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Pesticide Metabolism
Zbl. Mikrobiol. 137 (1982), 325-328 [From Atomic Energy Establishment, Cairo, Egypt]
Pesticide Metabolism. III. Fate of 14C-Cyolane by Soil Fungi M. R. E. BAHlG, Y. A. EL-ZAWAHRY, and D. WAFA With one figure
Summary The metabolism of Cyolane in soil fungi, i. e., Rhizoctonia solani, Penicillium chrysogenum, and Trichoderma viride was studied, using 14C-Cyolane, labelled in the imino-carbon position. At least 6 metabolites could be separated in addition to the parent compound. Four metabolites were identified as the imino-dithiolane ring, thiocyanate ion, conjugated metabolite, and mono or desethylated Cyolane. The imino-dithiolane ring constituted the major metabolite, especially in Penicillium chrysogenum (47.5 % of the total metabolites).
Zusammenfassung Beim Abbau von an der Imino-Carbon-Position markiertem 14C-Cyolan (Phospholan) durch die Bodenpilze Rhizoctonia solani, Penicillium chrysogenum und Trichoderma viride wurden neben der Ausgangssubstanz mindestens sechs Metabolite festgestellt. Vier Metabolite wurden identifiziert als Imino-Dithiolan-Ring, das Thiozyanat-Ion, ein konjugierter Metabolit und mono- oder desathyliertes Cyolan. Der Imino-Dithiolan-Ring ist der Hauptmetabolit, insbesondere bei P. chrysogenum (47.5 % aller Metabolite).
Cyolane (Phospholan) , 2-diethoxy phosphinyl imino-1.3-dithiolane, is a systemic organo-phosphorus insecticide, widely used for the control of white flies, lygus bugs, tribs, leaf miners, flea beetles, and leaf-feeding larvae. In Egypt Cyolane has been applied and accepted for controlling the cotton leaf worm Spodoptera littoralis (MOSTAFA et al. 1974, SALAMA et al. 1973, 1974, 1975) and the bean fly, a wide-spread agromyrrid pest on beans and other legumes. The metabolic fate of Cyolane, labelled in the imino-carbon position, has been studied in the rat and cotton leaf worm (BAHIG and WAFA 1980, and KAPOOR et al. 1977). The aim of the present work was to study the fate of 14C-Cyolane as affected by some common soil fungi, i.e., Rhizoctonia solani, Penicillium chrysogenum, and Trichoderma viride.
Materials and Methods HC-Cyolane, labelled in the imino-carbon position, was kindly supplied by American Cyanamide Company. 30 ml aliq)1ots of sterilized Czapek-Dox medium at pH 5 were added to HC-labelled Cyolane (1.5 mg/lOO ml), specific activity 5,uCi/mg, and seven-day-old culture of either Rhizoctonia 8olani, Penicillium chrysogenum or Trichoderma viride was incubated at 28°C for 2 weeks. Control flasks were prepared free from fungi, but with all other conditions maintained.
326
M.
:R. E.
BAHIG
et al.
At the end of the incubation period the developed mats were filtered and thoroughly washed with distilled water for several times. The radioactivity in the combined clear media was measured direotly, using liquid scintillation counterNE 8310/1/2 (with external standards, scintillator based on dioxane). Mycelial mats were homogenized, and an aliquot was counted by liquid scintillation counter. Isolation and identification of metabolites For determination of metabolites and unchanged parent, the aqueous fractions were coneentr'ated in a rotary evaporator. The concentrates were separated, using paper chromatography (Whatman No. I filter paper); thin· layer chromatography on precoated plates (5 X 20 cm) with 0.25 mm silica gel 60 (Merck) was also used. The imino-dithiolane ringl) and thiocyanate ions (KSCN) were used as authentics, localization was done by spraying with special reagent (sodium azide), specific for sulphur-containing compounds (LEDERER and LEDERER 1957) . Thin-layer plates and paper chromatogr ams were scanned with G. M. radioscanner.
