Soil & Sediment Contamination, 17:111, 2008Copyright Taylor & Francis Group, LLCISSN: 1532-0383 print / 1549-7887 onlineDOI: 10.1080/15320380701741263

Determination of Organochlorine Pesticidesfrom Agricultural Soils Using Pressurized

Liquid Extraction

D. VEGA MORENO,1 Z. SOSA FERRERA,1J.J. SANTANA RODRIGUEZ1, E. POCURULL AIXAL `A,2AND F. BORRULL BALLARIN2

1University Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain2University Rovira i Virgili, Tarragona, Spain

A method to determine thirteen organochlorine pesticides from soils samples has beendeveloped using pressurized liquid extraction (PLE) and gas chromatography electron-capture detection. The variables optimized have been: type of solvent, number of cycles,temperature, time, flush volume and pressure. The optimal conditions have been hexane-acetone (1:1), 1 cycle, 50C, 60% of flush volume and 1500 psi.

The results obtained indicate that most of these compounds can be recovered quanti-tatively with RSDs lower than 10% and detection limits ranged between 0.3200 ngkg1for the pesticides studied. Under the optimized conditions, the proposed method has beenapplied to the analysis of agricultural soils. Aldrin and p,p-DDT have been found inmost of these samples at low gkg1 levels.

Keywords Organochlorine pesticides, soils, pressurized liquid extraction, gaschromatography-electron capture detection

IntroductionDuring more than half a century organochlorine pesticides have been used as insecticidesin agriculture and for the control of vector-borne diseases. Developed countries bannedthese compounds in 1970s, but some countries have continued to use DDT, especially forpublic health applications such as malaria control (Kusvuran and Erbatur, 2004; StockholmConvention, 2001). Organochlorine pesticides can be found in the environment, in spiteof their prohibition, due to the long-term persistence of them in soil (Shivaramaiah et al.,2002; Chen et al., 2002; Nawab et al., 2003; Megharaj et al., 2000).

Their lipophilic nature, hydrophobicity and low chemical and biological degradationrates have led to their accumulation in biological tissues and subsequent magnification ofconcentrations in organisms progressing up the food chain with consequences for humans(Mwevura et al., 2002; Maroni et al., 2000). For these reasons they are listed as US Envi-ronmental Protection Agency (EPA) priority pollutants (US EPA, 1979). Consequently, it

Daura Vega thanks the Spanish Ministry of Education and Science for her PhD Student Grant(FPU).

Address correspondence to E. Pocurull Aixala`, Department of Analytical Chemistry and OrganicChemistry, University Rovira i Virgili, Carrer Marcell Domingo s/n, 43007 Tarragona, Spain. E-mail:eva.pocurull@urv.net

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2 D. Vega Moreno et al.

is very important to know the residual concentration of organochlorine pesticides in soilsafter having been used for decades to study the intake of these pesticides in the harvest andconsequently in humans.

The techniques used for the extraction of organochlorine pesticides from soils areSoxhlet extraction (Boer and Law, 2003), which requires high amounts of organic solventsand long analysis time, ultrasonic extraction (Boer and Law, 2003), microwave assistedsolvent extraction (MASE) (Esteve-Turillas et al., 2004) and more recently pressurizedliquid extraction (PLE) (Hussen et al., 2006).

PLE is an extraction technique that combines elevated temperature and high pressure.It represents a viable alternative to the conventional techniques such as soxhlet and ultra-sonic extraction due to its lower organic solvent consumption and its lower extraction time(Helaleh et al., 2005).

The aim of this work is to develop a method to determine thirteen organochlorine pesti-cides in agricultural soils using pressurized liquid extraction (PLE) and gas chromatographywith electron-capture detector (ECD) (Mwevura et al., 2002; Aguilar et al., 1999). Someworks have applied PLE to extract some organochlorine pesticides from soils; however,none of them have applied PLE to a wide group of them. All samples were collected inEbros delta where organochlorine pesticides are used to mainly grow rice, although somevegetables are also cultivated.

