1 15 40 Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 077 Folder The folder containing the original item. 1980 Series The series number of the original item. Series III Subseries IV Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Date A point or period of time associated with an event in the lifecycle of the resource January 26 1984 Title A name given to the resource Transcript of Proceedings: Royal Commission on the Use and Effects of Chemical Agents on Australian Personnel in Vietnam, 26 January 1984 Subject The topic of the resource Australian Royal Commission Hearings herbicide toxicology pesticide toxicology dioxin ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/705d330b601bd6f010265578c4d03efa.pdf b4e95d1fe9cf25954eae9a55134fb242 PDF Text Text Item ID Number ones Bababunmi, Enitan A. Corporate Author Report/Article TltlB Toxins and Carcinogens in the Environment: An Observation in the Tropics JOUmal/BoOk TltlO Journal of Toxicology and Environmental Health Year Month/Day Color D Number of Images 9 DOSCrlptOn NotOS ^'vin ^- Young filed this item under the category "DDT/Human Toxicology and Environmental Fate" Wednesday, April 11, 2001 Page 1188 of 1242 �TOXINS AND CARCINOGENS IN THE ENVIRONMENT: AN OBSERVATION IN THE TROPICS Department of Biochemistry, School of Medicine, University of Ibadari, Ibadan, Nigeria The incidence of primary liver cancer in the countries of tropical Africa is the highest in the world. There, is a growing belief that the relatively high prevalence of hepatocellular carcinoma in Niyeria may have a multiple chemical factor etiology in such forms as food contaminants, herbal leas, and environmental chemicals. Major chemical toxins and carcinogens that have been identified so far in the tropical environment include supotoxin, cycasin, mushroom toxin, capsaicin, oxalic acid, prussic acid, fluotooleic acid, N-nitroso compounds, aflatoxin, palmotoxin, pyrrolizidine alkaloids, quinine, DOT, and eye/ornate. INTRODUCTION During the past two decades, there has been an increasing awareness of the hazards to human and animal health from various chemical substances Jthat occur in the tropical environment. There is evidence to indicate that azarious health problems, including some forms of cancer, have their origin in the presence of toxic chemicals in medicines, herbal residues, pesticides, foods, and drinks. Some of these problems are common to countries throughout the world, while others are peculiar to Africans who live in the tropics. For example, altogether the incidence of primary liver cancer in adult males is • the highest in the world in African countries such as Nigeria, Uganda, and Mo/ambique (IARC, 1971). The primary objective of the present review is to focus attention on the presence of toxic chemicals in foods and in the environment of tropical African countries, particularly Nigeria, which is the most populous country in Africa. Some of these toxins have been shown to possess carcinogenic (or mutagenic) properties in various biological systems. However; in a large number of cases, adverse effects of some of these toxic substances on human health are not known. It is hoped that this paper will have the important effects of stimulating more research into the special toxicological problems that arc of concern to people living in the tropics. This article was written during the author's tenure as an ICRCTT Fellow of UICC (1977) at the Department of Biochemistry and King's College, University of Cambridge, Cambridge, [England. Requests for reprints should he sent to fcnitan A. Bababunmi, Department of Biochemistry, School'of Medicine, University of Ibadan, Ibadan, Nigeria. 691 journal oi Toxicology and Environmental Health, 4:691-699,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/040691-09$2.25 �692 E. A. BABABUNMI ENDOGENOUS FOOD TOXINS Endogenous food toxicily is widespread in tropical Africa. Nicholls ct al. (1961) dealt with the various types.of tropical foods that carry toxins. The review of Crampton and Charlcsworth (1975) adequately covers the occurrence of food toxins in the nontropical world. Hypoglycin This toxin is contained in the unripe fruit of the food plant, Blighia sapida. In Nigeria the fruit is called isin, whereas it is commonly known as ak.ee in Jamaica. There are two types of this toxin, A and B. Although both types are biologically active, the A type (/J-mcthylenecyclopropylalaninej is the more toxic. Very little work has been done on the chemistry and the biological function of the B type. Dioscorine This toxic chemical is sometimes referred to simply as dioscorea toxin; it is an alkaloid that is present in Dioscorea hispida. The main toxic species of these tropical yams of West Africa are D. hispida, D. dumctorum, D. sansibarensis, and D. bulbifem. Since the isolation and identification of the related alkaloid dehydrodioscorine by Bevan and H.rst (1958), scientists have not looked into the existence of these or other structures in the other species of the wild yams. Sapotoxin Some tropical foodstuffs such as soybean, breadfruit, tomato, melon, orange, and groundnut contain some sapotoxin, which, at high concentrations, has drastic effects on humans. The toxin can cause gastroenteritis and produce paralysis of the nerve centers. Sapotoxin is a nitrogen-free glycosidc. Cycasin Cycasin occurs in plants of the family Cycadaceae, which are indigenous to tropical and subtropical regions (IARC, 1972). The biologically active moiety of cycasin is the aglycone melhyla/oxymelhanol. Cycad seeds are used as medicine in some parts of Africa, Indochina, and India. Feeding of a cycad diet has been shown to induce malignant tumors of the liver in the rat, mouse, hamster, fish, and guinea pig (IARC, 1972). Mushroom Toxin A large number of mushroom species are edible, but certain species that arc eaten in the tropics are poisonous. Examples of toxins elaborated by these species are agaritine (Ayaric'us bisporus toxin) and champigeon (A. hortemis toxin). Muscarine and Amanita phalloictes toxin have been reported to be toxic by Nicholls el al. (1961). �TOXINS AND CARCINOGENS IN THE TROPICS 693 Names for different species of mushrooms are descriptive in many pails of I he tropics. In the western state of Nigeria, these names give an indication either of the habitat, morphology, and texture or of the growth habitat of the fungi (Oso, 1975). Corprinus ephemerus, a fungus that grows on dunghills, appears at night or early in the morning, and within a very short time the pilcus is fully expanded. However, it deliquesces in the sun. It is considered poisonous by the Yoruba people of Nigeria, and local doctors use it in the preparation of some (harms. (Extracts of the fungus should be tested for mutagenicity. Capsaicin (Red Pepper Toxin) The substance is the active principle of the plants Capsicum annum and C. frutescens. It is a powerful irritant and a skin blister. Although these plants are rich in vitamin C, excessive feeding on them can be dangerous. Species of pepper such as Piper nigrum contain alkaloids and volatile oils that are toxic to both animals and humans. Halogeton Toxin (Oxalic Acid) Plant species such as Halogelon ylomeritus, Celosia argentae, Amaranthus candatus, Celosia laxa, and Talinum are used as food in tropical Africa, especially on the west coast. These vegetables contain significant levels of oxalic acid (Oke, 1967). There are conflicting data on the lethal dose for humans. However, Oke showed that an average healthy Nigerian would consume about 6 g of oxalic acid daily, on the basis that 50 g of fresh vegetables could be consumed at a meal. Cyanogen (Prussic Acid) Tropical plants, cassava, mai/ie, and sugar cane are good sources of cyanogenetic glycosides such as linamarin and dhurrin. Cassava (manioc; Man/hot ulilissima} is the most widely grown of all tropical root crops. It is mainly a carbohydrate food with a very low protein content. In West Africa, manioc flour (gari). has become a major diet. In the West Indies the dried flour is called farina. All over the tropics it is used as food for the young and adults in one form or another. The en/yme linasc liberates prussic acid (HCN) from linamarin. HCN is toxic to many species of animals, including humans. The production of HCN varies with the variety of the plant and the conditions of cultivation (Osuntokun et a!., 1969). Although significant amounts of HCN arc said to occur only in the bitter variety of cassava, there is in fact no clear differentiation between the sweet and hitler strains. The fact that the cortex of the root contains the highest concentrations of the toxin provides biological protection for the plant against invading insects. Chronic cyanide intoxication by laboratory animals has resulted in neural damage in the guinea pig, rabbit, sheep, and cat. �694 E. A. BABABUNMI Fluoruoleic Acid The seeds of the West African plant Dichapctalum toxicarum contain fluoroeleic acid and some minute amounts of shorter-chained fluoro acids. These fluoro compounds arc toxic.'Local, doctors in the countries of West: Africa often administer the seed extracts in an attempt to produce loss of motor activity, loss of sensation, and sometimes death (Peters el al., I960). Free oleic acid uncouples oxidative phosphorylation (Pressman and Lardy, 1956). /V-Nitroso Compounds .Dimclhylnilrosaminc (DMN) and diethylnilrosamine (DEN) have been detected in measurable quantities in several alcoholic beverages in Nigeria (joaquim, 1973). Bababunmi et al. (1977) reviewed the extent of contamination of these drinks by the two carcinogens. There is some evidence that the formation of these nil.rosamines involves bacterial action. FUNGAL TOXIC CONTAMINANTS Fungus-infected foodstuffs are the cause of many types of food poisoning (see Kadis et al., 1972). In the tropics, a Variety of fungal species have been reported to be involved in some toxicity syndromes. Notable examples are Asperyi/lus, Penicil/ium, Stachybotrys, Trichoderma, f-'usarium, Pseudomonas, and .Helminthosporiurn species. The most ubiquitous in Nigeria are Aspergillus and Pcnicillium. Many strains of each of these fungi are toxigenic. Among the common toxic metabolites of the aspergilli arc aspergillic acid, flavacol, j3-nilropropionic acid, kojic acid, sterigrnatocystin, ochratoxin, aspet toxin, aflaloxin, and palmotoxin. Penicillium elaborates the mycotoxins patulin, islandiloxjn, lutcoskyrin, rugulosin, cifrinin, frcquentic acid (citreomycctin), gliotoxin, costaclavine, and citreviridin. Of these, aflatoxin has been studied most extensively, mainly because of its potent carcinogenic properties. A comctabolile of aflaloxin, palmotoxin, has been the subject of investigation for some years in this laboratory. Many other naturally occurring toxins (Table 1) that are known should be tested for carcinogenictty. Aflatoxin The literature on the biochemistry, toxicity, carcinogenicity, and mutagenicity of aflatoxin is enormous (Goldblatt, 1969; Wogan, 1975a, 1975b; IRAC, 1976), The discovery of aflatoxin in the tropics (Asplin and Carnagnan, 1961) as a contaminant of human and animal foodstuffs (groundnuts) aroused the interest of scientists all over the world because of i t . health ha/aids and possible economic effects on the producers of these foods. Nigeria is one .of the world's major exporters of groundnuts. Other tropical foods that arc vectors of aflatoxin are beans, corn, rice, cocoa, and wheat. �TOXINS AND CARCINQGtNS IN lilt. TROPICS 695 TABLE 1. Sonic Known Naturally Occurring Toxins Toxic sulislance Occurrence in l-'usaric acid Fiisariuni oxysp oiium Pcriconin T toxin llclminthosporo'sidc Tabtoxin Javanicin Pcriconia circ'inatu 1 lei min tli ospvrium may dis Hvlniitithosporium sqcchari Pseudomonus coronal aciens f-'usarium solan! Possible human exposure through Tomato, sorghum, maize Sorghum Maize Sugarcane Tobacco Maize Aspergillus flavus, the main source of aflatoxin, is common in air and soil, It will grow on agricultural products and food materials in a favorable environment with a relative humidity of 70-90% and a minimum temperature of about 10°C. In general, the growth of A. flavus can be correlated with the production of aflatoxin except at high temperatures, 40-50°C. !n different regions of Muranga in Kenya (Hast Africa), mean aflaloxin levels of about 0.25 ppm in food and 0.1 mg/l in beer have been detected (IARC, 1972). When common food preparations of Nigeria's principal food crops were sampled from local market stalls and assessed for aflatoxin contamination by conventional techniques, the aflatoxin content was not less than 0.5 ppm in any of the foods (Bababunmi, 1976). Several industrialized countries such as the United States, Denmark, Britain, and Italy consider a level of aflatoxin of the order of 0.25 ppm as dangerous and the contaminated food as unconsumable. Although the proportional contribution of agriculture to the Nigerian economy continues to fall, it will continue to be the single most important sector in the economy for a long time (Aboyadc, 1971). The export value of cocoa ranks second to that of oil in Nigeria. In 1974, Nigeria's foreign trade was $3.462 billion. If oil accounted for 80% of the export value in the 1974-1975 fiscal year, other export products such as cocoa, groundnuts, and palm products should account for about $700 million. Therefore, if aflatoxin contamination in this class of export commodities is not eliminated, Nigeria's foreign reserves may diminish continuously. Palmotoxin Isolation of two additional fluorescent toxins from cultures of A. flavus on unfcrmcnled palm sap (a common West African wine) obtained from a variety of Elacis yuineensis was reported by Bassir and Adekunle (1968). Toxicity titrations of pal rnolox iris B 0 and G0 on 6-d-old White Rock chick embryos indicated that B 0 is as toxic as aflatoxin B t . Recent results of Uwaifo ct al. (1977) suggest that the structures of the palmotoxins could be hetetocyclic and may be similar to those of the aflatoxin family (Asao et al., 1965). Comparative mulagenicily studies by �696 E. A. BABABUNMI Uwaifo el v.al. (1978) show that palrnotoxin B() induces microlesions thai consist of point mutations, in Ames' tester strains of Salmonella typhimurium. However, the ratio of the mutagcnicity of aflatoxin fi, to that of palmotoxin B0 is about 6:1. In Nigeria and several developing African countries, several facilities and preservation techniques for agricultural products are quite inadequate. The combination of this unfortunate situation, the natural warm and moist weather, a dirty environment, human error, and ignorance is conducive to the growth of A. f/avus and consequently to the elaboration of mycotoxins (such as aflatoxin and palrnotoxin) on agricultural commodities. It seems to me, therefore, that the problems associated with mycotoxin contamination of food and agricultural products will remain in the developing tropical regions of the world for some time, at least.in the foreseeable future, unless very drastic control measures a' - c initialed. HERBAL RESIDUES For years, herbalists and local doctors in tropical Africa have used herbs and their concoctions to treat various human diseases (Dal/.iel, 1948). In modern times, countries such as Nigeria and Ghana have intensified their search for authentic medicinal plants ;ind their active principles. Apart from their use as local medicines, many toxic plant species are used as food in many parts of West Africa. Many chemical compounds have been isolated from useful plants of West Africa and characteri/cd in their pure forms. In this respect, scientists in the Department of Chemistry of the University of Ibadan have contributed immensely to the knowledge of the chemistry of active principles in plants. Toxicological and other biological studies of these chemicals are, however, scanty. Miller and Miller (1976) staled that the plant genera Crotolaria, Senecio, Laburnum, and Heliotropium have long been known to contain carcinogenic substances (IARC, 1976), some of which arc pyrroli/idine alkaloids. •; • FOREIGN TOXIC CHEMICALS Environmental toxins of this class exist in such forms as medicines, pesticides, and food additives. With the gradual emergence of some tropical African countries (for example, Nigeria) from the underdeveloped to the developing slate, environmental pollution and the presence of industrial materials such as those used in the processing and packaging of foods are potential sources of toxins. Common examples in this calegory are ioni/ing radiation, plastici/ers, adhesives, paraffins, printing inks, and irea'cd papers. Well-rccogni/.cd environmental toxic (or carcinogenic) chei lical substances in this tropical area of the world include quinine (antirnalarial drug), DDT (insecticide), and cyclarnate (food additive). �TOXINS AND CARCINOGENS IN THt TROPICS 697 Quinine Malaria is a disease that occurs throughout the tropical and subtropical countries. It is actually a group of diseases characterized by recurrent attacks of fever, anemia, and enlargement of the spleen. Malaria can also occur in temperate climates if the environmental temperature is right for the protozoan species (e.g., Plasmodium falciparum) to complete their life cycle in the female Anopheles mosquitoes. The parasite lives in the red blood cells. There are three forms of the malaria parasite in humans corresponding to malignant tertian, benign tertian, and quartan malaria. In the tropics the most common malaria is the malignant tertian, although the other two varieties have been identified in a very few cases. Chloroquinc, mcpacrine, and quinine arc drugs that are very effective in rapidly destroying the parasite in the blood. Chloroquinc (Nivaquine) is the most widely used antimalarial drug and has been reported to be the safest. Mcpacrine (Atebrin) can be given only intramuscularly and is not often used. Quinine is the oldest of all the antimalarial medicines. It is also the quickest acting. For many years quinine was the only drug available for the treatment of malaria. Although quinine has some toxic side effects, it is still used, especially for cases that are resistant to other drugs. Quinine was the first alkaloid isolated from the bark of the Cinchona tree. A single oral dose of about 8 g is regarded as fatal for an adult man. Quinine poisoning usually results in nausea, headache, visual disturbances, nervous system and cardiovascular system disorders, and respiratory arrest. DDT The insecticidal properties of DDT are well known (IARC, 1974). This compound has been extensively used as an insecticide and produced commercially for this purpose since 1943, when a low-cost production technique was developed. It has been widely used for the control of numerous insect pests—for example, as a mosquito larvicide and as a residual spray for eradicating malaria in the tropics. DDT is distributed by the World Health Organi/ation throughout the world for the prevention of yellow fever, sleeping sickness, and malaria. Apart from these uses, quantities of DDT are used for the treatment of peppers, onions, soybeans, groundnuts, cowpeas, and sweet potatoes, in storage. Tropical countries such as the Upper Volta and Ghana use at least 500 kg of DDT annually for agricultural purposes. In 1973, research on the environmental effects of pesticides in the tropics was carried out at the International Institute for Tropical Agriculture (IITA), Ibadan. The study was concerned with the effects of DDT (used as a crop protector) on the fertility of agricultural soil. Cowpca (Vigna unyuiculata var. Prima), which is a high-yielding legume, was selected in the IITA study because it requires regular pesticide applications and also because of its growing �698 C. A. BABABUNMI importance in tropical agriculture. Since 1970, DDT has been restricted to uses other than on human and animal foodstuffs in the more advanced countries. The hepatocarcinogenicity of DDT on oral administration has been amply demonstrated in several strains of mice. Liver cell tumors were produced in both male and female mice, and an increased tumor incidence was reported in some other organs. The most frequent tumor types were leukemia, rcticulum cell sarcoma, carcinoma of the lungs, and hemangioendothclioma (IARC, 1974). Cyclamate Calcium cyclamate (cyclohexylsulfamic acid calcium salt) is still used as a nonnutritivc sweetener in a large number of soft drinks in many African countries. The use of cyclamic acid as a sweetener has been banned in several industrialized' countries because the compound was suspected of being a bladder carcinogen in the rat. CONCLUSION Apart from foods, beverages, and medicines, there are other sources of potential toxins and carcinogens that are introduced by humans into the tropical environment, especially in the cities, in such forms as narcotics and atmospheric pollutants. With the arrival of various industries in big African cities, inhalation of dust, vapors, and exhausts presents a new form of danger. Epidemiologic appraisal of these factors is lacking. There is a need to estimate the total load of toxins and carcinogens in the tropical environment. REFERENCES Aboyadc, O. 1971. Nigeria's economy. In Africa South of the Sahara, 1st ed., pp. 558-563. London: Luropa. Asao, T., Buchi, G., Abdel-Kader, M. M., Chang, S. 13., Wick, E. I . and Wogan, G. N. 1965. The ., structures of aflaloxins B and G,. ]. Am. Chum. Soi: 87:882-886. Asplin, [-". D. and Carnaghan, K. 13. A. 1961. The toxicity o\ certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Kec. 73:1215-1219. Bababunrni, Li. A. 1976. Excretion of af'latoxin in the urine of normal individuals and patients with liver diseases in Ibadan (Nigeria). In Di'tuclifin and 1'revcnt/on of Cancer, ed. H. E. Nieburgs. New York: Ueker. Babdbunni, L:. A., Uwaifo, A. O., and Bassir, O. 1977. Hepatocarcinogeris in Nigerian foodstuffs. World Rev. Nutr. Did 28: article 44. Bassir, O. and Adckunle, A. A. 1968. Two new metabolites of Aspcnjillus lluvus (Link). FF.BS Lett. 2:23-25. Bevan, C, W. 1.. and Hirst, ). 1958. A convulsuil alkaloid of Dioscoreu dunu'torurn.' Chcm. Ind, 4: 103. . Crampton, K. l:. and Charlesworlh, I . A. 1975. Occurrence of natural toxins in food. Or. Mvd. B II. 31:209-213. Dal/:iel J. M. 1948. Useful plants of west tropical Africa. In i-loia of West Tropical Africa, cds. J. hutchinson and J. M. Dal/iel. London: Crown Agents for the Colonies. �TOXINS AND CARCINOGENS IN Tilt TROPICS 699 Goldblalt, L A. 1969. Allatoxln. New Yoik: Academic Press. IARC. 1971. Liver Cancer. Sci. I'ubl. no. I. l.yon: International Agency for Research on Cancer. IARC. 1972. Evaluation of Carcinogenic Risk of Chemicals to Man, vol. 1. Lyon: International Agency for Research on Cancer. IARC. 1974. Chemical Carcinogencsis l.'ssays. Sci. Publ, no. 10. Lyon; International Agency for Research on Cancer. IARC. 1976. Evaluation of Carcinogenic Risk of Chemical* to Man, vol. 10. Lyon: International Agency lor Research on Cancer. Joaquim, K. .1973. Nilrosamine contamination of some Nigerian beverages. Ph.D. thesis, Ibadan University. Kadis, $., Ciegler, A., and Ajl, S. J. 1972. In Microbkil Toxins: A Comprehensive Treatise, vol. 8, Fungal Toxins. New York: Academic Press. Miller, j. A. and Miller, !"..• C. 1976. Carcinogens occurring naturally in foods. Fed. Proc. 35:1316-1321. Nicholls, L., Sinclair, H. M., and Jelliffe, D. B. 1961. Tropical Nutrition and Dietetics. London: Bailliere, Tindall & Cox. Oke, O. L. 1967. Oxalic acid in plants and in nutrition. World Rev. Nutr. Diet. 8:262-303. Oso, B. A. 1975. Mushrooms and the Yoruba people of Nigeria. Mycologia 67:311-319. Osuntokun, B. O., Monekosso, G. L., and Wilson, J. 1969. Cassava diet and a chronic degenerative neuropathy. An epidemiological study. Niger, j. Sci. 3:3-15. Peters, R. A., Hall, R. J., Ward, P. l:. V., and Sheppard, N. 1960. The chemical nature of the toxic compounds containing fluorine in the seeds of Dkhapelalum toxicarium. Biochcm. J. 77:17-23, Pressman, B. C. and Lardy, II. A. 1956. Effect of surface active agents on the latent ATP-asc of mitochondria. Biochim. B/ophys. Ada 21:458-466. Uwaifo, A. O., Emcrolu, C. O., and Bassir, O. 1977. Comparative study of the fluorescent characteristics ol solutions of aflatoxins and palmotoxins in chloroform. /. Agric. Food Chem. 25:1218-1220. Uwaifo, A. O., Emcrolc, G. O., Bababunrni, E. A., and Bassir, O. 1978. Comparative rnutagcnicity of palmotoxin U0 and aflatoxin B,. In press. Wogan, G. N. I975a. Mycotoxins. Annii. Rev. Pliarmacol. 15:437-451. Wogan, G. N. 1975b. Dietary lactors and special epidemiological situations of. liver cancer in Thailand and Africa. Cancer Rex. 35:3499-3502. Received March 12, 1978 Accepted April 3, 1978 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 047 Folder The folder containing the original item. 1188 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Bababunmi, Enitan A. Source A related resource from which the described resource is derived Journal of Toxicology and Environmental Health Date A point or period of time associated with an event in the lifecycle of the resource 1978 Title A name given to the resource Toxins and Carcinogens in the Environment: An Observation in the Tropics Subject The topic of the resource heath effects pesticide toxicology ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 037 Folder The folder containing the original item. 0800 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Kay, Kingsley Source A related resource from which the described resource is derived Environmental Research Date A point or period of time associated with an event in the lifecycle of the resource 1973 Title A name given to the resource Toxicology of Pesticides: Recent Advances Subject The topic of the resource pesticide toxicology ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 037 Folder The folder containing the original item. 0811 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Kimbrough, Renate D. Source A related resource from which the described resource is derived Archives of Environmental Health Date A point or period of time associated with an event in the lifecycle of the resource 1972-08-01 Title A name given to the resource Toxicity of Chlorinated Hydrocarbons and Related Compounds Subject The topic of the resource pesticide toxicology animal testing chloracne ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 037 Folder The folder containing the original item. 0810 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Kimbrough, Renate D. Source A related resource from which the described resource is derived CRC Critical Reviews in Toxicology Date A point or period of time associated with an event in the lifecycle of the resource 1974-01-01 Title A name given to the resource The Toxicity of Polychlorinated Polycyclic Compounds and Related Chemicals Subject The topic of the resource pesticide toxicology pesticide properties animal testing ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/a43524e853447ff25b9c6cdce1df9cc7.pdf eac9e207abbff5e41591d8b44990dc3b PDF Text Text 00172 Author CorporatG Author Kotchmar, George S,, Jr. Flame, Incendiary, and Explosives Division, Air Force Armament Laboratory, Eglin AFB, Florida Metabolism of High Concentrations of the Organophosphorus Insecticide Phorate Applied Foliariy to Selected Plant Species Journal/Book Title Year MOIltll/Day Color February n 54 Project No. 5066 Friday, January 05, 2001 Page 172 of 194 �AFATL-TR-71-22 THE METABOLISM OF HIGH CONCENTRATIONS OF THE O R G A N O P H O S P H O R U S INSECTICIDE PHORATE APPLIED FOLIARLY TO SELECTED PLANT SPECIES PYROTECHNICS BRANCH FLAME INCENDIARY, AND EXPLOSIVES DIVISION TECHNICAL REPORT AFATL-TR-71-22 F E B R U A R Y 1971 Approved for public release; distribution unlimited, AIR FORCE ARMAMENT LABORATORY AIR FOtCE SYSTIMS COMMAND • UNITED STATES AIM FORCE EGLIN AIR FORCE BASE, FLORIDA �The Metabolism of High Concentrations of the Organophosphorus Insecticide Phorate Applied Foliarly to Selected Plant Species George S. K o t c h m a r , Jr., Capt, U S A F Billy C. W o l v e r t o r t E l i z a b e t h I. Soothe Sandra M. L e f s t a d Approved for public release; distribution unlimited, �FOREWORD The active Air Force project directly related to the information discussed in this report is Exploratory Development Project 5066. Requests for further detailed information or any comments on this report may be referred to Air Force Armament Laboratory (DLI), Eglin Air Force Base, Florida 32542. Statistical analyses were performed by Booz-Allen Applied Research. The use of trade names is for identification purposes only and does not constitute endorsement by the United States Air Force. This report has been reviewed and is approved. r ^ ^ { t^ —~z—/- te FRANKIN C. p M E S w i o n e l , USAF KLI Chief, Flame, Incendiary and Explosives Division �ABSTRACT Gas chromatographic and enzymatic analyses (cholinesterase-inhibition SthSr«re used to monitor the metabolism of the organophosphorus insect dde 0,0-diethyl S-[(ethylthio)methyl] phosphorod thioate (Pforate) Ippfied fofiarly to three economically important plants (Homestead tomato, Siley sorg m" and Honey sorghum). The resulting data provided guide ines in predicting toxicity and persistence of metabo ite residues £r high ^binr^r^ei^i^ Ice d'on ass plates located adjacent to treated Plants, indicated the formation of toxic phorate metabolites was without the influence of biological substrates within the plants. There were no statisticaljy s gnificant differences with respect to the rate of increase of cholinesterl e- n ibition percentage values between the sorghum and glass plates, the rate of formation of anticholinesterase oxidized metabolites was predominantly through chemical oxidation on the leaf surface and not by K enzymecatalysis, or at least, the oxidation occurred at_such a ratfas tfmask the enzyme catalysis. The large droplet size in the application^ phorate resulted in higher toxic residue values, especially on the surface of the plant, than would normally be expected. Approved for public release; distribution unlimited. m (The reverse of this page is blank) ��TABLE OF CONTENTS Section I II Title Page INTRODUCTION 1 EXPERIMENTAL PROCEDURES 4 MATERIALS AND METHODS 4 STATISTICAL ANALYSES 9 III RESULTS AND DISCUSSION 12 IV SUMMARY AND CONCLUSIONS 40 REFERENCES 43 �LIST OF FIGURES Figure 1 Title Page Plant Metabolism Pathway of Phorate (Based on Reference 5) 2 Horse Serum Cholinesterase Inhibition Versus Concentration of 0,0-Diethyl S-[Ethylsulfinyl)methyl] Phosphorothiolate.. 