This article was downloaded by: [University of Glasgow] On: 05 January 2015, At: 06:15 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Bioavailability to 14
rats of bound [ C] pirimiphos‐methyl in stored wheat a
b
S.U. Khan , S. Kacew & W. Matthews
c
a
Centre for Land and Biological Resources Research , Agriculture Canada , Ottawa, Ontario, K1A 0C6, Canada b
Department of Pharmacology, School of Medicine , University of Ottawa , Ottawa, Ontario, K1H 8M5, Canada c
ADAS Central Science Laboratory , Ministry of Agriculture, Fisheries and Food , Slough, Berks, SL2 7HJ, Great Britain Published online: 13 Nov 2008.
To cite this article: S.U. Khan , S. Kacew & W. Matthews (1992) 14
Bioavailability to rats of bound [ C] pirimiphos‐methyl in stored wheat, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 27:4, 355-367 To link to this article: http://dx.doi.org/10.1080/03601239209372786
Downloaded by [University of Glasgow] at 06:15 05 January 2015
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
J. ENVIRON. SCI. HEALTH, B27(4), 355-367 (1992)
BIOAVAILABILITY TO RATS OF BOUND [14C] PIRIMIPHOS-METHYL IN STORED WHEAT
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Key words: Wheat, 14 C-Pirimiphos-methyl, Bound residues Bioavailability S.U. Khan, S. Kacew+, and W. Matthews ++ Centre for Land and Biological Resources Research, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 + Department of Pharmacology, School of Medicine University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 ++ ADAS Central Science Laboratory, Ministry of Agriculture, Fisheries and Food, Slough, Berks, Great Britain SL2 7HJ
ABSTRACT Stored wheat t r e a t e d with r a d i o l a b e l l e d pirimiphos-methyl ( 0 - 2 - d i e t h y l amino-6-methyl-pyrimidin-4-yl 0,0-dimethyl p h o s p h o r o t h i o a t e ) formed bound
(nonextractable) 14C residues. Supercritical fluid extraction, gas chromatography and mass spectrometric techniques were used to identify and quantitate the 14C bound residues in wheat grains. The amount of bound 14 C residues present after 28 weeks of storage was about 9.9% of the applied radioactivity. Pirimiphos-methyl accounted for 80% of the bound residue. Grain-bound residues were fed to rats for 5 days. After a total period of 8 days a substantially large percentage of the administered bound14C residues (72.9%) was eliminated in urine while feces contained only 17.9%. Bound pirimiphos-methyl in wheat grain was metabolized in rats by processes involving hydrolysis, N-dealkylation and O-demethylation. The results indicate that wheat-bound residues of pirimiphos-methyl are highly bioavailable to the rat and may possess a toxicological potential as manifested by a significant reduction in body weight gain.
355 Copyright © 1992 by Marcel Dekker, Inc.
356
KHAN, KACEW, AND MATTHEWS INTRODUCTION Pirimiphos-methyl (0-2-diethylamino-6-methyl-pyrimidin-4-yl O,0-dimethyl
phophorothioate),
"Actellic' 1 -^ i s a broad spectrum i n s e c t i c i d e used t o
control a vide range of stored grain p e s t s .
I t has low mammalian t o x i c i t y and
i s applied d i r e c t l y to grains, where i t controls pests by both contact and vapor a c t i v i t y . I t has been shown that under normal conditions for grain
Downloaded by [University of Glasgow] at 06:15 05 January 2015
storage, pirimiphos-methyl i s degraded only slowly and p e r s i s t s (= 70%) on stored grains for longer than 24 weeks (Leahey and Curl, 1982).
A half-life
of 43 weeks for piriraiphos-methyl on wheat grain maintained under normal storage conditions has been reported (Bengston e t a l . , 1975). Wheat grain 14 o treated with I C] pirimiphos-methyl and stored a t 21 C for 8 months was found t o contain 78% of the r a d i o a c t i v i t y recoverable as parent compound (Rowlands, 1975).
In a subsequent study (Rowlands and Wllkin, 1975) i t was
observed t h a t approximately i o % of the r a d i o a c t i v i t y was bound to wheat and was unextractable except by digesting. Rowlands, 1980 reported about 40% loss 14 0 of [ C] pirimiphos-methyl from wheat stored a t 20 C a f t e r 6 months. I t 14 was also observed that about 15% of the applied C was not recoverable by solvent extraction. Leahey and Curl, 1982 also reported t h a t wheat t r e a t e d 14 with [ C] pirimiphos-methyl and stored for 6 months contained up to 13% bound r a d i o a c t i v i t y of the i n i t i a l l y applied 14 C. In the foregoing studies the nature of the bound residues was not determined. The Joint FAO/IAEA Division of Huclear Techniques in Food and Agriculture i n i t i a t e d a research programme i n 1986 to evaluate the safety of bound pesticide residues in food producing crops and stored grains.
The study
emphasized b i o a v a i l a b i l i t y and t e s t i n g for toxicological p o t e n t i a l i n r a t s fed materials containing bound p e s t i c i d e residues.
I n a previous publication we
reported that the bound residues in stored wheat treated with the i n s e c t i c i d e deltamethrin were highly bioavailable to the r a t (Khan e t a l . , 1990).
We now
report the r e s u l t s of our investigation on the b i o a v a i l a b i l i t y and metabolism in the r a t of bound
C residues in stored wheat treated with [ C] 14 C residues in stored wheat was
pirimiphos-methyl. The nature of bound also determined.
