LIBRARY STATE PLANT BOARD December 1948 E-763 United States Department of Agriculture Agricultural Research Administration Bureau of Entomology and Plant Quarantine TESTS WITH DDT ON HONEY BEES IN SMALL CAGES By A. W. Woodrow, Division of Bee Culture 1/ The rapidly increasing use of DDT against insects attacking crops visited by honey bees in their quest for pollen and nectar has caused much concern. Some serious losses of bees following extensive field applications of DDT have been reported, but for the most part this material seems to cause less injury than arsenic. However, the general effectiveness of DDT for destroying many species of insects raises the question whether DDT is also toxic to honey bees. Experiments on the toxicity of DDT to caged adult bees are reported by Eckert. (1), Filmer (2), and Hoist Q)« The experiments summarized in this paper give additional information. It is recognized that the behavior responses of bees in small cages differ from those of bees in normal colonies engaged in field activities. In addition to the confinement, the lack of a queen, combs, and colony organization, as well as changes in food, caged bees are subjected to treatments they would not undergo in their usual environment. Neverthe- less, cage experiments often give fundamental information that cannot be obtained otherwise. Experimental Procedure Experiments were conducted over a period of about 1 year with adult bees of unknown ages taken from the combs of their colony whenever required. Variations in bees used may have affected the uniformity of results of the experiments, but no differences directly attributable to this factor were observed. The bees were placed in cages 3 l/8 inches square and 5/8 inch deep, made of wooden frames covered on the under side with l/8-inch-mesh galvanized hardware cloth, and on the top with 12-mesh enameled wire screen. The wirw-cloth covers and bottoms of the cages were held in place by saw kerfs cut in the wooden side pieces, and they could be removed by sliding them horizontally. With this arrangement the bottom or floor could be opened for filling the cage with bees, or a treated floor, usually a tin or cardboard square of the same dimensions, could be substituted. The caged bees were kept on a laboratory table and were fed a 50-percent sugar sirup except as noted. Inverted specimen vials with a l/l6-inch hole in the cap were used as feeders. The temperature of the laboratory was maintained within the range at which bees remain active, usually 70° to 90 F. The relative humidity varied with daily and seasonal fluctuations. 1/ In cooperation with the University of California, O Q i - 2 - The studies included the toxicity of DDT in the food or when brought into external contact with the bees. The bees were fed DDT in sugar sirups queen-cage candy, or pollen paste; sprayed with DDT suspensions or solution 8 | dusted with DDT in talcj or brought into contact with surfaces which had been previously sprayed or dusted with DDT preparations. Special attention was given to external contact, since that appears to be the most likely method of poisoning from the use of DDT on agricultural crops. Technical DDT was used in the preparation of all materials used for feeding or spraying. The following preparations were used for the feeding tests: 1. A DDT suspension was prepared by dissolving DDT in hot 95-per- cent ethyl alcohol at the rate of 1 gram of DDT to 20 ml. of alcohol and then pouring the solution into cold sugar sirup. In some experiments the alcohol was removed from the sirup by evaporation before it was fed, to avoid alcoholic poisoning. Sirup containing DDT was fed individual bees with a micro syringe. 2. DDT was added to queen-cage candy and to pollen paste to the desired concentrations. Materials for spraying were prepared as follows! 1. DDT was dissolved in hot 95-percent ethyl alcohol at the rate of 1 gram to 20 ml. and then added to cold water to form a suspension. 