Daminozide (Alar) Breakdown During Thermal Processing of Cherry Products T0 Yield, Unsymmetrical Dimethyl— Hydrazine (Udmh)'." "

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3 129300 r \ LIBRARY Ilium“. State University

This is to certify that the

dissertation entitled

DAMINOZIDE (ALAR) BREAKDOWN DURING THERMAL PROCESSING OF CHERRY PRODUCTS T0 YIELD, UNSYMMETRICAL DIMETHYL— HYDRAZINE (UDMH)'." "

presented by

CHARLES RICHARD SANTERRE

has been accepted towards fulﬁllment of the requirements for Ph.D. degmin FOOD SCIENCE & ENVIRONMENTAL TOX.

Date March 24, 1989

MS U is an Afﬁrmative Action/Equal Opportunity Institution 0-1277 1

‘~.¢

O’Ntw , record. PLACE IN RETURN isﬂeckout from your TO AVOID FINES retur on or b ore date d .

DATE DUE-ADA15- I DATE DUE

MSU I. An Affirmative Action/Equal Opportunity Institution email-HM DAMINOZIDE (ALAR) BREAKDOWN DURING THERMAL

PROCESSING OF CHERRY PRODUCTS TO YIELD,

UNSYMMETRICAL DIMETHYLHYDRAZINE (UDMH).

Charles Richard Santerre

A DISSERTATION

Submitted to Michigan State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science a Human Nutrition

1989 “‘3X

5072 ABSTRACT

DAMINOZIDE (ALAR) BREAKDOWN DURING THERMAL PROCESSING OF CHERRY PRODUCTS TO YIELD, UNSYMMETRICAL DIMETHYLHYDRAZINE (UDMH).

Charles Richard Santerre

Daminozide is applied to tart and sweet cherries to improve fruit

color, texture and abscission at harvest. This growth retardant is a

system ic compound which is distributed throughout the fruit matrix.

Thermal processing of fruit containing daminozide produces a hydrolysis

product, unsymmetrical dimethylhydrazine (UDMH), which is a cancer

suspect agent.

Daminozide was added to a 50 mM NaH2P04-24% sucrose solution at 12.5

ppm (w/w) to determine the influence of heating, pH and processing

conditions on hydrolysis of daminozide to UDMH. Daminozide fortified

solutions at three pH levels (pH 3.0; 3.6; 4.2) were processed by two

regimens. For the first regimen, no. 303 X 404 cans were filled (150 g)

with the daminozide fortified solution, sealed, heated for 5, 10, 15

min. at 100°C, and cooled. For the second regimen, cans were filled

(150 g) with the daminozide fortified solution which was then heated to 80°C and held for 0, 5, or 10 min. The cans were immediately sealed,

heated in 100°C water for 5 min. and cooled. Daminozide concentrations were not decreased at a detectable level in any of the heated solutions. For the daminozide solutions which were heated following can closure, the concentration of UDMH increased with heating time.

Approximately 5 ppb UDMH was produced for every 5 min. of heating in

100°C water. However, UDMH concentration was not directly related to heating time for solutions which were heated prior to can closure. This was due to volatilization of UDMH which countered daminozide degradation during heating in the open cans. For the three pH levels, a maximum concentration of UDMH occurred for solutions adjusted to pH 3.6.

Tart (Montmorency) and sweet (Napolean and Schmidt) cherry trees were treated 2 wk after full bloom with 3.4 and 6.8 lbs. daminozide/acre at 3 commercial orchards in northwestern Michigan. Daminozide residues in freshly harvested fruit and processed cherry products were less than

13 ppm and 10 ppm, respectively. UDMH residues in freshly harvested fruit and processed cherry products were less than 10 ppb and 500 ppb, respectively. Processing of both sweet and tart cherries reduced the levels of daminozide. However, thermal processing increased the levels of UDMH in canned cherry products. DEDICATION

To my parents, Betty and Roland

and siblings

Michael, Annette, Jeanine, Suzette,

Celeste, Roger, Mary, Phyllis,

James and Peter.

-iv- ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to all the people who have supported me in my academic affairs. To my father, Roland, who intrigued me with carpentry, mechanics, and electronics at an early age. To my mother, Betty, who would never tire of questions from a 12 year old as he read through her nursing microbiology text. To my major professor and friend, Dr. Jerry N. Cash, a person of integrity who has earned my respect by his actions. His trust, support, and guidance have helped me to grow as a professional and more importantly, as a person.

I shall always be in his debt. To his wife, Stella Cash, who taught me of Southern hospitality even though I am and will always be a Darn

Yankee. To Dr. Matt Zabik, who extended my horizons past the laboratory walls. He has taught me to cut through the tripe and seek excellence in every phase of my life. To Dr. Maurice Bennink, who molded me during my undergraduate years. His outstanding teaching ability and concern for students was a pivotal factor which led me down this road. To Dr.

Pericles Markakis, who supported my efforts with grace and sophistication. To Dr. James Pestka, for constructive feedback during my studies.

In particular, I wish to thank Dr. Gilbert Leveille and Dr. Don

Coffey. The influence which they have had on my career can never be put into words. One has shown me the breadth of a career and the other has shown me the importance of a sense of humor in achieving my career goals. I would also like to thank the many graduate students who have endured the journey with me. These include Mark McLellan, David Huang,

Touran Cheraghi, Mohammed Kenawi, Ramidan Habiba, Annie Chai, Nirmal

Sinha, John Kallas, Amir Zaman, Ahmad Shirazi, Terri Biggerstaff and many others.

Finally, I would like to thank Maggie Conner, who helped prepare this dissertation. She has been a good friend for the past 10 years.

-v‘i- TABLE OF CONTENTS

£5.95

LIST OF TABLES ...... ix LIST OF FIGURES ...... x

INTRODUCTION ...... 1 LITERATURE REVIEW ...... 3 A. Daminozide as a Plant Growth Retardant ...... 3

l. Daminozide in Apple and Pear Production ...... 8

2. Daminozide in Tart and Sweet Cherry Production ...... 11

3. Economice Importance of Daminozide ...... 13

B. Daminozide Toxicity ...... 14

C. UDMH Toxicity ...... 17

1. Absorption, Transport and Excretion of UDMH ...... 17

2. Metabolism of UDMH ...... 19

3. Mutagenicity of UDMH ...... 25

4. Carcinogenicity of UDMH ...... 26

5. Influence of UDMH on Nervous Function ...... 27

List of References ...... 37

CHAPTER I - The Influence of pH and Heating Time on the

Decomposition of Daminozide (Alar) and the

Formation.of Unsymmetrical Dimethylhydrazine

(UDMH) ...... 4S

-vii- 2292

Abstract ...... 46

Introduction ...... 47 Materials and Methods ...... 49 Results and Discussion ...... 53

Conclusions ...... 57 References ...... 58

Acknowledgement ...... 59

CHAPTER 2 - Daminozide (Alar) and Unsymmetrical Dimethyl-

hydrazine (UDMH) Residues in Tart and Sweet

Cherries and Processed Cherry Products ...... 72

Abstract ...... 73 Introduction ...... 74 Materials and Methods ...... 76 Results and Discussion ...... 81

Conclusions ...... 89

References ...... 90

Acknowledgement ...... 92

-viii- LIST OF TABLES

Ease

CHAPTER 1

Table 1. Analysis of variance (AOV) table describing the

influence of pH and heating time on the

concentration of UDMH in canned samples which were closed prior to heating (100°C) ...... 60

Table 2. Analysis of variance (ADV) table describing the

influence of pH and heating time on the

concentration of UDMH in canned samples which were heated before (80°C) and after (100°C) can

closure ...... 61

CHAPTER 2

Table 1. Daminozide (Alar) and 1,1-dimethylhydrazine (UDMH)

residues in tart cherries and cherry products ...... 93

Table 2. Daminozide (Alar) and 1,l~dimethylhydrazine (UDMH)

residues in sweet cherries and cherry products ...... 94

-1x- LIST OF TABLES

Page

CHAPTER 1

Table 1. Analysis of variance (AOV) table describing the

influence of pH and heating time on the

concentration of UDMH in canned samples which were closed prior to heating (100°C) ...... 60

Table 2. Analysis of variance (AOV) table describing the

influence of pH and heating time on the

concentration of UDMH in canned samples which were heated before (80°C) and after (100°C) can

closure ...... 61

CHAPTER 2

Table 1. Daminozide (Alar) and 1,1-dimethylhydrazine (UDMH)

residues in tart cherries and cherry products ...... 93

Table 2. Daminozide (Alar) and 1,1-dimethylhydrazine (UDMH)

residues in sweet cherries and cherry products ...... 94

-ix- LIST OF FIGURES

£392

LITERATURE REVIEN ...... 3

Figure 1. Chemical structure of daminozide ...... 3

Figure 2. Biosynthetic pathways for gibberellin synthesis

(Dennis et al., 1965) ...... 6

Figure 3. Decomposition of daminozide to produce l-ldimethyl-

hydrazine ...... 17

Figure 4. Stoichiometric conversion of 1,1—dimethylhydrazine

to fomaldehyde ...... 23

Figure 5. Biosynthesis and degradaton of gamma-amino-

butyric acid ...... 29

Figure 6. Compartmentalization of gamma-aminobutyric

acid in the nerve ending ...... 30

Figure 7. Forms of Vitamin 85 ...... 31 2199

Figure 8. Reaction of pyridoxine with 1,1—dimethylhydrazine ...... 34

CHAPTER 1.

Figure 1. Thermal decomposition of daminozide to yield

unsymmetrical dimethylhydrazine (UDMH) ...... 62

Figure 2. Reaction between unsymmetrical dimethylhydrazine

and salicylaldehyde to produce salicylaldehyde

dimethylhydrazone ...... 63

Figure 3. Mass spectral fragmentation of salicylaldehyde

dimethyl hydrazone (MW - 160) ...... 64

Figure 4. LC chromatogram of daminozide (RT = 3.26 min.) ...... 65

Figure 5. GC/MS, SIM of m/e - 120, 123, 153, 164 for a 10

ppm salicylaldehyde dimethylhydrazone standard ...... 66

Figure 6. GC/MS standard curve of 1,1-dimethylhydrazine

(UDMH) from 0-500 ppb ...... 67

Figure 7. GC/MS standard curve of 1,1-dimethylhydrazine

(UDMH) from 0-10 ppm ...... 68

-x‘i- Figure 8. UDMH residues in samples which were heated (100°C) following can closure. Bars with the

same letter are not significantly different at

the Tukey’s "0.01 level ...... 69

Figure 9. UDMH residues in samples which were heated (80°C)

prior to can closure in addition to 5 min. heating (100°C) following closure. Bars with the

same letter are not significantly different at

the Tukey’s "0.05 level ...... 70

Figure 10. Influence of pH on the production of UDMH ...... 71

CHAPTER 2.

Figure 1. Daminozide residues in fresh and processed tart

cherry products from location 1 ...... 95

Figure 2. Daminozide residues in fresh and processed tart

cherry products from location 2 ...... 96

Figure 3. Daminozide residues in fresh and processed tart

cherry products from location 3 ...... 97

-xii- Figure 4. Unsymmetrical dimethylhydrazine (UDMH) residues in

fresh and processed tart cherries from location 1 ...... 98

Figure 5. Unsymmetrical dimethylhydrazine (UDMH) residues in

fresh and processed tart cherries from location 2 ...... 99

Figure 6. Unsymmetrical dimethylhydrazine (UDMH) residues in

fresh and processed tart cherries from locatoin 3 ...... 100

-xiii- INTRODUCTION

Daminozide has recently come under great public scrutiny for several reasons. Daminozide was developed in the early 1960’s and approved for use on food plants in 1968. The timing for registration of this agricultural chemical is important for two reasons. First, the grassroots political movement to protect the environment and our food supply was increasing in the late 1960’s, along with public awareness of cancer. This movement was the driving force behind the establishment of the Environmental Protection Agency (EPA) in 1970 with responsibilities for regulating pesticide usage. The Food and Drug Administration (FDA), the U.S. Department of Agriculture (USDA) and the Bureau of Alcohol,

Tobacco and Firearms (BATF) would be responsible for regulating pesticides which occur in foods. The division of responsibility for regulating pesticides caused dramatic changes in the interpretation of the Federal Food and Drug and Cosmetic Act (including the Delaney

Clause) and the Federal Insecticide, Fungicide and Rodenticide Act.

Current technologies have led the EPA to use a ’de Minimis’ interpretation of carcinogenic compounds in foods in order to avoid strict interpretation of the Delaney Clause, which prevents all carcinogens (even insignificantly occurring ones) from being added to foods. The agency now accepts compounds which pose a risk of cancer of less than 1:1 mil. to the consumer and uses a risk-benefits approach when regulating pesticides. Second, the increased interest in food safety has caused greater evaluation of pesticides prior to registration. Hence, testing protocols and analytical technologies were

1 improved to give a better estimation of human exposure to pesticides.

In addition, standard methods were established to determine the toxicity of pesticides. Presently, regulations for approving pesticides require many years of testing and significantly greater resources to determine crucial parameters including acute and chronic toxicity, environmental fate, food residue and metabolite levels and human exposure risks.

Daminozide was approved for use on agricultural crops prior to the extensive development of the regulatory protocols which are in use today.

In the early 1980’s, it was reported that daminozide could degrade to form unsymmetrical dimethylhydrazine (UDMH), a cancer suspect agent. Unfortunately, methods for determining daminozide and UDMH residues in the ppm or ppb range were not available. In addition, the toxicity of daminozide and UDMH in 2 year chronic exposure animal studies was not determined. These factors have permitted consumer advocacy groups to play on the ’fear of cancer’ of the public to demand removal of daminozide from agricultural use. Daminozide may prove to be the first ’domino to fall’ in a trend to eliminate agricultural chemicals from our food system regardless of their safety.

The research described in this dissertation investigated the influence of pH and heating time on the decomposition of daminozide using standard food processing conditions. Subsequently, the residues of daminozide and UDMH were measured in fresh sweet and tart cherries and processed cherry products. The measurement of these residues will permit estimation of exposure levels of UDMH and daminozide for cherries and cherry products. LITERATURE REVIEW

A. Daminozide as a Plant Growth Retardant

Daminozide (B995, Alar, Kylar, B-Nine, succinamic acid 2,2-dimethyl

hydrazide) belongs to a class of compounds known as "growth retardants". Growth retardants include, ”all chemicals that slow cell

division and cell elongation in shoot tissues and regulate plant height

physiologically without formative effects" (Cathey, 1964). Daminozide,

molecular weight of 160, is very water soluble and stable in plants and

soil but breaks down during thermal processes such as steam

sterilization of soil (Cathey, 1964). The chemical structure of

daminozide is given in Figure l.

0 ll / 1CH 3 CFl'--—-C---FN1---N 2 \ l (Ii-l3

CH2--C-———OH 1| 0

DAMINOZIDE

Figure 1. Chemical structure of daminozide.

This compound, introduced in 1962 (Unrath, 1969), is used in the production of fruits, vegetables and ornamental plants. Approximately

80% of the daminozide produced is used for apple production with

3 significant amounts also being used on peanuts, sweet and tart cherries, lettuce, cranberries, blueberries, pears, and grapes to improve crop yield and product quality (Uniroyal Chemical Co.). The influence of daminozide on plant growth is species and cultivar specific (Fisher and

Looney, 1966). In this review, information will be presented regarding the physiological and structural responses of apples, pears, tart and sweet cherries to this growth retardant. In addition, research involving other plant species will be used to demonstrate the metabolic changes which occur following treatment with daminozide.