MATERIALS AND METHODS Chemicals Pirimiphos-methyl l a b e l l e d with
14
C i n t h e 2 p o s i t i o n of t h e pyrimidine
ring ( s p e c i f i c a c t i v i t y 57 mCi/mmol) was purchased from Tracerco ICI Ltd., Cleveland, Ohio, USA. The a n a l y t i c a l grade m a t e r i a l was obtained from B r i t i s h
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
357
Greyhound Chromatography and Allied Chemicals Ltd. and had a purity of 98.1%.
Downloaded by [University of Glasgow] at 06:15 05 January 2015
All solvents were of pesticide grade and were used as received.
N
T
=^
N(C2H5)2
Pirimiphos-Methyl
Grain Treatment and Storage Pesticide-free English wheat of the variety "Mission" with an i n i t i a l moisture content of 13.2% was treated with [14C] pirimiphos-methyl (100 uCi/kg) and technical pirimiphos-methyl to achieve a concentration of 4.5 ppm. The insecticide was dissolved in diethyl ether and applied dropwise to 500 g batches of grain whilst this was being stirred. The treated grain was placed in a separate glass j a r which was sealed and tumbled end over end for 1 hour. The grain was spread on a tray in a fume hood draught to allow the ether to evaporate and was then returned to the j a r which was resealed and tumbled for a further two hours. The storage temperature of 15°C was chosen as an estimate of the mean temperature attained by a field scale bulk of grain. The grain was stored for 28 weeks. The grain used as control was similarly stored except i t was not treated with the insecticide. Extraction of Stored Grains The wheat grain was extracted with methanol using the method developed by the Committee for Analytical Methods for Residues of Pesticides and Veterinary Products in Foodstuffs (Anon., 1980). An additional sonication step was used to ensure complete extraction. The extracted material after drying at room temperature contained 1.5% H,0.
358
KHAN, KACEW, AND MATTHEWS
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Grain-Bound Residues A portion of the extracted grain was combusted to 14 CO. to determine 14 14 the total bound C. A second portion was used to release C-bound residues by supercritical fluid extraction (SFE), Fig. 1. The supercritical fluid extractor (Suprex Model SFE-5O, Suprex Corp., Pittsburgh, PA) consisted of a 250-mL syringe pump, a control module for control of the SFE system, an extraction oven, a 5-mL extraction vessel containing 2g of extracted wheat grains, and a four-part valve connected with the outlet restriction (fused s i l i c a tubing, 50-um i.d.) that was inserted into glass tubes containing methanol. The supercritical fluid used was CO, and the extractions were performed a t a pressure of 280 atm. and temperature 110°C. The flow rate of mobile phase CO. was 1 mL/min. The extraction was carried out for 1 h and the material was collected in methanol. The remaining portion of the extracted grain was used for feeding rats (Fig. 1). Feeding Study Four male Sprague-Dawley rats weighing approximately 210g each were housed individually in metabolism cages which permitted separate collection of urine and feces. The rats were conditioned for 4 days t o a daily diet of Purina Laboratory Chow. The feed was then replaced for 5 days with the methanol-extracted material containing 14 C bound residues (22,105dpm/g). Four control rats were fed the extracted untreated wheat material. Total feed consumed by each rat in 5 days was about 70g. The rats were then maintained on regular rat chow for an additional 3 days. The urine and feces were collected daily for 8 days. The weight of each rat was also recorded on a daily basis during the course of the experiement. After 8 days the animals were sacrificed by decapitation, selected organs were excised and weighed, and a portion of these organs was frozen in liquid nitrogen for radioassay (Fig. 2). Preparation of Samples and Analysis ( i ) Wheat grain. The SFE extract was concentrated to a small volume and analyzed by thin layer chromatography (TLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). ( i i ) Feces. The feces from rats were extracted with methanol until further extraction did not result in any extractable 14 C. The methanol extract was concentrated to a small volume,
Downloaded by [University of Glasgow] at 06:15 05 January 2015
w o
Wheat Grain
"
I
1
Treat with Radioiabeled Pirimiphos-methyl (100 uC1/ka, 4.5 ppm)
[| a
Store at 15*C *C for 28 Weeks
«
Ground and Extract with Methanol
I
I
Extractabie l4 C-Res1dues
Nonextractable (8ound) "C-Res1dues
, I
L JL
, I
Combustion Supercritical Fluid Feeding to Rats 14 C0, l4 Extraction (B1oava1lab1Hty of bound "C) (Total bound C) (Identification of bound l4C) FIG. 1. Schematic diagram for the grain treatment with [I4C] p1r1m1phos-methyl and determination of bound M C residues.
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Methanol Extracted Stored Wheat Containing Bound I4C Feed to Rats for 5 Days (70 g/rat/5 days) Collect Urine and Feces, Record Weight of Each Rat on a Dally Basis f o r 9 Days
1 Urine
Organs TC
Combustion l4 CO,
Feces Lie
Extract with Methanol
1
1
Hydrolysis
Extractable
Nonextractable
Methylatlon
T
1 GC, GC-MS
GC, GC-MS
I TLC, GC GC-MS
>jC
I LSC
Combustion l4 C0 (Total'l4C)
FIG. 2. Schematic diagram for the Identification of bound 14C residues 1n stored wheat treated with [ 14 C] p1r1m1phos-methyl.