2. DDT was dissolved in xylene to the desired concentrations* 3. Bnulsions of DDT were prepared with Triton X-100 (an aralkyl polyether alcohol), xylene, and water, according to common formulations. Sprays were applied with a small atomizer modified to use com- pressed air from the tank of a knapsack sprayer. This arrangement en- abled the application of small quantities of spray and provided a con- tinuous flow of air for spraying. For some of the experiments the caged bees were exposed to dried DDT residues on tin surfaces 3 1/8 inches square, by substituting the sprayed tin for the floor of the cage for a definite period and then re- placing the screen floor. For dusting, DDT diluted with talc was used. A 10-percent stock dust was diluted further with talc when less concentrated dusts were required. Where sulfur end DDT mixtures were used, sulfur was added to DDT dust in talc. Dust was at first applied from a small duster with the same source of air pressure as for spraying. In later experiments two other methods were used, as follows: - 3 - 1. The dust was poured into the cage onto the bees and then distrib- uted by shaking the cage. 2. The dust was placed on a cardboard 3 l/8 inches square and dis- tributed evenly by rubbing it with a small piece of smooth paper. The treated cardboard was then used as the floor in the cage. The last method was the most satisfactory one tried. Tests in which a sprayed tin floor was placed under the bees for definite periods showed much variability because of inadequate dosage control and because bees often remained in other parts of the cage during part of the exposure period. For the same reason larger dosages were used than would be present under usual field conditions. Response of Caged Bees to DDT Tremors and increased activity followed by paralysis are character- istic symptoms ofi DDT poisoning among caged bees. DDT in the food or in direct external contact with the body produces parti cally the same response. Extremely light doses of DDT may have little noticeable effect upon the bees. When they are affected by DDT, however, symptoms usually may be observed within a few hours after contact. Some bees completely re- cover from the mild tremors and restlessness produced by light dosages. Heavier dosages bring on intense tremors and frantic movements followed by paralysis and finally death, in which the victim usually lies on its side or back. Such violent response is often immediate, particularly among bees sprayed or dusted directly, and frequently they sting one an- other causing death. The time required to kill bees treated with DDT varies according to the dosage. With heavy dosages some may die within 1 to L, hours, and frequently all are dead within 18 hours. When the mortality is high, most of the bees die the first day. Bees that survive the first 2 days in good condition usually continue to be unaffected. Response to the same dosage varies considerably in different lots of bees. The factors governing this variation have not been determined, although disturbance at the time of exposure to DDT may be an important one. Bees dusted with DDT diluted with talc and those dusted with talc alone respond at once with increased activity and frequently resort to fighting. They immediately begin to clean the dust from their bodies and drop it. Many of the bees hang from the cover of their cage by their forelegs and remove the dust with their other legs. The visible dust may be removed within an hour unless the percentage of DDT is high enough to cause paralysis immediately. Later the bees and the cages appear to be completely free of the dust. - A- Effect of DDT in the Food The results of the experiments with bees fed DDT in sugar* sirup are summarized in table 1* The experiments in which individual bees were fed DDT in sugar sirup demonstrate that small quantities are poisonous when ingested with the food. Because the suspensions settled, it is believed that the bees actually ingested smaller quantities of DDT than are given in the table. Minimum lethal dosages were not determined. When the bees were fed collectively, the comparatively rapid settling of the DDT in suspension unquestionably greatly increased this concentra- tion at the feeding point. In other experiments 0.1 percent of DDT killed nearly all bees feeding on it in 24. hours, and 0.01 percent killed many bees* Pollen paste containing 5 percent of DDT fed to caged bees (table 2) killed them all in 2 days, and queen-cage candy with only 0*5 percent of DDT was equally lethal. Over 