ST ATI i:;OARD April 1942 . E-565 2-PHENYLAZOANILINE AS AN INSECTICIDE By M. C. Swingle, J. B. Gahan, and A. M. Phillips,!/ Division of Control Investigations Curing 1940 and 1941 at the Sanford, Fla. , laboratory a large number of synthetic organic compounds were tested as insecticides against 16 species of insects. One of the most promising compounds tested was ^-phenylazoaniline (^-aminoazobenzene) . It was therefore selected for further investigation with regard to its general effectiveness as a spray on truck-crop plants. Most of these investigations were made at Sanford, during 1940 and 1941. The compound (Ce Hs N: NCg H4 NH2) is preferably called ^-phenylazo- aniline, although it is often referred to as 2-aminoazobenzene. It is a yellow crystalline solid when pure, but the color of commercial samples may vary from yellow to dark brown. The compound is only slightly soluble in water but is soluble in ether, acetone, and hot ethyl alcohol. A pure grade of ^-phenylazoaniline was used in these tests. The derris standard used in comparative tests contained 4.5 percent of rotenone and the pyrethrum standard 0.33 percent of pyrethrin I and 0.38 percent of pyrethrin II. 2-Phenylazoaniline has been tested on several species of insects by different workers. In 1930 McAllister and Van Leeuwen (5) reported rather poor results with the compound when used as a spray against newly hatched codling moth larvae. Later Fink and coworkers (2) reported the compound toxic to mosquito larvae at 20 parts per million in water, and therefore not particularly promising compared with other compounds reported in the same paper, Bushland (1) used the compound in jar tests with young screw- worm larvae and reported it nontoxic by the method used. On the basis of the results of Fink and coworkers, application for an insecticide patent was made by D. L. Vivian and H. L. J,. Kaller (7), of 1/ A. M. Phillips was transferred to the Division of Fruit Insect Investigations July 1, 1941. 'd^ this Bureau. This patent describes not only 2-Fhenylazoaniline but also other compounds having two homocyclic nuclei joined by an azo linkage and having one or more amino groups. The compound was received from the Division of Insecticide Investiga- tions for general testing, as described in a previous publication (6) . These tests were designed to obtain preliminary evidence of toxicity, and then to study in more detail the amount of dust or concentration of spray required for satisfactory control of various pests, the utility of the material as a spray on tender foliage, and something of its residual effec- tiveness when exposed to certain factors of weathering. Insects Tested Since many organic compounds are specific in their toxicity, it was considered that a few tests on a large number of species of insects would be more useful and informative than many replicated tests on a few species. The compound was therefore tested on 16 species representing 4 orders. The majority of the tests were made on leaf-feeding lepidopterous larvae. Nearly full grown larvae were generally used because they are more resistant to insecticides than smaller individuals. The various species used in these tests were as follows: Insect Foliage or grain treated American cockroach (_Periplanet_a americana (L.)) None Cabbage looper (Autographa brass icae (Riley)) Collards Cabbage webworm (Hellula undalis (F.)) do. Colorado potato beetle (Leptinotarsa decemlineata (Say)) Potato Cowpea weevil (Callosobruchug maculatys (F.)) Peas (Whippoorwill) Cross-striped cabbage worm (Evergestis lisiosalis (Guen.)) Collards Diamondback moth (Plutella maculipennis (Curt.)) do. Greenhouse leaf tier (Phlyctaenia rubigalis (Guen.)) do. Hawaiian beet webworm (Hymenia fascialis (Cram.)) Swiss chard and beet Imported cabbage worm (Pieris rapae (L.)) Collards Kelonworm (Diaphania hyalinata (L.)) Pumpkin Rice weevil (Sitophilus oryza (L.)) Wheat Southern arm^/worm (Prodenia eridania (Cram.)) Collards Southern beet webworm (Pachyzancla bipunctalis (F.)) Swiss chard and beet Termites (Reticulitermes sp.) None Yellow woolly bear (Diacrigia virginica (F.)) Collards