8 3 Cholinesterase Inhibition of Homestead Tomato..... 13 4 Cholinesterase Inhibition of Wiley Sorghum 14 5 Cholinesterase Inhibition of Honev Sorqhum (Phorate Applied in May) 15 6 Comparison of Percent Cholinesterase Inhibition for Three Varieties of Plants. 17 7 Percent Cholinesterase Inhibition, Homestead Tomato 19 8 Effects of Passage of Time upon Percent Cholinesterase Inhibition, Homestead Tomato 20 2 9 Percent Cholinesterase Inhibition, Wiley Sorghum 10 Percent Cholinesterase Inhibition, Honey Sorghum.. 11 Percent Chlinesterase Inhibition, Wiley and Honey Sorghum.. 23 12 Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley Sorghum(March) and Honey Sorghum(April).. 24 13 Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) 26 Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) 27 14 21 22 15 Percent Cholinesterase Inhibition, Glass Plates 16 Comparison of Percentage Cholinesterase Inhibition for Glass Plates and Wiley Sorghum 33 Comparison of Percentage Cholinesterase Inhibition for Gl ass PI ates and Honey Sorghum 34 17 VI 32 �LIST OF TABLES Table I II III Title Page Plant Parameters Employed With High Concentrations of Phorate. 5 Efficiency of Extraction Technique for Phorate With Tomato and Sorghum 6 Measurement Days by Species and Experiment. 10 IV Gas Chromatographic Analysis for Phorate From Tomato and Sorghum. ,,. V Average Percent Cholinesterase Inhibition, Homestead Tomato 12 VI VII VIII IX Average Percent Cholinesterase Inhibition, Wiley Sorghum (March) and Honey Sorghum (April). Average Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May). Gas Chromatographic Analysis for Phorate from Glass Plates and Sorghum. " ?R ^ *' Persistence of Phorate Oxygen Analog Sulfoxide Equivalent in Sorghum. ™ vii (The reverse of this page is blank) ��SECTION I INTRODUCTION The research reported in this study constitutes part of a program to elucidate toxicological and ecological hazards associated with repetitive aerial applications and spills of organophosphorus insecticides. Toxicological hazards may exist in plant foliage even after environmental persistence studies determine the absence or safe level of the parent insecticide. These hazards result from the conversion of the parent insecticide to toxic oxidized metabolites. The rate of this conversion determines the nature and magnitude of the toxic residues in the plant tissues. The use of organophosphorus insecticides is increasing because of their wide spectrum of effectiveness (comparable to the chlorinated hydrocarbons) and their short residual action in water, soils, and plants. Any accident involving ultra-low-volume formulations of insecticides could result in a per-unit-area concentration that would be detrimental to agronomic plants because of unacceptable residue levels. For this reason, data relating the tolerance of agronomic plants to repetitive aerial applications of ultra-low-volume formulations of organophosphorus insecticides are of interest to military pest control programs. Guidelines were desired for predicting toxicity and persistence of metabolite residues in plants after application of high concentrations of the insecticide. Insecticides in or under considerations for the Air Force inventory include malathion (dithiophosphoric derivative), naled (phosphoric acid derivative), fenthion (thiophosphoric acid derivative with sulfide linkage), and Dursban^(thiophosphoric acid derivative). Phorate, 0,0-diethyl S-[(ethylthio) methyl] phosphorodithioate, was the insecticide selected because it is a model compound containing oxidation sites analogous to those found in sulfur-containing organophosphorus insecticides. Its toxicity, expressed as an oral 105^ value, is more tnan 100 times as great as the most toxic insecticide presently used by the militaryO). Thus, persistence data on toxic metabloties of phorate should provide a model system of maximum toxic residues on foliage wnich should be unapproachable for insecticides presently used by the military wnen applied at the same concentration. Furthermore, previous studies^' ^» "> ^J have elucidated the plant metabolism pathway (Figure 1) of the insecticide along with solvent-partitioning functions of phorate and its metabolites(2, 4). Conclusive research data are not available concerning insecticide pnytotoxicity; however, it is known tnat plant species exhibit a wide range of tolerance to applications of organophosphorus insecticides ^»'t. 1 �(CH3CH20)PSCH2SCH2CH3 S 0 (CH3CH20)2PSCH2SCH2CH3 0 ^^ II I *r (CH3CH20)2PSCH2SCH2CH3 N. 0 0 X 11 * (CH3CH20)2PSCH2SCH2CH3 (CH3CH20)2PSCH2SCH2CH3 Figure 1. Plant Metabolism Pathway of Phorate (Based on Reference 5} The basis for this phytotoxic resistance or susceptibility is not clear. The experimental procedure used in this study allowed an investigation into the role performed by individual plant chemistries in metabolizing organophosphorus insecticides. Studies of the morphological effects caused by highly concentrated foliar applications of mevinphos and methyl demeton on selected plant species indicated that, in general, brpadleaf plants were more susceptible to the insecticides than were grasses^ 7 /. Severe morphological injuries were observed on soybean, cotton, and tomato plants one day after foliar treatment, w h i l e seven days were required before comparable injuries were noted on corn and sorghum. Coleman and Dean^°' found that resistance of sorghum to methyl parathion was genetically controlled in their studies with a resistant and a susceptible variety, Wiley and Honey, respectively. Thus, differential phytotoxicity to organophosphorus insecticides can be expected between plant species as well as w i t h i n a given species. Differences do occur in the rates of metabolism of insecticides in different plant species. A study^) of the plant metabolism of Di-Syston® (dithioSystoxdD) and phorate indicated that the rates of a reaction may vary slightly from one plant species to another and according to the stage of growth, but the data obtained may be used as a guide to the relative proportions of the metabolites present at intervals after application. In �another study w y the effects of temperature and plant species upon the rates of metabolism of systemically applied Di-Syston®were considered, and s i m i l a r results were obtained. Phorate differs from Di-Syston® due to the presence of an ethylene rather than a methylene group in its side chain. Thus, this study attempted to relate the metabolism of the insecticide to phytotoxic damage among plant species and within a plant species. Oxidation can increase both the water solubility and the anticholinesterase activity of organophosphorus insecticides^"'. The logical approach for monitoring anticholinesterase compounds (toxic phosphorus esters) formed during the metabolism of phorate was a cholinesterasei n h i b i t i o n method. Phorate alone is too weak an inhibitor to be detected in micro amounts, but when applied to plants, it is very rapidly converted to potent anticholinesterase agents. The final unhydrolyzed metabolite (phorate oxygen analog sulfone) in the oxidation series (Figure 1) is the most active i n h i b i t o r ' ^ ' . The 150 value (molarity of i n h i b i t o r which results in 50 percent of the activity of the control) of phorate is approximately 250 times that of the phorate oxygen analog s u l f o n e ' ^ ) . One day after the application of phorate to corn, the residue of phorate sulfoxide, which has an 159 value approximately 1/100 that of phorate, was more than three times that of phorate^. �SECTION II EXPERIMENTAL PROCEDURES 1. MATERIALS AND METHODS a. Apparatus. A Son/all Omni-mixer^ was used for macerating plants. A gas chromotograph equipped with a flame photometric phosphorus detector and a digital integrator was employed in the phorate analyses. A recording pH stat was used to determine cholinesterase activity. b. Reagents and Solvents. Standards, supplied by the American Cyanamid Company, were technical grade phorate (Thimet® ) of 90-percent purity, analytical grade phorate of 97.8-percent purity, and 94-percent phorate oxygen analog sulfoxide containing 6-percent phorated oxygen analog sulfone. Reagents were anhydrous sodium sulfate, certified A . C . S . ; acetylcholine perchlorate (a "rare and fine" chemical from K and K Laboratories, Inc.); sterile filtered horse serum (Colorado Serum Co.); sodium chloride (crystals), analytical reagent grade; sodium chloride (granular), U.S.P. grade; sodium hydroxide pellets, analytical reagent grade; and potassium hydrogen phthalate, certified A.C.S. acidimetric standard. Solvents were certified A.C.S, acetone, certified A.C.S. hexanes, and vegetable oil. c. Plant Parameters. The representative broadleaf plant selected was Lycopersicon esculenturn mill, var. Homestead 24 (tomato), and the grasses were Sorghum vulgare Pers. var. Wiley (sorghum), and Sorghum vulgare Pers. var. Honey (sorghum). The two varieties of sorghum were selected to represent a variety (Wiley) that was resistant to an organophosphorus insecticide and one (Honey) that was susceptible. The plants were grown in a clear glass greenhouse with a minimum night temperature of 60° to 65°F and a maximum day temperature of 95° to 100°F. Seeds were planted in a soil consisting of a 7:3:1 mixture of sandy loam, peatmoss, and perlite with four pounds of dolomitic limestone and one pound of superphosphate added per cubic yard of soil. The pH of the soil was 6.5. Each tomato plant was transplanted to an individual four-inch plastic pot at the age of four weeks. The sorghum experimental unit consisted of 10 plants per four-inch plastic pot. A 15-15-15 liquid fertilizer was applied bi-weekly. A 2 percent or 1 percent solution of phorate (0.2 milliliter or 0.1 milliliter of technical grade phorate dissolved in 10 mi Hi liters of vegetable oil medium) was applied foliarly as 0.01 milliliter droplets with microsyringe to the tomato and sorghum at the concentrations shown in Table I. This procedure allowed a uniform and exact application to all �TABLE I. PLANT PARAMETERS EMPLOYED WITH HIGH CONCENTRATIONS OF PHORATE Species Age, Weeks Homestead Tomato Date of Application 8 August1969 19 November 1969 Phorate, Concentration, pprrr lb/Ab 19,839 16,630 2.19 1.84 Wiley Sorghum 4 3 2 March 1970 20 May 1970 8,753 (c) 1.26 (c) Honey Sorghum 4 3 8 April 1970 20 May 1970 8,461 (d) 1.22 (d) Concentration of phorate solution based on gas chromatographic analysis with analytical grade phorate. "Concentration applied to plant based on leaf area (pounds of active ingredient per acre). c Same concentration applied as 2 March 1970, but no gas chromatographic analysis. "Same concentration applied as 8 April 1970, but no gas chromatographic analysis. the plants. The levels of phorate applied were adjusted to the maximum amount that would result in minimal visible damage. To insure similarity in plant size at each application, the phorate was applied after three to six weeks of initial plant growth. The varying lengths of time between planting and insecticide application were to compensate for seasonal variation in plant growth. Table I shows the age of the plants with the date of application of phorate. During the March (Wiley sorghum) and April (Honey sorghum) portions of the experiment, the phorate in the vegetable oil medium was applied to glass plates located adjacent to the treated and control plants. d. Extraction Technique. The plants to be sampled were severed at soil level, sectioned, and rinsed in a 400-milliliter beaker containing 25 mi 11 iliters of hexanes and 25 mi Hi liters of acetone in distilled water (60 percent by volume) to remove any residues remaining on the plant surface. The solvent mixture, followed with the plant material, was poured into a cup for the Sorvall Omni-mixer®. The beaker was rinsed with 5 mi 11iliters of hexanes and then by 5 mi 11iliters of acetone in distilled water (60 percent by volume). After the plant material was thoroughly macerated, the macerate was filtered through three layers of 5 �cheesecloth into a separatory funnel. The cup which had contained the macerate was rinsed with 10 milliliters of hexanes and then by 10 millilitars of acetone in distilled water (60 percent by volume). The rinsings were added to the separatory funnel followed by 15 milliliters of a saturated sodium chloride solution which aided in separating the organic and aqueous phases. Acetone was removed from the aqueous layer by use of a rotary evaporator attached to a water aspirator. Complete removal of the acetone was essential because of its ability to inhibit cholinesterase. The aqueous layer was filtered (to remove minute pieces of plant material) through Whatman No. 2 paper into a 50-mi Hi liter volumetric flask and brought to volume with distilled water. The organic phase was placed over 10 grams of anhydrous sodium sulfate, filtered into a 50-mi11iliter volumetric flask, and brought to volume with hexanes. If the samples could not be analyzed immediately, they were stored at 5°C. The aqueous layer was analyzed for cholinesterase-inhibiting metabolites and breakdown products. Gas chromatographic analysis of the organic phase for phorate allowed monitoring of the rapidity of breakdown and oxidation. The efficiency of the extraction technique, based on the recovery of phorate in the organic phase, is shown in Table II. TABLE II. EFFICIENCY OF EXTRACTION TECHNIQUE FOR PHORATE WITH TOMATO AND SORGHUM Species Date of Experiment Homestead Tomato August November Wiley Sorghum March Honey Sorghum Apri 1 Efficiency of Extraction, Percent Controls3 Insecticide-Treated Plants^ 3 3 66.2 87.7 32.4 63.2 63.3) [ Qla ss 75.9) pla tesc 46.7 51 .7 ^Concentration of phorate applied to plant placed through extraction scheme. b Phorate-treated plants from day 0 placed through extraction scheme. c Glass surface residues from day 0 placed through extraction scheme. e. Cholinesterase-Inhibition Method. The advantages of using cholinesterase-inhibition methods for determining organophosphorus residues are that (a) the sensitivity is far greater than for chemical methods and (b) the method is particularly suitable when the insecticide undergoes 6 �changes in the plant to produce metabolites with a high inhibitory activity(10,11}_ One of the disadvantages is the lack of specificity or inability to distinguish among different types of cholinesterase inhibitors found within the plant. The persistency of toxic metabolites and breakdown products in the respective plant species was monitored using an automated pH stat method(12) to determine cholinesterase activity. The cholinesterase inhibition for each species and glass plate sample was measured immediately after the application of the insecticide and on various succeeding days for one month. Control plants were treated with only the corresponding amounts of vegetable oil and were similarly measured. The experiment was repeated for each species. The cholinesterase activity of the prepared water sample was recorded with a recording pH stat. A sample (0.2 milliliter tomato or 0.4 milliliter sorghum sample) was placed in a microbeaker containing 5 milliliters of a 0.154-molar saline solution (9.000 grams of analytical reagent grade sodium chloride in 1000 mi Hi liters distilled water) and 0.5 milliliter horse serum. The microbeaker solution was heated to 37.5°C and adjusted to pH 8.0 prior to addition of 0.3 milliliter of the cholinesterase enzyme substrate, 0.110-molar acetylcholine perch!orate (0.675 grams crystalline substrate in 10 milliliters distilled water). The titrant, 0.0100N sodium hydroxide.was standardized with 0.0100N potassium acid phthalate (0.20423 grams acid in 1000 milliliters distilled water). Normal-activity curves (no samples added) and control-activity curves (aqueous samples from control plants) were obtained prior to measuring cholinesterase activity for the aqueous samples from phoratetreated plants. Cholinesterase inhibition of the phorate metabolites and breakdown products was expressed as a percentage value obtained from a ratio of cholinesterase activity (expressed in units of micromoles of acetylcholine hydrolyzed per minute per milliliter of horse serum) of the samples from the phorate-treated plants to samples from the control plants. A second percentage of cholinesterase inhibition was calculated using the normal activity value as the base. f. Calibration Curve. To equate the percentage of cholinesterase inhibition to the concentration of the metabolite extract, a log-linear plot was made of the phorate oxygen analog sulfoxide equivalent, in parts per million (ppm) of 0,0-diethyl S-[(ethylsulfinyl) methyl] phosphorothiolate, versus the percentage of cholinesterase inhibition. Figure 2 contains the calibration curve based on 0.4 milliliter samples of the standard parts-per-million solutions of the phorate oxygen analog sulfoxide. Sample sizes of 0.2 milliliter gave cholinesterase inhibition percentages that were approximately half the values shown in Figure 2. �109 a 76 5 4 uu Ci I—I X o u. 2- co .9- uu 1 !?• .6.5.4- .3.2- JO- 20 Figure 2. 30 50 60 HORSE SERUM CHOIIMESTERASE INHIBITION (PERCENT) Horse Serum Cholinesterase Inhibition Versus Concentration of 0,0»Diethyl S-[(Ethylsulfinyl)methyl] Phosphorothiolate �g. Gas Chromatographic Analysis. Gas chromatography was used to detect unaltered phorate in the organic phase from the plant extracts. A gas chromatograph equipped with a flame photometric phosphorus detector was employed. A six foot by one-fourth inch stainless steel column containing Chromport X X X , 80/90 mesh, coated with 3 percent SE-30, and conditioned for 24 hours at 220°C, was used for the phorate analysis of the August tomato extracts. A retention time of 105 seconds was recorded with the inlet temperature set at 250°C, the column-oven temperature at 200°C, and the detector temperature at 235°C. Gas flow rates in cubic centimeters per minute were nitrogen 80, hydrogen 150, air 20, and oxygen 20. A six foot by one-fourth inch glass column containing Chromosorb id , 80/90 mesh, coated with 3 percent QV-1 and conditioned for 24 hours at 210°C was used for the phorate analysis of the November tomato extracts, March Wiley sorghum extracts, April Honey sorghum extracts, and glass plates. A retention time of 130 seconds was recorded with the inlet temperature set at 245°C, the column-oven temperature at 190°C, and the detector temperature at 185°C. Injection sample sizes 1 for August tomato extracts were 5 microliters, and all others were 2.7 microliters. Concentrations of phorate present in the August analysis were determined by integration of peak area by a digital integrator. Concentrations of all other analyses were determined by peak height. All determinations were expressed in parts per million based on standard curves. 2. STATISTICAL ANALYSES. Before any of the data were analyzed, all of the cholinesterase inhibition percentages were transformed using the arc sine formula: 8 = 2 arcsin P where 6 is the new variable to be analyzed and P is the percent of cholinesterase inhibition divided by 100. Tnis transformation minimized and stabilized the variation of the data and created the homogeneity of variance that was essential to the use of the analysis of variance technique, and the other statistical procedures used for the analysis of these experiments. The results were stated in percentage notations for presentation throughout the report. It was not possiole to analyze all of the cholinesterase inhibition percentages on an experiment-wide basis because the measurements of the three experimental species were taken on various and differing days �during the month following application of the insecticide. To make comparisons that were valid and meaningful, it was necessary to select those days on which all species under consideration were measured. Table III shows the days of measurement for each species. During the first week TABLE III. HEASUREMENT DAYS BY SPECIES AND EXPERIMENT Days After Application 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 Homestead Tomato August November X X X X X X Wiley Sorghum March May Honey Sorghum April May X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X following application of the insecticide, only those measurements that were made on the same day were compared; during the next three weeks, measurements that were made on contiguous days were also included in the comparison. 10 �Only one measurement of cholinesterase inhibition was made on each of the days for the Homestead tomato experiments and for the March and April Wiley and Honey sorghum experiments. It was not possible, therefore, to test for significance of the interaction between seasonal effects and the number of days following application. Since this interaction would have been used to test the seasonal and time-passage effects individually, an unduly large interaction would have masked significance of the main effects, Paired t-tests with the same or contiguous days' results were used to circumvent this possibility. When nonsignificance of the paired values was determined, an analysis-of-variance technique was used to test for significance of the passage of time upon the cholinesterase-inhibition percentage. In those cases in which these results were significant, Duncan's new multiple range test was used to identify where the differences did, in fact, exist. In the absence of significance, means were computed, and the effects of the passage of time upon the cholinesterase inhibition were studied. Two replicates of measurements were made for the May Wiley and Honey sorghum plants, and it was, therefore, possible to use the analysis of variance technique when only these data were involved in comparisons. Initially, all testing was conducted at the 95-percent probability level (significant). When this proved significant, subsequent testing was conducted at the 99-percent probability level (highly significant). 11 �SECTION III RESULTS AND DISCUSSION Ultra-low-volume formulations of insecticides used by the military involve the aerial application of a low-volume concentrate (0.75 to 10 ounces per acre, undiluted). Phorate is applied with normal formulations at rates of 0.5 to 3 pounds of active ingredient per acre. Table I presents the application rates within this range. However, the defined, directed application of phorate to the leaf surface without spraying resulted in a maximum interface between insecticide droplet (10 microliters) and leaf area. This large droplet size represented the application of high concentrations of phorate; the droplet contained more phorate (0.25 milligram) and is larger than that ordinarily found following routine application of insecticides. The optimum3 diameter for insecticide spray droplets is in the range of 20 microns'' '. The percentages representing the efficiency of the extraction technique employed (Table II) are minimum values because of the volatility of phorate. Phorate is lost from the soil by volatilization and about 25 percent of the loss occurs in the first hour after treatment^'^/. Similar results would be expected on the leaf surface, therefore, the length of time between treatment and initial extraction of the plant would result in a value of phorate present that is slightly less than that which was applied. The time before initial extraction was approximately the same in all cases; however, the extraction of the August tomato after application of phorate during the late morning heat probably accounts to some degree for the lower value in extraction efficiency. Phorate was applied to the other plants in the early morning. A comparison of the percentage of cholinesterase inhibition obtained using the normal-activity value and the percentage obtained using the control-activity value as a base is shown in Figures 3 and 4 for Homestead tomatoes and for Wiley sorghum for each of the two applications of insecticide. A comparison for Honey sorghum is shown in Figure 5 for the May application; however, the April control plants were not usable due to contamination. The results of paired t-tests in analyzing the differences between percentages for each species allowed the use of all the cholinesterase-inhibition percentages based on the normal activity value. Cholinesterase-inhibition values obtained showed no correlation with plant weight. A comparison of the percentages of cholinesterase inhibition during the month following application of the insecticide for each of the three plants, using the analysis of variance technique, indicated that the average for the Homestead tomato was significantly lower than those for 12 �/D fe CJ 50 - uj ex. O t~~t K h~, — UJ -J < nr .? j ( fc to o t~t GO 2:. uj t ^ —c > ---* rf t,^ »/ '• .