' Compoxitional and Phyxiologzkal ' Rexlbonses of the Cottan Plant t0 the System ie I nreeticzkles Sc/omdan and Demeton gepfwnéa i955 ‘V551’ Bulleltn’ s21 TEXAS AGRICULTURAL EXPERIMENT STATION R. D. LEWIS. D | R E c T o R. C o L L E GE S T A T | o N, TEXAS SUMMARY Current systemic insecticides are organophosphorus compounds which, when applied to a porti 0f a plant, are distributed throughout the plant and kill insects feeding on any part. Cotton plants grown in solution cultures containing schradan accumulated the insecticide in su cessively lower concentrations in leaves, roots, bolls, petioles and stems. Relatively low concentratio of schradan stimulated vegetative development but higher concentrations were phytotoxic to both ve etative and fruiting activity. A Increased concentrations of chlorophyll and carotenoid pigments were directly correlated with sct radan treatment. It is suggested that this additional photosynthetic potential was related to increas in plant dry weights which occurred with the non-phytotoxic concentrations. ‘ As the nitrogen level in the nutrient solution was decreased, schradan was found to accumulate, the plant in significantly larger quantities. A similar but weaker trend was noted when the phosph us level was varied. The level of external potassium had little or no effect on schradan absorption. ' Plants grown in phosphorus-deficient solutions made significantly greater growth with than wit out added schradan in non-phytotoxic concentrations. Marginal leaf burning characterizing the l; phosphorus plants was not evident on those receiving schradan. e Neither schradan nor demeton, when applied to cotton as a foliar spray or when the former was t: sorbed by the roots, affected the viability of the seed produced. The schradan-treated plants produ seed which tended to be higher in protein and lower in oil than the untreated plants, while the dei ton-treated plants produced seed which were higher in oil and lower in protein. There was no eff - on phosphorus accumulation. ' Young bolls contained only 1 percent of the total schradan accumulated by the cotton plant, there was no evidence of increasedaccumulation through the addition of boron and sucrose. Emb ~ from plants grown continuously on schradan contained 1 to 10 p.p.m. of the insecticide. Attempts‘ determine the demeton in treated cotton plants by the cholinesterase inhibition method were not :_ cessful because of the presence of a natural inhibitor in the boll, possibly gossypol. A 5 to 12-fold” hancement of schradan was found after passage through the plant when measured by cholineste i activity. t Soluble nitrogen in the plant increased as the schradan concentration in the nutrient solution. f increased, but there was no effect on the protein fraction. At the highest level of insecticide used, t1 was a slight increase in phosphorus and a highly significant reduction in total sugars. The effect schradan on starch, which was increased substantially by the two higher concentrations, appeared ited to the root tissues. CONTENTS Page _ summary ________________________________________________________________________________ __ 2 Foliar Application of Schrndan and Demeton A Introduction 3 and Their Influence on Seed Properties and Re .e f """""""" "' '''''''''''''''''''''''''''''''' " 3 Chemiflll COHIPOSitiOII --------------------------------------------- v1 w o ........................................................... .. -. S d I d ............................................................. History of Development ............................................. .. 3 Gee .n EX i Absorption by Plants ________________________________________________ n 4 0311111113 10R ----------------------------------------------------------- -- Leaves ..................................................................... ._ 4 Pl """"""""""""""""""""""""""""""""""""""" " Stems 4 rotem ------------------------------------------------------------------ .- . Rt t """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" " 4 Ceebehydtetee ------------------------------------------------------ ' ' ' ' ' ‘ ' ' ' ’ ‘ ‘ ‘ ' ' ' ' ‘ ' ' ‘ ' ' ‘ ' ' ' ' ' ' ‘ ' ' ' ' ' ‘ ' ‘ ' ' ' ' ' ' ' ' ' ' ' ’ ' ' ‘ ' ' ' ' ‘ ' ' ' ' ' ‘ ' ' ' ' ' ' " 4 and Boron ______________________________________ u . Transfjgtgg; """""""""""""""""""""""""""""""""""""""""""" i‘ 4 Reeiddei Tcxicity edd Insecticide Ceittedt--~--. Persistence and Distribution of Schradan ........... .. I . _ _______________________________________ n 4 Mode of Actwn of schradan Influence of Schradan on Growth and ' Analytical MGthOdS --------------------------------------------------------------- -- 5 Development _____________________________________________________________ , schradan ------------------------------------------------------------------------- -- 5 Nutritional Factors Associated with the c Carbohydrates _______________________________________________________________ _. 5 Absorption and Accumulation of Schradan ...... Nitrogen _________________________________________________________________________ __ 5 Nutritive Value of Schradan Phosphorus ............ Oil ____________________________________________________________________________________ -. 5 Metabolic Intoxification of Schradan ..................... .. Other Constituents ....................................................... .. 5 Attempts to Determine Residual Insecticides inf Chloroplast Pigments .................................................. .. 6 the Developing B0“ by cholinesterase Act“.- 6 ity Measurements ............................................... Culture of Plantls": """""""""""""""""""""""""""""""" " 6 Chloroplast Pigments _____________________________________________ __ Nlltrl-lillt S0 utlon ---------------------------------------------------------- -- Influence of schradan on the Chemical Sdutlo" Culture ------------------------------------------------------------ -- 6 Composition ........................................................... .. Insecticides Used ------------------------------------------------------------------- -- 6 Discussion ......................................................................... ... Experimental Procedure ...................................................... .. 6 Literature Cited ------------------------------------------------------- ----- --.' i ompositionctl ctncl Physiological Responses of the Cotton Plant l to the Systemic I nsecticicles Schradan ctnclDemeton 1 JOSEPH HACSKAYLO and DAVID R. ERGLE* SYSTEMIC INSECTICIDE IS A COMPOUND WHICH ‘ absorbed and translocated by actively growing Slants in quantities sufficient to kill insects feed- 1 on a part remote from the point of application. ese materials not only have a residual effect, ut they also make toxic to insects portions of the lant heretofore left unprotected by the conven- onal sprays and dusts. Since growing plants e continually producing new growth, insect pro- r tion is afforded the new growth by systemic ; tion from the older treated plant parts. Schradan and demeton are the only systemic secticides approved for use on food crops at the esent time; others are being tested for possible lease as safe insecticides. There are many ma- rials which may serve as excellent systemic in- ticides, but their high mammalian toxicity loperties eliminate them as plant protection emicals. Most of the early studies concerning these ma- rials were associated mainly with the biochem- l and toxicological problems relating to their e. Very little work has been done to establish ‘ at influence these materials have on the phy- logy of plants in general. _ The purpose of this investigation was to ob- 'n a broad spectrum of the growth and physio- ical responses of the cotton plant to schradan V» demeton, with most emphasis being placed on v former. REVIEW OF LITERATURE History oi Development . Although the subject of systemic insecticides pears to be relatively new, attempts to produce temic effects in plants from chemicals actually te back to the 17th century. The techniques .1 monly employed previous to the 1930’s con- fl ed of either introducing chemicals into the nslocation stream by hypodermic injection or ‘ filling bored holes in tree trunks with the de- d chemicals. The substances used were usu- _ apted from a dissertation in plant physiology submit- ,» to the faculty of the Graduate School of the A&M llege of Texas in partial fulfillment of the require- nts for the degree of doctor of philosophy. nt physiologists, Entomology Research Branch and eld Crops Research Branch, Agricultural Research Ser- e, U. S. Department of Agriculture, and Department j Plant Physiology and Pathology, Texas Agricultural f eriment Station, College Station, Texas. ally heavy metal complexes and phenolic deriva- tives. Since few favorable results were obtained, these methods for insect control were not gener- ally accepted. Craighead and St. George (6) gave an excellent review of this subject up to 1938. Neiswander and Norris (27) reported that sodium selenate was toxic to mites and aphids when present in plants upon which these insects were feeding. This method of insect control also was eliminated since selenium, even at relatively low concentrations, was found to be very toxic to higher animals. A major stimulus in the development of sys- temic insecticides came in 1935 when Schrader (33), working in Germany, noted that the esters of B-fluoro-ethyl alcohol had strong contact in- secticidal action. These compounds have the for- mu ae: F-CH2-CH2-0\ F-CHQ-CH F ,8» a wmg-cna-o F-CHQ-CHZ Additional studies by Schrader and Kueken- thal (cited in 33) showed that the methylals of B-fluoro-ethyl alcohol could penetrate actively growing plants and