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ISSN: 2380-2391
Journal of Environmental Analytical Chemistry
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14C-Prothiofos Residues in the Presence of Deltamethrin and Dimilin Pesticides in Cotton Seeds and Oils, Removal of Prothiofos Residues in Oils and Bioavailability of its Bound Residues to Rats

Hassan Abdel-Gawad1*, Fathia Mahdy1, Adly Hashad1, Soliman M Soliman1 and Ebtsam A Ahmed2

1Applied Organic Chemistry Department, National Research Centre, 33 El Bohouth St., Dokki, Giza, 12622 Egypt

2Organic Chemistry Department, Faculty of Science, Helwan University, Egypt

*Corresponding Author:
Dr. Hassan Abdel-Gawad
Department of Applied Organic Chemistry
National Research Centre
33 El Bohouth St., Dokki Giza, 12622 Egypt
Tel:
2-01063157828
E-mail: abdelgawadhassan@hotmail.com

Received date: May 24, 2015; Accepted date: June 26, 2015; Published date: June 30, 2015

Citation: Abdel-Gawad H, Mahdy F, Hashad A, Soliman SM, Ahmed EA et al. (2015) 14C-Prothiofos Residues in the Presence of Deltamethrin and Dimilin Pesticides in Cotton Seeds and Oils, Removal of Prothiofos Residues in Oils and Bioavailability of its Bound Residues to Rats. J Environ Anal Chem 2:145. doi: 10.4172/2380-2391.1000145

Copyright: © 2015 Abdel-Gawad H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Ethyl-1-14C-prothiofos and some of its degradation products have been prepared for the present investigation. Cotton plants were treated with 14C-prohiofos alone and in presence of deltamethrin and dimilin pesticides under conditions simulating local agricultural practice. 14C-residues in seeds were determined at harvest time about 75% of 14C-activity was associated with oil. The ethanol soluble 14C-residues accounted for 6% of the total seed residues after further seeds extraction, while the cake contained about 18.5% of the total residues in case of prothiofos only. The bound residues increased in presence of deltamethrin and dimilin pesticides and amounted to 30% and 34% of the total residues, respectively. About 89% of the 14C-activity in the crude oil could be eliminated by simulated commercial processes locally used for oil refining. Chromatographic analysis of cotton oil extracts (acetonitrile layer) revealed the presence prothiofos oxon, O-ethyl phosphorothioate, O-ethyl S-propyl phosphorothioate, des-propylthio prothiofos, O-ethyl phosphoric acid as the main degradation products, besides to one unknown compound in addition to the parent compound, in case of prothiofos alone. The same degradation products are found in case of prothiofos and deltamethrin and prohiofos and dimilin except compounds O-ethyl S-propyl phosphorothioate and des-propylthio prothiofos. Chromatographic analysis of ethanol extract revealed the presence of O-ethyl phosphorothioate, despropylthio prothiofos and O-ethyl phosphoric acid as free metabolites. The conjugated metabolite was liberated by acid hydrolysis and identified as 2,4-dichlorophenole which was detected by color. When rats were fed the extracted cake for 72 hours, the bound residues were found to be bioavailable. The main excretion route was via the expired air (49%), while the 14C-residues excreted in urine and feces were (34% and 9.7%), respectively. The radioactivity detected among various organs accounted to 6.1%. Chromatographic analysis of urine was determined.

Keywords

14C-Prothiofos; Residues; Cotton seed oil; Refining processes;Bioavailability

Introduction

Cotton (Gossypium hirsutum L.) is a cash crop for more than 20 million farmers in developing countries of Asia and Africa. It is mainly cultivated to meet the basic requirement for cotton fabrics. Cotton seed is a valuable by-product of the cotton plant and for every kg of cotton fiber, 1.65 kg of cotton seed is produced. Cotton seed contains approximately 18-25% of oil and 20-25% high quality protein [1].

Cotton seed oil is rich in tocopherols which inhibits rancidity development and thus contribute to its stability resulting in a longer shelf life for the product. Cotton seed oil is naturally hydrogenated oil and is suitable for heart due to the presence of palmtic, stearic, mysteric, oleic, linoleic and linoleinic fatty acids in sufficient quantities. Cotton seed oil has also gained importance in food preparation due to its higher smoke point (about 232°C) compared to other cooking oils and is good for frying food articles [2].

