702
INDUSTRIAL AND ENGINEERING CHEMISTRY
rain, but there is often an optimum ratio of adhesive to toxicant that is required to obtain best results. Although spreading coefficients and viscosity measurements may serve to predict the performance of a spray in regard to wetting power and initial retention, rain resistance seems t o require an actual rain, or washing test, for its evaluation. LITERATURE CITED (1) (2) (3) (4)
Cupples, H. L., IND. ENG.CHEM.,27, 1219-22 (1935). Cupples, H. L., J . Agr. Research, 63, 681-6 (1941). Garman, Philip, Conn. Agr. Expt. Sta., BUZZ.485 (1945). ~ ~L. T.,and ~ Richardson, h ~c. H,, J~. E ~, ~ ~~ t ~ ~35,. ~ 911-14 (1942).
W. D.. and Feldman. A. J.. J . Am. Chem. SOC.. . , Harkins. . 44.. 2665-85 (1922).
(5)
Vol. 40, No. 4
(6) Holland, E. B., Dunbar, C . O.,and Gilligan, G. M., Mass. Am. Expt. Sta., Bull. 252 (1929). (7) Hopperstead, S. L., Agr. Chemicals, 2, 24-6 (1947). (8) Hoskins, W. M., and Wampler, E. L., J . Econ. Entomol., 29,13443 (1936).
(9) Isely, D., and Horsfall, W. R., Ibid., 36, 751-6 (1943). (10) Marshall, James, Wash. Agr. Expt. Sta., Bull. 350 (1937). (11) Moore, William, Minn. Agr. Expt. Sta., Bull. 2 (1921). (12) O’Kane, W. C., Westgate, W. A., Glover, L. C., and Lowry, P. R., N. H. Agr. Expt. Sta.. Tech. BUZZ.39 (1930). (13) Vermorel, V., and Dantony, E., Compt. rend., 154, 1300-2 (1912). (14) White, R. P., N. J. Agr. Expt. Sta., Bull. 611 (1936). Frank, and Hartzell, Albert, Contrib. BoWe Thompson ~ (15) l ,WilCoxon, , Inst., 3, 1-12 (3931). (16) Woodman’R * ” Ind‘, 49193-8T (1930)* RECEIVED November 22, 1947.
Biological Methods of Testing Insecticides Harold H. Shepard Insecticide Testing Laboratory, United States Department of Agriculture, Washington, D . C. Biological methods of testing insecticides are utilized to discover promising new materials in screening operations, to develop specific uses and formulations for practical application, to make fundamental studies of relative toxicity and mode of action, to provide quality control in commercial production and governmental regulation,‘and to supplement chemical analyses with biological assays. Because of the variables which must be controlled when the reactions of living organisms are measured, a thorough understanding of insect biology is necessary for the most effective biological testing of insecticides.
I
N RECENT years intense interest has been aroused by the successful development of synthetic organic insecticides capable of being produced in large quantities by the chemical industry. As a result tens of thfousands of new compounds are now being scrutinized by biological methods for possible insecticidal value. SCREENING
Biological methods of testing insecticides may be classified appropriately on the basis of the objectives to be gained in their use. Given a series of new compounds entirely unknown as t o their potentialities in this field, it is necessary fist t o screen out those which show sufficient promise t o be studied further. This process of screening is accomplished by a variety of techniques, many of which are tests directed toward specific practical applications rather than ltoward insecticidal activity in general. As a consequence, it is likely that valuable insecticidal chemicals are overlooked because the screening test applied is not sufficiently broad. The injection of test fluids into the blood stream of 8 common large insect such as the American cockroach is a recommended preliminary or basic screening technique. This method eliminates the influence of differences in surface activity, penetrability, and other properties of the body wall, digestive tract, or respiratory system of difTerent kinds of insects. Such factors are largely responsible for the specificity of insecticidal action and in the early stages of studying a series of compounds should be bypassed. Equipment for injection of insects may be a tuberculin syringe and a calibrated micrometer head, or it may be a calibrated capillary tube drawn out to a fine tip and equipped with a rubber tube or bulb for the application of pressure (fa).