o
3
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
361
Downloaded by [University of Glasgow] at 06:15 05 January 2015
an aliquot was radioassayed and the remaining portion was analyzed by TLC, GC and GC-MS. The insoluble fecal material containing bound 14 C residues was air-dried and combusted to 14 CO for the determination of the 14 14 unextractable C residues. Total C content of feces was obtained by summation of the two values, ( i i i ) Urine. An aliquot of urine was radioassayed by scintillation counting. The remaining urine was brought to just dryness under a stream of dry nitrogen, dissolved in methanol, and analyzed by TLC, GC and GC-MS. Samples of SFE extract of control wheat grain, feces and urine from rats fed control wheat grain were processed as described for the treated samples. Radioassav Radioactivity of appropriate aliquots of the SFE extracts, urine and other liquid samples was measured in a Packard Model 3320 scintillation spectrometer. The solid samples (ground wheat material, feces, animal tissues) were assayed by combusting in a Packard sample oxidizer, Model 306, followed by liquid scintillation counting (LSC) with an external standard and correcting data for quenching. Radioactive spots or zones on TLC plates were located and counted by a Berthold Beta camera LB292. For purpose of quantitation each radioactive region was scraped from TLC plate, a portion was placed into a scintillation vial for direct counting. TLC plates were also examined under ultraviolet light. Chromatographic Procedures ( i ) TLC. Precoated s i l i c a gel GF chromatopiates, with 1.0mm gel thickness (Analtech, Inc., Newark, DE) were used for thin-layer chromatography. The solvent system used was toluene-hexane-acetic acid (15:3:2). Aliquots of the radioactive extracts were chromatographed on a 20 x 20cm plate. Authentic standard of pirimiphos-methyl was also chromatographed on the same TLC plate for preliminary identification. The radioactivity zones were located on the developed plate, each radioactive zone was scraped off the plate, extracted with methanol, and analyzed by LSC to determine the total extractable 14 C. The extractable material was then further analyzed by GC and GC-MS. ( i i ) GC-MS. Gas chromatography was performed using a Varian Model 6000 fitted with a thermionic specific detector and a s i l i c a Megabore column
Downloaded by [University of Glasgow] at 06:15 05 January 2015
362
KHAN, KACEW, AND MATTHEWS
(30m x 0.535mm i.d.) coated with DBWAX. The oven temperature was programmed at 5°C/min from 100°C t o 220°C. The detector and injector port temperatures were 290 C and 200 C, respectively. Helium was used as c a r r i e r gas a t a flow r a t e of 20 mL/min. A calibration curve for reference standard was constructed by p l o t t i n g quantity injected against peak height. Under the GC conditions described pirimiphos-methyl showed a peak a t a retention time of 18.3 min. The i d e n t i t i e s of the compounds were confirmed by comparing the GC retention times with those of authentic samples, cochromatography, and f i n a l l y by GC-MS. A high-resolution mass spectrometer, Model VG2AB-2F, connected to a Varian GC Model 3700 was used. The mass spectra were recorded a t 70 eV. Hydrolysis of Conjugates in Urine Urine was heated with 3N HC1 for 2 h i n an o i l bath maintained a t 115-120°C. The solution was cooled and evaporated to dryness to remove HC1. The residual radioactivity was taken up i n a small amount of methanol, derivatized with freshly prepared diazomethane and analyzed by GC and GC-MS.
RESULTS AND DISCUSSION Quantity and nature of grain-bound residues Following a storage period of 28 weeks, wheat grain contained 9.94% of the t o t a l applied radiolabelled pirimiphos-methyl as bound C. The 14 identity of the bound C residues was determined by a combination of SFE technique and chromatographic analyses. I t was observed i n the preliminary experiments that SFE of the solvent extracted control [untreated wheat grain samples] f o r t i f i e d with radiolabelled pirimiphos-methyl (54323dpm/g, 5.2ppm), 14 resulted i n a recovery of 90-93% of C residue i n methanol. GC analysis of the material released by SFE showed the presence of about 93% of the i n t a c t parent compound i n the extract indicating that the insecticide was stable under the experimental conditions used. In treated grain, i t was shown that the amount of radioactivity released and collected i n methanol amounted to about 89% of the t o t a l bound C residues. TLC showed one radioactive spot a t Rf 0.76; similar to that of pirimiphos-methyl.
Radioassay of the methanol extract of t h i s radioactive 14 band accounted for 89.3% of the t o t a l C i n the SFE extract. GC analysis
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
363
of the extract showed a major peak at a retention time of 18.3 min which was identical with the retention time of pirimiphos-methyl. A GC-MS of the peak exhibited molecular ion at m/z 305 confirming the presence of pirimiphos-methyl.
This indicates that the wheat-bound residue i s primarily
in the form of the parent compound (approx. 0.36ppm).
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Bioavailabilitv to r a t s Rats fed solvent extracted untreated wheat grains (control) gained weight during the course of the experiment.
Their average weights at the s t a r t and
termination of the experiment were 210gm and 265gm, respectively. Rats fed 14 wheat containing bound C for 5 days consumed the feed but gained very l i t t l e weight (210g to 214g) during this period. However, during the succeeding 3 days, when the r a t s were fed t h e i r regular chow, the weight was quickly restored to control levels (= 260gm). The presence of bound pirimiphos-methyl in diet appears to have affected the weight gain in r a t s . Ho apparent toxic symptoms in the t e s t r a t s were observed during the course of the experiment. 14 Table 1 shows the excretion of C i n feces and urine following feeding of r a t s with stored wheat grains containing bound radiolabelled pirimiphosmethyl.
Most of the radioactivity was eliminated at the end of the 5th day
(79.9%), with approximately 64.9% in urine and 15.0% in feces. After 8 days, 14 cumulative C in the urine and feces was 72.9% and 17.9% of the t o t a l administered bound 14 C, respectively. Extraction of some radioactivity from the fecal material with methanol (Table 2) suggests that the extracted
C, or part of i t , might have been
bioavailable and ultimately reached the i n t e s t i n e through the b i l e .
The role
of i n t e s t i n a l microflora in releasing firmly bound 14C should also be taken into account.