Prothiofos (I), (tokuthion, O-(2,4-dichlorophenyl) O-ethyl S-propyl phosphorodithioate) is an organophosphorus insecticide effective for the control of leaf-eating caterpillars, pseudoccus sp., thrips, cockchafer larvae, cutworms in a range of crops including vegetables, fruit, maize, sugar cane, sugar beet, tea, tobacco, ornamentals and canola [3]. Prothiofos is readily metabolized in rats, [4] insects [5] and plants [6] via oxidative desulfuration of the P=S moiety to P=O, cleavage of P-O-aryl and P-S-alkyl linkages, and conjugation of liberated 2,4-dichlorophenol.

The pest infestation in cotton causes heavy losses and remains a major bottleneck in its economic cultivation. To reduce the losses caused by insect pests, the main reliance has been on the use of insecticides. Due to the change in pest complex over the years and severe damage caused by American bollworm (Helicoverpa armigera Hubner), specifically, the number of sprays have increased from 4-7 in 1980’s to around 15-20 in 2000’s. The farmers quite often, apply more number of sprays at higher doses, at shorter intervals and sometimes use mixtures of insecticides at their own level [7].

It is crucial to monitor pesticide residues by using various analytical methods and techniques. Colorimetry [8], capillary electrophoresis (CE) [9], mass spectrometry (MS) [10], gas chromatography (GC) [11], high-performance liquid chromatography (HPLC) [12], gas chromatography-triple quadruple mass spectrometry (GC–MS/MS) [13], thin-layer chromatography coupled with different detectors [14], and spectral techniques [15] are the most commonly employed methods for trace environmental analysis of  pesticides.

The use of pesticides can result in the accumulation of their residues in seeds, oil and cake. The residues in cake reach the milk through lactating animals and eventually into human body [16-18]. Since people are at risk from the pesticide residues which may be present in the oil, so it is important to determine the removal of pesticide residue in oils. However, a very little information is available on residual fate and metabolism of prothiofos in cotton plants. The objective of the present study were (i) to determine and identify of prothiofos residues in cotton seeds and oils (ii) to deduce the effect of oil refining processes on the level and nature of 14C-labelled prothiofos residues in crude cotton seed oil and (iii) to investigate the bioavailability of its bound residues in rats.

Materials and Methods

The Radiochemical

14C-Prothiofos labeled at carbon atom of ethyl group was prepared in one vesseled reaction according to Abdel-Gawad et al.[6] The synthesized 14C-prothiofos had specific activity (0.74 MBqg-1) and the radiometric purity was greater than 98% Figure 1.

environmental-analytical-chemistry-ethyl-prothiofos

Figure 1: Synthesis of 14C-ethyl prothiofos.

Non-labelled prothiofos and some of its degradation products were synthesized for comparison purposes according to Abdel-Gawad et al. [6] such as; prothiofos oxon (O-(2,4-dichlorophenyl) O-ethyl S-propyl phosphorothioate) (II), O-ethyl phosphorothioate (III), O-ethyl S-propyl phosphorothioate (IV), despropyl thioprothiofos (V), O-ethyl phosphoric acid (VI), O-ethyl S-propyl phosphordithioate (VII), and 2,4-dichlorophenol (VIII). The structures of these compounds are shown in Figure 2.

environmental-analytical-chemistry-prothiofos-insecticide

Figure 2: Main degradation products of prothiofos insecticide.

Field experiments

Plants treatment with pesticides: Pesticide-free Gossypium hirsutum L. seeds (Var. Giza 86 were obtained from Agricultural Research Centre, Cairo). The seeds were cleaned from any dockage and impurities before cultivation. Sound whole seeds of cotton were cultivated under normal field conditions in a controlled, isolated field area. Irrigation, fertilization, hand weeding were performed and soil management were conducted as usually practiced in the field. A control field plot and of similar soil characteristics to the experimental plot, was cultivated as in the experimental plot and kept untreated with pesticides. Shortly at flowering stage, leaves of plants were treated twice, 21 days apart, with 14C-prothiofos alone (4 mg/plant each time) and in presence of deltamethrin (3.6 mg/plant) and dimilin (1.6 mg/ plant) pesticides. Samples of cotton plant were collected manually at harvest time. The cotton seed samples were air-dried and delinted to get cotton seed for preparation of oil and cake and determination of radioactivity.