• " « • ' lc b UJ CO '-=C CJ fti K UJ •< r™"- ••—— • 0 U o^ UJ -5 • CONTROL 25 § O NORMAL 1 I I § o 1 0 I 1 1 1 1 1 1 2 4 6 S 10 12 14 U U 20 22 1 1 24 26 21 TIME FROM APPLICATION WS) a. _ Phorate Applied in August 75 iUj | — 50 C±- 8 r^ 0 ft S^ A«s UJ UJ S oo t-i S^ uu o 0 8 • >-~< UJ m ° 8 8 0 8 — h- Oi c/) -< UJ •—• 3: o j. -25 • CONTROL 1 0. I I I I 1 1 1 2 4 6 6 10 12 14 U O NORMAL I 1 1 18 20 22 1 I 24 26 TIME FROM APPLICATION [VMS] b. Phorate Applied _in November Figure 3. Cholinesterase Inhibition of Homestead Tomato 13 2B �/p s- § • A UJ CJ c* 50 UJ O UJ »—1 —1 - » § • 8 » o 8 25 e CO V) ^— 1 1 1 ?5 t—< CO UJ 5ur§ 5t CO CJ tf <c UJ — n U to UJ t—1 —1 • CONTROL -5 — O NORMAL o CJ 1 1 1 1 1 1 f I 6 -25 i 1 1 1 1 « /O 12 14 16 H 20 TIME FROM APPLICATION (PAKS) 22 24 26 28 1 26 21 a. Phorate Applied in March /5 * 8i | 0- O UJ t—t —J \~ •< 50 8 • 8 * ' o • 881$ 8 » 8 UJ o e* UJ 25 t-i CJ CO CO b-< 1—t UJ W "<t UJ •— 1- UJ t—< —1 o 5 — '.r • CONTROL -5( 3: o O NORMAL i ^c 0 i 2 4 i 6 1 B 1 10 1 12 1 14 1 16 1 U 1 20 1 22 1 24 TIME FROM APPLICATION b. Phorate Applied in May Figure 4. Cholinesterase Inhibition of Wiley Sorghum 14 �72 !s UJ 75 ^* o CM UJ (X "~" 50 — 25 i ii o S 8 * • ^k ^k ^ ^fc. - 8 O t—1 — PQ •< 8 8 •t ^2 "» 8 ^h t—t CJ (-1 UJ UJ *-< C/) C/) 5 - So UJ CM UJ t—t —4 O -C o 0 _ 1 * CONTROL O WORMAL I 1 1 i I t I i I i t i I g 10 12 J4 76 JS 20 22 24 26 28 TIME FROM APPLICATIOW (PA^S) Figure 5. Cholinesterase Inhibition of Honey Sorghum (Phorate Applied in May) 15 �the two varieties of sorghum. Figure 6 shows the inhibition percentage values for each of the three plants; the lower level for the tomato plants is evident. Therefore, for statistical purposes, data from the tomato plant experiments were analyzed independent of the sorghum data. Gas chromatographic analysis of the hexanes phase after extraction from tomato and sorghum indicated less than one ppm phorate present by the sixth day (Table IV). A determination of residues of-phorate and five of its netab- TABLE IV. GAS CHROMATOGRAPHIC ANALYSIS FOR PHORATE FROM TOMATO AND SORGHUM Day Tomato August November Concentration of Phorate, ppm Sorq lum March (Wiley) April (Honey) 0 128 42 16 18 1 140 34 9-12 9 28 6-7 5-6 9 <3 1 2 3 23 4 <1 5 4 6 1 <1 elites from various parts of corn plants (treated with one pound of the.insecticide per acre) indicated that phorate was essentially gone in 14 days(4). During this determination, phorate was recovered from fortified samples with 96 percent efficiency using a Soxhlet apparatus in an eight-hour extraction technique. The higher values of phorate in the August tomato samples, as compared to the November samples, were due to the concentration of the sample to 10 milliliters instead of the 50-milliliter final volume of all other samples. Small differences in the data are probably due to the slight variations in extraction efficiencies for each plant. The disappearance of phorate proceeds at approximately the same rate in each plant variety. 16 �75 A D A A 9 50 — O 9 • o A D nc '--' O g «-< o _ =5 °° 0 • * A | > • UjS C/) CO UJ CM ^ A H * • o 00 — D A UJ (X *vl S UJ Q A UJ PuJ t—< —J CO •< n A 1 P * (* ^9 r 5 0 o o —1 o o 0 - HOMESTEAD TOMATO , 0 1 2 i 1 4 i | 6 i 1 8 O.VO I/EMBER WILE/ SORGHUM I3 •AUGUST * MARCH ^ MA/ HO/JE/ SORGHUM i 1 i 1 i 10 12 • APRIL 1 i 1 14 16 D MA/ t 1 20 i 1 IS i TIME FROM APPLICATIO/V (3A/S) Figure 6. Comparison of Percent Cholinesterase Inhibition for Three Varieties of Plants 1 22 i | 24 I 2< �The results of a cholinesterase-inhibition analysis for the Homestead tomato plants for all of the days measured for the August and November applications of insecticide are shown in Figure 7. An analysis of comparable daily measurements in August and November indicated no significant differences between the two application dates or by the number of days after application. The average percentages of cholinesterase inhibition for the days used in the comparison are given in Table V and Figure 8 and are not significantly different at the 95 percent probability level. TABLE V. AVERAGE PERCENT CHOLINESTERASE INHIBITION, HOMESTEAD TOMATO Days After Application Cholinesterase Inhibition, Percent 21,22 27.1 6 24.4 12,13 23.9 4 21.4 9,10 15.9 1 12.4 0 11.3 Figures 9 and 10 show the percentages of cholinesterase inhibition for Wiley and Honey sorghum, respectively. Analyses of the possible daily comparisons for each variety indicated significant differences between the March and May data for the Wiley sorghum and highly significant differences between the April and May data for the Honey sorghum. Similar analyses of those cholinesterase-inhibition percentages which were obtained on the same or contiguous days for Wiley sorghum in March and Honey sorghum in April indicated no significant differences between the two varieties for those two months (Figure 11). An analysis of the differences in cholinesterase-inhibition percentages with respect to the number of days after application of phorate to sorghum during March and April indicated highly significant differences. Table VI shows average daily percentages. In Figure 12, the average values with associated letters are shown graphically. The cholinesterase-inhibition percentage peak was reached by the ninth day following application of phorate, and an 18 �14J C_> {* U-i o « I4J CO O *-» CO U3 2 UU UJ •< CO UJ o o AUGUST APPLICATIOM WOI/&MBER APPLICATION 70 72 74 16 18 TIME FROM APPLICATION (VAVS) Figure 7. Percent Cholinesterase Inhibition, Homestead Tomato O �75 UJ UJ PL. \~ UJ CO t— t\3 25 UJ O )—< UJ CO •<C C_> UJ •< UJ a: «—< —i o » -5 10 n TIME FROM APPLICATION Figure 8. H 14 (VMS) 20 Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Homestead Tomato 24 26 �uj UJ h- UJ t~> —J CQ • < t~< O UJ V) uj »oo O UJ MARCH APPLICATION • APPLICATIOW JO 72 H 16 TIME FROM APPLICATION (VAVS] Figure 9, Percent Cholinesterase Inhibition, Wiley Sorqhum O �fe UJ o Be: UJ R- CQ ™ UJ2 APRIL APPLICATIOW MAV APPLICATION Figure 10. O TIME FROM APPLICATION {DAYS} Percent Cholinesterase I n h i b i t i o n , Honey Sorghum �95 WILEV SORGHUM MARCH APPLICATION HOWE/ SORGHUM APRIL APPLICATION O -25 10 72 14 U TIME FROM APPLICATION (PA/S) Figure 11. Percent Cholinesterase Inhibition, Wiley and Honey Sorghum �Cxi UJ R- 1- UJ 1-1 •—I CO •< l-« CJ K CJ UJ C* V- < TIME FROM APPLICATION Figure 12. Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley Sorghum (March) and Honey Sorghum (April) �average peak level of 63.9 percent Inhibition was maintained until the eighteenth to twenty-first day, when it began decreasing. By the twentythird to twenty-fifth day it had decreased to 31.5 percent. TABLE VI. Days After Application AVERAGE PERCENT CHOLINESTERASE INHIBITION, WILEY SORGHUM (MARCH) AND HONEY SORGHUM (APRIL) Cholinesterase Inhibition , Percent Remarks (Common letter indicates no significant difference at 99-percent Probability Level) 11 67.9 a 16,18 62.5 ab 9 61.1 ab 14 51.0 be 4 49.1 be 7 49.0 be 21,20 42.1 c 25,23 31.5 d 2 18.5 e 0 10.2 e 1 9.8 e Figure 13 shows the percentages of cholinesterase inhibition for Wiley and Honey sorghum on various days during May. The analysis indicated highly significant differences only with respect to the number of days after application; neither the species of sorghum nor the species/day interaction yielded results which indicated any significant effect. The average percentages for the various days after application are shown in Table VII. All averages that are not significantly different at the 99 percent probability level have a common letter. In Figure 14, the average values with associated letters are shown graphically. May Wiley and Honey sorghum plants reached a peak percentage 25 �o H- Uj CQ < 5: <s> 2: t-t UU li? Lu a: Ol l-t UU o WILE/ SORGHUM O WNEV SORGHUM n u TIME FROM APPLICATION Figure 13. Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) �UJ (X o l— ul t-i ~j B3 • < »—* O r\> i60 >-' ty uj Si UU Oi h- •< o o -5 1 I I 1 I 1 1 1 1 1 1 1 n 1 1 1 1 n 1 1 1 i 20 1 1 1 1 24 TIME FROM APPLICATION (PAW) Figure 14. Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) 1 1 26 1 �of cholinesterase inhibition by the fourth day and maintained an average peak level of 64.5 percent for the remainder of the month. TABLE VII. AVERAGE PERCENT CHOLINESTERASE INHIBITION, WILEY AND HONEY SORGHUM (MAY) Days After Application Cholinesterase Inhibition, Percent Remarks (Common letter indicates no significant difference at 99-percent Probability Level ) 14 68.8 a 12 68.7 a 8 65.2 a 7 65.1 a 19 64.5 a 4 64.0 a 5 63.1 a 16 60.6 a 21 60.4 a 2 28.9 b 0 -1.5 c With no significant differences existing between the two varieties of sorghum in May or during continuous experiments in March and April, it appeared that the metabolism of high concentrations of phorate proceeded at the same rate in each variety; however, distinct visible differences occurred between the two plant varieties. Despite slight initial visible damage, both sorghum and tomato recovered from insecticide damage within 30 days of treatment. The preliminary data did show, however, that neither variety of sorghum recovered from insecticide damage when exposed to the same concentration of phorate as was used on the tomato plants. This is indicative of the 28 �effects of a high concentration of phorate due to the large droplet size, since any visible damage at the rates used in this experiment would not be expected. With the tomato, minor c u r l i n g of the leaves was observed and many plants had a loss of apical dominance indicating that possibly, at the concentrations of phorate used, there was a change in the auxin content or hormonal distribution w i t h i n the plant. The first injury symptoms for both sorghum varieties were characterized by a localized bleaching of the blade pigments ( i . e . , chlorophyll) to a yellow-green coloration with a s l i g h t l y flaccid condition. These necrotic blotches were more distinct with Honey sorghum as they acquired a red-brown coloration. This characteristic color difference was apparent in the aqueous samples, as Honey samples were much darker than those of Wiley. The flaccid condition was more evident with Honey sorghum. The more v i s i b l e damage to sorghum compared to tomato in preliminary experiments, with both exposed to the same concentration of phorate, may appear to contradict the research reported in Reference 7. However, that report notes that (a) conclusions regarding the possible effects of organophosphorus insecticides, except mevinphos and methyl demeton, could not be made since the experiment was a fixed-effects model and not a random selection of possible organophosphorus insecticides; and (b) differences in plant response (susceptibility or resistance) could be accounted for by a postulation that differences may be related to leaf area interception of the insecticide. The reason for existing differences between March/April and May sorghum can only be postulated. Peak percentages of cholinesterase i n h i bition by the fourth day (64.5 percent) in May versus the n i n t h day (63.9 percent) in March/April indicate a more rapid oxidation of phorate to anticholinesterase metabolites. A s i g n i f i c a n t factor may be the relatively higher mean temperatures in May. In an evaluation of the effects of environmental temperature on Di-Syston©systemically applied to cotton leaves, the oxidation of the s u l f i d e , Di-Syston®, to the sulfoxide occurred so rapidly at temperatures above 70°F that only traces could be detected, even at intervals as short as one hour after treatment'**) The major^component in the leaves during the one-week experiment was the Di-Syston®sulfoxide. The rate of disappearance of sulfoxide was increased approximately 1.86 times for each 10°C rise in temperature (energy of activation of 10 kcal/mole). The i n i t i a l oxidation of phorate in cotton leaves was less rapid than the oxidation of Di-Syston®with traces of phorate found up to three days, although these never.exceeded 5 percent of the total radioactivity in the labeled experiment'^). It was also noted^ that the rate of oxidation of the sulfoxides in the oxidation series (Figure 1) was measurably slower for phorate than for Di-Syston® The sum of the rate constants for the disappearance of phorate sulfoxide due to oxidation is h a l f that of Di-Syston®sulfoxide. This indicates that phorate sulfoxide was probably the major metabolite during the first two weeks of this investigation. Also, when a l f a l f a seed was treated with 29 �treated with phorate or Di-Systorr% the effectiveness for aphid control varied by as much as several weeks depending on the rate of plant grow The rate of metabolism is slower in cooler weather, and the slower the plant growth, the longer the persistence of toxic residues. Tomato behaved similarly to the May sorghum. Tomato maintained a mean percentage value of 19.5 throughout the experiment; sorghum maintained a constant mean value throughout the month after, the third day. Thus, the results indicated that the metabolism of high concentrations of phorate proceeded at approximately the same rate in each species and between plant varieties. These results were not in complete harmony with those from earlier experiments. In previous metabolism studies of phorate and Di-Syston®on various plants such as cotton, alfalfa, lemon, and bean, it was found that the rates of reaction may be expected to vary slightly among pjant species and according to the stage of growth^). Later,, studies\°' specifically oriented toward the metabolism of Di-Syston^in a * variety of plant species, indicated that at 70°F, the metabolism of Di-Syston sulfoxide and hydrolytic decomposition of the toxic products occurred two to three times faster in tomato leaves than in cotton leaves. Differences in results and insecticide application parameters indicated the experimental data resulted from the chemical nature of phorate on the plant surface without the influence of biological substrates. Application methods^ 5 > included topical application of 5 to 25 microliters of insecticide to the base of a young plant or placement of isolated leaves in a water dispersion (0.1 percent solution) of the insecticide to permit the study of the rates of metabolism uncomplicated by the continual accumulation of translocated material. The method of application used in this work was foliar with a definite quantity of phorate aoplied as larae droplets to each intact plant. An earlier study' '^/ with Systox^(similar to phorate in structure) showed that the chemical nature of the surface washes from fruit treated with thiono- and thiolo-isomers changed rapidly upon exposure to light and air. Exposure of the isomers to light and air under controlled conditions on glass plates, uncomplicated by biological substrates, resulted in a surprisingly rapid conversion of the Systox®isomers into compounds which appeared to be chromatographically similar to those found within the plant tissues. Another study'''' showed that the action of air and sunlight on surface residues of Systox®isomers has a rapid effect and appears to promote their oxidation in the same sequence as found in vitro with hydrogen peroxide and in plant tissues. Thin films of phorate exposed to ultraviolet light or sunlight and air gave similar results^» ^8, 19, 20) ^ Exposure to sunlight on paper, glass, and leaf surfaces indicated that the initial stable residues of phorate may not be the original compound or its simple oxidation products; prolonged exposure resulted in the formation of more polar compounds^' 0 '. Results of ultra- 30 �violet irradiation of phorate on the surface of a liquid suggested that the oxidation products are the sulfoxide and sulfone of the parent compound, with the sulfone showing greater persistency and the s p]foxide being in greater quantity during the early stages of irradiation''^» ^0). To substantiate the concept that the experimental results in these investigations were without the influence of biological substrates within the plants, the same concentrations of phorate were applied to glass plates as were applied to the treated plants. The glass plates were located on the greenhouse bench adjacent to the control and treated plants. This was done with Wiley and Honey sorghum, in March and April, respectively, Table VIII shows a comparison of the gas chromatographic data for the plants and glass plates. Within 48 hours, phorate could no longer be detected on the glass plates; the same rapid disappearance was noted with the plants—approximately one ppm detected after 96 hours. TABLE VIII. GAS CHROMATOGRAPHIC ANALYSIS FOR PHORATE FROM GLASS PLATES AND SORGHUM Day Phorate Concentration, ppm March Aoril Wiley Sorghum Glass Plates Honey Sorghum Glass Plates 0 16 22 18 26 1 9-12 5-6 9 5 2 6-7 5-6 4 <3 1 <1 6 The cholinesterase-inhibition percentages obtained from the glass plates for the March and April experiments (Figure 15) were compared with the values obtained for the treated plants (Figure 11). The plots are very similar, with the glass plate cholinesterase-inhibition values being significantly higher on all days considered. However, most noteworthy is that the data through the twentieth day following application exhibited logarithmically linear trends (99 percent probability level) with no quadratic tendencies for both the sorghum and glass plates in March and April. The best-fitting straight lines have been plotted (Figures 16 and 17), and the equations for each of the lines are: 31 �99. Sr hUJ O R3 •< UJ (A) ro CO UJ CJ I UJ o CJ 25 O * Figure 15. n APRIL APPLICATION u TIME FROM APPLICATION (PAKS) Percent Cholinesterase Inhibition. Glass Plates �99.5 UJ CJ c*: o •— l~> UJ co to t"-t 3= <Z 50 *—» UJ CJ SORGHUM, MARCH APPLICATION O GLASS PLATES, MARCH APPLICATION 2 7 TIME FROM APPLICATION (PAVS) (LOGARITHMIC SCALE) Figure 16. Comparison of Percentage Cholinesterase Inhibition for Glass Plates and Wiley Sorghum I 24 29 �99.5 UJ UJ UJ _J t—1 I— •< uj II UJ kV) UJ o • HONEV SORGHUM, APRIL APPLICATION OGLASS PLATES, APRIL APPLICATION I I I I 4 7 3 TIME FROM APPLICATION (VMS) (LOGARITHMIC SCALE) Figure 17. Comparison of Percentage Cholinesterase Inhibition for Glass Plates and Honey Sorghum 79 24 29 �Wiley sorghum, March Y = 0.597 + 0.484 log (X + 1) Glass plate, March Y = 0.922 + 0.626 log (X + 1) Honey sorghum, April Y = 0.489 + 0.480 log (X + 1) Glass plate, April Y = 0.917 + 0.621 log (X + 1) where Y is the arc sine transformation of the percent cholinesterase inhibition and X is the number of days after the application of phorate in vegetable oil. An analysis of comparison of the linear plots gave significant results. The percentage of cholinesterase inhibition increased at the same rate for both varieties of sorghum. The percentages of cholinesterase inhibition for Wiley and Honey sorghum increased at the same rate as did those for the glass plates. The percent of variation explained by the linear trend is as follows: Wiley Sorghum 78.6 Glass Plate, March 89.3 Honey Sorghum 88.2 Glass Plate, April 92.4 The rate of formation of cholinesterase-inhibiting compounds appeared the same between the glass plates and sorghum plants. It appeared that, at least at high concentrations of phorate, formation of anticholinesterase oxidized metabolites was predominantly through a chemical oxidation on the leaf surface, and not plant enzyme catalysis. This took place at least at such a rate as to mask enzyme catalysis. With low concentrations of phorate within sorghum blades, methods similar to those described in References 5 and 8 could distinguish any difference in the rates of metabolism between the two varieties of sorghum. The higher cholinesterase inhibition values for the glass plates, (Figure 15) in comparison with the sorghum (Figure 11), can be attributed to the higher extraction efficiency with the glass plates. However, the phorate plant residue analysis by gas chromatography indicated the presence of phorate 48 hours after it could no longer be detected in samples from 35 �the glass plates. Although the cholinesterase-inhibition analysis Indicates the same rapid oxidation of phorate on leaf and glass plate, phorate could be present witnin certain portions of the leaf, e.g., within stomatal pores, and hence, within a potentially low-oxygen environment. This could explain the gas chromatographic data. An examination of roots resulted in no detectable cholinesterase inhibitors. Metabolism studies with lemon leaves showed the presence of large amounts of intact Di-Syston® accompanied by very slow conversion to other oxidative products(8). This suggested that the oil-soluble esters were being protected from aqueous hydrolysis by the oil content of the leaves. This was confirmed bv a radioautograph which showed nearly all of the radioactivity from P^2 Di-Syston® translocated into a lemon leaf is confined to the oil glands. A similar radioautograph of Systox®-thiol-isomer translocated into lemon leaves showed that, most of the radioactivity was located in the aqueous tissues of the plants^''. These differences were correlated with the relative water solubilities of the compounds, i.e., Di-Syston® 66 ppm and Systox®thipl-isonner 3900 ppm. The water solubility of phorate was recorded as 85 With the rate of formation of cholinesterase inhibitors the same en glass plates and sorghum leaf surfaces, the lower cholinesterase-inhibition percentage values for tomato (Figure 6) are difficult to explain. The values are lower than expected with tomato having received a concentration of phorate double that received by sorghum. Since the gas chromatographic data for tomato and sorghum agree, the distinct differences in cholinesteraseinhibition percentage values could have resulted from poorer efficiency in recovering oxidized metabolites from tomato compared to sorghum. The similarities in graphic plots for May sorghum (Figure 14) and tomato (Figure 8) would support this rationale. Evaluation of the cholinesterase-inhibition percentage values with the calibration curve resulted in values relating the concentration of toxic residues present in and on the plant foliage. Fourteen days after application of phorate to Honey sorghum in April, sample preparation of a foliar rinse of the surface of the plant accounted for approximately 50 percent of the total cholinesterase inhibition of the plant. A lack of detection of phorate within seven days indicated that residues of the oxidized metabolites in sorghum occurred to a very large degree via oxidation of phorate on the leaf surface, absorption within the leaf, and possible translocation within the plant. This is not in agreement with the previous studies presented in References 5 and 9. Other researchers have postulated(S) that the relative rates of absorption and translocation of phorate and Di-Syston® increased as the experiment proceeded because of the formation of more water-soluble oxidative metabolites in the subcuticular layers of olant tissue around the region of application. In Reference 9, the postulation is that the parent compounds were fairly persistent on the surface of the leaves but were metabolized rapidly once they had penetrated. �These variations can be explained by the differences in application method and in insecticide concentration: i.e., a 5 microliter topical application to the base of the stem of a cotton plant versus a 0.2 milliliter application of a 2 percent solution of phorate in vegetable oil to the blades of sorghum. It must be remembered that as the toxic metabolites are forming, they are concurrently being hydrolyzed to nontoxic phosphoric or thiophosphoric acids. Though the oxygen-analogs of phorate can inhibit cholinesterase activity more than their thionpohosphate precursors, they appear to have a higher degree of instability'^' ^'• The phosphorus is considerably more electrophilic in the P=0 compounds, thus weakening the P-S ester bond and facilitating hydrolysis and accelerating phosphorylation of the enzyme'^). Consequently, the presence of relatively large amounts of a highly oxidized metabolite in a plant would result in higher cholinesterase inhibition and higher apparent residue values than would an equivalent amount of a metabolite with less cholinesterase activity in another plant. The higher cholinesterase-inhibition values mean the presence of metabolites which are easily hydrolyzed, resulting in an overall faster rate of metabolic detoxification. The concentrations of phorate metabolites in tomato and sorghum were expressed in parts per million (ppn.) as phorate oxygen analog sulfoxide equivalent via a cholinesterase-inhibition method of analysis. The calibration curve for the residue method is given in Figure 2. It is independent of plant material analyzed and of the sample preparation technique. It reflects none of the losses that may occur in the various steps of sample preparation. A sample calculation follows the formula: .v = parts per million of phorate oxygen analog sulfoxide equivalent in sample analyzed. Where w is the phorate oxygen analog sulfoxide equivalent obtained in the analyses, micrograms. v is the aqueous extract in the determination, mi Hi liters. V is the total solvent in sample extraction, milliliters. W is the sample extracted, grams (fresh weight). The toxic residues present in tomato foliage were based .on the average weight of tomato plants initially after application of phorate (six weeks) and at the conclusion of the experiment (nine weeks), 6 grams and 28 grams, respectively. Thus, residues in the tomato foliage ranged from 1.1 to 5.3 ppm phorate oxygen analog sulfoxide equivalent. 