1 15 137 https://www.nal.usda.gov/exhibits/speccoll/files/original/f7dec7f13438a6e941502d236acb5f7d.pdf d5c0136a345740e328a4041a78dbd405 PDF Text Text Item ID Number: 00090 Author Young, Alvin L. CorpOPatB Author Environics and Flame Munitions Branch, Flame, Incendiary and Explosives Division, Air Force Armament Laboratory, Eglin AFB, Florida Roport/Artido Tltlfl Ecological Studies on a Herbicide-Equipment Test Ares (TA C-52A) Eglin AFB Revervation, Florida; Final Report: January 1967 to November 1973 Journal/Book Title Year 1974 Month/Day January Color Number of Images 144 DOSGrlptOn NOteS Program No. 5154-02 Friday, December 01, 2000 Page 90 of 91 �TECHNICAL REPORT AFATL-TR-74-12 ECOLOGICAL STUDIES ON A HERBICIDE-EQUIPMENT TEST AREA (TA C-52A) EGLIN AFB RESERVATION/FLORIDA JANUARY 1974 FINAL REPORT: January 1967 to November 1973 Approved for public release; distribution unlimited. ENVIRONICS AND FLAME MUNITIONS BRANCH FLAME, INCENDIARY AND EXPLOSIVES DIVISION AIR FORCE ARMAMENT LABORATORY AIR FORCE SYSTEMS COMMAND » UNITED STATES AIR FORCE EGLIN AIR FORCE BASE, FLORIDA �UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered) READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 1. REPORT NUMBER 2. GOVT ACCESSION NO 3. RECIPIENT'S CATALOG NUMBER AFATL-TR-74-12 4. T I T L E (and Subtitle) 5. TYPE OF REPORT & PERIOD C O V E R E D Final Report January 1967 - November 1973 ECOLOGICAL STUDIES ON A HERBICIDE-EQUIPMENT TEST AREA (TA C-52A) EGLIN AFB RESERVATION, FLORIDA 6. P E R F O R M I N G ORG. REPORT N U M B E R 8. CONTRACT OR GRANT NUMBERfs.) 7. AUTHORfs.) Alvin L. Young, Captain, USAF, Ph.D. 9. PERFORMING O R G A N I Z A T I O N NAME AND ADDRESS 10. PROGRAM ELEMENT, PROJECT, TASK AREA & WORK UNIT NUMBERS Flame, Incendiary and Explosives Division. (DLIP) Air Force Armament Laboratory Eglin Air Force Base, Florida 32542 5154-02 12. REPORT DATE 11. CONTROLLING OFFICE NAME AND ADDRESS January 1974 Air Force Armament Laboratory Air Force Systems Command Eglin Air Force Base, Florida 32542 13. NUMBER OF PAGES 141 14. MONITORING.AGENCY NAME & ADDRESSf/f different from Controlling Office) IS. SECURITY CLASS, (of this report) Unclassified Air Force Systems Command (SDWC) Andrews Air Force Base Washington D. C. 20334 15a. DECLASSIFICATION/DOWN GRADING SCHEDULE 16. DISTRIBUTION STATEMENT (of this Report) Approved for public release; distribution unlimited. 17. D I S T R I B U T I O N STATEMENT (of the abstract entered In Block 20, It different from Report) 18. SUPPLEMENTARY NOTES Available in DDC 19. KEY WORDS (Continue on reverse side If necessary and Identity by block number) Algal Survey 4-Amino-3,5,6-Trichloropicolinic Amphibians Animal Survey Studies Bioassay Acid Blue Defoliant Testing Birds 2,4-Dichlorophenoxyacetic Acid Dimethylarsinic Acid 20. ABSTRACT (Continue on reverse side if necessary and Identify by block number) This report attempts to answer the major questions concerned with the ecological consequences of applying massive quantities of herbicides (346,117 pounds), via repetitive applications, over a period of eight years, 1962 - 1970, to an area of approximately one square mile. Moreover, the report documents the persistence, degradation, and/or disappearance of the herbicides from the Test Area's soils and drainage waters and their subsequent effects (direct or indirect) upon the vegetative, faunal, and microbial communities. The active ingredients of the four military herbicides (Orange, Purple, White, and Blue) sprayed on Test Area C-52A were 2,4-dichlorophenoxyacetic acid (2,4-D), (continued) FORM 73 DD , JAN 1473 EDITION OF 1 NOV 65 IS OBSOLETE UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered) �IIMPI SECURITY CLASSIFICATION OF THIS PAGEfHTien Data Entered; Item 19. Continued Ecological Investigations Fish Herbicide Herbicide Equipment Test Grid Histology Mammals Microbial Survey Military Defoliation Program Necropsy Orange Purple Reptiles TCDD Teratogenic Test Area C-52A, Eglin AFB Reservation 2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,4,5-Trichlorophenoxyaetic Acid Vegetative Coverage Survey White Item 20. 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), and dimethylarsinic acid (cacodylic acid). It is probable that the 2,4,5-T herbicide contained the highly teratogenic (fetus deformina) contaminant 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Significant herbicide residues were found in 1969. However, analysis of soil cores in 1971 indicated residues of 2,4rD, 2,4,5-T, picloram, and arsenic to be in the parts per billion range; no TCDD was found at this detection limit. Soil samples collected in June and October 1973 showed TCDD (or a TCDD-like chemical) levels ranging from less than 10 parts per trillion (ppt) to 710 ppt. Direct effects of the herbicides on the vegetative community were temporary. With the disappearance of residue, vegetative succession was initiated. By 1973, the majority of the test area could not be distinguished from control sites. Analysis of sample animal populations indicated that no histological or gross abnormalities were present in adults or their progeny. UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGEfWhen Data Entered) �PREFACE The Air Force project directly related to the information in this report is Air Force Systems Command Project 5154-02, Ecological Survey of Test Area C-52A, Eglin AFB Reservation, Florida. This report documents five years of ecological investigations performed between 1967 and 1973. The assistance provided during portions of this report by the Air Force Environmental Health Laboratory, Booz-Allen Applied Research, Dow Chemical U.S.A., United States Air Force Academy, United States Department of Agriculture, University of Alabama, University of Florida, and Vitro Services is gratefully acknowledged. Information on the test grid monitoring system and types and amounts of defoliants disseminated on Test Area C-52A from July 1962 to April 1969 was obtained from Armament Development and Test Center working papers, "Defoliant History of Test Area C-52A", by Helen Biever. After April 1969, this same information was obtained from Vitro Services, Vitro Corporation of America. Information on soils of Test Area C-52A was obtained from a July 1969 soil survey of Eglin AFB Reservation prepared by the Soil Conservation Service of the United States Department of Agriculture. This technical report hasjpeen reviewed and is approved. F. RAMON BONNANO, Lt Colonel, USAF Chief, Flame, Incendiary and Explosives Division �LIST OF CONTRIBUTORS Kenneth M. Ayers, Captain, USAF, VC, Staff Veterinarian, Veterinary Pathology Division, Armed Forces Institute of Pathology William J. Cairney, Captain, USAF, BSC, Assistant Professor of Life Sciences, Department of Life and Behavioral Sciences, United States Air Force Academy Robert W. Clegern, Captain, USAF, BSD, Ph.D., Research Entomologist, Air Force Environmental Health Laboratory John H. Hunter,Ph.D., Field Research and Development Specialist, Dow Chemical, U.S.A. P. Jeffery Lehn, B.A., Biologist, Somerville, New Jersey John W. Sigler, 1 Lt, USAF, M.S., Project Scientist, Flame Incendiary and Explosives Division, Air Force Armament Laboratory Charles E. Thalken, Major, USAF, VC, Assistant Professor of Life Sciences, Department of Life and Behavioral Sciences, United States Air Force Academy William E. Ward, Lt Colonel, USAF, Ph.D., Tenure Professor, Department of Life and Behavioral Sciences, United States Air Force Academy Edwin A. Woolson, Ph.D., Pesticide Chemist, Pesticide Degradation Laboratory, United States Department of Agriculture Alvin L. Young, Captain, USAF, Ph.D., Associate Professor of Life Sciences, Department of Life and Behavioral Sciences, United States Air Force Academy �TABLE OF CONTENTS Section Page SUMMARY I 5 INTRODUCTION 1. Description of Geographical and Environmental Factors 2. Description of the Test Facility 3. Descriptions of Samplings Grids and Herbicide Deposition 4. Description of Pesticides 10 10 12 19 23 BIOASSAY AND CHEMICAL RESIDUE STUDIES OF THE SOILS OF TEST AREA C-52A 1. Synopsis of Bioassay Research, 1969 - 1970 2. Synopsis of Bioassay Research, 1970 - 1971 3. Results and Discussion 4. Conclusions 5. Chemical Analyses of Soil Cores 28 28 31 33 42 42 III STUDIES OF THE VEGETATION OF TEST AREA C-52A 1. Synopsis of Taxonomic Studies, 1966 - 1969 2. Synopsis of Growth of Sand Pine, 1969 - 1970 3. Synopsis of Histological Study of Yucca, 1970 4. Current Vegetative Succession Studies 52 53 54 56 57 IV STUDIES OF THE ANIMALS OF TEST AREA C-52A 1. Synopsis of Qualitative Animal Surveys, 1970 - 1973 2. Current Studies on Animals 3. Materials and Methods 4. Results and Discussion 5. Conclusions 76 76 78 82 83 91 V INSECT DENSITY AND DIVERSITY STUDIES ON TEST AREA C-52A 92 1. Synopsis of Previous Research, May - June 1971 92 2. Materials and Methods 92 3. Results and Discussion 93 4. Summary and Conclusions 109 II VI VII AQUATIC STUDIES OF TEST AREA C-52A 1. Synopsis of Previous Research, 1969 2. Current Studies of Aquatic Organisms 3. Conclusions 110 110 116 125 STUDIES ON THE MICROFLORA OF TEST AREA C-52A 1. Synopsis of Previous Research, 1967 - 1970 2. Current Studies on Microflora 3. Literature Review 4. Materials and Method 5. Results and Discussion 6. Current Studies on Survey of Aquatic Algae 7. Conclusions 3 (The reverse of this page is blank) 128 128 130 130 133 135 135 139 ��SUMMARY In support of programs testing aerial dissemination systems, a one square mile test grid on Test Area (TA) C-52A, Eglin AFB Reservation, Florida, received massive quantities of military herbicides. The purpose of these test programs was to evaluate the capabilities of the equipment systems, not the biological effectiveness of the various herbicides. Hence, it was only after repetitive applications that test personnel began to express concern over the potential ecological and environmental hazards that might be associated with continuance of the test program. This concern led to the establishment of a research program in the fall of 1967 to measure the ecological effects produced by the various herbicides on the plant and animal communities of TA C-52A. This report documents 6 years of research (1967 - 1973) on TA C-52A and the immediately adjacent streams and forested areas. This report attempts to answer the major questions concerned with the ecological consequences of applying massive quantities of herbicides (346,117 pounds), via repetitive applications, over a period of 8 years (1962 - 1970) to an area of approximately one square mile. Moreover, the report documents the persistence, degradation, and/or disappearance of the herbicides from the soils and drainage waters of TA C-52A, and the subsequent effects (direct or indirect) of the herbicides upon the vegetative, faunal, and microbial communities. The active ingredients of the four military herbicides (Orange, Purple, White, and Blue) sprayed on TA C-52A were 2,4-dichlorophenoyxacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), and dimethylarsinic acid (cacodylic acid). It is probable that the 2,4,5-T herbicide contained the highly teratogenic (fetus deforming) contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). From 1962 to 1964, 92 acres of the test grid received 1,894 pounds 2,4-D, 2,4,5-T per acre while, in 1964 to 1966, another 92 acres received 1,168 pounds per acre. From 1966 to 1970, a third distinct area of over 240 acres received 343 pounds per acre of 2,4-D and 2,4,5-T, and 6 pounds per acre of picloram; and from 1969 to 1970, this same area received 53 pounds per acre of cacodylic acid (28 pounds per acre of arsenic as the organic pentavalent form; calculated on weight of Blue applied per acre). From the rates of herbicides that were applied during the years of testing spray equipment, it was obvious that TA C-52A offered a unique opportunity to study herbicidal persistence and soil leaching. Yet the problem of how best to assess the level of herbicide residue was a difficult one. The herbicides could be chemically present but because of soil binding might not be biologically active. Thus, both bioassay techniques and analytical analyses were employed. The first major bioassay experiment was conducted in April 1970. By considering the flightpaths, the water sources, and the terracing effects, it was possible to divide the one square mile test grid into 16 vegetation areas. These areas formed the basis for the random selection of 48 soil cores taken from the surface to a depth of 3 feet. Soybean bioassays indicated that 27 of the 48 cores were significantly different from control cores (95% probability level). The results indicated that soil leaching or penetration was much more prevalent along the dissemination flightpaths than in other areas of the test grid. Efforts to quantitate (chemically) the bioassay were confined to only the top 6 inch increment because of withincore variations. By considering that all phytotoxic effects resulted from 2,4-D and 2,4,5-T, the average value for the top 6 inches of the eight cores showing greatest herbicide concentration was 2.82 ppm (parts per million) herbicide. Chemical analyses of soil cores collected from the eight sites showing greatest phytotoxic concentrations were performed in December �1970. Results indicated that the maximum concentration of either 2,4-D or 2,4,5-T was 8.7 ppb (parts per billion). A 1970 analysis of soil cores from areas receiving the greatest quantities of Blue indicated maximum arsenic levels of 4.70, 1.30, and 0.90 ppm, respectively, for the first three 6 inch increments of the soil profile. These same increments were again collected and analyzed in 1973, and the levels of arsenic were 0.85, 0.47, and 0.59 ppm for the three consecutive 6 inch increments. Leaching of the arsenical from the soils may have occurred. In November 1969, picloram analysis of soil cores from areas receiving greatest quantities of White indicated maximum levels of 2.8 ppm picloram present in the 6 to 12 inch increment. An analysis of the same sites in 1971 indicated the picloram had leached further into the soil profile but concentrations were significantly less (ppb). The analyses of soil cores in 1971 showed no residue of TCDD at a minimum detection limit of less than 1 ppb, even in soils previously treated with 947 pounds 2,4,5-T per acre. However, data from soil analysis (via mass spectrometry) of four total samples collected in June and October 1973 indicated TCDD levels of <10, 11, 30, and 710 parts per trillion (ppt), respectively. These levels were found in the top 6 inches of soil core. The greatest concentration (710 ppt) was found as a sample form the area that received 947 pounds 2,4,5-T in the 1962 - 1964 test period. A comparison of vegetative coverage and occurrence of plant species on the one square mile grid between June 1971 and June 1973 showed that areas with 0 to 60% vegetative cover in 1971 had a coverage of 15% to 85% in June 1973. Those areas having 0 to 5% coverage in 1971 (areas adjacent to or under flightpaths used during herbicide equipment testing) had 15% to 54% coverage. The rate of coverage seemed to be dependent upon soil type, soil moisture, and wind. There was no evidence to indicate that the existing vegetative coverage was directly related to herbicide residue in the soil: some dicotyledonous or broadleaf plants that are normally susceptible to damage from herbicide residues occurred throughout the entire one square mile grid except in a few irregularly spaced barren areas. The square-foot transect method of determining vegetative cover indicated that the most dominant plants on the test area are the grasses - switchgrass (Panicurn virgatum). woolly panicum (Panicum lanuqinosum). and the broadleaf plants - rough buttonweed (Diodia teres). poverty weed (Hypericum gentianoides). and common polypremum (Polypremum procumbens). In 1971, 74 dicotyledonous species were collected on the one square mile grid; in 1973, 107 dicotyledonous species were collected. All of the plant species collected were pressed, mounted, and placed in the Eglin AFB Herbarium. An evaluation of the effects of the spray equipment testing program on faunal communities was conducted from May 1970 to August 1973. The extent of any faunal ecological alteration was measured by assessing data on species variation, distribution patterns, habitat preference (and its relationships to vegetative coverage), and the occurrence and incidence of developmental defects as well as gross and histologic lesions in postmortem pathological examinations. A total of 73 species of vertebrate animals (mammals, birds, reptiles, and amphibians) were observed on TA C-52A and in the surrounding area. Of these 73 species, 22 were observed only off the grid, 11 were observed only on the grid, and 40 were observed to be common to both areas. During the early studies, no attempts were made to quantitate animal populations in the areas surrounding the grid; however, in 1970 preliminary population studies by trap-retrap methods were performed on beach mouse (Peromyscus polionotus) populations for a 60 day period to confirm the hypothesis that it was the most prevalent species on the grid. The hypothesis was supported by the capture of 36 beach mice from widely distributed areas on the grid, except in areas with less than 5% vegetation. Eight pairs of eastern harvest mice were taken to the laboratory and allowed to breed. Six of the eight pairs had litters totaling 24 mice. These progeny were free from any gross external birth defects. During February - May 1971, population densities of the beach mouse were studied at eight different locations on the grid and in two different areas off the grid which served as controls. Populations were estimated �on the basis of the trap-retrap data. There was no difference in mouse population densities in herbicide treated and untreated control areas affording comparable habitats. All indications were that any population differences in other animal species between the test area and the surrounding area were due to differences caused by the elimination of certain plants, and therefore, certain ecological niches, rather than being due to any direct toxic effect of the herbicides on the animal populations present on TA C-52A. During the last day of the 1971 study, 9 mice were captured and taken to the laboratory for postmortem pathological examination. There were no instances of cleft palate or other deformities. Histologically, liver, kidney, and gonadal tissues from these animals appeared normal. In the 1973 study, several different species of animals were caught, both on and off the test grid. These included beach mice (Peromyscus polionotus), cotton mice (Perorrjyscus gossypinus), eastern harvest mice (Reithrodontomys humulis), hispid cotton rats (Signodon hispidus), six-lined racerunners (Cnemidophorus sexlineatus), a toad (Bufo americanus), and a cottonmouth water moccasin (Ankistrodon piscivorus). A total of 89 animals were submitted to the Armed Forces Institute of Pathology, Washington, D. C., for complete pathological examination including gross and microscopic studies. Liver and fat tissue from 70 rodents were forwarded to the Interpretive Analytical Services, Dow Chemical, U.S.A., for TCDD analyses. The sex distribution of the trapped animals was relatively equal. The ages of the animals varied, but adults predominated in the sample. No gross or histological developmental defects were seen in any of the animals. Several of the rats and mice from both groups were pregnant at the time of autopsy. The stage of gestation varied from early pregnancy to near term. The embryos and fetuses were examined grossly and microscopically, and no developmental defects or other lesions were observed. Gross necropsy lesions were relatively infrequent and consisted primarily of lung congestion in those animals that had died from heat exhaustion prior to being brought to the laboratory. The organ weights did not vary significantly between the test and control animals when an animal with lungs and kidneys showing inflammatory pathological lesions was removed from the sample. Histologically, the tissues of 13 of the 26 control animals and 40 of the 63 animals from the test grid were considered normal. Microscopic lesions were noted in some animals from both groups. For the most part, these were minor changes of a type one expects to find in any animal population. One of the most common findings was parasites. A total of 11 controls and nine grid animals were affected with one or more classes of parasites. Parasites may be observed in any wild species, and those in this population were for the most part incidental findings that were apparently not harmful to the animal. There were exceptions however; protozoan organisms had produced focal myositis in one rat and were also responsible for hypertrophy of the bile duct epithelium in a six-lined racerunner. Moderate to severe pulmonary congestion and edema was seen in several rats and mice. All of these animals were found dead in the traps before reaching the laboratory, and the lung lesions were probably the result of heat exhaustion. The remainder of the lesions in both groups consisted principally of inflammatory cell infiltrates of various organs and tissues. These lesions were usually mild in extent and although the etiology was not readily apparent, the cause was not interpreted as toxic. The analysis of TCDD from the rodents collected in June and October 1973 indicated that TCDD or a chemically similar compound accumulated in the liver and fat of rodents collected from an area receiving massive quantities of 2,4,5-T. However, based on the pathological studies, there was no evidence that the herbicides produced any developmental defects or other specific lesions in the animals sampled or in the progeny of those that were pregnant. The lesions found were interpreted to be of a naturally occurring type and were not considered related to any specific chemical toxicity. �In 1970, beach mice were not found on the more barren sections of the grid (0 to 5% vegetative cover). However, some areas of the grid had a population density that exceeded that of the species most preferred habitat as reported in the literature. In 1973, in an attempt to correlate distribution of the beach mouse with vegetative cover (i.e., habitat preference) a trapping-retrapping program of 8 days duration was conducted. The majority of animals (63) were found in areas with 5% to 60% vegetative cover: Within this range, the greatest number of animals trapped (28) was from an area with 40% to 60% cover. A similar habitat preference has been observed along the beaches of the Gulf Coast. In this study, it appeared that the beach mouse used the seeds of switchgrass (Panicum virgatum) and woolly panicum (Panicum lanuginosum) as a food source. Trapping data from 1971 and from 1973 were compared to determine whether an increase in the population of beach mice had occurred. The statistical evidence derived from that study showed that the 1.64 beach mice per acre population (based on the Lincoln Index for 1973) was slightly higher than the 0.8 and 1.4 mice per acre reported for a similar habitat. The population of beach mice was also higher in 1973 than in 1971 in the area of the test grid. The apparent increase in beach mouse population on the grid for 1973 over 1971 was probably due to the natural recovery phenomenon of a previously disturbed area (i.e., ecological succession). Some areas of the test grid have currently exceeded the preferred percentage of vegetative coverage of the beach mouse habitat, and other areas were either ideal or fast developing into an ideal habitat. If the test grid remains undisturbed and continues toward the climax species, a reduction in the number of beach mice will probably occur simply due to the decline of preferred habitat. A 1973 sweep net survey of the Arthropods of TA C-52A resulted in the collection of over 1,700 specimens belonging to 66 insect families and Arachnid orders. These totals represented only one of five paired sweeps taken over a one mile section of the test grid. A similar study performed in 1971 produced 1,803 specimens and 74 families from five paired sweeps of the same area using the same basic sampling techniques. A much greater number of small to minute insects were taken in the 1973 survey. Vegetative coverage of the test area had increased since 1971. The two studies showed similarities in pattern of distribution of arthropods in relation to the vegetation, number of arthropod species, and arthropod diversity. Generally, the 1973 study showed a reduction of the extremes found in these parameters during the 1971 study. This trend was expected to continue as the test area stabilizes and develops further plant cover, thus allowing a succession of insect populations to invade the recovering habitat. Two classes of aquatic areas are associated with TA C-52A; ponds actually on the one square mile area and streams which drain the area. Most of the ponds are primarily of the wet weather type, drying up once in the last 5 years; however, one of the ponds is spring fed. Three major streams and two minor streams drain the test area. The combined annual flow of the five streams exceeds 24 billion gallons of water. Seventeen species of fishes have been collected from the major streams and three species from the spring fed pond on the grid. Statistical comparisons of 1969 and 1973 data of fish populations in the three major streams confirm a chronologically higher diversity in fish populations. However, the two control streams confirm a similar trend in diversity. Nevertheless, from examining all of the aquatic data, certain observations support the idea that a recovery phenomenon is occurring in the streams draining TA C-52A. These observations are difficult to document 8 �because of insufficient data. For example, in 1969 the southern brook lamprey (Ichthyomyzon gagei) was never collected in one of the streams immediately adjacent to the area of the grid receiving the heaviest applications of herbicides; however, in 1973 this lamprey was taken in relatively large numbers. These observations may or may not reflect a change in habitat due to recovery from herbicide exposure. Residue analyses (1969 to 1971) of 558 water samples, 68 silt samples, and 73 oyster samples from aquatic communities associated with drainage of water from TA C-52A showed negligible arsenic levels. A maximum concentration of 11 ppb picloram was detected in one of the streams in June 1971, but this level had dropped to less than 1 ppb when sampled in December 1971. TCDD analysis of biological organisms from streams draining TA C-52A or in the ponds on the test area were free from contamination at a detection limit of less than 10 parts per trillion. In analyses performed 3 years after the last application of 2,4-D and 2,4,5-T herbicides, the test grid exhibited population levels of soil microorganisms identical to those in adjacent control areas of similar soil and vegetative characteristics not exposed to herbicides. There were increases in Actinomycete and bacterial populations in some test site areas over levels recorded in 1970. This was possibly due to a general increase in vegetative cover for those sampling sites and for the entire test grid. No significant permanent effects could be attributed to exposure to herbicides. Data on aquatic alga populations from ponds on the one square mile grid (previously exposed to repetitive applications of herbicides) indicated that the genera present were those expected in warm, acid (pH 5.5), seepage, or standing waters. �SECTION I INTRODUCTION The Eglin AFB Reservation has served various military uses, one of them having been the development and testing of aerial spray equipment (e.g., herbicide spray equipment). It was necessary for this equipment to be tested under controlled conditions that were as near to being realistic as possible. For this purpose a testing installation was established in 1962 on the Eglin Reservation with the place of direct aerial application restricted to an area approximately one mile square within Test Area C-52A (TA C-52A) in the southeastern part of the reservation. In support of programs testing aerial dissemination systems, TA C-52A received massive quantities of military herbicides. The purpose of these test programs was to evaluate the capabilities of the equipment systems, not the biological effectiveness of the various herbicides. After repetitive applications, personnel involved with the test program expressed concern about potential ecological and environmental hazards that might be associated with continuance of these test programs. This concern led to the establishment of an "Environmental Pollution Control and Monitoring System Task Team". One of the purposes of this report is to document the efforts of this task team and other personnel who were assigned to or were associated with the Air Force Armament Laboratory and the Armament Development and Test Center. Their efforts should serve as an indication of the interest and concern on the part of the Air Force for pollution abatement as an integral part of weapon systems development. In view of the controversy associated with the use of herbicides, TA C-52A offers a unique opportunity for evaluating the ecological effects of repetitive applications of herbicides. Data obtained during the past six years of research, plus the current research effort, may be of significance in dictating future programs involving herbicides in military programs, civic action applications, and the public acceptance of herbicides for continued use in weed and brush control programs. 1. DESCRIPTION OF GEOGRAPHICAL AND ENVIRONMENTAL FACTORS a. General Area The Eglin AFB Reservation is located in Northwest Florida where it occupies a portion of Santa Rosa Island, Okaloosa Island, the southeastern part of Santa Rosa County, the southern half of Okaloosa County, and the southwestern quarter of Walton County. It covers an area of approximately 750 square miles. To the south the Reservation is adjacent to Choctawhatchee Bay and the Gulf of Mexico, while to the north and east it is bordered roughly by the Yellow River and Alaqua Creek. The Reservation lies on generally level or gently rolling terrain, all under 300 feet elevation and sloping to sea level on the west and south. It is drained by small tributaries of the Yellow River and Alaqua Creek and by smaller streams that flow directly into Pensacola Bay and Choctawhatchee Bay. The valleys of these streams often are steep sided and terminate abruptly. The soil of most of the Reservation consists of somewhat excessively drained, deep, acid sands of the Lakeland series. In the stream bottoms, and particularly along the Yellow River, the soils are much more heavily organic. b. Test Area C-52A Test Area C-52A is located in the southeastern part of the Eglin Reservation. It covers an area of approximately three square miles (Figure 1-1) and is a grassy plain surrounded by a forest stand that is dominated by longleaf pine (Pjnus palustris). sand pine (Pinus clausa), and 10 �c 138 HARPSITE TO C 106 A B C D E QA LABORATORY TEST GRID 300 FOOT TOWER CONCRETE APRON CONTROL TOWER CONTRAVES INACTIVE ASKAN1A SPOTTING TOWER CONTROL =—= = 5,280 FEET BLDG. RAVED ROAD CUtf R040 ==== SAM) ROAD O TOWER MTERAN6E BOUNDARr LINE Figure 1-1. Map of Test Area C-52A, Eglin AFB Reservation, Florida �turkey oak (Quercus laevis). The actual area for test operations which occupies an area of two square miles, is a cleared area occupied mainly by broomsedge (Andropogon virginicus). switchgrass (Panicum virgatum), and low growing grasses and herbs. Much of the center of the range was established prior to 1960, but the open range as it presently exists was developed in 1961 and 1962.Figures l-2(a)and I-3 are aerial photographs of the one square mile test grid and the immediate adjacent area as it appeared on 16 March 1971 and 14 June 1973, respectively. The test grid is approximately 93 feet above sea level with a water table of six to ten feet. The major portion of this test area is drained by five small creeks whose flow rates are influenced by a 60.4-inch average annual rainfall (Table 1-1). The average temperature for the area is 64.9°F (Table I-2). The average maximum and minimum temperatures (°F) by month for the test area are shown in Table I-3. For the most part, the soil of the test grid is a fine white sand on the surface changing to yellow beneath. The profile composition for a typical 3-foot soil core is shown in Table I-4. The soils of the range are predominantly well drained, acid sands of the Lakeland Association with 0 to 5% slope. Figure l-2(b) shows the location of the Lakeland, Chipley, and Rutledge sand series of the Lakeland Association as found on the one square mile grid. The Lakeland sand that covers most of the grid area forms excessively drained thick deposits that extend to a depth of about seven feet. This sand is characteristically very dry, even with 60 inches of annual rainfall. The Chipley sand is moderately well drained,and the water table in this soil may rise to within 20 to 40 inches of the surface for three months during the year. The Rutledge sand is poorly drained, strongly acid (pH 4.5 to 5.0) soil. The water table in this sand is within ten inches of the surface for several months during the year. 2. DESCRIPTION OF THE TEST FACILITY a. Description Test Area C-52A, the southern most portion of the TA C-52 range complex, is a 3 square mile cleared area on which a one square mile micrometerological and aerosol/particulate sampling grid was located (Figure 1-1). Test Site C-1, on the western edge of TA C-52A, was the control center for operation of the sampling instrumentation, grid support and test data assessment. Test Site C-102 at TA C-52 Central, provided cinetheodolite time-space-position support and fixed and mobile communications for TA C-52A mission aircraft control. This test area was closed January 1971. b. Capabilities and Uses Test Area C-52A was used for assessing the dissemination and deposition characteristics of aerially delivered liquid and particulate materials from spray tanks and other systems of a similar nature. Micrometeorological conditions existing below 300 feet over the test area were continuously described by the Automatic Meteorological Data Acquisition and Processing System (AMDAPS) which included wind, temperature, and dew point sensors on a 300-foot tower at grid center and wind sensors on 12-foot masts located at each of the four corners of the one square mile grid. A complex of defoliant-grids, intersecting near the central AMDAPS tower and oriented to eight major compass headings, provided 16 discrete sampling grids which could be selected for the most advantageous wind conditions prior to and during mission time. These grids employed glass plates and Kromekote cards for physical collection of test materials in droplet form. Each of the 250 permanent sampling stations of the TA C-52A basic grid array employed a wide variety of sampling devices including the above but were also equipped with individual commercial power and sequencing control lines for remote operation of automatic vacuum type samples which collected small particle and aerosol test materials. These sampling stations were arranged on 400-foot centers to form the one square mile grid (see sampling station array in Figure I-4). Remotely controlled, battery operated, portable samplers were also available to gather data in special purpose grid configurations anywhere in a 10 square mile area. 12 �(a) Photograph of the One Square Mile Grid Taken at 5,000 Feet Above Ground Level on 16 March 1971 Figure I-2. Test Area O52A �'-:**. £ * ' >£$ 'A i£%^ & p.f\3 4 S 6 f'^9 l y % *" '/ #"*• ^^/\ ". **. * % ¥\v; '% LAKELAND SAND * <\ v* K> CHIP LEY SAND * v *'/* r*- < '* *i * v/ • " t- YI * \ f %»\ t RUTLEDGE SAND * . '-*^ *, '*• ,» ^V/ % »\V" 'f*-> ',,j * (b) Soil Types of the One Square Mile Grid on Test Area C-52A Figure 1-2. Concluded 14 �Figure 1-3. Photograph of the One Square Mile Grid Taken at 4,300 Feet Above Ground Level on 14 June 1973 15 �TABLE 1-1. ANNUAL TOTAL PRECIPITATION FOR EGLIN AFB AND NICEVILLE, FLORIDA, FROM 1964 TO 1969 YEAR PRECIPITATION, Inches EGLIN AFB NICEVILLE 1964 68.10 72.68 1965 61.85 65.29 1966 51.10 66.95 1967 62.76 73.05 1968 31.68 42.33 60.01 68.96 55.92 64.88 1969 3 Average a Average of the two locations is 60.40 inches TABLE I-2. AVERAGE MONTHLY TEMPERATURES (°F) FOR FOUR YEARS AT NICEVILLE, FLORIDA YEAR JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC 1966 45.4 50.1 56.1 65.3 73.8 76.1 81.7 79.3 77.1 67.7 56.6 49.6 1967 49.4 48.9 60.8 70.6 72.2 79.2 78.7 72.4 63.5 55.2 56.8 65.4 1968 48.7 45.9 54.5 67.9 72.3 79.9 79.2 81.5 75.5 66.6 55.0 49.3 1969 49.8 52.0 52.1 66.9 73.2 80.5 81.2 78.1 75.2 69.6 54.5 49.2 Average3 48.3 49.2 55.9 67.7 72.9 78.9 80.2 78.3 72.8 64.8 55.7 53.4 3 Average temperature for 4 years is 64.9° F 16 �TABLE 1-3. AVERAGE MAXIMUM AND MINIMUM TEMPERATURES (°F) BY MONTH FOR FOUR YEARS (1966 - 1969) TAKEN FROM THE SIX FOOT LEVEL AT THE CENTER TOWER OF TEST AREA C-52A, EGLIN AFB, FLORIDA MONTH8 TEMPERATURE, °F MINIMUM 40.05 40.55 48.15 59.96 62.67 68.38 68.82 71.04 66.90 54.24 46.09 42.08 January February March April May June July August September October November December a MAXIMUM 58.86 60.69 68.32 81.52 82.15 86.04 87.48 87.71 84.06 77.41 68.03 61.67 Average temperature for 4 years is 65.54°F TABLE 1-4. SOIL PROFILE (6-INCH INCREMENTS) FOR TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA 8 DEPTH, Inches CLAY, % O.M., %b C.E.C.C SAND, % SILT, % 1 -6 91.6 4.0 4.4 0.46 1.19 6- 12 90.1 4.3 5.6 0.20 0.81 12- 18 92.1 4.3 3.6 0.20 0.73 18- 24 92.9 3.5 3.6 0.00 0.69 24-30 93.1 2.8 4.1 0.07 0.69 30- 36 92.8 3.6 3.6 0.07 0.69 a As determined by the Soils Department, University of Florida, Gainesville, Florida. Soil sample taken within 50 feet of K-9 permanent sampling station. '-'Percent organic matter. C C.E.C. (cation exchange capacity) is the ability of a cation to be displaced or exchanged from the soil by another cation. The cation exchange capacity of a typical greenhouse potting soil is 11.43. A soil with a cation exchange capacity of 1 can "bind" or "fix" 10 ppm of a given cation (s). 17 �2 O A O 3 4 5 6 9 10 II 12 13 14 O O O O O O O O O O B O O O O O O O O O O O O O O c o o o o o o o o o o o o o o D O O O O O O O O O O O O O O E O O O O O O O O O O O O O O F O O O O O O O O O O O O O O G O O O O O O O O O O O O rv_i-_ O — 0- 1 T( TR 400 FEET H O O O O O O O O O O O O O J O O O O O O O O O O O O O O K O O O O O O O O O O O O O O L O O O O O O O O O O O O O O M O O O O O O O O O O O O O O N O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 500 1000 1500 2000 GRAPHIC SCALE-FEET Figure 1-4. Location of the Permanent Sampling Stations on the One Square Mile Grid 18 �Fixed and portable illuminated flight line markers were available for missions during hours of darkness. A Quantitative Assessment Laboratory facility, collocated with the Test Site C-1 control center, received the collected samples and performed appropriate chemical analysis or biological assays providing data for assessment of test item performance. Micrometeorological data from TA C-52A was both recorded at the AM DAPS master station located at Test Site C-1 and transmitted to the main base Staff Meteorologist Control Center, Eglin AFB, via teletype on a 24-hour basis. 3. DESCRIPTIONS OF SAMPLING GRIDS AND HERBICIDE DEPOSITION Descriptions of the sampling grids located on TA C-52A and individual mission data including herbicide and total gallons sprayed have been compiled by Biever . a. Grid Descriptions Figure I-5 shows the location of the various herbicide grids that were located on TA C-52A. The original sampling grid (Grid!) for spray equipment testing became operational in June 1962. It consisted of four intersecting straight lines in a circular pattern, each line being at a 45° angle from those adjacent to it. This grid was discontinued after two years. It was located immediately south of the one square mile grid. The second sampling grid (Grid 2) consisted of three parallel lines intersected at right angles by another set of three parallel lines. These lines were 800 feet apart, thus forming four equal quadrants. The southwest corner of this grid corresponded to the southwest corner of the one square mile grid. The parallel line grid was operational during the period May 1964 to November 1965. The third sampling grid (Grid3) consisted of three concentric circles, with respective diameters of 1200, 1600, and 2000 feet. This grid was located in the northeast quadrant of the one square mile grid. The concentric circles grid was operational between October 1967 and April 1968; however, difficulty in interpreting data from this sampling array caused use of this grid to be discontinued. The fourth sampling grid (Grid 4) is a one square mile grid, the center of which was marked by a 300-foot tower. This was the last testing grid used on TA C-52A and its inwind and crosswind sampling arrays extended into Grid 2 and Grid 3. Figures I-6, I-7, I-8, and I-9 show various views of Grid 4 at the time the grid was under construction. The two inwind and four crosswind sampling arrays of Grid 4 became operational in May 1968. Each inwind array consisted of three parallel rows spaced 400 feet apart, with 297 sampling stations per row. The aircraft flight path crossed the midpoints of the sampling lines. The crosswind sampling arrays consisted of three parallel rows 400 feet apart, with 253 sampling stations per row. b. Deposition Rate The total amounts of chemicals (including herbicides, insecticides, oils, and simulants) applied to TA C-52A are shown in Table I-5. All of these materials were disseminated during the period from June 1962 to December 1970 (Figure 1-10). The total pounds of actual herbicides ^Defoliant History of Test Area C-52A, Working Papers, Vitro Corporation of America and Armament Development and Test Center, Eglin AFB, Florida, December 1969. 19 �1 A * 2 • 3 • 4 • 5 • 6 • 7 • 8 • * UOO feet 400 feet Boundary of major aircraft flight paths over grid Boundary of minor aircraft flight paths over grid Grid Boundary 2000 feet Figure 1-5. Location of Test Grids and Major Flight Paths Used during Dissemination of Herbicides Over Test Area C-52A 20 �Figure 1-6. A View of Grid/Number 4 Looking from the Southeast to the Northwest, 1964 Figure 1-7. A View of Grid Number 4 Looking from East to West, 1964 21 �Figure 1-8. A View of Grid Number 4 Looking from North to South, 1964 Figure I-9. A View of the Western Portion of Grid Number 4 Looking from the Southeast to the Northwest, 1964 22 �14,000 ' C=3 PURPLE rUH FUEL OIL • • ORANGE nnnni WHITE 10,000 - ECS BLUE - c _o j? 6,000 - Ul - 5 . - O ^ 2,000 3 p 3 1 - 1962 / a_ ilJa_ .1 63 64 65 66 67 68 ^ / 69 70 YEAR Figure 1-10. Annual Dissemination of Herbicides on Eglin AFB Test Area C-52A • deposited on the test area (total of all grids) is shown in Table I-6. The approximate deposition rate of herbicides, pounds active ingredient per acre, for each grid is shown in Table I-7. 4. DESCRIPTION OF PESTICIDES As closely as possible the equipment utilized on TA C-52A was tested under realistic yet controlled conditions. Most testing programs involving military herbicides and insecticides actually included the pesticides themselves rather than simulants. The low toxicity associated with these pesticides was the salient justification for such action. 4 a. Orange Orange was a reddish-brown to tan colored liquid soluble in diesel fuel and organic solvents, but insoluble in water. One gallon of Orange contained 4.21 pounds of the active ingredient of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.41 pounds of the active ingredient of 2,4,5trichlorophenoxyacetic acid (2,4,5-T). Orange was formulated to contain a 50:50 mixture of the n-butyl esters of 2,4-D and 2,4,5-T. The percentages of the formulation were: n-butyl ester of 2,4-D free acid of 2,4-D n-butyl ester of 2,4,5-T free acid of 2,4,5-T inert ingredients (e.g., butyl alcohol and ester moieties) 49.49% 0.13% 48.75% 1.00% 0.62% Some of the physical, chemical, and toxicological properties of Orange are listed in Table I-8. 23 �TABLE 1-5. APPROXIMATE TOTAL VOLUME OF HERBICIDES, INSECTICIDES, AND/OR SIMULANTS APPLIED TO TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, 1962- 1970 CHEMICAL GALLONS DISSEMINATED 19,807 Orange Purple3 White Blue Stull Bifluidb Fuel Oil Orange Simulant0 Malathion Insecticide 16,164 4,172 4,395 1,716 10,863 1,460 215 Total 58,792 a Purple was a mixture of n-butyl 2,4-D (50%), n-butyl 2,4,5-T (30%), and isobutyl 2,4,5-T (20%). The isobutyl portion was included as a measure to depress the freezing point of 2,4,5-T. This mixture was eventually replaced by Orange. "Stull Bifluid consisted of Orange (85%) plus a chemical additive, which when mixed in the spray system pump during agent dissemination produced a gel defoliant. C 0range simulant consisted of glycerine (68%), sodium thiosulfate (16.8%), and water (15.2%). TABLE I-6. TOTAL POUNDS OF ACTIVE INGREDIENTS OF HERBICIDES DISSEMINATED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1962 - DECEMBER 1970 HERBICIDE POUNDS ACTIVE INGREDIENT 2,4-D 169,292 2,4,5-T 160,948 Picloram 2,253 13,624 Cacodylic Acid and Sodium Cacodylate 24 �TABLE 1-7. APPROXIMATE DEPOSITION RATE OF HERBICIDES APPLIED TO TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA TEST GRID 3 1 GRID AREA b , Acres 92 HERBICIDE (POUNDS ACTIVE INGREDIENT/ACRE) 2 584 (1964-1966) 3 92 30 (1967) 4 CJ7 92 240 183 (1968-1969) PICLORAM CACODYLIC ACID 8 (1967) 2,4-D 2,4,5-T 947 947 ( 1962-1 964)d (1962-1964) 11 (1968) 6 (1969-1970) 53 (1969-1970) ARSENIC 0 584 (1964-1966) 160 (1968-1969) 6 28 a The test grids are described in text ^In actuality, grids 2 and 3 fall within the confines of the 640 acre grid 4. However, the positioning of the test arrays on grid 4 has resulted in most of the herbicide being disseminated within a 240 acre area, with only slight infringement on the original sites of grids 2 and 3. °Pounds per acre of arsenic as the organic pentavalent form; calculated on weight of Blue applied per acre. °Years when the majority of the herbicide was applied. �TABLE 1-8. PHYSICAL, CHEMICAL, AND TOXICOLOGICAL PROPERTIES OF THE THREE MAJOR HERBICIDES AND ONE MAJOR INSECTICIDE CHEMICAL SPECIFIC DENSITY (25C)a VJSCOSITY CENTIPOISE (23C) MOLECULAR MASS WEIGHT OF FORMULATION (Ibs/gal) SOLUBLE WEIGHT ACTIVE IN INGREDIENT WATER (Ibs/gal) RELATIVE SPECIFIC TOXICITY TOXICITY FOR WHITE RATS (mg/kg) Orange 1.282 43 618 10.7 8.62 No Low White 1.120 125 1,173 9.4 2.54 Yes Very Low 3,080 Blue 1.324 14 296 10.9 3.10 Yes Very Low 2,600 Malathion 1.232 36 328 10.3 9.74 No Low 1,375 NJ a As determined by the Air Force Armament Laboratory 566 �b. White White was a dark brown, viscous liquid that was soluble in water but insoluble in organic solvents and diesel fuel. One gallon of White contained 0.54 pound of the active ingredient of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2.00 pounds of the active ingredient of 2,4-D. White was formulated to contain a 1:4 mixture of the triisopropanolamine salts of picloram and 2,4-D. The percentages of the formulation were: triisopropanolamine salt of picloram triisopropanolamine salt of 2,4-D inert ingredient (primarily the solvent triisopropanolamine) 10.2% 39.6% 50.2% Some of the physical, chemical, and toxicological properties of White are listed in Table I-8. c. Blue Blue was a clear yellowish-tan liquid that was soluble in water, but insoluble in organic solvents and diesel fuel. One gallon of Blue contained 3.10 pounds of the active ingredient dimethylarsinic acid (cacodylic acid). Blue was formulated to contain both cacodylic acid (as the free acid) and the sodium salt of cacodylic acid (sodium cacodylate). The percentages of the formulation were: cacodylic acid sodium cacodylate surfactant sodium chloride water antifoam agent 4.7% 26.4% 3.4% 5.5% 59.5% 0.5% Some of the physical, chemical, and toxicological properties of Blue are listed in Table I-8. It should be noted that cacodylic acid and sodium cacodylate contain arsenic in the form of the pentavalent, organic arsenical. This form of arsenic is essentially nontoxic to animals as can be noted by the LD§Q value for white rats. Of the total formulation, 15.4% is arsenic in the organic form, only trace quantities are present in the inorganic (toxic) form. d. Malathion Malathion insecticide (0,0-dimethyphosphorodithioate) was a clear brown to colorless liquid withia slight characteristic odor. The ultra-low-volume (ULV) formulation was very slightly soluble in water (145 ppmw). Malathion ULV had a minimum purity of 95%. One gallon of ULV malathion contained 9.74 pounds active ingredient and 0.51 pound inert ingredients. Some of the physical, chemical, and toxicological properties of malathion are listed in Table I-8. 27 �SECTION II BIOASSAY AND CHEMICAL RESIDUE STUDIES OF THE SOILS OF TEST AREA C-52A From the rates that were applied during the years of testing spray equipment, it was obvious that Test Area C-52A at Eglin AFB Reservation offered a unique opportunity to study herbicidal persistence and soil leaching. Yet, the problem of how best to assess the level of residue was a difficult one. The herbicides could be chemically present but because of soil binding might not be biologically active. Moreover, as noted in Section I, many chemicals were applied to the test area, and a biological assessment might be the result of two or more chemicals interacting. Thus, both bioassay techniques and analytical analysis were employed. The results of the bioassay studies by A. L. Young and J. H. Hunter have not been published; however, the methodology developed and the results obtained have played a major role in understanding the ecological succession of the plant and animal communities on the test area. Thus, a detailed synopsis of their work is included in this report. 1. SYNOPSIS OF BIOASSAY RESEARCH, 1969 - 1970 In the late summer of 1969, six 5-foot cores were randomly collected from an area known to intersect spray flight paths used for missions involving the herbicide designated as Orange. The samples were taken to the laboratory and subsampled for bioassay and analytical results. The bioassay technique employed the use of soybean (variety Clarke 63) for detecting phenoxy-herbicide residue. The experiments were conducted under greenhouse conditions. No standard herbicide concentrations were included; instead, cores from treated areas were compared to control cores and the relative differences noted. Comparisons for each depth were made against control plants for the particular depth. These initial bioassay studies indicated two things. First, significant concentrations of herbicides (or phytotoxic materials) were present on the test grid; and second, these herbicides were definitely leaching or penetrating into the soil (at least to a depth of 3 feet). Moreover, the bioassay analysis indicated different relative concentrations of herbicides both between cores and within a given core. As noted earlier, the area of interest was an area greater than one square mile. Obviously, this area was too large to completely bioassay or to subject to chemical analyses. Therefore, it was decided to find the areas of greatest herbicide concentration and follow up with detailed bioassay and chemical analyses. To find these specific areas, it was necessary to design an experiment that would allow inferences about herbicidal persistence for the entire test area. Consulting statisticians assisted in designing the experiment and in analyzing the results. In order to properly evaluate herbicidal persistence and soil leaching, a vegetation chart of the test grid was prepared on 26 March 1970. The greatest amounts of vegetation were found near the water sources of the grid. There were two areas that supported very dense vegetation. A terracing effect of diminishing amounts of vegetation away from these two areas was apparent. The effects of repeated spray could be seen along the flightpaths most frequently used in test programs. In these strips, vegetation occurred only near the water sources and even there it was scant. By considering the flightpaths, the water sources, and the terracing effects, it was possible to divide the test grid into 16 vegetation areas. These areas formed the base for the random selection of soil samples. The statistical null hypotheses that were to be investigated included the following: 1. There were no herbicide concentration differences among the soils of the various vegetative areas. 28 �2. There were no differences in herbicide content among soil depths down to 3 feet. 3. There were no interactions between the vegetative areas and the soil depths. In order to conduct an experiment that would provide reliable evidence with respect to these hypotheses, three random 3 foot soil cores were taken from each of the vegetative units and three from a control area, an area 0.2 mile northwest of the square mile grid. Figure 11-1 shows the sites for the random sampling of these soil cores. These cores provided the replication for the experiment. Because of the time involved in taking the soil cores and the possible effects of the soil drying out if left unplanted for several days, it was necessary to apply the technique of blocking over the days of soil core removal and planting. The experiment was initiated 1 April 1970. Again the bioassay organism was soybean (five seeds per cup and one cup per 6 inch increment of soil core), and the experiment was conducted in a greenhouse. A series of standards for herbicide Orange was included in this experiment (range of standards was 0.25 to 4.00 ppm Orange). All standards were prepared in soil taken from the top 6 inch increment of control soil. The results indicated that therewereherbicidal persistence and leaching; of the 48 treatment cores collected and bioassayed, 27 cores were significantly different from control cores (95% probability level). The results indicated that soil leaching or penetration was much more prevalent along the dissemination flightpaths than in other areas of the test grid. Moreover, there were differences among the soils of the various vegetative areas within a given flightpath. Likewise, differences were found in herbicide content among the increments of many of the soil cores. This was probably due to both the elapsed time since herbicide application and to such factors as rainfall frequency and organic matter content of the soil. It is interesting to note that there were no statistical evidences of differences between wet and dry soils that received approximately the same amounts of herbicide. Efforts to quantitate the bioassay were confined to only the top 6 inch increment because of within-core variations. By considering that all phytotoxic effects were from Orange, the approximate concentration was 2.82 ppm herbicide. This was an average value for the top 6 inches of soil core for the eight cores showing greatest herbicide concentration. In reference to the statistical null hypothesis, all three were rejected: (1) there were differences in herbicide concentration between cores; (2) there were differences in herbicide content within cores; and, (3) there were interactions between the sampling areas and the soil depths (this indicated nonuniformity in soil strata). Sixteen of the soil cores (one from each vegetation type) were subsampled for arsenic concentration (hence, a measure of Blue). The arsenic was extracted by a cold-acid extraction technique and analyzed by atomic absorption spectrophotometry. Four of the 16 locations contained arsenic levels above 1.0 ppm in the top 6 inches of soil. A further analysis for arsenic in the soil profile indicated that arsenic readily (and almost uniformly) leached throughout the soil profile. In areas receiving repetitive applications of Blue, the top 6 inches of soil contained arsenic levels of 1.4 ppm, and the additional 6 inch increments down to 5 feet contained from 0.70 to 1.2 ppm arsenic. From the bioassay study, it was evident that some areas of the test grid contained high levels of phytohormonal herbicide residue (i.e., residue showing plant responses similar to those caused by 2,4-D; 2,4,5-T; and picloram). Thus, 5 foot cores were collected from two areas (dry soils) exhibiting highest herbicide residue. Each core was divided into 6 inch increments, placed in amber bottles, and immediately shipped to the United States Department of Agriculture, Pesticide Degradation Laboratory, Beltsville, Maryland, for analysis of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). No TCDD was found in either soil core (the Pesticide .Degradation Laboratory reported a detection limit capability of 0.0005 ppm TCDD). 29 �f B F G H J K M N 1 2 3 8 4 9 10 11 12 Figure 11-1. Collection Sites for the Random Sampling of 3 Foot Soil Cores For Residue Analyses 30 13 14 �2. SYNOPSIS OF BIOASSAY RESEARCH, 1970 - 1971 a. Introduction A follow-up bioassay experiment of six of the field locations studied in the previous bioassay experiments was initiated in December 1970. Three of the samples were selected because they showed leaching to a depth of 36 inches, two because of leaching to 30 inches, and the remaining sample because of leaching to 18 inches. In addition, two samples were obtained from grid 1 (the 1962 - 1964 grid). These samples had not been previously bioassayed, but because of prior history, it was expected that data on persistence could be obtained from both a bioassay and a chemical analysis. Since the preliminary work indicated that although equal amounts of Orange were introduced into soil cores, the varying organic composition of the soil at different depths influenced the amount of Orange available to the plants. This investigation attempted to determine if differences in amounts of herbicides and in soil composition do affect plant growth and, if so, the magnitude of this effect. This information was then used to estimate the concentration of Orange in the selected sites. b. Method and Materials In order to calculate the effects of herbicide concentration upon plant growth and to study the effects of soil depth on herbicide activity, a soil core was taken from a control site 0.2 mile northwest of the one square mile test area. This core was collected in 6 inch depth segments down to 3 feet. After being dried, sieved, and weighed, each depth-segment unit was divided into seven parts and treated so that soil samples from each unit contained the following concentrations of Orange: 0.028 part per million (ppm), 0.057 ppm, 0.113 ppm, 0.226 ppm, 0.454 ppm, and 0.908 ppm; one soil unit was not treated and so had 0.0 ppm concentration of Orange. These soil samples were then used as standards. Each of these samples was thoroughly mixed and distributed among six cups. Five soybean seeds were planted in each of three of the cups and five cucumber seeds were planted in each of the remaining three cups for each concentration. All bioassays were conducted in an ISCO E-3 environmental chamber maintained at a diurnal temperature regime of 90° to 70°F, a diurnal humidity regime of 65% to 85%, and a 14-hour daylength. The length of the cucumber plants from the root tip to the end of the epicotyl was measured after 6 days. The soybeans were harvested 10 days after planting, and the root length of each plant was recorded. Samples of soil were also collected from six selected sites on the test grid. These locations were coded using the coordinate of the grid marker and the direction with respect to that marker in which the cores were taken. These six locations were B-14 SW, C-9 SW, J-3 NE, M-8 SW, N-12 NW, and 0-7 NE. A composite of three soil cores, 2-1/2 feet apart, was collected from each site at a distance 50 feet from the designated grid marker and on an imaginary line perpendicular to the specified direction for sampling. These cores were collected in 6-inch segments down to 3 feet, and the soil from the corresponding depth segments for each of the three cores collected from a specified site were throughly mixed. A control soil sample was collected in a similar manner from the same location that the soil for the standards was taken. The soil from each site/depth segment-unit was distributed among six cups and a bioassay was conducted in the same manner as that described above for the Orange concentration standards. Again both cucumber and soybeans were used as the test organisms. This same procedure was used to collect and bioassay soil samples from sites 50 yards south of grid markers O-5 and 0-8 which are on the southern border of the present test area (and in the old grid 1 area). Control soil was collected for this bioassay also from the same site as the other controls and the standards, and soybeans and cucumbers were used as the test organisms. 31 �In addition, soil samples from many locations on the test grid were collected and analyzed for organic matter content. Samples were analyzed by the use of a muffle furnace (total combustion of organic matter). c. Statistical Methods General. Except as noted, statistical analyses were conducted to test for significant differences at the 0.95 probability level. If a significant difference was indicated at this level, further testing was conducted at the 0.99 probability level. Wide variations .occurred among the measurements of the plants within the individual cups. The seeds actually belonged to two populations: (1) those seeds that would germinate under proper conditions and (2) those seeds that would not germinate. It was impossible to determine which population an individual seed belong to in all cases, and the extreme values (zero length when a seed did not germinate) would have biased the results had the arithmetic mean been used as a representative cup value. Therefore, the median measurement of the plants within a cup was used as the cup value, and the cup became the experimental unit. Calibration of Standards. An analysis of variance (ANOVA) technique was employed to study the effects upon the plants grown in the soils treated with the standard concentrations of the defoliant Orange. This was done to determine those concentrations which affected plant growth in a significantly different manner. Also, it was hypothesized that because of soil composition variations with depth, a different percentage of the applied defoliant would be bound to the soil or in other ways unavailable to the plants. If this were the case, different standard curves would have to be developed for each depth group. A test for homoscedasticity indicated unacceptable differences in variances. The transformation: x = Iog10 (x' + 1) Where : x = the transformed data x' = the original cup value provided homogeneity of variance. Both the concentration levels and the depth increments were arbitrarily selected and thus are parametric factors. Since each corresponding site/depth-segment was treated alike, the experiment has a cross-classified design, and the statistical model can be expressed: Yijk = a + c, + dj + (cd)jj + e jjk Where: a = the overall effect Cj = the effect of the itn concentration (i = 1,2,...,m) dj = the effect of the jtn depth-segment (j = 1,2,...,n) 32 �Gjjk = the error factor for the ktn replicate of the itn concentration and the jtn depth-segment yp = the cup value for the ktn replication of the itn concentration and the jtn depth-segment The ANOVA technique employed was based on this model. In each instance that a significant difference was indicated between the levels of a factor or when a significant interaction was indicated, Duncan's new multiple range test was used to separate the levels into homogenous groups. A mathematical expression of the form y = a + bx which approximated the relationship between the defoliant concentration and the cup value was determined for each homogenous group by the method of least squares. To find such a relationship a correlation study was undertaken which compared the standard data and square root and logarithm transformations of both the concentration and cup values. Once the proper form of the data was selected, regression analysis was used to study the mathematical expressions. This included tests of the following hypotheses: (1) The data does not fit the curve (lack of fit test). (2) One regression line can be used for all the depth groups. (3) The regression coefficients for the different depth groups are equal. (4) If all the regression coefficients are equal, the elevations (Y-intercepts) are equal. Analysis of the Grid Samples. The concentration of Orange present in the soil from each site/depth-segment taken from the testing area was estimated by using the calibration curves developed for the standard concentrations. The bioassay of the standard Orange concentrations and the bioassay of the grid soil samples were done at different times; therefore, an adjustment of the data was necessary to eliminate the bias and confounding introduced by this procedure. Since control plants were grown with each group, the grid soil bioassay data was weighted according to the ratio of the average of the controls for the test area data to the average of the zero standard concentration data. (A paired-observations t-test of these groups indicated they were significantly different at the 0.95 probability level.) 3. RESULTS AND DISCUSSION a. Calibration of Standard Curves The results of the ANOVA was the same for both soybeans and cJcumbers. The interaction effect between concentration and depth-segments was non-significant; therefore, this effect was pooled with the residual term. The effects of both concentration and depth were highly significant (at a probability level greater than 0.99). These ANOVA tables are presented in Table 11-1 along with the means and the results of Duncan's new multiple range tests. The multiple range tests indicated that for both soybeans and cucumbers each concentration level was significantly different from every other concentration level, and the range of the standard curve could include all the concentration levels tested, i.e., from 0.0 ppm to 0.908 ppm. The 33 �TABLE 11-1. ANALYSIS OF VARIANCE.- ORANGE STANDARDS Concentration 6 5.3013 0.8836 152.343 3.0419 0.5070 144.869 Depth 5 0.6875 0.1375 23.71a 0.7185 0.1437 41.069 30 0.1195 0.0040 0.1620 0.0054 1.93 Residual 84 0.5389 0.0064 0.2314 0.0028 Adj Error 114 0.6584 0.0058 0.3934 0.0035 Cone, x Depth --- Indicates significance at the 0.99 probability level. DUNCAN'S NEW MULTIPLE RANGE TEST Soybean Data Results Cone. Mean Values: 1.06 0.97 0.92 0.80 0.73 0.59 0.43 Depth Mean Values: 0.85 0.78 0.74 0.71 0.91 0.70 Cucumber Data Results Cone. Mean Values: 1.17 1.04 0.99 0.93 0.83 0.75 Depth Mean Values: 0.93 0.91 0.87 0.85 1.07 0.69 0.85 Note: Common underscoring indicates homogenous groups at 0.95 probability level. depth-segment groupings for soybeans were somewhat different that those for cucumbers. At the 0.95 probability level, three distinct depth groups were indicated from the cucumber data set. The groupings indicated by the soybean data set were not as clearcut, and the 18 to 24 inch depth-segment could be placed into two different groups. Since the cucumber data set indicated that this depth-segment should be placed in the last (deepest) depth group, a similar decision was made in the case of the soybean data. These depth groupings are as follows: Depth, inches Group Cucumber Soybean I 0 to 6 0 to 6 II 6 to 18 6 to 12 III 18 to 36 12 to 18 IV --- 18 to 36 In the correlation analysis, the necessity for transformation of both the Orange concentration values and cup values were studied. In addition to the untransformed data, square root and logarithmic transformations were included. The correlation matrices for both the soybean and the cucumber data sets are presented in Table II-2. In both cases, the best correlation (soybeans: -0.8896; cucumbers: -0.8335) was logiQ(cup value + 1) x vconcen1:ration (PPm) 34 �TABLE 11-2. CORRELATION MATRICES CUP VALUES CONCENTRATION (ppm) NO TRANS LOGin(x+y) SQUARE ROOT No Trans: -0.7423 -0.7761 -0.9353 Log10(x+y) -0.8405 -0.8637 -0.8896 Square Root -0.8085 -0.8363 -0.8758 No Trans: -0.6547 -0.6908 -0.7703 Log10(x+y) -0.7399 -0.7728 -0.8335 Square Root -0.7049 -0.7385 -0.8094 SOYBEAN DATA CUCUMBER DATA The method of least squares was used to determine the best fitting linear expression of the relationship between these two transformations for each of the four depth groups identified for the soybean standards and the three depth groups identified for the cucumber standards. These preliminary mathematical equations along with the square root of the percent of deviations from the mean explained by the regression equation (correlation coefficient) are presented in Table II-3. TABLE II-3. PRELIMINARY MATHEMATICAL RELATIONSHIPS FOR STANDARD CONCENTRATIONS CORR: COEFF. DEPTH, in. REGRESSION EQUATION I 0 to 6 Y = 1.15- 0.59X 0.86 II 6 to 12 Y = 1.11 - 0.64X 0.86 III 12 to 18 Y = 1.06 - 0.69X 0.98 IV 18 to 36 Y = 1.01 - 0.72X 0.98 GROUP SOYBEAN DATA CUCUMBER DATA I 0 to 6 Y = 1.27 - 0.49X 0.86 II 6 to 18 Y = 1.13- 0.52X 0.87 III 18 to 36 Y = 1.06 - 0.50X 0.92 Where: Y = lo 3^rj(cup value + 1) x- 2 \concentration 35 �F-tests were employed to further analyze these results as follows: Hypothesis Tested Soy Bean Data (1) The data does not fit the curve: Depth Group I Depth Group II Depth Group III Depth Group IV F-Value Cucumber Data a 55.49 353.92 a 420.24 a 1,184.46 (2) One regression equation can represent all depth groups a 58.14 271.84 a 320.00 a a 11.26 (3) All the regression coefficients are equal 14.00 0.98 a (4) The elevations are equal a 28.39 0.04 a 36.82 Indicates significant at the 0.99 probability level; hypothesis rejected. Except for the hypothesis that all the regression coefficients are equal (which was not rejected), these hypotheses were rejected at the 0.99 probability level for both the soybean and cucumber data sets. Thus within each data set, the slope of the regression equations for the different depth groups is equal. However, rejection of the last hypothesis indicates that the intercept for each depth group is different. A common regression coefficient was computed for each data set, and the final equations are as follows: (These equations are plotted in Figures II-2 and II-3). Depth Group Regression Equation for Root Lengths Soybeans Cucumbers I Y = 1.18467 - 0.68076-X Y = 1.27936 - 0.50374-X II Y = 1.12705 - 0.68076-X Y = 1.12775 - 0.50374-X III Y = 1.05610 - 0.68076-X Y = 1.06402 - 0.50374-X IV Y = 0.99721 - 0.68076-X Where: Y = log-|Q(cup value + 1) 2 X = >/ concentration Tppm) and 0.0 <X < VO908 b. Analysis of Grid Samples Using the relationships expressed in the previous paragraph, estimates were made of the concentration of herbicide Orange in the soil samples taken from the defoliant testing area. These estimates along with the limits of the 95% confidence intervals are presented in Table II-4. Analysis for organic matter content are presented in Table II-5. A comparison of organic matter content within selected cases is presented in Table II-6. 36 �DEPTH GROUP 1 O) o (0 c (Q E o z UJ ORANGE (ppm) Trans.: Figure II-2. Concentration Calibration Curves for Soybean Bioassay [see text for description of depth groups] 37 �DEPTH GROUP 1 O) o O.9 (0 c CO 0.7 E u * 0.5 O z UJ 0.3 0. 0.8 ORANGE (ppm) Trans. = Figure II-3. Concentration Calibration Curves for Cucumber Bioassay [see text for description of depth groups] 38 �TABLE 11-4. CONCENTRATION ESTIMATES OF ORANGE AND 95% CONFIDENCE INTERVAL LIMITS VIA BIOASSAY ANALYSES Cucumber Data Soybean Data Site Depth Segment Lower Limit Concentration Estimate (ppm) Unper Limit Lower Limit B-14 1 0.0000 .0113 .1638 0.0000 .0032 .1526 B-14 2 0.0000 .0730 .3413 0.0000 0.0000 .0358 B-14 3 0.0000 .0138 .0593 0.0000 0.0000 .0363 B-14 4 .0003 .0130 .0450 0.0000 0.0000 .0344 B-14 5 .0012 .0175 .0528 0.0000 0.0000 .0341 B-14 6 .0148 .0475 .0987 0.0000 .0000 .0341 C-9 1 .0693 .3034 .7030 .0087 .1652 .5183 C-9 2 .0535 .2975 .7389 0.0000 .0161 .0932 C-9 3 .0765 .1579 .2684 0.0000 .0079 .0723 C-9 4 .0344 .0791 .1420 0.0000 0.0000 .0333 C-9 5 .0344 .0791 .1420 0.0000 0.0000 .0326 C-9 6 .0293 .0712 .1315 0.0000 .0003 .0389 ,1-3 1 .0814 .3297 .7447 .0136 .1851 .5532 .1-3 2 .0214 .2094 .5912 0.0000 .0050 .0632 J-3 3 .0263 .0805 .1645 0.0000 .0025 .0540 .1-3 4 .0057 .0297 .0725 0.0000 0.0000 .0391 J-3 5 0.0000 .0017 .0196 0.0000 0.0000 .0387 J-3 6 0.0000 0.0000 .0105 0.0000 0.0000 .0393 M-8 1 .0814 .3297 .7447 .0017 .1260 .4473 M-8 2 .0222 .2120 .5957 0.0000 .0032 .0567 M-8 3 .0700 .1485 .2562 0.0000 ..0098 .0778 M-8 4 .0367 .0824 .1464 0.0000 0.0000 .0331 39 Concentration Upper Estimate (ppm) Limit �TABLE 11-4. CONCLUDED Cucumber Data Soybean Data Site Depth Segment M- 8 5 Lower Limit .47 07 M- 8 6 .0426 .0911 N-12 1 •0814 .3297 .7447 N-12 2 •0183 .1993 5738 0.0000 .0044 . 6 3 0 1 N-12 3 •°567 -1289 .2304 0.0000 .0039 .0594 N-12 4 •0691 .1282 .2054 0.0000 .0005 .0406 N-12 5 .0985 .1672 .2540 0.0000 0.0000 .0339 N-12 6 •0593 .1147 .1883 0.0000 0.0000 .0326 0- 7 1 •°169 -1718 .4885 0033 .1377 .4689 0- 7 2 •°183 -19" .5738 0.0000 .0111 .0812 0- 7 3 .0441 .1097 .2046 0.0000 .0262 .1151 0- 7 4 •0344 . 7 1 0 9 .1420 0.0000 .0022 .0507 0- 7 5 .0247 .0640 A217 0.0000 .0007 .0423 0- 7 6 •0293 .0712 <1315 0.0000 0.0000 .0326 0- 5 1 0.0000 .0321 .0184 .2020 .5826 0- 5 2 0.0000 .Oi96 0.0000 .0209 . 0 0 1 4 0- 5 3 0.0000 .ooio .0259 .0025 .0508 . 6 5 1 0 0- 5 4 0.0000 . 0 8 0 1 .0202 000 .00 .06 02 .56 02 0- 5 5 o.oooo .0102 0.0000 .0116 .0806 0- 5 6 0.0000 .0012 .0180 0.0000 .0252 .1110 0- 8 1 0.0000 .0249 .2040 0.0000 .0047 .1611 0- 8 2 0.0000 .0029 .1489 0.0000 0.0000 .0385 0- 8 3 o.oooo 0.0ooo .0178 0.0000 0.0000 .0347 0- 8 4 000 .00 o.oooo . 1 3 0 0 0.0000 0.0000 .0369 0- 8 5 o.oooo o.oooo ,0103 0.0000 0.0000 .0370 0- 8 6 0.0000 .0007 . 1 0 0 6 0.0000 0.0000 .9354 Concentration Upper Estimate (ppm) Limit .95 08 <1675 157g .2225 >2142 o.oooo 40 Lower Limit Concentration Upper Estimate (ppm) Limit 000 .00 000 .00 .33 04 0.0000 0.0000 -0330 .0005 .1136 .4243 �TABLE 11-5. PERCENT ORGANIC MATTER OF SOIL FROM TEST AREA C-52A [Samples Collected between December 1970 and June 1971] SAMPLE A-11 B-8 B-14 C-11 D-7 E-3 E-6 F-12 J-1 L-10 N-5 N-8 N-12 0-2 CONTROL (0.2 mile NW of grid) C-9 C-9 C-9 C-9 C-9 C-9 G-11 G-11 G-11 G-11 G-11 0-7 0-7 0-7 0-7 0-7 0-7 CONTROL (0.2 mile NW of grid) a DEPTH, Inches 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 0-6 6-12 12-18 18-24 24-30 30-36 0-6 6-12 12-18 18-24 24-30 0-6 6-12 12-18 18-24 24-30 30-36 0-6 6-12 12-18 18-24 24-30 30-36 ORGANIC MATTER' 0.68° 1.05a 1.13a 0.55a 0.87a 0.73b 0.37b 1.31a 0.85a 0.68b 0.76a 0.41b 0.72a 0.45b 1.25b 1.99 0.96 0.71 0.72 0.67 0.87 0.41 1.65b 1.99 1.04 1.61 1.49 1.06 0.57 0.52 0.56 0.62 0.54 0.59 2.03a 1.35a 0.86a 0.73a 0.83a 0.71a Average of three replicates. b Determined by the Wakley-Black wet digestion method; all others analyzed by weight loss after 30 minute heating in tared crucible. c Samples designated by the closest permanent sampler station. Samples taken 50 feet from sampler. 41 �TABLE 11-6. PERCENT ORGANIC MATTER WITHIN TREATMENT AND CONTROL CORES TAKEN FROM TEST AREA C-52A DEPTH, inches CONTROL ORGANIC MATTER, % G-11 C-9 0-7 Oto 6 2.03 0.96 1.99 0.57 6 to 12 1.35 0.71 1.04 0.52 12 to 18 0.86 0.72 1.61 0.56 18 to 24 0.73 0.67 1.49 0.62 24 to 30 0.83 0.87 1.06 0.54 30 to 36 0.71 0.41 0.96 0.59 4. CONCLUSIONS The varying effects of different concentrations of herbicide Orange and the changes in soil composition associated with different depths are clearly indicated by the ANOVA and multiple range tests on the bioassay of the standard concentrations. Thus, it should be expected that the same concentration of Orange in different types of soils will be reflected by different plant growth patterns. An obvious disparity exists between the soybean data and cucumber data estimates of Orange concentration at various test area sites. Confounding occurred because parts of this experiment were conducted at different times without statistical balancing. The attempt to adjust for those differences by weighting the data to reflect the differences in the control plants was not successful. Evidently the differences in the environments could not be explained by such a simple adjustment. 5. CHEMICAL ANALYSES OF SOIL CORES As a result of the bioassay analyses previously discussed, those soil samples collected in November 1969 and in April and December 1970, which caused the greatest growth inhibition, were analyzed chemically for 2,4-D; 2,4,5-T; picloram; arsenic; and the contaminant TCDD. a. Methods and Materials A 25 to 50 gram soil sample was weighed, acidified, and extracted with 1:1 hexane: acetone (see Figure II-4). The hexane:acetone was made basic and the aqueous phase saved for extraction by ether after acidification, butylation via boron trichloride, and the subsequent determination of 2,4-D and 2,4,5-T. The hexane phase was gently shaken repeatedly with sulfuric acid until the sulfuric acid was clear. The hexane phase was then condensed and the extract representing 50 - 100 mg of soil was injected on a 5% OV-225 gas chromatographic (GC) column. The GC was equipped with a Ni"^ electron capture detector. If a peak was found within.+. 10% of the retention time of TCDD, the sample was irradiated with ultraviolet light for 16 hours. Column chromatography was also employed. A gas chromatograph trace of the TCDD samples is shown in Figure II-5. This figure shows an unaltered and a spiked soil sample before and after ultraviolet light treatment for three different soil samples. In no case was any TCDD detected. Notice also that the added TCDD was completely destroyed by UV irradiation. 42 �CONDENSE 5 ml EXTRACT 50 g SOIL HEXANE ACETONE 1 GO EXTRACT WATER PHASE KOH, H20 HCI ETHER EXTRACT IRRADIATE TCDD DESTROYED ETHER n - BuOH BCI3 INJECT 10,\a= 100 mg LLDb = 0.001 ppm DETERMINE BUTYL ESTERS a b c Lambda = A LLD = Lower Limit of Detection p value = Partition Value Figure ii-4. p - VALUE 0 0.51 ±0.06 HEXANE: ACETONITRSLE Analysis Flow Chart for the Extraction of 2,4-D; 2,4,5-T; and TCDD from Lakeland Sand �50 mgSOIL INJECTED -UNALTERED SOIL 5 ppb TCDD ADDED I N 160 POUNDS 2,4,5-T/A APPLIED 5 ppb TCDD ADDED I UNALTERED SOIL I UV UV \ 584 POUNDS 2,4,5-T/A APPLIED \ \ 5 ppb TCDD ADDED UNALTERED SOIL i UV 1 2 UV < 3 4 5 6 CONTROL SOIL Figure II-5. Gas Chromatograph Traces of TCDD Samples. (The Traces Represent 50 mg of Extracted Soils from Grids 2 and 4. See Table I-7) 44 �The soils were analyzed for arsenic using 6 mis of 1:1 h^SC^/HCK^ per 10 g, followed by reductive distillation and development of a molybdenum blue color. Nine soil cores (to the 36-inch depth) were analyzed for picloram by Dow Chemical U.S.A. The minimum detection limit for picloram was 5 ppb. Additional soil samples were collected in June and October 1973 from near sampler sites C-9, F-6, and 0-7 and from the center of Grid 1 located approximately 1000 feet south of sampler station 0-7 (see Figure I-5). These soil samples were analyzed for TCDD by the Interpretive Analytical Services, Dow Chemical,. U.S.A., Midland, Michigan. The method of analysis reported by the Interpretive Analytical Services is as follows: 10 gms of soil were extracted with 1:1 acetone-hexane, and the hexane recovered from the extract by the addition of water. (J. Assoc. Offic. Anal. Chemists 56. 728 (1973). (Initially, the soil extracts were subjected to separation procedures involving only silica gel column chromatography, but too many interferences were found for best sensitivity. Hence, all samples were treated by a modification of the techniques developed by Baughman and Meselson, "An Analytical Method for Detecting Dioxin", National Institute of Environmental Health Sciences Conference on Dibenzo-dioxins and Dibenzo furans, Research Triangle Park, NC, April 1973, published in Evnironmental Health Perspectives, No. 5, August 1973). The modified procedure is as follows: Hexane extracts from above washed with successive 10 ml portions of concentrated H2S04 until H2SC>4 is colorless (4 - 5 washings). Hexane evaporated to 0.5 ml, and passed through a silica gel column. Dioxins eluted from the column and 1:5 benzene-hexane. Eluate evaporated to a small volume, and taken up in 0.5 ml hexane. (This step is in addition to the Baughman-Meselson procedure, and was found necessary to achieve best sensitivity.) Hexane solution from above placed on an Al 20o column. PCB's eluted with 1:4 CC^-hexane. Dioxin eluted with 1:4 CH2Cl2-hexane. Eluate evaporated to small volume and injected directly into an LKB-9000 gas chromatography-mass spectrometer combination. Column Conditions: 6 ft x 1/8 inch stainless steel packed with 3% OV-3 on 80/100 mesh Gaschrom. Z, isothermal at 230°C. Retention time ~ 6.5 min. Detector: mass spectrometer set to monitor m/e = 320 and 322 simultaneously. b. Results and Discussions Table II-7 shows the results of all analyses for soils collected during the period 1969 through 1971. Notice the persistence of 2,4-D and 2,4,5-T in soil core J-3NE from April to December 1970. However, it should be emphasized that if no herbicide degradation had occurred, a concentration of approximately 80 ppm 2,4,5-T might be expected within the top 6 inches (see Table II-7, 45 �assuming the weight of a one acre 6-inch increment weighs 2 million pounds). Notice that cores M-8SW, N-12SW, and 0-5S show leaching of 2,4-D, and 2,4,5-T. This particular region of the grid received 1,168 pounds per acre of Orange from 1964 to 1966. Moreover, 0-5S may have received heavy concentrations of herbicides when Grid 1 was in use (1962 to 1964). Table II-8 compares chemical and bioassay data for the same core (M-8S), collected in December 1970 and subsampled for analyses. These data suggest that chemical analysis for 2.4-D and 2,4,5-T alone may not account for all the biologically active phytotoxic components (e.g., degradation products and/or fuel oil residue). Note that the trend is reasonably similar between methods of analysis. No TCDD residues were found by the Pesticide Degradation Laboratory in any of the soil samples at a minimum detection limit of less than 1 ppb. Recent analyses of drums of Orange in storage suggest that the average concentration of TCDD in Orange may be 2 ppm . If it is assumed that all the 2,4,5-T sprayed on TA C-52A was contaminated with 2 ppm TCDD, then approximately 0.5 pound of TCDD was disseminated in this test area. However, the analyses of TCDD in the part per trillion (ppt) range would be required for detection of potential residue. The results of soil samples collected in June and October 1973 and analyzed by Interpretive Analytical Services, in the parts per trillion range, did in fact indicate the presence of TCDD or a TCDD-like chemical compound. These data are shown in Table 11-10. The greatest concentration of TCDD was found in the soil core from the center of Grid 1. This grid received 0:1 the average 947 pounds 2,4,5-T per acre in the period 1962-1964. The levels detected in the 6 to 12 and 12 to 18 inch depths were probably due to contamination at the time of sample collection. These data suggest that TCDD or a TCDD-like compound may persist for an extended period of time. Moreover, it would appear that the Orange (Purple) disseminated on this test grid was significantly contaminated with this compound. Significant levels of picloram were found in November 1969 near Sampler K-9. Notice that at this date the residue was confined to the top 12 inches. However, by May 1970 picloram may have moved to the lower increments within the soil profile. Table II-7 also shows the levels of arsenic found in selected soil cores. From the data in this table, it is evident that there is no appreciable build-up of arsenic in the soil. Perhaps this is due to leaching or possibly to the reduction and volatilization of dimethylarsine from the cacodylic acid. These observations are further verified by data on arsenic levels from sites J-3NE, C-9SW, K-9N, and 0-8S collected and analyzed in June 1973. Table II-9 compares the arsenic levels from these sites collected in 1970 and again in 1973. c. Conclusions Small amounts (in parts per billion) of 2,4-D, 2,4,5-T, and picloram were found persisting on the test area in June 1971. The last application of Orange was December 1969, while the last application of White was May 1970. The last application of Blue was in September 1970; nevertheless, no significant build-up of arsenic has been noted. However, leaching of the arsenical from the soils may have occurred. Significant TCDD residue (or a TCDD-like compound) has been detected in the parts per trillion range from Grid 1. o ^Personal communication with Dr. Walter Melvin, February 1973, Air Force Environmental Health Laboratory, Kelly AFB, Texas. 46 �TABLE 11-7. RESULTS OF CHEMICAL ANALYSES FOR PICLORAM; 2,4-D; 2,4,5-T; 2,3,7,8-TCDD; AND ARSENIC IN SOIL SAMPLES FROM EGLIN AFB RESERVATION TEST AREA C-52A SOIL SAMPLE3 DEPTHb TCDDd, ppb ARSEN!Cd, ppm PICLORAMC, ppb 2,4-D, ppb 1e 34,21 1.2 2.8 <1.0 2.24 2 21, 11 7.0 2.0 <1.0 0.86 3 <10, 6 0.1 0.8 <1.0 0.90 4 <10, 5 0.1 0.6 <1.0 0.52 5 <10, <5 0.1 0.9 <1.0 0.62 6 <10,< 5 0.1 0.3 <1.0 0.54 1 ND 1.7 1.2 <1.0 0.55 2 ND 1.7 1.0 0.34 3 ND 0.1 1.0 <1.0 <1.0 4 ND 0.1 1.0 <1.0 0.41 5 ND ND ND <1.0 ND 6 ND 0.1 0.7 <1.0 0.52 2,4,5-T, ppb NOVEMBER 1969 K-9N 6 APRIL 11 370 T3RE" " 0.41 10 DECEMBER 1970 1 <10 <0.1 2.4 <0.1 4.70 2 <10 <0.1 1.8 <0.1 1.30 3 <10 <0.1 1.1 <0.2 0.90 4 <10 <0.1 0.7 <0.2 0.55 5 J3NE <10 <0.1 1.0 <0.2 1.13 <0.1 0.3 <0.2 0.90 ND 1.6 5.9 <1.0 3.21 2 ND 1.1 0.1 <1.0 0.48 3 ND 0.1 0.7 <1.0 0.20 4 ND 0.1 0.4 <1.0 0.27 5 ND 0.1 0.3 <1.0 0.27 6 ND 0.1 0.4 <1.0 0.20 6 <10 1 6 APRIL 1970 J-9SE 47 �TABLE 11-7. CONTINUED SOIL SAMPLE3 b DEPTH PICLORAMC, ppb 2,4-D, ppb 2,4,5-T, ppb TCDDd, ppb ARSENICd, ppm 10 DECEMBER 1970 1 <10 <0.1 0.6 <0.1 0.55 2 <10 2.4 0.4 < 0.1 0.55 3 <10 0.6 0.4 <0.1 0.55 4 <10 <0.1 0.2 <Q.2 0.58 5 <10 <0.1 <0.1 <0.3 0.41 6 <10 <0.1 <0.1 <0.1 0.48 1 <10 <0.1 1.8 <0.4 1.64 2 <10 <0.1 1.2 <0.3 0.88 3 <10 <0.1 0.3 <0.2 0.48 4 <10 <0.1 0.3 <0.1 0.48 5 <10 <0.1 <0.1 <0.2 0.55 6 <10 <0.1 0.4 <0.4 0.52 1 <10 5.6 7.0 <0.2 0.90 2 <10 5.8 1.4 <0.2 0.48 3 <10 7.6 2.8 <0.2 0.34 4 <10 15.0 5.6 <0.1 0.41 5 <10 5.0 2.8 <0.5 0.34 6 <10 13.2 6.8 <0.2 0.55 1 <10 7.6 2.2 <0.2 1.86 2 <10 10.0 1.0 <0.2 1.05 3 <10 11.8 1.2 <0.2 0.76 4 <10 4.8 1.4 <0.3 0.69 5 <10 6.0 2.6 <0.2 0.69 6 B-14SW <10 7.0 3.0 <0.2 0.76 10 DECEMBER 1970 C-9SW 10 DECEMBER 1970 M-8SW 10 DECEMBER 1970 N-12SW 10 DECEMBER 1970 0-5S 1 ND 0.8 1.2 <0.1 0.90 2 ND 0.6 0.6 <0.1 0.80 3 ND 0.6 1.2 <0.2 0.76 4 ND 0.6 0.6 <0.2 0.41 5 ND <0.1 8.4 <0.7 0.69 6 ND 0.6 1.4 <0.1 0.55 48 �TABLE 11-7. SOIL SAMPLE3 DEPTH b PICLORAMC, ppb CONTINUED 2,4-D, ppb 2,4,5-T, ppb TCDDd, ppb ARSENICd, ppm 10 DECEMBER 1970 0-7NE 1 <10 ND 6.6 <0.2 2 <10 2.8 0.2 <0.3 3 <10 3.6 0.4 <0.2 4 <10 0.8 2.6 <0.2 5 <10 5.6 2.6 < 0.2 6 <10 1.2 0.2 <0.6 1.52 0.76 0.76 0.62 0.62 0.62 10 DECEMBER 1970 1 1.2 <0.1 ND <0.1 2.6 <0.1 ND <0.1 0.8 <0.1 4 ND <0.1 0.6 <0.1 5 ND <0.1 1.2 <0.1 2.70 0.58 0.62 0.20 0.41 6 ND <0.1 0.6 < 0.2 0.07 1e ND 0.1 8.7 <1.0 1 ND 0.1 3.2 <1.0 2 17,7 ND ND ND 3 <10, 5 ND ND ND 4 11, <5 0.1 0.1 <1.0 4 ND 0.1 ND <1.0 5 <10,<5 10, 5 ND ND ND 6 0.1 0.9 1.0 6 13 MAY 1971 G-9N <0.1 3 13 MAY 1970 K-9N ND 2 0-8S ND 0.1 1.0 1.0 3.94 4.25 ND ND 0.41 0.41 ND 0.41 0.48 2 92, 160 ND ND ND ND 10 DECEMBER 1970 CONTROL 1 <10 <0.1 <0.1 <0.2 0.55 2 <10 <0.1 <0.1 <0.3 3 <10 <0.1 <0.1 <0.2 4 <10 <0.1 <0.1 <0.4 5 <10 <0.1 <0.1 < 0.4 6 <10 <0.1 <0.1 < 0.5 0.41 0.55 0.24 0.41 0.48 r ° f 8 APRIL 1971f 49' �TABLE 11-7. CONCLUDED a Samples designated by the nearest permanent air sampler station on the one square mile test grid. All samples were taken 50 feet from a certain air sampler, except control site was 0.4 mile from the one square mile grid. b Samples taken with a core borer in 6 inch increments. Depth 1 = 0 to 6 inches; 2 = 6 to 12 inches; 3 = 12 to 18 inches; 4 = 18 to 24 inches; 5 = 24 to 30 inches and 6 = 30 to 36 inches. Each increment was uniformily mixed prior to sampling for chemical analysis. G Picloram analysis was performed by International Research and Development Corporation and/or The Dow Chemical Company; Dow Chemical Method ACR 69.10, modified. ^Analysis performed by E. A. Woolson, Pesticide Degradation Laboratory, United States Department of Agriculture, Beltsville, Maryland. e Two samples from same depth were taken 10 feet apart. f,Control soil for picloram analysis taken 8 April 1971, and other analyses performed after 10 December 1970 sampling. TABLE II-8. A COMPARISON OF CHEMICAL AND BIOASSAY DATA FOR SOIL CORE M-8a COLLECTED DECEMBER 1970 DEPTH, Inches CHEMICAL ANALYSIS, ppbb BIOASSAY ANALYSIS0 LOWER LIMIT, UPPER LIMIT, ppb ppb 80 745 0-6 12.6 6-12 7.2 22 596 12-18 10.4 70 256 18-24 20.6 37 146 24-30 7.8 48 168 30-36 20.0 43 158 a Sample collected 50 feet southwest of air sampler station M-8. b Total concentration of 2,4-D and 2,4,5-T. c Data from soybean bioassay (see Table II-4) 50 �TABLE 1 -9. A COMPARISON OF ARSENIC LEVELS IN SOIL CORES COLLECTED IN 1970 AND 1973 FROM TA C-52Aa LOCATION nCDTU Utr I n, J-3NE C-9SW b 1970 1973 1970^ 1973 ON! 1970 1973 0-6 4.70 0.85 1.64 1.68 3.94 0.62 2.70 1.46 6-12 1.30 0.47 0.88 0.56 4.25 0.54 0.58 0.42 12-18 0.90 0.59 0.48 0.60 <MD 0.41 0.62 0.45 I nches a Analysis performed by E. A. Woolson, Pesticide Degradation Department of Agriculture, Beltsville, Maryland. 0-8S 1970 1973 Laboratory, United States "Samples collected 50 feet in the designated direction (e.g., NE) from the permanent air sampler station. C ND = not determined. TABLE 11-10. LEVELS (PARTS PER TRILLION) OF 2,3,7,8 TETRACHLORODIBENZOp-DIOXIN (TCDD) IN SOIL FROM TA C-52A COLLECTED IN JUNE OR OCTOBER 1973a DEPTH, Inches 0-6 6-12 12-18 18-24 24-30 30-36 LOCATION C-9 <10b ND ND ND ND ND F-7 11 ND ND ND ND ND 0-7 GRID 1 30 <10 <10 <10 <10 <10 710 140 72 <10 <10 <10 a Method described in text. '-'Lower limit of detection in parts per trillion TCDD. c Probable interference from excessive organic matter. 51 CONTROL <20C <10 <10 <10 <10 <10 �SECTION III STUDIES OF THE VEGETATION OF TEST AREA C-52A The first studies of Test Area C-52A were those concerned with vegetation. Testing of aerial spray equipment began in June 1962, and following heavy applications of materials in 1963 and 1964, vegetation surrounding the test site showed changes suggestive of herbicidal damage. In the fall of 1966, concern about the extent of this damage led to the establishment of a contract with the University of Florida, Gainesville, Florida. The purpose of the contract was to conduct a taxonomic study that would quantitatively measure changes in density of vascular plants in the area adjacent to the test grid (References 111-1, III-2, and III-3). Observations of tree growth rings in those reports prompted studies (Reference III-4) concerned with assessment of spray drift upon the forest trees adjacent to the test area. A third study (Reference IM-5) was concerned with the histological examination of a plant species growing in the flight lines on the test grid. Synopses of Reference 111-1 to IM-5 are included in this section. The last report, and the one most concerned with the current research effort (Reference III-6), will be summarized and referred to in this report within the section on current studies. References: 111-1. Ward, D. B.: Ecological Records on Eglin AFB Reservation - - the First Year. AFATL-TR-67-157, Air Force Armament Laboratory, Eglin Air Force Base, Florida, 1967. Unclassified. III-2. Ward, D. B.: Ecological Records on Eglin AFB Reservation - - the Second Year. AFATL-TR-68-147, Air Force Armament Laboratory, Eglin Air Force Base, Florida, 1968. Unclassified. III-3. Ward, D. B.: Ecological Records on Eglin AFB Reservation - - Conclusions. AFATL-TR-70-55, Air Force Armament Laboratory, Eglin Air Force Base, Florida, 1970. Unclassified. III-4. Hunter, H. H. and B. M. Agerton: Annual Diameter Growth of Conifers Adjacent to Eglin Reservation Test Area C-52A as Related to the Testing of Defoliant Spray Equipment. AFATL-TR-71-52, Air Force Armament Laboratory, Eglin Air Force Base, Florida, 1971. Unclassified. III-5. Sturrock, T. T. and A. L. Young: A Histological Study of Yucca filamentosa L. from Test Area C-52A, Eglin Reservation, Florida. AFATL-TR-70-125, Air Force Armament Laboratory, Eglin Air Force Base, Florida. 1970. Unclassified. III-6. Hunter, J. H. and A. L. Young: Vegetative Succession Studies on a Defoliant-Equipment Test Area, Eglin AFB Reservation, Florida. AFATL-TR-72-31, Air Force Armament Laboratory, Eglin Air Force Base, Florida. 1970. Unclassified. 52 �1. SYNOPSIS OF TAXONOMIC STUDIES, 1966 - 1969 a. General Observations In the fall of 1966, turkey oak, Quercus laevis. adjacent to the test clearing was severely affected, apparently by herbicide driftage; and the large proportion of the trees of this species gave the entire forest an abnormal aspect. Upper branches of all trees had apparently been completely defoliated, and many or even most of the twigs were killed. On the lower trunks a proliferation of small branchlets had appeared; in all cases, these branchlets began growing in the spring of 1965, as determined by the number of bud scale scars present. Blue-jack oak, Quercus incana. and sand live oak. Quercus germinata. were also heavily damaged near the test clearing but appeared undamaged as little as 1.1 miles from the clearing in an area where turkey oak still exhibited signs of damage. Longleaf pine, Pinus palustris. and sand pine, Pinus clausa. adjacent to the test clearing appeared unaffected. Needle length and internode length were normal, as compared with similar plants away from the test area. A sampling was made of the growth rates of four tree species as indicated by width of annual growth rings. Blue-jack oak, turkey oak, and long-leaf pine produced growth rings that appeared to vary independently of spray applications to the adjacent TA C-52A. Sand pine seemingly showed a positive correlation with a marked increase in growth in the years immediately after the 1963 - 1964 period of heavy spray application. Extensive observation of the vegetation in all directions from TA C-52A indicated a rapid disappearance of damage attributable to spray driftage. The maximum distance that damage was definitely detected was 5 miles, seen on trees at the upper end of Range 52, north of the test area. Observations made later in this project failed to disclose damage at any distance from the test area that was not attributable to the 1963 - 1964 period of activity. Following a testing period in 1969, particular attention was given to the oaks, pines, and herbaceous plants in various directions from TA C-52A. A few sand pines along the south edge of the test area had visible abnormalities in their early spring growth, but this had largely disappeared by early August. Other vegetation appeared normal, suggesting that herbicide driftage, if any, from the 1969 series of tests was significantly less than in earlier years. b. Quantitative Sampling In the fall of 1966, a program was devised to permit the quantitative measurement of changes in density of stand of vascular plants in the area adjacent to TA C-52A. Since changes in the herbaceous plants might be more subtle and more difficult to detect than those in the larger woody plants, an experimental design was developed that would disclose differences of slight magnitude but of statistical significance. Five stations were selected in apparently homogeneous woodland east of the test area, and at each station four parallel transects of 50 meters were laid out at right angles to an imaginary 53 �radius from the center of TA C-52A. The first of these sets of transects was immediately adjacent to the test clearing, and the fifth was at a distance of 2.0 miles. All plants intercepted by the transects were counted and listed. The resulting data were interpreted by a conventional analysis of variance and F-test. The 2157 plants of 54 species intercepted by the 1000 meters of transect were found (with a few exceptions) not to differ significantly in their frequency with distance from the test area. This was taken to mean that the supposedly higher spray driftage near the test area had not had a significant effect on the viability of individuals of most species. A few species were found to differ significantly with distance from the test area. Eleven species fluctuated significantly in number of individuals at varying distances from the test area, but upon examination, these differences were found to result from natural heterogeneities in the supposedly homogeneous woodland. They could not be attributed to spray effects. Three species were found to be absent near the test area and to increase significantly away from this area. These species were the small, upright legume, I^h_rQais..nQoJiriL the persimmon, Djospyros virgmiana. and the weeping haw, Crataegus lacrimata. Again, since the legume is a perennial with a deeply buried rootstock and the other two are trees which would have left evidence in the form of dead trunks, it is probable that these species were not exterminated by spray driftage near the test area, but rather reflect an original and natural heterogeneity in their distribution. Only in the case of four species was there clear evidence of influence by proximity to the test area. The number of individuals of four herbs - hjyp.gricym gentianoides, Solidago odora. Warea sessilifolia, and Rhynchosia cytisoides - decreased significantly away from TA C-52A. More meaningfully, the number of individuals of these species increased significantly adjacent to the test site. These four species are noted for their ability to rapidly colonize cleared land. The observed variation in number of individuals is best explained by these species utilizing the opening in the canopy resulting from the spray-induced loss of foliage by the oaks. By early August 1969 the oaks in the area of the five transect stations again had normal foliage. At that date, it was not possible to find individuals of the Hypericum. the Warea. or the Rhynchosia. and the Solidago was very reduced in number. The presumption is that the spray-induced damage of 1963 - 1964 to the forest surrounding the test site had largely disappeared and that by 1969 the vegetation of the area was again essentially normal. 2. SYNOPSIS OF GROWTH OF SAND PINE, 1969 - 1970 a. General Comments One of the observations noted in Reference 111-1 was that the growth of sand pine, Pin'us cjausa, was seemingly related (positive correlation) to the periods of heaviest testing of spray equipment. This observation prompted an investigation, conducted between March 1969 and June 1970, into the growth of tree rings. The species of trees selected for sampling were sand pine, longleaf pine, P. palustrjs. and turkey oak, Quercus Jaeyjs. A total of 18 sand pines were cut along with two longleaf pines and two turkey oaks. Sand pines were cut at ground level, and all others were cut 2 to 3 feet above ground level. A cross section was cut from the end of each trunk and was sanded so that the annual growth rings could be measured - to the nearest 0.1 mm. 54 �An examination of the relative amount of annual herbicide delivery on TA C-52A revealed that the greatest amount of defoliant drift probably occurred in the following increasing order of years: 1967, 1966, 1962, 1965, 1969, 1968, 1964, and 1963. This order is based primarily on the amount, type, and formulation of materials sprayed per year. The amount of drift and defoliant damage that occurred would also have depended upon climatic and flight conditions. For example, probably very little drift damage occurred in 1967 because relatively little testing was done and all testing occurred during winter months (the time of a reduced level of plant growth). Likewise, damage would have been expected in 1964, not only because of the many Orange missions, but also because all testing occurred from May through July and some of the missions were flown at altitudes greater than the usual 150 to 200 feet. b. Results An initial examination of the annual diameter growth data indicated that possibly the growth of some trees was directly or indirectly stimulated by spray drift from defoliant testing. However, an analysis of the data did not substantiate this hypothesis. Growth obtained by trees during individual years of testing was often no greater than growth obtained in some previous year before defoliant spraying started. When the total annual diameter growth for individual pines during years of defoliant testing was compared with the same number of years before testing, no significant differences were apparent. Five of nine treatment trees (those close to TA C-52A) and three of six control trees made less total growth during the years of testing. For longleaf pine, one sample showed a decrease and the other an increase between 1962 and 1968. Several of the pines located close to TA C-52A made less annual growth in 1963 - 1964 (period of expected greatest damage) when compared to the annual diameter growth of other years of defoliant testing. However, the reduced amount of growth did not seem to be related to defoliant spraying because three control trees also grew less in 1963 than any other year between 1962 and 1969. In addition, the amount of diameter growth in 1963 for trees close to TA C-52A was often no less than the diameter growth for some year previous to defoliant testing. Difficulties were encountered in measuring annual diameter growth in turkey oak. The older increments could be easily differentiated, but the last three to five increments were not discernable. Gross observations of the diameter growth of the turkey oak sampled close to TA C-52A indicated the tree had grown less during the years of defoliant testing. As discussed in Reference III-3, the annual diameter growth of two turkey oaks that had grown within a few hundred yards of TA C-52A was measured, but without a clear indication that annual diameter growth was inhibited by defoliant testing. The greatest amount of tree defoliation around TA C-52A had been noticed on turkey oak, and a reduction in annual diameter growth was expected to be most evident in individuals of this species. However, the small amount of sampling showed no indication of a general reduction of annual diameter growth during the years of testing. An attempt was made to relate annual diameter growth to rainfall and temperature. An analysis of average, maximum, and minimum monthly temperatures recorded since 1949 at IMiceville, Florida, revealed a high degree of uniformity for the same months from year to year. Rainfall for each month, however, varied greatly over the years; and many attempts 55 �were made to discover what combinations of monthly rainfall data correlated with annual growth. While the combination of April and May rainfalls yielded the highest correlation of any two other months, only one sample showed a significant probability (95 percent confidence level) of this rainfall being related to annual diameter growth. Therefore, the rainfall data available could not be used to explain the annual growth patterns of the trees observed. 3. SYNOPSIS OF HISTOLOGICAL STUDY OF YUCCA, 1970 a. General Comments The military herbicides Orange (2,4-D and 2,4,5-T) and White (2,4-D and picloram) function as growth regulators in their herbicidal behavior. A study was undertaken to determine whether structural (histological) changes were evident in a plant species (Yucca filamentosa) found in a high herbicide residue area of TA C-52A. Observations have confirmed that the largest bulk of the various chemicals used in the testing of aerial defoliation spray equipment was released and fell within the instrumented test area (TA C-52A). As a result of these repetitive applications, many plant species (i.e., the dicotyledonous plants) were selectively eliminated. The vast majority of the remaining plant life was monocotyledonous with the only distinct plant association being broomsedge (Andropogon virginicus). switchgrass (Panicum virgatum). and yucca (Yucca filamentosa). Field observations indicated that yucca was the most persistent species occuring in the flight-line areas of the test grid. Observations on gross morphology indicated no differences between plants occurring on and off the test grid; however, crownal areas (the plant part at the soil surface) appeared to be different. Specimens of yucca were selected from areas of high herbicide residue and from an area sufficiently distant from the treated area to preclude contact with the herbicides and/or herbicide residues. Tissue samples obtained from the crown area of these plants were prepared for microscopic observation. b. Results Both cross and radial sections of treated and control plants were examined. The control plants were used to establish the normal development of this species. Yucca is different from most monocotyledonous plants in that it has some secondary growth and develops a peridermlike structure. In addition, repeated divisions of parenchyma cells and the suberization of their products produces tangential bands of cork progressing inward and including some of the fibrovascular bundles which have an uncharacteristic forked habit of growth. All of these structures were quite evident in both the control and the treated samples. Based on these studies, there is no apparent difference in the formation of structures in Yucca filamentosa specimens obtained from an area subjected to repetitive applications of military herbicides and specimens from an area not receiving the herbicides. Both samples followed the normal structure for this species, as described in the scientific literature. While these observations were conducted on a very small sample of the plants which did persist in the heavily treated area, no evidence of malformations was found. A larger sample and many more observations of different tissues of the plants might detect abnormalities in this 56 �species under these conditions, and it is also possible that a larger sample of untreated plants would similarly produce individuals with malformations - - such anomalous structures are often found in many species of plants grown under different environmental conditions. 4. CURRENT VEGETATIVE SUCCESSION STUDIES a. Introduction The first complete survey of the vegetation existing on the herbicide test grids was initiated in 1971 (Reference III-6). The 1971 survey established a base line from which future observations or surveys could proceed to determine the rate and type (plant species involved) of plant succession on the test grids. The June 1973 studies of vegetation are a continuation of the 1971 survey and provide precise data and photographs to illustrate changes that have occurred during a 2-year period. b. Materials and Methods In May 1971, the one square mile grid (Figure I-4) was divided into 169 sections (each 400 by 400 feet). The percentage pla.nt cover in each 160,000 ft^ section was visually estimated, and a vegetative coverage map resulted. In June 1973, the same technique was used to construct another vegetative coverage map. Coverage was ranged into five classes as follows: Class O = 0 to 5% cover, Class I = 5 to 20% cover, Class II = 20 to 40% cover. Class III = 40 to 60% cover. Class IV = 60 to 80% cover, and Class V = 80 to 100% cover. In June 1971, three 400 by 400 foot sections from each coverage class were randomly selected for a detailed collection of dicotyledonous (broadleaf) plant species. A diagonal transect starting 20 feet within the northwest corner of each section was walked to the southeastern boundary. Plants were collected along the transect, and the results were tabulated for the number of dicotyledonous plants occurring in each section. A control area 0.2 mile northwest of the one square mile grid and an area in the center of the plot formerly occupied by Grid 1 were also surveyed. In June 1973, each of these areas was again surveyed by the same method. A square-foot analysis was performed on (1) 15 additional 400 by 400 feet sections, (2) the control area used in 1971, (3) a new control area west of the one square mile grid, and (4) a 160,000 ft^ section in the center of area occupied by Grid 1. The additional 15 sections were randomly selected, and within each section, nine areas, each measuring one square foot, were analyzed (Figure 111-1). The square foot sampling sites were selected by dividing the 400 by 400 foot sections into three strips, each 133 feet wide. A line was drawn in center of each strip and three one-square-foot areas were selected by generating d-j, ^ anc' ^3 as distances to be walked. The distances were generated by a random number generator which included constraints that assured one sampling area from each one-third of the transverse with the sampling area being random within the one-third area. After each distance was walked-off, the metal square-foot measuring device was placed, and the percent coverage for each plant species was visually estimated. d. Results and Discussion The 1971 and 1973 vegetative coverage maps are shown in Figures III-2 and III-3, respectively. Table 111-1 shows the percent coverage that each vegetative class occupied in June 1971 and in June 1973. 57 �-Figure 111-1. Square-Foot Measuring Device in Use During Vegetation Survey - June 1973 TABLE 111-1. PERCENT OF VEGETATIVE COVER OCCUPIED BY VEGETATIVE CLASS FOR THE 1 SQUARE MILE GRID VEGETATIVE CLASS JUNE 1971 JUNE 1973 0 (0 to 5%) 4% 0% I (5 to 20%) 14% 4% II (20 to 40%) 29% 12% III (40 to 60%) 25% 18% IV (60 to 80%) 21% 42% V (80 to 100%) 4% 23% 58 �80 to 100% 40 to 60% 5 to 20% 60 to 80% 20 to 40% 0 to 5% 8 A 9 10 11 12 13 14 B C D E F G H J M N O Figure III-2. Vegetative Coverage of the One Square Mile Grid on Test Area C-52A, May 1971 59 �80 to 100% 40 to 60% 5 to 20% 60 to 80% 20 to 40% 0 to 5% 12 13 14 Figure III-3. Vegetative Coverage of the One Square Mile Grid on Test Area C-52A, June 1973 60 �After a 2 year period, no vegetative class O areas were judged to remain on the one square mile grid. All class O areas had developed into class I or II areas. The greatest change in number of individual areas occurred from class II to IV (14% of total) and class III to IV (17% of total). For those areas with less cover than class V, 19 class IV areas, two class III areas, four class II areas and four class I areas did not change in percent cover during the 2 year interval. Examples of vegetative cover changes are illustrated in Figures III-4 to 111-13 by comparing the 1971 and 1973 photographs of identical areas. All of these photographs represent changes from one coverage class to another, except for area IM-5 (Figures III-6 and III-7). Although an increase in vegetative coverage can be seen for N-5, this increase was not enough to place the area in Class II. Figure 111-14 and Figure 111-15 illustrate an increase in number of shrubs for a Class IV area. In 1971, data on number of dicotyledonous or broadleaf species were assembled, and the same 400 by 400 feet areas were again surveyed in 1973. Table III-2 shows the increase in numbers of broadleaf plants after 2 years. The numbers of broadleaf plants are still significantly increasing in all areas, including Control Area 1. As is expected, the greatest percent change occurs in those areas with the smallest amount of vegetation because these areas have open sections where seeds can germinate free of competition from other plant species. However, these areas are relatively dry, windblown sites, and vegetative succession will continue to be relatively slow as compared to areas on the grid that have more poorly drained soils, and therefore, more available soil moisture. An indication of relative rate of succession for different areas of the one square mile grid is illustrated by observing the speed with which class 0, I, II, and III areas change to other classes. For example, all 1971 class 0 areas on the northern section of the grid are now in class II, but 1971 class O areas in the southern portion of the grid only changed to class I in the 2 year interval. From June to September 1971, 74 dicotyledonous species were collected on the one square mile grid, and 33 additional species were found during the June 1973 survey. Table III-3 contains a complete list of all species that have been found on the grid. The relative frequency of occurrance of all new species collected in 1973 was rare or infrequent except for Euphorbia maculata and Polygonella gracillis. Plants that were found in Control Area 2 but not found on the grid were rosinweed (Silphium ovatifolium). hairy bedstraw (Galium pilosum). sun flower (Helianthus sp.), and flax (Linum floridanum). Because the square-foot analysis technique is a more accurate method of determining vegetative cover, the results of the two methods are compared in Table III-4. This shows that visual estimations of 400 by 400 foot areas were 8% to 30% higher than the more accurate square-foot technique. However, class rankings for the two methods are the same except for areas found to have 40% to 60% vegetative cover by the square-foot analysis. The most significant comparison of the two methods is that which shows Control Site 2 and Grid 1 area to be in class III instead of class IV. As a result of the square-foot analysis, the most important plants on the grid in terms of coverage are the grasses, switchgrass (Panicum virgatumHFigure 111-16), and, woolly panicum (£. lanuginosum) (Figure 111-17). These two grasses were found to comprise from 44% to 64% of the existing coverage for all vegetative classes. The most important dicotyledonous plants are rough buttonweed (Diodia teres) (Figure 111-18), poverty weed (Hypericum gentianoides) (Figure 111-19), and common polypremum (Polypremum procumbens). These three dfcots occupy from 3% to 17% of the existing cover in all classes on the grid. 61 �*&%'•••'• ****$*£ * ~Zff' .'•*^i,"*: 4,%'T"^'W;*'i ^ j>«*r-*,'**'L,}^^T~^?fc'^V'&2.> *-. ..*'. 2r •&&*"*•%' '-»--'-„-.-•:.. ~ * ,*-*•.'• * , -'^w" -v r •»Awi«>'A*i:<ii«r^-** i .;.=, ••"j^, -^' v"„-.-..^.. 4 i.v /•-, ^ *»*» ^ ^,.v ? .-»•* v : v : •"4,:-^ / . i-^^'^S-'^ijatt.-, . c: ^>ti-ir ^L^** . • > -. .." * - . " ' • : :.'-f -- -^' ' /'-4^ -"**-*- « r-. - Apfc's-tu:'»" *.*u.i .T ~ .. jal:<^ . ^-,AA^«.. • ;::-.*»• • Figure IM-4. Area N-8, June 1971, Looking SE from Samp,er N-; 8 ; '\ , '*? • i •,:; :^ -^ ^* "* \ -^ <• * - .^ -y^ i-t # Figure II.-5. Area N-8, June 1973, Same View as Figure IM-4 After 2 Years 62 »«^ �Figure IH-6. Area N-5, June 1971, Looking SE from Sampler N-5 Figure 111-7. Area N-5, June 1973, Same View as Figure HI-6 After 2 Years 63 �Figure 111-8. Area L-10, June 1971, Looking SE from Sampler L-10 Figure III-9. Area L-10, June 1973, Same View as III-8 After 2 Years 64 �Figure 111-10. September 1971 View of North-South Flightpath on One Square Mile Grid from Sampler A-9 Figure 111-11. June 1973 View from Same Position as Figure 111-10 After 2 Years 65 �.*,,!«Vi v;;v '•£ <*. ,t>*' Hs^-r Figure 111-12. April 1969 Looking South Near Sampler G-5 Figure 111-13. July 1973, Same View as Figure III-12 After 4 Years 66 �Figure 111-14. Area D-7, June 1971, Looking SE From Sampler D-7 Figure 111-15. Area D-7, July 1973, Same View as Figure 111-14 After 2 Years (Notice increase in number .-of shrubs after 2 years) 67 �Figure 111-16. Switchgrass (Panicum virgatuml Figure 111-17. Woolly panicum (Panicum lanuginosum) 68 �Figure 111-18. Poorjoe or Rough Buttonweed (Diodia teres) Figure 111-19. Poverty Weed (Hypericum gentianoidesl 69 �TABLE II 1-2. VEGETATIVE COVER CHANGES AND DICOTYLEDONOUS PLANT SPECIES CHANGES BETWEEN JUNE 1971 AND JUNE 1973 400 BY 400 FT AREAS SURVEYED CLASS RANKING 1971 1973 NUMBER OF DICOTYLEDONS COLLECTED 1971 1973 PERCENT INCREASE 1971 TO 1973 B-8, M-7, N-8 0 I, II % a 10 100 L-10, N-5, M-2 I I, II, III a 6 a 13 116 II IV a 13 a 23 76 D-9, K-13, J-1 III IV a a27 59 D-7, C-6, E-3 IV V a 19 a 37 94 E-6, F-12, H-11 V V a24 a 37 54 Grid I Area IV IV 17 27 59 Control Area 1 V V 28 39 39 Control Area 2 ND IV ND 44 ND D-12, A-11, C-11 17 a Average of the three 400 by 400 ft areas 70 �TABLE 111-3. SPECIES OF DICOTYLEDONOUS SHRUBS AND HERBS COLLECTED ON TA C-52A ONE SQUARE MILE GRID IN SUMMER 1971 AND 1973 SPECIES COMMON NAME VEGETATIVE CLASS AND FREQUENCY OF OCCURENCE SHRUBS *Baptisia ellipitica *Baptisia hirsuta Callicarpa americana Diospyros virqiniana Ilex qlabra Ilex opaca Lespedeza sp. Pinus clausa Pinus palustris Quercus laevis Quercus sp. Quercus sp. Quercus sp. Quercus sp. wild indigo wild indigo American beautyberry common persimmon gal I berry American holly sand pine longleaf pine turkey oak V; rare V; rare IV; rare IV; infrequent V; infrequent IV; infrequent III; rare IV; rare IV; rare III, IV, V; frequent V; infrequent IV; infrequent III, IV; infrequent IV, V; infrequent HERBS Acanthospermum australe Achillea millefolium Aqaloma discoidalis * AgaJims djyar icata. Ambrosia artemisiifolia *Arenaria caroliniana Asclepia humistrata *Asclepias tuberosa *Asvrum hvpericoides *Baccharis halimifolia *Balduina anqustifolia Biqelowis nudata Cassia fasciculata Centella asiatica *Chenopodium sp. Chrysobalanus oblonqifolius *Chrysoma pauciflosculosa *Chrysopsis aspera Chrvsopsis graminifolia Chrvsoosis mixta *Clitoria fragrans Cnidoscolus stimulosus Crotalaria maritima Crotalaria sagittalis paraquay bur common yarrow foxclove common ragweed Carolina sandwort common milkweed butterfly weed St Andrews cross groundsel baccharis partridgepea senna goosefoot gopher apple golden-aster grassleaf golden-aster golden-aster pigeonwings risky treadsoftly rattlebox arrow Crotalaria *Collected only in June 1973 71 III; frequent III; infrequent II, III; frequent IV, V; infrequent II, III, V; frequent II, IV, V; rare IV; rare IV; rare IV; rare IV, V; rare V; infrequent 0,l,ll,lll; frequent V; rare V; rare III, V; frequent V; rare II; infrequent II; infrequent V; rare III, IV; infrequent III, IV, V; infrequent V; rare �TABLE 111-3. CONTINUED SPECIES COMMON NAME VEGETATIVE CLASS AND FREQUENCY OF OCCURENCE HERBS Continued *Croton arqvranthemus Croton qlandulosus *Cuphea so. Diodia teres Erechtites hieracifolia *Eriqeron annuus Erioqonum tomentosum Eupatorium capilifolium * Euphorbia maculata Euphorbia supina Raf. Froelichia floridana Galactia microphylla Gnaphalium falcatum Gnaphalium obtusifolium Gnaphalium purpurem Hedyotis procumbens Hedvotis uniflora *Heterotheca subaxillaris Hypericum qentianoides *Hypericum myrtifolium *Kalmia harsuta *Kraqia virqinica Lechea patula *l_echea villosa *Liatris secunda *Liatrisqracilis Lithospermum caroliniense Lobelia brevifolia Lugwigia virgata Lupinus diffusus Lupinus nuttallii Molluqo verticillata Oxalis stricta Paronychia patula Petalostemon caroliniense Phlox flotidana *Physalis heterophylla Pluchea rosea Polyqala nana Polyqala polyqama Polygala sp. *Pnl\/nnnellq gracilis tropic croton cuphea rough buttonweed fireweed annual fleabane wild buckwheat dogfennel spotted spurge prostrate spurge Florida snakecotton milkpea cudweed fragrant cudweed purple cudweed camphor telegraph plant poverty weed St Johns-wort sandhill kalmia pinweed hairy pinweed pinkscale gayfeather slender gayfeather Carolina gromwell lobelia false loosestrife sandhills lupine carpetweed yellow woodsorrel nail wort prarie-clover Florida phlox clammy groundcherry bachelor button bitter polygala polygala jointweed *Collected only in June 1973 72 V; rare III; rare IV; rare all classes; common V; infrequent IV,V; frequent II, IV; infrequent II, III, IV; frequent 1, 1 1, 1 V,V; frequent I; infrequent I; infrequent 1 1, 1 1 1; infrequent IV; infrequent IV,V; frequent 1 1 1, IV; infrequent V; rare V; rare IV; rare II, III, IV; frequent V; infrequent V; rare IV; rare 1 1 1,IV; frequent IV; rare V; infrequent V; infrequent l,lll; infrequent V; frequent V; rare 0,l,lll; infrequent I; rare I; rare III; rare 1, 1 1, 1 1 1; frequent I; infrequent II; infrequent V; rare V; rare IV,V; infrequent II, III, IV,V; frequent III; infrequent 1. 