Analysis of Pesticides in Food and Environmental Samples - Chapter 8 pdf

Tables 8.1 through 8.3 summarize representativepublished papers on the analysis of pesticides in soil using those extraction techniques.8.2.2.1 Herbicides Analyses of herbicide residues

8 Determination of

Pesticides in Soil

Consuelo Sánchez-Brunete, Beatriz Albero,

and José L Tadeo

CONTENTS

8.1 Introduction 208

8.2 Sample Preparation 208

8.2.1 Sampling and Preparation of Soil Samples 208

8.2.2 Extraction 209

8.2.2.1 Herbicides 209

8.2.2.2 Insecticides and Fungicides 210

8.2.2.3 Multiresidue 212

8.2.3 Cleanup 213

8.2.3.1 Herbicides 213

8.2.3.2 Insecticides and Fungicides 215

8.2.3.3 Multiresidue 215

8.2.4 Derivatization 215

8.2.4.1 Benzonitriles 215

8.2.4.2 Glyphosate 215

8.2.4.3 Phenoxy Acid Herbicides 216

8.2.4.4 Phenylureas 216

8.2.4.5 Sulfonylureas 216

8.2.4.6 Carbamates 216

8.3 Determination of Pesticide Residues 217

8.4 Application to Real Samples 221

8.4.1 Benzonitriles 221

8.4.2 Glyphosate 221

8.4.3 Sulfonylureas 222

8.4.4 Carbamates 222

8.4.5 Organophosphorus 222

8.4.6 Pyrethroids 222

8.4.7 Pyrimethanil and Kresoxim-methyl Fungicides 223

8.4.8 Multiresidue 223

8.5 Future Trends 223

References 225

8.1 INTRODUCTION

Pesticides may reach the soil compartment by different ways Direct soil application

is normally employed for the control of weeds, insects, or microorganisms, the use

of herbicides being a typical example Pesticides may also reach the soil indirectly,when the pesticide fractions applied to the aerial part of plants (to controlweeds, crop pests, or diseases) drop to the soil during application, or lixiviate fromthe crops Other ways the pesticides reach the soil are by transportation from adifferent compartment, e.g., with the irrigation water, or by atmospheric deposition.Once in the soil, pesticides may undergo a series of transformation anddistribution processes These transformation processes may have a biotic or abioticorigin and cause the degradation of pesticides through several mechanisms, such asoxidation, reduction, or hydrolysis The distribution of pesticides can be originated byvarious processes, such as volatilization, leaching, runoff, and absorption by plants Inthese processes, the physical–chemical properties of pesticides and the adsorption–desorption equilibrium in soil are the main factors involved Figure 8.1 shows the mostimportant pathways of pesticide distribution and transformation in soil

The fate of pesticides and their degradation products in soil will depend ondifferent factors, such as the agricultural practices, the climate, and the type of soil.Pesticides and their degradation or transformation products may cause toxic effects

to man and the environment, making necessary to evaluate if their application maycause an unacceptable risk Consequently, many developed countries have regulatedthe pesticide use in agriculture [1,2]

8.2 SAMPLE PREPARATION

8.2.1 SAMPLING ANDPREPARATION OFSOILSAMPLES

The plough layer of soil (0–20 cm) is generally sampled for the determination

of pesticides in this compartment Nevertheless, other layers may be sampled at

Photodegradation

Transformation Volatilization

different depths to study the distrib ution of these compo unds in soil and, in addit ion,soil solution may be sometim es samp led to know the bioavailab ility of pesticide s.After field samp ling, soil is usual ly air dried and sieve d through a 2 mm mesh inthe laboratory The n, soil samp les are placed in closed glass fl asks and stored frozenuntil the analys is of pesticide s.