90 percent of these bees were killed in 17 hours. The greater apparent toxicity of the DDT in queen-cage candy is due to the fact that it was eagerly consumed, whereas little of the pollen paste was actually ingested. Most of the pollen paste was discarded and dropped from the cage by the bees. All bees were provided with water throughout the experiment* At the end of the first day the remaining live bees were fed 50-percent sucrose sirup. Effect of DDT Sprays and Spray Residues Some results obtained with DDT sprays are given in table 3« Most of the bees sprayed directly with xylene or water containing 1 percent of DDT died within 6 hours. The same sprays applied to the wood of their cages had no effect, presumably because much of it was absorbed by the wood. Sprays containing 4 percent of DDT applied to the floors of the cages killed nearly, all bees the first day. In later experiments (not reported in table) 1-percent sprays were equally effective. A sprayed tin surface was placed underneath untreated cages of bees to determine whether the DDT had any fumigating effect on the bees. The screen floor of the cage was about 3/32 inch above the sprayed surface. The bees did not appear to be affected in any way. On the sixth day the screen was removed, and the bees were allowed to come in contact with the sprayed surface. All bees died within 48 hours. No bees were observed attempting to remove spray residues within their cages. There was no indication that they could even detect the spray deposit. The experiments summarized in table 4 show the relation of spray dosage and the mortality of bees exposed to the residues. The 1-percent concentration was generally very toxic with exposures of 10 minutes or - 5 - more, whereas concentrations of 0.5 percent or less were much less so. The 0.1-percent sprays were comparatively safe for the bees. Concentration was more important than dosage and more important, within limits, than the exposure period. Table 5 shows the influence of the time of exposure on mortality. In an experiment to determine the persistence of DDT residual sprays, several lots of bees (3 cages in each lot) were exposed consecutively to surfaces bearing residues from single sprayings with a 1-percent DDT suspension* The results from two dosages--0.5 gram and 0.06$ gram per cage— are «hown in table 6. Only two bees in the first five lots exposed to the 0.5-gram dosage survived, but the 0. 065-gram dosage was appreciably effective only against the first lot. Since this experiment was carried out over a short period, the reduction in toxicity was not due to oxidation or evaporation. Effect of DDT Dusts Table 7 gives results of dusting caged bees with 10-percent DDT. The bees reacted violently to the treatment, becoming extremely active immediately. They cleaned the dust from their bodies. Nearly all the bees were helpless or dead in 3 to 5 hours. Sufficient DDT remained in the DDT-dusted cages to kill bees placed in them later. In the DDT-dusted cages that were restocked without further treatment all the bees were killed in 2 days. After a second restocking, the mortality was low. It has been reported that few bees may be found in fields of blossoming alfalfa recently dusted with DDT where bees were plentiful before dusting. Then after a few days bees again are numerous. Some observers have attributed this circumstance to the death of the bees in the field at dusting time, and others believe that the bees working in the field are driven from it by the dust. The tests summarized in table 8 were designed to show whether DDT is repellent to bees. Bees were placed in cages having only half the floor sprayed or dusted with DDT, so that the bees were free to move away from the treated area. Those placed in cages with half the floor sprayed with DDT walked upon the dried-spray surface as freely as on the unsprayed. There was no indication that they avoided the sprayed surfaces. Nearly all were helpless in 5 hours. On the other hand, in cages with half the floor dusted with DDT, the bees often walked into the treated area but then immediately turned away. Dust clinging to the feet was cleaned off and discarded. This type of repellency was more pronounced with DDT than with talc alone. In spite