1 I.I V; frequent �TABLE 111-3. CONCLUDED SPECIES COMMON NAME VEGETATIVE CLASS AND FREQUENCY OF OCCURENCE common polypremum IV; infrequent V; rare IV; frequent V; frequent V; rare V; rare V; rare 1, 1 1, IV; frequent V; rare III; infrequent IV,V; rare 1 1, 1 1 1, IV; frequent V; rare IV,V; infrequent 1 1, 1 V,V; frequent I V,V; frequent 0,l; rare III; rare III; rare all classes; common IV; rare V; rare V; rare V; rare IV; infrequent 1 1 1, IV; infrequent 1 1 1, IV; infrequent HERBS Concluded Polypremum procumbens *Pterocaulon undalatum Rhexia alifanus *Rhexia lutea * Rhexia mariana * Rhexia salicifolius * Rhexia virginiana Rhvnchosia aalactioides Rhynchosia reniformis Rubussp. *Rudbechia hirta Rumex acetosella *Sabatiaangularis Schrankia microphylla *Smilax sp. Sophronanthe hispida Stylisma villosa Stylosanthes biflora Tephrosia so. Tithymalus soaerospermus Tragia linearifolia Tragia smallii *Traqia sp. * Verbena carnea Vernonia angustifolia Wahlenberaia merginata Wareasessilifolia meadowbeauty yellow meadowbeauty Maryland meadowbeauty common meadowbeauty pinebarrenpea dollarleaf rhynchosia blackberry blackeyed coneflower red sorrel rosegentian littleleaf sensitive brier greenbrier twin pencilflower common euphorbia noseburn noseburn noseburn verbena ironweed rockbell *Collected only in June 1973 73 �TABLE 111-4. DATA COMPARISON OF PERCENT VEGETATIVE COVER BY VISUAL OBSERVATION OF OVERALL PLOT VERSUS THE SQUARE-FOOT TRANSECT METHOD, 1973 DATA VEGETATIVE CLASS/SITE VEGETATIVE CLASS CRITERIA VISUAL ESTIMATE9 SQUARE-FOOT TRANSECTb Class I 5 to 20% 19 11 Class II 20 to 40% 29 19 Class III 40 to 60% 60 41 Class IV 60 to 80% 76 67 Class V 80 to 100% 89 80 Control Site 2C 75 45 Grid 1 d 75 47 a Average of 12 estimates b c Average of 27 transects Located 0.1 mile west of Sampler E-1 d Center section of Grid 1 located 1000 feet south of Sampler 0-7 74 �e. Conclusions A comparison of vegetative coverage and occurrence of plant species on the one square mile grid between June 1971 and June 1973 has shown that areas with 0 to 60% vegetative cover in 1971 have a coverage of 15% to 85% in 1973. Those areas having 0 to 5% coverage in 1971 (areas adjacent to or under flightpaths used during herbicide equipment testing) now have 15% to 54% coverage. The rate of change in the coverage seems to depend on soil type, soil moisture, and wind; there is no evidence to indicate that existing vegetative coverage is in any way related to herbicide residue in the soil. Dicotyledonous or broadleaf plants that are normally susceptible to damage from herbicide residues presently occur throughout the entire one square mile grid. 75 �SECTION IV STUDIES OF THE ANIMALS OF TEST AREA C-52A In May 1970, a survey was initiated to determine the animal species composition of the sprayequipment testing grid on TA C-52A and within the adjacent 11 square mile area. The purpose of this survey was to determine the extent of faunal ecological alteration that occurred in the test area due to repetitive applications of military herbicides. It was expected that application of military herbicides would temporarily alter the faunal ecology of an area, primarily due to the changes in the vegetation. It had been postulated that the animals living in a sprayed area would either be killed outright by herbicides or would receive doses via water or food that would affect their reproductive processes. Laboratory studies dealing with the teratogenic and embryotoxic effects of TCDD, a contaminant found in 2,4,5-T, have been reported (Reference IV-1). It had also been suggested that animals would totally avoid a sprayed area either due to the lack of food, the offensive appearance or taste of the vegetation, or odors produced by the herbicides or their degradation products. The objectives of this animal survey were to determine species variation, distribution patterns, migration, and relative population sizes as found on the test grid or immediately adjacent to it. Methods of study included early morning, midday, and night field trips for identification and collection of mammals, birds, reptiles, and amphibians. Many species collected were brought into the laboratory where they were photographed and either preserved or mounted, and these now serve as a reference collection to facilitate identification for subsequent studies. The results of the 1970 animal survey were reported in Reference IV-2. A synopsis of the 1972 report and comments concerning its correlation to the 1973 studies are included in this report. 1. SYNOPSIS OF QUALITATIVE ANIMAL SURVEYS, 1970 - 1973 A total of 18 mammal species were observed off the test grid with 12 of these species also being found on the grid. All of the animals sighted on the grid used the area for foraging or as a source of drinking water except the beach mouse and the hispid cotton rat, which were using the area as their habitat. The hispid cotton rat was first seen on the grid during the 1973 study. Table IV-1 lists the mammals observed both on and off the grid. The most important economic population in the area was the deer herd. Night field trips yielded average counts of from 24 to 36 deer on the grid and within the immediate area. Close inspection of aquatic areas on the grid during early morning field trips revealed extensive activity the previous nights. In addition to the deer herd a sizable herd of feral hogs earlier crossed with Russian Boars, also inhabited the area. The hogs frequented the marshy areas, drinking and rooting for food. During the spring of 1970, a red fox was frequently observed close to the grid and its den was found approximately 100 yards from the edge of the grid. Five kits were found in the den and based upon gross observations, they appeared healthy and normal. The most common rodents found off the grid in 1970 along the streams that drain the area were the cotton mouse and the hispid cotton rat. In the fields surrounding the grid, the eastern References: IV-1. Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency, 7 May 1971 IV-2. Pate, B. D., R. C. Voigt, P. J. Lehn, and J. H. Hunter: Animal Survey Studies of Test Area C-52A, Eglin AFB Reservation, Florida. AFATL-TR-72-72, Air Force Armament Laboratory, Eglin AFB, Florida, April 1972. Unclassified. 76 �TABLE IV-1. MAMMALS FOUND ON THE 1 SQUARE MILE GRID AND .WITHIN THE ADJACENT 11 SQUARE MILE AREA SPECIES AREA WHERE OBSERVED COMMON NAME ON GRID Canis familiaris wild dog Dasypus novemcinctus armadillo Didelphis marsupialis opossum Geomvs pinetis southeastern pocket gopher Lynx rufus bobcat Mephitis rnephitis striped skunk Odocoileus virginianus whitetail deer Oryzomys paulustris rice rat Peromyscus gossypinus cotton mouse Peromvscus polipnotus beach mouse Reithrodontomys humulis eastern harvest mouse Procyon jotqr racoon Sciurus carolinensis eastern gray squirrel Sciurus niger eastern fox squirrel Sigmodon hispidus Sus scrofa hispid cotton rat Sylvilagus floridanus eastern cottontail rabbit Vulpes fulva red fox OFF GRID b,c b,c b,c b,c wild pig a Species found on or off grid for first time, 1973 data b Dominant species; sighted during 80% of the field trips, 1973 data c Dominant species; sighted during 80% of the field trips, 1970 data 77 �harvest mouse was common. Eight pairs of the eastern harvest mouse were taken into the laboratory and allowed to breed. Six of the eight pairs had litters totalling 24 offspring which were normal in size and free from any apparent birth defects. During the 1973 study, only two cotton mice and two eastern harvest mice were found. The cotton mice were caught near a stream draining the grid, and the eastern harvest mice were captured on the grid. There were eight beach mice captured off the grid in areas along streams and in open fields. The most common rodent species on the grid was the beach mouse. Trapping studies during the summer of 1970 showed that this species was widely distributed throughout the grid, except in areas with less than 5% vegetative cover. In the 1973 study, the beach mouse was found predominantly in the areas of 5% to 60% vegetative cover. At least 25 species of birds were observed in the area immediately adjacent to the grid or feeding within its boundaries. Many more species than those listed in Table IV-2 are found in the more densely forested areas near the outer limits of the 2 mile radius. In 1970, seven species of water birds and waders were sighted repeatedly in the aquatic areas on or off the grid. The most common birds on the grid were the meadow lark and the mourning dove. It seems significant that all birds sighted, with the single exception of a grasshopper sparrow (caught in a live animal trap) were medium to large species. In I973, the first sightings of red-wing blackbirds and little blue heron occurred on the grid. In 1970, the meadow lark was the predominant species of bird found, while in 1973, frequent and repeated sightings of night hawks, bobwhite quail, Mississippi kites, mourning doves and meadow larks were reported. Eighteen species of reptiles were collected or observed, with 10 species recorded on the grid and 12 species from the surrounding area (Table IV-3). Differences in faunal species composition on and off the grid due to vegetation differences can best be illustrated with the reptiles. Those species that are adaptable and occupy a variety of niches were found both on and off the grid in large numbers. The dominant species on the grid was the six-lined racerunner, and it was also one of the dominant species in the wooded area surrounding the grid. Those species whose habitat is characterized by definite vegetative type cannot adapt to the open habitat of the grid. The green anole and southern fence lizard are two of these. There are also species which occur in the forest areas but are more plentiful in the open areas, such as the eastern coachwhip. In 1973, the first softshelled turtle was seen on the grid. Twelve species of amphibians were collected (Table IV-4). The amphibian population on the grid centered mainly around the aquatic areas with the exception of the two toad species, which were also found in the dry areas. There were breeding populations throughout most of the year in the aquatic areas on the grid: the southern cricket frog, the southern toad, the oak toad, the barking tree frog, the southern leopard frog, and the squirrel tree frog. The slimy salamander is one of the dominant species in the surrounding forest but does not occur on the grid, presumably because of its need for sufficient moist ground cover. The squirrel tree frog and the hog-nosed waterdog were first reported on the grid in the 1973 study. 2. CURRENT STUDIES ON ANIMALS In the 1970 animal survey, 73 species of vertebrates were observed on and off the test grid. The most frequently observed species on the grid was the beach mouse Peromyscus polionotus 78 �TABLE IV-2. BIRDS FOUND ON THE 1 SQUARE MILE GRID AND WITHIN THE ADJACENT 11 SQUARE MILE GRID SPECIES COMMON NAME AREA WHERE OBSERVED ON GRID Accipiter striatus velox Aqelaius phoeniceus OFF GRID sharp-shinned hawk red-wing blackbird a + + Ammodramus savannarum grasshopper sparrow Bubulcis ibis cattle egret Botaurus lentiginosus American bittern Buteo jamaicensis red -tailed hawk Buteo liniatus red-shouldered hawk eastern green heron Butorides virescens virescens Caprjmulgus vociferus eastern whippoorwill Casmerodius acbus egretta American egret Cathartes aura turkey vulture Chordeiles minor night hawk b+ b+ Co!jn us. virginlanus bobwhite quail b+ b+ Coragyps atratus black vulture Con/us brachyrhynchos American crow Florida caerulea little blue heron Elanoides forficatus forficatus swallowtail kite Falco sparverius sparrow hawk Ictinia misisipoiensis Mississippi kite Sturnella maqna meadow lark b,c + b+ Turdus migratorius robin Zenaidura macroura Unidentified Duck mourning dove b+ b+ Unidentified Goose Unidentified Grebe a Species found on grid for the first time, 1973 data boominant species; sighted during 80% of the field trips, 1973 data c Dominant species; sighted during 80% of the field trips, 1970 data 79 �TABLE IV-3. REPTILES FOUND ON THE 1 SQUARE MILE GRID AND WITHIN THE ADJACENT 11 SQUARE MILE AREA SPECIES COMMON NAME AREA WHERE OBSERVED ON GRID Agkistrodon piscivorus eastern cottonmouth Alligator mississippiensis American alligator Anolis carolinensis carolinensis green anole Cnemidophorus sexlineatus six-lined racerunner Coluber constrictor priapus southern black racer Crotalus adamanteus eastern diamondback rattlesnake Elphae guttata guttata corn snake Ferox sp. soft-shelled turtle Heterodon platyrhinos eastern hognose Lampropeltis doliata doliata scarlet kingsnake Lygosoma laterale ground skink Masticophis flagellum flagellum eastern coachwhip Matrix sipedon pictiventris Florida water snake Pituophis melanoleucus mugitus Florida pine snake Pseudemys scripta scripta yellow-bellied turtle Sceloporus undulatus undulatus southern fence lizard Sistrurus miliarius barbouri dusky pigmy rattlesnake Sternotherus minor minor loggerhead musk turtle OFF GRID a + b,c a Species found on grid for the first time, 1973 data ^Dominant species; sighted during 80% of the field trips, 1970 data c Dominant species; sighted during 80% of the field trips, 1973 data 80 b,c �TABLE IV-4. AMPHIBIANS FOUND ON THE 1 SQUARE MILE GRID AND WITHIN THE ADJACENT 11 SQUARE MILE AREA SPECIES COMMON NAME AREA WHERE OBSERVED ON GRID Acris sryllus gryllus southern cricket frog Bufo quercicus oak toad Bufo terrestris southern toad Eurycea bislineata cirrigera southern two-lined salamander Gastrophryne caroljnensis eastern narrow-mouthed toad Hermidactylium sccutatum four-toed salamander Hyla qratiosa barking tree frog Hyla squirella squirrel tree frog Necturus beyeri hog-nosed waterdog Plethodon glutinosus glutinosus slimy salamander Rana clamitans clamitans bronze frog Rana pipiens/ sphenocephala southern leopard frog b+ a,b+ a Species found on grid for first time, 1973 data bA breeding population 81 OFF GRID b+ �and the six-lined racerunner Cnemidophorus sexlineatus. These two species were suggested as candidates for future studies of population distribution. During the months of February, March, and May 1971 a trapping study was performed on the test area for three, 4-day periods in each month (Reference IV-3). A total of 38 beach mice were captured during the three trapping periods. Thirty beach mice were captured during the February-March periods, and of these, six from the test grid and three from a control area were examined for gross deformaties. Sections of liver, kidney, and gonads were free of abnormalities, and no cleft palates were observed. The primary purpose of the present study was two fold. First (Test Program I), animals were to be obtained for examination of gross and microscopic lesions, since it has been reported (Reference IV-1) that TCDD produces teratogenic and embryotoxic effects under certain experimental conditions. Second (Test Program II), the trapping survey discussed in the previous paragraph was to be expanded in an attempt to correlate habitat preference for the most prevalent mammal observed on the grid in order to determine if the population distribution is related to vegetative cover. 3. MATERIALS AND METHODS Traps used for this study were Havahart traps (Havahart Traps, Department 1, P.O. Box 551, Ossining, N.Y. 10562) numbers 0 and 1 for small mammals. Traps were baited with peanut butter and oatmeal. In June 1973, Test Program I was initiated by placement of traps on the square mile grid in two patterns. At first, one trap was placed in every other plot (400 by 400 foot areas) in every other row. For example, Row A had one trap each in plots 1, 3, 5, 7, 9, 11, and 13. Row C had one trap each in plots 2, 4, 6, 8, 10, and 12. Four to eight traps were placed in these areas. A total of 90 traps were emplaced. Traps were checked daily and the trapping duration was one week. A second portion of Test Program I utilized four sampling plots with distinct physical characteristics and involved the placement within each plot of 25 traps in five rows of five traps each, 20 paces apart, in each plot. Historically, these areas were exposed to low or high concentrations of herbicide . Traps were checked daily, and the trapping was carried out for 7 days. In October 1973, a third portion of Test Program I was conducted on Grid 1 exclusively (Figure I-5). Grid 1 was divided into equal quadrants North-South and East-West, and was numbered upper left (Area 1), lower left (Area 2), lower right (Area 3), and upper right (Area 4). Twenty-five traps were placed in each area in two rows of 10 traps per row and one row of five traps with 15 paces between traps. The rows were located at 250, 500, and 750 feet from the center of the grid on the ordinate. Traps were checked daily for 7 days. Mice, rats, and reptiles were taken to the laboratory for gross examination and prepared for histologic examination. The majority of the animals were alive on arrival but some had succumbed to the intense heat and confinement in the trap. Live animals were subjected to a euthanasic procedure using ether. All animals were photographed, weighed, measured, and examined for develqpmental defects such as cleft palate, cleft lip, polydactyly, and micro-ophthalmia. All internal organs were examined for gross lesions, and individual organ weights were recorded. Representative sections of each tissue were placed in ^Personal .communication with Donald King, Department of Zoology, University of Minnesota, Minneapolis, Minnesota. 82 �neutral 10% buffered formalin and processed for microscopic study by the Veterinary Pathology, Washington, D.C. 20305. All remaining control and grid rat liver tissue and mouse fat and liver tissues were collected, placed in clean glass jars, frozen, and sent to the Interpretive Analytical Services Laboratory, Dow Chemical U.S.A., for TCDD analysis. The method of analysis was as described in Section II with the following exceptions: Ten grams of tissue were added to 10 ml ethanol and 20 ml 40% aqueous KOH and refluxed 2 hours. The resulting mixture was extracted with four 10 ml portions of hexane. The hexane extracts were combined, subjected to h^SC^ extraction and the same subsequent steps as in analysis of the soil samples. Recovery studies using blank fish, beef liver, and soil averaged 70+% at the 10 to 25 part per trillion level. In Test Program II, eight mouse traps were placed by computer randomization in areas which were classified according to vegetative coverage. Five vegetative areas were classified as follows: 5% to 20% coverage; 20% to 40% coverage; 40% to 60% coverage; 60% to 80% coverage; 80% to 100% coverage (see Section III, Figure III-3). Three of the 400 by 400 foot plots for each of Classes 1 to 5 were chosen at random. Eight pairs of coordinates were generated using the library random number generator for the Wang 720C programmable calculator. Each trap was placed in accordance with these coordinates. A total of 120 traps were utilized. Animals thus trapped were ear tagged with size 1, sequentially numbered, fingerling tags (National Band and Tag Company, Newport, Kentucky, 41071) and species, sex, and trap location was recorded. They were examined for external abnormalities and released. Traps were checked daily during the 8 days this study was conducted, and records were kept of original capture and the recapture of individual animals. 4. RESULTS AND DISCUSSION During Test Program I, several different species of animals were caught, both on and off the test grid (Table IV-5). The sex distribution of the trapped animals was 23 male and 14 female beach mice, eight male and eight female cotton rats, two female eastern harvest mice, one male and one female cotton mice, ten male and seven female six-lined racerunners, one male eastern cotton mouth, and one male toad. TABLE IV-5. TOTAL NUMBER AND LOCATION OF ANIMALS COLLECTED DURING 1973 TEST PROGRAM I CLASS COMMON NAME OFF GRID ON GRID Mammalia beach mouse cotton rat eastern harvest mouse cotton mouse 8 10 0 2 42 6 2 0 six-lined racerunners eastern cottonmouth 4 1 13 0 toad 1 0 Reptilia Amphibia 83 �The age of the rodents was determined by histological examination of the gonads based on the presence or absence of sperm or ova (gametes) in the gonads. Animals with gonads showing gametogenesis were classified as adults and those with gonads showing no gametogenesis were classified as immature. The age of the animals varied, but adults predominated in the sample, 55 adults, 33 immature. Nine pregnant mice and five pregnant rats were found in the adult female animals. The stage of gestation varied considerably from early pregnancy to near term. Fifty-four embryos and fetuses were examined grossly and microscopically. No developmental defects or other lesions were seen. Gross necropsy lesions were relatively infrequent in the test population and consisted primarily of lung congestion in those animals that died prior to being brought to the laboratory. No developmental defects were seen in any of the adult animals. Histologically, the tissues of 13 of the 26 control animals and 40 of the 63 animals from the test grid were considered normal. Microscopic lesions were noted in some animals from both groups. For the most part, these were minor changes of a type that would be expected in any animal population. One of the most common findings was parasites. A total of 11 controls and 9 grid animals were affected with one or more classes of parasites. These are summarized in Table IV-6. Parasites may be observed in any species, and those in this population were for the most part incidental findings that were apparently not harmful to the animal. There were exceptions however. Protozoan organisms had produced focal myositis in one rat and were also responsible for hypertrophy of the bile duct epithelium in a six-lined racerunner. Moderate to severe pulmonary congestion and edema were seen in several rats and mice. All of these animals were found dead in the traps before reaching the laboratory, and the lung lesions were probably the result of heat stroke. The remainder of the lesions in both groups consisted principally of inflammatory cell infiltrates of various organs and tissues. They were usually mild in extent, and although the etiology was not readily apparent, the cause was not interpreted as toxic. It was highly improbable that any of the mice trapped during this study were alive during the final phase of herbicide dissemination (September 1970), although the life span of the beach mouse has been reported to be 5 years in captivity (Reference IV-3). A portion of the grid population was certainly made up of offspring of these animals present in 1970. Emigration from, or immigration to, the test grid could occur, especially on the fringe areas, since it has been reported that the area traveled by an individual beach mouse during its daily activities may extend to 5 acres (Reference IV-4). An analysis of the ratios of organ weight to body weight, and organ weight to body length for mice captured off the grid versus mice captured on the grid was conducted within the severe constraints of limited data (June 1973 data). Female mice were not considered due to the fact all References: IV-3. Benton, A.H. and W. E. Werner, Jr. Field Biology and Ecology. McGraw Hill, New York, 1966. IV-4. Andrewartha, H.G. Introduction to the Study of Animal Populations. University of Chicago Press, 1961. 84 �control females were pregnant and showed large individual body weight and organ weight variations. Only two of 11 female mice from the grid were pregnant. There were five control males (three mature and two immature) and 18 males (ten mature and eight immature) captured on the grid. Complete organ data were available only for 13 of the 18 grid males (nine mature and 4 immature). It is recognized that the mature and immature mice will likely show different characteristics; however, combination of these two groups was necessary to produce any reasonable sample size. The t test for unpaired samples was used on 16 different factors (Table IV-7). These factors are as designated below: FACTOR DESCRIPTION A = Total organ weight/body length B = Total organ weight/body length C = Sum of lung, heart, kidney, and brain/body weight D E = = Sum of lung, heart, kidney, and brain/body weight Lung weight/body weight F = Heart weight/body weight G = Spleen weight/body weight H = Liver weight/body weight I = Kidney weight/body weight J = Brain weight/body weight K = Lung weight/body length L = Heart weight/body length M = Spleen weight/body length N = Liver weight/body length 0 = Kidney weight/body length P = Brain weight/body length Formula for the procedure used: sx 2 ~N~2 r r I 9 iA sx.,^ 1 sx., ITf (2X1)2+ Ml w.2 ~X2 -2 Where IS!-) + N 2 ¥ 2 85 <SX2>"1 N2 rN 1 + N 2 i . N 1N2 J �TABLE IV-6. PARASITES FOUND IN RODENTS COLLECTED FROM CONTROL AND TEST AREA SITES, JUNE 1973 LOCATION Control Test Area a NUMBER OF ANIMALS EXAMINED NUMBER OF PARASITES ANIMALS EFFECTED NEMATODES CESTODES PROTOZOAN? 20 3 11 9 1 5 50 g 4 0 7 Animals trapped on Grids 2, 3, and 4 TABLE IV-7. SUMMARY OF RESULTS OF ORGAN WEIGHT TO BODY WEIGHT, AND ORGAN WEIGHT TO BODY LENGTH FOR MICE OFF THE GRID VERSUS MICE ON THE GRID (HEOn-Grid and Off-Grid Samples are from the Same Population) v = 16 FACTOR t P (exceeding t. Given H) A 0.1863 0.43 B 0.8859 0.19 C 0.9750 0.17 D 0.1025 0.45 a b E 1.6618 F 0.2750 0.38 G 1.1025 0.14 H 0.7077 0.06 (0.05) 0.25 a b I 2.2228 J 0.2363 0.41 K 0.6500 0.27 L 0.5659 0.29 M 0.4979 1.0214 0.32 1.1034 0.14 1.1647 0.13 N 0 P 0.02 (0.10) 0.16 Significant at p < 0.05 "Value when control animal with lung and kidney lesions is removed from sample 86 �When the ratios of average body weight to average organ weight of various visceral organs were compared between the male mice captured on the grid and the male mice captured off the grid it was found that on the average, the control animals had lung and kidney weights that varied significantly at the 95% confidence level. The lung variation just being significant. When this information was compared to the pathological work-up, it was found that one male control animal had multifocal subacute pneumonitis and multifocal subacute nephritis. When this animal was removed from the sample, the ratio of kidney weight/body weight between the control and grid animals no longer varied significantly. The lung weight/body weight variation became slightly more significant. It is felt this variance is due to the difference in ratio of mature to immature animals between the two groups, i.e., controls 3:2 compared to 9:4 for the grid animals. The analyses for TCDD from rodents collected in June and October 1973 are shown in Table IV-8. An initial interpretation would be that TCDD does in fact accumulate in liver and fat of tissue from rodents living on the test grid. Data from soil analysis (Table 11-10, Section II) confirm the presence of TCDD in soils of the test area. Discrepancy of levels of TCDD between soils and tissues suggest the potential for bio-magnification of this compound. These data do not correlate with previously published research (Reference IV-5). Such levels encounted in the animals reported herein would be suspect of teratogenic or pathologic abnormalities. Such abnormalities, however, were not encountered in this study. It would appear that analytically, via mass spectrometry, the chemical detected is of a very similar nature to TCDD, but biologically does not behave in the manner characterized for TCDD (Reference IV-5). TABLE IV-8. CONCENTRATION (PARTS PER TRILLION) OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) IN LIVER AND FAT SAMPLES FROM RODENTS COLLECTED FROM CONTROL AND TEST SITES ON TA C-52A, JUNE OR OCTOBER 1973a RODENT TISSUES CONTROL LOCATION GRIDS 2, 3,4 Rats Liver, Fat" <20 210 Mice Liver, Fatb <20 300 GRID 1 No Sample0 540d a Analysis for TCDD was performed by the Interpretive Analytical Services, Dow Chemical U.S.A., Midland, Michigan "Tissues represent a composite from all animals collected at the respective location c Rats do not frequent dry areas. d Sample collected in October 1973. Reference: IV-5. Conference on Dibenzodioxins and Dibenzofurans, Environmental Health Perspectives, Experimental Issue Number Five, September 1973, National Institute of Environmental Health Science, Research Triangle Park, North Carolina. 87 �The beach mouse was reported in Reference IV-2 as the most common rodent species on the grid in 1970. Observations in the field indicate that the beach mouse remains the most common rodent on the grid. The 1970 study (Reference IV-2) also indicated that the beach mouse was widely distributed throughout the grid except in areas of less than 5% cover. In an attempt to correlate distribution of the beach mouse with vegetative cover, a second test program (Test Program II) was initiated with a total of 83 animals being trapped during an 8 day period, 28 June to 3 July 1973. The majority of animals (63) were found in areas with 5% to 60% vegetative cover; within this range, the greatest number of animals trapped (28) was from an area with 40% to 60% cover (Figure IV-1). A similar habitat preference has been observed along the beaches of the Gulf Coast (Reference IV-5). In this study, it appears that the beach mouse utilizes the seeds of switchgrass, (Panicim virgatum) and woolly panicum (Panicum lanuginosum) for a food source, and these are two of the most dominant plants on the grid (see photographs in Section III). Seed husks of these plants have been observed in areas of mouse activity. It is possible that another prominant plant, broomsedge bluestem (Andropogon virginicus). also provides food for the beach mouse. In an attempt to compare the trapping data from 1971 with those data obtained in the 1973 study and, hence, to determine whether an increase in the population of beach mice has occurred, the following assumptions were made: a. It was assumed that the traps, the bait, and the methods employed for setting and placing the traps were equally as effective and similar in the 1971 and 1973 studies. b. It was assumed that the density of trap placement was equally as effective and similar in the 1971 and 1973 studies. c. It was assumed that traps should be no further apart that approximately 1-1/2 times the mean random travel distance of the animal being trapped. For beach mice, this distance is approximately 300 feet (Reference IV-5). d. On the other extreme, it was assumed that the traps should not be so dense as to impede animal movement nor disrupt animal habits. All trapping experiments involved in this study were conducted within these extremes. In order to produce an estimate of the population density of the beach mouse in the herbicide treated area, it was necessary to determine what portion of that area was effectively surveyed. It was also necessary to normalize the areas sampled for comparison based on the mean random travel of the beach mouse from the 1973 recapture data. A tabulation was made of the distances between the trap where initial capture occurred and the trap location of the recapture farthest from the initial capture point. These distances represent the distances that the mice from the sample were known to have traveled and were assumed to be random samples from the population of habitat radii. The longest radius observed was 3,200 feet, the next longest was 285 feet, and the shortest was 45 feet. The 3,200-foot distance was disregarded as a freak occurence because such a number appeared only once out of a sample 88 �30 S 20 <D I 15 01 5- 20 40 60 100 Vegetative Cover, % Figure IV-1. Relationship of Animals Trapped to Vegetative Cover of 18 and it is more than 11 times as large as the next largest distance which is, itself, only 6.3 times as large as the smallest distance observed. The mean distance, 194 feet, of the remaining 17 distances was used as the average habitat radius. Circles with 194-foot radii were drawn using the traps of 12 randomly selected 400 by 400 foot plots as centers. The envelope area around the traps was estimated using a triangle, rectangle, or circle according to the fit which, by inspection, appeared to be the best. The average of the 12 areas measured was 8.3 acres with a standard deviation of 1.69, a maximum of 10.45, and a minimum of 5.17. The estimate, 8.3 acres, was used as the effective survey area for each group of eight traps. a. Variation of the Lincoln Index The Lincoln Index may be stated mathematically as: Pi = - R) Where PI = first population estimate 89 �N-| = pre-census sample N2 = total census sample R = number of recaptures Using the catch of 26 June and considering only the tagged animals, NI = 13. Using the catch of 27 June, N2 = 18 and R = 1. According to this information, the first estimate is PI = 222. Enough uncertainty is introduced, however, such as how a dead animal should be counted in the pre-census sample, that an alternative method of estimation was desirable. The following equation was used to calculate the second estimate of the population. 8 RJ + 1 'max " ' Where N.|i = the catch from the ith day N2 + 1 = the catch from the (i + 1)th day RJ + 1 = the recaptures from the (i + 1)th day which were captured on the (ith)day also The values for RJ + 1 were corrected for the influence of mice no. 18, recaptured six times; no. 27. recaptured four times; no. 28, recaptured four times; no. 41, recaptured four times; no. 45 recaptured three times; and no. 50, recaptured three times. These mice were considered to be trap addicts. From the data available, P2 = 152. A third method was used for calculating P% in which the first catch was considered to be the pre-census and the second catch the census. A computer program to simulate the trapping of mice was developed with inputs of average sample size, Sa, and true population size, Pt. Twenty-five runs were made at specific values of Sa and Pt, and the estimated population was calculated with the Lincoln Index and averaged over the 25 trials. The average recapture number, Ra was also calculated. Pt was changed until Ra was equal to the observed recapture rate and Sa was equal to the observed sample size. Pt at these values is considered to be the best estimate of the population, i.e., Pt is the number most likely to produce the results which were observed in the real case. P% by this method was 191. This is not grossly different from the 222 and 152 estimated from P-| and Po. The average of P l f P2, and Po is probably the best overall estimate of the population. This average is approximately 189 total population - approximately 1.64 mice per acre. 90 �5. CONCLUSIONS Based on the pathologic findings of the Test Program I study, it was concluded that there was no evidence that the herbicide contaminant in question (TCDD) had produced any developmental defects or other specific lesions in the animals sampled or in the progeny of those that were pregnant. The lesions found were interpreted to be of a naturally occurring type and were not considered related to any specific chemical toxicity. The organ to body weight and organ to body length comparisons for the grid versus the control animals did not vary significantly when age and pathological lesions were considered. Chemical analysis of composite rodent liver and fat tissue indicated that there was an accumulation of TCDD-like chemical in tissue. If these data are valid (an assumption that may be challenged), what is the source of the TCDD? Seeds of switchgrass (Panicum virgatum) were found in abundance in the stomachs of beach mice. Samples of such seed collected from the test area were analyzed for TCDD. Results indicated no residue of TCDD at a minimum detection limit of less than 10 parts per trillion. Based on information provided by the Test Program II study, it was concluded that the beach mouse forms a natural, integral part of the ecosystem of the Lakeland Sand Complex utilizing the dominant plants on the grid for food. The beach mouse continues to inhabit areas of 5% to 60% cover, with a preference for areas of 40% to 60% vegetative cover. This is indeed similar to the habitat preference of the beach mouse in other locations. The statistical evidence derived from the Test Program II study shows that the 1.64 beach mice per acre population (based on the Lincoln Index for 1973) is slightly higher than the 0.8 and 1.4 mice per acre found on Santa Rosa Island (Reference IV-6). It was also concluded that the population of beach mice was higher in 1973 than in 1971 in the area of the test grid. Even though the first trial of the 1971 data reflected a higher count of mice per acre trapped, the low capture count on the second trial in 1971 indicates a lower actual population based on the Lincoln Index assumptions than the 1973 data. The apparent increase in beach mouse population on the grid in 1973 over 1971 was probably due to the natural recovery phenomenon of a previously disturbed area. Some areas of the test grid have already exceeded the preferred percentage of vegetative coverage of the beach mouse habitat, and other areas are either ideal or fast developing into an ideal habitat. If the test grid remains undisturbed and continues toward the climax species, a decline in the number of beach mice will probably occur simply due to his habitat preference. Reference: IV-6. Blair, W. F., Population Structure, Social Behavior, and Environmental Relations in Natural Populations of the Beach Mouse (Peromyscus polionotus leucephalus). Contribution from the Laboratory of Vertebrate Biological, University of Michigan, Number 48:1-47, June 1951. 91 �SECTION V INSECT DENSITY AND DIVERSITY STUDIES ON TEST AREA C-52A During 1970 and 1971, an initial survey of the arthropod populations of Test Area C-52A, Eglin Air Force Base, Florida, was accomplished, and the results were published in Reference V-1. 1. SYNOPSIS OF PREVIOUS RESEARCH, MAY - JUNE 1971 (Reference V-1) A sweep net survey of the insects on a 1 mile linear transect of Test Area C-52A resulted in the collection of more than 1,800 specimens belonging to 74 insect families and two non-insect arthropod orders. Eighteen of the taxa collected accounted for 97 percent of the collection, and of these, six taxa accounted for 72 percent of the collection: order Araneida (spiders), insect families Cicadellidae (leafhoppers), Elaterjdae (click beetles), Asilidae (robber flies), Hygaeidae (lygaeid plant bugs), and Pentatomidae (stink bugs). Spiders and robber flies are carnivores, stink bugs are carnivores or herbivores, and the other families are herbivores. As plants were eliminated by the herbicides, those insects which fed specifically upon those plants disappeared; however, no direct effects of residue on the insects were observed. The objective of the present study was to duplicate the techniques of the 1971 study as closely as possible in order to evaluate populations along the same grid line 2 years later. Qualitative and quantitative comparisons were drawn to indicate the changes in variety and number of arthropods (especially insects) that had become established on the grid since the aerial dispersal tests were terminated in 1970. 2. MATERIALS AND METHODS The techniques used in the study discussed in this report were the same as those outlined in Reference V-1 except as discussed in the following paragraphs. Time and manpower limitations precluded general, non-systematic sampling of the grid, therefore the bulk of this comparative study was based on sweep net surveys along sampler row 8 of the test area. This allowed a quantitative comparison of results while non-systematic net sampling of grid areas did not lend itself to such analysis. A total of five paired sweep net surveys were performed on the mornings of June 14, 16, and 18; these dates being approximately 2 years and 2 weeks after the study discussed in Reference V-1. A given "paired sweep" (200 sweeps made by 2 individuals using 15-inch diameter nets) was taken across the grid and then back to the starting point. The simultaneous sweeps were 20 feet apart. At the end of each 400 foot transect, rather than using killing jars, the net contents were emptied into a paper bag into which a vial of ethyl acetate had been placed. These bags were then tightly folded and placed in a large sack that was carried out to the same taxonomic level as in the 1971 study. Exceptions to this classification scheme included the listing of Acalypterate muscoid flies as a group and the listing of certain Reference: V-1. Valder, S. M.: Insect Density and Diversity Studies on Test Area C-52A, Eglin AFB Reservation Florida. AFATL-TN-72-4, Air Force Armament Laboratory, Eglin AFB, Florida. January 1972. Unclassified. 92 �undetermined insects only to order. Table V-1 represents a full listing of the arthropods found in the sweep net survey. The undetermined insects (112 specimens) for the most part were either immatures or only partial specimens. The identifications were based on information in Reference V-2. Finally, due to time limitations, only one full set of sweep net survey samples had been identified at the time of this report. Therefore, while the 1971 report discussed the results of five 2-mile sweeps of sampler row 8 (essentially 10 replicates), the present study considers data only from one 2-mile sweep of the same sampler row (essentially two replicates of each 400 foot transect of the row). Representative specimens of the identified samples are in the reference collection of the Biological Studies Branch, USAF Environmental Health Laboratory, Kelly AFB, Texas. 3. RESULTS AND DISCUSSION Those taxa or arthropods that were collected in numbers exceeding one percent of the total number of specimens collected are listed in Table V-2. This table was formatted for comparison with Table V-3 (from Reference V-1). (The various taxa are treated as families for simplicity.) Such a comparison indicated that even though the 1973 data are based on only one sweep of the grid, the total number of identified arthropods equaled 1,614 as compared to a total of 1,803 specimens from five sweeps in 1971. These data would then indicate that if the one 1973 sweep were representative of all five sweeps taken during this second study, the total number of insects caught would likely be greater than four times the number taken during the 1971 study. Further analysis of the data indicates that the 1973 survey found great numbers of very small insects as compared to the 1971 study. The majority of the Chrvsomelid beetles were very small insects, and the Qcalvptrate muscoids, Psocoptera, Thysamoptera, Sminthuridae, Ocarine, and Chalcidoidea also fall into this small to minute category. Table V-1 represents a listing of all arthropods collected during the 1973 survey, and is compared to Table V-4 (from Reference V-1). Reference V-1, however, lists not only those arthropods collected in 1971, but also those groups only observed in 1970 and 1971. Therefore, the tables are not fully comparable either in taxa listed or in the number of specimens reported. Comparison of this second set of tables again shows a relatively large number of small insects found in the 1973 study. This discrepancy may simply represent a difference in sampling/separation techniques or it may indicate an influx of populations of these smaller arthropods as the vegetation and other environmental characteristics of the transects have developed since the spray program was terminated. Figures V-1 and V-2 represent arthropod/vegetation comparisons on Test Area C-52A for both the 1971 and 1973 surveys. There exists a similarly vegetative distribution, and a slightly greater plant coverage is indicated in 1973. The extreme differences in the numbers of arthropods found on the transects during the 1971 study are shown as being reduced in 1973, and further replication as well as time would likely reduce these differences more. Comparisons of the arthropod populations have to take into consideration the fact that Figure V-1 is based on the total observed and collected specimens from Table V-4, while Figure V-2 is derived from only the identified specimens of a single sweep of the 1973 study. Therefore, although the graphs representing the number of arthropod "families" in both figures are relatively similar, and there is a tendency toward reduction of extreme differences in the 1973 transect data, further discussion and comparison might be spurious. Similar comments pertain to comparisons of the arthropod diversity graphs, even though basic Reference: V-2. Pate, B. D., P. J, Lehn, R. C. Voigt, and J. H. Hunter: Animal Survey Studies on Test Area C-52A, Eglin AFB Reservation, Florida. AFATL-TR-72-72, Air Force Armament Laboratory, Eglin AFB, Florida. April 1972. Unclassified. 93 �TABLE V-1. ANTHROPODS COLLECTED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1973 TAXON COMMON NAME CLASS: ARACHNIDA ORDER: Araneida (Spiders) ORDER: Acarine (Mites) TOTAL SPECIMENS 144 46 NUMBER OF SPECIMENS COLLECTED ON TRANSECT AB BC CD DE EF FG GH HJ JK KL LM MN NO 4 9 9 2 16 2 18 3 36 27 18 7 1 1 7 6 6 2 8 3 2 1 1 4 1 3 CLASS: INSECTA ORDER: Coleoptera (Beetles) 275 Specimens collected Anthicidae Antlike Flower Beetles 3 1 Carabidae Ground Beetles Chrysomelidae Leaf Beetles 219 Cicindellidae Tiger Beetles 7 Coccinellidae Lady Beetles 1 Curculionidae Snout Beetles 5 1 Elateridae Click Beetles Blister Beetles Mycetaeid Fungus Beetles Shining Flower Beetles 12 1 1 Meloidae Mycetaeidae Phalacridae Tenebrionidae Darkling Beetles 7 Undetermined larvae and adults 6 1 1 1 1 84 35 2 1 1 5 69 18 1 1 1 1 1 2 2 1 1 1 2 53 1 1 1 1 1 12 1 11 2 1 1 2 1 1 2 3 14 12 1 ORDER: Collembola (Springtails) 53 Specimens collected Sminthuridae 2 7 3 7 9 1 �TABLE V-1. ARTHROPODS COLLECTED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1973 (Continued) NUMBER OF SPECIMENS COLLECTED ON TRANSECT TOTAL SPECIMENS AB BC CD DE EF FG GH HJ JK KL LM MN NO ORDER: Diptera (Flies) 165 Specimens collected TAXON COMMON NAME Acalyptrate Asilidae Muscoids Robber Flies Bombiliidae Bee Flies 1 Cecidomyiidae Gall Midges 3 1 1 14 2 Ceratopogonidae Biting Midges 2 2 6 6 21 14 7 3 7 1 8 3 3 1 1 1 1 1 2 Muscid Flies 2 1 1 Pipunculidae Bigheaded Flies 3 1 1 Sarcophagidae Flesh Flies 4 Syrphidae Flower Flies 2 Tachinidae Tachina Flies 10 Tipulidae Crane Flies Midges Culicidae en 9 5 1 Chironomidae CO 86 Mosquitoes Muscidae Undetermined Adults 1 2 1 5 4 1 1 1 1 1 Burrowing Mayflies 33 2 2 4 4 6 1 1 1 5 1 ORDER: Hemiptera (True Bugs) 183 Specimens collected Corimelaenidae Corizidae Corimelaenid Bugs Grass Bugs 6 3 1 1 ORDER: Ephemeroptera (Mayflies) 1 Specimen Collected Epnemeridae 1 1 1 2 1 1 1 1 2 2 4 4 2 �TABLE V-1. TAXON ARTHROPODS COLLECTED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1973 (Continued) COMMON NAME TOTAL SPECIMENS NUMBER OF SPECIMENS COLLECTED ON TRANSECT AB BC CD DE EF FG GH HJ JK KL LM MN NO ORDER: Hemiptera (True Bugs) Continued Lygaeidae Lygaeid Bugs Miridae Plant Bugs 6 1 33 2 Nabidae Damsel Bugs 33 Neididae Neidid Bugs 1 Pentatomidae Stink Bugs 1 2 7 1 3 1 13 3 5 1 3 2 5 1 7 2 4 3 1 1 2 3 1 1 12 6 11 1 Assassom Bugs 19 Scutellerid Bugs 13 2 Usidetermined Nymphs 68 2 2 1 2 3 1 10 3 3 6 1 1 10 2 4 3 Homoptera (True Bugs) 454 Specimens collected Aleyrodidae Whiteflies 1 Aphidae Plantlice 5 Cercopidae Spittlebugs Cicadellidae Leafhoppers Scale Insects Membracidae 6 1 Scutelleridae Coccoidea Fulgoridae 1 1 Reduviidae ORDER: 4 Fulgorid Planthoppers Treehoppers 1 1 43 1 400 1 21 3 1 27 3 81 2 1 25 41 3 1 32 1 4 28 27 1 1 36 1 4 28 31 3 22 1 8 18 �TABLE V-1. ARTHROPODS COLLECTED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1973 (Continued) TAXON COMMON NAME TOTAL SPECIMENS NUMBER OF SPECIMENS COLLECTED ON TRANSECT AB BC CD DE EF FG JGH HJ JK KL LM MN NO ORDER: Hymenoptera (Bees, Wasps, Ants) 167 Specimens collected Andrenidae 2 Bethylidae Bethylids 2 Braconidae Braconid Wasps 2 Chalcidoidea Cynipoidea Chalcids Dryinidae CO Mining Bees Dryinids Formicidae Ants Halictidae Ichneumonidae Sweat Bees Ichneumon Wasps 9 1 Mutillidae Pompilidae Velvet Ants Spider Wasps 3 2 Undetermined Adults 5 Gall Wasps 40 1 1 1 1 1 2 3 7 1 3 1 99 1 3 1 9 6 3 1 2 1 28 8 10 9 4 15 4 2 1 1 3 4 2 4 10 1 1 1 1 12 1 1 2 1 1 1 1 4 2 1 1 1 1 2 1 ORDER: Neuroptera (Nerve Winged Insects) 1 Specimen collected Myrmeleonidae Antlions 1 1 1 ORDER: Lepidoptera (Butterflies and Moths) 13 Specimens collected Microlepidoptera Several Families Noctuidae Owl Moths 2 1 �TABLE V-1. ARTHROPODS COLLECTED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1973 (Concluded TAXON COMMON NAME TOTAL SPECIMENS NUMBER OF SPECIMENS COLLECTED ON TRANSECT AB BC CD DE EF FG GH HJ JK KL LM MN NO ORDER: Odonata (Dragonflies and Damselflies) 12 Specimens collected Coenagrionidae Damselflies 12 2 3 7 5 1 13 3 1 6 7 3 11 3 3 2 3 7 1 7 1 5 2 3 1 ORDER: Psocoptera (Psocids) 66 Specimens collected Family not determined 66 2 7 10 15 6 3 1 2 2 1 ORDER: Orthoptera (Grasshoppers and Crickets) 92 Specimens collected Acrid idae Gryllidae Grasshoppers 36 Crickets 34 Mantidae Mantids Phasmidae Walkingsticks 14 1 Tettigoniidae Katykids 2 1 1 2 6 1 1 7 1 1 5 ORDER: Thysanoptera (Thrips) 54 Specimens collected Family not determined TOTAL ARTHROPODS TOTAL IDENTIFIED ARTHROPODS 54 1 1726 57 1614 55 9 6 2 19 9 1 81 253 190 128 357 184 104 92 70 248 185 123 339 172 98 77 1 4 92 118 2 37 33 81 103 33 30 �TABLE V-2. TAXA COLLECTED IN NUMBERS EXCEEDING ONE PERCENT OF THE TOTAL SPECIMENS COLLECTED3, JUNE 1973 FAMILY COMMON NAME Cicadellidae Leafhoppers Chrysomelidae Leaf Beetles 219 13.6 38.4 Araneida Spiders 144 8.9 47.3 Formicidae Ants 99 6.1 53.4 Acalyptrate Muscoid Flies 86 5.3 58.7 Psocoptera Psocids 66 4.1 62.8 Thysanoptera Thrips 54 3.3 69.4 Sminthuraidae Springtails 53 3.3 72.3 Acarina Mites 46 2.9 72.3 Cercopidae Spittlebugs 43 2.7 75.0 Chalcidoidea Chalcid Wasps 40 2.5 77.5 Acrid idae Grasshoppers 36 2.2 79.7 Gryllidae Crickets 34 2.1 81.8 Miridae Plant Bugs 33 2.0 83.8 Nab idae Damsel Bugs 33 2.0 85.8 Reduviidae Assassin Bugs 19 1.2 87.0 a Total equals 1,614 identified specimens: NUMBER PERCENT CUMULATIVE COLLECTED OF TOTAL PERCENT OF TOT A Ic 24.8 400 24.8 1 percent of the total equals 16 specimens "As discussed in the text, several of the taxa represent ordinal or super family levels of classification rather than family. c Cumulated percent of total is derived by the progressive summation of the figures in the percent of total column. 99 �TABLE V-3. TAXA COLLECTED IN NUMBERS EXCEEDING ONE PERCENT OP THE TOTAL SPECIMENS COLLECTED3, JUNE 1971 PERCENT OF TOTAL FAMILY-COMMON NAME CUMULATIVE PERCENT OF TOTALb Cicadelliclae - leaf hoppers 31.7 31.7 Araneida - spiders '(order) 18.6 50.3 Lygaeidae - lygaeid bugs 7.7 58.0 Elateridae - click beetles Pentatomidae - stink bugs 4.7 62.7 4.5 67.2 Asilidae - robber flies 4.2 71.4 Nabidae - damsel bugs 3.9 75.3 Acrididae - grasshoppers 3.2 78.5 Reduviidae - assassin bugs 2.7 81.2 Sphecidae - sand wasps 2.6 83.8 Tenebrionidae - darkling beetles 2.4 86.2 Chrysomelidac - leaf beetles 2.2 88.4 Scutelleridae - scutellerid bugs 2.1 90.5 Coenagrionidae - dragonflies 1.4 91.9 Halictidae - sweat bees 1.4 93.3 Mydaidae - mydas flies Tettigoniidae - katydids 1.3 94.6 1.3 95.9 Myc e t oph i 1 i dae - mycetophilid flies 1.0 96.9 Total equals 1803 specimens: 1 percent of the total equals 18 .specimens Cumulated percent of total is derived by the progressive summation of the figures in the percent of total column 100 �TABLE V-4. INSECTS AND ARACHNIDS COLLECTED OR OBSERVED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, JUNE 1971 ARACHNIDS ORDER Araneida Phalagida NUMBER OF SPECIMENS COLLECTED ON TRANSECT COMMON NAME Spiders Harvestmen TOTAL SPECIMENS 355 1 AB BC CD 4 3 25 DE EF FG GH HJ JK KL LM MN NO 8 30 188 26 28 7 4 8 2 2 NUMBER OF SPECIMENS COLLECTED ON TRANSECT FAMILY COMMON NAME TOTAL SPECIMENS AB BC CD DE EF 1 FG GH HJ JK KL LM MNJ NO ORDER: COLEOPTERA (BEETLES) 206 Specimens Collected Anthicidae 1 Buprestidae Ant like Flower Beetles Seed Beetles Metallic Wood Borers Carabidae Ground Beetles Cerambycidae Long Horned Beetles 4 1 Chrysomelidae Leaf Beetles Cicindellidae Tiger Beetles Coccinellidae Curculionidae j-j Lady Beetles Bruchidae Snout Beetles 1 1 43 2 1 2 4 4 1 1 1 1 1 8 6 Click Beetles Gyrinidae" Blister Beetles Tumbling Flower Beetles 6 Passalidae Passalid Beetles 4 4 2 4 2 4 2 10 3 Mordellidae 6 Whirligig Beetles Meloidae 5 1 Predacious Diving Beetles Elateridae 1 1 10 Dytiscidae" 1 Sighted but not collected in 1971 'sighted or collected in 1970 84 12 10 15 5 13 10 2 5 2 1 2 4 1 1 7 3 �TABLE V-4. CONTINUED NUMBER OF SPECIMENS COLLECTED ON TRANSECT FAMILY ORDER: TOTAL SPECIMENS COMMON NAME Scarabaeidae Scarab Beetles Staphylinidae Rove Beetles Tenebrionidae Darkling Beetles ORDER: BC CD DE EF FG GH HJ KL LM MN NO 7 3 6 9 3 2 6 JK 1 2 1 43 1 1 2 1 1 1 1 2 2 10 3 3 4 9 2 1 14 2 1 DERMAPTERA (EARWIGS) 1 Specimen Collected Forficulidae ORDER: 1 Forficulid Earwigs DIPTERA (FLIES) 211 Specimens Collected Anthomyiidae Anthomyiidid Flies 14 Asilidae o ro AB COLEOPTERA (Continued) Robber Flies 76 1 4 4 8 11 10 4 2 8 3 2 Midges 7 1 • 4 Blow Flies 1 Bibionidae March Flies Bombiliidaea>b Bee Flies Calliphoridae Chironomidae Chloropidae" Culicidaea'u Mosquitoes Dolichopodidae Long-Footed Flies Drosophilidae Mycaidae Vinegar Flies Fungus Gnats Mydas Flies Muscidae Muscid Flies Pipunculidae Bigheaded Flies Mycetophilidae 4 4 Chloropid Flies 3 16 2 2 3 1 18 23 : 17 3 1 4 1 3 2 1 1 16 1 2 2 2 2 9 1 1 3 2 1 4 |.. _ 1 �TABLE V-4. CONTINUED !NUMBER COMMON NAME FAMILY ORDER: TOTAL SPECIMENS AB BC OF SPECIMENS COLLECTED ON TRANSECT CD Tabanidae Sepsid Flies Flower Flies Horse Flies, Deer Flies Tachinidae Tachina Flies Tipulidae Crane Flies 1 1 Tripetidae Trypetid Flies 4 Syrphidae ORDER: 11 3 9 1 GH HJ JK KL LM MN NO 5 1 2 9 7 1 1 1 ) 1 | 3 I 1 HEMIPTERA (TRUE BUGS) 390 Specimens Collected Belastomatidae CO FG DIPTERA (FLIES) (Continued) Sepsidae o EF DE Giant Water Bugs Coreidae Coreid Bugs 3 2 Corimelaenidae Corimelaenid Bugs 5 5 Cydnidae Cydnid Bugs 2 Gerridae* Water Striders Lygaeidae Lygaeid Bugs Miridae Nabidae Plant Bugs Xeididae Neidid Bugs Nepidae N'otonectidae Water Scorpions Backswimmers Pentatomidae 1 138 2 4 38 10 19 40 6 19 1 1 8 15 3 ,3 9 14 2 7 5 4 18 1 | 71 1 2 Stink Bugs 82 2 Reduviidae Assassin Bugs 49 1 Scutelleridae Scutellerid Bugs 37 Damsel Bugs 7 1 2 4 1 7 22 1 11 2 3 13 14 1 12 ft 1 13 6 2 4 1 1 7 2 1 �TABLE V-4. CONTINUED NUMBER OF SPECIMENS TOTAL SPECIMENS AB BC CD DE EF FAMILY COMMON NAME ORDER: HOMOPTERA (TRUE BUGS) 360 Specimens Collected 1 4 Aphidae Plantlice 1 2 1 Cercopidae Spittlebugs 9 Cicadellidae 10 30 46 54 41 Leafhoppers 343 a b Coccidae ' Scale Insects Fulgoridae 1 Fu Igor id Planthoppers Membracidae 3 Treehoppers ORDER: HYMENOPTERA (BEES, WASPS, ANTS) 125 Specimens Collected Apidae 1 Apid Bees Bombidae" Bumble Bees 1 1 1 2 Braconidae Braconid Wasps 11 Chalcididae 2 Chalcids Chrysididae 1 Cuckoo Wasps Cynipidae Gall Wasps 2 Formicidae 1 2 12 Ants Halictidae 2 1 Sweat Bees 3 1 25 Ichneumonidae Ichneumon Wasps 3 Megachilidae Leafcutting Bees 2 1 Mutillidae Velvet Ants 4 3 Pamphiliidae 1 1 Webspinning Sawflies 5 Pompilidae Spider Wasps 6 COLLECTED ON TRANSECT FG GH HJ JK KL 1 1 1 1 1 3 80 21 29 10 10 2 LM MN NO 2 2 10 1 1 1 1 1 1 3 1 2 2 6 12 2 5 1 1 1 2 1 1 1 2 1 1 1 2. 1 �TABLE V-4. CONTINUED NUMBER OF SPECIMENS COLLECTED ON TRANSECT TOTAL AB BC CD DE EF FG GH HJ JK KL LM MN NO COMMON NAME SPECIMENS FAMILY ORDER: HYMENOPTERA (BEES, WASPS, ANTS) (Continued) 1 1 Scoliidae Scoliid Wasps 1 2 4 6 2 2 2 1 Sphecidae Sand Wasps 46 6 20 4 Tiphiid Wasps 4 Tiphiidae Carpenter Bees Xylocopidae ORDER: ISOPTERA (TERMITES) Observed Only Rhinotermitidae Subterranean Termites ORDER: LEPIDOPTERA (BUTTERFLIES AND MOTHS) 38 Sp-cimens Collected Danaidae Milkweed Butterflies Geometridae ' Geometrid Maths a D Lycaenidae ' Blues and Coppers Hesperiidae ' Skippers Microlepidopter* Several Families 23 5 3 Noctuidae 1 1 Owl Moths a b Nyrophalidae ' Brushfooted Butterflies Papilionidae ' Swallowtail Butterflies Pieridae*'11 Sulfurs * ~ Psychidae Bagworm Moths Pyralidae Pyralid Moths 14 3 1 2 1 i 'Several families in this group, but identified no further �TABLE V-4. CONCLUDED NUMBER OF SPECIMENS COLLECTED ON TRANSECT THTiT FAMILY ORDER: Green Lacewings Brown Lacewings Ant lions CD DE EF FG GH HJ JK KL LM 1 1 MN 5 NO 1 8 Dragonflies Damselflies Dragonflies Damselflies Dragonflies 1 25 2 21 15 1 1 1 1 14 ORTHOPTERA (GRASSHOPPERS AND CRICKETS) 74 Specimens Collected Acrididae Grasshoppers Gryllidae Crickets Gryllotalpidae* '"Mole Crickets Mantidae Mantids Tettigoniidae Katykids Trydactylidae Pygmy Mole Crickets ORDER: BC ODONATA CDRAGONFLIES AND DAMSELFLIES) 40 Specimens Collected Aeshnidae Coenagrionidae Corduliidae0 Lestidaeb Libel lulidae ORDER: AB NEUROPTERA (NERVE WINGED INSECTS) 9 Specimens Collected Chrysopidae Hemerobaeidae Myrmeleonidae ORDER: SPECIMENS COMMON NAME TRICHOPTERA (CADDISFLIES) Family not determined 58 Observed Only 1 6 1 6 8 16 1 3 6 3 6 4 2 4 23 7 1 4 7 7 1 4 1 5 1 1 1 1 1 �s? o < 100. cc 90 — t- 40 - £ 20. 2 > 0 70 ZP- ?P-i nnni1ft 40 Ti n 30 AB BC CD DE EF 500. 450. Q 400 . FG GH HJ JK 40 KL LM MN NO 52 i | l § 350. K 300. I £ 250. < 200 . u. 150 O . 100 . o<trt A 50.' 50 0 "n i 90 !0 > 80. R u 60- AB r J£ \ BC n 96 Ifl V 147 CD DE 1fi| FiFiflrin EF FG GH jJ4_ HJ JK KL LM MN NO 48 U) % 50. I £ Q O O 40- fE CC 20. u. O 10. 31 M. 22. -18 2i AB BC ^1 nnnf 5 113 n 17 CD DE EF FG GH HJ JK 17 , KL LM MN NO 9. U 8. t 7 6.9 •*__ - (A 5.9 Q 4.4 0 cc z KCC 4.6 5- 4 4.2 — „„ « ' •JL - 3.8 iZ 4.1 f" 3. P i 0 2 1 - 0 AB BC CD DE EF FG GH HJ JK KL LM MN NO 400 FOOT GRID TRANSECTS ON SAMPLER ROW 8 Figure V-1. Arthropod/Vegetation Comparisons on TA C-52A, 1971 Study 107 �Ul 0 K Ul 10Q. > 0 ui 90 80 6Q 3D 60_ UL Bj §' ff ° I1V13D3A ^ 70 OS, SO p i 40 40 JK KL LM 98 77 81 J23. HJ JK KL LM 40 AH BC CD DE EF FG GH HJ nnnn* MN NO MN NO CO O 400. 2 339 §300. X N 248 < 200. 21 LL 0 n ft d ioo- ' 55 z o AB BC CD DE n EF 172. FG GH to Ul i £ 40. 0 2 0 cc 36 29 fl 30 - X I- 20 . K nL Ul > _ 27 ^ 27 BC CD DE EF FG 76- __ 5.6 5.1 § ££J KL LM MN NO 5.6 !^ 4.4 3- cc JK pa 4.2 Q 4- MM GH HJ 6.8 •— 5_ Q 21 121 19 AB > H tt 28 31 m . n "~ 4.6 ^^ f*1 A r^ 2.9 ~* I 2 . 1 1. AB BC CD DE EF FG GH HJ JK KL LM 2.4 MN NO Hn 400 FOOT GRID TRANSECTS ON SAMPLER ROW 8 Figure V-2. Arthropod/Vegetation Comparisons on TA C-52A, 1973 Study 108 �similarities exist. The diversity index used was that of Margalef: In N Where d = diversity S = number of Taxa N = number of specimens In = natural log This formula has been used in previous diversity studies on Test Area C-52A (References V-3 and V-4), and it is used elsewhere in the present report. The greatest diversity of Arthropods as indicated by this formula was in transect GH in both surveys, even though the adjacent transect FG showed equal or greater amounts of vegetation (due to the presence of standing water in this area). The greater diversity in GH is due to a large number of taxa relative to the total number of Arthropods present. This large number of different organisms is likely due to the existence of a wider variety of available niches in transect GH, which includes influences of the adjacent aquatic area, dry sandy areas, and areas more disturbed by man. (The central sampling tower of the test area is located in this transect.) Other factors would be of interest in comparisons of the 1971 and 1973 data, such as the relationship of Arthropod population biomass to vegetative cover. While this biomass-vegetation comparison would ideally show a close correlation, two factors make it impractical. First, the influence of randomly caught large insects (especially the grasshoppers) on biomass data would be quite confounding until a great deal of replication had produced a representative sampling of these animals. Second, the 1971 insect survey considered factors other than biomass, so there is no direct basis for comparisons. Many of the other topics that were discussed in the 1971 study hold equally true at this time. Experimental biases of the sweep net technique factors affecting plant distribution, and plant-insect relationships are discussed in Reference V-1. 4. SUMMARY AND CONCLUSIONS A sweep net survey of the Arthropods of Test Area C-52A on the Eglin AFB Reservation resulted in the collection of over 1,700 specimens belonging to 66 insect families and Arachnid orders. These totals represent only one of five paired sweeps taken over a one-mile section of the test grid. A similar study performed in 1971 produced 1,803 specimens and 74 families from five paired sweeps of the same area using the same basic sampling techniques. A much greater number of small to minute insects were taken in the 1973 survey. Vegetative coverage of the test area had increased since 1971. The two studies showed similarities in distribution pattern of vegetative Arthropod numbers, number of Arthropod \ varieties, and Arthropod diversity. Generally, the present study showed a reduction of the extremes found in the above parameters in the 1971 study. This result is expected to continue as the test area stabilizes and develops further plant cover, thus allowing a succession of animal populations to invade the recovering habitat. References: V-3. Boror, D. J. and D. M. DeLong: An Introduction to the Study of Insects. New York, Rinehart. 1952. V-4. Lehn, P. J., A. L. Young, N. A. Hamme, and B. C. Wolverton: Studies to Determine the | Presence of Artificially Induced Arsenic Levels in Three Freshwater Streams and its Effects of I Fish Species Diversity. AFATL-TR-70-81, Air Force Armament Laboratory, Eglin AFB, Florida. August 1970. Unclassified. 109 �SECTION VI AQUATIC STUDIES OF TEST AREA C-52A One of the major parameters involved in the process of herbicide movement and/or persistence in soils is the adsorptive capacity of the soil. The adsorptive capacity, or the cation exchange -capacity (i.e., the ability of a cation to be displaced or exchanged from the soil by another cation), is closely associated with the inorganic colloids (e.g., clay particles) and organic colloids (e.g., organic matter) of the soil. A soil with a large cation exchange capacity could bind within its colloidal system a large concentration of herbicide. Soils with a low cation exchange capacity do not retain cationic herbicides (e.g., cacodylic acid or sodium cacodylate), and thus, soil leaching of these herbicides would be expected. From June 1969 to October 1970, 4,395 gallons of military herbicide Blue were disseminated on TA C-52A (Table I-7). Approximately 13,624 pounds of cacodylic acid and sodium cacodylate were sprayed onto an area of less than one square mile. The soil of the test area has a low cation exchange capacity of approximately 0.8 mg exchangeable cation per 100 g of soil (Table I-4), while the annual precipitation of the area is high (Table 1-1). Data from the analyses of soil cores for arsenic (Table II-9) confirm the movement and/or disappearance of arsenic from the test grid. Moreover, Table II-7 suggests that picloram, a component of the herbicide White, has moved within the soil profile and is apparently rather residual in nature. Test Area C-52A is drained by five streams: Mullet, Trout, Basin, Grassy, and Pucker Creeks (Figure VI-1). The combined annual flow from these streams exceeds 24 billion gallons of water. However, only Mullet, Trout, and Basic Creeks are closely associated with the test grid. The mean daily flow rate for these three streams is shown in Table VI-1. As previously noted, studies on the movement of arsenicals and picloram indicated the possibility of herbicides contaminating the three freshwater stream communities draining the test grid. Since arsenical residues may concentrate in the tissue of fish, and particularly in the tissue of oysters, studies were conducted in 1969 and 1970 to determine (1) whether arsenic residues were entering the streams from the test grid and (2), if so, whether these residues were having adverse effects on the fish populations in the streams or were accumulating in oysters found at the mouth of streams adjoining Choctawhatcheee Bay. Synopses of these studies (Reference VI-1) are included in this report. 1. SYNOPSIS OF PREVIOUS RESEARCH, 1969 a. Fish Study To assess the effects of possible arsenic residues, a diversity index study of the fish populations of Mullet, Trout, and Basin Creeks was initiated 3 months prior to the aerial spraying of Blue and continued for approximately 4 months after spraying. Of the three streams under investigation, Trout Creek seemed the most likely to receive herbicide residues from the grid area. The headwaters of the stream are at the bottom of steepReference: VI-1. Lehn, P. Jeffery, A. the Presence of Artifically on Fish Species Diversity. Force Base, Florida, 1970. L. Young, N. A. Hamme, and B. C. Wolverton: Studies to Determine Induced Arsenic Levels in Three Freshwater Streams and its Effects AFATL-TR-70-81, Air Force Armament Laboratory, Eglin Air Unclassified. 110 �1 MILE LITTLE BASIN CREEK \ ) BASIN SPRAY FLIGHT PATH |BAS IN BAYOU EAGLE CRtEK CHOCTAWHATCHEE BAY Figure VI-1. Map of Test Area Showing Streams in Relation to Test Grid and Location of Sampling Stations Used in Arsenic Monitoring Study 111 �TABLE VI-1. CHARACTERISTICS OF SAMPLING SITES ON STREAMS DRAINING TEST AREA C-52A NAME Temperature, Range, °C Mean PH Width, Feet Depth, Feet Bottom Material Mean Flow Rate, gal/day Basin Creek 16-23.5 5.8 to 12 to 4 Sand 39,073,000 Mullet Creek 14-23.0 6.0 to 10 to 2 Sand 3,648,000 Trout Creek 13-23.5 6.1 to 15 to 2 Sand 5,870,000 Little Basin Creek3 (Control Stream) 15-22.5 6.0 to 8 to 3.5 Sand 3,450,000 a Does NOT drain TA C-52A sided bayheads adjacent to the edge of the grid and directly in line with the lower extremities of the repeatedly used spray flightpath (Figure VI-1). From its headwaters, the stream flows approximately 2 miles directly south into Choctawhatchee Bay. As the stream nears the bay, it deepens to several feet and has a heavy deposit of leaves and other organic matter on the bottom. Mullet Creek has portions of its headwaters originating in steep-sided bayheads within 0.5 mile of the west boundary of the spray grid and flows south for approximately 2.5 miles into Choctawhatchee Bay, deepening near its mouth with a heavy deposit of leaves and other organic matter on the bottom (Figure VI-1). The headwaters of Basin Creek originate several miles to the north of the spray grid. The stream flows southeast within 0.25 mile of the northeast corner of the grid and joins with a small tributary originating at the north margin of the grid, continues east for approximately 3.5 miles, and turns south for 2.25 miles emptying into Basin Bayou and Choctawhatchee Bay (Figure VI-1). Six sampling stations were established on the three streams: One on Mullet Creek, two on Basin Creek, and three on Trout Creek (Figure VI-1). The selection of sampling station locations was determined mainly by their accessibility, variation of habitat within the station, and apparent fish populations. Because of the number of stations and time involved, they were not all sampled on the same day. On each sampling date, observations were made in an effort to detect any gross changes in the population levels of the following selected benthic organisms: crayfish (Orconectes sp.). dragonfly naiad (Gomphus sp.), freshwater snail (Neritian sp.), and an unidentified immature freshwater clam. Observations were also made to detect any morphological effects that may have occurred to eelgrass (Vallisneria americana). the only species of vascular aquatic plant common to all stations. 112 �Fish were collected with a variety of seines ranging in length from 4 to 15 feet and in mesh size from 1/8 to 1/4 inch. All represented habitats within each station were sampled randomly, and the time of day that the samples were taken was also varied. For the first several weeks of the survey, the fish were returned to the stream after the total catch was made and counted; however, for the remainder, and majority, of the survey, the fish were preserved in 10% formalin and counted in the laboratory. In conjunction with the stream sampling, two ponds on the test grid were sampled using dip nets. b. Residue Sampling Samples were routinely collected at 11 stations on the streams and in Choctawhatchee Bay after each rainfall following herbicide missions, or, if no missions had been flown, samples were collected monthly. Water from these streams was sent to the Regional Environmental Health Laboratory, Kelly AFB, Texas, where it was analyzed for arsenic. Detritus (bottom) samples were taken monthly with an Eckman dredge at three randomly selected water sampling locations. After appropriate pretreatment, these were assayed in the same manner as the water samples. In addition to water and detritus sampling, oysters were used to monitor changes in arsenic level. Because these mollusks are filter feeders, the arsenic content of their bodies was correlated with that of their environment. Oyster racks were established in Choctawhatchee Bay at the mouths of Basin, Trout, Grassy, Mullet, and Rucker Creeks. A control rack was also located in the bay at the mouth of Eagle Creek, which does not drain the grid area (Figure VI-1). Each rack contained approximately 2,000 oysters in the 1 to 3 inch diameter range, and these were sampled periodically. The small size of the oysters was intended to discourage removal from the racks. Samples obtained from the racks were frozen and taken to the laboratory for analysis. There the sample was acidified, hydrolyzed, and neutralized before undergoing standard atomic absorption analysis for arsenic (Reference VI-2). Water samples were collected for picloram analysis from a small bayhead of Basin Creek just north of sampler station A-11. The bulk of herbicide White was disseminated on Grid 3, with the remainder being sprayed on Grid 4 (see Section I, Figure I-5 and Table I-7). Thus, most of picloram was probably concentrated around the northern portion of the one square mile area. Other water samples for picloram analysis were collected in the bayhead of Long Creek, which is located approximately 3 miles northwest of the one square mile grid and which has a water source not associated with TA C-52A. c. Results Twenty-one species of fishes were collected, with three species occurring within the boundaries of the one square mile grid and 20 species from the surrounding streams (Table VI-2). Habitat and spatial isolation seemed to be the major limiting factors on the grid. Reference: VI-2. Hamme, N. A., A. L. Young, J. H. Hunter: A Rapid Analysis of Soil and Water by Atomic Absorption. AFATL-TR-70-106, Air Force Armament Laboratory, Eglin Air Force Base, Florida, 1970. Unclassified. 113 �TABLE IV-2. FISH SPECIES FOUND IN PONDS AND DRAINAGE AREAS OF THE ONE SQUARE MILE GRID AND IN BASIN, MULLET, AND TROUT CREEKS SPECIES AND COMMON NAME AREAS WHERE COLLECTED ON GRID 1 Amhlnplitfis rupflstris - snnthfirn rnnk hass - 2. Anguilla rostrata - American eel OFF GRID +B +BT 3. Aphredoderus sayanus - pirate perch - +BT 4. Elassoma okefenokee - Okefenokee pigmy sunfish +T 5. Erimyzon sucetta - lake chubsucker +* 6. Esox arpericanus - red-fin pickerel - +B 7. Esox niger - chain pickerel - +B 8. Etheostoma edwini - brown darter - +BT* 9. Fundulus nottj - starhead topminnow - +T 10. Gambusia affinis - mosquito fish - +BMT* 11. Ichthyomyzon gagei - southern brook lamprey - +BM 12. Ictglurus natalis - yellow bullhead + - 13. Lepomis punctatus - spotted sunfish + +BMT 14. Micropterus punctulatus - spotted bass - +T 15. Minytrema melanops - spotted sucker - +B 16. Notropis hypselopterus - sailfin shiner - +BMT* 17. Notropis texanus - weed shiner - +B 18. Noturus funebris - black madtom - +T 19. Noturus gyrinus - tadpole madtom - +T 20. Noturus leptacanthus - speckled madtom - +BMT* 21. Percina nigrofasciata - blackbanded darter - +BMT* * Denoted large population in area. B = found in Basin Creek M = found in Mullet Creek T = found in Trout Creek 114 - �The lake chubsucker was abundant in one of the ponds on the grid but was not found in the three streams within a 2 mile radius of the center of the grid, however, the species occurs several miles downstream in more sluggish waters. The employment of a diversity index (i.e., a statistical comparison of the fish populations before and after the spray missions, representing a time period of 8 months) showed a population change in one fish species at one of the six stations studied. This change, however, was probably due to an unidentified variable (e.g., variation in collecting techniques) rather than to arsenic residue. The arsenic analyses for 588 water samples and 68 silt samples were negligible (less than 1 ppm and not significantly different from control streams). A comparison of arsenic contents of 73 oyster samples taken from sampling stations established in Choctawhatchee Bay showed no significant differences from control samples taken elsewhere in the bay at the 95% probability level (1.32 ppm arsenic versus 1.45 ppm). The results of water samples collected from Basin, Trout, and Long Creeks, and analyzed for picloram content are shown in Table VI-3. Picloram residues were still being detected in the small bayhead north of sampler station A-11 as late as December 1971. The last mission with herbicide White was in May 1970. TABLE IV-3. RESULTS OF CHEMICAL ANALYSIS OF WATER SAMPLES FOR PICLORAM, 1971 DATA SAMPLE LOCATION DATE COLLECTED PICLORAM8, ppb Basin Creek, North of Sampler Station A-11 in NE Corner of one square mile grid 11 Jun 1971 11 Trout Creek, South of Sampler Station 0-11 in SE Corner of one square mile grid 11 Jun 1971 2.4 Basin Creek, same as June 1971 location 3 Dec 1971 11, 9.4 Trout Creek, same as June 1971 location 3 Dec 1971 1.4 Control; Long Creek, approximately 3 miles from one squane mile grid 11 Jun 1971 <0.1 a Analysis performed by the Dow Chemical Company; Method ACR 68-14 115 �2. CURRENT STUDIES OF AQUATIC ORGANISMS The objectives of the current studies were (1) to reaccomplish the 1969 - 1970 aquatic studies and to compare population and diversity data, (2) to accomplish an in-depth survey of the aquatic organisms in the test grid ponds, and (3) to obtain samples of aquatic vertebrates from streams and grid ponds for arsenic and TCDD residue analyses. a. Methods and Materials Sampling of the ponds on TA C-52A was accomplished using dip nets and, where aquatic vegetation permitted, a 4 by 15 foot seine and a variable mess gill net. Sampling of the ponds was performed twice with identical collection methods employed both times. Tadpoles (Rana pipens subsp. sphenocephala Hyla gratiosa) and lake chubsuckers (Erimyzon sucetta) were frozen for arsenic and TCDD residue analyses. Those aquatic organisms caught only for species diversity and relative quantity were preserved in 10% formalin. Tadpoles were also collected at a control pond (north of TA C-52A) and were frozen for TCDD residue analysis. Sampling of the streams draining TA C-52A (Mullet, Trout, and Basin Creeks) and the control stream (little Basin Creek) was accomplished also using the 4 by 15 foot seine. Approximately 100 yards of each stream was worked for 2 hours. The identical sampling technique was employed, and each stream was sampled three times. (This technique was that described in Reference VI-1). Species collected only for diversity and relative quantity were preserved in 10% formalin. Crayfish (Ordonectes sp.), speckled madtoms (Noturus leptacanthus). brown and blackbanded darters (Etheostoma edwini) and (Percina nigrofasciata) and any larger fish, e.g., redfin pickerel (Esox americanus) and spotted sunfish (Lepomis punctatus). were frozen for subsequent analysis of TCDD and arsenic residue. The selection for residue analyses of the crayfish and smaller fish species was based on the fact that they are bottom feeders or primary/secondary consumers and thus likely to ingest organic matter containing TCDD and arsenic. The larger fish were selected for residue analysis because they had been in the stream for a longer time and were predators, filling niches at the top of the aquatic food web - hence, a greater likelihood of residue accumulation taking place if bio-magnification was occurring. In addition to these species, oysters were collected for arsenic analysis from the mouth of Mullet and Trout Creeks where they drain into Choctawhatchee Bay. The samples collected for TCDD analysis were sent to the Interpretive Analytical Services Laboratory, Dow Chemical U.S.A., while the samples collected for arsenic analysis were sent to the Pesticide Degradation Laboratory, United States Department of Agriculture. The analysis of arsenic was by atomic absorption of arsine generated with N2BH4All of the streams that were sampled for fish were also sampled for aquatic invertebrates. Benthic samples were taken near the stream margins and in mid-stream at each station using a modified Surber Sampler with number 15 mesh. The margins were covered with a thin layer of organic debris and entangled with the root systems of neighboring plants, while the center of the stream bed was composed almost entirely of sand. The sampler was sunk about 6 inches into the stream bottom with the net on the downstream portion; then, the sand and debris enclosed by the sampler were placed in the net - going down about 6 inches into the stream bottom. The netting was taken to a deep spot on the stream and washed so that sand and debris would pass out through the netting. The remaining contents in the mesh were transferred into an enamel pan. The debris was examined, and all invertebrate organisms were removed and placed in plastic bottles containing water from the stream. The bottles were labled and taken to the laboratory. There, the organisms were placed in boiling water for 5 minutes, transferred to containers with 70% ethanol solution, classified, and counted. 116 �Ten-foot strip samples of the aquatic areas of the grid were taken by using an insect net to make a 10 foot linear scoop along the bottom of the pond. The debris collected was then sorted for invertebrate organisms. Figure IV-2 shows the location of the major bodies of water at the time of survey, June 1973. It should be noted that the first 6 months of 1973 were abnormally high in rainfall, and thus, the 1973 survey showed more water on the grid than observed in 1969 to 1971. The three aquatic invertebrate samples were taken from the ponds located near sampler stations F-7, F-13, and G-13. Biological specimens were forwarded to Dow Chemical U.S.A., Midland, Michigan for determination of TCDD levels. Analysis was accomplished using a modification of the technique of Baughman and Meselson (Reference VI-3). b. Results and Discussion Table VI-4 compares those fish species caught in the streams (draining TA C-52A) in 1969 (Reference VI-1) and those caught in 1973. The methods of collection and the sampling stations were the same for both studies. In order to compare the fish populations caught in 1969 with fiose caught in 1973, three assumptions were made: (1) That fish caught per sampling is proportional to the total fish population at that site, so long as the methods employed are sufficiently similar. (2) That the sampling methods remained sufficiently similar to justify assumption one during all seining operations both in 1969 and 1973. (3) That the frequency distribution of fish caught per sampling is approximately normal. The data for fish populations per sampling for 1969 and 1973 can be shown as: Sampling Period Number of Observations Mean Number of Fish Per Sampling Standard Deviation Before Spraying Blue (Mar 1969) 36 84 29.6 After Spraying Blue (Oct 1969) 16 84 49.2 1973 Sampling 13 141 60.2 As can be seen from these data, the fish caught per sampling before and immediately after the dissemination of Blue in 1969 remained constant. Moreover, a significant increase in fish caught per sampling occured in 1973 as compared to 1969. If the control stations (Little Basin and Fox Creeks) are compared for population changes during this time period, the following data are obtained. Control Stations Little Basin Creek Fox Creek 1969 Means Per Sampling 81 83 1973 Means Per Sampling 94 84 Reference: VI-3. Report Number IAS-405, Dow Chemical U.S.A., Midland, Michigan 117 �N I 8 5 9 10 11 12 13 14 "W B • G H r • • * ' • * sr * - •* r^ • • TOWER X • • I ——W» V M N Figure VI-2. Location of Water and Major Drainage Ditches on the One Square Mile Grid of TA C-52A, 1973 Data 118 �TABLE VI-4. FISH SPECIES COLLECTED IN 1969 AND 1973 FROM THREE STREAMS DRAINING TA C-52A AND A CONTROL STREAM COMMON NAME TROUT CREEK MULLET CREEK BASIN CREEK LITTLE BASIN^ 1969 SPECIES 1973 1969 1969 1973 1969 1973 1973 Notropis hvpselopterus sailfin shiner +° + + + + + + + Gambusia affinis mosquito fish + + + blackbanded darter + + + Percina niorofasciata + + + + + + + + + + Etheostoma edwini brown darter + + + spotted sunfish speckled madtom + + + + + + + + + + Lepomis punctatus Noturus leptacanthus + + + + + + + + + + + Ichthvomvzon gagei southern brook lamprey -c + + + + + + + Notropis texanus weed shiner + + Esox niqer chain pickerel + — Aphredoderus savanus pirate perch + + + + + + Esox americanus redfin pickerel — + Anquilla rostrata American eel + — Minvtrema melanops spotted sucker — + .+ + + + + — a Control Stream D Species Present (+) Species Absent (— ) G + + _u — — + + �TABLE VI-4. CONCLUDED SPECIES southern rock bass TROUT CREEK MULLET CREEK BASIN CREEK 1969 Ambloplites rupestris Muqil ceohalus COMMON NAME 1969 1969 — — common mullet Ictalurus natalis yellow bullhead Micropterus punctulatus spotted bass + to o Control Stream 1973 1969 1973 — + + + — 1973 + — a 1973 LITTLE BASIN3 + — �There is no significant change in the fish populations at the control sites. There are insufficient data on other variables (e.g., nutrient fluctuations), on other environmental factors, or on food chain growth data to warrant pinpointing the direct cause of the fish population increase other than that it appears to be associated with the general recovery phenomenon of vegetation, animal, and insect populations as noted in other sections of this report. The species diversity was determined by the same method employed in the 1969 study (Reference VI-1). The mean diversity for 1969 (before and after the spraying of Blue) and 1973 for the control sites are: Control Sites Number of Samplings Mean Diversity Standard Deviation Variance Before Blue, 1969 36 0.9779 0.3049 0.0930 After Blue, 1969 16 1.3286 0.4903 0.2404 1973 Sampling 13 1.5934 0.1952 0.0381 The 1973 sample size of 13 may be too different from that of 1969 to compare diversity indices. The dependence of the diversity index (d) on the number of samples taken (N) may exist in such a way as to bias d when large or small values of N are used. If the diversity index is plotted as a function of N (using actual data) then the difference in d values before and after spraying Blue (and hence, the 1973 data) is too greatly dependent on N to use without either correcting for the sample size difference or re-sampling (thus using nearly the same sample sizes) A correction technique was employed. A description of this method is included and is in fact an analysis of the diversity of species using Monte Carlo normalized diversity indices. In attempting to make comparisons between d values it appeared desirable to factor out the influences of N by making all N's the same. A small simulation was undertaken in which the observed frequency of species was assumed to be the expected value. A sample size of 80 was chosen as the common sample size because it is near the mean of the actual sample taken. Then, using the observed distribution to establish the probability of the occurrence of each species, 80 "fish" were drawn from the population. This closely simulated the process in which fish are captured until exactly 80 were caught in each sample and then the specimens classified and the data tabulated. One source of error for the simulation is the fact that the number of species, S, cannot exceed the S value for the observed case; i.e., if a species did not appear in the original sample, then the probability of its appearance in the "redrawn" sample is zero. This error, however, should be insignificant in cases where the original sample size was 30 or larger. In a further effort to make comparisons of diversity more meaningful, the expected values of sample size, Ne, and expected number of species, Se, were calculated assuming that the variety of fish life had not decreased; i.e., the d value now is no worse than the d value for the time period before the herbicide was applied. A comparison of Ne and Se were made with the respective observed values N0 and S0. 121 �The equations used are: and S e = d log e (N + 1 ) The d values were calculated for the redrawn samples. The only tendency, if any, was for the diversity index to increase after spraying. Linear correlation coefficients between d and average sample size compared to distances of the sampling stations from the center of the spray area were very small and insignificant. The Se and Ne values for 1969 both before and after spraying were compared to those for 1973 using the two control sites to establish the expected diversity index. In both cases S0 was 10 to 20 percent higher than Se, and Ne was grossly larger than NQ. These observations both tend to imply that the collecting sites considered to be within the spray zone are richer in fish life than the control sites outside the spray zone. By using the correction technique on the mean diversity for the control sites only, a chronologically higher diversity in fish is evidenced from before spraying Blue in 1969, through the after spray period, to the 1973 sampling. Control Sites Before Blue 1969 Diversity After Blue 1969 Diversity 1973 Diversity Little Basin 1.324 1.806 1.770 Fox Creek 1.138 1.212 1.580 This same trend was noted for the streams draining'the test area. However since the control sites are assumed to be either too far away from the grid area to be affected by the herbicides or are experiencing the recovery phenomenon noted for TA C-52A, no significant changes are evident in the diversity of fish life from 1969 to 1973. In order to compare the species proportions for the 1969 data to the 1973 data, the following assumptions were made: (1) That the average of the percentages of a given species found in the sample is a reasonable estimate of the actual percentage of that species in the fish population in the stream. (2) That the percentages of rare species found in the samples are not valid for comparisons because sample sizes are not large enough for a sufficiently high confidence in the percentages. 122 �Using these assumptions, only the two most common species were compared from the 1969 and 1973 data for significant changes. The rare species, therefore, were treated as part of the general diversity analysis. Table VI-5 compares the 1973 mean percentages for the sailfin shiner (Notropis hypselopterus) and the mosquito fish (Gambusia affinis) to the 1969 data of before and after spraying of Blue and the combined mean for 1969. The significance of the differences was tested using the t test. The sailfin shiner had a significant decrease in its proportion of the fish population in 1973 as compared to the before spray (March 1969) fish populations, but the difference was not significant when 1973 data were compared to after spray data (October 1969). For the overall comparison, however, of 1973 data to 1969 data (combined) no significant difference existed. If data for the sailfin shiner are compared only for the control sites a significant decrease occurs in the percent of the population between the March 1969 data and the 1973 data. However, such a significant decrease does not occur when 1973 data is compared to October 1969 data. Control Station March 1969 Means 1973 Means October 1969 Means Little Basin 0.869 0.623 0.788 Fox Creek 0.928 0.714 0.758 As a result of these data, it is apparent that some factor (unrelated to the herbicide or recovery phenomenon) was at work between the spring of 1969 and the fall of 1969. As a result, it is more prudent to assume no significant proportional changes existed between spring 1969 and 1973 for the most abundant species. The three aquatic areas on the grid were observed to be areas of 80% to 100% vegetative cover with the vegetation chiefly composed of grassy plants. The pond at station F-7 is located on Rutledge Sand, and the ponds at F-12 and G-13 have Chipley Sand underlying them. The F-7 pond had a pH reading of 5.51 and was heavily congested with algae and aquatic grasses. The two other ponds had a pH reading of 6.39 and were much less overgrown with aquatic grasses and algae. Both of the ponds were known to be intermittent; partially drying up once in the last 5 years. An alligator was sighted in F-7 pond and two 6-inch lake chubsuckers (Erimyzon sycetta) were taken from it. In the east grid ponds (F-12 and G-13), sightings were made of turtles, but no fish were taken from these ponds. The results of bottom sampling these ponds for specimens of invertebrate are shown in Table VI-6. The dominant order is Odonata. Without exception, the members of this order are predacious; therefore, their supply in these ponds must be relatively extensive. The Serber sampling of the streams is shown in Table VI-7. Fox Creek yielded very few aquatic organisms, either invertebrate or vertebrate. The yields of invertebrates were so few, in fact, that Fox Creek was considered too different from Basin, Trout, or Mullet Creeks to effectively serve as a control stream. Perhaps the low yield in organisms in Fox Creek is related to its depth (mean depth of 18 inches with pools reaching to five feet) and/or swiftness. The majority of the organisms found in the other three streams were caddis fly larvae and snails. The caddis fly larvae are omnivores, while the snails are herbivores. Presumably, there is an extensive food web associated with these invertebrates that was not sampled by the Serber sampler. 123 �TABLE VI-5. POPULATION CHANGES IN THE TWO MOST COMMON FISH SPECIES (ALL SITES ARE GROUPED TOGETHER) 1969 AND 1973 DATA SPECIES DATE NUMBER OF OBSERVATIONS MEAN STANDARD PROBABILITY OF DEVIATION CHOOSING SAMPLES WITH MEANS HAVING THIS OR GREATER DIFFERENCE Notroois hypselopterus Mar 1969 8 0.79 0.117 0.0 1a Oct 1969 8 0.58 0.172 0.94 16 0.69 0.179 0.91 Jun 1973 5 0.51 0.161 Mar 1969 8 0.102 0.101 0.92 Oct 1969 8 0.188 0.132 0.99 16 0.145 0.122 0.96 5 0.182 0.194 Combined 1969 Gambusia affinis Combined 1969 Jun 1973 a 95% level of significance TABLE VI-6. NUMBER OF AQUATIC INVERTEBRATE SPECIMENS COLLECTED FROM BOTTOM SAMPLING THREE PONDS ON TEST AREA C-52A8 ORDER LOCATION OF PONDSb COMMON NAME F-7 F-13 G-13 Coleoptera scavenger beetle 0 1 0 Hemiptera backswimmers giant water bugs 3 0 10 1 6 0 Odonata dragonflies/ damsel flies 10 2 8 17 1 9 Trichoptera caddis flies 1 0 0 16 37 16 Total Specimens a Each sample represents three collections with a 1 -square foot Serber Sampler "Ponds designated by the closest permanent sampler station 124 �TABLE VI-7. NUMBER OF AQUATIC INVERTEBRATE SPECIMENS COLLECTED FROM BOTTOM SAMPLING THE STREAMS DRAINING TEST AREA C-52A8 ORDER COMMON NAME CREEK FOXb MULLET TROUT BASIN 10 11 10 1 5 8 1 1 19 1 0 48 96 15 0 0 Annelida aquatic earthworms Coleoptera Decopoda beetles crayfish Gastropoda snails Odonata dragonflies 0 0 2 2 Plecypoda freshwater clams 0 0 9 0 Trichoptera caddis flies 51 75 158 49 115 202 202 53 Total Specimens a Ten-foot strip sample from bottom of pond using a 15-inch insect net '•'Control station The results of residue analysis for arsenic in aquatic organisms are shown in Table VI-8. The level of arsenic in the oysters svas considerably lower than those values reported for oysters in 1970 - 1971 (average of 0.28 ppm arsenic versus 1.32 ppm, respectively). The lower levels of arsenic in 1973 may be due to the employment of different analytical procedures or to the increased stream flow noted this year, and hence, to a greater purging of the arsenic by the large volumes of freshwater entering Choctawhatchee Bay. No control tadpoles were analyzed for arsenic content, but presumably the level of arsenic was probably higher than it would be in control samples. This would be evident from data of the arsenic levels in 1973 grid soils as shown in Table II-7 (Section II). The results of residue analysis of aquatic organisms for TCDD are shown in Table VI-9. The analysis were performed by the Interpretive Analytical Services, Dow Chemical U.S.A., Midland, Michigan. The duplicate samples were analyzed independently by high resolution mass spectrometry (see Section II for methods and materials). Duplicate samples of all biological specimens were also submitted to the Pesticide Degradation Laboratory, Agricultural Environmental Quality Institute, Beltsville, Maryland, for an independent check on results. However, none of the methods employed by the Degradation Laboratory could lower the limit of detection below 0.1 - 0.2 ppb TCDD. 3. CONCLUSIONS From examining the data, certain observations support the idea that a recovery phenomenon is occurring in the streams draining TA C-52A. These observations are difficult to document because of insufficient data. For example, in 1969 the southern brook lamprey (Ichthyomyzon gagei) was never collected in Trout Creek, yet in 1973 it was taken in relatively large numbers. It now appears that the lamprey is breeding in Trout Bayhead south of sampler station 0-11. Moreover, all of the specimens of lamprey collected this year in Trout Creek are immature 125 �TABLE VI-8. CONCENTRATION OF ARSENIC IN BIOLOGICAL SPECIMENS COLLECTED ON OR ADJACENT TO TA C-52A BIOLOGICAL SPECIMEN3 (Common Name) LOCATION COLLECTED (June 1973) Oyster Mouth of Trout Creek Oyster Mouth of Mullet Creek 0.44 0.12 Blue Crab Mouth of Trout Creek 0.32 Blue Crab Mouth of Mullet Creek 0.32 b CONCENTRATION OF ARSENIC ( ^ g As/gram Fresh Tissue) Crayfish Little Basin Creek Crayfish Trout Creek Black-banded Darters Little Basin Creekb 0.29 0.30 0.75 Black-banded Darters Trout Creek 0.15C Speckled Madtom Little Basin Creekb 0.30 Speckled Madtom Trout Creek 0.38 Redfin Pickerel Trout Creek 0.23 Tadpoles Grid Pond (F-7) 1.47 a Samples for analysis were either aliquots of homogenates or the entire homogenate depending on sample size. "Control Samples c Part of the tissue was lost in digestion of sample. TABLE VI-9. CONCENTRATION OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) IN BIOLOGICAL SPECIMENS COLLECTED ON OR ADJACENT TO TEST AREA C-52A BIOLOGICAL SPECIMEN9 (Common Name) LOCATION COLLECTED Cub Sucker Crayfish Crayfish Oyster Rock Bass Spotted Sunfish Spotted Sunfish Tadpole Tadpole Mouth of Trout Creek Trout Creek Control Mouth of Trout Creek Trout Creek Trout Creek Controlb Grid Pond (F-7) Control CONCENTRATION OF TCDD (parts per trillion) a <10 < 10 <10 <10 < 10 <10 <10 <10 <10 AII samples were run in duplicate and analyzed independently by high resolution mass spectrometry. b Control locations are noted in text. 126 �indicating that the population was recently established (within the past two years). However, statistical comparisons of 1969 and 1973 data confirm a chronologically higher diversity in fish populations for even the control streams. Thus, the presence of the lamprey may or may not reflect a change in the habitat due to recovery from herbicide exposure. The data on picloram in waters draining from the test grid would support the need for production studies of these streams. However, a review of toxicological data, for picloram (Reference VI-4) suggests that concentrations of 1000 ppb do not seem to effect aquatic organisms. Moreover, the lack of baseline data and adequate control streams would probably make such studies futile or of doubtful value. It is apparent from the results of samples analyzed for TCDD that representative organisms living in streams draining Test Area C-52A or in the ponds on the test area were free from TCDD contamination at a lower detection limit of less than 10 ppt. These data are not unexpected knowing the low solubility of TCDD in water and its apparent lack of movement in the soil profile (Reference IV-1). Reference: VI-4. Pimentel, David: Ecological Effects of Pesticides on Non-Target Species. Executive Office of the President, Office of Science and Technology, June 1971. 127 �SECTION VII STUDIES ON THE MICROFLORA OF TEST AREA C-52A The soil persistence of herbicides is influenced by many environmental and biological factors. Perhaps one of the most important of these is that of the presence or absence of microorganisms. In an area such as Test Area C-52A, Eglin AFB Reservation, where the soils were subjected to repetitive applications of four different herbicides (2,4-D, 2,4,5-T picloram, and cacodylic acid) over a period of 8 years (1962 - 1970), the organisms had to either adapt to the presence of the chemicals or be adversely affected (i.e., reduction in population). For this reason, studies were initiated to examine population levels of various microflora found occurring on the one square mile grid. The initial studies '° were conducted from 1967 to 1969. These studies provided data on the soil algal populations and are included as a synopsis in this report. In June 1970, a survey was conducted® of the soil bacterial, fungal, and Actinomycete populations found at specific sites on the grid and in control areas. The data from this study (identified in this report as the 1970 study) have been the basis for comparisons in the current study. In addition, the current studies also include a preliminary examination of aquatic algae found in the ponds in the center of the one square mile grid. 1. SYNOPSIS OF PREVIOUS RESEARCH , 1967 - 1970 In 1967, three areas were soil sampled for algal flora. Area I was Grid 1 located immediately south of the present one square mile grid. This area received a total accumulative concentration of 1,894 pounds of 2,4-D,and 2,4,5-T from June 1962 through July 1964. Area II was Grid 2 located in the southwest portion of the present grid. This area received a total accumulative concentration of 1168 pounds of 2,4-D and 2,4,5-T from May 1964 through September 1966. Area III was a control area and was located 3 miles northwest of the present grid. (See Figure I-5). Samples were taken from two levels in the soil. The first level included the surface litter and the first centimeter of soil. The second level samples included an amalgam of the soil between one and 15 cm. Two methods of culture were used. In the first, sterile filter paper was placed in sterile Petri dishes, after which approximately 10 gm of the sample soil were added. The cultures were moistened with sterile Bristol's solution and placed under fluorescent lights with an intensity of 300 Ht-candles. The second method of culture preparation was identical to the first, except an additional piece of sterile filter paper was placed directly on the soil and moistened with the nutrient solution. The number of algae was found to be low but no significant differences could be noted between the sprayed area and plots that had not received herbicides or only minimal amounts due to drift (Table VI1-1). Only green and bluegreen algae were considered for identification. A total of 38 organisms were identified (Table VII-2). At least one species of Chlamydomonas. Chlorococcum. Chlorella. Micrococcus. Nostoc. Oscillatoria and Schizothrix was in every sample. In the majority of cases, Chlorococcum. Nostoc. and Schizothrix were represented by two or more species. Most of the other algae were located sporadically through the sampling period, and few were not universally distributed in all samples. A species of Sponiococcum was the only alga found repeatedly in a single location. The most frequently located alga was Schizothrix calcicola. 4 Arvik, J. H.: Soil Algae of the Eglin AFB Defoliant Test Range and the Response of Selected Species to Militacy Herbicides. Air Force Armament Laboratory Unpublished Data. 1969. Unclassified. 5Arvik, J. H. and J. H. Hunter. Soil Algae of a Herbicide Test Area, Eglin AFB, Florida, and -the Response of Selected Species to Military Herbicides. Air Force Armament Laboratory Unpublished data. 1971. Unclassified. 6 Hunter, J. H. Soil Microorganism Study of TA-C52A, Eglin AFB, Florida. Air Force Armament Laboratory Unpublished Survey. 1970. Unclassified. 128 �TABLE VIM. NUMBER OF SOIL ALGAE FOR GRAM OF SOIL FROM GRIDS 1 AND II, TEST AREA C-52A, AND THE CONTROL AREA, 1967 DATA SAMPLING AREA SOIL pH SURFACE ( 0 - 1 cm) CORE (1 - 15 cm) Grid I (Area I) 2,360a 820a Grid II (Area II) 5.2 2,243 567 Control a 5.4 5.3 2,468 570 Data are averages of three samples and three replications taken 30 days apart from September 1967 through November 1967. TABLE VII-2. SOIL ALGAE FOUND ON OR NEAR TEST AREA C-52A, EGLIN AFB RESERVATION CHLOROPHYTA Characium ambiguum Herm Characium sp. Chlamydomonas pyrenoidosa Deason and Bold Chlamydomonas typica Deason and Bold Chlorella vulgaris Beyer Chlorella sp. Chlorococcum ellipsoideum Deason and Bold Chlorococcum Jiplobionticujm Hern Closteridium sp. Cylindrocystic brebissonii Meneg. Euglena sp. Homidium subtillissimum Mattox and Bold Hormidium flaccidum Mattox and Bold Protococcus viridis C. A. Agardh. Spongiococcus bacillaris Naeg. Ulothrix tenerrima Kuetz. Zygogonium ericetorum Kuetz. CYANOPHYTA Anacystis marina Drouet and Daily Arthrospira brevis (Kuetz.) Drouet Calothrix parictina (Naeg.) Thuret. Coccochloris aeruginesa Drouet and Daily Coccochloris peniocystis Drouet and Daily Fischerella ambigua (Naeg.) Gom. Microcoleum lyngbyaceus (Kuetz.) Crouan Microcoleus vaginatus (Vauch.) Gom. Nodularia sp. Nostoc commune Vauch Nostoc ellipsosporum (Desmaz.) Raben. Nostoc muscorum Ag. Oscillatoria lutea Ag. Oscillatoria submembranaceae Ard. and Straff Porphyrosiphon Natarisii (Menegh.) Gom. Rivularia sp. Schizothrix arenaria (Berk.) Gom. Schizothrix calcicola (Ag.) Gom. Schizothrix friezii (Ag.) Gom. T29 �2. CURRENT STUDIES ON MICROFLORA In the June 1970 study, soil samples were selected for microbial analysis from sites which had been separated into four general areas on the basis of a prior bioassay experiment to determine phenoxy herbicide residues. The four areas were as follows: (a) relatively moist soil with high residue, (b) relatively moist soil with low residue, (d) relatively dry soil with high residue, and (d) relatively dry soil with low residue. The lowest fungal counts were obtained from Area c: 4 x 103 to 7 x 103 propagules per gram of soil. The highest fungal counts were obtained from Area b: 4 x KT' to 1 x 1CP propagules per gram of soil. Actinomycetes and bacteria were considerably higher on wet sites than on dry sites, but the amount of herbicide residue did not seem to affect the count. The objective of the present study was to analyze the microflora in the C-52A Test Grid (population levels of bacteria, fungi, and Actinomycetes, and identification of predominant genera) and then compare the results with a microfloral analysis of an adjacent non-treated area (control) and with a similar C-52A Test Grid analysis carried out in the 1970 study. 3. LITERATURE REVIEW The general role of microorganisms in the biodegradation of herbicides has been thoroughly investigated and well documented. As part of the study, a literature review was conducted of the items listed in Table VII-3. Most research has centered around the biodegradation of various commercial herbicides by fungi and bacteria, both alone and in combination (Table VII-3, items 2 to 9, 11 to 14, 16, 17, 20, 21, and 27 to 31). In many such efforts, various soils have been used to effect the breakdown of herbicides with little attention paid to the microbial genera involved in the process. Total numbers, rather than types of organisms, have been stressed (Table VII-3, items 12 to 14, 16, 17, 20, 21, 27, and 30 to 32). Pfister has investigated the role of soil organisms in biodegradation of pesticides, especially the breakdown of halogenated hydrocarbons (Table VII-3, item 27). In that study, soil fungi, Actinomycetes, and bacteria are implicated in the breakdown of various pesticide compounds. Kearney has extensively studied the role of microorganisms in the soil degradation of halogenated hydrocarbons and has likewise concluded that they do function in the breakdown of these products. Kaufman, Kearney, Sheets, and Beall have been involved in elucidating the role of total bacterial count and individual enzymes in the breakdown process (Table VII-3, items 20 to 23). Bollag has researched the biochemical transformation of pesticides by soil fungi, and considers the fungi to be an important group of organisms in this transformation process (Table VII-3, item 7). He implicates specific organisms, particularly Geotrichum candidum, in connection with polymerization of an aniline compound to an azo-derivative. Tyagny-Ryadno, in determining the effect of heroicides on the microflora and agrochemical properties of the soil, concludes that some herbicides actually increased bacterial populations (2,4-D was a notable exception). He states also that some herbicides promote the growth of fungi, but seem to inhibit Actinomycetes. In his experiments, Simazine caused the greatest increase in populations of Clostridium pasteurianum (Table VII-3, item 32). In investigating the action of 2,4-D on Azotobacter spp. in sugarcane soils, Colmer concludes that the population levels of these organisms are not affected if herbicide application rates are within those normally recommended for sugarcane (Table VII-3, item 1). Johnson and Colmer have included in their work the effect of 2,4-Diand 2,4,5-T on metabolic activities of various .bacteria including specific Azotobacter organisms, Bacillus cereus and Pseudomonas fluorescens (Table VII-3, items 11, 18, and 19). Johnson and Colmer have determined that concentrations of 2,4-D less than 5,000 ppm have little effect on Pseudomonas fluorescens. 130 �TABLE VII-3. LITERATURE REVIEWED IN 1973 STUDY 1. Adams, A. P. and A. M. Wachinski. 1973. Microbial Assessment of Soil Samples from Desert Soil Treated with 2,4-D/2,4,5-T herbicide. Presented to the Ad-hoc subcommittee of the U. S. Scientific Advisory Board. 22 - 23 February 1973. 2. Alexander, M. and M. I. H. Aleem. 1961. Effect of Chemical Structure of Microbial Decomposition of Aromatic Herbicies. Ag. and Food Chem. 9:44-47. 3. Audus, L. J. 1950. Biological Detoxification of 2,4-dicblorophenoxyacetic Acid in Soils: Isolation of an Effective Organism. Nature 166:356. 4. Audus, L. J. and K. V. Symonds. 1955. Further Studies on the Breakdown of 2,4-dichlorophenoxyacetic Acid by a Soil Bacterium. Ann. App. Biol. 42:174. 5. Bell, G. R. 1960. Some Morphological and Biochemical Characteristics of a Soil Bacterium Which Decomposes 2,4-D. Can. J. of Microbio. 3:821-840. 6. Bell, G. R. 1960. Studies on a Soil Achromobacter which Degrades 2,4-dichlorophenoxyacetic Acid. Can. J. of Microbiol. 6:325-337. 7. Bollag, J. M. 1972. Biochemical Transformation of Pesticides by Soil Fungi. CRC Critical Reviews in Microbiology. 2(1):35-37. 8. Bollag, J. M., G. G. Briggs, J. E. Dawson, and M. Alexander. 1968. Enzymatic Degradation of Chlorocatechols. J. Agr. Food Chem. 16(5):829-833. 9. Bollag, J. M., C. S. Helling, and M. Alexander. 1968. Enzymatic Hydroxylation of Chlorinated Phenols. J. Agr. Food Chem. 16(5):826-828. 10. Cairney, W. J. 1973. Summary of Results: Populations of Soil Microorganisms from Desert Soil Treated with 2,4-D/2,4,5-T Herbicide. Presented to Ad-hoc subcommittee of the U. S. Scientific Advisory Board. 22 - 23 February 1973. 11. Colmer, A. R. 1953. The Action of 2,4-D upon the Azotobacter of Some Sugarcane Soils. Applied Microbiology 1:184-187. 12. Fletcher, W. W. 1966. Herbicides and the Bio-activity of the Soil. Invitation lecture given at Wageningen at meeting organized by the Dutch Working Party for Weed Control. 22 March 1966. 13. Fletcher, W. W., and J. E. Smith. Herbicides and Soil Microbes. New Scientist 24:527-528,. 14. Hirsch, P. and M. Alexander. 1960. Microbial Decomposition of Halogenated Propionic and Acetic Acids. Can. J. of Microbiol. 6:241-249. 15. Hunter, J. H. and A. L. Young. 1972. Vegetative Succession Studies on a DefoliantEquipment Test Area, Eglin AFB Reservation, Florida. AFATL-TR-72-31. Air Force Armament Laboratory, Eglin Air Force Base, Florida. February 1972. Unclassified. 16. Jensen, H. L. 1957. Decomposition of Chloro-substituted Aliphatic Acids by Soil Bacteria. Can. J. of Microbiol. 3:151-164. 131 �TABLE VI1-3. Concluded. 17. Jensen, H. L. 1957. Decomposition of Chloro-organic Acids by Fungi. Nature 180:1416. 18. Johnson, E. J. and A. R. Colmer. 1955. I. The Effect of 2,4-dichlorophenoxyacetic Acid on some Phases of the Nitrogen Metabolism of Bacillus cereus. Applied Microbiology 3:123-126. 19. Johnson, E. J. and A. R. Colmer. 1955. II. The Effect of 2,4-dichlorophenoxyacetic Acid on some Phases of the Nitrogen Metabolism of Pseudomonas fluorescens and the Microorganisms of a Soil Suspension. Applied Microbiology 3:126-128. 20. Kaufman, D. D., P. C. Kearney, and T. J. Sheets. 1963. Simazine: Degradation by Soil Microorganisms. Science 142:405. 21. Kaufman, D. D., P. C. Kearney, and T. J. Sheets. 1965. Microbial Degradation of Simazine. J. Agr. Food Chem. 13:238. 22. Kearney, P. C. 1965. Purification and Properties of an Enzyme Responsible for Hydrolyzing Phenylcarbamates. J. Agr. Food Chem. 13:561. 23. Kearney, P. C., D. D. Kaufman, and M. L. Beall. 1964. Enxymatic Dehalogenation of 2,2-dichloroproprionate. Biochem. Piophys. Research Commun. 14:29. 24. Lamartinere, C. A., L. T. Hart, and A. D. Larson. 1969. Delayed Lethal Effect of 2,4-dichlorophenoxyacetic Acid on Bacteria. Bull. Environ. Contam. Toxic. 4(2): 113-119. 25. Magee, L. A. and A. R. Colmer. 1955. III. The Effect of some Herbicides on the Respiration of Azotobacter. Applied Microbiology 3:288-292. 26. Pate, B. D., R. C. Voigt,. P. J. Lehn, and J. H. Hunter. 1972 Animal Survey Studies of Test Area C-52A, Eglin AFB Reservation, Florida. AFATL-TR-72-72. Air Force Armament Laboratory, Eglin Air Force Base, Florida. April 1972. Unclassified. 27. Pfister, R. M. 1972. Interactions of Halogenated Pesticides and Microorganisms: A Review. CRC Critical Reviews in Microbiology 2(1): 1-34. 28. Rogoff, M. H. and J. J. Reid. 1956. Bacterial Decomposition of 2,4-dichlorophenoxyacetic Acid. J. Bacteriol. 71:303-307. 29. Smith, N. R., V. T. Dawson, and M. E. Wenzel. 1945. The Effect of Certain Herbicides on Soil Microorganisms. Soil Sci. Soc. Amer. Proc. 10:197-201. 30. Steenson, T. A. and N. Walker. 1956. Observations on the Decomposition of Chlorophen^ oxyactic Acids by Soil Bacteria. Plant and Soil. 8:17-32. 31. Thiegs, B. J. Fall 1962. Microbial Decomposition of Herbicides. Down to Earth. 18(z):7-10. 32. Tyagny-Ryadno, M. G. 1967. Effect of Herbicides on the Microflora and Agrochemical Properties of the Soil. Trudy Kamenetzpodolsk. sel',-khoz. Inst. 9:43-48. 132 �Two recent studies, however, have indicated that the 2,4-D/2,4,5-T herbicide combination has a short term effect on levels of soil microorganisms, especially bacteria (Table VI1-3, items 1 and 10). Analyses of desert soil to which herbicide had been applied three months earlier have revealed that bacteria levels are still considerably reduced from levels in control soil samples. Fungal levels were also affected but not to the same degree as the bacteria. 4. MATERIALS AND METHOD Samples were taken from the C-52A Test Grid on 13 June 1973. Eight sampling sites were selected to correspond with those sampled in the 1970 study. Three samples were taken from each of the eight 400 by 400 foot grid areas according to the pattern in Figure VII-1. Samples were taken from depths of 0 to 6 inches and from 6 to 12 inches at each site. Four control samples were taken from the same depths in an area 1/4 mile distant from the C-52A site, but similar to it in soil and vegetative cover. The control area was upwind from prevailing wind patterns, upstream from natural test grid water drainage, and never subjected to concentrated herbicide application. In selecting areas for sampling within the 400 by 400 foot grid squares, an attempt was made to sample sites with varying vegetative cover. A system was devised to approximate cover which employed a rank ordering of the sites from 0 to 5; 0 indicated a 0 to 5% vegetative cover, 1 a vegetative cover of 5 to 20%, 2 a cover of 20 to 40%, 3 a cover of 40 to 60%, 4 a cover of 60 to 80%, and 5 a cover of 80 - 100%. In obtaining the samples, a shovel was used to bare a slightly more than one foot deep vertical cross section of soil. The side of the cross section was marked at the 6 and 12 inch points. Soil was skimmed from the side of the hole, first from the 0 to 6 inch depth, then from the 6 to 12 inch depth. Samples were placed in plastic bags and labelled. The soil was kept at 4°C for no more than 2 days before plating on media for microorganism analysis. Three media were used to enumerate microorganisms. Potato dextrose agar medium plus Tergitol NPX (100 ppm) and chlorotetracycline (40 ppm) was used for maximum development of soil fungi. Nutrient agar plus 150 ppm Actidione was used for development of bacteria. Sodium caseinate medium (DIFCO Actinomycete Isolation Agar) plus 50 ppm Actidione was used for determination of Actinomycetes. Thirty grams of each sample to be analyzed were blended with 300 ml of sterile distilled water for one minute. Dilution series were made using subsequent sterile distilled water blanks to achieve dilutions of 10"^, 10 , and 10" . Dilutions were dispensed in three media in sterile petri plates with three replicates per dilution for each sample. All plated samples were incubated at 25°C. Potato dextrose agar plates were examined for fungi after 3 days. Nutrient agar plates were examined for bacteria after 4 days, and the sodium caseinate agar plates were examined for Actinomycetes after 6 days. Counts were made from each plate and predominant organisms were isolated in pure culture for subsequent identification. In addition to enumeration of microorganisms, 10 samples (0 to 6 inch depth) were analyzed for water content. Samples were selected on the basis of Hunter's previous estimations of relatively high or relatively low water content of a given area of the C-52A Grid and on the basis of relative vegetative cover (0 to 5). Samples tested were as follows: (1) five from relatively high moisture areas, (2) two from areas with a vegetative cover of 1, (3) one from an area with a vegetative cover of 3, (4) two from areas with a vegetative cover of 5, (5) five samples from relatively low moisture areas, (6) three from areas with zero vegetative cover, and (7) two from areas with a vegetative cover of 1. 133 �A I I B J I C D I I 1 E 123 I 1 i 1 F 3 21 1 G 32, 1 \ I X T H _ x 2 2 1 l 1 ,1 12 | 3 1 K 1 I 1 L 3'' 1 1 23 1 IV 1 1 M 12=1 n 1 2 3 A E> 6 7 1 1 8 g 1C) 11 12 Figure VI1-1. Schematic of the Test Area C-52A Grid Showing Soil Sampling Pattern (also see Figure I-5) 134 13 14 �5. RESULTS AND DISCUSSION The average number of organisms per gram of soil are shown in Tables VI1-4 and VI1-5. Table VI1-4 indicates the average number of bacteria, fungi, and Actinomycetes for each grid location sampled. Table VII-6 indicates the average number of organisms per gram of soil in terms of relative vegetative cover. Table VI1-5 is a summary of the data from the 1970 study arranged for comparison with Table VII-4. Table VII-7 is a summary of the data from the 1970 study arranged for comparison with Table VII-6. Tables VII-8 and VII-9 show water content and percent organic matter, respectively, for each of the samples analyzed. There were no large differences in the numbers of Actinomycetes, bacteria, or fungi between the sampling sites on the grid for the 0 to 6 inch depth. Comparing these data with the 1970 population levels shows an increase in the average number of Actinomycetes in the J-9 and K-2 locations and an overall increase in the number of bacteria in all sampling areas. In the K-2 area, particularly, the number of bacteria per gram of soil shows an order of magnitude increase over the 1970 level. This increase might be partially explained by the marked increase in overall vegetative cover around J-9 and K-2 since 1970. Differences in microorganism levels in 1970 correlated to an extent with vegetative cover, the lower populations existing where cover was minimal (see Table VII-7). The 1973 data (Table VII-6) shows a significant increase in microorganisms in poorly covered areas. The 1973 data indicates no strong correlation between vegetative cover and microorganism populations. Control areas had population levels similar to those found for the grid. Predominant bacteria isolated from the test grid were Bacillus sp. and Pseudomonas sp.. Predominant fungi were Penicillium spp., Aspergillus spp., and Fusarium spp.. In addition, Nigrpspora sp., Helminthosporium sp.f Pullulariasp., and Curvularia sp. were recovered. The predominant Actinomycetes were Strepjomyces sp. and Nocardia sp. Although number of organisms from the 6 to 12 inch depth were not tabulated for this report, the numbers of fungi were approximately 40 to 50% reduced from the corresponding 0 -to 6 inch depth averages. The average numbers of Actinomycetes and bacteria were about the same as those from the corresponding 0 to 6 inch depth. Water content varied very little in the 10 samples tested; the range being 0.35% to 1.22%. The average for all samples was 0.54%. There was no correlation between microorganism population levels and the slight differences in water content. Organic matter variation was also minimal . Percent organic matter variations did not correlate with differences in microorganism populations. 6. CURRENT STUDIES ON SURVEY OF AQUATIC ALGAE The role of phytoplankton in the productivity of both soil and aquatic ecosystems is well documented. Algae have been identified (Reference VI1-1) as being important in the initial 7 This confirms data from personal communication between A. L. Young with the Department of Life and Behavioral Sciences, United States Air Force Academy, Colorado, 1973. Reference: VII-1. Shields, L. M., and L. W. Burrell, 1964. Algae in Relation to Soil Fertility, Bot. Rev. 30:90-128. 