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Tóxicos/Intoxicações

Metodologia avalia níveis de resíduos de pesticidas em tomates

13/01/2005

O Brasil é o sexto maior produtor mundial de tomate, item de destaque na alimentação humana. Seu cultivo, sujeito a diversos tipos de infestações de pragas, é constantemente bombardeado com inseticidas e fungicidas, que garantem alta produtividade. Por causa disso, o governo brasileiro estabelece níveis de tolerância para os resíduos de pesticidas que permanecem no tomate, mas que nem sempre são respeitados pelos produtores. Uma metodologia desenvolvida por pesquisadores da Universidade Regional do Noroeste do Estado do Rio Grande do Sul (Unijuí) e da Universidade Federal de Santa Maria (Ufsm) consegue determinar até níveis bem baixos de pesticidas presentes no tomate, utilizando apenas um solvente para detectar múltiplos tipos de pesticidas, diferentemente dos métodos tradicionais. O estudo foi publicado na última edição da revista científica Journal of the Brazilian Chemical Society.

O método foi aplicado por Anagilda Gobo, química da Unijuí, e seus colegas, em tomates obtidos em vários mercados de Santa Maria e Ijuí, ambas no Rio Grande do Sul, entre 23 de dezembro de 2000 e 26 de janeiro de 2001. Das amostras coletadas, apenas uma continha resíduos do pesticida methamidophos bem acima (2,4 mg/kg) dos níveis estabelecidos pela legislação brasileira (0.3 mg/kg). "Isso significa que não há uma preocupação adequada em obedecer as boas práticas de agricultura por parte de alguns produtores, causando exposição do trabalhador e resíduos tóxicos nos tomates", afirmam os autores do artigo. Quantidades acima das permitidas representam risco potencial à saúde dos consumidores.

Nozomu Makishima, agrônomo da Embrapa Hortaliças, informa que o cultivo da batata e do tomate recebe a maior quantidade de princípios ativos (substâncias ativas que conferem eficácia aos pesticidas) de inseticidas e fungicidas. No caso do tomate são 52 diferentes, sendo que alguns são produzidos por mais de um fabricante, totalizando 150 à disposição do agricultor. Geralmente, as aplicações são semanais, por um período de 5 a 6 meses até a colheita, o que equivale a 24 ou 30 aplicações, sendo que este número pode aumentar nos períodos de chuva. Makishima lamenta que a legislação brasileira não obrigue os produtores a fazer um controle das aplicações. "O ideal seria uma legislação que obrigasse as grandes centrais de distribuição de hortaliças e frutas a fazer uma amostragem para verificar a presença de resíduos [de pesticidas em seus produtos], pelo menos uma vez por ano", sugere o pesquisador.

A Companhia de Entrepostos e Armazéns Gerais de São Paulo (Ceagesp) é um dos únicos locais a ter um programa de monitoramento dos níveis de resíduos de pesticidas em frutas e hortaliças. O pesquisador da Embrapa enfatiza que o problema é mais sério nas feiras livres, onde fica ainda mais difícil fazer este controle. Anagilda Gobo ressalta que além do município de Ijuí, que fez um trabalho com a Prefeitura Municipal para monitorar alguns resíduos de pesticidas em determinadas hortaliças e frutas em feiras livres, a cidade de Porto Alegre também faz um controle destes resíduos em uma parceria entre sua Central de Abastecimento S.A. (Ceasa), a Associação Riograndense de Empreendimentos de Assistência Técnica e Extensão Rural (Emater) e a Secretaria Estadual da Saúde.