Preliminary Petri-Dish Tests Preliminary tests were made in Petri dishes to establish insecticidal action and to determine the species most susceptible to the compound. The tests were made by feeding insects leaf sections that had been rather heavily dusted with the pure compound, as described in a previous publication (6) . Cust was applied to both sides of the leaves, and the deposit was determined - 3 - cpproximately by weirhin^ a small metal plate dusted 'together with the leaves. Two leaves were thus treated for each species and placed in two Petri dishes containing a total of 25 larvae. After 48 or 72 hours an examination was made for m9rtality of the larvae and an estimate of the amount of feeding. Similar tests were run at the same timo en standard insecticides recognised as being effective against the came species of insects. As the dishes were closed and the larvae able to crawl over the dusted leaves, mortality might result from fumigation, contact, or feeding. Preliminary tests against 12 species are reported in table 1. Ihe results presented for the cross-striped cabbage v.orm, the Hawaiian beet webworm, the imported cabbage worm, the melonworm, the southern armj;worm, and the southern beet webworm are averages of three replications; those for the other insects represent only one or two tests each. Against four species — the Colorado potato beetle, the cross-striped cabbage worm, the Hawaiian beet webworm, and the melonworm — £-f henylazoaniline was about as effective as the standard insecticide used. In each case a deposit of 70 to 100 micrograms per square centimeter caused either high mortality or very limited feeding. The compound was also toxic to the imported cabbage worm, the southern armyworm, and southern beet webworm but inferior to the standard insecticide. It was highly effective against only one out of the five cabbage insects tested. This apparent resistance of cabbage insects was also evident in the tests made on 1,4-diphenyl semicarbazide (3). As these insects are usually rather susceptible to insecticides, their common resist- ance to these compounds is of interest from a ph^^siological standpoint. Volatility Tests The results of the preliminary tests in table 1 establish the fact of toxicity to certain insects but do not give much information on the mode of toxic action. To help clarify this point and aid in the planning of subsequent investigations, the volatility of the compound was determined under certain atmospheric conditions. A glass slide dusted with the powdered chemical was weighed before and after exposure to atmospheric conditions (not sunlight) on a screened porch for 5 days-. -This procedure showed not more than 3 percent of the deposit volatilized within 5 days, which is ample stability for use as a stomach insecticide on foliage. It was also evident that the compound was not sufficiently volatile to act as a fumigant . This conclusion v/as checked, hov/ever, by a fumigation test on the melonworm, the cross-striped cabbage worm, and the southern armjrworm by isolating some of the powdered chemical in the lids of Petri dishes containing larvae feeding on untreated foliage. These tests verified the conclusion that £-phenylazoaniline did not act as a fumigant in the tests recorted in table 1. - 4 - Table 1. — Toxicity of ^-phenylazoaniline 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-Phenylazoaniline Standard insectici de Insect Deposit Kill 2 days in . 3 days Feeding on last day Deposit Kill in Feeding on 2 days 3 days last day Micrograms Percent Percent Micrograms Percent Percent per sq. cm. per sq. cm. Derris Cabbage looper 75 43 47 Moderate Cabbage webworm 75 110 2 28 10 Moderate Trace 170 56 Ncne Colorado potato 110 83 Trace 125 100 Trace beetle Cross-striped 55 73 84 Moderate 55 66 S6 Trace cabbage worm 100 81 96 do. 100 78 S6 do. 155 96 96 Trace 152 78 94 do. 200 S6 100 do. 1S8 87 S6 do. Diamondback moth 133 18 Moderate 200 100 None Hawaiian beet 55 54 72 Moderate 55 67 81 Trace webworm 100 77 88 Trace 100 74 88 do. 153 76 90 do. 150 77 90 do. 200 80 97 do. 200 84 96 None Imported 55 9 45 Moderate 55 88 97 Trace cabbage