37 �Residue persistence in sorghum (Table X) was higher. The May sorghum had concentrations with a range of 0 to 20.2 ppm. The average residue value after the second day was 17.9 ppm. The March/April sorghum had concentrations with a range of 2.4 to 18.5 ppm. The fourfold increase in residues by the ninth day is comparable to that found by Bowman and Casida'^' in considering the persistence of phorate-P 2 and its metabolites in vegetable crops. The total anticholinesterase activity of greenhouse pea plants, sprayed with phorate at one pound per acre, increased for about the firslv four days and then declined, but inhibitors persisted for 20 to 30 ^ ' Foliage application of 0,0-diethyl S-[(isopropylthio) methyl] phosphorodithioate to pea plants resulted in the appearance of anticholinesterase metabolites within one day and persistence of such metabolites in high concentration for at least nine days with detectable amounts present for 21 days^'. The results of this investigation were comparable: the main difference was higher residue levels. In crops treated with phorate., the ultimate toxic residues are present in a fractional part per million'". When applied to corn at a rate of one pound per acre, phorate was essentially gone in 14 days, while very low levels of its sulfoxide and sulfone (0.1 ppm or less) persisted through the 28-day experimental interval. At harvest time, the plant was essentially 4 free of insecticide, less than 0.01 ' The concentration of phorate metabolite residues present, though high, would probably be at a safe level by harvest time. The ultimate toxic metabolites present in harvest time residues are dependent upon both the interval between application and harvest and the method of application. Older plants having phorate applied at high rates would definitely have to be monitored for toxic residues. The lower residue values for tomato foliage are due in part to a larger daily plant weight—approximately threefold that of sorghum. The result could be a more rapid metabolism and hydrolysis and provides another possibility for the cholinesterase-inhibition percentage values being lower for tomato than for sorghum. Generally, oxidation. in plants never increases the toxicity of an application significantly^"'. However, the large residue values obtained in this study indicate that the toxicity is increased considerably on the surface of the plant when high concentrations are involved. A number of researchers, in speculating upon potential residue problems after various methods of treatment with systemic insecticides, have concluded that persistence curves should be 8 established on different crops grow under different environmental conditions' '. Military application of insecticides at normal rates results in residues which can be monitored with guidance from available literature. Residue breakdown of these organophosphorus insecticides is usually rapid with no persistency problems. However, the result of repetitive 38 �aerial application or spillage of insecticides in cropland areas may result in concentrations higher than usual. This study indicates exposure studies of the respective insecticides on glass plates alone under different environmental conditions would serve as a guide in predicting residues from high concentrations of insecticides. TABLE IX. PERSISTENCE OF PHORATE OXYGEN ANALOG SULFOXIDE EQUIVALENT IN SORGHUM Time From Application, Day 0 1 2 4 5 7 8 9 11 12 14 16 18 19 20 21 23 25 a Concentration, ppm March /April 3 2.4-5.3 2.4-5.3 2.4-5.3 10.3-14.5 10.3-14.5 Mayu 0 8.3 17.1 19.7 18.7 18.5 12.6-18.5 16.0-18.5 10.3-14.5 12.6-18.5 12.6-18.5 16.6 20.2 18.0 15.3 10.3-11.9 10.3-11.9 6.9-7.9 6.9-7.9 17.0 Concentration range is the result of considering the standard deviation for the average plant weight for the entire experimental period in May; this is due to the lack of plant weight values for March/April. Results are averages of two samples, three replications each. 3 Results are averages of four samples, three replications each. 39 �SECTION IV SUMMARY AND CONCLUSIONS Data on the metabolism of foliar applications of high concentrations of the organophosphorus insecticide phorate on Homestead tomato and Wiley and Honey sorghum are reported. The investigation of phorate metabolism, monitored by gas chromatographic and enzymatic analysis, produced the following results: 1. The cholinesterase activity values obtained showed no correlation with plant weight. 2. The disappearance of phorate appeared to proceed at the same rate in each plant species and variety; phorate disappeared more quickly from glass plates than from March/April sorghum under the same experimental parameters. 3. No significant differences were apparent between the two varieties of sorghum in May or during continuous experiments in March and April. It appeared that the formation of anticholinesterase metabolites, after high foliar applications of phorate, proceeded at the same rate in each variety although distinct visible differences occurred between the Wiley and Honey sorghum. 4. The peak percentages of cholinesterase inhibition from sorghum samples by the fourth day in May versus the ninth day in March/April indicated more rapid oxidation of phorate to anticholinesterase metabolites at higher temperatures. 5. The phorate metabolism in tomato was similar to metabolism in the May sorghum; however, actual comparison of percentage values of cholinesterase inhibition during the month for each of the three plants indicated that the average for the Homestead tomato was significantly lower than those for the two varieties of sorghum. 6. The percentage values of cholinesterase inhibition for the Wiley and Honey sorghum increased at the same rate as for the glass plates, indicating that the rate of formation of anticholinesterase-oxidized metabolites was predominantly through chemical oxidation on the leaf surface and not by plant enzyme catalysis; this surface oxidation took place at least at such a rate as to mask enzyme catalysis. 7. The larger droplet size in application technique resulted in higher toxic-residue values for phorate metabolites, especially on the surface of the plant, than would normally be expected. 40 �This study was initiated to find a basis for predicting toxicity and persistence of metabolite residues in plants after application of high concentrations of sulfur-containing oraanophosphorus insecticides during military spray operations. The results indicate that exposure studies of high concentrations of insecticides on glass plates alone, under different environmental conditions, would serve as a guide in predicting residues from repetitive aerial application or spillage of insecticides used by the military in cropland areas. Such studies with a controlled environment would yield toxic-residue-persistence data under various conditions for high concentrations of insecticides. 41 (The reverse of this page is blank) ��REFERENCES 1. Thomson, W.T. Agricultural Chemicals, Book I. Insecticides, Acaricides, and Ovicides. Thomson Publications, Davis, California, 1967. 2. Bowman, J.S. and J.E. Casida. Metabolism of the Systemic Insecticide 0,0-Diethyl S-Ethylthiomethyl Phosphorodithioate (Thimet) in Plants. J. Agr. Food Chem. 5: 192-197, 1957. 3. Bowman, J.S. and J.E. Casida. Further Studies on the Metabolism of Thimet by Plants, Insects, and Hammals. J. Econ. Entomol. 51:838-843, 1958. 4. Bowman, M.C., M. Beroza, and J.A. Harding. Determination of Phorate and Five of Its Metabolites in Corn. J. Agr. Food Chem. 17:138-142, 1969. 5. Metcalf, R.I., T.R. Fukuto, and R.B. March. Plant Metabolism of Dithio-Systox and Thimet. J. Econ. Entomol. 50:338-345, 1957. 6. Coleman, O.H. and J.L. Dean. Inheritance of Resistance to Methyl Parathion in Sorgo. Crop Sci. 4:371-372, 1964. 7. Wolverton, B.C., W.J. Wallace, A.L. Young, and D.D. Harrison. Studies on the Systemic Uptake of Toxic Phosphorus Esters by Plants. Morphological Effects of Foliar Applications of the Organophosphate Insecticides Mevinphos and Methyl Demeton on Selected Plant Species. Air Force Armament Laboratory Technical Report AFATL-TR-69-116, Eg!in Air Force Base, Florida, September, 1969. 8. Metcalf, R.L., H.T. Reynolds, M. Winton, and T.R. Fukuto. Effects of Temperature and Plant Species upon the Rates of Metabolism of Systemically Applied Di-Syston. J. Econ. Entomol. 52:435-439, 1959. 9. Heath, D.F. Metabolism in Plants and Soils. Jm Organophosphorus Poisons, Anticholinesterases and Related Compounds edited by D.F. Heath. Pergamon Press, Mew York, 1961. 10. Archer, T.E. Enzymatic Methods. Jm Analytical Methods for Pesticides, Plant Growth Regulators, and Food Additives, Volume I edited by G. Zweig. Academic Press, New York, 1963. 11. Sutherland, G.L., P.A. Giang, and T.E. Archer. Thimet. I_n Analytical Methods for Pesticides, Plant Growth Regulators, and Food Additives , Volume II edited by G. Zweig. Academic Press, New York, 1964. 43 �12. Nabb, D.P. and Florence Whitfield. Determination of Cholinesterase by an Automated pH Stat Method. Arch. Environ. Health. 15:147-154, 1967. 13. Himel, C.M. The Optimum Size for Insecticide Spray Droplets. J. Econ. Entomol. 62:919-925, 1969. 14. Young, A.L. and B.C. Wolverton. Military Herbicides and Insecticides. Air Force Armament Laboratory Technical Note AFATL-TN-70-1, Eglin Air Force Base, Florida, January, 1970. 15. Reynolds, H.T., T.R. Fukuto, R.L. Metcalf, and R.B. March. Seed Treatment of Field Crops with Systemic Insecticides. J. Econ. Entomol. 50:527-539, 1957. 16. Metcalf, R.L., R.B. March, T.R. Fukuto, and M.G. Maxon. The Nature and Significance of Systox Residues in Plant Materials. J. Econ. Entomol. 48:364-369, 1955. 17. Fukuto, T.R., R.L. Metcalf, R.B. March, and M.G. Maxon. Chemical Behavior of Systox in Biological Systems. J. Econ. Entomol. 48:347-354, 1955. 18. Cook, J.W. and R. Ottes. Note on the Conversion of Some Organophosphate Pesticides to Less Polar Compounds by Ultraviolet Light. J. Assoc. Offie . Agr. Chemists. 42:211-212, 1959. 19. Mitchell, T.H., J.H. Ruzicka, J. Thomson, and B.B. Wheals. The Chromatographic Determination of Organophosphorus Pesticides. Part III. The Effect of Irradiation on the Parent Compounds. J. Chromatog. 32:17-23, 1968. 20. Ruzicka, J.H., J. Thomson, and B.B. Wheals. The Gas Chromatographic Examination of Organophosphorus Pesticides and Their Oxidation Products. J. Chromatog. 30:92-99, 1967. 21. Metcalf, R.L., R.B. March, T.R. Fukuto, and M.G. Maxon. The Behavior of Systox-isomers in Bean and Citrus Plants. J. Econ. Entomol. 47:1045-1055, 1954. 44 �DISTRIBUTION LIST AFSC (DLSW) (SDWM) (SGP) ARPA (TECH INFO) DDR&E (CHEM TECH) (TECH LIB) SAAMA (SFQT) 2 3 3 1 1 5 5 AIR UNIVERSITY LIB HQ DA OACSFOR (FOR CM SR) OPERATIONS RSCH GRP EDGEWOOD ARSENAL (SMUEA-TD-S) (SMUEA-RPRE (2)) (SMUEA-CC) (SMUEA-QS) (SMUEA-CCCR) (SMUEA-TSTI-L) (SMUEA-D) (SMUEA-TS-CF) 1 1 1 1 2 1 1 1 1 1 1 (WPNS DEV & ENGR LABS) ENGR R&D LABS (TECH DOC CTR) ABERDEEN PRV GD (TECH LIB) DESERET TEST CENTER (TECH LIB) CBR AGENCY (CSGSB-ST) USA CHEMICAL SCHOOL (AJMCL-A) NAV AIR SYS COMD (AIR-532G) USN WEAPONS LAB 2 2 1 4 1 1 2 2 USN RESEARCH LAB (CODE 6140) 4525 FTR WPN UG (FWOA) 6570TH AMRL (HEF) DDC 12AF (DMEME) DL DLIP SSLT TAWC (DOD) (DIM) SOC (DFS) HQ USAF (AFRDPA) TAC (DOO-S) SOF-DOR 319SOS-DM TAWC-CB USAFETAC USAF ENVIRONMENTAL HEALTH LAB (Kelly AFB TX) 1 1 1 12 1 1 50 2 1 1 1 2 2 2 2 1 1 1 45 �DISTRIBUTION LIST (Concluded) USAF ENVIRONMENTAL HEALTH LAB (McClellan AFB CA) 1 USAFEL AFWL (DEE) USAFA (DFLS) DUGWAY PROVING GROUND (TECH LIB) ONR (CODE 440) HQ USACDC (NBC BRANCH) ASD (ENYS) DLOS CDCLNO 1 2 2 1 1 1 1 1 2 46 �UNCLASSIFIED Security Classification DOCUMENT CONTROL DATA - R & D (Security classification of title, body of abstract and indexing annotation must be entered when the overall report is ORIGINATING A C T I V I T Y (Corporate author) Flame, Incendiary, and Explosives Division Air Force Armament Laboratory Eqlin Air Force Base, Florida 3 classified) . REPORT SECURITY CLASSIFICATION UNCLASSIFIED 26. GROUP* REPORT TITL.E THE METABOLISM OF HIGH CONCENTRATIONS OF THE ORGANOPHOSPHORUS INSECTICIDE PHORATE APPLIED FOLIARLY TO SELECTED PLANT SPECIES D E S C R I P T I V E NOTES (Type- ot report and inclusive dates) Final Report (May - December 1970) 5 AUTHORCSI (First name, middle initial, Imxt name) George S. Kotchmar, Jr., Capt, USAF3 Billy C. Wolverton, Elizabeth.E. Boothe, Sandra M. Lefstad 6 REPORT D A T E 7«. T O T A L NO. OF PAGES February 1971 8«- C O N T R A C T OR G R A N T NO. &. PROJECT NO. 5066 53 \7t. NO. OF REFS 1 21 9a. ORIGINATOR*^ REPORT NJUMBERfSt AFATL-TR-71-22 9b. OTHER REPORT NO (si (Any other numbers that may oe assigned this report) C. d. 1O. DISTRIBUTION S T A T E M E N T Approved for public release; distribution unlimited. M- S U P P L E M E N T A R Y NOTES Available in DDC 12- SPONSORING M I L I T A R Y A C T I V I T Y Air Force Armament Lauoratory Air Force Systers Command Eglin Air Force Base, Florida 32542 ABSTRACT Gas chromatographic and enzymatic analyses (cholinesterase-inhibition method) were used to monitor the metabolism of the organophosphorus insecticide 0,0diethyl S-[(ethylthio)methyl] phosphorodithioate (phorate) applied foliarly to three economically important plants (Homestead tomato, Wiley sorghum, and Honey sorghum). The resulting data provided guidelines in predicting toxicity and persistence of metabolite residues for high concentrations of insecticides employed by the military. An attempt was also made to relate the metabolism of the insecticide to phytotoxic damage among and within plant species. The data indicated that no plant-variety-dependent distinction exists in the formation of toxic phorate metabolites as shown by in vitro anticholinesterase activity recorded over a four-week period. Further investigation, with the same high concentrations of phorate placed on glass plates located adjacent to treated plants, indicated the formation of toxic phorate metabolites was without the influence of biological substrates within the plants. There were no statistically significant differences with respect to the rate of increase of cholinesterase-inhibition percentage values between the sorghum and glass plates; the rate of formation of anticholinesterase oxidized metabolites was predominantly through chemical oxidation on the leaf surface and not by plant enzyme catalysis, or at least, the oxidation occurred at such a rate as to mask the enzyme catalysis. The large droplet size in the application of phorate resulted in higher toxic residue values, especially on the surface of the plant, than would normally be expected^ DD FORM 1 NOV 65 1473 UNCLASSIFIED Security Classification �UNCLASSIFIED Security Classification 1 14. K EY LINK A LINKS LINK' C WORDS ROLE WT ROLE WT Phorate 0,0-diethyl S-[(ethylthio)methyl] phosphorodithioate Organophosphorus Insecticides Plants Insecticide Residues Insecticide Metabolism UNCLASSIFIED Security Classification ROL E W T � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 016 Folder The folder containing the original item. 0172 Series The series number of the original item. Series II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Kotchmar, George S., Jr. Billy C. Wolverton Elizabeth E. Boothe Sandra M. Lefstad Description An account of the resource Corporate Author: Flame, Incendiary, and Explosives Division, Air Force Armament Laboratory, Eglin AFB, Florida Date A point or period of time associated with an event in the lifecycle of the resource 1971-02-01 Title A name given to the resource The Metabolism of High Concentrations of the Organophosphorus Insecticide Phorate Applied Foliarly to Selected Plant Species Subject The topic of the resource pesticide testing pesticide toxicology Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 056 Folder The folder containing the original item. 1512 Series The series number of the original item. Series III Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Wilson, James G. Source A related resource from which the described resource is derived Down to Earth Date A point or period of time associated with an event in the lifecycle of the resource 1973 Title A name given to the resource Teratological Potential of 2, 4, 5 -T Subject The topic of the resource pesticide toxicology congenital birth defects animal testing TCDD ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/3115a0b0721d97ce1ccc804a998ccf6d.pdf 38777d4fb47a0a6bdbd237f831c9d4c0 PDF Text Text Item ID Number °1185 Author Anonymous Corporate Author Report/Article Tltlfl Technology Notes JOUrnal/BOOk TitlB World Review of Pest Control Year 1969 Month/Day Color n Number of bnages 1 DeSOriptOU Notes Alvin L Youn 9 f'led tnis item under the category "DDT/Human Toxicology and Environmental Fate" Wednesday, April 11, 2001 Page 1185 of 1242 �or Technology noted W.VIN t. YOUNG DDT and man's health It is a strange fact that, in their consideration of the long' term hazards to man of exposure to the chlorinated hydrocarbons, the Advisory Committee on Poisonous Substances used in Agriculture and Food Storage, made no mention of the men subject to high and continuous exposure in the manufacture of these compounds. Dr Hayes, in his contribution to the Royal Society discussion of the toxicity of pesticides to man,1 concluded that real assurance about the possible long-term effects of small repeated doses may be gained by studying the effects of larger doses given over a briefer period. Hence the results of a clinical and chemical study of men with an intensive occupational exposure to DDT, carried out by Laws, Curley and Biros," arc of high interest. This study was made on thirty-five men with eleven to nineteen years of work in a factory that has produced DDT continuously and exclusively since 1947, now producing an average of six million pounds per month. The content of DDT, its isomcrs and metabolic products, in the men's fat ranged from 38 to 647 ppm; the average for the general population of this area is 8 ppm. From these figures and the excretion of DDA in the urine, it was estimated that the mean daily intake of DDT by the twenty men with high occupational exposure was 17-5 to 18 mg per man per day; the average for the general population was 0-04 mg per man per day. Neither medical history, physical examination, routine clinical tests nor chest x-ray revealed any ill effects attributable to this massive exposure to DDT. 1 Hayes, W. J. Jr, 1967, Proc. R. Soc. B., 187, 101. 2 Laws, E. R., Curley, A. and Biros, F. J., 1967, Arch, environ. Health, 16, 766. International Congress of Plant Protection The Seventh International Congress of Plant Protection will be held in Paris from September 21st to 25th, 1970. The objectives of the Congress are those of previous Congresses, the sixth of which was held in Vienna in 1967, though the seventh Congress will not be concerned with the chemistry of pesticides, but rather their general characteristics. Section A, dealing with economic studies, will include the significance of crop losses due to pests, the economic aspects of pest control, the implementing of crop protection measures and methods of investigation. Section B comprises: (I) prophylactic methods including sanitation, the use of resistant varieties, regulatory methods; (2) mechanical and physical methods such as thermal therapy, irradiation, etc; (3) chemical methods such as the use of pesticides, chemosterilants, attractants, repellents and inhibitors; (4) biological methods; (5) integrated control, prognosis and warnings. Section C is for the study of the consequences of pest control including the risk of residues and their effects on wild life and the problems of pesticide resistance. Section D is concerned with general procedures for the application of control measures. The Congress is sponsored by the Socie'te' Francaise de Phytiatrie et de Phytopharmacie, from whom further particulars may be obtained, the address being 57, Boulevard Lannes, 75-Paris XVP, France. Technological economics of pest control and crop protection The Symposium arranged under the above title by the Pesticides Group of the Society of Chemical Industry was mentioned in an earlier Technology Note (Spring 1968, p. 5); further details are now available. The symposium will open, on the evening of September 77 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 047 Folder The folder containing the original item. 1185 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Source A related resource from which the described resource is derived World Review of Pest Control Date A point or period of time associated with an event in the lifecycle of the resource 1969 Title A name given to the resource Technology Notes Subject The topic of the resource DDT pesticide toxicology health effects ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 048 Folder The folder containing the original item. 1235 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Witter, Robert F. Description An account of the resource Corporate Author: Technical Development Laboratories, Technology Branch, Communicable Disease Center, Bureau of State Services, Public Health Service, U.S. Department of Health, Education, and Welfare Date A point or period of time associated with an event in the lifecycle of the resource 1959 Title A name given to the resource Reprint: Protection of Shade Trees with DDT and Its Relation to Human Health Subject The topic of the resource CDC agricultural exposure human testing pesticide toxicology ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/e787c6e13e415c42d0bd84b559613312.pdf 9fce70516f1fe3304a9e588fd0c482b5 PDF Text Text Item ID Number 01190 Author Corporate Author U. s - ArlT|y Medlcal Bioengmeenng Research and Devel RBDOrt/ArtidO Tltto Report: Problem Definition Studies on Potential Environmental Pollutants, VII. Physical, Chemical, lexicological, and Biological Properties of DDT and Its Derivatives ' Journal/Book Title Year 1979 Month/Day Color rj Number of Images ^ DQSCrlptQn NDtQ8 Alvin Li Young filed this item under the category "DDT/Human Toxicology and Environmental Fate" Technical Report 7906 Wednesday, April 11, 2001 . Page 1190 of 1242 �Technical Report 7906 . / •o* CO PROBLEM DEFINIT70N STUDIES ON POTENTIAL ENVIRONMENTAL POLLUTANTS VII. PHYSICAL, CHEMICAL, TOXICOLOGICAL, AND BIOLOGICAL PROPERTIES OF DDT AND ITS DERIVATIVES JUNE 1979 O Prepared for the OFFICE of the PROJECT MANAGER for CHEMICAL DEMILITARIZATION and INSTALLATION RESTORATION by US ARSSY MEDICAL BIOENGINEERING RESEARCH and DEVELOPMENT LABORATORY ForlDfitrlck Frederick, BSD 21701 Edited by _W.. .JQickinson Burrows , _Ph . D . David R ,-Gogley , -Ph . D . Ph.D. Jack C. Dacre, Ph.D. jGeoffrey Woo4ard, Ph^ APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED US ARMY MEDICAL RESEARCH and DEVELOPMENT COMMAND FortDetr&k Frottertok, RSD217C1 81 1 28 00* �NOTICE Disclaimer The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. *» Disposition Destroy this report when it is no longer needed. to the originator. Do not return it �Unclassified SECURITY CLASSIFICATION Of THIS I*ACE READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 1. REPORT NUMBER (fc 2. COVT ACCESSION NO. 3. R E S I E N T ' S CATALOG NUMBER Technical Report 7906 •, V J^ROBLEM DEFINITION STUDIES ONJPOTENTIAL j JNVIRONME^TAL POLLUTANTS, vii/PHYSICAL, ^CJBEMICAL,: ^JXiqOLOGICAL.AKD JIOLOGICAL PROPERTIES OF DDJ 'ID ITS DERIVATIVES^ ; ~~". - «. PERFORMING ORG. REPORT t^MBER 8. CONTRACT OR GRANT NUMBERf»; Dickinson/Burrows /T fw. Dick: I /OS David R./Cogley R r ^~f William G./Light r~" Jack C/ Dacre Geoffrey/Woodard 1 NAME AND ADDRESS O. PROGRAM ELEMENT. PROJECJ^T ASK AREA « WORK UNIT N U M B S ^ ^ U.S. Army Medical Bioengineering Research and Development Laboratory, ATTN: 'SGRD-UBG, Fort Detrick, Frederick, MD 21701 H. CONTROLLING OFFICE NAME AND ADDRESS U.S. Army Medical Research and Development Comtnan^ ATTN: SGRD-RMS Fort Detrick, Frederick, MD 21701 *i"*"tv- » L L ^^^- L j-^IP. , IIU J.J. / VJX 14. MONITORING AGENCY NAME 6 AOORESSfl/ dlltettnt trom Controlling Olllce) IS. SECURITY CLASS, fof Hit* rcporQ UNCLASSIFIED I5«. OECLASSIUCATION/OOWNGRADING 16. DISTRIBUTION STATEMENT (at thlt Rtpott) Approved for public release; distribution unlimited 17. DISTRIBUTION STATEMENT (ol th* mbtli-tst nnterfd In Block 30, It dlllorent from Rupert) 18. SUPPLEMENTARY NOTES 19. KEY WORDS (Contlnu* on rmvtrl* ila* It ne«»*»«iy and Identity by block number) Amphibians Analytical Methods Bioconcentration Biotransformation Birds Carcinogenesis DDD DDE DDT • Fish Invertebrates Mammals Man Metabolism Microorganisms iV\A BSTH ACT fCoaUaa* *a »»v»r»» •£<*. H n<KnH*JU]r nod Idmlllr fcr Wocfc numfcwj This report is a data base of physical, chemical, toxicological, and biological properties of DDT. Sufficient data are presented to allow a preliminary assessment of the human health impacts and ecological impacts of DDT at sites within the United States where DDT is found. Thia report is intended to be used with chemical analysis data of DDT concentrations in water, sediments, soils, and biota at particular sites of interest to develop a qualitative assessment of potential hazard. DD , KK. W3 EO moHo F , M o V «,so OSO L E T E unclassifed SeCOmTY CLASS! FICATtOH Of THIS PAGE (Mrm D*if £nf»»»d) �ACKNOWLEDGMENT The contributions of David R. Cogley and William G. Light to this report were per f ormedyndfir U.~^~7trmjtsMedical Research and Development Command Contract No. 4SAMD17~77-C-705o't^ Walden Division of Abcor, Inc., Wilmington, MA. The human TWltrorrog JTlection of the manuscrilpt was reviewed by Dr. Marcus Mason (Walden consultant) of Shrewsbury, MA. Guidance for the .:: ecological impacts section was provided by Professor Charles F. Wurster (Walden" consultant.) of the State University of New York at Stony Brook. Geoffrey Woodard ^s consultant to the Environmental Protection Research Division, U.S. Army Medical Bioengineering Research and Development Laboratory. . Editorial assistance was provided by Franklin Research Center under Contract No. DAMD17-79-C-9129, Marsha Hall, principal editor. -1- �TABLE OP CONTENTS . . • • -..:-...-• - Page ACKNOWLEDGMENT . .. . . ., . . . . . . . . .... . .. . . . , , . . . 1 .. ... LIST OF FIGURES . .."... 3 LIST OF TABLES .. . . . ... . . .:v."V". . ... . . . i . "4 I. INTRODUCTION . . • 5 Requirement for Report. Format ..........'......... . Scope .. Objective ................. . . . . . . . . " II. ALTERNATIVE NAMES III. .. 7 PHYSICAL AND CHEMICAL PROPERTIES . . . 8 Composition of Technical DDT Analysis 10 10 ^ . MAMMALIAN TOXICOLOGY 10 Human Exposure Laboratory Animals V. »• VI. STANDARDS AND CRITERIA FOR DDT Air ....... Drinking Water and Food Water for Aquatic Life VII. EFFECTS OF DDT ON A MODEL ECOSYSTEM Manufacturing Practices . . . Composite Hypothetical Site Observed DDT Concentrations Predicted Effects Decontamination Objectives VIII. 10 13 ENVIRONMENTAL CONSIDERATIONS Behavior in Soil, Water, and Air ...' Degradation Bioaccumulation and the Food Chain Effects on Terrestrial Animals. Effects on Aquatic Organisms Effects on Microorganisms REFERENCES . 5 5 6 7 15 .... 15 18 19 21 25 41 41 ..... 41 41 42 42 ...... 42 43 43 47 52 53 �LIST OF FIGURES IV-1 Biodegradation of DDT. ... IV-2 Further Degradation Products of DDT and DDE V-l Examples of DDT Bioaccumulation. . V-2 Models of Bioaccumulation Pathways .......... 23 V-3 Effects of DDT on Frog Tadpoles 35 VII-1 Map of Model DDT Plant Site. ........... . . 44 VII-2 Biological Effects of Dietary DDT for Typical Receptors ....................... -3- ..... ...... ..... . . 16 ...... 17 ..... 22 ............ 50 �LIST OF TABLES 1-1 Pollutants at Pine Bluff Arsenal 1II-1 Selected Physicochemical Properties of Components of Technical DDT ......... 11 IV-1 Toxicity of DDT to Man 12 IV-2 Acute Oral Toxicity of DDT for Animals ; V-l Acute Toxicity of DDT to Birds V-2 . 13 .. 24 Concentrations of DDE in Bird Eggs Resulting in 10% Reduction in Normal Shell Thickness ...... 26 Acute Toxicity of p,p'-DDT to Fishes by Static Bioassay 27 V-4 Sac-Fry Mortality for Various Fish Species 31 V-5 Acute Toxicity of p,p'-TDE to Fishes 33 V-6 Toxicity of DDT to Tadpoles. . . 34 V-7 Behavior and Morphological Abnormalities of Tadpoles Exposed to DDT 36 V-8 Toxicicy of p.p'-DDT to Arthropods 37 V-9 Toxicity of DDT Isomers to Mosquito Larvae 39 V-10 Toxicity of p,p'-TDE to Arthropods 40 VII-1 Area and Wildlife Contamination Levels for Various Sites 45 Environmental Concentrations of DDT at the Model Site 48 lexicological Effects of DDT on Typical Wildlife as a Function of Concentrations of DDT in Water, the Diet, and Tissues 49 Predicted Effects on Wildlife. . 51 V-3 VII-2 VII-3 VII-4 -4- �I. INTRODUCTION Requirement for Report The U.S. Army Toxic and Hazardous Materials Agency (USATHAMA), formerly the Office of the Project Manager for Chemical Demilitarization and Installation Restoration, has identified an initial list of substances requiring assessment because of their actual or potential presence in the environment outside the boundaries of Pine Bluff Arsenal (PBA), Arkansas (Table I-l).1 The U.S. Army Medical Bioengineering Research and Development Laboratory (USAMBRDL) has divided the list into logical units for problem definition studies. Substances used in pyrotechnic devices are treated in two reports.*»' Thiodiglycol and elemental phosphorus have been assessed previously in reports by Rosenblatt e_t_ al_.