Agricultural Control Chemicals (1950)

AGRICULTURAL CONTROL
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.fw001
CHEMICALS
Collected Papers from the Symposia on Economic Poisons presented before the Division of Agricultural and Food Chemistry of the American Chemical Society at the 115th national meeting in San Francisco, March 28 to April 1, 1949, and the 116th national meeting in Atlantic City, September 18 to 23, 1949
AMERICAN CHEMICAL SOCIETY 1155 Sixteenth Street, N.W. Washington 6, D. C.
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.fw001
Copyright April 1950 by AMERICAN CHEMICAL SOCIETY AU Rights Reserved
INTRODUCTION
T h e g r o w t h i n t h e n u m b e r of s y m p o s i a presented a t meetings of the A M E R I C A N C H E M I C A L S O C I E T Y has been r a p i d i n t h e last several years. T h e i m p o r t a n c e of these s y m p o s i a is w i d e l y recognized. T h e y p r o v i d e o p p o r t u n i t i e s t o r e v i e w a n d e v a l u a t e scientific a n d technological a d v a n c e m e n t s a t periodic i n t e r v a l s . I n a n e r a w h e n research is e x p a n d i n g a t a r a p i d rate a n d scientists a n d technologists are finding i t i n c r e a s i n g l y difficult t o keep abreast w i t h even a s m a l l p o r t i o n of the p u b l i s h e d l i t e r a t u r e , periodic s y m p o s i a p e r f o r m a m o s t useful purpose. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.fw001 O c c a s i o n a l l y , however, s y m p o s i a are of s u c h h i g h l y specialized n a t u r e t h a t o n l y a r e l a t i v e l y s m a l l percentage of the readers of a n y one of the established A . C . S . j o u r n a l s w o u l d be served b y j o u r n a l p u b l i c a t i o n . T h e p r o b l e m s of p u b l i s h i n g such s y m p o s i a i n t h e i r e n t i r e t y i n the regular j o u r n a l s are increasing, p a r t i c u l a r l y so w h e n t h e d e m a n d continues u n a b a t e d f o r e d i t o r i a l pages t o report the results of o r i g i n a l research. N o t i n f r e q u e n t l y , too, s y m p o s i a c o n t a i n a large n u m b e r of papers t h a t d o n o t f a l l w i t h i n t h e e d i t o r i a l scope of t h e established A . C . S . p u b l i c a t i o n s . I n these instances m a n y papers are released f o r p u b l i c a t i o n elsewhere, a n d m a n y m a y n o t appear i n p r i n t i n a n y established j o u r n a l . T o satisfy t h e obvious need of p r o v i d i n g a m e d i u m f o r t h e p u b l i c a t i o n of h i g h l y specialized s y m p o s i a i n t h e i r e n t i r e t y , the editors of Industrial and Engineering Chemistry, Analytical Chemistry, a n d Chemical and Engineering News proposed t o the B o a r d of D i r e c t o r s of
t h e A M E R I C A N C H E M I C A L S O C I E T Y t h e e s t a b l i s h m e n t of a n A D V A N C E S
IN C H E M I S T R Y S E R I E S . P e r m i s s i o n w a s r e a d i l y received f r o m t h e B o a r d of D i r e c t o r s o n J u n e 4, 1949.
T h e v o l u m e s of A D V A N C E S I N C H E M I S T R Y S E R I E S w i l l be n u m b e r e d
consecutively. I t is expected t h a t t h e A D V A N C E S I N C H E M I S T R Y S E R I E S w i l l be recognized as a n i n t e g r a l p a r t of t h e p e r m a n e n t l i t e r a t u r e , a n d w i l l be covered b y the a b s t r a c t j o u r n a l s .

N u m b e r one of A D V A N C E S I N C H E M I S T R Y S E R I E S , "Agricultural
C o n t r o l C h e m i c a l s / ' i s a c o l l e c t i o n of papers f r o m t h e S y m p o s i a o n E c o n o m i c Poisons, organized u n d e r t h e c h a i r m a n s h i p of J . L . S t .
American Chemical Society Library

1155 16th St., N.W. Washington, D.C. 20036
John and presented before the Division of Agricultural and Food

Chemistry of the A M E R I C A N C H E M I C A L S O C I E T Y at the spring 1 9 4 9 meeting of the A M E R I C A N C H E M I C A L S O C I E T Y in San Francisco and the
fall meeting in Atlantic C i t y . This first number is a perfect illustration of the desirability of establishing a special medium for the publication of certain symposia presented at A M E R I C A N C H E M I C A L S O C I E T Y meetings. Only a relatively small percentage of the papers can be said to be strictly chemical i n nature. M a n y deal with such subjects as the toxicological effects of economic poisons, public health aspects, etc. Only through the
establishment of the A D V A N C E S I N C H E M I S T R Y S E R I E S was it possible
to present the symposia in their entirety. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.fw001 In introducing this series we are highly optimistic in our belief that it will help to solve at least some of the present publication problems facing the A M E R I C A N C H E M I C A L S O C I E T Y .
WALTER J. MURPHY
vi
Objectives and Scope

J. L. ST. JOHN
Agricultural Experiment Stations, State College of Washington, Pullman, Wash.
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch001
Further information is needed on the fundamental chemistry, mechanism of action, and toxicity of the newer economic poisons. An outline is presented of a research program that emphasizes the breadth and scope of the problem and the variety of research and educational needs.
D u r i n g the organization of these symposia much interest i n the field of agricultural control chemicals was evident. I t is difficult adequately to express appreciation to those who have so generously responded with both suggestions and papers. A broad field of topics was included i n the suggestions received. I t became evident i n attempting to organize the suggested topics into a program of titles that it would be imperative to limit the field to be covered. The symposia were thus confined to certain phases of the field of insecticides. M a n y other topics i n addition to those covered by the titles i n these symposia were proposed, and may form the basis for future symposia. Other suggestions included the broad and important topic of formulation, which may many times have an important relation to the effectiveness of the economic poison for the purpose for which it is designed, may be modified to prepare a given pesticide for different uses, and may also influence its toxicity to warm-blooded animals. A report of work on methods of application and their relation to effectiveness was suggested, including much work on the use of concentrated sprays. The fundamental chemistry, especially of the newer economic poisons, is of primary importance. T h e mechanism of action of the various types of economic poisons and the relation of structure to toxicity of insects are of fundamental interest. Chemical versus biological methods of evaluation should be presented. Performance methods of evaluation of these chemicals have been given careful consideration by several workers. E m phasis was placed by several workers on the need for much additional information on various aspects of the problem regarding the use of D D T , 2,4-D, and other pesticides. There is direct importance i n studies on the metabolism of D D T . The toxicity and the physiological action of insecticides, fungicides, rodenticides, and herbicides on plants are of basic importance. The toxicity of treated plants to animals, and the toxicity of treated plants and animals to humans and to wildlife are of practical concern. A long-range consideration of the effect of sprays on both plant and human nutrition and its relation to public health is of direct concern. The hazards i n field application and methods of protecting operators should be reported i n detail and further research should be emphasized. The relative toxicity, especially of the newer compounds, to various pests and species is of practical importance, as are also differences i n species tolerance to D D T and other sprays. This is also of direct concern i n relation to parasites and predators. There is interest i n synergistic action and the effect of spreaders, stickers, and related products. The effect and use of repellents and attractants have received attention. The spray residue problem has been emphasized for 30 years. I t should receive continued intensive attention from a variety of viewpoints, including its importance i n both
1
ADVANCES IN CHEMISTRY SERIES
fresh and processed fruits and vegetables. The possibility of soil contamination, which was earlier emphasized with the lead arsenate sprays, should also be explored i n evaluat ing new sprays. Information already accumulated regarding this problem should be pre sented i n the immediate future. It has been stated that 4 0 % of the economic poisons are used i n the F a r West. I t , therefore, seemed fitting that an extensive Symposium on Economic Poisons should be organized for the western meeting (San Francisco, M a r c h - A p r i l 1949) of the AMERICAN CHEMICAL SOCIETY . This was followed by the Atlantic C i t y symposium, September 1949. A symposium on fungicides will be presented at the Philadelphia meeting i n A p r i l 1950, and another on oil sprays at the September 1950 meeting of the Society. Stanley B . Freeborn of the University of California, i n the September 1948 issue of Agricultural Chemicals, has forcefully discussed research needs from the standpoint of the experiment stations, and pleads for an intensive attack on pesticidal problems through an organized fundamental type of research. Such an attack needs continued expansion to all research i n the broad field of economic poisons as well as i n agriculture. The National Research Council has recognized the importance of the food protection problem and the need for an organization to provide technical counsel on the utilization of new materials i n the production and processing of foods. The desirability of cooperative action b y industry, government, and other research organizations i n providing the scien tific guidance required for the protection of the food supply has been emphasized. The field of these very important chemicals covers a very broad scope. A n organized consideration of all phases of the problem is needed i n developing a research program. F r o m this the most critical research needs should be selected and emphasized. T h e fol lowing outline presents one type of an organized picture of the breadth and scope of the problem, and the variety of research and educational needs. Chemistry plays a major role i n this work.
Research Program on Economic Poisons

I. Safety and Hazards 1. Toxicology and Pharmacology A. A more detailed outline appears in Food and Drug Quarterly, September 1949, including chemical and biochemical studies and acute, chronic, and allergic toxicity 2. Medical and Public Health A. Symptoms and diagnosis, acute and chronic B. Antidotes C. Methods of treatment 3. Use and Application A. Precautions and safety measures B. Formulation C. Dusting (1) Machine (2) Airplane D . Bait exposure stations for mammal poison E. Secondary poisoning hazards 4. Consumption, Foods and Feeds A. Spray residue (1) Spray load at harvest (2) Removal methods (a) Fresh fruits and vegetables (b) Processed foods (3) Relation of effectiveness and limitation of application to residue load (4) Keeping quality and flavor (5) Processing breakdown (6) Effect on nutritive values of human and animal food 5. Soils and Plants A. Soil toxicity B. Plant toxicity and nutrition (A and include immediate and delayed injury) 6. Manufacturing, Safety and Precautions 7. Chemistry and Analytical Methods A. Fundamental
ST. JOHNOBJECTIVES AND SCOPE
II.
III.
B. Methods of analysis for trace amounts in food and toxicological material Education A. Public press B. Distributors C. Research men, extension, county agents D . User, professional and general E . Physicians F. Manufacturer and field representatives Effectiveness and Efficiency of Pest Control 1. Entomological, Rodenticidal, Fungicidal, Herbicidal A. Effectiveness of newer pesticides for different pests under different climatic conditions in different areas B. Efficiency of spray application C. Limitations in use, effectiveness, safety D . Species and developed resistance E . Ecological relationships 2. Chemical A. Formulation and compatibility B. Coverage C. Mode of action Engineering 1. Design of Equipment for Efficient and Safe Application A. Ground equipment (1) Stationary and portable (2) Pumps, spray nozzles, pipe lines, pressures B. Airplane equipment 2. Development of Methods of Application 3. Decontamination 8.
This is a very broad and extensive program that should be of both direct scientific and practical value to manufacturers, food processors and the food industry, food consumers and general public, and governmental and industrial research. It should emphasize the value and real benefit to mankind of the use of the newer economic poisons in pest control, even though their improper use may in some cases be somewhat hazardous, necessitating regulation and adequate precautions. Undue emphasis m a y have been given to certain hazards in the use of pest control chemicals and insufficient emphasis to the value and benefits resulting from their use. Further public emphasis should be placed on the important role which these chemicals have played in safeguarding the health of man and animals, and on the important contribution which they have made i n reducing tremendous economic losses due to pests of various kinds. Attention is directed to the papers in this volume which do place emphasis on the value and benefit to m a n k i n d . M o r e rapid, effective, and sound progress will be made i n the introduction of new economic poisons if we proceed cautiously. T o o hasty release of such products may boomerang and delay the progress of pest control for many years, with an adverse effect on the various groups involved. T h e inauguration and establishment of a research foundation to make possible a more comprehensive and organized study of the multiplicity of problems involved i n this rapidly expanding field is needed. Such a research foundation would act as a coordinating agency and as an organizational clearinghouse for all research in this field, and thus markedly promote efficiency and maximum productiveness of time and funds. Such a foundation would (1) collect, organize, and interpret information through n u merous subcommittees ; (2) determine the most critical needs in a research program and point out where priorities should be placed ; (3) arrange a subsidy program to promote research in the critical areas ; and (4) encourage educational programs through special committees. Other essential parts of the program as it is progressively developed may be implemented as rapidly as facilities and funds permit.
Subdivision on Economic Poisons

Interest in economic poisons has resulted in the formation of a new subdivision within the Division of Agricultural and Food Chemistry of the AMERICAN CHEMICAL SOCIETY.
This was authorized at the Atlantic C i t y meeting (September 1949) of the Society, and organization of the subdivision has subsequently been effected. T h e purpose of the subdivision as outlined by the committee is to promote interest, research, and publications on the chemistry of pest control materials, and to provide a means for the exchange of information and ideas i n this field. The interests of this group can be defined broadly to include ai phases of the chemistry of pest control materials. This encompasses chemical work on substances used to control, mitigate, or destroy pests of all kinds, such as insecticides, insect attractants and repellents, fungicides, herbicides, bactericides, rodenticides, and substances for related uses. Work dealing mainly with the application of these materials and their biological properties properly belongs in the fields of entomologists, plant pathologists, plant physiologists, bacteriologists, agronomists, rodent control specialists, etc., and are not of p r i mary interest to the new subdivision. The group will furnish a forum for research workers i n chemistry and other scientific fields to discuss problems of mutual interest, and develop a better concept of the many problems involved through the exchange of information and viewpoints. There has been much discussion regarding the name which should be used to identify the new subdivision and the field of work covered. A referendum is now i n progress to assist i n the selection of a suitable name which is both inclusive and exclusive. Because a decision will not be made until the fall (1950) meeting of the Society, the use of the name "Economic Poisons" is being continued for the subdivision, at least until that time. Thanks and appreciation are expressed to the many individuals and groups whose generous cooperation and help have made possible the organization of both the symposia and the new subdivision of the Society. The subdivision on economic poisons appreciates the privilege of presenting the p a pers included i n the San Francisco and the Atlantic C i t y symposia as the first number of
the new A M E R I C A N C H E M I C A L SOCIETY A D V A N C E S IN CHEMISTRY SERIES.
World Use of Economic Poisons

STEPHEN S. EASTER Food and Agriculture Organization of the United Nations, Washington, D. C.
This paper points out a few simple but fundamental reasons why expansion of the world use of economic poisons is being seriously retarded. These remarks are based on personal observations in twenty different countries of North America, South America, Europe, and Africa, and on contacts with technical men in as many more countries.
It is obvious that the world use of economic poisons can be greatly expanded; no a t tempt will be made to say how much. The use of pesticides is frequently associated with the spectacular nature of a pest or the urgency of meeting its attacks. Grasshopper or locust campaigns are, generally speaking, easy to sell to the administrative powers because these periodic pests destroy all the crops attacked. Quick action must be taken to save the crops. O n the other hand, continual annual crop losses from other pests may, over a period of years, equal or exceed those caused b y locusts, but these losses are accepted as a matter of course because only a part of the crop is lost each year. I n 1945 three Central American countries imported 38,500,000 pounds of copper sulfate-and only 75,000 pounds of all other pesticides. T h e copper sulfate was used almost exclusively i n the preparation of Bordeaux mixture for the control of the sigatoka disease of bananas. Without this control measure, the bananas could not have been grown profitably. During 1948 i n the same countries the imports of certain other pesticides increased greatly because of the need for locust control. I t is to be expected that, after the locust outbreak subsides or is controlled, these imports will drop again. The use of economic poisons has expanded i n the field of public health since their indispensability has been shown i n the control of insect-borne diseases. T h e organic insecticides, especially D D T , are now being used extensively i n the control of malaria, yellow fever, dysentery, and other diseases. Their use i n this field will certainly expand for years to come. Lea Hitchner of the National Agricultural Chemicals Association estimates an annual use of $200,000,000 i n pesticides for the United States. I n spite of the fact that the United States has been using more pesticides than any other country, the amount used has increased steadily. There is still a likelihood of expansion here. The introduction of the Colorado potato beetle into various European countries has created a potential market for insecticides. Its recent spread into Germany and Poland has been followed b y a marked increase i n the use of various kinds. The past history of this insect indicates that steadily increasing amounts of insecticides will be needed annually i n order to control i t . More examples could be given to show potentially greater use of economic poisons, but all would show the same trend. The manner i n which the world chemical industry quickly produced vast tonnages of D D T during the war was amazing. E v e n more so was the efficiency achieved i n production, so that entomologists had an insecticide available at a price which permitted its
5
use. The chemical industry is still far ahead of the entomologists. New insecticides are being produced more rapidly than the entomologists can test them. The same can be said for other pesticides used to combat plant pathogens or weeds. The new chemicals must be properly tested and evaluated before being released as pesticides. M u c h cooperative work is needed. After all, D D T was only a chemical entity for over 60 years before its insecticidal properties were discovered. Today, i n most of the world, application of the presently known good pesticides is needed far more than discovery of other new ones.
Work of the FAO

These observations relating to the use of economic poisons have been made through the work of the Food and Agriculture Organization of the United Nations. F A O is an autonomous body of 58 nations concerned with all phases of food, agriculture, forestry, and fisheries throughout the world. It was conceived because, i n peace or war, the majority of the world's peoples are not amply fed and on the premise that food is basic to human well-being and world peace. F A O is not a relief agency. It does not purchase or distribute food products, although it is much concerned with food deficits and food surpluses. Neither is it a research agency, though it does try to enlist the interest of scientists i n its fields and coordinate their efforts in the solution of pressing problems. Finally, of course, it is not a political organization, but operates as an international extension agency on a strictly impartial basis. A t the present time many nations are sharing their food with other less fortunate n a tionsless fortunate because of the devastation of war, drought, poor crops, or other reasons. There seems to be no other immediate solution. B y sharing the scientific knowledge now available for increasing food production, the devastated and underdeveloped countries can be enabled to increase their own food production. It is true that some nations will probably always be partially dependent on imports of food, but i m proved methods of production, processing, and storing will reduce the degree of dependency. The dissemination of technical information is a major responsibility of F A O . It may be done i n various ways, according to the problem. F A O is primarily concerned with problems relating to food. Entomology is so closely associated with the production and saving of food that this phase of work is obviously i m portant to the Agriculture Division of F A O . The field of agricultural entomology i n the world is so broad that an extremely large staff would be required if F A O attempted to cover the entire field. The entomological staff is small and its coverage is limited. However, it is not the purpose of the staff to conduct research or to participate i n action programs. A few specific problems of world-wide interest have been selected for study. These i n clude the pests of stored food, the Colorado potato beetle, the Moroccan locust, and the field insects of China. I n addition, advice may be given on control of introduced insects. The technical knowledge of America and western Europe is drawn upon freely. The task of F A O is to assist the member nations, especially those least developed; consequently, this section becomes a kind of international entomological extension service.
Stored Food
Attention has been focused by F A O on the tremendous losses in stored food over the world caused by insects, rodents, and fungi. International meetings have been held on the subject i n Washington, D . C , London, England, Florence, Italy, and Cali, Colombia, where technicians from many countries have gathered to study the problem and exchange information on means of solving it. I n addition, field trips have been made to different countries to study grain storage. As a result of these meetings and field trips some progress has been attained. The potential use of economic poisons i n this specialized field may be stated as follows : The use of fumigants will increase, but no faster than the construction of improved storage facilities which will permit the efficient use of fumigants. Residual insecticides will i n crease i n use for many years, but the total tonnage will not be high. I n a few countries,
EASTERWORLD USE OF ECONOMIC POISONS
insecticides are being added directly to grains to ensure protection against insect attack. A trend i n this direction could result i n a great increase i n the use of economic poisons. Two factors are delaying i t at this timecost and hazard to the consumers. The millers resist any addition of foreign material to the grain because they have to take i t out again. I n the small subsistence-producing nations this method appears to hold much promise. A general use of rodenticides i n grain storage plants would not require many tons of chemicals and would not be much of a factor i n expanding the world use of economic poisons. The heating of grain i n storage has been controlled by the use of certain chemicals added to the grain, but the cost is still too high for practical use. The fungi are controlled by drying and there is little likelihood of a market here for economic poisons for many years to come. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch002
Costs
Further remarks regarding costs of insecticides or inadequacy of equipment used are generally applicable to the whole field of pesticides and are not limited to their use in grain storage only. W h y is the world use of economic poisons being retarded? The price paid for the pesticide b y the ultimate consumer is frequently prohibitive i n importing countries. The quality of the pesticides obtained from the major producing countries is excellent, but this quality is of little value if the price prevents its use. A few examples may be quoted to demonstrate this point. In one European country where field insecticides are not generally used but are very promising, 5 0 % wettable D D T of American manufacture was purchased i n midyear 1948. The retailer was a large cooperative with presumably favorable prices to consumers, yet the price to the consumer i n that country, at the official rate of exchange, was roughly $1.50 per pound. A t the same time this identical material was selling f.a.s. New Y o r k at $0.23 per pound. It is granted that the price per pound must be higher to compensate for freight, handling, selling costs, etc., but there is no justification for an i n crease of 500%. The inevitable result is either the refusal of the imported material as an aid to agriculture or, i n case of emergencies, a trend toward establishment of small local manufacturing plants which are made secure i n their inefficiency by protective tariffs. The end result is the same to the consumerprohibitive prices. The example given for Europe can be repeated for numerous other countries and on other economic poisons. The differential may be as great i n a few places. I t is not found on all poisons. The correction of this one factor lies mainly i n the sales organizations responsible for supplies to the foreign markets.
Equipment for Pesticide Application

Pesticides and the equipment with which they are applied are so fundamentally associated that i t is astonishing to see how often these two items are separated. Pesticides are frequently sold i n a foreign area with complete disregard of the available means of application; yet the whole future market depends mainly upon the proper distribution of the materials. The want of suitable equipment can be a very serious factor i n retarding the use of economic poisons. There are many examples of equipment being sold i n areas for which i t is not suited. The unsuitability may be a lack of trained operators, a lack of basic knowledge of the equipment's possibilities, equipment inappropriate to the terrain, or, occasionally, equipment so highly specialized that i t cannot be adapted to the general use for which i t is needed. I n general, simple equipment can best be used to demonstrate the value of the use of pesticides. One specific piece of equipment, which had to be mounted on a vehicle, was extensively sold for locust control i n an underdeveloped country. The roads i n this area were not passable to trucks and the machine was mounted on a jeep. The machine was too heavy for a jeep, so the net result was a broken-down jeep and a piece of specialized equipment shaken to pieces b y travel over almost impassable terrain. A d d to this the complete lack
of operators with mechanical training and a lack of appreciation for dosage per acre, and the final result is either of two things : When the equipment is working the amount of i n secticide applied per acre greatly exceeds the needs, and when the equipment is not working a large crew of workers and other vehicles are inactive for long periods. Simple oneman dusters would have done a far better job at a greatly reduced cost. The value of the insecticide also would have been better demonstrated. In some areas where grapes are raised on a large scale the use of small one-man sprayers is well established for the control of disease. In Southern Europe, for instance, these sprayers have been designed for the use of i n expensive fungicides, typified by home-mixed Bordeaux mixture. Neither the nozzles nor the guns are suitable for the use of high-priced organic insecticides or fungicides. I n order to get the proper application of the new pesticides, suitable nozzles and guns must be used. A t present the flow rates are too high and the spray patterns are far too coarse to give desirable coverage with such insecticides as wettable D D T or benzene hexachloride. The guns are frequently a plain quarter-turn valve, rather than a simple gun with a mechanism for permitting the quick release and cut-off of flow. The only justification offered for a quarter-turn valve i n place of a gun is the very minor saving i n the original cost. This is not good economics, because the savings i n the original investment are quickly lost by the wasted pesticides of a few days' field operation. Theoretically, when spraying fruit trees the operator always turns off his quarter-turn valve when walking from tree to tree. I n practice, however, this is frequently not done because both the operator's hands are occupied otherwise. It becomes only too obvious that many of the designing engineers have not used, under practical conditions, the equipment they have designed. Equipment of this type is being sent into South America. There are many other small factors that affect the operation of pesticide-applying equipment. There is, unfortunately, a tendency on the part of many people to forget the basic reason for sprayingthe control of plant pests. The chemical industry should keep i n mind that pesticides, no matter how excellent their quality, or how abundant their quantity, are useless in a warehouse and are of value only after they are applied i n the field. The means of application is therefore a very fundamental consideration i n the expansion of economic poisons. I n recent years there has been a great increase i n the degree of cooperation between the engineers designing equipment and the scientists who are concerned with application. The lack of technicians i n underdeveloped countries is a serious handicap i n the i m mediate expansion of the use of economic poisons. The salesmen representing the producers commonly tend to exaggerate the merits of their products when selling i n the foreign market. Claims are made without scientific investigation to substantiate them. Such practices are particularly harmful because of the lack of technicians. Help is needed from technicians who can work on the problems i n other countries, not at home. There is, for example, a very large potential market for an insecticide that will control leaf-cutting ants. These insects are one of the major entomological problems from Mexico to the Argentine. A simple cheap method of control is needed. There are several promising leads, but almost nothing is being done to find such a method. The labels of pesticides going into foreign markets should receive careful attention to guard against misunderstanding the directions. There is likely to be a language difficulty, and even i n the foreign language the directions should be clear and concise. A n d the change to the metric system should not be overlooked. M u c h misunderstanding can be avoided by sending technical men to give first-hand information where export is desired. Some customer service in foreign markets by technicians would pay excellent dividends in the increased use of pesticides. The world use of economic poisons can be expanded. There are simple basic factors retarding this expansion: prohibitive consumer prices, lack of equipment with which to make proper field application, lack of skilled personnel in countries needing the benefits of economic poisons, exaggerated claims of nontechnical salesmen, and difficulties involved i n labeling the export products.
Insecticides in Agriculture
S. A. ROHWER
Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, Washington, D. C.
There is general agreement that destructive insects cause tremendous losses; the figure currently used is $ 4 , 0 0 0 , 0 0 0 , 0 0 0 for an average year. Insect control presents many problems, the simplest of which is the correct concentration and rate of application. For development of insecticides cooperation of chemists, entomologists, and toxicologists is essential.
Estimates of losses caused by insects have been assembled by different workers at various times and i n various ways, but none have attempted to consider all the destructive species. N o t all insects are destructive; i n fact, many kinds are beneficial. N o summary has been made of the value of insects. A i l estimates of losses have dealt largely with the destructiveness of the better known important species that attack crops and products needed by man. The losses reported i n earlier estimates were restricted to comparatively few species. Large as these were, they were much less than the currently used figure of $4,000,000,000 for an average year. The wide difference between the estimated losses of today and those of some fifty years ago is due to many factors. Some introduced insects have become important destructive pests since the earlier estimates were made. Agricultural conditions have changed during this period and many native species are now causing heavy losses to crops which replaced their native hosts. I n storing and handling our agricultural products we have not taken steps to prevent losses. Some current practices make the products more susceptible to attack by insects and encourage multiplication of the pests. The public has become insect-conscious and rejects products which are infested. This increases loss and raises the standard for control. W o r m y apples, insect fragments i n cereals and canned products, are no longer acceptable. M a n y other changes i n our habits and methods of agriculture could be cited to explain the wide difference i n estimates that have been made of the losses that insects cause to agriculture. Irrespective of the method used i n preparing estimates of these losses, all investigators agree that the losses are tremendous. A few examples to illustrate this destructiveness have been taken from a report of hearings before a Congressional committee (1).
Crop Cotton Corn Stored small grains Balsam, fir, and spruce Ponderosa pine Apples Citrus Beans Onions Tobacco Cattle Pest Boll weevil Earworm Corn borer Weevils and moths Spruce budworm Black Hills beetle Codling moth California red scale Mexican bean beetle Onion thrips Hornworm Cattle grubs Period 1909-29 1946 1945 1946 1947 1910-20 1947 1940-44 1943-44 1944 1944 1944 1940-44 Average Annual Loss Dollars 256,015,000 319,349,000 140,000,000 37,000,000 600,000,000 4,500,000 15,000,000 50,000,000 10,000,000 5,502,000 14,500,000 84,073,000 160,000.000
10
Benefits from Insect Control

Important as estimates of losses may be, information on the prevention of losses is of greater interest and significance. Measures developed by entomologists for the control of many important pests involve the use of several different procedures: following certain farm practices, using cultural methods, distributing natural enemies, applying insecticides, and using mechanical devices. Information on how control may be attained has been disseminated by many means, and an effort has been made to encourage farmers and others to use them. The extent of use has varied greatly and i t is well recognized that full advantage is not taken of what is now known on how to prevent losses caused by insects. A few illustrations of controls requiring the use of insecticides will show the benefits that have been derived where control measures have been applied. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch003 I n 1947 growers i n Louisiana who applied measures to control wireworms increased the yield of cane b y 20 tons per acre and the acre yield i n sugar b y 3500 pounds. I n 1947 the yield of potatoes treated with D D T insecticides increased as much as 3 0 % above the yield i n untreated fields. In U t a h the production of alfalfa hay has been nearly doubled i n areas where insecticides were applied ; and i n areas where alfalfa seed was produced the yield has increased by 6 0 0 % b y the timely application of insecticides. I n the latter case i t meant the difference between a loss and handsome profit. In 1948 the use of D D T insecticides for the control of the hornfly so increased the production of milk and meat that the county agents i n several states estimated the benefits to be worth $54,000,000. The 1946 cooperative control program against grasshoppers saved crops on 5,666,000 acres at an estimated value of $41,000,000. F o r each dollar spent $52 worth of crops was saved. The aerial application of D D T insecticides to 413,000 acres protected^ 1,500,000,000 board feet of lumber at an estimated stumpage value of $4,600,000. The estimated cost of protecting apples from codling moth is $25,000,000it permits marketing a crop with an average annual value (1931-35) of $110,000,000. Without control the crop would be largely unmarketable. I n the years 1947 and 1948 the growers of lima beans i n a single county i n California increased their returns b y $7,500,000 by controlling wireworms.
Cost of Insect Control

Satisfactory figures on the annual expenditure by growers for insecticides are not available. This varies from season to season, depending on several factors i n addition to the intensity of insect infestations. I t has been estimated that for the calendar year 1934 farmers and others paid about $25,000,000 for the more commonly used insecticides; for the same materials the cost i n 1948 would have been approximately $28,000,000. During this 14-year interval the use of agricultural insecticides had more than doubled. The estimated expenditure for insecticides i n 1948 approximated $60,000,000. Interest i n and appreciation of the benefits of insect control are thus apparent. Expenditures for insecticides alone are not an accurate index of the cost of insect control. M a n y control practices do not involve the use of chemicals. Where chemicals are used considerable labor is required. T o this must be added operation, maintenance, and investment costs of equipment needed to apply them. When these are taken into account the cost of control increases several times. I n 1938 Hyslop (2) estimated that the cost of controlling the more important insect pests was $142,927,000.
Problems of Insect Control

The control of insects is no easy or simple task. E v e n for those species that are well known and for which control measures are fairly standardized, many things have to be considered. I n cases where the suitable insecticide is known, there is need for accuracy i n using the correct concentration and rate of application. T h a t is usually the simplest part of the operation. T i m i n g the application may mean the difference between success and
ROHWERINSECTICIDES IN AGRICULTURE
11
failure. Proper application is also essential. N o t infrequently the task of control is further complicated by the presence of several pests. This may require knowledge of the compatibility of several materials, some of which may be applied at the same time, others a few days later. W i t h these and other factors, and the variability that is associated with ail biological activity, there is need to keep i n mind the effect the materials may have on the plant or animal and the product which is being protected. Are the materials safe and usable for the purpose for which they were intended? is an ever-present question. Entomologists have had these and various other questions before them i n developing measures for control. They are fully aware that answers cannot be determined i n short order, and that the solution of a problem for one area does not mean that the answer is applicable to all parts of the country. Experimental work has to be done when the insects are present and during the limited active period of the stage of development when the treatment may be applied. This frequently means that experimental work can be done only during a few weeks within a year. Standard control practices developed within these limitations have taken all the v a r i ous factors into consideration. Entomologists recognize the inadequacy of many of the recommended control measures. They have initiated the effort to develop new and more effective insecticides and welcomed the interest and assistance of others. The recent accelerated development of new insecticides has opened new fields and done much to stimulate public interest i n insect control. M u c h more needs to be learned than to know that the new materials will k i l l insects, before their true worth can be determined. We must determine many things. Knowledge of the effect on beneficial insects, wild life, soil, and machines is important. Of greater importance is: Are they safe for the operator and for those who handle and use the product?
Cooperation in Development of Insecticides

M a n y of the new insecticidal chemicals are complex organic materials with long and complicated names. There is need for simple common names. Without simplified terminology costly losses may result from relatively minor errors i n nomenclature. Coined names must be protected if they are to remain available for general use, and must be defined with sufficient accuracy to assure that a standard product will be marketed. Selection of such names requires cooperative consideration b y many agencies. Chemists can make important contributions to this undertaking. The complexity of the new insecticidal chemicals brings many other problems. S y n thetic organic chemicals are not effective against all pests. There is a marked selectiveness i n action even between closely related species of insects. Some insects have already developed resistance to some of the newer materials. The idea of insects developing resistance to certain chemicals is not new. The over-all principle is well established i n a few cases. The early development of flies resistant to D D T , a chemical which had been highly and universally effective for fly control, came as a surprise. Other cases of resistance to D D T are being indicated, and at least one kind of mite has developed resistance against another of the newer chemicalsparathion. W i t h chemicals that are as highly toxic to insects as some of the newer ones are, other problems arise i n the development of methods for their use. When only a small amount of a chemical is needed, ways must be devised to apply i t . The first of these is to determine how the chemical can be formulated into an insecticide. This is not a task for the chemist alone, because not a l l solvents, carriers, and diluents, even though suitable from the physical-chemical point of view, are equally effective entomologically. Here the chemist and entomologist must work together. The more closely and understandingly they cooperate the sooner the answer will be developed. When the new materials are developed, there is still the need to determine that they can be effectively and properly applied. This requires investigations i n the field of application equipmentsprayers, dusters, etc. The standard machines for handling large volumes of insecticides are not suitable where the dosage is reducedas i t can beto amounts as low as a gallon of finished insecticide per acre.
12
The development of new insecticides means even more. It requires appraisal of the effect of the material on the operator i n all stages of use. It involves knowledge of the amount of residue that may remain on, or in, the part of the product that is used as food for man or beast and the effect of such residue on their health. Leadership i n such studies belongs to the toxicologist. The chemist, however, has a very important relation to these problems. He must supply the method for analysis and for removal of insecticidal residues. F o r some of the new insecticidal chemicals the entomologist has accurate information on their effect on insects. Suitable, satisfactory methods of analyses of the chemical and its residues await determination.
Outlook for Insect Control

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch003 The field of insect control is expanding; the need for it is better recognized and a p preciated. Agricultural development will i n future depend on pest control much more than i n the past, and it will be a salient part of the production and protection of agricultural commodities. Chemicals are essential for the control of many major pests. It is highly important that chemists whose part i n the cooperative effort is of increasing i m portance, increase their interest i n and understanding of the wide variety of problems. Entomologists welcome the opportunity to work closer with the chemists. The author, for one, is delighted with the expanding opportunities for developing the cooperative teams needed to find sound, safe, effective, economical answers to the numerous problems of insect control.
Seasonal Market for Insecticides

I n general, the use of insecticides is seasonal, and the demand for them fluctuates greatly because the intensity of insect infestation differs widely i n various years. Peak seasons of infestations may be separated by years. The market for insecticides is, therefore, highly fluctuating; and success i n the business of producing and marketing insecticides depends i n no small measure on the ability to have the insecticide available at the time and the place needed. Production and marketing of insecticides offer a greater challenge to management and judgment than do many other fields i n the production of chemicals.
Literature Cited
(1) Committee on Appropriations, House of Representatives, hearings before subcommittee on Department of Agriculture Bill for 1949. (2) Hyslop, "Losses Occasioned by Insects, Mites, and Ticks in the United States," Bur. Entomol. Plant Quarantine, Bull. E-444 (1938).
Some Problems in the Use of Newer Economic Poisons

G. F. MacLEOD
Sunland Industries, Inc., Fresno, Calif.
Some of the economic problems that confront the chemical industry and some practical problems of agriculture and society in general, resulting from the use of newer economic poisons, are discussed. Above all else, more facts are needed upon which to base both present procedures and future studies.
N o adequate discussion of even a single major problem associated with the newer chemicals can be offered without neglecting equally important and related problems. Actually, the titles of the papers presented i n this symposium reflect the complexity of the situation resulting from the rapid development of sharp chemical tools for combating pests. This paper discusses, first, some of the economic problems that confront the chemical industry and, secondly, some practical problems of agriculture and society i n general, which are associated with these newer products. There may be little that is new i n such a procedure, other than to bring well known facts into a relationship that appears to be somewhat vague. There is a recognized need for a clearer understanding between research workers i n public agencies and the men whose task i t is to make available the products that are needed i n a progressive society. The struggle to maintain a working equilibrium becomes more intense as the numbers of products multiply and the speed of development is increased.
Economic Problems
The costs of developing and producing new economic poisons are well known to the men of industry. T o the average research worker, particularly i n agriculture, these costs are merely words. They are, however, definite segments of the problem of availability and use. N o social contribution has been made by either institutional research worker or chemical manufacturer until people have found a use for a product and are willing to purchase i t because i t will do something safely, more efficiently, at less cost, or more agreeably than compounds previously used. The newer insecticides and fungicides have these qualifications. Basic manufacturers of chemicals and local formulators have some mutual problems and each group has difficulties peculiar to itself. The costs of screening new products, trying to find one useful compound among thousands that prove to be useless, the expenses of pilot plant operations, conversion to plant production, product control, getting product acceptance, working out details of insurance, and safety precautions as well as general advertising, sales, and administrative costs give the basic manufacturer many a headache. The continuous fluctuation i n prices of raw materials, machinery made obsolete b y new products or processes, new packaging requirements, provisions for meeting the nearly 500 laws under which the industry operates, all contribute to the ultimate cost that a consumer must pay.
13
14
The local formulator who purchases concentrated products from the manufacturer has his problems. M o r e frequently than not the advent of a new product catches him with a stock of some older chemical which the new one will replace. Lead and calcium arsenates, rotenone, pyrethrum, sabadilla, and others a l l have lost some usage to the newer economic poisons. Costs such as these cannot be passed on to consumers because of that grand American institution, competition. Concentrates of new chemicals are always expensive, but they frequently change rapidly i n price. Picture the position of a small but locally important mixer of insecticides remote from sources of supply, who suddenly finds himself with a substantial i n ventory of a new chemical purchased at 15 to 2 0 % above a newly announced price. H i s competitive position becomes what is known as "poor." W i t h many of the newer economic poisons manufactured i n the East, west coast formulators find themselves at a disadvantage with respect to both freight rates and supplies. .Everyone realizes the almost impossible task of estimating the amounts of insecticides or fungicides that will be used i n a given year, particularly where new compounds are concerned. There is yet to be discovered a formula by which the annual guesstimate of "how m u c h " can be solved. The end result is that most smaller, local mixers play conservative and sacrifice possible savings i n carload prices, supplies, and sales, by purchasing l.c.l. lots. This is, of course, doubly true where costly compounds are concerned. These factors all enter into the ultimate price to the consumer. The local manufacturer shares with the basic supplier the problems of machinery changes, safety precautions for operating personnel, developing useful local formulations, product control, product acceptance, and the usual administrative, advertising, sales, and delivery cost. Furthermore, these secondary manufacturers, despite ever-increasing help from the more progressive manufacturers, must work out many problems of labels, containers, and compliance with local laws or regulations concerning manufacture and sale of new formulations. A l l this takes manpower, time, and money. If these local institutions are to continue filling the long-established needs which their services have merited, as evidenced b y the demand for the goods they produce, the costs of those services must be reflected i n the price the consumer pays. The increasing demand for personnel adequately trained to cope with the use of new chemicals does not lessen the costs. A l l these facts and many more are well known to any commercial man i n the insecticide or fungicide industry today. When agricultural scientists and advisers talk with growers, they frequently overlook many of these factors. On the other hand, the men of industry frequently forget some of the complex problems associated with the widespread use of potent chemicals. The processes of nature cannot be hurried and most of the knowledge we have is relatively fragmentary. Practically every known agricultural practice is subject to continuous scientific scrutiny, with the result that periodic revisions are almost the rule. If our oldest concepts are so subject to change, how very little we must know about these five-year-old marvels. I t is natural for everyone involved to be concerned with the possible hazards which may be incurred as a result of volume use of newer economic poisons.
Harmful Residues
One of the first considerations i n the use of any chemical is possible accumulations of harmful residues i n soils. Evidence proves beyond any doubt that many of the newer compounds remain i n the soil for at least 5 years. H o w much longer they may persist, time alone can determine. We know that both selenium and molybdenum can be picked up from soils by plants, which thus become extremely toxic to animals, even when plants themselves are apparently unharmed. Often a given piece of land may be treated safely as far as one crop is concerned but another crop may be injured. F o r example, potatoes will tolerate large amounts of D D T i n the soil as a means of controlling wireworms which are extremely destructive; melons, on the other hand, are severely injured by excessive D D T i n the soil. There can be no assurance i n many cases as to the ultimate use of any
MacLEODSOME PROBLEMS IN THE USE OF NEWER ECONOMIC POISONS
15
piece of land, so the implications are clear. A similar situation exists i n the case of sweet potatoes, which may be treated with benzene hexachloride (hexachlorocyclohexane) soil applications, whereas white potatoes are very susceptible to taste changes from even small amounts of the chemical i n the soil. The long-time effects of chemicals i n soils on microorganisms are of sufficiently serious import to slow down the most enthusiastic sales manager. The widespread use of economic poisons has a definite impact on the animal complex on the face of the earth which provides our sustenance. Already we have seen the use of D D T for codling moth control on apples result i n a relatively minor pest becoming a serious threat. The same material used as a wonder spray for fly control now fails, after a couple years of common usage, with the appearance of new, resistant strains of flies. Bees and other pollinating insects as well as helpful predators or parasites may be decimated and their important aid be lost by untimely or improper use of most of the newer insecticides. The danger to domestic animals, including pets, is an important hazard i n the use of all newer economic poisons. Drift dusts or sprays from carelessly applied materials may set back the useful development of many valuable chemicals. Only recently have authorities finally decided that we should slow down on the use of D D T on cows until we know more about the occurrence of the chemical i n milk, butter, and steaks. Our sportsmen and, incidentally, a major economic factor i n our pleasant way of lifethe fish, game, and wildlife activitiesare part and parcel of the problem of chemical usage i n forests and streams. We need continued and expanded investigations of the effects of the newer pesticides on wild life. There is a tremendous financial investment i n our agriculture. T h e place which this country occupies i n world leadership today is due i n part to our food-producing capacity. T o maintain that position at a time when portentous predictions are rife regarding an overpopulated world, we need ever-improved chemical aids, but we must, at the same time, impair neither the quantity nor quality of our orchards and vineyards. Frequently i t takes 10 to 20 years or even more before we see the ultimate effects of continued usage of chemicals on plants. The financial and social losses which can result from improper or unduly hasty use of chemicals should temper profits with caution.
Effect on Man
Finally, new and little-known chemicals present hazards to man himself, no less complex than the biological enigmas of agriculture. Plant workers, commercial pest control operators, dealers and feed store employees, growers, farm hands, food industry workers, and consumers all have the mutual problem of both acute and chronic toxicity. W i t h i n the past two years we have seen marked strides i n the fields of study which concern themselves with the toxicity to man of all the newer economic poisons. I t is self-evident that a compound designed to k i l l one living organism will affect other living things. W h a t we most need to know is the margin of safety between those organisms which we would k i l l and those which we would keep unharmed. M o s t of the newer economic poisons are actually less toxic than those which we have used i n volume for years. Cyanide, arsenic, strychnine, and nicotine have a much more fatal ring to the average person than D D T , benzene hexachloride, chlordan, or parathion. I t is what we do not know rather than what we know which concerns us most about the newer compounds.
Investigation, Education, Regulation

Obviously, some definite responsibilities must be met. They can best be met b y i n vestigation, education, and regulation i n that order. Investigation is a job for both i n dustry and government. I t is the cornerstone upon which education and regulation must be laid. Agricultural workers i n soil sciences, pathology, and entomology are well i n the forefront from the standpoint of investigation of the newer chemicals. They are aware of the values of such farm aids and the need for them. T h e plant physiologists have not kept abreast of the effects of these same compounds on plants. The establishment of both
laboratories of their own and fellowships at institutions proves the sincere interest of i n dustry i n helping to solve the many problems involved. There is a great need for more work on the pharmacology and toxicology of the new economic poisons with respect to both man and animals. The problem of educating the users of these new tools is a joint one, facing both i n dustry and the public instructional agencies. If and when clear-cut answers to the many questions that arise are forthcoming from investigators, the educational aspects of the problems at hand will be solved with great rapidity. Y o u cannot inform others until you yourself know the answers. It is impossible to legislate safety. The death rate from automobile accidents should prove that. Furthermore, no one can regulate or legislate wisely with insufficient facts at hand. Specific legislation regarding individual compounds is unnecessary and i l l advised; such rules and regulations will create more problems than they will solve. There is a defi nite need for some control over unscrupulous or misinformed persons who, perhaps u n wittingly, would expose themselves and the public to needless waste or hazards. A l l reputable industrial groups recognize this and would be the first to demand such protec tion for themselves. There now are adequate laws for this purpose; they need unification and simplification, not multiplication. The problems of proper usage of our newer, sharper chemical tools require closer i n tegration of efforts and a clearer understanding among the many groups involved. Above all else we need more facts upon which to base both present procedures and future studies. Continued and increasing contact and exchange of ideas together with intelligent plan ning of effort can speed our knowledge and our progress. There is more opportunity and more satisfaction i n our personal share of any task if we try to understand how our work meshes with that of other groups.
Labeling Requirements for Insecticides and Other Economic Poisons under Federal Law
W. G. REED
Insecticide Division, Livestock Branch, U. S. Department of Agriculture, Washington, D. C.
The Federal Insecticide, Fungicide, and Rodenticide Act affords added protection against the dangers inherent in the use of newer means of pest control. The manufacturer has greater responsibility for furnishing specific information as to how the product can be used effectively but without damage to the user, crops or animals, or the public.
T h e Federal Insecticide, Fungicide, and Rodenticide A c t regulates the marketing i n interstate commerce of insecticides, fungicides, rodenticides, and herbicides. Instruments and contrivances intended for trapping, destroying, repelling, or mitigating insects or rodents, or destroying, repelling, or mitigating fungi, are also covered b y the act. This law, which was enacted on June 25,1947, superseded the Insecticide A c t of 1910. The old law covered only insecticides and fungicides and did not control the marketing of such products as effectively as does the new law. T h e new legislation became effective for devices on June 25, 1947, for rodenticides and herbicides on December 25, 1947, and for i n secticides and fungicides on June 25, 1948. Tremendous progress has been made i n the development of pest-control measures i n recent years, and with i t has come an increased interest by the general public i n pest control. People are beginning to realize that it is no longer necessary to put up with pests that annoy man and carry disease, or continue to suffer heavy economic losses caused b y the many pests that attack livestock, growing crops, and forest and other agricultural products. Research has provided better tools and methods than were ever before available for combating these pests and research is continuing to improve them. Unfortunately, however, many of the chemicals used i n pest control are highly toxic to man and animals. Great care must be exercised i n handling the more toxic materials to prevent injury to man, useful animals, and vegetation. Contamination of foods with harmful residues must at all times be avoided. N o doubt highly toxic substances will always have an important place i n pest control operations, but they need to be used with great care, precision, and discrimination. The new law is intended to afford the public added protection to avoid the dangers inherent i n the use of these new and more effective means of pest control. I t places upon the manufacturer greater responsibility for furnishing the user specific information as to how the product shall be used to be effective i n controlling pests, and at the same time cause no damage to the user or his crops or animals. Containers of insecticides and other economic poisons must bear labels showing the name or brand of the article, the net contents, the name and address of the manufacturer or distributor, an ingredient declaration, an appropriate warning or caution statement, including the word " P O I S O N " (in red), the skull and crossbones, and a n antidote statement on highly toxic materials. Adequate directions for use must accompany each economic poison. Devices subject to the act are not required to bear labels; but if they
17
18
do, no false or misleading representations may be made on labels or i n accompanying literature.
Registration of Economic Poisons

Before an insecticide or other economic poison may be marketed legally i n interstate commerce, it must be registered with the U n i t e d States Department of Agriculture. T h i s procedure brings the product to the attention of the department before i t is tried out on the public. It gives the department an opportunity to examine the proposed claims and to question those which are likely to cause injury, before the product goes on the market, i n stead of waiting, as under the old law, until the product has been marketed, a sample of i t analyzed and tested (which might take many months), and court action instituted. T o obtain registration, an application, together with the formula and proposed labeling, must be submitted to the department. This material is closely scrutinized b y scientific personnel i n the various fields involved and, if the proposed claims appear warranted and the submitted material is otherwise i n compliance with the law, a notice of registration is issued. If the article is such as not to warrant the proposed claims, or if i t and the labeling or other material required to be submitted do not comply with the law, the applicant is notified of the deficiencies and given an opportunity to make necessary corrections. If he then insists, i n writing, that such corrections are not necessary and requests that the article be registered, the law requires that i t be done under protest. B e cause of the higher penalties that are imposed if a person or firm is found guilty i n court of marketing a misbranded or adulterated product that has been registered under protest, there is little interest on the part of manufacturers and distributors i n obtaining this type of registration. E v e n though a product is registered unconditionally, the responsibility of the person or firm marketing i t is i n no way changed thereby. Under the law, registration cannot be used as a defense for the commission of any crime prohibited b y the law. Furthermore, registration can be canceled at any time if, upon the basis of later information, it appears that the labeling, previously accepted, does not give the protection intended by the law. It is the policy of the Insecticide Division, when labeling for a new economic poison is submitted for registration, to require reasonably conclusive evidence that the material can be used safely and effectively if the accompanying directions and precautions are followed. The marketing of pest-control materials before the hazards involved i n their intended uses are known can be a dangerous procedure. If reasonable doubt exists as to effectiveness or safety when proposed directions and precautions are followed, preparations should be handled only on an experimental basis. Shipments for experimental use only are exempt from the penalties of the law if used under the supervision of any federal or state agency authorized by law to conduct research i n the field of economic poisons, or by others if a permit has been obtained from the department before shipment. Before a new chemical is first offered for federal registration, the manufacturer has often carried on considerable research and spent large sums of money developing i t to the point where he considers i t ready to market. Generally, some tests have been made by federal and state research agencies, but these tests have not been conducted on a large enough scale or over a sufficient period of time to permit specific recommendations as to all the potential uses for the product or the care needed i n handling it. I n most instances there is considerable information available from one source or another concerning the material's effectiveness for some purposes. The acute oral toxicity to the usual laboratory animal is frequently fairly well known, but adequate data, concerning chronic toxicity are in many cases lacking. Chronic toxicity studies require long periods to complete and are very costly. When sufficient information is available concerning a substance to justify releasing i t to the general public, labeling containing adequate directions for use and necessary precautionary statements should be prepared and affixed to each package. Users of economic poisons should be educated to follow directions and observe precautions i n every instance
REEDLABELING REQUIREMENTS UNDER FEDERAL LAW
19
unless otherwise advised by competent authorities. M a n y of the new complex economic poison formulations must be handled like precision instruments. Careless use invariably leads to trouble. , E v e n when the manufacturer has carried out extended studies of his product, practical use and further experimental work will sometimes uncover faults or previously u n suspected dangers. I n such cases, appropriate changes should be made on labels without delay.
Degree of Toxicity
The regulations for the enforcement of the Federal Insecticide, Fungicide, and Rodenticide A c t set specific dosages b y which formulations must be judged to determine the exact labeling requirements for only the "highly toxic" class of substances. Briefly, the highly toxic category is limited to that group of the economic poisons which will kill 5 0 % or more of the laboratory animals (mice, rats, or rabbits) when the substance is administered at the dosages and i n any one of the manners described below, when ten or more animals of each species are used for assay : 1. Orally, 50 mg. per kg. 2. B y inhalation, 200 parts of a poison per 1,000,000 parts of air b y continuous inhalation for 1 hour or less. 3. B y skin contact (rabbits only), 200 mg. per kg. by continuous contact with bare skin for 24 hours or less. Economic poisons which have acute toxicity of varying degrees below the highly toxic' category have been segregated somewhat arbitrarily into three additional groups. T h e first might well be called the one requiring a " W A R N I N G : M A Y B E F A T A L I F S W A L L O W E D " type of label, and includes mixtures which are less hazardous than those i n the "highly toxic" group but are still toxic enough to be somewhat hazardous to handle. M a n y economic poison formulas which are not toxic enough to require the word " P O I S O N " (in red), the skull and crossbones, and an antidote statement are capable of causing injury and must carry suitable precautionary labeling for public protection. The second group is one i n which formulations having minor hazards are placed. This class may be characterized b y " C A U T I O N : H A R M F U L I F S W A L L O W E D " type of designation. The final group is the rather small class of compounds which have a relatively low toxicity and require no precautionary statements. I n this class are a number of products for which claims of absolute safety cannot be used logically because some injury to beneficial plants or animals remains. These major categories are based largely on acute toxicity records, but the significance of chronic poison hazards to the adequacy of " w a r n i n g " and "directions-for-use" labeling is given full consideration whenever it is a factor for public protection. N o specific wording for precautionary labeling has been felt to be necessary or a d visable, but certain patterns of suitable statements are being prepared by a number of groups, notable among which are the Labels and Precautionary Information Committee of the Manufacturing Chemists' Association and the Committee on Toxicity and A n t i dotes of the Association of Economic Poisons Control Officiais. The Insecticide Division maintains close liaison with these groups and others interested i n the problem, and acceptable recommendations are being worked out. Labeling will, of course, never be a static proposition, because initial warnings and directions for use will be subject to change as more precise information on economic poison formulations becomes available.
Federal and State Regulations

One of the difficulties encountered b y producers of economic poisons has been the preparation of labeling which will comply with the laws and regulations of both the Federal Government and the various states i n which the product is marketed. This difficulty has been aggravated by differences i n interpretation by different enforcement officials. The Insecticide, Fungicide, and Rodenticide A c t recognizes this difficulty and authorizes the
20
administrative authorities to cooperate with state regulatory agencies i n carrying out the provisions of the law and i n securing uniformity of regulations. F u l l advantage is being taken of this authorization through both cooperative arrangements for enforcement work and discussion with representative groups of state officials. I n consultation with the Executive Committee of the Association of Economic Poisons Control Officials, a set of interpretations of the federal regulations has been prepared and published, so that the manufacturer and all others concerned can be governed accordingly. The closer cooperation between federal and state officials is tending to iron out differences and make the marketing of useful economic poisons on a nation-wide basis a much easier job.
Registration and Labeling of Economic Poisons in California

ALLEN B. LEMMON
Bureau of Chemistry, California State Department of Agriculture, Sacramento, Calif.
In almost 50 years of regulating the sale of economic poisons, the state of California has developed legislation to prevent sale of worthless products, to provide for adequate labeling, and to assure users that the products correspond to guarantee. Each product must be registered before it can be sold legally in the state, and conformity to guarantee is determined by analysis of samples.
Because the protection afforded users of economic poisons under the federal law applies only to materials shipped i n interstate commerce, state laws must provide adequate protection to users of economic poisons shipped solely within the state, and provide cooperative enforcement of the federal law. California, one of the first states to recognize the need for control, enacted legislation i n 1901 governing the sale of insecticides. This was later broadened to include, under the term "economic poisons," a l l materials used for pest control. I n the almost 50 years of regulating these materials i n California the law, which is the Economic Poisons Article of the Agricultural Code, has been modified from time to time, but the basic purpose has been to prevent sale of worthless products, to provide for adequate labeling, and to assure users that the products correspond to guarantee.
Registration
The law provides that each product must be registered before it can be sold legally i n the state. Registration acts as a screen by which worthless and untried products are withheld from the market. Registration may be refused, after hearing, for any economic poison which is of little or no value for the purpose for which it is intended, or which is detrimental to vegetation (except weeds), to domestic animals, or to the public health and safety when properly used, and there may be required such practical demonstration as may be necessary to determine the facts. When only a few standard chemicals were used i n pest control, it was relatively easy b y referring to published information to determine whether a product could be expected to do what was claimed for it. W i t h the development of the many new chemicals, such as D D T , benzene hexachloride, chlordan, tetraethyl pyrophosphate, and parathion, it has been necessary to adjust procedures to evaluate their usefulness and hazards. Although the experiment stations and other official agencies are still relied upon for unbiased data concerning these new materials, there has developed a trend for manufacturers to carry on their own research. M a n y manufacturers have extensive investigational staffs and the data they present are given careful consideration i n connection with registration of a new chemical. A manufacturer must not experiment at the expense of a user, but there is no objection to his giving away free samples for trial to develop information.
21
22
To be of most value to users of economic poisons, the label must carry adequate directions for use, proper statement of ingredients, and any necessary cautions. T h e directions for use must not only inform the user how to mix the material and the proper dosage to apply against a particular pest, but also must list the pests against which i t is effective. I t is important that the ingredient statement show both the name and percentage of each active ingredient, in order that a user may determine the relative worth of the products offered him. B o t h the federal and state laws permit options whereby the manufacturer is not required to disclose the amount of each active ingredient, but a few manufacturers take advantage of this, as it is good salesmanship to point out how much of a particular ingredient is present.
Precautions
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch006 Not all pest control materials are as poisonous to human beings as strychnine, sodium arsenite, hydrogen cyanide, and mercuric chloride, but almost every one presents some hazard when it is improperly handled. There are many types of hazards, and all must be considered if accidents and damage are to be avoided. There may be dangers during applicationfor example, a material may be flammable or i t may produce vapors or mists that are toxic to the operator. There may be dangers to the area treatedfor example, i t may injure valuable plants, or livestock, or wildlife. Danger is still possible after the material is applied. Residues on forage crops or food crops may be deleterious, or water supplies may be contaminated. Furthermore, danger may still lurk long after the job has been done. Disposition of empty containers presents a very serious danger i n the case of some distinctly poisonous chemicals such as sodium fluoroacetate, sodium arsenite solution, and lead arsenate. Particular care is necessary i n disposing of empty containers which have held 2,4-D herbicides, to avoid damage to valuable plants. Care is needed to make certain that leftover portions of material are not set aside i n improperly labeled containers or stored where they may cause injury years later. A n unlabeled container of a hazardous chemical is like a loaded gunor worse, i t is like a gun that isn't known to be loaded.
Conformance with Guarantee

When an economic poisons product is accepted for registration, the manufacturer is authorized to sell it i n the state. Official samples are drawn b y inspectors to determine if a product corresponds to guarantee. Approximately 8000 different economic poisons are registered for sale i n the state and it is possible, with present facilities, to sample and analyze only about one fifth of these materials i n any one year. Samples are always drawn from original containers that have not previously been opened, and they may be drawn from supplies i n the hands of manufacturers, dealers, or users. Reports of analyses are sent to the registered manufacturer, any dealer involved, and any user involved. A t the end of each year, reports of all samples are published i n a special publication of the department. Copies are available without charge upon request.
Analysis of Samples
The problems of regulatory chemistry were relatively simple i n the early days, or at least they now seem so i n retrospect. The analytical chemist was faced with only a few compoundsParis green, lead arsenate, lime-sulfur solution, nicotine dusts, and petroleum oil sprays. The complexity of chemicals used for pest control began to increase more rapidly 10 or 15 years ago, particularly with the development of many organic compounds designed for specific uses. Then the intensified research conducted during the war i n creased the number and types of pest control materials almost explosively. Twenty years ago there were 1600 products registered for sale i n California, ten years ago there were 3600, and today there are 8000. Applications for registration of approximately ten new products are filed every day. Although many of the new products contain but one active ingredient mixed with a solvent or a dust diluent, it is not uncommon for a product to contain five or six active i n -
LEMMONREGISTRATION AND LABELING OF ECONOMIC POISONS IN CALIFORNIA
23
gredients and several inert ingredients. T o complicate matters still further, the accelerated development and production of pest control materials frequently present the analytical chemist with a complex mixture to analyze before anything has been published concerning the chemistry of the new ingredients i t contains. The " u n k n o w n " samples i n a quantitative chemistry class were unknown mixtures of known compounds. A n official sample of a pest control product is actually an unknown mixture of unknown compounds. The guaranteed analysis offers a suggestion of the composition, but the chemist cannot assume that i t is correct. Each development i n pest control materials made b y the research chemist constitutes a challenge to the scientific skill and ingenuity of the analytical chemist. Methods devised for examination of certain mixtures of ingredients are seldom of sufficient general interest to warrant publication, although typewritten copies circulate freely between regulatory offices and the manufacturers concerned. The technical literature of analytical chemistry is continually consulted for assistance i n examination of pest control materials, and the author's laboratory regularly contributes data on problems of general interest. During the past year or two, papers have been published i n Analytical Chemistry on analysis of products containing D D T , benzene hexachloride, 2,4-dichlorophenoxyacetic acid, and phenothiazine. Papers have been published i n the Journal of the Association of Official Agricultural Chemists by chemists serving as referees i n development of analytical techniques. Some methods of less general interest are included i n an annual publication issued by the bureau, presenting results of analyses of official samples of economic poisons.
Bioassay
Examination of official samples is usually restricted to chemical analysis and determination of significant physical characteristicsfor instance, the guaranteed fineness of some dusts, and the boiling range and viscosity of petroleum spray oils. A bioassay laboratory is also maintained for examination of products for which chemical analysis is not possible or practicalfor example, no chemical method is yet available for examination of red squill, commonly used as a rat poison. The potency of such products can be determined only by feeding carefully apportioned dosages to a series of white rats. Colonies of houseflies, cockroaches, clothes moths, and other similar pests are maintained for testing the efficacy of different types of products. Cooperation of the chemical laboratory and the bioassay laboratory has not only been found effective i n examining products officially sampled, but i t has been of service i n developing analytical methodsfor example, bioassay with houseflies may readily determine traces of toxic chemicals left behind i n the separation of components by crystallization, distillation, or other means.
Misuse
Economic poisons are probably more closely regulated than any other class of material generally sold. I n addition to registration required before a product can be sold, and continuous sampling and investigation after i t is once put on the market, each reported case of injury is studied to determine if the material was at fault or if there was misrepresentation made i n recommendations for use of the product. Two cases of reported injury that were of particular interest come to mind. A grower reported that he had purchased lime-sulfur solution, but that i t was not labeled and the odor was not quite right. H e had sprayed seventeen acres of peaches and wondered if there was something wrong with the material, as it had seemed to burn his face more than usual when he sprayed it. Investigation revealed that he had accidentally been sold an unlabeled drum of sodium arsenite solution. L u c k i l y , he had done a poor job of spraying, i t had rained, and the injury was not as serious as would be anticipated when sodium arsenite solution is used at the rate normal for lime-sulfur dormant spray. The seller of the unlabeled material paid reasonable damages to the farmer and, after settling i n court, will probably never again sell an unlabeled drum of a pest control material. Another interesting case is that of a manufacturer who recommended use of 2,4-D weed killer as a selective spray for control of weeds i n young carrots. H e had previously
24
restricted sale of his material for control of weeds i n lawns, but wished to extend his business. Apparently he believed that if petroleum oil selective weed killers could be used on carrots, 2,4-D should also be suitable. Twenty acres of carrots were a total loss and a hearing was called for the manufacturer to show cause w h y his product should not be refused registration for having been sold for a purpose for which i t was not intended. The manufacturer paid the farmer for his damage, which amounted to about $2000 and promised henceforth to obey all provisions of the law, and the matter was settled. Sale of a deficient or misbranded economic poison is a violation of law, for which the seller is subject to prosecution as a misdemeanor. The maximum penalty is a fine of $500 or 6 months i n jail. Repeated violations are cause for revocation of registration. This is a very drastic penalty and is seldom required. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch006
Summary
E a c h insecticide, fungicide, rodenticide, herbicide, or other pest control product must be registered with the State Bureau of Chemistry as an economic poison before being offered for sale i n California. Registration may be refused, after hearing, for a product that is of little or no value for the purpose intended, or that is detrimental to vegetation (except weeds), to domestic animals, or to the public health and safety, when properly used. Labels should bear adequate directions for use, proper statement of ingredients, and any precautions or warnings necessary for proper use of the product. New chemicals for pest control are not permitted to be sold until data are developed to demonstrate that they can be used i n accordance with proper directions without undue hazard. About 8000 different economic poisons are registered for sale i n the state. Samples are drawn where materials are found offered for sale throughout the state and analyses are made to determine conformity to guarantee.
State and Municipal Health Department Requirements for Use of Common Residual Insecticide Sprays
EDWARD L. HOLMES and LLOYD J. SALATHE
American Institute of Baking, Chicago, III.
A questionnaire requesting the regulations applicable to the use of DDT and chlordan in food-manufacturing establishments was sent to 244 city and state health departments. It yielded 188 or 75% replies, 164 or 87% of which revealed the absence of any regulations of use of these products. Four state and twenty city health departments reported specific regulations. No regulatory actions were reported as resulting from the use of residual sprays and no instance of an injury or unfavorable results directly attributable to the use of residual sprays was reported. DDT and chlordan sprays may be used legally under carefully controlled conditions where there is no possibility of the food product becoming contaminated, except in Tennessee.
T h e r e is much confusion among food manufacturers concerning municipal and state restrictions on the use of D D T and chlordan residual sprays i n their plants. The attitude of the Federal Food and D r u g Administration revolves basically upon two sections of the federal law: Section 402(a), " A food shall be deemed to be adulterated if i t bears or contains any poisonous or deleterious s u b s t a n c e . . o r " i f i t bears or contains any added poisonous or added deleterious substance which is unsafe within the meaning of Section 406 " The applicable portion of Section 406 is : " A n y poisonous or deleterious substance added to any food, except where such substance is required i n the production thereof or cannot be avoided b y good manufacturing practice, shall be deemed to be unsafe for the purposes of the application of Clause 2 of Section 402(a) ; but when such substance is so required or cannot be so avoided, the administrator shall promulgate regulations . . .. " N o valid argument can be presented alleging that D D T and chlordan spray i n manufactured food products cannot be avoided. Therefore, this requires an interpretation of the Federal Food and D r u g viewpoint to be that no D D T or chlordan will be tolerated i n a manufactured food product. On the other hand, nothing i n this federal act prohibits the use of D D T and chlordan sprays, provided they do not actually contaminate the product. B u t i n addition to considering the federal viewpoint, food manufacturers must also follow state and municipal regulations. T o determine the attitude of such agencies, 244 questionnaires were sent to all state health departments and to health departments of cities whose population exceeds 50,000. The inquiries were : 1. Does your department have any regulations or restrictions concerning the use of residual sprays i n food manufacturing establishments? 2. Name the residual sprays regulated. 3. Describe briefly the type of regulation. 4. Have these laws or regulations actually been invoked i n any regulatory action?
25
26
5. H a s your department any record of injury or experience with unfavorable effects resulting from the use of these materials i n food manufacturing establishments? 6. If you have not done so already, are you considering issuing any regulations for the use of these products? Of the 188 replies received, 158 stated that these agencies have no regulations governing the use of D D T and chlordan, but 38 added that they are considering the issuance of such regulations. Presumably, each department i n this group of 158 depends upon the "standard adulterations clause," embodied i n most food laws, which prohibits the presence of any poisonous, deleterious, filthy, or decomposed substance i n a food product, as the means of regulating the use of residual sprays.
Control of Residual Sprays

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch007 Of the thirty departments which state they do have regulations controlling the use of residual sprays, six specifically stated that they rely upon the standard adulteration section of their food law. The remaining 24 agencies, including four state departments, have the following regulations: The state of California has a spray residue law (Agricultural Code, Section 1011) which sets tolerances for certain toxic substances including D D T . I n addition, the use of a l l residual sprays i n food manufacturing establishments is prohibited if its use can be avoided b y good manufacturing procedures (California Health and Safety Code, Section 6471). Administratively, a l l such materials are viewed with disfavor except those which have been proved harmless. The cities of Berkeley, L o n g Beach, San Jose, Glendale, Pasadena, and Sacramento follow the state regulations. California apparently permits the use of D D T and presumably other residuals under carefully controlled conditions, as evidenced by the existence of a California State Department of Public Health poster entitled "Use of D D T i n Food Establishments." This poster suggests that D D T be employed under carefully controlled conditions, which include the use of a nonmisting compressed air spray, limited to wetting surfaces remote from food supplies, utensils, and open flames under adequate ventilation. The oil solutions must not be allowed to remain i n contact with the skin. Although Colorado relies on standard contamination laws, the city and county of Denver specifically limit the use of residual sprays to those conditions i n which foodstuffs, utensils, and equipment are covered and protected. M i s t i n g sprays may not be used i n food preparation areas but are permitted i n dining rooms of eating establishments. Indianapolis regulates the use of D D T b y requiring that food and utensils be protected during spraying. This is required under the authority vested i n the health officer to regulate the use of harmful substances, including residual sprays. In Wichita, K a n . , residual sprays m a y be used after notifying the C i t y Health Department and taking protective measures approved b y the health department. Detroit, M i c h . , controls the use of D D T b y a regulation which requires that a l l food be removed from the area being sprayed. Kansas C i t y , M o . , Health Department restricts permission for the use of residual sprays to D D T until further studies have been made. Nevada's Department of Health prohibits the use of sprays on a l l surfaces with which the food product may come i n contact. This includes even pyre thrum sprays. I n Charlotte, N . C , a l l residual sprays are regulated, i n that they m a y be used only after establishments have closed for the day. A l l food must be protected during the spraying operation. The Portland, Ore., Bureau of Health prohibits the use of toxic chemicals that might come i n contact with food or food surfaces. The New Y o r k State Department of Agriculture and Markets relies on the standard adulteration section of its law, but additionally advises that the department is "very fussy" regarding the use of poisonous material i n food-handling establishments, particularly where poisons might come i n contact with the food. I n New Y o r k C i t y the standard adulteration section also applies and additional requirements are aimed at offering protection to the operator during the spraying project. Akron, Ohio, permits the use of " D D T or other efficient and safe insecticide . . . i n and around the premises . . . of food-selling establishments and, when used, all food shall be so protected that it will not be contaminated. . . . "
HOLMES AND SALATHESTATE AND MUNICIPAL REQUIREMENTS
27
The city of H a m i l t o n , Ohio, considers that a l l these substances are regulated under its law, which forbids the use of poisonous substances where persons, animals, or fowl may be affected. Tennessee's Department of Agriculture prohibits the use of D D T because its Food and D r u g L a w considers a food adulterated if i t m a y have become contaminated. This is possible, i t is alleged, when D D T is used. Knoxville's Health Bureau, on the other hand, permits the use of residual sprays, provided the food is "protected from contamination, and the food must be pure and wholesome." The Dallas, Tex., Health Department permits the use of D D T and chlordan, under the restriction that i t not come i n contact with food or utensils. The Milwaukee, Wis., Health Department regulates the use of D D T , chlordan, and the gamma isomer of benzene hexachloride b y permitting their use only when food, food working surfaces, and equipment are covered during the spraying process so as to prevent spray from being deposited thereon. I n each of the above instances, except the state of Tennessee, the regulations are designed to restrict the use of residual sprays i n such a way that they will not contaminate the food. This is a positive approach, with the same viewpoint as those departments which consider their adulteration law as controlling. The noted exception may well be based on a misunderstanding aimed at restricting the indiscriminate spraying of materials containing residual sprays i n a fog or mist, as is the common practice with the harmless type of sprays, essentially pyrethrum. The proper application of residual sprays contemplates the use of nonmisting, nondripping spray heads directed at nonproduct zone surfaces, which accomplishes the job more efficiently than a paint brush. A s a matter of fact, the Federal Food and D r u g Administration has recently been quoted as frowning upon the use of D D T i n dairy barns. This has been taken as a discouraging indication b y those interested i n the use of residual sprays. However, i t is almost certain that this opinion contemplates use under conditions peculiar to dairy barns. N o state or city had actually taken any regulatory action except Tennessee, where the food products i n two establishments were condemned, presumably after a residual spray had been applied. The details of the spraying were not given. In reply to the request for a record of injury or experience involving unfavorable effects from the use of these materials, there were several nonspecific comments but no definite instances of ill effects.
Use of Residual Spray Materials in a Typical Food Industry

EDWARD L. HOLMES and LLOYD J. SALATHE
American Institute of Baking, Chicago, III.
The plant inspection program of the American baking industry has shown that 80% of sanitation problems can be avoided by good housekeeping. Proper use of residual spray material, such as DDT and chlordan, will control casual invadersroaches, ant, flies, silverfish, dermestids, fungus beetles, and meal worms without contamination of food products.
Following passage of the Federal Food, D r u g , and Cosmetic A c t of 1938, the baking industry established a broad program of education i n sanitation among its manufacturing members. M u c h of this program has centered about the activities of the American I n stitute of Baking, which is the scientific advisory foundation maintained by the baking industry to accomplish just this objective. The first concrete idea materialized from the industry's program was the realization that approximately 8 0 % of sanitation problems can be avoided i n a baking establishment if good housekeeping is maintained. This means: (1) proper building maintenance, with provision for adequate rodentproofing, screening, and removal of interior structural harborages for insects as well as rodents; (2) good storage practices, which include the development of an inspection system for all incoming ingredients, storage away from walls and i n stacks of such size as to permit easy inspection, proper turnover of ingredients, and proper handling of damaged goods; and (3) generally good housekeeping practices, adequate cleanup of flour-handling equipment, overhead proofers, and removal of flour dust throughout the plant, cleaning of machinery and working areas to remove accretions, proper cleaning of mobile equipment, proper maintenance of floors, maintenance of generally good appearance, and removal of unused equipment from production or i n gredient storage areas. Unfortunately, no bakery so far has been able to maintain these criteria of good housekeeping to absolute perfection, although i t is not possible to say that 100% maintenance of such criteria would keep a bakery absolutely free from insect infestation. The American Institute of Baking, i n advising the industry, feels that i t must regard baking establishments as subject to constant danger from casual invaders. This theory has been well borne out by the facts developed during the plant inspection program. A t present, the American Institute of Baking is inspecting some 150 to 200 bakeries per year. Such inspections involve painstaking appraisal of the conditions of infestation within the plants over a period of 3 or 4 days by an individual scientifically trained b y years of experience i n this work. During the year 1948 approximately 100 bakeries were so inspected.
Invading Insects
Results of these inspections have shown that the following insects must definitely be regarded as a casual invasion threat :
28
HOLMES AND SALATHEUSE OF RESIDUAL SPRAY MATERIALS
29
1. Roaches, of all four common varieties. The Oriental variety comes from outside the plant and invades individually wherever possible b y getting i n through broken screens, open doors, etc. The brown-banded roach has no particular habitat and flies into the plant i n areas where i t is common. The German and American roaches ordinarily come i n with cartons of ingredients. 2. The common ants, particularly the Pharoah and Thief varieties, are common invaders of a bakery. 3. Flies of all types are common invaders. However, the housefly and vinegar fly or gnat are the two most common varieties. These w i l l invade the plant under their own power. 4. Silverfish and firebrats come into the plant i n cartons and incoming paper stocks. They are particularly likely to infest returnable cartons from old deliveries. 5. Dermestids. The two most common invaders are the carpet beetle and the drugstore beetle or cigaret beetle. The latter, although not exactly identical species, are comparable i n their habitat and habits. They are all likely to fly into the plant. 6. Fungus beetles, another family that feeds upon decomposed flour and other cereal products, are brought into the plant i n ingredient containers or they mayflyi n . 7. M e a l worms. The three most common are the light meal worm, dark meal worm, and lesser meal worm. M a n y other insects are occasional casual invaders; the ones described are merely those most commonly seen. Ordinarily, when one or two specimens of an insect species invade a clean bakery, nothing is seen of them until they have bred and developed into a definite focus of subsequent infection; i n other words, until there is a colony i n the place where the original invader sought harborage. I t has been common practice to seek out the colonies visually and spray them with a contact spray. This contact spray killed the adults and larva which i t touched, but all too often left eggs and pupae to develop u n harmed. Experience i n the baking industry since World W a r I I has shown that proper use of residual spray materials such as D D T and chlordan will most effectively control these casual invaders. When housekeeping i n a bakery is maintained as near perfect as possible, the application of a residual layer of toxic insecticide on areas upon which casual invaders are most likely to travel i n seeking harborage will effectively k i l l the invader individual before i t has an opportunity to nest. Experience has borne out this theory i n a general way, and specific data are now being" collected which will describe i t i n more scientific terms.
Application of Sprays
B o t h D D T and chlordan are used i n colorless, odorless, deobase-type solvent : D D T in 5 % solution and chlordan i n 2 % solution. The oil solvent is used because i t is a nonconductor and because experience has shown that the crystals from a film of oil solvent solution adhere more firmly to the surface sprayed. A pressure-type spray tank, either a hand pump or mechanical source of air pressure, is used, with a special nozzle which gives a fan-shaped nonmisting spray. A special dripless valve is used. Several valves and nozzles which meet these requirements are on the market. The appropriate areas are sprayed with this fan-shaped painting spray stream, so that the surface glistens with the wet film but there is not sufficient quantity to run down. Experience has shown that this will leave approximately 200 mg. of D D T per square foot or an equivalent amount of chlordan. This procedure is recommended by the U . S. Public Health Service i n its spraying technique for residences and food establishments treated i n its program of spraying D D T only for malaria control. I t has not as yet recommended chlordan for this use. Specifications for the sprayer nozzles procedure can be obtained from this source. The following areas of the bakery are treated with such a spray : screens, the window and door jambs, areas of wall immediately adjacent to windows and doors, all walls up to a height of 3 feet, the corners and areas of the floor next to the wall to a distance of 3 feet from it, light cords, electrical switchboxes, connection boxes and electrical motors, space behind sinks, space below the floor joists and the basements, where the ends of the joist are sprayed for a distance of 2 feet from the wall, bottom of the storage skids, the lockers, be-
30
hind and on the top and i n the insides, the areas around urinals and stools i n lavatories, and floor drains generally. Objection has been raised to the use of D D T and chlordan i n food establishments because of the possibility that mists may spread onto the product zone of equipment and onto surfaces of food or ingredient mixes. Experience has shown that the use of the paint spray nozzle effectively prevents this. A s large a pressure is used as will lay a flat stream of liquid; there is no mist and should be no drip. Admittedly, other sprays are used i n baking establishments, and special precautions must be taken to keep D D T or chlordan solutions from being confused with these. A sound sanitation program for a baking establishment provides that only specific employees apply the D D T or chlordan spray. The spray materials are kept properly labeled and locked up with the spraying equipment, and only employees authorized to use it have keys to this equipment. The employees applying the equipment should be provided with rubber gloves and masks, although experience has shown that they will not consistently use masks and do not use rubber gloves. They are instructed, when undertaking an assignment of this character, to be sure that there is adequate ventilation i n the room and all windows are open, and they are told to wash their hands immediately, should any spray solution spill on them. So far, i n many thousands of applications, no i l l effects have been reported i n baking establishments following this procedure. In its program of bakery sanitation, the institute feels that it is allowing for control of insect infestation to the extent of about 2 0 % of its effort i n the use of these sprays. They give a tool that protects from possibility of casual invasion. This feeling of security is checked upon constantly by reinspections and plants that have followed this procedure faithfully, combined with the good housekeeping required, have had no infestations. Infestation of the interior of flour handling and other bakery equipment, which might arise from an entirely different source than the casual invader, requires different techniques and this problem is not a part of this paper.
Conclusions
The baking industry feels that it needs residual sprays of the character of chlordan and D D T . I t has no fear of contamination of the finished product, for there is far less danger from contamination here than there has been i n the past from such poisons as sodium fluoride powders, which were used for many years without adverse publicity, despite infrequent food poisonings from their use. Residual sprays such as chlordan and D D T are needed i n the production of bakery goods and are an essential factor i n the production of these goods free of insect sources of filth. However, there is no need to establish a tolerance or acceptance of any amount of D D T i n bakery goods. N o residual sprays are to be employed i n any way i n any area where they could contaminate either ingredients or the finished product. This is i n contrast to the agricultural uses of these products, where they are necessarily incorporated i n a slight degree i n the finished product. Whatever is decided regarding agricultural use should not enter into the question of use i n food-manufacturing establishments.
Pharmacology and Toxicology of Parathion

LLOYD W. HAZLETON and EMILY G. HOLLAND
Hazleton Laboratories, Falls Church, Va.
To determine the toxicity of parathion to warm -blooded animals, tests were made with mice, rats, guinea pigs, rabbits, and dogs. Greatest hazards are associated with insecticidal use, but symptoms of toxicity may be detected well in advance of severe toxic actions.
Karathion (0,0-diethyl O-p-nitrophenyl thiophosphate) is primarily of interest as an i n secticide. Preliminary reports on some of the pharmacological and toxicological aspects of this organic phosphate have been presented by DuBois, Doull, and Coon (4), Hagan and Woodard (6), and Hazleton and Godfrey (8). A method for its chemical estimation has been presented b y Averell and Norris (2). These preliminary investigations indicated that parathion functioned as a cholinesterase inhibitor, was rapidly absorbed following dermal application, and was relatively toxic to warm-blooded animals, and atropine was only moderately effective as an antidote. More recently Lehman (9) presented a comparison of D D T and several other insecticides, including parathion. As indicated i n the paper, many of the data were preliminary at the time of presentation and are subject to re-evaluation with additional experience. D u B o i s et al. () have contributed studies directed primarily at the anticholinesterase activity of parathion (93% purity). The purpose of the present paper is to present certain aspects of the pharmacology and toxicology i n greater detail and to report the results of chronic toxicity experiments. The material used i n these investigations was Thiophos 3422 parathion supplied by the A m e r i can Cyanamid Company. The material was of technical grade, 95 to 9 7 % purity, specific gravity 1.2537. Parathion is a heavy brown-colored liquid with a characteristic odor. I t is almost insoluble i n water but is soluble i n organic solvents, including propylene glycol. Wherever the term "wettable powder" is used it indicates a preparation of parathion a d sorbed onto Attapulgus clay to represent the percentage indicated. " D u s t s " are prepared from the wettable powder b y further dilution with pyrophyllite.
Experimental
Acute Toxicity. T h e L D following oral administration of parathion, either i n propylene glycol solutions or i n aqueous suspensions of the 1 5 % wettable powder, has been determined for rats, mice, and guinea pigs. The lethal dose was approximated for rabbits and dogs. The results of these experiments are summarized i n Table I . Statistical evaluation was b y the method of Wilcoxon and Litchfield (11). These data indicate that female rats are significantly more susceptible to the action of parathion than are males, i n either sex the propylene glycol solution is more toxic than the wettable powder, i n propylene glycol the slopes of the dose response curves for the two sexes deviate from parallelism, and guinea pigs and rabbits are relatively resistant to its toxic action. I n mice and guinea pigs the sex difference was not great and the data were combined. Following oral administration of lethal doses of parathion the animals ex hibited peripheral muscular twitching followed by labored breathing, salivation, lach5 0
31
32
rymation, convulsions, depression, and death. Prompt and apparently complete recovery from noniethal doses is indicative of reversibility of the action. Depending on the dosage and the preparation, death may result i n from 1 to 24 hours. I n general, animals that survive more than 24 hours may be expected to recover, and there is no apparent latent toxicity. Technical parathion held i n the conjunctival sac of rabbit eyes produces local irritation and a marked myosis which is spontaneously reversible i n 24 hours. N o systemic effects were observed. For these experiments mongrel male dogs were given parathion orally by means of capsules containing the 1 5 % wettable powder. A single dose of 25 mg. per k g . was lethal in about 3 to 5 hours. T e n milligrams per kilogram caused severe symptoms of toxicity and refusal to eat, and after three daily doses this animal succumbed. After 5 mg. per k g . one dog showed such severe effects that the dose was not repeated, and the animal succumbed i n about 36 hours. These three dogs are included i n Table I as acute deaths. The symptoms were comparable to those seen i n other species, and autopsy revealed only gastrointestinal irritation and pulmonary hemorrhage. Table I. Summary of Acute Oral Toxicity of Parathion in Mice, Rats, Guinea Rabbits, and Dogs Pigs,
(Values in body of table represent number of deaths/number tested. Maximum observation period was 48 hours. A l l doses are as parathion) Dog Guinea Pig Rabbit Rat Mouse Propylene 15% wet- 15% wetPropylene Propylene 15% wet15% wettable glycol table table glycol table glycol 1 powder 10 and powd*;r powder powder 1 and 10 1 1 100 capsule 1 and 2 M M M and F F M F M M and F M and F Sex Animal Solvent, mg./tnl. Dose, Mg./Kg.
1.0 1.5 2.0 3.0 4.0 5.0 7.5 8.0 10.0 12.0 12.5 15.0 17.5 20.0 22.5 25.0 30.0 35.0 40.0 50.0
LDso, mg./kg
3/20 5/20 11/38 10/16 25/26 15/20 16/20 /io 3/3
6/
0/6 1/14 2/14 5/7 7/14 4/6 6/7 2/2 5/5 2/2
4/11 6/11 7/11 4/4 i/io 10/19 4 /io 14/20 16/20 9/10 7/8 9/11
0/10 2/10 9/17 10/17 4/7 /7 7/7
0/1 0/1 0/1 1/1 o/i 0/1 o'/i 1/1
6/ii 0/2 6/20 6/32 25/45 5/13 33/34 10/12 6/6 21.0
3/8 7/13 14/17 6/12 9/11
6/
2/2 2/2 9.3
2/2
6.0
5.0
1.75
12.5
3.5
Method of Wilcoxon and Litchfield (il).
Dermal Absorption. T o determine the t o x i c i t y of parathion following dermal application, the method of Draize, Woodard, and Calvery (3) was followed. Variables considered i n the design of these experiments were concentration as a factor of area, solvent, exposure time, and number of exposures. I n some cases the wettable powder was applied i n the dry form, while i n other cases sufficient water was added to produce a viscid paste. A l l doses i n the table are presented as milligrams per kilogram of parathion, re gardless of the concentration or solvent. A s the concentration of parathion i n the propylene glycol solutions is increased, i t follows that the area covered b y the solution is decreased. T h a t this is a factor i n toxicity is indicated by the greater toxicity of the 10 mg. per m l . solution than the 50 mg. per m l . solution. This relationship appears to be true also of the various dry preparations, i n that the 1 % powder is somewhat more toxic than the 1 5 % . The addition of water to convert the powder to paste does not appreciably influence the toxicity. I n comparable concen trations the wettable powder formulation is less toxic than the propylene glycol solution.
HAZLETON AND HOLLANDPHARMACOLOGY AND TOXICOLOGY OF PARATHION
33
This is evidenced b y a definite shift to higher doses, as shown i n Table I I . Acute symp toms develop within a few hours, the most obvious being salivation, lachrymation, exophthalmia, peripheral tremors, and diarrhea. When these are severe the animal usually succumbs within 12 to 18 hours following application. Table II. Toxicity of Parathion Following Dermal Application to Rabbits
(Skin of abdominal area was closely clipped and parathion preparation applied under rubber sheeting. A l l doses are as parathion. " D u s t suspension" is an aqueous suspension of 15% wettable powder comparable to a spraying application of 3 pounds per 100 gallons) Propylene Dose, Mg./Kg. Exposure Time, Hours 24 24 24 24 1 24 24 24 24 24 24 24 24 1 1 24 1 No. of Expo1 1 1 5 1 1 1 1 1 1 1 1 1-4 3-5 1 1 Technical Mg^/ML_ 10 50 200 0/1 1/1 .. 0/3 .. 0/3 1/3 1/2
$
1
?!
Dust Suspension, 1% Dust 15% Dust 25% Dust 3 to 800 D r y Plaster Dry Plaster D r y Plaster 15% 25%
10.0 15.0 20.0 25.0 37.5 40.0 40.0 50.0 65.0 100.0 150.0 200.0 250.0 300.0 500.0 600.0 750.0 1000.0
..0/1 . . 1/2 . . .. ..
o/i
0/3
::
0/3
0/4 0/4
0/1 0/1 1/1
1/1
1/2 1/1
0/3
o/i
2/3 1/1
0/3 1/1. 0/3 3/4 3/3 1/3 2/3
Chronic Toxicity. R A T S . I n these experiments albino rats of a standard strain were placed i n individual cages with wire-mesh floors elevated above the droppings. Water was offered ad l i b . The basic control diet consisted of a commercial, finely powdered dog meal. Parathion was added to the basic diet b y incorporating carefully prepared parathion dust i n the appropriate quantities. T o ensure thorough mixing, each batch was mixed i n a M c L e l l a n batch mixer. E a c h week the animals were weighed and the food consumed during the previous week was determined. The residual food was discarded and a freshly prepared food was offered. Occasional assays of the residual food indicated that the loss of parathion during the week was negligible. Initially, dietary levels as high as 1000 p.p.m. were offered, but the rats refused to accept levels of 250 p.p.m. or higher, and died of starvation with no detectable food consumption. W i t h 10 male rats serving as control, similar groups of rats were placed on dietary levels of 50 and 100 p.p.m. F r o m the group receiving 100 p.p.m. two rats were lost after 8 and 17 days, respectively, and this group was, therefore, considered to have been repre sented b y eight animals. A t 50 p.p.m. the animals showed no evidence of toxicity, but at 100 p.p.m. there were occasional periods of peripheral tremors and irritability during the first several weeks of the experiment. Thereafter the animals appeared to be normal. These experiments were continued for 2 years. Summaries of body weights, food con sumption, and survival are presented i n Tables I I I and I V . The mortality indicated i n Table I V is relatively low for all groups. When survival is calculated on the basis of the number of days each rat lived i n terms of the theoretical survival of a l l rats for the 2-year period, the results indicate 8 7 % survival for the controls, 9 5 % survival for the 50 p.p.m. group, and 9 3 % for the 100 p.p.m. group. This indicates that the majority of the animals were lost relatively late i n the 2-year period. I t is probable that the difference i n either mortality or survival is not significant. W i t h the upper tolerable limits thus established, three additional groups of 20 male rats each were started. One group served as control, one group received 10 p.p.m., and one 25 p.p.m. Shortly after the second series of experiments was started a minor epidemic of respiratory disease was responsible for several deaths, particularly i n the control group, and these animals were eliminated from further consideration, because experiments of this type are designed to cover infantile rats i n the 60- to 70-gram weight range and i t was i m possible to replace them. After 93 weeks these feeding experiments were continuing with out apparent undesirable effects. The data are also summarized i n Table I I I .
34
I n view of the observed difference i n acute susceptibility between rats of the two sexes, it became desirable to study the chronic toxicity of parathion i n female rats. T o initiate such studies, two or three immature females were placed i n a cage with one male and a l lowed to remain until pregnancy became obvious, at which time they were placed i n i n dividual cages but continued on the diet until birth of offspring. The results from this series are also summarized i n Table I I I , the blank areas representing the period of parturition and rearing of litters. A l l females of the control and 10 p.p.m. groups produced living litters, and all but one of the 50 p.p.m. group. A s soon as the litters were weaned, the parent females were placed i n individual cages and returned to the original parathion diet. A t the time of report there had been no significant mortality, although the groups receiving parathion showed some retardation of growth. This appears to be associated with a reduction i n food intake and there is no gross evidence of toxicity. Female rats were maintained over a considerable period of time on a diet containing 100 p.p.m. but continually manifested evidence of toxicity and presented an unthrifty appearance. I t is evident, therefore, that chronic toxicity follows a sex pattern comparable to the acute toxicity. Table III. Summary of Body Weight and Food Consumption after Various Intervals of Feeding
(Parathion was added to diets in form of carefully prepared dust. Dietary levels in p.p.m. are as parathion. All values are average for number of survivors. Omissions in female series cover periods of parturition and lactation) Males Males Females Control 10 P . P . M . 25 P . P . M . Control 50 P . P . M . 100 P . P . M . Control 10 P . P . M . 50 P . P . M . Food, Food, Food, Food, Food, Food, Food, Food, Food, Week Wt., _g./ Wt., g . / Wt., g . / Wt., g . / Wt., g . / Wt., g . / Wt., g . / Wt., g . / Wt., g . / No. g. wk. g. wk. g. wk. g. wk. g. wk. g. wk. g. wk. g. wk. g. wk. 0 4 6 7 8 14 15 i?
3
4
66
..
69
..
62
..
66
..
70
..
69
. .
63 128 160
.. ..
61 125 167
64 108 139
. . ..
226
143
242
1
158
4 0 2 3 3 3 3 3
228
9 4 6 8 8 9 4 2 5 5 5 4 1 1 1 1 1 1 1
141
3 4 3 2 3 2 7 8 9 0 8 9 2 6 3 3 3 3 3 3 3 7 7 8 0
240
1 2 4 6 4 9 0 7 7 0 1 1 1 1 1 1 1 3 3 2 1 1 1 2
151
4 3 4 3 5 0 3 3 3 3 3 3 3 3 1 3 5 7 8 9 9
249
1 1
t
2 I?
72 80 88
9
?o? ?I 222 ? ? i f i g U% J i ? 2 2 21 22 21? } 2 22 ?2 i 2? } S?

