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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
Deltamethrin residues in milk and tissues of lactating dairy cows a
a
M. Humayoun Akhtar , Claude Danis , H. a
Locksley Trenholm & Kenneth E. Hartin
a
a
Centre for Food and Animal Research, Research Branch , Agriculture Canada , Ottawa, Ontario, K1A 0C6 Published online: 21 Nov 2008.
To cite this article: M. Humayoun Akhtar , Claude Danis , H. Locksley Trenholm & Kenneth E. Hartin (1992) Deltamethrin residues in milk and tissues of lactating dairy cows, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 27:3, 235-253 To link to this article: http://dx.doi.org/10.1080/03601239209372777
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.
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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/termsand-conditions
J. ENVIRON. SCI. HEALTH, B 2 7 ( 3 ) , 2 3 5 - 2 5 3 ( 1 9 9 2 )
Downloaded by [University of Auckland Library] at 12:28 02 March 2015
DELTAMETHRIN RESIDUES IN MILK AND TISSUES OF LACTATING DAIRY COWS M. Humayoun Akhtar, Claude Danis, H. Locksley Trenholm and Kenneth E . Hartin Centre for Food and Animal Research, Research Branch, Agriculture Canada, Ottawa, Ontario K1A 0C6
Key
words: Deltamethrin, Residues, Pyrethroids, Milk
ABSTRACT Lactating dairy cows were fed deltamethrin (2 or 10 mg kg-1 feed) for 28 consecutive days and deltamethrin residues measured in milk and tissues. Deltamethrin residues were higher relative to dose administered. The
order of relative concentrations of deltamethrin in tissues, measured 1,
4, and 9 days after the last dose was: renal fat > subcutaneous fat > forequarter muscle > hindquarter muscle > liver > kidney. Depletion of deltamethrin residues in milk was very rapid indicationg the half-life of the insectide of about 1 day. Trace amounts of deltamethrin metabolites 3-
235 Copyright© 1992 by Marcel Dekker, Inc.
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AKHTAR ET AL.
(2,2-dibromovmyl)-2,2-dimethylcyclopropane carboxylic acid ( < 0.0235 ppm) and 3-phenoxybenzoic acid ( < 0.034 ppm) were also detected in milk and
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tissues of treated cows.
INTRODUCTION Deltamethrin [(s)-o-cyano-3-phenoxybenzyl-cis-(lR,3R)-3-(2¿dibromovinyl)-2^-dimethylcyclopropanecarboxylate] is a potent insecticide with a broad spectrum activity towards a wide variety of insects that infest crops, animals, and vegetation. The technical product, DecisR, contains 98% deltamethrin (FAO, 1981). The metabolic fate of deltamethrin has been reported in vivo in rats (Ruzo et al., 1978), mice (Ruzo et al, 1979), laying hens (Akhtar et al., 1985), as well as in vitro using mouse liver microsomes (Shono et al., 1979), and cow and chicken liver homogenates (Akhtar, 1984). When lactating cows were dosed orally with 14C labeled (gem dimethyl or benzyl) deltamethrin, the insecticide was absorbed poorly from the rumen (Akhtar et al. 1986). Absorbed deltamethrin was secreted in milk at trace levels only, and did not accumulate in edible tissues to any significant extent. The Canadian Prairies often face grasshopper infestations (Research Branch Report 1984, 1985) which result in serious economic losses. Deltamethrin is registered for grasshopper control on forage and cereal crops at rates of 5.0 -7.5 g active ingredient (AI)/ hectare (ha) using
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
237
ground applicators (Hoechst Canada Inc., 1983). Aerial application of deltamethrin on pasture and rangeland at 7.2 g AI/ha resulted in 65% reduction in grasshopper density by the 4th day after application.
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Deltamethrin residues on forage and Utter determined within 6 hr after application were 2.19 and 0.79 ppm, respectively (all values are on dry matter basis [DM]) (Johnson et al. 1986). To assess the safety of deltamethrin residues to lactating cows feeding on treated forage in the present study 2 and 10 ppm of deltamethrin was added to the daily diet. The objective of the study was to determine residues of deltamethrin and its two major metabolites, namely 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylic add (Br2CA) and 3-phenoxybenzoic acid (3-PBadd) in milk, liver, kidney, fat (subcutaneous, renal) and meat (fore and hind quarter) for these two intakes. The level of 2 ppm is closer to that expected (2.19 ppm, Johnson et al. 1986) in the freshly treated pasture; whereas a 10 ppm is an exaggerated level to determine the safety at five times the daily intake.