*»* and Dacre and Rosenblatt;* a separate report on these substances specific to PBA has been deferred indefinitely. DDT is considered separately here because (a) it is neither military-unique nor installation-unique; (b) there is an overwhelming amount of information available in the published literature; and (c) most pertinent data have been suiwnarized in review articles. The present report deals exclusively with DDT, its isomers and metabolites. The format of this report departs from that of previous reports in this series2"*'7»' because it incorporates both the data base and site-specific TABLE I-l. POLLUTANTS AT PINE BLUFF APSENAL8 *" DDT Thiodiglycol Phosphorus (white) Auramine Benzanthrone 1,4-Di-p-toluidinoanthraquinone 1,4-Diamino-2,3-dihydroanthraquinone 1-Methylaminoanthraquinone a. As provided in Reference 1. -5- �considerations for a hypothetical installation. There are two reasons, for this approach. First, Redstone Arsenal (RSA) also has major DDT contamination, and USATHAMA personnel have indicated that RSA data are as important to their mission as PBA data. Second, contamination surveys and corrective measures were initiated at both PBA and RSA while this report was in preparation. Thus, in view of the continuous output of new data, it appeared neither practical nor useful to analyze site data for either installation. Instead, a hypothetical site has been created (Section VII) to illustrate the qualitative relationships of DDT levels in water, sediment, and biota to effects of DDT on health and the environment. Quantitative considerations for this site are derived from fragmentary data available for PBA and RSA at the time this study was initiated. This section may be used to estimate the potential ecological effects of DDT waste disposal relative to past known or postulated declines in wildlife populations as well as to the lower DDT concentrations in soil and water resulting from cleanup operations. An important caveat must be given here. Concentrations of DDT in soil, sediment, water, and biota, and the toxic effects predicted therefrom, have been derived using concentration factors, i.e., the ratio of DDT in sediment to DDT in water, DDT in biota to DDT in water, etc. To do so is strictly valid only if these concentration factors represent true equilibrium or steady state values. In very few cases are data sufficient to make such a distinction, and for this reason, soil, sediment, water, or food chain concentrations predicted to lead to a particular toxic effect may be in error by an order of magnitude. This report is ecologically oriented. Mammalian toxicology and human health effects of DDT have been exhaustively reviewed in a 1979 document of the World Health Organization (WHO).' Some representative data are included in the present report, but investigators concerned with human health aspects of DDT (and the tradeoff between health benefits and hazards) should refer to the WHO text. The volume of data on environmental effects of DDT has obliged USAMBRDL to exercise considerable and arbitrary selectivity in choice of material to review. For the most part, data relevant to the environments of south central Arkansas and northern Alabama have been collected. The ecological literature has been surveyed systematically through mid-1976 and selectively thereafter. Because of the availability of many definitive reviews, efforts concentrated on surveying the literature of the last ten years, and few references published prior to 1970 were retrieved. In the case of aquatic organisms, a search was conducted not only for DDT, but also for the seven isomers and metabolites detected in the soil of PBA—p»p'-, o,p'-, and m,p'-DDT; p,p'- and o,p'-TDE (ODD); and p,p'- and o,p'-DDE—and for in,p'-TDE, m,p'-DDE, DDMU, and DDMS, metabolites not detected at PBA but judged likely to be present (see Fig. IV-1 for structures). Throughout this report, DDT (unprefixed) refers to the technical product, sometimes designated DDTR in the literature. -6- �Objective The objective of this report is to provide, to those charged with assessment and amelioration of DDT contamination at Army installations, guidance on the health and environmental hazards of DDT and the ecological consequences of various actions. II. ALTERNATIVE NAMES DDT is the name approved by the International St&iviards Organization for the technical product of which p,p'-DDT is the predominant component. As used in the present report, DDT refers to the technical product or any of ten isomers or degradation products listed below. DDT trade names: Anofex, Arkotine, Chlorophenothane, Dicophane, Estonate, Gesarol, Guesarol, Neocid, Zerdane. p,p'-DDT: 1 , l'-(2,2,2-trichloroe<.-.hylidene)bis[4-chloro]benzene; o,o-bis(pchlorophenyl)-B,8,0-trichlorethane; 2,2-bis(p-chlorophenyl)-!,!,1trichloroethane; 4,4'-dichlorodiphenyltrichloroethane; l,l,l-trichloro-2,2bis(p-chlorophenyl)ethane. o,p'-DDT: l-chloro-2[2,2,2-trichloro-l-(4-chlorophenyl)ethyl]benzene; 1,1,1-trichloro~2-(o-chlorophenyl)-2-(p-chlorophenyl.)ethane. m,p'-DDT: l-chloro-3[2,2,2-trichloro-l-(4~chlorQphenyl)ethyl]benzene; 1,1,1-trichloro-2-(m-chlorophenyl)-2-(p-chlorophenyl)ethane. p,p'-DDD: l,l'-(2,2-dichloroethylidene)bis[4-chloro]benzene; l,l-dichloro-2,2-bis(p-chlorophenyl)ethane; p,p'-TDE. o,p'-DDD: l-chloro-2l2,2-dichloro-l-(4-chlorophenyl)ethyl]benzene; l,l-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane; mitotane; o,p'-TDE. m,p'-DDD: l-chloro-3[2,2-dichloro-l-(4-chlorophenyl)ethyl]benzene; l,l-dichlorq-2-(m-chlorophenyl)-2-(p-chlorophenyl)ethane; n,p'-TDE. p,p'-DDE: l,l'-(2,2-dichloroethenylidene)bis[4-chloro]benzene; l,l-dichloro-2,2-bis(p-chlorophenyl)ethylene. o,p'-DDE: l-chloro-2[2,2-dichloro-l-l(4-chlorophenyl)ethenyl]benzene; 1,l-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethylene. DDMU: l,l'-(2-chloroethenylidene)bisl4-chloro}benzene; l-chloro-2,2-bi8(p-chlorophenyl)ethylene. DDMS: l,l'-(2-chloroethylidene)bis[4-chloro]benzene; 2-chloro-l,l-bis(pchlorophenyDethane, -7- �PHYSICAL AND CHEMICAL PROPERTIES1* III. p,p'-DDT: Chemical Abstracts Service Registry Number: 50-29-3 Toxic Substances List: KJ33250 Wiswesser Line Notation: GXGG YR DG&R DG Molecular Weight: 354.48 Molecular Formula: Structural Formula: CC1. o.p'-DDT: Chemical Abstracts Service Registry Number: Toxic Substances List: KH7910000 *" Wiswesser Line Notation: GXGG YR BG&RDG Molecular Weight: 354.48 Empirical Formula: Structural Formula: -K 789-02-6 �p,p'-TDE (DDD): . Chemical Abstracts Service Registry Number: 72-54-8 Toxic Substances List: KI0700000 Wiswessor Line Notation: Molecular Weignt: GYGYR DG&R DG 320.0 Empirical Formula: Structural Formula: .CHC1. p,p'-DDE Chemical Abstracts Service Registry Number: Toxic Substances Lint: KV9450000 Wiswesser Line Notation: GYGUYR DG&R DG Molecular Weight: 318.0 Empirical Formula: Structural Formula: CCL /"*~~~\ ci—(\ /V-c- -9- 72-55-9 �Composition of Technical DDT DDT is the name approved by the International Standards Organization for the technical product of which p,p'-DDT is the predominant component. Pure p,p'-DDT is a colorless crystalline solid, whereas the technical material takes the form of a white or cream-colored waxy solid or amorphous powder. Technical DDT is a mixture of isomers containing 65 to 80% p,p'-DDT and up to 14 other components. The major impurities are o,p'~DDT (15 to 21%); p,p'-TDE (>4%; l-(p-chlorophenyl)-2,2,2-trichloroethanol (>1.5%); traces of o,o'-DDT and m,p'-DDT; and traces of bis(p-chlorophenyl)sulfone. On exposure to sunlight or alkaline conditions, p,p'-DDT is converted to stable p,p'-DDE, which may constitute a significant fraction of any environmental sample. Physicochemical properties of the pure substances comprising technical DDT are summarized in Table III-l. Analysis No attempt has been made to review analytical methods for DDT. Approved methods for detection and estimation of DDT and its derivatives in environmental samples (soil, sediment, water, and tissues) have been compiled by the U.S. Environmental Protection Agency, and are subject to frequent revision. l ; »l * IV. MAMMALIAN TOXICOLOGY Human Exposure was introduced in 1945 for t^e control of malaria mosquitoes. It is a highly potent contact poison of the nervous system in insects. It is very stable, so it persists, offering continuous protection for many months after a single application. During World War II, DDT was widely used to prevent insect vector-borne disease among troops, prisoners, and refugees. DDT vas .applied directly to the skin and clothing in concentrations as high as 25% in powder form. Despite these massive exposures, very few, if any, authentic cases of human poisoning have been observed as a result.1* DDT is moderately toxic to man by oral administration; Table IV-1 gives dosages and expected or observed effects in man. Laws £t £l. "» ls conducted extensive tests on 35 individuals employed in the manufacture of DDT who had been exposed from 16 to 25 years (21 years median) to amounts up to 18 mg per person per day. Phyi ical examinations, medical histories, and liver function tests failed to reveal any evidence of an untoward effect on human health. Experimental work on human volunteers has not produced convincing evidence that DDT is harmful to man at exposure levels 100 times those likely to be encountered in the workplace or environment.1""1* Despite extensive studies over the past 30 years, the exact mechanism of DDT1 s toxic action in man is still uncertain. Based upon studies primarily -10- �TABLE tlt-l. SELECTED PtnrSICOCHEHICAL PROPERTIES OF COMPONENTS OF TECHNICAL DDT Pure Substance! Comprising Technical DDT Property Description Melting point Boilidg point Solubility Molecular .sight Molecular formula Volatility Chenical . reactivity and atability p,p'-DDT o,p'-DDT White, crystalline solid Colorless crystal* 74.2*C 108.5*C 185* Practically insoluble in water Water, 0.085 ng/1 at (1 vg/1), moderately soluble in 25'C; soluble in fat hydroxylie and polar solvent!, readily and most organic solvents soluble in most aromatic and chlorinated solvents 354.5 354.5 Vapor pressure • 1.9 x 10"' Torr at 20*C Dehydrochlorinated at temperatures Stable in concentrated above its melting point into sulfuric acid ethylene derivative (DDE), a reaction catalyzed by ferric and aluminum chloride and by UV light. In solution, it is readily dehydtochlorinated by alkalis or organic bases; otherwise it is stable being unattccked by acid and alkaline permanganate and by aqueous acids and alkalis. With technical DDT.-dehydrochlorination may proceed at temperatures »« low as 50*C p,p'-TDE (ODD) p.p'-DDE Colorless crystals 109'-110*C White, crystalline solid 88.4*0 Similar to p,p'-DDT Hater, 0.12 mg/1 at 25*C; soluble in fat snd most organic solvents 320.0 318.0 Similar to p.p'-DDT, but it is more slowly hydrolyzed by alkalis Stable in concentrated sulEuric acid. It may be oxidized to p.p'-di'-Morobenzophenone•, a r« ction catalyzed by UV radiation �in laboratory animals, using relatively massive doses, it has been speculated that DDT affects the metabolism of some of the biogenic substances in the central nervous system and some of the carbohydratemetabolizing enzymes in the uterus, kidney cortex, and liver. The microsomal enzyme systems in the liver and possibly other tissues are increased when exposure levels become sufficiently high.17 The occurrence of enzyme induction in man at current environmental exposure levels has not been established. Human exposure to DDT has resulted in no reported cases of cancer or other neoplasms, although carcinogenesis has been demonstrated in some laboratory animal species. Feeding DDT to men for nearly 2 years did not result in tumors,18 and no tumors were found in men whose occupation w&s the manufacturej formulation, or application of DDT." (However, the latency period for appearance of cancer in humans may exceed the 35 years since DDT was introduced.) The U.S. Environmental Protection Agency*' has estimated an upper-limit lifetime cancer risk of 1 in 10^ for males consuming 7.3 x 1CT* tng/day (10 ng/kg/day) of DDT. This estimate is derived from the observation that Jewish males in Israel have higher fat levels of DDT than males in New York State (16.S3 versus 9.04 ppra) and that the lifetime incidence of nervous system cancer is correspondingly higher (1.1 versus 0.5%). It is based on the assumption that cancer resulting from DDT it^estion will be expressed in humans solely in the nervous system and on tie admittedly unsupported corollary that the excess incidence of .nervous system cancer results solely from excess DDT consumption. TABLE IV-1. TOXICITY OF DDT TO MANa Dosage (mg/kg/day) mg 70-kg person Remarks Unknown'' — Fatal 16-286b 1,100-20,000 Vomiting at higher doses, convulsions in some 6-10b 400-700 Moderate poisoning in some 0.5 35 Tolerated. Periods lasted 21 months with volunteers, 6.5 years with workers 0.25 (inhalation ?) 18 Tolerated by workers for 19 years a. Adapted from Jukes.1* b. Precise dosage unknown. -12- �Laboratory Animals Acute Toxicity.' Data on the acute toxicity of DDT to mammals are summarized in Table IV-2.*1 These data indicate that the short-term toxicity to mammals is moderate to high, depending on the mode of ingestion, and that DDT i • generally more efficiently absorbed from the gastrointestinal tract when dissolved in an oil vehicle. TABLE IV-2. ACUTE ORAL TOXICITY OF DDT FOR ANIMALS8 » tug/kg Species Water Suspension or Powder Oil Solution Rat 500-2,500 113-450 Mouse 300-1,600 100-800 Guinea pig Rabbit 2,000 275 250-560 300-1,770 Cat 100-410 Dog >300 From Hayes.*1 Careinogenicity. Carcinogenesis experiments have been performed in which rodents were fed DDT at concentrations ranging from 2 to 1,650 pptn.**"'1 There appear to be wide ranges in susceptibility to DDT-induced carcinogenesis for different mammalian species and strains. Other studies have found that no increase in tumors was induced by feeding DDT to golden hamsters,11 and no tumors vere induced in a small number of dogs and monkeys.1* DDT, TOE, and DDE were tested in the National Cancer Institute Bioassay Program. The summary of their results follows.11 -13- �"Bioajsays of technical-grade DDT, TDE, and p,p'-DDE for possible carcinogenicity were conducted using Osborne-Mendel rats and B6C3F1 mice. Each compound was administered in the feed, at either of two concentrations, to groups of 50 male and 50 female animals of each species. Twenty animals of each species and sex were placed on test as controls for the bioassay of each compound. The time-weighted average high and low dietary concentrations of DDT were, respectively, 642 and 321 ppm for male rats, 420 and 210 ppm for female rats, 44 and 22 ppm for male mice, and 175 and 87 ppm for female mice. The time-weighted average high and low dietary concentrations of TDE were, respectively, 3294 and 1647 ppm for male rats, 1700 and 850 ppm for female rats, and 822 and 411 ppm for male and female mice. The time-weighted average high and low dietary concentrations of DDE were, respectively, 839 and 437 ppm for male rats, 462 and 242 ppm for female rats, and 261 and 148 ppm for male and female mice. After the 78-week dosing period there was an additional observation period of up to 35 weeks for rats and 15 weeks for mice. "There were significant positive associations between increased chemical concentration and accelerated mortality in female mice closed with DDT and in both sexes of rats and in female mice dosed with DDE. This association was not demonstrated in other groups. There wr.s, however, poor survival among control and dosed male mice used in th.5 bioassays of DDT and DDE. In all car.es adequate numbers of animals in all groups survived sufficiently long to be at risk from late-developf • •} tumors. "When those male rats receiving TDE and their controls were combined within each group so that the numerators of the tumor incidences represented those animals with either a follicular-cell carcinoma or a follicular-cell adenoma of the thyroid, the incidence in the low dose group was significantly higher than that in the control. There was a significant positive association between the concentration of DDE administered and the incidences of hepatocellular carcinomas in male and female mice.. Among dosed rats and mice no other neoplasms occurred in statistically significant incidences when compared to their respective control groups. "Under the conditions of these bioassays there was no evidence for the carcinogenicity of DDT in Osborne-Mendel rats or B6C3F1 mice, of TDE in female Osborne-Mendel rats or B6C3F1 mice of either sex, or of p,p'-DDE in Osborne-Mendel rats, although p,p'-DDE was hepatotoxic in Osborne-Mendel rats. The findings suggest a possible carcinogenic effect of TDE in male Osborne-Mendel rats, based on the induction of combined follicular-cell carcinomas and follicular-cell adenomas or the thyroid. Because of the variation of these tumors in control male rats in this study, the evidence does not permit a more conclusive interpretation of these lesions. p,p'-DDE was carcinogenic in B6C3F1 mice, causing hepatocellullar carcinomas in both sexes." -14- �Mutagenicity. The fact that DDE is tnutagenic in mammalian cells" and DDT is not suggests that the proximate carcinogen is DDE, a metabolite of DDT. It has been shown that chlorinated hydrocarbon carcinogens, such as carbon tetrachloride and dieldrin, are negative in the standard Ames test. These materials presumably require metabolic activation, possibly dehalogenation, for mutagenic activity. Because the Ames test includes only metabolic activation mediated by the liver microsomal system and dehalogenation is not so mediated, it is reasonable that pure DDT is negative in the Ames test. Metabolism. The principal pathways for DDT metabolism are depicted in Fig. IV-1, with lesser pathways presented in Fig. IV-2. It is important to note that DDD and DDE arise by independent mechanisms and that DDE is relatively inert. Hence, environmental DDT samples will show increasing percentages of DDE with time where use of DDT has been discontinued. Equivalent metabolites arising from the o,p'-DDT isomer in technical DDT also appear in residues. The biological transformation of DDT is further discussed in the following section of this report. V. ENVIRONMENTAL CONSIDERATIONS The literature on the toxicology, ecology, environmental fate, and bioaccumulation of DDT is extensive and has been comprehensively reviewed elsewhere, notably by Brown,'* Edwards,** Matsumura," Tahori,*7 White-Stevens," and Wurster and coworkers."'112 Information on the environmental fate of DDT and the bioaccumulation of DDT in the food chain is summarized in the following subsections. Behavior in Soil, Water, and Air To summarize, factors affecting the behavior of DDT in soil, water, and air include low water solubility, ease of adsorption on soil, chemical reactivity (p,p'-DDT conversion to p,p'-DDE), low vapor pressure, and ease of uptake by plants and animals. When present in soil, DDT tends to remain for years, acting as a long-lived reservoir for gradual release to surface waters and biota. When present in surface waters, DDT is assimilated rapidly by aquatic organisms and i,;. accumulated in the food chain. Evaporation into the atmosphere also occurs. Atmospheric transport leads to low (background) concentrations over wide geographic areas. Worldwide, rainwater DDT levels fall in the range from 0.018 to 0.066 ppb.' Although practically insoluble in water, DDT readily adsorbs to particulate material in aquatic systems. In addition to accumulation through the food chain, DDT may be incorporated into aquatic organisms by direct contact with DDT-containing water or through ingestion of particulate matter containing DDT. DDT may enter an aquatic ecosystem by physical, chemical, or biological transport. Atmospheric transport and erosion of contaminated solids appear to be the most frequent routes. Eventually, the DDT tends to reach the -15- �DDE R-C-R (1 cc.i t R— CH p __* R-CH-R —-»-R—C— R . 1 1 CCI, CHCIj DDT DDD - »^ R— CH — R a R—C— R »~ II CHCI 1 CH2C! II CH, DDMU DDMS DDNU R-CH-R —-*- R— CH— R —-^- R^CHj— R —-»- R— CH— R CH2OH COZH DDOH DDA OH DPM DBH Fig. IV-1. Biodegradation of DDT (R=C,H4CI) -*•"-§-' O DBP �R—CH—R I CC1, DDT OH t -R-C-R I CGI, and Kelthane R-CH—R I CN DDCN CCb OCli DDE Fig. tV-2. Further Degradation Products of DDT and DDE (R-C.H«CI) CCI, �water surface where it can co-distill with water and reenter the atmospheric cycle. As noted earlier, DDT is converted to DDE by sunlight. Various organisms also convert p,p'-DDT to p,p'-TDE and p,p'-DDE, the latter being the most abundant DDT compound in the environment. For the purpose of this review, the three compounds are considered collectively, unless specified otherwise. The amount of DDT that runs off into water bodies depends on the degree of slope of the ground, the fineness of the ao'j.. ind th«» degree of vegetation cover.*8 Water transport of DDT depe: v. on .•rision runoff because DDT is strongly adsorbed to soil particles. Dv. SVfo.uiMjs so tightly bound to soil particles that it does not rea-'ily le.;ci LM'JV groundwater.** Nonpolar compounds such as DDT either reach Che aqt-.-r,/r sink adsorbed onto soil particles in the runoff or, when directly .ip\>!Leil '<.c water, become adsorbed onto the suspended matter. When a pond was treated with DDT at 0.02 '. vi, an Tfective concentration for mosquito control, the DDT disappeared from the water after 3 weeks and was found in the mud for 8 weeks after the treatment.** Greater amounts of DDT reach the bottom of a water body when the sedimenting material is composed of fine particles.*5 The stability of DDT in soil has been studied by Guenzi and Beard, who have also reviewed the subject.**'** The rates and products of degradation are dependent on temperature, oxidation-reduction potential, and moisture content of the soil. In aerobic soils, DDT is converted to DDE by a predominantly chemical process.** In anaerobic soils, the products are TDE and its transformation products.***** In dry aerobic soils, DDT is stable; loss is very slow by either degradation or volatilization.*'**7 Degradation Reviews by.Fries** and Rhead51 summarize much of our knowledge concerning the natural degradation of p,p'-DDT. A proposed scheme for partial biodegradation of DDT is presented in Fig. IV-1. Although the metabolites have all been identified, the pathway depicted must be considered only representative because no single organism has been found to produce all the metabolites (with the possible exception of Aerobacter aerogenes'*), and it is likely that different organisms emphasize different pathways. TDE is by far the most prevalent metabolite of bacteria and fungi, whereas phytoplankton species produce small amounts of DDE only. Only TDE has been isolated from the intestinal microflora of the northern anchovy (Engraulis mordax).* * Two other minor products of microbiai degradation of DDT are Kelthane and DDCN (Fig. IV-2), although the latter may result in part from chemical degradation. It should be emphasized that complete biodegradation of DDT proceeding via a series of hydrodechlorination steps, as in Fig. IV-1, requires both anaerobic and aerobic conditions. -Itt- �Fish that have received DDT by intravenous injection,** feeding,** cr uptake from water produce TDE and DDE in various proportions in addition to some DDMU. Brook trout receiving intramuscular DDT are reported to produce only DDE.*' -DDT administered to lobsters (Homerus americanus) by intravascular or oral routes is converted to TDE, DDE, and DDA.*7 Sheridan has shown that DDT concentrated from the water is converted to TDE and DDE in the hepatopancreas of the blue crab, Callinectes sapidus.** Lower aquatic invertebrates convert DDT to TDE, DDE, and other metabolites, but daphnids are reported to produce only DDE.** Zinck and Addition have noted that p,p*-DDE is probably a metabolic dead end.*' However, ringhydroxylated metabolites of DDE, shown in Fig. IV-2, have been isolated from the fat of the guillemot (Uria algae) and grey seal (Halichoerus grypus).* * Fries has reviewed data indicating that o,p'-DDT is degraded to o,p'-TDE by mechanisms and rates similar to those for p,p'-DDT.*° It is likely that the degradation pathways presented in Fig. 1V-1 are followed. DDT may also undergo chemical degradation. Photolysis is reported to convert DDT to TDE, DDE, DBF, and p,p'-dichlorobiphenyl, and heat also converts DDT to TDE and DDE. DDT is unchanged after 8 weeks in river water.'1 Bioaccumulation and the Food Chain % The direct accumulation of DDT from water may, in certain cases, make the additional uptake from food insignificant. The algae and bacteria in water are very efficient concentrators of DDT; their small size, and consequently high surface-to-mass ratio, results in rapid and thorough adsorption.** For example, bacteria concentrated DDT from 1 ppb in water to 1,140 to 3,400 times that within 30 minutes,'* and freshwater algae concentrated DDT from 1 ppm in water to 130 to 270 ppm in their cells within 1 week.'8 When exposed to DDT in water at concentrations between 50 and 100 ppt for 3 days, aquatic arthropods achieved increases in concentration ranging from 3,000 to 114,000 times.'* When exposed to DDT in salt water for 2 weeks, the Atlantic croaker concentrated 0.1 ppb by 40,000 times.'* Brown trout exposed to 2.3 ppb and given DDT-free food for 3 weeks concentrated the DDT in their tissues by 3,000 times." DDT, applied onca at the rate of 1 Ib/acre (1.12 kg/ha), persisted in the soil of Maine forests with little change throughout a 9-year period.'* Robins li"v .- in the forest had higher DDT levels than those in surrounding areas, indicating a period of continuous availability of residues through the food chain, as shown in the following table: -19- �Robin Body DDT Concentration (ppm) Time of Analysis 13.53 4.50 3.55 0.47 1 year after treatment 3 years after treatment 9 yearn after treatment Untreated areas DDT applied to a forested area in Montana at the rate of 0.5 Ib/acre (0.56 kg/ha) resulted in the following concentrations in the blue grouse.** Concentration in Fat (ppm) Time of Analysis 80 22 18 Within 1 week of spraying 1 year after spraying 2 years after spraying Predatory or fish-eating birds usually have higher DDT residues than seed-eaters. Alaskan peregrine falcons, which feed primarily on birds, contained far higher residues than the small birds in their area.i?«** Scaup, which feed more heavily upon animal material than mallards, accumulated residues that were 2 to 4 times as great when both were placed on a DDT-treated marsh for the same periods of time.'* Various small mammals were collected in Maine forests after a single application of DDT at the rate of 1 Ib/acre (1.12 kg/ha).'* In the year of treatment, shrews, mice, and voles contained an average of 15.6, 1.1, and 1.1 ppm, respectively. The relative differences between shrews and the mice and voles prevailed throughout the years after treatment. In the same areas, mink, which are carnivorous feeders like the shrews, accumulated higher totcl DDT residues (8.5 ppm) in the first year of treatment than hares (0.08 ppm). For areas treated seasonally with DDT, residues in small mammals increased and decreased seasonally in relation to the treatment times. Food Chain. The bioaccumulation of DDT in the food chain is primarily a consequence of its stability and high fat solubility. In the food chain, energy is transferred from one trophic level to another. In general terms, -20- �only about IOZ of the energy in one troph.lc level will be transferred to the next level, and the rest will be respired or released as wastes.•' Chemicals that are preferentially taken up by living organisms and stored for extended periods, svh as DDT and its derivatives, tend to be concentrated in the foou chain. Examples of DDT bioaccunulation in the food chain**"71 are displayed graphically in Fig. V-l. A review of the extensive literature on aquatic and terrestrial food chains is given by Brown.