The addition of know n amounts of pesticide s to blank soil samp les is a norm alpractice to study the recover y of these compo unds However , the recover y

of p esticides from soil may be different in fresh ly spiked than in aged soil samp les.Pesticides in soil may undergo trans formatio n processes that lead to the form ation

of bound resi dues, which cannot be extra cted even after exhaust ive extra ctionwith organi c solve nts The use of refere nce soil samp les with certi fied concent rations

of the studied pesticide s is recom mende d for the valid ation of the analytical methods,but these refere nce mat erials are dif ficult to prepar e an d maintai n and are avail ableonly for a few pesticide s

The liquid –solid extractio n (LS E) of p esticides from soil is general ly carri ed out byorganic solvents Two techni ques have been widely used, the shaking a nd filtermethod and the Soxhlet extractio n method These class ical analyt ical techniqueshave the advant age of being simple and low cost met hods, but they are timeconsum ing, laborious, difficult to automate, and nonsel ective methods In addit ion,they suffer from vario us disad vantages, such as the use of large volume o f organicsolvents and the need of cleanu p steps

Several modern analytical techniques have been developed to overcome theseproblems Accelerated solvent extraction (ASE), also named pressurized liquid extrac-tion (PLE), is a fast technique that uses low volumes of solvents and can be automated,although the high temperatures used to accelerate the process may degrade somepesticides Supercritical fluid extraction (SFE) uses fluids above their critical tempera-ture and pressure In these conditions, supercritical fluids behave similar to liquids,

CO2 being widely employed because of its reduced cost and low critical temperature(318 C) and pressure (73 atm) Microwave-assisted extraction (MAE) is also a fasttechnique that is able to extract multiple samples at the same time, but the extractionvessels are expensive and must be cooled at room temperature before opening.Ultrasonic or sonication assisted extraction with various organic solvents has alsobeen employed to extract pesticides from soil A miniaturized technique based on thesonication assisted extraction in small columns (SAESC) has been recently developed

in our laboratory In this method, the soil sample located in a small column is placed

in an ultrasonic water bath, wherein pesticides are extracted with a low solventvolume, assisted by sonication Tables 8.1 through 8.3 summarize representativepublished papers on the analysis of pesticides in soil using those extraction techniques.8.2.2.1 Herbicides

Analyses of herbicide residues in soil have been frequently performed because ofthe wide application of these compounds Initially, polar herbicides, such asbenzonitriles and phenoxy acids, were extracted from soil with organic solvents of

low–medium polarity at acidic pH, using manual or mechanical shaking or tion For less polar herbicides, such as triazines, chloroacetamides, and dinitroani-lines, organic solvents such as acetone, ethyl acetate, methanol, and acetonitrile,alone or in mixtures with water, were commonly used.

sonica-More recently, a considerable reduction in solvent consumption has been achieved

by miniaturizing the scale of sample extraction In addition, MAE and SPME havebeen successfully applied to the extraction of various herbicides from soil MAE is atechnique with a reduced consumption of solvent, which is normally acetonitrile ormethanol, alone or in mixtures with water, and solid-phase microextraction (SPME)eliminates the need of solvent and an ulterior cleanup step is not needed

In multiclass herbicide analysis, soil samples were generally extracted with apolar or medium polarity solvent, such as acetone or acetonitrile PLE is a newtechnique used successfully for the extraction of herbicides, such as triazines andphenoxy acids, using water and acetone as solvents

8.2.2.2 Insecticides and Fungicides

Conventional methods have been widely used in the extraction of organochlorine(OC) insecticides from soil, although the use of new extraction techniques has

TABLE 8.1

Extraction Methods of Herbicides from Soil

Shaking Benzonitriles, phenoxy acids Low –medium polarity, acidic pH [3 –6]

Dinitroanilines Acetonitrile –water (99:1, v=v) [7] Phenoxy acids, glyphosate Water, basic pH [8 –10]

Sonication Phenoxy acids, pyrimidines Water, basic pH [26,27]

Multiclass Cyclohexane –acetone (3:1, v=v) [29]

Triazines Water –methanol (1:1, v=v) pH 7 [35]

SAESC, sonication assisted extraction in small columns; PLE, pressurized liquid extraction; MAE, microwave-assisted extraction; SPME, solid-phase microextraction.

increased during the last years In the PLE, the soil sample is placed in acartridge and extracted with mixtures of acetone and hexane The use of MAEhas also increased because of the good recoveries obtained Moreover, headspaceSPME has been successfully used to determine OC insecticides in soil withlimits of detection (LOD) similar to other extraction techniques.

