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Fenarimol
12 Fenarimol Ε. W . D A Y , J R . AND Ο . D . D E C K E R CI
a-(2-Chlorophenyl)-a-(4-chlorophenyl)-5-pyrimidinemethanol
I. GENERAL A. Empirical Formula C 1 7H 1 2C 1 2 N 2 0 (Mol. wt. 331.2). B. Alternate Names The chemical was first tested under the code number EL-222. The American National Standards Institute and the British Standards Institute have accepted fenarimol as the c o m m o n (generic) name for the chemical. Rubigan® and Bloc® are registered t r a d e m a r k s of Elanco Products Co., a Division of Eli Lilly and C o . for formulated products of fenarimol. C. Source of Analytical Standard Elanco Products Co., a Division of Eli Lilly and Co., P.O. Box 1750, Indianapolis, Indiana 46285. D. Biological Properties 1. FUNGICIDAL PROPERTIES
Fenarimol is a foliarly applied fungicide that provides control of m a n y economically important diseases of plants, including the powdery mildews 173 ANALYTICAL METHODS FOR PESTICIDES AND PLANT GROWTH REGULATORS, VOL. XIII
Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-784313-2
174
Ε. W . DAY, JR. AND O. D . DECKER
on apple, cherry, cucurbits, grape, pepper, rose, and t o m a t o . It has also demonstrated activity against apple scab, against turf diseases such as dollar spot and brown patch, and against the leaf spot diseases of cherry, peanut, soybean, strawberry, and sugar beet. The mechanisms by which fenarimol inhibits fungal growth have been demonstrated by Ragsdale and Sisler (1973) and by Buchenauer (1977) to involve inhibition of the formation of the fungal sterol, ergosterol. 2.
TOXICITY
The acute oral L D 5 0 values of technical fenarimol in mice and rats are ~ 4 5 0 0 and ~ 2 5 0 0 mg/kg, respectively. Oral doses of 200 mg/kg had no adverse effects on beagle dogs. N o dermal effects have been observed on rabbits, and instillation of 68 mg into rabbit eyes caused only transient irritation. Fenarimol has been shown to be nonmutagenic in laboratory tests and was not teratogenic in rabbits at doses u p to 35 mg/kg. When mice were given diets containing u p to 600 p p m of fenarimol for 24 m o n t h s , no toxicologically significant effects were noted. Fenarimol produced no signs of toxicity when fed to bobwhite quail and mallard ducks at 6550 p p m for 5 days. The 96-hour L C 5 0 values for bluegill and rainbow trout were 5.7 and 4.1 mg/liter, respectively. E a r t h w o r m s were not affected by 100 p p m fenarimol in soil, and the chemical was not toxic to adult honeybees when fed at 100 ppm. E. History The fungicidal properties of fenarimol were first reported by Brown et al (1975). Fenarimol is protected by U.S. Patent N o . 3818009, which was issued on June 18,1974, and by patents issued in a number of other countries. F. Physical Properties Pure fenarimol is a white crystalline solid that melts at 117-119°C. It is soluble in organic solvents such as acetone, acetonitrile, benzene, chloroform, and methanol, but only slightly soluble in hexane. Its solubility in 7 water is 13.7 p p m at p H 7 and 25°C, and its vapor pressure is 2.2 χ 10" Torr at 25°C. The log of the o c t a n o l - w a t e r partition coefficient is 3.69. G. Chemical Properties 1. METHOD OF SYNTHESIS
Fenarimol is synthesized by a patented process that involves the reaction of 5-bromopyrimidine with the appropriate ketone in a cryogenic reactor.
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175
2. STABILITY
Fenarimol is stable to hydrolysis at p H 3,6, and 9 for 4 weeks at temperatures of 25, 37, and 52°C. It is thermally stable through its melting point but begins to decompose above 225°C. It is susceptible to photolytic degradation in aqueous solution and as a thin film on a solid surface resulting in the formation of a large n u m b e r of p h o t o p r o d u c t s . H. Formulations Fenarimol is available in several types of formulations, including emulsifiable concentrates (4 and 12%), wettable powders (6 and 12%), and an aqueous suspension concentrate (12%). F o r m u l a t e d fenarimol has been found to be physically compatible with a large number of agricultural chemicals in tank-mixes, including insecticides, other fungicides, growth regulators, and foliar nutrients.
II. ANALYSIS A. Formulations 1. REVIEW OF METHODS
G a s chromatography (GC) is used for the determination of fenarimol in technical material and formulations.
176
Ε. W. DAY, JR. AND O. D. DECKER 2. RECOMMENDED METHOD—GC
a.