** These studies are based on measurements of the DDT content in the environment (e.g., soil and water) as well as measurements of the DDT content in tissues of various wildlife species. It would be advantageous and would simplify an environmental assessment if it were possible to relate the concentrations of DDT in the environment (viz., in soil and water) to the toxicological impact on wildlife by using established factors for DDT bioaccumulation and translocation through the food chain. Once the bioaccumulation factors were determined, it vould be possible to relate toxicological effects at dietary concentrations to soil and water concentrations. This relation could be represented by bioaccumulation pathway models, such as those shown in Fig. V-2, The bioaccumulation factors given in Fig. V-2 were estimated from limited actual data for the purpose of demonstration and should be considered hypothetical. Although attempts have been made to predict mathematically the behavior of DDT introduced into the environment,7* the predictive capacity and utility of these models suffer from the enormous complexity of the environment. Due to the many concomitant variables (e.g., environmental site differences, species and strain differences, wide ranges in DDT base concentrations, and different lipid/water partition coefficients and equilibrium factors), it is not possible to establish categorically DDT bioaccumulation factors that have a reasonable level of significance for all ecosystems of the world. It is important to consider each environmental setting individually. Effects on Terrestrial Animals Mammals. No information was retrieved concerning the effects of DDT on mammalian wildlife. As noted in Section IV, acute toxicity for mammals is low in terms of likely environmental concentrations. Data from laboratory studies of mice indicate that teratogenesis and carcinogenesis could result in mammalian wildlife exposed to DDT, but this has not been confirmed by field studies. Likewise, there is no field evidence to indicate DDTassociated reproductive failure in mammals. The high fat solubility of DDT may pose a threat to hibernating insectivores and other mammals that are exposed to high levels of dietary DDT and that release large amounts of DDT to the bloodstream from body fat during periods of high activity and scant food supply. Such DDT releases have been observed for bats containing certain chlorinated hydrocarbon insecticides in their tissues and might also occur for mammalian carnivores. -21- �sea water (0.1 ppb) sea water (0.1 ppb) ' sea water (1 ppb) 40 days oyster (7ppm) "7555 hooked mussel (0.126 ppm) 1.260X hard shell clam (0 6ppm) 600X ' sea water ppb) 11 fish species "(12-20 ppm) phytoplankton estuarlne waters dppb) 2QOX tidal marsh surface water (0.3 ppm) lake sediments (0.014 ppm) pond (0.02 ppm) water (1 ppm) ... . »_ d'tcn 30X 3.4 ppm drywt. «^.. Insects ^..Insects (0.41 ppm) (0.41 ppm) 22QX fish (is ppm) <AV <ftY 10X 22X 75 ppm drywt. - porpoise (100 ppm) ^. 4 species of algae (220 ppm) Fig. V-1. Examples of DDT Bioaccumulation -22- 4-89 ppm wetwt j^. risn _»^__»""» g^flsh j^.8""8 (3.8-5.8 ppm) ?xx (90 ppm, muscl (3.8-5.8 ppm) 2OTV (9°. ppm. muscle ^' 2,411 ppm, fat) ^ rainbow trout (4.15 ppm) black bullhead (3.11 ppm) crayfish (1.47 ppm) 150X ay 1Q-100X ... »». sediment __^ vegetation _^. J«J • v (4ppm) 13X birds �Amphibians (tadpoles) w»t«r ?l "S. 200X '\ A— ~_k»> tarlnolrti fla if 7" sediments —' Fish and Birds yf plants -*«- birds »*. fish water sediments soil 300X 10X —£^ birds {predatory) 20X worms Land-Dwelling Mammals (herblvoi es and ctrnlvores) soil , 20X *~ plants 25X mammals (herbivore) j mammals Carnivore) Insects worms SOX birds /^" Fig. V-2. Models of Bloaccumulatlon Pathways -23- �Birds. DDT and its metabolites are universally distributed so that exposure is essentially continuous, and few, if any, birds are free from these compounds. Although the acute toxicity of DDT to birds is low, direct toxic effects occur due to bioaccumulation of DDT in birds and in their food. The most serious hazard of DDT to birds is that of decreasing their reproductive capacity through eggshell thinning. It is estimated that as little as 67 ppb of DDE (the proximate agent) in the diet can cause a substantial increase in embryo mortality due to eggshell failure. The many instances of bird kills in woodlands sprayed with DDT are believed to be due to secondary poisoning by the oral route and not to contact poisoning.**-•• However, the direct lethal toxicities of DDT to birds are low, as indicated in Table V-l. TABLE V-l. ACUTE TOXICITY OF DDT TO BIRDS7 » Speoies Dosage Route Toxic Effects Mallard Pheasants Coturnix Sandhill cranes Oral , capsule Oral , capsule Oral , capsule Oral, capsule LD5Q > 2,240 mg/kg LD5Q a 1,296 mg/kg LDso s 841 mg/kg LD50 > 1,200 mg/kg Mallard Pheasants Bobwhites Coturnix Oral, 5 Oral , 5 Oral , 5 Oral , 5 LC50 LCso LCso LCso Pheasants p,p'-DDT, oral Technical DDT, oral days days days days » 850-1,200 ppm * 300-700 ppm » 600-1,000 ppm • 400-600 ppm LC5Q - 550 ppm LCso * 935 PPm The direct toxic effects of DDT to birds accompany bioaccumulation in the birds' food. Although bioaccumulation is most pronounced for precatory birds, it also can be significant for birds lower on the food chain. i?or example, soil contaminated with 5 to 10 ppm DDT is sufficient for earthworms to pick up 50 to 200 ppm, which could result in a lethal dose for a robin (ca. 3 rag)."* High residues of DDT in bird fat and other tissues can be mobilised to become lethal if the birds are starved or hyperactive."* These processes reduce the adipose fat and release DDT into the body circulation to concentrate in the nervous system. House sparrows with DDT residues of 800 ppm in body fat displayed no adverse physiological signs if well fed, but died if not well fed; the DDT mobilization engendered tremors that further reduced fat and sent lethal concentrations into nerve and brain.•*>•* The minimum content of DDT in the brain at which death occurs is 50 ppm for American robins and 60 ppm for house sparrows,'1 while it is 14 ppm for female ring-necked pheasants." -24- �Concerning reproductive e f f e c t s , a 30% decline in breeding pairs'of the fish-eating osprey on the coast jf Connecticut in 1963 was found to be associated with a high body content of DDT residues, especially DDE, 111 *** The reproductive failure was later related to a reduction of the eggshell' thickness due to contamination of the eggs by DDT and its metabolites. Feeding experiments with mallards showed that 40 ppm of DDE in the diet resulted in frequent shell cracking, leading to 40% embryo mortality and 75% reduction in duckling production.* 5 A concentration of 20 ppm of DDT in the diet of mallards resulted in 20% reduction in eggshell thickness.** A concentration of 10 ppm of DDE in the diet caused 25% shell thinning in the American sparrow hawk, 8 7 13% in the screech owl,'* and 18 to 29% in the black duck." DDT in the diet of pheasants had little or no effect on egg production or fertility, but hatchability and chick survival were reduced at concentrations of 100 ppm or more." In bobwhite quail on a diet containing 100 ppm, egg production was normal, but fertility and hatchability were reduced, and chick survival was eventually zero.*' In addition, high dietary doses of DDT have reduced sperm production in cockerels'* and the bald eagle. 9 * It is generally accepted that DDE is the major shell-thinning factor, because a linear inverse relationship between shell thickness and DDE content of the egg has been demonstrated for the prairie falcon, herring gull, doublt crested cormorant, brown pelican, and peregrine falcon.'**** In general, whenever the residues induced eggshell thinning more than 10% below the normal thickness, that bird population would decline.'* Concentrations of DDE that elicit this effect in various species of birds are listed in Table V-2. The bird prey for one population of peregrine falcons have whole body residues of 0.3 to 6*0 ppm DDE, whereas the f.it and eggs of the falcons contain 560 and 15 ppm DDE, respectively.* 7 This concentration factor of 2.5 to 50 for eggs, combined with an observed concentration of 8 ppm in peregrine falcon eggs for onset of reproductive failure (Table V-2). corresponds to a dietary limit of 0.16 to 3.2 ppm. If the same concentration factor is arbitrarily assumed for other birds, then the dietary threshold for reproductive failure would fall in the range of 1.6 to 32 ppm for the great blue heron, 0.05 to 1.0 for the osprey, and 0,02 to 0.4 for the brown pelican. Based on the latter two birds being fish-eaters, it appears that substantially lower levels of DDE (and hence DDT) in fish may be required to assure the survival of these birds than to protect human health. Effects on Aquatic Organisms Because the proportions of the various isomers and metabolites of DDT in different environmental samples are quite distinct, and because the toxicological data base for aquatic organisms is large, every effort has been made to identify the toxic effects associated with each specific isomer or metabolite throughout this section. -25- �TABLE V-2. CONCENTRATIONS OF DDE TN BIRD EGGS RESULTING IN 10% ' REDUCTION IN NORMAL SHELL Tl'.iCKNESS Bird Species DDE Concentration in Eggs (ppm wet weight) Double-crested cormorant Reference 20 95 Prairie falcon 7 96 Brown pelican 1 97 t r e a t b l u e heron 80 71 Herring gull 70 71 A t l a n t i c gannet 25 71 White pel lean 10 71 Fish-eating osprey 2.4 99 Alaskan peregrine f a l c o n 8 68 Fish. The acute toxicity of p,p'-DDT to fishes has bee.i reviewed by Pimentel71 and others. 18C ~ I •* Some representative data are presented in Table V-3, which shows that the 96-hr LC50 for most fishes falls between 1 and 20 wg/1. Fish and Wildlife Service investigators at the Fish-Pesticide Research Laboratory in Columbia, Missouri, report 96-hr LCs^'s in this range for 18 common freshwater fishes.110 They also report that p,p'-DDT is roughly three times as toxic to bluegills (Lepotnis macrochirus) at 7°C as at 24°C. Macek notes that for most common formulations containing DDT and other pesticides, acute toxicities to bluegills are additive.11* The low LC5Q valiies may be due to the rapid uptake and concentration of DDT in fish. For example, brown trout exposed to 2 ppb DDT can concentrate it about 500 times in the gill tissues and about 3,000 times in the muscle.*' The gills of 2-lb brown trout pass about 700 liters of water per day. 11 * In addition, certain fish, such as catfish, appear to be fairly tolerant to DDT under laboratory conditions, whereas in a natural setting they may succumb through bottom-feeding at the sediment level. Sublethal concentrations of DDT to adult fish may lower their reproductive success because DDT accumulates in egg yolk and kills the fry shortly after they hatch from contaminated eggs.'*»102 The DDT is passed into the egg yolk, the embryo develops and hatches, and at the stage of -26- �TABLE V-3. ACUTE TCXICITY OF p,p'-DDT TO FISHES BY STATIC BIOASSAY Temp. Species Rainbow trout Salmo gairdneri Exposure Time (hr) LCso (ug/O 7 (5-10)a 3.8 (3.4-4.3) 1.72 (1.42--2.09) 28 0.26 Reference 104 105 106 107 108 13 16 12.9 96 96 96 96 360 Brown trout Salmo trutta 13 96 2 U-3) 104 Brook trout Salvelinus fontinalis 13 96 7.4-11.9 106 Cutthroat trout Salmo clarki 13 96 0.85-1.37 106 13 9-11 13 96 96 96 13 96 0.68 110 18 24 23 96 96 96 8 (6-10) 2.2 ( . - . ) 1826 7 104 105 111 Redear sun fish Lepomis nticrplophus 18 . 96 5 (3.9) 104 Largemouth bass Micropterus salmoides 18 96 2 (1-3) 104 18 24 96 96 96 Coho salmon Oncorhynchus kisutch Chinook salmon Oncorhynchus tshawytschak > c Bliiegill Lepomis macrochirus Goldfish Carassius auratus Carassius carassius -27- 11.3-18.5 13 4 (3-6) 21 (14-30) 9.8 (7.3-13.2) 25 106 109 104 104 105 107 �TABLE V-3. (Cont.) Exposure Time (hr) LC50 (ug/D Species Temp, CO Carp Cyprinufl carpio 18 96 10 (7-13) 104 Fathead minnow Pimephales promelas 18 96 19 (13-27) 104 18 24 26 96 96 24 16 (9-28) 13.5 (9-20) 104 105 111 Black bullhead Ictalurus me las 18 96 5 (3-7) , 104 Yellow perch Perca flavescens 18 96 9 (7-11) 104 Channel catfish Ictalurus punctatus Mosquitofish Gambusia af finis 34 Reference 96 96 20 27 112 111 Guppy Poecilia reticulata 96 3 112 Mozambique mouthbreeder Tilapia mossambica 96 7 112 Aholehole Kuhlia sandvicensis^ 96 3.9 112 Nehu Stolephorus purpureus^ 12 1.0 112 96 0.53 ( . 8 0 8 ) 03-.4 0.9 1.13 110 Striped bass Roccus (Morone) saxatilis^ Roccus (Morone) saxatilis^ 17 96 -28- �TABLE V-3. (Cont.) Temp. Species Shiner perch Cymatogaster aggregate Exposure Time (hr) LC5Q (wg/O Re f erence 13 17 96 96 7.6 0.45 114 110 Dwarf perch Micrometrus minimus^ Micrometrus minimus'' » c 13 18 96 96 4.6 0.26 114 110 White seaperch Phanerodon furcatus^« c 19 96 0.74 110 English sole Parophrys vetulus^* 0 16 96 0.91 110 Pacific staghorn sculpin Leptocottus armatus"*0 19 96 0.98 110 Rubberlip seaperch Rhacochilus toxotes^« c 19 96 1.01 110 Goby Acinthrogobius flavimanusb» c 19 96 2.40 110 19 19 19 19 19 24 48 96 120 144 Speckled sanddab C i th ar i ch t h y s 8tigmaeus^» c a. b. c. d. • 10.0 7.2 3.7 1.7 0.9 Numbers in parentheses are 95% confidence interval. Seawater. Dynamic bioassay. Brackish water. -29- 110 110 110 110 110 �final yolk sac adsorption after hatching, the fry will die if the DDT concentration in the yolk is sufficiently high. 11T » l l i This phenomenon was first observed in the lake trout of Lake George, New York; 1 1 ' and later at Ja-jper, Alberta; 1 4 * Lake Taupo, New Zealand; 121 Lake Michigan; 1 * 2 Sebago Lake, Maine; 111 and other locations.'* Data for studies in these areas are listed in Table V-4 and indicate that DDT concentrations in water a» low as 0.004 ppb can cause a significant increase in sac-fry mortality. No reports were recovered describing systematic studies of the chronic effects of DDT on life stages of fishes. A DDT concentration of 5 mg/1 has been shown to result in 4(!% mortality of carp embryos reared j.n vitro.' 2 * Exposure of Atlantic salmon (Salmp sf.lar) eggs to 50 pg/1 of DDT at gastrulation retards behavioral development in the newly hatched alevins. 1 2 * The coughing frequency in juvenile coho salmon was found to be enhanced significantly after 4 days' exposure at a sublethal concentration of 5 ug/1. 1 ** High sublethal (0.3 to 3 ug/1) levels of DDT have been found to result in loss of glycogen and other pathological changes in the liver of 7 zebrafish (Br^chydanio rgrio) and, to a much lesser extent, of guppy. 1 2 Interrupted exposure of salmon;d fishes to high sublethal concentrations of DDT is reported to raise the lower lethal temperatures, alter the temperature selectivity,12diminish learning ability, and affect the central nervous system in general. ** 1 ** Continuous exposure to 110 Mg/^? for 4 days is said to alter the exploratory1'1 and locomotor *2 behavior of goldfish (Carassius auratus). Desaiah e_t al. have presented evidence for 50? or greater inhibition of activity of mitochondrial Mg2+ ATPase, an important energy-linked enzyme, in brain homogenates of fathead 1 minnows chronically exposed to DDT at a level of 0.5 ug/1 for 266 days. " There is also a substantial, although lesser, drop in gill Na-K-ATPase activity. The latter enzyme functions in osmoregulation in marine fishes, and in this regard, Leadem e£ ajU have found that seawater-acclimated rainbow trout receiving 2.75 tng/kg DDT/48 hr in their diet exhibit 1 impaired osmoregulation as well as inhibition of gill Na-K-ATPase activity. '* Kinter £t al. have reported similar disruption of osmoregulation in two marine species, mummichog (Fundulus heteroclitus) and American eel (Angui11a rostrata), at lethal DDT concentrations. 111 Weisbart and Feiner report that goldfish (C. auratus) exposed to DDT at a level of 17.5 to 35 wg/1 exhibited no clear" evidence for impaired osmoregulation. 1 " This agrees with the observation of Leadem e£^l_. that osmoregulation is unimpaired by DDT in the diet of the freshwater rainbow trout. • The 90-dose (30-day) oral LD5Q for juvenile coho and chinook (Oncorhynchus tshawytscha) 7 salmon have been reported as 64 and 27.5 ing/kg/day, respectively. 1 ^ Sublethal 1oral doses may result in loss of light discrimination in rainbow trout. '* -30- �TABLE V-4. SAC-FRY MORTALITY FOR VARIOUS FISH SPECIES Fish Species DDT Cone. in Eggs (ppm) Effect Estimated DDT Cone. in Water* (ppb) Reference Lake trout 3-355 Fry containing more than 3 ppm died at the time of final adsorption of the yolk sac 0.03 119 Brook, rainbow, and cutthroat trout >0.4 30 to 90% sac-fry mortality >0.004 120 Rainbow trout 5 45% sac-fry mortality 0.05 121 Coho salmon 1.1-2.8 15 to 75% sac-fry 0.011-0.028 mortality, respectively 122 The DDT concentrations in water were estimated using a concentration factor of 100,000. The factor was based on data from a study with fathead minnows reared in 2 ppb DDT for a 9-month period. DDT concentrated in their eggs to more than 100,000 times the water concentration.1** This is the only long-term study giving both egg and water concentrations that could be found in the literature. Fragmentary evidence indicates that o,p'-DDT is less toxic to fish than p,p'-DDT. The 96-hr LC5Q for goldfish (C. auratus) t as measured by Ginsburg, 1 ** is 1.0 mg/1 for o,p'-ODT, compared with about 0.06 mg/1 for the p,p'-isomer. Gardner reports that brook trout fingerlings are unharmed by 24-hr exposure to o,p'-DDT at a concentration of 0.05 mg/1, although there is a noticeable effect on temperature selection at 0*02 mg/1, i.e., cooler water is preferred by exposed fish. 1 ** According to Alabaster, the 24-hr LC5Q for harlequin fish (Rasbora heteromo_rj)ha) ie 30 ug/1 for o,p'-DDT, compared with 13 jig/1 for the~p,p"'-"Isomer.l* * No information was retrieved for m,p'-DDT. -31- �Toxicity data for p,p'-TDE (ODD) have been reviewed by McKee and Wolfe. 1 " Fragmentary evidence, presented in Table V-5, indicates that TDK is highly toxic to fishes, although perhaps a half ovder of magnitude less toxic than p,p'-DDT. Gardner has demonstrated that high sublethal levels of TDE affect temperature selection by fingerling brook trout. 12 ' He further reports that brook trout are unharmed by 24-hr exposure to o,p'-TDE at a concentration of 50 yg/1, although there is some effect on temperature selection at 10 pg/1. 1 ** No information was retrieved for m,p'~TDE. Gardner has found that brook tr< u; •".•..-• 'mharmed by exposure to 50 -;g/l of p,p'-DDE for 24 hours and that there la almost no effect on temperature selection. 1 ** Applegate et_ al_. re >orc' Chat v^inbow trout, bluegills, and the larvae of sea lampreys -(Pt£: y .;>I • *},"',.*•'.>• "£ arc ""affected by 24-hr ; exposure to DDE at 5 mg/1 and 5: ' " o t h t j . s " report 96-hr LC^Q'B of 10 to 100 ng/1 for bluegills and rai'•);-w trout at 24° and 13°C, respectively. 11 * No information was retrieved for m,p'-DDE. Reptiles. No quantitative toxicity data were recovered, but Stickel has stated that the box turtle population of a Maryland forest was not noticeably affected by DDT applied at a dosage of 2 Ib/acre (2.2 kg/ha). 1 ** Evidence both for and against loss of reptiles through land application of DDT is summarized by McKee and Wolfe. 1 ** Direct treatment of ponds at DDT concentrations of 2 ppm or more has killed water snakes and turtles. 1 ** In the Brazos River floodplain of Texas, where cottonfields had been heavily treated with DDT, the average residues in the fat bodies of aquatic snakes were DDE, 510 ppm; TDE, 1.5 ppm; and DDT, 16.0 ppm. 1 * Tine DDT residues in the brain did not exceed 1.5 ppm, and fat-body residues in terrestrial snakes were much lower than in aquatic snakes.1** In vitro treatment of cellular fractions from various tissues of six species of terrestrial turtles resulted in negligible to substantial inhibition of Mg2*-, (Na + , K*)-, and (Na+, K + , Mg2+)-dependent ATPase at DDT levels of 2 to 76 mg/l. 1 *** 1 ** Similarly, in vitro treatment of cellular fractions from various tissues of the red-eared turtle, Chrysemys scripta elegans, resulted in negligible to substantial inhibition of ATPase at TDE or DDE levels of 2 to 76 mg/1. 1 ** Amphibians. For tadpoles of Fowler's toad (Bufo woodhousii fowleri) and the chorus frog (Pseudacris triseriata), Sanders reports 24-hr LC5Q habitat water values of 2.4 and 1.4 mg/1, respectively, 1 * 7 whereas a 96-hr LC5Q of 0.27 mg/1 for bullfrog tadpoles is reported by Carter and Graves. 1 1 1 Another reference gives a 96-hr LC$Q of 0.8 mg/1 for 5-week-old tadpoles of P. triseriata and 0.74, 1.0, 0.1, and 0.03811 mg/1 for B. woodhoasii tadpoles of 1, 4 to 5, 6, and 7 weeks, respectively. * These data are summarized in Table V-6. A lethal concentration of 0.15 mg/1 is given for Bufo bufo tadpoles. 1 ** Some relative and highly ambiguous toxicity assessments based on DDT application data have been provided by Pimentel 71 and Cooke. 1 ** -32- �TABLE V-5. Species ACUTE TOXICITY OF p,p'-TDE (DDD) TO FISHES Temp. CO Exposure Time (hr> LC50 (pg/1) Goldfish3 Reference 1,000 100 <2,600 15,000 100 110 Channel catfish8 20 18 96 96 Bluegill* 24 96 Striped bassc Morone saxatilis 17 96 Brook trout Salvelinus fontinalis 10 24 45 128 Rainbow trout 3 13 96 43-93 110 Fathead minnow3 18 96 1,000-10,000 110 largetnouth bassa 18 96 39 110 Walleye 3 18 96 10-100 110 a. b. c. d. 30b >10 2.5 <1.6-4)d 100 110 113 Species not given. Toxicity threshold. Bioassay in saline water. Numbers in parentheses are 95Z confidence interval. Field studies showed that 0.1 kg DDT/ha applied as an emulsion did not kill tadpoles, but 1.0 kg DDT/ha achieved 80% mortality in two days.1** The toxic effects on frog and toad tadpoles o_ DDT sprayed in the field at 0.4 to 0.5 kg/ha is given by Cooke.1*' The DDT was sprayed on the water surface,.as would be appropriate to kill mosquito larvae. Five water sites were monitored. DDT concentrations in surface water and in water at a depth of 20 cm decreased as the size of the water body increased, to the extent that DDT was not detected (<0.02 ppb) in water from the two larger sites. The DDT residue concentrations and the behavioral and morphological abnormalities of the tadpoles for the three smaller sites are summarized in Table V-7. The residues were measured one day after spraying. It is important to note that virtually all of the DDT sprayed was taken ui> by algae or incorporated elsewhere within only 3 days. Hence, the increases in -33- �the DDT levels in the tadpoles after the third day may be due to direct ingeoiion of DDT-contaminated algae. A schematic diagram of behavioral abnormalities versus time after spraying DDT is given in Fig. V-3. Average DDT concentrations are derived from data of Table V-7 assuming that there is a linear gradient of concentration with depth. TABLE V-6. : Species TOXICITY OF DDT TO TADPOLES Exposure Time (hr) LC5Q (tng/1) Reference Bullfrog 96 0.27 111 Chorus frog 24 96 1.4 0.8 147 110 Fowler's toad 24 2.4 147 (1 week) (4-5 weeks) (6 weeks) (7 weeks) 96 96 96 96 0.74 1.0 0.1 0.038 110 110 110 110 For tadpoles of Pseudacris triseriata and Bufo exposed to p,p'-TDE, 96-hr LC50's of 100 to 1,000 and 18 jig/1, respectively, have been reported. 11 ' No other information concerning isomers or metabolites was retrieved. Invertebrates. For the most part, only references dealing with nontarget species were retrieved. Toxicity data for arthropods, taken from Pimentel's review, 71 Malina's review, 1 " and some recent papers, are summarized in Table V-8, which shows that marine and freshwater species demonstrate about the same order of acute sensitivity to DDT as fishes, although ostracods appear to be more resistant. There is also evidence for impaired reproductive capability in ostracods, 15 ' brine shrimp, 1 ** and Daphnia at sublethal levels. 1 ** Ingested DDT has been shown to be harmful to crayfish (Procambarus clarkii), blue crabs (Callinec tes sapiduii), *» T »*" and fiddler crabs (Uca pugnax),'* * but the reported data are not readily quantified. Larvae of two caddisflies (Hydropsyche pellucidula and H. instabilis) have been found to construct irregular webs when exposed to DDT at sublethal levels (2.5 jig/1). 1 '* In field studies, it was found that when an unprotected stream was sprayed directly with 1 Ib/acre (1.1 kg/ha), nymphs of all species of mayflies were exterminated and larvae of every species o caddisfly were affected to some extent. 1 * . -34- �Normal k /" Estimated Average DDT Concentrations In the Water 17.5 ppb £ 2.26 ppb ——• 0.57 ppb Reslgnet Dead 4 6 8 1 0 Days After Spraying DDT Fig. V-3. Effects of DDT on Frog Tadpoles -35- 12 14 �TABLE V-7. Total Water Residues One Day After Spraying3 (ng/1) 20 cm below Surface BEHAVIOR AND MORPHOLOGICAL ABNORMALITIES OF TADPOLES EXPOSED TO DDT DDT Levels in Tadpoles (vg/g) Behavioral and Morphological Abnormalities Day 1 Small Ditch 36.2 Day 14° 7.90 6.52 None left Franticc None 3.27 None left Frantic None 0.70 0.77 0.24 Some normal, Some frantic None 0.64 0.38 Frantic None 1.16 0.83 0.52 1.23 1.07 1.97 Frantic Frantic None None Behavior Day 14 Day 3 Day 2 0.99 Surface Day 1 1.21 Site Abnormalities Behavioral Abnormalities Behavior Abnormalities __ Larger 10.5 17.5 4.9 Bitch Fool 2.7 1.9 Resigned All survivors-abnormal No survivors snouts, 4/8 dead tadpoles had tails laterally curled to left Some frantic, All survivors-abnormal No survivors some resigned snouts 4 normal, Few normal, None 1 moribund most frantic, few resigned 13 normal. Most frantic, 2 dead tadpoles with 3 moribund upturned tails few resigned Frantic Frantic None None Normal Mormal a. Other than the 0.19 wg/1 from below-surface sample of small ditch taken on Day 3, no residues were detected in water samples on Days 3 and 14, ...cone. <0.02 wg/1; spraying was uneven; two large sites had no detectable (<0.02 vg/1) DDT in water. b. Increases probably due to eating DDT-contaminated algae. c. ?raittic • hyperactivity, greatly excited, frencied Resigned » passive, submissive Progressive stages of abnormal behavior. Moribund - dying. / (All showed slow rite of metamorphosis.) — 1 downcurved in body and tail 1 with abnomial snout , 2 dovncurved None None �TABLE V-8. TOXICITY OF p,p'-DDT TO ARTHROPODS Species Exposure Time (hr) EC50 or LC50 (wg/l) Reference Sand shrimp 24 3 71 Seed shrimp Cypridopsis vidua 48 54 151 Glass shrimp Palaemonetes kadiakensis Grase shrimp Stonefly Pteronarcella badia Classenia sabulosa Pteronarcys californica , Acroneuria pacifica Waterflea Daphnia pule* Daphnia magr. a Simocephalus serrulatus 4.2 2.3 48 96 12 24 151 110 71 •/ 12 1.9 16 10 3.5 41 19 100 7.0 180 24 96 24 96 96 24 48 96 96 96 0.36-3.6 4 1 0.67 0.4 48 48 96 366 48 71 110 71 150 110 71 71 150 110 150 71 151 150 152 71 Os traced Cyprinotus incongruena Cypridopsia vidua 48 48 l,300a 230a 153 153 Brine shrimp Artemia selina 48 46a 154 -37- �TABLE V-8 (Cont.) Exposure Time (hr) Species Crayfish Procambarus acutas Orconectes nais (10-week) Reference 48 96 96 •** Gammarus fasciatus 22.5 1.0 151 110 4.7 4.0 151 110 24 48 96 48 96 Am phi pod Gammarus lacustris 155 108 110 48 96 Sowbug Asellus brevicaudus 3(7. 2)c 0.24 30 48 96 Damseifly Ishnui-a verticalis Hermit crab3 EC50 or LCjo (pg/D 4.7 2.1 1.0 3.6 3.2 71 71 110 151 110 71 7 1.85 110 96 Purple shore crab Hemigrapsus nudus *-• Market crab Cancer magister'"" 24 96 ' 4.6 110 48 3.3-10 156 » * • Brown shrimp Crangon crangon a. Species not given. b. Extrapolated from author's data. c. Value in parentheses for crayfish acclimated to natural, DDT-contarainated water of an unspecified concentration. -38- �Although mollusks are not so readily killed by DDT, the growth of eastern oysters is reported to be reduced significantly (and reversibly) at a level of 0.1 wg/1, 7 1 and survival of the larvae of the American oyster (Cr88803trea virginica) is diminished by 202 at a level of 25 yg/1. 