Determination of Atrazine, Acetochlor, Clomazone, Pendimethalin and Oxyfluorfen in Soil by a Solid Phase Microextraction Method

Abstract

A solid phase microextraction (SPME) method for simultaneous determination of atrazine, acetochlor, clomazone, pendimethalin and oxyfluorfen in soil samples was developed. The method is based on a combination of conventional liquid-solid procedure and a following SPME determination of the selected pesticides. Initially, various microextraction conditions, such as the fibre type, desorption temperature and time, extraction time and NaCl content, were investigated and optimized. Then, extraction efficiencies of severalsolvents (water, hexane, acetonitrile, acetone and methanol) and the optimum number of extraction steps within the sample preparation step were optimized. According to the results obtained in these two sets of experiments, two successive extractions with methanol as the extraction solvent were the optimal sample preparation procedure, while the following conditions were found to be most efficient for SPME measurements: 100 μm PDMS fibre, desorption for 7 min at 2700C, 30 min extraction time and 5% NaCl content (w/v). Detection and quantification were done by gas chromatography-mass spectrometry(GC/MS). Relative standard deviation (RSD) values for multiple analysis of soil samples fortified at 30 μg/kg of each pesticide were below 19%. Limits of detection (LOD) for all the compounds studied were less than 2 μg/kg.

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Determination of Atrazine, Acetochlor,
Clomazone, Pendimethalin and
Oxyfluorfen in Soil by a Solid Phase
Microextraction Method
Rada Đurović, Jelena Gajić-Umiljendić and Tijana Đorđević
Institute of Pesticides and Environmental Protection, 11080 Belgrade, Banatska 31b, Serbia
(radda@ptt.rs)
SUMMARY
A solid phase microextraction (SPME) method for simultaneous determination of atra-
zine, acetochlor, clomazone, pendimethalin and oxyfluorfen in soil samples was devel-
oped. The method is based on a combination of conventional liquid-solid procedure and
a following SPME determination of the selected pesticides. Initially, various microextrac-
tion conditions, such as the fibre type, desorption temperature and time, extraction time
and NaCl content, were investigated and optimized. Then, extraction efficiencies of several
solvents (water, hexane, acetonitrile, acetone and methanol) and the optimum number of
extraction steps within the sample preparation step were optimized.
According to the results obtained in these two sets of experiments, two successive
extractions with methanol as the extraction solvent were the optimal sample preparation
procedure, while the following conditions were found to be most efficient for SPME meas-
urements: 100 μm PDMS fibre, desorption for 7 min at 2700C, 30 min extraction time and
5% NaCl content (w/v).
Detection and quantification were done by gas chromatography-mass spectrometry
(GC/MS). Relative standard deviation (RSD) values for multiple analysis of soil samples for-
tified at 30 μg/kg of each pesticide were below 19%. Limits of detection (LOD) for all the
compounds studied were less than 2 μg/kg.
Keywords: Solid phase microextraction; Pesticides; Soil
Pestic. Phytomed. (Belgrade), 23 (2008) 265-271
Pestic. fitomed. (Beograd), 23 (2008) 265-271
UDC: 632.95:543.393
Scientific paper * Naučni rad
INTRODUCTION
One of the most time-consuming and difficult tasks
in pesticide residue chemical analysis is the extraction
and purification of target analytes from sample matri-
ces, particularly from such highly complex ones as soil.
Generally, routine procedures such as liquid-liquid ex-
traction (LLE), soxhlet extraction and solid phase ex-
traction (SPE) are time-consuming, tedious, require
large quantities of organic solvents and are often rel-
atively expensive. Therefore, recent trends in sample
preparation have focused on developing simpler, fast-

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Rada Đurović et al.
er, more reliable and cost-efficient methods by reduc-
ing analysis time and solvent consumption. Solid phase
microextraction (SPME), as a technique combining the
extraction and concentration processes into one step, is
an example of such development.
SPME is a simple, selective and efficient sorption/
desorption method, based on the analytes’ distibution
between the sample matrix and extraction medium.
