Rev. Fac. Agron. (LUZ). 2005, 22: 57-66

Persistence of Organophosphorus Malathion and Chlorpiryphos insecticides in Guava (Psidium guajava L.) J. Sánchez2, G. Ettiene3, I. Buscema3 y D. Medina3 Instituto Universitario de Tecnología de Maracaibo (IUTM). Departamento de Química, Facultad de Agronomía. Universidad del Zulia, Apdo. 15205. 1

2

Abstract In this work was evaluated the malathion and chlorpiryphos persistence in physiologically mature guavas, cultivated in the Canaima farm, Mara county, Zulia State in an area of 147 m2 with three repetitions and a witness. The applied dose of the commercial formulations were 2 L/ha (malathion) and 1.5 L/ha (chlorpiryphos). The residual levels were measured from day 0 to 15. The extraction was optimized with acetate of ethyl-acetone (90:10) and the quantification using capillary gas chromatography coupled to the detecting Nitrogen-Phosphorous (NPD), being obtained percentages of recovery of 81.50% ± 8.22 and 90.50% ± 5.83 with coefficients of variation of 10.09 and 6.44%. The detection limits were 0.0204 to malathion and 0.0165 mg/kg, for chlorpiryphos. The dissipation of the insecticides followed a first order kinetics with times of half life of 0.29 and 2.35 days, malathion‘s dissipation was 100% after the third day and chlorpiryphos, after the eleventh day. In times of tolerance Codex, the limit time in the application before harvesting was 2 days for malathion and 3 days for chlorpiryphos. Key words: Organophosphorus Insecticide, Guava, Persistence, gas chromatography.

Introduction One of the main fruit-growing crop on Zulia state is guava (Psidium guajava L.), specifically on Mara county, where producers in order to protect their crops against pest that attack them (as mites, flies of the own

fruit and white fly) made constant applications of organophosphorus insecticides every seven days approximately (9). This activity might cause that the residuals levels of these products could be present in guava

Received 10-29, 2002 z Accepted 4-30, 2004 2 Author’s e-mail: [email protected]

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Sánchez et al. fruits when sowing them, however, on the market are not common the harmful effect on taste (18). The World Health Organization (OMS) and the Food and Agriculture Organization of the United Nations (FAO) established a program about alimentary norms, called “Codex Alimentarius”, which establishes the maximum limits of residues (LMRs) in food, and controls the insecticides residues that result by the authorized employ on these foods. This quality control should be applied for the commercialization of agriculture products in both national and international aspect, this application and the strict follow of rules would guarantee that agriculture products arrive in the market with low levels of insecticides residues under LMRs (4, 11). It is therefore necessary to determine the time that must take between the insecticide application and harvest. This waiting time is in function of the type of harvest, type of insecticide, toxicity, residual power, action mechanism and the meteorological conditions of the crop’s area (10, 18). The situation can be delicate if is considered that guava plants (Psidium guayaba L.), have fruits in different physiological phases and the crop is generally constant twice

or three times a week, because in some cases guavas are sowed without respecting the waiting time that must oscillates from 15 to 21 days. This reality justifies the necessity of knowing the persistence of Ops insecticides in guava fruits (Psidium guayaba L.), under the environmental conditions of Mara parish, Zulia state. Research of Ops insecticides persistence have been done in other countries on tomato and pepper samples (17) and in other matrixes, as well as water of the river (15), must, and grape wine (7). Inside the analytical techniques commonly employed for the detection and quantification of Ops insecticides on agriculture products, are the capillary gases chromatograph stacked on the nitrogen-phosphorus detectors (NPD) (19) and flame photometric FPD (3), liquid chromatography of high resolution (HPLC) and gases chromatography stacked on the mazes spectrometry (8). The aim of this research was to determine the evaluation of the persistence of organophosphorus malathion and chlorpiryphos insecticides in physiologically ripe guava (Psidium guayaba L) under the environmental conditions of Mara county, employing capillary gas chromatograph with NPD detection.

Materials and methods The persistence study was made on guava plants (Psidum guajava L.) sowed on Canaima farm, located on “El Derrote” area, on the right margin of “El Derrote” drain, Luis de Vicente

parish, Mara county, Zulia state. Eight (8) plants were randomized selected, sowed in a distance of 7x7 correspondent to an area of 147 m2. A total of three replications were done.