worm 100 20 45 do. 100 90 94 do. 155 37 76 do. 155 93 S6 do. 200 49 75 Trace 200 £8 100 do. Melonworm 55 43 79 Moderate 55 37 71 Traoe 100 63 83 Trace 100 49 80 do. 150 60 92 do. 155 70 92 do. 200 73 97 do. 200 70 90 do. Lead arsenate Southern 55 26 36 Moderate 55 65 96 Trace armyworm 100 46 70 do. 100 84 99 do. 155 62 89 Trace 155 93 100 do. 200 66 96 do. 200 S7 100 do. Southern beet 50 7 26 Moderate 50 26 55 Moderate webworm 100 22 45 do. 100 26 64 do. 155 33 58 Trace 155 24 71 Trace 200 35 64 do. 200 53 78 do. Yellow woolly bear 215 33 Moderate 166 32 Mcdeiatft Pyrethrum Greenhouse leaf 133 18 Moderate 280 83 Modeiat» tier -.5 - Screen-Cage Tests With the toxicity of £-phenylazoaniline established with regard to certain insects, a study was made of the effectiveness of spray deposits on potted plants in cylindrical screen cages. For this purpose the coir.pound was made up as a spray at concentrations of 8, 4, 2, and 1 pound to 100 gallons of water, and each concentration was applied to two plants with a compressed-air spray gun until the spray began to drip from the leaves. When the plants were dry, 15 larvae were confined on each plant by a cylin- drical screen cage. The treated plants were then placed out of doors in a sheltered location rnd examined at two-day intervals for larval mortality and an estim.ate of the feeding on the plants. Of the various wetting agents tried, the most satisfactory was saponin. The spray was prepared by grinding the weighed portion of £- phenylazoaniline in a mortar with a measured quantity of a saponin solution (equivalent to 1/8 pound of saponin per 100 gallons of spray) and then adding water gradually to obtain the desired concentration. The spray thus for.ned remained well in suspension, with occasional agitation, and adhered well to foliage. Spray suspensions of p-phenylazoaniline were tested against five species of insects. The results, presented in table 2, are averages of three replicated tests. The compound was very effective against the cross- striped cabbage worm and the melonworm, being about equal to derris, which is very effective on these species. Against the Hawaiian beet webwor.Ti ^-phenylazoaniline appeared to be slightly more effective than derris at all concentrations. It was definitely inferior to lead arsenate against the southern armyworm but slightly superior to this insecticide against the southern beet webworm. Phytotoxicity Tests An experimont was next made to determine the tolerance of certain tender truck-crop plants to spray deposits of ^-phenylazoaniline. The spray was prepared at concentrations of 4 and 8 pounds per 100 gallons of water, with saponin, and applied to small field plots of bean, collards, pumpkin, and swiss chard. After 10 days a second application vras m.ade, and the final results were recorded 20 days after the first application. From 4 to 12 plants of each kind were used with each concencration of spray. 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 and collards, but they did cause slight injury to pumpkin and swiss chard. A few spot burns were noted on the swiss chard, and moderate to severe burning of the old leaves resulted on pumpkin. The two applications of the 8-100 spray caused no injury to collards and only very slight injury to the old leaves on bean. The injury to swiss chard and pumpkin was similar to that described for the 4-100 spray. Table 2. - 6 - -Toxicity of ]D-phenylazoaniline as compared with a standard insecticide when applied as sprays to potted plants infested with nearly full grown larvae of several insects "■*" Ccncentration of insecticide Tests p-Phenylazoaniline _Sta ndard insecticide Insect Feeding en sixth day Kill after Feedin sixth g en day Kill after __ 2 days 4 days 6 days 2 days _ 4. days ■ ■6_days Pounds per Number Percent Percent Percent Percent Percent Percent 100 gallons Derris Cross-striped 8 1 Trace 61 S7 100 Trace 83 100 100 cabbage worm 4 1 do. 70 S3 100 do. 83 100 100 2 1 do. 58 83 100 do. 68 100 100 1 1 do. 58 83 86 do. 48 57 £6 Hawaiian beet 8 3 Trace 26 79 94 Trac3 22 57 63 w ebworm 4 3 do. 23 67 85 do. 11 51 68 2 3 do. 7 32 77 do. 6 38 