remain unchanged for long periods of time in the plant’s system. The for- mula for this compound is: and /O'cH2'cH2'F c112 ‘O - c112 - cs2 - F Since all of the foregoing compounds proved to be very toxic to warm-blooded animals, their use as systemic insecticides was abandoned. Another important compound synthesized was fluoro-phosphoric-acid-di-dimethylamide in 1941. The formula is: 0 (c3372 - "x1: F (CH3)2 - N/ Remarkable systemic properties were noted for this material. However, its toxic properties par- tially eliminated its use as a plant protection chem- ical. Since the mode of action of these materials was obscure at that time, Schrader was interested in determining whether fluorine was responsible for the toxic properties exhibited by these com- pounds. The above compound was placed in an aqueous solution of HCl in an effort to replace 3 the fluorine atom, and the following reaction en- sued: 0 - 0 (ca ) - N " " N - (ca ) HCl 3 2 \,P _ O _ P,/ 3 2 * 0 (cn ) - N " 2 3 2 \\P I (CH3)2 - N N - (CH3)2 - F + non (cn3)2 - n" 21E‘ The new compound proved to be octamethyl pyro- phosphoramide (OMPA, Pestox, schradan) . This chemical not only had excellent systemic properties but has recently been approved for use on certain crop plants, including cotton, to con- trol spider mites and aphids. Still another important systemic formulated was the diethyl thiophosphoric acid ester of ethyl thioglycol ether. This compound was marketed under the name of Systox (demeton) and has the proposed formula: s CH3 _CH2_\l| ,»P - 0 - CH2 - CH2 - s - cu on cn3 - cue - o 2 3 Absorption by Plants Absorption of systemics by plants occurs through leaves, stems, roots and seed. Reynolds (31) and Ripper (32) presented excellent reviews on this subject. Leaves The penetration of systemics into leaves was shown to occur by Kuekenthal (cited in 33) and all subsequent investigators. The rate of uptake varies with the age, type of plant and environ- mental factors. Heath and Llewellyn (cited in 32) found that visible light and near infra-red greatly increases the uptake of systemic insecti- cides, perhaps being associated with carbohydrate content of the plant. David (7) reported that 69 percent of the schradan applied to leaves of bean was absorbed in 14 hours, and Metcalf and March (24) found that 50 percent enter citrus leaves in 24-48 hours. t Stems Application of hanane to the bark of coffee trees was shown by Bond (1) to be very effective in controlling mealy bugs. J eppson (21) report- ed very effective control of aphids and mites on citrus by trunk applications of schradan. Met- calf and March (24) state that application of schradan to the base of orange seedlings was much more effective than solution culture treat- ments. Roots The plant root system is capable of readily ab- sorbing systemic insecticides from soil or solution culture. Casida et al. (3) reported that the pea plant absorbs significantly greater amounts of schradan from phosphorus deficient nutrients. Metcalf and March (24) grew lemon seedlings in solution culture containing 360 p.p.m. schradan and found that the distribution of the insecticide in leaves, stems and roots was 69, 20 and 11. per- 4 cent, respectively. These and many other inv gators have shown that systemics absorbe. the roots are translocated to all parts of the pl‘ Seed Ivy et al. (18) showed that cotton seed ab schradan in sufficient quantities to provide , and aphid protection for several weeks after p ing. Other investigators confirmed these 1 ings. "I Translocation When schradan was applied to the soil, ‘l ing and Metcalf (37) found that the sys moved to the younger leaves of the valentine -, The rate of upward movement in the stem‘ measured and found to be approximately 2 per hour. ' By the use of girdling experiments, w, (38) reported that the movement of P32 S' (demeton) in the lemon plant was principa the phloem when applied to the stem. Whe labeled Systox was applied above the girdles little downward movement was detected, l considerable upward movement resulted. p Systox was applied below the girdle, very; upward or downward movement resulted. ,7 indicates that the phloem is the chief aven transport when stem applications are made. . rate of movement in the lemon plant was- mated to be 2.5 cm. per hour. Metcalf and Reynolds (cited in 31) sp Acala cotton with P32 Systox and OMPA dan) and found that after 15 days, 2.3 perc the Systox and 19 to 24 percent of the OMP " translocated into the new growth. On root such as sugar beets and carrots, large amou OMPA were translocated into the fleshy tap while Systox remained in the aerial porti the plants. ‘ Metcalf (25) reported that cotton sprayed with P32 schradan at the rate of 1 per acre accumulated 167 p.p.m. schradan g cottonseed cake and 109 p.p.m. in the raw-l days after application. This indicates tha siderable amounts of this material are t, cated to the seed. ' Mode of Action of Schrcrdan In pure form, schradan is not toxic to" mals or insects. When applied to the skin I ministered orally, a subsequently formed 1 olic intermediate has been shown to be ve i to mammals (8). Estimates have indica -| 280 to 560 mg. would be lethal to man; ho daily doses of 25 mg. have been adminis Q humans for 3 weeks for treatment of My‘ gravis, giving beneficial results and produ pronounced toxic symptoms. _ In animals, schradan is metabolized liver to produce an active intermediate causes the inactivation of cholinesterase. result, acetylcholine accumulates and a w; '0n of the para-sympathetic nervous system en- '1 es, which, if severe enough, causes death of the nimal. q Since some insects are not susceptible t0 schra- qn poisoning, it has been postulated that resis- . qnt insects lack the enzyme system to convert A hradan to an active metabolite. It was reported 8) that the fore-gut, mid-gut, hind-gut, fat ‘dy, nerve tissue and cuticle of a resistant cock- ach could convert schradan, consequently the ove hypothesis should be amended. O’Brien and encer (28) proposed that resistant insects con- ‘irt most of the OMPA in the fat body, conse- ently little ever reaches the nerve cord un- ' anged. The relative inactivity of the converted hradan may be due to the inability of the me- ~ f1 olized material to penetrate the membrane ihich invests the nervous system. The half life monochloro-schradan is 40 minutes and if the hradan metabolite is similar, its half life may ‘ of the same order. Plants likewise are capable of converting sch- an to an anti-cholinesterase agent, which, ac- rding to Casida et al. (5), is identical to the ani- 1 and insect metabolite. Since plants have no 3 ous system, no toxic response similar to that und in mammals resulted from schradan. A The proposed plan (5) for the production of l‘ active intermediate is as follows: 0 O $ a o o " (c113), - 1K; ",n - (c1135 ‘U2 02+ - _ P . 1 _ / _ / \ .- (0835 n \N (6113),, (c113)2.- n n - (c113)2 schradan Monophosphoz-amide oxide of Schradan- r 2 2 we“ <2. <2 N» .,n\ ,n\ R\ I \ _ [P-O- CH3 4' HCl lP-O-P CH3 HCl n’ ‘u (ca) non \n cn non 33ml l 2H3P0“ .. _ 3 2 _ / 3 cs3 + ncno + 011311112 HCl This theory proposes that the phosphoramide 'de selectively combines with cholinesterase re- _ing in inactivation of the enzyme. .0 It also was reported by Casida et al. (2) that radan is capable of inhibiting chrymotrypsin. ANALYTICAL METHODS Schradan An adaptation of the method employed by Cas- f et al. (3) was used for the chemical determi- __'on of schradan. Approximately 0.5 gm. of id ground plant material was placed in a mor- containing 10 ml. of distilled water and und with pure quartz sand. After the plant ple had been thoroughly macerated, the water act was decanted into a 50 ml. volumetric k. The extraction procedure was repeated - times. The resulting plant extract and res- =0 was made to volume and centrifuged. Suit- able aliquots were made to 19 ml., to which was added 1 ml. of a 2 normal solution of NaOH, and hydrolyzed by heating in a water bath at 80° C. for 30 minutes. The hydrolysate was cooled and decanted into a separatory funnel containing 30 ml. of chloroform and the mixture shaken for 1 minute. After standing for a few minutes, the chloroform layer was collected in a 40 ml. cen- trifuge tube and centrifuged for 5 minutes at 2,000 r.p.m. Twenty ml. of the chloroform was introduced into a 12-inch pyrex test tube contain- ing several glass beads and evaporated nearly to dryness in a water bath under the hood. The resi- due was wet ashed with diluted H2804 and H202. The phosphorus content was determined and the amount present multiplied by 7.87 to give the amount of schradan present in the original ali- quot. Cholinesterase activity, using a purified en- zyme preparation (acetylcholinesterase, manufac- tured by Winthrop-Stearns, Inc.) was measured manometrically by using the Warburg apparatus as outlined by Casida et al. (3). Carbohydrates Determinations of the carbohydrate fractions were by methods given in detail by Eaton and Rigler (9). Nitrogen Total nitrogen was determined by the semi- micro Kjeldahl method (14). Soluble nitrogen was determined similarly and included all, of the nitrogenous compounds extractable from dried plant tissue with water at 80° C. Insoluble nitro- gen was obtained by the difference of the above two fractions. Nitrate nitrogen present in the water soluble fraction was estimated colorimetri- cally by the phenoldisulfonic acid method (14). q Oil Suitable quantities of seed were ground to pass a 20-mesh screen in a Wiley mill and dried in an oven at 80° C. for 24 hours. The material was weighed then introduced into an alundum thimble and extracted in a Soxhlet apparatus for 24 hours with petroleum ether. The petroleum ether was evaporated, the residual oil