Materials and Methods

Materials

The organochlorine pesticides studied were: -hexachlorocyclohexane (HCH), -HCH, lin-dane ( -HCH), heptachlor, heptachlor-endo, -endosulfan, -endosulfan, aldrin, dieldrin,endrin, p,p-DDD, p,p-DDE and p,p-DDT. All pesticides were purchased from Riedel-deHaen (Seelze-Hannover, Germany) with a purity higher than 98% and were prepared bydissolving appropriate amounts of the commercial products in hexane to obtain a concen-tration of 1 gL1 and stored in amber bottles at 4C. Working solutions were prepared byfurther diluting the initial concentrations with acetone. The internal standard was endosulfansulphate (99.9% purity), which was also supplied by Riedel-de Haen.

Hexane 99% of PAR quality (for pesticide analysis residue) was obtained from SDS(Barcelona, Spain). Acetone for gas chromatography and aluminium oxide 90 active neutralwere supplied by Merck (Barcelona, Spain). The aluminium oxide was heated at 120C inthe oven for 24 hours before its use. Dichloromethane, for pesticide residue analysis, wasobtained from Scharlau (Barcelona, Spain).

Helium (99.995% quality) and nitrogen (99.995% quality) were supplied by CarburosMetalicos (Tarragona, Spain).

Instrumentation

Chromatographic analyses were performed on a Hewlett-Packard (HP, Palo Alto, CA, USA)5890 Series II gas chromatograph equipped with a split/splitless injector, which was usedin the splitless mode and a 63Ni electron-capture detector. A merlin microseal high-pressureseptum from Hewlett-Packard and a splitless liner were used. A Hewlett-Packard HP-5MS(cross-linked 5%-phenyl-methylpolysiloxane) of 30 m 0.25 mm with a 0.25 m filmthickness was used to separate the analytes. Chromatographic data were recorded using theHP CHEMSTATION.

Determination of Organochlorine Pesticides 3

The samples were extracted by PLE procedure using an ASE 200 accelerated solventextraction system (Dionex, Sunnyvale, CA, USA) equipped with an 11 ml stainless steelextraction vessel.

Procedures

Sampling and Soil Pretreatment. Six agricultural soil samples were collected in DeltasEbro zone, in the mouth of the most important river in the northeast of Spain. Organochlorinepesticides are used in this agricultural area to mainly grow rice. These samples were air-dried at room temperature for more than 2 weeks, ground in a mortar with pestle, sievedthrough a 125 m sieve and stored in the refrigerator at 4C before analysis. The organicmatter contents of these soils were determined by the Sauerlandt method (organic matteroxidation by potassium dichromate and sulphuric acid) (Sauerlandt and Berwecke, 1952).Spiked soils were prepared by mixing with an organochlorine solution in acetone withenough volume for covering all the sample to ensure homogeneity. After spiking, soilswere stirred to enable enough contact between compounds and matrix.

Pressurized Liquid Extraction. Soil samples were extracted using PLE procedure (Poppet al., 1997; Zhuang et al., 2004). 5 g of the pre-treated soil were thoroughly mixed withaluminium oxide and filled into 11 ml stainless steel extraction cell. Different solventmixtures of hexane, acetone and dichloromethane were studied as extractants in differentproportions, and hexane-acetone (1:1, v/v) was the optimum. The volume of the extractionvaries depending on the selected conditions during PLE procedure. The selected operatingconditions were as follows: extraction temperature, 50C; extraction pressure, 1500 psi;preheating period, 5 min; static extraction, 5 min; solvent flush, 60% of the cell volume;nitrogen purge, 300 s; final extraction volume, 14.5 ml; and number of extraction cycles, 1.

The extract was filtered through a 0.45 m syringe-driven filter (Phenomenex,Barcelona) and 10 ml were added, together with the internal standard, in a volumetricflask before GC-ECD analysis. The volume of the internal standard added was considerednegligible.