I t was observed that the proportion of extracted
C
increased somewhat near the termination of the experiment. The nonextractable 14 C residues i n feces (Table 2) demonstrate the i n d i g e s t i b i l i t y of the matrix of wheat grains containing bound
C residues and 50 to 65% of
radiocarbon i n i t i a l l y present in the feces remained in a t i g h t l y bound form. Radioactivity in selected tissues determined following the s a c r i f i c e of r a t s was very low.
The highest C residues were found i n the liver (0.2% of the administered bound 14 C) whereas kidney, heart and lungs contained 14 only trace amount of C residues (< 0.05%). Examination of r a t organs revealed no histopathological a l t e r a t i o n s .
364
KHAN, KACEW, AND MATTHEWS
TABLE 1. ELIMINATION OF RADIOCARBON IN14 THE URINE AND FECES OF RATS FED STORED WHEAT GRAINS CONTAINING BOUND [ C]PIRIHIPH0S-HETHYL % of bound 14C Consumed/rat
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Days a f t e r treatment
1 2 3 4 5 6 7 8
Cum % of dose
Urine
Feces
0.3 12.6 17.3 18.0 16.7 5.7 1.4 0.9
0.4 3.0 3.3 4.8 3.5 2.4 0.3 0.2
0.7 16.3 36.9 59.7 79.9 88.0 89.7 90.8
Recovered In urine Recovered In feces
TABLE 2.
72.9 17.9
EXTRACTABLE AND NONEXTRACTABLE 14C IN FECES
Days after treatment
1 2 3 4 5 6 7 8
% Of
14
C In feces
Extractable
Nonextractable
37.7 35.1 41.4 40.2 42.6 42.5 44.8 50.1
62.3 64.9 58.6 59.8 57.4 57.5 55.2 49.9
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
365
The elimination of 14 C residues in urine indicates that most of the bound 14 C pirimiphos-methyl fed to rats was bioavailable. There i s also an apparent negative effect on the body weight gain suggesting a toxicological potential.
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Nature of urinary and fecal radioactivity Most of the urinary radioactivity (90% 14 C) remained at the spotting origin of the TLC system used. In addition, a weak radioactive zone at R, 0.48 (10% 14 C) was detected. The material at R. 0.0 was scraped off the plate, extracted with methanol and hydrolyzed with HC1 as described above. Ether extract of the hydrolyzed material contained most of the radioactivity 14 14 (> 85% of the total C in urine) but the partitioning of C into organic phase was not reproducible, possibly due to the presence of other coextractives. GC analysis of the methyl derivative of the ether extracted material showed four peaks at the retention times of 14.0, 14.2, 15.3 and 18.3 min. GC-MS of these peaks showed molecular ions at m/z 139, 167, 195 and 305, respectively indicating the presence of compounds IV, I I I , I I , VI and/or VII (Figure 3). The GC peak at 18.3 min corresponded to the retention time of the parent compound ( I ) . Furthermore, the GC-MS of this peak exhibited a molecular ion at m/z 305. Since the presence of I in the radioactive region at Rf 0.00 is very unlikely (Pirimiphos-methyl Rf = 0.76), i t appears that the parent compound was dealkylated to yield compounds VI and/or VII. Methylation of these compounds prior to GC and GC-MS converted them to pirimiphos-methyl. GC analysis of the methanol extract of the radioactive zone at Rf 0.48 showed a peak at a retention time of 27.3 min. A GC-MS of this material gave a peak at m/z 277 indicating the presence of compound V. The data indicate that bound pirimiphos-methyl in wheat grain released on digestion was metabolized by hydrolytic cleavage, N-dealkylation and 0-demethylation. The products formed were present in urine in conjugated forms. TLC of methanol extractable 14 C residues from feces showed only one radioactive zone at Rf 0.00. Methanol extracted 92% of this material from the TLC plate. GC and GC-MS analysis of the methylated material confirmed the presence of compound II and trace amounts of IV. However, a major portion of radioactivity In the extract remained unidentified. The results of this study indicate that bound residues in stored wheat treated with pirimiphos-methyl are present mostly in the form of the parent
366
KHAN, KACEW, AND MATTHEWS
R1
R2
I
^O-CH 3
C2HS_
C2H5_
II
H
C2H5_
C2HS_
HI
H
H
C2H5_
IV
H
H
H
H
C2HS_
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Compound
R3
Lo-H
FIG. 3 .
VI
^O-CH3
C^Hs—
C2H5—
VII
^O-H
C2H5_
C2H5_
Chemical structures of pirimiphos-methyl and metabolites.
compound. They a r e h i g h l y b i o a v a l l a b l e t o the r a t and may possess a toxicological potential.
More research w i l l be required t o provide
information on b i o l o g i c a l a c t i v i t y by t e s t i n g parameters such a s hematology and blood biochemistry including c h o l i n e s t e r a s e a c t i v i t y .
REFERENCES
Anon, Analyst, 105, 515-517 (1980). Bengston, M., Cooper, L.M. and Grant-Taylor, F.J., Anim. S c i . , 32, 51-78
(1975).
Queensl. J . Agric.
BOUND [14C] PIRIMIPHOS-METHYL IN STORED WHEAT Khan, S.U., Kacew, S. and Akhtar, M.H., J . Agric. Food Chem., 38, 1077-1082 (1990). Leahey, J . P . and Curl, E.A., P e s t i c . Sci. 1 2 , 467-474 (1982). Rowlands, D.G., Residue Rev., 58, 113-155 (1975).
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Rowlands, D.G. and Wilkin, D.R., Report of Director Pest Infestation Control Lab. Slough, U.K. 1971-73 (1975). Rowlands, D.G., Report No. 54. Slough, U.K. (1980).