MATERIALS AND METHODS Chemicals. Pestiddes and reagent grade solvents were used. Deltamethrin was supplied by Hoechst Canada Inc. Pure Br2CA and 3PBacid were available from previous studies (Akhtar et al., 198S, 1986). Stock solutions of deltamethrin, (i) 2.88 g in 100 mL acetone for 2 ppm, and (ii) 14.44 g in 100 mL acetone for 10 ppm were prepared. Daily feed
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was prepared by treating 200 g grain with 0.5 mL volume of either of the two stock solutions. Feed for control cow was treated in the same way with same volume of acetone. In order to remove acetone, the containers
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were allowed to stand at room temperature. Fortified feed was prepared 2 or 3 days prior to use. Animal Treatment. Seven cows with a daily milk production of 10.3-13.8 kg were used in this study. They were kept in individual stalls and fed to appetite a standard dairy ration for lactating cows for 14 days, while daily feed intake was recorded. Based on an average daily intake of 14.4 kg (DM) during this acclimatization period, the cows on 2 and 10 ppm treatment received 288 mg and 1.4 g, respectively, deltamethrin daily in feed. The cows were milked twice daily at 06:00 and 16:00 h. Seven cows were divided into three groups: control (1), 2 ppm (3), and 10 ppm (3) treatment for a 28-day test period. Each cow was fed treated grains immediately after morning and evening milking. A standard total mixed ration was fed after cows had consumed all the treated grains. At each milking, the milk was weighed and sub-sampled. Daily composited samples were analyzed for fat by infrared milk analyses system (Multispec IR. Analyser Mark Π, Whetdrake, York England) on weekly basis. Subsampled daily milk samples were stored at 4°C for residue analyses. The control and one cow from each treatment group were slaughtered at 24 h after the last treatment with deltamethrin. Subsequently, one cow from each pesticide-fed group was killed at 4 and 9 days after the last
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
239
treatment. Each cow was examined for gross pathological changes, samples of subcutaneous and renal fat, front and hind quarter muscles, liver and kidney were removed and stored at -20°C until analyzed.
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Extraction,
(i) Milk: An aliquot (25 mL) of sub-sampled milk, in
duplicate, after pH adjustment to about 2 and addition of 2 mL of 5% aqueous potassium oxalate was shaken gently with 40 mL of ethanol-ether (1:3, v/v) in a 250 mL Mixxor (Scientific Products and Equipment). The organic phase was removed, and the aqueous phase was vigorously shaken with ethanol-ether (4 χ 40 mL). The organic phase was reduced in volume to ca 5 mL on a rotary evaporator at 40°C. The residue was quantitatively transferred into a centrifuge tube, and the flask was rinsed with acetone (3 χ 3 mL). Acetone was evaporated under a stream of Nj, the residue extracted with hexane ( 5 x 5 mL), passed over NajSQ,, and concentrated to 2mL (in Tissues: A 20 g portion, in duplicate, of tissues (muscle, liver and kidney) was cut into small pieces (ca 5 mm), and homogenized in a Waring blender with 50 mL ethanol-ether (1:3) and 2 mL of 0.1 Ν HC1, and the homogenate filtered. The residue was again cut into small pieces and blended with the extracting solvent (4 χ 30 mL). The filtrate was concentrated to ca 5 mL, and the residue was quantitatively transferred into a centrifuge tube, and the flask was rinsed with acetone. Organic solvent was evaporated under a stream of N2, the residue extracted with hexane ( 5 x 5 mL), dried over NajSO^ and the volume reduced to 2 mT.,
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(iii) Fat:
An aliquot (20 g), in duplicate, was cut into small pieces (about
5 mm) and blended with 50 mL ethanol-ether (1:3) and 2 mL of 0.1 Ν HC1. The organic phase was decanted and the residue was re-extracted
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with ethanol-ether (4x) 30 mL The fíltrate was concentrated to about 5 mL, the residue dissolved in 25 mL hexane, and the mixture filtered through pre-washed anhydrous NajSCV Partition. The hexane extracts were shaken with acetonitrile ( 4 x 2 mL), and the acetonitrile layer was evaporated to near dryness. The hexane phase was discarded. Clean-up. To remove residual lipids, a gel permeation clean-up technique was used. The column, a 100 mL buret (i.d. 15 mm), was packed with a glass wool (prewashed with eluting solvent) plug, and 15 g of Sephadex LH-20 (Pharmacia) previously soaked in 60 mL 2-propanol for 48 h. The column was washed with an additional 50 mL 2-propanol. Acetonitrile residues obtained after partitioning, as detailed above, was dissolved in 1 mL of 2-propanol, and placed in the column and eluted with 2-propanol. The first 50 mL of eluant was discarded, while the next 125 mL fraction collected. It was shown by gas chromatography (GC) in the preliminary experiment and radiochemical analyses that spiked 14C deltamethnn, Br2CA and 3-PBacid completely eluted with 2-propanol in the 65-100 mL fraction from the column. Furthermore, no tailing was observed in the next 25 mL fraction. However, to ensure complete recovery of the residues, 125 mL fractions were collected.