60 1 3 Moderate 4 34 50 Moderate 2 23 37 Melonworm 8 1 Trace 73 100 100 Trace 30 56 95 4 1 do. 64 100 100 do. 23 74 98 2 3 Moderate 26 52 85 do. 11 45 86 1 3 do. 9 28 58 Moder ite 1 18 61 Lead arsenate Southern 8 3 Trace 6 42 43 race 71 98 100 army worm 4 3 do. 0 20 46 do. 63 87 99 2 3 Moderate 2 7 26 do. 60 86 £4 1 3 do. 0 3 24 do. 16 30 52 Southern beet 8 3 Trace 37 78 £6 Trace 10 25 49 webworm 4 3 do. 27 57 75 do. 5 22 C3 2 3 Normal 5 13 31 do. 0 3 19 1 3 do. 2 8 17 Moc:erate 0 2 10 Field-Laboratory Tests A limited study was made of the effect of exposure or weathering on deposits of p-phenylazoaniline on foliage. For this purpose a spray was made up in proportions equivalent to 8 pounds of p-phenylazoaniline and 1/8 pound of saponin to 100 gallons of water and applied with a knapsack-type sprayer to 12 nearly full grown beet plants growing in an outdoor garden. A similar plot was treated with an 8-100 derris (4.5 percent rotenone) spray, and a third plot was left unsprayed as a check. When the plants were dry, six leaf samples (about 2 inches square) were cut at random from each plot and placed in as many Petri dishes for infesting in the laboratory. Five Hawaiian beet webworm larvae (fifth-instars) were placed in each dish, which was held at room temper- ature in the laboratory for observation for 72 hours. At the end of 48 and 72 hours the dishes were examined for mortality of the larvae and an estimate of the feeding on the - 7 - leaf sections. Similar samples were cut from the plants and fed to larvae in the laboratory every 2 days for 10 days. During this period the sprayed plots were exposed to all weather conditions other than rainfall, from which they were protected by covering. The results of these tests (table 3) show that a residue of g- phenylazoaniline remained effective against the Hawaiian beet webworm for about 4 days on beet plants but lost its effectiveness thereafter. As the compound is not very volatile, this loss in effectiveness would seem to have been caused either by growth of the plant exposing fresh tissue or by a change in the residue through the action of sunlight or other weathering factor. Its effectiveness, however, was equal to that of derris, which caused little mortality after the first 2 or 3 days although causing some repellency throughout the 10-day period. Table 3. — Results of tests with leaf samples taken at 2-day intervals from beet plants sprayed in the garden with p-phenylazoaniline and derris and fed to nearly full grown larvae of the Hawaiian beet webworm in Petri dishes een P-Ph Feedin enylazoaniline Derris 14. 5| rotejionel Feeding Check (unsprayed) . Time betvi Feeding spraying and after Kill after - after Kill aft. II_r_ after _Kill_after_=_ sampling 3 days 2 days 3 days 3 days 2 days 3 days 3 days 2 days 3 days Days Percent Percent Pe rcent Percent Percent Percent 0 Trace 30 73 Trace 43 67 Normal 0 0 2 do. 63 83 do. 50 73 do. 6 6 4 do. 10 40 do. 3 10 do. 0 3 6 Moderate 16 33 Moderate 0 3 do. 0 0 8 do. 6 30 do. 6 10 do. 0 0 10 do. 0 13 do. 3 10 do. 0 3 Miscellaneous Tests A preliminary test of ^-phenylazoaniline was made on the American cockroach by placing 10 nearly full grown nymphs in a 6-inch battery jar on a disk of paper rather heavily dusted with the pure compound. As the paper disk covered the entire bottom area of the jar, the nymphs were certain to come in contact with the powdered chemical. A film of vaseline about the inside upper edge of the jar prevented the escape of the roaches. The jar was kept at room temperature in the laboratory. Under these, conditions a deposit of 170 micrograms of dust per square centimeter killed none of the roaches in 3 days. A slightly heavier deposit of sodium fluoride killed 70 percent of the roaches under the same conditions. p-Phenylazoaniline was therefore not effective against the American roach, under these condi- tions at least. Tests were also made against the cowpea weevil and the rice weevil to find out the possible use of the compound as a grain protectant. A variety called Whippoorwill pea was used with the cowpea weevil, and wheat was used with the rice weevil . The