1 15 11 Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 049 Folder The folder containing the original item. 1263 Series The series number of the original item. Series III Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Goring, C. A. I. J. W. Hamaker D. A. Laskowski Source A related resource from which the described resource is derived Chemicals, Human Health and the Environment: A Collection of Dow Scientific Papers, Volume 1 Date A point or period of time associated with an event in the lifecycle of the resource April 9 1973 Title A name given to the resource A Proposed Approach to PR 70-15 Guidelines Subject The topic of the resource EPA pesticide testing ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 048 Folder The folder containing the original item. 1246 Series The series number of the original item. Series III Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Source A related resource from which the described resource is derived Bulletin of the World Health Organization Date A point or period of time associated with an event in the lifecycle of the resource 1962 Title A name given to the resource Measurement of the Exposure of Workers to Pesticides Subject The topic of the resource industrial exposure pesticide testing study protocol ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 048 Folder The folder containing the original item. 1195 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Source A related resource from which the described resource is derived Food and Cosmetics Toxicology Date A point or period of time associated with an event in the lifecycle of the resource 1971 Title A name given to the resource Reduction of Pesticide Residues in Human Adipose Tissue with Diphenylhydantoin Subject The topic of the resource adipose tissue testing human testing pesticide testing ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/d84c18225cc860ed084c3c1c298f0c95.pdf 2624455df9ce42ce184cefedb29a7862 PDF Text Text Item ID Number °1186 Author Adamovic, V. M. Corporate Author Lower Level Phenomena of DDT Cumulation in Female Abdominal Fatty Tissue JOIirnal/BOOk Title Year Arhiv za Higijenu Rada 1973 Month/Day Color Number of Images n 4 Alvin L. Young filed this item under the category "DDT/Human Toxicology and Environmental Fate" Wednesday, April 11, 2001 Page 1186 of 1242 �hig. rada. 24 (1973) 303. LOWER LEVEL PHENOMENA O F JJDJ C U M U L A T I O N I N F E M A L E ABDOMINAL FATTY T I S S U E f ^-V. M. A n fl M n v i ^ and B o R K A S O K I d Institute for Health Protection of Serbia, and Department for Sanitary Chemistry, Beograd The authors found a significantly higher level of DDT storage in the fatty tissue samples of men than in women (mean pp'DDE was 12.8 ppm and 10.2 ppm respectively). By comparing the excretion of organochlorine insecticides in the milk of breast-feeding mothers with their total daily DDT intake by food, they registered considerably greater quantities of pp'DDE eliminated than ingested. By excluding the nondietetic DDT intake the authors explain this phenomenon by hormonal factors i. e. by increased mobilization of the stored DDT metabolites during the lactation period. The lower DDT storage found in women may also reduce the mean residue values related to the general population. Irrespective of its restricted use the presence of DDT in the human environment will continue for some time yet to influence to a very great extent man's existence and of course, his health. The conclusions arrived at meetings of experts (1) show that DDT will continue to be used for controlfing the vectors of certain diseases and even in the control of various agricultural pests. Taking into account the persistence of these compounds on one side and the quantities that have been used so far and that will be used in the future on the other, it is not hard to conclude that man's contact with DDT remains inevitable, whatever guarantees may be prescribed. Past investigations of the toxioological, cancerogenic, mutagenic and other properties of DDT have yielded so many data that it may well be claimed that DDT is the most widely checked compound to date (2, 3). Even so, present knowledge does not offer a realistic idea of its effect upon man. Very little is known about the daily effect of small quantities "Of DDT on the general population, except that it is stored in the adipose 'tissue (4) and that it is thus present in all organs of vital importance. These effects can be evaluated by periodical determinations of the concentration of stored DDT and its degraded products in the human adipose tissue and blood or serum, and correlated with the data on the �304 V. M. AlMMOVld, BORKA SOKI(5 daily intake of DDT with food. Some authors have found the concentration of stored DDT to be on a steady downward line and it is considered that the measures undertaken will further diminish its excretion (2). Among numerous data of this kind there are some which suggest the existence of a significantly higher level of DDT, especially of pp'DDE, in males than in females. We found the same in 1967/68 after having investigated 100 samples of adipose tissue from the inhabitants of Belgrade and Serbia proper (5): the mean value of the total DDT depot was 10.2 ppm in women as compared to 12.8 ppm in men, the total mean value for the general population amounting to 11.5 ppm. This difference of about 2—2.5 mg/kg of adipose tissue is to be found in papers published before (6) and after (7, 8) our report, although there have been papers (9) in which no difference was found. The existence of a difference has been explained in several ways, one being the relative complexity of female hormonal interrelationships which could conceivably result in an increased microsomal enzyme activity and a subsequent body burden reduction. This may also be interpreted in part by a differing female pattern of fat deposition and the likelihood of males having greater environmental exposure to pesticides. However, in the course of our examinations of the factors contributing to increased concentrations of organochlorine insecticides in infants (10) we found some interesting data which might help to explain this phenomenon too. Namely, in investigating the daily intake of organochlorine insecticides by infants we also determined the entire daily intake of these insecticides with food by breast-feeding mothers and the daily excretion with milk. We found a significant disproportion between the total daily intake of DDT and its excretion as illustrated in Table 1. From the above data the following may be concluded: a) breast-feeding mothers excreted considerably greater quantities of pp'DDE with milk than they took in with food; b) the disproportion was far greater in mothers of twins (A and B) than in mothers of a single child (persons C); c) similar occurrences were not found in the case of other organochlorine insecticides, although we found (5) that the levels of stored djeldrin likewise differed according to sex, which was not suggested by other authors. Since the experiment was carried out under controlled conditions and since the factor of non-dietetic DDT intake could be ignored on this occasion, obviously a powerful mobilization of the stored pp'DDE and other DDT derivatives takes place during the lactation period i, e., the surplus of DDE excffeted with milk becomes more apparent. Knowing that the »good old practice* of breast-feeding is still maintained in some parts of the world, that in some regions this lactation .period lasts up to six — nine months, that a balance in subsequent storage of DDT is not �LOWER LEVEL PHENOMENA OF DDT CUMULATION 305 Table 1 Total daily excretion of organochlorine insecticides in human milk compared to their total daily intake with food (expressed in y/day person) O Person Mean quantity of milk ml/day ffi « £ G, CJ ffl « W Q O I < Kj O & a H Q Q a H Q Q « 01 a. o 5H oQ HQ .s •a S A 1.590 8 22 828 16 154 9 1.078 9 B 1.249 3 14 745 14 179 9 1.033 3 C 527 1 4 67 3 25 2 105 <1 9 317 65 20 112 59 266 9 Total daily intake with food (2.660 g) The result represents the mean value for the observed period of seven consecutive days. A and B are mothers breast-feeding twins C are mothers with one child. attained at once and that such cases figure among the examined subjects from the general population, we may assume that such samples can significantly contribute to a decrease of the mean value for one group of examined subjects and thereby explain the phenomenon of reduced storage of DDT in women. This should be borne in mind when assessing the results of epidemiological studies of OC insecticide exposure. References 1. Deichmann, W. B,: Arch. Toxikol., 29 (1972) 1. 2. Report of the Secretary's Commission on Pesticides and Their Relationship to Environmental Health. U.S. Dept. of Health, Education and Welfare, 1969. 3. Kagan, Yu. S., Pudel-Ossipova, S. I., Khaikina, B, J., Kuzminskaya, U. A., Kouton, S. D.: Res. Rev., 27 (1969) 43. 4. Howell, D. E.: Proc. Okla. Acad. Sci., 29 (1948) 31. 5. Adamovit, V. M., Hus, M.t Sindjic, M., Dukid, V.; Hrana i ishrana, 11 (1970) 6. Zavon, M. R., Hine, H. C., Parker, E. D.: J.A.M.A., 193 (1965) 181. 7. Watson, M., Benson, W. W., Gabica, J.: Pestic. Monit. J.( 4 (1970) 47. 8. Wyltie, /., Gabica, J., Benson, W. W.: Pestic. Monit. J., 6 (1972) 84. 9. FiSerova-Bergerova, V,, Radomski, J. £., Davtes, J. E., Davies, J. H.: Ind. Med. Surg., 36 (1967) 65. 10. Adamovic, V. M., Soktt, B., Petrovic, O.: Ernahrungsforschung, 16 (1971) 579. �306 V. M. ADAMOVIC, BORKA SOKI<; SaZetak POJAVA MANJE KUMULACIJE DDT-ja U ABDOMINALNOM MASNOM TKIVU 2ENA Autori su naSli znacajno viSu razinu pdlaganja DDT-ja u masnom tkivu muSkaraca nego onorn u zena (srcdnja vrijednost pp'DDE iznosila je 12.8 ppm za muSkarce odnosno 10.2 ppm za zenc). Usppredujuci izlucivanje organoklornih insckticida u mlijeku zcna-dojilja s kolicinom DDT-ja uncsenog hranom, izmjerili su znatno viSe eliminiranih rezidua no 5to bi odgovaraio ingestiji insekticida. Isklju£ivSi drugi put apsorpcijc izuzev hranom, autori tumafie tu pojavu hormonskim ciniocima, tj. povecanom mobilizacijom deponiranih metabolita DDT-ja za vrijcine laktacijskpg razdoblja. Smanjeno odlaganje DDT-ja u zena bit cc da utje(?.e i na srednje vrijcdnosti deponiranih metabolita za cjelokupnu populaciju nckog podrucja. Odjel sanitarne kernije, Zavod za zaStitu zdravlja Srbije, Beogmd � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 047 Folder The folder containing the original item. 1186 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 Adamovic, V. M. Sokic Borka Source A related resource from which the described resource is derived Arhiv za Higijenu Rada Date A point or period of time associated with an event in the lifecycle of the resource 1973 Title A name given to the resource Lower Level Phenomena of DDT Cumulation in Female Abdominal Fatty Tissue Subject The topic of the resource human testing pesticide testing ao_seriesIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 043 Folder The folder containing the original item. 1020 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 Rivers, Jerry B. William L. Yauger, Jr. Howard W. Klemmer Source A related resource from which the described resource is derived Journal of Chromatography Date A point or period of time associated with an event in the lifecycle of the resource 1970 Title A name given to the resource Simultaneous Gas Chromatographic Determination of 2, 4 -D and Dicamba in Human Blood and Urine Subject The topic of the resource pesticide properties pesticide testing human exposure ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/c8d45f6a1ae447cf1ee7f5cc9b3b5c30.pdf 09987213933822e963f6186b366cb26c PDF Text Text °0309 Item ID Number Author CorpOratB Author Office of the Federal Register ROpOPt/ArtMO TitlO pro JOUmal/BOOk Title Federal Register Year 1978 Month/Day Ju| 31 Color Posed Tolerances for the Pesticide Chemical Malathion v n Number of linages 2 DeSOrlOtOU NOtBS Found in a file labeled: "Correspondence Relating to the First Use of Military Aircraft for the Dessimination of Insecticides" Monday, January 22, 2001 Page 309 of 341 �' Dated: July 24,1978. DOUGLAS D. CAMPT, Acting Director, Registration Division. Statutory Authority: Section 408(e) at the Federal Food, Drug, and Cosmetic Act [21 U.8.C. 348a<e)3. It is proponed that part 180, Subpart c, § 180.208 be revised in Its entirety by editorially revising the section into an alphabetized columnar listing and by alphabetically inserting the toleraice of 0.5 ppm on satflower seed, as follows: § 180.298 Methidnthion; tolerances for residues. Tolerances are established tor residues of the insecticide methidathton (0,0-dimethyl phosphorodithioate, Sfnt&r with 4-(mercaptomethyl)"2-methoxy-AM,3,4-thiadla!solin-8"On8> in or on the following raw agricultural commodities: Commodity: Alfalfa AHalft, lw ..„.,... OiOVCf, ,.,.„.,.,„„.,.,., »ll,t.4...(. Ctovor, hay Cottonseed............ ,„ Qrapoftuit ,.,...„. 8 8 .-..«....HIM... ,.„ 8 6 .3 3 „... Onm, hay ...„,» Feaehca........ ... . 3 09 .08 PotfttOGJI,.,....,...,..,.,....,..,,...,,.,.,. .......... ,„ ....... .„, ' Sorghum, f Braghum, forage.-.. Sorghum. fi's.in ..........., Sunflower seeds .......... ,. ,2 t 8 3 .0 CPR Doc. 7S-.31016 Plied 7-3ft-78; 8:« am] [$560.01] [40 Cft Pert 180] ^/ EW 7E1881/P81; FBL S34-4] PtO»» TOLiRAKCi COS THI PESTICIDE CHIMSCAl MALATHiOM Tolorcncos few f *tij;!rf« Chamktib in or on Sow Ayrktil- AGENCY: Office of Pesticide Programs, Environmental Protection Agency (EPA). ACTION: Proposed rule. SUMMARY: This notice proposes that the insecticide nmlathion be weed on wild rice. The proposal was submitted by the Interregional Research Project No. 4. This amendment to the regulations would establish a maximum permissible level for residues of malathion on wild rice. , DATE: Comsiersts must be received by August 30, 1OT8. 1 Comma ister Section, Techr ..***• Division (WH-569), Off/estlcide Programs, EPA, Room ,:01, East Tower, 401 M Street SW., Washington, D.C. 20460. POP, FURTHER INFORMATION CONTACT; Mrs. Patricia Critchlow, Registration Division (WH-567), Office of Pesticide Programs, EPA, 202-756-2516. SUPPLEMENTARY INFORMATION Dr. C.C. Compton, Coordinator, Interregional Research Project No. 4 (IR4), New Jersey State Agricultural Experiment Station, P.O. Box 231, Rutgers University, New Brunswick, N.J. 08903, on behalf of the IR-4 Technical Committee and the Agricultural Experiment Station of Minnesota has submitted a pesticide petition (PP 7E1881) to the EPA. This petition request that the Administrator propose that 40 CPR 180.111 be amended by the establishment of a tolerance for residues of the insecticide malathion (0,0-dimethyl dithlophosphate of dlethyl mercaptoaucciiiate) in or on the raw agricultural commodity wild rice at 8 parts per million (ppm). The data submitted in the petition and other relevant material have been evaluated. The toxicological data considered in support of the proposed tolerance ^Included two 2-year rat feeding studies, 'one with a no-observableeffect level (NOEL) of 100 ppm; the other showing chollnesterase-lnhibition at 100 ppm but no systemic effects at 1,000 ppm; a one-generation rat reproduction study in which reproductive effects were observed at 4.000 ppm, the only level tested; a negative neurotoxicity study; a negative single dose (900 milligrams (mgVkilogram (kg) of body weight (bw)) Intraperitoneal teratology study in rats; rat and mouse oral lethal dose (LDW) tests; two negative mutagenicity tests using microbial assay systems; and a 47-day human feeding study with an NOEL at 0.2 mg/kg bw/day. Based on this last study and using a safety factor of 10, the acceptable daily intake (ADI) is 0.03 rag/kg bw/day. The maximum permissible intake for a 80-kg man la 1.2 mg/day. Tolerances have previously been established for residues of malathion on a variety of raw agricultural commodities at levels ranging from 135 ppm to 0.1 ppm. Food additive tolerances have also been established for malathion residues on raisins at 12 ppm and in safflower oil at 0.6 ppm. Peed additive tolerances have been established for malathion residues in dehydrated citrus pulp at 50 ppm and in nonmedicatedi cattle feed concentrate blocks at 10 ppm. On a theoretical besis, the total maximal residue contribution (TMRC) of thoe« tolerances, exceeds the ADI; however, total diet surveys show that Over a 4-year period the actual exposure to malathion, was not more than 0.00013 mg/kg bw/day, which is less than 1 percent of the ADI. The increment of human exposure due to the tolerance on wild rice would be negligible, and thus, the increment In risk, if any, is acceptable. The metabolism of malathion is adequately understood, and an adequate analytical method (gas chromatography using a flame photometric detector) is available for enforcement purposes. The following studies are currently lacking: oncogenicity studies in two mammalian species using currently acceptable protocols, a multlgeneration reproduction study, and a teratology (oral) study. However, it has been determined that the proposed tolerance can be established because: (1) Tolerances currently exist for malathion on a majority of food and feed items in the United States and (2) the use of malathion on wild rice would not significantly Increase human exposure to malathion residues. There Is no reasonable expectation of residues In eggs, meat, milk, or poultry as delineated in 40 CFR 180.6(a)(3>. The pesticide is considered useful for the purpose for which a tolerance is sought, and it is concluded that the tolerance of 8 ppm established by amending 40 CFR 180.111 will protect the' public health. It is proposed, therefore, that the tolerance be established as set forth below. Any person who has registered, or submitted an application for the registration of a pesticide under the Federal Insecticide, Fungicide, and Rodenticide Act which contains any of the ingredients listed herein may request, within 30 days after publication of this proposal in the FEDERAL REGISTER, that this rulemaklng proposal be referred to an advisory committee in accordance with section 408(e) of the Federal Food, Drug, and Cosmetic Act. Interested persons are invited to submit written comments on the proposed regulation. The comments must bear a notation indicating both the subject and the petition/document control number, "PP7E1881/P81." All written comments filed in response to this notice of proposed rulemaklng will be available for public inspection in the Office of the Federal Register Section from 8:30 a.m. to 4 p.m. Monday through Friday. STATUTORY AUTHORITY: Section 408(e) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 346*(e». FE0etAl HE©OTfft, VOL 43, NO. 147-~MON0AY, JULY 31, �33265 PROPOSE RULES Dated: July 24,1978. DOUGLAS D. CAMPT, Acting Director Registration Division, It is proposed that part 180, subpart C, § 180.111 be amended by alphabetically inserting the tolerance of 8 ppm on wild rice In the table to read as follows: Section 180.111 Malathion; tolerances for residues. Commodity: Port* per million Rice, wild.... IFR Doc. 78-21016 Filed 7-28 78; 8:45 am] [4110-84] DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service [42 CFK Part 23] NATIONAL HEALTH SERVICE CORPS Subpart A—Assignment of National Health Ssrvice Corp* Personnel AGENCY: Public Health Service, HEW. ACTION: Notice of proposed rulemaking. SUMMARY: These proposed regulations prescribe the requirements for the assignment of National Health Service Corps personnel under Section 333 of the Public Health Service Act (42 U.S.C. 254f) to public or nonprofit private entities to provide health services in or to a health manpower shortage area designated under Section 332 of the Public Health Service Act. DATE: Comments must be received August 30, 1978. ADDRESSES: Written comments, preferably in triplicate, should be addressed to the Director, Division of Policy Development, Bureau of Community Health Services, Health Services Administration, room 6-17, 5600 Fishers Lane, Rockville, Md. 20857. All comments received will be available for public inspection and copying at the above address, weekdays (Federal holidays excepted) between the hours of 8:30 a.m. and 5 p.m. FOR FURTHER CONTACT: INFORMATION Fitzhugh S. M. Mullan, M.D., Director, National Health Service Corps, Bureau of Community Health Ser- vices, Room 6-05, Parklawn Building, 5600 Fishers Lane, Rockville, Md. 20857, 301-443-4434. SUPPLEMENTARY INFORMATION: On October 12,1976, a new section 333 was added to the Public Health Service Act (42 U.S.C. 254f) by Pub. L. 04484, the Health Professions Educational Assistance Act of 1976. This section allows the Secretary to assign, pursuant to regulations, members of the National Health Service Corps to public and nonprofit entitles to provide health services In or to a health manpower shortage area. Based upon the enactment of this new section 333, the Assistant Secretary for Health, Department of Health, Education, and Welfare, proposes to revoke the existing part 23, subpart A, and add a new Subpart A entitled "Assignment of National Health Service Corps Personnel." A notice of intent to issue regulations for this program was published in the FEDERAL REGISTER on May 20, 1977 (42 FR 25992). Interested persons were invited to comment on the issues raised and several comments were received. The regulations establish the conditions applicable to the assignment of National Health Service Corps personnel to a public or nonprofit private entity to provide health services in or to a health manpower shortage area. In the interest of streamlining regulations and reducing regulatory burden, minute program particulars have not been set forth In the regulations. For this reason, most of the comments received on the notice of Intent were not pertinent to the development of these regulations, but will be considered by the Department in developing program policies. Following Is a brief summary of the major features of the proposed regulations: (1) In approving applications for assignment, section 23.5(b) provides that if two eligible entities, one located In the health shortage area and one not located in the area but having a demonstrated Interest in It, submit applications for assignment of National Health Service Corps personnel to provide health services to the area, spe-.. clal consideration will be given to the entity located In such health manpower shortage area. This special consideration was adopted in response to the several public comments received suggesting that such a consideration would promote greater community involvement between the approved entity and the individuals receiving health services. (2) Section 333(c) of the Public Health Service Act requires that the Secretary take into consideration four factors in assigning National Health Service Corps personnel to entities v/ith approved applications. The four factors are (i) Need of the health manpower shortage area, (ii) Use of physician assistants, nurse practitioners, or expanded function dental auxiliaries, (ill) Willingness of the individuals within the health manpower shortage area to assist and cooperate with the National Health Service Corps and (iv) Comments of professional societies serving the health manpower shortage area. In implementing this statutory requirement, the regulations provide that the Secretary will utilize a weighted-value system in which weights will be assigned to the four statutory factors, with the greatest weight being assigned to the first factor listed above and weights being assigned to the remaining factors in descending order. Based on this approach, approved applications will be assigned to priority categories. Personnel assignments will then be made to entitles In accordance with these priority categories to the extent possible consistent with the statutory mandate that in assigning personnel to provide health services to a health manpower shortage area, the Secretary shall seek to assign to an area personnel with those characteristics which will increase the probability of their remainIng to serve the area upon completion of the assignment period. (3) The proposed regulations require that individuals receiving services from assigned National Health Service Corps personnel be charged on a fecfor-scrvlce or other basis at a rate to be approved by the Secretary. Fees are to be based upon the cost of delivering services and on fees charged for similar services by similarly situated practitioners and facilities. The proposed regulations further provide that no individual will be denied health services based upon his inability to pay for such services. With respect to this inability to pay, §23.9 of the proposed regulations states that those individuals with annual incomes at or below the "CSA Income Poverty Guidelines" (45 CFR 1060.2) may receive services at a reduced charge. Individuals who have annual incomes above the "CSA Income Poverty Guidelines" but which do not exceed 200 percent of such CSA levels will also receive health services at reduced charge. Charges will be made, however, for services to Individuals to the extent that payment will be made by a third party which is authorized or under legal obligation to pay such charges. (4) The statute requires In section 334(a)(3) that the National Health Service Corps site repay the Federal Government for the costs involved in providing assigned National Health Services Corps personnel. The statute also provides that repayment may, under certain specific circumstances, FEDERAL REGISTER, VOL. 43, NO. 147—MONDAY, JULY 31, 1978 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 020 Folder The folder containing the original item. 0309 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/. Description An account of the resource Corporate Author: Office of the Federal Register Source A related resource from which the described resource is derived Federal Register Date A point or period of time associated with an event in the lifecycle of the resource July 31 1978 Title A name given to the resource Proposed Tolerances for the Pesticide Chemical Malathion Subject The topic of the resource pesticide testing pesticide properties https://www.nal.usda.gov/exhibits/speccoll/files/original/61abe0a593ddec01560c1d1658a4d339.pdf b2cdea012ae9de4b978e37bf74848498 PDF Text Text °0308 Item ID Number Author Corporate Author Office of the Federal Register Report/Article TitlB Proposed Tolerances for the Pesticide Chemical Malathion; Proposed Tolerances for the Pesticide Chemical 5-ethoxy-3-trichloro-methyl-1,2,4-thiadiazole JOUmal/BOOk Title Federal Register Year 1978 Month/Day March e Color n Number of Images 4 DeSOrlptOU Notes Found in a file labeled: "Correspondence Relating to the First Use of Military Aircraft for the Dessimination of Insecticides" Monday, January 22, 2001 Page 308 of 341 �PROPOSED RULES land submitted to the Regional Administrator a proposed revision of the Maryland State Implementation Plan for the attainment and maintenance of national ambient air quality standards (NAAQS). The proposed revision was defined in an Order issued by the Maryland Secretary of the Department of Health and Mental Hygiene on July 8,1975. The proposal consisted Of a request to grant an exception to the Westvaco Corp., Luke, Md., from Maryland's sulfur-in-fuel regulation (10.03.36.04B) which requires the use of fuel containing 1% orless sulfur by weight. The purpose of this request for an exception was to allow the Westvaco Corp., to burn coal with a higher sulfur content. At the same time the Westvaco Corp., would be allowed to emit up to 58 tons/day of sulfur dioxide (SO,) from all fuel burning equipment. The State of Maryland justified this request with the following: 1. Westvaco desires to remain on coal firing, but lias been unable to secure an adequate supply of 1 percent sulfur coal. ~ 2. Desulfurization alternatives were not yet available to the company. 