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Determination of sugar cane herbicides in soil and soil treated with sugar


									                                                                             Talanta 77 (2008) 701–709

                                                                  Contents lists available at ScienceDirect

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Determination of sugar cane herbicides in soil and soil treated with sugar
cane vinasse by solid-phase extraction and HPLC-UV
Carolina Lourencetti a , Mary Rosa Rodrigues de Marchi b , Maria Lúcia Ribeiro a,∗
    Organic Chemistry Department, Chemistry Institute, São Paulo State University - Unesp, P.O. Box 355, Araraquara, SP 14801-970, Brazil
    Analytical Chemistry Department, Chemistry Institute, São Paulo State University - Unesp, P.O. Box 355, Araraquara, SP 14801-970, Brazil

a r t i c l e           i n f o                            a b s t r a c t

Article history:                                           This work reports on the development and validation of a small-scale and efficient SPE-HPLC-UV method
Received 24 March 2008                                     for the simultaneous determination of the most used herbicides (diuron, hexazinone, and tebuthiuron)
Received in revised form 2 July 2008                       applied to soil and soil treated with sugar cane vinasse (soil-vinasse) in areas where sugar cane crops
Accepted 4 July 2008
                                                           are grown in the state of São Paulo, Brazil. The analytical procedure was optimized for solvent extraction
Available online 16 July 2008
                                                           and HPLC-UV conditions. Extraction and clean-up were combined in a single step employing solid-phase
                                                           extraction, avoiding sophisticated techniques, organic–solvent–water mixtures and consequently a longer
                                                           concentration step. Recovery studies with soil and soil-vinasse samples spiked at two herbicides lev-
                                                           els (around 0.25 and 2.0 mg kg−1 ) and sample stability (sample frozen for 20 days before analysis) were
Vinasse                                                    applied as parameters to control the efficiency of the method. Good accuracy and precision were achieved
Solid-phase extraction                                     with average recoveries ranging from 78% to 120% and relative standard deviations less than 10% through-
HPLC-UV                                                    out the whole recovery test. The method’s limit of detection ranged between 0.025 and 0.050 mg kg−1 for
                                                           diuron, hexazinone, and tebuthiuron in soil and soil-vinasse. The feasibility of this method was applied to
                                                           determine the herbicide half-lives (t1/2 ) in soil and soil-vinasse in a laboratory study. Sugar cane vinasse
                                                           added to soil increased the degradation of diuron and tebuthiuron (p < 0.05), reducing the t1/2 from 80 to
                                                           7 days and 128 to 73 days, respectively. This method is presented as an alternative which could be applied
                                                           to assess herbicide behavior in soil in order to prevent water contamination and to contribute to establish
                                                           pesticide limits in soil.
                                                                                                                                © 2008 Elsevier B.V. All rights reserved.

1. Introduction                                                                               The degree to which each process will contribute to the overall
                                                                                              loss of the pesticide is in turn dependent on the physicochemical
   Brazil is one of the world leaders in the production of sugar cane,                        properties of each pesticide (e.g., water solubility, adsorptive affin-
sugar, and fuel alcohol (ethanol) [1], which has been considered as                           ity), characteristics of the soil (e.g., pH, organic content, biomass
a renewable alternative for conventional fossil fuels [2]. The sugar                          and redox status), environmental conditions (e.g., temperature and
cane monoculture requires a large amount of pesticides, and the                               moisture), and management practices (e.g., application rate and
herbicides represent approximately 56% of the total dollar value of                           formulation type) [6].
the pesticide business in Brazil [3], and these are the most widely                               The determination of pesticide behavior in soil has been pre-
employed class of pesticide applied as a pre- and post-emergent                               sented as an alternative to prevent superficial and groundwater
weed control agent to improve sugar cane crop yields.                                         contamination since it is the first step to detect and alert to possible
   It has been claimed that only 1–3% of the agricultural pesticide                           cases of water contamination [7].
application reaches the site of action [4]. In the soil, the fate of                              The development and application of methodologies to deter-
the pesticide is controlled by the chemical, biological and physi-                            mine pesticides in soil are challenging tasks as a result of some
cal dynamics of this matrix. These processes can be grouped into                              of the aspects encountered, such as the pesticide concentration in
those that affect persistence, including chemical and microbial                               the soil, which can be high or extremely low; a great variety of
degradation, and those that affect mobility, involving adsorption,                            pesticides can cover a wide range of polarities; a strong binding
plant uptake, volatilization, wind erosion, run-off and leaching [5].                         of the analytes to the soil; and there is also a lack of analytical
                                                                                              standards for the degradation products formed [5]. Other factors
                                                                                              that can affect the efficacy of a method are factors which involve
    ∗ Corresponding author. Tel.: +55 16 33016664; fax: +55 16 33016692.                      the soil itself, such as: (1) the amount of organic matter present
      E-mail address: (M.L. Ribeiro).                                      in soil, and (2) the compounds present in the soil which could

