Residue levels of endosulfa¡, endosulfan sulphate, glyphosate, and aminomethylphosphonate (AMPA) in green turgescent immature seeds of glyphosate-tolerant and non-tolerant soy beans were studied in Argentina. To establish the incidence of pesticides spraying, one control and two plots that  varied in glyphosate and endosulfan treatment in two differents fields, located in Entre Rios province (Argentine) were studied Endosulfan, glyphosate and AMPA are present in inmadure soybean and analysis revealed that the concentration of endosulfan residues were similar between soy varieties, but statistical differences were observe between the two treatment.Fil: Lorenzatti, Eduardo Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Maitre, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Lenardon, Argelia Maria Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Lajmanovich, Rafael Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Peltzer, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Anglada, Marta Monica. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias; Argentin

Anglada, Marta Monica

Lajmanovich, Rafael Carlos

Lenardon, Argelia Maria Lucia

Lorenzatti, Eduardo Antonio

Maitre, Maria Ines

Peltzer, Paola

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

 Volume 13 – No.7 – 2004  REPRINT  pp. 675 - 678 PESTICIDE RESIDUES IN IMMATURE  SOYBEANS OF ARGENTINA CROPLANDS  Eduardo Lorenzatti - Maria Ines Maitre - Lenardón Argelia  Rafael Lajmanovich - Paola Peltzer - Marta Anglada     Angerstr. 12 85354 Freising - Germany Phone: ++49 – (0) 8161-48420 Fax: ++49 - (0) 8161-484248 Email: parlar@psp-parlar.de www.psp-parlar.de © by PSP Volume 13 – No 7. 2004   Fresenius Environmental Bulletin    675  PESTICIDE RESIDUES IN IMMATURE  SOYBEANS OF ARGENTINA CROPLANDS  Eduardo Lorenzatti1, Maria Ines Maitre1, Lenardón Argelia1,  Rafael Lajmanovich2,3, Paola Peltzer2 and Marta Anglada4 1 Instituto de Desarrollo Tecnológico para la Industria Química, INTEC, Güemes 3450, (3000) Santa Fe, Argentina  2 Instituto Nacional de Limnología, INALI (3016) Santo Tomé, (Santa Fe), Argentina  3Escuela Superior de Sanidad, FBCB, Paraje El Pozo s/n, (3000) Santa Fe, Argentina 4Facultad de Ciencias Agropecuarias. UNER. Oro Verde, Entre Ríos, Argentina     SUMMARY Residue levels of endosulfan, endosulfan sulphate, glyphosate, and aminomethylphosphonate (AMPA) in green turgescent immature seeds of glyphosate-tolerant and non-tolerant soy beans were studied in Argentina. To establish the incidence of pesticides` spraying, one control and two plots that varied in glyphosate and endosulfan treatments in two different fields, located in Entre Ríos Province (Argentina), were studied. Endosulfan, gly-phosate and AMPA are present in immature soybeans and analyses revealed that the concentration of endosulfan residues were similar between soy varieties, but statistical differences were observed between the two treatments.     KEYWORDS:  Immature soybean, pesticide residues, endosulfan, glyphosate.    INTRODUCTION Despite most soybeans are harvested at the mature stage and further processed into a variety of products for human or animal uses, in certain areas of the world, such as oriental countries, considerable quantities are harvested at the immature stage for direct human consumption [1]. The immature soybeans refer to those harvested at 80 % of maturity and their seeds have low dried matter [1, 2]. This maturity level is equivalent to R6 stage [3]. The immature soybean is often used directly as hu-man food in Asia, because of the high level of nutrients, carbohydrates, vitamins, and isoflavones. Actually, soy is a global staple food and more than 110 million tons of beans are produced, mainly in the United States, Brazil, Argentina, and China [4]. In soy production, a pre-planted application with gly-phosate ((N-(phosphonomethyl)glycine) [5] is recommended, followed by one or two applications of this herbicide on  pre-flowered glyphosate-tolerant (GT) soybeans [6], but anyone on conventional (CN) soybeans [7]. Moreover, an organochlorine pesticide (endosulfan) is also applied twice or even more times in the seeding stage to control sucking, chewing and boring insects and mites [8, 9].  