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
241
Each packed column was used for 10 samples before being recharged with a fresh packing. It was noted that each column could be used for up to 20 additional samples.
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Analyses.
The eluants from the Sephadex column were evaporated to
dryness, and the residues were redissolved in acetone and transferred into centrifuge tubes. The acetone was removed and residues dissolved in 2 mL hexane for GC analyses. The hexane solution was divided into two equal portions of 1 mL each. One portion was analyzed directly by GC for deltamethrin while the second portion was derivatized for the analyses of Br2CA and 3-PBacid. (a) Derivatization. The acid metabolites were converted into trichloroethyl derivatives by the method of George and McDonough (1975) with slight modifications. The volume of the reactants DCC-pyridine and 2,2,2trichloroethanol were reduced in half while the reaction time was doubled. Changes in reaction conditions did not alter the yield, but produced fewer interfering substances. The derivatives were extracted with hexane and subjected to clean-up technique as detailed below: The clean-up was carried out on a disposable Pasteur capillary pipet (16 cm χ 0.8 cm i.d.) containing a cotton plug, 5 cm of deactivated Florisil (10% H2O), which was topped with 1 cm of 25% cellulose in decolorizing carbon in that order. The column was prewashed with hexanedichloromethane. The first 3 mL (1st fraction) was discarded, and the next
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two fractions of 4 mL (2nd fraction) and 8 mL (3rd fraction) each were collected separately for analysis of Br2CA and 3-PBacid derivatives. In the preliminary studies, it was shown that the trichloroester of 3-
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PBadd eluted completely in the 4 mL (2nd fraction), while Br2CA ester was exclusively in the 8 mL fraction (3rd fraction). The eluant from the Sephadex Column could not be analyzed directly for Br2CA and 3-PBacid by GC on a 3% SE-30 or a 5% OV-210 column without prior derivatization. The methyl derivative of 3-PBacid was not ECD-sensitive and required approximately 1/ig of material to provide 44% recorder response when a flame ionization detector (FID) was used. The ECD-sensitive trichloroethyl derivatives were prepared by treating the extracts with DCC-pyridine and trichloroethanol. As expected, the trichloroethyl ester of Br2CA was much more sensitive than that of 3PBacid. For example, the ester equivalent to 168 pg of Br2CA gave a 29% recorder response, where 33% recorder response was obtained from 4 ng of 3-PBacid. Recoveries of Br2CA and 3-PBacid were calculated by comparing the GC peak height of the respective esters. The short column clean-up technique that employed deactivated Florosil (10% water), and hexane-dichloromethane as eluting solvent was fast, efficient and reproducible. During the clean-up, the two esters were collected separately since the vast differences in their ECD responses made it difficult to estimate 3-PBacid in the presence of Br2CA. Use of the large sample required to detect the 3-PBacid ester caused merger of the two peaks.
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243
(b) Gas Chromatographv (GC). A Perkin-Elmer Sigma 1 gas Chromatograph system equipped with a ö Ni electron capture detector (ECD) was used under the following conditions: (i) glass column 1.82 m x
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4 mm (i.d.) packed with 5% OV-210 on 80-100 mesh Gaschrom Q; injector, column and detector temperatures were at 275, 245 and 400°C, respectively; carrier gas was 5% argon-methane, flow rate was 35 mL/min for deltamethrin, and (ii) glass column 3% SE-30 on 80-100 mesh chromosorb WHP (1.5 m χ 4 mm (i.d.); injector, column and detector were at 275, 165 and 400°C, respectively; carrier gas flow rate was 50 mL/min for trichloroethyl esters of Br2CA and 3-PBacid. (c) Gas-Chromatography - Mass Spectrometry (GC-MS). The GC-MS analyses were carried out on a Finnigan mass spectrometer (Model MAT 312). The MS was connected to the INCOS data system. The spectra were recorded in electron impact mode at 70 eV.