0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
702                                                 C. Lourencetti et al. / Talanta 77 (2008) 701–709

interfere in the extraction or quantification steps. Nowadays, many             2.2. Apparatus
organic products have been applied to soil in order to improve soil
conditions, such as municipal solid waste compost [8], sludge com-                 A high-performance liquid chromatograph (Waters, Milford,
post [9], and sugar cane vinasse [10,11], but the influence of these            MA, USA), equipped with two solvent delivery pumps (Model 501),
additives to the behavior of pesticides in soil is still not totally           manual injector with a 20 l loop (Model UK), UV–vis absorbance
known.                                                                         detector (Model 485) and a reporting integrator (Model 746) was
    Andreu and Picó [5] reviewed the most relevant analytical                  used to determine the diuron, hexazinone and tebuthiuron. A stain-
methods to determine pesticides and their transformation prod-                 less steel analytical column Gemini C18 (150 mm × 4.6 mm i.d.,
ucts in soil, regarding a discussion about the steps involved                  5 m; Phenomenex) was employed. The mobile phase consisted
in method development, such as matrix preparation, extraction,                 of a mixture of methanol and water (45:55, v/v) and was delivered
clean-up, fractionation and determination. In this review, Soxhlet             in isocratic mode at a flow rate of 1.0 ml min−1 . Before using, the
is appointed as one of the most frequently used techniques since               mobile phase was passed through a 0.45 m membrane filter from
it has been adopted in many standardized analytical methodolo-                 Millipore (Bedford, MA, USA) and degassed in an ultrasonic bath.
gies to determine pesticides in soil. However, this technique uses             Simultaneous pesticide detection was performed at 247 nm and all
drastic conditions that have often broken the structural integrity of          measurements were carried out at room temperature.
thermolabile pesticides, and requires much time and solvent con-
sumption. Sonication and shaking are other traditional techniques              2.3. Procedure
for organic analytes, but these also consume large quantities of sol-
vent, and are labor intensive. Modern technologies, including the              2.3.1. Sample collection and treatment
use of new sources of energy, have been described. However these                   One composite non-agricultural soil sample (total of 10 kg) was
new extraction procedures, based on instrumental techniques,                   taken at different points, from 0 to 20 cm depth, in a regular area
such as microwave-assisted extraction (MAE), supercritical fluid                located in Araraquara City, São Paulo State, Brazil. The texture of
extraction (SFE), pressurized fluid extraction (PLE), and subcriti-             the soil was 19.2% sand, 58.1% clay and 22.7% silt. Two laboratory
cal water extraction (SWE), have been tested to facilitate sample              samples (3 kg) were reduced by quarting and air-dried at room
pre-treatment [5,12], they require special equipments. Small-scale             temperature.
methods have brought about a combination of extraction and clean-                  Five liters of soil sample were treated with sugar cane vinasse
up steps into one step, using a chromatographic column prepacked               (750 ml) (150 m3 ha−1 ). This dose corresponds to the regular appli-
with sorbents, using [13] or not using sonication [14].                        cation dose in sugar cane crops in the Araraquara region. The
    The extraction methods described in literature to determine                soil-vinasse sample was thoroughly mixed to assure complete
herbicides applied to sugar cane crops have been carried out                   homogeneity and was air dried at room temperature for 3 days.
by shaking [15–18], accelerated solvent (ASE) [19], sonication                 Soil and soil-vinasse samples were reduced to approximately 1 kg
[20], pressurized fluid (PFE) and Soxhlet [21]. However, the                    by quarting and sieving through a 0.84 mm sieve.
simultaneous determination of the most utilized herbicides in                      Soil and soil-vinasse, containing approximately 20 g dm−3 of
