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Talanta 77 (2008) 701–709 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta 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,∗ a Organic Chemistry Department, Chemistry Institute, São Paulo State University - Unesp, P.O. Box 355, Araraquara, SP 14801-970, Brazil b 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 Keywords: concentration step. Recovery studies with soil and soil-vinasse samples spiked at two herbicides lev- Herbicides Soil 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) , which has been considered as ity), characteristics of the soil (e.g., pH, organic content, biomass a renewable alternative for conventional fossil fuels . 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) . the pesticide business in Brazil , 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 . application reaches the site of action . 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 . of the analytes to the soil; and there is also a lack of analytical standards for the degradation products formed . 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: email@example.com (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. doi:10.1016/j.talanta.2008.07.013 702 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 , sludge com- A high-performance liquid chromatograph (Waters, Milford, post , 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  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  or not using sonication . 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) , sonication Soil and soil-vinasse samples were reduced to approximately 1 kg , pressurized ﬂuid (PFE) and Soxhlet . 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 . 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) m−1 ¯ ¯ where B and A are the mean blank and mean analytical value, respectively. 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)). ¯ A S= (5) q 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 . 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 . 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 n ¯ 2 for these compounds, 251, 147 and 255 nm, respectively. The value (B 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 , 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 a 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 solvent. 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. a 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 vinasse 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 . 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.  emphasized that the contribution made by each of the loss validation . 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 . Giazomazzi and Cochet  alerted to the was pointed out by Andreu and Picó  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  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  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  ACN:H2 O ( =/ conditions); 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  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  (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  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  RP-amida C16, 5 m; MF: ACN:H2 O (50:50); 30 ◦ C) R.S.D.: Relative standard deviation; s, not described. a Solid-phase extraction. b Liquid–liquid extraction. c Gel permeation chromatography (60 g Bio-Beads® SX-3; chloroform-hexane, 50:50). d Pressurized ﬂuid extraction. e Pesticide multi-residue: hexazinone, tebuthiuron, alachlor, bromacil, and metribuzin. f Pesticide multi-residue: diuron, chlorotoluron, simazine, propizamide, and diﬂufenican. g Thin layer chromatography. 707 708 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 a 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 Acknowledgment 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 dent from the correlation coefﬁcients (r) listed in Table 6.  C. Bolling, N.R. Suarez, Sugar Sweetener Situation Outlook SSS-232 (2001) 14. ln[C] = ln[Ci ] + (−k)t (6)  J. Goldemberg, S.T. Coelho, P.M. Nastari, O. Lucon, Biomass Bioener. 26 (2004) 301. where C is the herbicide concentration in soil at time t, Ci the initial  R.S. Oliveira Jr., W.C. Koshinen, F.A. Ferreira, Weed Res. 41 (2001) 97. herbicide concentration in soil, k the dissipation rate constant, and  R. Hüskes, K. Levsen, Chemosphere 35 (1997) 3013.  V. Andreu, Y. Picó, Trends Anal. Chem. 23 (2004) 83. t is the time since treatment with herbicides.  K.D. Racke, M.W. Skidmore, D.J. Hamilton, J.B. Unsworth, J. Miyamoto, S.Z. The half-life values (t1/2 ) (Table 6), when 50% of the initial Cohen, Pure Appl. Chem. 69 (1997) 1349. amount of residues is left in the soil, were obtained from the regres-  A. Farran, A. Chentouf, J. Chromatogr. A 869 (2000) 481. sion between ln(C/Ci ), according to Eq. (7):  M.S. Cravo, T. Murakota, M.F. Giné, Rev. Bras. Ci. Solo 22 (1998) 547.  M.D. Webber, H.R. Rogers, C.D. Watts, A.B.A. Boxall, R.D. Davis, R. Scofﬁn, Sci. ln 2 Total Environ. 185 (1996) 27. t1/2 = (7)  E. Gloeden, R.C.A. Cunha, M.J.B. Fraccaroli, R.W. Cleart, Water Sci. Technol. 