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Journal of Food Protection, Vol. 47, No. 3, Pages 242-244 (March 1984) Copyright0, International Association of Milk, Food and Environmental Sanitarians

Daminozideand Unsymmetrical Dimethylhydrazine(UDMH) Residues in Fresh and Processed 'Red Delicious' Apples

K. S. RYMAL1*, W. A. DOZIER, JR.1, J. W. KNOWLES1, R. D. COSPER1 and R. B. REED2

Department of Horticulture andResearchData Analysis, Alabama Agricultural Experiment Station, Auburn University, Auburn, AlabamaDownloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/242/1656158/0362-028x-47_3_242.pdf by guest on 27 September 2021 36849

(Received for publication July 19,1983)

ABSTRACT with an LD50 (i.v.) of 60 mg/kg for dogs (16), and subacute Daminozide is a growth regulating substance widely used on ap­ doses produced mild kidney damage in rats (3). UDMH has ples and other fruit crops to affect various fruit and tree characteris­ also been shown to be a carcinogen in mice (12). Several in­ tics. Daminozide residues were determined in 'Starkrimson' Red vestigators have determined levels of daminozide in apple Delicious apples orchard-sprayed at both recommended and exces­ fruit sprayed at recommended levels and typical application sive rates, at recommended periods before expected harvest, and at times for several different regions (4,5,9,10). Shutak et al. several application times closer to harvest than recommended. Analyses were conducted on the whole raw fruit, including the peel­ (13) applied daminozide at several application dates and ing, and on applesauce processed from the peeled and cored fruit, found highest residues in fruit from trees sprayed 60 to 90 d immediately after harvest, and after cold storage periods of 30, 60 before harvest, whereas others (10) reported highest levels and 90 d. Residues in both the fresh and processed fruit were di­ in fruit sprayed 30 to 60 d before harvest. Residues in apples rectly proportional to the concentration of daminozide in the foliar were persistent in fruit in storage (10). Edgerton et al. (5) sprays, regardless of application date. The highest residues in both found residues of daminozide in tree buds during the dormant fresh and processed apples were in fruit from trees sprayed 14 to season in New York orchards, but no residues in similarly 28 d before harvest at all spray concentrations. Residues from all sprayed trees in North Carolina. spray concentrations and all application dates were persistent for When this study was initiated, daminozide was in the in­ 90 d at 0°C storage for fresh fruit and at 23°C storage for processed fruit. Only the residues in fruit sprayed with four times the recom­ itial risk investigation phase by the U.S. Environmental Pro­ mended level at 2 or 4 wk before harvest exceeded the U.S. En­ tection Agency (EPA) to determine whether a rebuttable pre­ vironmental Protection Agency tolerance level (30 ppm). Residues sumption against registration should be issued by the EPA. ranged from <2 to 32 ppm. Unsymmetrical dimethylhydrazine It has since been returned to the registration process and stan­ (UDMH) levels were directly proportional to the daminozide re­ dards for food uses are being developed. The standards when sidues in the applesauce. Average UDMH residues ranged from written will not necessarily constitute final action by the 0.03 ppm for fruit sprayed with the recommended rate of EPA. daminozide to 0.8 ppm for fruit with the highest daminozide re­ The objective of this research was to determine the effect sidues. of recommended and excessive rates of daminozide sprays on residue levels in fresh apples and applesauce.