Extraction of cotton seeds : The analysis procedures for 14C-residues in cotton seeds were shown in Figure 3. Dry cotton seeds were crushed and extracted with n-hexane for 12 hours using a Soxhlet apparatus. After evaporation of hexane under reduced pressure, radioactivity in the cotton oil was measured. The residue remaining after extraction (cake A) was further extracted with ethanol Figure 3. Aliquots of the hexane extract (oil) and the ethanol extract were used for determination of radioactivity.

environmental-analytical-chemistry-Analysis-procedures

Figure 3: Analysis procedures for 14C-residues in cotton seeds and oils.

The remaining cake (cake B) was air dried and digested by adding 1 mL Solusol (tissue and gel solubilizer) and digest samples at 40-50°C until tissue dissolves, decolorize with 30% H2O2 (1 mL) and add 70 μL glacial acetic to eliminate chemiluminescence [19] The radioactivity was counting using liquid scintillation counter.

Removal of pesticide residues from oils: Oil processing was conducted in four steps namely; alkali refining, bleaching, winterization and deodorization according to the flow-chart shown in (Figure 4) as previously reported [20].

environmental-analytical-chemistry-prothiofos-residue

Figure 4: Effect of refined processes on 14C-prothiofos residue levels in cotton seed oils.

Neutralization (alkali refining): Samples of the crude cotton oil ( 14.4 g) were heated at 85°C with continuous stirring for 30 minutes, and then neutralized by adding 2N NaOH and stirring was continued for another 30 minutes. The mixture was then centrifuged at 3000 rpm for 10 minutes. After separating it from the soap stock, the oil was washed with 100 mL hot distilled water several times using separating funnel until the washing was neutral. This process removed free fatty acids and other acidic material from oil and 14C-residue levels were determined.

Bleaching : Bleaching clay performs not only color removal but also the removal of trace metals, adsorption of phospholipids, soap and decomposition of oxidation products such as peroxides. Neutralized oil was heated to 60°C under vacuum and fuller earth (Tonsil, 0.15 g) was added. The mixture was then heated to 100-110°C on an oil bath for 30 minutes with continuous stirring. The oil was centrifuged and decanted at 3000 rpm for 10 minutes and 14C-residue levels were determined.

Winterization: The clear dry oil was winterized (cooled) at 5°C for 3 days and the high saturated glycosides which separated were removed by filtration or centrifuged at 3000 rpm for 10 minutes and then decanted and 14C-residue levels were determined.

Deodorization: Deodorization is the last major processing step in the refining of edible oil and is responsible for removing undesirable ingredients. The winterized oil was heated at 200-220°C on an oil bath while passing superheating steam (heating distilled water in a closed system at 2 atm. pressure). The deodorization was continued for 3 hours. The oil was then decanted by centrifugation or by separating funnel and 14C-residue levels were determined.

Bioavailability of bound residues in rats: Three months old, sexually mature white male rats (derived from Sprague-Dawlay strain) weighing 150 +10 g were purchased from animal house colony, Dokki, Giza Egypt. The animals were individually housed in glass metabolism cages that allowed separate collection of feces, urine and expired air. The rats were conditioned for two days to a daily diet consisting of standard feed mixed with extracted untreated cotton seeds for acclimation under the laboratory conditions (29 ± 3°C). The animals were kept without food for 24 hours and then fed the diet containing bound 14C-prothiofos residues for three days. To make the feed palatable, the extracted seeds were mixed thoroughly with an equal amount of white cheese and the paste was left to dry.

The respiratory carbon dioxide was trapped in 10% NaOH solution. Urine, feces and 14CO2 were collected separately for three days, and assayed for radioactivity. After the end of three days each rat was lightly anaesthized with ether and blood removed from the pumping heart. The animal was then killed with an overdose of ether, and samples from liver, kidney, fat, blood, lung and brain were collected and kept frozen till analysis of radioactivity by digestion and followed by LSC-counting.

Isolation and characterization of 14C-residues: Analysis of radioactive compounds was achieved by thin layer chromatography (TLC). Samples of crude oil at harvest time and after each refining process were partitioned between acetonitrile and hexane to remove the oil. The radioactive residues were almost completely retained in the acetonitrile layer. Analysis of crude and refined oil extracts were achieved by thin layer chromatography (TLC).

