V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2
555
tinuously in the form of fine bubbles, making a very large liquidgas interface for efficient absorption with a small volume of solution. It is not necessary to use the Warburg apparatus for this determination. Any other manometric device which will detect small volumes of gas is satisfactory. Continuous shaking of the vessels is not essential, but equilibrium is attained much more rapidly and reliably if a shaking mechanism is used. Gas flow rates over the plant tissue must be large enough to prevent excessive accumulation of carbon dioxide or ethylene. The former will suppress respiration and the latter may accelerate it. Reagents. Placing a bulb containing a small amount of nbutyl alcohol upstream from the absorber maintains a constant low concentration of alcohol in the absorbent solution by replacing the evaporation loss from the absorber. High concentrations of butyl alcohol (above 0.5%) must be avoided to prevent interference t ~ i t hthe subsequent release of the ethylene. For example, when 4% butyl alcohol was added to the mercuric perchlorate reagent, the value of the blank was doubled, presumably because of a change in the partial pressure of the alcohol when the reagents were mixed. BJ- the use of two absorbers in series, it was found that the absorbent solution must be a t least 0.25 M in mercuric ion for complete absorption of the ethylene. On the other hand, mercuric ion concentrations above this value gave much higher blank values. Other mercuric salts for absorption, and other chlorides for ethylene release have been tested for use in this procedure and found unsatisfactory. Solutions made up with mercuric acetate in acetic acid, mercuric sulfate in sulfuric acid, mercuric nitrate in nitric acid, and mercuric chloride in perchloric acid either did not absorb ethylene as efficiently as mercuric oxide in perchloric acid or produced a higher pressure change (blank) on addition of
hydrochloric acid. The same blank values were obtained with hydrochloric acid and Kith lithium chloride, but sodium, potassium, calcium, or barium chlorides produced much higher blanks. Hydrochloric acid was used as a matter of convenience and economy. LlTERATURE CITED
Biale, J. B., Ann. Rea. Plant Phvsiol., 1, 183 (1950). Chiistensen, B. E., Hansen, Elmer, and Cheldelin, V. H., IND. ENG.CHEM.,ANAL. ED.,11, 114 (1939). Davis, H. S.,Crandall, G. S.,and Higbee, W. E., Jr., Ibid., 3, 108 (1931).
Denny, F. E., Bot. Gaz., 77,322 (1924). Denny, F. E., Conlrib. Boyce Thompson Inst., 9,431 (1938). Dixon, Malcolm, “Manometric Methods,” Iiew York, Macmillan Co., 1943. Gross, C. R., Cornel1 Univ., Dept. Pomol., Mimeo., 1947. Hansen, Elmer, Bot. Gae., 103,543 (1942). Hansen, Elmer, and Christensen, B. E., Ibid., 101,403 (1939). Hansen, Elmer, and Hartman, Henry, Oregon Agr. Expt. Sta., Bull. 342 (1935). Harvey, R. B., Minnesota Agr. Expt. Sta., Bull. 247 (1928). Keller, R. N., Chem. Rev., 28, 229 (1941). Nelson, R. C., Plant Physiol., 12, 1004 (1937). Porritt, S. W., Sci. Agr., 31, 99 (1951). Pratt, H. K., and Biale, J. B., Plant Physiol., 19, 519 (1944). Scholander, P. F., Xiemeyer, H., and Claff, C. L., Science, 112, 437 (1950).
Stitt, Fred, Tjensvold, A. H., and Tomimatsu, Y . , AKAL.CHEM., 23, 1138 (1951). Umbreit, W. W., Burris, R. H., and Stauffer, J. F., “Manometric Techniques and Tissue Metabolism,” Minneapolis, Burgess Publishing Co., 1949. Walls, L. P., J . Pomol. Hort. Sci., 20, 59 (1942). 1-oung, R. E., Pratt, H. K., and Biale, J. B., Plant Phusiol., 26, 304 (1951).
RECEIVED for review June 28, 1951. Accepted December 6,
1951. A cooperative project of t h e Division of Subtropical Horticulture and the Division of Truck Crops, carried o u t in the laboratories of the former division.