Daminozide applied to the leaves of plants is readily absorbed by both monocots and dicots (Moore, 1968). Kilby et al. (1970) measured the rate of absorption of l4C-daminozide applied to the lower epidermis of Tung tree leaves. The labeled compound was detected in the petioles within one hour and in lateral branches, stems and roots within 24 hours. Peak absorption of 14C-daminozide occurred within 4 days with significant amounts present in all samples after 10 days. Domir (1980) injected l4C-daminozide into American Elm (ulmus amgrjcana L.) seedlings and observed rapid translocation to all parts of the plants. Chromatographic techniques were able to recover 79-87% of the test compound as unmetabolized daminozide. Attempts to recover additional label from the plant tissue were unsuccessful and no metabolites were observed or identified during the 21-day study.

Contrary to this finding, Zeevaart (1966) noted a single unidentified metabolite in cotyledon extracts from treated Eharbiti§_n11, strain

"Violet" plants. The metabolite was also measured in leaf extracts during the 33 days of sampling. No attempts were made to identify the minor component. Undurraga and Ryugo (1970) applied l4C-daminozide to almond seedlings and measured translocation. Much of the daminozide applied, remained on the leaf surface while the absorbed compound penetrated into the phloem and xylem. They found that, "when [daminozide] reaches a threshold value in roots, it tends to leak into the root medium. Once in the soil solution, it can be readily absorbed by the roots of adjacent plants." Daminozide was reported to lower membrane integrity and increase cytoplasmic permeability which results in leakage of sucrose into the xylem of treated plants. The sucrose leakage may account for depressed utilization of respiratory energy in treated plants. It is possible that daminozide allows fruiting bodies (apples, cherries, etc.) to compete more efficiently with shoots for available carbohydrate resources by inducing sucrose leakage.

Moore (1968) did not find metabolites in the extracts of treated

Zinnias, Briza_§p., Zga_§p. or Iaggtg§_sn. plants. He was able to extract only 9-12% of the radioactive compound applied to the plants.

In addition, as much as 1% of the labelled residue remained in the plant tissue following extraction. The 1% of ’bound’ compound may include a metabolically active metabolite of daminozide which is firmly bound to an active site.

Physiologically, daminozide was reported to inhibit shoot elongation by interferring with auxin production. Reed et al. (1965) attributed the retarding action of daminozide to the formation of the hydrolysis product, 1,1-dimethylhydrazine (UDMH). UDMH was demonstrated to inhibit, in vitro, the enzyme diamine oxidase which converts tryptamine to the auxin, indolacetaldehyde. Auxins are growth regulators which promote root formation and shoot elongation but inhibit bud formation. Unfortunately, Reed et al. (1965) did not show that UDMH is produced in their experimental model, and more importantly, they did not demonstrate that growth retardation could be prevented by treatment with an auxin spray (Zeevaart, 1966). Gibberellins are another class of growth regulators which function similar to auxins except that they promote bud formation. Gibberellins were shown to prevent many of the growth retardation effects of daminozide (Cathey, 1964). Dennis et al. (1965) attempted to demonstrate that daminozide interferred with gibberellin synthesis by inhibiting the conversion of trans-geranylgeranyl pyrophosphate to the gibberelin precursor, (-) kaurene. The biosynthetic pathway for gibberellin synthesis is given in Figure 2.

trans-geranyl geraniol i meval onate 9 .9 9 trans-geranylgeranyl pyrophosphate

(—) kaurene 1 (~) kauren-I9-ol i/ I gibberellins

Figure 2. Biosynthetic pathways for gibberellin synthesis (Dennis et al. 1965). Dennis et al. (1965) were unsuccessful in demonstrating lower (-)

kaurene levels following daminozide treatment.

Zeevaart (1966) demonstrated that inhibition of flower formation by

daminozide in Phagbitis_gil, strain "Violet”, could be completely

overcome by application of gibberellin A3. Treatment with the auxins,

indole-3-acetic acid (IAA) and naphthalene acetic acid (NAA) were

ineffective in overcoming daminozide growth retardation. Ryugo and

Sachs (1969) then demonstrated that daminozide inhibits the synthesis of

kauren-19-ol, a gibberellic acid precursor. They also observed that

daminozide does not hydrolyze to UDMH to any detectable degree. This

evidence is contrary to the theory of Reed et al. (1965), regarding the

physiological action of daminozide. Ryugo and Sachs (1969) found that

UDMH was not as effective as daminozide for retarding growth and floral

initiation when applied to immature almond ovules.

Daminozide applied to plants results in markedly different

manifestations which are dependent on species and cultivar. For

instance, daminozide applied at 2000 ppm to Tagetgs erecta L., var.

’Sovereign’ resulted in thicker leaves and larger root diameters

(McConnel and Struckmeyer, 1971). The internode lengths were 62% of the

control plants and cell number and size were reduced by this

treatment. When daminozide was applied to apple trees var. ’Cleopatra’

at 2000 ppm, smaller fruit were observed due to decreased cell number

per fruit and not due to decreased cell volume (Martin et al. 1967). In other reports, daminozide has been shown to have both anti-auxin

(inhibits shoot and root development) and auxin-like influence on plants. The auxin-like influence was seen when daminozide was applied to Dahlia pinnata Cav., at 2500 ppm. Treated plants had significantly greater weight and number of adventitious roots (Read and Hoysler,

1969). The anti-auxin influence was demonstrated when daminozide treatment resulted in decreased root weight of treated apple trees var.

”Delicious”. Root development was retarded to a lesser extent than terminal and new stem growth (Barden, 1967). Finally, daminozide was found to delay senescence of stored "Grand Rapids" leaf lettuce but was ineffective when applied to broccoli (Halevy et al. 1966).

Daminozide has had far ranging uses in crop production. When applied to Helianthgs annggs (sunflower), the growth retardant enhanced the ability of the plant to recover from severe drought conditions

(Martin and Lopushinsky, 1966). When applied to [jtjs Iabrgsga L., var.

’Concord’ (grapes), daminozide increased cluster size, total vine yield and fruit set during a year when fruit set was low (Tukey and Fleming,

1968). In addition, daminozide reduced the vegetative development of grape vines and thereby reduced the pruning requirements for vine maintenance. Unfortunately, as fruit number increased following spray application, fruit size decreased.

1. Daminozide in Apple and Pear Production

- Daminozide applied to immature McIntosh apples was detected within 4 hours in vegetative buds, cluster bases and seeds (Edgerton and

Greenhalgh, 1967). Absorbed daminozide was found to be chemically stable in Delicious apple trees during a 128 day study (Martin et al.

1964) and a 228 day study (Shutak et al. 1967). Rogers and Thompson

(1968) observed that daminozide applied for 4 consecutive years was ineffective in reducing terminal growth after 1 or 2 years. It is believed that reducing terminal growth will promote more efficient use of carbohydrate resources for fruit production. These researchers determined that daminozide was not phytotoxic to young trees when applied at concentrations less than 40,000 ppm. Daminozide is normally applied to apple trees at label recommendations of 2000 ppm. Along with its inhibition of terminal growth, daminozide was shown to increase leaf number and chlorophyll content of Golden Delicious, York Imperial and

Delicious apple trees (Halfacre et al. 1968). In addition, Halfacre and

Barden (1967), confirmed that treated leaves on Golden Delicious and

York Imperial trees had longer palisade cells and a "looser arrangement of mesophyll cells" which increased leaf thickness without increasing cell number. The increased leaf size following daminozide treatment did not alter the percent dry weight of leaves on apple rootstocks (Barden,

1967). Rather, daminozide acted to reduce the top/root ratio of whole apple trees.

Many researchers have investigated the influence of daminozide on apple fruit. Martin et al. (1967) applied five, 2000 ppm doses to

Cleopatra and Jonathan trees. There was no significant effect on fruit cell volume, P, K, Mg, Na, soluble solids or fruit color of Jonathan apples. There was a significant decrease in cell number and fruit size and a significant increase in Ca content for Cleopatra apples.

Treatment of McIntosh applies resulted in reduced growth rate of fruit within 2 weeks of application (Southwick et al. 1967). Daminozide applied at post-bloom dates had no influence on fruit set, total yield or red color of the fruit. Treatment did reduce flesh softening at harvest and preharvest drop. Reducing preharvest drop can greatly increase harvest yields in a commercial apple operation. Improvement of fruit texture can extend storage and reduce handling-damage of apples for fresh market. Southwick et al. (1967) found that treatment of IO

Cortland apples was effective in reducing scald of control atmosphere stored apples but did not influence brown core or decay of stored

McIntosh apples. Reduction of scald during storage and improved apple texture were also found for treated Red Delicious apples (Williams et al. 1964). The improvement in apple texture was associated with an increase in total pectin and a decrease in the water soluble pectin.

These changes in apple composition were shown to significantly extend the shelf life of regular cold stored (0°C) Red Delicious apples.

Varietal differences for the textural influence of daminozide were reported by Tukey and Camba (1971). Red Delicious apples were resistant to bruising and rupture injury, whereas, Golden Delicious apples were not different from controls. Both varieties had, ”greater resistance to fruit abscission and removal at harvest." The influence of daminozide to reduce fruit drop and improve texture was reported by Fischer and

Looney (1966) for Golden Delicious, Winesap, Spartan and McIntosh varieties. However, they found that treated apple trees had a significantly greater number of pygmy fruit at harvest and the overall fruit size was reduced. Greene et al. (1986) supported this finding with a trial using Mutsu, McIntosh, Cortland, Delicious and Spencer apples. In addition, they determined that fruit shape was altered which caused a reduction in the length/diameter ratio. Webster and Crowe

(1969) also reported that McIntosh apples had reduced fruit elongation, stem length and stem cavity depth following daminozide applications.

These physical changes contrast with those observed after treatment with gibberellin spray.

Daminozide was found to reduce terminal growth and increase fruit bud initiation of Magness pear trees (Rogers and Thompson, 1968). There 11 was no change in fruit size. Griggs and Iwakiri (1968) observed that treatment of Bartlett pear trees resulted in increased fruit size, improved color, decreased fruit set, decreased fruit soluble solids and reduced shoot elongation. Jerie (1972) treated Williams’ Bon Chretien pears and reported an increase in yield of 50-120%, a decrease in shoot number by 25-50% and a decrease in spurs for the growing year following autumn daminozide application.

2. Daminozide in Tart and Sweet Cherry Production

Until recent years, the cherry industry has relied extensively on migrant labor to harvest the cherry crop. However, the rising cost of labor, coupled with a diminishing work force, encouraged the cherry industry to pursue mechanical shaking as a suitable alternative to hand picking. Complete removal of fruit has proven to be a major problem when using mechanical harvesting. When harvesters generate enough force to harvest cherries, tree damage may occur and thus reduce cherry production in future years by decreasing the trees lifespan. Cain

(1967) has described a method for estimating the ”fruit removal force"

(FRF) required to mechanically harvest cherries. A chatillion push/pull force gauge was used to measure this FRF at different locations on a tree. Statistical methods were then used to predict mechanical FRF at harvest time at a 92-95% accuracy rate for this method. Bukovac et al.

(1969) found that Z-chloroethyl phosphonic acid (Ethrel) applied at concentrations less than 1000 ppm was effective for reducing the FRF of

Ergﬂg§_ggra§g§_L., var. "Montmorency" tart cherries. Concentrations of

Ethrel greater than 1000 ppm were found to be phytotoxic. Looney and

McMechan (1970) applied 2000 ppm daminozide at two weeks after full bloom (shuck split) to Montmorency trees. This chemical treatment was 11 was no change in fruit size. Griggs and Iwakiri (1968) observed that treatment of Bartlett pear trees resulted in increased fruit size, improved color, decreased fruit set, decreased fruit soluble solids and reduced shoot elongation. Jerie (1972) treated Williams’ Bon Chretien pears and reported an increase in yield of 50-120%, a decrease in shoot number by 25-50% and a decrease in spurs for the growing year following autumn daminozide application.

2. Daminozide in Tart and Sweet Cherry Production

(1967) has described a method for estimating the ”fruit removal force"

(1969) found that 2-chloroethyl phosphonic acid (Ethrel) applied at concentrations less than 1000 ppm was effective for reducing the FRF of

Ergng§_cgra§gs_L., var. "Montmorency" tart cherries. Concentrations of

Ethrel greater than 1000 ppm were found to be phytotoxic. Looney and

McMechan (1970) applied 2000 ppm daminozide at two weeks after full bloom (shuck split) to Montmorency trees. This chemical treatment was 12

found to advance red color development, reduce fruit acid levels and

reduce FRF at harvest. Unfortunately, the FRF was still too high for

efficient harvest of cherries. Subsequently, they applied 500 ppm

Ethrel one week prior to harvest to daminozide-treated trees and found

better fruit quality than trees treated with either compound alone.

Unrath et al. (1969) also reported that 1000 to 8000 ppm daminozide

applied at shuck split to Montmorency cherry trees decreased FRF, acid

levels and advanced fruit color. Furthermore, daminozide was found to increase fruit firmness and reduce fruit softening during mechanical

harvest. The improved color and texture were also evident in both

canned and frozen products. Regions with short growing seasons benefit

from accelerated fruit maturation induced by daminozide application.

Physical manifestations such as a significant decrease in internode

length and increase in flower bud initiation have also been observed for

cherry tree foliage. Daminozide may also shorten time-to-harvest of

cherries by 1 week.

Daminozide applied to Prgnus angm L., var. "Bing“ sweet cherries

three times at 10 day intervals (2000 ppm) following shuck split

resulted in reduced terminal growth and shorter internodes (Batjer et

al. 1964). There was also 80-100% more leaves and increased bloom during the season following application. There was no decrease in fruit

size at harvest as seen with treated apples and pears. Ryugo (1965)

applied 2000 ppm daminozide to Native Burlat, Bigarreau Moreau and Bing trees at shuck split. Residue levels measured in leaves 2 weeks

following application were 80, 30 and 100 ppm, for each of these respective varieties. Trees from each variety were also treated with

2000 ppm, 3 times within a 3 week interval following shuck split. The 13 residue level in Bing fruits at 22 days following the third application was 7 ppm. Ryugo (1965) concluded that daminozide treatment accelerated the rate of anthocyanin biosynthesis but did not alter the physiological maturity of the fruit. The degree of physiological maturity was assessed by noting that soluble solids and size were not altered by daminozide application. Looney (1969) further elucidated the influence of application date to fruit maturity. He found that soluble solids, fruit size and skin color were increased for Van sweet cherries when daminozide was applied shortly after full bloom. He theorized that daminozide advances sweet cherry maturity when applied at shuck split but delays maturity when applied shortly before harvest. Chaplin and

Kenworthy (1970) further confirmed the results of Looney (1969) for

Windsor sweet cherries. They reported that sweet cherries treated with

1000, 2000 or 4000 ppm daminozide at shuck split had advanced anthocyanin development by 2 weeks and advanced sugar accumulation by 1 week when compared to untreated fruit. Daminozide caused fruit to enlarge at a more rapid rate but that size may appear to be slightly smaller if compared on a harvest date basis to control fruit. The sugar content of fruit for all treatment levels was 25% greater than control fruit and there was no difference in fruit firmness, regardless of harvest date.