Urine was extracted with chloroform (chloroform 1) and the aqueous layer was then acidified with 2 N HCl and heated for 2 h at 100°C on a water bath and reextracted with chloroform three times. The combined chloroform was dried over anhydrous sodium sulfate, filtered off, and evaporated under vacuum (chloroform 2) to obtain the conjugated metabolites. Aliquots from both chloroform 1, 2 and ethanol layers (after hexane extraction) were analyzed by TLC on silica gel plates using suitable solvents

Residues were characterized by TLC on silica gel plates (20 × 20 cm; 0.25 mm thickness) with fluorescent indicator (Kiesel gel 60 F254, Merck, Germany). The solvent systems used were:

System 1: n-hexane:ethyl acetate 99.5:0.5 (v/v)

System 2: n-heptane:ethyl acetate 99:1 (v/v)

System 3: n-heptane only

Authentic samples were run alongside as references and spots were viewed under UV-light at 254 nm and by spraying the plates with a freshly prepared Hans-Isherwood reagent [21] or after preliminary spray with PdCl2 solution, the plates were subjected to I2 vapor to detect the compound by color [22,23]. To detected the phenolic compounds, the plate were developed in the above systems and sprayed with a freshly prepared solution of 1% ferric chloride (20 mL), 60 mL distilled water and 20 mL of 1% potassium ferricyanide, where blue spots against yellow background appeared [24] .

Radioactivity measurements: Radioactivity in oil (acetonitrile extracts), ethanol extracts, urine and other liquid samples were measured directly by LSC using a Packard Tri-Carb liquid scintillation spectrometer (Model 2300, Perkin Elmer Life Science, Boston, MA, USA) in vials using a dioxane-based scintillation cocktail comprised of dioxane (1 L), naphthalene (100 g), (PPO; 2,5-diphenyloxazole) (10 g) and POPOP; 1,4-di (2-(5-phenyloxazolyl))-benzene (0.25 g).

Cake, feces, ground cotton, and animal tissues (100 mg) were assayed for radioactivity by digestion using (1 mL Solusol (tissue and gel solubilizer), 1 mL 30% H2O2 and 70 μL glacial acetic at 40-50°C). The radioactivity was counted using a liquid scintillation counter. The internal standard technique was used for quench correction. Thin layer plates were divided in 1 cm increments, scraped into vials, eluted with methanol, covered with scintillator and counted.

Results and Discussion

14C-residues in seeds, oil and cake: To be able to assess the risk to the consumer from the ingestion of residues in food, field trials were performed in which prothiofos was applied in the normal quantities, the topical application of 14C-prothiofos on cotton plant led to the appearance of 14C-activity in the dry seeds. The 14C-residues in dry cotton seeds obtained from 14C-prothiofos treated plants amounted to 0.32% of the applied dose at harvest time. The percentage of residues increases to 0.46% and 0.49% in the presence of deltamethrin and dimilin pesticides, respectively Table 1. The insecticide was absorbed and translocated very slowly from the treated leaves to the seeds of investigated plant due to enzyme metabolic activity [20,25]. Following foliar application of soybean plants with 14C-pirimiphos-methyl [26] and cotton plants with 14C-malathion [27]. The aged insecticide residues in seeds amounted to 0.37% and 0.11% of the applied dose, respectively. The data obtained are in agreement with those reported by Singh et al. [28] in their studies on the persistence of ethion residues on cucumber. Though persistent insecticides are beneficial for controlling pests for extended periods, yet their residues in consumable parts of the crops may be harmful to the consumers when they exceed the Maximum Residue Limit (MRL) values [29].

Samples* Average 14C-residues
Seeds
Weight (g) (ppm)a %**
Prothiofos 59 1.30±0.11 0.32
Prothiofos anddeltamethrin 58 ±1.90 0.16 0.46
Prothiofosanddimilin 67  1.78 ±0.14 0.49

Table 1: 14C- Residues in seeds after treatment of cotton plants with 14C-prothiofos in presence of deltamethrin and dimilin pesticides at harvest time.