current use for this culture in the state of São Paulo, Brazil,                organic carbon, 3% of humidity and pH 5.0, were used to develop
which are diuron [(3-(3,4-dichlorophenyl)-1,1-dimethylurea)], CAS              and validate the method and for the degradation study of diuron,
number 330-54-1, hexaninone [(3-cyclohexyl-6-(dimethylamino)-                  hexaninone and tebuthiuron.
1-methyl-1,3,5-triazine-2,4(1H,3H)-dione], CAS number 51235-
04-2, both presented as a mixture in commercial products, and                  2.3.2. Spiked samples and extraction
tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N -                 Spiked soil and soil-vinasse samples were prepared by adding
dimethylurea), CAS number 34014-18-1, is very rare and no                      1.0 ml of a standard mixture of herbicides to 40 g of sample,
analytical method has been reported so far.                                    resulting in two spiked sample levels: one at 0.25, and the other
    This study takes into account the development and validation               at 2.0 mg kg−1 for hexazinone and tebuthiuron and 0.26 and
of a new analytical method employing solid-phase extraction and                2.57 mg kg−1 for diuron. In both cases, field application rates were
HPLC-UV to determine diuron, hexazinone and tebuthiuron in soil                used at the lowest and highest recommended doses. The spiked
and soil treated with sugar cane vinasse (soil-vinasse), and also the          samples were kept at room temperature for 24 h for total solvent
application of this method in a laboratory study to determine the              evaporation and after this, the extractions were carried out.
half-lives of these three herbicides in soil and soil-vinasse.                     The solid-phase cartridge packed with a reverse phase (C18)
                                                                               was previously conditioned by rinsing with 10 ml of methanol
2. Experimental                                                                (5 ml min−1 ) under vacuum before transferring 4 g of soil sample
                                                                               (dry weight) to the top of the cartridge (Fig. 1). During the condi-
2.1. Reagents                                                                  tioning, the cartridges were not allowed to be dried before sample
                                                                               addition, as recommended [22].
   Pesticide standards of hexazinone (99.9%) and tebuthiuron                       At the beginning of the experiments, two elution solvent sys-
(99.9%) (Riedel de Haën) were obtained from Sigma–Aldrich                      tems were compared as extraction solvents: 20 ml of methanol and
Laborchemikalien GmbH (United Kingdom) and diuron (97.5%)                      20 ml of acetone at 2 ml min−1 . For the method validation, methanol
from Ehrenstorfer GmbH (Augsburg, Alemanha). Stock solutions                   was used as the extraction solvent to determine diuron, hexazi-
(200 mg l−1 for hexazinone and tebuthiuron and 255 mg l−1 for                  none and tebuthiuron in soil and soil-vinasse samples. The eluent
diuron) and different working standard mixtures of pesticides                  was concentrated to a small volume (approximately 0.5 ml) with a
were prepared in methanol and were stored at −18 ◦ C. Ace-                     rotary vacuum evaporator at 40 ◦ C. The concentrated extract was
tone (Mallinckrodt Ultiam AR® , Paris, Kentucky), methanol and                 adjusted with methanol to 2.0 ml and stored at −18 ◦ C until anal-
acetronitrile (J.T. Baker, USA) were pesticide-residue analysis grade.         ysis. The 20 l aliquots were injected into the HPLC-UV system
HPLC-grade water was obtained from a Millipore water purification               ( = 247 nm) for analyses. After the method validation, evaluation
system (Milford, MA, USA). Solid-phase extraction (SPE) cartridges             of the analytes stability in frozen samples was carried out analyzing
AccuBOND II ODS-C18 (500 mg, 6 ml capacity) were purchased from                the spiked samples, the soil and the soil-vinasse, stored for a period
Agilent Technologies (United Kingdom).                                         of 20 days under refrigeration (−18 ◦ C).
                                                             C. Lourencetti et al. / Talanta 77 (2008) 701–709                                                703