3. Economic Importance of Daminozide

In a report to the Federal Insecticide, Fungicide and Rodenticide

Act Scientific Advisory Panel on September 26, 1985, Uniroyal Chemical

Co. Inc., commented on the economic impact of daminozide for the apple, cherry and peanut industry in the U.S. They reported that the net annual income resulting from use of daminozide for the food industries 14 which use daminozide is $214 million. Daminozide was applied to approximately 1/3 of the U.S. apple crop and use of this compound saves about $192 million in product, jobs and production costs. Presently,

(1989 estimates) daminozide is used on only 5% of the apple crop due to recent developments. Uniroyal states that daminozide used in apple production: 1) brings nonbearing young trees into bearing sooner; 2) reduces the requirement for pruning by reducing vegetative growth; 3) reduces the biennial fruit cycling of some varieties; 4) reduces preharvest fruit drop; 5) reduces water core development; 6) reduces cracking of Stayman apples; and 7) increases fruit size, red color and firmness.

Uniroyal Chemical, Co., also assessed the economic impact of daminozide use on cherries as $4 million. The two important benefits cited were; I) maintenance of fruit firmness; and 2) permiting easier removal of cherries by mechanical harvesting. Uniroyal Chemical Co., feels that since there are no acceptable alternatives to daminozide on the market, removal of this compound may have severe economic implications for the fruit and vegetable industry.

8. Daminozide Toxicity

Very little research has been reported regarding the toxicological properties of daminozide by independent research groups. The L050 and

No Observable Adverse Effect Level (NOAEL) for daminozide in rats

(species and source of information not reported) are 8400 mg/kg and

43,200 ppm (90 day feeding study), respectively (St. John et al. 1969).

St. John et al. (1969) fed 0.579 (25 ppm of diet) of l4C-daminozide in the feed for 4 days to a Holstein cow. Milk, feces and urine were 15 sampled to determine the fate of the labelled compound during 10 days of

sampling. They measured no daminozide in the milk but 11.9% and 81.4% of the dose were found in the urine and feces, respectively.

Toth (1977) provided a 20,000 ppm daminozide drinking water

solution, ad libitum, to 100 male and female Swiss albino mice from 6 weeks of age to death. The average daily intake for the male and female mice was 170 mg and 134 mg of daminozide, respectively. For mice averaging 309 body weight, the dosage received was approximately 5000 mg/kg/day for the duration of the study. The treatment solution contained an impurity (hydrolysis product) of 13 ppm 1,1-dimethylhydrazine (UDMH), a suspected carcinogen. Animals were necropsied following death or when found in poor health. Tumors were observed in blood vessels, lungs and kidneys at 73%, 73% and 5% for treated animals and 6%, 18% and 0% for control animals, respectively. Further examination indicated that lesions were angiomas and angiosarcomas of blood vessels, adenomas and adenocarcinomas of lungs and adenomas of kidneys. The life span of the treated mice declined significantly after

30 weeks of exposure. Toth, (1977) does not rule out the possibility that the UDMH impurity may be responsible for the carcinomas.

A teratologic assessment relating to daminozide exposure was reported by Khera et al. (1979). Female Wistar rats were paired with proven males. Twenty females with positive vaginal smears were then dosed orally with 0, 300, 600 or 1000 mg/kg/day daminozide at days 6 to

15 of gestation. Twenty-five fetal and maternal parameters were used to determine the teratological potential following sacrificing at day 22 of gestation. There was no significant influence detected as a result of daminozide exposure. 16

Tosk et al. (1979) used a strain of Salmgng11a_typh1mgrigm (TA 1530) to test the mutagenic capacity of daminozide. Previous studies have shown that strains TA 1535 and TA 100 were insensitive for measuring the mutagenic potential of hydrazines. Daminozide, applied at 5-20 mg/plate did not cause significant reversion of the histidine-requiring auxotroph and therefore was not shown to be mutagenic. In a report by Kimball (1977), daminozide fed to larvae of

Egyptian cotton leafworm resulted in sterilizaton of the adult. This finding may relate more to a toxic rather than a mutagenic response when considered relative to dosage.

In summary, the limited evidence suggests that daminozide is excreted in urine and feces with little metabolic conversion. Very large doses (Toth, 1977; i.e. 5000 mg/kg/day) have been shown to induce tumors in mice. There is a possibility that an impurity of UDMH was responsible for the tumor formation reported in this study. Daminozide doesn’t appear to be a teratogenic compound when given to Wistar rats and no mutagenic activity was observed in the Ames assay.

The furor over the safety of daminozide contaminated foods was brought about by the finding of Newsome (1980). He reported that daminozide present in applesauce during thermal processing could undergo hydrolysis to yield UDMH (Figure 3). 17

0 I II | /CH3 /CH3 CHZ—C—t—NH—N\ -—9 NHZ—N\

l | CH3 A CH3 CH2—‘ﬁ—‘OH uorm

omINozIoE

Figure 3. Decomposition of daminozide to produce 1,1-dimethylhydrazine.

The fortification of applesauce with 30 ppm daminozide yielded 1.53 ppm UDMH following 30 min. boiling or 1.92 ppm following 60 min. boiling. UDMH has been detected in tobacco and tobacco smoke at 0.1-147 ppb (Tosk, 1979; Toth, 1975; Schmeltz et al. 1977) and has also been studied in toxicological screening tests because it has been used as a rocket propellant by the U.S. military (Back et al. 1962).

C. UDMH Toxicity

1. Absorption, Transport and Excretion of UDMH

Smith and Clark (1971) applied 5-30 mmole/kg of UDMH to a 15 X 20 cm shaved area on the chest of anesthetized dogs. After 6 hr all of the dogs treated with 30 mmole/kg died following convulsions. UDMH was measured in the blood within 30 s of application and in urine within 15 min. The concentration of labelled compound in the urine was found to be proportional to the level in blood. This demonstrates that UDMH is rapidly absorped when administered topically. Smith and Clark (1971) 18 determined that 0.02% to 0.18% was recovered in the urine within 6 hours after 5 mM/kg and 20 mM/kg doses, respectively. Dost et al. (1966) administered 14C-UDMH at 0.13-1.33 mM /kg body weight i.p. to Sprague

Dawley rats. They determined that 50% was cleared in the urine within 2 days and 30% was respired as 14C02 in 10 hr when the rats were given a low dose (0.33 mM/kg; 20 mg/kg). Animals given a convulsive dose of UDMH (1.33 mM/kg; so mg/kg) respired only 13% as ”co2 after 20 hr This indicates that rats have the capacity to eliminate approximately 0.17 mM/kg of UDMH as 14C02 in 20 hr. The maximal threshold of excretion by the kidney was not reached even when convulsive doses were given to the animals. After 53 hr, 5-20% of UDMH was retained in the tissue. Back et al. (1952) administered so mg l4c-uoNH/kg body weight i.p. to 2 dogs and 6 cats and measured urinary output. Between 11.4% and

47.5% of the administered dose was collected in the urine within 5 hr.

They concluded that the kidney was able to excrete large amounts of UDMH and that the rate of excretion depends on the hydration of the animal.

They found that the urinary level was the most sensitive qualitative indicator of exposure. In another experiment, the distribution of UDMH in 9 major organs of 12 dosed (i.p.) female albino rabbits was determined. In 24 hr, 10-28% of the label was found in all body tissues examined. The concentrations measured in brain tissue were half the amount measured in the spleen, kidney or liver. A larger volume of blood in the latter organs may explain the differences observed. In general, no organ selectively accumulates UDMH in the body.

Smith and Castaneda (1970) reported that UDMH readily crosses the blood-aqueous humor barrier of the eye of male mongrel dogs. UDMH increased corneal opacity possibly by causing hydration of the 19 endothelium in the anterior chamber of the eye.

It is evident from these studies, that the polar nature and the small molecular size of UDMH allows for rapid absorption, transport and excretion. The rapid clearance of a compound results in reduced exposure time and generally reduced toxic influence. Current evidence does not suggest that UDMH is accumulated by any organs in the body.

2. Metabolism of UDMH

Wong (1966) studied the effects of hydrazine and UDMH on renal function. Hydrazine caused damage to the proximal tubular epithelium which reduced tubular resorption of glucose. 0n the other hand, UDMH given i.v. at 45 mg/kg body weight to anaesthized female mongrel dogs did not affect tubular resorption of glucose. Treated dogs had a slightly significant increase in creatinine clearance.

Cornish and Hartung (1969) gave 10-70 mg UDMH/kg/day (i.p.) for 21 days to female Sprague Dawley rats. One animal of ten survived at 70 mg/kg and 4 of 10 survived at 50 mg/kg. Animals treated with 50 and 70 mg/kg, showed signs of fat infiltration into the tubular epithelium of the kidney. In addition, animals treated with 50 mg/kg had elevated blood urea nitrogen (BUN) and serum glutamic-oxaloacetic transaminase

(SCOT) which are indiators of kidney damage. There was a strong diuretic effect observed with the 10 mg/kg dose throughout the 21 day study. Urine output was twice that of control rats.

Barth et al. (1967) also noted a diuretic effect following UDMH treatment. Male Sprague Dawley rats given a single dose of 80 or 100 mg

UDMH/kg body weight i.p., had a 3-fold increase in urine excretion during a 6 hr period. There was a marked decrease in serum potassium and calcium levels at l and 2 hr following injection, however, at 24 hr, '20 potassium returned to normal while calcium was significantly higher than controls. Studies have also been done to determine if UDMH is hepatotoxic.

Reinhardt et al. (1965) measured the levels of indicator enzymes to determine if hepatic changes have occurred following treatment. The enzymes monitored were lactic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase and glutamic dehydrogenase. Male Wistar rats were injected with a single dose of 80 mg UDMH/kg body weight. Enzyme activities were measured in serum and liver at 16, 24, 48 and 72 hr after administration. Carbon tetrachloride, a strong hepatotoxin, was included as a reference. The results indicated that UDMH does not cause liver necrosis but may alter the permeability of liver cells. UDMH did not produce any histologic alterations nor did it inhibit any of the enzymes examined. However, UDMH did cause a significant increase in serum activity of lactic dehydrogenase and malic dehydrogenase enzymes. These enzymes are good indicators of tissue damage.

Hawks et al. (1974) found evidence to indicate that UDMH has no effect on hepatic protein synthesis or ribosomal aggregation at 6 or 24 hr following administration to Wistar albino or B.D.1X rats. The incorporation of 3H leucine in ribosomal preparations was used to measure protein synthesis.

Smith and Clark (1971) reported that levels of glucose increased in blood and urine as a result of UDMH treatment. There was also a decrease in glutathione peroxidase activity of red blood cells (RBC) at

5 mmole UDMH/kg, no change in activity at 20 mmole /kg and increased activity at 30 mmole/kg. They found no change in reduced glutathione in

RBC’s at any level. Weeks et al. (1963) exposed dogs with UDMH vapor at 21

50, 200, 600 ppm for 5, 15 and 60 min. durations. They found no changes

in RBC, WBC and reticulocyte counts or in hematocrit, non-protein

nitrogen, glucose, bilirubin or in RBC cholinesterase determinations.

Multiple exposure at the higher dose caused vomiting, tremors,

convulsions and death. Dogs retained about 80% of the inhaled UDMH in

the respiratory tract.

Patrick and Back, (1964) gave 7 rhesus monkeys daily doses of 10 mg

UDMH/kg body weight for 20 injections. All animals were found to lose weight (0.2-0.8 kg) during the experiment. Many of the animals had

regained their initial weight by the forth week of the experiment.

There were no alterations in hemoglobin, hematocrit, WBC counts, differential counts and SGOT. Glucose was significantly elevated and

histological examination found a slight amount of lipid around the

central vein in the liver of one animal. In another animal, there were

lipid deposits around the tubular membrane of the kidney. Two monkeys

had fat infiltration in the heart.

Research data indicates that UDMH is not very toxic. What is the cause of death at high doses? It was previously mentioned that rats given a large dose of UDMH experienced convulsions within 2 hr and death within 5 hr (O’Brien et al. 1964). Convulsions included tonic, clonic and coordinate episodes. Animals which survived for 6 hr generally recovered. These UDMH induced seizures were thought to be due to interference with the metabolism of pyridoxine (vitamin 86). This vitamin is a cofactor in the metabolism of gamma aminobutyric acid

(GABA) which is a neurotransmission inhibitor. The inhibition of GABA synthesis will be covered later in more detail.

Wittkop et al. (1969) investigated the role of multifunctional 22 oxidases (MFO’s) in UDMH metabolism. UDMH was found to be a substrate for demethylase in rat liver microsomal preparations. The demethylase reaction requires NADPH and 02 and yields 176 mM of formaldehyde/5 mg protein/40 min. Cytochrome P450 metabolism of UDMH is limited by UDMH’s low lipid solubility.

Kato et al. (1969) correlated the rate of oxidative conversion by cyt. P450 of hydrazines to their lipid solubility. Because of its polar nature, UDMH, may undergo a conjugation, in yiyg, to increase its lipid solubility. Godoy et al. (1983) confirmed the findings of Wittkop et al. (1969) and reported that UDMH could be converted to formaldehyde by enzymatic processes in microsomal preparations. They found that non- enzymatic reactions could produce formaldehyde from UDMH. Non- enzymatically transformed UDMH, covalently binds to proteins but not to nucleic acids. The metabolism of UDMH by cyt. P450 is improbable because SKF525A and other cyt. P450 inhbitors were ineffective in altering microsomal conversions (Wittkop et al. 1969). Instead, a second microsomal system which incorporates a flavoprotein, FAD- containing monooxygenase was explored. Methimazole, a substrate for the

FAD-containing monooxygenase, strongly inhibited the oxidation of UDMH by rat and hamster microsomes (Prough et al. 1981). Therefore, UDMH is chemically transformed by non-cyt. P450 microsomal reactions. The oxidation of UDMH to formaldehyde is stoichiometric (1:1:1) with 02,

NADPH aand UDMH. The reaction shown in Figure 4, involves a diazine intermediate. 23

,.- . CH 3 l °”3\\ CH CH ' =”\ —> / N—szw—N

CH cn/ /\ CH onnzcur 3 :2 3 {J

CH a / 0" + / 3 / \ s CH- CH3 N"'2 3 CH2

H30

FORMALDEIIYOE HONOHETHYL HYDRAZINE (PROUGH ET AL.. 1981)

Figure 4. Stoichiometric conversion of 1,1-dimethylhydrazine to

formaldehyde.

UDMH reacts with bovine plasma amine oxidase (PAO) to form an enzymatically-inactive complex (Hucko-Haas and Reed, 1970). This inactive complex decomposes by first-order kinetics to yield an active enzyme and an unidentified compound which does not inhibit PAO.

Acute exposure to UDMH in animal systems has been explored using many models. Slonim (1977) found the LC50 for guppies exposed to UDMH 24

was lower in hard water than in soft water. Guppies exposed for 24 hr

gave a LC50 for hard and soft water of 78.4 mg/L and 82.0 mg/L,

respectively. Guppies exposed for 96 hr gave a L°50 for hard and soft

water of 10.1 and 26.5, respectively. This represents a difference of

250% at 96 hr of exposure. Aeration was slightly effective in reducing

the toxicity of hard water after 24 hr of exposure.