Gas Chromatography Analysis with ECD Detection. 2 l of the PLE extract were injectedinto GC by using a split/splitless injector heated at 250C. The flow-rate of the carrier gas,helium, was 2.8 ml min1 and nitrogen was used as make-up gas. The oven temperatureprofile was as follows: the initial temperature was 60C, which was increased to 160C at40Cmin1 and then to 205C at 1.8Cmin1. This temperature was held for 3 min. Thetotal run time was 30.5 min.

Results and Discussion

Optimization of PLE ExtractionA soil sample with 3.2% of organic matter content was used to optimize PLE parameters.A blank chromatogram of this sample showed that no peaks eluted at the same retentiontimes as organochlorine pesticides.

Firstly, we optimized the extraction solvent by using 0.5 g of sample spiked at700 gkg1, 1 cycle, 100C, 1500 psi, 5 min preheating time, 5 min static time, 60%flush volume and 300 s purge time as initial conditions. Four different solvent mixtureswere tested: hexane-acetone (3:1, v/v), hexane-acetone (1:1, v/v), hexane-acetone (1:2,

4 D. Vega Moreno et al.

Table 1Recoveries obtained for several extracting mixtures used in the PLE pro-

cess. H= hexane, A= acetone, D= dichloromethane

CompoundH A

3:1H A

1:1H A

1:2H D

1:1

-HCH 90 57 19 36-HCH 88 79 104 65 -HCH 22 68 29 42Heptachlor 51 111 77 9Aldrin 92 93 99 64Heptachlor-endo 73 86 105 6-endosulfan 27 7 Dieldrin 95 101 117 13p,p-DDE 130 111 131 83Endrin 68 99 117 -endosulfan 22 48 65 p,p-DDD 76 114 120 95p,p-DDT 60 121 86 52

(-): under detection limits of the method.RSDs < 11%.

v/v) and hexane-dichloromethane (1:1, v/v). Recoveries were best when hexane-acetone(1:1, v/v) was used (Table 1). In that case, recoveries higher than 68% were obtained forall compounds except for -HCH, -endosulfan and -endosulfan, whose recoveries were57%, 27% and 48%, respectively. To increase their recoveries, we optimized the numberof cycles by doing successive extractions to the same spiked sample. Recoveries from thesecond cycle were negligible, thus we selected one cycle as the optimum.

Then, we considered the extraction temperature to be optimized. Three different tem-peratures were tested: 50C, 75C and 100C. Figure 1 shows that higher recoveries wereobtained at 50C for most of the analytes. Moreover, at this temperature less noisy baselinewas obtained because extraction of interferences was minimized. Also degradation pro-cess was avoided, especially for the most labile compounds such as -HCH and -HCH,which are degraded to -HCH or p,p-DDD and p,p-DDT, which are degraded to p,p-DDE(Walters and Aitken, 2001).

The extraction time or static time was also optimized. We determined the recoveriesobtained for 5 min, 10 min and 15 min. The results, shown in Figure 2, indicated that5 min gave the best recoveries for the organochlorine pesticides under study. Recoveriesdecreased with the extraction time because the interferences increased and consequently,the determination was more difficult.

Flush volume was the next variable optimized and 60%, 100% and 150% of cell volumewere tested. Recoveries did not increase with a flush volume higher than 60%, thus it wasselected as the optimal. Then, pressure was optimized. For all compounds the recoveriesat 1000 psi were equal or lower than the recoveries with a pressure of 1500 psi. So, thepressure was fixed at 1500 psi.

In order to decrease detection limits, we tested higher sample amounts. Up to 5 g gavesimilar recoveries (see Table 2) than 0.5 g and no block of the extraction cell was ob-served. Therefore, it was selected for further experiments. Under these optimum extraction

Determination of Organochlorine Pesticides 5

Figure 1. Recoveries obtained by using different extraction temperatures in the PLE process.

conditions, good recoveries were obtained and minimum interferences were extracted, thusa clean-up step could be avoided.

Method Validation

The same sample used for PLE optimization was used to validate the method (a soil with3.2% organic matter content and without the analytes studied).