Principle
The fenarimol is dissolved in or extracted with an appropriate solvent and then assayed by G C using flame ionization detection ( F I D ) . b.
Reagents Methanol, analytical reagent. Chloroform, analytical reagent. Fenarimol standard, 1.0 mg/ml in chloroform.
c.
Apparatus
Gas Chromatograph, Hewlett-Packard Model 5790, equipped with a flame ionization detector, Model 7621A automatic liquid sampler, and Model 7123A 1-mV recorder, or equivalent G C system. The c h r o m a t o graphic column is 120 cm χ 3 m m i.d. glass tubing packed with 3 % OV-17 (w/w) on 100/120 mesh C h r o m o s o r b W — H P (Applied Science Laboratories, State College, Pennsylvania). The column is conditioned at 300°C for 16 hours under carrier gas purge prior to use. d.
Procedure i. Sample
Preparation
(a) Technical Material. Accurately weigh a 100-mg sample of technical material into a 100-ml volumetric flask. Dissolve in and dilute to volume with chloroform. (b) Emulsifiable Concentrate. Accurately weigh a sample equivalent to 100 mg fenarimol into a 100-ml volumetric flask. Dissolve and dilute to volume with chloroform. (c) Aqueous Suspensions. Accurately weigh a sample equivalent to 100 mg of fenarimol into a 100-ml volumetric flask. Disperse the sample with ~ 2 ml water. Dissolve and dilute to volume with methanol. (d) Wet table Powders. Accurately weigh a sample equivalent to 100 mg fenarimol into a 250-ml glass-stoppered Erlenmeyer flask. A d d exactly 100 ml chloroform and a magnetic stirring bar. Replace the stopper, place on a magnetic stirrer, and stir for at least 30 minutes. ii. Gas Chromatography
Conditions
Flash heater temperature: 290°C. Column temperature: 260°C.
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FENARIMOL
Detector temperature: 290°C. Nitrogen carrier flow rate: 35 ml/minute. Hydrogen flow rate: 35 ml/minute. Airflow rate: 350 ml/minute. Electrometer range: 10. Attenuation: as needed. Fenarimol retention time: ~ 4 minutes. iii.
Measurement
Inject ~ 2 μΐ of the standard and sample solutions into the gas Chromatograph. Measure the peak response for the fenarimol in each solution by an acceptable technique. iv.
Calculations
Calculate the percentage of fenarimol in the sample as follows : fenarimol (%) =
^ ^ x P
s
td
where Rs is the response for the sample, Rsid the response for the standard, C s td the concentration of the standard (mg/ml), W% the weight of the sample (mg), Vs the volume of the sample (ml), and P s td the purity (%) of the s t a n d a r d Prior to the measurement of experimental samples, the analyst should inject a series of graded levels of fenarimol to prepare a standard curve. The fenarimol standard should be injected periodically during the course of the measurement of a series of samples to ensure that the instrument response has not changed. v.
Discussion
The precision of the method is approximately 3 % at the 9 5 % confidence level. O n instruments not equipped with an autosampling device, it is preferable to use an internal standard. Dibenzyl phthalate has been found to be a suitable internal standard. B. Residue Analysis 1. RECOMMENDED METHOD
a.
Principle
Fenarimol is extracted from crops with methanol, from soil with hot aqueous methanol, and from water with dichloromethane. A n aliquot of the extract is purified by liquid-liquid partitioning and alumina column
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Ε. W. DAY, JR. AND O. D. DECKER
chromatography. Detection and measurement is by electron capture gas chromatography ( E C - G C ) . b.
Reagents Alumina, Alcoa F-20, deactivated by the addition of 4% water. 1-Chlorobutane, reagent grade, redistilled. Dichloromethane, reagent grade, redistilled. Ethyl acetate, reagent grade. Fenarimol, analytical standard, 50 ^g/ml stock solution in toluene. Methanol, reagent grade. Sodium chloride, 5% in deionized (DI) water. Sodium sulfate, anhydrous, reagent grade. Toluene, reagent grade, redistilled.
c.
