Kg of dry soil

C1-Benzothiophene 0.37 52 1
C3-Benzo~hiophene 0.28 398
Dibenzothiophene 0.40 566
C1 -Dibenzothiophene 1.57 2,108
C2-Dibenzothiophene 1.75 2,450
C2-NafLo(2,3-b)[hiophene 0.47 664
Total PAS 4.85 6,796

Rodgers et These compounds can be identified in diesel before the

hydrogenation process, which could be evidence that the spilled diesel was
unproccssed. This. in turn, could imply the presence of nitrogen compounds such
as carbazol and quinoline,1241found at a concentration of 0.5% in unproccssed oils.
However, these compounds were not detected in the analyzed soil. This could be due
to the fact that nitrogen con~poundscan by degraded by soil microorganisins such as
of the genus ~ s ~ u c l o ~ n o nautypical
s [ ~ ~ ~microorgai~isnlfound in contaminated soils.
Likewise, analyzing the results from Fig. 2, 45% of the aliphatic compounds
(approximately 49,280 ing TPHs/kg of dry soil) are lineal aliphatic hydrocarbons,
which are bcen reported as compounds that can be easily removed by microorgan-
i s ~ n s . [In
~ ~addition,
] of the PAHs that are present in contaminate soil, the methyl
substituted naphthalene and fluorene are found at a higher percentage (Table 3).
The presence of sulphur con~pounds,both aliphatic and PAHs, could be because
removal of some of these compounds occurred during weathering. mainly of
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Identification of Recalcitrant Hydrocarbons 1543

aliphatic hydrocarbons, PAHs and nonsubstituted sulphur compounds.[271the

recalcitrant being those that are strongly absorbed to soil particles or have low
solubility in aqueous media, such as the alkyl substituted PAHs and PASS.['^]
Furthermore, there is evidence that methyl-substituted PAHs are more toxic than the
nonsubstituted PAHS.['~' Nevertheless, there is a possibility that biodegradation
decreased due to the lack of nutrients.13"]
The amount of TPHs is a fundamental parameter in biodegradation. Although
there is evidence that it is possible to degrade similar hydrocarbons as those reported
in this study in 800 days, the hydrocarbon concentration is always lower than
50,000 mg TPHs/kg dry
It should be noticed that, although the origin of the contamination is unknown,
there is enough evidence that the hydrocarbon compounds determined in this soil
present low bioavailability due to their absorption to S O that even if
hydrocarbon degrading-bacteria are found, it would bc necessary to enhance
bioavailability and thus biological removal of these compounds to improve soil
condition^.["^

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Received November 17, 2003