Back and Thomas (1963) found a relationship between toxicity and age

of UDMH solution. Sprague Dawley rats were given an i.p. dose of UDMH

in a freshly prepared solution and solutions held 8, 12 and 27 days.

The LDso’s for the solutions were 125, 100, 78, <78 mg/kg,

respectively. This change in toxicity may be due to photodegradation of

UDMH solutions, which causes a change in solution color from clear to

yellow-orange. Rats exposed to 10% of the LD50 (10 mg/kg) had no

observable adverse effects from the treatment. Rats exposed to 50 mg/kg

experienced a slight drop in blood pressure for 1—3 min. Animals

exposed to 100 mg/kg experienced a 50% drop in blood pressure for 2 1/2

min. Rats exposed to UDMH vapor gave LC50’s of 252, 1410, 4010, 8230

and 24,500 ppm for 240, 60, 30, 15 and 5 min. exposure times,

respectively (Weeks et al. 1963). UDMH applied topically to a shaved

area of the chest of dogs for 6 hr, yielded a L050 of 20-28 mmole

UDMH/kg. This is 15-20 X the L050 for intravenous administration of

UDMH (Smith and Clark, 1971). The threshold dose for convulsions in

monkeys (species not reported) is 40 mg UDMH i.v./kg body weight (Back

et al. 1962). A dose of 100 mg/kg is lethal for most species of

animals. The symptoms observed following exposure to UDMH are dependent on the hydration of the animal, which influences hepatic clearance of UDMH. The pyridoxine status of the animal is also important. 25

Several cases of human exposure to UDMH vapor have arisen during the handling of UDMH in a military setting. Shook and Cowart (1957) observed two patients exposed to UDMH fumes following an accidental spillage. Both persons experienced choking and difficulty breathing.

Within 4 hr, both persons suffered from nausea and vomiting. Six months after exposure, neither individuals was found to have clinical symptoms. Two male patients exposed to Aerozine-SO (a UDMH containing solution) vapor had nausea, trembling, weakness, headache, hyperactive reflexes and sore throat (Frierson, 1965). Both were administered 200 mg (i.m.) pyridoxine hydrochloride and within 20 min. all symptoms except congestion and chest pain had subsided.

3. Mutagenicity of UDMH

With the rising incidence of lung cancer, research has focused on the carcinogenic agent(s) which are present in tobacco smoke. Schmeltz et al. (1977) reported finding between 60 to 147 ppb UDMH in tobacco samples. They attributed this level of UDMH to the herbicide, maleic hydrazide, which is used to reduce ”suckers” on tobacco plants. In addition, they attributed the presence of nitrosamine in tobacco to a reaction between UDMH and nitrite. Unfortunately, these researchers did not attempt to measure UDMH in tobacco which had not been treated with maleic hydrazide. Tosk et al. (1979) demonstrated that UDMH can occur naturally in tobacco which hasn’t been treated with maleic hydrazide.

Luinsky et al. (1968) confirmed that UDMH is a possible metabolite of dimethylnitrosamine (DMN). This finding led resarchers to investigate whether UDMH produces the mutagenic properties which were attributed to

DMN. Kimball (1977) reported that methyl derivatives of hydrazine can alkylate purines to yield 7-methylguanine. In contrast, Kruger et al. 26

(1970) used l4C-UDMH treatment on male Sprague Dawley rat liver and

Yoshida transplantation tumor to test for alkylation of purine bases.

They found very little incorporation of label and no 7-methylguanine was

detected. They concluded that UDMH is not a strong alkylating

intermediate of DMN. which is noted for its alkylation properties.

In a mutation assay using a histidine requiring auxotroph of

Salmonella typhimgrium (TA 1530), Tosk et al. (1979) found that UDMH did

not produce a significant number of revertants. Epstein et al. (1972)

used a dominant lethal assay with ICR/Ha Swiss mice to test UDMH

mutagenicity. In this assay, males are dosed for 8 weeks at subacute

levels (i.e. 25 and 63 mg/kg) then mated with 3 virgin females at weeks

1-3, 4-5 and 6-8. Females were sacrificed on day 12 or 13 from onset of

vaginal plugs (pregnancy). The mutagenic index was estimated by

dividing the number of early fetus deaths by the total number of

implants. UDMH did not have a mutagenic index which was signficantly

higher than the control. f

Wyrobek and Bruce (1975) reported that UDMH did not induce sperm

abnormalities in (C57BL X C3H)F1, mice during a 10 week subacute dosing of 10-100 mg/kg. They concluded that UDMH does not have any observable mutagenic properties when examined in this assay.

4. Carcinogenicity of UDMH

Virgin female Swiss mice (25) were dosed five times per week for 40 weeks by gavage with 0.5 mg UDMH (16 mg/kg/day) (Roe et al. 1967). UDMH was found to increase the incidence of pulmonary tumors which were mainly alveologenic or bronchiologenic adenomas or adenocarcinomas. After 50-60 weeks, 24 total tumors were observed in 4 of 9 surviving 27 mice. Kelly et al. (1969) administered 3.6 mg UDMH i.p. and 7.2 mg UDMH orally, to (BALB/c X DBA/2)F, mice, once per week for 8 weeks. They reported 1 tumor in 30 animals and 1 tumor in 25 animals for the 3.6 mg and 7.2 mg doses, respectively. Tumor incidence was not signficantly greater than for control animals. Toth (1973) treated Swiss mice (50 male and 50 female) with 0.01% UDMH in drinking water (given ad libitum) for the lifetime of the animals. Mice were necropsied following death or when found in poor health. The average intake of UDMH was 0.7 mg per animal per day or approximately 30 mg/kg for mice weighing 25-30 g.

Histological examination revealed a 79%, 71%, 10% and 6% incidence in blood vessel, lung, kidney and liver tumors, respectively. Untreated mice had an incidence of 2, II, 0 and 0% tumors, respectively. The average latent period for tumor formation was 59 weeks for females and

42 weeks for males. Toth, (1975) indicated that UDMH must be administered at a dose level 10 times greater than symmetrical dimethylhydrazine (SDMH) to induce tumors. In another investigation,

Toth (1977) administered 0.1% UDMH in the drinking water (ad libitum) to

Syrian golden hamsters (50 male and 50 female) for the lifespan of the animals. UDMH treatment reduced the lifespan of female but not male hamsters. Males and females were found to have tumors of the cecum at

30% and 20% and vascular tumors at 28% and 4%, respectively.

5. Influence of UDMH on Nervous Function

Fairchild and Sterman (1968) tested subconvulsive i.p. doses of UDMH

(7, 14 and 21 mg/kg) given to cats on excitatory and inhibitory mechanisms of the CNS. Cats were implanted with electrodes in the midbrain reticular activating system (RF) and the basal forebrain inhibitory area (BF). One of these sites was electrically stimulated at 28 the appropriate time while the cats attempted to retrieve food during a coordination experiment. As expected, stimulating the RF electrode, enhanced the cats performance time while stimulating the BF electrode, hindered the cats response time. A UDMH dosage of 7 mg/kg had little effect on the animals response to food. At the subconvulsive dosages of

14 and 21 mg/kg, UDMH increased the inhibition observed during BF stimulation. The researchers concluded that subconvulsive doses of UDMH have detectable CNS effects as measured by this experimental model.

In attempting to show the influence of UDMH on neurological responses, Goff et al. (1964) measured brain waves following UDMH administration. They gave 16 and 32 mg/kg doses (i.p.) to free-moving cats and a 64 mg/kg dose to anesthetized cats. UDMH was shown to act primarily at axodendritic synapses to increase somatosensory cortex negativity. This increase in negativity proceeds animal seizures by a few minutes. Free-moving cats suffered seizures at 162 min. following chemical administrations, whereas, anesthetized, paralyzed cats did not show seizures until 200 min. even when a double size dose was given.

Research indicates that UDMH causes seizures by inhibiting the production of GABA in the CNS. The discussion which follows will describe the function, synthesis and degradation of GABA and the role of

Vitamin 85.

GABA has been shown to be a potent inhibitor of nervous transmission by action on cortical cells (Krnjevic and Schwartz, 1966). This action is necessary for removal of cellular excitation following nervous transmission. Kelly and Krnjevic (1968) state that, "GABA has a strong depressant action on neurons in the cerebral cortex which is associated with an increase in membrane potential and conductance. The presence of 29 substantial amounts of GABA in the cortex has therefore led to the suggestion that GABA may be a cortical inhibitory transmitter."

GABA is synthesized from glutamate through the irreversible action of glutamic acid decarboxylase (GAD), which catalyzes the decarboxylation reaction. GABA is then cleared by a reversible reaction with GABA- glutamic acid transaminase (GABA-T) to produce succinate semialdehyde. Pyridoxine (Vitamin 85) is an important cofactor for both synthesis and degradation of GABA (Figure 5).

glutamate

Vitamin 86____;>. glutamic acid decarboxylase (GAD)

V gamma-aminobutyric acid (GABA)

Vitamin B6.___ﬁ;> GABA-glutamic acid transaminase (GABA-T) V/ succinate

semialdehyde i succinate

Figure 5. Biosynthesis and degradation of gamma-aminobutyric acid.

GAD has been shown to be bound to the axoplasm of nerve endings and

GABA-T is compartmentalized (Figure 6) in the mitochondrial matrix

(Salganicoff and De Robertis, 1965). There are two forms of GAD (I + 30

II) which are present in the CNS and kidneys (Haber et. al. 1970; Lowe et al. 1958; Haber et al. 1970).

009 Glutuinuic

Otoc

Glut . yiil e-Glinc atom IUIOMO 4

GUN

Perlkorion

(Salganicoff and De Robertis, 1965)

Figure 6. Compartmentalization of gamma-aminobutryic acid in the nerve

ending. 31

Vitamin 85 is a water soluble vitamin which is present as

pyridoxine(PY), pyridoxal(PAL) or phyridoxal phosphate(PALP). The later

is synthesized by the action of pyridoxal kinase as shown in Figure 7.

H 0 H 0 \ C’ I \C’ l 0

Ho Ho / l CH20H pmoox“ \ / CH2—O—P—OH KINASE " I I CH \ CH \ OH 3 N 3 N

PYRIDOXAL (PAL) PYRIDOXAL PHOSPHATE (PALP)

CH20H CH2NH2 Ho Ho / CHZOH / CH CH \ I | 2 CH3 N CH3 \ N

PYRIDOXINE (PN) pvmooxmme (PM)

Figure 7. Forms of Vitamin 86.

What is the mode of action of hydrazine compounds in causing

seizures and death? Several hydrazides were shown to inhibit GAD, glutamic aspartic transaminase, and L-cysteine disulfhydrase, all of which require pyridoxal phosphate as a cofactor (Killam and Bain,

1957). Administration of the hydrazide, semicarbazide, to adult white 32 rats confirmed that convulsions were correlated with inhibition of

GAD. Maynert and Kaji (1962) subsequently demonstrated that semicarbazide caused a reduction in whole brain GABA in Swiss albino mice. Researchers theorized that hydrazides would bind to B5 vitamers and cause inhibition of enzymes which require this cofactor. If hydrazides bind to B6 vitamers to reduce enzyme activity, why isn’t GAD and GABA-T inhibited equally? Baxter (1969) reported that as GAD is inhibited by thiosemicarbazide, GABA decreases even after GAD activity begins to increase. This can be explained by compartmentalization, since at the same time that GAD is strongly inhibited, GABA-T continues to breakdown GABA. Sze and Lovell (1970), also found compartmentalization to play a major role in the differential inhibition of GAD and GABA-T. They found that brain slices, obtained after a convulsive dose of thiosemicarbazide administration, gave no indication of GAD inhibition. Homogenization of brain tissue, however, caused extensive inhibition of GAD activity. This supports the possibility that compartmentalization of enzymes is responsible for the observed responses. Salganicoff and De Robertis (1965) stated that, "GAD is concentrated mainly in the nerve endings when it is important for GABA control. Inhibition of GAD could cause parallel decreases of GABA in synaptic cleft and whole brain. The GABA ion in the synaptic cleft is probably removed by transport through the cell membrane and GABA-T degrades in the mitochondria. An increased rate of transport could therefore produce an increased removal of GABA from synaptic cleft and whole brain. No increase in the amount of GABA-T enzyme per se should be required since the potential activity of this enzyme is normally several times greater than that of the GABA synthesizing system. 33

Inhibition of GABA-T wouldn’t necessarily cause an increase in GABA in

the synaptic cleft. Baxter (1969) had shown that thiosemicarbazide

treatment caused an increase in the rate of GABA diffusion out of the

brain which may suggest a change in membrane permiability had occurred

(Abrahams and Wood, 1970). Wood et al. (1975) investigated whether any

hydrazide tested would inhibit GABA-T preferentially over GAD. Although

several compounds were tested, there were none found which exerted

greater inhibitory effects on GABA-T. Horton et al. (1979) demonstrated

that hydrazides do not affect all regions of the brain similarly. For

instance, they found that methyl dithiocarbazinate reduced GABA

concentration in all regions of the brain except in the ventral

midbrain. In contrast, isonicotinic acid hydrazide reduced GABA levels

in all areas except the frontal cortex until 45 min. after

, administration. Clearly, there are regional differences exerted by the

various hydrazides. There are also intercellular as well as

intracellular differences due to the effects of hydrazides on: 1) GAD

activity; 2) GABA—T activity; and 3) membrane permeability.

To demonstrate that hydrazides caused enzyme inhibition by competing

for 85 vitamers, Maynert and Kaji (1962) administered pyridoxine and

pyridoxal to semicarbazide treated Swiss albino mice and Sprague Dawley

rats. They reported that pyridoxine delayed seizures, whereas,

pyridoxal exerted no protective effect from seizures. Wood and Peesker

(1972) found that 1.46 moles/kg pyridoxine administered to isonicotinic

acid hydrazide treated white leghorn cockerels, prevented seizures and

reduced the effects of hydrazide administration on GABA levels and GAD

activity but not on GABA-T activity. It appears that hydrazides reduce

GABA levels by binding vitamin B5 and thereby inhibit GAD. These 34

effects lead to seizures and death in animals treated with excessive

amounts of hydrazides.

To further confirm the theory of pyridoxine conjugation by

hydrazides, Williams and Abdulian (1956), measured the hydrazone of

pyridoxine and semicarbazide in the urine of treated dogs (Figure 8).

Furst and Gustavson (1967) gave convulsive doses of UDMH and the

corresponding hydrazone to Swiss albino mice. The UDMH-hydrazone proved

CH3\ /CH3 N CH20H CH3 L

Ho / CH 0H + NH __ / ___) H

CH \ s CH3 Ho 3 N / I CHZOH UDMH pvmooxms (PN) CH 3 \ N

PN - UDMH HYDRAZONE

Figure 8. Reaction of pyridoxine with 1,1-dimethylhydrazine.

to be more toxic than UDMH when three criteria were compared. These

include: 1) lag time before convulsions; 2) time until death; and 3)

L050.

What is the mode of action of hydrazones to induce convulsions?