Extraction is performed in a thin polymer film coat-
ing of a fused silica fibre, which is either immersed in a
sample (DM/SPME) or exposed to a headspace above
the sample (HS/SPME). After extraction, the fibre car-
rying the sorbed analytes is introduced into a gas chro-
matograph injector for thermal desorption (GC), while
in case of liquid chromatography (LC) the analytes are
desorbed by solvent elution.
So far, there have been scarce references to SPME
application for determining pesticides in soil. Most of
them are based on preparation of soil mixtures with dis-
tilled water and subsequent immersion of the SPME fi-
bre in the slurry (Magdic et al., 1996) or its exposure
to the gas phase above slurry (Ng et al., 1999; Castro et
al., 2001; Doong and Liao, 2001; Navalon et al., 2002;
Zhao et al., 2006; Fernandez-Alvarez et al., 2008). Some
researchers have suggested that the DM/SPME of a soil
organic extract obtained by solid-liquid extraction and
diluted with an appropriate amount of water is the most
reliable soil SPME method (Prosen and Zupancic-Kralj,
1998; Bouaid et al., 2001; Lambropoulou and Albanis,
2004). Their results indicate that this approach is more
sensitive and provides both higher recoveries and bet-
ter linearity. Most of these proposed methods, howev-
er, focus on simultaneous determination of pesticides
belonging to only one or two pesticide groups. To our
knowledge, there is actually only one report on SPME
determination of pesticides that belong to several pesti-
cide groups (chloroacetanilide, pyrethroid, organochlo-
rine and organophosphorus compounds) (Fernandez-
Alvarez et al., 2008). This method is based on head-
space analysis of soil samples wetted with ultrapure wa-
ter (50%, v/w).
As no previous studies are known to us dealing with
the DM/SPME determination of pesticides of different
pesticide groups, the intent of this study was to develop a
rapid and simple DM/SPME method for simultaneous
determination of 5 compounds having distinct chemical
structures and belonging to different pesticide groups.
The main parameters affecting DM/SPME procedures,
such as the fibre type, temperature and time of desorp-
tion, extraction time and NaCl content, as well as the
extraction efficiencies of several solvents (water, hexane,
acetonitrile, acetone and methanol) and the optimum
number of extraction steps within the sample prepara-
tion step were investigated and optimized.
MATERIAL AND METHODS
Reagents and materials
The pesticides chosen for this study were: clom-
azone, acetochlor, oxyfluorfen and pendimethalin
(Dr Ehrenstorfer), and atrazine (Syngenta) (Table 1).
Stock solutions (1 g/L) of each pesticide standard were
prepared by dissolving the weighed amount in acetone
(J.T. Baker, Deventer, Holland). The solutions were
stored at -18ºC. Working standard mixed solutions
(10 mg/L and 1 mg/L of each compound) were pre-
Table 1. Pesticides studied and some of their physico-chemical propertiesa
Tabela 1. Pesticidi i neke njihove fizičko-hemijske osobinea
Pesticide
Pesticid
Chemical group
Hemijska grupa
Mrb
(g/mol)
Water solubility
Rastvorljivost u
vodi (mg/L)
33
223
0.116
0.3
1100
Log Kowc
Hd
(Pam3/mol)
Atrazine
Acetochlor
Oxyfluorfen
Pendimethalin
Clomazone
aData cited from refs. – Podaci preuzeti iz referenci (Pesticide Manual, 2000-2001; www.sitem.herts.ac.uk/aeru/footprint/en/
index.htm, 2008)
bMolecular weight – Molekulska masa
cPartition coefficient between n-octanol and water (as the log value) – Particioni koeficijent između n-oktanola i vode
(predstavljen preko logaritamske vrednosti)
dHenry’s constant – Henrijeva konstanta
Triazine 215.7
269.8
361.7
281.3
239.7
2.5
4.14
4.47
5.18
2.5
1.5 x 10-4
3.83 x 10-1
9.40 x 10-2
2.73 x 10-3
4.19 x 10-3
Chloroacetamide
Diphenyl ether
Dinitroaniline
Isoxayolidinone

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Pestic. fitomed. (Beograd), 23 (2008) 265-271
pared weakly by diluting individual stock solutions
with acetone and storing at 4ºC. Water standard so-
lutions (25 μg/L) were used for optimizing the SPME
method. Highly purified deionized water (Purelab
Option – R7, Elga, UK) was used to dilute the mixed
acetone solutions. Sodium chloride (99.5% purity) was
purchased from Merck (Darmstadt, Germany) and
hexane, acetonitrile and methanol from J.T. Baker
(Deventer, Holland).