About 75.4% of 14C-activity in dry seeds was found to be associated with oil (hexane extract). The ethanol soluble 14C-residues accounted for 5.9% of the total seed residues, while the cake contained about 18.5% of the total residues as non-extractable or bound residues in case of prothiofos only. The percentage of bound residues increases when deltamethrin and/or dimilin pesticides were used in combination with prothiofos and amounted to 30% and 33.7%, respectively. The recovery percent of the applied radioactivity ranged from 98.3-99.7% as shown in Table 2. When two compounds are mixed, there are basically four types of interactions that change the efficacy of pesticide combinations, they can either be additive effects or synergistic responses (potentiating) or antagonism or enhancement in an insect species. If a mixture is potentiating, it is a useful tool in enhancing control efficacy and combating insecticide resistance. If a mixture is antagonistic, it should not be used, because it will reduce the efficiency of pest control and aggravate the resistance problem [30,31]. It is worthy to mention that Zayed et al.[32] found that repeated applications of pesticides might enhance the release of 14C-bound residues in their study on the impact of repeated pesticide applications on the binding and release of methyl 14C-monocrotophos and U-ring labeled 14C-carbaryl to soil matrices under field conditions. On the contrary, Abdel-Gawad et al. [33] reported that ethion produced a good potentiating with deltamethrin and dimilin, respectively. The 14C-ethion residues decrease in presence of deltamethrin and dimilin pesticides. The number of degradation products increases in presence of deltamethrin than with dimilin. The bound residues decrease in presence of deltamethrin and dimilin pesticides than ethion alone.

Samples* Oil hexane (ppm)a Ethanol (ppm)a Cake (ppm)a % recovery
Prothiofos 0.98± 0.091 0.077±0.002 0.24± 0.02 99.7
Prothiofos anddeltamethrin 1.20 ± 0.100 0.110 ± 0.008 0.57 ± 0.05 98.9
Prothiofosanddimilin 1.05 ± 0.092 0.100 ± 0.006 0.60 ± 0.03 98.3

Table 2: 14C- Residues in extracted seeds after treatment of cotton plants with 14C-prothiofos in presence of deltamerthrin and dimilim pesticide at harvest time.

Effect of refining processes

Commercial processing procedures led to a gradual decrease in the total amount of 14C-residues in oils as shown in Figure 5. The complete refined oil from treated seeds lost about 89% of the total 14C-residues in crude oil. This decrease could be attributed to alkali hydrolysis, effect of adsorption, effect of heat and/or evolved 14CO2 gas. On the other hand, the alkali treatment, bleaching and winterization eliminated 53%, 58% and 63% of the originally present radioactivity in oil, respectively. These results agree with Miyahara and Saito [34] who found that in some organophosphorus insecticides such as malathion, dichlorvos and chlorpyrifos, the deodorizing process was effective in removing pesticide residues due to decomposition and volatilization. This also agreed with the findings of Ruiz Me´ndez et al. [35] who reported that pesticide residues (simazine) were highly eliminated in olive oil during the deodorizing step. Tayaputch et al. [36] found that during the processing of crude soybean oil, alkali treatment removed 50%, while deodorization reduced the residues of 14C-prothiofos by further 25%. Also, the present results seem to be in agreement with those reported by Zayed et al. [37,38] who showed that the amounts of both 14C-carbofuran and 14C-pirimiphos-methyl residues in soybean oil decreased to 16% and 25% through the refining processes respectively. Working on cotton oil grown with 14C-aldicarb, 63% of the residues in cured oil were also found to be lost during the refining processes [16]. The effect of processing methods used for oil refining on 14C-Zineb residues was studied. Obvious reduction was detected by neutralization (31.7%) and was increased by further bleaching (34.1%), winterization (41.46%) and deodorization (63.6%) methods [39]. Research on sunflower seeds and oil treated with endosulfan and lindane were exceeded the MRLs value in sunflower seeds at harvest [40].

environmental-analytical-chemistry-refining-processes

Figure 5: Effect of refining processes on 14C-prothiofos residues in cotton seed oil.

Identification and characterization of radioactive degradation products in oil

The Rf values and the average concentration of 14C-prothiofos and its degradation products in cotton oil extract at harvest time are shown in Table 3. Chromatographic analysis of cotton oil extracts (acetonitrile layer) revealed the presence prothiofos oxon (II), O-ethyl phosphorothioate (III), O-ethyl S-propyl phosphorothioate (IV), despropylthio prothiofos (V), O-ethyl phosphoric acid (VI) as the main degradation products, besides to one unknown compound in addition to the parent compound (I), in case of prothiofos alone. The same degradation products are found in case of prothiofos and deltamethrin and prothiofos and dimilin except compounds (IV) and (V).