Organophosphorus (OP) pesticides are compounds highly polar and soluble

in water that have been extracted from soil by shaking with organic solvents such

as methanol Other new techniques, such as SPME, are now frequently used forthe extraction of these compounds in soil samples

Carbamates were initially extracted from soil by conventional methodsusing mechanical shaking with different solvents SFE and MAE were afterwardssuccessfully applied to soil as a practical alternative to traditional methods In recentyears, analysis by means of SAESC has obtained good results

TABLE 8.2

Extraction Methods of Insecticides and Fungicides from Soil

Pyrethroids Isooctane –Dichloromethane (15:85, v=v) [47]

Multiclass-insecticides CO 2 –3%methanol [55] PLE Organochlorines Acetone –hexane (1:1, v=v) [56 –58]

Organochlorines Acetone –hexane (1:1, v=v) [59]

Pyrethroid insecticides are a class of natural and synthetic compounds thatare retained in soils because of their high lipophility and low water solubilityand extracted from soil samples by sonication with organic solvents, alone or inbinary mixtures Investigations with fortified samples showed that good and similarrecoveries of these compounds were obtained with MAE and SFE.

The analysis of multiclass mixtures of insecticides was initially carried out

by Soxhlet or shaking methods with low or medium polarity solvents SFE with CO2modified with methanol and SAESC with ethyl acetate are other techniques used morerecently

The analysis of fungicides in soil was initially accomplished by classicalextraction methods, such as the shaking andfilter method using acetone or ethylacetate The ultrasonic assisted extraction and SPME have been other techniquesused more recently for the determination of fungicides in soil samples

8.2.2.3 Multiresidue

Reliable multiresidue analytical methods are needed for monitoring programs ofpesticide residues in soil The classical procedure for pesticide extraction from soilwas to shake soil samples with an organic solvent, ethyl acetate or acetonitrile, alone

or in mixtures with water, being the most widely used solvents

SFE with carbon dioxide containing 3% methanol, as a modifier used to improverecoveries of polar pesticides, has been employed for the multiresidue extraction ofpesticides having a wide range of polarities and molecular weights SFE using CO2isessentially a solvent-free extraction wherein the carbon dioxide is easily removed atatmospheric pressure

TABLE 8.3

Multiresidue Methods of Pesticide Extraction from Soil

Shaking H, I, F Acetonitrile –water (70:30, v=v) [68]

Recentl y, a modi ficati on of the SAESC has be en used for the sim ultaneousdetermin ation o f different classes of pesti cides The good reprod ucibi lity anddetection limit s achiev ed with this method allow its appli cation to the moni toring

of pesti cide resi dues in soil [76]

SPME has been mainly used for the extractio n of pesticide s from aqueoussamples; howe ver, head space SPM E has been recent ly used for the determin ation

of p esticides volat ilized from soil The appli cation of MAE for the extractio n ofpesticide residues is incre asing in the last years and together with o thermodern techniques, such as sonicatio n and PLE, are the most wi dely used methods

at presen t

8.2.3 C LEANUP

Soil samp le extra cts, obtained with an y of the methods described earlier, general lycontain a consi derabl e a mount of other compo nents that may interfere in thesubseq uent analys is Therefor e, the deter minati on of pesticide s at resi due levelfreque ntly requires a furt her cleanu p of soil extra cts Liquid –liquid parti tion (LLP)between an aqueous and an organi c phase, at modul ated pH in some cases, has beenthe most commo n first step in the cleanup of extracts An alte rnative cleanu ptechnique is column chromatography, using reverse or normal phases, in whichpesticides are separated from interferences by elution with a solvent of adequatepolarity Tables 8.4 through 8.6 summ arize the cleanu p procedu res empl oyed in thedetermination of pesticides in soil

8.2.3.1 Herbicides

Phenoxy acid herbicides are normally formulated as amine salts or esters, whichare rapidly hydrolyzed in soil to the acidic form Cleanup techniques for the

TABLE 8.4

Cleanup Techniques Used in the Analysis of Herbicides

SPE-polymer Benzene –hexane (1:9, v=v) [8,10]