The fibres used (Supelco, Bellefonte, PA, USA)
were: 100 µm polydimethyl-siloxane (PDMS) and 85
µm polyacrilate (PA). Before use, the fibres were con-
ditioned in the gas chromatograph injection port as
recommended by the manufacturer. A magnetic stir-
rer (Roth RCT Basic, Germany) and 8 x 3 mm stirring
bars were used to mix the samples during extraction.
Extraction was performed in 4 ml vials (Supelco).
An uncontaminated soil sample originating from
Kikinda was used in the study. The main physico-chem-
ical properties of this soil were: pH (H2O) = 8.39; or-
ganic matter content = 3.17%; sand content = 73.96%;
silt content = 22.60%; clay content = 3.44%. The soil
was air dried and sieved (2 mm) before using.
Polypropylene centrifuge tubes with caps (50 ml)
(Sarstedt, Germany), filter papers 1PS, 150 mm diam-
eter (Watman Int. Ltd., Maidstone, UK) and a centri-
fuge (UZ 4, Iskra, Slovenia) were used in the soil ex-
traction procedure.
Instrumentation
A gas chromatograph-mass spectrometer (GC/MS)
as a detection device (CP–3800/Saturn 2200, Varian,
Australia) with 30 m x 0.25 mm x 0.25 µm VF-5ms
column (Varian) was used. The GC was programmed
as follows: initial temperature was 120ºC, then in-
creased to 170ºC at 8ºC/min and held for 4.5 minutes,
increased to 280ºC at 9ºC/min and held for 5.5 min-
utes. Helium was used as the carrier gas and its flow
rate was 1.1 ml/min.
The ion trap mass spectrometer was operated in the
electron impact/selected ion monitoring (EI/SIM)
mode. The ion trap and transferline temperatures
were set to 220ºC and 250ºC, respectively. One spe-
cific pesticide ion was selected for detection and quan-
tification, while a second one was used for confirma-
tion. The ions inspected were as follows: 200 (215) for
atrazine, 204 (125) for clomazone, 223 (146) for ac-
etochlor, 252 (317) for oxyfluorfen and 252 (191) for
pendimethalin.
Optimization of DM/SPME analysis
DM/SPME conditions, such as the fibre type, desorp-
tion temperature and time, extraction time and NaCl
content, were investigated and optimized using 4 ml of
aqueous solution containing 25 μg/L of each pesticide.
The following SPME conditions were found to be
the most efficient for simultaneous extraction of the se-
lected pesticides: 100 µm PDMS fibre, desorption for 7
min at 270ºC, extraction time of 30 min, and 5% NaCl
content (w/v).
Soil extraction optimization
Efficiency of the method optimized for SPME of
aqueous solutions was tested in the analysis of soil sam-
ples. In that part of the study, sub-samples of 8 g were
placed in polypropylene centrifuge tubes and fortified
at 30 μg/kg level of each pesticide using 1 mg/L mixed
standard solution. The spiked samples were homoge-
nized for 15 min using a mehanical stirrer and left for
24 hours prior to further analysis.
The extraction efficiencies of various solvents (wa-
ter, hexane, acetonitrile, acetone and methanol) and
the optimum number of extraction steps were deter-
mined by the following procedure: soil samples were
extracted with 15 ml of solvent for 30 min using a
mehanical stirrer and then centrifuged for 15 min at
4000 rpm. The extract was filtered and evaporated to
dryness at 35ºC using a rotary evaporator (Devarot,
Elektromedicina, Slovenia). The residues were redis-
solved in 1 ml of acetone, and 0.2 ml of these solutions
were each diluted with water to a final volume of 10 ml
for DM/SPME measurements. The presence of organ-
ic solvent (2%) was so prevented from affecting SPME
measurements and fibre life (Eisert and Levsen, 1995a,
1995b; Urruty and Montury, 1996; Hernandez et al.,
2000; Lambropoulou and Albanis, 2004).