3. Such an exception would not cause violation of federal or state standards in the vicinity of the plant. The State of Maryland submitted proof that a public hearing with adequate public notice was held on May 26, 1975, pursuant to 40 CPR Part 51, Requirements for Preparation, Adoption, and Submittal of State Implementation Plans. The State of Maryland, on behalf of the Westvaco Corp., submitted a modeling analysis intended to demonstrate that the exception requested would not result in violations of NAAQS for S0». Upon review of the analysis, EPA determined that it was incorrect in a number of respects and underpredipts the impact of SO, emissions. Therefore EPA concluded that the analysis did not provide a demonstration that air quality standards would be attained or maintained if the exception were to be approved as a SIP revision. Subsequent meetings were held with the State of Maryland and Westvaco in attempts to resolve differences between the company's modeling approach in support of the request for exception and that which EPA believes to be appropriate. Although some minor differences were resolved, EPA maintained that, overall, the modeling analysis did not demonstrate that the SO, limitations specified in the exception request will not violate the NAAQS for SO,. Accordingly, EPA informed the State of Maryland, in a :letter dated February 28, 1977, that the exception request was uhapprovable as a revision of the Maryland SIP. On November 18, 1977, the State of Maryland submitted to the Regional Administrator an Amendment to the aforementioned Secretarial Order. This submittal alters the originally proposed revision of the SIP in that the Westvaco Corp. would be limited to 49 tons/day of SO, instead of 58 tons/day. Also, under the terms of the Amendment, the Westvaco Corp. consents to its installing two meteorological stations and additional SO, monitors. The stated purposes of the additional equipment is to provide information regarding air quality levels and to verify the adequacy of the 49 ton/ day emission limitation as determined by the Company's modeling analysis, or to establish with any new data, a different emission limitation. After evaluating the revised submittal, EPA maintains that the modeling analysis, including the most current revision dated June 14, 1977, does not demonstrate that the 49 ton/day SO, limitation will not violate NAAQS for SO,. This determination is not altered by an evaluation of information contained in a February 2, 1978 report forwarded directly to EPA by Westvaco. Thus the agency believes that the revised request for an exception on behalf of the Westvacp Corporation is also unapprovable as a revision of the Maryland SIP. The State of Maryland submitted proof that a public hearing on the Amendment to the Secretarial Order of July 8, 1975 was held on October 7, 1977, after adequate public notice, pursuant to 40 CPR Part 51, Requirements for Preparation, Adoption and Submittal of State Implementation Plans. This notice is to advise the public of receipt of Maryland's request for an exception, and to request public comment on it. Only comments received before April 5,1978, will be considered. The Administrator's decision to approve or disapprove this proposed revision will be based on whether it meets the requirements of Section 110 of the Clean Air Act and EPA regulations in 40 CPR Part 51. Copies of the proposed revision, together with supporting documentation and correspondence, are available for public Inspection during normal business hours at the offices of: tion Agency, Region III, Curtis Building, Sixth and Walnut Streets, Philadelphia, Pa. 19106, ATTN: AHOOGMD. (42 U.S.C. 7401 et seq.) Dated: February 27,1978. JACK J. SCHRAMM, Regional Administrator. IFR Doc. 78-5707 Piled 3-3-78; 8:45 am] [1505-01] [40 CFR Part 141] [PRL 851-5] INTERIM PRIMARY DRINKING WATER REGULATIONS . Control of Organic Chemical Contaminants in Drinking Water Correction In FR Doc. 78-3414 appearing at page 5756 in the issue for Thursday, February 9,1978, on page 5777, second column, (c) of § 141.12 should read as follows: (c) Total trihalomethanes [the sum of the concentrations of bromodichloromethane, dibromochloromethane, tribromomethane (bromoform) and trichloromethane (chloroform)]." ....0.10 mg/1 [6560-01] [40 CFR Part 180] [PRL 863-4; PP 7E2001/P64] PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FROM TOLERANCES FOR PESTICIDE CHEMICALS IN OR ON RAW AGRICULTURAL COMMODITIES Proposed Tolerances for The Pesticide Chemical Matothion AGENCY: Office of Pesticide Programs, Environmental Protection Agency (EPA). U.S. Environmental Protection Agency, ACTION: Proposed rule. Region III, Air Programs Branch, Curtis Building, Sixth and Walnut Streets, Phila- SUMMARY: This notice proposes that delphia, Pa. 19106, ATTN: Mr. Harold the insecticide malathion be used on Frankford (3AK11). birdsfopt, trefoil. The proposal was Maryland State Bureau of Air Quality and submitted by the interregional ReNoise Control, 201 West Preston Street, search Project No. 4. This amendment Baltimore, Md. 21201, ATTN: Mr. George to the regulations would establish a Perreri. Public Information Reference Unit, U.S. En- maximum permissible level for resivironmental Protection Agency, 401 M dues of malathion on birdsfoot trefoil. Street SW., Washington, D.C. 20460. DATE: Comments must be received on All comments should be addressed or before April 5,1978. to: ADDRESS COMMENTS TO: Federal Mr. Howard R. Helm (3AH10). Chief, Air Register Section, Technical Services Planning Branch, Environmental Protec- Division (WH-569), Office of Pesticide FEDERAL REGISTER, VOL 43, NO. 44-ttONDAY, MARCH 6, 1*78 �9164 PROPOSEP RULES Programs, EPA, Room 401, East Tower, 401 M Street SW7, Washington, D.C. 20460. FOR FURTHER INFORMATION CONTACT: Mrs. Patricia Critchlpw, Registration Division (WH-561?), Office of Pesticide Programs, EPA, 202-755-2516. SUPPLEMENTARY INFORMATION: Dr. C. C. Compton, Interregional Research Project No. 4, New Jersey State Agricultural Experiment Station, P.O. Box 231, Rutgers University, New Brunswick, N.J. 08903, on behalf of the IR-4 Technical Committee and the Agricultural Experiment Station of New York has submitted a pesticide petition (PP 7E2001) to the EPA. This petition requests that the Administrator propose that 40 CFR 180.111 be amended by the establishment of a tolerance for residues of the insecticide malathion (O.O-dimethyl dithiophosphate of diethyl mercaptosucclnate) in or on the raw agricultural commodity birdsfoot trefoil at 135 parts per million (ppm). The data submitted in the petition and all other relevant material have been evaluated. The pesticide is considered useful for the purpose for which the tolerance is sought, and it is concluded that the tolerance of 135 ppm established by amending 40 CFR 180.111 will protect the public health. The toxicologlcal data considered in support of the proposed tolerance Included rat and mouse oral acute toxicity determinations, recombinant assay and reversion assay mutagenicity studies, a delayed neurotoxocicity study, a single-dose intraperitoneal teratology study, a one-generation reproduction study, two 2-year rat-feeding studies, and a 47-day human-feeding study. Since birdsfoot trefoil is not a human food but a substitute forage crop for alfalfa on which a tolerance is already established and since tolerances are also established in meat, milk, and meat byproducts, the Toxicology Branch concludes that there will be no increase in residues contributed to the human diet resulting from the proposed use. Oncogenic studies in two species, a multigeneration reproduction study, and a teratology (feeling) study, are lacking. An adequate analytical method (gas chromatography using electron capture detection) is available for enforcement purposes, and the nature of the residues is adequately delineated. Tolerances have previously been established (40 CFR 180.111) for residues of malathion on a variety of raw agricultural commodities ranging from 135 ppm to 0.1 ppm. There is no reasonable expectation of residues in eggs and poultry from the proposed use. The established tolerances for residues in meat arid milk are adequate to cover secondary resi- dues resulting from the proposed use as delineated in 40 CFR 180.6(a)(2). It is proposed, therefore, that the tolerance be established as set forth _, , - . i ' - 'Any person who has registered, or submitted an application for the registration of a pesticide under the Federal Insecticide, Fungicide, and Rodenticide Act which contains any of the in-' gradients listed herein may request, on or before April 5, 1978, that this rulemaking proposal be referred to an adTisory committee in accordance with section 408(e) of the Federal Food, are invited to submit written comments on the proposed regulation. The comments must bear a notation indicating both the subject and the petition/document control number, "PP7E2001/P64". All written comments filed in response to this notice of proposed rulemaking will be available for public inspection in the office of the Federal Register from 8:30 a.m. to 4 p.m. Monday . *i- _.._i. 1-u.ij,... ... • through Friday. Dated: February 27, 1978. It is proposed that Part 180, Subpart C, sectiQn 180.111 be revised in its entirety by editorially reformatting the section into an alphabetized columnar listing and alphabetically inserting the new tolerance of 135 ppm on birdsfoot as lOllUWS.. as follows ^ i . §180.111 Malathion; tolerances ffor residues, Tolerances are established for residues of the insecticide malathion (0,0-dimethyl dithiophosphate of diethyl mercaptosuccinate) in or on the following raw agricultural commodities: Commodity Alfalfa (PRE-H).............................. Almond hulls (PRE-H) ............. „ Almonds (PRE- and POST-H)....... Apples (PRE-H) Apricots (PRE-H) Asparagus (PRE-H)........ Avocadoes (PRE-H) Barley, grain (PRE- and POST-H) Beans (PRE-H) Beets (including tops) (PRE-H) Be^ts, sugar, roots (PRE-H) Beets, sugar, tops (PRE-H) Birdsfoot trefoil, forage (PRE-H)...... Birdsfoot trefoil, hay (PRE-H) Blackberries (PRE-H) Blueberries (PRE-H) Boysenberrles (PRE-H).................. Broccoli (PRE-H) Brussels sprouts (PRE-H) ..... Cabbage (PRE-H)..................................... Carrots (PRE-H)................................... Cattle, fat (PRE-S) Cattle, meat1 (PRE-S) ......... ..>........ Cauliflower (PRE-H)........,.„„..„„..,......... Celery (PRE-H)...... 136 8 8 cowpea, nay (PRE-H> cranberries (PRE-H) 135 135 a corn, fresh (including sweet K+CWHRHPRE-H).............................. *"• ****? <P°T-H).......................«••»- Dandelions (PRE-H) Dates <PRE-H> Dewberries (PRE-H) Eggplants (PRE-H) 8 °l a Figs (PRE-H) 1 Goats, mbyp- ( p R E . . . Goats, meatorapes (PRE-H) ora^ (PRE-H) .. Grass, hay (PRE-H) .................................... P&rts psf million 135 50 8 8 8 8 8 8 8 8 1 8 135 135 8 8 8 8 8 8 8 4 4 8 8 4 ....... Limes (PRE-H) ................................... ...-. Loganberries (PRE-H) _ .............................. Lupine, hay (PRE-H).................. ................ Lupine, seed (PRE-H) ................................. Lupine, straw (PRE-H) ..."........ .................... Macadamla nuts (PRE-H)......................... Mangos (PRE-H)..................„...„„......,......., Melons (PRE-H) ........................................... cows)., (from application to dairy etarineRE-H) ., oats, grain (PRE- an'd"p'osT-H")'"""!""»] okra (PRE-H) .............................. .........,. ..... Onions (Including green onions) (PREH) ...... * ................... .. ....... ........ ......... *............ Oranges (PRE-H) .................................. .. ..... Papayas (PRE-H) ......................................... Parsley (PRE-H) ................................. .„„..,. Parsnips (PRE-H)................................. ....... Passion fruit (PRB-H) ................................ Peaches (PRE-H) ......................................... Peanut, forage (PRE-H) ....................... ...... Peanut, hay (PRE-H).............'.............— ... Peanuts (PRE- and POST-H) ................... Pears (PRE-H).................................„ ........... Peas (PRE-H).............................. ................. Peayine, hay (PRE-H) ................................. Peavlnes (PRE-H)................................. ....... Pecans (PRE-H) ........................................... Peppermint (PRE-H) „.... ............................ Peppers (PRE-H)........................................ Pineapples (PRE-H)..............................„.,. Plums (PRE-H) ......................................... ... Potatoes (PRE-H).....................................-. Poultry, fat (PRE-S) ................................. Poultry, mbyp1 (PRE-S).............. ............... Poultry, meat- (PRE-S) .............................. Prunes (PRE-H) ........................................... Pumpkins (PRE-H)..................................... Quinces (PRE-H) ............ ., ........................... Radishes (PRE-H) ........,»,wv...........„,„...„ FEDERAL REGISTER, VOL. 43, NO. 44—MONDAY, MARCH «, 1978 S a 135 135 4 i Lentils (PRE-H) Lespedeza, nay (PRE-H) MUt fat 4 4 ! 4 Hogs, mbyp -(PRE-S) Kale (PRE-H) Kohlrabi (PRE-H) Kumquats (PRE-H) Leeks (PRE-H);.,. Lemons (PivE-H) * 8 g a Horses, mbyp '(PRE-S) STATUTORY AUTHORITY: Sec. 408(e), Federal Pood, Drug, and Cosmetic Act (21 U.S.C. 345a(e)>. »...,• CoUards (PBE-H) .................................... ..... Corn, forage (PBE-H) ................................. Horses, meat* (FRE-S) Registration Division, o „«..... 8 i cherries (PEEchestnuts (PRE-H) 4 8 8 8 8 8 8 135 8 8 8 135 8 135 1 8 8 o.s I I & 8 8 8 1 8 8 8, 8 j 135 j 135 • 8) 8 j 8 j 8 ( 8 j 8' 8 8 8 8 8 4 4 4 8 8 8 8 �9165 PROPOSED RULES -•-. Partsper Commodity million Raspberries (PR£-H) 8 n Bice, grain (PRE- and POST-H) t Rutabagas (PRE-H) „ „. 8 Rye, grain (PRE- arid POST-H) 8 Safflowcr, seed (PRE-H) O.Z Salsify (including tops) (PRE-H) 8 Shallots (PRE-H) , ' 8 : Sheep, fat (PRE-S) 4 Sheep, mbyp > (PRE-S) 4 Sheep, meat' <PRE-S) 4. Sorghum, forage (PRE-H) " 8 Sorghum, grain (PRE-and POST-H j.— ' 8 Soybeans (dry and succulent) (PRE-H)... . 8 Soybeans, forage (PRE-H) 135 Soybeans, hay (PRE-H)... 135 Spearmint <PRE-H) ™ 8 Spinach (PRE-H) 8 Squafh, summer and winter (PRE-H).... 8 Strawberries (PRE-H) 8 Sweet potatoes (PRE-H) 1 Swiss chard (PRE-H) 8 Tangerines (PRE-H) 8 : Tomatoes (PRE-H) 8 Turnips (Including tops) <PRE-H) 8 Vetch, hay (PRE-H) 135 Vetch, seed (PRE-H) „ 8 Vetch, straw (PRE-H) i 135 Walnuts (PRE-H) 8 Watercress (PRE-H) — 8 Wheat, grain <PRE- and POST-JO 8 •The tolerance level shall not be exceeded in any cut of meat or in any meat byproduct from cattle, goats, hogs, horses, poultry, or sheep. ' IFB Doc. 78-5716 Piled 3-3-78; 8:45 am] SUPPLEMENTARY INFORMATION: The California State Dept. of Food and Agriculture, Agricultural Chemicals and Feed, 1220 N Street, Sacramento, Calif. 95814, has submitted a pesticide petition (PP 6E1747) to the EPA. The petition requests that the Administrator propose that 40 CFR Part 180 be amended by the establishment of a tolerance for combined residues of the fungicide 5-ethoxy-3-trichloromethyl-l,2,4-thiadiazole and its monoacid metabolite 3-carboxy-5ethoxy-l,2,4-thiadazole in or on the raw agricultural commodity avocados at 0.15 part per million (ppm). The data submitted in the petition and other relevant material have been evaluated. The pesticide is considered useful for the purpose for which the tolerance is sought. The toxicology data considered in support of the proposed 0.15 ppm tolerance were a twoyear rat and dog-feeding study with no-effect levels (_NEL) of 80j>pni based on systemic effects in the rat study and 100 ppm based on the dog study, a three-generation rat reproduction study with an NEL of 80 ppm, and an oral lethal dose (LDSO) study in rats. [6560-01] There is no reasonable expectation of residues in eggs, meat, milk, or C40 CFR Parti 80] poultry as delineated in 40 CFR [PRL 863-5; PP 6E1747/P62] 180.6(a)<3). The nature of the residue is adequately understood, and adeTOLERANCES AND ' EXEMPTIONS quate analytical methods (electron FROM TOLERANCES FOR PESTICIDE capture gas-liquid chromatography CHEMICALS IN OR ON RAW AGttland liquid chromatography using a UV . detector) are available for enforceCULTURAL COMMODITIES ment purposes. An interim tolerance Proposed Tolerances for the Pesticide has previously been established (40 CFR 180.319) for residues of the subClieinical5ieTli«xy=3=fncril9roject fungicide on cottonseed at 0.3 m*thyl-l,2,4-th1adiazo1e ppm. AGENCY: Office of Pesticide ProA second oncogeniclty study and a grams, Environmental Protection teratogenicity study are lacking. HowAgency (EPA). ever, based on: (1) The above information considered by the Agency; (2) the ACTION: Proposed rule. fact that there is no expectation of reSUMMARY: This notice proposes that sidues on avocados; and (3) the insiga tolerance be established for residues nificance of avocados in the diet, it is of the fungicide 5-ethpxy-3-trichloro- concluded that the tolerance of 0.15 methyl-l,2,4-thiadiazole. The proposal ppm established by amending 40 CFR was submitted by the California State Part 180 would protect the public Dept. of Pood and Agriculture. This health. Should any amendment will establish a maximum op to show adverseinformation develeffects from the permissible level for residues of the fungicide on man or the environment, > subject fungicide on avocados. the petitioner has agreed not to conDATE: Comments must be received on test a revocation of the tolerance by or before April 5,1978. this Agency. It is proposed, therefore, ADDRESS COMMENTS TO: Federal that the tolerance be established as Register Section, Technical Services set forth below. Division (WH—569), Office of PestiAny person who has registered, or cide Programs, EPA, Rm. 401, East submitted an application for the regisTower, 401 M Street SW., Washington, tration of a pesticide under the Federal Insecticide, Fungicide, and Rodenti-" D.C. 20460. FOR FURTHER INFORMATION cide Act which contains any of the ingredients CONTACT: . . t or before listed herein may request, on April 5, 1978 that this ruleMrs. Patricia Critchlow. Registration making proposal be referred to an adDivision (WH-567), Office of Pesti- visory committee in accordance with, cide Programs, Environmental Pro- section 408(e) of the Federal Food, tection Agency, 202-755-2516. ' - Drug, and Cosmetic Act. Interested persons are invited to submit written comments on the proposed regulation. The comments must bear a notation indicating both the subject and the petition/document control number, "PP6E1747/P62". All written comments filed in response to this notice of proposed rulemaking will be available for public inspection in the Office of the Federal Register from 8:30 a.m. to 4 p.m., Monday through Friday. Dated: February 27,1978. DOUGLAS D. CAMPT, Acting Director, Registration Division. (Sec. 408(e), Federal Pood, Drug, and Cosmetic Act <21 U.S.C. 34«a(e)>.) It is proposed that Part 180, Subpart C, be amended by adding the new § 180.370 to read as follows: § 180.370 5-Ethoxy-3-trichlorometli}l-l,2,4tbiadiazole; tolerances for residues. Tolerances are established for combined residues of the fungicide 5ethoxy - 3 - trichloromethyl - 1,2.4"thiadiazole and its monoacid metabolite 3-carboxy-5-ethoxy-l,2,4-thiadiazole in or on the following raw agricultural commodities: Commodity: Paris per million Avocados , ,..,„ 0.15 IFR Doc. 78-5717 FJled £ 3-78; 8:45 *m] [4910-14] DEPARTMENT OF TRANSPORTATION Coast Guard [46 CFR Part 4] [COD 77-036] VESSEL REPORTING REQUIREMENTS AGENCY: Coast Guard, DOT. ACTION: Proposed rule. SUMMARY: The Coast Guard is considering amending the Marine Investigation regulations in order to eliminate dual reporting requirements imposed upon certain vessels. Vessels that do not hold a Certificate of Inspection and that are either numbered under the provisions of the Federal Boat Safety Act of 1971, or used by their operators for recreational purposes are presently subject to dual reporting requirements for accidents or casualties. This proposed amendment will eliminate the undue burden currently imposed upon the .owners of these vessels. DATES: Comments must be received on or before April 23,1978. ADDRESSES: Comments should be submitted to Commandant (G-CMC/ FEDERAL REGISTER, VOL 43, NO. 44—MONDAY, MARCH 6, 1978 �9166 PROPOSED RULES 81) (COD 77-036), U.S. Coast Guard, Washington, D.C. 20590. Comments will be available for examination at the Marine Safety Council (G-CMC/ 81), Room 8117, Department of Transportation, Nassif Building, 400 Seventh Street SW., Washington, D.C. 20590. ' . ' . / . FOR FURTHER INFORMATION CONTACT: Captain George K. Greiner, Marine Safety Council (G-CMC/81), Room 8117, Department of Transportation, Nassif Building, 400 Seventh Street SW., Washington, D.C. 20590, 202426-1477. SUPPLEMENTARY INFORMATION: Interested persons are invited to participate in this proposed 'rulemaking by submitting written views, data, or arguments. Each comment should include the name and address of the person submitting the comment, reference this notice (CGD 77-036), identify the specific section of the proposal to which each comment applies, and give the reasons for each comment. All comments received before the expiration of the comment period will be considered before final action is taken on this proposal. No public hearing is planned but one may be held at a time and place to be set in a later notice in the FEDERAL REGISTER if requested in writing by an interested person raising a genuine issue and desiring to comment orally at a public hearing. DRAFTING INFORMATION The principal persons involved in drafting this proposal are: Lieutenant Anthony Regalbuto, Project Manager, Office of Merchant Marine Safety, and Edward J. Gill, Jr., Project Attorney, Office of the Chief Counsel. DISCUSSION OF THE PROPOSED REGULATIONS An operator of a vessel that does not hold a^ Certificate of Inspection and that is either numbered under the provisions of the Federal Boat Safety Act of 1971, or used by the operator for recreational purposes, is required by 33 CFR 173.55 to submit Coast Guard Form CG-3865, Boating Accident Report, or an equivalent form prescribed by state authorities whenever the vessel or equipment is involved in any occurrence specified in 33 CFR 173.55. If such a vessel is involved in a casualty which meets the reporting criteria in 46 CFR 4.05-1, then the operator is also required to submit Coast Guard Form CG-2692, Report of Vessel Casualty or Accident and Coast Guard Form CG-924E, Report'of Personal Injury. The reporting criteria for 46 CFR 4.05-1 and 33 CFR 173.55 overlap whenever a vessel or its equipment is involved in a casualty or accident where there is loss of life, injury causing any persons to remain inca- pacitated for a period in excess of 72 hours, or actual physical damage to property in excess of $1,500.00 In consideration of the foregoing, it is proposed that part 4 of Title 46 of the Code of Federal Regulations be amended by adding a new §4.01-3 to read as follows: . § 4.01-3 Reporting exclusion. Vessels subject to 33 CFR 173.51 are excluded from the requirements of Subpart 4.05. (Sec. 10, 18 Stat. 128, as amended (33 U.S.C. 361); R.S. 4462, as amended (46 U.S.C. 416); Sec. 17, 54 Stat. 166, as amended (46 U.S.C. S26p); Sec. 6(b)(l), 80 Stat. 937 (49 U.S.C. 1655(b)(l); 49 CFR 1.46(b».) NOTE.