1 3 2
3 8 1 3
39
413 426 427
149 127 134
432 436 450
135 117 128
417 432 434

4
149 122 128

0
1 0 4
, ,,
,
11/16
391 393 398 407

7
129 133 121 105
180 191 .. 183 .. 182 110 129 187 108 185 141 150 326 150 217 115 206 115 201 105 143 214 127 205 108 53 125 226 123 207 126 123 375 123 257 141 236 112 210 116 I 261 1 0 241 106 212 101 253 88 222 93 401 148 398 141 404 161 395 151 '.'. '. 412 136 402 133 418 113 397 111 .. .. '.'.
4 7 2 9 0 1 3 9 1 9 4 4 1 3 5 3 5 2 1 3 5 2 2 9 1 2 9 3 7 1 1 3 3 2 1 1 7 3 7 7 2 3 6 2 1 4 2 3 7 7 1 3 2 8 0 1 2 5
154
222
147
4 2 5
119 385 121

5/8
Survival
7/10
14/16
6/10
8/10
4/6
9/9
5/8*'
Table IV. Summary of Survival and Food Consumption Data for Rats Receiving Basic Laboratory Diet, and Basic Laboratory Diet to Which Were Added 50 and 100 P.P.M. Parathion
Rat Days Parathion, P.P.M. Control 50 100 No. of Rats Start Finish 10 10 8 6 8 5 % Mortality
o f
Average Weight, G . Start Finish 66 70 69 407 425 385
Theoretical 7290 7290 5832
Actual 6154 6934 5415
% f survival
0
40 20 38
87 95 93
Total Food and Parathion Consumed, G . Control 50 100 132,955 103,284
Food Consumed Av./rat/day, Total, g. g. 105,534 132,948.35 103,273.67 17.15 19.17 19.07
Parathion Consumed Av./rat/day, Total, g. g. 6.65 10.33 0.001 0.002
U p o n completion of the 2-year experimental period a l l surviving male rats i n the initial groups receiving 50 and 100 p.p.m., and the corresponding control group, were sacrificed b y exsanguination through a cardiac puncture following light ether anesthesia. A n autopsy was performed on each rat immediately following death. The liver, kidneys,
35
adrenals, testes, spleen, heart, lungs, and brain were removed and weighed. I n addition to these tissues, the thyroid glands were examined and perirenal and peritoneal fat was removed. I n most of the animals the nasal and ear passages were exposed for examination. A portion of the blood was oxalated for hematological examination. Samples of the tissues were placed i n formalin for histopathological examination and the remainder, plus blood and fat, were saved for chemical analysis. These animals at the time of sacrificing presented an extremely good general appearance for rats of their advanced age. There were a few cases of improper balance which is suggestive of middle ear infection, and a few animals showed gross evidence of nasal or upper respiratory tract infection, but none was of a severe nature. Some of the animals on the 50 and 100 p.p.m. levels exhibited slight alopecia on the lower portions of the body and legs; however, the skin over these areas appeared to be i n excellent condition with no evidence of scales or dermatitis, and the condition was of such a mild nature that it was not visible when the animals were i n their normal position. Gross examination at autopsy revealed the majority of the animals to be i n very good condition, although there were occasional variations from the normal i n specific organs. The most general observation, which occurred without exception, was the presence of hemorrhagic and consolidated areas of the lungs, a condition always observed i n animals of this age, and gross examination revealed no significant difference i n the appearance of the various groups. N o peritoneal fluid was found i n any of the animals. The incidence of pleural fluid was negligible i n the control and 50 p.p.m. groups. The 100 p.p.m. group showed some pleural fluid i n four of the five survivors. I n two of these there was evidence of upper respiratory tract infection. The incidence of pus i n the ear was relatively high but apparently not associated with the outward symptoms of disturbance i n equilibrium noted, for one animal showing disturbed equilibrium was entirely free of ear infection while the other showed the presence of a greenish pus.
Correlation of Data
In general, there is reasonably good correlation in the data on individual organ weight for each animal. The range of weight and average for the liver, kidneys, adrenals, testes, spleen, and brain appeared to be comparable for all groups. There was evidence of slight cardiac enlargement i n the parathion-treated groups since, when expressed as per cent of body weight, the control was 0.0027% while the 50 and 100 p.p.m. groups were 0.0035 and 0.0036%, respectively. The range and average for the lungs were comparable with the exception of those from one rat i n the 50 p.p.m. group, which showed a localized abscess i n one lobe and multiple abscesses throughout the entire left inferior lobe. The hematological findings appeared to be well within normal ranges encountered in the experience of this laboratory. Histopathological examinations of random selected samples of lung, adrenal, testicle, spleen, stomach, heart, intestine, liver, esophagus, brain, colon, and kidney were made. The lung samples showed evidence of congestion. Occasional testicular atrophy was observed, both grossly and microscopically. A l l these changes, as well as any observed i n the other tissues, appear to be within normal range considering the age and consequent senility of the animals. Chemical analysis of the above tissues, plus abdominal fat and blood, by the method of Averell and Norris (2) was essentially negative. Occasional positive values were observed i n the control rats, as well as those on the parathion diets. T o test for tissue storage of parathion, female rats on 50 and 100 p.p.m. diet, and appropriate control rats, were allowed water while food was withdrawn. During the withdrawal period the behavior of the treated and control rats was typical of starvation with no evidence of parathion toxicity. Death was neither hastened nor delayed. DOGS . For chronic feeding experiments mongrel male dogs were given oral doses of the 1 5 % wettable powder i n hard gelatin capsules, 6 days per week. I t was found necessary to administer the dosage either with the meal or afterward because prior administration resulted i n loss of appetite and refusal to eat. The animals were weighed each week
36
and the daily dosage was adjusted according to the weight. A t intervals during the period blood samples were obtained for red and white counts, differentials, and hemoglobin determination. Although these showed some fluctuation, the values i n general were within normal limits. A t 2 mg. per kg. daily the dog lived for 3 weeks but continually showed toxic symptoms and was difficult to feed. A t 1 and 3 mg. per kg. per day the animals survived for 90 days. During the early stages of this period the animals were nervous and irritable and the dog receiving 1 mg. per kg. exhibited urinary retention and leucocytosis. During the last month both dogs were normal i n behavior. U p o n completion of the 90-day period, the animals were sacrificed for pathological examination and chemical analysis of the tissue for the presence of parathion. Neither dog showed evidence of gross pathology, absence of pulmonary hemorrhage being particularly noticeable. Histopathological examination revealed degenerative changes and cloudy swelling i n the liver, more severe with the lower dose. Other organs were normal except for evidence suggestive of a pneumonic condition i n the lungs. Fate. P r e l i m i n a r y investigations directed at adapting the method of A v e r e l l and Norris (2) to the analysis of animal tissues indicated that if precautions were taken to avoid emulsions the method could be used satisfactorily. Tissue samples of about" 5 grams were most convenient, and the usual reagent and tissue blanks were run simultaneously. Following the administration of an acutely lethal intravenous dose to a dog i t was found that parathion could be recovered from the urine, liver, bile, kidney, spleen, and lung. Following oral administration of a lethal dose to a dog (25 mg. per k g . wettable powder) tissues taken immediately after death analyzed as follows: no parathion recovered from bladder bile, liver, kidney, abdominal fat, saliva, or intestine; small quantities (2 to 7 p.p.m.) of parathion recovered from oxalated blood, spleen, lung, brain, and spinal cord. The urinary bladder was strongly contracted and no urine could be collected. The results of these two experiments indicate a universal distribution of parathion following acutely lethal doses. M a l e and female rats fed on 10, 50, and 100 p.p.m. food for several months showed no storage of parathion i n the abdominal fat, nor was any recovered from the lung, liver, spleen, brain, whole blood, or kidney. Two dogs which had received parathion orally for 90 days were sacrificed 24 hours after the last dose; the analyses were essentially negative. Small quantities were found i n the voided and bladder urine of both dogs, while the oxalated blood, serum, brain, liver, and lungs were completely negative. F o r the dog which had received 1 mg. per kg. of parathion per day the bile, spinal cord, spleen, kidney, and intestine were also negative ; the dog which had received 3 mg. per k g . per day showed traces i n the spleen, kidney, and intestine and a value of 13 p.p.m. for the spinal cord, although the brain was negative. These results are i n good agreement with those obtained following oral administration of acutely toxic doses, except for the one value for spinal cord, which does not seem to be i n agreement with the other tissues.
Discussion
A discussion of the results reported should take into consideration both the pharmacological and toxicological aspects per se, and their interpretation i n terms of the health hazards presented by parathion. There is satisfactory agreement between the acute toxicities reported by various laboratories and investigators, although results i n Table I tend to indicate that parathion is somewhat more toxic than the results reported elsewhere. Factors which may influence the acute toxicity are route of administration, the vehicle used i n administration, and the sex. I n common with other organic phosphates, parathion is readily absorbed following application to the skin. D r y formulation materially reduced the toxicity. These observations are not i n agreement with the data of Lehman (9) so far as the toxic dose is concerned. Because parathion is almost insoluble i n water, no comparison can be made between the aqueous solution and the dry powder. The dry formulation of parathion is less
37
toxic than the propylene glycol solution and moistening the dry preparation does not m a terially influence its toxicity. Long-term feeding experiments using rats and dogs contribute data of material significance i n both directly and indirectly evaluating the hazards of parathion exposure. During the first few weeks of the chronic feeding experiments i n rats reported above, each animal was consuming daily a quantity of parathion which, calculated on body weight, is equivalent to one or more lethal doses. A s noted, this level produced some toxic symptoms. A s weight increased the percentage of lethal dose intake was reduced proportionately and these symptoms completely disappeared. It is apparent, therefore, that following oral administration rats are capable of disposing of at least one lethal dose per day through such mechanisms as excretion, destruction, and storage. Chemical analyses of the urine and of the tissues at autopsy indicate that neither urinary excretion nor tissue storage is a significant factor i n the disposition of parathion. This observation has been substantiated by Lehman (10), who reports that parathion is not stored i n the tissues to an appreciable extent and that i t is rapidly destroyed b y the tissues of the body. The results of chronic feeding to dogs also substantiate these observations, and i n neither species could any significant histopathological damage be detected. Because the results of these chronic feeding experiments at maximum tolerated levels do not reveal any influence of parathion on behavior, survival, weight gain, food consumption, or tissue pathology i n male animals and only minor influence i n female rats, they are at considerable variance from the observations of D u B o i s et al., following repeated intraperitoneal injection. Selection of the intraperitoneal route of administration and female rats as the basis for generalization on the hazards of parathion does not appear to be justified. Such a design does not take into consideration the difference i n fate following a b sorption of drugs b y the oral and intraperitoneal routes, and a l l observers have noted the particular susceptibility of the female rats, whereas i n other animals this sex difference is not so apparent. B o t h the LD50 and the slope of the dose response curve are significantly different for male and female rats following oral administration of parathion i n propylene glycol. Although the above authors do not give confidence limits for their L D ^ s following intraperitoneal administration, the confidence limits following administration of parathion in propylene glycol orally to female rats are such that 7 5 % of the calculated LD50 would be well within the confidence limits and therefore not significant. When the daily dose was reduced to 12.5% of the calculated LD50 they reported no deaths after 20 days of administration. This is i n line with other observations on the ability of the rat to metabolize parathion and would not be indicative of cumulative hazards under any reasonable conditions of exposure.
Evaluation
F r o m a practical standpoint i t is entirely possible that parathion may be of clinical interest, but certainly its greatest hazards are associated with its insecticidal use. I n this respect i t is interesting to evaluate parathion according to a series of criteria postulated by Lehman (9) : Parathion is relatively toxic to insect life (Jf) as well as to mammals; therefore, under conditions of usage i t has a favorable ratio. Administration over a long period of time b y means of treated food produces no evidence of toxicity or accumulation i n doses which are acutely tolerated. Although a sex variation was observed i n rats, this is no greater following chronic exposure than following acute toxicity. Parathion is uniformly distributed throughout the body following acutely lethal doses, but there is no evidence of storage i n any of the tissues studied even after long terms of feeding. B o t h clinical and biological tests are available for the detection of parathion i n fractional parts per million, thus making the contamination of foodstuffs or the presence of residues readily detectable. Although published clinical reports on parathion are not available, field reports (7) indicate that the symptoms of toxicity are detected well i n advance of severe toxic actions.
38
These symptoms are consistent with the pharmacological action and usually appear i n the order of nausea, chest pains, blurred vision. Removal from exposure and symptomatic treatment have thus far been followed by rapid abatement of symptoms.
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) American Cyanamid Co., Tech. Bull. 2 (Dec. 1, 1948). Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753 (1948). Draize, J. H . , Woodard, Geoffrey, and Calvery, H . O., J. Pharm. Exptl. Therap., 82, 377 (1944). DuBois, K. P., Doull, John, and Coon, J. M . , Federation Proc., 7, 216 (1948). DuBois, K. P., Doull, John, Salerno, P. R., and Coon, J. M . , J. Pharm. Exptl. Therap., 95, 79 (1949). Hagan, E . C., and Woodard, Geoffrey, Federation Proc., 7, 224 (1948). Hamblin, D . O., American Cyanamid Co., personal communication. Hazleton, L . W., and Godfrey, Emily, Federation Proc., 7, 226 (1948). Lehman, A. J., Bull. Assoc. Food and Drug Officials, XII, 82 (1948). Lehman, A . J., communication to Natl. Canners Assoc., Feb. 16, 1949. Wilcoxon, F., and Litchfield, J. T., Jr., J. Pharm. Exptl. Therap., 96, 99 (1949).
Pharmacology and Toxicology of Some Important Economic Poisons

C. H. HINE
School of Medicine, University of California, San Francisco, Calif., and School of Public Health, University of California, Berkeley, Calif.
Some of the more important pharmacologic and toxicologic principles that have been highlighted by government, commercial, and university laboratories are outlined. Cooperative research by chemists and toxicologists is needed, directed toward the development of economic poisons possessing a greater margin of safety for man and animals.
T h e pharmacology and toxicology of certain economic poisons have been developed to a degree which surpasses investigations of any other class of nonmedicinal compounds. I n certain instances more is known concerning the site and mechanism of action, the absorption, distribution, and excretion of these substances than is known concerning some of the more commonly used pharmaceutical compounds. This has come about as a result of the conscientious recognition of the public health hazards which are inherent i n the economic poisons. This review outlines some of the more important pharmacologic and toxicologic principles which have been highlighted b y the work of the government, commercial, and university laboratories that have been concerned with this problem (8, 5,13,16).
Portals of Entry
A s far as man and animals are concerned, the economic poisons exert their harmful and deleterious effect after absorption and distribution through the blood stream. Relatively few agents are irritating or corrosive i n their action, and their effect on the intact skin may be considered secondary (5, 8). Toxic action of economic poisons is exerted by alteration i n physiologic or biochemical activity of various systems, organs, and cells. The portals of entry through which the poisons gain access to the body are determined largely by the nature of the exposure. The use of sprays and aerosols in dispersing media has greatly increased the hazards of absorption through the respiratory tract. A b sorption is influenced markedly by the physical properties of the particular compound. When the portal of entry is v i a the gastroenteric route, the compounds with high water solubility such as soluble arsenates, strychnine, thallium, and 1080 are much more hazardous. Absorption through the skin depends on high lipide solubility, and the ratio of fatwater solubilities may also be important with respect to gut absorption and transfer by the blood stream. Accidental poisoning i n children not infrequently occurs. Economic poisons, not properly labeled, are of great concern both for children and adults. The more extensive problem of poisoning i n man and domestic animals, however, arises from the ingestion of residual poisons remaining on agricultural products, on weeds, and i n impregnated soil
(1, 6). 39
40
Percutaneous absorption has been shown to be a definite hazard with the organic phosphates. I n animals, fantastically small quantities applied to the intact skin or the mucous membranes result i n absorption with fatal consequence. The work of Horton and others has indicated the inadvisability of incorporating benzene hexachloride (hexachlorocyclohexane) i n clothing for miticidal purposes. A s Woodard has pointed out, it is of paramount importance to determine the absorption, metabolism, excretion, and storage of a toxic compound i n mammals. Once having gained access into the body, the economic poison is carried by the blood stream and distributed throughout the tissues i n a manner regulated by the agent's physical and chemical activity. Eventually an equilibrium is established between absorption on the one hand and storage, detoxication, and excretion on the other. The relative rate of this reaction determines, i n part, the toxicity of the i n secticide or other poison. The majority of the fat-soluble compounds which are halogenated are absorbed through the skin or across mucous membranes, are distributed to the liver and other parenchymal organs, and may be stored i n the body fat i n a quantity which is determined by the characteristics of the lipoid matter of the animal and the dosage of the compound (6).
Toxicity
It is a first principle of toxicology that no chemical substance is a poison at all concentrations; toxicity occurs only when a critical concentration is reached within vital cells. Whether or not an economic poison will exert a particular deleterious effect depends on the relative rates of absorption as compared with detoxication and elimination, its i n herent toxicity, and the physiologic status of the organism. Poisoning may be acute, delayed, subacute, or chronic, depending upon the intensity and duration of exposure and the susceptibility of the species. Quantities of D D T may be stored i n the body i n amounts which, if taken i n an acute dose, would be rapidly toxic. This is true of lead and other agents, as well. However, conversely, the organic phosphates and nicotine, which exert an extremely deleterious effect when absorbed i n large quantities over a short period of time may, when absorbed i n lesser quantities over a considerable time, exert no appreciable untoward effects. Furthermore, the summation of repeated minimal injuries may result i n eventual serious damage to an organ or cell. The more highly developed cells of the body, such as are found i n the central and peripheral nervous systems and the conducting mechanism of the heart, are more sensitive to the toxic effects of economic poisons than are the less specialized cells of muscles, fat, and bone. It has been demonstrated with several of these agents that gross effects, such as retardation of growth and normal activity, may not occur at absorption levels which produce significant tissue damage. This emphasizes the necessity for more complete toxicologic studies, with special reference to histologie-pathologie changes. Tolerance i n insects to increasing amounts of D D T , B H C , and several of the inorganic agents, such as arsenic, is known to occur. However, i n animals, this phenomenon has not been demonstrated to the same degree. A b i l i t y of the cells of insects to function i n the environment of a toxic agent, without alteration of cellular function, is one of the most striking of biologic phenomena. This review does not attempt a discussion of detailed pharmacologic knowledge which has arisen concerning many of the principal agents. However, a simple approach can be made to the toxicology of these agents by classifying them according to the vital systems, upon which they exert their primary effect. If a toxic agent exerts a significant effect upon a particular tissue, organ, or system, i t will cause one of the following changes. (1) stimulation, (2) depression, or (3) degeneration. A summary of the principal effects resulting from the more common economic poisons is given i n Table I . It is true that the degree of system damage may be determined i n part by the nature of the exposure; thus, i n mammals, high concentrations of D D T may produce central nervous system stimulation and cardiac irregularities, while low concentrations, absorbed over a period of time, may produce damage primarily to parenchymal tissues (3). Generally, similar chemical types exert approximately similar qualitative physiologic
HINEPHARMACOLOGY AND TOXICOLOGY OF SOME IMPORTANT ECONOMIC POISONS
41
effects and pathologic changes; however, pathologic changes i n tissues are i n addition occasionally specific for particular agents. A n example of this is seen when the deleterious effects of D D T on the liver and muscle are compared with the action of D D D on the adrenal glands and of chlordan on the optic nerve. Table I. Chief Effects of Economic Poisons on Tissues, Organs, and Systems of Man
Stimulation Nicotine, strychnine, arsenic, halogenated hydrocarbons, organic phosphates, dinitrophenols, fluoroacetate (1080) Nicotine, organic phosphates Organic phosphates, ethylene dibromide, ethylene dichloride, ethylene chlorohydrin Effect Depression Nicotine Degeneration Arsenic, methyl bromide
Organ or System Central nervous system
Peripheral nervous system Lung Liver
.... .... .... Arsenic, phosphorus, D D T , benzene hexachloride, chlordan, toxaphene, carbon tetrachloride, D D , ethylene chlorohydrin Halogenated hydrocarbons .... Arsenic, methoxychlor, benzene hexachloride, ethylene chlorohydrin, carbon tetrachloride, D D T phenols Arsenic, phosphorus, fluorides, petroleum oils, calcium oxide and hydroxide, phenols Chlorates Fluorides, phosphorus Arsenic
Heart Kidneys
D D T , fluoroacetate (1080)
Gastroenteric tract Muscles Blood pigment Bone Hair Body as a whole Dinitrophenols Organic phosphates
.... .... .... .... .... ....
Exposure to a toxic agent does not necessarily result in poisoning. A definite concentration must result i n vital tissues before irreversible toxic effects occur. The most effective method of treating poisoning is to prevent its occurrence. This having failed, the sine qua non is removal of the unabsorbed portion, or alteration chemically or physically so that the absorbed portion does not remain i n contact with cells i n toxic amounts. Unfortunately, most of the antidotal treatment of poisoning from agents of economic importance is symptomaticthat is, physiologic i n nature. F o r example, the acute, excitatory effects of the halogenated hydrocarbons on the central nervous and cardiovascular systems predominate i n acute poisoning. Phnobarbital has proved to be useful for control of experimentally induced poisoning i n animals and is apparently the drug of choice i n human therapy (10). Procaine may be of value i n the treatment of cardiac arrhythmias which may arise i n man following exposure to 1080. A t present, no effective agent has been devised to combat the hyperglycemia and depletion of liver glycogen which result from acute poisoning with A N T U (4). The intense pulmonary edema and pleural effusion seen with this compound may respond to oxygen under increased pressures. Fortunately, the minimum toxic dose is high i n man, i n contrast to other species. The organic phosphate esters are among the most potentially dangerous economic poisons with which we have to deal. Fortunately, specific antidotes against the phosphates are now available. The inhibition of choline esterase and resulting excessive parasympathetic nervous stimulation which are caused b y these agents may be successfully combated b y means of magnesium salts and atropine (9). Unfortunately, i t has not been possible to develop an ideal economic poison which has a low order of toxicity for man and is effective over a wide variety of field conditions. Cooperative research on the part of chemists and toxicologists should be directed toward the development of economic poisons possessing a greater margin of safety for man and animals.
42
Bibliography
(1) Carter, R. H . , Ind. Eng. Chem., 40, 716 (1948). (2) Chenoweth, M . B., and Gilman, Alfred, J. Pharmacol., 87, 90-103 (1946). (3) Draize, J . H . , Woodard, Geoffrey, Fitzhugh, O. G., Nelson, . ., Smith, R. B . , Jr., and Cal very, H . O., Chem. Eng. News, 22, 1503 (1944). (4) Dubois, K . P., et al., Proc. Soc. Exptl. Biol. Med., 61, 102-4 (1946). (5) Dunn, J . E., Dunn, R. C., and Smith, B. S., Pub. Health Repts., 61, 1614-20 (Nov. 8, 1946); Reprint 2754. (6) Fitzhugh, O. G., Ind. Eng. Chem., 40, 704 (1948). (7) Hall, S. ., and Jacobson, Martin, Ibid., 40, 694 (1948). (8) Horton, R. G., Karel, L . , and Chadwick, L . E., Science, 107 (No. 2775), 246-7 (1948). (9) Jones, H . W., et al., Federation Proc., 7 (No. I), 231 (1948). (10) McNamara, B. P., and Krop, Stephen, J. Pharmacol., 92, 147-52 (1948). (11) McNamara, B. P., et al., Ibid., 88, 27-33 (1946). (12) Neal, P. ., Sweeney, T. R., Spicer, S. S., and von Oettingen, W. F., Pub. Health Repts., 61, (No. 12), 403-9 (March 22, 1946); Reprint 2698. (13) Neal, P. ., von Oettingen, W. F., Smith, W. W., Malmo, R. B., Dunn, R. C., Moran, . E . , Sweeney, T . R., Armstrong, D . W., and White, W. C., Pub. Health Reports, Supplement 177 (1945). (14) Philips, F . S., and Gilman, Alfred, J. Pharmacol., 86, 213-21 (1946). (15) Philips, F. S., Gilman, Alfred, and Crescitelli, F . N . , Ibid., 86, 222-8 (1946). (16) Stammers, F . M . G., and Whitfield, F . G . S., Bull. Entomol. Research, 38, 1-73 (1947).
New Safe Insecticides

HERMAN WACHS, Dodge & Olcott, Inc., Bayonne, N. J., HOWARD A. JONES, U. S. Industrial Chemicals, Inc.,
Baltimore, Md., and LAWRENCE W. BASS, U. S. Industrial Chemicals, Inc., New York, . Y.
In the search for safe insecticides, hundreds of products have been prepared and subjected to screening tests against insects. This paper presents information on six materials related to piperonyl butoxide, tested in combination with pyrethrins. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch011
W o r l d food shortages lend added weight to the importance of safe insecticides. M o r e than 5 % of all cereal food is destroyed i n storage b y insects. The millions of tons lost each year are equal roughly to the volume of grains i n international trade. Protection of stored grains is only one aspect of the use of insecticides i n food conservation. Control of insects on growing crops, on livestock, i n food processing plants, i n storage, and i n the household assumes new significance i n view of the necessity for more adequate production of food stuffs. Insecticides that combine high efficiency against insects with complete safety to man and other warm-blooded animals are therefore a mighty weapon to ensure food supplies for the world. During World W a r I I popular interest i n insecticides was centered on the spectacular usefulness of D D T i n combating abnormal exposure of troops and civilian populations to insect pests. This important and versatile material has become almost a household synonym for "insecticide." N o w that we are engaged i n a war against hunger, however, public health authorities are becoming increasingly conscious of the fact that the protection of food products properly demands a high degree of safety i n any insecticides that are used for this purpose, a property that most of the existing commercial synthetic products do not possess. The ideal insecticide should meet the following exacting list of requirements : Toxicity to undesirable insects and lack of toxicity under conditions of use to desir able insects Complete safety for man and other warm-blooded animals R a p i d knockdown action toward insects with minimum recovery Stability on storage and exposure Solubility i n suitable solvents Freedom from irritation, undesirable odor, color, and staining properties Volatility low enough to ensure effectiveness over an adequate period of time Cost i n economic range I n the search for safe insecticides the authors have prepared hundreds of new prod ucts and subjected them to preliminary screening tests against insects. T h a t part of their work dealing with methylenedioxyphenyl derivatives was prompted b y the original fun damental studies of 0 . F . Hedenburg, with whom they have collaborated i n this field. T w o materials of this typepiperonyl butoxide (I) and piperonyl cyclonene (II)have re cently been introduced commercially. These products have definite insecticidal properties in themselves, but show their maximum efficiency toward insects and other arthropods when used i n combination with pyrethrins. Furthermore, they are at least as nontoxic
43
44
toward warm-blooded animals as pyrethrum, which is recognized as the safest insecticide in common use.
H 0
2
<
\CH2CH2CH3
CH OCH2CH OCH CH OC4H9

2 2 2 2
Piperonyl butoxide (7) (Propylpiperonyl)(butyl) diethylene glycol ether

COOC2H5
<
d)HCO \ VC V-H TL C K sH n
CH CO
2
\jr
/OCH
CH CH CAlkyl
2
0\J
CH CAlkyl N>-\J Mixture

2
II Piperonyl cyclonene (3, 8) In continuing efforts toward the development of other nontoxic insecticides, m a n y new products are being synthesized and tested. The direction of the synthetic work i s guided b y the theory that the insecticidal activity of a given substance is due to the c o m bined influence of a toxic nucleus and modifying auxiliary groups. T o illustrate the theory, this paper presents information on six materials related to piperonyl butoxide, when tested i n combination with pyrethrins.
Screening Tests on Insecticides

Experimental results obtained on these products by various screening procedures are summarized i n Tables I to I V . Detailed description of these tests on insects and discus sion of their relative practical usefulness for evaluating insecticides will be given i n a sub sequent publication (). I n this screening program on seven materials and the necessary standards of reference about 38,000 houseflies and over 5000 other insects were used. These tests not only represent different techniques, but also supply some basic i n formation regarding the use of the insecticides under practical conditions. Because they are screening tests, i t is desirable to use insects that are commonly employed b y many dif ferent laboratories, i n order to give a comparative evaluation of the materials. This p r o Table I. Tests against Houseflies by Peet-Grady Methods
Test solutions. 300 rag. of material + 30 mg. of pyrethrins per 100 ml. Standard solutions. 30 and 100 mg. of pyrethrins per 100 ml. 100 houseflies per test. Averages of 19 tests Material (Propylpiperonyl) (butyl) diethylene glycol ether (I) (piperonyl butoxide) Modification of auxotoxic group (Propylpiperonyl) (methyl) ethylene glycol ether (V) (Propylpiperonyl) (methyl) ether (VI) (Propylpiperonyl) (decyl) ether (VII) (Propylpiperonyl) (benzyl) ether (VIII) Modification of toxophoric nucleus (Methylnaphthylmethyl) (butyl) diethylene glycol ether (III) (Dichlorobenzyl) (butyl) diethylene glycol ether (IV) Pyrethrins, 30 mg .per 100 ml. Pyrethrins, 100 mg. per 100 ml. % Knockdown 3 min. 5 min. 10 min. 46 49 35 31 35 29 37 27 60 65 68 55 47 51 43 51 44 93 93 78 68 73 66 79 68 92 74 27 24 26 14 17 19 43 % Mortality (of Flies Down) in 24 Hours
WACHS, JONES, AND BASSNEW SAFE INSECTICIDES
45
Table II.
Peet-Grady method. Turntable method. Loop method.
Tests against Houseflies
Residue method.
Test solutions. 300 mg. of material plus 30 mg. of pyrethrins per 100 ml. Standard solution. 100 mg. of pyrethrins per 100 ml. 100 houseflies per test. Averages of 19 tests Test solutions. 150 mg. of material plus 15 mg. of pyrethrins per 100 ml. Standard solution. 100 mg. of pyrethrins per 100 ml. 100 houseflies per test. Averages of 16 tests Test solutions. 300 mg. of material plus 30 mg. of pyrethrins per 100 ml. Standard solution. 175 mg. of pyrethrins per 100 ml. 50 houseflies per test. Averages of 6 tests Dosage 0.00023 ml. per housefly Test deposits. 10 mg. of material plus 1 mg. of pyrethrins per square foot Standard deposit. 20 mg. of pyrethrins per square foot 100 houseflies per test. Averages of 4 tests Material % Mortality in 24 Hours Peet-Grady Turntable Loop Residue (I) 74 74 27 24 26 14 17 43 69 81 8 7 6 6 4 35 98 99 28 13 25 16 18 53 74 93 <1 32 1 <1 1 47
(Propylpiperonyl) (butyl) diethylene glycol ether (piperonyl butoxide)
Modification of auxotoxic group (Propylpiperonyl) (methyl) ethylene glycol ether (V) (Propylpiperonyl) (methyl) ether (VI) (Propylpiperonyl) (decyl) ether (VII) (Propylpiperonyl) (benzyl) ether (VIII) Modification of toxophoric nucleus (Methylnaphthylmethyl) (butyl) diethylene glycol ether (III) (Dichlorobenzyl) (butyl) diethylene glycol ether (IV) Pyrethrins, 100 mg. per 100 ml. Pyrethrins, 175 mg. per 100 ml. Pyrethrins, 20 mg. per square foot
Toble III.
German cockroaches. Red flour beetles.
Tests by Residue Method

mg. of material plus 2 mg. of pyrethrins per square foot 5 mg. of pyrethrins per square foot Averages of 10 tests mg. of material plus 1 mg. of pyrethrins per square foot 5 mg. of pyrethrins per square foot Averages of 10 tests German Cockroaches, % Mortality in 24 Hours 77 80 21 37 52 28 27 22 Red Flour Beetles, % Knockdown in 24 Hours 75 79 34 75 52 23 67 85
Test deposits. 20 Standard deposit. 10 insects per test. Test deposits. 10 Standard deposit. 20 insects per test. Material
(Propylpiperonyl) (butyl) diethylene glycol ether (I) (piperonyl butoxide) Modification of auxotoxic group (Propylpiperonyl) (methyl) ethylene glycol ether (V) (Propylpiperonyl) (methyl) ether (VI) (Propylpiperonyl) (decyl) ether (VII) (Propylpiperonyl) (benzyl) ether (VIII) Modification of toxophoric nucleus (Methylnaphthylmethyl) (butyl) diethylene glycol ether (III) (Dichlorobenzyl) (butyl) diethylene glycol ether (IV) Pyrethrins, 5 mg. per square foot
Table IV.
Tests against Mexican Bean Beetle Larvae by Settling Tower Method

Test dusts. 0.5% material + 0.05% pyrethrins + 0.25% Standard dust. 0.75% rotenone 10 larvae per test. Averages of 12 tests Dosage of dust. 0.283 gram per square foot rotenone
Material Piperonyl cyclonene (II) (Propylpiperonyl) (butyl) diethylene glycol ether (I) (piperonyl butoxide) Modification of auxotoxic group (Propylpiperonyl) (methyl) ethylene glycol ether (V) (Propylpiperonyl) (methyl) ether (VI) (Propylpiperonyl) (decyl) ether (VII) (Propylpiperonyl) (benzyl) ether (VIII) Modification of toxophoric nucleus (Methylnaphthylmethyl) (butyl) diethylene glycol ether (III) (Dichlorobenzyl) (butyl) diethylene glycol ether (IV) Rotenone, 0.75%
48 Hours % dead and dead moribund
72 Hours % dead and dead moribund
78 43 52 28 14 22 19 8 18
98 83 94 56 32 44 43 30 40
90 60 73 32 19 29 29 15 32
96 84 93 58 32 43 38 25 45
46
cedure is necessary to provide a standard measure of the effectiveness of a given substance, and to give other workers data that they can compare with their results on other insecticides. The majority of the tests were carried out against houseflies, because they are the most commonly used insects in standard test procedures, and they are comparatively easy to rear i n the laboratory. Four tests were employed against houseflies: the standard Peet-Grady method ( i ) ; the turntable method (2) ; the loop method, i n which the material is applied to individual female houseflies, by means of a calibrated wire loop (9) ; and a residual deposit test, i n which flies are exposed to glass plates 25 hours after they have been sprayed with acetone solutions of the products. Because of the importance of safe insecticides i n the protection of foods, additional tests have been run against German cockroaches, red flour beetles, and Mexican bean beetle larvae. The residual deposit test was used on the first two insects, and a dust test on the bean beetles. The procedure for the latter method is to incorporate the materials into dusts which are distributed on bean leaves (6) ; piperonyl cyclonene, which is s u perior to butoxide against this insect, was included i n this series of tests.
Chemical Structure and Insecticidal Activity

Experimental work carried out in these laboratories during recent years has been based on the theory that insecticides owe their activity to a toxic nucleusthe toxophorethe properties of which may be modified by auxiliary radicalsthe auxotoxes. This nomenclature is suggested by the names of analogous functions i n dyestuffs, the chromophore and auxochrome groups. A striking illustration of the effect of chemical structure on insecticidal properties is provided by the data given i n this paper on compounds related to piperonyl butoxide. According to the above theory, the methylenedioxyphenyl nucleus present i n this substance is the toxophore. The materials selected for comparison show the reduction i n toxicity produced, first, by modifying the toxophore, and, second, by substituting different groups for the auxotox radical. A t the present time, from the point of view of toxicity to insects combined with low toxicity to warm-blooded animals, there is no sound chemical basis for the selection of toxophoric nuclei. This point is made clear by the data presented for the methylnaphthylmethyl (III) and dichlorobenzyl (IV) analogs of piperonyl butoxide, both of which contain the diethylene glycol b u t y l ether group, which is considered to be the auxotoxic group according to the theory stated above. E a c h of these nuclei would be expected to show strong insecticidal properties i n structures of this type, but the findings prove that these products are generally ineffective. U n t i l our knowledge of the influence of structure on i n secticidal activity has been greatly extended, the discovery of potent new toxophoric n u clei seems bound to depend on chance.
CO
CH
:Q
2 2 2
CH OCH CH20CH CH OC4H9

2
CH OCH CH20CH CH OC H9
2 2 2 2 4
III
IV
The improvement of a toxophoric nucleus by modification of the auxotoxic groups comes through painstaking research. This aspect of the development of a valuable insecticide is illustrated by the comparison of four materials (V, V I , V I I , and V I I I ) closely related to piperonyl butoxide, selected from a large number of homologous series which the authors have studied. These substances are analogs of piperonyl butoxide i n which the diethylene glycol b u t y l ether auxotoxic group has been replaced by ethylene glycol methyl ether, methyl ether, decyl ether, and benzyl ether groups. Generalizations regarding the effect of different auxotoxic groups cannot be made safely, because their influence may
WACHS, JONES, AND B A S S N E W SAFE INSECTICIDES
47
well be dependent on the nature of the toxophoric nucleus. Product V is shown b y the authors' data to have insecticidal activity approximating that of piperonyl butoxide, but has the disadvantage of being more volatile. Products V I , V I I , and V I I I are generally low in activity.
CH OC H
2 1 0
2 1
CH OCH C H
2 2 6
VII
VIII
Although compounds containing the methylenedioxyphenyl group are usually considered solely as synergists or activators when used in combination with pyrethrins, materials of the type of piperonyl butoxide also have definite insecticidal properties in themselves for example, against houseflies b y the Peet-Grady method piperonyl butoxide alone at 2 grams per 100 ml. gave an average 24-hour mortality of 7 5 % in 20 tests, and b y the turntable method at 5 grams per 100 ml. gave an average 24-hour mortality of 7 8 % i n 20 tests. B y comparing these results with those in Table II, it will also be seen that combinations of piperonyl butoxide with pyrethrins exhibit a marked degree of activation. Thus, products such as these are true insecticides which also have the property of showing activation when combined with materials such as pyrethrins. These substances are normally used i n combination with pyrethrins in order to obtain maximum efficiency at an economical cost level.
Application Studies on Insecticides

When a given material has been demonstrated b y screening techniques to have potential value as an insecticide, there then arises the still broader question of how to evaluate its usefulness for specific applications under practical conditions. T h e n this information must be extended b y development of suitable commercial formulations, with due regard to their effectiveness and safety in the hands of the average user, who is the ultimate link in the efficient use of insecticides. Finally, i n order to prove the safety of a given substance to man and other warmblooded animals, exhaustive study must be made of its acute andmost importantits chronic toxicity. In the case of piperonyl butoxide, for example, about 3 years have a l ready been spent on these studies, and the results of the acute .toxicity tests have been published (4). A 2-year study of chronic toxicity is nearing completion and the results, which have demonstrated the high degree of safety of piperonyl butoxide both alone and in combination with pyrethrins, will be published at an early date. These experiments with laboratory animals have involved the daily ingestion of doses in food at different levels throughout the life of the animals or for periods of 1 to 2 years, and also i n successive generations. In addition to observations of the general health of the animal, information on gross and microscopic pathology has been accumulated, the latter through the agency of a competent outside expert. N o t the least important aspect of all such studies is wholehearted cooperation with federal and practicing toxicologists specializing in this field.
Acknowledgment
The authors are indebted to H . O. Schroeder, H . H . Incho, D . H . Moore, J . D . C a p r a rola, and R . E . Newberry of the Research and Development Laboratories, U . S. Industrial
American Chemical Society Library 1155 16th St., N.W. Washington. D.C 20036
48
Chemicals, Inc., who conducted the biological tests and assisted in the compilation of the data.
Literature C i t e d (1) (2) (3) (4) (5) (6) (7) (8) (9) Anon., Soap Sanit. Chemicals, "Blue Book," pp. 207-10, 1947. Campbell, F . L . , and Sullivan, W. N . , Soap Sanit. Chemicals, 14 (6), 119-25, 149 (1938). Hedenburg, O. F., and Wachs, H . , J. Am. Chem. Soc., 70, 2216-17 (1948). Sarles, M . P., Dove, W. E., and Moore, D . H . , Am. J. Trop. Med., 29, 151-67 (1949). Schroeder, H . O., Incho, H. H., and Moore, D . H . , manuscript in preparation. Swingle, M . C., Phillips, A. M . , and Gahan, J . B., J. Econ. Entomol., 34, 95-9 (1941). Wachs, H . , Science, 105, 530-1 (1947). Wachs, H . , and Hedenburg, O. F . , J. Am. Chem. Soc., 70, 2695-7 (1948). Wilson, C. S., J. Econ. Entomol., 42, 423-8 (1949).
Spray Residues on Food Crops and Their Relation to Total Food Consumption
R. H. ROBINSON
Oregon Agricultural Experiment Station, Corvallis, Ore.
It may be estimated conservatively that less than 10% of the food we eat is contaminated with harmful spray residues. These observations, correlated with the relatively low amounts of various pesticide residues found on food crops and the immeasurable trace that may be on a single service of the food, emphasize the little danger to health from spray residues.
I he wide publicity given D D T and the many new pesticides that have been developed during the past few years has made the public aware of the widespread use of spray chemicals. M a n y misstatements about the poisonous properties of these new products have caused abnormal concern and fear over the possible presence of excessive amounts of spray residues on food crops. This concern is a natural one, because the public does not realize the very small amount of the insecticidal residue that may remain on a single service of food, which usually does not exceed 0.25 pound i n weight. N o r does the public realize that only a relatively small percentage of the food we eat has ever been treated with a spray or dust of a specific pesticide. M o s t pesticidesthat is, insecticides and fungicidesare poisonous to humans as well as to the pest that they control if ingested in sufficient quantity. This is true of the older insecticides such as nicotine, the arsenicals, and the fluorine compounds that have been i n common use for many years, as well as the new organic compounds such as D D T . Spray residues on food products must be kept below amounts that may result i n a health hazard to man. T h e possible hazard to human health, however, from eating food that carries excessive amounts of spray residues is one not of acute toxicity because of the deadly poisonous nature of the spray chemical, but of chronic complications. There never is enough pesticide remaining on a single service of food to cause a temporary illness, let alone a fatality. This m a y be exemplified from the observations of N e a l and v o n Oettingen (1), who fed a man subject a single dose of 770 mg. of D D T i n olive oil at one time and 475 mg. of D D T at another time. A thorough physical examination before and after the ingestion of the D D T failed to reveal any untoward effects whatsoever. B y comparison, 770 mg. is more than 1500 times the amount of D D T that may be found on a service of food that has received spray treatment during the growing season. The danger to health from food carrying trace amounts of a pesticide is from the cumulative effect of eating poison spray continuously over a period of time. Our problem then is to adjust and recommend spray programs for the control of each pest, so that there will not be enough of the spray chemical on the food crops to cause health complications. This is not an impossible task. Entomologists and plant pathologists must learn the minimum amount of each pesticide that w i l l give effective control of the pest, having in mind the need of the longest period of time possible between the last application of the pesticide and harvest. This time period will permit maximum losses of the pesticide by decomposition, volatilization, and erosion and will also lower the proportional amounts
49
50
because of increase i n weight of the food crop b y growth after the last spray treatment. A few results showing correlation of these factors are given i n Oregon Experiment Station circulars (#). Furthermore, limited studies indicate that the processing treatment given prior to freezing or canning lowers the residue on some food crops. Investigations of amounts of pesticide residues on crops have been under way for several years at the Oregon Agricultural Experiment Station. Analytical data on the amounts of spray residues on food crops before and after one or more spray or dust applications, on the food at harvest, and after the usual heat and washing treatment given prior to freezing or canning show that relatively small quantities of pesticides are present on the food as it is consumed b y the public. Residue studies of the newer organic insecticides include D D T , D D D , D M D T , and parathion as they may be used to control pests on v a r i ous fruits and vegetables. Table I indicates the amounts of spray residues remaining on several crops, after specifically controlled spray or dust treatments have been applied. Table I.
Apples
Spray Residues on Food Crops at Harvest

DDT, P.P.M. 1.0 2.0 3.0 3.0 1.0 2.0 3.0 3.0 6.4 2.6 1.1 0.7 Trace Trace Negative 1.8 Parathion, P . P . M . 2.9 0.09 0.01 0.08 0.21 0.04
Hood River, Oregon fruit district
Pears
Asparagus Beans Corn Peas Tomatoes
34% of samples 28% of samples 18% of samples 20% of samples above Medford, Oregon fruit district 30% of samples 34% of samples 13% of samples 22% of samples above 5.0% D D T dust, 30 lb. per acre, 4 days before harvest Same dust treatment, then washed and blanched for freezing 3.0% D D T dust, 30 lb. per acre, 4 days before harvest Same treatment, canned by usual cannery process 5.0% D D T dust, 30 lb. per acre, 2 weeks before harvest 3.0% D D T dust, 50 lb. per acre, 1 week before harvest 3.0% D D T dust, 30 lb. per acre, 7 weeks before harvest 3.0% D D T dust, 30 lb. per acre, 1 week before harvest
Apples
Beans Cauliflower
0.5 lb. of 25% parathion to 100 gal., after 2nd application 0.5 lb. of 25% parathion to 100 gal., 2 applications, last one 4 weeks before harvest 0.5 lb. of 25% parathion to 100 gal., 2 applications, last one 9 weeks before harvest 0.5% parathion dust, 50 lb. per acre, 7 days before harvest 0.5% parathion dust, 50 lb. per acre, 7 days before harvest Same dust treatment, then washed and blanched for freezing
The determination of D D T residue on apples grown i n the Hood River fruit district and on pears at Medford is carried on i n branch laboratories established i n those areas. The majority of samples selected for analyses are suspected of carrying higher amounts of residue than the average because of the spray program used or because the last application of insecticide was made within a few weeks of harvest. A s indicated b y Table I , about 8 0 % of all the samples analyzed carried 3.0 p.p.m. or less of D D T during the past harvest season. Only about 2 0 % of the samples showed residues above 3.0 p.p.m.; six samples showed residue deposits slightly above 7.0 p.p.m. The amounts of pesticide residues on vegetables after dust treatments are variable, depending upon the surface area of the vegetable. Italian broccoli and leafy vegetables will retain more residue than beans on a weight basis. The few analyses given i n Table I indicate that vegetables properly treated with D D T dust carry relatively small amounts of the insecticide at harvest time. A further lowering of the amount of residue has been found after the washing and blanching prior to freezing. F o r example, asparagus found to carry 6.4 p.p.m. of D D T at harvest shows only 2.6 p.p.m. after washing and blanching. A similar decrease was observed after beans received the blanching treatment. C o r n and peas that had received heavy treatments of D D T dust during the growing season showed a trace of the insecticide due to contamination b y the mechanical handling of the vegetables prior to canning. Analyses after canning or freezing gave negative results for the same vegetables. Determination of parathion deposits on fruits and vegetables indicates such small amounts present that there should not be a residue problem with this insecticide. P a r a thion apparently volatilizes or decomposes so rapidly that less than 0.10 p.p.m. of residue
ROBINSONSPRAY RESIDUES ON FOOD CROPS AND RELATION TO CONSUMPTION
51
remains on the food crop at harvest. Furthermore, limited studies indicate that when vegetables carrying parathion residue are blanched or otherwise heated, more than 7 5 % of the parathion is decomposed b y the treatment. Further studies on the effects of blanch ing and canning will reveal to what extent various other pesticides may undergo decom position. Chronic health complications that may develop from eating food that contains traces of harmful spray residues depend upon the amount present and the extent to which daily or recurrent ingestion of the contaminated food continues. N o t a l l foods included in the daily diet carry spray residue. Based upon statistics obtained from The Western Canner and Packer and the U . S. Department of Agriculture (8, 4\ approximate percentages of various foods eaten per person i n the United States are given in Table I I . Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch012 Table II. Percentage of Foods Eaten per Person in United States %
19.5 11.9 5.6 5.7 20.5 2.7 1.4 3.9 0.9 0.3 0.3 1.8 0.6 75.1 2.5 5.8 2.4 0.3 0.2 0.3 0.3 2.9 1.1 4.9 2.5 1.7 24.9
Not contaminated with spray Flour, cereal, bread, etc. Meats, fish, poultry Fats, butter, margarine, etc. Sugars, candy, jellies, etc. Potatoes Potatoes, sweet Peas Corn Carrots Beets Pumpkin Onions Cucumbers Usually sprayed Beans Tomatoes Cabbage Asparagus Peppers Cauliflower and broccoli Spinach, lettuce, etc. Apples Pears Oranges Grapefruit Other fruits
About 7 5 % of the food we eat contains no spray residue whatsoever; the other 2 5 % may or may not have been sprayed during its production. Some food crops may have been treated with D D T i n one part of the country, but with nicotine or rotenone insecti cide i n another production area. Consequently, possibly half or even less of a certain crop grown throughout the United States may have received spray or dust treatments with this one pesticide. Again, a large proportion of crops such as apples, pears, and peaches are peeled before canning or freezing and therefore no spray residue is present i n the product. Furthermore, a large proportion of the citrus fruits are ingested as juice and the consumer is not exposed to the residues on the peel. Accordingly, it m a y be estimated that less than 1 0 % of the food we eat is contaminated i n any way with harmful spray residues. These statistics, correlated with the relatively low amounts of various pesticide residues found on food crops and the immeasurable trace that may be on a single service of the food, emphasize that there is little, if any, danger to health from spray residues.
Literature Cited
(1) (2) (3) (4) Neal, P. ., and von Oettingen, W. F., Med. Ann. Dist. Columbia, 15, No. 1, 15-19 (January 1946). Robinson, R. H . , Oregon Expt. Sta., Information Circ. 412, 413 (1947). U . S. Dept. Agr., Annual Crop Report, 1947. U . S. Dept. Agr., Misc. Pub. 550 (1944).
PUBLISHED as technical paper 615 with the approval of the Director of the Oregon Agricultural Experi ment Station, Oregon State College. Contribution of Department of Agricultural Chemistry.
Public Health Aspects of Agricultural Chemicals

HERBERT K. ABRAMS
Department of Public Health, State of California, Berkeley 2, Calif.
A well planned public health program is needed for the safe exploitation of agricultural chemicals. This should embody research on toxicity, cooperation of public and private agencies for exchange of information and promulgation of control measures, labeling of economic poisons, adequate inspection, and education in occupational and consumer health.
In 1900, rural people were generally healthier than people who lived i n the city (7). F i f t y years ago the length of life among white males i n the rural areas was ten years, and among white females, seven and a half years, longer than that of the same groups who lived i n the city. Since this time, health conditions have improved markedly i n urban centers but more slowly i n rural areas, so that today the life expectancy of rural and urban populations i n the United States is approximately the same. Undoubtedly a major reason for this development is the improvement of medical care and public health services i n the cities, while the rural areas have lagged i n this respect. Coincidentally, agriculture and allied activities have become highly industrialized. Today the farm is a 'factory i n the field." It is perhaps not generally known that accidental deaths are much higher i n the rural areas than i n the cities. F o r example, i n 1948 there were 55 accidental deaths per 100,000 agricultural workers as contrasted with 16 per 100,000 manufacturing workers. I n addition to accidents, the farmer is the special target of many other occupationally associated conditions, such as the diseases transmitted b y contact with plants and animals; conditions resulting from excessive exposure to sunlight, such as actinic dermatitis and skin cancer; and skeletal difficulties, such as arthritis, myositis, and others resulting from strenuous physical labor plus exposure to the elements. Finally, w i t h the enormous strides made i n recent years i n the sciences, farming has now become a mass production industry, using practically every type of chemical. Today no significant agricultural production is achieved without the aid of chemical adjuncts.
1
Importance of Agricultural Chemicals

Reports of occupational diseases sent to the California Department of Public Health reveal strikingly that it is "one w o r l d " so far as hazards to health among occupational groups are concerned. The same chemical may cause illness to the factory worker who manufactures it, to the truckers and stevedores who handle and transport it, to the farmer who uses the material, to the packers and canners of the food treated with i t , and to the person who consumes the foods that may be contaminated with i t . There are 80,000 kinds of insects i n N o r t h America, at least 5000 of which are known to be of economic importance. The estimated damage to food products caused by insects annually i n the United States amounts to $2,000,000,000. The insecticide industry i n the
52
ABRAMSPUBLIC HEALTH ASPECTS OF AGRICULTURAL CHEMICALS*
53
United States has a total value of about $15,000,000. I n 1948, 35,000,000 pounds were produced of one insecticide a l o n e D D T (9). There are about 25,000 plant diseases i n the United States, including 10,000 of economic importance, and these cost the nation about $4,000,000,000 a year. The annual wholesale value of a l l agricultural chemicals i n the United States is about $185,000,000. The Bureau of Chemistry of the State Department of Agriculture i n California reported during the fiscal year 1946-47 the registration of 7717 economic poisons. Pesticides are only one group of chemicals i n use i n agriculture. There are now hormones either to accelerate or retard the growth of plants. Livestock are being fattened with the aid of chemicals. Soil and seeds are treated with chemicals. New fertilizers are being developed. M a n y other applications are being discovered. A t the outset, one must note the fact that the public health values of the agricultural chemicals far outweigh the health hazards i n their use. The conquest of the insect-borne plagues with the aid of insecticides can now be foreseen. Agricultural chemicals make possible greatly increased production and preservation of food. The Malthusian philosophy of starvation is being discredited effectively with the aid of modern chemistry. Society can and should use the agricultural chemicals with complete safety to health. It can be assumed that almost any material that is poisonous to insects is poisonous to man, the main variables being the dose and relative susceptibilities of the species. The agricultural chemicals, like other chemicals, reach the body b y inhalation, ingestion, and contact with the skin and mucous membranes. They may cause acute and chronic diseases which vary from superficial skin and mucous membrane inflammations to serious and sometimes fatal systemic conditions. Other speakers on this program describe the toxicological and pharmacological action of various of these poisons. I t is appropriate, however, that a representative of a public health department should discuss the public health aspects. When illness and death occur as a result of overexposure to these poisons, the public health industrial hygienist is called upon to investigate and devise controls. The Bureau of A d u l t Health of the California State Health Department has i n recent years investigated and devised controls for a great variety of economic poisons. These include fumigants such as methyl bromide and the cyanides, preservatives such as sulfur dioxide, the pesticides, and others such as ethylene chlorohydrin used to accelerate the sprouting of seed potatoes. F r o m another aspect, the Bureau of Food and Drugs, also a part of the State Health Department, is vitally interested i n the prevention of adulteration of foods by these poisons. I t is essential today that the public health agencies cooperate very closely with the agricultural agencies, the farmer, the manufacturer of chemicals, and others concerned i n the proper handling of this problem.
Hazards to Public Health

The arsenicals are today not the public health problem they once were. However, many lessons were learned from the experience with them. I n 1940, the U . S. Public Health Service reported its study on the effect of lead arsenate exposure on orchardists and consumers of sprayed fruit (8). I t found, i n a large group of consumers of the apples treated with lead arsenate, that there was a slight increase in the amount of lead and arsenic i n the blood and urine, although no harmful effects from this could be demonstrated at that time. However, lead arsenate constituted a definite health hazard to sprayers i n the apple orchards of Washington (1). The U . S. Public Health Service investigators (8) stated, "Although sprayers who apply lead arsenate spray intermittently did not appear to be adversely affected, top consideration should be given to the protection of the health of men who mix or apply lead arsenate sprays every working day of the season." They recommended that protective equipment be provided for mixing tanks at which people work on a full-time basis. Other investigators recommended also that the orchard industry set up an " a u t h o r i t y " with responsibility for providing an adequate health protection program for orchard workers (1). These recommendations apply essentially to similar operations for any of the economic poisons.
54
W i t h the exception of nicotine, the agricultural chemicals derived from plants are probably among the least hazardous to man (6). Nicotine itself is one of the most efficient insecticides. Nicotine can cause serious poisoning or death if swallowed, inhaled, or ab sorbed through the skin. The halogenated hydrocarbons comprise the largest significant group of pesticides. M o s t extensive investigation of toxicology has been done with D D T and i t illustrates well the ramifications of a public health problem with agricultural chemicals. D D T can cause toxic manifestations by any route of administration, and a number of cases of poisoning have been reported when large amounts of the material have been ingested. I t is possi ble, too, that many reported cases of D D T poisoning were i n reality caused b y the solvent in which the material was prepared rather than b y the active insecticide itself. B u t a more important health aspect of D D T is the fact that it is stored i n the body fat at a level four to ten times that of the dietary intake. It is secreted i n the m i l k of cows, goats, dogs, and rats (2). Other animals fed this milk show toxic symptoms. The D D T i n the milk appears to be concentrated i n the butterfat portion and therefore is transferred to the butter. So today the American people are consuming D D T i n their m i l k and butter as well as i n other agricultural produce. This may be especially important i n the case of infants and children because of their high consumption of milk. I n animals the w i t h drawal of food with consequent consumption of body fat produced characteristic D D T nervous symptoms. This effect might be important i n cases of human starvation or i l l ness i n which there is an appreciable storage of D D T i n the body. Howell (4) recently re ported finding 17 p.p.m. i n a lipoma removed from an individual who had worked with D D T and ingested it i n milk and other foods over a period of about four years. N o clinical effects were noticed i n the patient. One can only speculate on the possible hazard should such an individual be forced b y starvation or illness to metabolize his body fat.
Organic Phosphates
These observations have motivated the U . S. Department of Agriculture and Bureau of Food and Drugs recently to take steps banning the use of D D T i n spraying of dairy barns and of dairy and meat cattle. The organic phosphates are the newest large group of insecticides, the major com pounds being tetraethyl pyrophosphate and parathion. These have all been shown to be extremely poisonous to animals, and i n the brief period i n which they have been used they have demonstrated their toxicity to human beings. I n the first half of 1949, there were at least five fatalities reported, one i n California. Persons exposed to these materials may display marked contraction of the pupils to the point of blindness, shortness of breath with a feeling of tightness of the chest, headache, and other symptoms depending upon the severity of exposure. These effects result, i n part at least, from the destruction of cholinesterase b y parathion with consequent building up in the body of excessive amounts of acetylcholine. Because the phosphates are readily absorbed through the skin and are hazardous from exposure b y any route, prevention of poisoning includes avoiding contact with the bare skin and avoidance of inhalation of the chemicals. I t is recommended also that workers change their clothes completely and bathe with soap and water after every use of this material. Particular caution is indicated on the part of pilots engaged i n airplane spraying because of the effects of the organic phosphates on the eyes. Parathion presents special problems because of its translocation into the plant. The agricultural chemicals i n general are more dangerous to handle i n solution than i n wettable powder or dust form. Parathion and nicotine are the only exceptions to this as they seem to be about equally toxic i n wet or dry form. One overriding problem is the lack of knowledge concerning the effects of exposure to small amounts of these chemicals over a long period of time. Cancer must be regarded as a possible hazard i n the handling of some of the agricultural chemicals and the solvents i n which they are prepared. Cancer of the skin has been reported i n persons i n contact with arsenicals used i n fruit spraying and sheep dipping (3, ). Multiple organ malignancies
A B R A M S PUBLIC HEALTH ASPECTS O F AGRICULTURAL CHEMICALS
55
were observed i n animals exposed to 2-acetaminofluorine, a material originally under i n vestigation for use as an insecticide (10). Public Health Program for Control of Economic Poisons A well planned public health program is needed for the safe exploitation of the agricultural chemicals. Such a program should embody the following major features : Research. M a n y economic poisons are released on the market prior to adequate research i n their toxicity. Too often human injury is the stimulus to research which should have been done before the material was used. There is need for the training of more personnel i n the fields of toxicology, pharmacology, and occupational health. There is an u r gent need for the augmentation and improvement of present research facilities. There is a need for the better coordination and free exchange of information between private and public research groups. A l l the above could be greatly facilitated b y the establishment of a national science foundation with adequate financial support and broad representation of the groups concerned. Cooperation of Private and Public Agencies. The federal, state, and local public health agencies, the agricultural agencies, and the chemical manufacturers and distributors should maintain a close liaison for the exchange of information and the prompt promulgation of public education and control measures. Legal Controls. These include a labeling law requiring a l l economic poisons to be clearly labeled according to content and amount of each ingredient, with appropriate statements on first aid measures and precautions; and an adequate system of inspection for the presence of poisonous residues on the surface of, or within, the foods placed on the market. It is recommended also that the states adopt legislation consistent with the federal laws. Education i n the occupational health a n d consumer health aspects must be extended. Public health agencies in collaboration with the agricultural and food and drug manufacturers' agencies should disseminate appropriate educational material for farmers and allied workers, for the employees engaged in handling and manufacturing economic poisons, for the medical and allied professions, and for the public generally. Literature Cited
(1) Farner, L . M . , Yaffe, C. D., Scott, N . , and Adley, F . E., J. Ind. Hyg. Toxicol., 31, 162 (May 1949). (2) Fitzhugh, O. G., Ind. Eng. Chem., 40, 704 (1948). (3) Franseen, C. C., and Taylor, G. W., Am. J. Cancer, 22, 287 (1934). (4) Howell, D. E., paper presented before Oklahoma Academy of Science, 1949. (5) Hueper, W. C., "Occupational Tumors and Allied Diseases," p. 17, Springfield, Ill., C. C. Thomas, 1942. (6) Lehman, A . J . , "Toxicology of the Newer Agricultural Chemicals," U . S. Food and Drug Administration, Washington, D . C., 1948. (7) Mott, F., and Roemer, M . , "Rural Health and Medical Care," New York, McGraw-Hill Book Co., 1948. (8) Public Health Service, Bull. 267 (1941). (9) Webster, R. L . , State College of Washington, Circ. 64 (December 1948). (10) Wilson, R. H . , DeEds, F., and Cox, A . J., Jr., Cancer Research, 1, 595-608 (1941).
Benefits and Hazards of Insecticides to Public Health

W. J. HAYES, JR., and S. W. SIMMONS
Communicable Disease Center, Public Health Service, Federal Security Agency, Savannah, Ga.
Striking improvements in the control of insect-borne diseases in recent years are largely due to DDT. Improvements in agricultural insect control are due to inorganic insecticides, insecticides of vegetable origin, DDT, and an ever-widening range of new organic insecticides. Use of promising new insecticides must wait on knowledge of their toxicity.
T h e effectiveness of some of the new insecticidal agents against an extremely wide v a r i ety of disease vectors has made possible the control of many important diseases which previously defied all practical efforts at control and has brought within the realm of possibility the ultimate eradication of certain diseases. Diseases which careful scientific study indicates are subject to control b y insecticides include malaria, plague, epidemic typhus, murine typhus, and enteritis due to Shigella. Diseases which w i l l probably be subject to control b y insecticides but have not yet been adequately tested include sandfly fever, dengue, urban yellow fever, bartonellosis, cutaneous leishmaniasis, Chagas' disease, filariasis, trench fever, and louse-born relapsing fever. Some of the virus encephalitides sleeping sickness, and visceral leishmaniasis may also be susceptible of control.
;
Control of Malaria and Plague

More data showing epidemiological statistics for insecticide-treated areas and for control areas have been published on malaria than on any other disease. W i t h few exceptions, these data indicate a remarkable degree of control surpassing anything reported previously. F o r example, after a single year of operations, Viswanathan and Rao (31), working i n Bombay Province, India, were able to report control of 7 4 % or better based on spleen and parasite rates of children and adults i n sprayed as compared to control villages in the Kanara District and 3 0 % or better i n the less severely affected Dharwar District. The infant parasite rate, which more nearly reflects new cases of malaria, was reduced b y 9 4 % i n the K a n a r a District and apparently b y 100% i n the Dharwar District. (These examples are very incomplete. Since the preparation of this paper, a much more extensive rsum of disease control with insecticides has been presented at the meetings of the American Society of Tropical Medicine, Memphis, Tenn., November 6 to 9, 1949. The rsum will be published.) Essentially similar results have been reported i n more limited studies i n Panama, where a reduction i n the parasite rate from 5 2 % i n the control area to 14.8 i n the treated area was obtained (27), and i n Puerto Rico, where the rate was reduced from 5.8 to 0 . 9 % in one year (12, 25). I n Panama and Costa Rica, Macready (15) reported that the m a laria rate of hospitalized employees was reduced on the average b y 5 3 % for each 1000 a d missions. I n Peru, the parasite rate was less than 1% after treatment, where it had
56
HAYES AND SIMMONSBENEFITS AND HAZARDS OF INSECTICIDES TO PUBLIC HEALTH
57
ranged from 11 to 2 8 % before the use of D D T (4). I n a small area of the Volturno River Valley i n Italy, the parasite rate was reduced from 21 to 1% i n one year (1). A n improve ment of about 5 0 % was noted i n Veneto Province i n Italy following treatment with D D T (23). Good results have been reported i n New Guinea (2) and i n the New Hebrides Is lands (33), especially among military personnel. I n Mauritius, i n the Indian Ocean, a carefully controlled study showed the parasite rate of the general population to be reduced b y two thirds and that of the children by one half (26). In the United States, the incidence of malaria is low and it has been falling gradually for many years. However, the current large program of house spraying conducted by the states i n cooperation with the Communicable Disease Center is believed to be increasing that rate of fall. Coupled with auxiliary techniques, the house spraying program, if con tinued, has an excellent chance of eradicating malaria from the continental United States i n the not too distant future. The first control of plague through the use of D D T was accomplished by the U . S. A r m y i n D a k a r i n November and December 1944 and i n Casablanca i n J u l y 1945 (8). The outbreak i n D a k a r had been active since A p r i l 20, 1944, and its complete control was apparently almost entirely due to the universal application of D D T i n the native quarters to persons, beds, floors, walls, and premises generally. Following this experience, D D T was used more promptly i n Casablanca. Macchiavello (13, 14) considers, on the basis of his experience i n Peru, that the use of D D T followed by the use of 1080 (sodium fluoroacetate) may be the method of choice i n the control of epidemics of bubonic plague. Pollock (20) using D D T alone successfully controlled an epidemic of plague i n Haifa i n J u l y 1947.
Control of Typhus
Probably the most dramatic accomplishment i n public health i n the last quarter of a century was the control of major epidemics of louse-borne typhus i n densely populated, heavily infested populations under wartime conditions. This accomplishment was made possible by a great body of research before World W a r I I and b y the efforts of at least a dozen military and civilian agencies during that war. Methods of louse control with M Y L (pyrethrins, 0.2%; N-isobutylundecylenamide, 2.0%; 2,4-dinitroanisole, 2.0%; and phenol S, 0.25%) and D D T powders were first field tested i n the Near Eastern and African theaters and finally proved i n the epidemics i n Naples (3, 24), i n German concentration camps (, 7), and i n K o r e a and Japan (21). Although immunization was of value espe cially i n protecting expert personnel carrying out control work, it has been pointed out by Bayne-Jones and others that it was the judicious use of insecticidal powders that actually stopped the epidemics. It has been estimated that there were 5,000,000 cases of typhus i n Russia alone following World War I. B y contrast, the number of civilian cases i n the Near East, N o r t h Africa, Italy, Germany, Korea, and Japan during W o r l d W a r I I ap pears to have been less than 500,000. The murine typhus control programs of the individual states, i n cooperation with the United States Public Health Service, probably represent the largest unified attack on murine typhus ever undertaken. Since actual operations were begun i n September 1945, a total of 1413 tons of 1 0 % D D T dust has been applied i n 1,105,006 premises treatments in 156 counties of 10 states. Control of the tropical rat flea, Xenopsylla cheopsis, the chief vector of murine typhus, has been 8 0 % or better under field conditions. A s a result of the programs, murine typhus has decreased more rapidly i n the treated counties where it was originally more frequent. There has, during the same period, been a decrease i n the offi cially untreated counties which,originally suffered less from the disease. This may be ex plained, i n part, by D D T treatment, ratproofing, rat eradication, and general sanitation carried on i n the ' 'untreated" counties b y the local government without state support or by private enterprise and, i n part, by a natural decline i n the disease due to unidentified causes. The decrease i n murine typhus i n 61 counties of 9 states dusted each year, 1946 to 1948, was 8 4 % for the first 10 months of 1948 based on the first 10 months of 1945 before treatment was begun. The comparable figure for the officially untreated counties was 6 1 % . The rapid control of small outbreaks of typhus transmitted b y lice but presumably of the murine variety has been reported (6,17).
58
Control of Other Diseases

The control of diarrheal diseases through the control of flies b y insecticides has been noted i n association with several large scale programsfor example, the antimalaria program i n Greece (30) and the antisandfly program i n M a l t a (22). The only program of fly control b y insecticides, i n which the effects on diarrheal diseases were carefully followed and subjected to statistical analysis, was carried out by the Public Health Service i n towns i n the lower R i o Grande Valley. This study showed that a significant reduction i n the amount of infection, disease, and death resulted from the degree of fly control which was obtained. The effect on infections due to Shigella was greater than that on infections
due to Salmonella (32).
Several other diseases have already apparently responded to vector control with i n secticides, although the conditions i n which the outbreaks occurred did not permit complete statistical evaluation of the control. Control of sandfly fever i n M a l t a was reported by Semple (22). Hertig and Fairchild (9) have noted the virtual cessation of new cases of both cutaneous leishmaniasis and bartonellosis following sandfly control with D D T i n Peru. There is every reason to suppose that dengue, urban yellow fever, and filariasis would respond to control of their mosquito vectors and that Chagas' disease and trench fever and louse-borne relapsing fever would also respond. W i t h somewhat more reservation, one may predict the control of some of the virus encephalitides, trypanosomal sleeping sickness, and visceral leishmaniasis. Public health officers are concerned with the control of arthropods as vectors of h u man disease. Important as disease control may be, food production is even more important. Biologically, organisms are more often and more completely limited b y their food supply than b y disease. The total health of our population is affected b y anything which reduces our food supply. The chemical control of food-destroying insects is, therefore, a necessary part of any survey of the benefits of insecticides to public health. Of course, the methodology and conduct of such control are not the responsibility of the public health profession, but the necessity of such control must be considered here, just as the necessity for maintaining the health of farmers and their families must be considered in a comprehensive outline of agriculture. Just how much human food is saved from insect destruction is difficult to estimate. I t is thought that 1 0 % or more of the total crop in this country with an annual value of about $13,000,000,000 is lost through insect damage (11, 16). The damage to stored food products alone is said to average 5 % even i n countries with well developed technical services (10). The earlier estimates of losses may be too low i n view of the increased production which may now actually be obtained by the judicious use of i n secticides (18). The toxicity of a substance is its capacity for causing injury, whereas the hazard of a substance is the probability that such injury will actually occur. Factors that influence the capacity of a given compound to cause injury include its residual action; its acute toxicity; its subacute and chronic toxicity (including not only the accumulation of the toxin or its metabolites in the tissues but also the accumulation of tissue damage with or without the accumulation of the toxin) ; its solvent and particle sizes, as used; its relation to diet; and the timing of its application. The toxicity of a compound may vary for different species, for different ages, and for the two sexes. Factors that influence hazard include not only all the aspects of toxicity but also the method of use, the degree of warning, such as odor given by the compound itself ; the degree of education of the public ; and many other subtle factors. There is nothing basically new i n our present problem, but there are a great variety of new insecticidal compounds and formulations available, and there is a greatly increased demand for their use. Just as the benefits of insecticides to public welfare fall into two classes, so also do the hazards fall into two groups : first, those which are traditionally and legally the responsibilities of local, state, and federal health officers, and, second, those which are traditionally and legally the responsibilities of other authorities. The two groups are often impossible to separate. Of direct interest to the public health professions are the following:
HAYES AND SIMMONSBENEFITS AND HAZARDS OF INSECTICIDES TO PUBLIC HEALTH
59
Acute poisoning i n the household b y actual ingestion or food contamination Acute poisoning of handlers and operators Acute poisoning i n industry Subacute or chronic poisoning of consumers through residues i n fruit, vegetables, meat, or dairy products or through improper household use Subacute or chronic poisoning of handlers and operators Subacute or chronic poisoning i n industry (Health officers may also be notified when household pets are poisoned.) Of equal importance to the public health are hazards of acute or chronic poisoning of farm animals and poultry; insecticide residues i n fruit, vegetables, meat, or dairy products; phytotoxicity; reduction of soil fertility; and disturbance of the balance of nature through selective destruction of wildlife. These latter hazards are largely the responsibilities of agriculturalists and conservationists but are mentioned here for completeness. The great interdependence of public health and agriculture can be no better illustrated than by the problem of insecticide residues i n food. Compounds, vehicles, and spraying schedules adapted for insect control must be selected by the agriculturalist i n such a way as to ensure an acceptable product at harvest. However, once the product is harvested and offered for human food, it becomes the concern of various public health agencies. The health of the agricultural operator who applied the insecticide is also a matter for medical and public health attention. The benefits which have been pointed to i n disease control are largely due to D D T . The benefits which have been pointed to i n agricultural insect control are due to the inorganic insecticides, to insecticides of vegetable origin, to D D T , and to an ever-widening range of new organic insecticides. I n both fields, and particularly i n disease control, the use of several promising new insecticides must be severely limited or entirely omitted because so little is known of their toxicity that they cannot be used with confidence. A t least the following groups are interested, i n one capacity or another, i n the toxicity and hazard presented by insecticides : the public health and medical profession; the chemical industry; the agricultural industry, including farmers and state and federal departments of agriculture (registration and labeling) ; the Food and D r u g Administration and similar state authorities (prohibition of contaminated food i n commerce) ; the food industry, including canners, frozen food packers, and green grocers; the armed services; v a r i ous research groups; and the public generally. Broadly speaking, the most important factors i n the safe use of economic poisons are adequate information and appropriate care b y all persons having any contact whatsoever with the poisons. Safety may be promoted but not ensured b y voluntary control, such as the self-discipline of industry; by legal control, such as regulation of sale, labeling, and distribution ; and b y economic control, such as the refusal of food processors to buy from farmers or dealers food containing excessive residues for which no adequate method of decontamination is known. The principal features of the Federal Insecticide, Fungicide, and Rodenticide Act have been briefly and clearly presented b y Perry (19) ; the full text and interpretations of the act are also available (28, 29).
Conclusions
Because the benefits of insecticides to the public health both i n a restricted and i n a broad sense are very great, i t should be our purpose to use the new as well as the older compounds i n such a way as to avoid injury and at the same time obtain the maximum benefits. Some think that efforts should be made to simplify the nomenclature and to encourage industry to do proper toxicological research and supply methods for analysis before a new compound is submitted for registration. The study of antidotes should be encouraged. I t is clear that research i n the health hazards as well as the benefits of insecticides must be intensified and that the efforts of different laboratories should be better coordinated. Education of industry, of handlers and operators, of the medical and public health profession, and of the public must be advanced.
60
Literature Cited
(1) Aitken, T . H . G., J. Natl. Malaria Soc., 5, No. 3, 169-87 (1946). (2) Bang, F . G., Hairnston, N . G., Maier, J., and Roberts, F., Trans. Roy. Soc. Trop. Med. Hyg., 40, No. 6, 809-22 (1947). (3) Bayne-Jones, S., Am. Assoc. Advancement Sci., Sect. Med. Sci., Rickettsial Diseases of Man, 1-15 (1948). (4) Corradetti, ., Pub. Direccion General Salud Publica (Peru), (Aug. 7, 1947). (5) Davis, W. ., Am. J. Hyg., 46, No. 1, 66-83 (1947). (6) Davis, W. ., Malo-Juvera, F., and Lira, P. H . , Ibid., 39, No. 2, 177-88 (1944). (7) Gordon, J . E., Am. Assoc. Advancement Sci., Sect. Med. Sci., Rickettsial Diseases of Man, 16-27 (1948). (8) Gordon, J . E., and Knies, P. T., Am. J. Med. Sci., 213, No. 3, 362-76 (1947). (9) Hertig, M . , and Fairchild, G. B., Am. J. Trop. Med., 38, No. 2, 207-30 (1948). (10) Hitchner, L. S., Ind. Eng. Chem., 40, 679 (1948). (11) Hyslop, J . ., U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, E-444 (July 1938). (12) Link, V. B., J. Natl. Malaria Soc., 6, No. 2, 124-30 (1947). (13) Macchiavello, ., Am. J. Pub. Health, 36, No. 8, 842-54 (1946). (14) Macchiavello, ., Mostajo, B., and Mostajo, B., Jr., Bol. oficina sanit. panamer., 25, No. 12, 1097-100 (1946). (15) Macready, S. D., Florida Anti-Mosquito Assoc., 16th Annual Meeting, pp. 74-8 (1947). (16) Metcalf, C. L . , and Flint, W. P., "Destructive and Useful Insects," 2nd ed., New York, M c Graw-Hill Book Co., 1939. (17) Ortiz-Mariotte, C., Malo-Juvera, F., and Payne, G . C., Am. J. Pub. Health, 35, No. 11, 1191-5 (1945). (18) Pepper, . B., Ind. Eng. Chem., 40, 708-9 (1948). (19) Perry, D . P., Assoc. Food Drug Officials, U . S., Quart. Bull., 12, No. 2, 64-8 (1948). (20) Pollock, J . S. M c K . , Trans. Roy. Soc. Trop. Med. Hyg., 41, No. 5, 647-56 (1948). (21) Scoville, A . B., Jr., Am. Assoc. Advancement Sci., Sect. Med. Sci., Rickettsial Diseases of Man, 28-35 (1948). (22) Semple, A . B., Medical Officer, 79, No. 4, 35-7 (1948). (23) Sepulchri, P., Riv. malariol., 26, No. 4, 163-82 (1947). (24) Soper, F . L., Davis, W. ., Markham, F. S., and Riehl, L . ., Am. J. Hyg., 45, No. 3, 305-34 (1947). (25) Stephens, P. ., and Pratt, M . D., Science, 105, 35 (1947). (26) Tonking, H . D . , and Gebert, S., Med. Health Dept., Mauritius, Central Lab. Pub., No. 40 (1947). (27) Trapido, H . , Am. J. Trop. Med., 26, No. 4, 383-415 (1946). (28) U . S. Dept. Agr., Production and Marketing Administration, Interpretations of Regulations for Enforcement of Federal Insecticide, Fungicide, and Rodenticide Act, Service and Regulatory Announcements, No. 167 (1948). (29) U . S. Dept. Agr., Production and Marketing Administration, Regulations for Enforcement of Federal Insecticide, Fungicide, and Rodenticide Act, Service and Regulatory Announce ments, No. 166 (1948). (30) Vine, J. M . , Proc. Roy. Soc. Med., 40, No. 13, 841-8 (1947). (31) Viswanathan, D . K., and Rao, T . R., Indian J. Malariology, 1, No. 4, 503-42 (1947). (32) Watt, J . , and Lindsay, D . R., U . S. Pub. Health Service, Pub. Health Repts., 63, No. 41, 131934 (1948). (33) Yust, H . R., J. Econ. Entomol., 40, No. 6, 762-8 (1947).
The Physician's Role in the Pesticide Problem

BERNARD E. CONLEY and JAMES R. WILSON
American Medical Association, Chicago, III.
The host of new synthetic organic pesticides presents a variety of problems to the practicing physician because of the lack of basic fundamental information on these chemicals. The American Medical Association has organized a committee on pesticides to consider the following problems of economic poisons and to coordinate information and make it available to physicians and other interested persons or groups: safe standards of use, development of prophylactic and antidotal measures, voluntary industry controls, standardization of nomenclature, and professional and public education.
T h e introduction of the newer economic poisons has created potential health hazards which, although not entirely new, are of such magnitude that the practicing physician must give serious consideration to them i n order to safeguard the public welfare. T h e seriousness and extent of these problems are reflected i n the increasing amount of attention which is being devoted to them over the radio and i n the press. Unfortunately some of this publicity has been ill-advised and not always i n accordance with the facts. Consequently, i n this atmosphere of misinformation and misunderstanding, considerable confusion on the part of physicians, as well as the general public, regarding the safety of these materials has resulted. This confusion, if allowed to go unattended, may jeopardize the benefits to the health and economic welfare of the country which a more enlightened attitude would ensure. Since chemists have played such a prominent part i n the development of pesticides, the public health problems associated with this situation and the approach undertaken by the medical profession to assist i n their resolution may be of interest.
Practitioner's Problems
The bewildering host of new synthetic organic chemicals presents a variety of complex problems to the medical practitioner, many of which are entirely new to h i m . The basis for these problems lies i n the lack of essential information about the many ways i n which pesticides seem to influence physiologic function and the apparent indifference and carelessness with which many of these new and untried compounds are handled and used. Frequently the physician is called upon to render professional advice or service on health problems involving these toxicants, only to find that there is little or no information upon which to base a rational conclusion. I n other instances, he must piece together facts from a variety of widely scattered sources. Too often this information is of a limited nature and not applicable to the situation at hand or is so controversial and inconclusive i n character as to render a reliable judgment impossible. A s a consequence, the physician must resort to symptomatic measures which are often unsatisfactory because of the difficulty of relating symptoms to causative agent. A t the same time, he is
61
62
faced with the problem of warning the public without alarming it about the dangers inherent i n these materials. These and related problems are being encountered by the practicing physician with increasing frequency because of the wider and oftentimes injudicious use of the newer pesticidal toxicants. The need for information has been apparent for some time. H o w ever, the equal need for the coordination and integration of the existent information, so that it may become readily available to all who should have it, has not received adequate attention to date.
Toxicology Problems
The wide gaps i n present knowledge of the physiologic and phytologic properties of the new organic toxicants are all too apparent to anyone familiar with these poisons. Physicians need fundamental information on the mechanism of action, rate of destruction or excretion, and the long-term effects of the compounds on tissue structures. M e d i cal practitioners have little information on secondary biological factors such as diet, susceptibility of age groups, individual sensitivity, and the presence of pre-existing organic changes i n relation to their influence on the ultimate toxicity of these chemicals. I n addition, many factors other than the acute or chronic toxic properties of the basic ingredient must also be evaluated. Such factors as the toxicity of the carrier, diluents and other so-called inert ingredients, the existence of additive or potentiating activity of these materials i n combination with the basic ingredient, particle size i n the case of aerosols, and the intrinsic dangers of different types of application demand further study. U n t i l this information is available, the medical profession will take a d i m view of those preparations which have labels implying that the product may be used with relative freedom and safety, but at the same time carry a disclaimer which states, i n part, that the buyer assumes a l l the risks of handling and use whether i n accordance with directions for use or not. That much of this information is actively being sought i n private, industrial, state, and governmental research laboratories is to the credit of those individuals who have recognized the need and are actively seeking solutions to these pressing questions. The medical profession recognizes that the orderly accumulation of information of this nature is both time-consuming and expensive. However, it also recognizes that i n most instances the acquisition of this information should have preceded, rather than followed, the release of the chemical. Undoubtedly many of the toxicologic problems which have been encountered could not have been anticipated. Nevertheless, the absence of such information should have tempered the enthusiasm with which some of these materials were introduced. In common with other aspects of the toxicology problem, physicians are embarrassed by the absence of adequate information to guide them i n the assessment of the hazards of food contamination. Untried agents of complex composition have been rapidly introduced i n a multitude of different formulations for agricultural purposes. Oftentimes these preparations were employed i n excessive amounts far beyond the indications for use set forth by the manufacturer. This resulted i n a series of economic misadventures which caused a wave of hysteria and overrestrictive legislation. Exaggerated claims for usefulness by some salesmen and distributors have compounded the initial harm to a point where the potential usefulness of the newer agricultural chemicals is being seriously threatened. The medical profession views with equal alarm the abuses that have occurred with these materials and the retaliatory measures which threaten overregulation. These abuses have compelled food processors to reject fruit and vegetable crops which have been treated with certain of the newer materials. I t has also prompted health officials to confer with government and technical workers on means of establishing measures for the protection of the public. The economic and health significance of this problem is so acute that immediate control measures are imperative. What the nature of control measures should be can Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch015
CONLEY AND WILSONTHE PHYSICIAN'S ROLE IN THE PESTICIDE PROBLEM
63
only be determined by the willingness with which all groups enter into the solution of the problem. Voluntary control should and can be the measure of choice provided standards for the development, release, and distribution of pesticides are adopted which offer the same type of protection to the consumer that exists for drugs and other inherently dangerous substances. Uniform standards for the pretesting of new compounds which would thoroughly evaluate their relative safety and effectiveness under all circumstances of ordinary use must be established. Factors such as the effects on nutritive value, appearance, palatability, and storage qualities of food products, acute and chronic toxicity, practical methods for the detection and decontamination of residues, and the medicolegal aspects of quality control, labeling, and tolerances must be investigated. Advertising claims and promotional activities should be confined within the limits that the available information indicates are justifiable and reasonable. This type of control should extend not only to agricultural chemicals but for all types of bulk and packaged preparations which are offered for sale as pesticides. N o better standard than the old, well worn axiom, " i t ' s better to be safe than to be s o r r y / ' could be used by the pesticide industry i n the development and distribution of its products.
Education of the Public

Aside from the apalling lack of basic toxicologic information on pesticides, the need for public enlightenment i n these matters is one of the most pressing problems which confronts the practicing physician. The education of the consumer, be he farmer, industrial handler, or domestic user of economic poisons, to their intrinsic toxic properties is of immediate importance. The consumer must be psychologically conditioned to the fact that he is using inherently dangerous compounds. Individually, he must be trained to read labels and follow directions for use to avoid unnecessary risks i n handling or application i n the home or i n his work. H e must be educated in order to discount the claims of overenthusiastic salesmen and certain newspaper scientists' reports of new worlds conquered. H e must also be informed that new compounds are not "miracle workers" or "wonder drugs," but highly selective chemicals which are specific i n their action. Lastly, the consumer must be told where to seek reliable information when questions about use confront h i m . The problems of educating the public i n such matters are many and diverse. I t will require not only the support of medical people but that of all individuals who have a social conscience. The interest and sincere efforts of all scientific workers are needed to simplify and hasten this much needed program of public education.
Committee on Pesticides
The practicing physician, because of his unique position i n community life, has the opportunity to create a -better understanding of the uses and limitations of these pesticides. Recognizing the physician's responsibility i n this aspect of the problem, the A m e r i can Medical Association has held two meetings at its headquarter offices to determine i n what way the association and its membership can best make a contribution. A conference on the health hazards of pesticides was called on November 3, 1948, to assess the health hazards created b y the newer economic poisons and to exchange ideas and i n formation relative to the public health problems they have created. About forty individuals attended this meeting representing government agencies, academic centers, trade associations, manufacturers, toxicologic laboratories, and state public health departments. It was exceedingly apparent from this meeting that there is much to be learned about the uses and limitations of pesticides. However, i t was more apparent that an equal need exists for the coordination and integration of the existent information so that i t may become readily available to physicians and others. T o fill this gap and to provide this much needed service, a committee on pesticides was proposed. A n exploratory meeting of this committee was recently held to determine i n what
64
manner i t could best serve physicians and the public. worthy for immediate consideration:
1. PROMOTION OF S A F E STANDARD OF U S E .
The following projects seemed
the criteria of safety b y which the older pesticides were judged do not always hold for the newer preparations which have been introduced. New methods of application, new fields of usefulness, and more frequent use b y untrained individuals have increased the need for establishing standards for the measurement of the immediate and long-term hazards to the user of pesticides. Considerable concern has been expressed i n government and medical circles about the expanded use of the newer pesticides without a full knowledge of their potential dangers. Consequently, i t is imperative that the areas of probable health hazards be outlined and precautions broadcast relative to their use under these circumstances. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch015 stimulation of laboratory and clinical research on prophylactic and treatment measures for many of the newer economic poisons is much needed at the present time. Considerable laboratory and animal work remains to be done before investigations can be undertaken. The therapeutic trials committee of the Council on Pharmacy and Chemistry of the has had considerable experience i n the organization of impartial clinical trials on remedial agents, and its knowledge and facilities would be offered freely i n such matters. H o w ever, at the present time, the field of study is so broad that these investigations must be restricted to individual agents which show promise of wide acceptance and use. the desired method for regulating problems which have their roots i n industry. I t is de sired that this approach be undertaken and its many possibilities exhausted before resort is made to other measures. Sincere efforts i n this direction will win the cooperation and support of a l l other interested groups. The committee will aid and encourage this a p proach b y bringing health problems to industry's attention and will suggest possible ways for their correction. initials, numbers, and combinations thereof for pesticides contributes to misuse and errors i n the handling of these preparations. T h e coining of common or generic names for pesticides is a recent and commendable innovation i n this field of science. I t presents many problems which require the services of many groups. The many years of experi ence of the Council on Pharmacy and Chemistry i n the development of generic names for drugs is at the disposal of the committee i n its consideration of nomenclature problems. of new information bearing on the health aspects of pesticides w i l l be undertaken b y the committee. I n this phase of its work i t w i l l keep i n close contact with state and government health services i n an effort to establish comprehensive records of poisonings and the circumstances of their occurrence; i t w i l l suggest means to avoid recurrence. The assembled information w i l l be available to a l l who have a use for such data. on health benefits derived from the wise use of pesticides and the health hazards associated w i t h their misuse is greatly needed. The educational facilities of the American M e d i c a l Association, consisting of ten professional journals and the lay publication, Today s Health, w i l l be available to the committee. I n addition, the inquiry service which is conducted b y the various departments, councils, and bureaus of the American Medical Association w i l l be coordinated i n order that letters of committee interest will receive their mutual attention.
1
I t becomes increasingly apparent that
2.
FOSTER DEVELOPMENT
OF PROPHYLACTIC AND ANTIDOTAL M E A S U R E S .
The
3.
STIMULATE V O L U N T A R Y CONTROL.
A S previously indicated, v o l u n t a r y control is
4.
ASSIST IN STANDARDIZATION OF N O M E N C L A T U R E .
T h e profusion of trade names,
5.
A C C U M U L A T E AND E V A L U A T E N E W INFORMATION .
T h e compilation and appraisal
6.
U N D E R T A K E A N INTENSIVE EDUCATIONAL PROGRAM .
A program of information
Preliminary work on several of these projects has already been i n progress for over a year. These initial efforts will be expanded and intensified when the committee on pesti cides becomes activated i n the near future. However, pursuance of this program will require the support and cooperation of all segments of science and industry. Advisory bodies from the various interested groups will be called upon for recommendations and suggestions on problems which fall within their field. I n turn, the assistance of this committee will be available, not only to the medical profession, but to all other groups i n any direction i n which the committee may be useful.
Determining New Insecticides in Formulations and Residues

H. L. HALLER Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, Washington, D. C.
Three groups of synthetic organic insecticides have come into commercial use in the past few years chlorinated hydrocarbons, phosphorus-containing compounds, and piperonyl derivatives. Methods for determining composition and purity of commercial grades have been developed, but only a few procedures are available for their detection and estimation in dusts, oil solutions, emulsion concentrates, and aerosols. When more than one insecticide is present in a mixture, even less is known concerning accurate determination of each component. More precise methods for determining their amount and possible degradation products are needed.
f T h e ultimate usefulness i n economic entomology of a compound possessing outstanding insecticidal properties can be determined only after numerous problems have been solved. These problems include determining the acute and chronic toxicity of the product to man, to cattle, and to other farm animals. The effect of the product on vegetation, soils, beneficial insects, and wildlife must also be ascertained. Solutions to these and related problems can be expedited b y physical and chemical studies of the product. Methods of analysis are especially important. N o t only is it necessary to have methods that will permit the analysis of the technical or commercial grade of the insecticide, but procedures should be developed for the detection and estimation of the product i n dusts, wettable powders, solutions, emulsion concentrates, and aerosols. Methods are also needed for determining the product i n combination with other insecticides and fungicides and i n spray residues. Because some new insecticides are absorbed i n animal tissues and found i n dairy products, quantitative methods must be found for determining the insecticide i n them. The importance of methods of analysis for new insecticides is evidenced b y the fact that during the past two years industry and government have cooperated i n developing methods for two of themtetraethyl pyrophosphate and benzene hexachloride (1,2,3,4,5,6hexachlorocyclohexane) (37, 45). Among the more important synthetic organic compounds that have been developed commercially as insecticides during the past several years are certain chlorinated hydrocarbons, phosphorus-containing compounds, and piperonyl derivatives. The chlorinated hydrocarbons that have been of most practical interest are D D T , T D E ( D D D ) , methoxychlor (methoxy analog of D D T ) , benzene hexachloride, chlordan, and toxaphene (chlorinated camphene). Although methoxychlor contains oxygen, i t is included i n this group because of its close relationship to D D T . Of the organic phosphorus-containing products, hexaethyl tetraphosphate, tetraethyl pyrophosphate, and parathion have received greatest attention. The more important piperonyl compounds are piperonyl cyclonene, piperonyl butoxide, and propyl isome.
65
66
Numerous investigators have reported the entomological results obtained with these synthetic organic compounds against many different insects, both agricultural and household. I n this paper some of the analytical procedures that have been developed for these compounds and formulations of them are assembled and reviewed. The methods of analysis for the chlorinated hydrocarbons may be divided into five classesdetermination of total organic chlorine, determination of hydrolyzable or labile chlorine, colorimetric methods, physical methods, and bioassays. The last mentioned is beyond the scope of this manuscript and is not considered. Determinations of Total Organic Chlorine Chlorine makes up 5 0 % or more of the weight of all the chlorinated hydrocarbons under consideration except methoxychlor, which contains 3 0 % . Therefore, methods based on the determination of total chlorine appear appropriate. Such methods are not specific, however, and any other chlorine-containing organic compound will interfere. I n one procedure that has been widely used, the sample, after suitable treatment, is refluxed with sodium and isopropyl alcohol, after which the solution is diluted with water and the inorganic chloride is determined by standard methods (13, 54)- The method has been adopted by the Association of Official Agricultural Chemists (29, 30) as a tentative one for technical D D T and for dusts, oil solutions, and aqueous emulsions of D D T , for use in the absence of other chlorine-containing compounds. The National Association of I n secticide and Disinfectant Manufacturers has also accepted the total-chlorine method for the analysis of these preparations (28). Essentially the same procedures have been described by Donovan (22), of the Insecticide Division of the Production and Marketing Administration, for technical D D T and various commercial D D T products containing no other compounds interfering with the chlorine determination. Carter (12), of the Bureau of Entomology and Plant Quarantine, has adapted the total-chlorine method to the analysis of mixtures of D D T and benzene hexachloride i n the following manner: After determination of total chlorine in the mixture, the D D T is estimated b y the Schechter-Haller colorimetric method (47), half this value is subtracted from the total chlorine (because D D T contains 5 0 % of chlorine), and the difference is calculated as benzene hexachloride. This procedure gives no indication of the amount of the gamma isomer of benzene hexachloride. The total-chlorine method has been used extensively in the determination of spray residues of the chlorinated hydrocarbons (56). Usually the kind of insecticide applied has been known, and by means of the proper factor the chlorine values could be calculated to the insecticide originally used. This calculation is not entirely valid, as the determinations do not differentiate between the insecticide and its degradation products or other contaminants containing organic chlorine. The values obtained by the total-chlorine method are useful, however, because they indicate the magnitude of the residue and the analysis can be made i n a short time with standard laboratory equipment. A procedure that has been widely used for spray residues is the separation of the residue from the sample by extraction with an organic solvent, usually benzene. After most of the solvent has been removed, the residue is treated with sodium and isopropyl alcohol and the chloride ion is estimated by standard methods. Carter (10) has determined i n this manner D D T residues on a number of crops, and he has recommended the adoption by the Association of Official Agricultural Chemists of the method as a tentative one for D D T (11). Koblitsky and Chisholm (42) have determined D D T i n soil samples by the sodiumisopropyl alcohol procedure after removing the D D T by extraction with an azeotropic mixture of two volumes of benzene and one volume of isopropyl alcohol. The total-chlorine method for determining residues of benzene hexachloride, chlordan, and toxaphene has also been used (55) i n experiments where it was known that these i n secticides had been applied. W i t h benzene hexachloride, which is known to give off-flavor to some crops, it has not been demonstrated that a relation between organic chlorine values and off-flavor exists. I n fact, i n most cases where off-flavor was attributed to benzene hexachloride, it has not been possible to detect organically bound chlorine.
HALLERDETERMINING NEW INSECTICIDES IN FORMULATIONS AND RESIDUES
67
The total-chlorine method for residues of the chlorinated hydrocarbons has also been applied to animal tissues, milk, and dairy products (9). As i n the spray-residue deter minations, the method does not differentiate between the insecticide and metabolites. Recently it was shown that when D D T , benzene hexachloride, or toxaphene is fed or applied to cattle, such organic chlorine residue as may be present i n the fatty tissues con sists essentially of unchanged insecticide. Carter (12) demonstrated their presence b y separating the fats and other oxygenated products with sulfuric acid-sodium sulfate mix ture and determining total chlorine. I n experiments with D D T Schechter (46) demon strated its presence i n fatty tissue and i n butterfat b y the Schechter-Haller colorimetric method (47). The residues were then tested for toxicity to houseflies i n comparison with the known insecticides of the same concentration. I n both cases the known insecticide gave the same mortality as the residue. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch016
Determinations of Hydrolyzable Chlorine

A l l six of the chlorinated hydrocarbons yield part of their chlorine on treatment with ethanolic alkali, but methods of analysis involving this reaction have been developed only for D D T and benzene hexachloride. This type of determination is somewhat more specific and subject to less interference than the total-chlorine method, but it must be used with discretion. When it is applied to D D T , one of the chlorine atoms is removed to form hydrogen chloride; with benzene hexachloride 3 moles of hydrogen chloride are formed. Gunther (34) was the first to develop a method using this principle for the determination of D D T i n dust and spray residues. The original procedure has been modified by substitut ing 4.5 ammoniacal methanol for potassium hydroxide i n ethyl alcohol and carrying out the reaction at 45 C. instead of refluxing the solution (5). This modification has been successfully employed b y Gunther (33) for the determination of D D T residues on many different crops. L a C l a i r (44) and Solo way et al. (50) have modified the dehydrohalogenation reaction to permit the determination of , ' - D D T i n dusts and oil solutions containing technical D D T . The reaction between the base and the halide is carried out at 20 to 30 C , as Cristol (16) has found that under proper conditions at this temperature the , ' - D D T reacts completely, whereas the ,'- and most of the impurities react only slightly. The Association of Official Agricultural Chemists' (3) tentative method for the determina tion of the purity of , ' - D D T , which employs the dehydrohalogenation procedure, has been modified b y Fleck (30) so that the reaction is carried out at 25 C. instead of under reflux conditions. Unlike D D T , T D E does not lose one mole of hydrogen chloride when heated with ferric chloride, but rearranges to form an isomeric compound (31). It may be possible to develop this observation into an analytical method to differentiate between the two products or to detect the one i n the presence of the other. The dehydrohalogenation reaction has been applied by Goldenson and Sass (32) to the determination of benzene hexachloride in impregnated cloth. Howard (39) proposed the reaction for the determination of benzene hexachloride residues i n food, and Barlow (6) used it for benzene hexachloride i n the blood of cattle. L a C l a i r (43) has employed the dehydrohalogenation reaction to determine the gamma isomer of benzene hexachloride i n the technical product and i n dust mixtures. Two identical samples are dissolved i n 9 5 % ethyl alcohol and treated with 1 ethanolic potassium hydroxide at 0 C. for 15 and 50 minutes, respectively. The 15-minute period is sufficient to dehydrochlorinate most of the alpha and the delta isomers without appre ciably affecting the gamma. In 50 minutes the gamma isomer is also dehydrochlorinated. The beta isomer does not react under these conditions, and usually the epsilon isomer is present i n quantities too small to interfere seriously.
Colorimetric Methods
Methods based on color reactions have been published for several of the chlorinated hydrocarbon insecticides. Although most colorimetric methods are much more specific
68
than chlorine determinations, care must be exercised i n their use, as analogs and closelyrelated compounds may produce the same or similar colors. I n general, colorimetric methods are used mostly for spray-residue determinations and for qualitative tests of the insecticide i n various formulations. More experimental work has been done with D D T than with a l l the other five chlo rinated hydrocarbons combined, probably because D D T was the first of the group found to have insecticidal value. Carter (10) has summarized the several colorimetric methods for D D T . The one proposed by Stiff and Castillo (51), as modified b y Claborn (14), and the one b y Schechter and Haller (47) have probably been most widely used. I n the Stiff and Castillo method, when the D D T is heated i n pyridine solution containing xanthydrol and potassium hydroxide, a red color develops which is proportional to the quantity of D D T present. The reaction is sensitive to 10 micrograms. As T D E does not give a color with this reagent, Claborn (14) has proposed the reaction for the determination of D D T i n the presence of T D E . H e has also shown that for the development of the color the amount of water i n the pyridine is critical. The colorimetric method for D D T developed b y Schechter et al. (49) is based on i n tensive nitration of D D T with the formation of a tetranitro derivative. The product ob tained with the ,' isomer gives an intense blue color upon addition of sodium methylate, whereas the ,' derivative produces a violet-red color. A s the method permits the esti mation of as little as 10 micrograms and none of the degradation or metabolic products of D D T interfere, i t is especially useful for work on spray residues and biological specimens. Modifications of the method that are suitable for determining D D T i n fatty materials such as milk, butter, meat, and eggs have been described b y Schechter et al. (48) and Clifford (15). Tressler (53) has adapted i t for determining D D T or its residues i n canned foods. The nitration method is more specific for D D T than any other proposed. W i t h the ex ception of T D E , which gives an almost identical color, none of the other chlorinated hydrocarbons interferes. Recently a colorimetric test for methoxychlor residues was proposed b y Fairing (27). The methoxychlor sample is treated with alcoholic potassium hydroxide, the reaction product is extracted with ether, the ether is removed, and the residue is treated with con centrated sulfuric acid. A n intense cherry-red color is developed. N o other insecticide has been found to interfere, and the reaction is sensitive to about 5 micrograms of meth oxychlor. Satisfactory colorimetric methods for benzene hexachloride, chlordan, and toxaphene are not available and are urgently needed. Davidow (19), of the Food and D r u g Administration, has described a colorimetric method applicable to technical chlordan. The method is based on the observation that when technical chlordan is heated with a mixture of diethanolamine and methanolic potassium hydroxide, a purple color is produced. When known amounts of this insecticide were added to cabbage, pears, and fresh and rancid rat fat, recoveries of 74 to 104% of the insecticide were obtained. However, because two crystalline isomers of chlordan isolated from the technical product do not give a colored reaction product with the reagent, further investigation of the method is being made. The red color obtained when technical chlor dan is heated with pyridine, alcoholic alkali, and ethylene glycol monoethyl ether, as de scribed b y A r d (2), likewise fails with the crystalline isomers of this insecticide. A method that is stated to be applicable to residues of benzene hexachloride (20) is based on the fact that benzene hexachloride yields essentially 1,2,4-trichlorobenzene on dehydrohalogenation with alkali. This product possesses a characteristic absorption band in the ultraviolet, which permits its quantitative determination.
Physicochemical Methods
Methods utilizing characteristic physical properties have been developed for several chlorinated hydrocarbon insecticides. Daasch (18) has used infrared spectroscopy for the analysis of benzene hexachloride. B y this means i t is possible to determine the gammaisomer content, as well as that of the other isomers of technical benzene hexachloride, pro vided the product is substantially free of the higher chlorinated cyclohexanes.
HALLERDETERMINING NEW INSECTICIDES IN FORMULATIONS AND RESIDUES
69
The gamma isomer of benzene hexachloride can also be determined b y polarography (24, 40)' The method is based on the fact that, under the conditions used, the gamma isomer is the only one of the five isomers that is reduced at the dropping mercury elec trode. B o t h the infrared spectroscopic method and the polarographic method require special instruments. When instruments for both are available, the latter method seems to be pre ferred. Neither method has been found to be applicable to spray residues. Physical methods based on absorption spectra i n the infrared or ultraviolet have also been suggested for determining D D T . The method proposed b y Herriott (38) for the ,' isomer is based on the fact that in ethyl alcohol solutions it absorbs ultraviolet light very slightly at a wave length of 250 . After dehydrochlorination b y dilute alcoholic sodium hydroxide the reaction product absorbs strongly at this wave length. The increase i n ab sorption is measured and compared with the value obtained with a similarly treated standard solution. Downing et al. (23) have shown that infrared spectroscopy can be used for the chemical characterization of technical D D T , including detection of the several isomers and impurities, and the quantitative estimation of D D T content. Neither method appears to have had widespread application. A method for determining the gamma isomer of benzene hexachloride b y partition chromatography has been developed b y Aepli et al. (1). Nitromethane and n-hexane are used as the partition solvents, and silicic acid is the supporting medium. The method a p pears to be useful for routine product analyses. A n accuracy of about 2 % of the actual gamma isomer present is claimed. A cryoscopic method for determining the gamma isomer of benzene hexachloride, developed b y Bowen and Pogorelskin (8), is based on the fact that the freezing point of a compound is lowered b y the presence of dissolved impurities. The method is useful for benzene hexachloride preparations of higher gamma-isomer content than the usual tech nical grade containing 10 to 1 2 % of the gamma isomer. The method is rapid and requires only simple, readily available equipment. Cristol et al. (17) have based a method for the determination of ,'-DOT i n tech nical D D T on the fact that the ,' isomer is almost insoluble i n 7 0 % aqueous ethyl alcohol and the ,' isomer is soluble. The method has not been tried with mixtures of the other chlorinated hydrocarbons. A mass-isotope dilution method for determining the gamma isomer of benzene hexachloride, i n which gamma-hexadeuterobenzene hexachloride is used as a tracer mole cule and the dilution is determined b y use of infrared spectrophotometry, has been de veloped b y Trenner et al. (52). Impurities have no effect on the accuracy of this method.
Analysis of Organic Phosphorus Compounds