135 �TABLE VII-4. AVERAGE NUMBER OF ORGANISMS PER GRAM OF SOIL FOR EACH GRID LOCATION SAMPLED (0 - 6 INCHES DEPTH) 1973 GRID LOCATION ACTINOMYCETES BACTERIA FUNGI C-13 394,000 870,000 58,750 G-13 366,670 722,500 30,000 G-7 255,000 630,000 24,375 H-8 290,000 980,000 60,000 J-9 360,000 575,000 39,909 K-2 285,000 428,000 55,000 L-6 353,330 ND 20,710 0-7 363,000 468,000 32,166 TABLE VII-5. AVERAGE NUMBER OF ORGANISMS PER GRAM OF SOIL FOR EACH GRID LOCATION SAMPLED (0 - 6 INCHES DEPTH), 1970 GRID LOCATION ACTINOMYCETES BACTERIA FUNGI C-13 440,000 ND 23,400 G-13 612,000 ND 78,750 G-7 460,000 173,000 74,500 H-8 536,000 224,000 27,500 J-9 21,465 ND 17,968 K-2 54,333 29,000 11,366 L-6 276,000 ND 10,315 0-7 NOT SAMPLED IN 1970 136 �TABLE VII-6. AVERAGE NUMBER OF ORGANISMS PER GRAM OF SOIL (0 - 6 INCH DEPTH), VEGETATIVE COVER, 1973 VEGETATIVE COVER ACTINOMYCETES ~\ BACTERIA FUNGI 5 310,000 1,015,000 50,000 4 300,000 1,070,000 110,000 722,500 30,000 3 1,860,000 2 283,330 890,000 32,166 1 357,000 416,000 30,800 0 326,360 529,000 25,800 CONTROL 5 235,000 ND 48,370 CONTROL 4 370,000 810,000 55,000 CONTROL 3 303,000 740,000 51,000 TABLE VII-7. AVERAGE NUMBER OF ORGANISMS PER GRAM OF SOIL (0 - 6 INCH DEPTH), VEGETATIVE COVER, 1973 VEGETATIVE COVER ACTINOMYCETES BACTERIA FUNGI 5 460,000 173,000 74,500 5 612,000 3 536,000 2 1 ND 78,750 440,000 224,000 ND 23,400 54,333 29,000 11,366 0 21,465 ND 17,968 0 276,000 ND 10,315 137 27,500 �TABLE VI 1-8. WATER CONTENT OF TEN C-52A SOIL SAMPLES (0 TO 6 INCH DEPTH) SAMPLE VEGETATIVE COVER GRID LOCATION PERCENT WATER 1 5 G-13 1.22 2 1 C-13 0.45 3 5 C-13 0.63 4 3 G-13 0.52 5 1 C-13 0.48 6 0 K-2 0.35 7 0 L-6 0.40 8 1 J-9 0.44 9 0 0-7 0.41 10 1 J-9 0.47 TABLE VII-9. ORGANIC MATTER OF SIX C-52A SOIL SAMPLES (0 TO 6 INCH DEPTH) SAMPLE VEGETATIVE COVER GRID LOCATION PERCENT ORGANIC MATTER 1 0 K-2 0.75 2 0 J-9 0.81 3 1 J-9 4 1.19 G-13 4 a a 5 5 C-13 6 3 G-13 a 2.58 4.29 1.88 Samples taken from directly beneath a clump of panicum grass and contained root material. 138 �ecological succession of barren areas. Other investigations (Reference VI1-2) have indicated the effects pesticides have on algae (Reference VI1-3). Grab samples were obtained from the pond located near the one square mile grid. Two types of samples were collected: Sample One was a collection of the suspended and precipitated algal material in the pond and Sample Two was a collection of the dense algal mat which occurred just beneath the surface of the water. The one liter samples were returned to the laboratory for algal genera identification. Seven genera of algae were identified from the samples collected (Table VI1-9). All genera were present in both samples: Sample Two being predominantly Zygnema and Sample One being predominantly Zygnema and Tripjoceras. The seven genera represent two divisions, Chlorophyta, the green algae, and Chrysophyta, the yellow-green or yellow-brown algae. Data collected during the course of this study included some physical data. Of interest specifically with respect to the aquatic algae is the pH which was found to be 5.51, or slightly acid. Genera represented in the samples collected are those expected to be found under conditions of this type^. The previous study, conducted from September to November 1967 was more extensive than the current study and encompassed the periodicity of algal species with time. In the previous study, genera representative of two divisions were found. Representative of Chlorophyta (green algae) and Cyanophyta (blue-green algae) were identified. The present study, based on a single collection time, would be expected to identify less diversity of orders and families. No genera were found to be common to the two studies. The previous study, however, addressed only Chlorophyta (green) and Cyanophyta algae (blue-green). These data do not however necessarily indicate changes in algal populations as the previous.study dealt exclusively with soil populations. 7. CONCLUSIONS In tests performed 3 years after the last application of 2,4-D/2,4,5-T herbicide, the Test Area C-52A grid, Eglin AFB Reservation, exhibits a population level of soil microorganisms identical to that in an adjacent control area of similar soil and vegetative characteristics not exposed to massive quantities of herbicide. There are increases in Actinomycete and bacteria populations in some test site areas over levels recorded in 1970. This is possibly due to a general increase in vegetative cover for those sampling sites and for the entire test grid. No significant permanent effects could be attributed to the presence of herbicides. Data on aquatic algae populations from ponds previously exposed to repetitive applications of herbicides indicate that the genera present are those expected in warm, acid (pH 5.5), seepage, or standing waters. o °Personal Communication with R. Lynn, Utah State University, Department of Botany, Logan, Utah, 1973. References: VII-2. Schluter, M. 1966. Investigations of the Algacidal Characteristics of Fungicides and Herbicides. Int. Rev. Gestamten Hydrobiol. 51:521-541. VII-3. Wolf, F. T. 1962. Growth Inhibition of Chlorella Induced by 3-amino, 1,2,4, Triazole, and its Reversal by Purines. Nature, 193:901-902. 139 �TABLE VII-10. AQUATIC ALGAE* FROM PONDS OF TEST AREA C-52A DIVISION CLASS ORDER FAMILY GENERA Chlorophyta Chlorophyceae Zygnematales Desmidiaceae Closterium Cosmarium Triploceras Chlorophyta Chlorophyceae Zygnematales Zygnemataceae Z.ygnema Chlorophyta Chlorophyceae Tetrasporales Palmellaceae Asterococcus Chrysophyta Bacillariophycaeae Pennales Suborder: Fragilarineae Fragilariaceae Asterionella Chrysophyta Bacillariophyceae Navjculaceae Navicula Naviculineae *Smith, G. M. 1950. The Fresh-Water Algae of the United States. McGraw-Hill. 2nd. Ed. 709 pp. �INITIAL DISTRIBUTION AFSC (SOW) AFSC (VN) AF (RDP) DDR&E (Env & Life Sciences) DDR&E (Tech Lib) USA FA (DFLS) AF (SAFILE) AFLC (MMNO) AFLC (DS) DDC Deseret Test Center (Tech Lib) EA (SAREA-TS-L) (Tech Lib) EA (SAREA-CL-V) USAMC (AMCRD-WB/AFSC-SDWC) OOAMA (MMNO) SAAMA (SFQT) USNWC (Tech Lib) USNWL (Tech Lib) USDA (Agriculture Resch Serv) CINCPAC (J3A1) USAF(Environ Health Lab) ARL (ARC) USDA (ARS) USAF (SAMI) ASD (ENYS) AUL (AUL-LSE-70-239) TRADOC (TAWC-DO) AFATL (DL) AFATL (DLOSL) AFATL (DLIF) 2 2 2 10 5 200 10 2 10 12 5 2 5 10 2 2 2 2 25 5 5 5 5 1 1 1 1 1 2 100 141 (The reverse of this page is blank) �� 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 010 Folder The folder containing the original item. 0090 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 Young, Alvin L. Description An account of the resource <strong>Corporate Author: </strong>Environics and Flame Munitions Branch, 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 1974-01-01 Title A name given to the resource Ecological Studies on a Herbicide-Equipment Test Area (TA C-52A) Eglin AFB Reservation, Florida; Final Report: January 1967 to November 1973 Subject The topic of the resource ecological impact herbicide application dioxin https://www.nal.usda.gov/exhibits/speccoll/files/original/37800aa88b67610cc31f3dc0000978c7.pdf a1031b643d1d5aae74d7bc08affff15a PDF Text Text Item ID Number: Author Corporate Author 00104 Young, Alvin L. USAF School of Aerospace Medicine/Epidemiology Division and the USAF Occupational and Environmenlal Health Laboratory, Brooks AFB, Texas Roport/Artitio TltlO Persistence, Bioaccumulation and Toxicology of TCDD in an Ecosystem Treated With Massive Quantities of 2,4,5-T Herbicide Journal/Book Title Year 1979 Month/Day September Color Number of Images 24 DOSOrlptOn NOtOS SAP/CIS Document 791336; AFSC Document 79-890 Friday, December 08, 2000 Page 104 of 106 �PERSISTENCE, BIOACCUMULATION AND TOXICOLOGY OF TCDD IN AN ECOSYSTEM TREATED WITH MASSIVE QUANTITIES OF 2,4,5-T HERBICIDE ,. C. E. Thalken and D. D. Harrison* USAF School of Aerospace Medicine/Epidemiology Division and the USAF Occupational and Environmental Health Laboratory Brooks Air Force Base, Texas Abstract and Presentation Symposium on the Chemistry of Chlorinated Dibenzodioxins and Dibenzofurans DIVISION OF PESTICIDE CHEMISTRY AMERICAN CHEMICAL SOCIETY 178th ACS National Meeting 1 3 - 1 4 September 1979 Washington DC * Address Inquiry: Major Alvin L. Young, USAF, Ph.D. Environmental Sciences Consultant Epidemiology Division USAF School of Aerospace Medicine Brooks AFB, San Antonio, TX 78235 Phone: 512 536-3471/2411 SM-/OIS noriFn.NT 7913.36 AFSC nnCUMRN'T 7P-890 ��ABSTRACT PERSISTENCE, BIOACCUMULATION AND TOXICOLOGY OF TCDD IN AN ECOSYSTEM TREATED WITH MASSIVE QUANTITIES OF 2,4,5-T HERBICIDE* A. L. Young, C. E. Thalkcn and D. D. Harrison USAF School of Aerospace Medicine/Epidemiology Division and the USAF Occupational and Environmental Health Laboratory Brooks Air Force Base, Texas 2 Field investigations were conducted during 1973-1978 on a 3.0 Km military test area (Test Area C-52A, Eglin Air Force Base, Florida) that received 73,000 kg 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) herbicide during the period 19621970. No residues of 2,4,5-T were detected (detection limit of 10 ppb) in any soil samples collected during 1971-1972. However, residues of the contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were still present in 1978. During the period 1974-1978, 54 soil samples were collected to a depth of 15 cm from thoughout the test area. per trillion (ppt). TCDD levels ranged from <10 to 1,500 parts The median concentration was 30 ppt while the mean was 165 ppt. The ecological survey extending over a five-year period documented the * presence of at least 123 different plant species, 77 bird species, 71 insect families, 20 species of fish, 18 species of reptiles, 18 species of mammals, 12 species of amphibians and 2 species of mollusks. At least 250 biological samples were analyzed for TCDD, including 30 species of animals. found in any of the plant species examined. No TCDD was However, TCDD was found in nine species of animals including two rodent species: beachmouse (300-2,^-00 ppt, liver) and hispid cotton rat (<10-210 ppt, liver); three species of birds: meadowlark (100-1,020 ppt, liver), mourning dove (50 ppt, liver), and Savannah *Abstract, American Chemical Society, Division of Pesticide Chemistry, Washington, D.C., 10-15 September 1979. �-2- sparrows (69 ppt, liver); three species of fish: spotted sunfish (85 ppt, liver) mosquitofish (12 ppt, whole body) and sailfin shinner (12 ppt, whole body), and one reptile, the six-lined racerunner (360-430 ppt, muscle). Gross pathology was done on all species collected for TCDD residue analyses. I-Iistopathological examinations were performed on over 300 buach- mice or hispid cotton rats from the test area and a control field site. Examina- tions were performed on the heart, lungs, trachea, salivary glands, uhymus, liver, kidneys, stomach, pancreas, adrenals, large and small intestine, spleen, genital organs, bone, bone marrow, skin and brain. Initially, the tissues were examined on a random basis without the knowledge of whether the animal was from a control or test area. All microscopic changes were recorded, including those interpreted as minor or insignificant. The tissues were then reexamincd on a control and test basis, which demonstrfited that the test and control mice could not be distinguished histopathol^gically. Similar histo- pathological studies were conducted on the.- fish and racerunner, and again no significant abnormalities were found. �PERSISTENCE, BIOACCUMULATION AND TOXICOLOGY OF TCDD IN AN ECOSYSTEM TREATED WITH MASSIVE QUANTITIES OF 2,4,5-T HERBICIDE A. L. Young, C. E. Thai ken and D. D. Harrison USAF Occupational and Environmental Health Laboratory Brooks Air Force Base, Texas Concern over the level of contamination of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) herbicide by the teratogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has prompted discussion on the safety of using 2,4,5-T in forest and rangeland environments. Although numerous reports have recently appeared in the scientific literature, most of these deal with effects of 2,4,5-T and TCDD in laboratory systems. In general the effects and mode of action of TCDD on laboratory animals can be characterized by a relatively small number of clinical signs. It is reported that a single oral dose (25 ug TCDD/kg) caused an actual weight loss for one week in young female rats and young male rats receiving the same dose had significantly decreased weight gain over a two week period. Slight thymic atrophy, related to TCDD dose levels, was a common finding in young mice receiving a single oral dose (50 ug TCDD/kg), while severe thymic atrophy in young mice receiving a single oral dose of TCDD (150 jug TCDD/kg) or four separate oral doses (25 ug TCDD/kg x 4) was reported. A single oral close of TCDD (50 jjg TCDD/kg) in young adult rats and (3>ig TCDD/kg) in young guinea pigs caused severe thymic atrophy. At these same dose levels slight to severe centrilobular liver necrosis and degeneration of parenchymal cells in mice, rats and guinea pigs, together with ceroid pigment deposits and hepatic �porphyria in mice given four oral doses of TCDD (25 ug TCDD/kg) at weekly intervals were seen. Acute death in guinea pigs has occurred following a single (3 "jug TCDD/kg) oral dose of TCDD. A recent report indicated that four doses of TCDD (25 iig TCDD/kg) given at weekly intervals to young mice induced the production of <S-aminolevinic acid (ALA) synthetase and hepatic porphyria. Laboratory data for rodents strongly suggest a correlation between histological lesions in the liver and lymphatic system and the amount of TCDD ingested. Unfortunately, data relating to any actual effects on wild populations in their natural habitat are lacking. The problem of finding a field site where a wild population of rodents has been exposed to significant quantities of TCDD is improbable because of (1) low levels of TCDD «0.1 ppm) found in currently produced phenoxy herbicide, and (2) low rates of 2,4,5-T applied for brush control on rangelands or for reforestation (1.1 to 2.2 kg/ha). This report, however, documents the effects of residual TCDD on selected animal populations inhabitating a unique test site: a site previously treated with massive quantities of 2,4,5-T herbicide and located on the Eglin Air Force Base Reservation, Florida. The Eglin Reservation has served various military uses, one of them having been development and testing of aerial dissemination equipment in support of military defoliation operations in Southeast Asia. It was necessary for this equipment to be tested under controlled situations that would simulate actual use conditions as near as possible. For this purpose an elaborate testing installation, designed to measure deposition parameters, was established on the Eglin Reservation with the place of direct aerial application restricted to an area of approximately 3.0 km2 within Test Area C-52A in the southeastern part of the reservation. Massive quantities of herbicide, used in the testing of aerial defoliation spray equipment from 1962 through 1970, were released and S �fell within the instrumented test area. The uniqueness of the area has promoted continued ecological surveys since 1967. As a result, few ecosystems have been so well studied and documented. MATERIALS AND METHODS Description of Field - Test Area C-52A (TA C-52A) covers an area of approximately eight square kilometers and is a grassy plain surrounded by a forest stand that is dominated by longleaf pine (Pinus palustris), sand pine (Pinus clausa), and turkey oak (Quercus laevis). The actual area for test operations occupies an area of approximately three square kilometers and is a cleared area occupied mainly by broornsedge (Andropogon virginicus), switchgrass (Panicurn virga turn), woolly panicum {Panicum lanuginosum) and low growing grasses and herbs. Much of the center of the range was established prior to 1960, but the open range as it presently exists was developed in 1961 and 1962. The test grid is approximately 28 m above sea level with a water table of six to ten feet. The major portion of this test area is drained by five small creeks whose flow rates are influenced by an average rainfall of 150 cm. The mean annual temperature for the test area is 19.7°C while the mean annual relative humidity is 70.8 percent. For the most part, the soil of the test grid is a fine white sand on the surface, changing to yellow beneath. The soils of the range are predominantly well drained, acid sands of the Lakeland Association with 0 to 3 percent slope. A typical three-foot soil core contained approximately 92 percent sand, 3.8 percent silt, and 4.2 percent clay with an organic matter content of 0.17 percent, an average pH of 5.6, and a cation exchange capacity of 0.8. �Although the total area for testing aerial dissemination equipment was approximately 3,0 km^, the area actually consisted of four separate testing grids. The primary areJwas located in the southern portion of the testing area and consisted of 37 ha instrumented grid. This was the first sampling grid and was in operation in June 1962. It consisted of four intersecting straight lines in a circular pattern, each being at a 40° angle from those adjacent to it. Although this grid was discontinued after two years it received the nost intense testing program. From 1962 to 1964, this grid (called Grid I) received 39,550 kg of 2,4-dichlorophenoxyacetic acid (2,4-D) and 39,550 kg of 2,4,5-T. The herbicide was disseminated as the water insoluble n-butyl and iso-butyl esters (their military code names were Orange and Purple). Two other testing grids were sprayed with 2,4,5-T. Grid II was an area of 37 ha and located immediately north of Grid I. Grid II received 15,890 kg 2,4,5-T from 1964 through 1966. Grid IV was the latest and final Grid established on Test Area C-52A. It was approximately 97 ha and received 17,440 kg 2,4,5-T from 1968 through 1970. Both\ Grids II and IV received Herbicide Orange (a 50:50 mixture of the n-butyl esters of 2,4,-D and 2,4,5-T). Despite excellent records as to the number of missions and quantity of herbicide per mission, there was no way to determine the exact quantity of herbicide deposited at any point on the instrumented grid. The first extensive soil sampling for residues of herbicides was initiated in 1969 for Grid I (five years after the last mission) and in 1970 for Grids II and IV. At that time traces (parts per billion) of 2,4,5-T were detected in soils of Grid I and in parts per million for Grids II and IV. Analyses for TCDD were initiated in 1972. By midsummer 1973 analysis of soil samples indicated that TCDD was detected only in the top 15 cm of soil (e.g.., analysis of soil cores at 15 cm increments to a depth of one meter indicated no detectable TCDD in increments �below 15 cm. Therefore, fifty four sites on three Grids were sampled for TCDD in the top 15 cm increment. One of the sites was also subsampled at increments of 0-2.5, 2.5-5, 5-10 and 10-15 cm. Analysis of soil samples for TCDD was accomplished by three different commercial laboratories. Vegetation - Detailed studies of the vegetative composition of Test Area C-52A were conducted in 1971, 1973 and 1976. Transect analyses were conducted on all test grids. Frequencies and densities of monocotyledonous and dicotyledonous species were determined. Representatives of all plant species were collected and mounted. Photographic records of numerous sites were maintained through the years of study. Animals - Studies of the animals began in 1970. However, detailed investi- gations of the beachmouse, hispid cotton rat, and six-lined racerunners were conducted in 1973 and 1974. The beachmouse was further studied in 1975 and 1978. The birds were studied in 1974 and 1975. The insect studies were conducted in 1971 and 1973, while the aquatic communities were initially examined in 1970 and again in 1973 and 1974. List of species, description of habitats and diets and residue analysis were conducted throughout all years of study. RESULTS Slides: 1. Test Area C-52A 2. Application Rates of 2,4,5-T 3. Types of Ecological Investigations 4. Survey of Species 5. Concentration of TCDD in Test Grid Soils 6. Disappearance of TCDD from Soils of Grid I 7. Investigations of Bird Species 8. Investigations of Insect Families �9. Investigations of Mammals 10. Studies of the Beachmouse - Control Area 11. Studies of the Beachmouse - Test Grid I 12. Mean Liver Weights of Pregnant Females 13. Histopathological Examination of Beachmice DISCUSSION The data suggest that TCDD may persist for long periods of time in the environment. However, caution must be exercised in making such a statement. As noted from the slides, it was probable that Grid I received highly contaminated herbicide. The herbicide was most likely produced in the 1950s or early 1960s and thus was subjected to preparation treatment different from those controlled procedures subsequently used. A conservative estimate for TCDD contamination may be 8 ppm in the formulation. Using the 8 ppm figure for all of the herbicide applied to Grid I, then the amount of TCDD applied would have been at a concentration equal to 12,267 ppt TCDD in the top 15 cm of soil. At least, this has declined to 710 ppt in about 8 years. This is a loss of about 95 percent. Thus, the apparent high residue is probably due to the massive quantities applied rather than to the resistance of TCDD to biological and/or physical degradation. The levels of TCDD in the livers of beach mice and birds collected from the test grids substantiated bioaccumulation of TCDD; i.e., an accumulation of TCDD jjl an organism from its environment. In general, levels of TCDD in the livers were no greatg1 than the most concentrated zones of TCDD in the soil; there are no data from this study to support biomagnification of TCDD; i.e., an increase in concentration of TCDD in successive organisms ascending the trophic food chain. �The only significant lesions seen on histopathologic examination of 255 adult and fetal beach mice were two instances of moderately severe multifocal, necrotizing, hepatitis and a single mouse with severe venous ectasia of the renal veins in one kidney. All other lesions were of the minor or insignificant type, normally observed in microscopic surveys of large numbers of field animals. The absence of liver lesions (necrosis and porphyria) in animals that had liver levels of TCDD from 300 ppt to 1,500 ppt is most significant in view of the massive quantities of both 2,4,5-T and TCDD that must have been applied to the test site. Moreover, a report of a previous study of this area, which terminated in the summer of 1970, indicated that a significant population of beach mice were inhabiting the test site. The average life-span of a related species, Peromyscus mam'culatus, has been recorded to be less than five months and only a few mice lived the full potential of three or more years. A single female beach mouse is capable of producing eighty or more young under laboratory conditions with litters being born at approximately 26 day intervals. It is further reported that beach mice on Santa Rosa Island, Florida(within 32 km of Test Area C-52A), may have produced 10 generations per year. At this frequency the animals collected in 1974 on Grid I may be 40 generations removed from the population first noted in 1970. However, a more conservative estimate would be 6 generations per year (giving a female 60 days to reach sexual maturity), for a total of 24 generations. It must be stressed that the populations of beach mice noted in 1970 were probably subjected to much greater levels of residual TCDD in the soil than those animals collected in subsequent years. The absence of pathological signs in these mice indicated that TCDD was neither mutagenic (somatic or germinal) nor carcinogenic in the field at the concentrations noted and during the life �span of the animals studied. Since none of the 67 fetuses examined from animals captured on the test grid showed teratogenic defects it must also be concluded the levels of TCDD encountered failed to induce observable developmental defects. �TEST AREA C-5? A EGLIN AIR FORCE BASE, FLORIDA , A TEST RANGE USED IN THE DEVELOPMENT OF DEFOLIATION SPRAY EQUIPMENT FOR SOUTHEAST ASIA , HERBICIDFS SPRAYED ON THE TEST AREA, 1962-1970, �Yu-Rraph 2 TEST GRIDS AND QUANTITIES OF 2,4,5-T APPLIED TO TEST AREA C-52A, EGLIN AFB FL GRID AREA (HA) YEARS 2,4,5-T (KG) 1 II 37 1962- 1964 39, 550 37 1964- 1966 15, 890 IV 97 1968- 1970 17,440 TOTAL 72, �Vu-tyrapu i ECOLOGICAL INVESTIGATIONS, TEST AREA C-52A 1973 - 1978 SOIL RESIDUES: 2,4,5-T, TCDD TERRESTRIAL ECOSYSTEMS VEGETATION ANIMALS VERTEBRATE INVERTEBRATE MICROORGANISMS AQUATIC ECOSYSTEMS ANIMALS VERTEBRATE INVERTEBRATE MICROFLORA �Ya-r-raph ECOLOGICAL SURVEY, 1973 - 1978 TEST AREA C-52A NUMBER OF SPECIES 123 77 71 20 18 18 12 2 ORGANISMS PLANTS BIRDS INSECT FAMILIES FISH REPTILES MAMMALS AMPHIBIANS MOLLUSCS 170 BIOLOGICAL SAMPLES ANALYZED FOR TCDD �Yu-* CONCENTRATION OF TCDD (PPT) IN TEST GR1D SOILS GRID NO. SAMPLES" RANGE MEDIAN MEAN I 22 <10 - 1,500 110 326 II 6 < 10 - 470 30 117 IV 26 <10 - 150 19 27 * 0 - 1 5 C M INCREMENT �Vu--; ra;)li 6 DISAPPEARANCE OF TCDD FROM SOILS OF GRID I (PARTS PER TRILLION) PLOT* NUMBER AUGUST 1974 JANUARY 1978 1 1,500 420 2 610 300 3 1,200 580 4 270 100 5 440 400 MEAN 804 360 •FIVE SUBSAMPLES FROM EACH 1-M2 PLOT COMPOSITED (0-10 CM DEPTH) �INVESTIGATIONS OF BIRD SPECIES Test Area C-52A 77 Species Observed DOMINANT SPECIES TCDD RES I DUE ANALYSIS (PPT) No. Samples* 'Composites from at least 6 birds Liver 100-1,020 Stomach 10 2 Liver 50 Stomach 10 1 Liver 69 1 Savannah Sparrow 3 1 Mourning Dove Range 1 Southern Meadow/lark Organ Stomach 84 Mean 440 �Vu-srmh INVESTIGATIONS OF INSECTS Test Area C-52A 71 Families Observed FAMILY TCDD Residue Analysis (ppt) Grasshoppers ND (3)* Crickets 26 Composite of Soil/Plant Insects 40 * Detection Limit �Vu-graph i' INVESTIGATIONS OF MAMMALS, TEST AREA C-52A SPECIES TCDD RES I DUE ANALYSIS (PPT) ORGAN CONCENTRATION DETECTION LIMIT DEER FAT LIVER KIDNEY ND ND ND 4 5 4 OPOSSUM FAT LIVER ND ND 10 10 RABBIT LIVER PELT ND ND 8 2 COTTON RAT LIVER 10 - 210 BEACHMOUSE LIVER PELT 300 - 1, 500 130 - 140 �10 STUDIES OF THE BEACHMOUSE, PEROMYSCUS POL10NQTUS Grid 1, Test Area C-52A, Eglin AFB FL LOCATION Maturity, Sex YEAR 1973 1974 1975 1978 Tota_l_ CONTROL AREA Mature Male Female Immature Male Female Fetuses 4 3(3) 11 8(3) 3 3(1) 2 2(2) 1 0 1 2 0 0 0 0 2 2 12 11 3 5 31 Total ( ) Number of Pregnant Females Fetuses/Pregnancy = 3.4 20 16(9) 71 �C \u- T r.-,i>. Li STUDIES OF THE BEACHMOUSE, PEROMYSCUS POLIONOTUS Grid 1, Test Area C-52A, Eglin AFB FL LOCATION Maturity, Sex TEST GRID 1 Mature Male Female Immature Male Female Fetuses 1973 18 15(6) 8 1 25 YEAR 1974 1975 14 9(6) 3 4 9 1978 7 6(6) 7 3 12 46 36(22) 6 3 21 7 6(4) Total ( ) Number of Pregnant Females Fetuses/Pregnancy = 3.1 Total 24 11 67 184 �Vu-n, rrnn 12 MEAN LIVER WEIGHTS (MG) OF PREGNANT BEACHMICE TEST AREA C-52A LOCATION YEAR Control 1973 1974 LIVER WEIGHT (MG) 1975 1978 Gridl 929 765 934 919 1973 1974 1975 1978 1,247 1,019 1,109 1,101 STATISTICALLY SIGNIFICANT! �Vu-praph 13 HISTOLOGICAL PARAMETERS HEART LUNGS TRACHEA SALIVARY GLANDS THYMUS LIVER KIDNEYS STOMACH PANCREAS ADRENALS LARGE/SMALL INTESTINE SPLEEN GENITAL ORGANS BONE BONE MARROW SKIN BRAIN ALL MICROSCOPIC CHANGES RECORDED. TEST AND CONTROL MICE COULD NOT BE DISTINGUISHED. � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 012 Folder The folder containing the original item. 0104 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 Young, Alvin L. C.E. Thalken D.D. Harrison Description An account of the resource <strong>Corporate Author: </strong>USAF School of Aerospace Medicine/Epidemiology Division and the USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas Date A point or period of time associated with an event in the lifecycle of the resource 1979-09-01 Title A name given to the resource Persistence, Bioaccumulation and Toxicology of TCDD in an Ecosystem Treated With Massive Quantities of 2,4,5-T Herbicide Subject The topic of the resource soil survey animal testing Eglin AFB dioxin https://www.nal.usda.gov/exhibits/speccoll/files/original/dd4280761c30ba23ac1c8e04d3d1a0a7.pdf 72b4a89c20932245745139584e7e6195 PDF Text Text 00187 Young, Alvin L. USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas ReOOrt/ArtJCiB Tlth Herbicide Orange Site Treatment and Environmental Monitoring: Summary Report and Recommendations for Naval Construction Battalion Center, Gulfport, MS 1979 November Color n 46 Friday, January 05, 2001 Page 187 of 194 �Report OiHL HERBICIDE ORANGE SITE TREATMENT AND ENVIRONMENTAL MONITORING SUMMARY REPORT AND RECOMMENDATIONS FOR NAVAL CONSTRUCTION BATTALION CENTER GULFPORT MISSISSIPPI November 1979 Approved for public release; distribution unlimited USAF Occupational and Environmental Health Laboratory Aerospace Medical Division (AFSC) Brooks Air Force Base, Texas 78235 �S11H TREATWHf MID ENVIRQHiffililAL MQHITORIMG AND FOR NAVAL CONSTRUCTION BAOTM.ION November 1979 . Prepared for AIR FORCE IiOGISTICS COMMAND WB OH ��- .. JECURlTV Ct A»riCATiON OF THi« SEAS INSTRUCTIONS BEFORE COMPt-ETlNG FORM REPORT DOCUMENTATION PAGE 2, OOVT ACCfSflON NO. «fHT*S CATALW tttttllf• OIHL-fR-79-169 S. TYPE OF REPORT * CEHlOO COVEHIO *. TITLE Herbicide Orange Site Treatment and Environmental final Monitoring* Sawary leport and !eeoiui»ndatlons for Naval Construction Battalion Center, i, PBMPemuita ens. RSPDMT Gulfport MS I CONIHACT 9» HAIT . Alvin L. Young, Major, USAF Charlea 1. fsalkan, Lieutenant Colonel, USA?,-VC Williaa J. Gainsay, Major, QSAF, BSC onaAiiiAfiON MMII *«§ USAP Occupational and Environmental Health laboratory iroofes Mr Force Base, Itoas 78235 ^ ^ i, g^/i,jimraMis' I, ItiPQllT BAT• I H, COttT«Ot.lilN« OF^tet NAMI ANO ABPflltll USAF Occupational and Environmental Health Laboratory Brooks Mr Force Base, Texas 78235 Moveaber 197! 36 Ti, »OllrTOlHS8 AOSNCY MAMI ft AODMBSSfJl dilftt^i Irom Ooatrenini Olllef) !•• MCUWTT CUASI, f*f Unclassified It. ITATIMtNT f*l » for public release *, distribution unlimited •>, DtlTNilUTION STATSM8NT (el tfe* *fe«if*«t fnitrti in Mae* M, I lUPPLEMIMTAIiy NOTIt , I. KEY WONBI CCenltniM MI tmmtm «)<*• // n»s««»«ry «nd id«n(//y by M«eft nu»b«o aquatic studies bioaeovmulation biod»fradation of herbicides biodegradation of TCDD chlorinated phenols ecological effects 2,4-dicnlorophenoxyacetic acid {2,4-D) environmental aonitoring herbicides Herbicide Orange dloxin Orange phenos^ herbicides PACER HO 0, ABfrftACT fCenthMM on «»««• *!4t It n*a***«y »nd ia»nlHy kr Moo* mambtr) Snviroiu»ntal surveys of the soils, plants and the aquatic system in and around a 12-acre Herbicide Orange storage area at Gulfport MS were conducted from 1970 through 1979. The major objectives of the surveys were to (1) determine the magnitude of Herbicide Orange contamination on the storage area| (2) determine the fate of the phenoxy herbicides 2,4-D and 2,4,5-T, their phenolic degradation products and fCDD in soils of the storage area?{3) monitor movements of residues from the storage area into adjacent water, sediments and biological organisms! and (4) recommend managerial techniques for niniaizing the impact EDITION Of 1 NOV ft IS OMOLETE Unclassified CtAStlfICATtOM Or THIS PAOC �HCURtTy CI.»SSIFIC*"nOH OF THJkf AqgftHJMQ D»t* soil microbial studies TCDD 2,3»7,8HMtr«ehlorodibenso-p-dloxin (TCDD) 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) 20. of the herMcldes and TCDD residues on the ecology and human populations adjacent or near the storage area. High levels"of TCDD (e.g., 100-200 parts per billion [ppb]) were associated with spill sites on the herbicide storage area. Sediment samples from the storage area contained 2.7 to 3.6 ppb TCDD and biological organisms closely associated with the sediment contained 0.14 to 7.2 ppb TCDD. Water staples collected in the same area were negative £or TCDD at a detection level of 0,02 ppb. Two of five off-base samples were positive for TCDD (ft crayfish and a sediment sample both contained 0.02 ppb TCDD). The primary recommendation is that the 12-acre Herbicide Orange storage area be left undisturbed permitting the continuation of "natural" degradation of the herbicides and TCDD. It is recommended that the area be restricted and that efforts be immediately undertaken to minimize future erosion of contaminated soil into the ditches. The prevention of soil and silt movement from the area may be accomplished by stabilizing the ditch banks, constructing silt catchments within the ditches and constructing a silt retaining pond prior to the stream leaving the NCBC. Unclassified SECURITY CLASSIFICATION OF THIS PAQEfffftwi Dm* �PURPOSE The report was prepared to present senior Mr Force leaders the latest available data In the continuing environmental monitoring studies of a 12-acre storage area on the Naval Construction Battalion Center (NCBC), Gulfport MS, ftie area had been used for the long-term storage of approximately 8 0 0 0 gallons of Herbicide Orange from mid-1968 to 4,0 mid-1977, SASIC HISTORY . Since 1970,, various Air Force and contract laboratories have been conducting environmental surveys and analyses of the soils, plants, and the aquatic system in and around the Herbicide Orange storage area. As some leaking became evident and as more information became available on the toxic contaminant 2,3,7,8-tetrachlorodibenzo'~p-dioitin (TCDD) contained in the herbicide, more extensive monitoring programs were conducted, fhe entire inventory was redrummed in 1972 and checked for leaks continuously thereafter. In the summer of 1977, the herbicide was transferred to a specially equipped ship and destroyed by at-sea incineration dwinf Project PACER HO. fhe Air Force Plan and the 1PA permits for the disposal of the herbicide committed the Air Force to a follow-on storage site reclamation and environmental monitoring program, fhe major objectives of this program were to (1) determine the magnitude of Herbicide Orange contamination in the storage area; *Updated to include data received 3 Dec 1979 subsequent to report preparation, �(2) determine the soil persistence of the pheonxy herbicides 2f4-dichJ.orophenoxyacetic acid (2,4-D) and 2,4,5-T, their phenolic degradation products and TCDD in soils of the storage area; (3) monitor for potential movement of residues from the storage area into adjacent water, sediments and biological organisms; and (4) recommend managerial techniques for minimizing any impact of the herbicides and TCDD residues on the ecology and human populations adjacent or near the storage area. STORAGE SITE CWTAMIHATIOti AND FATE The monitoring approach used to determine storage site contamination consisted of analyzing soil samples selected from 42 different sites within the storage area. Sampling points were selected in groups depending upon whether a spill of the herbicide had occurred in that area or not. Previous studies had shown that residue did not appreciably move within the acid soil or significantly penetrate the impervious concrete-stabilized hardpan located approximately six inches below the soil surface. Soil samples were also analyzed for microorganisms. The results indicated that approximately 15% of the 12-acre site is significantly contaminated with Herbicide Orange and TCDD. Levels of 2,4-D and 2,4,5-1" in the samples, which were greater than 100,000 parts per million (ppu) in July 1977, have decreased to one-third that level in IS months. Data from spill sites monitored for this same time period also suggested that TCDD levels are decreasing but at a slower rate. The soil penetration of the herbicides was low while penetration of TCDD was negligible. Sterilization of the soil did not occur; rather, certain microflora proliferated under high levels of herbicides. �RESIDUE MOTEMBI1 IMTO ADJACENT AlffiAS To monitor for potential movement of residue from the storage area, soil and biological samples were collected from the drainage ditch directly adjacent to the site. A Novenbar 1978 analysis of this nearby on-base drainage ditch found positive TCDD residues [o.14-3.6 parts per billion (ppb)]. She TCDD movement was presumably caused through soil erosion from the annual (Jan-June) heavy rain season {approximately 60 in). Drainage ditches carry heavy rain from the storage site and other parts of the bas« into Ixsng Beach Canal 11» approximately 9,000 feet from the site. The canal runs from the city of Long Beach through the base carrying municipal surface drainage, and until July 1978, carried treated sewage materials. The canal eventually runs into Turkey Creek approximately 12,000 feet from the storage site. Due to the November 1978 findings, further samples were collected at varying distances from the site in January, February, and June 1979. Following extensive and difficult analyses in contract laboratories, the results were received in September, November, and December 1979, The results confirmed the November 1978 data and indicated slightly higher levels (sediment levels of 1.7-3.6 ppb and biological levels of 0.14-7.2 ppb). Water samples collected in the same area were negative for TCDD at a detection level of 0.02 ppb. TCDD appears to move only as a part of soil sediment. Sediment and biological sauries taken downstream at 3,000, 7,000, 9,000 and 12,000-feet from the site indicated that some TCDD residue was now present but at very low levels. A crayfish collected at 9,000 feet and numerous fish collected at 12,000 feet were analyzed with .032 ppb the highest level detected. This figure of .032 ppb is three times lower than the Pood and Drug iii �Administration suggested maximum permissible level of 0.1 ppb. With present "state-of-the-art" detection limits, readings as low as these in biological samples have only been considered reliable in recent months. RECOMMENDATIONS To control the now verifiable but very low levels of residue, the report recommends the following actions: - Stabilize drainage ditch banks to prevent water erosion during heavy seasonal rainstorms. - Construct siltation traps in the drainage system allowing for greater silt catchment prior to drainage water leaving the base. - Leave the storage area in its present undisturbed state and continue to limit access so that the "natural" degradation of the herbicide and its TCDD continue to occur. - Allow the continued growth of native vegetation in the contaminated storage area and drainage ditches since this plant community inhibits water erosion. - Continue sampling to ensure that preventive actions do control contamination. - Develop follow-on reserach to determine possible methods for returning the storage area to full and beneficial use. iv �PREFACE This technical report represents the culmination of a two-fear environmental monitoring program of an area previously used for the long-term storage of Herbicide Orange at the Naval Construction Battalion Center. The study was conducted by personnel of the United States Mr Force Occupational and Environmental Health Laboratory, Brooks Mr Force Base, Texas and the United States Mr Force Academy, Department of Chemistry and Biological Science, USAF Academy, Colorado. Funds for this program were provided by Air Force Logistics Command through the San totonio Mr Logistics Center, Directorate of Fuels, Kelly Air Force Base, Texas, fhe report was prepared for the Mr Force Logistics Conaand, Wright-Patterson &FB, Ohio. �Acknowledgements Analyses of herbicides, phenols, and soil TCDD were perforaed by Dr B. Mason Hughes, Mr W.H. McClennen, Mr L.H. Wojcik and Mr F,D. Hilematn, Flaranability Research Center, the University of Utah, Salt lake City Uf 84108. The analyses of ethers and isooctyl esters of trichlorophenol and herbicides were conducted by Or E.L. Arnold, formerly of the Clinical Sciences Division, USAF School of Aerospace Medicine, Brooks AFB TX 78235. High resolution GG-MS analysis of TCDD in selected biological and sediment samples was performed by Dr M.L. Gross, Mass Spectrometry Laboratory, University of Nebraska, Lincoln NE 68588. The assistance of Mr Tom Murphy, Epidemiology Division, USAF School of Aerospace Medicine, in statistically analyzing herbicide data is gratefully acknowledged, The assistance of Mrs Joyce Kidd, Secretary to the Vice Commander, USAF Occupational and Environmental Health Laboratory, in typing and editing this report is gratefully acknowledged. WILWMI E. MABSOM, Colonel, USAF, BSC Commander VI �IMTBOOUCTIOII During the sooner'of 1977 the United States Air force (0SAF) disposed of 2.22 million gallons of Herbicide Orange by high temperature incineration at sea. This operation, Project PACER HO, was accomplished under the very stringent criteria set forth in an tF.S. Environmental Protection Agency (IPA) ocean dumping permit. Among the numerous conditions of thi« UPA-approved disposal operation was the requirement for the USAF to conduct extensive environmental and occupational monitoring of the land-transfer/loading operations, shipboard incineration operations and subsequent storage site reclamation and environmental monitoring. Details of the proposed site monitoring programs were documented in April 1977 by the Air Force Logistics Command (AFLC) in a programming plan for the disposal of Herbicide Orange ( ) In this plan, AFLC proposed that 1. soil samples from the storage sites at both the Naval Construction Battalion Center (NCBC), Gulfport MS, and Johnston Island (JI), Pacific Ocean, be Qollnated and analyzed for Herbicide Orange after the completion of transfer operations, These analyses were to aid in the establishment of a •chadule for future monitoring. The site monitoring program would be flexible to requirements generated by construction of any facility on the storage site and would be concluded upon mutual agreement of all agencies involved. In July 1977, following the completion of the PACER HO dedrunming and subsequent site clean-up operations at NCBC, the USAF Occupational and Environmental Health Laboratory (USAF OEHL) initiated an extensive site monitoring program, fhe objectives of this program were: 1. To determine the magnitude of Herbicide Orange contamination on th« storage site. •. �2. To determine the soil persistence of the two phenoxy herbicides contained in Herbicide Orange and a. dioxin contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 3. To monitor for any movement of residues from the site into adjacent water, sediments and biological organisms. 4. To recommend techniques for managing the storage area with the ultimate goal of returning the area to full beneficial unrestricted use. HISTORICAL BACKGROUND (GENERAL) In April 1970, the Secretaries of Agriculture; Health, Education and Welfare; and the Interior, jointly announced the suspension of certain uses of the herbicide 2,4,5-trichlorophenoxyacetic acid ( , , - ) These 2457. suspensions resulted from published studies indicating that 2,4,5-T was a teratogen. Subsequent studies revealed that the teratogenic effects had resulted from a toxic contaminant in the 2,4,5-T, identified as 2,3,7,8-tetrachlorodibenzo-p-diojcin (TCDD). Subsequently, the Department of Defense suspended the use of Herbicide Orange [a mixture of 2,4,5-T and 2,4-diehlorophenoxyacetic acid C2,4-D)] in South Vietnam. At the time of the suspension, the Air Force had an inventory of 1.37 million gallons of Herbicide Orange in South Vietnam and 0.85 million gallons at the Haval Construction Battalion Center, Gulfport MS. In September 1971, the Department of Defense directed that the Herbicide Orange in South Vietnam be returned to the United States and that the entire 2.22 million gallons be disposed of in an environmentally safe and efficient manner. �The 1.37 million gallons were moved from South Vietnam to Johnston Island, Pacific Ocean, for storage in April 1972,' HISTORICAL BACKGROUND (NCBC) Craig (2), in a historical review of herbicides for Southeast Asia noted that the storage of Herbicide Orange became an item of significant importance with the temporary suspension placed on all uses of Herbicide Orange by the Assistant Secretary of Defense on 15 April 1970. Prior to 1970, shipments of herbicides into and out of the Mobile Outport and the Naval Construction Battalion Center were handled in a routine manner. As the herbicide inventory began to accumulate in Southeast Asia, the San Antonio Air Logistics Center, Directorate of Fuels (SA ALC/SF), Kelly AFB TX, discontinued shipments from the port of embarkation to Southeast Asia in 1963 to avoid exposing large quantities of herbicides * to possible damage by enemy action. The SA ALC then had to determine disposition of the product at the port and that scheduled for delivery. Bather than return the product to the manufacturer and suspend delivery to the port, SA ALC decided to arrange for the product to be temporarily placed in storage. Since the Mobile Outport, Mobile AL, was routinely used as the port of embarkation for herbicides, this was the logical place for the temporary storage. It was anticipated at that time that the storage period would be about six months. Herbicides were sent to the Mobile Detachment for storage between April and June 1968, and were removed from storage between September and December 1968. Except for �one shipment to Southeast Asia during September 1968, herbicides removed from this storage site were used only to fill equipment test requirements at Iflin AFB PL. On 26 June 1968 an Interservice Support Agreement was made by and between SA ALC and NCBC, to provide services related to receiving and storing approximately 50,000 18-gauge, 55-gallon drums of herbicide. The agreement was effective for the two-year period 1 July 1968 - 1 July 1970. It was to be reviewed annually by both parties. Input of herbicides to Gulfport began in July 1968. Additional Interservice Support Agreements were made in 1970 and 1972. Storage was considered a better alternative than the return to the manufacturer where storage charges would have been more expensive, lite NCBC agreed to receive and store the drums of herbicide and remove from storage quantities of drums as designated by SA ALC while SA ALC agreed to provide personnel in support of this operation. This was modified in July 1968 to reimburse NCBC for material and supervisory personnel salaries. The Gulfport outside storage area was about two miles from the docks, with convenient access to the railroads. It was fenced and isolated from public traffic. The NCBC provided surveillance personnel as well as a controlled access. It was planned and set up for long-term storage. To provide good drainage, 2 x 6-inch dunnage (creosoted lumber) was laid on a hard surface and drums, positioned horizontally with the bung closure pointing outward, were stacked in double rows, three high, in pyramidal fashion. The number of drums in each single row, bottom to top, was 55, 54, and 53. To allow inspection of the bungs, there was an 18-inch walking space between each double row. �HOC urns the only Continental Onited States (COOTS) storage facility used daring the last half of FT69 and through Pf70. The Mobile Outport intransit storage facility was not used after Deeeatoer 1968 when the last drums of herbicide were moved to NCBC. At the end of FY70 there were 833,855 gallons of Herbicide Orange in storage at NCBC. Except for a small quantity stored at iglin AFB FL for test purposes, Gulfport was the CONUS storage point. & few damaged drums were received at NCBC with leaks around the bung closures because the seals had vibrated loose. In such cases the producer was notified to supply new bung closures. NCBC personnel took the corrective action. Usually the leaks could be stopped by removing the cover and tightening the bung or replacing the bung gasket. When damaged leaking drums were spotted while in storage, they were redrumtad by the people on duty. It was discovered that a herbicide moistened area usually appeared on the drum two or three weeks before noticeable loss occurred, and the contents could be saved by transferring it to a new drum when the damp area was noted. In May 1971, during an inspection of the inventory, it was noted that deterioration of some of the drums had required HCBC personnel to redrum the product. As drums were removed from the stacks, indications of additional leaking drums became apparent. Previously, leaking had been attributed to breakdown of the bung seals used in the drum closures or an occasional seam leak. Now there were indications of leaks starting in the drum surfaces. During 1972, military personnel moved, inspected, and redrummed as required, the entire inventory of approximately 15,400 drums. Thereafter, an intensive drum surveillance program was initiated �in which all drums were routinely inspected and moved or redrummed as required. The drum surveillance program was continued until May 1977 when Project PACER HO began. The observations in 1971 and 1972 that drums were deteriorating prompted AFLC to task the USAF Environmental Health Laboratory (EHL/K) , Kelly MB TX and the Department of Chemistry and Biological Sciences (USAF/DFCBS) , USAFA CO, to undertake a cursory chemical and biological monitoring program of the storage site. & review of these efforts is provided in a subsequent section of this report. DESCRIPTION OF JHEBBICIEB Pour military herbicides were stored for various lengths of time at NCBC. These herbicides were code-named Herbicides Orange, Orange II, Blue and White. Herbicides Blue and White were intermittently stored at NCBC during 1968 and 1969. However, all stores of these materials were shipped to South Vietnam. Since these two herbicides (Blue and White) were only briefly stored at NCBC, site monitoring programs did not include these materials. The herbicide inventory that underwent long-term storage was comprised of primarily Herbicide Orange (approximately 13,855 drums) and a relatively small quantity of Orange II (1,545 drums) . Young, et al. (8) have described these herbicides. 1. Herbicide Orange Orange was a reddish-brown to tan colored liquid, soluble in die se 1 fuel and organic solvents, but insoluble in water. One gallon or Orange theoretically contained 4.21 pounds (Ib) of the active ingredient of 2,4-0 and 4.41 Ib of the active ingredient of 2,4,5-T. Orange was formulated to contain a 50s 50 mixture of the n-butyl esters of 2,4MJ and 2,4,5-T. The percentages of the formulation typically weres �n-twityl ester of 2,4-D free acid of 2,4-D n-butyl ester of 2,4,5-T free acid of 2,4,5-T 49.49 0,13 48.75 1.00 in*rt ingredients {e.g., butyl 0.63 alcohol and ester moieties) 2. Herbicide Orange II Orange II was a formulation similar to Orange with the only difference being the substitution of the iaooctyl eater of 2,4,5-T for the n-butyl e«ter of 2,4,5-T. The physical, chemical, and toxicological properties of Orange II were similar to those of Orange. Orange IX was produced solely by one chemical company. A detailed analyses of the inventory of Herbicide Orange and Orange II stored at NCBC was prepared in 1975 by Hughes, et al. (4) and Fee, et al (3) A summary of manufacturers and TCDD contents is presented in Table 1. SUMMARY Of SAKL? EJiViaOHMENTAL MONITORING PROGRAMS As early as 1970 the Air Force was expressing its concern about the possible adverse environmental impact of the storage of Herbicide Orange at NCBC, Gulfport MS. Environmental scientists from Eglin AFB visited the storage site at the request of SA ALC/SP and conducted an environmental survey of the plant and aquatic animal community in and around the herbicide storage cite. No significant environmental problems were noted at that time. In 1972, members of the OSAP Environmental Health Laboratory, Kelly AFB TX (EHL/K), conducted an environmental survey of the storage area and also found no significant environmental problems. �TJBL! 1. Identification Data on Herbicide Orange Stocks Stored at the Naval Construction Battalion Center, Gulfport MSa Manufacturer Analysis Total Number Transportation b Seepienee of Drums *TCDDC Control No. (TCN) Mo. with Same TCN (ppm) Hercules Co 9 6 8156 0 0 44 01 Hercules Co 9464 8192 001 14 Diamond Co PY9461 7165 0001AA 18 60 14. 2e Diamond Co PY9461 8156 001AA 11 421 8.62f Thompson Hayward Co 9463 8155 X032 8 1 500 2,152 <0.05 n&a 1,546 0.32 0.12 Dow Chemical Co 9463 8155 X052 10 6,976 Thompson Co 9463 7184 X011 3 46 HA Thompson Co 9463 8155 X012 5 808 0.17 Monsanto Co FY9463 7163 X0001XX 4 563 NA Monsanto Co PY9463 8183 X002XX 6 2,185 15,257 a SOURCEs 7.62 Pee, et al. (3). Each separate purchase of herbicide was designated by a separate TCN G Tetrachlorodibenzo-p-dioxin (TCDD) content. Results reported in this column are the average of six samples collected from six different barrels of Herbicide Orange having the same TCN. d Not Analysed. ^Average value of five samples: 12, 17, 12, 15, 15. Other sample value was 0 0 with recheeJcs. .7 ^Average value of four samples: 8.0, 8.1, 8.7, and 9.7. Other two samples each averaged <0.05 with rechecks. *0n the bajis of 280 samples of Herbicide Orange taken from the Gulf port inventory, the weighted mean concentration of TCDD was 2.06 ppm. �In July 1974, members from the OSAF Academy Department of Chemistry and Biological Sciences conducted an extensive survey and ecological assessment of the herbicide storage area and collected soil, water, and biological samples. There was considerable evidence of herbicide contamination within th« storage area itself (i.e., visual evidence of leaks and •pill* on the soil)i however, there was no evidence that any of the material had been carried from the storage area by the surface drainage system. Soil samples collected between the stored drums, on the banks of the drainage system and silt deposits at various points in the drainage ditches had no detectable levels of herbicide at the 1 part per million (ppm) level, One soil sample was taken only six feet from the drums where prior leakage had been detected as evidenced by discoloration of the soil surface. Hater samples from the drainage ditches had no detectable levels of herbicide at the 50 parts per billion (ppb) level. One of the water samples did, however, contain hydrocarbon residues apparently from washing operations in the area. The presence o£ the fuel in the water gave the stream an oily appearance which may have lead some people to conclude that a herbicide residue was present. The biologicals (frogs, tadpoles, minnows) that were collected were not analyzed because there was no evidence that the aquatic drainage system was contaminated at that time. Upon gross examination no abnormalities were seen in any of these aquatic specimens. A complete survey of the flora surrounding the storage area was also completed during the July 1974 visit by the USAF Academy personnel. Plant damage of a herbicidal-nature (twisting and bending of leaves and stems) was noted on two plant species as far as 85 yards west (downwind) of the drum storage site. �In December of 1974 Dow Chemical Interpretive Analytical Services reported the first known TCDD positive soil sample frent between the rows of barrels on the storage site. Two soil samples were analysed. One sample had nondetectable levels at a detection limit of 4 parts per trillion (ppt) while the second soil sample was positive for TCDD at IS ppt. During the period of August 1974 to October 1976 representatives of the EHL/K made 11 trips to the Naval Construction Battalion Center to monitor pilot plant activities, drum rinse studies and conduct environmental monitoring including the collection of water samples from the herbicide storage area drainage ditches. Water sample values for 2,4-D had a range of average mean value of 0.15 ppb to 409.4 ppb; the 2,4,5-T range of average mean values for water was 0,3 ppb to 519.4 ppb and a 1976 TCDD positive sample that had an average mean value of 7.7 ppt. Sediment samples collected from the drainage area contained 2,4-D in a range of average mean values of 0.04 ppm to 0.24 ppm; the 2,4,5-T range of average mean values for sediment was 0.04 ppm to 0.42 ppm. All sediment samples for TCDD were negative} however, the analytical laboratory could not establish a level of detection for TCDD because of interferences. In the October 1976 report it was noted that of the 26 water samples analyzed, 13 were reported as containing more than 10 ppb herbicide. However, at the base discharge sample point leading off base, there were no water samples analysed that exceeded this lower detection limit of 10 ppb. Also, of the 23 water samples that were analyzed for TCDD, there was only one that had a positive reading and that sample was collected near the storage area. TCDD. Samples collected further downstream had no detectable The detection limit in these samples was 0.01 ppb. These results indicated that although some herbicide was entering the drainage system, 10 �it was not leaving the base and most likely was being held in the bottom sediments of the drainage ditch system. Visual observations of the drainage ditch system indicated that there. were no deleterious effects being exerted on the biotic community and that fish, frogs, snakes and other normal fauna and flora seemed to flourish. Only two of the sediment samples analyzed exceeded 1 ppm herbicide. These samples were collected near- the storage area. The sediment samples collected near the base discharge point never exceeded the 1 ppa herbicide leval and no fCDB was ever detected in any of these sediment samples. However, the analytical laboratory could not establish a level of detection for TCDD because of interferences. Soil sample data in October 1976 was not sufficient to make an interpretation as to the degree of severity of the herbicide contamination of the soil. Recommendations from the October 1976 EHIi/K report weres 1. The levels of Herbicide Orange (HO) in the ambient air were not high enough to create any concern about any on- or off-base exposure. Thi« was also borne out by the biomonitoring that had been performed during the Agent Chemical Inc (ACI) operation at NCBC. If the TCDD analytical result* were viewed as upper limits, as suggested by the analytical laboratory [Wright State University {WSU)], then there was no need for concern. 2. There was no indication of any off-base discharge of TCDD in the water or sediment samples. 3. Quarterly environmental monitoring surveys should be continued. 4. There is need for a comprehensive sailing program of the soil in the HO storage area to permit a better evaluation of the degree and extent of contamination by both HO and TCDD. 11 �In January 1976, members from the USAF Academy, Department of Chemistry and Biological Sciences,conducted an extensive aquatic and soil survey of the herbicide storage area. During this survey, many soil, sediment and biological samples were collected from throughout the storage area and the surface drainage system. These samples were frozen and archived as baseline samples should the need arise to evaluate similar types of samples during or after the dedrumming operation. Selected samples frost this collection were later analyzed in 1978. Data from these samples are incorporated into the Results and Discussion Section of this report. USAF OEHL SITE MONITORING PROTOCOL Four problem areas were apparent in the design of a study: 1. Over 25 individual chemical components in Herbicide Orange had been identified [Hughes, et al. ( ) . Should or could a monitoring 4] program include all of these components? The low percentage in content of most of these components combined with their known low toxicity and/or rapid biodegradability (e.g., butanol, toluene and xylene) suggested that only the principle herbicides (acid and ester formulations of 2,4-D and 2,4,5-T), their major breakdown products (di- and trichlorophenol) and TCDD should be followed. 2. What criteria should be used to determine the number and location of sampling sites on an area of approximately 12 acres? Spills, due to handling of the drums during dedrum operations (during and prior to PACER HO) or to leakage (prior to PACE! HO), could have occurred almost anywhere on the storage area over the eight-year period. Certainly, the persistence and fate of individual herbicides, phenols or dioxin might be determined if a technique could be used to determine old spills from new spills. 12 �3. What factors associated with'the actual storage Urea at NCBC will have influenced the penetration of herbicides/TCDD into the •oil profile? This problem would certainly influence the depth of sampling that would be required, 4. In an "ideal" monitoring program, some method would be required to determine a minimum level of residue that could be considered biologically and ecologicallf acceptable, i.e., a "no significant effect" residue level. Should this no effect level be based upon soil micro- organisms, surface vegetation or some other criterion? Previous environmental studies in 1974 and 1976 by Young, 19), and Ifoung, et al. (10), showed that movement of the herbicide components of Herbicide Orange and the TCDD contaminant was low, suggesting that both lateral movement and soil penetration of the water-insoluble Herbicide Orange and TCDD would be minimal. Thus, surface sampling, e.g., the top three inches (S cm) of soil, should constitute the primary sampling depth. As noted above, the depth of routine sampling was of major concern in designing the residue monitoring program. Young, et ai. ( 0 had shown that 1} neither the herbicide components of Orange nor the TCDD had appreciably moved in the soil during biodegradation studies at Eglin AFB PL or the APLC test lange Complex, Hill AFB OT. However, these studies had involved soils treated with herbicides by using a hand sprayer and at concentrations greatly below those encountered in spills. Certainly some of the spills that had occurred at NCBC were "old" spills and the effects of time (years) on these spills was essentially unknown. Another factor in sampling depth was that the soil in the outdoor storage areas of NCBC had been treated in the 1940s with cement and compacted ( ) This treatment had created a 6-12 inch (15-30 1. en) layer of hardened stabilized soil. This "hardpan" was relatively 13 �impervious to water and presumably herbicide; however, in 1977, the hardpan was 3 to 6 inches (8-15 cm) below surface due to the addition of soil and gravel during the intervening years. This upper layer of soil was primarily sandyloam in texture. Selected sites where heavy spills had apparently occurred had also been treated with a 2 inch (5 cm) layer of oyster shells. All of these factors influenced the decision to select only one depth as the primary sampling depth which was the top three inches (8 cm). In July 1977, a preliminary sampling study was initiated. This consisted of assessing the heterogenity of the soils on the sites and the heterogenity of the herbicide concentrations. Twelve sites were selected for sampling; six were in areas of obvious spills and six in areas that showed no spill. Not only were the spills discernible by sight but also by smell. Winston and Ritty (7) had previously found that the olfactory senses can detect a butyl enter formulation of 2,4,5-T at levels of 0.4 ppb. The results of this fir»t sampling after PACER HO are shown in Table 2. Significant concentrations of herbicides, phenols and TCDD were detected in soils from spill sites. The variation in concentrations and in the portion of acids to ester* suggested that the spills were from different time periods. Accordingly, a more extensive protocol was proposed for future sampling. 197§ fROTOCQE The sites selected within the storage area for monitoring of residue were determined by whether a spill had occurred or not occurred at that specific location. The basis for determining a spill was whether a herbicide stain was discernible (heavy, light, absent) and whether a herbicide odor was detectable (strong, mild, absent). Thus, within the Storage Area numerous location* were found that had a heavy stain ajid strong odor (labeled 8/H, presumably representing a recent spill)? a light stain and 14 �2. Concentration parts per Million, of total herbicides, total phenols, and TCDD in 12 soil samples collected July 1977 from the Herbicide Orange Storage Area, Naval Construction Battalion Center, Golfport MSa Location total Herbicides (ppm) Total Phenols13 (ppm) TCDD Spill Sitesc 1 51,600 132,400 3 5 8 10 11 37,350 34,840 117,060 Mean = 95,000 78,040 42,395 87 109 166 96 303 152 (5) 90 019 .00 0.6310 »008) (.049 0.1900 0.0185 MA. 0.2371(4) 4- 0.2718 Mo Spill Sitesd 2 4 6 7 9 12 •f 12.4 0.7 0.2 0.1 0.6 0.2 0.2 0.3 + 0.2 NA NA NA MA HA NA a Analysis by the Flasmability Research Center, The university of Utah, Salt Lake City OT. Air Force Contract No. 561178C0062. Report submitted 17 May 1 7 . 99 "Total herbicides refers to concentrations of acid and all esters detected of 2,4-D and 2,4,5-f. °Total phenols refers to concentrations of dichlorophenol and trichlorophenol. %he sample consisted of a cube (3x3x3 inches) of soil removed from the center of an area designated spill or no spill. 8 HA • Mot Analyzed. ( ) refers to number of samples included in obtaining the means and standard deviation. %D • Not Detected at the detection limit specified in parenthesis. 15 �mild odor (labeled L/L, presumably representing an older spill); and no stain and no odor (labeled O/O, presumably representing an uncontaminated area). Fourteen replications of each treatment were then randomly selected to represent the storage area (thus a total of 42 permanently marked sampling locations). Twelve of these locations had been tentatively located and marked on 28 July 1977 with the remaining 30 located and marked on 17 January 1 7 with sampling being conducted on these dates, as well 98 as 6 November 1978. In collecting the soil samples, a 3-inch square was marked, 6 inches away from the site marker pin. At each sampling tine, soil was taken from a different "point of the compass" with reference to the marker pin to insure a fresh and undisturbed profile. At the designated site, a 3x3x3-inch cube of soil was removed with a ceramic spatula which was rinsed with acetone between uses to prevent carryover of residue and microorganisms. Wherever possible, sediment samples were collected from the drainage ditches in a similar manner. CHEMICAL ANALYSES Each soil sample consisted of approximately 200 grans and was placed into new glass jars ( 0 ml) appropriately labeled and transported to the 40 laboratory where they were uniformly mixed and subsampled. The subsample used for chemical analysis was immediately frozen. The remaining sample was used for microbial studies (see Microbial Analyses). All soil samples collected from NCBC in July 1977, January 1978 or November 1978 were submitted for chemical analyses to the Flantmability Research Center, University of Utah, Salt Lake City UT. Each soil sample was analyzed for the esters and acids of 2,4-D and 2,4,5-T. In addition, each sample was analyzed for diand trichlorophenols (intermediate degradation products of 2,4-D and 16 �2,4,5-7) and selected samples analyzed for TCDD. & brief description of the netted employed in the analyses has been published ( ) 5. MICROSIAL ANALYSES SubBttRf>le8 of all soils were sent to the Department of Chemistry and Biological Sciences, USAF Academy CO for microbial analyses. Ml samples were analyzed for total populations of actinomycetes, fungi and bacteria. In addition, Jcey species presumably responding to the presence of herbicides were identified. The method employed in the microbial analyses has been previously described by Young ( ) It was hoped that quantitative and 9. qualitative studies of the microorganisms from each of the treatment classes used in association with residue data would permit an establishment of a no effect level. CTSULTO AMD DISCUSSIONS, Of HERBICIDl AMP MICBQBIAI* DATA A summary of the analytical results for the 42 sites sampled in January and November 1978 is shown in Table 3. A statistically significant decrease in the levels of total herbicides and total phenols was found to occur between the two dates. There was also a downward trend in TCDD levels, but it was not statistically different {P.05), This trend'in decreasing levels of TCDD (as well as in herbicides and phenols) is even more pronounced when the July 1977 data (Table 2) are compared to the 1978 data (Table 3). Unfortunately, because of differences in site delineation between 1977 and 1978, data for spills vs no spills between the two years cannot be "paired" and statistically analyzed. Nevertheless, the data suggest that TCDD may be degrading within the time period of this study (18 months). Data on the soil penetration of the herbicides, phenols, and TCDD are shown in Table 4. This site (site 17) was a site where a herbicide spill 17 �TABLE 3. Mean concentrations, parts per million, of total phenols and TCDD in soils collected in January and November 1 7 frost selected sites on the Herbicide 98 Orange Storage Area, Naval Construction Battalion Center, Gulfport MS* Location Number of Sites Sampled*3 Total Herbicides (ppit)c Total Phenols (ppm)a 14 14 32af 36* 3.5a 04 .0 TCDD (pp«) "Ho" Spills ( / )e 00 Jan 78 78 ND(4) "Old" Spills ( / ) LL Jan 78 Kov 78 14 14 1,2020. 4920 86a 230 14 14 51,2850 30,0050 437tt 2530 O.G3641(3) 003(} .483 "New" Spills (H/B) Jan 78 Nov 78 026(0a .041) 014(1a .441) a Samples analyzed by the Flammability Research Center, The University Of Utah, Salt Lake City OT. Mr Force Contract Mo. 561178C0062. Reports submitted 17 May 1979 and 7 November 1 7 . 99 Each soil sample consisted of a cube of soil (3x3x3 inches) removed adjacent to a designated marker. °fotal herbicides refers to the concentration of acid and all esters of both 2,4-D and 2,4,5-T. %otai phenols refers to total concentration of both dichlorophenol and tr ichlorophenol . e The coding O/O, L/L and H/H are described in the text. Means within columns within subtitles followed by the same letters are not significantly different at the 0.05 probability level. For the statistical analyses, the Wilcoxon Paired-Sample Test was used. A test for a one- tailed hypothesis with paired samples was used in the procedure for nonparametric data since it could not be assumed that the levels of residue detected were from a normal distribution and it was expected that the residues would decrease with time. See Reference 11. Detected? the number of samples analyzed is in parentheses. The detection limit was generally 0.0002 ppra ( 0 ppt) . 20 n NA-Mot Analyzed. %he number within parentheses refers to number of positive samples used in calculations of the means. In L/L sites, the other 11 samples were either ND or not analyzed; in H/H sites the remaining samples were HD. 18 �TABUS 4. Penetration of herbicides, phenols and TCDD in soil collected June 1979 from a site (Nuaber 17, H/H) where a herbicide spill occurred in 1977 on the Herbicide Oranfe Storage Area, Haval Construction lattalion Center, Gulf port MSa Description of Siteb Soil Depth (Inches) Total Total Herbicides (Pl»»}c Phenols (ppa)d KDD (ppm) Surface Layer 0-3 61,650 365 0.325 Above Hardpan 3-6 34,690 95 0,340 Within Hardpan 6-9 1,620 48 0.021 Within Hardpan 9-15' 322 " 11 MDe *Sanf»les analyzed by the Flaamability Research Center, fhe Uniwrsity of Utah, Salt lake City OT. Mr Force Contract No. 561178C0062. Report submitted 7 Marorober 1979. See text for description of flardpan. C fotal herbicides refers to concentration of acid and all esters of both 2-4D and 2,4,5-T. total phenols refers to total concentration of both dichlorophenol and trichlorophenol. e ttot Detected. The detection limit was 0.00048 pp» ( 8 ppt) for this 40 sample. 19 �had occurred during the PACER HO Operation in Jane 1977. The soil core was collected in June 1979; thus, a period of at least two years had elapsed from date of spill to date of sampling, A decrease in concentration of residue occurred with depth. The hardpan (soil stabilized with cement at least 30 years earlier) was relatively impervious to any residues, despite the high annual rainfall (60 inches) received in this geographic location. These data suggest that soil penetration of residue as a route for contamination of subsurface water will be negligible. Some additional observations of the residue data that may influence future monitoring programs concern the nature of the remaining residues. Although most of the sites, where high levels of residues have been found, have been associated with a spill of Herbicide Orange, two of the sites contain significant levels of the isooctyi esters of 2,4-D and 2,4,5-T. These data suggest that Orange II was spilled at these sites rather than Orange. Whereas the butyl esters of 2,4-D and 2,4,5-T have rapidly hydrolyzed in the soil, the data from Orange II sites show little or no degradation of the isooctyi esters over the two-year period, especially the isooctyi esters of 2,4,5-T. In addition, in these two sites detailed studies of the reiidue indicate the presence of an apparently very stable isooctyi ether of 2,4,5-trichlorophenol. Unpublished data by Arnold* of the studies on soils treated with Orange II in 1972 and collected six years later, have shown negligible degradation in the isooctyi ether of 2,4,5-trichlorophenol. The stability of this ether has permitted its use in confirming the actual concentration of herbicide in the soil at the time of treatment. It may be possible to use this "marker" ether to date selected spills at NCBC, *E.L. Arnold, August 1979. Analysis of Herbicide Orange Components in Selected Soil Samples. USAPSAM/NGP, Brooks APB TX. Report submitted to USAF OEHL. 20 �Data from the micrcbial analyses of soil samples collected from the storage area in July 1977 and January and November 1978 are shown in Tables 5 and 6. Although the biological activity was high in all three treatment areas ( / , L/L, and H/H) trends in populations were discernible. The 00 July 1977 data in fable 5 indicate the impact that activities associated with Project PACER HO may have had on the storage area. During PACER HO, not only did personnel and vehicular traffic disturb the entire site, but when the operation was complete, the site was leveled and a layer of oyster shells was placed in selected sites where spills of herbicide and fuel oil had occurred. The bacteria were especially affected} note that the July 1977 levels in either no spill or new spill sites were ituch lower than the other two dates. However, these data may also reflect both an effect of PACER HO and a lag-phase effect in the adaptation of the bacteria to herbicide. The highest levels of bacteria were found in highly herbicidecontaminated sites (January 1978). Of the several bacterial genera isolated and identified, Psuedoponas spp. predominated in samples with the highest levels of herbicides. Levels of fungi decreased both with time and herbicide concentration. Only 50 percent of the H/H sites in January or November 1978 had detectable levels of fungi, and then, as noted in Table 6, they were not always of genera found in O/O or control soils. Proliferation of certain organisms could indicate their ability to metabolize or co-metabolize herbicide or herbicide degradation products or it could indicate elimination or inhibition of natural competitors. Specific metabolic activity studies using the predominant organisms would be necessary to determine their exact role (if any) in biodegradation. 21 �TABLE 5. Microbial population levels (number of organisms per gram of soil) in soils collected in July 1977, January and November 1978 from selected sites on the Herbicide Orange Storage Area, Naval Construction Battalion Center, Gulfport MSa Fungi, xlO5 Number of Sites Bacteria, xlO7 "No" Spills ( / )b 00 Jul 77 Jan 78 Nov 78 6 14 14 29.7 45.6 40.2 29.6 Old Spills (L/L) Jan 78 Nov 78 14 14 41.8 36.3 1 .2 ( ) 0 8 4.2 ( ) 8 6 14 14 15.4 49.4 34.6 28.6 (5) 7.7 ( ) ? 6.1 (7) Location tsr 7.8 6.2 New Spills (K/H) Jul 77 Jan 78 SOV 78 Control*1 Jan 78 1 38 3.0 Sov 78 I 35 3.2 a Microbial analyses conducted by Department of Chemistry and Biological Sciences, USAF Academy CO. Final report received August 1979. **fhe codling 0/0, L/L and H/H are described in text. c The number within parentheses refers to number of samples where colonies could be counted. Fungi in soils contaminated with herbicide frequently showed no growth after 7 days or growth was random. Control taken in open grassy area one mile from Storage Area. 22 �TABLE 6. Fungal genera found in soils collected from selected sites in 197? and 1978 on and off the Herbicide Orange Storage Area, Naval Construction Battalion Center, Gulfport MSa Predominant Genera Off-Site Control On Site o/o VL Aspergillus spp. Panici Ilium spp. Gunninghamella spp. Zygorhynehus sp. Alternaria sp. Mycelial Molds Candida^ spp. Rhodotorula sp. Geotrichum sp. Triehoderma spp. Muoor. spp. Rhizopus sp • Absidia sp. X X X X X X X X X X X X X X X X X X X X X A X X X X X X A X Miorobial analyses conducted by Department of Chemistry and Biological Sciences, OSAF Academy CO. Final report received August 1979. ft» ceding 9/0, L/L and H/H refer to no spill ( / ) old spill 00, (L/L) and new spill (H/H) and are further described in text. 23 *Jf / f ^ t* I /H I X �AQUATIC SYSTEM MOIilTORIHG FOR TCDD RESIDUE, 1 7 - 9 9 9717 The extreme toxicity associated with 2,3,7,8-TCDB (Reference 8) and its occurrence as a contaminant in 2,4,5-T (and hence Herbicide Orange) dictated that it must be the focus of any residue monitoring study. The location of the NCBC in relation to the major population center of Gulfport MS and to the associated aquatic system is shown in Figure 1. Previous ecological studies on the environmental fate of TCDO by Young ( ) 9 and Young, et al. ( 0 suggested that aquatic drainage systems could be 1) contaminated by water erosion of soil particles containing TCDD. The herbicide storage area is drained by a series of snail ditches that connect into a single ditch immediately adjacent to the area. This larger ditch is fed by other small ditches as it transversea the property of the NCBC. Zn an effort to obtain baseline data on TCDD in this aquatic system, archived biological samples (collected in the immediate storage area and frozen in January 1976) were analyzed in November 1978 and found positive for TCDD residue. Thereafter, additional environmental samples were collected in January, February and June 1979 at varying distances downstream from the storage area. These designated Aquatic Sampling Sites are shown in Figure 2, Aquatic Site III was located at the NCBC perimeter. Aquatic Site IV was at a culvert discharge from the drainage ditch into Long Beach Canal Number 1. Aquatic Sampling Site V was at the confluence of the canal and Turkey Creek. The analytical results from some of these environmental samples were received in September and November 1 7 . 99 A summary of all available TCDD residue data for the aquatic system draining from the storage area is shown in Table 7. It should be again noted that TCDD data in Tables 2, 3 and 4 are presented as parts per million (ppm). Aquatic monitoring studies detected residue levels in 24 �4» O O -H 3 *J C (0 g« o «o ® r-i +J 15 111 > -H 4 O 3 0 CO C S ffi 5° •P C I I tie Is H 14 (D S +J e 9 V Il l rt i & 25 �ci4e M O 4) 41 « §« •H £8 re i-t -ft C 4J •H A (0 i) 31 •H «H 0) -P .si ^ «• D» 3 n. jjj S J3 io (0 rt o * "51 §• a> «g ««4 « O « • tt a 1 tie s H M \ \ V \ 26 M 10 �TABLE 7. Summary of results (parts per billion) for TCDD residue studies in water, sediments anil biological organisms associated with drainage from the Herbicide Orange storage area, Naval Construction Battalion Center, Gulfport IB* Aquatic Sampling Site Distance from Storage Area (Feet) Water Cppb) Maximum Concentration in Sediments (ppb) I Immediate Area ND 3.6 Biologicals (ppb) 0.14-3,5fc 1.6 -7.2 II III 3,000 NAd 7,000 NA 0.01 005 .4e IV 9,000 NA 0.02 0.02f V 12,000 NA ND ND 0.2-2.2 ND9 The analyses for TCDD were conducted by the University of Nebraska, Mass Spectroroetry Laboratory, Lincoln HE, under Mr Force Contract Ho. P0561178C0063 and the Oniversity of Utah, Salt Lake City Of, under Air Force Contract No. 5S1178C0062. Reports submitted 6 September 1979 from the University of Nebraska and 17 May 1979 and 7 November 1979 from the University of Utah. m » Not Detected. Detection limit varied with the sample. All water samples were analyzed by the university of Utah and the detection limit was 0.02 ppb. Sediment samples from Sites I, II and V were analyzed by the University of Utah by low resolution GC-MS where the detection limit was 0.5 ppb. Sediment sauries from Sites III and IV were analyzed by the Dniversity of Nebraska by hifh resolution GC-MS where the detection limit was O.OOS ppb. All biological samples were analyzed by the University of Nebraska and the detection limit ranged from approximately 0.05 to 0.005 ppb, °First sample set collected in January 1976 and analyzed and reported in January 1979; second sample set collected in January 1979 and reported in September 1979. NA - Not Analyzed. This value is an average for a single biological, a crayfish, which was analyzed twice. The mean detection limit was 0.01 ppb. This value was for a single biological, a crayfish, which was analyzed twice. The mean detection limit was 0.008 ppb. A single biological sample, a composite of mosquitofish, was analyzed three times. The sample was considered negative at a mean detection limit of 0.007 ppb. 27 �parts per billion (ppb) and pacts per trillion (ppt). Thus, the average mean level of TCDD in storage site soils (spills) in July 1977 was 237 ppb (0.237 'pftt, see Table 2) j 206 ppb in January 1978 and 144 ppb in November 1978 (see Table 3). Data in Table 7 in very low parts per billion are two orders of magnitude below levels in the storage area soils. Water Samples - Surface Drainage System Herbicide storage Area h total of 61 surface drainage system water samples were collected (Aquatic Sampling Site I) during the history of the project. One sample collected in 1976 was positive at an average mean value of 7.7 ppt TCDD. All remaining samples were negative for TCDD at detection limits ranging from 5-37 ppt. Water Samples - Potable Water System and Wells on the NCBC h total of 36 potable water system and well water samples taken during th« history of the project have contained no detectable levels of TCDD at detection levels as low as 10 ppt. Sediment Samples Two of eight sediment samples collected (Aquatic Sampling Site I) in the immediate surface drainage system of the herbicide storage area in June 1979 were positive for TCDD at levels of 2.7 ppb and 3.6 ppb. Of the remaining six samples, five contained no detectable TCDD at a detection limit of 2 ppb. The sixth sample contained no TCDD at a 37 ppb detection limit. The maximum positive value for this location is shown in Table 7. Two sediment samples have been collected from Aquatic Sampling Site ZZ. These samples were collected in June 1979 and were found negative for TCDD at a detection limit of 0.5 ppb. 28 �Two sediment samples have been collected from Aquatic Sampling Site III (located at the NCBC perimeter}, One of these samples was collected in February 1979,- the other in June 1979. The June sample (data reported in November 1979) was negative for TCDD at a, detection limit analysis of 0.5 ppb [low resolution Gas Chromatography-Mass Spectroraetry (GC-MS}], while the February sample (data reported in September 1979} was positive for TCDD at a level of 0.01 ppb (high resolution GC-MS analysis). the datum from the February sample is reported in Table 7. One sediment sample collected in February 1979 off-base, 9,000 feet from the herbicide storage area (Aquatic Sampling Site IV), in the drainage system leading away from the herbicide storage area and the NCBC, was positive for fCDD at 0.02 ppb with a lower detection limit of 0.01 ppb (report received September 1979). One additional sample collected from the same area (Aquatic Sampling Site IV), in June 1979 contained no detectable TCDD, when the detection limit was 0.5 ppb (report received November 1979). A single sediment sample was collected from Aquatic Sampling Site v. The sample was collected in June 1979 and analyzed by low resolution GC-MS. The sample was found negative for TCDD at 0.5 ppb. Biological Samples Aquatic biological samples (snails, fish, tadpoles, crayfish, and insects) collected over the past three years from the drainage ditch serving the immediate herbicide storage area (Aquatic Sampling Site I)» contained TCDD levels that ranged between 0.14 ppb and 7.2 ppb (Table 7). Aquatic biological samples (snails, tadpoles, fish and crayfish) collected over the past three years from the drainage ditch 3,000 feet 29 �downstream from the herbicide storage area (Aquatic Sampling Site II), contained TCDD levels that ranged between 0.2 ppb and 2.2 ppb. A large crayfish was collected in January 1979 and the muscle tissue and intestine trace separately analyzed. The intestine was found to contain 1.1 ppb TCDD, while the muscle tissue contained 0.0? ppb TCDD. A crayfish sample collected in February 1979, 7,000 feet downstream from the herbicide storage area (Aquatic Sampling site III), just before the drainage system exited the NCBC property, contained 0.045 ppb TCDD. A crayfish sample collected in February 1979, 9,000 feet downstream from the herbicide storage area (Aquatic Sampling Site IV), offbase in the drainage system serving NC1C was found to contain 0.02 ppb TCDD. A mosquitofish sample collected in February 1979, 13,000 feet downstream from the herbicide storage area (Aquatic Sampling Site V), in the off-base drainage system, contained no detectable TCDD at a detection limit of 10 ppt. 30 �Environmental studies of an area on the Naval Construction Battalion Center, previously used for the storage of Herbicide Orange from mid-1968 through mid-1977 were conducted during the period 1970 through 1 7 . The 99 following are conclusions from those studies: ' 1, .approximately 1-2 acres of the 12-acre area are contaminated with Herbicide Orange and its associated dioxin. 2. Levels of 2,4-0 and 2,4,5-T herbicides in selected saaples from the top three inches of soil profile were greater than 100,000 ppmdaean 78,040 ppn) in 1977, bat rapidly decreased to one-third that level in 18 months. 3. No accurate estimate of TCDD persistence is possible from these studies. However,, data from spill sites monitored for 18 months suggest that TCDD levels are decreasing. 4. Soil penetration of the herbicides was low while soil penetration of TCDD was very low but measurable. 5. Soil sterilization did not occur as a result of Herbicide Orange contamination. 6. Proliferation of certain microflora occurred under high levels of herbicide (specifically members of the fungal order Mucorales, white nonsporulating mutants, soil yeasts, and Pseudomonas spp.) 7. Yeast and Pseudomonas spp. predominate in samples with highest levels of herbicide, 8. Proliferation of certain organisms could indicate: a. ability to metabolize HO or degradation products. b. Ability to co-metabolize HO or degradation products. c. Elimination/inhibition of natural competitors. 31 �9. The low solubility of TCDD in water would suggest that its solubility in water alone could not account for the levels of TCDD found in the drainage ditch sediment. 10. The movement of TCDD from the storage sites is primarily through soil erosion, especially that caused by water. 11. Organisms that come into direct and intimate contact with TCDD-contaminated soil generally become contaminated themselves, (A wide variety of organisms have been examined.) 12. TCDD was found in a crayfish collected on base 3,000 feet downstream from the storage site. Levels in the intestine were 1.1 ppb, levels in muscle tissue were only 0.07 ppb. Movement of contaminated soil from the storage area downstream may have resulted in the contamination of crayfish. However, crayfish are highly mobile and nay have migrated from the storage area to the point of capture. 13. TCDD was found in two samples (1 sediment and 1 biological) collected off-base of NCBC. Although the levels of TCDD were extremely low (20 parts per trillion in each sample), it is apparent that some contamination from the storage area has occurred. Contamination from the storage area is not yet extensive and can be controlled. RECOMMENDATIONS The principle recommendation for management of the 12-acre area at the Naval Construction Battalion Center, formerly used as a storage area for Herbicide Orange, is that the area be left undisturbed permitting the continuation of "natural" degradation of the herbicides and TCDD, Specific recommendations to prevent further movement of contaminated soil from the area include: 32 �1. Limiting access to the storage area, and preventing motor vehicle traffic froa crossing the area and potentially "tracking" TCDDcontaminated soil particles to other parts of the installation. 2. Preventing water erosion wherever possible by stabilizing the drainage ditch banks with concrete or asphalt material. The ditch banks should be slightly elevated on the contour to allow pooling of water from the storage area prior to entering the ditch creating an initial siltation catchment. The ditches should be allowed to have plant growth in them to slow the movement of water and allow for more silt catchment, In several places along the ditch drainage system concrete dams should be constructed to slow water movement and provide a wide shallow overflow {in effect creating snail siltation ponds in the ditch drainage system). 3. Constructing one or two larger siltation ponds in the drainage system prior to the drainage water leaving the base. 4. Allowing native vegetation to invade the storage area and establish a plant coaanunity to help prevent both wind and water erosion, 5. Developing a research protocol to determine possible methods for returning the storage area to full beneficial use. This protocol might include techniques to: a. decontaminate TCDD-laden soils. b. increase TCDD degradation rates. c. characterize the distribution and effects of TCDD in the aquatic environment. 33 �LITERATURE CITED 1. Anonymous. 1977. Air Force Logistics Command Programming Plan 75-19 for the Disposal of Orange Herbicide. San Antonio Mr Logistics Center, Kelly AFB TX. Annex 8, pp 2-4. 2. Craig, D.A. 1975. Use of Herbicides in Southeast Asia. Historical Report. San Antonio Air Logistic Center, Directorate of Energy Management, Kelly AFB TX. 58 p. 3. Fee, D.C., B.M. Hughes, M.L. Taylor, T.O. Tiernan and C.E. Hill. 1975. Analytical Methodology for Herbicide Orange, vol lit Determination of Origin of OSAP Stocks. Technical Report ARL-TR-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB OB. 36 p. 4. Hughes, B.M., D.C. Fee, M.L. Taylor, T.O. Tiernan, C.E. Bill and R.L.C. Wu. 1975. Analytical Methodology for Herbicide Orange. Vol Xi Determination of Chemical Composition, Technical Report ARL-TR-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB OH. 365 p. 5. Httfh**, B.M., F.B. Hileaan, i,H. Wbjeik and W.H. MeClennen. 1979. A rapid method for the analysis of low levels of Herbicide Orange (butyl **t*rs of 2,4-D ami 2,4,5-T), 2,4-ST 2,4,5-T, dichlorophenol, trichlorophenol and tetrachlorodibenzo-p-dioxin (TCDD) in environmental samples. Division of Analytical Chemistry, American Chemical Society. Abstract, 177th ACS Rational Meeting, Honolulu HI. 6. Hummel, R.A. 1977. Clean-up Techniques for the Determination of Parts Per Trillion Residue Levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDB). Journal of Agricultural and Food Chemistry 25(5)s1049-1053. 7. Winston, A.W. and R.M. Ritty. 1971. What Happens to Phenoxy Herbicides When Applied to a Watershed Area. Industrial vegetation Management 4(1):12-14. 8. Young, A.L., J.A. Calcagni, C.E. Thalken and J.W. Tremblay. 1978. The Toxicology, Environmental Fate and Human Risk of Herbicide Orange and its Associated Dioxins. Technical Report OEHL-78-92. USAF Occupational and Environmental Health laboratory, Brooks AFB TX. 247 p. 9. Young, A.L. (Ed). 1974. Ecological studies on a herbicide-equipment test area (TA C-52A), Eglin AFB Reservation, Florida. Technical Report AFATL-TR-74-12. Air Force Armament Laboratory, Eglin AFB FL. 141 p~. 10. Young, A.L., C.E. Thalken, E.L. Arnold, J.M. Cupello and L.G. Cockerham. 1976. Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the Environment: Summary and Decontamination Recommendations. Technical Report USAFA-TR-76-18. Department of Chemistry and Biological Sciences, USAF Academy CO. 41 p. 11. Zar, J.H. 1974. Biostatistical Analysis. I»renti0e-Hall Inc., Edgewood Cliffs NJ. pp 124-126. 34 �ADDENDUM Additional residue data from selected biological samples collected June 1979 were received 3 December 1 7 . These data are shown in Table A-l. 99 Vh«M data offer additional support of the previous conclusion, that TCDD from the Herbicide Orange storage area is present in selected biological samples obtained outside the boundary of the Naval Construction Battalion Center. 35 �TABLE A-l. Summary of results (parts'per billion) for TCDD residue in biological organisms collected June 1 7 fj?o» the 99 drainage system associated with the Herbicide Orange storage area, Naval Construction Battalion Center, Gulfport MS* Aquatic Sampling Distance iron Site Storage Area Mature of Sample Concentration Detection of Limit TCDD (ppb) (ppb) 11 3,000 Composite: Crayfish/Fish 015 .7° 0.035 III 7,000 Composite: Crayfish/Fish Turtle ( a ) Ft 0081 .8* HD® 0.010 0.035 IV 9,OOO Composite: Crayfish/Fish 001 .3f 0.017 V 12,000 Composite: Crayfish/Fish Frog (whole body) 000 .2 006 ,0 008 .0 O.OOS *fhe analyses for TCDD were conducted by the University of Nebraska, Mass Spectrometry Laboratory, Lincoln HE, under Mr Force Contract No. F056118C0063. Report submitted 3 December 1 7 . 99 This composite sample and subsequent composite samples in this table consisted of mosquitofish and snail crayfish. Q Average of three analyses. dAverage of two analyses. eND * not detected. Average of two analyses. 36 � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 017 Folder The folder containing the original item. 0187 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 Young, Alvin L. Charles E. Thalken William J. Cairney Description An account of the resource <strong>Corporate Author: </strong>USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas Date A point or period of time associated with an event in the lifecycle of the resource 1979-11-01 Title A name given to the resource Herbicide Orange Site Treatment and Environmental Monitoring: Summary Report and Recommendations for Naval Construction Battalion Center, Gulfport, MS Subject The topic of the resource biodegradation Mississippi dioxin Pacer Ho https://www.nal.usda.gov/exhibits/speccoll/files/original/4add1471606266da07270d34132a966f.pdf 18d963859085ea24a26bf603f01acbda PDF Text Text P: AuttlOT 00190 Young, Alvin L. Report/Article Title Typescript: The Military Use of Herbicides in South Vietnam, 1962-1971, 16 May 1983 Journal/Book Title Year Color Number of Images 000 ° u 2 Summary of history of herbicide use, including types and amounts, in Vietnam, Friday, January 05, 2001 Page 190 of 194 �THE MILITARY USE OF HERBICIDES IN SOOTH VIETNAM, 1962-1971. Research and development on phenoxy herbicides began in the early 1940s, when nost of the initial phytotoxic screening programs and the development of explication technologies were sponsored by the DoD. The herbicide, 2,4,5-T, was first commercially produced in the United States in 1947. During the years from 1961 through 1969, the DoD procured 53 million pounds of this herbicide (approximately 34 percent of the total US production) for use in the Republic of Vietnam (SMN). However, 8.9 million pounds of that amount were not sprayed in Vietnam, but were destroyed by at-sea incineration in 1977. The first sustained DoD operational use of herbicides was initiated during the Vietnam Conflict (Operation RMICB HMO) and the first shipment of herbicides used in KftNCH HAND was received at Tan Son Nhut Mr Base, (RVN), on 9 January 1962. The use of these compounds was intended to accoaplish two objectives: (1) the defoliation of vegetation to improve visibility and thus decrease the risk of ambush, and (2) the destruction of enemy crops. Four 2,4,5-T containing herbicides were used by the military during the period 1962-1970. These four included: (1) Herbicide Purple (used from 1962 through 1964) n-butyl n-butyl iso-butyl 2,4-D 2,4,5-T 2,4,5-T 50% 30% 20% (2) Herbicide Pink (used from 1962 through 1964) n-butyl iso-butyl 2,4,5-T 2,4,5-T 60% 40% (3) Herbicide Green (used from 1962 through 1964) n-butyl 2,4,5-T 100% (4) Herbicide Grange (used from early 1965 through 15 April 1970) n-butyl n-butyl 2,4-D 2,4,5-T 50% 50% Analyses of archived samples of Herbicides in 1972 suggested that the mean concentration of TCDD may have been approximately 33 ppm (Range: 17 to 47 ppm TCDD) for Herbicide Purple while archived samples of Herbicide Orange had a mean concentration of approximately 2 ppn (Range: <0.02 to 15 ppm TCDD). In addition, two other herbicides were widely used in KVN. These were Herbicide Blue, an organic arsenical formulated fron the sodium salt of cacodylic acid, and Herbicide Wiite, a water soluble triisopropanolamine salt formulation of 2,4-D and picloram. The amounts of the various herbicides used in RVN from January 1962 through February 1972 are shown in Table 1. �Table 1. QUANTITIES OP AM) TCDD IN EMS, JAN 1962-PEB 1972 CHEMICAL 2,4-D 2,4, 5-T TCDD 55,940,150 44,232,600 368 Picloram Cacodylic Acid Herbicide Total 3 , 04 1 , 800 3,548,710 106,763,260 Ninety-six percent of the 2,4,5-T disseminated in RVN was contained in Herbicide Orange; the remaining 4 percent in Herbicides Green, Pink, and Purple. However, Herbicides Green, Pink and Purple contained approximately 40 percent of the estimated amount of TCDD disseminated in WN. Green, Pink and Purple were sprayed as defoliants on less than 90,000 acres from 1962 through 1964, a period when only a small force of U.S. military personnel were in WM. Ninety percent of all the Herbicide Orange (containing 38.3 million pounds of 2,4,5-T and 203 Ib of TCDD} was used in defoliation operations on 2.9 million acres of inland forests and mangrove forests of 8VN. Most of the herbicide used in RVN was sprayed from aircraft. RANCH HAND aircraft, the C-123, disseminated 88 percent of all herbicide. Helicopters and ground application equipnent used by personnel from all branches of the U.S. Armed Forces applied the remaining 12 percent, primarly Herbicide Blue, to maintain visibility around base perimeters. Concurrent with the change to Herbicide Orange, the scope of aerial use shifted from four aircrews on temporary assignments, to 36 permanently assigned aircrews, and additional support personnel. Following the announcement in October 1969 that the administration of 2,4,5-T to pregnant rodents caused an increase in the rate of congenital abnormalities, the DoD confined Herbicide Orange spray operations to nonpopulated areas and in April 1970, all uses of the 2,4,5-T containing herbicides were halted. Other non-2,4,5-T herbicides continued to be used until June 1971 and Operation BANCH HAND was officially deactivated in October 1971. In March 1972, all remaining stocks of 2,4,5-T containing herbicides were removed from IWN, and transported to Johnston Island, Pacific Ocean, for open storage (Project PACER IVY), and Environmental Protection Agency (EPA) suspended many uses of herbicides containing 2,4,5-T because an epidemiologie study in the United States attributed abortogenic (miscarriage) effects to its use. � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 017 Folder The folder containing the original item. 0190 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 Young, Alvin L. Title A name given to the resource Typescript: The Military Use of Herbicides in South Vietnam, 1962-1971, 16 May 1983 Subject The topic of the resource Ranch Hand dioxin herbicide application https://www.nal.usda.gov/exhibits/speccoll/files/original/471d7083ad383802b5903b6eadf0fbec.pdf 4223a7930cb9ecb9cc5da02099f70aec PDF Text Text Item ID Number: 00191 Author Young, Alvin L. Report/Article Title Typescript: Chapter 2: The Military Use of Herbicides in Vietnam, "Massive Quantities of Herbicides Were Applied by the United States in a Tactical Operation Designed to Reduce Ambushes and Disrupt Enemy Tactics" Journal/Book Title oooo Month/lay Number of Images 23 Typescript for Chapter 2 in A.L. Young and G.M. Regigani, eds., Agent Orange and Its Associated Dioxin: Assessment of a Controversy (Amsterdam: Elsevier, 1988} Friday, January 05, 2001 Page 191 of 194 �CHAPTER 2 "MASSIVE QUANTITIES OF HERBICIDES WERE APPLIED BY THE UNITED STATES IN A TACTICAL OPERATION DESIGNED TO REDUCE AMBUSHES AND DISRUPT ENEMY TACTICS" THE MILITARY USE OF HERBICIDES IN VIETNAM A. L. YOUNG The introduction of herbicides in 1962 into the armed conflict in Vietnam represented an application of a new technique for modern warfare. Their use in a defensive role was for defoliation. roles was for crop denial. Their use in offensive Today, fifteen years after the last spray mission, these herbicides are at the center of intense scientific debate involving not only medical but also legal, political and ecological issues. This chapter reviews the historical and operational concepts and some potential human exposure considerations involving the military use of herbicides in the Southeast Asian Conflict. Herbicides Used in South Vietnam Synthesis technology, efficacy data, and field application techniques were developed for the two major phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-5-trichlorophenoxyacetic acid (2,4,5-T) during World War II at Fort Detrick, Frederick, Maryland (12). II, the commercial use of these two revolutionized American agriculture. "synthetic" Following World War organic herbicides In 1950, more than 4.5 million kilograms (kg) of these materials were used annually for weed and brush control in the United States. used (13). By 1960, in excess of 16 million kg were �In May 1961, the Office of the Secretary of Defense requested the Fort Detrick personnel to determine the technical feasibility of defoliating jungle vegetation in the Republic of Vietnam ( ) 7. By early Fall, 1961, 18 different aerial spray test 8 (defoliation and anticrop) had been conducted with various formulations of commercially-available herbicides. The choice of these herbicides was based upon the chemicals that had had considerable research, proven performance, and practical background at that period in time ( ) 4. Also, such factors as availability in large quantity, costs and known or accepted safety in regard to their toxicity to humans and animals were considered (12). The defoliation and anticrop mixtures of herbicides. results of these tests were effects could be obtained with two significant different The first was a mixture of the n-butyl esters of 2,4-D and 2,4,5-T and the iso-butyl ester of 2,4,5-T. code-named "Purple." that This mixture was The second "military herbicide was code-named "Blue" and consisted of the acid and sodium salt of cacodylic acid. The colored bands which were painted around the center of the 208-liter drums served as aid to the identification by support personnel ( ) 1. The first shipment of Herbicides Purple and Blue was received at Tan Son Nhut Air Base, Republic of Vietnam, on 9 January 1962. These were the first military herbicides used in Operation RANCH HAND, the tactical military project for the aerial spraying of herbicides in South Vietnam ( ) 1. Two additional phenoxy herbicide formulations were received in limited quantities in South Vietnam and evaluated during the first two years of Operation RANCH HAND. These were code-named Pink and Green. By January 1965, two additional military herbicides, code-named Orange and White, had been evaluated and brought into the spray program. Herbicide Orange replaced all uses of Purple, Pink, or Green, and eventually became the most widely used military herbicide in South Vietnam (7). The composition of the three major herbicides in South Vietnam were as follows: �1. Herbicide Orange Orange was a reddish-brown to tan colored liquid soluble in diesel fuel and organic solvents, but insoluble in water (15). One liter of Orange theoretically contained 510 grams of the active ingredient of 2,4-D and 530 grams of the active ingredient of 2,4,5-T. Orange was formulated to contain a 50:50 mixture of the n-butyl esters of 2,4-D and 2,4,5-T. The percentages of the formulation typically were: n-butyl ester of 2,4-D free acid of 2,4-D n-butyl ester of 2,4,5-T free acid of 2,4,5-T inert ingredients (e.g., butyl alcohol and ester moieties) 49.49 0.13 48.75 1.00 0.62 2. Herbicide White White was a dark brown viscous liquid that was soluble in water but insoluble in organic solvents and diesel fuel (15). One liter of white contained of 65 grams of the active ingredient 4-amino-3,5,6- trichloropicolinic acid (picloram) and 240 grams of the active ingredient of 2,4-D. White was formulated to contain triisopropanopamine salts of picloram and 2,4-D. a 1:4 mixture of the The percentages of the formulation were: triisopropanolamine salt of picloram triisopropanolamine salt of 2,4-D inert ingredient (primarily the solvent triisopropanolamine) 3. 10.2 39.6 50.2 Herbicide Blue Blue was a clear yellowish-tan liquid that was soluble in water, but insoluble in organic solvents and diesel fuel (15). One liter of Blue contained 370 grams of the active ingredient hydroxydimethyarsine oxide (cacodylic acid). Blue was formulated to contain cacodylic acid (as the free acid) and the sodium salt of cacodylic acid (sodium cacodylate). The �percentages of the formulation were: cacodylic acid sodium cacodylate surfactant sodium chloride water antifoam agent 4.7 26.4 3.4 5.5 59.5 0.5 As previously noted, not all of the herbicides used in South Vietnam were used throughout the entire 10 years (1962-1971) encompassed by the Department of Defense defoliation program. In addition, 2,4,5-T formulations used early in the program are believed to have contained higher levels of the toxic contaminant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin the time periods shown in Table 1 can be differentiated on the basis of specific herbicides used and the mean dioxin content (15). TABLE 1 - THE DIFFERENTIATION OF THE THREE TIME PERIODS DURING THE US MILITARY DEFOLIATION PROGRAM IN SOUTH VIETNAM AND MEAN DIOXIN CONTENT PERIOD HERBICIDES USED MEAN DIOXIN CONTENT* January 1962 June 1965 Purple, Pink, Green Blue 32** 0 July 1965 June 1970 Orange White, Blue 2+ 0 July 1970 October 1971 White, Blue 0 *Found only in 2,4,5-T containing formulations, **Value based on analyses of five samples. +Value based on the analyses of 488 samples. Herbicide Orange was the most extensively used herbicide in South Vietnam (5). Orange accounted for approximately 40.5 million liters of the 67 million liters of herbicide used (Table 2). June 1970. It was used from mid-1965 to However, as noted in Table 2, Orange was not the only 2,4,5-T containing herbicide used in the defoliation program ( ) 6. of Purple, Pink, and Green, all containing through mid-1965 (5,15). Small quantities 2,4,5-T were used from 1962 In subsequent sections of this document, the term "Herbicide Orange" will refer to all of the 2,4,5-T containing herbicides �used in Vietnam (Purple, Pink, Green, and Orange). TABLE 2 - NUMBER OF LITERS OF MILITARY HERBICIDE PROCURED BY THE US DEPARTMENT OF DEFENSE AND DISSEMINATED IN SOUTH VIETNAM DURING JANUARY 1962 - OCTOBER 1971(+) Code Name Herbicide Quantity Period of Use Orange 2 ,4-D; 2,4,5-T 40,295 ,000 1965 -1970* White 2 ,4-D; Picloram 21,321 ,000 1965 -1971** Blue Cacodylic Acid 4,353 ,000 1962 -1971** Purple 2 ,4-D; 2,4,5-T 549 ,000 1962 -1965 Pink 2 , 4,5-T 46 ,600 1962 -1965 Green 2 , 4,5-T 31 .400 1962 -1965 Total 67,015 ,400 +Source: Craig ( ) 6. *Last fixed-wing mission of Orange 16 April 1970; last helicopter mission of Orange 6 June 1970. **Last fixed-wing mission 9 January 1971; all herbicides under US control stopped 31 October 1971. The data in Table 2 were obtained by Craig (6) in 1975 from examination of procurement records maintained by the San Antonio Air Logistic Center, Kelly Air Force Base, Texas. The completeness of the records is unknown. A log of herbicide applications was maintained by the United States Military Assistance Command, Vietnam, and these "paper records" became the source documents for the National Academy of Sciences (NAS) 1972-74 study of the effects of herbicides in South Vietnam (5). records into the HERBS Tape. 6,100 herbicide 71,672,760 The HERBS Tape contained data on approximately missions with liters. As procurement documents The NAS computerized these noted an estimated in Table 2, herbicide expenditure Craig could only on 67,015,400 liters. account of for The National Academy of Sciences (5) estimated that their record searches and/or incomplete records accounted for 86% of the RANCH HAND missions. In 1985, the United States Army and Joint Services Environmental Support Group, a joint (Army, Air Force and Navy) military group of military record specialists completed an �exhaustive search of the military records of the Vietnam era. This unit constructed a data base of 2,394 additional military herbicide missions in Vietnam that were either improperly/incompletely documented in the HERBS Tape or were missions unrecorded. An additional 557 RANCH HAND fixed-wing missions were identified/verified. Tape contain spraying. Of significance, the SERVICES HERBS data on helicopter, backpack and other types of ground When the two tapes (HERBS and SERVICES HERBS) were combined, 8,930 missions were identified and 72,740,400 liters of herbicide were reported (Table 3). TABLE 3 - HERBICIDE MISSION DATA FROM COMBINING HERBS TAPE (1974) AND THE SERVICES HERBS TAPE (1985), 1961 - 1971 * Herbicide Number of Missions Total Number of Liters Orange 4,698 44,953,560 White 2,194 20,616,860 Blue 981 4,712,920 Unknown** 965 2,339,460 92 117,600 8,930 72,740,400 Other (Purple, Green, etc) Total *Data combine all fixed-wing, helicopter, and ground application missions . **Records document herbicide mission but do not identify specific herbicide. Use Patterns of Individual Herbicides Each of the three major herbicides specific uses. (Orange, White, and Blue) had About 90 percent of Herbicide White was applied in defoliation missions. It was not recommended for use on crops because of the persistence of picloram in soils. Because the herbicidal action on woody plants was usually slow, full defoliation did not occur for several months after spray application. Thus, it was an ideal herbicide for use in the inland forests in areas where defoliation was not immediately required, �with Orange or Blue. Herbicide Blue was the herbicide missions involving cereal or grain crops. of choice for crop destruction Approximately 50 percent of all Blue was used in crop destruction missions in remote or enemy controlled areas with the remainder being used as a contact herbicide for control of grasses around base perimeters (9). Approximately 85 percent of all Herbicide Orange was used for forest defoliation and it was especially effective in defoliating mangrove forests. Eight percent of Herbicide Orange was used in the destruction of broadleaf crops (bean, peanuts, ramie, and root or tuber crops). percent was used around The remaining 7 base perimeters, cache sites, waterways, and communication lines. Table 4 shows the number of hectares sprayed with herbicides in South Vietnam within the three major vegetational categories. TABLE 4 - THE NUMBER OF HECTARES TREATED IN SOUTH VIETNAM, 1962-1971, WITH MILITARY HERBICIDES WITHIN THE THREE MAJOR VEGETATIONAL CATEGORIES* Vegetational Category Hectares Treated** Inland forest 1,080,970 Mangrove forests 127,750 Cultivated crops 105.260 Total 1,313,980 *Source: National Academy of Sciences, 1974 (5). **Areas receiving single or multiple coverage. Certain portions of South Vietnam were more likely subjected to defoliation. Herbicide expenditures for the four Combat Tactical Zones of South Vietnam are shown in Table 5. obtained from HERBS Tape. to have been These data were The distribution of defoliation missions was mapped by the National Academy of Sciences (5) and is shown in Figure 1. Figure 2 is a map of Vietnam identifying the provinces and major cities. �TABLE 5 - HERBICIDES EXPENDITURES IN SOUTH VIETNAM, 1965-1971: A BREAKDOWN BY COMBAT TACTICAL ZONE* Herbicide Expenditure (liters) Orange White Blue Combat Tactical Zones CTZ I 8,516,360 1,374,000 1,127,900 CTZ II 9,534,400 2,759,300 1,790,300 CTZ III 20,094,600 14,076,400 1,112,800 4.644.200 1.646.500 234.700 (includes Saigon) CTZ IV 42,789,500 Subtotals 19,856,200 Grand Total *Source: 4,265,700 66.911.400 HERBS Tape In addition to the herbicides, numerous other chemicals were shipped to South Vietnam in 208-liter drums. These included selected fuel additives, cleaning solvents, cooking oils, and a variety of other pesticides. The insecticide malathion was widely used for control of mosquitoes and at least 1,514,000 liters of it was used from 1966 through 1970. smaller quantities of lindane and DDT were used throughout the war in Southeast Asia. In addition, much in ground operations The distribution of the herbicides within Vietnam after their arrival did not occur randomly. About 65 percent was shipped to the 20th Ordnance Storage Depot, Saigon, and 35 percent was shipped to the 511th Ordnance depot, Da Nang. Military Aircraft and Vehicles Used in the Dissemination of Herbicides Numerous aircraft were used in the air war in Vietnam, but only a few of these aircraft were used for aerial dissemination of herbicides. "work horse" of aircraft was carriage. Operation RANCH HAND was the C-123, "Provider." adapted to receive a modular spray system The This cargo for internal The module (the A/A 45 Y-l) consisted of a 3,785 liter tank pump, and engine which were all mounted on a frame pallet. An operator's console �was an integral part of the unit, but was not mounted on the pallet. booms (3.8 cm in diameter and 6.7 m in length) engine nacelles toward the wing tips. Wing extended from outboard A short tail boom (7.6 cm in diameter, 6.1 m in length) was positioned centrally near the aft cargo door. Each aircraft normally had a crew of three men: the pilot, co-pilot (navigator), and flight engineer (console operator). During the peak activity of RANCH HAND operations (1968-1969), approximately 30 UC-123K aircraft were employed. However, many other squadrons of non-RANCH HAND C- 123 aircraft were routinely used throughout South Vietnam in transport operations. The control of malaria and other mosquito-borne diseases in South Vietnam necessitated an extensive aerial insecticide application program in order to control these vector insects. from 1966 through 1972, three C-123 aircraft were used to spray malathion, an organophosphate insecticide. These aircraft could be distinguished from the herbicide-spraying aircraft because they were not camouflaged. These aircraft routinely sprayed insecticide adjacent to military and civilian installations, as well as in areas where military operations were in progress, or about to commence. Approximately 10 to 12 percent of all herbicides used in South Vietnam was disseminated by helicopter or ground application equipment. Generally, helicopter crews were not assigned to herbicide spray duties on a full-time basis and rotated the spraying duties with other mission requirements. The military UH-1 series of helicopters, deployed by the Air Force, the Army, and Navy units, generally sprayed the herbicides. The most common spray systems used were the H1DAL and AGRINAUTIGS units. These units were installed in or removed from the aircraft in a matter of minutes because they were "tied down" to installed cargo shackles and aircraft modifications were not required for their use. Each unit consisted of a 760-liter tank and a collapsible 9.8 m spray boom. The unit was operated by manual controls to control the flow valve and a windmill brake. helicopter had three crew members. Generally, each �A summary of the aircraft used in herbicide and insecticide operations is shown in Table 6. TABLE 6 - MILITARY AIRCRAFT USED IN THE DISSEMINATION OF HERBICIDES AND INSECTICIDES IN SOUTH VIETNAM Aircraft Camouflaged Chemical Disseminated UC-123/UC-123K Yes All Herbicides UC-123K No Malathion Helicopter Air Force UH-1 "*\ Army UH-1B/UH-1D.H-34Y Yes Navy H-19 Orange, Blue J Various ground delivery systems were also used in South Vietnam for control of vegetation in limited areas. mounted on vehicles. turbine. One unit that was routinely used was the buffalo It developed a wind blast with a velocity up to 240 km/h at 280 m^/minute volume. essentially Most of these units were towed or When the herbicide was injected into the air blast, it was "shot" at the foliage. The buffalo turbine was useful for roadside spraying and applications of perimeter defenses. The herbicides of choice in these operations were Blue and Orange. Mission Concepts The objectives of the defoliation and anticrop programs in South Vietnam have been thoroughly reviewed by Huddle (11) and others (2,3,5,7). It is the objective of this section to elaborate only on the background and mechanics of a "typical" herbicide mission that would have influenced the degree of exposure to herbicides by aircrew and/or ground personnel. The following scenario of events or "standard operating procedures" has been compiled from the literature and interviews by Young et al (15), and is captured in photographs in Figures 3 through 2%. 10 �1. Each of the 11 different companies that manufactured military herbicides packed them in new ICC 17C 208-liter 18 gauge steel drums for shipment to Southeast Asia. shipment of Blue. Until 1967, lined drums were used only for However, because of the results of compatibility tests, lined drums were also used to ship White beginning in 1967. 2. Each herbicide drum was marked with a 7.6 cm color-coded band around the center to identify the specific military herbicide. This marking was initially a 30 cm band, but was changed to a 7.6 cm band in March 1966. 3. Shipping time from the arrival of the herbicide at a US port until it arrived in South Vietnam varied from 47 to 52 days. 4. About 10 out of every damaged or defective state. 10,000 drums shipped were received in a This represented a damage rate of 0.1 percent. About 50 percent of these damaged drums leaked as a result of punctures or split seams. These were caused by improper loading and defective drums. Forklifts operated by stevedores also caused punctures. Redrumming was accomplished at the ports. 5. About 65 percent of the herbicide was shipped to the 20th Ordnance Storage Depot, Saigon, and 35 percent was shipped to the 511th Ordnance Storage Depot, Da Nang. Under the normal handling procedures, drums were unloaded at Da Kang and Saigon from the cargo vessel directly into semi-trailers and were placed in an upright position. were driven to the various units The trailers of the 12th Air Commando Squadron (primarily at the bases of Da Nang, Phu Cat, or Bien Hoa) for disposition. 