Daminozide applied as foliar sprays affects both the veg­ etative and fruit development of apples and is used to im­ MATERIALS AND METHODS prove certain physical characteristics that are of economic Field application importance in producing and marketing apples (1,2,7,9,14). Daminozide was applied to 18-year-old 'Starkrimson' Red Delicious However, questions have been raised concerning the hazard apple trees on seedling rootstock at the Chilton Area Horticulture Substa­ to humans of exposure to daminozide and unsymmetrical di­ tion, Clanton, AL. The treatments were applied as fine mist foliar sprays methylhydrazine (UDMH) which is suspected as a contamin­ to the point of run-off with a high pressure hand-held spray gun. Spraying ant in commercial daminozide spray products or as a break­ was done so no spray materials drifted to other treated or untreated tree plots. The field design was a randomized complete block with four replications down product in thermally processed apple products (11). of whole tree plots. Daminozide was applied at (OX), at the recommended Daminozide has been implicated in the production of tumors rate of 1.5 lb of 85% wettable powder per 100 gal of spray solution or 1500 in mice (15). Acute toxicity studies with UDMH and related ppm (IX), and at excessive rates of 3 lb or 3000 ppm (2X), and 6 lb or 6000 hydrazines have shown that UDMH is a convulsant poison ppm (4X) per 100 gal of spray solution. Treatments were applied to separate tree plots 21 d after full bloom (125 d before harvest) or at 56, 42, 28, 14 or 0 d before harvest. The 125- or 56-d spray dates were the alternate dates 'Department of Horticulture. recommended on the daminozide (Alar 85) label. The fruit were randomly 'Research Data Analysis. sampled from each treatment on August 25 (146 d after full bloom) and

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984 DIAMINOZIDE AND UDMH RESIDUES IN APPLES 243 stored at 0°C for 0, 30,60 and 90 d before fresh fruit analysis for daminozide a split plot in time imbedded in the factorial. The two processing types (raw residue or processing and analysis for daminozide and UDMH residues. vs. cooked) added a further split to the design such that the entire experiment was characterized as a 4 x 6 factorial with an imbedded split-split plot; all Processing arranged in a randomized complete block design. An appropriate analysis Apple samples from each storage treatment were hand-peeled, cored and of variance was performed which showed that neither storage time nor the diced. Diced apples (850 g) were mixed with sugar (162 g) and water interaction of storage time and type of processing significantly affected (44 g), and cooked in an open kettle for 20 min. Cooked apples were daminozide residue. Therefore, data from all storage times were pooled and blended 1 min (to simulate commercial pulping), filled into pint (473 ml) a response surface was fit to the daminozide residues for each processing jars, processed at 100°C for 25 min, spray cooled, and stored at 23°C until type. A stepwise multiple regression procedure was used in which terms analyzed. Weights of sugar, water added and water evaporated during cook­ were successively eliminated from an nth order polynomial model until all ing were monitored carefully so that applesauce sample weights could be remaining terms were highly significant (P<0.01). corrected for residue calculations. RESULTS AND DISCUSSION Residue analysis Daminozide residues were determined by alkaline hydrolysis, distillation Residues in both the raw fruit and the processed of the resulting dimethylhydrazine, and spectrophotometric measurement of the colored complex formed with pentacyanoamine ferrate according to the applesauce were directly proportional to the concentration of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/242/1656158/0362-028x-47_3_242.pdf by guest on 27 September 2021 method of Lane (S). Ten apples from each treatment sample were randomly daminozide in the foliar sprays (Tables 1 and 2). There was selected and a one-eighth section (including skin, flesh and core) was taken no significant difference between residue levels in the raw from each fruit. The sections were weighed and combined in a blender with fruit samples, which included the peeling, and the processed one-half their weight of water. Thirty grams of raw apple homogenate or applesauce, which did not include the peeling. Although the undiluted applesauce were used for analyses. Weights were corrected for residue calculations. The pH in each distillate was adjusted to pH 5 im­ data in Tables 1 and 2 appear to differ, statistical analysis mediately before color development. The absorbance was measured at 490 (not shown) determined that there was no difference at the and 600 nm in a Perkin Elmer model 124 spectrophotometer, using a previ­ 5% level of probability. This is especially noteable for the ously untreated sample in the reference cell of the double-beam instrument. 0-d treatment (fruit sprayed the day of harvest), indicating UDMH determinations on applesauce of selected samples were done ac­ that spray material was absorbed through the skin within cording to the method of Newsome (11) in the laboratories of the Bureau of Chemical Safety, Ottawa, Ontario, Canada. 24 h (time from harvest to analysis) and that no significant amount of spray material remained in the skin. Statistical analysis Residue levels were highest at all spray concentrations in The various combinations of time and rate of daminozide application fruit from trees sprayed 14 to 28 d before harvest. We may formed a 6 x 4 factorial experiment arranged in a randomized complete conclude that spray materials were translocated to the fruit block experimental design. The various storage times for apples represented