Of the three organic phosphorus insecticideshexaethyl tetraphosphate, tetraethyl pyrophosphate, and parathionthe first two have been shown to be mixtures (36) that contain tetraethyl pyrophosphate as the principal active ingredient. Several methods have been proposed for the determination of this compound i n the commercial products (25, 35). A l l are based on the separation of the tetraethyl pyrophosphate from the related ethyl phosphates, followed b y its hydrolysis to diethyl orthophosphoric acid and titration with standard alkali. B o t h hexaethyl tetraphosphate and tetraethyl pyrophosphate are soluble i n water and are rapidly hydrolyzed to monoethyl and diethyl orthophosphoric acid. This rapid hydrolysis to nontoxic products greatly limits the duration of the in-* secticidal effectiveness of tetraethyl pyrophosphate, but i t also eliminates the danger of toxic residues on the crops treated. The only method that has been described for the assay of technical grades of para thion and its formulations is that of Bowen and Edwards (7). The method makes use of the polarograph. The electrolysis is carried out i n an acetone-water solution with potas sium chloride as the electrolyte and gelatin as the suppressor. A n accuracy of 1 % is obtained. F o r spray residues of parathion Averell and Norris (4) have developed a method that is sensitive to about 20 micrograms. The method is based on the reduction of the nitro
70
group to the amine, diazotization, and coupling with iV-(l-naphthyl)ethylenediamine, which gives an intense magenta color with an absorption peak at 555 . Edwards (26) has found that commercial samples of benzene, which is commonly used for stripping resi dues from plant material, give an identical color by the method. The interfering sub stance can be removed by distillation of the benzene. The oxygen analog of parathion, diethyl p-nitrophenyl phosphate, gives the same color as parathion b y the method. This oxygen analog is stated to be much more toxic to warm-blooded animals than parathion, and i t has been suggested that parathion is converted to the oxygen analog on exposure to the air. However, no definite evidence has been adduced that this compound accompanies spray residues of parathion, and laboratory experiments have failed to confirm the reac tion.
Analysis of Piperonyl Compounds

Of the three piperonyl compounds that have received considerable commercial atten tion as insecticides, a method of analysis is available only for piperonyl butoxide (41). This product gives a blue color on treatment with a reagent comprising tannic acid i n a mixture of phosphoric and acetic acids. Satisfactory results can be obtained i n the pres ence of small amounts of pyrethrins, but larger amounts tend to obscure the color. A modification of the method (21) which overcomes this difficulty is the removal of the pyrethrins by saponification with alcoholic sodium hydroxide prior to carrying out the test.
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) Aepli, O. T., Munter, P. ., and Gall, J . F., Anal. Chem., 20, 610 (1948). Ard, J . S., Ibid., 20, 858 (1948). Assoc. Official Agr. Chemists, "Official and Tentative Methods of Analysis," 6th ed., 1945. Averell, P. R., and Norris, M . V . , Anal. Chem., 20, 753 (1948). Baier, W. E., Edmonds, E . J . , Wilson, C. W., Elliot, M . I., and Gunther, F . ., Science, 104, 376 (1946). Barlow, F., Nature (London), 160, 719 (1947). Bowen, C . V., and Edwards, F. I., Jr., Anal. Chem., in press. Bowen, C . V., and Pogorelskin, . ., Ibid., 20, 346 (1948). Carter, R. H . , Ibid., 19, 54 (1947). Carter, R. H . , J. Assoc. Offic. Agr. Chemists, 30, 456 (1947). Ibid., 32, 353 (1949). Carter, R. H . , unpublished report. Carter, R. H . , and Hubanks, P. E., J. Assoc. Offic. Agr. Chemists, 29, 112 (1946). Claborn, . V., Ibid., 29, 330 (1946). Clifford, P. ., Ibid., 30, 337 (1947). Cristol, S. J . , J. Am. Chem. Soc., 67, 1494 (1945). Cristol, S. J . , Hayes, R. ., and Haller, H . L . , Ind. Eng. Chem., Anal. Ed., 17, 470 (1945). Daasch, L . W., Anal. Chem., 19, 779 (1947). Davidow, B., paper presented at meeting of Association of Official Agricultural Chemists, Washington, D . C., October 1948. Davidow, B., paper presented at meeting of Federation of American Societies for Biology, Detroit, Mich., April 1949. Davidson, J . C., and Terrell, H . D., paper presented at Philadelphia, Pa., meeting of AMERICAN
C H E M I C A L SOCIETY, Jan. 20, 1949.
(22) Donovan, C. G., Soap and Sanit. Chem., 22 (6), 165 (1946). (23) Downing, J . R., Freed, M . V., Walker, J . F . , and Patterson, G. D., Ind. Eng. Chem., Anal. Ed., 18, 461 (1946). (24) Dragt, G . , Anal. Chem., 20, 737 (1948). (25) Dvornikoff, M . N . , and Morrill, H . L . , Ibid., 20, 935 (1948). (26) Edwards, F . I., Jr., Ibid., 21, 1415 (1949). (27) Fairing, J . D., Agr. Insect. Fungicide Assoc. News (Dec. 31, 1948). (28) Fiero, G . W., Soap and Sanit. Chem., 23 (10), 147 (1947). (29) Fleck, E. E., J. Assoc. Offic. Agr. Chemists, 30, 319 (1947). (30) Ibid., 31, 368 (1948). (31) Fleck, E . E., J. Org. Chem., 12, 708 (1947). (32) Goldenson, J . , and Sass, S., Anal. Chem., 19, 320 (1947). (33) Gunther, F . ., Hilgardia, 18, 297 (1948). (34) Gunther, F. ., Ind. Eng. Chem., Anal. Ed., 17, 149 (1945). (35) Hall, S. ., and Jacobson, M . , Agr. Chemicals, 3 (7), 30 (1948).
HALLERDETERMINING NEW INSECTICIDES IN FORMULATIONS AND RESIDUES (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49)
71
(50) (51) (52) (53) (54) (55) (56)
Hall, S. ., and Jacobson, M . , Ind. Eng. Chem., 40, 694 (1948). Haller, H . L . , Agr. Chemicals, 2 (9), 26 (1947). Herriott, R. M . , Science, 104, 228 (1946). Howard, . H . , Analyst, 72, 427 (1947). Ingram, G . B., and Southern, . K., Nature (London), 161, 437 (1948). Jones, . ., private communication. Koblitsky, L . , and Chisholm, R. D., J. Assoc. Offic. Agr. Chemists, 32, 781 (1949). LaClair, J . B., Anal. Chem., 20, 241 (1948). LaClair, J . B., Ind. Eng. Chem., Anal. Ed., 18, 763 (1946). Rohwer, S. ., Chem. Eng. News, 26, 2356 (1948). Schechter, M . S., unpublished report. Schechter, M . S., and Haller, H . L . , J. Am. Chem. Soc., 66, 2129 (1944). Schechter, M . S., Pogorelskin, . ., and Haller, H . L., Anal. Chem., 19, 51 (1947). Schechter, M . S., Soloway, S. B., Hayes, R. ., and Haller, H . L., Ind. Eng. Chem., Anal. Ed., 17, 704 (1945). Soloway, S. B., Schechter, M . S., and Jones, . ., Soap and Sanit. Chem., Blue Book, 18th ed., 215 (1946). Stiff, . ., and Castillo, J . C., Science, 101, 440 (1945); J. Biol. Chem., 159, 545 (1945); Ind. Eng. Chem., Anal. Ed., 18, 272 (1946). Trenner, N . R., Walker, R. W., Arison, B., and Buhs, R. P., Anal. Chem., 21, 285 (1949). Tressler, C. J . , Jr., J. Assoc. Offic. Agr. Chemists, 30, 140 (1947). Umhoefer, R. R., Ind. Eng. Chem., Anal. Ed., 15, 383 (1943). Wichman, H . J . , J. Assoc. Offic. Agr. Chemists, 31, 349 (1948). Wichman, H . J . , Patterson, W. I., Clifford, P. ., Klein, A . K., and Claborn, . V., Ibid., 29 , 188 (1946).
Mass-Production Techniques for Estimation of Parathion Residues

F. A. GUNTHER and R. C. BLINN
University of California Citrus Experiment Station, Riverside, Calif.
The magenta color reaction for parathion has been adapted to mass-production techniques for quantitative estimation of parathion residues in and on certain fruits, vegetables, and miscellaneous substrates.
Experimental batches of parathion (0,0-diethyl O-p-nitrophenyl thiophosphate) were made available to many entomological research centers in early 1947 (through the courtesy of the American Cyanamid Company), for evaluation as to insecticidal potential in an integrated program of testing against many species of insects and mites. Requisites of entomological practice in the field include companion chemical estimations of deposits afforded by the various dosages, adjuvant mixtures, and additive combinations. Details of a very sensitive method for the chemical estimation of parathion, as developed by Averell and Norris (i), were made available at the same time. The presumed gross chemistry of the analytical procedure is shown in Figure 1.
Figure 1.
Brilliant magenta dye Presumed Chemistry of Color Reaction for Parathion
E.K.C. 4835 is N-(l-naphthyO-ethylenediamine dihydrochloride. Recent chromatographic evidence indicates that the final colored reaction product from technical grade parathion may be a mixture of at least three and possibly four components.
The resultant magenta color is remarkably intense per unit of parathion; this, and other characteristics, indicated the ready adaptability of this method to the quantitative estimation of parathion i n and on fruits, vegetables, foliage, and woody tissues, and i n soil. The present paper reports upon endeavors to define or elaborate upon the parameters
72
GUNTHER AND BLINNMASS-PRODUCTION TECHNIQUES FOR PARATHION
73
restricting the applications of this method, and upon certain refinements pertinent to mass production techniques for parathion estimations, as based upon 2 years' work and nearly 3000 estimations upon approximately 50 different substrates. These techniques are designed for the efficient handling of large numbers of analyses involving a variety of substrates.
Analytical Procedures
F r o m 8 to 30 pounds [or 120 leaves, if foliage U)} of the field sample are used; pene tration samples are dissected or otherwise suitably treated so as to afford several pounds of the desired anatomical portion of the field sample. [The size of the field sample and its method of collection vary tremendously for different materialse.g., olives vs. alfalfa hay. These two parameters are under statistical investigation, but the paucity of reliable and sufficiently extensive data precludes formal publication of field sampling and processing techniques for materials other than leaves (4). A manual of processing procedure (5) has been mimeographed for guidance of personnel, based upon empirical observations; as data are accumulated, it will be revised and expanded periodically. ] After being coarsely chopped or shredded, the material so obtained is sampled b y quarters to afford 454 grams of analytical sample. This pound of sample is placed i n a spe cial stainless steel container with 908 m l . of technical benzene and 25 m l . of 1 0 % hydro chloric acid solution, then minced thoroughly with a high-speed cutter on a drill press, as previously described (3). After being capped tightly with N o . 300 M . S . T . cellophane u n -
400 Figure 2.
500 WAVELENGTH
600 IN mp
700
Dyed Parathion and Dyed Aniline

Parathion, Xmax. 560 \, cmax. 39,091 Aniline, Xmax. 560 , emax. 40,859 In 6 0 % ethyl alcohol, pH 1.2
der a cork gasket (3), the stainless steel can is tumbled end over end at 58 r.p.m. for 30 minutes. ["Strip" samples are tumbled from 2 to 30 minutes, depending upon their n a ture; leaf and certain other penetration samples may be tumbled for as long as 4 hours (see for details).] F r o m 200 to 300 m l . of the resulting benzene extract are then col-
74
lected by dcantation through Sharkskin filter paper, and stored i n a tightly closed 500m l . bottle at 3 C . for subsequent estimation of parathion content. W i t h i n 48 hours, the sample is analyzed. T o the bottle, at room temperature, are added 20 grams of an intimately ground 2 to 1 mixture of clay (Attaclay, available from the Attapulgus Clay Company, Philadelphia, Pa.) and Hyflo Super-Cel, with shaking. After the resulting suspension has settled, an aliquot (usually 100 m l . This volume will vary with the amount of parathion present i n the original sample, and must be predetermined for each sample type and field treatment.) of the clear supernate is pipetted into a 500-ml. bottle, and evaporated quickly to a moist residue at 80 to 90 C . with the aid of a gentle current of impinging air.
Figure 3.
MICROGRAMS OF PARATHION Standard Curves for Dyed Parathion
In 2 0 % ethyl alcohol (7) In 6 0 % ethyl alcohol In acetone For 2 0 % ethyl alcohol, 304.2 X +607.6; for 6 0 % ethyl alcohol, = -824.3 X + 1647.2; foracetone,E = -350.7 X +715.5. Ordinate is % transmittance X 1 0
_ 1
T o the bottle are then added 10 m l . of 9 5 % ethyl alcohol, 10 m l . of water, 2 m l . of 5 A hydrochloric acid solution, and approximately 0.5 gram of powdered zinc, and the re sulting mixture is maintained at 80 to 90 C . for 10 minutes. This reduction mixture is filtered at once through Sharkskin paper into a 100-ml. volumetric flask, and the filter cake is washed with three 7-ml. portions of water. Exactly 1 m l . of 0.25% sodium nitrite solution is then added to the combined filtrates with shaking, followed after exactly 10 minutes by 1.0 m l . of 2.5% ammonium sulfamate solution with shaking. After another 10-minute period 2.0 m l . of a 1% solution of A-(l-naphthyl)-ethylenediamine dihydrochloride (Eastman K o d a k Company, N o . 4835) are added, with shaking. The magenta color indicative of parathion develops at this stage; after a 10-minute development 50 ml. of 9 5 % ethyl alcohol and 2 m l . of 3 A hydrochloric acid solution are added, and the mix ture is adjusted to volume with water. If cloudy, the final mixture is extracted i n the volumetric flask with two 5-ml. portions of petroleum ether (30 to 60 or 60 to 70 C ) .
T
GUNTHER AND
BLINNMASS-PRODUCTION TECHNIQUES FOR
PARATHION
75
The final optically clear solution is evaluated as to intensity of color at 555 , and with slit width at 0.02 to 0.04 mm., on a Beckman spectrophotometer adjusted to 100% transmittance with an untreated sample freshly processed i n parallel with the unknown sample. F r o m a standard calibration curve (see Figure 3), the concentration of parathion may readily be ascertained, and an appropriate factor converts this value to micrograms of parathion present i n the original field sample. Calibration The original procedure of Averell and Norris (1) may be followed, using either para thion distilled under high vacuum or reduced parathion repeatedly recrystallized from dilute hydrochloric acid solution. The use of ordinary technical grade parathion is not recommended for precise comparison purposes, because it has been the authors' experience that batch and manufacturer variation in both quality and composition of product may be considerable. High-vacuum distillation of parathion requires equipment not always available, and the securing of sufficiently pure crystalline reduced parathion, without seed material, is likely to be a time-consuming and difficult process. For precise calibration and the preparation of a standard curve, therefore, carefully purified aniline or aniline hydrochloride may be used, inasmuch as the visible absorption spectrum obtained thereby (after dyeing) is spectrophotometrically indistinguishable from that of reduced parathion (after dyeing), as shown in Figure 2. The resultant magenta color attains 87.50% of its maximum intensity at 555 within 6 minutes at room temperature, 92.61% within 9 minutes, 96.03% within 15 minutes, and 99.43% within 30 minutes. A t 5 0 % transmittance the ratio of purified aniline to technical grade parathion is 0.958; as the purity of the parathion is increased this ratio approaches unity. Thus, the determination of this ratio could conceivably be used as a criterion of purity for technical grade parathion. T y p i c a l standard curves for a technical grade of parathion are shown i n Figure 3. Beer's law is followed within the range 10 to 400 micrograms of parathion per milliliter of dyed solution. Deviation becomes apparent outside this range, under the conditions of test. Table I. Typical Blank Values of Various Fruits and Vegetables
(Parts per million of parathion) Apples, Rome Beauty Beans, string Cabbage Carrots CeleryCorn, sweet Grapes, seedless (Thompson) Hay, alfalfa Lemons, Eureka Olives, Mission Oranges, navel Surface Pulp Total Total Tubers Foliage Total Kernels Husks Total Air dried Peel Pulp Seeded Peel Pulp Processed peel (stock feed) Orange oil Twigs Peel Pulp Processed peel (stock feed) Orange oil Fruits plus caps Surface 0.00 0.01 0.01 0.20 0.04 0.00 0.12 0.04 0.02 0.03 0.00 0.00 0.00 0.06 0.10 0 00 0.50 2.50 0.09 0.35 0.07 0.30 1.60 0.10 0.10
Oranges, Valencia
Strawberries Walnuts (unhusked)
For consistent, comparable results it is essential that a fresh sample blank be proc essed and otherwise be treated identically with each current batch of field samples. Whenever possible, this blank specimen should be from the same locality and should have the same previous spray (or other) history as the actual samples. Frequently, fruit and vegetable parts possess benzene-extractable pigments or other substances not removable by the decolorizing treatment utilized. I n order to eliminate such background interfer-
76
ences, the dyed solution from the sample blank is used to preset the spectrophotometer at 100% transmittance immediately prior to running the actual samples corresponding to that blank. When the field sample is sufficiently large, there is ordinarily little if any difference between the colors at 555 developed or present i n blank samples from the same locality; blank samples from different localities may, however, exhibit demon strably different absorption characteristics at this wave length when processed as de scribed. I n Table I are listed some typical blank values (sample blank transmittance cor rected for reagent blank transmittance at 555 , converted to parts per million of para thion).
Preparation of Samples
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch017 As pointed out by Averell and Norris (1), this colorimetric procedure for estimating parathion is extremely sensitivefor example, as little as 2 to 3 micrograms of parathion per sample will afford a visible color, and the presence of as little as 0.2 microgram of parathion per sample is spectrophotometrically demonstrable. I t thus becomes essential to avoid contamination of the sample at every step. Extreme measures are required to accomplish this end, for on exploratory material a negative or borderline sample could easily be trace-contaminated so as to appear to contain misleading quantities of para thion. The following aids are recommended in eliminating contamination :
Scrub all utensils, containers, working surfaces, and equipment with warm 10% trisodium phosphate solution after each sample. Wherever possible, rinse each utensil and piece of equipment with acetone and benzene. Because parathion residues adhere so tenaciously to glass surfaces, subject all glassware to the following sequence of washes: warm 10% trisodium phosphate solution, distilled water, acetone, and benzene. Rinse stored glassware with benzene again just prior to use. Never bring containers of parathion or parathion dusts into the room where these prepara tions or analyses are being performed. If parathion-containing extracts are spilled, hose down the floor at once. Cover all table areas and other working surfaces with wrapping paper or other disposa ble material ; replace after processing each sample. Keep blank field samples separate from treated samples, both when in transit from the field and when in refrigerated storage awaiting processing. Separate component parts of some field samples in the fielde.g., carrots from carrot tops i f they are to be analyzed separately for parathion content. As an illustration of this point, airplane-dusted carrots were put through a commercial washer and divided into two lots. One lot was topped and stored 4 days in a paper bag; the untopped lot was treated similarly but topped during the normal processing. The tops analyzed 1.2 p.p.m. of parathion. The fieldtopped carrots analyzed nil parathion, whereas the lot untopped during storage analyzed 0.2 p.p.m. of parathion. Exercise scrupulous care during dissection of the samples to avoid even trace carry-in con tamination. Specific techniques must be evolved and verified for each type of fruit or vegetable [see Carman et al. (2) and Gunther (5) ]. T o illustrate, the following procedure has been found satisfactory for apple penetrations. A clean, sharp knife is forced axially one third through the apple, and the fruit is cracked open by twisting the knife. That portion of the apple pulp un touched by the knife is scraped out with the aid of a sharpened melon bailer. With care, the flesh can be scraped gently from areas inside the skin so as to leave ultimately an intact paperthin translucent shell of skin freed from adhering pulp. Accidental puncture of the skin with the scraping tool means discarding the remainder of that apple and recleansing the tool. Unless proved otherwise, even refrigerated (3 C.) storage of field samples may not lessen the disappearance (migration, degradation, metabolization, or other routes) of parathion con tained in certain tissuesfor example, frequently 10 to 20% losses of parathion content have been experienced after storage at 3 C. for 5 days. Expeditious processing of all samples is thereby indicated.
Size of Subsample. A l t h o u g h i t is experimentally obvious that, w i t h m a n y fruits and vegetables, an arbitrary 8- to 30-pound field sample is hardly adequate for true representation of a field plot, practical considerations i n large scale field operations delimit the size of the sample. Exploratory studies indicate that 64 mature Valencia oranges per sample, when picked i n a consistent manner with 16 fruits from each of 4 randomized normal trees, suf fice to give fair (10%) agreement between replicates; such a sample ordinarily weighs from 18 to 24 pounds. Very cursory tests with Thompson seedless grapes demonstrate
77
several hundred per cent variation between the deposits on 5-pound replicated samples of small bunches selected at random throughout the treated vineyard. Nonetheless, when such a field sample is suitably subdivided into pooled component parts and subsampled b y quarters into pound portions, there will i n general be excellent analytical agreement between the pound subsamples or "aliquots." Subsamples of less than a pound may contain insufficient parathion to afford readily obtained aliquots of extract containing amounts of the insecticide optimum to the accuracy of the present method. Compounded dusts and other concentrated forms, of course, require less material but also extraordinarily painstaking sampling and subsampling techniques so as to maintain the bulk homogeneity originally present. The inhomogeneity of aged insecticide-containing dust mixtures will be dealt with in a subsequent report. Choice of Solvent. A s indicated b y Averell and Norris (1), and independently confirmed b y the authors, technical benzene is a superior stripping solvent for parathion residues. I t is almost completely miscible with technical grade parathion at room temperatures, it is universally available and low i n cost, i t is readily volatile, it fails to contribute to storage decomposition (6), it is a good solvent for plant oils and waxes, and it is immiscible with water. On the other hand, benzene is highly flammable and its vapors are very toxic to human beings, especially as a chronic toxicant even i n small doses. Because of these hazardous features, certain commonly used solvents other than benzene were investigated for this purpose. Solubility tests with purified parathion indicated that i t is at least 30 volume % soluble i n technical grade acetone, benzene, carbon tetrachloride, dioxane, ethyl acetate, and ethyl alcohol. Cost and other considerations (1) limited the field to acetone, benzene, and dioxane. Residues remaining from the evaporation of dioxane i n terfered with the development of the desired deep magenta color, affording instead a pale yellow-orange. When 50- to 200-ml. volumes of the other two solvents containing from 76 to 279 micrograms of technical grade parathion at five levels were evaporated and run through the analytical procedure, benzene afforded slightly more reliable results with a recovery range of 82 to 9 6 % (average 89%), whereas the recoveries from acetone were within the range 76 to 9 5 % (average 86%). I n both sets of data, as the quantity of parathion increased from 76 to 279 microgramsregardless of volume of solventthe analytical recovery decreased i n an irregular manner. These facts suggest that variable recoveries of parathion from the evaporation procedure, as used routinely, should be expected. I n general, however, the recovery data did not demonstrate a clear-cut distinction between acetone and benzene for the purpose at hand. A slow steady loss of parathion in proportion to the volume of either solvent evaporated (1) was not noted, which would indicate that the rate of evaporation is also important. The final decision as to solvent was determined by certain incidental properties of the parathion. Wet Extraction Techniques. I t may readily be demonstrated that suitably d i s persed parathion is short-lived. F o r example, a thin film of a 2 5 % wettable powder exposed i n a laboratory environment may show little or no parathion, b y analysis, after 14 days (7) ; ground citrus peel containing 12 p.p.m. of field-applied parathion will analyze considerably less insecticide after being dried to constant weight at 65 C. in a convection oven; and there is marked dissipation of parathion from treated fruits even at 3 C . Such observations indicated that the drying and extraction techniques (4) previously used for residue and penetration studies could not be employed with parathion. The wet extraction techniques herein described were therefore devised to circumvent drying operations. Acetone as the solvent was thereby eliminated from consideration because most of the contemplated field samples were high i n water content and would thus have required excessive quantities of solvent for adequate extractione.g., orange juice, plums, ripe peaches, etc. As a rule of thumb, 2 m l . of benzene per gram of analytical sample serve to afford adequate extraction with minimum emulsification difficulties, so that the addition of emulsion breakers is usually unnecessary i n order to obtain sufficient clear extract for analysis. Experiences have indicated that occasionally less benzene will suffice; on the
78
other hand, Thompson seedless grapes and ripe freestone peaches require 4 m l . per gram. The optimum volume should be determined for each type of material (5). Purifying the Benzene. Occasionally a d r u m of technical grade benzene is e n countered, the contents of which will develop a pronounced pink color when sub jected to the analytical procedure. A typical transmittance-wave-length curve from a 250-ml. specimen of such processed benzene is shown in Figure 4. B y comparison with
700 500 600 WAVELENGTH IN my Figure 4. Typical Transmittance-Wave-Length Curve for Processed Unknown Constituent of Technical Grade Benzene 400 Figure 2, it will be seen that the major maxima coincide at 550 to 560 . The nature of this interfering contaminant is not known, but it is readily extractable into aqueous h y drochloric acid (7). T o eliminate the tedium of testing and tracing every lot of benzene, 25 m l . of 1 0 % hydrochloric acid solution are routinely added to the sample just prior t a mincing. This procedure has effectively removed such solvent interference from consid eration. Table II. Interference of Liner Materials with Analysis
Indicated Micrograms of Parathion 77.2 77.2 Dissolved (no color) 240.5 30.1 No color No color 50.0 66.5 Disintegrated (no color)
100
Material Stock solution Cellophane Lucite Neoprene Nylon Oilcloth, white Rubber Velon Vinyl acetate (polymer) Waxed paper
Subdividing the Sample. W e t processing requires unusually thorough s u b d i v i sion of the sample, to permit efficient extraction by a water-immiscible solvent. The machine developed for this purpose has been described (3) ; its design was based upon that of the familiar and efficient Waring Blendor. Originally, samples were minced i n added water, but emulsions difficult to break were frequently formed during the subsequent stripping process, whereas if the sample were minced directly i n benzene as the fluid me dium these emulsification phenomena were minimized. Adequate mincing i n benzene is a hazardous operation and should not be entrusted to untrained personnel. Forced ventila tion of the entire mincing area is essential; benzene vapors are both toxic and flammable. Container Closure. T h e stainless steel containers (3) of material minced i n b e n -
79
zene are capped with a double thickness of N o . 300 M . S . T . cellophane topped with a buffer gasket of 0.125-inch cork sheeting, and then the steel l i d is bolted on. M a n y types of liner material were tested. Cork liners were unsatisfactory because of contamination, and metal foils were too expensive for large-scale operations. A number of materials were evaluated b y being immersed i n 100 m l . of a stock solution of parathion i n benzene for 1 hour, with subsequent analysis of the resulting benzene extracts. Some of these materials, and the analytical results obtained, are presented i n Table I I . The N o . 300 M . S . T . cellophane is sufficiently thick to resist reasonable tearing and is waterproof; each cellophane liner is used only once, then discarded. The authors have found it convenient to purchase this cellophane i n precut 6 X 12 inch sheets i n lots of 10,000 (available from the Zellerbach Paper Company, Los Angeles, Calif.). D o w n - D r a f t H o o d . T o protect personnel from benzene vapors, i t is recommended that all the pouring, container closing, and final dcantation and bottling operations be performed on an efficient down-draft hood (4). T o increase efficiency in inserting and removing the bolts on the steel cans, a reversible air-driven screwdriver has been suspended on a counterweight over the down-draft hood.
Figure 5. Automatic Stripper.
Drum-Type Stripping Machine with Automatic Controls A photograph of the drum-type stripper is shown in Figure 5.
E a c h of the four vanes of the drum contains four compartments, 6 X 6 X 10 inches in inside dimensions. One of the steel cans fits snugly into such a compartment; the wooden blocks stacked to the right of the apparatus i n the photograph are used to wedge 2-quart M a s o n jars (for leaf stripping) into the compartments. Each vane l i d , opening to the front, is fastened shut b y two suitcase catches. The drum itself is fastened b y means of welded collars to a 1-inch steel shaft suspended on 4-inch bronze sleeve bearings. This shaft is coupled to a heavy-duty, 30-to-l gear reducer, which i n turn is driven through a
80
coupling by a 0.5-hp., 1725 r.p.m., explosionproof motor. Thus, the drum revolves at 58 r.p.m., a speed sufficient to induce a violent surge effect within the stripping container. Accessories include a remote-control, explosionproof switch, and a built-in electric time clock. This last feature is very convenient, for with i t the operator can set the stripper to run any predetermined length of time without attention. The pulley system visible above the driving mechanism allows the operator to hand-position the drum for loading and u n loading the vanes. Efficiency of Stripper. There are extant nearly as m a n y stripping devices or manual stripping procedures as there are entomological laboratories. Regardless of the means whereby residue stripping is achieved, consistent and assiduous application of any one technique by one group of workers will presumably lead to comparable analytical re sults within that laboratory. However, this constancy will not hold when the attempt is made to compare analytical results originating i n different laboratories where different techniques are employed, because the efficiency of the stripping operation will delimit the ultimate analytical value even though the* analytical techniques themselves are standard. If rigid comparisons of data from various groups are to be valid, therefore, stripping tech niques must be standardized.
20
40
60
80
100
120
140
160
180
Figure 6. Time-Efficiency of Stripping Apparatus in Extracting Parathion from Orange Leaves

A. After 7 days; After 11 days Original strip; B. First restrip; C. Second restrip
Adequate comparisons of the efficiencies of the many types of stripping devices i n existence have never been made ; actually, such comparisons are not necessary, nor is it necessary to consider the purchase or construction b y each group of a standardized piece of stripping equipment. Rather, it is proposed that the efficiency of each such apparatus be determined as the time of stripping required to attain equilibrium of concentration of the insecticide between extracting solvent and substratei.e., when the plot of quantity of insecticide in solvent vs. time levels off. Then, if all stripping operations i n a given ap-
GUNTHER AND BUNNMASS-PRODUCTION TECHNIQUES FOR PARATHION
81
paratus were extended to twice this minimum time, all samples i n all apparatus or procedures would be positively overstripped and thus in comparable condition. This statement is based upon the fact that the ratio of solvent to solute i n question is ordinarily so large (of the order of 1,000,000 to 1) that an eventual equilibrium is unavoidable. To this end, the authors have determined the time-efficiency of their apparatus, as demonstrated by the data plots in Figure 6. The substrate was Valencia orange leaves, with 2500 leaves per sample selected i n a carefully prescribed manner (4). The trees involved were field sprayed i n a conventional manner with 4 pounds of a 2 5 % wettable powder of parathion, then sampled after 7 days and again after 11 days. E a c h sample was mixed thoroughly and subsampled into 125leaf units i n 2-quart Mason jars. T o all units were added 250 m l . of benzene each, and they were sealed, stripped for various lengths of time, then restripped with fresh benzene, again for various lengths of time. The strip solutions were analyzed i n the usual manner. F r o m Figure 6 it is apparent that the stripping apparatus approaches the equilibrium distribution within 15 to 20 minutes; therefore i n accordance with their proposed standardization, the authors utilize 30- to 40-minute stripping intervals i n routine practice. It is further demonstrated i n this figure that one strip per leaf sample is adequate for routine purposes, with the initial equilibrium at approximately 8 0 % extraction. These curves also demonstrate the incidental 5 0 % loss of parathion after the additional 4 days i n the field for the second sample. Similar data have been secured for the other plant parts studied to date. M i n c e d
82
citrus peel, for example, requires 30 minutes to attain the 8 0 % equilibrium condition and, therefore, 1 hour is routinely utilized for the overstrip standardization. Residues on 2pound analytical samples of whole Rome Beauty apples are apparently extracted well within a 30-minute interval, as shown i n Table I I I . These four samples were stripped separately, to afford an indication of the typical variation between field deposits, under the conditions of the experiment. Table III.
Sample No. B-196 B-192
Stripping Interval for Rome Beauty Apples

Parathion, P . P . M . , after 30 min. 180 min. 7.1 13.6 13.0
7.7
Storage of Extracts. After being filtered, the decanted extracts are stored, i n bottles capped with waxed paper liners, i n a refrigerator set for 3 C . E v e n at this low temperature, with most of the benzene frozen, a loss of the contained parathion must be assumed to occur unless proved otherwise Pure parathion in benzene solution can be stored without loss almost indefinitely in the dark at this temperature, but technical grade parathion in a benzene extract of plant material may behave differently. For example, benzene extracts of apple pulp (total) have lost 3 0 % of their contained parathion i n 5 days at 3 C , and benzene solutions of orange oil have exhibited 2 0 % losses in 10 days. On the other hand, certain leaf extracts are apparently stable at this temperature. ^ Room-temperature environments for any but pure benzene solutions are not to be recommended. Because of weekly surges of sample receipts, it is frequently necessary to store large n u m bers of samples and sample extracts for short periods of time. Under such circumstances, mechanical failure of the refrigerators could be serious. The authors have therefore fitted their refrigerators with red warning lights and thermostats set to turn the lights on at 10 C . Because of the unknown nature of this possible disappearance of parathion during refrigerated storage, it is strongly recommended that all extracts be analyzed within 48 hours after preparation.
Analysis of Samples
The actual analytical procedure is essentially that of Averell and Norris (1), the m a jor modifications being the addition of steps to eliminate the interfering amounts of proteinaceous and waxy or oily extractants resulting from general utilization of larger samples. Some minor modifications have necessarily been introduced in adapting their procedure to mass-production efficiency. To preserve the continuity of the present report, however, the detailed analytical method has been presented in its entirety. Decolorization. T h i s procedure, adapted from (1), will remove most green and some orange-to-red substances from the benzene solutions, but will not remove appreciable parathion in the concentrations commonly encountered. Evaporation of Samples. Six samples of usually 250 m l . each are simultaneously evaporated to a moist residue in a water bath, shown in Figure 7, which is maintained at 80 to 90 C . The exhaust manifold is connected with the house vacuum. W i t h the assemblies shown a gentle stream of air is drawn over the surface of the evaporating solution and out the vacuum line ; the air inlet tube is lowered frequently so as to maintain a rippled surface on the benzene extract. I n this manner, 250 m l . of benzene can be evaporated completely i n 10 minutes or less. Although Averell and Norris (1) suggest that the final evaporation of the last 10 m l . be accomplished with the aid of a jet of air at room temperature, so as to avoid excessive losses of parathion, the apparatus shown in Figure 7 accomplishes the final evaporation without such "excessive" losses. This conclusion is supported b y the data i n Table I V , with losses of about 2 0 % i n all cases. D a t a for both acetone and benzene are again i n cluded to re-emphasize the interchangeability of the two solvents for this utility. The two
83
volumes of solvent were chosen to represent the extreme range most frequently encountered i n these laboratories. Table IV. Evaporation Losses of Parathion with Water Bath at 80 to 90 C. vs. Air Jet at Room Temperature
Solvent Acetone Acetone Benzene Benzene Acetone Acetone Benzene Benzene Solvent, Ml. 1.0 1.0 1.0 1.0 250 250 250 250 How Evaporated Air jet Air jet Air jet Air jet Water bath Water bath Water bath Water bath % Loss 21.9 23.9 19.2 20.6 18.0 16.9 19.2 20.4 112.1 112.1 106.0 106.0 112.1 112.1 106.0 106.0
Parathion, Micrograms
Reduction of Parathion. This reduction is carried out i n the same k i n d of bottle and i n the same bath as shown in Figure 7. Such a tall bottle, i n contrast to a beaker or short flask, maintains reflux conditions and thus prevents excessive losses of alcohol. A s indicated b y Averell and Norris (1) and verified by the authors, this procedure will reduce quantitatively several milligrams of parathion in the time specified. Ordinarily six reductions are carried on simultaneously. Development of Color. This procedure is exactly as described by Averell and Norris (1).
0.4
0.6
0.8
1.0 PH
1 . 2
Figure 8. Effect of Varying pH on Intensity of Color of Dyed Parathion

A. pH of original dyed sample without dilution
Removal of Proteinaceous "Clouds." W i t h the unchanged method of A v e r e l l and Norris (1) the final colored solution of 50.0 m l . may contain as much as 2 0 % ethyl alcohol. M a n y plant materials, particularly apple pulp, yield proteinaceous substances to the benzene during extraction; if the aliquot of extract represents more than 40 to 50 grams of sample, these substances will be present i n this final solution as a cloud of solid particles. These substances, whatever their nature, appear to be completely soluble i n 6 0 % ethyl alcohol. Thus, the authors recommend the addition of 50 m l . more of ethyl alcohol at this stage, the addition of more hydrochloric acid to maintain p H , and final dilution to 100.0 m l . with water. The resulting solution will be optically clear except for oily or waxy substances, which are removed in the next step. Removal of Oils and Waxes. T w o extractions with 5-ml. portions of petroleum ether (30 to 60 C . or 60 to 70 C.) have sufficed to eliminate interfering oils and waxes in suspension. Such extractions are conveniently carried out in the 100-ml. volumetric flask after final dilution and mixing. For the evaluation of color only a small portion of the 100 m l . will be utilized; therefore, if necessary, some of this solution can be discarded
84
at this point so as to accommodate the 5 m l . of petroleum ether i n the volumetric flask. After being vigorously mixed b y shaking with the colored solution, the petroleum ether is conveniently removed with the aid of a small pipet and rubber bulb. Neither visible nor spectrophotometric evidence has ever been secured to indicate that any of the dye is transferred to the petroleum ether. If, however, for other reasons it is deemed undesirable to extract oils and waxes at this stage, the extraction m a y be performed similarly just prior to the addition of the sodium nitrite solution. Extraction at this earlier stage is best accomplished with 30 to 60 C . petroleum ether, for the 60 to 70 C . fraction will remove significant amounts of the reduced parathion hydrochloride. This point is illustrated b y the data i n Table V . I n each instance, 194 micrograms of the reduced amine i n 43 m l . of water at p H 3.1 were extracted twice with 5-ml. portions of the petroleum ether, then color development and evaluation were carried out i n the usual manner.
WAVELENGTH IN mp
Figure 9. Transmittance-Wave-Length Curves for Dyed Parathion in 2 0 % Ethyl Alcohol

pH2.25; pH 1.68; ---pH1.40; pH 10.9
85
p H and Color. A v e r e l l and N o r r i s (1) stressed the fact that this colored solution is p H sensitive, although i t would seem to be well buffered. The authors have evaluated certain effects of minor changes i n p H , and have found that the absorption maximum is not shifted from p H 0.2 to p H 2.25, and that the intensity of color is not affected appre ciably within the p H range 0.2 to 1.4. When a solution containing 104.6 micrograms of parathion as the magenta dye was adjusted to various p H values, and the transmittance was determined at 555 , the transmittance held essentially constant below p H 1.4, as shown i n Figure 8. Table V. Extraction of Reduced Parathion Hydrochloride by Petroleum Ethers from Aqueous Solutions
Petroleum Ether, C .
0
% Loss 6.4 6.4 20.6 20.0
30-60 60-70
WAVELENGTH IN mji
Figure 10. Transmittance-Wave-Length Curves for Dyed Para thion in 6 0 % Ethyl Alcohol
pH 1.90; pH 2.30; pH 2.80; pH 10.5
86
T a u t o m e r i s m of the D y e . T h e complete visible absorption spectra were secured for dyed parathion at several p H values i n both 20 and 6 0 % ethyl alcohol. These curves are plotted i n Figures 9 and 10. T h e appearance of an isobestic point i n both instances indicates that tautomeric forms of the dye are involved (&). 20% vs. 60% E t h y l A l c o h o l . A s m a y be seen from Figures 9 and 10, the absorp t i o n m a x i m a i n both are at 558 .
Interfering Substances
A n y nitro- or amino-aromatic compound may be considered a potential source of i n terference until demonstrated otherwise. For example, D N - 1 1 1 in benzene was processed in the usual manner. During the reduction step a pink color developed which was insol uble in petroleum ether; addition i n proper sequence of the dye-producing reagents re sulted i n the development of a deep purple color within 10 minutes. D y e d aniline is spectrophotometrically indistinguishable from dyed parathion, within the visible range. However, when purified cyclohexylamine (7.6 X 1 0 ~ M) is subjected to the analytical procedure there is no color development.
5
10
WAVELENGTH IN mil
Figure 1 1. Transmittance-Wave-Length Curves for Dyed Parathion and p-Nitrophenol in 6 0 % Ethyl Alcohol at pH 1.2
Parathion; p-Nitrophenol
p-Nitrophenol also interferes, as may be seen from Figure 11. D y e d parathion exhib its maximum absorption at 5 5 8 ( x . 3 9 , 0 9 1 ) after 10 minutes, whereas dyed p-nitrophenol peaks at 5 9 4 ( . 9 6 0 0 ) . The latter dye requires more than a 10 -minute de velopment period to attain maximum intensity of color, but some color is developed within 10 minutes. There is sufficient overlap of the two curves to warrant the use of a spectrophotometer i n routinely evaluating the colored solutions obtained from technical grade parathion. T o this end, the authors recommend that the wave length 555 be
ma
max
GUNTHER AND BLINNM ASS-PRODUCTION TECHNIQUES FOR PARATHION
87
adopted for the colorimetric estimation of parathion, with particular reference to parathion i n and on plant products. E v e n though the p-nitrophenol color may not develop fully before colorimetric evaluation of a sample, the use of a spectrophotometer will m i n i mize this possibly variable interference.
Acknowledgment
This work and any practicable results derived therefrom have been encouraged and expedited throughout b y the American Cyanamid Company. Additionally, the authors wish to express appreciation for aid i n various phases of the work to M r s . M . E l l i o t M i l l e r , Charlotte M c H a l e , Jacquelyn Sellers, L . D . Anderson, M . M . Barnes, G . E . Carman, J . H . Barkley, J . C . Ortega, C. R . Shafer, and H . U . Meyer of these laboratories, and to W . E . Baier and C. W . Wilson of the California F r u i t Growers Exchange, Ontario.
Summary
The magenta color reaction for parathion has been adapted to mass-production tech niques for quantitative estimation of parathion residues i n and on certain fruits, vege tables, and miscellaneous substrates. F r o m examination of the mechanisms of this color reaction, and from experiences gained i n running nearly 3000 analyses on 50 different sub strates, it has become apparent for such applications that: 1. Storage of field samples at room or refrigerator temperatures (3 C.) may be equiv alent to leaving i n the field with regard to persistence and migration of the contained parathion. 2. E i g h t pounds of field sample quartered to 1 pound of parts are adequate for many of the materials examined. 3. Benzene is the best single solvent for stripping or extracting the contained para thion. 4. Wet processing techniques, with usually 2 m l . of benzene per gram, are essential. 5. The resulting benzene strips or extracts frequently will not tolerate storage even in the frozen state. 6. R a p i d removal of solvent at low temperatures is essential. 7. Chlorophyll and some red constituents should be removed prior to color develop ment. 8. F i n a l dilution to 6 0 % ethyl alcohol eliminates protein clouds and extraction with petroleum ether removes oily and waxy substances. 9. Reasonably close p H control is indicated. 10. Spectrophotometry evaluation of color at 555 is recommended. The efficiency of the machinery employed is discussed, pertinent supporting analyti cal data are presented, and the sensitivity of the color reaction, leading to unbelievably easy contamination, is stressed. Overprocessing i n all steps is recommended as a means of nullifying inherent variations in the efficiencies of different types of processing appara tus used by other workers.
Literature Cited
(1) Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753 (1948). (2) Carman, G. E., Ewart, W. H . , Barnes, M . M . , and Gunther, F . ., ADVANCES IN CHEMISTRY
SERIES 1, 128 (1950).
(3) (4) (5) (6) (7) (8)
Gunther, F. ., Anal. Chem., 21, 748 (1949). Gunther, F . ., Hilgardia, 18, 297 (1948). Gunther, F . ., University of California Citrus Experiment Station, mimeo, 1948. Horsfall, J. L., correspondence. Norris, M . V., personal communication. Weissberger, Arnold, "Physical Methods of Organic Chemistry," 1st ed., V o l . II, pp. 775-6, New York, Interscience Publishers, 1946.
Station.
P A P E R 627, University of California Citrus Experiment
Mass Estimation of DDT Surface and Penetration Residues

F. A. GUNTHER and M. ELLIOT MILLER
Techniques for processing large numbers of DDT surface and/or penetration samples whereby precision is in no way sacrificed for speed, involve the utilization of several mechanical devices, including a revolving drum-type stripping machine and an adapted drill press. The efficiency of these mechanized techniques in terms of per cent residue removed and number of samples processed per day is given and compared with the efficiency of hand processing as previously done in this laboratory. The dehydrohalogenation method is the primary analytical procedure used and the permanent setup expediting the analysis of 40 to 50 samples per day is described. Results of analyses of duplicate surface and penetration samples agree within 0.5 microgram per sq. cm. or 0.5 p.p.m.
W i t h the advent of D D T as an important insecticidal material, much work has been required to study its persistence i n the field and its tolerance b y animals and plants. For the past 5 years this laboratory has been engaged i n estimating residues on approximately 20,000 surface and penetration samples. These included a wide variety of m a terials, such as alfalfa, fruits, vegetables, nuts, leaves, twigs, soil, peat moss, wooden slats, galvanized iron, and tar paper. Techniques had to be developed to handle this large volume and variety of samples routinely and expeditiously. Hand Processing Until 1947 a l l samples were processed b y hand, with benzene as the stripping solvent because of its high efficiency and ready availability. W i t h leaf surface residues, the measured leaves were placed i n a 2-quart w ide-mouthed Mason jar with 150 m l . of benzene. A gasket of three thicknesses of heavy waxed paper was used under the tightly screwed l i d . (The wax dissolved from the exposed part of the gasket does not interfere with the subsequent analysis.) The jar was then shaken 20 times vertically, inverted, and again shaken 20 times vertically, and finally shaken 20 times horizontally with rotation. T h e benzene extract so obtained was filtered, 100 m l . more of benzene were added to the jar of leaves, and the process was repeated. A l l filtering operations were done over a specially constructed down-draft slotted floor hood (3), as the normal updraft hood does not remove the heavy benzene vapors adequately, and discomfort and injury to the technician may result. The recovery of D D T was 98 to 100%, as indicated b y restripping the sample and analyzing the second strip solution for D D T . A n experienced technician could process 35 samples a day by this method. W i t h fruit surfaces, 8 pounds of fruit weighed to the nearest ounce were washed b y a jet of benzene from a calibrated wash bottle, using 15 m l . per fruit. The very fine stream was directed at the fruit while it was rotated on an ice pick over a 6-inch funnel fitted with
T
88
GUNTHER AND MILLERMASS ESTIMATION OF DDT RESIDUES
89
Figure 1.
Permanent Apparatus Assembly for Quantitative DDT Estimations
fluted Sharkskin filter paper. The recovery of D D T was the same as for leaf surfaces and again 35 samples could be turned out per day. I n fruit penetration studies 8 pounds of fruit were first thoroughly scrubbed with warm 1 0 % trisodium phosphate solution and then rinsed thoroughly with distilled water. Citrus fruits, if depth of penetration into the peel was of interest, were peeled i n longitudinal sections with a buttonhook peeler and the albedo or white portion was separated from the flavedo or colored portion. T h e separated peel was placed i n pie tins lined w i t h waxed paper and dried i n a forced draft oven at 65 C . for 16 hours. The dried peel was then crushed and steeped for 48 hours i n a measured volume of benzene sufficient to cover the sample. If, on the other hand, only the total amount of D D T i n the peel was of interest, the fruit was halved and juiced on a power juicer. T h e pulp was removed, the peel sliced, and the sample dried and treated as before. Thin-skinned fruits, such as apples, pears, and avocados, were peeled with a vegetable peeler, cores or seeds were removed, and the pulp was sliced i n thin slices. P u l p and peel were then dried and treated i n the same way as the citrus peel. The steeping completed, the samples were filtered through Sharkskin filter paper and the volume of benzene recovered was noted. The recovery of D D T by this method was 93 to 9 6 % (3), as confirmed b y exhaustively extracting a drained sample with benzene and analyzing the extract for D D T . T w o samples per operator could be prepared for drying i n 1 d a y ; however, 3 days elapse between the start and finish of the processing when done i n this way. When the penetration study was conducted on leaves, they were dried, crushed, and extracted exhaustively with benzene i n a Soxhlet apparatus i n the usual manner.
Mechanical Processing
For the past 2 years the output of analyses has been increased greatly, particularly with penetration studies, b y utilization of wet-processing techniques and development of several new pieces of apparatus. This equipment includes the revolving drum-type stripping machine and especially adapted drill press used also for parathion studies. A detailed description of this press has been published (#), and the stripping machine and accessory equipment have been described (5). Leaves are stripped for 30 minutes i n the usual 2-quart wide-mouthed M a s o n jars. Instead of a waxed paper gasket, however, 2 squares of N o . 300 M . S . T . cellophane topped by a square of white nylon are used, as the benzene would disintegrate the wax paper during this longer stripping period. The entire 250 m l . of benzene are added at once, the
90
Figure 2.
Fisher Filtrator Assembly for Quantitative DDT Estimations
lids tightened, and the jars wedged firmly i n the compartments by means of wooden blocks. The rest of the processing is carried out as before. Actually the stripping opera tion is 9 5 % complete i n 5 minutes. A l l samples are overprocessed, however, so that re sults will be comparable to those of other workers who overstrip regardless of their strip ping method. B y using the stripping machine 60 samples may be processed per day as compared with 35 per day by hand stripping. Recovery of D D T was shown to be the same i n both cases. In fruit and vegetable penetration studies, after the samples have been washed and peeled i n a manner prescribed for each type, they are placed i n the metal cans originally developed for parathion studies and minced on the drill press. A double layer of cello phane again serves as the contact gasket. These cans are also used now i n stripping off fruit surface residues. A 1-pound sample of peel segments taken by quarters from the peel of an 8-pound sample of oranges is minced for 1 minute with 2 m l . of benzene per gram of peel and stripped for 30 minutes on the mechanical stripping machine. This again represents overstripping. It has been found empirically that this amount of ben zene is sufficient for most fruits and vegetables. After stripping, the samples are filtered through Sharkskin filter paper and the volume of benzene recovered is noted. W i t h these improved techniques 16 samples can be completely processed i n one day by one operator. Recovery of D D T is the same as for dry processing. Soil samples are handled by shaking 2 pounds of soil, the moisture content of which has been determined for subsequent correction to dry weight, with 1.5 m l . of benzene per gram of soil for 1 hour i n the metal cans on the mechanical stripper. Benzene solutions of D D T may be stored indefinitely at 3 C . Leaves and fruit or vegetables awaiting processing for D D T estimation may be stored at 3 C . as long as the fruit and leaves are i n good condition.
Analytical Procedures
The analytical procedures used for quantitatively estimating the D D T are the dehydrohalogenation method (4) developed by Gunther and the modification of Baier et al. (1) involving the use of 4.5 ammonia i n methanol instead of 1 alcoholic potassium hydroxide as the dehydrohalogenating agent. The latter method is used for samples con taining large quantities of oils, such as occur in certain penetration studies and for samples containing sulfur. Because the modified method involves a 16-hour incubation period, it is used only when the other method proves inadequate.
GUNTHER AND MILLERMASS ESTIMATION OF DDT RESIDUES
91
A permanent setup has been made to expedite the handling of a large number of analyses (8). The benzene strip or extract solutions are evaporated nearly to dryness i n 500-mL standard-taper Erlenmeyer flasks on 3 units of a 6-unit variable heat extraction apparatus hot plate (see Figure 1). Evaporation is hastened b y directing a jet of air at the surface of the benzene, gentle enough to avoid spattering when maintained 0.5 inch above the surface of the liquid. The benzene vapors are removed through a manifold connected t o the house vacuum. On this apparatus 250 m l . of sample can be reduced to a volume of about 5 m l . i n 10 minutes.
Figure 3. Titrator Assembly for Quantitative DDT Estimations T o the moist residue from the evaporation are added approximately 50 m l . of 1 ethanolic potassium hydroxide solution from a graduated cylinder. T h e flasks are placed on the 3 remaining hot plates of the apparatus, fitted with reflux condensers, and allowed to reflux gently for exactly 15 minutes as timed b y a stop clock. Three digestions and 3 evaporations may be carried on simultaneously (see Figure 1). The digestion com pleted, the flasks are disconnected from the condensers and 100 m l . of distilled water are added r a p i d l y to each from an automatic Machlett pipet i n order to stop the reaction. Three drops of phenolphthalein indicator solution are added and the solution is neutral ized with 2 nitric acid solution from another automatic pipet (Figure 1). E x a c t l y 25 m l . of a saturated solution of C.P . barium nitrate are added with swirling, to precipitate any fatty acids resulting from saponification as barium salts. After standing at room temperature for 5 minutes or longer to allow some coagulation of the barium salts, the flask contents are filtered with gentle suction through a double thickness of Sharkskin filter paper on a battery of 4 Fisher Filtrators into 400-ml. beakers (Figure 2). I n order to remove water-soluble chlorides, i t is imperative that the filter paper be washed thoroughly prior to filtering operations. T w o drops of concentrated sulfuric acid are added to the clear or faintly turbid filtrate and the samples are titrated electrometrically on a Leitz G . & D . electrotitrator (Figure 3). This method of titration has the advantage of giving accurate and easily determined end points, no matter what
92
the color of the solution. per day.
It is easily possible for one operator to titrate 40 to 50 samples
Conclusion
Through the use of these techniques, accuracy has not been sacrificed for speed. Results from analyses of duplicate surface and penetration samples agree within 0.5 p.p.m. or 0.5 microgram per sq. cm., as the case may be. Obviously, the larger the amount of D D T present the greater the accuracy. Using all this special equipment, the cost per analysis is about $1.25, including labor and normal depreciation of equipment. The advantages of mechanized over hand processing are so great, however, that the equip ment is considered well worth while. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch018
Literature Cited
(1) Baier, W. E., Edmonds, E . J . , Wilson, C. W., Elliot, M . I., and Gunther, F. ., Science, 104, (2) Gunther, F. ., Anal. Chem., 21, 748 (1949).
(3) Gunther, F. . , Hilgardia, 18, 2 9 7 - 3 1 6 (1948). 376 (1946).
(4) Gunther, F. ., Ind. Eng. Chem., Anal. Ed., 17, 149 (1945).
(5) Gunther, F. ., and Blinn, R. C . , A D V A N C E S IN CHEMISTRY SERIES, 1, 72 (1950). PAPER 626, University of California Citrus Experiment Station, Riverside, Calif.
Microbioassay of Insecticide Residues in Plant and Animal Tissues

W. M. HOSKINS and P. S. MESSENGER
University of California, Berkeley, Calif.
Small shell vials closed by a screen top have been used as expo sure chambers in evaluating the toxicity of organic insecticides to houseflies and in bioassaying for contamination of plant or animal matter with these substances. Treatment of a chloroform extract containing hexachlorocyclohexane or DDT with strong sulfuric acid and passage of the chloroform extract containing parathion through an absorption column greatly diminish the interference due to extractives from the plant or animal tissues. Data for LD to female houseflies are given for parathion, -hexachloro cyclohexane, and p,p'-DDT in terms of micrograms per vial and per square centimeter. Female flies require approximately twice the dosage needed for males. The small vial method is extensively used in following the development of resistance.
50
S e v e r a l organic insecticides have come into wide use recently before chemical methods for their analysis had been developed, and i n some cases interfering reactions have pre vented use of known analytical methods i n the presence of plant or animal tissues. T h e importance from the legal and public health viewpoints of minute traces of residual i n secticides i n foodstuffs makes quantitative determination imperative. Hence, i n this emergency recourse has been taken to methods of bioassay. This, of course, is a very old practice with many drugs of plant or animal origin, but until a few years ago bioassay with insecticides was limited to the so-called screening of chemicals to find those having possible insecticidal value and to the standardization of fly sprays. Neither of these uses necessarily involved determination of the amount of insecticide present. W i t h the advent of D D T , determining its persistence on walls sprayed for control of mosquitoes and flies became very important. A simple metrod of analysis involving exposure of mosquitoes to the sprayed surface and comparison of mortality to that caused by known deposits of D D T was widely used (9). F o r the determination of D D T residues i n plants and animals, total organic chloride provided a fairly satisfactory analytical method from the start, and the colorimetric method of Schechter and Haller (6) is extremely delicate and widely applicable. This is not the case with hexachlorocyclohexane, often called benzene hexachloride or B H C , whose highly insecticidal gamma isomer has defied chemical analysis when present among the other isomers or other organic compounds containing chlorine. A pioneer study of the distribution of this substance i n the tissues of rats to which it had been fed was made b y Laug (3). H e evaporated ether extracts i n Erlenmeyer flasks, so that a deposit was left over the bottom. Female houseflies were confined i n the flasks, and the mortalities after 20 hours were compared to those obtained with known amounts of 7 -hexachlorocyclohexane. Because most of the inner surface of the flask was untreated, the flies were out of contact with the toxicant during a n unknown fraction of the exposure period. The exposure period was so long that the insects had to be fed dur93
94
ing the period to prevent injury from lack of food and water. Laug found that some "tissue m o r t a l i t y " was caused b y extractives from liver and other organs, and he noted that i n the presence of much fat the toxic effect of 7 -hexachlorocyclohexane was so decreased that less than 10 p.p.m. could not be estimated with certainty. I t seemed that these two difficulties might be greatly lessened by using a much smaller exposure chamber whose entire inner surface could be treated, and that the interference from fat or organ extractives might be lessened by appropriate treatment. The method described was designed with these two objectives i n mind.
Apparatus and Procedures

The exposure chamber is a flat-bottomed shell vial, 2.0 cm. i n diameter b y 4.0 cm. long. The open end is heated in the flame and lightly pressed upon a flat surface to give a rolled edge projecting both outward and inward. O n the outside this holds i n place a metal cover (Figure 1), and on the inside it prevents spilling of the insecticidal solution
Figure 1.
Small Vial Exposure Chamber with Screen Top in Place
when the vial is rolled as described below. The metal cover is a thin sheet of copper or aluminum 6 cm. long and just wide enough for its curled sides to fit snugly around the outer rolled edge of the vial but permitting the vial to slide when pushed. T w o circular holes 2 cm. across are i n the cover. One is open and soldered; over the other is a piece of sufficiently fine wire screen to restrain the test insect. Such chambers have the important advantage that a l l trace of toxicant from a previous test can be removed readily by solvents and detergents, and because they are both cheap and easy to make, any number desired can be kept on hand. The inner surface of a vial is treated with the insecticidal coating b y introducing a measured volume, usually 5 or 10 m l . , of standard solution or tissue extract and placing the vial i n n air bath oven at 70 C. with care to avoid local overheating until the volume is reduced to about 1 m l . The vial is then removed and rolled b y hand while the remaining solvent evaporates, care being taken to secure an even distribution over the sides and bottom. A s soon as the vial is cool, the cover is put on with the open hole over the vial. Flies or other test insects may then be introduced as desired. This exposure chamber is not restricted to any species of test insect and the extract of tissue suspected of containing a given insecticide may, of course, be prepared i n any desired manner. The use of the common housefly, Musca domestica, as test insect and
HOSKINS AND MESSENGERMICROBIOASSAY OF INSECTICIDE RESIDUES
95
procedures that have been found useful i n bioassay of 7 -hexachlorocyclohexane and of parathion may be taken as examples. A colony of houseflies is maintained i n the laboratory i n general conformity with the official procedure for the Peet-Grady test (5). When needed, a group between the ages of 3 and 5 days is anesthetized b y chilling or with carbon dioxide, and females are taken in a group of 25 and allowed to revive completely in a test tube plugged with cotton and laid on its side. Then the plug is removed and the mouth of the test tube is quickly placed just within the open hole i n the cover of a treated vial. B y a quick shake the flies are thrown into the vial. The cover is slid to bring the screen over the vial, and the assembly is laid on its side for the duration of the exposure. The practice is to lay the vials parallel to the source of light. This is not important with houseflies, but may be critical with i n sects that are strongly phototropic. Observation has shown that the flies crawl over a l l parts of the surface to which they have access, but they tend to remain on the lower side wall. N o tendency to go to the untreated screen has been noted. Because the screen has an over-all area almost exactly one tenth of the total area, the loss i n sensitivity on this account is slight. After exposure for 30 minutes, each group of flies is released into a small observation cage and given sugar and water. T o avoid environmental effects they should be kept at the same temperature, humidity, and light conditions as those of the rearing room, but to avoid possible effects upon the breeding stock from volatile toxicants a l l exposed flies should be kept i n a separate room. After 24 hours, mortality is determined. W i t h a small proportion of the individuals this must be somewhat arbitrary, according to the criteria that insects too helpless to crawl are counted as dead, and those that crawl or fly more or less normally are counted as surviving. Schechter et al. (7) i n their studies on D D T in milk found that interfering extractives are largely removed b y treatment with strong sulfuric acid. Furman and Hoskins (2) found the same treatment effective when hexachlorocyclohexane was the contaminating substance, and i t has been applied i n later work to numerous extracts of plants and a n i mals. The procedure now used when either D D T or hexachlorocyclohexane is suspected is as follows: The chloroform extract obtained i n any desired mannere.g., b y mixing i n a W a r i n g Blendoris shaken thoroughly with an equal volume of 1 0 % sodium sulfate i n concen trated sulfuric acid. . After the acid layer is withdrawn, the treatment is repeated twice with a 1 to 1 mixture of concentrated and 2 0 % fuming sulfuric acids. Rinsing with water, then with saturated sodium carbonate solution, and finally with water again gives a chloro form solution that can be evaporated i n the test vial. The temperature is not allowed to rise, because much charring will occur and there is danger of destroying part of the insecti cide. Parathion is a relatively unstable compound and the drastic treatment with sul furic acid cannot be used. Interfering extractives from some plants can be removed w i t h out loss of parathion b y passing the chloroform solution through a short column contain ing 2 parts of Attaclay plus 1 part of Hyflo Super-Cel, followed b y rinsing with an equal volume of pure chloroform. D D T also passes through such a column without loss. T h e solution is then evaporated i n a test vial as described before.
Results
T o illustrate the results obtained b y this method, a few data are selected from i n vestigations on the contamination of chicken flesh with 7 -hexachlorocyclohexane and of cabbage with parathion. These cases are especially interesting because with the chicken flesh the liquid fats alter contact with the hexachlorocyclohexane and also cause consider able mortality of flies and because with cabbage chemical analysis of parathion is diffi cult on account of very high blanks with untreated samples when the usual colorimetric method is used. Figure 2 shows the standard dosage-mortality lines for 7 -hexachlorocyclohexane (line 4 ) , parathion (line C), and , ' - D D T (line F) plotted according to the probit-log dosage procedure of Bliss (1). Line is the corresponding dosage-mortality line resulting when
96
7 -hexachlorocyclohexane i n the presence of chloroform extract of chicken flesh is put through the acid treatment. The small displacement to the left, signifying greater effect from a given quantity of insecticide than when the material is tested alone, may be due to 7.0 65 h 6.0 g 5.5
0
00
at.
MO ALIT
11 il
4.5 4.0 h 3.5 I 3.0

h"
0.2 0.3 0.5 Figure 2. A. B. C. D. E. F.
1.0
2.0 3.0
5.0
10 15 20 30
DOSAGE, PER VIAL Dosage-Mortality Lines for Female Houseflies with 7 -BHC, Parathion, and ,'-DDT
7-BHC 7-BHC plus extract of chicken flesh after acid treatment Parathion Parathion plus cabbage extract Parathion plus cabbage extract after passage through absorption column ,'-DDT
some extractives not removed b y the sulfuric acid, for L a u g (3) noted that fats reduced evaporation of the comparatively volatile 7 -hexachlorocyclohexane. The arrow indicates the mortality found with 5 m l . of an extract of chicken flesh corresponding to the pres ence of 2.5 micrograms of 7 -hexachlorocyclohexane. Because this was an aliquot of 100 m l . used i n extracting 75 grams of flesh the original contamination m a y be calculated to be 2.5 X 1 0 0 / 5 75 = 0 .67 p.p.m. of 7 -hexachlorocyclohexane. Direct exposure of flies to a chloroform extract of contaminated chicken flesh gave no definite result be cause they became smeared i n the liquid fat. This effect was completely eliminated b y the acid treatment. I n the case of parathion plus cabbage extractives the dosage-mortality line (D) is so far to the right of that for straight parathion that analytical results based upon the latter as a standard would be entirely erroneous. Thus the L D M ' S are 0 .56 and 14.0 micro grams per vial, respectively, or a 25-fold greater dosage is required i n the presence of the cabbage extractive. Of course, line D could be used as the standard for analysis, but probable differences among various kinds of cabbage make this an unsafe procedure. T h e great loss i n sensitivity is another serious objection to use of line D for reference. Passage of a chloroform extractive solution plus parathion through the adsorption column de scribed above resulted i n line E, which can be used as the reference line. Actually, i t agrees closely with the original standard dosage-mortality line, C. Using this method, no
HOSKINS AND MESSENGERMICROBIOASSAY OF INSECTICIDE RESIDUES
97
difficulty has been encountered i n assaying parathion residues on cabbage and allied plants. The residues never exceeded a few hundredths part per million. A device i n which insects may be exposed to known amounts of a chemical may be used i n ascertaining comparative toxicitiese.g., i n screening. Figure 2 gives a simple example of this, for i t shows that the L D ' s for parathion, 7 -hexachlorocyclohexane, and , ' - D D T to adult female houseflies are 0.56, 2.7, and 6.7 micrograms per vial, respec tively. Because the treated surface has an area of 28.0 sq. cm., these dosages amount to 0.020, 0.096, and 0.239 microgram per sq. cm., respectively. When an attempt was made to determine the L D / s for the other isomers of hexachlorocyclohexane no mortality re sulted with deposits up to 10,000 micrograms per v i a l . These deposits are easily visible, and, i n fact, were partly flaked from the glass surface b y the activity of the confined flies. M a l e houseflies are more susceptible than females to certain insecticidese.g., p y rethrins (8). Such differences may be determined with ease and precision b y the small v i a l method. Thus with , ' - D D T the L D ' s for male and female houseflies are 3.7 and 6.7 micrograms per vial, respectively. W i t h 7 -hexachlorocyclohexane they are 1.4 and 2.7 micrograms per vial. Thus the females require approximately twice the dosage needed for the males. This difference is only partly accounted for on a weight basis, for the aver age weights of the flies used were: males 12 mg. and females 18 mg. The reproducibility achieved with the small vial method may be illustrated b y a typical set of data obtained with 7 -hexachlorocyclohexane and each sex of houseflies. Successive trials gave the following mortalities.
5 0 5 ( 5 0
Mortality
7-BHC, 7/Vial
1 1.5 2 3 4
Males
8,11,18,21,3,8,9,10,10,13 Av. 11.1 40,56,62,66,47,54 Av. 54.2 88,91,93,94,97,89,93,93,95 Av. 91.4 .. ..
Females
5,11,14,5,8,15 A v . 9.7 63, 71, 77, 52, 58, 59, 66 Av. 63.7 85,91,93,90,93,93,95 Av. 91.4
A matter of very great interest is the increase i n resistance to D D T that has been shown recently b y houseflies i n various parts of the world. This is often so marked that practical use of D D T is no longer feasible. A n especially resistant strain, found i n south ern California b y M a r c h and Metcalf, is called the Bellflower strain for purposes of identi fication (4). They reported that no residual deposit of D D T gave 100% k i l l . B y the small vial method not over 2 5 % k i l l of these female flies could be obtained with several thousand micrograms per v i a l . Further tests with various naturally occurring and selected resistant races are i n progress.
Discussion
The methods described for handling chicken flesh and cabbage have been used for several other products i n which hexachlorocyclohexane or parathion contamination was suspected, but doubtless occasions will arise i n which these procedures will be inappli cable. Modification i n the preparation of extracts, however, does not affect the useful ness of the small exposure chamber. Because b y this small vial method a test may be made with amounts of a few micrograms or less, the term "microbioassay" is appropriate. The effect of failure to extract the toxicant completely from a tissue is diminished b y a d d ing known amounts to representative samples of the same kind of uncontaminated tissue when determining the reference dosage-mortality line. There is always the possibility that a chemical present i n some part of the body after ingestion will be harder to extract than when freshly mixed with the tissue. Hence, precautions such as fine subdivision, long extraction, and use of a really good solvent should always be taken. W i t h some insecticides the physical state of a residual deposit has a pronounced effect upon its toxic effect. This is especially true of technical D D T , which tends to remain in viscous droplets when released from solution i n volatile solvents. These may remain
98
for weeks if undisturbed, but they crystallize instantly when touched b y a moving insect. The deposit within a small vial may be observed closely, under a microscope if desired, and this phenomenon may be followed i n detail. The flaking off of very heavy depositse.g., of -, -, or 7 -hexachlorocyclohexane or of ,'-sets a limit to the amounts to which insects can be exposed i n the vials. In this respect the present procedure differs sharply from those i n which the toxicant is applied to a piece of filter paper or other porous material. Under such conditions enor mous amounts may be employed, but a large percentage is completely removed from con tact with the test insects and dosages so determined for 5 0 % or any other mortality have little meaning. Accessibility of the deposit to the insects b y contact is the chief feature of the small v i a l method, but fumigant action cannot be eliminated entirely. I n the case of D D T this has been found to be unimportant, for flies kept i n the vials out of contact with the sur face are not affected. B u t with 7 -hexachlorocyclohexane or parathion there is a notice able toxic effect. If the vials are stood upright, laid on the side, or hung upside down, there is a decrease i n the mortality produced i n the order given. T h e position on the side has been adopted because i t avoids extremes and because practical use of an insecticide often involves limited but not accentuated ventilation.
Acknowledgment
The valuable assistance of . H . Fullmer is gratefully acknowledged.
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) Bliss, C. C., Science, 79, 409-10 (1934). Furman, D. P., and Hoskins, W. M . , J. Econ. Entomol., 41, 106-7 (1948). Laug, E . P., J. Pharm. Exptl. Therap., 93, 277-81 (1948). March, R. B., and Metcalf, R. L., Division of Entomology, Citrus Experiment Station, Univer sity of California, News Letter 38 (1949). National Association of Insecticide and Disinfectant Manufacturers, "Soap Yearbook," pp. 1836, 1948. Schechter, M . S., and Haller, H . L., J. Am. Chem. Soc., 66, 2129-30 (1944). Schechter, M . S., Pogorelskin, . ., and Haller, H . L . , Anal. Chem., 19, 51-3 (1947). Simanton, W. ., and Miller, A . C., J. Econ. Entomol., 30, 917-21 (1938). Simmons, S. W., U . S. Pub. Health Service, Publ. Health Repts., Supplement 186, 3-20 (1945).
C O N T R I B U T I O N from the laboratory of insect physiology and toxicology, Division of Entomology and Parasitol ogy, University of California.
Toxicity of Spray Residue of Fresh and Processed Fruits and Vegetables

ALBERT HARTZELL
Boyce Thompson Institute for Plant Research, Inc., Yonkers 3, . Y.
Bioassay with mosquito larvae for the detection of insecticide residues in fresh and processed fruits and vegetables is feasible, subject to the limitation that the untreated natural product is in itself nontoxic to the larvae at the dilutions tested.
T h e purpose of this work was to determine the toxicity to mosquito larvae of insecticide spray residues. T h a t certain insecticides are translocated i n plants (4, 5) adds impetus to this study. Fresh orchard fruit sprayed or dusted w ith preparations containing parathion (0,0-diethyl O-p-nitrophenyl thiophosphate), tetraethyl pyrophosphate ( T E P P , H E P P ) , D D D [2,2-bis(p-chlorophenyl)-l,l-dichloroethane], D D T [2,2-bis(pchlorophenyl)-l,l,l-trichloroethane], chlorinated camphene, and basic lead arsenate were shipped from California to Yonkers, . Y . , by air express for bioassay.
r
Materials and Methods

As mosquito larvae are relatively easy to kill with insecticides, any toxic spray residue is likely to be detected. T w o species of mosquito larvae were used, the yellow fever mosquito (Aedes aegypti L.) and the southern house mosquito (Culex quinquefasciatus Say). Tests with the southern house mosquito were made essentially according to the method of Campbell, Sullivan, and Smith (1), except for the kind of food supplied and size of containers used. The eggs (furnished through the courtesy of C. H . Bradley) were shipped weekly from Orlando, F l a . , to Yonkers, . Y . , via air mail, and as soon as received, they were placed in tap water i n 4-liter beakers at room temperature. The eggs hatched within 24 hours. When the larvae had hatched, powdered dog biscuit was added at the rate of 100 mg. per liter. The following day 125 mg. of blood albumen were dissolved i n 150 m l . of water, and added at the rate of 125 mg. per liter to beakers containing larvae. Thereafter powdered dog biscuit and blood albumen were fed on alternate days in amounts specified above. Larvae 5 days old were used for testing. Test tubes (25-ml. capacity) containing ten larvae each i n the solution to be tested and the controls i n tap water alone were placed in an oven at 30 = = 1 C. overnight (20 hours). The solutions containing larvae were poured into porcelain dishes and living and dead larvae were counted. Tests were run i n duplicatei.e., two tubes containing 10 larvae each. If any dead larvae were found i n a check, the tests were repeated. Tests were made at four or more concentrations. Each series of tests was repeated on a different day. Larvae of the yellow fever mosquito, which are shallow feeders, were reared at room temperature by methods similar to those used i n culturing Anopheles mosquito larvae (2, 6). Filter papers containing the eggs (furnished through the courtesy of R . E . Heal, Merck & Co., Inc., Rahway, N . J.) were placed i n tap water i n shallow porcelain pans (12 X 7 inches, and 2 inches deep). The eggs hatched within 24 hours. When the larvae had hatched, powdered dog biscuit was added daily at the rate of 100 mg. per liter of water.
99
100
Larvae 3 days old were used in testing. Tests were conducted in the same manner as with the southern house mosquito. The fresh fruit was ground in a meat chopper, and the juice was pressed out and diluted with tap water at ratios of 1 to 7.5, 1 to 15, and 1 to 30. The processed baby food was also diluted with tap water at the same ratios as the ground fresh fruit.
Results
Apricots, prunes, and peaches from sprayed trees were tested on the larvae of Aedes aegypti for the toxicity due to spray residues. The apricots were sprayed on April 1 and May 6 and the fruit was harvested on or about August 9. The samples were tested during the month of August at dilutions of 1 to 7.5, 1 to 15, and 1 to 30. Apricot samples from trees sprayed with D D T (50% wettable) at the rate of 1.5 pounds per 100 gallons of water were toxic at all three dilutions tested. Samples from trees sprayed with D D D (50% wettable) at the rate of 2 pounds per 100 gallons of water were toxic at dilutions of 1 to 7.5 and 1 to 15. Apricot samples from trees sprayed with parathion (25% wettable) at the rate of 2 to 3 pounds per 100 gallons of water were also toxic at dilutions of 1 to 7.5 and 1 to 15. The peach trees (cling) were sprayed on June 16 with a single application of tetraethyl pyrophosphate, and the fruit was harvested on or about July 2. Tests were made during the month of July. Both treated and check peaches were unripe when tested. It was found that unripe unsprayed peaches were toxic to mosquito larvae at dilutions of 1 to 7.5 and 1 to 15, but at the dilution of 1 to 30 neither the treated nor the the check peaches showed toxicity. It was not possible to distinguish between the toxicity of treated and check at dilutions of 1 to 7.5 and 1 to 15 by the method of ranking described by Wilcoxon (7).
AGE OF SOLUTION IN DAYS
Figure 1.
Stability of Parathion
In addition to the tests made on peaches and apricots, samples of prunes from trees that had been sprayed with parathion, D D T , D D D , basic lead arsenate, and toxaphene at the rate of from 1 to 2 pounds of these insecticides per 100 gallons of water were tested on larvae of Aedes aegypti. The trees had been sprayed on April 20 and June 16, 1948. The fruit was harvested on or about September 10. Prunes from trees that had been treated with 1 quart of tetraethyl pyrophosphate and 12 pounds of sulfur dust per acre on June 15, and harvested about July 6, were tested on larvae of the above named species. None of the prune samples tested in this study exhibited any significant toxicity to mosquito larvae as compared with the unsprayed check.
HARTZELLTOXICITY OF SPRAY RESIDUE
101
Strained processed peas and peaches containing known quantities of parathion ranging i n concentrations from 0.25 to 10 p.p.m. were diluted with tap water at ratios of 1 to 7.5, 1 to 15, and 1 to 30, and tested on larvae of Aedes aegypti. K i l l s of 100% were obtained at all concentrations tested. The average L D o of aqueous parathion solutions was 0.003 p.p.m. [a value comparable to that obtained by Gleissner (8)], as compared with 0.055 p.p.m. for processed peas and 0.0053 p.p.m. for processed peaches. Strained processed peas containing known quantities of gamma isomer of benzene hexachloride (-hexachlorocyclohexane) were nontoxic, whereas strained processed peaches gave kills of 100% at the same dilutions. The p H values of the processed peas and peaches used i n these tests were 6.0 and 3.7, respectively, indicating that the toxin is not destroyed i n an acid medium. The L D of an aqueous solution of the gamma isomer of benzene hexachloride was approximately 0.1 p.p.m. A n aqueous solution containing 1 part i n 200,000,000 of parathion gave 5 0 % k i l l to southern house mosquito larvae (Culex quinquefasciatus Say). Parathion solutions did not lose any toxicity on standing for a month at room temperature (Figure 1). A t the end of 2 months, however, the solutions lost their toxicity to mosquito larvae.
5 5 0
Table I.
Toxicity to Aedes aegypti Larvae of Processed Baby Food"

(Known quantities of insecticides added before processing) Concn. before Dilution, P.P.M. 10 5 2 10 5 2 10 5 2 Dilution Ratio 1:15 1:30 1:7.5 1:30 1:7.5 1:15 Peaches, Strained, % Dead Peas, Strained, % Dead 4 6 0 0 0 0 56 5 0 0 0 6 0 0 0 7 15 0 0 0 9 5 0 0 0 0 0 0 0 0 0 4 20 100 100 100 14 10 5 6 0 0 0 100 100 100 0 6 0 0 100 100 100 0 0 0 0
Insecticide TEPP BHC Methoxychlor Check
4
0 0
% moisture in strained % moisture in strained peas ranged from 83.7 to 87.5; total solids, 12.6 to 16.3. peaches ranged from 63.3 to 73.5; total solids, 26.5 to 39.7. Moisture and total solids determinations by Theo Svolos.
Literature Cited
(1) Campbell, F . L . , Sullivan, W. N . , and Smith, C. R., J. Econ. Entomol., 26, 500-8 (1933). (2) Deonier, C. C., Hinchey, E., and Incho, H . H . , U . S. Dept. Agr., Bur. Entomol. Plant Quarantine E-733, Part III, 7-9 (1947). (3) Gleissner, B. D., private communication. (4) Grainger, M . M . , "Absorption of Parathion through the Root System of Plants and Its Effect upon Insects Infesting Them," thesis, Cornell University, February 1948. (5) Grainger, M . M . , and Lieby, R. W., Agr. Chemicals, 4 (2), 34-5, 79-81, 83, 85 (1949). (6) Granett, P., and Haynes, H . L., Proc. New Jersey Mosquito Exterm. Assoc., 31, 161-8 (1944). (7) Wilcoxon, Frank, Insecticide and Fungicide Section, Stamford Research Labs., American Cyan amid Co., Stamford, Conn., "Some Rapid Approximate Statistical Procedures," 1948.
Some Poisonous Residue Factors in Use of Two New Organic Insecticides

G. S. HENSILL and L. R. GARDNER
California Spray-Chemical Corporation, Richmond, Calif.
Laboratory and field research work has developed methods for using two new organic insecticides with freedom from poison residue. With pure gamma isomer of hexachlorocyclohexane proper formulation and timing of application are the essence of success. On some crops applications must be made before fruits or heads form; on others applications may be made to within 2 or 3 weeks of harvest, and in others, within a few days of harvest. Commercial usage with proper timing and application has resulted in no poison residues or undesirable taste residues. With proper usage, dosage, and application the pure gamma isomer of hexachlorocyclohexane leaves no residues that would constitute a health hazard or produce off-flavors in food products. The unstable nature of tetraethyl pyrophosphate makes treatment of crops up to harvest time possible. This chemical is therefore an effective agricultural insecticide against many pests.
T h e introduction of the use of D D T (dichlorodiphenyltrichloroethane) as an insecticide during World W a r I I initiated a revolution i n the problems of insecticide residues. The resulting changes have been so far-reaching that today, only a few years later, most major insecticides are new synthetic organic chemicals. Prior to the introduction of D D T the major insecticides were inorganic chemicals, except for some few relatively expensive organics of plant origin such as nicotine, pyrethrum, and rotenone. Two of the most important synthetic organic insecticides which have been introduced following D D T are the technically pure gamma isomer of hexachlorocyclohexane (lindane) and tetraethyl pyrophosphate. The technically pure gamma isomer was developed as the result of investigational work on the insecticide, benzene hexachloride, which was first used i n England and France during World W a r I I and was used i n the United States on agricultural crops at the same time. I t was found to have insecticidal value equal to D D T i n most respects and better i n others. Considerable residue action was evident, and i t was toxic to a wider range of insects than was D D T , and had vapor action not evident with D D T . Of five or more isomers, the only one that is appreciably insecticidally active is the gamma isomer, which occurs i n various percentages, usually 12 to 1 3 % , depending on methods of manufacture. This mixture of isomers results i n a compound of strong and persistent odor, mostly due to the beta isomer, which odor is retained by some fruits, vegetables, and animal tissues after treatment for insect infestations. Research work soon showed the possibility of producing the pure or technically pure gamma isomer. This production was finally accomplished on a large commercial scale and the compound has now been introduced as a large-volume insecticide on the American market. Tetraethyl pyrophosphate was first manufactured i n Germany about 1940 as hexa102
HENSILL AND GARDNERRESIDUE FACTORS IN NEW ORGANIC INSECTICIDES
103
ethyl tetraphosphate or the hexaethyl ester of tetraphosphoric acid. This compound and its manufacturing methods were discovered i n Germany by chemists with the allied occupation forces and the information was brought to the United States. Research work on this chemical finally indicated that i t contained as the active agent tetraethyl diphosphates which are generally described as tetraethyl pyrophosphate, and this is now well established i n the insecticide and chemical industry. As the value of these two new chemicals for insecticides became more evident, the need for extended experimental and test work was definitely established. I t was necessary to determine chemical formulas, work out analytical methods, obtain knowledge of various physical and chemical characteristics, and complete evaluation of insecticidal action as well as toxicity and effect of residues. Toxicity was concerned with not only insects but humans and other warm-blooded animals. Residual studies included information on persistence and type and amount of residue. This information, once accumulated, must be correlated with similar information on other insecticides.
Pure Gamma Isomer of Hexachlorocyclohexane

Investigational work on the pure gamma isomer of hexachlorocyclohexane involved both laboratory and field tests. The pure gamma isomer was found to have retained practically all of the insecticidal value of the parent commercial benzene hexachloride containing 12 to 13% gamma isomer. The three-way action of contact poison, stomach poison, and vapor action poison was also evident with the pure gamma isomer. I n addition, methods of manufacture were found which retained the insecticidal action of the parent compound and yet removed the objectionable odor, thus making available a fine chemical for killing insects. According to Lehman (#), toxicity tests i n the laboratory with small animals such as rats and dogs indicated that the refined or technically pure gamma isomer has a mean lethal dose of 125 mg. per kg. of body weight. This gives i t an acute oral toxicity about twice that of D D T . Nicotine is twelve times and tetraethyl pyrophosphate sixty times as toxic as gamma isomer. On the same basis arsenic is about four times as toxic. A c cording to Lehman, pure gamma isomer shows much less tendency toward storage i n body tissues than does D D T . Pure gamma isomer is stored i n body fat at a level about equal to the amount of dietary intake; D D T is stored i n body fat at a level four to ten times that of the dietary intake. T h e pure gamma isomer of hexachlorocyclohexane shows practically no dermal toxicity on skin application. Its chronic toxicity is four times less than D D T . Laboratory studies showed the pure gamma isomer of hexachlorocyclohexane to have residual life equivalent to that of the normal mixed isomers. The material does not break down i n normal storage, as do mixed isomers. It was also established i n laboratory work that this product could be readily formulated into dusts, wettable powder, or liquid formulations. Liquid formulations were more readily made with this than with the commercial benzene hexachloride, because of the higher concentration of the gamma isomer. Field test work with technically pure gamma isomer of hexachlorocyclohexane has been extensive and involved and is being continued. I t was necessary to know such factors as insecticidal value i n field applications as compared to other insecticides, as well as residual life, residue from the poison standpoint, and residual taste or odor factors. These factors have been worked out on numerous crops and some of the results are dealt with i n this paper. Because the pure gamma isomer was found to be effective on insects in the soil as well as on insect infestations on plants, its residual life i n soil of all types and effects on tuber and root crops were also of major importance. The residual toxicity of the pure gamma isomer was found to be equivalent to that of ordinary commercial benzene hexachloride. Commercial usage has shown that the residual action is effective for a longer time with dust or wettable powder spray applications than with emulsive solvent-type formulations. T h e over-all residual life of the chemical is on the order of 4 to 8 days as compared to 14 to 21 days for D D T . T h i s is, of course, adequate residual life for good insect control i n most cases^ and the shorter
104
life and the smaller amount used make the pure gamma isomer especially important because of freedom from residue problems. Despite the fact that i t has a low vapor pressure, 0.00001 m m . at ordinary temperature and 0.2 m m . at the temperature of boiling water (1), i t is an effective fumigant and its volatility is one of the major reasons for its disappearance from a plant crop (Figure 1). M o r e important than this is the high po tency to insects, which is approximately ten times that of D D T . This means that lower dosages are used, thus greatly reducing any residue problem.
30
100 TEMPERATURE, F.
200
Figure 1. Vapor Pressure of Gamma Isomer of HexachJoro cyclohexane and DDT T o date, within the scope of the writers' information, there has been no residual deposit or poison residue recovered from treated fruits or vegetables, where proper formu lations and amounts of the pure gamma isomer have been used not later than 2 weeks prior to crop harvest. Likewise, there is no known record of poisoning to man or animals from applying the insecticide or eating food treated with the insecticide. Freedom from poisonous residue and undesirable taste i n the use of the pure gamma isomer of hexa chlorocyclohexane is achieved therfeore by proper formulation, timing, and application of insecticide treatments. The pure gamma isomer has been found to be an excellent soil insecticide for control of most common soil insects. Persistence of the material i n the soil is longer than on plant surfaces. T h e possibility of flavoring of root and tuber crops grown i n infested soil is also a problem and this has been worked out i n the following manner: First, light dosages such as 0.125 to 0.25 pound of gamma isomer per acre may be used i n soils to be planted to potatoes, which are probably the most sensitive crop as regards imparting of flavor, or to other tuber crops. Such treatment has not been found to impart unfavorable taste to most root crops. Second, the soil may be treated with a heavier dosage, such
105
as 0.25 to 0.5 pound of gamma isomer per acre, and a nonaffected crop planted for that season. So far this procedure has not produced undesirable results i n most tuber or root crops grown during the season following treatment (Table I ) . Table I.
Gamma Isomer, Lb./Acre 0.125 0.25 Check untreated (second year) Taster 1 None None None
Pure Gamma Isomer Taste Test on Potatoes

Taster 2 None None None Taster 3 None Slight None Objectionable Taste Taster 4 Taster 5 None Slight None Slight None None Taster 6 None Slight None Taster 7 None None None Taster 8 None None None
The application of pure gamma isomer to plants for insect control has created similar problems, i n that undesirable taste might be imparted to mature fruits or vegetables (Table I I ) . Table II.
Gamma Isomer, Lb./Acre -25 0,5 Check untreated
Pure Gamma Isomer Taste Test on Canned Tomatoes

Taster 1 None None None Objectionable Taste Taster 2 Taster 3 None None None None Slight None Taster 4 None None None
This factor is handled by applying the chemical not later than 30 days before harvest on crops that might hold some residual taste. O n other crops i t has been possible to use the chemical to within 2 weeks of harvest without retaining undesirable taste. This point is largely one of varieties, so that it becomes necessary to specify on labels which crops must be treated before fruits or heads form, which can be treated up to 2 or 3 weeks prior to harvest, and which have no particular time limit. T h e following fruits and vegetables are among those satisfactorily treated. I n the eastern United States apples of the Delicious variety treated 10 days prior to harvest with pure gamma isomer showed no trace of off-flavor at harvest time. Dusts and sprays applied to carrot seedlings produced no traces of off-flavor i n the mature vegetables. Several tasters could not differentiate between peaches sprayed within a few days of harvest and check fruit. Celery i n Florida sprayed twice, once within 6 weeks of harvest and once within 30 days of harvest, was canned and put through a severe series of tests.
TIME OF HYDROLYSIS, HOURS
10 20 30
Figure 2.
Tetraethyl Pyrophosphate Persistence Test
Filter papers treated with solution and exposed to Drosophila
106
Reports by the National Canners Association showed negative results on both taste tests and analytical tests for residual chlorides. Asparagus treated and canned i n a manner comparable to the celery also gave completely negative results. It has thus been possible to work out satisfactory applications of this chemical on many crops without the danger of poison or other undesirable residues on the har vested crops. The use of pure gamma isomer of hexachlorocyclohexane on livestock has also been worked out. I t has been found possible to use the wettable powder formulation dispersed i n water as a spray on livestock for control of flies, lice, and ticks. Proper dosage and application must be used, of course, but this is again indicative of the safety factor of this insecticide. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch021
Tetraethyl Pyrophosphate
E a r l y work i n the laboratory and i n the field soon established the fact that there would be no poison residue factor with this chemical, owing to its rapid decomposition. W i t h i n a relatively few hours the chemical broke down into diethyl phosphoric acid
DAYS WEATHERING 1 2 3 4 5
50
150
= 5
900
J|-
Figure 3.
Tetraethyl Pyrophosphate Persistence Test
Fly cage sprayed with tetraethyl pyrophosphate and weathered in laboratory. Flies introduced at stated intervals
and finally ethyl alcohol and phosphoric acid, both of which are nonpoisonous i n rela tively large amounts, especially i n view of the low dosage used (Figures 2 and 3). A l though this chemical was found to be highly toxic i n its pure form to both insects and warm-blooded animals, the rapid hydrolysis on exposure to air or moisture eliminated the
107
poison residue problem. This has been verified b y field usage experience and laboratory tests. I n the laboratory the chemical was put into water at the ordinary spray dilution of 1 to 800 and after 24 hours' standing the treated water was used as drinking water for test animals. There were no reactions, evidence of poison, or undesirable effects on any animals as a result of these tests, even with long feeding periods. I t was not possible to differentiate between test animals and check animals by any of the customary tests. Formulations of tetraethyl pyrophosphate as an emulsive concentrate proved to be relatively complex, considering the apparent ease of formulation. Because of the u n stable nature of the chemical, dust formulations were considered impossible. I t was found after extensive research work that a dust which would be stable for 10 days to 2 weeks could be made with a specially selected and processed filler. Field test work with the chemical has consisted of many tests and a large number of commercial applications i n both spray and dust forms. Insecticidal action has been satisfactory i n all cases where materials have been properly applied. N o toxic residue has been found on any treated plants or food crops, which include most varieties of crops. A sample of hops which had been treated with tetraethyl pyrophosphate showed a negative chemical analysis. The plant material was also extracted and the extract added to the drinking water of test animals and sensitive insects. The animals and insects that drank this treated water for several days showed no reaction. W i t h the sensitive insects i t would have been possible to detect even a few parts per million. I n addition, there have been extensive commercial field applications of the chemical i n dust and spray form to crops such as apples, pears, grapes, celery, broccoli, Brussels sprouts, and others up to within a few days of harvest; there has been no detectable poison residue on any of the crops. The lack of poison residue with use of tetraethyl pyrophosphate is due to the fact that it hydrolyzes within a few hours of application, breaking down into transient nonresidual and nonpoisonous chemicals. Thus i t is possible to use tetraethyl pyrophosphate well up to harvest time of food products without danger of residual poison on crops. The fact that the chemical is used i n extremely small amounts is a definite advantage i n respect to freedom from poison residue.
Literature Cited
(1) Balson, E. W., Trans. Faraday Soc., 43, 54-60 (1947). (2) Lehman, A . J . , "Toxicology of the Newer Agricultural Chemicals," reprint from conference of 42nd annual convention of National Canners Association, Atlantic City, N . J., Jan. 17, 1949.
Selenium Residue on and in the Peel of Washington Apples

KENNETH C. WALKER
Washington Agricultural Experiment Stations, Wenatchee, Wash.
The average selenium residue on and in the peel of 30 samples of Jonathan, Delicious, and Winesap apples with no history of selenium sprays was found to be 0.001 p.p.m. The selenium residue on and in the peel of Jonathan and Delicious apples that had received one or more sprays of selenium ranged from 0.020 to 0.155 p.p.m. The addition of 1 quart of light grade petroleum oil increased the selenium residue. When single-tree plots were sprayed twice with selenium sprays significantly more selenium on and in the peel was found than in the unsprayed fruit, but there was significant difference due to variety or to position of sample on tree.
T h e literature on the distribution of " n a t u r a l " selenium i n the soils, the absorption b y vegetation, the toxicity of compounds of either natural or applied selenium to man and animals, and the use of selenium as a spray for insect control is voluminous and no a t tempt is made to cover i t here. The literature has been well reviewed fairly recently (#). Very little has been published on the increase i n selenium content of apple peelings, due to the application of selenium-bearing sprays. A proprietary selenium compound, known as Selocide, has been proposed for the control of Pacific mite, Tetranychus pacificus M c G . , and European red mite, Paratetranychus pilosus C a n . and Fanz., i n the apple-producing areas of the state of Washington. Selocide has been used on an experimental basis and to a limited extent on a commercial basis. Selocide is reported (4) to be a 3 0 % solution of a mixture of potassium hydroxide, ammonium hydroxide, sulfur, and selenium i n the proportions corresponding to the e m pirical formula ( K N H S ) S e . The commercial material contains 48 grams of selenium per liter, or approximately 6.4 ounces per gallon. The reactions that occur when a concentrated solution of Selocide is diluted with water to prepare a spray mixture, i n the presence of carbon dioxide and oxygen of the air, may be represented as follows :
4 6
4 ( K N H S ) S e + 10CO (from air) + 1 5 0 (from air) + H 0 >

4 5 2 2 2
1 0 K C O (soluble) + 2 0 N H + 10H S + 10SO + 4Se (metallic) + H 0

2 3 3 2 2 2
The Selocide spray residue remaining on the fruit is red metallic selenium (3). Selocide is not compatible with acid lead arsenate ( P b H A s 0 ) under normal spray program conditions, but is compatible with cryolite (Na3AIF ) (a). Selocide is compatible with D D T [2,2-bis(p-chlorophenyl)-l,l,l-trichloroethane] and has been used with D D T for the control of mites and Codling moth, Carpocapsa pomonella L . , during the 1947 and 1948 growing seasons.
4 6
108
WALKERSELENIUM
RESIDUE ON AND IN THE PEEL OF WASHINGTON APPLES
109
Methods and Procedure

Selenium on and i n the peel of apples was determined b y the procedure of the Association of Official Agricultural Chemists (1). Samples of approximately 1 k g . of whole fruit were hand-peeled with a Devault peeler and the parings were digested as directed (1 29.60). Samples, with no selenium on or i n the fruit, were prepared for analysis and known quantities of selenium were added. These were analyzed b y the above noted methods. The quantity of selenium recovered and the percentage recovery are presented i n Table I .
7
Table I.
Added, 7
Recovery of Selenium Added to Apple Parings

Recovered, 7 59.8 58.8 78.4 82.2 119.0 121.2 198.0 194.2 Recovery, % 99.6 98.0 98.0 102.8 99.7 101.0 99.0 99.4 Average recovery (8) 99.4 Range 98.0 to 102.8
60 60 80 80 120 120 200 200
Selenium in Unsprayed Fruit

T o establish the level of selenium on and i n the peel of fruit not sprayed with selenium, samples were collected from thirty different commercial growers whose insect-control program contained no selenium sprays. Samples of approximately one-half box (0.5 bushel) were taken at random from incoming trucks at the fruit packing warehouses. Only fruit from growers whose spray programs were definitely known b y the field representatives of the warehouses were selected for sampling. T e n samples per variety, of Jonathan, Delicious, and Winesap from ten different growers, were taken for analysis. The half-box samples were taken to the laboratory and subsampled for analysis. A l l subsamples were properly labeled and stored at 32 F . for 3 to 4 months before being analyzed. The selenium residue on and i n the peel of the non-selenium-sprayed fruit is presented i n Table I I . Table II.
Variety
0
Selenium Residue on and in Peel of Non-Selenium-Sprayed Fruit

No. of Fruit per Sample 7 to 11 5 to 8 6 to 11 Total Weight of Fruit per Sample, Grams 1000 to 1102 1020 to 1160 982 to 1112 Average Selenium b, P.P.M. 0.001 0.0006 0.001 Selenium Range, P.P.M. 0.000 to 0.005 0.000 to 0.004 0.000 to 0.004
Jonathan Delicious Winesap
10 samples per variety analyzed in duplicate. b Whole fruit basis.
The samples reported i n Table I I carried a very low residue level of selenium on and in the peel. The average value of 0.001 p.p.m. for the thirty samples was less than the variation within varieties and less than the variation between duplicates. The selenium residue of 0.001 p.p.m., the average, can be considered as a possible trace of selenium on and i n the peel.
Selenium in Sprayed Fruit

T o establish the level of selenium on and i n the peel of selenium-sprayed apples, samples were collected from five commercial Jonathan apple orchards and eight commercial Delicious apple orchards b y representatives of the packing warehouses and submitted to the laboratory for analysis. The sampling and analytical procedure used for these samples was identical to that used for the samples reported i n Table I I . Sufficient fruit was available for duplicate analysis of most of the samples.
110
The results obtained from these samples and the dates of the selenium sprays are presented i n Table I I I . Table III.
Sample Weight, Grams 1052 1002 1016 1039 924 1082 1010 1050 1090 1036 1004 1178 1102 1048 1070 1064 1058 1028 1072 1011 1062 1128 1090 1118 1022 1020 1072
Selenium Residue on and in Peel of Selenium-Sprayed Fruit