                                                                                        where m is the number of analytical values (Ai ) and n is the number
                                                                                        of the blanks values (Bi ). The degree of freedom (f) = m + n − 2.

                                                                                                       n                2
                                                                                                          (A       ¯
                                                                                                                 − A)
                                                                                                       i=1 i
                                                                                         ˆA =                                                                 (4)

                                                                                                ¯     ¯
                                                                                        where B and A are the mean blank and mean analytical value,
                                                                                           The sensitivity of the analytical method (S), which means
                                                                                        the change in signal value per change of concentration, can be
                                                                                        estimated from the mean analytical value and from the lowest
                                                                                        fortification level (q) (Eq. (5)).
                                                                                        S=                                                                    (5)

                                                                                        2.3.4. Degradation study
                Fig. 1. SPE column packed with soil [24,modified].                          The study of herbicide degradation in soil to determine the
                                                                                        half-life was conducted under laboratory conditions according
                                                                                        to specifications given by the Organization for Economical Co-
2.3.3. Quality control and method validation                                            operation and Development, 2002 [24]. An aliquot of 0.4 ml of
    For each herbicide determined by HPLC-UV, the range of her-                         a methanolic solution was applied to both the soil and the soil-
bicide concentrations (0.25–12.7 mg l−1 ) was appropriated to the                       vinasse at doses of 1.61 mg kg−1 for diuron, 0.374 mg kg−1 for
recommended dose usually applied to sugar canes crops for                               hexazinone and 1.03 mg kg−1 for tebuthiuron. These doses cor-
this kind of soil (approximately 0.774–1.714 mg kg−1 for diuron,                        respond to the highest agricultural dose for sugar cane crops
0.251–0.364 mg kg−1 for hexazinone and 0.735–1.103 mg kg−1 for                          cultivated in the kind of soil used in this study. Soils samples (50 g)
tebuthiuron). Quantitative measurements were obtained using                             placed into aluminum boxes (210 cm3 of capacity) were previously
external standard calibration curves. Three injections were per-                        incubated for 5 days in a totally darkened incubator. Temperature
formed for each calibration point.                                                      (30 ◦ C) and humidity of soil (at 60–75% of its maximum water hold-
    Two different fortification levels were considered for the                           ing capacity) were kept constant during the entire period of this
method validation step, one low at approximately 0.25 mg kg−1 and                       experiment.
one high at approximately 2.0 mg kg−1 . Also, soil and soil-vinasse                        All soils were thoroughly stirred to complete homogeneity after
spiked samples were processed to asses the influence of aging and                        the addition of the water and the herbicides. The aluminum boxes
freezing on the extraction efficiency, which were performed by                           were lightly closed to allow air exchange. The soil moisture was
accuracy and precision determination.                                                   controlled by weighing the boxes containing the soils periodically
    The accuracy and precision of the extraction procedure were                         and by replacing any losses by adding deionized water.
carried out by extracting replicate spiked sample (n = 5). These two                       The method described above was employed to quantify the her-
parameters were expressed in terms of the percentage recovery                           bicide residue contents at intervals of 0, 3, 5, 7, 14, 21, 28 and 50 days
and the percentage relative standard deviation (R.S.D.), respec-                        for the diuron, hexazinone and tebuthiuron treatment. For this, at
tively. The specificity of the assay was established analyzing                           each fixed time, three replicates of each treatment were frozen dur-
the soil and soil-vinasse samples without standard addition. The                        ing 2 days in order to minimize the action of soil micro-organisms
chromatograms were visually inspected for interfering chromato-                         and to avoid differences in the samples after taking them out of
graphic peaks from the sample matrix substances.                                        the incubator. Before doing the analyses, the samples were kept in
    The limits of quantification (LOQ) and detection (LOD) of this                       ambient temperature until defrosted.
method were calculated according to the Thier and Zeumer cri-
teria [23]. The LOQ was determined as the lowest concentration                          3. Results and discussion
of the compounds that gives a response that could be quantified
with an R.S.D. of less than 20% and a recovery of at least 70%. The                     3.1. Optimization of chromatographic separation
LOD (Eq. (1)) was estimated from recovery experiments by the
equations:                                                                                  Both the mobile phases, methanol:water and acetonitrile:water,
                                                                                        are the most used mixtures applied in analytical methods reported
          2tf,95 ˆ com                                                                  for diuron, hexazinone and tebuthiuron determination in differ-
LOD =                                                                         (1)
               S                                                                        ent matrices [15,25–27]. However acetonitrile:water (30:70, v/v)
                                                                                        resulted in a faster chromatographic elution when compared
   The standard deviation ( ˆ com ) (Eq. (2)) is computed from the
                                                                                        with methanol:water (45:55, v/v), this mixture was avoided with
standard deviation of the blank signal ( ˆ B ) (Eq. (3)) and from the
                                                                                        regard to the elevated cost and elevated toxicity of acetonitrile.
standard deviation ˆ A (Eq. (4)), estimated during the experiment
                                                                                        This first chromatographic optimization was obtained employing
with the lowest fortification level.
                                                                                          = 254 nm, the most common value used for the simultaneous
                                                                                        determination of hexazinone and tebuthiuron [16,18,20,27].
             (m − 1) ˆ A + (n − 1) ˆ B                                                      To optimize the sensitivity of the HPLC-UV, absorbance spec-
ˆ com =                                                                       (2)
                   m+n−2                                                                trums were produced for diuron, hexazinone and tebuthiuron and
                                                                                        the following wavelength values were obtained as maximum values
                        ¯    2                                                          for these compounds, 251, 147 and 255 nm, respectively. The value
              i=1 i
                      − B)
ˆB =                                                                          (3)       247 nm resulted in the best sensitivity for simultaneous detection.
                n−1                                                                     Isocratic elution (Fig. 2A) was applied instead of the gradient elution
704                                                               C. Lourencetti et al. / Talanta 77 (2008) 701–709

                       Fig. 2. HPLC-UV chromatograms ( = 247nm) of standard mixture (10.0 mg l−1 ) using isocratic (A) and gradient (B) mode elutions.