Results and Discussion Table I shows the recovery and distribution of radioactive metabolites between the soil fungi mycelia and media. The retained radioactivity in mycelia was 2-14 % of the applied dose in the different species used, and this percentage is comparable to those obtained with other commonly known organophosphorus insecticides (ZAYED et al. 1964, MOSTAFA et al. 1972a, and MOSTA,FA et al. 1972b). Chromatographic separation of the hydrolytic products in the different fungi media revealed the presence of at least 6 metabolites in addition to the parent compound. Table 1. Distribution of 14C-Cyolane degradation products on three soil fungi after 14 days of growth at 28°C Organism
Rhizoctonia solani TriclwdB1'ma vil'ide
Penicillium chrysogenum
Recovery (%) Medium
Mycelium
92.00 98.00 85.93
8.00 2.00 14.07
Table 2 presents the percentage of the different radioactive degradation products of 14C-Cyolane from the three fungi species used. It is olear that Penicillium chrysogenum is the most active organism, since 93 % of the radioactivity in the media were hydrolytic metabolities of Cyolane and only 7 % was unchanged insecticide. This high activity of Penicillium was reported by MOSTA,FA et al. (1972b) ; they stated that Penicillium notatum could metabolize 76 % of the applied Malathion after 10 days of incubation. Radioactive measurements of aqueous fraction of Trichoderma viride contained 66 % of the applied Cyolane dose as hydrolytic products. Rhizoctonia solani was found to be the least active organism, 55 % of the radioacting in the aqueous fraction was identified as unchanged Cyolane (MosTAFA, et al. 1972b). 1) Kindly supplied by American Cyanamide Company
327
Pesticide Metabolism Table 2. Percentage1 ) and Rr values of He-labelled metabolites Substance2)
Percentage
R.8olani
Rf
P. chrysogenum
T. viride
System A
B
Cyolane
55.5
7
34.00
0.88
0.94
Mono or desethylated Cyolane
10.5
4
8.5
0.8
0.82
Imino-dithiolane ring
8.0
47.5
28.5
0.45
0.75
Thiocyanate ion
9.5
16.5
14
0.11
0.42
Conjugated metabolites
15.5
18.0
12
0.0
0.12
Unknown!
1
5
3
Unknown II
0.25
2
0.36
1) Percentage of metabolites was estimated from TLC and paper chromatographic studies (100% = total hydrolytic products). 2) Spots of reference compOlmds and metabolic products were located on chromatograms with sodium azide solution. A B
= =
Benzene : Methanol: Acetic acid (79: 14: 7) Benzene: Methanol: Formic acid (25: 25: 1)
From the characterization of the different hydrolytic products, it is clear that the P-N bond of Cyolane is hydrolyzed, leading to the imino-dithiolane ring. Cleavage of this ring may occur to give thiocyanate ion (WlLLIAMS 1959), or the ring may form conjugated metabolite (Fig. 1). The percentage of formation of the thiocyanate ion and the conjugate is comparatively lower in these organisms than in the rat (BA,;IIIG 1980). The conjugated metabolite, on separation and hydrolysis, using 8 N H 2S04 at 40°C for 8 hours and extraction with ether, we got by chromatographic separation and using standards, the imino-dithiolane ring and thiocyanate ion, due to the partial cleavage of the ring (Fig. 1). In addition, mono or desethylated Cyolane may be present in comparatively low percentage (4-10 %). There are 2 other unknown metabolites (unknown I and II) that could not be identified due to their low percentage. It can be concluded that nearly all the Cyolane metabolites, obtained in this study, are similar to those reported in our previous investigation in the rat and cotton leaf worm, but in different metabolic rates (BAIDG and WAFA 1980a, b).
II: " / 5]
01
(Cz H:;Iz-PtN=c " 1
I'e - cyalone
S
HYDROLYSIS
t
* / S]
- - - HN=C" S
J
Imino dlihiolune ring PRESENT III THE NEOlA
""".I""" - IS eN!
Fig. 1. Possible metabolites of HC-Iabelled Cyo1ane by soil fungi.
I1INOR Conjugaled mel.holites
~
328
M.