TABLE 1. Effect of time and rate of spraying on daminozide residue (ppmf content of 'Red Delicious' apples. Application time (days before harvest) Rate (PPm) 125 56 28 14 0 0b 0.35c 0.45 0.65 0.49 0.45 1500 1.79 3.97 7.70 5.77 3.76 3000 3.74 10.46 15.71 13.81 6.10 6000 6.02 21.16 28.80 30.29 12.43 "Residue values are means of four replications and four storage periods. Apple sample slices included peels and core. Residue calculations were based on weight of apple slices only. bResidues from 0 rate (unsprayed) trees represent carryover from the previous year's sprays. No spray drift reached unsprayed trees and the analysis method eliminated response from substances other than daminozide. degression analysis: y = 1.903 + 0.404 D + 4.020 R - 0.011 D2 + 0.0001 D3 + 0.0769 DR - 0.0008 D3R. y = residue value (ppm); D = days before harvest; R = rate of application (ppm); R2 = 0.88. All terms highly significant (p<0.01). TABLE 2. Effect of time and rate of spraying on daminozide residue (ppmf content of processed 'Red Delicious' apples. Application time (days before harvest) Rate (ppm) 125 56 28 14 0 0b 0.88c 0.79 0.93 0.72 0.85 1500 2.14 3.88 6.27 5.65 2.31 3000 3.45 7.17 11.30 11.87 4.02 6000 5.29 16.92 26.28 26.02 5.49 "Residue values are means of four replications and four storage periods. Applesauce was prepared from peeled and cored apples. Residue calculations were based on apple flesh weight only. bResidues from 0 rate (unsprayed) trees represent carryover from the previous year's sprays. No spray drift reached unsprayed trees and the analysis method eliminated response from substances other than daminozide. "Regressionanalysis:y = 1.902 + 0.404D + 4.020R - 0.011 D2 + 0.0001D3 + 0.0769DR - 0.0008D3R. y = residue value (ppm); D = days before harvest; R = rate of application (ppm); R2 = 0.88. All terms highly significant (P<0.01).