Weighed and the percent composition calculated. Other Constituents Boron was determined colorimetrically as out- lined by Hatcher and Wilcox (16). A modified Wolf procedure was followed for the determina- tion of phosphorus as outlined by Hall (14). Potassium determinations were made as fol- lows: 1 gram of dried plant material was ashed in a muffle furnace for 1 hour at 250° C., follow- ed by 4 hours at 550° C. Five ml. of HCl and 5 ml. of distilled water were added to the ash after cooling to room temperature. The solution was then brought to a volume of 50 ml. and filtered through number 12 Watman filter paper. The potassium content was determined with the Beck- 5 man flame photometer as outlined in Instrumental Methods of Analysis (39). Chloroplast Pigments The chlorophylls and carotenoids were deter- mined quantitatively on fresh leaves as given by Loomis and Schull (22) and Shertz (34, 35). Ab- sorption spectra also were determined on the ex- tracted chlorophylls and carotenoids by the Beck- man spectrophotometer. CULTURE OF PLANTS Nutrient Solution The basal nutrient solution used during these investigations contained millimolar concentrations of salts as follows: 6 Ca (NO3)2.4 H2O, 4 KNOB, 1 KH2PO., 1 KCI, 2 MgSO4.7H2O, and 1 NaCl. . The following trace elements in parts per million also were supplied: 5 p.p.m. boron, 0.5 p.p.m. manganese, 0.05 p.p.m. zinc, 0.01 p.p.m. copper and 5 p.p.m. iron as sodium sequestrian. Solution Culture Five-gallon glazed earthenware jars were fit- ted with removable wooden lids containing ten 1- inch holes, through which the plant root systems were suspended into the nutrient solutions. Ab- sorbent cotton was packed around the stem in each hole to support the plants. A continuous supply of air was forced through gas-diffuser stones to insure adequate aeration in the root zone. The pH of the culture solution was main- tainedat 5.8 throughout the growth of the plants by additions of nitric acid. INSECTICIDES USED The schradan used throughout this work was manufactured by the Monsanto Chemical Com- pany and has the following composition: Active ingredients ................... .- 90 percent Octamethyl pyrophosphoramide---- 70 percent Related organic phosphates ........ -- 20 percent Inert ingredients .......................... -- 10 percent Technical grade demeton (Systox), the other insecticide used in this series of investigations, was produced by the Chemagro Corporation. SEED INDEX AND CHEMICAL COMPOSITION OF WHOLE SEED AND SEED FRACTIONS FROM C EXPERIMENTAL PROCEDURE Foliar Applications of Schradan and Demeton ~ Their Influence on Seed Properties and Chemical Composition Since dementon and schradan can persist ‘ plants up to 3 and 6 weeks, respectively, it W0 be beneficial to determine (a) their translocat to, and persistence in, the cotton seed, and ( their effect on seed properties and chemical l; position. The following experiment was, the fore, conducted to investigate these effects .. was performed in the greenhouse during the F of 195:3. Five groups of Stoneville 2B cotton plants 1 . sisting of four plants per group were grown 3-gallon glazed jars containing fertile Hous Black clay. Beginning with the appearance the first flower, four of the groups were spra‘ weekly from September through December one of the following: . 1. 0.2 percent schradan 2. 0.2 percent schradan plus 2 percent sucrose and 40 p.p.m. boron 3. 0.1 percent demeton 4. 0.1 percent demeton plus 2 percent sucrose and 40 p.p.m. boron ' 5. Untreated The boron and sucrose additions were primarily to determine whether they enha translocation of the systemics from the leav the bolls, as had been observed with 2,4-D The spray applications were disconti when dehiscence of the first boll occurred. f; maturity, the seed cotton was collected, gi and the seed were examined for effects of. treatments on their chemical composition, vi ity and insecticide accumulation. These i are summarized in Table 1. Seed Index .. The seed index is defined as the weig grams of 100 seeds. Both schradan and de i reduced the weight of the seed as compared the seed of the untreated plants. The incof 4 TABLE 1. _ PLANTS SPRAYED WITH DEMETON AND SCHRADAN ALONE. AND IN COMBINATION WITH SUCROSE: BORON Seed index . Protein (weight oi mo) O/il. (NHs x 5J3) Phosphorus. Carbolgydrates. gms- o % o o S d Wh l Wh 1 Wh l Treatment coegts Embryos seeode segde Embryos segde Embryos Y??? Embryos Embryos — — — — — — — — — — Data expressed on a dry weight basis — — — — — — — Control 4 8 6.4 11 2 22.9 35.6 26.4 38.7 0.668 1.066 6.69 Schradan 4 4 5.6 l0 0 21.6 34.1 27.1 40.2 .698 1.050 5.73 Schradan plus boron - and sucrose 4.2 5.3 9.5 25.0 39.0 22.5 32.6 .688 1.021 7.28 Demeton 4.4 5.4 9.8 24.4 38.0 24.4 36.2 .694 1.046 6.96 Demeton plus boron and sucrose 4 3 5.1 9 4 25.1 39.9 21.7 32.9 .603 .950 8.31 6 'on of sucrose and boron into the sprays caused further decrease. To determine whether this uction was in the seed coat, embryo, or both, yices were obtained on these seed fractions. . ere a reduction in the weight of the whole resulted, there also was a corresponding re- ction in the weights of both the seed coats and _bryos. ylnnination 1 One hundred seed obtained from plants in each itment were planted in flats of sand in the yeenhouse. Germination counts made 2 weeks ter indicated that there was no significant in- uence of treatment on seed viability. Germina- 1» percentages were: control, 98; schradan, 0; schradan plus sucrose and boron, 94; de- ‘ton, 94'; and demeton plus sucrose and boron, -' A Schradan alone caused a slight reduction in l content of the seed but demeton increased the lfrom 35.6 (embryo basis) to 38.0 percent. Ad- ‘tion of sucose and boron to each of the insecti- de sprays caused an increase in oil content; both, compared wih the controls and systemics alone. otein _ Schradan increased slightly the protein con- of both the whole seed and embryo, but de- eton caused a decrease. In each instance, the dition of sucrose and boron to the sprays re- F lted in protein reduction, as compared with both 1 e control and insecticides alone. - rbohydrates The plants sprayed with demeton plus sucrose ‘ A d boron produced seed which contained the fghest carbohydrate level, while those sprayed 'th schradan plus sucrose and boron were next. emeton alone caused a slight increase and sch- - dan alone caused a slight decrease over the con- 1 0lS. hosphorus and Boron . A decrease in total seed phosphorus was found [J the demeton plus sucrose and boron treated lants, while the other treatments remained es- 'ntially the same. There was no significant in- _ uence of treatment on boron accumulation in I e seeds. esidual Toxicity and Insecticide Content i" Seed from each of the treatments were germ- f ated in flats of sand and the seedlings infes- d with cotton aphids, Aphis gossypii Glov. and ider mites, Tetrcmychus tumidus Banks. None I ‘j the seedlings was toxic to the test insects. " hemical tests for schradan also were negative. Persistence and Distribution oi Schradan As previously mentioned, schradan-treated lants may be toxic to aphids and mites for per- 'ods up to 6 weeks. Since the length of residual TABLE 2. SCHRADAN CONTENT OF 28-DAY COTTON PLANTS GROWN IN SOLUTION CULTURE Leaves Stems Roots Schradan Per Per Per Per Per Per in gram total gram total gram total Entire nutrient dry dry dry dry dry dry plant solution wt. wt. wt. wt. wt. wt. P.p.m. A11 values expressed in mg. per gram dry weight 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10 0.09 0.12 0.04 0.03 0.05 0.02 0.17 100 0.97 1.21 0.13 0.08 0.40 0.18 1.47 1000 8.73 9.16 1.08 0.55 4.59 1.61 11.32 toxicity is inadequately defined, an experiment was performed in the greenhouse to furnish in- formation concerning the exact length of time re- quired to render non-toxic to mites and aphids a known initial amount of this insecticide, when present in a vigorously growing cotton plant. Cotton seed of the Empire variety were plant- ed in metal flats containing sterilized builders sand on April 17, 1954. On May 1, 1954, 10 seed- lings were transplanted into each of twenty 5- gallon glazed earthenware jars containing nutri- ent solution. The plants were then separated into four groups consisting of 5 jars each (50 plants per group). Two weeks later, schradan was ad- ded to the solutions of each group to produce treatments consisting of 0, 10, 100 and 1,000 p.p.m. After the plants had remained in the treated solutions for 7 days, three plants from each jar per treatment were selected at random, partition- ed into leaves, stems and roots, and eachfraction weighed. Harvests were made in three replica- tions. The plant portions were then placed in a forced draft oven and dried for 24 hours at 78° C. The dried material was weighed and ground in a Wiley mill to pass an 80-mesh screen, then stored in a stoppered bottle. The total schradan content was determined on the harvested plant material and the amount pres- ent per plant calculated. Table 2 shows that the plants grown in 0, 10, 100 and 1,000 p.p.m. sch- radan contained 0.00, 0.17, 1.47 and 11.32 mg. per plant, respectively. Table 3 shows that ap- proximately 80 percent of the schradan absorbed through the root system by young cotton plants accumulates in the leaves, 5 to 16 percent in the stems and 12 to 14 percent in the roots. Concurrently with the plant harvest, one plant from each of four replicates per treatment was removed, the root system thoroughly washed with TABLE 3. SCHRADAN DISTRIBUTION IN 28-DAY COTTON PLANTS GROWN IN SOLUTION CULTURE Schrm Leaves Stems Roots dan in Total Schradan Total Schradan Total Schradan nutrient dry distri- dry distri- dry distri- solution wt. bution wt. bution wt. bution Gms. P.p.m. ‘X, Gms. ‘Y, Gms. ‘Z, 0 1.28 0.00 0.68 0.00 0.45 0.00 10 1.27 72.05 0.69 16.14 0.42 11.80 100 1.25 82.30 0.64 5.45 0.45 12.35 1000 1.05 80.93 0.51 5.87 0.35 14.20 TABLE 4. PERSISTENCE OF SCHRADAN IN THE COTTON FOR COTTON APHID POPULATION TO INCREASE PLANT AS MEASURED BY THE NUMBER OF DAYS REQ I, Schradan treatment (p.p.m.) 1 0 l0 00 1000 \ Du s Aphid Percent Aphid Percent Aphid Percent Aphid Percent T Y count mortality count mortality count mortality count mortali 0 112 0.00 113 0.00 111 0.00 117 0.00 3 114 1.791 81 28.32 38 65.77 0 100.00 i 6 267 138.391 135 19.461 2(93)2 98.19 0(103)2 100.00 ' 10 972 767.851 481 334.511 70 26.31 0(99)2 100.00 14 3 1617 1130.971 226 137.891 46 53.54 l 17 3 3 1125 1084.201 185 76.761 t 2U 3 3 3 366 269.691’ 24 3 3 3 1190 1202.201 . 