Another approach t o the screening of potential insecticides is a sorting scheme made up of a series of combinations of cage tests which indtcate fairly well whether a compound has much promise as a stomach poison, a contact insecticide, a fumigant, or a repellent, or various combinations of these (23). The steps in the scheme read like a miller’s flow sheet. I n the end one can say for what particular purpose a promising compound should be tested further. Settling tower methods are commonly utilized for either dusts or sprays, both in preliminary screening operations and in the more fundamental study of practical formulations where carriers and additives may all influence the behavior of the primary toxicant compound. I n the use of a settling tower the object is to produce as uniform a cloud of dust or spray particles as possible, so that a number of test objects (such as leaves, glass slides, or insects) will receive simultaneously a fairly uniform deposit of insecticide. The insecticide is dispersed by means of an air line opening sometimes at the top of the tower ( 2 4 , sometimes at the bottom (12). I n all cases a check on dosage is made by weighing pieces of glass or paper of known area exposed t o the same deposit as the‘ test leaves or insects. By modifying the equipment and standardizing the procedure it is possible t o reproduce deposits within reasonable errors between different sprayings or dustings, or to vary dosage in a predetermined manner. Considerable advantage is claimed for the horizontal or Hoskins type of spray tunnel. I n this apparatus a mist is intrcduced at one end, while below the other end is attached a cage of insects upon which the spray settles. Being sprayed horizontally, the insecticide appears t o fall in a manner resembling actual practice more than when a perpendicular tower is employed. For this type of work results possess a high degree of uniformity (18). For the sake of even distribution of insecticides many methods involve turntables (f4)and conveyers upon which test objects are exposed to a spray from precision sprayers installed in B fixed position (9). Results, however, are usually not so reproducible as in a settling tower. Many organic compounds have been tested by the Siegler appleplug technique, whereby newly hatched codling moth larvae must penetrate a spray residue if they are t o eat their way into their normal food, the fruit of the apple (81,26). Because of differences in the susceptibility of various groups of these larvae,
’
April 1948
I N D U S T R I A L AND E N G f N E E R I N G C H E M I S T R Y
lead arsenate is usually employed as a stahdard of comparison. Although this appears to be a specialized technique, it is valuable as a rather general screening method because both stomach poisons and contact insecticides will produce positive results. Volatility of a compound suggests its investigation for use where fumigant action is an asset. This may be in space fumigation or in the treatment of stored products or soil; it may be in such preparations as dusts for aphid control and roost paints for application against poultry lice. Generally the dosage of a volatile compound is based on the cubic space treated rather than the deposit of insecticide per unit area or the amount consumed by a n insect. For their behavior in the absence of soil, grain, and other absorptive materials, potential fumigants are tested in large glass jars (20) or in chambers connected t o a measured flow of the gas (6). The armed forces in the late war required effective insect repellents to protect them from insect-borne diseases. As a result large numbers of potential repellents were screened and much interest continues in this field. I n testing repellency the chemical must overcome the attractiveness of the insect's food source. I n order to test mosquito repellents human subjects therefore play an important part. The various species of mosquitoes and other biting insects differ in thek reaction to repellents. I n fact, repellency tests are extremely sensitive and the test insects must be checked constantly against a standard of comparison to avoid errors in interpretation of results. COMMERCIAL DEVELOPMENT
After a compound has been shown to be sufficiently promising to warrant further study, specific uses are sought and then formulations of the compound developed which will best serve those uses. Although laboratory information is of the utmost importance, extensive comparative tests based on such fundamental knowledge must be carried out under practical conditions. These are set up first on a small scale, as in gross feeding tests in screen cages, then enlarged by steps until finally tests are made on a full-sized commercial scale with power equipment. Not only must the new compound control a given insect pest as efficiently as insecticides already in use, but it must present no undue hazard to the crop or other material treated or to man or animals which may be exposed to the insecticide.
'103
the surface of an insect a measured droplet of toxkant with a calibrated micropipet and record the time to the initial reaction or until death takea place (15). Recently the amount of spray which adheres to an insect while flying through an insecticidal mist has been studied and correlated with the natural activity of the insect and with the activating capacity of certain insecticides (6, 7 ) . Whereas the insecticidal action of contact sprays is often reported in terms of the strength of spray (as milligrams per milliliter of spray) that kills under a given set of conditions, the measurement of the actual amount of insecticide in contact with, or at least adhering to, the insect should be a more fundamental figure. Dosages of aerosol mist sufficiently small to obviate the need for a relatively large test room are accurately measured by spraying the aerosol against a swinging shutter in which a small hole allows the mist to pass through into the test chamber during a fraction of a second for each swing (11). In fumigant. research little attention is paid t o the actual quantity of gas which passes into the respiratory system of the insect. In some studies, however, the absorption of gas by the insect body as a whole has been measured (4). The order of toxicity of a series of compounds, all applied in the same manner, is likely t o differ considerably for different species of insects. The specific action of insecticides is one of the most potent reasons for a thorough-going test routine in the development of new insecticides, and of fresh uses and new formulations for the older materials. For instance, the imported cabbage worm is easily controlled by dusts containing rotenone, whereas the cabbage looper, another caterpillar infesting the same fields of cabbage, is difficult to control with this insecticide. In laboratory tests using nearly full-grown caterpillars and determining the percentage kill on th8 fourth day after treatment, 0.15% rotenone killed 84% of the imported cabbage worms, whereas fully 20 times the strength of rotenone was required to kill only 64y0 of the cabbage loopers. Another example involves two rather closely related species of beetles, the striped cucumber beetle and the spotted cucumber beetle. I n laboratory tests in which the mortality of the adult beetles was determined on the fourth day after treatment, only 0.16T0 rotenone killed 75% of the striped species, but 4.0% rotenone (25 times stronger) killed practically none (only 4%) of the spotted beetles. The concentrations cited here would not be effective under field conditions.