No. of Fruit per Sample 8 7 6 7 7 7 8 7 8 7 8 6 6 9 9 7 7 5 5 7 8 6 6 6 6 6 6 Selenium, P.P.M. Jonathan Apples 0.03 0.02 0.03 0.05 0.03 0.06 0.07 0.08 0.06 0.07 Delicious Apples 0.03 0.03 0.04 0.04 0.02 0.05 0.07 0.09 0.03 0.11 0. 10 0.09 0.12 0.12 0.16 0.14 0.13 Av. (27) 0.07 Range 0 .02 to 0.16 0.03 0.04 0.03 0.06 0.06 0.10 0.11 0.14 May 5 M a y 28 M a y 5 and June 1 M a y 5 and June 28 M a y 5 and June 1 and 28 M a y 5 and July 9 May 13 and June 14 May 13 and June 14
c
Average Selenium, P.P.M. 0.03 0.03 0.05 0.07 0.06
Dates Selenium Sprays Applied b May 5 June 1 M a y 6 and June 18 M a y 5 and 28 M a y 6 and June 18
Whole fruit basis. b All spray programs consisted of 1 pint of Selocide plus D D T per 100 gallons. Sprays applied on June 14 had 1 quart of light grade petroleum oil per 100 gallons (California state specification).
c
Statistical analysis of the data i n Table I I I shows the increase in selenium on and i n the peel, over the samples i n Table I I , to be highly significant. The amount required for significance at the 0.01 level was found to be 0.003 p.p.m. The selenium residue on and in the peel is roughly proportional to the number of applications and the length of time between spraying and harvesting. The addition of 1 quart of light grade petroleum oil significantly increased the selenium spray residue remaining on and i n the peel.
Effect of Variety and Location of Sample on Tree

Single-tree plots of Jonathan, Delicious, and Winesap apples were sprayed on June 2 and 28 with 1 pint of Selocide and 1 pound of actual D D T per 100 gallons. Samples of one full box were selected from the north, south, east, and west sides and from the top of the tree to determine the effect of prevailing winds, amount of sunshine, etc., on the selenium residue. These samples were subsampled i n the laboratory, stored, and analyzed by the same procedure as the samples i n Table I I . The results are presented i n Table I V . Table IV.
Variety" Jonathan Delicious Winesap
Effect of Variety and Location of Sample on Tree

No. of Fruit per Sample 8 to 11 6 to 9 7 to 12 Weight of Fruit per Sample, Grams 1016 to 1138 1006 to 1126 1000 to 1140 Av. Selenium Residue, P.P.M.6 0.16 0.12 0.11 Av. (30) 0.13 Range 0.06 to 0. 17 Range in Selenium Residue, P.P.M. 0.14 to 0.17 0.10 to 0.16 0.08 to 0.17
5 samples per variety analyzed in duplicate. b Whole fruit basis.
WALKERSELENIUM RESIDUE ON AND IN THE PEEL OF WASHINGTON APPLES
111
Statistical analysis of the data i n Table I V shows the selenium residue on and i n the peel to be highly significant over the residue on and i n the peel of nonsprayed apples. There is no significant difference between varieties. Selenium residue is evenly distributed on the trees.
Acknowledgment
Appreciation is expressed to the M c L a u g h l i n - G o r m l e y - K i n g Company for assistance in conducting this work.
Literature Cited
(1) Assoc. Offic. Agr. Chemists, "Official and Tentative Methods of Analysis," 29.57, 1945. (2) Chilean Nitrate Educational Bureau, New York, "Bibliography of Literature on Minor Ele ments and Their Relationship to Plant and Animal Nutrition," 4th ed., Vol. 1, pp. 747-63, 1948. (3) Frear, D. . H . , "Chemistry of Insecticides and Fungicides," 2nd ed., p. 49, New York, D . Van Nostrand Co., 1948. (4) Gnadinger, C. B., Ind. Eng. Chem., 25, 633 (1933). (5) Moore, J . , J. Econ. Entomol., 34, 116 (1941).
Fruit Surface Residues of DDT and Parathion at Harvest

M. M. BARNES, G. E. CARMAN, W. H. EWART, and F. A. GUNTHER
Surface residues of DDT and parathion at various times during the season and at harvest were determined for apples, pears, peaches, oranges, and lemons. Low level surface residues of parathion on apples were not carried over into cider. Harvest residues on fresh fruit are distinguished from residues present in food at the time of consumption which are included under the designation ultimate residues.
W i t h reference to the surface deposits accruing from orchard application of insecticides, certain characteristics which enhance their value as implements i n the chemical control of many species of insectsphysical persistency and chemical stabilitymay also be conducive to the contamination of the harvested fruits with potentially deleterious residues. Supplementary to the need for data on the acute and chronic toxicity of these i n secticides to man and domesticated animals is the requirement for information concerning the magnitudes of the deposits that are present on or in foodstuffs as harvest or ultimate residues following commercial usage. The widespread commercial use of D D T [ 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] and the potentialities of the more recently developed parathion (0,0 -diethyl 0-pnitrophenyl thiophosphate) have placed emphasis on such investigations concerning these compounds. The Food and D r u g Administration has not as yet held hearings concerning the establishment of formal tolerances for D D T or parathion on fresh produce or i n processed foods. F o r apples and pears, an informal tolerance for D D T of 7 p.p.m. has been announced (3). [These hearings were i n progress at the time of publication.]
Scope, Definitions, and Methods

This investigation is a portion of a general experimental program being carried out b y the University of California Citrus Experiment Station on the fate of insecticide residues (2) and methods of removing them (5). The discussion presented herein involves tree fruits and is largely restricted to a consideration of "surface residues." This term has been defined (5) to refer to residues present above the cuticle (extrasurface residues) and to deposits that may be incorporated i n the cuticle (cuticular residues). The quantities reported as surface residues are those present i n the solvent following standardized extraction procedures and include both extrasurface and cuticular residues. Little is known as to what extent insecticide residues may be redistributed through the epidermal layer i n the process of solvent extraction or stripping. While most of the data presented are representative of harvest residues on fresh fruit, some consideration is also given to residues present i n processed food. It is considered
112
BARNES et /.FRUIT SURFACE RESIDUES OF DDT AND PARATHION AT HARVEST
113
appropriate to refer to residues present i n food at the time of consumption as "ultimate residues" (5). D D T was determined b y the dehydrohalogenation method (4). Parathion analyses were made b y the magenta color reaction of Averell and Norris (1) as modified b y Gunther and B l i n n (6). Two compounds or degradation products thereof which may cause the development of interference colors i n the magenta color reaction may be encountered i n surface residues resulting from commercial spray or dust applications. The first of these, dicyclohexylamine dinitro-o-cyclohexylphenate, is i n widespread commercial use, whereas the second, a dinitrocaprylphenylcrotonate, is involved at present only i n experimental and semicommercial usage. Blank corrections were provided for all sets of analyses. Samples for estimates of surface residue parameters on apples and pears were taken from three trees selected for representative size and shape among those of the experimental orchard. E a c h of the three samples consisted of 30 fruits. Six fruits were taken from each of four tree quadrants composing three fourths of the tree height and six from the top one fourth of the tree. These samples were generally taken before and after the penulti mate and ultimate orchard applications and at harvest. Samples for analyses of cider were taken from juice expressed with a hydraulic cider press. Samples of fruit for pressing were selected at harvest from a series for which parallel analyses for surface and pulp residues were made. E a c h of the triplicated peach samples was constituted b y selecting three fruits from each of eight trees. Citrus varieties were sampled b y selecting one fruit from each quadrant of seven trees. Replicate samples were taken from other sets of seven trees. Sample routing and manipulation have been described (6, 7). Table I. Surface Residues of DDT on Rome Beauty Apples at Harvest
0
Technical Compound , Lb./100 Gal. 1 0.5 0.5 0.5 0.5 1

a
No. of Applications 3 4 4 5 6 6
Interval, Days& 75 50 50 40 40 60
Fresh Weight, Entire Fruit, P.P.M. 1.0 1.5 0.3 2.0 3.8 5.2
As wettable powder containing 50% technical D D T . b Since final application.
Table II.
Technical Compound", Lb./100 Gal. 2 1 1 0.5 0.5 0.5 0.5
Surface Residues of DDT on Bartlett Pears at Harvest

No. of Applications 1 1 2 2 2 3 4 Interval, Days*> 110 110 70 70 85 40 14 Fresh Weight, Entire Fruit, P.P.M. 0.9 0.4 2.2 1.6 0.9 1.2 2.7
As wettable powder containing 50% technical D D T . b Since final application.
Results
The amounts of surface deposits resulting from commercial and experimental applica tions of D D T and parathion were ascertained on apples, pears, peaches, oranges, and lemons. Applications were made with conventional high pressure spraying equipment, utilizing manually operated guns or semiautomatic booms, and with two types of air blast sprayers. Surface residues of D D T on apples resulting from experimental applications applied in schedules comparable to commercial usage ranged from less than 0.5 to 2.0 p.p.m. at harvest. Schedules of application involving higher spray concentrations and greater fre quency of application than are at present required i n commercial practice resulted i n most cases i n residues of less than 7 p.p.m. T y p i c a l harvest residues are presented i n Table I.
114
On pears, experimental applications of D D T applied in commercial schedules resulted in surface residues ranging from less than 0.5 to approximately 3 p.p.m. T y p i c a l harvest residues are shown i n Table I I . Intervals between application and sampling of apples retaining weathered surface residues of parathion ranged between 21 and 70 days. A t harvest, these residues were 0.05 p.p.m. or less, following each of 13 varied schedules of application (Table I I I ) . (These values are derived from samples containing an optimum amount of parathion for analytical precision.) Low level surface residues on apples were not carried over as u l t i mate residues i n cider (Table III). This fact supports the contention that there is close association of the parathion deposits with the waxlike coating of the fruit.
Table III.
Technical Compound , Ounces/100 Gal.
0
Surface Residues of Parathion on Apples

Fresh Weight, Entire Fruit, P.P.M. Pretreat- Posttreatment ment 0.02 0.01 0.04 0.02 0.09 0.09 0.3 0.3 0.4 0.4 1.3 1.0 0.5 1.2 0.5 0.3 0.8 0.6 1.4 0.6 2.2 0.9 Fresh Weight, Entire Fruit, P.P.M. Surface Cider
Variety Rome Beauty
Application 3rd 4th 3rd 4th 3rd 4th 1st 1st 2nd 3rd 2nd 3rd 3rd 4th 2nd 3rd
Interval, Days& 21 25 21 25 21 25 27 36 27 36 27 36 27 36
Interval, Daysb
Conventional Sprayer 1 2 4 2 4 1 2 2 4 41 41 41 70 70 29 29 29 29 0.02 0.02

c
Delicious
0.01
0.01 0.02 0.01 0.03 0.02 0.09 0.05
0.05 Trace Trace

c
0 .01 c o.'oi c
0.02' 00* 00<*
0.'05
Speed Sprayer Delicious (400 gal./ acre) Delicious (200 gal./ acre) Delicious (100 gal./ acre)
a
2 4 4 8
1st 1st 1st 1st
0.3 0.4 0.5 0.2
22 22 22 22
0.01 0.01 0.04 0.01
As wettable powder supplied by American Cyanamid Co. containing 25% technical parathion. & Since previous treatment. Harvest. Values reported for cider are from duplicate analyses of juice expressed from entire fruits of same series bearing indicated surface residues at harvest.
c d
On pears (Table I V ) parathion residues resulting from ten varied schedules did not exceed 0.08 p.p.m. at harvest. (These values are derived from samples containing an optimum amount of parathion for analytical precision.) Intervals between the terminal application and harvest ranged from 7 to 33 days. Surface residues of parathion on peaches were 4- to 15-fold higher than for comparable schedules on apples or pears, possibly because of the higher initial deposits retained on the more retentive surfaces of these fruits. Surface residues of D D T on peaches were also higher than those which would be expected to result from comparable schedules on apples and pears. T y p i c a l residue values for peaches are shown i n Table V . As compared with the residues of D D T present within citrus peel (#), the relative significance of surface residues of D D T on citrus fruits is diminished by the fact that these are readily removed i n large proportion b y the usual packinghouse processing (5). On oranges, typical surface residues of D D T at harvest ranged from less than 0.5 to approximately 2.5 p.p.m. following single applications. Under conditions of multiple a p plications and shorter intervals between treatment and sampling, a higher range of surface residues was found. Equivalent or larger amounts were generally present within the peel (#). Representative surface residues are presented i n Tables V I and V I I .
BARNES et /.FRUIT SURFACE RESIDUES OF DDT AND PARATHION AT HARVEST
115
Table IV.
Technical Compound", Ounces/100 Gal. 2 2 4 8 Application 2nd 3rd 4th 3rd 4th 3rd 4th 2nd 4th 1st 2nd 1st 1st 1st
Surface Residues of Parathion on Bartlett Pears

Interval, Days 6 28 27 21 27 21 27 21 23 22 Fresh Weight, Entire Fruit, P . P . M . PretreatPosttreatment ment 0.01 0.01 0.03 0.02 0.04 0.03 0.08 0.01 0.01 0.3 0.5 0.7 0.8 1.1 2.6 2.1 0.3 0.2 0.2 0.3 0.1 0.2 0.3 Interval, Days & 7 33 33 33 7 Fresh Weight, Entire Fruit, P.P.M. 0.08
Conventional Sprayer, Single Gun

c
o.
oi
c
0.02 0.04 0.06 0.02
Conventional Sprayer, Broom Guns 2

c
Conventional Sprayer, Fixed Nozzle Vertical Boom 2 28

c
Spray Duster (Master Fan Type) 8 gal./acre) 1 2 4

a
27
0.03 Speed Sprayer
22 25 25 25
Trace 0.00 Trace Trace
As wettable powder containing 25% technical parathion. 6 Since previous treatment. Harvest.
c
Table V.
Attenuation of Posttreatment Surface Residues of Parathion and DDT on Lucas Beauty Peaches
Technical Compound, Ounces/100 Gal. Interval, Days 7 14 Fresh Weight, Entire Fruit, P . P . M . 4.5 10.9 16.1 1.5 3.7 7.6 1.0 1.8 4.4 30 0.2&
0.6b 2.7b
Parathion Parathion DDT

C
4 8 8
As wettable powder containing 25% technical parathion. b Harvest. As wettable powder containing 50% D D T .
c
Table VI.
Attenuation of Posttreatment Surface Residues of DDT on Citrus Following Single Applications

Technical Compound, Lb./100 Gal. 2.0 2.0 1.0 1.2 17.1 8.9 2.8 2.2 Interval, Days" 141 2 141 2 Fresh Weight, Entire Fruit, P . P . M . Valencias Lemons Navels 2.16
2.3b
Type Formulation Wettable powder Kerosene solution Kerosene solution Light medium oil solution
a
1.56 0.26
16.2 4.9 3.0 2.1
23.9 14.0 3.2 2.7
1.7 2.3 0.2 1.3
Application, 10/1/45. b Harvest.
Table VII.
Surface Residues of DDT on Valencia Oranges at Harvest Following Sequence Applications

Applications 6/7/46
0
5/9/46 S S S
a
8/9/46
Interval, Days 6 117 88 88 25
Fresh Weight, Entire Fruit, P.P.M. 2.3 5.8 8.4 6.3
v 's
2
Kerosene-Velsicol AR-60 (95-5) 3 gallons, technical D D T 2 lb., and blood albumin spreader 0.25 lb. per 100 gallons of water applied as full coverage spray (S) or outside coverage spray O A S). 6 Since application.
On lemons, surface residues at harvest ranged from 2 to 3 p.p.m. (Table V I I I ) , some what larger amounts being generally present within the peel {2).
116
Table VIII. Attenuation of Surface Residues of DDT on Eureka Lemons

Technical Compound , Lb./lOO Gal.
0
Interval, Days 23 35 44 Fresh Weight, Entire Fruit, P . P . M .
84
2 15.8 6.4 ... 2.9b 2 ... 7.8 ... 2.36 Kerosene-Velsicol AR-60 (95-5) 3 gallons, technical D D T 2 lb., and blood albumin spreader 0.25 lb. per 100 gallons of water. 6 Harvest.
Studies on oranges (Table I X ) have shown that surface residues of parathion are also of relatively low magnitude i n comparison with the quantities found within the peel (#). Surface residues on these fruits decreased rapidly (5), reaching values of 0.01 p.p.m. or less at harvest following a single application at required dosages. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch023 Table IX. Surface Residues of Parathion on Navel Oranges at Harvest
0
Technical Compound , Lb./lOO Gal. 1 1.25 1.5 1
No. of Applications 1 1 1 2
Interval, Days ^ , ^ . . . 0 Fresh Weight, Entire Fruit, P . P . M . 0.01 0.02 0.09 0 20

1 1 U x A A 1 4
As wettable powder containing 25% parathion. First application 9/15/47, second application 10/15/47.
Acknowledgment
The authors are indebted to the American Cyana'mid Company for supplies of the wettable powder of parathion used i n these trials.
Literature Cited
(1) Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753 (1948). (2) Carman, G . E., Ewart, W. H . , Barnes, M . M . , and Gunther, F . ., ADVANCES IN CHEMISTRY SERIES, 1, 128 (1950). (3) Food and Drug Administration, Federal Security Agency, Trade Correspondence Letter 3-A (Nov. 5, 1945). (4) Gunther, F . ., Hilgardia, 18, 297 (1948). (5) Gunther, F . ., Barnes, M . M . , and Carman, G . E., ADVANCES IN CHEMISTRY SERIES, 1, 137 (1950). (6) Gunther, F . ., and Blinn, R. C., Ibid., 1, 72 (1950). (7) Gunther, F . ., and Miller,, M . E., Ibid., 1, 88 (1950).
P A P E R 624, University of California Citrus Experiment Station, Riverside, Calif.
DDT and Parathion Spray Residues on Apples

W. E. WESTLAKE and JACK E. FAHEY
Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, Beltsville, Md.
The tests reported were conducted in 1948 on apples growing in the Yakima Valley in the Pacific Northwest and in the Mississippi Valley, to determine the magnitude of parathion and DDT spray residues at harvest. The climates and spray schedules differ markedly in the two areas; consequently, spray residues also differ, and are larger in the Mississippi Valley than in the Yakima Valley.
T h e use of insecticides on the edible parts of plants presents the problem of removal or avoidance of excessive toxic residues on the harvested crops. This paper shows the magnitude of residues that may result from the application of sprays containing parathion and D D T insecticides. I n 1948 tests were made on apples growing i n the Y a k i m a Valley i n the Pacific N o r t h west and i n the Mississippi Valley. The climates of these two areas differ strikingly. The Mississippi Valley is characterized b y moderately heavy precipitation during the growing season, the average rainfall being 3.5 to 4.3 inches per month. The Y a k i m a Valley, on the other hand, is very arid, the average precipitation being less than 1 inch per month during the summer, and is completely dependent upon irrigation for orchard growth. Spray schedules also differ i n the two areas. I n the Mississippi Valley the orchards are sprayed more often, and the interval between the last spray and harvest is shorter, than i n the Y a k i m a Valley. Spray residues at harvest may be expected to differ also.
Methods
Spray schedules applied on experimental plots at the Y a k i m a . Wash., and Vincennes, Ind., laboratories of the United States Bureau of Entomology and Plant Quarantine were studied to determine the magnitude of parathion and D D T spray residues at harvest. The parathion sprays were prepared from 2 5 % wettable powder and the D D T sprays from 5 0 % wettable powder, except i n one series of tests, when a 2 5 % D D T wettable powder was used. A l l spray treatments were planned and made by members of the Division of F r u i t Insect Investigations. Conventional hydraulic sprayers were used in this work. The studies in the Y a k i m a Valley were made on two varieties of apples and those i n the Mississippi Valley on five varieties. Duplicate samples of 1500 and 2000 grams of fruit were employed for each analysis at Vincennes and samples of 1000 to 1500 grams at Yakima. Parathion analyses were made by the method of Averell and Norris (1). D D T residues were determined at Vincennes b y the total-chlorine method of Wichman et al. (4), and at Y a k i m a by the colorimetric method of Stiff and Castillo (5), as modified by the Food and D r u g Administration (#).
117
118
Parathion Residues
Table I shows the parathion residues on Delicious and Winesap apples i n the Y a k i m a \ alley immediately after the last spray application and at intervals until harvest. P a r a thion was used at two concentrations, both as close as possible to the minimum necessary to give the desired control. Consequently, the residues found were comparatively low and dropped to 0.1 p.p.m. or less i n approximately 2 weeks.
r
Table I.
Parathion Residues on Delicious and Winesap Apples, Yakima Valley

No f' Sprays
G
Plot No. 7
Treatment (Active Ingredient in 100 Gal.) DDT, Parathion, ounces lb. 1 1 25 1 25 1 25 0.5
Parathion Found, P . P . M . Delicious Apples July 29 Aug. 16 Oct. 11 0.90 0.09 0.07 1.10 0.40 0.30 0.05 0.05 0.03 0.90 0.04 0.24 03 0.36 10 Aug. 5 Winesap Apples Aug. 11 Aug. 17 Oct. 18
10 11
1 25 0 6 0 6 0 6 0
0.5 0.5 0.5
12
1 25 1 25
On Delicious apples the initial residues were 0.9 and 0.4 p.p.m. i n plots 7 and 10, respectively, both of which were sprayed with 1.25 ounces of parathion. T h e difference between the two plots was consistent throughout the individual trees sampled. T h e spray mixture used on plot 7 also contained D D T , while that used on plot 10 contained only parathion. These plots showed the same relative magnitude of residues 18 days after spraying, and at harvest, 74 days after the spraying. Plot 11, sprayed with 0.6 ounce of parathion, showed an initial residue of 0.3 p.p.m. Initial residues on the Winesap apples were somewhat higher than those on Delicious. Six days later approximately one third of the residue had been lost, and 12 days after the spraying the deposits were down to 0.10 and 0.03 p.p.m. for the respective strengths. A t harvest little more than a trace of parathion was found on the fruit. Table I I shows the parathion residues on Golden Delicious apples i n the Mississippi Valley immediately after the final spray application and after 25 and 38 days of weathering. Five plots received six parathion sprays and a sixth plot received parathion i n only the last two sprays. Table II. Parathion Residues on Golden Delicious Apples, Mississippi Valley
(Six sprays on all plots; final spray Aug. 5) Treatment (Active Ingredient in 100 Gal.) D D T , ounces Parathion, ounces 1 2 4 4 8 (2 sprays) (4 sprays) 8 4 8 12 12 Parathion Found, P . P . M . Sept. 13 Aug. 30 0.19 0.24 0.37 0.36 0.88 0.42 0.10 0.15 0.20 0.15 0.42 0.25
No. 27 30 25 28 24 21
Aug. 5 1.09 3.49 3.04 3.40 6.14 3.20
W i t h the exception of plot 30, the residues after the final spraying show a good correlation with the amount of parathion applied. T h e loss of residue was very rapid, however. The results i n plot 21, which received parathion only i n the last two sprays, as
WESTLAKE AND FAHEYDDT AND PARATHION SPRAY RESIDUES ON APPLES
119
compared with those i n plots 25 and 28, which received the same amount of parathion i n all six sprays, show that the first four parathion sprays had little effect on the residue at harvest. Table I I I shows the parathion residues on Jonathan and Starking Delicious apples from seven spray plots i n the Mississippi Valley. Identical treatments were used on both varieties. The Starking variety showed a slightly lower parathion residue than the Jonathan. The difference was not great, however. I n general, the residue after the final spraying was proportional to the concentration of parathion i n the spray mixture. The exception is plot 4, sprayed with 2 ounces of parathion with nicotine-bentonite-oil, which shows a residue approximately equal to that obtained on plots sprayed with 4 ounces of parathion, alone or i n combination with D D T (plots 11 and 12). The residue 2 weeks after spraying was only one quarter to one third of that found immediately after spraying, and 46 days after spraying only one plot (No. 14 sprayed with 8 ounces of parathion) showed a residue significantly i n excess of 0.1 p.p.m. Table III. Parathion Residues on Jonathan and Starking Delicious Apples, Mississippi Valley
(Six sprays, except where otherwise indicated) (Active iigredient in 100 Gal.) Parathion, ounces D D T , lb.
e
No. 9 11 12
Parathion Found, P . P . M . Jonathan Apples Starking Delicious Apples Sept. 14 July 30 Aug. 13 Aug. 13 Sept. 14 July 30 0.53 1.41 1.23 0.12 0.41 0.33 0.03 0.11 0.09 0.32 1.22 0.99 0.07 0.24 0.23 0.03 0.07 0.04
1 1 4 0.5 4 0.5 (Sprays 3 and 6) (In sprays 1, 2, 4, 5) 1.5 (In sprays 1, 2, 4) 1 (In spray 6) 4 (In sprays 4 and 6) (Sprays 3 and 5 nitted) 8 2 (With nicotine-bentonite-oil) 2 12 (In sprays 1 to 4)
15
1.65
0.39
0.11
1.22
0.20
0.07
14 4 13
3.80 0.99 0.10
1.06 0.23 0.00
0.28 0.02 0.00
3.39 1.08 0.12
0.73 0.20 0.01
0.20 0.03 0.00
(In sprays 5 and 6)
Final parathion spray on July 28 except plot 13, which received final spray on June 16.
Plots 12 and 13 afford a comparison of the harvest residues from first- and secondbrood sprays. O n plot 13, which was sprayed on M a y 11 and 20 and June 3 and 16, the parathion residue was only 0.1 p.p.m. on July 30 and none i n subsequent samplings. On plot 12, which received two sprays on J u l y 7 and 28, the residue on Jonathan apples was 1.23 p.p.m. on July 30 and 0.09 on September 14. Table I V gives the data from seven plots of Winesap and Rome Beauty apples i n the Mississippi Valley. The spray schedules are similar to those used for the plots included in Table I I I , except that an additional parathion spray was applied on plots 9, 11, 14, and 4 on August 19, and the final harvest sample was taken on October 5. Only on the plot that was sprayed seven times with the 8-ounce strength of parathion (plot 14) d i d the spray residue at harvest approximate 0.1 p.p.m. The data presented indicate that parathion spray residues weather very rapidly on apples. Sprays applied to the fruit prior to J u l y 1 would not be expected to leave more than a trace of parathion on the fruit at harvest. The harvest residues vary i n magni tude according to the concentration of parathion i n the spray mixture. Adhesives such as nicotine-bentonite-oil, although increasing the initial residue of a given spray mixture, do not show an appreciable effect on the final harvest residue. When the concentration of parathion is not greater than 4 ounces in 100 gallons of spray, and the final spraying is done not less than 40 days before harvest, the parathion residue at harvest, under the conditions of the experiments, does not exceed 0.1 p.p.m.
120
Table IV.
Parathion Residues on Rome Beauty and Winesap Apples, Mississippi Valley

(Seven sprays, except where otherwise indicated) Date of Final Parathion Spray Aug. 19 Aug. 19 July 28 Parathion Found, P . P . M . Winesap Apples Rome Beauty Apples July 30 Aug. 20 Oct. 5 July 30 Aug. 20 Oct. 5 0.45 0.28 0.14 0.01 0.01 0.01 0.72 0.46 0.16 0.02 0.04 0.03
Plot No. 9 1 12 1
Treatment (Active Ingredient in 100 Gal.) Parathion, ounces D D T , lb. 4
1. 1 8 4 0.5 (In sprays 3 and 6) 1 (In sprays 1, 2, 4, 5, 7) 15 .. 1.5 (In sprays 1, 2, 4) 1 (In spray 6) 4 (In sprays 4 and 6, sprays 3, 5, 7 omitted) 14 8 4 2 (With nicotine-bentonite-oil) 13 2 0.75 (In sprays 1 to 4) 1 (In sprays 5, 6, 7)
1.16
1.28
July 29
1.55
0.14
0.04
1.83
0.27
0.06
Aug. 19 Aug. 19 June 16 0.10
0.49 1.13 0.00
0.09 0.05 0.00 0.12
1.03 1.39 0.01
0.12 0.05 0.00
DDT Residues
Table V shows the D D T spray residues at harvest on Delicious and Winesap apples in the Y a k i m a Valley. M o s t of the Delicious plots received two sprays containing D D T , the last one 97 days before harvest. The Winesap plots received four sprays containing D D T , the last spray 74 days before harvest. The harvest residues i n the Winesap plots were significantly higher than those i n the Delicious plots, but were less than one half the proposed tolerance of 7 p.p.m. of D D T . Table V. DDT Residues on Delicious and Winesap Apples, Yakima Valley
(One to three sprays) Plot No. 4 7 Treatment (Active Ingredient m 100 Ual.) Parathion, ounces D D T , lb. .25 1.25 1.25
1.25
No. of Sprays 2 1 1 1 1 3 1 2 1 1 1 1 3 1 1 1
Date of Last D D T Spray July 7 July 7 Aug. 5 June 16
D D T Found, P . P . M . Winesap apples, Delicious apples, Oct. 11 Oct. 18 1.0 1.0 2.7 1.4
1 1 0.5
10 11
0.5 1.25 1 (1 lb. benzene hexachloride technical + 2 quarts oil) 0.6 1 0.6 0.5 0.6 0.5 1 (1 lb. benzene hexachloride + 2 quarts oil) 1.25 0.5 1.25 o'.
i' '
July 7 Aug. 5
1.5 3.3
12
July 7
1.7
Table V I shows the D D T residues at harvest on plots i n a Golden Delicious orchard i n the Mississippi Valley. A l l plots received six sprays containing D D T . The harvest residue i n plot 1, i n which a 5 0 % D D T wettable powder was used, was only slightly less than where the same amount of D D T i n a 2 5 % wettable powder was applied i n plot 2. Reduction of the concentration of actual D D T i n the spray mixture (plots 3 and 4) resulted i n lower residues at harvest. Table V I I shows the residues of D D T at harvest i n the Mississippi Valley on Jonathan and Starking Delicious apples on which a six-spray schedule was used. A l l plots except plot 8 were sprayed six times with D D T at 8 ounces to 1 pound per 100 gallons. Plot 8 received only four sprays, three containing 1.5 pounds and one containing 1 pound
WESTLAKE AND FAHEYDDT AND PARATHION SPRAY RESIDUES ON APPLES
121
Table VI.
DDT Residues on Golden Delicious Apples, Mississippi Valley

(Six sprays on all plots; final spray Aug. 5; analyses Sept. 13) Treatment (Active Ingredient in 100 Gal.) & Parathion, ounces D D T , ounces .. 12 (No adhesive or spreader used on 1 plot) .. 12 8 (No adhesive or spreader) 2 4
Plot No.
a
D D T Found, P.P.M. 7.9 8.8 7.1 3.7
1 2 3 4
Plots 1 and 3 represent averages of 3 plots each. 6 50% wettable powder used in plot 1 and 25% wettable powder in other plots.
of D D T . I n plots 2 and 3, adhesives were included i n all sprays, and harvest residues approximated 10 p.p.m. of D D T even though only 8 ounces of D D T were used. I n a l l other plots the harvest residues were less than 7 p.p.m. of D D T except i n plots 7 and 8, i n which they were 7.8 and 7.3 p.p.m., respectively, on the Starking apples. I n general, the harvest residues were proportional to the amount of D D T applied i n the last cover spray.
Table VII.
DDT Residues on Jonathan and Starking Delicious Apples, Mississippi Valley

(Six sprays on all plots; final spray on July 28) Treatment (Active Ingredient in 100 Gal.) Parathion, ounces D D T , ounces 8 (With lead arsenate-Bordeaux-oil or nicotinebentonite-oil) 8 (With nicotine-bentonite-oil) 1 (Av. of 5 plots, no adhesive or spreader) 4 8 1 (In sprays 1, 2, 4, 5) 4 8 (In s prays 3 and 6) 2 12 (In sprays 1 to 4) 1 (In sprays 5 and 6) 1.5 (In sprays 1, 2, 4) 4 (In spray 6, sprays 3 and 5 omitted) D D T Found, P . P . M . Starking Delicious Jonathan apples, apples, Aug. 31 Sept. 2 10.2 7.8 10.3 6.4 3.7 4.3 10.0 6.8 4.7 5.6
Plot No.
5.6
7.8
6.5
7.3
Table VIII. DDT Residues on Rome Beauty and Winesap Apples, Mississippi Valley
(Seven sprays on all plots; final spray on Aug. 19; analyzed on Oct. 5) Plot No. 2 3 4 5 6 Treatment (Active Ingredient in 100 Gal.) Parathion, ounces D D T , ounces (With 8 lead arsenate-Bordeaux-oil or nicotine-bentonite-oil) 8 (With nicotine-bentonite-oil) 16 (Av. of 5 plots, no adhesive or s preader) 4 8 16 (In sprays 1 2, 4, 5, 7) 4 8 (In sprays 3 and 6) 2 12 (In sprays 1 to 4) 16 (In sprays 5, 6, 7) 24 (In sprays 1, 2, 4) 4 16 (In spray 6, sprays 3. 5. 7 omitted) D D T Found, P . P . M . Winesap Rome Beauty apples apples 9.1 12.1 9.5 6.7 8.6 11.8 11.1 10.4 6.9 9.8
11.3
11.2
5.1
6.8
122
Table V I I I shows the residues of D D T at harvest on Rome Beauty and Winesap apples i n the Mississippi Valley. The plot treatments are the same as for Jonathan and Starking Delicious apples (Table V I I ) except that a seven-spray schedule was used. T h e residues at harvest shown i n Table V I I I are greater than those i n Table V I I . A com parison shows that when six cover sprays of D D T are applied without adhesives the harvest residues are approximately 7 p.p.m. or slightly more. If, however, seven cover sprays are applied, the residues may exceed 9 p.p.m. of D D T , unless the concentration is reduced to less than 1 pound of D D T i n 100 gallons.
Summary
I n 1948 spray schedules used experimentally i n the Y a k i m a Valley i n the Pacific Northwest and i n the Mississippi Valley were studied to determine the magnitude of the parathion spray residues during the interval between the last spray and harvest. D D T residues were also studied and results of analyses made at harvest are reported. The spray schedules studied i n the Y a k i m a Valley included at least three sprays containing not more than 1.25 ounces of parathion in 100 gallons. Harvest residues were less than 0.1 p.p.m. of parathion. I n the Mississippi Valley the studies included sprays containing as much as 8 ounces of parathion i n 100 gallons. When 4 ounces or less of parathion were used, and no spray was applied less than 40 days before harvest, parathion residues were generally less than 0.2 p.p.m. Increasing the concentration of parathion i n the spray mixture or decreasing the time interval between the last spray and harvest sometimes resulted i n heavier residues. Spray schedules using 1 pound or less of D D T i n 100 gallons i n one to four sprays were studied i n the Y a k i m a Valley. D D T residues were well below the proposed toler ance of 7 p.p.m. i n all treatments studied. A four-spray schedule with 74 days between the last spray and harvest resulted i n a residue of only 3.3 p.p.m. of D D T at harvest. Spray schedules with as much as 1.5 pounds of D D T i n 100 gallons were studied i n the Mississippi Valley. The number of sprays containing D D T was as high as seven, six being applied i n most of the treatments. A six-spray schedule i n which 1 pound of D D T was used, without any adhesive, resulted i n harvest residues approximating or slightly i n excess of 7 p.p.m. of D D T . When seven sprays were u s e d * D D T residues i n some treatments were considerably i n excess of 7 p.p.m. The use of adhesives, such as nicotine-bentonite-oil or lead arsenate-Bordeaux-oil, increased the D D T residues at harvest.
Literature Cited
(1) (2) (3) (4) Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753 (1948). Food and Drug Administration, unpublished. Stiff, . ., and Castillo, J . C., Science, 101, 440 (1945). Wichman, H . J . , Patterson, W. I., Clifford, P. ., Klein, A. K., and Claborn, . V.,J.Assoc. Offic. Agr. Chemists, 29, 188-218 (1946).
Parathion Spray Residue on Soft Fruits, Apples, and Pears

KENNETH C. WALKER
Washington Agricultural Experiment Stations, Wenatchee, Wash.
The amount of parathion spray residue on soft fruits is roughly proportional to the length of time between date of application and date of analysis. Parathion spray residue was lost from the surface of Delicious apples at the rate of 80 to 85% in 12 to 13 days and 93 to 100% in 30 to 32 days. The rate of loss was the same for 1-pound as for 4-pound concentrations. Fifty-five samples, collected from commercial orchards, were analyzed. No significant relationship was found between the number of days between spraying and analysis and the parathion residue. There was no significant difference in parathion residue due to the concentration of the spray mixture used. All residues were only a fraction of 1 p.p.m.
Parathion (0,0-diethyl O -p-nitrophenyl thiophosphate) is an ester of thiophosphoric acid with the empirical formula C10H14NO5PS. It is a high boiling deep-brown to yellow liquid, some samples of which possess a characteristic odor. Its boiling point has been calculated to be 375 C . or higher, at 760 m m . pressure ; its refractive index is n^ 1.15360 ; specific gravity is 1.26. The vapor pressure is 0.0006 m m . of mercury at 24 C . The technical grade has a purity of approximately 9 5 % .
5
Parathion is very slightly soluble i n water (20 parts per million), but is completely miscible i n many organic solvents including esters, alcohols, ketones, ethers, aromatic and alkylated aromatic hydrocarbons, and animal and vegetable oils. It is practically i n soluble i n such paraffinic hydrocarbons as petroleum ether, kerosene, and refined spray oils (about 2%) unless a mutual solvent is used (1). Parathion has been recommended i n the state of Washington (3) for the control of various species of mites and orchard aphids. Wettable powders, containing 15 or 2 5 % of parathion, have been used at concentrations ranging from 0.25 to 4 pounds per 100 gallons of water on an experimental basis and from 0.25 to 0.5 pound on a commercial basis.
Methods and Procedure

Parathion spray residue on the surface of the fruit was determined b y the method of Averell and Norris (). Samples of sufficient size of firm fruit completely to fill 1-gallon wide-mouthed glass jars were selected for analysis. The parathion was removed from the surface of the fruit with benzene (redistilled) in an end-over-end type tumbling machine at a speed of 72 revolutions per minute. A l l samples were washed i n this manner for a period of 0.5 hour. Reagent blanks and u n sprayed fruit blanks were run with all samples. A l l results, as reported, have been cor rected for reagent and fruit blanks.
123
124
Soft Fruit
Although parathion has not been recommended to date for insect control on soft fruits, there are indications that some injurious insects may be controlled b y applications of parathion. Experimental sprays of 1 pound of the 2 5 % wettable powder per 100 gal lons of water were applied to soft fruits to determine the amount of parathion residue that would remain on the, surface at harvest time. The parathion residue at harvest time is presented i n Table I . Table I. Parathion Residue on Surface of Soft Fruits at Harvest Time
Variety Days between Last Spray and Analysis 14 25 36 43 75 79 14 20 38 47 72 28 60 7 30 55 75 97 38 38 53 Surface Parathion, P.P.M. 0.30 0.08 0.08 0.08 0.00 0.00 0.40 0.21 0.00 0.01 0.02 0.03 0.00 1.63 0.80 0.03 0.00 0.01 0.57 0.76" 0.00
Bing cherry
Morpark apricot Tilton apricot
Yakamine Italian prune Elberta peach
Golden Jubilee peach J. H . Hale peach

a
Two sprays of 2 pounds of 25% wettable powder per 100 gallons.
Parathion, used for the control of injurious insects on soft fruits, would be applied more than 30 days prior to harvest. Infestation of the various potential insects is relatively early i n the spring. Where sprays of 1 pound of 2 5 % wettable powder per 100 gallons of water were applied more than 30 days before harvest, the parathion residue at harvest time on B i n g cherries, M o r p a r k and Tilton apricots, Yakamines, Italian prunes, Elberta and J . H . Hale peaches ranged from 0.00 to 0.08 p.p.m., as shown i n Table I . When less than 30 days were allowed to elapse between spraying and analysis, the parathion residue on the above soft fruits ranged from 0.30 to 1.63 p.p.m. Increased concentrations of parathion applied to Golden Jubilee peaches resulted i n a higher parathion residue. The amount of parathion residue was roughly proportional to the length of time be tween date of application and date of harvest. Table II.
Variety Bartlett pear Bartlett pear D'Anjou pear D'Anjou pear Jonathan apple Delicious apple Winesap apple
a a
Parathion Residue on Pears and Apples

South side 0.00 0.04 0.03 0.00 0.02 0.02 0.03 Area Sampled and Parathion Residue, P . P . M . East West side side Top Average 0.00 0.04 0.04 0.00 0.01 0.01 0.04 0.03 0.00 0.05 0.04 0.01 0.01 0.04 0.01 0.02 0.04 0.00 0.03 0.03 0.04 0.01 0.02 0.04 0.01 0.02 0.02 0.03 Range 0.00 0.00 0.03 0.00 0.01 0.01 0.02 to 0.03 to 0.04 to 0.05 to 0.04 to0.03 to 0.03 to 0.04
North side 0.01 0.02 0.04 0.02 0.02 0.04 0.02
Plots sampled twice.
Apples and Pears

Single tree plots of Bartlett and D ' A n j o u pears and Jonathan, Delicious, and Winesap apples were sprayed on June 2 and 28 with 1 pound of 2 5 % wettable para-
WALKER PARATHION SPRAY RESIDUE ON SOFT FRUITS, APPLES, AND PEARS
125
thion powder and 2 pounds of 5 0 % 2,2-bis-p-chlorophenyl-l,l,l-trichloroethane ( D D T ) . Samples of one full box (1 bushel) were selected from the north, south, east, and west sides and from the top of the tree. These samples were subsampled i n the laboratory and analyzed b y the same procedure as the samples i n Table I . The results are presented i n Table I I .
12
22
30
JULY
Figure 1.
Loss of Parathion Residue under Field Conditions
Statistical analysis of the data i n Table I I shows no significant difference between varieties or between positions on the tree. The average parathion residue on a l l varieties is equal to or slightly less than the variation between samples.
Loss of Parathion Residue under Field Conditions

Spray programs of 1 and 4 pounds of 2 5 % wettable parathion powder per 100 gallons of water were applied to Delicious apples on July 2 (plots 1 and 2). Samples were taken as soon as the fruit became dry and at 10- to 13-day intervals for a period of 32 days. These plots were sprayed again on August 3 with the same mixtures and resampled over a 30day period (plots 3 and 4). The results and the percentage of loss of parathion are shown in Table I I I and Figure 1. A l l plots lost from 80 to 8 5 % of their parathion residue from the surface of the fruit i n 12 to 13 days and from 93 to 100% i n 30 to 32 days. Plots 3 and 4 were sprayed later i n the season (August 3) than plots 1 and 2 (July 2), and the slightly smaller loss of parathion residue from the surface of the fruits at the later date may be due to a decrease i n volatility of parathion because of lower day and night temperatures.
Relation of Concentration of Spray Mixture to Residue

Cover sprays, at concentrations ranging from 0.25 to 4 pounds of the 2 5 % wettable parathion powder per 100 gallons of water, were applied to Delicious apples. These sprays were applied on June 7, J u l y 2, and August 3. The residue samples were selected at harvest time and held i n cold storage (32 F.) for a period of time and then analyzed. The elapsed period of time between spraying and analysis was 90 days. The results are presented i n Table I V . Under normal insect infestations (mites and aphids) encountered i n the state of Washington, a spray program of not more than three cover sprays at a concentration of not more than 0.5 to 1 pound of the 2 5 % wettable powder per 100 gallons of water should give
126
very excellent control of the insects. Normally, a period of 90 days between the last ap plication and harvest would not elapse (45 to 60 days would be expected). The parathion residue at harvest time resulting from a program of 0.5 to 1 pound of 2 5 % wettable powder applied 45 to 60 days before harvest could be expected to be 0.10 p.p.m. or less. The application of sprays with a concentration i n excess of 0.5 to 1 pound (25%) would result i n no measurable increase i n insect control, waste of materials, and higher parathion spray residues. Table III.
Plot No.
Parathion Residue and Rate of Loss under Field Conditions

Parathion Residue, P.P.M. 5.1" 1.0 0.1 0.0 19.1 3.2 1.1 0.8 4.7 0.7 0.4 0.3 18.9 3.5 2.0 1.4 Days between Spraying and Analysis 0 12 20 32 0 12 20 32 0 13 21 30 0 13 21 30 Loss, 0 80 98 100 0 83 94 96 0 85 91 94 0 81 89 93
36
46
Sprayed July 1. 6 Sprayed August 3.
Table IV.
Relation of Concentration of Spray Mixture to Residue

Concentration", Lb. 4 3 2.56 2 1 0.25 Parathion Residue, P.P.M. 0.60 0.19 0.18 0.18 0.08 0.01
Plot No. 1 2 3 4 5 6
Pounds of 25% wettable parathion powder per 100 gallons of water. 6 Four cover sprays applied June 7 and 23, July 2, and August 3.
Parathion Spray Residue on Commercially Sprayed Apples

Fifty-five samples of applesJonathan, Delicious, Rome Beauty, and Winesap were collected from commercially sprayed orchards that had received a single parathion spray and where the spray program was known. These samples were analyzed b y the pre viously described method and the results are presented in Table V . Table V. Parathion Spray Residue on Commercially Sprayed Apples
No. of Samples 29 5 21 Days between Spraying and Analysis 32- 81 (av. 55.5) 33- 79 (av. 54.0) 29-78 (av. 51.9) Parathion Residue, P.P.M. 0.00-0.14 (av. 0.041) 0.00-0.07 (av. 0.010) 0.00-0.07 (av. 0.026)
Spray Program V2 L b . 15% wettable powder/100 gallons water /% Lb. 15% wettable powder/100 gallons water V 4 L b . 15% wettable powder/100 gallons water
1
Regression analysis comparing the number of days from spraying to analysis and the parathion spray residue show no significant relationship. The difference i n parathion residue due to the V -, and y - p o u n d applications was not significant. A l l residues were only a fraction of 1 p.p.m.
3 4
WALKERPARATHION
SPRAY RESIDUE ON SOFT FRUITS, APPLES, AND PEARS
127
Acknowledgment
Appreciation is expressed to W . J . O ' N e i l l and E . W . A n t h o n of the D i v i s i o n of E n t o mology, State College of Washington, for their k i n d assistance i n the field.
Literature Cited
(1) American Cyanamid Co., Tech. Bull. 2 (1949). (2) Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753 (1948). (3) Washington State College Extension Service, Bull. 279, revised (February 1949),
Absorption of DDT and Parathion by Fruits

G. . CARMAN, W. H. EWART, M. M. BARNES, and F. A. GUNTHER
Commensurate with the need for information on the effectiveness of new organic insecticides has been the need for determining the magnitude and distribution of toxic residues on and in edible produce. Absorption of insecticide residues of DDT and parathion by fruit was investigated. Specific techniques for the physical separation of component fruit parts with minimization of sample contamination are described. DDT residues were analyzed by the dehydrohalogenation method. The magenta color reaction as modified by Gunther and Blinn was used for parathion residues. Results indicated the presence of DDT and parathion in the peel but not in the pulp of harvested citrus fruits. The relatively rapid penetration of the toxicants was followed by a slower loss with the retention of appreciable amounts over long periods of time. Neither DDT nor parathion was found in the pulp of apples, pears, or peaches following treatment with standard dosages in sequence applications. Spectrographic examination of transmission-wave length curves of the benzene extractives of DDT-treated and parathion-treated navel oranges in comparison with curves for parent compounds indicated definite shifts in absorption bands.
Although various workers (6, 18, 20, 21) have reported on the mammalian toxicity of l,l,l-trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT) and 0,0 -diethyl 0-p-nitrophenyl thiophosphate (parathion), more detailed studies with these toxicants are being conducted by numerous pharmacological groups because available information indicates that certain hazards may be associated with the use of these materials as economic poisons. In addi tion to the possible dangers from exposing personnel to these toxic materials in the course of handling or making insecticide applications, the retention of deposits on edible products or the accumulation of residues by translocation may be objectionable or hazardous. A s a means of clarifying the problems incident to this aspect of food contamination, agri cultural chemists and entomologists have recognized the desirability of projecting parallel studies to determine the magnitude and location of D D T and parathion residues on or in treated produce. In many instances the problem of surface contamination has been principally studied {8-6, 7-10, 16,17, 19, 22). However, early in the D D T studies on citrus fruits, it be came evident that appreciable amounts of this insecticide penetrated into certain compo nents of the peel. Following this disclosure, suitable techniques were investigated and an evaluation of the penetration of D D T and later of parathion into various kinds of fruit was undertaken. The studies reported herein consider for the most part the occurrence of penetration following the practical uses of these insecticides on specified crops and, to a limited ex tent, the ultimate fate of the penetrated compounds. They may additionally serve to indicate the nature of the problems which may be associated with other uses. The final interpretation of these results will be dependent on the fuller elaboration of pharmaco128
CARMAN et /.ABSORPTION OF DDT AND PARATHION BY FRUITS
129
logical studies and on the subsequent clarification of the need for legal tolerances for resi dues of these specific toxicants or their degradation compounds on consumer products.

These studies were limited to work with navel and Valencia oranges, lemons, grape fruit, apples, pears, and peaches. Preliminary results have also been obtained with avo cados, grapes, olives, plums, and certain vegetable crops. Representative fruit samples were assembled from trees which had been treated with experimentally formulated technical grades of D D T and parathion or with commer cially available formulations of these toxicants. A l l insecticide applications were made with power equipment typical of that used i n commercial practice. As a means of mak ing direct comparisons and of assuring greater control over those variables, inherent i n field studies, most fruit samples were taken from test plots i n experimentally treated grove areas. The experimental groves were located i n Los Angeles, Orange, Riverside, San Ber nardino, San Diego, Tulare, and Ventura counties of California. Approximately eight pounds of fruit constituted each analytical sample (11, 14)', the number of fruits per sample varied from ten to thirty. Fruits were selected at random from within a peripheral band around the tree 3 to 6 feet above the ground. Individual samples were constituted with fruits from six to eight trees and replicate samples were taken from different groups of trees. I n some cases, samples of deciduous fruits were col lected from three trees and additionally involved a portion of fruit from the upper quarter of the tree. Duplicate, or more generally triplicate, samples were utilized for analyses. A l l fruits for penetration studies were collected i n paper bags, which were immediately stapled to ensure sample integrity. The sample preparations and quantitative estimations of residues were completed as soon after the fruits were sampled as proved practicable (14) Standardized procedures were adopted with regard to sample preparation, recovery of toxicant, and chemical assay. I n order to determine the nature and magnitude of pene trated residues, i t was necessary to disassociate all extra-surface residues. The techniques originally developed to effect this separation and which were used i n most of the D D T penetration studies have been described b y Gunther (11). Certain modifications which have been developed subsequently i n connection with the parathion studies are described in detail below since this phase of penetration studies assumes singular importance (see also 14)Citrus Fruit Types. T h e method previously described (11) consisted essentially of scrubbing the fruits w i t h a w a r m 1 0 % trisodium phosphate solution, rinsing w i t h distilled water, h a l v i n g each fruit, a n d reaming the juice a n d pulp from each half with a power juicer. Pieces of pulp adhering to the insides of the individual hemispheres of peel were carefully scraped free and combined with the remainder of the pulp and juice. Independent analyses were then completed on the discrete peel and pulp-juice samples. Whenever desirable the flavedo and albedo components of the peel were separated with peeling tools, and each was pooled and analyzed. This method proved inadequate i n the parathion studies because of trace contami nations and the following procedure of sample preparation was adopted : Approximately 8 pounds of fruit were scrubbed manually i n warm 1 0 % trisodium phosphate solution. One hemisphere of peel was then removed from each fruit, using a household-type peeler. F r o m the pooled segments of peel a 1-pound subsample was used for processing. To obtain the pulp sample a circle of peel approximately 1.5 inches i n diameter was lifted from the center of the remaining hemisphere of peel on each fruit and a decontami nated N o . 15 cork borer with a serrated cutting edge was forced through the fruit antipodally i n such a manner as to avoid a l l flavedo peel. The albedo-bearing ends of each extracted plug of fruit pulp were carefully removed with the aid of a clean razor blade. After being pooled, a 1-pound subsample of the pulp was utilized for processing. The samples of citrus peel obtained b y either method may have contained trace amounts of extra-surface D D T residues as differentiated from subsurface or penetrated
130
residues (2, 18). Although difficult to establish experimentally the empirical evidence indicated that the washing of fruits with warm trisodium phosphate solution was nearly quantitative i n removing or degrading D D T extra-surface residues. The extra-surface residues of parathion shortly after application appear to be n i l ; the amounts obtained by stripping procedures apparently were extracted from the waxlike cuticle of fruits {2). I n the preparation of citrus pulp samples by this revised method the likelihood of contamination appeared to be eliminated providing the tools and work area were free of contaminants and the manipulations were carefully executed. However, the samples so prepared do not contain any of the pulp from the area immediately adjacent to the peel and to that extent are not totally representative. P o m e F r u i t Types. A s with citrus fruit types, the method of sample preparation was modified for the parathion studies. I n the earlier studies the D D T - t r e a t e d apples a n d pears were scrubbed i n a warm 1 0 % solution of trisodium phosphate, and all the peel was removed from the water-rinsed fruit with a household-type potato peeler. The pooled samples of peel and pulp were then processed independently to re cover the contained toxicant for subsequent estimation. I n the parathion studies and the more recent D D T studies, the fruits were split in half by cutting axially one third through the fruit with a broad-bladed knife and then twisting the blade to one side. The core and pulp, exclusive of the part touched by the knife blade, were carefully scraped out with sharpened melon bailers. As much of the pulp as possible was removed but in the event the skin was punctured the fruit was discarded and the scraping tools decontaminated. The pulp taken from the individual fruits of the sample was pooled for subsequent processing. Separate samples of fruit were used for surface residue estimations (12). Stone F r u i t Types. V e r y preliminary studies of D D T or parathion penetration into peach fruits were completed. T h e pulp samples were prepared b y immersing the intact fruits i n boiling water for 1 minute, slipping the skins off, rinsing thor oughly w i t h water and removing the seeds. Subsequent processing of the fruit components and extraction of the contained toxi cants have been described (11, 12, H, 15). Analyses for D D T residues have been made with the dehydrohalogenation method (11, 15). The magenta color reaction of Averell and Norris (1) as modified by Gunther and B l i n n (14) was used to analyze for parathion residues. Appropriate fruit blanks were run with each set of analyses. The results of all determinations are expressed as parts per million based on fresh weight of analyzed substrate. When dissection of fruits was involved i n the preparation of the samples, the values reported represent the parts per million of toxicant based on fresh weight of the indicated component only and not of the weight of the whole fruit. A s separated i n these studies the peel of most citrus fruits constitutes approximately one sixth the weight of the whole fruits.
Results
The summarized data presented in Tables I to V I I indicate the nature of the results which have been obtained in certain of these studies. It is not intended that these be i n terpreted as absolute values since cogent limitations exist not only i n the techniques of Table I. Immediate Posttreatment Residues of DDT in Peel of Citrus Fruits
(Sampled 48 hours after application) Technical Grade DDT/100 Gal., L b . 2.0 2.0 1.0 1.2 0.6 Navel oranges 5.6 13.9 3.9 17.1 11.8 .. D D T Valencia oranges 5.8 16.4 10.7 6.3 5.9 Eureka lemons 5.6 12.2 7.5 13.1 7.8
Type Formulation Wettable powder Kerosene solution Kerosene solution Light medium oil solution Light medium oil solution
Based on fresh weight of peel only.
CARMAN et /.ABSORPTION OF DDT AND PARATHION BY FRUITS
131
sampling and sample preparation but also i n the methods of recovery by extraction and i n the quantitative procedures of estimation. For example, in these studies the over-all efficiency of the D D T dehydrohalogenation method from sampling through calculated results appears to be approximately 9 0 % whereas that for parathion is probably not more than 6 0 % (14, 15). On the other hand, the data may help to characterize the nature and extent of the problem imposed by the use of these or similar materials and to indicate the relative effects of some of the factors i n penetration phenomena. Consistent with the definition of terms adopted for the discussion in this series of papers of integral phases of the residue studies being conducted by the Division of E n t o mology, University of California Citrus Experiment Station (2, 13-15), the following distinctions are noted: Residues may be specified as pretreatment, posttreatment, har vest, or ultimate. The latter refers to the residue on or i n foodstuffs, whether fresh or processed, at the time of consumption (2, 13). The location of residues with reference to fruit parts may be extra-surface (external to the cuticle) or subsurface. Subsurface residues may be differentiated with reference to actual location as cuticular residues or specified intracarp residues. Residues i n the cuticular layers or in any of the cellular structures or matrices are herein indicated as subsurface (penetrated) residues (2, 13). Citrus F r u i t s . T h e recovery of demonstrable amounts of D D T i n the peel of oranges and of lemons indicated the necessity for detailed studies w i t h regard to the effect of dosage, formulation, number of applications, method of application, fruit development at time of application, and other factors. W i t h i n the precision of the methods utilized D D T has never been recovered from the pulp portions of D D T - t r e a t e d citrus fruits. Results typical of the amounts found as subsurface D D T i n the composite peel of citrus fruits are collated in Tables I and I I . The infiltration of D D T into the peel was rapid, and it persisted there over long periods during the development and m a turing of the fruits. The magnitude and distribution of penetrated D D T residues were influenced by the specific nature of the formulation applied. Dissection of orange and lemon peel into subsamples of flavedo (outer peel) and albedo (inner peel) showed in subse quent analyses that in all cases most or all the D D T was present i n the flavedo. I n a limited number of instances relatively small amounts of D D T were recovered i n the albedo tissues, particularly when the fruits were sprayed with D D T formulations involving large amounts of kerosene or heavier fractions of petroleum oils. Table II. Harvest Residues of DDT in Peel of Citrus Fruits
(Averages for all samples) Fruit Development at Time of Last Application Approaching maturity Intermediate Post blossom -P.P.M.6 D D T 11.3 10.7 4.7 4.7 7.3 16.7 22.0 25.3 25.3 36.7
Spray Schedule , No. Applications

0
Valencia oranges 1 2 3 Navel oranges 1 2 3
12.7 5.3 13.3 12.7
All sprays were formulated by dissolving 2 pounds of technical grade D D T (8 grams D D T / 1 0 0 ml. solvent) in 3 gallons of kerosene-Velsicol AR-60 (95-5) and emulsifying the solution with 4 ounces of blood albumin spreader in 100 gallons of finished spray. b Based on fresh weight of peel only.
The use of D D T at application rates indicated i n Table I I represents the maximum dosage that has been considered for commercial use on citrus. The largest current uses of D D T on citrus involve single applications at the rate of 0.75 pound of technical grade compound per 100 gallons of spray mixture. The amount of D D T found in the peel of oranges harvested from 2 to 7 months after treatment with these dosages has ranged from 0.6 to 4.6 p.p.m., fresh weight of peel. Varietal differences with regard to the amount of D D T present i n the peel of har vested fruits sprayed at different times during the development of the fruit suggest the
132
possibility of determining i n subsequent investigations certain of those factors limiting the infiltration of D D T into fruit tissues (Table I I ) . Studies with parathion-treated citrus fruits have also shown no parathion to be present i n the endocarpal, or pulp segment, portions of the fruit (Tables I I I , I V , and V I ) . Since the magenta color reaction for parathion is extremely sensitive, i t is believed that even trace quantities are not present i n the pulp. The rapidity of the subsurface penetration of parathion into the peel of citrus fruits and the persistence of these residues i n the peel is demonstrated i n Tables I I I through V I I . Table III. Posttreatment Residues of Parathion in Citrus
(Sprayed with 4 pounds of 25% parathion wettable powder per 100 gallons water 4-26-48)
Sampled after, No. Days 1 2 4 8 10 14 17 53
- P . P . M . ParathionEureka Lemons Valencia Oranges Peel & Peel b Pulp Pulp

0 0 c
12.4 9.8 9.1 6.2 4.4 2.9 2.7 3.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
15.9 11.8 10.5 8.9 5.1 4.6 2.8
0.0 0.0 0.0 0.0 0.0 0.0
Valencia oranges approaching maturity and lemons mature when sprayed. b Based on fresh weight of peel only. Based on fresh weight of entire fruit.
c
Table IV.
Harvest Residues of Parathion in Valencia Oranges

105 Time after Application, Days 140 170 P . P . M . Parathion 2.1 4.6 4.5 0.0 0.0 0.0 2.0 3.9 4.3 0.0 0.0 0.0 230 &
Technical Grade Parathion/ 100 Gal., L b . Peel 0.5 0.75 1.0 Pulp<* 0.5 0.75 1.0
c
4.4 0.0 0.0 0.0
0.0
Applied October 28, 1947, as 25% wettable powder. b Fruit fully mature when sampled. Based on fresh weight of peel only. < * " Based on fresh weight of entire fruit.
c
Table V.
Harvest Residues of Parathion in Peel of Mature Citrus Fruit

(Average for all samples) Technical Grade Parathion per 100 Gal., L b . 0.25 0.5 0.75 1.0 1.25 P . P . M . b Parathion0
Variety Navel oranges Valencia oranges Eureka lemons Marsh grapefruit
0.125 0.1 0.1
1.5 8.1
1.7 0.7
2.5 1.9 2.1
4.2 3.5
5.2 4.3 2.6 4.9
6.4
Applied as 25% wettable powder. b Based on fresh weight of peel only.
Preliminary tests have shown that the amount of parathion penetrating into the peel of navel oranges is not markedly altered by drenching the tree with water sprays 1 day after treatment. Table I I I shows that much of the parathion initially present as subsurface residue is lost 10 to 20 days after treatment, possibly by reissuance and volatilization. Metabolism within the fruit tissues has not been indicated i n preliminary studies (see subsequent section). The parathion residues persisting i n the peel over long periods of time tend to remain at a somewhat constant level (Table I V ) . Thus the gradient of parathion residues i n peel with increased dosages shown i n Table V suggests that the initial depth and distribution of penetrated parathion may be dependent on the steepness of the concentration gradient which existed immediately after application (Tables I I I and I V ) . I n some instances slight varietal differences are indicated (Table V ) , but the de-
CARMAN et aiABSORPTION OF DDT AND PARATHION BY FRUITS
133
velopment of the fruit of each variety at the time of treatment is probably a more limiting factor (Table V I I ) . Since generally higher temperatures prevailed i n the field during the last week of August and the first week of September i n 1948 than during the corresponding period i n 1947 the somewhat higher penetration values reported i n samples taken at that time i n 1948 (Table V I ) m a y be a consequence of generally higher fruit temperature. tors m a y be operative. Other fac
Table VI.
Harvest Residues of Parathion in Mature Navel Oranges Technical Grade Parathion/100 . Gal., L b . 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.05 0.09 0.09 0.18 0.98
(Sampled during January and February of year following time of application) Parathion Formulations Date of Application 8-16-48 9-30-48 11-11-48 8-13-48 8-13-48 8-20-47 8-24-48 8-26-48 8-30-48 9-10-48 9-10-48 9-30-48 8-13-48 8-19-48 8-24-48 8-29-47 9- 2-48 9- 3-48 9-10-48 9-16-48 9-22-48 9-30-48 10-11-48 10-22-48 10-28-48 11-11-48 11-11-48 6-15-48 8-24-48 8-26-47 9- 3-48 9- 3-48 10- 9-47 10-15-48 10-22-48 9- 9-48 9- 9-48 9-11-48 9- 9-48 9-14-48 P . P . M . Parathion Peel Pulp*
a
25% wettable powder
In light medium oil 0.25% 0.5% 0.5% 1% Technical grade compound emulsified Based on fresh weight of peel only.
1.6 2.1 1.4 2.0 2.2 1.0 3.0 3.7 2.3 2.5 2.5 3.3 3.5 3.7 6.5 2.0 5.8 5.8 6.7 4.5 4.1 3.3 4.6 4.1 2.3 4.2 3.8 1.1 7.5 4.0 6.1 6.4 2.7 6.4 4.7 0.3 0.7 0.8 1.3 10.5
0.0 0.0 0.0 0.0
b.b 0.0 0.0 o.b
b.b o.b 0.0 0.0 b.b 0.0 0.0 0.0 0.0 0.0 0.0 0.0
& Based on fresh weight of entire fruit.
Table VII.
Posttreatment and Harvest Residues of Parathion in Peel of Citrus FruitsEffect of Seasonal and Multiple Applications (All sprays with 4 pounds of 25% parathion wettable powder per 100 gallons water) Application Dates 8-17-48 Date Sampled 11-19-48 1- 11-49 2- 11-49 11-19-48 1- 11-49 2- 11-49 11-19-48 1- 11-49 2- 11-49 11-19-48 1- 11-49 2- 11-49 11-19-48 1- 11-49 2- 11-49 Navel oranges 1.3 1.1 0.7 11.7 6.8 4.9 13.5 17.7 10.6 13.6 13.8 10.4 12.2 7.7 6.4 P . P . M . Parathion& Valencia Eureka oranges lemons 0.5 0.4 0.4 8.3 8.8 9.8 20.4 18.8 17.0 26.6 18 9.5 8.4 9.1 4.0 1.0 0.5 7.5 6.2 5.2 13.1 9.0 6.4 13.3 6.9 5.8 1.6 2.7
6-14-48* Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed
10-29-48
Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed
Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed Sprayed
Fruits small when sprayed.
& Based on fresh weight of peel only.
134
The importance of formulation on the penetration of parathion into the peel of citrus fruit is indicated i n Table V I , the emulsified technical grade compound entering i n great est amount. This suggests the possibility of being able to formulate parathion in a man ner which might effectively prevent the penetration of the compound into citrus peel but an attendant difficulty might be that its insecticidal effectiveness would be lessened. P o m e a n d Stone F r u i t s . Following the application of D D T and of parathion as wettable powders i n control schedules, neither compound has been recovered from the pulp portions of apples, pears, and peaches. A m a x i m u m of six spray applications of D D T wettable powder and four spray applications of parathion wettable powder were involved i n these studies. As high as 0.4 p.p.m. of D D T and 1.7 p.p.m. of D D T were found in the pulp of apple and pear fruits, respectively, following sea sonal treatments with five to six applications of D D T formulated in a petroleum oil frac tion.
Nature of Persisting Subsurface Residues

A n exploratory spectrophotometric examination of subsurface extractants obtained from fully ripe Rome Beauty apples, previously sprayed with a deliberate overdosage of a parathion wettable powder, revealed a shift i n the absorption maximum of the dyed product to the right of that found for the dyed technical parathion. Since the composition of both the dyed technical parathion and the dyed carefully purified parathion has not been elucidated, it was not possible to attempt an interpretation
6000
400
220 240 260 280 300 WAVELENGTH IN mp
320
500 WAVELENGTH
IN
600 mji
700
Figure 1. Transmission-Wave-Length Curves for Colorless Benzene Extractives from Peel of DDT-Treated Navel Oranges
sample Untreated sample A . For comparison purposes ultraviolet ab sorption spectrum of carefully purified p,p'-DDT, in ethyl alcohol, has been plotted as upper solid line with different ordinate
Figure 2. Transmission-Wave-Length Curves for Dyed Benzene Extractives from Peel of Parathion-Treated Navel Oranges
sample Sample A ; dyed para thion ; in 6 0 % ethyl alcohol, pH 1.2. Instru ment was set at 100% transmission prior to each transmission reading with untreated sample; numbers refer to X x.
ma
CARMAN ef al ABSORPTION OF DDT AND PARATHION BY FRUITS
135
of this displacement toward longer wave lengths. Present information indicates that there may exist at least three and possibly four colored components in each mixture. Because of continued interest i n the i n situ degradation products of both D D T and parathion, preliminary spectrophotometric evidences, which are also indicative of struc tural alterations and/or changes i n composition, have been secured with extracts of navel oranges previously treated with standard formulations of these compounds. Approximately 5 months prior to sampling of fruits the trees were sprayed with the equivalent of 2 pounds of technical grade D D T per 100 gallons i n an emulsified kerosene solution or with the equivalent of 0.75 pound of technical grade parathion per 100 gallons in a wettable powder preparation. W i t h the duplicate D D T - t r e a t e d samples and one untreated sample, the benzene ex tracts were evaporated and the residue redissolved in ethyl alcohol and evaporated again. This procedure was repeated several times to remove the last traces of benzene. The final residue was dissolved in low-boiling (30 to 60 C.) petroleum ether and chromatographed on a column of activated alumina which afforded a colorless eluate. After removal of solvent the resulting residue was triturated with boiling ethyl alcohol, cooled, filtered, and spectrographed i n a Beckman spectrophotometer. The resulting transmission-wave-length curves are shown in Figure 1. The duplicate samples show markedly different absorption characteristics from those of the untreated sample,-particularly i n the region 225 to 260 . Further comparisons with the absorption spectrum of carefully purified ,'- in ethyl alcohol demonstrate complete absence of fine structure in the region 260 to 280 for the D D T - t r e a t e d samples. F r o m the noncoincidence of the sample maxima with those of the D D T curve i t may be concluded that very little if any D D T is left in the samples but that fragments of the original D D T molecule may be present. W i t h the duplicate parathion-treated samples and one untreated sample, the ben zene extracts were processed in the usual manner and subjected to the dyeing procedure. Their transmission-wave-length curves are shown in Figure 2 with a companion curve for dyed parathion. W i t h reference to the curve for dyed parathion, shifts toward longer wave lengths in all maxima and minima are apparent with the greatest displacement in the region 400 to 440 . The intensities of absorption change proportions between sample and reference curves. The fact that the transmission-wave-length curve of dyed parathion is composite pre cludes the possibility of interpreting these observations at the present time. These displacements were considered sufficiently interesting to warrant the develop ment of more exact techniques to elucidate the nature of these persisting subsurface resi dues. Such studies are 'currently in progress.
Acknowledgment
The authors are indebted to the American Cyanamid Company and the Monsanto Chemical Company for supplying the parathion materials used i n these studies.
Literature Cited
(1) Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753-6 (1948). (2) Barnes, M . M . , Carman, G. E . , Ewart, W. H . , and Gunther, F. ., ADVANCES I N C H E M I S T R Y S E R I E S , 1, 112 (1950). (3) Borden, A. D., Hoskins, W. M . , and Fulmer, . H . , Blue Anchor, 24, 19 (1947). (4) Carter, R. H . , J. Assoc. Offic. Agr. Chemists, 30, 456 (1947). (5) Carter, R. H . , and Hubanks, P. E., Ibid., 29, 112 (1946). (6) Draize, J. H . , Woodard, G., Fitzhugh, O. G., Nelson, . ., Smith, R. B., and Calvery, H . O., Chem. Eng. News, 22, 1503 (1944). (7) Ebeling, W., J. Econ. Entomol., 38, 689 (1945). (8) Fahey, J . E., and Rusk, H . W., J. Assoc.Offic.Agr. Chemists, 30, 349 (1947). (9) Fleck, . E., Ibid., 30, 319 (1947). (10) Frear, D . . H . , and Cox, J . ., Food Packer, 27, 64, 78 (1946). (11) Gunther, F . ., Hilgardia, 18, 297 (1948). (12) Gunther, F . ., Univ. Calif. Citrus Expt. Sta., rept. (February 1948).
136
(13)

Gunther, F . ., Barnes, M . M . , and Carman, G . E . , A D V A N C E S I N CHEMISTRY SERIES, 1, 137
(14) (15) (16) (17) (18) (19) (20) (21) (22)
(1950). Gunther, F . ., and Blinn, R. C., Ibid., 1, 72 (1950). Gunther, F . ., and Miller, M . E., Ibid., 1, 88 (1950). Hough, W. S., Virginia Fruit, 33, 1 (1945); 34, 128 (1946). Manalo, E . D., Hutson, R., and Benne, . J . , Canning Trade, 68, 9, 22 (1946). Neal, P. ., Soap Sanit. Chemicals, 21, 99 (1945). Reiber, H . G., and Stafford, . M . , Calif. Agr. Expt. Sta., Circ. 365, 104 (1946). Smith, M . I., and Stohlman, E . F . , U. S. Pub. Health Repts., 59, 984 (1944). Ibid., 60, 289 (1945). Wickman, H . J . , Patterson, W. I., Clifford, P. ., Klein, A . K . , and Claborn, . V.,J.Assoc. Offic. Agr. Chemists, 29, 188 (1946). 625, University of California Citrus Experiment Station, Riverside, Calif.
PAPER
Removal of DDT and Parathion Residues from Apples, Pears, Lemons, and Oranges
F. A. GUNTHER, M. M. BARNES, and G . E. C A R M A N University of California Citrus Experiment Station, Riverside, Calif.
Alkaline and halogen-carrier media, such as sodium silicate, trisodium phosphate, ferric chloride, sodium carbonate and bicarbonate, and alkaline soaps were used for chemical removal of DDT deposits. Me chanical removal was sought by using emulsifying agents, detergents, pressure sprays, and scrubbing brushes. Solvent removal attempts included the use of kerosene, mineral oil, xylene, and polymethylated naphthalenes. With apples and pears, sodium sili cate frequently proved superior, removing as much as 90% of the residual surface DDT, and an alkaline soap effected significant removal from oranges. No experimental treatment has afforded significant re moval of parathion from treated fruits.
T h e widespread commercial use of D D T and the anticipated use of parathion indicated the practical value of investigating the feasibility of removing their harvest residues without injury to the fruits. A distinction has been drawn (1) between "harvest" and " u l t i m a t e " residues. The former term is being defined as the surface or penetrated residue of insecticide at the time of harvest, whereas the latter designation refers to the residues i n or on the foodstuff, whether fresh or processed, at the time of consumption. Thus, there exist harvest sur face and penetration or harvest total residues, as well as ultimate surface, penetration, and total residues. Where necessary, component parts of the foodstuff m a y be specified to delimit this concept further. Begun i n 1944 with D D T and i n 1947 with parathion, the present report includes analytical data secured from certain chemical, mechanical, and solvent actions on apples, pears, lemons, and oranges. I n the absence of established tolerances for these two insecticidal materials, i t is hardly possible to interpret the significance of many of these data with respect to consumer hazard. Although there are a number of published reports of the magnitude of [harvest] D D T residues, particularly on apples (4, 5, 7, 8, 14, 15, 18, 19), grapes (), oranges (10), and pears (2, 5,18), and of terminal residues i n canned products (15,17), only a few publica tions have been concerned either with the nature of the residual deposits (6, 7,11,16) or with their removal (2,14,18) prior to consumption. Hough (14), for example, reported that 1.3% hydrochloric acid solution, and dilute solutions of sodium silicate, trisodium phosphate, IN-181-P (a powder containing 5 0 % sodium lauryl sulfate, as supplied b y . I . d u Pont de Nemours & Company, Inc., Wilmington, Del.), and a Santomerse a l l failed to remove readily the D D T deposits on mature apples. (The Santomerses are the
137
138
salts of a homologous series of substituted aromatic sulfonic acids, supplied b y the M o n santo Chemical Company, St. Louis, M o . ) Similarly, Borden et al. (2) recorded the conclusion that D D T applied w i t h spray oils is not removed easily. So far as the authors are aware, there have been no published results of attempts to remove parathion residues from harvested fruits. Because D D T is readily dehydrohalogenatable, chemical expedition of deposit removal was sought through the use of alkaline and halogen-carrier media, such as sodium silicate, trisodium phosphate, ferric chloride, sodium carbonate and bicarbonate, and strong soaps. Mechanical removal was sought through the use of a variety of emulsifying agents, detergents, pressure sprays, and scrubbing brushes. Solvent removal attempts included the use of kerosene, mineral oil, xylene, and a mixture of polymethylated naphthalenes. F o r purposes of coherence and clarity, methods and results with apples and pears are presented separately from those with oranges.
Methods with Apples and Pears

Apples. T h e Rome Beauty apples used i n the wash tests were sampled from trees that had received varying amounts of D D T mixtures i n as many as six cover sprays. Duplicate or triplicate samples of 30 apples each were taken at random for the residue analyses from the fruit passed through each experimental wash mixture. Additional lots of 30 washed apples each were placed i n cold storage for subsequent examinations. U n less otherwise indicated, all washing tests were r u n i n a flood-type washer of recent design (a B A D D washer with a heated prewash tank unit, a n unheated main tank unit, a water rinse tank unit, and a velour roller dryer unit, manufactured b y the Bean-Cutler Division, Food Machinery Corporation, San Jose, Calif.). Surface deposits of D D T were determined as described (10, 12) on samples taken just before and immediately after the washing treatments. Pears. Triplicate samples of 20 Bartlett pears each were used and a fourth sample was placed i n cold storage for subsequent examination, as with the apples. Washing trials were performed i n a standard type washer (an Ideal fruit washer with an unheated main tank unit and a water rinse tank unit, M o d e l W K A , manufactured b y the Ideal Grader and Nursery Company, H o o d River, Ore.).
Results with Apples and Pears

D D T on Apples. E x p l o r a t o r y investigations i n 1944 w i t h the flood-type tandem washer indicated t h a t D D T surface residues of from 3.3 to 9.5 p.p.m. could readily be reduced 82 to 9 8 % b y a number of materials, b o t h alone and i n combination. These materials included sodium silicate solution (70 to 82 pounds of the 58.5 Baume solution per 100 gallons), hydrochloric acid (1.25%), ferric chloride solution (1 ounce per 100 gallons), sodium hypochlorite solution (3 pints of 4 % per 100 gallons), M e r m a i d Soap (a proprietary, buffered sodium salt of the fatty acids i n cottonseed oil foots, as supplied by the Los Angeles Soap Company, Los Angeles, Calif.) (4 pounds per 100 gallons), t r i sodium phosphate (4 pounds per 100 gallons), and IN-181-P (1 ounce per 100 gallons). Solution temperatures ranged from 50 to 110 F . i n a variety of wash sequences. A l though combinations of IN-181-P washes and sodium silicate washes appeared to be the most efficient, all the washes reduced the residues appreciably. I n some instances, slight visible deposits remained i n the blossom and stem ends following the washing treatment. Washing tests subsequent to 1944 were designed to evaluate more precisely the efficiency of the sodium silicate solution, but other materials were considered sufficiently promising to be included. I n Table I are summarized the data for the sodium silicate washes, at 80 pounds per 100 gallons, with water only i n the main tank, for a variety of types of spray deposits. I n practically all cases the residual D D T was reduced to less than 1 p.p.m. fresh weight. However, some of the apples used i n the 1944 tests carried as high as 9.5 p.p.m. of D D T as surface residue, whereas subsequent deposits prior to washing averaged about 2 p.p.m. F r o m these and supplemental tests i t would seem that such small harvest residues of D D T
GUNTHER, BARNES, AND
CARMANREMOVAL OF
DDT
AND
PARATHION RESIDUES
139
can readily be reduced by at least 5 0 % with a number of washing materials including so dium silicate (80 pounds per 100 gallons, 65 to 100 F . ) , trisodium phosphate (4 pounds per 100 gallons, 108 F . ) , and IN-181-P (1 ounce per 100 gallons, 65 F . ) . Tennessee ball clay was the only extending material which when i n composition with D D T appeared to interfere appreciably with the removal of D D T surface residues by sodium silicate washes. None of these washing treatments occasioned apparent fruit injury or decreased storage life. D D T on Pears. E a r l y experiments indicated t h a t the conventional hydrochloric a c i d b a t h , as used for the r e m o v a l of lead arsenate residues, afforded p a r t i a l D D T residue r e m o v a l b y v i r t u e of mechanical action o n l y f o r example, a surface residue of 1.0 p . p . m . was reduced to 0.6 p . p . m . b y such treatment, but the residue i n the calyx o n l y was untouched (15 p.p.m., fresh weight of calyx o n l y ) . Supplemental wash tests w i t h other materials afforded the residue data collected i n T a b l e I I . Table I. Removal of DDT Surface Residues from Rome Beauty Apples with Sodium Silicate Washes
Dosage Spray Treatments 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. D D T (c.p.)-amorphou8 silica (35-65) D D T (aerosol)-amorphous silica (40-60) D D T (tech.)-amorphous silica (50-50) D D T (tech.)-amorphous silica (25-75) D D T (tech.)-amorphous silica (121/2-871/2) D D T (tech.)-amorphous silica (50-50), remilled D D T (tech.)-calcium silicate (50-50) D D T (tech.)-ball clay (50-50) D D T (tech.)-kaolin clay (50-50) Gesarol A K - 4 0
a
Sprays, Lb. 1.4 1.4 1.4 2.8 5.6 1.4 1.4 1.4 1.4 1.4
D D T Surface Residues on Fruits, P . P . M . Wet Weight Washed Unwashed 80 F . 100 F . 0.9 2.0 1.7 1.8 2.0 2.0 3.1 1.6 2.7 1.7 1.9 0.5 0.7 0.1 0.4 0.6 0.2 0.6 0.2 0.2 1.2 0.6 0.6 0.5
D D T Surface Residue Removed 80 F . 100 F . 75 59 85 65 87 38 59 76 68 89 80 65 89 70 90 94 25 78 65 75
0.3
0.7 0.4 1.0 1.1 0.4 0.6
Av.
Wettable powder with 40%
DDT.
Table II.
Wash Tests for Removal of DDT Surface Residues from Bartlett Pears
Materials Lb./100 Gal.
e
D D T Surface Residues, P . P . M . & Unwashed Washed 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 1.9 3.3 2.3 2.4 2.4 2.8 3.2 3.0 2.7 2.7 2.6 2.8
'Pi
Residue Removed 74 54 68 67 67 61 56 58 62 62 64 61
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
1.
Trisodium phosphate Trisodium phosphate Triton X - 1 0 0 IN-181-P Ivory soap Mermaid soap Sodium silicate Ferric chloride C.P. Hydrochloric acid Mineral seal oil Triton X-100 Treatment 9 followed by 7 Xylene Triton X-100 Treatment 11 followed by 7
c
16 8 1.67 ounces 1 ounce 2 6 80 4 ounces 5 quarts 1 gal. 0.5 ounce 2 quarts 0.5 ounce
....
Fruits exposed to wash for approximately 20 seconds. & Each figure represents average of 3 analyses, except 1, 5, and 9 where figure represents one analysis only. Liquid, anhydrous nonionic emulsifier, Rohm & Haas Co., Philadelphia, Pa.
The xylene emulsion wash caused injury i n the lenticel areas of the fruits. Fruits washed i n the mineral seal oil emulsion retained a considerable oil deposit even when washed subsequently with sodium silicate and exhibited considerable shrinkage while i n storage. Further wash tests with apples and pears have not been extensive because the magni tudes of typical D D T harvest residues suggest that no appreciable difficulty will be en countered i n bringing fruits sprayed with the lower dosages under the provisional toler ance for D D T residues on these fruits. Parathion on Apples and Pears. A l t h o u g h parathion deposits appear to be less
140
persistent t h a n those f r o m D D T , the ready sorption of p a r a t h i o n b y certain fruit metabolites indicates t h a t late seasonal a p p l i c a t i o n on early harvested varieties of apples a n d pears could result i n measurable amounts of p a r a t h i o n as ultimate resi dues. Washing tests for these trials were made i n a flood-type washer with one wash and one rinse chamber. T h e results presented i n Tables I I I and I V suggest that parathion residues (from wettable powders) are not readily removed with the materials and equipment e m ployed; support is thus secured for the authors* contention that with many substrates parathion residues are actually subsurface and exist i n situ i n close association with the oily and waxy constituents of the plants. Of these materials, kerosene and mineral oil decreased the storage life of the fruits. Table III. Wash Tests for Removal of Parathion Residues from Rome Beauty Apples*
Materials Trisodium phosphate Velsicol A R - 6 0 (0.1% Triton X-100) Mineral seal oil Blood albumin spreader Triton X-100 IN-181-P* Kerosene Blood albumin spreader
e t
Gal./100 Gal. 61b. 1 1 1 ounce 1 ounce 3 ounces 1 1 ounce
Parathion in Total Fruit&, P . P . M . Unwashed Washed 2.5 2.5 2.5 1.0 '2.5 2.5 2.5 1.5 1.5 1.7 0.8 1.9 2.0 2.4
% Removed 40 40 32 20 24 20 4
Temperature of wash water 50 F . wash 25 seconds, rinse 10 seconds. b Entire fruits macerated for analysis representing total parathion. Mixture of polymethylated naphthalenes. Added in maximum amount without excessive foaming.
c
Table IV.
Wash Tests for Removal of Parathion Residues from Bartlett Pears and? Rome Beauty Apples*'*
Parathion Surface Residues Series l Series 2 Series 3/ Bartlett Bartlett Green Rome Pears Pears Beauty Apples Re Re Re moved, moved, moved,. P.p.m. P.p.m. P.p.m. % % % 0.07 0.32 0.27 15 0.23 0.03 57 0.30 7 0.04 43 0.25 0.28 12 0.04 43 0.17 37 0.30 6 0.21 22 57 0.03 0.33 15 57 0.03 0.23 0.30 26 0.03 57 0.20 57 0.21 22 0.03 120.28 22" 0.04 43 0.25 0.25 7 60.04 43 33 0.30 0.18
0 d e
Material No treatment (unwashed) Trisodium phosphate Trisodium phosphate Santomerse 1 Trisodium phosphate Trisodium phosphate Santomerse 1 Sodium triphosphate Sodium acid pyrophosphate Sodium acid pyrophosphate Tetraeodium pyrophosphate Tetraeodium pyrophosphate Tetrasodium pyrophosphate Santomerse 1 Trie odium phosphate Triton X-100 Tetraeodium pyrophosphate Tetrasodium pyrophosphate Santomerse 1 Sodium silicate
Lb./100 Gal. 8 J 2 ounces?J 16 16 ) 2 ounces/ 8 8

8
Approxi mate p H >io >10 >10 >10 9 4 7 9 9 >10 10 10 >10
4
4
\ 36 m l . /
8
2 ounces/
\ \
8
8
2 ounces/ 80
0.23 0.22 0.28 0.20
15 19 26
0.03 0.04 0.05 0.03
57 43 29 57
0.22 0.24 0.27 0.21
31 25 16 34
Temperature of wash water 68 F . , wash 35 seconds, rinse 25 seconds. & Surface residue values reported to second decimal largely because of close agreement between duplicateanalyses and because amount of parathion in each sample was within range for maximum accuracy of analytical method. Therefore, ratios reported retain possible significance. Each figure represents average of 2 analyses. Fruits of this series sprayed with 4 ounces of actual parathion per 100 gallons specifically for this test. * Fruits of this series sprayed with 2 ounces of actual parathion per 100 gallons July 18 and Aug. 10, sampled! Aug. 26. / Fruits of this series sampled after fourth cover of 4 ounces of actual parathion per 100 gallons. Maximum amount without excessive foaming.
d
Methods with Lemons and Oranges

D D T . W i t h both the Eureka lemons and the Valencia oranges, trees were sprayed i n a conventional manner with conventional high pressure equipment, and with the follow ing mixtures:
GUNTHER, BARNES, AND C A R M A N R E M O V A L
O F DDT AND PARATHION RESIDUES
141
I . T e n pounds of A K - 2 0 (a wettable powder with 2 0 % D D T ) and 4 ounces of a casein spreader per 100 gallons of water. I I . A light-medium oil containing 4 grams of technical grade D D T per 100 m l . used at 1.67%, plus 4 ounces of a blood albumin spreader, emulsified i n 100 gallons of water. I I I . A kerosene containing 4 grams of technical grade D D T per 100 m l . used at 3 % , plus 4 ounces of a blood albumin spreader, emulsified i n 100 gallons of water. Three field boxes of each fruit for each treatment were picked at random from the treated trees 2 days after spraying. One box of each sample was stripped with the washbottle technique and the D D T obtained thereby was estimated b y the dehydrohaiogenation method (10). T h e remaining boxes of treated fruit were subjected to the normal citrus packinghouse treatments, which include a thorough machine scrubbing with M e r maid soap (at Ontario, Calif., by-products plant of the California F r u i t Growers E x change). After having been so processed, these fruits were stripped and analyzed as before. Parathion. T h e available evidence suggests t h a t parathion is soluble i n m a n y p l a n t oils a n d waxes. W i t h citrus fruits, i t is clear t h a t t o p i c a l l y applied parathion preparations q u i c k l y lose a part of their parathion essentially b y a transfer into the surface waxes a n d then into the o i l gland containing tissues of the fruits. F o r example, samples stripped either b y the wash-bottle technique (10) or b y a machine technique (18) 24 hours after treatment always exhibit little or no parathion, whereas total peel values for the same fruits may be relatively high i n parathion content (see also 1,8). O i l expressed from the peel of parathion-treated oranges may contain from 65 to 9 0 % of the parathion originally present i n that peel. Prescrubbing of treated citrus fruits with warm 1 0 % trisodium phosphate solution does not alter appreciably the amount of parathion obtainable b y surface stripping (10). I t is concluded that there is no true measurable extrasurface residue of parathion on fruits such as citrus shortly after application, and that surface stripping, b y either laving or brief steeping techniques, merely extracts some of the insecticide from subsurface regions. T h e fact that citrus waxes are completely miscible with benzene lends credence to the postulate that the parathion does not linger i n the wax layers but migrates rapidly through the cuticle. T h i s migration and a t tendant further redistribution of subsurface parathion are under investigation. I t would seem, therefore, that particularly with o i l - and wax-soluble insecticides the older concepts of surface residues on plant tissues should be revised i n terms of extrasurfacei.e., above the cuticleand subsurfacei.e., within or below the cuticle residues. The latter would i n turn be subdivided into cuticular residues and various i n t r a carp residues.
Results with Lemons and Oranges

D D T . I n T a b l e V are shown the results of attempts to remove D D T surface residues f r o m treated lemons and oranges b y the standard packinghouse processing. F r o m these data i t is apparent t h a t such D D T surface residues are readily removable. T h e ultimate fates of penetrated D D T (8) or field-decomposed surface residues of D D T (1,12) have not been determined, but work along these lines is being continued. Table V. Removal of DDT Surface Residues from Lemons and Oranges with Standard Packinghouse Processing
Fruit Lemons Treatment* I II III I II III D D T Surface Residue before Processing P.p.m. 7 / s q . cm.
6
D D T Surface Residue after Processing P.p.m. 7 &/sq. cm. 0.4 0.5 1.9 0.0 0.0 0.3 0.4 0.5 1.9 0.0 0.0 0.3
12.5 1.0 4.6 9.1 2.5 2.3
19.3 1.4 4.8 8.4 2.1 2.0
Oranges
See text for explanation of field treatments. Each value represents average of duplicate determinations.
142
Parathion. A t present i t has not been found possible to remove p a r a t h i o n f r o m the peel of citrus fruits.
Acknowledgment
Besides companies specifically mentioned i n the text, the authors are indebted for donations of materials to the American Cyanamid Company, the Geigy Company, and t h e Pennsylvania Salt Manufacturing Company. T h e y also wish to express indebtedness to W . E . Baier, of the California F r u i t Growers Exchange, for arranging to process the D D T treated lemons and oranges, and to M . E l l i o t M i l l e r and R . C . B l i n n of these laboratories for most of the analytical work..
Summary
Because D D T is readily dehydrohalogenatable, chemical expedition of deposit r e moval was sought through the use of alkaline and halogen-carrier media, such as sodium s i l i cate, trisodium phosphate, ferric chloride, sodium carbonate and bicarbonate, alkaline soaps, and others. Mechanical removal was sought by using a variety of emulsifying agents, detergents, pressure sprays, and scrubbing brushes. Solvent removal attempts included the use of kerosene, mineral oil, xylene, and polymethylated naphthalenes. W i t h apples and pears, sodium silicate frequently proved superior, removing i n some cases 9 0 % of the residual surface D D T , and a n alkaline soap (standard packinghouse treatment) effected significant removal from oranges. None of the experimental treatments has afforded significant removal of parathion from treated fruits. A satisfactory chemical attack on parathion residues has not been achieved, possibly because these residues are actually subsurface.
Literature Cited
(1) Barnes,M.M.,Carman, G. E., Ewart, W. H . , and Gunther, F .A.,ADVANCES IN CHEMISTRY (2) Borden, A. D., Hoskins, W. M . , and Fulmer, . H . , The Blue Anchor, 24, 19 (1947). (3) Carman, G. E . , Ewart, W. H., Barnes, M . M . , and Gunther, F. ., ADVANCES IN CHEMISTRY (4) (5) (6) (7) (8) (9) (10) (11) (12) (14) (15) (16) (17) (18) (19) Carter, R. H., J. Assoc. Offic. Agr. Chemists, 30, 456 (1947). Carter, R. H., and Hubanks, P.E.,Ibid., 29, 112 (1946). Ebeling, W., J. Econ. Entomol., 38, 689 (1945); 40, 628 (1947). Fahey, J. E . , and Rusk, H . W., J. Assoc. Offic. Agr. Chemists, 30, 349 (1947). Fleck, . E . , Ibid., 30, 319 (1947). Frear, D. . H . , and Cox, J. ., Food Packer, 27, 64, 78 (1946). Gunther, F. ., Hilgardia, 18, 297 (1948). Gunther, F. ., J. Econ. Entomol., 41, 895 (1948). Gunther, F. ., Mimeo, Univ. of Calif. Citrus Experiment Station, February 1948. Hough, W. S., Virginia Fruit, 33, 1 (1945); 34, 128 (1946). Manalo, G. D., Hutson, R., and Benne, . J., Canning Trade, 68, 9, 22 (1946). Parkin, . ., and Green, . ., Nature, 155, 668 (1945). Tressler, C. J . , J. Assoc. Offic. Agr. Chemists, 30, 140 (1947). Walker, K . C., Proc. Am. Soc. Hort. Sci., 51, 85 (1948). Wichmann, H . J . , Patterson, W. I., Clifford, P. ., Klein, A . K., and Claborn, . V., J. Assoc. Offic. Agr. Chemists, 29, 188 (1946).
SERIES , 1, 128 (1950). SERIES , 1, 112 (1950).
(13) Gunther, F . ., and Blinn, R. C., ADVANCES IN CHEMISTRY SERIES , 1, 72 (1950).
PAPER 623, University of California Citrus Experiment Station, Riverside, Calif.
Synthesis and Development of Parathion and Related Compounds

J. T. CASSADAY, JOHN H. FLETCHER, J. C. HAMILTON , INGENUIN HECHENBLEIKNER, . I . HOEGBERG, B. J. SERTL, and J. T. THURSTON
1
American Cyanamid Company, Stamford, Conn.
The investigations carried out and the many problems solved in the synthesis and development of parathion and related compounds are reviewed. Equations are given to illustrate the procedures used in this work.
P a r a t h i o n is the generic name of a compound that was first prepared b y German chem ists and designated as E-605. I n this country many trade names have been used. I n 1946 a report b y the British technical investigators M a r t i n and Shaw (6) was released i n the United States, disclosing among other insecticides a compound called p-nitrophenoxy thiophosphoric acid diethyl ester. A s a result of a publication b y M a s t i n , Norman, and Weilmuenster (7) i n 1945, i n which the name diethyl chlorothionophosphate was used, the compound as first prepared b y United States chemists was designated as diethyl pnitrophenyl thionophosphate.
S
(C H Q) P Q<(
2 5 2
^>NQ
(1)
Whether an arrow or a double bond is used between the phosphorus and sulfur is a much-discussed question. B o t h are probably correct. In view of the large amount of field testing that was being carried out during 1947 and the possibilities of publications, i t was desirable to have a common name as well as an approved chemical name for this product. A s a result of meetings with members of the AMERICAN CHEMICAL SOCIETY'S Committee on Nomenclature, Spelling, and P r o nunciation the chemical name 0,0-diethyl O-p-nitrophenyl thiophosphate was adopted. B y a series of similar conferences with members of the U . S. Department of Agriculture (in particular Haller and Rohwer), the name " p a r a t h i o n " was approved. Those par ticipating i n the selection of this common name proposed by Rohwer and concurring i n its suitability included committees representing many scientific societies.
Physical Properties
Physical Properties of Parathion
Color Melting point, C. Boiling point, C. n ' d
0
27
Pale yellow 6 . 1 0 . 1 corrected 157-162 at 0.6 mm. 1.53668 1.263
The technical material has a brown color, a refractive index that may differ slightly i n the third place, and a density that is approximately the same as that of the pure material. Parathion is soluble i n water to the extent of about 20 to 25 p.p.m. I t is completely
1
Present address, Yale University, New Haven, Conn.
143
144
miscible with many organic solvents, including esters, alcohols, ketones, ethers, aromatic and alkylated aromatic hydrocarbons, and animal and vegetable oils. I t is very slightly soluble i n paraffinic hydrocarbons such as petroleum ether, kerosene, and refined spray oils. The compound is very slowly hydrolyzed i n water. Hydrolysis rates were re ported i n August 1948 by Peck and co-workers (9), who stated that 5 0 % hydrolysis occurs at 25 C . after 120 days i n saturated distilled water solutions or i n 1 sulfuric acid. The time required to reach 5 0 % hydrolysis i n a saturated lime solution is reduced to 8 hours. Hence parathion appears to be very stable to hydrolysis except under alkaline conditions.
Toxicology
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch028 Studies on the pharmacological effect of parathion i n animals have been i n progress for almost 2 years at the Hazleton Laboratory, Falls Church, V a . (4). Some phases of the work, such as long-range feeding tests, are continuing. Tests have been carried out on toxicity by dermal application, inhalation of dust, exposure to aerosol mists, exposure to vapors, and chronic toxicity. Test animals have included rabbits, guinea pigs, rats, mice, and dogs. Acute Oral Toxicity of Parathion
Form of Administration Albino rats Albino mice Guinea pigs Propylene glycol solution Wettable powder in water Propylene glycol solution Wettable powder in water Propylene glycol solution Mean Lethal Dose, Mg./Kg. 5.0 12.5 6.0 21.0 9.3
Schrader (12) stated that i n its pure form, Bladan is ten times as toxic to mammals as E-605 (parathion). ' ' B l a d a n " is the name given by the Germans to a material which they thought to be hexaethyl tetraphosphate. Actually, this material is a mixture, the insecticidally active ingredient of which is tetraethyl pyrophosphate. Tests to determine the long-range effect on animals that ingest a sublethal dose of parathion have been i n progress for more than 18 months. Although definite conclusions cannot be drawn at this time, average food consumption and weight gains are comparable to the controls. I n all these tests the amount of material remaining on the food was over 50 times that anticipated i n residual quantities on food for consumption by humans. Experimental and commercial experience with formulations containing parathion applied as sprays or dusts has covered two seasons. I n a few cases where protective measures were not taken, temporary nausea, headache, or other effects were experienced when individuals were sub jected to unusual exposure to vapors, dusts, or mists. I t is believed that chronic toxicity hazards are greater from D D T than from parathion, even though the acute oral mean lethal dose of D D T is 250 mg. per k g .
Preparation of Parathion and Its Intermediates