Pest Infestation Laboratory, MAFF,
367
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Bioavailability to 14
rats of bound [ C] pirimiphos‐methyl in stored wheat a
b
S.U. Khan , S. Kacew & W. Matthews
c
a
Centre for Land and Biological Resources Research , Agriculture Canada , Ottawa, Ontario, K1A 0C6, Canada b
Department of Pharmacology, School of Medicine , University of Ottawa , Ottawa, Ontario, K1H 8M5, Canada c
ADAS Central Science Laboratory , Ministry of Agriculture, Fisheries and Food , Slough, Berks, SL2 7HJ, Great Britain Published online: 13 Nov 2008.
To cite this article: S.U. Khan , S. Kacew & W. Matthews (1992) 14
Bioavailability to rats of bound [ C] pirimiphos‐methyl in stored wheat, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 27:4, 355-367 To link to this article: http://dx.doi.org/10.1080/03601239209372786
Downloaded by [University of Glasgow] at 06:15 05 January 2015
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
J. ENVIRON. SCI. HEALTH, B27(4), 355-367 (1992)
BIOAVAILABILITY TO RATS OF BOUND [14C] PIRIMIPHOS-METHYL IN STORED WHEAT
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Key words: Wheat, 14 C-Pirimiphos-methyl, Bound residues Bioavailability S.U. Khan, S. Kacew+, and W. Matthews ++ Centre for Land and Biological Resources Research, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 + Department of Pharmacology, School of Medicine University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 ++ ADAS Central Science Laboratory, Ministry of Agriculture, Fisheries and Food, Slough, Berks, Great Britain SL2 7HJ
ABSTRACT Stored wheat t r e a t e d with r a d i o l a b e l l e d pirimiphos-methyl ( 0 - 2 - d i e t h y l amino-6-methyl-pyrimidin-4-yl 0,0-dimethyl p h o s p h o r o t h i o a t e ) formed bound
(nonextractable) 14C residues. Supercritical fluid extraction, gas chromatography and mass spectrometric techniques were used to identify and quantitate the 14C bound residues in wheat grains. The amount of bound 14 C residues present after 28 weeks of storage was about 9.9% of the applied radioactivity. Pirimiphos-methyl accounted for 80% of the bound residue. Grain-bound residues were fed to rats for 5 days. After a total period of 8 days a substantially large percentage of the administered bound14C residues (72.9%) was eliminated in urine while feces contained only 17.9%. Bound pirimiphos-methyl in wheat grain was metabolized in rats by processes involving hydrolysis, N-dealkylation and O-demethylation. The results indicate that wheat-bound residues of pirimiphos-methyl are highly bioavailable to the rat and may possess a toxicological potential as manifested by a significant reduction in body weight gain.
355 Copyright © 1992 by Marcel Dekker, Inc.
356
KHAN, KACEW, AND MATTHEWS INTRODUCTION Pirimiphos-methyl (0-2-diethylamino-6-methyl-pyrimidin-4-yl O,0-dimethyl
phophorothioate),
"Actellic' 1 -^ i s a broad spectrum i n s e c t i c i d e used t o
control a vide range of stored grain p e s t s .
I t has low mammalian t o x i c i t y and
i s applied d i r e c t l y to grains, where i t controls pests by both contact and vapor a c t i v i t y . I t has been shown that under normal conditions for grain
Downloaded by [University of Glasgow] at 06:15 05 January 2015
storage, pirimiphos-methyl i s degraded only slowly and p e r s i s t s (= 70%) on stored grains for longer than 24 weeks (Leahey and Curl, 1982).
A half-life
of 43 weeks for piriraiphos-methyl on wheat grain maintained under normal storage conditions has been reported (Bengston e t a l . , 1975). Wheat grain 14 o treated with I C] pirimiphos-methyl and stored a t 21 C for 8 months was found t o contain 78% of the r a d i o a c t i v i t y recoverable as parent compound (Rowlands, 1975).
In a subsequent study (Rowlands and Wllkin, 1975) i t was
observed t h a t approximately i o % of the r a d i o a c t i v i t y was bound to wheat and was unextractable except by digesting. Rowlands, 1980 reported about 40% loss 14 0 of [ C] pirimiphos-methyl from wheat stored a t 20 C a f t e r 6 months. I t 14 was also observed that about 15% of the applied C was not recoverable by solvent extraction. Leahey and Curl, 1982 also reported t h a t wheat t r e a t e d 14 with [ C] pirimiphos-methyl and stored for 6 months contained up to 13% bound r a d i o a c t i v i t y of the i n i t i a l l y applied 14 C. In the foregoing studies the nature of the bound residues was not determined. The Joint FAO/IAEA Division of Huclear Techniques in Food and Agriculture i n i t i a t e d a research programme i n 1986 to evaluate the safety of bound pesticide residues in food producing crops and stored grains.
The study
emphasized b i o a v a i l a b i l i t y and t e s t i n g for toxicological p o t e n t i a l i n r a t s fed materials containing bound p e s t i c i d e residues.
I n a previous publication we
reported that the bound residues in stored wheat treated with the i n s e c t i c i d e deltamethrin were highly bioavailable to the r a t (Khan e t a l . , 1990).
We now
report the r e s u l t s of our investigation on the b i o a v a i l a b i l i t y and metabolism in the r a t of bound
C residues in stored wheat treated with [ C] 14 C residues in stored wheat was
pirimiphos-methyl. The nature of bound also determined.
MATERIALS AND METHODS Chemicals Pirimiphos-methyl l a b e l l e d with
14
C i n t h e 2 p o s i t i o n of t h e pyrimidine
ring ( s p e c i f i c a c t i v i t y 57 mCi/mmol) was purchased from Tracerco ICI Ltd., Cleveland, Ohio, USA. The a n a l y t i c a l grade m a t e r i a l was obtained from B r i t i s h
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
357
Greyhound Chromatography and Allied Chemicals Ltd. and had a purity of 98.1%.