RESULTS AND DISCUSSION No overt sign of ill effects (watery eyes, diarrhea) was observed in the cows when fed orally 2 or 10 ppm of deltamethrin in regular diet for 28 consecutive days. Further, the daily feed intake and milk production were not affected during the course of this investigation. Also, no gross pathological changes were observed when the cows were slaughtered. Deltamethrin, Br2CA and 3-PBacid were effectively extracted from the sample matrix by ethanol-ether (1:3, v/v). Deltamethrin, Br2CA and 3-
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tu CO ζ
o CL CO UJ OC
oc UJ
o oc
o υ UJ
ce
0
TIME Figure 1. Gas chromatograms of milk extracts of cows fed (A) Control diet, (B) a known amount of deltamethrin added to the extract of Control milk, (C) Control milk fortified with 0.001 ppm deltamethrin, (D) 2 ppm deltamethrin, (E) 10 ppm deltamethrin.
I
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
245
TABLE 1 Average Recoveries of Deltamethrin and Metabolites from Biological Samples.
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Recovery (%)a Biological Samples Added ppm.
Milk.
0.01 0.005 0.001 0.027
101±13 103±15 71± 4
S. fat
Renal fat Hind muscle Fore muscle liver
68±15
103± 17
100+10
Kidney
72± 30
91±30
b
83± 7
82± 10
b
Br2CA 0.0235
81± 8 68± 11
61± 15
80+8 3-PBadd
0.034 a b
77± 1 62±10
61±15
52+8
63+7
b
b
Average of 2 or 3 replicates. Could not be ascertained due to interference peaks.
PBacid were collected in a single fraction from gel permeation chromatography. The GC of milk extracts are shown in Figure 1. Recoveries of deltamethrin and the metabolites from fortified biological samples (milk and tissues) are recorded in Table 1. At 1 part per billion fortification level, the recovery from milk ranged between 67-75%. The recovery values for deltamethrin are based on the ratio of GC peak heights with standard. The response of deltamethrin under the GC condition detailed was linear between 26-428 pg. The detection limit of deltamethrin in the extracts was highly dependent on the column and detector
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conditions. It was observed that repeated injection of biological extracts resulted in decreased sensitivity due to fat content in the extract. To maintain the column at its top performance, re-conditioning of column and
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clean-up of ECD were required often. The trichloroethyl esters of Br2CA and 3-PBacid gave well defined peaks on a 3% SE-30 column at 165°C. The relative retention time for Br2CA ester was 0.87 times of the 3-PBacid ester. The response of Br2CA ester was linear between 94 and 396 pg; whereas for the 3-PBacid ester it ranged between 680 and 3400 pg (3.4 ng). The mass spectra of the trichloroethyl esters Br2CA exhibited peaks at m/z 251, 253 and 255 (two bromine atoms pattern; molecular ion (M+-CO2CH2CC13). A similar fragmentation pattern was reported for the trichloroethyl ester of chlorine analogue Q2CA (George et al., 1977). Mass spectra of the trichloroethyl ester of 3-PBacid had a molecular ion at m/z 344 with a distinctive pattern for three chlorine atoms. In addition, the spectrum also had peaks at m/z 309 (M+-CO2CH2CC13). Deltamethrin residues were determined in milk at various times during the feeding period. Table 2 showed that deltamethrin residues were secreted into milk within 24 h after the commencement of feeding. More deltamethrin was secreted in milk of cows fed large quantities of deltamethrin. For example, the average deltamethrin residues in milk at its peak was < 0.008 and 0.027 ppm for 2 and 10 ppm dietary level, respectively. These observations are in agreement with the results
DELTAMETHRIN RESIDUES I N LACTATING DAIRY COWS
247
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TABLE 2 Residues of Deltamethrin in the Milk of Lactating Dairy Cows fed Deltamethrin in Diet for 28 Consecutive Days at 2 or 10 ppm Level. Residues 0
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
Deltamethrin residues in milk and tissues of lactating dairy cows a
a
M. Humayoun Akhtar , Claude Danis , H. a
Locksley Trenholm & Kenneth E. Hartin
a
a
Centre for Food and Animal Research, Research Branch , Agriculture Canada , Ottawa, Ontario, K1A 0C6 Published online: 21 Nov 2008.