E. E. BAHIG et aI., Pesticide Metabolism
References BAHIG, M. E. E., and WAFA, D.: Pesticide metabolism. I. Fate of 14C.Cyolane in rat. Isotope and Rad. Res. (accepted for publication). - - Pesticide metabolism. II. Degradation of 14C·Cyolane in the cotton leaf worm. Die Naturwissenschaften (in Press). KAPOOR, I. P., and ROGER, C. B.: Absorption, excretion, and metabolism of Cyolane (Phospholan), systemic insecticide (diethoxy phospholan) dithioimidocarbonic acid, cyclic ethylene ester, in the rat. J. Agric. Food Chem. 25 (1977), 413-417. LEDERER, E., and LEDERER, M. A.: Review of principles and application. Elsevier Publ. Comp., Amsterdam 1957, 315. MOSTAFA, I. Y., FAKHR, I. M. I., BAHIG, M. R. E., and EL-ZAWAHRY, Y. A.: Metabolism of organophosphorus insecticides. XIII. Degradation of Malathion by Rhizobium spp. Arch. MikrobioI. 86 (1972a), 221-224. BAHIG, M. R. S., FAKHR, I. M. I., and ADAM, Y.: Metabolism of organophosphorus insecticides. XIV. Malathion breakdown by soil fungi. Z. Naturforsch. 27 (1972b), 1115-1116. FAKHR, I. M. I., and EL-ZAWAHRY, Y. A.: Metabolism of organophosphorus insecticides. XV. Translocation and degradation of 32P-malathion in bean and cotton plants. "Comparative studies of food and environmental contamination". International Atomic Energy Agency, Vienna 1974, 385-392. SALAMA, A. M., MOSTAFA, I. Y., and EL-ZAWAHRY, Y. A.: Insecticides and soil microorganisms. I. Effect of dipterex on growth of Rhizobium legumino8arum and Rhizobium trifolii as influenced by temperature, pH and type of nitrogen. Acta BioI. Acad. Sci. Hung. 24 (1973), 25-30. - - Insecticides and soil microorganisms. II. Effect of Dipterex on nodule formation in broad bean and clover plant under different manurial treatments. Acta BioI. Acad. Sci. Hung. 25 (1974), 239-246. - - Insecticides and soil microorganisms. III. Fate of 14C-labelled Dipterex as affected by two nodule-forming Rhizobium spp. and roots of their respective leguminuous host plants. Acta BioI. Acad. Sci. Hung. 26 (1975),1-7. WILLIAMS, D. T.: Detoxication mechanisms. New York 1959. ZAYED, S. M. A. D., MOSTAFA, I. Y., and HASSAN, A.: Organische P-haltige Insecticide im StoffwechseI. VII. Umwandlung von 32P-markiertem Dipterex durch Microorganismen. Arch. MikrobioI. 51 (1964),188-121. Authors' Address: M. R. E. BAHIG, Y. A. EL-ZAHWAHRY, and D. WAFA, Middle Eastern Regional Radioisotope Center for Arab Countries, Malaeb El Gamma Street, Dokki, Cairo, Egypt.
Pesticide Metabolism. III. Fate of 14C-Cyolane by Soil Fungi M. R. E. BAHlG, Y. A. EL-ZAWAHRY, and D. WAFA With one figure
Summary The metabolism of Cyolane in soil fungi, i. e., Rhizoctonia solani, Penicillium chrysogenum, and Trichoderma viride was studied, using 14C-Cyolane, labelled in the imino-carbon position. At least 6 metabolites could be separated in addition to the parent compound. Four metabolites were identified as the imino-dithiolane ring, thiocyanate ion, conjugated metabolite, and mono or desethylated Cyolane. The imino-dithiolane ring constituted the major metabolite, especially in Penicillium chrysogenum (47.5 % of the total metabolites).
Zusammenfassung Beim Abbau von an der Imino-Carbon-Position markiertem 14C-Cyolan (Phospholan) durch die Bodenpilze Rhizoctonia solani, Penicillium chrysogenum und Trichoderma viride wurden neben der Ausgangssubstanz mindestens sechs Metabolite festgestellt. Vier Metabolite wurden identifiziert als Imino-Dithiolan-Ring, das Thiozyanat-Ion, ein konjugierter Metabolit und mono- oder desathyliertes Cyolan. Der Imino-Dithiolan-Ring ist der Hauptmetabolit, insbesondere bei P. chrysogenum (47.5 % aller Metabolite).
Cyolane (Phospholan) , 2-diethoxy phosphinyl imino-1.3-dithiolane, is a systemic organo-phosphorus insecticide, widely used for the control of white flies, lygus bugs, tribs, leaf miners, flea beetles, and leaf-feeding larvae. In Egypt Cyolane has been applied and accepted for controlling the cotton leaf worm Spodoptera littoralis (MOSTAFA et al. 1974, SALAMA et al. 1973, 1974, 1975) and the bean fly, a wide-spread agromyrrid pest on beans and other legumes. The metabolic fate of Cyolane, labelled in the imino-carbon position, has been studied in the rat and cotton leaf worm (BAHIG and WAFA 1980, and KAPOOR et al. 1977). The aim of the present work was to study the fate of 14C-Cyolane as affected by some common soil fungi, i.e., Rhizoctonia solani, Penicillium chrysogenum, and Trichoderma viride.