Linear range, calculated by internal standard calibration and doing direct injection, wasgood for most compounds between 1 gL1 and 50 gL1, with correlation coefficientshigher than 0.995. Adjusting for recovery efficiency, a concentration in soil about 3 gkg1

Figure 2. Recoveries obtained at different extraction times in the PLE process.

6 D. Vega Moreno et al.

Table 2Validation data for the PLE/GC-ECD method developed

N CompoundRecovery

(%)RSD(%)

LOD(ngkg1)

1 -HCH 100 8.4 302 -HCH 74 7.5 303 -HCH 64 5.7 304 Heptachlor 98 5.4 305 Aldrin 89 2.9 306 Heptachlor-endo 81 1.8 157 -endosulfan 77 11.0 158 Dieldrin 98 7.1 39 p,p-DDE 95 4.8 3

10 Endrin 99 4.9 311 -endosulfan 80 5.2 0.312 p,p-DDD 99 8.9 0.313 p,p-DDT 103 7.4 200

and 145 gkg1, can be quantified. Limits of detection of the entire method were calculateddoing a signal noise ratio of 3 (Lindsay, 1992) and ranged between 0.3 and 200 ngkg1.Repeatability, expressed as relative standard deviation, RSD(%) (n= 4), was calculated fora soil sample spiked with 70 gkg1 of each compound. RSDs were lower than 10% inmost cases. Results are shown in Table 2.

The chromatogram obtained for the mixture of pesticides extracted of spiked soil with70 gkg1 is shown in Figure 3. It can be observed that the selected chromatographicconditions allow a good separation of analytes and a short analysis time.

The influence of organic matter content on recoveries was studied (see Figure 4). Fivesoils not contaminated with the compounds studied were spiked at 70 gkg1 (individualconcentration), a typical contamination level on agricultural soils (Kantachote et al., 2001),and stored in the dark at room temperature for 24 hours before analysis. The organic mattercontent was determined by Sauerlandt method and ranged from 0.6% to 11%. Recoveriesdecreased with the organic matter content probably due to the interaction with humic sub-stances present at higher concentration in soils with high organic matter content (Huanget al., 2003). However, no linear relationship could be established between these two fac-tors. Consequently, calibration by standard addition should be done when environmentalsoil samples with different organic matter content are analyzed. As example, Figure 4 showsthe recoveries for 3 different soils. It can be observed that recoveries decrease about 15%when organic matter content increases from 0.64% to 2.32% and they decreases more than20% when organic matter content is 7.88%.

Finally, the method was validated by analyzing a certified reference soil (CRM814-050). Because recoveries obtained with spiked samples may not be representative of thosefound with native compounds, spiked analytes are generally lightly coated on the surfaceof the matrix whereas native ones can be strongly adsorbed inside the matrix. This canbe explained by the diffusional and the kinetic limitations of the sorption process, andthe several interactions, which may have been simultaneously established between nativeanalytes and the matrix (Dupeyron et al., 1997).

Determination of Organochlorine Pesticides 7

Figure 3. Chromatogram of an extract of a spiked soil after PLE procedure. Chromatographic con-ditions as described in the text. The numbering refers to Table 2.

Due to the high concentration of the certified soil, 1 g was analyzed by the method devel-oped. Table 3 reports the pesticides present in the certified soil and gives the correspondingcertificated values, the confidence intervals, the prediction intervals and the concentrationsfound experimentally. All concentrations were between the prediction interval except for-endosulfan, dieldrin, endrin and p,p-DDT.

Figure 4. Recoveries obtained for all compounds using 3 types of spiked soils with different organicmatter content.