Downloaded by [University of Glasgow] at 06:15 05 January 2015
All solvents were of pesticide grade and were used as received.
N
T
=^
N(C2H5)2
Pirimiphos-Methyl
Grain Treatment and Storage Pesticide-free English wheat of the variety "Mission" with an i n i t i a l moisture content of 13.2% was treated with [14C] pirimiphos-methyl (100 uCi/kg) and technical pirimiphos-methyl to achieve a concentration of 4.5 ppm. The insecticide was dissolved in diethyl ether and applied dropwise to 500 g batches of grain whilst this was being stirred. The treated grain was placed in a separate glass j a r which was sealed and tumbled end over end for 1 hour. The grain was spread on a tray in a fume hood draught to allow the ether to evaporate and was then returned to the j a r which was resealed and tumbled for a further two hours. The storage temperature of 15°C was chosen as an estimate of the mean temperature attained by a field scale bulk of grain. The grain was stored for 28 weeks. The grain used as control was similarly stored except i t was not treated with the insecticide. Extraction of Stored Grains The wheat grain was extracted with methanol using the method developed by the Committee for Analytical Methods for Residues of Pesticides and Veterinary Products in Foodstuffs (Anon., 1980). An additional sonication step was used to ensure complete extraction. The extracted material after drying at room temperature contained 1.5% H,0.
358
KHAN, KACEW, AND MATTHEWS
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Grain-Bound Residues A portion of the extracted grain was combusted to 14 CO. to determine 14 14 the total bound C. A second portion was used to release C-bound residues by supercritical fluid extraction (SFE), Fig. 1. The supercritical fluid extractor (Suprex Model SFE-5O, Suprex Corp., Pittsburgh, PA) consisted of a 250-mL syringe pump, a control module for control of the SFE system, an extraction oven, a 5-mL extraction vessel containing 2g of extracted wheat grains, and a four-part valve connected with the outlet restriction (fused s i l i c a tubing, 50-um i.d.) that was inserted into glass tubes containing methanol. The supercritical fluid used was CO, and the extractions were performed a t a pressure of 280 atm. and temperature 110°C. The flow rate of mobile phase CO. was 1 mL/min. The extraction was carried out for 1 h and the material was collected in methanol. The remaining portion of the extracted grain was used for feeding rats (Fig. 1). Feeding Study Four male Sprague-Dawley rats weighing approximately 210g each were housed individually in metabolism cages which permitted separate collection of urine and feces. The rats were conditioned for 4 days t o a daily diet of Purina Laboratory Chow. The feed was then replaced for 5 days with the methanol-extracted material containing 14 C bound residues (22,105dpm/g). Four control rats were fed the extracted untreated wheat material. Total feed consumed by each rat in 5 days was about 70g. The rats were then maintained on regular rat chow for an additional 3 days. The urine and feces were collected daily for 8 days. The weight of each rat was also recorded on a daily basis during the course of the experiement. After 8 days the animals were sacrificed by decapitation, selected organs were excised and weighed, and a portion of these organs was frozen in liquid nitrogen for radioassay (Fig. 2). Preparation of Samples and Analysis ( i ) Wheat grain. The SFE extract was concentrated to a small volume and analyzed by thin layer chromatography (TLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). ( i i ) Feces. The feces from rats were extracted with methanol until further extraction did not result in any extractable 14 C. The methanol extract was concentrated to a small volume,
Downloaded by [University of Glasgow] at 06:15 05 January 2015
w o
Wheat Grain
"
I
1
Treat with Radioiabeled Pirimiphos-methyl (100 uC1/ka, 4.5 ppm)
[| a
Store at 15*C *C for 28 Weeks
«
Ground and Extract with Methanol
I
I
Extractabie l4 C-Res1dues
Nonextractable (8ound) "C-Res1dues
, I
L JL
, I
Combustion Supercritical Fluid Feeding to Rats 14 C0, l4 Extraction (B1oava1lab1Hty of bound "C) (Total bound C) (Identification of bound l4C) FIG. 1. Schematic diagram for the grain treatment with [I4C] p1r1m1phos-methyl and determination of bound M C residues.
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Methanol Extracted Stored Wheat Containing Bound I4C Feed to Rats for 5 Days (70 g/rat/5 days) Collect Urine and Feces, Record Weight of Each Rat on a Dally Basis f o r 9 Days
1 Urine
Organs TC
Combustion l4 CO,
Feces Lie
Extract with Methanol
1
1
Hydrolysis
Extractable
Nonextractable
Methylatlon
T
1 GC, GC-MS
GC, GC-MS
I TLC, GC GC-MS
>jC
I LSC
Combustion l4 C0 (Total'l4C)
FIG. 2. Schematic diagram for the Identification of bound 14C residues 1n stored wheat treated with [ 14 C] p1r1m1phos-methyl.