Substances Rf in systems Metabolites residues (ppm)
Sys.1 Sys.2 Sys.3 Prothiofos Prothiofosand deltamethrin Prothiofosanddimilin
Prothiofos (I) 0.56 0.47 0.21 0.067 0.21 0.36
Prothiofosoxon (II) 0.01 0.60    0.36 0.286 0.25 0.33
O-ethyl phosphorothioate (III) 0.21 0.52 0.45 0.192 0.24 0.00
O-ethyl S-propyl phosphorothioate (IV)  0.43 0.41 0.32 0.174 0.00 0.19
Des-propylthioprothiofos (V) 0.54 0.49 0.30 0.058 0.00 0.00
O-ethyl phosphoric acid (VI) 0.77 0.65 0.60 0.066 0.32 0.04
Unknown 0.85 0.45 0.65 0.086 0.09 0.08

Table 3: The amount and Rf values of 14C- prothiofos and its active degradation products in extracts of cotton seeds and oil in presence of deltamethrin and dimilin pesticides at harvest time.

Chromatographic analysis of ethanol extract (and ethanol layers [after hexane extraction] were analyzed by TLC on silica gel plates using suitable solvents) revealed the presence of O-ethyl phosphorothioate (III), despropylthio prothiofos (V) and O-ethyl phosphoric acid (VI) as free metabolites in addition to a conjugated metabolite. This was liberated by acid hydrolysis and identified as 2,4-dichlorophenole (VIII), the latter compound was detected by color.

Metabolism of pesticides may involve a three-phase process. In Phase I metabolism, the initial properties of a parent compound are transformed through oxidation, reduction, or hydrolysis to generally produce a more water-soluble and usually a less toxic product than the parent. The second phase involves conjugation of a pesticide or pesticide metabolite to a sugar, amino acid, or glutathione, which increases the water solubility and reduces toxicity compared with the parent pesticide. Generally, Phase II metabolites have little or no phytotoxicity and may be stored in cellular organelles. The third phase involves conversion of Phase II metabolites into secondary conjugates, which are also nontoxic [41].

The suggested pathway for the degradation of 14C-prothiofos in crude and refined cotton seed oil is shown in Figure 6. The formation of these degradation products suggest that prothiofos is degraded in cotton plants via three pathways. One consists of oxidation of P=S to P=O analogue, the second is hydrolysis to 2,4-dichlorophenol which is further conjugated with sugar in the plant, and the third is cleavage of the propyl group lead to the formation of despropylthio compounds. Katagi and Mikami [42] noted that the metabolism of organophosphorus pesticides in plants have revealed cleavage of the P-O-aryl linkage and O-dealkylation to be among the most predominant metabolic pathways. Enzymatic and acid hydrolysis release an aglycon and direct spectroscopic analyses of metabolites implied that 14C-prothiofos primarily underwent cleavage of the P-Oaryl linkage or hydroxylation of the aryl methyl group similarly to other organophosphorus pesticides in plants. These metabolites have been detected in human urine [43] and in rats [4].

environmental-analytical-chemistry-cotton-seed-oils

Figure 6: Proposed pathways for degradation of 14C-prothiofos in cotton seed oils.

The degradation products of 14C-prothiofos in refined cotton seed oil (hexane extract) were illustrated in Table 4. In addition to the parent compound (I), prothiofos oxon (II), O-ethyl phosphorothioate (III), O-ethyl phosphoric acid (VI) and O-ethyl S-propyl phosphorodithioate (VII) were identified as the main degradation products, besides to one unknown compound. Their amounts decreased during the refining step. Abdel-Gawad and Hegazi [20] studied the fate of 14C-ethyl prothiofos insecticide in canola seeds and oils and found the presence of the parent compound together with three metabolites which were identified as prothiofos oxon, O-ethyl phosphorothioate and O-ethyl S-propyl phosphorothioate, besides one unknown compound.

Substances Rf in systems Metabolites concentration (ppm)
Sys.1 Sys.2 Sys.3 Crude oil A D
Prothiofos (I) 0.56 0.47 0.21 0.067 0.03 0.010
Prothiofosoxon (II) 0.01 0.60    0.36 0.286 0.19 0.060
O-ethyl phosphorothioate (III) 0.21 0.52 0.45 0.192 0.07 0.010
O-ethyl S-propyl phosphorothioate (IV)  0.43 0.41 0.32 0.174 0.04 0.000
Des-propylthioprothiofos (V) 0.54 0.49 0.30 0.058 0.00 0.000
O-ethyl phosphoric acid (VI) 0.77 0.65 0.60 0.066 0.03 0.007
O-ethyl S-propyl phosphordithioate (VII) 0.68 0.65 0.35 0.000 0.05 0.010
Unknown 0.85 0.45 0.65 0.086 0.01 0.007

Table 4: 14C- prothiofos and its degradation products in cotton seed oil after subjecting to commercial processing procedures.

