INDUSTRIAL AND ENGINEERING CHEMISTRY
April 1948
TABLE IV. D D T
4
.
7
...
17 11
0. ..8.
... ...
8
16 12
0.6
Fodder (2 Dusts) P.P.M. D D T In fat I n muscle 1.8 ... 1.3 ... 2.8 0.0 0.7 4.3 ... 2.2
... ...
consumed, on an average, 32 pounds of silage per head daily. When slaughtered on the 108th day, samples of shoulder muscle and kidney fat were removed for analysis. The results, shown in Table IV, indicate that significant amounts of D D T do accumulate in the fat of steers fed silages which contain less than 10 p.p.m. of DDT. Extremely small concentrations of D D T were found in the lean meat from these steers. It was also found that the steers fed on the husk and cob silage retained the greatest amount of D D T in spite of the fact that the silage, when fed, contained only about 1 p.p.m. of DDT, or roughly 0.1 as much D D T as fed to the steers in the first lot of silage. When canning corn is cut from the cob much of the germ remains on the cob. It seems likely that the high oil content of the germ aids the absorption and retention of the small amount of D D T present in the silage.
TABLEV.
Based on these experiments, feeding corn silage from dusted fields to beef Husk and Cob animals creates no appreciable hazard to P.P.M. D D T human beings who later eat the meat I n fat In muscle 24 ... from such animals. The small amounts 12 ... of D D T that are absorbed are largely 17 0.5 4 0.1 deposited in inedible portions of the 27 0.7 body fat and only negligible amounts are 17 ... found in the muscle tissue. The high-DDT sweet corn silage from plots dusted four times was also fed to a group of dairy cows a t the rate of about 32 pounds per cow daily for a period of 100 days. Composite samples of the milk were analyzed a t intervals with the results shown in Table V. Again extremely small quantities of D D T were secreted in the milk.
TISSUES OF STEERSFEDON CORNSILAGE
Fodder (4 Dusts) P.P.M. D D T I n fat I n muscle
Steer No. 1 2 3 5 Average
IN
D D T IN MILK FROM Cows FED CORN SILAGE CONTAININQ 5 TO 10 P.P.M. D D T Days on Expt. 31 42 65 85 100
711
SUMMARY
The results of this work indicate that DDT insecticides can be used under practical conditions for the control of pea aphids and corn borers without detrimental effects to livestock fed on the treated crop residues. Extremely small amounts of D D T find their way to the meat and milk of these animals; this indicates a negligible hazard to human health. However, as yet federal and state agencies have not set official tolerance levels for D D T in foods for human consumption, except for apples and pears. This fact should be considered when suggestions regarding the use of D D T on feed crops are made to farmers. LITERATURE CITED
D D T in Milk, P.P.M (1) Schechter,
M. S., Soloway, S. B., Hayes, R. A,, and Haller.
H. L., IND.ENG.CHEM.,17,704-9 (1946). (2) Wilson, H. F., Allen, N. N., Bohstedt, G., Betheil, J., and Lardy, H. A,, J. Econ. Entomol., 39,801 (1947). RECEIVED November 22, 1947.
Metabolism of Chlorinated HydrocarbonInsecticides Geoffrey Woodard, Bernard Davidow, and Arnold J. Lehman Food and Drug Administration, Federal Security Agency, Washington 25, D . C .
A
summary of the present knowledge concerning the metabolism, fate, and excretion in mammals of the six newer chlorinated hydrocarbon insecticides is given. These six are diohlorodiphenyltrichloroethane, dichlorodiphenyldichloroethane, methoxychlor, benzene hexachloride, chlordan, and chlorinated camphene. An understanding of the mammalian metabolism and fate of these compounds necessary for evaluation of the possible hazards involved in their use in insecticidal applications, when exposure to man and animals will result, is far from complete.
A
INTEGRAL part of the determination of the toxicity of any compound is a study of the fate of that compound in the body. An understanding of how the compound is absorbed, how it is metabolized, how it is excreted, and whether or not it is stored in any tissue is necessary for the evaluation of the possible hazard connected with the use of the compound. Such an understanding is especially needed in the case of insecticides to which man and animals will be exposed. With the introduction of D D T work was undertaken in several laboratories t o elucidate the
metabolic fate of this insecticide in mammals. As each new insecticide was made available, the inevitable question arose regarding its metabolic fate in mammals. Including DDT, there are now six chlorinated compounds generally available as insecticides. The over-all picture of our present knowledge concerning their absorption, accumulation, fate, and excretion may be summarized as follows:
DDT
Compound
Dlohlorodiphenyldichloro-
ethane (TDE DDD) Methoxychlor (methoxy analog of DDT) Benzene hexachloride (BHC) Chlordan Chlorinated camphene
Absorbed
+
+ + +
++
Accumulated
+
Excreted in Urine Unchanged Changed
-
-I-
+
-
?