To cite this article: M. Humayoun Akhtar , Claude Danis , H. Locksley Trenholm & Kenneth E. Hartin (1992) Deltamethrin residues in milk and tissues of lactating dairy cows, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 27:3, 235-253 To link to this article: http://dx.doi.org/10.1080/03601239209372777
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.
Downloaded by [University of Auckland Library] at 12:28 02 March 2015
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/termsand-conditions
J. ENVIRON. SCI. HEALTH, B 2 7 ( 3 ) , 2 3 5 - 2 5 3 ( 1 9 9 2 )
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DELTAMETHRIN RESIDUES IN MILK AND TISSUES OF LACTATING DAIRY COWS M. Humayoun Akhtar, Claude Danis, H. Locksley Trenholm and Kenneth E . Hartin Centre for Food and Animal Research, Research Branch, Agriculture Canada, Ottawa, Ontario K1A 0C6
Key
words: Deltamethrin, Residues, Pyrethroids, Milk
ABSTRACT Lactating dairy cows were fed deltamethrin (2 or 10 mg kg-1 feed) for 28 consecutive days and deltamethrin residues measured in milk and tissues. Deltamethrin residues were higher relative to dose administered. The
order of relative concentrations of deltamethrin in tissues, measured 1,
4, and 9 days after the last dose was: renal fat > subcutaneous fat > forequarter muscle > hindquarter muscle > liver > kidney. Depletion of deltamethrin residues in milk was very rapid indicationg the half-life of the insectide of about 1 day. Trace amounts of deltamethrin metabolites 3-
235 Copyright© 1992 by Marcel Dekker, Inc.
236
AKHTAR ET AL.
(2,2-dibromovmyl)-2,2-dimethylcyclopropane carboxylic acid ( < 0.0235 ppm) and 3-phenoxybenzoic acid ( < 0.034 ppm) were also detected in milk and
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tissues of treated cows.
INTRODUCTION Deltamethrin [(s)-o-cyano-3-phenoxybenzyl-cis-(lR,3R)-3-(2¿dibromovinyl)-2^-dimethylcyclopropanecarboxylate] is a potent insecticide with a broad spectrum activity towards a wide variety of insects that infest crops, animals, and vegetation. The technical product, DecisR, contains 98% deltamethrin (FAO, 1981). The metabolic fate of deltamethrin has been reported in vivo in rats (Ruzo et al., 1978), mice (Ruzo et al, 1979), laying hens (Akhtar et al., 1985), as well as in vitro using mouse liver microsomes (Shono et al., 1979), and cow and chicken liver homogenates (Akhtar, 1984). When lactating cows were dosed orally with 14C labeled (gem dimethyl or benzyl) deltamethrin, the insecticide was absorbed poorly from the rumen (Akhtar et al. 1986). Absorbed deltamethrin was secreted in milk at trace levels only, and did not accumulate in edible tissues to any significant extent. The Canadian Prairies often face grasshopper infestations (Research Branch Report 1984, 1985) which result in serious economic losses. Deltamethrin is registered for grasshopper control on forage and cereal crops at rates of 5.0 -7.5 g active ingredient (AI)/ hectare (ha) using
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
237
ground applicators (Hoechst Canada Inc., 1983). Aerial application of deltamethrin on pasture and rangeland at 7.2 g AI/ha resulted in 65% reduction in grasshopper density by the 4th day after application.
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Deltamethrin residues on forage and Utter determined within 6 hr after application were 2.19 and 0.79 ppm, respectively (all values are on dry matter basis [DM]) (Johnson et al. 1986). To assess the safety of deltamethrin residues to lactating cows feeding on treated forage in the present study 2 and 10 ppm of deltamethrin was added to the daily diet. The objective of the study was to determine residues of deltamethrin and its two major metabolites, namely 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylic add (Br2CA) and 3-phenoxybenzoic acid (3-PBadd) in milk, liver, kidney, fat (subcutaneous, renal) and meat (fore and hind quarter) for these two intakes. The level of 2 ppm is closer to that expected (2.19 ppm, Johnson et al. 1986) in the freshly treated pasture; whereas a 10 ppm is an exaggerated level to determine the safety at five times the daily intake.