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984 244 RYMALETAL. from the foilage within 14 d after application, because re­ ACKNOWLEDGMENTS sidues in fruit sprayed the day of harvest were significantly We gratefully acknowledge the collaboration of Dr. W. H. Newsome, lower than those sprayed 14 d before harvest. Furthermore, Food Research Division, Bureau of Chemical Safety, Ottawa, Ontario, less of the daminozide was metabolized in that short period Canada, in the UDMH analyses. (14 or 28 d) before harvest than from earlier sprays. This research was funded by a grant from the Southern Region Pesticide Impact Assessment Program, U.S. Department of Agriculture, CSRS. All daminozide residues were persistent up to 90 d of stor­ Alabama Agricultural Experiment Station Journal Series No. 11-83473. age for fresh fruit and for applesauce processed from the stored fruits (data not shown). In spite of the excessive concentration rates of daminozide REFERENCES applied as foliar sprays (up to four times the rate recom­ 1. Batjer.L. P., andM.W. Williams. 1966. Effects of N-dimethyl amino mended to produce the desired biological effects) and appli­ succinic acid (Alar) on watercore and harvest drop of apples. J. Am. cation times closer than recommended to harvest (including Soc.Hort.Sci. 88:76-79. the day of harvest), residues in only those fruit sprayed with 2. Batjer, L. P., M. W. Williams, and G. C. Martin. 1964. Effects of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/242/1656158/0362-028x-47_3_242.pdf by guest on 27 September 2021 four times the recommended rate 2 wk before harvest ex­ N-dimethyl amino succinic acid (B-Nine) on vegetative and fruit char­ acteristics of apples, pears, and sweet cherries. J. Am. Soc. Hort. Sci. ceeded the tolerance level (30 ppm) published by the EPA 85:11-16. (6). Residue levels in fruit from trees sprayed at the recom­ 3. Cornish, H. H., and R. Hartung. 1969. The subacute toxicity of 1,1-di- mended levels and times were less than 5 ppm. methylhydrazine. Tox. Appl. Pharm. 16:62-68. UDMH levels in the applesauce were directly proportional 4. Edgerton, L. J., C. R. Unrath, C. G. Forshey, and D. J. Lisle. 1977. to the daminozide residues in the thermally processed Persistence of daminozide residues in apple spurs under different con­ ditions. HortScience 12:242-243. applesauce (Table 3). The EPA has not set tolerance levels 5. Edgerton, L. J., M. L. Rockey, H. Arnold, and D. J. Lisk. 1967. Col- for UDMH in food products. However, the levels of UDMH iformetric determinations of Alar residues in apples. J. Agr. Food determined in samples of applesauce made from fruit from Chetn. 15:812. trees sprayed according to the Alar-85 label directions were 6. Environmental Protection Agency, Office of Pesticide Programs. exceedingly low (less than 0.05 ppm). UDMH levels in 1983. Tolerances and exemption from tolerances for pesticide chemi­ cals in or on all raw agricultural commodities. 40 Code of Federal Reg­ applesauce with daminozide residues approaching the EPA ulations, Part 180-246. EPA, Washington, DC. tolerance levels of 30 ppm still averaged < 1 ppm. 7. Fisher, D. V., ajid N. E. Looney. 1966. Growth, fruiting and storage It would appear from this study that if the label directions response of five cultivars of bearing apple trees to N-dimethyl amino for daminozide use are followed, residues in apples or succinic acid (Alar). J. Am. Soc. Hort. Sci. 90:9-19. applesauce will not exceed the presently established toler­ 8. Lane, J. R. 1967. Analytical methods for pesticides, plant growth reg­ ulators and food additives, vol. 5. Academic Press, New York. pp. ance. UDMH makes up only a small portion of the residues, 499-505. even in processed applesauce. 9. Looney, N. E., D. V. Fisher, and J. W. W. Parson. 1967. Some effects of annual applications of N-dimethyl amino succinic acid (Alar) to ap­ ples. J. Am. Soc. Hort. Sci. 91:18-24. TABLE 3. Effect of time and rate of spraying on UDMH residue 10. Looney, N. E., K. Williams, and G. A. Wardle. 1972. Factors in­ (ppm) content of processed 'Red Delicious' apples. fluencing the level of succinic acid 2,2-dimethylhydrazide residues in Daminozide apple fruits. J. Am. Soc. Hort. Sci. 97:323-325. rate Application time (days before harvest) 11. Newsome, W. H. 1980. Determination of daminozide residues on (ppm) 125 56 14 foods and its degradation to 1,1 -dimethylhydrazine by cooking. J. Agr. FoodChem. 28:319-321. a 0 <0.01 <0.01 <0.01 12. Roe, F. J., G. A. Grant, and D. M. Millican. 1967. Carcinogenicity 1500 0.03 0.02 0.13 of hydrazine and 1,1-dimethylhydrazine for mouse lung. Nature 6000 0.11 0.38 0.80 216:373-376. 13. Shutak, V. G.,C. E. Olney, and T.W.Kerr. 1969. Succinic acid 2,2- "Residues in fruit from 0 rate (unsprayed) trees represent carryover dimethylhydrazide residues after application to fruit trees. J. Am. Soc. from the previous year's sprays. No spray drift reached unsprayed Hort. Sci. 92:63-66. trees and the analysis method eliminated response from substances 14. Sullivan, D. T., and F. B. Widmeyer. 1970. Effects of succinic acid other than daminzide. 2,2-dimethylhydrazide (Alar) on bloom delay and fruit develoment of Delicious apples. HortScience 5:91-92. 15. Toth, B., L. Wallcave, K. Patil, Schemltz, andP. Hoffman. 1977. In­ duction of tumors in mice with the herbicide succinic acid 2,2-di­ methylhydrazide. Cancer Res. 37:3497-3500. 16. Witkins, L. B. 1956. Acute toxicity of hydrazine and some of its methylated derivatives. A.M. A. Arch. Ind. Health 13:34-36.

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984