Bioassay of 1,2,3,4,5,6-Hexachlorocyclohexane (lindane) Some Factors Influencing the Contact of Chemical and Test Insect and Methods for Standardizing the Process W. M. HOSKINS, .I. IX. WITT,
AYD
W. R. ERWIN, University of Culiforniu, Berkeley, Calif.
Quantitative determination of residual insecticides in foodstuffs is becoming increasingly important because of extensive use of new and highly toxic organic compounds. Specific chemical methods often are unknown and biological assay must be used. The present report describes procedures for use of houseflies in bioassay for residues of lindane (1,2,3,4,5,6-hexachlorocyclohexane) and for removal of extractives from plant or animal tissues that interfere with the test. The residual lindane is collected upon the interior surface of a shell vial in which the flies are confined. Uniform contact be-
A
PREVIOUS comniunication (.$) described a method for microbioassay of insecticide residues in plant or animal tissues. It n-as found that the standard dosage-mortality curve obtained by adding known amount of insecticide to the produce being analyzed might show either higher or lower mortality for a given amount than when only pure insecticide was used directly. Thus the oils of egg yolk caused an additional mortality, but waxes such as those of cabbage greatly decreased mortality.
tween the insects and the chemical is secured by addition of a small amount of light oil to the vial. By use of the improved bioassay, very small residues of lindane and several other insecticides have been determined in oils and other fatty products that cannot be handled by customary procedures. A dependable and highly sensitive bioassay is useful not only for residue analysis but for determination of distribution of insecticides among body tissues of insects. I t is especially valuable in study of the distribu tion and degradation of absorbed insecticides by plants and resistant insects.
Uncertainty in the response of the test insects t o known amounts of an insecticide will cast doubt upon any result obtained with unknown samples, irrespective of how perfectly these may have been prepared and purified. Hence a more detailed study was undertaken of several factors which might have an effect upon contact between insecticide and test insects when persistent deposits are used, as in the small vial technique. The more obvious of these are the solid or liquid nature of the deposit,
ANALYTICAL CHEMISTRY
556
its uniformity over the exposed surface, length of exposure, portion of the insect's body reached, and opportunity for loss of the adhering insecticide. The present report gives data on these subjects, on use of the pickup of insecticide as a quantitative measure of contact, and on limited results obtained with processes for removing interfering extractives from samples used for analysis. MATERIALS AND METHODS USED
The commercial preparation of y-benzene hexachloride, known as lindane, was used as a typical insecticide in most of the work and DDT, aldrin, tetraethyl pyrophosphate, and parathion were used less extensively. Because of its inertness toward strong acids, favorable action in adsorptive columns, and easy removal during preparation of the toxic surface, the solvent of choice was petroleum ether, C.P. grade, distillation range 35' to 60' C., but chloroform, C.P. grade, and thiophene-free benzene also have been used. The exposure chambers were the shell vials previously described. These were rotated through 180" a t the mid-point of the test to increase uniformity of contact with the interior surface The acid treatment of extracts, the flies, and methods of rearing and handling them were the same as described before. In establishing the standard dosage-mortality curves and in determining the effect of the various processes thereon, known quantities of the chosen insecticide were added to the solvent before carrying through the treatments. I n many cases contaminants such as oils or other materials were added to simulate the situation with field samples and most of the work reported refers to such lahoratory contaminations, as their nature may be varied as desired. Extracts of practical samples could be used without treatment when only small amounts of fatty materials were present, as in the case of spinach tested for external residue. Usually either acid treatment or passage through a column, or both, was needed. The appearance of the vial after a treated extract has been evaporated is a fairly reliable indication of whether the treatment has removed enough of the interfering materials to permit an accurate bioassay. If a film is barely perceptible to the unaided eye and it proves to be a mobile liquid-e.g., by stirring under the microscope with the point of a pin-the conclusion may be drawn that conditions are favorable. A clearly obvious film indicates either more oil or a semisolid, perhaps a solid, deposit. If it appears to f l o when ~ the vial is tipped, there is too much oil for a bioassay. A solid film readily shows its state Then scratched. If very thin, addition of 1 or 2 cu. nim. of light oil in a little petroleum ether and re-evaporation are worth trying, for a liquid film may result. With more solid this is impossible. Choice between acid treatment and passage through a column is somewhat arbitrary, though waxes are usually resistant to acid but easy to remove in a column, whereas many oils show the opposite behavior As adsorptive solids the following materials have been used: Attaclay (Attapulgus), Hyflo