seeds were treated by shaking 15 grams in a flask with a weighed amount of the insecticide until thoroughly mixed. The treated grain was then placed in a Petri dish containing 30 adult weevils and held at room temperature for 3 days. A concentration of 1 part of p-phenylazoaniline to 1,000 parts by weight of peas caused no mortality within 3 days, although in a similar test derris killed 100 percent of the weevils in 2 days. A concentration of 1 part of g-phenylazoaniline to 200 parts of wheat killed none of the rice weevils within 3 days. Derris. in a similar test, killed 36 percent of the weevils. It is apparent that g- phenylazoaniline is not toxic to these weevils under the conditions of the experiment. The compound was also tested as a soil treatment against termites by a method described by Hockenyos (4) . A weighed quantity of the insecti- cide, according to the concentration desired, and 40 grams of sandy soil were ground in a mortar until thoroughly mixed and then poured into a 150-cc. beaker containing about 12 cc . of water and a little tissue paper. After the dry soil had absorbed the water, 30 adults or large nymphs of the worker caste were placed in the beaker, which was held for 3 days in a cabinet at 80°F. Under these conditions it was found that a concentration of 1-200 of the insecticide in soil killed only 50 percent of the termites in 2 days and a 1-1,000 concentration had practically no effect. Pyrethrum at 1-200 killed 100 percent of the termites within 48 hours. Summary p-Phenylazoaniline (g-aminoazobenzene) was tested against 12 species of leaf-feeding insects in comparison with standard insecticides generally used with these species. In preliminary tests as a dust on foliage the com- pound was about as effective as the standard insecticide against the Colorado potato beetle, the cross-striped cabbage worm, the Hawaiian beet webworm. and the melonworm. A volatility test showed the compound to be practically nonvolatile over a period of 5 days, which precludes the possibility of fumigating action. Spray concentrations of 8. 4. ?.. and 1 pound per 100 gallons, with saponin added as a dispersing agent, were applied to potted plants and tested against five species of insects confined by screen cages. In these tests 2-phenylazoaniline was equal or superior to the standard insecticide against the cross-striped cabbage worm, the Hawaiian beet webworm. and the melonworm. Spray concentrations of 8 and 4 pounds per 100 gallons applied to small field plots of tender truck crops caused slight to moderate injury to Swiss chard and pumpkin and little or no injury to bean and collards. Small field plots of beet plants were sprayed with an 8-100 suspen- sion of £-phenylazoaniline and leaf samples taken from the plots at 2-day intervals for a period of 10 days and fed to Hawaiian beet wetv/orms in the laboratory. The residue was effective for about 4 days but became less effective thereafter. e-Phenylazoaniline was ineffective against the American roach, the cowpea weevil, the rice weevil, and termites when tested by the methods described. Literature Cited (1) Bushland, Raymond C. 1940. The toxicity of some organic compounds to young screwworms. Jour. Econ. Ent. 33: 669-676. (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. U. S. Bur. Ent. and PI. Quar. Cir. E-425, mimeographed. (3) Gahan, J. B., Swingle, M. C, and Phillips, A. M. 1941. 1.4"Diphenyl semicarbazide as an insecticide. U. S. Bur. Ent. and PI. Quar. Cir. E-549, mimeographed. (4) Hockenyos, G. L. 1939. Laboratory evaluation of soil poisons used in termite con- trol. Jour. Econ. Ent. 32: 147-149. (5) McAllister, L. C, Jr., and Van Leeuwen, E. R. 1930. Laboratory tests of miscellaneous chemicals against the codling moth. Jour. Econ. Ent. 23: 907-922. (6) Swingle, M. C, Phillips, A. M. , and Gahan, J. B. 1941. Laboratory testing of natural and synthetic organic sub- stances as insecticides. Jour. Econ. Ent. 34: 95-99, illus. (7) Vivian, D. L., and Haller, H. L. J. 1938. Insecticide. U. S. Patent 2,111,879; issued March 22. STAtE ."^''boabd UNIVERSITY OF FLORIDA illlillillllillllilllli 3 1262 09224 7443