PC1 + S > PSCI3
3
(2)
II
2 C H O N a + PSC1 > ( C H 0 ) P C l + 2NaCl
2 5 3 2 6 2
(3)
S
2 5 2 6 4 2 2 5 2
S
6 4 2
(C H 0) PCl + NaOC H N0 > (C H 0) POC H N0 + NaCl (4) Equations 2, 3, and 4 summarize the method proposed by the Germans for preparing parathion (compound E-605). Schrader (18) has reported that thiophosphoryl chloride was synthesized from phosphorus trichloride and sulfur by heating at 130 i n a lead-lined autoclave. Woodstock and Adler (14) carried out a similar reaction at 150 to 160 C .
CASSADAY et /.PARATHION AND RELATED COMPOUNDS
145
using alkali metal sulfides as catalysts, and were able to reduce the pressures that were normally necessary for the reaction. A s shown i n Equation 3, the alcoholic sodium ethoxide was combined with thiophosphoryl chloride at temperatures of 5 to 10 C , followed by removal of the sodium chloride b y filtration. The final step was carried out i n chlorobenzene during a 15-hour heating period. If this method of preparing diethyl chlorothiophosphate is used, other by-products may be formed which will enter into the final reaction unless the desired chloro ester is very carefully fractionated. Other methods of preparing thiophosphoryl chloride have been reported (10). PC1 + H S > PSC1 + 2HC1
5 2 3
(5)
3
P C l . 2 F e C l + 5S > 2FeCl + 2S C1 + PSC1

5 3 2 2 2
(6) (7)
F e P + 7SC1 > 2FeCl + PSC1 + 6S

2 2 3
In E q u a t i o n 6, a method is shown b y which, Woodstock and M c D o n a l d (15) report, thiophosphoryl chloride can be obtained. E q u a t i o n 7 shows a related method (16) for the preparation of this intermediate. Glatzel (8) has reacted iron and antimony chlorides with phosphorus pentasulfide to obtain the desired compound.
6FeCl
3
+ 2P S >
2 5 3
3FeCl
+ 4PSC1 + 3FeS
3 4
(8) (9) (10)
SbCl + P2S5 > SbPS + PSCls 3PCU + P2S5 > 5PSC1

3
Weber (8) states that the intermediate thiophosphoryl chloride can be obtained from phosphorus pentachloride, as shown i n E q u a t i o n 10. 5P0C1 + P S > P 0 + 5 P S a
3 2 6 2 6 3
(11) (12) ( 13)
6SOCI2 + 2P2S5
4 2
> 4PSC1 + 3 S 0 + 9S
3 2 5 3 2
3CC1 + 2P S > 4PSC1 + 3CS
Carius (8) replaced the oxygen i n phosphoryl chloride b y sulfur. This m a y be somewhat analogous to the replacement of oxygen i n a carbonyl group b y sulfur using phosphorus pentasulfide. Prinz (8) used thionyl chloride i n his reaction with phos phorus sulfide and D e F a z i (2) used carbon tetrachloride i n a very interesting preparation of this important intermediate. Methods of Synthesizing Diethyl Chlorothiophosphate. I n 1861 Carius (1) re ported Equations 14 to 17. /PS Oi/PS 0 l ( C H ) 3 + PCU > C1\(C H ) +
3 2 5 2 5 2
CI3PO
+ C1C H
2
(14)
S ( C H 0 ) P S + PCU > ( C H 0 ) P C l +
2 5 3 2 6 2 2 3 2 5 2 6 2 5
POCI3
+ C H C1
2 5 3
(15) (16)
TPS 0 /PS 0 t ( C H ) M e + PC1 > C1\(C H )2 + CIMe + C l P O S ]

2 5 2 2 5 2
s
POCI3
_ ( C H 0 ) P O J M e + PCI5 > ( C H 0 ) P - -Cl + M e C l +
(17)
The authors' interpretation of the reactions which he carried out is given immediately under the equation taken from the original article. I n 1945, M a s t i n , Norman, and W e i l muenster (7) reported the preparation of the compound as shown i n E q u a t i o n 18. S 2C H OH +
2 5
2 C 5 H 5 N + PSCI3
> (C H 0) PCI + 2C H N.HC1

2 5 2 6 6
(18)
146
T h e yield obtained was approximately 2 4 % . M u c h higher yields were probably obtained by Schrader's process (12) and b y Equations 19 to 27. S S
Il
2 5 2 2 2 5
II
2 2 2
2 ( C H 0 ) P S N a + 3C1 > 2 ( C H 0 ) P C l + 2NaCl + S C1 S

2 5 2 5 2 5 2
(19)
S
3
( C H 0 ) P S N a + PC1 > ( C H 0 ) P C l + PSC1 (?) + N a C l S

2 5 2 2 2 5 2
(20)
S
2 2
2 ( C H 0 ) P S H + 3C1 > 2 ( C H 0 ) P C l + 2HC1 + S C1
(21)
When chlorine reacted with sodium diethyl dithiophosphate i n the presence of an organic solvent, the desired compound was obtained in good yield. T h e reaction of phosphorus pentachloride on the sodium salt resulted i n the same product. Thiophos phoryl chloride is a probable by-product of this reaction. It was later found that diethyl dithiophosphoric acid could be chlorinated in an organic solvent without the intermediate preparation of the sodium salt. T h e purity and identity of diethyl chlorothiophosphate prepared b y new methods were checked b y reaction with phenylhydrazine according to the directions of M a s t i n , Norman, and Weilmuenster (7). S (C H 0) PCl + H NNH<^
2 5 2 2 2 5
S ^> > ( C H Q ) P H N N H < ^

2
^> + HC1
(22)
A general equation for the chlorination of certain dithiophosphoric acids and their salts can be written as follows: S
2 2 2
S
2 2
2 ( R O ) P S M + 3C1 > 2 ( R O ) P C l + 2MC1 + S C1 R = C H , C H , C H , iso-C H , C H , iso-C H , C H

3 2 5 3 7 3 7 4 9 4 9 6 5
(23)
= H , , N a
Using this general method, the chloro esters shown i n E q u a t i o n 23 were made. This method of preparation is a rather important discovery and the reaction is not a simple one. There are numerous methods of preparing the desired intermediate, which can be combined with sodium nitrophenoxide. S
2 5 2 6 4 2 2 5 2
S
6 4 2
(C H 0) PCl + NaOC H N0 > (C H 0) POC H N0 S S I! Base | | (C H 0) PCl + HOC H N0 > (C H 0) POC H N0

2 5 2 6 4 2 2 5 2 6 4
+ NaCl
(24)
+ HC1
(25)
T h e reaction shown in Equation 24 gives good yields, using both aqueous and non aqueous media, in a much shorter time than that reported b y Schrader (13). T h e inter mediate chloro ester can be combined directly with paranitrophenol using alkali metal salts in order to prepare sodium nitrophenoxide, which is used in situ. Another method which has been proposed for the preparation of parathion is shown in Equations 26 to 28. PC1 + N a O C H N 0 > C 1 P O C H N 0 + N a C l
3 6 4 2 2 6 4 2
(26) (27)
C1 POC H N0
2 6 4
+ 2NaOC H > (C H 0) POC H N0

2 6 2 5 2 6 4
+ 2NaCl
CASSADAY et al.PARATHION
AND RELATED C O M P O U N D S
147
S (C H 0) POC H N0
2 5 2 6 4 2
+ S
> (C H 0) POC H N0
2 5 2 6 4
(28)
It has been reported that the compound resulting from Equation 26 may be hazardous to distill (12). However, the compound resulting from Equation 27 has been prepared and attempts have been made to add sulfur to this phosphite i n order to obtain the desired thiophosphate. Similar reactions have been reported in the literature (3). A very vigorous reaction takes place. Another method is shown in Equations 29 and 30. S PSC1 + N a O C H N 0
3 6 4 2
^ C 1 P O C H N 0 + NaCl
2 6 4 2
(29)
's
C1 POC H N0
2 6 4 2
S
+ 2NaOC H
2 5
> ~(C H 0) POC H N0

2 5 2 6 4
+ 2NaCl
(30)
S (C H 0) PS + N a O C H N 0
2 5 3 6 4 2
>- ( C H 0 ) P O C H N 0
2 5 2 6 4
+ NaOC H
2
(31)
Still another method was reported b y Schrader (12) (Equation 31). Compounds prepared b y the general method of reaction of the chloro esters with the nitrophenol, either in the presence of base or b y using a salt of the phenol, are shown in Formula 32. S (RO ) POC H N0 -p
2 6 4 2
( 32 )
4 9 4 9 6 6
R = C H , C H , n - C H , i s o - C H , n - C H , iso-C H , C H
3 2 5 3 7 3 7
A l l these compounds could be prepared by this method. C H 0

2 5
^P^-OCeH.NO,-?
(33)
S (C H;0) POC H
2 2 6 5
(34)
S (C H 0) POC H CH -p (35) In Formula 33 is pictured a completely mixed ester of a thiophosphate in which all groups are different. T h e thiophosphoryl chloride method was used for preparing such compounds. It was of interest to see whether or not other compounds could be prepared b y the condensation of a dialkyl chlorothiophosphate with a phenol to give products such as shown in Formulas 34 to 36. S
2 5 2 6 4 3
(C H 0 ) POR
2 5 2
(36)
2
R =
< ^
V n H , <^~^>CH , <f^^>CI,
2 3
N0
N0
<f
V ~COOC2H5
T h e first compound of Equation 36 is not made b y the condensation procedure, but by the chemical reduction of a nitro group in parathion. Some compounds are rather difficult to obtain b y the general method. However, the next three compounds shown here were prepared b y this method.
American Chemical Society Library 1155 16th St, H.W. Washington, D.C. 20038
148
Oxygen Analogs of Parathion

Because the Germans had been interested i n the oxygen analog of parathion, it was of interest to see whether or not their claims were correct and what other oxygen ana logs might be made. In Equations 37 and 38, a convenient method is shown for pre paring diethyl chlorophosphate. T h i s method was reported b y the British investigators M c C o m b i e , Saunders, and Stacey () i n 1945. 3 C H O H + PCI,
2 5
( C H 0 ) P O H + C H C1 + 2HC1
2 5 2 2 5
(37)
(C H 0) POH + Cl
2 5 2 2
( C H 0 ) P C l + HC1
2 5 2
(38)
Diethyl chlorophosphate can also be prepared from ethyl alcohol and phosphoryl chloride i n the presence of base, but the method shown is reported to give better yields. T h e intermediate diethyl chlorophosphate has been condensed with nitrophenol i n a manner similar to that used for parathion (Equation 39). Base e o j *OC H N0 -CI + H O C H N 0 > ( C H ,
6 4 2 2 6 4
(C H 0) :
2 5 2
+ HC1
(39)
(C H 0) POC H
2 6 2 6 6
+ HNO3 (C H 0) POCeH N0
2 5 2 4 2
+ H 0
2
(40)
(C H 0) POCeH N0
2 5 2 4 2
+ HNO3
(G
O C e H N 0 -h ?
4 2
(41)
Because phenol is cheaper than nitrophenol, it might cost less to condense phenol with this chloro intermediate, followed b y nitration with nitric acid. T h i s method of preparing the oxygen analog can be used as shown i n Equation 40. Still another method of preparation is shown i n Equation 41. Formulas 42 to 44 give examples of phosphate esters related to parathion and pre pared b y the above methods. In some of these compounds the ethyl group has been changed, whereas i n others various substituents have been introduced into the benzene ring. A number of the structures shown have been reported b y the Germans (11-18). (RpJQ<^
2
^>NQ
4
(42)
R C H6, C3H7,7i-C H9 ( C H 0 ) P Q<^

2 6 2 3 2 2
^>R
2
(43)
R = H, CH , S0 NH , N 0 (C H 0) POR
2 5 2
II
(44) COOC H
2 5
R = CI -NO2
CASSADAY ef /.PARATHION AND RELATED COMPOUNDS
149
A large number of problems were encountered and a great many scientists were i n volved i n the development of parathion. Problems i n synthesis, engineering, and analy sis, as well as formulating and testing procedures were all solved. T h e compound is effective on a large number of crops and insects, and data sheets can be obtained giving recommended uses and precautions to be taken i n handling the material. I n spite of the fact that parathion is such an amazing insecticide, it cannot replace all other insecticides. A s a matter of fact, i t can be used beneficially with a number of other insecticides and fungicides. Excellent cooperation has been received from the United States Department of A g r i culture and state agricultural experiment stations i n carrying out field tests with this material. Without such cooperation this important economic poison would not be i n general lise at this time. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch028
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) Carius, Ann., 119, 289 (1861). De Fazi, Remo, Atti II Congresso naz. chim. pura applicata, 1926, 1293-4. Foss, Acta Chem. Scand., 1, 12 (1947). Hazleton and Holland, ADVANCEMENT IN CHEMISTRY SERIES, 1, 31 (1950). McCombie, Saunders, and Stacey, J. Chem. Soc., 1945, 380. Martin and Shaw, British Intelligence Objectives Sub-committee, BIOS Final Rept. 1095, Item 22 (1946). Mastin, Norman, and Weilmuenster, J. Am. Chem. Soc., 67, 1662 (1945). Mellor, "Comprehensive Treatise on Inorganic and Theoretical Chemistry," Vol. VIII, p. 1056, New York, Longmans, Green and Co., 1928. Peck, D . R., Chemistry and Industry, 1948, No. 33, 526. Plets, J. Gen. Chem. (U.S.S.R.), 8, 1296 (1938). Schrader, BIOS Final Rept. 714 (revised) (1947). Ibid., 1808 (1948). Thurston, U . S. Dept. Commerce, Office of Technical Services, FIAT Final Rept. 949 (PB -60890). Woodstock and Adler, J. Am. Chem. Soc., 54, 464 (1932). Woodstock and McDonald, U . S. Patent 1,811,602 (1931). Ibid., 1,848,813 (1932).
Organic Phosphorus Insecticides

S. A. HALL
In the field of organic phosphorus compounds there is a wealth of highly toxic compounds from which to pick a potential insecticide. The ultimate choice will be based not only on toxicity to a certain group of insect species, but on volatility, stability, safety in handling and applying, and freedom from plant injury, spray-residue and translocation hazards, and long-term toxicity to man and animals.
T h e new field of organic phosphorus insecticides was opened up during World W a r I I by Gerhard Schrader, a German chemist employed at the Elberfeld laboratories of I . G . Farbenindustrie (37, 38). Schrader, who had done earlier work on insecticides, was engaged primarily i n the search for chemical warfare agents. I n the preface to Schrader's report (38) M u m f o r d and Perren make the following statement regarding the organic phosphorus insecticides described i n the report: It is known that other substances of similar general type are still more toxic to higher animals, and i t is quite possible that workers undertaking synthetic studies i n this series may prepare a substance sufficiently toxic to constitute a real danger to themselves and others i n the vicinity. It is therefore recommended that stringent safety precautions should be taken when preparing and testing any compound of this type, unless i t has already been shown by appropriate tests to be comparatively harmless to higher animals. F r o m this statement i t may be inferred that Schrader's insecticides merged into toxic warfare agents or vice versa, depending on his point of approach. It is estimated from available reports (32, 38, 40, 44> 45) that Schrader synthesized well over 300 compounds containing organically bound phosphorus. Although the reports are notably lacking in completeness, i t appears that most of Schrader's compounds were screened for biological activity against aphids (species not given). Approximately 150 compounds tested as a spray at 0.2% concentration gave 90 to 100% mortalities of the aphids. Apparently some of his data are missing, or else only a portion of the compounds active at 0.2% were tested at lower concentrations. However, i t can be estimated that about 1 0 % of the compounds active at 0.2% continued to give 100% mortalities when diluted to 0.002%. T h i s will convey some idea of the extraordinary biological activity associated with this series of compounds. There is little evidence of any particular compound i n Schrader's series possessing a selective toxicity to insects. I n general (although there may be an exception), the compounds that are highly toxic to insects are also highly toxic to warm-blooded animals. The toxic organic phosphorus compounds act as powerful inhibitors of cholinesterase, an enzyme found predominantly i n the nervous tissue of animals, including insects. This enzyme hydrolyzes acetylcholine, which plays an essential role i n the transmission of nerve impulses. The toxicity of compounds i n this series can be largely accounted for on the basis of their anticholinesterase activity (7, 8,12,14, SI).
1 5 0
HALLORGANIC PHOSPHORUS INSECTICIDES
151
A t about the time Schrader was synthesizing new organic phosphorus compounds of extraordinary toxicity, M c C o m b i e and Saunders (29) i n England were intensively en gaged i n a similar endeavor. They investigated especially the dialkyl fluophosphates. Mackworth and Webb (31), who tested these compounds, found them to be highly potent inhibitors of horse serum cholinesterase. The most active ester, diisopropyl fluophosphate, which British investigators call diisopropyl fluorophosphonate ( D F P ) , was 30 times as active as the alkaloid eserine. I n their series of fluophosphates they found a correlation between the i n vitro inhibitory power of cholinesterase and toxicity. Webb (48) also found that the inhibitory effects of the alkyl fluophosphates are not confined to the enzyme cholinesterase. They may poison a range of esterases, some of which have no activity at all toward acetylcholine as a substrate. The inhibition b y fluophosphates is similar to that reported earlier b y Bloch and Hottinger () for tri-o-cresyl phosphate and tri-o-chlorophenyl phosphate. D u B o i s and M a n g u n (14) found that hexaethyl tetraphosphate exerts a strong inhibitory effect on mammalian and insect cholinesterase i n vitro and i n vivo. I t is of interest that hexa ethyl tetraphosphate and its principal active ingredient, tetraethyl pyrophosphate, were found b y Jansen et al. (25) to inhibit the enzyme acetylesterase, which is of plant origin. Brauer (7) has studied the cholinesterase-inhibiting activities of a series of organophosphorus compounds. H e has proposed a rule for the structural requirements of an organophosphorus inhibitor and also a mechanism for its inhibiting effect on the enzyme. D u Bois and associates (13) found parathion to be a strong inhibitor of cholinesterase i n rats. Nachmanson and co-workers (34) have pointed out that the quantity of cholinesterase present i n most organisms may vary considerably i n different species and even i n different tissues of the same species. Their studies indicate that an organisman insect, for exampledoes not begin to show toxic effects until 66 to 9 5 % of the enzyme has been inactivated. There also appears to be a very small differential between a nonlethal and a Table I. Properties of Organic Phosphorus Insecticides
Effectiveness" Concn. Kill of spray Density of soin., Solubility at aphids, Remarks and References 25 C . in Water % % Pale yellow, almost 100 Slight 1.2655 0.001 odorless oil; crys (15-20 p.p.m.) tallizes in long nee dles, m.p. 6 C . Technical product may have garliclike odor (2, 3, 17,19, 46) Practically odorless, 0.005 100 1.269* Slight reddish-yellow oil (sp. gr.) (0.25%) (4, 10, 38) 50 0.001 Odorless, colorless, 100 0.05 1.1810/ Completely hygroscopic oil. miscible Hydrolyzes rapidly. 1. 19010 Technical products (sp. gr. called T E P P and at 24) H E T P (10, 15, 21,
c
Compound Parathion
Boiling Point, C.
0
Refrac tive Index at 25 C . 1.5370

e
157-1626 0.6 mm.
Oxygen ana log of para thion Tetraethvl pyrophos phate
148-151* 1 mm. 173/1 m m . 104-110/ 0.08 mm. 135-1380 1 mm.
1.5060* 1.4170/ 1.41800
23, 60)
24,
37, 89,
47, 49,
Tetraethyl dithiopyrophosphate Tetraethyl monothiopyrophosphate Octamethyl pyrophosphoramide
110-113* 0.2 mm. 135/2 mm.* 110-117/ 0.3 mm. 118-122/ 0.3 mm.
1.4753*
1.196*
Slight
0.005
100
1.4466/
1.1833/
Slight (0.06%) Completely miscible
0.005
100
Yellow to colorless oil. Technical product may have unpleas ant odor (4, 22, 38) Colorless oil of u n pleasant odor (10,
22, 38)
1.4612/
1.1343/
0.05
100
Almost odorless, color less, somewhat vis cous oil. Systemic poison. Absorbed by living plant (10, 11,
86, 38)
Schrader's screening tests (88). b Fletcher et al. (18). Edwards and Hall (17). * Adler, Victor Chemical Works, Chicago, 111.
a c
* Schrader (38). f Hall et al. (22, 24). a Toy (47).
152
lethal dose. Their experiments with diisopropyl fluophosphate demonstrated that its toxicity must be attributed exclusively to the inactivation of cholinesterase. Because toxicity to insects is only one criterion of a potential insecticide, no attempt is made here to examine all of Schrader's toxic compounds. M u m f o r d and Perren (88) have indicated that some of these compounds may not be described for reasons of security. Only those compounds containing organically bound phosphorus which have already shown some promise of joining the family of insecticides are described here. A l l are highboiling liquids which, i n general, undergo thermal decomposition and hence must be dis tilled at reduced pressure. A s a further generalization the P - O - P linkage of these com pounds undergoes hydrolytic cleavage (more readily at alkaline p H ) , which results i n loss of effectiveness of the compound. Some properties of the compounds are listed i n Table I .
Parathion Parathion is the accepted trivial name for 0,0-diethyl O-p-nitrophenyl thiophos-
Q N-<(
2
y-
OC H
2
OC H
2
Parathion phate, which at the present time is the most prominent of the organic phosphorus i n secticides. I n contrast to the tetraethyl pyrophosphate type of insecticide, which h y drolyzes completely i n a day or two, parathion is reasonably stable. I t is the first of Schrader's stable anticholinesterases to be developed as an insecticide on a commercial scale. Its popularity may be due to this fact or to the possibility that parathion may actually possess a certain combination of desirable properties not shared b y any other compound i n Schrader's series. I t was first described as E-605 by Schrader (82,88,44,4&) who prepared i t as follows: S
II
PSCU + 2 C H O N a > (C H 0) PC1 + 2NaCl

2 5 2 6 2
(1)
S
2 6 6 4 2 2 6 2
S
6 4 2
(C H 0)JPC1 + N a O C H N 0 > (C H 0) PO C H N0 + N a C l (2) Chlorobenzene was the solvent preferred by the Germans for carrying out the reaction in step 2. The foregoing reaction scheme had been described i n a patent issued i n 1934 to Clemmensen (9), who prepared certain organic esters of thiophosphoric acid embodying both aromatic and aliphatic radicals. These esters are claimed as new compositions of matter useful as fire retardants. Parathion itself is not specifically described i n th patent. Fletcher et al. (18) have given details of its laboratory preparation by Schrader's method, i n which the yield i n step 1 was 5 0 % . I n step 2, with ethyl alcohol used as the solvent, a yield of 7 5 % of the vacuum-distilled ester was obtained. Water was also tried as the solvent in step 2, and the yield was 6 4 % of crude undistilled parathion. Its preparation on a larger laboratory scale has been described in Australian patents (2) of the American Cyanamid Company. H i g h yields of the crude ester were obtained i n step 2, in which various solvents, especially ketones, were successfully employed. Also described is a modification of step 2 i n which the diethyl chlorothiophosphate reacts with p-nitrophenol i n the presence of anhydrous sodium carbonate or other suitable alkali. N o directions are given i n the patents for preparing the diethyl chlorothiophosphate or other dialkyl chlorothiophosphates used to prepare analogs of parathion. Thurston (46) has recently reviewed the methods for preparing dialkyl chlorothiophosphates.
153
The Australian patents also contain descriptions of analogs, which include nitrophenyl thiophosphates possessing more than one nitro group. M a n y of the analogs are biologically active, and several exhibit high activities comparable with parathion. T h e o-nitrophenyl isomer, which is present to some extent i n crude parathion, is substantially less active than the p-nitrophenyl isomer (parathion itself). These patents disclose a synergistic effect of the ortho isomer with parathion. Pure parathion is a pale yellow, practically odorless oil, which crystallizes i n long white needles melting at 6.0 C . (17). I t is soluble i n organic solvents, except kerosenes of low aromatic content, and is only slightly soluble i n water (15 to 20 p.p.m. at 20 to 25 C ) . Peck (85) measured its rate of hydrolysis to diethyl thiophosphate and nitrophenate ions i n alkaline solutions. H e found that the reaction kinetics are first order with respect to the ester and to hydroxyl ion. I n normal sulfuric acid the rate of hydroly sis was the same as i n distilled water. Peck concluded that hydrolysis takes place b y two mechanismsa reaction catalyzed by hydroxyl ions and an independent uncatalyzed reaction with water. H e calculated that at a p H below 10 the time for 5 0 % hydrolysis at 25 C . is 120 days; i n the presence of saturated lime water the time is 8 hours. T h e over-all velocity constant at 25 C. is k = 0.047 [OH~] + 4 1 0 " m i n . " Averell and Norris (3) have developed an analytical method adapted to the deter mination of parathion i n spray or dust residues, which is sensitive to about 20 micrograms. I t is based upon the reduction of parathion with zinc to the amino compound, diazotization, and coupling with Bratton and Marshall's amine, which gives an intense magenta color with an absorption peak at 555 millimicrons. Bowen and Edwards (6) have used the polarograph to assay technical grades of parathion and its formulations. Parathion is coming into extensive use. Applied at concentrations ranging from about 25 to 600 p.p.m., it has been found highly effective against an imposing list of insect species (19, 46). The scope of safe application of this powerful insecticidal compound is not yet defined. If its high toxicity (13, 28) to man and animals does not stand i n the way of its use on food and fodder crops, it may indeed have a very wide field of application. The answer to the question of its ultimate usefulness must await the outcome of many large-scale field tests to cover all aspects of the spray-residue problem, including possible translocation (20) of the compound.
6 1
Oxygen Analog of Parathion

D i e t h y l p-nitrophenyl phosphate was designated b y Schrader (38) as E-600, the O C A
OC H
2
Oxygen analog of parathion immediate forerunner of his E-605, parathion. I n Germany, where the two esters were first compared for insecticidal use, parathion was preferred (32, 44) because E-605 was less poisonous to man and not so readily absorbed through the skin. D u B o i s and associ ates (18) have found the oxygen analog more toxic than parathion to rats. Schrader prepared the ester (88) i n 6 0 % yield b y reaction of sodium p-nitrophenate with diethyl chlorophosphate, using xylene as solvent for the reaction. H e made i t , but i n lower yields, from p-nitrophenol and diethyl chlorophosphate, using, respectively, pyridine and sodium cyanide as acceptors for hydrogen chloride. Schrader also pre pared i t i n 9 6 % yield b y nitrating diethyl phenyl phosphate at 0 C . or below. Under the conditions he used, Schrader claims that the nitro group is directed to the para posi tion. N o yield is given for the diethyl phenyl phosphate, which he presumably made from sodium phenate and diethyl chlorophosphate. Diethyl chlorophosphate may be prepared i n high yield (30) from diethyl phosphite and chlorine. D i e t h y l p-nitrophenyl phosphate (E-600) is an odorless reddish-yellow oil which
154
possesses physical properties and solubility characteristics similar to parathion. Its solubility (22) i n water is about 0.25% at 25 C . Schrader (38) dissolved 0.5 gram of the ester " b y vigorous shaking i n 10 liters of water." The solution, i t is reported, was sprayed with complete effectiveness on a cineraria plant infested with aphids (species not given). The statement is made i n one of the British B I O S reports (32) that E-600 but not E-605 is absorbed to a slight degree by the living plant. N o data are given. Coates (10) has calculated that the ester is about 300 times more stable to hydrolysis than tetraethyl pyrophospate. H i s value for the over-all velocity constant is = 0.52 [OH~] + 1 X 10~ m i n . B a l l and Allen (4) have found that diethyl p-nitrophenyl phosphate is the most effective of the organic phosphates against the housefly, milkweed bug, cock roach, and two species of aphids. The Bureau of Entomology and Plant Quarantine in preliminary tests has found it very effective against the European corn borer, armyworm, celery leaf tier, large milkweed bug, pea aphid, and two-spotted spider mite. The colorimetric method of Avereli and Norris (3) for estimation of parathion is also applicable to the oxygen analog, which gives a magenta color of identical absorption peak (16). The polarographic method of Bowen and Edwards (6) is also applicable to the analysis of this ester.
6 - 1
Tetraethyl Pyrophosphate Insecticides

The first of Schrader's organic phosphorus anticholinesterases to find rather largescale application as an insecticide i n Germany during World War I I was his so-called hexaethyl tetraphosphate (37), which the Germans called Bladan. Schrader obtained German and United States patents (39) on this material, and ascribed to i t a branchedchain structure. H a l l and Jacobson (24) found that i t is a mixture of ethyl polyphos-
(C H 0) P
2 5 2
(C H 0) P o H o) 0) :P (c H
2 5 2 2 55 22
!!
P=0 /
Schrader's hexaethyl tetraphosphate (structure hypothetical) phates containing as its principal active ingredient the compound tetraethyl pyrophos phate. Hexaethyl tetraphosphate was first made (89) by reaction of 3 moles of triethyl C H 0
2 5
OC H
2
\!l
/
C H 0
2 5
PO
11/
\
OC H
2 5
Tetraethyl pyrophosphate phosphate with 1 mole of phosphorus oxychloride (Equation 3); the reaction was sub sequently modified by increasing the molar ratio of triethyl phosphate to phosphorus oxychloride (Equation 4). Another process, patented by Woodstock (49), i n which phos phoric anhydride was used instead of phosphorus oxychloride is illustrated i n Equations 5 and 6. P O C l Procedure.
3
3(C H ) P0 +
2 5 3 4
POCI3 POCI3
> (C H ) P40
2 5 6 2 5 2
13
+ 3C H C1
2 5 2 5
(3) (4)
5(C H )3P04 +
2 5
> 3(C H )4P 0 + 3C H C1

7
155
P 0 Procedure.
2 5
2(C H ) P0
2 5 3
+ P2O5 > ( C H ) P 4 0
2 5 6
13
(5)
7
4(C H ) P0
2 5 3
+ P 0
2
3(C H ) P20
2 5 4
(6)
I n all cases the reaction products are mixtures of ethyl polyphosphates, and, on the basis of elementary analysis, they approximate the empirical formulas given i n the above equations. I n Equations 3 and 5 the product has been arbitrarily called hexaethyl tetra phosphate, which may contain 8 to 2 0 % of the active tetraethyl pyrophosphate. I n Equations 4 and 6 the products have been called technical tetraethyl pyrophosphate, which may contain up to 4 0 % of pure tetraethyl pyrophosphate. Hexaethyl tetra phosphate has also been made from phosphoric anhydride and diethyl ether b y a process recently patented b y Adler (1). Kosolapoff (26, 27) has proposed a mechanism for the reactions based upon addition of phosphoryl chloride or phosphorus pentoxide to the P O bond of triethyl phosphate to give intermediate phosphonium-type adducts which may form linear and cyclic poly esters b y thermal decomposition. H e has advanced the view that the actual tetraethyl pyrophosphate content of these complex mixtures may be very small and that the bio logically active ester may be generated from the cyclic polyphosphates b y partial h y drolysis. This theory may find some support i n the entomological experiments of Smith, F u l t o n , and L u n g (48) with tetraethyl pyrophosphate insecticides in methyl chloride applied as aerosols i n greenhouses. They found that, although the k i l l of spider mites correlated well with the tetraethyl pyrophosphate content of their samples, such was not the case with aphid mortalities. T h e hexaethyl tetraphosphate manufactured b y Schrader's original process was found more effective against aphids, suggesting that it contains toxic principles other than tetraethyl pyrophosphate. Smith and F u l t o n pointed out that with their mode of testing, the ethyl polyphosphate mixtures are i n an anhydrous condition until they leave the nozzle at the time of application. T h e aerosols thus differ from spray solutions, i n which at least some of the toxicity is lost by hydrolysis when the ethyl polyphosphates are dissolved i n water before application. Because aerosol application i n greenhouses is responsible for a large part of the production of this insecticide, there has recently been a trend back to the manufacture of hexaethyl tetraphosphate as originally produced about 3 years ago. The purified tetraethyl pyrophosphate is a colorless, odorless, water-soluble, hygro scopic liquid (24, 4?)- I t possesses a very high acute toxicity (28), exceeding that of parathion, and is rapidly absorbed through the skin. There is no spray-residue problem, however, for tetraethyl pyrophosphate hydrolyzes even i n the absence of alkali to non toxic diethyl phosphoric acid. H a l l and Jacobson (24) and T o y (47) have measured its rate of hydrolysis, which is a first-order reaction. Its half-life at 25 C. is 6.8 hours and at 38 C. is 3.3 hours. Coates (10) determined the over-all velocity constant at 25 C : k = 160 [ O H - ] + 1.6 " m i n . " T o y (47) has described an elegant method for preparing this ester as well as other tetraalkyl pyrophosphates, based upon the controlled hydrolysis of 2 moles of dialkyl chlorophosphate :
3 1
2 5 2 2 2 5 2
2 5 2
2 ( C H 0 ) P 0 C 1 + H 0 > ( C H 0 ) P O P ( O C H ) + 2HC1
The hydrogen chloride is removed either b y reduced pressure or b y salt formation with pyridine or sodium bicarbonate; the latter procedure gave high yields of the pure ester. T o y (47) also measured the hydrolysis rates and compared the toxicities of a series of tetraalkyl pyrophosphates. Of these tested, the tetraethyl ester was the most toxic to white mice. Several chemical-assay methods (15,23,50) for tetraethyl pyrophosphate were recently developed and applied b y seven collaborating laboratories to samples of representative commercial products and to a sample of purified tetraethyl pyrophosphate which served as a common standard. Concordant results, which correlated well with bioassay results,
156
were obtained. The methods used (21) are based upon the selective hydrolysis of the sample followed by separation of the tetraethyl pyrophosphate from acidic constituents. The methods differ only i n techniques of separation or hydrolysis. The ultimate usefulness of technical tetraethyl pyrophosphate depends largely on its rapid killing action, its compatibility with sulfur, and its freedom from residual toxicity hazards. O n the other hand, i t is not profitably employed where a certain amount of residual toxicity is desirable or necessary to k i l l a particular insect species. Tetraethyl pyrophosphate has found wide use as an aerosol to control pests on greenhouse vege tables and flower crops. Because i t leaves no residual toxic vapors, i t offers a definite advantage from the standpoint of safety to the greenhouse operator. I n addition, there is evidence that its use i n greenhouses has a stimulatory action on plant growth, particu larly on rose plants, which have produced ' 'larger leaves with a deeper green color, longer and heavier stems, and more flowers" (42).
Thio Analogs of Tetraethyl Pyrophosphate

Tetraethyl dithiopyrophosphate has recently been made available i n experimental
0
2
OC H
2
M l
C H 0
2 6 / /
1 1 /
\>0
2
Tetraethyl dithiopyrophosphate quantities and has been tested more widely than the tetraethyl monothiopyrophosphate.
C H 0
2 5
OC H
2
M l
/
C H 0
2 5
I ! /
\
OC H
2 5
Tetraethyl monothiopyrophosphate Schrader (38) i n a brief description characterized tetraethyl pyrophosphate as "not completely water-stable," the monothio analog as "water-stable," and the dithio analog as "lime-stable." The monothio analog was prepared (22) i n 6 7 % yield as follows : O
2 6 2
S
2 6 2 2
S
2 2
( C H 0 ) P C 1 + K O J > ( O C H ) >
(C HO) P(00 )
+ KC1
As a solvent for this reaction, anhydrous methyl ethyl ketone was found satisfactory. Coates (10) determined the rate of hydrolysis of the monothio analog as approximately one fifth that of tetraethyl pyrophosphate under similar conditions. T h e dithio analog has been prepared (22) i n 9 0 % yield from diethyl chlorothiophosphate, water, and p y r i dine in a modification of the reaction T o y (47) used to make tetraethyl pyrophosphate : S II 2(C H 0) PC1 + H 0
2 5 2 2
Pyridine
2 5 2
S II
S II
2 5 2 5 5
> ( C H 0 ) P O P ( O C H ) + 2C H N.HC1 at room temp. B a l l and Allen (4) found that the dithio analog compared favorably with tetraethyl pyrophosphate. Other preliminary tests i n the bureau have indicated that the dithio analog is very effective against the European corn borer, large milkweed bug, celery leaf tier, army worm, and two-spotted spider mite. Smith (4I) has tested both analogs as aerosols i n greenhouses. I t is too early to assess the value of these analogs, but they may fit into the insecticide picture as compounds of intermediate stability to hydrolysis, lying somewhere between tetraethyl pyrophosphate and parathion, i n the region of tox-
157
icity desirable for the control of insects but not of sufficient duration to constitute a hazard to man.
Octamethyl Pyrophosphoramide
Toxic compounds that can be absorbed to a marked degree b y a living plant through either its roots or its leaves have been called by British investigators systemic insecticides. Schrader (38) first found this peculiar property i n certain acetals of 2-fluoroethanol and bis-(2-fluoroethoxy)methane, as well as i n certain compounds of his organic phosphorus series, notably bis(dimethylamido)fluophosphate and octamethyl pyrophosphoramide. (CH ) N
3 2
O N(CH )
3
(CH ) N N(CH ) Octamethyl pyrophosphoramide

3 2 3 2
The latter compound has the practical advantage over the former of not containing fluorine, which might on repeated application accumulate i n the soil and poison i t . Schrader (38) prepared the pyrophosphoramide as follows : (CH ) N
3 2
O N(CH )
3
\
A (CH ) N
3 2
y
+ NaO ^(CH,), \ (CH ) N
3 2
O N(CH )
3
/
/ (CH ) N
3 2
/
(CH ) N
3 2
\
N(CH,)
2
O N(CH )
3
3 2
HCl
-f
C H 0Y
2 6
(CH ) N ^NiCHi), N o details are given for scheme A . Presumably one could use the phosphoryl chlo ride instead of the fluoride. Scheme B , i n which ethyl chloride is formed, was run i n boiling xylene using equimolar quantities of the reactants. Michaelis (33) has partially described the preparation of starting materials from secondary amines with phosphorus oxychloride and also ethyl dichlorophosphate. Schrader (38) obtained alkyl and amido fluophosphates by reaction of the corresponding chlorophosphates with sodium fluoride in aqueous or alcoholic solution. Octamethyl pyrophosphoramide is a colorless oil, completely soluble i n water, ben zene, acetone, and many other common organic solvents except the paraffinic hydrocarbons. Its hydrolysis rate has not been measured, but i t appears stable i n the absence of alkali. I n England, this systemic insecticide has been used to control aphids on hops. There i t has been calculated that only a negligible quantity of the poison ultimately may find its way into the beer made from the hops. Despite calculations of this sort, the use of octa methyl pyrophosphoramide on food or fodder crops i n this country is definitely not to be recommended. However, i t may prove useful if properly applied to control certain i n sects, especially those attacking ornamental plants, such as rosebushes, and possibly on the cotton aphid and grape phylloxera. T h e compound has only recently been made available experimentally.
Recent Developments
Since the presentation of this paper i n M a r c h 1949 at San Francisco there have been several new developments i n the field of organophosphorus insecticides. M e t h y l H o m o l o g of P a r a t h i o n . 0 , 0 - D i m e t h y l O-p-nitrophenyl thiophosphate
158
has been manufactured i n Germany for insecticidal formulations to supplement or replace parathion. The methyl homolog (22) is a white crystalline compound melting at 3 5 C . Preliminary tests indicate that it is a somewhat less potent insecticide than parathion but may prove less hazardous to apply than parathion, which has caused a number of cases of acute poisoning and several deaths of individuals who handled and applied i t without sufficient precautions. Tetraisopropyl Pyrophosphate. T h i s ester, a water-white l i q u i d , has been pre pared by T o y (47) i n 9 4 % yield from diisopropyl chlorophosphate and water i n the presence of pyridine. The ester is insecticidal and is about one tenth as toxic to white mice as tetraethyl pyrophosphate. A n insecticidal dust of much greater stability than tetraethyl pyrophosphate dust can be formulated from tetraisopropyl pyrophosphate, inasmuch as the tetraisopropyl ester hydrolyzes at approximately V oth the rate at which the tetraethyl ester breaks down i n the presence of moisture. Octamethyl Pyrophosphoramide and Analogs. D a v i d a n d K i l b y (11) have re cently published details of Schrader's synthesis, which gives a good over-all yield of octa methyl pyrophosphoramide. Also included i n this paper are data on its absorption by the roots and leaves of a living plant, its phototoxicity, and mode of action of insects infesting the plant. Ripper and associates (86) give entomological data on this insecti cide and results of its action on plants and on warm-blooded animals. They point out that its systemic properties enable i t to reach insects which are extremely difficult to kill by direct contact application and that its persistence enables i t to be used early i n the development of an aphis outbreak without fear of reinfestation. Analogs of octamethyl pyrophosphoramide which act as systemic insecticides are the isomeric symmetrical (I) and unsymmetrical (II) diethyl bis (dimethylamido)pyro phosphates :
5
(CH ) N
3 2
M l
po- -p I
11/
N(CH )
3
(CH ) N
3 2
M l
O OC H
2
CTsO^
OC H
2
(CH ) N
3 2
POP II
1 1 /
OC H
2
Conclusions
I n the field of organic phosphorus compounds uncovered by Schrader there is a wealth of highly toxic compounds from which to pick a potential insecticide. The u l t i mate choice, however, will not be based solely on the toxicity of the compound to a certain group of insect species. Other important factors are volatility, stability, safety i n h a n dling and applying, freedom from plant injury, freedom from spray-residue and trans location hazards, and safety from the standpoint of its long-term chronic toxicity to man and animals. A s a determining factor for its use, the cost per pound of an organic phos phorus insecticide may not be very important. T o compare the cost per pound of para thion, for example, with that of D D T on a realistic basis, one must bear i n mind that parathion is effectively applied at much lower concentrations (2, 19, 46). These potent new organic phosphorus insecticides may not be used, i n general, to control insects affect ing man and animals (household pests, cattle and sheep pests, etc.) because of their ex treme toxicity to warm-blooded animals (18, 28). However, because they are effective over a very wide range of insect species at concentrations so low as to be almost fantastic, the potential usefulness of this class of insecticides, if properly applied, needs no emphasis.
Literature Cited
(1) (2) (3) (4) (5) Adler, H . , U . S. Patent 2,462,057 (1949). American Cyanamid Co., Australian Patents 16,371, 16,372, 16,807, 17,162 (1947). Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753-6 (1948). Ball, H . J., and Allen, T . C., J. Econ. Entomol., 42, 394-6 (1949). Bloch, H . , Helv. Chim. Acta, 26, 733-9 (1943); Hottinger, ., and Bloch, H . , Ibid., 26, 142-55 (1943).
HALLORGANIC PHOSPHORUS INSECTICIDES (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50)
159
Bowen, C. V., and Edwards, F. I., ADVANCES IN CHEMISTRY SERIES, 1, 198 (1950). Brauer, R. W., J. Pharm. Exptl. Therap., 92, 162-72 (1948). Chadwick, L . E., and Hill, D . L., J. Neurophysiol.,10, 235-46 (1947). Clemmensen, E . , U . S. Patent 1,982,903 (1934). Coates, H . , Ann. Applied Biol., 36, 156-9 (1949). David, W . A. L., and Kilby, . ., Nature, 164, 522-3 (1949). Dayrit, C., Maury, C. H . , and Seevers, M . H . , J. Pharmacol. Exptl. Therap., 92, 173-86 (1948). DuBois, K . P., Doull, J . , Salerno, P. R., and Coon, J . M . , Ibid., 95, 79-91 (1949). DuBois, K . P., and Mangun, G. H . , Proc. Soc. Exptl. Biol. Med., 64, 137-9 (1948). Dvornikoff, M . N . , and Morrill, H . L., Anal. Chem., 20, 935-6 (1948). Edwards, F . I., unpublished communication. Edwards, F . I., and Hall, S. ., Anal. Chem., 21, 1567 (1949). Fletcher, J . H . , Hamilton, J . C., Hechenbleikner, I., Hoegberg, E . J . , Sertl, B. J . , and Cassaday, J. T., J. Am. Chem. Soc., 70, 3943-4 (1948). Gleissner, B. D., Wilcoxon, F . , and Glass, . H . , Agr. Chem., 2 (10), 61 (1947). Granger, M . M . , and Leiby, R. W., Ibid., 4 (2), 34 (1949). Hall, S. ., J. Assoc. Offic. Agr. Chemists, 32, 377-83 (1949). Hall, S. ., unpublished data., Hall, S. ., and Jacobson, M . , Agr. Chem., 3 (7), 30-1 (1948). Hall, S. ., and Jacobson, M . , Ind. Eng. Chem., 40, 694-9 (1948). Jansen, E . F., Nutting, M . D . F., and Balls, A. K., J. Biol. Chem., 175, 975-87 (1948). Kosolapoff, G . M . , personal communication. Kosolapoff, G . M . , Science, 108, 485-6 (1948). Lehman, A. J . , Bull. Assoc. Food & Drug Officials, 12 (3), 82-9 (1948). McCombie, H . , and Saunders, B. C., Nature, 157, 287-9 (1946). McCombie, H . , Saunders, B. C., and Stacey, G. J . , J. Chem. Soc., 1945, 380-2. Mackworth, J . , and Webb, E. C., Biochem. J., 42, 91-5 (1948). Martin, H . , and Shaw, H . , BIOS Final Rept. 1095, U . S. Dept. Commerce, Office of Technical Services, PB-78244 (1946). Michaelis, ., Ann., 326, 129-258 (particularly p. 179) (1903). Nachmanson, D., Rothenberg, . ., and Feld, . ., J. Biol. Chem., 174, 247-56 (1948). Peck, D . R., Chemistry & Industry, 1948, No. 33, 526. Ripper, W. E., Greenslade, R. M . , and Lickerish, L . ., Nature, 163, 787-9 (1949). Roark, R. C., U . S. Bur. Entomol. Plant Quarantine, E-721 (1947). Schrader, G. (as presented by S. A. Mumford and E. A. Perren), BIOS Final Rept. 714(Revised), U. S. Dept. Commerce, Office of Technical Services, PB-87923 R (1945). Schrader, G., German Patent 720,577 (1942); U . S. Patent 2,336,302 (1943). Schrader, G., U . S. Dept. Commerce, Office of Technical Services, PB-73754 (1946). Smith, F . F., unpublished communication. Smith, F. F., Brierly, P., and Fulton, R. ., Proc. Am. Soc. Hort. Sci., 51, 327-32 (1948). Smith, F. F., Fulton, R. ., and Lung, P. H . , J. Econ. Entomol., 41, 624-30 (1948). Tanner, C. C., Greaves, W. S., Orrell, W. R., Smith, . K . , and Wood, R. E. G., BIOS Final Rept. 1480, U . S. Dept. Commerce, Office of Technical Services, PB-81638 (1946). Thurston, J . T . , FIAT Final Rept. 949, U. S. Dept. Commerce, Office of Technical Services, PB-60890 (1946). Thurston, J . T . , paper presented at meeting of . Y . Section, A M . CHEM. SOC., New York, . Y., Jan. 22, 1949. Toy,A.D. F., J. Am. Chem. Soc., 70, 3882-6 (1948). Webb, E . C., Biochem. J., 42, 96-8 (1948). Woodstock, W. H . , U . S. Patent 2,402,703 (1946). Wreath, A. R., and Zickefoose, E . J . , Anal. Chem., 21, 808-10 (1949).
Chemistry and Toxicity of Some Organofluorine Insecticides

W. T. SUMERFORD
Communicable Disease Center, Public Health Service, Federal Security Agency, Savannah, Ga.
The chemistry, insecticidal, activity and toxicity of the major organofluorine insecticides are reviewed. In the ten years since the discovery of DDT opened up a new field of endeavor for the chemist, biologist, and toxicologist, activity in the field of fluorine-containing insecticides has been great.
T h e discovery of the phenomenal insecticidal activity of l,l,l-trichloro-2,2-bis(pchlorophenyl)-ethane ( D D T ) , which occurred less than 10 years ago, opened up a new field of endeavor for the chemist, biologist, and toxicologist. The activity i n this field is considerable, and a portion of i t has been directed toward efforts-to locate useful insecticides among the fluorine-containing compounds. Some of the fluorine compounds known to be insecticidal were re-evaluated, other compounds were tested biologically for the first time, and new compounds were prepared to be subjected to such tests. Elemental fluorine, inorganic combinations of fluorine, and organofluorine compounds are known to be general systemic poisons (74). Fluorine itself appears to be about as toxic as hydrogen cyanide to certain species of insects, with the effective insecticidal concentration lying between 100 and 1000 p.p.m. (81). A number of inorganic fluorine compoundse.g., volatile fluorine compounds (76), metallic fluorides, fluosilicates, and fluoaluminateshave been employed for some 50 years mainly as stomach poisons for household and agricultural insects (15). Furthermore, the assertion has been made that any inorganic compound containing fluorine will mothproof wool (16). More recently i m proved methods for introducing fluorine into organic molecules (1, 73) have provided samples of an increased number of fluorinated compounds for the determination of their toxicity toward insects. These developments would seem to justify a review of the pertinent information which has been published on the organofluorine insecticides, with special reference to the methods for their synthesis and to their biological activities. Such hybrids as the hydrofluorides (108), fluosilicates (100), and fluosulfonates (98) of aromatic amines and the fluoborates of some organic acids (99), although known to be active insecticides, are not included. For the purpose of this review, the compounds considered are distributed among the following sections: l,l,l-trichloro-2,2-bis(p-fluorophenyl)-ethane, analogs of 1,1,1trichloro-2,2-bis(p-fluorophenyl)-ethane, and miscellaneous organofluorine compounds. The chemistry, insecticidal activity, and toxicity of these groups of compounds are considered i n the order given. M u c h of the work i n synthetic insecticides, especially the biological portion, is necessarily of an exploratory character, and hence will have to be supported before appraisals become final.
Chemistry
l,l,l-Trichloro -2,2 -bis(^-fluorophenyl)-ethane. A t present, the most i m portant organofluorine insecticide is l,l,l-trichloro-2,2-bis(p-fluorophenyl)-ethane
160
SUMERFORDCHEMISTRY AND TOXICITY OF SOME ORGANOFLUORINE INSECTICIDES
161
( D F D T ) , the ,'-difluorine analog of D D T . D F D T was not specifically mentioned i n the original United States patent (86) issued to Paul Muller and assigned to J . R . Geigy, A . - G . However, i t is reported to have been used i n large quantities i n Germany (under the name Fluorogesarol) as an all-purpose insecticide as early as 1944 (64). D F D T can be prepared by methods similar to those used to produce D D T i . e . , by condensing two molecular equivalents of fluorobenzene with chloral i n the presence of concentrated sul furic acid. T h e fluorobenzene required for the reaction was obtained i n Germany b y the fission of nitrogen from phenyldiazonium fluoride which resulted from diazotizing aniline in the presence of copper and hydrofluoric acid (106). The laboratory methods that have been used to prepare D F D T are given i n outline form in Table I . The selected items of information were not uniformly available. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch030 Table I .
CClsCHO, Moles 1 16 0.1 0.42 c 1.1 1.1 0.8 0.23 0.10 0.53 0.1/
a C C
Synopsis of Laboratory Methods for Preparing (p-fluorophenyl)-ethane

PhF, Moles 2 2 0.2 0.84 2.0 2.0 2.0 0.72 0.20 0.117 0.25 Condensing Agent Moles H2SO4 HSO3CI H S04.H 0 H SCM H SOi H2SO4 H S04 H S(V
2 2 2 2 2 2
1,1,1-Trichloro-2,2-bis-
Order of Mixing"
Conditions Temp., Time, C. Hours
Yield,
M.P., C.
0
Reference (20) (39) (86) (65) (107) (10) (10) (10) (10) (109) (60) (S3)
8 20
H S0 PhF
2 2
30-50 45 ' 25 0 10-20 30 Cool 5 30 Cooled
36 2 "2 12 2 10 "2 20 12 12
67 28 50 81 76 68 73
4' 4 '
3.4 0.35 1.8 0.11 1
H S04 H S04 Acid

2
b.8 160-80 43-4 41-2 26 42-3
HSOjCl HSOCI HSO3CI H S04

2
HSO3CI HSO3CI HSO3CI H S04

2
40 ' b.7-8 172-6 45-6
Compound listed in this column added to mixture of other reactants. b Chloral ethylate used in place of free chloral in this reaction. Chloral hydrate used in place of free chloral in this reaction. d 60 ml. of 2 0 % fuming H S 0 4 added during course of reaction. 50 ml. of fuming HSC>4 added during course of reaction. / 1,2,2,2-Tetrachloroethyl ether used in place of chloral.
c 2 E 2
T A B L E I . I t is probable that all the indicated yields can be improved. However, using these data as a criterion, it appears that the chloral can be replaced by its hydrate or alcoholate and that a moderate excess of fluorobenzene favors the reaction. There is one report that aluminum chloride brings about this condensation (34), but here, as with D D T , the choice condensing agents are concentrated sulfuric acid (with or without the addition of oleum), and chlorosulfonic acid (84)- A moderate temperature and prolonged stirring, which must also be vigorous, increase the yield. D F D T is usually obtained in the laboratory as an almost-white, gummy material with an odor resembling that of ripe apples. (A recent sample of technical grade D F D T supplied through the courtesy of W . M . Lee, Pennsylvania Salt Manufacturing Company, Philadelphia, P a . , met this description and melted at 35-37C.) I n some instances, refrigeration is necessary to bring about solidification. This product is a mixture of several isomers of l,l,l-trichloro-2,2-bis(p-fluorophenyl)-ethane from which the p,p'isomer, boiling point, 133-34 C., melting point 42-43 C., can be separated by distillation at reduced pressures (10). The recrystallization of crude D F D T is attended by considerable loss due to its high solubility i n the common organic solvents. Dissolving the low-melting crude mixture i n ethyl alcohol and adding water to the point of incipient precipitation followed by cooling raised the melting point to 40 (109). Recrystallization from ethyl alcohol produced needle-shaped crystals, melting point 44-45 (79), and from methanol (twice) crystals melting at 45 (78). (The oil from which these crystals were obtained had a boiling point at 1 mm. of 133-134 and a refractive index of 1.5707 at 20 C.) The vapor pressure of D F D T (0.5 m m . of mercury at 178) (79) is reported to be some fifteen times that of D D T (3). I n a recent study i n this laboratory, Goette found that a 400 mg. per square foot deposit of D F D T on protected glass panels lost 9 5 % of its weight in 9 weeks
162
as compared to a 1 5 % loss for D D T (36). This has considerable influence on the ac tivity of D F D T as a residual insecticide. B o t h D D T and D F D T are extremely insoluble in water, but D F D T (79) is approximately ten times as soluble as D D T (61) i n such solvents as mineral oil, kerosene, dibutyl phthalate, Velsicol A R - 6 0 (a mixture of polymethylnaphthalenes supplied by the Velsicol Corporation, Chicago, 111.), carbon tetra chloride, xylene, o-dichlorobenzene, and cyclohexanone. These solvents are commonly used as vehicles for insecticidal formulations, thus giving D F D T an advantage over D D T in this respect. Like other 2,2-diaryltrichloroethanes, D F D T undergoes dehydrohalogenation i n the presence of a base to yield l,l-dichloro-2,2-bis(p-fluorophenyl)-ethylene. The rate of this reaction has been found to be directly proportional to the temperature, and the rate con stant for D F D T is approximately one seventh that for D D T at ordinary temperatures (18, 110). This ethylene derivative has been oxidized by the use of chromic anhydride to ,'-difluorobenzophenone, a sample of which did not depress the melting point of an authentic sample prepared by a different route (10). D F D T has been subjected to the usual analysis for its halogen content, but no spe cific method has been worked out for its determination, nor has an investigation been made of its response to the known colorimetric reactions of D D T . Analogs of l,l,l-Trichloro -2,2 -bis(^-fluorophenyl)-ethane. T h e broad a n d powerful insecticidal a c t i v i t y of D D T appears to a marked degree i n several com pounds related to i t i n structure. Because the a c t i v i t y of D F D T against certain species of insects compares very favorably w i t h that of D D T , several analogs of D F D T have been prepared for biological testing. Some of these analogs were o b tained b y reactions corresponding to those used to prepare D F D T , while others were prepared directly from the D D T or D F D T molecules themselves. T h e reactions which have been used to prepare the D F D T analogs are outlined i n Table I I . A l l available information is included. T A B L E I I . Several of the reactions outlined i n Table I I were discussed under the chemical reactions of D F D T . I n general, the D F D T analogs listed here were prepared in the laboratory for the purpose of producing a sample of the material for biological test ing rather than to study the several reactions involved or to improve the yields. T h e latter were usually omitted i n the literature. Samples of l,l,l-trifluoro-2,2-bis(p-fluorophenyl)-ethane, and the corresponding bis(p-chlorophenyl) derivative were obtained b y applying Henne's fluorination method to D F D T and D D T , respectively. I n both instances a mixture of the mono-, d i - , and trifluoro compounds was obtained, but the desired trifluorinated material was separated by fractional recrystallization from methanol and cooling with a mixture of dry ice and acetone. The reactions used to prepare 2,2-bis(p-fluorophenyl)-l,l-dichloroethane and 1,1,1tribromo-2,2-bis(p-fluorophenyl)-ethane are reminiscent of those used for D D T .
Insecticidal Activity
It has been demonstrated that the organofluorine compounds are capable of killing insects as either stomach or contact poisons. Some of the more volatile members of this group have a degree of fumigating action. However, most of the investigations of this group have been directed toward a determination of their activity as contact poisons. W i t h the possible exception of D F D T , too few of the organofluorines have been tested under controlled conditions to permit a definite evaluation of their usefulness even i n this one field. It is generally agreed that the contact-insecticidal activity of the D D T type of com pound depends on at least one toxic component and the C C 1 group or some other lipoidsoluble group for penetration. Beyond this point, there is a lack of agreement as to the exact mechanism b y which the contact insecticides exert their action. Although a lipoid-soluble group characterizes many contact insecticides, simple o i l solubility of a compound is not always a criterion of activity. Busvine (14) tested a series of D D T analogs and found that solubility i n oil was not essential to activity. K i r k w o o d
3
163
Table II. Synopsis of Laboratory Methods Used to Prepare Analogs of 1,1,1 -Trichloro-2,2-bis(p-fluorophenyl)-ethane Having Insecticidal Interest
Reactant Product (p-FC H ) CHCF (p-ClC6H ) CHCF3 (p-ClC H ) CFCFCl & (p-ClC H ) CHCF Cl (p-FC H ) CHCHCl
6 4 2 3 4 2 6 4 2 2 6 4 2 2 6 4 2 2 6 4 2 2
Reactant Mole Mole HF HF

a
(p-FCeH ) CHCCl3 (p-ClC H ) CHCCl

4 2 6 4 2
0.94 0.11 (0.2)
Excess Excess (0.1)
(p-ClCeH ) CHCCl PhF

4 2
SbF CHCl CH(OEt)

3 2
(p-FC H ) C=CCl (p-FC H ) CHCC1=CC1

6 4 2 6 4 2 3
(p-FC H ) CHCCl PhF

6 4 2
(p-FC H ) CHCBr ( p - F C H ) (p-ClC H )CHCCl < (p-FC H ) CHOH (p-FCeH ) CO

6 4 6 4 3 6 4 2 2 4
PhF (p-FC H ) C=CCl

6 4 2
KOH/EtOH CHC1 CHC1= CC1 CBr CHO

2 2 3
CrOj
Condensing Agent Moles HgO HgO H S0 .H 0 Oleum (26%)

2 4 2
Order of Mixing HF HF Acid
Conditions Temp., Time, hours C. 20-30 20-30 160 + Cool 20-30 Reflux 5 2* 3 ' 15
M . P., C. 78-80 64-65 89-90 77 42-42.5

b.0.2
Reference
(65) (65) (91) (85) (10) (85) (34) (95) (79) (10)
0.22 0.25 4.5 18 ml.
A1C1
Aici
H2SO4
149-150 34-36 106-107
120' '
0
1.'25
Liquid hydrogen fluoride. b Presumably prepared by addition of fluorine to dehydrochlorinated product of D D T . In Chemical Abstracts and in English summary of article (91). Body of paper refers to compound as ,'dichlorodiphenyldifluorodichloroethane, but gives no analytical data. d Synthesis of this compound not given in abstracts, but similar hybrids have been prepared via l-trichloro-2-chlorophenylethanol (103). Reported as prepared by R. Picard, University of Illinois.
c
and Phillips (66) showed that both D D T and l,l,l-trifluoro-2,2-bis(p-chlorophenyl)ethane are very soluble i n fats, but only D D T is insecticidal. When fed under the same condition to rats, the D D T accumulated i n the perirenal fat of rats, while the noninsecticidal analog did not. The approximate tenfold increased solubility i n fixed oils of D F D T over D D T fails to increase its insecticidal activity i n anywhere near this ratio. These findings support the earlier postulation that the oil-water distribution coefficient of a com pound is more important i n this respect than its solubility i n oil (70). There are several theories concerning the mechanism by which the toxic component of a contact insecticide exerts its action. The toxicity has been credited to the condensed chlorobenzene system, which is also lipophilic i n character (70). If this is the explanation, D F D T and the other compounds depending on the fluoroaryl system for toxicity would be expected to be less toxic than the corresponding chloro compounds, for fluorobenzene is generally less toxic than chlorobenzene. A second theory advanced by M a r t i n and W a i n (77) is based on the observation that D D T and certain other related insecticides easily lose hydrogen chloride, which presumably affects vital centers of the insect. The results of some recent work of Cristol with numerous halogenated insecticides detract from this theory (17). D D T and D F D T , having similar insecticidal activities, differ considerably i n the rate at which they undergo dehydrohalogenation. Finally, a study of D D T on several enzyme systems disclosed that i t sig nificantly inhibited phosphatidase, possibly through an affinity for the cholesterol of the lipoid membrane of the cell (71). The comparative effect of D F D T on phosphatidase has not been reported.
164
Efforts have been and are being made to standardize testing procedures for evaluating insecticides (41, 88), but considerable variations still exist in the methods, and results therefrom are not always uniform. A description of the various methods requires too much space to be given here. I n general, the test insects are placed on a surface or held i n an atmosphere with known concentrations of the insecticide. I n other procedures, the insects are treated directly with a prepared spray or dust of a known strength of the compound under investigations. L a r v i cidal activity is determined by placing samples of the larvae i n water or aqueous preparations containing serial dilutions of the product to be tested. After the various types of exposure, the test insects are usually placed under optimum conditions for recovery and so held for stated observation periods to determine the knockdown and/or mortality percentages i n a given group. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch030 Busvine, using adult lice and bedbugs, obtained more precise results from the spray method (followed by forced contact on a filter paper surface) than from an exposure to the same insecticide i n the form of a prepared dust (H). The surfaces used for the contact tests range from those peculiar to the insects' habitat to those easily provided in the laboratorye.g., glass and filter paper. Fresh deposits of several contact insecticides gave higher kills of fruit flies on glass than on filter paper (93). Contrarily, a D D T deposit on dry bamboo, bark, rusty metal screen, and pine plywood remained toxic to adult female mosquitoes longer than did a similar deposit on new sheet metal, glass, tile, palmetto thatch, and new metal screen (28). The variations i n testing apparatus, insect species, dosages, exposures, and criteria of activity render difficult the appraisal of individual or groups of insecticides. Tables I I I to X I have been arranged in an effort to show the comparative activity of the organofluorine insecticides, principally D F D T , against a variety of organisms by including a reference standard compound, usually D D T . It is recognized that it would be advantageous to compare the chlorinated and fluorinated pairs, and this is done in so far as the sketchy data permit. Table III. Comparative Insecticidal Activity of DDT and DFDT against Insects of Diptera Order
DDT
Insects Adults Fruit fly Fruit fly Fruit fly Housefly Fruit fly Fruit fly Fruit fly Housefly b Fruit fly Fruit fly Fruit fly
Fly
A.
Mortality DFDT 23 36 63 99 11 100 100 100 50.5 81.2 96.8 4 +

c
Dosage 0.04 7/sq. cm. 0.08 7/sq. cm. 0.16 7/sq. cm. 0.25% D D T , 1% D F D T " 2.5 mg. % 25 mg. % 100 mg. % 200 mg./sq. ft.<* 0.6 mg./10 ml. 0.85 mg./10 ml. 1.20 mg./10 m l .
e
Experimental Period, Hours 24 24 24 0.5 18 18 18 0.1
Reference
(79) (79) (79) (92) (U) (11) (11) (27) (98), (9sy (98) (28)< (21> U0)> (soy (92> (89> (89)
(C.
vomiloria)
quadrimaculatU8
35 52 78 38 65 71 89 60 49 53.5 56.8 3+
c
Larvae
A. A. C. C. quadrimaculatus quadrimaculatus quinquefa8ciatu8 apicalis apicalis
60 100 100 50 100 100
5 85 100 50 100 100
P.P.M. 0.005 0.005 D D T 0.01 D F D T 0.06 0.01 to 0.05 0.025 0.025
24/ 48 24 72* 72
Pupae
C.
a
kill. b When same dosages were tested against female flies having some degree of D D T resistance, mortality percentages were 44 and 86 for D D T and D F D T , respectively, with 7-day-old deposits, but with 18-day deposits, mortality percentages were approximately the same with both insecticides. Female flies; kill was 100 and 95%, respectively, for D F D T and D D T against male flies. Deposit 7 days old when tested; with 18-day-old deposits, mortality for D F D T remained at 100% and that for D D T rose to 93%, both percentages based on female flies. Concentration of solution drained from vials in which mortalities were determined. / At 48 hours, same dosage of D D T gave 95% mortality and D F D T gave 10% mortality.
c e
With 0.025% of added pyrethrins to each, D D T (0.1%) gave 73% kill and D F D T (0.2%) gave 91%
T A B L E I I I . I t is obvious from the data i n Table I I I that the housefly and the mosquito, i n both the adult and larval stage, are susceptible to insecticides of the D D T type. However, the extravagant claims that D F D T is far superior to D D T as a contact insecticide against flies are not borne out b y the results of controlled laboratory tests. T h e Peet-Grady testing technique used b y P r i l l (92) would indicate that i n the presence of added pyrethrins D D T is definitely superior to D F D T when applied as a spray. O n the other hand, D F D T gave higher percentage kills than D D T when flies were placed under a Petri dish and held i n contact with deposits of the compounds on glass surfaces. A comparison of the activity of these compounds against adult mosquitoes has not been reported. The testing techniques used with mosquito larvae, usually the addition of calculated quantities of the larvicide previously dissolved i n a water-miscible solvent, are somewhat more comparable. However, the results here are conflicting. T h e A. qvxjdrimoxulatus larvae are killed within 24 hours b y either D D T or D F D T at concentrations somewhat
1 6 5
Table IV.
Comparative Insecticidal Activity of DDT and DFDT against Insects Classified by Order
Insect DDT 50 70 81 100 78 29 80 65 75 62 50 60 55 0 37 100 100 100 50 100 12 50 0 76 90 6 0 < *
Mortality DFDT 50 100 91 100 63 24 100 30 35 66 50 100 52 100 80 55 100 100 50 37 86 50 0 99 93 100<*
Dosage 0 . 3 % for D D T 1.4% for D F D T
Experimental Period, Hours
Referen
Anoplura Body lice Coleoptera Vegetable weevils Flour beetles Soldier beetles Grain weevil Flour beetle Lepidoptera Cabbage looper Arctiid caterpillar Oakworm (larvae) Flour moth Hemiptera Bedbugs Milkweed bugs Large milkweed bugs Orthoptera German cockroach German cockroach Homoptera Red scale crawlers Hymenoptera Red ants Honey bees Thysanoptera Greenhouse thrips Greenhouse thrips Dermoptera Forficula Acarina Citrus mites Citrus mites Rat mites Rat mites Isopoda Pillbug
(14)
96 120 20 120-168 120-168 20 96 96 40
(79) (79) (78) (12) (12)
85 7/sq. cm. 15 7/sq. cm. 10 mg./63.3 sq. cm. 2.5 mg. % D D T 12.5 mg. % D F D T 2.5 mg. % D D T 12.5 mg. % D F D T 10 mg./sq. cm. A d lib. 85 7/sq. cm. 2.5 mg. % D D T 12.5 mg. % D F D T 0.53% D D T 5.0% D F D T 2500 7/sq. cm. 2.5 mg. % D D T 12.5 mg. % D F D T
a
(78) (79) (79) (12)
(14)
96 40 48 48 504
b c (79) (12)
2500 7/sq. cm. 2.5 mg. % D D T 12.5 mg. % D F D T 1-1000 100 7/sq. cm. 1000 7/sq. cm. 0.001% D D T 0.006% D F D T 0.005% 0.125 mg./sq. cm. <10% D D T < 1% D F D T 1-100 0.1 mg./sq. cm. 200 mg./sq. foot 0.25%
(79)
(12)
(79)
(79) (79) (80)
24 69
(79) (78) (80)
24 0.5 0.5 58
(79) (87) (29) (78)
Values obtained with oil spray. As dusts, D D T and D F D T gave slightly better results; this preparation of D F D T was outstandingly active against bedbugs. b D D T produced 100% mortality in 48 hours and D F D T in 16 hours. D D T produced 100% mortality in 20 hours and D F D T in 5 hours. < * Values determined on basis of direct contact insecticidal activity. D F D T showed superiority to D D T against other species of Isopoda.
c
less than those used i n field larviciding. Concentrations lower than these gave erratic results. I t is reported that the culicine larvae are more resistant than the anopheline larvae to D D T (26), but it is not yet possible to draw a distinction here between D D T and DFDT. T A B L E I V . Riemschneider (95) reported that a dust with 1 % D F D T was equal to 1 0 % D D T against lice, flies, and bedbugs, but apparently these ratios do not hold when the compounds are compared in preparations other than dusts (H). Busvine was forced to use a higher concentration of D F D T to obtain the same degree of kill against lice and bedbugs. Regardless of the concentration required, it is known that D F D T acts more rapidly against these insects (12). Among the Coleoptera, the soldier beetles were most susceptible to D D T and D F D T , with small dosages producing comparatively rapid kills. I n general, D F D T was superior to D D T against all insects of this order. D F D T closely resembles D D T i n its activity against the two adult members of the Lepidoptera where they were compared, but D D T is somewhat more active against the larvae of two other insects i n this order. Based on the restricted number of species involved, it appears that D F D T is superior to D D T against the German cockroach, European earwig, citrus mites, and rat mites with an outstanding advantage i n the case of the cockroach. The marked susceptibility of the German cockroach to D F D T has been noted by several workers. D F D T ' s effectiveness against the cockroach may depend on a combination of its high toxicity and increased v a por pressure which permits contact with this particular insect. B o t h compounds show about the same degree of activity toward red ants and honey bees, but D D T is superior against red scale crawlers and greenhouse thrips. Table V.
Insect Diptera Flies Flies Fly Anoplura Lice Lice Lice Coleoptera Grain weevil Potato beetles Weevil Lepidoptera Flour moth Clothes moth Hemiptera Bedbugs Bugs Hymenoptera Ants Ants Corrodentia Book lice Acarina Mite larvae
a
1 6 6
Approximate Insecticidal Activity of DFDT against Insects

Degree of Activity 7 times as effective as D D T 1% equal to 10% D D T Superior to D D T Moderate 1% equal to 10% D D T Equal to D D T Superior to D D T Approaches D D T Much superior to D D T Equal to D D T Superior to D D T 1% equal to 10% D D T Moderate Better than D D T Better than D D T Superior to D D T Equal to D D T Reference
(106) (96) (86) (107) (96) (23) (86) (94) (23) (28) (86) (96) (19) (23) (86) (86) (23)
Tineola
biselliella.
T A B L E V . The expressions used i n the second column of Table V to indicate the relative potency of D D T and D F D T are exact quotations from the respective reports. The sevenfold activity against houseflies credited to D F D T by the Germans actually represents the comparative activity of Gix, the active constituent of which is D F D T . I t is possible that the other constituents of G i x acted as a synergist for the D F D T . Alessandrini (2) reports the isolation from G i x of a compound melting at 9 9 to 100 C , which obviously is not D F D T . A crude sample of D F D T , a sample of recrystallized D F D T , a n d a sample of the impurities separated i n the re crystallization of D F D T were found by F a y
167
and Buckner (27) to have the same contact toxicity toward houseflies. Proverbs and Morrison (93) report D F D T to be a better poison than D D T for fruit flies, which is i n opposition to the claim b y K i r k w o o d and Dacey (65) that D F D T is two fifths as effective here. Although most of the values i n Table V are only approximate, they are i n general agreement with the more precise values obtained with the same species of insects. Table V provides values for three insects not appearing i n earlier tablesnamely, potato beetles, cnethocampidae, and book lice. The potato beetle is more susceptible to D D T , and the cnethocampidae and book lice are more susceptible to D F D T . Table VI. Comparative Knockdown Rate and Power of a Series of Organofluorine Compounds %
Insect
3 0
Compound (p-FC H ) CHCCl

6 4 2
Dosage 0.2% 200 mg./sq. foot 1 mg./sq. cm.

7 c
Knockdown, Hours
Knock down
Reference
(92) (27) () (79) (79) (79) (79) (79) (79) (79) (79) (79) (79)
Housefly Housefly 6 Housefly Blow fly Fruit fly Soldier beetle Fruit fly Fruit fly Fruit fly Fruit fly Fruit fly Fruit fly Fruit fly
0.17 0.10 Slower than D D T 2

e
91 1 6 < * 100 50 100 50 50 50 50 50 50 50
(p-FC H ) CHCHCl (p-FC H ) C=CCl A (p-FC H ) CHCCl=CCl A (p-FC H ) CHCF (p-ClC H ) CHCF (p-FC H )CHOHA (p-FC H ) CO^
6 4 2 2 6 4 2 2 2 6 4 2 6 4 2 3 6 4 2 6 4 6 4 2 a
/Sq. 15 150 15 15 15 15 15 15 15
Cm. 1/ 1.5 3.5 6 24 5 6 24 24
Pyrethrins, 0.025 gram/100 ml., were added. Under identical conditions, D D T at 0.1% gave 85% knock down. A 1% spray of D F D T without added pyrethrins gave 99% knockdown in 10 minutes. i > Mixed sample of male and female flies. c This deposit was 7 days old, but an 18-day-old deposit gave approximately same results. d D D T gave no knockdown under the same conditions. D D T required 18 hours to give same knockdown. / D D T required 8 hours to give same knockdown. D D T required 24 hours to give same knockdown. h Chlorine analogs required 24 hours to give 50% knockdown.
e
T A B L E V I . The knockdown power of the organofluorine insecticides has been deter mined principally against members of the Diptera order and b y a limited number of workers. There is general agreement that D F D T acts more rapidly than D D T , at least against those species with which they have been compared. P r i l l (92) found that twice the amount of D F D T compared to D D T was required to give the same knockdown against houseflies when tested b y a space spray technique with added pyrethrins. The forced contact method of F a y and Buckner (27) revealed that without added pyrethrins D F D T was a more powerful knockdown agent than D D T . The dehydrohalogenation of D F D T materially reduced its knockdown rate, but the resulting ,'-difluorodiphenyldichloroethylene derivative is more rapid i n action than the analogous trichloropropylene compound. The fact that the propylene compound maintained some activity suggests that its increased weight might explain the difference i n activity. Substitution of the chlorines on the alkane bridge of D D T b y fluorine gave a sloweracting knockdown, and the rate was not restored b y the further substitution of fluorine for chlorine i n the ring. T A B L E V I I . There was no significant difference between the potency of o-chlorofluorobenzene and p-bromofluorobenzene toward codling moth larvae, but o-bromofluorobenzene and p-chlorofluorobenzene must be tested to determine whether the detect able difference is due to the difference i n halogen substitution or to position isomerism. The complete replacement of chlorine i n the D D T molecule b y fluorine gives (pF C H ) 2 C H C F , a compound with greatly decreased toxicity toward fruit flies and thrips and probably other species. This reduced activity is matched b y the low activity of ( p - C l C H ) 2 C H C F 3 which, when contrasted to the potency of D D T and D F D T , shows that the attenuation is caused by the replacement of the three chlorine atoms on the alkane bridge. The corresponding substitution of only one chlorine atom by fluorine gave a compound having as much activity as D D T against the chafer beetle.
6 4 3 6 4
168
Table VII.
Insecticidal Activity of Some Miscellaneous Organofluorine Compounds

Physical Constants, C. B.p. 138-140 B.p. 152
a
Compounds o-FCeH Cl
4
Chem. Ref.
(97) (113) (65)
p-FCeH Br
4
(p-FCeH ) CHCF
4 2
M . p . 79-80
a
(p-ClCeH ) CHCFj
4 2
M.p. 64-65
(66)
( p - F C e H ) C H C H C l & M . p . 77
4 2 2
(85)
Insect Codling moth larvae Codling moth larvae Greenhouse thrips Fruit flies Greenhouse thrips Fruit flies Flour beetles Flies Grain weevils Cnethocam pidae Greenhouse thrips Ants Book lice Fruit flies Chafer beetle Flies Bedbugs Lice
Order Lepidoptera Lepidoptera Thysanoptera Diptera Thysanoptera Diptera Coleoptera Diptera Coleoptera Lepidoptera Thysanoptera Hymenoptera Corrodentia Diptera Coleoptera Diptera Hemiptera Anoplura Thysanoptera Isopoda Diptera Coleoptera Lepidoptera Thysanoptera Hymenoptera Corrodentia
Degree of Activity 91% toxic to larvae 100% toxic to larvae 52% mort, from 0.1% in 24 hours Vioo that of D D T 34% mort, from 0.1% in 24 hours Vioo that of D D T 15 /sq. cm. gave 2% mort, in 120 hours Equal to D D T Inferior to D D T Inferior to D D T 59% mort, from 0.01 % in 24 hours Superior to D D T Superior to D D T Not active Equal to D D T l ^ ^ e q u a l to 10%
C
Biol. Ref.
(22)
(22)
(79) (ID (79) (ID (79) (86) (86) (86) (79) (85) (85) (ID (91) (95) (95)
(p-ClCeH ) CFCFCl ( p - C l C e H ) C H C F C l M.p. 89-90 (p-FCeH )(p-ClCeH )CHCCh

4 2 2 4 2 2 4 4
(11) (91) (95)
l^êqual l^êqual
to to
10% 10%
(95)
(p-FCeH ) C=CCl
4 2 2
M.p. 42-42.5
(10)
Greenhouse thrips Woodlice Flies Grain weevils Cnethocam pidae Greenhouse thrips Ants Book lice Book lice
(p-FCH ) CHCCl= CC1

4 2 2
B.p., 0.2 mm., 149-150
(85)
100% mort, from 1% in 24 hours 10 mg./63.3 sq. c m . affected 50%<* Inferior to D D T Equal to D D T Inferior to D D T
(79)
(78) (85) (85) (86)
31% mort, from 1% (79) in 24 hours (85) Equal to D D T (85) Inferior to D D T 67% mort, from 1.0% (p-FCeH ) CHOH M . p . 34-36 (79) (79) in 24 hours (p-FCeH )CH(OH)Fruit flies Diptera (ID (ID Vs to A as active as CCh DDT Homoptera (75) Bean aphis CH (FCH CH 0) 100% mort, from 2 g. (4) larvae/ after 5 days* Lepidopterous Lepidoptera 100% mort, from 0.5 (4) larvae/ g. after 21 days* Weevils/ Coleoptera 0% mort, from 1.0 g. (4) after 28 days* Thysanoptera 17% mort, from 0.1% (79) M.p. 106-107 (p-FCeH ) CO (10) Greenhouse thrips in 24 hours Diptera (22) FCH C0 HA. M . p . 33 (111) A. quad. 0.1 p.p.m. gave 10% larvae mort. 24 hours (22) 0.1 p.p.m. gave 65% mort. 48 hours Homoptera FCH C0 CH CHEtB.p., 2 mm., (47) Aphids (47) 100% mort, from (CH ) CHIJ 65-68 0.01% soin. (112) Codling moth Lepidoptera (104) p-FCeH NH B.p. 185-189 larvae 90% toxic to larvae Anoplura 1% gave 100% knock B.p., 7.5 mm., (67) (p-FCeH ) S * Body lice (24) down in 1 hour 165-167 Anoplura 0.1% gave 88% Body louse (25) mort. J eggs 50% mort, in 48 hours A. quad. Diptera (22) larvae from <1 p.p.m. Anoplura 0.1% gave 62% p-CH CH4SOF M . p . 43-44 (43) Body louse (25) mort. eggs Moderately superior to corresponding chlorine analogs in this particular test. f> Moderately inferior to corresponding chlorine analog in this particular test. Paralyzing effect decreased, but toxicity remained unchanged. Exposure time 68 hours; under same conditions 16% were affected by D D T . Systemic poison, applied in soil supporting plants. / Killed on tick beans, cauliflower, and wheat in order given. Degree of control proportional to dosage; smaller dosage gave less control. Prepared by hydrolyzing methyl fluoroace tate (from corresponding iodoacetate and A g F at 170) with barium hydroxide. 1 Tested as sodium salt. i Prepared by heating ethyl fluoroacetate with 2-ethylhexanol at 160 in presence of p-toluenesulfonic acid monohydrate. * Prepared by reducing p-fluorobenzenesulfonyl chloride with H I (60% yield). l Same concentration in pyrophyllite gave 95% mortality.
4 2 a 4 2 2 2 2 2 4 2 2 2 2 2 2
2 3
169
The replacement of the one remaining hydrogen atom i n the bridge of ( p - C l C H ) C H C F C I 2 gave an inactive compound against the fruit fly, the only insect tested. This is precedented by the known inactivity of ( p - C l C e H ^ C C l C C U . The following halogenated hydrocarbons, ( p - F C H ) C H C H C l and ( p - F C H ) ( p C 1 C H ) C H C C 1 , have a close structural relationship to D D D , D D T , and D F D T and pre sumably as a consequence share the high insecticidal activity of these compounds. l,l-Difluorodiphenyl-2,2-dichloroethylene, produced b y the dehydrochlorination of D F D T , is almost devoid of activity, at least against thrips and woodlice. The activity of the analogous l,l-dichloro-2-chloro-3,3-difluorodiphenylpropene is even less against thrips, but the latter compound is said to be as active as D D T against grain weevils and ants. The fluorinated carbinols, ( p - F C H ) C H ( O H ) C C l and ( p - F C H ) C H O H , and the ketone corresponding to the latter have not been tested very widely. The carbinol with the trichlorinated carbon atom is more promising structurally, and its activity against fruit flies warrants additional testing. The absorption of the ether bis (2-fluoroethoxy) methane from the soil and its trans location i n plants in a concentration sufficient to k i l l insects is a recent discovery hereto fore observed only i n some selenium compounds and i n the insecticidal esters of some polyphosphoric acids (4). Whether this property is shared by other organofluorine com pounds will doubtless be determined for the purpose of studying plant metabolism and of evaluating any practical application. Monofluoroacetic acid offers little promise as a mosquito larvicide, but 2-ethylhexyl monofluoroacetate is a very powerful aphicide. The high toxicity of the fluorinated lower aliphatic acids and their esters to vertebrates probably precludes their use as insecticides except under highly controlled conditions. The toxicity of the fluorinated acids to the vertebrates is attributed to their interference with an enzyme system, but the possibility of finding one with a high specificity for insects is not excluded. Difluorodiphenyl disulfide, when tested on woolen swatches, is a n outstanding knockdown agent and toxicant for body lice. Several compounds with p-fluorinated phenyl radicals have exhibited the same order of activity against lice, but too few com pounds with the disulfide linkage have been tested to warrant an appraisal of its effect here.
6 4 2 6 4 2 2 6 4 6 4 3 6 4 3 6 4 2
Table VIII.
Miscellaneous Organofluorine Compounds Credited with a Degree of Activity against Some Specific Insect
Physical Constants, C.
0
Compound CHF CH,(CH )ioCH F o-CH CeH4F6 l,2,4-(CH,),CeH F 4,4 -FCeH4.C H4Fcd p-FC H40H FCH C0 H 3.4- N 0 F C e H C 0 H p-CH CeH4S0 F 1.5- CioHe(S0 F) (3,4-Cl CeHi)S0 (4-FC H NH)
S 2 2 8 2 , 6 2 2 2 8 2 8 2 2 2 2 2 e 4
Chem. Ref.
(44) (8) (46) (36) (6) (118) (HI) (106) (48) (43), P. (48)
Insect Clothes moths Housefly Red scale Moths Codling moth Moths Moths Moths Moths Moths Moths
Order Lepidoptera Diptera Homoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera
Biol. Ref.
(60) (9) (19) (60) (50) (61) (60) (60) (69) (59) (48)
B.p. - 8 2 . 2 B.p. 114 M.p. 26 M.p. 88-89 B.p. 186-188 M.p. 33 M . p . 121-122 M.p. 43-44 M . p . 203
Prepared in 80% yields by heating bromoform with mixture of antimony chloride and mercuric chloride at 100. b Prepared by treating o-toluenediazonium sulfate with hydrofluoric acid. Prepared by eliminating 2 atoms of bromine from 2 molecules of p-bromofluorobenzene by means of metallic sodium. * This compound, used as 1% aqueous solution of sodium salt, was toxic to moth and highly toxic to codling moth.
e
T A B L E V I I I . I t is difficult to conceive of fluoroform, which is chemically and physio logically inert, having toxicity to moths unless i t acts as a mechanical asphyxiant. The activity of monofluorododecane against houseflies is probably explained on the basis of the alteration i n the physical properties of the hydrocarbon molecule which already possesses a degree of toxicity toward this species. Of a series of fluorinated benzenes and fluorinated toluenes tested with and without
170
added hydrocyanic acid against red scale, only o-fluorotoluene could be rated as moderately toxic. The balance of the compounds i n Table V I I I support the earlier statement that any compound containing fluorine is toxic to moths. These fluorinated hydrocarbons, phenols, acids, sulfonic acids, and sulfones probably act against moths as stomach poisons. The fluorosulfonic acid derivatives and the sulfone with a fluorinated substituent were key compounds whose toxicity to moths laid the groundwork for the deductions which led to the synthesis and testing of D D T as an insecticide. T A B L E S I X AND X . A description of the methods used to determine the insecticidal activity of the compounds listed i n Tables I X and X is not available. The compounds are classified i n both tables on the basis of their chemical-structures, with a view to providing a guide for future searches for insecticides among organofluorine compounds. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch030 Table IX. Fluorinated Hydrocarbons, Acids, and Acid Derivatives Patented as General Insecticides
Fluorinated hydrocarbons Monofluorobutane Difluorobutane a, a, a-Trifluorotoluene 1,2-Difiuoro-1,1-diphenylethane o-Trifluoromethylbenzal fluoride 1,1,2,2-Tetrafluoro-l ,2-dinitroethane 1 -Chloro-1,2,2-trifluoro-1,2-dinitroethane 1,2-Dichloro-1,2-difl uoro-1,2-dinitroethane Fluorinated acids and acid derivatives Fluorostearic acidic 9,10-Difluorostearic acid** Monofluoroundecanic a c i d Sodium monofluoroleate Methyl fluoroundecanate&
e a
Reference
(63) (65) (37) (69)
(101) (42) (42) (42) (102) (102) (102)

(53) (63)
Nontoxic to housefly. b Prepared by treating corresponding unsaturated compound, in carbon tetrachloride, with hydrogen fluoride at 0-10. Metallic salts of esters of this acid with hydroxyethanesulfonic acid are described as insecticides (68). Calcium salt of difluorostearic acid of unspecified composition is described in (53). Used in form of salt.
c d e
Table X.
Miscellaneous Organofluorine Compounds Patented as General Insecticides

Organofluorine Compounds
a
Reference
(53) Fluoro-octadecyl alcohol Trifluoromethylbenzaldehyde (64) (90) Dichloroethylfluorosilane (90) Chloroethyldifluorosilane (90) Ethyltrifluorosilane (90) Dichlorofluorophenylsilane (90) Chlorodifluorophenylsilane (90) Trifluorophenylsilane (90) Diethoxyfluorophenylsilane (90) Difluoromethylphenylsilane (90) Chlorofluorodiphenylsilane (90) Difluorodiphenylsilane (53) Methyl iV-fluorobutyryl-iV-methylaminoacetate (66) b JV-Monofluorostearoylglycine (66) JV-Difluorostearoyltaurine (58) Diethylaminomonofluorophosphoric acid (68) Diethylaminodifluorophosphoric acid (58) Diethylaminomonofluorothiophosphoric acid (57) Methanesulfonyl fluoride (62) 1-Piperidinesulfonyl fluoride (62) N, JV-Bis (2-chloroethyl)sulfamyl fluoride (62) JV-Hexyl-iV-methylsulfamyl fluoride (52) JV-Dodecyl-iV-methylsulfamyl fluoride (52) iV-Carbomethoxymethyl-iV-methylsulfamyl fluoride (52) N, JV-Bis(2-cyanoethyl)sulfamyl fluoride (56) Phenyltrifluoromethyl sulfone (56) p-Tolyltrifluoromethyl sulfone (56) p-Chlorophenyltrifluoromethyl sulfone (56) p-Nitrophenyltrifluoromethyl sulfone (66) m-Bis(trifluoromethylsulfonyl) -benzene (52) A^-Methyl-iV-phenethylsulfamyl fluoride (62) 4-Morpholinesulfonyl fluoride Prepared by treating trifluoromethylbenzal fluoride with sulfuric acid. b This patent described monofluorostearyl alcohol-sulfone as having insecticidal activity. Prepared by reacting diethylaminophosphoric compounds, containing one halogen replaceable by F , with potassium fluoride or antimony fluoride.
c c c a c
171
Toxicity
The physiological effects of the fluoride ion were reviewed by M c C l u r e (74) i n 1933. Lehmann (72) published an article on the toxicity of aromatic fluorine compounds i n 1928. However, i t has been impossible to investigate the toxicity of the organofluorine compounds as thoroughly as that of the organochlorine compounds. Some of the fluorinated hydrocarbonse.g., trifluoromethane (44), 1,1-difluoroethane (45), and difluorodichloromethane (82)are known to be chemically inactive and physiologically inert. Stepwise replacement of the chlorine atoms i n chloroform by fluorine yields the dichlorofluoromethane and chlorodifluoromethane, neither of which is as toxic as the original substance (7). I n a few instances, the introduction of fluorine into certain compounds containing oxygen, nitrogen, or phosphorus increases the toxicity of such compounds to a spectacular degree. The rodenticide monofluoroacetic acid has an L D i n certain species of warm-blooded animals of less than 1 mg. per k g . of body weight (49). The esters of certain fluorinated aliphatic acids (75) are likewise very toxic, as are the a l k y l esters of fluophosphoric acid (63, 68). The toxicities of these compounds greatly exceed that of the fluoride ion, and they are thought to function through their interference with an enzyme system. The substitution of chlorine or fluorine i n the methyl radical of ra-toluidine increases the toxicity of the parent compound to frogs, but fluorine increases it about 5 0 % over chlorine. m-Trifluorotoluidine is a quick-acting and lasting narcotic for frogs (72). In contrast to the toxicity of these fluorinated oxygen and nitrogen-containing compounds, the products from trifluorinated methyl ketones and amines are reported to have low acute toxicities (62). The fluorocarbons are insoluble i n water, alcohol, and hydrocarbons (38). The toxicity of insecticides to vertebrates is frequently a deciding factor i n determining where and under what conditions, if any, they can be used. Therefore, the tox5 0
Table XI.
Comparative Toxicity of DDT and DFDT and Some Miscellaneous Organofluorine Insecticides to Vertebrates
Animal Mice Mice Mice Mice Mice" Mice
a
Compound (p-ClCeH ) CHCCl (p-FC H4) CHCCl3

4 2 6 2 3
Dosage, Mg./Kg. 200 900 250 1000 150-500 250-1000 400 440
Route Adm. Oral Oral Oral Oral Oral Oral per os

per os
No. of Animals 15 10 20 20 7 4
Mortality 47 40 60 45 60 0 50 50 Equal
Reference
(HO)
(110)
(818) (32) (88) (88) (23) (23) (96) (96) (32) (82) (82) (32) (32) (32) (32) (82) (ID
(p-ClCeH4) CHCCl (p-FCH ) CHCCl

2 4 2 3
(p-ClC6H ) CHCCl (p-FCeH ) CHCCl

4 2 4 2 3 4 2 4 2 3
(p-ClCeH ) CHCCl, (p-FCeH ) CHCCl (p-ClC H ) CHCCl (p-FC H ) CHCCl

6 4 2 6 4 2 3 6 4 2 4 2 3 3
Mice Mice Mice Mice White rats White rats White rats White rats White rats White rats White rats White rats Mice Mice Mice
2 3 3
(p-ClC H ) CHCCl (p-FCeH ) CHCCl (p-ClCeH ) CHCCl (p-FCeH ) CHCCl (p-ClC H ) CHCCl (p-FC H ) CHCCl (p-ClCeH ) CHCCl (p-FC H ) CHCCl
4 2 4 2 3 6 4 2 6 4 2 3 4 2 6 4 2 3
250 250 500 500 1000 1000 1500 1500 2200 & 22006 2200 2.5 10 P.P.M. 0.1 1.0 0.01 0.125
Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral Ear
10 10 10 10 10 10 10 10 4 4 4 24 1 10 10 10 10
20 0 80 0 90 30 100 70 75-100 75-100 0 58 100 100 100 100 100
(p-ClC H ) CHCCl (P-FC H ) CHCC1 (p-FCeH ) CHCF

6 4 2 6 4 2 4 2 3 2 2 2 2 2
(11)
FCH C0 H FCH C0 CH CHEt(CH ) CH (p-ClC H ) CHCCl (p-FC H ) CHCCl

6 4 2 6 4 2 3 4 2 4 2 3
White rats Rabbit Goldfish Goldfish

Gambusia Gambusia
(11) (114) (47)