1 Percent increase. 2 Reiniestation. 3 Complete infestation. de-ionized water, and transplanted into a 5-gal- lon glazed earthenware jar containing only nutri- ent solution. Two plants in each treatment were infested with cotton aphids, Aphis gossypii Glov. and the other two infested with spider mites, Tet- ranychus tumidus Banks. Since these plants were taken from the same substrate as the plants har- vested and analyzed for schradan, it was assum- ed that the plants used in the persistence study had the same insecticide content as the harvested plants. As the insecticide content of the plants in- creased, insect protection was found to extend over a longe period of time. The maximum time limit for aphid and mite protection was found to be approximately 18 and 14 days, respectively (Tables 4 and 5). To determine the distribution of schradan in cotton plants having young bolls, the following experiment was performed in the greenhouse dur- ing the summer of 1954. Two groups of Empire cotton plants were grown in solution culture as described earlier in this section, except that only one plant was cul- tured in each jar. Enough schradan was added to produce a treatment consisting of 100 p.p.m. On the ninth week, the plants were harvested and separated into old leaves, young leaves, petioles, flowers plus bracts, bolls plus bracts (1 to 10 days old), stems and roots. The fresh weight, TABLE 5. PERSISTENCE OF SCHRADAN IN THE COTTON PLANT AS MEASURED FOR SPIDER MITE POPULATION TO INCREASE dry weight and schradan content were deter I ed on all of these fractions. ‘ When schradan was expressed as mg. per ~ of dry weight, the insecticide accumulated in 5 cessively lower amounts in old leaves, yo leaves, roots, flowers plus bracts, bolls plus bra petioles and stems, Table 6. 1 Approximately 80 percent of the total .. radan absorbed was found in the leaves. t, old leaves contained more of the insecticide t y the young leaves when expressed either as J per gm. of dry weight or as percent distributi The roots contained the next highest amount, the insecticide in terms of total dry weight. radan accumulated in slightly smaller amou in the fruiting fractions than in the Woody A TABLE 6. DISTRIBUTION OF SCHRADAN IN 9-WEEK- COTTON GROWN IN NUTRIENT SOLUTI CONTAINING 100 P.P.M. SCHRADAN I Schradan Per gram Per total Schrad PM“ P“ dry wt. dry wt. distributt Mg. Mg. ‘X, Old leaves 0.52 12.80 65.95 ~. Young leaves 0.35 3.77 19.43 Roots 0.15 1.79 9.20 ' Flowers plus bracts 0.12 0.22 1.12 I Bolls plus bracts 0.12 0.19 0.98 l (1 to 10 days) Petioles 0.05 0.35 1.82 » Stems 0.04 0.29 1.15 '77 Total 19.41 A BY THE NUMBER or DAYS mso rj Schradan treatment (p.p.m.) 1 0 10 00 1000 k Spider mite Percent Spider mite Percent Spider mite Percent Spider mite Percent I Days count mortality count mortality count mortality count mortality; 0 75 0.00 50 0.00 48 0.00 49 0.00 Z 3 94 12.531 46 8.00 41 14.58 3 93.88 ‘ 6 171 128.001 55 1.001 33 31.25 2(52)2 95.92 10 438 484.001 108 116.001 152 216.661 771 24.071 , 14 1210 1513.331 880 1760.001 1025 2035.411 92 88.881 17 3 3 3 304 562.961 ‘ 2D 3 3 3 1223 2164.141 24 3 s s 3 1 Percent increase. 2 Reinfestation. 3 Complete infestation. 8 it‘; ons when expressed 0n a percent distribution SIS. Influence of Schradan on Growth and Development ._ Empire cotton seed was planted in quartz sand ntained in 16 tall four-gallon glazed earthen- p jars on April 24, 1954. Nutrient solutions ere supplied to each of the jars. On May 15, p weeks after germination, the plants were thin- to one per jar and the remaining plants sep- grated into four groups consisting of four jars Each group was then supplied with two . iters of nutrient solution containing either 0, 10, 00 or 1,000 p.p.m. schradan. The solutions were - hanged weekly. Supplementary de-ionized water as added to the cultures as needed to maintain ufficient moisture conditions. g The flowers were tagged and dated at anthe- is, while the bolls were tagged and dated at de- iscence. The tags from shed bolls were collec- f daily and recorded. The experiment was ter- inated on August 27 , 1954, and the seed cotton rvested, ginned, and its fiber and seed proper- ies determined. j The plants grown on the 0 and 10 p.p.m. treat- l ents showed no noticeable phytotoxic symptoms, hile those on 100 p.p.m. schradan developed ne- rotic flecking and slight cupping of the lower jves. The terminal leaves of the plants grown l cultures containing 1,000 p.p.m. schradan were rk green and appeared to be healthy. Marginal and interveinal necrosis, accompanied by consid- I able cupping of the lower leaves, occurred after in first Week on plants at this level of schradan. flfter the second week, severe necrosis and ab- ission of the lower-most leaves continually oc- l urred. The effects of schradan supply upon its con- in the foliage, vegetative and reproductive haracters, and on dry-weight production, are 'ven in Table 7. At the two higher schradan levels, there was decrease in the number of flowers produced and _ in increase in the number of leaves abscised. lants on the highest treatment level developed ore nodes but shorter main stems. An increase I dry weight was apparent over the control lants in those grown in 10 and 100 p.p.m. sch- dan and a decided decrease was evidenced in ABLE 7. INFLUENCE OF SCHRADAN SUPPLY ON LEAF j INSECTICIDE CONTENT, REPRODUCTIVE CHAR- ACTERS, AND DRY-WEIGHT PRODUCTION I hmdan content and Schradan in nutrient solution (p.p.m.) narzansnsmranaummnrmm 8 plant character 0 10 100 1000 g. schradan in leaves 0.00 2.13 13.19 41.16 l. o. flowers produced 33.7 33.5 30.2 12.7 o. leaves abscised 12.0 91.2 12.7 21.2 o. main stem nodes 22.7 24.0 23.7 26.7 eight oi main stem (cm.) 111.5 115.7 116.5 97.2 weight (gms.) 891.7 117.61 105.2 36.82 Significant at 5% level. Significant at 1% level. $ O ppm SOHRADAN IO PW" SOHRADAN - I00 ppm SOHRADAN - I000 99m SCHRADAN ' 8 I O >909‘ ll *5 IQ O I '6 . I T\x—-"”E 2 3 4 5 - 6 WEEKS FROM START U FLOWERNG l Figure 1. Influence of schradan on flower production and flowering time in cotton. the 1,000 p.p.m. plants. The total schradan con- tent of the leaves was found to be correlated with the supply. Figure 1 shows that flower production was not only reduced at the two higher levels, but also was extended over a longer period of time. There was no difference between the 0 and 10 p.p.m. plants in flower production or flowering date. Since cotton usually produces flowers until a full fruit load is set, the bolls on the 0 and 10 p.p.m. plants were located on the lower-most fruit- ing branches, while those on the 100 p.p.m. plants were distributed on fruiting branches along the entire main stem axis (Figure 2). An average of only one boll per plant developed on those grown on the highest insecticide treatment (1,000 p.p.m.). Not only were there fewer flowers produced and bolls set on the 100, and 1,000 p.p.m. treat- ment levels, but there also was an increase in the percent boll shed (Table 8). Relative fruitful- ness, as measured by Eaton (10), was inversely correlated with schradan supply. Even though there were more bolls produced on the 10 p.p.m. Figure 2. Fruiting activity oi cotton grown on 0. l0. 100 and 1.000 p.p.m. schradan. 9 TABLE 8. INFLUENCE OF SCHRADAN .ON SHEDDING AND RELATIVE FRUITFULNESS OF COTTON-l Repmdudive Schradan in nutrient solution (p.p.m.) character 0 10 " 100- 1000 No. flowers produced 1.35 134 121 51 No. bolls set 46 50 38 3 Percent bolls shed 65.92 62.69 68.59 94.12 Relative Iruitfulnessz 5.3 4.3 3.7 1.0 lResults based on four plants per treatment. 2Number bolls per 100 gms. fresh weight of leaf and stem tissue. ' level, there was a reduction in relative fruitful- ness over the controls because of the increase in dry weight shown in Table 7. There was no difference in maturation time of any of the bolls in any of the treatments. Total seed cotton and the seed cotton per boll were reduced as the level of schradan in the nu- trient solutions increased (Table 9). There was no apparent influence of schradan on the seed in- dex, but the lint index increased slightly with in- creasing treatment up to the 100 p.p.m. level. The lint index at 1,000 p.p.m. was below the other treatment levels, but higher than the controls. TABLE 9. INFLUENCE OF SCHRADAN ON THE YIELD OF SEED COTTON AND THE SEED AND LINT INDICES Item Schradan in nutrient solution (p.p.m.) 0 10 100 1000 Values as means per plant Total seed cotton, gms. 56.50 54.10 40.30 4.30 Seed cotton per boll, gms. 4.89 4.33 4.13 4.27 Seed indexl 14.90 14.70 15.00 14.402 Lint index?’ 