?
+ + tI
6?
i i
1
That each of these insecticides is absorbed from the gastrointestinal tract follows directly from the fact that animals can be severely poisoned or killed following oral administration of the compounds in corn oil solutions. Accumulation of D D T in the fat of animals and its appearance in milk have been amply demon-
712
Vol. 40, No. 4
INDUSTRIAL AND ENGINEERING CHEMISTRY
strated by many investigat,ors (5, 9, 12). I n this laboratory it was shown ( 2 , 11) that both dichlorodiphenyldichloroethane and the isomers of benzene hexachloride also accumulate in the fat of animals ingesting these insecticides. Whether methoxychlor, chlordan, and chlorinated camphene are accumulated still remains t,o be definitely established. D D T and dichlorodiphenyldichloroethane normally do not appear in the urine of animals to which they are fed. The fact that DDT is absent, from uncont'aminated urine samples has been checked (4,6, 7 ) . When D D T is fed to rabbits a large part of the organically bound chlorine excreted in the urine appears in the acid fraction. Upon isolation, this material was shown by White and Sweeney (10) to be p-dichlorodiphenyl acetic acid (DDA),. The identification of this metabolite as p-dichlorodiphenyl acetic acid was demonstrated by means of an x-ray diffraction comparison with synthetic dichlorodiphenyl acetic acid. Subsequently two neutral metabolites were isolated which upon either acid or alkaline hydrolysis yielded dichlorodiphenyl acetic acid ( 7 ) . The nature of these conjugates is unknown. Man (6) and dogs also excrete dichlorodiphenyl acetic acid in the urine. Dichlorodiphenyldichloroethane and dehydrohalogenated D D T yield urinary metabolites which appear in the acidic fraction and give red colors with the Schecht,er-Haller color reagent (8). These colors are ident,ical to that produced by dichlorodiphenyl acetic acid a n d , ,t,herefore, this metabolite is presumed to be dichlorodiphengl acet,ic acid. One of the sites of D D T metabolism appears to be the liver. This is based on t,he observation that D D T disappears from ho'mogenized portions of the livers of rabbits injected intravenously with emulsions of DDT. I n order to obtain livers containing small amounts of DDT, the insecticide was injected intravenously in an aqueous Tween 20 emulsion a t a dose of 25 mg. per kg. The rabbits were sacrificed in about 30 minutes and the whole livers were ground in a mechanical blender. Aliquots of the blends were incubated a t 37" C. for various periods of time and then analyzed by the Schechter-Haller procedure (8). The results of the averages of five samples were: Time of Incubation a t 37O C., Hours 0
2to 3 4to 8 17 t o 24
Decrease in DDT, Micrograms per Gram 154 146 142 116
The fate of methoxychlor in the body is unknown a t present. In preliminary studies unchanged methoxychlor and the correspondingly substituted acetic acid were not found in the urine. Apparently the met,aholism of this compound involves more profound changes in the molecule than occur with DDT. A study of the fate of benzene hexachloride in the animal body is complicated by the fact that this insecticide is composed of a niixture of isomers. Slthough the gamma isomer is responsible for the major insecticidal activity of benzene hexachloride, the alpha isomer predominates in the technical product. Therefore, the alpha isomer was chosen for first study. This isomer as well as t,he delta, beta, and gamma isomers is easily dehydrohalogenated with dilute alkali t.o form mixtures of trichlorobenzenee, the predominant member of which is 1,2,4-trichlorobenzene. Coupled with this observation is the fact that cyclohexanc compounds tend to be dehydrogenated in the' animal body to the corresponding benzene compounds as has been shown, for example, by Bernhard (1) and by Friedmann (3). They observed t,hat hexahydrobenzoic acid is dehydrogenakd by the dog to benzoic acid. Following this line of reasoning, a search xas mad- for 1,2,4-trichlorobenzene or a metabolite of 1,2,4-trjchlorobenzene in t,he urine of rabbits inject,ed intravenously mlth an aqueous Tween 20 emulsion of the alpha isomer. I n the meantime it was established that following the oral administ,ration of 1,2,4-trichlorobenzene to rabbits, there appeared, in the phenolic fract,ion of the urine, a phenol which gave the ultraviolet absorp-