As técnicas para determinação de resíduos de organofosforados - freqüentemente utilizados em plantações de tomates - utilizam a cromatografia gasosa com um detector de ionização de chamas (FID, em inglês) e a espectrometria de massas (técnicas também utilizadas em testes antidoping) e que podem medir a extração de resíduos múltiplos, para baratear os custos e melhorar a produtividade nos laboratórios. Um dos problemas, no entanto, é o excesso de uso de vários solventes orgânicos que acabam prejudicando as análises em laboratório. O método proposto pelos pesquisadores das universidades gaúchas usa apenas um solvente para determinar níveis de seis pesticidas distintos empregados em tomates, tornando-o simples, rápido, eficiente e com custo reduzido, características que facilitariam a detecção da presença de resíduos. A química afirma que o procedimento desenvolvido pode ser empregado para a determinação de pesticidas organofosforados em outras culturas, "desde que cada laboratório faça a sua própria validação de acordo com os equipamentos disponíveis", recomenda.

 
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J. Braz. Chem. Soc., Vol. 15, No. 6, 945-950, 2004.

Printed in Brazil - ©2004 Sociedade Brasileira de Química

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Article

* e-mail: rzanella@base.ufsm.br

Development and Validation of Methodology for the Determination of Residues of

Organophosphorus Pesticides in Tomatoes

Anagilda B. Goboa, Márcia H. S. Kurzb, Ionara R. Pizzutti b, Martha B. Adaimeb, Renato Zanella*,b

a Departamento de Biologia e Química, Universidade Regional do Noroeste do Estado do Rio Grande do Sul ,

98700-000 Ijuí - RS, Brazil

b Departamnto de Química,Universidade Federal de Santa Maria, 97105-900 Santa Maria - RS, Brazil

Os pesticidas organofosforados são freqüentemente aplicados no cultivo de tomate no Brasil.

No presente trabalho uma metodologia analítica foi desenvolvida e validada para a quantificação de

resíduos dos pesticidas organofosforados acefato, chlorpyrifós, malation, metamidofós and paration

metílico em tomate, empregando Cromatografia Gasosa com Detector de Nitrogênio e Fósforo (GCNPD).

A possibilidade de ocorrência de efeito matriz foi estudada. As curvas analíticas, preparadas

nos extratos da matriz, foram lineares de 0,006 até 0,80 mg L-1. Os estudos de precisão forneceram

resultados com RSD <15%. As recuperações dos pesticidas, obtidas com as curvas preparadas no

extrato da matriz, foram entre 88 e 118%. Com o procedimento proposto foram obtidos limites de

quantificação entre 0,0132 e 0,135 mg kg-1.

The organophosphorus pesticides are frequently applied in tomato cultivation in Brazil. In the

present work an analytical methodology for quantification of the organophosphorus pesticides:

acephate, chlorpyrifos, malathion, methamidophos and parathion-methyl residues in tomatoes was

developed and validated using Gas Chromatography with a Nitrogen-Phosphorus Detector (GCNPD).

The possibility of a matrix effect was studied. Analytical curves prepared in an extract of the

matrix were linear from 0.006 to 0.80 mg L-1. The precision studies supplied results with RSD

<15%. The recoveries of the pesticides calculated from the curve prepared in the matrix extract were

between 88 and 118%. With the proposed procedure quantification limits between 0.0132 and 0.135

mg kg-1 were obtained.

Keywords: pesticide residues, organophosphorus, GC-NPD, tomato

Introduction

The cultivation of tomatoes in Brazil started during

the 50’s and 60’s. Starting from 1972 there was a very large

increase in the total production, mainly in the State of São

Paulo.1 Today, Brazil is one of the world’s largest producers

of tomatoes, occupying ninth place in the ranking, with a

production, in 1997, of 2.6 million tons and median

production of 44 t ha-1. Tomato is a food with an expressive

content of vitamins and mineral salts, being considered

one of the most important vegetables to be used in human

feeding. However, it is a very demanding crop, due to the

occurrence of several possible infestations from planting,

throughout development and until harvest.2 One of the

factors that contributes to the high productivity is the

pesticides use, mainly insecticides and fungicides.

Inadequate or incorrect pesticides use, done to control of

pests that attack the tomato crop, can represent a serious

potential risk to consumer health, because pesticides can

leave persistent residues in foods and in the environment.2

Thus analysis of pesticide residues is of great importance,

because it allows determining if the residues are within

the established tolerance limits.