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Rev. Fac. Agron. (LUZ). 2005, 22: 57-66 Insecticides were applied following the established by the manufacturer according to dose, and taking previsions established by COVENIN norms (5). The used doses were: 2 L/ha of 57 Malathion, equal to 114 g of active/ha ingredient, and 1.5 L/ha for Pirinet (Chlorpiryphos). Correspondent to 6.7 g of active/ha compounds. A dilution of 400 L/ha was applied, and in function of the effective area of the essay were applied 8 liters of mix, that had 0.030 L of Malathion and 0.020 L of Chlorpiryphos. An hour later of the application of commercial formulas, samples of guava were taken collecting from 2 to 3 fruits by plant, which is equal to approximately 2 kg of harvested guavas in eight (8) plants. These samples corresponded to day zero (0). Subsequently, inter-daily samples were taken in days: one, three, five, seven, nine, eleven, thirteen and fifteen, for a total of nine samples. Previous to the application, a white sample was taken to verify that there were not insecticides residues in fruits. Samples were recollected in black, clean and dry polyethylene bags, were labeled and taken to the laboratory for the chromatographic analysis. It is important to mention that during the evaluated period, were not registered precipitations in the unit. Purity standards of insecticides were employed (Dr. Ehrenstorfer Gmbh, Germany), malathion (98.5%) and chlorpiryphos (99.2%), to prepare base solutions of 1000 µg/ml of each insecticide in ethyl acetate HPLC grade (j.T. Baker), which were prepared by dilution, calibration solutions in ethyl

acetate HPLC grade (J.T. Baker) and sprinkled solutions in methanol HPLC grade. The employed acetone as extraction solvent was HPLC grade (J.T. Baker), the sulfate of anhydrous sodium and the phosphate triphenyl (99% pure), used as internal standard was analysis grade (Riedel de Häen). The chromatographic determinations were done on a gas chromatograph, Auto System (PerkinElmer), equipped with a Nitrogenousphosphorus detector, an automatic sampler (Perkin-Elmer), a capillary column of 30 m x 0.53 mm ID x 1.2 µm of covering thickness of 5% phenyl methyl silicone (ALLTECH). The chromatograms registered and the areas interaction of peaks, were made with a personal computer, equipped with a software (Turbochrom Navigator 4, Perkin Elmer). The injector and the detector were used at 250 and 280ºC respectively. Samples were injected with the splitless method. The temperature program of the oven was: 60ºC by 0.80 min, slope 1:40ºC/in until 160ºC, slope 2: 3.5ºC/in until 230ºC, slope 3: 8ºC/min until 280ºC, sustained by 1 min. Helium as dragging of gas was employed at 10 mL/min, and hydrogenous and air as detector of gas at 1.70 and 100 mL/min, respectively, all with high purity (AGA de Venezuela). The detector was 1.0 mV. The quantification was done by the internal standard. The extraction of organophosphorus insecticides was initially made according to the extraction procedure described by Molero et al. (17) for tomato samples, with two modifications. 100 g of guava sample were

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Sánchez et al. homogenized on an electrical mixer. Sub samples of 4 grams were weighted, were added 10 mL of ethyl/ acetone acetate (90:10), 2 grams of anhydrous sodium sulfate analytical grade, and 0.2 grams of sodium chloride analytical grade, the mix was stirred for 10 minutes, and then were centrifuged at 2000 rpm for 4 minutes. From the superior surface of the extract, 2 mL were separated from the extract, and were transferred to a vial. The internal standard was added (TPP) and 1 ìL was injected twice on the gases chromatograph. The efficiency of the extraction technique was determined calculating the recovery percentages. Samples of white guavas were sprinkled with a methanolic solution of two organophosphorus insecticides in concentrations between 0.2 and 8.0 ìg/ mL and were submitted to the described extraction procedure. The detection limit was calculated according to the EPA criteria

(Environmental Protection Agency of United States): Y – YB = 3SB (16). Where Y, is the signal of the significantly different instrument to the white signal or the ground signal, YB is the white signal and SB, is the standard deviation of white. For the statistical calculus of the detection limit of malathion and chlorpiryphos, base solutions were prepared in concentrations between 0.010 and 0.040 µg/mL, which were injected three times on the gases chromatograph. Mean times (t1/2) of malathion and chlorpiryphos concentrations were calculated employing the following expression (2):

The time of mean life is the time where the insecticide concentration is the same to the half of the initial concentration. K value is obtained from the degradation curve, K is the slope of the line.

