Triazine Herbicides - ACS Publications - American Chemical Society

Chapter 12

Downloaded by STANFORD UNIV GREEN LIBR on October 1, 2012 | http://pubs.acs.org Publication Date: May 14, 1998 | doi: 10.1021/bk-1998-0683.ch012

Use of a Multiresidue Method for the Determination of Triazine Herbicides and Their Metabolites in Agricultural Products

1

2

John R. Pardue and Rodney Bong

1

Southeast Regional Laboratory for Food and Drug Admnistration, 60 Eighth Street, NE, Atlanta, GA 30309 Minneapolis District Laboratory, Food and Drug Administration, 240 Hennepin Avenue, Minneapolis, MN 55401

2

325 samples of both domestic and imported agricultural products were analyzed using a method which will determine 19 triazine herbicides and 3 metabolites. The method consisted of blending a composite portion with methanol andfiltering,followed by partitioning into methylene chloridefroman aqueous saline solution. The organic extract was then evaporated and the herbicides isolated using solid-phase extraction (SPE) with a strong cation exchange (SCX) cartridge prior to gas chromatography and nitrogen-phosphorus detection (NPD). The only triazine detected was simazine found in ten samples of orangesfromtrace levels to 0.035 ppm. Duplicate and triplicate recoveries were conducted for four selected herbicides in each of the 10 commodities examined in the surveys. These recoveries, spiked at 0.1 ppm, averagedfrom82.5% to 104.6%. Triazines are widely used chemicals applied for season long weed control as both selective and non selective herbicides (7). In 1987 atrazine was used at the rate of 100 million pounds and was estimated by EPA to be the most widely used pesticide (2). Symmetrical triazine herbicides are six memberedringcompounds containing 3 cyclic nitrogen atoms and 2 amino groups external to the ring. Substitutions at the sixth position of the ring and of the amino groups produce a large number of compounds with herbicidal activity. Substitution at the six position with -CI yields atrazine, simazine, and propazine; substitution with -OCH yields atraton, prometon, and simeton; and with -SCH yields ametryn, dipropetryn, and simetryn. The amino groups attached to the ring are usually of the form - N H C J i ^ and the alkyl group attached is usually either ethyl or isopropyl or, lessfrequently,tertiary butyl. 3

3

U.S. Government work. Published 1998 American Chemical Society In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

123


124 Loss of one or both of the alkyl groups (CnH^+j) produces metabolites which were studied and shown to be recovered by the method used in the surveys (3). The desalkyl metabolites of atrazine have been found in surface and ground water (4,5) and in soil (6-9). While these metabolites are listed as atrazine metabolites, an examination of their structures reveal that they are the same as those produced by the dealkylation of simazine and propazine. The structures and relationship between atrazine, simazine, propazine, and three desalkyl metabolites are illustrated in Figure 1. These metabolites are potentially important as residues in food products. They are included in the tolerances in determining total chlorotriazine residues for atrazine in certain grasses and millet, and as residues for simazine in bananas andfish(JO). This paper describes pertinent steps of the screening method and the results of two Food and Drug Administration surveys for triazine herbicides and selected metabolites conducted in FY 1995 and FY 1996. Materials and Method Reagents and Materials. Solvents were pesticide grade obtained from Baxter, Burdick and Jackson Div., Muskegon, MD. Reference standards were obtained from U. S. Environmental Protection Agency, Pesticide and Industrial Chemicals Repository, Research Triangle Park, NC; Crescent Chemical Co., Hauppauge, NY; and Ultra Scientific, North Kingstown, NY. SPE tubes were Supelclean LC-SCX, 3 mL obtained from Supelco, Inc., Bellefonte, PA. Sample Collection and Preparation. Both imported and domestically grown food samples were collected for triazine analysis in nine different Food and Drug Administration (FDA) districts or regions located throughout the United States. The first survey contained 232 samples consisting of apples, bananas, cherries, corn, grapefruit, grapes, olives, oranges, pears, and plums and were collected and analyzed during FY 1995. Because of the special interest by FDA in those foods which are consumed by children and infants, an additional 93 samples were analyzed during FY 1996. These products consisted of apples, bananas, grapes, oranges, pears, and plums. Samples were composited at the collecting district or regional laboratory according to PAM I, Table 102-a (77), frozen, and sent to the Minneapolis laboratory for analysis. Compositing consisted of grinding the entire commodity after removing and discarding any stems, stalks, and/or crown tissue which might be present. Extraction and Cleanup. A 100 g portion of each composited sample was extracted with methanol andfiltered.A portion of thefiltrateequivalent to 50 g of sample was diluted with water and saturated salt solution, and then, extracted twice with methylene chloride. The methylene chloride extracts were dried by passing them through sodium sulfate into a 500 mL Kuderna-Danish evaporator. The methylene chloride was concentrated on a steam bath to about 2 mL, 25 mL hexane added, and the solvent mixture reconcentrated leaving only hexane. This solution was then diluted to 10 mL with hexane prior to the cleanup step. A one mL aliquot of the hexane solution was placed onto a previously washed Supelclean LC-SCX tube. The tube was again washed with methylene chloride and acetone to remove crop co-extractives. Any triazine compounds present were then