mode (Fig. 2B) as it resulted in a better chromatographic resolu-                            (10 ml) was used to evaluate the remaining residues in soil after
tion and avoided time stabilization required for the gradient elution                        the first extraction. No compounds of interest were detected in the
mode.                                                                                        extract of the second extraction, thus a volume of 20 ml was enough
                                                                                             to extract all herbicides at the highest spiked level studied. How-
3.2. Method development and validation                                                       ever the extraction efficiency of both solvents was satisfactory, with
                                                                                             an average recovery from 92% to 107% and a R.S.D. lower than 7%,
    Under the chromatographic conditions described, calibration                              methanol was select as the extraction solvent to carry out the vali-
curves were constructed using external standard method calibra-                              dation method, since the chromatogram obtained using methanol
tion. Good linearity (range: 0.25–12.7 mg l−1 ) and good correlation                         was cleaner than the one obtained using acetone (Fig. 3). These
coefficients (r2 > 0.999) were achieved for all herbicides. The reten-                        results show that it is possible to avoid organic–solvent–water
tion time and detector response precision were determined over                               mixtures and consequently, a long concentration step can be elim-
intra- and inter-day periods injecting standards solutions at the                            inated.
beginning and between sample injections. Satisfactory results were                              Fig. 4 shows the chromatographic data obtained for herbicides
obtained and these are presented in Table 1.                                                 extracted from spiked soil-vinasse compared with soil-vinasse
    Environmental extracts contain high proportions of co-                                   (sample control) and the standard solution. Sugar cane vinasse
extracted materials which may deteriorate the HPLC system and                                constituents did not interfere in the analyses. Average recov-
affect the analysis results. Therefore, these co-extracted sub-                              ery (78–104%) and R.S.D. (5–7%) were considered satisfactory
stances should be avoided during extraction and clean-up steps                               for the recovery experiments with spiked sample at 2.0 mg kg−1
and removed prior to quantification by HPLC. The influence of                                  for hexaninone and tebuthiuron and at 2.57 mg kg−1 for diuron.
vinasse constituents, a dark brown effluent with high organic mat-                            Vinasse addition to soil only affected the potassium content, which
ter and salt contents, on the chromatographic analysis for diuron,                           increased almost 30-fold. Neither the pH, nor the organic matter
hexazinone and tebuthiuron had not been described in literature                              content suffered modification (Table 2).
before now. Taking this into account, this method was also vali-                                A procedural chemical and sample control were checked to
dated for soil treated with sugar cane vinasse, trying to simulate                           assure the absence of interfering compounds. The chromatographic
similar conditions present in the environment. Considering this                              data confirmed the selectivity of the proposed method for diuron,
aspect, preliminary experiments were conducted in order to select                            hexaninone and tebuthiuron, and presented no interference from
the solvent to extract these herbicides from soil. By substitut-                             the matrix during the analysis. The clean-up step, coupled with the
ing organic–solvent–water mixtures, methanol and acetone were                                extraction step, resulted in the elimination of possible substances
tested as extraction solvents. These experiments were carried out                            that could interfere during the identification and quantification of
employing the procedure described and the extraction efficiency of                            the target analytes.
both solvents was evaluated realizing a recovery study at the level                             Due to the lack of certified natural-matrix materials contain-
of the most highly spiked soil sample. A second fraction of solvent                          ing relevant pesticides in soil [5], as in this case of the mixture of

Table 1
Retention time (tR ), calibration data, repeatability and inter-days precision of the herbicides analyzed by HPLC-UV

Herbicides              tR (min)          Calibration data                                  Repeatibilitya (R.S.D., %)                Inter-days precisiona (R.S.D., %)

                                          Equation                        r2                tR            Peak area                   tR           Peak area

Hexazinone              11.8              y = 158,550x − 12,468           0.9996            0.8           1.9                         1.0          2.2
Tebuthiuron             13.5              y = 128,826x − 10,975           0.9996            1.3           1.3                         1.6          1.6
Diuron                  30.6              y = 218,321x − 24,522           0.9996            0.7           1.6                         0.8          1.8
      Relative standard deviation of retention time and peak area (n = 15); x: concentration (mg l−1 ); y: detector response (HPLC-UV).
                                                              C. Lourencetti et al. / Talanta 77 (2008) 701–709                                                         705

Fig. 3. HPLC-UV chromatograms ( = 247 nm) of: (A) standard mixture (5.0 mg l−1 ) [hexazinone (1), tebuthiuron (2) and diuron (3)]; spiked soil sample at 2.0 mg kg−1
(hexazinone and tebuthiuron) and 2.57 mg kg−1 (diuron) extracted with methanol (B) and acetone (D); control soil sample, methanol (C) and acetone (E) as extraction

