STATE ti^^.i^l L;UAKU E-550 September 1941 £-AMINOAZOBENZENE HYDROCHLORIDE AS AN INSECTICIDE By A. M, PhilliFS, M. C. Swingle, J. B. Gahan, and E. R. McGovran, Division of Control Investigations While making toxicological studies on a large number of organic compounds (5), the authors found that ^-aminoazobenzene hydrochloride (C 12H1 1N3HCI) was toxic to certain insects. This material is closely re- lated chemically to other azo compounds having insecticidal value that were mentioned in a recent patent by Vivian and Haller (6) . Para- aminoazobenzene (^-phenylazoaniline) was found effective against mosquito larvae by Fink et al . (2) but was found ineffective against screwworms by Bushland (1) . Para-aminoazobenzene hydrochloride is a crystalline solid with a purplish-brown color which may vary considerably with the purity or physical state of the sample. It is slightly soluble in water and in acetone and moderately soluble in alcohol. When properly prepared the compound can be applied to foliage either as a dust or as a spray. Although the com- pound is difficult to suspend in water, a satisfactory spray suspension can be made with certain agents. Para-aminoazobenzene hydrochloride is commercially available, but at present it is too expensive!/ for practical use as an insecticide. However, if it is found to be an effective insecti- cide under practical conditions, the cost possibly could be reduced to com- pete with the insecticides that are now available. In common with other azo compounds, g-aminoazobenzene hydrochloride is highly colored and v/ill stain the skin or clothing. Spray deposits are readily visible on foliage but have no special resistance to weathering because of this staining quality. 1/ Approximately $1.75 per pound in small lots in 1940. EB 1 Insects Tested Preliminary tests were made on the following species of insects; Insect Foliage American cockroach (P erjplane t a americana (L.)) None Cabbage aphid (B revico ryne b rassi c ae (L.)) Collards Cabbage looper (A ut r grapha b rassicae (Riley)) Do. Colorado potato beetle (Leptinotarsa decem lineata (Say) ) Potato Cowpea weevil ( Callc sobruchus macul atus (F.)) None Cross-striped cabbage worm ( Ever g;estis ri mcsali s (Guen.)) Collards Fall webworm (Hyphantria cunea (Drury)) Pecan Hawaiian beet webworm (Hymenia fas cialis (Cram.)) Swiss chard Imported cabbage worm (Pieris r apae L.) Collards Melonworm (Dia phani a hyilinita (L.)) Pumpkin Rice weevil S tc^hilus or^za (L.)) Wheat (seeds) Southern armyworm (Prcdenia eri dania (Cram.)) Collards Southern beet webworm (Pac'nyz ancla bipunctalis (F.)) Swiss chard Termites ( Retic u literm es sp.) None Yellow woolly bear (Diacrisia vi rgini ca (F.)) Collards All the leaf-feeding insects, as well as the cockroach, the rice weevil, and the cowpea weevil, were reared in the laboratory by the methods described in a previous paper (3) . The termites were collected in the field and brought into the laboratory for testing. The cabbage aphid was tested as a natural infestation on collard plants in the garden. Preliminary Tests Preliminary tests of dusted foliage were made to determine the fact of toxicity and the species against which the compound was effective. For the tests on the leaf-feeding insects a settling dust chamber (4) was used to apply the compound to excised leaves. The dusted leaves were then placed in Petri dishes in which approximately 25 nearly full grown larvae were con- fined. After 48 and 72 hours examinations were made for mortality of the in- sects and an estimate of the feeding. Parallel tests were made with leaves dusted with the standard insecticide for the respective species. The derris sample used contained 4.5 percent of rotenone. The results, which are given in table 1, show £-aminoazobenzene hydro- chloride to be effective against most of the insects and in some cases as effective as the standard insecticide. Only the cabbage looper and the fall web.vorm were unaffected by this compound. It showed considerable