(89) (89) (89) (89)
(p-ClCeH ) CHCCl (p-FCeH ) CHCCl

a
White-footed mouse, Peromyscus leucopus. b Dosage given over a period of 6 days.
172
icity of organofluorine insecticides is not only of academic interest, but also of extreme practical importance. The acute oral toxicity of D D T and D F D T for several species of laboratory animals is compared in Table X I . T A B L E X I . The substitution of fluorine for chlorine on the two rings i n D D T r e duces the acute oral toxicity to a significant degree. The acute toxicity of D F D T appears to be of the order of one fourth to one third that of D D T to mice and white rats. A t the dose levels at which symptoms were seen with both compounds, D D T appeared to act more rapidly. Replacement of the phenyl chlorine atoms of D D T by fluorine reduces the toxicity to a greater degree than by bromine or iodine, but to a lesser degree than b y tert-bxityl, methoxy, butoxy, amyloxy, and acetoxy groups (110). Failure to relate the toxicity of a series of D D T analogs to their solubility i n oil and their dehydrohalogenation rates led von Oettingen and Sharpless (110) to investigate the metabolic fate of this series of compounds. D F D T , the least toxic of .the ,'-dihalogen analogs of D D T , represents the strongest halogen-carbon bond. D D T and D F D T showed the expected high toxicity toward fish, but on the basis of the preliminary results D F D T appears to be one fifth to one tenth as toxic as D D T . Goldfish were more resistant to both compounds than was the wild gambusia. Riemschneider (96) purposely swallowed both D D T and D F D T , and reports that no essential pharmacological differences could be detected in the two compounds. There has been an increased exploration i n the organofluorine compounds since 1940, when an authoritative and comprehensive article giving the toxicity of 551 miscellaneous organic compounds to young screwworms did not include a single compound containing fluorine (13). Acknowledgment A large percentage of the data on miscellaneous organofluorine compounds which are listed in Tables I X and X was taken from (31). A paper by Metcalf (79) provided a good measure of the data on the comparative activity of these compounds listed i n Tables I I I , V , V I , and V I I . The writer is indebted to his associates, R . W . F a y , H . P . Nicholson, and Thomas B . Gaines for the activity and toxicity data credited to them i n Tables I I I , I V , and X I , and to M a r y B . Goette for some physical measurements on D F D T . Eugene Odum of the University of Georgia supplied the information on the relative toxicity of D D T and D F D T to the white-footed mouse. Literature Cited
(1) Adams, R., et al., "Organic Reactions," Vol. II, pp. 49-93, New York, John Wiley & Sons, 1944. (2) Alessandrini, M . E., Rend. ist. super, sanit, 10, 807-12 (1947). (3) Balson, E . , Trans. Faraday Soc., 43, 54 (1947). (4) Bennett, S. H . , and Martin, H . , Ann. Rept. Agr. Hort. Research Sta., Univ. Bristol, pp. 147-56 (1947). (5) Bishopp, F . C., J. Econ. Entomol., 39, 456 (1946). (6) Blatt, A . H . , "Organic Syntheses," Coll. Vol. 2, pp. 188-90, New York, John Wiley & Sons, 1943. (7) Booth, H . S., and Bixby, . M . , Ind. Eng. Chem., 24, 637-41 (1932). (8) Bousquet, . W., Graves, G . D., and Salzberg, P. L . (to . I. du Pont de Nemours & Co.), U . S. Patent 20,869 (reissue 1938). (9) Bowen, C . V., and Smith, L . E., U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Div. In secticide Invest., "Synthetic Organic Compounds Patented for Use as Substitutes for Pyrethrum," Mimeo (February 1944). (10) Bradlow, H . L., and VanderWerf, C. ., J. Am. Chem. Soc., 69, 662-4 (1947). (11) Browning, H . C., et al., Can. J. Research, 26D, 282-300 (1948). (12) Ibid., pp. 301-6. (13) Bushland, R. C., J. Econ. Entomol., 33, 669-76 (1940). (14) Busvine, J. R., J. Soc. Chem. Ind., 65, 356-60 (1946). (15) Carter, R. H . , and Busbey, R. L . , U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Mimeo. Pub. E-466 (1939). (16) Clark, C. O., J. Soc. Dyers Colourists, 59, 213-15 (1943). (17) Cristol, S. J., ADVANCES IN CHEMISTRY SERIES, 1, 184 (1950).
173
(18) Cristol, S. J . , J. Am. Chem. Soc., 67, 1494-8 (1945). (19) Cupples, H . L., Yust, H . R., and Hiley, J . , Econ. Entomol.,29, 611-18 (1936). (20) Curtis, F . J . , Davis, F . C., Smadel, J . E., Southworth, Hamilton, and Volwiler, E. H., U . S. Dept. Commerce, Office of Publication Board, Rept. 237, 61 (1945). (21) Deonier, C. C., Jones, . ., Haller, H . L . , Hinchey, E . , and Incho, . H . , Soap, 22, No. 11, 118-19, 139 (1946). (22) Deonier, C. C., Jones, . ., and Incho, H . H . , J. Econ. Entomol., 39, 459-62 (1946). (23) Domenjoz, R., Helv. Chim. Acta., 29, 1317-22 (1946). (24) Eddy, G . W., and Carson, . B., J. Econ. Entomol.,39, 762-7 (1946). (25) Ibid., 41, 31-6 (1948). (26) Eide, P. M . , Deonier, C. C., and Burrell, R. W., Ibid., 38, 537-41 (1945). (27) Fay, R. W., and Buckner, Annette, unpublished results. (28) Fay, R. W., Cole, E . L . , and Simmons, S. W., Soap Sanit. Chemicals, 24, 130 (1948). (29) Fay, R. W., and Cullens, Doris, unpublished results. (30) Fay, R. W., and Pal, R., unpublished results. (31) Frear, D . . H . , "Catalog of Insecticides and Fungicides," Vol. I, "Chemical Insecticides," Waltham, Mass., Chronica Botanica Co., 1947. (32) Gaines, T . B., unpublished results. (33) Geigy Colour Co., Ltd., Balaban, I. E., and Manchester, Frank, Brit. Patent 585,007 (1947). (34) Geigy Colour Co., Ltd., Balaban, I. E., and Sutcliffe, Frank, Ibid., 603,685 (June 6, 1948). (35) German Patent 96,153 (1897). (36) Goette, M . B., and Sumerford, W. T . , unpublished results. (37) Goodhue, L . D., J. Econ. Entomol., 35, 533-6 (1942). (38) Grosse, . V., and Cady, G . H . , Ind. Eng. Chem., 39, 374 (1947). (39) Hall, S. ., U . S. Dept. Commerce, Office of Publication Board, Rept. 306, 4-5 (1945). (40) Haller, H . L., Ind. Eng. Chem., 39, 467-73 (1947). (41) Hartzell, ., Haynes, J . L . , and Connola, D . P., Contrib. Boyce Thompson Inst., 15, 131-40 (1948). (42) Hass, . B., and Whitaker, A . C., U . S. Patent 2,447,504 (1948). (43) Heilbron, I. M . , "Dictionary of Organic Compounds," Vol. I II, p. 775, London, Oxford Uni versity Press, 1943. (44) Henne, A . L . , J. Am. Chem. Soc., 59, 1200 (1937). (45) Henne, A . L . , and Renoll, M . W., Ibid., 58, 887-9 (1936). (46) Holleman, A. F . , and Beekman, J . W., Rec. trav. chim., 23, 238 (1904). (47) Horsfall, J . L . (to American Cyanamid Co.), U . S. Patent 2,409,859 (1946). (48) Huismann, Johann, and Schweitzer, Hugo (to I. G . Farbenindustrie), German Patent 506,988 (1930). (49) Hutchens, J . O., et al., J. Pharmacol. Exptl. Therap., 95, 62-70 (1949). (50) I. G . Farbenindustrie, Brit. Patent 333,583 (1930). (51) Ibid., 335,547 (1930). (52) Ibid., 457,119 (1936). (53) Ibid., 458,179 (1936). (54) Ibid., 466,007 (1937). (55) I. G . Farbenindustrie, French Patent 799,432 (1936). (56) Ibid., 800,303 (1936). (57) Ibid., 804,545 (1936). (58) Ibid., 807,769 (1937). (59) I. G . Farbenindustrie, German Patent 450,418 (1927). (60) Iris, R. C., and Mendiazbal, G . D., Rev. inst. salubridad y enfermedad trop. (Mexico), 8, 63-8 (1947). (61) Jones, . ., Fluno, H . J . , and McCollough, G. T., Soap, 21, 110-15, 155 (1945). (62) Jones, R. G., J. Am. Chem. Soc., 70, 143-4 (1948). (63) Kilby, . ., and Kilby, M . , Brit. J. Pharmacol., 2, 234-40 (1947). (64) Kilgore, L . B., Soap, 21, 138-9, 169-71 (1945). (65) Kirkwood, S., and Dacey, J . R., Can. J. Research, 24B, 69-72 (1946). (66) Kirkwood, S., and Phillips, P. H . , J. Pharmacol. Exptl. Therap., 87, 375-81 (1946). (67) Knapp, W. ., private communication. (68) Koelle, G . B., and Gilman, ., J. Pharmacol. Exptl. Therap., 95, 172 (1949). (69) Krefft, . T . (to I. G . Farbenindustrie), Brit. Patent 452,656 (1936). (70) Lager, P., Martin, H . , and Mller, P., Helv. Chim. Acta, 27, 892-928 (1944). (71) Lager, P., Pulver, R., Montigel, C., Wiesmann, R., and Wild, H . , "Mechanism of Intoxication of D D T Insecticides in Insects and Warm-Blooded Animals," p. 7, New York, Geigy Co., 1946. (72) Lehmann, F., Arch. exptl. Path. Pharmakol., 130, 250-5 (1928). (73) McBee, E . T., et al., Ind. Eng. Chem., 39, 236-436 (1947). Review. (74) McClure, F . J., Physiol. Rev., 13, 277-300 (1933). (75) McCombie, H . , and Saunders, B . C., Nature, 158, 382-5 (1946). (76) Marcovitch, S., J. Econ. Entomol.,35, 288-9 (1942). (77) Martin, Hubert, and Wain, R. L . , Nature, 154, 512-13 (1944).
174
(78) Martin, Hubert, and Wain, R. L . , "University of Bristol, Annual Report, Agricultural and Horticultural Research Station," pp. 121-40, 1944. (79) Metcalf, R. L . , J. Econ. Entomol., 41, 416-21 (1948). (80) Ibid., p. 877. (81) Metcalf, R. L . , and Lindgren, D . L . , Ibid., 41, 522 (1948). (82) Midgley, Thomas, and Henne, A . L . , Ind. Eng. Chem., 22, 542 (1930). (83) Morrison, F . O., Can. J. Research, 21D, 37-75 (1943). (84) Mosher, H . S., Cannon, M . R., Conroy, . ., Van Strien, R. E . , and Spalding, D. P., Ind. Eng. Chem., 38, 916-23 (1946). (85) Mller, Paul, Helv. Chim. Acta, 29, 1560-80 (1946). (86) Mller, Paul (to J . R. Geigy, A.-G.), U . S. Patent 2,329,074 (1943). (87) Nicholson, H . P., and Cullens, Doris, unpublished results. (88) Odum, E . P., unpublished results. (89) Odum, E . P. and Sumerford, W. T . , Science, 104, 480-2 (1946). (90) Pletcher, D . E., and Nutting, H . S., U . S. Patent 2,436,777 (1948). (91) Pouterman, E . , and Girardet, ., Experientia, 2, 459 (1946). (92) Prill, . ., Synerholm, M . E., and Hartzell, ., Contrib. Boyce Thompson Inst., 14, 341-53 (1946). (93) Proverbs, M . D., and Morrison, F . O., Can. J. Research, 25D, 12-41 (1947). (94) Raucourt, M . , and Viel, G., Compt. rend. acad. agr. France, 34, 328-30 (1948). (95) Riemschneider, R., Sddeut. Apoth.-Ztg., 87, 62 (1947). (96) Riemschneider, R., Z. Naturforsch., 2B, 245-6 (1947). (97) Rinkes, I. J . , Chem. Zentr., II, 1432 (1914). (98) Roark, R. C., "Index of Patented Mothproofing Materials," U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Div. Insecticide Invest., 125 (1931). (99) Roark, R. C., "Second Index of Patented Mothproofing Materials," U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Div. Insecticide Invest., Mimeo, 109 (1933). (100) Roark, R. C., and Busbey, R. L., "Third Index of Patented Mothproofing Materials," U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Div. Insecticide Invest., 104 (1936). (101) Scherer, Otto (to I. G . Farbenindustrie), German Patent 670,833 (Jan. 27, 1939). (102) Scherer, Otto, Platz, Carl, and Sll, Julius (to I. G . Farbenindustrie), Ibid., 621,977 (1935). (103) Schneller, G . H . , and Smith, G . B. L., J. Am. Chem. Soc., 70, 4058 (1948). (104) Siegler, E . H . , Munger, F., and Smith, L . E., U . S. Dept. Agr., Circ. 523 (1939). (105) Slothouwer, J. H . , Rec. trav. chim., 33, 335 (1914). (106) Smadel, J . E., and Curtis, F . J . , U . S. Department Commerce, Office of Publication Board, Rept. 240, 5 (1945). (107) Stephenson, O., and Waters, W. ., J. Chem. Soc., 1946, 339-43. (108) Sttter, Herman, German Patent 485,101 (1929). (109) Sumerford, W. T., J. Am. Pharm. Assoc., Sci. Ed., 36, 127-8 (1947). (110) Von Oettingen, W. F., and Sharpless, . E . , J. Pharmacol. Exptl. Therap., 88, 400-13 (1946). (111) Von Richter, Victor, "Organic Chemistry," Vol. I, p. 335, New York, Elsevier Publishing Co., 1947. (112) Wallach, O., Ann., 235, 267 (1886). (113) Wallach, O., and Heusler, Fr., Ibid., 243, 226 (1888). (114) Ward, J. C., and Spencer, D . ., J. Am. Pharm. Assoc., Sci. Ed., 36, 59-62 (1947).
Alkali-Stable Polychloro Organic Insect Toxicants, Aldrin and Dieldrin

REX E. LIDOV, HENRY BLUESTONE, and S. BARNEY SOLOWAY, Julius Hyman & Company,
Denver, Colo., and CLYDE W. KEARNS, University of Illinois, Urbana, III.
The chemistry and general properties of two new alkali-stable insect toxicants, aldrin and dieldrin, are discussed, and the general properties of these materials are given. Entomological data serving to illustrate the magnitude of the insect toxicity of these compounds are included. Aldrin is an alkali-stable, relatively nonresidual insect toxicant with activity equal to or greater than the activity of -hexachlorocyclohexane. Dieldrin is an alkali-stable in sect toxicant with high activity and high persistence. Its period of residual activity is equal to or greater than that of DDT. In general, its activity level is somewhat higher than that of aldrin.
W i t h the advent of D D T it became possible to think in terms of the eradication of insect pests instead of their control only. Although very lethal organic toxicants such as the pyrethrins and rotenone had been previously employed, their instability under normal conditions of use limited their utility. D D T is only the first of a new group of toxicants, members of which were discovered or invented independently almost simultaneously i n various parts of the world. Of these, the group of halogenated hydrocarbons, which includes D D T , toxaphene, 7 -hexachlorocyclohexane, and chlordan, is the best known. Although some members of the group possess undesirable properties which severely limit their practical importance, others, like chlordan, are many times more toxic than D D T toward many insect species and possess no disadvantage not common also to D D T and other members of the group. U n t i l now, however, no compound has been available which combined high potency with the long period residual activity characteristic of D D T . One of the greatest practical disadvantages possessed b y the hitherto commonly used halogenated hydrocarbon insect toxicants has been the extreme ease with which they are dehydrohalogenated b y alkaline reagents and b y many metal halides. This of itself is serious, but even more serious is the fact that simultaneously these compounds lose all or almost all of their insecticidal activity. I n fact, so marked is this reaction pattern that some investigators have attempted to devise theories of insecticidal activity based on the ability of these compounds to lose hydrogen chloride and to correlate the degree of activ ity with the ease, under various experimental conditions, with which dehydrohalogenation could be induced.
Chemical and Physical Properties

During the past 30 months two new compounds, aldrin and dieldrin, possessing great usefulness as insect toxicants, have been devised, synthesized, and studied i n the laboratories of Julius H y m a n & Company. B o t h compounds are completely impervious
175
176
to the action of alkaline reagents, i n either aqueous or alcoholic media. Under conditions of practical use they likewise appear unaffected by acidic reagents. Despite this fact, they possess insecticidal activity equal to or greater than that of the best halogenated insect toxicants known or used. A t the time of original presentation of this paper Compounds 118 and 497 had not yet been named. Since then the Interdepartmental Committee on Pest Control of the U . S. Department of Agriculture has adopted the names aldrin and dieldrin, respectively, for (a) " a n insecticidal product having not less than 9 5 % of its principal constituent, the chemical 1,2,3,4,10,10 - hexachloro - l,4,4a,5,8,8a - hexahydro -1,4,5,8 - dimethanonaphthalene. . ." and (6) " a n insecticidal product having not less than 8 5 % of its principal constituent, the chemical 1,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro-l,4,5,8-dimethanonaphthalene. . ." " A l d r i n (recrystailized)" and "dieldrin (recrystallized)" are defined to refer to products containing not less than 9 9 % of the above-named chemicals. Because continued use of the names 118 and 497 can only lead to confusion, the names aldrin and dieldrin have been adopted for use i n the nonentomological portions of this paper to refer to the pure chemical compounds, with the recognition that such usage does not exactly correspond with that officially adopted. Chemically, aldrin is 1,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexahydro-l,4,5,8-dimethanonaphthalene and possesses, in planar representation, the structure :
The planar representation shown serves, i n part, to obscure somewhat the complexity of the structural problem involved. Actually, four stereoisomers are represented by the simple planar representation shown; it is not yet known with certainty which is the one corresponding to aldrin. Physically pure aldrin is a white crystalline solid with the properties set forth in Table I. Table I. Physical Properties of Aldrin
Mild
Melting point. 104-104.5 C. Odor. Substantially odorless at room temperature. pinelike odor when warm Solvent Methanol Acetone Benzene Hexane Base oil (deobase) Water
Solubility of Aldrin at 30 C , Grams/100 M l . 9 159 350 98 89 Insoluble
A l d r i n exhibits i n many instances the chemical behavior expected on the basis of its structure. Thus, it is readily attacked by halogens to yield the expected halides.
Aldrin +
Cl
IIDOV et al.ALDRIN AND DIELDRIN
177
Chlorine adds to form the frans-dichloride; bromine adds, i n carbon tetrachloride solution, to give a mixture of a s - and rans-dibromides ; this addition can be directed wholly to the trans derivative b y conducting the bromination i n a mixed phase reaction i n aqueous suspension. The double bond i n the unchlorinated ring, i n the presence of acidic catalysts, adds a variety of reagents of the type H Y , illustrated i n Table I I . Table II. Reaction of Aldrin with Reagents HY
Cl H H Y Halide ion Product Halide Ester 0 Ether
Aldrin + H Y
>
RC \ RO
M a n y other additions, similar i n type, are possible. Although acidic reagents can bring about alterations i n aldrin, such reactions proceed only i n the presence of strong acids or strongly acidic catalysts i n the homogeneous phase and hence are without signifi cance for the conditions under which insecticides are normally utilized. Because aldrin contains the bicyclo-(2.2.1)-heptene ring structure, it reacts typically with phenyl azide to form a phenyldihydrotriazole derivative. This reaction is of impor tance i n that it provides the basis for an analytical method for determining aldrin (dis cussed more fully in 2). The double bond i n the unhalogenated ring is readily attacked b y oxidizing agents. Chromic acid i n acetic acid, and potassium permanganate i n alkaline solution, oxidize the compound to the expected dicarboxylic acid, 4,5,6,7,8,8-hexachloro-3a,4,7,7a-tetrahydro1,3-dicarboxy-4,7-methanoindane. CI
Aldrin + K M n O , CI / \ COOH
Potassium permanganate i n neutral solution and lead tetracetate give rise, respec tively, to the anticipated glycol and to its diacetate. B y far the most interesting oxidation of aldrin, at least from the present viewpoint, is the oxidation with per acids. This oxidative process, which occurs normally, produces 6,7-epoxy-6,7-dihydroaldrin, the compound now called dieldrin. Dieldrin is then 1,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro-l,4,5,8-dimethanonaphthalene :
Dieldrin, which, when pure, is a white crystalline solid, possesses the physical prop erties shown i n Table I I I .
178
Table III.
Physical Properties of Dieldrin

175-176 C. Odor. Odorless Solubility of Dieldrin, Grams/100 Grams 26 C. 0 C. 4.9 54.0 75.0 7.7 4.3 3.4 35.4 36.9 2.5 1.3
Melting point. Solvent Methanol Acetone Benzene Hexane Base Oil (Std. oil 10) Water
Insoluble
While dieldrin will, under suitable conditions, exhibit many of the expected reactions of epoxy compounds, it is a remarkably stable oxide. Thus, in its preparation the presence in the oxidizing solution of 1% or more of sulfuric acid i n no way affects it. However, when dissolved i n b u t y l ether and refluxed with concentrated solutions of the halogen acids, dieldrin reacts typically to yield the halohydrin.
The somewhat unexpected inertness of dieldrin toward mineral acids has great practical significance; despite its structure it remains, under practical conditions of use, insecticidally active under both acidic and alkaline conditions.
Insect Toxicity
The data of Table I V give a concise general summary of the relative toxicity of these two compounds to a number of common insect pests. I n each case technical chlordan is used as the standard of comparison and accordingly is assigned a base value of 10. I n some instances similar data for other of the common toxicants are included in order better to enable visualization of the relative activity levels. (In Table I V and the tables which follow 7 -C H Cl6 represents the gamma isomer of hexachlorocyclohexane.) A s the data i n this table are presented the figures given represent relative a c t i v i t y ; as activity increases the relative activity figure also increases. Thus, the first line of the table indicates that a l drin is approximately four times as effective against the housefly as is chlordan and that D D T is only about one third as effective as is chlordan.
6 6
Table IV.
Relative Toxicity of Aldrin and Dieldrin to Insects

Chlordan Aldrin 40 60 60 70 140 160 68 40-50 100 40 80 60 Dieldrin 120 300 60 96 200 70 DDT 3 Toxaphene 2 T-CeHeCle 40 30 40 45 30 12.5 6-7 3-4 40
Housefly German roach American roach Black carpet beetle Milkweed bug Squash bug Confused flour beetle Differential grasshopper (adults) Fall webworm Imported cabbage worm Chinch bug Plum curculio Red spider mite Mexican bean beetle
10 10 10 10 10 10 10 10 10 10 10 10
80
+ +
_ -
LIDOV ef al.ALDRIN AND DIELDRIN
179
Kearns, Weinman, and Decker rate the more common halogenated insect toxicants in the following order of decreasing toxicity (7) : dieldrin, aldrin, heptachlor, 7 -hexachlorocyclohexane, chlordan, toxaphene, and D D T . This rating follows as the result of rather extensive tests on ten species of insects and is believed to represent, in general, the order of their relative activity. In order to evaluate the actual magnitude of the insect toxicity of aldrin and dieldrin and the utility of these new compounds, additional data must be considered. ( M a n y of the data herein presented have been obtained from letters and other unpublished communications. The details of the entomological investigations thus represented will, in most instances, be published i n appropriate journals by their authors.) Kearns, Weinman, and Decker determined the dosages of a number of the chlorinated compounds, dissolved in 9 5 % ethyl alcohol, required to produce a 5 0 % mortality of the housefly (7). The values obtained, expressed as micrograms of toxicant per gram of fly weight, are listed in Table V . Table V. LD Values on Houseflies
50
(Based on topical application of some chlorinated compounds dissolved in 95% ethyl alcohol. Values expressed as micrograms of toxicant per gram of fly weight") Compound DDT Chlordan T-CeHeCU Aldrin Dieldrin
a
LDso, y 20.5 4.0 2.9 1.6 1.1
Data from results of tests made by W. N . Bruce, assistant entomologist, Illinois Natural History Survey.
The Dacus fly (Dacus oleae, olive fly), a pest producing serious damage to olives and one related to other flies that attack a variety of fruits, is also highly susceptible to aldrin
(6).
Weinman and Decker list the L D o values against adult grasshoppers (M. differentialis) for a number of the more common toxicants, both as contact poisons and as stomach poisons, giving the values shown in Table V I {lJf).
5
Table VI.
LD Values for Single Compounds Tested for Contact and Stomach-Poison Effect against M. differentialis Adults
50
(Expressed as micrograms of toxicant per gram of grasshopper weight) Compound DDT Toxaphene Chlordan T-CeHeCle Aldrin Dieldrin Contact 9380 61 9.8 3.4 1.8 1.4 Stomach Poisons 2579 91.5 12.0 6.7 2.3 3.7
Weinman and Decker studied the toxicity of aldrin to young grasshoppers on V ^th acre plots surrounded on a l l sides by untreated check plots. The small size of the test plots and the "high pressure' ' of grasshoppers from the surrounding untreated areas made it impossible to secure control under these circumstances. Nonetheless, a significant reduction i n the number of grasshoppers i n the control plots was obtained even after 5 days with application of aldrin at the very low dosage rate of 0.05 pound per acre (0.75 mg. per square foot). F o r the control of locusts, grasshoppers, and crickets, 2 to 4 ounces of aldrin per acre, applied as a spray to host crops, yield highly effective results. The 4-ounce dosage is necessary under adverse control conditions such as prevail where vegetation is dry and most of the insects are i n advanced stages of growth (9). The effectiveness of the insect toxicant aldrin to a wide variety of cotton pests has been determined through a large number of field tests. A s a result of these tests, recommendations have been published suggesting the use of a mixture of 2.5% aldrin and 5 %
180
D D T at 10 pounds per acre to control the bollworm, boll weevil, cotton fleahopper, tarnished plant bug, rapid plant bug, and some species of cutworms and thrips. (On cotton just up it was found that only 0.07 pound per acre of aldrin, applied as an emulsion, was necessary for the control of cutworms and thrips.) Significantly, no increase i n aphid population was noted following the use of the a l d r i n - D D T mixture ( 4 ) . Tests of dieldrin against cotton pests have not been as extensive, but the Mississippi 1950 Cotton Insect Control Recommendations state that " i t kills a larger proportion of immature weevils i n squares than any other insecticide tested thus far and is considered very promising for cotton insect control" ( 4 ) . A l d r i n and dieldrin have been found similarly highly effective against a wide variety of other economically important insects. In Georgia, Savage and Cowart determined the relative effectiveness of several commercial insecticides for eradication of plum curculio on peaches (10). Their data are summarized i n Table V I I . A similar order of effectiveness was found for the adult curculio. Table VII.
Toxicant Acid lead arsenate Chlordan Parathion Aldrin
Plum Curculio Larvae Emergence

Pounds per 100 Gal. 2 1.5 0.45 0.75 % Infestation 41.59 4.08 1.87 0.30
Studies on the control of various species of ants show conclusively that aldrin consistently gives control when applied to infested turf at the rate of 1 ounce per 1000 square feet (4 ounces of 2 5 % wettable powder in 200 gallons of water). This dosage is only half of that required when chlordan is the toxicant employed (11,12). Several reports on the use of aldrin to combat other soil-infesting pests indicate its high order of effectiveness for this purpose. I n a series of field tests on Japanese beetle grubs, chinch bugs (12), white grubs (16), and tropical earthworms (15), aldrin has been found to be the most effective material tested. Against wireworms, preliminary work i n dicates aldrin to be effective when applied to the soil as a side dressing at the rate of 1.5 pounds per acre (3). One series of experiments also indicates aldrin to be at least twice as effective as chlordan in combatting the cabbage maggot (13). The foregoing comments and data, although far from complete, serve to indicate the high degree to which aldrin and dieldrin possess the property of insect toxicity. These m a terials have been tested as toxicants for more than one hundred insect genera. Although broad generalizations are unsatisfactory and inadequate, it is clear that to a large number of insect species these two compounds are the most toxic halogenated hydrocarbons yet available.
Residual Activity
A l d r i n and dieldrin show high toxicity to insect life, but differ greatly with respect to the length of time during which they exhibit residual activity. A l d r i n , like chlordan, exhibits residual effectiveness under field conditions for somewhat less than 3 weeks. E v e n when aldrin is applied at the uneconomical and unnecessary rate of 5 pounds per acre, leafy material so treated exhibits only slight insect toxicity after 3 weeks. A l d r i n , therefore, falls into that class of materials which exhibit pronounced initial toxicity but relatively short residual action. The situation with respect to dieldrin is altogether different. N o insect toxicant hitherto available, with the exception of D D T , has been characterized b y the possession of insect toxicity which continued for long periods after its application. I n this respect, d i eldrin is unique i n that, i n addition to its high order of insect toxicity, it possesses a span of residual activity comparable to that of D D T . The comparative residual activity of deposits of D D T and dieldrin, using the common housefly as the test insect, is illustrated i n Table V I I I . F o r purposes of comparison, both chlordan and aldrin are included i n the tabulations. This material is taken from the p a per b y Kearns, Weinman, and Decker (7).
LIDOV et al.ALDRIN AND DIELDRIN
181
The residual effectiveness against flies of a number of formulations of insect toxicants was studied by investigators of the U . S. Public Health Service. Over a 2-month period the formulations containing dieldrin were found to give the best results (S). Roaches have also been used i n studying the length of time for which deposits of dieldrin are active (7). Table I X gives some of the data obtained using the German roach as the test insect. I n this case, the toxicants were applied at the rate of 1 mg. per 1000 sq. cm. of surface ; the table lists the mortalities obtained when the insects were left on surfaces of various ages for periods of 24 and 48 hours. D D T is not included in this tabulation, because it is inactive at the dosage rates tested. Table VIII. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch031 % Mortality of Houseflies
(24 hours after a 30-minute exposure to a deposit of 50 mg. per sq. foot of some chlorinated insecticides at various intervals after application. Each figure based on three replicates of 125 flies each. Compounds deposited on glass plates") Age of Residues, Days 21 28 Compound Chlordan Aldrin DDT Dieldrin 93.4 83.8 73.7 100.0 58.9 30.0 41.1 100.0 Per Cent Mortality 1.8 17.9 19.1 4.6 11.5 95.7 35
71.1
4.3 64.3
Data from results of tests made by W. N . Bruce, assistant entomologist, Illinois Natural History Survey, and G . F . Ludvik, special research assistant, Illinois Natural History Survey and Illinois Agricultural Experiment Station.
Table IX.
Residual Toxicity of Chlorinated Insecticides to Adult Male German Roaches
(1 mg./1000 sq. cm. deposits at various intervals after application. Percentage dead and moribund after exposure periods of 24 and 48 hours) Age Deposit, Days 21 28 24 Compound Chlordan 7-CeHeCle Aldrin Dieldrin 100 100 100 100 100 100 100 100 24 14 22 100 92 18 100 100 48 24 48 Exposure Period, Hours 24 48 24 48 Per Cent Dead and Moribund 0 0 0 100 0 0 0 100 0 0 0 100 0 0 0 100 24 42 48 24 49 48
46
100
24
90
The formulation of aldrin and dieldrin can be readily accomplished i n normal fashion; no difficulty has been encountered i n incorporating these materials into dusts, wettable powders, or emulsifiable concentrates.
Phytotoxicity
The evaluation of new materials intended for use as insect toxicants requires consider ation of the toxicity of such materials to other forms of life. The question of phytotoxicity can be disposed of very rapidly. The evidence available indicates that even when applied i n gross overdosage, neither aldrin nor dieldrin is harmful to plants. Thus, for example, the application of aldrin to the soil at the rate of 100 pounds per acre led to no apparent inhibiting effect on the germination or growth of corn or of cucumbers (16). Other more extensive investigations conducted by Bauer and D a h m , using i n some experiments soil treatment at the rate of 100 pounds per acre and in others up to seven applications of aldrin to growing plants, at the rate of 0.5 pound per acre per application, on thirteen varieties of plants showed no abnormal effects, except that lettuce seed germination was slightly reduced and bean plant emergence was delayed 2 days; tobacco plants on the treated plots developed more rapidly and bloomed earlier than did the tobacco plants grown on the untreated plots (1). Bauer and D a h m report further that the vegetables grown under these conditions possess no foreign taste or objectionable odor. These work-
182
ers further demonstrate the complete absence of aldrin i n most of the plants so raised and the presence of only trace quantities of aldrin i n soybeans and tomatoes similarly grown. Bauer and D a h m state that "the application of Compound 118 to the soil at the rate of 100 pounds per acre represents a dosage that is probably from 20 to 50 times that required for practical insect control."
Mammalian Toxicity
M u c h work remains to be done before all aspects of mammalian toxicity of aldrin and dieldrin are completely known. However, the work already completed has established many important facts. When administered in edible oil to albino rats, aldrin demonstrates an acute L D ranging from 40 to 50 mg. per k g . of body weight. M u c h chronic toxicity work is i n progress. Albino rats on a daily diet containing 75 p.p.m. of aldrin incorporated in an edible oil continued normal i n all respects after 6 months. Cattle, sheep (pregnant ewes), and lactating cows with suckling calves were wintered on alfalfa hay sprayed with 0.5 pound of aldrin 8 days before harvest. The hay i n these tests was baled for storage. None of the animals fed on this hay developed abnormal symptoms, nor was evidence of damage found on autopsy of sacrificed individuals. Furthermore, aldrin could not be detected in the milk from the lactating cows (8). Dieldrin administered i n edible oil to albino rats demonstrates an acute LD50 toxicity of 50 to 55 mg. per kg. The chronic toxicity of dieldrin has not been established, a l though tests are now i n progress to determine its possible hazard to animals sprayed or dipped in formulations containing varying percentages. It has been established experimentally that rats are considerably more resistant to the action of dieldrin than rabbits. Among farm animals exposed to spray formulations containing dieldrin, swine proved to be the most resistant and very young calves proved to be the most susceptible. Preliminary results of work in this field indicate that dieldrin is less toxic to young calves than are some other comparably effective chemicals when applied i n a similar manner, although dieldrin is definitely more residual i n action against the insect parasites affecting farm animals.
5 0
Recommended Applications
A l d r i n exhibits only moderate persistence and evaporates completely under field conditions in somewhat less than 3 weeks. Consequently, if the simple precaution of applying aldrin not later than 3 weeks before crop harvest is observed, the possibility of u n desired toxicant residue on harvested crops should be very slight. It is recommended that aldrin be considered, on an experimental basis, both as a crop insecticide and for the control of subterranean pests. On crops intended for human and animal food no application should be made later than 3 weeks before the harvest of these crops is planned. Although dieldrin has been found effective against all the insects susceptible to the action of aldrin, it should not at present be recommended for use against any pests in situations where its residue might constitute a hazard on edible foods or on forage crops. I t is suggested that dieldrin be employed experimentally, especially for the control of flies, mosquitoes, cotton insects, forestry pests, termites, pests i n soil, pests of lumber products, cloth-eating insects, and industrial pests not actually infesting food products. Dieldrin should be considered wherever extended residual effectiveness is advantageous.
Summary
Two new insect toxicants, aldrin and dieldrin, provide new halogenated insect toxicants with an extremely high order of toxicity toward insects, combined, for the first time, w i t h complete stability to alkalies. Under all the usual conditions of use these new toxicants are also stable to acids. D a t a illustrate the order of magnitude of the insecticidal activity of these materials and their utility. A l d r i n is a relatively nonresidual material, in contrast to dieldrin which, because of its high persistence, exhibits prolonged residual activity.
LIDOV et al.ALDRIN AND DIELDRIN
183
Literature Cited
(1) Bauer, C. L . , and Dahm, P. ., "Field Plot Studies on Compound 118 (Aldrin) in 1949," 61st Annual Meeting, Am. Assoc. Econ. Entomol., Tampa, Fla., Paper 107, Dec. 13 to 16, 1949. (2) Danish, . ., and Lidov, R. E., ADVANCES IN CHEMISTRY SERIES, 1, 190 (1950). (3) Dogger, J . R., Insect Control Conference with Industry, Univ. Wisconsin, Wireworms Bull., Jan. 11, 1950. (4) Dunnam, E. W., Hamner, A . L . , Lyle, C., and Murphree, L . C., State Plant Board of Missis sippi, State College, Miss., "1950 Cotton Insect Control Recommendations," December 1949. (5) Federal Security Agency, Pub. Health Service, Atlanta, Ga., Communicable Disease Center Bull., 58 (October, November, December 1949). (6) Hadjinicolaou, J . , Reconstructionist, 7, 10 (May 15 to 31, 1949). (7) Kearns, C. W., Weinman, C. J . , and Decker, G . C., J. Econ. Entomol.,42 (1), 127-34 (February 1949); data presented at Am. Assoc. Econ. Entomol. meeting, New York, December 1948. (8) Kitselman, C. H . , Borgmann, A . R., and Dahm, P. ., "Toxicological Studies of Compound 118 (Aldrin) on Large and Small Animals," 61st Annual Meeting, Am. Assoc. Econ. Entomol., Tampa, Fla., Paper 108, Dec. 13 to 16, 1949. (9) Medler, J . T., Insect Control Conference with Industry, Univ. Wisconsin, "Grasshopper Control in Alfalfa Seed Fields with Low Volume Sprays," Bull., Jan. 11, 1950. (10) Savage, E . F., and Cowart, F . F., Georgia Expt. Station, "Report of 1949 Spraying Experiments for Control of Plum Curculio and Other Insects on Peaches," Sept. 29, 1949. (11) Schread, J. C., Conn. Agr. Expt. Sta., Circ. 173 (1949). (12) Schread, J . C., J. Econ. Entomol., 42 (3), 499-502 (June 1949). (13) Sciaroni, R. H . , Lange, W. H . , and Carlson, E. C., Agr. Extension Service, San Mateo County, and Division of Entomology and Parasitology, Davis, Calif., "Suggestions Regarding the Control of the Cabbage Maggot in San Mateo County." (14) Weinman, C. J . , and Decker, G . C., J. Econ. Entomol., 42 (1), 135-42 (February 1949); data presented at Am. Assoc. Econ. Entomol., New York, December 1948. (15) Westchester-Connecticut Turf Improvement Assoc., Tropical Earthworm Project, Second 1949 Progress Report. (16) Wolcott, G . N . , Agr. Expt. Sta., Univ. Puerto Rico, Rio Piedras, Puerto Rico, "Effectiveness of Hyman 118 against White Grub of Puerto Rico."
Insecticidal Activity and Dehydrochlorination Rates of Some Polychloro Insecticides

STANLEY J. CRISTOL
University of Colorado, Boulder, Colo.
The hypothesis that the insecticidal activity of various polychloro insecticides is due in large measure to the ability of the compound to liberate hydrogen chloride at the site of action of the insecti cide is examined. Reaction-rate constants for elimination of hydrogen chloride with ethanolic alkali were compared with insecticidal activity against Anopheles quodrimaculatus larvae and with Hammett substituent constants for eighteen analogs of DDT; the data lend no support to any hypothesis in which significant correlation between dehydrochlorination rate and insecticidal activity is required. Considerations of toxicity toward other in sects also show no correlation, as do similar comparisons with isomers of benzene hexachloride and polycyclic compounds related to chlordan.
T h e problem of the relationship between constitution and insecticidal activity i n or ganic compounds is one of great fascination to chemists and biologists, and any corre lation that might be found to exist between chemical reactivity and biological activity would be of outstanding importance. F o r this reason, the hypothesis of M a r t i n and W a i n (16, 17) that the insecticidal activity of various polychloro insecticides is due i n large measure to the ability of these materials to liberate hydrogen chloride at the site of action of the insecticide requires careful consideration. This hypothesis was based on early investigations on D D T analogs, where i t was observed that the relative biological activities of D D T [ ( p - C l C H ) C H C C l ] , its related olefin [ ( p - C l C H ) C = C C l ] , D D D (or T D E ) [ ( p - C l C 6 H ) C H C H C l ] , and the hexachloro compound [ ( p - C l C H ) C C l C C l ] seemed to bear a relationship to the abilities of these compounds to lose hydrogen chloride to alkali. This hypothesis has been criticized by Busvine (2,3), Domenjoz (10), Muller (18), and C a h n (4). Domenjoz and M u l l e r have shown that there is no direct correlation between activity toward a variety of insects i n a number of compounds of the type A r C H C C l and the amount of hydrogen chloride liberated under standard conditions. Busvine attempted a correlation for similar compounds between activity toward lice and bedbugs and this author's reaction-rate constants (2, ) for second-order elimination with ethanolic alkali and found that no statistically significant correlation exists. I t appeared to the author some years ago that, irrespective of the mechanism of the toxic action of D D T , there might be a correlation of structure and toxicity i n analogous compounds. Hammett (13) has shown that the rate and equilibrium constants of over 50 side-chain reactions of meta and para substituted aromatic compounds may be corre lated with the so-called substituent constant , according to the equation log k log ko = , where k and k are rate (or equilibrium) constants for substituted and unsubstituted compounds, respectively, is the reaction constant giving the slope of the linear relationship, and is the substituent constant, which is determined by the nature and
6 4 2 3 6 4 2 2 4 2 2 6 4 2 3 2 3 0
184
CRISTOLINSECTICIDAL ACTIVITY AND DEHYDROCHLORINATION RATES
185
position of the substituent and is independent of the reaction studied, being defined as the difference between the logarithms of the ionization constants of the substituted and u n substituted benzoic acids. This relationship is effective whenever the meta or para substituents affect the reactivity of the compounds by virtue of their electronic effects, and satisfactory relationships are not found i n cases where steric interactions are involved, as, for example, i n the reactions of ortho substituted compounds. Thus the observation of such a relationship is good evidence of the relative unimportance of steric factors i n re activity. Accordingly, i t was felt that such a correlation might be observed with reactions of the trichloroethyl group of D D T and its analogs. Elimination of hydrogen chloride with ethanolic alkali is such a reaction, and a satisfactory correlation was found for the secondorder reaction-rate constants for elimination with ethanolic alkali (S). O n the assump tion that the toxic reaction of D D T also involved a reaction of the trichloroethyl group without steric involvement of the ring substituents, a similar correlation was attempted between the insecticidal activity and the substituent constants. Results of insect mor tality were available from the work of the entomologists and chemists of the Orlando labo ratory of the Bureau of Entolomogy and Plant Quarantine for larvae of the common malaria mosquito (Anopheles quadrimaculatus Say) and have since been published (9).
2
_i
-1
-2
*-3
_0
C O
-4
- 5
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
Figure 1. Relation among Log k of Dehydrochlorination Reaction, Median Lethal Dosage to Mosquito Larvae, and Substituent Constant for Trichloroethyl Compounds
# Median lethal dosages Rate constants
The data used are given i n Table I . The elimination rate constants included were determined at 20 C. (5). The toxicity to mosquito larvae, given as median lethal dosages (concentration i n parts per million of water required to cause 5 0 % mortality i n 48 hours), was estimated from the data of Deonier et al. (9) and is probably reproducible to within 30%.
The logarithms of the elimination rate constants were plotted against substituent con stants to give the lower line of Figure 1, and the logarithms of the median lethal dosages were treated similarly to give the upper line. The straight lines were constructed by the method of least squares. The reaction-rate data fit a line with the equation
log k = 2.661 2.938
1 8 6
A statistical test of these data gave a correlation coefficient of 0.968 (19), with only 0.834 required for 1% significance. Thus the elimination rates are functions dependent Table I. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch032 Dehydrochlorination-Rate Constants and Insecticidal Activity of DDT Analogs
Dehydrochlorination Rate Constant, 10* k, L./Sec./Mole A. Di-Br Di-Cl H , CI Di-F Di-H Di-CH Di-ieri-butyl Di-CH 0
3 3
Toxicity to Larvae LDso, P.P.M. 0.001 0.002 0.008 0.007 0.4 0.007 20 0.007
a
Para Substituents
A.
quad.
Hammett Substituent Constant,
Diaryltrichloroethanes 0.464 0.454 0.227 0.124 0.000 -0.340 -0.394 -0.536 0.464 0.454 0.124 0.000 -0.340 -0.536
3470 2480 301 303 36.9 10.9 10.7 9.18 B. 776 568 71.0 14.3 4.33 3.36 C.
Diaryldichloroethanes 0.0016 0.001 0.16 0.46 0.0256 1 (est.) 6 Diarylmonochloroethanes 0.076 0.06 0.36
Di-Br Di-Cl Di-F Di-H
D 1 C -H 3
Di-CH,0 Di-Br Di-Cl Di-CH,
a
127 91.0 1.0 (est.)
(9). (8).
1^1
-0.6 -0.4
t
-0.2
i 0.0
i 0.2
1
0.4
t
0.6
Figure 2. Relation among Log k of Dehydrochlorination Reaction, Median Lethal Dosage to Mosquito Larvae, and Substituent Constant for Dichloroethyl Compounds
# Median lethal dosages Rate constants
CRISTOLINSECTICIDAL ACTIVITY AND
DEHYDROCHLORINATION RATES
187
upon side-chain reactions and relatively independent of steric factors i n the para substit uants. The best line relating insect mortality and substituent constant has the equation
log L D
5 0
1.87 -
1.69
and the poorness of fit can be noted from Figure 1. These data have a correlation coeffi cient of only 0.505 with 0.707 required for 5% significance. It is apparent from the data in Figure 1 that fit to a curve seems no more valid than the linear relationship. Attempts to find statistically significant relationships of the biological data were not successful when correlation was made with dehydrochlorination rates directly or with dosages i n molar units rather than weight units. Hence, it appears unlikely that any relationship exists between dehydrochlorination rate or any other reaction of the trichloroethyl groups without consideration of the steric involvement of the para substituents in the D D T analogs. In the course of work on the mechanism of elimination reactions, the author and his co-workers have measured reaction-rate constants for the second-order elimination of hydrogen chloride from six dichloroethyl compounds of type A r C H C H C l and three monochloroethyl compounds of type A r C H C H C l (7). Samples of each of these m a terials were furnished to the Bureau of Entomology and Plant Quarantine for insecticidal testing, and the author is indebted to C. C. Deonier and I. H . Gilbert for permission to use certain of their data i n this paper. The rate constants and larvicidal results are given i n Table I.
2 2 2 2
10
20
30 40 TIME, H O U R S
50
60
70
Figure 3.
Solvent 9 5 %
Rate of Chloride Production from Chlordan Constituents

ethyl alcohol, 0.04 M sodium hydroxide, 0.005 M halide, 45.7 C
Treatment of the data on the dichloroethyl compounds is indicated i n Figure 2. Here again there is excellent correlation between substituent constants, , and the reactionrate constants, fitting the line
log k = 2.456 3.430
and no statistically significant correlation between substituent constant and insecticidal activity, the best straight line having the form
log L D
5 0
2.50 -
1.43
These data lead to the conclusion that, contrary to the hypothesis of M a r t i n and Wain, there is no statistically significant relationship between dehydrochlorination rate and insecticidal activity i n D D T analogs, at least as regards activity against A. quadrimaculatus larvae. Similar comparisons of insecticidal data against adult houseflies (11,12),
adult yellow fever mosquitoes (Aedes aegypti) (11, 12), codling moth larvae (12), corn borer larvae (12), and screw-worm larvae (12) do not fit the dehydrochlorination rate h y pothesis. The isomers of benzene hexachloride (1,2,3,4,5,6-hexachlorocyclohexane) also elimi nate the elements of hydrogen chloride to alkali, and comparative data regarding the proposed hypothesis are available. Table I I gives reaction-rate constants for dehydro chlorination (6) and comparative toxicities to larvae of A. quadrimaculatus (12). Here, of course, the compounds differ only in stereochemistrythat is, i n spatial arrangement of theâtomsand there is no apparent relationship between reactivity and toxicity. M a r t i n (16) has also suggested such a relationship i n the chlorinated poly cyclic com pounds such as chlordan. Accordingly the author has measured the reactivities of the Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch032 Table II. Dehydrochlorination-Rate Constants and Toxicity to Anopheles quadrimaculatus Larvae of Isomers of Benzene Hexachloride
Isomer Alpha Beta Gamma Delta
a
1 8 8
Rate Constant, (20) , L./Sec./Mole

a
Dosage, P.P.M. 2.5 100 0.01 2.5
% Mortality b, 48 Hours 92 40 100 62
0.170 3 X 10- 0.0446 Fast
(6).
Table III.
Relative Effective Dosages of Polycyclic Compounds toward Houseflies

Compound Relative Effective Dosages (Technical Chlordan = 0.01) 0.0043 0.0056 0.017 0.020 0.0040 0.0028
Heptachlor (I) Octachlor (IIA) active isomer (IIB) inactive isomer Nonachlor (III) 118 (IV) 497 (V) Data from R. E . Lidov, Julius Hyman and Co.
various constituents of technical chlordan (14) toward ethanolic sodium hydroxide (Figure 3). The compounds referred to i n the figure are: heptachlor, 1,4,5,6,7,8,8-heptachloro3a,4,6,6a-tetrahydro-4,7-methanoindene (I); octachlor, l,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7-methanoindene, of which two isomers are known (IS), one (IIA) more active insecticidally than the other ( I I B ) ; and nonachlor, 1,2,3,4,5,6,7,8,8-nonachloro-2,3,3a,4,7,7a-hexahydro-4,7-methanoindene (III) (the stereochemistry of the com pounds has not been reported).
Cl C l - -CI
y
CL
Cl
Cl-
H -Cl H II / \ H H
|\ci
CU
Cl
Cl-
-ci
Cl H III / \ H Cl
Kci
l/H
Cl
/ \ H Cl
Cl
cr
In reactions with 0.04 M ethanolic sodium hydroxide at 46 C. the insecticidally more active octachloro isomer, I I A , and the nonachloro compound, I I I , reacted much more rapidly than the heptachloro compound, I, and the less active octachloro compound, I I B , showed no measurable reaction i n 43 hours. I n refluxing 1 M ethanolic alkali, the octa chloro compound, I I B , is reactive. The relative toxicities of these compounds toward houseflies (I4) are given i n Table I I I . Here again there is no correlation between chlo-
CRISTOLINSECTICIDAL ACTIVITY AND DEHYDROCHLORINATION
RATES
189
ride ion formation rate and relative toxicity, heptachloro compound I being the most effective insecticidally, yet of intermediate reactivity. The recent announcements of the insect toxicants Compound 118, 1,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexahydro-l,4,5,8-dimethanonaphthalene (IV) (1, 15), and C o m pound 497, l,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro-l,4,5,8-dimethanonaphthalene (V) (15) are of further interest. The official name aldrin has been desig nated for material containing at least 9 5 % of I V , and dieldrin for material containing at least 8 5 % of V .
Cl 01- -01 H H H - -H CL H H H H
CI
CL
Cl
01- -Cl
I H - -H > 0
H H
cr
Cl
Cl
IV
These compounds are much more toxic than chlordan (Table III), and yet are stable toward alkaline reagents (15), being unable to eliminate hydrogen chloride without the formation of a double bond at a bridgehead carbon atom. Thus i n this type of compound the conclusion must again be reached that dehydrochlorination with alkali and insectici dal activity have no systematic relationship. Biological systems are, i n general, specialized as regards steric factors. Although the biological chemistry involved i n the action of the chlorinated insecticides is still u n known, the data of this paper suggest the relative importance of steric factors i n the action.
Acknowledgment
The author wishes to acknowledge the aid of the Office of N a v a l Research, U . S. N a v y Department, for support of the work on elimination rates; of H . L . Haller, E . F . Knipling, W . V . K i n g , C . C . Deonier, and I . H . Gilbert of the Bureau of Entomology and Plant Quarantine, U . S. Department of Agriculture, for the use of unpublished insecticidal data; and of Rex E . L i d o v of Julius H y m a n & C o . for information regarding the polycyclic insecticides and for research samples.
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) Anon., Chem. Eng. News, 26, 3854 (1948). Busvine, J . R., J. Soc. Chem. Ind., 1946, 356. Busvine, J . R., Nature, 156, 169 (1945). Cahn, R. S., Chemistry & Industry, 1948, 291. Cristol, S. J., J. Am. Chem. Soc., 67, 1494 (1945). Ibid., 69, 338 (1947). Cristol, S. J . , Meek, J. S., Hause, N . L., Quant, A. J., Miller, H . W., and Eilar, K . R., Ibid., submitted for publication. Deonier, C. C., private communication. Deonier, C. C., Jones, . ., Haller, H . L . , Hinchey, E., and Incho, . H., Soap Sanit. Chemi cals, 22 (11), 118 (1946). Domenjoz, Helv. Chim. Acta, 29, 1317 (1946). Gilbert, I. H . , private communication. Haller, H . L . , Ind. Eng. Chem., 39, 467 (1947). Hammett, "Physical Organic Chemistry," Chap. VII, New York, McGraw-Hill Book Co., 1940. Lidov, R. E., private communication. Lidov, R. E . , Bluestone, H . , Soloway, S. B., and Kearns, C., A D V A N C E S I N C H E M I S T R Y S E R I E S , 1, 175 (1950). Margin, H . , J. Soc. Chem. Ind., 65, 402 (1946). Martin, H . , and Wain, R. L., Nature, 154, 512 (1944). Mller, P., Helv. Chim. Acta, 29, 1560 (1946). Snedecor, "Statistical Methods," 4th ed., p. 149, Ames, Iowa, Iowa State College Press, 1946.
Colorimetric Method for Estimating Small Amounts of Aldrin (Compound 118)

A. A. DANISH and REX E. LIDOV
Julius Hyman & Company, Denver, Colo.
A colorimetric procedure is described for the determination of small amounts of Compound 118 ( 1,2,3,4,10,10-hexachloro - 1,4,4a,5,8,8a - hexahydro - 1,4,5,8 - dimethanonaphthalene). Reaction with phenyl azide to form a dihydrotriazole derivative and subsequent treatment with diazotized dinitroaniline in strongly acid medium produce an intense red color. Amounts of the insect toxicant of 10 to 40 micrograms in the final 10-ml. aliquot are readily determined with a spectrophotometer. Commonly used insect toxicants do not interfere.
T h e synthesis of an alkali-stable highly potent insect toxicant, tentatively called C o m pound 118, has been announced recently (2). Structurally Compound 118 is 1,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexahydro-l,4,5,8-dimethanonaphthalene (I). T h e chemist r y and applications of this material have been discussed b y Lidov, Bluestone, Soloway, and Kearns (3). The availability of Compound 118 for wide scale experimentation i n the field of agricultural, household, and public health uses makes necessary a method for determining minute amounts such as would be present i n spray or dust residues on plants and i n biological fluids and tissues. A n analytical procedure based on the bicycloheptene structure of Compound 118 offers a more or less specific method, and is the subject of this paper. Compound 118 I Cl H Cl Compound 118-phenyldihydrotriazole II H
II + dinitrophenyl diazonium salt > colored compound (III) Phenyl azide reacts at the double bond of the halogen-free bicycloheptene ring of Compound 118 to form a phenyldihydrotriazole derivative (118-phenyldihydrotriazole, II). This type of reaction has been discussed b y Alder and Stein (1). The triazole derivative, i n turn, produces an intensely colored substance, I I I , with a diazonium compound under the experimental conditions' devised for this analysis. The reactions leading to the formation of the colored substance have not yet been fully elucidated, but i t appears that opening of the triazole ring as well as coupling is involved. The chemistry of the color formation w i l l be discussed i n another publication.
190
DANISH AND LIDOVESTIMATION OF COMPOUND 118
191
The aforementioned series of reactions provides a basis for a colorimetric analytical method for Compound 118 in which the commonly used agricultural chemicals do not interfere. The procedure described herein permits the estimation of as little as 10 micrograms of Compound 118, and has been successfully applied to the analysis of this insect toxicant i n insecticidal dusts, i n film residues on glass and paper, i n human and animal urine, and i n mixture with other insecticides. Application of this procedure to the determination of Compound 118 i n milk and i n spray and dust residues on plants appears promising. Method The analytical procedure for Compound 118 consists of the following basic steps: Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch033 Quantitative formation of 118-phenyldihydrotriazole (II) by heating Compound 118 with an excess of phenyl azide liquid, i n absence of solvent, and then removing unreacted azide with a mechanical vacuum pump. (Because of the small amounts of 118 and phenyl azide reacting, the presence of conveniently measured amounts of solvent makes the rate of dihydrotriazole formation too slow to be practicable.) Treating the triazole residue i n alcoholic hydrochloric acid with an excess of diazotized 2,4-dinitroaniline, and then acidifying strongly to produce an intensely red colored compound (III). (The presence of hydrochloric acid is essential. Other strong acids, in the absence of hydrochloric acid, do not lead to the desired color formation.) Evaluation of the developed red color, using a spectrophotometer and a standard transmittance-concentration curve from data on known amounts of purified 118-phenyldihydrotriazole. Preparation of Standard Optical Transmittance Curve of 118-Phenyldihydrotriazole Purified 118-phenyldihydrotriazole, prepared from Compound 118 and phenyl azide, was used for plotting a standard optical transmittance curve. A stock solution of the triazole i n hexane was made up and diluted to various strengths, and 1.0-ml. aliquots of the diluted solutions were carried through the procedure described below. The transmittance of the colored solutions obtained from 10 to 50 micrograms of the 118-phenyldihydrotriazole was plotted against concentration to make a standard curve. I n subsequent analyses, the amount of Compound 118 is readily calculated from the amount of dihydrotriazole formed. Absorption curves of the colored solutions were run on a Beckman spectrophotometer using 1.00-cm. quartz cells. A typical curve, shown i n Figure 1, has an absorption m a x i mum at about 515 millimicrons. The Coleman Junior spectrophotometer was used for routine determinations of Compound 118 throughout this study. Preparation of Standard 118-Phenyldihydrotriazole Curve Reagents. P H E N Y L A Z I D E (freshly distilled), boiling point 49-50 C , at 5 m m . The phenyl azide was prepared according to the method of Lindsay and Allen (4). COMPOUND 118. A material recrystallized from methanol and melting at 100-102 is satisfactory. A sample of crystalline Compound 118 may be obtained from Julius H y m a n & Company, R o c k y M o u n t a i n Arsenal, Denver, Colo. refluxing a solution containing 10.0 grams of Compound 118 and 4.0 grams of phenyl azide in 30 m l . of hexane for 40 minutes. The dihydrotriazole begins to precipitate out during the refluxing period. The white crystalline solid is collected b y suction filtration, recrystallized once from 1 to 5 benzene-ethyl alcohol, and dried i n a vacuum desiccator. Melting point is 174 C . (decomposes). (The melting point is determined b y plunging the capillary tube containing the triazole i n the melting point bath at 164 and heating at the rate causing a 2 rise i n temperature per minute.) P u r i t y may be determined by total nitrogen analysis (theory is 8.67%). Other reagents used are: 2,4-dinitroaniline, E a s t m a n ; sodium nitrite, finely powdered, reagent grade; concentrated hydrochloric acid; acetone, C.P .; phosphoric acid,
CRYSTALLINE 1 18- PHENYLDIHYDROTRIAZOLE . T h e dihydrotriazole is prepared b y
192
8 5 % , C.P .; absolute ethyl alcohol; concentrated sulfuric acid, C.P .; hexane, redistilled (Skellysolve B , boiling point 60 to 70); and sulfuric acid, 2 to 1, made b y mixing 2 v o l umes of concentrated sulfuric acid with 1 volume of distilled water. ethyl alcohol, 1 volume of concentrated hydrochloric acid, and 4 volumes of 2 to 1 sulfuric acid. It is made up just before use. (In the equation for calculation of results, this solution is referred to as Solution B.) DIAZOTIZED 2,4- DINITROANILINE . A slight modification of Schoutissen's procedure is employed (5). A solution of 1.5 grams of 2,4-dinitroaniline i n 30.0 m l . of concentrated sulfuric acid is prepared by heating to 90. The solution is cooled to 0 and then 0.7 gram of finely powdered sodium nitrite is sifted onto the sulfuric acid solution at 0. After standing for 1 to 2 hours, at a temperature below 15, the sodium nitrite dissolves. To the resultant solution, 40.0 m l . of 8 5 % phosphoric acid are added with stirring. The temperature should not exceed 40 during the phosphoric acid addition. After standing at room temperature for an additional 2 hours, the preparation is ready for use. The final solution is pale yellow and is stable at room temperature for at least a week. O n prolonged standing the reagent solution darkens progressively to a deep orange, and should be discarded. 0.1000 gram of 118-phenyldihydrotriazole is dissolved in 5 m l . of acetone and enough hexane is added to make 1000 m l . One milliliter contains 100 micrograms. 118 is dissolved i n hexane to make 1000 m l . of solution. E a c h milliliter contains 100 micrograms of Compound 118. Apparatus. A Coleman J u n i o r M o d e l 6 A spectrophotometer, w i t h tubular cells 9 m m . i n diameter, is used (Catalog N o . 6-302). Beakers; 10 to 1000-ml. volumetric flasks; 1-ml. volumetric pipet, serological pipet; steam bath; vacuum desiccator; vacuum pump; and water aspirator are also required. Procedure. F i v e aliquots of the triazole stock solution of 5, 10, 15, 20, and 25 ml. are introduced into 50-ml. volumetric flasks. E a c h aliquot is diluted to 50 m l . with hexane, making working standards equivalent to 10, 20, 30, 40, and 50 micrograms of t r i azole per m l . , respectively. Using a volumetric pipet, 1 m l . of each working standard is
STOCK STANDARD SOLUTION OF COMPOUND 118. E x a c t l y 0.1000 gram of Compound STOCK STANDARD SOLUTION OF COMPOUND 118- PHENYLDIHYDROTRIAZOLE . Exactly COLOR D I L U T I N G SOLUTION . This is prepared by mixing 5 volumes of absolute
193
quantitatively transferred to a cylindrical spectrophotometer cell and 1.0 m l . of hexane, which serves as a blank, is placed i n another cell. T w o drops of phenyl azide (30 to 40 mg.) are added to each cell, care being taken that none of the azide liquid strikes the glass walls. The hexane is evaporated completely at room temperature b y placing the cells i n a vacuum desiccator attached to a water aspirator. The evaporation requires 10 to 20 minutes. A drop of oily residue remains. This evaporation is handled conveniently b y placing the cells i n a beaker containing a 2.5-cm. level of mineral oil at room temperature and transferring the whole to the desiccator. The oil bath prevents excessive cooling of the hexane solution during the evaporation. After complete removal of solvent, the vacuum is broken gently and the colorimeter cells are then heated i n an oven at 75 to 80 C . for exactly 30 minutes. I t is during this heating period that, i n an actual analysis, any Compound 118 is quantitatively converted to the triazole. The excess phenyl azide is then removed i n vacuo at 1 to 2 m m . b y almost com pletely immersing each cell i n a beaker of water at 45 to 50 and attaching each cell d i rectly to the vacuum pump for 3 minutes. The nearly colorless film residue at the bottom of the cell is now ready for coupling with diazotized dinitroaniline. The film residue is dissolved i n 5.0 m l . of absolute ethyl alcohol and then 1.0 m l . of concentrated hydrochloric acid and 0.3 m l . of diazotized dinitroaniline solution are added. A serological 1.0-ml. pipet, graduated i n tenths, is used to measure out the diazotized d i nitroaniline solution. The solutions are mixed well and allowed to stand for 20 minutes, during which an orange color develops. Finally, 3.7 m l . of 2 to 1 sulfuric acid are added slowly to each solution to make a final volume of 10 m l . The solutions are mixed well and allowed to stand for at least 3 minutes. A n intense red color is produced at this point. I n the equations for calculation of results this solution is called Solution A . The blank solution is used to set the galvanometer index line at 100% transmittance at 515 m i l l i microns, and the transmittance of each red solution is measured. The standard curve is prepared b y plotting per cent transmittance against concentration expressed as micro grams of 118-phenyldihydrotriazole per 10 m l .
Determination of Compound 118 in Hexane Solutions

Reagents and apparatus are the same as those used i n the preparation of the standard curve. Procedure. A hexane solution of C o m p o u n d 118 is diluted or concentrated so as to bring the 118 content within a range of 15 to 150 micrograms per m l . I n cases where the hexane solution requires concentration, the evaporation is carried out i n a beaker on a steam bath with a gentle stream of air passing over the surface. The concentrated or d i luted solution of 118 is washed with hexane into a volumetric flask and made up to volume with the hexane washings. One milliliter of the adjusted Compound 118 solution is pre cisely measured into a spectrophotometer cell, 2 drops of phenyl azide are added, and the dihydrotriazole is quantitatively formed and then treated with diazotized dinitroaniline to produce the red color as i n the preparation of the standard curve. A blank, starting with 1.0 m l . of hexane and 2 drops of azide, is run at the same time. The final blank solution is set at 100% transmittance and the transmittance of the test solution is then measured. Reading from the standard curve, one obtains the number of micrograms of triazole.
Calculation of Results
The percentage of 118 present i n the samples analyzed can be obtained b y means ot the following equations.
% Compound 118 = /volume of Solution A +\ micrograms of dihydrotriazole ( volume of Solution ) read from standard curve \ volume of Solution A ' grams of test sample X 10,000
a l i
r a t i o
194
The 0.754 factor represents the ratio of the molecular weight of Compound 118 to that of the 118-phenyldihydrotriazole. Thus: Micrograms of Compound 118 = micrograms of triazole X 0.754 Using this conversion factor, the standard 118-phenyldihydrotriazole curve may be replotted, if desired, to read directly i n micrograms of Compound 118. I n that case the calculation becomes :
% Compound 118 = /volume of Solution A + micrograms of Compound 118 I volume of Solution ^ X aliquot ratio read from standard curve \ volume of Solution A grams of test sample X 10,000

The factor i n parentheses is omitted when Solution A is not diluted with Solution B . The aliquot ratio is the ratio of the total volume of the solution containing Compound 118 to the volume of that solution taken for analysis. The conversion of micrograms to grams requires a factor of 10 The conversion of the analytical result to a percental basis requires multiplication b y 10 . The figure 10,000 present i n the denominators of the equations above accounts for these two factors.
2
Notes on Procedure
On heating, phenyl azide decomposes partially to give nonvolatile products which form colors with diazonium compounds. T o minimize the presence of these impurities, the quantity of phenyl azide is limited to 2 drops i n the triazole formation step. If the color intensity of the final solution corresponds to more than 50 micrograms of triazole (15% transmittance), dilution of the sample and blank with color diluting solu tion will give a transmittance value within a more desirable range for color intensity meas urement. In equations for calculation of results the color diluting solution is called Solu tion B . Should i t be found that more than 250 micrograms of dihydrotriazole had been present before dilution, the entire analysis should be repeated, after readjusting the con centration of Compound 118 test solution to 20 to 40 micrograms per m l . , using hexane as the diluent. This step is necessary because the amount of phenyl azide i n the first determination may have been insufficient for quantitative conversion of all the C o m pound 118 to the dihydrotriazole. On the other hand, should the transmittance be equiva lent to less than 20 micrograms of dihydrotriazole,. the analysis should also be repeated, starting with 2 or 3 m l . of the hexane solution of 118 and 2 drops of phenyl azide, evapo rating the solvent i n a vacuum desiccator, and continuing the determination as i n the preparation of the standard curve. The transmittance readings should be taken within 5 to 10 minutes after red color development. The developed red color is stable on standing up to at least 2 hours when working with crystalline dihydrotriazole only; however, when phenyl azide is used, as i t must be i n an actual analysis for Compound 118, some of its nonvolatile thermal decom position products develop colors on standing. It appears that the decomposition of phenyl azide is significantly accelerated b y direct exposure to light. Consequently, during all steps of the procedure i n which phenyl azide is present, including that of its removal i n vacuo, exposure of the reaction mixtures to direct light must be avoided. The phenyl azide reagent should be stored i n the cold and distilled on the day it is used in order to minimize extraneous color formation. Complete removal of excess phenyl azide after triazole formation is necessary to en sure reproducible results. F r o m the manipulative standpoint, the critical step lies i n the vacuum evaporation of the solvent from the solution of Compound 118 and the two drops of phenyl azide. Care must be observed that no foaming or undue chilling occurs during the evaporation; undue chilling may cause some Compound 118 to crystallize out of contact with the phenyl azide and prevent quantitative formation of the dihydrotriazole.
195
The application of vacuum and its release must at all times be gradual, so that none of the crystalline Compound 118 or its dihydrotriazole is swept out b y a surging air stream. A two-way stopcock may be conveniently used for this purpose. The aliquots of Compound 118 solution should be pipetted at the same temperature as that at which it was made up to volume.
Effect of Evaporative Concentration of Hexane Solutions of Compound 118

Hexane was found to be a convenient extracting solvent for insecticidal dusts containing Compound 118. To determine whether any loss of Compound 118 occurs on evaporation, a stock solution of Compound 118 i n hexane was made up, and aliquots containing 20 to 250 micrograms of Compound 118 were measured into tall-form beakers and diluted to 250 m l . with hexane. The solutions were evaporated to 10 m l . on a steam bath with a jet of air passing gently over the liquid surface. Following the same procedure, solutions of C o m pound 118 of varying concentrations were reduced to convenient final volumes. After concentration, i n the manner described, the Compound 118 content of the final concentrates was determined. The data of Table I show a 2 to 6 % loss of Compound 118, depending on the volume of hexane evaporated. The loss of Compound 118 is roughly 2 to 3 % for each 250 m l . of hexane evaporated. Where a 94 to 9 8 % recovery is not sufficiently accurate for the objectives sought, a correction can be applied to compensate for evaporative loss. Table I. Recovery of Compound 118 after Evaporative Concentration of Hexane
Added 20 50 100 125 150 200 250 1000 500 100 Compound 118, y Recovered 19 49 97 120 142 188 241 920 460 97.5 Recovery, 95 98 97 96 95 94 96
Hexane Solution Concentrated, from 250 to 10 ml.
500 to 50 ml. 500 to 25 ml. 100 to 10 ml.
92 92 97.5
The complete procedure was tested by analyzing hexane solutions containing known amounts of Compound 118. The recoveries, shown in Table I I , average about 96.5%. Table II.
Taken for Analysis, 7 10 15 20 25 30 35 40
Analyses of Known Amounts of Compound 118

Found Present, 7 8.8 14.5 19.3 24.2 28.8 35.4 40.0 Recovery, % 88.0 96.5 96.5 96.0 96.0 101.0 100.0
To test further the accuracy of the method, synthetic mixtures of Compound 118 with its insecticidally active epoxide derivative, Compound 497, were prepared (2). The results, shown i n Table I I I , indicate that Compound 118 can accurately be determined i n the presence of gross amounts of structurally related material. Table III. Analyses of Known Amount of Compound 118 in Compound 497
Compound 118 Found, % 4.5 16.6 25.8
Compound 118 Added to Compound 497, % 3.8 16.0 25.3
196
This colorimetric procedure is applicable to the estimation of Compound 118 i n cow's urine. T o 100-gram batches of 1-day-old samples of cow's urine were added 0.05, 0.1, and 0.5 mg. of Compound 118 i n acetone to give 0.5, 1.0, and 5.0 p.p.m., respectively. The urines were then extracted with two 50-ml. batches of hexane. Occasional emulsions were broken b y centrifuging. The hexane extracts were dried with anhydrous sodium sulfate, filtered, evaporatively concentrated, and analyzed for Compound 118 as described under Procedure. The results of these analyses are shown i n Table I V . Similar experiments with human urine gave slightly better recoveries. Table IV.
Compound 118 Added, %
Recovery of Compound 118 Added to Cow's Urine

Compound 118 Found, P . P . M . Uncorrected for blank Corrected for blank 0.20 0.35 0.62 0.60 1.10 1.00 5.45 5.36
a a
Recovery Corrected for Blank, % 80 82 90 93 98 97
0 0 0.5 0.5 1.0 1.0 5.0 5.0 Calculated as Compound 118.
0.40 0.41 0.90 0.93 4.91 4.84
For analysis of Compound 118 i n commercial dusts or wettable powders, a preliminary extraction with hexane is necessary. A Soxhlet apparatus containing a sample large enough to be representative is satisfactory. A minimum of 30 minutes' extraction time has been used, and 1 hour is recommended.
Interferences
The commonly used organic insect toxicants do not interfere i n the analysis of C o m pound 118 b y this new procedure. Hexane solutions of chlordan, D D T , methoxychlor, hexachlorocyclohexane ( B H C ) , and toxaphene treated according to the procedure for determining Compound 118 gave a pale yellow color similar to that of the blank. Dimalone [bicyclo-(2.2.1)-5-heptene-2,3-dicarboxylic acid dimethyl ester] and Octacide 264 [the Af-octyl imide of bicyclo-(2.2.1)-5-heptene-2,3-dicarboxylic acid] do produce a red color with an absorption maximum in the same region as that obtained i n the analysis of Compound 118. However, because Dimalone is an insect repellent and Octacide 264 is a pyrethrum synergist, neither of these products is likely to be encountered i n commercial mixtures of Compound 118. The response to the colorimetric test for Compound 118 of some chemicals commonly used for insect control is listed i n Table V . Table V. Response of Some Substances Used for Insect Control to Color Test for Compound 118
Interfering Substances Repellent Pyrethrum synergist Noninterfering Substances Chlordan Methoxychlor Hexachlorocyclohexane (BHC) Toxaphene Compound 497
Dimalone
Octacide 264
Acknowledgment
The authors wish to acknowledge the assistance of Y u j i A . Tajima of this laboratory i n conducting the spectroscopic determinations and to Phebe Hines for aid i n carrying out the analyses.
197
Literature Cited
(1) Alder, K., and Stein, G., Ann., 485, 211 (1931). (2) Chem. Eng. News, 26, 3854 (1948). (3) Lidov, R. E . , Bluestone, H . , Soloway, S. B., and Kearns, C . W., ADVANCES I N C H E M I S T R Y S E R I E S , 1, 175 (1950). (4) Lindsay, R. O., and Allen, C. F. H . , "Organic Syntheses," Vol. 22, p. 96, New York, John Wiley & Sons, 1942. (5) Schoutissen, H . A. J . , J. Am. Chem. Soc., 55, 4535 (1933).
Polarographic Determination of O,O-Diethyl O-p-Nitrophenyl Thiophosphate (Parathion)

C. V. BOWEN and FRED I. EDWARDS, JR.
Parathion may be determined quantitatively by means of the polarograph. The electrolysis is carried out in an acetone-water solution with 0.05 potas sium chloride as the electrolyte, and 0 . 0 1 % gelatin as the suppressor at 25 0.5 C. An accuracy of 1% is obtained. Several commercial products were analyzed.
T h e only reported method for the estimation of 0,0-diethyl O-p-nitrophenyl thiophos phate (parathion) is a colorimetric procedure () based upon the reduction of the nitro group to an amino group with subsequent diazotization and coupling with iV-(l-naphthyl)ethylenediamine to produce a color that may be measured. This procedure has had a p plication i n the determination of spray and dust residues where 9 0 % recovery is satisfactory, but is not suitable for use i n the assay of technical materials. Consequently, reliable and sensitive methods of analysis are greatly needed for this new and highly toxic material i n insecticidal formulations. Because the reduction occurs so readily with zinc in the above-mentioned procedure and nitrobenzene (6) was the first organic compound t o be investigated with the polarograph, it was considered probable that parathion would be easily reduced at the dropping mercury electrode and thus be determined b y this means. Apparatus A Sargent M o d e l X X I polarograph was used i n this investigation. The reduction was carried out i n an -type electrolysis cell with a saturated calomel reference cell i n one arm (5). A thermostatically controlled water bath maintained the cell at 25 = * = 0.5 C . During the recording of the polarogram the air stirrer was stopped i n order to eliminate vibration and the heating system was disconnected at the bench outlet to remove the possibility of stray current effect (3). I t was observed that other operating electrical appliances, such as hot plates on the same bench, had a stray current effect on the polarograph. Preparation of Standard Curves The 0,0-diethyl O-p-nitrophenyl thiophosphate used i n the preparation of the standard curves was obtained b y isolation from a high-purity technical parathion accord ing to the method devised by Edwards and H a l l (#). It was a crystalline material that melted sharply at 6 C . The physical constants were in agreement with those published by Fletcher et al. (4). A sample of 0.4863 gram of this purified 0,0-diethyl O-p-nitrophenyl thiophosphate was dissolved i n acetone to make 1 liter of standard solution. A 20-ml. aliquot, con taining 9.73 mg., was placed i n a 100-ml. volumetric flask and 30 m l . of acetone were added. Then 0.35 gram of potassium chloride and 0.6 gram of acetic acid were dissolved in about 25 m l . of water and added to the acetone solution; 0.01 gam of gelatin was dissolved i n a few milliliters of water b y warming, cooled, and added to the above, and the
198
BOWEN AND EDWARDS-POLAROGRAPHIC DETERMINATION OF PARATHION
199"
solution was brought to the mark with water. (This solution is 0.05 with respect to p o tassium chloride, and 0.1 with respect to acetic acid, and contains 0.01% of gelatin and 5 0 % of acetone i n water.) The acetic acid was added to prevent any hydrolysis of the ester during the electrolysis. The sample side of the cell was emptied and rinsed by means of suction without being removed from the thermostat bath. The used mercury was retained i n the suction flask. The cell was rinsed well with acetone and then with a portion of the sample solu tion before being filled with the sample solution. Prior to the electrolysis oil-pumped nitrogen was bubbled through the sample solution for 10 minutes to remove dissolved oxygen. The nitrogen was passed through a 1 to 1 acetone-water solution before i t reached the sample solution. F o r electrolysis the dropping mercury electrode was placed firmly in the cell and the polarograph set to record the wave at 0 to 1.5 volts at a sensi tivity of 0.020 microampere with maximum damping. Waves were recorded i n duplicate for 0.020-, 0.030-, and 0.040-microampere sensitivity to allow for considerable leeway i n the size of the sample. The sensitivities of the polarograph refer to the microamperes corresponding to 1-mm. deflection of the recorder. Polarographic waves were obtained i n the same manner for 7.29-, 4.86-, and 2.43-mg. samples of parathion. Figure 1 shows the average wave height for from 2 to 10 determinations at each concentration plotted against the concentration to give the standard curves.
1 2
3 4 5 6 7 8 PARATHION - m g / ml.
10
Figure 1.
Standard Curves for Parathion Determination
Sensitivity, microampere per millimeter. A, 0.020; , 0.030; C, 0.040
After some of the standardization polarograms had been obtained, i t was decided that considerable time could be saved b y using an aqueous stock solution of twice the nor mality of potassium chloride and acetic acid desired i n the sample to be analyzed instead of weighing these materials for each determination. The gelatin was weighed fresh each day.
Analysis of Commercial Products

Technical parathion samples were analyzed i n the same manner as the standard samples (Table I). F o r dust formulations at least 1 gram was taken for a sample and
200
ADVANCES
IN CHEMISTRY SERIES
made to volume with acetone to obtain an extract containing approximately 1 mg. per m l . of parathion based on the manufacturer's claims. The sample was shaken intermittently for 1 hour, and allowed to stand for 10 minutes, and a portion was centrifuged i n a glass-stoppered tube until clear. A n aliquot of this clear solution to contain approximately 10 mg. was taken and the procedure described was followed. The recovery of parathion from dusts b y this procedure was checked by extracting two of these commercial dusts i n a Soxhlet apparatus. The results were found to be within the limits of accuracy of the method, as shown i n Table I I . Dusts of known parathion content prepared i n this laboratory were analyzed after Soxhlet extraction, and the recovery as shown i n Table I I was found to give results also within the limits of accuracy of the method. Table I. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch034 Analysis of Commercial Samples of Technical Parathion
Sample No. 1 Parathion Found, % 84.7 83.9 85.3 Av. 84.6 92.3 91.5 92.0 Av. 91.9 98.1 97.5 97.7 Av. 97.8 93.1 92.0 93.2 Av. 92.8 94.4 93.5 95.3 Av. 94.4
Table II.
Material Commercial dusts Sample 1 (25%)
Analysis of Parathion Formulations

Parathion Present, % ... Parathion Found Soxhlet extraction, % ... Flask extraction, % 24.0 24.0 24.1 A v . 24.0 24.0 24.1 24.0 A v . 24.0 0.97 0.92 0.94 Av. 0.94 24.0 23.7 23.9 Av. 23.9 23.9 23.7 23.8 A v . 23.8 9.7 9.7 9.8 9.7
Sample 2 (25%)
...
Sample 3(1%)
...
24.4 24.1 24.4 Av. 24.3 ...
Wettable powders Sample 1 (25%)
...
...
Sample 2 (25%)
...
Synthetic dusts Sample 1
23.3 23.3 23.6 Av. 23.4 ...
10.0
Av. Sample 2 12.2 12.2 12.0 12.1 A v . 12.1 29.2 29.3 29.3 Av. 29.3
Sample 3
29.1
BOWEN AND EDWARDSPOLAROGRAPHIC
DETERMINATION OF PARATHION
201
Discussion
Normal curves for the polarograms were obtained with the technical materials as well as with the purified sample. The decomposition potential of 0.30 volt and a half-wave potential of 0.39 volt were obtained against the saturated calomel electrode. p-Nitrophenol, which is a major contaminant of the technical parathion, does not re duce at the dropping mercury electrode until after the parathion has been completely re duced, and consequently does not interfere with the curve obtained i n the analysis. The decomposition and half-wave potentials for p-nitrophenol under the conditions for the determination of parathion were found to be 0.45 and 0.68 volt, respectively. Diethyl p-nitrophenyl phosphate, the oxygen analog of parathion, was investigated to ascertain whether it would interfere, if present, i n the determination of parathion. It was found, however, under the conditions used i n this method to have a decomposition potential of 0.37 volt and a half-wave potential of 0.47 volt. A mixture consisting of one-third parathion and two-thirds oxygen analog, instead of giving the anticipated broken wave beginning at the decomposition voltage for para thion, gave a normal curve with a decomposition potential of 0.34 volt. This i n d i cates an interference if small amounts of the oxygen analog should be present, a situation not likely to occur with present methods of synthesis (4) The polarographic method of analysis of parathion as described here has an accuracy of 1%, and 2 mg. of ,-diethyl O-p-nitrophenyl thiophosphate per 100 m l . of solution are apparently a minimum concentration for the sensitivities investigated. However, the polarograph used is equipped with resistors, so that a sensitivity of 0.003 microampere per millimeter may be used. A t this sensitivity it would be possible to obtain a sufficient wave height to determine parathion at a concentration of less than 1 p.p.m.
Literature Cited
Averell, P. R., and Norris, M . V., Anal. Chem., 20, 753-6 (1948). Edwards, F . I., and Hall, S. ., Ibid., 21, 1567 (1949). English, F . L . , Ibid., 20, 889-91 (1948). Fletcher, J. H . , Hamilton, J . C., Hechenbleikner, I., Hoegberg, . I., Sertl, B. J., and Cassaday, J. T . , J. Am. Chem. Soc., 70, 3943-4 (1948). (5) Kolthoff, I. M . , and Lingane, J . J . , "Polarography," New York, Interscience Publishers, 1946. (6) Shikata, M . , Trans. Faraday Soc., 21, 42-52 (1925). (1) (2) (3) (4)
Effect of Acetic Acid on Recovery of Ethylene Dibromide from a California Soil

W. J. HANSON
Great Western Division, The Dow Chemical Company, Seal Beach, Calif.
In the evaluation of ethylene dibromide as a soil fumigant, a sensitive procedure was needed for determining concentrations lower than 0.2 mg. per cubic inch in soil. When the method of Brenner and Poland was used, addition of a small quantity of acetic acid considerably increased the percentage of ethylene dibromide recovered.
the evaluation and development of ethylene dibromide (1,2-dibromoe thane) as a soil fumigant or disinfectant for the sugar beet wireworm Limonius californicm (Mann) and various nematodes, i t was desirable to study its behavior i n the soil. This chemical is usually ejected into the soil at the rate of 2 to 4 gallons per acre from shanks 12 inches apart and 6 inches deep. If 2 gallons of ethylene dibromide were evenly distributed, with out loss, i n the top foot of soil, the amount i n each cubic inch would be 0.217 mg. H o w ever, i t is unlikely that the distribution would be uniform. Accordingly, i t was necessary to develop a procedure sensitive enough to determine concentrations of ethylene dibro mide lower than 0.2 mg. per cubic inch before studying the manner i n which i t distributes itself i n soil. Dillon, Young, and Lucas (3) described a method for determining dibromobutanes i n 9 9 % methanol. D i l l o n (2) expanded this work to include a procedure for determining ethylene dibromide. Brenner and Poland (1) described a procedure i n which they deter mined 1.000 mg. of ethylene dibromide with a recovery of 65.8%. Equations 1 and 2 show the main reactions that occur. C H Br + 31- > C H + 2Br" + I "
2 4 2 2 4 3
(1) (2)
I - + 28,0s 31- + S 0
3 4 6
When 0.50 mg. of ethylene dibromide was added to 70 grams of air-dried soil and sub sequently removed b y steam distillation, it was not possible to detect it b y their procedure. Inasmuch as the soil used was slightly alkaline ( p H 7.70) i t was believed that some of the triiodide produced i n the soil distillate b y Reaction 1 would be lost. T h e addition of a small quantity of acetic acid to this distillate considerably increased the percentage of ethylene dibromide recovered i n the analysis (Table I ) . However, the addition of acid aids the oxidation of iodide ion to triiodide ion b y oxygen of the air according to E q u a tion 3. 4H+ + 6 1 - + 0 21," f 2 H 0 (3)
2 2
B y adjusting the acid concentration i t was possible to minimize both Reaction 3 and the loss of triiodide ion and thus modify the procedure of Brenner and Poland (1) sufficiently to obtain a 59.6% recovery when 0.125 mg. of ethylene dibromide had been added to 70 grams of air-dried soil. Larger quantities could be recovered more com pletely.
2 0 2
HANSONRECOVERY OF ETHYLENE DIBROMIDE
203
Table I.
Effect of Adding Acid in Analysis of Ethylene Dibromide in Soil

Acetic Acid Added, Ml. 0.15 0.15 0.15 0.15 0.00 0.00 0.15 0.15 0.00 0.00 0.15 0.15 0.00 0.00 0.15 0.15 0.10 0.20 0.30 0.40 0.15 0.15 0.332 0.330 0.123 0.149 0.507 0.517 0.328 0.394 0.699 0.719 0.910 0.910 0.913 0.912 1.18 1.17 Ethylene Dibromide Re covered (Cor rected for Blank), Mg. 0.073 0.076 0.163 0.160 Ethylene Dibromide Equivalent to Blank, M g . 0.055 0.060 0.047 0.055 0.068 0.055 0.049 0.066 0.066 0.049 0.047 0.060 0.068 0.049 0.047 0.066 0.068 0.088 0.088 0.088 0.074 0.066
Ethylene Dibromide Added, Mg. 0.125 0.250 0.500 0.500 0.750
Recovery, 58.4 60.8 65.2 64.0 0.0 0.0 66.4 66.0 16.4 19.9 67.6 68.9 32.8 39.4 71.7 73.7 74.0 74.0 74.2 74.1 78.7 78.0
0.750 1.00 0.975 1.23
1.50
Reagents
Ethylene Dibromide. Technical material was washed three times with concen trated sulfuric acid, rinsed with water, and dried over calcium chloride. I t was then dis tilled through 1 foot of glass beads. The fraction boiling between 130.2 and 131.2 C . was used. E v e r y third day an aqueous solution was prepared by weighing approximately 0.5000 gram of ethylene dibromide into 2000.0 grams of water. I t was stored i n a dark bottle to minimize light decomposition. Acetic Acid, glacial, U . S . P . , 9 9 . 5 % . Potassium Iodide. Iodate-free reagent grade was screened so that a l l crystals used were between 2 and 5 m m . i n diameter. Starch Indicator. F i v e grams of soluble starch were dissolved i n 1 liter of dis tilled water. A few drops of toluene were placed on top of the solution as a preservative. Potassium Iodate. A n a l y t i c a l reagent grade was dried for 4 hours at 110 C . i n the oven. Approximately 0.00300 solution was made b y making 100.0 m l . of 0.1500 solution and diluting a 10.00-ml. aliquot to 500.0 m l . Sodium Thiosulfate. A 0.00250 solution was made b y dissolving 0.63 gram of sodium thiosulfate and 0.10 gram of sodium carbonate per liter of freshly boiled water. It was standardized each day against approximately 0.00300 potassium iodate solution. Ethyl Alcohol. F o r m u l a 3A, 9 5 % e t h y l alcohol denatured w i t h 5 % commercially pure m e t h y l alcohol. Soil. A p p r o x i m a t e l y 5 pounds of air-dried H a n f o r d fine sandy loam soil were selected from a lima bean field i n an area where a l l plants had been killed b y wireworms. The p H of a water slurry of this soil was 7.70. I t was screened through a 20-mesh sieve, blended, and stored i n a closed can.
Procedure
In some of this work soil samples were desired from fields that had been treated with ethylene dibromide. Samples were taken with a thin-walled, cylindrical, sampling tube which took cores 1.0 inch i n diameter and 1.0 inch long. Three cores were placed i n each 250-ml. widemouthed Erlenmeyer flask. I n the laboratory the rubber-stoppered flask was shaken to break up the cores and 5 m l . of water were added immediately before analysis. However, i n this preliminary work to standardize a procedure, a 70.0-gram portion of air-dried soil was weighed into each flask. W i t h a 50-ml. buret the quantities of aqueous ethylene dibromide solution necessary to give 0.125 to 1.500 mg. of this chemical
204
were measured into each sample of soil. Enough water was added to bring the total volume of solution and water added to the soil to 10 m l . The ethylene dibromide and water were distilled from the soil b y heating i t for 1.5 hours i n an oil bath maintained between 125 and 130 C . A blank containing no ethyl ene dibromide was run simultaneously with every three samples, a l l of which were heated in a common oil bath. Each water-cooled condenser was modified by bending the inlet down and fitting it with a rubber stopper which accommodated the wide-mouthed flasks. The outlet of each condenser was bent so that it dipped below the surface of 50 m l . of ethyl alcohol contained i n a 250-ml. receiving flask with a standard-taper neck. It was necessary to avoid drafts and to maintain the temperature of the bath as constant as possible; otherwise, alcohol would climb up the condenser from the receiving flask. When this began to happen it was usually necessary to lower the receiving flask just long enough to allow air to enter the condenser. A t the end of the distillation the condenser was rinsed by forcing 15 m l . of 3 A ethyl alcohol through it and into the distillate by connecting a separatory funnel to the upper condenser opening with a rubber tubing and blowing the alcohol through. The re ceiving flask was removed and 0.15 m l . of glacial acetic acid was added with a 0.200-ml. serological pipet. After addition of a boiling chip, the flask was fitted to a standardtaper condenser and placed on an electric hot plate, and the distillate was boiled several minutes before 5 grams of potassium iodide crystals were introduced b y dropping them down the condenser. Twenty-five milliliters of ethyl alcohol were added to wash down any potassium iodide crystals that stuck i n the condenser. The solution was refluxed gently for 2.5 hours. A t the end of this period the solution was removed from the condenser while still hot and titrated immediately with 0.002500 sodium thiosulfate before any appreciable oxygen could be absorbed and oxidize iodide ion to triiodide ion. The disappearance of the yellow color of triiodide ion against a white background was used for the end point. These solutions usually had a slight brown tint at the end point, which was assumed to be organic matter distilled over from the soil. Accordingly, the blank was usually titrated first and its final color was used as a standard end point color for the other three solutions run with it. The results were corrected by subtracting the amount of thiosulfate used for the blank run parallel with each determination. Calculations. M l . of thiosulfate X 93.94 = m g . of ethylene dibromide.
Results and Discussion

It was necessary to analyze the soil samples as soon as possible to avoid loss of ethyl ene dibromide. The results shown i n Table I demonstrate the value of adding acetic acid i n this procedure. Nothing was gained b y adding more than 0.15 m l . to each reaction medium. The error introduced by Reaction 3 was further diminished b y flushing out much of the oxygen from the refluxing system b y boiling the contents before adding the potassium iodide. Crystals between 2 and 5 m m . were used because smaller ones had a tendency to stick to the walls of the wet condenser. E a r l y i n this work it was noticed that crystals of potassium iodide which were not washed down were often partly oxidized to iodine, which worked down into the flask and caused high results. Thus i t was important to wash all the potassium iodide down the condenser with water. Crystals of potassium iodide were used rather than a solution because this avoided the possibility of such a solution being oxidized to contain triiodide ion. The disappearance of the faint yellow color of triiodide ion was used to determine the end point of the titration with sodium thiosulfate. This gave better results than were obtained when the solution was diluted with sufficient water to use the starch t r i iodide end point i n the presence of ethyl alcohol. I n a method subject to both oxidizing and reducing side reactions i t was necessary to run a blank containing everything but ethylene dibromide with each set of three analy ses run i n parallel. Possible inaccuracies resulting from impurities i n the ethyl alcohol or other reagents were also corrected b y the blank. However, blanks were not always foolproof. Carelessness i n treating any flask differently from those run parallel with i t resulted i n errors. Accordingly, all runs were at least duplicated.
HANSONRECOVERY OF ETHYLENE DIBROMIDE Acknowledgment
205
The author wishes to express his gratitude to Robert P . Huston for his assistance i n the experimental work and to Joseph M . Rule for helpful suggestions. Literature Cited (1) Brenner, M . W., and Poland, G. L., Ind. Eng. Chem., Anal. Ed., 10, 528-9 (1938). (2) Dillon, R. T., J. Am. Chem. Soc., 54, 952-60 (1932). (3) Dillon, R. T., Young, W. G., and Lucas, H . J., Ibid., 52, 1953-64 (1930).
Multiple Responsibilities of the Insecticide Chemist

DON D. IRISH
The Dow Chemical Company, Midland, Mich.
The chemist should recognize the requirements for a new agricultural chemicaleffectiveness, economy, specificity, and safety. His responsibilities are to effect cooperation with all contributing researchers, increase specificity of action, make available the most practical materials we have today, and develop new chemicals which better fulfill the over-all requirements.
T i m e s change. The transition from one basic way of thinking to another takes place gradually, but the realization that we have gone through such a metamorphosis sometimes comes rather suddenly. We realize today that we are i n a new era of insecticide chemistry. I t might be called the D D T era as compared with the former lead arsenate era; it would be better perhaps to call i t the era of specific organic insecticides as compared with the past era of shotgun inorganic insecticides. F r o m a different point of view, we also recognize a parallel change i n thinking. Perhaps this new era i n insecticide chemistry should be dubbed the era of conscious responsibility. If we are to recognize our responsibilities i n introducing a new chemical substance with potential usefulness i n the agricultural industry, we must find some way of classifying its basic requirements and reducing them to the simplest terms. When we have these basic requirements well i n hand, we can more readily recognize our responsibilities. There are many difficulties i n attempting to classify all the complex requirements of an insecticide according to a simple scheme, but they may be arbitrarily placed under four simple headings: that i t be effective, economic, specific, and safe. These terms cover a multitude of detailed problems.
Effectiveness
This requirement is obvious, but one point should be recognized. The term "effect i v e " refers to practical control and not necessarily to k i l l . The aim is the maximum of quality of product and maximum productivity of the host plant through the prevention of damage by the pest. If we can accomplish this by interference with the activities of the pest, such as prevention of multiplication or prevention of invasion, i t is as satisfactory as the kill of the individual insect.
Economy
This subject is more complicated than it at first seems. This requirement may be defined as the lowest cost to society per unit of high quality agricultural product over several years. B y requiring a period of several years, we take into account the possibility that the chemical may affect the potential productivity or quality through a sustained effect on the host plant or its environment. I t must be calculated on the basis of the
206
IRISHMULTIPLE RESPONSIBILITIES OF THE INSECTICIDE CHEMIST
207
quality and cost to society per unit of the final agricultural product. The cost per pound of the insecticidal chemical is obviously not the answer, nor is the cost per acre. Other factors i n the economy of production of an insecticide material are often overlooked. E a c h product represents a major, capital investment i n productive capacity. Although i t is necessary to take advantage of scientific developments, so that our agricultural industry will have the best possible protection of its product, we should not jump into a new development without taking into account all the factors involved i n its use and in so doing place a capital investment i n manufacturing capacity which must be discarded in a short time to be replaced by another large capital investment. The end result would be a n over-all increase i n cost of agricultural chemicals, and an over-all increase i n cost of the final agricultural product. The long-term economy represented b y the introduction of a new substance into the agricultural industry is not always recognized i n a l l its ramifications b y the chemist.
Specificity
This is the most difficult and varied requirement for a new insecticide. I t is desirable to control a pest insect with the least possible undesirable effect on any other factor i n the complex biological system within which we are operating. The practical agricultural field man will inform us immediately that i t is necessary to control a wide variety of organisms. F r o m a practical point of view, i t is desirable to have a chemical substance which i n one application will control a great many different pest organisms. A s we broaden the spectrum of effectiveness against pest organisms, we are at the same time broadening the spectrum of activity against a wide range of other organisms, many of which are beneficial and necessary to the biological balance. Among the beneficial organisms is always the host plant and there are other plants of desirable nature i n the immediate area. There are desirable insects, which are necessary for the pollination of the host plant or of other plants in the area, and there are organisms which are highly beneficial as predators or parasites i n controlling undesirable pest insects. The effect of D D T on the predators and parasites of the red mite is well known. The effect of insecticides on pollinating insects did not begin with D D T but has long been with us. The need for greater specificity must always be qualified b y economic and practical usability. *
Safety
Although the requirement for safety to the operator i n handling an agricultural chemical and safety to the final consumer of the agricultural product is too obvious to require argument, the matter of attaining this safety is not so obvious. I n handling and applying agricultural chemicals, the problem is not solely the toxicity of the substance to man. The practical question iscan i t be handled b y reasonable operating procedures without encountering difficulties due to health hazards? A few materials are so low i n toxic effect to man and animals that an individual is not likely to get into trouble with them under ordinary conditions of use. Piperonyl butoxide, methoxychloro, and the di(4-chlorophenoxy)methane (Neotran) would probably fall i n this category i n the insecticide field. This is the direction i n which we should attempt to move with new insecticidal materials; however, we must always balance off this requirement with the requirements of effectiveness and economy. I t seems unreasonable to limit the use of a material that has the effectiveness, economy, and specificity desired simply because it has a fairly high toxicity to man, if i t can be handled without hazard to the operator. Our decision then should be based on the practicability of handling a material i n a way that will not represent a hazard to the operator. We must consider the effect on the quality of the agricultural product and particularly the existence of any residue. Such residue, if any, must present no hazard to the final consumer of the agricultural product, whether man or animal.
The Chemist's Responsibilities

W h a t responsibilities do these factors place upon us i n developing a future program? The chemist today recognizes more than ever the necessity for taking into account all these
208
requirements when designing a molecule which he wishes to introduce into the agricultural industry. If, however, the chemist is attending to his job of synthesizing a new organic chemical i n the chemical laboratory, he does not have time to become thoroughly acquainted with the many biological problems introduced by a new chemical substance in a complex, biological system such as the agricultural industry. He must depend to a great degree upon the biologists who are working i n that field for this information. This i m plies a much greater cooperation between the chemist and the biologist i n future developments. Our first responsibility i n designing our future program is to effect the greatest possible cooperation between a l l the groups that are in a position to contribute to this very important fieldthe chemist, the biologist, and the practical man i n the agricultural field. Present demands require moving i n the direction of greater specificity for the purpose of improving effectiveness and at the same time reducing as far as practicable the side effects on other desirable living organisms i n the system. F r o m other biological fields, we can glean the fact that it is possible to control a particular species of organism with high specificity. E a c h species of organism has within itself certain biological mechanisms which are unique to that species of organism. We know enough about specific antagonists for certain biological systems to know that with the proper knowledge of these systems, we could control an individual species of organism i n the presence of a great variety of other organisms. We can look forward to such developments i n the very near future. One of the best illustrations of high specificity is di(4-chlorophenoxy)methane (Neotran), which has been used for some time commercially i n the field, principally for the control of the citrus red mite. So far there has been no indication of undesirable effect upon pollinating insects or on desirable predators or parasites. There are advantages in moving i n the direction of greater specificity for the action of agricultural chemicals, but this desirable characteristic must be balanced with the effectiveness and practical economy of today. Our second responsibility is to recognize the possible advantages of increased specificity for our insecticidal chemicals, qualifying this with the best balance of the practical needs of the agricultural industry. Because of the requirements of circumstances, it is impossible to wait until the ideal combination which should be met in the development of a new insecticide is attained. We are faced today even more than i n the immediate past with the necessity of protecting our food and fiber crops from depredations. Our third responsibility is to make available the best material we have today, which will aid i n the production of our most critically needed food and fiber crops. A t the same time we must be looking for new materials. Year by year there is an i n crease i n crop concentration. This is a natural "enrichment culture" for increasing pests. There seems also to be some increase i n resistance of the insects to older insecticides. This means running hard to stay i n the same place, but we must not stay i n the same place. We must drive ahead. Therefore, our fourth and last responsibility is to develop much better materials i n the future, materials with the best balance of the four major requirements: effectiveness, economy, specificity, and safety,
Responsibilities of the Chemist in the Development of Insecticides