Fig. 4. HPLC-UV chromatograms ( = 247nm) of: (A) standard mixture (5.0 mg l−1 ) [hexazinone (1), tebuthiuron (2) and diuron (3)]; (B) spiked soil and (D) soil-vinasse
sample at 2.0 mg kg−1 (hexazinone and tebuthiuron) and 2.57 mg kg−1 (diuron); (C) control soil sample; (E) control soil-vinasse sample. Methanol as extraction solvent for
all chromatograms.

herbicides studied in this work, the method was validated using                             Detection limits obtained for diuron, hexazinone and tebuthi-
spiked samples of soil and soil-vinasse at two levels, and thus, the                     uron in soil and soil-vinasse are summarized in Table 3. The LOQ
lowest and highest recommended dose for sugar cane crops were                            was determined as the lowest concentration of the compound that
contemplated.                                                                            gives a response that could be quantified with an R.S.D. of less than

Table 2
Soil and soil-vinasse properties

                    P (mg dm−3 )        O.M. (g dm−3 )       O.C. (%)        pH          Ka           Caa         Mga      P.A.a       T.E.B.a       C.E.C.a        B.S. (%)

Soil                2                   20                   1.2             4.9          0.4         9           2        32          11            43            26
Soil-vinasse        3                   20                   1.2             5.0         11.7         8           4        31          24            55            44

P: Phosphorus, O.M.: organic matter, O.C.: organic carbon, K: potassium, Ca: calcium, Mg: magnesium, P.A.: potential acidity, T.E.B.: total exchangeable bases, CEC: cation
exchange capacity and B.S.: base saturation.
    Presented as mmol dm−3 .
706                                                            C. Lourencetti et al. / Talanta 77 (2008) 701–709

Table 3                                                                                      From our knowledge, the presented method is the first study for
Method detection limits for diuron, hexazinone and tebuthiuron in soil and soil-
                                                                                          the simultaneous determination of these three widely employed
                                                                                          herbicides applied to soil used for sugar cane crops. Most of the
Herbicides              Method detection limits (mg kg−1 )                                compared methods involve laborious extraction and clean-up pro-
                        Hexazinone               Tebuthiuron             Diuron           cedures or they require special apparatus when considering the
Soil                    0.025                    0.040                   0.042
                                                                                          pressurized fluid extraction (PFE) technique. On the other hand,
Soil-vinasse            0.030                    0.050                   0.035            some described methods did not show important analytical param-
                                                                                          eters, such as limit of detection and quantification, which are
                                                                                          necessary for the validation of an analytical method [30–32]. The
                                                                                          proposed method shows good precision and accuracy, as do the
20% and a recovery of at least 70%. So the LOQ value for diuron,
                                                                                          other cited methods.
hexazinone and for tebuthiuron was 0.26, 0.25 and 0.25 mg kg−1 ,
respectively. These values are in agreement with the advised val-
ues established for some soil-pesticide levels in the state of São                        3.3. Degradation study
Paulo [28].
    The accuracy and precision were considered adequate to recover                            Herbicides are a widely applied class of pesticides employed in
diuron, hexazinone and tebuthiuron in soil and soil-vinasse sam-                          agriculture and they are causing concern regarding the potential
ples at both lowly and highly spiked levels, with recoveries ranging                      contamination of ground and surface waters, which has been sys-
from 81% to 119% and an R.S.D. lower than 10% (Table 4). In this                          tematically reported in literature [27,33–38]. Knowledge regarding
recovery experiment, spiked samples were extracted 24 h after the                         the fate of pesticides and monitoring studies have been focused on
herbicides were added to soil.                                                            assessing the exposure of these to humans and the environment
    The elapsed time between sample collection and laboratory                             [4,39]. Pesticide dissipation in soil is an important parameter to
sample processing is an important aspect in the analysis of the                           estimate the persistence of pesticides in the environment. Racke et
pesticide residues, and it also should be considered during method                        al. [6] emphasized that the contribution made by each of the loss
validation [29]. Soil samples can be frozen until they are required                       mechanisms, such as microbial degradation, chemical hydrolysis,
for analysis, but is necessary to define the time that samples will                        photolysis, volatility, leaching and surface runoff, to the overall dis-
remain stable and that analytes concentrations will not be affected.                      sipation is generally assessed by conducting laboratory and/or field
In a previous study, spiked samples with hexazinone and tebuthi-                          studies.
uron at 1.25 mg kg−1 were frozen for 3, 10 and 20 days before                                 Taking into account that diuron, hexazinone and tebuthiuron
analysis. Good preliminary results were obtained and a second                             have been detected in groundwater samples from different coun-
experiment was performed to evaluate the influence of storing soil                         tries [40–43], and the fact that the assessment of their behavior
and soil-vinasse samples, which contained the three target her-                           in soil is an important contribution to understand the pathway to
bicides at two levels (the low and the high level applied in this                         avoiding water contamination, the degradation of herbicides in soil
study) and were frozen for 20 days at the aforementioned herbi-                           and the influence of vinasse addition to soil was evaluated in this
cide concentrations. Similar to the recovery experiments employed                         study employing the analytical method presented above.
on non-frozen spiked samples, good results were also obtained                                 The soil degradation experiment carried out with soil and soil-
(Table 4) for the frozen ones. This experiment showed that it is                          vinasse under laboratory conditions indicated that the behavior of
possible to keep soil and soil-vinasse samples at the spiked sample                       diuron, hexazinone and tebuthiuron varied between themselves
levels studied frozen for 20 days without any modification of the                          and for different kinds of treatment (Fig. 5).
herbicide concentrations.                                                                     However the degradation of diuron gave a more representive
    As can be seen in Table 4, a greater recovery was obtained for                        result when compared with that of hexazinone and tebuthiuron
all herbicides at the low spiked level, except for hexazinone and                         (Fig. 5), its principal product of biodegradation, 3,4-dichloroaniline,
tebuthiuron in frozen soil. This fact can be justified by the possi-                       exhibits a higher toxicity, and it is also persistent in soil, water
ble formation of herbicides bound to residues in soil. This factor                        and groundwater [44]. Giazomazzi and Cochet [44] alerted to the
was pointed out by Andreu and Picó [5] as one of the challeng-                            fact that biodegradation and toxicological studies must not be only
ing aspects related to the development of analytical methods to                           focused on the disappearance of a polluting agent that could not
determine pesticides in soil.                                                             be mineralized and transformed into another compound, but also
    A comparison of the proposed method with other analytical                             on the intermediate degradation products in order to define the
methods previously presented in literature for the determination                          real environmental impact of a pollutant, since the intermediate
of diuron, hexazinone and tebuthiuron in soil is shown in Table 5.                        compounds may be more toxic than the initial compound itself.