Among the pesticides more used for the control of pests

in the cultivation of tomatoes in the Southern area of Brazil,

the organophosphorus pesticides, such as: acephate [0,Sdimethyl-

phosphoroamidothioate], chlorpyrifos [O,Odiethyl-

O-(3,5,6-trichloro-2-pyridylphosphoro-thioate)],

malathion [O,O-dimethyl-S-(ethyl-1,2-dicarboethoxy)

phosphorodithioate], methamidophos [O,S-dimethylphosphoramidothioate]

and parathion-methyl [O,Odimethyl

O-(4-nitrophenyl)phosphorothioate], whose

chemical structures are presented in Figure 1, stand out.

946 Gobo et al. J. Braz. Chem. Soc.

The technique most often used for the determination

of residues of organophosphorus pesticides in tomatoes is

Gas Chromatography (GC) using Nitrogen-Phosphorus

(NPD), Flame Photometric (FPD) or Mass Spectrometric

(MS) Detectors. The tendency in the analysis of pesticide

residues in foods is centered on procedures of multiresidue

extraction, thus having a reduction of analysis costs and

increased productivity in the laboratories, especially

because, for the control of different pests, many different

pesticides may be used. In the specific case of the

organophosphorus pesticides, the diversities of the

physical-chemical properties of the several compounds

represent a problem for the development of an analytical

methodology using only one extraction procedure.3,4

For the determination of pesticides in fruits and

vegetables several extraction procedures have been used

and, in many cases, there is also the need for purification

and pre-concentration.3 The extraction techniques more

used for pesticides in fruits and vegetables are extraction

with solvent, supercritical fluid extraction (SFE) and solid

phase matrix dispersion (SPMD).4-7 In agreement with the

literature, one of the solvents more indicated for pesticide

extraction in fruits and vegetables is ethyl acetate in the

presence of anhydrous sodium sulphate or acetone,

followed by partition with dichloromethane and petroleum

ether.4,8,9

This paper describes the development and validation

of a methodology applying extraction with solvent and

analysis by GC-NPD for the determination of residues of

organophosphorus pesticides used routinely in tomato

cultivation. Another propose of the present study was to

determine the influence of the matrix in the chromatographic

response of organophosphorus pesticides.

Experimental

Instrumental

A Varian 3400 gas chromatograph (Palo Alto, USA)

with CX 8200 autosampler was used for the gas chromatography,

equipped with a splitless 1079 injector at

220 °C, with a glass liner of 0.5 mm i.d. using an injected

volume of 1 µL; a 30 m x 0.25 mm x 0.25 µm DB-1701

capillary column (J & W Scientific, Folsom, USA) and a

nitrogen-phosporous detector (300 °C, bead current

3.200 A, flow-rate of the detector gases: make-up (nitrogen)

28 mL min-1, hydrogen 5.5 mL min-1 and synthetic air

174 mL min-1). The carrier gas was nitrogen at 1.7 mL min-1

and the temperature program of the column oven was 60 °C

(3 min), 15 °C min-1 up to 200 °C, then 3 °C min-1 up to

225 °C (1 min). Data acquisition was through a Star 4.5

Workstation (Varian). A Varian 3800 gas chromatograph

(Palo Alto, USA), also equipped with a 1079 injector. All

other conditions are the same as used with the Varian 3400.

Reagents, solvents and reference pesticide standards

All solvents used were of pesticide residue grade from

Mallinckrodt, (Phillipsburg, USA). The anhydrous sodium

sulfate, analytical grade from Merck (Rio de Janeiro,

Brazil), was previously heated at 650 °C for 4 hours. The

sodium chloride, analytical grade, was from Merck

(Darmastadt, Germany); the dimethyldichlorosilane was

from Sigma-Aldrich Chemie (Steinheim, Germany) and the

Extran was from Merck (Rio de Janeiro, Brazil),

The reference pesticide standards of acephate,

chlorpyrifos, malathion, methamidophos and parathionmethyl

were obtained from Dr. Ehrenstorfer (Augsburg,

Germany). The analytical stock solutions of each pesticide

were prepared in ethyl acetate and stored in amber flasks

maintained at -18 °C.