Results and discussion The recovery research of the two insecticides mentioned in this study, in guava samples (Psidium guajava), showed an efficiency of 10.09 and 6.44%. These results indicate an acceptable precision of extraction employing the mix of ethyl/acetone acetate (90:10) and gas chromatography, resulting efficient for the analysis of both organophosphorus insecticides, which would allow to process a huge number of samples simultaneously, with short analysis times (approximately 50 minutes for

four samples), and allow the application in laboratories of control quality of fruits and others, that are commercialized nationally and internationally. Figure 1 shows a common chromatograph of a guava extract (Psidium guajava) sprinkled with malathion and chlorpiryphos in levels between 0.05 and 2.0 mg/g. Malathion persistence The average concentration of malathion residues in guava in day zero, an hour after the application was of 0.41 ± 0.28 mg/kg. In day 1 was

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Rev. Fac. Agron. (LUZ). 2005, 22: 57-66 3 Sing of the detector 1 2

Time (min) Figure 1. Common chromatogram of the determination of organophosphorus insecticides of a guava extract (Psidium guajava). (1) Malathion, retention time 18.385 min. (2) Chlorpiryphos, retention time 19.531. (3) Triphenyl phosphate, retention time 28.182 min (internal Standard) observed a fast reduction of the residues concentration of Malathion until 0.04 ± 0.02 mg/kg (figure 2). This behavior might be because malathion is a contact insecticide that dissipates very rapidly maybe due to volatilization (13). In day 3, were not detected residues of malathion. Similar results were obtained for malathion in persistence research in tomatoes sowed in farm, and in must and grape wine (7, 19). The dissipation velocity of malathion in guava, followed a kinetic of first order (figure 3) and a time of mean life of 0.29 days. The equation of lineal regression for the applied doses was: Y= 0.8916-2.3273t, with a R 2 =1,000, with a probability of statistical significance P≤0.01. When are compared the registered malathion concentrations

between the moment of the application (day 0) and day 1, can be observed that the correspondent value to day zero, (0.41 mg/kg) is under LMRs for guava, which is of 0.5 mg/kg and over admissible daily ingestion (IDA) that correspond to 0.02 mg/kg On the other hand, the found residues in day 1 kept over IDA. For day 3, there were not registered malathion residues over the detection limit of the method (0.0165 mg/kg) which allow to deduce that from this moment and on the fruit has values of this insecticide, under the critical values established in the Codex Alimentarius, and therefore does not represent any danger for consumers. (6) Chlorpiryphos persistence The average concentration of Chlorpiryphos in day zero an hour later of the application was of 0.63 ± 0,40 mg/ kg, observing a strong descending of the

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Sánchez et al.

mg/kg concentration

0,7 malathion

0,6

chlorpiryfos

0,5 0,4 0,3 0,2 0,1 0 Day 0

Day 1

Day 3

Day 5

Day 7

Day 9

Day 11 Day 13 Day 15

Days after the application

Figure 2. Tendency in the reduction of the residues concentration of Malathion and Chlorpiryphos in guava. concentration to 0.081 ± 0.052 mg/kg in day 1, corresponding to a 87% of dissipation in relation to the concentration in day zero. From day 3, concentrations continued reducing, registering a concentration of 0,027 ±

0,012 mg/kg, that correspond to a 96% of dissipation respect to the initial concentration. The dissipation of the product in a gradual way, during days 5, 7 and 9 where were registered concentrations of 0.016, 0.015 and

0,0

ln(concentration mg/kg)

y = -2,327x - 0,892 -1,0

R2=1

-2,0

-3,0 -4,0 -5,0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Days after the application

Figure 3. Demonstration of kinetic of first order in the degradation of Malathion in guava.