5.42 5.79 5.98 5.632 1 Weight in grams oi 100 seed. 2 Results based on 64 seed. 3 Weight in grams of lint per 100 seed. The fiber data (Table 10) show a uniform trend toward a reduction in the length and fine- ness by schradan at the 100 and 1,000 p.p.m. level. The oil, phosphorus, protein and schradan con- tents of the seed embryos obtained from plants grown in each of the treatment levels were de- termined. Table 11 shows that oil decreases and protein increases as the treatment level increases up to 100 p.pm. The oil content of the embryos in the 1,000 p.p.m. treatment level was essenti- ally the same as the 100 p.p.m., however, the amount of protein in the 1,000 level was less than TABLE 12. COMPOSITION OF NUTRIENT SOLUTIONS AS MILLIMOLES AND MILLIEQUIVALENTS PER LITER1 l‘ INFLUENCE OF SCHRADAN ON FIBER P . TABLE 10. g ERTIES OF COTTON Fiber property > Schrifidan Length Fensitlh P'(inene 4 . s reng micr . nultrient Upper hall Mean (100 lb.per grams p so u lon mean (in.) (in.) sq. in.) inch) P.p.m. — — — Values as means per plant — —; 0 1.08 0.87 3.45 a 10 1.08 0.87 95 3.70 ; 100 1.06 0.85 94 4.00 1000 0.95 0.78 93 —1 llnsufficient cotton for test. the 100, but more than the 10 p.p.m. treatme No consistent trend was found in the phosphol content even though a slight increase in schra was detected in the oil-free residue as the tr, ment level increased. a Nutritional Factors Associated with the Absorption and Accumulation of Schradan’, It has been reported (3) that increasing le of phosphorus in the substrate cause correspo ing reductions in the amounts of schradan f sorbed through the root system of the pea pl- Since this work involved using levels up t0 times the amount commonly employed in nutri solutions, an experiment was designed to de mine whether the various nitrogen, phospho and potassium levels more commonly used W0 influence the insecticide uptake by the c0 g plant. TABLE 11. OIL. PHOSPHORUS. PROTEIN AND SCHR l,” CONTENT OF COTTON SEED EMBRYOS TAINED FROM PLANTS GROWN IN S n; CULTURES SUPPLIED WITH NUTRIENT S. TION CONTAINING VARYING AMOUNTS . _. ' SCHRADANl Schradan A in on Phos- Protein S85 nutrient phorus (NHa x 5.13) residu-l solution ; P.p.m. ‘X, ‘Z, "/0 P.p. -~ 0 36.4 1.145 34.3 0 10 35.5 1.118 35.4 1.3 _ 100 34.9 1.167 36.7 4.8 ‘E 1000 34.8 1.156 35.9 9.6 l; 1 Values expressed on a dry-weight basis. On June 8, 1954, 1-week-old Empire c0 seedlings were transplanted to solution cult containing three levels of N, P and K (Table 1 Each treatment contained 200 p.p.m. schra, The plants were harvested on June 22, 11954. _ 11 T°tal mini‘ Millimoles I Element equivalents ; of N. P or K Ca(NO.'.)2 KNO3 CaCl2 KH2PO4 KCl NGNOS NaH2PO4 MgSO4 Na fi high 16.0 6.0 .0 1.0 1.0 2.0 1 F ' Nitrogen med. 4.0 4.0 6.0 1.0 1.0 2.0 1 low 1.0 1.0 6.0 1.0 4.0 2.0 1 high 1.00 0.0 4.0 1.00 1.00 2.0 1 Phosphorus med. 0.25 6.0 4.0 0.25 1.65 2.0 1 low 0.0625 6.0 4.0 0.06 1.94 2.0 l high 0.000 0.0 4.0 1.0 1.000 . 2.0 1 f Potassium med. 1.500 6.0 1.0 0.500 4.0 2.0 l‘, low 0.375 6.0 0.375 4.0 1.0 2.0 l lPlus: 5 p.p.m. boron; 0.5 200 p.p.m. schradan. l0 p.p.m. manganese; 0.05 p.p.m. zinc; 0.01 p.p.m. copper"; 5 p.p.m. iron as sodium sequestrian." The plants grown in the high N-P-K solutions e growing vigorously at the termination of experiment and produced the greatest amount dry weight per plant (Table 13). The plants wn on the medium and low nitrogen, phos- rus and potassium series produced less growth the corresponding elemental content decreased. ttling was evident on leaves of the low and ium nitrogen plants; the leaves of the plants Wn on medium and low phosphorus and po- ium remained dark green. Chemical analyses for nitrogen, phosphorus potassium were made on plants harvested m each series. The amounts present in the nts were correlated with the external elemen- supply (Table 14). As the nitrogen supply i reased, there occurred an increase in schradan tent when expressed as mg. per gm. of dry ight. A similar but weaker trend was noted en the phosphorus supply was varied. The of external potassium had little or no effect y; schradan absorption. Nutritive Value of Schradan Phosphorus Since schradan contains 21.7 percent phos- irus, an experiment was devised to determine ether any of the phosphorus liberated from ‘A metabolized schradan in the plant can be uti- in growth. i‘ Cotton seedlings were transplanted into nu- lent solutions containing the following treat- nts: complete nutrient solution (81 p.p.m. P), ‘ rient solution minus P, and minus P nutrient ution plus schradan equivalent to the phos- orus content of the plus P solution (31 p.p.m.). * The plants were harvested 27 days later and ¢ctioned into leaves, stems and roots. The dry ight and total phosphorus content of each tis- 7e fraction was determined. The control plants were growing vigorously i, the termination of the experiment. Marginal rning of the leaves was evident on the minus P nts, but did not occur on the schradan-treated nts. As measured by the amount of dry weight pro- iced, the plants grown in the schradan solutions yoduced less growth than the controls, but more CENT MOISTURE OF 28-DAY-OLD COTTON I LE 13. HEIGHT. FRESH AND DRY WEIGHT AND PER- PLANTS GROWN IN SOLUTION CULTURES FOR 21 DAYSl A atment Height t wgitlght Moisture T Cm. Gms. Gms. ‘Yo gh N-P-K 19.5 9.17 0.99 90.29» idium N 19.4 9.42 0.79 90.79 N 17.9 7.09 0.95 90.99 ldium 1> 20.1 9.90 0.94 90.59 2w 1> 19.9 9.99 0.79 90.55 clium x 20.2 9.52 0.91 90.49 ow K 19.1 7.15 0.71 90.07 l values as means per plant. TABLE 14. NITROGEN. PHOSPHORUS. POTASSIUM AND SCHRADAN CONTENTS OF 28-DAY-OLD COT- TON PLANTS GROWN IN SOLUTION CUL- TURES FOR 21 DAYSl Treatment Nitrogen pphhogis Potassium Schradan High N-P-K 4.19 0.686 3.125 0.160 Medium N 3.82 0.662 3.295 0.1682 Low N 3.59 0.650 3.195 0.1863 Medium P 4.18 0.654 3.195 0.167 Low P 4.27 0.525 3.280 0.170 Medium K 4.14 0.694 3.175 0.164 Low K 4.20 0.695 2.392 0.165 1 All values as percent of dry weight. 2 Significant at 5% level. a 3Significant at 1% level. than the minus P plants (Table 15). Analyses for total phosphorus established the presence of a greater amount of phosphorus in the schradan- treated plants than in the minus P plants. How- ever, the schradan-treated plants contained much less phosphorus than the controls. Metabolic Intoxification of Schradan Hall et al. (12) found that chloroform extracts of bean plants treated with schradan were 700 times more effective in inhibiting cholinesterase activity than the original insecticide. Casida ct al. (3) reported that pea plants produce an 18 times enhancement of schradan as measured by chol- inesterase activity. Zeid and Cutcomp (41), how- ever, were unable to corroborate these findings. De Petri-Tonelli and March (30) and Ivy et al. (18) using insect bioassay found no evidence of intoxification of schradan in the bean and cotton plant, respectively, and concluded that these plants merely serve as carriers for the insecti- c1 e. Since the reports on this matter are controver- sial, an experiment similar to the one performed by Casida et al. (3) on the pea plant was con- ducted with the cotton plant. On September 9, 1954, 1-week-old Empire seed- lings were transplanted into four 5-gallon glazed jars containing nutrient solutions. On Septem- ber 20, 1954, schradan was added to each jar to produce treatment of 0, 10, 100 and 1,000 p.p.m. One week later, one plant from each treatment was removed, washed with de-ionized water, and macerated in a mortar containing Ringers buffer solution and a little quartz sand. The brei was TABLE 15. DRY WEIGHTS AND PHOSPHORUS CON- TENTS OF 34-DAY-OLD COTTON PLANTS AFTER 27 DAYS ON PLUS AND MINUS P AND SCHRADANl High P Low P Schradan Plant part Dry 5 P Dry P Dry P weight weight weight Gms. ‘Y, Gms. ‘X, Gms. ‘Z, Leaves 1.78 1.438 0.48 0.149 0.74 0.219 Stems, petioles .89 1.216 .10 .123 .22 .152 Root .83 1.441 .32 .125 .46 .180 Entire plant 3.50 4.095 .90 .397 1.42 .551 1 A11 values expressed on a dry-weight basis. 11 | | | r I I I o = Homogencne from schradan treated plants x = Schradun added to plant homogenate PEI? CENT llVH/B/T/O/V Cl?! R) OI & (ll m O O O O O 74 \ 5 >¢ 1 1 l 1 l l | Q 5O IOO I50 200 250 360 3250 400 450 560 550 500 CONCENTRATION OF SCH RADAN lN ppm Figure 3. Metabolic intoxitication of schradan as meas- ured by cholinesterase (Che.) activity before and after passage through the cotton plant. strained through a muslin cloth and the tissue- free homogenate was made to 50 ml. with the buf- fer solution. Schradan was added to similar un- treated plant homogenates to produce concentra- tions of 0, 10, 100, 300 and 500 p.p.m. Chemical and anti-cholinesterase activity determinations were made on all of the plant extracts and the data plotted (Figure 3). It was shown by chemical analysis that the homogenates from plants grown on 10, 100 and 1,000 p.p.m. schradan contained 2, 8 and 86 p.p.m. of the insecticide when diluted to 50 ml., and caused 4.8, 13.5 and 44.3 percent inhibition of cholinesterase activity, respectively. On the other hand, the schradan added to the untreated plant homogenates in the amounts of 10, 100, 300 and 500 p.p.m. inhibited the enzyme by 3.6, 14.5, 28.6 and 43.1 percent, respectively. _ Since 2 p.p.m. schradan from the treated plant homogenate produced a 4.8 percent inhibition and a 3.6 percent inhibition resulted in the untreated plant homogenate to which 10 p.p.m. schradan were added, the former was approximately 12 times as effective in inhibiting cholinesterase ac- tivity. At the highest concentration used, 86 p.p.m. of the insecticide in the treated plant homo- genate produced approximately the same amount of enzyme inhibition as 500 p.p.m. that were ad- ded to the untreated plant homogenate. These re- _ E GOSSYPOL Z SCHRADAN _ lllllllll DEMETON (D O Q O 3 U7 0 | C” O I 45 O 30- PEI? GENT INHIBIT/ON Olie m O '6 I no . oo CONCENTRATION m ppm Figure 4. IN VITRO inhibition of cholinesterase activity by schradan. demeton and gossypol. 