—The Coast Guard has determined that this document does not contain a major proposal requiring preparation of an Economic Impact Statement under Executive Order 11821, as amended, and OMB Circular A-107. i Dated: March 1,1978. O. W. SlLER, Admiral, U.S. Coast Guard, Commandant IFR Doc. 78-5799 Piled 3-3-78; 8:45 am] [4910-22] Federal Highway Administration [49 CFR Part 393] [BMCS Docket No. MC-82; Notice No; 78-5] PARTS AND ACCESSORIES NECESSARY FOR SAFE OPERATIONS Proposed Rulemaking AGENCY: Federal Highway Administration, DOT. ACTION: Notice of Proposed Rulemaking. SUMMARY: There is concern that the Federal Motor Carrier Safety Regulations (FMCSR) which are applicable to users of commercial vehicles in interstate and foreign commerce, and the Federal Motor Vehicle Safety Standards which are applicable to manufacturers of new motor vehicles and new vehicle equipment do not conform in certain instances resulting in the appearance of inconsistency. This document proposes changes to the Federal Motor Carrier Safety Regulations to coordinate the FMCSR and the FMVSS, and also to resolve the inconsistencies in the regulations. DATES: Comments' must, be received on or before August 3,1978. ADDRESSES: BMCS Docket Number MC-82, Bureau of Motor Carrier Safety, Federal Highway Administration, Room 3402, 400 Seventh Street SW., Washington, D.C. 20590. All comments and suggestions received will be available for examination at the above address between 7:45 a.m. and 4:15 p.m. .e.s.t., Monday through Friday. FOR FURTHER CONTACT: INFORMATION Mr. D. W. Morrison, Chief, Vehicle Requirements Branch, Bureau of Motor Carrier Safety, 202-426-1700; or Mrs. K. S. Markman. Attorney, Office of the Chief Counsel, 202426-0786, Federal Highway Administration, 400 Seventh Street SW., Washington, D.C. 20590. Office hours are from 7:45 a.m. to 4:15 p.m. e.s.t., Monday through Friday. SUPPLEMENTARY INFORMATION: The Federal Motor Carrier Safety Regulations apply to users of commercial vehicles in interstate or foreign commerce, while the Federal Motor Vehicle Safety Standards apply to manufacturers of new vehicles and vehicle equipment. Concern has been expressed that the two sets of regulations do not conform in certain instances, resulting in the appearance of inconsistency. The principal reasons for the issuance of this notice are to coordinate the FMCSR and the FMVSS and to resolve inconsistencies in the requirements where they may exist. In instances in which _the two sets of regulations, FMCSR and FMVSS, are not complementary, confusion could arise among commercial vehicle owners, operators, and manufacturers as to which set of regulations should be adhered to. The possibility exists that a vehicle may be manufactured in accordance with FMVSS requirements but once that same vehicle is put in operation, it may be in violation of a particular FMCSR. In order to eliminate possible discrepancies, it is recommended that 49 CFR 393.45, 393.46, 393.60 and 393.80 be amended. In § 393.45(a)(6), the proposed change is the inclusion of FMVSS 106 as a requirement for commercial vehicles in use. In § 393.46, the addition of a new paragraph, §393.46(e), is proposed. The new paragraph, (e), would require compliance with FMVSS 106 where applicable for vehicles manufactured on and after April 1, 1979. In § 393.60, the proposed change would amend §393.60(a) by deleting the American Safety Standard Code year of "1964" and substituting in its place "1966" and supplements through March 1969. Changing the Safety Code reference date in the subject FMCSR would bring BMCS requirements into line with those of NHTSA. Presently, the FMCSR, following 1964 Safety Code guidelines, requires fewer samples of glass to" be used in vehicles' windows be subjected to the prescribed tests. The newer guidelines, on which the NHTSA regulations are based, include FEDERAL REGISTER, VOL. 43, NO. 44—MONDAY, MARCH 6, 1978 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 020 Folder The folder containing the original item. 0308 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/. Description An account of the resource Corporate Author: Office of the Federal Register Source A related resource from which the described resource is derived Federal Register Date A point or period of time associated with an event in the lifecycle of the resource March 6 1978 Title A name given to the resource Proposed Tolerances for the Pesticide Chemical Malathion; Proposed Tolerances for the Pesticide Chemical 5-ethoxy-3-trichloro-methyl-1,2,4-thiadiazole Subject The topic of the resource pesticide testing pesticide properties Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 018 Folder The folder containing the original item. 0199 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/. Description An account of the resource Corporate Author: United States Air Force Academy, Colorado Date A point or period of time associated with an event in the lifecycle of the resource 1971 Title A name given to the resource Faculty Publications, 1971: Papers, Books, Chapters, Reports, and Talks by Members of the Faculty and Research Staff Subject The topic of the resource pesticide testing Eglin AFB ecological fate bibliography https://www.nal.usda.gov/exhibits/speccoll/files/original/a43524e853447ff25b9c6cdce1df9cc7.pdf eac9e207abbff5e41591d8b44990dc3b PDF Text Text 00172 Author CorporatG Author Kotchmar, George S,, Jr. Flame, Incendiary, and Explosives Division, Air Force Armament Laboratory, Eglin AFB, Florida Metabolism of High Concentrations of the Organophosphorus Insecticide Phorate Applied Foliariy to Selected Plant Species Journal/Book Title Year MOIltll/Day Color February n 54 Project No. 5066 Friday, January 05, 2001 Page 172 of 194 �AFATL-TR-71-22 THE METABOLISM OF HIGH CONCENTRATIONS OF THE O R G A N O P H O S P H O R U S INSECTICIDE PHORATE APPLIED FOLIARLY TO SELECTED PLANT SPECIES PYROTECHNICS BRANCH FLAME INCENDIARY, AND EXPLOSIVES DIVISION TECHNICAL REPORT AFATL-TR-71-22 F E B R U A R Y 1971 Approved for public release; distribution unlimited, AIR FORCE ARMAMENT LABORATORY AIR FOtCE SYSTIMS COMMAND • UNITED STATES AIM FORCE EGLIN AIR FORCE BASE, FLORIDA �The Metabolism of High Concentrations of the Organophosphorus Insecticide Phorate Applied Foliarly to Selected Plant Species George S. K o t c h m a r , Jr., Capt, U S A F Billy C. W o l v e r t o r t E l i z a b e t h I. Soothe Sandra M. L e f s t a d Approved for public release; distribution unlimited, �FOREWORD The active Air Force project directly related to the information discussed in this report is Exploratory Development Project 5066. Requests for further detailed information or any comments on this report may be referred to Air Force Armament Laboratory (DLI), Eglin Air Force Base, Florida 32542. Statistical analyses were performed by Booz-Allen Applied Research. The use of trade names is for identification purposes only and does not constitute endorsement by the United States Air Force. This report has been reviewed and is approved. r ^ ^ { t^ —~z—/- te FRANKIN C. p M E S w i o n e l , USAF KLI Chief, Flame, Incendiary and Explosives Division �ABSTRACT Gas chromatographic and enzymatic analyses (cholinesterase-inhibition SthSr«re used to monitor the metabolism of the organophosphorus insect dde 0,0-diethyl S-[(ethylthio)methyl] phosphorod thioate (Pforate) Ippfied fofiarly to three economically important plants (Homestead tomato, Siley sorg m" and Honey sorghum). The resulting data provided guide ines in predicting toxicity and persistence of metabo ite residues £r high ^binr^r^ei^i^ Ice d'on ass plates located adjacent to treated Plants, indicated the formation of toxic phorate metabolites was without the influence of biological substrates within the plants. There were no statisticaljy s gnificant differences with respect to the rate of increase of cholinesterl e- n ibition percentage values between the sorghum and glass plates, the rate of formation of anticholinesterase oxidized metabolites was predominantly through chemical oxidation on the leaf surface and not by K enzymecatalysis, or at least, the oxidation occurred at_such a ratfas tfmask the enzyme catalysis. The large droplet size in the application^ phorate resulted in higher toxic residue values, especially on the surface of the plant, than would normally be expected. Approved for public release; distribution unlimited. m (The reverse of this page is blank) ��TABLE OF CONTENTS Section I II Title Page INTRODUCTION 1 EXPERIMENTAL PROCEDURES 4 MATERIALS AND METHODS 4 STATISTICAL ANALYSES 9 III RESULTS AND DISCUSSION 12 IV SUMMARY AND CONCLUSIONS 40 REFERENCES 43 �LIST OF FIGURES Figure 1 Title Page Plant Metabolism Pathway of Phorate (Based on Reference 5) 2 Horse Serum Cholinesterase Inhibition Versus Concentration of 0,0-Diethyl S-[Ethylsulfinyl)methyl] Phosphorothiolate.. 8 3 Cholinesterase Inhibition of Homestead Tomato..... 13 4 Cholinesterase Inhibition of Wiley Sorghum 14 5 Cholinesterase Inhibition of Honev Sorqhum (Phorate Applied in May) 15 6 Comparison of Percent Cholinesterase Inhibition for Three Varieties of Plants. 17 7 Percent Cholinesterase Inhibition, Homestead Tomato 19 8 Effects of Passage of Time upon Percent Cholinesterase Inhibition, Homestead Tomato 20 2 9 Percent Cholinesterase Inhibition, Wiley Sorghum 10 Percent Cholinesterase Inhibition, Honey Sorghum.. 11 Percent Chlinesterase Inhibition, Wiley and Honey Sorghum.. 23 12 Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley Sorghum(March) and Honey Sorghum(April).. 24 13 Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) 26 Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) 27 14 21 22 15 Percent Cholinesterase Inhibition, Glass Plates 16 Comparison of Percentage Cholinesterase Inhibition for Glass Plates and Wiley Sorghum 33 Comparison of Percentage Cholinesterase Inhibition for Gl ass PI ates and Honey Sorghum 34 17 VI 32 �LIST OF TABLES Table I II III Title Page Plant Parameters Employed With High Concentrations of Phorate. 5 Efficiency of Extraction Technique for Phorate With Tomato and Sorghum 6 Measurement Days by Species and Experiment. 10 IV Gas Chromatographic Analysis for Phorate From Tomato and Sorghum. ,,. V Average Percent Cholinesterase Inhibition, Homestead Tomato 12 VI VII VIII IX Average Percent Cholinesterase Inhibition, Wiley Sorghum (March) and Honey Sorghum (April). Average Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May). Gas Chromatographic Analysis for Phorate from Glass Plates and Sorghum. " ?R ^ *' Persistence of Phorate Oxygen Analog Sulfoxide Equivalent in Sorghum. ™ vii (The reverse of this page is blank) ��SECTION I INTRODUCTION The research reported in this study constitutes part of a program to elucidate toxicological and ecological hazards associated with repetitive aerial applications and spills of organophosphorus insecticides. Toxicological hazards may exist in plant foliage even after environmental persistence studies determine the absence or safe level of the parent insecticide. These hazards result from the conversion of the parent insecticide to toxic oxidized metabolites. The rate of this conversion determines the nature and magnitude of the toxic residues in the plant tissues. The use of organophosphorus insecticides is increasing because of their wide spectrum of effectiveness (comparable to the chlorinated hydrocarbons) and their short residual action in water, soils, and plants. Any accident involving ultra-low-volume formulations of insecticides could result in a per-unit-area concentration that would be detrimental to agronomic plants because of unacceptable residue levels. For this reason, data relating the tolerance of agronomic plants to repetitive aerial applications of ultra-low-volume formulations of organophosphorus insecticides are of interest to military pest control programs. Guidelines were desired for predicting toxicity and persistence of metabolite residues in plants after application of high concentrations of the insecticide. Insecticides in or under considerations for the Air Force inventory include malathion (dithiophosphoric derivative), naled (phosphoric acid derivative), fenthion (thiophosphoric acid derivative with sulfide linkage), and Dursban^(thiophosphoric acid derivative). Phorate, 0,0-diethyl S-[(ethylthio) methyl] phosphorodithioate, was the insecticide selected because it is a model compound containing oxidation sites analogous to those found in sulfur-containing organophosphorus insecticides. Its toxicity, expressed as an oral 105^ value, is more tnan 100 times as great as the most toxic insecticide presently used by the militaryO). Thus, persistence data on toxic metabloties of phorate should provide a model system of maximum toxic residues on foliage wnich should be unapproachable for insecticides presently used by the military wnen applied at the same concentration. Furthermore, previous studies^' ^» "> ^J have elucidated the plant metabolism pathway (Figure 1) of the insecticide along with solvent-partitioning functions of phorate and its metabolites(2, 4). Conclusive research data are not available concerning insecticide pnytotoxicity; however, it is known tnat plant species exhibit a wide range of tolerance to applications of organophosphorus insecticides ^»'t. 1 �(CH3CH20)PSCH2SCH2CH3 S 0 (CH3CH20)2PSCH2SCH2CH3 0 ^^ II I *r (CH3CH20)2PSCH2SCH2CH3 N. 0 0 X 11 * (CH3CH20)2PSCH2SCH2CH3 (CH3CH20)2PSCH2SCH2CH3 Figure 1. Plant Metabolism Pathway of Phorate (Based on Reference 5} The basis for this phytotoxic resistance or susceptibility is not clear. The experimental procedure used in this study allowed an investigation into the role performed by individual plant chemistries in metabolizing organophosphorus insecticides. Studies of the morphological effects caused by highly concentrated foliar applications of mevinphos and methyl demeton on selected plant species indicated that, in general, brpadleaf plants were more susceptible to the insecticides than were grasses^ 7 /. Severe morphological injuries were observed on soybean, cotton, and tomato plants one day after foliar treatment, w h i l e seven days were required before comparable injuries were noted on corn and sorghum. Coleman and Dean^°' found that resistance of sorghum to methyl parathion was genetically controlled in their studies with a resistant and a susceptible variety, Wiley and Honey, respectively. Thus, differential phytotoxicity to organophosphorus insecticides can be expected between plant species as well as w i t h i n a given species. Differences do occur in the rates of metabolism of insecticides in different plant species. A study^) of the plant metabolism of Di-Syston® (dithioSystoxdD) and phorate indicated that the rates of a reaction may vary slightly from one plant species to another and according to the stage of growth, but the data obtained may be used as a guide to the relative proportions of the metabolites present at intervals after application. In �another study w y the effects of temperature and plant species upon the rates of metabolism of systemically applied Di-Syston®were considered, and s i m i l a r results were obtained. Phorate differs from Di-Syston® due to the presence of an ethylene rather than a methylene group in its side chain. Thus, this study attempted to relate the metabolism of the insecticide to phytotoxic damage among plant species and within a plant species. Oxidation can increase both the water solubility and the anticholinesterase activity of organophosphorus insecticides^"'. The logical approach for monitoring anticholinesterase compounds (toxic phosphorus esters) formed during the metabolism of phorate was a cholinesterasei n h i b i t i o n method. Phorate alone is too weak an inhibitor to be detected in micro amounts, but when applied to plants, it is very rapidly converted to potent anticholinesterase agents. The final unhydrolyzed metabolite (phorate oxygen analog sulfone) in the oxidation series (Figure 1) is the most active i n h i b i t o r ' ^ ' . The 150 value (molarity of i n h i b i t o r which results in 50 percent of the activity of the control) of phorate is approximately 250 times that of the phorate oxygen analog s u l f o n e ' ^ ) . One day after the application of phorate to corn, the residue of phorate sulfoxide, which has an 159 value approximately 1/100 that of phorate, was more than three times that of phorate^. �SECTION II EXPERIMENTAL PROCEDURES 1. MATERIALS AND METHODS a. Apparatus. A Son/all Omni-mixer^ was used for macerating plants. A gas chromotograph equipped with a flame photometric phosphorus detector and a digital integrator was employed in the phorate analyses. A recording pH stat was used to determine cholinesterase activity. b. Reagents and Solvents. Standards, supplied by the American Cyanamid Company, were technical grade phorate (Thimet® ) of 90-percent purity, analytical grade phorate of 97.8-percent purity, and 94-percent phorate oxygen analog sulfoxide containing 6-percent phorated oxygen analog sulfone. Reagents were anhydrous sodium sulfate, certified A . C . S . ; acetylcholine perchlorate (a "rare and fine" chemical from K and K Laboratories, Inc.); sterile filtered horse serum (Colorado Serum Co.); sodium chloride (crystals), analytical reagent grade; sodium chloride (granular), U.S.P. grade; sodium hydroxide pellets, analytical reagent grade; and potassium hydrogen phthalate, certified A.C.S. acidimetric standard. Solvents were certified A.C.S, acetone, certified A.C.S. hexanes, and vegetable oil. c. Plant Parameters. The representative broadleaf plant selected was Lycopersicon esculenturn mill, var. Homestead 24 (tomato), and the grasses were Sorghum vulgare Pers. var. Wiley (sorghum), and Sorghum vulgare Pers. var. Honey (sorghum). The two varieties of sorghum were selected to represent a variety (Wiley) that was resistant to an organophosphorus insecticide and one (Honey) that was susceptible. The plants were grown in a clear glass greenhouse with a minimum night temperature of 60° to 65°F and a maximum day temperature of 95° to 100°F. Seeds were planted in a soil consisting of a 7:3:1 mixture of sandy loam, peatmoss, and perlite with four pounds of dolomitic limestone and one pound of superphosphate added per cubic yard of soil. The pH of the soil was 6.5. Each tomato plant was transplanted to an individual four-inch plastic pot at the age of four weeks. The sorghum experimental unit consisted of 10 plants per four-inch plastic pot. A 15-15-15 liquid fertilizer was applied bi-weekly. A 2 percent or 1 percent solution of phorate (0.2 milliliter or 0.1 milliliter of technical grade phorate dissolved in 10 mi Hi liters of vegetable oil medium) was applied foliarly as 0.01 milliliter droplets with microsyringe to the tomato and sorghum at the concentrations shown in Table I. This procedure allowed a uniform and exact application to all �TABLE I. PLANT PARAMETERS EMPLOYED WITH HIGH CONCENTRATIONS OF PHORATE Species Age, Weeks Homestead Tomato Date of Application 8 August1969 19 November 1969 Phorate, Concentration, pprrr lb/Ab 19,839 16,630 2.19 1.84 Wiley Sorghum 4 3 2 March 1970 20 May 1970 8,753 (c) 1.26 (c) Honey Sorghum 4 3 8 April 1970 20 May 1970 8,461 (d) 1.22 (d) Concentration of phorate solution based on gas chromatographic analysis with analytical grade phorate. "Concentration applied to plant based on leaf area (pounds of active ingredient per acre). c Same concentration applied as 2 March 1970, but no gas chromatographic analysis. "Same concentration applied as 8 April 1970, but no gas chromatographic analysis. the plants. The levels of phorate applied were adjusted to the maximum amount that would result in minimal visible damage. To insure similarity in plant size at each application, the phorate was applied after three to six weeks of initial plant growth. The varying lengths of time between planting and insecticide application were to compensate for seasonal variation in plant growth. Table I shows the age of the plants with the date of application of phorate. During the March (Wiley sorghum) and April (Honey sorghum) portions of the experiment, the phorate in the vegetable oil medium was applied to glass plates located adjacent to the treated and control plants. d. Extraction Technique. The plants to be sampled were severed at soil level, sectioned, and rinsed in a 400-milliliter beaker containing 25 mi 11 iliters of hexanes and 25 mi Hi liters of acetone in distilled water (60 percent by volume) to remove any residues remaining on the plant surface. The solvent mixture, followed with the plant material, was poured into a cup for the Sorvall Omni-mixer®. The beaker was rinsed with 5 mi 11iliters of hexanes and then by 5 mi 11iliters of acetone in distilled water (60 percent by volume). After the plant material was thoroughly macerated, the macerate was filtered through three layers of 5 �cheesecloth into a separatory funnel. The cup which had contained the macerate was rinsed with 10 milliliters of hexanes and then by 10 millilitars of acetone in distilled water (60 percent by volume). The rinsings were added to the separatory funnel followed by 15 milliliters of a saturated sodium chloride solution which aided in separating the organic and aqueous phases. Acetone was removed from the aqueous layer by use of a rotary evaporator attached to a water aspirator. Complete removal of the acetone was essential because of its ability to inhibit cholinesterase. The aqueous layer was filtered (to remove minute pieces of plant material) through Whatman No. 2 paper into a 50-mi Hi liter volumetric flask and brought to volume with distilled water. The organic phase was placed over 10 grams of anhydrous sodium sulfate, filtered into a 50-mi11iliter volumetric flask, and brought to volume with hexanes. If the samples could not be analyzed immediately, they were stored at 5°C. The aqueous layer was analyzed for cholinesterase-inhibiting metabolites and breakdown products. Gas chromatographic analysis of the organic phase for phorate allowed monitoring of the rapidity of breakdown and oxidation. The efficiency of the extraction technique, based on the recovery of phorate in the organic phase, is shown in Table II. TABLE II. EFFICIENCY OF EXTRACTION TECHNIQUE FOR PHORATE WITH TOMATO AND SORGHUM Species Date of Experiment Homestead Tomato August November Wiley Sorghum March Honey Sorghum Apri 1 Efficiency of Extraction, Percent Controls3 Insecticide-Treated Plants^ 3 3 66.2 87.7 32.4 63.2 63.3) [ Qla ss 75.9) pla tesc 46.7 51 .7 ^Concentration of phorate applied to plant placed through extraction scheme. b Phorate-treated plants from day 0 placed through extraction scheme. c Glass surface residues from day 0 placed through extraction scheme. e. Cholinesterase-Inhibition Method. The advantages of using cholinesterase-inhibition methods for determining organophosphorus residues are that (a) the sensitivity is far greater than for chemical methods and (b) the method is particularly suitable when the insecticide undergoes 6 �changes in the plant to produce metabolites with a high inhibitory activity(10,11}_ One of the disadvantages is the lack of specificity or inability to distinguish among different types of cholinesterase inhibitors found within the plant. The persistency of toxic metabolites and breakdown products in the respective plant species was monitored using an automated pH stat method(12) to determine cholinesterase activity. The cholinesterase inhibition for each species and glass plate sample was measured immediately after the application of the insecticide and on various succeeding days for one month. Control plants were treated with only the corresponding amounts of vegetable oil and were similarly measured. The experiment was repeated for each species. The cholinesterase activity of the prepared water sample was recorded with a recording pH stat. A sample (0.2 milliliter tomato or 0.4 milliliter sorghum sample) was placed in a microbeaker containing 5 milliliters of a 0.154-molar saline solution (9.000 grams of analytical reagent grade sodium chloride in 1000 mi Hi liters distilled water) and 0.5 milliliter horse serum. The microbeaker solution was heated to 37.5°C and adjusted to pH 8.0 prior to addition of 0.3 milliliter of the cholinesterase enzyme substrate, 0.110-molar acetylcholine perch!orate (0.675 grams crystalline substrate in 10 milliliters distilled water). The titrant, 0.0100N sodium hydroxide.was standardized with 0.0100N potassium acid phthalate (0.20423 grams acid in 1000 milliliters distilled water). Normal-activity curves (no samples added) and control-activity curves (aqueous samples from control plants) were obtained prior to measuring cholinesterase activity for the aqueous samples from phoratetreated plants. Cholinesterase inhibition of the phorate metabolites and breakdown products was expressed as a percentage value obtained from a ratio of cholinesterase activity (expressed in units of micromoles of acetylcholine hydrolyzed per minute per milliliter of horse serum) of the samples from the phorate-treated plants to samples from the control plants. A second percentage of cholinesterase inhibition was calculated using the normal activity value as the base. f. Calibration Curve. To equate the percentage of cholinesterase inhibition to the concentration of the metabolite extract, a log-linear plot was made of the phorate oxygen analog sulfoxide equivalent, in parts per million (ppm) of 0,0-diethyl S-[(ethylsulfinyl) methyl] phosphorothiolate, versus the percentage of cholinesterase inhibition. Figure 2 contains the calibration curve based on 0.4 milliliter samples of the standard parts-per-million solutions of the phorate oxygen analog sulfoxide. Sample sizes of 0.2 milliliter gave cholinesterase inhibition percentages that were approximately half the values shown in Figure 2. �109 a 76 5 4 uu Ci I—I X o u. 2- co .9- uu 1 !?• .6.5.4- .3.2- JO- 20 Figure 2. 30 50 60 HORSE SERUM CHOIIMESTERASE INHIBITION (PERCENT) Horse Serum Cholinesterase Inhibition Versus Concentration of 0,0»Diethyl S-[(Ethylsulfinyl)methyl] Phosphorothiolate �g. Gas Chromatographic Analysis. Gas chromatography was used to detect unaltered phorate in the organic phase from the plant extracts. A gas chromatograph equipped with a flame photometric phosphorus detector was employed. A six foot by one-fourth inch stainless steel column containing Chromport X X X , 80/90 mesh, coated with 3 percent SE-30, and conditioned for 24 hours at 220°C, was used for the phorate analysis of the August tomato extracts. A retention time of 105 seconds was recorded with the inlet temperature set at 250°C, the column-oven temperature at 200°C, and the detector temperature at 235°C. Gas flow rates in cubic centimeters per minute were nitrogen 80, hydrogen 150, air 20, and oxygen 20. A six foot by one-fourth inch glass column containing Chromosorb id , 80/90 mesh, coated with 3 percent QV-1 and conditioned for 24 hours at 210°C was used for the phorate analysis of the November tomato extracts, March Wiley sorghum extracts, April Honey sorghum extracts, and glass plates. A retention time of 130 seconds was recorded with the inlet temperature set at 245°C, the column-oven temperature at 190°C, and the detector temperature at 185°C. Injection sample sizes 1 for August tomato extracts were 5 microliters, and all others were 2.7 microliters. Concentrations of phorate present in the August analysis were determined by integration of peak area by a digital integrator. Concentrations of all other analyses were determined by peak height. All determinations were expressed in parts per million based on standard curves. 2. STATISTICAL ANALYSES. Before any of the data were analyzed, all of the cholinesterase inhibition percentages were transformed using the arc sine formula: 8 = 2 arcsin P where 6 is the new variable to be analyzed and P is the percent of cholinesterase inhibition divided by 100. Tnis transformation minimized and stabilized the variation of the data and created the homogeneity of variance that was essential to the use of the analysis of variance technique, and the other statistical procedures used for the analysis of these experiments. The results were stated in percentage notations for presentation throughout the report. It was not possiole to analyze all of the cholinesterase inhibition percentages on an experiment-wide basis because the measurements of the three experimental species were taken on various and differing days �during the month following application of the insecticide. To make comparisons that were valid and meaningful, it was necessary to select those days on which all species under consideration were measured. Table III shows the days of measurement for each species. During the first week TABLE III. HEASUREMENT DAYS BY SPECIES AND EXPERIMENT Days After Application 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 Homestead Tomato August November X X X X X X Wiley Sorghum March May Honey Sorghum April May X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X following application of the insecticide, only those measurements that were made on the same day were compared; during the next three weeks, measurements that were made on contiguous days were also included in the comparison. 10 �Only one measurement of cholinesterase inhibition was made on each of the days for the Homestead tomato experiments and for the March and April Wiley and Honey sorghum experiments. It was not possible, therefore, to test for significance of the interaction between seasonal effects and the number of days following application. Since this interaction would have been used to test the seasonal and time-passage effects individually, an unduly large interaction would have masked significance of the main effects, Paired t-tests with the same or contiguous days' results were used to circumvent this possibility. When nonsignificance of the paired values was determined, an analysis-of-variance technique was used to test for significance of the passage of time upon the cholinesterase-inhibition percentage. In those cases in which these results were significant, Duncan's new multiple range test was used to identify where the differences did, in fact, exist. In the absence of significance, means were computed, and the effects of the passage of time upon the cholinesterase inhibition were studied. Two replicates of measurements were made for the May Wiley and Honey sorghum plants, and it was, therefore, possible to use the analysis of variance technique when only these data were involved in comparisons. Initially, all testing was conducted at the 95-percent probability level (significant). When this proved significant, subsequent testing was conducted at the 99-percent probability level (highly significant). 