o
3
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
361
Downloaded by [University of Glasgow] at 06:15 05 January 2015
an aliquot was radioassayed and the remaining portion was analyzed by TLC, GC and GC-MS. The insoluble fecal material containing bound 14 C residues was air-dried and combusted to 14 CO for the determination of the 14 14 unextractable C residues. Total C content of feces was obtained by summation of the two values, ( i i i ) Urine. An aliquot of urine was radioassayed by scintillation counting. The remaining urine was brought to just dryness under a stream of dry nitrogen, dissolved in methanol, and analyzed by TLC, GC and GC-MS. Samples of SFE extract of control wheat grain, feces and urine from rats fed control wheat grain were processed as described for the treated samples. Radioassav Radioactivity of appropriate aliquots of the SFE extracts, urine and other liquid samples was measured in a Packard Model 3320 scintillation spectrometer. The solid samples (ground wheat material, feces, animal tissues) were assayed by combusting in a Packard sample oxidizer, Model 306, followed by liquid scintillation counting (LSC) with an external standard and correcting data for quenching. Radioactive spots or zones on TLC plates were located and counted by a Berthold Beta camera LB292. For purpose of quantitation each radioactive region was scraped from TLC plate, a portion was placed into a scintillation vial for direct counting. TLC plates were also examined under ultraviolet light. Chromatographic Procedures ( i ) TLC. Precoated s i l i c a gel GF chromatopiates, with 1.0mm gel thickness (Analtech, Inc., Newark, DE) were used for thin-layer chromatography. The solvent system used was toluene-hexane-acetic acid (15:3:2). Aliquots of the radioactive extracts were chromatographed on a 20 x 20cm plate. Authentic standard of pirimiphos-methyl was also chromatographed on the same TLC plate for preliminary identification. The radioactivity zones were located on the developed plate, each radioactive zone was scraped off the plate, extracted with methanol, and analyzed by LSC to determine the total extractable 14 C. The extractable material was then further analyzed by GC and GC-MS. ( i i ) GC-MS. Gas chromatography was performed using a Varian Model 6000 fitted with a thermionic specific detector and a s i l i c a Megabore column
Downloaded by [University of Glasgow] at 06:15 05 January 2015
362
KHAN, KACEW, AND MATTHEWS
(30m x 0.535mm i.d.) coated with DBWAX. The oven temperature was programmed at 5°C/min from 100°C t o 220°C. The detector and injector port temperatures were 290 C and 200 C, respectively. Helium was used as c a r r i e r gas a t a flow r a t e of 20 mL/min. A calibration curve for reference standard was constructed by p l o t t i n g quantity injected against peak height. Under the GC conditions described pirimiphos-methyl showed a peak a t a retention time of 18.3 min. The i d e n t i t i e s of the compounds were confirmed by comparing the GC retention times with those of authentic samples, cochromatography, and f i n a l l y by GC-MS. A high-resolution mass spectrometer, Model VG2AB-2F, connected to a Varian GC Model 3700 was used. The mass spectra were recorded a t 70 eV. Hydrolysis of Conjugates in Urine Urine was heated with 3N HC1 for 2 h i n an o i l bath maintained a t 115-120°C. The solution was cooled and evaporated to dryness to remove HC1. The residual radioactivity was taken up i n a small amount of methanol, derivatized with freshly prepared diazomethane and analyzed by GC and GC-MS.
RESULTS AND DISCUSSION Quantity and nature of grain-bound residues Following a storage period of 28 weeks, wheat grain contained 9.94% of the t o t a l applied radiolabelled pirimiphos-methyl as bound C. The 14 identity of the bound C residues was determined by a combination of SFE technique and chromatographic analyses. I t was observed i n the preliminary experiments that SFE of the solvent extracted control [untreated wheat grain samples] f o r t i f i e d with radiolabelled pirimiphos-methyl (54323dpm/g, 5.2ppm), 14 resulted i n a recovery of 90-93% of C residue i n methanol. GC analysis of the material released by SFE showed the presence of about 93% of the i n t a c t parent compound i n the extract indicating that the insecticide was stable under the experimental conditions used. In treated grain, i t was shown that the amount of radioactivity released and collected i n methanol amounted to about 89% of the t o t a l bound C residues. TLC showed one radioactive spot a t Rf 0.76; similar to that of pirimiphos-methyl.
Radioassay of the methanol extract of t h i s radioactive 14 band accounted for 89.3% of the t o t a l C i n the SFE extract. GC analysis
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
363
of the extract showed a major peak at a retention time of 18.3 min which was identical with the retention time of pirimiphos-methyl. A GC-MS of the peak exhibited molecular ion at m/z 305 confirming the presence of pirimiphos-methyl.
This indicates that the wheat-bound residue i s primarily
in the form of the parent compound (approx. 0.36ppm).
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Bioavailabilitv to r a t s Rats fed solvent extracted untreated wheat grains (control) gained weight during the course of the experiment.
Their average weights at the s t a r t and
termination of the experiment were 210gm and 265gm, respectively. Rats fed 14 wheat containing bound C for 5 days consumed the feed but gained very l i t t l e weight (210g to 214g) during this period. However, during the succeeding 3 days, when the r a t s were fed t h e i r regular chow, the weight was quickly restored to control levels (= 260gm). The presence of bound pirimiphos-methyl in diet appears to have affected the weight gain in r a t s . Ho apparent toxic symptoms in the t e s t r a t s were observed during the course of the experiment. 14 Table 1 shows the excretion of C i n feces and urine following feeding of r a t s with stored wheat grains containing bound radiolabelled pirimiphosmethyl.
Most of the radioactivity was eliminated at the end of the 5th day
(79.9%), with approximately 64.9% in urine and 15.0% in feces. After 8 days, 14 cumulative C in the urine and feces was 72.9% and 17.9% of the t o t a l administered bound 14 C, respectively. Extraction of some radioactivity from the fecal material with methanol (Table 2) suggests that the extracted
C, or part of i t , might have been
bioavailable and ultimately reached the i n t e s t i n e through the b i l e .
The role
of i n t e s t i n a l microflora in releasing firmly bound 14C should also be taken into account.
I t was observed that the proportion of extracted
C
increased somewhat near the termination of the experiment. The nonextractable 14 C residues i n feces (Table 2) demonstrate the i n d i g e s t i b i l i t y of the matrix of wheat grains containing bound
C residues and 50 to 65% of
radiocarbon i n i t i a l l y present in the feces remained in a t i g h t l y bound form. Radioactivity in selected tissues determined following the s a c r i f i c e of r a t s was very low.