Phenylureas SPE- florisil Ethyl ether –n-hexane (1:1, v=v) [23,24] Pyrimidines SPE-alumina Ethyl ether –n-hexane (1:2, v=v) [15] Triazines SPE-polymer Methanol–ethyl acetate (7:3, v=v) [35] Multiclass LLP-SPE- florisil-alumina Dichloromethane –diethyl ether [21] LLP, liquid –liquid partition; SPE, solid-phase extraction.

purification of soil extracts include liquid–liquid partitioning, at basic or acidic pH,and column chromatography using various adsorbents (Florisil, alumina, or silica gel).These cleanup processes are time consuming and large quantities of solventsare generally required Therefore, minicolumns and cartridges, which reducethe solvent consumption and the analysis time, have replaced conventionalchromatographic columns Various organic solvents with different polarity, such

as methanol, dichloromethane, or other intermediate polarity solvents, have beenused to elute phenoxy acid herbicides from cleanup columns In recent years, newpolymeric packing materials have been developed

The cleanup of triazine herbicides in soil extracts has been carried out by SPEwith alumina or Florisil and various mixtures of organic solvents have been used foreluting these compounds

TABLE 8.5

Cleanup Techniques Used in the Analysis of Insecticides and Fungicides

Insecticides

Organochlorines SPE-alumina Hexane –ethyl acetate (7:3, v=v) [44]

SPE-carbon Hexane –ethyl acetate (80:20, v=v) [57] SPE-florisil Heptane–ethyl acetate (1:1, v=v) [58]

Pyrethroids SPE- florisil Hexane –ethyl acetate (2:1, v=v) [60,61]

Fungicides

Strobilurins SPE- florisil Toluene-ethyl acetate (20:1, v =v) [39] LLP, liquid–liquid partition; SPE, solid-phase extraction; MISPE, molecularly imprinted solid-phase extraction.

TABLE 8.6

Cleanup Techniques Used in the Multiresidue Analysis of Pesticides

H, I, F LLP Petroleum ether-diethyl ether (1:1, v =v) [68]

H, I, F, A SPE-polymer Dichloromethane –methanol (1:1, v=v) [74]

H, herbicides; I, insecticides; F, fungicides; A, acaricides; LLP, liquid –liquid partition; SPE, solid-phase extraction.

In the analysis of multiclass herbicide mixtures, the cleanup of soil extracts hasbeen carried out by SPE on Florisil or alumina, after LLP.

8.2.3.2 Insecticides and Fungicides

In general, extracts from soil samples have been cleaned up by means of graphic columnsfilled with alumina or Florisil as adsorbents and pesticides havebeen eluted with nonpolar or low polarity solvents (hexane, ethyl acetate) In somecases, more hydrophobic sorbents, such as carbon, have been used for low polarityinsecticides In addition, LLP of soil extracts between immiscible solvents is amethod sometimes used Moreover, solid-phase extraction with molecularlyimprinted polymers (MISPE) is a novel selective method that has been used for theanalysis of OPs in soil and proved to be a good tool for their selective extraction

chromato-In the analysis of multiclass insecticide mixtures, good recoveries have beenobtained using reversed-phase C18 cartridges and methanol as eluting solvent

8.2.3.3 Multiresidue

Analysis of complex mixtures of pesticides in soil is a difficult problem because ofthe presence of a wide variety of compounds with different physical–chemicalproperties

In modern analytical techniques, the classical methodology for the cleanup

of extracts, based on LLP, has been replaced by miniaturized techniques forresidue analysis that are less solvent consuming SPE is a technique widely used todetermine pesticide residues in soil after their extraction with water or aqueousmixtures of organic solvents Octyl and octadecyl-bonded silica sorbents have beenfrequently used in the analysis of nonpolar and medium polarity pesticides in soilextracts

The thermal instability and low volatility of some pesticides make analysis by gaschromatography (GC) difficult Consequently, methods of analysis based on GCrequire, in some cases, the derivatization of pesticides to increase their volatility

In addition, pesticide derivatives are sometimes prepared to enhance the response of

a pesticide to a specific detector in GC or high-performance liquid chromatography(HPLC) analyses

8.2.4.1 Benzonitriles

The derivatization of the hydroxyl group usually involves perfluoroacylationwith heptafluorobutyric anhydride to form perfluoroacylated derivatives, which aredetermined by GC [6]

8.2.4.2 Glyphosate

This compound is very polar and has a high solubility in water so direct ation by GC or HPLC is difficult Derivatives for HPLC determination are prepared

determin-to improve the pesticide response and pre- or postcolumn reactions have beenused with this aim In postcolumn derivatization, the reaction is produced witho-phthalaldehyde (OPA) and mercaptoethanol and in precolumn derivatization9-fluorenylmethyl chloroformate (FMOC-Cl) is used to form fluorescent derivativeswith an improvement in the chromatographic determination [9].