SuperCel (Johns-Manville), 200mesh silica gel (Davison Chemical Corp.), and alumina (Eimer & Amend adsorption grade). Various procedures were used to secure favorable adsorption. The solids were used in tubes 9 mm. in inside diameter and were added with gentle suction and with tapping of the tube until a column 10 em. high resulted, supported on a small cotton plug. After the column had been rinsed with petroleuni ether or other solvent, the dried extract and excess solvent or other developing liquid were put through under sufficient air pressure to give a flow of 2 to 3 ml. per minute; this usually was 1 to 3 pounds per square inch. Benzene, chloroform, and nitromethane (Eastman grade) were used as auxiliary solvents to modify the behavior of adsorption solids. The effluents were collected in 25- to 200-ml. portions, which were evaporated in a warm air oven a t 70" C. to about 5 ml. This volume was transferred with rinsings to an exposure vial and taken to dryness. RESULTS
Effect of Nature of Deposit upon Pickup and Mortality. Examination under the microscope shows that insecticides may de-
posit in widely different forms even from pure solvents. Thus lindane from petroleum ether or benzene forms chunky crystals, but from chloroform flat plates predbminate with a few small platelets. Bioassay of various deposits showed that the large flat plates are but slightly tosic to houseflies with exposure of an hour or less. The effect is probably due to the few small pieces picked up by the insects, as a second group of flies introduced into the same vial gives no sign of toxic action, even though several times the LDSo dose is still present, Crystal size and shape of lindane are favorable from petroleum ether or benzene and when the deposit is well distributed over the surface of the evposure vials, consistent mortality results, though the pickup is usually much lower than from liquid films (Figure 1). Crude benzene hexachloride tends to give an apparently amorphous deposit from all solvents. Part of this is picked up readily by flies, which accounts for the reproducible dosage-mortality curves found in the earlier work (4). Pure and technical p,p'-DDT will persist for a time as sniall liquid droplets on the surface, but when a fly walks through them, the droplets change to minute crystals. Contact is readily made and reproducible dosage-mortality curves are obtained without difficulty from fresh deposits Older deposits consist of crystals of irregular size range and inconsistent mortalities often result, especially if the original deposit was irregularly distributed. Those insecticides which normally exist in the liquid state, such as tetraethyl pyrophosphate or parathion, are readily contacted : some lack of uniformity in distribution has no serious effect upon mortality with an active insect such as the housefly, but doubtless would lead to large errors n-ith a more sluggish one.
''I 95
/
A
vv 0,
5I 1
0.1
/
PB
P
0.2 0.5 1.0 2.0 5.0 10.0 AMOUNTS OF LINDANE IN MICROQRAMS PER VIAL
Figure 1. Dosage Mortality Lines A. B.
2 cu. mm. of oil per vial No added oil
The deposition of insecticide during evaporation of the solvrnt does not automatically give a uniform distribution; in fact, the tendency is to form windrows alternating with relatively bare areas, despite every effort t o prevent this by rolling the vial. Inclusion of a few micrograms of an oil-soluble dye such as OilSoluble Red enables the uniformity of deposit to be judged with the unaided eye. Experience has shown that uniform distribution is much e sier to secure when estracts from some plant or animal tissues are evaporated than when pure lindane solutions are used. This is due to oily substances which tend to spread more or less uniformly over the interior surface of the vials and which keep small amounts of lindane or other chlorinated hydrocarbons in solution so crystals usually do not appear. Uniform deposits of technical
1.5
.
2
2
1.4
5 a
1.3
y
1.2
-
4 0
5
v)
0 0 K
2
-
5
0
a 2
I,l.
1.0-
0.9 0.8
-
0.7
-
0.b0.5
-
in light Volck oil, as expressed in terms ofthe percentage removed from the surface of the vials by 25 flies during exposure periods of varying length. The average resultp from four to nine tests are shown in Table I. The amounts remaining in the exposure chambers were deterinined by extracting several vials and evaporating into one or by adding a known amount of lindane to the residual amount in each. Comparison of the mortality obtained with the standard dosage-mortality curve enables the residual amounts to be calculated. The tivo methods agreed very closely, but the second is preferable because the residual amounts in single vials may be determined rather than an average from several. From the experiments summarized in Table I, the con-
” A
ditions of 1 cu. mm. of oil per vial and GO minutes’ exposure were adopted as most satisfactory. The consistency of results with these conditions is illustrated by five runs which gave the following percentages of pickup from the vials: 89, 87, 80, 8-1, 94;average 88. The per cent pickup found in changing from 1 or 2 to 10 cu. mm. of oil declines only slightly and hence strict cont,rol of volume is not critical over this range. The conclusion may be drawn therefore that ahereas 1 t o 2 cu. nini. of oil very greatly improve distribution and pickup as compared n-ith
a I n this run combined residual auionnts of lindane caused such low inortalities t h a t only a maximum could be ca!culated. Hence pickup probably exceeded 84%.