Tapia and Pasantes (1971) found a correlation between convulsive

episodes and PALP-gamma glutamyl hydrazone formation following L-

glutamic acid-gamma-hydrazide administration. The hydrazone was

determined to decrease pyridoxal phosphate in whole brain and thereby

cause convulsions. This relationship was further elucidated by Perez De 35

La Mora et al. (1973), who reported the inhibition of pyridoxal kinase by pyridoxal phosphate-gamma-glutamyl hydrazone. Inhibition of pyridoxal kinase causes a reduction in pyridoxal phosphate levels. Pyridoxal phosphate concentration was shown to correlate with GAD activity, in £129. Therefore, hydrazones function to reduce GABA levels by inhibiting pyridoxal kinase activity. Hydrazides, on the other hand, bind to B5 vitamers to inhibit GAD activity and reduce GABA levels.

Both hydrazones and hydrazides lead to convulsions and death when given at high doses.

UDMH administered to male Sprague Dawley rats (1.6 mM/kg) with and without simultaneous pyridoxine, pyridoxal or pyridoxal phosphate injection (0.5 mM/kg) (Medina, 1963), caused moderate inhibition of GAD but only slight inhibition of GABA-T. There was a significant decrease in GABA concentration. UDMH plus pyridoxal or pyridoxal phosphate caused a decrease in GAD activity and GABA concentration but not GABA-T activity. Pyridoxine administered simultaneously with UDMH resulted in less GAD inhibition and no change in GABA concentration of GABA-T activity. The time to onset of seizures was shortened from 90 to 30 min. in animals treated with pyridoxal or pyridoxal phosphate. Medina

(1962) reported that 85 vitamers such as pyridoxine and pyridoxamine were effective in preventing seizures, whereas, pyridoxal tended to potentiate seizures. He found the restoration of GAD activity, from greatest to least to be; pyridoxal phosphate; pyridoxine phosphate; pyridoxal; pyridoxine; pyridoxamine. It is possible that pyridoxal phosphate has the highest affinity for GAD, or the least affinity for

UDMH. It is also possible that compartmentalization is responsible for this phenomena. Medina (1962) reported the order of inhibition of GAD 36 as follows; UDMH > pyridoxal 1,1-dimethylhydrazone > pyridoxal phosphate

> 1,1-dimethylhydrazone. Hydrazones was measured in the brain homogenates from treated Sprague Dawley rats. Geake et al. (1966) administered the LDIOO dose (120 mg/kg) to Sprague Dawley rats. This was followed by intracerebreal injections of 85 vitamers. The intent of giving intracerebral injections was to circumvent the blood-brain barrier influences. Rats given pyridoxine between 0.5-1 mg at the onset of seizures, survived. Animals given pyridoxine at a greater or less dosage, experienced seizures. A dose of 0.25-1 mg pyridoxal was also effective in preventing convulsions and death. A dose of 0.03-0.17 mg pyridoxal phosphate prevented death in 18 of 23 rats but was not effective in preventing convulsions. They believed that delaying of 36 treatment was necessary to allow some UDMH to be excreted in the urine. In addition, depletion of 36 prior to further administration will reduce the amount of hydrazone which is formed. Hydrazones may prove to be more toxic and cleared slower than UDMH. Pyridoxine may be more effective than other 85 vitamers because it does not readily form hydrazones following UDMH administration. LIST OF REFERENCES

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THE INFLUENCE OF pH AND HEATING TIME ON THE DECOMPOSITION 0F DAMINOZIDE (ALAR) AND THE FORMATION OF UNSYMMETRICAL DIMETHYLHYDRAZINE (UDMH).

Charles R. Santerre The Influence of pH and Heating Time on the Decomposition of

Daminozide (Alar) and the Formation of Unsymmetrical Dimethylhydrazine (UDMH).

ABSTRACT

Daminozide was added to 50 mM NaH2P04 - 24% sucrose solution at 12.5

ppm (w/w) to determine the influence of heating, pH and processing

conditions on the decomposition of this pesticide.

Daminozide-fortified solutions (pH 3.0; 3.6; 4.2) which were canned and heated (100°C) for 0-15 min., contained 0-23 ppb of the decomposition product, 1,1-dimethylhydrazine (UDMH). UDMH production was directly related to heating time. Daminozide-fortified solutions which were heated (80°C) in an open container for 0-10 min., then canned and heated (100°C) for 5 min. Due to vaporization, UDMH concentration was not directly correlated to heating time. UDMH concentration was greatest in the solutions at pH 3.6 for both heated in an open-container or a closed-container. Daminozide concentrations were not decreased greater than 1.5 ppm (limit of detection) from 12.5 ppm for any of the experimental treatments.

INTRODUCTION

Daminozide (succinamic acid 2,2-dimethylhydrazide) is a plant growth regulator which is used in the production of apples, peanuts, cherries, pears, blueberries, cranberries, and grapes to improve crop yield and product quality (Unrath, 1969), This pesticide was introduced in 1962 and registered for use on food crops in 1968. In 1980, Newsome (1980) demonstrated that daminozide can hydrolyze during the thermal processing of fruit products to form unsymmetrical dimethylhydrazine (UDMH; Figure

1). Concern over the safety of foods containing UDMH has prompted studies to determine the toxicity of UDMH and the levels of UDMH in processed food products which contain daminozide residues.

Newsome (1980) fortified applesauce with 30 ppm daminozide then boiled the sauce in an open container for 30 or 60 min., and measured

1.53 ppm and 1.92 ppm UDMH, respectively. Unfortunately, analytical interferences prevented replication of this study by other researchers

(Uniroyal Chemical. Co., Inc., 1984). Furthermore, the duration of heating which was applied to the apple sauces was not representative of commercial practices. Further research was required to determine the dynamics of daminozide decomposition and UDMH formation so that human exposure to UDMH could be estimated.

Montmorency cherry trees treated with 3.4 and 6.8 lbs. active ingredient/acre produced fruit containing <13 ppm daminozide and <10 ppb

UDMH (see results Chapter 2). Processing of these cherries into pie filling reduced daminozide residues to <5 ppm but increased UDMH to as high as 100 ppb. These data indicate that processing of treated fruit

47 48 products can have a significant effect on daminozide and UDMH residues.

A sensitive assay using GC/MS to determine daminozide concentrations

in foods was reported by Conditt et al. (1988). In this procedure, daminozide is first hydrolyzed with boiling sodium hydroxide to form

UDMH. UDMH is then distilled into a collection vessel containing

salicylaldehyde to form salicylaldehyde dimethylhydrazone (SDH; Figure

2). GC/MS, selected ion monitoring (SIM) is then used to determine SDH concentration following extraction with methylene chloride. Free UDMH

is generally present at much lower levels than daminozide and will only

slightly affect the daminozide determinations. Free UDMH can be determined by eliminating the daminozide hydrolysis step and adding salicylaldehyde directly to the sample prior to methylene chloride extraction. The method of Conditt et al., (1988) is time consuming (30 min./distillation) and subject to sample loss if the distallation system

in not completely sealed. To avoid these problems 3 LC method was developed to assay for daminozide which would be acceptable for samples which contain few interfering compounds.

The objectives of this study were to determine the influence of heating time and pH on the decomposition of daminozide and the formation of UDMH in a canned sucrose syrup. The processing conditions were designed to predict the reaction kinetics of UDMH formation in food samples following standard processing regimens. An additional objective was to determine the influence of heating daminozide fortified samples prior to can sealing. MATERIALS & METHODS

Forty eight liters of 0.05 M NaH2P04 buffered solution was prepared by combining 331.2 g NaH2P04-H20 (Mallinckrodt Inc., Paris, KY) with 48

L of distilled-deionized water. Sixty kilograms of 24% sucrose solution was prepared by adding 14.4 kg sucrose (GR grade; EM Science, Cherry

Hill, NJ) to 45.6 kg of 0.05 M NaH2P04 solution. Three solutions were prepared from the buffered sucrose solution by adjusting the pH to Pb

3.0, 3.6, 4.2. These pH values were selected to cover the range of pH’s for processed fruits such as apples and cherries. A 20 kg volume of this buffered-sucrose solution was adjusted to pH 3.0 by adding 70 mL of

1N NC]. A 20 kg volume of this buffered-sucrose solution was adjusted to pH 3.6 by adding 14 mL of 1N HCl. A 20 kg volume was adjusted to pH

4.2 by adding 4 mL of 1N NaOH. Each solution (i.e. pH 3.0, 3.6 and 4.2) was fortified with 100 mL of daminozide stock solution (125 g daminozide

(Aldrich Chem. Co., Milwaukee, WI)/500 mL distilled-deionized (H20) to give a final concentration of 12.5 ppm daminozide in a 0.05 M NaH2P04,

24% sucrose solution.

The daminozide-fortified solutions were filled (450 g per can) into

No. 303 enamel-coated cans (Continental Can Co.), lidded and sealed.

Twenty cans per pH level were then divided into 4 groups and immersed in rapidly boiling water for 1) 0 min.; 2) 5 min.; 3) 10 min.; and 4) 15 min. Following heating, cans were immediately cooled in 15.5°C water for 1 hr and placed in 3.8°C storage prior to daminozide and UDMH analysis.

Fifteen cans per pH level were filled (450 g daminozide-fortified

49 50 solution per can), heated to 80°C, held for I) 0 min.; 2) 5 min.; and 3) 10 min., lidded and sealed. Following closure, cans were immersed for 5 min. in rapidly boiling water, then cooled in 15.5°C water and stored at

3.8°C until daminozide and UDMH analysis. To compensate for evaporative losses, cans were opened and the fill weight was adjusted to 450 g with distilled-deionized water just prior to residue analysis.

Daminozide Determination

Canned samples were opened (adjusted to 450 9 weight with distilled- deionized water if heated prior to can closure), and adjusted to pH 4.2 with 1N NaOH. Five milliters were filtered through a 0.45 X 10'6 m filter (Type HA; Millipore Inc., Bedford, MA) attached to a Luer-lock syringe. Daminozide samples and standards were separated and quantified using High Performance Liquid Chromatography (HPLC) with UV detection using the following parameters; Alltech Carbohydrate IOU column

(Alltech Inc., Deerfield, IL), 300 mm x 4.1 mm i.d.; Anspec Model 2200 pump, flow rate . 1.5 mL/min.; Milton Roy, Spectro Monitor 3100 detector, Settings: 0.01 AUFS, 0.50 5 (response time), 218 nm

(wavelength); Injector: Rheodyne Model 7125 (20 X 10'°L loop); solvent: 0.05 M NaH2P04/methanol (90:10) degassed and filtered (0.45 X

10'6 m Millipore filter; Type HA); Integrator: Spectra Physics Model

SP4270, chart speed - 1.27 cm/min.; Syringe: Hamilton #705 (blunt tip), 30 X 10'°L injection volume. Three cans for each heating time and pH level were assayed two times to provide 3 replicates and 2 subsamples per replicate.

Daminozide standards were prepared at 25, 10, 7.5, 5.0, 2.5 and 0 ppm daminozide concentrations by diluting a 1000 ppm daminozide stock 51 solution (100 mg daminozide crystals/100 mL 0.05 M NaH2P04 - 24% sucrose solution (pH . 4.2) to a 100 ppm daminozide stock solution (0.5 mL of

1000 ppm daminozide in 500 mL volumetric flask). Further dilutions were prepared by diluting the 100 ppm daminozide stock solution with the 0.05 mM NaH2P04 -24% sucrose solutions (pH - 4.2).

UDMH Determination

Canned samples were opened (adjusted to 450 9 weight with distilled- deionized water if heated prior to can closure) and a 150 mL aliquot was combined with 150 X 10’6 L salicylaldehyde (Aldrich Chem. Co.) (neat) in a 38 x 200 mm culture tube and sealed with a teflon-lined screw cap

(Kimax). Culture tubes plus sample were shaken, placed in a 50°C water bath for 90 min., and cooled to 3.8°C overnight. To each tube, added 3 g of sodium chloride, sealed and shaken. The samples were then extracted two times with 4 mL methylene chloride (Aldrich Chem. Co) which contained 0.5 mL 4-nitroanisole (Aldrich Chem. Co.)/1000 mL as an internal standard. After each addition of methylene chloride plus 4- nitroanisole, samples were sealed and shaken vigorously for 30 sec. The bottom layer was removed using a pasteur pipet and combined with 1 g

NaZSO4 (anhydrous; Aldrich Chem. Co.) as a drying agent. Samples were then analyzed using gas chromatography/mass spectrometry (GC/MS). GC/MS equipment and parameters were as follows: Gas Chromatograph - Delsi Di 700, parameters; oven temperature - 180°C, isothermal interface temperature - 250°C, source temperature - 250°C; column: 08-1; 30 m - fused silica, phase thickness . 0.25 X 10'6 m; carrier gas: helium; injection: on-column (2 X 10'°L). Quadrapole Mass Spectrometer -

Nermag RIO-10C; parameters; electron ionization (E1), positive ion 52 mode, selected ion monitoring; primary pressure; 1.3 x 10’3 Torr; IE =

0.198 amps; e’ - 70 eV; focal - -94.6; ions . -15.5; external - +39.1;

multiplier - -2.81; polarizing voltage - -4.85V; monitor: Tektronix

4012; Printer: Okidata 250 printer; computer: Digital PDP 11/70, data

acquisition delay time , 2 min.

Four spectral mass units were used to quantify UDMH and the internal

standard, 4-nitroanisole. The mass spectrum of salicylalhehyde

dimethylhydrazone (MW - 160) is shown in Figure 3. The mass with the

greatest intensity is the molecular ion peak m/e - 164 and the most

intense fragment is the [N - 44] m/e . 120. For 4-nitroanisole, the

parent peak is found at m/e - 153 with the most intense fragment at m/e

- 123. Retention times for salicylaldehyde dimethylhydrazone and 4-

nitroanisole are 4:37 min. and 3:30 min., respectively. The ratio of

m/e 164/153 was used to determine concentration of UDMH.

UDMH standards were prepared fresh daily at 500, 250, 100, 50, 25

and 0 ppb levels. A 10,000 ppm solution was prepared by mixing 1 mL

UDMH (Aldrich Chem. Co.) in a 100 L volumetric flask with 0.05 M NaH2P04

- 25% sucrose solution (pH - 4.2). Serial dilutions of 10,000 ppm, 100

ppm and 5 ppm were made. Final concentrations of UDMH were prepared

from the 5 ppm stock solution using the buffered sucrose diluent.

Both sets of UDMH analysis data (i.e. samples heated following can

closure and samples heated before and after can closure) were

statistically analyzed using a two treatment, randomized complete block

(RCB), factorial design with replicates (2). Tukey’s w was used to determine significant differences between treatment means. RESULTS AND DISCUSSION

Daminozide Analyses

In the liquid chromatographic (LC) analysis, daminozide appeared as

a descending shoulder of the large sucrose peak (Figure 4). The

retention time of daminozide was 3:26 min. (44 s after sucrose) and

could not be resolved further from the sucrose peak by changing the

solvent polarity or flow rate. In order to reduce the variability of

this analytical method, each of three replicate samples was analyzed

twice. The sensitivity of the LC procedure was determined to be i 1.5

ppm daminozide for these experiments.