Extraction procedure

The tomato samples were first washed and homogenized

in a 2 L industrial blender. Then 25 g of sample

was weighed into a 250 mL glass flask with cap and 50 mL

of ethyl acetate and 2.5 mL of a 10% solution of sodium

chloride were added. The extraction flask was shaked for

25 min, 35 g of sodium sulphate was added and the flask

was shaked another 10 min. After standing for 5 min an

aliquot of sample was transferred to an autosampler vial

with cap and submitted to analysis.

Validation of the method

The validation procedure investigated the following

parameters: analytical curve and linearity, detection limit

and quantification limit, precision (repeatability and

intermediate precision) and accuracy. The linearity was

studied during the construction of the analytical curves

obtained using analytical solutions of the mixture of the

Figure 1. The chemical structures of the organophosphorus pesticides.

947 Development and Validation of Methodology Vol. 15, No. 6, 2004

pesticides prepared in pure solvent and prepared in the

extract of the matrix in the concentration range from 0.006

to 0.81 mg L-1. The studies to evaluate the recovery of the

pesticides were made in tomato samples without pesticides

residues, fortified with an analytical solution containing

the pesticides under study, at two different levels. Each

concentration level was extracted and analyzed three times.

The study of repeatability of the instrument was evaluated

with three injections in the chromatographic system for

each level of concentration of the analytical solutions in

pure solvent and in the extract of the matrix. The

intermediate precision tests were carried out in another

laboratory, using a Varian 3800 gas chromatograph with

the same column. The chromatographic conditions used

for this test were similar to those optimized in the

development of the proposed method. To determine

detection limit (LOD) and quantification limit (LOQ),

analytical solutions of the pesticides, prepared in both

pure solvent and in the matrix extract, were used. The

injections to determine LOD were made in decreasing order

of concentration to reach a peak area three times higher in

relation to the noise of the baseline at the retention time of

the peak of interest.10 For the LOQ determination the

concentration obtained in the determination of LOD was

considered, being multiplied by 3.3 times.

Results and Discussion

In the optimization of the separation of the pesticides,

two capillary columns of different polarities (DB-5 and

DB-1701) were tested. The DB-5 column was not

considered efficient for the separation of the chlorpyrifos

and parathion-methyl, because these peaks elute very close

together with most temperature programs. The DB-1701

column was considered more efficient and it was used for

the determination of the selected pesticides.

The temperature of the injector and the internal

diameter of the silanized glass liner were also evaluated.

The best results were obtained with an injector temperature

of 220 °C and with a liner of 0.5 mm d.i., by virtue of

having a reduced contact surface when compared with the

one of 2 mm d.i.. Figure 2 shows a typical chromatogram

of a analytical solution of the pesticides methamidophos

(0.23 mg L-1), acephate (0.23 mg L-1), chlorpyrifos (0.049

mg L-1), parathion-methyl (0.099 mg L-1) and malathion

(0.029 mg L-1), prepared in an extract of the matrix.

Detection limit and quantification limit

In agreement with the data presented in the Table 1, it

is observed that methamidophos and acephate prepared in

pure solvent have LOD and LOQ concentration values

higher than the values obtained from the solutions prepared

in the extract of the matrix. As already discussed in others

papers,5,6 these compositions exercise the main effect,

because when injected in pure solvent the pesticides

undergo larger adsorption in the injection system, while

when injected in the extract of the matrix, they undergo

the protecting effect of the matrix and a larger amount is

transferred to the column. For the other pesticides studied

this difference is not too expressive.

Figure 2. Chromatogram of organophosphorus pesticides prepared in an extract of the matrix: methamidophos (tR = 11.2 min), acephate (tR =

13.2 min), chlorpyrifos (tR = 17.9), parathion-methyl (tR = 18.1 min) and malathion (tR = 18.5 min). For conditions, see text.