6. Normally the contents of the drums were transferred into blocked F-6 trailer tanks through a suction tube without removing the full drums from the semi-trailers. 78 drums of herbicide. the total inventory, Each F-6 trailer held 16,300 liters or about If blocked F-6 trailer tanks could not accomodate the drums were stacked in pyramidal needed. 11 style until �7. The transfer of the herbicides from the 208-liter steel drums to storage tanks or aircraft tanks required some precautionary measures. Personnel charged with the supervisory responsibilities of handling the herbicides were indoctrinated in appropriate safety precautions including the use of gloves and face shields as needed. Personnel handling the chemicals were encouraged to "take normal sanitary precautions and to maintain personal cleanliness and to avoid skin and eye contact with the material. Contaminated clothing were to be washed before re-use. Spillage on the skin or in the eyes was to be rinsed copiously with clean water. 8. When the herbicide was pumped from the drums into the F-6 trailers about 2 to 5 liters remained in the drum. Hence the drum was placed on a drain rack and the "drippings" were collected from many drums in a pan-type receptacle and used for spraying base perimeter areas. 9. Empty drums were generally given to the military forces (Vietnam, U.S. and Free World Military Assistance Forces) for use as barriers in defensive positions. The drums were filled with sand or concrete and used in the construction of bunkers or in foundations for runways and barbed wire perimeters. 10. Surface areas contaminated by spillage of the herbicides were flushed with diesel fuel or water with diversion of the drainage into settling basins or pits for incorporation into the soil. 11. The F-6 trailers were tied to plumbing and pumps so that the herbicide could be delivered to the aircraft without moving the trailers. 12. As previously noted, Blue and White were not. a gummy substance formed. Orange was insoluble in water, while When Orange was mixed with either Blue or White, The F-6 trailers were therefore color-coded to correspond to the drum color-codes and used exclusively for the herbicide to which the code applied. 13. The aircraft spray tanks, positioned in the center of the airplane, and the spray system were purged before the type of herbicide carried was changed. Particular attention had to be given to sequences 12 �involving Blue and White. A mixture of these two herbicides resulted in the formation of a precipitate consisting of the sodium salt of 2,4-D. 14. Most of the personnel involved in the actual handling of the herbicide drums were Vietnamese. However, a USAF flight mechanic or crew chief was responsible for insuring that the aircraft was properly loaded and the spray system functional. operator for the spray unit. A flight mechanic was also the console The pilot and co-pilot were officers while the flight mechanics, crew chiefs and other ground support personnel were enlisted men. 15. For record keeping purposes a herbicide "mission" consisted of several aircraft; if only one aircraft was used the operation was termed a sortie. All missions within a target formed a project. 16. Aircraft takeoffs were normally before sunrise. From a tactical point of view, the arrival of the aircraft at the target area just prior to sunrise permitted the aircraft to approach the target from the direction of the rising sun. enemy ground fire. This afforded some degree of protection from From the standpoint of herbicidal action, application by aerial spray was most effective if accomplished prior to 0800 hours while inversion conditions existed, in the absence of precipitation, and while the wind was calm or not exceeding a velocity of 8 knots. This insured the proper settling of the spray on the target area. 17. Within the aircrafts, it was not uncommon to have herbicide leakage from around the numerous hose connections joining the spray tank and pumps with the wing and aft spray booms. In hot weather, the odor of herbicide within the aircraft was decidedly noticeable. Periodically, the spray tank and console were removed (especially with the portable A/A 45Y-1 system) and the interior flushed with surfactant or soap and with water. Because of the corrosive nature of some herbicides, it was necessary for the aircraft to also be repainted periodically. 18. In the 1966 through 1968 day was often co'mmon. period, more than one sortie per For example, during the first six months of 1968, 13 �the 24 UC-123B aircraft assigned to RANCH HAND averaged approximately 39 sorties per day. Exposure Considerations: Applications and Environmental Parameters There were relatively few military operations that involved the handling of herbicides by military personnel. A review of operations involving Herbicide Orange in South Vietnam from January 1962 to April 1970 revealed that there were essentially three groups of military personnel potentially exposed contaminant. to Herbicide Orange and its associated dioxin These three groups were: 1. "OPERATION RANCH HAND" defoliation program. personnel actively involved in the This group included aircrew members and maintenance and support personnel directly assigned to the RANCH HAND squadrons. 2. Personnel assigned to selected support functions that may have resulted in exposure to Herbicide Orange. This group included, for example, personnel who sprayed herbicides, using helicopters or ground application equipment; personnel who may have delivered the herbicides to the units performing the defoliation missions; aircraft mechanics who were specialized and occasionally provided support to RANCH HAND aircraft; or, personnel who may have flown contaminated C-123 aircraft, but were not assigned to RANCH HAND (e.g., during the Tet Offensive, all RANCH HAND aircraft were reconfigured to transport supplies and equipment, and were assigned to non-RANCH HAND squadrons). 3. Ground personnel who may have been inadvertently sprayed by defoliation aircraft or who, during combat operations, may have entered an area previously sprayed with Herbicide Orange. The total number of US military personnel exposed to Herbicide Orange is not known. Approximately 1,250 RANCH HAND personnel were exposed in direct support of the defoliation operations; however, there are no data on the number of non-RANCH HAND personnel who may have been exposed. The actual number of people may be in the thousands since at least 100 helicopter spray equipment units were used in South Vietnam, and most 14 �military bases had vehicle-mounted and backpack spray units available for use in routine vegetation control programs. The number of military ground personnel who may have inadvertently been sprayed with Herbicide Orange during combat operations is not known. Approximately 10 percent of South Vietnam was sprayed with herbicides, and most of this area was contested and/or controlled by enemy forces. unpopulated and forested ( 4 . 1) Most areas sprayed were remote, Because of the dense canopy cover, the target of the defoliation operation, the amount of herbicide penetrating to the forest floor would have been small. The exposure of personnel could have occurred by essentially three routes: 1. Percutaneous absorption and inhalation of vapors/aerosols by direct exposure to sprays. 2. Percutaneous absoprtion and inhalation of vapors by exposure to treated areas following spray application, and 3. Ingestion of foods contaminated with the material. The chemical and physical characteristics of Herbicide Orange and the spray, as it would have occurred following dissemination from a C-123, are important factors in assessing relative exposure to the herbicides and TCDD. Table 7 reviews the pertinent chemical and physical characteristics of Herbicide Orange. Table 8 reviews both the application parameters of the spray system used in the UC-123K aircraft and the characteristics of the spray itself. Generally, herbicides were sprayed in the early morning or late afternoon, so as to minimize the effects of air movement on particle dispersion. 15 �TABLE 7 - PERf INENT CHEMICAL AND PHYSICAL CHARACTERISTICS OF HERBICIDE ORANGE+ Formulation Concentrated 1 Kg ai/liter* Water Insoluble Density - 1.28 Vapor Pressure 3.6 x 10"4 mm Hg at 30 °C NBE** 2,4-D : 1.2 x 10"4 NBE 2,4,5-T : 0.4 x 1 ' 04 TCDD : 4.68x 10"7 Viscous 40 centipoises at 20°C Noncorrosive to metal Deleterious to paints, rubber, neoprene Long Shelf life +Source:Young et al, (15,16) *Kilograms active ingredient (2,4-D and 2,4,5-T) per liter. **NBE - Normal Butyl ester. TABLE 8 - APPLICATION PARAMETERS AND SPRAY CHARACTERISTICS OF THE C-123 MODULAR INTERNAL SPRAY SYSTEM Aircraft speed 130 KIAS* Aircraft altitude 50 m Tank volume 3,785 liters Spray time 3.5-4 minutes Particle size: 100 microns: 1.9% 100-500 microns: 500 microns: 76.2% 21.9% 87% impacted within 1 minute 13% drifted or volatilized Mean particle volume 0.61 microliters Spray swath 80+6 meters Mean deposition 28 liters/hectare Total area/tank 130 hectares Source: Darrow et al, (8) and Harrigan *Knots indicated air speed 16 (10) �Ground combat forces normally would not have been expected to have entered a previously treated area for several weeks after treatment, during which time numerous environmental factors would have reduced the potential for exposure to military personnel. An indepth review of the environmental fate of Herbicide Orange and TCDD concluded that the vast majority of the phenoxy herbicides (15). would have impacted forest canopy, the intended target The proposed scenario was as follows: Rapid uptake (e.g., within a few hours) of the ester formulations of 2,4-D and 2,4,5-T would have occurred following aerial application. Most of the herbicide probably would have undergone rapid degradation (weeks) within the cellular matrix of the vegetation. However, some of the herbicide may have remained uniaetabolized and would have been deposited on the forest floor at the time of leaf fall. action would Soil microbial and/or chemical likely have completed the degradation process. Herbicide droplets that impacted directly on soil or water would have hydrolyzed rapidly (within hours). Biological and nonbiological degradative processes would have further occurred to significantly reduce these residues. Some volatilization of the esters of 2,4-D and 2,4,5-T would have occurred during and immediately after application. The volatile material most likely would have dissipated within the foliage of the target area. Photodecomposition of TCDD would have minimized the amount of biologically active volatile residues moving downwind of the target area. Accumulation of phenoxy herbicides in animals may have occurred following ingestion of treated vegetation. The magnitude of this accumulation would have rapidly declined after withdrawal from treated feed. Most TCDD sprayed into the environment during defoliation operations would have probably photodegraded within 24 hours of application. The TCDD that escaped photodegradation would probably have entered the soilorganic complex on the forest floor following leaf fall. processes would residues. have further reduced the bioavailability Soil chemical of the TCDD Bioconcentration of the remaining minute levels of TCDD may have 17 �occurred in liver and fat of animals ingesting contaminated vegetation or soil. However, there are no field data available that indicate that the levels of TCDD likely to have accumulated in these animals would have had a biological effect. Report on Exposure Assessment by the Agent Orange Working Group In 1981, the President of the United States established the White House Agent Orange Working Group (AOWG). While the AOWG does not conduct any research, it is charged with being the overall coordinator, clearinghouse, and evaluator of the Federal research effort. In January 1986, the AOWG directed its Science Panel (a panel of senior scientists from ten Federal agencies) Orange to review pertinent and/or information its associated dioxin, on veteran exposure to Agent to examine additional pilot data developed by the U.S. Army and Joint Services Environmental Support Group, and to evaluate the feasibility of a scientifically valid epidemiologic study where the cohorts were selected on the basis of military records. The conclusions of the AOWG Science Panel were reported (16), and are quoted below: The Science Panel concluded that the U.S. Army's Environmental Support Group has sought and obtained all military records pertinent to the use of herbicides in Vietnam. The environmental Support Group staff is trained and qualified to have expertly reviewed and abstracted the records appropriate to exposure assessment. From a thorough review of these military records, it appears that considerable misclassification of the individual's exposure status is possible; i.e., we found no way, based on military records, to verify an individual's exposure to herbicide or dioxin. Two issues were specifically recognized as influencing the degree of misclassification: a. Unit Dispersion - On a substantial number of days, personnel in combat units eligible for the Agent Orange Study were not located together as a unit, rather they were dispersed geographically up to 20 kilometers on the same day. b. Incomplete Records - The most complete records for herbicide applications in Vietnam are the "HERBS TAPES," records of the missions of OPERATION RANCH HAND. These tapes, originally computerized by the National Academy of Sciences in the early 1970s, were 'supplemented recently by the SERVICES HERBS TAPES which provide additional data on perimeter applications (including 18 �helicopter and ground application missions). Expert opinion suggested that an unknown, but apparently large proportion of firebase perimeter spray operations were never recorded. The degree to which these "unrecorded" operations may have influenced exposure is unknown. After extensive review of military records during the past two years, it was apparent that the majority of veterans had never been within two kilometers of a sprayed area within a week of herbicide application. Additional pilot data reviewed at this time confirmed this finding, and the paucity of clearly exposed combat veterans makes it questionable whether a sufficient number can be assembled to conduct an epidemiological study of the type originally designed. It is clear from the available data that health studies designed to assess the effects of Agent Orange and its associated dioxin can be done on more appropriate populations than those identified through military records; e.g., industrial workers and commercial herbicide applicators. Recent advances in analytical chemistry may make it feasible to identify chemical (e.g., 2,3,7,8-TCDD) or biological (DNA adducts) markers that will permit a more reliable exposure assessment. RECOMMENDATION: This Science Panel recommends that any study of ground troops, which is dependent upon military records for the assessment of exposure to herbicides, not be conducted without an additional method to verify exposure. Conclusions The documentation on the use of herbicides in the military conflict in Southeast Asia from 1962 through 1971 is extensive. Nevertheless, the records were never intended to serve as the basis for health studies or litigation activities. Health studies of the effects of the phenoxy herbicides are difficult enough under conditions of normal agricultural use, but they become more complex when conducted with cohorts briefly exposed more than two decades ago under conditions of war in a tropical environment. 19 �References 1. Brown, J.W. 1962. Vegetational spray tests in South Vietnam. U.S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland. 119 p. Available from the defense Documentation Center, Defense Logistics Agency, Cameron Station, Alexandria, Virginia, DDC Number AD 476961. 2. Buckingham, W.A. Jr 1982. Operation Ranchhand: The Air Force and Herbicides in Southeast Asia, 1961-1971. Office of Air Force History, Washington, D.C. 253 p. 3. Cecil, P.F. 1984. The Air Force Ranch Hand Project in Southeast Asia: Operations and Consequences. Ph.D. Dissertation, Texas A&M University, College Station, Texas. 327 p. 4. Coates, J.H., L.M. Sharpe, and H. Pollack. 1962. The Present Status of Chemical Control of Vegetation in Relation to Military Needs. Technical Notes 62-68. Institute for Defense Analyses, Department of Defense, Washington, D.C. 30 p. 5. Committee on the Effects of Herbicides in South Vietnam. 1974. Part A. Summary and conclusions. National Academy of Science, Washington, D.C. 398 p. 6. Craig, D.A. 1975. Use of Herbicides in Southeast Asia. Historical Report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 p. 7. Darrow, R.A., K.R. Irish, and C.E. Minarik. 1969. Herbicides Used in Southeast Asia. Technical Report SAOQ-TR-69-11078. Directorate of Air Force Aerospace Fuels, Kelly AFB, Texas. 60 p. 8. Darrow, R.A., G.B. Truchelut, and C.M. Bartlett. 1966. OCONUS Defoliation Test Program. Technical Report 79. Crops Department, Biological Sciences Laboratory, U.S. Army Biological Center, Fort -Detrick, Frederick, Maryland. 126 p. 9. Fox, Roger P. 1979. Air Base Defense in the Republic of Vietnam, 1961-1973. Office of Air Force History, Washington, D.C., pp 55-78. 10. Harrigan, E.T. 1970. Calibration Test of the UC-123K/A/A45Y-1 Spray System. Technical Report ADTC-TR-70-36. Armament Development and Test Center, Eglin AFB, Florida. 160 p. 11. Huddle, F.P. 1969. A Technology Assessment of the Vietnam Defoliant Matter - A Case History. Report to the Subcommittee on Science Research and Development of the Committee on Science and Astronautics. U.S. House of Representatives, Ninety-first Congress. Prepared by the Science Policy Research Division, Legislative Reference Service, Library of Congress, Washington, D.C. 73 p. 12. Irish, K.R., R.A. Darrow and C.E. Minarik. 1969. Information Manual for Vegetation Control in Southeast Asia. Miscl. Public. 33. Department of the Army, Fort Detrick, Frederick, Maryland. 71 p. 13. Peterson, G.E. 1967. Hist. 41:243-253. The discovery and development of 2,4-D. Agr. 14. Tschirley, F.H. 1969. Defoliation in Vietnam - The ecological consequences of the defoliation program in Vietnam are assessed. Science 163:779-786. 20 �15. Young, A.L., J.A. Calcagni, C.E. Thalken and J.W. Tremblay. 1978. The toxicology, environmental fate, and human risk of Herbicide Orange and its associated dioxin. Technical Report OEHL-TR-78-92. USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas. 247 p. Available from National Technical Information Center, Springfield, Virginia. 16. Young, A.L. (Chairman), 1986. Report of the Agent Orange Work Group Science Subpanel on Exposure Assessment. Domestic Policy Council Agent Orange Work Group, Department of Health and Human Services, Washington, DC, 350 p. 21 �LIST OF FIGURES Figure 1. The 1974 National Academy of Sciences Computerized Map of Vietnam Showing Defoliation Missions, 19651971. Figure 2. A 1972 Map of South Vietnam Showing Provinces and Major Cities. Figure 3. Slide 1. Standard Military Transportation labelling of a 208-liter drum of Herbicide Figure 4. Slide 2. Agent Orange was procurred from eight different chemical companies and was shipped by railroad from the manufacturer to the ports at Mobile or Gulfport, Mississippi. Figure 5. Slide 3. Generally, it took 50 days for the herbicide to be shipped by sea from the United States to the ports at Saigon or DaNang, Republic of Vietnam. Orange II, produced in the late 1960's contained the isoctyl ester of 2,4,5-T in place of the n-butyl ester. Figure 6. Slide 4. At the docks in Vietnam, the herbicide drums were transferred to military cargo trailers for transport to the RANCH HAND Squadrons. Figure 7. Slide 5. Normally the contents of the drums were transferred to holding tanks (F-6 trailers or other storage tanks) or transferred directly to the aircraft. Figure 8. Slide 6. Typically, 3-5 liters of herbicide remained in the drums after pumping. This was recovered through the use of drain tanks. The residual herbicide was frequently sprayed aroung base perimeters. Figure 9. Slide 7. Empty drums were frequently discarded to a "drum dump" until disposal was accomplished. The dump at Bien Hoa, 1968, is shown in the photograph. Figure 10. Slide 8. Although most empty drums were destroyed and used in the construction of runways and bunkers, it was not uncommon to see drums used for other purposes. The photograph shows the construction of a portable shower made from 208-L drums. Figure 11. Slide 9. The "work horse" of Operation RANCH HAND was the C-123 aircraft. The photograph is of "Patches" an aircraft that survived hundreds of herbicide or insecticide missions during its seven years of service during in Vietnam. �-2Figure 12. Slide 10. The A/A 45Y-1 Internal Defoliant Dispenser was a moduler spray system for internal carriage in the C-123 aircraft. The module consisted of a 3.785liter tank, pump, and engine mounted on a frame pallet. An operator's console was an integral part of the unit but was not mounted on the pallet. Figure 13. Slide 11. The RANCH HAND C-123 aircraft had spray booms mounted under each wing and behind the cargo door. Each wing boom was 6.5m in length and mounted with 16 nozzles. Figure 14. Slide 12. A typical RANCH HAND Crew consisted of a pilot and Co-pilot (officers) and a flight mechanic (enlisted) who served as a console operator for the spray unit. Figure 15. Slide 13. aircraft. A typical RANCH HAND mission consisted of 3-5 Figure 16. Slide 14. RANCH HAND Aircraft timed the arrival of the aircraft at a target site just prior to sunrise, thus, permitting the aircraft to approach the target from the direction of the rising sun. This afforded some degree of protection from enemy ground fire. Figure 17. Slide 15. Defoliation missions along canals and roadways wre common targets. The photograph shows a defoliated area associated with a canal. Figure 18. Slide 16. The inland forests of Vietnam were defoliation mission targets aimed at dislocating enemy forces. This "swath" was defoliated by at least a five-aircraft formation spraying a distance of almost 13 Km. The herbicides of choice for such an operation were either White or Orange. Figure 19. Slide 17. The U.S. Army's UH-1 helicopter was ideal for base perimeter spraying of herbicides. Figure 20. Slide 18. The typical spray system for helicopters consisted of a 750-liter tank, spray boom and windmill pump. Figure 21. Slide 19. Helicopter spraying Agent Blue on the tall elephant grass on a base perimeter. Figure 22. Slide 20. Military personnel in a area defoliated by herbicides. Typically, personnel did not enter a defoliated area until the effects of defoliation were apparent and visibility within the area was significantly enhanced. � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 017 Folder The folder containing the original item. 0191 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 Young, Alvin L. Title A name given to the resource Manuscript: Chapter 2: The Military Use of Herbicides in Vietnam, "Massive Quantities of Herbicides Were Applied by the United States in a Tactical Operation Designed to Reduce Ambushes and Disrupt Enemy Tactics" Subject The topic of the resource Ranch Hand herbicide application dioxin https://www.nal.usda.gov/exhibits/speccoll/files/original/3f4bef0956a64c1c127a1b2894821266.pdf 3181b7d44f6de47aef9776417196ede7 PDF Text Text Item ID Number 00193 Author Young, Alvin L. Corporate Author Department of Chemistry and Biological Sciences, USA Report/Article TitlO Fate of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCCD) in the Environment: Summary and Decontamination Recommendations Journal/Book Titlo Year Month/Day Color Number of Images October [J 49 Monday, January 22, 2001 Page 205 of 341 �USAFA-TR-76-18 FATE OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIQXlN (TCDD) IN THE ENVIRONMENT: SUMMARY AND DECONTAMINATION RECOMMENDATIONS CAPTAIN ALVIN L. YOUNG MAJOR CHARLES E. THALKEN LT COLONEL EUGENE L. ARNOLD CAPTAIN JAMES M. CUPELLQ MAJOR LORRIS G. COCKERHAM DEPARTMENT OF CHEMISTRY AND BIOLOGICAL SCIENCES USAF ACADEMY, COLORADO 80840 OCTOBER 1976 APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED Prepared for: HEADQUARTERS AIR FORCE LOGISTICS COMMAND WRIGHT-PATTERSON AIR FORCE BASE, OHIO 4S433 DEAN OF THE FACULTY UNITED STATES AIR FORCE ACADEMY COLORADO 80840 �Editorial Review by Lt Colonel J. M. Shuttleworth Department of English and Fine Arts USAF Academy, Colorado 80840 This research report is presented as a competent treatment of the subject, worthy of publication. The United States Air Force Academy vouches for the quality of the research, without necessarily endorsing the opinions and conclusions of the author. This report has been cleared for open publication and/or public release by the appropriate Office of Information in accordance with AFR 190-17 and DODD 5230.9. There is no objection to unlimited distribution of this report to the public at large, or by DDC to the National Technical Information Service. This research report has been reviewed and is approved for publication. PHILIP J/QZRDLET Colonel, USAF Vice Deah of the Faculty Additional copies of this document are available through the National Technical Information Service, U. S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22151. �UNCLASSIFIED SECURITY CLASS'FICATION OF THIS »AGE (When Data Entered) READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 1. REPORT NUMDER 2. GOVT ACCESSION NO 3. RECIP'FN'T'S CATALOG NUMBER USAFA-TR-76-18 4. TITLE (and Subtitle) 5. TYFE OF REPORT ft PERIOD COVERED Fate of 2,3,7,8-TetracMortdibenzo-p--dioxin (TCED) in the Environment: Surtmary and Decontamination Recxxtmendations Summary Report 6. PERFORMING ORG. REPORT NUMBER 8. 7. AUTHOR?.) Alvin L. Young, Capt, USAF, PhD; Charles E. Thalken, Maj, USAF, VC, DVM, MS; Eugene L.Arnold, LtCpl, USAF, BSC, PhD; James M, Cupello, Capt, USAF, PhD; Lorris G. CocTcerham, Maj, USAF, MS CON1 RACT OR GRANT NUMBERf»> 10. PROGRAM F.LEMENT. PROJECT, TASK AiHTA ft WORK UNIT NUMBERS 9. PERFORMING ORGANIZATION N A M E AND ADDRESS Department of Chemistry and Biological Sciences DFCBS-R USAF Academy, Colorado 80840 t. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE October 1976 Department of Chemistry and Biological Sciences DFCBS-R USAF Academy, Colorado 80840 13. NUMBER OF PAGES 14. MONITORING AGENCY NAME & ADDRESSf// different from Controlling Otlice) 15. SECURITY CLASS, (of thla report) 44 UNCLASSIFIED 15«. DECLASSIFICATION/DOWNGRADING SCHEDULE 6. DISTRIBUTION STATEMENT (ot thla Report) Approved for public release: distribution unlimited. 7. DISTRIBUTION STATEMENT (of the abstract entered In Block 20, II different from Report) 8. SUPPLEMENTARY NOTES 9. KEY WORDS (Continue on reverse aide It necessary and Identity by block number) Animal SUTVey; Aquatic Studies; Bic>accumulation; Biodegradation of Herbicides; Biodegradation of TCDD; Ecological Effects; 2,4-dichlorophenoxyacetic acid (2,4-D); Fish Studies; Herbicide; Histopathology; Insect Studies; Maranals; Necropsy; Orange; Reptile Study; Soil Microbial Studies; TCDD; Teratogenic; 2,3,7,8-tetxochlorodibenzo-pdioxin (TCDD); Test Area C-52A, Eglin AFB Reservation; 2,4,5-trichlorophenoxyanifl \rf f ^ * *rf__ jfr f T Vegetative Succession. • —• irtffrYftrr H-hM HiVfAM ^r^-VT^_ _* ^y VMV^ ^ * ^- - «''*^^ *' -. *"^ ' " — -..,..-— ———.... , —, 0. ABSTRACT (Continue on rovora* aide (/ ri «vt** «ry ant/ I ((entity by bjpck number) . . /msmr\\ i* U.^ Studies on the fate of 2,3,7,8-tetrachiorodibenzo-p-dioxin (TCDD) have been conducted on biodegradation plots and field test areas that have received massive quantities of Orange herbicide (a 50:50 mixture of the n-butyl esters of 2,4lichlorophenoxyacetic acid [ , 4 D and 2,4,5-trichlorophenoxyacetic acid 2'-] 2,4,5-T]). From the studies reviewed in this report, it is apparent that 1) TCDD may persist (in biotic and abiotic components) for long periods of time when initially present at extremely high concentrations on the soil surface, 2) TCDD will accumulate in the tissues of rodents, reptiles, birds, fish, and W DD , FORM73 1473 JAN M >l EDITION OF 1 NOV 65 IS OBSOLETE Mg F i rs UNCLASSIFIED �SECURITY CLASSIFICATION OF THIS PAGEfWhen Data Entered) 20. Abstract (Continued) insects when these organisms are exposed to TCDD contaminated soils (however, the levels of TCDD in the tissues apparently do not exceed the levels of TCDD found in the environment), (3) organisms tolerate, i.e., based on no observed deleterious effects, soil levels between 10-1,500 ppt TCDD, (4) TCDD is degraded by soil microorganisms, especially when in the presence of other chlorinated hydrocarbons, (5) TCDD is degraded in the presence of sunlight, (6) movement of TCDD in the abiotic portions of the environment can be by wind or water erosion of soil particles, but leaching by water alone does not appear to occur, and (7) TCDD is probably not readily released or degraded in the environment when bound to activated coconut charcoal. SECURITY CLASSIFICATION OF THIS PAGE(Wien Date Entered) �TABLE OF CONTENTS Title Page Introduction 1 Soil Incxjrporation/Biodegradation Studies 6 Fate of TCDD in an Ecosystem Geographical and Vegetative Features Sampling Grids and Herbicide Deposition Preliminary Ecological Studies Soil Studies of TCDD Residues Rodent Studies Trapping Data/Histopathology Liver and Pelt Analysis Burrow and Diet Studies TCDD Laboratory Uptake Experiment Hepatic Ultrastructural Study Reptile Studies TCDD in Aquatic Organisms TCDD in Birds of TA C-52A Vegetative Succession Studies on TA C-52A 17 ... 18 18 18 .21 23 23 25 25 26 27 29 30 31 32 Laboratory and Greenhouse Experiments with TCDD 34 Recormiendations 39 �LIST OF TABLES Number 1 2 3 Page Analyses of the Top 15-on Layer From Each of the Soil Biodegradation Sites 7 Descriptions of Three Biodegradation Studies Involving Use of Herbicide Orange 8 Concentrations of Herbicide Orange and TCDD in Plots Originally Treated with 4,480 kg/ha, AFLC Test Range Complex, Utah, at Various Sampling Dates After Application. (TCDD in parts per billion) 9 4 Concentrations of Herbicide Orange and TCDD in Plots Originally Treated with 4,480 kg/ha, Garden City, Kansas, at Various Sampling Dates After Application. (TCDD in parts per trillion) . 9 5 Concentrations of Herbicide Orange and TCDD in Plots Originally Treated at 4,480 kg/ha, Eglin AFB, Florida, at Various Sampling Dates After Application 10 6 Movement of Herbicide Orange and TCDD in a Soil Profile, Eglin AFB, Florida. (TCDD in parts per trillion) 12 7 Comparison of Herbicide Orange Degradation Rates in Plots at the Eglin AFB, Florida, Site, Receiving Either Herbicide, Herbicide Plus Soil Amendments, or Herbicide Plus Amendments and Charcoal .14 8 Approximate Amounts of 2,4-D and 2,4,5-T Herbicides Applied to Test Area C-52A, Eglin AFB Reservation, Florida 9 10 11 19 Concentration of TCDD in Soil Profile (1974) of Grid I, Test Area C-52A, Eglin AFB, Florida 22 Numbers of Beach Mice Collected During the 1973 and 1974 Studies of Test Area C-52A 22 Concentration (Parts Per Trillion) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Liver and Pelt Samples from Beach Mice, Peromyscus polionotus, Collected from Control and TCDD-Exposed Field Sites, 1973 and 1974 24 11 �UST OF TABLES (Continued) Nottogr 12 13 14 Page Concentration (Parts Per Trillion) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Liver and Pelt Samples from Beach Mice, Peronyscus polionotus, Dusted with Alumina Gel Containing No TCDD (Control) or 2.5 Parts Per Billion TCDD (Test) 28 Concentration (Parts Per Trillion) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Composite Samples of Viscera or Trunk from Six-Lined Racerunners, Cnemidophorus sexlineatus, Collected from Control and TCDD-Exposed Field Sites 28 Degradation of TCDD (Parts Per Trillion) in a Greenhouse Experiment, Eglin AFB, Florida 37 111 �INTRODUCTION The heterocyclic organic molecule 2,3,7,8-tetrachlorc-dibenzop-dioxin (TCDD) has received a great deal of attention in the last 6 years because of its highly toxic properties and the possibility of it being widespread in the environment by the use of products made from trichlorophenol, especially the herbicide 2,4,5trichlorophenoxyacetic acid (2,4,5-T). Although TCDD may occur as a contaminant in products made from trichlorophenol, the levels of TCDD found in any given lot of trichlorophenol is dependent upon the manufacturing process. TCDD may be produced as a by-product during an alkaline hydrolysis reaction when the temperature for making 2,4,5-trichlorophenol from tetrachlorobenzene exceeds 160°C. However, there is less likelihood of TCDD formation in the manufacturing process which starts with phenols and chlorinates them to form trichlorophenol since little or no heat is required in this reaction. Public interest in TCDD originated in 1970 when the herbicide 2,4,5-T was implicated as a potential teratogen in pregnant rats ( ) Later tests indicated that the teratogenesis may have been 1. caused by 27 ± 8 ppm of TCDD present as a contaminant in the 2,4,5-T. As more data have been obtained (2), it has become apparent Courtney, K.D., D.W. Gaylor, M.D. Hogan, J.L. Falk, R.R. Bates, and I. Mitchell. Teratogenic evaluation of 2,4,5-T. Science 168:864-866, 1970. 2 Schwetz, B.A., J.M. Norris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Oner son, and C.G. Gerbig. Toxicology of chlorinated dihenzo-pdioxins. Biviron. Hlth. Perspect., Experimental Issue No. 5:87-100, September 1973. �that TCDD is one of the most toxic chemicals known; the oral LD5Q for many animal species is in the range of micrograms per kilogram. Purthermore, the known effects of TCDD include anorexia, severe weight loss, hepatotoxicity, hepatoporphyria, vascular lesions, chloracne, gastric ulcers, and teratogenicity ( ) The 2. hazard posed by the presence of even a small amount of this substance in the environment has therefore been of concern. For a person or animal to be poisoned with TCDD, a rare set of circumstances would be required. Since present production methods are able to reduce the TCDD level to less than 0.1 ppm, it is unlikely that contaminated 2,4,5-T herbicide or even contaminated trichlorophenol would be implicated in such a poisoning. Nevertheless, two accidental poisoning episodes involving TCDD have been recently reported. In 1975, Carter et al. (3) identified TCDD as the apparent cause of an outbreak of poisoning in humans, horses, and other animals on a horse breeding farm in eastern •H .• , Missouri in 1971. Exposure to TCDD followed the spraying of contaminated industrial waste oil on riding arenas for dust control. An investigation concluded that a hexachlorophene (made from trichlorophenol) factory in southwestern Missouri had accumulated distillate residues containing 306 to 356 ppm TCDD. It was this distillate residue that was subsequently disposed of via a salvage oil company and sprayed on the horse arenas. 3 Carter, C.D., R.D. Kiiribrough, J.A. Liddle, R.E. Cline, M.M. Zack, Jr., W.F. Barthel, R.E. Koehler, and P.E. Phillips. Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188:738-740, 1975. �The second incident of TCDD poisoning occurred in July 1976 in Seveso, Italy ( ) The source of the TCDD was a chemical 4. factory that produced trichlorophenol through the alkaline hydrolysis of tetrachlorobenzene. When the temperature in a steamheated reaction vessel rapidly increased, a.safety disk ruptured sending a plume of trichlorophenol, TCDD anici other products 30 to 50 m high above the factory. The cloud apparently rose into the air, cooled, and came down over a cone-shaped area about 2 km long and 700 m wide. An area of 110 hectares (ha) was evacuated after hundreds of animals had died and many people had reported skin disorders. Several measurements of TCDD on vegetation in an area adjacent to the factory were in the 1 to 15 ppm range, with one reading as high as 51.3 ppm. An Italian government commission (5) recommended: "removal of topsoil to a depth of 10 cm in an area of 113 ha, the. dismantling of all buildings in the Seveso area, and the total disruption of all wildlife." The need for data on the fate of TCDD in the environment is not confined to solving problems associated with the above two incidents. During the latter portion of the last decade, a program of aerial application of herbicides was conducted in Southeast Asia by the United States Air Force. In 1969, at the conclusion of this program, considerable amounts of herbicide were left unused. 4 Rawls, R.L., and D.A. O'Sullivan. Italy seeks answers following toxic release. Chem. Engr. News 54(35):27-35, August 23, 1976. Itay, A. Toxic cloud over Sevesco. Nature 262(5570):636-638, August 19, 1976. �One of the herbicides used extensively in this project was a herbicide designated "Orange" which was formulated as a 50:50 mixture of the n-butyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-T. In 1970, approximately 2.3 million gallons of this material was placed in storage by the Air Force. An analysis of TCDD in the Orange herbicide stocks (6) indicated that the range in concentration was 0.1 to 47 ppm TCDD. The weighted average concentration of TCDD for the 42,015 55-gallon drums of herbicide was 1.859 ppm. Because of the TCDD concentration, the herbicide could not merely be declared surplus and disposed of on the agricultural markets. Many methods have been evaluated for disposing of this material. However, regardless of the final method selected for its disposition, the storage sites where the material is currently stored (Naval Construction Battalion Center, Gulfport, Mississippi, or Johnston Island, Pacific Ocean) will need to be decontaminated. At the request of Headquarters, Air Force Logistics Command, Wright-Patterson AFB, Ohio, in April 1972, the Department of Chemistry and Biological Sciences, United States Air Force Academy, initiated studies on herbicide Orange and TCDD. The objectives of these studies were: (1) to investigate soil incorporation/ biodegradation as a disposal method for herbicide Orange; (2) to investigate the ecological effects associated with past uses of Department of the Air Force. Disposition of orange herbicide by incineration. Final Environmental Statement, November 1974, pp. 36-37. �herbicide Orange; and (3) to investigate the soil persistence and food chain accumulation of TGDD. This report documents the available data on TCDD from these studies. Furtherxnore, using these data, recommendations for decontamination of an area exposed to TCDD are presented. �SOIL INCORPORATION/BIODEGRADATION STUDIES One potential method proposed for the disposal of herbicide Orange was subsurface injection or soil incorporation of the herbicide at massive concentration rates. The premise for such studies was that high concentrations of the herbicides and TCDD would be degraded to innocuous products by the combined action of soil microorganisms and soil hydrolysis. In order to field test this concept, biodegradation plots were established in three climatically different areas of the United States; Northwest Florida (Eglin AFB), Western Kansas (Garden City), and Northwestern Utah (Air Force Logistics Command Test Range Complex). A comparison of the soils of the three sites is given in Table 1. The Utah site had a mean annual rainfall of 15 on, while the Kansas and Florida sites had 40 and 150 cm, respectively. Table 2 describes the experimental protocol for the three sites to include when the plots were established, the method of herbicide incorporation, the experimental design and the initial calculated herbicide concentration, ppm, at the time the plots were established. Further details on the experimental protocol can be obtained from Young, Arnold and Wachinski ( ) 7. Tables 3, 4, and 5 compare the rate of disappearance of TCDD with that of Orange herbicide for selected plots at the Utah, Young, A.L., E.L. Arnold, and A.M. Wachinski. Field studies on the soil persistence and movement of 2,4-D, 2,4,5-T, and TCDD. Appendix G. Department of the Air Force. Disposition of orange herbicide by incineration. Final Environmental Statement, November 1974. �TABLE 1. ANALYSES OF THE TOP 15-CM LAYER FROM EACH OF THE SOIL BIODEGRADATION SITES ORGANIC MATTER (%) SAND (%) SILT (%) CLAY (%) 5.6 0.5 91.6 4.0 4.4 Garden City, KS^ 7.0 1.7 37 42 21 Silt loam AFLC Test Range Complex, UT0 7.8 1.4 27 53 20 Clay loam LOCATION pH Eglin AFB, FLa SOIL DESCRIPTION Sandy loam located on Test Area C-52A, Eglin AFB Reservation, Florida T>lots located on the Kansas Agricultural Experiment Station, Garden City, Kansas °Plots located 75 miles west of Salt Lake City, Utah �TABLE 2. LOCATION Eglin AFB, Florida CO Garden City, Kansas DESCRIPTIONS OF THREE BIODEGRADATION STUDIES INVOLVING USE OF HERBICIDE ORANGE DATE ESTABLISHED 2 Apr 1972 10 May 1972 AFLC Test 2 Oct 1972 Range Complex, Utah METHOD OF INCORPORATION TREATMENT CALCULATED INITIAL HERBICIDE CONCENTRATION (PPM)C 4,480 kg Herbicide/haa 4,480 kg Herbicide/ha, plus soil amendments^ 4,480 kg Herbicide/ha plus soil amendments and activated charcoal 5,000 5,000 Preplant Incorporate (Rototiller) 2,240 kg Herbicide/ha 4,480 kg Herbicide/ha 1,000 2,000 . Simulated Subsurface Injection (8 cm band width) 1,120 kg Herbicide/ha 2,240 kg Herbicide/ha 4,480 kg Herbicide/ha Simulated Subsurface Injection (30 cm band width) 5,000 10,000 20,000 40,000 of herbicide calculated as active ingredient. Herbicide injected at 10-15 cm level or preplant incorporated in the 0-15 cm level. All plots duplicated. xhe amendments included 4.5 kg lime, 13.5 kg organic matter, and 1.4 kg fertilizer (12:4:8 for N,P,K, respectively) uniformly mixed within the top 0-30 cm of soil in the plot. °Contained in the top 0-15 cm layer. �TABLE 3. CONCENTRATIONS OF HERBICIDE ORANGE AND TCDD IN PLOTS ORIGINALLY TREATED WITH 4,480 KG/HA, AFLC TEST RANGE COMPLEX, UTAH, AT VARIOUS SAMPLING DATES AFTER APPLICATION. (TCDD IN PARTS PER BILLION) DAYS AFTER APPLICATION TOTAL HERBICIDE3 (PPM) TCDD ., (PPMxlO ) 282 8,490 15.0 637 4,000 7.3 780 2,260 5.6 1,000 2,370 3.2 1,150 1,150 2.5 a Composite sample from replicated plots, 0-15 on increment TABLE 4. CONCENTRATIONS OF HERBICIDE ORANGE AND TCDD IN PLOTS ORIGINALLY TREATED WITH 4,480 KG/HA, GARDEN CITY, KANSAS, AT VARIOUS SAMPLING DATES AFTER APPLICATION. (TCDD IN PARTS PER TRILLION) DAYS AFTER APPLICATION TOTAL HERBICIDE3 (PPM) TCDD3 , (PPMxlO~°) 8 1,950 ~b 77 1,070 225 189 362 210 600 40 659 a 490 <:L —b —b —b 42 Composite sampling from replicated plots, 0-15 cm increment HSbt determined �TABLE 5. CONCENTRATIONS OF HERBICIDE ORANGE AND TCDD IN PLOTS ORIGINALLY TREATED AT 4,480 KG/HA, EGLIN AFB, FLORIDA, AT VARIOUS SAMPLING DATES AFTER APPLICATION DAYS AFTER APPLICATION TOTAL, HERBICIDE (PPM) TCDDa , (PPMxlO~b)C 5 4,897 375 414 1,866 250 513 824 75 707 508 46 834 438 -b 1,293 <10 b — Composite sample from the plot containing only herbicide (i.e., no lime, organic matter, or fertilizer added). Sample from the 0-15 cm increment. Analysis not completed. °TCDD in parts per trillion. 10 �Kansas, and Florida sites, respectively. Although the number of analyses have been limited, the data have indicated that TCDD (and phenoxy herbicide) degrade more rapidly in the Kansas soils (Ulysses Silt Loam) than in the Florida soils (Lakeland Sandy Loam), and least rapidly in the Utah desert soils (Lacustrine Clay Loam). The levels of TCDD and herbicide in a soil profile from one of the Bgl.in AFB, Florida, biodegradation plots are shown in Table 6. Initially (e.g., day 414), the data indicate that both the herbicide and the TCDD may be leaching down into the lower soil increments. However, note that the analysis for herbicide in a soil profile obtained on day 557 shows no leaching. The method of collecting soil samples, i.e., by the use of a soil auger contaminated the lower soil increments whereas the trenching technique showed no contamination. The analysis of soil profiles at all three locations for biodegradation indicated that neither the herbicide nor the TCDD appreciably penetrated below the 15-30 cm level. Thus, we believe that the disappearance of the herbicide and the TCDD can be attributed to the action of soil microorganisms, rather than leaching. Data for TCDD are not available at this time (analysis in progress) on the influence of soil amendments (e.g., lime, fertilizer, and organic matter) on the degradation of TCDD in the Eglin AFB, Florida, biodegradation study. However, preliminary indications are that the addition of these amendments in these Florida soils does appear to slightly enhance herbicide degradation. On the other hand, the presence of activated coconut charcoal in 11 �TABLE 6. MOVEMENT OF HERBICIDE ORANGE AND TCDD IN A SOIL PROFILE, EGLIN AFB, FLORIDA. (TCDD IN PARTS PER TRILLION) DAYS AFTER APPLICATION3 4l4b 557C HERBICIDE (PPM) DEPTH (CM) HERBICIDE (PPM) 0-15 1,866 250 824 15-30 263 50 11 30-45 290 <25d <10d 45-60 95 <25d <10d 60-75 160 <25d <10d 75-90 20 <25d <10d TCDD ,. (PPMxlO ) a Composite sample from the plot containing only herbicide r~ Increments obtained by use of a soil auger having cup dimensions of 7.6 x 15.2 on, diameter and length, respectively £i Increments obtained by use of porcelain spatula from the side of 90 cm deep trench < T)etection limit 12 �the Eglin plots, at the 12 on level, prevented degradation of the herbicide and probably also prevented degradation of the TCDD. These data are shown in Table 7. In no instance can it be shown that TCDD levels reached a non-detectable level (less than 10 parts per trillion) within the designated time periods (see Tables 3, 4, and 5). Although biodegradation appears to reduce the level of herbicide and TCDD, the data did not follow simple exponential decay curves. For the mixture 2,4-D and 2,4,5-T herbicides, disappearance was rapid initially, but slowed substantially in the later portions of the test period. With this type of decay kinetics, meaningful half lives are difficult to calculate; however, a reasonable estimate appears to be in the range of 150-210 days. The degradation of TCDD followed a similar decay pattern. However, it appears at this time that the decreased rate of degradation of TCDD as a function of time may be even more pronounced than for the herbicides. One might speculate that the enzymes responsible for herbicide metabolism are inducible and also are involved in TCDD breakdown. If this is the case, it is not surprising that TCDD metabolism slows or ceases when the initial massive concentrations of herbicide are removed. Microbial studies have been conducted in the biodegradation plots in both Utah and Florida ( , ) For the Utah plots, 89. Q Stark, H.E., J.K. McBride, and G.F. Orr. Soil incorporation/ biodegrada.tion of herbicide orange. Vol I. Microbial and baseline ecological study of the U.S. Air Force Logistics Command Test Range, Hill AFB, Utah. Document No. DPG-FR-C615F, US Army Dugway Proving Ground, Dugway, Utah 84022, February 1975. 13 �TABLE 7. COMPARISON OF HERBICIDE ORANGE DEGRADATION RATES IN PLOTS AT THE EGLIN AFB, FLORIDA, SITE, RECEIVING EITHER HERBICIDE, HERBICIDE PLUS SOIL AMENDMENTS, OR HERBICIDE PLUS AMENDMENTS AND CHARCOAL TREATMENT HERBICIDE PLUS HERBICIDE PLUS AMENDMENTS 3 HERBICIDE AND CHARCOAL" AMENDMENTS DEPTH (CM) DEPTH (CM) DEPTH (CM) DAYS AFTER 0-15 15-30 0-15 15-30 0-15 15-30 APPLICATION (PPM) (PPM) (PPM) (PPM) (PPM) (PPM) 5 4,897 302 5,703 232 3,074 134 98 4,280 580 5,422 <50 414 1,866 263 2,015 193 2,767 c <50 c 463 1,217 222 c 824 11 161 c c 557 1,796 c 707 508 <10 c c 2,660 c <50 c 834 438 <10 184 <10 c c 1,293 <10 <10 <10 <10 1,556 <10 amendments included 4.5 kg lime, 13.5 kg organic matter, and 1.4 kg fertilizer (12:4:8 for N,P,K, respectively) uniformly mixed within the top 0-30 cm of soil in the plot. A 1 cm layer of activated coconut charcoal was applied to the trench prior to application of the herbicide. ^ °Not determined. 14 �samples were taken three times throughout the year (summer, winter and spring, 1973-1974), and nacrobial species present (bacteria, actlncmycetes and fungi) were determined. Bacterial counts were higher for soils with greater concentrations of the herbicide and with greater moisture content, but the herbicide, in any concentration, had no significant effect on the mycoflora. For the Florida plots (9), soil samples were taken from all plots in June and August 1974, and in April 1975. Although bacterial and fungal levels were similar for control plots or plots receiving either herbicide or herbicide plus the soil amendments lime, fertilizer, and organic matter, the levels were significantly higher in the plots receiving the activated charcoal. Microorganisms tended to be concentrated in the level which contained the charcoal (0-15 cm), but greatly reduced in number at depths immediately below the charcoal. This effect of increasing the number of microorganisms may have been due to adsorption of growth promoting substances (e.g., nutrients and water) on the surface of the charcoal particles. Although the number of organisms were greater in these plots, the level of herbicide residue was also greatest (Table 7). Apparently, the binding of the herbicide by the charcoal prevented it from being degraded by the microorganisms. These two microbial studies have shown that the application of 2,4-D and 2,4,5-T at massive rates (5,000-40,000 ppm) not only Q Cairney, W.J. Determination of soil microorganism populations in the Eglin AFB, Florida, biodegradation plots. Department of Chemistry and Biological Sciences, United States Air Force Academy, CO, 1975, unpublished. 15 �does not sterilize the soil, but indeed stimulates the growth of certain rnicroflora. That these bacteria, actinomycetes and fungi are proliferating to such an extent indicates that they are probably using the herbicide and TCDD as a carbon source (the exception being the charcoal plots at Eglin), and, as such, are conjxibuting to their degradation. 16 �FATE OF TCDD IN AN ECOSYSTEM The biodegradation plots offered little opportunity to evaluate the ecological effects associated with the use of herbicide Orange or to investigate food chain acramulation of TCDD. Although studies were conducted on the microorganisrns, plants and dominant resident vertebrate and invertebrate animals on and adjacent to these plots (8,9), they were limited to studies of less than 0.5 ha. Therefore, concurrent with the biodegradation studies/ an investigation was initiated on the much larger ecosystem (terrestrial and aquatic) of a unique military test area in Northwest Florida. In support of programs testing aerial dissemination systems, Test Area (TA) C-52A, Eglin AFB Reservation, Florida, received massive quantities of military herbicides. The purpose of these test programs was to evaluate the capabilities of the equipment systems, not the biological effectiveness of the various herbicides. Nevertheless, after several applications, test personnel began to express concern over the potential ecological and environmental hazards that might be associated with continuance of the test program. This concern led to the establishment of a research program in the fall of 1967 to measure the ecological effects produced by the various herbicides on the plant ccnniunity of TA C-52A (10). Ward, D.B. Ecological records on Eglin AFB Reservation—the first year. AFATL-TR-67-157, Air Force Armament Laboratory, Eglin AFB, Florida, 1967. 17 �Geographical and Vegetative Features In 1962, the Armament Development and Test Center (ADTC), Eglin AFB, Florida, established an elaborate testing installation designed to measure deposition parameters of aerially applied herbicides on the Eglin Reservation. The direct aerial application 2 was restricted to an area approximately 2.6 km within TA C-52A in the southeastern part of the reservation. The entire test area 2 covers approximately 5 km and is a grassy plain surrounded by a forest stand that is dominated by long leaf pine (Pinus palustris), sand pine (Pinus clausa), and turkey oak (Quercus laevis). The actual area of test flight paths and herbicide application is in 4 a mechanically cleared area now occupied mainly by broomsedge (Andropogon virginicus), switchgrass (Panicum virgatutn), and other low growing herbaceous vegetation. Sampling Grids and Herbicide Deposition Four separate test grids were established on the 2.6 km2 test area during the 1962 through 1970 testing period. Table 8 indicates the approximate total amount of herbicides (active ingredients) applied on each test grid and the time periods of those applications. Preldmnary Ecological Studies The first in depth animal survey was initiated on the herbicide equipment test grids and surrounding area in 1970 ( 1 . This 1) T>ate, B.D., R.C. Voight, P.J. Lehn, and J.H. Hunter. Animal survey of test area C-52A, Eglin AFB Reservation, Florida. AFATLTR-72-72, Air Force Armament Laboratory, Eglin AFB, Florida, April 1972. 18 �TABLE 8. APPROXIMATE AMOUNTS OF 2,4-D AND 2,4,5-T HERBICIDES APPLIED TO TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA TEST GRID GRID AREA (HECTARES) 1 37.25 39,540 (1962-1964)a 39,540 (1962-1964) 2 37.25 15,885 (9416) 16-96 15,885 (1964-1966) 3 37.25 4 97.0 HERBICIDES (KILOGRAMS ACTIVE INGREDIENT) 2,4-D 2,4,5-T 1,263 (97 16) 19,959 (9817) 16-90 19,959 (9817) 16-90 When the major portion of the herbicide was applied. 19 �survey was conducted during the time that aerial spray equipment was actively being tested. The purpose was to determine species variation and distribution patterns on the test grids and surround2 ing 28.5 km . Of the 86 species of vertebrate animals observed or collected, it was concluded that the beach mouse (Beromyscrus polionotus) and the six-lined racerunner (Cnemidophorus sexlineatus) were present in sufficient numbers to conduct population studies. In the spring of 1973, analyses of random soil samples from the test area indicated that low levels (e.g., parts per billion or parts per trillion) of TCDD were persisting in areas (i.e., the flight paths) that had received repetitive aerial applications of 2,4,5-T. Based on the beach mouse populations and the residue information a study was initiated in the summer of 1973 to obtain rodents for analysis of TCDD in body tissues and for examination of TCDD in body tissues and for examination of gross and microscopic evidence of teratogenic and mutagenic effects. A trapping survey was also conducted to study habitat preference of the beach mouse to determine if population distribution was related to vegetative cover. Data from these studies (12) indicated a correlation between the levels of TCDD in rodent liver and soil; however, there was no evidence of toxic histopathology in any rodent tissue. It was also found that indeed the population distribution was related to vegetative cover. *oung, A.L. Ecological studies on a herbicide-equipment test area (TA C-52A) Eglin AFB Reservation, Florida. AFATL-TR-74-12, Air Force Armament Laboratory, Eglin AFB, Florida, 1974. 20 �In the sunnier of 1974 a team of military and civilian scientists undertook a more extensive investigation of the numerous components of the ecosystem of TA C-52A. Using the information from previous studies, they obtained data on the fate of TCDD in soils, rodents, reptiles, aquatic organisms, birds, and vegetation. These results have been published by Young, Thalken and Ward (13), and are summarized in the following sections of this report. Soil Studies of TCDD Residues Soil samples (the top 0-15 cm increment) were collected from all four of the test grids on the test area and analyzed for TCDD. With the exception of Grid I, TCDD residues were in the range of <10 (minimum detection limit) to 30 parts per trillion (ppt, 1x10-12) Soil analysis of 20 separate samples from Grid I detected levels of TCDD in the range of <10 to 1,500 ppt. This wide fluctuation in TCDD concentrations was attributable to the locations of the actual flight paths on the test grid ( . . not all of the 37 ha ie, received the same amount of aerially applied herbicide). It was • also apparent that the massive amounts of herbicides applied to this area in 1962-1964 contained very high levels of the TCDD contaminant. Further analysis of a duplicate soil core, obtained from a site having 110 ppt TCDD, indicated that TCDD was stratified within this top 0-15 cm of soil (Table 9). Young, A.L., C.E. Thalken, and W.E. Ward. Studies of the ecological impact of repetitive aerial applications of herbicides on the ecosystem of test area C-52A, Eglin AFB, Florida. AFATLTR-75-142, Air Force Armament Laboratory, Eglin AFB, Florida, 1975. 21 �TABLE 9. CONCENTRATION OF TCDD IN SOIL PROFILE ( 9 4 17) OF GRID I, TEST AREA C-52A, EGLIN AFB, FLORIDA SOIL PROFILE GRID I APPLICATIONS OF HERBICIDES ( 9 2 1 6 ) 16-94 PARTS PER TRILLION ( P ) TCDD PT 0-2.5 on 150 2.5-5 on 160 5-10 on 700 10-15 on 44 Below (15-90 on) None detectable TABLE 10. NUMBERS OF BEACH MICE COLLECTED DURING THE 1973 AND 1974 STUDIES OF TEST AREA C-52A CONTROL 1973 1974 TOTAL Male 5 12 17 Female 5 10 15 12 11 33_ Fetuses Subtotal 65 TEST 1973 1974 TOTAL Male 26 17 43 Female 18 13 31 Fetuses 25 9 34 Subtotal 108 TOTAL 173 22 �Rodent Studies TRAPPING DATA/HISTOPATHOLOGY. In the 8 weeks of trapping beach mice during the summer of 1973 and 6 weeks during the summer of 1974, 106 specimens were collected from Grid I. Many of the females were pregnant at the time of collection, providing 67 fetuses for examination. Table 10 indicates the numbers of males, females and fetuses collected from Grid I during 1973 and 1974. The only significant lesions seen on histopathologic examinations of 173 adult and fetal beach mice were two instances of moderately severe, multifocal, necrotizing, hepatitis (one test, one control animal) and a single test mouse with severe venous ectasia of the renal veins in one kidney. All other lesions were of the minor or insignificant type normally observed in microscopic surveys of large numbers of field animals. The absence of liver lesions (necrosis and porphyria) in mature animals that had liver levels of TCDD from 20 ppt to 1,300 ppt (Table 11) is most significant in view of the massive quantities of both 2,4,5-T and TCDD that must have been applied to the test site. There was no evidence to indicate that TCDD was mutagenic nor carcinogenic in the field at the concentrations noted in Table 11. None of the 34 fetuses examined from animals captured on the test grid showed teratogenic defects. This leads one to the conclusion that the levels of TCDD encountered in the field failed to induce observable developmental defects. An analysis of the organ to body weight ratios of each of the control (males and females) and 23 �TABLE 11. COSfCENTRATiai (PARTS PER TRILLION) OF 2,3,7,8-TETr'RACHLORODIBENZOP-DTOXIN (TCDD) IN LIVER AND PELT SAMPLES FROM BEACH MICE, PEROMySCUS IE. IOUONOTLJS, COLLECTED FROM CONTROL AND TCDD-EXPOSED FIELD S T S , 1973 AND : PELT TREATMENT SEX Control 1973 Male and Female 20a ND13 Control 1974 Male 51 40a Control 1974 Female 83 40a Grid I to .fc- YEAR 1973 Male and Female 540 NDb Grid I 1974 Male Grid I 1974 Female a Minimum level of detection determined LIVER 1,300 960 130 140 �test (males and females) using the Wilcoxon Rank Sum Test indicated no statistical differences between field control males and field test males nor field control females and field test females (P^O.05). LIVER AND PELT ANALYSIS. The presence of TGDD in the liver samples of both male and female mice collected from the control site in 1974 may have been due to high levels in one or more specimens in the pool of samples. Mice from the test area could have migrated to the periphery of the grid and wandered into the area designated as control. The closest point from the control site to the test area was 200 m. However, it is emphasized that a mouse (or mice) could have been contaminated in this way, and thus have contaminated pooled samples analyzed for TCDD. Therefore, the use of these data as truly control data must be viewed with caution. The levels of TCDD in the liver of beach mice collected from Grid I substantiated bimccumulation of TCDD; i.e., an accumulation of TCDD in an organism from its environment. In general, levels of TCDD in the livers were no greater than the most concentrated zones of TCDD in the soil. There are no data from these studies to support biomagnification of TCDD; i.e., an increase in concentration of TCDD in successive organisms ascending the trophic food chain. BURROW AND DIET STUDIES. In all burrows that contained mice a consistant finding was a plug of soil pushed up into the tunnel within the first 2.5 to 25 on of the entrance. Frequently an "escape tunnel" would extend from the nest area to within 2.5 to 25 �15 on of the soil surface. Although the concentration of TCDD on the pelts of beach mice from the test area was only 10-15 percent of that In their livers, Table 11, it was apparent that the mice were continually contaminating themselves as they repeatedly moved in and out of their burrows. The soil data, Table 9, substantiated the presence of a zone of TCDD within the region of the tunnel entrance. Likewise, the location of the escape tunnel suggested that even the nest itself may contain detectable levels of TCDD. An examination of the plant and insect litter within the nests indicated that the beach mouse diet was made up of about 90 percent seeds (based on caryopsis hulls) and about 10 percent insects (based on insect exoskeletons and wings). Four composite seed samples were analyzed for TCDD with no TCDD being detected in any sample (at a minimum detection limit of 1 ppt TCDD). The insect remains are currently being analyzed for TCDD. TCDD LABORATORY UPTAKE EXPERIMENT. Twenty-two beach mice from the designated control area were brought into the laboratory and divided into a "control" group of 10 animals and dusted with 100 mg of alumina gel 10 times over a period of 28 days while the "test" group of 12 animals was dusted with 100 mg alumina gel containing 2.5 ppb TCDD 10 times over the 28 day period. Table 12 indicates control levels and test levels of TCDD in the composite liver samples and on the composite pelt samples of the alumina gel and alumina gel plus TCDD dusted animals. These animals were given complete histopathologic examinations at the completion of the experiment with no differences being 26 �seen between control and test animals. An analysis of the organ to body weight ratios of each of the control males to test males and control females to test females using the Wilcoxon Rank Sum Test indicated a statistical difference involving only the spleens of control male and test male animals at the 95 percent confidence level. This difference was not supported by either histopathological evidence nor by morphometric data as indicated in the Hepatic Ultrastructural Study section which follows. HEPATIC ULTRASTRUCTURAL STUDY. After the liver was removed from 30 beach mice (15 control and 15 from the test area) and weighed, a section was taken from the center of the median lobe. Representative electron micrographs were made from the liver tissue of each animal and the data obtained from each micrograph using a stereology technique. This method of quantitative analysis of the cell ultrastructure used morphometric procedures based on the techniques developed by Weibel et al. (14), as modified by Buchanan (15) With this method, a transparent grid of intersecting lines was placed at random over the micrographs and all the line intersections (points) which were over the required cell structures were counted. All of the points lying over the mitochondria, the damaged (swollen and ruptured) mitochondria, the granular vfeibel, E.R., G.S. Kistler, and W.F. Scherle. Practical stereological methods for morphometric cytology. J. Cell. Biol. 30: 23-38, 1966, Buchanan, G.M. Effect of high dietary molybodenum on rat adrenal cortejc. Unpublished thesis. University of Colorado, Boulder, CO, 1973. 27 �TABLE 12. CONCENTRATION (PARTS PER TRILLION) OF 2,3,7,8TETRACHLORPDIBENZO-P-DIOXIN (TCDD) IN LIVER AND PELT SAMPLES FROM BEACH MICE, PEROMYSCUS POLIONOTUS, DUSTED WITH ALUMINA GEL CONTAINING NO TCDD (CONTROL) OR 2.5 PARTS PER BILLION TCDD (TEST) TREATMENT SEX Alumina Gel Malea Female Alumina Gel + TCDD Male3 Femalea LIVER PELT NDb NDC NDb NDC 125 45 125 89 a Male and female livers composited for analysis Minimum detection level - 10 ppt detection level - 8 ppt TABLE 13. CONCENTRATION (PARTS PER TRILLION) OF 2,3,7,8TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN COMPOSITE SAMPLES OF VISCERA OR TRUNK FROM SIX-LINED RACERUNNERS, CHEMIDOPHORUS SEXLINEATUS, COLLECTED FROM CONTROL AND TCDD-EXPOSED FIELD SITES LOCATION VISCERA Control Site NDa Test Site 360 TRUNK 370 a Minimum detection limit - 50 ppt Tiinimum detection limit - 40 ppt 28 �endoplastnic reticulum (RER) and the agranular endoplasmic reticulum (SER) were then counted. The total area of the cytoplasm was then measured in the same manner. The volume fraction of each structure was determined to be the ratio between the point count of that structure and the total point count of the cytoplasm. In this manner the ratio of mitochondria volume to cytoplasm volume of the hepatic parenchyma! cell was determined for each animal, as was the ratio of damaged mitochondria volume to total mitochondria volume, RER to cytoplasm, SER to cytoplasm, and RER to SER. Using these volume fractions or ratios as quantitative measurements of the structures in question, the hepatic parenchymal cells from treated animals were compared with those from control animals. Similar data were collected from 22 mice brought from the field into the laboratory and exposed to 30 days of external dusting with alumina gel (with or without 2.5 ppb TCDD) . Analysis of the morpheme-trie data using the Wilcoxon Rank Sum Test indicated no statistical differences between field control and field treatment animals, nor were there statistical differences between the control and treatment animals of the dusting study Reptile Studies ANALYSIS OF REPTILE TISSUE. Chemical analysis for TCDD in body parts of the six-lined racerunner indicated significant levels of TCDD in both the visceral mass and in the trunk, Table 13. 29 �Gross post-mortem examinations were performed on 19 racerunners collected from either a control site or from Grid I with no evidence of gross abnormalities seen in any of the specimens. TCDD In Young, Lehn and Mettee (16) conducted species diversities and food chain studies in two aquatic ecosystems draining TA C-52A. Erosion of soil occurred in to a pond on the test area and in to a stream irrmediately adjacent to the area. TCDD levels of 10-35 ppt were found in silt of the aquatic systems, but only at the point where eroded soil entered the water. Species diversity studies of the stream were conducted in 1969, 1970, 1973 and 1974. Insect larvae, snails, diving beetles, crayfish, tadpoles, and major fish species from both aquatic systems were analyzed for TCDD. Species diversity studies indicated no significant change in the composition of ichthyofauna between these dates or a control stream. Concentrations of TCDD (12 ppt) were found in only two species of fish from the stream, sailf in shiner (Nbtropis hypselopterus) and mosquitofish (Gambusia affinis) . The sample of mosquitofish consisted of bodies with heads and tails removed. Two samples of sailf in shiner were analyzed; one containing viscera only and the other bodies less heads, viscera, and caudal fins. Only the viscera contained TCDD. Samples of skin, muscle, gonads, and gut were obtained from spotted sunfish (Lepomis punctatus) Young, A.L., P.J. Lehn, and M.F. Mettee. Absence of TCDD toxicity in an aquatic ecosystem. Weed Sci. See. Amer. Abst. 107, p. 46, 1976. 30 �from the test grid pond. Levels of TCDD in those body parts were 4, 5, 18, and 85 ppt, respectively. Gross pathological observations of the sunfish revealed no significant lesions or abnormalities. TCDD In Birds of TA C-52A Bartleson, Harrison, and Morgan (17) have conducted an extensive survey of the birds of TA C-52A. Between March 1974 and February 1975, they visited study areas twice each week at various times of day and night, and observed a total of 77 species of birds. Of this number, 44 species were observed on Grid I, the area of greatest TCDD residue. The remaining birds were seen in the surrounding clearing and bayheads projecting into the clearning. A small collection of specimens was made for species identification and for TCDD analysis. Only three species could be classified as residents which nest on the test grids. These were southern meadowlark (Sturnella magna), morning dove (Zenaidura macroura), and bobwhite quail (Colinus virginianus). TCDD residues were found in meadowlark livers (100-1,020 ppt) and in the stomachs and stomach contents (46 ppt) of these same birds. An analysis of liver and fat tissue from doves indicated concentrations of 50 ppt. An analysis of seed in the crop of the doves showed no detectable residue of TCDD. Two routes of TCDD contamination Bartleson, F.D., D.D. Harrison, and J.D.-Morgan. Field studies of wildlife exposed to TCDD contaminated soils. AFATL-TR-75-49, Air Force Armament Laboratory, Bglin AFB, Florida, 1975. 31 �were proposed for these birds. The first was through the process of dusting and subsequent ingestion of contaminated soil while preening. A second possible route was through the ingestion of soil-borne insects from the test grid; an analysis of a single composite insect, sample indicated a concentration of 40 ppt TCDD. Vegetative Succession Studies on TA C-52A TCDD analysis of vegetation has been limited to seed samples collected in support of the rodent diet study. No TCDD was found in four samples of seeds collected from vegetation on Grids I or II. The minimum level of detection was 1 ppt. A vegetative succession study has been conducted by Young and Hunter (18) to document the re-vegetation of an area denuded first by mechanical means and then by hundreds of applications of phenoxy herbicides. Nine months (June 1971) after the last defoliant-equipment test mission, a detailed survey of the vegetation was initiated. The area was divided into a grid of 169 sections (each 122 by 122 m), and within each section the percentage vegetative coverage was visually ranked as Class 0, 0-5 percent; I, 5-20 percent; II, 2040 percent; III, 40-60 percent; IV, 60-80 percent; and V, 80-100 percent. Three sections within each class were selected at random and surveyed for dicotyledonous plants. An unsprayed area 0.32 km northwest of the test area was also surveyed. In June 1973, each Young, A.L., and J.H. Hunter. A long-term field study of vegetative succession following repetitive application of phenoxy herbicides. Weed Sci. Sec. Amer. Abst. 1977. 32 �of these areas was again surveyed, but in addition/ a square-foot 2 (0.093m ) analysis technique was performed in 15 additional sections. These sections were randomly selected and within each 2 section, nine areas, each 0.093m , ware analyzed for species composition and ground cover density. Both methods of vegetative survey were repeated in June 1976. The number of dicotyledonous species increased from 74 in 1971 to 107 in 1973, and to 123 in 1976. In 1971, 20 percent of the test area had less than 20 percent vegetative cover, while 26 percent of the test area had more than 60 percent vegetative cover. In 1976, no sections had less than 20 percent vegetative cover, but over 73 percent of the test area had a cover of more than 60 percent. The major grass species were Panicum yirgatum and Panicum lanuginosum. The major dicotyledon was Diodia tores in 1971, but was replaced by Chrysqpsis graminifolia in 1976. These data demonstrate the rapid invasion of dicotyledonous species despite the unusually heavy applications of phenoxy herbicides. As a concluding remark, Test Area C-52A, Eglin AFB, Florida, has offered a unique opportunity to examine the effects of longterm, low-level exposure of biological systems to TCDD. Perhaps when the herbicide 2,4,5-T (contaminated by TCDD) was first applied to the test area (1962-1964), the levels of TCDD that accumulated on the soil may have been sufficiently high to be toxic, although there is no mention of animal deaths in the records of test missions for this area. It is of interest to note that in the 33 �Italian TCDD episode, an estimated 0.9 to 4.5 kg TCDD were 2 disseminated on an area of 1.4 km . This is approximately equal to 6.5 to 32 g/ha. Grid I on Test Area C-52A probably received between 0.07 and 1.86 kg TCDD on an area of 37 ha, or approximately 2 to 50 g TCDD/ha over a 2-year period. This range of values was arrived at using the arithmetic mean and maximum concentration of TCDD contamination of the herbicide Orange presently in the United States Air Force inventory. 34 �LABORATORY AND GREENHOUSE EXPERIMENTS WITH TODD Two additional studies have been conducted in support of the previous investigations of TCDD in field ecosystems. One of these studies has been conducted by Cupello and Young (19) on the potential uptake from soil of 14C-TCDD by plants. In this study, 2,240 kg active ingredient Orange herbicide/ha, containing 14 ppm 14C-TCDD, was placed beneath the soil surface in specially designed growth boxes containing 100 plants of Sorghum (Sorghum vulgare) per box. The plants were grown under controlled environmental conditions for 9 weeks; 14-hour photoperiod, diurnal temperature of 35 ± 2°C and 15 ± 1°C, and a relative humidity of 60 and 85 percent, day and night, respectively. On day 64 the plants were cut at the soil surface, ground in a Wiley Mill, and extracted with hexane in a Soxhlet Extraction apparatus for 4 hours. The TCDD in the extract was then concentrated by using the Dow Chemical Company Analytical Method ML-AM 73-97. The "TCDD concentrate" was quantitatively transferred to a scintillation vial using benzene, and 15 cc of Aquasol added to it. Analysis of the counting data from a liquid scintillation counter indicated no significant uptake of hexane extractable 14C-TCDD activity in the plant material. An analysis was also performed on the plant tissue prior to hexane extraction, and after hexane extraction for 4 hours. These plant samples were combusted in a 19Cupello, J.M., and A.L. Young. Radiochemical bioassay of TCDD uptake in plant material. Department of Chemistry and Biological Sciences, United States Air Force Academy, CO, 1976, unpublished. 35 �Packard Model 306 sample oxidizer, the OCL collected in Packard Carbo-Sorb, this solution diluted in Packard Permafluor-V, and the filler counted in a liquid scintillation counter. These data indicated the presence of sufficient C activity in the unextracted plant material to be equivalent to approximately 430 ppt TCDD in the plant tissue. This activity was not significantly reduced by hexane extraction. This relatively high 14C activity in the plant tissue could be explained by one of at least four hypotheses. It could represent the presence of (1) bound (non-hexane soluble) TCDD, (2) a soil biodegradation product of TCDD that was taken up and bound by the plant, (3) a metabolic breakdown product of the TCDD that was formed after incorporation of the TCDD into the plant/ or (4) a contaminant in the original C TCDD stock solution that eventually found its way into the plant either as the original contaminant or as a metabolic of it. A second study has been conducted by Bartleson, Harrison, and Morgan (17) on the effect of tilling TCDD contaminated soil. One cubic meter of soil was collected from Grid I, TA C-52A, and removed to the laboratory. Four samples were taken from the uniformly mixed soil, analyzed and found to contain 1,100 ppt (2 samples) and 1,300 ppt (2 samples) TCDD. The contaminated soil was placed in two groups of four pots (20 cm deep and 20 cm in diameter). The four pots in each group were treated as follows: two were left outside and exposed to natural elements, and two were placed in a greenhouse and watered with a nutrient solution. 36 �One of the two containers in each location was left undisturbed, and the other was stirred (tilled) weekly with a spatula. This stirring was not complete, and soil in the bottom of the pots was relatively undisturbed. The soil in each of the pots was emptied into a clean tray and mixed thoroughly before samples were collected and analyzed for degradation of TCDD. The data shown in Table 14 suggest that sunlight, tilling and perhaps increased temperatures (associated with the greenhouse) may promote more rapid degradation of TCDD. There also may be an additive effect from use of nutrients. 37 �TABLE 14. DEGRADATION OF TCDD (PARTS PER TRILLION) IN A GREENHOUSE EXPERIMENT, EGLIN AFB, FLORIDA LENGTH OF EXPOSURE 0 (PPT) 9 weeks (PPT) 23 weeks (PPT) Tilled 1,200 1,100 520 Untilled 1,200 1,000 530 Tilled 1,200 640 460 Untilled 1,200 810 530 TREATMENT Full Sunlight3 Greenhouse Samples placed outside of greenhouse Samples watered with a nutrient solution 38 �RECOMiyENDATIONS FOR DECONTAMmTION OF TCDD EXPOSED FIELD SITES Although there are many potential options for the decontamination of an area exposed to TCDD (see reference 4), data provided in this report would suggest that one of the most feasible options would be soil incorporation/biodegradation. The data base used in selecting this option is as follows: 1. TCDD may persist (in biotic and abiotic components) for long periods of time when initially present at extremely high concentrations on the soil surface. 2. TCDD will accumulate in the tissues of rodents, reptiles/ birds/ fish/ and insects when these organisms are exposed to TCDD contaminated soils (however, the levels of TCDD in the tissues apparently do not exceed the levels of TCDD found in the environment). 3. Organisms tolerate/ i.e./ based on no observed deleterious effects, soil levels between 10-1,500 ppt TCDD. 4. TCDD is degraded by soil microorganisms, especially when in the presence of other chlorinated hydrocarbons. 5. TCDD is degraded in the presence of sunlight. 6. Movement of TCDD in the abiotic portions of the environment can be by wind or water erosion of soil particles, but leaching by water alone does not appear to occur. 7. TCDD is probably not readily released or degraded in the environment when bound to activated coconut charcoal. 39 �Specific Re<xirntrendations In locations where accidental TCDD contamination covers Significant geographical area, e.g., many hectares, an in situ biodegradation program may be most effective in reducing levels of TCDD residues. Incorporation of organic material, lime, and fertilizer to enhance microbial activity may be advantageous. The biodegradation site should be tilled frequently so as to expose residue to sunlight. Watering of the site is recommended to reduce wind movement of contaminated particles and to enhance biodegradation. In locations where a limited area has been exposed to accidental contamination of TCDD, the top 0-15 cm of soil should be removed and taken to an isolated area where biodegradation procedures can be conducted. Similar treatments should be applied to these plots as would be for an in situ program. Protective clothing should be worn by all site personnel. The contaminated clothing should be incinerated at an approved incinerator. Following use, all equipment should be rinsed with an organic solvent (e.g., diesel fuel) to remove TCDD residue. The solvent containing TCDD residue may be collected in activated coconut charcoal and either incinerated or placed in an approved sanitary landfill, although if a sufficiently isolated land area is available, biodegradation may be feasible. It should be noted that some TCDD residue will remain in a contaminated site. However, research on the ecosystem at Test Area C-52A, Eglin AFB, Florida, indicated that organisms do have 40 �a tolerance to low levels of TCDD. Therefore, in those areas having soil residues below 1 ppb, further efforts to decontaminate the area are not practical. These areas should, however, be fenced and posted to prevent livestock and human exposure. 41 � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 017 Folder The folder containing the original item. 0193 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 Young, Alvin L. Charles E. Thalken Eugene L. Arnold James M. Cupello Lorris G. Cockerham Description An account of the resource <strong>Corporate Author: </strong>Department of Chemistry and Biological Sciences, USAF Academy, Colorado Date A point or period of time associated with an event in the lifecycle of the resource 1976-10-01 Title A name given to the resource Fate of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCCD) in the Environment: Summary and Decontamination Recommendations Subject The topic of the resource biodegradation ecological fate soil decontamination Eglin AFB https://www.nal.usda.gov/exhibits/speccoll/files/original/bbe3dd5ae7a23003154d445c92e1fa7b.pdf 2829adad1af30d0df77eb7a31551d451 PDF Text Text Item ID Number 0031G Author Young, Alvin L. Corporate Author Manuscript: Meeting Notes: Meeting with Dr. Terry Biery and Lt. Col. George Rowcliffe at AF/Pest Control Board Meeting, WRAMC, Washington, D.C., 13 September 1979 Journal/Book Title Year 000 ° Month/Day Color Ll Number of Images 4 Desoripton Notes Monday, January 22, 2001 Page 316 of 341 �13 £c ���- TV L. &jgtfo '. ^r A,<"oa ^i r^€^oUoi^j0etc LA& ^r'csQ*^ V � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 020 Folder The folder containing the original item. 0316 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 Young, Alvin L. Title A name given to the resource Manuscript: Meeting Notes: Meeting with Dr. Terry Biery and Lt. Col. George Rowcliffe at AF/Pest Control Board Meeting, WRAMC, Washington, D.C., 13 September 1979 Subject The topic of the resource spray equipment Ranch Hand aircraft https://www.nal.usda.gov/exhibits/speccoll/files/original/5d84ecb95d12f547894d9de9574b8f30.pdf fe418b5e13879987ea4c99062e3fe187 PDF Text Text Item ID Number °0318 Author Young, Alvin L. Corporate Author RepOrt/ArtlClB TitlB Letter: To Mrs. Cleary from Alvin L. Young Regarding Use of Herbicides in Southeast Asia Journal/Book Title Year 000 ° Month/Day Color '] Number of Images 5 Descripton Notes Monday, January 22, 2001 Page 318 of 341 �ALV1N L YOUNG Dear Mrs. Cleary Aerial spray of herbicides has been conducted in Southeast Asia since January 1962, and as of 31 December 1967, 2,21^,600 acres have been treated. Herbicide operations, in particular, are one of the more controversial aspects of the conflict in Southeast Asia due to the limited knowledge of the general, public. The. most important point one must realize to understand the defoliation process is that the chemicals currently in use do not affect the'soil and attack only the plant or tree on which it is sprayed. Dr. Charles E. Minarik of the Plant Science Laboratory at Ft. Detrick, Md. has gone on record with the position that defoliation in Vietnam is essentially no more of a threat to the balance of nature than the spraying of vegetation along power lines, railways, and highways in the United States. The chemical herbicides which are being used by the Republic of Vietnam in clearing out jungle growth to reduce the hazards of ambush by Viet Cong bandits have been used commonly in the United States and other countries for the past 15 years by farmers, ranchers and home owners. tThe two herbicides -- known as 2,4-D and 2,U,5~T -- are used extensively in most countries of both the free world and the communist bloc for selective control of undesirable vegetation. These chemicals are better for vegetation control than other compounds of a similar nature because they are not harmful to people, animals, soil or water. They �are described by research scientists as "non-toxic -- even when fed to cows and livestock at rates exceeding those used for vegetation control." The importance of the use of herbicides in agricultural production is universally recognized. In fact, Premier Krushchev, in.reporting to the U.S.S.R. Central Committee on March 5, 1962, stressed the importance of herbicides and called for production on an industrial basis. The two chemicals, 2,^-D and 2,4,5-T, are now in regular use, particularly for weed control, in rice paddies, other field and horticultural crops, and rangeland, in Asian countries such as Burma, Thailand, Philippines, Republic of China, Japan, India, Indonesia, Australia and Hew Zealand. The herbicides are being used by the government of the Republic of Vietnam in the guerrilla warfare with the Viet Cong in order to increase visibility on the ground and from the air. At low rates of application, the herbicides wither the leaves and cause them to fall from the plants, but jungle plants usually regrow in about 30 days. At higher rates of application, the herbi- • 'cides will cause defoliation, kill the top growth of brush, plants and trees, and prevent'regrowth for a year or more. The government of South Vietnam has been very thorough in its investigations and decisions to use the chemicals 2,^-D and 2,4,5-T. Scientists first set up a list of standards to be used in selec* ting any chemicals that might be used extensively in the country. �All the areas sprayed in South Vietnam are first mapped and then selected after careful study. Scientists have long known that the herbicides 2,k-D and 2,4,5-T are not toxic to people or animals. The two chemicals are registered in the United States by the Department of Agriculture for use on food crops, in rivers and ponds, and on rangelands where livestock ^ graze. They are available for purchase throughout the United States and are labeled nonpoisonous. Records on the production of the herbicides during the past 15 years show that workers in the chemical manufacturing plants have not been adversely affected. There has been no documentable death or injury from association with the chemicals.. The same is true in the use .of these chemicals in Vietnam. The South Vietnamese are also learning that the herbicides can be most useful for them. In addition to getting rid of -weeds, the farmers are now harvesting the trees of the sprayed junglelands and using the weed for fuel. Previously the farmers were afraid to enter the jungles because of the threat to their lives from ambush by the Viet Cong, , After harvesting the wood, the farmers are clearing sorae of the land for agricultural use. In fact, the farmers have been so enthusiastic about the clearing of lands that the government of Vietnam has asked them not to harvest wood too soon afte,r chemical spraying because this practice reduces the effectiveness of the herbicides in controlling the vegetation. • �The herbicidal.effects of 2,4-D and 2,^,5-T were discovered and published in 1 ^ Commercial manufacture of the chemicals -was 9. developed in the United States bet-ween 1 ^ and 19Vf. 9 During the 15 years since 19^-7> U.S. production of the two herbicides has averaged about 25 million pounds (11.3 million kilograms) per year. The herbicides are used on an average of 30 million acres (12 million hecares) in the past 15 years, while most of this land •was under cultivation. The herbicides are used in the United States to control weeds in cereal crops such as corn, wheat, oats, rice, barley and grain sorghums; for control of weeds on rangeland where livestock graze; for clearing ponds and streams of undesirable plant growth, such as water hyacinths; on lawns and parks to kill weeds where pets and children play; to kill poison ivy and other poisonous plants in wooded. areas near camp sites; and for killing weeds, such as ragweed \ and heavy-pollen producing plants, which are a threat to good health or create allergies in some people. In a study conducted from 15 August 1967 to 1 December 1967, the Midwest Research'Institute of Kansas City, Missouri concluded that: "There is no question that the greatest short-term and long-term direct ecological consequence of using herbicides in Vietnam or anywhere else is'the destruction of vegetation. It should be borne in mind that as long as soil sterilization is not an objective, destruction of vegetation by herbicides is a selective process and that denuded earth �does not occur. Furthermore, the end result of the use of the herbicides from an ecological standpoint represents a process that is common after uncontrolled fires, or thw wild regrowth of abandoned fields, that is, the ecosystem is set ck to an earlier sere." It is a pleasure to be of assistance to you in providing this information. � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 020 Folder The folder containing the original item. 0318 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 Young, Alvin L. Title A name given to the resource Letter: To Mrs. Cleary from Alvin L. Young Regarding Use of Herbicides in Southeast Asia Subject The topic of the resource herbicide application ecological impact Ranch Hand https://www.nal.usda.gov/exhibits/speccoll/files/original/5c56b76a52235883573a7e79bc378e2f.pdf 2abd017e641043200a441dac5938a007 PDF Text Text Item ID Number °1165 Author Young, Alvin L. United States Air Force Occupational and Environmental Report/Article Title The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange and its Associated Dioxin Journal/Book Title Year Month/Day Color Oct ber ° D Number of Images 263 DeSCrlptOU NOtBS A'v'n *-• Young filed this item under the category "Human Exposure to Phenoxy Herbicides and TCDD" Thursday, April 05, 2001 Page 1165 of 1180 �• 4 MTlftrO A-L. eVdL Report OEHLTR-78-92 r ^j-—-' j •••4>^ USAF OEHL TECHNICAL REPORT THE TOXICOLOGY, ENVIRONMENTAL FATE, AND HUMAN RISK OF HERBICIDE ORANGE AND ITS ASSOCIATED DIOXIN Alvin L. Young, Captain, USAF John A. Calcagni, Lieutenant Colonel, USAF, MC Charles E. Thalken, Lieutenant Colonel, USAF, VC James W. Tremblay, Major, USAF, BSC October 1978 Final Report I Approved for public release; distribution unlimited PREPARED FOR: The Surgeon General United States Air Force Washington, D.C. 20314 USAF Occupational and Environmental Health Laboratory Aerospace Medical Division (AFSC) Brooks Air Force Base, Texas 78235 �NOTICES This report has been released to the National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, for sale to the general public. *** Qualified requestors may obtain copies of this report from Defense Documentation Center (DDC), Cameron Station, Alexandria, Virginia 22314. *** This technical report has been reviewed and is approved for publication. WILLIAM E. MABSON, Colonel, USAF, BSC Commander �UNCLASSIFIED S E C U R T t V ' t V A S S I F I C A T I O N OF T H I S P A G E (When Data Entered) READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 2. GOVT ACCESSION NO 1. REPORT NUMBFR 3. RECIPIENT'S C A T A L O G NUMBER USAF OEHL - 7 8 - 9 2 i t " . ; ':" C-inrt Subtitle) 5. TYPE OF REPORT & PERIOD COVERED The Toxicology, Environmental Fate and Human Risk of. • i , Herbicide Orange and Its • Associated. Dioxin - • , Fin; I 6. PERFORMING ORG. REPORT NUMBER 3 ?. AUTHOR,,, /\yvin L. young, Captain, USAF John A. Calcagni, Lieutenant Colonel, USAF, MC Charles E. Thai ken, Lieutenant Colonel, USAF, VC : James U. Tremblay, Major. USAF, BSC 8. C O N T R A C T OR G R A N T NUMBERfa.) 9. P E R F O R M I N G O R G A N I Z A T I O N N A M E AND ADDRESS 10. PROGRAM ELEMENT, PROJECT, TASK A R c A & WORK UNIT NUMBERS US Air Force Occupational and Environmental Health Laboratpry Brooks AFB TX 78235 11. 12. CONTROLLING OFFICE NAME AND ADDRESS REPORT DATE October 1978 The Surgeon General US Air 'Force 13. NUMBER OF PAGES Washington, DC 20314 14 M O N I T O R I N G A G E N C Y N A M E 4 ADDRESS^/ different Iron Controlling Olficv) 15. S E C U R I T Y CLASS, (at this report) Unclassified 1Sa. DECLASSIFI CATION/ DOWN GRADING SCHEDULE 16. D I S T R I B U T I O N S T A T E M E N T (ol this Report) Approved for public release; distribution unlimited. 17. D I S T R I B U T I O N STATEMENT (ol the abstract entered In Block 30, It different 18. tram Report) S U P P L E M E N T A R Y NOTES Authors: A.L. Young, PhD J.A. CaVcagni, MD C.E. Thalken, DVM J.W. Tremblay, P.E. 19. KEY WORDS (Continue on reverse aide II nocessary and identity by block number) clorinated phenols 2,4-dichlorophenoxyacetic acid (2,4-D) dioxin environmental monitoring herbicides Herbicide Orange phenoxy herbicides Pacer HO Ranch Hand South Vietnam toxicity - animal toxicity - human 20. A B S T R A C T fContinue on reverse aide If necnssary and Identify by block number) The use of herbicides in South Vietnam between 1962 and 1971 was reviewed, including the nature and quantities of herbicides used, their handling and application.' Emphasis was placed on Herbicide Orange, a 50:50 mixture of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), with its associated contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The atrisK US military population in South Vietnam was defined to establish the potential for exposure in handling "and application of Herbicide Orange. The environmental fate of the phenoxy herbicides and TCDD was reviewed to evaluate E O . T I O M O F I - . ' 6 5 I S OBSOLETE U N C L A S S I F I FD SS.CURITY CLASSI f : » iT, r;) A G E (Wien Deta Entt-rr ' �UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAOBftWiwi £>•<« Entered) Item 19. Key Words (cont): 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) Item 20. Abstract (cont): the potential for human risk associated with exposure to areas previously treated with Herbicide Orange. The occupational and environmental aspects of the project to incinerate at sea 2.22 million gallons of Herbicide Orange durinc the summer of 1977 were summarized to assess the potential for human exposure in handling large quantities of the material. Scientific data were reviewed on incidents and episodes involving suspected poisoning of humans or animals by phenoxy herbicides or TCDD. Literature dealing with animal toxicology and the effects of human exposure to 2,4-D, 2,4,5-T and TCDD was reviewed to correlate exposures with symtomatology. iv UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGEfHTion Data Entered) �PREFACE The use of herbicides in support of tactical military operations in South Vietnam from 1961 to 1971 has had (and continues to have) a negative impact on the use of pesticides by numerous facets of our society. fjrior to the Vietnam conflict, herbicides were considered invaluable to agriculture, innocuous to human life and of little environmental concern. Today, seven years after the last herbicide mission in Vietnam, these same herbicides are the center of scientific debate involving not only ecological but also medical, legal and political issues. The United States Environmental Protection Agency (EPA) has recently issued a Notice of Rebuttable Presumption Against Registration (RPAR) of pesticides containing one of these "Vietnam" herbicides, while at the same time some Veterans of the Vietnam Conflict have reported medical problems they claimed were the result of herbicide exposure while assigned to military duties in Vietnam. In April 1978, the Surgeon General of the United States Air Force (USAF) tasked personnel'of the USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas with updating previous scientific assessments of possible adverse effects to human health resulting from exposure to the herbicides, especially Herbicide Orange used in South Vietnam during the Vietnam Conflict. The present report was assembled using the latest available published scientific information, previously unpublished data, and observations from medical and scientific personnel intimately associated with the herbicides in question. The report reviews the use of phenoxy herbicides in Vietnam, their environmental fate, and pertinent animal and human toxicological studies. In addition, a description is given of the 1977 military operation for the disposal of Herbicide Orange emphasizing the facets of environmental monitoring and industrial hygiene. The document concludes with an assessment of the risk to human health following exposures to the phenoxy herbicides. Special emphasis was placed upon the chemistry, environmental fate and toxicology of the trichlorophenoxy herbicide contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The authors are indebted to Kenneth C. Back, PhD, Chief Toxicology Branch, Toxic Hazards Division, United States Air Force 6570th Aerospace Medical Research Laboratory, Wright-Patterson AFB Ohio for his critical review of-this document and the many recommendations he made to improve the qualify of the discussions on toxicology. Special thanks are expressed to Rodney W. Bovey, PhD, United States Department of Agriculture", Texas A&M University, College Station, Texas, for his assistance in providing copies of the foreign literature on the phenoxy herbicides. �The services of many staff members and consultants of the United States Air Force Occupational and Environmental Health Laboratory are acknowledged. Special acknowledgement is made to Mrs Joyce G. Kidd who served as the general editor, and to the following typists who willingly worked numerous overtime hours in preparing this manuscript: Ruth S. Bledsoe; Yolanda Carrisalez; Irma Ledesma; Lorraine M. Polonis; Nancy L. Ragan and Trina Roark. �CONTENTS Page PREFACE v CHAPTER I .THE USE OF HERBICIDES IN SOUTH VIETNAM I. INTRODUCTION 1-1 II. THE HERBICIDES USED IN SOUTH VIETNAM A. Historical B. Descriptions of the Herbicides Used in Operation RANCH HAND 1-1 1-1 1-3 C. Quantities of Herbicides Sprayed in South Vietnam D. Land Area Sprayed with Herbicides in South Vietnam III. THE AIRCRAFT, SPRAY SYSTEMS AND MISSION CONCEPT IN OPERATION RANCH HAND A. Historical B. Spray Systems and Characteristics of the RANCH HAND Aircraft C. Mission Concepts 1-8 1-11 I-11 1-11 1-14 1-15 IV. PERTINENT DEPLOYMENT AND BIOLOGICAL FACTORS OF THE HERBICIDES 1-18 A. Use Patterns of Individual Military Herbicides B. Canopy Penetration of Defoliants « V. 1-18 1-20 ESTIMATED QUANTITIES OF INDIVIDUAL CHEMICALS SPRAYED IN SOUTH VIETNAM A. Herbicide Orange and its Components 2,4-D, 2,4,5-T and TCDD B. Military Projects that Involved Handling Herbicides Orange, Purple, Pink or Green VI. SUMMARY 1-21 1-29 1-29 LITERATURE CITED 1-32 LIST OF TABLES 1. Selected physical, chemical and toxicological properties of the three major military herbicides used in South Vietnam, 1962 - 1971. VI 1 1-5 �2. Number of gallons of military herbicide procured by the U.S. Department of Defense and disseminated in South Vietnam during the period January 1962 - December 1964. 1-9 3. Estimated number of gal of military herbicide procured by the U.S. Department of Defense and disseminated in South Vietnam during the period January 1965 - February 1971. 1-10 4. Comparison of data from three sources of the estimated number of acres treated in South Vietnam during the period of January 1962 - February 1971. Data make noi allowance for multiple coverage. 1-12 5. The number of acres treated in South Vietnam, 1962 - 1971, with military herbicides within the three major vegetational categories. Data represent areas receiving single or multiple coverage and for 90 percent of all areas treated. 1-13 6. Concentration, ppm, of TCDD in samples of Herbicides Orange and Purple. 1-23 7. Composition, percent, of selected samples of Herbicide Orange in relation to military specifications. 1-27 8. Estimated quantities of herbicides and TCDD disseminated in South Vietnam from January 1962 - February 1971. 1-28 9. Data on the major military projects involved in the handling and/or spraying of Herbicides Orange, Purple, Pink or Green in support of military programs in South Vietnam. 1-30 LIST OF FIGURES 1. Chemical structure and nomenclature of the major herbicides used in South Vietnam, 1962-1971. Formulas A and B comprised Orange, C and D - White, and E was Blue. 1-6 2. Structure and physical/chemical characteristics of 2,3,7,8- tetrachlorodibenzo-p-dioxin, TCDD or dioxin. VII I 1-22 �CHAPTER II DISPOSAL OF HERBICIDE ORANGE Page I. INTRODUCTION 11-1 II. HISTORICAL BACKGROUND II-l III. DESCRIPTION OF LAND-BASED OPERATIONS A. NCBC, Gulfport MS B. Johnston Island I1-2 II-3 II-4 * IV.. LAND-BASED OPERATIONS MONITORING PROGRAMS A. Monitoring Equipment and Procedures B. Analytical Procedures and Methodologies V. LAND-BASED MONITORING RESULTS II-5 II-6 II-7 II-8 A. NCBC, Gulfport MS II-8 B. Johnston Island 11-10 VI. SUMMARY AND CONCLUSIONS 11-15 LITERATURE CITED , 11-19 LIST OF TABLES 1. Results of industrial hygiene air samples collected inside the dedrumming facility Project PACER HO NCBC, Gulfport MS, 24 May 10 June 1977. II-9 2. Results of ambient air samples collected at Gulfport MS, Project PACER HO, 24 May - 10 June 1977. 11-11 3. Results of industrial hygiene air samples collected inside the dedrumming facility, Project PACER HO Johnston Island, first loading 27 July - 5 August 1977 11-12 4. Results of industrial hyigiene air samples collected inside the dedrumming facility Project PACER HO, Johnston Island, second loading, 17-23 August 1977. 11-13 5. Results of industrial hygiene "breathing zone" samples collected inside the dedrumming facility Project PACER HO, Johnston Island. IX 11-14 �6. Results of downwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. 7. Results of upwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. 11-16 11-17 CHAPTER III ENVIRONMENTAL FATE OF 2,4-D, 2,4,5-T AND TCDD I. THE ENVIRONMENTAL FATE OF THE PHENOXY HERBICIDES III-l III-l IJ.I-4 III-6 THE ENVIRONMENTAL FATE OF TCDD 111-7 A. Analytical Limitations 111-7 B. Laboratory Studies of TCDD C. Field Studies of TCDD III-7 III-ll D. Environmental Production of TCDD E. Photodegradation of TCDD III. III-l A. Physical/Chemical Factors Influencing Disappearance of Herbicide B. Biological Degradation of the Phenoxy Herbicides C. Accumulation and Metabolism of Phenoxy Herbicides in Animals II. INTRODUCTION 111-20 111-21 IV. SUMMARY LITERATURE CITED II1-22 III-24 LIST OF TABLES 1. Concentrations of TCDD, parts per trillion, in the Herbicide Orange biodegradation plots, AFLC Test Range, Utah, four years after applications, 111-15 2. Concentration of TCDD in soil profile of Grid 1, Test Area C-52A, Eglin AFB, Florida. 111-17 LIST OF FIGURES 1. Semi-loaarithmic olot of soil concentrations (parts per million) of herbicide in Herbicide Orange biodegradation studies at Eqlin AFB, Florida, and Hill AFB, Utah. TIT-13 �2. Semi-logarithmic plot of soil concentrations (parts per trillion) of TCDD in Herbicide Orange biodegradation studies at Eglin AFB, Florida, and Hill AFB, Utah. 111-14 CHAPTER IV THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS I. II. INTRODUCTION IV-1 REVIEW OF 2,4-D TOXICITY IN ANIMALS . A. The Acute and Short-Term Toxicity Potentials of 2,4-D IV-3 IV-3 B. The Subacute and Chronic Toxicity Potentials of 2,4-D . . IV-5 C. Absorption, Distribution and Excretion of 2,4-D D. Embryotoxic, Fetotoxic and Teratogenic Potentials of 2,4-D IV-17 E. Carcinogenic and Tumorigenic Potentials of 2,4-D IV-20 F. Mutagenic and Cytogentic Potentials of 2,4-D in Animals IV-23 REVIEW OF 2,4,5-T TOXICITY IN ANIMALS IV-26 A. The Acute and Short-Term Toxicity Potentials of 2,4,5-T III. IV-16 IV-26 B. The Subacute and Chronic Toxicity Potentials of 2,4,5-T C. Absorption Distribution and Excretion of 2,4,5-T IV-26 IV-31 D. Embryotoxic, Fetoxic and Teratogenic Potentials of 2,4,5-T IV-36 E. Carcinogenic and Tumorigenic Potentials of 2,4,5-T IV-46 F. Mutagenic and Cytogenic Potentials of 2,4,5-T IV-47 IV. REVIEW OF TCDD TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of TCDD IV-50 IV-50 �B. The Subacute and Chronic Toxicity Potentials of TCDD IV-52 C. Absorption Distribution and Excretion of TCDD IV-56 D. Embryotoxic, Fetoxic and Teratogenic Potentials of TCDD IV-61 E. Carcinogenic and Tumorigenic Potentials of TCDD F. Mutagenic and Cytogenic Potentials of TCDD IV-71 SUMMARY OF THE LITERATURE REVIEW OF THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS IV-72 A. 2,4-D IV-72 B. 2,4,5-T IV-73 C. TCDD IV. IV-63 IV-74 • LITERATURE CITED IV-76 LIST OF TABLES 1. Summary of literature data on the no-effect, LD^Q and LD-iuu levels of the acute toxicity of 2,4-D in nn animals IV-6 2. Summary of literature data on the subacute and chronic toxicity of 2,4-D in animals IV-13 3. Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4-D in animals IV-21 4. Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4-D in animals IV-24 5. Summary of literature data on the no-effect LD50 and LD-inn levels of the acute toxicity of 2,4,5-T in animals IV-27 6. Summary of literature data on the subacute and chronic toxicity of 2,4,5-T in animals IV-32 7. Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T in animals IV-41 8. Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4,5-T in animals XII IV-48 �9. Summary of literature data on the no-effect, 1050 'and levels of the acute toxicity of TCDD for animals IV-53 10. Summary of literature data on the subar.ute and chronic toxicity of TCDD in animals IV-57 11. Summary of literature data on the embryotoxic, fetoxic and teratogenic potentials of TCDD in animals IV-64 12. Summary of literature data on the carcinogenic and tumorigenic potentials of TCDD in animals IV-69 CHAPTER V 2,4,5-T/TCDD EPISODES I. II. INTRODUCTION V-l INDUSTRIAL EXPERIENCES V-2 A. Industrial Processes B. Industrial Episodes ' V-2 . V-5 III. VIETNAM EPISODE IV. , EASTERN MISSOURI HORSE ARENA EPISODE V. THE SEVESO, ITALY EPISODE VI. V-12 V-17 V-19 GLOBE, ARIZONA EPISODE V-21 VII. THE SWEDISH LAPLAND EPISODE V-24 VIII. THE AWAMUTU, NEW ZEALAND EPISODE IX. DISCUSSION OF LITERATURE AND CONCLUSIONS X. SUMMARY V-26 V-28 V-32 LITERATURE CITED V-33 LIST OF TABLES 1. Total United States production and use of 2,4,5-T herbicide for the period 1961 through 1969. V-3 2. Industrial incidents associated with the manufacture of chlorinated phenols. V-7 XTM �3. Some clinical features observed in cases of chloracne associated with production of 2,4,5-T and other chlorinated phenols. V-10 LIST OF FIGURES 1. Synthesis scheme for production of the n-butyl ester 2,4,5-T (NBE 2,4,5-T) and site where formation of TCDD may occur. V-4 CHAPTER VI HUMAN EFFECTS OF HERBICIDE ORANGE I. II. INTRODUCTION . VI-1 PHARMACODYNAMICS VI-1 A. B. C. D. VI-1 VI-1 VI-2 VI-4 Percutaneous Entry of Phenoxy Herbicides Ingestion of Phenoxy Herbicides Tissue Analyses for the Phenoxy Herbicides Pharmacodynamics "of TCDD III. ADVERSE EFFECTS VI-4 A. Limitations of Referenced Studies B. Phenoxy Herbicides That Do Not Contain TCDD C. Trichlorophenol (TCP), 2,4,5-T and TCDD D. Cancer VI-12 VI-27 CONCLUSIONS VI-28 A. Pharmacodynamics B. Effects of the Herbicides VI-28 VI-29 C. Effects of TCDD IV. VI-4 VI-6 VI-30 V. SUMMARY VI-30 LITERATURE CITED VI-31 LIST OF TABLES 1. Levels (part per million) of phenoxy herbicides in human tissue or body fluid following ingestion of fetal dose. VI-3 2. TCDD levels in a human body. VI-3 3. Distribution of symptoms in 292 workers employed in the production of the amine salt and the butyl ester of 2,4-D VI-7 xiv �4. Distribution of adverse effects in case reports following the ingestion of non-TCDD containing phenoxy herbicides. VI-9 5. Distribution of reported adverse effects following exposure of field workers and applicators to 2,4-D. VI-11 6. Organ systems reported affected after occupational exposure to PCP, TCP, 2,4,5-T or TCDD. VI-13 7. Signs, symptoms, and disorders reported after occupational exposure to TCP, 2,4,5-T or TCDD. VI-14 8. Special clinical studies following occupational exposure to TCP, 2,4,5-T or TCDD. VI-15 9. Organ systems reported affected after exposure to TCP and TCDD following an industrial accident. VI-17 10. Signs, symptoms and disorders reported after exposure to TCP and TCDD following an industrial accident. VI-18 11. Special clinical studies after exposure to TCP and TCDD following an industrial accident. xv VI-19 �CHAPTER I THE USE OF HERBICIDES IN SOUTH VIETNAM I. INTRODUCTION The introduction of herbicides in 1962 into the armed conflict in Vietnam represented an application of a new technique for modern warfare. Their use in a defensive role was for defoliation. Their use in offensive roles was for food crop denial. The herbicides most widely employed were the phenoxyacetic acids. They were extensively used for almost a decade throughout the forested, semi-populated, regions of South Vietnam. Assessments -of their ecological impact in South Vietnam have been published (see Chapter V). An assessment of the effects of herbicides on the human indigenous populations of South Vietnam has also been conducted (11). No assessment has been made of potential adverse human effects of the phenoxy herbicides or the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on personnel of the U.S. Military forces. Adverse human effects in military personnel due to the herbicides or the contaminant would be predicated on the assumption that an exposure occurred. The presence of military spray aircraft or the observation that drums of herbicide were stored on a military installation, or even smelling the odor of "herbicides" in the air does not necessarily constitute an exposure to the herbicide per se. An exposure would have had to involve physical contact for a sufficient period of time to permit the chemical(s) to penetrate the body. This chapter examines those factors that would have influenced the likelihood of such exposures. They include: 1. the nature of the herbicides used in South Vietnam, 2. the nature of the herbicide applications, 3. the procedures employed in the handling of the herbi- cides, and 4. the quantities of individual chemicals sprayed in South Vietnam. Detailed examinations of these "parameters" are reported in the following sections. II. THE HERBICIDES USED IN SOUTH VIETNAM A. Historical The discovery and early history of the phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) have been reviewed by Peterson (31). Peterson noted 1-1 �that the effectiveness of these plant growth regulators as "herbicides" was determined in mid-1944 field trails at Beltsville and Camp Detrick (now Fort Detrick), Maryland. The outstanding effectiveness of these two herbicides in controlling the growth of broad-leaved plants and weeds, coupled with their apparently low mammalian toxicity and low application rates, resulted in their rapid acceptance in world agriculture. Peterson (31) reported that the annual production of 2,4-D alone exceeded 14,000 pounds in 1950 and 36,000,000 pounds in 1960. Irish et al (26) and Darrow et al (14) have documented the early military use of the phenoxy herbicides. They reported that the earliest aerial spray trials (conducted in military aircraft) occurred in 1944 and 1945. Three different mixtures of 2,4-D were used in these early tests. Although herbicides were not used in tactical military operations in World War II, a small program for screening potential herbicides for military use continued after the War. By 1951, personnel at Fort Detrick had determined that the vegetationcontrol chemicals of choice were mixtures of the butyl esters of 2,4-D and 2,4,5-T. In 1959, the Crops Division, Fort Detrick, conducted the first large-scale military defoliation effort at Fort Drum, New York. This project involved the aerial application of the butyl esters of 2,4-D and 2,4,5-T to approximately four square miles of vegetation. Its success prompted the Office of the Secretary of Defense (OSD) in May 1961 to request that the Crops Division determine technical feasibility of defoliating jungle vegetation in the Republic of Vietnam. As part of a project to evaluate herbicides and defoliation techniques (Project AGILE) in Southeast Asia, Brown (7) conducted eighteen different aerial, spray tests (defoliation and anticrop) with various formulations of commercially .available herbicides. The choice of these herbicides was based "upon the chemicals that had had considerable research, proven performance, and practical background. Also, other factors had to be considered, such as availability in large quantity, costs and known or proven safety in regard to their toxicity to humans and animals" (7). The results of these tests were that significant defoliation and anticrop effects could be obtained with two different mixtures of herbicides. The first was a mixture of the n-butyl esters of 2,4-D and 2,4,5-T and the iso-butyl ester of 2,4,5-T. This mixture was code-named "Purple". The second "military" herbicide was code-named "Blue" and consisted of the acid and sodium salt of cacodylic acid. The colored bands which were painted around the center of the 55-gallon drums served as aid to the identification by support personnel. Brown (7) reported that the first shipment of Herbicides Purple and Blue was received at Tan Son Nhut Air Base, Republic of Vietnam, on 9 January 1962. These were the first military herbicides used in Operation RANCH HAND, the tactical military project for the aerial spraying of herbicides in South Vietnam. Two additional phenoxy herbicide formulations were received in limited quantities in South Vietnam and evaluated during the first two years of Operation 1-2 �RANCH HAND. These were code-named Pink and Green and will be described in the subsequent section. By January 1965, two additional military herbicides had been evaluated and brought into the spray program. These were code-named Orange and White, and are also described below. Herbicide Orange replaced all uses of Purple, Pink, or Green and eventually became the most widely used military herbicide in South Vietnam. In April 1970, the Secretaries of Agriculture; Health, Education and Welfare, and the Interior jointly announced the suspension of certain uses of 2,4,5-T. These suspensions resulted from published studies indicating that 2,4,5-T was a teratogen. Subsequent studies revealed that the teratogenic effects had resulted from a toxic contaminant in the 2,4,5-T, identified as 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD). 'Subsequently, the Department of Defense suspended the use of Herbicide Orange (4). At the time of the suspension, the Air Force had an inventory of 1.37 million gallons of Herbicide Orange in South Vietnam and 0.85 million gallons at the Naval Construction Battalion Center (NCBC), Gulfport, Mississippi. In September 1971, the Department of Defense directed that the Herbicide Orange in South Vietnam be returned to the United States and that the entire 2.22 million gallons be disposed of in an environmentally safe and efficient manner. The 1.37 million gallons were moved from South Vietnam to Johnston Island, Pacific Ocean for storage in April 1972. B. Descriptions of the Herbicides Used in Operation RANCH HAND The following military herbicides were used in South Vietnam in Operation RANCH HAND. The first three were extensively used in both defoliation and anticrop programs. Only limited quantities of the herbicides Purple, Pink or Green were used in South Vietnam, and then primarily during the 1962-1964 time period. 1. Herbicide Orange Orange was a reddish-brown to tan colored liquid soluble in diesel fuel and organic solvents, but insoluble in water. One gallon (gal) of Orange theoretically contained 4.21 pounds (lb) of the active ingredient of 2,4-D and 4.41 lb of the active ingredient of 2,4,5-T. Orange was formulated to contain a 50:50 mixture of the n-butyl esters of 2,4-D and 2,4,5-T. The percentages of the formulation typically were: n-butyl ester of 2,4-D free acid of 2,4-D n-butyl ester of 2,4,5-T free acid of 2,4,5-T inert ingredients (e.g., butyl alcohol and ester moieties) 1-3 49.49 0.13 48.75 1.00 0.62 �Some of the physical, chemical, and toxicological properties of Orange are listed in Table 1. The structures of the n-butyl esters of 2,4-D and 2,4,5-T are shown in Figure 1. 2. Herbicide White White was a dark brown viscous liquid that was soluble in water but insoluble in organic solvents and diesel fuel. One gal of White contained 0.54 Ib of the active ingredient of 4-amino-3,5,6trichloropicolinic acid (picloram) and 2.00 Ib of the active ingredient of 2,4-D. White was formulated to contain a 1:4 mixture of the triisopropanolamine salts of picloram and 2,4-D. The percentages of the formulation were: triisopropanolamine salt of picloram triisopropanolamine salt of 2,4-D inert ingredient (primarily the solvent triisopropanolamine) 10.2 39.6 50.2 Some of the physical, chemical, and toxicological properties of White are listed in Table 1. The structures of the triisopropanolamine salts of 2,4-D and picloram are shown in Figure 1. 3. Herbicide Blue Blue was a clear yellowish-tan liquid that was soluble in water, but insoluble in organic solvents and diesel fuel. One gal of Blue contained 3.10 Ib of the active ingredient hydroxydimethyarsine oxide (cacodylic acid). Blue was formulated to contain both cacodylic acid (as the free acid) and the sodium salt of cacodylic acid (sodium cacodylate). The percentages of the formulation were: cacodylic acid 4.7 sodium cacodylate surfactant 26.4 3.4. sodium chloride water antifoam agent 5.5 59.5 0.5 Some of the physical, chemical, and toxicological properties of Blue are listed in Table 1. The structure of the sodium salt of cacodylic acid is shown in Figure 1. It should be noted that cacodylic acid and sodium cacodylate contained arsenic in the form of the pentavalent, organic arsenical. This form of arsenic was essentially nontoxic to animals as can be noted by the LD^Q value for white rats. Of the total formulation, 15.4 percent was arsenic in the organic form, only trace quantities were present in the inorganic form. The term Herbicide Blue was first applied to powdered cacodylic acid in 1961 through 1964. This first Herbicide Blue contained 65 percent active ingredient 1-4 �TABLE 1. Selected physical, chemical and toxicological properties of the three major military herbicides used in South Vietnam, 1962 - 1971.a- Herbicide Code Name Orange White Blue a Molecular Mass Specific Density, 25°C Viscosity, Centipose, 23°C Weight Total Acid Ester Equivalent Ib/gal Ib/gal Soluble in Water Specific Tpxicity for Mhite Rats mg/kgb. Relative Toxicity 589 1.28 43 10.7 8.62 No 1,173 1.12 125 9.4 2.54 Yes 3,080 Very Low 296 1.32 14 10.9 3.10 Yes 2,600 Very Low 566 Low Source: (35) Milligrams of the herbicide per kilogram of body weight of the test animal lethal to 50 percent of white rats. �n-butyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D) B. n-butyl ester of 2,4,5-trichlorophenoxyacetic acid (^,4,5-T) 0 0-CH0C-0~ +NH[C H ^ 3 6 03 Cl D. triisopropanolamine salt of 2,4-D - 0 -4- C-0 NH[C_H OHl 36 3 triisopropanolamine salt of 4-amino-3,5, 6-trichloropicolinic acid (picloram) CH - As - 0 Na' sodium salt of hydroxydimethylarsine oxide (cacodylic acid; FIGURE 1. ' Chemical structure and nomenclature of the major herbicides used in South Vietnam, 19621971. Formulas A and B comprised Orange, C and D - White, and E was Blue. 1-6 �cacodylic acid and 30 percent sodium chloride and was mixed in the field with water (7, 14). 