Many researchers (Allen, 1980; Conditt et al., 1988; Dicks, 1971;

FDA, 1975; Lane, 1970; Newsome, 1980) have analyzed for daminozide in

food and environmental samples. None of these researchers have applied

LC techniques to determine daminozide residues. Instead, these researchers have hydrolyzed daminozide with heat and alkalai to produce

UDMH. UDMH was then reacted with trisodium_pentacyanoamine ferroate

(FDA, 1975; Lane, 1970) or salicylaldehyde (Allen, 1980; Conditt et al.,

1988) and determined colorimetrically or spectrometrically. Another approach was to oxidize UDMH with selenium dioxide to yield formaldehyde which was then determined colorimetrically with 2-hydrazino- benzothiazole. Another approach has been derivatization of UDMH with penta-fluorobenzoyl chloride to produce 1,1-dimethyl-2-2-bis

(pentafluorobenzoyl) hydrazine which was determined with GC (Newsome, 1980).

There were no statistical differences (p = 0.01) in the daminozide

53 54

concentrations between samples heated in open or closed cans and those

samples which were unheated at any of the pH levels (i.e. pH - 3.0; 3.6

and 4.2). The 12.5 ppm daminozide which was added to the buffered-

sucrose solutions, was not degraded in any significant (p-0.05) amount

by heating at 100°C for 15 min. Daminozide was stable in buffered-

sucrose solution during the experimental conditions and less than 1.5

ppm daminozide was decomposed during the heating intervals applied.

Since the food matrix has a major influence on the heating and cooling

dynamics of canned food products, the degradation of daminozide may be

different in another food matrix for the same time-temperature conditions.

Theoretically, for every 1 ppm daminozide hydrolyzed in a closed

system, 375 ppb UDMH is generated. As discussed in the following

section, all of the samples in this experiment had less than 25 ppm

UDMH. This means that less than 1 ppm of daminozide was degraded during

this experiment. In order to detect changes in the daminozide

concentration of less than 1 ppm 3 more sensitive assay would be

required.

UDMH Determination

A mass spectral scan of a 10 ppm UDMH solution is shown in Figure

5. The retention time of 4-nitroanisole (internal standard) and UDMH were 3:30 min. and 4:37 min., respectively. Samples containing less

than 10 ppm UDMH did not have a peak at m/e - 120, due to limited

intensity of this mass fragment. Concentrations were calculated directly from the area ratios of m/e 164 : m/e 153. Standard curves for

0-500 ppb and 0-10 ppm UDMH are shown in Figures 6 & 7. The correlation coefficients for the linear regression of these curves is 0.995. 55

UDMH production was significantly (p - 0.01) influenced by the

interaction between heating time and pH for samples which were sealed prior to heating (Table 1). There was significantly greater UDMH concentration in samples at pH 3.6 than samples at pH 3.0 or pH 4.2

(Figure 8). This suggests that daminozide is hydrolyzed to UDMH at an optimal pH in food products which are heated. There was an increase in UDMH of about 5 ppb for every 5 min. at 100°C.

The level of UDMH found in these samples is much lower than the level reported by Newsome (1980). He measured 600 ppb UDMH in applesauce which was fortified with 30 ppm daminozide and boiled in an open container for 10 min. After 20 min. of boiling, 1.25 ppm of UDMH was detected in the applesauce. There is no indication that the level of UDMH was assayed prior to heating. However, the level of UDMH, extrapolated to 0 min. of boiling, was 200 ppb. There appears to be a large amount of residual UDMH in the daminozide stock solution or applesauce. The UDMH concentrations measured, represent a 40-fold increase in daminozide decomposition rate compared to the data presented here. The difference between our results may be attributed to the analytical methods used to determine UDMH concentration or to differences in the sample matrix and processing conditions.

Daminozide-treated samples, which were heated to 80°C, held for 0,

5, 10 min., then lidded, sealed and heated in 100°C water for 5 min., had significantly (p . 0.01) higher concentration of UDMH than control samples (Figure 9). The samples adjusted to pH 3.6 had greater concentrations of UDMH than samples adjusted to pH 3.0 or 4.2. A slightly higher concentration of UDMH was measured for closed and open heated samples at the pH 3.6 level for each experiment (Figure 10). 56

Lowest concentrations of UDMH were measured at pH 3.0 for both processing regimens.

There was no difference in UDMH accumulation between samples heated for greater than 5 min. prior to can closure. This differs from the samples which were lidded and sealed prior to thermal processing. Since

UDMH is an extremely volatile compound, it appears that further heating of daminozide-treated solutions will cause UDMH evolution as rapidly as

UDMH is generated. In contrast, daminozide-treated solutions which are heated following can closure have accumulated more UDMH than open- container heated samples. CONCLUSIONS

LC can be used to determine daminozide concentration if greater than

1.5 ppm sensitivity is acceptable and interferring compounds are absent. Sensitivity of 1.5 ppm was achieved when a 24% sucrose solution was the sample matrix. Residual daminozide is only slightly degraded by

15 min. of heating (100°C) in a closed system or 10 min. of heating

(80°) in an open system, followed by 5 min. of heating (100°C) in a closed system. Reducing the processing time where possible will insure minimal daminozide degradation. Daminozide was found to be only slightly degraded during processing when dissolved in solutions adjusted to pH 3.0, 3.6 and 4.2.

UDMH production in a closed system was affected by the pH of the solution (pH 3.6 gave maximum UDMH) and the heating duration. UDMH accumulated at an approximate rate of 5 ppb forevery 5 min. of heating at 100°C. UDMH was evolved when daminozide fortified solutions were heated in an open system. Accumulation of UDMH was not directly related to heating time in an open system.

57 REFERENCES

Allen, J.G. I980. Daminozide residues in sweet cherries, and their determination by colorimetric and gas-liquid chromatographic methods. Pets. Sci. 11:347.

Conditt, H.K., J.R. Baumgardner and L.M. Hellmann. 1988. Gas chromatographic/mass spectrometric determination of daminozide in high protein food products. J. Assoc. Off. Anal. Chem. 71:735.

Dicks, J.W. 1971. Determination of aminozide residues in Chrysanthemum tissues. Pest. Sci. 2:176. Food and Drug Administration. 1975. Method of Analysis for Alar Residue in Various Crops. From “Pesticide Analytical Manual," Vol. 2.. FDA, Rockville, MD. Pest. Reg. Sec. 180-246.

Lane, J.R. 1970. General Alar Reside Method. Uniroyal Chemical Co. pp. 1-4.

Newsome, H.H. 1980. Determination of daminozide residues on food and its degradation to 1,1-dimethylhydrazone by cooking. J. Agric. Food Chem. 28:319.

Santerre, C.R. 1989. Daminozide and unsymmetrical dimethylhydrazone residues in tart and sweet cherries and processed cherry products. Unpublished Data.

Uniroyal Chemical Co. 1984. Personal Communication W/ M.D. Parkins, Ph.D.

Unrath, C.R., A.L. Kenworthy and C.L. Bedford. 1969. The effect of Alar, succinic acid 2,2-dimethylhydrazone, on fruit maturation, quality, and vegetative growth of sour cherries. Prunus cerasus L., cv. "Montmorency". J. Am. Soc. Hort. Sci. 94:387.

58 ACKNOWLEDGEMENTS

Thanks to Yen Ling (Annie) Chai and Dr. Salah Selim for assistance in sample preparation and analysis. Special thanks to Margaret Conner for assistance with manuscript preparation.

59 60

Table 1. Analysis of variance (AOV) table describing the influence of pH and heating time on the concentration of UDMH in canned samples which are closed prior to heating (100°C).

Source dfs, SS .MS F-Valge Sign.

Rep 1 0.37 0.368 0.15 n.s. pH Level (A) 2 126.35 63.174 25.85 .000

Heating Time (B) 3 974.74 324.912 132.96 .000

A X B 6 184.01 30.668 12.55 .000 Error 11 26.88 2.444

Table 2. Analysis of variance (AOV) table describing the influence of pH and heating time on the concentration of UDMH in canned samples which are heated before (80° C) and after (100° C) can closure.

Source df SS HS F-Value Sign.

Rep 1 8.34 8.343 2.00 n.s pH Level (A) 2 53.01 26.507 6.37 .014 Heating Time (B) 3 515.81 171.936 41.32 .000 A X B 6 92.16 15.359 3.69 .029

Error 11 45.78 4.161

° 1 II I CH3 CH3 CHZ—C—T—NH—N< -——> ""2_ <

l I C” 3 ‘43 CH 3 CH2 ﬁ 0" mm 0

DAMINOZIDE

Figure 1. Thermal decomposition of daminozide to yield unsymmetrical dimethylhydrazine (UDMH). 63

0 II [C H 3 CH HC=N—-N \CH ,~—~\ . —-—> H C H 3

UDMH SALI CYLALDEHYDE SALICYLALDEHYDE DINETHYLHYDRAZONE

Figure 2. Reaction between unsymmetrical dimethylhydrazine and salicylaldehyde to produce salicylaldehyde dimethylhydrazone. 64 41+

ji'

160 1;;

160).

STANDARD

= 149 nJll.n

PP"

(MW n .

140

. 134 .1?11 n

IIIjjIl‘jljjII'UIUI'U

20 I

120

dimethylhydrazone

INETFNLHVORRZDNE lllénI}‘n

D 1 ll

100 44,

m/e IJ

salicylaldehyde 31

of I.

ICVLRLDEHVOE 32

['4 78

SRL

804:40.

RT also:

fragmentation

60 151?.

6801?

spectral g?I21 I

‘ljjl‘l‘l'I'i'U‘iﬁI1jr1l1‘tj1j'l‘UﬁU—U'T

Mass

1002=

AIISNHINI 3AIIV138 Figure 65

"-1H

"I PS: IIHII (‘11 21'

pi 3H: 1.1:. ("'1

‘v'l

78 INDEX

t BC Ii:- U‘I L WI N

13 M M (13 In 11':

H HREH Ch 0‘1 I33 13:: 14:. 14:. ITI

121:: RUN L .,—( 11:1 LEI"

ET ['1] 1 131 It rd

:1 8.

F- I

RT L] '1 Lu

"".1 .- HT 1 0"1 f- “ x-i

METHOD ~.—'1 ' .35 ID GI

HREHZ it-i

II I! J":

1. .1 - OVERRHNGE Lu 2 I‘- .1 CE Z l- (I: D I HHS l—.

INPUT

PEHK# Q l-e FILE

Figure 4. LC chromatogram of daminozide (RT = 3.26 min.). 66

TIC=0

R103164

RIG-153

RIC‘IZ3

RIC‘IZO standard.

6:00

5:30 dimethylhydrazone

5:00 salicylaldehyde

4:37

(min.)

4:30 ppm

TIME

a for

4:00

RETENTION

164

llllllllljiililllilTjIIItllrrrllﬁ

153,

123,

3'30

120,

m/e

3300 SIM

GC/MS,

2833

2:30

606208

1122815 374656

1396508

696576

02'

002-

1002'

1002-

Figure V""]IIIIj—]IIIIIIII

‘

AIISNBINI BAIIVTBU

y = 1.53118-3 + 3.435394!

F102 = 0.995 0.20

153)

m/e

164

(mle 0.10

Ratio Area l n I a l A l n l a 0.00 0 100 200 300 400 500 600

Unsymmetrical Dimethylhydrazine (ppb)

Figure 6. GC/MS standard curve of 1,1-dimethylhydrazine (UDMH) from O-SOO ppb. 68

y -.- - 5.9123e-2 + 0.250331: n52 = 0.995

153)

m/e

164

(m/e

Ratio Area or..1..i..1..1.1 .. 0 2 4 6 8 1i) 112 114

Unsymmetrical Dimethylhydrazine (ppm)

Figure 7. GC/MS standard curve of 1,1-dimethylhydrazine (UDMH) from 0-10 ppm. 69

30 Tukey's w 1. 4.85 . 0

Heaﬂng Thne I 0min.@1OOC

(ppb) E 5min.@1000 El 10min.@iOOC 15min.@1OOC

UDMH

pH Level

Figure 8. Unsymmetrical dimethylhygrazine (UDMH) residues in samples which were heated (100 C) following can closure. Bars with the same letter are not significantly different at the Tukey's "0.01 level. 7O

20 Tukey's w - 4.49 .05 d d 15 ' :2: ' g; Heaﬂng Thne E I 0 min.

(ppb) bc 3 = E 0 min. @ 800 1° F 2:: egg . E g :3 Cl 5 m1n.@ 800 é: E E 10 min. @ 800

UDMH 5 E g g

o E E .: 3 (i 13.6 44 2 pH Level

Figure 9. Unsymmetrical dimethylhydrazine (UDMH) residues in samples which were heated (80 8) prior to can closure in addition to 5 min. heating (100 0) following can closure. Bars with the same letter are not significantly different at the Tukey's w0 05 level.

(ppb)

—0— Closed Cans

—l— Open Cans

Dimethylhydrazine

1,1-

l I l l l

3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4

pH Level

Figure 10. Influence of pH on the production of UDMH. CHAPTER 2

DANINOZIDE (ALAR) AND UNSYMMETRICAL DIMETHYLHYDRAZINE (UDNH) RESIDUES IN TART AND SHEET CHERRIES AND PROCESSED CHERRY PRODUCTS

Char1es Richard Santerre

72 Daminozide (Alar) and Unsymmetrical Dimethylhydrazine (UDMH) Residues

in Tart and Sweet Cherries and Processed Cherry Products

ABSTRACT

Daminozide is applied to tart and sweet cherry trees to improve

fruit color, texture and abscission at harvest. This growth retardant

is a systemic compound which penetrates into the fruit matrix. Thermal processing of fruit containing daminozide produces the hydrolysis product, unsymmetrical dimethylhydrazine (UDMH), which is a cancer suspect agent.

Tart and sweet cherry trees were treated with 0, 3.4 and 6.8 lbs. daminozide/acre at 3 commercial orchards in northwestern Michigan at 2 weeks after full bloom. Daminozide residues were less than 13 ppm in fresh fruit and less than 10 ppm in processed cherry products. UDMH residues were less than 10 ppb in fresh fruit and less than 503 ppb in processed cherry products. Processing of both sweet and tart cherries reduced the levels of daminozide. However, thermal processing increased the levels of UDMH in canned cherry products.

73 INTRODUCTION

Michigan ranks number one, in the U.S., in the production of tart cherries which are predominantly processed into pie filling. In 1988,

Michigan produced 120.4 million kilograms or 73.9% of the total U.S. tart cherry crop (Michigan Department of Agriculture Statistics,

1988). Michigan ranks fourth in the U.S. for the production of sweet cherries which are sold fresh or processed (i.e. canned maraschino cherries, pie filling etc.). In 1988, Michigan produced 14.5 million kilograms or 15.2% of the total U.S. sweet cherry crop (MDA Statistics,

1988).

Harvest yields of tart and sweet cherries are dramatically improved following application of the growth regulator daminozide. This compound increases yields during mechanical shake-harvesting by promoting fruit abscission (Looney and McMechan, 1970). By maintaining fruit firmness at harvest and increasing the yield during mechanical harvest, it has been estimated that daminozide usage has an economic impact of $4 million on the U.S. cherry industry (Uniroyal Inc., 1985).