In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


2

5

2

Simazine

5

5

NH 2

\

^

Desisopropylatrazine

Ν

XX

çi

2

N

2

^

Çl

N

3

^

NHCH(CH )2

Desethylatrazine

y / '

2

H N

N / ^ N

Cl

Propazine

(CHjJzHCHN^Si^SfflCHiCTbîz

XX

3

NHCH(CH )2

NH

XX

Cl

Atrazine

N

Diaminochloro-s-triazine

2

S

HN

5

C H HN

N/4N

Çl

Figure 1. Structure and relationship between simazine, atrazine, propazine and their desalkyl metabolites.

2

C H HN

^

C H HN^N^NHC H

N/^-xN

Cl


^

126 eluted from the tube with a solution of IN NH OH/methanol (1+ 3) into a pH 6.5 phosphate buffer. The buffer solution was extracted twice with methylene chloride, methylene chloride evaporated to dryness, and the residue dissolved in 2.0 mL acetone. This solution was then analyzed by gas chromatography. Step by step descriptions of this method and the method used to analyze the the metabolite, diaminochloro-s-triazine, are published in the original paper (3). Because only low amounts (less than 0.04 ppm) of the parent herbicides and none of the mono-dealkylated metabolites were detected in any of the samples, the method to determine diaminocWorc^s-triazine was not used. The basic steps of this procedure and those of the method used for the surveys are illustrated in Figure 2. The method for diaminochloro-s-triazine was developed because it was found that only a very small amount of this compound was recovered by the original procedure. The major differences in the two methods are (1) the reduction of water and removal of methanol prior to partitioning diaminochloro-s-triazine into the organic solvent and (2) substituting ethyl acetate for methylene chloride as the partitioning solvent. Both of these changes were taken because of the high solubility of the metabolite in the aqueous methanol solution. Recoveries show that diaminochloro-s-triazine remains in the methanolic layer when it is extracted with methylene chloride (3).


4

Gas Chromatographic Analysis. Analysis was done using a HP 5890A with 19234B/C nitrogen-phosphorus detector (Hewlett-Packard Co., Wilmington, DE); DB17, 30 m X 0.53 mm with 1 μπι film thickness (J&W Scientific, Folsom, CA). Temperatures (°C) were as follows: inlet, 220; detector, 220; column programmed from 150 to 230 at a rate of 4°C/min and afinalhold time of 12 min. Gas flows (mL/min) were as follows: helium carrier, 15; helium auxiliary, 35; air, 90; hydrogen, 3.5. Retention times relative to atrazine for the 22 compounds tested in the original study rangedfrom0.76 for melamine to 2.94 for hexazinone. With these conditions, atrazine elutes in 10 minutes. Mass Spectral Analysis. Confirmation of detected residues was with an HP 5995 Quadrapole instrument using electron impact mass spectroscopy. Column and column conditions were the same as for the gas chromatographic analysis except a 0.25 mm DB-17 column was used. Results and Discussion. Triazine residues were found in 10 samples of the 325 samples examined during the surveys. Tables I and II list the number and types of samples analyzed during the two fiscal years. All residues detected were simazine found in domestically grown oranges. One positivefindingof 0.035 ppm and four trace levels (less than 0.01 ppm) were found in the 25 orange samples examined during FY 1995. One positive of 0.030 ppm and four trace levels were found in 20 orange samples analyzed during FY 1996. These samples were composited by grinding the whole oranges. They were then analyzed on the whole basis, therefore, it was not determined if the residues were on the peel, pulp, or juice. These positivefindingswere well below the established tolerance for simazine of 0.25 ppm in oranges (10).