Materials and Methods HC-Cyolane, labelled in the imino-carbon position, was kindly supplied by American Cyanamide Company. 30 ml aliq)1ots of sterilized Czapek-Dox medium at pH 5 were added to HC-labelled Cyolane (1.5 mg/lOO ml), specific activity 5,uCi/mg, and seven-day-old culture of either Rhizoctonia 8olani, Penicillium chrysogenum or Trichoderma viride was incubated at 28°C for 2 weeks. Control flasks were prepared free from fungi, but with all other conditions maintained.
326
M.
:R. E.
BAHIG
et al.
At the end of the incubation period the developed mats were filtered and thoroughly washed with distilled water for several times. The radioactivity in the combined clear media was measured direotly, using liquid scintillation counterNE 8310/1/2 (with external standards, scintillator based on dioxane). Mycelial mats were homogenized, and an aliquot was counted by liquid scintillation counter. Isolation and identification of metabolites For determination of metabolites and unchanged parent, the aqueous fractions were coneentr'ated in a rotary evaporator. The concentrates were separated, using paper chromatography (Whatman No. I filter paper); thin· layer chromatography on precoated plates (5 X 20 cm) with 0.25 mm silica gel 60 (Merck) was also used. The imino-dithiolane ringl) and thiocyanate ions (KSCN) were used as authentics, localization was done by spraying with special reagent (sodium azide), specific for sulphur-containing compounds (LEDERER and LEDERER 1957) . Thin-layer plates and paper chromatogr ams were scanned with G. M. radioscanner.
Results and Discussion Table I shows the recovery and distribution of radioactive metabolites between the soil fungi mycelia and media. The retained radioactivity in mycelia was 2-14 % of the applied dose in the different species used, and this percentage is comparable to those obtained with other commonly known organophosphorus insecticides (ZAYED et al. 1964, MOSTAFA et al. 1972a, and MOSTA,FA et al. 1972b). Chromatographic separation of the hydrolytic products in the different fungi media revealed the presence of at least 6 metabolites in addition to the parent compound. Table 1. Distribution of 14C-Cyolane degradation products on three soil fungi after 14 days of growth at 28°C Organism
Rhizoctonia solani TriclwdB1'ma vil'ide
Penicillium chrysogenum
Recovery (%) Medium
Mycelium
92.00 98.00 85.93
8.00 2.00 14.07
Table 2 presents the percentage of the different radioactive degradation products of 14C-Cyolane from the three fungi species used. It is olear that Penicillium chrysogenum is the most active organism, since 93 % of the radioactivity in the media were hydrolytic metabolities of Cyolane and only 7 % was unchanged insecticide. This high activity of Penicillium was reported by MOSTA,FA et al. (1972b) ; they stated that Penicillium notatum could metabolize 76 % of the applied Malathion after 10 days of incubation. Radioactive measurements of aqueous fraction of Trichoderma viride contained 66 % of the applied Cyolane dose as hydrolytic products. Rhizoctonia solani was found to be the least active organism, 55 % of the radioacting in the aqueous fraction was identified as unchanged Cyolane (MosTAFA, et al. 1972b). 1) Kindly supplied by American Cyanamide Company
327
Pesticide Metabolism Table 2. Percentage1 ) and Rr values of He-labelled metabolites Substance2)
Percentage
R.8olani
Rf
P. chrysogenum
T. viride
System A
B
Cyolane
55.5
7
34.00
0.88
0.94
Mono or desethylated Cyolane
10.5
4
8.5
0.8
0.82
Imino-dithiolane ring
8.0
47.5
28.5
0.45
0.75
Thiocyanate ion
9.5
16.5
14
0.11
0.42
Conjugated metabolites
15.5
18.0
12
0.0
0.12
Unknown!