Subsequent to its registration in 1963 by the U.S. Food and Drug

Administration (FDA), daminozide was shown to undergo hydrolysis during thermal processing (Newsome, 1980). This hydrolysis produces the cancer-suspect agent, unsymmetrical dimethylhydrazine (UDMH). Several researchers have implicated UDMH as a potential carcinogen in rodent trials (Roe et al. 1967; Toth, 1973). Investigators are currently attempting to assess the risks of UDMH exposure in laboratory studies using animal models. Concurrent with these studies, the U.S. 74 75

Environmental Protection Agency (EPA) has requested data regarding the levels of daminozide and UDMH residues in a number of crops including fresh cherries and processed cherry products. There is concern that cherries containing daminozide residues will have elevated levels of

UDMH following thermal processing. Newsome (1980) fortified applesauce with 30 ppm daminozide and reported a yield of 1.53 ppm UDMH after boiling the sauce for 30 min. and 1.92 ppm UDMH after boiling for 60 min. Cherry products which are processed in this manner include tart cherry pie filling and canned sweet cherries.

Very little has been reported concerning the levels of daminozide in cherry trees or fruit following application. Ryugo (1965) applied 2000 ppm daminozide to Hative Burlat, Bigarreau Moreau and Bing sweet cherry trees at shuck split (2 weeks after full bloom). Residue levels in leaves at 2 weeks after application were 80, 30 and 100 ppm, respectively. Bing trees were also treated with 2000 ppm, 3 times within a 3 week interval following shuck split. The residue level of daminozide in fruit at 22 days following final application was 7 ppm. Since these trees were treated much later than indicated on label specifications, it would be expected that residues in the harvested fruit would be significantly higher than fruit treated as required in label recommendations. When daminozide was originally approved for use on cherry trees, the EPA set a tolerance of 55 ppm for residues in fresh tart cherries and 30 ppm in fresh sweet cherries.

The objectives of this study were: 1) to determine the residues of daminozide and UDMH on tart and sweet cherries from commercial orchards; and 2) to determine how thermal processing of tart and sweet cherries affects the levels of daminozide and UDMH. MATERIALS AND METHODS

Daminozide Application

Daminozide (Alar-85) was applied at shuck split (2 weeks after full bloom) to Montmorency (Prunus cerasus) tart cherry trees and dark, sweet Schmidt or light, sweet Napolean (Prunus ayium) cherry trees between

June 1-6, 1984. Alar-85 is a formulation of 85% daminozide and 15% inert ingredients. Trees were located at three grower locations in the

Traverse City, Michigan region. Daminozide was applied by growers using air-blast sprayers at 0, 4 lbs. formulation/acre (3.4 lbs. active ingredient/acre) or 8 lbs. formulation/acre (6.8 lbs. active ingredient/acre), as specified on label requirements. Additional pesticide sprays were also applied to reduce damage from birds, insects and fungus. Sweet and tart cherries were harvested using mechanical- shake harvesters and immediately immersed in cold water. A 2.3 kg sample of cherries was removed and packaged in a 8549 Cryovac plastic bag prior to immersion in water and frozen (-23°C). This sample was labeled as the fresh unpitted cherries. The remaining cherry samples were placed in containers containing cold water and ice during transport to Michigan State University’s, Fruit and Vegetable Processing Facility.

Tart Cherry Processing

Harvested Montmorency cherries were stored in a cooler (3.8°C) overnight. Cherries were held in running water for 4 hr., drained and mechanically pitted using a small (8 X 20 cup) Dunkley Pitter

(Kalamazoo, MI). A 2.3 kg sample of pitted cherries was packaged in a

Cryovac plastic bag and frozen (~23°C) for later analysis.

76 77

Hater Pack Process Three hundred and fifty grams of freshly pitted tart cherries were filled into enamel coated No. 303 cans and covered with boiling water.

The filled cans were "exhausted" by heating to a core temperature of

80°C, sealed and boiled for 8 min. Following this, cans were immediately cooled in 10°C water and dried.

Sugar (5 + 1) Pack Preparation Pitted tart cherries (approx. 4.5 kg) were filled into a plastic bag lined-containers and covered with 0.9 kg of sucrose (i.e. 5 + 1 - 5 lbs. cherries plus 1 lb. sugar). Plastic bags were sealed and containers were immediately frozen (-23°C).

Cherry Pie Filling and Pie Preparation

A cold slurry of the starch "Thermtex" (National Starch & Chemical

Co., Bridgewater, NJ) was prepared by mixing 565 g starch with 6.6 L of cold water. This slurry was combined with 2.8 kg sugar, 76.8 mL of 50% citric acid solution, 20.2 9 salt and 134 mL of red dye #40 solution.

The starch solution was stirred constantly during heating to 80°C. Nine kilograms of freshly pitted tart cherries were added and the mixture was again heated to 80°C, then filled into enamel coated No. 303 cans and sealed. The cans were boiled for 12 min., cooled in 10°C water and dried. Two cans of prepared pie filling were poured into frozen pie crusts (Sysco Inc.) and baked at 204°C for 5 min. Pie crusts were initially prepared by baking crusts for 8 min. at 204°C. Pies were cooled, packed in plastic bags and frozen (-23°C).

Sweet Cherry Processing

Harvested Schmidt and Napoleon sweet cherries were stored in a cooler (3.8°C) overnight, then placed in running water (10°C) for 1 78 hr. A 2.3 kg sample of unwashed cherries was collected at harvest, packaged in a 8549 Cryovac plastic bag and frozen (-23°C) for residue analysis. Cherries were removed from the water and mechanically pitted as previously described. A 2.3 kg sample of pitted cherries was frozen (-23°C) for residue analyses.

Canned, Sugar Pack Preparation

Three hundred grams of pitted sweet cherries were placed in enamel coated No. 303 cans and covered with a boiling 25° brix (25%) sucrose solution. The cans were ”exhausted" by heating the internal temperature to 80°C, sealed and boiled for 10 min. Cans were cooled in 10°C water and dried. Daminozide Analysis

Daminozide residues were determined by Biospherics, Inc. (Rockville,

MD) using a method which hydrolyzes daminozide to UDMH, then derivatizes

UDMH with salicylaldehyde to form salicylaldehyde dimethylhydrazone

(Conditt et al. 1988). The hydrazone was then quantified using GC/MS with selected ion monitoring at m/e 164 (parent) and m/e 120 (~NMe2).

The detection limit was 10 ppb based on a 50 9 sample. Recoveries range from 74-142% for 0.01-5.0 ppm fortifications of daminozide. UDMH Analysis

UDMH was determined using an Isotope Dilution GC/MS method developed by Uniroyal Chemical Co., Inc. (Bethany, NY). Analysis were performed by Hazleton Labs, Inc. (Madison, WI). UDMH was converted to 1,1- dimethyl-2,2-bis pentafluorobenzoyl chloride (PFBC). The complex was then quantified using GC/MS. The calibration curve was generated by derivatizing known concentrations of 15N-UDMH with known concentrations of 14N-UDMH with PFBC. Ratios of m/e at 404 and m/e at 405 were then

79 obtained. Samples were then fortified with 15N-UDMH and derivatized.

The limit of detection for this method was 1 ppb.

Preparation of calibration curve.

To separate aliquiots of cherry products, 25 ppb UDMH-15M was added. Samples were then fortified with UDMH-14" at 1, 10, 15, 25, and

50 ppb.

Samples were derivatized and analyzed by GC/MS; chemical ionization

(methane gas); negative ion detection as described below for cherry samples. The ratio of m/e - 404 to m/e - 405 was used to determine the equation for the calibration curve relating l4N/ISN to the ion response ratio for m/e - 404 and m/e - 405. UDMH residues were calculated using the following equation;

UDMH (ppb) - [(R)*(C) - AI/l - (R)*(B) where; R - ratio (m/e 404/m/e 405) A - amount of 14N in 15N B - amount of 15N in 14N

C . constant

Preparation of cherry samples.

A sample of 20 g pitted cherries (for pie filling, both cherries and sauce were used) product was weighed into a 50 mL conical centrifuge tube and 2 mL of 1N HCl was added. This sample was fortified with 25 ppb UDMH-15M (w/w) and homogenized at 0°C with a polytron homogenizer

(30 s on, 1 min. off; 3 cycles). The polytron head was removed from the centrifuge tube and rinsed with a minimum amount of distilled water into the tube. The sample was centrifuged at 4500 X g for 1 hr and the 80

supernatant liquid was decanted into a second 50 mL centrifuge where the

volume was determined. One half this volume was removed for analysis.

A 10 mL sample of cherry juice was fortified with 250 ng UDMH-[2-

15N] (w/w; calculated as free base) in a 125 mL erlenmeyer flask. A 4 mL PFB Cl reagent (0.05 mL PFBC/mL methylene chloride) aliquot was added

along with 10 mL 2 M K2C03. The top of the flask was covered with

aluminum foil and shaken for 3 hr on a rotating shaker. After shaking,

the contents of the flask were decanted into a 30 mL separatory funnel containing 5 mL saturated aqueous NaCl. The erlenmeyer flask was rinsed with 4 mL methylene chloride and added to the separatory funnel which was shaken and the phases were allowed to separate. The methylene chloride was drawn off and saved while the aqueous layer was re- extracted with 4 mL methylene chloride. The methylene chloride extracts were combined and added to Na2504 and filtered into a 50 mL round bottom flask. The Na2504 was rinsed with 5 mL methylene chloride and filtered

into the 50 mL round bottom flask. The volume of the methylene chloride was reduced to less than 1 mL (but not to dryness) on a rotary evaporator. The residue was quantitatively transfered to a 1 mL volumetric flask and diluted to volume with methylene chloride. This final solution was then analyzed by GC/MS.

RESULTS AND DISCUSSION

Tart Cherry Daminozide Residue Analysis Daminozide residues, for tart cherries harvested from location 1,

ranged from 3.1 ppm for untreated cherries to 7.2 ppm for cherries

treated with 3.4 lbs. Alar/acre and 13.0 ppm for cherries treated with

6.8 lbs. Alar/acre (Figure 1). Daminozide residues were detected in the

untreated fruit from all three locations (Table l). Contamination of

untreated fruit may be due to broadcast drift during spray

application. Daminozide residues in untreated fruit may also be due to carry-over from previous growing years because this compound has been

shown to be persistent and reasonably stable in bark and root tissue

(Edgerton and Greenhalgh, 1967; Dozier et al. 1984). Daminozide is not

as stable in soil as in plant structures. Dannals et al. (1974) has

shown that daminozide in soil is rapidly degraded by microorganisms

under greenhouse conditions.

Daminozide residues, from cherries harvested at location 2, ranged

from 1.2 ppm for untreated samples to 0.7 ppm for cherries from trees treated with 3.4 lbs. Alar/acre and 0.8 ppm for samples from trees treated with 6.8 lbs. Alar/acre (Figure 2). Daminozide residues from this location were dramatically lower than residues on cherries from locations I and 3 (Table l). Sprays for the three locations were applied on three different days and the trees in location 2 may have been washed by a rainstorm shortly after Alar application. However, the elevated residue level on the untreated samples is difficult to explain.

Daminozide residues on the cherries harvested from location 3 were

81 82

0.5, 7.1 and 13.0 ppm for the untreated, 3.4 lbs. and 6.8 lbs. Alar per acre, respectively (Figure 3). The residue levels for locations 1 and 3 are very similar (Table 1). None of the freshly harvested cherries had residues as high as the 1986 U.S. EPA tolerance of 55 ppm (40 CFR,

Section 180.246) for tart cherries.

Daminozide residue analyses of the pitted cherries indicated that pitted cherries have less residue than freshly harvested cherries (Table 1). Since Daminozide is a systemic compound which is readily absorbed 9“ by the fruit, it is less likely that washing of cherries prior to i pitting will cause a dramatic reduction in concentration. However, Q. daminozide is a water soluble compound which can diffuse out of cherries during a 4 hr precooling. This is a normal field practice following harvest which improves the firmness of cherries. Removal of the cherry stone during pitting in addition to fluid loss, may have also contributed to the daminozide reduction. Pitted cherries from location

1 had daminozide levels of 0.9, 5.3 and 6.4 ppm for the untreated, 3.4 and 6.8 lbs. Alar/acre, respectively. These levels are approximately half of those found on fresh cherries. Pitted cherries from location 2 had daminozide residues less than 0.2 ppm for all treatments. These levels are also less than those measured on fresh fruit. Pitted cherries from location 3 had residue levels which were comparable to pitted samples from location 1. Daminozide concentrations were 0.5, 5.3 and 5.8 ppm for untreated, 3.4 and 6.8 lbs. Alar/acre, respectively.

Only sugar pack (5 + l) cherries from locations 2 and 3 were analyzed for daminozide residues. Daminozide concentrations for the sugar pack samples were similar to concentrations in pitted cherries.

The residues for location 2 were 2.0, 1.1 and 0.9 ppm for untreated, 3.4

and 6.8 lbs. Alar/acre, respectively. It is unusual that the control

samples have higher residue levels than the treated samples. The

residues for location 3 were 1.2, 4.8 and 6.0 ppm for untreated, 3.4 and

6.8 lbs. Alar/acre, respectively.

Daminozide residues for the water pack samples were also similar to

pitted cherries. The concentrations of pesticide on cherries from

location 1 were 0.4, 4.2 and 9.4 ppm for the untreated, 3.4 and 6.8 lbs.

Alar/acre, respectively. Residues on the water pack cherries from

location 2 were slightly higher than those found on the pitted

cherries. The residues were 0.8, 0.5 and 0.7 ppm for the untreated, 3.4

and 6.8 lbs. Alar/acre, respectively. Residues from location 3 water

pack samples were similar to those for the pitted cherries.

Concentrations of residues on the water pack samples were 0.9, 4.0 and

5.9 ppm for the untreated, 3.4 and 6.8 lbs. Alar/acre, respectively.

Daminozide residues in the tart cherry pie filling were less than

those found in pitted cherries and much less than those measured on

fresh cherries. Residues in pie filling from location 1 were 0.7, 2.7

and 4.2 ppm for untreated, 3.4 and 6.8 lbs. Alar/acre, respectively.

Pesticide residues in pie filling from location 2 were 1.1, 0.3 and 0.3 ppm for untreated 4 and 8 lbs Alar/acre. Finally, the residues in samples from location 3 were 1.1, 0.8 and 0.1 ppm for the untreated, 3.4 and 6.8 lbs. Alar/acre, respectively. The residues from the third location are much less than residues found on fresh tart cherries.

Uniroyal Chemical Co., Inc. (1986) has reported to the U.S.

Environmental Protection Agency (EPA) that daminozide residues from 24 samples received from across the U.S. (including those data reported here), averaged 1.87 ppm in cherry pie filling. 84

The final step in the processing of tart cherries was the

preparation of pie from the canned pie filling. Analyses of residues in

prepared pies would give a good estimate of human exposure to persistent

residues. The residues in cherry pie from location 1 were 0.7, 0.8 and

3.6 ppm for the untreated, 3.4 and 6.8 lbs. Alar/acre, respectively.

These levels are comparable to concentrations found in the canned pie

filling.

Residues in pie from cherries grown in location 2 were 0.8, 0.7 and

0.6 ppm for untreated, 3.4 and 6.8 lbs. Alar/acre, respectively. The

residues in pie from cherries grown in location 3 are quite a bit higher than residues in the canned pie filling. The residues found in the pies were 1.2, 2.4 and 3.4 ppm for untreated, 3.4 and 6.8 lbs. Alar/acre, respectively.

When evaluating daminozide residue data, it should be noted that the analytical regimen requires hydrolysis of daminozide to UDMH.