11 �SECTION III RESULTS AND DISCUSSION Ultra-low-volume formulations of insecticides used by the military involve the aerial application of a low-volume concentrate (0.75 to 10 ounces per acre, undiluted). Phorate is applied with normal formulations at rates of 0.5 to 3 pounds of active ingredient per acre. Table I presents the application rates within this range. However, the defined, directed application of phorate to the leaf surface without spraying resulted in a maximum interface between insecticide droplet (10 microliters) and leaf area. This large droplet size represented the application of high concentrations of phorate; the droplet contained more phorate (0.25 milligram) and is larger than that ordinarily found following routine application of insecticides. The optimum3 diameter for insecticide spray droplets is in the range of 20 microns'' '. The percentages representing the efficiency of the extraction technique employed (Table II) are minimum values because of the volatility of phorate. Phorate is lost from the soil by volatilization and about 25 percent of the loss occurs in the first hour after treatment^'^/. Similar results would be expected on the leaf surface, therefore, the length of time between treatment and initial extraction of the plant would result in a value of phorate present that is slightly less than that which was applied. The time before initial extraction was approximately the same in all cases; however, the extraction of the August tomato after application of phorate during the late morning heat probably accounts to some degree for the lower value in extraction efficiency. Phorate was applied to the other plants in the early morning. A comparison of the percentage of cholinesterase inhibition obtained using the normal-activity value and the percentage obtained using the control-activity value as a base is shown in Figures 3 and 4 for Homestead tomatoes and for Wiley sorghum for each of the two applications of insecticide. A comparison for Honey sorghum is shown in Figure 5 for the May application; however, the April control plants were not usable due to contamination. The results of paired t-tests in analyzing the differences between percentages for each species allowed the use of all the cholinesterase-inhibition percentages based on the normal activity value. Cholinesterase-inhibition values obtained showed no correlation with plant weight. A comparison of the percentages of cholinesterase inhibition during the month following application of the insecticide for each of the three plants, using the analysis of variance technique, indicated that the average for the Homestead tomato was significantly lower than those for 12 �/D fe CJ 50 - uj ex. O t~~t K h~, — UJ -J < nr .? j ( fc to o t~t GO 2:. uj t ^ —c > ---* rf t,^ »/ '• .• " « • ' lc b UJ CO '-=C CJ fti K UJ •< r™"- ••—— • 0 U o^ UJ -5 • CONTROL 25 § O NORMAL 1 I I § o 1 0 I 1 1 1 1 1 1 2 4 6 S 10 12 14 U U 20 22 1 1 24 26 21 TIME FROM APPLICATION WS) a. _ Phorate Applied in August 75 iUj | — 50 C±- 8 r^ 0 ft S^ A«s UJ UJ S oo t-i S^ uu o 0 8 • >-~< UJ m ° 8 8 0 8 — h- Oi c/) -< UJ •—• 3: o j. -25 • CONTROL 1 0. I I I I 1 1 1 2 4 6 6 10 12 14 U O NORMAL I 1 1 18 20 22 1 I 24 26 TIME FROM APPLICATION [VMS] b. Phorate Applied _in November Figure 3. Cholinesterase Inhibition of Homestead Tomato 13 2B �/p s- § • A UJ CJ c* 50 UJ O UJ »—1 —1 - » § • 8 » o 8 25 e CO V) ^— 1 1 1 ?5 t—< CO UJ 5ur§ 5t CO CJ tf <c UJ — n U to UJ t—1 —1 • CONTROL -5 — O NORMAL o CJ 1 1 1 1 1 1 f I 6 -25 i 1 1 1 1 « /O 12 14 16 H 20 TIME FROM APPLICATION (PAKS) 22 24 26 28 1 26 21 a. Phorate Applied in March /5 * 8i | 0- O UJ t—t —J \~ •< 50 8 • 8 * ' o • 881$ 8 » 8 UJ o e* UJ 25 t-i CJ CO CO b-< 1—t UJ W "<t UJ •— 1- UJ t—< —1 o 5 — '.r • CONTROL -5( 3: o O NORMAL i ^c 0 i 2 4 i 6 1 B 1 10 1 12 1 14 1 16 1 U 1 20 1 22 1 24 TIME FROM APPLICATION b. Phorate Applied in May Figure 4. Cholinesterase Inhibition of Wiley Sorghum 14 �72 !s UJ 75 ^* o CM UJ (X "~" 50 — 25 i ii o S 8 * • ^k ^k ^ ^fc. - 8 O t—1 — PQ •< 8 8 •t ^2 "» 8 ^h t—t CJ (-1 UJ UJ *-< C/) C/) 5 - So UJ CM UJ t—t —4 O -C o 0 _ 1 * CONTROL O WORMAL I 1 1 i I t I i I i t i I g 10 12 J4 76 JS 20 22 24 26 28 TIME FROM APPLICATIOW (PA^S) Figure 5. Cholinesterase Inhibition of Honey Sorghum (Phorate Applied in May) 15 �the two varieties of sorghum. Figure 6 shows the inhibition percentage values for each of the three plants; the lower level for the tomato plants is evident. Therefore, for statistical purposes, data from the tomato plant experiments were analyzed independent of the sorghum data. Gas chromatographic analysis of the hexanes phase after extraction from tomato and sorghum indicated less than one ppm phorate present by the sixth day (Table IV). A determination of residues of-phorate and five of its netab- TABLE IV. GAS CHROMATOGRAPHIC ANALYSIS FOR PHORATE FROM TOMATO AND SORGHUM Day Tomato August November Concentration of Phorate, ppm Sorq lum March (Wiley) April (Honey) 0 128 42 16 18 1 140 34 9-12 9 28 6-7 5-6 9 <3 1 2 3 23 4 <1 5 4 6 1 <1 elites from various parts of corn plants (treated with one pound of the.insecticide per acre) indicated that phorate was essentially gone in 14 days(4). During this determination, phorate was recovered from fortified samples with 96 percent efficiency using a Soxhlet apparatus in an eight-hour extraction technique. The higher values of phorate in the August tomato samples, as compared to the November samples, were due to the concentration of the sample to 10 milliliters instead of the 50-milliliter final volume of all other samples. Small differences in the data are probably due to the slight variations in extraction efficiencies for each plant. The disappearance of phorate proceeds at approximately the same rate in each plant variety. 16 �75 A D A A 9 50 — O 9 • o A D nc '--' O g «-< o _ =5 °° 0 • * A | > • UjS C/) CO UJ CM ^ A H * • o 00 — D A UJ (X *vl S UJ Q A UJ PuJ t—< —J CO •< n A 1 P * (* ^9 r 5 0 o o —1 o o 0 - HOMESTEAD TOMATO , 0 1 2 i 1 4 i | 6 i 1 8 O.VO I/EMBER WILE/ SORGHUM I3 •AUGUST * MARCH ^ MA/ HO/JE/ SORGHUM i 1 i 1 i 10 12 • APRIL 1 i 1 14 16 D MA/ t 1 20 i 1 IS i TIME FROM APPLICATIO/V (3A/S) Figure 6. Comparison of Percent Cholinesterase Inhibition for Three Varieties of Plants 1 22 i | 24 I 2< �The results of a cholinesterase-inhibition analysis for the Homestead tomato plants for all of the days measured for the August and November applications of insecticide are shown in Figure 7. An analysis of comparable daily measurements in August and November indicated no significant differences between the two application dates or by the number of days after application. The average percentages of cholinesterase inhibition for the days used in the comparison are given in Table V and Figure 8 and are not significantly different at the 95 percent probability level. TABLE V. AVERAGE PERCENT CHOLINESTERASE INHIBITION, HOMESTEAD TOMATO Days After Application Cholinesterase Inhibition, Percent 21,22 27.1 6 24.4 12,13 23.9 4 21.4 9,10 15.9 1 12.4 0 11.3 Figures 9 and 10 show the percentages of cholinesterase inhibition for Wiley and Honey sorghum, respectively. Analyses of the possible daily comparisons for each variety indicated significant differences between the March and May data for the Wiley sorghum and highly significant differences between the April and May data for the Honey sorghum. Similar analyses of those cholinesterase-inhibition percentages which were obtained on the same or contiguous days for Wiley sorghum in March and Honey sorghum in April indicated no significant differences between the two varieties for those two months (Figure 11). An analysis of the differences in cholinesterase-inhibition percentages with respect to the number of days after application of phorate to sorghum during March and April indicated highly significant differences. Table VI shows average daily percentages. In Figure 12, the average values with associated letters are shown graphically. The cholinesterase-inhibition percentage peak was reached by the ninth day following application of phorate, and an 18 �14J C_> {* U-i o « I4J CO O *-» CO U3 2 UU UJ •< CO UJ o o AUGUST APPLICATIOM WOI/&MBER APPLICATION 70 72 74 16 18 TIME FROM APPLICATION (VAVS) Figure 7. Percent Cholinesterase Inhibition, Homestead Tomato O �75 UJ UJ PL. \~ UJ CO t— t\3 25 UJ O )—< UJ CO •<C C_> UJ •< UJ a: «—< —i o » -5 10 n TIME FROM APPLICATION Figure 8. H 14 (VMS) 20 Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Homestead Tomato 24 26 �uj UJ h- UJ t~> —J CQ • < t~< O UJ V) uj »oo O UJ MARCH APPLICATION • APPLICATIOW JO 72 H 16 TIME FROM APPLICATION (VAVS] Figure 9, Percent Cholinesterase Inhibition, Wiley Sorqhum O �fe UJ o Be: UJ R- CQ ™ UJ2 APRIL APPLICATIOW MAV APPLICATION Figure 10. O TIME FROM APPLICATION {DAYS} Percent Cholinesterase I n h i b i t i o n , Honey Sorghum �95 WILEV SORGHUM MARCH APPLICATION HOWE/ SORGHUM APRIL APPLICATION O -25 10 72 14 U TIME FROM APPLICATION (PA/S) Figure 11. Percent Cholinesterase Inhibition, Wiley and Honey Sorghum �Cxi UJ R- 1- UJ 1-1 •—I CO •< l-« CJ K CJ UJ C* V- < TIME FROM APPLICATION Figure 12. Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley Sorghum (March) and Honey Sorghum (April) �average peak level of 63.9 percent Inhibition was maintained until the eighteenth to twenty-first day, when it began decreasing. By the twentythird to twenty-fifth day it had decreased to 31.5 percent. TABLE VI. Days After Application AVERAGE PERCENT CHOLINESTERASE INHIBITION, WILEY SORGHUM (MARCH) AND HONEY SORGHUM (APRIL) Cholinesterase Inhibition , Percent Remarks (Common letter indicates no significant difference at 99-percent Probability Level) 11 67.9 a 16,18 62.5 ab 9 61.1 ab 14 51.0 be 4 49.1 be 7 49.0 be 21,20 42.1 c 25,23 31.5 d 2 18.5 e 0 10.2 e 1 9.8 e Figure 13 shows the percentages of cholinesterase inhibition for Wiley and Honey sorghum on various days during May. The analysis indicated highly significant differences only with respect to the number of days after application; neither the species of sorghum nor the species/day interaction yielded results which indicated any significant effect. The average percentages for the various days after application are shown in Table VII. All averages that are not significantly different at the 99 percent probability level have a common letter. In Figure 14, the average values with associated letters are shown graphically. May Wiley and Honey sorghum plants reached a peak percentage 25 �o H- Uj CQ < 5: <s> 2: t-t UU li? Lu a: Ol l-t UU o WILE/ SORGHUM O WNEV SORGHUM n u TIME FROM APPLICATION Figure 13. Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) �UJ (X o l— ul t-i ~j B3 • < »—* O r\> i60 >-' ty uj Si UU Oi h- •< o o -5 1 I I 1 I 1 1 1 1 1 1 1 n 1 1 1 1 n 1 1 1 i 20 1 1 1 1 24 TIME FROM APPLICATION (PAW) Figure 14. Effects of Passage of Time Upon Percent Cholinesterase Inhibition, Wiley and Honey Sorghum (May) 1 1 26 1 �of cholinesterase inhibition by the fourth day and maintained an average peak level of 64.5 percent for the remainder of the month. TABLE VII. AVERAGE PERCENT CHOLINESTERASE INHIBITION, WILEY AND HONEY SORGHUM (MAY) Days After Application Cholinesterase Inhibition, Percent Remarks (Common letter indicates no significant difference at 99-percent Probability Level ) 14 68.8 a 12 68.7 a 8 65.2 a 7 65.1 a 19 64.5 a 4 64.0 a 5 63.1 a 16 60.6 a 21 60.4 a 2 28.9 b 0 -1.5 c With no significant differences existing between the two varieties of sorghum in May or during continuous experiments in March and April, it appeared that the metabolism of high concentrations of phorate proceeded at the same rate in each variety; however, distinct visible differences occurred between the two plant varieties. Despite slight initial visible damage, both sorghum and tomato recovered from insecticide damage within 30 days of treatment. The preliminary data did show, however, that neither variety of sorghum recovered from insecticide damage when exposed to the same concentration of phorate as was used on the tomato plants. This is indicative of the 28 �effects of a high concentration of phorate due to the large droplet size, since any visible damage at the rates used in this experiment would not be expected. With the tomato, minor c u r l i n g of the leaves was observed and many plants had a loss of apical dominance indicating that possibly, at the concentrations of phorate used, there was a change in the auxin content or hormonal distribution w i t h i n the plant. The first injury symptoms for both sorghum varieties were characterized by a localized bleaching of the blade pigments ( i . e . , chlorophyll) to a yellow-green coloration with a s l i g h t l y flaccid condition. These necrotic blotches were more distinct with Honey sorghum as they acquired a red-brown coloration. This characteristic color difference was apparent in the aqueous samples, as Honey samples were much darker than those of Wiley. The flaccid condition was more evident with Honey sorghum. The more v i s i b l e damage to sorghum compared to tomato in preliminary experiments, with both exposed to the same concentration of phorate, may appear to contradict the research reported in Reference 7. However, that report notes that (a) conclusions regarding the possible effects of organophosphorus insecticides, except mevinphos and methyl demeton, could not be made since the experiment was a fixed-effects model and not a random selection of possible organophosphorus insecticides; and (b) differences in plant response (susceptibility or resistance) could be accounted for by a postulation that differences may be related to leaf area interception of the insecticide. The reason for existing differences between March/April and May sorghum can only be postulated. Peak percentages of cholinesterase i n h i bition by the fourth day (64.5 percent) in May versus the n i n t h day (63.9 percent) in March/April indicate a more rapid oxidation of phorate to anticholinesterase metabolites. A s i g n i f i c a n t factor may be the relatively higher mean temperatures in May. In an evaluation of the effects of environmental temperature on Di-Syston©systemically applied to cotton leaves, the oxidation of the s u l f i d e , Di-Syston®, to the sulfoxide occurred so rapidly at temperatures above 70°F that only traces could be detected, even at intervals as short as one hour after treatment'**) The major^component in the leaves during the one-week experiment was the Di-Syston®sulfoxide. The rate of disappearance of sulfoxide was increased approximately 1.86 times for each 10°C rise in temperature (energy of activation of 10 kcal/mole). The i n i t i a l oxidation of phorate in cotton leaves was less rapid than the oxidation of Di-Syston®with traces of phorate found up to three days, although these never.exceeded 5 percent of the total radioactivity in the labeled experiment'^). It was also noted^ that the rate of oxidation of the sulfoxides in the oxidation series (Figure 1) was measurably slower for phorate than for Di-Syston® The sum of the rate constants for the disappearance of phorate sulfoxide due to oxidation is h a l f that of Di-Syston®sulfoxide. This indicates that phorate sulfoxide was probably the major metabolite during the first two weeks of this investigation. Also, when a l f a l f a seed was treated with 29 �treated with phorate or Di-Systorr% the effectiveness for aphid control varied by as much as several weeks depending on the rate of plant grow The rate of metabolism is slower in cooler weather, and the slower the plant growth, the longer the persistence of toxic residues. Tomato behaved similarly to the May sorghum. Tomato maintained a mean percentage value of 19.5 throughout the experiment; sorghum maintained a constant mean value throughout the month after, the third day. Thus, the results indicated that the metabolism of high concentrations of phorate proceeded at approximately the same rate in each species and between plant varieties. These results were not in complete harmony with those from earlier experiments. In previous metabolism studies of phorate and Di-Syston®on various plants such as cotton, alfalfa, lemon, and bean, it was found that the rates of reaction may be expected to vary slightly among pjant species and according to the stage of growth^). Later,, studies\°' specifically oriented toward the metabolism of Di-Syston^in a * variety of plant species, indicated that at 70°F, the metabolism of Di-Syston sulfoxide and hydrolytic decomposition of the toxic products occurred two to three times faster in tomato leaves than in cotton leaves. Differences in results and insecticide application parameters indicated the experimental data resulted from the chemical nature of phorate on the plant surface without the influence of biological substrates. Application methods^ 5 > included topical application of 5 to 25 microliters of insecticide to the base of a young plant or placement of isolated leaves in a water dispersion (0.1 percent solution) of the insecticide to permit the study of the rates of metabolism uncomplicated by the continual accumulation of translocated material. The method of application used in this work was foliar with a definite quantity of phorate aoplied as larae droplets to each intact plant. An earlier study' '^/ with Systox^(similar to phorate in structure) showed that the chemical nature of the surface washes from fruit treated with thiono- and thiolo-isomers changed rapidly upon exposure to light and air. Exposure of the isomers to light and air under controlled conditions on glass plates, uncomplicated by biological substrates, resulted in a surprisingly rapid conversion of the Systox®isomers into compounds which appeared to be chromatographically similar to those found within the plant tissues. Another study'''' showed that the action of air and sunlight on surface residues of Systox®isomers has a rapid effect and appears to promote their oxidation in the same sequence as found in vitro with hydrogen peroxide and in plant tissues. Thin films of phorate exposed to ultraviolet light or sunlight and air gave similar results^» ^8, 19, 20) ^ Exposure to sunlight on paper, glass, and leaf surfaces indicated that the initial stable residues of phorate may not be the original compound or its simple oxidation products; prolonged exposure resulted in the formation of more polar compounds^' 0 '. Results of ultra- 30 �violet irradiation of phorate on the surface of a liquid suggested that the oxidation products are the sulfoxide and sulfone of the parent compound, with the sulfone showing greater persistency and the s p]foxide being in greater quantity during the early stages of irradiation''^» ^0). To substantiate the concept that the experimental results in these investigations were without the influence of biological substrates within the plants, the same concentrations of phorate were applied to glass plates as were applied to the treated plants. The glass plates were located on the greenhouse bench adjacent to the control and treated plants. This was done with Wiley and Honey sorghum, in March and April, respectively, Table VIII shows a comparison of the gas chromatographic data for the plants and glass plates. Within 48 hours, phorate could no longer be detected on the glass plates; the same rapid disappearance was noted with the plants—approximately one ppm detected after 96 hours. TABLE VIII. GAS CHROMATOGRAPHIC ANALYSIS FOR PHORATE FROM GLASS PLATES AND SORGHUM Day Phorate Concentration, ppm March Aoril Wiley Sorghum Glass Plates Honey Sorghum Glass Plates 0 16 22 18 26 1 9-12 5-6 9 5 2 6-7 5-6 4 <3 1 <1 6 The cholinesterase-inhibition percentages obtained from the glass plates for the March and April experiments (Figure 15) were compared with the values obtained for the treated plants (Figure 11). The plots are very similar, with the glass plate cholinesterase-inhibition values being significantly higher on all days considered. However, most noteworthy is that the data through the twentieth day following application exhibited logarithmically linear trends (99 percent probability level) with no quadratic tendencies for both the sorghum and glass plates in March and April. The best-fitting straight lines have been plotted (Figures 16 and 17), and the equations for each of the lines are: 31 �99. Sr hUJ O R3 •< UJ (A) ro CO UJ CJ I UJ o CJ 25 O * Figure 15. n APRIL APPLICATION u TIME FROM APPLICATION (PAKS) Percent Cholinesterase Inhibition. Glass Plates �99.5 UJ CJ c*: o •— l~> UJ co to t"-t 3= <Z 50 *—» UJ CJ SORGHUM, MARCH APPLICATION O GLASS PLATES, MARCH APPLICATION 2 7 TIME FROM APPLICATION (PAVS) (LOGARITHMIC SCALE) Figure 16. Comparison of Percentage Cholinesterase Inhibition for Glass Plates and Wiley Sorghum I 24 29 �99.5 UJ UJ UJ _J t—1 I— •< uj II UJ kV) UJ o • HONEV SORGHUM, APRIL APPLICATION OGLASS PLATES, APRIL APPLICATION I I I I 4 7 3 TIME FROM APPLICATION (VMS) (LOGARITHMIC SCALE) Figure 17. Comparison of Percentage Cholinesterase Inhibition for Glass Plates and Honey Sorghum 79 24 29 �Wiley sorghum, March Y = 0.597 + 0.484 log (X + 1) Glass plate, March Y = 0.922 + 0.626 log (X + 1) Honey sorghum, April Y = 0.489 + 0.480 log (X + 1) Glass plate, April Y = 0.917 + 0.621 log (X + 1) where Y is the arc sine transformation of the percent cholinesterase inhibition and X is the number of days after the application of phorate in vegetable oil. An analysis of comparison of the linear plots gave significant results. The percentage of cholinesterase inhibition increased at the same rate for both varieties of sorghum. The percentages of cholinesterase inhibition for Wiley and Honey sorghum increased at the same rate as did those for the glass plates. The percent of variation explained by the linear trend is as follows: Wiley Sorghum 78.6 Glass Plate, March 89.3 Honey Sorghum 88.2 Glass Plate, April 92.4 The rate of formation of cholinesterase-inhibiting compounds appeared the same between the glass plates and sorghum plants. It appeared that, at least at high concentrations of phorate, formation of anticholinesterase oxidized metabolites was predominantly through a chemical oxidation on the leaf surface, and not plant enzyme catalysis. This took place at least at such a rate as to mask enzyme catalysis. With low concentrations of phorate within sorghum blades, methods similar to those described in References 5 and 8 could distinguish any difference in the rates of metabolism between the two varieties of sorghum. The higher cholinesterase inhibition values for the glass plates, (Figure 15) in comparison with the sorghum (Figure 11), can be attributed to the higher extraction efficiency with the glass plates. However, the phorate plant residue analysis by gas chromatography indicated the presence of phorate 48 hours after it could no longer be detected in samples from 35 �the glass plates. Although the cholinesterase-inhibition analysis Indicates the same rapid oxidation of phorate on leaf and glass plate, phorate could be present witnin certain portions of the leaf, e.g., within stomatal pores, and hence, within a potentially low-oxygen environment. This could explain the gas chromatographic data. An examination of roots resulted in no detectable cholinesterase inhibitors. Metabolism studies with lemon leaves showed the presence of large amounts of intact Di-Syston® accompanied by very slow conversion to other oxidative products(8). This suggested that the oil-soluble esters were being protected from aqueous hydrolysis by the oil content of the leaves. This was confirmed bv a radioautograph which showed nearly all of the radioactivity from P^2 Di-Syston® translocated into a lemon leaf is confined to the oil glands. A similar radioautograph of Systox®-thiol-isomer translocated into lemon leaves showed that, most of the radioactivity was located in the aqueous tissues of the plants^''. These differences were correlated with the relative water solubilities of the compounds, i.e., Di-Syston® 66 ppm and Systox®thipl-isonner 3900 ppm. The water solubility of phorate was recorded as 85 With the rate of formation of cholinesterase inhibitors the same en glass plates and sorghum leaf surfaces, the lower cholinesterase-inhibition percentage values for tomato (Figure 6) are difficult to explain. The values are lower than expected with tomato having received a concentration of phorate double that received by sorghum. Since the gas chromatographic data for tomato and sorghum agree, the distinct differences in cholinesteraseinhibition percentage values could have resulted from poorer efficiency in recovering oxidized metabolites from tomato compared to sorghum. The similarities in graphic plots for May sorghum (Figure 14) and tomato (Figure 8) would support this rationale. Evaluation of the cholinesterase-inhibition percentage values with the calibration curve resulted in values relating the concentration of toxic residues present in and on the plant foliage. Fourteen days after application of phorate to Honey sorghum in April, sample preparation of a foliar rinse of the surface of the plant accounted for approximately 50 percent of the total cholinesterase inhibition of the plant. A lack of detection of phorate within seven days indicated that residues of the oxidized metabolites in sorghum occurred to a very large degree via oxidation of phorate on the leaf surface, absorption within the leaf, and possible translocation within the plant. This is not in agreement with the previous studies presented in References 5 and 9. Other researchers have postulated(S) that the relative rates of absorption and translocation of phorate and Di-Syston® increased as the experiment proceeded because of the formation of more water-soluble oxidative metabolites in the subcuticular layers of olant tissue around the region of application. In Reference 9, the postulation is that the parent compounds were fairly persistent on the surface of the leaves but were metabolized rapidly once they had penetrated. �These variations can be explained by the differences in application method and in insecticide concentration: i.e., a 5 microliter topical application to the base of the stem of a cotton plant versus a 0.2 milliliter application of a 2 percent solution of phorate in vegetable oil to the blades of sorghum. It must be remembered that as the toxic metabolites are forming, they are concurrently being hydrolyzed to nontoxic phosphoric or thiophosphoric acids. Though the oxygen-analogs of phorate can inhibit cholinesterase activity more than their thionpohosphate precursors, they appear to have a higher degree of instability'^' ^'• The phosphorus is considerably more electrophilic in the P=0 compounds, thus weakening the P-S ester bond and facilitating hydrolysis and accelerating phosphorylation of the enzyme'^). Consequently, the presence of relatively large amounts of a highly oxidized metabolite in a plant would result in higher cholinesterase inhibition and higher apparent residue values than would an equivalent amount of a metabolite with less cholinesterase activity in another plant. The higher cholinesterase-inhibition values mean the presence of metabolites which are easily hydrolyzed, resulting in an overall faster rate of metabolic detoxification. The concentrations of phorate metabolites in tomato and sorghum were expressed in parts per million (ppn.) as phorate oxygen analog sulfoxide equivalent via a cholinesterase-inhibition method of analysis. The calibration curve for the residue method is given in Figure 2. It is independent of plant material analyzed and of the sample preparation technique. It reflects none of the losses that may occur in the various steps of sample preparation. A sample calculation follows the formula: .v = parts per million of phorate oxygen analog sulfoxide equivalent in sample analyzed. Where w is the phorate oxygen analog sulfoxide equivalent obtained in the analyses, micrograms. v is the aqueous extract in the determination, mi Hi liters. V is the total solvent in sample extraction, milliliters. W is the sample extracted, grams (fresh weight). The toxic residues present in tomato foliage were based .on the average weight of tomato plants initially after application of phorate (six weeks) and at the conclusion of the experiment (nine weeks), 6 grams and 28 grams, respectively. Thus, residues in the tomato foliage ranged from 1.1 to 5.3 ppm phorate oxygen analog sulfoxide equivalent. 