Sabadilla and Ryania - American Chemical Society

Chapter 16

Downloaded by STANFORD UNIV GREEN LIBR on August 8, 2012 | http://pubs.acs.org Publication Date: December 14, 2006 | doi: 10.1021/bk-2007-0947.ch016

Photolysis of Two Pesticides Used by Organic Farmers: Sabadilla and Ryania 1

Joseph D . Rosen and Xuejun

1,2

Zang

1

Department of Food Science, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901 Current address: NeoPharm, Inc., 1850 Lakeside Drive, Waukegan, IL 60085 2

Solar irradiation o f the major components of the "organic" pesticides sabadilla and ryania was studied in aqueous solution. One of the sabadilla components, veratridine, degraded slowly when exposed to sunlight. Another sabadilla component, cevadine, was stable. The major components o f ryania (ryanodine and dehydroryanodine) also decomposed slowly in sunlight. The major products resulted from photohydrolysis.

Introduction Under current United States Department of Agriculture National Organic Program regulations organic farmers may use some insecticides under certain conditions. These insecticides include pyrethrum, rotenone, sabadilla and ryania. Sabadilla is a broad-spectrum insecticide made by grinding seeds of Schoenocaulon officinale A . Gray, a member of the lily family which grows mainly in the Andes Mountains in Mexico, Guatemala and Venezuela. It is a mixture of alkaloids whose structures are shown in Figure 1. Ryania is the dried powder of roots, leaves and stems of Ryania speciosa which grows in the northern part of South America and the Amazon Basin. It consists mainly of two components, ryanodine and dehydroryanodine, whose structures are shown in Figure 2. There are no E P A tolerances for either sabadilla or ryania so they may be used in any amounts that the grower needs to prevent insects from 222

© 2007 American Chemical Society In Crop Protection Products for Organic Agriculture; Felsot, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


MW

R

1 veratridine

673

2 cevadine

CH,

591 CH

3

3 cevine

H

0

4 cevacine

0 509

551

CH ( 3

5 sabadine

B

CH

537 3

Figure 7. Structures and molecular weights (MW) of sabadilla components

destroying his/her crop. Even i f there were tolerances, there would be no way to enforce them as there is no approved multiresidue procedure to determine these pesticides in food. We previously reported the development of a sensitive multiresidue procedure for pyrethrum, rotenone, sabadilla and ryania in food based on high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (APCI/MS) (7). In addition to the paucity of approved analytical methods for these pesticides, there is very little information on their chronic toxicity, their endocrine disrupter properties, and their possible enhanced effects on children.

In Crop Protection Products for Organic Agriculture; Felsot, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

224 OH HO OH

CH

>

3

CH ' 3

H

H


21

ryanodine (MW=493)

H

dehydroryanodine (MW=491)

Figure 2. Structures and molecular weights of major components of ryania.

Nor is there much information available on the environmental fate of these materials. This report will provide preliminary results on the effect of exposure of sabadilla and ryania to sunlight in aqueous solution.

Experimental

Chemicals Ryania (a mixture consisting of 53% dehydroryanodine and 47% ryanodine), veratrine (a mixture consisting of 59% cevadine, 38% veratridine and 3% other alkaloids), veratridine, veratric acid (3,4-dimethoxybenzoic acid) and pyrrole-2-carboxylic acid were purchased from Sigma Chemical, St. Louis, MO.

Instrumentation H P L C and A P C I / M S instrumentation and conditions have been published earlier (1). Gas Chromatography/Mass Spectrometry (GC/MS) instrumentation consisted of a Varian Model 3400 Gas Chromatograph (Varian Associates, Sunnyvale, C A ) interfaced to a Finnigan ITS Magnum Ion Trap Detector (Finnigan M A T , San Jose, C A ) . A 30 m χ 0.25 mm id DB-1 fused-silica capillary column (0.25 μπι film thickness) was used. The G C was temperatureprogrammed from 60°-210°C at 7.5°C/min and from 210°-260°C at 5°C/min.


225


Helium carrier gas velocity was 1 mL/min. Filament emission current and electron multiplier voltage were 21 μΑ and 1700 V , respectively. A Waters 600 Multisolvent Delivery System connected to an Applied Biosystems 1000S Diode Array Detector was used to determine the U V spectra of veratridine, cevadine, ryanodine and dehydroryanodine. A Rayonet Merry-go-round Photchemical Reactor (Southern New England Ultraviolet Co., Hamden CT) containing 16 U V 23W lamps, emitting U V light above 300 nm was used for the photolysis of pyrrole-2-carboxylic acid.