Therefore, the daminozide measurements reported do not only include UDMH from daminozide but also includes free UDMH. .Data presented in the next section will indicate that UDMH is present in much lower concentrations and therefore, does not greatly affect daminozide analyses. UDMH Residues in Tart Cherries and Products

UDMH concentrations varied greatly for processed cherry products, with a range from <1 to 503 ppb (Table 1). UDMH residues for fresh cherries from location 1 were 6.1, 2.9 and 6.1 ppb for untreated, 3.4 and 6.8 lbs. Alar/acre, respectively (Figure 4). UDMH residues for location 2 were <1, 2.8 and 9.3 ppb for untreated, 4 and 8 lbs.

Alar/acre, respectively (Figure 5). Cherries from location 3 had residues of <1, 1.2 and 1.0 for untreated, 3.4 and 6.8 lbs. Alar/acre, 85 respectively (Figure 6). The residues of UDMH on fresh cherries were generally lower than for many of the processed cherry products.

UDMH residues for the sugar pack (5 +1) were determined for locations 2 and 3 only and no measurements were made for untreated samples. The UDMH for sugar packed cherries from location 2 were 47.0 and 55.0 ppb for the 3.4 and 6.8 lbs. Alar/acre, respectively. Residues from location 3 were 7.0 and 8.0 ppb for the 3.4 and 6.8 lbs. Alar/acre, respectively. The variability in levels for these samples may be due to many factors relating to sample preparation, cleanup and analysis.

Consistent results were found for cherries processed in the water pack. There appears to be a strong correlation between daminozide concentration and UDMH concentration. Hater pack cherries from location

2 had UDMH concentrations of <1, 199.0 and 503.0 ppb for the untreated,

3.4 and 6.8 lbs. Alar/acre, respectively. The daminozide concentrations for this location and for the given application rates were 0.4, 4.2 and

9.4 ppm, respectively. It appears that for every 1 ppm daminozide, 50-

60 ppb of UDMH are formed in the water pack samples. This is also apparent with samples from location 3 where the UDMH and daminozide residues were 11.0, 115.0 and 286.0 ppb; 0.9, 4.0 and 5.9 ppm for the untreated, 3.4 and 6.8 lbs. Alar/acre, respectively. UDMH residues from cherries grown at location 2 were 8.7, 9.0 and 13.0 ppb for the previously mentioned application rates, respectively. The elevated levels of UDMH in the sugar pack (5 + 1) and water pack samples is likely due to hydrolysis caused by thermal processing. UDMH is an extremely volatile gas which would evolve during heating.

Unfortunately, these cherry products are heated following can closure which prevents UDMH evolution. 86

Unexpectedly, the UDMH residues in the cherry pie filling were generally lower than those determined in the water pack samples. This may be due to greater thermal processing prior to can filling and closure. The UDMH residues in pie filling prepared from cherries grown at location 1 were 1 ppb for both application rates. UDMH residues for location 2 were 3.0 and 4.0 ppb and those for location 3 were 50.0 and

99.0 ppb for the 3.4 and 6.8 lbs. Alar/acre treated cherries. Uniroyal

Chemical Co., Inc. (1986) has reported that 24 samples of pie filling from several regions of the U.S. (including data presented here), contained an average of 105.7 ppb UDMH. This value is higher than observed for the Michigan samples.

Cherry pies prepared from the canned pie filling had UDMH residues of <1, 51.4 and 59.5 ppb for the 3 application rates of 0, 3.4 and 6.8 lbs. Alar/acre at location 1. Cherry pie from cherries grown at location 2 had UDMH resiudes of 1.4, <1 and <1 ppb while cherry pie from location 3 had residues of 1.0, 51.1 and 55.6 ppb for the 0, 3.4 and 6.8 lbs. Alar/acre. There appears to be a high degree of variation between samples when observing UDMH concentrations. Consumers would be exposed to less than 60 ppb UDMH (based on filling weight) when consuming a cherry pie.

It appears from this research that thermal processing of daminozide increases the formation of the hydrolysis product, UDMH. Since UDMH is a volatile compound which would evolve prior to can closure, thermal processing following closure may cause even greater concentrations of

UDMH in processed products. Sweet Cherry Daminozide Residue Analysis

Two varieties of sweet cherries, Napoleon and Schmidt, were grown 87

and harvested from the same Traverse City, MI location. The daminozide

residues for fresh Schmidt sweet cherries were 0.8, 5.9 and 13.0 ppm for

the untreated, 3.4 and 6.8 lbs. Alar/acre, respectively (Table 2).

Residues on fresh Napoleon sweet cherries were 0.41, 7.7 and 10 ppm for

the three treatment levels, respectively. It appears that the

daminozide residues for sweet cherries are very similar to residues for

the tart cherries from locations 1 and 3. Uniroyal Chemical Co., Inc.,

(1985) reported an average concentration of 9 ppm in sweet cherries

harvested across the U.S. (including this data).

Daminozide residues were measured in the canned, syrup pack

samples. The residues were 1.6, 6.3 and 9.6 ppm for Schmidt sweet

cherries and 1.6, 3.0 and 4.0 ppm for Napoleon sweet cherries for the

untreated, 3.4 and 6.8 lbs. Alar/acre, respectively. The concentrations

of daminozide in these samples is similar to the concentrations found in

the tart cherry water pack samples from locations 1 and 3. These

residues are well below EPA tolerance levels of 30 ppm for sweet

cherries.

Sweet Cherry UDMH Residue Analysis

UDMH levels for Schmidt sweet cherries were determined to be less than 1 ppb for the fresh cherries at all treatment levels.

Unfortunately, the levels of UDMH were not measured in the syrup pack

samples.

The UDMH reSidue levels for the 3.4 and 6.8 lbs. Alar/acre for the fresh Napoleon sweet cherries were <1 and 1.6 ppb, respectively. The

UDMH levels for the syrup packed samples were 12.0 and 30.0 ppb for the

3.4 and 6.8 lbs. Alar/acre samples, respectively. It appears that sweet cherries processed in the syrup pack have UDMH levels related to the 88 amount of daminozide residue in the sample and degree of thermal processing.

In a recent market basket survey, (Uniroyal Chemical Co. Inc., 1985)

it was estimated that sweet and tart cherries comprise 0.1% of the daily

U.S. diet. 1f 8 ppm is considered as the average daminozide residue on tart and sweet cherries and 11.8% of the total crop is sprayed with the growth retardent, than the intake of this plant growth retardant is 1.65 ug/1.5 kg diet/day/60 kg person. When calculated for all commodities which receive Alar applications (i.e apples, tomatoes, peanuts, cherries, grapes, etc.), the estimated intake is 47.4 ug/l.5 kg diet/day/60 kg person. This estimate is 83 times less than would be obtained if the EPA tolerance levels were used to estimate intake.

These estimates of residue levels and dietary intakes will be important for determining the acceptable exposure for humans from the toxicological studies which are currently being reported. CONCLUSIONS

Daminozide residues were much lower in both sweet and tart cherries

than permitted by EPA tolerance levels. The residues in processed cherry products were lower than for fresh cherries. Our study was not

able to determine the reason for reduced residues following soaking and

pitting. Soaking fresh cherries in water may cause daminozide to leach out of the cherry matrix and increasing the soak time, prior to pitting, may reduce daminozide levels even further. The reduced levels of daminozide in processed cherry products may permit a lowering of the tolerance levels. UDMH residues were detectable in fresh sweet and tart cherries and thermal processing was shown to increase the content of

UDMH in canned cherry products. In order to reduce the levels of UDMH

in processed cherry products, it may be necessary to further reduce daminozide residues on fruit which are thermally processed.

Toxicological screening will assist in determining the safe level of

UDMH in food products.

89 REFERENCES

Conditt, M.K., Baumgardner, J.R. and Hellmann, L.M. 1988. Gas chromatographic/mass spectrometric determination of daminozide in high protein food products. J. Assoc. Off. Anal. Chem. 71:735.

Dannals, L.E., Puhl, R.L. and Kucharczyk, N. 1974. Dissipation and degradation of Alar in soils under greenhouse conditions. Arch. Environ. Contam. & Tox. 2:213.

Domir, S.C. 1980. Fate of daminozide and malic hydrazide in American Elm (Ulmus americana L.). Pesticide Science, 11:418.

Dozier, H.A., Rymal, K.S. and Knowles, J.R. 1984. Carryover residue levels of Alar in apple trees sprayed the preceding spring and summer. Alabama Agric. Exper. Station 31:9.

Edgerton, L.J. and Greenhalgh, W.J. 1967. Absorption, translocation and accumulation of labeled N—dimethylaminosuccimic acid in apple tissues. J. Am. Soc. Hort. Sci., 91:25.

Looney, N.E. and McMechan, A.D. 1970. The use of 2-chloroethylphosphonic acid and succinic acid 2,2-dimethyl hydrazine to aid in mechanical shaking of sour cherries. J. Am. Soc. Hort. Sci., 95:452.

Michigan Department of Agriculture. 1988. Michigan Agriculture Statistics. p.4

Newsome, H.H. 1980. Determination of daminozide residues on food and its degradation to 1,1-dimethylhydrazine by cooking. J. Agric. Food Chem., 28:319.

Roe, F.J.C., Grant, G.A. and Millican, D.M. 1967. Carcinogenicity of hydrazine and 1,1-dimethylhydrazine for mouse lung. Nature, 216:375.

Ryugo, K. 1965. Persistence and mobility of Alar (B-995) and its effects on anthocyanin metabolism in sweet cherries, Prunus avium. J. Am. Soc. Hort. Sci., 88:160.

Toth, B. 1973. 1,1-Dimethylhydrazine (unsymmetrical) carcinogenesis in mice. Light microscopic and ultrastructural studies on neoplastic blood vessels. J. Nat’l. Cancer Institute, 50:181.

Uniroyal Chemical Co., Inc. 1985. Special Report: Alar growth regulant. Important information concerning a special registration review by the Environmental Protection Agency. Bethany, CN., pp. 1-8.

90 91

Uniroyal Chemical Co., Inc. 1986. Daminozide/UDMH Market Basket Survey: Sampling time 11. Personal Communication. Bethany, NY. pp 2-7.

U.S. Food and Drug Administation. 1986. Summary U.S. Food and Drug Administation’s FY 86 Assignment ”Survey for Daminozide Residues in Selected Fruits”. Washington, D.C. pp. 1-7. ACKNOWLEDGEMENTS

Sincere appreciation is extended to Uniroyal Chemical Co., Inc.,

Michigan Cherry Commission, Wilderness Foods, George McManus, Stanek

Farms, Larry Bradford, Don Gregory, Conway Smith, Jim & Fred Hawley,

Larry Esch for product and supplies. Special thanks also to Chris

Herald, Margaret Conner, David Ankrapp and Chuck Kesner for assistance.

Tart Application Cherry Rate Treatment Harvest Fresh Pitted 5 + 1 Water Pie Logation (lbs. a.i./A) Date Date Frozen Frozen Pack Pack Filling

Alar UDMH Alar Alar UDMH Alar UDMH Alar UDMH (m) (ppb) (pm) (PM) (9») (pp!) (ppb) (m) (M) OM90 7 17-84 3.1 6.1 08349 on? - - 0.4 <1 0.7 - d’ N O O D D e 7-17-84 7.2 2.9 0 o 199.0 2.7 (1 7 17 84 13.0 6 I - - 9.4 503.0 4.2 <1 HM" No.6 F490 0000 wan “00 CO N75.” No.4 H

6 4 84 7-26-84 0 i”

6-4-84 7-26-84 0 6 4 84 7-26-84 F0 Hoe-c ~00 00000 cum ind-no 0mm mac

6 6 84 7-30-84 0 9 11.0 93 5i 0 0 0 6-6-84 7-30-84 0 0 4.0 115.0 6 6 84 7-30-84 H 5 9 286.0

12.0

30.0

(ppb)

UDMH

Syrup

Pack

1.6

3.0

9.6

6.3

Alar

(ppm)

Canned

1.6

(I (I

(ppb)

UDMH

Frozen

Fresh 0

7.7

5.9

0.8

Alar

10.0

13.

(m)

Date

7-23-84

7-23-84 7-23-84

7-23-84

7-23-84 Harvest

3; H

(Date I

6-1-84

6-1-84 '0 Treatment

V“) 4 o O .8

OM40 0:00

g.i./A)

Rate

Application (lbs.

Schmidt Schmidt

Schmidt Napolean Napolean Variety Napolean

15 E Growing Location #1 3. E . . E g 10 . g 8 fresh cherries :9 ’ E= 72 p lited cherries g = :5: Z water pack as i E E g El plefllling IS 5:- _§_7 g g E prepared pie 8 . '5' g é E - E a % o (u .l- 5= =E / o in = f'" /%

0 3.4 6.8 Application Rate (lbs. Alar a.i. IAcre)

Figure 1. Daminozide residues in fresh and processed cherry products from location 1.

15 E Growing Location #2 .3:

3 fresh cherries a 10' pitted cherries E (D sugarpack 0) a: water pack 3 lflefflflng ‘fi 5' :nepanxipka Bus-am O .E E a . ° o~ e 5w fil%m_

0 3.4 6.8 Application Rate (lbs. Alar a.i. IAcre)

Figure 2. Daminozide residues in fresh and processed tart cherry products from location 2.

,_. 15 E Growing Location #3 .3; * a

3 10 _ E. 5 fresh cherries :3 § pitted cherries 3 g I sugar pack a: = 5 water pack 0) E E f/ D pieiilllng '0 = =y33 / 'ﬁ 5 5 5%? a prepared pie ‘3 == ==é§ﬁ¢2 -- ‘3 = =// £5 =:= ...... ::%@5%2=/:2vi/ £3 55 1“ Eiéé g? g 3%: % 0 3.4 6.8 Application Rate (lbs. Alar a.i. lAcre)

Figure 3. Daminozide residues in fresh and processed tart cherry products from location 3.

_ Growing Location #1 , E 500 , g

3 400 " a 5 fresh cherries g f /// sugar pack :9 300 - é a water pack 3; . g; a! lflefflflng , HE C] Ifle :1:a: 200 - 7 é a % r 3 100 - g a

0 3.4 6.8 Application Rate (lbs. Alar a.i. IAcre)

Figure 4. Unsymmetrical dimethylhydrazine (UDMH) residues in fresh and processed tart cherries from location 1. 99

100 Growing Location #2 ‘8. 80 - :1 V » B fresh cherries 3 E sugarpack 3 60 '7 :2 g B waterpack g} 22 E; I! [Nefﬂﬂng a: .40 - 22 2; E] {we a: . 2 2 g 2 2 = 20 ' 2 2

o‘_=. a H E E 1__

(I 3.14 6.13 Application Rate (lbs. Alar a.i. I Acre)

Figure 5. Unsymmetrical dimethylhydrazine (UDMH) residues in fresh and processed tart cherries from location 2.

100

300 Growing Location #3 7 * %

(ppb) Z g irxzrcgeJSes 200 ' ? Z water. pack ; ’// pleillllng

Residues E] [ﬁe a?‘5 100 ‘

UDMH r a » A i .

o -— W -— O 3.4 6.8 Application Rate (lbs. Alar a.i. IAcre)

Figure 6. Unsymmetrical dimethylhydrazine (UDMH) residues in fresh and processed tart cherries from location 3. nICHIan STATE UNIV. LIBRARIES "HIWW"llll”IWINllllﬂllllllllllWHIHIIIHIHI 31293007884194