Effect of Length of Exposure. The effect of time of exposuie is shon-n in Figure 2 in terms of the L D & values calculated fioni dosage-mortality lines such as d in Figure 1. The rapid drop within the first hour and relatively slow change thereafter iiidicate an advantage for the 60-minute period unless longer periods are equally convenient. As noted earlier, each generation of flies may vary slightly in response, but the variation with esposure follows the same pattern for all.
558 Use of Pickup in Calculation of LDbe. From the data presented in Table I and Figure 2, L D ~ values o may be calculated in terms of lindane actually removed from the vials by the flies. Thus for a 30-minute exposure, the LDSO is 0.9 microgram of lindane per vial containing 25 flies, and 78% of the total deposit was picked up. Hence the LD50 per fly is (0.9 X 0.78)/25 = 0.028 microgram. Similar calculations give 0.027 microgram per fly for 60 minutes’ exposure and 0.023 microgram per fly for 120 minutes’ exposure. Some less definite data for 10 minutes’ exposure indicate that the percentage pickup is about 70 and accordingly the LD50 is 0.039 microgram per fly. These figures may be compared with those for topical application of acetone solutions of lindane to the ventral thorax of houseflies. With no oil present the topical L D ~ was Q found to be 0.045 microgram per fly and with 0.03 cu. mm. oil per fly (corresponding to about 2.5 CU. mm. per vial) it v a s 0.03 microgram of lindane per fly. Thus in terms of material actually placed on the fly, application chiefly to the tarsi as in the vial method is more effective than to the thorax. LOSS of Toxicant after Exposure. When insecticides are used as contact poisons, the amount which remains on the body and continues t o be available for toxic action viill be affected by the insect’s movements after leaving the treated area. Thus nith the small vial method an exposure of 60 minutes folloived by confinement in a wire cage for 24 hours resulted in an LDsa of 0.76 microgram of lindane per vial as shown in Figure 2. If the exposure is prolonged to 240 minutes, the LDEo drops to 0.48’microgram. If exposure is 60 minutes but the flies are then transferred to a clean vial for 180 minutes more, the LDSOis 0.63 microgram, It is obvious that the free ventilation in the Lvire cage and opportunity to wipe off a part of the adhering toxic oil result in the highest LDM value. Longer confinement allows more penetration of the toxicant , so a markedly lower LD50 results. Transfer to a clean vial instead of an open cage gives less chance to wipe off adhering toxicant and probably adds a fumigant effect. Further light on the importance of volatility was secured by placing flies within a small wire cage which was fixed inside a treated vial but kept away from the walls or bottom. With varying dosagesof lindane in the vial it m-as possible toestablish an L D ~ of o approximately 7 micrograms of lindane per vial for this purely fumigant action. With a similar arrangement, D D T v a s nontoxic for any amount per vial and exposure up to 24 hours. Purification of Extract with Sulfuric Acid. All factors having to do with improving the reproducibility and sensitivity of the standard dosage-mortality curve also affect bioassay of insecticidal residues in plant or animal tissues. Extracts of these tissues usually contain some relatively nonvolatile fatty material, which may aid in obtaining uniform distribution and high pickup in the same manner as the added spray oil. However, this cannot be depended upon, for the amount and nature of these extractives are extremely variable, as illustrated by very toxic liquid oil from chicken fat (3)and solid nontoxic wax from cabbage Tvhich almost completely prevents contact with any residual insecticide ( 4 ) . It is preferable t o remove these interfering materials and then add the optimum amount of a suitable liquid to secure good distribution and pickup. For this purpose fuming sulfuric acid is often satisfactory when the contaminating insecticide is not affected as with DDT or lindane (4). It is, of course, entirely useless mith the organic phosphates, sulfites, and other oxidizable materials. Even with the stable insecticides the arid treatment is satisfactory only for certain extracts and does not remove heavy plant waxes such as those of cabbage or pears. It has been successful with several kinds of contaminated produce-e.g., cream from cows’ milk containing technical benzene hexachloride and the following all contaminated with lindane: chicken skin, flesh, and liver; soils containing considerable organic matter; peanuts; and alfalfa. As a numerical example the latter may be cited. For the group of flies used, the LDSOwas 0.85 microgram of lindane per vial. With D to 2.5 extract from 30 grams of dry alfalfa present, t h e L D ~rose micrograms and after treatment with sulfuric acid this decreased