repellent action to most of the insects but usually did not kill so rapidly as did the standard insecticide. ^ In the preliminary tests this compound was not effective against termites, roaches, the rice weevil, the cowpea weevil, and the cabbage aphid; consequently no further tests were made with these species. - 3 Table 1. — Toxicity of E-aminoazobenzene hydrochloride as compared with a standard insecticide when dusted on foliage and fed to nearly full grown larvae of several species confined in Petri dishes p-Aminoazobenzene hydrochloride Standard insecticide Insect Deposit Feeding on third day Micrograms per sq.cm. Cabbage looper 240 Moderate Colorado potato 230 None beetle Cross-striped 55 Moderate cabbage worm 100 do. 155 do. 197 Trace Hawaiian beet 52 Moderate webworm 101 Trace 154 do. 200 do. Imported 55 Normal cabbage worm 100 do. 155 Moderate 200 do. Melonworm 54 do. 100 do. 154 Trace 200 do. Fall webworm Southern armyworm Southern beet webworm 230 Trace 55 Moderate 100 Trace 153 do. 200 do. 52 Moderate 102 do. 155 Trace 200 do. Kill in — Deposit Feeding on 2 days 3 days third day Percent Percent Micrograms per sq.cm. Derris 94 49 80 60 88 64 84 59 86 27 57 37 68 64 86 50 82 14 45 14 53 35 71 35 65 46 80 46 79 62 90 72 98 Kill in — 2 days 3 days Percent Percent 71 39 90 47 86 49 93 24 55 20 57 29 57 18 67 110 Trace 47 — 110 do. 100 ~ 55 Moderate 66 S6 100 Trace 78 96 152 do. 82 94 197 do. 88 96 52 Moderate 62 81 101 Trace 68 89 152 do. 72 92 200 do. 66 93 55 do. 88 97 100 do. 91 95 155 do. 93 96 200 do. 98 100 54 do. 43 77 100 do. 55 84 152 do. 68 93 201 do. Lead arsenate 69 93 80 Normal 7 — 53 Moderate 49 95 98 Trace 84 99 155 do. 63 99 195 do. 92 100 50 Moderate 22 55 93 Trace 31 71 155 do. 31 62 198 do. 47 73 Yellow woolly bear 490 76 92 185 LIBRARY STATE PLANT BOARD 100 Fumigation Tests in Petri Dishes Since with certain species the preliminary tests showed a high mor- tality with only a trace of feeding, tests were subsequently made to de- termine if this mortality was due to a fumigating action of the compound. Leaf sections with approximately 25 larvae each were placed in Petri dishes, and about 0.5 gram of the compound between two pieces of filter paper was press3d into the top of each dish to prevent the insects from coming in con- tact with it. As no fumigating action occurred within 48 hours, it was as- sumed that the high mortalities in the previous tests were not caused by fumigation. Phytotoxicity Tests After the toxicity of £-aminoazobenzene hydrochloride to certain in- sects had been established, an experimert was made to determine the toler- ance of certain tender truck-crop plants to spray deposits of this com- pound. The spray was prepared at concentrations of 4 and 8 pounds per 100 gallons of water, and applied to bean, swiss chard, squasl" , collard, and tomato plants. After 7 c'ays a second application was made, and the final results were recorded 14 cajs after the first application. Five plants of each variety were used with each concentration of ^pray. The plants were protected from showers and at night to prevent the spray residue from being washed off. The two applications of the 4-100 spray caused no injury to bean, Swiss chard, collard, or tomato plants in 14 cays, but caused moderate in- jury to squash plants in 10 days. One application of the 8-100 spray caused slight sturtin^ of the bean plants end curling of the leaves in 7 days, moderate burning of swiss chard in 5 days, and moderate to severe curling and browning of the squash leaves in 7 days. No injury resulted to collard pla'^ts in 14 days at this concentration and only slight injury to some leaves on tomato plants. Laboratory Cage Tests Tests were next made to determine the toxicity of ^-aminoazobenzene hydrochloride to insects when applied as a spray on potted plants. Several methods of making a spray suspension were tried, but tl^e most satisfactory spray contained saponin as a dispersing agent. A weighed portion of the dry compound was ground