8.2.4.3 Phenoxy Acid Herbicides

Because of their highly polar nature and low volatility, they cannot be directlydetermined by GC and have to be derivatized to their corresponding esters Severalderivatization procedures have been applied to make phenoxy acid herbicidesamenable to GC analysis

The carboxylic group is converted to the corresponding methyl ester byreacting with diazomethane [5,22] or by alternative less toxic methods such asesterification with methanol using an acid catalyst such as boron trifluoride [3] orwith trimethylphenylammonium hydroxide [32] The sensitivity towards electron-capture detection can be improved by using bromine–iodine to obtain the brominatedmethyl esters [5] or by reacting with pentafluorobenzyl bromine to obtain thehalogenated aromatic esters [4,26]

8.2.4.4 Phenylureas

The analysis by direct GC of these compounds is difficult because of their thermalinstability caused by the NH group Phenylureas decompose in the sample inlet portand produce several peaks in the chromatogram (phenyl isocyanates)

Several analytical methods have been developed based on the possibility toobtain stable derivatives for GC determination, such as alkyl, acyl, and silylderivatives Other derivatization mode for phenylureas is the ethylation with ethyliodide and hydrolysis to N-ethyl derivatives [14]

8.2.4.5 Sulfonylureas

Gas chromatographic analysis of sulfonylureas is difficult owing to theirstrongly polar nature Pentafluorobenzyl derivatives, which have enhanced detectionproperties, have been used since the method is more sensitive than with ethyl ormethyl derivatives [17]

8.2.4.6 Carbamates

Carbamates are thermally decomposed into the corresponding phenols and methylisocianate HPLC methods for carbamates are preferred over GC determinationand they are based on postcolumn basic hydrolysis to release methylamine, whichsubsequently reacts with the OPA reagent to form isoindol derivatives, which aredetermined byfluorescence (FL) detection [49]

8.3 DETERMINATION OF PESTICIDE RESIDUES

Gas and liquid chromatography are the most widely used analytical techniques forthe determination of pesticide residues in soil Thermal stability and volatility are themain characteristics that a pesticide must possess in order to be suitable for gaschromatographic analysis Initially, GC was performed with short glass or steelcolumns packed with a stationary phase; however, nowadays fused silica capillarycolumns are almost exclusively employed The stationary phases used are usuallypolysiloxanes with different functional groups to increase the polarity

Table 8.7 summarizes the GC methods used to determine pesticide residues insoil Electron-capture detection (ECD) is adequate for halogenated compounds or

TABLE 8.7

GC Methods Used for the Determination of Pesticide Residues in Soil

ECD, electron-capture detector; NPD, nitrogen –phosphorus detector; FPD, flame photometric detector;

MS, mass spectrometry; EI, electron impact; NCI, negative chemical ionization; MS =MS, tandem mass spectrometry LOD, limit of detection.

those that contain electronegative atoms such as oxygen or sulfur, pyrethroids and

OC pesticides being typical examples A chromatogram of a mixture of fungicidesanalyzed by GC–ECD is depicted in Figure 8.2 On the other hand, the determination

of pesticides that contain nitrogen or phosphorus atoms, such as triazines and OPpesticides, has been carried out with nitrogen–phosphorus detection (NPD) or flamephotometric detection (FPD) Atomic emission andflame ionization detectors havealso been employed in the determination of pesticide residues in soil

Although these selective detectors allow quantitating residues at trace levels, theconfirmation of the identity is achieved by mass spectrometry (MS) coupled to GC.The ionization technique most commonly used in GC–MS analysis is electronimpact (EI), which produces characteristic ion fragments of compounds that are

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319, 2002 With permission.)