MATERIALS AND METHODS Chemicals. Pestiddes and reagent grade solvents were used. Deltamethrin was supplied by Hoechst Canada Inc. Pure Br2CA and 3PBacid were available from previous studies (Akhtar et al., 198S, 1986). Stock solutions of deltamethrin, (i) 2.88 g in 100 mL acetone for 2 ppm, and (ii) 14.44 g in 100 mL acetone for 10 ppm were prepared. Daily feed
238
AKHTAR ET AL.
was prepared by treating 200 g grain with 0.5 mL volume of either of the two stock solutions. Feed for control cow was treated in the same way with same volume of acetone. In order to remove acetone, the containers
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were allowed to stand at room temperature. Fortified feed was prepared 2 or 3 days prior to use. Animal Treatment. Seven cows with a daily milk production of 10.3-13.8 kg were used in this study. They were kept in individual stalls and fed to appetite a standard dairy ration for lactating cows for 14 days, while daily feed intake was recorded. Based on an average daily intake of 14.4 kg (DM) during this acclimatization period, the cows on 2 and 10 ppm treatment received 288 mg and 1.4 g, respectively, deltamethrin daily in feed. The cows were milked twice daily at 06:00 and 16:00 h. Seven cows were divided into three groups: control (1), 2 ppm (3), and 10 ppm (3) treatment for a 28-day test period. Each cow was fed treated grains immediately after morning and evening milking. A standard total mixed ration was fed after cows had consumed all the treated grains. At each milking, the milk was weighed and sub-sampled. Daily composited samples were analyzed for fat by infrared milk analyses system (Multispec IR. Analyser Mark Π, Whetdrake, York England) on weekly basis. Subsampled daily milk samples were stored at 4°C for residue analyses. The control and one cow from each treatment group were slaughtered at 24 h after the last treatment with deltamethrin. Subsequently, one cow from each pesticide-fed group was killed at 4 and 9 days after the last
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
239
treatment. Each cow was examined for gross pathological changes, samples of subcutaneous and renal fat, front and hind quarter muscles, liver and kidney were removed and stored at -20°C until analyzed.
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Extraction,
(i) Milk: An aliquot (25 mL) of sub-sampled milk, in
duplicate, after pH adjustment to about 2 and addition of 2 mL of 5% aqueous potassium oxalate was shaken gently with 40 mL of ethanol-ether (1:3, v/v) in a 250 mL Mixxor (Scientific Products and Equipment). The organic phase was removed, and the aqueous phase was vigorously shaken with ethanol-ether (4 χ 40 mL). The organic phase was reduced in volume to ca 5 mL on a rotary evaporator at 40°C. The residue was quantitatively transferred into a centrifuge tube, and the flask was rinsed with acetone (3 χ 3 mL). Acetone was evaporated under a stream of Nj, the residue extracted with hexane ( 5 x 5 mL), passed over NajSQ,, and concentrated to 2mL (in Tissues: A 20 g portion, in duplicate, of tissues (muscle, liver and kidney) was cut into small pieces (ca 5 mm), and homogenized in a Waring blender with 50 mL ethanol-ether (1:3) and 2 mL of 0.1 Ν HC1, and the homogenate filtered. The residue was again cut into small pieces and blended with the extracting solvent (4 χ 30 mL). The filtrate was concentrated to ca 5 mL, and the residue was quantitatively transferred into a centrifuge tube, and the flask was rinsed with acetone. Organic solvent was evaporated under a stream of N2, the residue extracted with hexane ( 5 x 5 mL), dried over NajSO^ and the volume reduced to 2 mT.,
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AKHTAR ET AL.