ANALYTICAL CHEMISTRY to 1 0 microgram. All three dosage-mortality lines were parallel. Either the line for acid-treated extract or, with less accuracy, the line for uncontaminated lindane could be used for calculating contamination of samples from the field. Another example is afforded by chicken liver from fowls exposed to lindane. Tengram samples may be cleaned so effectively by the acid treatment that the dosage-mortality line coincides with that for straight lindane. In Figure 1 points 1 and 2 indicate mortalities obtained with two portions of a ceitain liver extract. These correspond to 0.86 and 0.80 microgram of lindane v-hich rame from a 6-gram sample of liver and a 3-gram sample to which 0.35 microgram of lindane had been added. Hence the parts per million may be calculated as 0.86 t 6 = 0.14 and (0.80 - 0.25) t 3 = 0.18. Purification of Extract by Passage through Adsorption Column. The acid process offers considerable opportunity for mechanical loss of toxicant during the numerous manipulations involved and, of course, only very resistant chemicals can avoid serious destruction. To avoid this difficulty and if possible to devise p. general procedure free from the disadvantages of using a powerful acid, a search has been made for a chromatographic column which would remove the interfering extractives but permit the insecticide to pass through, The procedure was to pass a solution of lindane or of lindane plus plant extractives through a column, and t o test portions of the emerging liquid for toxicity to flies by the small vial method as described, Petroleum ether was used as the standard solvent. If the lindane came through readily-e.g., in the first 50 m1.-it was certain that the column would not retain lindane from any other solvent, since, both theoretically and by test, petroleum ether on account of its highly nonpolar nature is less efficient in removing lindane than any other among many solvents tested. Hence columns which did not hold back lindane in petroleum ether \vi11 be of limited usefulness when a considerable amount of oil accompanies the lindane, but they may hold back plant pigments and heavy waxes as in the case of ilttaclay. If a given solid retained lindane with petroleum ether but liberated it when a more polar solvent-e.g., benzene or nitromethane-was added, it n as then used with extractives present. The behavior of the adsorbents with straight lindane may be described first. As obtained, the materials contained varying amounts of water and retained lindane poorly even from petroleum ether solution. They were activated by heating a t 500” C. for 6 hours and then kept in a desiccator. Loading into the tubes was done with minimum exposure to atmospheric moisture. Because Attaclay allowed very slow flow, it was mixed with an equal volume of Hyflo SuperCel, which by itself has almost no absorptive po\ver but increases rate of flow with the Attaclay. This mixture allon-s all lindane to pass in the first 50 to 100 ml. of petroleum ether and hence was useless except that it holds back eigment and some heavy wax (4). Hence a short column of Attaclay and SuperCel was very useful ahead of the silica in the runs described later. All lindane solutions passing through alumina columns were greatly reduced in toxicity. Either the lindane is adsorbed too tightly for complete removal by the polar solvents benzene or nitromethane or it is degraded to a harmless derivative. With activated silica no lindane comes through in 200 ml. of petroleum ether or cyclohexane but all is removed by 200 ml. of 5% benzene in petroleum ether or by 150~ml.of this solvent saturated mith nitromethane. Every plant or animal product presents a different problem, but perhaps extremes with regard to amount and kind of interfering extractives are offered by olive oil and dry alfalfa hay. The results obtained n-ith these when purposely contamined with lindane will serve to illustrate the work with adsorption columns. When a petroleum ether solution of 2 ml. of commercial olive oil plus lindane is passed through an activated silica column, the first 50 ml. contain no lindane but about half the oil. Further solvent up to about 200 ml. brings through no lindane and but little additional oil. Addition of 200 ml. of petroleum ether saturated with nitromethane brings through the lindane and about 15% of the original oil; a heavy yellow oil containing no
V O L U M E 24, NO. 3, M A R C H 1 9 5 2 lindane follows in further portions of the mixed solvent. Thus most of the interfering substances can be separated from the lindane and a bioassay can be made successfully if the original contamination was 5 p.p.m. or over. For lower contamination, the product from the column must be treated u i t h acid as described earlier. The first 50 ml. of a petroleum ether extract of 10 to 25 grams of alfalfa plus lindane after passage through an activated silica column contain a small amount of oil but no lindane and very little more solute follows in an additional 150 ml. Substitution of petroleum ether saturated 15 ith nitromethane brings through the lindane in about 200 ml. nith a noticeable amount of a hard wax. Petroleum ether plus 5% benzene brings the lindane m ith very little wax in about 200 nil. With 10% benzene, all lindane is contained in 150 ml. but more wax is present. A short fore column of Attaclay miill retain much of the wax. Hence for alfalfa, the preferable procedure is to pass through 50 ml. of petroleum ether followed by 200 ml. of 570 benzene solution.