37 �Residue persistence in sorghum (Table X) was higher. The May sorghum had concentrations with a range of 0 to 20.2 ppm. The average residue value after the second day was 17.9 ppm. The March/April sorghum had concentrations with a range of 2.4 to 18.5 ppm. The fourfold increase in residues by the ninth day is comparable to that found by Bowman and Casida'^' in considering the persistence of phorate-P 2 and its metabolites in vegetable crops. The total anticholinesterase activity of greenhouse pea plants, sprayed with phorate at one pound per acre, increased for about the firslv four days and then declined, but inhibitors persisted for 20 to 30 ^ ' Foliage application of 0,0-diethyl S-[(isopropylthio) methyl] phosphorodithioate to pea plants resulted in the appearance of anticholinesterase metabolites within one day and persistence of such metabolites in high concentration for at least nine days with detectable amounts present for 21 days^'. The results of this investigation were comparable: the main difference was higher residue levels. In crops treated with phorate., the ultimate toxic residues are present in a fractional part per million'". When applied to corn at a rate of one pound per acre, phorate was essentially gone in 14 days, while very low levels of its sulfoxide and sulfone (0.1 ppm or less) persisted through the 28-day experimental interval. At harvest time, the plant was essentially 4 free of insecticide, less than 0.01 ' The concentration of phorate metabolite residues present, though high, would probably be at a safe level by harvest time. The ultimate toxic metabolites present in harvest time residues are dependent upon both the interval between application and harvest and the method of application. Older plants having phorate applied at high rates would definitely have to be monitored for toxic residues. The lower residue values for tomato foliage are due in part to a larger daily plant weight—approximately threefold that of sorghum. The result could be a more rapid metabolism and hydrolysis and provides another possibility for the cholinesterase-inhibition percentage values being lower for tomato than for sorghum. Generally, oxidation. in plants never increases the toxicity of an application significantly^"'. However, the large residue values obtained in this study indicate that the toxicity is increased considerably on the surface of the plant when high concentrations are involved. A number of researchers, in speculating upon potential residue problems after various methods of treatment with systemic insecticides, have concluded that persistence curves should be 8 established on different crops grow under different environmental conditions' '. Military application of insecticides at normal rates results in residues which can be monitored with guidance from available literature. Residue breakdown of these organophosphorus insecticides is usually rapid with no persistency problems. However, the result of repetitive 38 �aerial application or spillage of insecticides in cropland areas may result in concentrations higher than usual. This study indicates exposure studies of the respective insecticides on glass plates alone under different environmental conditions would serve as a guide in predicting residues from high concentrations of insecticides. TABLE IX. PERSISTENCE OF PHORATE OXYGEN ANALOG SULFOXIDE EQUIVALENT IN SORGHUM Time From Application, Day 0 1 2 4 5 7 8 9 11 12 14 16 18 19 20 21 23 25 a Concentration, ppm March /April 3 2.4-5.3 2.4-5.3 2.4-5.3 10.3-14.5 10.3-14.5 Mayu 0 8.3 17.1 19.7 18.7 18.5 12.6-18.5 16.0-18.5 10.3-14.5 12.6-18.5 12.6-18.5 16.6 20.2 18.0 15.3 10.3-11.9 10.3-11.9 6.9-7.9 6.9-7.9 17.0 Concentration range is the result of considering the standard deviation for the average plant weight for the entire experimental period in May; this is due to the lack of plant weight values for March/April. Results are averages of two samples, three replications each. 3 Results are averages of four samples, three replications each. 39 �SECTION IV SUMMARY AND CONCLUSIONS Data on the metabolism of foliar applications of high concentrations of the organophosphorus insecticide phorate on Homestead tomato and Wiley and Honey sorghum are reported. The investigation of phorate metabolism, monitored by gas chromatographic and enzymatic analysis, produced the following results: 1. The cholinesterase activity values obtained showed no correlation with plant weight. 2. The disappearance of phorate appeared to proceed at the same rate in each plant species and variety; phorate disappeared more quickly from glass plates than from March/April sorghum under the same experimental parameters. 3. No significant differences were apparent between the two varieties of sorghum in May or during continuous experiments in March and April. It appeared that the formation of anticholinesterase metabolites, after high foliar applications of phorate, proceeded at the same rate in each variety although distinct visible differences occurred between the Wiley and Honey sorghum. 4. The peak percentages of cholinesterase inhibition from sorghum samples by the fourth day in May versus the ninth day in March/April indicated more rapid oxidation of phorate to anticholinesterase metabolites at higher temperatures. 5. The phorate metabolism in tomato was similar to metabolism in the May sorghum; however, actual comparison of percentage values of cholinesterase inhibition during the month for each of the three plants indicated that the average for the Homestead tomato was significantly lower than those for the two varieties of sorghum. 6. The percentage values of cholinesterase inhibition for the Wiley and Honey sorghum increased at the same rate as for the glass plates, indicating that the rate of formation of anticholinesterase-oxidized metabolites was predominantly through chemical oxidation on the leaf surface and not by plant enzyme catalysis; this surface oxidation took place at least at such a rate as to mask enzyme catalysis. 7. The larger droplet size in application technique resulted in higher toxic-residue values for phorate metabolites, especially on the surface of the plant, than would normally be expected. 40 �This study was initiated to find a basis for predicting toxicity and persistence of metabolite residues in plants after application of high concentrations of sulfur-containing oraanophosphorus insecticides during military spray operations. The results indicate that exposure studies of high concentrations of insecticides on glass plates alone, under different environmental conditions, would serve as a guide in predicting residues from repetitive aerial application or spillage of insecticides used by the military in cropland areas. Such studies with a controlled environment would yield toxic-residue-persistence data under various conditions for high concentrations of insecticides. 41 (The reverse of this page is blank) ��REFERENCES 1. Thomson, W.T. Agricultural Chemicals, Book I. Insecticides, Acaricides, and Ovicides. Thomson Publications, Davis, California, 1967. 2. Bowman, J.S. and J.E. Casida. Metabolism of the Systemic Insecticide 0,0-Diethyl S-Ethylthiomethyl Phosphorodithioate (Thimet) in Plants. J. Agr. Food Chem. 5: 192-197, 1957. 3. Bowman, J.S. and J.E. Casida. Further Studies on the Metabolism of Thimet by Plants, Insects, and Hammals. J. Econ. Entomol. 51:838-843, 1958. 4. Bowman, M.C., M. Beroza, and J.A. Harding. Determination of Phorate and Five of Its Metabolites in Corn. J. Agr. Food Chem. 17:138-142, 1969. 5. Metcalf, R.I., T.R. Fukuto, and R.B. March. Plant Metabolism of Dithio-Systox and Thimet. J. Econ. Entomol. 50:338-345, 1957. 6. Coleman, O.H. and J.L. Dean. Inheritance of Resistance to Methyl Parathion in Sorgo. Crop Sci. 4:371-372, 1964. 7. Wolverton, B.C., W.J. Wallace, A.L. Young, and D.D. Harrison. Studies on the Systemic Uptake of Toxic Phosphorus Esters by Plants. Morphological Effects of Foliar Applications of the Organophosphate Insecticides Mevinphos and Methyl Demeton on Selected Plant Species. Air Force Armament Laboratory Technical Report AFATL-TR-69-116, Eg!in Air Force Base, Florida, September, 1969. 8. Metcalf, R.L., H.T. Reynolds, M. Winton, and T.R. Fukuto. Effects of Temperature and Plant Species upon the Rates of Metabolism of Systemically Applied Di-Syston. J. Econ. Entomol. 52:435-439, 1959. 9. Heath, D.F. Metabolism in Plants and Soils. Jm Organophosphorus Poisons, Anticholinesterases and Related Compounds edited by D.F. Heath. Pergamon Press, Mew York, 1961. 10. Archer, T.E. Enzymatic Methods. Jm Analytical Methods for Pesticides, Plant Growth Regulators, and Food Additives, Volume I edited by G. Zweig. Academic Press, New York, 1963. 11. Sutherland, G.L., P.A. Giang, and T.E. Archer. Thimet. I_n Analytical Methods for Pesticides, Plant Growth Regulators, and Food Additives , Volume II edited by G. Zweig. Academic Press, New York, 1964. 43 �12. Nabb, D.P. and Florence Whitfield. Determination of Cholinesterase by an Automated pH Stat Method. Arch. Environ. Health. 15:147-154, 1967. 13. Himel, C.M. The Optimum Size for Insecticide Spray Droplets. J. Econ. Entomol. 62:919-925, 1969. 14. Young, A.L. and B.C. Wolverton. Military Herbicides and Insecticides. Air Force Armament Laboratory Technical Note AFATL-TN-70-1, Eglin Air Force Base, Florida, January, 1970. 15. Reynolds, H.T., T.R. Fukuto, R.L. Metcalf, and R.B. March. Seed Treatment of Field Crops with Systemic Insecticides. J. Econ. Entomol. 50:527-539, 1957. 16. Metcalf, R.L., R.B. March, T.R. Fukuto, and M.G. Maxon. The Nature and Significance of Systox Residues in Plant Materials. J. Econ. Entomol. 48:364-369, 1955. 17. Fukuto, T.R., R.L. Metcalf, R.B. March, and M.G. Maxon. Chemical Behavior of Systox in Biological Systems. J. Econ. Entomol. 48:347-354, 1955. 18. Cook, J.W. and R. Ottes. Note on the Conversion of Some Organophosphate Pesticides to Less Polar Compounds by Ultraviolet Light. J. Assoc. Offie . Agr. Chemists. 42:211-212, 1959. 19. Mitchell, T.H., J.H. Ruzicka, J. Thomson, and B.B. Wheals. The Chromatographic Determination of Organophosphorus Pesticides. Part III. The Effect of Irradiation on the Parent Compounds. J. Chromatog. 32:17-23, 1968. 20. Ruzicka, J.H., J. Thomson, and B.B. Wheals. The Gas Chromatographic Examination of Organophosphorus Pesticides and Their Oxidation Products. J. Chromatog. 30:92-99, 1967. 21. Metcalf, R.L., R.B. March, T.R. Fukuto, and M.G. Maxon. The Behavior of Systox-isomers in Bean and Citrus Plants. J. Econ. Entomol. 47:1045-1055, 1954. 44 �DISTRIBUTION LIST AFSC (DLSW) (SDWM) (SGP) ARPA (TECH INFO) DDR&E (CHEM TECH) (TECH LIB) SAAMA (SFQT) 2 3 3 1 1 5 5 AIR UNIVERSITY LIB HQ DA OACSFOR (FOR CM SR) OPERATIONS RSCH GRP EDGEWOOD ARSENAL (SMUEA-TD-S) (SMUEA-RPRE (2)) (SMUEA-CC) (SMUEA-QS) (SMUEA-CCCR) (SMUEA-TSTI-L) (SMUEA-D) (SMUEA-TS-CF) 1 1 1 1 2 1 1 1 1 1 1 (WPNS DEV & ENGR LABS) ENGR R&D LABS (TECH DOC CTR) ABERDEEN PRV GD (TECH LIB) DESERET TEST CENTER (TECH LIB) CBR AGENCY (CSGSB-ST) USA CHEMICAL SCHOOL (AJMCL-A) NAV AIR SYS COMD (AIR-532G) USN WEAPONS LAB 2 2 1 4 1 1 2 2 USN RESEARCH LAB (CODE 6140) 4525 FTR WPN UG (FWOA) 6570TH AMRL (HEF) DDC 12AF (DMEME) DL DLIP SSLT TAWC (DOD) (DIM) SOC (DFS) HQ USAF (AFRDPA) TAC (DOO-S) SOF-DOR 319SOS-DM TAWC-CB USAFETAC USAF ENVIRONMENTAL HEALTH LAB (Kelly AFB TX) 1 1 1 12 1 1 50 2 1 1 1 2 2 2 2 1 1 1 45 �DISTRIBUTION LIST (Concluded) USAF ENVIRONMENTAL HEALTH LAB (McClellan AFB CA) 1 USAFEL AFWL (DEE) USAFA (DFLS) DUGWAY PROVING GROUND (TECH LIB) ONR (CODE 440) HQ USACDC (NBC BRANCH) ASD (ENYS) DLOS CDCLNO 1 2 2 1 1 1 1 1 2 46 �UNCLASSIFIED Security Classification DOCUMENT CONTROL DATA - R & D (Security classification of title, body of abstract and indexing annotation must be entered when the overall report is ORIGINATING A C T I V I T Y (Corporate author) Flame, Incendiary, and Explosives Division Air Force Armament Laboratory Eqlin Air Force Base, Florida 3 classified) . REPORT SECURITY CLASSIFICATION UNCLASSIFIED 26. GROUP* REPORT TITL.E THE METABOLISM OF HIGH CONCENTRATIONS OF THE ORGANOPHOSPHORUS INSECTICIDE PHORATE APPLIED FOLIARLY TO SELECTED PLANT SPECIES D E S C R I P T I V E NOTES (Type- ot report and inclusive dates) Final Report (May - December 1970) 5 AUTHORCSI (First name, middle initial, Imxt name) George S. Kotchmar, Jr., Capt, USAF3 Billy C. Wolverton, Elizabeth.E. Boothe, Sandra M. Lefstad 6 REPORT D A T E 7«. T O T A L NO. OF PAGES February 1971 8«- C O N T R A C T OR G R A N T NO. &. PROJECT NO. 5066 53 \7t. NO. OF REFS 1 21 9a. ORIGINATOR*^ REPORT NJUMBERfSt AFATL-TR-71-22 9b. OTHER REPORT NO (si (Any other numbers that may oe assigned this report) C. d. 1O. DISTRIBUTION S T A T E M E N T Approved for public release; distribution unlimited. M- S U P P L E M E N T A R Y NOTES Available in DDC 12- SPONSORING M I L I T A R Y A C T I V I T Y Air Force Armament Lauoratory Air Force Systers Command Eglin Air Force Base, Florida 32542 ABSTRACT Gas chromatographic and enzymatic analyses (cholinesterase-inhibition method) were used to monitor the metabolism of the organophosphorus insecticide 0,0diethyl S-[(ethylthio)methyl] phosphorodithioate (phorate) applied foliarly to three economically important plants (Homestead tomato, Wiley sorghum, and Honey sorghum). The resulting data provided guidelines in predicting toxicity and persistence of metabolite residues for high concentrations of insecticides employed by the military. An attempt was also made to relate the metabolism of the insecticide to phytotoxic damage among and within plant species. The data indicated that no plant-variety-dependent distinction exists in the formation of toxic phorate metabolites as shown by in vitro anticholinesterase activity recorded over a four-week period. Further investigation, with the same high concentrations of phorate placed on glass plates located adjacent to treated plants, indicated the formation of toxic phorate metabolites was without the influence of biological substrates within the plants. There were no statistically significant differences with respect to the rate of increase of cholinesterase-inhibition percentage values between the sorghum and glass plates; the rate of formation of anticholinesterase oxidized metabolites was predominantly through chemical oxidation on the leaf surface and not by plant enzyme catalysis, or at least, the oxidation occurred at such a rate as to mask the enzyme catalysis. The large droplet size in the application of phorate resulted in higher toxic residue values, especially on the surface of the plant, than would normally be expected^ DD FORM 1 NOV 65 1473 UNCLASSIFIED Security Classification �UNCLASSIFIED Security Classification 1 14. K EY LINK A LINKS LINK' C WORDS ROLE WT ROLE WT Phorate 0,0-diethyl S-[(ethylthio)methyl] phosphorodithioate Organophosphorus Insecticides Plants Insecticide Residues Insecticide Metabolism UNCLASSIFIED Security Classification ROL E W T � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 016 Folder The folder containing the original item. 0172 Series The series number of the original item. Series II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Kotchmar, George S., Jr. Billy C. Wolverton Elizabeth E. Boothe Sandra M. Lefstad Description An account of the resource Corporate Author: Flame, Incendiary, and Explosives Division, Air Force Armament Laboratory, Eglin AFB, Florida Date A point or period of time associated with an event in the lifecycle of the resource 1971-02-01 Title A name given to the resource The Metabolism of High Concentrations of the Organophosphorus Insecticide Phorate Applied Foliarly to Selected Plant Species Subject The topic of the resource pesticide testing pesticide toxicology https://www.nal.usda.gov/exhibits/speccoll/files/original/355890c6af5da1f145114408fa767656.pdf b1c89d4bddbc724805f64a142582eca8 PDF Text Text Item ID Number: 00101 Author Corporate Author Simmon, Vincent F. Stanford Research Institute, Menlo Park, California Report/Article TltlB Evaluation of Seleclod Pesticides as Chemical Mutagens 'In Vitro' and 'In Vivo' Studies Journal/Book Title Year ™n Month/Day Ma v Color Number of Images 252 DOSCriptOn NOtBS Conlracl No. 68-01 -2458; EPA-600/1 -77-028 Friday, Doccrnbcr 08, 2000 Pago 101 of 106 �S immon , V . F . , 1977 F.va-luation oC selected pesticides as chemical mutagcns 'In vito' and 'In vivo1 studies I Technical Information Service •- *- *"*"^it" "'^ "^^ ^* "Iff™1* ™1* PL268&4Z _ Evaluation of Selected Pesticides as Chemical AAutagens 'In vitro' and 'In vivo' Studies a .- ' . • _ - . . _ . . _ . 1. Stanford.Research Institute, Menlo Park, Calif • - - • - - - r. • - c z .: - ' I "-- - Prepared for Health Effects Research Lab, Research Triangle Park, N C May 77 AEROM EDICAL LIBRARY APR 21 1980 DOCUMENT'S �:(:C!(,MiCAL REPOR1 •. A 1 ,:-asc read Instructions on the reverse be}u.'f completing) i REPORT NO J:PA-600/1-77-028 4. T I T L E AND S U B T I T L E 5 REPORT DATE May 197/ EVALUATION OK SELECTED PESTICIDES AS CHEMICAL MUTAGENS In Vitro and In Vivo Studies 6. PERFORMING ORGANIZATION CODE B. PtHFORMING ORGANIZATION RfPOHT NO. 7 AUTMOR(S) Vincent F. Silicon, Ann D. Mitchell and Ted A. .Torgenson LSU-3493 iO-'pROGHAM ELEMENT NO". 9>EFirORMlNG ORGANIZATION NAME AND ADDRESS Stanford Research Institute Menlo Park, California 94025 1EA615 TTCONtH'AflT^eHANi^NO. * 68-01-2458 12. SPONSORING AUKNCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED HeaJth Effects Research Laboratory - RTF, NC Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, N.C, 27711 iSTs u P PL'EMFNTARYIVIOTES" "" [^"ABSTRACT " " " 14. SPONSORING AGENCY CODE 600/1.1 ~ Twenty pesticides being reviewed as part of the EPA Substitute Chemical Program were studied for mutagenic activity by several _Ln vitro and in vivo test procedures. The pesticides reviewed were: monocrotophos, bromacil,, ca^codyllc acicf, captan, chlorpyrifos, dinoseb, DSMA, fenthion, folpet, azinphos-methyl, malathion, methomyl, monuron^ MSMA, parathion, parathion-methyl, quintozene (PCNB), phorate, simazine, and trtfluralin. Ten of the twenty compounds were evaluated in_vivp by the mouse dominant lethal test. All twenty compounds were tested in vitro. None of the ten compounds tested in the mouse produced a dominant lethal response. Ten of the twenty compounds were mutagenic in one or more, in vitro assays. Two were mutagenic in all of the in vitro assays: captan and folpet. KEY WORDS AND DOCUMENT ANALYSIS 7. b.lDENTIFIERS/OPEN ENDED TERMS DESCRIPTORS pesticides in vivo analysis in vitro analysis Diutagtms COS AT I Held/Group 06 F 06 T 19. SECURITY CLASS (This Keportl 19. DISTRIBUTION S T A T E M E N T 21. NO. __UNCLAS^IFIED RELEASE TO PUBLIC 20. SECURITY CLASS (Thispage) REPRODUCED EV EPA Form 2220-1 (9-73) c. NATIONAL TECHNICAL INFORMATION SERVICE U. S. DEPARTMENT OF COMMERCE SPRINGFIELD, VA. 22161 22. P'RICE ��NOTICE THIS DOCUMENT HAS BEEN R E P R O D U C E D F R O M T H E BEST C O P Y F U R N I S H E D U S B Y THE SPONSORING AGENCY. ALTHOUGH IT IS R E C O G N I Z E D THAT CERTAIN PORTIONS ARE I L L E G I B L E , IT IS BEING R E L E A S E D IN THE INTEREST OF MAKING A V A I L A B L E AS MUCH I N F O R M A T I O N AS POSSIBLE. �RESEARCH REPORTING SERIES Research reports of the Office of Research and Development, U.S. Environmental Protection Agency, have been grouped intc nine series. These nine broad categories were established to facilitate further development and application of environmental technology Elimination o< traditional grouping was consciously planned to foster technology transfer and a maximum interface in related fields. The nine series are: 1. 2. 3. 4. 5. 6. 7 8. 9. Enviroimenta! Health Effects Research Environmental Protection Technology Ecological Research Environmental Monitoring Socioeconomic Environmental Studies Scientific and Technical Assessment Reports (STAR) Interagency Energy-Environment Research and Development "Special" Reports Miscellaneous Reports This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RESEARCH series. This series describes projects and studies relating to the tolerances of man for unneaithful substances or conditions This work is generally assessed from a medical viewpoint, including physiological or osychological studies. In addition to toxicology and othe- medical specialities, study areas include biomedical instrumentation and health research techniques utilizing animals — but always with intended application to human health measures. This document is available to the public through the National Technical information Service, Springfield, Virginia 22161. �EPA-600/1-77-028 May 1977 EVALUATION OF SELECTED PESTICIDES AS CHEMICAL MUTAGENS in Vitro and In Vivo Studies By Vincent F. Simmon, Ann D. Mitchell, and. Ted. A. Jorgenson Stanford Research Institute Menlo Park, California 94025 Contract No. 68-01-2458 Project Officer Michael D. Waters Environmental Toxicology Division Health Effects Research Laboratory Research Triangle Park, N.C. 27711 U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF RESEARCH AND DEVELOPMENT HEALTH EFFECTS RESEARCH LABORATORY RESEARCH TRIANGLE PARK, N.C. 27711 10. �DISCLAIMER This report has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that tha contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. it �FOREWORD The. many benefits of our modern, developing, Industrial society are accompanied by certain hazards. Careful assessment of the relative risk of pxisting and new man-made environmental hazards is necessary for the establishment of sound regulatory policy. These regulations serve to enhance the quality of our environment In order to promote the public health and welfare and the productive capacity of our Nation's population. The Health Effects Research Laboratory, Research Triangle Park, conducts a coordinated environmental health research proBr#:n in toxicology, epidemiology, and clinical studies using human volunteer Kiibjects. These studies address problems in air pollution, non-ionizing radiation, environmental, carcinogenesis and the toxicology of pesticides as well as other chemical pollutants. The Laboratory develops and revises air quality criteria documents on pollutants for which national ambient; air quality standards exist or are proposed, provides the data for registration of new pesticides or proposed suspension of those already in use, conducts research on hazardous and toxic materials, and Is preparing the health basis for non-ionizing radiation standards. Direct support to the regulatory function of the Agency is provided in the form of expert testimony and preparation of affidavits as well as expert advice to the Administrator to assure the adequacy of health care and surveillance of persons having suffered imminent and substantial endange.rment of their health. This report describes the testing of a series of twenty technical grade pesticide chemicals for genotoxic properties by use of a battery of JLn yjltro and .In vivo methods. The battery includes tests for gene and chromosomal mutations and primary damage to DNA as measured by effects on DNA repair recombination. Since DNA is chemically similar in all species, test results from a variety of cells and organisms are relevant in assessing the potential genetic hazard of pesticide chemicals in humans. John H. Knelson, M.D. Director, Health Effects Research Laboratory \ �ABSTRACT Twenty pesticides being reviewed as a part of the EPA Substitute Chemical Program were studied for mutagenic activity by several in vivo and lii vlcro test procedures. Ten of Che twenty compounds were evaluated in vivo by the mouse dominant lethal test. All twenty compounds were tested by the following _in_ vitro procedures: Unscheduled DNA synthesis (UDS) In human fibroblasts (WI-38 cells); reverse mutation in Salmonella typhlmur:Lum strains TA1535, TA1537, TA1538, and TA100 and in Escherlchla.coll. WP2; mitotlc recombination in the yeast Saccharomyces cereylslae D3; and preferential toxlclty assays In DNA repairproficient and -deficient strains of ]J. coH (strains VO110 and p3478, respectively) and Bacillus subtilis (strains H17 and MAS, respectively). None of the ten compounds tested in the mouse produced a dominant lethal response. Ten of the twenty compounds were mutagenic in one or more ^n vitro assays. Two were mutagenic in all of the in vitro assays: captan and tolpet. In a heritable translocate on study in mice, under the experimental procedures employed, captan at 5000 ppm in the diet of male mice for 8 consecutive weeks produced a heritable mutagenic event in F^ generation male mice. iv �CONTENTS LIST OF TABLES Hi SUMMARY 1 TWENTY PESTICIDES EVALUATED BY SRI FOR MUTAGENIC ACTIVITY 3 INTRODUCTION 4 DOMINANT LETHAL TEST IN THE MOUSE 6 General Experimental , , Animals anil Chemicals Determination of Acute Toxlclty Maximum Tolerated Dose Study Treatment Levels Administration of the Compounds Test Groups Necropsy and Evaluation Results and Discussion MAMMALIAN JEN VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS General Experimental Cell Culture Dilution of Compounds Controls UDS Assays Interpretation of Results MICROBIOLOGICAL ASSAYS 6 6 6 7 7 7 8 8 9 9 12 12 13 13 13 13 14 15 17 General 17 Experimental 18 Salmonella typhimurlinn Strains TA1535, ~TAi537," TA1538,"and TA100 18 JEscherlchia coll WP2 19 Escherlchla coU W3100/p3478 and Bacillus 'subtllls H17/M45 20 jjaccharomjrces cerevlslae D3 21 Aroclor 1254-Stlmulated"~MetaboHc Activation System . . . 22 Results and Discussion 22 DISCUSSION 25 REFERENCES 26 APPENDIX A: Mutageneals Studies of Pesticide Compounds - Mouse Heritable Translocation Test - Captan 17.1 �TABLES DOMINANT LETHAL TEST 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Chi-Square Test of the Fertility Index - Monocrotophos . . . . 