FUNDAMENTAL RESEARCH
QUALITY CONTROL
A third objective of testing methods of mounting present-day interest is in the field frequently spoken of as insect toxicology. I t involves first the relative toxicity of chemical compounds on the basis of controlled dosage with respect t o insects and secondly the mode of action of those compounds upon the physiological processes of insects. An underitanding of the fundamental action of insecticides should form the basis first for more effective choice of compounds to be tested and then for more effective development of practical formulations for compounds of proved merit. The study of relative toxicity depends upon techniques by which measured doses are applied under controlled conditions to insects either hdividually or in standard groups. Methods by which individual doses of stomach poisons are administered orally ibclude ihjection directly into the esophagus of the insect (10)and feeding ih leaf sandwiches (3), in solid baits (19),and i n solutions which the iwect drinks (16). The feeding and drinkihg habits of the particular species with which dne is concerned must be well hderstood. Many insects do not respond well after beitlg handled or refuse to feed except a t ihfrequent intervals. Oral doses should usually be calculated t o tinit body weight, as is done in toxicological work with larger animals: To determine the relative toxicity of contact insecticides, screening techniques such as the settling tower methods are further refined. Another method is to apply to a given area on
The control of insecticide quality in the commercial production and the governmental regulation of insecticides is a fourth field for which test methods are developed. Perhaps the best known of these is the Peet-Grady method (17), a standard technique for. the evaluation or grading of commercial oil-base fly sprays (1). Tests under specified conditions of fly-rearing and testing are carried out with a standard comparison insecticide which is reformulated each year. To indicate how closely results may agree by this method, the comparison insecticides for two recent successive years gave, respectively, 43 and 44% kills of houseflies at the same dosage level. A method for the evaluation of cockroach sprays, which i a also rather widely used, employs the same standard, the Official Test Insecticide of the National Association of Insecticide and Disinfectant Manufacturers. Groups of 20 male German roaches &re sprayed in standard equipment. A series of tests is averaged to determine the killing efficiency of the test material in relation to that of the comparison insecticide tested a t the same time (8). The advent of dichlorodiphenyltrichloroethane (DDT) has brought about the development of a new type of contact insecticide, the residual type which kflls insects that alight on treated surfaces in contrast to the type which must be directed at the insect itself. Methods of making comparative tests of the persistence of residual effectiveness at the present date differ widely, but all inbolve the exposure of houseflies and sometimes
704
INDUSTRIAL AND ENGiNEERING CHEMISTRY
other insects to treated surfaces (13). An effort is being made t o select the most logical approach to a standard evaluation of such surface treatments (8). The commercial popularity of aerosol insecticides has likewise brought about a demand for a standard test method as a means of evaluating the performance of aerosol bombs. Most methods now in use simply require a test room in which to apply the aerosol against actively flying insects. BIOLOGICAL ASSAY
Still another objective of insecticide testing is biological assay as a supplement to chemical analysis. Such assays have been proposed for the gamma isomer of benzene hexachloride, and have been utilized as 3, check on chemical results in the study of the nature of t et raet hylpyro phosphat e. CONTROL OF VARIABLES
,
Other variables than the species of insect may be inherent in the insect or they may be a matter of environment. Related to the organism are the stage of the insect Tyith respect to life cycle, age within each stage, weight, and sex-all factors which can affect profoundly the results of controlled tests. Environmental factors of greatest importance are temperature, moisture or humidity, and quality and quantity of food. A thorough understanding of insect biology is therefore necessaiy for the most effective work in testing insecticides. Although the nonbiologist is likely to overlook the importance of variation in the insects used for test purposes, the biologist frequently underrates his ability to control the variables in biological test material. The' precision of individual assays is greater when the homogeneity of the test population is increased and &s the procedure is further standardized. The degree to which control of variation in test insects and method should be attempted depends upon the refinement which results warrant. Increased control requires more time and equipment. I t increases the quality of results at the expense of quantity. On the other hand, it provides more reliable data with fewer tests involving smaller numbers of organisms.
Vol. 40, No. 4
.LITERATURE CITED (1) hnon., S o a p Sanit. Chemicals, Blue Book, pp. 207-10, 1947. ( 2 ) Bottimer, L. J., S o a p S a n k Chemicals, 21 (12), 151-9 (1945).