Apparatus
Grinding and blending equipment. Gyratory shaker, New Brunswick Model G 3 3 , or equivalent. Water bath, 80°C. Chromatography columns, 250 χ 14 m m i.d., equipped with a 250-ml solvent reservoir and removable Teflon stopcock, or equivalent. Rotary vacuum evaporator, Rinco. Gas Chromatograph, equipped for glass on-column injection and electron capture detection. d. Gas Chromatography
Conditions
Instrument: Hewlett-Packard Model 5713A (or equivalent) equipped 6 3 with a N i electron capture detector. Column: glass, 120 cm χ 2.0 m m i.d., packed with 2% OV-17 on C h r o m o s o r b W — H P 80-100 m e s h ; alternate packings are 5% XE-60 or 5% Dexsil 300 on C h r o m o s o r b W — H P 80-100 mesh. Column temperature: 230°C Injector temperature: 250°C. Detector temperature: 300°C. Carrier gas: 10% methane in argon. Carrier flow rate: 50-60 ml/minute. Electrometer : attenuation set to provide 30% full-scale deflection from the injection of 0.4 ng of fenarimol. The retention time of fenarimol is about 4.5 minutes under the above conditions.
12.
e. Experimental i. Sample
FENARIMOL
179
Procedures Preparation
(a) Soil. Mix the soil sample in a suitable blender, adding dry silica sand as needed if the soil is too moist to flow freely. Weigh a 50-gm sample into a pint mason jar, and add 200 ml of m e t h a n o l - w a t e r (3:1 v/v). Cover the jar with a watch glass, m a r k the liquid level, and heat for 30 minutes in an 80°C water bath. Remove jar from heat, allow to cool, add m e t h a n o l - w a t e r (3:1 v/v) to the mark, and mix well. Allow the particles to settle. Transfer a 10-ml aliquot of the supernatant extract to a 125-ml separatory funnel. (b) Fresh Fruit and Vegetable, Pomace, Sauce. C h o p and blend the sample to provide a homogeneous mixture. Weigh a representative 100-gm sample into a pint mason jar, and add 120 ml of methanol. F o r dried pomace, use a 25-gm sample and 200 ml of methanol. Shake on a gyratory platform shaker for 15 minutes at an oscillating speed sufficient to provide a vigorous " s w i r l " to the liquid (about 300 rpm). Allow the solids to settle, and transfer by pipette a 20-ml aliquot of the supernatant extract to a 250-ml separatory funnel. If the extract is turbid, pass a portion through W h a t m a n N o . 1 filter paper before making the transfer. (c) Raisins. Weigh a representative 100-gm sample into an 8-ounce mason jar, add 80 ml of deionized water, and blend on a Hamilton-Beach (Model 262 or equivalent) blender. Quantitatively transfer the blended sample to a pint mason jar with the aid of 120 ml of methanol. Swirl for 15 minutes on a gyratory shaker, and allow solid particles to separate. If the extract is turbid, pass about 40 ml through a W h a t m a n N o . 1 filter paper. Transfer a 20-ml aliquot of clear extract to a 250-ml separatory funnel. (d) Juice, Wine. Weigh a 10-gm sample, and quantitatively transfer to a 125-ml separatory funnel. (e) Water. Place 25-ml aliquot or any other desired volume in an appropriately sized separatory funnel. (f) Dry Tissue (Cereal Grains, Beans, Hay, Straw). Weigh a 25-gm sample into a pint mason jar. Estimate the,amount of moisture in the sample, and add sufficient methanol to bring the total liquid volume to 200 ml. F o r example, if moisture is estimated at 2 0 - 6 0 % , add 190 ml of methanol, and if less than 20% add 200 ml of methanol. Swirl the mixture for 15 minutes on a gyratory shaker, and allow the solids to settle. Decant about 40 ml of supernatant liquid through a W h a t m a n N o . 1 filter and transfer a 20-ml aliquot of filtrate to a 250-ml separatory funnel.