559
method for determining pickup is necessary. A procedure based upon simultaneous pickup of an added dye is now being studied. I n the rather brief examinat'ion of various oils for use in the vials, certain differences in their behavior were noted. As extractives from plant and animal tissues differ greatly in physical and chemical properties, it is probable Chat they also differ in readiness of contacting insect integument. Hence they may be picked up to varying degrees. -2bsorption of toxicant into the body from an adhering layer is influenced by the solubility according to the principle clearly stated by Ferguson ( 2 ) . For a given concentration of a toxic solute in a series of solvents, the system closest t o saturation-Le., in which the solute is least solublewill be the most toxic, since the toxicant will have the strongest tendency to escape by moving into the organism where its effects can be exerted. Conversely, high solubility viill result in little tendency for passage into t'he organisms. This theory presupposes Tests of efficiency of recovery of lindane through a silica column that the solvents themselves do not penetrate and carry toxicant s h o f that approximately 75% of 1 microgram of lindane in the along. In the case of extractives encountered in residue deterextract from 10 grams of alfalfa-Le., original contamination of minations, doubtless variation occurs in both dissolving power 0.1 p.p.m.-is recoverable. If the original conditions are 10 and ability to penetrate. Of course, material of a fatty nature on micrograms of lindane on 25 grams of alfalfa, almost complete the exterior of an insect's cuticle may dissolve in the applied solurecovery is possible. tion and further complicate the situation. A petroleum ether extract of asparagus gives a heavy deposit As stated earlier, a primary requisite of a satisfactory bioassay of wax in the exposure vials. Activated silica holds back this is rrproduci1)ility of t'he st,andard dosage-mort,ality curve. The wax but allons aldrin (1,2,3,4,10,10-hexahydro-1,4,5,&diforegoing Tvork has sholvn that inclusion of a small amount of methanonaphthalene) to come through completdy in about 75 light oil will change the conditions for pickup from that of a solid rnl. Hence the silica column u a s used for determination of aldrin deposit, often irregularly distributed, to that of a thin liquid film. contamination in asparagus grown in soil to which the insecticide This is beneficial in two ways: by increasing uniformity of deposit had been applied. Because flies are fairly sensitive to this comover the surface of the exposure chamber and by enabling a high pound (L& = 1.4 micrograms per vial) it would be possible to percentage of the toxicant to be transferred to the bodies of the determine contaminations much loner than the least one found, test insects. Both functions increase mortality from a given which was 0.08 p.p,ni. Houseflies are relatively insensitive to amount of toxic residue and hence make the method more sensitetraethyl pyrophosphatp by the vial method of exposure (LD,a = tive as well as more accurat,e. 6 to 7 mirrograms per vial), but the procedure was used successA second requisite of a satisfactory bioassay is the reduction fully for determination of relatively heavy external contaminaof plant or animal contaminants extracted along with the residual tions on several kinds of plants such as green leaves of beans, insecticide. Preferably, this removal is done completely, though squash, and alfalfa which did not give enough extractives to intera small amount of contaminant either in liquid or solid state may fere. Some results of hiomay for parathion were mentioned in the not interfere xith the proper action of the added light oil. The earlier report (4). Highly activated columns have not been size of a sample \Those extract can be concentrated in a vial and used with either of these organic phosphate insecticides, and the used directly is governed by the amount of extractives which repossibilities for improving the procedures remain to be examined. main after treatment', and hence t.he relative sensitivity as expressed in parts per million detectable is controlled by t,he efDISCUSSION ficiency of the purification process. Sometimes the c0nt.aminant.s do not interfere a t all, as when very high residue permits use of a The pickup of poison is very important in any bioassay, as very small sample, or their amount and physical state correspond there is an assumption underlying all techniques that a reproducito the required