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Rev. Fac. Agron. (LUZ). 2005, 22: 57-66 0.012 mg/kg, corresponding to a reduction in the product concentration in fruit of 97, 46, 97, 61 and 98.0%. In day 11, was registered an average concentration of residues of 0.011 ± 0,001 mg/kg equal to a dissipation percentage of the product of 98.25%. For the subsequent thirteen and fifteen days, were not detected residues of the product in samples of analyzed fruits (figure 2). It is important to mention that the dissipation velocity of insecticides varies according to some factors, some of these are: humidity, air, light, temperature, volatilization, wash, way of action of the insecticide in the plant (systemic or of contact) and the metabolic activity of the plant (18, 20). Chlorpiryphos is classified as an insecticide of contact, but has a slightly penetration power in the tissue without translocated in the interior of the plant (9, 14). Therefore, it is probably that this might be the reason that persist

under the research conditions for 13 days, and does not dissipate in guava fruit in the period of time that normally dissipate others organophosphorus insecticides of contact, as in malathion and parathion (7, 17). This behavior is justified because systemic insecticides penetrate the fruit, suffer in less magnitude the degraded effects of the environment that are around the plant, that is the reason that these persist for a long time. The dissipation velocity of chlorpiryphos in guavas followed a kinetic of first order (figure 4) with a time of mean life of 2.32 days. The equation of lineal regression of the applied doses was: Y= -1.8702-0,2981t, with a R 2 = 0.6979, and with a probability of statistical significance P≤0.01. When comparing chlorpiryphos concentrations registered from the moment of the application (day zero) until day eleven, can be observed that

0,0

ln(concentration mg/kg)

-1,0 y = -0,299x - 1,87 R 2 = 0,6979

-2,0 -3,0 -4,0 -5,0 -6,0 0

1

2

3

4

5

6

7

8

9

10 11

12

13 14

15

Days after the application

Figure 4. Demonstration of kinetic of first order in the degradation of Chlorpiryphos in guava.

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Sánchez et al. all registered concentrations are over the established IDA as critical level by the Codex Alimentarius for guava fruits (0.010 mg/kg). When comparing the average concentrations of the insecticide residues with LMRs established in 0.5 mg/kg can be observed that from level 3 (0.027 mg/ kg), residues were found under the established critical limit, it means, according to these results from level 3 these fruits can be commercialized, when interpreting the expressed in the Codex Alimentarius (6). The analysis of the obtained results for mean life of insecticides in guava, shows that chlorpiryphos insecticide behaved as a systemic insecticide, so persisted much longer than the contact insecticide malathion. The turkey test, applied to mean concentrations of two insecticides measured in guavas, showed significant differences (P≤0.05). Insecticides residues and fruits quality of guava In function of the obtained results, and considering that food quality and

in this case guava fruits, can be affected by external agents that in some cases can cause different pathologies in consumers, depending on the concentration and the frequency the consumer eats the fruit (12) and after comparing the obtained results with the established LMRs by the Codex Alimentarius, can infer that guava quality is affected by external agents that contaminate it, in this case chlorpiryphos residues keep eleven days after the application, being the critical phase the three first days, due to insecticides levels are over LMRs (o,5 mg/kg). However, until day 11, are registered values over IDA (0.01 mg/kg). In the case of malathion for day 1, the obtained residual levels (0.04 mg/ kg) are under LMRs (0.5 mg/kg). After the second day, under the research conditions, must not present risky situations when consuming fruits that were treated with malathion, and therefore the fruit quality, from the point of view of insecticides residues, is not affected from this moment.

Conclusion The dissipation of these two insecticides, measured in guava (Psidium guajava) followed a kinetic of first order. For only one insecticide application on plants, the time of mean life was of 0.29 days for malathion and 2.35 days for chlorpiryphos. When comparing the insecticides concentrations with the established

LMRs in Codex Alimentarius under the research conditions, after the third day, none of the insecticides affect the quality of the fruit, due to malathion is completely dissipated, and the residual levels of chlorpiryphos are under LMRs, therefore, the consumption of these fruits must not be a risk for the consumer.