Solar irradiation Aqueous solutions (75 mg/L) of veratridine and ryania were exposed to sunlight in stoppered quartz tubes (Ace Glass, Vineland, NJ) on the roof of our laboratory building between April 22 and July 12, 1998 between the hours of 10AM and 4 P M . There were only 44 days of exposure during this period as samples were not exposed on days when little sunlight was expected. An aqueous veratrine solution (20 mg/L; equivalent to 7.6 mg/L veratridine and 11.8 mg/L cevadine) was exposed for 22 days between August 18-September 15, 1998 between 10AM and 4 P M , again, only on sunny days. Controls during both exposure periods consisted of the same concentrations of pesticides in the same size test tubes but wrapped in aluminum foil. Samples were analyzed periodically by removing 250 μ ί aliquots. The aliquots were combined with 10 μΐ, of 100 ppm aqueous caffeine solution and analyzed by H P L C / A P C I / M S (7).

Determination of the Major Photolytic Products of Veratridine and Ryania Solution A Supelco Envi-18 cartridge (6mL) was conditioned with 6 mL of methanol and then 6 mL of water. The irradiated veratridine solution (after acidification to pH=3) was loaded onto the cartridge and passed through at a flow rate of 1-2 mL/min. The cartridge was dried under vacuum for five minutes, and then 3 m L methanol eiuate was collected. The eluate was then dried under a nitrogen stream and treated with diazomethane (generated from l-methyl-3-nitro-lnitrosoguanidine and sodium hydroxide using the method of Quin and Hobbs [2]). N O T E : D I A Z O M E T H A N E IS TOXIC, MUTAGENIC A N D C A R C I N O G E N I C A N D S H O U L D B E G E N E R A T E D O N L Y IN A W E L L F U N C T I O N I N G H O O D . Finally, the reaction mixture was dried under a nitrogen stream to remove the excess diazomethane. Ten μΐ, of 40 ppm internal standard mixture solution (l,4-dichlorobenzene-d naphthalene-d , acenaphthene-dio, phenanthrene-dio, chrysene-di ) was added, and the volume 4>

2


8

226 was adjusted to 250 μ ι with acetone. The acetone solution was analyzed by G C / M S . Veratric acid aqueous solution (75 ppm) and pyrrole 2-carboxylic acid aqueous solution (75 ppm) were also derivatized by following the above procedure.

Alkaline hydrolysis of veratridine and ryania Five mL of veratridine aqueous solution (75 ppm) was adjusted to p H 11 with N H O H (20-22% w/w), and stored at room temperature for one day (5). The solution was extracted with three 10-mL portions of methylene chloride. The extracts were evaporated to 1 mL under reduced pressure on a flash evaporator (Buchler Instruments, Fort Lee, NJ), and then dried in a gentle stream of nitrogen. The hydrolyzed product was then dissolved into 250 μί, of water, and 20 μί. was injected into H P L C / A P C I / M S . Five mL of ryania aqueous solution (75 ppm) was adjusted to p H 12 with 2 N N a O H and then was heated at 110°C for three hours. The hydrolysis solution was neutralized with acetic acid, and 20 μί. was directly injected into HPLC/APCI/MS.


4

Results and Discussion

Solar Degradation of Veratridine and Cevadine It took about 44 days for an aqueous solution (initial concentration: 75 ppm) of veratridine to photodegrade to 48% of its initial concentration. There was only very limited degradation in the control sample. We were unable to obtain commercial samples of any of the other sabadilla components (Figure 1) but we were able to obtain veratrine, which is a mixture of cevadine (59%) and veratridine (38%). Exposure of a 20 ppm aqueous solution of veratrine (11.8 ppm cevadine and 7.6 ppm veratridine) to sunlight for 22 days resulted in a loss of 50 % of the veratridine and only 10 % o f the cevadine. Analysis o f the controls indicated 98% and 94% of the veratradine and cevadine were present at the end of the experiment, respectively. Given the imperfect quantification of our methods, it is reasonable to conclude that very little, i f any, degradation of veratridine and cevadine occurred during storage or at the elevated temperatures on our roof. It is also reasonable to conclude that while veratridine undergoes photolysis in sunlight, cevadine does not. The U V absorption spectrum of veratridine had peaks at 222, 264 and 294 nm, while the U V absorption spectrum for cevadine showed a peak at 225 nm. Since the earth's ozone layer absorbs all wavelengths below 286 nm, it is reasonable to expect that cevadine