The highest C residues were found i n the liver (0.2% of the administered bound 14 C) whereas kidney, heart and lungs contained 14 only trace amount of C residues (< 0.05%). Examination of r a t organs revealed no histopathological a l t e r a t i o n s .
364
KHAN, KACEW, AND MATTHEWS
TABLE 1. ELIMINATION OF RADIOCARBON IN14 THE URINE AND FECES OF RATS FED STORED WHEAT GRAINS CONTAINING BOUND [ C]PIRIHIPH0S-HETHYL % of bound 14C Consumed/rat
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Days a f t e r treatment
1 2 3 4 5 6 7 8
Cum % of dose
Urine
Feces
0.3 12.6 17.3 18.0 16.7 5.7 1.4 0.9
0.4 3.0 3.3 4.8 3.5 2.4 0.3 0.2
0.7 16.3 36.9 59.7 79.9 88.0 89.7 90.8
Recovered In urine Recovered In feces
TABLE 2.
72.9 17.9
EXTRACTABLE AND NONEXTRACTABLE 14C IN FECES
Days after treatment
1 2 3 4 5 6 7 8
% Of
14
C In feces
Extractable
Nonextractable
37.7 35.1 41.4 40.2 42.6 42.5 44.8 50.1
62.3 64.9 58.6 59.8 57.4 57.5 55.2 49.9
BOUND [I4C] PIRIMIPHOS-METHYL IN STORED WHEAT
365
The elimination of 14 C residues in urine indicates that most of the bound 14 C pirimiphos-methyl fed to rats was bioavailable. There i s also an apparent negative effect on the body weight gain suggesting a toxicological potential.
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Nature of urinary and fecal radioactivity Most of the urinary radioactivity (90% 14 C) remained at the spotting origin of the TLC system used. In addition, a weak radioactive zone at R, 0.48 (10% 14 C) was detected. The material at R. 0.0 was scraped off the plate, extracted with methanol and hydrolyzed with HC1 as described above. Ether extract of the hydrolyzed material contained most of the radioactivity 14 14 (> 85% of the total C in urine) but the partitioning of C into organic phase was not reproducible, possibly due to the presence of other coextractives. GC analysis of the methyl derivative of the ether extracted material showed four peaks at the retention times of 14.0, 14.2, 15.3 and 18.3 min. GC-MS of these peaks showed molecular ions at m/z 139, 167, 195 and 305, respectively indicating the presence of compounds IV, I I I , I I , VI and/or VII (Figure 3). The GC peak at 18.3 min corresponded to the retention time of the parent compound ( I ) . Furthermore, the GC-MS of this peak exhibited a molecular ion at m/z 305. Since the presence of I in the radioactive region at Rf 0.00 is very unlikely (Pirimiphos-methyl Rf = 0.76), i t appears that the parent compound was dealkylated to yield compounds VI and/or VII. Methylation of these compounds prior to GC and GC-MS converted them to pirimiphos-methyl. GC analysis of the methanol extract of the radioactive zone at Rf 0.48 showed a peak at a retention time of 27.3 min. A GC-MS of this material gave a peak at m/z 277 indicating the presence of compound V. The data indicate that bound pirimiphos-methyl in wheat grain released on digestion was metabolized by hydrolytic cleavage, N-dealkylation and 0-demethylation. The products formed were present in urine in conjugated forms. TLC of methanol extractable 14 C residues from feces showed only one radioactive zone at Rf 0.00. Methanol extracted 92% of this material from the TLC plate. GC and GC-MS analysis of the methylated material confirmed the presence of compound II and trace amounts of IV. However, a major portion of radioactivity In the extract remained unidentified. The results of this study indicate that bound residues in stored wheat treated with pirimiphos-methyl are present mostly in the form of the parent
366
KHAN, KACEW, AND MATTHEWS
R1
R2
I
^O-CH 3
C2HS_
C2H5_
II
H
C2H5_
C2HS_
HI
H
H
C2H5_
IV
H
H
H
H
C2HS_
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Compound
R3
Lo-H
FIG. 3 .
VI
^O-CH3
C^Hs—
C2H5—
VII
^O-H
C2H5_
C2H5_
Chemical structures of pirimiphos-methyl and metabolites.
compound. They a r e h i g h l y b i o a v a l l a b l e t o the r a t and may possess a toxicological potential.
More research w i l l be required t o provide
information on b i o l o g i c a l a c t i v i t y by t e s t i n g parameters such a s hematology and blood biochemistry including c h o l i n e s t e r a s e a c t i v i t y .
REFERENCES
Anon, Analyst, 105, 515-517 (1980). Bengston, M., Cooper, L.M. and Grant-Taylor, F.J., Anim. S c i . , 32, 51-78
(1975).
Queensl. J . Agric.
BOUND [14C] PIRIMIPHOS-METHYL IN STORED WHEAT Khan, S.U., Kacew, S. and Akhtar, M.H., J . Agric. Food Chem., 38, 1077-1082 (1990). Leahey, J . P . and Curl, E.A., P e s t i c . Sci. 1 2 , 467-474 (1982). Rowlands, D.G., Residue Rev., 58, 113-155 (1975).
Downloaded by [University of Glasgow] at 06:15 05 January 2015
Rowlands, D.G. and Wilkin, D.R., Report of Director Pest Infestation Control Lab. Slough, U.K. 1971-73 (1975). Rowlands, D.G., Report No. 54. Slough, U.K. (1980).
Pest Infestation Laboratory, MAFF,
367