4. Herbicide Orange II Orange II was the code-name of a formulation similar to Orange with the difference being the substitution of the issocytl ester of 2,4,5-T for the n-butyl ester of 2,4,5-T, The physical, chemical, and toxicological properties of Orange'II were similar to those of Orange. Orange II was produced solely by one chemical company. Approximately 950,000 gal of Oramge II were shipped to South Vietnam during 1968 and early 1969 (12). How much Orange II was returned to Johnston Island from South Vietnam in April 1972 was not determined. 5. Herbicide Purple Purple was first formulated in the mid-1950s time period. It was used in the Camp Drum, New York, defoliation test in 1959 (26). The formulation was a brown liquid soluble in diesel fuel and organic solvents but insoluble in water. One gal of Purple contained 8.6 Ib of the active ingredients 2,4-D and 2,4,5-T. The percentages of the formulation were: n-butyl 2,4-D 50 n-butyl 2,4,5-T 30 iso-butyl 2,4,5-T 20 The physical, chemical, and toxicological properties of Purple were similar to those described for Orange. 6. Herbicide Pink Pink was a formulation of 2,4,5-T used extensively in early RANCH HAND operations (7) and in the defoliation test program of 1963 and 1964 in Thailand (15). Pink was a mixture of the n-butyl and iso-butyl esters of 2,4,5-T. No data were available on the physical, chemical, or toxicological properties of Pink- However, Darrow et al (15) reported that it contained 8.16 Ib active ingredient per gal. The percentages of Pink formulation were: n-butyl 2,4,5-T 7. 60 iso-butyl 2,4,5-T 40 Herbicide Green Green was a single component formulation consisting of the n-butyl ester of 2,4,5-T. It was used in limited quantities in the 1962-1964 period (3). However, the only reported use of Green 1-7 �was in an evaluation program of herbicides for use against manioc and [(7), and correspondence between personnel of the Air Force Armament Laboratory, Eg!in AFB, Florida, and personnel of the Crops Division, Fort Detrick, Maryland, dated 5 Sep 63]. No data were available on physical, chemical, or toxicological properties of Green. Brown (7) reported that Green contained the same amount of active ingredient as Pink. 8. Other Herbicides Used in South Vietnam In addition to evaluating Herbicides Purple, Pink and Green, Brown (7) also evaluated Dinoxol, a mixture of 20 percent each of the butoxy ethanol esters of 2,4-D and 2,4,5-T; Trinoxol, 40 percent butoxy ethanol ester of 2,4,5-T; Diquat, 6,7-dihydrodipyridol (l,2-a:2'5 l'-C) pyrazidinium dibromide; and small quantities (grams) of 16 different chemicals. The latter chemicals were applied on native grasses and bamboo at the Saigon Navy Yard. Darrow et al (14) reported that small quantities of soil-applied herbicides were used on base camp perimeters, mine fields, ammunition storage areas, and other specialized sites requiring control of grasses and woody vegetation. The soil-applied herbicides evaluated for use in South Vietnam included Bromacil, 5-bromo-3-sec-butyl-methyluracil; Tandex, (3,3dimethyluneido) pheny1 -tert-butylcarbamate; Monuron, 3-(p-chlorophenyl)-!. 1-dimethylurea; Diuron, 3-(3,4-dichlorphenyl)-l, 1-dimethylurea; and Dalapon, 2,2-dichloropropionic acid. C. Quantities of Herbicides Sprayed in South Vietnam The estimated number of gal of the various military herbicides sprayed in South Vietnam from 1962 through 1971 have been obtained by examination of procurement and disposition records. The data obtained from these sources were compared to other sources when available; e.g., actual tactical mission records. Table 2 presents a summary of the available data on the number of gal of herbicides Blue, Green, Pink and Purple procured and disseminated in South Vietnam between January 1962 and December 1964. (3) Table 3 gives a comparison of the estimated number of gal procured and disseminated in South Vietnam between January 1965 and February 1971 as reported by the National Academy of Science (11), Westing (34), and Craig (12). The discrepancies in total herbicide quantity between the three sources occurred because Craig's data were from procurement records only, while the NAS and Westing data are based on both records and estimates. The latter two reports used different assumptions in calculating the total herbicide volume. These included such factors as spray line data (length and width of the spray swath), rate of application (1.5 or 3 gal/acre, and the amount of herbicide disseminated during a mission, 1-8 �TABLE 2. Number of gallons of military herbicide procured by the U.S. Department of Defense and disseminated in South Vietnam during the period January 1962 - December 1964.a Military Herbicide Gallons of Formulation Pounds Active Ingredient Blueb 5,200 10,000 Greenc 8,208 66,980 Pinkc 122,792 1,001,980 Purple 145,000 1,180,300 281 ,200 2,259,260 Total a Source document: Memorandum for Assistant Secretary of Defense from the Office of the Under Secretary, Department of the Air Force, Washington, D.C.; dated December 15, 1961. Subject: Summary of Current Status Project "RANCH HAND" Chemicals. (3) e was procured as a fine white hygroscopic powder which contained 65 percent cacodylic acid (active ingredient), 30 percent sodium chloride, 3 percent sul fates and 2 percent water. Approximately 290 1b of powder were mixed with 100 gallons of water (6). Thus, a total of 5,200 gal of Blue were probably disseminated in South Vietnam (primarily by the HIDAL Spray System). c d Pink and Green contained approximately 8.16 Ib active ingredient per gal (7). Purple contained approximately 8.14 Ib active ingredient per -gal (see Section V.A.3., Chapter I, p 1-26) 1-9 �TABLE 3. Estimated number of gal of military herbicide procured by the U. S. Department of Defense and disseminated in South Vietnam during the period January 1965 - February 1971. Military Herbicide Craig, 1974 ( 1 2 ) a NAS Report, 1974 (11 )b Westing, 1976 ( 3 4 ) c Orange 10,645,904 11,266,929 11 ,712,860 White 5,632,904 5,274,129 5,239,853 Blue 1,144,746 1,137,470 2,161 ,456 17,423,554 18,936,068 19,114,169 Total Data compiled from procurement and disposition records maintained by the San Antonio Air Logistics Center, Directorate of Energy Management, Kelly Air Force Base, Texas. The data for expenditures of Herbicide Orange, White, and Blue were based on procurement and delivery records for late FY 64 through FY 72, less those quantities of herbicides returned to Johnston Island in April 1972 or retained in the Continental United States. b See Table III C-l of the referenced National Academy of Science report (11) c See Table 3.3 of the referenced report. 1-10 �D. Land Area Sprayed with Herbicides in South Vietnam As noted, in Section C above, the estimate of acreage sprayed with herbicides was often based on spray line data and/or the quantity of herbicide expended. The National Academy of Science (11) discussed these parameters as they applied to the HERBS tape, the major source of all mission maps and tabulations of herbicide operations in South Vietnam for the period August 1965 through February 1971. Table 4 presents a comparison of data from three sources on the estimated number of acres treated in South Vietnam from January 1962 through February 1971. Included in these figures are the same areas of land counted more than once if they were sprayed more than once. Table 5 is a comparison of the data for acreage sprayed within the three major vegetational categories. These data have been corrected for multiple coverage. The National Academy of Science Report (11) concluded that herbicides were sprayed on 10.3 percent of the inland forests of South Vietnam, 36.1 percent of the mangrove forests, and 3 percent of the cultivated lands or approximately 8.6 percent of .the total land area in South Vietnam. Westing (34) estimated that approximately 10 percent of South Vietnam was sprayed. III. THE AIRCRAFT, SPRAY SYSTEMS AND MISSION CONCEPT IN OPERATION RANCH HAND Almost all herbicide used in South Vietnam was sprayed from aircraft. Irish et al (26) have described some ground delivery systems for herbicides, but noted these were used primarily for control of vegetation on minefields and perimeter defenses. U.S. military personnel were responsible for operating and maintaining the aircraft used in Operation RANCH HAND. The number and types of aircraft, their load capacity, ease of loading, and the spray system employed in them, all were important factors in determining the number of personnel required in performing the herbicide missions. Standard procedures were adopted in all herbicide handling phases of the operation. This section, then, reviews the aircraft factors where military personnel were likely to have physically contacted the herbicides. A. Historical The first aerial spray trials for herbicides were conducted by the military in 1944 and 1945 (14, 26). These early tests were accomplished using the U.S Army Chemical Corps M-10 smoke tanks hanged externally on a B-25 aircraft. By 1953, the U.S, Air Force had accomplished prove-out and acceptance testing of the large-capacity (1,000-gal) spray system known as the Hourglass or MC-1 Spray System. In 1960 and 1961, Air Force personnel assigned to the Special Aerial Spray Flight, La ITley AFB, Virginia, acquired two MC-1 Spray Systems 1-11 �TABLE 4. Comparison of data from three sources of the estimated number of acres treated in South Vietnam during the period of January 1962 February 1971. Data make n£ allowance for multiple coverage. ACRES TREATED YEAR NAS Report (11) Irish et ail. (26) Westing (34) MEAN 1962 NAa 5,68T 5,724 5,703 1963 NA 24,947 24,920 24,934 1964 NA 93,842 93,869 93,856 1965 75,50lb 221 ,559 221 ,552 221,555 1966 608,106 842 ,764 845,263 765,378 1967 1,570,114 1,707,758 1,707,784 1 ,661 ,885 1968 1,365,479 1 ,330,836 1 ,696,337 1,464,217 1969 1,365,754 NA T, 519,606 1,442,680 294,925 NA 252,989 273,982 1,259 NA 3,346 2,303 1970 1971 Total of Mean = 5,956,493 a Data not available (NA) ^Data for period August 65 through December 65. 1-12 �TABLES. The number of acres treated in South Vietnam, 1962 - 1971, with military herbicides within the three major veqetational categories. Data represent areas receiving single or multiple coverage and for 90 percent of all areas treated. ACRES TREATED Vegetational Category NAS Report, 1974 01 )a Westing, 1976 (34 )b Inland Forest 2,670,000 2,879,000 Mangrove Forest 318,000 746,000 Cultivated Crops 260,000 595,000 3,248,000 4,221,000 Total a See page II1-39 of the referenced report. b See Table 3.6 of the referenced report, Data for Inland Forest was woody subtotal, less acreage for mangrove forest. 1-13 �and modified them to spray insecticide and to interface with the newly acquired Fairchild-Hiller C-123 air transport. Irish et al (26) noted that in October 1961, six C-123 aircraft were made available to the USAF Tactical Air Command with a high-priority directive to install the MC-1 Spray System. Fabrication was accomplished-expeditiously and on 7 January 1962, three of the configured-aircraft arrived at Tan Son Nhut Air Base, Republic of Vietnam. During the early months of 1962, the C-123/MC-1 system and the HIDAL (Helicopter, Insecticide Dispersal Apparatus, Liquid) were evaluated for dissemination characteristics. The aircrews were members of the Special Aerial Spray Flight, Langley AFB, and were on temporary duty to South Vietnam as part of Operation RANCH HAND. Air Force,personnel engaged in the herbicide program did not receive permanent change of station assignments until 1964. In late 1962 and early 1963, an intensive RDT&E (Research Development, Testing and Evaluation) program was initiated between the Crops Division, Fort Detrick, and the Air Force Armament Laboratory, Eglin AFB, Florida, to provide improvements in spray system components in support of RANCH HAND (26). Concurrently, operational employment of the spray capability by the RANCH HAND units was intensified steadily with time and availability of resources. B. Spray Systems and Characteristics of the RANCH HAND Aircraft Tests and evaluations of aircraft and spray systems were conducted on the calibration grids on Test Area C-52A, Eglin AFB, Florida (6, 14, 22, 27, 35) and on the calibration grid at Pran Buri, Thailand (11, 13, 15). The C-123/MC-1 spray configuration was initially calibrated to spray 1 to 1.5 gal of herbicide per acre (gal/A) (14, 26). Thus, in 1962 and 1963 the herbicide missions conducted using this initial system resulted in the dissemination of herbicide at this lower rate. The numerous modifications and extensive evaluations of the equipment configurations at Eglin AFB and Pran Buri did not result in equipment changes for Operation RANCH HAND until 1964 (14). . Darrow et al (14) and Irish et al (26) have reported that in early 1964 the rate of 3 gal/A was obtained at first by making double passes with the aircraft but by late 1964 the modifications were complete and the system was capable of spraying 3 gal/A in a single pass. The modified 1,000-gal C-123/MC-1 spray system was capable of depositing 3 gal/A on swaths 240 feet wide when spraying at an airspeed of 130 knots at a 150 feet altitude. Two 20-hp pumps were needed to achieve the required flow rate of 430 gal/min of Purple (26). The HIDAL spray system was capable of deposits of 1.5 gal/A when flown inwind at 55 knots and at an altitude of 100 feet (26). The tank volume for this system was 200 gal. 1-14 �In early 1966, following its development, the A/A 45Y-1 Internal Defoliant Dispenser, replaced the MC-1 in all C-123 aircraft. However, completion of calibration tests and performance characteristics for this spray system did not occur until 1968 (16, 22, 27). The A/A 45Y-1 defoliant dispenser was a modular spray system for internal carriage in cargo aircraft. The module consisted of a 1,000 gal tank, pump, and engine (20 hp) mounted on a frame pallet. An operator's console was an integral part of the unit but was not mounted on the pallet. The C-123 aircraft had wing booms 1.5 inches in diameter and 22 feet long extending from the outboard engine nacelles toward the wing tips. A short tail boom 3 inches in diameter was positioned centrally near the aft cargo door. There were 16 nozzles on each wing boom and eight on the tail boom. The nozzles were check valve bodies with 3/8-inch orifices (no nozzle tips). The system was capable of spraying at the rate of 240 gal/min, which, when released at 150 feet altitude at 130 knots airspeed produced a swath 260120 feet wide with a mean deposit of 3 gal/A in a coarse spray having an MMD (mass median diameter) of 320 to 350 micron (p). Spraying time was approximately 3.5 to 4 minutes, which was adequate to dispense 950 gal of chemical on a spray line about 8.7 statute miles (14 km) in length. In order to achieve predictable deposits, it was recommended that the missions be conducted under inversion to neutral temperature situations and calm wind conditions. Craig (12) has reported that each aircraft had a crew of 3 men: the pilot, co-pilot (navigator), and flight engineer (console operator). However, observers (Vietnamese and American) frequently accompanied the aircrews on herbicide missions (32). C, Mission Concepts The objectives of the defoliation and anticrop programs in South Vietnam have been thoroughly reviewed by Huddle (23) and others (11, 14, 34). It is the objective of this section to elaborate only on the background and mechanics of a "typical" herbicide mission that would have influenced the degree of exposure to herbicides by aircrew and/or ground personnel. The following scenario of events or "standard operating procedures" has been compiled from reports by Craig (12), Darrow et al (14), Irish e't al (26) and the National Academy'of Science Report (11). 1. Each of the 11 different companies that manufactured military herbicides packed them in new ICC 17C 55-gal 18 gauge steel drums for shipment to Southeast Asia (12). Until 1967, lined drums were used only for shipment of Blue. However, because of the results of compatibility tests, lined drums were also used to ship White beginning in 1967. 1-15 �2. ^ach herbicide drum was marked with a three-inch color-coded band around the center to identify the specific military herbicide. This marking was initially a 12-inch band, but was changed to a 3-inch band in March 1966. 3. Shipping time from the arrival of the herbicide at a U.S. port until it arrived in South Vietnam varied from 47 to 52 days. 4. About 10 out of every 10,000 drums shipped were received in a damaged or defective state. This represented a damage rate of 0.1 percent. About 50 percent of these damaged drums leaked as a result of punctures or split seams. These were caused by improper loading and defective drums. Forklifts operated by stevedores also caused punctures. Redrumming was accomplished at the ports. 5. About 65 percent of the herbicide was shipped to the 20th Ordnance Storage Depot, Saigon, and 35 percent was shipped to the 511th Ordnance Storage Depot, Da Nang. Under the normal handling procedures, drums were unloaded at Da Nang and Saigon from the cargo vessel directly into semi-trailers and were placed in an upright position. The trailers were driven to the various units of the 12th Air Commando Squadron (primarily at the bases of Da Nang, Phu Cat, or Bien Hoa) for disposition. 6. Normally the contents of the drums were transferred into blocked F-6 trailer tanks through a suction tube without removing the full drums from the semi-trailers. Each F-6 trailer held 4,298 gal or about 78 drums of herbicide. If blocked F-6 trailer tanks could not accommodate the total inventory, the drums were stacked in pyramidal style until needed. 7. The transfer of the herbicides from the 55-gal steel drums to storage tanks or aircraft tanks required some precautionary measures. Personnel charged with the supervisory responsibilities of handling the herbicides were indoctrinated in appropriate safety precautions including the use of gloves and face shields as needed. Personnel handling the chemicals were encouraged to "take normal sanitary precautions and to maintain personal cleanliness and to avoid skin and eye contact with the material. Contaminated clothing were to be washed before re-use. Spillage on the skin or in the eyes was to be rinsed copiously with clea1" water" (14). 8. When the herbicide was pumped from the drums into the F-6 trailers about 0.5 to 1.5 gal remained in the drum. Hence the drum was placed on a drain rack and the "drippings" were collected from many drums in a pan-type receptacle and used for spraying base perimeter areas. 1-16 �9. Empty drums were given to the military forces (Vietnam, U.S. and Free World Military Assistance Forces) for use as barriers in defensive positions. The drums were filled with sand or concrete and used in the construction of bunkers or in foundations for runways and barbed wire perimeters (12). 10. Surface areas contaminated by spillage of the herbicides were flushed with diesel fuel or water with diversion of the drainage into settling basins or pits for incorporation into the soil. 11. The F-6 trailers were tied to plumbing and pumps so that the herbicide could be delivered to the aircraft without moving the trailers. 12. As previously noted, Orange was insoluble in water, while Blue and White were not. When Orange was mixed with either Blue or White, a gummy substance formed. The F-6 trailers were therefore color-coded to correspond to the drum color-codes and used exclusively for the herbicide to which the code applied. 13. The aircraft spray tanks, positioned in the center of the airplane, and the spray system were purged before the type of herbicide carried was changed. Particular attention had to be given to sequences involving Blue and White. .A mixture of these two herbicides resulted in the formation of a precipitate consisting of the sodium salt of 2,4-D. 14. Most of the personnel involved in the actual handling of the herbicide drums were Vietnamese. However, a USAF flight mechanic or crew chief was responsible for insuring that the aircraft wa^ properly loaded and the spray system functional. A flight mechanic was also the console operator for the spray unit. The pilot and co-pilot were officers while the flight mechanics and crew chiefs were usually enlisted personnel. 15. For record keeping purposes a herbicide "mission" consisted of several aircraft; if only one aircraft was used the operation was termed a sortie.' All missions within a target formed a project. 16. Aircraft takeoffs were normally before sunrise. From a tactical point of view, the arrival of the aircraft at the target area just prior to sunrise permitted the aircraft to approach the target from the direction of the rising sun. This afforded some degree of protection from enemy ground fire. From the standpoint of herbicidal action, application by aerial spray was most effective if accomplished prior to 0800 hours while inversion conditions existed, in the absence of precipitation, and while the wind was calm or not exceeding a velocity of 8 knots. This insured the proper settling of the spray on the target area. 1-17 �17. Within the aircraft, it was not uncommon to have herbicide leakage from around the numerous hose connections joining the spray tank and pumps with the wing and aft spray booms. In hot weather, the odor of herbicide within the aircraft was decidedly noticeable. Periodically, the spray tank and console were removed (especially with the portable A/A 45Y-1 system) and the interior flushed with surfactant or soap and with water. Because of the corrosive nature of some herbicides, it was necessary for the aircraft to also be repainted periodically. 18. In the 1966 through 1968 period, more than one sortie per day was often common. For example, during the first six months of 1968, the 24 UC-123B aircraft assigned to RANCH HAND averaged approximately 39 sorties per day. IV. PERTINENT DEPLOYMENT AND BIOLOGICAL FACTORS OF THE HERBICIDES The previous section dealt with those factors that would Influence the frequency of "physical contact" with liquid forms of the herbicide. As noted, the individuals most likely to be exposed to liquid herbicide were those charged with transport, handling, and disseminating responsibilities. This section will deal with some factors that would have influenced the likelihood of contact with the herbicides once they had been sprayed. A. Use Patterns of Individual Military Herbicides 1. Herbicides Orange, Orange II, Purple, Pink and Green Herbicides Orange, Orange II, Purple, Pink and Green were effective defoliants and herbicides on a wide array of woody and broadleaf herbaceous species. Grasses, bamboos, and other monocoiyledonous plants were less affected. The effects of these military herbicides on the forests of South Vietnam has been well documented (5, 9, 11, 13, 14, 21, 29, 32, 34). Darrow (13), and Harrow et al (14, 15) showed that at the normal use rates (3 gal/A) these herbicides, when applied to mixed woody vegetation, caused a browning and discoloration of the foliage within a period of one or two weeks. Foliage of the more susceptible species turned brown rapidly, and subsequent leaf drop occurred over a period of one to two months. Under tropical conditions, maximum defoliation occurred two to three months after the spray application. At 3 gal/A the maximum average defoliation in a single or multiple canopy was 88 and 75 percent respectively for rainy season application, or 82 and 67 percent respectively for dry season application. Under tropical forest conditions, satisfactory levels of defoliation persisted for four to twelve months or more. The National Academy of Science (11) reported that from August 1965 through February 1971, 2,962 herbicide mission^ (out Q? a total of 6,237 missions for all herbicides and all use:.) were for forest defoliation uring Orange. These 2,962 missions �accounted for 90 percent of all Herbicide Orange (including Orange II) used in South Vietnam. Likewise 90 percent of all Purple, Pink and Green sprayed in South Vietnam was for forest defoliation (26). Orange and Orange II (and Purple) were also used in control of broadleaf crops (8, 11, 34). For convenience and simplification in programming crop destruction and defoliation targets, application rates were routinely 3 gal/A (14). Annual crops; e.g., beans, gourd, jute, peanuts, and ramie, were rapidly killed by an application of Orange. Root or tuber crops; e.g., manioc, potatoes, taro, and yams, showed great reduction in yield when treated with Orange during early growth stages. Perennial and woody tropical crops; e.g., jackfruit, papaya, castor bean, and mango were susceptible to Herbicide Orange (14). From August 1965 through February 1971, crop destruction missions with Orange accounted for 8 percent of the Herbicide Orange applied (11). The remaining 2 percent of Herbicide Orange used in South Vietnam was used around base perimeters, cache sites, waterways, and communication lines (11). 2. Herbicide White Herbicide White was effective principally on broadleaf herbaceous and woody plants. Conifers (pine trees) were especially susceptible to White. However, the herbicidal action on woody plants was slow and full defoliation did not occur for several months after spray application (14). Since White was water soluble, it was frequently used in field situations where drift was to be held at a minimum (e.g., near rubber plantations). White was sprayed during 1,324 defoliation missions (21 percent of all missions) and of the total volume of White used in South Vietnam, 99 percent was for defoliation (11). The remaining one percent of White was used primarily in base perimeter applications. White was not recommended for use on crops because1' of the persistence of picloram in soils (14). 3. Herbicide Blue Herbicide Blue was the herbicide of choice for other crop destruction missions; e.g., on cereal or grain crops. For crop destruction missions, the basic rate of 3 gal/A was used. Helicopter applications were usually at the rate of one gal/A for control of grain crops. (15). Forty-nine percent of all Blue (580,000 gal) was used in crop destruction missions conducted from August 1965 through February 1971 (11). The remaining Blue was used in defoliation or in control of grass around base perimeters pi)- As a defoliant, Blue caused a rapid browning or desiccation with accompanying shriveling and leaf fall. Noticeable browning or discoloration was evident in one day, with maximum defoliation occurring within two to four weeks (14). 1-19 �B. Canopy Penetration of Defoliants As previously noted, 90 percent of all Herbicide Orange (and probably Purple, Pink and Green) was for defoliation in the forests and mangroves of South Vietnam. The quantity of herbicide that reached the forest floor is not known. However, such factors as canopy composition and time of season of application would have influenced this value. Huddle (23) recorded the following 1968 statement by Dr C.E. Minarik (Dr Minarik was at that time Director, Plant Sciences Laboratories, Fort Detrick, Maryland): "Three gallons per acre is employed. We would prefer to use less if we could get uniform deposition, but in these dense jungle areas where there may be 300 tons of vegetation per acre, this is the minimal effective volume. The three gallons contain 24 pounds of herbicide on an acid basis. Thus high dosage rate is also a requirement since much of the vegetation consists of trees 100 to 150 feet tall." In the evaluation tests of the C-123/A/A 45Y-1 Spray System, Harrigan (22) and Klein and Harrigan (27) found that in mass distribution studies (following aerial dissemination) 87 percent of the Orange Herbicide intercepted by collecting devices had a mass median diameter between 100 and 500u. The mean diameter was 367u. Harrigan (22) concluded that with altitude delivery conditions at 130 knots and 150 feet altitude, most of the Orange released would have settled onto the forest canopy in a swath approximately 260^20 feet wide within which effective defoliation was produced. Hurtt and Darrow (25) showed that the minimum biological effective deposition rate under the climatic conditions of South Vietnam was 1.0 gal/A at a mass median diameter of 350y. The minimum biological effective rate was defined as "that rate which promoted leaf fall and inhibited growth" (25). In canopy penetration studies, Tschirley (33) found (with phenoxy herbicide formulations similar to Orange) that the volume of spray reaching lower sampling levels varied proportionately with the amount deposited on the top line above the canopy. On the average, about 21 percent of the spray penetrated the upper canopy and about 6 percent penetrated to ground level. He also found that the percentage penetration remained relatively constant for drop densities greater than about 100 per square inch. Spray drops having mass median diameters of 400 to 500y would approximately equal 100 drops per square inch. Moreover, the percent spray penetration through forest canopies was inversely related to canopy density (33). 1-20 �No data were available on the number of defoliation missions conducted during the wet or dry seasons. However, as noted earlier, defoliation of forest canopy was greatest during the rainy season, when the vegetation was in full-leaf and actively growing. V. ESTIMATED QUANTITIES OF INDIVIDUAL CHEMICALS SPRAYED IN SOUTH VIETNAM For this report, the total quantities of individual chemicals become important only in reference to their potential association with dose and duration'of exposure to the population at risk. Although the National Academy of Science (11) primarily defined the population at risk as Vietnamese (especially Montagnards), our concern at this time is with U.S. military forces. The chemicals of concern are 2,4-D, 2,4,5-T and TCDD. The extreme toxicity of TCDD, however, makes it the prime chemical of concern. The toxicology of these chemicals is discussed in detail in Chapters IV and VI. The previous scientific assessments of these chemicals as applied in South Vietnam are addressed in Chapter V. A. Herbicide Orange and its Components 2,4-D, 2,4,5-T and TCDD 1• Concentrations of TCDD in Orange, Purple, Pink and Green Figure 2 shows the structure of TCDD and gives a brief description of some of its physical and chemical characteristics. Table 6 shows the available data on the concentration of TCDD (parts per million, ppm) in samples of Herbicides Orange and Purple. As noted, the mean concentration of the surplus Herbicide Orange remaining after termination of its use in South Vietnam was a value derived from data on the analyses of 492 samples. Some of these data have been previously published (4, 11). Craig (12) has reported that the Orange Herbicide maintained at the Naval Construction Battalion Center (NCBC), Gulfport, Mississippi, was probably authorized and procured during the 1968-69 fiscal year. The Orange returned from Vietnam in 1972 (to Johnston Island) was procured no earlier than late FY 64, since the first shipment of Orange did not arrive in Vietnam until early 1965, and a six months lead time was typical. Note that the mean TCDD concentration was 1.91 ppm for the Johnston Island inventory and weighted means of 1.77 and 2.11 for samples analyzed from the NCBC inventory. It is important to note the range of TCDD concentration of TCDD in both surplus Orange inventories. The maximum concentration of TCDD in Orange samples collected at NCBC was 15 ppm, while the maximum concentration of TCDD reported in samples from Johnston Island was 47 ppm. Only 4 of 200 samples from Johnston Island exceeded TCDD levels found in the NCBC inventory (4). The values of these 4 samples were 17, 22, 33, and 47 ppm (4). 1-21 �A. Structure 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) B. Physical Characteristics molecular weight melting point, °C decomposition point, °C C. 322 303 - 305 980 - 1,000 Chemical Characteristics Solubility, grams/liter ortho-dichlorobenzene chlorobenzene Orange Herbicide benzene chloroform acetone 1.40 0.72 0.58 0.57 0.37 0.11 . normal-octanol 0.05 lard oil methanol water 0.04 0.01 2 x 10 FIGURE 2. Structure and physical/chemical characteristics of 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD or dioxin. 1-22 �TABLE 6. Concentration, ppm, of TCDD in samples of Herbicides Orange and Purple.3 Number of Samples Orange Purple Range of TCDD (ppm) Johnston Island b Inventory, 1972 200 (4)c 0.05-47 1.91 Johnston Island Inventory, 1974 10 0.07-5.3 1.68 NCBC, Gulfport d Inventory, 1972 42 0.05-13.3 1.77 NCBC, Gulfport Inventory, 1975 238 0.02-15 2.11 Source of Samples Mean TCDD, Concentration (ppm) Eglin AFB Archived Sample 45 Eglin AFB Inventory, 1972 0.04 The Weighted Mean Concentration of TCDD in Orange = 1.98 ppm Analyses for TCDD performed by Interpretive Analytical Services, Dow Chemical U.S.A., Midland Michigan; Aerospace Research Laboratories, Wright-Patterson AFB, Ohio; and The Brehm Laboratory, Wright State University, Dayton Ohio. Surplus Herbicide Orange was shipped from South Vietnam to Johnston Island for storage in April 1972. c Four of 200 samples may have been Herbicide Purple, see text. d The Naval Construction Battalion Center (NCBC) Gulfport, Missippi served as a storage site for Surplus Herbicide Orange from 1969 to 1977. e Herbicide Purple was extensively used in the evaluation of aerial spray equipment on Test Area C-52, Eglin Air Force Base Reservation, Florida, 1962-1964. 1-23 �Only one sample of Herbicide Purple has been analyzed (see Table 6). The age of the sample was not known except that it was representative of the Purple applied to Grid 1 (ARPA Grid), Test Area C-52A, Eglin AFB, Florida (Personal information, A.L. Young, and references 34 and 36), and thus, may have been from the 1962-1964 time period. The 1971 Report on 2,4,5-T by the Executive Office of the President (18) presented data on TCDD concentrations found in the analysis of Technical 2,4,5-T from one manufacturer. The data were for samples manufactured yearly from 1958 through 1969, and ranged from 1 to 32 ppm. The highest levels of TCDD were found in samples manufactured in 1965 (32 ppm) and 1968 (25 ppm). If these two samples had been used in formulating Herbicide Orange, the concentrations in the Orange would have been 16 and 12.5 ppm, respectively. If the lowest TCDD containing samples for the same two years would~Fave been used (i.e., samples containing 5 and 1 ppm TCDD) the concentrations in the Orange would have been 2.5 and 0.5 ppm, respectively. The one sample of Purple reported in Table 6 contained 45 ppm. Thus, the Technical 2,4,5-T used in that sample may have contained 90 ppm TCDD. When the Orange Herbicide was shipped to Johnston Island from South Vietnam, redrumming of the herbicide in South Vietnam was accomplished as necessary (12). The project (PACER IVY) involved U.S. military personnel. One of the individuals participating in the redrumming operation at Da Nang (redrumming also occurred at Phu Cat and Bien Hoa) has stated that drums of Purple were found (although fewer than 20) and redrummed into Orange-banded drums (personal communication, Or Michael D. Neptune, now with the U.S. Environmental Protection Agency, Washington, D.C.). In addition, an analytical chemist involved in the analyses of Orange samples for 2,4-D and 2,4,5-T, reported finding significant quantities (15 percent) of the iso-butyl ester 2,4,5-T in a few of the samples collected from Johnston Island (unpublished data, personal communication, Dr Eugene L. Arnold, now with the Clinical Sciences Division, USAF School of Aerospace Medicine, Brooks AFB, Texas). Thus, the 4 samples of Orange Herbicide containing TCDD concentrations greater than 15 ppm, may have been Purple. If these were, in fact, from drums of Purple, then the mean concentration of TCDD in 5 samples of Purple would have been 32.8 ppm. The mean value of 32.8 ppm may or may not represent the TCDD concentration of the Herbicide Purple used in South Vietnam from 1962 through 1964. Data from the 1971 Report on 2,4,5-T (18) suggests that Purple manufactured in 1958 through 1963 would have had a mean concentration of approximately 5 ppm (4.711.2 as the mean and standard deviation for the 6 samples reported for the years 1958 through 1963). However, the persistence of TCDD in soils of the two grids used for the testing and evaluation of the early RANCH HAND spray systems may indicate that Purple indeed had concentrations of TCDD from 17 to 47 ppm. Young (35) and Young et al (37) reported finding concentrations of 710 parts per trillion TCDD in the top 6 inches (15 cm) of soil collected in 1974 from the equipment-testing 1-24 �grid known to have received at least 1,894 Ib of Purple per acre during the 1962 through 1964 period. The test grid had received 16,164 gal of Purple. On an adjacent test grid, 1,168 pounds of Orange per acre had been disseminated during the 1964-1966 programs evaluating the A/A 45Y-1 Spray System. The'levels of TCDD in the soil treated with Orange at comparable depth was 30 parts per trillion. All soil samples were analyzed in 1973. Young et al (36) have reported the half-life of TCDD to be less than one year when in the presence of the phenoxy herbicides. Persistence data suggested that the levels of TCDD in Purple and Orange were significantly different. Further evidence of this is recorded by the National Academy of Science (11) for TCDD residue found in the soils of the Pran Buri Calibration Grid. They reported finding levels from <0.0012 to 0.233 ppm TCDD in the top 6 inches of soil from this testing ground. They concluded that since the grid had received approximatly 1,000 Ib/A 2,4,5-T in 1964-65, the original concentration of the TCDD in Orange would have ranged from <3 to 50 ppm. The NAS Committee (11) assumed that the material applied to the Pran Buri Calibration Grid was Orange. Darrow et al (15), responsible for the original calibration studies, reported that the Pran Buri Calibration Grid received 6,000 gal Purple, 3,800 gal Pink (all 2,4,5-T) and only 825 gal Orange. Since the majority of herbicide applied on this grid was either Purple or Pink, it further supports the contention that the four high-TCDD-containing samples from Johnston Island were Purple. Accepting the mean concentration of TCDD in Purple as 32.8 ppm and recognizing that Pink and Green contained essentially twice the active ingredient (8.16 Ib acid equivalent 2,4,5-T per gal) as Purple (4.0 Ib acid equivalent 2,4,5-T per gal), the mean concentration of TCDD in Pink and Green would have been twice that of Purple, or 65.6 ppm. Also, from the above discussion, it can be concluded with reasonable certainty that the weighted mean concentration for aVl_ Herbicide Orange sprayed in South Vietnam was 1.98 ppm: individual lots may have contained higher (<15 ppm) or lower (>_ 0.02 ppm) concentrations of TCDD, but the weighted mean was 1.98 ppm. 2- Concentrations of 2,4-D and 2,4,5-T in Orange The original military specifications for Herbicide Orange were published on 19 July 1963 as specifications MIL-H-51158 (MU) and MIL-H-51147 (MU). As written in the specifications, for the n-butyl ester of 2,4-D: "The total acid equivalent of the herbicide shall be not less than 78 nor more than 80 percent when tested as specified. The free acid content of the herbicide shall not be greater than 1.0 percent." For the n-butyl ester of 2,4,5-T the specifications noted: "The total acid equivalent of the herbicide shall be not less than 80 nor more than 82 percent when tested as specified. The free acid content of the herbicide shall not be 1-25 �greater than 1.0 percent." Orange was to be a 50:50 mixture of the products from the two specifications. These specifications were updated on 7 November 1966. Fee et al (20) and Hughes et al (24) have extensively analyzed*Herbicide Orange samples (from Johnston Island and NCPC, Gulfport, Mississippi) for composition. Table 7 .is a comparison of different manufacturers' lots for percent composition. Although the actual mean composition varied from the "theoretical" specification, the analytical method employed to test the total acid equivalent of the herbicide permitted some fluctuation in content. The parent acid portion of the herbicide molecule will remain as the herbicidally active portion of its respective ester form, while the ester appendage to the parent acid form will serve to satisfy some additional properties, such as decreased water solubility and increased surface penetration and/or translocation. The butyl ester of 2,4-P contained 79.4 percent acid 2,4-D and the butyl ester of 2,4,5-T contained 80.2 percent acid 2,4,5-T. From the data in Table 7, the mean actual weight of active ingredient per gal of Orange was 4.14 and 4.00 pounds for 2,4-D and 2,4,5-T, respectively. These values have been accepted also for the active ingredients in Herbicide Purple. 3. Quantities of Herbicides and TCDD Disseminated in South Vietnam •~-~~ Using data in Tables 2 and 3 (herbicide procurement records), Table 6 (mean TCDD concentration in Orange) the value of 32.8 ppm TCDD for Purple,the value of 65.6 ppm TCDD for Pink and Green, and Table 7 (mean, actual composition of Herbicide Orange), an estimate of the quantities of herbicides and TCDD disseminated in South Vietnam from January 1962 through February 1971, can be determined. These "estimated" quantities are in Table 8. The National Academy of Science Committee on the Effects of Herbicides in South Vietnam (11) estimated that between 220 and 360 pounds of TCDD were released over South Vietnam during the period August 1965 to February 1971. The estimate of 368 pounds in Table 8 for TCDD falls very close to their estimate. The important difference is that 143 pounds of the TCDD reported in Table 8 (or approximately thirty-nine percent of all the TCDD) was contained in Purple, Pink, and Green and was sprayed on 90,000 acres in Vietnam from 1962 through 1964» a time period when only a small force of military personnel were in South Vietnam. Herbicide Orange was sprayed on 3.5 million acres from 1965 through 1970. However, 90 percent of the Orange was sprayed on 2.9 million acres of inland forests and mangrove forests. 1-26 �TABLE 7. Composition, Percent, of Selected Samples of Herbicide Orange in Relation to Military Specifications. NCBC Inventory Number9 ASN 10 ASN 14 Mean Composition Approximate Military Specification^ Component ASN 8 Number of Gallons 123,695 383,955 145,860 Level of TCDD <0.02 ppm 0.30 +0.06 ppm <0.02 ppm n-Butyl ester 2,4-D 42.6% 46.2% 43.7% 44.2% 49.5% n-Butyl ester 2,4,5-T 39.3 44.9 42.2 42.1 48.8 Other Butyl esters of chl orophenoxyaceti c acids 7.96 4.01 ^ Octyl esters of "-1 chlorophenoxyacetic acids 5.76 0.25 Acid, 2,4-D 0.78 0.19 Acid, 2,4,5-T 0.84 Inert Ingredients0 2.76 9.05 7.0 *• 2.0 — 0.65 0.5 0.1 0.13 0.78 0.6 1.0 4.32 3.62 3.6 0.6 t—t Selected samples of Herbicide Orange were collected from the surplus inventory maintained at the Naval Construction Battalion Center (NCBC), Gulfport,, Mississippi. Samples represented lots produced by different manufacturers. Analyses for TCDD and samp]_e composition were performed by the Aerospace Research Laboratories, Wright-Patterson AFB, Ohio. [_ See Reference by Hughes et al. ( 4 . ] 2)] ^Military specifications for manufacture of Herbicide Orange were based on Specifications MIL-H-51147A (MU) and MIL-H-51148A (MU) dated 7 Nov 1966. c lnert ingredients included butanol, toluene, butylchloride, dichlorophenol, trichlorophenol, butoxydichlorobenzene, and butoxytrichlorobenzene. �TABLE 8. Estimated quantities of herbicides and TCDD disseminated in South Vietnam from January 1962 - February 1971. Chemical Pounds 2,45-Da 55,940,150 2,4,5-Tb 44,232,600 TCDDC 368 Picloram 3,041,800 Cacodylic Acid6 3,548,710 Total of Herbicides 106,763,260 2,4-D was an active ingredient in Herbicides Orange, Purple and White. From data in Table 7, the acid equivalents for 2,4-D in Herbicide Orange and White were calculated to be 4.14 Ib/gal and 2.00 Ib/gal, respectively. The acid equivalent for 2,4-D in Herbicide Purple was assumed to be 4.14 Ib/gal. 2,4,5-T was an active ingredient in Green, Pink, Purple and Orange. Approximately 276,000 gal of Green, Pink and Purple were sprayed in South Vietnam prior to 1965, when it was replaced by Herbicide Orange. Herbicides Green and Pink contained 8.16 Ib/gal 2,4,5-T. Herbicides Purple and Orange contained 4.00 Ib/gal 2,4,5-T (Table 7). G The mean TCDD concentration in Herbicide Purple was estimated at 32.8 ppm. The mean TCDD concentration in Herbicides Pink and Green was estimated at 65.6 ppm. The mean TCDD concentration in Herbicide Orange was estimated at 1.98 ppm. Picloram was an active ingredient of Herbicide White. e Cacodylic acid was the acitve ingredient of Herbicide Blue. The Herbicide Blue formulation contained 15.4 percent arsenic in the pentavalent organic form. The value includes 10,000 Ib cacodylic acid disseminated in South Vietnam from 1962-1964. 1-28 �B. Military Projects that Involved Handling Herbicides Orange, Purple, Pink or Green. Herbicide Orange was first manufactured in late 1964. It arrived in Vietnam for use in Operation RANCH HAND in early 1965. Prior to Orange, Herbicides Purple, Pink and Green were used but in far less quantities and on a limited area. All of the quantities of Orange returned from Johnston Island in 1972 and those stored at the Naval Construction Battalion Center since late 1968 were destroyed by at-sea incineration in 1977. From the first aerial spray test in 1961 through the incineration project in 1977, numerous U.S personnel directly handled the herbicide in support of specific project goals. Table 9 was assembled after an extensive search of available documents and from personal contact with eleven different individuals that had participated in one or more of the listed projects. Other than Operation RANCH HAND the most extensive handling of Herbicide Orange occurred during Project PACER HO. At the time of this latter project, analytical techniques were sufficiently developed to permit the environmental monitoring of TCDD at the parts per trillion level during all stages of the project. These data permitted an assessment of the actual exposure of personnel involved in the handling of the herbicide. Chapter II is devoted to Project PACER HO, the disposal of the surplus Herbicide Orange. VI. SUMMARY The choice of herbicides used in South Vietnam in Operation RANCH HAND, 1962-1971, was based upon those herbicides that had been widely used in world agriculture, shown to be effective in controlling a broad specturm of vegetation, and proven safe to humans and animals. * The major herbicides used in South Vietnam were the phenoxy herbicides 2,4-D and 2,4,5-T. These two herbicides were formulated as the water insoluble esters and code-named by the military as Purple, Orange, Pink and Green. A water soluble amine formulation of 2,4-D was used in Herbicide White. Two other herbicides were extensively used by the military, picloram (in White) and cacodylic acid (in Blue). An estimated 107 million pounds of herbicides were aeriallydisseminated on 6 million acres in South Vietnam from January 1962 through February 1971. Approximately 94 percent of all herbicides sprayed in Vietnam were 2,4-D (56 million pounds or 53 percent of total) or 2,4,5-T (44 million pounds or 41 percent of total). The 44 million pounds of 2,4,5-T contained an estimated 368 Ib of the toxic contaminant, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD or dioxin). Ninety-six percent of all 2,4,5-T was contained in Herbicide Orange; the remaining 4 percent in Herbicides Green, Pink and Purple. . However, Herbicides Green, Pink and Purple contained approximately 40 percent of the estimated amount of TCDD disseminated in South Vietnam. 1-29 �TABLE 9. Project Data on the major military projects involved in the handling and/or spraying of Herbicides Orange, Purple, Pink or Green in support of military programs in South Vietnam. Dates Brief Description Selected References Project AGILE 1960-1968 Selection of herbicides, and development and evaluation of defoliation techniques. Brown, 1962 (7) Coates et a!, 1962 (10) Darrow et al, 1966 (15) Demaree and Creager, 1968(16) Operation RANCH HAND 1962-1971 Aerial spraying of herbicides in South Vietnam. Anonymous, 1961 (3) Fair, 1963 (19) Ellison, 1967 (U) Darrow et al, 1969 (14) Huddle, 1969 (23) McConnell, 1970 (29) USAF Projects 2525, 5172 5186, 5957 1962-1970 Development and testing of aerial spray equipment Biever, 1969 (6) CO o Redrumming and movement of surplus herbicide from South Vietnam to Johnston Island Craig, 1975 (12) 1972-1977 Maintenance of herbicide inventory and research on options for disposal Young, 1974 (35) Anonymous, 1974 (4) Lavergne, 1974 (28) Newton, 1975 (30) Young, et al, 1976 (37) 1977 Dedrumming of herbicide inventory and at-sea incineration of Herbicide Orange Ackerman et a l , 1978 (1) Project PACER IVY 1971 AFLC Project on Disposition of Herbicide Orange Project PACER HO Klein and Harrigan, 1969(27) Harrigan, 1970 (22) �Green, Pink and Purple were sprayed as defoliants on less than 90,000 acres from 1962 through 1964, a period when only a small force of U.S. military personnel were in South Vietnam. Ninety percent of all the Herbicide Orange (containing 38.3 million pounds of 2,4,5-T and 203 Ib of TCDD) were used in defoliation operations on 2.9 million acres of inland forests and mangrove forests of South Vietnam. The handling, transport and storage procedures employed for the herbicide generally precluded physical contact with the herbicides by most military personnel assigned to Operation RANCH HAND. However, flight mechanics (console operators for the internal spray systems) and crew chiefs (responsible for loading the aircraft) were the most likely military personnel exposed to the herbicides. The methods employed in spraying the herbicides and the geographical areas designated for dissemination of the herbicides generally precluded direct physical contact with the herbicide by military personnel assigned to other military programs. 1-31 �CHAPTER I LITERATURE CITED 1. Aekerman, D.G., H.J. Fisher, F.J. Johnson, R.F. Maddalone, B.J, Mathews, E.L. Moon, K.H. Scheyer, C.C, Shin, and R.F. Tobias, 1978. A£-4ea ^nc^tneAa-tuw oft HcAb-tcu.de Orange onboard the. M/T l/a£canai. Environmental Protection Technology Series EPA-600/2.J8-Q86. Office of Research and Development. U.S.- Environmental Protection Agency, Research Triangle Park, North Carolina. 263 p. 2. Advisory Committee on 2,4,5-T. 1971. Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency. 76 p. 3. Anonymous. 1961. Memorandum for Assistant Secretary of Defense. Subject: Summary of current status project "RANCH HAND" chemicals. Department of the Air Force, Office of the Under Secretary, Washington, D.C. Win. 4 p, 4. Anonymous. 1974, Disposition of Orange Herbicide by incineration, Final Environmental Statement. Department of the Air Force, Washington, D.C. 737 p, 5. Bethel, J.S., K.J. Turnbull, D. Briggs, and J. Flores. 1975. Military defoliation of Vietnam forests. Am&t-tcan F0<te-i£6 81(1):26-30, 56-61. 6. Biever, H. 1969. Defoliant history of Test Area C-52A. Working Papers. Armament Development and Test Center, Eglin AFB, Florida. December 1969. 7. Brown, J.W. 1962, Uefle&t£t0na£ Apbcuj te4tt> -in South U.S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland. 119 p. Available from' the Defense Documentation Center, Defense Logistics Agency, Cameron Station, Alexandria, Virginia, DDC Number AD 476961. 8. Carrier, Hickey's in South Science, J.M. 1974. The location of herbicide missions and Informants in South Vietnam. The Effects of Herbicides Vietnam, Part B. Working Papers. National Academy of Washington, D.C. 15 p. 9. CAST. 1975. Effects of herbicides in Vietnam and their relation to herbicide use in the United States. Council for Agricultural Science and Technology. Report No. 46. Department of Agronomy, Iowa State University, Ames, Iowa. 14 p. 1-32 �10. Coates, J.H., L.M. Sharpe, and H. Pollack. 1962. The 4,to£iL6 oft ehenu.ca£ e.on&io£ ofi vegetation -in le&ttuw to need-i. Technical Notes 62-68. Institute for Defense Analyses, Department of Defense, Washington, D.C. 30 p. 11. Committee on the Effects of Herbicides in South Vietnam. 1974. Part A. Summary and conclusions. National Academy of Science, Washington, D.C. 398 p. 12. Craig, D.A. 1975. Use of Herbicides in Southeast Asia. Historical Report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 p. 13. Darrow, R.A. 1973. Foliage characteristics and defoliation/ herbicidial responses in a Thailand Forest. Weed Sex.. Soc. Am. Ab*#l. 66, pp 29-30. 14. Darrow, R.A., K.R. Irish, and C.E. Minarik. 1969. HeA.b.icxxie-6 U&ed -in Soutkmut Aaxa. Technical Report SAOQ-TR-69-11078. Directorate of Air Force Aerospace Fuels, Kelly AFB, Texas. 60 p. 15. Darrow, R.A., G.B. Truchelut, and C.M. Bartlett. 1966. OCONUS de^o-tcatuM tut p/tog/iam. Technical Report 79. Crops Department, Biological Sciences Laboratory, U.S. Army Biological Center, Fort Detrick, Frederick, Maryland. 126 p. 16. Demaree, K.D. and R.A. Creager. 1968. Defoliation tests in 1966 at Base Gagetown, New Brunswick, Canada. Technical Memorandum 141. Department of the Army, Fort Detrick, Frederick, Maryland. 17. Ellison, R. 1967. C-123s defoliate jungle stronghold of Viet Cong. Aviation Week and Space Technology 86(19) :82-86. 18. Executive Office of the President. 1971. Report on 2,4,5-T. A report of the Panel on Herbicides of the President's Science Advisory Committee. C.M. MacLeod, Chairman. Office of Science and Technology, Executive Office Building, Washington, D.C. 69 p. 19. Fair, S.D. 1963. No place to hide. How defoliants expose the Viet Cong, Asuny 14:54-55. 20. Fee, D.C., B.M. Hughes, M.L. Taylor, T.O. Tiernan, and C.E. Hill. 1975. Ano£t/-ttca£ Methodology faofi HeA.bx.cx.de 0/wuage. l/o£. II. V&teAmination o$ Onig-in o& USAF S-tocfc6. Technical Report ARL-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB, Ohio. 30 p. 21. Flamm, B.R., and J.H. Cravens. 1971. Effects of war damage on the forest resources of South Vietnam. J. Poie^u/ 69(11):784-789. 1-33 �22. Harrigan, E.T. 1970. Catibtuvtian Jut oh the. UC-123K/A/A45y-l Sptai/ Sy*te.m. Technical Report ADTC-TR-70-36. Armament Development and Test Center, Eglin AFB, Florida. 160 p. 23. Huddle, P.P. 1969. A Technology Assessment of the Vietnam Defoliant Matter - A Case History. Report to the Subcommittee on Science Research and Development of the Committee on Science and Astronautics. U.S. House of Representatives, Ninety-first Congress. Prepared by the Science Policy Research Division, Legislative Reference Service, Library of Congress, Washington, D.C. 73 p. 24. Hughes, B.M., D.C. Fee, M.L. Taylor, T.O. Tiernan, C.E. H i l l , and R.L.C. Vlu. 1975. Analytical Methodology iofi HeA.bi.<Ude. Oiange,. Vol. I. V<LteAmina£ian o^ Chemical Compo&ition. Technical Report ARL-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB, Ohio. 357 p. 25. Hurtt, W., and R.A. Darrow. 1968. &ioloai.o.al eXXecttueneiA oX Stult SifilLud and Osianae.. Technical Report AFATL-TR-68-122. Air Force Armament Laboratory, Eglin AFB, Florida. 31 p. 26. Irish, K.R., R.A. Darrow and C.E. Minarik. 1969. manual fax. vegetation control in Bouuth<La&t k&ia. Miscl . Public. 33. Department of the Army, Fort Detrick, Frederick, Maryland. 71 p. 27. Klein, R.E., and E.T. Harrigan. 1969. CompasuAon Tut 06 Ve.Kolia.nt!>, Technical Report ADTC-TR-69-30, Vol. I. Armament Development and Test Center, Eglin AFB, Florida. 356 p. 28. Lavergne, E.A. 1974. Study oh teaAlbilitu oh HeAbicMie. O^ianpe. c.hlo>u.noluAJA . Technology Series Report EPA-600/2-74-006. Office of Research and Development. Environmental Protection Agency, Washington, D.C. 67 p. 29. McConnell, A.F. 1970. Mission: RANCH HAND. - MJI UYiivvuitg Re.vi.ew 21(2):89-94. 30. Newton, M. 1975. Environmental impact of "Agent Orange" used in reforestation tests in Western Oregon. Weed Sex.. S<?c. Am., Abstr. 144, 52 p. 31. Peterson, 6.E. 1967. The discovery and development of 2,4-D. Ag*. Hlt>t. 41:243-253. 32. Tschirley, F.H. 1969. Defoliation in Vietnam - The ecological consequences of the defoliation program in Vietnam are assessed. Science. 163:779-786. 1-34 �33. Tschirley, F . H . 1968. Reiponie ofa &iopic.at and bmb&Lopic.aJL woody p£aitt6 to c.kmic.aJL -fiea£rnen£6 . Research Report CR-13-67. Agricultural Research Services, U . S . Department of Agriculture, Washington, D . C . 197 p. 34. Westing, A.H. 1976. Ec.otoQ-ic.aJL consequence* o& the. second Indochina. Wo/i. Stockholm International Peace Research Institute. Almgrist and Wiksel Internation, Stockholm, Sweden. 119 p. 35. Young, A.L. 1974. Ec.otoQic.aJL AtudieA on a keAbi.oJ.de. - equipment teAt oA.ua, (TA C-52A). Air Force Armament Laboratory, Eglin AFB, Florida. 141 p. 36. Young, A . L . , C . E . Thalken, E . L . Arnold, J . M . Cupello, L . G . Cockerham. 1976. fate. o& 2,3,7,S-te£uiQ.hlo'LOdA.be.nzo-p-dioiu.n (TCW) -en tke. nwiA.onm2.nt', Aummany and dzzontamination H.e.commz.ndatiom, . Technical Report USAFA-TR-76-18. Department of Chemistry and Biological Sciences, USAF Academy, Colorado. 41 p. 37. Young, A . L . , C.E. Thalken, and W . E . Ward. 1975. S^udcei o& the. <LC.oloQic.aJL impact o& ^epetctcue aerial apptication* o& heAbiciideA on the. <Lc.o*yAtw ofi TeAt AA.ea C-52A, Egtin AFB, fi.oni.da. Technical Report AFATL-TR-75-142. Air Force Armament Laboratory, E g l i n AFB, Florida. 127 p. 1-35 �CHAPTER II DISPOSAL OF HERBICIDE ORANGE I. INTRODUCTION During the summer of 1977 the United States Air Force (USAF) disposed of 2.22 million gallons (gal) of Herbicide Orange by high temperature incineration at sea. This operation, Project PACER HO, was accomplished under very stringent criteria of U.S. Environmental Protection Agency (EPA) ocean dumping permits. Numerous conditions of these EPA permits required the USAF to conduct extensive environmental and occupational monitoring of the land-transfer/loading operations and shipboard incineration operations. The results of EPA permit compliance monitoring for ship board operations are reported elsewhere (1). The purpose of this chapter is to summarize the historical background leading to Project PACER HO, to briefly describe the land-transfer operations and to present a summary of industrial hygiene and ambient air monitoring accomplished during the land-based operations. At the time of this writing not all occupational and environmental monitoring data are -available; thus, the final reports of land-based monitoring for project PACER HO have not yet been published. II. 'HISTORICAL BACKGROUND In April 1970, the Secretaries of Agriculture; Health, Education and Welfare, and the Interior jointly announced the suspension of certain uses of 2,4,5-T. These suspensions resulted from published studies indicating that 2,4,5-T was a teratogen. Subsequent studies revealed that the teratogenic effects had resulted from a toxic contaminant in the 2,4,5-T, identified as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Subsequently, the Department of Defense suspended the use of Herbicide Orange (3). At the time of the suspension, the Air Force had an inventory of 1.37 million gal of Herbicide Orange in South Vietnam and 0.85 million gal at the Naval Construction Battalion Center (NCBC) Gulfport Mississippi. In September 1971, the Department of Defense directed that the Herbici.de Orange in South Vietnam be returned to the United States and that the entire 2.22 million gal be disposed of in an environmentally safe and efficient manner. The 1.37 million gal were moved from South Vietnam to Johnston Island, Pacific Ocean, for storage (Project PACER IVY) in April 1972. The average concentration of TCDD in the Herbicide Orange was about 2 parts per million and the total amount of TCDD in the entire Herbicide Orange stock was approximately 44.1 pounds. Various techniques of destruction and recovery of the herbicide were investigated from 1971 to 1974 (AFLC Project on Disposition of Herbicide Orange). Destructive techniques included soil biodegradation, high temperature incineration, deep well injection, burial in underground nuclear test cavities, sludge burial and microbial reduction. Techniques to recover a useful product included use, return to manufacturers, fractionation and chlorinolysis. II-l �Of these techniques, only high temperature incineration was sufficiently developed to warrant further investigation. The other methods were rejected because of several considerations, including long lead times for development, inadequate assurance of success, and the lack of industrial interest. In December 1974, the USAF filed a final environmental impact statement (3) with the President's Council on Environmental Quality on the disposition of Herbicide Orange by destruction aboard a specially designed incineration vessel in a remote area of the Pacific Ocean, west of Johnston Island, The EPA held a public meeting in February 1975 to consider an ocean incineration permit application submitted by the USAF in accordance with the Marine Protection, Research and Sanctuaries Act of 1972 as amended, 33 U.S.C. 1401 et seq. During this meeting, testimony was presented which indicated that techniques for chemically reprocessing the herbicide to remove unacceptable quantities of TCDD might have been developed. The EPA indicated that the option for reprocessing should be further explored as a means of disposition prior to making a decision to destroy the herbicide via incineration (7). Subsequently, the USAF undertook an investigation into the feasibility Of reprocessing Herbicide Orange. Pilot plant studies were conducted from the fall of 1975 to July 1976 on selective activated carbon adsorption of TCDD from herbicide. This reprocessing method was shown to be technically and environmentally feasible; however, a feasible and environmentally acceptable method of safely disposing of the TCDD-laden activated carbon was not demonstrated. The USAF concluded in February 1977 that the option of reprocessing was not feasible, timely or cost effective since a technique for the ultimate disposal of the activated carbon was not currently available Or anticipated in the foreseeable future. Consequently, on 9 March 1977, the USAF requested reconvening the EPA public hearings. As a result of the public hearing held on 7 April 1977, the EPA issued a research permit to the USAF and Ocean Combustion Services, B.V. (OGS) (6). This permit authorized the transport of the Herbicide Orange from the Naval Construction Battalion Center, Gulfport MS to a designated site in the North Pacific Ocean for the purpose of atsea incineration in accordance with the provisions of the Marine Protection, Research and Sanctuaries Act of 1972, as amended. The vessel contracted for the at-sea incineration was the Dutch-owned ship, M/T Vulcanus, a ship registered in Singapore and previously used in the North Atlantic Ocean and the Gulf of Mexico to destroy chlorinated hydrocarbon wastes (12). A total of three herbicide loadings were required to incinerate the total stocks of Herbicide Orange: one loading from Gulfport MS and two loadings from Johnston Island. III. DESCRIPTION OF LAND-BASED OPERATIONS The operations at both storage sites were similar in many ways. At both sites, the 55-gal drums of Herbicide Orange were transported II-2 �short distances from their storage location to a centralized facility. The herbicide was drained from the drums and transferred to the M/T Vulcanus. Following emptying, the drums were rinsed with diesel fuel, and subsequently crushed. The rinsing from empty drum cleaning was combined with the herbicide and transferred to the ship for later incineration at sea. A. NCBC, Gulfport MS The centralized dedrumming facility at the NCBC was a temporary, enclosed facility measuring approximately 35 feet by 35 feet with an interior ceiling height of approximately 10 feet. A ventilation system capable of providing approximately 57 air changes per hour was equipped with in-line activated charcoal filters to reduce vapor emissions to the outside air. Within this enclosed facility were four identical processing lines. Each line consisted of a self-closing entry door to admit full drums, a roller conveyor along which drums were moved in an upright position, a position equipped with a heavy duty electrically operated deheading cutter, a suction wand to remove the greatest portion of the herbicide from a deheaded drum, a spray device beneath the conveyor over which the deheaded and emptied drum was inverted and rinsed with two gal of diesel fuel, a commercial, heavy duty drum crusher and a selfclosing exit door through which the crushed drums were passed. Once each drum was deheaded the con.tents were removed by the suction wand, leaving approximately three gal of liquid in the drum. The drum was then manually inverted and the remaining herbicide was collected in an open trough beneath the conveyor. Each drum was permitted to drain into the same trough for a minimum period of five minutes after which it was sprayed with two gal of diesel fuel, allowed to drain while still inverted for a minimum of two minutes, and then crushed end-to-end to approximately one-third its original volume. The rinsed and crushed drum was passed through the exit door and stacked with all other crushed drums. The liquid herbicide from the suction wands, and the herbicide and diesel fuel rinsing from the below-grade, open trough were pumped to 10,000 gal capacity rail tank cars. Air displaced from the tank cars during filling was filtered through an activated charcoal filter. The rail cars were moved along a rail spur approximately two mi.les to a dockside location where the herbicide was transferred to the incinerator ship, M/T Vulcanus. Displaced air from the ship's cargo' tanks was also filtered through activated charcoal. A total of 15,480 drums of Herbicide Orange was processed in this fashion at the NCBC between 24 May 1977 and 10 June 1977. Two 8hour shifts of approximately 55 men each accomplished the dedrumming/transfer operations. These men were all USAF officers/technicians from the five Air Logistics Center of the Air Force Logistics Command located at Kelly AFB, Texas; Hill AFB, Utah; Robins AFB, Georgia; Tinker AFB, Oklahoma and McClellan AFB, California. All workers were provided daily changes of II-3 �freshly laundered work clothes and men working within the dedrum facility were provided protective clothing including cartridge respirators, face shields, rubber aprons and rubber gloves. With only few exceptions the men rotated through all jobs involved in the dedrumming/transfer operations. All personnel were given pre-operational and post-operational physical examinations consisting of a complete medical history, complete neurological examination and the following laboratory procedures: 1. Complete hemoglobin, including hematocrit and platelet count 2. Prothombin time 3. Serum lipids 4. Serum glutamic oxaloacetic transaminase (SGOT) or 5. Serum glutamic pyruvate transaminase (SGPT) 6. Serum bilirubin 7. Blood glucose 8. Complete urinalysis 9. Chest x-ray B. Johnston Island The centralized dedrum facility at Johnston Island was a temporary, open facility measuring approximately 30 feet by 90 feet consisting of a concrete pad, roof and moveable canvas walls to exlude rain. This open facility was located adjacent to the Herbicide Orange storage site on the northwest end of Johnston Island. Nearly constant east winds ranging from 10 to 20 miles per hour provided natural ventilation and carried released vapors away from occupied areas. Two processing lines consisting of fabricated metal racks and open troughs were located in the west two-thirds of the facility. The east one-third contained pumps and drive-through for fuel trucks that were used to transport the dedrummed herbicide to the M/T Vulcanus. Full drums of herbicide were transported to the dedrum facility in sets of four using forklifts equipped with specially designed clamps. The drums were placed on the inclined metal racks in four groups of 12 drums each. Each set of 12 drums was handled independently by the dedrumming crew. Once a set of 12 drums was on the rack and the forklifts had withdrawn, a crew member would punch one hole near the top of each inclined drum as a vent hole to allow the crew's supervisory personnel to check the contents. Any drums containing other than Herbicide Orange were removed from the line and held for further testing. Three or more closely spaced holes were then punched in the bottom of each drum and the contents allowed to drain into II-4 �the open troughs. Once the herbicide had stopped flowing from the drums, they were allowed to drain for a five minute period after which the interior of each drum was rinsed twice with a total of two gal of diesel fuel. The diesel fuel rinsing drained into the open troughs, combining with the herbicide. After the 12 drums in each set had drained for a minimum of two minutes they were transported to a nearby drum crusher which consisted of a large weight suspended between two vertical I-beams. One drum at a time was crushed along its longitudinal axis and when approximately 30 drums had been crushed they were removed, banded, and stacked together near the crusher. The liquid herbicide and diesel fuel rinsing from the drums flowed into the two open troughs to a below-grade sump. The material was pumped from this sump into modified fuel tankers that transported 3,000 gal lots to dockside where the material was pumped aboard the M/T Vulcanus. A total of 24,795 drums of herbicide was processed in this fashion between 27 July 1977 and 23 August 1977. Two 10-hour shifts of approximately 50 men each were used. The workers were civilian employees of a contractor engaged to perform the dedrumming operations. USAF officers monitored all operations. As at NCBC, all workers were provided daily changes of freshly laundered work clothes, and men working within the dedrum facility were provided protective clothing consisting of cartridge respirators, face shields, rubber aprons, rubber gloves and boots. Unlike at NCBC, men on each crew remained in the same job through the dedrumming/transfer operations. A requirement of employment was preand post-operational physical examinations similar to those given the workers at the NCBC. IV. LAND-BASED OPERATIONS MONITORING PROGRAMS Detailed plans for environmental and occupational monitoring at both sites are contained in Annexes 4 and 5, kJtii Fo/ice. LciQiAticA Command VnoQfummhiQ Plan 75-19 faon the. V<it>pa&cut o& Oi&nge HeA.bi.cu.de (2). This section outlines only the industrial hygiene and ambient air monitoring programs conducted at each site. These aspects of the environmental and occupational monitoring at each site were very similar. Essentially, the same equipment, methods and procedures were used at both sites. The only significant difference between the two operations was that all sampling at the NCBC site was accomplished by members of the US Air Force Occupational and Environmental Health Laboratory (USAF OEHL), Brooks AFB, Texas, while all sampling at the Johnston Island site was conducted by Battelle Columbus Laboratories (BCL), Columbus, Ohio,under contract to the USAF. An environmental engineer from the USAF OEHL served as Project Officer and monitor of the BCL contract. In general, the industrial hygiene sampling program consisted of daily air samples within the dedrum facilities with rapid analysis (approximately 24-hour turn around time) for 2,4-D and 2,4,5-T. Samples collected for analysis of TCDD were analyzed after-the-fact. The ambient air sampling at various locations and distances from the dedrum II-5 �facilities included samples for 2,4-D, 2,4,5-T and TCDD analyses, as well as biomonitoring using rapidly growing tomato plants as indicator organisms. Pre-operational and post-operational background sampling was also accomplished. A. Monitoring Equipment and Procedures Two different methods were employed for industrial hygiene and ambient air sampling for 2,4-D, 2,4,5-T and TCDD. These procedures have been developed and field tested by the USAF OEBL. 1. 2,4-D and 2,4,5-T . , Sampling for 2,4-D and 2,4,5-T was accomplished utilizing Chromosorb*R' 102 as an adsorption medium, a granular polymer well suited for collection of chlorinated hydrocarbon vapors in air (10,11). The polymer was packed in micro-pipet tubes which were then wrapped in new aluminum foil and stored in rubber stoppered test tubes. The sampling apparatus consisted ofRa Mine Safety Appliance Model G Personnel Sampling Pump., The Chromosorb^ ' 102 tubes were connected to the pumps with Tygon^' or latex rubber tubing. A flow rate of 0.50 liters/minute (1/min) for periods ranging from five to ten hours was used, yielding an air sample volume of approximately 150 to 300 liters. This sampling time corresponded to the length of approximately one-half shift and was expected to yield sufficient adsorption efficiency to permit easy analysis. Flow rates were checked hourly with a calibrated rotameter to insure that 0,50 1/min flow rate was maintained. Where possible the pumps were maintained on constant "high" recharge by providing connections to available 110-volt power supply. When the Chromosorb(R' 102 tubes were removed from the field for lab analysis the individual tubes were wrapped in aluminum foil and returned to their respective rubber stoppered test tubes. 2. TCDD Air sampling for TCDD was accomplished using benzene as a collection medium. The sampling apparatus consisted of a train of four Greenberg-Smith impingers. The first two impingers were fritted and each contained approximately 350 ml of benzene. The third and fourth impingers were modified by removal of the fritts and contained activated carbon to adsorb vaporized benzene. The two benzene impingers were wrapped with aluminum foil providing a light barrier that would prevent any photodecomposition of the TCDD collected in the sample. Following the four impingers, an in-line paper filter was attached with TygonW tubing to prevent carbon particles from entering the Mi Hi pore pump. The pumps were operated directly from 110-volt AC power and the flow rate was one 1/min. The duration of sampling ranged from three to five hours, yielding an air sample volume from 180 to 300 liters. Flow rates were checked hourly using a calibrated rotameter and total volume of air sampled was calculated from these hourly flow rates. The maximum running time of five hours was dictated by ambient temperatures ranging from 71 to 92 degrees F and the saturation limitations of the carbon to adsorb the benzene vapors. Samples were removed from the sampling sites with II-6 �impinger trains intact in special wooden holders. The benzene was drained into new brown glass jars in a "clean" laboratory area. The impinger glassware was rinsed with benzene into the sample container to collect any materials adhering to the impinger walls. All impinger glassware was rinsed three times with acetone and once with benzene prior to reuse in the field. 3. Biomonitoring Immature, rapidly-growing, potted tomato plants, Lycop&ti>J.con ej>c.vJte.Yvt(m, ranging in size from 6 inches to 18 inches were used as indicator organisms for detecting the presence of Herbicide Orange vapors in air at various locations around the land-based dedrumming, transfer and loading operations. Young tomato plants are known to be very sensitive to phenoxy herbicide vapors (9). The symptoms typical of exposure to Herbicide Orange vapors, known as epinastic growth, is described as a curling and/or twisting of the apical portions of the plants. Depending on level of exposure these symptoms would appear within 24-hours after exposure. Normal procedures included observations, at least once daily, of the tomato plants to record the presence of the epinastic growth symptoms and to water the plants. Relative rating scales were used to describe the levels of damage noted. It was not possible to quantitate the levels of vapor exposure, but the extent to which low parts-pertrillion (ppt) herbicide vapor levels were carried by prevailing winds could be determined. B. Analytical Procedures and Methodologies The analytical procedures and methodologies used throughout Project PACER HO were developed, refined, tested and repeatedly used throughout the variety #f field exercises conducted by the USAF OEHL over the five year period from 1972 to 1977. 1. 2,4-D and 2.4,5-T Analysis of Chromosorbv(R^ 102 air samples was provided by ' two different laboratories. In the case of the NCBC, samples were analyzed by the U.S. Department of Agriculture Laboratory, Gulfport MS under an interservice agreement. All Johnston Island air samples for 2,4-D and 2,4,5-T were analyzed by the staff of the Battelle Columbus Laboratory team. The methods for analyses of herbicide will be reported elsewhere (4,5). 2. TCDD The Brehm Laboratory, Department of Chemistry, Wright-State University, Dayton Ohio, analyzed all benzene impinger samples for TCDD as well as many other types of samples and substrates in support of Project PACER HO. The Brehm Laboratory has been under contract with the USAF for II-7 �several years and has developed unique analytical capabilities in trace analysis for TCDD in a variety of substrates. The analytical methods employed for this project by the Brehm Laboratory have recently been published (8). V. LAND-BASED MONITORING RESULTS Detailed results of environmental and occupational monitoring at both sites will be reported elsewhere (4,5). This section outlines only the industrial hygiene and ambient air monitoring results for each site. Suffice to say that all other available data have indicated that there were no adverse environmental impacts on air, water or land resources at either site as a result of land-based dedrumming, transfer operations. A. NCBC, Gulfport MS The results of the industrial hygiene and ambient air monitoring programs at the NCBC are summarized below: 1. Industrial Hygiene The industrial hygiene air sampling results for 2,4-D, 2,4,5-T and TCDD are presented in Table 1. Five operational industrial hygiene samples were collected during each shift from the four corners within the enclosed dedrum facility. Four of these samples were for 2,4-D, 2,4,5-T using Chromosorb'R) 102, while the fifth sample was a benzene impinger in one corner of the facility collected for TCDD analysis. The ChromosprbW 102 and benzene impinger samplers were placed in low traffic areas near the four corners of the enclosed facility to prevent interference with work activity within the facility. As shown in Table 1, vapor concentrations of the n-butyl esters of 2,4-D and 2,4,5-T ranged from 7.76 - 141.15 ug/m3, respectively. The uniformity of concentrations of herbicide vapors within the dedrum facility is demonstrated by the lack of significant variability of 2,4-0/2,4,5-T data among the four sampling locations. All noted levels were well below the time weighted average Threshold Limit Value (TLV) of 10,000 yg/m3 for either 2,4-D or 2,4,5-T as adopted by the American Conference of Governmental Industrial Hygienists (ACGIH). No TCDD was detected in any of the 27 benzene impinger samples. The minimum detectable concentrations for TCDD ranged from 22.4 to 35.9 ng/m3. 2. Ambient Air Ambient air samples for 2,4-0/2,4,5-T and TCDD analysis were collected from three different locations. In addition, 29 groups of four tomato plants each were positioned around the dedrumming/transfer operations. The results of these monitoring efforts are presented below: a. 2,4-D, 2,4,5-T and TCDD. Sampling stations at two locations on the NCBC were established, one at the base fire station approximately 900 feet SW of the dedrum facility and one at the PACER HO Operation Center approximately 1,500 feet E of the dedrum facility. A II-8 �TABLE 1. Results of industrial hygiene air samples collected inside the dedrumming facility Project PACER HO NCBC, Gulfport, MS, 24 May - 10 June 1977. Sample Location Dedrum Facility Naval Construction Battalion Center (NCBC) • SE Corner NE Corner SW Corner NW Corner 28 28 14 14 NBEa2,4-D (yg/m3) Range Std Dev Mean 8.7-141.15 31.45 52.99 7.86-136.35 34.55 53.72 7.76-134.9 36.25 54.58 15.18-105.11 27.01 51.5 NBEa2,4,5-T (yg/m3) Range Std Dev Mean 5.52-65.11 14.98 26.40 5.70-76.36 18.57 29.93 3.01-79.62 21.02 32.39 7.59-51.31 12.79 25.93 0 0 0 Parameter No. of Samples TCDD No. of Samples Mean a 27 K NDb NBE is normal-butyl ester. ^ND is non-detectable at minimum detectable concentrations that ranged from <22.4 to <35.9 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 yg/m3. (See text) II-9 �third location on the wharf approximately 300 feet north of the ship loading point was also sampled. The results of analyses of these samples are presented in Table 2. As expected, the levels of 2,4-D, 2,4,5-T were significantly (45 to 150 times) lower than were found within the dedrum facility. Filtering of exhaust air from the facility, downwind diffusion/ dispersion of released vapors, and the lack of any significant spillage of herbicide outside the facility no doubt accounted for these significantly lower levels. No TCDD was detected at any of the three ambient air sampling stations with the minimum detectable concentrations ranging from approximately 22 to 55 ng/m3. b. Biomonitoring. Tomato plants were placed in groups of four in two concentric rings around the dedrum facility at 500 feet and 1000 feet distances. Moderate to severe plant damage was noted along the axis of prevailing winds in the inner ring (500 feet). Slight to moderate plant damage was noted in the corresponding outer (1000 feet) ring. In addition, several sets of four plants were set up along the NCBC perimeterfence. In two cases test plants along the base perimeter showed only minimal damage. One set of plants was also placed on the dock 300 feet inland from the loading operations. No damage was noted at this location. B. Johnston Island The results of the industrial hygiene and ambient air monitoring programs at Johnston Island are summarized below. There were two distinct loading operations during the Johnston Island phase of the project. The first dedrum/transfer (first loading) operation was conducted from 27 July 1977 to 5 August 1977, and the second loading from 17 August 1977 to 23 August 1977. 1. Industrial Hygiene The industrial hygiene sampling of the Johnston Island operations differed from the sampling at the NCBC. The facility was larger and open to natural ventilation and the dedrum operations were far different as described earlier. Because of these and other factors the industrial hygiene sampling program was modified to include true "breathing zone" samples for 2,4-D, 2,4,5-T from selected worker positions. In general, there were three worker positions evaluated using the Chromosorb\R' 102 tubes. These positions were selected after an analysis of all positions revealed that these worker locations represented the greatest possibility of receiving a significant exposure. The first was the position occupied by those workers who punched the vent holes in each drum. When the vent holes were punched internal pressure in many drums was released, and there was a possibility of elevated exposures to workers in these positions. The second worker position evaluated was that occupied by the workers who punched the several drain holes in each drum, and the third position was the operator of the sump pump. In the latter two cases, these workers were close to open troughs of flowing Herbicide Orange. In addition to these "breathing zone" samples, air samples within the dedrum facility were also collected for ^,4-D, 2,4,5-T and TCDD. Tables 3, 4 and 5 present the results of these sampling programs. 11-10 �TABLE 2. Results of ambient air samples collected at Gulfport MS, Project PACER HO, 24 May - 10 June 1977. Sample Location NCBC, Gulf port, MS Parameter No. of Samples Fire Station Ops Center Wharf 28 29 30 NBEa2,4-Diugym3l Range Std Dev Mean 0.09-5.76 1.20 1.17 0.13-3.88 1.00 1.09 0.07-2.41 0.53 0.52 NBE a 2,4,5-T (yg/m 3 ) Range Std Dev Mean 0.04-3.36 0.85 0.52 0.34-1.97 0.49 0.34 0.01-1.45 0.32 0.21 TCDD No. of Samples Mean 27h NDb 27. NDb 23 h NDb NBE is normal butyl ester. } ND is non-detectable at minimum detectable concentrations that ranged from <21.9 to 55.2 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 yg/m3. (See text) 11-11 �TABLE 3. Results of industrial hygiene air samples collected inside the dedrumming facility* Project PAGER HO Johnston Island* first loading 27 July - 5 August 1977. Sample UeatiSn Dedfum Facility deHhStbfi Islands First Loading Parameter SW Corner NW Corner E Wall 3 3 3 NBE a 2,4-D (ug/m3) Range Std Dev Mean 12.8-16,0 1,77 14.84 4.79-13*33 7,30 9.99 0.50-2.58 1.37 1.03 NBla2,.4,5-t (ug/m3) Ramge Std Dev Mean 192-8.84 1.05 8J2 2.26-8.28 3.24 4.58 - 0 0 No, of Samples TGDD No. of Samples Mean NDb a NBE is normal butyl ester, bND is non-detectable ait ifiihlfiiufri deteetSble concentrations that ranged from <8.06 to <13.89 rig/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 vig/m3. (Sfee text) 11-12 �TABLE 4. Results of industrial hygiene air samples collected inside the dedrumming facility Project PACER HO, Johnston Island, second loading, 17 - 23 August 1977. Sample Location Dedrum Facility Johnston Island, Second Loading Parameter SW Corner No. of Samples NBEa2,4-D (ug/m3) NW Corner 1 1 18.78 6.60 7.35 2.27 5 NDb 0 NBEa2,4,5-T (uq/m3) TCDD No. of Samples Mean NBE is normal butyl ester. 'ND is non-detectable at minimum detectable concentrations that ranged from <6.64 to <23.41 ng/m3. 11-13 �TABLE 5. Results of .industrial hygiene "breathing zone" samples collected inside the dedrumming facility Project PACER HO, Johnston Island. p'arsfrtete'r ' Sample' locations Dedrum Facility Johnston Island, (See Text) Veftt Drain Pump Punchers Ptine fret's O^efatof First Leading (27 July - 5 August 1977} Nd. of Samples NBEa2,4-D (ug/m3) Range Std Dev Mean NBEa2.,4,5-T (ug/m3) Range Std Dev Mean 8 10 5 2.14-30.8 8.35 17.92 7.64-19.18 5.73 19.18 . 6.11-26.78 8.18 14.36 0. 57-16. t 4.52 8.70 3.79-13.6 2.95 9.54 2.43-11.48 3.61 ' 6.32 Second Loading (17 - 23 Atigust 1977) No., of Samples 12 7 NBEa2,4-D (yg/m3) Range Std Dev Mean 8.38-40.28 10.47 23.20 .NBEa2,,4,5-T 4yg/m3J Range Std D£v Mean 6.49-22.22 6.06 13.-21 0 15.96-38.0 8.53 23.04 - 8.82-22.53 5.20 13.68 - NBE is ndrltial butyl ester. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10*000 yg/m3. (See text) 11-14 �The levels noted within the dedrum facility at Johnston Island were on the order of two to five times lower than those noted at the NCBC, Gulfport MS. These lower concentrations probably resulted from much greater dilution by natural ventilation of the open facility at iiohnston Island. Needless to say, the noted levels of 2,4-D and 2,4,5-T were well below the ACGIH TLV of 10,000 ug/m3. No TCDD was detected in any of the samples analyzed. 2. Ambient Air Ambient air samples for 2,4-0/2,4,5-T and TCDD analyses were collected from three different locations. In additon, 14 groups of four tomato plants were positioned at selected locations around the dedrum/transfer operations. The results of these monitoring efforts follow. a. 2,4-0/2,4,5-T and TCDD. One downwind and two upwind sampling stations were established. The downwind site was located approximately 300 feet west of the dedrum facility. The two upwind sites were the fire station approximately 4,000 feet SE and the weather station approximately 6,000 feet ESE of the dedrum facility. The results of downwind and upwind ambient air sampling sites are presented in Tables 6 and 7, respectively. As was expected, the levels of 2,4-0/2,4,5-T noted at the downwind site were somewhat lower than those levels noted within the dedrum facility. The relatively higher levels noted for the second loading as compared to the first loading are not explainable. These levels, however, are well below the TLV. No TCDD was detected in any of these samples. b. Biomonitoring. Tomato plants were placed at 14 biomonitoring stations on Johnston Island. Four of these sites were downwind of the dedrumming facility and the remaining ten locations were all upwind. Throughout the two periods of dedrumming operations all the downwind sites displayed slight to severe herbicide induced damage. There was only slight damage noted on two days at one of the upwind sites. The results of the tomato plant bioassay indicate that during the dedrumming operations concentrations of Herbicide Orange did not occur upwind of the dedrumming facility at sufficient concentrations to affect the tomato plants. VI. SUMMARY AND CONCLUSIONS As part of the environmental and occupational monitoring programs, the USAF accomplished industrial hygiene and ambient air sampling of all land-based dedrumming/transfer operations of Project PACER HO, the USAF project to dispose of 2.22 million gal of Herbicide Orange. The results of these sampling programs revealed that under the worst case noted, the levels of 2,4-D and 2,4,5-T vapors were well below the TLV for each of these materials. The noted levels were at least two and in most cases three orders of magnitude below the TLVs. TCDD was not detected in any air samples. 11-15 �TABLE 6. Results of downwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. Downwind Ambient Air Sampling Parameter No. of Samples NBEa2,4-D (yg/m3) Range Std Dev Mean NBEa2.3»5-T Cug/m3) Range Std Dev Mean First Loading (27 Jul-5 Aug 77) Second Loading (17-23 Aug 77) 14 1.92-25.5 5.99 6.21 5.79-32.67 7.73 12.51 0.82-17.1 4.33 3.27 1.89-14.0 3.46 5.12 TCDD No, of Samples Mean NDC NBE is normal butyl ester. ND is non-detectable at minimum detectable concentrations that ranged from <11.68 to <21.0 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 pg/m3. (See text) 11-16 �TABLE 7. Results of upwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. Wharf Station Weather Station First Loading'3 No. of Samples NBEa2,4-D (jjg/m3) Range Std Dev Mean NBEa2,4,5-T (jjg/m3) Range Std Dev Mean TCDD No. of Samples Mean a 11 Second 0 Loading 11 First 13 Loading 11 7 Trace-0.67 0.39 0.25 ND-2.54 0.77 0.23 0 0 Trace 0.34 0.10 0 0 0 0 1 NDd 1 NDe 0 0 Trace-1.09 0.42 0.29 Second Loadi ngc 0 0 NBE is normal butyl ester. b First Loading 27 July - 5 August 1977. C 5econd Loading 17-23 August 1977. d ND is non-detectable at the minimum detectable concentration of <8.52 ng/m3. e ND is non-detectable at the minimum detectable concentration of <20.34 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 yg/m3. (See text) 11-17 �Biomonitoring using tomato plants revealed that low-level vapors of Herbicide Orange were dispersed and diffused downwind of the land-based dedrumming/transfer operations at both sites. No adverse environmental impact resulted from these operations. Approximately 200 personnel carried out the dedrumming activities at the NCBC, Gulfport MS and at Johnston Island. Comparisons of available pre- and post-operational medical examinations of military personnel involved have revealed no apparent physical effects as a result of these activities. II-18 �CHAPTER II LITERATURE CITED 1. Ackerman, D.G., H.J. Fisher, R.J. Johnson, R.F. Maddalone, B.J. Mathews, E.L. Moon, K.H. Scheyer, C.C. Shin, and R.F. Tobias. 1978. At-4ea -tnc-tneAotton ofa HeAb4.cJ.de. Orange onboard the. M/T l/u£canoA. Environmental Protection Technology Series EPA-600/2-78-086. Office of Research and Development. U.S. Environmental Protection Agency, Research Triangle Park, North Carolina. 263 p. 2. Anonymous. 1977. A/iA Force Log<it>ticA Command programnujtg p£an 75-19 fan the. di&po&aJL o<j Orange Herfa.tc-t.de. San Antonio Air Logistics Center, San Antonio, Texas. Annex 4, pp 1-17, Annex 5, pp 1-23. 3. Anonymous. 1974. fl-iipo-A-ctcon o& Orange HeAb^cu.de by -imu.neAott.on. Final Environmental Statement. Department of the Air Force, Washington, D. C. 737 p. 4. Anonymous. 1977. Land-bo6ed env-tronmentat monitoring at Johnston Uland. Parts I and I I . Project PACER HO. USAF Contract No. F08635-76-D-0168 Battelle Columbus Laboratories, Columbus, Ohio. IH press. 5. Anonymous. 1978. Lewd-boused env-tAonmentod monitoring at tke. Navat Co»t6.t>t.uCxfcton Bouttation CwteJi, GutfipoKt, Mx6i4-c4AxCpp/c.. Technical Report of the U.S. Air Force Occupational and Environmental Health Laboratory, Brooks AFB, Texas. Jji press. 6. Anonymous. 1977. Mo/toie Pio.£ecxtt.on, Reieotch, and Act (Ocean Pampxjag) Re^ eaA.cn peAm-ct No. 770VH001R, United State* Protection Agency, Washington, D. C. , 15 p. 7. Anonymous. 1975. Ocean dumping, receipt of application and tentative determination. U.S. Environmental Protection Agency. Reg-cAteA 40(57): 13026- 13028. 8. Erk, S.D., M.L. Taylor and T.O. Tiernan. 1978. Env/^ionmenta£ moyUtotsing -en con/unctcon w^t^i -tnctneAa^tcon o& HeAb-tttde Orange at *ea. Activities of the Brehm Laboratory, Wright State University Dayton, Ohio. Presentation to the 1978 National Conference and Exhibition on Control of Hazardous Material Spills, Miami, Florida. 31 p. 9. M u l l i s o n , W . R . 1951. The tomato as a test plant for growth regulators. Bot. Gaz. 112:521-524. 10. Thomas, T.C. and J . N . Seiber, 1974. Chromosorb(R) 102, an efficient medium for trapping pesticides from air. Bu££. Env-cAon. Contain. and ToKicol. 12(1): 17-25. 11-19 �11. Thomas, T,C. and J.W. Jackson. 1978. A technique for sampling 2,4-D; 2,4, 5-T herbicides from air. J. A-UL VoUi-. Control tin press. 12. Wastler, T.A, , C,A. Offutt, C.K. Fltzsilflmons and P.E, Des 1975. V4J>p0Aa£ Ojf 0tycM.o£ki0JUn& um/tfci by 4.nc*Ln&t£Utin Environmental Protection Tedhnglociy Series EPA«430/9-7 Office ef Water and Hazardous Materials. Environmental Proteetion Agehcy* Washifigtdn, D,C, 223 p; II-20 �CHAPTER III ENVIRONMENTAL FATE OF 2,4-D, 2,4,5-T AND TCDD I. INTRODUCTION Chapter I was devoted to the topics of types and quantities of herbicides sprayed in South Vietnam and their handling and application. Emphasis was placed on those factors that may have influenced human exposure to the herbicides prior to actual spray applications. This chapter will focus primarily on the fate of the phenoxy herbicides sprayed in South Vietnam and on the contaminant TCDD. This is appropriate since 94 percent of all herbicides disseminated in South Vietnam were phenoxy herbicides (53 percent 2,4-D and 41 percent 2,4,5-T). The extreme toxicity of the contaminant, and its associated biological effects, require that all available data be reviewed in an attempt to determine the potential adverse human effects this compound may have had on the population at risk in South Vietnam. What happens to the individual compounds physically, chemically and biologically in the environment will significantly influence the route of exposure, the duration of exposure and the total dose (or level) of that exposure to the population at risk. Again, as noted in Chapter I, the population at risk will be confined to personnel of the U.S. military forces. The expression of units of weight, area, or volume has not been standardized between various publications cited in this Chapter. II. THE ENVIRONMENTAL FATE OF THE PHENOXY HERBICIDES A. Physical/Chemical Factors Influencing Disappearance of Herbicides 1. Fate in Air Harrigan (27) reported that in a test program evaluating the dissemination characteristics of the A/A 45 Y-l Spray System, the mean recovery of Herbicide Orange by ground sampling methods from six missions flown under operational parameters typically used in South Vietnam was 87 percent. The remaining 13 percent may have been undetected due to sampling technique or may havev failed to impact the sampling array due to drift or volatility. The mean particle size for the six missions flown was 367 micron (y). Harrigan (27) 1n the above test program with Herbicide Orange, found the following droplet size distribution in the mean percent mass recovered: Particles less than lOOy Particles 100 to 50Qy Particles greater than 500y III-I 1.9 percent 76,2 percent 21.9 percent �The recovery of 87 percent of the Herbicide Orange disseminated is in agreement with Plimmer (50) who reported that deposition of 80 percent of particles greater than 200u in size takes place in short downwind distances, whereas those of diameter less than 5y may drift for miles. The aerial application of Herbicide Orange also presented an opportunity for volatilization since spray drops evaporate during their fall. This was recognized by Grover et al (25), who examined the relative potential for drift of volatile and nonvolatile formulations of 2,4-D under conditions of typical agricultural application. The ground application system employed by Grover et al resulted in only 2.8 percent of the total spray having a particle size less than 200y. The mass of the formulation drifing- as droplets was similar (3 to 4 percent) for either the volatile (n-butyl ester) or nonvolatile (dimethylamine) formulation of 2,4-D. However, for the butyl ester, in addition to droplet drift, within the first 30 minutes after spraying 25 to 30 percent of the material was collected as vapor drift in air samplers up to 246 feet (ft) downwind from the point of application. The data by Grover et al (25) may suggest that although high percentages of Orange particles were intercepted by the vegetation, a significant amount of the material may have rapidly volatilized and moved in the air within the jungle canopy. This is in accord with what Brown (12) had first proposed in 1962 when he recommended the use of the esters of 2,4-D and 2,4,5-T for defoliation in South Vietnam. Better effect can be achieved on a susceptible tree if all its leaves receive a few drops of chemical as opposed to only one side or only the very top of the tree receiving all the chemical. In this connection, forms of the chemical known as volatile esters were requested subsequently in order to achieve more uniform coverage within a forest canopy. 2. Fate on Vegetation Approximately 85 percent of all the 2,4-D and 2,4,5-T sprayed in South Vietnam was'with the C-123/A/A 45 Y-l Spray System [estimate based on data by Irish et al (31), National Academy of Science (15), Craig (16), and Chapter I.] Klein and Harrigan (36) found that in five standard Orange missions the statistical mean value for maximum swath width having a deposition rate that would result in acceptable defoliation was 260t 20 ft. Thus, a typical 1,000 gallon (gal) sortie in South Vietnam would have effectively defoliated an area of approximately 346 acres (A). Data by Tschirley III-2 �(58) suggested that a multicanopy forest would intercept at least 94 percent of all the spray droplets. It is therefore reasonable to assume that if the entire 1,000 gal of Orange fell within the 346 A area, 940 gal of Orange would have been deposited on the canopy vegetation, and 60 gal deposited at ground-level on the soil or small herbaceous understory. The actual ground-level deposition may then • have been 0.17 gal/A or 1.4 pounds (Ib) of 2,4-0/2,4,5-T per acre (60 gal/346 A = 0.17 gal/A x 8.14 Ib active ingredient/gal = 1.4 Ib 2,4-0/2,4,5-T per A). In the United States, mixtures of these phenoxy herbicides are routinely applied at 2 Ib/A. If time after application was the same, then military personnel moving through defoliated forests in South Vietnam probably would have encountered the same amount of herbicide as would a rancher in the United States walking through defoliated brush-infested ranch land. Once the herbicide is intercepted by the vegetation, numerous physical and chemical barriers influence the amount of herbicide that is absorbed, transported and accumulated. In Volume 2 (Weed Control) of a special series on the principles of plant and animal pest control, the National Academy of Science (49) reviewed the physical and chemical barriers which intervene between application of a herbicide and its ultimate effect on the plant. They found that, in general, both the upper and lower leaf surfaces absorb herbicides. Usually, the lower epidermis is penetrated more readily, but not all areas of either surface are equally permeable. The penetration of the phenoxy herbicides into most foilage is by diffusion through the cuticle (cuticular entry). Warm temperatures that are not excessive and high humidity may actually promote the entry. Because the cuticle and the cell walls upon which the cuticle is deposited contain chemically nonpolar materials that are slightly electronegative, nonpolar herbicides (e.g., Orange and Purple) tend to be absorbed into leaves faster than polar herbicides. Cuticular penetration by the esters of 2,4-D or 2,4,5-T may occur within 30 minutes of their application. 3. Fate in Soils Hamaker (26) has reviewed the physical and chemical factors that influence fate of herbicides in soil. These include soil adsorption, hydrodynamic dispersion and diffusion, adsorption dynamics and evapotranspiration. The phenoxy herbicides, for example, have low adsorption coefficients and thus tend to leach in a soil profile. The actual amount of leaching, however, will vary from soil to soil, mainly in response to the organic carbon content. Moreover, only the herbicide free in the soil water will be carried down by descending water. Crosby (18), in reviewing nonbiological degradation of the phenoxy herbicides in soil, reported that the isopropyl, butyl and isooctyl esters of 2,4-D had a half-life of about 100 hours (h) III-3 �in neutral soil water (although hydrolysis was almost instantaneous in the presence of a base or a suspension of any of several soils at pH 7.0-7.5). Moreover, many of the phenoxy herbicides, e.g., 2,4-D, will readily undergo oxidation, reduction and substitution (notably hydrolysis) in aqueous solutions when activated by sunlight in air. The end product of the photodegradation of 2,4-D is humic acid (17). Although 2,4,5-T absorbs some ultraviolet light in sunlight, the amount is small and this herbicide is relatively unreactive (only 7 percent was hydrolyzed in 48 h). However, the presence of ferric salts or zinc oxides in the soil water will result in an increase in photolysis rate (17). Another nonbiplogical factor that determines the soil persistence of the phenoxy herbicides is their tendency to volatilize from the soil complex. Plimmer (50) noted that some volatilization will occur whether or not water is evaporating from the soil. However, a reduction in soil moisture content will decrease the pH of soil. This will favor the undissociated form of 2,4-D and 2,4,5-T and their potential for vapor loss may be increased. B. Biological Degradation of the Phenoxy Herbicides 1. Fate in Plants Loos (40) has recently reviewed the degradation of phenoxy herbicides in plants. In general, because of the widely different degradative pathways, the phenoxy herbicides do not persist in plants. However, Muzik (44) has reported that in some plants, for example, tomato, unmetabolized 2,4-D may be bound to cellular membranes and persist for two or three months. 2. Fate in Soils There is considerable evidence available to show that the phenoxy herbicides are rapidly decomposed in soils (5). Goring et al (23) in reviewing principles of pesticide degradation in soil noted that 2,4-D may undergo at least 6 different types of oxidation reactions, 1 reductive reaction, 1 hydrolytic reaction and 4 conjugative reactions. Because of this ability to readily undergo transformation, 2,4-D has been classed as a non-persistent pesticide since the estimated time required for 50 percent to disappear from soil was <0.5 months. However, 2,4,5-T has been classed as a slightly persistent pesticide since the time required for 50 percent disappearance was 0.5 to 1.5 months. III-4 �If 2,4-D were applied to a moist loam soil under summertime temperature at a rate of 0.5 to 3 pounds/acre (Ib/A), it would disappear in 7 to 30 days (37). If applied at rates of 4 to 55 Ib/A, it would probably disappear in one to three months (22). If 2,4-D were applied to the soil at a concentration of 500 ppm and disappeared at a rate proportional to the breakdown of 55 Ib/A, the calculated time would be 5.6 years. However, there is evidence that a more realistic time for inactivation of 500 ppm would be less (4). Persistence of 2,4,5-T in soils is usually two to three times longer than 2,4-D (22), and very few organisms have been identified as having the ability to breakdown the 2,4,5-T molecule (2). Newton (46) has calculated from studies on the kinetics of degradation by microorganisms that 2,4,5-T has a half-life of seven weeks in the forest floor. Investigations by Winston and Ritty (59) and Reigner et al (51) indicated that both 2,4-D and 2,4,5-T are decomposed to form carbon dioxide, inorganic chlorides and water; objectionable chlorophenols are not end-products of this decomposition. Further supporting evidence has been provided by Reinhart (52). The upper half of a 60 acre timber watershed in northern West Virginia was logged and treated with 2,4,5-T ester to kill all vegetation. The volume of herbicide that was applied was 1,325 gal on 30 acres (418 liters/ha). Almost 790 gal of this were potential contaminating materials: about 740 gal of diesel oil and 50 gal of a commercial formulation of 2,4,5-T (313 pounds acid equivalent). Reinhart found n£ odor contaminants (phenols or catechols) in the numerous water samples taken from the stream draining the treated watershed. In relation to the effects of herbicides on the soils of South Vietnam, the National Academy of Science published a report by Blackman et al (11) on persistence and disappearance of herbicides in tropical soils. The 1974 report stated a number of general conclusions, namely: 1. The behavior of herbicides in the soils of South Vietnam was similar to that reported for soils elsewhere. 2. Only where 2,4-D and 2,4,5-T were applied in very massive doses; e.g., at the Pran Buri Calibration Grid in Thailand at rates in the magnitude of 1,000 Ib/A, were there still residues (10 years following application) in concentrations above the threshold likely to induce phytotoxic symptoms in some plant species. 3. When applied to mangrove soils at total doses approaching 10 Ib/A of 2,4-D and of 2,4,5-T, the level of herbicide residue at the end of 30 weeks had no effect on the establishment of two major mangrove species. 4. In geographical areas subjected to one or two military herbicide missions 1.5 years before sampling, no soil phytotoxic residues could be detected. III-5 �5. Soils that received a directed application of Herbicide Orange at the rate of 27 Ib/A safely supported the growth of crops sensitive to 2,4-D or 2,4,5-T four to six months following application. 6. Claims that the herbicides rendered the soil sterile were without any foundation. Byast and Hance (14) have studied the degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Although care must be exercised in extrapolating laboratory results to field situations, their results suggested that the four Vietnamese soils studied were inherently capable of degrading 2,4,5-T at levels roughly twice the rate of military application in Vietnam. In support of feasibility tests for the soil disposal of surplus Herbicide Orange, the Air Force established a field study in 1972 on the Air Force Logistics Command Test Range, Hill Air Force Base, Utah. The study consisted of replicated plots subsurface injected with concentrations of either 1,000, 2,000, or 4,000 Ib herbicide/A. Soil samples were taken by Stark et al (56) three times throughout 1973, and microbial species present (bacteria, actinomycetes and fungi) were determined. Bacterial counts were higher for soils with greater concentrations, of the herbicide and with greater moisture content; i.e., those samples collected in midwinter from the 4,000 Ib/A plots. Herbicide Orange, in any concentration, had no significant effect on mycoflora. Arnold et al (4) monitored the herbicide levels in these plots. They sampled the plots on eight occasions from 1973 through 1975 and determined the concentrations of the n-butyl esters and free acids of both 2,4-D and 2,4,5-T. They suggested that at such massive application rates (soil concentrations greater than 10,000 ppm) and in an alkaline desert environment, the half-life of 2,4-0 and 2,4,5-T appeared to be in the range of 150 to 210 days. The cooperative studies by Stark et al (56) and Arnold et al (4) have shown that the application of 2,4-D and 2,4,5-T at massive rates not only did not sterilize the soil, but indeed stimulated the growth of certain microflora, and this stimulation may have contributed to the degradation of the herbicide. C. Accumulation and Metabolism of Phenoxy Herbicides in Animals A detailed review of the toxicity, distribution and fate of 2,4-D and 2,4,5-T in animals is provided in Chapter IV. Some general observations on the metabolism of the phenoxy herbicides have recently been published by Leng (39). She reported that residues of the phenoxy herbicides in treated food or feed crops were readily absorbed in the gut of animals and were excreted rapidly in the urine, largely as unchanged phenoxy acid. Some conjugation occurred, particularly at higher dosage levels, but the basic structure of the herbicide was III-6 �not readily altered in animals. The ether linkage can be cleaved by bacterial action in the rumen but the rate of cleavage depended on the chemical structure of the phenoxy compound. The rate of clearance of residues from the body was dependent upon dosage level, particularly if the renal threshold was exceeded. Leng (39) concluded that residue levels were considerably lower in muscle, milk, and cream than in liver and kidney, but that all residue levels rapidly declined after withdrawal of animals from treated feed. Residues of phenol metabolites were present in milk, liver and kidney of animals fed high doses of 2,4-0 and 2,4,5-T. III. THE ENVIRONMENTAL FATE OF TCDD A. Analytical Limitations Statements on the fate of TCDD in the environment are predicated upon the detection in environmental substrates. Prior to 1973, the detection limit for TCDD, was 0.1 ppb for soils and 0.05 ppm for biological tissue (60). As noted by Kearney et al (35) and Dost et al (23), a 1 Ib/A application of 2,4,5-T containing 0.1 ppm TCDD applied directly to the soil could result in a maximum of 0.1 parts per trillion (ppt) in the top 15 cm of soil. Likewise, Baughman and Meselson (9) have calculated that environmental monitoring of food chains for buildup of TCDD would require a level of detection of 1 ppt. For a 1 gram sample of biological tissue, this would require a limit of detection of 1 picogram (pg) (10-^2 gram). Highly sophisticated instrumentation is required to obtain these low detection limits. However, another one of the limiting factors, even with appropriate instrumentation, has bee,n the need for cleanup techniques applicable to a wide variety of environmental samples. Recently (1977), Hummel (30) has reported on a technique suitable for permitting the detection of ppt residue levels of TCDD. Using this technique, Hummel has analyzed a wide array of environmental substrates. These have included analyses of whole fish, fish muscle, rat and mouse liver, mouse pelts, bird liver and stomach, insects, diving beetles, seeds, soil, water, and bovine and human milk. Largely due to the analytical limitations" noted above, the quest for environmental data on TCDD began with laboratory experiments. The use of radiolabeled preparations were invaluable in these studies. There has been considerable interest placed on the analysis of TCDD in field samples; e.g., fish and human milk from South Vietnam (7), bovine fat, liver and milk from the Western United States (3,41), and rice from Arkansas (30, 55). B. Laboratory Studies of TCDD Two model ecosystem studies (33, 42) have been conducted in an attempt to simulate the mode of entry of TCDD into water with the III-7 �subsequent exposure of several organisms representing parts of natural food chains. These systems were not designed to determine the effects of TCDD on the organisms but rather, how does TCDD behave when subjected to likely environmental conditions. Matsumura and Benezet (42) introduced 14C-TCDD in the form of residues on sand into an aquatic ecosystem containing brine shrimp, mosquito larvae and fish. The results indicated that the rate of pick-up was extremely low in brine shrimp and fish under the experimental conditions, Mosquito larvae, which were bottom feeders, showed a faster rate of TCDD pick-up. They concluded that because of TCDD's low solubility in water and its low partition coefficient in liplds, it was not likely to accumulate in as many biological systems as DDT. Isensee and Jones (33) exposed several organisms to ' C-TCDD for up to 31 days to determine the distribution and bioaccumulation potential in the aquatic environment. TCDD accumulation by all organisms was directly related to water concentration (0.05-1330 ppt) and ranged from 2.0 x 104 to 2.6 x 104 times the water concentration for snail, mosquito fish and daphnids and averaged 4.9 x 103 for duckweed, algae and catfish. No metabolities of TCDD were found in submerged soil, water, snails, mosquito fish or catfish. Isensee and Jones further noted that most (85-99 percent) of the 14C-TCDD originally added to the ecosystem remained in the soil at the end of the experiment. Total recovery for the ecosystem averaged 92.2 percent, indicating that TCDD was very stable during this study. From the model ecosystem data, it has been concluded that TCDD is taken up by an organism and retained (bioaccumulation). The accumulation results in TCDD concentrations in the environment (bioconcentration). The food chain studies do not suggest, however, that TCDD is biomagnified; i.e., organisms at successive trophic levels do not exhibit an ascending order of TCDD concentrations in their tissues. Neither of the model ecosystem studies reported toxic effects from the bioconcentration of the TCDD. Both studies, however, were of short duration and the water concentration was generally low, although in one experiment by Isensee and Jones (33) the water concentration exceeded 1 ppb and'mosquito fish and catfish accumulated concentrations greater than 1.4 ppm TCDD for 3 and 6 days, respectively. Miller et al (43) conducted chronic toxicity tests to assess the hazard to aquatic organisms exposed to TCDD in water or food. They evaluated three species of fish: guppies, coho or silver salmon, and the rainbow trout; and three aquatic invertebrates: a snail, a worm and mosquito larvae. Their conclusions were that TCDD in water or food was toxic to fish. The effects of exposure for 2496 h of young salmon to TCDD in water at levels greater than 23 ng/g (23 ppb) were irreversible, and death resulted in 10-18 days. Duration of exposure was less important than level of exposure except as threshold response level was approached. The critical exposure III-8 �period was somewhat less than 24 h in static water toxicity tests in which TCDD concentrations changed markedly with time. Small fish were more sensitive than large fish on an equivalent exposure level basis. TCDD in food at 2.3 ppm markedly reduced growth of young rainbow trout (10/aquaria) exposed to 6.3 yg TCDD per tank per week for 4 weeks. TCDD at 0.2 ppb had no effect on pupation of the mosquito larvae, but reduced the reproductive successes of the pulmonate snail and the oligochaete worm. Morris and Miller (48) have conducted additional bioassay tests with guppies. Exposure of guppies to concentrations of TCDD equal to or greater than 0.1 ppb for 120 h caused complete mortality in approximately 30 days. Duration of survival was significantly and positively correlated with body lengths. Beatty et al (10) administered larval and adult forms of the American bullfrog doses of TCDD varying from 25 to 1,000 yg/kg. Doses of TCDD as high as 1 mg/kg failed to have any significant effect upon survival or completion of metamorphosis in tadpole. Doses of TCDD up to 500 yg/kg had no effect on survival of adult frogs. Histopathological examination of various tissues from the metamorphosed tadpoles and adult frogs revealed no abnormalities. In one of the first laboratory studies of TCDD in soil, Helling (28) found that TCDD was immobile when evaluated by soil thin-layer chromatography. In laboratory leaching studies, Matsumura and Benezet (42) found that virtually no TCDD leached from soil columns of sand or sandy loam. Kearney et al (34) have determined the persistence of TCDD after 20, 40, 80, 160 and 350 days in Hagerstown and Lakeland soils receiving 1, 10 and 100 ppm TCDD. After 1 year, 56 and 63 percent of the originally applied TCDD was recovered in the Hagerstown and Lakeland soils, respectively. Thus, the half-life was estimated to be about 1 year. Furthermore, TCDD could not be detected after 70 days in soils receiving 10, 100 or-1,000 ppm 2,4,5-trichlorophenol, suggesting that TCDD was not biosynthesized by microbial condensation reactions. The long half-life of TCDD suggested to Kearney et al (34) that it was not readily metabolized by soil microorganisms. This observation was in keeping with what Matsumura and Benezet (42) found. They evaluated 100 microbial strains, which had previously shown the ability to degrade persistent pesticides, for their ability to degrade TCDD. Only 5 of 100 organisms showed some ability to degrade this compound, suggesting that microbes capable of degrading TCDD were rather rare in nature. Helling et al (29), in reviewing the previous studies, concluded that persistence of TCDD was not surprising since it is an insoluble, nonpolar, chlorinated molecule, devoid of biologically labile functional groups. III-9 �Isensee and Jones (32), in laboratory studies determined the uptake of TCDD from soil by two crop species. Lakeland Sandy loam, a soil with low adsorptive capacity, was treated with ^C-T at the rate of 0.10 and 0.06 ppm, respectively. Oats or soybeans were grown in this soil and their tops were harvested at intervals to maturity. All tissue '^C-activity was expressed on the basis of the original compound. Oats and soybeans accumulated in their tissue less than 0.15 percent of the TCDD present in the soil. Isensee and Jones (32) also evaluated the fate ot TCDD when applied to foliage. Uniform quantities of '^C-TCDD were applied to the center leaflet of the first trifoliate leaf of 3-week-old soybean plants. The first leaf blade of 12-day-old oat plants was treated with '4C-TCDD only. Results indicated that TCDD was not translocated beyond the treated leaflet. An average of 94 percent of the TCDD remained on soybean leaves for 21 days, but the amount continuously decreased on oat leaves. Although Isensee and Jones suggested that volatilization was a key factor in the disappearance of TCDD from foliage, Crosby and Wong (19) have suggested that photodegradation of the dioxins was a plausible explanation. In an uptake study similar to that of Isensee and Jones (32), but using sorghum, Cupello and Young (20) found that the rate of uptake of TCDD from a Ulysses sandly loam soil was approximately one millionth of one percent of the amount of TCDD in the soil. Nash and Beall (45) have recently (1978) completed a study on the fate of TCDD in the plants, soil, water and air of a microagroecosystem. Tritium-labeled TCDD at concentrations of 44 or 7,500 ppb was applied to a bluegrass turf microagroecosystem using an emulsifiable concentrate form of the isooctyl ester of 2-(2,4,5trichlorophenoxy) propionic acid (Silvex) as a carrier. They found that: 1. TCDD concentrations in water leached through soil were below the analytical detection limit (10~'6 g/g water). 