(iii) Fat:
An aliquot (20 g), in duplicate, was cut into small pieces (about
5 mm) and blended with 50 mL ethanol-ether (1:3) and 2 mL of 0.1 Ν HC1. The organic phase was decanted and the residue was re-extracted
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with ethanol-ether (4x) 30 mL The fíltrate was concentrated to about 5 mL, the residue dissolved in 25 mL hexane, and the mixture filtered through pre-washed anhydrous NajSCV Partition. The hexane extracts were shaken with acetonitrile ( 4 x 2 mL), and the acetonitrile layer was evaporated to near dryness. The hexane phase was discarded. Clean-up. To remove residual lipids, a gel permeation clean-up technique was used. The column, a 100 mL buret (i.d. 15 mm), was packed with a glass wool (prewashed with eluting solvent) plug, and 15 g of Sephadex LH-20 (Pharmacia) previously soaked in 60 mL 2-propanol for 48 h. The column was washed with an additional 50 mL 2-propanol. Acetonitrile residues obtained after partitioning, as detailed above, was dissolved in 1 mL of 2-propanol, and placed in the column and eluted with 2-propanol. The first 50 mL of eluant was discarded, while the next 125 mL fraction collected. It was shown by gas chromatography (GC) in the preliminary experiment and radiochemical analyses that spiked 14C deltamethnn, Br2CA and 3-PBacid completely eluted with 2-propanol in the 65-100 mL fraction from the column. Furthermore, no tailing was observed in the next 25 mL fraction. However, to ensure complete recovery of the residues, 125 mL fractions were collected.
DELTAMETHRIN RESIDUES IN LACTATING DAIRY COWS
241
Each packed column was used for 10 samples before being recharged with a fresh packing. It was noted that each column could be used for up to 20 additional samples.
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Analyses.
The eluants from the Sephadex column were evaporated to
dryness, and the residues were redissolved in acetone and transferred into centrifuge tubes. The acetone was removed and residues dissolved in 2 mL hexane for GC analyses. The hexane solution was divided into two equal portions of 1 mL each. One portion was analyzed directly by GC for deltamethrin while the second portion was derivatized for the analyses of Br2CA and 3-PBacid. (a) Derivatization. The acid metabolites were converted into trichloroethyl derivatives by the method of George and McDonough (1975) with slight modifications. The volume of the reactants DCC-pyridine and 2,2,2trichloroethanol were reduced in half while the reaction time was doubled. Changes in reaction conditions did not alter the yield, but produced fewer interfering substances. The derivatives were extracted with hexane and subjected to clean-up technique as detailed below: The clean-up was carried out on a disposable Pasteur capillary pipet (16 cm χ 0.8 cm i.d.) containing a cotton plug, 5 cm of deactivated Florisil (10% H2O), which was topped with 1 cm of 25% cellulose in decolorizing carbon in that order. The column was prewashed with hexanedichloromethane. The first 3 mL (1st fraction) was discarded, and the next
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two fractions of 4 mL (2nd fraction) and 8 mL (3rd fraction) each were collected separately for analysis of Br2CA and 3-PBacid derivatives. In the preliminary studies, it was shown that the trichloroester of 3-
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PBadd eluted completely in the 4 mL (2nd fraction), while Br2CA ester was exclusively in the 8 mL fraction (3rd fraction). The eluant from the Sephadex Column could not be analyzed directly for Br2CA and 3-PBacid by GC on a 3% SE-30 or a 5% OV-210 column without prior derivatization. The methyl derivative of 3-PBacid was not ECD-sensitive and required approximately 1/ig of material to provide 44% recorder response when a flame ionization detector (FID) was used. The ECD-sensitive trichloroethyl derivatives were prepared by treating the extracts with DCC-pyridine and trichloroethanol. As expected, the trichloroethyl ester of Br2CA was much more sensitive than that of 3PBacid. For example, the ester equivalent to 168 pg of Br2CA gave a 29% recorder response, where 33% recorder response was obtained from 4 ng of 3-PBacid. Recoveries of Br2CA and 3-PBacid were calculated by comparing the GC peak height of the respective esters. The short column clean-up technique that employed deactivated Florosil (10% water), and hexane-dichloromethane as eluting solvent was fast, efficient and reproducible. During the clean-up, the two esters were collected separately since the vast differences in their ECD responses made it difficult to estimate 3-PBacid in the presence of Br2CA. Use of the large sample required to detect the 3-PBacid ester caused merger of the two peaks.