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Literature cited 1. Adnan, I, Al Samarice, Kloud A. M. Shaker and Mabriouk A. AlBassomy 1988. Residue level of three organophosphorus insecticides in sweet pepper grow in commercial greenhouses. Pestic. Sci 22:189-194.

9. International Group of National Associations of Manufacturers of Agrochemical Products. (GIFAP). 1988. Normas para el empleo seguro y eficaz de los insecticidas. Bélgica.

2. Antonious, G. and Snyder, J. 1994. Residues and half-lives of Acephate, Methamidophos and Pirimiphos-Methyl in leaves and fruit of greenhouse-grown tomatoes. Bull. Environ. Contam. Toxicol. 52: 189-194.

10. International Regulatory Aspects for Pesticide Chemicals. 1981. Toxicity profiles CRC Press Inc Florida, 2nd edición. 11. Juran , J. M. and Gryma, F. M. 1997. Manual de control de calidad. Vol 1, 4a edición. Mc Graw Hill. México.

3. Bicchi, C., D’Amato, A. and C. Balbo. 1997. Multiresidue method for quantitative Gas Chromatographic determination of pesticide residues in Sweet Cherries. J. of AOAC Int. 80 (6):1281-1286.

12. La Dou, J. 1995. Medicina Laboral. Manual Moderno. México, D.F. 13. Liapis, K. G. Milladis, and P. ApladaSarlis. 1994. Persistence of Monocrotophos residues in green house tomatoes. Bull. Environ. Contam. Toxicol. 53: 303-308.

4. Centro de Comercio Internacional. UNCTAD/GATT.1995. Control de calidad en la Industria Alimentaria. Manual de Introducción. Ginebra.

14. Martínez, I. 1974. Toxicología Ambiental de los Insecticidas. Facultad de Agronomía La Universidad del Zulia. Trabajo de Ascenso.

5. Comisión Venezolana de Normas Industriales (COVENIN). 1992. Residuos de plaguicidas en Alimentos. Definiciones y Terminología. Caracas. Venezuela.

15. Medina, D., A. Prieto, G. Ettiene, I. Buscema, A. Abreu de V. 1999. Persistence of organophosphorus pesticide residues in Limón River waters. Bull. Environ. Contam. Toxicol. 63:39-44.

6. FAO/OMS. CODEX ALIMENTARIUS.1993. Residuos de Plaguicidas en Alimentos. Programa conjunto FAO/OMS sobre Normas Alimentarias. Comisión del Codex Alimentarius. Vol 2.

16. Miller, J.C. and J.N. Miller. 1993. Estadística para Química Analítica. 2da Edición. USA. Iberoamericana Addison-Wesley.

7. Ettiene, G., A. Prieto, D. Medina, I. Buscema, L. Sandoval, and L. Hunda. 1996. Residuos de Insecticidas Organofosforados en Mosto y Vino de Uvas. Rev. Téc. Ing. Univ. Zulia. 20 (3): 223-230.

17. Molero D. 1996. Evaluación de residuos de plaguicidas organofosforados en tomates. Trabajo de grado para optar al título de Magíster Scientarium en Ciencias del Ambiente. Facultad de Ingeniería La Universidad del Zulia.

8. Fillion, J., F. Sauvé, and J. Selwyn. 2000. Multiresidue Method for the Determination of Residues of 251 Pesticides in Fruits and Vegetables by Gas Chromatography/ Mass Spectrometry and Liquid Chromatography with Fluorescence Detection. J. of AOAC Int. 83 (3): 698-713.

18. Palenzuela, J.1990. Efectos de plaguicidas en la fisiología de frutas y hortalizas. Editorial. Limusa. México.

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Sánchez et al. 19. Prieto, A., D. Molero, G. González, I. Buscema, G. Ettiene, and D. Medina. 2002. Persistence of methamidophos, diazinon and malathion in tomatoes. Bull. Environ. Contam. Toxicol. Vol. 69:479- 485.

20. Primo, Y. and D. Carrasco. 1977. Química Agrícola II, Plaguicidas y Fitorreguladores. Editorial Alhambra, S.A. Primera edición. Capitulos 1 y 4. España.

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