1
5
3
Unknown II
0.25
2
0.36
1) Percentage of metabolites was estimated from TLC and paper chromatographic studies (100% = total hydrolytic products). 2) Spots of reference compOlmds and metabolic products were located on chromatograms with sodium azide solution. A B
= =
Benzene : Methanol: Acetic acid (79: 14: 7) Benzene: Methanol: Formic acid (25: 25: 1)
From the characterization of the different hydrolytic products, it is clear that the P-N bond of Cyolane is hydrolyzed, leading to the imino-dithiolane ring. Cleavage of this ring may occur to give thiocyanate ion (WlLLIAMS 1959), or the ring may form conjugated metabolite (Fig. 1). The percentage of formation of the thiocyanate ion and the conjugate is comparatively lower in these organisms than in the rat (BA,;IIIG 1980). The conjugated metabolite, on separation and hydrolysis, using 8 N H 2S04 at 40°C for 8 hours and extraction with ether, we got by chromatographic separation and using standards, the imino-dithiolane ring and thiocyanate ion, due to the partial cleavage of the ring (Fig. 1). In addition, mono or desethylated Cyolane may be present in comparatively low percentage (4-10 %). There are 2 other unknown metabolites (unknown I and II) that could not be identified due to their low percentage. It can be concluded that nearly all the Cyolane metabolites, obtained in this study, are similar to those reported in our previous investigation in the rat and cotton leaf worm, but in different metabolic rates (BAIDG and WAFA 1980a, b).
II: " / 5]
01
(Cz H:;Iz-PtN=c " 1
I'e - cyalone
S
HYDROLYSIS
t
* / S]
- - - HN=C" S
J
Imino dlihiolune ring PRESENT III THE NEOlA
""".I""" - IS eN!
Fig. 1. Possible metabolites of HC-Iabelled Cyo1ane by soil fungi.
I1INOR Conjugaled mel.holites
~
328
M.
E. E. BAHIG et aI., Pesticide Metabolism
References BAHIG, M. E. E., and WAFA, D.: Pesticide metabolism. I. Fate of 14C.Cyolane in rat. Isotope and Rad. Res. (accepted for publication). - - Pesticide metabolism. II. Degradation of 14C·Cyolane in the cotton leaf worm. Die Naturwissenschaften (in Press). KAPOOR, I. P., and ROGER, C. B.: Absorption, excretion, and metabolism of Cyolane (Phospholan), systemic insecticide (diethoxy phospholan) dithioimidocarbonic acid, cyclic ethylene ester, in the rat. J. Agric. Food Chem. 25 (1977), 413-417. LEDERER, E., and LEDERER, M. A.: Review of principles and application. Elsevier Publ. Comp., Amsterdam 1957, 315. MOSTAFA, I. Y., FAKHR, I. M. I., BAHIG, M. R. E., and EL-ZAWAHRY, Y. A.: Metabolism of organophosphorus insecticides. XIII. Degradation of Malathion by Rhizobium spp. Arch. MikrobioI. 86 (1972a), 221-224. BAHIG, M. R. S., FAKHR, I. M. I., and ADAM, Y.: Metabolism of organophosphorus insecticides. XIV. Malathion breakdown by soil fungi. Z. Naturforsch. 27 (1972b), 1115-1116. FAKHR, I. M. I., and EL-ZAWAHRY, Y. A.: Metabolism of organophosphorus insecticides. XV. Translocation and degradation of 32P-malathion in bean and cotton plants. "Comparative studies of food and environmental contamination". International Atomic Energy Agency, Vienna 1974, 385-392. SALAMA, A. M., MOSTAFA, I. Y., and EL-ZAWAHRY, Y. A.: Insecticides and soil microorganisms. I. Effect of dipterex on growth of Rhizobium legumino8arum and Rhizobium trifolii as influenced by temperature, pH and type of nitrogen. Acta BioI. Acad. Sci. Hung. 24 (1973), 25-30. - - Insecticides and soil microorganisms. II. Effect of Dipterex on nodule formation in broad bean and clover plant under different manurial treatments. Acta BioI. Acad. Sci. Hung. 25 (1974), 239-246. - - Insecticides and soil microorganisms. III. Fate of 14C-labelled Dipterex as affected by two nodule-forming Rhizobium spp. and roots of their respective leguminuous host plants. Acta BioI. Acad. Sci. Hung. 26 (1975),1-7. WILLIAMS, D. T.: Detoxication mechanisms. New York 1959. ZAYED, S. M. A. D., MOSTAFA, I. Y., and HASSAN, A.: Organische P-haltige Insecticide im StoffwechseI. VII. Umwandlung von 32P-markiertem Dipterex durch Microorganismen. Arch. MikrobioI. 51 (1964),188-121. Authors' Address: M. R. E. BAHIG, Y. A. EL-ZAHWAHRY, and D. WAFA, Middle Eastern Regional Radioisotope Center for Arab Countries, Malaeb El Gamma Street, Dokki, Cairo, Egypt.