in a mortar, and then small amounts of a solution of saponin in water were added to make a paste. When the compound and saponin were thoroughly mixed, water was added to produce the concentration desired, as the effect on each insect was tested at 8, 4, 2, and 1 pound of insecti- cide to 100 gallons of water. The saponin was used at the rate of 1/8 pound per 100 gallons. This spray was satisfactory when used on collard, swiss chard, and pumpkin plants. For each test the spray was applied to two potted plants with a com- pressed-air spray gun. When the leaves were drv, 15 nearly full grown - 5 - larvae were confined with each plant in a cylindrical screen cage. With each test of 2-aTiinoazobenzene hydrochloride a similar test was made with a standard insecticide. Three examinations were made at 2-day intervals for mortality of the insects and an estimate of the feeding on the plants. The results of these spray tests on the cross-striped cabbage worm, the Hawaiian beet webworm, the melonworm, the southern armyworm, and the southern beet webworm are given in table 2. Para-aminoazobenzene hydrochloride was about as ef- fective as derris (4.5 percent rotenone) against the cross-striped cabbage worm, slightly more effective than derris against the Hawaiian beet webworm, and about equal to derris against the melonworm. At the 8-100 concentration it was almost as effective as lead arsenate against the southern beet webworm and the southern army- worm. Against the southern armyworm, however, there was a definite decrease in tho effectiveness of £-aminoazobenzene hydrochloride at the lower concentration. Table 2. — Toxicity of 2 aminoazobenzene hydrochloride as compared with a standard insecti- cide when applied as sprays to potted plants infested with nearly full grown larvae of several insects Cone jentration of secticide Tests p-Aminoazol Feeding on sixth day Denzene hydrochloride LI after— Standard insectic ide Insect Feeding on sixth day Ki 11 after— inj 2 days 4 days 6 days 2 days 4 days 6 days Pounds per Numbe - Pet. Pet. Pet. Pet. Pet. Pet. 100 gallons Derris Cross-striped 8 3 Trace 64 89 100 Trace 63 100 100 cabbage worm 4 do. 54 91 99 do. 68 97 100 2 Moderate 31 72 92 Moderate 59 90 97 1 do. 27 67 90 do. 48 79 97 Hawaiian beet 8 3 Trace 26 64 93 Trace 27 65 82 webworm 4 do. 14 59 92 do. 18 54 63 2 Moderate 3 28 69 do. 8 32 65 X do. 3 20 43 Moderate 17 31 Melonworm 8 2 Trace 35 94 100 Trace 26 84 93 4 do. 25 83 96 do. 19 77 98 2 1 Moderate 6 63 76 do. Lead 3 arsenat 70 9 93 Southern 8 3 Trace 23 37 91 do. 76 98 100 armyworm 4 do. 5 20 83 do. 71 90 100 2 Normal 1 2 do. 41 72 8D 1 do. 1 1 3 Moderate 16 30 52 Southern beet 8 4 Trace 16 54 84 Trace 39 47 64 webworm 4 do. 11 33 57 do. 30 51 66 2 Normal 2 7 20 Moderate 3 20 33 1 do. 1 7 25 do. 1 9 21 Field Cage Tests Para-aminoazobenzena hydrochloride '.vas also tested against the melon- worm on garden plots of young pumpkin plants, under normal weather conditions except that the plants were protected from showers and at night. Three plots of three plants each were used. The first plot was sprayed with 2-aminoazobenzene hydrochloride, the second with derris, and the third was left unsprayed as a check. The spray was used at a concentration of 8 pounds per 100 gallons of water and applied with a compressed-air gun fitted to a knapsack sprayer. Each plant in each plot v;as then infested with melonv/orm larvae and confined in a cylindrical screen cage. The cages were examined at 2-day intervals for mortality of the insects and an estimate of the feeding on the plants. In this test ^-aminoazobenzene hydrochloride was as effective against the melonworm on the fourth day as was derris on the eighth day. At this time 83 percent of the larvae were dead, and there was only a trace of feed- ing. Slight burning was noticed on the plants on the fourth day, but it was not severe enough to interfere with the tests. Field-Laboratory Tests The effect of weathering other than direct rainfall on the toxicity of spray deposits of £-aminoazobenzene hydrochloride was determined over a IC-day period on three plots of seven plants in an outdoor garden. One plot v.