2. TCDD concentrations on grass were initially 20 ppt (10-12 g/g grass), but after four weeks were at or below 1 ppt. The half-life was approximately six days. 3. TCDD concentrations in or on soil were less than 0.2 ppt and most (80 percent) was near the soil surface (0-2 cm). 4. TCDD concentrations in air were (immediately after application) less than 100 fg/m3 (femtogram - 10~'5g/m3) and after four weeks decreased to <3 fg/m^. 5. TCDD, or its degradation products, concentrations in earthworms were less than 0.3 ppt. 111-10 �6. The major repositories for TCDD were the soil and thatch. Nash and Beall (45) concluded that volatilization (approximately 10 percent) of TCDD was a major pathway of dissipation from the microagroecosystem chamber. However, once TCDD was volatilized it dechlorinated in the direct sun and apparently even in shade outdoors or when the sun was filtered with glass in the chambers. Thus, TCDD is sensitive to photodechlorination in the vapor phase even without the presence of ultraviolet light. C. Field Studies of TCDD 1. Residue in Aquatic Ecosystems Several monitoring studies for TCDD in aquatic organisms have been conducted. Baughman and Meselson (8) reported finding TCDD concentrations of 70 to 810 parts per trillion (ppt) in fish from rivers of interior Vietnam and concentrations of 18-79 ppt in fish, and shellfish along the seacoast of South Vietnam. Their samples were collected in 1970 and analyzed 2-1/2 years later by their method and instrumentation. Zitko (65) and Zitko et al (66) did not detect dioxins in a wide assortment of aquatic organisms collected from the St. John River, New Brunswick or the Bay of Fundy, Canada. Their detection limits, however, were between 0.1 and 1.0 ug/g of tissue. Shadoff et al (55) have examined fish (bass and catfish) from a reservoir in a rice-growing region of Arkansas, where 2,4,5-T had been used annually for more than 20 years. Likewise, fish (walleyes and catfish) were obtained from a reservoir in West Texas where 2,4,5-T had been used for brush control over the past 20 years. No TCDD was detected in any of the samples with a minimum range of 10 ppt. Young et al (62) reported on species diversities and food chain studies conducted in two aquatic ecosystems draining a unique one-square mile military test area (Test Area C-52A, Eglin AFB, Florida) that received 161,000 pounds 2,4,5-T and 170,000 pounds 2,4-D herbicide during the period 1962-1970. Significant levels (10710 parts, per trillion) of TCDD were found in 1973 within the top six inches of the test area soil. Erosion of soil occurred into a pond on the test area and into a stream immediately adjacent to the area. TCDD levels of 10-35 ppt were found in 1974 in silt of the aquatic systems, but only at the point where eroded soil entered the water. Species diversity studies of the stream were conducted in 1969, 1970, 1973 and 1974. Insect larvae, snails, diving beetles, crayfish, tadpoles and major fish species (by body parts) from both aquatic systems were analyzed for TCDD. Species diversity studies indicated no significant change in the composition of ichthyofauna between these dates or a control stream. Concentrations of TCDD (12 ppt) were found in only two species of fish from the stream, sail fin III-ll �shiner and mosquito fish. The sample of mosquito fish consisted of bodies with heads and tails removed. Two samples of sailfin shiner were analyzed: one containing viscera only and the other bodies less heads, viscera and caudal fins. Only the viscera contained TCDD. Samples of skin, muscle, gonads, and gut were obtained from spotted sunfish!, from the test grid pond. Levels TCDD in those body parts were 4, 4, 18 and 85 ppt, respectively. Grass pathological observa tions Qf the sunfish revealed no significant lesions or abnormal itit r.. 2. Residues 1n Sails * The National Academy of Science (15) reported finding TCDD concentrations of <1.2 to 23.3 parts per billion (ppb) in soil of the Pran Buri Calibration Grid (Thailand), an area used in calibrating RANCH HAND aerial equipment. Wool son et al (60) found no residues in 1971 in Lakeland sand which had received 947 Ib/A of 2,4,5-T during 1962-1964. These unusually high doses resulted from testing of aerial application equipment at Eglin AFB, Florida. Although analysis of the applied material was not conducted, 2,4,5-T made prior to 1968 probably contained enough TCDD to be detected throughout the 1-yard of soil profile sampled. Wool son et al suggested that the lack of detectable residue was due probably to its decomposition on or in the soil and/or to its transportation by wind erosion. Young et al (64) conducted four years of field studies on the persistence of Herbicide Orange and TCDD when applied at massive rates to soils. Herbicide Orange "biodegradation" plots were established in Utah (Air Force Logistics Command Test Range) and in Florida (Eglin AFB Reservation) using simulated subsurface injection techniques to place the herbicide 4 to 5 inches beneath the soil surface in bands 2.5 or 6 inches wide for Utah or Florida, respectively. An application rate of 4,000 Ib herbicide/A resulted in initial TCDD residues of approximately 148 ppb and 0.375 ppb in the Utah and Florida plots, respectively. Figure 1 is a semi-logarithmic plot of the soil concentration of Herbicide Orange while Figure 2 is a semilogarithmic plot of the soil concentration of TCDD in the same field tests. Using Figures 1 and 2, the half-life data were calculated as 300 and 220 days for Orange, and 320 and 230 days for TCDD for Utah and Florida, respectively. It should be emphasized again that these data were from field plots where the herbicide and TCDD were injected as highly concentrated herbicide in narrow bands beneath the soil surface. Data on soil penetration of TCDD within the soil profile of Utah biodegradation plots receiving either 1,000, 2,000 or 4,000 Ib/A are shown in Table 1 (Unpublished data: Young, A.L., and E.L. Arnold. 1978. Report on TCDD soil penetration studies, USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas). Note that in Table 1, 98 percent of all TCDD was detected in the 0-6 inch increment of soil, the increment into which the herbicide was applied. Even in the plots receiving 4,000 Ib/A, the TCDD detected in the 6-12 111-12 �Hill AFB, Utah .Eg!in AFB, Florida 10,000 c o t. O) Q . 03 Q . 1 ,000 u -S O) a: -M (O 4-> C cu u c o o (X) 100 r ?1 200 400 600 800 1,000 Time (Days After Incorporation) FIGURE 1. Semi-logarithmic plot of soil concentrations (parts per million) of herbicide in Herbicide Orange biodegradation studies at Eg!in AFB, Florida, and Hill AFB, Utah. Source: Reference (64 ) 111-13 1,200 �20,000 Hill AFB, Utah Eglin AFB, Florida 10,000 *<• 1,000.. Q . in •»-> i. (T3 Q. Q O O c O £ •4-> C O) O c O O 100-. O 00 10 200 400 600 800 1,000 Time (Days After Incorporation) FIGURE 2. Semi-logarithmic plot of soil concentrations (parts per trillion) of TCDD in Herbicide Orange biodegradation studies at Eglin AFB, Florida, and Hill AFB, Utah. Source: Reference (.64). 111-14 1,200 �TABLE 1, Concentrations of TCDD, parts per trillion, in the Herbicide Orange biodegradation plots, AFiC Test Range, Utah, four years after applications.3 Original Rate of Herbicide Orange Applied Depth (inch) 1,000 Ib/A 2,000 Ib/A 4,000 Ib/A 0 -6 650 1600 6600 6 - 12 11 90 200 12 - 18 NAb NAb 14 a Samples collected 6 November 1976. Plots established 5 October 1972. ^Samples not analyzed. Source: Unpublished data (Young, A. L., and E. L. Arnold. 1978. Report on TCDD soil penetration studies. USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas). 111-15 �inch increment may have been there because of the mass movement of the herbicide at the time of application rather than through the movement of percolating water. These penetration data are similar to those reported by Young et al (64) for the Florida biodegradation plots (noted earlier) although the Florida site received an annual rainfall of 60 inches (vs 10 Inches annual rainfall in Utah). Young et al (63) reported TCDD data from soil analyse-* of the Eglin AFB, Florida, Spray Equipment Calibration Grid (Grid 1 5 Test Ana C-52A). As noted in Chapter I, this grid received 1,894 pounds of Purple per acre during the 1962 through 1964 period. TCDr concentrations in a soil profile from samples collected ten years after the last application of Purple are shown in Table 2. 3. .Residues in Animals The current search for TCDD in beef fat and liver in the United States may provide an indication of the possible fate of TCDD in South Vietnam. In September 1974, the Environmental Protection Agency established a Dioxrn Implementation Plan which consisted of a short term monitoring program (Part I) and a broad research plan which would take 4 to 5 years to complete (Part II) (3). Part I of the program was initiated in February 1975. The guiding principles for the sample program were: (a) the samples should be representative of beef actually prepared for human consumption and (b) the samples should be from cattle grazed on lands treated with 2,4,5-T. Control samples were to be taken from cattle grazed on non-treated areas within the same state. Between February and March 1975, 85 beef fat (peritoneal and kidney) and 43 liver samples were collected (3). Approximately 25 percent of these samples were collected from non-treated areas. One laboratory prepared all sample extracts, and identical aliquots were sent to all participating analytical laboratories. In June 1976, analytical results for these samples were announced by the EPA Dioxin Project Manager (53). TCDD was present (range of 20-60 ppt) in a small percentage (3.5 percent) of the beef fat samples taken from cattle with a known exposure of 2,4,5-T. All of the beef liver samples analyzed were negative, at a detection limit of 10 ppt TCDD. Phase II of the Dioxin Implementation Plan began in 1978, with the intended goal of providing EPA with information on the range and possible bioaccumulation of TCDD in the environment (3). Analyses of human fat and liver tissue and human milk, and additional samples of beef fat and liver were to recieve the highest priority. Mahle et al (41) have recently completed a surveillance of bovine milk samples from the states of Oklahoma, Arkansas and Missouri. Twenty-five samples were collected from cows grazing on pastures on rangeland treated with normal applications of 2,4,5-T. These samples and control samples were analyzed for TCDD by gas chromatography-mass spectroscopy (GC/MS). They found no TCDD in bovine milk 111-16 �TABLE 2. Concentration of TCDD in soil profile of Grid 1, Test Area C-52A, Eg!in AFB, Florida.3 Depth of (inch) Parts per Trillion (ppt) TCDD 1 1 -2 160 2 -4 700 4 -6 44 6-36 a 150 NDb Grid 1 received 1,894 pounds of Herbicide Purple per acre during 19621964. The soil samples were collected and analyzed in 1974. detected, minimum detection limit - 10 ppt. Source: Young et al . (63). 111-17 �from control or treated areas with a detection limit of 1 ppt. In reforestation tests in Western Oregon, Newton and Snyder (47) applied Herbicide Orange at the rate of 2-4 Ib/A. Analysis of resident mountain beaver captured inside the treated area two months after treatment showed no TCDD in livers, with a minimum detection limit of 3 ppt, and the animals appeared to be in good health in all respects. Wool son et al (60) examined extracts of 19 bald eagles from locations throughout the United,States for TCDD and higher dioxin residues. No dioxins were detected at a minimum detection limit of 50 ppb. Baughman (7) analyzed samples of human milk for TCDD from areas of South Vietnam heavily treated with 2,4,5-T during the military herbicide program. Levels of 40-50 ppt in human milk were found in samples collected in 1970 and analyzed four years later. Shadoff et al (55) analyzed samples of human milk obtained from mothers residing near the North Concho River Basin of West Texas, an area where large acreages of the watershed had been sprayed repetitively with 2,4,5-T herbicides for brush control over the past 20 years. No TCDD was found in any of the milk samples at a minimum detection limit below 10 ppt. 4. Air Force Studies Chapter I and earlier sections of this chapter have referenced studies conducted on the Spray Equipment Calibration Grids, Test Area C-52A, Eglin AFB, Florida. The soil residue studies and the aquatic studies have previously been described. Test Area C-52A offered a unique opportunity to follow the fate of TCDD in the many components of the ecosystem. Young (61), Young et al (62, 63, 64), and Bartleson et al (6) have reported on various investigations conducted on this test area. The following is a brief synopsis of the magnitude of the contamination and the subsequent effects upon the wildlife of the test area. In addition to these references, data by Young, Thalken and Harrison (unpublished - USAF Occupational and Environmental Laboratory, Brooks AFB, Texas) of recent investigations at the test site have been incorporated into the synopsis. Field investigations were conducted during 1973-1978 on the 3.0 km2 test area containing 4 different calibration grids that received a total of approximately 73,000 kg 2,4,5-T and 77,000 kg 2,4-D during the period 1962-1970. No residues of 2,4,5-T or 2,4-D were detected (detection limit of 10 ppb) in any soil samples collected during 1971-1972. However, residues of the contaminant, TCDD, were still present in 1978. Fifty-four soil samples were collected to a depth of 015 cm from throughout the test area. TCDD levels ranged from <10 to 111-18 �1,500 parts per trillion (ppt). The median concentration was 30 ppt while the mean was 165 ppt. The ecological survey extending over a five-year period documented the presence of more than a 123 different plant species, 77 bird species, 71 insect families, 20 species of fish, 18 species of reptiles, 18 species of mammals, 12 species of amphibians and 2 species of molluscs. At least 170 biological samples were analyzed for TCDD, including 30 species of animals. No TCDD was found in any of the plant species examined. However, TCDD was found in nine species of animals including two rodent species: beachmice (300-1,500 ppt, liver) and hispid cotton rat (<10-210 ppt, liver); three species of birds: meadowlark (100-1,020 ppt, liver), mourning dove (50 ppt, liver), and Savannah sparrows (69 ppt, liver); three 'species of fish: spotted sunfish (85 ppt, liver), mosquito fish (12 ppt, whole body), and sail fin shiner (12 ppt, whole body), and one reptile, the six-lined racerunner (360-430 ppt, muscle). Gross pathology was done on all species collected for TCDD residue analyses. Histopathological examinations were performed on over 300 adult or fetal beachmice or hispid cotton rats from the test area and a control field site. Examinations were performed on the heart, lungs, trachea, salivary glands, thymus, liver, kidneys, stomach, pancreas, adrenals, large and small intestine, spleen, genital organs, bone, bone marrow, skin and brain. Initially, the tissues were examined on a random basis without the knowledge of whether the animal was from a control or test area. All microscopic changes were recorded including those interpreted as minor or insignificant. The tissues v/ere then reexamined on a control and test basis, which demonstrated that the test and control mice could not be distinguished histopathologically. Similar histopathological studies were conducted on the fish and racerunner, and again no significant abnormalities were found. As a concluding remark, Young et al (64) noted that Test Area C-52A offered a unique opportunity to examine the effects of long-term, low-level exposure of biological systems to TCDD. As previously noted, histopathological examination in body organs from adult and fetal beachmice revealed only lesions which are normally observed in microscopic surveys or large numbers of field animals. The absence of liver lesions in animals that had liver levels of TCDD from 200 to 1,500 ppt was most significant in view of the quantities of TCDD that must have been applied to the test site. Although these pathologic studies were initiated in 1973, beachmice had been collected from the test area as early as 1970 for gross pathological observations. They believed the animals examined in 1973-1974 from Grid 1, the area of greatest contamination (having received 1,894 pounds of Purple per acre in 1962-1924) may have been between 24 and 40 generations removed from the mouse population first noted in 1970. Thus, these studies conducted on the mice of Test Area C-52A suggested that long-term, low-level exposure to TCDD under field conditions may in fact not be teratogenic, mutagenic nor carcinogenic. 111-19 �D. Environmental Production of TCDD In 1971, Buu-Hoi et al (14) reported that small quantities of TCDD were formed upon the pyrolysis of 2,4,5-T acid, its butyl ester, or from vegetation defoliated by these products. In their article, Buu-Hoi provided mass spectral data for TCDD (compound I in his text). In reference to these spectral data, they stated (as translated from French): There is no need to use the precise analytical techniques described in the foregoing in the case of pyrolysis of 2,4,5-trichlorophenoxyacetic acid (500-600°), because simple fractional sublimation of the pyrolysate, prewashed in diluted aqueous soda, will yield about 5 percent of compound (1). This yield is increased to 15 percent as a result of the pyrolysis of trichloro-2,4,5 sodium phenoxyacetate. The conclusion (and this has been verified) is that quantities of "dioxin" (I) are formed during the combustion, more or less forced, of materials coming from plants pretreated by 2,4,5trichlorophenoxyacetic acid, and its derivatives (2,4,5-trichlorobutyl phenoxyacetate, the base of the "Orange" defoliant, leads naturally to free acid as a result of hydrolysis attributable to humidity, or to bacterial or fungal degradation), and this is all the more so because alkaline ash appears as a result of such combustion. One then can conceive the possibility of danger, in the long or short term, to public health in areas such as South Vietnam where the people use materials that are principally of plant origin, and are local, as fuels in their homes (wood, charcoal, dry leaves and branches), and which, as a result of the intensive defoliation that took place since 1964* could contain 2,4,5-trichlorophenoxyacetic acid. In 1972, Saint-Ruf (54), a colleague of Buu-Hoi, reported on the formation of "dioxin" from the pyrolysis of Si 1 vex. He reported that although the quantity of TCDD was less than that observed from the pyrolysis of 2,4,5-T, it was nevertheless sufficiently important to render the use of Silvex extremely dangerous for man and animals, especially in areas where treated vegetable matter was likely to be used as domestic foodstuff. The data of Buu-Hoi et al (13) and Saint-Ruf (54), and their conclusions* were challenged by Langer et al (38) in 1973. Langer et al investigated conditions which might produce dioxins from salts and esters of 2,4-D, 2,4,5-T and Silvex. No dioxins were detected even when the sodium salts of 2,4,5-T and Silvex were heated to 300°C and 111-20 �3500C, respectively. However, if a mixture of 0.25 gram (g) 2,4,5-T acid, 2 g HoO and 10 g koC03 was refluxed at 100°C for 3 hr, then heated at 200°C for 15 hr, then at 400°C for 43 hr, a total yield of 0.13 percent TCDD could be detected. Furthermore, Lanaer et al found that the mass spectrum reported by Buu-Hoi et al (and used by Saint-Ruf) for TCDD was in fact not the mass spectrum of TCDD. They suggested that the mass spectrum obtained by Buu-Hoi et al was that of a polymeric matter similar to that found in their own studies. Langer et al concluded that it was extremely unlikely that dioxin {TCDD) could be produced in the field by burning plant material treated with 2,4-D, 2,4,5-T, Silvex or their derivatives. Recently (1977), Stehl and Lamparski (57) reported finding small amounts of TCDD in trapped residue from self-supported fires of grass and paper treated with different compounds containing 2,4,5-T. Under controlled, but as "natural" as feasible conditions, they analyzed the combustion products of the grass or paper after treatment with 13.3 kg 2,4,5-T per ha (12 Ib/A). Stehl and Lamparski felt that the most meaningful way to express their data was in parts per trillion (ppt) of TCDD formed per parts per million (ppm) of 2,4,5-T burned. The average of all their experiments was 0.6 ppt of TCDD formed per 1 ppm of 2,4,5-T burned. The TCDD burden added to the environment by the combustion of natural materials treated with 2,4,5-T would be no . larger than 1 ppt of TCDD per 1 ppm of 2,4,5-T residue burned. Ahling et al (1) has reported similar results when 2,4,5-T residue on wood chips is burned at 500°C; 6 ppt of TCDD formed per 1 ppm 2,4,5-T residue burned. They suggested that this would correspond to a formation of about 1 microgram (yg) TCDD per m2 in forest fire directly after application of the herbicide formulation. However, Cutler (21) recently (1978) has suggested that the burning of forested areas treated with 2,4,5-T may be of little concern, since TCDD decomposes at temperatures above 800°C (and 2,4,5-T decomposes at temperatures above 500°C), considerably below the temperatures of 1200°C or more achieved in the field with a free exchange of air. E. Photodegradation of TCDD In perhaps what can be termed as one of the most significant studies on the environmental degradation of TCDD, Crosby and Wong (19), in 1977, found that herbicide formulations (including Orange) containing known amounts of TCDD and exposed to natural sunlight on leaves, soil or grass, lost most or all of the TCDD in a single day, due principally to photochemical dechlorination. Despite the known persistence of pure TCDD, it was not stable as a contaminant in thin herbicide films exposed to outdoor light. Crosby and Wong (19) have established three requirements for significant dioxin breakdown in the environment; namely, dissolution in a light-transmitting film, the presence of an organic hydrogendonor such as a solvent or pesticide and ultraviolet light. They 111-21 �noted that all three conditions are normally met during the practical application of 2,4,5-T or other TCDD-containing chemicals. Thus, their data suggested that environmental residues of TCDD often will be considerably less than previously expected. Nash and Beall (45) concluded from their studies of the fate of TCDD in a microagroecosystem chamber, that once TCDD was volatilized, it dechlorinated in the direct sun and apparently even in shade outdoors or when the sun was filtered with glass in the chambers. They concluded further that TCDD was sensitive to photodechlorination in the vapor phase even in the absence of ultraviolet light. IV. SUMMARY Available data indicate that the vast majority of the phenoxy herbicides would impact forest canopy, the intended target. Rapid uptake (e.g., within a few hours) of the ester formulations of 2,4-D and 2,4,5-T would occur. Most of herbicide probably would undergo rapid degradation (weeks) within the cellular matrix of the vegetation. However, some of herbicide may remain unmetabolized and would be deposited on the forest floor at the time of leaf fall. Soil microbial and/or chemical action would likely complete the degradation process. Herbicide droplets that impacted directly on soil or water would probably hydrolyze rapidly (within hours). Biological and nonbiological degradatiye processes would further occur to significantly reduce these residues. Some volatilization of the esters of 2,4-D and 2,4,5-T would occur during and immediately after application. The volatile material most likely would dissipate within the foliage of the target area. Photodecomposition of TCDD would minimize the amount of biologically active volatile residues moving downwind of the target area. Accumulation of phenoxy herbicides in animals may occur following ingestion of treated vegetation. The magnitude of this accumulation would likely be at nontoxic levels. Herbicide residues in animals would rapidly decline after withdrawal from treated feed. Most TCDD sprayed into the environment during defoliation operations would probably photodegrade within 24 hours of application. Moreover, recent studies suggest that even within the shaded forest canopy, volatilization and subsequent photodecomposition of TCDD would occur. Since translocation into vegetation would be minimal, most TCDD that escaped photodegradation would enter the soil-organic complex on the forest floor following leaf fall. Soil chemical and microbial processes would further reduce TCDD residues. Bioconcentration of the remaining minute levels of TCDD may occur in liver and fat of animals ingesting contaminated vegetation or soil. However, there are no field data available that indicate that the levels of TCDD likely to accumulate in these animals would have a biological effect. 111-22 �The environmental generation of TCDD from 2,4,5-T residues, through thermal or photolytic processes, would be highly unlikely and of no consequence. 111-23 �LITERATURE CITED CHAPTER III 1. Ahling, B., A. Lindskog, B. Jansson and G. SundStrom. 1977. Formation of polychlorinated dibenzo-p-dioxins and dibenzofurans during composition of a 2,4,5-T formulation. Ckuma&ph&m 33:461-468. 2. Aly, O.M. and S.p. Faust. 1964. Studies on the fate of 2»4-D and ester derivatives in natural surface waters. J. Ag/w.c.. Food Chem. 126.541-546. 3. Anonymous. 1977. flioxot: Position document. (Draft) Dioxin Working Group. April 1977. U.S. Environmental Protection Agency; Washington, D.C. Mim. 17 p. 4. Arnold, E.L., A.L. Young and A.M. Wachinski. 1976. Three years of field studies on the soil persistence and movement of 2,4-D, 2,445-T and TCDD. Weed SCA.. Soc. Am. Meet. Atotfc. 206. p 86. 5. Audus, L.J. 1960. faioAabiotogJjCLOut breakdown. o&ft.eA.b-ccx.dei-in 4o^tA. P 1-19. In_ Herbicides and the soil. E.K. Woodford and G.R. Sugar (Eds.). Blackwell Sci. Pub, , Oxford, England. 6. Bartleson, D.D., O.D» Harrison, and J.D. Morgan. 1975. Stadia ol WiJtttJUAe. exposed to TCW zon&min&tiid 4o^ta. Technical Report AFTL-TR-75-49. Air Force Armament Laboratory, Eglin Air Force Base, Florida. 53 p. 7. Baughman, R.W. 1976. Tetrachlorodibenzo-p-dioxins in the environment. High resolution mass spectrometry at the picrogram level. P-U-6. Ab^^t. Int. 36(7):3380B. 8. Baughman, R.W. and M.S., Meselson. 1973. An analytical method for detecting TCDD (dioxin): Levels of TCDD in samples from Vietnam. EnviAdn. Health P&upa&t, 5:27-35. 9. Baughman, R.W. and M.S. Meselson. 1973. An improved analysis for tetrachlorodibenxo-p-dioxins. Advdn. Chem. SeA. 120:92-104. 10. Beatty, P.W., M.A. Helscher, and R.A. Neal. 1976. Toxicity of 2,3,7,8-tetrachlorodibenxo-p*dioxin in larval and adult forms of Rana catesbelana. 6«££. Envvwn* Cdnfaw. Taxi-tol, 16(5) :578-581 . 11. Blackman, G.E.,, J,D. Fryer, A. Lang and M. Newton. 1974. The effects of herbicides in South Vietnam. Part B» Working PapersPersistence and disappearance of herbicides in tropical soils. Nat. Acad. Sci., Washington, D.C. 56 p. 111-24 �12. Brown, J.W. 1962. l/egexUttoiaa£ bpnay ttete in South Vietnam. U.S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland, 119 p. Available from the Defense Documentation Center, Defense Logistics Agency, Cameron Station, Alexandria, Virginia, DDC Number AD476961 . 13. Buu-Hoi, N.P., G. Saint-Ruf, P. Bigot and M. Mangane. 1.971. Preparation, properties and identification of dioxin (2,3,7,8tetrachlorodibenzo-p-dioxin) in the pyrolysate of defoliants containing 2,4,5-T and its esters and in contaminated vegetation. C.R. Hebd. Seances Acad. Sex.., Se/t. 0. 273:708-7111. (French) 14. Byast, T.H. and R.J. Hance. 1975. Degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Bo££. Envision. Contam. Tovuiat. 14(1): 71 -76. 15. Committee on the Effects of Herbicides in South Vietnam. 1974. Part A. Summary and conclusions. National Academy of Science, Washington, D.C. 398 p. 16. Craig, D.A. 1975. U6e orf HeAbx.cx.de* in Southeast Mia. Historical Report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 p. 17. Crosby, D.G. 1976. Herbicide. pkotade.c.ompo*ition. P 835-890. In. Herbicides - Chemistry, Degradation and Mode of Action. Vol. 2. P.C. Kearney and D.D. Kaufman (Eds.). Marcel Dekker, Inc., New York. 18. Crosby, D.G. 1976. Nonbio£ogic.cLt de.Qtiadcvtion o& heAbicideA in tk<L boiJL. P 65-97. _In_ Herbicides - Physiology, Biochemistry and Ecology. Vol. 2. L.J. Audus (Ed.). Academic Press, New York. 19. Crosby, D.G. and A.S. Wong. 1977. Environmental degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Science 195:1337-1338. 20. Cupello, J.M. and A.L. Young. 1976. Radiochemical bioassay of TCDD uptake in plant material. Extract. Annual Research Progress Report No. 11. Dean of Faculty, United States Air Force Academy. 22 p. 21. Cutler, M.R. 1978. Remarks to the National USDA/EPA Symposium on the use of herbicides in forestry. USDA-SEA, Washington, D.C. 7p. 22. DeRose, H.R. and A.S. Newman. 1947. The comparison of the persistence of certain plan growth-regulators when applied to soil. PA.OC. Sci. Soa. Am. 12:222-226. 23. Dost, F. , J. Witt, M. Newton and L.A. Norris. 1975. Statement on 2,4,5-T and TCDD. J. fofi. 73(7) :410-412. 111-25 �24. Goring, C.A.I., D.A. Laskowski , J.W. Hamaker and R.W. Meikle. 1975. Psu-ndipleA o& pe6£tcxde d&gsuidation -in Ao-it. P 135-172. In_ Environmental Dynamics of Pesticides. R. Haque and V.H. Freed (Eds.) Plenum Press, New York. 25. Grover, R. , J. Maybank and X. Yoshida. 1972. Droplet and vapor drift from butyl ester and dimethyl ami ne salt of 2,4-D. Weed Sex. 20(4): 320-324. 26. Hamaker, J.W. 1975. The, witeA.pi&tation ofi *oi£ le.ac.kt.ng expetxmentb. P 115-133. ln_ Environmental Dynamics of Pesticides. R. Haque and V.H. Freed (Eds.). Plenum Press, New York. 27. Harrigan, E.T. 1970. CaUbxation TdAt o& tkz UC-123K/A/M5V-1 Sp>uu/ SyAt&m. Technical Report ADTC-TR-70-36. Armament Development and Test Center, Eg! in AFB, Florida. 160 p. 28. Helling, C . S . 1971. Pesticide mobility in soils. II. Applications of soil thin-layer chromatography. P/toc. SoJJt Sex. See. Am. 35(5):737-743. 29. Helling, C.S., A.R. Isensee, E.A. Woolson, P.D.J. Ensor, G.E. Jones, J.R. Plimmer and P.C. Kearney. 1973. Chlorodioxins in pesticides, soils and plants. 3. tnvJUion. Qual. 2(2) :171-178. 30. Hummel, R.A. 1977. Clean-up techniques for the determination of parts per trillion residue levels of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). J. Ag^c. Food Chm. 25(5) : 1049-1053. 31. Irish, K.R., R.A. Darrow and C.E. Minarik. 1969. InfiotLmcution manual fan. v&geJution c.on&ioi Ln SouuthnaAt faia. Miscl. Public. 33. Department of the Army, Fort Detrick, Frederick, Maryland. 71 p. 32. Isensee, A.R. and G.E. Jones. 1971. Absorption and translocation of root and foliage applied 2,4-dichlorophenol , 2,7-dichlorodibenzop^dioxin, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Ag/txc. Food Ckm. 19(6):1210-1214. 33. Isensee, A.R. and G.E. Jones. 1972. Distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic model ecosystem. BnuAon. Sex. Techno£. 9(7) :668-672. 34. Kearney, P.C., E.A. Woolson and C.P. Ellington, Jr. 1972. Persistence and metabolism of chlorodioxins in soils. Envision, Sex.. Uchnol. 6(12):1017-1019. 35. Kearney, P.C., E.A. Woolson, A.R. Isensee and C.S. Helling. 1973. Tetrachiorodibenzodioxin in the environment: Sources, fate, and decontamination, tnv-uion. H&alth PvAApe&t. 5:273-277. 111-26 �36. K l e i n , R . E . and E . T . Harrigan. 1969. Comp<vuAon Tut o Technical Report ADTC-TR-69-30, Vol. I. Armament Development and Test Center, Eglin AFB, Florida. 356 p. 37. Klingman, G.C. 1961. Sons, Inc., New York. 38. Langer, H . G . , T.P. Brady and P.R. Briggs. 1973. Formation of dibenzodioxins and other condensation products from chlorinated phenols and derivatives. EmuAon. Health P&t6pee£. 5:3-7. 39. Leng, M.L. 1977. Companative. m&taboti&m o& phznozy k<>Abi.cA.d<Lt> <in cwxjTia&6. P 53-76. Jj^ Fate of Pesticides in the Large Animal. Academic Press, Inc., New York. 40. Loos, M.A. 1975. Phe.noxyalkano-ic. acMt>. P 1-128. In_ Herbicides Chemistry, Degradation and Mode of Action. Vol. 1. P . C . Kearney and D.D. Kaufman ( E d s . ) . Marcel Dekker, Inc., New York. 41. Mahle, N . H . , H.S. Higgins and M.E. Getzendaner. 1977. Search for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin in bovine m i l k . Ball. EmuAon. Contam. Tazicol. 18(2) :123-130. 42. Matsumura, F. and H . J . Benezet. 1973. Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Envision. Health PeA6pee£. 5:253-258. 43. M i l l e r , R.A., L.A. Norris and C.L. Hawkes. 1973. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic organisms. Env^ion. Health Pnupuct. 5:177-186. 44. Muzik, T.J. 1976. Influence o& e.nv4Aonme.ntal factor on toKiCsity to plant*. P 203-247. j£ Herbicides - Physiology, Biochemistry and Ecology. Vol. 2. L.J. Ausus (Ed). Academic Press, New York. 564 p. 45. Nash, R.G. and M.L. Beall, Or. 1978. EnviJiom\e.ntal dlbtfu-bution aft 2,3,7,$-te&ULC.hlotLO(LLb<>.nzo-p-dioXsin (TCVV) apptiid with 4-cCvex to tunfa <in m*ioAoa.QSLoe,c.oAyAtm. Final Report EPA-1AG-D6-0054, Agricultural Environmental Quality Institue, U . S . Department of Agriculture, Beltsville, Maryland. 46. Newton, M. 1971. Disappearance of 2,4,5-T from forest ecosystems. Weed Sex.. Soc. Am. Meet. Abttn. 57. pp-30. 47. Newton, M. and S.P. Synder. 1978. Exposure of forest herbivores to 2,3,7,8-tetrachloro-p-dioxin (TCDD) in areas sprayed with 2,4,5-T. Bull. EnviAon. Con-tarn. Tozicoi. (In Press) Weed control: <u a .icx.ence. 421 p. 111-27 John Wiley and �48. Norris, L . A , and R.A. Miller. 1974. The toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in guppies (Poecilia reticulatus Peters). Eatt Env.iAon. Contam. TOXA,O.O£. 12(1):76-SO. 49. Palm, C . E , (Chairman). 1968. Weed Control, Vol. 2. Principles of Plant and Animal Pest Control. Nat. Acad. Sei,, Washington, D . C . 471 p, 50. Plimmer, J,R. 1976. Vet&tA&Uy, P 891-934. In Herbicides Chemistry, Degradation and Mode of Action. P,c71<earney and D.D. Kaufman (Eds,), Vol. 2. Marcel Dekker, Inc., New York, 51. Reigner, I . e . , w . E . Sopper and R . R , Johnson. 1968. W i l l the use of 2,4,5-T to control streamside vegetation contaminate public water supplies? J, FOA, 66(12) :914-918. 52. Reinhart, K . G . 1965. Herbicidal treatment of watersheds to increase water yield. N. East Weed Contr. Conf. Proc. 19:546-551. 53. Ross, R . T . 1976. 2,4,5/Dioxins: Analytical Collaborators Meeting, June 15, 1976. June 25, 1976, Memorandum of the United States Environmental Protection Agency; Washington, D . C . p 3, 54. Saint-Ruf, 6. 1972, Formation of dioxin in the pyrolysis of sodium a-(2,3,7,8-trichlorophenoxy)'propionate. No^u/tM^am chosen 59(12) :648. (French) 55. Shadoff, L.A,, R,A, Hummel and L. Umparski, 1977. A search for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an environment exposed. annually to 2,4,5-triehlorophenoxyacetic acid ester (2,4,5-T) herbicides. 8u££, Env-cton. Contam. Tou.o.oi. 18(4) :478-485. 56. Stark, H . E . , J.K. McBride and G.F. Orr, 1975. Soil biodtQfm.dcuU.on o£ H&ib-ctiide. 0/tang&. 1. bti.cAob<iai and <Lc.atoQ-ic.ak 4tudy a£ the. U.S, MA. FoA.ce Log<u>£icA Command TeAt HIU MA Poize. 6o4e, U-tofe. Final Report, TECOM Project No. 5-CO213'00'015, U.S, Army Dugway Proving Ground, Dugway, Utah. 73 p. 57. Stehl, R.K, and LL Umparski, 1977. Combustion of several 2,4,5trichlorophenoxy compounds: Formation of 2,3,7,8-tetrachlorodibenzop-dioxin. Science 1 97 ( 4307) :1Q08- 1009, 58. Tschirley, F.H. 1968. Reiponie ofi tfiopicjaJi and Au.b&iap<ic.at woody plante to chmic.aUi- Vieatinnnte , Research Report CR-13-67. Agricultural Research Services, U.S. Department of Agriculture, Washington, D.C. 197 p, 59. Winston, A,W., Jr. and P.M, Ritty. 1972. What happens to phenoxy herbicides when applied to a watershed area. Ind, l>eg. Manage. 111-28 �60. Woolson, E.A., P.D.J. Ensor, W.L. Reichel and A.L. Young. 1973. Dioxin residues in lakeland sand and bald eagle samples. Advan. Ckem. Svi. 120:112-118. 61. Young, A.L. 1974. Ecological &tudi&> on a heAb-icide. equipment tut ouita (TA C-52A) Egtin AFB ReAeA.vatt.an, Florida. Tech Rep. AFATL-TR-74-12. Air Force Armament Laboratory, Eglin Air Force Base, Florida. 141 p. 62. Young, A.L., P.J. Lehn and'M.F. Mettee. 1976. Absence of TCDD toxicity in an aquatic ecosystem. Weed Sex. Sec. Am. Meet. Afa-6-tt. 107. p 46. 63. Young, A.L., C.E. Thalken and W.E. Ward. 1975. Studies o& tke. ecological impact o& sie.pe£itivn a&ual appLic&tionA of, h<Lnbtcid(L& on tho, <tcoAyt>tw o& tut ouzo. C-52A, Egtin AFB, FloiLda. Technical Report AFATL-TR-74-12. Air Force Armament Laboratory, Eglin AFB, Florida, and Department of Chemistry and Biological Sciences. U.S. Air Force Academy, Colorado 80840.- 127 p. 64. Young, A.L., C.E. Thalken, E.L. Arnold, J.M. Cupel lo and L.G. Cockerham. 1976. Fate otf 2,3,7,B-t<i&iachlosiodibe.nzo-p-dAQxJ.n (TCW) -en tke. znvJJionmtLnt: Sumnasiy and de.contam<ination ie.comme.ndationA. USAFATR-76-18. Department of Chemistry and Biological Sciences, USAF Academy, Colorado 80840. 41 p. 65. Zitko, V. 1972. Absence of chlorinated dibenzodioxins and dibenzofurans from aquatic animals. 8o££. EmuAoia. Contain. Topical. 7(2/3):105-110. 66. Zitko, V., 0. Hutzinger and P. U.K. Choi. 1972. Contamination of the Bay of Fundy-Gulf of Maine area with polychlorinated biphenyls, polychlorinated terphenyls, chlorinated dibenzodioxins, and dibenzofurans. EnvxAon. Health PeAAp&ct. 1:47-50. 111-29 �CHAPTER IV ' THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS I. INTRODUCTION This review cites a major portion of the world scientific literature dealing with the toxicological aspects of 2,4-D, 2,4,5-T and TCDD in various laboratory and domestic animal species. The primary purpose was to provide a broad overview of research investigations performed to date. With this information, a more critical evaluation could be made of reported human exposures to actual and theoretical levels of 2,4-D, 2,4,5-T and TCDD as presented in other chapters of this report. In an attempt to organize this chapter the following format and sequence was followed. Each of the compounds in question was reviewed for a) acute and short-term toxicity; b) subacute and chronic toxicity; c) absorption, distribution and excretion data; d) embryotoxic, fetotoxic and teratogenic potentials; e) carcinogenic and tumorigenic potentials; and f) mutagenic and cytogenetic potentials. Where possible, the cited data were tabu! ari zed for ease of comparison and interpretation and a summary of the tabular data presented in the text. In the review of acute and short-term toxicity the primary effort was directed toward finding references establishing for each compound a no effect dose, a dose lethal to 50 percent (LD50), and a dose lethal to 100 percent (LDiOO) of the laboratory or domestic animal species studied. After the acute toxic doses were known the subacute and chronic levels were then addressed. The highest "no effect" level of repeated dosing as well as the lowest repeated dosing level causing symptoms of toxicity were collected. These two sections were followed by discussion of cited literature dealing with the absorption, distribution in various body compartments and tissues, and excretion of the 2,4-D, 2,4,5-T and TCDD. In a 1977 review of the TeAatogenxc E^e.c.t& oft Wilson (145) stated: Many chemicals with which man comes into contact are known to be overtly or potentially harmful, causing structural or functional change immediately and these effects are recognized as acute toxic responses to these chemicals. On the other hand, chemicals known to be overtly or potentially harmful, causing structural or functional change and effects, only, after some intermediate time or considerable lapse of time following exposure, are recognized IV-1 �as chemicals producing a chronic toxic response. This latter group with its subacute effects is where most of the chemicals fall the at interfere with reproduction. Reproduction effects are rarely the first or only toxic manifestations, but occasionally an embryo or fetus in ut&io is the primary or the only individual expressing the effects of the toxic material, indicating that the conceptus may have extraordinary sensitivity to certain chemicals or compounds. These so called teratogens may be naturally occurring or manufactured materials and despite its sequestered location deep within the maternal body, the embryo or fetus sometimes receives a toxic, i.e., teratogenic dose, albeit only a small fraction of the maternal dose. Dencker (31) in the introduction to his 1976 study TVcAAue Localization o& Some. T0Aatog<Lm> at EanJiy and Late. Gestation ReJLat&d to f<Ltai Ejects stated: Our knowledge concerning the mechanisms by which chemicals cause fetal damage is very sparse, and only in a few instances have generally accepted theories been presented. Most often there is no specific effect for a given chemical; rather it seems. that one agent produces a wide spectrum of mal forma tions - which indicates a nonspecific mechanism of action. Moreover, different chemicals may often produce the same type of malformation. This confusing aspect may partly be explained by the fact that the different organs have certain sensitive periods; in their development, when they are especially susceptible to external influences. With these comments in mind the literature dealing with embryotoxic, fetotoxic and teratogenic potentials of 2,4-D, 2,4,5-T and TCDD was reviewed to provide as much detail as possible as to the number of animals used, reproductive state, route of administration and toxic response as well as dose and formulation of each compound being tested. A Similar approach was used to review the carcinogenic, tumorigenic, mutagenic and cytogenetic potentials for each of the compounds. Care was taken to try and establish numbers of test animals, method and route of administration and the specific effect of a particular formulation or purity of 2,4-D, 2,4,5-T or TCDD on those animals. Where specific animal data were not available, studies dealing with animal tissue cultures or bacterial mutant strains were referenced. For simplicity in format, dosage levels of the various chemicals were expressed as mg/kg, but it should be understood that this refers to milligrams of a specific chemical or formulation per kilogram of body weight of the test animal unless otherwise specified. IV-2 �II. REVIEW OF 2,4-D TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of 2,4-D The following review of 2,4-D toxicity was based primarily on three review articles: Rowe and Hyman (115); Dalgaard-Mikkelsen and Poulsen (29); and the International Agency For Research on Cancer (IARC) Monograph, Vol 15 (66) although other recent publications on the subject have been cited. Bucher (18) in 1946 was among the first to report the results of experiments with small animals using 2,4-D. Temporary myotonia lasting from eight to twenty-four hours or more following a single injection of 150 to 250 mg/kg was observed in mice, rats, rabbits and dogs. In 1947, Hill and Carlisle (63) published the results of acute oral studies, following single doses of 2,4-D and found the U^Q for mice to be 375 mg/kg; for rats, 666 mg/kg; for rabbits, 800 mg/kg; and for guinea pigs, 1,000 mg/kg. The largest single oral dose administered to monkeys without serious after-effect was 214 mg/kg or 428 mg/kg given intraperitoneally. An oral, plus an intraperitoneal injection in monkeys for a total dose of 500 mg/kg 2,4-D caused nausea, vomiting, lethargy, muscle incoordination and head drop. These workers observed that all species reacted similarly and that there were no significant differences in potency between crude and purified preparations, or between the sodium or ammonium salts. Deaths from large doses were apparently due to ventricular fibrillation. When death was delayed, myotonia, stiffness of extremities, ataxia, paralysis and coma were observed. Parenteral administration of 150-200 mg/kg 2,4-D caused symptoms of myotonia in mice. In those acutely intoxicated, dilatation of the blood vessels of lungs, liver and kidneys was observed by Bucher (18). Rats and guinea pigs administered lethal doses of 2,4-D exhibited congestion of the viscera. Enlarged, swollen, kidneys and microscopically .massive cloudy swelling of the proximal convoluted tubules with cast formation was noted by Hill and Carlisle (63). Florsheim and Velcoff (43) reported a decrease in both thyroid and body weights in male rats given single subcutaneous injections of 2,4-D at 100 mg/kg. Guseva (57) found the LDso for the subcutaneous injection of 2,4-D in mice to be 220 mg/kg. At 10-100 mg/kg 2,4-D in rats and mice no impairment of motor activity was seen nor did those doses alleviate strychnine spasms. In cats, 20-30 mg/kg 2,4-D given intravenously was hypotensive and that effect was not impaired by atropinization. 'Baker et al (6) administered 112 grams (g) of grass mixed with horsemeat and dog meal divided over three consecutive meals to two IV-3 �healthy one-year-old mongrel dogs. The grass had been treated two days earlier with the equivalent of 4 pounds per acre (Ib/A) of 2,4-D butyl ester, which was twice the recommended rate. The dogs readily ate the food and no ill effects were observed during the following 96 hours. Each animal was then treated with 500 mg/kg 2,4-D in a single oral dose. No deleterious effects were seen in the next 96 hours of observation. One animal, killed and necropsied at 96 hours post administration, failed to reveal any macroscopic lesions and the other animal remained healthy for 82 days following the second treatment, at which time the experiment was terminated. In dogs, Drill and Hiratzka (33) found that toxic symptoms were often delayed up to six hours following a single oral administration of lethal doses of 100, 250 and 400 mg/kg 2,4-D. The deaths were delayed and occurred two to nine days after the compounds were administered; The acute oral LDgg for (98.5 percent purity) 2,4-D was in the range of 100 mg/kg or higher. Death appeared to be due in most cases to hepatic congestion or pneumonia. Pathological changes were limited to the gastrointestinal tract, lungs and liver, and followed the development of anorexia, weight loss and myotonia (33). Dogs exhibited more evidence of hepatic congestion and moderate hepatic necrosis than was seen in other animals studied by Bucher (18). Drill and Hiratzka (33) concluded that the no effect level for a single oral dose of 2,4-D in dogs was 25 mg/kg. In a study by Shavgulidze et al (125), the single oral LDioO of 2,4-D sodium salt in sheep was 900 mg/kg. Death occurred in 2-4 days following the clinical signs of asthenia, depression, ataxia, hypothermai, dyspnea, muscle paralysis, anorexia and intense photophobia in those animals dosed at 500-1,000 mg/kg. The no effect single, oral dose of 300-400 mg 2,4-D was detoxified in 9-12 days with no traces remaining in the tissues. McLennan (88), reported on the accidental oral administration of 2,4-D in two cows. He noted that the death of one animal occurred within 12 hours following a calculated dose of 150-188 mg/kg. The toxic dose for the animal that survived, was calculated at between 105 and 132 mg/kg. In contrast Rowe and Hymas (115) noted that the 1050 of 2,4-D for cattle ranged between 500 and 2000 mg/kg body weight while a single dose of 1000 mg/kg may or may not cause illness. Rade-leff (110) cited a report in which cattle given one dose of 250 mg/kg 2,4-D showed signs of toxicity. In calves six to eight weeks old, Bjorklund and Erne (15) found that single doses of 100 to 200 mg/kg 2,4-D produced reversible signs of toxicity. Toxic symptoms summarized by Rowe and Hymas (115) included the following general observations in animals treated with acute toxic doses of 2,4-D: loss of appetite, loss of weight, depression, roughness of coat, general tenseness and muscular weakness particularly of the posterior quarters. Post mortem findings usually included irritation of the stomach of small animals and of the abomasum of ruminants, minor evidence of liver and kidney injury and in some instances congestion of the lungs. IV-4 �From data presented in Table 1, the acute 1059 as a rule was in the order of 300-800 mg/kg 2,4-D for rats, mice, guinea pigs, rabbits and cats. Analyses of data from the limited available studies supported the conclusion that the dog may be slightly more susceptible to oral doses of 2,4-D than other animals. Monkeys, sheep and cattle appeared to be somewhat more tolerant. The experiments referenced in Table 1 have also provided information on the acute oral administration of various salts and esters of 2,4-D as pure chemicals and as commercial preparations. No significant differences in the toxicity of the salt and ester forms of 2,4-D were seen when compared to the free acid (63,115). Hill and Carlisle (63) stated: In any assessment of the acute toxicity of a chemical based on data obtained from laboratory animals it should be borne in mind that considerable variation in species susceptibility may occur and that the data obtained cannot always be translated into toxic doses for humans. In the studies referenced in this section it can be concluded as Hill and Carlisle did in their studies that: all of the laboratory animals tested reacted in a similar fashion from signs and symptoms which developed and from the pathological lesions which were present at autopsy. Assuming that man is no more resistant or susceptible than the rabbit or monkey, then the largest tolerated dose for a 75 kg man would be 15 grams of 2,4-D. With the exception of dog and monkey, all of the laboratory animals used in the cited references lacked the vomiting reflex so that they were unable to relieve themselves of irritating material by vomiting. The experiments conducted in monkeys indicate that the material is a gastric irritant in large doses, so that the possibility of the occurrence of acute poisoning in humans would seem relatively remote because of the large dose which man could presumably tolerate. Assuming that man is no more susceptible than the most susceptible animal test, the mouse, then the calculated oral LDso for man would amount to approximately 28 grams (63). It is generally accepted that the oral LD5Q of 2,4-D for man is around 500 mg/kg while the accepted LD^Q of aspirin for man is around 1,500 mg/kg. B. The Subacute and Chronic Toxicity Potentials of 2,4-D Repeated once or twice daily, subcutaneous injections of 50 to 90 mg/kg 2,4-D in mice for three weeks to ninety days did not elicit a characteristic chronic syndrome of toxicity or any notable histological IV-5 �TABLE 1 . Animal Number Used Mouse 450 Summary of literature data on the no-effect, and LD levels of the acute toxicity of 2,4-D in animals Route of Administration DoseToxicity Single Dose mg/kg Reference § Gavage LD 375a'b 63 NSC Intraperitoneal LD 375a 63 NS Subcutaneous LD 220a 57 NS Subcutaneous LD 280b 18 NS Oral in olive oil LD 368a 115 NS Oral in olive oil LD 541d 115 NS Oral in corn oil LD 713e 115 NS Oral LD 380f 81 50 50 50 50 50 50 50 50 Rat L 150 Gavage LD 666b 63 NS Intraperitoneal LD 666b 63 NS Oral in water LD 805b 115 NS Oral in olive oil LD 375a 115 NS Oral in olive oil LD 700d 115 NS Oral in corn oil LD 620e 115 NS Oral LD 1 ,500f 119 NS Oral LD 2,000b 119 NS Oral LD 900f 81 125 Gavage LD 1 ,000b 63 NS Intraperitoneal LD 666b 63 NS Oral in water LD NS Oral in olive oil 50 50 50 50 50 50 50 50 50 Guinea Pig u 50 50 IV-6 50 LDcn 551-2000b 469a 115 115 �Table 1 continued NS Oral in olive oil LD50 550° H5 NS Oral in corn oil LD 50 848e 115 70 Gavage LD 50 800 63 NS Intraperitoneal LD 50 400b 63 NS Intravenous LD50 400b 63 NS Oral in corn oil LD 50 424e 115 NS Oral LD 50 820 81 • Oral LD 100d 33 1 Mh/2 F Oral No effect 25a 33 2 Oral No effect 500' 6 Oral No effect 214a 63 Intraperitoneal No effect 428b 63 Oral LD 900 125 No effect 300-400b 125 Rabbit Cat Dog 4 Fg 50 Monkey Sheep NS 100 Oral LD 100 Oral Cattle LD 50 150-188' (a calculated dose) 500-2000^ 115,132 a 2,4-D acid Sodium salt of 2,4-D C NS - number of animals in study not stated or unavailable from literature source. Isopropyl ester of 2,4-D e Mixed butyl esters of 2,4-D IV-7 �Table 1 continued Butyl ester calculated as 2,4-D 9 F - Female h M - Male n 20% w/v amine salt of 2,4-D in aqueous solution J Form not stated in available literature source IV-8 �changes. Levels of 70 mg/kg or more retarded growth, probably by reducing food intake. Mice undergoing this treatment became pregnant and bore apparently normal litters (18). Guseva (57) found that 22 daily subcutaneous injections of 0.1 mg 2,4.-D in mice caused no toxic effects. In subacute studies with rats, Hill and Carlisle (63) fed a diet containing 1,000 mg 2,4-D/kg for 30 days without severe harmful effects. Some visceral congestion and kidney edema with degenerative changes in the tubules were noted. No adverse effects were seen in groups of 5 or 6 young female rats given 2,4-D by intubation five times a week for four weeks at doses of 3, 10, and 30 mg/kg in olive oil. At doses of 100 mg/kg 2,4-D, varying degrees of gastrointestinal irritation, slight cloudy swelling in the liver and depressed growth rates were noted. At 300 mg/kg 2,4-D, the animals failed rapidly and died. The principle lesion observed at post mortem was a severe gastrointestinal irritation. In another study, matched groups of five, young, adult, female rats were placed on diets containing 100, 300, 1,000, 3,000 and 10,000 mg 2,4-D/kg of diet for 113 days. The no effect levels were 100 and 300 mg/kg of diet. At 1000 mg/kg, adverse effects were characterized by a depressed growth rate, excessive mortality, slightly increased liver weights and slight cloudy swelling of the liver. The animals on the 3,000 and 10,000 mg/kg levels in their diets were destroyed after twelve days as they were not eating and were rapidly losing weight. Increased liver and kidney weights were noted with unstated minimal pathological changes (115). No drastic damaging effects were noted when male Long-Evans rats were given 2,4-D equivalent to 2-5 g/kg over a 4 to 7-week feeding period. Response to herbicide treatment was dependent on animal age and on the duration of time that the chemical was fed. Little or no effect was noted on liver weight. Herbicide-induced enlargement of the liver was associated with increases in most of the major cellular components on a per liver basis. Isolated liver nuclei were 20-30 percent more active in the -cn-v-c^to RNA synthesis than in the control nuclei (22). Schwetz et al (122) found that oral doses of 12.5 to 87.5 mg/kg 2,4-D did not adversely affect the weight gain of rats during pregnancy. In a preliminary study, non-pregnant rats tolerated 75 mg/kg 2,4-D for 10 days while 100 mg/kg killed two rats and produced overt signs of toxicity in three survivors, Hansen et al (58) conducted a study with rats starting at 3 weeks of age, using groups of 25 female and 25 male animals, fed 0, 5, 25, 125, 625 or 1,250 mg 2,4-D/kg of diet for 2 years. During the study no significant differences in survival rates between controls and test animals were noted. The mean body weights of the different groups of IV-9 �males and females and the organ-to-body weight ratios for liver, kidney, heart, spleen and testes were not significantly different (P>0.025). The only exceptions were in two male rats, one at 625 mg/kg, and a second at 125 mg/kg dosage level in the diet. These two animals had slightly enlarged spleens. Mean values for hemoglobin, hematocrit, and total white blood cell count of controls and of rats at each dose level, at the same time interval, were similar and within normal range. The maximum no effect level for the rats in this study was greater than l»250 mg 2,4D/kg of diet, Hansen et al (58) in another study fed 0, 100, 500 or 1,5QQ mg 2,4-D/kg of diet to groups of 20 male and 20 female rats, t^o effect was observed at the 100 and 500 mg/kg of diet levels. At the 1,500 mg/kg level, there was no effect on fertility nor on the average number of pups per litter; however, significant effects on the average number of pups weaned and also on their weaning weights were noted. The no effect level is at least 500 mg/kg but less than 1,500 mg/kg of 2,4-D in the diet. Bjorklund rats at 1,000 mg/1. same dose level for health and diarrhea and Erne (15) administered 2,4-D in drinking water to Progeny from treated females were maintained on the 2 years with signs of growth inhibition, poor general as the main effects. Kay et al (72) found no significant adverse effects in a study using 112 New Zealand strain albino rabbits where 15 ml of each of three commercially available formulations of 2,4-D (dimethylarrrfne salt and the isooctyl and butyl esters) were administered 5 times a week for 3 weeks to the intact and abraded skin at 0.626 percent and 3.13 percent concentrations. Body weights, survival, hematological values, clinical chemistry values and organ/body weight ratios were all within normal ranges. Local skin inflammatory reactions occurred in all groups of animals including controls. This was especially severe in those applications where the 2,4-D esters were diluted with an unspecified oil. The water dilutions of all three forms produced less local skin inflammation. Historically, the treated animals had an increased incidence and severity of subepithelial fibre-sis and accompanying mononuclear infiltration in the skin. No peripheral or central nervous system tissues or microsections of other tissues disclosed any adverse findings. Hansen et al (58) conducted a study using groups of 3 male and 3 female beagle dogs being fed 0, 10, 50, 100 or 500 mg/kg 2,4-D in the diet (96.7 percent pure, with no detectable TCDD by GLC with a sensitivity of 1 mg/kg) for 2 years, starting at 6-8 months of age. Twenty-eight dogs surviving the 2-year period were clinically normal, in fair to good condition, with a no effect level greater than 500 mg/kg in the diet. One female at the 100 mg/kg level was emaciated at the end of the experiment; however* no significant lesions were noted. A male animal that died after 10 months on the study at the 10 mg/kg 2,4-D lev/el, shbwed a slight atrophy o,f the testes and moderate depletion of cellular elements in other tissues. IV-10 �Drill and Hiratzka (33) orally dosed (via capsule in a piece of canned dog food) adult mongrel dogs of both sexes with either 2, 5 or 10 mg/kg 2,4-D 5 days a week for 13 weeks. The 2,4-D was a commercial product of 98.5 percent purity (label stated). All dogs survived this study and no significant symptoms of toxicity were seen and no changes in body weight, organ weights, or blood count were noted. In a separate study, three of four dogs given daily doses of 20 mg/kg 2,4-D died between days 18 and 49. The signs observed in these animals differed somewhat from those seen in the acute studies. The chronically treated animals displayed stiffness of hindlegs and ataxia, weakness, difficulty in chewing and swallowing and occasionally bleeding from the gums. Weight loss occurred after 7-12% days and a terminal fall in lymphocyte count occurred prior to death. * The authors stated, "Death during the repeated administration of 2,4-D was not related to pathological changes in the liver, kidneys, or other organs examined." Seabury (123) treated three dogs experimentally infected with histoplasmosis, by intravenous injections of sodium 2,4-D at the rate of 1.17, 2.6, and 3.2 mg/kg per injection for 32-37 days without evidence of chronic toxicity. Bjorklund and Erne (15) treated young pigs at varying intervals up to 103 days with 50, 100 or 300 mg/kg of the commercial triethanolamine salt or butyl ester of 2,4-D. Exhibited symptoms of intoxication and pathology were analagous to those seen in laboratory animals. Clinical signs of anorexia and retarded growth were found in one animal given 51 doses of 50 mg/kg triethanolamine salt over 103 days. Pigs fed 500 mg/kg of diet triethanolamine salt of 2,4-D for up to 12 months developed locomotor disturbances of increasing severity after about one month. Animals sacrificed after 2-12 months had normal organ weights and no gross pathological changes. Clinical chemistry observations included lowered hemoglobin and hematocrit values, elevation of glutamic-oxaloacetic transaminase and reduced albumin and albumin: globulin ratios in the treated animals [see IARC Monograph, Vol 15 ( 6 ] 6). Shavgulidze (125) observed transient hematological changes in sheep receiving daily doses of 18 mg/kg 2,4-D sodium salt for 120 days. Mitchell et al (90) fed a cow 5.5 g of 2,4-D acid daily for 106 days with no apparent harmful effects on the health or milking performance. Post mortem examinations revealed no pathological changes in the liver, kidneys or body fat. By biological assay the presence of 2,4-D was demonstrated in the blood serum; however, 2,4-D was not found to be secreted in the milk nor was it found in the blood serum of a calf fed milk from this cow. Palmer (99) found that yearling steers needed to be given 15 daily doses of 250 mg/kg of the alkanolamine salt of 2,4-D before signs of toxicity occurred. He found that 112 daily doses of 50 mg/kg of this 2,4-D salt had no deleterious effect on the steers. IV-11 �D stated: Rowe and Hymas (115) in reviewing the chronic toxicity of 2,4The results of repeated oral administrations indicate that 2,4-D can be tolerated without adverse effects in doses only slightly smaller than those which cause toxic effects when given only once. This fact demonstrates that 2,4-D has a low degree of chronic toxicity. The same general observations of toxicity were noted in animals receiving chronic toxic doses of 2,4-D, as were seen in animals given single toxic doses. These were loss of appetite, loss of weight, depression, roughness of coat, general tenseness, and muscular weakness particularly of the posterior quarters. Post mortem findings usually included irritation of the stomach and gastrointestinal tract of small animals and abomasum of ruminants with only minor evidence of gross and histopathological injury in the liver and kidneys. Study of the data presented in Table 2 indicated that mice tolerate subcutaneous injections of 2,4-D at 50-70 mg/ka with no effect, while 70-90 rug/kg retards growth. Rats tolerated 1,000-1,250 mg/kg 2,4-D in their diet and 75 mg/kg orally without toxic effects. At levels of 1,000 mg/kg 2,4-D in the water and 1,500 mg/kg in the diet and 100 mg/kg orally, toxic signs were noted. Rabbits showed no gross differences between test and control animals, as far as skin irritation, when 3.13 percent solutions of various formulations of 2,4-D were placed on their intact or abraded skin. Dogs tolerated 500 mg/kg diet or 10 mg/kg orally with no toxic signs, while 20 mg/kg caused death in three of four animals. Oral doses of 300 to 500 mg 2,4-D/kg of diet were toxic to pigs. Rowe and Hymas (115) stated: Cattle demonstrate a similar susceptibility to 2,4-D as do the small laboratory animals. Cattle are distinctly more tolerant of 2,4-D than are dogs. Cattle can probably tolerate 30-50 mg/kg/day [(99)] for long periods without adverse effects. Daily doses of 100-250 mg/kg (99) would have to be continued for a week or longer to cause ill effects in cattle. A single dose of 500-1,000 mg/kg is not likely to cause problems; however, if repeated, serious effects and deaths are likely to occur. The chronic toxicity of 2,4-D did not differ greatly from the acute toxicity. At only slightly lower doses the same general signs, symptoms, and pathology were seen. Hansen et al (58) made the following statement on chronic exposure of humans to 2,4-D residues. (The cited values were for 1971. They have now been lowered slightly; however, in this case they were used to establish a worst-case situation.) IV-12 �TABLE 2 . Summary of literature data on the subacute and .chronic toxicity of 2,4-D in animals Route of Administration Effect Dose NSa 1-2 daily s.c. injections for 3 weeks to 90 days No effect 50-70 mg/kgb 18 1-2 daily s.c. injections Retarded growth 70-90 mg/kgb 18 NS Mouse No. Used NS Animal 22 daily s.c. injections No effect 0.1 mg/injc 57 NS 30 days in diet No severe effect 1000 mg/kg dietb 63 6Fd 5 doses/wk for 4 wks by intubation No effect 30 mg/kge 115 5 doses/wk for 4 wks by intubation Liver, G.I. growth effect 100 mg/kge 115 5 doses/wk for 4 wks by intubation Fatal in days 300 mg/kge 115 5 F 113 days in diet No effect 300 mg/kg diet6 115 5 F 113 days in diet Liver and growth effects, deaths 1000 mg/kg diet 115 Slight effect Total 2-5 g/kgc No effect 75 mg/kg' Rat 6 F 6 F 44 Mf 5 F 4 or 7 wks in diet 10 daily doses via stomach tube Referent t- 22 122 �Table 2 contumed 10 daily doses via stomach tube 2 died, overt toxicity m 3 100 mg/kgc 25 F/25 .M In diet for 2 yrs No effect 1250 ing/kg d i e t 58 W F/20 M In diet for 3 generation reproduction study in adults No effect 500 rag/kg diet 0 58 No effect on fertility or litter size. Lower no. pups weaned, lowered weight 1500 mg/kg diet 0 58 growth inhibition, poor health, diarrhea 1000 rag/ 1 water 15 No effect at gross exam. Some histopath effects in 2,4-D/oil treated animals. 3.13% solution9 72 5 f 20 F/2D M NS Rabbit 22 F/22 M In diet for 3 generation reproduction study in adults In drinking water for 2 yrs. 5 times/wk for 3 wks to intact and abraded skin Dog 122 3 F/3 M In diet for 2 yrs No effect 500 nig/kg diet c 58 3 M Oral dose via capsule 5 days/wk for 13 wks No effect on gross 10 mg/kg 33 Death in 3 at 18-49 days. Severe signs in 1 animal surviving 20 rag/kgc 33 1 F/3 M Oral dose via capsule 5 days/wk for 13 wks �Table 2 continued 1.1 mq/kg° 2.6 mg/kgj 3.2 mg/kg° 123 123 123 Toxicity, anorexia, retarded growth 300 mg/kgh 15 Locomotor problems, normal organ weights, no gross pathology 500 mg/kg1 15 Transient hematological and biochemical changes 18 mg/kg 3F Daily I.V. doses for 32 days at two higher levels, 37 days at lower level No effect NS Oral doses up to 103 days Pig NS In diet up to 12 months Sheep NS Daily oral doses for 120 days Cattle 125 1 Daily oral dose for 106 days No effect 5.5 gc 90 NS, S 15 daily oral doses Toxicity 250 mg/kg1 99 NS, S 112 daily oral doses No effect 50 mg/kg1 99 I en NS - number of animals in study not stated or unavailable from literature source Sodium salt of 2,4-D C 2,4-D acid F - Female e Butyl ester calculated as 2,4-D f M - Male ^Sodium salt, isooctyl ester and butyl ester of 2,4-D each applied separately on individual animals under the conditions described and concentration listed. Amine salt and butyl ester of 2,4-D used separately in animals at dose indicated. Vmine salt , Female lactating b S - Steer �Adequate data are not available to enable one to state conclusively what the total level of 2,4-D residues may be in foods ingested by the human population. An estimate of the greatest amount that might possibly be invested cart be made by use of the legal tolerances established by the FDA for 2*4-0 in various crops. They are 5 mgVkg pri 4 fruit Crops (apples^ citrus fruits* pe^ars and qUirices} aHd 0;5 mg/ky 6h 4 fcjraih crops (barley'} bats, fy'e ana wheat); if it is assumed that all the crop fbr Which a tdie^aHce exists always carried the" maximum amount of 2j4-D permitted; it can bis calculated that approximately Ch3 mg/kg of 2*4-0 Would be cbntribut^d to the total diet (fruit crops = 6 pertertt of the dietary intake of man ahd grain drops = 9 percent); tohe'h the maximum estimated human exposure to 2,4-D via the diet is cbtiipared to the dbsages given rats in the pr~e'sent study, it is apparent that there is an extremely wide margin of safety between 0.3 mg/kg of diet in man and the Ij250 mg/kg of diet fed to rats; C; Absorption, Distribution and Excretion of 2*4-0 Different degrees of sdditim 2,4-D poisoning were produced by Elb artd Yiitalo (35) in adult male Sprague-Dawley rats when 250 mg/kg • 2,4-0 was administered &y subcutaneous injection-. After varioU's intervals the cOncen'tratibn of intravenous 14fe-2,4-D Was cbm'pafed to the level fburtd iti the e^referbspihai fluid (CSF) and brairti At 4.5 hours 'when the sodium 2 i4-D, radioactivity in plasilia had diminished to 67 percent of control levels* ah 11-fold increase in the brain and a 39-fold increase i'n the CSF were s^fert compared to a 4.5 fold increase in the liver. All physiological and t'oxied logical parameters of this 1977 study had not been fully analyzed; however, during acute 2,4-D poisoning, the levels found in the brain were greatly increased. This increase appeared to be closely associated with the toxic symptoms. In ratsi pigs and cal'vies, 2^4-D administered in doses of 50-100 mg/kg brail y as salts ty&?$ readiiy absorbed arid eHifriMt&di mainly in the ail uHriev Wth.JpjAsflft hajf-li'ves varying from 3-12 hMrs .(^-,3^). The rate of 2-i4-D elimfnatib'n ih rats was dosage dependent-. Fbl Vowing administration of 14C-2-,4-D-, Khartna ahd Faftg (73) fouWd radioactivity in all organs and tissues examined [see IARC monograph i Vol 15 (66)]. Berridt arid Koschier (9) using J G - labeled 2-,4-D in rat and rabbit renal ddrtical tissue sliees-, JLH vijtio, noted that 2,4-D was transporlfecl. By the ciassitil retii.! organic aMon transport process j howeVer-, 'otfter 'mechanisms of tra;ns'p'ort may also have b'een involved. This study tnay h^Vp 'ek'pVafn brYe of tfe frtech'an'isms contributing to the relatively ! ;f%^j el dls'app'e'aran'c^ '6f 2\4-D 'frdm iftbst species and th'e loW levels of bi'oVO'gical deposition of this compbund. IV-16 �The esters of 2,4-D were hydrolyzed in animals and the phenoxy acids were excreted predominantly as such in the urine of rats after oral administration, although a minor portion of them may have been conjugated with the amino acids glycine and taurine and with glucuronic acid (54). No 2,4-dichlorophenol was detected, however, in the urine of C57BL/6 mice treated subcutaneously with 2,4-D or its butyl or isooctyl esters. The rates of disappearance from plasma of 2,4-D and its butyl and isooctyl esters following single subcutaneous injections of 100 mg/kg of the compounds to female C57BL/6 mice were: butyl ester > isooctyl ester > 2,4-D (147), [see IARC monograph, Vol 15 (66)]. After oral administration of 0.05 mg/kg 2,4-D to rats, Fedorova and Bel ova (42) found that traces were detected in the milk of lactating animals for six days. Within 24 hours after administration of 2,4-D to pregnant rats, 16.8 percent of the dose was detected in the uterus, placenta, fetus and amniotic fluids, [see IARC monograph, Vol 15 (66)]. Bjorklund and Erne (15) found that 2,4-D passed the placental barrier in pigs. Clark et al (23) fed 2,4-D acid (99 percent purity) to groups of 3, adult beef cattle and adult sheep at levels of 0, 300, 1,000 and 2,000 mg/kg of feed for 28 days. Animals were killed and tissues sampled one day after the last dose, others one week later. Residues of the 2,4D and its phenol metabolites were determined in muscle, fat, liver and kidney. Muscle and fat contained the lowest levels while kidneys and liver contained the highest residue level. Withdrawal from treatment for one week before killing resulted in a significant reduction in tissue residue levels. With the exception of the kidneys, 2,4-D residues averages less than 1 mg/kg in the tissue analyzed. The kidney tissue level averaged 7.82 mg/kg with 0.37 mg/kg present after a 7-day withdrawal period. No 2,4-D was detected in fat or muscle of any animals at a detection limit of 0.05 mg/kg. All treated animals showed some anorexia, weight loss or poor weight gain depending on the level 2,4-D present in the feed, due to lowered palatability. During the 7-day withdrawal period, feed consumption in all groups returned to normal. 2,4-D was rapidly eliminated from animals, mainly in the urine with plasma half-lives of 3-12 hours following a single dose. Generally, it accumulated in animal tissues when given at high doses or repeated lower doses. However, these residues declined rapidly with a half-life of 1 to 2 weeks. Because of its excretion by the kidney, kidney tissue levels were as much as twenty times greater than the level seen in other organs and tissues. D. Embryotoxic, Fetotoxic and Teratogenic Potentials of 2,4-D The embryotoxic, fetotoxic and teratogenic potentials of 2,4-D appeared to be extremely variable with observable effects dependent upon concentration, degree of purity and method of administration with some effects only occurring with doses that approached maternal toxicity. IV-17 �Bionetics Research Laboratories (12, 13, 14) reported that either the acid* or the isopropyl, butyl and isooctyl esters of 2,4-D, administered orally or subcutaneously at days 6-14 of gestation, increased the incidence of anomalous fetuses among BL6, AKR and C3H strains of mice but not among B6AK arid AIHa strains. No single strain showed a positive response to all formulations. No single formulation caused a positive response among all strains of mice. Thus, the reported effects were highly strain-specific, in addition, the Bionetics study involved parenterai administration using dimethyl sulphoxlde (DMSO) as a vehicle, which complicated the interpretation of the data* since DMSO has been shown to be a teratogen in several species of laboratory animals when administered by the route used in the Bionetics study [see Schwetz et al (122)]. Schiller (118) found no difference in fertility (defined as the number of rats weaned per female mated) of test and control animals in one experiment where rats were fed potatoes which had been treated with 2,4-D. In a combined second and third experiment, fertility of the P, Fp Fo and Fo generations was 7.2, 5.8, 6.8 and 6.1 for controls versus 7.2* 7.1, 5.4 and 5.1, respectively, for test rats. The differences between control and test groups were not significant (P>0.05). The content of 2,4-0, its form, or purity in the potatoes was not given. When 1,000 mg/1 2,4-D was given throughout pregnancy to S'pragueDawley rats via the drinking water, the gestation and parturition were normal. The litter size was not significantly reduced and no anomalies were seen in the pups (15). Hansen et al (58) stated that in unpublished work performed by T.B. Gaines and R.D, Kimbrough, female rats were fed 2,4-D acid at 0, 1,000, and 2,000 mg/kg of diet for 95 days, mated with untreated males and continued on their respective diets throughout gestation and lactation, At the highest dosage level, females gave birth to pups that were small at birth and 94 percent died before weaning. Some deaths also occurred in pups of females fed the lower level. Starting with rats three weeks of age, groups of 25 female and 25 male animals were fed for two years either 0^ 5* 25, 125, 625 or 1,250 mg 2,4-D/kg of diet. No significant effects on growth rate, survival rate, organ weights or hematologic values were noted (58). Hansen et al (58) also noted in a three generation, six litter rat reproduction study, no deleterious effect of dietary 2,4-D acid at 100 or 500 mg/kg was evident. At 1,500 mg/kgi, however, 2,4-D, while apparently affecting neither fertility of either six nor litter size, sharply reduced the percent of pups born surviving to weaning and the weights of weanlings. I* studies by Schwetz et al (122), the acid of 2,4^0, the propylene glycol butyl ether ester of 2,4-D and the isooctyl ester of 2,4-D were evaluated for effects on fetal development, neonatal growth IV-18 �and survival when administered at 12.5, 25, 50, 75 and 87.5 rag/kg orally to pregnant Sprague-Dawley (Spartan strain) rats during organogenesis (days 6-15 of gestation). Fetuses were delivered by Caesarean section on day 20 of gestation and were examined grossly, measured and weighed. Fetotoxic responses seen at high dose levels 50, 75 and 87.5 mg/kg included subcutaneous edema, delayed ossification and wavy ribs. Teratogenic responses were not seen at any dose level. 2,4-D did not affect fertility, gestation, lactation or viability of the newborn. The esters of 2,4-D decreased viability of the newborn and lowered lactation indices (Lactation Index: pups weaned/pups alive on day 4 X 100). In a second part of the experiment in which litters delivered naturally, 2,4-D and its esters had little or no effect on fertility, gestation, viability or lactation indices. There were no observable effects on neonatal growth and development. Khera and McKinley (74) observed minimal 2,4-D induced fetopathy and an increased incidence of skeletal anomalies in rat pups following single daily oral doses of 100-150 mg/kg 2,4-D from days 6 to 15 of gestation. The observed skeletal defects did not appear to be incompatible with postnatal survival. Following treatment of dams with the acid of 2,4-D and the butyl and isooctyl esters of 2,4-D, weight gain and viability of the offspring were within control limits. The findings of their study suggested that postnatal parameters were unrelated to the teratologic potential of the chemicals. No consistent embryotoxic effects were noted when 2,4-D acid was administered orally to hamsters at doses of up to 100 mg/kg on days 6-10 of gestation (24). Binns and Johnson (10) showed that 2,4-D did not have a teratogenic potential in sheep. Starting one day after breeding ewes were given 2 g/day of 2,4-D acid in an alfalfa meal/water mixture via stomach tube for 30, 60, or 90 days. No clinical signs of toxicity nor histopathologic lesions were seen in the ewes and no congenital anomalies nor histopathologic lesions were seen in the lambs. Ewes were reported to have had increased rates of stillbirths and bucks displayed reduced sexual activity and decreased sperm quality when pastures were grazed soon after treatment with 3 Ib/A of the 2,4- • dichlorophenoxybutric acid (2,4-DB) (116). When a diet containing 500 mg/kg 2,4-D was fed to a sow during the entire pregnancy, the sow was anorexic and the newborn piglets were underdeveloped and apathetic with 10/15 dying within 24 hours. When the survivors were continually fed 2,4-D at 500 mg/kg of diet until they were 8 months of age marked growth depression, persistent anemia and moderate degenerative changes of the liver and kidneys were noted (15). Erne (40) fed pregnant reindeer birch leaves that had been sprayed with a mixture of 2,4-D and 2,4,5-T at a daily dose of 1 mg IV-19 �phenoxy herbicide per kg body weight. There were no clinical or histopathological changes noted in any of the female reindeer and no fetal anomalies were seen. From the data presented in Table 3 the no effect level for embryotoxic, fetotoxic and teratogenic signs in the rat was approximately 1,000 mg/1 of the sodium salt of 2,4-D, while the no effect level from 2,4-D acid in the rat diet ranges from 1,250 to 1,500 mg/kg of food. Oral doses of 2,4-D acid and the butyl and isooctyl esters cause no effect at daily doses of 87.5 mg/kg. At 100 to 150 mg/kg 2,4-D acid and esters produced embryo and fetotoxic responses in rats and hamsters. In pigs 500 mg 2,4-D acid/kg diet caused the sow to be anorexic and produced weakened piglets with 10 to 15 dying within one day after birth. When the five surviving piglets were fed the same diet for eight months they showed a marked depression in growht. Sheep have tolerated oral doses of 2,4-D acid for 30-90 days at 2 grams per day levels, while reindeer experienced no adverse effects from daily oral doses of 1 mg/kg for 30-54 days. E. Carcinogenic and Tumorigenic Potentials of 2,4-D Studies of the carcinogenic properties of 2,4-D in mammalian biological systems are limited at best. However, in an extensive study by Innes et al (67), the tumorigencity of some 130 test compounds were tested in mice. Included in the test compounds were the 2,4-D acid and the isopropyl, butyl and isooctyl esters of 2,4-D. They were given orally, at a daily dosage rate of 46.4 mg/kg. An additional test using the dosage rate of 100 mg/kg for 2,4-D acid was included. These doses were given by stomach tube starting at 7 days of age and continued until the mice were 4 weeks of age. After weaning, the test compounds were mixed directly into the diet and the same dosage rate maintained for approximately^ 18 months of observation. The tumor incidence in any group or combination of groups in which 2,4-D was tested was not significantly different from that in control animals. Groups of male and female mice were given single subcutaneous injections of 215 mg/kg 2,4-D in dimethyl sulphoxide (DMSO) on the 28th day of life and observed up to 78 weeks of age. Tumor incidences in any group or combination of groups were not significantly different from that in controls. No increase in the incidence of tumors was observed in similar groups of mice treated with single subcutaneous injections of 21.5 mg/kg butyl or 100 mg/kg isopropyl esters of 2,4-D, both 99 percent pure. Mice treated with 21.5 mg/kg isooctyl ester of 2,4-D, 97 percent pure, had 5/17 females of one strain developing reticulum-cell sarcomas (12). Walker et al (141) demonstrated that six, daily, intraperitoneal, injections of highly purified 2,4-D (99.0 percent) at the rate of 62 mg/kg effectively inhibited development of the Ehrlich ascites tumor maintained in BALB/c mice. IV-20 �TABLE 3. Animal Rat Number Used . PFa NSb Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4-D in amimals Route of Administration Response Dose fteferen< 1000 mg/1 water0 15 Diet for 95 days then mated and Small birth wt. 94% died continued through gestation before weaning. and lactation. 2000 mg/kg dietd 58 Some reduction in birth wt. Some deaths in pups. PF NS Drinking water during pregnancy. No effect 1000 mg/kg dietd 58 25 Fe ro In diet for 2 yrs. No effect 1250 mg/kg dietd 58 25 Mf In diet for 2 yrs. No effect 1250 mg/kg dietd 58 PF NS In diet 3 generations. Six litter reproduction study. No effect 500 mg/kg diet No effect on fertility of either sex nor litter size. Reduced % of pups born and surviving to weaning - lowered weaning weights. 1500 mg/kg diet No effect on fertility, gestation, lactation or viability of newborn. 87.5 mg/kg9 122 Fetotoxic as edema, delayed ossification, wavy ribs. No teratogenicity. 87.5 mg/kg- 122 19 PF 119 Fetuses Daily oral dose - days 6-15 of gestation. Daily oral dose to females days 6-15 of gestation. H 58 58 �Table 3 continued PF NS Daily oral dose - days 6-15 of gestation Minimal fetopathy, increased , skeletal anoma'Mes 150 mg/kg PF NS Daily oral dose - days 6-15 of gestation No consistent embryotoxic effects 100 mg/kg1 24 Via stomach tube 30, 60 or 90 days No effect 2 g/dayl 10 In diet throughout pregnancy, Female anorexic. 10 of 15 piglets died in 24 hrs. 500 mg/kg diet 15 Growth depression, anemia, moderate liver and kidney lesions. 500 mg/kg diet0 15 No effect 1 mg/kg 40 74 Hamsters Sheep PF Pig 1 PF 5 Newborn ro ro In diet for 8 months Reindeer 15 PF In diet for 1 - 1 . 5 months PF - pregnant female NS - number of animals in study not stated or unavailable from literature source c Sodium salt of 2,4-D d 2,4-D acid p F - Female f M - Male 9 2,4-D acid or molar equivalents of propylene glycol butyl ether ester of 2,4-D or the isooctyl ester of 2,4-D 2,3-D acid or butyl or isooctyl esters of 2,4-D �Hansen et al (58) studied groups of 25 male and 25 female Osborne-Mendel rats that were fed for two years on diets containing 2,4D at 0, 5, 25, 125, 625 or 1,250 mg/kg levels. The 2,4-D was 96.7 percent pure and contained no detectable levels of 2,7-dichloro or 2,3,7,8tetrachlorodibenzo-p-dioxin (limit of sensitivity of method of analysis was 1 mg/kg). No target organ tumors were observed and the individual tumor types were randomly and widely distributed and of the type normally found in aging rats of that strain. Statistical analysis of the randomly distributed tumor types indicated a tendency for the proportion of females with tumors to increase with 2,4-D dosage and a trend toward dose related increases in the proportion of males with malignant tumors. The number of treated rats with malignant tumors over controls was found only in males receiving the highest dosage level. A review of the summary of the literature on the carcinogenic and tumorigenic potentials of 2,4-D in animals presented in Table 4, revealed that 2,4-D acid, and the isopropyl, butyl and isooctyl esters of 2,4-D did not adversely affect nor increase the incidence of tumors in test animals when fed at levels of 46.4 to 100 mg/kg of diet to mice or 1,250 mg/kg of diet to rats for 18 to 24 months. Those tumors that did occur were not necessarily in target organs and were the type tumors normally seen in aging laboratory animals of the species and strain being studied. Single subcutaneous injections of 21.5 to 215 mg/kg of 2,4-D acid, isopropyl and butyl esters of 2,4-D in DMSO did not produce carcinogenic or tumorigenic responses in male or female mice, A single subcutaneous injection of 21.5 mg/kg of the isooctyl ester of 2,4-D in DMSO did produce an increased incidence of reticulurn-cell sarcomas in treated female mice. It should be noted that DMSO itself is now considered to be a potential carcinogen. At 62 mg/kg, 2,4-D acid injected intraperitoneally in mice inhibited the development of Ehrlich ascites tumor being maintained in mice. F. Mutagenic and Cytogenetic Potentials of 2,4-D in Animals Most of the mutagenic studies of 2,4-D have been conducted in bacterial cultures or in plant and animal tissue cultures; however, Styles (133) investigated the cytotoxic effects of 2,4-D on .01 u-cvo and Jin v-cfio test systems and found no increase in mutation rate and no evidence of mutagenicity in the test rats. He found serum from orally dosed rats was not mutagenic to Sa&none&ta. typkunusuium. Complete details of this study were not readily available. Pilinskaya (101) observed that treatment of cultured human lymphocytes with 2.5 X 10~7 M (0.02 yg/ml) 2,4-D increased the number of chromatid aberrations (single acentric fragments) and, to a lesser extent, the chromosomal aberrations (paired acentric fragments). In mice, Pilinskaya (101) found toxic concentrations (100-300 mg/kg) of 2,4-D administered as a single oral dose significantly increased the frequency of aberrant metaphases (2-4 fold) with single fragments being the aberration seen. IV-23 �TABLE 4 Animal Number Used Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4-D in animals Route of Administration M - F NSe Single subcutaneous injections in DMSO No effect 46.4 mg/kg dietc 67 100 mg/kg diet 67 No effect 215 mg/kgd 12 No effect h 18 Ma/18 FD of Stomach tube, beginning at 7 two hybrid days of age for 21 days, then strains in diet for 18 months Dose No effect Mouse Response 21.5 mg/kgf 12 Reference 9 No effect f\ f J_ . T . T 6 daily . intraperitoneal injections Rats 25 M/25 F Diet for 2 years 12 5/17 females developed reticul urn-cell sarcomas ro 100 mg/kg 21.5 mg/kgh 12 C O m» / I «^ Erlich ascites tumor No target organ tumors, random type tumors normally seen in aging rats M - Male F - Female C 2,4-D acid and isopropyl, butyl and isooctyl esters of 2,4-D 2,4-D acid e NS - number of animals in study not stated or unavailable from literature source 1250 mg/kg diet Butyl ester of 2,4-D ^Isopropyl ester of 2,4-D h lsooctyl ester of 2,4-D 58 �Jenssen and Renberg (68) found there was no detectable increase of micronuclei in the erythrocytes of mouse bone marrow after intraperitoneal administration of 100 mg/kg 2,4-D. Because of the high experimental resolution power of the test system used in this study it was particularly suitable for the detection of weak chromosome breaking activity of 2,4-D in mammals. The lack of penetration of 2,4-D into the cells was in accordance with the rapid excretion that is known to occur in the mammalian body. This experiment did 'not in the authors opinion, consititute a reliable measure of the mutagenic potential of 2,4-D; however, in practice the lack of penetration of this substance into the cells indicated it does not constitute a cytogenetic hazard to man. Epstein et al (37) found that 2,4-D did not increase dominant lethal mutations in mice when given as a single intraperitoneal injection of 125 mg/kg or when given orally on five successive days for a total dose of 75 mg. In host-mediated assays Zetterberg et al (146) using strains TA1530 and TA1531, or Sac.c.kaAomyc&> D4, found no mutagenic effects in the organisms when host adult male mice were given 6 mg 2,4-D (200 mg/kg) by gavage. Bongso and Basrur (17) exposed embryonic bovine kidney cells and bovine peripheral blood cells, /en v-ttao, to concentrations of 1-1,000 iag/ml 2,4-D for 6-96 hours resulting in stimulation of mitosis. ChromosoMid' aberrations were not detected in the peripheral blood cells, but nucleolar irregularities and polyploid mitotic stages were observed in the kidney cells. Andersen et al (4) evaluated 110 herbicides for their ability to induce point mutation in one or more of 4 different microbial systems. The herbicide 2,4-D was included in this study. The authors did not state the purity of the compounds being tested. The 2,4-D did not cause point mutations in these microbial systems in comparison with known mutagens such as 5-bromouracil or 2-aminopurine. These observations of no mutagenicity of 2,4-D in Eiah<yu.dUa c.oti WP2 her+ or her" or in S<t£mone££a typhimuruwn strains TA1535, TA1536, TA1537 or TA1538 were also confirmed in works by Nagy et al (94), Shirasu (126) and Shirasu et al (127). A review of the literature on the mutagenic and cytoger.ic potentials of 2,4-D in animals generally supported the premise that 2,4-D was not highly cytotoxic in laboratory animals. It did not increase mutation rates nor stimulate a mutagenic response in rats and mice. In various Ln vJutiio and Jin vuvo test systems 2,4-D did cause chromatid aberrations and nucleolar irregularities in cultured human lymphocytes, bovine kidney cells and tissues of mice given single toxic doses. No mutagenic responses were seen in several studies using microbial systems for the detection of mutagenic and cytogenic responses to 2,4-D. IV-25 �III. REVIEW OF 2,4,5-T TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of 2,4,5-T Detailed accounts of the experimental procedures used to study the acute and short-term toxicity potentials of 2,4,5-T were not available, References to acute toxicity of 2,4,5-T in small laboratory animals referred to a summary article on toxicological information on 2,4-D and 2,4,5-T by Rowe and Hymas in 1954 (115). Their literature review made reference to the 1953 work of Drill and Hiratzka (33) where acute and chronic oral toxicity studies on 2,4-D and 2,4,5-T were conducted with dogs. Apparently, the earlier research with small laboratory animals dealt primarily with 2,4-D, although some 2,4,5-T studies conducted in 1950 discussed effects on horses, dairy and beef cattle, sheep, swine and chickens immediately pastured on freshly treated alfalfa. Unfortunately, Rowe and Hymas did not detail the methodology for obtaining all the data that appeared in their article. Table 5 presents the available data from the literature dealing with the acute toxicity of 2,4,5-T in laboratory animals. Drill and Hiratzka (33) administered commercial 2,4,5-T of 98.9 percent purity (TCDD level not stated) in a single oral dose of 50, 100, 250 and 400 mg/kg to a total of 10 adult mongrel dogs of both sexes, The 400 and 250 mg/kg dosages were given to individual male dogs and both died in 2 and 3 days, respectively. One of four female animals died at the 100 mg/kg dose level; however, no other signs of toxicity were noted. All three males and one female in the 50 mg/kg dosage level survived with no signs of toxicity. The acute oral LDso for 2,4,5-T acid was in the range of 100 mg/kg or higher for dogs. Toxic doses of this level produced only mild signs of muscle spasticity. Bjbrklund and Erne (15) fed single oral doses of 100 mg/kg 2,4,5-T to pigs causing anorexia, vomiting, diarrhea and ataxia. At autopsy, hemorrhagic enteritis and congestion of the liver and kidney were found [see IARC Monograph, Vol 15 (66)]. Study of the data in Table 5 indicated that the various forms of 2,4,5-T all fall in the same range of acute toxicity for mice, rats, guinea pigs and rabbits. The dog appeared to be somewhat more susceptible, The LDso values for 2,4,5-T and its common derivatives were in the range of 380 to 940 mg/kg for the small laboratory animals. When given orally to dogs, even in fatal cases, 2,4,5-T produced only weak signs in the form of ataxia and stiff movements of the hindlegs. B. The Subacute and Chronic Toxicity Potentials of 2,4,5-T Highman et al (62) conducted a study using 978 mice including control animals. On days corresponding to days 6 through 14 of pregnancy, groups of pregnant and nonpregnant CD-I mice and male and nonpregnant IV-26 �TABLE 5 . Animal Mouse Number Used Summary of literature data on the no-effect LD5Q and LD1QO levels of the acute toxicity of 2,4,5-T in animals Route of Administration Dos e-Toxi city Single Dose nig/ kg Reference NSa Mb Oral in olive oil LD50 389C 1 15 NS Fd Oral in olive oil LD50 e 551 1 15 NS F Oral in corn oil LD50 940f 1 15 NS M Oral in olive oil LD50 500C 1 15 NS M & F Oral in ol i ve oil LD 50 495e 115 NS F Oral in corn oil LD50 481f 1 15 NS F Oral in ol i ve oil LD50 7509 1 15 NS M & F Oral in olive oil LD50 381c 1 15 NS F Oral in olive oil LD50 449e 1 15 NS F Oral in corn oil LD en 750f 1 15 NS M Oral in corn oil LD 712' 115 Rat ro Guinea Pig Rabbit 50 �Table 5 continued 1 M Oral in capsule LD 2501 33 4 F Oral in capsule LD 100C 33 1 Dog Oral in capsule F 3 M 100 50h No effect 33 Pig NS Oral Anorexia, vomiting, diarrhea, ataxia, hemorrhagic enteritis, liver and kidney congestion. 100 NS - number of animals in study not stated or unavailable from literature source. b M - Male C 2,4,5-T acid ro oo F - Female e lsopropyl ester of 2,4,5-T f Mixed butyl esters of 2,4,5-T 9 Mixed amyl esters of 2,4,5-T One of four animals died on 7th day 15 �female dihybrid cross Fg mice received, by gavage, 2,4,5-T acid doses ranging from 30 to 140 mg/kg. Some groups received a technical preparation of 2,4,5-T (97.9 percent pure, containing < 0.05 mg/kg TCDD or a purified preparation of 2,4,5-T (99 percent pure, containing < 0.05 mg/kg TCDD). Mice killed when they became moribund and at 1, 2, 4, 6, 8 and 11 days after beginning treatment. Sick or moribund mice sacrificed after 2-9 doses of 2,4,5-T often showed severe myocardial lesions, hypocellularity of the bone marrow and depletion of lymphocytes in the thymus, spleen, or lymph nodes. They also showed marked hematologic and blood chemistry changes. Treated mice remaining healthy showed few or no lesions and no blood chemistry changes, but often developed a mild anemia attributable to a hemolytic effect of 2,4,5-T. The incidence of animals becoming moribund was less than 1 percent in the CD-I mice, including those given 140 mg/kg, and ranged from 53 to 82 percent in groups of male and female F2 mice receiving 120 mg/kg 2,4,5-T. The incidence of moribund mice tended to be higher in male than in female F2 mice and in those given the purified compound. The findings of this study indicated that impairment of maternal health by severe lesions early in gestation were not the primary cause of an increased incidence of fetal abnormalities observed in mice given 2,4,5-T. The lesions appeared to be due primarily to 2,4,5-T, rather than to contaminants in the technical preparation. Finally, the importance of using more than one strain of mouse in toxicological studies was vividly illustrated. Highman et al (60) using 378 pregnant dihybrid cross F£ female mice gave either 60 or 120 mg/kg 2,4,5-T via gavage on days 6 through 14 of gestation. Both technical (97.9 percent pure with less than 0.005 mg/kg TCDD) and a more purified preparation (99 percent pure with less than 0.005 mg/kg TCDD) of 2,4,5-T were used in this study. Mice were killed when they became moribund and at 6, 24, and 30 hours, as well as at 4, 6, 8 and 11 days after beginning treatment. Mice given 60 mg/kg and many given 120 mg/kg 2,4,5-T appeared normal at the time they were terminated either in early or late pregnancy and showed few or no pathologic changes. Mice that became ill or moribund often showed severe lesions and few survived past 11 days. The histopathological lesions included myocardial rarefaction and necrosis, thymus cortical atrophy, splenic atrophy and hypocellularity in bone marrow and lymph nodes. Groups of 10 male and 10 female rats per test dose were fed for 90 days on diets containing 2,4,5-T at the daily dosage levels of 0, 3, 10, 30 or 100 mg/kg body weight. The 2,4,5-T acid was from commercial production and contained less than 1 mg/kg TCDD. No effects were noted in the animals fed 3, 10 or 30 mg/kg doses. Changes found in both sexes fed 100 mg/kg included depression in body weight gain, slight decrease in food intake and elevated serum alkaline phosphatase levels. Male rats at this dose had slightly increased serum glutamic-pyruvic transaminase levels and slight decreases in red cell counts and hemoglobin. Inconsistent hepatocellular swelling was observed upon histopathological examination in some livers. [See World Health Organization (WHO) Monograph 71.42 (143), and IARC Monograph, Vol 15 (66)]. IV-29 �Groups of 10 male and 10 female rats per test dose were fed for 90 days on diets containing 0, 100, 300, 1,000 an0 3,000 mg/kg of food, of the mono-, di-, and tripropylene glycol butyl either esters of 2,4,5-T. The 2,4,5-T acid equivalent was 62 percent. No effect levels were 100 and 300 mg/kg of diet. At 1,000 mg/kg level slight cloudy swelling of the parenchyma! cells with central lobular necrosis was noted in two of ten animals examined. Kidney weights were increased wtfth mild cellular changes noted such as cloudy swelling of renal tubular epithelium. At 3000 mg/kg a significant retardation in growth was noted in males but not females, liver and kidney weight increased in males, livers were large and light in color in both sexes, generalized cloudy swelling of liver cells and slight central lobular necrosis and cloudy swelling of renal tubular epithelium [see WHO Monograph 71.42 (143) and IARC Monograph, Vol 15 (66)]. Konstantinova (81) conducted experiments with pregnant rats using 12-13 animals per treatment group and dosing them via stomach tube for the entire gestation period with 0.01, 0.1, 0.42 and 4.2 mg 2,4,5T/kg body weight. The butyl ester of 2,4,5-T was used in this study; however, source and purity were not stated in the translation. No effects were noted at the 0.01 mg/kg dose. The threshold level in this study was at the 0.1 mg/kg dose. At 0.42 mg/kg a general toxic effect on pregnant females was noted and embryotoxic effects were of an irregular character and difficult to evaluate. The 4.2 mg/kg produced a significant increase in total embryo fatalities, a decrease in the number of live offspring (average one less per female) and a decrease in the weight of the offspring. Rip and Cherry (111) fed a group of 12, four-week-old, male, Long-Evans rats, analytical standard grade 2,4,5-T (containing no detectable TCDD at a sensitivity of 0.05 mg/kg) mixed with the diet at a rate of 10 mg/animal/day for 1-11 days. Feeding of the 2,4,5-T caused liver enlargement with no effect on the weight of the kidneys, spleen, or body weight of the animal. Increases in relative liver weight were dose dependent and were observed after the first or second feeding. Enlargement was associated with substantial increases in total RNA and total protein per liver. The increases were not restricted to any particular subcellular fraction, but appeared to represent a general induction of RNA and protein synthesis. Total DNA content per liver was not affected. The enlargement response was reversible on the removal of the 2,4,5-T from the diet. This increase did not appear to be directed toward the synthesis of 2,4,5-T metabolizing enzymes. 2,4,5-T did not stimulate production of enzymes known to be produced by hepatotoxic compounds. This suggested that 2,4,5-T did not have a strong hepatotoxic activity and in fact it demonstrated activity similar to a structurally related compound chlorophenoxyisobutyrate (CPIB) which, like 2,4,5-T, induced liver enlargement and stimulated RNA and protein synthesis while inducing a strong self-metabolizing influence in the liver. IV-30 �Drill and Hiratzka (33) administered 2,4,5-T acid via capsule to adult mongrel dogs, five days a week over a 13 week period. There was 1 male and 1 female in the 0, 2 and 5 mg/kg groups, a male and 2 females in the 10 mg/kg group and 2 males and 2 females in the 20 mg/kg group. All dogs in the 0, 2, 5 and 10 mg/kg dosage levels survived'the 90-day test period. At 20 mg/kg the dogs died between days 11 and 75. The no effect level was 10 mg/kg per day. The histopathological examination of the 20 mg/kg dosed dogs was not remarkable and did not reveal a morphological cause of death. The prominent effects were weakness, slight stiffness in the hind legs, difficulty in swallowing food and in one dog, bleeding from the gums. Study of the data in Table 6 indicated that the various forms of 2,4,5-T fell in the same general range of chronic toxicity for mice, rats and sheep. The exception to this statement were data presented by Konstantinova (81) using the butyl ester of 2,4,5-T of unstated purity. In mice there appeared to be a definite strain difference in susceptibility to 2,4,5-T toxicity. The no effect level in mice ranged from 30-120 mg/kg with an overlapping of adverse effects from 60-140 mg/kg in various strains of mice. Rats appeared to be tolerant to about 10 times the 2,4,5-T calculated dose when administered as mg/kg of diet as opposed to mg/kg body weight of the animal. The no effect level for rats fed 2,4,5-T chronically were approximately 30 mg/kg body weight and 300 mg/kg diet. Threshold toxicity levels for rats were approximately 100 mg/kg body weight and 1,000 mg/kg diet. C. Absorption, Distribution and Exretion of 2,4,5-T Single subcutaneous administration of 100 mg/kg 2,4,5-T to mice resulted in 23 percent of the dose being recovered in the body over a 24hour period (147). In rats, 85.8 percent of a single intravenous dose of 100 mg/kg was found in the urine within 6 days (117). Single oral doses to rats of 100 mg/kg of the triethanolamine salt of 2,4,5-T were readily absorbed, distributed and eliminated; excretion was primarily via the kidneys (38). Seven days after oral administration of 50 mg/kg 2,4,5-T (99.6 percent pure) to rats, 56-69 percent of the dose was recovered in the urine; 70-85 percent of the recovered dose was unchanged 2,4,5-T, and approximately 15-30 percent was found as the glycine and taurine conjugates and as 2,4,5-trichlorophenol; the two conjugates were excreted in nearly equal amounts (16, 55). Similar results were obtained in mice, except that the quantity of the taurine conjugate was greater (54). The biological half-life of 5 mg/kg 2,4,5-T administered orally to dogs (77 h) was longer than that in rats (4.7 h). When the dose of 2,4,5-T to rats was increased to 200 mg/kg the biological half-life was prolonged to 25 h, indicating that the excretory capacity of the animals could be exceeded (104). IV-31 �TABLE 6. Animal Number Used Summary of literature data on the subacute and chronic toxicity of 2,4,5-T in animals Route of Administration Reference Dose Varied by strain 30-140 mg/kgd 62 <1% moribund 53-82% moribund 111 or moribund No effect 140 mg/kgd 120 mg/kgd 60 mg/kgd 90-120 mg/kgd 62 62 62 62 Varied 60-120 mg/kgd 60 Most all animals outwardly6 normal 60 mg/kgd 60 Many animals outwardly normal Mouse Effect 120 mg/kgd 60 Daily dose per animal in feed for 90 days No effect 30 mg/kg 66 Daily dose per animal in feed for 90 days Anorexia, depressed weight gain 100 mg/kgf 66 90 days in diet No effect 300 mg/kg9 66 . 978; F PFD Mc Gavage, dosed daily for 6-14 days, corn oil vehicle CD-I strain F£ dihydrid NCTR strain CRBL strain 378; PF Gavage, dosed daily for 6-14 days, corn oil vehicle CO no Q Rat 10 M/10 F 10 M/10 F 10 M/10 F �Table 6 continued 10 M/10 F 90 days in diet 10 M/10 F 90 days in diet Toxicity; no deaths due to treatment; histopath changes were noted 1000 nig/kg diet9 66 Growth retardation in males not females', no deaths due to treatment; histopath changes were noted 3000 mg/kg diet9 66 12 or 13/PF Stomach tube, daily dosing, entire gestation. No effect 0.01 mg/kg 8T 12 or 13/PF Stomach tube, daily dosing, entire gestation. Threshold level 0.1 mg/kg 81 12 or 13/PF Stomach tube, daily dosing, entire gestation. Irregular effect on dam and fetuses . 0.42 mg/kg 81 12 or 13/PF Stomach tube, daily dosing, entire gestation. One less live pup per female, ^ toxic signs noted. 4.2 mg/kg 81 I co co 12 M In diet to provide daily dose indicated Liver enlargement only 10 mg/kg Via capsule for 90 days Via capsule for 90 days Oral for 35 days No effect All died No effect 10 mg/kgv 20 mg/kgJ 100 mg/kg 1 111 Dog Sheep 1 M/l F 2 M/2 F NSk F - Female PF - Pregnant female C M - Male 33 33 29m �Table 6 continued d Both technical and purified 2,4,5-T acid containing <0.05 and 0.005 mg TCDD/kg. e Histopathological information presented in text. 2,4,5-T acid from commercial production containing <1 mg TCDD/kg. 9 Mono-, di-, and tripropylene glycol butyl ether esters of 2,4,5-T, 62% 2,4,5-T acid equivalent, h Butyl ester of 2,4,5-T, purity not stated. Analytical standard grade 2,4j5-T acid containing <0.05 mg TCDD/kg. •Commercial 2,4,5-T acid, 98.9% purity, TCDD level not stated. NS - number of animals in study not stated or unavailable from literature source. Form not known. < CO m From table in reference (29): �Similar results were obtained with single intravenous injections of 5 or 100 mg/kg 14C-2,4,5-T in rats, where 2.3 and 7.6 percent of the radioactivity were excreted in the feces, respectively, suggesting that at the higher dose biliary excretion of 2,4,5-T and/or Hs degradation products was involved in the overall elimination of 2,4,5-T from the body (117). Marked differences in the pharmacokinetics of 2,4,5-T were seen with different species, ages and doses: clearance of 2,4,5-T from the plasma and body of dogs, mice and man was slower than that in rats. The volume of distribution after a single oral dose of 5 mg/kg also differed: in man, 0.079; in rats, 0.14; and in dogs, 0.22 I/kg (49, 104). A single dose of 100 mg/kg 2,4,5-T to pregnant mice was almost entirely eliminated in 72 h; however, after 4 daily administrations of the same dose, 2,4,5-T accumulated in maternal tissues and fetuses, and by 48 h 2,4,5-T was still detectable throughout the fetuses (34). No radioactivity was found in NMRI strain mouse embryos in an early stage of gestation after administration of ^C-2,4,5-T to the dams. When given in late gestation, the fetal tissue had a level similar to that in maternal tissue (83). Selective uptake of 2,4,5-T into the yolk sac epithelium and absence of placental transfer in early pregnancy were effects similar to those seen with trypan blue in mouse embryos (84). No radioactivity was detected in hamster embryos in a late stage of gestation after similar administration of 2,4,5-T to the dams (31).* The biological half-life of l4C-2,4,5-T was significantly longer in newborn than in adult rats (41, 64). Radioactivity was found in all tissues examined as well as in milk and fetuses after a single oral administration of 0.17-41 mg/kg l4C-2,4,5-T to pregnant rats (41). Care must be taken when making all inclusive or generalized statements on the absorption, distribution and excretion of 2,4,5-T due to the demonstrated and marked differences in the pharmacokinetics of 2,4,5-T seen in laboratory animals. Such variables as age, dosage levels, routes of administration and chemical formulations all contributed to variations in response. Single doses of 2,4,5-T appeared to be rather quickly eliminated, primarily unchanged, via the urine and feces in a few hours up to about 7 days. High doses and repeated lower doses of 2,4-D or 2,4,5-T accumulated in animal tissues. The liver appeared to take a more active role in the metabolism and excretion of higher or chronic doses of 2,4,5-T than when single lower level doses were administered. IV-35 �D. Ernbryotoxic, Fetotoxic and Teratogenic Potentials of 2,4,5-T When reviewing the literature dealing with the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T, care must be taken to note the levels of TCDD contamination that may have been present in the 2,4,5-T tested. The TCDD contamination may very well have ranged from undetectable levels, using analytical technology available at the time, to 30 ppm or more. In an extensive 1977 review article of the teratogenic effects of environmental chemicals, Wilson (145) stated that 2,4,5-T had been intensively examined in pregnant animals of six different species. A low level of teratogenicity had been demonstrated in three rodent species: rats, mice and hamsters. Tests in pregnant rabbits, sheep and rhesus monkeys have been negative. Wilson discussed these studies in detail in the test, Handbook o& JnnatotoQg (144). Doses of more than 30 mg/kg 2,4,5-T (containing <0.1 mg/kg TCDD) increased the frequency of cleft palates in some strains of mice. When similar doses were administered to pregnant mice on days 6-15 of gestation some fetal growth retardation was observed (13). Courtney et al (26) first reported that under laboratory conditions 2,4,5-T was implicated as being teratogenic and fetotoxic. The 2,4,5-T used in the study was later found to contain 30 mg/kg TCDD. The 2,4,5-T was administered either orally or subcutaneously at a dose rate of 113 mg/kg per day on days 6-14 of gestation in C57B1/6 mice and days 6-15 in AKR mice. Oral administration caused an increased incidence of cleft palate and fetal mortality in both strains and cystic kidneys in the C57B1/6 mice. Subcutaneous injection resulted in significant increases in the incidence of cleft palate and cystic kidneys in the embryos of both strains of mice and evidence of fetal mortality in the C57B1/6 mice. Roll (112) found embryotoxic and teratogenic effects in NMRI mice exposed to 2,4,5-T, containing 0.05 ppm TCDD, administered orally at 20 to 130 mg/kg daily from 6 to 15 days of gestation. At 90 or 130 mg/kg/day the percentages of resorptions and/or dead fetuses were markedly increased relative to the controls. These levels also produced maternal toxic effects. Dose related reductions in fetal weight were observed at levels of 20 mg/kg/day and above. Cleft palate increased among fetuses exposed to 35 mg/kg/day or more. The teratogenic no effect level in mice for this particular sample of 2,4,5-T was considered to be 20 mg/kg/day. This was later confirmed with specially prepared samples of 2,4,5-T with no detectable (<0.02 mg/kg) amount of TCDD (112, 113). Neubert and Dillmann (96) found that samples of 2,4,5-T acid containing less than 0.02 mg/kg TCDD produced embryotoxic effects in NMRI mice in the form of fetal weight reductions at levels as low as 10 and 15 mg/kg per day, given orally from day 6 to 15 of gestation. The butyl ester of 2,4,5-T showed similar embryotoxic effects in mice when administered IV-36 �in the same manner. Cleft palates were produced using single doses of 2,4,5-T acid at 150-300 mg/kg. The maximum teratogenic effect was seen when mice were dosed on day 12 or 13 of gestation. A potentiation of the teratogenic effects (cleft palate) of 2,4,5-T and TCDD was obtained when teratogenic doses of one of the substances was combined with threshold doses of the other. However, for clear-cut potentiation of the effect of 30-60 mg/kg 2,4,5-T acid more than 1.5 mg/kg TCDD was required. When the level of TCDD drops below 1 mg/kg it was predicted that there would be no additional contribution to the embryotoxic effects of 2,4,5-T (i.e., cleft palate) in NMRI mice. It was concluded that in some of the 2,4,5-T preparations there must have been other contaminants present which exaggerated the teratogenic effect to some extent. Such contaminants may have been present in more than trace amounts. For example, 2,4,5trichlorophenol, did not contribute significantly to the teratogenic effect. The effect of 2,4,5-T and TCDD were studied in random bred CD-I and inbred DBA/2J and C57B1/6 strains of mice by Courtney and Moore (27). Two different samples of 2,4,5-T and one sample of TCDD were used. The 2,4,5-T technical grade contained 0.5 mg/kg TCDD and the analytical grade contained less than 0.05 mg/kg TCDD. Compounds were administered subcutaneously from day 6 to day 15 of pregnancy as solutions in 100 percent dimethyl sulfoxide (DMSO) in a volume of 100 yl/animal/injection. Both samples of 2,4,5-T and TCDD produced cleft palate in all three strains of mice when 2,4,5-T was administered at levels of 100 mg/kg and TCDD at 3 ug/kg. Kidney malformations were produced by both 2,4,5-T samples in CD-I mice and TCDD produced marked kidney anomalies in all mice strains. When 100 mg/kg 2,4,5-T and 1 yg/kg TCDD were administered in combination to CD-I mice, the activity was not potentiated at the dose levels employed. Bage et al (5) injected NMRI mice subcutaneously with 50 and 110 mg/kg 2,4,5-T containing less than 1.0 ppm dioxin on each of days 6 through 14 of gestation. At 110 mg/kg 2,4,5-T was teratogenic, causing cleft palates, rib and vertebrae anomalies as well as being fetotoxic causing 25 to 35 percent resorptions. Highman et al (61) recently reported that it was possible to detect a retardation in renal alkaline phosphatase in fetal kidneys from fetuses of mice given doses of 2,4,5-T by gavage at the rate of 60-120 mg/kg on days 6-14 of pregnancy. This retardation in renal alkaline phosphatase levels was suggested as the cause for the delay in renal functional development and indirectly supported the view that 2,4,5-T caused retarded development, rather than true teratogenesis. In this study a reduction of fetal weight and an increase in the incidence of cleft palate were seen in fetuses from treated females. Frohberg et al (45) administered 0, 20, 40, 80 and 120 mg/kg 2,4,5-T acid or butoxyethyl ester containing <0.1 mg/kg dioxin, by the oral route to NMRI mice. Oral doses of 80-120 mg/kg 2,4,5-T acid or 120 IV-37 �mg/kg butoxyethyl ester were required to produce 3 malformations and fetal deaths. In the inhalation experiments, 216 mg/m of the butoxyethyl ester showed a slight maternal toxic and fetotoxic and teratogenic effect. Ten exposures to 374 mg/m killed 5 of 15 dams, while 392 mg/m3 from day 11-15 of gestation was toxic for the dam and caused fetal deaths. Sparschu et al (130) orally administered commercial grade 2,4,5-T containing 0.5 mg/kg TCDD, to rats in daily doses of 50 and 100 mg/kg on days 6 to 15 and 6 to 10 of pregnancy, respectively. At the lower dosage level minimal fetal effects were seen with a slightly higher incidence of delayed ossification of the skull bones being observed. The higher level was toxic to the dams and caused a high incidence of maternal deaths. Only 4 of 25 rats survived with three showing complete, early, fetal resorptions and one had a litter of 13 viable fetuses which showed toxic effects but no evidence of teratogenic anomalies. Khera and MeKinley (74) found that 2,4,5-T, containing less than 0.5 mg/kg TCDD, induced some fetopathy and increased the incidence of skeletal anomalies in Wistar rats following single daily oral doses of 100-150 mg/kg on days 6-15 of gestation. The butyl ester produced no grossly observable teratologic effects when given at doses of 50 or 150 mg/kg. Various formulations of 2,4,5-T given to pregnant female rats demonstrated that a teratologic potential existed, in the form of skeletal anomalies, when repeated doses of 100 mg/kg or greater were given. At 25 mg/kg 2,4,5-T negative results were noted while at 50 mg/kg effects were noted but were not significant (P=0.05) when compared to control animals. The butyl ester produced no grossly observable anomalies and no adverse effects on the postnatal survival when pregnant females were treated at 50 and 150 mg/kg. Three of 8 females died at the 150 mg/kg dose. Skeletal deformities noted were not incompatible with life and no adverse change in reproductive performance or behavioral characteristics were detected. In the authors opinion, th£ predictive value of postnatal studies in relation to the detection of the teratogenic potential of test compounds may not be raliable on its own. Courtney et al (26) found that when 4.6, 10 or 46.4 mg/kg/day of 2,4,5-T was given orally on days 10-15 of gestation to Sprague-Dawley rats, kidney anomalies and other embryotoxic signs were seen at all levels. Some rat fetuses were reported to have had hemorrhagic gastrointestinal tracts. At the highest level there was a 60 percent fetal mortality and a higher incidence of abnormalities in the survivors. Courtney and Moore (27) reported that in CD rats, 2,4,5-T orally administered at 10, 21.5, 46.4 and 80.0 mg/kg was neither teratogenic nor fetotoxic. Prenatal administration of 2,4,5-T did not effect the postnatal growth and development of the CD rat. Sokolik (129) orally administered 2,4,5-T acid at dosage levels of 100 and 400 mg/kg per day and the butyl ester of 2,4,5-T at dosage levels of 50 and 200 mg/kg per day to rats on days 1 to 14 or 1 to 16. The purity of the 2,4,5-T in either form was not given. At 100 mg/kg IV-38 �2,4,5-T produced embryos with a combination of deformities including absence of the lower jaw, changes in the hind limbs and exophthalmos. At the level of 400 mg/kg one embryo was found with tridactyly of the upper limb combined with syndactyl, while another embryo had brachydactylia of the upper limb. Both levels of the butyl ester of 2,4,5-T were more toxic than 2,4,5-T acid, causing 30 percent embryonic mortality at 200 mg/kg. The lower dose of 50 mg/kg caused high mortality among the embryos as well. At the higher level the butyl ester induced cleft palate, hydronephrosis, hydrocephalus and extensive gastrointestinal hemorrhages along with hind limb brachydactylia. Cleft palate was the primary anomaly seen at the 50 mg/kg dosage level. Sokolik (129) concluded that the identical teratogenic action of the two preparations was probably attributable to the presence of dioxin, while the quantitative differences between the effects were attributable to differences in the concentration of the dioxin. Konstantinova (81) conducted experiments in white rats where 2,4,5-T butyl ester, purity not stated, was given orally to pregnant females for the entire period of the pregnancy at 0.01, 0.1, 0.42 and 4.2 mg/kg. The lowest level found to cause no effect was 0.01 mg/kg in the water. The threshold level was considered to be 0.1 mg/kg, with 0.42 mg/kg showing a general toxic effect on the pregnant female rat; however, the changes noted in the embryos had an irregular character. The highest dose level, 4.2 mg/kg, had a general toxic effect causing nervous system dysfunction in the female rats, changes in peripheral blood and a relative ( increase in the weight of internal organs. The embryotoxic effects were increased embryo deaths, lowered offspring weight, hydrocephaly and peritoneal cavity hemorrhages. In FW49 rats given daily oral doses of 25 to 150 mg/kg of either the TCDD-free or commercial grade 2,4,5-T (<0.1 ppm TCDD) showed no evidence of teratogenic effects (113). Emerson et al (36) confirmed the lack of teratogenic and fetotoxic effects of 2,4,5-T when containing only 0.5 ppm TCDD and when given in daily doses, by gavage, at the levels of 1, 3, 6, 12 or 24 mg/kg in Sprague-Dawley rats. Moreover, doses up to 24 mg/kg 2,4,5-T containing 1 mg/kg TCDD had no teratogenic effect in rats when given on days 6-15 of gestation. King et al (76) found no cleft palates when 93 embryos of Sprague-Dawley rats were injected in uteAo with purified 2,4,5-T on any one day ranging from 12 to 16 days of gestation at dosages of 50 to 125 yg/embryo. Two cleft palates were produced when technical grade 2,4,5-T was injected on day 15 of gestation into 118 embryos using the same techniques. In the control rats 45 females delivered 442 normal fetuses, with a 3.5 percent resorption rate and average liter size of 9.8 Commercial samples of 2,4,5-T containing TCDD in concentrations of 0.1, 0.5, 2.9 or 45 mg/kg caused fetal death and teratogenic effects IV-39 �in Syrian golden hamsters when given orally on days 6 through 10 of pregnancy at dosage levels of 20, 40, 80 or 100 mg/kg. As the dosage of 2,4,5-T increased and the TCDD content elevated, the effects were also increased. Pure 2,4,5-T containing no detectable TCDD produced no malformations when the dosage level was less than 100 mg/kg. Absence of eyelids (bulging eyes) and delayed ossification of the skull and exencephaly accounted for the main teratological abnormalities caused by 2,4,5-T containing TCDD. Hemorrhagic gastrointestinal tracts in the hamster fetuses appeared to be directly related to 2,4,5-T administration and could not be clearly linked to dose level of the compound or the dioxin content* These hemorrhages along with a marked edema noted in some of the fetuses reflected a toxic effect on fetal organs as opposed to a teratological effect (24). Gale and Perm (47) gave pregnant golden hamsters intravenous doses of 2,4,5-T on day 8 of gestation at the level of 2 mg/kg and found a 9 percent resorption rate in test animals compared to a 6 percent resorption rate in control animals. No malformed embryos were detected in this study; however, the purity of the 2,4,5-T was not given. New Zealand rabbits given oral doses of 0, 10, 20 or 40 mg/kg 2,4,5-T (containing <0,5 mg/kg TCDD) on days 6-18 of pregnancy showed no evidence of embryotoxic or teratogenic effects in their offspring (36). Dougherty et al (32) found that technical grade 2,4,5-T, containing 0.05 mg/kg TCDD was not teratogenic in rhesus monkeys, Macaco. mu£at£a, when given at 0, 0.05, 1.0 or 10 mg/kg, nor did 1t interfere with normal development of the young. Groups of 10 pregnant females were treated dally with stomach tube from days 22-38 of pregnancy. There was no evidence of toxicity to the females at these levels. In the 1971 Report of the Advisory Committee on 2,4,5-T (2), a preliminary study was cited where pregnant rhesus monkeys were orally dosed with 2,4,5-T, containing 0.05 mg/kg TCDD, at levels of 5, 10, 20 and 40 mg/kg three times weekly for 4 weeks between days 20-48 of pregnancy. After 100 days of gestation 12 fetuses were removed by hysterectomy and examined. All were found to be developmental^ normal and their weight range was not significantly different than the control animals of the same age. B1nns and Balls (11) found no congenital deformities 1n lambs from ewes daily fed 100 mg/kg 2,4,5-T add or the propylene glycol butyl ester of 2,4,5-T from the 14th to the 36th day of gestation. A third group of ewes fed 113 mg/kg of 2,4,5-T also showed no congenital deformities when fed at different periods during gestation. A summary of the literature on the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T in animals is presented in Table 7. In reviewing the literature it was evident that embryotoxic and teratogenic responses occurred in some strains of mice* rats and hamsters when repeated oral doses of 20 to 400 mg/kg 2,4,5-T was administered. Embryotoxicity and teratogenic studies in pregnant rabbits, sheep and rhesus monkeys have been negative. The embryotoxic and teratogenic potentials IV-40 �TABLE 7 Animal Number Used Mouse NSa PFb NS PF C57B1/6 strain AKR strain Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T in animals Route of Administration Response Dose Daily oral dose, days 6-15 of gestation Increased frequency of cleft palate in some strains. Fetal growth retardation. >20 trig/kg0 13 113 mg/kge 26 Daily oral or s.c. dose, days 6-14 of gestation Oral increased cleft palate and fetal mortality, both strains. Reference Cystic kidneys in C57B1/6 s.c. increased incidence of cleft palate and cystic kidneys in both strains. Increase in fetal mortality in C57B1/6 strain. NS PF Daily oral dose, days 6-15 of gestation No effect 20 mg/kgr 112 Cleft palate 35 mg/kg9 112 Marked increase in resorptions and "4" dead fetuses. 