Ε. W. DAY, JR. AND O. D. DECKER
180 ii. Partitioning
Cleanup
A d d 50 ml of 5% aqueous sodium chloride to the extract in the separatory funnel. Extract with two 20-ml portions of dichloromethane, and combine the extracts in a 125-ml boiling flask. Extracts may be passed through a bed of anhydrous sodium sulfate if phase separation is incomplete. Rinse the bed with dichloromethane, and add the rinsings to the boiling flask. Evaporate the dichloromethane to dryness using a rotary vacuum evaporator and a 4 0 - 4 5 ° C water bath. iii. Alumina Column
Procedure
Place a pledget of glass wool in the b o t t o m of a 14-mm i.d. glass chromatographic column. A d d 15 ml of 1-chlorobutane, and t a m p the glass wool with a stirring rod to eliminate air bubbles. Add 10 gm (13 ± 0.5 ml) of standardized (see Section II, B, 1, f) alumina through a funnel. A d d 5-10 ml of 1-chlorobutane, stir with a rod, and rinse down the sides of the column with additional solvent. After the alumina has settled, add about 1.5 cm layer of anhydrous sodium sulfate, taking care to avoid disturbing the alumina surface. Again, rinse the sides of the column with 1-chlorobutane, and drain the solvent to the t o p of the sodium sulfate layer. Transfer the residue in the boiling flask from Section II, B, 1, e (ii) above to the column, using two 5-ml portions of 1-chlorobutane. Allow each addition of solution to pass onto the column at a flow rate of 3 - 5 ml/minute. Rinse the sides of the column with an additional 5 ml of 1-chlorobutane, and drain to the top of the adsorbent. Wash the column with 40 ml of 1chlorobutane-ethyl acetate (9:1 v/v), followed by 50 ml of 1-chlorobutanemethanol (99:1 v/v). Discard all eluates to this point. Elute the fenarimol from the column with 65 ml of 1-chlorobutane-methanol (99:1 v/v), collecting the eluate in a 125-ml boiling flask. Evaporate the eluate solution to dryness, using a rotary vacuum evaporator and a 4 0 - 4 5 ° C water bath. Dissolve the residue in toluene (1.0 ml for crop and water samples and 5.0 ml for soil samples), mix thoroughly, and proceed with the G C measurement. iv. Controls and
Recoveries
Assay a standard recovery and a control sample with each set of experimental samples. The following fortifications are suggested: Soil: 0.1 ppm, 5.0 ^g fenarimol to 50 gm control soil. Freshfruit, vegetables, pomace, sauce:0.0\ p p m , 1.0μgto 100 gm control sample. Raisins: 0.01 p p m , 1.0 μg to 100 gm control sample. Juice, wine, water: 0.01 ppm, 0.1 ^g to 10 ml control sample. Dry crops and tissue: 0.04 ppm, 1.0 μg to 25 gm of control sample.
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FENARIMOL
181
If a control sample is unavailable, process a system recovery sample (all reagents except sample) that simulates the desired recovery level. v. Gas
Chromatography
Prepare a standard curve by injecting solutions of fenarimol in toluene into the gas Chromatograph. A concentration range of 0.025-0.25 //g/ml and injection volumes of 5 μΐ are suggested. Inject the toluene solutions of processed control, recovery, and experimental samples. A n automatic sampler-injector is recommended for more reproducible results. Measure the response of the fenarimol peak, and determine its concentration in the toluene solutions from the standard curve. vi.
Calculations
Calculate results for fenarimol from the following equations : recovery (%) =
(Cr -
fenarimol (ppm) =
C C) M ( 1 0 0 ) Wx
CsV{DA(\00) ^ [ r e c o v e r y (%)]
where C r , C c , and C s are the concentrations of fenarimol (pgjmX) in the final solutions from recovery, control, and experimental samples, respectively; V{ is the final volume (ml); A is the aliquot factor from the initial extraction; W is the weight of fenarimol added for recovery (/ig); D is the final dilution factor if a n y ; and Ws is the weight of sample (gm). f.
Discussion
Each batch of deactivated alumina should be standardized prior to use. Prepare a column as described in Section II, B, 1, e, iii, and add 0.1 μg of fenarimol to the top of the column. Elute the column with 10-ml portions of 1-chlorobutane-methanol (99:1 v/v), and assay each fraction for fenarimol content. Adjust elution volumes in Section II, B, 1, e, iii if necessary to collect a m i n i m u m of 90% of the added fenarimol. The procedure described usually yields recoveries in excess of 90% at the levels indicated. The limit of detection is about 0.002 p p m for fresh fruits, vegetables, pomace, raisins, juice, and wine. F o r dry pomace and crops, the limit is about 0.01 p p m , and in soil, it is about 0.02 ppm. The procedure for water samples can usually be simplified by eliminating the alumina column step. The dichloromethane extract is dried, evaporated to dryness, and the residue dissolved in toluene for G C measurement. Fenarimol may also be extracted from water with toluene followed by direct injection of an aliquot of the extract into the gas Chromatograph.
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Ε. W. DAY, JR. AND O. D. DECKER
The limit of detection is dependent on the water : toluene ratio, a n d a 10-ml to 5-ml ratio will result in a limit of about 10 p p b . N u a r i m o l (see Chapter 13, this volume) can be used as an internal standard for fenarimol analysis if improved precision is desired.