of the repellency, however, sufficient DDT was picked up by the bees to kill them in less than 18 hours. Relation Between Dust Concentration and Mortality An experiment was conducted in hope of finding a concentration of DDT dust which would not be harmful to bees but still of value in the control of injurious insects. As shown in tables 9, 10, and 11, various concentrations of DDT dusta were applied directly to the bees in the - 6 - cages until they were well covered with it. Dust was applied only to the floors of the cages in the tests shown in table 12, and the bees were exposed to the dust presumably only by walking in it. The 0.1- and 0.5- percent concentrations failed to kill even after 5 days of exposure. Except in the experiment summarized in table 9, where the reasons for difference are obscure, 1-percent DDT also was practically harmless, and it seems likely that under field conditions a somewhat more concentrated dust, perhaps 2.5 percent, would cause little mortality among bees because of the shorter period of exposure and the smaller quantities used. How- ever, 5- and 10-percent or more concentrated DDT dusts are very toxic to bees, even in small quantities, and unless care is used in their applica- tion to avoid the contamination of blossoms visited by bees, losses may be expected. There was no indication that sulfur in the dust mixture (table 12) had any effect on mortality within the cages. The lowest possible concentration of dust that will give reasonably satisfactory control should be used. Perhaps something less than com- plete control of harmful insects must be accepted for the sake of pre- serving beneficial pollinating insects. If dust of low concentration were used, more frequent applications could be applied with less danger to the bees. This practice would also minimize poisonous residues present at time of harvest. Relative Toxicity of DDT in Food, Spray, and Dust When fed in sugar sirup, from 6 to 12 micrograms of DDT per bee (table 1) was poisonous. Somewhat less than 12 micrograms per bee was sufficient to kill half the bees to which it was fed. Queen-cage candy containing 0.5 percent of DDT (table 2) killed 96 percent of the bees feeding on it within 21+ hours. Each bee consumed on an average 10.7 mg. of the candy, or 53*7 micrograms of DDT. Obviously the median lethal dose in queen-cage candy is much below this figure. As previously pointed out, 5 percent of DDT was required in pollen paste to kill as many bees as were killed by 0.5 percent of DDT in queen-cage candy because the bees did not eat the pollen paste readily. In general the more concentrated spray residues were more toxic than less concentrated ones, even though the dosage was the same. For example, 0.125 gram of 1 -percent DDT spray killed about three times as many bees as did 0.25 gram of 0.5-percent spray, although each presumably supplied 1.25 mg. of DDT (table 4). In a eomparison between sprays and dusts it is clear that sprays had greater residual action than dusts, but in initial action there seemed to be little difference. A spray deposit of 5 mg. of DDT per cage (table 6) showed much greater residual action than a dust deposit (table 7) contain- ing 25 mg. of DDT. Both were heavy dosages. The spray adhered tenaciously to the treated surfaces, whereas the dust was easily displaced. In initial action 50 mg. of 2.5-percent DDT dust per cage killed about half the bees (table 12). About the same result was obtained with 0.5 gram of a 0.5-percent spray (table A), and 0.125 gram of a 1-percent spray killed nearly 90 percent of the bees. - 7 - As was true of sprays, greater quantities of dust of low DDT content did not increase the kill as much as did increases of DDT concentration. Summary Bees in small cages were fed, sprayed, or dusted with various prep- arations of DDT and the resulting behavior and mortalities were observed. Immediate tremors and greatly increased activity, followed by paralysis and death in which the victim usually lies on its side or back, are characteristic symptoms of DDT poisoning among caged bees. Some affected bees recover from light dosages. Many bees were killed by 6 micrograms of DDT in sugar sirup when fed individually and by concentrations as low as 0.01 percent when