(3) Campbpll, F. L., and Filmer, R. S., 4th Inte~nat.Congr. Entomol.. Trans. I I (1928), 523-33 (1929). (4) Carpenter. E. L.. and Moore, Wm., J . Econ. Entomol., 31, 2705 (1938). ( 5 ) Cotton, R. T., Pub. Am. Assoc. Advancement Sci., 20, 144-51 (1943). (6) David, W. A. L., Bzrll. Entomol. Research, 36,373-93 (1946). ( 7 ) David, W. A.L., - T a t w e , 155 (3929), 204 (1945). (8) Doner, M. W., S o a p s a n i t . Chemicals, 23 ( G ) , 139-43, 193 (1947). (9) Hansberry, Roy, P u b . Am. Assoc. Adzancement Sci., 20, 85-94 (1943). (10) Hansherry, Roy, Middlekauff, W. W., and Norton, L. B., J . Econ. Entomol. 33,511-17 (1940). (11) McGovran, E. R., and Fales, J. H., U. S.Dept. Agr., Bur. Entomol. Plant Quarantine, Circ. ET-239 (1947). (121 McGovran, E. R., and Maser, E. L., Ibid.,ET-208 (1943). (13) Monro, H . A. U.. Beaulieu, A. A . , and Delisle, R., S o a p Sanit. Chemicals, 23 (8), 123-9, 143-5 (1947). (14) O'Kane, W. C., Glover, L. C., and Blickle, R. L., New HampshireA4gr.Expt. Sta., Tech. Bull. 76 (1941). (15) O'Kane, W.C., Walker, G. L., Guy, H. G., and Smith, 0. J . , Ibid., 54 (1933). (16) Pearson, A. M., and Richardson, C. H., J . Econ. Entomol., 26, 486-93 (1933). (17) Peet, C. H., and Grady, A. G., Ibid. 21, 612-17 (1928). (18) Richardson, C. H., Pub. Am. Assoc. AdvancemenbSci., 20, 126-36 (1943). (19) Richardson, C. H . , and Seiferle, E. J., J . Econ. Entomol., 32, 297-300 (1939). (20) Shepard, H. H., Lindgren, D. L., and Thomas, E. L., Univ. Minnesota Agr. Expt. Sta., Tech. Bull. 120 (1937). (21) Siegler, E. H., and Munger, Francis, J . Econ. Entomol., 26, 43845 (1933). (22) Sieglcr, E. H., M'unger, Francis. and Gahan, J. B., Ibid. 27,11402 (1934). (23) Swingle, M. C., Pub. Am. Assoc. Advancement Sci., 20, 82-4 (1943). (24) Swingle, M. C., Phillips, A . hl., and Gahan, J. B., J . Econ. Enfomol., 34, 95-9 (1941). RECEIVED Kovember 22, 1947.
Use of DDT Insecticides on Food Products 0. Garth Fitzhugh ' Food a n d Drug Administration, Federal Security .4gency, Washington, D . C .
*
Because small amounts of DDT in animal food cause the storage of large amounts in animal products which are used in enormous quantities by man, the question of the safety of DDT on and in food products becomes critically important. Experiments with rats fed DDT over a period of 2 years are discussed.
T
HE general availability and effectiveness of DDT as an insecticide introduce the possibility of its widespread occurrence in food products. The most serious source of danger from ' the use of D D T is the repeated ingestion of small amounts that cling to forage, fruits, and vegetables that have been treated with this insecticide. The possibilities of acute poisoning in man from the consumption of food products sprayed with DDT are so limited that incidences of acute intoxication can be expected to occur only from gross carelessness. Damage to the livers of animals fed repeated doses of DDT has been observed extensively in laboratory animals (3, 4,5 ) . This damage usually consists of hypertrophy of the centrolobular
hepatic cells, increased oxyphilia of their cytoplasm with a slight hyaline appearance, and peripliei a1 segregation, basophilia, and increased size of the hepatic cell cytoplasmic gianules. In the more marked examples on higher-dosage schedules, there are focal hepatic cell necrosis and distortion of lobular architecture. The livers of chronically fed rats are enlarged as much as 60% and slices from these livers studied by standard Warburg procedure show a 40% decrease in oxygen utilization (3). The level of DDT intake which shows histopathological lesions in animals is much below that which shows significant gross effects such as retardation of growth and hyperexcitability. The author has observed slight damage to the livers of rats on levels of DDT in the diet as low as 10 p.p.m. However, no gross effects have occurred in rats fed less than 400 p.p.m. DDT. The pathological lesions observed have revealed wide individual variations in susceptibility to poisoning in a given animal species. Nevertheless, the type of lesion has been consistent throughout the different species. The storage of DDT in the tissues, especially in the fatty