-as sprayed with an 8-100 coiicentratior of £-aminoazobenzene hydrochloride, another with derris at the same concentration, ard a third was an unsprayed check. When dry, six leaves were taken at random from each plot and fed to 30 nearly full grown melonworm larvae in Petri dishes in the laboratory. Similar samples from the plots were tested at 2-day intervals for 10 dety^: The results are shown in table 3. In these tests ^-aminoazobenzene hydrochloride was more effective than derris against the melonworm, as shown by the retarded feeding for the first 2 days and the slightly greater toxicity. The compound was approximately equal to derris in toxicity or the eighth and tenth days. The reduced ef- fectiveness after the fourth day was probably due to the rapid growth of the plants after the spray was applied, for during the IC-day period the plants more than doubled in size, v/hich would tend to spread the spray deposit over twice the leaf area. - 7 - Table 3. — Results of tests with leaf samples taken at 2-day intervals from plants sprayed in the garden with E-aminoazobenzene hydrochloride and the standard insecti- cide and fed to nearly full grown larvae of the melonworm in Petri dishes E-Am inoazobenzene Stan 3ard insecti- Time hydrochlor Lde cide (derris) Chec Feeding after' " i (unsprayed) between Feeding after _-Kill_after-=__ 2 days 3 days Feeding after Kill a fter— Kill after— spraying 2 days 3 days 2 days 3 days and 3 days 3 days . 3 days sampling P^ys Percent Percent Percent Percent Percent Percent ■ Trace 19 61 Moderate 30 ■ Normal 3 2 do. 40 83 do. 36 60 do. 3 3 4 Moderate 40 70 do. 23 56 do. 30 36 6 do. 36 73 do. 3 10 do. 3 6 8 do. 16 30 do. 13 23 do. 10 do. 6 46 do. 16 . . 46 _ do. 6 16 Summary . In laboratory tests e-aminoazobenzene hydrochloride, a commercially available organic compound, was found to be toxic' to a number of insect pests. In preliminary tests on 15 species gpod results were. obtained against all. but 2 species, although it did not usually kill so rapidly as did the standard insecticides. No fumigating action was shown in t'e'sts with this compound. In laboratory cage tests where the compound was applied as a spray to potted plants, the results with certain species were similar to those obtained in Petri-dish tests. In field cage tests ^-^^li'^'^^zobenzene hydro- chloride was as effective against the melonworm on the fourth day as was derris on the eighth day. In the field-laboratory test also it was more effective than derris, both materials being considerably less effective after 10 days of weathering than on the date of application. A 4-100 spray did not injure any of the tender truck-crop plants used in the tests except squash, but an 8-100 spray caused some injury to most of the plants used. The compound was not toxic to termites, the American cockroach, the rice weevil, the cowpea weevil, and the cabbage aphid in preliminary tests. UNIVERSITY OF FLORIDA lliilillliillilillil 3 1262 09230 4012 Literature Cited (1) Bushland, Raymond C. 1940. The toxicity of some organic compounds to young screw- worms. Jour. Eccn. Ent. 33: 669-76. (2) Fink. D. E., Smith, L. E. , Vivian, D. L. , and Claborn, H. V. 1938. Toxicity tests with synthetic organic compounds against culicine mosquito larvae. Bur. Ent. and PI. Quar. Cir. E-425, mimeographed. (3) Swingle, M. C, Gahan, J. B., and Phillips, A. M. 1941. Laboratory rearing of certain leaf-eating insects. Jour. Econ. Ent. 34: 90-95, illus. (4) Swingle, M. C, Phillips, A. M., and Gahan, J. B. 1941. Laboratory testing of natural and synthetic organic substances as insecticides. Jour. Econ. Ent. 34: 95- 99, illus. (5) , Gahan, J. B., and Phillips, A. M. 1941. Phthalonitrile as an insecticide. Bur. Ent. and PI. Quar. Cir. E- 548, mimeographed. (6) Vivian, D. L., and Haller. H. L. J. 1938. Insecticide. U. S. Patent 2,111.879, issued March 22.