spray oil, which then can be omitted. However, ble fraction of the active material reaches the test organisms in differences in dissolving power and ease of penetration into the any given case, for otherwise it would be impossible t o compare reinsects may be involved. I n general, removal of all contaminants sults for an unknown with the reference data from known quanis preferable. tities. A bioassay in which a large percentage of the toxicant For the immediate purpose of improving bioassay by the use of reaches the test insects is influenced strongly by the number of liquid films cont'aining the toxicant, it is important to determine the latter used and would be more sensitive with few insects. how extractives affect pickup and what effect treatment has upon Large numbers are desirable for statistical reasons and a comthis behavior. As an example, the poor pickup from solid waxy promise must be made. In the present work this was set a t 25 infilms can be overcome a t least in part by passage of the solution sects per test. With most residue determinations there is no through an Attaclay column. Further work designed to deterscarcity of toxicant and high pickup is of advantage chiefly bemine which plant and animal tissues yield extractives that cause cause use of minimum volume of extract results in less interpoor pickup, the best solvent, and the most effective means for ference. However, another important use of bioassay is deterremoving interfering material without loss of the toxicant is in mination of distribution of a toxicant among the tissues, secreprogress. tions, or excretions of an organism-e.g., storage of lindane in the tissues of resistant flies. Here the highest possible sensitivity is SUMMARY needed, By combining data on total dosage per test nith per cent pickup The small vial method for bioassay of insecticidal residues it is possible to determine LDS0)s or any other mortality dosage per of lindane with houseflies as test insects has been improved by individual insect as illustrated by the data cited. Thus the moradding a minute quantity of light spray oil which is nontoxic by talities from contact with deposits may be compared directly with itself and has the functions of spreading the toxicant in solution topical application. Measurement of dosage in terms of that over the inner surface of the vials and enabling a large and conactually reaching the test organisms is preferable t o the usual sistent fraction of the total deposit to reach the test'insects. The expression in terms of concentration or amount in the surrounduniformity of contact and high pickup contribute to consistency ings. I n order t o be practical for deposits of insecticides, a simple of mortality. The same procedure has been used successfully in
560
ANALYTICAL CHEMISTRY ACKNOWLEDGMENT
preliminary tests with tetraethyl pyrophosphate, parathion, and aldrin. Extracts of plant or animal tissues contain oily or waxy extractives which often interfere with the test. If the film contains enough oil to run visibly when the vial is tilted, the flies will suffer moderate to high mortality without any insecticide. If the deposit is solid, pickup will be low and most of the toxicant will not reach the flies. The interfering materials can be removed in whole or in part by treatment of the extract with strong sulfuric acid or by passage of the extract through a suitable column of solid adsorbent. Promising results have been obtained with lindane residues passed through silica columns when petroleum ether was employed as solvent and petroleum ether saturated with nitromethane or containing 5 % benzene was added to remove the lindane.
The California Spray Chemical Corp. has given generous financial support to this work. LITERATURE CITED
(1) Fairing, J. D., and Phillips, 1%’. F., “Colorimetric Determination of Small Quantities of Benzene Hexachloride,” Divisions of Ag-
ricultural and Food Chemistry and Analytical Chemistry, 119th Meeting, AM. CHEM.SOC., Boston, 1951. (2) Fereuson. J.. Proc. Row. Soc. London. (B)127. 387-404 (1939). (3j Furman, ‘D.’P., and Bankowski, R.’ A.[ J . i c o n . Entokol., ’42, 980-2 ... - (1949). \----,
(4) Hoskins, W. M., and Messenger, P. S., Advances in Chem. Series, KO.1, 93-8 (1951). RECEIVEDfor review May 28, 1951. Accepted October 18, 1951. Presented in part before the Divisions of Agricultural and Food Chemistry and Analytical Chemistry, Symposium on Method8 of Analysis for Micro Quantities of Pesticides, a t the 119th Meeting of the AMERICAN CHEMICAL SoCIETY, Boston, Mass.