RESULTS AND DISCUSSION
DM/SPME optimization
Different experimental parameters that affect SPME
measurements were optimized using spiked water sam-
ples. Optimization was done by a well-structured step-
by-step approach including choice of a most suitable
SPME fibre, determination of optimal desorption tem-
perature and time, extraction time and NaCl content.

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Rada Đurović et al.
Because polydimethyl-siloxane (PDMS) and poly-
acrylate (PA) fibres have been most throughly studied,
and frequently described as the most efficient for pesti-
cide extraction (Lambropoulou et al., 2000; Doong and
Liao, 2001; Sakamoto and Tsutsumi, 2004; Fernandez-
Alvarez et al., 2008), these fibres were chosen for our
study. In order to determine the optimum desorption
temperature and time, half-hour extraction procedures
were performed at ambient temperature. In the first
set of experiments, desorption temperature was varied
from 265 to 285ºC with 5 min desorption time. After
that, desorption time was varied from 5 to 9 min at the
chosen optimal desorption temperature. Between two
measurements, desorption of a blank fibre was done
each time to ensure that no residual compounds were
present on the fibre. For the PA fibre, desorption for 7
min at 280ºC was found to be optimal, while the cor-
responding optimum for the PDMS fibre was 7 min at
270ºC. Finally, the PDMS fibre was found to be more
appropriate for the mixture of selected pesticides and
was therefore chosen for further work.
Time dependence of the amount of analytes extracted
by the fibre was investigated at intervals ranging from
10 to 60 min. The results indicate that 30 min extrac-
tion time was enough to reach sorption equilibrium for
atrazine, 45 min for acetochlor and 50 min for cloma-
zone. On the other side, the 60 min interval was insuf-
ficient for pendimethalin and oxyfluorfen to overtake
the sorption equilibrium. This is in line with the es-
tablished fact that high molecular weight compounds,
due to their low diffusion, and compounds that have
low water solubility (higher affinity toward the SPME
fibre) need longer extraction times to overtake equilib-
rium (Pawliszyn, 1997; Valor et al., 2001). Considering
the pesticides' molecular weights, water solubility and
log Kow shown in Table 1, it is evident that our results
are in accordance with the rules mentioned.
Although extraction using equilibrium time is rec-
ommended, some proposed theoretical models for ex-
planation of the SPME process have indicated that
quantification is possible before a sorption equilibri-
um is reached (Ai, 1997, 1998; Đurović et al., 2007),
so that a 30 min extraction time, for practical reasons,
was chosen in the following experiments. The time pe-
riod of 30 min has been found enough time to provide
sufficient analytical sensitivity for all compounds stud-
ied. Additionally, this interval was in accordance with
the chromatographic run time (in our case 28.47 min),
which ensured a maximum sample throughput when
manual extraction was applied.
An addition of salt to a sample would decrease
the solubility of some analytes in the aqueous phase,
which stimulates their movement into the fibre coating
(Pawliszyn, 1997). For that reason, the effect of ionic
strength on the SPME process was studied by adding
different amounts of NaCl to the water mixed stand-
ard solutions (0, 2.5, 5, 10 and 15% (w/v)).
The results (Figure 1) indicate that ionic strenght af-
fects SPME efficiency in different ways and that the
yield of SPME depends on the nature of each pesti-
cide. Thus, based on compounds behaviour, consider-
ing their logKow values and solubility (Table 1), they can
be classified into two groups. The first group includes
compounds whose extraction efficiencies decrease as
the percentage of NaCl added to the solution increas-
es. This group consists of the more hydrophobic pesti-
cides, such as pendimethalin and oxyfluorfen, which
have high log Kow values (5.18 and 4.47, respectively)
and low water solubility (0.3 and 0.116 mg/L, respec-
tively) (Table 1). The second group of compounds is
made up of pesticides whose extraction yields increased
with the increase of NaCl content. These compounds
are characterized by high solubility in water and/or low-
er log Kow values, as in the case of atrazine, clomazone
and acetochlor (Table 1). Figure 1 shows the effect of
ionic strength on analytical signals for atrazine and oxy-
fluorfen as the representative pesticides of each group.