Recent research reports and articles on the agronomic and environmental benefits and risks of GT-soybean have been published [10]. In this context, soybean has been recognised to have a very significant contribution in nutri-tion and human health suggesting that consumption of soybean products is associated with lower blood choles-terol, gives protection against cardiovascular disease, and reduces risk of certain cancers and osteoporosis [11].  On the other hand, preliminary studies observed that pesti-cides` residues usually used in soy cultivation are present in milk, dairy products, and wild fauna [12-15] and have negative effects on human and wildlife health [16]. Therefore, we analyzed pesticide residues present in immature soybeans (IS) and compared GT-soybean vs. CN-soybean. In addition, a new technique was used to evaluate endosulfan, endosulfan sulphate, glyphosate, and alpha-amino-3-hydroxy-5-methylisoxazolepropionate (aminometh-ylphosphonate, AMPA). The study was conducted to as-sess whether the use of glyphosate on GT-soybean and endosulfan on GT- and CN-soybean affect the residues` concentration of pesticides in immature seeds. This study is part of a major project, which investigates the persistence of pesticides used in agricultural production and the bio-magnification of residues through the highest food chain.    MATERIALS AND METHODS Spray treatments The study was conducted at the Entre Ríos province (Argentina) in two experimental fields located at Sauce Pinto (31°52’S; 59°30’W) and Diamante (31°58’S; 60°32’W) © by PSP Volume 13 – No 7. 2004   Fresenius Environmental Bulletin    676 through the soy seasons from November 2000 to March 2001. During the study the average minimum and maxi-mum temperatures ranged between 17.9 °C - 32.3 °C in 2000, and 30.4 °C - 19.2 °C in 2001. GT-soybean in Dia-mante (20 ha) and CN-soybean in Sauce Pinto (30 ha), both seeding in December. Glyphosate (Roundup, Mon-santo, Argentina) was applied seven days after seeding (2500 g ha-1) on GT-soybean, and no spraying on CN-soybean.  In each experimental field were considered three plots, Plot 1: endosulfan free (control plot), Plot 2: endosulfan applied once on soy 92 days after seeding, and Plot 3: two endosulfan sprayings on 60th and 92nd day after seeding (R6 stage). In all the cases the spraying solution was applied at 1000 g ha-1. The endosulfan (Agarcros, Argentina) was prepared by appropriate dilution with water of the commercial herbicide Piastra, 350 g litre-1.  Sampling procedure For the study, both soybean crops were harvested at random 100 days after seeding from the field at the R6 stage of development. Replicate bean samples were col-lected in each plot and analyzed separately to determine the pesticide residues (α+β-endosulfan, endosulfan sul-phate, glyphosate, and AMPA). Immediately after picking each sample, the beans were packed in polyethylene bags, transported to the laboratory to be worked up or frozen at – 18 °C within 90 min.  Analytical procedure Twenty grams of samples of immature beans were ex-tracted as follows: weighed sample was macerated in a ho-mogeneizer at 17,000 rpm. with ultra-pure water (100 ml) to extract glyphosate and AMPA, transferred into a centri-fuge tube with 10 ml ultra-pure tap water and then 50 ml ethyl acetate were added. After been shaken for 10 min, the samples were centrifuged at 7,000 rpm for 10 min at room temperature.  