W. H. TISDALE and JONATHAN W. WILLIAMS
Grasselli Chemicals Department, E. I. du Pont de Nemours & Company, Inc., Wilmington, Del.
The chemist must work with the entomologist, the toxicologist, and others concerned in the formulation of new and better insecticides, or in the improvement of old ones. All formulations must be tested to determine their insecticidal efficiency, as well as their toxicity to warm-blooded animals, before manufacture on a large scale is begun.
T h e successful development of insecticides requires a broad and thorough knowledge of the ever-changing and unfilled needs for better products, and of the fields of chemistry, physics, engineering, and biology that pertain to the development of better insecticides. The research chemist must understand and appreciate teamwork. The development of insecticides depends on close cooperation of the research, manufacturing, and analytical chemists with the entomologists, toxicologists, and others who m a y be directly or i n directly interested i n the program. I t is especially important that the research chemist have a thorough knowledge of organic chemistry and of the methods of organic synthesis. H e should also have a sufficient knowledge of biology to enable him to understand the biochemical structures and functions of insects and their plant and animal hosts on which the insecticides are to be used. Such information is helpful i n understanding the action of insecticides and may also point the way to the discovery of more effective and safer chemicals for insecticidal use. The chemist should have a general knowledge of chemical research i n other laboratories, from which products of insecticidal value may be obtained. Needs for Better Insecticides More effective and safer insecticides are needed, i n spite of the extensive progress made i n recent years. The chemist should familiarize himself with the needs i n the agricultural, storage, household, livestock, and industrial fields. The weak and strong points of the products i n use should be understood. Some of the commonly used products need better formulations, or better methods of application with the use of more effective supplements. Better products should replace some of those now i n use. Statistical evidence of ample potential should be available before work on a problem is begun. Several factors may govern the fitness of a product for a given use. Changes i n biological factors may render the insecticide unsuited for use. A new variety of the host, or crop plant, may be injured b y the insecticide, or a strain of the insect that is resistant to the chemical may evolve as a result of continued use. I n such cases a modification of the insecticide to render i t more effective, or a replacement, must be found. Certain insecticides, including the arsenicals and some of the newer chlorinated compounds, although they are safe when applied to foliage, accumulate i n the soil to the extent of eventually causing injury to crops. A new insecticide must be compatible with other insecticides, fungicides, and supple-
2 0 9
210
merits with which it may be used. This involves the need for a knowledge of all such compounds that come into extensive use. For some important insect pests there are still no satisfactory chemical controls. Such problems should be given due consideration i n the development program. M a n y of these problems appeared to be solved with the discovery of D D T , benzene hexachloride (hexachlorocyclohexane), and some of the more recent insecticides. Further studies of the toxicity of some of these products to warm-blooded animals have raised the important question of the advisability of continuing their use where food and feed products are concerned. Considerable attention is being centered on rinding safer analogs, such as T D E and methoxychlor, and new and better insecticides.
Synthetic Organics
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch037 Synthetic organics dominate the field of new insecticides. M u c h time and money have been spent i n attempting to determine the chemical structures of rotenone, pyrethrum, and other natural insecticides, and to reconstruct them through synthesis. A great deal has been learned about the chemical structures of these compounds, but little success has been attained toward synthesis. Nicotine and, recently, a pyrethrumlike compound are exceptions, but the synthesis of nicotine is too expensive to be practical. The basic information obtained has possibly been helpful i n directing the thoughts of the chemist to the synthesis of entirely new compounds. A l l available information that offers possibilities must be used. However, what appears to be logical theory may not prove up i n the selecting of chemicals for insecticidal purposes. The chemical structure that, according to such theory as can be drawn from available knowledge, should be effective often proves ineffective, whereas structures considered ineffective under the same system of theorizing may prove to be effective. Much still depends on the " c u t and t r y " method. The chemist should explore the fields of chemicals with structures related to compounds known to be effective. H e should also study new classes of structures. Safety appears to lie i n numbers. The failure of several members of a new class of compounds does not prove that there are no effective members i n the group. Only certain of the analogs of D D T are good insecticides. E v e n D D T is not effective on some species of insects. Only the gamma isomer of benzene hexachloride is highly effective. The possibility of finding an insecticide that is safe to plants and warm-blooded a n i mals and universally effective appears remote. However, investigations have uncovered a few very promising products. A n outstanding example is methoxychlor, one of the analogs of D D T , which is far less toxic to warm-blooded animals than D D T . Poisonous insecticides are being used extensively. However, safety to plants and warm-blooded a n i mals, including man, should be one of the leading goals of the development program. The statement that " i t is hard to find a chemical that w i l l k i l l protoplasm and not k i l l protoplasm" overlooks the fact that some chemicals have properties that enable them to reach the living protoplasm of one organism and not that of another. M a n y factors may be involved. The chemist should be prepared to take full advantage of such differences. A thorough understanding of the chemical and biological actions involved is valuable.
Formulation
The effective use of insecticides depends to a large extent on formulations prepared for or adjusted to specific uses. Seldom is an insecticide used i n its pure or technical form. I n order to do an efficient job of formulating insecticides, the chemist should know how they are to be used and b y what type of machine they are to be applied. Is the insecticide to be used as a spray, mist, fog, dust, aerosol, or dip? W i l l i t be applied to plants, v a l u able animals, or humans? W i l l i t come i n contact with humans or products that may be used for food b y humans or valuable animals? W i t h this information at hand, suitable carriers, solvents, and wetting, emulsifying, spreading, penetrating, stabilizing, and sticking agents may be selected for trial. A n extensive knowledge of possible supplements is desirable. The supplements must be compatible with the active chemical and with other
TISDALE AND WILLIAMSRESPONSIBILITIES IN DEVELOPMENT OF INSECTICIDES
211
supplements used. The use of synergists may be considered. T h e finished formulations must be stable i n storage and compatible with other chemicals, including other insecticides and fungicides with which they may be used. This involves a knowledge of the chemistry of fungicides and other chemicals that may be used i n conjunction with the insecticide. The preparation of formulations may involve the principles of physics and mechanics. T h e preparation of emulsions, fine particle materials, etc., constitutes an important part of insecticide development. A knowledge of the kinds of equipment used i n the preparation of formulations enables the chemist to handle the job with greater facility. Formulation is not confined to the preparation of suitable mixtures of new chemicals. M a n y old or established insecticides have been improved and i n some cases have been adapted to new uses through the development of new and suitable formulations. T o see and take advantage of these opportunities, the chemist must be familiar with the weak and strong points of the products i n use. Close cooperation with entomologists is necessary. A l l formulations should be thoroughly tested i n the laboratory and/or greenhouse to determine their insecticidal efficiency. Products that survive reasonably severe preliminary tests are ready for field trials. Extensive chemical work often is required to readjust the formulations as the laboratory and field trials are i n process. I t may take months or even years to arrive at a suitable formulation for some uses after the chemical is known to have outstanding merit. E v e n when a formulation has been proved satisfactory for a certain use, much remains to be done i n the development of suitable formulations for other uses and i n following through with the effective product. I t may be desirable to develop means of removing residues of the product from treated surfaces.
Toxicology
Among the most important characteristics of an insecticide is its safety to humans. In recent years the United States Government, state authorities, and commercial organizations have given special and extensive attention to toxicity problems and to the safe use of toxic products. N e w insecticides are being thoroughly studied b y competent toxicologists. Unfortunately, some of the outstanding new chlorinated compounds are not as safe as they were once thought to be. I t has been necessary to modify or even withdraw claims concerning their safety to humans and specify methods of safe use, but advantage is still being taken of the original claims of safety i n some Latin-American countries, for instance, where poisonous products are recommended for use on stored grain and other food products. In order to avoid hazards to the chemist and his associates as well as to the user and consumer of treated products, a chemical showing sufficient promise should be subjected to acute toxicity tests b y a competent authority i n its early stages of development. Further experimental work should be governed b y the outcome of these tests. If toxic, i t should be handled with care. If symptoms of poisoning do not occur, the product should still be handled with reasonable care until chronic toxicity studies can be made. Such studies are expensive, and, as a rule, elaborate chronic studies are not considered necessary until it is reasonably certain that the product w i l l be put into use. A t this stage the question of public health becomes an important factor. Further toxicological studies, including those of chronic nature, may be required, so that effective directions for safe use may be issued. Attention should be given to the safety of any new formulations of the product once i t is i n use. New or modified formulations of old poisonous insecticides should be considered and the necessary directions for safe use be given. This may or may not involve the need for toxicological investigations.
Manufacture
After a product has passed the critical tests as an insecticide and a preferred process of manufacture has been decided upon, full information concerning use, potential raw m a terials, and process is the basis for the preparation of firm cost estimates. These estimates may cover production on a small or pilot plant scale, or on a large scale. If estimates are favorable, the process information is passed along to the manufacturing unit which, as a
212
rule, prepares for manufacture on a pilot plant basis. The research chemist should m a i n tain close cooperation with the manufacturing unit to ensure getting the process properly established for uniform production of a high-grade material. The analytical chemist should make regular checks on quality of production. When this pilot plant product goes into limited commercial use, the entomologists and chemists concerned should follow the results closely, to be sure that the product is up to standard i n a l l respects under different environmental conditions. Its stability under different storage conditions should be studied. Compatibility under conditions of use with other products with which it may be used is important. Everything possible should be done to assure its success at this point and to be sure that it merits large scale production. If the product survives the pilot plant stage of development and sufficient potential use is indicated, it passes to large scale production, where the responsibility shifts largely to the manufacturing and analytical chemists to produce a uniform, high-grade product. Interest of the research chemist should continue through cooperation with the manufacturing chemist, the entomologist, and the technical sales service men with the purpose of maintaining high efficiency and correcting any unforeseen weaknesses in the product.
Pest-Control Chemicals in the Production of Food

L. S. HITCHNER
National Agricultural Chemicals Association, Washington, D. C.
The use of agricultural chemicals makes possible higher yields and food products of higher quality and contributes more than any one other factor to lower production costs. At the same time the farm has been made a virtual factory in a rural setting.
I oday the agricultural chemical industry has made available, through chemical research, the largest selection i n history of agricultural chemicals for the control of pests. These agricultural chemicals are making possible higher yields and products of higher quality. I n this day of potential surplus they are contributing more than any one single factor to lower production costs.
Effect of Agricultural Chemicals on Crops

Potato yields, on a national basis, have been increased one third, largely because of more effective pest control. After 10 years of research on wire worm control i n California, the application of recommended agricultural chemicals on lima beans to control this pest added $7,500,000 i n i n come to growers i n 1948. Only i n recent months has i t been known that this same pest reduces the production of sugar cane i n some areas b y nearly half; consequently i t is now possible to double the yield i n those areas b y controlling this one insect. The production of livestock and livestock products has been greatly increased through the effective application of agricultural chemicals, as evidenced i n the control of insects and diseases on and around dairy cattle, which has resulted i n many instances of increased milk production ranging between 15 and 2 0 % . I t has been reliably estimated that the treatment of beef cattle for insect pests has resulted i n additional gains of 50 pounds per head of beef animals treated. The dollar value of these increases i n both beef and milk has been estimated at $54,000,000 per year. Several examples demonstrate the effectiveness of agricultural chemicals i n the production of fruit. The use of a fungicide i n the production of A n j o u pears has increased production 300,000 bushels a year. The apple crop of the nation, representing an investment b y commercial growers of about $750,000,000 i n land and trees alone, and with a crop averaging $240,000,000 to $250,000,000 a year during the past 5 years, is entirely dependent on the use of agricultural chemicals to control insects and diseases. I n the states of M o n t a n a and Wyoming, some 2700 acres of grassland were treated in 1949 as a part of the grasshopper control program. Competent estimates show that this program resulted i n saving half of the grass grown on the treated lands from destruction b y grasshoppers. The grass saved could produce 11,000,000 pounds of beef on the hoof. T h e soil conservation value is also very evident, because were the land to be laid bare, i t would be subject to damage during the period required to re-establish grass cover.
213
214
Stored grain losses caused b y insects throughout the world total approximately 6,000,000 bushels a year. The development of new chemical methods for determining weevil infestation and the increased application of chemicals for the control of other pests are rapidly reducing these staggering losses. Grain and corn production i n the United States has been tremendously increased in many areas through the application of agricultural chemicalsfor grain, increases have been recorded ranging from 25 to 100% through the use of weed control chemicals. Corn production has been increased from 200 to 300 pounds of green ear corn per acre through the control of the corn borer and corn earworm. The 1948 farm value of agricultural products i n New Jersey amounted to $320,750,000. Included i n this production are the following : Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch038
Vegetable products White potatoes Nursery and greenhouse Tree fruits Berries Total $ 51,200,000 19,800,000 17,500,000 7,000,000 6,000,000 $101,500,000
None of these crops, the value of which is one third of all New Jersey agricultural production, could be produced without agricultural chemicals. Another phase of New Jersey agriculture involving the use of agricultural chemicals can be added to this total, including the production of grains valued at $21,900,000, and poultry, eggs, and dairy products valued at $65,400,000. Hence, the importance to agriculture i n New Jersey of agricultural chemicals to control insects and diseases is abundantly evident. The analysis of other sections of the country produces evidence which parallels that reported for New Jersey. Another side to this picture, however, deserves careful consideration b y chemists. A t the present time many phases of pest control are being severely criticized b y the A m e r i can M e d i c a l Association, segments of the food industry, and consumer groups. The agricultural chemical industry fully appreciates its responsibility for proper labeling and sound education to promote the intelligent selection and proper use of various chemicals, and to guard the health of workers and of those using the materials as well as the consumer. In this educational program the industry is working in cooperation with the U . S. D e partment of Agriculture, the state land grant colleges, the U . S. Public Health Service, and many other governmental and private agencies. Never before has a grower had such a wide range of materials from which to make his selection, ranging from those which create no residue problem, to products which, because of their chemistry, require various degrees of precaution i n handling and usage. J . G . Townsend, chief, Division of Industrial Hygiene, U . S. Public Health Service, speaking in September 1949 before the annual meeting of the National Agricultural Chemicals Association, said: U n t i l a decade ago, the farm may have been considered outside the pale of industrial hygiene. Today, however, the industrialization of agriculture and the advent of new insecticides and other chemicals have made the farm a virtual factory i n a rural setting. On the farm, as well as in the factory, dangerous chemicals may be used safely with proper precautions. If manufacturers, processors, and distributors had to relinquish certain substances because they are toxic, American industry would be i n a primitive stage. Likewise, the farmer need not bow to economic pests, but can use to the fullest advantage the potent new insecticides if he is scrupulous in protecting himself. The establishment of a permanent section on agricultural chemicals in the AMERICAN CHEMICAL SOCIETY can contribute much in the public interest. The more discussion and intelligent consideration given to problems arising from the usage of pest-control chemicals, the better able industry will be to provide agriculture with effective pest-control chemicals which increase the efficiency of production and make possible the continuance of an abundant supply of wholesome food.
Military Characteristics of Insecticides

FREDERICK W. WHITTEMORE, JR.
Preventive Medicine Division, Office of The Surgeon General, Department of the Army, Washington, D. C.
A high degree of insecticidal activity per unit weight, effectiveness against many species of insects, prolonged residual effect combined with rapid knockdown, compatibility with various vehicles, and availability under wartime conditions are among the most important military characteristics of insecticides. No present insecticide is entirely satisfactory.
The properties of insecticides are, i n military parlance, called characteristics. M a n y of these characteristics are of importance i n both military and civilian usage, but because of the vast difference between military and civilian operations, certain properties of insecticides become extremely important to the armed services, and i n many instances are more important to the military economy than to the civilian economy. Possibly the most important single military characteristic is a high degree of insecticidal activity per unit weight. The necessity for this requirement may be more fully appreciated when i t is realized that approximately 3 tons of equipment and supplies per individual soldier are necessary during the assault stages of an invasion, and 0.75 ton of supplies and equipment per man is necessary to maintain one combat soldier for one month i n the field. W i t h this vast amount of equipment and supplies for each i n d i vidual soldier, the weight of each specific item needed for his support must be critically scrutinized. Prior to the discovery of D D T , with its high degree of insecticidal activity, it was necessary to include far larger weights of insecticides than at present. Although further improvements may be made i n the direction of increasing the toxicity of insecticides per unit weight of the pure chemical, i t is not believed that this requirement can be changed appreciably. Future research should be directed, not toward finding insecticides of higher insecticidal activity per unit weight, but toward finding insecticides that fulfill more adequately the other military characteristics discussed below.
Effectiveness
Another extremely important military characteristic is effectiveness against many different species of insects without the development of resistant strains. E v e r y insecticide that must be added to the military list of supplies geometrically increases the difficulties of procurement and distribution. A t the present time, nineteen different insecticides and insect repellents and four different rodenticides are issued by the A r m y Quartermaster. These figures do not include the different formulations of insect repellents issued under the same stock number. The three basic insect repellents are dimethyl phthlate, Indalone, and Rutgers 612. These repellents are issued either alone or i n various combinations, further complicating the supply situation because of the variation i n efficiency of these substances against different species of mosquitoes i n different parts of the world. Although all insecticide containers are adequately marked when they are filled at the factory, extreme difficulty has been encountered i n identifying many insecticides following
215
216
long storage in the field under adverse conditions. E v e n under the best conditions the supply problem may become very complicated in overseas areas because of the necessity of utilizing unskilled foreign labor i n supply dumps. In many instances this may result in the storage in the same stockpile of items that differ from each other only i n percentage composition. The results of this situation can be readily foreseen. A requisition for a specific item is occasionally filled with an item which, at first glance, appears to be the one requested but, actually, was formulated for a different purpose and is either inadequate or dangerous to use for the intended purpose. A specific example of the hazards inherent in this situation may be found i n the case of insecticide space spray composed of 1% D D T , 0 . 1 % pyrethrins, or 2.5% thiocyanate in deodorized kerosene and 5 % residual-effect D D T , both of which are issued in 5-gallon steel drums. Obviously, if a requisition for residual-effect D D T were to be filled with space spray, the application of the solution as a residualeffect compound would be of little or no value. Under some conditions, when stocks have been exposed to such adverse weather conditions that all gross identifying marks have been removed from the containers, the assumption has been made by the untrained native laborers that all unidentifiable cans of the same size contained the same material. Were it possible to have just one insecticide for all military purposes, such a situation could easily be avoided. It is also necessary, from the military point of view, that the insecticides supplied to troops i n the field be readily convertible into end-use items. It should not be necessary to go through an involved procedure in preparing insecticides for field utilization. For example, the earliest lots of D D T that were received i n the N o r t h African theater were of the consistency of beeswax, and were extremely difficult to get into solution. It was necessary to process all this material through meat grinders, of the hamburg variety, requisitioned from the civilian economy before this material could be satisfactorily dissolved i n Diesel oil. Such a situation complicates field operations unnecessarily, and should be avoided in the development of future insecticides.
Residual Effect and Knockdown

Another extremely important military characteristic is prolonged residual effect combined with rapid * 'knockdown. " Although, at the present time, we have some chemicals which give a comparatively long residual effect, and other chemicals which give a relatively quick knockdown, the military still require more prolonged residual effect and more rapid knockdown in their insecticides. Although D D T has a prolonged residual effect, and is extremely valuable in the control of such insects as mosquitoes and sand flies of the Phlebotomies type, improvements are needed in the speed of knockdown, particularly of mosquitoes. There is an appreciable time interval after the mosquito first comes i n contact with D D T before its biting reflexes are no longer inhibited. This factor is not so important with insects such as the Phlebotomus-type sand fly, because this group of insects makes extremely short flights of 2 to 3 yards and then stops to rest. Residual D D T applied to breeding areas and buildings will adequately prevent sand flies from entering buildings and biting personnel. M o s quitoes, insects capable of sustained flight, may conceivably enter a building, come i n contact once or twice with D D T , and still bite and infect a person with malaria before they are killed by the D D T . A t the present time we have no chemical that combines both quick knockdown and prolonged residual toxicity for use against all insects of medical importance. Still another requirement is that the concentrated form of the insecticide readily combines with various types of vehicles. A t the present time, pure D D T can be dissolved only in organic solvents, and it has been necessary to provide a 5 0 % water-dispersible D D T powder when water was to be used as the vehicle. W i t h the advent of 9 0 % water-dispersible D D T , it may be possible to utilize this material i n the preparation of both water suspensions and organic solutions of the chemical. The ideal chemical i n this respect would be one that could be shipped as 100% dust, and could be readily diluted with inert dust, water, and organic solvents i n the field.
WHITTEMOREMILITARY CHARACTERISTICS OF INSECTICIDES
217
Another extremely important military requirement that is not usually associated with civilian requirements is stability i n prolonged storage under adverse conditions. For example, the first lots offlyspray that were shipped to N o r t h Africa, i n 1942, contained pyrethrum as the principal active ingredient. I t was not possible to store this m a terial under cover and much of it remained under constant exposure to the intense tropical sun, undoubtedly deteriorating very rapidly. Covered storage facilities are the exception rather than the rule i n a combat theater of operations, and all insecticide compounds furnished to the military forces should withstand this type of storage.
Availability
The last, but by no means the least, important of the military characteristics is availability under wartime conditions. Materials are classified as strategic, critical, and noncritical i n this respect: A strategic material is one that must be imported into the United States; a critical material is one that is available i n the United States but, because of either limited plant facilities or excessive demands, becomes nonavailable; a noncritical m a terial is one that is readily available i n . sufficient amounts under wartime conditions. A n insecticide to be of value to the military forces must be readily available under wartime conditions, must not be dependent upon the importation of certain essential materials, and must not impose a burden upon plant facilities i n the United States. The only military characteristic which has been met adequately at the present time is that of a high degree of insecticidal activity per unit weight. The other requirements are not being met adequately by the insecticides i n commercial production at this time and a vast amount of research is necessary before the armed forces can be furnished with an insecticide which is entirely satisfactory for military needs.
Development and Use of Synthetic Organic Insecticides

CHARLES E. PALM
Cornell University, Ithaca, . Y.
The synthetic organic insecticides offer great advances in insect control. Their high degree of toxicity and specificity to certain species are noticeable features. Hazards to health in manufac ture, formulation, and application are primary considerations. New methods of application are being developed. Official guidance on residue hazards will aid entomologists and farmers in developing application programs. Resistance to insecticides is of growing importance. The effect of pesticides on naturally occurring beneficial insects is causing heretofore unimportant forms to become pests of major concern. The search for the solution to the insecticide problem is a specialized and coopera tive venture among a team of scientists trained in many fields.
Frequently it helps us to understand a current problem if we review what has taken place i n previous years. This is particularly true with the present situation in the field of synthetic organic insecticides. The use of plant extracts for insect control dates into antiquity; the use of Paris green as an insecticide for control of the Colorado potato beetle in 1867 probably marks the beginning of the modern era of chemical control of injurious insects. The develop ment of lead arsenate followed later i n the nineteenth century for gypsy moth control. The commercial production of nicotine insecticides, the production of calcium arsenate at the time of the first world war, and the use of fluorine, arsenical, and cyanide compounds, as well as other inorganic chemicals for insect control, were important steps i n pest con trol. These chemicals were applied largely by dilute high pressure sprays or dusts. The concern over fluorine, lead, and arsenical residues in the 1920's and 1930's seems to have tied i n with greater interest i n the development and use of the botanical insecti cides, pyre thrum, rotenone, and nicotine. E v e r y effort was made to supplement the effectiveness of these materials through formulation, combinations, or, as i n the case of lead arsenate, by adding deposit builders, spreaders, and other similar materials. Progress with application equipment continued along established lines with streamlining of equip ment, increased pressures, and similar measures. Farmers developed a background of experience in the use of these insecticides through the years and knew fairly well what to expect i n terms of hazards of use and performance. The developing problem of harm ful residues at harvest time alerted growers, entomologists, and the consuming public to the need for research in the production of safer insecticides.
Recent Developments
A l l of us have witnessed developments of the past decade when a second world war engulfed most of the civilized world. Insect control was of prime importance to the pro tection of the armed forces against insect vectors of disease organisms as well as to the pro218
PALMDEVELOPMENT AND USE OF SYNTHETIC ORGANIC INSECTICIDES
219
duction and protection of food and fiber. The American farmer was called upon to i n crease production beyond all previous limits. Shortages existed i n the supply of almost all insecticides and application equipment. The loss of the D u t c h East Indies to Japan early i n the war shut off the principal supply of rotenone to the Allies. I n spite of every conceivable handicap, industry and government teamed up to aid the farmer to make the excellent production record which is so well known to everyone. The advent of D D T brought about revolutionary practices in economic entomology. Its effectiveness for many pests was nothing short of miraculous. The uncertainty of use and residue hazards brought about precautions for grower application. Control of many insects became feasible for the first time because of the effectiveness of D D T as well as the small amounts of the insecticide needed. It is still amazing to watch a plane applying one gallon of insecticide solution per acre of forest, at the rate of 100 acres per minute, with results of near eradication of a pest like the gypsy moth. Technology i n the field of synthetic organic insecticides and application equipment has recently made unbelievable strides. To the uninitiated, i t seemed certain that D D T was almost the final answer to all our insect problems. Following D D T , other chlorinated hydrocarbons were introduced, including benzene hexachloride (hexachlorocyclohexane), chlordan, toxaphene, and others. More recently, there have been the organic phosphates including parathion, tetraethyl pyrophosphate., and hexaethyl tetraphosphate, several of the dinitro compounds, and many others still i n the research and development stages. The rapid development of the chemical industry i n producing new insecticides left all, including the farmer, without a background of use experience with these materials, which for the most part w ere more potent i n killing insects than anything that had ever been used. Is i t any wonder then, that the average farmer and entomologist became and are still confused? Close working relationships have of necessity developed with this rapid introduction of new insecticides. N o longer can an insecticide be recommended for use on the basis of its insect-killing properties alone. The chemists, toxicologists, pharmacologists, physiologists, manufacturers, and others are all contributing to the story.
r
Problems with New Materials What are some of the problems that exist today relating to the development and use of new insecticides? Obviously there are hazards i n manufacturing, formulating, and applying these chemicals. Doubtless the first two processes are under more constant supervision than the last. M a n y farmers and farm workers are careless about reading precaution labels before handling and applying insecticides. The use of a mask or respirator, rubber gloves, and other protective clothing with some of the newer materials is not, unfortunately, general practice i n the field. Part of the negligence has been blamed on D D T , because few if any of the possible difficulties in the use of D D T insecticides ever m a terialized. I t is now difficult to make many farm workers realize that all the new chemicals are not i n the same safety category, particularly if they or their neighbors have applied one of the phosphate compounds, for example, without suffering i l l effects. The three deaths reported from use of parathion in the field i n the summer of 1949 are creating more respect for the material among growers than all the words of warning could ever do. In brief, the problem of educating the farmer on safety precautions to be observed in applying insecticides is not solved. The very nature of the over-all problem has made i t impossible for an agency of government like the Food and Drug Administration to establish official guidance in terms of the residue hazards and degree of contamination that might be permitted if a given chemical must be used for pest control i n a production program. Through its research, the F D A has been extremely helpful i n giving guidance during this critical period. New Y o r k farmers, like others throughout the nation, are anxious to know what levels of contamination will be considered safe on fruits, vegetables, and other commodities, i n order to guide their choice and use of insecticides. Certainly, the entomologist will be glad to receive this guidance i n making recommendations. The hearings by the Food and D r u g Administration to deal with residue hazards i n the use of insecticides deserve the sup-
220
port of all who have data bearing on the problem and an interest in i t . I t is a very t a n gible illustration of the important role of toxicologists and pharmacologists on the team.
Variation in Insect Species

Insects, like other living organisms, show variation within species. This has long been recognized i n so far as structure, size, and color are concerned. I t has been demonstrated from a physiological viewpoint where two strains, seemingly identical structurally, behave differently i n their habits and seasonal history. Insect toxicologists have consistently demonstrated the variation i n individual susceptibility of a population of an i n sect species to a uniform dosage of an insecticide. Variation among living organisms is a basic concept of biology. I t is not startling, then, that we should find the development of resistant or tolerant strains of a species after continued use of an insecticide. M u c h is being said about the development of resistance to insecticides, and i n a number of i n stances data substantiate the claim. I t is also likely that resistance will be blamed i n many cases of faulty timing, poor application, or poor materials.
Insect Resistance to Insecticides

The development of resistant strains of an insect to a given insecticide is not new. Melander (7) i n 1914 pointed out that the San Jose scale in Washington had developed a resistance to lime-sulfur sprays. Recently Babers (1) of the Bureau of Entomology and Plant Quarantine brought together an excellent evaluation and summary of the literature dealing with the development of insect resistance to insecticides; he lists 111 references to work on this phenomenon. Quayle (11, 12), working i n California, noted resistance of the California red scale, black scale, and citricola scale to hydrocyanic acid gas. Hough (5) first called attention to the developing resistance of the codling moth to arsenicals. I n South Africa a blue tick, Boophilus decolor'atus, was found b y D u Toit (3) and others to be resistant to arsenic. Boyce and Persing (2) reported resistance among the citrus thrips, Scirtothnps citri, to tartar emetic sprays. K n i p l i n g (6) found that the larvae of the screw worm fly, Cochliomyia americana, could acquire resistance to phenothiazine. Mosna (9) i n Italy reported a variety of mosquito resistant to D D T . Missiroli (8) also working i n Italy reported on the failure of D D T to control houseflies i n 1945 and 1946, owing to resistance to D D T . W i l son and Gahan (13) developed a laboratory strain of houseflies resistant to D D T and several other insecticides. These examples from the literature compiled by Babers indicate that resistance to different materials is developing among different species and i n several localities. I t may become more extensive with organic insecticides because of their more widespread use as well as the greater number of chemicals that will be applied i n the field.
New Pest Species and Resistance in New York

Several examples of resistance have been observed i n New Y o r k . The codling moth has long been considered the N o . 1 pest of commercial apple production. I n the Hudson River Valley, Chapman of the New Y o r k Agricultural Experiment Station staff reported one orchard i n 1930 where codling moth could not be controlled with the usual lead arsenate schedule. Since that time practically the entire orchard area of the Hudson Valley has developed a codling moth problem which lead arsenate will not handle satisfactorily. The same experience was noted i n western New Y o r k , except that it began a few years earlier. Harman (4), reporting to the Horticultural Society i n 1945, indicated that i n spite of everything growers could do i n areas of western New Y o r k , inability to control codling moth made apple growing unprofitable except for the very high prices being received for fruit at that time. The number of cover sprays increased to as many as six i n a season. Deposit builders, stickers, spreaders, oil added as an ovicide, nicotine, and other materials supplementing lead arsenate comprised the only weapons until D D T proved to be so tremendously effective i n codling moth control. Thus far there has been no sign
PALMDEVELOPMENT AND USE OF SYNTHETIC ORGANIC INSECTICIDES
of a reduction i n efficiency of D D T for control of the codling moth. Spray practices of applying the minimum effective dosages of lead arsenate for economy and residue considerations may have favored the development of resistant strains. The greenhouse red spider, more properly called the two-spotted spider mite, has developed a resistance in New Y o r k to two chemicals, an ammonium potassium selenosulfide compound, marketed as Selocide, and parathion. W i t h both Selocide and parathion excellent initial kills were obtained. The experience with parathion is still i n progress. Blauvelt of the Cornell University staff reports some cases where parathion aerosols no longer will effectively kill spider mites in greenhouses. There are strong indications that the rapid development of parathion-resistant populations i n some ranges was associated with the use of a marginal program of treatments. This allowed a considerable build-up of populations between treatments and thus may have afforded favorable conditions for the selective survival and increase of resistant individuals or strains originally present i n small numbers. On certain ranges where cooperative trial programs with parathion aerosols have been carried on for over two years, no significant increase in resistance to parathion has yet developed where applications were made often enough to keep the mite population at a very low level from the start. The short life cycle and more or less continuous breeding of the spider mites in greenhouses favor a more rapid development of resistance than is possible with a slower breeding species like the codling moth. Another interesting case is the growing feeling among farmers that a 0.75% rotenone dust is no longer effective against Mexican bean beetledosage of 1% seems to be required. There are also complaints that D D T wettable powders are not as effective against the potato flea beetle as i n former years. Such circumstantial evidence cannot be accepted as proving resistance to insecticides, but it bears watching and investigation. New Y o r k has experienced a rather widespread breakdown in housefly control with the use of D D T . Schwardt of the Cornell University staff first noticed this failure of D D T in 1948. I n 1949 the fly problem was very bad. Farmers, remembering the exceptional control of the past few years with D D T residual sprays, were greatly disturbed when D D T was first withdrawal from use i n dairy barns because of the danger of D D T contamination in milk. Methoxychlor under conditions in 1949 did not measure up to the performance of D D T in other years; neither did D D T . Lindane (gamma isomer of hexachlorocyclohexane) has been hailed by many dairymen as the successor to D D T , and by some farmers the question is raised"What is the successor of lindane to be?" Along with development of resistance has come the destruction of important natural enemies of pest species, which has been attributed to field use of insecticides and certain fungicides. Pickett (10) recently presented evidence to show that the development of the oyster shell scale problem i n N o v a Scotia orchards is related to the use of sulfur fungicides. The scale was present as an occasional pest of apples for over half a century, but beginning i n 1930 it built up to dangerous levels. Sulfur was shown to destroy the parasitic wasp and the predatory mite responsible under natural conditions for keeping the pest under control. Prior to 1930 lime sulfur was used as a fungicide and it served also as an insecticide for the scale. W i t h the change to mild elemental sulfurs about that time, the oyster shell scale population built up to injurious levels, because the milder sulfurs continued to kill the parasites and predators but did not kill the scales. Pickett further recorded a high build-up of the European red mite on apples when mild sulfurs were used as a fungicide as opposed to copper fungicides. Again sulfur is charged with destruction of parasites and predators of the red mite. The same correlation between copper and sulfur fungicides and the degree of codling moth infestation is presented by Pickett over a 5-year period i n N o v a Scotia. H e thinks possibly sulfur has reduced the natural enemies of the codling moth to a greater extent than copper fungicides.
2 2 1
Upsetting Balance in Nature

D D T has been blamed b y some for upsetting the balance i n nature. Certainly the balance i n nature was upset by many factors long before D D T , not the least offender being man. We recognize specific problems, however, where the use of D D T for the control of one pest has been associated with the rise to prominence of other pests. One example
222
from experience i n New Y o r k is the red-banded leafroller. The incidence of this pest is closely correlated with the use of D D T as a codling moth insecticide. I t is possible that the natural enemies of the leafroller which were not disturbed b y lead arsenate were killed b y D D T . The red-banded leafroller is now a major pest capable of destroying an entire apple crop. Similarly, the two-spotted spider mite became an economic problem in many New Y o r k orchards following the use of D D T for codling moth control. This species was never considered as an orchard pest prior to the use of D D T . Although the reasons for this development are not completely explained, it again is probable that para sites and predators of the mite have been reduced. The European red mite on apples has increased i n importance i n New Y o r k with the use of D D T . Y e t research workers have not been able to say that the rise in populations of this species is directly attributable to the use of D D T . Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch040
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) Babers, F. H . , U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Bull. E 7 7 6 , 1-31 (1949). Boyce, A . M . , and Persing, C. ., Calif. Agr. Expt. Sta., News Letter, 2 1 (1942). Du Toit, R., Graf, H . , and Bekker, P. M . , S. African Vet. Med. Assoc. J., 1 2 , 50-8 (1941). Harman, S. W., Proc. . Y. State Hort. Soc., 1 9 4 5 , 46-53. Hough, W. S., J. Econ. Entomol., 21, 325-9 (1928). Knipling, E . F., Ibid., 35, 63-4 (1942). Melander, A. L., Ibid., 7, 167-72 (1914). Missiroli, ., Riv. parassitol., 8 (2/3), 141-69 (1947). Mosna, E . , Ibid., 8 (2/3), 125-6 (1947). Pickett, A . D., Can. Entomol., LXXXI, 67-76 (1949). Quayle, H . J . , Calif. Univ. J. Agr., 3 , 333, 358 (1916). Quayle, H . J . , Hilgardia, 11 (5), 183-210 (1938). Wilson, H . G., and Gahan, J. B., Science, 1 0 7 , 276-7 (1948).
Toxicological Investigations by Industry

JOHN H. FOULGER
Haskell Laboratory of Industrial Toxicology, Wilmington 98, Del.
Consideration of the many factors involved in the collection and use of toxicological data on chemicals and the types of problems posed in manufacturing, marketing, transportation or usage suggests that, in general, information required by manufacturers to conform to statutes is best gathered in laboratories established by industrial companies, individually or cooperatively. The final correlation and interpretation of data in terms of human hazard are best made by individuals or committees qualified in medicine as well as in pharmacology and toxicology.
T h e rapid accumulation of statutes, regulations, and ordinances controlling the manufacture, transportation, labeling, and use of chemicals is making acute the problem of obtaining adequate information on the health hazards which these chemicals might afford under various situations. This growth i n the mass of legislative regulation is due to a trend in public thought, which has occurred simultaneously with the increase i n industrialization of the United States. Before the chemical industry i n this country started its mushroomlike expansion, the country was already fairly highly industrialized. The inevitable problems of employer-employee relationships and fair compensation for i n juries to workmen led to dissatisfaction with the ponderous, expensive methods of the common law by which each particular case was compared with a pyramid of precedent. In many fields, statutory law replaced common law and by 1909 several states already had passed occupational injury acts governing the compensation of workers for injuries resulting from accidents within their plants. A s more and more workers were exposed to new and untried chemicals i n manufacture or in usage, it was realized that in addition to the possibility of an acute injury, there was possibility of chronic disease; the time of onset of the disease could not be determined usually and, therefore, could not be used to designate the date of a specific accident. The compensation laws concerning accidents were, therefore, either extended by clauses covering occupational diseases, or other laws were passed specifically concerning occupational diseases. This trend has been particularly important since 1920. The obligation of a manufacturer or marketer of hazardous chemicals to warn his customer of the hazard was still subject to decisions under common law, except where the introduction of chemicals into foods was governed by the Food and Drug A c t of 1906, and the interstate transportation of certain hazardous chemicals was regulated, particularly with respect to containers, by the Interstate Commerce C o m mission regulations. Probably the greatest impetus to statutory regulation has come from the necessity for an industrial population to conserve its food supply. The use of chemicals as insecticides quite often demands a degree of exposure to the user which would not be tolerated in the manufacture of the same chemical. The Federal Insecticide, F u n gicide, and Rodenticide A c t of 1947, plus state acts regulating and registering insecticides, emphasize this situation. A t the present time, the list of statutes and regulations governing the manufacture, transportation, and use of new chemical products includes the following: Twenty-four
223
224
states have occupational disease laws, which either mention specific chemicals as the cause of disease or which give blanket coverage against injury from chemicals; thirty-nine states and the Federal Government have laws regulating the sale of insecticidesof these, the Federal A c t , twenty-nine states, and the Territory of Hawaii require registration of the insecticide and control labeling, including literature used i n sales; three states have laws governing labeling of industrial chemicals; Massachusetts has special laws on benzol and carbon tetrachloride; Interstate Commerce Commission regulations govern the transportation of hazardous chemicals, and many states and municipalities have more specific laws and regulations concerning the use of certain materials i n residential areas or the storage of hazardous materials; M a r y l a n d has this year passed a law requiring the labeling of paints and finishes on children's toys or furniture, contact with which might be injurious to the child. The Federal Food, Drug, and Cosmetic A c t regulates not merely chemicals introduced deliberately into foods, or used in drugs or cosmetics, but also those employed i n food containers. N o r is this the complete picture. The agitation for control of stream pollution and the smog incidents at Donora and Los Angeles have resulted i n the consideration during the last year of some two hundred state regulations concerning stream and atmospheric pollution. Lest this total picture of accumulated regulation of almost all phases of the chemical industry should lead the general public to believe that chemicals as such are an important cause of death or injury in the United States, a few facts are of value. Table I shows data rearranged from reports i n "Accident Facts," 1949 edition, National Safety Council. Actually, acute death due to chemicals occurred in some 3500 cases, but of these a great proportion were either the result of exposure to motor vehicle exhaust, sewer gas, or i l l u minating gas, or were due to injudicious usage of barbiturate medicaments. Eliminating these causes, which are obviously not attributable to lack of w arning by manufacturers or to any factors which could i n any way be controlled by chemical manufacturers, there were about 1000 deaths i n the United States in 1947 due to chemicals. The favorite national weapon, the automobile, which is relatively uncontrolled by legislation, would accumulate an equal " b a g " in less than two national holiday week ends.
r
Table I.
Heart disease Cancer Cerebral hemorrhage Accidents Nephritis Pneumonia Tuberculosis Premature birth Diabetes Congenital malformations
Comparative Data on Causes of Death in 1947 (1 )

460,580 189,811 111,725 99,579 80,288 54,172 48,064 41,053 37,515 20,315 Automobile Home Occupational Others 32,697 34,500 17,000 11,940
Absorption of poisonous gas Motor vehicle exhaust Utility gas Other sources of carbon monoxide Other poisonous gases Acute poisoning by liquid or solid"
1938 285 1009 354 290 1504
More than 25% due to barbituric acid and derivatives.
D a t a on nonfatal injuries or chronic diseases caused by chemicals are very difficult to collect. F a r too often, i t is gathered from reports of compensation commissions or court decisions i n which legal rather than scientific "proof" prevails. One might venture the guess that at least as many injuries and chronic conditions are caused b y synthetic medicaments used improperly, or without adequate medical control, as are caused b y the ordinary products of the chemical manufacturing industry. It might be debatable whether this really good record of chemicals is due to the efficiency of regulatory laws. Certainly i n the field of industrial health, the industrial disease acts have led to a very great improvement i n the picture, but whether regulationsfor example, on labeling, however adequate they may appear to be on paperhave actually served to protect the general user of hazardous chemicals is questionable. F a r too often, the practice of the user is to employ the product first and then read the label if he gets into trouble. Despite the possibility of dispute over the reason for the good record of chemicals as a
FOULGERTOXICOLOGICAL INVESTIGATIONS BY INDUSTRY
225
source of death or injury, it is quite probable that there will be a continual increase i n regulation. Changes i n policyfor example, of the Food and D r u g Administrationhave already led to the possibility that a chemical incorporated in a food container and having, coincidntally, antimold or bactericidal properties, but not used i n the container for those properties, can, if i t should enter the food i n the container, be subject to regulation. Arbitrary changes of so-called "maximum allowable concentrations" of atmospheric contaminants may completely alter the effect of state occupational disease laws. If both common law and statutory obligations incurred b y a manufacturer of new chemicals are considered, there is a "cradle to grave" coverage with respect to health hazards, for obligations now arise with the birth of the chemical i n the research laboratory and continue through its life in mass production, packaging, transportation, on to its final conversion into a new substance or final distribution or dissipation by the ultimate user. M u c h confusion existsand much vague legislation has resultedfrom careless use of the term "poison." T o the uninitiated, it would seem that a simple classification of new materials into "poisonous" and "nonpoisonous" would satisfy all needs, and further that such classification is a simple matter of laboratory tests. This idea is erroneous, because there is not only the question of whether the materials are inherently poisonous (whatever definition is used for poison), but also whether, under the conditions of human contact contact of respiratory or digestive tracts, or skin, or mucous membraneto be expected in any phase of their use, there is a significant health hazard. A particular chemical may be intrinsically highly toxic, but because of its manner of manufacture and use, the hazard may be minimal. The defining of hazard must be based on an assessment of all factors involved i n a particular situationthe intrinsic acute or chronic physiological action of the chemical, its physical properties, and the conditions of its use. I n the interests of clarity, the term "poison" should be discarded for the term "hazardous chemical." I n tracing the life of a new chemical, a definition of the intensity and nature of hazard is necessary at the general stages of development shown i n Table I I . Table II.
Stage Research: small scale production of crude chemical and its purification; study of properties and possible uses Semiworks: moderately large scale Design of new plant
Evaluation of Hazardous Chemicals

Purpose of Information Protection of research staff; decision as to suitability for desired use or selection of least tdxic chemical from a number with equal technical possibilities Decision as to possible health hazards in mass production; preliminary information on type of hazard which might exist in mass manufacture Safety of equipment; necessity for ventilation, safe atmospheric concentration; selection of suitable physical protective devices for workers (respirators, protective clothing, showers) ; procedure for medical treatment in emergency if this requires special equipment Decision as to health services needed at plant; plan of preventive medicine (including selection of workers according to expected degree of exposure and methods of periodic check on health) Continual check on efficiency of protection; treatment in emergencies Classification of product under I . C . C . regulations Adequate labeling information to protect customers' workers; advice when mode of use involves new form or degree of hazard Adequate labeling and use information either to conform to statute or to fulfill common law obligations; special requirements of federal and state insecticide or pure food laws Fundamental research on new techniques for study of hazards to animals and men
Before operation of new plant During operation Transportation Use by industrial customer Final user (general public) At all stages
To this outline must be added the need for information on the safe disposal of industrial wastes containing commercially insignificant, but perhaps physiologically important, residues of the compound. If such disposal involves production of new substances for example, when wastes are burnedthe hazard from these substances needs definition. The information needed at each of these stages in the life history of the compound relates, first, to hazards involved i n a specific use and, second, to the hazard to humans, not to laboratory animals. The needed information, therefore, can be gained adequately only by designing laboratory experiments with animals to cover the pertinent conditions of the specific use. Finally, this information can only be interpreted adequately b y those versed i n normal and abnormal human physiology and pathology. The interpretation of
226
the degree of hazard which the chemical affords to industrial workers selected by a rigid pre-employment examination and followed thereafter by periodic studies under a preventive medical program may be quite different from the interpretation in terms of the hazard to a customer's workers, who are not selected or studied medically, or to the general public, who may be suffering from a variety of diseases which could be complicated by exposure to a chemical which is harmless to those in good health. The final interpretation, therefore, can seldom be made adequately by those not qualified i n medicine.
Collection of Data on Health Hazards

To collect data for the assessment of health hazards at the several stages of development and use described here, physical chemistry, biochemistry, physiology, toxicology, pathology, and clinical medicine are all employed, plus an intimate knowledge of manufacturing procedures and specific methods of use. B u t these are not used efficiently if isolated i n separate departments. The whole program of investigation must be planned and timed with the closest possible correlation of all fields of knowledge. Development of chemical and physical methods of identification or measurement of a chemical contaminating the atmosphere must be based on knowledge of the range of concentrations which may be of significance i n the health hazard. The biochemical studies of the fate of the chemical i n the body must be designed to be directly applicable, especially i n details of technique, to humans. The medical knowledge applied in the assessment of the entire picture must not stop short at an understanding of diagnosis and treatment, but must i n clude a clear concept of health as an entity with degrees varying from optimal to those bordering on disease. Briefly, the research organization must be a closely knit, cooperative unit in which each field of science functions as a dynamic part of the whole and not as an isolated entity. Such an organization was assembled i n the medical division of the A r m y Chemical Corps at Edge wood Arsenal, M d . , during the recent war and operated, in the writer's opinion, as one of the most valuable medical research groups ever assembled. The type of organization defines the training required of the senior members at least. Among them should be represented the disciplines of physics, biochemistry, physiology, pharmacology and toxicology, and pathology and clinical medicine. They should constitute (2) " a team of scientists, each a specialist i n his own field, but each possessing a thoroughly sound and trained acquaintance with the fields of his neighbors, all i n the habit of working together, or knowing one another's intellectual customs, and of recognizing the significance of a colleague's new suggestion before it has taken on a full formal expression. The mathematician need not have the skill to conduct a physiological experiment, but he must have the skill to understand one, to criticize one, and to suggest one. The physiologist need not be able to prove a certain mathematical theorem, but he must be able to grasp its physiological significance and to tell the mathematician for what he should look." Above all, they should never forget that their ultimate decisions must be made i n terms of human health. The junior staff of such research organizations should consist of the technical assistants and untrained laboratory helpers and animal caretakers needed to facilitate the functioning of the whole organization. Currently, toxicological problems for the chemical manufacturing industry are occasionally studied i n special laboratories established b y particular corporations but more frequently either i n departments of biochemistry or pharmacology attached to university medical schools or by commercial laboratories, but seldom is the whole pattern of investigation, needed to cover all phases of manufacture and use of a chemical, set up i n one place. Seldom, also, do those undertaking investigations have the required close contact with the technical or medical problems of industry. I n wartime, it is possible to establish such research organizations; in peacetime, the scientists needed for similar problems are scattered widely among medical schools and isolated in separate departments of those schools. Therefore, the responsibility of the manufacturer of chemicals for obtaining adequate information on the materials which he uses and sells can best be fulfilled b y toxicological research laboratories within industry. These could be established either by i n dividual corporations or, when financial considerations make that difficult, by groups of
FOULGERTOXICOLOGICAL INVESTIGATIONS BY INDUSTRY
227
corporations having similar interests. Since, in the final analysis, the decision as to hazard from a given compound is a medical one, the toxicological laboratory should be a part of, or closely associated with, the medical organization of the company or group. F u r ther, there should be freedom of exchange of scientific knowledge between such research laboratories so that the industry as a whole may benefit from information gained by each and duplication of effort may be eliminated. Twenty years ago this program would have been regarded as the dream of an idealist ; it is no longer an ideal but a very real, immediate need. Manufacturers of chemicals cannot inform the public adequately on the health hazards from the materials which they make and sell unless they, themselves, have adequate information. I n the absence of proper education of the public, hysterical, ignorant, or malicious gossip will lead to legislative regulation, which may be harmful to progress. Two examples of such legislation, or attempts at legislation, have appeared within the last 12 months: Section 323 of Article 27 of the Annotated Code of Maryland, 1939 edition, states: It shall be unlawful for any person to manufacture, sell, or offer to sell any toy or plaything, including children's furniture, decorated or covered with paint or any other m a terial containing lead or other substance of a poisonous nature, from contact with which children may be injuriously affected, unless such article shall contain a label affixed thereto stating in clear and unambiguous language that the said paint, or other material, contains lead or other substance of a poisonous nature. This law, which is a criminal law, is so loosely worded that it is at the moment i m possible to establish criteria for paints, varnishes, or lacquers to be placed on children's furniture or toys to be sold in M a r y l a n d . Further, the M a r y l a n d law has little protective value, since anyone can repaint children's furniture or toys with any material regardless of its suitability for the purpose and its inherent toxicity. This law, as it stands, places a heavy and complex burden on the manufacturer and marketer of children's toys and furnitureand necessarily on the manufacturer of paints, lacquers, and varnishes. T h e M a r y l a n d law has greater significance as a piece of troublesome legislation in that in the courts of the state of Maryland, the jury is the judge of the law as well as the facts under the law, and since under criminal law each case would be argued on its own merits, two different juries might reach contradictory decisions on the same set of circumstances. Again, during the last 12 months, there has been strong agitation for an increase of severity i n the labeling of carbon tetrachloride i n the state of California. Two deaths were caused i n the city of San Francisco b y grossly improper use of carbon tetrachloride, which had been removed from its original container. The original container apparently had been properly labeled according to the opinion of competent authorities. Public agitation i n the city of San Francisco demanded that carbon tetrachloride be labeled ' 'poison," and that, therefore, i t carry an antidote. I t is a principle of good labeling that the word "poison" should not be used indiscriminately lest its frequent sound i n the ears of the public lead to a dangerous dilution of its value. I t is a standard principle of toxicology that one cannot prescribe an antidote for materials which are hazardous b y inhalation. B o t h i n the M a r y l a n d and the San Francisco situations, adequate and proper public knowledge of the problem would have had great value. Undoubtedly the establishment of toxicological research organizations will be costly, but the cost will be much less than the cost of not establishing them. There are many difficulties to be faced, but none is greater than the practical difficulties which manufacturers of modern synthetic chemicals continually face i n their visions of new products or in their attempts at more economic production of well known products.
Literature Cited
(1) National Safety Council, Chicago, "Accident Facts," 1949. (2) Weiner, Norbert, "Cybernetics," p. 9, New York, John Wiley & Sons, 1948.
Toxicological Action and Metabolic Fate of Chlordan

. F. STOHLMAN and M. I. SMITH
National Institutes of Health, Bethesda, Md.
In rabbits under light amytal anesthesia, chlordan has no direct effect on the blood pressure, but produces a type of respiration having many characteristics in common with Cheyne-Stokes type. The generalized tremors, opisthotonus, tonic and clonic convulsions, produced by chlordan were decreased or abolished and respira tion restored to normal by suitable injections of the sodium salts of amytal, phenobarbital, and pentothal. The LD of chlordan, which was about 20 mg. per kg. on intravenous administration to intact rabbits, was increased to about 60 mg. per kg. through the antidotal action of the barbiturates. An unidentified chlorine containing degradation product with acidic properties was re covered from the urine of rabbits treated with chlordan. Ap proximately one third of its chlorine content was set free on hy drolysis at 100 C. with sodium hydroxide in either absolute alcohol or in water.
50
It was previously shown (4) that the oral L D o of chlordan i n rats was 200 to 250 mg. per kg., i n rabbits about 300 mg. per kg., and on intravenous injection i n rabbits i n ween 20 about 20 mg. per kg. I t was also shown (4) that the chronic toxicity of chlordan i n both rats and rabbits was greater than that of D D T under comparable conditions. The purpose of this paper is to give some of the results obtained i n a study of the antidotal action of barbiturates i n chlordan poisoning, and of the metabolic fate of chlor dan.
5
Experimental I n this work the chlordan used was taken from the same lot employed i n previous work (4). The effects of this compound on blood pressure and respiration were studied i n rabbits under barbiturate anesthesia of light to moderate depth. F o r t y milligrams per k g . were the usual dose of barbiturate, administered intravenously. Chlordan was administered 25 to 40 minutes later intravenously as a 4 0 % solution i n Tween 20. A t this time animals having received sodium amytal were under slightly deeper anesthesia than those having received equivalent amounts of either sodium phnobarbital or sodium pentothal. Additional amounts of the barbiturates were given, when the animals developed severe general tremors and respiratory symptoms, i n order to determine their antagonistic action against chlordan. The antidotal action of the barbiturates was also investigated i n intact rabbits by the intravenous administration of both chlordan and the barbiturate. Sixty milligrams per kg. of chlordan were injected as a 4 0 % solution i n Tween 20 at a uniform rate over a period of 3 minutes. The sodium salts of the barbiturates were injected slowly as a 2.5% solution i n distilled water i n two doses. The first dose was given 2 to 5 minutes preceding
228
STOHLMAN AND SMITHTOXICOLOGICAL ACTION AND METABOLIC FATE OF CHLORDAN
229
the chlordan administration and the second, 5 to 15 minutes following i t . A l l animals were observed a minimum of 30 days.
CC-C 5
Cft-C 6 Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch042
1,2,4,5,6,7,8,8-OCTACHLORO2,3,3a,4,7,7a-HEXAHYDR0-4 7METHAN0-INDENE
>
Structural Formula of Chlordan, CioHeCIs The metabolic fate of chlordan was studied i n rabbits by analysis of the relative chlorine content of chlordan added to normal rabbit's urine and of the chlorine content of the urinary excretory product. The method of analysis was similar to the one previously used (2, 4)- I n addition, hydrolysis of chlordan and of the urinary excretory productswas carried out b y adding solid sodium hydroxide to saturation to a 10-ml. solution of these substances i n hot absolute ethyl alcohol. The mixture was refluxed for 3 hours in a round-bottomed flask immersed i n boiling water and the amount of inorganic chlorine determined. Hydrolysis was similarly carried out with solutions of the respective substances i n aqueous sodium hydroxide.
Results
The effects of chlordan on the blood pressure and respiration of rabbits under sodium pentothal are illustrated i n Figure 1. The injection of 60 mg. per kg. of chlordan produced a gradual drop i n blood pressure of about 20 m m . of mercury, starting during the injection and continuing for about 3 minutes after the injection (Figure I, A). I t then rapidly returned to the preinjection level. Since a similar drop i n blood pressure was produced b y the injection of an equivalent amount of the solvent, Tween 20, alone, it appears that chlordan has little if any direct effect on the blood pressure. Initially there was an increase i n respiratory rate with a slight decrease i n amplitude. This was followed b y a marked increase i n respiratory rate and amplitude with a simultaneous onset of generalized tremors. Cheyne-Stokes type of respiration followed (Figure 1,B). This consisted of alternating cycles of rapid and deep respirations with short periods of apnea, during which slight temporary elevations i n blood pressure may be seen. These cycles lasted from several minutes to 1.5 hours i n different experiments. M i l d to moderate tremors occurred more or less continuously, whereas the more severe tremors occurred i n cycles corresponding to the periods of apnea i n the Cheyne-Stokes cycle. The antagonistic effects of sodium pentothal on the blood pressure and the respiratoryeffects of chlordan are illustrated i n Figure 1, C. T h e intermittent severe tremors with concomitant Cheyne-Stokes type of respiration produced by the injection of chlordan were decreased or entirely abolished, and both the respiratory rate and depth were restored to near normal. A dose of barbiturate sufficient to diminish the tremors appreciably and to restore the respiration to normal also produced a temporary drop of 10 to. 40 m m . of mercury i n blood pressure.
230
Figure 1. Kymographic Tracing Showing Effects of Chlordan on Blood Pressure and Respiration of Rabbit under Light Pentothal Anesthesia and Antagonistic Action of Sodium Pentothal
From top down: respiration, blood pressure, injection signal, and base line, time in 5 seconds; A = 60 mg./kg. chlordan, intravenously; = 6 minutes later, Cheyne-Stokes respiration; C = 15 mg./kg. sodium pentothal 15 minutes later (respiration restored to normal and lowered blood pressure due to temporary action of pentothal); D = 20 minutes after pentothal injection T h e effects of chlordan on the blood pressure and respiration of rabbits under sodium pentothal, in general, were characteristic of those obtained under sodium amytal and so dium phnobarbital, with some variation in intensity and length of duration.
Table I.
Antidotal Action of Barbiturates in Chlordan Poisoning in Rabbits

a
Barbiturate Used, M g . / K g . Before Barbiturate chlordan Amytal N a Pentothal Na Phnobarbital N a Controls Controls
After chlordan 30 15 15-50 30-40 0 0
Chlordan, Mg./Kg. 60 40 60 60 30 20
No. D i e d / No. Used 1/13 0/3 8/14 5/14 10/10 17/23
20 25-30 20-30 20-30 0 0
Administered intravenously.
T h e antidotal action of the barbiturates in intact animals is shown in Table I . A l l three barbiturates in the amounts given were effective i n decreasing the severity of the intermittent general body tremors, opisthotonus, extensor rigidity, salivation, and in the prevention of convulsions. In some cases the symptoms were entirely abolished through out the critical periodthe first 2 hours following chlordan administration. When deaths did occur they were generally delayed beyond the time of occurrence of death i n the nontreated groups ( 4 ) . T h e most effective antidotal action, however, was obtained with sodium amytal. T h e shorter acting sodium pentothal had considerably less antidotal effect under the conditions of these experiments. This may have been due to its relatively shorter duration of action, as a result of which the effects of chlordan were not antagonized over a sufficiently long period of time. T h e intermediate antidotal effect obtained with the long-acting phnobarbital is probably due in part to its relatively more shallow depth of anesthesia and slower action. T h e preliminary dose of the barbiturate was given in order to make the administration of the full dose of chlordan possible. Without this the severity of the tremors and convulsions developing during the injection made the complete
STOHLMAN AND SMITHTOXICOLOGICAL ACTION AND METABOLIC FATE OF CHLORDAN
231
Table II.
Fractionation of Organically Bound Chlorine Excreted in Urine of ChlordanPoisoned Rabbits

Extracted, M g . With ether from acid urine, p H 3.8 From ether with dilute alkali, p H 8.6 55.0 (83.8%) 0 With ether from aqueous alkali after acidification to p H 3.8 55.0 (83.8%) 0 Chlorine Split Off , ^ydrolysis at 100 C , % of Total Alcoholic Aqueous NaOH NaOH
n 0
Experimental Control plus 228 mg. chlordan
65.6 194.5 (85.3%)
38.2 44.3
34.5 6.9
injection of the relatively large doses of chlordan difficult or impossible. As a result of the antagonistic action of these barbiturates, rabbits tolerated up to approximately three times the L D dose of chlordan. The analysis of urinary excretion of organically bound chlorine by chlordan-poisoned rabbits is shown i n Table I I . Apparently all the organic chlorine excreted i n the urine of chlordan-poisoned rabbits is extractable with ether from urines acidified to p H 4.0 or lower. Only part of it was extracted from urines acidified to p H 6.0, and relatively little was ex tracted from alkaline urines. Nearly all of the organic chlorine-containing constituents (83.8%) were extracted from ether with dilute alkali, and i n turn were again re-extracted from aqueous alkali with ether after acidification. I n contrast, chlordan added to normal rabbit urine could be extracted from either an acid or an alkaline urine with ether, to the extent of about 8 0 % of the amount added. Moreover, this was not extractable with d i lute alkali from the ether phase. About one third of the total organic chlorine i n the u r i nary excretory product was split off with either alcoholic or aqueous sodium hydroxide, whereas only about 7% of the total chlorine was removed from chlordan by aqueous so dium hydroxide under comparable conditions. These data indicate that the urinary ex cretory product probably consists of one or more acidic chlorine-containing degradation products of chlordan, which are water soluble at p H 8.6 or higher. It seems possible that the labile chlorines i n positions 5 and 6 may be split off i n the body, followed by the oxi dation of the corresponding carbons.
5 0
Discussion
The barbiturates are effective against convulsions induced experimentally from all central locations, the cerebrum, medulla, or spinal cord. They may be used clinically as well as experimentally to suppress most, if not all, varieties of convulsions of central origin (3). Since they are effective i n the prevention of the tremors, tonic and clonic convulsions, and i n the restoration of normal respiration from the Cheyne-Stokes type, produced by chlordan, it appears that these symptoms may have their origin in the central nervous system. The antidotal action of the barbiturates is probably limited to the effects of chlordan on the nervous system. They most likely have no beneficial antagonistic action against the delayed parenchymatous degenerative changes produced by chlordan (4). Therefore, they are primarily only of possible value i n acute poisoning i n which severe stimulation of the central nervous system may be the primary cause of death. The conversion i n the animal body of at least some of the water-insoluble chlordan to a water-soluble degradation product must facilitate the elimination of the poison through its excretion into the urine by the kidneys. Moreover, the degradation of chlordan as shown i n the present experiments may be a mechanism for its detoxification, as i n the case of D D T (1). Only the isolation of the degradation product, its identification, and a study of its toxicity can determine this point.
Literature Cited
(1) Smith, M . I., Bauer, H . , Stohlman, E . F., and Lillie, R. D., J. Pharmacol. Exptl. Therap., 88, 359 (1946). (2) Smith, M . I., and Stohlman, E . F., U . S. Pub. Health Service, Rept. 59, 984 (1944). (3) Sollmann, . ., "Manual of Phartnacology," 7th ed., p. 681, Philadelphia, W. B. Saunders Co., 1948. (4) Stohlman, E . F., Thorp, W. T . S., and Smith, M . I., Arch. Ind. Hyg. Occupational Med., 1, 1319 (1950).
DDT in Eggs and Tissues of Chickens Fed Varying Levels of DDT

MELVIN J. BRYSON , C. I. DRAPER, JOSEPH R. HARRIS, CLYDE BIDDULPH, D. A. G R E E N W O O D , L. E. HARRIS, WAYNE BINNS, M. L. MINER, and L. L. MADSEN
1
Utah Agricultural Experiment Station, Logan, Utah
The concentration of DDT in eggs and tissues of chickens fed a mash made with DDT-treated alfalfa hay is reported. The amount of DDT in the fat, muscle, and eggs was correlated with the DDT intake.
I he use of D D T to control lygus bugs (Lygus elisus V a n D ; Lygus hesperus K n i g h t ) and alfalfa weevil (Hypera postica Gyll.) i n the production of seed alfalfa has been demonstrated by Lieberman (10), Smith and Michelbacher (17), and Lieberman and Hare (11). Alfalfa hay is also markedly improved b y the use of this insecticide (7). This has resulted in widespread use of the compound. W i t h the dissemination of D D T dusts and sprays over large forage crop areas, i t is important to know the effect of the insecticide on livestock and poultry. A series of studies has been conducted in the authors laboratories to gain information on this problem. When D D T is fed to animals, even i n small quantities, there is an accumulation of the compound i n the tissues, particularly the fat. Telford and Guthrie (18), Orr and M o t t (13), Woodward et al. (20, 21), and Laug and Fitzhugh (9) have demonstrated that D D T will accumulate i n certain tissues and i n milk fat of domestic and laboratory animals. Marsden and B i r d (12) found that D D T was toxic to turkeys i n concentrations above 0.075% of the diet, and that turkeys fed the insecticide for 7 to 8 weeks stored D D T i n their fat at concentrations ranging from 4 to 8 times that i n the diet. R u b i n et al. (14) reported that hens fed 0.062% D D T in their diet for 12 weeks showed reduced egg production with lowered hatchability. A t one half this concentration there was a detrimental effect on egg production, but hatchability was not seriously affected. The hens were killed by doses of 0.125% D D T . The insecticide was found i n the eggs i n quantities much smaller than i n the body fat. Harris et al. (8) have shown that D D T will accumulate i n considerable quantities i n the fat of lambs fed D D T - t r e a t e d hay. Small amounts of the insecticide were found i n other tissues. Carter and Fitzhugh (2, 5) have summarized the results of experiments on domestic
1
Table I.
Ground wheat Bran Ground barley Fish meal Soybean oil meal Salt Ground limestone Bone meal Fish liver oil Vitamin concentrate
1
Basal Mash
25 24.25 25 10 10 1 1.75 2.25 0.25 0.5
Present address, Department of Biochemistry and Nutrition, Agricultural and Mechanical College of Texas, College Station, Tex.
232
BRYSON ef al.DDT IN EGGS AND TISSUES OF CHICKENS
and laboratory animals i n which D D T was found to have accumulated in small amounts in the tissues. Carter et al. have shown (3) that cooking meat from D D T - t r e a t e d animals did not seriously change its D D T content and (4) that the insecticide accumulated in the fat of pigs fed beef that contained D D T . The purpose of this work was to determine the extent of accumulation of D D T i n eggs and the tissues of chickens during and after a period of feeding a mash made with D D T - t r e a t e d alfalfa hay.
2 3 3
Design of the Experiment

The alfalfa hay used i n making the mashes i n this experiment was from the same field as that described b y Harris et al. (1, 8). This alfalfa was dusted with 0, 1, 2, and 4 pounds of technical D D T per acre, which was approximately the same amount used b y hay producers. The harvested hay was ground into meal and a mash was made for l a y ing hens, containing 1 5 % alfalfa meal. D D T was also added to mash made from u n treated alfalfa meal at levels of 0, 50, 100, and 200 p.p.m. to determine a possible margin of safety for feeding D D T - t r e a t e d alfalfa. A total of 48 White Leghorn pullets approximately 6 months of age were used i n the experiment. Thirty-two pullets were selected at random from the original 48 birds, and divided into four groups of 8 birds each. One group was then assigned to each mash made with the hay dusted with 0, 1, 2, and 4 pounds of technical D D T per acre. The remaining 16 pullets were divided into four groups of 4 birds each, and were assigned to mashes containing 0, 50, 100, and 200 p.p.m. of D D T . This mash was made by thoroughly mixing an ether solution of D D T with 1 pound of mash and then mixing this with 19 pounds of mash (see Table I ) . The mash fed was made by mixing 1 5 % alfalfa meal with 8 5 % of the basal mash. This was available to the hens at all times. I n addition, the hens were fed a grain mixture once daily. This was made up of 5 0 % wheat, 4 0 % barley, and 10% oats. A l l hens were i n excellent laying condition at the start of the experiment. E a c h hen w as assigned at random to a compartment of a laying battery. The eggs were collected daily and were kept under refrigeration until they were ana lyzed for their D D T content. Feed consumption and production records were kept for each hen. The data on weight gains, food consumption, and egg production will be pub lished at the end of a 3-year feeding period. A t the end of one year of feeding D D T - t r e a t e d mash, 16 of the hens were killed and their leg and breast muscle and fatty tissues were analyzed for D D T . During this time 8 hens had died of leukemia complex, and their death was not attributable to the D D T . A t the beginning of the second year 24 of the original 48 hens were still on the experiment. I n order to replace those hens that were killed and those that had died, 24 more 6-monthold pullets were assigned at random to the eight groups of the experiment, making a total of 48 hens at the beginning of the second year.
r
The D D T residue of the hay was determined b y the total chloride method of U m hoefer (19) as described b y Harris et al. (8). The eggs and tissues were analyzed b y the method of Schechter.ei at. (16) with the modifications described b y Harris et al. (7). T h e eggs were sampled b y mixing the yolks and the whites of a l l the eggs from one hen for one month i n a Waring Blendor. A 50-gram sample of the eggs was placed i n a 250-ml. centri fuge bottle and 75 m l . of 9 5 % ethyl alcohol and 50 m l . of Skellysolve were added. The Table II.
D D T
DDT Content of Eggs of Chickens Consuming Mash Containing DDT-Treated Hay (1947-1948)
Months after Beginning D D T Feeding 9 10 D D T in Eggs, P . P . M . 1.7*2.0 1.7*2.0 0.91 2 2.0*2.0 2.1*1.6 1.8*0.8 1.6*1.3 3.7*4.9 2.4*2.5 3.3*1.6 4.1*1.0 2.5*1.2 8
Applied D D T Residue, P . P . M . per Acre, L b . On hay In mash6 0 0 0 1 15 2.3 2 22 3.3 4 42 6.3 Each value is average * S.D. of 6 to 8 hens. b D D T content of mash by calculation.
a
234
addition of the ethyl alcohol served to precipitate the protein and to prevent the for mation of emulsions with the Skellysolve B . The D D T was extracted from the egg m i x ture b y the Skellysolve B . After centrifuging, the Skellysolve was easily separated from the mixture. F r o m this point the analysis was carried out as with milk i n the method of Schechter et al. (15). A l l colorimetric readings were made on the Beckman quartz spectrophotometer.
Results and Discussions

None of the chickens showed i l l effects from the ingestion of D D T . The mortality of the 48 hens used was 15% over a period of a year, which is below the average mortality for laying hens in this area. The deaths were not associated with D D T feeding. There was no significant difference in live weight gain between any of the hens on any of the D D T treatments or between any of the D D T levels and the control chickens. The amount of D D T present on the treated hay and in the eggs for the first year of the experiment is shown in Table I I . The D D T residue on the alfalfa hay varied from 0 to 42 p.p.m. The undusted hay contained no D D T and the dusted hay contained amounts that increased progressively with the quantity of D D T applied. Small amounts of D D T that appeared i n the eggs of the control chickens might be explained by t h e hens scattering the mash during feeding to other compartments i n the laying battery. The presence of D D T i n small amounts does not detract from the significance of the other data. W i t h an increase of D D T in the feed there is generally an increase of the com pound i n the eggs. The D D T content of the eggs from chickens fed 0, 50, 100, and 200 p.p.m. i n the mash during the first year is presented in Table I I I . The eggs of the 12 control chickens were analyzed as one group; therefore, the results in both Tables I I and I I I are the same for the control chickens. The amount of D D T found i n the eggs of the chickens fed mash containing D D T i n most cases is roughly proportional to the amount of D D T ingested.. Table III. DDT Content of Eggs of Chickens Consuming Mash Containing DDT (1947-1948)
.
8 y
. ... Months after Beginning D D T Feeding D D T Applied r to Mash, . , P.P.M. D D T in Eggs, P . P . M . 0 1.7*2.0 1.7*2.0 50 7.5*8.7 7.7*6.9 100 13.3*5.8 18.7*8.4 200 65.6*25.2 57.1 * 5 . 9 Each value is average * S.D. of 2 to 4 hens. l> Value of one hen consistently high during 3-month period.
0.9*0.8 23.30& 18.3*10.6 43.5*12.8
In Table I V are tabulated the concentrations of D D T found i n thetissues of the chickens killed after one year of feeding. The concentration of D D T was markedly higher in the fat than in the diet. Only small amounts of the insecticide were found in the breast and leg muscle tissues. I n each case the fatty tissues contained D D T at markedly higher levels than the eggs. Table IV.
D D T Applied per Acre, Lb. 0 1 2 4 D D T Added to Mash, P.p.m.
DDT Content of Chickens Killed at End of 1 2 Months of Feeding DDT (1947-1948)

D D T Residue, P . P . M . On hay In mash 0 15 22 42 50 100 200 0 2.3 3.3 6.3 No. of Chickens 3 2 3 4 1 1 2 Leg, P.P.M. 0.3 0.5 0.7 1.0 1.8 11.6 Breast, P.P.M. 0.2 0.1 0.8 0.4 0.2 0.5 4.0 Fat, P.P.M 30.0 29.8 44.1 103.6 502.6
The concentration of D D T in the eggs during the second year is shown i n Tables V and V I . During the first month of the second year D D T was not fed. However, 24 of the hens that had been on the experiment during the first year still had D D T present i n their
BRYSON et al.DDT IN EGGS AND TISSUES OF CHICKENS
235
eggs. The 24 new pullets did not have D D T i n their eggs during the first month, but thereafter the level of D D T was about the same as that in the eggs during the first year. Table V. DDT Content of Eggs of Chickens Consuming Mash Containing DDT-Treated Hay (1948-1949)
a
DDT DDT6 Applied Residue per in Acre, Mash, Lb. P.P.M. 0 1 2 4
Months after Beginning D D T Feeding 3 4 5 6 7 D D T in Eggs, P . P . M .
0 0.30.6 0.30.5 0.30.6 0.20.4 0.70.8 0.20.3 0.30.8 0.40.5 0.10.4 2.30.6 2.20.6 3.61.0 2.60.8 2.90.8 2.3 3.3 1.00.4 2.10.6 0.80.3 3.40.5 3.40.7 3.10.9 6.3 6.3 0 . 2 0 . 3 0 . 5 0 . 4 4 . 3 1 . 5 2.7*1.5 8 . 2 1 . 9 7 . 9 1 . 9 8 . 3 2 . 8 Each value is average S.D. of 6 to 8 hens. b D D T content of mash by calculation. D D T not fed during first month. 16 hens were from experiment the preceding year D D T in their eggs. 16 new pullets did not have D D T in their eggs during first month.
a c
0.80.7 1.20.4 2.60.8 3.10.9 2.70.9 3.11.3 7.62.0 6.92.2 and still contained
Table VI.
DDT Content of Eggs of Chickens Consuming Mash Containing DDT (19481 949)*
Beginning D D T Feeding 5 6 " 7 8 9 in Eggs, P . P . M . 0 . 7 0 . 8 0 . 2 0 . 3 0.3*0.8 0 . 8 0 . 7 1.20.4 23.0 5 . 6 19.8 0 . 8 22.6 1 . 8 18.5 2 . 1 21.0 0 . 9 56.1 2 . 2 5 0 . 5 6 . 8 4 6 . 7 6 . 9 3 2 . 2 2 . 9 4 7 . 3 3 . 3 6 6 . 5 2 . 7 64.1 8 . 1 5 1 . 9 7 . 6 4 2 . 9 3 . 7 5 8 . 2 4 . 1
DDT in Months after Mash, lb 2 3 4 P.P.M. DDT 0 0.30.6 0.30.5 0.30.6 0.20.4 50 1.1 1 . 1 15.9 3 . 3 1 0 . 5 8 . 8 100 3 . 2 5 . 5 1 1 . 2 * 8 . 9 2 9 . 0 1 . 0 2 9 . 3 4 . 7 200 6 . 6 9 . 3 7 . 7 3 . 0 2 8 . 9 4 . 5 4 8 . 4 1 6 . 4
Each value is average * S.D. of 3 to 4 hens. 6 D D T was not fed during first month. 8 hens were from experiment of preceding year and still contained D D T in their eggs. 8 new pullets did not have D D T in their eggs during first month.
Rubin et al. have shown that 1250 p.p.m. of D D T in the diet of laying hens for 12 weeks produced toxic symptoms and death. I n the work reported here the highest level of D D T used was 200 p.p.m. The authors did not observe toxic symptoms at this or lower levels. The results of this study indicate that when alfalfa is dusted with D D T and the har vested hay is used in making a mash for chickens, D D T will appear i n considerable quan tities in the tissues and eggs of chickens consuming such mash. The daily intake of D D T in this study was not sufficient to produce toxic symptoms. However, if the same level of D D T is consumed for a long period of time, D D T may accumulate i n sufficient concentra tion to produce harmful effects in the chickens, or in a human consuming the meat or eggs. Fitzhugh and Nelson (6) have shown that the withdrawal of food from animals fed a high level of D D T produced characteristic D D T tremors; D D T - t r e a t e d animals made sick by infection also showed characteristic D D T tremors. They attributed these effects to the fact that the animals were metabolizing their body fat, which contained large amounts of D D T , at an increased rate during starvation or infection. This effect could be impor tant in cases of human infection or starvation if there was a large storage of D D T i n the fat tissue.
Summary
Alfalfa was dusted with 0, 1, 2, and 4 pounds of technical D D T per acre as an insecti cide and the harvested hay was ground into meal. A mash was made for laying hens containing 1 5 % alfalfa meal. D D T was also added to mash made with untreated alfalfa hay at 0, 50, 100, and 200 p.p.m. Four groups of 8 hens each were fed the mash made with the field-dusted hay and four groups of 4 hens each were fed the mash containing 0, 50, 100, and 200 p.p.m. of D D T . The D D T residue of the field-treated hay was 0.0, 15, 22, and 42 p.p.m. for the 0, 1, 2, and 4 pounds of D D T per acre treatment, respectively. During the first year of feeding the mash, D D T was present i n the eggs at a maxi mum concentration of 1.7, 2.1, 3.7, and 4.1 p.p.m. from the hens consuming the mashes
236
containing alfalfa treated with 0, 1, 2, and 4 pounds of D D T per acre, respectively. T h e eggs from the hens consuming mashes containing 0, 50, 100, and 200 p.p.m. of D D T c o n tained maximum amounts of 1.7, 23.3, 18.7, and 65.6 p.p.m. of D D T , respectively. The highest concentration of D D T was found i n the fat of the chickens killed after being fed D D T for 1 year. The average amount present was 30.0, 29.8, 44.1, 103.6, and 502.6 p.p.m. when hay treated with 1, 2, and 4 pounds of D D T per acre and 100 and 200 p.p.m. of D D T , respectively, was fed. M u s c l e tissues varied from 0.1 to 11.6 p.p.m. The concentrations of D D T i n the eggs during the second year of feeding the D D T treated mash were generally slightly higher than the first year.
Literature Cited
(1) Biddulph, C., Bateman, G. Q., Bryson, M. J., Harris, J. R., Greenwood, D. ., Binns, W.,
Miner, M . L . , Harris, L . E . , and Madsen, L . L . , A D V A N C E S I N CHEMISTRY SERIES, 1, 237
(2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)
(1950). Carter, R. H . , Ind. Eng. Chem., 40, 716 (1948). Carter, R. H . , Hubanks, P. E . , Mann, H . D., Alexander, L . M . , and Schopmeyer, G . H., Science, 107, 347 (1948). Carter, R. H . , Hubanks, P. E., Mann, H . D., Zeller, J . H . , and Hankins, O. G., J. Animal Sci., 7, 509 (1948). Fitzhugh, O. G., Ind. Eng. Chem., 40, 704 (1948). Fitzhugh, O. G., and Nelson, . ., J. Pharmacol. Exptl. Therap., 89, 18 (1947). Harris, L . E., Biddulph, C., Greenwood, D . ., Binns, W., Miner, M . L., and Madsen, L . L . , unpublished data. Harris, J . R., Biddulph, C., Greenwood, D. ., Harris, L . E., Bryson, M . J., Binns, W., Miner, M . L., and Madsen, L . L., Arch. Biochem., 21, 370 (1949). Laug, E. P., and Fitzhugh, O. G., J. Pharmacol. Exptl. Therap., 87, 18 (1946). Lieberman, F . V., U . S. Dept. Agr., Bur. Entomol. Plant Quarantine, Bull. E-658 (1945). Lieberman, F . V., and Hare, Q. ., Ibid., Bull. E-697 (1946). Marsden, S. J . , and Bird, H . R., Poultry Sci., 26, 3 (1947). Orr, L . W., and Mott, L . O., J. Econ. Entomol., 38, 428 (1945). Rubin, M . , Bird, H . R., Green, N . , and Carter, R. H . , Poultry Sci., 26, 410 (1947). Schechter, M . S., Pogorelskin, . ., and Haller, H . L., Anal. Chem., 19, 51 (1947). Schechter, M . S., Soloway, S. B., Hayes, R. ., and Haller, H . L., Ind. Eng. Chem., Anal. Ed., 17, 704 (1945). Smith, R. F., and Michelbacher, A. E., J. Econ. Entomol., 39, 638 (1946). Telford, H . S., and Guthrie, J . E., Science, 102, 647 (1945). Umhoefer, R. R., Ind. Eng. Chem., Anal. Ed., 15, 383 (1943). Woodward, G., and Ofner, R. R., Federation Am. Soc. Exptl. Biol., Federation Proc., 5, 215 (1946). Woodward, G., Ofner, R. R., and Montgomery, C. M . , Science, 102, 177 (1945).
R E S E A R C H supported in part by a research grant from the Division of Research Grants and Fellowships, N a tional Institutes of Health, U . S. Public Health Service, and published with the approval of the director of the Utah Agricultural Experiment Station.
DDT in Milk and Tissues of Dairy Cows Fed DDT-Dusted Alfalfa Hay
CLYDE BIDDULPH, G. Q. BATEMAN, M. J. BRYSON, J. R . HARRIS, D. A. GREENWOOD, WAYNE BINNS, M. L. MINER, L. E. HARRIS, and L. L. MADSEN
Utah Agricultural Experiment Station, Logan, Utah
Untreated alfalfa hay and hay dusted with technical DDT were fed to 16 Holstein cows during 2 years DDT appeared in the milk within 4 days after the feeding of DDT-treated hay and the amount gradually increased until a fairly constant level was reached. More DDT was found in milk and tissues of cows that consumed hay treated with higher levels of DDT. DDT persisted in the milk for 4 months and dis appeared approximately 6.5 months after feeding of DDT-treated hay was discontinued.
revious studies have shown that D D T appears in the milk and body fat of dogs (18), in the milk of rats and goats (11), in the tissues, especially fatty tissues, of rats (5, 6) and rabbits (4), i n the milk and tissues of dairy cows (1, 10), and i n the tissues (2) and milk (7) of sheep following the ingestion of D D T or of feeds containing D D T residues. D D T persists i n the milk of cows for relatively long periods of time after they have been sprayed with D D T for the control of flies (3). Inasmuch as these studies established the accumulation of D D T i n the fatty tissues and its appearance i n the milk following the ingestion of relatively large doses of D D T , the question arose as to whether the milk of dairy cows consuming alfalfa hay with low D D T residues contained sufficient D D T to be hazardous to animals and humans consum ing the milk. The problem assumed greater importance because of the practice i n this and other areas of dusting forage alfalfa with D D T for the control of alfalfa weevil and other insects and the feeding of such alfalfa hay to dairy cows. This paper reports data obtained following feeding of D D T - d u s t e d alfalfa hay to H o l stein dairy cows. M i l k , blood, and tissues were analyzed for their D D T content during two different years of feeding of the D D T - d u s t e d hay.

Alfalfa hay was obtained from a 44-acre unirrigated field at Petersboro, Cache County) U t a h . Thefieldwas divided into sixteen 1-acre plots with intervening untreated buffer strips of sufficient width (150 feet) to prevent drift of the D D T dust from one plot to another at the time of application. A block of four 1-acre plots received the following treatments during the summer of 1947 : no D D T , 1 pound, 2.pounds, and 4 pounds of tech nical D D T per acre, and no D D T , 0.5 pound, 1 pound, and 2 pounds of technical D D T per acre during the summer of 1948. These treatments were repeated on the remaining three blocks of plots, so that a total of four 1-acre plots received each treatment. T h e treatments within each block were assigned at random. The D D T dust i n a pyrophyllite carrier was applied with a power duster when a sub237
238
1 1
1\ ~~\ i
-i
1
f - r~
\/
\
I1
\ \
w s cn
c o n s u n linff i
lb. 1)DT-t eated hav
t 1
1
I
/
1
/
t 1
C( ws consuming 2 lb.
D D T t r e a t e d
toy
\
cows consuming 1 lb. DDT-treated hay \
\ \ \ \
/
t
/ /
nwc e n n a i m i n o rn o DDT-treated hav
^\
\
>
t
lan. 20
off DDT hay
May 13
Sept16
DAYS
Figure 1.
DDT in Milk of Cows Fed Alfalfa Hay Dusted with Technical DDT (1948)
stantial number of alfalfa weevil (Hypera postica Gyll.) larvae were present on the alfalfa. The number was determined by sweeping each of the plots with a hand net and counting the larvae obtained from each plot. These data will be published elsewhere. The alfalfa used was second crop i n 1947 and first crop i n 1948. I t was cut when the majority of the plots dusted with D D T were judged to be i n the tenth-bloom stage. The alfalfa within each plot was allowed to sun-cure, and was then baled and hauled tothe college barns for storage. Eight cows were chosen from the college experimental herd each year, two cows being assigned to hay from each of the field treatments. M i l k and blood samples were obtained from each cow before the experiment started (January 21, 1948, and November 24, 1948), and these samples were analyzed for D D T . On the day after the cows began eating the control and experimental hays (November 1948), and on the fourth day after (January 1948), milk samples were obtained and analyzed for D D T . Samples were then collected and analyzed at 3-day intervals until the concentration of D D T in the milk had reached a relatively constant level, after which samples were collected at weekly or longer intervals until the experiments were terminated several months later (Tables I and I I and Figures 1 and 2). Feeding of alfalfa hay was discontinued on M a y 13, after 113 days, the first year of the experiment, at which time the cows went to pasture. Sampling and analysis of the milk for D D T continued, however, until September 16. During the second year, the feeding of the alfalfa hay commenced on November 24 and continued until February 13, a period of 81 days. Between this time and the time the cows went to pasture ( M a y ) , they were fed untreated hay from the college fields. Blood and milk samples were collected the second year before, during, and following the period of feeding the treated alfalfa hay. The D D T content of the tissues and milk was determined by the colorimetric method of Schechter et al. (8, 9) as modified by the authors (2). The amount of D D T residue on the hay was determined b y the total chloride method of Umhoefer (12). Cow H u 29 (fed hay treated with 2 pounds of D D T per acre) was slaughtered on M a y 20, 1948, one week after experimental feeding was discontinued. Kidney and mesenteric
BIDDULPH ef al.DDT IN MILK AND TISSUES OF DAIRY COWS
239
fat, muscle, and liver were analyzed for D D T content. I n 1949 cows H u 36 (fed hay treated with 1 pound of D D T per acre) and H u 329 (fed untreated hay) were slaughtered at the close of the feeding period and the tissues were analyzed for their D D T content as before. Cow 139 (fed hay treated with 1 pound of D D T per acre) was slaughtered on June 29, 1949, approximately 4 months after the feeding of the D D T - t r e a t e d hay was dis continued, and the tissues were likewise analyzed for their D D T content. In addition to the alfalfa hay, the cows received a grain ration which consisted of a mixture of 8 0 % barley and 2 0 % molasses dried beet pulp, to which were added 2 % steam bone meal and 1% fine hay salt (sodium chloride). This ration was fed at the rate of 0.75 pound of grain per day for each pound of butterfat produced during the previous week. If the butterfat production dropped below 0.8 pound per day, the grain ration was routinely discontinued. Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch044
1! |3
4
i
J
10
I
20
I
30
I
40
I
50
I
60
I
70
l. I
8 * 90 Feb. 13
100 1 1 0 1 2 0 1 3 0
Nov. 24
Figure 2.
off DDT hay
DAYS
1 + 0 1 5 0 1 6 0 1 7 0 1 8 0 190 200 0 220 230 June 21
'
'
DDT in Milk of Cows Fed Alfalfa Hay Dusted with Technical DDT (1948-49)
In order to determine whether there was sufficient D D T in the milk to produce effects in rats, milk from the cows was separated and the cream was incorporated into a synthetic diet which was fed to a group of rats for a period of 4 months. Butter was churned from some of the cream and this was likewise incorporated into a diet and was fed to another group of rats. These data will be published elsewhere.
Results and Discussion

The amount of D D T present in the milk of the cows during the winter of 1948 is presented in Table I and Figure 1. N o D D T was present in the milk of any of the cows at the beginning of the experiment. However, within 4 days after the feeding of D D T treated hay was begun, D D T appeared i n the milk. The amount gradually increased until a fairly constant level was reached before the feeding of D D T - t r e a t e d hay was dis continued. Calculation of the average amount of D D T present i n the milk throughout the period of feeding of the D D T - t r e a t e d hay shows that the amount i n the milk was de pendent upon the amount applied to the hay (1 pound of D D T applied, 1.7 p.p.m. in the milk; 2 pounds of D D T applied, 3.3 p.p.m. in the milk; and 4 pounds applied, 7.1 p.p.m. in the milk). One of the control cows (A 133) had a trace of D D T in her milk shortly after the feeding of the alfalfa hay was begun. Inspection of the manger of this cow revealed an opening through which i t was possible for her to obtain a small amount of hay from the adjoining manger, and the attendant observed this cow obtaining such hay (treated with 2 pounds of D D T per acre). After the manger was repaired, the amount of D D T i n the milk of this cow decreased, but a trace of D D T persisted even after the cow freshened sev eral months later (see Table I ) . The other cows, 139 and H u 106, came to the end of their lactation period after 3 to 3.5 months on experiment. After freshening there was still a trace of D D T present i n
240
Table I.
DDT in Milk of Cows Fed Alfalfa Hay Dusted with DDT (1948)
(P.p.m. of D D T ) No D D T A 133 Hu 88 Av. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.6 0.0 0.4 0.0 0.3 0.0 0.6 0.0 0.9 0.0 1.1 0.0 1.4 0.0 1.2 0.0 1.2 0.0 0.5 0.0 0.5 0.0 0.3 0.0 0.3 0.0 0.4 0.0 0.3 0.0 0.2 0.0 0.2 0.0 0.2 0.0 0.2 0.0 0.4 0.0 0.1 0.0 0.2 0.0 0.2 0.0 Dry 0.0 Dry 0.0 Dry 0.0 Dry 0.0 Dry 0.0 Dry 0.0 Dry 0.0 Fresh 0.0 0.1 0.0 0.1 0.0 0.1 Killed 0.1 0.1 0.1 0.0 0.0 0.0 0.0 0.2 0.3 0.2 0.1 0.3 0.4 0.5 0.7 0.6 0.6 0.2 0.2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.05 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.05 0.05 0.1 0.1 0.1 0.1 1 Lb. D D T per Acre Hu 39 139 A v . 0.0 1.4 0.3 0.5 2.0 2.2 1.3 0.5 2.5 1.3 0.8 2.6 2.2 1.9 2.3 0.6 2.6 l'. 2.7 1.6 1.9 1.3 2.6 1.8 0.5 0.5 0.3 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.2 0.2 0.1 0.05 0.1 0.05 0.05 0.0 0.7 0.4 1.8 1.7 1.5 1.6 1.1 0.8 2.1 2.4 2.7 1.8 2.2 2.2 2.1 1.8 2.9 3.0 3.8 2.6 2.7 Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Fresh 0.4 0.5 0.3 0.3 0.2 0.0 1.0 0.3 1.1 1.8 1.8 1.4 0.8 1.6 1.7 1.6 2.6 2.0 2.0 2.2 1.3 2.2 2.9 2.3 3.2 2.1 2.3 1.3 2.6 1.8 0.5 0.5 0.3 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.2 0.2 0.3 0.3 0.2 0.2 0.1 2 Lb. D D T per Acre Hu 29 Hu 106 Av. 0.0 2.2 0.6 0.7 2.6 3.8 1.1 1.1 3.7 3.9 4.9 4.4 3.8 2.4 3.4 3.4 4.5 4.1 5.5 4.9 4.5 4.8 6.3 7.2 Killed 0.0 1.7 0.4 0.6 3.0 2.9 1.0 2.2 3.0 2.2 3.3 3.8 3.6 3.5 2.9 3.3 2.4 36 4.5 3.6 4.5 4.1 4.3 5.4 1.5 1.7 1.1 Dry Dry Dry Dry Dry Dry Dry Fresh 0.4 0.3 0.2 0.2 0.2 0.3 0.0 1.9 0.5 0.6 2.8 3.3 1.0 1.6 3.0 2.9 3.6 4.3 4.0 3.6 2.6 3.3 2.9 4.5 3.8 5.0 4.2 4.5 4.4 5.3 6.3 1.5 1.7 1.1 4 Lb. D D T per Acre 180 199 Av. 0.0 2.2 1.3 1.6 8.0 8.1 5.4 6.1 8.7 9.0 8.7 12.5 8.1 6.8 9.8 10.0 11.5 9.7 12.5 11.5 11.3 11.9 9.6 12.3 4.2 2.8 1.9 2.0 1.1 1.0 0.8 0.5 0.6 0.5 0.4 0.4 0.3 0.4 0.3 0.3 0.3 0.0 2.2 0.7 1.5 5.1 4.4 4.3 2.8 5.2 6.0 6.5 7.3 7.8 4.4 5.0 5.0 7.6 7.3 7.2 9.0 8.3 7.1 6.7 8.0 7.5 3.1 1.6 1.9 1.5 1.3 1.1 0.9 0.9 0.6 0.5 0.6 0.6 0.5 0.2 0.4 0.4 0.0 2.2 1.0 1.5 6.5 6.2 4.8 4.4 6.9 7.5 7.6 9.9 7.8 6.2 5.9 7.4 8.8 9.4 8.4 10.7 9.9 9.2 9.3 8.8 9.9 3.6 2.2 1.9 1.7 1.2 1.0 0.8 0.7 0.6 0.5 0.5 0.5 0.4 0.3 0.3 0.3 0.3
Day (Jan. 21) 4 7 11 13 16 19 -22 25 28 31 34 37 40 43 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 162 169 176 183 190 197 204 211 225 232 239 (Sept. 16)
a
0.4 0.3 0.2 0.2 0.2 0.3
* * M a y 13, cows off hay, to pasture.
Table II.
DDT in Milk of Cows Fed Alfalfa Hay Dusted with DDT (1948-49)
(P.p.m. of D D T ) 0.5 L b . D D T per Acre Av. 181 H u 83 1 L b . D D T per Acre H u 36 139 A v . 0 0 0.2 1.1 0.8 0.5 1.6 1.5 0.9 0.4 1.1 0.4 1.3 2.1 1.9 2.6 2.4 2.0 0.6 0.8 0.1 0.1 0.3 0.4 Killed 0 0 0.1 1.5 1.3 0.7 1.6 1.3 0.7 1.2 1.1 0.4 1.2 2.3 3.1 1.6 2.3 0.5 0.3 0.1 0.2 0.2 0.2 0.0 0.1 0.1 0 0 0.2 1.3 1.1 0.6 1.6 1.4 0.8 0.8 1.1 0.4 1.3 2.2 1.9 2.9 2.0 2.2 0.6 0.6 0.1 0.2 0.3 0.2 0.0 0.1 0.1 2 L b . D D T per Acre H u 106 b A 133 Av 0 0 0.3 1.9 1.0 1.3 2.1 2.1 1.1 1.5 1.7 1.7 1.5 2.0 4.0 3.5 2.5 2.1 1.4 0.2 0.2 0.5 0.5 0.5 0.2 0.1 0.2 0 0 0.2 1.6 2.6 1.0 2.6 2.7 1.2 2.1 1.5 1.9 2.9 2.7 3.7 5.6 5.4 2.8 1.5 0.6 0.9 1.2 1.4 1.2 0.5 Dry Dry 0 0 0.3 1.8 1.8 1.2 2.4 2.4 1.2 1.8 1.6 1.8 2.2 2.4 3.9 4.6 4.0 2.4 1.5 0.4 0.6 0.9 1.0 0.9 0.4 0.1 0.2
Day 0 (Nov. 24) 1 2 6 10 13 16 19 22 25 33 40 47 54 61 69 75 81 (Feb. 13) 82 91 96 103 110 131 145 161 180 209 (June 21)
a
No D D T Hu 85 Hu329 Av.
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 0.05 0 0 0 0.7 0.8 0.8 0 0 0 0.3 0.8 0.6 0 0 0 0.4 0.5 0.5 0 0 0 0.8 1.0 1.1 0 0 0 0.6 0.6 0.6 0 0 0 0.3 0.4 0.4 0 0 0 0.3 0.4 0.4 0 0 0.6 0.3 0.5 0 0 0 0 0.6 0.6 0.6 0 0 0 0.7 0.6 0.7 0 0 0 0.8 1.3 1.1 0 0 0 1.5 1.4 1.3 0 0 0 1.4 2.2 1.8 0 0 0 1.4 0.9 1.2 Last day that D D T - treated hay was fed 0 0 0 0.5 0.5 0.3 0 0.3 0 0 0 0.2 0.2 0 0 0 02 0.2 0 0 0.1 0 0.1 0.2 0.2 0 0 0 0 0.2 0.2 0.2 0 0 0 0 Dry 0.4 0.4 0 0 0 Dry 0.2 0.2 Killed 0 Dry 0.1 0.1 0 0 0 Dry 0.1 0.1
Cow received hay dusted with 1 lb. D D T per acre during 1948 (Table I). b Cow received hay dusted with 2 lb. D D T per acre during 1948 (Table I). Cow received hay dusted with no D D T during 1948 (Table I).
BIDDULPH et /.DDT IN MILK AND TISSUES OF DAIRY COWS
241
the milk of each 4 to 4.5 months later. This would seem to indicate that the D D T is re leased into the blood stream from the storage sites at a slow rate. This is further indi cated b y the presence of traces of D D T i n the milk of a l l the cows that consumed D D T treated hay for more than 4 months after the ingestion of the hay was discontinued. Cows A 133, H u 106, and 139 were all used during the second year's feeding trials. Reference to Table I I shows that at the beginning of the feeding period (November 24, 1948) there was no D D T present i n the milk of any of these. Thus, the D D T had disappeared com pletely from the milk within approximately 6.5 months after the feeding of the D D T treated hay was discontinued. The amount of D D T present i n the milk of the cows during the second winter (194849) is given i n Table I I and Figure 2. Three of the cows used the previous winter (A 133, H u 106, and 139) were used again; the remaining cows were new to this project. T h e results obtained are essentially like those of the previous winter. During the second winter hay treated with 0.5 pound of D D T per acre was substituted for hay treated with 4 pounds. Otherwise the experimental procedure was the same as during the first winter. As before, a greater quantity of D D T was found in the milk of the cows that consumed the hay treated with the higher levels of D D T . Calculation of the average amount of D D T present i n the milk during the feeding period shows a close agreement in the amount of D D T present during the two years, where i t is possible to make such a comparison. For example, the average amount of D D T present in the milk of the cows consuming hay treated with 1 pound of D D T per acre during 1948 was 1.7 p.p.m.; i n 1948-49, 1.3 p.p.m. Following the 2-pound treatment i n 1948, 3.3 p.p.m. of D D T were present i n the milk, whereas i n 1948-49 2.2 p.p.m. of D D T were present. The average amount of D D T pres ent i n the milk of the cows consuming hay treated with 0.5 pound of D D T per acre during the feeding period was 0.7 p.p.m. Considering all the possible sources of variation in the experiments, the agreement in the data from the two years seems fairly good. The amount of D D T residue present on the alfalfa hay fed to the dairy cows during the two years is given i n Table I I I . A greater D D T residue was present on the hay the second year than during the first. A possible explanation of this difference is that during the harvest i n the summer of 1947 the hay was rained on several times after i t was cut and before i t was dry enough to bale, whereas i n the summer of 1948 the hay was not rained on between cutting and baling. Whether this difference in the curing and handling of the hay is the only reason for the difference in D D T residue is not known. The method of application and calibration of the power duster and calculation of the dosage used each year was the same. The details of these procedures will be published elsewhere. Table III. Average DDT Residue on Alfalfa Hay Fed to Dairy Cows and Average DDT Appearing in Milk during Period of Feeding
1948-49 D D T residue, D D T in milk, p.p.m. p.p.m. 0 0 9.0 0.7 19.2 1.3 30.0 2.2
1947-48 D D T Treatment, D D T residue, D D T in milk, Lb./Acre p.p.m. p.p.m. None 0 0.05 0.5 .. 1 9.6 1.7 2 12.1 3.3 4 36.0 7.1 DDT-treated hay obtained from adjoining manger.
a
The hay used during the first winter was analyzed b y both the colorimetric and total chloride methods (8, 9, 12). The results obtained with the total chloride method were less variable than those obtained with the colorimetric ; consequently, the former method was adopted. I n one third of the samples, the values obtained with the two methods were the same. I n the remaining two thirds of the samples, the values with the colori metric method were approximately 2 0 % below those of the total chloride. However, analysis of the control samples by both methods revealed the absence of D D T i n the u n treated hay, thus indicating that organic compounds containing chlorine were absent in the untreated hay. Analysis of the blood of the cows for D D T before the feeding of D D T - t r e a t e d hay was
242
begun both years showed that no D D T was present. After the D D T - t r e a t e d hay had been fed for 113 and 81 days, analysis showed an average of 0.1, 0.1, and 0.2 p.p.m. of D D T in the blood of cows consuming hay treated with 1, 2, and 4 pounds of D D T per acre, re spectively. These trace amounts of D D T i n the blood were found at the same time the D D T concentration was highest in the milk. Three of the cows used were slaughtered at the end of the feeding period, and a fourth was killed 4 months later. The amount of D D T present i n various tissues at these times is presented in Table I V . It is evident that large amounts of D D T are present i n the fatty tissues, whereas there is little present in muscle, liver, or kidney. Apparently its presence i n the fat, and the slow release into the blood stream from these storage sites, account for the persistence of the D D T i n the milk for long periods of time after the feed ing of the D D T - t r e a t e d hay was discontinued. Cow 139 still had 3 p.p.m. of D D T i n her fat 4 months after the feeding of the D D T - t r e a t e d hay was discontinued. It was not possible to obtain a milk sample at this time, for the cow had ceased lactating some time before she was slaughtered. Table IV. DDT in Tissues of Cows at End of Feeding Period
No. of Days Fed Hay 81 81 113 81 Mesenteric fat 0 21.4 89 3 Kidney fat 0 19.3 90 3 _ _ _ D D T , P.P.M. Muscle 0 0.4 1.3 0.1 Liver 0 0.2 1.1 0 Kidney 0 0.1 0 '
Cow Hu 329 Hu 36 Hu 29 139

a
D D T Added to Hav Consumed, Lb./Acre 0 1 2 1
Slaughtered approximately 4 months after feeding of DDT-treated hay was discontinued.
D a t a showing daily feed consumption and milk and butterfat production of the cows for the two years are given i n Table V . During the period of feeding, there was no evi dence that the cows receiving D D T - t r e a t e d hay were injured i n any way, or that their milk production was affected. Apparently the amount of D D T ingested daily and the release of D D T from the fatty tissues did not provide sufficient D D T in the blood stream to affect feed consumption or milk production. Table V. Average Daily Feed Consumption and Milk and Butterfat Production of Dairy Cows
Cows A 133 H u 88 H u 39 139 H u 29 H u 106 180 199 H u 329 H u 85 H u 83 181 H u 36 139 H u 106 A 133 No. of Days Fed 113 113 113 113 113 113 113 113 81 81 81 81 81 81 81 81 Hay Consumed. Lb. 32.2 33.2 33.4 29.6 32.9 30.9 23.2 36.2 1948-49 0 0.5 1 2 44.8 40.7 40.1 41.0 40.6 45.4 41.2 43.9 4. .8 4 .6 4. ,1 4, .7 5, .3 7. .3 6, .9 7, .4 25. .2 24 .3 19, .2 22 .5 29. .5 42 .6 36 .6 35 .6 0. .84 0 .81 0 .72 0 .95 0. .96 0 .36 1. .27 1, .22 3.3 3.3 3.8 4.2 3.3 3.2 3.5 3.4 Grain Consumed, Lb. 4. .9 5, 5 7. .0 0 .6 4. .5 5 .0 5, .8 7, .7 Milk Produced, Lb. 25. .3 31 .7 33 .2 11. .6 20 2 26 .0 26 .4 37 .1 Butterfat Produced Lb. 0 .86 1 .04 1, .25 0 .39 0 .87 0 .96 0 .79 1 .37
D D T Added to Hay, Lb./Acre 0 1 2 4
%
3.4 3.3 3.7 3.4 4.3 3.7 3.0 3.7
1947-48
Three cows calved after the feeding of the hay was discontinued (Table I). The preg nancy of the cows was apparently normal and there were no abnormalities i n any of the calves. This indicated that the level of D D T was not sufficiently high to affect the cows or calves during the period of gestation. The results of these experiments show that D D T accumulates in the fatty tissues of cows, and that it is present in the milk i n varying amounts, the amount being dependent
BIDDULPH ET AL.DDT IN MILK AND TISSUES OF DAIRY COWS
243
upon the quantity of D D T ingested. Furthermore, sufficient residue remains on alfalfa hay after it has been dusted with the amount of D D T recommended to control insects, to cause substantial quantities of D D T to appear i n the milk and fat. Smaller amounts of D D T persist i n the fat and milk for at least 4.5 months after the ingestion of the D D T treated hay has been discontinued. The possible toxicity of such milk and fat to humans would seem to be dependent upon a number of factors, among which are the amount of D D T applied to the forage, the frequency and total length of time during which the treated hay is ingested b y the cow, the amount and frequency of consumption of such milk and fat b y humans, and possibly the age of the humans consuming these products.
Summary
Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch044 Alfalfa hay that had been dusted with varying amounts of technical D D T for insect control and similar untreated hay were harvested and fed to 16 Holstein dairy cows during two years. D D T appeared i n the milk promptly after the cows consumed the D D T - t r e a t e d hay. The concentration gradually increased until maximum amounts of 2.2, 3.8, 7.2, and 12.5 p.p.m. of D D T were obtained i n the milk of cows consuming hay dusted with 0.5, 1, 2, and 4 pounds, respectively, of D D T per acre. The average D D T residue on the hay var ied from 9 to 36 p.p.m., there being a greater residue on the hay for a given treatment the second year than there was the first. D D T persisted i n the milk for 4 months after the feeding of the D D T - t r e a t e d hay was discontinued. I t disappeared approximately 6.5 months after discontinuance of the feeding of treated hay. Four cows had D D T i n their milk at the end of their lactation period, and after calving traces of D D T were still present i n their milk. The blood was analyzed for D D T before the cows were placed on the D D T - t r e a t e d hay and again before the hay was discontinued. N o D D T was found before and a maxi mum of 0.2 p.p.m. at the close of the feeding period. Four cows were slaughtered and the tissues were analyzed for D D T . The analysis showed a maximum of 89 p.p.m. of D D T i n mesenteric fat, 90 p.p.m. i n kidney fat, 1.1 p.p.m. i n the liver, 1.3 p.p.m. in muscle, and 0.1 p.p.m. i n kidney tissues. The greatest amount of D D T was present i n the tissues of cows consuming the hay dusted with the higher levels of D D T .
Literature Cited
(1) Allen, . N., Lardy, . ., and Wilson, H . F., J. Dairy Sci., 29, 530 (1946). (2) Harris, J . R., Biddulph, C., Greenwood, D . ., Harris, L . E., Bryson, M . J . , Binns, W., Miner, M . L., and Madsen, L . L., Arch. Biochem., 21, 370 (1949). (3) Howell, D . E., Cave, H . W., Heller, V. G., and Gross, W. G., Dairy Sci., 30, 717 (1947). (4) Laug, E . P., J. Pharmacol. Exptl. Therap., 86, 332 (1946). (5) Laug, E . P., and Fitzhugh, O. G., Ibid., 87, 18 (1946). (6) Ludewig, S., and Chanutin, ., Proc. Soc. Exptl. Biol. Med., 62, 20 (1946). (7) Madsen, L. L., unpublished data. (8) Schechter, M . S., Pogorelskin, . ., and Haller, H . L . , Anal. Chem., 19, 51 (1947). (9) Schechter, M . S., Soloway, S. B., Hayes, R. ., and Haller, H . L., Ind. Eng. Chem., Anal. Ed., 17, 704 (1945). (10) Shephard, J. B., Moore, L . ., Carter, R. H . , and Poos, F. W., Dairy Sci., 32, 549 (1949). (11) Telford, H . S., and Guthrie, J . E., Science, 102, 647 (1945). (12) Umhoefer, R. R., Ind. Eng. Chem., Anal. Ed., 15, 383 (1943). (13) Woodward, G., Ofner, R. R., and Montgomery, C. M . , Science, 102, 177 (1945).
R E S E A R C H supported in part by a research grant from the Division of Research Grants and Fellowships, N a tional Institutes of Health, U . S. Public Health Service, and published with the approval of the director of the Utah Agricultural Experiment Station.
2,4-Dichlorophenoxyacetic Acid (2,4-D) as a Selective Herbicide