8 D. Vega Moreno et al.

Table 3Certified reference values and concentrations obtained by the method developeda

Compound Reference S.D. Confidence Prediction ConcentrationValueb (%) Interval Interval found

-HCH 313 77.7 276350 141485 338 33.1-HCH 265 55.1 234295 142388 256 6.2 -HCH 341 83.8 298384 155527 385 42.7Heptachlor 113 19.5 102123 69156 95 16.7Aldrin 105 9.23 99.5110 84125 96 1.77-endosulfan 297 76.3 260333 128465 110 8.7Dieldrin 190 28.7 174206 126254 271 25.6p,p-DDE 245 31.3 227264 175316 255 34.1Endrin 296 49.3 271322 187406 140 40.8endosulfa 297 61.7 265329 160434 297 51.4p,p-DDD 335 71.3 303368 176494 393 32.1p,p-DDT 263 44.2 241284 165361 153 6.4

aAll values are expresed in gkg1bThe pesticides values in the sample were certified by USEPA SW846 (3rd edition) method 8081A

(Pesticides by GC).

Then, the influence of aging time on recoveries was studied. Decreasing recoveries re-sulting from aging of matrices is a well-known phenomenon (Kiflom et al., 1999; Hawthorneet al., 1999). The analytes present in recent soil samples are more easily extracted than thosethat have had a longer contact time. This can be explained by how the analytes are incor-porated by adsorption (short periods) (Dec and Bollag, 1997). The former phenomenonoccurs at the early stages of sorption, where H-bonding and Van der Waals forces prevail.On the other hand, sequestration involves sorption at remote microsites within the soilmatrix (Kopinke et al., 1995).

In order to study the aging effect, we spiked a soil with 70 gkg1 and analyzed it at 24hours, 1 week, 2 weeks and 6 weeks. A portion of this soil was stored at room temperaturein dark and another portion in the refrigerator at 4C before extraction. Figure 5 shows the

Figure 5. Recoveries obtained with the aging time.

Determination of Organochlorine Pesticides 9

Table 4Organochlorine pesticides found in analyzed soils (gkg1)

Compound Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6

HCH n.d. n.d. 0.7 n.d. n.d. n.d.HCH 40.6 n.d. n.d. n.d. n.d. n.d.Aldrin n.d. n.d. 1.3 4.5 < loq 0.2endosulfan n.d. n.d. n.d. 15.2 n.d. n.d.Dieldrin n.d. n.d. 15.0 3.5 15.3 < loqp,p-DDE n.d. n.d. 8.1 5.9 10.3 n.d.p,p-DDD n.d. n.d. n.d. 16.4 n.d. n.d.p,p-DDT n.d. n.d. 2.1 10.9 0.2 n.d.

Loq: limit of quantification.

recoveries obtained for the different pesticides stored at room temperature with the agingtime.

It can be seen that recoveries decrease with the aging time for most of compounds,which could be explained by the sorption process. It mainly occurs during the first week andthen recoveries keep almost constant. This can be the reason why recoveries of the certifiedreference soil for some compounds are lower than the value expected. Recoveries for thesoil stored in the refrigerator showed the same behavior as room temperature samples butwith lower decrease of the recoveries.

Applications

The performance of the method was tested by analyzing 6 soils samples from the EbrosDelta zone, where organochlorine pesticides are used for agricultural purposes. Pesticides

Figure 6. Chromatogram corresponding to soil 4 sample from Ebros Delta. The numbering refersto Table 2.

10 D. Vega Moreno et al.

found are shown in Table 4. -HCH, heptachlor, heptachlor-endo, endrin and -endosulfanwere not found in any soil and the pesticides found in a higher number of samples were aldrin,dieldrin, p,p-DDE and p,p-DDT, but at low g Kg1 levels. -HCH was the pesticide foundat higher concentration, 41 gkg1, but it was only found in one soil. Figure 6 shows thechromatogram corresponding to soil 4.

ConclusionsThirteen organochlorine pesticides considered as priority pollutants by EPA have been ex-tracted from soils using PLE procedure, with gas chromatography and electron-capturedetection after the establishment of the optimum conditions. The method developed rep-resents a one step procedure, which allows the simple, fast and selective determinationof organochlorine pesticides in soils. Moreover, this method detects low concentrations oforganochlorine pesticides in soils up to 0.3 ngkg1. The recoveries obtained and RSD havebeen satisfactory. The method developed has demonstrated to be suitable for the extractionof organochlorine pesticides in soils and provides a promising and viable alternative withclassical extraction techniques.

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