tion spectrum of 2,4,5-trichlorophenol and which also behaved the same as 2,4,5-trichlorophenol on a partit,ion chromatogram. Extracts of urine from rabbits given the alpha isomer of benzene hexachloride either orally or intravenously, were separated into neutral, phenolic, and acidic fractions. S o unchanged alpha isomer was found in the neutral fractions. I n two of eight rabbits there did appear a material which gave the same ultraviolet absorption curve as 1,2,4-trichlorobenzene. No 2,4,5trichlorophenol, which might be expect>ed if 1,2,4-trichlorobenzene were an intermediary mebbolite, was found in the phenolic fractions in subsequent experiments. However, acidic fractions were obtained which gave charact,eristic absorpt,ion curves in the ultraviolet, but variable absorption curves in the infrared. The picture appears to be one of a highly variable metabolism of the alpha isomer. Since analytical methods for determining chlordan and chlorinated camphene in biological tissues are not. yet available, very little is known regarding their metabolism or fate in the body. Very little attention has been directed toward the importance and meaning of the metabolic fate of the insecticides other than t,heir accumulation in various tissues in the body. There appears to be some relation between storage and metabolism. A chlorinated hydrocarbon which is easily metabolized to a water-soluble compound which can be excreted in the urine should accumulate in fatty tissue to a lesser extent than a chlorinated hydrocarbon metabolized only with difficulty. Furthermore, there should exist an equilibrium between absorption of an insecticide, on the one hand, and storage and excret,ion, on the other. Such an equilibrium condition is indicated in the case of D D T by the fact, that the amount of D D T stored in rats t,ends to level off to a constant value for each const'ant level of intake. It' was found in rats that this constant level was reached after about 50 dags of feeding. The storage values of DDT in the fat of male rats on three different levels of intake, after 54, 72, and 90 days of feeding are shown; the storage levels became stat,ic a t each diebary level: DDT D
Mg./Kg.
2.6
5.6
11.2
~
~ Storage ~ , Values of DDT, Miorograme per Gram Fat 54 days 72 days 90 days
320 530
350
960
820
510
360 550
880
From a consideration of these storage data and from what is known regarding the metabolism of DDT, i t seems reasonable to hypothesize that there is a critical level of intake of D D T below which no appreciable storage will take place since i t will all be metabolized and excreted. If the other chlorinated hydrocarbon insecticides behave similarly, t8here should be a threshold level of intake for each one below which no appreciable t,issue storage will take place. The determination of such levels for each insecticide would be of paramount, importance in assessing its probable hazard to man and animals. LITERATURE CITED
(1) Bernhard, K., 2. physiol. Chem., 248, 286 (1937). (2) Davidow, B., Hagan, E. C.. and Woodard, G., Federation Proc., t o be published. (3) Friedmann, E., Biochem. Z . , 35,49 (1911). (4) Laug, E. P., J . Pha~macol.Erptl. Therap., 86, 332 (1946). (5) Laug, E. P., and Fitzhugh, 0. G., Ibid.. 87,18 (1946). ( 6 ) Teal, P. X., Sweeney, T. R., Spicer. S. E-.,and von Oettingen, W. F., Pub. Health Repts., 61, 403 (1846). (7) Ofner, R. R., and Calvery, H . 0.. J . Pharmacal. Ezptl. Therap., 85, 363 (1945). ( 8 ) Schechter, M. S., Soloway, S.B., Hayes, R. 4.,and Haller, H. L., IND. EKG.CHEM.,A s . 4 ~ED., . 17,704 (1945). (9) Telford, H. S., and Guthrie, J. E'., Science, 102, 647 (1945) (10) White, W. C., and Sweeney, T. R., P u b . Health Reptts., 60,66 (1945). (11) Woodard, G., Davidow, B., and Selson, A. A,, FederationProc., to be published. (12) T1700dard, G., Ofner, R. R., and Montgomery, C. M., Science, 102, 177 (1945).
._
RECEIVED November 22, 1947.