fed collectively. Queen-cage candy containing 0.5 percent of DDT killed all bees to which it was fed. In pollen paste 5 percent of DDT was necessary to kill all bees. Bees sprayed with DDT in xylene or water were immediately affected, and all died in less than a day. DDT had no apparent fumigating effect on caged bees. Residues on tin surfaces sprayed with 1 percent of DDT in water killed most of the bees exposed to them for 10 minutes or more. With 0.5-per- cent sprays mortality was greatly reduced, and 0.1-percent sprays were comparatively safe for bees. Concentration apparently was of more impor- tance than quantity of spray applied and within limits than length of the exposure period. DDT-water spray residues, although not repellent, were persistent in effect, a single application killing nearly all bees in five lots consec- utively exposed to it. Bees dusted with 10-percent DDT or placed in dusted cages were killed, and some cages retained sufficient DDT to kill bees placed in them later. The dust, however, was not so persistent as spray residues. The bees removed DDT dust and talc from their bodies and also from their cage soon after dusting. Those dusted with 10-percent DDT died. Bees were not observed to clean dried-spray residues from their cage. Dusts containing 5 or 10 percent of DDT killed practically all bees that came in contact with them, whether the bees were dusted directly or only exposed to dusted surfaces. Dusts with 2.5 percent of DDT were less toxic, and those of lower strength usually had little effect. - 8 - Literature Cited (1) Eckert,' J. E. 1945. The effect of DDT on honeybees. Jour. Econ. Ent. 38: 369-374. (2) Filmer, Robert S., and Smith, Charles L. 1944* DDT as a contact poison for honeybees. Jour. Econ. Ent. 37« 537. (3) Hoist, E. C. 1944. DDT as a stomach and contact poison for honeybees. Jour. Econ. Ent. 37t 159. Table 1. — Mortality of caged bees fed DDT suspended in sugar sirup. t Quantity , of DDT , Cages i t . . Mortality in « 1 Bees f fed , i i i ^y , 2 days "T" t 5 days Number Number Percent Percent Percent Bees fed individually 6 micrograms 12 30 to 36 60 3 3 2 3 Bees 62 9.7 54 35.2 20 80.0 36 88.9 fed collectively ad libitum 14.5 44.4 85.0 88.9 29.0 55.6 100 100 0.5 percent 1 89 93.3 Checks 98.9 100 Water only Pure sirup 1 5 102 0 129 .8 45.1 .8 100 3.9 Table 2. — Mortality of caged bees fed pollen paste and queen-cage candy containing DDT. Material fed t i , Cages i i , Bees i , Mortality in — i » t i i , 1 day | 2 days f 5 days Number Number Percent Percent Percent Pollen paste: 5 percent of DDT 10 487 99.6 100 - 0.5 percent of DDT 10 503 7.2 44.3 48.1 No DDT 10 530 5.1 13.0 14.3 Queen-cage candy: 0.5 percent of DDT 10 448 96.0 100 - No DDT 6 322 0 0 0 Water only 5 360 20.6 32.2 32.2 - 9 - Table 3. — Mortality of bees sprayed with DDT or placed in sprayed cages or close to sprayed surfaces. Treatment t • i Mortality in — . Object sprayed ,DDT • Diluent "' Cages t ' Bees ' i i 1 day J 2 days f 5 days Percent Number Number Percent Percent Percent Bees 1 Water 5 423 100 m. - Xylene 1 53 100 - - Tin floor of cage 4 Water 3 167 99.4 99.4 100 Xylene 4 424 100 - - Wood of cage 1 Water 1 89 1.1 1.1 1.1 Xylene 1 78 0 0 1.8 Tin under cage with screen floor 1 Water 2 109 0 0 0 Untreated 5 520 0 0 0 Table 4.*— Mortality of bees exposed to spray residues of DDT applied at various dosages to tin floors of cages. DDT - 1 t Cages t i 1 Bees '- i i Mortality in i - Concentrati on t Dosage , 1 day » 2 days < 5 days Percent Gram Number Number Percent Percent Percent 1.0 0.5 49 2845 82.5 83.9 84.2 .25 51 3100 75.0 80.4 84.2 .125 25 1385 74.0 83.0 87.3 .0625 27 1464 68.0 72.8 76.8 .5 .5 22 1143 52.6 58.1 61.2 .25 21 1183 26.4 27.2 29.7 .125 30 1580 14.5 14.7 17.3 •1 .5 37 2260 11.7 16.5 19.5 .25 40 2322 2.5. 