From the clarified supernatant, an aliquot from the organic layer (40-45 ml) was taken and filtered through anhydrous sodium sulfate, concentrated to 1-2 ml by rotary evaporator (Büchi, Switzerland or similar) at 40 °C and analyzed by gas chromatography (GC) for endosulfan and endosulfan sulphate. Aliquots of water layer (5ml), filtered by using 0,45 µm pore size, was analyzed for glyphosate and AMPA by high performance liquid chro-matography (HPLC).  GC and GC/MS analysis: Varian; model 3400, with elec-tron-capture detector (ECD) Ni63, DB-5 capillary column 30 m x 0.25 mm ID (J&W Scientific CA, USA), injector 220 °C, oven 250 °C and ECD detector 320 °C. Mass spec-tra were made with a Varian 2000 chromatograph-mass selective detector (MSD) combination, equipped with an HP-5 capillary column (30 m x 0.25 mm ID) to confirm GC results (operational conditions: oven 210°C, injector 230 °C, ion trap mass spectrometer operating in electron ionization (EI) mode and using MS/MS option.  HPLC analysis: Waters 600 with IC-Pack ion-exclusion 50 Å - 7µm (7.8 x 150 mm) column; mobile phase phosphoric acid 0.05% at 55 °C, fluorescence de-tector at excitation 339 and emission 345 nm, and Mille-nium 2000 data processor software.  Chemical pesticide standards, α and β-endosulfan, endosulfan sulphate (99.0% purity) glyphosate and AMPA (99.2% purity, Applied Science, USA) were used.  Statistical analysis Analyses of variance (ANOVA) were carried out on the data obtained experimentally (residue concentrations) to evaluate the influence of soy transgenic and non-trangenic variety (variety factor) and number of endosul-fan spraying on soy samples (treatment factor).   RESULTS AND DISCUSSION In order to obtain the calibration linearity, standard solutions at 0.2, 0.5, 1.0, 10.0 and 50.0 µg litre-1 were used for endosulfan isomers (α and β) and endosulfan sulphate, with dieldrin as internal standard. Nevertheless, 0.5, 1.0, 2.0 and 8.0 µg litre-1 of glyphosate and 0.45, 1.0, 2.0, and 3,6 µg litre-1 of AMPA were used. Three repli-cates of each concentration were analyzed and detection limits calculated. The data revealed no statistical differ-ences in the intercept and slopes obtained for each one of the five analytes studied (p > 0.05). Recovery studies were performed using clean soy-bean samples (20 g) fortified with two concentrations of endosulfan and endosulfan sulphate, (20 and 40 µg kg-1) glyphosate and AMPA (3.2 and 6.0 mg kg-1). A rapid and efficient method for simultaneous extrac-tion and quantification of pesticide residues has been applied for the analysis of immature soy seeds (IS), in a routine pesticide residue laboratory [18, 23]. Good recov-eries were obtained for endosulfan isomers, endosulfan sulphate, glyphosate and AMPA (92.3%; 96.1%; 92.1%; 95.1%, respectively) with a relative standard deviation (RSD) lower than 11.7%. The detection limits were found to be 5 10-4 mg/kg for endosulfán group and 5 10-3 mg/kg for the other herbicides and metabolites.  Residues found in the IS samples after one and two endosulfan applications on GT- and CN-soybeans are shown in Table 1. The residues of endosulfan applied as spray on the plants, were found in the immature beans, but endosulfan sulphate was no detected (n.d.). The effect of “variety” was calculated only for endo-sulfan residues. This effect was not significant to endosul-fan residues, because analysis of variance showed no sig-nificant difference between GT- and CN-soybeans (p = 0.8832). On the contrary, “treatment” significantly affected endosulfan concentrations (p < 0.01), but the effect was not significant for glyphosate residues (Table 2), as expected. © by PSP Volume 13 – No 7. 