12 sults indicate the formation of a cholinester’ inhibitor within the cotton plant which is more = fective than schradan per se. - Attempts to Determine Residual Insecticides the Developing Boll by Cholinesterase i Activity Measurements The results in the section on Metabolic Int) ification of Schradan have shown that a meas x of residual schradan in young cotton plants co ‘ be obtained by using the enzyme cholinester Since no suitable chemical method for the dete J ination of demeton has been devised, it w; thought that the use of this enzyme also mi provide a measure of accumulation of both ins’ ticides. A preliminary field experiment was l formed in the spring and summer of 1954 to l termine Whether demeton and schradan accu lated in the developing boll in measurable qu tities as determined by cholinesterase activity. On June 29, 1 week after flowering had s V ed, field-grown cotton plants in 30-foot rows wt sprayed with 800 ml. of one of the following y secticide treatments: schradan 0.2 percent t1 percent) ; demeton 0.1 percent (50 percent) ; control (untreated). .- Two days after spraying, previously tag bolls, 2 to 8 days old, were collected, washed =§ frozen. About 1 week later, the bolls were f; ed and the sap expressed in a Carved hydra a press. A portion of the sap was added to an etylcholine-cholinesterase system and the h: matic activity determined manometrically. i Natural inhibitors present in the bolls " found to impair the cholinesterase activity, drastically that no conclusions on the accum tion of schradan or demeton could be deduced _l Since gossypol may be present in the boll amounts up to 1.2 percent (11), it seemed of terest to know what its influence would be on ’ cholinesterase enzyme system. Solutions containing 0, 10, 100 and 1,000 r’ of gossypol, demeton and schradan were pre ed and the inhibition of the enzyme system termined on each material. ’ Demeton caused a 10 percent inhibition of - linesterase at 1 p.p.m. and 84 percent inhibi at 10 p.p.m. (Figure 4). Schradan, however, duced only a 23 percent inhibition at 100 p._ and 62 percent at 1,000 p.p.m. Gossypol als hibited cholinesterase, being 6 perceent at, p.p.m. and 93 percent at 1,000 p.p.m. When} percent inhibition was plotted against the l‘ the concentration, a straight line relationshil- isted between concentrations of 1 to 10, 10 to and 10 to 1,000 p.p.m. for demeton, schradan gossypol, respectively. Chloroplast Pigments During the course of this project, it wa ticed that plants treated with schradan app to produce greener leaves than those of the BLE 16. DRY WEIGHTS AND CONCENTRATIONS OF ~ C H L O R O P H Y L L AND CAROTENOIDS IN _LEAVES OF PLANTS GROWN 15 DAYS IN NUTRIENT SOLUTIONS WITH 0. l0. 100 OR 1.000 P.P.M. OF SCHRADAN I C111 h 11 Caroten- Dry weight ' .~ o crop _Y_ aids per 10 A, M1111moles1 fl, plants. gms. 1T - -— - — Results oi dry-weight basis — — — — — 0 0.97 1.08 0.37 22.3 10 1.03 1.15 .44 26.5 .1 100 1.09 1.22 .43 25.1 l, 1000 1.22 1.36 .50 18.0 fhlorophyll a (C55H12O5N4Mg). tated plants. Since an increase in dry weight ults in plants grown in non-toxic concentra- ns of this insecticide, an increase in chlorophyll " d photosynthesis was indicated. With the fore- .'ng in mind, chlorophyll and the associated ca- tenoid pigments were determined. gThe chlorophylls and carotenoids were deter- _ ed on fresh leaves which were obtained from grown as reported in the section on Per- tence and Distribution of Schradan. 1 As the treatment level increased, there was an rease in chlorophyll content of the leaves; how- er the percent of carotenoids in the 100 p.p.m. {e1 was slightly less than in the 10 p.p.m., but feater than the controls (Table 16). i Since it was clear that an increase in chloro- yll resulted with increasing schradan treat- nt, absorption spectra were determined on jse two pigments to establish their identity def- tely. a Figures 5 and 6 show that the characteristic ks of chlorophylls and the carotenoids, respec- “ely, were obtained. Two additional peaks were nd, one at 460 mu, and the other at 590 mu, ythe 1,000 p.p.m. chlorophyll curve. Influence oi Schradan on the Chemical 1 Composition ; Various chemical fractions were determined the dried plant material which had been col- ed and stored in stoppered bottles as described A- 0 ppm SCHRADAN x = I00 ppm SCHRADAN ‘ 9 = I000 ppm SCHRADAN ‘ \X._A _ = I . . =.:3*?-°<¢>f2 . . . 4W42s 45o 47s soo szs sso 51s e00 62s sso - WAVE LENGTH m "my 1 T s15 10o ._. — _ n-u- Figure 5. Absorption spectra oi the saponiiied chloro- lls extracted irom plants treated with schradan. A 1.02s - /\‘ - N\ A \/ El-OZO" %/-\ o - 0 ppm scMRAoAu “ o, o , x = I00 ppm SCHRADAN E Z \ A= aooo ppm SCHRADAN‘ ‘g o |.o|s - / - O g / 3 l.OlO ‘é . O 40o 42s 45o 4"rs soo slzs sso sis soo WAVE LENGTH IN my Figure 6. Absorption spectra oi the carotenoids (caro- tene and xanthophylls) extracted from plants treated with schradan. in the section on Persistence and Distribution of Schradan. The carbohydrate data (Table 17) show that total sugars in the leaves and stems increase as the treatment levels increase, except at the high- est concentration of schradan, where a decided reduction is apparent. It appears noteworthy that the control roots contained more total sugars than the roots of the 10 p.p.m. plants, but less than those harvested from the 100 p.p.m. schra- dan series. The roots of the 1,000 p.p.m. plants contained the least amount of total sugars of any treatment. The highest starch value in the leaves was found in plants grown in 10 and 100 p.p.m. of schradan, being followed in decreasing order by 1,000 p.p.m. and the controls. There was a de- crease in starch content of the stems as the treat- ment level increased, except at the highest con- centration of the insecticide. The starch content of the ro-ots followed the same order of accumula- tion as the total sugars in respect to treatment, ex- cept a high accumulation of starch was evident at the 1,000 p.p.m. level. TABLE 17. CARBOHYDRATE FRACTIONS OF COTTON PLANTS GROWN IN SOLUTION CULTURE CON- TAINING SCHRADAN Carbohydrate iractions Plant Schradan Total part treatment sugars Starch Total P.p.m. —Values as percent of dry weight- 0 1.70 7.08 8.78 Leaves 10 1.83 9.56 11.39 100 1.84 9.44 11.28 1000 1.162 7.77 8.93 0 2.98 4.00 6.98 Stems 10 3.27 3.57 6.84 100 3.841 2.911 " 6.75 1000 1.502 5.13 6.63 0 3.30 2.12 5.42 Roots 10 2.75 1.201 3.95 100 3.66 4.672 8.33 1000 0.732 4.182 4.91 1 Significant at 5% level. 2Signiiicant at 1% level. 13 TABLE 18. NITROGEN FRACTIONS. TOTAL PHOSPHORUS AND SCHRADAN CONTENTS OF COTTON PLANTS GROWN SOLUTION CULTURE CONTAINING SCHRADAN -~. Total Total . Plant Schradan No N Soluble Insoluble Total _, part treatment 3' nitrogen nitrogen nitrogen phosphorus schrada . P.p.m. — — — — — — — — Values as percent oi dry weight — — — — — — -+ 0 0.020 0.890 . 4.458 0.862 0.000 = Leaves 10 .019 .875 3.570 4.445 .902 .009 ’ 100 .018 .956 3.566 4.522 .940 .097 _ 1000 .0061 1.3372 3.392 4.729 1.1211 .873 p 0 0.183 1.414 2.086 3.500 0.567 0.000 I Stems 10 .195 1.564 1.924 3.488 .580 .003 a 100 .175 1.637 1.685 3.322 .554 .008 f 1000 .188 1.635 1.9‘11 3.546 .570 .108 _ 0 0.134 1.146 2.026 3.172 0.870 0.000 i Roots 10 .131 1.1251 2.562 3.687 .935 .004 1 100 .129 1.0281 2.593 3.621 .872 .018 i 1000 .1121 2.592 3.568 1.0131 459 a 1 Significant at 5% level. zSignificant at 1% level. Table 18 shows that the soluble and total nitro- gen fractions in the leaves increased as the treat- ment level increased, but no difference was found in the insoluble nitrogen fraction, except at the 1,000 p.p.m. level, where a decline was noted. Ni- trate nitrogen in the leaves and roots was inverse- ly correlated with treatment, while the total phos- phorus in the leaves and roots increased as the treatment level increased. The accumulation of schradan in the leaves, stems and roots was found to be correlated with the supply of schradan in the substrate. DISCUSSION Cotton plants, when grown to maturity in so- lution cultures containing a gradient of schradan content for varying absorption periods, accumu- lated approximately 80 percent of the insecticide in the leaves. Metcalf and March (24) reported similar findings for the lemon plant. Of the to- tal schradan detectable in the cotton plant, very small quantities were found in the roots and less in the stems and petioles. Only 1 to 3 percent of the total schradan was found in the young bolls and flowers, indicating that either small amounts are translocated to the fruiting structures, or that the insecticide is rapidly metabolized at reproduc- tive sites. If other systemic insecticides accumu- late in plant parts in the same proportions as shown for schradan, the control of leaf-feeding insects is very promising. Those that feed in other plant portions, such as fruiting structures, may be difficult to control by these insecticides. Although no translocation studies were con- ducted on schradan in this series of investiga- tions, it appears that the xylem is the chief ave- nue of acropetal movement. The basipetal trans- port should perhaps be investigated further, even though reports (19, 31) have shown that the downward movement results quite readily, possi- bly in the phloem. In no case were the seed, which were harves- ted from plants sprayed weekly with either de- meton or schradan, found to contain measurable amounts of the insecticides. Chemical determi- 14 nations for schradan, and insect bioassay on. seed extracts or of the seedlings, were foun be negative for both insecticides. Even seed ,, tained from the plants grown in solution cult ' containing continuous supplies of schradan found to have a maximum of 10 p.p.m. on‘ highest level (1,000 p.p.m.). No seed were f0 to produce seedlings which were toxic to mit aphids. Metcalf (25), however, showed that ton plants sprayed with P32 schradan at the of 1 pound per acre accumulated 167 p.p.ml this insecticide in the oil-free residue 