REFERENCES Brown, I. F., Taylor, H. M., and Hall, H. R. (1975). Proc. Am. Phytopath. Soc. 2, 31. Buchenauer, H. (1977). Ζ. Pflanzenkr. Pflanzenshutz 84, 286. Ragsdale, Ν. N., and Sisler, H. D. (1973). Pesticide Biochem. Physiol 3, 20.
a-(2-Chlorophenyl)-a-(4-chlorophenyl)-5-pyrimidinemethanol
I. GENERAL A. Empirical Formula C 1 7H 1 2C 1 2 N 2 0 (Mol. wt. 331.2). B. Alternate Names The chemical was first tested under the code number EL-222. The American National Standards Institute and the British Standards Institute have accepted fenarimol as the c o m m o n (generic) name for the chemical. Rubigan® and Bloc® are registered t r a d e m a r k s of Elanco Products Co., a Division of Eli Lilly and C o . for formulated products of fenarimol. C. Source of Analytical Standard Elanco Products Co., a Division of Eli Lilly and Co., P.O. Box 1750, Indianapolis, Indiana 46285. D. Biological Properties 1. FUNGICIDAL PROPERTIES
Fenarimol is a foliarly applied fungicide that provides control of m a n y economically important diseases of plants, including the powdery mildews 173 ANALYTICAL METHODS FOR PESTICIDES AND PLANT GROWTH REGULATORS, VOL. XIII
Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-784313-2
174
Ε. W . DAY, JR. AND O. D . DECKER
on apple, cherry, cucurbits, grape, pepper, rose, and t o m a t o . It has also demonstrated activity against apple scab, against turf diseases such as dollar spot and brown patch, and against the leaf spot diseases of cherry, peanut, soybean, strawberry, and sugar beet. The mechanisms by which fenarimol inhibits fungal growth have been demonstrated by Ragsdale and Sisler (1973) and by Buchenauer (1977) to involve inhibition of the formation of the fungal sterol, ergosterol. 2.
TOXICITY
The acute oral L D 5 0 values of technical fenarimol in mice and rats are ~ 4 5 0 0 and ~ 2 5 0 0 mg/kg, respectively. Oral doses of 200 mg/kg had no adverse effects on beagle dogs. N o dermal effects have been observed on rabbits, and instillation of 68 mg into rabbit eyes caused only transient irritation. Fenarimol has been shown to be nonmutagenic in laboratory tests and was not teratogenic in rabbits at doses u p to 35 mg/kg. When mice were given diets containing u p to 600 p p m of fenarimol for 24 m o n t h s , no toxicologically significant effects were noted. Fenarimol produced no signs of toxicity when fed to bobwhite quail and mallard ducks at 6550 p p m for 5 days. The 96-hour L C 5 0 values for bluegill and rainbow trout were 5.7 and 4.1 mg/liter, respectively. E a r t h w o r m s were not affected by 100 p p m fenarimol in soil, and the chemical was not toxic to adult honeybees when fed at 100 ppm. E. History The fungicidal properties of fenarimol were first reported by Brown et al (1975). Fenarimol is protected by U.S. Patent N o . 3818009, which was issued on June 18,1974, and by patents issued in a number of other countries. F. Physical Properties Pure fenarimol is a white crystalline solid that melts at 117-119°C. It is soluble in organic solvents such as acetone, acetonitrile, benzene, chloroform, and methanol, but only slightly soluble in hexane. Its solubility in 7 water is 13.7 p p m at p H 7 and 25°C, and its vapor pressure is 2.2 χ 10" Torr at 25°C. The log of the o c t a n o l - w a t e r partition coefficient is 3.69. G. Chemical Properties 1. METHOD OF SYNTHESIS
Fenarimol is synthesized by a patented process that involves the reaction of 5-bromopyrimidine with the appropriate ketone in a cryogenic reactor.
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FENARIMOL
175
2. STABILITY
Fenarimol is stable to hydrolysis at p H 3,6, and 9 for 4 weeks at temperatures of 25, 37, and 52°C. It is thermally stable through its melting point but begins to decompose above 225°C. It is susceptible to photolytic degradation in aqueous solution and as a thin film on a solid surface resulting in the formation of a large n u m b e r of p h o t o p r o d u c t s . H. Formulations Fenarimol is available in several types of formulations, including emulsifiable concentrates (4 and 12%), wettable powders (6 and 12%), and an aqueous suspension concentrate (12%). F o r m u l a t e d fenarimol has been found to be physically compatible with a large number of agricultural chemicals in tank-mixes, including insecticides, other fungicides, growth regulators, and foliar nutrients.
II. ANALYSIS A. Formulations 1. REVIEW OF METHODS
G a s chromatography (GC) is used for the determination of fenarimol in technical material and formulations.
176
Ε. W. DAY, JR. AND O. D. DECKER 2. RECOMMENDED METHOD—GC
a.