1 ' 1 Annelids are so insensitive to DDT intoxication as to present a dietary hazard to predator organisms. The 96-hr 1050 for the buffalo leach (Hirudinari manillgnsis) exceeds 100 mg/l, 1 '* and tubeficid worms (Branchiura sowerbv_i"> are said to exhibit no mortality after 72 hours at a level of 4 mg/l and 21°C, although they are completely destroyed when exposed to the same concentration at 4.4° and 32.2°C. l f l The extrapolated 96-hr LC5o for a planarian (Polycel.is felina) is 1.26 mg/l at-6.5°C."* Earlier data for invertebrates have been reviewed by McKee and Wolfe, 1 1 * and some additional toxicity data are contained in Reference 108. Fragmentary evidence, presented in Table V-9, indicates that o,p'- and m,p'-DDT may be less acutely toxic to mosquito larvae than the p.p'-isomer. No information concerning nontarget species was retrieved. TABLE V-9. TOXICITY OF DDT ISOMERS TO MOSQUITO LARVAE Anopheles quadrimaculatus* » • » » » • • Isomer p,p'-DDT 24-hr LC50 (Mg/D 48-hr LC50 (Wg/l) Aedes aegypti'** 96-hr LCso (wg/1) < 2.5 2.5 11 350 o,p'-DDT 15 10 ra,p'-DDT 15 <10 a. 4th instar. Data relating the acute toxicity of p,p'-TDE to arthropods are summarized in Table V-10. Comparison of Tables V-8 and V-10 reveals that for many arthropods TDE is equal to or greater in tox;city than DDT. McKee and Wolfe have reviewed pesticide application data and note that the larvae of Chaoborus (phantom midge) and gnats are "controlled" at 13 tc 14 vg/l and chironomid (midge) larvae are temporarily eliminated.1** With a 96-hr LCso of 740 vg/1, TDE is slightly more toxic to the freshwater planarian Polycelis felina than DDT.1** -39- �The 96-hr LC50 of p,p'-DDE to the freshwater planarian Polycelis felina is 1.23 mg/1, only slightly more than the corresponding value for DDT."* No further information was retrieved concerning isomers or metabolites. TABLE V-10. ACUTE TOXICITY OF p.p'-TDE (ODD) TO ARTHROPODS Species Exposure Time (hr) EC50 or LC50 (ug/D Reference Amphipod Gammarus lacustris Gammarus fasciatus 96 96 Sowbug Asellus brevicaudus 96 Water flea Paphnia magna Daphnia pulex Simocephalus nerrulatus 72 48 AS 0.1« 3.2 4.5 167 108 108 96 72 0.68 O.la 108 167 Mosquito (4th instar) Anopheles quadrimaculatus 24 2 168 Stonefly Pteronarcys californica 96 380 1C8 Glass shrimp Palaemonetes kadiakensis 0.64 0.86 10 a. Sublethal effects. -40- 108 108 108 �Effects on Microorganisms Luard has reviewed, in part, the literature on DDT toxicity to freshwater and marine phytoplankton, and notes evidence for a wide range of sensitivities. 1 *' Other data are contained in Reference 108. A few marine species exhibit inhibition of photosynthesis at 1 to 10 yg/1, but in general there is no e f f e c t on growth at levels below 100 ug/1 (see also Pimentel 7 1 ). A recent study shows an even higher level of resisti.--** in Euglena.' 7 * Bacteria also appear to be resistant to DDT. The growth of Bacillus megaterium in nutrient medis is unaffected by 100 mg/1 of DDT, although the death rate of resting cells is measurably enhanced at 1 mg/1. 1 ** Growth of Azotobacter^ chrooc occum is said to be unchanged in the presence of 400 mg/1. l7z The growth rated of Pseudomonas fluorcscens and Staphylococcug aureus, but not Escherichia coli, are noticeably inhibited at 50 mg/1. 1 '' It is probably safe to assume that microorganisms will be unaffected by p,p'-DDT at levels selected to protect fish and invertebrates. The chernolithotrophic nitrofier, Nitrobactor agilis, is completely v inhibited by TDE at a concentration of 10 tng/l and measurably inhibited at 0.1 mg/1. 17 * % DDE (as well as DDT) at a concentration of 10~6 to 10~5 M (0.35 to 3.5 mg/1) is said to inhibit photosynthetic electron transport in the green algae Codium fragile and Chaetomorpha area^ and in isolated chloroplasts. 1 ** DDE is reported to be more toxic than DDT to the marine dinoflagellate Exuviella baltica, causing significant growth inhibition at levels as low as 0.1 u g / 1 . 1 7 3 N o other information concerning isomers or metabolites was retrieved. *" VI. STANDARDS AND CRITERIA FOB DDT Air Threshold limit values for the workroom environment: 17 * Time-weighted average: 1 Short-term exposure limit: 3 ink in g Water and Food Allowable daily intake:* 0.005 mg/kg/day Maxiuium concentration in fish and agricultural products for interstate commerce: 177 5 ppra �Water f or Aquat ic Lif e - EPA recommended criterion:20 0.00023 l»g/l (24-hr average) and 0.00041 (not to be exceeded at any time) VII. EFFECTS OF DDT ON A MODEL ECOSYSTEM A model ecosystem is used here to illustrate the effects of DDT waste product disposal at U.S. manufacturing sites operated from the mid-1940's to the late 1960's. Data that would accurately and completely define the extent of the hazards resulting from DDT contamination at particular sites are not available. Thus, a hypothetical site was created to demonstrate an approach for relating toxicological and ecological data to levels of contamination and to demonstrate the types of data required for establishing such a relation. The model site was developed from limited data available from actual contaminated sites*7*""1*11 and from hypothetical circumstances (such as geology and hydrology) offered for the purpose of demonstration. The following topics are considered: manufacturing practices, composite hypothetical site, observed DDT concentrations, predicted effects, and decontamination objectives. Manufacturing Practices The contaminated areas of primary concern are those in the vicinity of sites previously used for the manufacture of DDT, typically following World War II until the late 1960's. As a result of manufacturing, handling, and disposal practices prevalent then, large quantities of DDT and its isomers and analogs were conveyed by surface water runoff through drainage ways into traversing E-treams that empty into lakes and major rivers. Depending on the manufacturing site, the methods of DDT handling and storage, and the time manufacturing ceased, theie are wide ranges of possible levels of site contamination. During the manufacturing period, it is possible that tons of DDT in th« form of blocks were present on the ground surface, readily accessible to leaching. After the plants were closed, massive quantities of DDT were either disposed of in burial sites and landfills, destroyed by incineration, or simply left on the ground surface. The DDT residues in areas surrounding manufacturing sites built up over the years as process water containing DDT was discharged to settling ponds or ditches. Analyses of soil, sediment, water, and biological samples showed that undegraded DDT at some sites was being leached to surrounding areas. For example, fish caught in a major river about one mile from a contaminated site contained as much as 500 ppm DDT, two orders of magnitude greater than the maximum concentration allowable for interstate commerce. Biological surveys of the streams in contaminated areas indicated that species diversity is adversely affected in these areas."* -42- �Due to DDT's low solubility in water, the most highly contaminated areas other than DDT storage or burial sites are streambed sludges. This is because the waterways leading from a plant act primarily as carriers for suspended DDT, which settles out in the streambeds. In addition, areas that are not vegetated pose a particular problem since erosion by wind and rain can carry land contaminants into surface waters. Similarly, open du.nps of waste DDT can be constantly errded by surface runoff. Composite Hypothetical Site A map of a composite hypothetical site is given in Fig. VII-1. In later sections of this report, the effects (approximate) of the site configuration on the environmental impacts of various DDT concentrations are considered. The features of the composite hypothetical site are: 1. the DDT manufacturing site discharging to a large drainage ditch 2. a train of shallow lakes and wetlands containing food fish and surrounded by natural areas 3. ipring flooding, periodically causing redistribution of sediments 4. ultimate drainage of DDT-containing waters into the river, which is open to boating and fishing 5. the possibility of free movement of fish and other wildlife from lakes to and from the river. Thus, there are wetland areas where DDT in sediments can persist over many years. There are also physical and biological mechanisms for the periodic redistribution of DDT in the environment. Finally, DDT can enter wildlife and human food chains in many ways. Observed DDT Concentrations Concentrations of DDT in soil, sediment, and various water bodies as well as in various wildlife species are listed in Table VII-1 as a function of the downstream distance from the DDT plant. For simplification, it is assumed that the waste DDT that is buried or landfilled is located at distance zero and, because the principal carrier of DDT is water, that concentrations of DDT in water and underlying sediment are indicative of the level of contamination at each downstream distance. (The referenced data are those for actual areas surrounding DDT plants. Some of these data, however, correspond to samples collected and analyzed more than; 15 years ago and, thus, may not be representative of present conditions in the areas. These data, possibly out of date, are included -43- �1 1 ^(^••••ftTijr^WJtWR*.**-*^"'* "' "*-'^'^ /'nv.f~'"in'' Drainage Ditch Direction of Spring Floods ^ X \ \ Flp. VIM. Map of Model DDT Plant Site -44- �TABU! VII-1. AREA AHD WILDLIFE COOTAHTHAT1OH LEVELS FOR VARIOUS SITES Approximate Distance from Plant Site (*ilea) 0 Approximate Location Ground surface of abandoned production ' area Median Sampled in Area Reference Concentration of DOT in Area* Open dumps of vaste TOT, 100 Ib Reference Concentration of OOT° <PP») Huscle Fat 1001 1002 0 Baseoent of abandoned buildings Blocks of DDT covered vith clay, 3 tont 0 * ; Wildlife Species Stapled Domsatic »ever in plant Water 181 6.0 ppb 0 Drainage ditch Water 181 2.7 ppb 005 -. Drainage ditch Sediscnt 181 7 , 0 ppa 000 O.I Clo«*d drainage ditch Topsoil 181 110 p!» 1 Cloaed drainage ditch Top*oil 181 0.1 pp* 1 Strea* A Water Sedinent 181 2.3 ppb 1 0 0 pp» .0 2 Lake Water Sedinent 1 ppb 100 pp» Crow Rabbit Oposaim Fox Fish Fish Birds Fish Deer Birds Oterbivores) Birds (carnivores) Fish 179 179 179 179 183 184 184 182 183 40 1 23 27 314 20-200 10 300 0.2 2 20 200 750 17 240 SO 800-2.817 50 500 100 ,0 �TABLE VTt-1. Approximate Distance from Plant Sice (niles) Approximate Location Kediim Sampled in Area Reference (Coot.) Concentration of DDT in Area* Wildlife Species Sampled Reference Mannals 3 JS- 3 Lake Bayou Water Sediaent 180 180 Creek6 Water Sediwent 3 River Water Sediment 0.1 ppb 5 PP" 181 0.03 ppb 1 ppo Shad Carp Bass Catfish Ban SunfUh Bluettill Fish Birds (herbivores) Birds (carnivores) Mamuls a. Wf solubility in water • 1.2 F 50 0.5 ppb IS ppa 4 b. FDA tolerance for fish • 5 p»»« . e. Hypothetical data. 5 0.5 ppb 6 ppra Water Sediscnt Concentration of DDTb <PP»> Muscle Fat 182 182 182 184 ISA 184 184 178 71 29 6 112 112 7 35 412 O.OJ 1.0 0.5 1 10 S �here to demonstrate an approach for relating toxicological and ecological effects to levels of DDT contamination.) The concentrations of DDT in wildlife at various distances downstream from the hypothetical site are listed in Table VI1-2, along with the concentrations of DDT in water bodies and sediments at these distances. It can be seen that the high concentrations in water and sediment at the shorter distances are reflected in high concentrations in the tissues of the species sampled. Conversely, the concentrations at a distance o£' 5 miles approach the average levels in t.he United States. For these data, the differences between concentrations found in muscle and fat were estimated from actual measurements. Predicted Effects The ultimate objective of this analysis is to predict potential site-specific environmental impacts of DDT contamination. The predictions, in their simplest form, relate environmental impacts to concentrations of DDT in soil and water. With such information and analyses of DDT in soil (sediment) and water samples, one can estimate impacts of environmental contamination and the benefits of cleaning up the soil and water to known levels. Preceding sections of this report provide evidence that currently available literature data are sufficient to relate environmental impacts to four types of exposure information: DDT concentrations in an affected organism, dietary DDT levels, acute or chronic; doses of DDT, and the DDT concentration in water (for aquatic species). If these four types of information can be related to soil and water concentration data, the objective will be met. USAMBRDL has devised a procedure for estimating safe exposure levels, called preliminary pollutant limit values (PPLVs), from laboratory or field dat/» to protect the health of humans and other animals." >"7 This procedure assumes an equilibrium (or steady state) relationship for a pollutant distributed among soil or sediment, water, and biota. However, as is evident in Tables VII-1 and VII-2, sediment:water and fish:water ratios vary with distance from the model site. Apparently, the PPLV algebra fails .for DDT concentrations that approach the water solubility limit. Thus, an alternative procedufe is required to relate health effects to environmental contaminant levels. For the model site, field data on concentrations of DDT in soil, water, and biota are adequate to predict health and environmental effects in qualitative terms. Data presented earlier on the toxicological effects of DDT on wildlife are summarized in Fig. VII-2 and Table VI1-3. Predicted impacts of DDT contamination at the model site are summarized in Table VII-4, which was derived from data presented in Tables VII-2 and VII-3. Acute toxicity is predicted to be a problem for predatory and fish-eating birds, sensitive fish species, and sensitive amphibian species at distances up to 2 miles from the DDT plant. Very sensitive fish species might be affected over the next few miles. No animal species are predicted to suffer acute toxicity symptoms at greater distances. -47- �TABIS VII-2. EWIROWBEOTA1. COHCENTRATIONS OF DDT AT THE HODEt SITE (pp») Distance fro* Plant Site (ailes) Suple Water Reference 181 1 2 5 0.001 0.00003 i, 090(440 )« 100(100) 1(33) 2(2) Sediasnt 0.0023 Average U.S. Levels 0.000008-0.000144 Reference 16 0.05(1.7) 0.17 Kuicle Ti*s«e Birdt Predatory end fish-eating Son- predatory OB 179,184 Pith 182-184,186 KaBMU 179,184 179.184 750(330) 25(11) 20(20) 1(33) 300(130) 200(200) 100(3,300) 13(5.7) 5(5) 0.5(17) 50(50) 1(33) 500(500) 10(330) F«t Birds Predatory «nd fish eating Non-predator* Fi»h 182-184,186 1,800(780) 1,000(1,000) 500(16.000) Maaoals 179,184 130(57) 50(50) 5(170) a. Values in parentheses are non-steady state concentration factors tines 10~3. 2.3 It �TABLE VII-3. TOXICOLOGICAL EFFECTS OF DDT ON TYPICAL WILDLIFE AS A . FUNCTION OF CONCENTRATIONS OF DDT IN WATER, THE DIET, AND TISSUES Medium Water Diet Tissues Approximate Concentration Species 0.01 ppb 1-20 ppb 5 ppb Fish Fish Amphibians Lethal to sac-fry Lethal Lethal Birds Mammals Birds Mammals Eggshell thinning Possible carcinogenic effects Lethal Lethal Fish Birds Lethal to sac-fry Eggshell thinning 0.15 ppm 2 ppm 600 ppm 200 mg/kg 1-5 ppm (eggs) 1 ,>pm (egga) Effect Reproductive failure is expected for predatory and fish-eating birds and for fish at distances up to 5 miles and more from the DDT plant site. Repopulation of the model site with these species is to be expected only for those species with some accessible breeding populations at distances sufficient to avoid DDT-associated reproductive failure. In other words, fish and predatory birds may be found at the model site, but it is unlikely that sensitive species hatched within 5 miles of the DDT plant. Mammals are generally much more resistant to DDT than birds or fishes. Even so, it is not improbable that fish-eating mammals, e.g., otters, could ingest toxic quantities of DDT, considering the high dietary levels (Table VII-1). Their intake might, for example, exceed the 20 ppm DDT reported to cause teratogenic or embryotoxic effects in mice. Lower levels could conceivably induce cancer, but this would not be ecologically significant because cancer from a weak carcinogen, such as DDT, would be expected to afflict only senescent individuals. Fish taken for human food within 5 miles of the site are virtually certain to exceed the 5 mg/kg limit established by the Food and Drug Administration. For average daily consumption of 18.7 g of such fish, tVie associated lifetime cancer risk, by the EPA's method,2' exceeds 1 in 1,000. For consumption of fish containing 50 mg DDT/kg, the associated risk would exceed 1 in 100. (Note, however., that EPA considers the cancer risk from DDT ingestion derived from epidemiological data to represent an upper bound. The actual risk may be substantially lower.) -49- �LC» (short-term) s LCw (long-term) _ *c Not lethal - Chromosome abnormalities <D « ? Teratogenic ; or embryotoxlc effects c £ Eggshell thinning, increased embryo mortality i O © A D 5 Possible carcinogenic effects _L 0.1 0.4 1.0 4.0 Amphibians Fish Birds Land-dwelling mammals I 10 40 100 I 400 1,000 Concentration of DDT in Diet (ppm) Fig. VII-2. Biological Effects of Dietary DDT Typical Receptors 4,000 �TAiLE VII-4. PREDICTED EFFECTS ON WILDLIFE Distance from DDT plant Site (miles) i, t-« i Effect . 0-2 2-5 ; Acute toxicity for Predatory and fish-eating birds, sensitive fish, sensitive amphibians Very sensitive fish Reproductive failure due to Eggshell thinning in predatory and fish-eating birds, dc»th of fish sac-fry Eggshell thinning in predatory &id fisheatv.tg birds, death of fish sac-fry Eggshell thinning in predatory and fisheating birds, death of fish sac-fry �\ Decontamina t ion Ob j ect i ves In lieu of PPLV's, maximum environmental DDT levels for protection of wildlife have been calculated directly from model site data (Table VII-1). These are 0.1 ppb in water and 4 ppb in sediment for predatory and fish-eating birds and 0.05 ppb in water and 2 ppb in sediment for fish. The critical effect for birds is eggshell thinning leading to reproductive failure. Available data suggest that for sensitive birds, such as the brown pelican, DDT concentrations greater than 1 ppm in the egg can cause a significant decline in reproductive success. Other studies suggest that DDT levels in bird fat are approximately 40 time« the levels in bird eggs. Table VII-2 shows that the ratio of DDT in fat of predatory birds to DDT in water is approximately constant and falls in the ranjje of 300,000 to 500,000. Assuming a ratio of 400,000, it is calculated that a DDT level in water not to exceed 0.1 j t> will provide a safe litnit of 40 ppm in fat and 1 ppm in eggs. This corv spends to a sediment concentration of about 4 ppb. For less sensitive species, safe concentration limits will be higher. (It should be emphasized th«t these calculations assume that measured DDT concentrations are equilibrium or 7* steady-state levels. If not, derived values could be in error by an order of magnitude or greater.) For fish, the critical effect is mortality of sac-fry, which c*n occur at DDT concentt-i.tions of about 1 ppm in fish eggs (Table V-4) and estimated corresponding 'concentrations of 0.01 ppb in water and 0.4 ppb in sediment (assuming a sediment-to-water ratio of 4 ) Data of Table 0. VII-1 indicate that reproductive success could be expected at di»t«nct« ^greater than 5 miles from the model marufacturing site. To establish engineering goals for cleanup efforts, benefitt to wildlife and humans are predicted to occur for any degree of cleanup from present levels uown to 2 ppb in sediments (0.05 ppb in water). These concentrations are so low, and the area of dispersal so great, that it may be better to focus on cleanup efforts giving the greatest reduction of total mass of DDT, accepting the fact that decreases to ppb levels in sediment will have to come through biodegradation. Regular monitoring of DDT levels in fish and waterfowl will provide a measure of restoration. -52- �VIII. REFERENCES 1. Letter, AMCPM-DRR, Subject: Installation Restoration Directed Actions (December 1, 1975). 2. Dacre, J.C., W.D. Burrows, C.W.R. Wade, A.F. Hegyeli, T.A. Miller, and D.R. Cogley (eds.), "Problem Definition Studies on Potential Environmental Pollutants: V. Physical, chemical, lexicological, and Biological Properties of Seven Chemicals Used in Pyrotechnic Compositions," TR 7704, U.S. Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD (October 1979, in press). 3. Burrows, W.D., J.C. Dacre, and D.R. Cogley (eds.), "Problem Definition Studies on Potential Environmental Pollutants: VI. Preliminary Assessment of Environmental Effects of Seven Substances Used in Pyrotechnic Compositions at Pine Bluff Arsenal," MR 28-79, U.S. Army Medical Bioengineeririg Research and Development Laboratory, Fort Detrick, Frederick, MD (November 1979). 4. Rosenblatt, D.H., T.A. Miller, J.C. Dacre, I. Muul, and D.R. Cogley (eds.), "Problem Definition Studies on Potential Environmental Pollutants: I. Toxicology and Ecological Hazards of 16 Substances at Rocky Mountain Arsenal," TR 7508, U.S. Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD, AD B039661 (December 1975.), 5. Rosenblatt, D.H., T.A. Miller, J.C. Dacre, I. Muul, and D.R. Cogley , (eds.), "Problem Definition Studies on Potential Environmental Pollutants: II. Physical, Chemical, Toxicological, and Biological Properties of 16 Substances," TR 7509, U.S. Army Medical Bioengineering Research and Development Laboratory, Fott Detrick, Frederick, MD, AD A030428 (December 1975). 6. Dacre, J.C. and D.H. Rosenblatt, "Mammalian Toxicology and Toxicity to Aquatic Organisms of Four Important Types of Waterborne Munitions Pollutants - An Extensive Literature Evaluation," TR 7403, U.S. Army Medical Bioengineering Research and Development Laobratory, Aberdeen Proving Ground, AD 778725 (March 1974). 7. Miller, T.A., D.H. Rosenblatt, J.C. Dacre, D.R. Cogley, and J.L. Welch, "Problem Definition Studies on Potential Environmental Pollutants. III. Toxicology and Ecological Hazards of Benzene; Toluene; Xylenes; and p-Chlorophenyl Methyl Sulfide, Sulfoxide, and Sulfone at Rocky Mountain Arsenal," TR 7604, U.P. Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD, AD B039662 (June 1976). -53- �8. Miller, T.A. , D.H. Rosenblatt, J.C. Dacre, J.G. Pearson, R.K. Kulktirni, J.L. Welch, D.R. Cogley, and G. Woodard, "Problem Definition Studies on Potential Environmental Pollutants. IV. Physical, Chemical, Toxicological, and Biological Properties of Benzene; Toluene; Xylenes; and p-Chlorophtnyl Methyl Sulfide, Sulfoxide, and Sulfone," TR 7605, U.S. Array Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD, AD A040435 (June 1976). 9. World Health Organization, Environmental Health Criteria 9; DDT and Its Derivatives, World Health Organization, Geneva, Switzerland _ 10. DDT and Associated Substances, IARC Monograph No. 5 (1974). 11. Thompson, J.F. (ed.), Analysis of Pesticide Residues in Human and Environmental Samples, A^Cqmpilat ion of Methods Selected for Use in Pesticide Monitoring Programs , U.S. Environmental Protection Agency, Health Effects Research Laboratory, Environmental Toxicology Division, Research Triangle Park, NC, June 1977. 12. Bontoyan, W.R. , Chairman of Editorial Committee, Manual of Chemical Methods for Pesticides and Devices, U.S. Environmental Protection Agency, Toxic Substances Division, Building 306, Room 101, Beltsville, MD (no date). 13. Laws, E.R., Jr., W.C. Maddrey, A. Cur ley, and V.W. Burse, "Long T<irm Occupational Exposure to DDT," Arch. Environ. Health 27:318-321 (1973). 14. Jukes, T.H., "Insecticides in Health, Agriculture and the Environment," Naturwissenschaften 61:6-16 (1974). 15. Laws, E.R. , Jr., A. Curley, and F.J. Biros, "Men With Intensive Occupational Exposure to DDT," Arch . Environ. Health 15:766-775 U967). 16. Metcalf, R.L. and J.J. McKelvey, Jr. (eds.), "The Future for Insecticides: Needs and Prospects," Advan . Environ ._ Sc i . Techno! . 6 (1976). 17. Hdrina, P.D., R.L. Singhal, and G.M. Ling, "DDT and Related Chlorinated Hydrocarbon Insecticides: Pharmacological Basis of Their Toxicity in Maeuaals," Adv. Pharmacol. Chemother. 12:?l-88 (1975). — 18. Hayes, V.J., E.D. Dale, and C.I. Pirkle, "Evidence of Safety of Long-Term High Oral Doseo of DDT for Man," Arch. Environ. Health 22:119 (1971). -54- �19. U.S. Environmental Protection Agency, DDT; A Review of__Scient:ifi(! and Economic^AspectB of the Decision to Ban Its Use as a Peatxcidj, Document EPA-540/1-75-022, WashingtonT DC (1975). 20. U.S. Environmental Protection Agency, 44 FR 56649, pp. 56649-56651 (October 1979). 21. Hayes, W.J., Jr., "Pharmacology and Toxicology of DDT," in Muller, P. (ed.), DDT; The Insecticide Dichlorodiphenyltrichloroethane *nd_ Its Significance, Birkhauser Verlag, Basel, Switzerland, Vol. 2, pp. 9-247 (19597. 22. Innes, J.R.M., "Bioassay of Pesticides and Industrial Chemicals for Tumorigenicity in Mice. A Preliminary Note," J. Natl. Cancer Inst. 42:1101 (1969). 23. Powers, M.B., R.W. Voelker, W.A. Olson, and W.M. Weatherholty, "Bioassays of DDT, TDE, and p,p'-DDE for Possible Carciriogenicity," National Cancer Institute, Carcinogenesis Technical Report Series, No. 131 (1978). 24. Rossi, L., M. Ravera, F. Repetti, and L. Santi, "Long-Terra Administration of DDT or Phenobarbital-NA in Wistar Rats," Int. J. Cancer 19:179 (1977). 25. Shabad, L.M., T.S. Kolesnichenko, and T.V. Nikonova, "Transplacental and Combined Long-Term Effect of DDT in Five Generations of A-Strain Mice," Int. J. Cancer 11:688 (1973). 26. - Tarjan, R. and T. Kemeny, "Multi-Generation Studies on DDT in Mice," Food Cosaiet. Toxicol. 7:215 (1969). 27. Terracini, B., R.J. Cabral, and M.C. Testa, "The Effects of Long-Term Feeding of DDT to BALB/c Mice," Int. J. Cancer 11:747 (1973). 28. Thorpe, E. and A.I.T. Walker, "The Toxicology of Dieldrin (HEOO): II. Comparative Long-Term Oral Toxicology Studies in Mice with Dieldrin, DDT, Phenobarbitone, B-B4C and -BHC," Food Cosmet. Toxicol. 11:433 (1973). 29. Tomatis, L., V. Turusso, R.T. Charles, and M. Biocchi, "The Effect of Long-Term Exposure to DDE and DDD on CF-1 Mice," J. Natl. Cancer Inst. 52:883 (1974). 30. Tomatis, 1,., V. Turusso, N. Day, and R.T. Charles, "The Effect of Long-Term Exposure to DDT on CF-1 Mice," Int^ J. Cancer 10:489 (1972). -55- �31. Turusov, V.S., N.E. Day, L. Tomatis, E. Gati, and R.T. Charles, "Tumors in CF-1 Mice Expooed for 6 Consecutive Generations to DDT," J. Natl. Cancer Inst. 51:983 (1973). 32. Agthe, C., H. Garcia, P. Shubik, L. Tomatio, and E. Wenyon, "Sr.udy of the Potential Carcinogenicity of DDT in Syrian Golden Hamsters," Proc...'Soc. Ejcp. Biol. Med. 134:113 (1970). 33. McCann, J., E. Choi, E. Yamasaki, and B.N. Ames, "Detection of Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals," Proc. Nat. Acad. Sci. USA 72:5135 (1975). 34. Brown, A.W.A., Ecology of Pesticides, John Wiley & Sons, New York (1978). 35. Edwards, C.A. (ed.), Environmental Pollution by peatAcides, Plenum Press, London, (1973). 36. Matsumura, F«, "Effects of Pesticides on Wildlife," in Toxicology of Insecticidea, plenum Press, New York, pp. 355-401 (1975). 37. Tahori, A.S. (ed.), Fate of Pesticides in Environment, Gordon and Breach Science Publishers, New York (1971). 38. White-Stevens, R. (ed.), Pest i c id e sin the Env ironment, Vol. 1, Parts I and II, Marcel Dekker, Inc., New York (1971). 39. Woodwell, G.M., C.F. Wurster, and P.A. Isaacson, "DDT Residues in an East Coast Estuary: A Case of Biological Concentration of a Persistent Insecticide," Science 156:821 (1967). 