127

Parent herbicides and majority of metabolites Blend samples with methanol and filter.


I Combine portion of filtrate with saline solution, water and partition into methylene chloride.

1 Evaporate methylene chloride to dryness and dissolve residue in hexane.

I Pass portion of hexane solution through S C X SPE tube and elute with pH 6.5 buffer solution.

I Extract buffer solution with methylene chloride.

I Evaporate methylene chloride to dryness and dissolve residue in acetone.

1 Analyze by G C with NP detection.

Desethyldesisopropylatrazine Blend samples with methanol and filter.

i Add saline solution to portion of filtrate and extract with methylene chloride. Discard methylene chloride.

1 Evaporate methanolic layer to small volume and partition into ethyl acetate. Evaporate ethyl acetate to dryness and dissolve residue in acetone.

I Pass portion of acetone solution through S C X SPE tube and elute with pH 6.5 buffer solution.

I Extract buffer solution with ethyl acetate.

1 Evaporate ethyl acetate to dryness and dissolve residue in acetone.

I Analyze by G C with NP detection.

Figure 2. Flow diagram illustrating basic steps of method used for the determination of triazine herbicides and their mono dealkylated metabolites as compared to the method developed to determine diaminochloro-s-triazine.


128 Table I. Samples analyzed during FY 1995.


No. Import Samples

Total No. Samples

No. Domestic Samples

Apples Bananas

25 25

9 0

16 25

Cherries Com

25 25

25 10

0 15

Grapefruit Grapes Olives Oranges Pears Plums

12 25 25 25 25 20

8 5 0 20 19 13

4 20 25 5 6 7

232

109

123

Commodity

Total

Principal Countries of Origin for Imports Chile Mexico, Ecuador, Panama Thailand, Mexico, Canada Israel Chile, Greece Spain, Greece, Italy Italy Chile Chile

Table II. Samples analyzed during FY 1996. Commodity

Total No. Samples

No. Domestic No. Import Samples Samples

Apples Bananas

10 20

8 0

2 20

Grapes

20

6

14

Oranges Pears Plums

20 20 3

18 10 3

2 10 0

Total

93

45

48

Principal Countries of Origin for Imports Chile, Canada Mexico, Ecuador, Panama Mexico, Chile, Greece China Chile

The simazine residues found at 0.035 and 0.030 ppm were confirmed by GC/MS using electron impact, selected ion monitoring. Ion ratios at mass units 201, 186, and 173 for the sample residues were matched with those of the reference standard simazine analyzed under the same conditions. For the FY 1995 survey, each commodity was fortified twice (on separate days) at the 0.1 ppm level with four triazines. The four triazines selected for the recovery studies were three having different substitutions at the sixth position (-C1, -OCH , SCH ) of the triazine nucleus and one of the metabolites of atrazine. Atrazine, 3

3


129 secbumeton, and simetryn were the three herbicides chosen for the recovery studies, and desethylatrazine was the metabolite. These same four compounds were selected for single recovery determinations in apples, bananas, grapes, and oranges for the FY 1996 survey. Table ΙΠ lists the cumulative recoveries which were obtained for both FY 1995 and for 1996. The recovery values for the 1996 survey are the third value in each grouping. Table ΙΠ. Combined FY 1995 & 1996 Recovery Data for Selected Triazine Herbicides^