90-130 mg/kg9 112 �Table 7 continued Daily oral dose, day 6-15 of gestation Fetal weight reduction 10-15 mg/kg f ' h 96 Single dose during midgestation Cleft palate, maximum teratogenic effect day 12 or 13 of gestation 150-300 mg/kg 96 Daily oral dose, days 6-15 of gestation Cleft palate 30-60 mg/kg1 96 NS PF CO-1 strain OBA/2J strain C57B1/6 strain s,c. days 6-15 of gestation in a solution of DMSO at 100 yl/animal/injection Cleft palate, all three strains 100 rag/ kgJ 96 NS PF s.c. days 6-14 of gestation 50 mg/kgK 5 NS PF Kidney malformations in CO-1 strain No effect 110 mg/kg1 Teratogenic,cleft palate, rib and vertebrae anomalies, fetotoxi c ro 5 1 NS PF Savage, daily,days 6-14 of gestation Retardation in renal alkaline phosphatase in fetal kidneys, no true teratogenes i s, reduced fetal weight, cleft palate 60-120 mg/kg MS Daily oral dose,days 6-15 of gestation Toxic to females, malformations and fetal death 80-120 mg/kg 120 rag/kg1 45 Inhalation of aerosol for 10 exposures Slight maternal toxicity, fetotoxic, teratogenic 216 mg/m 3,n 45 PF m 61 �Table 7 continued Inhalation of aerosol for 10 exposures 5-15 females died 374 mg/m 3,n 45 Inhalation of aerosol for 5 exposures Toxic to females, fetal deaths 392 mg/m 3,n 45 Daily oral dose,days 6-15 of gestation Minimal fetal effects 50 mg/kg 130 Daily oral dose, days 6-10 of gestation Toxic to females, high maternal death, 4 of 25 survived, 3 had complete fetal resorptions, 1 had a litter of 13 live fetuses, toxic but no anomalies 100 mg/kg 130 Daily Oral dose,days 6-15 of gestation Fetopathy and skeletal 100-150 mg/kgp anomalies No effect at 50 and 100 mg/ 5fl , 0 ma/kaq , 9/ 9 kg. 150 mg/kg killed 3 of 8 females. 4.6, 10 or 46,4 Kidney anomalies, embryotoxi c mg/kge Rat 25, PF per test group NS PF Wistar strain Daily oral dose,days 6-15 of gestation NS PF Sprague-Dawley strain Daily oral dose,days 10-15 of gestation 74 74 26 At 46.4 mg/kg, 60% fetal mortality, many abnormalities in survivors NS PF Daily oral dose,days 1-14 of gestation Many deformities 100 mg/kg 129 Many limb abnormalities 400 mg/kg 129 �Table 7 continued Daily oral dose, days 1-16 of gestation Embryo mortality, cleft palate 50 mg/kg 129 30% embryo mortality and many anomalies 200 mg/kgs 129 No effect 0.01 mg/kgs 81 Threshold level 0.1 mg/kg 81 Toxic to female, irregular embryotoxic effect 0.42 mg/kgs 81 Toxic to female, nervous signs, embryo deaths 4.2 mg/kgs 81 Daily oral dose during pregnancy No effect No effect 25-150 mg/kgC5t 1-24 mg/kg0 Sprague-Dawley strain Gavage, daily during pregnancy No effect 24 mg/kgu 36 93 embryos Sprague-Dawley strain One -ui uteA.o injection on any one day from 12-16 days of gestation No effect 50-125 yg/kg' 76 Daily oral dose, days 6-10 of gestation No effect NS PF NS PF Daily oral dose throughout pregnancy NS PF 113 36 Golden Hamsters NS PF <100 mg/kg Fetal death, teratogenic NS PF Single intravenous dose on day 8 of gestation 20-100 mg/kg w No malformed embryos, 9% resorption - Test 6% resorption - Control 2 mg/kg r 2424 47 �Table 7 continued Rabbit NS PF Daily oral dose on days 6-18 of gestation No effect 40 mg/kgu 36 Daily stomach tube dose from 22-38 days of gestation No effect 0.05, l.O9 or 10 mg/kg 32 NS PF 3 oral doses weekly for 4 weeks between days 20-48 of gestation No effect on 12 fetuses removed by hysterectomy at 100 days gestation 5,10,20, 40° mg/kg 1 NS PF Daily dose from 14-36 day of gestation No effect 100 mg/kgr'x 11 NS PF Dosed at various periods of gestation No effect 113 mg/kg' 11 Monkey 10, PF per group Sheep en NS - number of animals in study not stated or unavailable from literature source. PF - pregnant female C 2,4,5-T acid, containing <0.1 mg/kg TCDD. s.c. - subcutaneous injection. e 2,4,5-T acid containing 30 mg/kg TCDD. f 2,4,5-T acid containing <0.02 mg/kg TCDD. 9 2,4,5-T acid containing 0.05 mg/kg TCDD h Butyl ester of 2,4,5-T, containing <0.02 mg/kg TCDD. ''z^.S-T acid, containing 1.5 mg/kg TCDD. J 2,4,5-T acid, technical grade, containing 0.5 mg/kg TCDD or analytical grade, containing <0.05 mg/kg TCDD. k 2,4,5-T acid, containing <1.0 mg/kg TCDD. 1 2,4,5-T acid containing <0.05 mg/kg TCDD. m'2,4,5-T acid, containing <0.1 mg/kg TCDD. n ButoxyethyTester of 2,4,5-T, containing <0.1 mg/kg TCDD. °2,4,5-T acid, containing 0.5 mg/kg TCDD. P 2,4,5-T acid, containing <0.5 mg/kg TCDD. q Butyl ester of 2,4,5-T, containing <0.5 mg/kg TCDD. r 2,4,5-T acid, purity not stated. s Butyl ester of 2,4,5-T, purity not stated. ^ 4 5 acid, free of TCDD. Detection level not stated. ,,^ ^2,4,5-T acid, containing 1.0 mg/kg TCDD. v Purified 2,4,5-T acid, purity not stated. w'?,4,5-T acid with oil, 0.5, 2.9 or 45 mg/kg TCDD. K Propylene glycol butyl ester of 2,4,5-T. �of 2,4,5-T in susceptible animals varied with the content of the contaminant TCDD. Levels of TCDD greater than 1 mg/kg were required to enhance the embryotoxic and teratogenic potential of 2,4,5-T. E. Carcinogenic and Tumorigenic Potentials of 2,4,5-T The industrial production of 2,4,5-T always results in some TCDD contamination, although admittedly at very low levels (<0.01 ppm) with current technology. Nevertheless, in the following review, the effects of various levels of TCDD associated with the 2,4,5-T being tested must always be considered. Innes et al (67) and the Bionetics Research Laboratories (12) reported that in groups of male and female mice receiving commercial 2,4,5-T (98 percent pure) there were no increases in any type of tumor in any group or combination of groups when compared to control animals. The treated mice were given 2,4,5-T at the dosage level of 21.5 mg/kg in 0.5 percent gelatin by stomach tube at seven days of age daily up to 28 days of age, followed by 60 mg/kg of diet until the mice were 78 weeks of age. Muranyi-Kovacs et al (92) conducted a two month study in XVII/G and C3HF mice. Beginning at six weeks of age the mice were given 2,4,5-T (containing <0.05 ppm dioxins) in the drinking water at a dosage of 100 mg/1. Following the initial two month treatment the exposure was continued throughout the animals life span by mixing 2,4,5-T directly with the diet at a concentration of 80 mg/kg. The average survival times for the XVII/6 mice was 555 days in 20 treated males and 632 days in 19 treated females, compared to 516 days in 32 control males and 40 control females. No significant differences were found in the incidences of tumors in the XVII/G strain of mice between the treated and control mice. The XVII/G strain of mice have a known high spontaneous incidence of lung tumors. In test groups of 22 male and 25 female C3HF mice studied, the average survival times were 523 days in treated males and 621 days in treated females, compared to 641 days in 43 control males and 661 days in 44 control females. The total number of tumors was 13/22 in treated males and 13/15 in treated females, which was significantly different from that in the female controls of 9/44 (P<0.01). No significance was seen when test males with tumors were compared to control males with a tumor Incidence of 22/43. The C3HF strain of mice has a known high spontaneous incidence of hepatomas. In a 1968 study (12) groups of 18 male and 18 female mice from two different crossbred strains were given single subcutaneous injections of 98 percent pure, 2,4,5-T at a dosage level of 215 mg/kg in DMSO at 28 days of age and observed up to 78 weeks of age. Tumor incidences in treated mice of any groups or combination of groups were not significantly different from any groups or combination of groups of control animals that numbered 141, 154, 157 and 161. The control animals were either untreated or were injected with DMSO, 0.5 percent aqueous gelatin or corn oil. IV-46 �Walker et al (141) demonstrated that six daily intraperitoneal injections of highly purified 2,4,5-T (99.0 percent) at the rate of 62 mg/kg effectively inhibited development of the Ehrlich ascites tumor being maintained in BALB/c mice. When the dosage of 2,4,5-T was increased to 80-85 mg/kg per day for six injections, the extent of inhibition of tumor development was doubled. From data presented in Table 8 it appeared that 2,4,5-T was not carcinogenic in most strains of mice tested at the oral dosage ranges of 21.5 mg/kg or 60 to 100 mg/kg in the diet or drinking water. Single subcutaneous doses of 2.5 mg/kg 2,4,5-T did not induce tumor formation in mice and 62 to 85 mg/kg 2,4,5-T in six daily intraperitoneal injections actually inhibited Ehrlich ascites tumor development being maintained in BALB/c mice. The only exception noted was the results reported by Muranyi-Kovacs et al (92), where treated C3HF female mice had a significantly higher incidence of tumors than did the control females. These authors stated: The carcinogenesis observed in our experiments should be attributed to 2,4,5-T per se. Nevertheless, a problem in assessing the significance of this effect was the choice of statistical analysis. Since the average survival time was different in some experimental groups, the choice of the experimental animal in assessment of carcinogenic potential is very important. For practical reasons rodents, particularly mice, are often used without scientific justification for such a choice. The problem of species specificity in the metabolism of chemical carcinogens is a known variable. The work by Gehring et al [ 4 ) on 2,4,5-T showed that the (9] kinetics of excretion of 2,4,5-T was extremely variable from one species to another. The half-life of 2,4,5*T in the plasma after a dose of 5 mg/kg was found to be 4.7 h in the rat, 77 h in the dog and 23 h in man. So the mouse being a rodent may not be the ideal experimental model for testing the carinogenicity of 2,4,5-T. Muranyi-Kovacs et al (92) further noted that in their opinion 2,4,5-T should be placed in the C group of chemical substances whose activity has been insufficiently assessed and in C2 and C3 priority groups requiring additional data, Implying that further testing in greater numbers of animals and in other species such as the rat and the dog was necessary. F. Mutagenic and Cytogenetic Potentials of 2,4,5-T As with 2,4-D, most of the mutagenic studies involving 2,4,5-T have been conducted in bacterial cultures or in plant and animal tissue cultures; however, Styles (133) investigated the cytotoxic effects of 2,4,5-T on -in vivo and Jin vi&io test systems and found no increase in IV-47 �TABLE 8. Animal Number Used Mouse . 18 Ma/18 FD of two hybrid strains 20 M/19 F XVH/G strain Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4,5-T in animals Route of Administration Stomach tube, beginning at 7 days of age for 21 days, then in diet for 18 months Starting at 6 weeks of age for 60 days in drinking water, then in diet for life span Response No effect Dose . Reference 21.5 mg/kgc by stomach tube 60 mg/kg diet0 No effect 100 mg/ld for 60 days 80 mg/kg diet 5 i 22 M/25 F C3HF oo Starting at 6 weeks of age for 60 days in drinking water, then in diet for life span No effect in males, more tumors treated in females than in controls 12, 67 12, 67 92 H 92 100 mg/ld for 92 60 days 80 mg/kg diet H 18 M/18 F Single subcutaneous injections No effect 215 mg/kge in DMSO 8 sex not stated BALC/c Six daily intraperitoneal injections Inhibited Ehrlich ascites 62 mg/kg 92 tumor Doubled inhibition *M - Male 3 F - Female "2,4,5-T acid from a commercial source, TCDD level and purity not stated 80-85 mg/kg 12 141 f 141 2,4,5-T acid, containing <0.05 mg/kg TCDD "2,4,5-T acid, 93 percent pure, in dimethyl sulphoxide. 2,4,5-T acid, 99 percent pure, TCDD level not stated. �mutation rate and no evidence of mutagenicity in the test rats. He found serum from orally dosed rats was not mutagenic to SatmonMa. typhunufu.im. However, complete details of this study were not available. Jenssen and Renberg (68) found there was not a detectable increase of micronuclei in the erythrocytes of mouse bone marrow after intraperitoneal administration of 100 mg/kg 2,4,5-T containing less than 1 mg/kg TCDD. Because of the high experimental resolution power of the test system used, it was particularly suitable for the detection of weak chromosome breaking activity of 2,4,5-T in mammal cells. The lack of penetration of 2,4,5-T into the cells was in accordance with the rapid excretion that is known to occur in the mammalian body. This experiment did not, in the authors opinion, constitute a reliable measure of the mutagenic potential of 2,4,5-T; however, in practice, the lack of penetration of this substance into the cells indicated it did not constitute a cytogenetic hazard to man. In an abstract Buselmaier et al (19) reported on a large number of pesticides evaluated for mutagenic activity in mice with the hostmediated assay and to a smaller extent the dominant lethal method. These test systems took into account the mammalian metabolism and covered two different spectra of mutations: point mutations and the dominant lethal mutations which were thought to be the result of chromosomal aberrations. Back mutation systems of SaJLmonntta typhMnusuum G46 His" and SeA/uttut mot.ce4ce.n4 a21 leu" and Se/tAatLa. matce6cen4 a31 His" were used. In the host-mediated assay there was no significant increase in mutation rates after unspecified levels of subcutaneous injections of the acid or nbutyl ester of 2,4,5-T. All spot tests for this herbicide Jbi vWio was also negative. When the n-butyl ester, unspecified purity, was given to test mice by a single intraperitoneal injection, at a dose of 100 mg/kg, no increases in dominant lethal mutations were seen. Da'rving and Hultgren (30) reported that commercially available 2,4,5-T, with a TCDD concentration guaranteed on the label to contain less than 0.1 mg/kg, affected chromosomal and reproductive mechanisms in bone marrow cells from two different strains of mice. The authors concluded, however, that chromatid inter- or intra-exchanges were never observed. The study was not carried-out for sufficient time to demonstrate the effects on future generations of somatic cells. Majumdar and Hall (85) investigated the effect of 2,4,5-T -. containing no detectable TCDD, on male and female Mongolian gerbils. Test animals ranging from 50-80 days of age were given 5 consecutive daily intraperitoneal injections of 50, 150, 250, 350 or 500 mg/kg. No effects were seen on the chromosomes of bone marrow cells at doses of 150 mg/kg or less. At levels of 250 mg/kg and above, significant increases in chromatid gaps, chromatid breaks and chromatid fragmentation were observed. No exchange figures or isochromosome gaps or breaks were reported. IV-49 �Fujita et al (46) conducted studies to examine the cytogenetic effects of high purity 2,4,5-T (0.09 mg/kg TCDD) at levels of 10-' to 10-14 M on human lymphocytes in vWio. Breaks, deletions and rings were observed. Chromatid breaks increased with increasing concentrations of 2,4,5-T; however, it was not possible to distinguish if this effect was due to cellular toxicity or to a potential genetic alteration. Andersen et al (4) evaluated 110 herbicides for their ability to induce point mutation in one or more of 4 different microbial systems. The herbicide 2,4,5-T was included in this study. The authors did not state the purity of the compounds being tested. The 2,4,5-T did not cause point mutations in these microbial systems in comparison with known mutagens such as 5-bromouracil or 2-aminopur1ne. These observations of no mutagenicity of 2,4,5-T in E&che/tichia. aotL WP2 her* or her" or in Salmonella. typhirrwuum strains TA1535, TA1536, TA1537 or TA1538 were also confirmed in works by Nagy et al (94), Shirasu (136) and Shirasu et al (127). A review of the literature on the mutagenic and cytogenic potentials of 2,4,5-T in animals generally supported the premise that 2,4,5-T, like 2,4-D, was not highly cytotoxic in laboratory animals. The herbicide did not increase mutation rates nor stimulate a mutagenic response in rats and mice. In various in \)Wio and in vivo test systems 2,4,5-T did cause chromatid aberrations in cultured human lymphocytes and affected the chromosomes and reproductive mechanisms in mouse and hamster bone marrow cells. It was not determined whether these affects were due to cellular toxicity or to a potential for genetic alteration of future generations of somatic cells. No mutagenic responses were seen in several studies using microbial systems for the detection of mutagenic and cytogenic responses to 2,4,5-T. IV. REVIEW OF TCDD TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of TCDD Studies on the extremely high acute toxicity of TCDD, the most toxic of the chlorinated dibenzo-p-dioxins, have been conducted by Carter et al (21), Greig et al (53), Gupta et al (56), Harris et al (59), King et al (76), Kociba et al (77), McConnell et al (87), Schwetz et al (121), Vos et al (140) and Zinkl et al (148). Schwetz et al (121) noted that perhaps the most striking fact about TCDD was its ability to cause death after a single'oral dose at levels as low as 0.6 yg/kg in male guinea pigs or 1000 yg/kg in the dog. Lethal doses to rabbits were in the same dose range with either oral (115 yg/kg), intraperitoneal (>252 yg/kg), or skin (275 yg/kg) administration. In mice, single oral doses of 1 to 130 yg/kg produced some deaths, however, no dose-response relationship was established. Schwetz et al (121) noted that approximately half the deaths in mice occurred between 13 and 18 days after treatment. IV-50 �Poland and Kende (108) considered TCDD to be one of the most potent low molecular weight toxins and teratogens known. They noted that most poisons act rapidly and kill by impairing the physiologic function of the nervous system. TCDD in contrast, is a "cellular poison." In the rat, deaths appeared to have resulted from hepatic necrosis and ensued weeks after a single oral dose. Harris et al (59), Schwetz et al (121), and Vos et al (140) also noted TCDD produced hepatic cell necrosis that was the probable cause of death in the rats in their studies. They also noted that in mice and guinea pigs, hepatic cell necrosis and liver insufficiency occurred only minimally. Putnam and Courtney (109) treated female Wistar rats with single oral doses of 100 yg/kg TCDD and found it caused a biphasic decline in body weight with a cessation of food and water consumption and urine production. The first phase started immediately after dosing and lasted 7-10 days followed by a recovery from 4-6 days during which time the rats ate and drank and regained about 10-15 percent of their body weight. This was followed by a second phase which occurred at 16-24 days after treatment with a weight loss of about 15-30 percent. If the loss of body weight exceeded 30 percent the rats usually died. Daily administration of water, electrolyte solution, or a balanced liquid diet did not alter or reverse the biphasic response. Cunningham and Williams (28) treated groups of 12 to 16 weanling male Wistar rats with single oral doses of 0 or 10 yg/kg TCDD. This was close to the lethal dose for when this amount was given orally to rats each day in a preliminary experiment, all died within 2 to 4 days. The lowest level in a single dosage that caused an increase in liver weight of rats in the preliminary study was 0.1 yg/kg. The TCDD had no effect on the rate of incorporation of ^H-acetate into liver lipids; however, it may have restricted the transport of lipids out of the liver. The storage of lipids reached a maximum at about 3 days after the TCDD was given and was accompanied by a significant increase in the incorporation of 14C-leucine into liver proteins. The increased synthesis of all proteins may have resulted from an induction of liver enzymes by TCDD. Harris et al (59) found the mortality pattern was very near the same in rats and guinea pigs when TCDD was given as a single oral dose or divided into daily or weekly doses over a 4 to 5 week period. He noted that this mortality pattern could be interpreted as demonstrating a cumulative toxicity from the TCDD. In rats, guinea pigs and mice, changes in the weight of the thymus appeared to be the most sensitive indicator of TCDD exposure according to work by Harris et al (59). These decreases in thymus weight occurred with doses of TCDD that had no effect on body weight. Van Miller et al (137) produced high levels of TCDD in the skin of rhesus monkeys by giving a single intraperitoneal injection of 400 yg/kg TCDD and produced clinical signs of alopecia and acne. IV-51 �A summary of the literature on the LDcn levels of the acute toxicity of TCDD for animals is presented in Table 9. TCDD was found to be an extremely toxic compound with an oral LDso range of 0.6 yg/kg in male guinea pigs to 115 yg/kg male and female rabbits. Male rats appeared to be more sensitive than females when TCDD toxicity was studied in the Sherman (Spartan) strain rat. In rabbits, essentially similar dosage levels of TCDD caused death following either intraperitoneal, oral or skin administration. Limited studies on dogs suggested that tltsy were less sensitive to TCDD than were the other laboratory animal species studied. In all species studied however, reduction in body weight was a common finding following TCDD treatment while other signs of toxicity were species dependent. B. The Subacute and Chronic Toxicity Potentials of TCDD Subacute and chronic doses of TCDD produced a variety of toxic effects, including hepatic necrosis, thymic atrophy and lesions of the myocardium in rats (20, 56), thymic atrophy, depletion of lymphoid organs and hemorrhage and atrophy of adrenal zona glomerulosa in guinea pigs (56) and hepatic necrosis in rabbits (120). The main target organs of TCDD in rats, guinea pigs and mice appeared to be the liver and thymus (56, 69, 70, 139, 140). The degree of hepatic involvement appeared to be dose dependent and the severity of the changes produced varied between species (56). A single oral dose of 126 yg/kg TCDD resulted in loss of body weight and death with an enlarged fatty liver after 21 days in C57B1/6 mice. A progressive necrotic centilobular liver lesion was seen (71). Vos and Moore (139) studied pre- and postnatal effects of TCDD in*groups of 5, 6 and 5 pregnant C57B1/6 mice dosed at 0, 2 or 5 yg/kg TCDD on days 14 and 17 of gestation and postnatally on day 1, 8 and 15. All neonates were weaned on day 23 and used for a skin graft experiment. This treatment resulted in a severe depletion of lymphocytes in the thymic cortex of the offspring. Cellular immunity was impaired and allograft rejection times were prolonged. Murray et al (93) conducted a three generation reproduction study to evaluate the effects of chronic, low-level ingestion of TCDD in Sprague-Dawley rats administered daily doses of 0, 0.001, 0.01 or 0.1 yg/kg provided via the diet for 90 days. No signs of toxicity were noted in either male or female rats during the TCDD feeding study. Vos et al (140) found the most significant findings in both mice and guinea pigs treated with sublethal doses of TCDD were in the lymphoid system where there was a supression of cell mediated immunity at doses of 2 and 5 yg/kg TCDD. Thigpen et al (134) found that low levels of TCDD did not produce overt clinical or pathological changes, however, these low levels IV-52 �Summary of literature data on the no-effect, LD5Q and levels of the acute toxicity of TCDD for animals TABLE 9 . Animal Number Used Route of Admin. Dose-Toxicity Single Dose yg/kg Reference >50 59 1-130 T21 Mouse 10 CD-I strain C57Bl/6Sch strain LD NSa Oral A few sporadic deaths 29 Mb C57B1/6 strain Oral LD 150 50 M NS T ) Oral Intraperitoneal LD 120C 138 5 M Oral No effect 8 121 5 M Oral No effect 16 121 10 M Oral LD 32 121 Oral 25 M Sherman (spartan) strain LD 22 121 Oral LD 45 121 100 100 50 Rat NS F strain 100 50d 50d �Table 9 continued Guinea Pig NS M Oral NS M Hartley strain 50 .6 2.1 121 121 Rabbit NS M/F 5 M/F 5 M/F 5 M/F 5 M/F New Zealand albino Oral Topically to skin Intraperitoneal Intraperitoneal Intraperitoneal LD 50e LD 50e No effect 2 of 5 died 3 of 5 died 115 121 275 121 32 121 >252 121 500 121 300 121 3000 121 30 121 100 121 <70 87 Dog 2 M 2 M 2 F 2 F Beagles 100 No effect No effect 1 F Rhesus I en Oral Oral Oral Oral Oral LD No effect LD Monkey 50f NS - Number of animals in study not stated or unavailable from literature source M - Male 3 "3H-TCDD A calculated LD50 cn "Responses to individual doses when ID™ could not be calculated Correlated the acute LD™ of TCDD with the clinical and pathological manifestations - not true calculated '50 LD50 �reduced host defenses. When 1 ug/kg was given orally once a week for 4 weeks to mice before infection with SaJtmon&JUa be/m, an increased mortality and decreased time from infection to death was noted. Weissberg and Zinkl (142) and Zinkl et al (148) noted hematological changes in mice, rats and guinea pigs treated with TCDD including lymphopenia and thrombocytopenia at dosage rates of 0.004 to 10 ug/kg for various repeated doses. Goldstein (50) gave TCDD orally to mice once a week over a 4 week period at a dosage of 25 ug/kg and found a 2,000-fold increase in the levels of 8- and 7-carboxyporphyrins in the liver. In a 13-week feeding study by Kociba et at (77), Sprague-Dawley rats of both sexes were given 0.001 or 0.01 ug/kg TCDD five days per week. A slight increase in relative liver weight occurred in those animals receiving 0.01 ug/kg TCDD. A steady state concentration of TCDD was attained in body tissues by the end of the study. Vos and Moore (139) in a pre- and postnatal study, treated groups of 5, 4 and 6 pregnant Fisher-334 rats with 0, 1 or 5 ug/kg TCDD prenatally on days 11 and 18 of gestation and postnatally on days 4, 11 and 18 via gastric intubation. Most of the neonates in the 5 ug/kg group 'died. Only the spleens of 25-day-old male animals from the 0 and 1 ug/kg groups were used for immunologic studies. At 1 ug/kg the pups had a depressed body and spleen weight. At 5 ug/kg, in those pups that survived, the body and spleen weights were depressed and the thymus was severely affected with marked depletion of lymphocytes in the thymic cortex. Cellular immunity was impaired with allograft rejection times being prolonged. Schwetz et al (121) found that solutions of 0.04 ug TCDD/ml of benzene was acnegenic in a rabbit ear bioassay study where the solution was applied to the inside of the ear 5 days per week for four weeks. Norback and Allen (98) fed fat, containing unspecified concentrations of chlorinated dibenzo-p-dioxins in the diet, to Macaco, mulatta monkeys for 100 days and found the monkeys developed alopecia, subcutaneous edema, anemia, progressive leukopenia and hypoproteinemia. Enlargement of the liver, hydropericardium, gastric hyperplasia and ulceration as well as hyperplasia of the lymph tissue and bone marrow was noted in the treated monkeys. Allen et al (2) found that female rhesus monkeys given a diet containing 500 ng/kg TCDD for 9 months became anemic within 6 months and pancytopenic after 9 months of exposure. Marked thrombocytopenia was associated with widespread hemorrhage. Death occurred in five of the eight animals between months 7 and 12 of the experiment at total IV-55 �exposure levels of 2-3 yg/kg TCDD body weight. At autopsy, in addition to the hemorrhage, there was a distinct hypocellularity of the bone marrow and lymph nodes. Death of these monkeys was attributed to complications from the severe pancytopenia. McNulty (89) fed one rhesus monkey a diet containing 2 yg/kg TCDD and another monkey a diet containing 20 yg/kg TCDD. The first animal died within 76 days, while the second animal died in 12 days. McNulty noted that although responses in two animals scarcely provided data for a dose-response curve, two conclusions could be drawn: (a) a total TCDD dose of less than 10 yg/kg of body weight accumulated over a few weeks period, and (b) young rhesus monkeys were among the most TCDDsusceptible animals of those that have been tested. A summary of literature data on the subacute and chronic toxicity of TCDD in animals is presented in Table 10. Subacute and chronic doses of TCDD produced a variety of toxic effects, including hepatic necrosis in mice, rats and rabbits; thymic atrophy in mice, rats and guinea pigs with adrenal gland hemorrhages and depletion of lymphoid organs also being seen in guinea pigs. Repeated oral doses of 0.001 to 10 yg/kg TCDD for four to 13 weeks did not significantly affect weight gain nor were signs of toxicity noted in mice and rats. Suppressed immune responses and changes in liver enzymes were noted, however, in mice. Repeated doses of TCDD as low as 1 yg/kg caused guinea pigs to become moribund and repeated doses of 0.04 yg/kg decreased lymphocyte counts. Rabbits developed acne of increasing severity when doses of 0.04 to 400 yg/kg were applied repeatedly to the internal surface of the ear. A total oral dose of 2-3 yg/kg over a nine month period produced severe hematological changes and death in rhesus monkeys. C. Absorption, Distribution and Excretion of TCDD Following a single oral administration of 50 yg/kg ^C-TCDD to rats, Piper et al (102, 103) found that almost 30 percent was eliminated in the feces during the first 48 h. The half-life for the disappearance of 14c activity from the body was 17.4 ± 5.6 days. After this time the excretion of ^C activity via the feces was from 1-2 percent per day. As the l^C-TCDD was absorbed into the body tissues most of the activity was localized in the liver and fat at levels about 10 times higher than that in other tissues. A total of 53.2 percent of the dose was eliminated via the feces and 13.2 percent via the urine, while 3.2 percent was expired into the air when measured over a 21 day period. Rose et al (114) found that, following daily oral administration of 0.01, 0.1 or 1.0 yg/kg l^c-TCDD five times per week for seven weeks to Sprague-Dawley rats» the major route of excretion was via the feces. Urine contained 3-18 percent of the cumulative dose of 14C activity after the seven week treatment. The half-life of 14C activity in the rats studied was 23.7 days. IV-56 �TABLE 10. Animal Mouse Number Used Summary of literature data on the subacute and chronic toxicity of TCDD in animals Route of Administration 377 Ma Once per week by gastric tube C57Bl/6JFh for 4 weeks (J67) strain Specific Pathogen free Effect Dose No effect on weight gain 0.5, 1, 5 and 10 yg/kg 134 Significant decrease in weight gain 20 ug/kg 134 Reference NSC F CD-I 134 1 yg/kg and Significant increase in mortality of mice challenged greater wi th Salmonella bern 5-6 per group C57Bl/6Sch FD strain d C57B1/6 M strain 134 No effect on mice challenged 0.5 ug/kg wi th Salmonella bern en No effect, on mice challenged 0.5, 1, 5, 10 and 20 ug/kg with Herpesvirus suis 134 2 yg/kg Oral dose given days 14 and 17 of gestation and postnatal ly on day 1, 8 and 15 No effect on weight gain Single oral dose after 8 weeks of age Hematological changes at 1 week after dose; normal at 3 weeks 1, 10 or 50 yg/kg 2000 fold increase in carboxyporphyrins in the liver 25 yg/kg 12 M Oral dose once per week for C57B1/6 strain Four weeks Suppressed cellular immunity 2 or 5 yg/kg 140 140 143 50 �Table 10 continued Rat NS M/F Sprague-Dawley strain Daily oral dose for 90 days No signs of toxicity NS M/F Sprague-Dawley Daily oral dose, 5 days per week for 13 weeks No toxicity, slight increase 0.001 or 0.01 in relative liver weight at yg/kg 0.01 yg/kg NS F CO strain Daily oral dose for 30 days Liver enzyme changes and hematological changes 10 yg/kg 148 Weekly oral doses for 8 weeks Moribund at 3 to 5 weeks 1.0 yg/kg 148 Significant decrease in lymphocyte counts 0.04 yg/kg 148 Applied to inside of ear, 5 days per week for 4 weeks in a .1 ml volume Acne with increasing severity as dose was increased 0.04 to 400 yg/kg 121 NS Macaca mulatta Fed fat containing 64% mass tetrachlorinated compounds in diet for 100 days Multiple toxic signs Unknown 8 F Macaca mulatta Fed in diet for 9 months Hematologic changes, 5 animals died 500 ng/kg of diet 2-3 yg/kg total exposure 0.001, 0.01 or 0.1 yg/kg 93 77 Guinea Pig NS F Hartley strain en 00 Rabbi t Monkey 98 �Table 10 continued 2 Macaca mulatta d Fed in diet Death in 12 days Death in 76 days M - Male b F - Female C NS- Number of animals in study not stated or unavailable from literature source Ul 20 yg/kg diet 2 jig/kg diet 89 89 �Allen et al (2) treated 40 male Sprague-Dawley rats with a single intragastric dose of 50 yg/kg of 14C-TCDD. One-half of the animals died within 25 days, 25 percent being accounted for during the first 3 days. The total amount of radioactivity in the urine was 4.5 percent of the total dose, with the highest daily levels being excreted toward the end of the experiment. A large percentage of the remaining radioactivity was localized in the liver and of this over 90 percent was located within the microsomal fraction. Fries and Marrow (44) found the half-life to be 12-15 days in rats given 7 or 20 yg/kg Mc-TCDD of diet (equivalent to 0.5 or 1.5 ya/kg per day) for 42 days. *, Vinopal and Casida (138) administered 3H-TCDO by a single intraperitoneal injection to male mice at the LDso dose of 120 yg/kg and found that it was not measurably converted to water soluble products and was eliminated primarily in the feces. Traces of tritium activity were detected in the urine. A large proportion of the administered dose persisted in the unmetabolized form in the liver, partially concentrated in the microsomal fraction for 11 to 20 days after treatment. The 3H-TCDD was not metabolized by liver microsomal fractions from mice, rats or rabbits. Gasjewicz and Neal (48) studied the tissue distribution and excretion of 14C-TCDD in adult male guinea pigs for up to 15 days following its intraperitoneal injection of 2.0 yg/kg. On day 1 the highest levels of radioactivity were located in the adipose tissue 2.36 percent, adrenals 1.36 percent, liver 1.13 percent, spleen 0.70 percent, intestine 0.42 percent and skin 0.48 percent. 14 other tissues examined contained less All than 0.3 percent. The level of C-TCDD 1n the liver Increased to 3.23 percent on day 15. An increase in 14C-TCDD was also noted in the adrenals, kidneys and lungs while adipose tissue and skin decreased in radioactivity. For the 15 days of the experiment the total urinary and fecal excretion of radioactivity was less than 1 and 5 percent respectively. The effects of 1.0 yg/kg TCDD upon plasma levels of Na, K, Cl, 003, Fe, Ca, inorganic P, alkaline phosphatase, SGOT, SGPT, LDH, glucose, urea nitrogen, creatinine, uric acid, total protein, albumin, cholesterol, triglycerides and bilirubin were determined periodically up to 14 days and compared to pair-fed control animals. Statistically significant increases in plasma albumin, total protein, Fe, urea nitrogen, cholesterol and triglycerides were observed in the TCDD-treated guinea pigs. The primary route of excretion for TCDD in animals appeared to be the feces, with urinary excretion occurring at a much reduced rate. Liver and fat accumulated about 10 times higher levels of TCDD than did other body tissues. The half-life for TCDD in rats following a single or repeated exposure was 12-24 days after termination of treatment. Large proportions of an administered dose of TCDD remained unmetabolized in the liver microsomes and were slowly excreted over an extended period. IV-60 �D. Embryotoxic, Fetotoxic and Teratogenic Potentials of TCDD The embryotoxic and teratogenic effects of TCDD in mice have been described by Courtney and Moore (27), Neubert and Dillmann (96), Neubert et al (97), and Smith et al (128) where doses as low as 1-10 ug/kg» given in a single or repeated dose, caused significant increases in the frequency of cleft palate and kidney anomalies. Neubert (95), and Neubert et al (97) noted a dose-response relationship for producing cleft palates in mice with TCDD. They also observed increased incidences in the frequency of cleft palate in mice,1 apparently caused by the synergistic effect of combining 'sub-threshold and 'threshold1 levels of TCDD with similar low levels of other known teratogens such as the weak teratogen 2,4,5-T when administered during days 6-15 of gestation. Moore et al (91) confirmed that exposure to TCDD via the milk was a major factor in the development of renal hydronephrosis in mouse pups when the nursing dam received a single oral dose of 1, 3 or 10 ug/kg TCDD at parturition. This effect was also seen in mouse pups nursed by a foster mother treated with TCDD during pregnancy or at the time of parturition. The common etiology of these kidney anomalies, whether prenatal or postnatal, was TCDD interference with development of the metanephric kidney and/or subsequent maturation. The incidence and degree of hydronephrosis was a function of dosage and length of target organ exposure. The dose effecting 50 percent of the test organisms (£050) for cleft palate production in NMRI mouse pups was estimated by Neubert et al (97) to be 40 ug/kg TCDD per day. The no effect level during days 6 to 15 of gestation was estimated at 2 ug/kg TCDD per day with no pronounced fetal mortality occurring when 3 ug/kg TCDD was given from day 6 to day 15 of gestation. Becker (8) has concluded that the influence of a teratogenic substance closely related to the critical developmental period of a particular tissue or organ. After this critical period passed, damage to other tissues may have occurred even if no significant malformations were observed. Unspecified doses of TCDD produced an extremely fatty degeneration of the liver in adult female rats when they were treated on days 13 to 15 of gestation. Fatty inclusions were seen in the liver of embryos from these treated females; however, no structural anomalies were noted in any of the embryo livers. Courtney and Moore (27) produced cleft palates in three strains of mice by giving 1 or 3 ug/kg TCDD subcutaneously on days 6 to 15 of pregnancy, while Courtney (25) found TCDD to be the most fetotoxic and teratogenic of several dioxin compounds when given at 25, 50, 100, 200, and 400 ug/kg per day orally and 25, 50, 100, 200 ug/kg per day subcutaneously IV-61 �in CD-I mice on days 7 to 16 of pregnancy. Fetal mortality increased with the dose: up to 97 percent in orally treated dams and up to 76 percent in dams receiving subcutaneous administration of the highest levels of TCDD. Other anomalies observed were hydrocephalus, lack of eyelid formation (open eye) and clubfoot with edema and internal hemorrhages being noted in fetuses of dams receiving the highest doses. Smith et al (128) administered 0.001, 0.01, 0.1, 1.0 and 3.0 wg/kg TCDD per day to CF-1 mice by gavage from days 6 to 15 of pregnancy. Only at the 1.0 yg/kg dose was the percentage of resorption sites per implantation sites significantly higher than in the control animals. At 3.0 ug/kg, cleft palate occurred in 71 percent of the treated litters and at 1.0 yg/kg, 21 percent had cleft palate. Renal anomalies*occurred in 28 percent of the litters treated at 3.0 yg/kg and in 5 percent of the litters treated at 1.0 yg/kg. No significant anomalies were seen at the other dosage levels. Embryo lethal effects have occurred in rats under experimental conditions imposed by Sparschu et al (131). Courtney and Moore (27) have observed kidney anomalies in rats, while Khera and Ruddick (75) observed intestinal hemorrhages and general edema in rat fetuses when oral or subcutaneous doses ranging from 0.03 to 16.0 yg/kg TCDD were administered daily to dams on days 6 to 15 of gestation. Sparschu et al (131) administered 0.03, 0.125, 0.5, 2.0 and 8.0 yg/kg TCDD per day to Sprague-Dawley rats on days 6 to 15 of gestation. At 8.0 yg/kg per day all fetuses were resorbed. Fetal weights were significantly (p<0.05) depressed at the 0.125 and 2 yg/kg per day level. Internal hemorrhages were observed at the 0.125, 0.5 and 2.0 yg/kg per day level. No adverse effects were noted in the fetuses of dams treated at the 0.03 yg/kg per day level. The authors suggested that 0.03 yg/kg per day was the no effect level for fetal and embryotoxic effects in rats. Khera and Ruddick (75) studied the perinatal effects of TCDD in Wistar rats in a two part experiment by giving daily oral doses of 0.125, 0.25, 0.5 and 1.0 yg/kg TCDD on days 6 to 15 of pregnancy. Visceral lesions were observed at 0.25 yg/kg per day and above with slight decreases 1n fetal weight also being observed. Postnatal effects of prenatal exposure to TCDD were studied by allowing offspring of treated dams to be reared by untreated dams until weaning. At maternal levels of 0.5 and 1.0 yg/kg per day, reduced survival, lowered body weight and reduced reproductive ability in the offspring were observed. At levels of 0.125 yg/kg per day no fetotoxic effects were observed. In the second part of the Khera and Ruddick study (75), rats were treated with daily oral doses of 1, 2, 4, 8 and 16 yg/kg TCDD on days 6 to 15 of pregnancy. At doses of 1.0 and 2.0 yg/kg per day, visceral lesions, reduction in fetal weight, and lowering of the number of live fetuses per litter were observed. Doses of 1 yg/kg per day or more IV-62 �produced maternal toxicity with all doses of 4 ug/kg or more producing 100 percent embryo lethality. The fetotoxic no effect level in Wistar rats appeared to be 0.125 ug/kg per day with any level of 0.25 ug/kg per day or more on days 6 to 15 of pregnancy adversely affecting fetal rat development. Courtney and Moore (27) administered TCDD to CD rats at the rate of 0.5 ug/kg per day subcutaneously in solutions of 100 percent DMSO on days 6 to 15 of gestation. Kidney anomalies were seen in 67 percent of the litters of treated females. At this level, TCDD did not affect fetal mortality or fetal weight, nor were cleft palates observed in any of the fetuses. • A summary of literature on the etnbryotoxic, fetotoxic and teratogenic potentials of TCDD in animals is presented in Table 11. It was apparent that TCDD caused birth defects and embryo mortality. Repeated daily oral doses of 0.1 to 2 yg/kg in pregnant mice produced no effect on the embryos; however, 3 ug/kg was the threshold level for production of cleft palate and kidney abnormalities. Single or repeated oral doses of 6.5 to 40 ug/kg TCDD were required to produce cleft palate in 50 percent or more of some strains of mouse embryos being studied. Daily subcutaneous injections of 1 to 3 ug/kg TCDD produced cleft palate and kidney abnormalities in 50 percent or more of three different strains of mouse embryos studied. Repeated oral doses of 25 to 400 ug/kg TCDD produced increasing fetotoxic and teratogenic responses in mice. Repeated daily oral doses of 0.03 to 0.125 ug/kg TCDD produced no effect in some strains of rat embryos while repeated oral doses of 0.125 to 2 ug/kg TCDD depressed fetal weight, lowered fetal survival and caused internal hemorrhages in fetuses. Repeated daily oral doses of 4 to 8 ug/kg TCDD produced 100 percent fetal mortality in rats. Signs of embryo toxicity and fetal death occurred in rats more frequently than did any signs of teratogenicity. When teratogenic lesions did appear in rats, kidney abnormalities were more common than cleft palate. E. Carcinogenic and Tumorigenic Potentials of TCDD In a preliminary report by Toth et al (135), 50 ten-week old male random bred Swiss H/Riop mice received gastric intubations of 7 ug/kg TCDD in sunflower oil for 12 months. No tumors were observed in 19 mice receiving post mortem examinations at the end of the treatment period. The livers from three animals showed histological evidence of cirrhosis and eight animals had developed dermatitis and showed histological evidence of increased amyloid in the tissues. Weekly doses of 0.007 and 0.7 ug/kg TCDD were given for 12 months to similar groups of mice. No pathological lesions were observed in five animals killed two months after the end of treatment. All surviving mice were kept for life-span studies and observation for the development of tumors. IV-63 �TABLE 11. Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of TCDD in animals Mouse 700 total /PFa NHRI strain, 7000 fetuses examined 100 total/PF Route of Administration Response Dose yg/kg References Daily oral dose, days 6-15 of gestation No effect 96 CP - ED5Qd 2 (estimated) 3C 6.5 96 96 Daily oral dose, days 9-13 of gestation , Animal Number Used CP CP - ED5Qd 9C <9 96 96 Single oral dose, day 13 of gestation CP 15C 97 97 K D CP - Threshold CP 40 ED - 50 5C Single oral dose, day 11 of gestation 01 35 litters total from CD-I, DBA/2 J, and C57B1/60 strains CP Daily doses given subcutaneously on days 6-15 of gestation CD-I - CP effect, 1 litter only - CP, Threshold - CP, ED50 - KAe, Threshold - KA, ED5Q CP 15 ED * 50 DBA/ 20 - CP, Threshold 1 3C >3 c lc 1 -3 3C 97 97 27 27 27 27 27 - CP, ED5Q >3 - KA, ED5Q >3 27 27 27 27 C57B1/6J - CP, Threshold 3C >3 c 3C <3 27 27 27 27 - KA, Threshold - CP, ED50 - KA, Threshold - KA, ED50 c 3C �Table 11 continued 17 PF 19 PF Daily oral dose, days 7-16 of gestation Fetotoxic, teratogenic, increasing w/dosage up to 97% at highest dose 25, 50, 100, 200, and 400 25 Daily dose given subcutaneously on days 7-16 of gestation 31 PF CD-I strain Fetotoxic, teratogenic, increasing w/dosage up to 76% at highest dose 25, 50, 100, and 200 25 Daily oral dose, by gavage, days 6-15 of gestation No effect 0.1 128 Increased fetal resorption sites, 21% CP, 5% KA 1 128 71% CP, 28% KA 3 128 14 PF 18 littersf Single oral dose, day 10 of gestation No effect, CP 34% KA 1 91 16 littersf Daily oral dose, days 10-13 of gestation 1.9% CP 58.9% KA 1 91 14 littersf Daily oral dose, days 10-13 of gestation 55.4% CP 95.1% KA 3 91 NS9/fetuses Females given oral dose at parturition Renal hydronephrosis, 12, 71 or 75% depending on dose Daily oral dose, days 6-15 of gestation No effect 0.03 131 Depressed fetal weight 0.125 and 2 131 Internal hemorrhages in fetuses 0.125, 0,5 or 2 131 All fetuses died 8 131 cr> en ,il, 3 or 10 91 Pat 51 total/PF Sprague-Dawley (Spartan) strain �Table 11 continued 103 total/PF Daily oral dose, days 6-15 of gestation 0.25 75 0.5 and 1 75 Visceral lesions, reduced fetal weight, increased fetal death with maternal toxicity 1 and 2 75 100% embryo death en 75 Reduced fetal survival, lower body weight and lowered reproductive ability in progeny Daily subcutaneous dose, days 6-15 of gestation 0.125 Slight decrease in fetal wei ght 6 PF 48 fetuses CD strain No effect 4 75 No effect on fetal mortality 0.5 or CP 67% KA PF - Pregnant Female CP - Cleft palate b °Lowest dose with which a significant teratogenic effect has been produced. In some cases this is the only dose level tested and does not necessarily represent the lowest dose which could result in teratogenic effects.. EDg0 - Dose required to produce an effect in 50% of animals g KA - Kidney abnormalities f C57Bl/6 strain 9 NS - Number of animals in study not stated or unavailable from literature source Dose given in 100 percent dimethylsulfoxide solution (DMSO) 27 �Van Miller et al (136) recently reported the results of a two year study where ten groups of 10 male Sprague-Dawley rats were fed a laboratory diet-containing 0, 1, 5, 50, 500 or 1,000 yg/kg TCDD of food or 1, 5, 50 or 500 ng/kg TCDD of food for 78 weeks. All rats receiving the 50, 500 or 1,000 yg/kg TCDD of food died between the second and fourth week of treatment. In seven remaining groups, only one animals died before the 30th week and that death occurred in the 500 ng/kg TCDD of food at the 17th week. In the 1 and 5 yg/kg TCDD of food groups, all animals died between the 30th and 90th weeks of the experiment. The number of animals dead at the 95th week of the experiment were: 0 dose 6/10, 1 ng/kg 2/10, 5 ng/kg 4/10, 50 ng/kg 4/10, 50 ng/kg 4/10 and 500 ng/kg 5/10. Those animals surviving after the 95th week were killed and subjected to complete necropsy examinations. In all rats surviving past the 65th week laparotomies were performed and biopsies of any tumors were taken. After the 78th week on treated diets, the rats were placed on the same diet used to feed the control animals. Tumorigenie and toxic effects were observed in rats from the lowest six dosage groups. The overall incidence of neoplasms in these six experimental groups was 23/60 (38 percent) compared with 0/20 (0 percent) in both the 1 ng/kg and the control groups. Neoplastic nodules and cholangiocarcinomas of the liver were observed in 40 percent of the rats ingesting 5 yg/kg TCDD of food; two animals had both neoplastic nodules of the liver and cholangiocarcinomas, Van Miller et al (136) also found that tumors developed in 24/50 (46 percent) of the rats ingesting 5, 50 or 500 ng/kg TCDD-of food and 1 or 5 yg/kg TCDD of food, compared to none (0/10) in the control animals. The tumors seen were carcinomas of the ear duct, kidney and liver. Three retroperitoneal histiocytomas were described as metastasizing to the "lungs, kidney, liver and skeletal musculature." Three of the ten deaths which occurred in the 5 yg/kg TCDD of food dose group were attributed to aplastic anemia. One animal in the 500 ng/kg TCDD of food group had a severe liver infarction. Kociba et al (78) conducted a chronic study of TCDD toxic effects to Sprague-Dawley rats fed 0.1, 0.01 or 0.001 yg/kg TCDD daily for two years to groups of 50 rats of both sexes. Eighty-six animals of each sex served as controls. Discernible increases were noted in the incidence of hepatocellular carcinomas of the liver and of squamous cell carcinomas of the lung, hard palate/nasal turbinates and tongue in rats fed at the rate of 0.01 yg/kg. They also reported decreased incidences of pituitary, uterine, mammary gland, pancreatic and adrenal gland tumors at the 0.01 yg/kg level. The squamous cell carcinoma of the hard palate observed in one female rat receiving this dose was considered unrelated to TCDD treatment since a similar tumor had occurred in other unrelated studies. At 0.001 yg/kg TCDD, no significant lesions were seen in male rats and the only lesion of significance in female rats at the 0.001 yg/kg TCDD was swollen hepatocytes, considered to be a reversible lesion. IV-67 �Many chemically nonreactive carcinogens ai-e eiizymaLiu-a I ly converted to biologically active carcinogens. The enzyme aryl hydrocarbon hydroxylase (AHH) has been strongly implicated in this process (86). Kauri et al (32) studied AHH induction in human lymphocyte cultures by TCDD. The authors stated; TCDD itself is not a potent carcinogen in mice; however, the synergistic action of TCDD with 3-methylcholanthrer.e (MC) produces cancer in different strains of mice in direct proportion to the degree of elevation of the induced hy/droxylase activity and associated cytochrome content. Their study showed a positive correlation between basal enzyme activity and enzyme levels maximally inducible by either TCDD or MC. They also found that TCDO WAS about 40 to 60 times more potent than MC as an inducer of hydroxy/lase activity in cultured human lymphocytes. The implication, of TCDD in AHH inducibility had also been reported t>y Poland and Glover (105, 106) and Poland et al (107) in their studies on chick embryo livers. They found that all dioxins which were potent inducers have halogens at three of the four lateral ring positions and at least one noft-halagenateti carbon atom. When TCDD potency, as an inducer of hepatic AHW activity » was compared with that of MC by a computer bioassay technique, data reflected that TCDD may be 28,640 times as potent as MC on a molar basis. Allen et al (2) conducted a study in which female rhesus monkeys were fed diets containing 500 ppt TCDO for nine months. Anemia, thrombocytopenia and leuikapenia were the most debilitating changes noted. The altered, lymphopoiesis cotild be associated with immune suppression. Epithelial changes, including hypertrophy, hyperplasia, and metaplasia were reported in these TCDD exposed monkeys. A summary of the literature on the carcinogenic and tumori genie potentials of TCDD in animals is presented in Table 12. It was noted that in a preliminary study where O.Q07, O..Q7 and 7 yg/kg TCDD was given in weekly oral doses for 12 months to. mice* no tumors were produced. In "'ats, levels o>f 1 and! 5 wo/kg TCDO of diet and; 1, 5, 50 and 500 ng/kg 1CD0 of diet fed for 78 weeks produced an overall tumor incidence of 38 percent in the test amiraals. At 0.001 ug/kg TCDD, given via the diet to rats far 2 years, nS effect was produced. A level of 0.01 yg/kg TCDD given via the diet to rats* for 2 years, produced liver nodules and hyperplasia of the epithelium of the lungs. An increase in liver and lung carcinomas was seen when O.I ug/kg TCDD was fed to rats for 2 years via the diet. An interesting unexplained observation, however, was the reduction of pituitary, uterine, niaranary/,, pancreas and adrenal tumors. Monkey/s fed 500 n§/k§ TCDO of diet for 9 months did not develop tumors but died of marked hetnatological alterations. IV-68 �TABLE 12. Animal Number Used Summary of literature data on the carcinogenic and tumorigenie potentials of TCDD in animals Route of Administration Response Dose Gastric intubation weekly for 12 months starting with 10 week old animals No effect in 5 animals examined 2 months after treatment ended 0.007 ug/kgb 135 No effect in 5 animals examined 2 months after treatment ended 0.07 ug/kgb 135 No tumors in 19 animals examined at end of treatment 7 yg/kgb 135 All died in 2 to 4 weeks 50, 500 or 1000 ug/kg diet 136 All died in 30 to 90 weeks 1 and 5 yg/kg diet 136 50% dead at 95th week 500 ng/kg diet 136 50 ng/kg diet 136 Mouse 50 M per group Swiss H/Riop strain I CTi Reference Rat 10 groups of 10 M In diet for 78 weeks 38% tumors < 40% dead at 95th week 40% dead at 95th week '20% dead at 95th week No tumors 5 ng/kg diet 1 ng/kg 136 < 60% dead at 95th week Controls 136 �Tafrle 112 continued 10; Met fswr 2 years No, effect Live* - 540; ng/TOW' k§e Fat - 540 ng; JIQT lH€.reasedi urinary ex.Ofeti?<m erf? ptiqDfry/irins in; females Liver - noduTes 5® ararfcmaTs LQn! tag/kg Liver - 5,100 ng Fat - IJQQ n§ d Increaseefc incidence off eell carcinQmas evidence' ©f Ltterlnfi * mcanmary pancreas aiatdi adrenaJ Fat ~-J - 24,800 nig: TCBi^ltg; - ajtfO ng: o Monkey 8 Maeaca ff diet far § msrrtfos M - Male ""Preliminary report remaining animals to be kept for life span study and observation for tumor development wfthiro 6roanthis Pancytopenia after 5 months Rarked thrombocytapenia Tissue hemorrhages 5 of 8 died between 7 and 12 months Epithelial tissue changes ag/kg diet This is the dose supplied to each animal via the diet: 0.001 yg TCDD/kg body weight = 22 ng TCDD/kg diet 0.01 yg TCDD/kg body weight = 210 ng TCDD/kg diet 0.1 ug TCDD/kg body weight = 2200 ng TCDD/kg diet : F - Female "Terminal samples of liver and fat indicating accumulated levels of TCDD/kg of tissue after two years of treeiment at the respective dosage levels Total exposure, 2-3 yg/kg body weight �F. Mutagenic and Cytogenetic Potentials of TCDD Again, as with 2,4-D and 2,4,5-T, most of the mutagenic studies involving TCDD have been conducted in bacterial cultures or in plant and animal tissue cultures. Khera and Ruddick (75), however, have conducted dominant lethal tests in which male Wistar rats received TCDD, orally, at dosages of 4, 8 or 12 yg/kg per day for seven days. TCDD did not induce dominant lethal mutations during or in the 35 days following treatment. This 35 day period corresponded to the postmeiotic stages of spermatogenesis Green and Moreland (52) conducted a short-term investigation of several dioxins, using male Osborne-Mendel rats, to determine what potential these substances had to cytogenetic damage in rat bone marrow. In one study, all of the dioxins were tested via gavage in the rats for five consecutive days at 10 yg/kg per day. A second study involved TCDD being given separately by two routes. A single oral dose of 20 yg/kg TCDD or oral doses of 10 yg/kg TCDD for five consecutive days, and in other rats, single intraperitoneal doses of 5, 10 or 15 yg/kg TCDD were given. No evidence was found that any of the substances tested produced cytogenetic damage in the bone marrow of male rats under the conditions of the experiment. However, when rats of both sexes were treated twice weekly with TCDD at a dosage level of 4 yg/kg for 13 weeks, a significant increase in the number of chromosome aberrations was found by 6r,een (51). Hussain et al (65) evaluated the mutagenic activity of TCDD (99 percent pure) of three different microbial test systems. In the first study, TCDD significantly increased the incidence of reverse mutations in EAdieAdua. coti Sd-4 when 2 yg/ml TCDD caused the bacteria to change from streptomycin dependence to streptomycin independence. This dosage was the only dose at which mutations were clearly observed. In a second study, Hussain et al (65) examined reverse mutation from histidine dependence to histidine independence in So£mone££a typkmuA^u strains TA1530 and TA1532. TCDD caused positive changes in TA1532 strain but negative results were seen in TA1530 strain which indicated that TCDD may act as a frameshift mutagen in this bacterial strain. In a third study conducted by Hussain et al (65) slight prophage induction in E4cAa>u.cA-ta c.otl K-39 was observed. However, in this study the solvent DMSO was used which.itself causes cellular effects. Seiler (124) using plate assays to study the mutagenicity of TCDD found a positive response in Satmonatta. typkunufuum strain TA1532, doubtful responses in strains TA1531 and TA1534, and negative responses in strains G46 and TA1530. Metabolic activation systems were not included in any of these microbiological assays. Beatty et al (7) conducted a study with -en \>Ww cultures of the mammalian cell types Hela, Balb-3T3, virus (SV-40) transformed 3T3 IV-71 �mouse fibroblasts, human foreskin fibroblasts and human lymphocytes. In all cases TCDD added in a final theoretical concentration of 10'° to the culture medium prior to the addition of cells resulted in no significant inhibition of growth measured after a period of four days. Electron microscopic examination of the TCDD-treated cells did not reveal any changes in morphology as compared to untreated cells. Incubation of human fibroblasts and SV-101 cells with 14C-labelled TCDD showed that incorporation of the TCDD into the cells did occur. Kondorosi et al (79) found that TCDD did not impair the transfectivity of QB-RNA, thus confirming the assumption that TCDD did not react chemically with nucleic acid. Whatever mutagenic property it had must have occurred by the forming of a physical complex by "intercalation" in DNA, leading, to frameshift mutation. In summarizing the limited literature dealing with the mutagenic and cytogenic potentials of TCDD in animals, it was noted that daily oral doses of 4, 8, or 12 yg/kg TCDD given to rats for seven days did not induce dominant lethal mutations. Five daily oral doses of 10 yg/kg TCDD and a single oral dose of 20 yg/kg TCDD did not produce cytogenetic damage in bone marrow cells of male rats. Chromosome aberrations were detected when male and female rats were dosed twice weekly at 4 yg/kg TCDD for 13 weeks. Using microbiological systems, TCDD has been shown to induce mutagenic changes in some strains of bacteria. V. SUMMARY OF THE LITERATURE REVIEW OF THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS In summarizing the literature on the toxicity of 2,4-D, 2,4,5-T and TCDD in animals, the following general statements provided a concept of the overall toxicity of each compound as they related to each other and the effects they produced in experimental animal studies. Where possible, inclusive statements were given rather than individual species responses. A. 2,4-D 1. The LDcn for single oral doses of 2,4-D in animals ranged from 100-2,000 mg/kg with the majority of LDso values in the 300-800 mg/kg range. 2. Signs of chronic 2,4-D toxicity did not differ greatly from those seen in acute toxicity. No effect levels, seen when 2,4-D was given in repeated oral doses, ranged from 30 to 75 mg/kg. 3. Being a strong acid, 2,4-D was rapidly eliminated from the body mainly via the urine. The plasma half-life of a single oral dose was in the 3-12 h range. After high doses or repeated lower doses, 2,4-D accumulated in the tissues; with residue levels rapidly declining as evidenced by a half-life of 1 to 2 weeks. IV-72 �4. No teratogenic signs were seen in rats fed repeated doses of 1,250 to 1,500 mg/kg 2,4-D of diet, nor when repeated daily doses of 8.75 mg/kg were given. Embryo toxic and fetotoxic responses appeared in rats and hamsters at repeated daily oral doses of 100 to 150 mg/kg. 5. Tumors were not produced in mice fed 46.4 to 100 mg/kg 2,4D of diet nor in rats fed 1,250 mg/kg 2,4-D of diet for 18 to 24 months. Single subcutaneous injections of 21.5 to 215 mg/kg 2,4-D did not produce carcinogenic or tumorigenic responses in mice. 6. The 2,4-D was not highly cytotoxic in laboratory animals and did not cause increased mutation rates nor did it stimulate a mutagenic response in rats and mice. No mutagenic or cytogenic responses were seen in several studies using microbial systems for the detection of such toxicity. B. 2,4,5-T 1. The acute toxicity for 2,4,5-T was in the same general range as for 2,4-D in most animal species. The 1050 values for single oral doses of 2,4,5-T ranged from 380 to 940 mg/kg in small laboratory animals. 2. Chronic toxicity studies in mice using repeated oral doses of 30-120 mg/kg 2,4,5-T produced no effect. An overlapping of adverse effects were seen, however, in doses of 60-140 mg/kg 2,4,5-T, depending on the strain of mouse studied. The no effect level for rats orally administered repeated doses of 2,4,5-T was approximately 30 mg/kg while as much as 300 mg/kg of diet could be fed with no adverse effects being noted. Threshold toxicity levels for adverse effects of repeated oral doses of 2,4,5-T in rats was approximately 100 mg/kg or 1,000 mg/kg of diet. 3. Single doses of 2,4,5-T were eliminated in animals, primarily unchanged, via the urine and feces over a period of a few hours up to about 7 days. 4. It was evident that 2,4,5-T induced embryotoxic and teratogenic responses in some strains of mice, rats and in hamsters when repeated oral doses of 20 to 400 mg/kg were administered. However, doses of 20 to 150 mg/kg in the same laboratory animal species produced a negative or no effect response. This indicated a great species and strain variation in response to 2,4,5-T as well as the fact that the embryotoxic and teratogenic potential of 2,4,5-T varied with the concentration of TCDD present. Levels of TCDD greater than 1 mg/kg were required to enhance the embryotoxic and teratogenic potential of 2,4,5-T. Embryotoxicity and teratogenic studies in pregnant rabbits, sheep and rhesus monkeys have been negative. 5. In most strains of mice, oral doses of 21.5 mg/kg or repeated doses of 60 to 100 mg/kg in the diet or drinking water and single subcutaneous doses of 2.5 mg/kg of 2,4,5-T did not induce tumor formation. IV-73 �6. In animals, 2,4,5-T, like 2,4-D was not highly c>totoxic and did not increase mutation rates nor stimulate a mutagenic response in rats and mice. It produced, however, chromatid abnormalities in cultured human lymphocytes and affected the chromosomes and reproductive mechanisms in mouse and hamster bone marrow cells. These effects may have been due to cellular toxicity rather than genetic alterations. No mutagenic responses were seen in several studies using microbial systems for the detection of such toxicity. C. TCDD 1. TCDD was an extremely toxic material with a single oral dose LDgg range of 0.6 yg/kg in male guinea pigs to 115 yg/kg in rabbits. 2. Chronic toxicity was manifested by hepatic necrosis, thymic atrophy and depletion of lymphoid organs. In mice and rats, repeated oral doses of 0.001 to 10 yg/kg for four to 13 weeks produced a no effect response for weight gain and no signs of toxicity were noted. Repeated oral doses as low as 1 yg/kg caused guinea pigs to become moribund and a repeated dose of 0.04 yg/kg decreased lymphocyte counts. Acne of increasing severity was produced in rabbits when doses of 0.04 to 400 yg/kg were applied repeatedly to the internal surface of the ear. A total oral dose of 2-3 yg/kg over a nine month period produced severe hematological changes and death in rhesus monkeys. 3. The primary route of excretion for TCDD in animals appeared to be the feces, with urinary excretion occurring at a much reduced rate. Liver and fat accumulated about 10 times higher levels of TCDD than did other body tissues. The half-life for TCDD in rats, following repeated exposure, was 12-15 days after termination of treatment. 4. It was apparent that TCDD caused birth defects and embryo mortality. Repeated daily oral doses of 0.1 to 2 yg/kg TCDD in pregnant mice produced no effects on the embryos; however, 3 yg/kg was the threshold level for production of cleft palate and kidney abnormalities. Single or repeated oral doses of 6.5 to 40 yg/kg TCDD were required to produce cleft palate in 50 percent or more of some strains of mouse embryos. Daily subcutaneous injections of 1 to 3 yg/kg TCDD produced cleft palate and kidney abnormalities in 50 percent or more of three different strains of mouse embryos. Repeated daily oral doses of 0.03 to 0.125 yg/kg TCDD produced no effect in some rat strains while doses of 0.125 to 2 yg/kg TCDD depressed fetal weight, lowered fetal survival and caused internal hemorrhages in fetuses. When teratogenic lesions appeared in rats, kidney abnormalities were more common than cleft palate. 5. No tumors were produced in a preliminary study where 0.007, 0.07, or 7 yg/kg TCDD was administered to mice in weekly oral doses for 12 months. When levels of 1 and 5 yg/kg TCDD of diet and 1, 5, 50 and IV-74 �500 ng/kg TCDD of diet were fed to rats for 78 weeks, an overall tumor incidence of 38 percent was present in the test animals. No effects were produced when 0.001 yg/kg TCDD was given to rats via the diet for 2 years. A level of 0.01 yg/kg TCDD given via the diet for 2 years produced liver nodules and hyperplasia of the lung epithelium. A level of 0.1 yg/kg TCDD in the rats' diet for 2 years produced an increase in liver and lung carcinomas. Monkeys fed 500 ng/kg TCDD of diet for 9 months did not develop tumor but died of marked hematological alterations. 6. Daily oral doses of 4, 8 or 12 yg/kg TCDD given to rats for seven days did not induce dominant lethal mutations. Five daily oral doses of 10 yg/kg TCDD and a single oral dose of 20 yg/kg TCDD did not produce cytogenic damage to bone marrow cells of male rats. Chromosome abnormalities were noted in male and female rats dosed twice weekly at 4 yg/kg TCDD for 13 weeks. Using microbiological systems, TCDD has been shown to induce mutagenic changes in some strains of bacteria. IV-75 �CHAPTER IV LITERATURE CITED 1. Advisory Committee on 2,4,5-T. 1971. Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency. 76 p. 2. Allen, J.R., D.A. Barsotti, J.P. Van Miller, L.J. Abrahamson and J.J. Lalich. 1977. Morphological changes in monkeys consuming a diet containing low-levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. food Co4met. Tox^col. 15:401-410. 3. Allen* J.R., J.P. Van Miller and D.H. Norback. 1975, Tissue distribution, excretion and biological effects of [l^C] tetrachlorodibenzo-p-dioxin in rats. Food Co-dme*. Toxx.co£. 13:501-505. 4. Andersen, K.J., E.G. Leighty and M.T. Takahashi. 1972. Evaluation of herbicides for possible mutagenic properties. 3. Agt. Food Chew. 20(3):649-656. 5. Bage, G., E. 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IkivotnovodAtvo 34(2):73-74. (Russian) 117. Sauerhoff,. M.W., W.H. Braun, G.E. Blau and P.O. Gehring. 1976. The dose-dependent pharmacokinetic profile of 2,4,5-trichlorophenoxyacetic acid following intravenous administration to rats. Toxxco£. App£. PkaAmac.o£.. 36:491-501. 118. Schiller, K. 1964. Beeinflussung der Fertilit'at von Ratten durch Stoffe mit Sonderwirkung im Kartofelbau. 14(2):111-114. (German). 119. Schillinger, J.I. 1960. Hygienische Wertung von landwirtschaftlichen Erzeugnissen, angebaut unter Anwendung von Herbiziden. J. Hyg. EpUe.miol. 4:243-252. (Czech). 120. Schulz, K.H. 1968. Zur Klinik und Atiologie der Chlorakne. Arbeitsmed. Soz-uLtmed. M.b<i£ti>hyg. 3:25-29. (German). 121. Schwetz, B.A., J.M. Morris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Emerson and C.G. Gerbig. 1973. Toxicity of chlorinated dibenzo-p-dioxins. EMUAOPI. Heo£#i Pmpeet. 5:87-99. 122. Schwetz, B.A., G.L. Sparschu, P.J. Gehring. 1971. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and esters of 2,4-D on rat embryonal, fetal and neonatal growth and development, food CoAmnt. To?o.c.o£.. 9:801-807. 123. Seabury, J.H. 1963. Toxicity of 2,4-dichlorophenoxyacetic man and dog. M.ch. EmuAon. HeotCfc. 7:202-209. acid for 124. Seiler, J.P. 1973. A survey on the mutagenicity of various pesticides. Expe*x.mtui 29:622-623. 125. Shavgulidze, M.M., V.I. Nanobashvili and M.N. Mirianashvili . 1976. Toxicity of the herbicide 2,4-D. -Vzt<iru.naMycL 4:103-104. (Russian). 126. Shirasu, Y. 1975. Significance of mutagenicity testing on pesticides. Envlton. Qual. Satf. 4:226-231. 127. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada. 1976. Mutagenicity screening of pesticides in the microbial system. Mutation R&6. 40:19-30. IV-85 �128. Smith, F.A., B.A. Schwetz and K.D. Nitschke. 1976. Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in CF-1 mice. Tox^col. App£. 38:517-523. 129. Sokolik, I. Yu. 1973. Effect of 2,4,5-trichlorophenoxyacetic acid and its butyl ester on embryogenesis of rats. Byu£t. Efe^p. &to£. Med. 76(7):90-92. (Russian). 130. Sparschu, G.L., F.L. Dunn, R.W. Lisowe and V.K. Rowe. 1971. Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid on fetal development in the rat. Food Co4me£. To3u.co£. 9:527-530. 131. Sparschu, G.L., F.L. Dunn and V.K. Rowe. 1971. Study of the teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Food Commit. To3u.eo£. 9:405-412. 132. Stupnikov, A. A. 1966. Comparative toxicity of chemicals and mineral fertilizers. Translations on USSR Agr. No. 163, U. S. Dept. of Commerce, Joint Publications Research Service, Lesnoye Khozyaystvo 7:29-31. 133. Styles, J.A. 1973. Cytotoxic effects of various pesticides -in vivo and In vi&io. Matat. Re4. 21(1):50-51. 134. Thigpen, J.E., R.E. Faith, E.E. McConnell and J.A. Moore. 1975. Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infect. Immun. 12:1319-1324. 135. To'th, K. , J. Suga'r, S. Somfai-Relle and J. Bence. 1977. Carcino bioaAAay o^ the, keA.bi.cide., 2,4,5-tnic.h£oiaphe.nox.yy<ithanol (TCPE) with cU.MeAe.nt 2,3,7,8~teJ^chZaKa<iib<inzo-p-<LiQ.iu.n (cU.o\in) c.onte.nt. in SMU>& mice. \r\_ International Conference on Ecological Perspectives on Carcinogens and Cancer Control. Cremona, 1976, Basel, Karger AG (In press). 136. Van Miller, J.P., J.J. Lalich and J.R. Allen. 1977. Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8tetrachlorodibenzo-p-dioxin. ChwoAph&ie. 9:537-544. 137. Van Miller, J.P., R.J. Marlar and J.R. Allen. 1976. Tissue distribution and excretion of tritiated tetrachlorodibenzo-pdioxin in non-human primates and rats. Food Cotmet. Toxicol. 14:31-34. 138. Vinopal, J.H. and J.E. Casida. 1973. Metabolic stability of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mammalian liver microsomal systems and in living mice. M.oh. Emu/ton. Contain. Tox*cco£. 1:122-132. IV-86 �139. Vos, J.G. and J.A. Moore. 1974. Suppression of cellular immunity in rats and mice by maternal treatment with 2,4,7,8-tetrachlorodibenzo-p-dioxin. Int. M.ch. MZeAgy App£. Immanol. 47:777-794. 140. Vos, J . G . , J.A. Moore and J . G . Z i n k l . 1974. Toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice. Toxico£. App£. PhaAmacol. 29:229-241. 141. Walker, E . M . , Jr., R . H . Gadsden, L . M . Atkins and G . R . Gale. 1972. Some effects of 2,4-D and 2,4,5-T on Ehrlich ascites tumor cells Jun VM.VO and <in \jWio. Ind. Mecf. 41(l):22-27. 142. Weissberg, J.G. and J.G. Zinkl. 1973. Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin upon hemostasis and hematologic function in the rat. Environ. Health Pmpea*. 5:119-123. 143. World Health Organization. 1971. 1970 Evaluations on some pesticide residues in food. The Monographs. WHO/Food Add./71.42, pp 459-477. 144. W i l s o n , J . G . 1977. Envvtomzntal c.kzm.c.atb . P 357-386. In_ Handbook of Teratology. J . G . Wilson and F . C . Fraser ( E d s . ) . Vo-1 . 2. Plenum Press, New York. 145. W i l s o n , J . G . 1977. Teratogenic effects of environmental chemicals. Fed. Ptoc. 36 (5): 1698-1 703. 146. Zetterberg, G. 1977. Experimental results of phenoxy acids on microorganisms. ln_ Chlorinated Phenoxy Acids and their Dioxins: Mode of Action, Health Risks and Environmental Effects. C. Ramel , ( E d ) , Ecol. Bull. (Stockholm), 27: ( i n press). 147. Z i e l i n s k i , W . L . , Jr. and L. Fishbein. 1967. Gass chromatographic measurement of disappearance rates of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice. J. Ag/u.c. food Cfiem. 15:841-844. 148. Z i n k l , J . G . , J . G . Vos, J . A . Moore and B . N . Gupta. 1973. Hematologic and clinical chemistry effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Env&ion. H&altn PeAApe,c.t. 5:111-118. IV-87 �CHAPTER V 2,4,5-T/TCDD EPISODES I. INTRODUCTION The current controversy over the potential adverse human effects of 2,4,5-T and TCDD stem from a chain of events that occurred in the 1960s. The presence of TCDD as a contaminant, potent acnegen and acute toxin in the production of 2,4,5-trichlorophenol was documented in 1957 by Kimmig and Schulz (58). However, it was not until 1964 that concern over the levels of TCDD in 2,4,5-T herbicide was reported. In that year, the Dow Chemical Company experienced contamination problems during its expansion in production of 2,4,5-T to meet the requirements for Herbicide Orange by the U.S. military. They closed their production facilities and made extensive modification in the reaction conditions for the synthesis of trichlorophenol. By late 1965, the new technology developed by Dow Chemical Company permitted production of 2,4,5-T containing no more than 1 ppm TCDD (36). Simultaneously, in 1964, the National Cancer Institute contracted for a screening study of a number of pesticides to determine if they were tumorigenic, teratogenic or mutagenic. Among the pesticides evaluated was 2,4,5-T herbicide. By 1967-68, preliminary data on 2,4,5-T from this screening study indicated that 2,4,5-T was teratogenic (15). The data were apparently provided to the press prior to actual publication. [When the manuscript eventually appeared in the scientific literature, in 1970, it contained a footnote indicating that the original sample of 2,4,5-T used in the screening tests contained approximately 30 ppm TCDD (23).] The press releases in the U.S. on the teratogenicity of 2,4,5-T were occurring in the same time period that South Vietnamese newspapers were publishing reports of an alleged increased occurrence of birth defects in areas sprayed with Herbicide Orange. These releases elicited far-reaching reactions from governmental agencies, segments of the scientific community and various lay groups concerned with environmental problems (2). On October 29, 1969, the President's Scientific Advisor announced that a series of coordinated actions was being taken by several governmental agencies to restrict the use of 2,4,5-T herbicide. Additional animal experiments performed early in 1970 confirmed that pregnant mice did deliver some malformed offspring. The question then was one of whether, or to what extent, such animal data could be extrapolated to man. On April 14, 1970, the Secretary of Health, Education and Welfare (HEW) advised the Secretary of Agriculture that: "In spite of these uncertainties, the Surgeon General feels that a prudent course of action must be based on the decision that exposure to this herbicide may present an imminent hazard to women of child-bearing age." Accordingly, on the following day, the Secretaries of Agriculture; HEW, and Interior jointly announced the suspension of 2,4,5-T for "all uses around the home, recreation areas, and similar sites" and "all uses on crops intended for human consumption." Immediately thereafter, the Department of Defense suspended the use of Herbicide Orange in South Vietnam (7). V-l �Numerous incidents involving suspected 2,4,5-T/TCDD poisoning of humans or livestock have been reported since this initial controversy. The most recent alleged episode involved veterans of the Vietnam Conflict. In March 1978, WBBM, a CBS-owned television affiliate in Chicago, Illinois, aired a special report on "Agent Orange: Vietnam's Deadly Fog". In the film, a number of past episodes allegedly involving 2,4,5-T and TCDD were examined. This chapter will review the available scientific data on these and other episodes, including industrial episodes, assessments in South Vietnam and the incident that occurred in Seveso, Italy, in July 1976. The medical data on many of these episodes are reviewed in Chapter VI. The expression of units of weight, area, or volume has not been standardized between the various publications cited in this Chapter. II. INDUSTRIAL EXPERIENCES A. Industrial Processes The herbicide, 2,4,5-T, was first commercially produced in the United States in 1944 (79). The quantity o'f 2,4,5-T produced and used in the United States and in world agriculture increased steadily until 1968-69, after which a sharp decline in its use occurred. Table 1 shows total U.S. Production data for 2,4,5-T, and how it was subsequently used, during the period 1961 through 1969. Approximately 34 percent (53 million pounds) of the total U.S. production was procured by the Department of Defense for use in South Vietnam. However, 8.9 million pounds of the 53 million pounds were not sprayed in South Vietnam, but rather destroyed by at-sea incineration in 1977 (see Chapter II). During the same period, 1961 through 1969, 50.6 percent (78.1 million Ib) of the total U.S. 2,4,5-T production was used in domestic herbaceous and woody plant control programs. The synthesis scheme for the industrial production of 2,4,5-T herbicide is shown in Figure 1. Forth (40) has described two different processes for the manufacture of the herbicide. The "Dow" process is a pressureless, high temperature process (>160°C but <200°C) requiring the alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene to sodium trichlorophenate in the presence of ethylene glycol (an alcohol) and caustic soda (e.g., sodium hydroxide). The second process, the "Boehringer" process uses high pressure (19.5 atmospheres) but low temperature (157°C) conditions in the presence of methanol, caustic soda, and 1,2,4,5-tetrachlorobenzene. Both processes will result in the formation of sodium trichlorophenate. The sodium trichlorophenate can be acidified to form trichlorophenol or may be used directly in the production of 2,4,5-T by adding chloroacetic acid. The production of the n-butyl ester (NBE) of 2,4,5-T is accomplished by V-2 �TABLE 1. Total United States production and use of 2,4,5-T herbicide for the period 1961 through 1969.a Use Million Pounds Herbicides Green, Pink and Purple^ Percent of Total 1.6 1.04 Herbicide Orange0 51.4 33.27 Exports 23.4 15.15 Domestic Use 78.1 50.55 154.5 100.01 Total a Total production and export data were from The Pesticide Review, 1970 and earlier issues, U.S. Department of Agriculture, Agricultural Stabilization and Conservation Service, U.S. Government Printing Office, Washington D.C. Data expressed in acid equivalents. Data based on estimated number of gallons of Herbicides Green, Pink and Purple used in South Vietnam, 1962-1964. c Data based on estimated number of gallons of Herbicide Orange used in South Vietnam (10,645,904 gallons) plus the surplus 2,215,125 gallons remaining after termination of Operation RANCH HAND. V-3 �methanol or ethylene glycol caustic in«0 / socja Cl' " X "Cl 1 c n O - 180° C \" 160° 1,2,4,5-tetrachlorobenzene - where R = CH or OUCH Chloroacetic acid,^ sodium hydroxide sodium trichlorophenate > 230°C 0 0-CH^C-OH 2,4,5-T butanol, anhydrous hydrochloric acid 0-CH2C-0-CH2CH2CH2CH3 Cl n-butyl ester 2,4,5-T FIGURE 1. Synthesis scheme for production of the n-butyl ester 2,4,5-T (NBE 2,4,5-T) and site where formation of TCDD may occur. V-4 �esterification using butanol and anhydrous hydrochloric acid. TCDD is formed only, during the formation of the phenol. Dimerization of the sodium trichlorophenate to form TCDD will occur in the reaction vessel during the alkaline hydrolysis of 1,2,4,5-tetrochlorobenzene. Maintaining low temperatures, J160°-'I800C, will minimize the formation of TCDD. The reaction temperatures during the "Dow" process may become difficult to maintain. If the temperature of the hydrolysate rises above the normal ,180°C, an exothermic reaction occurs after any residual solvent, e.g., glycol, is removed by distillation. This reaction, attributed to the decomposition of sodium-2-hydroxethoxide, starts at a temperature of 230°C and continues to 410°C. The heat generated by this reaction assists in the formation of TCDD through the dimerization of two molecules of sodium trichlorophenate. The rapid temperature increase in the reaction vessel, results in a pressure increase; failure to release the pressure has resulted in some of the industrial accidents that have been reported (46). B. Industrial Episodes In the years since the first commercial production of 2,4,5-T herbicide (1946-47), there have been numerous industrial episodes involving exposure to TCDD (and/or other chlorinated dibenzo-p-dioxins). The exposure to TCDD normally occurred during the handling of contaminated intermediate products (e.g., trichlorophenol, TCP). Fifteen of 23 episodes recorded in the literature were apparently associated with this "occupational" exposure. However, on eight occasions, explosions occurred, generally during the production of sodium trichlorophenate, and personnel were exposed to TCDD at the time of the accident, during the clean-up of the accident or from subsequent contamination of the workshop environment. The first reported industrial accident occurred in Nitro, West Virginia, in 1949 (51). A total of 228 people were poisoned by the reactor residue during and/or immediately after the accident. No measures were taken to decontaminate the factories or to control the residue from the reaction vessel. In 1953, an accident occurred at a TCP factory in Ludwigshafen, Federal Republic of Germany (43). The 55 workers that showed chloracne and other acute toxic effects were exposed to the residue of the reaction vessel either during the accident or in the subsequent clean-up work. By 1957, Kimmig and Schulz (55) implicated TCDD as the causative agent of at least the chloracne seen in these workers. The most thoroughly documented episode of occupational exposure has been by Oirasek et al (54,55) and occurred in Czechoslovakia between 1965 and 1969. In 1965, two technicians developed chloracne during the evaluation of a new production process for the manufacture of 2,4,5-T. At the time, it was assumed that they were exposed because of careless work and defective equipment under the pilot plant conditions. During the following two years, after the plant became operational, an additional V-5 �76 people developed chloracne. An investigation and study of the entire problem finally resulted in a shutdown of the operation and abandonment of the production in 1968. Jirasek et al have carefully described the afflicted individuals and have continued to monitor their health since the onset of the disease. The above two episodes and other episodes involving occupational chloracne associated with the manufacture of chlorinated phenols are presented in Table 2. The clinical features of the affected cases described in the 23 episodes listed in Table 2 are described in Table 3. Note that most features observed were inconsistent with the exception of chloracne. Certainly, extended exposure to the major chemicals [TCP, pentachlorophenol (PCP), 2,4-D or 2,4,5-T] must have complicated the observed clinical features. In addition to TCDD, it should also be noted that TCP may also contain significant concentrations of hexachlorodibenzop-dioxin, while PCP may contain hexa-, hepta- and octachlorodibenzo-p-dioxin. An extensive review of occupational chloracne has been prepared by Crow (24) and Kimbrough (56). With one exception, men have been almost exclusively affected by the disease due to the occupational position they have historically maintained in the factories producing the chlorinated phenols. The few women and children that have been afflicted were exposed because of contact with clothing worn by the men. Goldmann (43) reported that a female animal nurse developed chloracne by contact with contaminated test animals. The one industrial incident where a large number of women were afflicted with chloracne was reported by Braun (17) in 1959. Braun examined 114 women and 9 men, who in the course of a year became afflicted with chloracne in a condenser factory where chloronaphthalenes of different degrees of chlorination were used as dip-waxes. The reporting of this incident is important because of contradictory statements in the literature on the differential sensitivity of different people (including^-sexes) to chloracne. From the examination of the women Braun concluded the following: 1. Age was of no importance within the range of 20 to 45 years. 2. Strongly adiposed persons were more likely to be afflicted. 3. Seborrhoic types with greasy skin and open pores and scars of previous Ac.ne vutg<wit> were sooner and more severely afflicted. 4. Non-affliction was definitely extremely rare under the circumstances. Only four women (2 of them sisters) with a very smooth and fine skin through which the veins showed bluish when they were at rest remained free from the alterations due to the disease. 5. The occurrence of chloracne had no real relationship to hair color and skin pigmentation. V-6 �TABLE 2. Industrial incidents Year Country associated with the manufacture of chlorinated phenols. Primary Production Source of Manufacturer/Location3 Productr Exposure Monsanto/ Nitro, West Virginia . / / Nordrhein, Westfalen / / TCP Explosion PCP, TCP 1952- West Germany 53 1953 West Germany Years from Number Incident to of Last Cases Observation0 Reference 228 4 Occupational 17 1 TCP Occupational 60 12 DUcrir 1 liyt:i / TCP Occupational 37 46 BSAF/ Ludwigshafen TCP Explosion 55 24 51, 43 Boehringer, Ingleheim/ Hamburg TCP, 2,4,5-T Occupational 31 9 58, 12 1956 France Rhone Poulenc/Grenoble TCP Explosion 17 2 30 1956 United States Diamond Alkalai/ Newark, New Jersey 2,4-D, 2,4,5-T Occupational 29 13 1956 United States tlUUKci/ TCP Occupational (?) 46 1960 United States Diamond TCP Occupational (?) 46 1949 United States 1949 West Germany 1952 West Germany 1954 West Germany Ch amv»ni~ \r t 51, 73 11 16, 68 �Table 2 (continued) 1962 Italy 1963 Netherlands 1964 USSR 1964 United States / 5 TCP Explosion Philips-Duphar/ Amsterdam / / TCP Explosion ' 50 2,4,5-T Occupational 128 Dow Chemical/ Midland, Michigan 2,4,5-T Occupational 60 6 38 TCP Occupational 78 6 54, 55 67 1965- Czechoslovakia Soolana/ 69 Rhone Poulenc/ Grenoble 47, 51 14 14, 26 51 50, 74 1966 France TCP Explosion 21 1968 United Kingdom Coalite and Chemicals TCP Products/ Bolsover, Derbyshire Explosion 79 9 51, 60 1970 Japan PCP, 2,4,5-T Occupational 25 3 64 1972 USSR TCP Occupational 1 1 81 2,4,5-T Occupational 50 40, 46 00 1973 Austria / / Linz Nitrogen 46 Un i-l-i: / worKs/ 1974 West Germany Bayer/Uerdingen 2,4,5-T Occupational 5 40, 46 1975 United States Thompson-Hayward/ TCP Occupational - 46 Kansas City, Kansas �Table 2 (continued) 1976 Italy ICMESA/Meda TCP Explosion 134 2 69, 80 a The name of the factory or company and its location was cited whenever it was available because considerable confusion exists in the published literature as to what incident is addressed. The absence of data indicates the information was not available. b TCP = trichlorophenol, PCP = pentachlorophenol Frequently individuals involved in an incident, and who were examined initially, may have also been examined at a later date. The years that lapsed from the exposure until the most recent examination are cited in this column. The absence of a number indicates that only an initial examination was reported in the referenced literature. �TABLE 3. Some clinical features observed in cases of chloracne associated with production of 2,4,5-T and other chlorinated phenols. Frequency observed Clinical Features Consistent Chloracne Additional Notes In worst cases, chest and inguinal area affected and scarring generally increased. Occasional Prophyria cutanea tarda Increased excretion of urinary uroporphyrin or coporporphyrin or both. -^ o Inconsistent Hyperpigmentation of the skin Usually prominent on face and consisted of grayish or brownish tone to the complexion. Hirsutism Noticeable between the outer edge of the eyebrow and the temple hair margin. Enlarged, tender liver Excessive mechanical fragility of the skin Neuromuscular symptoms Severe pains in the chest and pain and weakness in extremities �Table 3 (Continued) Mucous membrane irritation Itching of the eyes and frequent tearing, hyperemia of the nasal mucosa, and inflammation of the buccal mucosa. Irritability Nervousness and insomnia. �6. A simultaneous psoriasis or a systemic eczema in the previous case history, or a pregnancy, did not make any difference and had no perceptible effect on the course of the chloracne. One woman reported that she had noticed a worsening of her chloracne after her delivery. 7. During the menstrual period, the pimples on the cheeks of some of the women were temporarily more prominent. 8. Dirty and untidy women took ill sooner and more severely than those who placed great importance on cleanliness and hygiene. The persistence of dioxins in the environment of an industrial plant has been documented by Jensen (53). He reported on two cases of chloracne in employees of an outside contractor that had been working on a piece of equipment exposed (but thought to have been decontaminated) to TCDD three years earlier in an industrial explosion in Derbyshire, England in 1968. A young son of one of these employees also develeoped chloracne. The presumed source of the child's contamination was the father's working clothes. An episode involving the deliberate synthesis of TCDD has been reported by Oliver (65). This episode involved three young (male) scientists working with pure TCDD in the laboratory. Two of the men were exposed to the dioxin while attempting to synthesize it by heating trichlorophenol in an alkaline solution in the presence of a catalyst or by heating prepared potassium trichlorophenate in a closed system. Both men wore overalls and plastic gloves and allegedly took the utmost care to avoid inhalation or skin contact. The third man was a colleague of the other men and had been working with the diluted dioxin standards they had prepared. His work also had been done with the utmost caution and with special care to avoid personal contamination. Three clinical features were common to the three men, namely, chloracne, hyperpigmentation and hypercholesterolemia (increased levels of cholesterol). None of the three patients had evidence of acquired porphyria. However, two patients developed hirsutism (excessive facial hair) two years after the exposure. These same two also reported that when the hirsutism developed, other symptoms occurred, e.g., loss of appetite, oppressive headaches, and an unusual loss of vigor and drive with excessive fatigue. Oliver concluded from the evidence that those accidentally exposed to dioxin (TCDD) may be subject to delayed toxic effects for at least two years. III. VIETNAM EPISODE As noted in Chapter I, approximately 53 million Ib of 2,4,5-T were in the 13.2 million (gal) of Orange, Purple, Pink and Green procured by the Department of Defense. However, 8.9 million lb of 2,4,5-T were in the surplus Herbicide Orange. Thus, approximately 44 million pounds of 2,4,5-T were sprayed in South Vietnam from 1962 through 1970. As noted in Chapter I, an estimated 368 lb of TCDD were probably present in Herbicides Orange, Purple, Pink and Qreen. V-12 �Irish et al (52) have stated that among all the controversial subjects that were part of the conflict in Vietnam, the use of vegetationcontrol chemicals received an undue amount of publicity that was generally critical. They noted that the Department of Defense was not insensitive to the critics' pronouncements; justification for continuation of the RANCH HAND program had been periodically reviewed. The conclusions of all the evaluations prior to 1969 recognized that defoliation had reduced the incidence of ambushes, saved lives and disrupted enemy tactics. The issues of long-term ecological damage or potential adverse human health effects due to the herbicides were little discussed until the late 1960s. These issues when viewed in context with the realities of the military conflict were of minor concern, especially since the available scientific data did not support the justification for greater concern. It should be noted that in 1967 the Department of Defense had contracted with the Midwest Research Institute (MRI), Kansas City, Missouri, for an in-depth report on the assessment of ecological effects of extensive or repeated use of herbicides (49). Following its publication in December 1967, both the National Academy of Science (NAS) and the American Association for the Advancement of Science (AAAS) reviewed the document and concluded that MRI had "done a creditable job of assessing the scientific literature related to herbicides and their ecological effects." However, both organizations felt that the report represented "only a first step in investigating further the ecological effects of intensive use of herbicides" (3). Some of the conclusions that the MRI reported (49) included: 1. The greatest short-term or long-term direct ecological consequence of using herbicides in Vietnam or anywhere else is the destruction of vegetation. As long as soil sterilization is not an objective, destruction of vegetation by herbicides is a selective process, denuded earth does not occur especially in forest spraying. Furthermore, the end result of the use of herbicides from an ecological standpoint is that the ecosystem is set back to an earlier sere, i.e., an earlier stage of plant succession. 2. The long-term effects on wildlife may be beneficial or detrimental. Studies in other countries have shown that herbicidal treatment of forested areas improves wildlife habitat and is favorable to animal populations. The extent and pattern of herbicide treatment in Vietnam have no precedent; therefore, it is difficult to predict effects on wildlife with any accuracy. 3. The herbicides used in Vietnam will not persist at a phytotoxic (plant toxic) level in the soil for a long period of time. On the basis of the average temperatures and rainfalls in Vietnam, it would be reasonable to expect that the chlorophenoxy acid esters will be dissipated quickly. 4. The possibility of lethal toxicity to humans, domestic animals or wildlife by use of the herbicides used in Vietnam is highly unlikely and should not be a matter of deep concern. V-13 �5. Herbicides seldom persist in animal or insect tissues. Toxic transfer to the next higher animal in the food chain is minimal. In fact, biological concentration does not occur with most herbicides, since they are readily excreted from animals. In September 1968, the U.S. Department of State released an assessment of the ecological consequences of the defoliation program in Vietnam. Tschirley (75), a plant ecologist and the author of the Department of State report, published his assessment in Sconce (the Journal of the AAAS) in February 1969. The major conclusions reached by Tschirley after his fourweek visit to South Vietnam included: 1. The defoliation program has caused ecologic changes. These changes are not irreversible, but complete recovery may take a long time. Regeneration of the mangrove forest to its original condition is estimated to require about 20 years. 2. The effects of defoliation on animals is not known, but it does not appear to have been extreme. There is no evidence to suggest that the herbicide used in Vietnam will cause toxicity problems for man or animals. In March 1969, the Society for Social Responsibility in Science, sponsored a five-week trip, for two zoologists to Vietnam with the objective of supplementing Tschirley's observations. The subsequent report, written by Orians and Pfeiffer (66), was published in May 1970. Their conclusions included: 1. The ecological consequences of defoliation were severe, especially in areas receiving repetitive applications of defoliants. 2. Evidence was found of moderate to severe defoliation of trees and herbs in areas many miles removed from sites of application. 3. Little evidence of toxic effects of the herbicides to animals was found, although one report was received (through an interview) of many sick and dying birds and mammals in forests following defoliation. The report was not investigated. 4. No evidence was found that the herbicides had direct adverse effects on human health. The defoliation program however, has had tremendous psychological impact upon the Vietnamese people, and the crop destruction program may have impacted on the availability of food for women, children and elderly people in the highland regions of South Vietnam. The first Herbicide Orange and July 5, 1969 reports resulted reports of human birth defects allegedly attributed to appeared in Vietnamese newspapers between June 26, 1969 (2). The public and scientific furor caused by these in two surveys of South Vietnamese hospital records V-14 �conducted independently by Cutting et al (25) and Meselson et al (63). An evaluation of both documents in 1971 by an advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency (2) concluded with the following summary: Summarizing the Vietnam data on human embryotoxicity, it can be said that (1) the sample of births surveyed was from year to year a variable but usually very small fraction of the total number, (2) it was quite unrepresentative of the geographic and ethnic distributions, (3) the heavily sprayed and otherwise exposed areas were greatly underrepresented, and (4) the birth records were not trustworthy and, therefore, the rates of stillbirth, and especially of congenital malfoVmation, derived from them were equally unrealiable. For example, the overall congenital malformation rate found in South Vietnam, 4.91 per 1000 livebirths, is about half of what was reported in other studies in various parts of Asia, and possibly a quarter of what might actually exist at term. A further indication that the newborn children were not carefully examined is the absence of Down's syndrome in the list of specific malformations compiled by the Army survey [Cutting et al (25)] despite the fact that some Oriental populations have been reported to have an incidence of this condition not unlike that in Western populations. Finally there is, and can be, no precise knowledge or reasonable approximation of the exposure to 2,4,5-T (and hence, TCDD) experienced by pregnant Vietnamese women, including what amounts they ingested or absorbed and when this may have occurred during pregnancy. Thus, any attempt to relate birth defects or stillbirths to herbicide exposure is predestined to failure. It can only be concluded that the birth records that have been surveyed, and probably any that will be surveyed in the future, for South Vietnam for the period 1960-1970 cannot answer positively the questions about possible adverse prenatal effects following human exposure to 2,4,5-T. It must be emphasized, however, that the searches that have been made almost certainly would have revealed any marked increase in the incidence of birth defects or the introduction of a striking defect such as that produced by thalidomide. In spite of considerable effort, no such occurrences were found. Following the publication of the above two surveys, some additional reports of birth defects in South Vietnam were released. One of these was by Tung et al (77) of North Vietnam (Democratic Republic of Vietnam). They reported that out of a total of 903 South Vietnamese taking shelter in the North and grouped in hospitals and lodgings in Hanoi, 19 adult women, including 4 mothers, and 70 children between the ages of 6 and 14 had been directly hit by herbicidal sprays while living in South Vietnam. The report went on to state that of the above four mothers, two had given birth to children with Down's Syndrome (Trisomy 21). In addition, among V-15 �the 70 children between the ages of 6 and 14, numerous cases of deformations were evident, e.g., ocular lesions, exaggerated lumps on the forehead, valgus feet (i.e., feet that are bent outward) and a high frequency of chromosomal aberrations in lymphocytes and leucocytes. Following a summary of their data, Tung'et al (77) concluded by stating: Though still limited in number, our clinical observations confirm the results obtained on animals by American researchers. The massive and prolonged utilization of defoliants besides permanent ocular lesions, can cause chromosomic alterations among a population obliged to cling to ancestral soil and these alterations can provoke among* their progeny congenital malformations the importance of which remains to be determined. In the abominable history of wars, have we ever seen such an inhuman fate reserved for the survivors except in the case of atomic war? In reviewing the report by Tung et al (77), the Dow Chemical Company (8) noted that basically, the whole study was a result of a seemingly hit and miss clinical examination of some refugees from South Vietnam who had lived in regions where defoliants had been applied. There was no record of exposure except that most had been sprayed at one time or another with something. Dow further stated: "Trying to correlate cause and effect from the published data is completely frustrating and futile. There is no doubt that these authors saw some ill people, but to reach the conclusion that their problems were caused by 2,4,5-T rather than the ravages of war is speculation." A study similar to Tung et al (77) was reported by Rose and Rose (71) in 1972. They interviewed 98 refugees in Hanoi who claimed to have been repeatedly sprayed with defoliants while in South Vietnam. Abortions were reported for humans and domestic animals and monstrous births were said to have occurred. Deaths evidently occurred among human, domestic animals, fish and fowl. The charge by Tung et al that TCDD in 2,4,5-T was reponsible for much of the Down's Syndrome seen in South Vietnam was also made by Grumner as reported by Honoroff (48). Grumner, apparently of Rockstock University, German Democratic Republic, claimed to have observed high incidences of children with Down's Syndrome while on a trip through North and South Vietnam. Honoroff quoted Grumner: Provided that it be understood that this estimate cannot be anything but a cautious one, it may be assumed that there are at least 25,000 children with hereditary defects in South Vietnam. This does not include all the unborn babies whose mothers were sprayed during the missions that were flown in recent months. It does not include those who were stillborn, or died soon after birth, on account of their serious chromosomatic defects. Even V-16 �after the war, it will probably be possible to arrive at only an approximation of the entire scale of this crime since one will only be able to examine the survivors when the time comes. In 1973, Tung et al (78) reported an increase in the number of persons with primary liver cancer in proportion to all cancer patients admitted to Hanoi hospitals during the period 1962-1968 (790 liver cancer cases out of 7,911 cancer cases, 10 percent) as compared to the period 1955-1961 (159 liver cancer cases out of 5,492 total cancer cases, 2.9 percent), which was prior to the start of herbicide spraying. The authors attributed this increase to exposure as a result of the spraying of herbicides containing TCDD in South Vietnam during the 1960s [however, a recent IRAC monograph (50) noted that limitations in the reporting of the study make impossible an adequate assessment between the incidence of liver cancer and herbicide spraying in South Vietnam]. A further factor of importance has -been suggested by Ford et al (39). They noted that at least in Thailand, consumption of aflatoxin-contaminated food was highly correlated with liver cancers. Aflatoxin is a naturally occurring contaminant of cereal crops. In 1974, the National Academy of Science (NAS) (21) announced the results of studies conducted in South Vietnam in 1972 and 1973. The NAS Committee could find no conclusive evidence of association between exposure to herbicides and birth defects in humans. Available records of two major Saigon hospitals and evaluation of records in a third, as far as they went, showed no consistent pattern of association between rates of congenital malformations and annual amounts of herbicides sprayed. The Committee recognized, however, that the material was not adequate for definite conclusions. The Committee was also unable to confirm or deny reports that some humans (especially the Montapnards) and domestic animals became ill or died after exposure to herbicide sprays or after eating treated plants or drinking contaminated water. The Committee also attempted to assess the social, economic and psychological effects of the herbicide program. The impact of the program on the population "appeared relatively trivial as compared with other aspects of the upheaval in that country." Evidence was obtained that numbers of families moved away from their traditional homes because of the herbicide spray program but few were actually identified. In a letter of transmittal for the NAS report (21), the President of NAS stated: "On balance, the untoward effects of the herbicide program on the health of the South Vietnamese people appear to have been smaller than one might have feared." IV. EASTERN MISSOURI HORSE ARENA EPISODE In August 1972, the Missouri Division of Health, St. Louis, Missouri, and The Center for Disease Control, Atlanta, Georgia (59) reported an investigation of a horse arena in eastern Missouri where 54 of 57 horses exposed to the arena had died of an illness characterized V-17 �by skin lesions, severe weight loss and heptotoxicity. Birds, dogs, cats, insects and rodents were also found dead in and around the arena, and one 6-year-old girl exposed developed epistaxis, gastrointestinal complaints, and severe hemorrhagic cystitis (characterized by blood in the urine). Analysis of urine cultures for bacterial and viral pathogens was negative. Three other persons developed milder illnesses consisting primarily of transient headaches and nausea after exposure to the arena. The toxic substance(s) responsible for the illness was not at that time identified. In the investigation of the illness, Lobes et al (59) found that the outbreak coincided with treatment of the arena floor for dust control with approximately 2,000 gal of salvaged motor oil. The treatment occurred on May 26, 1971. On May 30, the stable owners reported that "hundreds" of birds were found dead on the floor of the arena barn. Within the next few weeks, cats, dogs, rodents and horses began to die. The four people cited above [2 adults and 2 children (both girls)] had more than occasional exposure to the arena barn during the six months following the oil spraying. These individuals were first examined in mid-August 1971, The report (59) also noted that similar horse illnesses and deaths occurred in two other horse arenas in the eastern Missouri area sprayed by the same salvage oil company. The three arenas had been sprayed within one month of each other. Subsequent to investigation, soil from all three arenas was excavated and disposed. No further problems occurred following the excavations. In 1974, laboratory analysis of soil samples taken from the initial arena implicated 2,4,5-trichlorophenol (TCP) and TCDD as the probable toxic substances (29). The actual levels of TCDD in these soils however were not published until 1975, when Carter et al (19) provided more details on the exposure and the probable source of the TCDD in the salvage oil. The horse arena soil was found to contain 31.8 to 33 yg of TCDD per gram (ppm) of soil. In addition, further investigations revealed that the sludge used to spray all three arenas came from a common storage tank at the salvage oil company. It was suspected that TCDD and TCP were in distillate residues collected by the salvage company from a hexachlorophene producer in southwestern Missouri. Between February 1971 and October 1971 the salvage oil company obtained and stored 18,000 gal of the distillate in a storage tank from which the sludge for spraying the three arenas was obtained. In late 1971 the hexachlorophene plant and subsequently the salvage oil company both discontinued operations. The residue remaining in the tank originally used to store the distillate residue at the plant site was sampled in 1974. It contained TCDD in concentrations of 306 to 356 yg/g (19). Case (20) has described some of the clinical studies performed on the horses involved in this episode. Kimbrough et al (57) has recently (1977) detailed the epidemiology and pathology associated with the poisoning episode. V-18 �Commoner and Scott (22) have reviewed the Missouri Horse Arena Episode in an attempt to provide consultative data to the Italian Government in the wake of the Seveso, Italy episode. Their,review focused on the human reactions (symptoms) to accidental TCDD exposure and the problem of soil degradation of TCDD. They also provided an excellent chronological account of the episode. Beale et al (13) have recently re-examined the young girl who had developed hemorrhagic cystitis following repeated exposure to TCDD in one of the horse arenas sprayed with the waste oil. In the 5-year interval since exposure, the patient had grown normally, and both her height and weight were above the 75th percentiles. Detailed physical, chemical and neurological examinations were also conducted and found to be normal. The same studies were done on the patient's sister and mother, exposed simultaneously, but less extensively to dioxin, and the results were also normal. Beale et al (13) concluded: "Our experience demonstrates that people exposed to dioxin can recover completely with no apparent sequela from the toxin. It remains to be determined whether -the exposure to dioxin in these children will result in abnormal pregnancies or affect their offspring." V. THE SEVESO, ITALY EPISODE Perhaps the most publicized chemical accident in modern times is the TCDD episode in Seveso, Italy. This episode has attracted worldwide interest and concern. Hundreds of scientists, physicians and veterinarians have participated in either on-site inspections, conferences, or consultations into the various facets of this episode. Although the Seveso, Italy episode did not involve 2,4,5-T herbicide, it did involve the production of trichlorophenol. The trichlorophenol was in this case used in the production of hexachlorophene. Nevertheless, this episode represents to many people the inherent danger associated with the industrial production of 2,4,5-T. Data on levels of TCDD found, the magnitude of the contamination and the extent of human and animal illness have just recently begun to appear in the scientific literature. The following scenario of the episode has been assembled from this literature. The episode of TCDD poisoning occurred on 10 July 1976 in Seveso, Italy, a small town 40 kilometers (km) north of Milan (40,46). The source of the TCDD was a chemical factory that produced trichlorophenol through the alkaline hydrolysis of tetrachlorobenzene (see Figure 1). When the temperature in a steam-heated reaction vessel rapidly increased, a safety disk ruptured sending a plume of trichlorophenol, TCDD and other products 30 to 50 meters (m) high above the factory. The cloud apparently rose into the air, cooled and came down over a cone-shaped area about 2 km long and,700 m wide. V-19 �The chemical plant involved was the Givaudan ICMESA (Swiss-owned) chemical plant. At the time of the incident, there were some 2,000 kg of reagents and reaction products in the reactor (sodium trichlorophenate, soda, sodium chloride, ethylene glycol, tetrachlorobenzene and secondary reaction products) (9). Based on determinations made by production officials it was estimated that 4,000-500 kg of reaction product was discharged into the atmosphere (9,27). The amount of TCDD dispersed with the other reaction products has been estimated to have ranged from 650 grams to 1,700 grams (27,69). A sample of the escaped product taken from the reactor head for analysis revealed the presence of 3.5 percent TCDD (35,000 parts per million TCDD) (9). The accident occurred on a Saturday. By the following Monday, a site inspection of the area revealed phytotoxic effects (brown discoloration and drop-like perforation of the leaves) for a distance of some 1,000-1,300 m in a triangle with a base of approximately 400 m and a vertex of 100 m centered on the factory (9). Several measurements of TCDD on vegetation in this area and areas adjacent to the factory were in the 1 to 15 ppm range, with one reading as high as 50 ppm (69). • Reggiani (69) reported that animals (birds, rabbits and chickens) were beginning to die 2-3 days after the accident. A few children and some adults who had been directly seized by airborne dust consisting of the reactor content were complaining of nausea and presenting skin lesions of various aspects and extension but mainly redness and swelling. Some of the children were hospitalized and the physicians in charge warned that beyond overt signs of injury pointing to the action of caustic material causing burns and blister formation, they also had to consider the possibility of a contact or ingestion of a still unknown quantity of TCDD. In the meantime numerous Italian laboVatories and the Givaudan Laboratories cooperated in mapping out the polluted zones, determining TCDD on soil, vegetation and buildings by gas chromatographic-mass spectrometric techniques (9,41,69). In addition, the Regional Veterinary Service assisted in drawing up the map, working from animal death patterns and TCDD levels in the liver of surviving animals. Highest TCDD levels were found in herbivorous animals (41). About 1,000 assays led to the area being divided into two zones. Giovanardi (42) reported that the first zone, Zone A, was a triangularshaped area covering approximately 1000 hectares (ha). This area, located south south-east of the ICMESA factory and downwind at the time of the accident, had estimated soil levels of TCDD greater than 0.001 ppm [Reggiani (69) later described this area as having TCDD levels greater than 10 ppb]. The 700 inhabitants of this area were evacuated in three stages, on 26 July, 28 July and 2 August 1976. In the second zone, Zone B, soil levels of TCDD were detectable but less than 0.001 ppm [Reggiani (69) defined the soil levels of TCDD as between 0.1 and 10 ppb]. This area covered approximately 250 ha and was divided between a large urban center and an extensive rural area with some small residential V-20 �aggregates (42). This area had a population of 4,900 and was not evacuated. For the people in Zone B, recommendations were issued to reduce the possibilities of exposure in particular for the children and the women (69). A third zone, Zone C or "Respect Zone," covering a total of about 1,430 ha was also delineated. Occasional concentrations of less than 0.1 ppb TCDD in soil were found in this zone. The population of this area was approximately 40,000 people (69). By late August 1976, an extensive surveillance system of the health of the population was established covering the acute and mid-term effects of the exposure as well as the long-term effects. General and special medical examinations, laboratory tests at given intervals, course and outcome of pregnancies, examinations of abortions, rate of stillbirths, followup of newborns, morbidity and mortality of the population, and a cancer registry were all set up to detect any abnormality of the health of the community for which an exposure to TCDD could be postulated. The medical health surveillance program was extended to 11 districts with a total population of 216,000 (9,14,37,69). Periodically, reports of clinical damage to the population of Seveso have appeared in the press and scientific literature (38,40,41, 46,80). However, the most complete analyses of health data have been recently published by Reggiani (69). He concluded: The Seveso accident has not revealed up to now toxic effects in humans, which have not been observed in other episodes. Chloracne, the typical skin lesion, has occurred in children with tendency to spontaneous and rapid healing. The peripheral nervous system has perhaps been attacked and reacted with subclinical signs of impairment. Signs of involvement of the liver without apparent functional disorders have occurred. No other organs or functions have been impaired. There has been no derangement of the gestation, no foetal lethality and loss, no gross malformations, no growth retardation at term and no cytogenetic abnormalities. The immunocapability of the population, not even of the,children with chloracne, has not been attained. VI. GLOBE, ARIZqNA_EPISODE_ Globe, Arizona was another site of possible human exposure to TCDD. In 1969, the U.S. Forest Service applied 3,680 Ib of 2 (2,4,5-trichlorophenoxy) propionic acid (Silvex) and 120 Ib 2,4,5-T in the Kellner Canyon-Russell Gulch spray project near Globe (76). The reports of harmful effects to animals and people from the spraying began during and immediately after the spray treatment. The complaints included damage to vegetation off the spray project area, deformed animals and human illnesses. Although the Forest Service investigated the allegations, many of the local citizens were dissatisfied with the V-21 �reports and the case continued to fester until, in February 1970, it attained national attention. Television newscasts showed deformed animals alleged to have been caused by the herbicides. On February 13, 1970, a public hearing was held in Globe. As Tune Ma.ga.zwie. (4) reported, the local veterinarian insisted that he had noticed nothing out of the ordinary in local animals. Doctors too were puzzled. Said one: "I keep trying to see the relationship between the spraying and the illnesses, but I have simply not found anything." T-unn (4) also reported that: "The investigators holding the public hearing ended up perplexed and incredulous. In a paranoid outburst, the investigators were accused of being impostors, really representatives of chemical manufacturers in clever disguise." To look further into this episode, The Office of Science and Education, USDA, established an investigating team to assess the allegations against the Kellner Canyon-Russell Gulch Spray Project. Tschirley et al (76) published the results of the investigating team following on-site inspections of the spray project area, February 16-20, 1970. Tschirley et al, attempted to assess numerous parameters that would contribute to a comprehensive assessment of the episode. Some of these parameters included: (1) assessment of herbicide damage to plants off the project area, (2) effects of plant diseases, (3) effects of air pollution, (4) residue analyses of soils, plants and animal tissue, (5) observations of fish and wildlife, (6) evaluations of the health of domestic animals and (7) interviews with many of Globe's citizen and physicians. Some of the conclusions reached by Tschirley et al (76) were: 1. There was clear evidence of drift of herbicide outside the project area. 2. There was evidence of woody plant mortality from root rot, and also visible damage to certain yard trees from several kinds of birds and insects. 