Finally, considering the results obtained for all pes-
ticides in this study, a 5% NaCl content was chosen as
optimal (Figure 1).
Figure 1. Effect of ionic strength on the analytical signal of
atrazine and oxyfluorfen
Slika 1. Uticaj jonske jačine rastvora na analitički signal kod
atrazina i oksifluorfena
0
20000
40000
60000
80000
100000
02,55 7,510 12,515
peak area – površina pika (counts)
w (NaCl) (%, w/v)
atrazine
oxyfuorfen

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Pestic. fitomed. (Beograd), 23 (2008) 265-271
Soil extraction optimization
Efficiency of the optimized SPME method was test-
ed by analysing soil samples. As mentioned before, the
DM/SPME of a soil organic extract obtained by con-
ventional solid-liquid extraction diluted with an appro-
priate amount of water was shown to be a more efficient
method than immersion of the SPME fibre in the slurry
of soil sample and distilled water (Prosen and Zupancic-
Kralj, 1998; Bouaid et al., 2001; Lambropoulou and
Albanis, 2004). Therefore the former approach was
chosen in the sample preparation step.
Extraction efficiencies of various solvents (water, hex-
ane, acetonitrile, acetone and methanol) and the opti-
mum number of extraction steps were determined by
a well-structured step-by-step approach. At first, the
most efficient solvent was chosen by applying a single
extraction procedure as described in MATERIALS
AND METHODS / Soil extraction optimization. In
general, for most of the selected pesticides, the recov-
eries obtained with methanol were higher than those
with other solvents, and methanol was therefore cho-
sen for further work. The next step was to determine
optimum extraction steps. Hence, the extraction of
spiked soil samples with methanol was repeated up to
four times under the same procedure. For most pesti-
cides studied, the best recoveries were achieved after
two extraction steps.
Finally, according to the results acquired in these two
sets of experiments (Figure 2), two successive extrac-
tions with methanol as the extraction solvent were cho-
sen as the optimal sample preparation procedure.
Validation of proposed method
Linearity of the developed method was tested in
a concentration range from 2 to 600 μg/kg. The ob-
tained arrangements and correlation coefficients (R)
for all pesticides under study are presented in Table 2.
It shows that the acquired correlation coefficients ex-
ceeded 0.996 for all compounds.
The limit of detection was computed as three times
the base line noise (S/N = 3) at the lowest detectable
concentration. LODs for all pesticides studied were
equal or less than 1.52 μg/kg (Table 2).
Precision and confidence of the developed meth-
od were determined by performing four consecutive
measurements of soil samples fortified at 30 μg/kg lev-
el. Both relative standard deviation (RSD) and recov-
ery values are presented in Table 2. The table shows that
RSDs for all pesticides under study were below 19%.
For most of the analyzed pesticides, the recovery val-
ues were higher than 68%. An explanation for the low-
er recoveries of pendimethalin may be the strong influ-
ence of soil matrix on the pesticide and/or an insuffi-
cient power of methanol as an extraction solvent in the
sample preparation step (Sparks, 1995).
The results presented suggest that the SPME method
can be used for efficient and selective extraction of pes-
ticides from complex matrix samples such as soil.
Figure 2. Dependence of extraction efficiency on: A) type of organic solvent and B) number of extraction steps, using the
most efficient solvent
Slika 2. Zavisnost efikasnosti ekstrakcije od: A) vrste organskog rastvarača i B) broja ekstrakcionih koraka pri korišćenju
najefikasnijeg rastvarača
0
25
50
75
100
recovery – prinos ekstrakcije (%)
acetochlor
atrazine
clomazone
oxyfuorfen
pendimethalin
water hexane acetonitrile
acetone methanol Imethanol II methanol IIImethanol IV