Semimicromethod for the Determination of Plant Sterols DICK WAGHORNE, Ontario Agricultural College, Guelph, Ontario, Canada AND
CHARLES D. BALL, Michigan State College, Edst Lansing, Mich. This work was undertaken to find a semimicromethod for the estimation of plant sterols, eo that the development of these sterols during the life of a plant could be studied. The method uses dichromate oxidation of the digitonides, followed by titration of the excess dichromate with ferrous solution, and yields results with a precision better than f3%. Samples containing as little as 0.1 mg. of total sterol are adequate for a single determination. The sensitivity of the method approaches that of the colorimetric methods and is unaffected by slight changes in the structure of the sterol molecule.
P
LANT sterols have been di.scussed from various viewpoints ( 1 , 4 , 8 ) ,but their development throughout the life cycle of a plant, beyond germination, has not been investigated. With this project in mind, a satisfactory method for the estimation of these mixtures of sterols was sought. The Liebermann-Burchard colorimetric method has the desired sensitivity and it has been adapted for use with plant sterols (19). It was not considered satisfactory in the present rrork because the saturated sterols give no color. Even the necessary isolation of a macro amount of the sterols from the particular plant species or the part of the plant under study, and the use of this material for the preparation of a working curve ( 5 , 19), are unsatisfactory because the ratio of saturated to unsaturated sterols may vary with the development of the plant. The gravimetric method dewribed by Wall and Kelly (19) offers a means of determining both saturated and unsaturated sterols but lacks the required sensitivity. Obtaining samples large enough to yield accurate results, up to 5 grams of air-dry tissue per determination, is often almost impossible for some plant parts. A survey of the literature revealed two methods for the determination of cholesterol, which might be adapted for the requirements of this study: a method developed by Rappaport and Klapholz (IS),which depends on precipitation of the sterol as the digitonide, followed by hydrolysis of the excess digitonin and determination of the reducing sugar subsequently liberated; and precipitation of the sterol as the digitonide, followed by
the dichromate oxidation of the entire precipitate as reported by Okey (11). Later attempts were made to improve Okey’s procedure for cholesterol (10, 18, 23), but from 1933 until the present it appears to have been abandoned. This was probably due to the development of the Liebermann-Burchard colorimetric method, which is satisfactory for cholesterol (14). Recently, pyridine and chlorosulfonic acid have been successfully used for the rapid precipitation of cholesterol as the pyridinium salt (16, 16). However, the classical digitonin precipitation of Windaus (21) was considered superior for this investigation for two reasons. First, the solubility of the pyridinium salts of the various plant sterols has not been investigated. Indeed, whether or not they all form precipitates under the conditions outlined is not known. On the other hand, Kith one reported exception ( 8 ) , all plant sterols isolated to date are known to form relatit ely insoluble digitonides (19). Secondly, the digitonide has a much higher molecular weight than the pyridinium salts and therefore this procedure should be considerably more sensitive, if the entire precipitate is to be oxidized. Schoenheimer, as reported by Sperry (17), developed a satisfactory method for the precipitation of cholesterol as the digitonide by the use of 80% ethanol as a precipitating medium. Under these conditions, or with higher water concentration, the precipitation is complete and is not affected by the excess of digitonin present. The authors have found that if the water concentration is increased to about 25% the precipitate, while still quantitative, becomes very sticky and hard to handle. The present work indicated that the reproducibility of Rappaport’s method was not satisfactory. Several modifications were tried and some improvement was noted, but only about 70% of the results were duplicable. The greatest difficulty appeared to be in the standardization of hydrolysis, especially when the digitonide itself was being hydrolyzed. Details of this work will be published elsewhere. Therefore, a modification of Okey’s method rr-as finally adopted. REAGENTS
All reagents were of analytical grade. Ethanol, 80%. was prepared by diluting 95% ethanol with - distilled water.. - ’ Digitonin solution, 1%. One gram of digitonin (Merck) was dissolved in 100 ml. of 80% ethanol with warming. No variability