3. Reports from wildlife specialists indicated no significant effects on birds, deer and other wildlife. 4. With the exception of soil from the site where the herbicide was loaded aboard the helicopter, no residues of 2,4-D, 2,4,5-T, Si 1 vex or TCDD were found in any of the substrates analyzed. 5. Information obtained from owners of livestock and observations of animals did not indicate any illnesses that do not commonly occur in other regions. No association was found between the herbicides and the deformed animals shown on the television newscasts. 6. Human illnesses had been reported by several residents in the Globe region. Many of the residents with complaints were interviewed V-22 �by a medical member of the panel. The complaints were those that commonly occurred in the normal population; no cases of chloracne were reported. One individual had an eye irritation from steam cleaning an empty herbicide drum. Nine doctors serving the area of Globe were interviewed and there was general agreement that there had been no significant increase in human illness related to the spraying. Tschirley et al (76) summarized their panel report by stating: "Significant in evaluating the Globe situation was the emotional peak of its inhabitants. The complaints offered were those occurring in normal populations, with many of them (especially in the adults) being quite subjective. With the exception of the skin rash and eye irritation experience by one subject, it is highly unlikely that the ailments described were related directly to the spraying. However, the psychosomatic effect of an aroused public very likely has played a role. It is also important to note that except for three subjects all of the complaints dated only from the June 1969 spraying, despite the Forest Service having sprayed the same area three other years." A subsequent report was published by Roan and Morgan (70) of analytical results of selected human tissue collected by Tschirley et al (76) and of an epidemiologic study of the hospital records. Roan and Morgan concluded: We cannot find any evidence that there was long-term exposure of residents of the Globe, Arizona area to chlorophenoxy herbicides, or significant contamination of water supplies in this area with 2,4-D, 2,4,5-T, Si 1 vex or metabolites of these herbicides. Nor have we found contaminants such as TCDD that may be associated with one or more of the above technical grade products. Statistics on reproductive mortality and morbidity for the period 1960 through the first six months of 1970, from one hospital serving this area, do not indicate any trends that are suggestive of adverse influences on human reproductive function that might be associated with herbicide use during the years 1965, 1966, 1968 and 1969. Even though the analytical data available to us apply only to the years 1969 and 1970, the rate of disappearance of these compounds in the environment leads us to believe that gross, protracted contamination was probably absent in prior years as well. Although samples of. human tissues and body fluids, obtained through the cooperation of the medical profession in the area, are few in number, we believe the analytical results (which were all negative) are very probably representative. V-23 �VII. THE SWEDISH LAPLAND EPISODE In the spring of 1970, Swedish newspapers reported an accumulation of sudden deaths of reindeer grazing in the Visttrask area of Lapland. Approximately 30 reindeer, mainly young animals, died within a week after a heavy, wet snowfall, without any previous signs of illness. It was also reported that about 10 reindeer cows aborted their fetuses. Examination of several reindeer by veterinarians showed inanition (empty stomachs). When given additional feed, the deaths stopped. The case was of particular interest since it was learned that the area where the reindeer grazed had been treated with a mixture of 2,4-D plus 2,4,5-T (2). Analyses performed on liver and kidney samples (33,35) from one of the cows and three aborted fetuses noted above indicated traces of 2,4-D (0.2 to 0.5 ppm) and 2,4,5-T (0.3 to 1.0 ppm). Tree leaves contained 25 and 100 ppm of 2,4-D and 2,4,5-T respectively. However, no herbicides could be found in the ground vegetation. Although it was generally accepted that the deaths of the reindeer were attributed to starvation rather than exposure to 2,4,5-T and/or TCDD, Erne (34) initiated a controlled experiment on female reindeer and phenoxy herbicides. Erne's experiment involved thirty pregnant reindeer, where half of the animals were given birch leaves from an area that had been aerially sprayed with one of the products that was used in the Visttrask area, the rest received untreated birch leaves. The average daily intake of leaves for both test and control groups was about 1 kg per animal, which for the test group corresponded to a daily dose of phenoxy acid of 1 mg/kg body weight. After the feeding experiment, the reindeer were sacrificed and necropsied just before the expected parturition. During the course of the investigation, no clinical hematological or chemical signs were observed of injurious effects attributable to the sprayed leaves. The necropsy of the sacrificed animals showed nothing at all remarkable. All were pregnant (except one in the control group) and all the embryos were alive and normally developed. In histological investigations of the female reindeer and the fetuses, no pathological changes were observed that could be attributed to the prolonged consumption of sprayed leaves as fodder. Thus, Erne (34) concluded that the toxic manifestations noted in the Lapland incident were probably not caused by ingestion of herbicides. Immediately following the report of reindeer deaths (and concurrent with press reports on alleged health effects from 2,4,5-T and TCDD in Vietnam), two cases of congenital malformations in human infants were also attributed to alleged exposure of pregnant women during application of phenoxy herbicides in Lapland forests (2). However, competent medical scientists at the Institute of Hygiene and the Teratological Laboratories of the Karolinska Institute of Stockholm and at the Institute of Human Genetics at Munster, Germany, were unable to find temporal or clinical evidence to suggest that the occurrence of these human birth defects was more than coincidentally related to the herbicide operations. V-24 �The publicity given to the Lapland incident resulted in additional reports of alleged adverse human health effects due to the phenoxy herbicides. For example, in early 1972, Swedish newspapers reported excess lung cancer mortality among railroad workers exposed to 2,4-D and 2,4,5-T. These reports prompted the Swedish National Board of Occupational Safety and Health to request an epidemiological evaluation of the stated excess mortality and its relation to herbicide exposure (10). The subsequent investigation as reported by Axel son and Sundell (10) in 1974 found that a slightly dose-dependent and significantly increased tumor incidence and mortality among workers exposed to the herbicide amitrol (3-amino-l,2,4-triazol) whereas those exposed to 2,4-D or 2,4,5-T had about normal tumor incidence and mortality. The study comprised 2,978 person-years at observation in the total cohort. The study has been recently reanalyzed with a case-control approach and through stratification on amitrol when considering the effect from phenoxy acids and vice versa (51). The results showed a possible and previously masked tumor inducing effect also from phenoxy acid. By 1976 an Intense debate was in progress in Sweden over the use of phenoxy herbicides. This debate prompted Harden (45) to examine the occupational history of 87 patients who had malignant mesenchymal tumors and who had visited the oncological clinic in Umea during the years 1970-76. Nine of the 87 patients were forestry workers, four worked in farming and forestry and six in sawmills or the pulp industry. The implication by Harden was that these 19 individuals were in occupations where exposure to phenoxy herbicides was relatively common. Based on the official statistics of Sweden, the expected fraction of tumors has been calculated for these occupations: the expentancy was 11 cases versus the 19 observed. Harden (45) however, cautioned making any conclusion about the possible casual connection between exposure to phenoxy acids and contaminants and the occurrence of malignant tumors, solely on the basis of the reported cases. In February 1977, the debate climaxed when the Royal Swedish Academy of Sciences organized a conference on "Chlorinated Phenoxy Acids and their Dioxins, Mode of Action, Health Risks and Environmental Effects" (31). The conference participants concluded that (1) there was no evidence that dioxins could be formed in nature, (2) there was no evidence of bioaccumulation of TCDD at levels of application used in Sweden and (3) that if the concentration of TCDD can be kept below 0.1 ppm in all phenoxy formulations the risks involved can be disregarded and the safety factors based on the phenoxy acids themselves. In the March 1978 WBBM television report on "Agent Orange: Vietnam's Deadly Fog", reference was made to a report from Sweden on birth defects (e.g., spina bifida) in children born to 65 women allegedly exposed to 2,4,5-T herbicide. The only reference to such an incident was that reported by Hailing (44) in 1977. Hailing studied the malformations V-25 �in children born to mothers exposed to hexachlorophene soap during early pregnancy. All of the mothers were employed as nurses in a hospital and thus came in contact with the hexachlorophene in performance of this job. A group of 65 children born to this group showed six slight malformations and five severe malformations, whereas only one slight case in 68 children was observed in the unexposed group. VIII. TE AWAMUTU, NEW ZEALAND EPISODE The New Zealand episode had many similarities to the episode in Sweden; once the initial report was publicized, additional cases were forthcoming. In January 1972, Sare and Forbes (72) reported the following in the New Zealand Medical Journal: "Sir, - Two babies, born within a month of each other at our local maternity hospital, had congenital defects incompatible with life. Both had a gross myelo-meningocele. Post-mortem was performed on only one and other congenital abnormalities were brought to light. What intrigued us was that the families concerned live on adjoining hilly country farms, where for several years aerial spraying has been carried out with a chemical called 2,4,5-T, designed to kill useless vegetation. Inquiries into the nature of this chemical revealed that it contains an impurity called dioxin, which is apparently one of the most powerful poisons ever discovered. It has been investigated in the United States, partially banned in all states, and totally in others. It was likewise banned in Vietnam when its potential danger was discovered " The suggested relationship between 2,4,5-T/TCDD and the two deformed babies quickly received national and international attention. Accusations that 2,4,5-T/TCDD were indeed responsible for the congenital defects soon appeared in articles in the United States (1, 32). The circumstances surrounding these cases at Te Awamutu were thoroughly investigated by a subcommittee of the Agricultural Chemicals Board of New Zealand (6). In the subcommittee report it was noted: The women who gave birth to deformed babies had both been exposed to 2,4,5-T during pregnancy, one person by assisting at the airstrip during spraying and the second person by helping to free the spray truck which was stuck on the property and was exposed to 2,4,5-T when spraying was done to lighten the load. It was not possible to ascertain the degree of exposure in either case. V-26 �The deformity common to both babies is spina bifida, caused by a failure of the end of the neural tube to close completely during early development. This deformity is one of the commonly occurring deformities, with overseas averages of about 1 per 1,000 total births. In New Zealand during the period 1964-70, 515 live births and 151 stillbirths affected with spina bifida were recorded. In the light of present embryological knowledge it may be stated that the neural tube is usually closed by the fourth week after conception and definitely by the sixth week. Medical records show that in one case, exposure to 2,4,5-T during the spraying operation occurred after the neural tube would have normally closed. It is concluded that in one of these cases the reported exposure to 2,4,5-T could not have caused the birth deformity. It is not possible to state definitely in the second case whether exposure to 2,4,5-T was in any way a factor causing the deformity, and thus the subcommittee was unable to arrive at any information of value to the general topic of 2,4,5-T toxicity to human foetuses. In April 1977, the New Zealand televison program "Dateline Monday" suggested that the occurrence of "clusters" of neural tube defects in the South Taranaki, Northland and Waikato areas of New Zealand were related to the use of 2,4,5-T (61). The New Zealand Department of Health, Division of Public Health, appointed a committee of experts to investigate the allegations. In the Committee report, McQueen et al (61) noted that the three "clusters" represented 20 cases of birth defects. Seven of the cases were anencephaly (congenital defect of the cranial vault) and 13 were spina bifida (congenital defect of the bony encasement of the spinal cord). McQueen et al noted that although this group of defects may well have occurred entirely by chance, the possibility of a common causal factor must be considered. After a thorough investigation of each of the 20 cases reported, McQueen et al (61) concluded: It is obvious from an inspection of the data for the three "clusters" that 2,4,5-T cannot reasonably be implicated in the causation of neural tube defects. It is true that in one or two cases there may have been some "exposure" to 2,4,5-T around the critical period. However, considering 2,4,5-T is the most used pesticide in New Zealand, this is'not unexpected. In short, the data permit the conclusion that there is no evidence to implicate 2,4,5-T as a causal factor in human birth defects. As a final note in relation to this episode, the following brief article appeared in the New Zealand Medical Journal (5): V-27 �Publicity on certain chemicals as causation of malformations of the human fetus has been widespread. Some of the publicity has been sensation mongering and not all the remarks from the profession have been in keeping with a balanced assessment of scientific evidence. It is proper that there should be intelligent public awareness of the various environmental hazards that may come from the use of chemicals in farming ....however, those who would write of their experiences in medical journals must remember that disasters are the staple of the sensation mongers in the news media industry. Until recent publicity there had been no suggestion that 2,4,5-T, which has been used for over 20 years in New Zealand, was responsible for congenital malfunctions either in man or in farm animals. It is the duty of the physicians (and scientists) who have any concern for science to attempt to make valid observations which can be repeated. In the problem at issue, fetal malformations are natures common mistakes which we have no desire to perpetrate or to increase, although they are the inevitable price that is paid for our place on the evolutionary scale. There are extensive gaps in our knowledge but they will be filled only by patient work. Unresolved problems of fetotoxicity can only be solved by accurate record keeping at all stages of pregnancy. IX. DISCUSSION OF LITERATURE AND CONCLUSIONS The episodes described in this chapter have provided much of our knowledge of the adverse effects to human health of the phenoxy herbicides, other chlorinated phenols and the associated dioxins. The only episodes however where TCDD was actually confirmed as a caustive agent were those involving some of the industrial accidents, the Eastern Missouri horse arena episode and the Seveso, Italy episode. Mercier (62) estimated that in the industrial accident in 1963 at the Philips-Duphar Company, Amsterdam, The Netherlands, up to 200g of TCDD were released into a factory hall. The incident in the horse arenas in Missouri may have involved 5,000g of TCDD (69). The quantity of TCDD involved in the Seveso, Italy episode has been estimated at 650-1,700g (69). In these three episodes, the TCDD was confined to a relatively limited area. The exposure of the people involved was from days (Philips-Duphar) to weeks (Seveso) to months (Missouri). Nevertheless, no human deaths were reported, although in both Missouri and Seveso, numerous animal deaths did occur. The clinical experience from these three episodes (and the other industrial episodes involving at least 1,000 individuals) support the opinion that patients without chloracne are extremely unlikely to have suffered the toxic effects of TCDD. In general, only in the most severe cases of chloracne has symptomatology persisted, admittedly for many years in a few instances. V-28 �The available scientific literature suggests that the episodes in Arizona, New Zealand and Sweden were primarily the result of emotionalism associated with zealous press coverage. Although each incident began subsequent to field applications of phenoxy herbicides, it was highly unlikely that the symptoms reported were attributable to actual pesticide or TCDD exposure. The behavior in the environment of 2,4,5-T and TCDD following normal field applications (see Chapter III) lends little credence to accusations that significant bioaccumulations occurred in humans to initiate the t-oxic symptoms reported. Furthermore, the absence of confirmed illness in domestic livestock or wildlife in these three episodes also addresses the issue of whether an actual toxic exposure occurred. Chapter IV defined the concentrations of herbicide and TCDD that were toxic to animals. The magnitude of the dosage required to elicite toxic symptoms in animals might be obtained only under the most extreme cases (e.g., spills or sequential repetitive applications). These extreme situations were not noted in the episodes in Arizona or New Zealand. The human responses associated with these episodes show a similarity to what occurred in Michigan involving exposure to polybrominated biphenyls (PBB). In 1973 and 1974, more than 10,000 Michigan farm residents were exposed to PBB when several hundred pounds were accidentally introduced into a nutritional supplement that was subsequently fed to numerous herds of dairy cattle. Budd et al (18) conducted an epidemiological study in an effort to determine whether or not exposure to PBB had caused illness in Michigan residents. Three groups were invited to participate in a prospective cohort study: (1) all persons who had been identified as living on PBB-contaminated farms at the time of quarantine; (2) all persons who had received food products directly from such farms; and (3) workers and their families who had been exposed occupationally to PBB in a chemical manufacturing plant. All subjects were administered a questionnaire requesting information on the occurrence in the years before and since 1973 of 17 symptoms and conditions potentially related to PBB. Venous blood samples were also obtained on the subjects. An evaluation of dose-response relationships revealed that symptom-prevalence rates were higher in persons with no detectable PBB in serum than in those with measurable quantities. These observations suggested that factors other than PBB absorption were responsible for the production of symptoms and that selection factors (e.g., selecting from a list of given symptoms by the subject) may have played an important role in the observed distribution of complaints. The episodes in Arizona, New Zealand and Sweden all occurred in the same time period; a period when numerous articles appeared in the world press on the alleged human health effects of Herbicide Orange and TCDD in South Vietnam. The effects these articles had on the actual episode can only be speculated. V-29 �The wide publicity that was given to the use of defoliants, especially Herbicide Orange in South Vietnam, appeared to have exceeded concerns of human health or the environment. Political issues may certainly have been a major reason for much of this publicity. Consider, for example, the data in Table 1 of this chapter; more 2,4,5-T and hence TCDD, was disseminated in the United States during the same period than in South Vietnam. If the assessment of canopy penetration is reasonably accurate in Chapters I and III,then the actual groundlevel deposition of Herbicide Orange in South Vietnam (1.4 pounds 2,4-0/2,4,5-T per acre) would have been approximately equal to the concentrations of herbicides encountered at ground-level following brush applications in the United States. The Committee on the Effects of Herbicides in South Vietnam of the the National Academy of Sciences (21) attempted to assess the effects of propagandists activities on the attitudes of the South Vietnamese towards the use of herbicides. The following statements are quotations from the 1974 report: Our findings indicate that there is a major dichotomy between,the views of the rural population and those of the urban middle-sector regarding the use of herbicides in SVN. Contrary to what might be expected, the herbicide missions are much less emotional issue among the peasants, who bore the brunt of the effects, than it is among urban intellecturals for whom it has become a symbol. Despite extensive propaganda and counter-propaganda campaigns waged by the RVN and the NLF, peasant views regarding herbicide effects seem to be based upon their own experience. The RVN stressed that herbicides were used as a military measure to deprive the guerrillas of their hiding places, that the herbicides might damage crops but could also have beneficial effects, and that people and livestock would not be adversely affected by spraying. NLF statements emphasized the dangerous nature of herbicides. They claimed that the chemicals caused the death of people as well as livestock and crops, resulted in increased numbers of miscarriages and stillbirths, and caused numerous diseases, especially leprosy and conjunctivitis. Further, it was said that the U.S. had deliberately introducted "chemical bacteria" into the spray which could penetrate peoples bodies and cause disease. The fact that the villagers did not appear to subscribe blindly to the propaganda claims of either side does not mean that they lacked political opinions nor that they were uninfluenced by information derived through the mass media. Rather it seems to mean that their opinions on this issue came mainly from their own observations. V-30 �The degree to which the above referenced propaganda influenced world opinion is illustrated by Dmitriyev (28) in articles published in 1974 in a Russian medical journal. The following quotation is a translation from that journal: Often in South Vietnam, chemical substances were used not only in the forest regions but also close to populated areas; this resulted in injury to a considerable part of the peaceful population. According to the data of the Provisional Revolutionary Government of the Republic of South Vietnam in 1961-1969, 1,293,000 persons were subjected to the effect of poisonous chemicals. In the first ten months of 1970, 185,000 cases of persons being poisoned were recorded. Three hundred persons died and a significant number of those injured became chronic patients. Persons injured by herbicides and defoliants noted perceiving a sharp odor of chlorine or DDT, sharp pain, burning in the nasopharynx and sneezing (91%), crying and vomiting (73%), headache and vertigo (38%), a burning sensation in the area of the eyelids and the skin (41%). These clinical symptoms were apparent after a 2.4-hour incubation period. Improvement in the patients, if they did not die, began after 3-4 days. However, they continued to suffer from asthenic symptoms in the form of sleeplessness, sexual weakness, and weakening of the vision. Similar quotations are available in American or European literature. Mercier (62), in reviewing the literature on TCDD for a conference in Milan, Italy in 1976, stated of the National Academy of Science Report (21): Considerable information is contained in a NAS report (1974) about the effects of herbicides, and especially 2,4,5-T, so-called "Agent Orange" and the contaminant TCDD on humans, animals and vegetation in Vietnam where they have been used during military herbicide operations. It contained reports of death to children, diarrhea, skin rashes looking like insect bites, and abdominal pain following spray missions. The use of the materials also significantly increased the incidence of congenital malformations among children. The point to be made is that the scientific studies that have been conducted in Vietnam; Globe, Arizona; Eastern Missouri, Sweden; New Zealand; Seveso, Italy; and the numerous industrial accidents do not document deaths of children or adults due to the herbicides or TCDD, nor do they substantiate increased incidence of congenital malformations among children. The reports published by North Vietnamese scientists provide insufficient data on which to draw contrary conclusions. V-31 �X. SUMMARY Increased industrial production of the phenoxy herbicides parallelled the rapid acceptance of these materials in world agriculture. The demands upon the industrial production however, resulted in at least 23 industrial incidents involving'over 1,100 people (almost all adult males). Although medical examinations were initially conducted on these individuals, few long-term studies are available. The use of herbicides by the United States military in South Vietnam precipitated numerous allegations of adverse health effects upon the human population. Review of the scientific literature of the few available studies conducted in Vietnam do not confirm the allegations. Episodes of TCDD poisoning in Eastern Missouri in 1974 and in Seveso, Italy in 1976 resulted in adverse effects to primarily women and children. Although the acute symptoms of poisoning have dissipated, long-term effects remain to be determined. Episodes of alleged poisoning from 2,4,5-T and TCDD in Globe, Arizona (1969-70), Sweden (1970) and New Zealand (1972) occurred in a period of time when intense publicity was given to the use of herbicides in South Vietnam. The available scientific studies of these incidents suggest that factors other than herbicide exposure may have been responsible for the symptoms reported. V-32 �CHAPTER V LITERATURE CITED 1. Adamson, L. 1974. Spray Now - Pay Later? EmuAon. 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Oliver, R.M. 1975. Toxic effects of 2,3,7,8-tetrachlorodibenzo-l ,4dioxin in laboratory workens. &Ut. J. Ind. Med. 32(1): 49-53. V-37 �66. Orians, G.H. and E.W. Pfeiffer. 1970. Ecological effects of the War in Vietnam. Science 168:544-554. 67. Pazderova, J., E. Lukas, M. Nemcova, M. Spacilova, L. Jirasek, J. Kalensky, J. John, A. Jirasek, and J. Pickova. 1974. Chronic poisoning by chlorinated hydrocarbons formed in the production ot 2,4,5-trichlorophenoxyacetate. P*AC. Lefe. 26(9):332-339. (Czech) 68. Poland, A. P., D. Smith, G. Metter, and P. Possick. 1971. A Health survey of workers in a 2,4-D and 2,4,5-T plant. M.c.k. 22:316-327. 59. Reqgiani, G. 1978. The estimation of the TCDD toxic potential in the light of the Seveso accidpnt. Pane*' presented at the 20th Congress of the European Society ot 1:0x1 coiogy. Berlin ^West), June 25-28, 1978. 70. Roan, C.C., and D.P. Morgan. 1972. Alleged effects on human health of the use of herbicides in the area around Globe, Arizona. Arizona Community Pesticides Studies Project, March 6, 1972. University of Arizona, Tucson, Arizona. Mim. 7 p. 71. Rose, H.A. , S.P.R Rose. 1972. Chemical spraying as reported by refugees from South Vietnam. Science 177:710-712. 72. Sare, W.M. and P.I. Forbes. 1972. Possible dysmorphogenic effects of an agricultural chemical: 2,4,5-T. Mew Zeo&md. Mc.d. J. 75(476): 37-38. 73. Suskind, R.R. 1976. A review of occupational exposures to dibenzop-dioxins. Presentation to a Conference on Dibenzodioxins/Dibenzofuran. November 18, 1976. Rougemont, N.C. 74. Telegina, K.A. and L.I. Bikbulatova. 1970. Affliction of the follicular apparatus of the skin in workers occupied in the production of butyl ether of 2,4,5-trichlorophenoxyacetic acid. l/e6^Cn. Vwmeutol. 44:35-39. 75. Tschirley, F.H. 1969. Defoliation in Vietnam. Science 163:779-786. 76. Tschirley, F.H., W. Binns, C. Cueto, B.C. Eliason, H.E. Heggestad, G.H. Hepting, P.F. Sand, and R.F. Stephens. 1970. Investigations of spray project near Globe, Arizona. Investigation conducted February 1970. U.S. Department of Agriculture, Office of Science and Education. Mim. 29 p. 77. Tung, T.T., T.K.anh, B.Q. Tuyen, D.X. Tra, and N.X. Hugen. 1971. Clinical effects of massive and continuous utilization of defoliants on civilians. lAtetnomeAe Studies 29:53-81. V-38 �78. Tung, T.T., T.T., An, N.D. Tarn, P.H. Phiet, N.N. Bang, T.T. Bach, H. vanSon and O.K. Son. 1973. Le cancer primaire due foie au Viet-nam. CklnuAQ^n 99:427-436. (French) 79. United States Tariff Commission. 1946. Synthetic organic chemicals: U.S. production and sales, 1944. Report No. 155, Second Series. U.S. Government Printing Office, Washington, D.C. 80. Walsh, J. 1977. Seveso: The questions persist where dioxin created a wasteland. Science 197(4308) :1064-1067. 81. Zelikov, A. Kh. and L.N. Danilov. 1974. Occupational dermatoses (acnes) in workers engaged in production of 2,4,5-trichlorophenol. Sov. Med. 7:145-146. (Russian) V-39 �CHAPTER VI HUMAN EFFECTS OF HERBICIDE ORANGE I. INTRODUCTION This chapter will discuss the human effects of Herbicide Orange. There has been considerable medical literature published on the constituents of Herbicide Orange, i.e., 2,4-D, 2,4,5-T, and the contaminant TCDD. The pharmacokinetics of these chemicals will be discussed along with their adverse effects. Most of the reports in the literature have involved occupational experiences. However, several episodes involving general populations from selected localities throughout the world will also be discussed. II. PHARMACODYNAMICS Little work has been done regarding the pharmacodynamics of 2,4-D, 2,4,5-T or TCDD in humans. The available studies are summarized below. A. Percutaneous Entry of Phenoxy Herbicides Feldmann and Maibach (27) in 1974 in studies using radioactive tracers showed that 2,4-D was able to penetrate the skin. Indirect evidence resulting from the numerous occupational exposures to 2,4-D and 2,4,5-T (and TCDD) in industry and herbicide spraying described later further supports percutaneous entry. B. Ingestion of Phenoxy Herbicides Kohli et al (46, 47) in two separate studies gave purified 2,4-D and 2,4,5-T in capsules to human volunteers. Each herbicide was orally administered to six men as the acid at a dose level of 5 mg herbicide per kg body weight (mg/kg). The 2,4-D was quickly absorbed and appeared in the plasma within one hour after ingestion. Seventy-five percent of the administered dose was excreted unchanged in the urine within 96 hours (h). The 2,4,5-T was also readily absorbed, being present in the plasma one hour after ingestion. After 96 h, 63 percent to 72 percent of the herbicides had been excreted unchanged by the kidney. Plasma levels peaked between seven and twenty-four hours for both 2,4-D and 2,4,5-T and the half-lives for plasma clearance were 33 and 18 h respectively. In a study by Saueroff et al (70) in 1977, five male humans ingested 5 mg/kg of 2,4-D. Essentially all was absorbed from the gastrointestinal tract. It was eliminated from the plasma with an average half-life of 11.6 hours and from the urine with an average half-life of 17.7 hours. Eighty-two and three tenth percent was excreted unchanged and 12.8 percent in a conjugated form for a 95.1 percent total recovery. Utilizing this rate of clearance, 99 percent of the steady state would be reached in about three days making body accumulation of repeated exposure unlikely. Gehring et al (28) in 1973 and Matsamara (52) in 1970 found similar results in comparable studies of 2,4,5-T. It should be noted VI-1 �that no short-term adverse effects were found in any of the above studies with 5 mg/kg being the highest dose. The fate of Silvex [2-(2,4,5-trichlorophenoxy) propionic acid] was studied by Saueroff et al (71). Seven men and one woman ingested a dose of 1 mg/kg. Peak plasma levels were reached two to four hours after ingestion. The Silvex was excreted in the urine both in the unchanged and conjugated forms. The mean recovery in the urine was 64 percent of the orally administered dose after 24 hours and 79.8 percent after 144 hours. Recovery of Si 1 vex in the feces accounted for not more than 3.2 percent of the administered dose. The half-life for plasma clearance was biphasic, 4.0 +. 1.9 h and 16.5 ±. 7.3 h for initial and terminal periods respectively. No adverse effects were noted. Park et al (60) described clinical and pharmacokinetic observations in a 39-year-old male following ingestion of the amine salts of 2,4-D and [2-(2-methyl-4-chlorophenoxy) propionic acid] (MCPP). Following forced alkaline diuresis, the plasma half-half of 2,4-D was greatly reduced from 220 to 4.7 h. The renal clearance of 2,4-D increased up to greater than 100-fold during the period of alkaline diuresis. I'lrinary recovery studies confirmed absorption of about 70 ml of the herbicide. ,fthough the patient initially demonstrated a mild proximal neuropathy and myopathy, full recovery occurred in two months. C. Tissue Analyses for the Phenoxy Herbicides Levels of phenoxy herbicides found in human tissue or body fluids following ingestion of a fatal dose are shown in Table 1. The data are reported in parts per million: it was assumed that all tissues (removed during the autopsy) were analyzed on a fresh weight basis and that 1 ml of blood or urine was equal to 1 g. Frequently, no description of the handling procedures was reported; thus, fluid may have been present within the organ (e.g., liver) at the time of the analyses. This fluid contamination may have resulted in high values for a given tissue. Coutselinis et al (20) in 1977, reported on the analyses of herbicide in the organs of a woman who died 16 hours after ingesting a large dose of a mixture of 2,4-0 and 2,4,5-T. Levels of the two herbicides found in selected tissues at the time of death are shown in Table 1. The formulation ingested was a mixture in a 3:2 ratio, 2,4-D to 2,4,5-T. Nielson et al (56) reported a more complete investigation of herbicide residue in body tissue resulting from an autopsy of a 23-year-old male who ingested 2,4-D. The levels of 2,4-D in parts per million for selected tissue are also shown in Table 1. The herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) has been associated with two deaths. Johnson and Koumides (39) reported the death of a 65-year-old man following the ingestion of 250 mg MCPA/kg body weight. VI-2 �TABLE 1 Levels (part per million) of Phenoxy Herbicides in Human Tissue or Body Fluid Following Ingesfion of Fatal Dosea Patiert Herbicide Coutselinis et al. (20) Young Wcmsn 2,4-D/ 2,4, 5-T "Large" Nielson et al. (56) 23 Yr Old Han 2,4-D Time Level of Herbicide (Parts Per Million) in Tissueb Ingestion Gastric Fatty - Death Blood Urine Liver Kidney ! Brain Spl een Muscle Heart Washings Tissue ;• 18 hrs 826 210 82 12 182 48 5 22 Dose (mg/kg) >80 Source i Dudley and Thapar (23) 669 264 183 63 13 2,4-D 76 Yr Old Man >2,000C 5 days 58 408 194 i 20 hrs 250 180 1 1 2,500 j 800 t I 440 20 hrs 230 970 i . i ! 32 Yr Old Han '• MCPA : I 118 93 ! MCPA 83 : 70 134 ( i 65 Yr Old Han Johnson and Koumides (39). Popham and Davies (63) 24 hrs 146 154 33 3,000 i I ^Tissue/fluid removed during autopsy. °Ip convert parts per million to mg/dVor to mg/100 ma, multiply tabulated values by 0.1 . e Tne 55 kg patient consumed one pint of a presumed ester formulation (kerosene-like, water insoluble formulation) of 2,4-D. tster formulations containing the least amount of active ingredient 2,4-D are two pound/gallon formulations. If a pint contained 113 g 2,4-D acid, then the dose would have been >2,000 mg 2,4-D/kg body weight. * �Popham and Davis (63) report the death of a 32-year-old man following a MCPA dose of 440 mg/kg. The levels of MCPA found in selected organs or body fluids following death are reported in Table 1. The high level of MCPA in the urine suggests that this herbicide, like 2,4-D, was rapidly excreted unchanged by the kidney. D. Pharmacodynamics of TCDD There is almost a complete lack of information concerning the Pharmacodynamics of the dioxins in man. In November 1977, Fanelli (2)-in a letter to the Mario Negri Institute of Pharmacologic Research in Italy found no TCDD in samples of the liver, mesenteric fat, or cerebral fluid in the necropsy of a woman who had been included in a follow-on study of the Seveso, Italy, TCDD episode. The lower limits of detection were 0*4 ng TCDD/ml of fluid and 0.25 ng/gm of tissue. The cause of death was not given. Reggiani (65) described the case of a 55-year-old woman who died of pancreatic carcinoma with liver involvement seven months after the Seveso episode. Children living with her suffered severe caustic burns of the skin and, subsequently, chloracne. Neither the patient nor the mother of the children developed chloracne. It is almost certain that the entire family ate food contaminated with TCDD. TCDD detected in the analysis of tissue obtained during the autopsy is shown in Table 2. No TCDD was found in the same tissues taken from autopsies of three persons who were certainly not related to a TCDD exposure. The samples were run concurrently with those of the case described above. III. ADVERSE EFFECTS A. Limitations of Referenced Studies There is considerable information in the world literature regarding the adverse effects of 2,4-D, 2,4,5-T, 2,4,5-trichlorophenol (TCP) and TCDD in humans. Most of it is the result of studies on worker experience, industrial accidents or individuals poisoning. Unfortunately, there are very few controlled studies and only generalizations can be made regarding a causeeffect relationship. In most cases all that can be said is that an association exists. There are several other important limitations of the studies that must be kept in mind when reviewing them. These include: 1. The populations were biased toward the adult male of working age. 2. Examinations were post-exposure, and therefore, pre-existing disease often was not known or reported. 3. Exposures frequently were to mixtures and, therefore, one cannot be certain which chemical produced which effect. 4. An accidental or intentional ingestion or an exposure from an industrial accident would result in a dose much higher than would be expected in the general population in the region of a herbicide spraying program. VI-4 �TABLE 2 TCDD Levels in a Human Body Date Sampl e b 28, 7, 7 Liver Limit of Origin Quantity Detection Recovery TCDDa 10 g 10 PPT 64% 0.15 PPB Autopsy 10 g 10 PPT 59% 1.84 PPB Pancreas Autopsy , Autopsy 5g 10 PPT 59% 1.04 PPB Fat Lung Autopsy 10 g 10 PPT 60% 0.06 PPB Kidney Autopsy 10 g 10 PPT 60% 0.04 PPB Brain Autopsy 10 g 10 PPT 60 0.06 PPB i a - Total body weight:, kg 70 - Calculated total amount at time of death: 40 yg b - Vacuum Generator Micromass Laboratory, Altrincham (Manchester, U.K.) Source: Reggiani (65). VI-5 �5. Although the routine occupational exposure would in most cases be at a dose rate lower than that of accidents, the exposure would be prolonged effectively raising the total dose. 6. The actual dose received in most instances was not known. B. Phenoxy Herbicides That Do Not Contain TCDD As was explained in a previous chapter, TCDD is a contaminant of phenoxy herbicides made from TCP. TCP is not a precursor of 2,4-D. This permits the evaluation of health effects of 2,4-D (or 2,4-D-like herbicides) as an entity separate from TCDD. 1. Experimental Exposure to 2,4-D There have been at least three reports of no-effect exposure where the precise dose was known. Assouly (4) in 1951 reported on a man who ingested 0.5 g of 2,4-D daily for three weeks without adverse effects. Kohli (46) in 1974 in his pharmacodynamic study of 2,4-D reported no effect after a single oral dose of 5 mg/kg. In 1962, Seabury (74) treated two cases of disseminated coccidiomycosis with 2,4-D. The first patient received a total of 40 mg of the sodium salt by intramuscular injection over a period of four days. The patient died on the fifth day without evidence of 2,4-D toxicity. In the second patient, approximately 13 g were given intravenously over a period of one month, the last 2 g in one dose. No adverse effects were noted. When the dose was increased to 3.6 g over a period of two hours the patient became semi-stuperous and exhibited fibrillary movements about the mouth and in both hands and forearms. The stupor deepened to a point where the patient responded only to painful stimuli. Forty-eight hours after the dose was given he returned to his pre-reaction state. There was no evidence of neurologic or muscular change in the next seventeen days after which he died from the primary disease. 2. Exposure in the Production of 2,4-D or MCPA Bashirov (8) in 1969, examined 292 workers including 44 women employed in the production of the amine salt and the butyl ester of 2,4-D. This report is of particular significance in that the butyl ester is the form found in Herbicide Orange. Table 3 shows the various responses along with the percentage of occurrence. Several organ systems were involved with emphasis on headaches, the asthenic syndrome, and gastrointestinal complaints. Fifty persons from the above group were selected for controlled studies involving the liver and stomach. Bashirov indicated that there were significant differences between the control and test groups in amount of gastric secretion and the antitoxin and carbohydrate functions of the liver. In addition, they noted a correlation between the length of service and the changes in the functional state of the stomach. The authors did not state their level of confidence. Telegina and Bikbulatova (78) in 1970 reported on 158 workers employed in the production of MCPA. Telegina and 'Bikbulatova found contact VI-6 �TABLE 3. Distribution of symptoms in 292 workers employed in the production of the amine salt and the butyl ester of 2,4-D. Percent of workers describing symptoms Symptoms 1. Weakness, fatigability, headaches 63 2. Asthenic Syndrome with vegetative dysfunction 61 3. Anorexia, bitter taste in mouth, dyspepsia abdominal pains, constipation 51.7 4. Vertigo 33 5. Dyspnea on exertion 26.7 6. Tachycardia, precordial pain 17.8 Source: Bashirov (.8) VIr7 �dermatitis or history of same in 55 individuals in the first examination and in 65 in a second examination a year later. Irritation of mucous membranes was also found in a majority of these individuals. 3. Accidental or Intentional Exposure to 2,4-D, MCPA, 2.4-DP or MCPP Another major group of persons exposed to 2,4-D or the analogs MCPA, 2,4-DP [2,4-dichlorophenoxy) propionic acid] and MCPP, are those involved with accidental or intentional ingestion of the substance. Table 4 is a summary of many such cases along with the estimated dose of herbicide (where available), major effects and outcome of the intoxication. Popham and Davies (63) reported the case of a 32-year-old man who ingested an estimated dose of 440 mg MCPA/kg. There were signs of severe meningoencephalitis including grand mal and focal seizures with death within hours. Necropsy showed no evidence of damage to the gastrointestinal tract, but the liver showed signs of early necrosis. The brain and meninges showed marked congestion but otherwise were normal. Johnson and Koumides (39) described a similar MCPA episode without the severe central nervous system signs and with death in hours. The dose was estimated at 250 mg/kg. Nielson et al (56) published a paper describing a 23-year-old man who committed suicide by ingesting an unknown amount of 2,4-D. Tissue analysis indicated, however, that at least 80 mg/kg must have been absorbed. Unlike the cases of Johnson and Koumides (39) and Popham and Davies (63), this subject was in good physical health prior to the ingestion. The others were suffering from chronic illnesses. There was evidence that this subject had at least one convulsive episode before dying, implicating the central nervous system. In the necropsy, small amounts of 2,4-D were found in the brain tissue (see Table 1). There was also evidence of degeneration of ganglionic cells in the brain. If the degeneration was due to 2,4-D and not hypoxia, it would have indicated that the cellular elements of the central nervous system were quite sensitive to 2,4-D as the tissue analysis showed the brain to have a much lower level of herbicide when compared with other organs of the body. Other episodes of poisoning by 2,4-D or MCPA have been reported by Jones et al (40), Berwick (12), Brandt (15), Dudley and Thapar (23), and Park et al (60). Findings, other than those involving the central nervous system, included abnormal enzyme levels, anemia, thrombocytopenia, skeletal myositis with myoglobinuria, myocardial irritability, loss of color vision, peripheral nervous system disorders, pulmonary edema, and renal disorders. The subject reported by Dudley and Thapar (23) died; the remainder survived with varying degrees of recovery. The case reported by Brandt (15) had a complete recovery after an estimated dose of 300 to 600 mg/kg; however, this individual had ingested a mixture of 2,4-D and 2,4-DP. The case reported by Berwick (12) was noteworthy because the individual involved accidentally ingested a dose of 110 mg 2,4-D/kg. The herbicide was formulated as the isooctyl ester of 2,4-D. Although the individual demonstrated numerous symptoms (Table 4), he fully recovered. VI-8 �TABLE Distribution of Adverse Effects in Case Reports Following the Ingestion of Non-TCDD Containing Phenoxy Herbicides c to c O O — 4_> .— 4J ._ *-* in >- — U 4) O *J OJ fD -C <U 4-1 O 4-1 X aj *- — i_ Q. (LI (0 Q- i/l — 4-t Q_ ••-» r— 4_l O I- ( U - C Q - O u i O <U O O (0 <0 O _c ra ^D ~ " I 0) *J 5 41tOmg/kg MCPA 1965 250mg/kg . ^ " 2 Q 4 toSI -C - 1 Nielson et al . (56) 1965 >80mg/kg 2.4-D Jones et al . (40) Berwick (12) g ( ? Z l G, O Z + 1967 <1 900mg/kg MCPA + 1970 HOmg/kg 2,4-D + + Brandt (15) 1971 300-600 mg/kg 2,4-D/ 2,4-DP Dudley and Thapar (2k) 11--A -2000mg/kg 2.4-D Total Number o f Reports Listing Effect Unkn 2.A-D/ MCPP 2 ^ (J (U d> 4-* a.— ^n < CL O -o 1_ 01 Death- + Death Death + •*- + + + + + * + + - + + 1 ) 8 2 + 2 Ful 1 Recovery + -t + Ful 1 Recovery + + + 6 Residual * * Peripheral Sensory Defect Death Full Recovery + 3 Outcome (_> | + 1377 - + + (fcn.) O 03 + •I- Park et ai. *j .— >-C 1 MCPA • Johnson and Koumides (39) tfl >- Q. 1964 Popham and Davies (63) Q o — Senses ion c •Q 0) -i-" 3 ^ 1 1 �The patient was routinely observed over a three year period and no signs of peripheral neuropathy occurred. 4. Exposure to 2,4-D in Spray Operations A fourth group of exposed individuals is those who were involved in spraying operations contacting either the spray or the liquid. Table 5 summarizes these cases where individuals were exposed to 2,4-D. In 1959, Goldstein et al (31) first reported on three patients who developed peripheral neuropathies manifested by pain, paresthesias and paresis. There had been previous skin contact with liquid 2,4-D indicating a probable percutaneous route of entry. Recovery from the neuropathy was incomplete for the three patients during the periods of observation which were 1, 2 and 3 years, respectively for a 65-year-old male, 50-year-old female and a 52-year-old male. The 65-year-old male was exposed during the course of spraying a field with an ester of 2,4-D wetting his arms and legs. He was reported to have been in ill health prior to exposure. The 50-year-old female was exposed twice, one year apart, to an ester of 2,4-D wetting hands and legs. The 52year-old male was exposed first when he spilled 60 ml 2,4-D ester on his arms and failed to wash it off. His second exposure was two months later, wetting his legs with the same formulation. In 1961, Monarca and di Vito (55) reported a case where the entry route may have been at least partially respiratory, the subject having stayed downwind during much of the spraying operation. The immediate toxic symptoms consisted of asthenia, autonomic hyperactivity, gastrointestinal irritation and alterations of the central nervous system. Some days later he developed a hemorrhagic enterocolitis. After a period of five months recovery was complete except for hyporeflexia of the lower limbs. Berkley and Magee (11) described the development of peripheral neuropathy in a 39-year-old farmer who had significant hand contact with 2,4-D. At the end of one year the only residual effect was mild hypoalgesia on the fourth and fifth fingers of the right hand. Todd (80) reported a case in which the subject presented with anemia and leukopenia as well as peripheral neuropathy. The subject had two separate contacts with liquid 2,4-D experiencing gastrointestinal symptoms each time. The neuropathy lasted almost two years. In 1966, Tsapko (81) reported headache, retrosternal pain, general weakness, vertigo, nausea, vomiting, and mild leukopenia in a group of field workers who entered an area immediately after it was sprayed with 2,4-D. Kotlarek-Haus et al (48) described an autoimmune hemolytic anemia in a pesticide applicator. However, DDT, Lindane and Fenthion were routinely sprayed by this individual, as well as was 2,4-D VI-10 �TABLE 5 Distribution of Reported Adverse Effects Following Exposure of Field Workers and Applicators to 2,4-D 4-1 VI to 4_t in — u O a Yc-> of if.^.. of Source Number numoer Episode Of Cases ? b~D i,t u Formulation Primarv — rrimary Rnnfp o r ( ) / nouie nf z Exposure O 3 nc/)i/l 2>. " OQ + 1<<55 3 Ester Percut Monarca anddi Vito (55) HoO 1 Sodium Sal t inhal o 0) (0 -C CL Q. _ O J: U3 ua) °-z 4-. O c— *> <U <0 a . * ->- — - 4J (/itO JEQ. >-O n j u QJ ra i- IQ z UuO 1 N/Ab Percut + Berkley and Magee (11) IS61 1 Amine Salt Percut Tsapko (81) 1S66 Group Sodium Salt Percut Wai Us et al. ( ? 8) 1?S6 1 N/A Inhal Paggiaro et al . (58) 1972 1 Ester Inhal Total Number of Reports Listing Effect Percut = Percutaneous; Inhal = Inhalation Formulation description not available. ">- 10 Q. O 41 _1-c -—o . c «Q. <g 1- z + + °1 >—O <u o i-v) -O Remarks + + + + + One case of neuropathy for 3 Yr 5 Mos Residual hyporeflexa 2 Yrs + 3 Vrs Full recovery but neuropathy lasted two Yrs 1 Yr + + + oju + + + ID e ° °- ° + + + «-^ ° •* + + Todd ( 0 8) b <u c C + Goldstein et a!. ( ! 3) a C Mild hyperalgesia in two finger N/A No comment 2 Yrs + + + Full recovery 1 Mo Ful 1 recovery �Sare (69) reported on a subject who complained of diplopia toward the end of days in which he sprayed 2,4-D. In 1974, Barthel (7) related three cases of pulmonary fibrosis in workers engaged in weed control programs using MCPA. It is more probable, however, that the fibrosis was related to the carrier substances which were slatemetal, kaolin, and talcum. In a letter to the editor, Taylor (76), reported a suicide in a young farmer who became depressed over an illness possibly resulting from exposure to 2,4-D and 2,4,5-T. The illness was not specified nor was it clear whether the depression was a primary response to the herbicides or entirely secondary to the illness. However, this was the only reference found in which a psychiatric disorder was attributed to 2,4-D. Paggiaro et al (58) described an individual intoxicated by inhalation of 2,4-D. The individual manifested headaches, constipation, urinary incontinence, myalgia, muscular hypotonia, proteinuria and tachyarrhythmia. Despite these numerous symptoms, the patient fully recovered in one month. Palva et al (59), in 1974, reported a case of aplastic anemia in a 64-year-old farmer after exposure to MCPA. Recovery was complete after five months. C. Trichlorophenol (TCP), 2,4,5-T and TCDD Since TCDD is formed in the production of TCP (see Chapter V), both TCP and 2,4,5-T are contaminated with TCDD. As a result, TCDD must be considered when discussing either TCP or 2,4,5-T. Although other dioxins are usually formed in the production of pentachlorophenol (PCP), small amounts of TCDD may also be produced and, therefore, exposure to PCP will be included in this section. 1. Industrial Exposure and Symptomatology Since the first commercial production of 2,4,5-T there have been numerous industrial episodes involving exposure to TCP, 2,4,5-T and TCDD. Chapter V discussed these industrial episodes in depth. Fifteen of the 23 episodes recorded in the literature were apparently occupational exposures that occurred during industrial production of chlorinated phenols. However, on eight occasions, explosions occurred and personnel were exposed during the clean-up of the accident or from subsequent exposure to an improperly decontaminated workshop. The symptomatology reported for various occupational episodes are presented in Tables 6, 7 and 8. Table 6 is a summary of the organ systems affected during episodes of occupational exposure to chlorinated phenols and/or TCDD. Table 7 is a summary of the signs, symptoms and disorders reported for these episodes. Table 8 is a summary of special clinical studies conducted in support of physical examinations given to selected VI-12 �TABLE 6 Organ Systems Reported Affected After Occupational Exposure to PCP, TCP, 2,4,5-T or TCDD <u z (1) u> .c 3 Q. O Source Chemical Baader and Bauer (6) Bauer et al. (9) PCP TCP/2,4,5-T Bleiberg et al. (14) TCP/2,4.5-T/2.4-D Po.land et al . (62) TCP/2,4,5-T/2.4-D Dugois et al. ( 4 2) TCP Phenoxy Acid TCP/2,4,5-T Hardell (33) Kimmig and Schulz (44) • c a 17 8 21 48 c <a o CP o. 3 20 PCP/2.4.5-T 10 23 76 PCP/2.4.5-T PCP/2,4,5-T PCP/2.4.5-T 53 + TCDD 2,4,5-T TCP Same p]ant as Bleiberg 1964 after improved conditions. Chloroform odor from skin. Same plant as Bauer 1961. 10 No significant difference in findings from control. 2 persons had porphyria without acne. Subjects taken from group examined in Jiracek et al. (37) 13 3 489 278 20 Comment Examined June 1950, 16 months after last exposure 31 workers exposed. Examined 5 years after exposure ceased. 7 17 31 3 Number of cases in which organ system affected1" a E O E O w ±J c/1 2,4,5-T Kramer ( 9 4) Jirasek et al, (37) Jirasek et a!. (38) Pazderova et al (61) Miura et al. (54) Oliver (57) Ter Beek et al. (79) Zelikov and Danilor (88) -Q E 3 Lab workers synthesizing TCDD. 4 40 36 11 24 10 0 10 Number entries in table reflect the number of cases in which a disorder of the organ system was reported. * -»• = Organ system involvement reported; however, number of cases not given. Numbers do not include cases represented by "+" and totals may represent some double counting due to overlap of studies by Jirasek et al . and Pazerova et al. �TABLE 7 Signs. Symptoms, and Disorders Reported After Occupational Exposure to TCP. 2,4,5-T or TCDD c O m to > > 10) -C U ~O 0) 4^ U . U 0) o> . — O> <B — .- (D I/1JI . - ! _ 1" u ~ 0)C Q ) > ~ ( 0 0) 4J 0) (0 T3 4->£ (0 >-<0 L - O (Din Z in- L. 4-1 V (0 CU1 <u • — £ 4J — l_ > . Ol 3 .— in Q.L. . _ 1O V) •— Q <u (0 -C c in »- — ( 3 (0 0 l _ ID .— C lf> Q. Q . d > X tt) (0 O ^ : (U 0) O O 0) (U J-> I/I (D Ifl C Ul — 0) OQ_Q S_ Source 8 4a Bauer et al . (9) 2 6 9 17 Poland et al. (62) 8 Dugois et al . (24) 4-J 4) 18 20 1 7 30 48 i + 22 i + + t (33) 9 5 87 Kimmig and Schulz (44) 31 + Kramer (49) Jirasek et al. (37) i | +• Pazderova et al. + + 12 + Jirasek et al . (38) 2 19 2 i + 78 + + 23 + 3 3 i + + (£]) + + Miura et al . (54) 2 \ 27 53 8 + + 1 1 1 + O l i v e r (57) 2 Ter Beek et al . (79) 1 + 1 + 17 6 15 18 0 47 75 2 3 1 + 1 3 Zelikov and Danilov (88) cases + : 4 3 Total number of reportedc 6 17 2 Q i 8 +b a »-.— CO 5 1 Hardell (0 O) •— C X— 11 3 Bleiberg et al . (14) -fc- 0 — ^ Baader and Bauer (6) < ( - C O - ( U Z Q U « _ >. — 3 •> • O >~l- fD u — + + 47 17 275 0 91 + 6 23 6 Number entries in table reflect the number of cases in which sign, symptom or disorder was reported. + = Sign, symptom or disorder reported but number of cases not given. c Numbers do not include cases represented by "+" and totals may represent some double counting due to the overlap to studies by Jirasek et al. and Pazderova et al. �TABLE 8 Special Clinical Studies Following Occupational Exposure to TCP, 2,4,5-T or TCDD Liver Funct Source Renal Funct Baader and Bauer (6) 6a 2 2 1 6 1 1 1 B 1 ood Elements 1 Poland et al. Proteins 2 Bauer and Schulz (9) Lipids (62) Kramer ( 9 . ^) a 0 2 7 1 k 5 ]k +b 11 11 Oliver (57) Total number of cases with abnormal study B 1 ood Pressure 6 Jaracek et al . (37) Pazderova et al . (61 )c EEG 6 Carbohydrates 37 8 9 3 28 5 18 **7 9 ' 13 15 Number entries in table reflect the number of cases in which the special study was reported as abnormal. °+ * Special study reported as abnormal but number of cases not given. C The results include studies reported in Jiracek et al. (AO). The two studies complement each other. - Numbers do not include cases represented by "+". 18 �individuals following or during occupational episodes. The data in these tables are probably representative of the over 520 individuals that were reported in Chapter V to have been medically examined following the various occupational episodes. Approximately 600 individuals were adversely affected by exposure to TCDD following eight reported industrial accidents (see Chapter V). These individuals were either involved in the accident, responsible for clean-up after the accident, or returned to work in the plant following the accident. Table 9 is a summary of organ systems affected after .an exposure to TCP and TCDD following these industrial accidents. Table 10 is a summary of the signs, symptoms and disorders noted in the individuals following exposure to TCP and TCDD. Table 11 is a summary*of the few available data on special clinical studies on those individuals involved in the industrial accidents. Armstrong et al (3) and Robson et al (67) have reported on extensive medical data (organ systems affected, symptoms, disorders and clinical examinations) from newborn infants exposed to sodium pentachlorophenate in a hospital episode of PCP poisoning. Since these data involved newborn infants and PCP in a hospital environment, they were not included in the Tables. The data in Tables 6 thru 11 list the effects reported in the industrial environment where TCDD may be produced in the course of trichlorophenol production. The absolute numbers must be looked upon with caution for the reasons expressed earlier. There were no controls and preexisting conditions in most cases were not described in the articles. This limitation is demonstrated nicely by the study of Reggiani in 1977 (64) on the workers of the ICMESA plant in Seveso, Italy. As will be explained in more detail later there appears to be minimal if any development of systemic disorders if chloracne or a history of the same is not also present (64, 65). (Personal communication: Crow, K. D., Princess Margaret Hospital, Swindon, England. Holder, B. B., Dow Chemical Company, Midland, Michigan.) Out of 176 ICMESA workers examined immediately after the accident and more thoroughly four weeks after, only one displayed a doubtful case of chloracne. Yet, there were 29 subjects with liver disorders, 28 with lower respiratory problems, and nine with disorders involving the heart. In this case, the caustic products and TCDD exited the plant through a stack resulting in minimal, if any, exposure to the workers in the plant. This is contrasted with other accidents where the formed material remained in the plant providing major exposure to TCDD. If the premise that chloracne will be present before or during the time systemic symptoms are present is accepted, the abnormalities seen in this case must be due to some etiology other than TCDD. It is a matter of speculation as to why the one worker developed chloracne. There are at least two possibilities. He may have had a low threshold or the chloracne may have preceded the incident, being present as a result of his routine work. This raises the question of how many of the systemic problems listed were also completely or partially unrelated to TCDD. From the data available, the question cannot be answered. VI-16 �TABLE 9 Organ Systems Reported Affected After Exposure to TCP and TCDD Following an Industrial Accident tn >- a> tfi <U i" O > 3 O > ui CO »- <0 U O > <1) Z I4) <1) </> <U — u — co +•> C •— +J U «o E (0 I - X ui i— c — ja Q. E >* i- i- Source in tO "O _a E 3 z 3 c .^ ~ ^ a) > • •— 1 - (/) a) c£ a) 2: c i- a> 3 o 1 O — ^ Dugois et al. (25) 21 83 83 13 1 Goldman U9,30) 42 42 6 1 Anonymous (2) (Seveso, Italy) 176 1 29 Suskind (75) 228 + + Number of cases in which organ system affected0 550 147 48 i- L. z z <u >- c <1> i/> — <u E i / i f D E - C E O E 10 — < l ) ( D o 3 u C ro ^ *-• -tJ .— j-i o 4-1 iCirt Uui 4JO1 to a/ > > ^ ( u > - 3 > » < u ) 0t Q.CO <co or o. u Q. 0) to Comments 21a Jensen and Walker (36) - 3 +b Includes family members of exposed worker 1 -xj a 7 4 1 6 6 28 35 1 3 + + 4 1 6 Includes 14-yr old son of employee Includes only workers at ICMESA Plant 9 + 2 1 6 10 1 3 Number entries in table reflect the number of cases in which a disorder of the organ system was reported. b + = Organ system involvement reported. c Number of cases not given. Numbers do not include cases represented by "+". �TABLE 10 Signs, Symptons and Disorders Reported After Exposure to TCP and TCDD Following an Industrial Accident C C <D E O in t/1 (U V) JT 0 ( a) Source U O <U •— — +j 0 0)O> »— 3 ( 0 3 1 - a) z ^ >- a) zz tn — (D °- Q) U) <U <U •— -C 1 . E <u = •*-* fl3 L . O °- O1 —i_ °-° 1 CD (75) Number of cases reportedc a ( •— C C u ** 4 J to "^ 6 O f C W — 0) § - ja L. <o 7 k2 3 1 5 + 5 ; + + 11 + + + + 0 Bk ]k 0 + + 1 6 7 + 00 ^ a. O +b 21 a 1 »3 D O •""" 13 Reggiani (64) Suskincf -*- i4-»tfl > - C l - Dugois, P. et al . (25) Goldmann (29,30) (X d) 1 Number entries in table reflect number of cases in which sign, symptom or disorder was reported. °+ = Sign, symptom or disorder reported. c Numbers do not include cases represented by "+". �TABLE 11 Special Clinical Studies After Exposure to TCP and TCDD Following an Industrial Accident Llver Funct Renal Funct Carbohydrates 13a 1 Goldmann ( 9 3 ) 2,0 + + Reggian! (64) +b Suskind (75) Total number of cases with abnormal studyc + Lipids 3 Source May, G. (53) 13 Blood Pressure + 17 + 1 3 a 0 ' 17 Number of entries in table reflect the number of cases in which the special study was reported as abnormal. ^Special study reported as abnormal but number of cases not given. c Numbers do not include cases represented by "+H. �Still, even with the limitations the data do allow for an evaluation of trends. Chloracne is by far the most common finding. Also appearing frequently are disorders involving the liver, nervous systems, and mental state, the latter primarily in the form of asthenia. Early symptoms such as respiratory tract and mucous membrane irritation as well as headaches and nausea probably result from the primary substance and not TCDD (75). Lipids are frequently evaluated, but due to the normal large day-to-day variation within an individual, the findings are difficult to evaluate. Chloracne, asthenia, and liver disease in the form of porphyria cutanea tarda will be discussed in greater detail. a. Chloracne. Chloracne is the hallmark of exposure to the highly chlorinated dibenzodioxins and dibenzofurans. Kimmig and Schulz (44, 45) in 1957 and Schulz in 1968 (73) showed that it was TCDD and not TCP that produced Chloracne. The history of chloracne since its first description in the late 1800's has been well documented, in numerous review articles (16, 21, 43, 44, 74, 77). The problem peaked about the time of World War II as the result of a large production of chlorinated napthalenes. It also has been a major problem with the polychlorinated biphenyls and the associated dibenzofurans, particularly in Japan. The incidence of chloracne has been decreasing as production techniques and housekeeping methods have improved resulting in a reduced level of TCDD. Chloracne is a disorder of the pilosebaceous mechanism with the overproduction of keratin in the sebaceous ducts. This results in the development of the comedone or blackhead seen in all types of acne. In mild cases this may represent the full extent of the disorder. However, the natural progression is the formation of cysts and in severe cases to the development of inflammatory lesions and scar formation. Inflammation, however, tends to be less prominent than that found in acne vulgaris (common or juvenile acne). Frequently associated with the chloracne are hyperpigmentation and hirsutism manifested by excessive facial and body hair. In the mildest cases acne may only appear in the area of the outer canthus of the eye and pre- and post-auricular regions. In somewhat more severe cases, the rest of the face and neck may be involved with a sparing of the nose. In even more pronounced cases, the trunk and extremities, except for the hands and feet, may be affected. A preferential site of involvement not usually seen in other forms of acne is the genital region. In the worst cases the skin of the entire body gives the appearance of a homogeneous covering of comedones and small cysts. Severity of the acne does not necessarily reflect the degree of exposure to TCDD (14). Acne may appear as early as two to three weeks after the first exposure; however, there may be a delay of several months. The delay could represent a time for the development of a skin burden of TCDD. (Personal communication: Holder B. B., Dow Chemical Company, Midland, Michigan.) This burden would represent a threshold below which acne does not appear. VI-20 �Oliver in 1975 (57) reported two laboratory workers who developed very greasy skin, one of whom developed acne. This picture is contrary to the usual finding in chloracne where the skin is typically very dry. (Personal communication: Crow, K. D., Princess Margaret Hospital, Swindon, England.) Why these workers developed the greasy skin cannot be explained and must at this time be considered an anomaly. Experience from the industrial episodes (and from the Seveso, Italy episode) confirm that mild chloracne may clear quickly (e.g., in months). Severe chloracne is known, however, for its recidivism. Cases with active lesions have persisted for up to fifteen years after exposure ceased (53). Many of the studies of systemic effects used populations presenting with chloracne as a point of entry. This probably is not a major weakness, however, because chloracne is one of the earliest indicators of disease (13). Nevertheless, it could have resulted in an artifically lowered incidence of systemic effects present without acne. b. Porphyria Cutanea Tardia. Porphyria cutanea tarda (PCT) is a disorder of heme pigment metabolism characterized by skin sensitivity, accumulation of excess pigment in the liver, and the build-up of the various porphyrin pigments. Skin findings include skin fragility, bullous lesions, pigmentation, and photosensitivity. It may be either hereditary or acquired. The latter is usually associated with hepatic disorders. Bleiberg et al (14), in 1964, discovered eleven cases of PCT in workers involved in the production of 2,4,5-trichlorophenol. In 1973, Pazderova et al (61) reported on twenty-three additional cases. Liver biopsies were obtained in five subjects and liver tissue from necropsies in two. All showed fluorescence under ultraviolet light indicating high levels of porphyrins. In 1971, Poland et al (62) studied the workers described by Bleiberg and noted only one asymptomatic urine uroporphyrin. Changes in the plant had greatly decreased the exposure to TCP and TCDD. The hyperpigmentation and skin lesions found in PCT are independent of those found in chloracne. Pre-existing liver disease appears to predispose a subject to PCT when challenged by another agent such as TCDD. Based on the majority of studies, systemic disease does not result unless chloracne is present either before or during the course of the disease. However, Bleiberg (14) found porphyria was present in two cases without acne, and Oliver (57) noted that one laboratory worker synthesizing TCDD developed rather severe systemic symptoms but never any acne. It is accepted that chloracne can result from external exposure to TCDD. The role of systemic absorption of TCDD in the development of chloracne has been a matter of debate. Similarly, it is even less clear what role percutaneous absorption of TCDD plays in the development of systemic disease. Regardless, the basic observation is true enough so that an etiology other than TCDD should be diligently searched for in any case where symptoms developed without acne. VI -21 �c. Asthenia. Many asthenic and other vegetative symptoms have been described in 2,4,5-T, TCP and TCDD intoxication. For purposes of this report, asthenia includes the following: headache, apathy, fatigue, anorexia, weight loss, sleep disturbances, decreased learning ability, decreased memory, dyspepsia, sweating, muscle pain, joint pain and sexual dysfunction. True pathology is closely interwoven with the depression which undoubtedly exists as a result of other disorders, particularly the disfigurement of chloracne, therefore causing difficulty in interpretation of these symptoms. This problem is well demonstrated in a report on polybrominated biphenyl (PBB) exposure in the April 7, 1978, issue of the Center for Disease Control MoibM<ti;y and mofttattty Weefe£</ Repoit (17). Several hundred pounds of PBB were accidently introduced into animal feed. Three cohorts were studied, the first involving all persons who had been identified by the Michigan Department of Public Health as living on PBB contaminated farms at the time of quarantine, the second including persons who had received food products directly from such farms, and the third included workers (and their families) who had been exposed occupationally to PBB in a chemical manufacturing plant. Two additional groups with low level PBB exposure were also evaluated. Highest PBB levels were found in those groups in whom one would expect the exposure to be the greatest. However, symptoms occurred most frequently in volunteers and in persons from nonquarantined farms with low level PBB contamination. Symptoms were least prevalent in quarantined farm families and in chemical workers, just the opposite of what one would expect. Symptoms and conditions included fatigue, rashes, joint pains, hepatitis, diabetes, benign tumors, and cancer. The point to be made is that signs and symptoms of asthenia are common and need not be related to chemical exposure. There is little question that asthenic symptoms can develop following TCDD exposure. In an early plant accident in which exposure is felt to have been massive, workers developed fatigue and severe muscle pain (75). Impotency was present. As it was one of the first such episodes, the symptoms of TCDD or TCP exposure had not been delineated, and therefore the effect of suggestion would have been minimal. It needs to be emphasized, however, that the exposure was massive and the symptoms did clear. One must be very careful in transposing the results of this accident to another where exposure was much less. One is on particularly tenuous ground if he attempts to attribute the symptoms to the exposure levels found in herbicide spraying. 2. Special Case Studies a. Exposure resulting from spraying operations. The study by Londono in 1966 (51) is of special interest in that he reported on five subjects who were involved in herbicide spraying as opposed to industrial exposure. The herbicides included the butyl ester of 2,4-D and the methyl ester of 2,4,5-T. All five developed chloracne. One of the workers manifested the acne seventeen days after the onset of spraying, three after two months, and one after eighteen months. No clinical systemic disease was reported, although liver function tests were mildly abnormal. b. Controlled study on 2,4,5-T plant workers. In contrast with the episodes just described, which were in effect case studies, Kramer in VI-22 �1970 and revised in 1974 (49) in an unpublished report described a control study on the health of employees exposed to 2,4,5-T at Dow Chemical Company. The control population of 4,600 non-exposed Dow employees did not vary significantly from the general population. Fifty clinical parameters were investigated including both history and laboratory studies. Parameters included were those which would be indicative of disorders of the central nervous system, mucous membrane irritation, pulmonary disease, cardiovascular disease, gastrointestinal and hepatic disorders, renal disease, asthenia, and psychiatric disorders. No significant differences were found between the study and control groups. 3. General Population Exposures The discussion up to this point has generally related to the occupational hazards of TCP, 2,4,5-T and TCDD. In recent years there has been considerable interest expressed by a number of groups concerning the public health aspects of the phenoxy herbicides and TCDD. The remainder of this section will concentrate on several incidents in which the general population was involved. Chapter V has provided more extensive details on each of these episodes. a. South Vietnam Episode. In the latter part of 1969 newspapers in South Vietnam reported that there were an unusually large number of malformed babies being born among the Montagnards, This was followed by a publication by Tung et al (85) of the Democratic Republic of Vietnam in T971 in which they reported 179 people who had lived in sprayed areas from two months to five years or had been in direct contact with the spray. He did not specify the type or composition of the spray material. Disorders were divided into three major groups: asthenia, ocular syndrome and genetic effects. The general asthenia was accompanied by insomnia, headache, sexual impotence and menstrual problems in females. A specific form of the asthenia, visual asthenia,was characterized by early onset of eye fatigue (5-15 minutes) when reading. The ocular syndrome consisted of the visual asthenia (mentioned above) as well as a decrease in visual acuity and corneal scarring. The genetic syndrome consisted of chromosomal alterations in seriously affected adults, congenital malformations (particularly Trisomy 21) in the new born, and unclassifiable multiple congenital malformations with multiple chromosomic alterations. In 1973, Tung et al (86) reported an increase in the number of persons with primary liver cancer in proportion to all cancer patients admitted to Hanoi hospitals during the period 1972-1968 (790 liver cancer cases out of 7911 cancer cases, 10 percent) as compared to the period 19551961 (159 liver cancer cases out of 5492 total cancer cases, 2.9 percent), which was prior to the start of herbicide spraying. The authors attributed this increase to exposure as a result of the spraying of herbicides containing TCDD in South Vietnam during the 1960's; however, a recent IARC monograph (34) noted that limitations in the reporting of the study make impossible an adequate assessment between the incidence of liver cancer and herbicide spraying in South Vietnam. VI-23 �A National Academy of Science (NAS) committee (18) was established in 1972 to investigate the effects of herbicides in Vietnam. in their report, published in 1974, they described an earlier study by Cutting et al (22) in 1970 reviewing congenital malformations, hydatidiform moles, and stillbirths in 22 hospitals in the Republic of Vietnam for the periods between 1960-1965 and 1966-1969. The first time period involved light spraying of Herbicide Orange, the second, heavy. Neither the NAS nor Cutting et al were able to demonstrate any influence of the herbicides on the development of these disorders. It must be pointed out that all studies in Vietnam were limited by poor and incomplete reporting, and most important by the politics of the area. Large segments of the population in question were not available to the investigating groups. b. Eastern Missouri Horse Arena Episode. Following the spraying in 1971 of three horse arenas in Lincoln County, Missouri, with salvage oil contaminated with TCDD, a number of people who worked or played in the arenas developed medical disorders (10, 19, 43, 50). The most serious war> a six-yearold girl who developed hemorrhagic cystitis and focal pyelonephritis. The urinary tract symptoms were preceded by headache, epistaxis, diarrhea and a general malaise. Her urinary tract symptoms cleared after a few days. Three months later examination was normal except for punctate hemorrhages of the bladder seen on cystoscopy. The father of the child had developed headaches and nausea while working in the arena. Her mother reported severe headache, nausea, diarrhea and abdominal pains, and arthralgia. A ten-year-old sister developed easy fatigability, epistaxis, headaches, abdominal pain, and diarrhea. All developed at least mild acne lesions (64). Follow-up studies on the mother and two children performed five years later were normal (10). Chloracne developed in two three-year-old boys who played in another arena (19, 43). One case lasted more than a year. Commoner and Scott (19) in 1976 mentioned one additional case of chloracne in a veterinarian who obtained samples in a third arena. The symptoms in all seven of the humans were relatively mild with the most severe being the hemorrhagic cystitis and focal pyelonephritis seen in the six-year-old girl. The symptoms had cleared on re-examination several years later. Exposure, at least to the four children must have been significant in that they regularly played in the soil of the arenas. Contrast this with the disastrous effects the dioxin had on the horses and other animals that were in the arena often for only a short period (43). This gives strong support to the contention that man is relatively resistant to TCDD or absorbs it to a much lesser extent. c. The Seveso, Italy Episode. The details of the Seveso, Italy, incident where an industrial accident resulted in the exposure of the general population to a cloud of TCP and other toxicants are described in the previous chapter. This incident is most significant in that it represents the first episode where a cross section of a community received a definite exposure to TCDD, although the degree of exposure can only be estimated. As in any such situation confusion reigns, and a great deal of information is passed VI-24 �consisting of a mixture of truths, half-truths, and untruths. This confusion was amplified by the fact that the caustic nature of the cloud produced serious irritative effects including skin burns in many of the people who came in direct contact with the cloud. Very early after the incident an organized program was established to follow the general populace to determine what if any effects, both long and short-term, resulted (26). The findings for the first two years have been reported and are summarized as follows (2, 64, 65, 83, 84): (1) Chloracne--A massive screening program was initiated in 32,000 school children below the age of ten. Seventy-nine cases of chloracne were confirmed, only eight of which were severe. Many of the victims were not present in the area until weeks or months after the accident, indicating that the TCDD remained in the environment outside the zone of evacuation at a level high enough to produce chloracne. Except for the eight severe cases, the acne cleared in a few months. Two years after exposure some of the severe cases still showed active lesions and had severe scarring (65). In October 1977, six new cases were found in children who returned to their homes after decontamination, bringing the total to 85. No systemic abnormalities have been found in the children with acne. It is of interest that nearly all cases of acne were found in children. There are several possible explanations" for this. A massive systematic, search for chloracne was undertaken for children under the age of ten. No such search was undertaken for adults. It may be that children are more sensitive than adults. This is a phenomenon frequently seen with chemicals and drugs. It may also simply be that the children's daily routine results in greater exposure. (2) Spontaneous Abortion and Fetal Malformation—Information concerning the birthrate, abortions and fetal malformations for the two ylars following the accident revealed no significant changes (2, 64, 65, 83, 84). There were several problems regarding the evaluation of the results. First and most important was the lack of reliable background data for the area. Worldwide figures and figures from the Lombardy region of which Seveso is a part were used. Data were also biased by the fact that therapeutic abortions were offered to women who were pregnant at the time of or immediately after the accident. Nevertheless, it was the opinion of the evaluators that significant increases in spontaneous abortions and fetal malformations could not be demonstrated. Chromosomal studies were performed on the fetuses of thirty pregnancies interrupted between August 13 and December 10, 1976. No abnormalities in number of pattern beyond the expected rate were seen (2, 64, 65, 83, 84). (3) Immunology—No differences in imrnunoglobulins and B lymphocytes were found between a study and a control group of children, even though twenty of the children in the study group had chloracne (65). Hospital admissions and disease classification data evaluation revealed no significant changes from the previous year. There was an increase in VI-25 �infectious diseases compared to the previous year, but this increase was also seen in the nonex.posed districts (2, 64, 65). (4) Summary—Except for the initial irritative effects of the caustic substances and the presence of eighty-five cases of chloracne, no adverse effects to the chemicals in the toxic cloud have been confirmed. It must be remembered, however, that in many instances the findings were not conclusive and that long-term effects such as cancer and hidden congenital malformations have not yet had time to manifest themselves. It will be several years before all the data are published on the Seveso incident. d. Globe, Arizona Episode. In 1969, a number of residents in the Globe, Arizona area alleged that numerous physical ailments resulted from the spraying of the surrounding area with 2,4-D, 2,4,5-T and Silvex. Symptoms and disorders mentioned included headaches, fatigue, chest and arm pain, worsening of pre-existing nasal allergies and asthma, loss of the sense of smell and taste, severe diarrhea, spasms of the arms and legs, anemia, irregular and painful menses, spontaneous abortions, fetal malformations and cancer (82). An investigation in 1970 by Tschirley et al (82) was unable to connect the disorders with definite exposure; however, he recommended further studies. As a result of this recommendation Roan and Morgan (66) in 1972 reported on the results of an epidemiological study of the hospital records in the area and a pesticide analysis of several body tissues and fluids including adipose tissue. The technique of analysis allowed minimum detection of TCDD at 2 ng/gm tissue and of 2,4,5-T, 2,4-D or Silvex at 0.01 ng/gm tissue. No 2,4,5-T, 2,4-D, Silvex or TCDD was found. They concluded that the probability of chronic human exposure in the area in question was very minimal. e. The Swedish Lapland Episode. As noted in Chapter V, this episode involved a series of public debates on the risks to humans (and animals) of the phenoxy herbicides, especially 2,4,5-T. In the March 1978 WBBM television report on "Agent Orange: Vietnam's Deadly Fog," reference was made to a report from Sweden on birth defects (e.g., spina bifida) in children born to 65 women allegedly exposed to 2,4,5-T herbicide. The only reference to such an incident was that reported by Hailing (32) in 1977. Hailing reported on the presence of malformations in children born to mothers exposed to hexachlorophene soap during early pregnancy. A group of 65 children born to this group showed six slight and five severe malformations. This contrasted with one slight malformation in 68 children born to a group of nonexposed mothers. It needs to be emphasized that the chemical in question was hexachlorophene and not phenoxy herbicide. f. Te Awamutu, New Zealand Episode. In 1972 Sare and Forbes (68) reported on two babies born within a month of each other in the same hospital, each presenting with meningomyelocoeles. They lived in farm country where spraying with 2,4,5-T had been routinely carried out for several years. The possibility that the malformations may have been related to the herbicide was suggested. Because of this report and other allegations that neural tube deformities were the result of 2,4,5-T exposure, a thorough, although retrospective, investigation of the problem was undertaken by the VI-26 �Department of Health in 1977. The investigating committee (1) concluded that there was no evidence to implicate 2,4,5-T as an etiologic factor. D. Cancer There are a number of individual case reports and geographically limited studies of herbicide workers, both in manufacturing as well as application, that suggest an associative relationship between exposure to either 2,4,5-T, TCP or TCDD and subsequent development of a variety of neoplasms. Of 75 workers exposed in a TCP factory accident in 1953, most were affected by chloracne, 42 were listed as severe. All of the 75 workers could be traced 25 years later and whfle the mortality rate was no higher than expected, there had been 6 deaths due to cancer versus the 4 that could have been expected from national averages. Three of the deaths were due to stomach cancer in the 60-69 year age group, which was significantly more than expected (35). One worker involved in an accident in a 2,4,5-T producing factory in the Netherlands in 1963 died of carcinoma of the pancreas in 1964 (35). Because of the extremely short time span between the exposure and the death, it is not likely that the two are related. In 1973 Tung (86) reported an increased incidence of hepatic cancer in Vietnamese allegedly exposed to the spray of Herbicide Orange. A lack of details in the reporting make evaluation of the claim difficult. Jirasek et al (37, 38) and Pazderova et al (61) reported the presence of two bronchiogenic carcinomas at ages 47 and 59 during the first five years of the follow-up of 75 workers occupationally exposed to 2,4,5-T and pentachlorophenol. They noted that only 0.12 lung cancer deaths were expected from national mortality statistics. Again, the latent period was short and no smoking statistics were given. In 1972, newspapers in Sweden reported an excess mortality due to lung cancer in railroad workers exposed to herbicides. As a result, Axelson and Sundell (5) initiated a controlled study and in 1974 reported that although there appeared to be an increased incidence with Amitrol there was no significant increase with 2,4-D or 2,4,5-T. However, a re-evaluation of the data indicated a possible and previously masked tumor inducing effect from the phenoxy acids (35). A similar study on workers involved with spraying 2,4-D and 2,4,5-T on brushwood in Finland showed no increase in overall mortality. There were, however, four cases of cancer in the age group younger than 45 years as opposed to the expected two(35). Hardell (33) reported that of 87 mesenchymal tumors seen from 1970-1976 in the Department of Oncology, Regional Hospital, Umea, Sweden, 19 had been in men whose employment (farmers and forestry workers) may have resulted in exposure to the phenoxy herbicides. The expected mesenchymal VI-27 �cancers for this group was eleven. Seven cases with known sporadic herbicide exposure 10-20 years before diagnosis were presented. Hardell noted the difficulty in establishing a causal relationship but suggested that the deviation from national averages for these relatively uncommon tumors could perhaps be linked to extensive use of the phenoxy herbicides in the Umea region. Five leukemia deaths have been reported in the area of Meda, Italy, since the Seveso episode in July 1976. No more than 1.4 were expected. One of the cases was found to predate the accident (35). Additionally, the interval from exposure to diagnosis appears to be too short to ascribe causation. The case of pancreatic carcin'oma in the 55-year-old woman described by Reggiani (65) and mentioned previously in the section on the pharmacodynamics of TCDD was also felt not to be related to the exposure to TCDD. To quote Reggiani "A causal relationship with the malignancy can be excluded owing to the lapse of time required by tumor growth to reach the size, weight and diffusion of this case. The exposure to TCDD has occurred at a time when the growth of the tumor had already reached the stage of occult spreading throughout lymphatic and blood vessels to the adjacent tissues and organs." (65) As noted above, these studies should be viewed only as preliminary evidence of a statistical relationship between exposure to the phenoxy herbicides, TCDD and TCP and subsequent cancer development. Except in cases such as the angiosarcoma caused by vinyl chloride where the type of cancer is rare and the association with exposure irrefutable, it is virtually impossible to differentiate a cancer caused by a specific chemical agent from a similar cancer caused by some other etiology. This is certainly true with the retrospective studies currently available and may be true even with meticulously controlled prospective studies. There are, however, a number of cohort studies either ongoing or planned which may help clarify the present uncertainty concerning the role of the phenoxy herbicides in cancer causation in humans (35). IV. CONCLUSIONS A. Pharmacodynamics 1. 2,4-D and 2,4,5.-T are readily absorbed via the cutaneous, and gastrointestinal routes, distributing throughout the body. The respiratory tract may also be a point of entry although of lesser importance, 2. Liquid phenoxy-herbicide contact to the skin can produce systemic reactions. 3. 2,4-D and 2,4,5-T have relatively short half-lives in the human body and persistent body burden is unlikely to develop, at least in short-term or intermittent exposures. VI-28 �Other than the knowledge that TCDD may enter the body ly, the pharmacokinetics of TCDD in man are essentially unknown. Sasud on the way ot;her_p«-c.t,icides are handled, it is reasonable to assume that t^e use of 2,^,5-T has resulted in considerable skin-liquid contact. In spite of this, reports of 2,4,5-T toxicity and therefore TCDD toxicity are minimal considering the degree of use. This may indicate that man is more resistant to the effects of 2,4,5-T and TCDD than other animals, but it could also indicate that percutaneous absorption is less. The apparent relative lack of toxicity or percutaneous absorption is further supported by the Missouri incident where there was a marked difference between the degree of toxicity in man and animals. B. Effects of the Herbicides 1. The use of 2,4-D and 2,4,5-T worldwide since the middle 1940s, with minimal reports of adverse effects indicate that they jre generally saf" chemicals if properly used. Large total doses or 2,4-D have been given tc humans in controlled circumstances without adverse effects. 2. The ne-vous system is p-: v - r icui at i,> sensitive to 2,4-D. If peripheral neuropathy developed following exposure to 2,4-D, it normally disappears in a matter of months. However, in some reported incidents, it "nd persist for on? to three years. 3. Symptoms present within the first few days after exposure are probably due to the herbicide and not TCDD. 4. Adverse effects of 2,4-D and 2,4,5-T should manifest themselves shortly after exposure. Symptoms arising for the first time, months to years after the last exposure are probably due to an etiology other than 2,4-D and 2,4,5-T, 5. The hematopoetic system may be an important target organ for 2,'-i-D in some people. C. Effects of TCDD 1. If there is not a history of chloracne, it is highly unlikely that systemic changes will be due to TCDD. However, the acne may be minimal and, therefore, the historical search must be meticulous. 2. The presence of active chloracne months to years after exposure does not necessarily mean continuing exposure. 3. Skin lesions of porphyria cutanea tarda are independent of those associated with chloracne. 4. The development of porphyria cutanea tarda following exposure to TCDD suggests an adverse liver response to the TCDD. VI-29 �5. Although asthenia is difficult to interpret, it probably represents a symptom of TCDD intoxication. 6. A rise in serum lipids may occur after exposure to TCDD. However, because of large individual variations, the finding is difficult to interpret. 7. Claims of carcinogens!s, teratogenesis, and mutagenesis in man have not been confirmed at this time for the phenoxy herbicides or TCDD. However, the topic remains open. • 8. The preliminary information from the Seveso episode and the study by Kramer on the health of 2,4,5-T workers indicate that incidental nonoccupational exposure to small amounts of TCDD is unlikely to produce symptoms. 9. The long-term effects of large acute doses of TCDD or small intermittent or chronic exposures are not known. V. SUMMARY The pharmacodynamics and adverse effects of the phenoxy herbicides, trichlorophenol and TCDD were reviewed, primarily through reports of occupational exposure and accidents as well as reported exposures to the general public. A number of organ systems may be involved if the dose is significantly high with emphasis on the skin, liver, CNS and peripheral nervous system. Adverse effects of 2,4-D and 2,4,5-T should manifest themselves shortly after exposure. Symptoms arising for the first time months to years after the last exposure are probably due to an etiology other than 2,4-D and 2»4,5-T. The hallmark of TCDD is chloracne and its absence makes it unlikely that systemic disorders present are related to TCDD. Asthenic and vegetative symptoms are often present in overexposure but are difficult to interpret. They would normally be expected to clear with time. There is no conclusive evidence at this time that the phenoxy herbicides or TCDD are mutagenic, teratogenic or carcinogenic in man. VI-30 �LITERATURE CITED CHAPTER VI 1. Anonymous. 1977. 2,4,5-T and Human Birth Defects. Report prepared in the Division of Public Health, Department of Health, New Zealand. Mim. 42p. 2. Anonymous. 1977. 28th Technical Report to the Seveso Authority. Mario Negri Institute of Pharmacological Research. Milan, Italy. November 1977. 3. Armstrong, R.W., E.R. Eichner, E.D. Klein, W.F. Barthel , J.V. Bennett, V. Jonsspn, H. Bruce, and I.E. Loveless. 1969. Pentachlorophenol poisoning in a nursery for newborn infants. II. Epidemiologic and toxicologic studies. J. PzdLLa&L. 75(2) -.317-325. 4. Assouly, M. 1951. Desterbants selectifes et substances de croissance. Apercu technique. Effet pathologique sur Thomme au cours de fabrication de Tester du 2,4-D. AtcA. Mo£. P/torf. 12:26-30. 5. Axelson, 0. and L. Sundell. 1974. Herbicide exposure, mortality and tumor incidence. An epidemiological investigation on Swedish railroad workers. Wo/ife, Enutton. , Heotth ll(l):21-28. 6. Baader, E.W. and H.J. Bauer. 1951. Industrial intoxication due to pentachlorophenol. Ind. Med. SotgeAt/ 20(6) : 286 -290. 7. Barthel, E. 1974. Pulmonary fibroses in persons occupationally exposed to pesticides. Z. &ikA.. ktmungAoig 141:7-17. (German) 8. Bashirov, A. A. 1969. The state of health in workers manufacturing the herbicides, the amine salt and the butyl ester of 2,4-D acid. (//uzcAebnoe t?e£o Wo. 10:92-95. (Russian) 9. Bauer, H., K. H. Schulz and U. Spieqeioerg. 1961. Occupational intoxication in tne manufacture of chiorophenol compounds. A/tcA. GzweA.be.path.ot. 18:538-555. (German). 10. Beale, M.G., W.i. Shearer, M.M. Karl and A.M. Roboon. 1977. Longterm effects of dioxin exposure. Ltr to Editor. Lancat(l) (8014) :748. 11. Berkley, M.C. and K.R. Magee. 1963. Neuropathy following exposure to a diethylamine salt of 2,4-D. A/ich. Intern. Med. 111:351-352. 12. Berwick, P. 1970. 2,4-Dichlorophenoxyacetic acid poisoning in man. Some interesting clinical and laboratory findings. 3. Am. Med. VI-31 �13. Birmingham, D.J. 1964. Occupational dermatology: current problems. 3:38-42. 14. Bleiberg, J., M. Wallen, R. Brodkin and I.L. Applebaum. 1964. Industrially acquired porphyria. M.c.h. Vejuncutat. 89:793-797. 15. Brandt, M.R. 1971. Herbatox poisoning, a brief review and a report of a new case. Ugtekn. La&g. 133(11) :500-503. (Danish) 16. Braun, W. 1970. Chloracne. TkeA.. Umck. 27(8) :541-546. (German) 17. Budd, M.L., N.S. Hayner, H.E.B. Humphrey, J.R. Isbister, H. Price, M.S. Reizen, G. van Amburg and K.R. Wilcox. 1978. Polybrominated biphenyl exposure - Michigan. Mo-tb. Matt. 27(14) :115-116, 121. 18. Committee on the effects of herbicides in South Vietnam. 1974. Part A. Summary and Conclusions. National Academy of Science, Washington, D.C. 398p. 19. Commoner, B. and R.E. Scott. 1976. Accidental contamination of soil with dioxin in Missouri: Effects and countermeasures. Center for the Biology of Natural Systems. Washington University; St. Louis, Missouri. Mim. 27p. 20. Coutselinis, A., R. Kentarchou and D. Boukis. 1977. Concentration levels of 2,4-D and 2,4,5-T in forensic material. Foimdxlc Science 10:203-204. 21. Crow, K.D. 1970. 56:79-99. Chloracne. Trans. St. John's Hosp. Vexmcutol. Sco. 22. Cutting, R.T., T.H. Phuoc, J.M. Ballo, M.W. Benenson and C.H. Evans. '1970. Congenital malformations, hydatidiform moles and stillbirths in the Republic of Vietnam, 1960-1969. Document No. 903.233. Government Printing Office, Washington, D.C. 23. Dudley, A.W. and N.T. Thapar. 1972. Fatal human ingestion of 2,4-D, a common herbicide. M.c.k. ?cuthol. 94(3):270-275. 24. Dugois, P., J. Mare'chal and L. Colomb. 1958. Chloracne caused by 2,4,5-trichlorophenol. hick. Mat Ptorf. 19:626-627. (French) 25. Dugois, P., D. Amblard, M. Aimard and G. Deshors. 1968. A collective and accidental Chloracne of a new type. Bo££e£tn de. to. Soc^e^e'Ctcn-tque do. VexmcutoioQle. <tt SyphJJUgHaphie. 75:260-261. (French) 26. Fara, G.M. 1976. Health surveillance program. Medical -Epidemiological . Commission. Milan, August 27, 1976. Mim. lOp. 27. Feldmann, R.J. and H.I. Maibach. 1974. Percutaneous penetration of some pesticides and herbicides in man. Tozicoi. App£. Phasunacoi. 28(1):126-132. VI-32 �28. Gehring, P.J., C.G. Kramer, B.A. Schwetz, J.Q. Rose and V.K. Rowe. 1973. The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral administration to man. TOJO.CO£. App£. VhaJwac.ot. 26:352-361. 29. Goldmann, P.J. 1972. Extremely severe acute chloracne due to trichlorophenol decomposition products. A contribution to the perna problem. Atfae^&med. Soz-ta6ned. Afi.b<ut&hyg. 7(1):12-18. (German) 30. Goldmann, P.J. 1973. Severe acute chloracne, a mass intoxication due to 2,3,6,7-tetrachlorodibenzodioxin. Huutaft.zt laJit. 24:149-152. (German) 31. Goldstein, N.P., P.H. Jones and J.R. Brown. 1959. Peripheral neuropathy after exposure to an ester of dichlorophenoxyacetic acid. J. Am. Med. A44oc. 171:1306-1309. 32. H a i l i n g , H. 1977. Suspected l i n k between exposure to hexachlorophene and birth malformed infants. LakafcUdninge.n 74:542-546. (Swedish) 33. Hardell, L. 1977. Malignant mesenchymal tumors and exposure to phenoxy acids - A clinical observation. Lafea/t£cdcu.ngen 74(33): 2753-2754. (Swedish) 34. International Agency for Research on Cancer. 1977. I ARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Vol. 15. Some ^umiQantA, the. koAb<icMizA 2,4-1? and 2,4,5-T, c.kio^inate.d and mit>c.<Mane.ouA induA&tijot chenu.co£a. Lyons, France. 35. IARC. 1978'. IARC Internal Technical Report No. 78/001. Coordination of epidemiological studies on the long-term hazards of the chlorinated dibenzo-dioxins/chlorinated dibenzofurans. Joint NIEHS/IARC Working Group Report. Lyon, France. 36. Jensen, N.E. and A.E. Walker. 1972. Chloracne: Three cases. Royal Soc. Meet. 65:687-688. P/coc. 37. Jirasek, L., J. Kalensky and K. Kubec. 1973. Acne chlorina and porphyria cutanea tarda during the manufacture of herbicides. Vesunatal. 48(5): 306-31 5. (Czech) 38. Jirasek, L. , J. Kalensky, K. Kubec, J. Pazderova and E. Lukas. 1974. Acne chlorina, porphyria cutanei. tarda, and other manifestations of general intoxication during the manufacture of herbicides. II. C&sfe. Vfwnalol. 49(3):145-157. (Czech) 39. Johnson, H.R.M. and 0. Koumides. 1965. A further case of M.C.P.A. poisoning. B/i. Meet. J. 2:629-930. 40. Jones, D.I.R., A.G. Knight and A.J. Smith. 1967. Attempted suicide with herbicide containing MCPA. M.c.k. Env<tiion. H&atth. 14:363-366. VI-33 �41. Kimbrough, R.D. 1972.. Toxicity of chlorinated hydrocarbons and related compounds. hick. Evwifian. Health 25(2) :125-131 . 42. Kimbrough, R.D. 1974. The toxicity of polychlorinated polycyclic compounds and related chemicals. CJut. Rev. Toiu,c.ol. 2:445-498. 43. Kimbrough, R.D. , C.D. Carter, J.A. Liddle, R.E. Cline and P.E. Phillips. 1977. Epidemiology and pathology of a tetrachlorodibenzodioxin poisoning episode. hick Env-inon H&cuLth 28:77-85. 44. Kimmig, J. and K.H. Schulz. 1957. Chlorinated aromatic cyclic ether as cause of so-called chloracne. Na£uAw^en4cha££en ,44:337-338. (German) 45. Kimmig, J and K.H. Schulz. 1957. Occupational acne caused by chlorinated aromatic cyclic ethers. V&ima£ol.OQ4.ca 115:540-546. (German) 46. Kohli, J.D. , R.N. Khanna, B.N. Gupta, M.M. Dhar, J.S. Tandon and K.P. Sircar. 1974. Absorption and excretion of 2,4-dichlorophenoxyacetic acid in man. Xeno b-iotic.a 4(2):97-100. 47. Kohli, J.D. , R.M. Khanna, B.N. Gupta, M.M. Dhar, J.S. Tandon and K.P. Sircar. 1974. Absorption and excretion of 2,4,5-trichlorophenoxyacetic acid in man. 'hich. Int. Phasunacodyn. TheA. 210:250-255. 48. Kotlarek-Haus, S., W. Dzierkowa-Borodej and B. Lawinska. 1971. Autoimmune hemolytic anemia after handling insecticides and herbicides with simultaneous detection of the Australian antigen (AT) in the serum, fotia. Hamcutol. 95(3)240-253. (German) 49. Kramer, C.G. 1970, revised 1974. Health of employees exposed to 2,4,5-T. Dow Chemical Company, Midland, Michigan. Mim. Unpublished data. 19p. 50. Lobes, L.A. , R.E. Koehler, W.F. 1972. Administrative report to Control (CDC) on toxic illness, EPI-72-13-2, U.S. Public Health Barthel , R.A. Feldman and J.V. Bennett. the director of the Center for Disease Lincoln County, Missouri, CDC No. Service, CDC, Atlanta, Georgia. 51. Londono, F. 1966. Occupational acne: Five cases produced by weed killers. Medicxjoo. Cwtanae. 3:225-232 (Spanish) 52. Matsumura, A. 1970. The fate of 2,4,5-trichlorophenoxyacetic acid in man. Jap. J. Ud. Hea&th 12(9):20-25. (Japanese) 53. May, G. 1973. Chloracne from the accidental production of tetrachlorobenzodioxin. B-t. J. Ind. Mad. 30:276-283. 54. Miura, H., A. Omori and M. Shibue. 1974. The effect of chlorophenols on the excretion of porphyrins in the urine. Sangyo Igafeu 16(6) :575,, (Japanese) VI-34 �area, G. and G, di Vito. 1961. Acute poisoning from weed killer ,4-dichlorophenoxyacetic acid), fotia Med. 44:480-485. 56. Hielson, K. , B. Kaempe and J. Jensen-Holm. 1965. Fatal poisoning in in man by 2,4-dichlorophenoxyacetic acid (2,4-D): Determination of the agent in forensic materials, kcta ?kamac.ol. TOJO.CO£. 22:224-234. 57. Oliver, R.M. 1975. Toxic effects of 2,3,7, 8-tetrachlorodibenzo-l ,4dioxin in laboratory workers. St. J. Ind. Meet. 32(l):49-53. 58. Paggiaro, P.L., E. Martino and S. Mariotti." 1974. A case of 2,4-dichlorophenoxyacetic acid (2,4-D) intoxication. Meci. Lav. 65 (3-4.) :128-135. (Italian) 59. Palva, H.L.A., 0. Koivisto and I. P. Palva. 1975. Aplastic anaemia after exposure to a weed killer, 2-methyl-4-chlorophenoxyacetic acid. Haemato£. 53(2) :105-108. 60. Park, J., I. Darrien and L.F. Prescott. 1977. Pharmacokinetic studies in severe intoxication with 2,4-D and mecoprop. Ctot. To>u.c.o£. 18:154-155. 61. Pazderoz, J., E. Lukas, M. Nemcova, M. Spacilova, L. Jirasek, J. Kalensky, J. John, A. Jirasek and J. Pickova. 1974. Chronic poisoning by chlorinated hydrocarbons formed in the production of 2,4,5-trichlorophenoxyacetate. Piac.. Ltk. 26(9) :332-339. (Czech) 62. Poland, A.P., D. Smith, G. Wetter and P. Possick. 1971. A health survey of workers in a 2,4-D and 2,4,5-T plant. A/icfo. Envision. \\<LaJWi 22:316-327. 63. Popham, R.D. and D.M. Davies. B*. Med. J. 1:677-678. 1964. A case of MCPA poisoning. 64. Reggiani, G. 1977. Medical problems raised by the TCDD contamination in Seveso, Italy. Presentation to the 5th International Conference on Occupational Health in the Chemical Industry (Medichem), San Francisco, California, September 5-10, 1977. 65. Reggiani, G. 1978. The estimation of the TCDD toxic potential in the light of the Seveso accident. Paper presented at the 20th Congress of the European Society of toxicology. Berlin (West), June 25-28, 1978. 66. Roan, C.C. and D.P. Morgan. 1972. Alleged effects on human health of the use of herbicides in the area around Globe, Arizona. Arizona Community Pesticide Study Project, March 6, 1972. University of Arizona, Tucson, Arizona. Mim. 7p. 67. Robson, A.M., J.M. Kissane, N.H. Elvick and L. Pundavela. 1969. Pentachlorophenol poisoning in a nursery for newborn infants. I. Clinical features and treatment. J. PzdMi. 75(2) :309-316. VI-35 �68. Sare, W.M. and P.I. Forbes. 1972. Possible dysmorphogenic effects of an agricultural chemical: 2,4,5-T. W.Z. Med. J. 75(476):37-38. 69. Sare, W.M. 1972. The weedicide 2,4-D as a cause of headaches and diplopia. N.A. Med. J. 75(478) :173-174. 70. Sauerhoff, M.W. , W.H. Braun, G.E. Blau and P.J. Gehring. 1977. The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man. Toiu.c.ot.ogy 8:3-11. 71. Sauerhoff, M.W., M.B. Chenoweth, R.J. Karbowski, W.H. Braun, J.C. Ramsey, P.J. Gehring and G.E. Blau. 1977. Fate of Silvex following oral administration to humans. J. ToiU.c.ol. Envision Hea&tk 3:941-952. 72. Schulz, K.H. 1957. Clinical and experimental studies on the etiology of chloracne. M.C/I. K&tn. Ex.p. V<ywatot. 206:589-596. (German) 73. Schulz, K.H. 1968. Clinical picture and etiology of chloracne. . Sozxu&ned. Atbexx&hyg. 3(2):25-29. (German) 74. Seabury, J.H. 1963. Toxicity of 2,4-dichlorophenoxyacetic acid for man and dog. A/tch. EmuAon. Health 7:202-209. 75. Suskind, R.R. 1978. Chlorinated hydrocarbon acne. Presentation at the American Occupational Health Conference, New Orleans, Louisiana, April 10-14, 1978. 76. Taylor, C.C. 1974. Chemical toxicity and mental disorder. Am. J. 131(5):609., 77. Taylor, J.S. 1974. Chloracne - A continuing problem. Cu£u 13:585-591 78. Telegina, K.A. and L.I. Bikbulatova. 1970. State of the skin in persons in contact with methoxone during its industrial production. l/ei£n. Vvwatol. VzneAol. 44(8):76-79 (Russian) 79. Ter Beek, R. , R. Bokhorst, M.V.D. Plas, K. Olsthoorn, P. Vergragt and G. Van Der Zwan. 1973. Dioxin, a dangerous contaminant in a much used herbicide. Cfiem. We.efefa£ 69(23) :57 (Dutch) 80. Todd, R.L. 1962. A case of 2,4-D intoxication. J. Iowa Med. Soc.. 52:663-664. 81. T.sapko, V.P. 1966. The herbicide 2,4-D as a health hazard in agriculture. GA&. Saute. 31:449-450. 82. Tschirley, F.H., (Chairman) W. Binns, C. Cueto, B.C. Eliason, H.W. Heggestad, G.H. Hepting, P.F. Sand and R.F. Stephens. 1970. Investigation of spray project near Globe, Arizona. Investigation conducted February 1970. Mim. , U.S. Dep Agric Office of Science and Education. Mim. 29p. VI-36 �83. Tuchmann-Duplessis, H. 1977. Embryo problems posed by the Seveso accident. Le. Conc.onfu> Medico£ No. 44, November 26, 1977. Translation. Mim. 17p. (French) 84. Tuchmann-Duplessis, H. 1978. Environmental pollution and offspring: With relation to the accident at Seveso. MecL &t Hyg. 36:1758-1766. (French) 85. Tung, T.T., T.K.A.B.Q. Tugen, D.X. Tra and N.X. Huyen. 1971. Clinical effects of massive and continuous utilization of defoliants on civilians. W.etnome4e Stu.di.tA. 29:53-81. 86. Tung, T.T. , T.T. An, N.D. Tarn, P.M. Phiet, N.N. Bang, T.T. Bach, H. vanSon and O.K. Son. 1973. Le cancer primaire du foie au Vietnam. e. 99:427-436. (French) 87. Wallis, W.E., A. Van Posnak and F. Plum. 1970. Generalized muscular stiffness, fasciculations and myokymia of peripheral nerve origin. A/icfc. NeuAo£. 22:430-439. 88. Zelikov, A. Kh and L.N. Danilov. 1974. Occupational derma toses (acnes) in workers engaged in production of 2,4,5-trichlorophenol . Sov. Meet. 7:145-146. (Russian) *U.S. GOVERNMENT PRINTING OFFICE: 1980-671-1H3/2S VT- 1 }? � 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 046 Folder The folder containing the original item. 1165 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 Young, Alvin L. John A. Calcagni Charles E. Thalken James W. Tramblay Description An account of the resource <strong>Corporate Author: </strong>United States Air Force Occupational and Environmental Health Laboratory, Aerospace Medical Division (AFSC), Brooks Air Force Base, Texas Date A point or period of time associated with an event in the lifecycle of the resource 1978-10-01 Title A name given to the resource The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange and its Associated Dioxin Subject The topic of the resource Ranch Hand biodegradation herbicide disposal herbicide 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 046 Folder The folder containing the original item. 1166 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 Young, Alvin L. Date A point or period of time associated with an event in the lifecycle of the resource 1980-05-01 Title A name given to the resource Use of Herbicides in South Vietnam, 1961-1971, Presentation/Proceedings Subject The topic of the resource herbicide properties Ranch Hand human 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 1167 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 Young, Alvin L. B. M. Shepard Source A related resource from which the described resource is derived Chemosphere 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 A Review of On-Going Epidemiologic Research in the United States on the Phenoxy Herbicides and Chlorinated Dioxin Contaminants Subject The topic of the resource VA congenital birth defects health studies 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 1168 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 Young, Alvin L. Source A related resource from which the described resource is derived Human and Environmental Risk of Chlorinated Dioxins and Related Compounds 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 An Overview of Laboratory and Waste Management Guidelines For Toxic Chlorinated Dibenzo-p-dioxins and Dibenzofurans Subject The topic of the resource toxic wastes herbicide disposal protective clothing safety 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 1169 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 Young, Alvin L. Source A related resource from which the described resource is derived Human and Environmental Risk of Chlorinated Dioxins and Related Compounds 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 Laboratory Safety and Waste Management Subject The topic of the resource toxic wastes herbicide disposal protective clothing safety 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 1170 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 Young, Alvin L. Han K. Kang Barclay M. Shepard Source A related resource from which the described resource is derived Environmental Science and Technology 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 Chlorinated Dioxins as Herbicide Contaminants Subject The topic of the resource Vietnam soft tissue sarcoma congenital birth defects veteran health and hygiene 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 <p style="margin-top: -1em; line-height: 1.2em;">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.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> 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. 1171 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 Young, Alvin L. Date A point or period of time associated with an event in the lifecycle of the resource 1983-10-01 Title A name given to the resource Manuscript: Analysis of Analytical Data on Dioxins and Furans in Human Adipose Tissue Subject The topic of the resource human testing adipose tissue testing breast milk testing ao_seriesIII