28 Average Implants per Pregnant Female - Monocrotophos 29 Average Dead Implants per Pregnant Female - Monocrotophos. . . 30 Chi-Square Test of the Death Index - Monocrotophos 31 Number of Dead Implants per Total Implants - Monocrotophos . . 32 Chi-Square Test of the Fertility Index - Bromacil 33 Average Implants per Pregnant Female - Bromacil 34 Average Dead Implants per Pregnant Female - Bromacil 35 Chi-Square Test of the Death Index - Bromacil 36 Number of Dead Implants per Total Implants - Bromacil 37 Chi-Square Test of the. Fertility Index - Captan 38 Average Implants per Pregnant Female - Captan 39 Average Dead Implants per Pregnant Female - Captan 40 Chi-Square Test of the Death Index - Captan 4i. Number of Dead Implants per Total Implants - Captan 42 Chi-Square Test of the Fertility Index - Folpet. . . 43 Average Implants per Pregnant Female - Folpet 44 Average Dead Implants per Pregr.ant Female - Folpet 45 Chi-Square Test of the Death Index - Folpet 46 Number of Dead Implants per Total Implants - Folpet 47 Chi-Square Test of the Fertility Index - Azinphos-Methyl . . . 48 Average Implants per Pregnant Female - Azlnphos-Methyl . . . . 49 Average Dead Implants per Pregnant Female - Azinphos-Methyl. . 50 Chi-Square Test of the Death Index - Azinphos-Methyl 51 Number of Dead Implants per Total Implants - Azinphos-Methyl . 52 Chi-Square Test of the Fertility Index - Malathion T.3 Average Implants per Pregnant Female - Malathion 54 Average Dead Implants per Pregnant Female - Malathion 55 Chl--Square Test of the Death Index - Malathion 56 Number of Dead Implants per Total Implants - Malathion . . . . 57 Chi-Square Teat of the Fertility Index - Parathion 58 Average Implants per Pregnant Female - Parathion 59 Average Dead Implants per Pregnant Female 60 Chi-Square Test of the Death Index - Parathion 61 Number of Dead Implants per Total Implants - Parathion . . . . 62 Chi-Square Tast of the Fertility Index - Parathion-Methyl. . . 63 Average: Implants per Pregnant Female - Parathion-Methyl. . . . 64 Average Dead Implants per Pregnant Female - Parathion-Methyl . 65 Chi-Square Test of the. Death Index - Pp.rathion-Methyl 66 Number of Dead Implants per Total Implants - Parathion-Methyl. 67 Chi-Square Test of the Fertility Index - Quiritozene (PCNB) . . 68 Average Implants per Pregnant Female - Quintozene (PCNB) . . . 69 vi �43 44 45 46 47 48 49 50 Average Dead Implants per Pregnant: Female Qulntozene (FCWB) .... Chi™3quare Test, of the Death Index - Quintozene (PCNB). . . . Number of Dead Implants per Total fmplants Qulntosene (PCND) Chi-Square Test of the Fertility Index - Phorate Average Implants per Pregnant E'.--,wile - Phorate Average Dead Implants per Pregnant Female - Phorate Chi-Sqisare Test of the Death Index -• Phorate Number of Dead Implants per Total Implants - Phorate 70 71 72 73 74 75 76 77 UDS ASSAYS 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation. DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay of Monocrotophos of Monocrotophos with of Bromacil of Bromacil with of Cacodyllc Acid of Cacodyllc Acid with of Captan of Captan with of Chloropyrifos of Chloropyrifos with of Dlnoseb of Dinoseb with of DSMA of DSMA with of Fenthlon of Fenthiori with of Folpet of Folpet with of Azinphos-Methyl of Azinphos-Methyl with of Malathion of Malathion with of Methomyl of Methomyl with , of Monuron vii 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 �76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation. ... DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay .of with Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of with Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation Monuron with MSMA MSMA with 1 Parathion Parathion with Parathion-Methy 1 Parathion-Methyl Qulntozene (PCNB) Qulntozene (PCNB) Fhorate Phorate with Simazine Simazine with Trifluralin Trifluralin with 103 104 105 106 107 108 109 110 Ill 112 113 114 115 116 117 MICROBIOLOGICAL ASSAYS 91 92 93 94 95 96 9? 98 99 .100 101 102 103 In Vitro Assays with Salmonella ^phjLmurium 118 Results" of Assays with Escerichia coli ~WP2 138 Mlcrobial Inhibition in~Egcerichia coll and Bacillus subtilis . ... 7~"."".. 148 .In Vitro_ Assays with Saccharomyces cerevisiae D3 Monocrotophos 150 In Vitro Assays with Saccharomyces Cjereyisiae D3 "Bromacil T .." . Y W "". " ' V 151 In Vitro Assays with .Saccharomyces cerevisiae D3 Cacodylic Acid. . . . . V ."."". ~. ."" T 152 1$. XiS,r,°- Assays with Saccharomyc.es .cereyj.si.ae D3 Captan 153 In Vitro Assays with .Saccharomyces cerevislAe D3 Chloropyrifos 154 _In Vitro Assays with Saiccharoiir/ces £ere\rlsiae D3 Dlnoseb 155 In Vitro Assays with Saccharoniy_ces. cejrwisiae D3 - DMSA . . . 156 In y_it:r<3 Assays with Saccjaromyces cerevisiae D3 Fenthlon ' 157 Iii Vitro Assays with jJaccharoiuyces cereyisiae D3 Folpet "."." . . " " . " . . .". . 7 158 In Vitro Assays with Saccharomyces. c.ej.evlsi3.?. D^ "Azinphios-Methyl . ."". .* - ". .""".".". ~~~~'~~ 159 In y_ltro_ Assays with Sa^cjtonniijrces cerevlslae^ D3 Malathion ".""".""."'. "7"."". ."".' ."".' 160 viii i �105 106 ^°7 108 109 110 111 112 113 114 Tn Vitrp_ Assays with ^ccharpmyces cer_eyiaiae D3 Methomyl ~. , In Vitro Assays with S&ircharqmyces £ereyisiae D3 Moiuiron , Jn Vitro Assays- with Sat^charqmjrces cerevlsiae D3 - MSMA . . . In Vitro Assays with Saccharom^cea cerevlBlae D3 Farathlon ~."".".. . . ." .7 In Vitro Assays with jtaccharomyces cerevlsiae D3 "" Parathlon-Methyl. . .".'. ~.~ .""."'.""." "i ~." "'. ."" In V_it.ro Assays with jJaccharomyces cerevlgiae D3 Quintozene (PCNB) . . . 7 . . ~ In Vitro Assays with 5a.ccl»arpmyces_ c_ereyl3iae_ D3 " Phorate T . . . . '." . ".". " In Vitro Assays with Saccharomyces cerevisiae_ D3 Simazine In X*-tr9 Assays with Saccharqmyces cerevirsiae D3 Trifluralin . , , . . " " . . . . " . T " " ' . " . " " " " " . ' ..' In Vitro Mutagenf.ais with .Salmouella tyjphlinur_ium. . . . . . . ix 161 162 163 164 165 166 167 168 169 170 �ACKNOWLEDGMENTS Stanford Research Institute wishes to thank the EPA project officers for their guidance and assistance during the course of this project. Dr. Robert E. McGaughy, Office of Research and Development, Washington, D.C., was Project Officer from the beginning of the project in June 1974 until July 1975. Project responsibility then was transferred to Dr. Ronald L. Baron, Health Effects Research Laboratory, Research Triangle Park, North Carolina, until January 1976. At that time, Dr. Michael D. Waters, now Chief of the Biochemistry Branch in the Environmental Toxicology Dlvlsi.cn, Health Effects Research Laboratory, Research Triangle Park, North Carolina, became Project Officer. Dr. Waters has continued in this project responsibility to the present. �INTRODUCTION The Federal Insecticide.. Fungicide, and RodenticJ.de Act designates Lhe Environmental Protection Agency as the governmental body responsible for the safety of all pesticides used in the United States. More recently, the Federal Environmental Pesticide Control Act (PL 92-516) strengthened EPA's regulatory responRihi.llt.les in the area of pesticides to include, ttitra- as well as Inter-state commerce. To be federally registered, a pesticide must h*ve beoii determined not to be hazardous to health or to the environment when used according to its labeling restrictions. Thus, relative to new law as well as to specific directives included in Public Law 9.1-135, 1973, EPA now is conducting a thorough review of rhe implications of using alternate chemicals, including older registered pesticides, for pest control. Ln the. pesticide review process, EPA emphasizes development of scientific criteria for evaluating the safety of compounds substituted for those penticides found to be hazardous. In addition to reviewing and evaluating the literature on pesticides and maintaining liaison with Industry and acadcmia, Che strategy program Includes laboratory studies to obtain additional data. One of these laboratory programs is directed toward gathering mutagenesis data on a selected -lumber of compounds. EPA's program is timely and responsive to one of the recommendations included in the President's Scientific Advisory Committee Report of September 1973, Chemlc a_ls_ and Health. In that document, the Committee recommended that "Regulatory agencies should take steps to insure that new scientific data raising the possibility of new or extended hazards from chemicals in use are subject to careful process of scientific, review for me.rir. interpretation." Development of methods for evaluating the rautagenic hazard of chemical compounds has advanced markedly in the last few years. In contrast to the undefined empirical tests used a short time ago, procedures now �available can detect chromosome breaks and other genetic changes caused by chemical stress. Mutant strains of microorganisms In cell culture and mammalian flbroblast cells In tissue culture are effective In vitro systems for .reliable detection of presumptive gene mutations, whereas the mammalian dominant lethal test is a recognized test for the assessment of chromosome damage to germinal cells.' Today many pesticide chemicals in commercial use have not been investigated adequately for their mutagenlc has:ard. With the public's increasing concern about possible pollution of our environment by chemicals, the widely used pesticides must be evaluated. In this project, SRI used test methods that are appropriate for these evaluations and that are in use by the scientific community. Under contract to EPA, SRI examined 20 pesticides for mutagenic activity using a combIntation of in vivo and in vitro mutagenicity assay systems. The 20 pesticides tested and their sources are listed in the ' following two tables. The assays used were the dominant lethal test in mice (only ten compounds); unscheduled DNA synthesis (UDS) in human fibroblasts (WI-38 cells); reverse mutation in SaJLmojiellji tvj^^miirium strains TA1535, TA1537, TA1538, and TA100 and in Esch^erlchia coll VF2; mitotic recombination in the yeast ^Sccharomyjce^ cerevisiae D3; and preferential toxicity assays in DNA repair-proficient and -deficient strains of IS. coll (strains W3110 and p3478, respectively) and Bacillus aubtjlls (strains H17 and M45, respectively. Based on positive responses in both Tier I (in vitro test) and Tier II (Droaophila) mutagenic studies, It was recommended that a heritable trenslocation test (Tier III) la the mouse be conducted �to further assess the matagenic potential of Captan. The results of these further studies are reported as Appendix A. The experimental procedures and results for the mammalian dominant lethal test, the UDS assay, and the microbiological assays are. described in the separate sections that follow. �IS VIVO MC IN VITRO MUTAGENES1S: SUMMARY DATA FOR E?ft PESTICIDES Positive Response, +; Negative Response, - Pesticide Mouse Dotiisanc Lethal* Salconella typhJJiuriiaat (His"1" Reversion) -HA +MA EacSerichia egli WF2 ( r '' Reversion) Ty1 -MA +MA Mocccrotophos bromacil Cacodylic. Acid Captan Chlor?yrlfos Dnos^S DbHA f<jnt!-.ion foipst Azlnphos-sechyl Halathion Konuron MSHA ?arathioE Parachlon-uechyl Qulntozene (FCNB) Phorate Sinazine Trifluralia • Only ten pesticides were tested by the dominant lethal procedure. t See page 170. T Marginally positive. Saecharomyces cerevlsiae (Mitotlc Recombination) -HA +MA Escheriehia coll (Selative Toxicicy) Bacillus surtills (Relative Toxicity) UDS (DMA Repair) -MA +MA �TWENTY PESTICIDES EVALUATED 3Y SRI FOR MUTAGENIC ACTIVITY Common Name* Monocrouophos Bromacil Cacodylic Acid Cap tar. Chiorpyrifos Dinoseb DSMA Fenthion Folpet Azinphos-methyl Malathion Me thorny 1 Monuron MSMA Parathion Par a t h ion-iae t hy 1 Quintozene (PCNB) Phorate Simazine Trifluralin Tradt Name of Compound Tested Manufacturer Batch or Lot Number Batch H, 9-SCL-77 E.I. DuPont de Nemours T80619/40 Hyvar Phyton 138 Phytar Ansul Chemical Company 5X640 Chevron Chemical Conpany Orthoslde 406 Dow Chemical Company MM-1 114-1 Dursban (603-D1) MM 200554 Dow Chemical Company PT emerge Ansul Chemical Company 8100 Ansar Chemogro 4-15-2026 Bay t ex Chevron Chemical Company SX579 Phaltan Chemogro 411-022S Guthlon American Cyanamid Company 40216006.300 Malathion 6602-82 E.I. DuPont de Nemours Latmate T-40817-20 E.I. DuPont de Nemours Telvar 170 H.C. Ansul Chemical Company Ansar Monsanto Chemical Company AD 1236 Niran Methyl Parathion Monsanto Chemical Company AD 0659 Technical Olin Mathieson Chemical Terrachlor Corporation American Cyanamid Company MC85 Thimet FL-740846 Primatol Ciba-Geigy Chemical Co. X-26290 Eli Lilly & Company Treflan Azodrin-5 Purity (%) Supplier 55.0 Manufacturer Shell Chemical Company 95.9 65.6 Technical 98.8 Battelle Baccelle Sattelle Battelle 97.7 Battelle Battelle Battelle Battelle Battelle Battelle 80.1 96.0 Technical Technical Technical 99.0 97.0 58.4 99.0 80.0 99.0 * Common name as approved by the International Organization for Standardization. 97.7 97.7 Battelle Battelle Battelle Battelle Battelle Battelle Battelle Battelle Battelle �DOMINANT LETHAL TEST IN THE MOUSE General In the dominant lethal test, the ten compounds under Investigation were fed in the diet to proven male breeder mice for 7 weeks. After this period, each male was mated with two adult virgin females for 7 days; these females were then replaced by two others for another breeding. The sequence was continued fof 8 weeks. This procedure emphasizes possible mutagenic effects on the male sperm, the normal female acting as a carrier to reveal in her offspring abnormalities that may have occurred in the male. We evaluated effects by examining the condition and state of fetal development during the middle to latter stages of gestation. Experimental Animals and Chemicals Adult ICR/SIM mice from a closed, random-bred colony were used for the acute toxiclty and maximum tolerated dose determinations as well as for the dominant lethal assay. These male and! female mice were supplied by Simonsen Laboratories, Gllroy, California. The males were 3- to 4-raonth-old proven breeders, and the females wore 10- to 12-week-oid virgin stock. At the direction of EPA, the Battelle Columbus Laboratories obtained the pesticides from the manufacturers and subsequently provided SRI with allquots for the studies reported here. Each pesticide was a "technical" grade product (or equivalent) and was provided in sufficient quantity for us to complete all aspects of the experimental program. Excess supplies were refrigerated or frozen, should they be needed fcr future reference. �Wo Investigated the solubility of each compound using water, propylene glycol, polyethylene glycol, corn oil, or carboxymeshylceLlulose to determine the. most appropriate vehicle for administration. Compounds were administered orally, by gavage for Che acute toxiclty (LD,-g) determinations, and via the diet Tor the maximum tolerated dose and dominant letha] studies. Determination of Acute Toxic ity_ Although acute toxiclty information on .some of the compounds was available in the literature, we conducted confirmatory tests on all to obtain an LD under our Laboratory conditions and for the 1CR/J1F.M strain of mouse. If no data were available, we conducted a preliminary range-finding test, followed by a determination of the oral Lft^Q. Maximum Tolerated Dose Study Based on the acute toxicity data and available information from the literature on dose levels known to cause adverse responses when administered in the diet, several dose, levels were selected and administered in the diet to adult male mice for 2 weeks. Treated males then were caged with two adult virgin females each for 7 days; these females were replaced by two others weekly for 2 weeks. The females were examined daily for the presence of vaginal (mating) plugs. At midterm of pregnancy, the females were sacrificed and examined for total implants, as well as for early and late fetal deaths. For this work, we defined a maximum tolerated dose as that dietary level which may produce up to a 20% weight loss, mild but transient clinical signs, no inhibition of breeding performance, and no mortality. Thus, these initial studies provided Information on changes in body weight, acceptability of the diet, clinical signs, mortality, and breeding performance. Treatment Levels For the dominant-lethal study, three dose levels were administered. The highest was the maximum tolerated dose or 5 g/kg (a maximum level �agreed on by EPA and SRI), whichever was lower. The intermediate and lower dosages were one-half and one-quarter of the highest dose, respectively. Administration of the Compounds Each pesticide was fed in the diet to adult male mice for 7 weeks. An appropriate amount of compound initially was dissolved or suspended In corn oil; then the compound-oil concentrate was added at a level of 3% to a finely ground commercial diet of known composition. The use of corn oil assured even distribution of the compound and prevented stratification of the tost material in an otherwise dry diet. Diets were pre- pared at 2-week Intervals and were refrlgeratwd at 4°C until fed to the animals. Fresh diet was placed in the. feed containers every other day to minimize the loss of compound through instability or volatility. Two reference control groups were included in this project. One was run at the beginning of each of the two dominant lethal series, five pesticides being run concurrently. In this manner, reference breeding and implant data were obtained at. two time periods, as was information on each shipment of research animals. Males In these groups were fed a finely ground commercial diet supplemented with corn oil at 3%. Control groups were treated in the same manner as the compound test groups. Two positive control groups were run concurrently wiLth each of the two series of five pesticide tests. For these groups, the known mutagen trlethylenemelamine (TEM) was administered as a single intraperitoneal inject. Ion of 0.2 mg/kg approximately 2 hours before the first mating. A commercial pelleted diet was available at all times. Each control and experimental test group contained 20 adult male mice. Af: the end of the 7 -week compound treatment period, each male was allowed to brer.d with Two virgin females over a period of 7 days. Femal.es wfico replaced weekly for 8 wpcks. 8 �Females wert sacrificed at midterm of pregnancy. A completu necropsy waa performed to determine If an Intercurrent infection was present; such a condition can induce preimplant&tion loss and early fetal deaths. At sacrifice, each female was scored for early fetal deaths, late fetal deaths, and living fetuses (all of which provide a LotaL implant score). The following parameters indicate effects in dominant lethal studies: Total implants (live Fetuses plus early and late fetal deaths), total <;.cad (early and late I'tif.al deaths), and dead implants per total Implants. Total imp]ants and dtad Implants were analyzed for significance by the t-test. Th« Jndex of dead implants per total implants was analyzed statistically by the t-test on arcsine- (or angular) transformed data, as described in Experimental Design (Theory_ and Apjgllcajclon) . index was computed for each female. Other parameters analyzed were the fertility and death indices. Rcsults and_ Discussion Single-dose oral acute toxicity data are as follows: Compound LD.50 Monocrotophos 17 mg/kg Bromacil 3.04 g/kg Captan > 15 g/kg Folpet > 10 g/kg Azinphos-methyl 15 mg/kg Malathion 1196 mg/kg Parathion This ' 17 mg/kg I'arathion-methyl 39 mg/'«.g Qulntozcne (FCNB) > 10 g/kg Phorate 6.59 mg/kg �After evaluating the acute toxicity data and those from subsequent maximum tolerated dose studies, we selected the following dosage levels for the dominant lethal studies: Compound Treatment Levels (E/kg_J: Dlet __ Monocrotophos Bromacil Captan Folpet Azinphos-methyl Malathion Parathion Parathion-methyl Quintozene (PCNB) Phorate 15, 30, 60 1250, 2500, 5000 1250, 2500, 5000 1250, 2500, 5000 20, 40, 80 1250, 2500, 5000 62.5, 125, 250 20, 40, 80 1250, 2500, 5000 5, 10, 20 Throughout the experiment, the biological criteria used to evaluate mutagenic effects in the mouse showed no consistent responses that could be attributed to treatment. Although we found occasional statistical differences between control and compound treated groups, they were random and did not suggest a time or dose-response effect. Summary data on the fertility index, implantations per pregnant female, dead implants per pregnant female, death index, and number of dead implants per total implants are presented by compound as follows: Tables 1 through 5, Monocrotophos; Tables & through 10, Bromacil; Tables 11 through 15, Captan; Tables 16 through 20, Folpet; Tables 21 through 25, Aalnphos-m«thyl; Tables 26 through 30, Malathion; Tables 31 through 35, Parathion; Tables 36 through 40, Parathion-Methyl; Tables 41 through 45, Quintozene (PCNB); and Tables 46 through 50, Phorate. Two copies of a description of the statistical analysis procedures used for dominant lethal tests -and computer printouts of the raw data and the statistical analyses are on file with the current Project Officer, Dr. Michael D. Waters, Environmental Toxicology Division, Health Effects Research Laboratory, EPA Environmental Research Center, Research Park, North Carolina 27711. 10 �The following statistical procedures were used: Chl-square cest of the fertility index; Armltage test Cor a linear trend in proportion for the fertility Index based on dose levels, based on .logarithms of the. dose Levels, and based on dose levels including the control group; t-test of the number of implantations in pregnant females; Regression fits of implantations on dose and log dose and with and without control group included; t-test of the (Freeman-Tukey transformed) preimplant.al.ion losses In pregnant females; t-test of the number of dead implants; Chl-square test of the death index; Arm.itage tost for a linear trend in proportion for the death index, based on dose levels with and without control group included and based on logarithms of Che dose levels; Probit analysis of the proportion of pregnant females with one or more dead implants; t-test of the (Freeman-Tukey transformed) number of dead implants (dead implants/total implants); Control group analyses of variances for number of pregnant females, number of implantations per pregnant female, preimplantation loss per pregnant female, number of dead implants per pregnant female, ratio of dead implants to total implants per pregnant ferwle; and t-test of the number of corpora lutea in pregnant females. Careful review and statistical evaluation of the data show that folpet, captan, parathion-methyl, parathion, phorate, malathion, bromacll, monocrotophos, quintozene (PCNB), and azinphos-methyl are not mutage.nLc in the. mouse by the dominant lethal test. �MAMMALIAN W VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS General Many mutagenic and carcinogenic agents have been shown to induce unscheduled DNA synthesis (UDS) in an ill yitjro tissue culture system of mammalian coll:-;. UDS is a form of mammalian repair synthesis that involves at least two processes. The first is Interaction of the agent with DNA, resulting in damage of the DNA. The second, which follows, is incopor- atton oC micleotldes to repair the DNA. UDS may be considered a fal-cly universal system because it occurs In a wide variety oi' mammalian cell types and because it has been observed in all stages of the- cell cycle (Gg, GI, G?, and M) other than S_, the normal DNA synthetic phase.?l3 (UDS is not observed during S_-phase be- cause tin; high level of Incorporation of nucleotides during the scheduled DNA synthesis obscures the relatively Low level of incorporation of nucleotide.s during unscheduled DNA synthesis.) An additional feature of UDS is that It may detect a level of DNA damage higher than that revealed by examination of chromosomeal aberrations'4 because some DNA repair results in little or no detectable change in chromosome morphology. For each compound tested, an in vitro metabolic activation system should be incorporated for a parallel series of UDS assays since some compounds may be ineffective in producing DNA damage unlpss they arc first activated by a microsomal preparation from a mamiiiai.i.-'1 P liv-M homoftenatc. The- ['DS rystrm we have developed Is unique in that, at the end or fvich assay, ON.', in axtractt'.d from human diplold flbroblasts (WI-38 cells) so that, the extent of repair may be expressed per unit of DNA. We have found that this. UDS assay system affords sensitivity and precision without sacrificing efficiency or economy. Under separate contact, NCI approved our use and validation of this system for the prescreening of chemical ;arclnogpns. With the approval of the EPA project officer, we used this system for testing the* 20 substitute pesticides, with and wi thou l wo i :ibo 11 c. ac t i va t ion. �Experimental Coll Culture WT-38 cells grown In T-25 tissue culture flasks were used Cor the UUS assays. Kepllcdte cultures of chase cells were initiated in Eagle's Hrisal Medium (BME) containing 10% (v/v) fetal calt serum and aureomycin, an antibiotic, specific for PPLO*. For 1 to 2 weeks preceding rlie UDS assays, the cells were grown in medium containing 0,5% serum. This pro- duced contact-Inhibited cells in synchronous cultures in the 01 phase of the mitotic cycle. To reduce further the possibility of incorporation of -'H-TdR by an occasional S-phase cell that might escape the contactinhiblLJon synchrony and thus obscure measurements of UDS, the cultures 2 were prelncubated for 1 hour with 10" M hydroxyurea (HU) before each assay, and 10""' M HU was iidded during each subsequent step of the assays. Di 1 u t ion o f Cpmpou nds Chemicals to be tested were made up immediately before use and were dJLuted in appropriate solvents (water, echanol, or DMSO), the final concentration of solvent being one that did not produce a cytDtoxic effect after repeated testing. Soniflcation and pH adjustments were used to ensure maximum solubility or even suspension of the stock solutions of the compounds. The highest concentration was diluted further in solvent and then in culture medium to give several log dilutions of each compound. All compounds were in apparent solution and within the physiological pH range when tested, except as otherwise noted in the tables. Controls The positive controls were 4-nitroquinoline-N-oxidc (4NQO), a compound that induces UDS in the absence of a metabolic activation system, and dimethylnitrosamine (DMN), a compound that induces UDS only with metabolic activation. The negative controls were the solvents diluted in culture medium. * As, an additional check against the presence of PPLO, which could incorporate tritlated thynddine (3H-TdR) and thus obscure measurements of UDS, stock cultures were analyzed monthly for the presence of PPLO. The results of these analyses were consistently negative. 