40. Wurster, C.F., "Chlorinated Hydrocarbon Insecticides and the World Ecosystem," Biol. Conserv. I(2):123 (1969). 41. Wurster, C.F., "DDT Proved Neither Essential Nor Safe," BioScience 23:105 (1973). 42. Wurster, C.F., "Effects of Insecticides," in Polunin, N. (ed.), The Environmental Future, MacMillan Press, Ltd., New York (1972). 43. Pionke, H.B. and G. Cheaters, "Pesticide-Sediment-Water Interactions," J. Environ. Qual. 2:29-45 (1973). 44. Bridges, W.R., B.J. Kallman, and A.K. Andrews, "Persistence of DDT and Its Metabolics in a Farm Pond," Trans. Am. Fish Soc. 92:421-427 (1963). 45. Bailey, T.E. and J.R. Hannum, "Distribution of Pesticides in California," J. Sanit._ F.ng. Div., Am. Soc. Civ. Eng. 93:27-43 (1967). -56- �46. Guenzi, W.D. and W.E. Beard, "Anaerobic Conversion of DDT to DDD and Aerobic Stability of DDT in Soil," Soil Sci. Soc. Am. Proc. 32(4):522-524 (1968). 47. Guenzi, W.D. and W.E. Beard, "Volatilisation of Lindaue and DDT From Soils," Soil Sci. Soc. Am. Proc. 34(3):443-447 (1970). 48. Guenzi, W.D. and W.E. Beard, "The Effects of Temperature and Soil Water on Conversion of DDT to DDE in Soil," J. Environ. Qua!. 5(3):243-246 (1976). 49. Guenzi, W.D. and W.E. Beard, "Degradation in Flooded Soil as Related to Temperature," J. Environ. Qual. 5(4):391-394 (1976). 50. Fries, G.F., "Degradation of Chlorinated Hydrocarbons Under Anaerobic Conditions," Advan. Chera. Ser. 111:256-270 (1972). 51. Rhead, M.M., "Fate of DDT and PCB's (Polychlorinated Biphenyls) in the Marine Environment," Environ. Chem. 1:137-159 (1975). 52. Wedemeyer, G., "Dechlorination of l,l,l-Trichloro-2,2-bis(p-chlorophenyDethane by Aerpbacte£ aerogenes. I. Metabolic Products," Appl. Microbiol. l5(3):569-574 (1967). 53. Malone, T.C., "In Vitro Conversion of DDT to DDD by the Intestinal • Microflora of the Northern Anchovy, Engraulis mordax," Nature 227:848-849 (1970). 54. Pritchard, J.B., A.M. Guarino, and W.B. Kintner, "Distribution, Metabolism, and Excretion of DDT and Mirex by a Marine Teleost, the Winter Flounder," Environ. Hea1th Perspect. June:45-54 (1973). 55. Young, R.G., L. St. John, and D.J. Lisk, "Degradation of DDT by Goldfish," Bui1. Environ. Contain.Toxicol. 6:351-354 (1971). 56. Zinck, M.E. and R.F. Addison, "The Effect of Temperature on the Rate of Conversion of p,p'-DDT to p,p'-DDE in Brook Trout (Salvelinus fontinalis)," Can. J. Biocheia. 53:636-639 (1975). 57. Guarino, A.M., J. B. Pritchard, J.B. Anderson, and D.P. Rail, "Tissue Distribution of 114C]DDT in the Lobster After Administration via Intravascular or Oral Routes or After Exposure from Ambient Sea Water," Toxicol. Appl. Pharmacol. 29:277-288 (1974). 58. Sheridan, P.F., "Uptake, Metabolism, and Distribution of DDT in Organs of the Blue Crab, Callinectes sapidus," Chesapeake Sci. 16:20-26(1975). -57- �59. Neudorf, S. and M.A.Q. Khan, "Pick-up and Metabolism of DDT, Dieldrin and Photodieldrin by a Fresh Water Alga (Ank is t rodesmua amalloxdes) and a Microcrustacean (Daphnia pulex)," Bull. Environ. Contarn. Toxicol, 13:443-450 (1975). 60. Sundstrom, G., B. Jennson, and S. Jensen, "Structure of Phenolic Metabolites of p.p'-DDE in Rat, Wild Seal and Guillemot," Nature 255:627-628 (1975). 61. Eichelberger, J.W. and J.J. Lichtenberg, "Persistence of Pesticides in River Water," Environ. Sci. Techno1. 5:541-544 (1971). 62. Johnson, B.T. and J.O. Kennedy, "Biomagnification of p,p'-DDT and Methoxychlor by Bacteria," Appl. Microbiol. 26:66-71 (1973). 63. Vance, B.D. and W. Drummond, "Concentration of Pesticide by Algae," J. Am. Water Works Assoc. 61:360-362 (1969). 64. Johnson, B.T., C.R. Saunders, H.O. Sanders, and R.S. Campbell, "Biological Magnification and Degradation of DDT and Aldrin by Freshwater Invertebrates," J. Fish. Res. Board Can. 28:705-709 (1971). 65. Hansen, D.J., Annual Report of the Gulf Breeze Biological Laboratory, 1965, U.S. Bur. Cotnm. Fish Circ. 247, pp. 10-11 (1966). 66. Holden, A.V., "A Study of the Absorption of C14-labelled DDT From Water by Fish," Ann. Appl. Biol. 50:466-477 (1962). 67. Cade, T.J., C.M. White, and J.R. Haugh, "Peregrines and Pesticides in Alaska," Condor 70:170-178 (1968). •• 68. Cade, T.J., J.L. Lincer, C.M. White, D.G. Roseneau, and L.G. Schwartz, "DDE Residues and Eggshell Changes in Alaska Falcons and Hawks," Science 172:955-957 (1971). 69. U.S. Dept. of Health, Education and Welfare, Report of the Secretary's Commission on Pesticides and Their Relationship to Environmental Health, U.S. Dept. HEW, Washington, DC (December 1969). 70. U.S. Environmental Protection Agency, Criteria Document - DDT (DDD, DDE), Washington, DC, EPA-440/9-76-010 (1976). 71. Pimentel, *)., Ecological Effects of Pesticides on Non-Target Species, Office of Science and Technology, U.S. Government Printing Office, W.shington, DC, No. 4106-0029 (June 197i). -58- �72. Gillett, J.W., J. Hill, A.V. Jarvinen, and W.P. Schoor, A Conceptual Model foir_the Movement of Pesticides Through the Environment,. U.S. Environmental Protection Agency, Washington, DC, EPA-660/3-74-024 (December 1974). 73. Barker, R.J., "Notes on Some Ecological Effects of DDT Sprayed on Elras « "J^Wildl. Manage. 22:269-274 (1958). 74. Cottara, C. and E. Higgins, "DOT: Its Effect on Fish and Wildlife," J. Econ. Entomol. 39:44-52 (1946). 75. Hickey, J.J. and L.B. Hunt, "Initial Songbird Mortality Following a Dutch Elm Disease Control Program," J. Hildl. Manage. 24:259-265 (1960). 76. Hotchkiss, N. and R.H. Pough, "Effect on Forest Birds of DDT Used for Gypsy Moth Control in Pennsylvania," J. Wildl. Manage. 10:202-207 (1946). 77. Hunt, L.B., "Song Bird Breeding Populations in DDT-sprayed Dutch Elm Disease Communities," J. Wildl. Manage. 24:139-146 (1960). 78. Hunt, L.B., "Kinetics of Pesticide Poisoning in Dutch Elm Disease Control,1' U.S. Fish Wildl. gerv. Circ. 226:12-13 (1965). 79. Robbins, C.S. and R.E. Stewart, "Effects of DDT on Bird population of Scrub Forest," J. Wildl. Manage. 13:11-18 (1949). 80. Wallace, G.J., W.P. Nickell, and R.F. Bernard, "Bird Mortality in th6 Dutch Elm Disease Program in Michigan," Cranbrook Inst. Sci. Bull. 41 (1961). 81. Bernard, R.F., "Studies on the Effect of DDT on Birds," Publ. Mus. Mich. State Univ. Biol. Ser. 2:155-191 (1963). 82. Bernard, R.F., "DDT Residues in Avian Tissues," J. Appl. Ecol. 3(Suppl.):193-198 (1966). 83. Hunt, E.G., "Studies of Pheasant-Insecticide Relationships," J. Appl. Ecol. 3(Suppl.):113-123 (1966). 84. Ames, P.L., "DDT Residues in the Eggs of the Osprey in the Northeastern United States and Their Relation to Nesting Success," J. Appl. Ecol. 3(Suppl.):87-97 (1966). 85. Heath, E.G., J.W. Spanna, and J.F. Kreitzer, "Marked DDE Impairment of Mallard Reproduction in Controlled Studies," Nature 224:47-48 (1969). -59- �86* Davison, K.L. and J.L. Sell, "DDT Thins Shells of Eggs From Mallard Ducks Maintained on Ad Libitum or Control-Feeding Regimens," Arch. Environ. Contam. Tpxicol. 2:222-223 (1974). 87. Peakall, D.B., J.L. Lincer, R.W. Risebrough, J.B. Pritchard, and W.B. Kinter, "DDE-Induced Eggshell Thinning: Structural and Physiological Effects in Three Species," Comp. Gen. Pharmaco1. 4:305-313 (1973). 88. McLane, M.A.R. and L.C. Hall, "DDE Thins Screech Owl Eggshells," Bull^Environ. Contam. Tpxicol. 8:65-68 (1972). 89. Longcore, J.R., F.B. Samson, and T.W. Whittendale, "DDE Thins Eggshells and Lowers Reproductive Success of Captive Black Ducks," Bui 1. Environ. Contam. Toxicol. 6:485-490 (1971). 90. Azevedo, J.A., E.G. Hunt, and L.A. Woods, "Physiological Effects of DDT on Pheasants," Calif. Fish Game 51:276-293 (1965). 91. DeWitt, J.B., "Chronic Toxicity to Quail and Pheasants of Some Chlorinated Insecticides," J. Agr ic. Food Chem. 4:863-866 (1956). 92. Albert, T.F., "The Effects of DDT on the Sperm Production of the Domestic Fowl," Auk 79:104-107 (1962). 93. Locke, L.N., N.J. Chura, and P.A. Stewart, "Spcrmatogenesis in Bald Eagles Experimentally Fed a Diet Containing DDT," Condor 68:497-502 (1966). 94. Hickey, J.J. and D.W. Anderson, "Chlorinated Hydrocarbons and Eggshell Changes in Raptorial and Fish-eating Birds," Science 162:271-273 (1968). 95. Anderson, D.W., J.J. Hickey, R.W. Risebrough, D.F. Hughes, and R.E. Christensen, "Significance of Chlorinated Hydrocarbon Residues to Breeding Pelicans and Cormorants," Can. Field Nat. 83:91-112 ( 9 9 . 16) 96. Fyfe, R.W., J. Campbell, B. Hayson, and K. Hodson, "Regional Population Declines and Organochlorine Insecticides in Canadian Prairie Falcons," Can. Field Na^. 83:191-200 (1969). 97. Bins, L.J., A.A. Belisle, and R.M. Prouty, "Relations of the Brown Pelicans to Certain Environmental Pollutants," PCStic. Monit. J. 7:181-194 (1974). 98. Keith, J.A. and I.M. Gruchy, "Residue Levels of Chemical Pollutants in North American Birdlife," Proc. 15th Int. Ornithol. Congr.t The Hague, The Netherlands, pp. 437-454""(1972)'. -60- �99. Wiemeyer, S.N., P.R. Spitzer, W.C. Krantz, T.G. Lament, and E. Cromartie, "Effects of Environmental Pollutants on Connecticut and Maryland Oapreys;" J. Wild I. Manage. 39:124-139 (1975). 100. McKee, J.E. and H.W. Wolfe, Water Quality^Criteria, 2nd edition, State Water Quality Control Board, Sacramento, CA (1963). 101. Katz, M., R.S. LaGore, D. Weitkamp, J.M. Cuunings, D. Anderson, and D.R. May, "Effects on Freshwater Fish," J. Water Pollut. Control Fed. 44:1226-1250 (1972). 102. Johnson, D.W., "Pesticides and Fishes - A Review of Selected Literature," Trans, /u.. Fish Soc. 97:398-424 (1968). 103. McKim, J.M., G.M. Chriotensen, J.H. Tucker, D.A. Benoit, and M.J. Lewis, "Effects of Pollution on Freshwater Fish," J. j/ater Pollut. Control Fed. 46:1540 ff (1974). 104. Macek, K.J. and W.A. McAllister, "Insecticide Susceptibility of Some Common Fish Family Representatives," Trans.Am. Fish Soc. 99:20-27 (1970). 105. e^ Ingham, B. and M.A. Gallo, "Effect of Asulam in Wildlife Species: Acute Toxicity to Birds and Fish," Bull. Environ. Contarn. Toxicol. 13:194-199 (197S). 106. Post, G. and T.R. Schroeder, "The Toxicity. of Four Insecticides to Four Salmonid Species," Bull. Environ... Contain. Toxicol. 6:144-156 (1971). 107. Bathe, R., L. Ultnann, and K. Sachsse, "Determination of Pesticide Toxicity to Fish," Schrifteher. Ver. Wasser Boden Lufthyg. Berlin*" Pah1em 37:241-256 (1973). 108. Committee on Water Quality Criteria, HAS-NAE, Water Quality Criteria 1972, U.S. Government Printing Office, Washington, DC, No. 5501-00520 (1972). 109. Schaumberg, F.D., T.E. Howard, and C.C. Walden, "A Method to Evaluate the Effects of Water Pollutants on Fish Respiration," Water Res. 1:731-737 (1967). 110. Julin, A.M., Fish-Pesticide Research Laboratory, Columbia, MO, letter to E. Bender, Ecological Research Office, Biomedical Laboratory, Edgewood Arsenal, MD (August 5, 1975). 111. Carter, F.L. and J.B. Graves, "Measuring Effects of Insecticides on Aquatic Animals," La. Agric. 16:14-15 (1972-3). �112. Nunogawa, J.N., N.C. Burbank, and R.H.F. Young, "The Relative Toxicities of Selected Chemicals to Several Speciej of Tropical Fish," Adyan. Water Pollut. Res. Proc. Int. Conf. 5th 2:HA14, 1-14 (1970). 113. Korn, S. and R. Earnest, "Acute Toxicity of Twenty Insecticides to Striped Bass, Morone saxatilis," Calif. Fish Game 60:128-131 (1974). 114. Earnest, R«D« and P.E. Benville, Jr., "Acute Toxicity of Four Organochlorine Insecticides toTwo Species of Surf Perch," Calif. Fish Game 58:127-132 (1972). 115. Macek, K.J., "Acute Toxicity of Pesticide Mixtures to Bluegills," Bui 1. Env iron. Cont&~. Toxicol. 14:648-652 (1975). 116. Holden, A.V., "Organochlorine Residues in Salraonid Fish," J. Appl. Ecol. 3(Suppl.):45-53 (1966). 117. Macek, K.J., "Reproduction in Brook Trout (_S_a.ly_elinuft font inalis) Fed Sublethal Concentrations of DDT," J. Fish. Res. Bd. Canada 25:1787-1796 (1968). 118. Macek, K.J., "Growth and Resistance to Stress in Brook Trout Fed Sublethal Levels of DDT," J. Fish. Res. Bd. Canada 25:2443-2451 (1968). 119. Burdick, G.E., E.J. Harris, J.H. Dean, T.M. Walker, J. Ske'd, and D. Colby, "The Accumulation of DDT in Lake Trout and the Effect on Reproduction," Trans. Am. Fish. Soc. 93:127-136 (1964). 120. Cuerrier, J., A. Keith, and E. Stone, "Problems with DDT in Fish Culture Operations," NaU Can. 94:315-320 (1967). 121. Dacre, J.C. and D. Scott, "Possible DDT Mortality in Young Rainbow Trout," N.Z.J. Mar. Freshwater Res. 5:58-65 (1971). 122. Johnson, H.E. and C. Pecor, "Coho Salmon Mortality and DDT in Lake Michigan." N. Am. Wildl. Nat. Res. Conf. Trans. 34:159-166 (1969). 123. Anderson, R.B. and W.H. Everhart, "Concentration of DDT in Landlocked Salmon in Sebago Lake, Maine," Trans. Am. Fish Soc. . 95:160-164 (1966). 124. Jarvinen, A.W., M.J. Hoffmann, and T.W. Thorslund, Significance to Fathead Minnows of Food and Water Exposure to DDT, Environmental Protection Agency, Duluth Laboratory Project No. 16AAK, Task A 010, Program Element 1BA021 (1974). 125. Malone, C.R. and E.G. Blaylock, "Toxicity of Insecticide Formulations to Carp Embryos Reared In Vitro," J. Wildl. Manage. 34:4v'-0-463 (1970). -62- �126. Dill P. A. and R.C. Saunders, "Retarded Behavioral Development and ftt^tired Balance in Atlantic Salmon (Salroo salar) Alevins Hatched iron Gastrulae Exposed to DDT," J_» Fish. Rea_. Board Can. 3:1936-1938 (1974). 127. Wei*, P., "Ultrastructural Changes Induced by Low Concentrations in tto* Livers of the Zebra fish and the Guppy," Chem. Biol. Interactions S J25-30 (1974). 128. Gardner, D.R. , "The Effect of Some DDT and Methoxychlor Analogs on Tenperature Selection and Lethality in Brook Trout Fingerlings," Pet tic . Biochem. Phy a iol . 2:437-446 (1973). 129. Aaderaon, J.M. , "Sublethal Effects and Changes in Ecosystems. Assessment of the Effects of Pollutants on Physiology and ," Proc. Roy. Soc. Londcn Ser. B. 111:307-320 (1971). 130. AsyJereon, J. M. and M.R. Peterson, "DL/T: Sublethal Effects on Breolc Trout Nervous Systems," Science 164:477-478 (1969). 131. De'/y, F.B., H. Kleerekoper, and J.H. Matis, "Effects of Exposure to Su&lethal DDT on the Exploratory Behavior of Goldfish (Carassius ," Water Re sour. Res. 2:900-905 (1973). 132. D«vy, F.B., H. Kleerekoper, and P. Gensler, "Effects of Exposure to £ijj> lethal DDT on the Locomotor Behavior of the Goldfish (Carassius •unit us.).," J. Fish. Res. Board Can. 29:1333-1336 (1972). 133. E>e*«iah, D. , L.K. Cutkomp, R.B. Koch, and A. Jarvinen, "DDT: Effect of Contir".ious Exposure on ATPase Activity J.n Fish, Pimephales pr&welas," Arch .Environ. Contain. Toxicol . 3:132-141 (1975T^ 134. iitedem, T.P., R.I>. Cambell, and D.W. Johnson, "Osmoregulatory E«*ponse to DPT and Varying Salinites in jjalnio gairdneri. I. Gill Jto-K-ATPase,:i Comp. Biochem. Physiol. 49A:197-205 (1974). 135. Kmter, W.B., L.S. Merkens, R.H. Janicki, and A.M. Guarino, "Studies on the Mechanism of Toxicity of DDT and Polychlorinated Bip-henyls (PCBs): Disruption of Osmoregulation in Marine Fish," Epyiron.Jealth Perapect. 1:169-173 (1972). 136. Wfisbart, M. and D. Feiner, "Subl?thal Effect of DDT on Osmotic and Ionic Regulation by the Goldfish Carassius auratus," Can. J. Zool. 52;739-744 (1974). 137. B'Jilet, D.R., M.E. Rasmusson, and W.E. Shanks, "Chronic Oral DDT Twxicity in Juvenile Coho and Chinook Salmon," Toxicol. Appl. pfesrmacol. 14:535-555 (1969). -63- �138. McNicholl, G. and W.C. Mackay, "Effect of DDT on Discriminating Ability of Rainbow Trout (Salmo gairdneri)," J. Fish. Res. Board Can. 32:785-788 (1975). 139. Ginsburg, J.M., "Comparative Toxicity of DDT Isomers and Related Compounds to Mosquito Lsrvae and Fish," Science 105:233-234 ( 9 7 . 14) 140. Alabaster, J.S., "Survival of Fish in 164 Herbicides, Insecticides, Fungicides, Wetting Agents rrvd Miscellaneous Substances," Int. Pest Control March/Apr?.l:29-35 (1969). 141. Applegate, V.C., J.H. Howell, A.E. Hall, Jr., and M.A. Smith, "Toxicity of 4,346 Chemicals to Larval Lampreys and Fishes," U.S. Fish Wildl. Serv. Spec. Sci. Rep. Fish. No. 207 (1957). 142. Stickel, L.F., "Wood Mouse and Box Turtle Populations in the Area Treated Annually with DDT for Pi"- i««ra," J. Wild!. Manage. 15:161-164 (1951). From Ref. 71. 143. Brown, A.W.A., "Insecticides and the Balance of Animal Populations," in Insect Control by Chemicals, John Wiley & Sons, New York, pp. 720-780 (1951). 14% Fleet, R.R., D.R. Clark, and F.W, Plapp, "Residues of DDT and Dieldrin in Snakeo from Two Texas Agro-ecosystems," BioScience 22:664-665 (1972). 145. Phillips, J.B. and M.R. Wells, "Adenosine 'Triphosphataae Activity in Liver, Intestinal Mucosa, Cloacal Bladder, and Kidney Tissue of Five Turtle Species Following In Vitro Treatment with l,l,l-Ti-ichloro-2,2-bis(p-chlorophenyl)ethane (DDT)," J. Agr. Food Chem. 22:404-407 (1974). »" " 146. Witherspoon, F.G., Jr. and M.R. Wells, "Adenosine Triphosphatase Activity in Brain, Intestinal Kucosa, Kidney, and Liver Cellular Fractions of the Red-Eared Turtle Following In Vitro Treatment with DDT, DDD and DDE," Bull. Environ. Contain. ToxicoU 14:537-544 (1975). 147. Sanders, H.O., "Pesticide Toxicities to Tadpoles of the Western Chorus Frog, Pseudacris triserlata, and Fowler's Toad, Bufo woodhousii fowleri," Copeia 2:246-251 (1970). Quoted in Ref. 71. 148. Ludemanh, D. and H. Neumann, "The Effect of Modern Insecticides on Freshwater Organisms," Anz. Schaedlingskd. 35:5-9 (1962). 149. Cooke, A.S., "The Effects of DOT, When Used as a Mosquito Larvicide, on Tadpoles of the Frog, Rana temporaria," Environ. Pollut. 5:259-273 (1973). -64- �150. Malina, J.F., Jr., "Toxicity of Petrochemicals in the Aquatic Environmenty" Water Sewage Works 10:456-460 ( 9 4 . 16) 151. Macek, K.J. and H.O. Sanders, "Biological Variation in the Susceptibility of Fish and Aquatic Invertebrates tf> DDT," Trans. Am. Fish. Soc. #9:89-90 (1970). 152. Maki, A.W. and H.E. Johnson, "Effects of PCB (Aroclor 1254) and p,p'-DDT on Production and Survival of Daphnia magna Strauss," Bull. Environ. Contam. Toxicol. 13:412-416 (1975). 153. Khudairi, S.Y.A-D. and E. Ruber, "Survival and Reproduction of Ostracods as Affected by Pesticides and Temperature," J. Econ. Entomol. 67:22-24 (1974). 154. Grosch, D.S., "Poisoning with DDT: Effect on Reproductive Performai.ce of Artetnia," Science 151:592-593 (1967). 155. Albaugh, D.W., "Insecticide Tolerances of Two Crayfish Populations (Procambarus acutuB) in South-Central Texas," Bull. Environ. Contam. Toxi~col. 8:334-338 (1972). ' 156. Hann, R.W., Jr. and P.A. Jansen, "Water Quality Characteristics of Hazardous Materials," Environmental Engineering Division, Texas A&M University, College Station, TX (no date). 157. Leffler, C.W., "Effects of Ingested Mirex and DDT on Juvenile Callinectes sapidu^ Rathburn," Environ. Iollut. 8:283-300 (1975). 158. Koenig, C.C., R.J. Livingston, and C.R. Gripe, "Blue Crab Mortality: Interaction of Temperature and DDT Residues," Arch. Environ. Contam. Toxicol. 4:119-128 (1976). 159. Odum, W.E., G.M. Woodwell, and C.F. Wurster, "DDT Residues Absorbed from Organic Detritus by Fiddler Crabs," Science 16« 576-577 (1969). 160. Decamps, H., K.W. Besch, and H. Vobis, "Effect of Toxic products on the Construction of Webs by Hydropsyche (Insecta, Trichoptera)," C^.R. Acad. Sci. Ser. D 276:375-378 (1973). 161. Davis, B.C. and H. Hidu, "Effects of Pesticides on Embryonic Development of Clams and Oysters and on Survival and Growth of the Larvae," U.S. Fish. yildl.Serv. Fish. Bull. 67(2)-.393-404 (1969). 162. Kimura, T., H.L. Keegan, and T. Haberkorn, "Dehydrochlorination of DDT by Asian Blood-sucking Leeches," Am. J. Trop. Med. Hyg. 16:688-690 (1967). 163. Naqvi, S.M.Z., "Toxicity of Twenty-three Insecticides to a Tubeficid Worm Branchiura sowerbyi from the Mississippi Delta," ,r._ Econ. Entomol. 66:70-74 (1973). -65- �164. Kouyoumjian, H.H. and R.F. Uglow, "Some Aspects of the Toxicity of p,p'-DDT, p,p'-DDE and p,p'-DDD to the Freshwater Planarian Pplycelis feUna (Tricladida)," Environ. Pollut. 7:103-109 (1974). 165. Haller, H.L., "Wartime Development of Insecticides," Ind. Eng. Chem. 39:467-473 (1947). 166. Cristol, S.J., H.L. Haller, and A.W. Lindquist, "Toxicity of DDT Isoraers to Some Insects Affecting Man," Science 104:343-344 ( 9 6 . 14) 167. Kemp, H.T., R.L. Little, V.L. Holoman, and R.L. Darby, Water Quality Criteria Data Book; Vol. 5, Effects of Chemicals on Aquatic Life, U.S. Government Printing Office, Washington, DC, 13050-HLA 09/73 (1973). 168. Deontier, C.C. and H.A. Jones, "TDE, l,l-Dichloro-2»2-bis(pchlorophenyl)ethane, as an Anopheline Larvicide," Science 103:13-14 (1946)., 169. Luard, E.J., "Sensitivity of Dunaliella an*' Pcenedesmus (Chlorophyceae) to Chlorinated Hydrocarbons," Phycologia 12:29-33 (1973). 170. Poorman, A.E., "Effects of Pesticides on Eiiglena gracilis. I. Growth Studies," Bull. Environ. Contain. Toxicol. 10:25-28 (1973). 171. Hicks, G.F., Jr. and T.R, Corner, "Location and Consequences of l,l,l-TrichlorO"2,2-bis(p-chlorophenyl)ethane Uptake by Bacillus me^aterium," A_ppl._M_icrobipl. 25:381-387 (1973). 172. Gil, I., J. Morales, A. Martin, C. Ruano, and F. Argones, "Effects of Some Pesticides on Azotobacter," Microbiol. Esp. 23:271-277 (1970); Chero. Abs. 75, 34405z (1971). 173. Collins, J.A. and B.E. Langlois, "Effect of DDT, Dieldrin and Heptachlor on the Growth of Selected Bacteria," Appl. Microbiol. 16:799-800 (1968). 174. Garretson, A.L. and C.L. San Clamente, "Inhibitory of Nitrifying Chemolithotropic Bacteria by Several Insecticides," J. Econ. Entomol. 61:285-288 (1968). 175. Powers, C.D., R.G. Rowland, R.R. Michaels, N.S. Fisher, and C.F. Wurster, "The Toxicity of DDE to a Marine Dinoflagellate," Environ. Pollut. 9:253-262 (1975). 176. American Conference .of Governmental Industrial Hygienists, Threshold Limit Values for Chemical Substances and physical Agents in tha Workroom Environment with Intended Changes for 1978, Cincinnati, OH, p. 14 (1978). -66- �177. Food and Drug Administration, "Action Levels for Poisonous or Deleterious Substances in Human Food and Animal Peed," Industrial Guidance Branch, Bureau of Foods (HFF-342), 200 C St. S.W., Washington, DC, p. 5 (June 1968). 178. "DDT Contamination Found in Tests," The Rocket ? 3 2 ) 9 (October 5, .(0! 1977). 179. Patuxent Wildlife Research Center, "DDT Analyses by Colorimetric Methods of Muscle and Fat Tissue from Wildlife Near Huntsville Spring Branch, Wheeler National Wildlife Refuge, Alabama," Patuxent Wildlife Research Center, Laurel, MD (February 14-24, 1964). 180. Pearson, J.G., E.S. Bender, D.H. Taormina, K.L. Manuel, P.P. Robinson, anri A.E. Asaki, "Effects of Elemental Phosphorus on the Biota of Yellow Lake, Pine Bluff Arsenal, Arkansas, March 1974-Ja»v:jry 1975," U.S. Army TR EO-TR-76077, Edgewood Arsenal, MD (December 1976). /. 181. Ward, F.P., C.F.A. pinkham, and J.G. Pearson, "Redstone Arsenal, Alabama, Installation Environmental Assessment Statement," Ecological Research Unit, Biomedical Laboratory, Edgewood Arsenal, MD (August 1972). 182. State of Alabama, Dept. of Conservation, Game and Fish Division, Water Pollution Report (February 5, 1971). 183. State of Alabama, Dept. of Conservation, Utilities Division, Utilities Lab Control (March 5, 1971). 184. U.S. Army Environmental Hygiene Agency, "Analytical Results of Biological Samples," Report No. 3, Aberdeen Proving Ground, MD (October 14, 1977). 185. Manuel, K.L., E.S. Bender, and J.G. Pearson, "Results of Aquatic Surveys at Pine Bluff Arsenal, Arkansas, September 1973-October 1974," U.S. Army TR EB-TR-76038, Edgewood Arsenal, MD (April 1976). 186. "DDT Residue Tests Ordered at Redstone," Huntsville Times, Evening Edition (July 25, 1977). 187. Cogley, D.R., J.C. Dacre, D.H. Rosenblatt, and W.D. Bvrrows, "Prioritiiation of Research Efforts on Environmental Chemicals: A Rationale for the Calculation of Preliminary Pollutant Limit Values for Soil and Water," presented at the 1979 National Conference on Hazardous Material Risk Assessment, Disposal and Management, Miami Beach, FL (April 25-27, 1979). -67- �DISTRIBUTION LIST Project No. 3E16272QA835/OQ/137 do, of Copies 5 12 •; ... ' . : • ' . , . • . ..,' ." :/.•-'..:. . •.. ,, • . ' • - """"•"%.-.;' US Army Medical Research and Development Command ATT,<: SGRD-RMS Fort Detrick Frederick, MO Zl.701 Defense Technical Information Center (DTIC) ATTtt: QTICrDUA Cameron Station Alexandria, VA 22314 1 Conmandant Academy of Health Sciences* US Army ATTrt: AHS-COM Fort Sam Houston, TX 78234 2 USAHBROL Technical Library 68 �m � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 047 Folder The folder containing the original item. 1190 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Description An account of the resource Corporate Author: U. S. Army Medical Bioengineering Research and Development Laboratory Date A point or period of time associated with an event in the lifecycle of the resource 1979-06-01 Title A name given to the resource Report: Problem Definition Studies on Potential Environmental Pollutants, VII. Physical, Chemical, Toxicological, and Biological Properties of DDT and Its Derivatives Subject The topic of the resource aquatic animals health effects pesticide toxicology ecological impact ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 074 Folder The folder containing the original item. 1933 Series The series number of the original item. Series III Subseries IV Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Sinclair, Ian Date A point or period of time associated with an event in the lifecycle of the resource 1982-12-01 Title A name given to the resource Report on the Use of Herbicides, Insecticides, and Other Chemicals by the Australian Army in South Vietnam Subject The topic of the resource herbicide testing herbicide disposal human exposure pesticide toxicology ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 084 Folder The folder containing the original item. 2108 Series The series number of the original item. Series III Subseries IV Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Report missing title page: Contents relate to pesticide exposure in Vietnam and Australia Subject The topic of the resource Australia pesticide toxicology human exposure Southeast Asia ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 048 Folder The folder containing the original item. 1231 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Stadelman, W. J. Source A related resource from which the described resource is derived BioScience Date A point or period of time associated with an event in the lifecycle of the resource 1973-07-01 Title A name given to the resource Record of Some Chemical Residues in Poultry Products Subject The topic of the resource agricultural exposure animal testing pesticide toxicology ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 040 Folder The folder containing the original item. 0913 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Moses, Marion Source A related resource from which the described resource is derived Environmental and Occupational Medicine Date A point or period of time associated with an event in the lifecycle of the resource 1983 Title A name given to the resource Pesticides Subject The topic of the resource industrial exposure pesticide properties pesticide toxicology agricultural exposure ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 043 Folder The folder containing the original item. 1019 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Robson, Alan M. J. M. Kissane N. H. Elvick L. Pundavela Source A related resource from which the described resource is derived Journal of Pediatrics Date A point or period of time associated with an event in the lifecycle of the resource 1969-08-01 Title A name given to the resource Pentachlorophenol Poisoning in a Nursery for Newborn Infants: I. Clinical Features and Treatment Subject The topic of the resource pesticide poisoning pesticide toxicology human exposure ao_seriesIII