Principle
The fenarimol is dissolved in or extracted with an appropriate solvent and then assayed by G C using flame ionization detection ( F I D ) . b.
Reagents Methanol, analytical reagent. Chloroform, analytical reagent. Fenarimol standard, 1.0 mg/ml in chloroform.
c.
Apparatus
Gas Chromatograph, Hewlett-Packard Model 5790, equipped with a flame ionization detector, Model 7621A automatic liquid sampler, and Model 7123A 1-mV recorder, or equivalent G C system. The c h r o m a t o graphic column is 120 cm χ 3 m m i.d. glass tubing packed with 3 % OV-17 (w/w) on 100/120 mesh C h r o m o s o r b W — H P (Applied Science Laboratories, State College, Pennsylvania). The column is conditioned at 300°C for 16 hours under carrier gas purge prior to use. d.
Procedure i. Sample
Preparation
(a) Technical Material. Accurately weigh a 100-mg sample of technical material into a 100-ml volumetric flask. Dissolve in and dilute to volume with chloroform. (b) Emulsifiable Concentrate. Accurately weigh a sample equivalent to 100 mg fenarimol into a 100-ml volumetric flask. Dissolve and dilute to volume with chloroform. (c) Aqueous Suspensions. Accurately weigh a sample equivalent to 100 mg of fenarimol into a 100-ml volumetric flask. Disperse the sample with ~ 2 ml water. Dissolve and dilute to volume with methanol. (d) Wet table Powders. Accurately weigh a sample equivalent to 100 mg fenarimol into a 250-ml glass-stoppered Erlenmeyer flask. A d d exactly 100 ml chloroform and a magnetic stirring bar. Replace the stopper, place on a magnetic stirrer, and stir for at least 30 minutes. ii. Gas Chromatography
Conditions
Flash heater temperature: 290°C. Column temperature: 260°C.
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FENARIMOL
Detector temperature: 290°C. Nitrogen carrier flow rate: 35 ml/minute. Hydrogen flow rate: 35 ml/minute. Airflow rate: 350 ml/minute. Electrometer range: 10. Attenuation: as needed. Fenarimol retention time: ~ 4 minutes. iii.
Measurement
Inject ~ 2 μΐ of the standard and sample solutions into the gas Chromatograph. Measure the peak response for the fenarimol in each solution by an acceptable technique. iv.
Calculations
Calculate the percentage of fenarimol in the sample as follows : fenarimol (%) =
^ ^ x P
s
td
where Rs is the response for the sample, Rsid the response for the standard, C s td the concentration of the standard (mg/ml), W% the weight of the sample (mg), Vs the volume of the sample (ml), and P s td the purity (%) of the s t a n d a r d Prior to the measurement of experimental samples, the analyst should inject a series of graded levels of fenarimol to prepare a standard curve. The fenarimol standard should be injected periodically during the course of the measurement of a series of samples to ensure that the instrument response has not changed. v.
Discussion
The precision of the method is approximately 3 % at the 9 5 % confidence level. O n instruments not equipped with an autosampling device, it is preferable to use an internal standard. Dibenzyl phthalate has been found to be a suitable internal standard. B. Residue Analysis 1. RECOMMENDED METHOD
a.
Principle
Fenarimol is extracted from crops with methanol, from soil with hot aqueous methanol, and from water with dichloromethane. A n aliquot of the extract is purified by liquid-liquid partitioning and alumina column
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Ε. W. DAY, JR. AND O. D. DECKER
chromatography. Detection and measurement is by electron capture gas chromatography ( E C - G C ) . b.
Reagents Alumina, Alcoa F-20, deactivated by the addition of 4% water. 1-Chlorobutane, reagent grade, redistilled. Dichloromethane, reagent grade, redistilled. Ethyl acetate, reagent grade. Fenarimol, analytical standard, 50 ^g/ml stock solution in toluene. Methanol, reagent grade. Sodium chloride, 5% in deionized (DI) water. Sodium sulfate, anhydrous, reagent grade. Toluene, reagent grade, redistilled.
c.