Commodity Desethylatrazine Apples Bananas Cherries Com Grapefruit Grapes Olives Oranges Pears Plums

90. 6 70. 1 88 .7 94 .0 84 .3 104. 7 82 .1 84 .2 72,.7 82 .1 60 .4 73 .6 79 .6 104..7

Fortification Level (ppm) Mean Recovery Std. Dev. CofF. of Variation

92.5 89.6 80.1 87.9 60.8 77.7 77.4 86.8 79.6 75.5

Recovery, % Atrazine Secbumeton 104.7 102.6 94.8 99.1 98.3 112.2 102.6 92.2 103.5 104.3 101.7 93.0 94.8 103.5 94.8 100.0 94.8 97.4 100.0 100.9 94.8 93.0 103.5 101.7

103.1 109.3 115.1 100.2 110.0 115.1 98.7 113.1 110.3 110.3 107.2 98.0 113.4 103.0 103.0 102.1 103.1 99.0 96.9 104.8 96.9 101.0 102.0 103.9

0.106

0.115

0.115

82.5% 11.1 13.5

99.4% 4.2 5.1

104.6% 5.5 5.3

Simetryn 94.1 102.0 102.9 97.1 102.2 110.8 98.0 110.8 102.0 101.0 101.0 91.9 27.5 51,0 96.5 94.1 93.1 71.6 94.1 96.1 97.1 95.1 110.3 111.3 0.102 93.8% 19.0 20.3

The statistical values shown for each of the four herbicides are based on pooled recovery data representing all commodities. Most of the recovery values were within the range reported in the original work. One notable exception was the low recovery for simetryn from grapes (27.5 and 51.0%) obtained during the 1995 survey. Recoveries for grapes were not conducted with the original work. For this reason, it was originally believed that the recoveries might be a peculiarity of the particular sample matrix and compound, and the results were not eliminated. When the recovery was repeated with the FY 1996 survey, however, a value of 96.5% was obtained, and it does not appear as if the sample matrix was the problem. The original low recoveries were probably due to unfamiliarity with the method. If these two low results are not used for the statistical evaluation of simetryn, the mean recovery is 98.8% for all commodities. The standard deviation is loweredfrom19.0 to 8.6, and the coefficient


130 of variationfrom20.3 to 8.7. These results bring the calculated values for each of the four compounds within the range reported in the original work (5). The recovery values and the results of these two surveys demonstrate that the methodology is excellent for determining triazine herbicides and their desalkyl metabolites in a variety of food commodities. Thefrequencyand levels of positive findings of these herbicides in the surveys indicate that there is a low dietary exposure rate.


Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Farm Chemicals Handbook '95; Meister, R. T.; Sine, C., Eds.; Meister Publishing Company, Willoughby, OH, 1995, Vol. 81, 32-33. Ware, W. W.; The PesticideBook;Thomson Publications, Fresno, CA, 1989, 112. Pardue, J. R. J. AOAC Int. 1995, 78, 856-862. Pereira, W. E.; Rostad, C. E.; Leiker, T. J.Anal.Chim. Acta. 1990, 228, 69-75. Thurman, E. M.; Goolsby, D. Α.; Meyer, M. T.; Kolpin, D. W. Environ. Sci. Technol. 1991, 25, 1794-1796. Steinwandter, H. J. Anal. Chem. 1991, 339, 30-33. Gorder, G. W.; Dahm, P. A. J. Agric. Food Chem. 1981, 29, 629-634. Karlaganis, G.; Von Arx, R. J. Chromatogr. 1991, 549, 229-236. Durand, G.; Forteza, R.; Barceló, D. Chromatographia 1989, 28, 597-604. Code of Federal Regulations, Title 40, Government Printing Office, Washington, D. C., 1994; Section 180. Pesticide Analytical Manual; McMahon, Β. M.; Hardin, N. F.,Eds.; Volumn I, 3rd Edition, Table 201-a.


Triazine Herbicides - ACS Publications - American Chemical Society

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