Table 1. LOD and LOQ (mg L-1) for the pesticides under study,

prepared in pure solvent and in an extract of the matrix

pesticide in pure solvent in extract of

the matrix

LOD LOQ LOD LOQ

acephate 0.080 0.62 0.0061 0.020

chlorpyrifos 0.0020 0.0066 0.00077 0.0025

Malathion 0.0039 0.013 0.0020 0.0066

methamidophos 0.080 0.33 0.0050 0.016

parathion-methyl 0.0027 0.0089 0.0020 0.0065

948 Gobo et al. J. Braz. Chem. Soc.

Analytical curves

Table 2 presents the results of the analytical curves

obtained with the analytical solutions prepared in pure

solvent and in solvent containing the matrix extract. From

the curves obtained, the model is linear with a determination

coefficient (r2) for the pesticides greater than 0.99,

considered satisfactory according by the literature,11,12

except for methamidophos, which presented an r2 value

slightly below 0.98 for the curve prepared in pure solvent.

The values obtained for the analytical curves with the

solutions prepared in the matrix extract demonstrated

satisfactory linearity for the pesticides, except for

methamidophos and acephate, for which a percentage

above 5% of the angular coefficient was obtained for some

intermediate concentrations. The analytical curves

obtained with the solutions in pure ethyl acetate showed

satisfactory linearity for chlorpyrifos. For methamidophos,

acephate, parathion-methyl and malathion, a percentage

above 5% of the angular coefficient was obtained for most

of the concentrations and this can be related to the matrix

effect. From the slope of the analytical curves presented in

the Table 2 can be concluded that the responses of the

NPD for the selected pesticides, when prepared in pure

ethyl acetate, are lower, compared with the responses for

the pesticides prepared in the matrix extract.

Precision (repeatability and intermediate precision)

Results of precision (Table 3) obtained at two

concentration levels in the interval of the linear range were

considered good, except for methamidophos at the lower

level, since they are within the limit accepted for the routine

application of a chromatographic method for pesticides,

where the precision should be ± 15%.13

The tests of intermediate precision of the method,

conducted in two different laboratories, were considered

satisfactory because they are within the values

recommended for precision (RSD ± 15%).13

Recovery

For quantification in the recovery studies both

analytical curves obtained with the analytical solutions

in pure solvent and in the extract of the matrix were used.

The results are presented in Table 3. The recoveries of the

pesticides obtained using the analytical curves with the

matrix extract are considered satisfactory for all the

concentrations studied because they are within acceptable

values, as described in the literature for chromatographic

methods applied for pesticide residues, which should be

between 70 to 120%, with values of RSD being ± 20%.14

Using the analytical curves obtained with the pesticides

Table 2. Parameters for the analytical curves obtained for the pesticides prepared in pure solvent and in the matrix extract

ethyl acetate matrix extract

Pesticide linear range(mg L-1) regressionequation r2 linear range(mg L-1) regressionequation r2

acephate 0.080 - 0.80 y = 69464x - 3467 0.9959 0.060 - 0.80 y = 160858x - 2899 0.9912

chlorpyrifos 0.017 – 0.17 y = 99136x - 73 0.9997 0.0060 - 0.086 y = 124023x + 58 0.9992

malathion 0.027 – 0.32 y = 80273x + 186 0.9962 0.027 - 0.30 y = 107170x - 259 0.9984

methamidophos 0.084 - 0.89 y = 40882x - 5033 0.9797 0.011 - 0.81 y = 67879x - 626 0.9977

parathion-methyl 0.027 – 0.27 y = 115799x - 1337 0.9942 0.0080 - 0.13 y = 136843x + 171 0.9997

y = peak area; x = pesticide concentration (mg L-1).