43 days“ ter application. The raw oil was found to con 107 p.p.m. but this amount was reduced to i p.p.m. after the refining process. : Casida et al. (3) found that schradan ab ed by pea seed is not completely metabolized - ing germination. Similarly, Ivy (19) noted p cotton seed soaked for 2 hours in solutions ~ taining as low at 0.5 percent schradan prod ' seedlings which were also toxic to aphids p mites. The present investigation established 10 p.p.m. schradan, when expressed on a f'- weight basis, are more than adequate to kill t_ insects in leaf tissue. In light of Metcalf’s (25) , one wonders why none of the seedlings ' duced from the seed obtained from plants -:_ in these experiments contained enough resi insecticide to kill mites and aphids. It is beli from the work performed in this series of ex ments that very little technical schradan reaches the seed in the original form. , " It was shown (19) that cotton plants ma toxic to mites and aphids for 6 weeks or t; when seed treatments employing schradan t used. In the persistence study performed i present investigation, the length of time req to render the schradan inactive was not p w: tional to the concentration in the plant. Alt ' a tenfold increase in schradan was found be ‘ successive treatments, the length of persis did not increase proportionally. Table 19 marizes the insect bioassay study and shows- the maximum time for insect protection .5 than 3 weeks, even though 100 times the a t usually applied for insect control was used. § TTABLE 19. SUMMARY OF TABLES 4 AND 5 SHOWING * PERSISTENCE TIME OF SCHRADAN IN COT- TON AS MEASURED BY COTTON APHID AND ' SPIDER MITE BIOASSAY Time required ior Time required tor Schradan . . . . aphid population spider mite popu- i Schradan per plant to increase lation to increase P.p.m. Mg. Days Days 0 0.00 — — 10 0.16 5-6 4 100 1.47 12 7 1000 11.32 18 14 l Several explanations may be offered for the apparent reduction in persistence time found in this experiment. Probably one of the most im- jportant was the rapid dilution of the systemic in gthe actively growing plant. Metabolic decomposi- ‘ tion of the insecticide probably took place rapidly ‘since these plants were growing vigorously dur- ng the experiment. Although not investigated, ne wonders how much, if any, of the systemic as lost through transpiration and guttation. l eath et al. (17) reported that the loss of schra- an from plants by evaporation, root excretion and washing out of leaves by rain was negligible. he same authors also state that the rate of loss Al; remarkably independent of plant species and he main reason for the disappearance of the in- cticide is metabolic decomposition. It has been mentioned previously that pure l, hradan is relatively non-toxic to cholinesterase , tivity; however, once it is metabolized by plants “f; 12, 23) and animals (8), drastic inhibition re- lts, being more severe in the latter. Hartley 15) discussed the intensification of schradan nd proposed that the metabolic intensification f this insecticide in plants is of little or no im- rtance in both insects and mammalin toxicol- a . Casida et al. (3), however, suggested that sects require the intermediary action of the lant in converting this systemic to an active me- bolite which is then ingested by the feeding in- it where it ultimately combines with cholines- rase. It was shown (30) that when insect bio- says were used to measure toxicity, the Valen- A e bean was not instrumental in increasing the xic properties of schradan. Although some of e insecticide was converted to a more reactive f» in the plant, it was suggested by the same thors that the unmetabolized schradan was con- frted to the active form after ingestion by the a - ect. l As shown in the section on Metabolic Intoxi- tion of Schradan, the quantitative determina- 3: of residual schradan in young cotton plants made by using cholinesterase activity meas- ements. Cholinesterase inhibition of schradan A er passage through the plant was 5 to 12 times re effective in inhibiting cholinesterase activ- than before it passed through the plant. These lues compare favorably with those obtained by gsida et all. (3) who report 18 fold enhancement ; schradan by the pea plant. 7 Attempts to determine the presence of schra- pn and demeton in young bolls by similar chol- inesterase activity measurements were not suc- cessful because of the presence of natural inhib- itors. Gossypol, a constituent of the boll, was an effective inhibitor of cholinesterase. The results of the development study, though more detailed, are not the first report of growth stimulation by the insecticide schradan. Casida et al. (3), without citing data, stated that low con- centrations of schradan increased the dry weight and moisture content of pea plants. Cotton seed- lings from seed soaked in dilute solutions of the insecticide were observed by Tsi (36) to be lar- ger than the controls. A similar response of plants to other related organophosphorus insecti- cides has also been reported (13). The beneficial effect has been ascribed to (a) the available nu- tritive phosphorus of schradan (36, 40), (b) the bond energy of the P-O-P linkage (29) or (c) the effect of the insecticide on some enzyme such as phosphatase (3). Data from the section on Nutritive Value of Schradan Phosphorus show that small amounts of phosporus liberated from the metabolized sch- radan were used in growth; its effects being re- flected in the increase in dry weight over phos- phorus deficient plants. It was apparent, how- ever, that the phosphorus supplied from schradan could not replace an equivalent amount of inor- ganic phosphate, although the phosphate released from the insecticide was used to some extent in the growth of the plant. The role of schradan as a supplemental source of phosphorus must be minor, because when sufficient quantities of» phos- phorus are available in the nutrient solution, the additional amounts as supplied by minute quan- tities of schradan in the 10 p.p.m. treatments are not sufficient to account for the observed stimu- lation in growth. Neither was there any signifi- cant difference in the total phosphorus content of the plants of the two treatments (0 and 10 p.p.m.) . Figure 7 shows plants grown in complete nu- trient solution and others grown in complete nu- Figure 7. Cotton plants at 35 days in complete nutrient solutions without 0 and with 10 p.p.m. schradan. Photo- graphed 15 days aiter addition of schradan. 15 trient solution with 10 p.p.m. schradan added. The treated plants are obviously larger than those grown on nutrient solution alone. Data of the current study suggest the chloro- plast pigments as a possible factor in explaining the stimulatory growth responses of some plants to organophosphorus insecticides. For, in the ab- sence of other inhibitory or limiting factors, it logically follows that one result of an increase in these important pigments would be accelerated photosynthesis and a corresponding increase in carbohydrates; which in turn could result in in- creased growth (dry weight) . This was indicated in cotton by the increase in the dry weights and photosynthetic pigments at the 10 and 100 p.p.m. concentrations of schradan over the control. That a still greater increase in these pigments at the 1,000 p.p.m. concentration was accompanied by a reduction instead of an increase in dry weight in- dicates, of course, that some other equally vital reaction of photosynthesis was at the same time greatly inhibited, or that carbohydrate oxidation was accelerated without accompanying growth. Although little is known about the mechanism of the phytotoxic action of schradan, there is evi- dence that in high concentrations it hinders the activity of the leaf phosphatase (3). Since the enzyme acts to hydrolyze the phosphate linkage of both high energy and ester phosphate com- pounds, inhibition of its activity would reduce carbohydrate utilization in cellular synthesis, re- gardless of a high photosynthetic potential, to a level too low to sustain normal growth. The noted beneficial effects of schradan on vegetative development and photosynthetic pig- ments did not extend to the reproductive activities of cotton. All concentrations reduced the yield of seed cotton to less than that of the control through either a reduction in boll size alone ( 10 p.p.m.) or a combination of this effect with (a) a reduction in number of flowers and (b) increase in boll shedding (100 and 1,000 p.p.m.). Eaton (10) considers the number of bolls oer gm. of fresh leaves and stems (relative fruitful- ness) to be a more critical measurement of the tendency of the cotton plant toward reproductive versus vegetative growth than is the percentage of flowers that develop into mature bolls. Fol- lowing this criterion, Table 8 shows that the rela- tive fruitfulness values decrease stepwise with each corresponding increase in concentration of schradan. Although a part of a consistent trend, relative fruitfulness was reduced by the 10 p.p.m. concentration of schradan and further by the 100 and 1,000 p.p.m. concentrations. Our data are not extensive enough to support a conclusion that significance is attached to the reduction at the 10 p.p.m. level in weight of seed cotton per plant, or weight of seed cotton per boll, Table 9. In laboratory tests with cotton seedlings, Ivy et al. (18) found that 6 p.p.m. of this insecticide in nutrient solution gave complete kill of spider mites and cotton aphids. Plants of the present 16 study were continusously supplied with schra, and, in all probability, they accumulated the l secticide in concentrations above those that required for adequate insect control. It has l regarded essential, notwithstanding, that inf, mation