13 �UPS Assays The contact-inhibited WI-38 cells were incubated at 37°C with log dilutions of the substitute pesticides and with 1 uCi/ml of 3H-TdR (sp act, 6.7 Ci/nungle) . For testing in the absence of metabolic activation, the cells were exposed simultaneously to the substitute pesticide and to 3H-TdR for 3 hours. For testing with metabolic activation, the cells were exposed to the substitute pesticide, to 3H-TdR, and to 500 mg/ml of the 9000 x jj supernatant' fraction of a liver homogenate from adult male. Swiss-Webster mice, with appropriate cof actors,* for 1 hour; then the cells were incubated with only %-TdR for an additional 4 hours. The shorter exposure time for metabolic activation testing was used to preclude cytotoxic effects of the liver homogenate preparation. Both approaches included a postincorporation incubation with un labeled thymldine. UNA was extracted from the cells by a modification of the PCA-hydrolysis procedure;5 one aliquot of the DMA solution was used to measure the DNA content, after reaction with diphenylamip.e,6 and a second aliquot was used for scintillation counting measurements of the extent of incorporation of 3H-TdR. Results were expressed as incorporated per unit of DNA and were compared with the background rate of incorporation. We have defined ns an acceptable assay one in which the response of the positive control, compound is predicted, within the 95% confidence limits, by regressions of average dpm/pg DNA VEJISUS average dpm/ug for background. The regressions that follow are based on data that we have acquired in previous testing: Typti oi; Testing Without metabolic activat 1 -n With M»-cabjllr activation ____ R*yjT.£?-§lPJ?-+. _________ YI - 696 + 17.45 (X)* Y, - 263 + 1.83 (X)* Sample ! • : - -£n). **£ 48 13 Correlation Coefficient (r) 0.7668 0.9639 *Nicotinamidc, 3.05 mg/ml; glucose-6-phosphate, 16.1 mg/ml; 5.08 mg/ml; NADP, 0.765 mg/mi. "^Regressions over a range of background dpm/ug DNA of 0 to 450. *Y! - Average dpm/ug DNA for 10~5 M 4NQO (positive control). Y^ = AVI rate dpoi/ng DNA for 5 x 10":? M ! M (positive control). )N X = Average dpm/ g DNA for background (negative control). �If the observed avecage level of incorporation for Lh<? po&JtIve ronrr"I compound is ouCalrle the 95% confidence limits of uhe regression, we assume that, some variation has occured in the experimental procedutr-.i and repeat the test. Interpretation of Results^ In a report to the National Cancer Institute, ' we presented the results of tests performed without metabolic activation on 40 compounds of known carcinogenlcity. We have analyzed these results using either the parametric One-Way Classifiction Analysis of Variance or the nonparametric Rruskal-Wallis One-Way Analysis of Variance, depending on which was more appropriate.* At f.he 99% confidence limits, all the ultimate carcinogens significantly elevate the Incorporation of JH-TdR into the DNA. The itoncarclnogonic compounds, with one exception, fail to elevate significantly the incorporation of •'H-TdR at r.hiu level of confidence. Thus, the 99% confidence Units of these statistical analyses apparently can be used with reasonable accuracy to predict the biological significance of the response to a chemical. The number of compounds we have tested with metabolic activation is insufficient to establish a correlation between statistical significance and biological significance. Therefore, we assumed that the 99% confid- ence levels of the analyses of variance used without metabolic activation also apply for test'ng with metabolic activation. Resu1ts_and Discussi on Tables 51 through 90 present the results of the UDS testing, with and without metabolic activation, of the 20 substitute pesticides. Tables 51 an) 57, the DNA repair synthesis assays of nonocrotophos, include detailed summaries of the cell culture and experimental conditions for these assays. The assays presented In the following tables (53 through 90) were conducted under similar conditions. In routine testing in the *If there is reason to believe that tho variances of each of the treatments in a test are equal (I.e., Bartlett's test of the variance is negative), the parametric analysis is the appropriate one. If the variances are not equal, the nonparamtitric analysis is the appropriate one. �absence of metabolic activation, six samples each are used for five log concentrations of each test compound and for the negative and positive controls. However, because of the expense of the metabolic activation preparations, for all compounds except bromacil we tested three replicate samples in the presence of metabolic activation and used three concentrations of the rest compound (selected on the basis of the testing without metabolJc activation). Based on the criteria for positive responses, we observed significant Increases in unscheduled DNA synthesis In the absence of metabolic activation afi.er exposure of the cells to only two substitute pesticides, monocrotcphos and parathion. In the presence of metabolic activation enzymes, KJgnlv i.cantly increased UOS was detected for five substitute pesticides: monocrotcphoa, captan, folpet, azinphos-methyl, and monuron. Co-nr-art-d with those of negative controls, the levels of 3H-TdR incnrpuration were greatly reduced in the absence of metabolic activation at the highest concentrations tested for captan, folpet, azinphos-methyl, and mouuron, the same four compounds that induced UDS only in the presence of metabolic activation. The reduced levels of incorporation may be interpreted as cytotoxlc effects or as inhibition of repair caused by the highest concentration of the test compounds. A similar effect was observed in the presence of mefabollc activation for only one compound, captan, and this was observed at a higher concentration than had been tested without metabolic activation. SLich et al.e have discussed the problem of cyto- toxlcHiy and possible inhibition of DNA repair systems by some chemicals and liciv-: <jtr-;ns«d that , whereas such factors may obscure measurements of UDS, oin.:ii a close relationship exists between concentrations that induce UDS and n«ct-.iNat ions thiit are cytotoxic or that inhibit repair. llcr.ausc of the cy to toxic or Inhibitory effects of the substitute pesri i.lilt's, ).l should not; hv: assumed without further testing that monocrotophos and parathion would be carcinogenic without metabolic activation or that the other four substitute pesticides that induced UDS in the presence of metabolic activation are procarcinogens. The positive UDS ti>sul£>-, indicate that chnse Filx substitute pesticides should be tested moi:e iMinnsively, with the testing to Include evaluations of the effects of cli: s.e •: Jiemiculs In in yfvo Moat's*; j . �MICROBIOLOGICAL ASSAYS General SRI examined twenty pesticides for mutagenicity by in yitjro microbiological assays with Salmonella ty£him_uriuM_ (TA1535, TA1537, TA1538, TA100), Escherichia coll WP2, repair-deficient: and -proficient strains of Bacilly_s subtilis and E. coil, and with the yeast Saccharomy_ces cereylsiae D3. An Axoclor l25<V™3timulated, rat-liver-hoim>gfinate metabolic activation system was included in each procedure, except the relative toxlcity assays, to provide metabolic steps that the bacteria are either incapable of conducting or that thfy do not carry out under the assay conditions. The purpose of this study WAS to determine whether the compounds elicited a mutagenic response In. microorganisms. The assay procedure with S_. ^phlinurium has been proven to be 85 to 90% accurate in detecting carcinogens as mutagens, and it has about the q same accuracy in identifying chemicals that are not carcinogenic. The assay procedure with S. cerevisiae is about 5C% accurate In detecting carcinogens as agents that increase mltotic recombination. E, coll WP2 and the microbial sensitivity assay are two additional methods of detecting tnutagnns. The combination of these four assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. 17 �Experimental Salmonella, typhlmurium Strains TA1535, TA1537^ TA1538. and TA100 The S. ^yphjLmurjLiim strains used at SRI were obtained from Dr. Bruce Ames of the University of California at Berkeley.10'12 All are histidine auxotrophs (hls~) by virtue of mutations in the histidine operon. In addition to the mutations in the histidine operon, the indicator strains have mutations in the lipopolysaccharide coat Crfa ) and deletions that covor a gene lavolvod in the repair of uv damage (uvrB~). The rfa~ mutation makes rhe strains more permeable to large molecules, thereby increasing their sensitivity to these molecules. The uvr_B~ mutation decreases repair of some types of chemically damaged DNA and thereby enhances Hei'ssit Ivity to some aiutagenlc chemicals. Strain TA1535 is + reverted to histidine prototrophy 0»is_ ) by many mutagens that cause basepair substitutions. t'rameshlft mutagens. Strains TA1537 and TA1538 are reverted by many TA1537 is more sensitive than TA1338 to mutation by some acridine and benzanthracenes, but the difference, is quantitative rather than qualitative. TA1GO is derived from TA1535 by the intro- duction of the R factor plasmld pKMlOl.13 The introduction of this plasmid, which confers ampleillin resistance to the strain, greatly enhances the sensitivity of the strain to some base-pair substitution mutagens. We have shown that mutagens such as benzyl chloride and 2-(2-furyl)-3-(5-nitro-2-furyl) acrylainide (known as AF2) can be detected in plate -rtssays by TALOO but not by TA1535. The presence of this plasmid ill so !r.;jk<vj -."XT'I, T.MOO sensitive to some frameshift mutagens—e.g., 1CR-191 , ben£o(.i)pyrene, aflatoxin B,, and 7,12~dlmethylbcnz(a) anthracene. A l l r.!i*' indJrat.or strains arc stored at -80°C. For each experiment, an inoculum From frnsen stock cultures is grown overnight at 37°C in a nutrLftnr >•":<'-\i consisting of 1% tryf.tone And 0.5% yeast extract. Afterstationary overnight growth, the cultures are shaken for 3 r.o 4 hours to ensure, optimal growth. Each culture is checked for sensitivity to crystal vjul ;t. Th-; presence of the rfa mutation makes the Indicator strnLns .'.;*•!.fit. Ivc to this dye, whereas tha parent strain, rfa , is not senHlt: ivt to the dye. However, the mutation is reversible, leading to �the accumulation of rtji cells in the culture. Therufore. t.b: ,.••)!' must be tested routinely to ensure their sensitivity to crystal violet. Each culture, also is tested by specific mutageus known to revert each tost strain (positive controls). To a sterile 13 x 100 mm test tube placed in a '43°C heating block, we add in. the following order: Assays in agar (1) 2 ml of 0.621 agar* ( ) 0.1 ml of indicator organisms 2 (3) 0.5 ml of metabolic activation mixture (optional) (4) Up ro 100 pi oL a solution of the test chemical,*'11 For negative controls, we use steps (1), (2), arid (3) (optional) and 100 jjl of the solvent used fcr the test chemical . This mixture is utirred gently and then poured onto minimal agar plates.i After the soft agar has set, the plates are incubated at 37°C for 2 days. The number of his revertants (colonies that grow on plates lacking a sufficient- amount of histldine to support colony formation) are counted and recorded. Some of the revertants are routinely tested to confirm that they are hls_+, require biotin, and are sensitive, to crystal violet (r_fa~). Escherichi_a_ col 1 _WP2 The E. coll WP2 (iiyr_A~) used in this project, was given to us Dr. D. McCalla. ' by \ procedure similar to the one used with Salmonella is used to measure the reversion of WP2 to tryptophan independence. How- ever, instead of containing a trace of tryptophan in the top agar, the minimal agar plates contain 1.25 g of oxoid broth per liter to provide * 0.6% agar contains 0.05 mM histldine and 0.05 iriM biotin. t Minimal agar plates consist of 15 g of agar, 20 g of glucose, 0.2 g of MgSO,.7 H20, 2 g of citric acid monohydrate, 10 g of K2HP04, and 3.5 g of NaHNltyPO^.H;>0 per liter. **Solvents used as appropriate include: water, dimethyl sulfoxide, cthanol, and ben/.ene. 19 �Che trace of tryptcphan required for enhancement of any mutagenic effect of the test chemical. Alternatively, reversion of the mutated tryptophan gene, WP2 may undergo a forward mutation in a tryptophan tRNA gene to obtain tryptophan independence. We do not distinguish experimentally between the true revert ants and the phenotyplc revertants (although the latter tend t.i- form smaller colonies). _H17/_M45 Thi> _:•__. CA.U strains W3110 and p3478 were obtained from Dr. H. Rosiprikrai-.? . ' Strain p3478 is a polA" derivative of strain W3110. It carries .! single, revertable mutation in a gene for a DNA polymerase; " ~t Gross dn<l Gross showed that this mutation Ls involved in DNA repair synltiesj :.' This uiutatlou increases the sensitivity of strain p3478 to chemicals that lead to alterations (damage) of the DNA. Therefore, we ran assay for chemicals that damage DNA by comparing the relative sensitivity of the two strains (p3478 and W3110) to the test chemical. The IJ. subtilijs strains HI 7 and M45 were obtained from Dr. Kada. 18 Strain H17 (rec+) is derived from H17 but is deficient in the genetic recombination mechanism necessary to repair DNA damage. Cells deficient in r.hJs repair mechanism are killed more oasily by chemical mutagens than arc wild-type cells (rec+). If the chemical is toxic to rec~ cells, b:H at the same concentration is not toxic to rec_+ cells, the chemical ;;V(. 'ftt-1 v i s a i:;utap.en. fiv.i--- •l-iT*- from frozen stocks are grown overnight in nutrient broth* :it '<•"< .,:'•; .•.'irtViog. V<: ?. ml of nutrient broth containing 0.6% agar 1s Tidod r'. i mi ot the Lest culture. The suspension is mixed and poured cnL--i .1 1 '.'. , containing nutr i.<.=ni. broth and 2% agar. After the soft agar has solidified, a sterile filter disc impregnated with the test chemical is placed in the center of the plate. The r:hve.--; •, : .- abated at 37°C for 15 hours, and the width of the zone of * Trypr•.-'!»•, 1%, and 0,5% yeast extract, supplemented with 5 jig of tlij-'tii v-v's:1 to pruvent selection of t:liy+ revcrtants. 20 �!-oxLcir.y or Inhibition of growth is then measured. We usually must test several concentrations of chemical to detect accurately differences Ln i:he zones '••£ growth inhibition because higher initial concentrations load to steep concentration gradients that may reduce the differences in growth inhibition of the two strains. The positive control for this assay is 1 nil. of l-phenyl-3,3-dimethyltriazene placed on the disc. A zone of approximately 40-ram width is observed (52 and 61 mm, respectively). of chloramphenlcol placed on a disc. An additional control is 30 pg Equal zones of inhibition are oxpected in all four strains (approximately 30 mm) since the toxlcity of this chemical does not depend on a mechanism that leads to i)NA damage. All assays are performed at least three times. Saccharomyces cereyisiae D3 The yeast 3. cerevlsiae D3 is a dip]old heterozygous for a mutation ^n an adenine-metabolizing enzymes.*' Cells homozygous for this mutation produce a red dye when grown on medium containing adenine. Adenine- vcquiring homozygotes can be generated from the heterozygotes by mitotic recombination. nation. Many mutagens increase the frequency of mitotic recombi- Mitotic recombination is Indicated by the development of colonies with red pigmentation, and the degree of conversion to this pigmentad colony indicates the mutagenlcity of a compound or its on metabolite. The Saccharomyces test strain from the liquid nitrogen is grown overnight .it 30°C with aeration in 1.0% tryptone and 0.5% yeast extract. The cells are washed twJce In 0.067M P04 buffer (pH 7.4) and resuspended In the same buffer at a concentration of 10 cells/ml. The _ln vitro yeast mitotic recombination assay in suspension consists of 5 x 10 washed, stationary-phase yeast cells in 1 ml of 0.067M I'O^ buffer (pH 7.4) and 50 rag/ml of'the test chemical (or a fraction of the concentration required to give 50% killing).. Incubated at 30° for 4 hours. The suspension is After incubation., the sample is diluted serially in sterile saline and plated on tryptone-yeast-agar plates. 21 �Plates of a 10~^ dilution are incubated for 2 days at 30°C, followed by 2 days at 4°C to enhance the development of the. red pigment indicative oh" adenine-negative homozygosity. To detect red colonies or red sectors, we scan l.lie plates with a dissecting microscope at 10 x magnification. Plates of a 10 dilution are incubated for 2 days at 30°C for determi- nation of the total number of colony-forming units. The in Vitro yeast Itotic recombination assay in suspension with metabolic activation is conducted as above with the addition of the. metabolic activation system to the incubation mixture. ArocLor 125/t-Stimulated Metabolic_Actiyation System Some' •.•arcinoganic mutagens (e.g., dlmethylnitrosamine) are inactive unless they are converted to their active form by being metabolized. Ames et a I. ,21 hav? coderib«d the metabolic, activation systems we use. Adult mnle mice are given a single 500-mg/kg intraperltoneal injection of A poiychlorinated biphenyl (Aroclor 1254).^ Four days after the injection, the animals' food is removed. On the fifth day, the mice are killed. The liver are removed aseptically and placed in prewelghed, sterile glass beakers. The organ weight, is determined, and all subsequent operations to th« metabolic, activation step are conducted in an ice bath, [he organ is washed in an equal volume of cold, sterile 0.15 M KC1 (I snl/g of wet. organ), minced with sterile surgical scissors in three VO'MSK.' • ' O.l.S K.C1, and homogenized with a Potter-Elvehjem apparatus. 'fie h'jirn-v'-.i.it? Is centrifuged for 10 minutes at 9000 x £, and the. superi: ii .'.is;, i ; in .-"TfJ .ir.d stored J.n liquid nitrogen. To the postmito- ciu-niuia.k .jMp.v^nii'.f ate added MgCl2» KC1, glucose-6-phosphate, TPN, and (•odium oV'?spi!jr.e {pH 7,4). Results _and_ Dtscussion A.i i " 1 o«< ;>i>3! icides submitted to SRI for examination were tested at icj.--: (.hi>:c times in tho microbiological assays. 1 Hiifz'.ed ^"iii *. are an average cf those experiments. 22 The results pre- �T.'ib1'. 11 i> •-•.•;ii;. !K i r s i i l i 1 ; uf ag;ir with Snlmonol la _tyj»li1Lniui"iuin. • •> i . :. n i u g i v , 1 .i.^-.- v.i In this liisridlne reverse •:•'.• .; t l o i assay system, two pesl:icides—rnptan and folpct were hu.'tiig:-nii-. I'oi oach chemical, we observed an increase in the number ot his'-'.:! ine independent revertants on strains TA1535 and TA100 but not on Jiir.iiny. TA98, TA1537, or TA1538. These results suggest that these u«>st •:: ld<?.-. can alkyJate DNA, causing mutations of the base-pair substitution type. This conclusion is consistent wiLh Llie rjiitagbnic activity of these compounds in assays with E. coli WP2 (Table 9?), which is sensitive no base-pair substitution mutagons. Although liver homogcnate activation was not required for mutagcnic activity, the mut.igenic activity was enhanced somwhnt with activation .it some doses. A toxic effect: (reduction of the: number of mutants) wt;.i observed at 'doses of 100 pg ot" each compound. Table 92 presents I he results of jssiiys with E. roli WP2. Essentially, thi 1 results were i d e n t i c a l to (.hose- obtained with J>. fyp!.1H!!ui:'iU!!: TA1515 ,-md TAIOO; «-aptan and Tolpnt were mutagenic, but. none of the other pesticides was mutagenic. Table 93 piesents the results oE the assays for microbial inhibition in repair-deficient arid-proficient strains of _B. .su_b_tj_li_s and K. .cpj.i. Folpet, captan, chloropyrifos, and dinoseb all gave toxic zones that were larger on the repair-deficient strains than on the repair-proficLent strains, indicating a mutagenic response. Toxic chemicals that do not act by damaging DNA (e.g., chlorampheiiico]) should give equivalent zones of toxicity. However, many if not all nutagens damage DNA and, if the damage is not repaired, can result in cell death. Thus, a given ciinreiitration of mut.agon may be toxic for a repair deficient strain but not for a str.iin the effectively it-pairs its DNA. Tables 94 through 1 1 3 present the results of the assays fur m i t o t i r recombination ir Saccharomyras ccrevisiae D3. A p o s i t i v e - response in t h i s assay IK ind iru tc-d by an increase- of more llian three-fold in the .'ib'.olute number of mitoM'c reroiisbi runts per m i l l i l i t c r a.s w e l l as in lhi> relative nunibi-r of i n i l o l i c re< oi:ib iiicint s per 10 survivors. l-'olpei. �captan, monocrotophos, cacodylic acid, and azinphos-methyl increased mitotic recombination significantly and are considered positive by these procedures. Methyl parathion gave a marginally positive response. Our results indicate that 7 of the 20 pesticides examined give positive responses in one or more of the four microbiological assay ps o...••'J'srea. Although a mutagenic response in a microorganisms does r-i-'.na that a chemical is a mutagen in humans, the combination of four separate assay system greatly enhances the probability of detecting PO'-M Maily h&zardous chemicals. Folpet and captan are mutagenic in :>i •. : ou, assay procedures. Chloropyrifos and dinosftb are positive in U: I'.'crobial sensitivity. Monocrotophos, cacodylic. acid, and azinphos3>s-••••,••!. ;tre pofiitlve in the yeast assays. �DISCUSSION Of the 20 pesticides tested for mutagenic activity, 9 were clearly mutagonic in one or morr i.n vitro assays. Of these. 9, 2 ware mutagenic in all the in vitro assays, but none of them produced a dominant, lethal response in the mouse. In the Salmonella assays., these chemicals caused base-pair substitution mutations but not fraine.shi.ft mutations. The absence of activity in the dominant lethal assay may be due r.o a lack of sensitivity of the mouse to these types of compounds; for example, N-methyl-N'-nitro-N-nitrosoguanidine and other alkylating agent*: that cause base-pair substitution mutations do not all cause dominant lethality. Anotho.r explanation for the absence of activity may be flint these pesticides did not reach the gonadal tissues In sufficient amounts to cause a mutagenic event. None of the other 6 pesticides was mutagenic in all the in vitro assays. The combination of assays used in this program is one means of identifying those pesticides that may present a rautagenic health hazard. Those that show positive responses in several experimental systems should be evaluated more, thoroughly before they are substituted for ocher pesticides already considered as a risk to the environment. Also apparent is that no one assay system is uniquely capable of detecting the spectrum of mutagenic events that different chemical, structures may cause. 25 �REFERENCES 1. W. T. Federer. Experimental Design (Theory and Application). The M,i. Vi! ii:ui Company, 1955. 2. b. .„.; -«i•J',evlc acid L. Tolraach. Response of synchronized populations of "sieLn. cells to ultraviolet Irradiation at selected stages of the generation cycle. Radiat. Res. 32, 327 (1967). 7 . f<. T.. W,' -s .mssen and R. B. Painter. Radiation-stimulated DNA iils in cultured mammalian cells. J. Cell Blol. 29, 11 (1966). '- , il. Stich and B. A. Lalshes. DNA repair and chemical carcinogens. Ii> |fat Sobtology Annual. H. L. loachim (ecL), Apple ton-CenturyCroris, NP-W York, 1973, pp. 341-376. 5. K.. £lgjo, H. Hennings, D. Michael, and S. H. Yuspa. Natural synchrony of newborn mouse epidermal cells in vitro. J. Invest. Derm. 66, 292-296 ( 1 6 ) .96. 6. K. Burton. A study of the conditions and mechanism of the dipheny.lamJ.ne reaction for the coloriinetric estimation of deoxgrlbonucleic acid. Biochem. J. 62_, 3.1.5-323 (1956). 7. A. D. Mitchell. Unscheduled DNA National. Cancer LSU-2735 (April Potential Prescreens for Chemical Carcinogens: Synthesis, Task 2. Final report prepared for Institute under Contract N01/CP-33394, SRI Project 1976). 8. H. F. Scich. D. Kleser, R.H.C. San, and B. A. Laishes. The Use of ONA i-op/iir in the identification of carcinogens, precarcinogens, in j •• strnet rLssue. Jjs Canadian Cancer Conference: Proceedings of •-!.: .•.-•'! •"a"..i:.iian Cancer Conference, Vol. 10. P. E. Scholefleld •v«j.y. Jinlvqrsity of 1'oronto Presss, Toronto, Canada, 1973, pp. 83-110. . :!<•«'. :••"., 5. f,hoi. v. Yamasaki, and B. N. Ames. Detection of ;.ai.<- .',H^.-,:N-:-, ,!•> nutagffjs in the Sa^lmorKilla taicroaome test: Assay of .iOO ,-.f.vcii..,-ils. Pro,. N.-iir. Ac..id~." ScT/"uSA" 7_2, 5135-5139. 10. B. N. Amcs, I'., G. Curney, J. A. Miller, arid H. Bartsch. Carcinogens as frameshlft mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic araine carcinogens. Proc. Nat. Acad. Scl. USA 69, 3128-3132 (1972). IS.. Si. • • AMKS., F . D . L«e, and W. E. Durston. An improved bacterial '•-":( .-; v •. t t-iii for thu detection and c. JLossif ication of oiutagens and ..ir.-'..,Tv,»ons. Proc. Nat. Acad. Sci, 'JPA 7_0, 782-786 (1973). 26 i �12. R. N. Aro«-is, F.. W, Ours ton, K. Yamasaki, and P. !. I.PP. O., .--. IP--.;, n ) are mutagGiis: A simple test system combining llvor homogenous for activation and bacteria for detection. Proc. Nat. Acad. Sri. USA 7Ci, 2281-2285 (1.973). .13. J. Mcl'-ann, N. E. Spingarn, J. Koburi, and B, N. Au.es. Thi* deJi.'.r? Ion of carcinogens as muthgens: Bacterial tester strains with R fp.ctor plasmids. Proc. Nat. Acad. Sci. USA 72, 979-983 (197.5). 14. B. A. Bridges. Simple, bacterial systems for detecting mut.agenJc agents. Lab. Pract. 21, 413-416 (1972). 15. D. R. McCalla and D. Voutsinos. Mutation Res. 26_, 3-16 (1974). 16. M. D. Anderson, E. E. Slater, and H. S. Rosenkranz. Rapid detection of mutageus and carcinogens. Cancer Res. Tl, 970-973 (1971). 17. J. Gross and M. Gross. Genetic analysis of an E. coll strain with a mutation affecting; DMA polymerase. Nature 22i, 1166-1168 (1969). 18. T. Kada. MntagenLclty testing of chemical? in microbial systems. New Methods In Environ. Chem. Toxicology 127-133 (November 1973). 19. F. K. Zimmermann anr. R. Schwaicr. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nItro-l-nitrosoguanldine and other alkylatJng agents in Saccharomy_ces cereyisiae. Mol. Gen. Genet. UK), 63-69 (1967). " 20. U. J. Rrusick and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives ^, 83-96 (1973). 21. I.. D. Kier, E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensat.es. Proc. Nat. Acad. Scl. USA 7_l_, 4159-4163 (1974). On mutagenlcity of nitrofurans. 22. fl. N. Ames. J. McCann, and E. Yamasaki. Methods for Detecting Cnri-i.iogens and Mutngcn.s with the £Salmone : lla/MaminaL lan-Microsome Mutagenicity Test. Mut. Res. TJL... 34"7~364 ~(1975). 27 �TRST OF THE FEBTILITY INDEX - MOKOCftOTC.'HO?. .1 DEGREE OF FREEDOM i--. a V?.«ICLE CONTROL ft N P»5 MTO PERT. INDEX O5S3 N N Pftii MYO 1? 74-10 iFERT. :*OEx CriliQ N if PR6 HTO 30 MG/KG iFCST. 2NDEX KG/KG TFM ChiSO n PHi *t iFcBT. CHIS3 h ft PRG MTO FERT. INDEX CHISG MULTIPLE TNEA7MIENT I 28 ill .70 0.05 23 40 ,57 .7 6 16 40 .45 4.14 .a 40 .40 *,!l« 29 40 .72 0.30 2 24 .C .65 O.Cfl Ji •0 .? 7 .98 20 49 .0 6 1.28 16 40 5*0 4,&6 * 27 39 .9 6 .03 3 23 4S .? 5 0.36 3C 40 .75 19 «C .*T .45 22 40 .5 5 0.30 32 40 .ec 3.7? .6 0 00 « 3? 40 .67 0.00 2S 40 39 40 .5 5 .4 8 IS 4fi .38 6.07* 2? 40 .7 6 .5 8 4.79*: 25 40 .63 0.00 ?1 40 .5? .20 30 40 .5 7 1.42 .03 23 40 .7 5 .8 0 25 38 .6 6 0.00 27 36 .71 .24 !8 .l 21 40 .52 4.91* 26 38 .4 7 .07 27 36 ."> .02 20 40 .50 2'7S 24 38 .3 6 .4 2 26 36 .? 7 • 02 .3 6 i £4 «Q .60 C.Ofl 33 6 ?» 