Apparatus
Grinding and blending equipment. Gyratory shaker, New Brunswick Model G 3 3 , or equivalent. Water bath, 80°C. Chromatography columns, 250 χ 14 m m i.d., equipped with a 250-ml solvent reservoir and removable Teflon stopcock, or equivalent. Rotary vacuum evaporator, Rinco. Gas Chromatograph, equipped for glass on-column injection and electron capture detection. d. Gas Chromatography
Conditions
Instrument: Hewlett-Packard Model 5713A (or equivalent) equipped 6 3 with a N i electron capture detector. Column: glass, 120 cm χ 2.0 m m i.d., packed with 2% OV-17 on C h r o m o s o r b W — H P 80-100 m e s h ; alternate packings are 5% XE-60 or 5% Dexsil 300 on C h r o m o s o r b W — H P 80-100 mesh. Column temperature: 230°C Injector temperature: 250°C. Detector temperature: 300°C. Carrier gas: 10% methane in argon. Carrier flow rate: 50-60 ml/minute. Electrometer : attenuation set to provide 30% full-scale deflection from the injection of 0.4 ng of fenarimol. The retention time of fenarimol is about 4.5 minutes under the above conditions.
12.
e. Experimental i. Sample
FENARIMOL
179
Procedures Preparation
(a) Soil. Mix the soil sample in a suitable blender, adding dry silica sand as needed if the soil is too moist to flow freely. Weigh a 50-gm sample into a pint mason jar, and add 200 ml of m e t h a n o l - w a t e r (3:1 v/v). Cover the jar with a watch glass, m a r k the liquid level, and heat for 30 minutes in an 80°C water bath. Remove jar from heat, allow to cool, add m e t h a n o l - w a t e r (3:1 v/v) to the mark, and mix well. Allow the particles to settle. Transfer a 10-ml aliquot of the supernatant extract to a 125-ml separatory funnel. (b) Fresh Fruit and Vegetable, Pomace, Sauce. C h o p and blend the sample to provide a homogeneous mixture. Weigh a representative 100-gm sample into a pint mason jar, and add 120 ml of methanol. F o r dried pomace, use a 25-gm sample and 200 ml of methanol. Shake on a gyratory platform shaker for 15 minutes at an oscillating speed sufficient to provide a vigorous " s w i r l " to the liquid (about 300 rpm). Allow the solids to settle, and transfer by pipette a 20-ml aliquot of the supernatant extract to a 250-ml separatory funnel. If the extract is turbid, pass a portion through W h a t m a n N o . 1 filter paper before making the transfer. (c) Raisins. Weigh a representative 100-gm sample into an 8-ounce mason jar, add 80 ml of deionized water, and blend on a Hamilton-Beach (Model 262 or equivalent) blender. Quantitatively transfer the blended sample to a pint mason jar with the aid of 120 ml of methanol. Swirl for 15 minutes on a gyratory shaker, and allow solid particles to separate. If the extract is turbid, pass about 40 ml through a W h a t m a n N o . 1 filter paper. Transfer a 20-ml aliquot of clear extract to a 250-ml separatory funnel. (d) Juice, Wine. Weigh a 10-gm sample, and quantitatively transfer to a 125-ml separatory funnel. (e) Water. Place 25-ml aliquot or any other desired volume in an appropriately sized separatory funnel. (f) Dry Tissue (Cereal Grains, Beans, Hay, Straw). Weigh a 25-gm sample into a pint mason jar. Estimate the,amount of moisture in the sample, and add sufficient methanol to bring the total liquid volume to 200 ml. F o r example, if moisture is estimated at 2 0 - 6 0 % , add 190 ml of methanol, and if less than 20% add 200 ml of methanol. Swirl the mixture for 15 minutes on a gyratory shaker, and allow the solids to settle. Decant about 40 ml of supernatant liquid through a W h a t m a n N o . 1 filter and transfer a 20-ml aliquot of filtrate to a 250-ml separatory funnel.