227 will not undergo appreciable photolysis in sunlight in aqueous solution. In the environment, however, photosensitization of cevadine by materials that absorb above 286 nm may occur. H P L C / A P C / M S determination of the extract from the veratridine irradiation sample showed two major peaks. The peak at 31.83 min was identical in retention time and mass spectrum to standard veratridine. The peak at 20.19 min had (M+H) ion at 510, suggesting that it was cevine (Figure 1). Cevine is a hydrolysis product of veratridine so we hydrolyzed the latter chemically. The major veratridine hydrolysis product exhibited the same retention time and mass spectrum as the major photodegradation product, strongly suggesting that cevine is one of the solar degradation products of veratridine. If veratridine undergoes photohydrolysis to cevine, it must also be converted to the acid portion of the ester. Treatment of the irradiation mixture with diazomethane resulted in the formation of a material identified by electron ionization G C / M S as methyl 3,4-dimethoxybenzoate. A material with identical G C / M S properties was obtained by treatment of commercially-obtained 3,4dimethoxybezoic acid (veratric acid). Thus, the two major solar photolysis products of veratridine are cevine and 3,4-dimethoxybenzoic acid (Figure 3). If sabadilla was a new insecticide, its


+

ΪΟΗ

RO OCH

OH

veratridine

H

3

3

Q — C O O H

3,4-dimethoxybenzoic acid R=3,4 -dimethoxybenzoyi

C H

3

O — ^

0

C

H

3

methyl 3,4 -dimethoxybenzoate

Figure 3. Photolysis of veratridine in water and chemical methylation of 3,4-dimethoxybenzoic acid.


228 manufacturer would have to provide toxicity and residue data as well as analytical procedures for its two major photoproducts.


Solar Degradation of Ryania Solar irradiation of ryania was examined at a concentration of 75 ppm. This solution contains 35 ppm ryanodine and 39 ppm dehydroryanodine. As can be seen from Figure 2, dehydroryanodine has the same structure as ryanodine except for the absence of 2 hydrogen atoms at carbons 9 and 21. After 22 days, approximately 72% ryanodine and 68% dehydroryanodine, respectively, remained. After a 44-day exposure, 39 and 41% ryanodine and dehydroryanodine, respectively, remained. There were no significant changes for the control groups.

Identification of Major Solar Degradation Products of Ryania Ryanodine and dehydroryanodine photoproducts were tentatively identified by H P L C / A P C I / M S in the negative ion mode. The retention times of ryanodine and dehydroryanodine were 22.37 and 21.29 min, respectively, while the retention times of their corresponding photoproducts were 11.63 and 9.16 min. Because A P C I in the positive ion mode did not exhibit (M+H) ions in either ryanodine or dehydroryanodine, we operated in the negative ion mode. Ryanodine exhibited an (M-H)" ion at m/z 492 while dehydroryanodine exhibited an (M-H)' ion at m/z 490. The photoproducts exhibited (M-H)" ions at m/z 399 and 397, respectively, suggesting that they were ryanodol and dehydroryanodol plausibly resulting from the loss of the pyrrole carboxylate moiety. Alkaline hydrolysis of ryania resulted in products having identical chromotograhic retention times as the photolysis products. Products from both reactions also exhibited identical H P L C / A P C I / M S spectra in both positive and negative ion modes, further providing very strong evidence for the structure of the photoproducts (Figure 4). Esterification of the ryania photolysate products and subsequent G C / M S analysis failed to find evidence for the presence of pyrrole-2-carboxylic acid, a material that would be expected to be formed as a result of the photohydrolysis of ryania. However, pyrrole-2-carboxylic acid was itself susceptible to rapid degradation in sunlight. After 1 hr of irradiation of pyrrole 2-carboxylic acid by U V light at wavelengths >300nm, 70% was lost, and after 4 hours none could be detected. +


229


Ryanodine (MW=493)

Ryanodol (MW=400)

Figure 4. Solar (upper ) and alkaline (lower) hydrolysis of ryanodine. Structure of dehydroryanodol is the same as ryanodol except for a double bond between carbon 9 and carbon 21.

References 1. 2. 3.

Zang, X.; Fukuda, Ε. K . ; Rosen, J.D. J. Agric. Food Chem. 1998, 46, 22062210. Quin, L . D.; Hobbs, M. E. Anal. Chem. 1958, 30, 1400-1405. Hare, J. D. J. Agric. Food Chem. 1996, 44, 149-152.


Sabadilla and Ryania - American Chemical Society

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