be developed which permits conclusions -@ the phytotoxic activity of this important inse ‘ cide. As the schradan in the nutrient solutions ' creased up to 1,000 p.p.m., there was a prop tional increase in the schradan content of the c ton plant. On the other hand, if the nitrogen 1e, in the solutions was varied from low to high, =; the schradan supply held constant, the absorpti of the insecticide decreased with each increase; _ nitrogen level. Casida et al. (3) had repo I" that increasing quantities of phosphorus in substrate caused a reduction in the amount‘ schradan absorbed by the pea plant. Within range of phosphorus concentrations supplied f plants in the present investigation, no signific difference could be found in the amount of sc i dan absorbed at the high (31 p.p.m.) and low’, p.p.m.) phosphorus levels. The data presen by Casida et al. (3) also show no significant r ference in the schradan absorbed by pea pla grown in 5 and 40 p.p.m. phosphorus levels, wh, are comparable with those of this study. Th, treatment containing 320 p.p.m. phosphorus d nitely reduced the uptake of the insecticide, be -\ significantly different from the 40 p.p.m. seri The external supply of potassium apparently 7 no influence on the absorption of schradan in t investigation. In light of the data presented by Casida et U (3) on the influence of phosphorus on the abso, tion of schradan and the information offered the present study concerning nitrogen, it may that the absorption of this insecticide is recip cal with the supply of anions in the substrate. ditional data, however, are required to substa ate this view. Although numerous chemical determinati were made on young cotton plants and on seed ‘ tained from treated plants, only minor differe » for the most part were found. It is notewo k that schradan caused an increase in the seed g tein over the controls, while a simultaneous re tion in oil resulted. Demeton, howeverfprodu a reversal of this trend. The explanation for , opposite effects of these two materials is not cl _ since one would anticipate that these two org phosphorus insecticides would influence like chemical systems in the plant in a similar ner. The leaves of plants grown in solution cult g which contained 10 and 100 p.p.m. schradan ,_ slightly higher total carbohydrates than the of control plants; however, this difference ‘ not statistically significant. The total su were reduced at the highest schradan level (g p.p.m), while starch was increased. The p 2' _tained on this level exhibited considerable city and the growth of the plants was drasti- , reduced over that of other treatments. Ad- nal work correlating respiration with carbo- pk rate content in the plant_is required to inter- _p data of this nature adequately. » Soluble nitrogen accumulated in the leaves of plants grown on the two highest levels of schra- dan. The amount of nitrogen detected was more than that present in the schradan fraction, indi- cating that additional soluble forms from cellu- lar synthesis were accumulating. Protein nitro- gen, however, was not affected. 17 10. 11. 12. 1 3. 14. 15. 16. 17. 18. 19. 18 LlTERATURE CITED Bond, J. A. B. Trunk absorption of a systemic chem- ical. Bull. Ent. Res. 44: 97-99. ,1953. Casida, J. E., Allen, T. C. and Stahmann, M. A. Re- action of certain octamethylpyrophosphoramide deriv- atives with chymotrypsin. J.A.C.S. 74: 5548. 1952. Casida, J. E., Chapman, R. K. and Allen, T. C. Re- lation of absorption and metabolism of OMPA by pea plants to available phosphorus. Jour. Econ. Ent. 45: 568-578. 1952. Casida, J. E. and Stahmann, M. A. Metabolism and mode of action of schradan. Agri. and Food Chem. 1: 883-888. 1953. Casida, J. E., Chapman, R. K., Stahmann, M. A. and Allen, T. C. Metabolism of schradan by plants and insects to a toxic phosphoramide oxide. Jour. Econ. Ent. 47: 64-71. 1954. Craighead, F. C. and St. George, R. A. Experimental work with the introduction of chemicals in the sap stream of trees for the control of insects. J our. For- estry 36: 26-34. 1938. David, W. A. L. Insecticidal action studies with bis- dimethylamino-phosphonous anhydride containing 32 phosphorus. Ann. Appl. Biol. 38: 508-524. 1951. DuBois, K. P., Doull, J. and Coon, J. M. Studies on the toxicity and pharmacological action of octamethyl- phosphoramide (OMPA, Pestox III)- Jour. Pharma- col. Expt. Therap. 99: 376-393. 1950. Eaton, F. M. and Rigler, N. E. Effect of light inten- sity, nitrogen supply, and fruiting on carbohydrate utilization by the cotton plant. Plant Physiol. 20: 380-411. 1945. Eaton, F. M. Influence of growth “hormones” on boll retention by cotton plants. Bot. Gaz. 111: 313-316. 1950._ Guthrie, J. D., Hoffpauir, C. L., Steiner, E. T. and Stansbury, M. F. Survey of the chemical composition of cotton fibers, cottonseed, peanuts, and sweet pota- toes. U.S.D.A., Agri. Res. Adm., Bureau of Agri. and Ind. Chem. 1944. Hall, S. A., Stohlman, J. W. III and Schechter, M. S. Colorimetric determinations of octamethylpyrophos- phoramide. Anal. Chem. 23: 1866-1868. 1951. Hall, Wayne C. Morphological and physiological res- ponses of carnation and tomato to organic phosphorus insecticides and inorganic soil phosphorus. Plant Phy- siol. 26: 502-524. 1951. Hall, W.C. An outline of methods and procedures commonly used in plant physiological research. Tex- as A. and M. College System. Mimeo. 1954. Hartley, G. S. Organo-phosphorus insecticides. The behaviour of schradan in the plant. Chem. and Ind. pp. 529-532. May, 1954. Hatcher, John T. and Wilcox, L. V. Colorimetric de- termination of boron using carmins. Anal. Chem. 22: 567-569. 1950. Heath, D. F., Lane, D. W. and Llewellyn, M. Studies on commercial octamethylpyrophosphoramide. “Octa- methylpyrophosphoramide in the living plant.” J our. Sci. Food and Agri. 2: 60-73. 1952. Ivy, E. E., Iglinski, W. Jr. and Rainwater, C. F. Translocation of octamethyl pyrophosphoramide by the cotton plant and toxicity of treated plants to cot- ton insects. Jour. Econ. Ent. 43: 620-626. 1950-. Ivy, E. E. Control of insects and spider mites by translocated compounds. Ph. D. Dissertation. Texas A. and M: College. 1951. 20. 21. 22. 23. 24. 25. 2'6. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. Ivy, E. E. Chemical characteristics of phosphl compounds to kill aphids and spider mites by s mic action. Agri. Chem. 137: 47-51. 1953. Jeppson, L. R., Josser, M. J. and Comolin, J. O. p trunk application as a possible method of using s mic insecticides on citrus. Jour. Econ. Ent. 45: 671. 1952. Loomis, W. E. and Schull, C. A. Methods in p physiology. lst Ed. McGraw-Hill Book Co., New York and London. 1937. Metcalf, R. L. and March, R. B. Studies of the l‘ of action of parathion and its derivatives and t toxicity to insects. Jour. Econ. Ent. 42: 721- 1949. ~ Metcalf, R. L. and March, R. B. Behavior of methyl pyrophosphoramide in citrus plants. J a Econ. Ent. 45: 988-997. 1953. . Metcalf, R. L. Radiotracers in study of systemic secticides. Agri. Chem. 9: 33-35. 1954. Mitchell, J. W., Dugger, Jr., W. M., and Gauch, ' ‘ Increased translocation of plant-growth modif substances due to application of boron. Science i 354-355. 1953. i Neiswander, C. R. and Norris, V. H. Introductio/ selenium into plant tissue as a toxicant for insects ‘ mites. Jour. Econ. Ent. 33: 517-525. 1940. A O’Brien, R. D. and Spencer, E. Y. Metabolism of tamethylpyro-phosphoramide by insects. Agri. Chem. 1: 946-950. 1953. Oesper, P. Sources of the high energy content energy-rich phosphates. Arch. Biochem. 27: 255- 1950. ‘l de Pietri-Tonelli, Pietro and March, R. B. Rela of the activation of schradan in plant tissues i5 toxicity to insects and mites. Jour. Econ. Ent. 902-908. 1954. i Reynolds, H. T. Entomological aspects of sys l pesticides. Agri. Chem. 9: 28-31. 1954. » Ripper, W. E. Systemic insecticides. Paper Q the Third International Congress of Crop Prot on Sept. 17, 1952 at Sorbonne, Paris. Pest C0 Ltd., Cambridge, England. .. Schrader, G. The development of systemic in. cides based on organic phosphorus compounds. . chen-Briefe, Bayer Pflanzen-Schutz-Nachric_ (English edition) 4: 161-170. 1952. a Shertz, F. M. The quantitative determinatio chlorophyll. Plant Physiol. 3: 323-334. 1928. Shertz, F. M. The extraction and separation of ' rophyll (a and b), carotin and xanthophyll, pre I ary to their quantitative determination. Plant , siol. 3: 211-216. 1928. Y Tsi, Chao-Seng. Protection against aphids by treatment. Nature 166: 909-910. 1950. * i Wedding, R. T. and Metcalf, R. L. Translocati radioactive octamethyl pyrophosphoramide in‘ valentine bean plants. Bot. Gaz. 114: 180-189. f Wedding, R. T. Plant physiological aspects use of systemic insecticides. Agri. and Food t 1: 832-835. 1953. Willard, H. 11., Merritt, L. L. and Dean, J. A. j mental methods of analysis. 2nd Ed. D. Von fl rand Co., Inc., Toronto, New York, and London. ,, Wolfenbarger, D. O. Nutritional value of phos insecticides. Jour. Econ. Ent. 41: 818-819. 19, Zeid, M. M. I. and Cutkomp, L. K. Effects ‘ass with toxicity and plant translocation of three I: phate insecticides. Jour. Econ. Ent. 44: T 1951. ' [Blank Page in Original Bulletin] State-wide Research The Texas Agricultural Experiment Station? is the public agricultural research agency; Location of field research units in Texas main- of the State of Texas‘ and 1s one oi nmei tained by the Texas Agricultural Experiment . Station and cooperating agencies parts Of lhG TGXCIS College System [N THE MAIN STATION, with headquarters at College Station, are 16 subject-matter departments, 2 se departments, 3 regulatory services and the administrative staff. Located out in the major agricultural I of Texas are 21 substations and 9 field laboratories. 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