Bioavailability in rats

Elimination and distribution of 14C-residues following feeding rats with the extracted cotton seeds (cake containing 12 μg equivalents per rat) for 72 hr are shown in Figure 7. It was observed that the major part of radioactivity was eliminated via expired air (49%) while radioactivity in urine and feces accounted for 34% and 9.7% of the ingested 14C-activity, respectively. Appreciable amount of 14C-residue (6.1%) were also detected in liver, kidney, heart, spleen, lung, blood and fat of treated rats. Bound 14C-prothiofos in cotton seeds proved to be highly bioavailable to rats. Similar observations were reported from studies on the bioavailability of soybean bound residues of pirimiphos-methyl [44] chloropyrofos [45] dichlorovos [46] and fenitrothion [47]. Also, the data obtained are in line with many other studies which indicate a moderate to high bioavailability of grain-bound 14C-pesticide residues in experimental animals [20,48-50].

environmental-analytical-chemistry-prothiofos-residues

Figure 7: Excretion and distribution of 14C-bound prothiofos residues in cotton seeds after feeding to rats for 72 hours.

Chromatographic analysis of urine extracts (chloroform layer) revealed the presence of four metabolites, which were identified as prothiofos oxon (II), O-ethyl phosphorothioate (III), O-ethyl S-propyl phosphorothioate (IV) and O-ethyl phosphoric acid (VI) as free metabolites in addition to one unknown substance. Compound 2 4-dichlorophenole (VIII) was detected as a conjugated metabolite and its concentration was 0.12 μg Table 5. This result agrees with some previous reports [20,51,52] which noted that in adult rats, prothiofos gave prothiofos oxon, O-ethyl phosphoric acid and 2,4-dichlorophenole. Fenitrothion insecticide was converted to phosphorothioate and dimethylphosphate. Malathion insecticide is converted to malathion mono-carboxylic acid and malathion dicarboxylic acid. Carbofuran, on the other hand, gave carbofuran phenol and 3-hydroxy carbofuran.

Metabolites Rf in systemsa Urine extract
Sys.1 Sys.2 Sys.3 Chloroform layer (µg)a
Prothiofosoxon (II) 0.01 0.60 0.36 1.50 0.11±
O-ethyl phosphorothioate (III) 0.21 0.52 0.45 0.09±1.02
O-ethyl S-propyl phosphorothioate (IV)  0.43 0.41 0.32 0.07±1.01
O-ethyl phosphoric acid (VI) 0.77 0.65 0.60 0.23 0.02±
Unknown 0.85 0.45 0.65 0.01±0.20

Table 5: 14C- Prothiofos and its degradation products in urine of male rats fed with cake containing prothiofos bound residues after 72 hours.

Conclusion

In conclusion; the joint action of deltamethrin and dimilin in combination with the organophosphate prothiofos was studied in cotton plants from Egypt by using radiolabeled insecticide. Prothiofos exhibited an antagonism with deltamethrin as well as Dimilin. The residues of prothiofos in cotton seed oil included, other than the parent compound, both free and conjugated metabolites. The major part of the insecticide residues (about 89%) could be eliminated during processing of the oil. Refining processes led to progressive degradation of the parent insecticide. The deodorization was found to be an efficient step for reduction of the insecticide residues. It was observed that alkali treatment and bleaching leads to significant losses of the pesticide residues in oil as well. The removal efficiency during the refining processes seems to depend upon the nature of the residue present. Data obtained emphasize the importance of studying the effect of oil refinement on reduction and/or elimination of aged pesticide residues in edible oils. The present results obtained indicate that cotton seedbound 14C-prothiofos residues are highly bioavailable to rats. It should be stressed that the presence of bound pesticide residues can no longer be ignored in the evaluation of toxicological hazards.

Acknowledgements

The authors are grateful to Prof. Dr. Bahira Hegazi, professor of Applied Organic Chemistry Department, National research Centre, for her kindness and support.

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