24 k (1991) 147.  E. Madejón, R. López, J.M. Murillo, F. Cabrera, Agric. Ecosys. Environ. 84 (2001) The addition of sugar cane vinasse to soil affected the diuron and 55. tebuthiuron degradation at a signiﬁcant level of p < 0.05 (t-student)  P. Richter, B. Sepúlveda, R. Oliva, K. Calderón, R.J. Seguel, J. Chromatogr. A 994 as a consequence of a possible increase of microbial activity, as (2003) 169.  C. Sanchez-Brunete, E. Miguel, J.L. Tadeo, J. Chromatogr. A 976 (2002) 319. reported by Prata et al. . Under the conditions of this study,  L. Polese, E.V. Minelli, E.F.G. Jardim, M.L. Ribeiro, Fresenius J. Anal. Chem. 354 diuron gave the greatest factor of degradation (11.4), followed by (1996) 474. tebuthiuron (1.8) and then hexazinone (1.6).  D.C. Bouchard, T.L. Lavy, J. Chromatogr. 270 (1983) 396.  S.G. Whisenant, W.P. Clary, J. Environ. Qual. 16 (1987) 397. A recent study determining tebuthiuron leaching and its half-  J. Lyndon, B.F. Engelke, C. Helling, J. Chromatogr. 536 (1991) 223. life in sugar cane ﬁelds in Brazil did not detect measurable residues  J.B. Fischer, J.L. Michael, J. Chromatogr. A 704 (1995) 131. of tebuthiuron in soil below a depth of 40 cm after 180 days from its  Y. Zhu, Q.X. Li, Chemosphere 49 (2002) 669. application . However laboratory studies do not elucidate the  A.E. Smith Jr., L.M. Shuman, N. Lokey, J. Agric. Food Chem. 32 (1984) 416.  Y. Zhu, K. Yanagihara, F. Guo, X.Q. Li, J. Agric. Food Chem. 48 (2000) 4097. overall behavior of a compound in an ecosystem, due to the multi-  J.S. Fritz, Soil-phase extraction, in: G. Laurent, S. Shapiro (Eds.), Encyclopedia of ples forces of dissipation and transport that are simultaneously at Analytical Science, second ed., Elsevier, Amsterdam, 2005, p. 604. work in ﬁeld conditions, laboratory investigations are often aimed  H.P. Thier, H. Zeumer, Manual of Pesticide Residue Analysis, Deutsche Forschungsgemeinschaft, Pesticide Commission, Verlag Chemie, Weinheim, to study isolated processes or isolated component of an ecosystem, New York, 1987, p. 433. presenting results not highly variable when compared with ﬁeld  Organization for Economical Co-operation and development, Aerobic and studies , as is the case in the assessment of the effect of vinasse anaerobic transformation in soil (OECD Guideline for Testing of Chemicals, 307), 2002, 17 pp. on the degradation of herbicides.  V.L. Ferracini, S.C.N. Queiroz, M.A.F. Gomes, G.L. Santos, Quim. Nova 28 (2005) 380.  P.S. Bonato, V.L. Lanchote, S.A.C. Dreossi, J. High Resolut. Chromatogr. Chro- 4. Conclusion matogr. Commun. 22 (1999) 239. ¸  S.H.G. Brondi, F.M. Lancas, J. Liq. Chromatogr. Relat. Technol. 27 (2004) 171. The developed method was demonstrated to be efﬁcient for the  http://www.cetesb.sp.gov.br/Solo/relatorios/tabela valores 2005.pdf (Last simultaneous determination of diuron, hexazinone and tebuthi- revision: November 2005).  M.P. Maskarinec, R.L. Moody, Storage and preservation of environmental sam- uron in soil and soil-vinasse, showing itself to be easy to operate and ples, in: L.H. Keith (Ed.), Principles of Environmental Sampling, American permitting the treatment of a reduced sample. It is an alternative to Chemical Society, United States of America, 1988, pp. 145–155. C. Lourencetti et al. / Talanta 77 (2008) 701–709 709  N.M. Brito, O.P. Amarante Jr., L. Polese, T.C.R. Santos, M.L. Ribeiro, Pesticidas:  M.B. Matallo, L.C. Luchini, M.A.F. Gomes, C.A. Spadotto, A.L. Cerdeira, G.C. Marin, Rev. Ecotoxicol. Meio Ambiente 12 (2002) 155. Pesticidas: Rev. Ecotoxicol. Meio Ambiente 13 (2003) 83.  E. Francotte, A. Davatz, P. Richert, J. Chromatogr. B 686 (1996) 77.  T.A. Albanis, D.G. Hela, T.M. Sakellarides, I.K. Konstantinou, J. Chromatogr. A 823  R. Causon, J. Chromatogr. B 689 (1997) 175. (1998) 59.  S.N. Hamlin, K. Belitz, S. Kraja, B. Dawson, Ground-water quality in the Santa Ana  D.A. Williamson, Water Pollut. Res. J. Canada 23 (1988) 434. Watershed, California: overview and data summary, Water Resources Investi-  J.L. Domagalski, N.M. Dubrovsky, J. Hydrol. 130 (1992) 299. gations Report, United States Geological Survey, United States, 2002, I–xi, pp.  W. Abke, H. Korpien, B. Post, Vom Wasser 81 (1993) 257. 1–137.  M.A.F. Gomes, C.A. Spadotto, V.L. Lanchote, Pesticidas: Rev. Ecotoxicol. Meio  R.L. Bengtson, H.M. Selim, R. Ricaud, Trans. ASAE 41 (1998) 1331. Ambiente 11 (2001) 65.  B.P. Wood, F. Gumbs, J.V. Headley, Commun. Soil Sci. Plant Anal. 33 (2002)  S. Giacomazzi, N. Cochet, Chemosphere 56 (2004) 1021. 3501.  F. Prata, A. Lavorenti, J.B. Regitano, V.L. Tornisielo, Rev. Bras. Ci. Solo 24 (2000)  M.C.P.Y. Pessoa, M.A.F. Gomes, M.C. Neves, A.L. Cerdeira, M.D. Souza, Pesticidas: 217. Rev. Ecotoxicol. Meio Ambiente 13 (2003) 111.  A.L. Cerdeira, M.D. Desouza, S.C.N. Queiroz, V.L. Ferracini, D. Bolonhezi, M.A.F.  H.F. Filizola, V.L. Ferracini, L.M.A. Sans, M.A.F. Gomes, C.J.A. Ferreira, Pesq. Gomes, M.A. Rosa, O. Balderrama, P. Rampazzo, R.H.C. Queiroz, C.F. Neto, M.B. Agropec. Bras. 37 (2002) 659. Matallo, J. Environ. Sci. Health Part B 42 (2007) 635.
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