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(b) Gas Chromatographv (GC). A Perkin-Elmer Sigma 1 gas Chromatograph system equipped with a ö Ni electron capture detector (ECD) was used under the following conditions: (i) glass column 1.82 m x
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4 mm (i.d.) packed with 5% OV-210 on 80-100 mesh Gaschrom Q; injector, column and detector temperatures were at 275, 245 and 400°C, respectively; carrier gas was 5% argon-methane, flow rate was 35 mL/min for deltamethrin, and (ii) glass column 3% SE-30 on 80-100 mesh chromosorb WHP (1.5 m χ 4 mm (i.d.); injector, column and detector were at 275, 165 and 400°C, respectively; carrier gas flow rate was 50 mL/min for trichloroethyl esters of Br2CA and 3-PBacid. (c) Gas-Chromatography - Mass Spectrometry (GC-MS). The GC-MS analyses were carried out on a Finnigan mass spectrometer (Model MAT 312). The MS was connected to the INCOS data system. The spectra were recorded in electron impact mode at 70 eV.
RESULTS AND DISCUSSION No overt sign of ill effects (watery eyes, diarrhea) was observed in the cows when fed orally 2 or 10 ppm of deltamethrin in regular diet for 28 consecutive days. Further, the daily feed intake and milk production were not affected during the course of this investigation. Also, no gross pathological changes were observed when the cows were slaughtered. Deltamethrin, Br2CA and 3-PBacid were effectively extracted from the sample matrix by ethanol-ether (1:3, v/v). Deltamethrin, Br2CA and 3-
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TIME Figure 1. Gas chromatograms of milk extracts of cows fed (A) Control diet, (B) a known amount of deltamethrin added to the extract of Control milk, (C) Control milk fortified with 0.001 ppm deltamethrin, (D) 2 ppm deltamethrin, (E) 10 ppm deltamethrin.
I
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TABLE 1 Average Recoveries of Deltamethrin and Metabolites from Biological Samples.
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Recovery (%)a Biological Samples Added ppm.
Milk.
0.01 0.005 0.001 0.027
101±13 103±15 71± 4
S. fat
Renal fat Hind muscle Fore muscle liver
68±15
103± 17
100+10
Kidney
72± 30
91±30
b
83± 7
82± 10
b
Br2CA 0.0235
81± 8 68± 11
61± 15
80+8 3-PBadd
0.034 a b
77± 1 62±10
61±15
52+8
63+7
b
b
Average of 2 or 3 replicates. Could not be ascertained due to interference peaks.
PBacid were collected in a single fraction from gel permeation chromatography. The GC of milk extracts are shown in Figure 1. Recoveries of deltamethrin and the metabolites from fortified biological samples (milk and tissues) are recorded in Table 1. At 1 part per billion fortification level, the recovery from milk ranged between 67-75%. The recovery values for deltamethrin are based on the ratio of GC peak heights with standard. The response of deltamethrin under the GC condition detailed was linear between 26-428 pg. The detection limit of deltamethrin in the extracts was highly dependent on the column and detector
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conditions. It was observed that repeated injection of biological extracts resulted in decreased sensitivity due to fat content in the extract. To maintain the column at its top performance, re-conditioning of column and
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clean-up of ECD were required often. The trichloroethyl esters of Br2CA and 3-PBacid gave well defined peaks on a 3% SE-30 column at 165°C. The relative retention time for Br2CA ester was 0.87 times of the 3-PBacid ester. The response of Br2CA ester was linear between 94 and 396 pg; whereas for the 3-PBacid ester it ranged between 680 and 3400 pg (3.4 ng). The mass spectra of the trichloroethyl esters Br2CA exhibited peaks at m/z 251, 253 and 255 (two bromine atoms pattern; molecular ion (M+-CO2CH2CC13). A similar fragmentation pattern was reported for the trichloroethyl ester of chlorine analogue Q2CA (George et al., 1977). Mass spectra of the trichloroethyl ester of 3-PBacid had a molecular ion at m/z 344 with a distinctive pattern for three chlorine atoms. In addition, the spectrum also had peaks at m/z 309 (M+-CO2CH2CC13). Deltamethrin residues were determined in milk at various times during the feeding period. Table 2 showed that deltamethrin residues were secreted into milk within 24 h after the commencement of feeding. More deltamethrin was secreted in milk of cows fed large quantities of deltamethrin. For example, the average deltamethrin residues in milk at its peak was < 0.008 and 0.027 ppm for 2 and 10 ppm dietary level, respectively. These observations are in agreement with the results
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TABLE 2 Residues of Deltamethrin in the Milk of Lactating Dairy Cows fed Deltamethrin in Diet for 28 Consecutive Days at 2 or 10 ppm Level. Residues 0