2004   Fresenius Environmental Bulletin    677  TABLE 1 Residues (mg/kg) in immature GT-soybean and CN-soybean. SD = standard deviation;  n.d. = not detected; Plot 1: endosulfan-free  (control plot); Plot 2: 92 days after seeding; Plot 3: 60 and 92 days after seeding (R6 stage); a, b indicate significant differences at p < 0.05.  Residues Soy Endosulfan spraying Endosulfan  Endosulfan sulphate Glyphosate  AMPA    mg/kg SD Mg/kg SD mg/kg SD mg/kg SD GT Plot 2 0.18 a 0.03 n.d. - 0.31a 0.04 0.33 0.03 GT Plot 3 0.24 b 0.04 n.d. - 0.30a 0.05 0.32a 0.04 GT Plot 1 n.d. - n.d. - n.d. - n.d. - CN Plot 2 0.19a 0.05 n.d. - n.d. - n.d. - CN Plot 3 0.23ab 0.04 n.d. - n.d. - n.d. - CN Plot 1 n.d. - n.d. - n.d. - n.d. -    TABLE 2 Effect of  number pesticide treatment on endosulfan and glyphosate residues. Effect of soy variety (transgenic or conventional) on endosulfan residues in immature soybeans are also shown; (p < 0.05).  Endosulfan Glyphosate  F “p” F “p” Treatment 7.96 0.0079 8.09 0.0120 Variety 0.02 0.8832 - -  - not calculated    Many studies were carried out to establish the curve of decline of the insecticide in vegetables for human consump-tion [17,18] Thus, Soto [19] and Lu [20] suggested that endosulfan has estrogenic effects on human estrogen-sensitive cells. We found that the GT-soybeans accumulated glyphosate in their immature seeds (0,3 - 0,31 mg/kg) and AMPA metabolites were also generated (0,32-0,33 mg/kg), whose toxicity is discussed elsewhere [21]. In CN-soybeans herbicide residues and corresponding metabolites were absent. The glyphosate and AMPA residues found in GT-soybean were quite similar, and higher than the limits permitted by European legislation (0.2 mg/kg) in the Codex Alimentarius [22], while the Federal Register of Argen-tina [23] contains no regulations. The α+β endosulfan residues (0.18 to 0.24 mg/kg) were found to be below those recommended by European legislation for dry soybean [22], but there is no regulation with regard to IS. Moreover, the endosulfan sulfate me-tabolite, which is known as degradation product in mature vegetables, was not detected in this study. The market outlook concerning IS shows increasing tendencies to be incorporated in Asian and American foods. In this context, an effort should be made to regulate the production, processing and marketing of IS. Similarly, it is necessary to study the possible replacement of endo-sulfan by a pyrethroid in order to reduce contamination and cost in the production of IS. In addition, the use of glyphosate should be reduced and the weeds extraction may be carried out manually in small fields and in coun-tries with low-cost workmanship standards, in order to reduce the toxic concentrations of biocides. Argentinean farmers seek to maximize production, on the one hand, and lower production costs, on the other, because the growing conditions vary considerably from one area to another. In deed, from farm to farm, produc-tion cost and corresponding savings can vary substantially in this context. Therefore, the disjunctive use of GT-soybean or CN-soybean depended on its world-wide dif-fering acceptance, taking into consideration also the cur-rent debate on transgenic herbicide-resistant soybean varieties and their ban, particularly in Europe.    REFERENCES [1] Liu, K. (1996) Immature soybean: Direct use for food. IN-FORM 7, 1217-1223. [2] Simonne, A., Smith, M., Weaver, D., Vail, T., Barnes, S. and Wei, C. (1994) Retention and Changes of soy isoflavones and carotenoids in immature soybean seeds (Edamame) during processing. J. Agric. Food Chem. 48, 6061-69. [3] Fehr, W., Caviness, C., Burnood, D. and Andpennigton, J., (1971) Stage of development descriptions for soybeans, Gly-cine max (L.) Merrill. Crop Science 11, 929-931. [4] Barnes, R. (2000) Why American Soybean Association sup-ports transgenic soybeans. Pest Management Science 56, 580-583.  © by PSP Volume 13 – No 7. 