J. H. QUASTEL
McGill University and Research Institute, Montreal General Hospital, Montreal, Canada
In view of the great agricultural importance today of the synthetic plant growth substances in controlling the growth of plants of economic importance, details of the history of this development of investigations on plant hormones are presented.
U u r i n g 1941 and 1942, two groups of workers i n England independently investigated the phytocidal effects of a number of plant growth substances, including 2,4-dichlorophenoxyacetic acid. N u t m a n , Thornton, and Quastel (27), working at the Rothamsted Experimental Station on the problem of whether root hair deformation can be induced by 2-indolylacetic acid and 1-naphthaleneacetic acid, found that these substances not only produced marked root hair deformation i n agar cultures of red clover but were highly toxic at a dilution of 1 i n 10 . The high toxicities of these substances suggested the possibility that such compounds might be used to control plant growth. Accordingly, experiments were made to determine whether toxicity would take place under soil conditions. It was found that the toxicity of 2-indolylacetic acid was retained i n sterile soil but was lost in normal soil, the growth substance obviously being broken down b y soil microorganisms. It had been known for some time that 2-naphthoxyacetic acid is a plant growth regulator producing abnormal formative effects on leaves, stems, flowers, and fruit (40). It had already been mentioned as an active substance i n this respect i n 1938 (16) and later investigations (6, 7, U, 44) provided further details of its activity. Further studies by Zimmerman and Hitchock (42) showed that halogenated phenoxyacetic acids have marked physiological activity i n plant growth, inducing formative effects. They noted that 2,4-dichlorophenoxyacetic acid was among the most active in influencing plant growth, inducing cell elongation of the tomato with concentrations as low as 0.0007% in lanolin. In view of these activities of 2-naphthoxyacetic acid and of the halogenated phenoxyacetic acids, and the likelihood that the chlorinated molecules would be relatively resistant to attack b y bacteria in the soil, investigations (27) were made of the toxicity of 2,4dichlorophenoxyacetic acid and other substances to plant growth i n agar culture and i n normal soil. Tests made i n October 1942 with red clover showed that 2,4-dichlorophenoxyacetic acid was much the most toxic of the compounds tested and, unlike the other substances, retained its toxicity to a satisfactory extent i n a normal soil. I t was then found that the lethal effect of 2,4-dichlorophenoxyacetic acid was selectivefor example, in Woburn soil, the substance was strongly toxic to clover and especially to sugar beets, even at a concentration of 1 p.p.m. Wheat, however, was not appreciably affected at this concentration, though marked toxicity appeared at 10 p.p.m. I n a Rothamsted soil, toxicity toward clover and wheat was less marked, although the action on sugar beets was again very striking. Investigations were made of the persistence of the toxicity of 2,4-dichlorophenoxyacetic acid i n unsterilized soils, both on storage and after leaching. It was found that there is persistence i n normal soils, sufficient to produce marked toxic
7
244
QUASTEL2,4-D AS A SELECTIVE HERBICIDE
245
effects, at a concentration of 1 p.p.m. of soil solution, equivalent to 1 part i n 4,000,000 of soil at 2 5 % moisture content. This represents, on a field scale, about 0.5 pound of 2,4-D per acre of soil to 6 inches depth. The results showed that 2,4-dichlorophenoxyacetic acid has the properties of a differential plant poison under soil conditions, and indicated its possible importance as a controller of plant growth i n agricultural practice. A report of the differential toxicity of 2,4-dichlorophenoxyacetic acid was first made to the secretary of the Agricultural Research Council on November 17, 1942. Publication (27) was not allowed until 1945. A second group of investigators, Slade, Templeman, and Sexton, working at the Jealott's H i l l Research Station, commenced their studies of the effects of growth substances on plants i n 1936. Templeman (31) had observed that high concentrations of the growth substances depressed seedling growth. Templeman, and M a r m o y (33) found in 1940 that application of 1-naphthylacetic acid at the rate of 25 pounds per acre to oats weedy with yellow charlock killed the charlock. The oats received only a slight setback and recovered fully. The effects of known plant growth substances were then surveyed. Substituted phenoxyacetic acids and naphthoxyacetic acids were found to be outstanding as plant inhibitors and certain of them were found to be fifty times as effective as 1-naphthaleneacetic acid. Further experiments showed that 4-chloro-2methylphenoxyacetic acid was one of the most active compounds tested and that it readily depressed the germination and early seedling growth of the corn buttercup, corn marigold, corn spurrey, and field poppy at concentrations that were without effect on cereals. 2,4-Dichlorophenoxyacetic acid was among the compounds found to be more active than 1-naphthylacetic acid. Slade et al. communicated the substance of their results i n November 1942 to the secretary of the Agricultural Research Council, but publication was not allowed until 1945 (7). After November 1942, joint experiments i n the field were carried out by the two groups of workers mentioned, i n cooperation with G . E . Blackman. I n 1943, a field trial showed that sodium 4-chloro-2-methylphenoxyacetate applied at the rate of 1 pound per acre gave 100% eradication of yellow charlock in spring oats without damaging the cereal and equally good results were obtained if application was made when the weeds were small or i n full flower (30). Later field experiments i n 1944 confirmed and extended these results. Blackman (8), working with a research team at the Imperial College of Science, London, subsequently found that the phenoxyacetic acid derivatives, among a variety of substances, alone showed promise for the control of perennial weeds.
Herbicidal Action
Herbicidal effects of 1-naphthylacetic acid (reduction of seed germination) were apparently noted by Templeman and Sexton (34) in October 1940, when a differential effect on the germination of yellow charlock and oats was observed. Field experiments on the herbicidal action of 2,4-dichlorophenoxyacetic acid were carried out in England i n A p r i l 1943 (8), though its differential effects on plant growth were known to the English investigators i n 1942. So far as the author is aware, the first statement of the herbicidal action of 2,4dichlorophenoxyacetic acid, made i n the United States, was i n a publication b y Hamner and Tukey (14) i n 1944. They sprayed 2,4-dichlorophenoxyacetic acid and 2,4,5trichlorophenoxyacetic acid on bindweed and obtained a complete k i l l down to the root tips. A t about the same time Hamner and Tukey (13) and M a r t h and Mitchell (24) demonstrated the effect of 2,4-dichlorophenoxyacetic acid as a differential herbicide on lawns. A l l plants growing on the lawns (except the grasses) were destroyed with apparently no noticeable i l l effect on the grasses. Templeman and Sexton, besides describing the effects of substances of the type a r y l - O C H C O O H on seed germination and growth (34), have pointed out the remarkable inhibitive effects of arylurethanese.g., isopropyl phenylcarbamateand related compounds i n very low concentrations on the germination of seedling growth of cereals (35). Lefevre (23) i n 1939 had already described the physiological effects of phenylurethane on wheat seedlings and considered the effects to be similar to those described for colchicine.
2
246
The latter substance, as is well known, interferes with nuclear division in plants. Templeman and Sexton (35) showed that, contrary to the effects of the phenoxyacetic acids, the arylurethanes destroy cereals more readily than the dicotyledonous plants. In America, early i n 1944, a project centered at Camp Detrick, M d . , was initiated for the study of the inhibitory activities of synthetic growth-regulating substances (86). B y August 1945 nearly 1100 substances had been tested. A t the research center at C a m p Detrick 1060 organic compounds were examined for their powers of selective plant i n hibition and more than 100 of the substances were found to have high growth-regulating activities. Reference may be made (11) to the series of papers from Camp Detrick for much valuable information on the effects of growth-regulating substances, especially 2,4dichlorophenoxyacetic acid, on plant growth, on the persistence of these substances i n soil, on the remarkable specific toxicity of 2,4,5-trichlorophenoxyacetic acid toward Irish potatoes, and on the action of isopropylurethane on plants. Kraus and M i t c h e l l have published an interesting paper (20) on the effects of a variety of substances including 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid on plant growth ; their experiments were carried out i n 1943. A very large literature on the herbicidal effects of 2,4-dichlorophenoxyacetic acid has now developed and excellent reviews have appeared. Allard, D e Rose, and Swanson (1), for example, have found 2,4-dichlorophenoxyacetic acid to inhibit germination of a number of species including kidney beans, wheat, and oats. Klingman (18) reports eradication of dandelion b y the same substance without harm to bluegrass. Kephart (17) describes the use of 2,4-dichlorophenoxyacetic acid i n killing dandelions, plantains, and other weeds i n turf and indicates the promise shown for destruction of perennial weeds such as bindweed and Canadian thistle (see 9, 25, 29, 32, 37, for further information on the weed-killing properties of the dichloro- and chloromethylphenoxyacetic acids).
Effects of Soil Conditions

Whether or not a given plant growth substance having toxic effects on plant growth will withstand soil conditions for 3 or 4 weeks is of decisive importance i n determining its importance i n agricultural practice. The toxicity of 2,4-dichlorophenoxyacetic acid on the growth of a variety of plants is not appreciably greater than that of 2-indolylacetic acid; yet the former is of great agricultural value and the latter of none. I n fact, resistance to attack b y microorganisms i n the field must be regarded as an indispensable property of a herbicide which is to be used i n agricultural practice. The results of experiments carried out i n water culture, sand, or sterile soils, while of great theoretical value, are of little practical importance until they have been assessed i n the field. N u t m a n , T h o r n ton, and Quastel realized this i n 1942 and their choice of 2,4-dichlorophenoxyacetic acid as a selective plant growth regulator was largely dependent on their observation of the relative resistance of 2,4-dichlorophenoxyacetic acid to attack i n the soil. O n the other hand, i t is important that the toxicity of the herbicide i n the field should not be lasting or cumulative; otherwise a general poisoning of the soil would ensue which would be detrimental to crop yield. M u c h work has been carried out recently on the velocity of detoxication of 2,4dichlorophenoxyacetic acid under a variety of soil conditionse.g., high soil moisture content, high temperatures, high p H , and high organic matter content (10,12,15,21,26). Lees and Quastel (22) i n 1946, using their soil perfusion technique, found that when soil is exposed to a compound, which is gradually metabolized, a process of enrichment (of microorganisms) i n the soil takes place, whereby the soil becomes more and more active i n breaking down the compound i n question. Ultimately a point of saturation occurs and at this point the rate of breakdown of the substance becomes maximal and is constant. Lees and Quastel and more recently Quastel and Scholefield (28) showed that in a biological process i n soil, the time progress curve of the process follows a characteristic pattern, showing an initial lag period succeeded b y an exponential rise, both significant of the rate of proliferation of microorganisms. After the initial stages a steady maximum rate of breakdown is reached, this being due to ''saturation" of the soil with the organisms involved.
QUASTEL2,4-D AS A SELECTIVE HERBICIDE
247
Audus (3) has followed the disappearance of 2,4-dichlorophenoxyacetic acid in soil using the soil perfusion technique. H e found, with initial concentrations of 10 p.p.m., little or no alteration of the concentration of 2,4-dichlorophenoxyacetic acid in the soil at 7 0 F . for at least 10 days. T h e n there commences a rapid rate of breakdown of 2,4dichlorophenoxyacetic acid typical of bacterial development, and in the 4 subsequent days all the 2,4-dichlorophenoxyacetic acid disappears. T h e soil at this point is enriched with organisms attacking the 2,4-dichlorophenoxyacetic acid, for if it is now perfused with another lot of 2,4-dichlorophenoxyacetic acid at the same concentration, this is com pletely broken down in 2 days. Moreover, if this is further perfused with a concentration of 2,4-dichlorophenoxyacetic acid ten times higher than the original perfusing concen tration, all the 2,4-dichlorophenoxyacetic acid disappears within 3 or 4 days. Higher initial concentrations of 2,4-dichlorophenoxyacetic acid give longer initial lags but ulti mately the substance is fully metabolized. T h e soil becomes highly enriched with or ganisms capable of rapid destruction of the herbicide. These results show that the 2,4-dichlorophenoxyacetic acid can remain stable, and exert its herbicidal effects, only during the initial lag phase before the rapid logarithmic phase of destruction takes place. This initial lag phase depends upon the previous treatment of the soil, particularly on whether it has already been exposed to 2,4-dichloro phenoxyacetic acid. Clearly, once a soil is enriched with 2,4-dichlorophenoxyacetic acid destroying organisms, the herbicide, if added to the soil, will have little or no value in the control of plants growing there.
Reversibility of Action of 2,4-Dichlorophenoxyacetic Acid

It is essential for any understanding of the mechanism of action of the plant growth substances to know if their inhibitive effects can be reversed. If these compounds have toxic effects because they combine irreversibly with cell constituents, their effects will be irreversible, and washing the plant tissue to remove the toxic agent will not result i n renewed growth of the plant. O n the other hand, if these agents act b y forming easily dissociated compounds with tissue constituents, whether they are enzymes or metabolites, washing the plant tissue to remove the toxic agent should result in renewed growth of the plant, so long as autolytic processes have not advanced too far during the period in which growth has not taken place. Audus and Quastel (, 5) have shown that with both 2,4-dichlorophenoxyacetic acid and another selective herbicide, coumarin, reversibility does take place. When a seedling, for example, has ceased to grow in coumarin solution for periods up to 2 or even 3 days, recovery can take place when the plant is transferred to a fresh nutrient solution. It is scarcely to be expected that the recovery will be more than partial, first, because it is u n likely that washing, however good, will remove all the toxic agent, and second, because, during the period in which growth is not occurring, autolytic processes must be taking place to the detriment of the subsequent growth. It has been found {2, 5) that recoveries from the inhibitive effects of coumarin and 2,4-dichlorophenoxyacetic acid are more com plete with certain plants than with others. T h e inhibitions of both germination and root growth of cress seeds treated with coumarin may be reversed by washing, or transference to fresh nutrient, so long as exposure is not too great. The facts indicate that the plant growth substances must effect their inhibitions b y forming loose combinations, or easily dissociated compounds, with enzymes or metabolites in the plant cell.
Action of p-Aminobenzoic Acid

Audus and Quastel (4) have found that the presence of p-aminobenzoic acid exerts an antagonism, though a relatively feeble one, to the root growth inhibitory actions of 2,4-dichlorophenoxyacetic acid, 2-chloro-4-methylphenoxyacetic acid, 2-naphthoxyacetic acid, and 2-indoleacetic acid. F o r any marked degree of antagonism the concentration ratio of p-aminobenzoic acid to the growth substance must be of the order of 1 0 , 0 0 0 . This relatively feeble action is in.marked contrast to the effect of p-aminobenzoic acid on the
American Chemical Society

1155 16th St, H.W. Washington, D.C 20038
Library
248
sulfonamide inhibition of bacterial growth. The physical conditions i n the plant, howevere.g., p H , cation concentrations, permeability factors, etc.may be so very different from those i n the bacteria that the observed effect of aminobenzoic acid m a y be of real significance and helpful i n elucidating the mechanism of action of 2,4-dichlorophenoxyacetic acid. I t is felt, nowadays, so far as bacteria are concerned, that the function of p-aminobenzoic acid lies i n its acting as a precursor of folic acid and that the sulfonamides act b y inhibiting this synthesis. I t is possible also that 2,4-dichlorophenoxyacetic acid may act b y inhibiting the formation i n the root cells of a metabolite essential for normal development.
Chemical Constitution and Herbicidal Effects

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch045 The results of the investigation of the effects on plant growth of a large number of organic substances, b y Koepfli, Thimann, and Went (19) and Zimmerman and his colleagues (39, 43, 44), seem to indicate that certain minimum requirements must be met to produce an active compound. I t (19, 38) is suggested that they are as follows: (1) a ring system or nucleus; (2) i n this ring a double bond; (3) a side chain; (4) i n the side chain a carboxyl group (or group that can be readily converted into a carboxlyl group) : and (5) a particular space relationship between the ring systems and the carboxyl group. Veldstra (38) has made a very useful summary of the physiological properties of a large variety of organic substances and develops the view that "the action of the growth substance takes place i n a boundary and that the ring system determines the degree of adsorption to that boundary, the carboxyl group performing the physiological function proper (influencing of the boundary potential) for which a definitely peripheral position of the group is required."
Conclusions
Although requirements such as those mentioned may be desirable or even essential for an active plant growth regulator, the mechanism of action of the substance i n the cell is still unknown. The suggestion that certain of these active molecules m a y exercise their effects b y combination with important thiol enzymes may be most fruitful. However, such requirements in constitution, while important for the production of a plant-growth regulator, may be inadequate for the formation of a herbicide working under field conditions. Indoleacetic acid and coumarin are both plant metabolites of high potency as plant growth regulators, but they are of little value as herbicides. T h e substances must be so constituted as to withstand immediate attack b y common soil organisms and yet so made up that they w i l l gradually undergo breakdown either spontaneously or b y stimulating the formation i n soil of organisms that can attack them. The possession of chlorine atoms i n the nucleus of 2,4-dichlorophenoxyacetic acid makes this substance relatively resistant to bacterial attack and i t is this property that helps to make i t so valuable. Investigations of the manner i n which molecules, easily broken down i n the soil, can be made more resistant to attack b y soil organisms are of the greatest importance i n the study of herbicides. The employment of a soil perfusion technique is of considerable value i n work of this description. Further work on these lines should help greatly to classify and extend our knowledge of the relations between chemical constitution and herbicidal effects i n the field.
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) Allard, De Rose, and Swanson, Botan. Gaz., 107, 575 (1946). Audus, New Phytologist, 47, 196 (1948). Audus, Plant and Soil, 2, 31 (1949). Audus and Quastel, Ann. Botany, 12, 27 (1948). Audus and Quastel, Nature, 159, 320 (1947). Bausor, Am. J. Botany, 26, 415, 733 (1939). Bausor, Reinhart, and Tice, Ibid., 27, 769 (1940). Blackman, Ibid., 155, 507 (1945). Brown and Carter, La. Agr. Expt. Sta., Bull. 402 (1946).
QUASTEL2,4-D AS A SELECTIVE HERBICIDE (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) Brown and Mitchell, Botan. Gaz., 109, 314 (1948). Chemical Warfare Service, Special Projects Division, Ibid., 107, 475 (1946). De Rose, Ibid., 107, 583 (1946). Hamner and Tukey, Ibid., 106, 232 (1944). Hamner and Tukey, Science, 100, 154 (1944). Hanks, Botan. Gaz., 108, 186 (1946). Irvine, Univ. Colo. Studies, 26, 69 (1938). Kephart, Agr. Eng., 27, 506 (1946). Klingman, Wyo. Agr. Expt. Sta., Bull. 274 (1946). Koepfli, Thimann, and Went, J. Biol. Chem., 122, 763 (1938). Kraus and Mitchell, Botan. Gaz., 108, 301 (1947). Kries, Ibid., 108, 510 (1947). Lees and Quastel, Biochem. J., 40, 803 (1946). Lefevre, Compt. rend. acad. sci. Paris, 208, 301 (1939). Marth and Mitchell, Botan. Gaz., 106, 224 (1944). Marth and Mitchell, Science, 104, 77 (1946). Mitchell and Marth, Botan. Gaz., 107, 276 (1945). Nutman, Thornton, and Quastel, Nature, 155, 500 (1945). Quastel and Scholefield, Ibid., 164, 1068 (1949). Seely, Proc. Wash. State Hort. Assoc., 1945, 21-4. Slade, Templeman, and Sexton, Nature, 155, 497 (1945). Templeman, Empire J. Exptl. Agr., 7, 76 (1939). Templeman, J. Ministry Agr., 53, 105 (1946). Templeman and Marmoy, Ann. Applied Biol., 27, 453 (1940). Templeman and Sexton, Proc. Roy. Soc., 133B, 300 (1946). Ibid., p. 480. Thomson, Swanson, and Norman, Botan. Gaz., 107, 478 (1946). Thornton, Colo. Farm. Bull., 8, No. 1 (1946). Veldstra, Enzymologia, 11, 97 (1944). Zimmerman, Ind. Eng. Chem., 35, 596 (1943). Zimmerman, Proc. Natl. Acad. Sci., 27, 381 (1941). Zimmerman and Hitchock, Contrib. Boyce Thompson Inst., 10, 481 (1939). Ibid., 12, 321 (1942). Zimmerman, Hitchock, and Wilcoxon, Ibid., 7, 447 (1935). Ibid., 10, 363 (1939).
249
Metabolic Products of Elemental Sulfur 35 Applied to Lemons as an Insecticide

FRANKLIN M. TURRELL and MARCELLA B. CHERVENAK University of California Citrus Experiment Station, Riverside, Calif.
Interference with conduction or radiation of heat from leaves or fruit during insolation increases the temperature of the plant part; linear temperature increases increase the rate of volatilization of elemental sulfur logarithmically; hydrogen sulfide production of sulfur-dusted lemons increases nearly linearly; and sulfur dioxide production of sulfur-dusted lemons increases linearly with increase in temperature. The sulfur content of the cell solution of the peel of sulfur-dusted lemons when determined as sulfate was shown to correlate with the increase in sulfur of "sulfur-burned" sides of peel of lemons injured on the tree. Using radioactive elemental sulfur, hydrogen sulfide, sulfur dioxide, and sulfuric acid to treat lemons incubated at insolation temperatures (41 to 4 4 C.) it was shown that radioactive hydrogen sulfide and/or sulfur dioxide was produced, that the percentage of sulfur determined as sulfate in lemon peel increased, and that the pH of water extracts of the peel decreased. The specific activities of the acid-soluble peel proteins were relatively high, whereas the specific activities of the alkali-soluble proteins were relatively low. There appeared to be a destruction of proteins in some of the treatments.
The drastic effect of high temperature and elemental sulfur on higher animals has been appreciated since biblical times. But the lethal effect of this combination on higher plants does not seem to have been recorded until the nineteenth century. Little work appears to have been done on the physiology of the injury to higher plants though the low cost of sulfur, its effectiveness in killing certain pests, and the increased toxicity of compatible pesticides in which it is used as a diluent still make its use desirable in spite of newer insecticides. The principal shortcoming of sulfurinjury to the plant during hot weatherperhaps may be overcome when the mechanism involved is understood. This paper discusses experiments that elucidate the mechanism in some respects. The groundwork on the toxicity of sulfur to lower plants was laid in this country by McCallan and Wilcoxon, Liming, and Young and Williams. These studies and those of European investigators have been reviewed well by Horsfall (4) and by Frear (2). In 1875 Pollacci (7) reported grape leaves treated with sulfur produced hydrogen sulfide gas. Recently it has been reported that hydrogen sulfide gas is produced by sulfurdusted lemons and oranges {22). This gas which emanates from sulfured plants is also produced by the reactions of ingredients in the widely used lime-sulfur sprays. St. John and Groves (9) have excellently reviewed the chemistry of these mixtures.
250
TURRELL AND CHERVENAKMETABOLIC PRODUCTS OF ELEMENTAL SULFUR 35
251
Thomas and Hendricks (13) found that sulfur dioxide was produced b y mild alkaline hydrolysis of plant tissues, and i t has been subsequently reported that sulfur dioxide is produced b y lemons treated with elemental sulfur (19). Atmospheric oxygen appears to accelerate hydrogen sulfide and sulfur dioxide formation by sulfur-dusted lemons. A n experiment i n which lemons were incubated i n a bottle which was opened hourly during the day yielded more hydrogen sulfide and more sulfur dioxide than when the bottle was kept closed at night, though the entire experiment was conducted i n the dark (18). Thomas and H i l l (16) showed that sulfur dioxide fumigation of alfalfa increased the sulfate content of the leaves. I n later work Thomas et al. (14) reported that the p H of alfalfa leaf tissue fluids was lowered 0.3 p H unit after fumigation with sulfur dioxide. Similar responses occur when lemons are incubated i n sulfur dioxide gas, or when they are treated with elemental sulfur or with hydrogen sulfide (18). Likewise lemons treated with dilute sulfuric acid gave similar responses. If a section cut through a small sulfur-burned area of a lemon injured on the tree is examined microscopically, coagulation of protoplasm and cell collapse are apparent. Also, the injured tissue stains abnormally dark with safranin indicating the protoplasm has become more acidic than i n normal tissue (18). Sides of the peel of lemons burned by sulfur on the tree were found to be higher i n total sulfate than were uninjured sides of the same peel. The high total sulfate content of the peel was subsequently found to be due i n part to soluble sulfate, as shown by analyses of the expressed cell solution (18). Small increases i n temperature cause the volatility of elemental ground sulfur to i n crease greatly. One report shows that volatility increased from 0.15, at 24 C , to 10.00 mg. per square cm. per 24 hours, at 48 C , or the amount of sulfur volatilized increased 66.6 times though the temperature only doubled (20). Thus i t may be surmised that the first effect of high temperature i n sulfur injury is the increased amount of sulfur vapor available to the fruit. Such a view appears to be fortified by the observation that hydrogen sulfide and sulfur dioxide gases were produced i n increasing amounts when sulfurdusted fruit were incubated at increasing temperatures (18). Under natural growing conditions, only lemon fruit and leaves that are exposed to direct sunlight are injured by sulfur dust. T h e question then arises as to what factors raise the temperature of the plant parts sufficiently to cause volatilization of sulfur and injury at one time and not at another (18). I n desert areas of southern California fruit are often injured but leaves are seldom injured by sulfur dust. I n coastal areas fruit burn is less marked but "leaf b u r n " may be acute. Where the air-vapor density is high, leaf temperatures i n the sun may sometimes become higher than fruit temperatures. The leaf, a better absorber of radiation and a better radiator than the fruit, has a higher surface-mass ratio and appears to be very sensitive to the "heat t r a p " effect of high vapor density; its temperature changes with great rapidity, but fruit temperature may lag until the danger period is passed (18).
Effect of Temperature
A i r temperature and vapor density are two factors which influence the rate of dissipation of radiant energy received by plants from the sun, and they determine i n a large measure the temperature of the plant part and consequently sunburn and sulfur burn (18), other factors, such as particle size of the sulfur, being constant. The temperature of that portion of the peel of a lemon i n direct sunlight is always warmer than the air, and usually warmer than leaves, providing that the vapor density of the air is moderate to low. Peel temperatures measured i n direct sunlight reached 127.7 F . when the air temperature registered 104.7 F . , a difference of 23 F . This suggests that during a warm summer day when air temperatures i n the shade reach 118 F . , peel temperatures of 141 F . might be expected. Such a temperature would probably result i n sunburn even though the fruit were not dusted with sulfur, but if dusted it would certainly result i n sulfur burn (18); sulfur burn occurs at about 10 F . lower temperature than sunburn (18).
252
The work described here was undertaken to determine what happens to the sulfur that is volatilized, and what is the source of the hydrogen sulfide, sulfur dioxide, and sul fate. T h e products formed b y lemons treated with elemental sulfur were employed i n radioactive form for the treatment of other lemons. Methods Mature yellow lemons were picked freshly from trees growing at the Citrus E x p e r i ment Station at Riverside prior to a given treatment, which resulted i n pickings at v a r i ous times over a year's period. Eureka lemons were used i n the treatments with elemen tal sulfur* and with sulfuric* acid. (Asterisks are employed to indicate radioactive atoms.) Lisbon lemons were used i n the treatment with hydrogen sulfide* and a m i x ture of both was used i n the treatment with sulfur* dioxide. Pickings were made on A p r i l 27, 1948, and January 1, A p r i l 7, and M a y 10, 1949, for the various treatments. Twenty-eight whole fruit, after dusting with elemental radioactive sulfur or after immersion i n 0.1 ( p H 1.14) radioactive H S * 0 for 3 hours, were placed i n large glass bottles connected to two gas absorption trains consisting of two smaller glass bottles on either side. Ground-glass joints were used throughout the apparatus. T h e first small bottle i n each train contained a phosphate-borate buffer to absorb sulfur dioxide, and the second small bottle i n each train contained 2 % zinc acetate solution for absorbing hydro gen sulfide (13). T h e gases were drawn off as formed, b y applying a vacuum on one side of the gas absorption train. When the radioactive gases ( S * 0 and H2S*) were a p plied, a closed system was employed i n which one gas train was closed off, while the other gas train was replaced b y a mercury manometer to allow for the accumulation and ex pansion of vapors during incubation of the fruit. A t the end of this period the manom eter was replaced b y a gas absorption train and the gases were drawn off. Constant i n c u bation temperature was maintained b y placing the large bottles i n a water bath kept at temperatures similar to the more moderate temperatures that the fruit would have a t tained outdoors i n the sun on the tree. L o n g incubation periods were employed (40 to 80 hours) i n order to permit the reactions to reach as near completion as possible. C o n trols consisted of 28 nontreated fruit which were incubated only, and 28 nontreated, nonincubated fruit.
2 4 2
Table I.
Starting Compound S* H S* S*Oj HjS*04
2 a
Constants in Treatments of Lemons with Sulfur and Sulfur Acids

35 35
Sp. A . of Starting Temp, of Experiments, Compounds, Counts/Sec./Mg.

F
Duration of Experi ments, Hours 68 37 89 43
Fruit Weight, Grams 2322 2841 2978 2352
Fruit Surface, Sq. C m . 2492 2884 3168 2698
Gas Pressure, C m . Hg 1.0 6.0 8.0 1.8 X X 10~ 10-1 "* 10^6
6
0.1107 0.458 1.784 64.7
106 111 111 111
Extrapolated from data given in Hodgman's Handbook of Physics and Chemistry. b Calculated osmotic pressure.
A i l gaseous sulfur products obtained as a result of incubation of sulfur-treated fruit were oxidized with alkaline hydrogen peroxide, precipitated as barium sulfate, and counted with a thin window Geiger counter. The peel and peel proteins were oxidized with mag nesium nitrate, the sulfur was precipitated as barium sulfate according to standard methods, and counted as i n the case of the gaseous products. Counting data, as r e ported, are fully corrected. The proteins were isolated as acid-soluble and alkali-soluble, using methods outlined b y Sinclair et al. (11) and Tupper-Carey and Priestly (17), and were purified b y three precipitations. Measurements of p H were made with a glass electrode on 25 grams of lemon peel macerated i n 100 m l . of distilled water i n a Waring Blendor, and cleared b y centrifugation. The p H ' s of muskmelon leaf extracts were determined on mature leaves of United States Department of Agriculture strains Nos. 5 and 11353, grown at Riverside. T h e
TURRELL AND CHERVENAKMETABOLIC
PRODUCTS OF ELEMENTAL SULFUR 35
253
p H values were obtained b y the method used for lemon peel except that 25 grams of leaves were macerated with 75 m l . of distilled water. Buffer curve titrations were made with 0.01027 hydrochloric acid on 25-ml. aliquots. The constants for the various treatments with radioactive elemental sulfur and with the radioactive sulfur acids are shown i n Table I .
Results
The radioactivity of the hydrogen sulfide produced by 28 lemons treated with radio active elemental sulfur dust, 28 lemons treated with radioactive sulfur dioxide gas, and 28 lemons treated with a radioactive sulfuric acid solution is expressed as per cent specific activity (Table I I ) , which for the purpose of this report is defined as: Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch046
% specific activity (Sp. A.) = No. of counts/second of product/Mg. BaSQ of product No. of counts/second of treating material/Mg. B a S 0 of treating material
4 4
Table II.
H S Production and Activity of Lemons Treated with Sulfur and Sulfur Acids and Incubated at 4 4 C.
35 2
35
Treatment H S*
2
Sp. A . H S / S p . A . Treatment, % (Treated and Incubated)

2
Treated and incubated 2,525 1,460 93,000
Total H S , / 2 8 Fruit Incubated only

2
Nonincubated 0
102.5 11.1 4.9
H S*CU Incubated at 41 C.
2
s*o
Table I I shows that 100% of the hydrogen sulfide* produced by sulfur*-dusted fruit was derived from the sulfur* applied, 1 1 % from the sulfur* dioxide treated fruit, and 5 % from the sulfuric* acid treated fruit. Thus, the higher the state of oxidation of the sul fur* applied, the more limited the production of radioactive hydrogen sulfide. N o hydro gen sulfide was obtained from the controls. The specific activity of the sulfur* dioxide was 14, 21, and 1 3 % for the elemental sulfur*, hydrogen sulfide*, and sulfuric* acid treated fruit, respectively (Table I I I ) . Thus about the same proportion (within experimental error) of sulfur* dioxide is derived from the radioactive elemental sulfur and sulfur acids applied i n the treatments. Table III. S0 Production and Activity of Lemons Treated with Sulfur and Sulfur Acids and Incubated at 4 4 C.
35 35
Treatment S* H S*
a 2
Sp. A . S 0 / S p . A . Treatment, % (Treated and Incubated)

2
Treated and incubated 266 247 184
Total SQ , / 2 8 Fruit Incubated only Nonincubated

2
s*o
2
14.4 21.0 13.0
0 0 0
0 0 0
H S*0*
a
Incubated at 41 C .
Table IV.
S0
Content and Activity in Peel of Lemons Treated with Sulfur Sulfur Acids and Incubated at 44 C.
35
4 4
35
and
Treatment S* H S* S*0 H S*0

a 2 2 2 a
Sp. A . S 0 (Peel)/ Sp. A . Treatment, % (Treated and Incubated) 77.1 51.1 5.6
S Determined as S Q on D r y Peel, % Treated and Incubated only incubated Nonincubated 0.0857 0.1500 0.2045 0.1068 0.0787 0.1067 0.1027 0.0742 0.0987 0.0779 0.0666
Incubated at 41 C.
254
I n all treated fruit the percentage of sulfur determined as sulfate i n peel increased over the controls (Table I V ) . Of the four different treatments, the sulfate having the highest percentage specific activity was where elemental sulfur* was applied. Specific activity was next highest i n the hydrogen sulfide* and lowest for the sulfur* dioxide treatment. The percentage of specific activities recovered as sulfates i n the exudates i n the v a r i ous treatments was slightly higher due perhaps to absorption of the externally applied activity, but otherwise they roughly paralleled the activities recovered i n the peel sulfates from the corresponding treatments (Table V ) . Table V. SO4 Exudation and Activity of Lemons Treated with Sulfur and Sulfur Acids and Incubated ot 4 4 C.
35 35
Treatment H S* S*0 H S*0

2 2 2 a
Sp. A . Exudate/ Sp. A . Treatment, % (Treated and Incubated) 82.7 60.0 7.5 40.8
SO4,
Treated and incubated 167 468 240 556
T/M1.
Incubated only 309 21 13
Nonincubated 0
'
Incubated at 41 C .
The p H values of lemon exudates and of extracts of the peels were lower i n the various sulfur treatments than i n the corresponding incubated or nonincubated controls (Table V I ) . The lower p H values of fruit treated with elemental sulfur and sulfur acids together Table VI. pH of Exudates and Water Extracts (1 to 4) of Peel of Lemons Treated with Sulfur and Sulfur Acids and Incubated at 4 4 C.
35 35

2 2 2 a
Treated and Incubated Exudate, Extract, pH pH 3.4 3.9 ... 3.3 3.7 3.5 3.3 3.8
Incubated Only Exudate, Extract, pH pH 3.5 ... ... 4.5 4.7 5.1 3.9 4.6
Nonincubated Extract, pH 5.3 5.3 5.5 5.0
Incubated at 41 C .
with the increase i n sulfate i n the peel (Table I V ) and that contained i n the exudate (Table V ) suggests that a fraction of the elemental sulfur or sulfur acids used i n the treat ments is converted b y lemon peel to free sulfur acids, possibly sulfuric acid if i t is not already i n that state of oxidation. Incubation of the fruit alone, however, increases the free acid or hydrogen ion concentration. T h e p H is further lowered i n the treatments with sulfur and sulfur acids, and ionization of the cell solution is increased as conditions favorable to sulfur injury are approached. Barron (1) has shown that sulfhydryl enzymes are inactivated i n water solution irradiated with x-rays because of the ionization of the water. H e postulated the formation of hydrogen peroxide b y combination of the ionized water and the dissolved oxygen i t contained. This mechanism may also function i n sulfur injury since catalase was shown to be inactivated b y elemental sulfur treatments (18) and reduced glutathione oxidized (18). Lemon juice protein has an isoelectric point at p H 5.6 to 5.7 (10) which is higher than the p H values of the cell solution of sulfurtreated fruit; lemon peel protein may also fall i n this category. The buffer capacity of lemon peel is very small, however, because of its comparatively low content of organic acids and their salts (12) and may be expected to reflect small changes i n p H . While i t is conceivable that an excess of bases i n the cell solution might be protective against mild sulfur burn, this possibility has not yet been tested. On the other hand, a small increase i n buffer capacity might reduce sulfur burn. A n example of this effect may be seen i n the buffer curves of the leaf sap i n two of the United States Department of A g r i culture's muskmelon varieties. N o . 5, which is susceptible to sulfur burn, has a buffer curve which lies 0.2 to 0.3 p H unit closer to the acid side than the buffer curve of the sulfur burn-resistant variety, N o . 11353 (Figure 1).
TURRELL AND CHERVENAKMETABOLIC
PRODUCTS OF ELEMENTAL SULFUR 35
255
A peel section of a lemon treated with elemental radioactive sulfur was killed i n for malin-acetic acid-alcohol mixture and then washed successively i n water, i n a graded series of ethanol-butanol-water mixtures from 10 to 9 5 % , 100% ethanol, and xylene. The insoluble substances remaining i n the section would be primarily the cellulose of the cell walls and alkali-soluble protein of the protoplasts, the acid-soluble protein having been washed out. Comparison of the photomicrograph and radioautograph of this peel section indicated that the alkali-soluble (acid-insoluble) proteins were weakly labeled with radioactive sulfur (21). Because of the indications of protein labeling i n the radioauto graph (Figure 3), the alkali-soluble and acid-soluble proteins were isolated from the peel i n the hydrogen sulfide*, sulfur* dioxide, and sulfuric* acid treatments. Radioautographs of tissue of fruit subjected to the latter three treatments, but killed i n formalin, are shown i n Figures 5, 7, and 9. Figures 2 to 9 are photomicrographs (X9.5) of radial sections of peel of lemons, and radioautographs of the same sections from fruit treated with elemental S and S acids. T h e right-hand sides of radioautographs correspond to left-hand sides of sections.
3 5 3 5
8.5
7.5
R
6.5
5.5 SUSC5 4.5
REST.
11353
3.5
20
Ml. /
30
HCI
40
50
Figure 1. Titration Curves of Leaf Tissue Fluids of Sulfur Burn-Susceptible and Sulfur Burn-Resistant Muskmelon Leaves
The alkali-soluble protein of the peel of lemons treated with hydrogen sulfide*, sulfur* dioxide, and sulfuric* acid contained radioactive sulfur, but the fruit treated with hydro gen sulfide* had a significantly lower per cent specific activity i n the alkali-soluble pro tein fraction than d i d the sulfur* dioxide or sulfuric* acid treated fruits (Table V I I ) . These results suggest that sulfur dioxide and sulfuric acid react with protein more directly, while hydrogen sulfide perhaps must be oxidized first, as indicated i n Table I I I . I t also appears (from Table V I I ) that the alkali-soluble protein may have been dismuted as the amounts isolated were less i n both the hydrogen sulfide* and sulfur* dioxide treated fruit than i n the incubated or nonincubated controls. Other evidence of dismutation has been obtained i n experiments where incubation at 60 C . was accompanied by the production of free ammonia (18), and the recovery of free ammonia and six amino acids i n the exu dates of incubated and sulfur-dusted fruits (18).
2 5 6
Figures 2 and 3. Peel Sec tion and Radioautograph from Elemental S Treat ment
35
Figures 4 and 5. Peel Sec tion and Radioautograph from H S* Treatment

2
Acid-soluble protein, like the alkaline-soluble protein, was labeled with radioactive sulfur by the several sulfur* treatments. The specific activity of the protein increased from 29 i n the hydrogen sulfide* to 5 2 % i n the sulfur* dioxide, to 6 8 % i n the sulfuric* acid treatment (Table V I I I ) . Here again, as i n the case of the alkali-soluble protein, there is an indication that hydrogen sulfide* may have required more processing b y the fruit than sulfur* dioxide, which i n turn required more processing than sulfuric* acid be fore i t reacted with the protein. Comparison of the specific activities in Tables V I I and V I I I indicates that the labeling of the acid-soluble protein is much heavier than the alkalisoluble protein. The percentage of acid-soluble protein decreased i n the peel i n the hydro gen sulfide* treatment and increased i n the sulfur* dioxide treatment. Sufficient data are not available to evaluate properly the latter shift, and further work must be done on this point, especially i n view of Barron's (1) findings which show a diminution of plasma albumins and an increase of -globulins i n the blood sera of x-ray-treated dogs.
Discussion
It is unfortunate that the results of these experiments could not have been predicted or anticipated prior to their completion, so that a l l of them might have been carried out simultaneously with the same k i n d of material, genetically and physiologically, and with the same incubation temperatures, incubation times, gas pressures, osmotic pressures, activities, and complete chemical analyses for a l l treatments. Because of these variables an attempt at further interpretation of the results may be hazardous, but because of sev eral possible factors that may offset the above-mentioned variables an attempt at further interpretations may be desirable. T h e differences i n concentration of the treating m a terials offer the greatest hazard, but if results of fumigation of plants with sulfur dioxide (IS) are used as a basis of judgment, the long incubation times tend to validate some dis-
TURRELL AND CHERVENAKMETABOLIC PRODUCTS OF ELEMENTAL SULFUR 35
257
Figures 6 and 7. Peel Sec tion and Radioautograph from S*02 Treatment
Figures 8 and 9. PeeljSection and Radioautograph from H 2 S * U 4 Treatment
Table VII.
Alkali-Soluble Protein Content and Activity of Peel of Lemons Treated with Sulfur and Sulfur Acids and Incubated at 4 4 C.
35 35
Treatment S* H S*
2
Sp. A . SO* (Protein)/ Sp. A . Treatment, % (Treated and Incubated) 2.7 31.8 24.1
Treated and incubated !05 0.0175 0.0253
Protein in Peel (Wet Wt.), % Incubated Nonincubated only !079 0.0311 !092 0.0483
H S*0 Incubated at 41 C .
2 4
s*o
Table VIII. Acid-Soluble Protein Content and Activity of Peel of Lemons Treated with Sulfur and Sulfur Acids and Incubated at 4 4 C.
35 35
Treatment
g*a
Sp. A . S04 (Protein)/ Sp. A . Treatment, % (Treated and Incubated)

29.0
Treated and incubated 0.0072 0.1646 0.1756
Protein in.Peel (Wet Wt.), % Incubated Nonincubated only
H S* S*0 H S*0
2 2 2
51.6 68.4
!0202
0.1468
0.0237 0.0955
Incubated at 41 C .
cussion. W i t h our present state of knowledge of sulfur metabolism i n plants i t is i m possible to explain the cell reactions which result i n the products formed when lemons are treated with sulfur or sulfur acids. The constitution of alkali- or acid-soluble proteins i n citrus is not known. Likewise, there is little information concerning the sulfur enzymes present i n citrus fruit. I n fact, the essentiality of sulfur to plants has been known only since the time of Liebig (3, 8).
258
However, Phinney (6) has suggested on the basis of experiments with mutants of Neurospora that the biosynthesis of cysteine involves the coupling of sulfate with an organic compound (presumably cysteine sulfinic acid) followed by reduction to sulfide. H e noted also that sulfate may be reduced stepwise to sulfide and may then enter the protein as such. B u t he observed this process proceeds less readily than when sulfate combines first with protein. F r o m Table I X it can be seen that the authors' experiments with radioactive sulfur seem to be i n accord with those of Phinney (6), i n so far as they are parallel. They suggest that elemental sulfur is first reduced to hydrogen sulfide. This reaction appears to be primarily one with reduced glutathione and results i n glutathione i n the disulfide form (18). However, the hydrogen sulfide formed in the other sulfur treatments must be largely a dismutation product. Hydrogen sulfide when produced by treatment of fruit with elemental sulfur possibly reacts with protein or cysteine and may then be oxidized to sulfate i n a series of steps from cysteine sulfenic acid to cysteine sulfinic acid to sulfate. According to Medes and F l o y d (5), these reactions occur in rats and appear to be enzymatically controlled except that cysteine sulfenic acid gives rise to cysteine sulfinic acid spontaneously. This process appears to occur i n plants (14) though the reverse process (6) appears to be the major one by which sulfate is utilized i n the plant. The experiments described here further indicate on the basis of the per cent specific activity of the products formed that hydrogen sulfide* is oxidized primarily to sulfate* which then combines with the protein. Likewise, they suggest that i n fruit treated with sulfur* dioxide the sulfur* dioxide is oxidized to sulfate*, and then the sulfate* is coupled with the protein. Sulfate* as i n sulfuric* acid, on the other hand, appears to react chiefly with the protein when applied directly to plant tissues. Table IX. Relative Specific Activities of Products from Treatments of Lemons with Sulfur and Sulfur Acids
35 35

2 2 2
H S*
2
Sp. A . Product/Sp. A . Treatment, % AcidS*0 soluble S*0 (peel) protein

4 2
Alkalisoluble protein Weak 3 32 24
Diff. p H Peel Extract (Incubated and Incubated and Treated) 1.0 1.6 0.6 0.8
102
4
1 5
14 21 13
77 51 6
29 52 68
N o evidence has been obtained as to the source of the sulfur* dioxide formed by the action of sulfur* and sulfur* acids on lemons but tentatively i t may be assumed, since sulfur* dioxide appears i n all the treatments and since i t has such a low activity, that i t may arise from sulfinic acid i n the formation of sulfate or that i t is a dismutation product of the protein which disappeared.
Acknowledgment
The authors wish to express their appreciation for the grants-in-aid made by the Texas Gulf Sulphur Company i n the early stages of this work, without which i t could not have been undertaken. The authors are also indebted to J . T . Middleton for the samples of muskmelon leaves. The radioactive sulfur 35 as H S * 0 was obtained from the Atomic Energy Commission, Oak Ridge, Tenn., and subsequently processed by Tracerlab, Boston, Mass.
2 4
Literature Cited
(1) Barron, E . S. G., U . S. Atomic Energy Commission, Tech. Inform. Div., Oak Ridge Directed Operations, Oak Ridge, Tenn., MDDC-484 (declassified 1946). (2) Frear, D . . H . , "Chemistry of Insecticides and Fungicides," New York, D. Van Nostrand Co., 1942. (3) Haynes, Williams, "Stone That Burns," New York, D . Van Nostrand Co., 1942. (4) Horsfall, J . G., "Fungicides and Their Action," Waltham, Mass., Chronica Botanica Co., 1945. (5) Medes, G., and Floyd, N . , Biochem. J., 36, 259 (1942). (6) Phinney, B. O., Rec. Gen. Soc. Am., 1 (17), 53 (1948). (7) Pollacci, E . , Gazz. chim. ital., 5 451 (1875).
TURRELL AND CHERVENAKMETABOLIC PRODUCTS OF ELEMENTAL SULFUR 35 (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)
259
Reed, H . S., "Short History of Plant Sciences," Waltham, Mass., Chronica Botanica Co., 1942. St. John, J . L . , and Groves, K., Proc. Wash. State Hort. Assoc.33rdAnn. Meeting, 128 (1938). Sinclair, W. B., Univ. Calif. Citrus Experiment Station, Project Notes (1935). Sinclair, W. B., Bartholomew, E . T., and Nedvidek, R. D., J. Agr. Research, 50, 173 (1935). Sinclair, W. B., and Eny, D. M . , Botan. Gaz., 108, 398 (1947). Thomas, M . D., and Hendricks, R. H . , J. Biol. Chem., 153, 313 (1944). Thomas, M . D., Hendricks, R. H . , and Hill, G. R., Plant Physiol., 19, 212 (1944). Thomas, M . D., and Hill, G . R., Ibid., 10, 291-307 (1935). Ibid., 12, 309 (1937). Tupper-Carey, M . , and Priestly, J . H . , Proc. Roy. Soc. (London), 9 5 B , 109 (1923). Turrell, F . M . , Plant Physiol., 25, 13 (1950). Turrell, F . M . , Scalacs, 3, 285 (1948). Turrell, F . M . , Science, 105, 434 (1947). Turrell, F . M . , and Chervenak, M . B., Citrus Leaves, 2 9 (4), 8 (1949). Turrell, F . M . , Cuneo, F., Slack, D., and Carns, H . , Calif. Citrograph, 28, 286 (1943).
P A P E R 629, University of California Citrus Experiment Station, Riverside, Calif.
Colorimetric Determination of Small Quantities of 1,1,1-Trichloro2,2-bis(p-methoxyphenyl)-ethane

J O H N D. FAIRING and HORACE P. WARRINGTON, JR. Beech-Nut Packing Company, Canajoharie, . Y.
In a sensitive and specific colorimetric method 1,1,1 -trichloro-2,2bis(p-methoxyphenyl)-ethane is extracted from plant or animal tissue, using benzene or petroleum ether as the solvent. The solvent is evaporated at room temperature by a current of air and the residue dehydrohalogenated with 2 % alcoholic potassium hydroxide. By petroleum ether extraction the resulting 1,1dichloro-2,2-bis(p-methoxyphenyl)-ethylene is removed from the reaction mixture. After the solvent is removed by air evapo ration the dehydrohalogenated methoxychlor is isolated from the nonsaponifiable portion of the fats and waxes by dissolving the residue in hot acetone, chilling, and filtering. After the acetone is removed by air evaporation, the residue is treated with 8 5 % sulfuric acid. This produces a red solution with an absorption maximum at 555 m, the intensity of which can be read on a colorimeter and is a function of the methoxychlor concentration. Beers law is obeyed over the range of 1 to 50 micrograms.
A need has arisen for a sensitive and specific method of estimating microgram quanti ties of the insecticide methoxychlor i n plant and animal products. Methoxychlor, l,l,l-trichloro-2,2-bis(p-methoxyphenyl)-ethane, is an analog of D D T and therefore procedures for the determination of D D T should be applicable to methoxychlor as well. Such has actually been found to be the case. The pyridine-xanthydrol method of Stiff and Castillo (4) and Claborn (1) will i n d i cate the presence of methoxychlor, but it has a low order of sensitivity and does not distin guish between methoxychlor and D D T . M u c h more promising is the method of Schechter and Haller (3), which depends upon the coupling of the tetranitro derivative of D D T with methanolic sodium methylate. Under conditions of intensive nitration methoxychlor will yield b y this procedure a red color with an absorption maximum at 535 . It may be generally stated that the Schechter-Haller procedure will distinguish methoxychlor from , ' - D D T , because the latter compound gives a blue color. H o w ever, ,'-DDT and various breakdown products of D D T yield red colors with absorption maxima close to that of methoxychlor, so that for practical purposes it is impossible to distinguish between them. It appears that the greatest value of the Schechter-Haller method applied to the determination of methoxychlor is in the analysis of materials whose previous spray his tory is unknown. The production of a red color i n such analysis indicates the possibility of the existence of methoxychlor, and warrants a further specific test. Upon treatment with alcoholic potassium hydroxide, methoxychlor will undergo
260
FAIRING AND WARRINGTONCOLORIMETRIC DETERMINATION OF METHOXYCHLOR
261
quantitative dehydrohalogenation. The dehydrohalogenated p r o d u c t reacts with 8 5 % sulfuric acid to produce a red complex of unknown c o m p o s i t i o n w i t h an absorption maximum at 555 (Figure 1). No other organic insecticide now i n use produces any color under similar conditions. Therefore, the method is specific for methoxychlor. Fats and waxes, however, yield strong brown colors which will completely mask the methoxychlor reaction. I n the method described this interfer ence has been reduced to a point where it introduces an error of less than 1% when the methoxychlor concentration is between 5 and 50 micrograms. Quantities of methoxy chlor of less than 1 microgram may be determined b y this method. There are six steps involved i n the procedure: 1. Extraction of the methoxy chlor from the material under ex amination with a suitable solvent. ~3S> 4O0 3 5 i o s S e o To 2. Dehydrohalogenation with WAVELENGTH- MLLIMICRONS alcoholic potassium hydroxide. 3. Separation of the dehydro Figure 1. Absorption of Red Complex halogenated derivative from the reac tion mixture with petroleum ether. 4. Further isolation of this material from the nonsaponifiable portion of fats and waxes with acetone. 5. Reaction of the dehydrohalogenated methoxychlor with 8 5 % sulfuric acid. 6. Spectrophotometry measurement at 555 of the optical density or per cent transmittance of the resulting color. I n order to prepare the methoxychlor for analysis, it must be removed from the m a terial under examination b y means of a solvent extraction. The procedures used for the stripping of D D T are applicable to methoxychlor (2, 5, 6). The solvent chosen should readily dissolve methoxychlor, be sufficiently volatile to be evaporated b y an air current at room temperature, and leave no residue upon evaporation that will interfere with the analysis. Benzene and petroleum ether satisfy these requirements rather well. I t is, however, essential that the solvents be absolutely pure. Reagent grade or C.P . chemicals are usually adequate for the initial extraction, but each batch should be checked to see that i t does not introduce an error into the determination. The extracted methoxychlor is isolated by evaporating the solvent with a current of air. N o measurable loss of methoxychlor has been found when this evaporation takes place at room temperature. However, precautions should be taken to avoid prolonged exposure to the air stream after the solvent has evaporated. Dehydrohalogenation is readily accomplished b y heating with 2 % potassium h y droxide i n 9 5 % ethyl alcohol. I n order to separate the dehydrohalogenated methoxychlor from the reaction mixture, the alcohol is allowed to evaporate, and the residue is taken up i n petroleum ether and washed with water. M o s t batches of even reagent grade petroleum ether contain substances which after contact with potassium hydroxide will yield
262
colors with sulfuric acid. I t is therefore essential to purify the petroleum ether used for this extraction. It is necessary to isolate the dehydrohalogenated methoxychlor from the nonsaponifiable portion of the fats and waxes. T o do this, advantage is taken of the fact that both methoxychlor and its dehydrohalogenated product are readily soluble in acetone, whereas fats are relatively insoluble. The residue is first dissolved i n hot acetone, and then the acetone is chilled to 15 C , which causes precipitation of the fats. After the fats are filtered off, the acetone is removed by evaporation. The intensity of color produced by a given a m o u n t of d e h y d r o h a l o g e n a t e d methoxychlor is dependent upon the acid concentration, as shown i n Figure 2. The optimum concentration is about 82.5%, with the intensity falling off rapidly at lower concentrations. A c i d at a nominal concentration of 8 5 % is used, so that the sensitivity of the reaction is not decreased by the absorption of small amounts of mois ture from the air. I t is difficult to prepare sulfuric acid of an exact concentration and even more difficult to keep such acid at a constant concentration for any length of time. Therefore it is advisable to run a standard of known methoxychlor content along with each set of samples being analyzed in order to correct for changes i n the acid strength. Because Beer's law is obeyed over the range of 1 to 50 micrograms of methoxychlor, i t is not essential to plot more than two or three points on the stand 90 80 70 60 ard curve during the daily recheck of the PERCENT SULPHURIC ACIO acid. The color develops slowly with acid of less than 8 2 % strength and not at a l l Figure 2. Effect of Acid Concentra with 6 0 % acid. The addition of water de tion on Color Intensity stroys the color. I n the absence of atmos pheric moisture the color is stable for several days. The reaction rate varies with different batches of acid. W i t h some lots the color is fully developed i n 15 minutes, but occasionally 60 or more minutes are required. A trace of ferric chloride added to the acid assures rapid color development. Analytical Procedure Reagents. Petroleum ether, C.P ., boiling range 35 to 60 C ; acetone, C.P., d r y ; a n d ethyl alcohol, C.P ., 9 5 % . E a c h reagent is purified before use b y eluting through a column of chromatographic a l u m i n a or b y shaking with activated charcoal and filtering. Benzene, C.P ., or petroleum ether, C.P ., for stripping. Potassium hydroxide (2%) i n 9 5 % ethyl alcohol, freshly prepared before use. Sulfuric acid, 8 5 % . The actual concentration may range between 82.5 and 8 8 % , but under no conditions should it be less than 82.5%. T o 1 liter of this are added 10 mg. or more of ferric chloride. Sodium chloride, saturated solution. Sodium sulfate, C.P ., anhydrous. Standard methoxychlor solution, 10 mg. of recrystallized methoxychlor made up to 1 liter with 9 5 % ethyl alcohol. Standard dehydrohalogenated methoxychlor solution is prepared from 44.71 mg. of recrystallized l,l-dichloro-2,2-bis(p-methoxyphenyl)-ethylene made up to 1 liter with 9 5 % ethyl alcohol. One milliliter of this solution is equivalent to 50 micrograms of methoxychlor.
The l,l-dichloro-2,2-bis(p-methoxyphenyl)-ethylene is prepared by refluxing recrystallized methoxychlor i n 9 5 % ethyl alcohol for 2 hours with an excess of potassium hydroxide. It is recrystallized from methanol.
2 6 3
Procedure
Extract the methoxychlor from the material under consideration with C.P . grade solvents (benzene, petroleum ether, etc.) that have been checked and found free of interfering substances. Shake stripping with about V a t h of its weight of anhydrous sodium sulfate. A small quantity of some filtering aid may be used if necessary to obtain a clear filtrate, but its use should be limited and it should be checked to see that no loss of methoxychlor occurs with the particular product under analysis. Filter the solution through rapid fluted filter paper. Transfer an aliquot of the filtrate containing between 5 and 50 micrograms of methoxychlor to a 125-ml. standard-taper Erlenmeyer flask. Remove the solvent with a gentle current of dry, oil-free air, preferably at room temperature but never over 35 C . A d d 50 m l . of 2 % potassium hydroxide i n 9 5 % ethyl alcohol. Connect a short length (12 cm.) of glass tubing equipped with a male standard-taper joint to the flask and immerse flask i n a boiling water bath until the volume of the liquid is reduced to about 5 m l . A v o i d prolonged heating after the alcohol has evaporated and run the next step as K > 20 30 40 50 soon as possible. I t is undesirable to hold this alcoholic solution overMICROGRAMS O F METHOXYCHLOR night. Figure 3. Standard Curve Transfer the contents of the flask to a separatory funnel with the aid of 200 m l . of water and 75 m l . of purified petroleum ether. A d d 10 m l . of saturated sodium chloride solution. Shake well, and separate and discard the aqueous layer. Wash two or more times with 200 m l . of water and 10 m l . of saturated sodium chloride solution, discarding the aqueous layers. Filter the petroleum ether extract through 1 or 2 cm. of anhydrous sodium sulfate contained i n a glass Buchner funnel equipped with a 30-mm. fritted-glass plate of coarse porosity. This filtration may be carried out rapidly without the aid of suction. (A small amount of filtering aid may be mixed with the sodium sulfate to remove plant pigments.) Wash the separatory funnel with 20 ml. of petroleum ether. Pass this washing through the filter and combine i t with the filtrate. Transfer the filtrate i n portions to a test tube and remove the solvent at room temperature, either with a current of air or by means of reduced pressure. When dry, add 5 m l . of dry acetone and heat from 2 to 4 seconds i n a boiling water bath to effect a complete solution of the entire residue. C h i l l the tube of acetone thoroughly, to 15 C . if possible. A t the same time chill some dry acetone for washing the filter. Filter the cold acetone rapidly into a glass-stoppered test tube b y means of suction through a 2- or 3-ml. micro Buchner funnel, fitted with a fritted-glass plate of medium porosity. Wash the tube and funnel with 3 m l . of cold acetone and combine i t with the filtrate. Evaporate the filtrate with a current of air. A v o i d excessive drying, as it makes the subsequent dissolving of the residue difficult; 0.01 to 0.05 m l . of moisture is frequently desirable. A d d 10 m l . of 8 5 % sulfuric acid (preferably b y means of an automatic pipet).
264
Stopper the tube with a glass stopper and shake well at intervals for 10 minutes. Let the tube stand for 15 minutes to 3 hours. If undissolved particles are i n suspension, filter through a plug of glass wool. This is generally unnecessary. Read the per cent transmittance at 555 . i n a colorimeter or spectrophotometer against 8 5 % sulfuric acid.
Preparation of Standard Curve

B y means of a microburet add 0, 0.1, 0.2, 0.5, 1, 2, and 5 m l . of the standard methoxy chlor solution to a volume of the stripping solvent corresponding to the average size of aliquot taken. Evaporate this mixture to dryness and carry i t through the above pro cedure.. Plot the resulting per cent transmittance semilogarithmically against concen tration (Figure 3). F r o m this standard curve calculate the original concentration of methoxychlor i n the unknown i n parts per million from the formula:
W Vo
X
WVx
x
'
where W = weight of residue i n micrograms determined from the standard curves, W = weight of sample in grams, V aliquot of solvent i n m l . , and V = original volume in m l . of solvent used for stripping.
0 x 0
If a series of determinations is to be made on the same type of material, i t is advisable to make a standard curve by adding known amounts of methoxychlor to a stripping of the material under consideration which is known to be free of methoxychlor. A n y slight variation introduced into the technique b y the presence of other extracted substances will be compensated for i n this way.
Discussion
The recovery of methoxychlor has been found to be quantitative from strippings of apples, green beans, peaches, carrots, celery, pears, peas, lamb fat, beef fat, pork fat, and milk. Table I shows the recovery from apples. Table I. Recovery of Methoxychlor from Benzene Solutions of Apple Wax
Methoxychlor Added, y 0 2.5 5.0 10.0 15.0 20.0 25.0 50.0 Methoxychlor Recovered, y 0 2.3 2.5 2.9 4.8 4.8 5.0 9.8 10.3 10.4 15.3 15.8 20.2 20.8 24.1 24.2 50.0 Av. Recovery.
%
92 100 116 96 96 100 98 103 104 102 105 101 104 96 97 100 100.6
The dehydrohalogenation proceeds rapidly with a yield of better than 9 8 % . The over-all yield of the entire procedure is better than 9 5 % . Because high yields are obtained, it is possible to prepare a standard solution of dehydrohalogenated methoxychlor to be used in the daily recheck of the calibration curve. The use of such a standard greatly reduces the time required and makes possible the frequent checking of the curve at several points with but little additional effort. One gram of methoxychlor is equivalent to 0.8942 gram of the dehydrohalogenated product.
265
In the authors' experience i t has not been possible to obtain a completely colorless blank, for even the most carefully purified reagents will yield a light yellow color. H o w ever, this color has no effect upon the determination of methoxychlor, for if the solvents have been properly purified this reagent blank will not lower the transmittance at 555 b y more than 0.25%. E v e n with the best technique, however, some interference will still be encountered from fats and waxes. W i t h the acetone separation method described this interference is reduced to a point where it seldom lowers the transmittance by as much as 1% and never by more than 3 % . I t has been found that the off-colors introduced b y any one type of biological material are remarkably constant and i t is therefore thoroughly practical to apply a correction for this interference. The over-all errors caused by solvents and fats reach significant proportions only when determinations are being made at the very limit of sensitivity of this test. The authors have been able to measure less than 1 microgram of methoxychlor with errors not in excess of 15%. For some time the acetone separation step was performed before dehydrohalogena tion. However, great difficulty was experienced i n preventing rather sizable losses, be cause some methoxychlor would dissolve in the precipitated fat. W i t h milk in particular these losses became prohibitive. After dehydrohalogenation most of the fat is saponified and removed, so that only a small nonsaponifiable portion remains to be separated. If any methoxychlor does dissolve i n this remaining fraction it is too small to be measured. Further separation of the methoxychlor from fatty contaminants may be accom plished b y diluting the acetone, after filtering, with an equal volume of water and again filtering. This step is seldom necessary, but when employed i t does provide a means for the removal of the last traces of fatty impurities. Because water will be present, it is necessary to extract the dehydrohalogenated methoxychlor from the solution with petro leum ether. This adds another step to the procedure. Such additional work is hardly warranted. Considerable variation is possible i n the technique of this procedure for the deter mination of methoxychlor. Several steps may be eliminated or shortened for rapid rou tine work, and with suitable biological materials this may be done without appreciably sacrificing the accuracy of the test.
Acknowledgment
The authors wish to thank . I . du Pont de Nemours & Company for supplying the samples of rriethoxychlor used i n this work, and M a r y L . Wilson for carrying out much of the routine experimental work.
Literature Cited
(1) Claborn, H . V., J. Assoc. Offic. Agr. Chemists, 29, 330 (1946). (2) Fahey, J . E., Cassil, C. C., and Rusk, H . W., Ibid., 26, 150 (1943). (3) Schechter, M . S., Soloway, S. B., Hayes, R. ., and Haller, H . L . . Ind. Eng. Chem., Anal. Ed., 17, 704 (1945). (4) Stiff, . ., and Castillo, J. C., Ibid., 18, 316 (1946). (5) Tressler, C. J., J. Assoc. Offic. Agr. Chemists, 30, 140 (1947). (6) Wichmann, H . J., Patterson, W. I., Clifford, P. ., Klein, A . K . , and Claborn, . V., Ibid., 29, 188 (1946).
Partition Chromatography in Analysis of Insecticide Formulations

THOMAS H. HARRIS
Livestock Branch, Insecticide Division, U. S. Department of Agriculture, Beltsville, Md.
A procedure for the determination of -benzene hexa chloride and DDT in benzene hexachloride-DDT-sul fur formulations, employing partition chromatography, is described. The procedure has also been applied to the assay of 1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane in technical methoxychlor. Results of separa tion of other insecticidal ingredients are discussed.
P a r t i t i o n chromatography, developed by M a r t i n and Synge (3) for the separation of amino acid derivatives, was employed by Ramsey and Patterson (4) for the separation of isomers of benzene hexachloride (1,2,3,4,5,6-hexachlorocyclohexane) i n the technical product. T h e work of Ramsey and Patterson was extended by Aepli, Munter, and Gall (1) i n the development of a quantitative method for the determination of -benzene hexa chloride. A modification of the method of Aepli and co-workers was recently published (2) . The essential feature of this modified method is the addition of a small quantity of dye, D and C Violet N o . 2 (l-hydroxy-4-p-toluinoanthraquinone), to the solution to be chromatographed. The dye forms a violet band on the column and serves as a visible marker for the position of the gamma isomer band on the column. Methoxychlor [l,l,l-trichloro-2,2-bis(p-methoxyphenyl)-ethane], benzene hexachlo ride ( B H C ) , chlordan, and toxaphene are chlorinated compounds of recognized impor tance i n the insecticide field. Other chlorinated compounds now undergoing-field testing experiments will, no doubt, soon be added to the list. The need for specific analytical methods is familiar to those concerned with the analysis of formulations containing one or more of the above insecticides. I n applying the modified partition chromatographic method to the determination of 7 -benzene hexachloride i n formulations of widely varying composition, this analytical technique appeared promising for the separation and determination of other insecticidal ingredients. I t was noted, for example, that l,l,l-trichloro-2,2-bis(p-methoxyphenyl)ethane (90 to 9 3 % of the technical product), 7 -benzene hexachloride, and technical D D T could each be separated and determined i n the mixture after chromatography on a single partition chromatographic column. A red dye, D and C R e d N o . 18 (1-xylylazoxylylazo2-naphthol), added to the solution, forms a red band on the column and serves to mark the exact position of the D D T band i n the same way that D and C Violet N o . 2 locates the 7 -benzene hexachloride. The present paper deals with certain separations that have been accomplished thus far and gives a detailed procedure for the determination of D D T and 7 -benzene hexachlo ride i n DDT-benzene hexachloride-sulfur formulations.
Apparatus
P a r t i t i o n Chromatographic C o l u m n . T h e column was constructed of standardwall boroslicate glass pipe b y the Scientific Glass Apparatus Company. I t is 83 cm.
266
HARRISPARTITION CHROMATOGRAPHY
267
long, 2.5 cm. i n inside diameter, and tooled at the top to fit a N o . 7 rubber stopper. A fritted-glass disk, 4 m m . thick and of medium porosity, is sealed i n place at the bottom of the column and about 5 cm. below the fritted disk is sealed the male part of a N o . 18/7 ball joint. The female part of the joint is pulled out i n a flame to form a short adapter. The joint is held together by a pinch clamp. Air-Pressure Reduction Valve. T h e column is operated b y air pressure. T h e valve used i n this work was purchased from the Southern Oxygen Company and was pro vided with a reduced pressure indicating gage reading i n pound units. T h e valve is con nected to the column by means of a length of pressure rubber tubing and a rubber stopper. Solvent Evaporator. T h e fractions are evaporated under reduced pressure obtained with an efficient and high-capacity water pump and with heating i n a water bath at 60 C . A glass manifold provides multiple vacuum connections and is constructed by sealing four glass tubes at equidistant points around a 50-ml. round-bottomed borosilicate glass flask. This permits the simultaneous evaporation of four fractions. The solvent is recovered by trapping i n a 800-ml. Kjeldahl flask immersed i n a Dewar flask containing a dry ice-acetone mixture. Photographs of both the column assembly and solvent evaporator have been published (2). Other apparatus includes a Waring Blendor, 1-quart mixing cup; 24 or more 125m l . Erlenmeyer flasks; two 10-ml. graduated cylinders; a 50-ml. glass-stoppered volu metric flask; and 10-ml. volumetric and 5-ml. serological-type pipets.
Reagents
n-Hexane, Phillips Petroleum Company, commercial grade. Nitromethane, Commercial Solvents Company, redistilled before use. Silicic acid, Mallinckrodt's reagent grade. M i x e d dye solution made by dissolving 25 mg. each of D and C Violet N o . 2 and D and C R e d N o . 18 i n 50 m l . of mobile solvent, stored i n a glass-stoppered bottle. Dyes are available from P y l a m Products Company, 799 Greenwich St., N e w Y o r k 14, . Y . Mobile solvent is a saturated solution of nitromethane i n n-hexane. T w o liters of n-hexane are shaken vigorously with an excess of nitromethane i n a glass-stoppered bot tle. The mobile solvent is decanted as needed.
Determination of -Benzene Hexachloride and DDT in Benzene Hexachloride-DDT-Sulfur Formulations

A number of official samples have been analyzed i n the insecticide laboratory of the Production and M a r k e t i n g Administration, which contained among the active ingredi ents 7 -benzene hexachloride, D D T , and sulfur i n the proportions of 3, 5, and 5 7 % , re spectively. The following detailed procedure was adopted after testing with authentic formulations of this type. Preparation of Sample. W e i g h a q u a n t i t y of sample sufficient to provide 0.750 gram of -benzene hexachloride and 1.25 grams of D D T , and extract the sam ple vigorously overnight i n a Soxhlet extractor with ethyl ether. This will remove total benzene hexachloride, D D T , and part of the sulfur. Evaporate most of the ether on the steam bath and the remaining few milliliters under vacuum. Extract dry residue re maining i n the flask (250-ml. standard taper) with mobile solvent, as follows: A d d 25 m l . of mobile solvent, heat just to boiling, and allow to cool to room temperature with oc casional shaking. Then decant the extract through a small, fast, conical filter paper into a 50-ml. volumetric flask containing 1 m l . of the mixed dye solution. M a k e a second hot extraction, using 10 m l . of solvent, and follow with two to three successive extractions with 5-ml. portions of solvent. Finally dilute to volume and mix. The extraction procedure described by Aepli, M u n t e r , and G a l l (1) may be used, if preferred, as an alternate extraction procedure for the residue after ether extraction. Preparation of Column. W e i g h 100 0.5 grams of silicic acid a n d transfer to a Waring Blendor. A d d 300 m l . of mobile solvent and with vigorous mixing add 55 m l . of nitromethane. After mixing for 15 seconds i n the blender, pour the mixture quickly into the column through a large glass funnel. A p p l y 8 pounds (3.6 kg.) of pressure to the column until the silicic acid-solvent boundary ceases to drop. Release the pressure, cautiously remove the rubber stopper, and, by means of a 25-ml. pipet, remove most of the upper solvent layer by gentle suction. The latter operation reduces the time required
268
in preparing the column. Again apply 8 pounds of pressure to the column and just force the solvent into the silicic acid. Release the pressure again cautiously and continue as directed under operation of column. Operation of Column. Pipet 10 m l . of the solution to be chromatographed a n d allow i t to flow slowly down the inside of the column without disturbing the surface of the silicic acid. Force the solution just into the column of silicic acid. Wash down the i n side of the column with 1 to 2 m l . of mobile solvent and force into the silicic acid by apply ing pressure. N o w fill the column up to within 1 inch (2.5 cm.) of the top with mobile solvent, but add the first 10 to 15 m l . very slowly, so that the surface of the silicic acid is not disturbed. This is done by using a pipet for the first portion. Insert the rubber stopper tightly i n the top of the column and apply 8 pounds of pressure. The separation of the two dyes will be noted soon after starting the column. Adjust the rate of flow through the column to 3 to 4 m l . per minute with the pressure regulator. Collection of Fractions. Begin to collect 10-ml. fractions b y using alternately two 10-ml. graduated cylinders when the red dye band is about 2 inches above the frittedglass disk. Pour the fractions cautiously into numbered 125-ml. Erlenmeyer flasks and wash the cylinder with a small quantity of n-hexane contained i n a dropping bottle. (This washing operation is necessary only at points where a cut is expected. Contamina tion of alternate fractions is thus minimized.) The small amount of sulfur which dissolves in the mobile solvent will be found concentrated i n one of the first fractions collected. The residue of sulfur will decrease to a negligible quantity i n the succeeding fractions and the first portion of the D D T will be recognized after evaporation of the fractions b y the appearance of a small amount of oily residue. This first fraction of D D T frequently precedes the red dye. Continue to collect 10-ml. fractions until only a trace of red dye remains on the column, then begin to collect 5-ml. fractions. Collect five 5-ml. fractions that contain no visible red dye and stop the column operation. Open the column, refill with mobile solvent, and continue the operation of the column to collect the -benzene hexachloride fraction. While the column is operating, work up the D D T fractions as directed below. A small portion of the D D T frequently comes off the column ahead of the red dye, but 94 to 9 8 % of the technical product is located within the red band. The cut be tween the D D T and benzene hexachloride will be indicated either by the absence of any residue after evaporation of one of the 5-ml. fractions or the appearance of a small amount of crystalline material which sublimes up around the neck of the flask, leaving no residue, and which is apparently the first portion of the heptachloro- and octachlorocyclohexane fraction. O n cooling and stirring with a stirring rod the D D T residues will crystallize readily. This offers another means of determining where to cut, for the quantity of crystalline residue will rapidly decrease to a negligible amount i n the succeeding flasks. When the cut is thus determined, combine the D D T fractions b y dissolving the residues with a minimum of n-hexane and pour into a weighed 125-ml. Erlenmeyer flask. E v a p o rate the solvent b y using the solvent evaporator. Then immerse the flask up to the neck in a water bath at 60 C . and evacuate with a high vacuum pump for 15 minutes. Remove the flask, wipe with a clean moist towel, and allow i t to attain equilibrium near the b a l ance for 10 minutes. Weigh and calculate the percentage of D D T i n the original sample. A second pumping is frequently necessary to attain constant weight. A t this point the residue is usually almost entirely crystalline. Table I. Determination of -Benzene Hexachloride and DDT in Benzene Hexachloride-DDT-Sulfur Formulations
a
Found, % Added, % Formula Source of DDT DDT 7-BHC tion 7-BHC Sulfur 7-BHC 50.6 50.1 1 50.0 0 50.0 Lindane& 2 Lindane 6 50 25.0 25.0 25.1 25.4 3 Lindane & 50 18.8 31.2 18.6 30.9 4 Lindane & 60 3.0 37.0 3.1 37.2 5 Technical B H C 0 12.9 20.8 13.0 20.3 6 Technical B H C 0 10.9 18.2 10.9 18.4 (18.5) 7 Technical B H C 0 11.6 11.1 11.8 11.2 ( l l . l ) 8 Technical B H C 60 11.0 18.8 11.4 18.5 9 Technical B H C 60 11.3 18.8 11.6 18.4 10 Technical B H C 60 10.9 15.7 10.7 15.6 7 - B H C in each technical B H C sample determined by partition chromatography. & The common name for the 7-isomer of benzene hexachloride of a purity of not less than 99%. Calculated from total chlorine determination on D D T fraction.
A c
c c
HARRISPARTITION CHROMATOGRAPHY
269
When a l l the violet dye is off the column, begin to collect and evaporate 10 -ml. frac tions. T w o or three fractions, which on evaporation yield little or no residue, will pre cede the appearance of the -benzene hexachloride. Continue to collect 10 -ml. fractions and evaporate until no more 7 -benzene hexachloride is obtained. Stop the column opera tion, dissolve the several 7 -benzene hexachloride residues with a minimum quantity of n-hexane, and pour into a tared 125 -ml. Erlenmeyer flask. Evaporate under vacuum, using the solvent evaporator, at 6 0 and finally at room temperature for 5 minutes with a high vacuum pump. Release the vacuum, wipe the flask with a clean, moist towel, and weigh after allowing the flask to come to equilibrium near the balance. Calculate the percentage of 7 -benzene hexachloride i n the original sample. Authentic formulations of benzene hexachloride-DDT-sulfur were analyzed, follow ing the above procedure, and the results are shown i n Table I .
I n preparation for the next experiment, invert the column and extrude the silicic acid b y applying gentle pressure. Clean the column with a long-handled brush, wash with water and acetone, and dry by attaching to a vacuum line. A s s a y of 1J J-Trichloro-2,2-bis(p-methoxyphenyl)-ethane in Technical Methoxychlor W i t h minor modificationthat is, shorter column and one half the reagentsthe procedure previously described was followed i n chromatographing solutions of technical methoxychlor. The l,l,l-trichloro -2,2 -bis(p-methoxyphenyl)-ethane was found to move down the column more slowly than 7 -benzene hexachloride, and i n one experiment the two compounds were separated and recovered quantitatively. The oily residue of 1 , 1 , 1 trichloro -2,2 -bis(p-methoxyphenyl)-ethane remaining after evaporation of the solvent crystallized readily and completely when the bottom of the flasks was scratched, and the purity of the combined residues was indicated by the melting point. The material melted at 87 to 88 C , which is the melting point reported in the literature for pure 1 , 1 , 1 trichloro -2,2 -bis(p-methoxyphenyl)-ethane. Table I I gives the percentages of 1 , 1 , 1 trichloro -2j2 -bis (p-methoxyphenyl) -ethane in the technical product found after chromato graphing different quantities of the technical material. Table II. Assay of l l l-Trichloro-2 2-bis(p-methoxyphenyl)-ethane in Technical Methoxychlor
/ / #
Detn. 1 2 3 4
Wt. of Material Chromatographed, Mg. 100 200 400 500
l,l,l-Trichloro-2,2bis(p-methoxyphenyl)-ethane Found, % 91.0 92.5 93.0 92.8
Notes on Procedure. I n p a r t i t i o n chromatography as well as adsorption experi ments, successful results require a certain amount of skill which is acquired after some practice. If the surface of the silicic acid is not flat, stir gently with a long stirring rod. T h e particles of silicic acid will settle evenly and a more horizontal band will be formed when the sample is introduced. The column should be clean and d r y ; otherwise, the silicic acid will tend to adhere to the inner wall. I n evaporating the fractions, do not allow the mobile solvent to boil. A two-way stop cock of 4-mm. bore inserted between the trap and pump will make i t convenient to stop the evacuation of the flasks if boiling occurs. If the system is tight, the fractions will continue to evaporate evenly and there will be no danger of loss of material through spat tering. Other Chromatographic Separations The method has been applied to the separation of other insecticidal ingredients. Chromatography of Pyrethrins Concentrate. F i v e hundred milligrams of con centrate consisting m a i n l y of the two pyrethrins and the two cinerins were dissolved
270
ADVANCES IN
CHEMISTRY SERIES
i n mobile solvent and chromatographed. Forty-three 10-ml. fractions were collected. The fractions which made up three bands on the column were combined and e v a p o rated, and the residues were tested w i t h DenigeV reagent. A positive test for c h r y s anthemum monocarboxylic acid was obtained on two residues, which separated widely on the column. Because p y r e t h r i n I and cinerin I are both esters of t h i s monocarboxylic acid, i t may be assumed that these two ingredients had been sepa rated. Chromatography of C h l o r d a n . A t t e m p t s to separate technical chlordan a n d D D T b y the above procedure were unsuccessful. M o s t of the chlordan came off the column with the D D T fraction. A solution of technical chlordan alone was separated into several fractions, some of which gave crystalline residues after evaporation of the solvent; however, the separations were not sharp.
Discussion
The formulations shown in Table I were made to contain approximately 3 parts of 7 -benzene hexachloride to 5 parts of technical D D T , because this is the ratio i n which they have been present i n many of the formulations encountered by the author. F o r these ratios the separation and recovery of 7 -benzene hexachloride and D D T are satis factory. However, if the ratio of 7 -benzene hexachloride to D D T is increased much beyond 3 to 5, a small amount of impurity from the technical benzene hexachloride con taminates the D D T fraction and leads to high D D T results. This is not the case with l i n d a n e - D D T mixtures, as will be seen by the results i n Table I. Because pure 7 -benzene hexachloride and D D T separate so widely on the column, it should be possible to separate and determine these two ingredients i n almost all proportions. The results shown i n Table I I were obtained on a recently manufactured sample of technical methoxychlor. The sharp separation of the l,l,l-trichloro-2,2-bis(p-methoxyphenyl)-ethane on the column and the purity of the material as evidenced by the melting point appear to recom mend it as a method of assay for the technical product. On the basis of results reported in this paper, it would appear that partition chromatog raphy will find other applications in analysis of insecticide formulations. The method possesses a certain degree of specificity i n that the partition behavior of the compounds being studied is a characteristic property.
Literature Cited
(1) (2) (3) (4) Aepli, O. T., Munter, P. ., and Gall, J. F., Anal. Chem., 20, 610 (1948). Harris, T. H . , J. Assoc. Offic. Agr. Chemists, 32, 684 (1949). Martin, A. J. P., and Synge, R. L. M . , Biochem. J., 35, 1361 (1941). Ramsey, L. L., and Patterson, W. I., J. Assoc. Offic. Agr. Chemists, 29, 337 (1946).
Organic-Chlorine Determinations as a Measure of Insecticide Residues in Agricultural Products

R. H. CARTER, R. H. NELSON, and W. A. GERSDORFF
Determination of organically bound chlorine is shown to be in general agreement with fly mortality tests of insecticide residues in agricultural products. Alfalfa hay that had received applications of toxaphene and beef fat from animals fed alfalfa hay containing toxaphene residues or sprayed with benzene hexachloride or DDT were used for tests.
T h e use of chlorinated hydrocarbon insecticides for the control of insects affecting field crops and farm animals has created interest i n the amounts of these materials that may be found i n products intended for human and animal consumption. Colorimetric methods (8, 6-10), some of which are specific, have been developed for the determination of D D T i n small amounts. F o r benzene hexachloride (hexachlorocyclohexane), chlordan, and toxaphene, however, specific analytical methods have not been developed, and their residues have been evaluated b y the determination of organically bound chlorine. The procedure comprises extraction of the insecticide residue from the sample with benzene or other suitable organic solvent, evaporation of the solvent, treatment of the residue with isopropyl alcohol and metallic sodium, and finally determination b y standard methods of the amount of chloride ion formed. Because the results obtained reflect the presence, not only of the insecticide i n quest i o n , but also of any of its decomposition products or other organic compounds containing halogen, confirmatory evidence of the identity and amount of the residues was desired. Therefore, studies to correlate the results of organic-chlorine determinations with insecticidal activity were undertaken.
Evaluation of Toxaphene Residues on Alfalfa Hay by Biological Tests

A sample of alfalfa hay from Bozeman, M o n t . , that had received two spray applications, each of 4 pounds of technical toxaphene per acre, was found by the method recommended by Carter and Hubanks (2) to contain 225 p.p.m. of organic chlorine, equivalent to 331 p.p.m. of toxaphene. This sample was obtained from a bale approximately 5 months after the last spray application. A similar sample of untreated hay was found to contain 1.2 p.p.m. of organic chlorine. Portions of the treated hay were extracted with benzene, and aliquots of the benzene solution were evaporated to dryness on the steam bath, with a gentle air current to remove the last traces of solvent. The residues were then taken up i n deodorized kerosene and tested against houseflies by the turntable method. For comparison, extracts were made up to contain the same amounts of technical toxaphene as were indicated by the chlorine determinations to be present i n the treated hay.
271
272
The results are reported i n Table I . The mortalities are the averages of seven repli cates, approximately 100 flies being used i n each test. Table I. Toxicity to Houseflies of Toxaphene Residues on Alfalfa Hay
Concentration of Residue or Toxaphene, Mg./Ml. 2 1 2 1 % Knockdown 10 10 15 15 % K i l l in One Day 81 56 89 65
Source of Extract Sprayed alfalfa * Technical toxaphene (standard)
The kills obtained from the residues from the sprayed alfalfa and from the known technical toxaphene are i n close enough agreement to support the assumption that the chemically determined material was toxaphene and that the concentrations deter mined were essentially correct.
Toxaphene Stored in Animai Fat

Samples of abdominal fat from a steer that had been fed hay containing toxaphene residues and from a steer that had been fed untreated hay were analyzed for their organicchlorine content by the method recommended by Carter (1). The organic-chlorine con tent of the fat from the treated steer in excess of the amount found in the untreated steer was equivalent to approximately 700 p.p.m. of toxaphene. Extracts of these fat samples were treated with sodium sulfate-concentrated sulfuric acid mixture and fuming acid by the method described by Schechter et al. () i n order to separate the organic-chlorine compound from the fatty materials. A n infrared spectrum from 7 to 15 microns on carbon disulfide solutions of the residues from the fat qualitatively identified the organic-chlorine compound as toxaphene. A l l the bands of toxaphene i n this spectral region were plainly seen i n the treated steer extract, whereas none of the ab sorption bands were visible i n the untreated steer extract. Different samples of chlorinated camphene containing from 62 to 7 2 % of chlorine all give the same infrared spectra. However, the toxicity to flies reaches a maximum at a chlorine content of 67 to 6 9 % and drops off rapidly below 6 0 % and above 7 2 % . From the results of both the infrared spectroscopic examination and the fly-toxicity tests given below, it is concluded that the organic-chlorine compound i n the fat was essentially u n changed toxaphene.
Toxaphene, Benzene Hexachloride, and DDT in Animal Fat

Samples of abdominal fat from a steer that had been fed on alfalfa hay containing toxaphene residues and from two steers that had been heavily sprayed, one with D D T and Table II. Toxicity to Houseflies of Residues of Beef-Fat Extracts Containing Chlorinated Hydrocarbon Insecticides
(One test per s ample) Extract. M g . per Flask 0 6.7 2.7 3.7 Sample No. 1 2 1 2 Known 2 Known 1 2 Known
1
Insecticide Fat only (checks) Toxaphene Benzene hexachloride (technical) DDT
1 hour 0 0 0 0 0 80 80 100 0 0 85
% Knockdown 3 2 hours hours 0 0 0 0 0 95 95 100 60 60 100 0 0 20 20 25 100 95 100 60 75 100
4 hours 0 0 28 65 60 100 100 100 85 90 100
% K i l l in 24 Hours 25 25 80 95 94 100 100 100 85 92 100
CARTER, NELSON, AND GERSDORFFORGANIC-CHLORINE
DETERMINATIONS
the other with benzene hexachloride, were found to contain several hundred parts per m i l lion of organic chlorine. Portions of the fat were treated by the Schechter-Haller () method to separate the toxicant residue. The residues were then subjected to biological assay with houseflies, i n comparison with the corresponding insecticides containing equiv alent amounts of organic chlorine. A modification of the Laug (4) technique was used. The residues were placed i n 500-ml. flasks, which were then turned on their sides. A p proximately 100 flies, immobilized b y chilling, were introduced into each flask. A s soon as the flies revived, the flasks were set upright and observations made on knockdown and k i l l . Food (skim milk) was provided on strips of cotton hung from the mouths of the flasks, which were covered with cheesecloth. The results are reported i n Table I I . The flasks used had narrow mouths, whereas the Laug technique specifies widemouthed beaker flasks. The small opening reduced air exchange and moisture condensed on the insides of the flasks. This condition is not good for flies and probably accounts for the mortality observed i n the checks. However, this effect had not shown up 5 hours after the tests were started, and by that time most of the flies i n the flasks containing insecti cides were down. These tests are qualitative rather than quantitative, but the data indi cate that the chemically determined quantities of the various insecticides in the fat extract were nearly equal i n toxicity to known samples at the same level. The tests reported i n this paper were intended only to show general agreement be tween the insecticide level calculated from organic-chlorine determinations and fly mor tality. It is also shown that organic-chlorine residues on alfalfa hay resulting from insecti cide applications of toxaphene and the organic-chlorine content of beef fat from animals fed alfalfa hay containing toxaphene residues or sprayed with benzene hexachloride or D D T approximate a true measure of the amounts of these compounds present.
2 7 3
Acknowledgment
Acknowledgment is made of assistance i n the chemical work b y A . C. Hazen, H . D . M a n n , and P . E . Hubanks. T h e infrared spectroscopic examination was made b y W . C . K e n y o n , Hercules Powder Company, Wilmington, D e l .
Literature Cited
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Carter, R. H . , Anal. Chem., 19, 54 (1947). Carter, R. H . , and Hubanks, P. E., J. Assoc.offic.Agr. Chemists, 29, 112 (1946). Claborn, . V., Ibid., 29, 330 (1946). Laug, P. L., J. Pharm. Expt. Therap., 86, 324 (1946). Schechter, M . S., Pogorelskin, . ., and Haller, H . L . , Anal. Chem., 19, 51 (1947). Schechter, M . S., Soloway, S. B., Hayes, R. ., and Haller, H . L., Ind. Eng. Chem., Anal. Ed., 17, 704 (1945). Stiff, . ., and Castillo, J. C., Ibid., 18, 316 (1946). Stiff, . ., and Castillo, J . C., J. Biol. Chem., 159, 545 (1945). Stiff, . ., and Castillo, J. C., Science, 101, 440 (1945). Wichmann, H . J . , Patterson, W. I., Clifford, P. ., Klein, A. K., and Claborn, . V., J. Assoc. Offic. Agr. Chemists, 29, 188 (1946).

Agricultural Control Chemicals (1950)

Uploaded by

Copyright:

Available Formats

Agricultural Control Chemicals (1950)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Agricultural Control Chemicals (1950)

Uploaded by

Copyright:

Available Formats

AGRICULTURAL CONTROL

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.fw001

AMERICAN CHEMICAL SOCIETY 1155 Sixteenth Street, N.W. Washington 6, D. C.

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.fw001

Copyright April 1950 by AMERICAN CHEMICAL SOCIETY AU Rights Reserved

consecutively. I t is expected t h a t t h e A D V A N C E S I N C H E M I S T R Y S E R I E S w i l l be recognized as a n i n t e g r a l p a r t of t h e p e r m a n e n t l i t e r a t u r e , a n d w i l l be covered b y the a b s t r a c t j o u r n a l s .

C o n t r o l C h e m i c a l s / ' i s a c o l l e c t i o n of papers f r o m t h e S y m p o s i a o n E c o n o m i c Poisons, organized u n d e r t h e c h a i r m a n s h i p of J . L . S t .

American Chemical Society Library

John and presented before the Division of Agricultural and Food

Objectives and Scope

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch001

ADVANCES IN CHEMISTRY SERIES

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch001

Research Program on Economic Poisons

ST. JOHNOBJECTIVES AND SCOPE

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch001

Subdivision on Economic Poisons

ADVANCES IN CHEMISTRY SERIES

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch001

World Use of Economic Poisons

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch002

ADVANCES IN CHEMISTRY SERIES

Work of the FAO

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch002

EASTERWORLD USE OF ECONOMIC POISONS

Equipment for Pesticide Application

ADVANCES IN CHEMISTRY SERIES

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch002

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch003

ADVANCES IN CHEMISTRY SERIES

Benefits from Insect Control

Cost of Insect Control

Problems of Insect Control

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch003

Cooperation in Development of Insecticides

ADVANCES IN CHEMISTRY SERIES

Outlook for Insect Control

Seasonal Market for Insecticides

Some Problems in the Use of Newer Economic Poisons

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch004

ADVANCES IN CHEMISTRY SERIES

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch004

MacLEODSOME PROBLEMS IN THE USE OF NEWER ECONOMIC POISONS

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch004

Investigation, Education, Regulation

ADVANCES IN CHEMISTRY SERIES

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch004

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch005

ADVANCES IN CHEMISTRY SERIES

do, no false or misleading representations may be made on labels or i n accompanying literature.

Registration of Economic Poisons

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch005

REEDLABELING REQUIREMENTS UNDER FEDERAL LAW

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch005

Federal and State Regulations

ADVANCES IN CHEMISTRY SERIES

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch005

Registration and Labeling of Economic Poisons in California

Published on January 1, 1950 on http://pubs.acs.org | doi: 10.1021/ba-1950-0001.ch006