Ε. W. DAY, JR. AND O. D. DECKER
180 ii. Partitioning
Cleanup
A d d 50 ml of 5% aqueous sodium chloride to the extract in the separatory funnel. Extract with two 20-ml portions of dichloromethane, and combine the extracts in a 125-ml boiling flask. Extracts may be passed through a bed of anhydrous sodium sulfate if phase separation is incomplete. Rinse the bed with dichloromethane, and add the rinsings to the boiling flask. Evaporate the dichloromethane to dryness using a rotary vacuum evaporator and a 4 0 - 4 5 ° C water bath. iii. Alumina Column
Procedure
Place a pledget of glass wool in the b o t t o m of a 14-mm i.d. glass chromatographic column. A d d 15 ml of 1-chlorobutane, and t a m p the glass wool with a stirring rod to eliminate air bubbles. Add 10 gm (13 ± 0.5 ml) of standardized (see Section II, B, 1, f) alumina through a funnel. A d d 5-10 ml of 1-chlorobutane, stir with a rod, and rinse down the sides of the column with additional solvent. After the alumina has settled, add about 1.5 cm layer of anhydrous sodium sulfate, taking care to avoid disturbing the alumina surface. Again, rinse the sides of the column with 1-chlorobutane, and drain the solvent to the t o p of the sodium sulfate layer. Transfer the residue in the boiling flask from Section II, B, 1, e (ii) above to the column, using two 5-ml portions of 1-chlorobutane. Allow each addition of solution to pass onto the column at a flow rate of 3 - 5 ml/minute. Rinse the sides of the column with an additional 5 ml of 1-chlorobutane, and drain to the top of the adsorbent. Wash the column with 40 ml of 1chlorobutane-ethyl acetate (9:1 v/v), followed by 50 ml of 1-chlorobutanemethanol (99:1 v/v). Discard all eluates to this point. Elute the fenarimol from the column with 65 ml of 1-chlorobutane-methanol (99:1 v/v), collecting the eluate in a 125-ml boiling flask. Evaporate the eluate solution to dryness, using a rotary vacuum evaporator and a 4 0 - 4 5 ° C water bath. Dissolve the residue in toluene (1.0 ml for crop and water samples and 5.0 ml for soil samples), mix thoroughly, and proceed with the G C measurement. iv. Controls and
Recoveries
Assay a standard recovery and a control sample with each set of experimental samples. The following fortifications are suggested: Soil: 0.1 ppm, 5.0 ^g fenarimol to 50 gm control soil. Freshfruit, vegetables, pomace, sauce:0.0\ p p m , 1.0μgto 100 gm control sample. Raisins: 0.01 p p m , 1.0 μg to 100 gm control sample. Juice, wine, water: 0.01 ppm, 0.1 ^g to 10 ml control sample. Dry crops and tissue: 0.04 ppm, 1.0 μg to 25 gm of control sample.
12.
FENARIMOL
181
If a control sample is unavailable, process a system recovery sample (all reagents except sample) that simulates the desired recovery level. v. Gas
Chromatography
Prepare a standard curve by injecting solutions of fenarimol in toluene into the gas Chromatograph. A concentration range of 0.025-0.25 //g/ml and injection volumes of 5 μΐ are suggested. Inject the toluene solutions of processed control, recovery, and experimental samples. A n automatic sampler-injector is recommended for more reproducible results. Measure the response of the fenarimol peak, and determine its concentration in the toluene solutions from the standard curve. vi.
Calculations
Calculate results for fenarimol from the following equations : recovery (%) =
(Cr -
fenarimol (ppm) =
C C) M ( 1 0 0 ) Wx
CsV{DA(\00) ^ [ r e c o v e r y (%)]
where C r , C c , and C s are the concentrations of fenarimol (pgjmX) in the final solutions from recovery, control, and experimental samples, respectively; V{ is the final volume (ml); A is the aliquot factor from the initial extraction; W is the weight of fenarimol added for recovery (/ig); D is the final dilution factor if a n y ; and Ws is the weight of sample (gm). f.
Discussion
Each batch of deactivated alumina should be standardized prior to use. Prepare a column as described in Section II, B, 1, e, iii, and add 0.1 μg of fenarimol to the top of the column. Elute the column with 10-ml portions of 1-chlorobutane-methanol (99:1 v/v), and assay each fraction for fenarimol content. Adjust elution volumes in Section II, B, 1, e, iii if necessary to collect a m i n i m u m of 90% of the added fenarimol. The procedure described usually yields recoveries in excess of 90% at the levels indicated. The limit of detection is about 0.002 p p m for fresh fruits, vegetables, pomace, raisins, juice, and wine. F o r dry pomace and crops, the limit is about 0.01 p p m , and in soil, it is about 0.02 ppm. The procedure for water samples can usually be simplified by eliminating the alumina column step. The dichloromethane extract is dried, evaporated to dryness, and the residue dissolved in toluene for G C measurement. Fenarimol may also be extracted from water with toluene followed by direct injection of an aliquot of the extract into the gas Chromatograph.
182
Ε. W. DAY, JR. AND O. D. DECKER
The limit of detection is dependent on the water : toluene ratio, a n d a 10-ml to 5-ml ratio will result in a limit of about 10 p p b . N u a r i m o l (see Chapter 13, this volume) can be used as an internal standard for fenarimol analysis if improved precision is desired.
REFERENCES Brown, I. F., Taylor, H. M., and Hall, H. R. (1975). Proc. Am. Phytopath. Soc. 2, 31. Buchenauer, H. (1977). Ζ. Pflanzenkr. Pflanzenshutz 84, 286. Ragsdale, Ν. N., and Sisler, H. D. (1973). Pesticide Biochem. Physiol 3, 20.