Table 4
Recoveries and R.S.D.s of the three studied herbicides from soil and soil-vinasse samples, no frozen and frozen (20 days)

Herbicides       Soil treatment      No frozen samples                                                        Frozen samples (20 days)

                                     Low level                           High level                           Low level                       High level

                                     Recovery (%)         R.S.D. (%)     Recovery (%)        R.S.D. (%)       Recovery (%)       R.S.D. (%)   Recovery (%)      R.S.D. (%)

Diuron           Soil                111 (101–119)        6               86 (81–90)         4                104 (98–112)       5             91 (87–92)       2
                 Soil-vinasse        115 (105–119)        5               86 (84–87)         1                116 (11–120)       3             86 (78–91)       6

Hexazinone       Soil                110 (108–114)        4             102 (98–106)         4                 97 (93–101)       4            101 (95–106)      4
                 Soil-vinasse        109 (105–114)        3              98 (95–99)          2                105 (102–110)      3             98 (90–104)      5

Tebuthiuron      Soil                111 (103–119)        6               93 (85–99)         6                100 (93–105)       5            100 (98–101)      1
                 Soil-vinasse        113 (106–119)        5               93 (91–95)         2                107 (100–112)      5             96 (87–102)      7

Low level: 0.25 mg kg−1 for hexazinone and tebuthiuron, and 0.26 mg kg−1 for diuron; high level: 2.0 mg kg−1 for hexazinone and tebuthiuron, and 2.57 mg kg−1 for diuron.
Recovery (n = 5) expressed as: mean (max–min). R.S.D.: Relative standard deviation.
Table 5
Comparison of literature methods and the present method for diuron, hexazinone and tebuthiuron determination in soil sample

Pesticides                                Sample (g)      Analytical procedure                                                                         Recovery (R.S.D., %)   LOD, LOQ (mg kg−1 )     Reference

                                                          Extraction                             Clean-up                     Analytical technique

Diuron, hexazinone, tebuthiuron              4            SPEa (C18; 20 ml MeOH)                 –                            HPLC-UV (EF: C18,        76–120, 1–10           0.025–0.05, 0.25–0.26   Proposed
                                                                                                                              5 m; MF: MeOH:H2 O                                                      method
                                                                                                                              45:55; = 247 nm)
Tebuthiuron                                 25            Sonication (20 ml MeOH:H2 O (55:45,    LLEb (40 ml diethyl ether)   HPLC-UV (EF: C18,        77–86 (4–5)            s                       [20]
                                                          v/v))                                                               10 m; MF: MeOH:H2 O
                                                                                                                              (45:55); = 254 nm)
Hexazinone, tebuthiuron                     20            Shaking (75 ml MeOH:H2 O (80:20)       GPCc                         HPLC-UV (EF: fenil,      98–102 (2)             0.005, s                [17]
                                                          1 h; 25 ml, 15 min                                                  4 m; MF: MeOH:H2 O