2004   Fresenius Environmental Bulletin    678 [5] WHO, (1994) World Health Organisation Environmental Health Criteria 150: Glyphosate. Geneva.  [6] Arregui, M. (2001) Manejo de agroquímicos en cultivos exten-sivos. Universidad Nacional del Litoral, Santa Fe, Argentina [7] CASAFE. (2001) Guía de productos fitosanitarios 2001. Cámara de Sanidad Agropecuaria y Fertilizantes. 9th, Buenos Aires, Argentina [8] Corres, C., Olivera, J. and Tornisielo, V. (2001) Avaliação de resíduos de endosulfan em matriz de vagem e soja para com-paração de dois sistemas de aplicação do produto formulado. Revista Instituto Adolfo Lutz 60 (2),  135-139. [9] WHO, (1984) World Health Organisation. Environmental Health criteria 40: Endosulfan. Geneva.  [10] Hin, C. , Schenkelaars, P. and Pak, G. (2001) Agronomic and environmental impacts of the commercial cultivation of gly-phosate tolerant soybean in the USA. Centre for Agriculture and Environment. Utrecjt, 64p. [11] Friedman, M. and Brandon, D. (2000) Nutritional and health benefits of soy proteins. Journal Agricultural Food Chemistry 49, 1069-1086. [12] Lenardón, A., Maitre, M. and Enrique de Carbone, S. (1994) Organochlorine pesticides in argentinian Butter.  Science To-tal Environment 144, 273-277. [13] Maitre, M., de la Sierra, P., Lenardón, A., Enrique, S. and Marino, F. (1994) Pesticide Residue levels in argentinian pasteurised milk. Science Total Environmental 155, 105-108. [14] Lorenzatti, E., Maitre, M., Lenardón A. and Serra, J. (2000) Serie de Ciencia e Ingeniería de Alimentos Vol II, Residuos de plaguicidas organoclorados en leche y derivados en Ar-gentina. Universidad Politécnica de Valenci, pp. 383-390. [15] Lajmanovich, R., Lorenzatti, E., de la Sierra, P., Marino, F. and Pelzer P. (2002) First records of organochlorine pesticide residues in amphibians of the mesopotamic region, Argen-tina. Froglog 54, 4-5. [16] Allsopp, M., Stringer, R. and Johnston, P. (1998) Persistent organochlorines in humans. Greenpeace Laboratories, Dept. Biological Sciences. University of Exeter. Principe of Road, Exeter. [17] Antonious, G., Buers, M. and Snyder, J. (1998) Residues and fate of endosulfan on field-grown pepper and tomato.  Pesti-cide Science 54, 61-67. [18] Arrebola, F., Egea-Gonzalez, F., Moreno, M., Fernandez-Gutierrez, A., Hernandez, M. and Martinez-Vidal, J. (2001) Evaluation of endosulfan residues in vegetables grown in greenhouses. Pest Managenment Science 57,  645-652.  [19] Soto, A., Chung, K. L. and Sonnenschein, C. (1994) The pes-ticides endosulfan, toxaphene and dieldrin have estrogenic ef-fects on human estrogen-sensitive cells. Environmental Health Perspectives 102, 380-383. [20] Lu Y., Morimoto K., Takeshita, T., Takeuchi, T. and Saito T. (2000) Genotoxic effects of endosulfan an  endosulfan on human hepG2 cells. Environment Health Perspective 108, 559-561. [21] Baylis S. A. (2000) Why glyphosate is a global herbicide: strengths, weaknesses and prospects. Pest Managagement Science 56, 299-308.  [22] FAO/WHO, (2003) Codex Alimentarius Commission. Codex committee on pesticide residues, Thirty-fifth session. Rotter-dam.  [23] U.S. EPA, (1996) Pesticide tolerances for glyphosate. 40 CFR parts 180 and 186. Rules And Regulations. Federal Register.                                 Received: February 23, 2004 Accepted: May 17, 2004   CORRESPONDING AUTHOR Eduardo Lorenzatti Instituto de Desarrollo Tecnológico  para la Industria Química INTEC Güemes 3450 3000 Santa Fe - ARGENTINA  e-mail: lorenzatti@ceride.gov.ar   FEB/ Vol 13/ No 7/ 2004 – pages  675 - 678 

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Pesticide Residues in Immature Soybeans of Argentina Croplands

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Pesticide Residues in Immature Soybeans of Argentina Croplands

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