Table 3. Recovery values and RSD for two different fortification levels, using the analytical curves prepared with pure solvent and in an extract

of the matrix

pesticide fortification from curves in pure solvent from curves in matrix extract

(mg kg-1) recovery (%) RSD(%) recovery(%) RSD(%)

acephate 0.080 250 27.0 99 11.01

0.20 252 21.61 118 4.37

chlorpyrifos 0.010 101 2.96 101 11.50

0.025 125 1.80 117 1.82

malathion 0.027 87 4.46 91 9.50

0.054 120 4.82 93 8.22

methamidophos 0.076 326 7.56 117 20.03

0.16 206 6.19 88 3.04

parathion-methyl 0.027 119 5.2 102 3.20

0.054 89 4.69 101 2.21

949 Development and Validation of Methodology Vol. 15, No. 6, 2004

prepared in pure solvent the recoveries for methamidophos

and acephate were outside the established patterns. Similar

results have been described in the literature5,6 and they

can be attributed to possible systematic errors in the

quantification of the pesticides because of the

characteristics of the solution in the injection of the

samples containing components of the matrix.

Application of the method developed

The method described here was applied to the analysis

of tomatoes sold in various markets in Santa Maria and

Ijuí, Rio Grande do Sul State (Brazil), in the period from

23 December 2000 to 26 January 2001 (the summer

season). In Brazil, the adopted lowest residue limit (LMR)

for tomatoes are: methamidophos, 0.3 mg kg-1, acephate,

chlorpyrifos and parathion-methyl, 0.5 mg kg-1, and

malathion, 3.0 mg kg-1.15 Of the eighteen collected samples,

just one presented residues (2.4 mg kg-1) of the pesticide

methamidophos above the LMR established in Brazil for

tomatoes. In the other samples, the residues of the

pesticides investigated were below the allowed LMR.

Acephate was found in four samples with values between

0.01 and 0.12 mg kg-1.

Conclusions

The results shown in this work indicate that the method

proposed for the simultaneous determination in tomatoes

of the six pesticides: acephate, chlorpyrifos, malathion,

methamidophos and parathion-methyl, each with different

polarities, is simple, fast and efficient. Validation of the

proposed method shows satisfactory parameters. The results

obtained with the pesticides in pure solvent and with the

real matrix extract were compared.

Although several analytical procedures for extraction

are described in the literature, this work opted for a

methodology using less glassware, to avoid losses due to

adsorption of the compounds during the extraction stage.

One of the advantages of the procedure is the use of a

single solvent during the whole process, which makes the

determination faster and less expensive. Selective

determination with the NPD does not require purification

of the extract, obtaining satisfactory recoveries at

concentrations below the maximum concentrations

permitted by Brazilian legislation. The heterogeneity of

the physical-chemical characteristics of the compounds is

unfavorable for the choice of a single adsorbent and does

not eliminate the problem completely, while such a cleanup

procedure elevates costs and analysis time. One of the

more practical alternatives to compensate the matrix effect

without elevating the cost of the analyses is to prepare the

analytical solutions for quantification in an extract of the

matrix. It is also indispensable that all components are

properly cleaned and that the GC injector liner is silanized,

because the matrix effect depends on the characteristics of

the pesticides, matrix type, concentration and cleanliness

of the chromatographic system.

The results obtained in the application of the method

for the analysis of tomato samples marketed in Southern

Brazil indicate the presence of acephate and

methamidophos residues. Although these pesticides are

highly toxic, they are frequently used for the control of

infestations in tomato cultivation. In one of the samples,

the level of the residue of methamidophos was well above

(2.4 mg kg-1) the maximum limit allowed by the Brazilian

legislation (0.3 mg kg-1) for tomatoes. This means that

there is not an appropriate level of concern in obeying

good agricultural practices on the part of some growers,

causing occupational exposure and toxic residues in

tomatoes that appear on the market.

Acknowledgements

Financial support and scholarship from FAPERGS,

CAPES and CNPq, Brazil, are gratefully acknowledged.

The authors thank Profª. Drª. Carol H. Collins, Unicamp

(Brazil) for comments during the preparation of this article.

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950 Gobo et al. J. Braz. Chem. Soc.

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Received: August 13, 2003

Published on the web: November 18, 2004


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