                                                                                                                                                                                                                  C. Lourencetti et al. / Talanta 77 (2008) 701–709
                                                                                                                              (50:50); teb = 254 nm,
                                                                                                                               hex = 249 nm)
Hexazinone and degradation products         50            Shaking (3 × 68 ml MeOH:H2 O (4:1))    2 ml lead acetate            HPLC-UV (EF: C18; MF:    s                      s                       [18]
                                                                                                                              ACN:H2 O ( =/
                                                                                                                               teb = 254 nm)
                                                                                                 SPEa (C18, MeOH)
Hexazinone, tebuthiurone                     5            PFEd (acetone; 1500 psi, 100 ◦ C)      Na2 SO4 (10 g)               GC–MS                    SPE: 86–107 (4–7)      s                       [21]
                                                          Soxhlet (250 ml acetone, 18 h)                                                               Soxhlet: 88–96 (3–8)
Diuronf                                   100             Shaking (200 ml acetone:H2 O           TLCg (silica gel)            GC-ECD/GC–MS             84–97 (s)              s                       [21]
                                                          (80:20), 1 h); LLEb (2 × 20 ml ethyl
                                                          acetate, 15 g NaCl)
Diuron and degradation products           200             Sonication (vol. MeOH = 2 times the    –                            HPLC-UV (EF: C18; MF:    s                      s                       [22]
                                                          weight soil) 40 ◦ C, 24 h                                           ACN:H2 O (35:65);
                                                                                                                               teb = 252 nm)
Diuron, metolachlor                          5            Shaking (8 ml acetone, 10 min)         –                            HPLC-DAD (EF:            70–96 (2–8)            s                       [23]
                                                                                                                              RP-amida C16, 5 m;
                                                                                                                              MF: ACN:H2 O (50:50);
                                                                                                                              30 ◦ C)

R.S.D.: Relative standard deviation; s, not described.
    Solid-phase extraction.
    Liquid–liquid extraction.
    Gel permeation chromatography (60 g Bio-Beads® SX-3; chloroform-hexane, 50:50).
    Pressurized fluid extraction.
    Pesticide multi-residue: hexazinone, tebuthiuron, alachlor, bromacil, and metribuzin.
    Pesticide multi-residue: diuron, chlorotoluron, simazine, propizamide, and diflufenican.
    Thin layer chromatography.

708                                                              C. Lourencetti et al. / Talanta 77 (2008) 701–709

Table 6
First-order equations and half-lives (days) obtained after fitting data of incubation study to a first order kinetic

                          Soil                                                                                Soil-vinasse
                                                                             a            2
                          Equation                               t1/2 (R.S.D.)            r                   Equation                                 t1/2 (R.S.D.)a           r2

Hexazinone                ln(C/Ci ) = 0.0559 − 0.0083t            84 (16)                 0.9497              ln(C/Ci ) = 0.0717 − 0.0131t             53 (5)                   0.9548
Tebuthiuron*              ln(C/Ci ) = −0.2713 − 0.0054t          128 (15)                 0.8755              ln(C/Ci ) = −0.2713 − 0.0054t            73 (7)                   0.9557
Diuron*                   ln(C/Ci ) = −0.3369 − 0.0086t           80 (3)                  0.9456              ln(C/Ci ) = −0.1207 − 0.0996t             7 (0.4)                 0.9753
      Relative standard deviation (n = 3).
      p < 0.05.

                                                                                              the use of sophisticated analytical methods that determine these
                                                                                              herbicides in soil, thus avoiding organic–solvent–water mixtures
                                                                                              and a long concentration step. It also increases the possibilities of
                                                                                              automation, economizing sample manipulation and analysis time.
                                                                                              The method’s quantification limits were similar to those estab-
                                                                                              lished as the advised values for some pesticides in Brazilian soil
                                                                                              (São Paulo State). Finally, the method was efficient to determine
                                                                                              the half-life of herbicides in soil and soil treated with sugar cane
                                                                                              vinasse. It was fast, efficient and robust as is required for the mon-
                                                                                              itoring of pesticides widely distributed in soil, which is especially
                                                                                              true for the monoculture of sugar cane.

Fig. 5. Dissipation rate of diuron, hexazinone and tebuthiuron in soil and soil treated
with sugar cane vinasse (n = 3).

                                                                                                C.L. wishes to thank CNPq (Conselho Nacional de Desenvolvi-
   The degradation of diuron, hexazinone and tebuthiuron in the                               mento Científico e Tecnológico) for the fellowship.
studied soil of Araraquara, with and without the addition of vinasse
at 30 ◦ C, tented to follow the first-order kinetics (Eq. (6)) as is evi-                      References
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