2.6 4.5 ■ .125 44 2571 1.3 1.6 4*4 .0625 8 626 .3 .6 1.6 None (untreated) 0 62 3498 .5 .9 2.3 - 10 - Table 5. — Mortality of bees exposed for various periods to spray residues of DDT applied to tin floors of cages* r_ — r™ " — 'T "' DDT • Exposure Cages Bees t Mortality in — concentration » i 1 day ' 2 days * 5 days - t t • ,1 • Percent Hours Number Number Percent Perp^it Percent 1.0 o.n 3 205 2.4 7.3 15.1 .17 3 160 80,6 90.0 91.3 .25 27 1505 37.8 41.3 49.0 .5 24 136i 62.1 72.4 76.3 1 24 14H 91.0 96.0 97.0 2 28 1696 88.9 92^7 94.2 3 12 637 89.5 92.9 94.7 4 13 716 95.5 99.6 99.7 6 3 144 100 8 6 362 99.7 99.7 99.7 y 9 597 99.7 100 - 0.5 .25 18 909 12.7 13,6 17.6 .5 21 1110 24.8 25.5 29.3 1 15 802 38.9 39.4 40.8 2 11 660 37.0 45.8 47.8 3 8 445 45.6 45.6 46.5 .1 .25 18 989 .7 1.1 3.7 .5 18 976 .6 1.1 3.9 1 18 869 3.2 3.5 6.2 2 23 1336 9.1 14.7 17.0 3 18 1092 5.4 6.1 9.9 4 17 1166 9.7 11.9 14.2 6 8 606 2.8 3.1 4.3 8 5 435 .7 .9 1.1 y 4 310 1.0 1.0 1.6 None (untreated) • 62 3498 .5 .9 2.3 1/ Continuous exposure. - 11 - Table 6. —Mortality of several lots of bees exposed consecutively to surfaces sprayed with 1-percent DDT. 1 Lot No. i Exposure i t t Bees i Mortality in t t 1 day i 2 days t 5 days Hours Number Percent Percent Percent At dosage i of 0.5 gram per cage 1 0.5 149 100 - - 2 2 176 100 — - 3 2 225 100 — — 4 2 220 98.2 99.1 99.1 5 8 168 100 _ — 6 2 230 30.4 35.2 36.1 7 2 204 0 0 .5 Series 1 At dosage of 0.0625 gram per cage 1 4 154 98.1 99.4 99.4 2 8 170 1.2 1.2 3.5 3 4 255 .8 .8 2.0 Series 2 1 6 ILL 100 - _ 2 8 U8 3.4 5.4 12.8 3 k 228 0 0 .4 Untreated checks: 1 (11 cages) - 604 .5 .7 .8 2 (2 cages) 3 (10 cages) - 116 0 0 5.2 • 738 .5 .7 1.4 - 12 - Table 7.~ Mortality of bees in cages dusted with 10-percent DDT and the residual effect of the DDT when such cages were again stocked with bees. 1 "f Treatment ' Cages * Bees i i t Mortality in — t t 1 day » t 2 days ' 5 days t Number Number Bees in cages heavily- dusted with 10- percent DDT 7 541 Percent 100 Percent Percent Cages restocked with bees without further treatment 267 52.1 100 Cages restocked with bees second time without further treatment 173 4.6 5.2 8.7 Bees in cages heavily dusted with talc 3 185 .5 .5 1.6 Undusted bees 3 273 .4 .4 1.1 Table 8. — Mortality of bees in cages having half the floor sprayed or dusted with DDT or dusted with talc. — - — 1 t Treatment , t Cages i i t Bees i t Mortality in — • 1 day 1 , ■ 1 2 days 1 5 days 1 Number Number Percent Percent Percent Sprayed with 1- percent DDT 2 136 100 - - Dusted with 10- percent DDT 2 120 100 - - DDT-dusted cages restocked with bees without futher treat- ment 2 163 2*4. 2.4 2.4 Heavily dusted with talc 1 76 0 0 2.6 Untreated checks 4 233 0 0 2.1 - 13 - Table 9.— Mortality of bees dusted with 100 mg.l/ of DOT in various concentrations in cages having tight tin floors. Concentration DDT of1 t t t i Cages t t Bees t i t t Mortality in . — (percent) 1 day- 2 days 5 days Number ftu«^ Percent Percent Percent 0.1 1.0 10.0 6 6 6 452 434 426 2.2 46.3 100 4.9 78.3 8.6 88.9 Checks: Talc Untreated 3 3 209 221 1 0 2.4 .9 3.9 3.2 \/ Equivalent to 140 lb. of dust falls upon the floor of the cage. per acre provided that all the dust Table 10.— Mortality of bees dusted with 200 mg. of DDT in various concentrations in cages having tin or screen floors. (5 cages used in each test) 10 in the check.) Concentration of , DDT ( (percent) , Type of cage floors t i r t , Bees ,- t > Mortality in — 1 day f i 2 days } 5 days 0.1 Screen Tin gUBfeex 478 447 Percent 2.1 1.6 Percent 2.1 1.6 Percent 4.2 2.5 .5 Screen Tin 487 458 1.8 3.1 2.1 4.1 2.5 4.4 1.0 Screen Tin 428 417 4.2 4.1 5.6 4.8 6.8 5.3 2.0 Screen Tin 417 uo 3.3 13.2 4.3 14.8 5.0 15.7 5.0 Screen Tin 388 428 85.8 100 '87.1 87.4 Untreated checks — .. 819 .6 .9 1.7 UNIVERSITY OF FLORIDA - 14 - 3 1262 09239 1969 Table 11. — Mortality of bees dusted with 250 mg. of DDT in various concentrations in cages having screen floors. (10 cages used in each test.) Concentre DDT tion of ' i it) • t Bees i t Mortality in — . (percei « 1 day 1 2 days i I i 5 days Number Percent Percent Percent 0.1 1.0 2.5 5.0 10.0 729 738 684 705 748 1.2 3.0 U.9 99.9 100 1.2 3.3 75.6 100 1.4 3.4 75.7 Untreated checks 742 .9 .9 1.1 Table 12.— Mortality of bees exposed continously to floors dusted with 50 mg. of DDT or DDT-plus sulfur in various concentrations. Concentratioi dust a of \ i r Cages i t ' Bees ' t i- t i Mortality in wmmt (percent) 1 day » t 2 days i i 5 days Number Number percent Percent Percent DDT 0.1 1.0 2.5 5.0 10.0 10 10 10 10 10 639 587 590 569 607 0.2 2.0 42.5 99.8 99.8 0.2 2.0 58.0 100 100 0.5 2.9 58.5 DDT 2.5 plus 75 sulfur 5 310 60.3 68.7 69.4 DDT 5.0 plus 50 sulfur 5 292 99.7 100 — Untreated checks 10 660 .2 .2 .8