38 .63 C.93 2? *0 .7 6 7 30 38 .11 0.00 25 40 .3 6 9 27 38 .71 0*00 23 40 .0 7 * I SIGNIFICANT AT P LT 0.05 INCREASED ABOVE COHTSOL 2.01 .&» • 02 22 �Table 2 AVERAGE IMPLANTS PER PREGNANT FEMALE - SONOCROTOPHOS WEEK CONTROL 74-10 15 HO/KG 7*-10 30 MG/KG 74-10 60 .2 MS/KG TEH MG/KG M ULTIPLE TREA TKENT 1 28-11.39 249/ 2.08 31.3 189/ 18-10.50 ISO/ 2 16-11 . 5 316/ 29-10.90 2 to 319/ 303/ 26.11.65 332/ 31-10.71 242/ 20-12.10 ISO/ 2 16-11 . 5 *93/ 27-13.85 3 24S/ 23.10.65 356/ 30-11.87 239/ 19-12.58 "I 267/ 2£-12.14 *1 34a/ 52.I8.97 4 309/ 27.11.44 314/ 2.25 51.6 274/ 2 . 2 45 21. le,6/ 15-11 .0? 266/ 2 . 9.35* 7 5 2T4/ 24.11.42 372/ 33.11.27 270/ 25.10.33 248/ 2!«1S .81 e 302/ 24-12.58 327/ 27-12.11 275, 23-11.96 25S/ 25*10 .2'J 7 346/ 30-11.53 285/ 25.11.40 25S/ 2l>12. 14 316/ 8 292X 27-10.81 313/ 28.11.18 228/ 20-11.40 291/ • •• I SIGNIFICANT AT P LT SIGNIFICANT AT P LT INCREASED ABOVE CONTROL 0.05 0.01 3S4/ 30-11.8? 2V3/ 27.10.11** 2 28-11 . 9 306/ 27-J1.33 24-12 .12 322/ 26.12.3f>**! *" �Table 3 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - rtQNOCROTOPHPS MCEK ?*-10 CONTROL 15 M6/K9 74-10 30 MO/KG 74-10 60 .3 M6/K6 TEW r MUIL.TIPLE TftEATHENl 1 S3/ 28* .6 4 2B/ 23" 2 9X 26* .31 3X 31* .10 *D 3 9X 23* .39 ;6X 30* .53 4 2X 27* .? 0 ?X 25* .28 * 5 ilX 24* .«* 22X 33* .? 6 1.22 lex 18* .6 5 iOX 16* .3 6 62X 29* 2.14" 1GX 20* .50 2X 16* .13 77X 27» 2. 8 ' 5* 19* .7 * 9X 22* .! * 87X 32* 27" .2 26X 22* 1.16* 9X 15* .0* 6* 11X 27* .41* \il 25* .8 4 *X 2l« .3 4 ?2X 30* ,73 25* ,4* 2*D 9' , 6 2iX 24* .35 \6X 2T* .9 5 14X 23* • 61 6X 17X 27* .3 6 7 30/ 30* 1.00 \\f 25* .* 4 4/ 21* .19 ex 28* .9 2 IIX 27* .41 a 19X 27* 24X 28* .6 8 7X 20* ,35 9X 24. .8 3 ii/ 2S- ,2 * • •• D .70 SISNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELCM CONTROL �Table 4 CHI-SQUAR2 TEST OF THE DEATH 1SDES - MONOCROT3PHOS 1 OEGRSS 07 KSEEDOM •EcK VEHICLE CONTROL N N *i)I PRO DEATH CMIS3 74-10 N N SCI PWG IS MS/KG DEATH I NOES 74-10 N H "KG CKISO 30 MG/KG DEATH INDEX CHiSS 00 MG/KG N . 1 .S 0 'O DEATH C ,. 5 r s we I fi OEATM MULTIPLE TREATftEN T 11 1 10 28 .6 3 0.00 2 7 26 .7 2 0.00 3 9 23 .9 3 0.00 11 4 2 27 .0? o.oo 7 23 .8 4 .35 7 18 .39 .01 6 ,6 .39 .04 25 ?9 .So 13.27 ** 3 31 .10 1.84 7 20 .35 ,07 2 16 .13 .52 26 e.7 .6 9 24.26 « 30 .It .01 7 19 .37 .03 6 2? .27 .28 25 32 .79 ?.C5 * . 25 .6 2 2.54 11 22 .50 * IS .6C 11.21 ** a 2~ .10 3.6? 12 25 .8 4 .21 3 2: .33 .of 10 30 .33 ,CO 23 .8 4 .52 5 25 • 2G 7.48 10 27" .37 2.36 7 28 .25 .02 9 27 .33 »GT a 2, .33 .27 26 .36 .03 9.20** 5 t 24 .33 0*00 11 33 .33 .00 6 15 24 .63 0.00 13 2T .8 4 .6 5 11 .? 2 0.00 8 25 .32 .02 4 21 .19 .9 0 .4 4 0.00 IS 28 .4 5 .17 7 20 .5 3 .12 7 8 ** a 30 12 27 SIGNIFICA.NT AT ? LT 0.01 10 �Table 5 SWttSH OF DEAD IMPLANTS PER TOTAL DfPLAKTS - MONOCS01X)?HCS •CEK CONTROL ?4-10 15 we/US ?4-i3 30 KG/KG ?*.- SO 40 «fe/SG TEN .2 MG/KG *H.(VTIPtE TREATMENT 1 13/ 319- ,0« 2 ?•< 303* .03 3 <?/ 245* .04 M/ £49" .11 5/ 332« ! / 356* & ,6i .04 10/ 1B9* .OS. 10/ 160* .6 0 *>2/ 316* .20** 10/ 24S« . 4 0 2/ 180* .01 77/ 293* .26** 9/ 267* .03 B7/ 348* .25** ll/ 266* .04* 9/ 239* .0* .02 2 . 274. 6' .09** 9/ 166* .05** .04 ze/ 372* .e« 12/ 2?o* .04 9/ 246* .04 2?/ 356* .6 0 21/ 3C2* .07 16/ 327* .OS 14/ 2T5* .ns 6/ 2S5» .0?**D IT/ 273= .6 0 ^ 3.0 / 3*6* .09 ll/ 285= .04 4/ 255* .02 a/ 316* .03 ll/ 306* .04 B 14/ 292* ,' 0 24/ 313* .6 0 T/ <2B* .03 9/ 29S> .03 ll/ 322- .03 4 2/ 309.- 5 i*./ 274« 6 * •• D 7/ 314. . Oi SIGNIFICANT AT P LT 0,05 SIGNIFICANT AT P LT O.C1 DECREASED BELOW CONTROL �Taole 6 CHI-SW.-AHE TEST OF THE FERTILITY IKDEX - BROMAC1L 1 DEGREE OF FREEDOM VEHICLE CONTROL N N PERT. PRG MTO INDEX CHI SO 74=06 N N PRG MTO 1230 HG/KG PERT. INDEX CMISO 7.-06 N N PRG MTC 2500 PERT. INDEX r»-Gi CHiiG 5303 Mli/KG N N PRG MTD PERT. IHOEX CHIS9 KG/KG TEM K, N PRG KTQ PERT. FNCKX CMISO MULTIPLE TREATMENT 1 26 40 .70 0.00 21 40 .2 5 1.90 23 40 .57 .87 29 40 .2 7 0.00 29 40 .72 o.co 2 26 40 .5 6 0.00 24 40 .to .OS 26 38 .8 6 .01 23 40 .63 0.00 27 39 ,9 6 .03 3 2.3 40 .57 0.00 29 40 .72 1.37 19 38 .50 .19 25 40 .63 .05 32 40 .SO 3.78 40 .3 6 .05 22 38 .8 5 .! 4 23 40 .70 0.00 27 •0 .6? .6 0 20 .9 .8 5 1.4? 4 27 40 .67 0.00 25 S 24 40 .0 6 0.00 31 40 .7 7 22 38 .00 27 40 .7 6 .2 2 30 40 .5 7 6 24 38 .63 0.00 31 40 .77 1.30 25 38 .6 6 0.00 27 40 .7 6 .03 27 38 .71 .2* 7 30 38 .79 0.00 29 40 .72 .16 22 38 .8 5 2.98 29 40 ,72 .16 27 36 .75 .02 8 27 38 .71 0.00 213 39 .72 .03 24 38 .63 27 *C .67 .01 26 36 .7? .02 .24 �Table 7 AVERAGE IMPLANTS PEH PHEGHJUfT FEMALE - CONTROL 74-S6 74-06 Z500 HG/K6 74-06 5000 MS/KG TEH MO/KG MULTIPLE TREATMENT i ?,!<¥/ 28• 11 .39 235," 21-1 i. I, 2T8/ 23.12.09 32B/ 29.11.31 316/ 29.10.90 * C-Ci-.- 6 26 .11 . 5 253' 24BJ0.54 264/ 26.10 . 2 9 304/ 25.12.16 293/ 2.08 71.5 3 24S/ 23.10 . 5 6 33SX 29.il.5S 22S/ * 19.1 i . 4 319/ 2 . 2 /6**I 51. 348/ 32.10.87 4 30*-' 4 27-11 . 4 2?Q/ 25.11*60 2&8/ 22=12.18 34 6/ 28-12.36 266/ 87- 9.85 * * S7*/ S«-11 .42 3$\/ 31*11.65 26 I/ 22*11 .d6 523/ 27-21.96 356/ 30*11.67 .-. .10 a/ 5 24.12 . 8 363/ 31" 11. Sf 294/ 7 25-11 . 6 276/ 27«10.30** 273/ 2?-10.11 ** 7 34S/ 30.11 ,53 35&/ 29.12.24 2S3/ 22.11 .50 357/ 2«i.U.31 306/ 27.11.33 3 292/ 27«10.61 318X 28.11.36 5 277/ 24.11 . 4 307/ 27-11.37 322/ 26*12.38 **I • S I G N I F I C A N T AT P LT 0.05 ** SIGNIFICANT AT P LT 0.01 I INCREASED A30VZ CONTROL �Table 8 AVERAGE DEAD IMPLANTS PEH PREGNANT FEMALE - BROMACIL *EEK CONTROL 74-06 1250 MG/KG 74-06 2500 *S/ivG 74-06 5000 MS/KG TEH .2 MG/KG MULTIPLE TRE* TMENT 1 28= .46 8/ 21 = .38 10X 23= .43 17/ 29= .9 5 62/ 29= 2.14** 2 e/ 26= .31 12/ 24= .0 5 a/ 26= .31 5/ 25= .20 77/ 27= 2 8 * . S . 3 Ln 13/ 9/ 23= .39 6/ 29= .21 12/ 19= .63 10/ 25= .40 87/ 32= 2,72** 4 2/ 27= .07 7/ 25= .28 16/ 22= . 3 * 7 * 15/ 29« . * 5 * 1 I/ 27= .41* 5 ll/ 24 = .6 4 12/ 31 = .9 3 15/ 22= .8 6 B/ 27= .30 22/ 36= .3 7 6 2i/ 24= .8 8 7/ 31 = .23 **D 16/ 25= .* 6 21/ 27" .78 17/ 27» .63 7 30/ 30 = 1.00 ll/ 29= .3 3 14/ 22= .4 6 14/ 29= .8 4 ll/ 27= .41 8 19/ 2. 7 15/ 28- .4 5 8/ 2. 4 .33 9/ 27= .3 3 ll/ 26= .42 .TO • S I G N I F I C A N T AT P LT 0 . 0 5 *» S I G N I F I C A N T AT P LT C . C ! D DECREASED BELOW CONTROL �Table 9 CHI-SQf*RE TEST OF THE DEVTH INDEX - BROMAC !I. 1 DEGREE OF FfiEEDOM VEHICLE CQKT90L N N «DT PSKi OEATh INDEX CHISd 74-Ofc N fc aJl PRG 7»-06 1250 MS/KG DEATH INDEX CMisa N N 25CQ HG/KG DEATH WDI PRG INDEX CHI so *.-06 5000 MQ/KG H N DEATH riDI PRG INDEX CHIS3 .2 MG/KG 7EM h N «DJ[ PHG DEATH INDEX CHISg MULTIPLE TREATMENT 1 10 28 .6 3 0,00 7 21 .33 .2 0 7 23 .30 .01 2 7 26 «S7 0.00 10 24 .42 .4 6 a 26 .31 3 9 23 .39 0.00 » 29 .£1 1.32 9 19 .47 .us * 2 27 .07 O.CC 6 2S • 24 I.b2 11 22 .50 9.20** 5 5 S* ,38 O'OO 12 31 .9 3 .4 0 12 22 .5 5 . 4 7 9.00 14 29 .8 4 .48 25 29 .6 8 13.27** .96 24.26** 5 25 .20 .06 26 27 6 25 .2« .? 6 25 32 ,->B 7.05** 14 28 .so 10. a** a 27 .30 3.Q7 8 27 .30 10 30 .33 .00 10 sf 7 2-16 9 27 .33 .07 26 .38 .03 .09 b 15 24 .3 6 o.oo 5 31 • 16 10.65**D 13 25 .52 .21 10 27 .37 2.J6 7 8 30 .7 2 0.00 7 29 .4 2 .01 10 22 .5 4 1.2* 12 29 .41 .64 8 12 27 .4 4 o.oe 11 28 .9 3 .01 7 24 .9 2 .70 7 27 .26 1.30 ** D SIGNIFICANT AT F LT C.01 DECREASED EELOW CONTROL 10 «3 �Table 10 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS • EEK 74-06 CONTROL 5000 MG/K5 TEM .2 «G/R5 MULTIPLE TREATMENT 1 13/ 319= 2 3 i ' 8/ 3039/ 245* 4 2/ 309, a/ .0. 2* 77/ 293- .6. 2* 10/ 319- .03 87/ 3iiis. .25** . 15/ 34e> ,04«* ll/ 266- .04* 323- .02 ?£/ 35*= .06 .05 21,' 278 = .OS 1 I/ ?73 = .06 .06 ! / 357* * .34 K/ 306- .04 9/ 307- .03 ! / 32?* ! .03 5/ 304 = .02*0 12/ 225- .05 7/ 290. .02 16/ 268- ,06** .03 is/ .) 16 .02 **D 16/ 294* 14/ 253* .03 .3 0 .5 0 .04 6/ 335. .01 ll/ 274. .04 12/ 361 = 6 2l/ 302= .07 7/ 368* 7 30/ 346* .9 0 ll/ 355* .03 a 19/ 292* .07 IS/ 3ia- .5 0 SIGNIFICANT AT P LT SIGNIFICANT AT P !_T DECREASED BELOW CONTROL 62/ 316. .02 17/ 323- .) 13 .03 5 * •« D . OS .04 284- 235- 12/ 253- .4 0 0.05 0,0! 10/ 278- a/ a/ 261- 277- a/ �Table 1). CHI-S41MKE TEST OF THE FERTILITY INDEX - C'IP'SA.S 1 CEGREE OF FREEDOM VEHICLE C'J.',TS3i. * N P3C 1TO FES', INOCS ChlSi. MG/Ki 7*-0? «=as -'••vo INDEX CilSO i-u/'Ki.- ?*-62 S N PHG MTU ! tH 1a F IMOEX Ch;S3 7«-oe N u PR G "ilD fc PERT. INDE? CHI SO t. F£RT. PBG MTO IMDCX CHISQ HULTIPUE TREATMENT oo 1 2J *Q »57 o,OC 34 40 .as 6.10*1 31 40 .7 7 2.?9 29 40 .72 1.37 29 40 .7? 1.37 2 a7 39 ,&<5 i.OO 30 40 .75 .10 36 40 .0 9 4.07*! 30 40 .75 .10 27 39 .9 6 .6 0 3 2* »0 .5 6 0.03 23 *C .s-* .21 3U 40 .5 7 .54 31 40 .77 .8 9 32 41 .89 1.57 * 2? to «S7 ;-.''C 31 3« .2 8 1.35 33 40 .2 6 l.*7 32 »0 .80 i.os 27 *0 .7 6 .6 0 i ?<* *0 »''? O.CO 26 3S .6 6 .02 30 40 .5 7 o.oo 26 38 .9 6 .2 0 30 40 . M 0.00 *> 2* 40 »7g 0.0-3 27 .] 7 .91 34 40 .as 1.20 30 38 .79 .16 27 M .71 .01 .71 .01 30 40 .5 7 0*00 S* 38 .8 6 .32 27 36 .5 7 .00 38 .7] v*3 2* 36 .? 7 .6 2 ' S * I i"? 1J *0 *0 7 .? .60 O.CO 0.00 SICNIFICAKT AT ?LT O.C3 INCREASED ABOVE CONTROL 27 31 3« 39 35 .7 8 .6 2 34 40 .9 8 -09 27 �Table 12 AVERAGE IMPLANTS PER PREGNANT FEMALE - C4PTAN sit*. CONTROL 7<.-Cc 12--0 M S / K G 74-02 MG/K& 2500 HG/KG TEH MULTIPLE TREATMENT i 25' 6. 23*11.52 3B7/ 3«=11.3B 339/ 31 = 10. <,* 310/ 29=10.69 3S6/ 29=:0.9C 2 305/ 27=11.30 3?6/ 30=10.93 375X 36=10. *2 3*2/ 30=: l.*0 293/ 27«1C.H5 3 268/ 26=10. 11 2S2/ 23=10.96 313/ 30=10.4.3 3*5 / 31=11.13 34A/ 32=10. «7 * ?RB/ ?7=10.67 32»/ 31=10. *5 3S9/ 33= lO"*" 333/ 32=10. *1 2fci>' 27= 9 8 . 5 5 33»/ 29=11. S2 2fi2/ 26=: 0.65 348/ 30=11.b O 298/ 26= 11. * A 35is/ 30=11.87 6 323/ 29=11. ! • 312/ 27=11.56 361/ 34=10.h2 3*0/ 30=11.33 273/ 27=10.11 30=10.97 3G9/ 26=11. AA 30n/ 27*11.33 3»»11.^ 301/ 27=11.15 322/ 26=12.38 7 3?3/ 29=11.!* 30V/ 27=11.** 329/ r 3= If 32=11.*! 3B2> 33 = 11.S>» 383/ • SIGNIFICANT A7 P LT 0.05 «• SIGNIFICANT AT P L7 0 . 0 1 �Table 13 AVERAGE OS*D IUPLAWTS FER PREGNANT FEMALE - «fct.K T + -02 CONTROL 2500 MG/XG 7,-Cc 533C -li/tt-i Ifrt «WLT£f>LE TREATMENT 19/ 31- .fcl 15.' 2i* ^2 M, 24« 2.14 ** .0 5 J7/ 36- .<•? !»/ 30* ,*7 77/ 27s 2.85 *• .70 16/ 30- .53 15/ 31- .8 4 67/ 32« 2.72 »* 12/ 31ft .39 ]S/ 33s .5 * ll/ 32« .3* ll/ 27, • »1 ?*/ 26= .92 9/ 30s .30 ll/ 26- .42 ?2/ 30s .73 .4,3 21' 34* .t>2 18.' 3C* .60 i?/ 27- •63 27 = -.Si 14/ 30= .*7 a/ 2*= .31 ll/ 27- .41 33= .35 !/ ! 3s 4 .32 7/ 27= il/ 26- i 23* . ?5 2 ** O «/ :\S 27s .*! Is/ 30 = 3 io/ 26s ,*2 16/ 23- * ;&/ 2T- . =9 5 i5/ 24- . '2 . t 9/ ?^s ,^> 13/ J'l ? 1 Ji. 2<!s .72 IS./ d n/ 32s ,»l 12/ 17,' • SIONIFICAKT AV P LT O.OS •• EISNIFICftNT AT P LT 0.01 3» = .50 .26 .42 �Table 14 CHI-SQUARE TEST 0? Thl DEATH IM3EX - CAPTAK 1 DEGREE OF FREEDOM VEHICLE CONTROL N UDI N DEATH PRG INDEX CHISQ 74-02 N N »DI PRG 12SO HO/K6 DEATH INDEX CHISQ 74-02 N N tfDI PRG ?50C MG/KQ DEATH INDEX CHISQ 74-02 N N yOI PRG 5000 MS/KG DEATH INOE« .? KG/KG N PRG CHlSa N SEAT*. INDEX CHISC MULTIPLE TREATMENT 1 a 23 .5 3 0.00 10 34 ,9 2 .2 0 12 31 .39 .00 12 29 .41 .4 0 25 29 .«* IS.**" 2 9 27 .33 0.00 8 30 .7 2 .07 12 36 .33 .07 !C 30 ,33 ,OS 26 27 .* 9 20.79" 3 •0 24 .8 3 •0.00 12 23 .2 5 .6 4 11 30 .37 .32 13 31 ,*? .00 25 32 <78 7.85" 4 11 27 .41 0.00 9 31 .6 2 .6 5 7 33 .21 :.as 6 32 .25 1.02 8 27 . 3<3 .32 .38 .6 0 7 30 .23 .37 li 26 .42 .00 10 30 .33 ,G1 .03 15 34 .44 lag!} ! 0 30 .33 .0* 1C 27 .37 .23 .0] ^ 26 .27 .71 4 27 .33 .12 .19 5 27 .19 1.15 10 24 .38 .03 5 fc 11 29 .* 3 0.00 10 26 •-2* n.on 9 27 .33 .4 4 .00 11 30 .37 • 24 .9 3 9 34 .26 8 29 7 12 29 .! * 0.00 12 27 8 11 32 • 34 0.00 a 33 ** SIGNIFICANT AV PLT O.Oi �Table !5 NUMBER Of DEAD IMPLANTS PER TOTAL IHPLASTS - C*PT'.>. 74-02 C3NTROL 2500 MS/KG 74-02 n S«C/X3 TE'M .? MULTIPLE TREATMENT ; .03 IT/ 367* .3* 19/ 3 9 3. ,6 0 15/ 310* .CS **/ 316= .20** £ *•- d/ 2&5« 11.' 315s .Ofc iS/ 326s .05 IT/ 375- .5 0 14/ 34?> .04 77/ 293* .iff* 2 Si/ 26Gn .06 IS/ 25?B .06 !«./ 313* .US ! / 3*55 .0* 87/ 3 8 4. .25** -' ib/ 2AAc . &'• IS/ 359. •0* ll/ 333" .03 ll/ 266= ,04 5 IS/ 33*3 .3* 9/ 348= .03 11,' 29»= .0* 22/ 356 = .06 16/ 3*3= .05 17/ 273.-: .6 0 fsi 12'* 3£» = 2»/ 28?- .04 .39 b •>/ 323s ,ft3 i3- 312= .0* 21/ 3*1. 7 2i/ 323» .Ci7 ! / 3Q9= 5 .5 0 I*/ 329. .11* a/ 305. ,C3 1 S .-' 30&. .04 - !.' -•}«!= '. .03 j2' 382= .03 ll/ 383. .03 7/ 30i= .Og ll/ 322" .03 • SIGNIFICANT AT P LT 0.05 •* SlSNieiCANT AT P LT 0 . 0 1 .06* �Table 16 CH1-SQUAR2 TEST OF THE FERTILITY INDEX - FOLFZT 1 DEGREE OF FHSEDOM • EEK VEHICLE C O N T R O L N N PRG MTD PERT. INDEX CMISU 74-03 N N PHIi MTJ 1250 MG/K6 (•tRT. INDEX CHIaQ 74-03 h N PRG MTU 2500 H6/KI. FE.RT. INDtX CHIiO 74-OJ N N HRii MTU SOO'j Mto/KG TEN F£RT. INDtJt CrilSQ h w PRG HTD fEKT. INDEX CHI Si Ul MULTIPLE TREATMENT 1 23 40 .57 0.00 <J7 4Q .67 .»e 29 40 .72 1.J7 3\j. 4U ,T5 £.01 <><» «>J .7? 1.37 2 27 39 .9 6 0.00 2U 40 .70 .03 23 40 .37 ,fe 3S> «C .8 8 £.99 27 39 .9 6 .6 0 3 26 40 .5 6 0.00 i-'b 40 .63 O.UO 31 4U .77 .SIB 32 tLi .60 1.57 32 40 ,60 1.57 4 27 40 .7 6 0.00 30 40 .5 7 .tf* 18 40 .5 4 3.£5 30 40 .75 .24 jfr no .6? .0= Q.OO 26 40 .65 .<!3 33 4Q .8 3 j9) .! 30 40 .75 0.00 O.OC id 4o .7s U.OO ^ 36 •7i .01 .72 U.OO JO 4Q .75 40 .2 7 0.00 3U 4g .75 O.UO 28 4u • 7o 29 40 .72 0.00 28 40 .70 0.00 27 4Q .67 . 0* 31 32 40 .BO U.OO e~> 40 .67 1.03 29 40 = ?2 , JS j'J 5 29 6 29 7 a 40 4Q .77 .Q7 27 jo .75 .30 4Q ," j .07 ?t J6 .?2 -2B �17 IMPLANTS PEB PREC-NAST FEMALE 74-03 CONTROL 1 Xt-js 23*ilnb? JOB/ 2 7 x > l . «l J24X - j>i>00 Kfi/Kb 29*11.1? . ZHG/.V.; 32flX 30*10,93 316X 29*10.90 £ :i?5/ 27x11.36 JO-/ 2a*10.Bb 267/ 23»ll.i7 393X 3!i»lS.14 293/ 27x10.35 3 fat/ 26x10.31 2Ti/ 2bxl0.tt4 33«/ 31x10. 11 327/ 32x10.22 348/ 32.10.87 4 28Ui' 2?BH'» A '' 29V/ 30* 9.97 18J/ l«»i'J.l7 J20/ 30*10«b7 26fc/ 27* SUSS % 33*/ 29*11. Si 333/ 3U*il.l9 31a/ 26«12.<i3 3<*9/ iS)ill,*0 35a/ 30*11.87 i 323/ 2V>il.i* 33<r/ 3J=ilt13 294/ 2b«10.30 J12/ 28=11,14 273/ 27x10.11 7 323/ 24*11.14 30 f / 2Hai3.V6 311/ 27*11.32 3b6/ 3<cll.4b 306/ 27il!.33 322' 26«12.38 15 3fel/ 32*11.*! 30 i'' • SIGKIFICAMT AT P LT O.Oi •« SIGNIFICANT AT V LT U.01 27*il.l5 34!>X 1 2 ** 11.^9 J46X X 30 11.53 �Table 16 AVERAGE DEAD IMPLANTS PER PREGN»T FLSIALE - POPPET CONT3CL 7*-OJ 12!>0 KG/KG 74.03 2*33 MS/ISO 7<t-93 5000 MULTIPLt TKtATMtNT 1 8/ 23. .35 It)/ 27* .37 25/ 29. • a* 13/ 2 ll/ 27. .! * lb/ 2Us .4 6 20/ 23* .87* 3 16/ 2. 6 .2 6 U/ 25, .52 19/ 31. .01 4 it/ 2 . 7 .9 5 16/ 30s .b.3 a/ 18. b 15/ 29, .2 5 IS/ 30= ,40 9/ 6 «/ 29, .31 }7/ 3o» .b? 7 21/ 2. 9 .72 !<;/ 28. .41 ll/ 27. 8 13/ 32 = • S I G N I F I C A N T AT P LT O.C5 •• S I G N I F I C A N T AT P LT 0.01 0 DECREASED BELOW CONTROL 30» .43 2i/ 3&» .60 T7/ 27. 2,05** ll/ 32« .34 87/ 32. 2.72** .4 4 7/ 30- 26« .JS io/ 35. . **> 22/ ?Ca .73 10/ 2. 8 .67 i3/ £«• .* • !. »' jTa .63 .43 10/ 27» .37 le/ 3i> 08 H/ 27* .*1 .41 20/ 29. .9 0 13/ ll/ 26« .42 30« c c3*D .43 62/ 29" i.14** :i/ z?a .*i �Table 19 CH!-Si3TARB TEST OF THE DEATH INDEX - FOLPET 1 DECREE OF FREiDOK 74-03 ¥E*!Cl.i CONT^l-L N N • 01 i 1DEATH INDEX -41SU ft N »Ul PhG iL)E«TH !NOEK CnliiU N N «01 ORb 2500 7»-Q3 *<t/f\i DEATH iNUtX N *> 5000 Mfa/Ati IDEATH IN-JtK CHi^Q CHlSU TEH M N »l): f>nG ULATn INDEX CHtSU "ULTIPUE TMtATMENT I e 23 .35 0.00 a 27 .30 .ul 13 29 .6 4 .<•» 10 30 .33 .03 25 £1 .6 6 12.49** 3S .43 .5 3 26 <7 .6 9 20.79*. 7.85** e .3 9 27 .33 S.i-0 13 *S .»a .Si 12 23 .2 6 i.iS 1= 10 26 .8 3 0.00 V <!<9 .6 3 ,«1 4 31 .29 .22 d 32 ,2i .67 2b Je .78 4 I: 27 .41 O.i'O 11 20 .3? ,UO 7 la .39 ,iiJ 7 30 .23 1.27 a j-7 .3G .32 5 11 29 •38 0.50 10 39 • 33 ic 35 .34 :'.-U 10 JO .. 31 .01 6 0 29 •28 u*cs 11 30 .37 .«2 il 26 .39 .43 V 28 •32 .01 10 l7 .37 .22 7 12 29 .41 000 6 <;B .29 .S>4 4 27 .3 3 .12 13 31 .42 .05 t ft .33 .12 c* .8 3 .00 a *• 11 32 •34 0.00 SIGSIFTCAHT AT PLT 0.01 4 2r .33 >t«l '4 0 » 16 26 29 .Ji .5 5 .' « 1.H9 11 30 OT .01 10 �Table 20 NUMBER OF OEAO IMPLANTS PER TOTAL IMPLA.STS - FOLPET fettK 7*-03 CONTROL 1250 M6/KG 7«-03 /4-U3 5000 M u / r G ,2MG/«b Ttl MULTIPLE TREATMENT 1 a/ 265* .03 10/ JOB- .03 »/ 324* . J8 i3/ 328 • .04 62/ 316- .20** 2 ll/ 305* .4 0 le/ 304* .06 20/ 257- .06* 21/ 390 • .05 77/ 293* . 6 *K 2 3 16/ 268. .06 1J/ 271. .05 I1*/ 33*« ,U6 ll/ 327 • .03 87/ 3 8 .* .25 t* * 16/ 288* .0* J6/ 29V= ,Q5 5 6 e/ 1H3- ,y» 7/ 320- .02 ll/ 266* • C4 .04 Z£f 356* .36 .04 n/ £73* .06 .0* lit 333* .04 9/ 318- .03 16/ 399 * «/ 323 = .03 I?/ J3»= .05 4 16/ ^ . .ub i3/ 3i2 15/ 334- E 7 21/ 323* .0? li/ 307. .04 10/ 311- .03 IS/ 356* .05 M/ 306> .0* a 13/ 381* .03 ll/ 301- .04 20/ 345- ,U6 13/ 346* .4 0 ll/ 322- .03 • •• S I G N I F I C A N T AT P LT 8.C5 S I G N I F I C A N T AT H LT 0.01 �Table 21 ikl-SQUABE fSST OF THE FERTILITY INDEX - AZINPBO:--fETFYi. 1 DEGREE OF FREEDOM HS/K6 *E"SCLt * * PRO * ! T) PERT. IWE* •lisa P*3 »T() i ••*?.£* c«isa T4-09 f4 N PR6 MTD MS/KG PERT , CHISQ INDEX 74-09 H M PRO MTO BC KG./HG PERT. 1MJEX CnlSQ Kl PR6 N PERT. CHISQ MULTIPLE TREATMENT ^ 1 ?8 43 .70 C-,09 34 ?6 40 .5 6 c.eo 29 oo 2 J S3 40 .57 3.00 33 40 •as .2 7 40 .2 8 28 .23 40 .70 .06 27 40 .67 0.00 29 40 .72 0.00 27 40 .7 6 0,00 27 40 .7 6 0.00 27 39 .9 6 .03 .72 1.37 24 3* .63 .OS 32 40 ,SS 3,72 21 36 .8 5 .35 27 40 .7 6 .6 0 .02 30 40 .75 1.42 27 J« • 7i .24 • »M*I 29 40 * 2? »0 .7 6 5.00 30 42 .75 .24 29 40 .72 .06 5 2* *0 .60 o.co 2* *0 .72 .9 8 26 49 .5 6 .05 20 34 .53 6 2* 33 .3 6 3.00 JO 40 .5 7 .79 27 40 .7 6 • 03 28 34 .82 7 3?, i8 • 79 0.00 27 40 .7 6 .6 7 25 40 .3 6 1.81 24 34 .71 .30 27 36 .75 .02 8 27 J5 .71 0.00 26 40 .5 6 «n 26 40 .5 6 .11 22 34 .5 6 .10 25 36 .72 .02 * I SIGNIFICANT AT F LT D-lr. INCREASED ABOVE CONTROL 2.41 �Table 22 AVERAGE IMPLANTS PZR PREGNANT FEMALE - A7IN7HOS-METHYL CONTROL 74.09 20 MG/KG 74-09 MULTIPLE »C 74»0<5 80 MG/KG MS/KG TREATMENT 1 319/ 28*11.39 375/ 34.11 .03 300X 28*10 ,71 306/ 27.11 .33 31*/ 29.iC.90 2 303/ 26*11.65 335/ 29.11 . 5 5 312/ 2r«n .56 310/ e7.il .*3 293/ 2V. 10. (55 3 ?45/ 23.10.65 379/ 33.11 . 8 * 338X 29.11 . 6 6 272/ 2«.l!.33 348X 32.10.87 * 309/ 27.11.44 367/ 30.12 . 3 2 369/ 2 . 2 7 * ! 9 1 . 2 * 247/ 21.1! .76 ?66/ 27. 9 8 * .5 5 ?74/ 24-11.42 33?/ 29*11 . 2 6 310/ 26*11 .92 228/ 20=11 .40 35S/ 30»11.S7 t, 302/ 24*12.Sa 345/ 33*1 i .50* 2?6/ 2 . 10. 2 ? 2" 323/ 2a-H • S** 27">/ 27*10.11 ** T 346/ 30*11.53 288/ 27.10.67 273X 2S«11 .12 262/ 24.10 .42 30<S/ 27.11.3J 8 292X 27.10.81 306/ 26.11 . " 293/ 26.11 .27 2SO/ 22.11 .3* 322/ 26.S2.3a **I • SIGNIFICANT AT P LT O.OS •• SIGNIFICANT AT P LT 0.0! I INCREASED ABOVE CONTROL �Table 23 AVERAGE L'EAD 1MPIANTS PER FSEuNAtT =EMALE - £0 CONTROL "lu/Ms 74-3? K6/".G 40 .? Mr,'KG MULTIPLE TREATMENT 1 !3/ 26- .46 13/ 3-.« ,36 3*/ 23« 1.21 * 19/ J7- .•JO «/ 29" 2.1* ** 2 <3' 26" .31 16/ 2%» .5 5 15/ ?T« 19/ ?7, ,?0*« T7/ 2T« 2.85** e/ ll/ 24> .6 4 8T/ 32. 2.72** T/ 2i» .33* !/ ! 27" .41 * 3 •»A 23. .35 31X 3 . 3 .4 9 4 ?/ 27* .0'-' 2i/ 30* .TO** 5 ll/ 24- .4fc IS/ 29* .52 t £i/ 24m ,63 15.' 3C-» ,33**D T 3C.' 30* I.GJ 8/ S7s« .30 e/ 3 '*" ,. 27. 13/ 26- ,60 ia/ .TO * SIGMFICAhT AT P Lf 5.05 •' SIGNIFICANT AT P LT 9.01 D DECREASED BELCH CONTROL 31 / 2. 9 .56 .20 29" l.oT «* 6/ 2e," .23 ll/ 20= .5 5 22/ 3ft- .73 IT/ 27« .3 6 9/ 2fia ,32«. E IT/ 27. .3 6 2 . .32 5 19/ 2. * .79 li/ 27. .41 8/ £2i .36 11.' 26* .".2 2b. .e j �Table I-. CHI-SQUARE TEST OF THE DEATH INDEX - AZINPHOS-METHY'.. 1 DEGREE OF FREEDOM VEHICLE CONTROL MEEK M s hDI PRO DEATH INDEX CMisa 74-09 N N WOI PRG 20 74-09 HO/KG DEATH INDEX CHI SO N N tfOI PRG 40 MG/K6 DEATfi INOEX 74-09 HO NO/KG .2 K N .01 PRG OEUTH INOEx N PRO DE*Th INDEX 11 27 .41 .01 25 29 .flfi 13.27** 26 27 N CKiSO CMISQ C*iSG MULTIPLE TREATMENT 1 £ 10 2B .6 3 0.00 12 34 .5 3 .05 14 28 .27 0.00 11 29 .6 3 . 4 3 3 27 .0 3 .01 12 27 .4 * 1.09 ,9ft 24.26*" 16 33 .8 4 .18 5 29 .17 2.11 a 24 .33 .01 25 32 .78 7.05** 14 29 .6 4 9.53** 7 21 .33 3.65 a 27 ,30 3.07 6 26 .3 2 .* 6 7 20 .35 .02 10 30 .33 .00 9 it .33 3.25 £ 28 .9 2 10 27 ,37 2.36 .6 0 7 25 .8 2 ,0* iO 24 .*2 .6 7 9 27 .33 .07 .03 a 26 .31 .5 S 6 22 .36 .8 0 1G 26 ,3fl .03 2 7 26 3 9 23 .9 3 0.00 4 2 27 .07 0.00 13 30 .3 * 5 9 24 .8 3 0.00 13 29 .»5 6 15 2* .3 6 0.00 a 30 .7 2 .27 0.00 7 27 .6 2 .*» 0.00 10 7 8 30 a 12 27 * ** D SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED SELOV CONTROL 26 .8 3 7.70** .07 5.61 **D .0 5 .6 6 4.73 *D �Table 25 TCMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - AZIXPH.Oi-y.r.THl;L. «££* ?*-S9 CCfeSQOL 7*-09 20 74-09 KG/KG 60 MULTIPLE TREATMENT IS/ 31Q" ,0* 13/ 3TS. .03 34/ 300» .11 la/ 306" .36 62/ 316« .20" S 1,' 303- .03 ii/ 335, .OS IS/ 312- .OS 19/ 310* .06 ?7/ 293« .26** 3 */ 2*5. .0* 31/ 379S .8 0 .02 ll/ 272s .0* 67/ 348a . 5 2 " 4 iV 339* .01 2 I/ 367s .36 " .03* ll/ 266> .04* S 11 / 2?»« .0* IS/ 337» .0* .OS 22/ 356m .06 6 21/ 302« .07 \0/ 3*5» .33 **3 .03**S 17/ 873a .06 7 30 / 3*fes 8 l$/ £92» S0<» 7 .O 6/ 2ACSic .03 13' 306= . 0* • •• SIGNIFICANT AT *> LT O.U5 SIGNIFICANT AT P LT 0.51 I) DgCKFA.-3! BELOH COKTROL a/ 336* 31X 369* . 8 0 " ?/ 2*7« .02 ll/ 22Ea .6 0 9/ 323« 8/ 2 8 7. .03 19/ 262: .07 ll/ 306= .04 IO/ 293- .3 0 6/ J5o» .03 ll/ 322« -03 6/ 3iO» IT/ 276. �Table 26 CHI-SQUARE TEST OF THE FERTILITY INDEX - MALATHION i DEGREE OF FREEDOM V E H I C L E CONTROL N P»3 N FERT. NTo INDEX CHISQ 74.07 N N PRG MTD 1250 MG/KS FERT, INDEX CM I SCI 74-07 N N PRG MTD 2500 KG/K6 FERT. IN3EX CM 1 56 74-07 5003 MG/KG N N PRG MTD FERT, INDEX CM I SO MG/KG TEM N PRG MTO FERT, INDEX CHISU MULTIPLE TREATMENT 40 .5 6 .06 29 40 .72 0.08 29 40 ,7? o,c* 24 40 .0 6 ,05 31 39 .79 1.40 27 y* .1 6 .03 .00 24 40 .0 6 o.oa 33 40 .32 4.82*1 32 40 oflO 3.72 .6 6 .01 22 38 .8 5 .41 * ' 40 .67 .06 27 40 .? 6 .6 0 36 .8 5 .01 22 36 .61 • 01 34 *0 .5 8 S.08*I 30 40 .5 7 1.42 36 .8 7 19 34 .6 5 .15 35 40 .38 5.02*1 27 38 .71 .4 2 32 40 ,80 .03 27 36 .75 .92 37 40 .92 4.72*1 2* 36 .? 7 .2 0 0.00 31 40 .77 .6 2 ,65 0.00 30 40 .75 .4 5 4C .57 0.00 23 38 .61 27 40 .7 6 0.00 25 38 5 24 40 .C 6 G.OC 21 6 24 38 .3 6 0.00 28 1 28 2 26 40 3 23 4 40 7 30 38 s 27 36 ** I .8 7 .9 7 .71 0.00 30 36 0.00 30 36 SIGNIFICANT AT P LT 0.05 INCREASED ABOV3 CONTROL 1.26 26 .3 8 .03 19 34 .6 5 3.39 .3 8 .6 9 16 34 .47 3.36 �T » D l e 27 A.^HAftE IMPLANTS PER PREGNANT FSMALi CONTROL 74-Q? 12SO 74-07 2SOO MS/>6 530S MG/KS !£•( H'JLTIPLE TREATMENT iit,' 2 JliJ/ 3 *" 28*11.39 e-S/ * 3l^/ 29^10.90 3:^ii.*s 293S 27.10.85 33.12.18 *I 348/ 3.08 21.7 27.11.11 266/ 27. 9.85* 2.10 21.5 366 / 34.ll.jS 3So/ 30.11.87 1 . 11.89 9 391/ 35-^.17 •* 2T3/ 27.10.11" 19.18.37*1 352/ 32.il. C3 3I56/ 27all.33 16.11.69 ?;,3 397 / 3 . 0 7 32 ? / ?6.12.3B**l 31--IU16 26' 9. 2.16 61.5 363/ 3C»O2.10 267/ 24.11.12 356/ 23.J0.65 291/ 23.12.22 *T 305/ 2»«12.71-I 402/ 359v £7.11.44 304/ 25.12,16 256 / 22=11.6* 300/ 5 £7<i.' 2«.11,*£ 23G/ 91x11,33 2*3/ n JC-3^ 2»«12.5« J1S/ 2A<£S.3&* 226/ f 3*o/ 3.153 01.. 337' 30.il. 23 23S/ S £9^/ 2.06 71.i 323' 30»:0,T7 187/ * SlGNiFICANT AT P LT 0.05 •• SISKIF1CANT AT f LT 0.01 I IKCiLASED ABOVE COMTBOL 26.11.46 32 1/ 36' 4, £4.11.07 �Tahl*- 28 AVERAGE 3E*D IMPLANTS PER PREGNANT FEMALE - MAHTHIOS CONTROL 74-07 125i> MS/KG 7*-C7 2500 CS/KG 7»-07 5000 MGXKG TEM .? MULTIPLE TREATMENT 2. 6 .31 13/ 23* .45 «f/ 29= 2.14*' 2. * .& * 11X 31* .35 77X 2?^ 2.65" .61 e/ nx l*x 24* ,58 I4X 33. .? * 87X 32* 2.72** .6 5" 8X 22* .36* 7X 2. 7 .6 2 !1X 27a .*i* ax 22- .36 ex 34* .2* 22X 3C= .73 19* 1.05 23X 35* .6 6 l^X 27= .63 1 13/ 28* .6 4 lex 31. .8 5 2 8/ 26* .31 12/ 30. .0 * 3 9X 23* 14X 23* 2. 5 J4X 2«* .6 * */ 21X 24* .8 8 21X 28* .5 7 20X 7 3, 0' 39* i.CS lax 30* .33 6/ 19* .32 i5/ 32= .«7 iiX 27. .! * a 19X 27. )9X 30s .63 7X 16. .** 32X 37. .6 8 11X 26< .*2 2X 5 it/ 6 27. .39 .07 A .70 SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 21. .19 �Ti-ble 29 CHI-SQUARE TEST OF THE DEATH INDEX - HALATHIOli 1 DEGREE OF FREEDOM 7i-07 VErtlCLE CONT*0-_ N N dtl »RG OEAT* INCil H N 0£»TH ^ftc- £sie)i c*>ise C.iiStJ 74-07 -250 KG/KG N N SDI p»e 5300 2500 KG/KG i INDEX N P«S h CrU5G Ml m IMCES CM ISO DEATH INOEX CHISO MULTIPLE TREATMENT • 1C- 28 .( 3i (i.OO 13 31 .42 .05 T 26 .27 .16 JO 29 .34 .03 25 29 .6 8 13.27 " 30 .27 .8 0 9 24 .6 3 .5 2 a 31 .6 2 .4 0 26 27 .6 9 24.26 ** 11 24 .6 4 .03 9 33 .27 .41 25 32 .7fl 7.05 •* £ 7 iS .7 8 3.03 a 3 9 23 .39 S.OO 7 23 ,30 ,1G » ^ 3'f ,C> P. 06 10 25 .40 6.04* 3 22 .3 S 1.24 5 2' .19 .6 6 e 27 .30 3.07 = 9 2* .3 ,9 9. CO 3 Si .14 2.01 6 22 .27 .18 7 34 .21 1.36 10 30 .33 .0 0 2* .2 6 0,00 11 £8 .30 1.93 6 19 .32 Z.41 16 35 ,«6 l.Oi 10 27 .3? 27 , " .07 26 .38 .03 t> ' 8 • ** i% 9 ji 30 27 .2? .*<> 5.80 T 0. CO 1* SICHIFICAXT AT P LT fi.05 SICSIFICAXT AT f LI 0.01 30 30 ,Z3 .47 0. 90 .01 4 19 .21 .vil 12 se .3 3 .: * a / .44 ,C7 16 37 .43 .33 10 16 2.36 �T a b l e 30 KUIIBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - KALATHIOS ?*-07 5000 MG/KG MULTIPLE TREATMENT 1 13/ 319. .4 0 18/ 346, .05 3/ 298* .93 13/ 321* .04 62/ 316. .20" 2 e/ 303* .03 12/ 363* .03 ll/ 267= .4 0 ll/ 356« .3 0 ?7/ £93, . 6 2 " 3 9/ 2*5. .0* 14/ 281. .5 0 I*/ 305. .05 !*/ *02« .03 37/ 3 8 4 . .25** 4 2/ 309. .01 • I*/ 304. .OS" 256 . .03 7/ 300* .02 ll/ 266, .0»* 5 ll/ 27*. .0* */ 236= .02*0 ,03 8/ 386* .02 22/ 3 6 5 , .6 0 6 21/ 302. .07 21/ 31B> ,07 . 9 0 23/ 391- .6 0 ' 7 273, ,/ .06 7 30/ 3*6. ,09 10/ 337. .03 &/ 25 3. .03 IS/ 352, .04 ll/ 306, .04 3 19/ 292. .07 19/ 323. .6 0 7/ 187* .0* 32/ 397* .08 ll/ 322. .03 Ln • SIGNIFICANT AT P LT 0.05 •• SIGNIFICANT AT P LT 0.01 D DECREASED BELOW CONTROL e/ a/ 2 3 * . 20/ 226* �Table 31 CHI-S«?.i*aE T-iSY OF THE FERTILITY INDEX l DECRSE OF ?iu£.£.XHi *EEK N id •I: "TO '' 74-01 a.2.5 MQ/Kn VEHICLE *£RT. INGES Crfisa h N FERY> PRS HTO !*OE* CHI so N N 125). MS/KG FERT. ?4-01 H PRO NTO INDEX CHisa 230. Mt/KCi N MTO FEfi?. CnlSJ Hi N PMG MIR FEHT. : MDF. x CHISO CO MULTIPLE TREATMENT ?3 «0 .57 C.OC 2S 40 .63 .05 27 40 .AT »*e 22 40 .55 u.OG i:4 *0 .? 7 i.37 £ ?7 i9 .6% 3£ 40 .an .7i 23 40 .57 .72 23 •a .57 .72 27 jS .*<» .6 0 J 26 40 .6% £3 tO .i.7 ,ci 20 40 .50 1.29 76 40 .65 .OSs 32 *3 .HP 1.57 « 27 »0 .67 o.co B.OO e.oo 25 40 .63 .05 23 40 .7 5 .8 4 aa 40 .70 0.90 27 «0 .• '7 .06 5 S<> 40 .72 O.flS 25 40 .63 .51 25 40 .63 .SI 30 40 ,75 0.00 30 «1 .7S 0.00 6 2* 40 ,72 ».co s.os 26 40 .70 0.10 23 40 .7 8 1.3.' 32 40 .ao .2S 2F J'» .71 .01 >9 4n .•?? .0* 21 4G .52 2.61 2* 40 ,7* .6 0 c~> 36 .* 7 t.OI 33 49 .»2 t.ta ?<» 40 .72 .28 30 »0 ,*S ,0? if6 36 .7? .no .?(> ' • it 32 44 41 .? 7 .9 8 �T a b l e 3? AVEKWE IMPLANTS PER PHSGNANT FEMALE - PA3ATHION MEEK CONTROL 74-01 62.5 MG/KO 7*-01 125. M 5 / « 6 74-C1 250, M<;/KI; TF« MULTIPLE THEATNfcNT 1 23-11.52 266/ 25=10 . * 6 3!*/ 27-11 .63 22*/ 23=10 .IS* 316/ 29=10.90 2 tr 265/ 305/ 27x11.30 337/ 32=10 . 3 5 250/ 23-10 ,H7 23d/ 3 23--1C . 5 291/ 27-50. BS 26-10.31 25 7 / 23=11 .17 2«6/ 20-12 .30 **I 277/ 26=10 . 5 6 3»8/ 3iT=iO.S7 3 S68/ VD 4 298X 27-10.67 277/ 25-11 . 8 0 253X 23-11 .UO 311/ 2S=M .11 266X 27= W.85 5 33»X 29=11.52 299X 25=11 . 6 « 285/ 26-11 . 0 4 339/ 3 30=11 . 0 35*i/ 33=11.87 6 323/ 29-11.14 322/ 28=11 . 0 5 259/ 23-11 .it, 331/ 32-10 .3* 27?/ 27-10.11 7 323/ 29-11. !•» 322/ 29=11 .10 236/ 21*11 . * 2 339/ 29-11 . 9 6 30<i/ 27-11.33 3. 3o = ll . ? 33?/ 26=:2. 3« 8 9 •• I 38l/ 32-11.91 393/ S I G N I F I C A N T AT P LT 0.05 S I G N I F I C A N T AT P LT 0.01 INCREASED ABOVE CONTROL 33-11 .91 336/ 29=11 .<>9 35.5X �Table 33 AVERAGE DEAD IMPLANTS PER PREQ1AMT FEMALE - FAKATHIIW WEEK CONTROL 74-Cl fe2.S US/KG 74-01 12*>. MG/KG 74-01 250. «G/Ku TEM .2 «G/*C, HW.TIPLE TREATMENT 1 G.' 23= .35 IS/ 25- .72 ll/ 2 ll/ 2T- .*! !«/ 32« .44 3 it..' 26* .62 7/ 23- .30 10/ 4 16/ £7- .9 5 9/ 25s .36 5/ S !«/ 29» .53 IV ?5« S52 & 9/ 2. 9 .31 1G/ 2 . .36 6 7 2i/ 29« .72 35/ 29« a IS/ 32- .41 13/ • SIGNIFICANT AT P LT 9.OS <• SIONIFICANT »T P LT 0.01 D DECREASED BELOW CONTROL 33* 10/ 27» 2320- .41 8/ .3 4 IT/ .50 e/ .36 6?/ 29- iJ.!4** 23- .74 77/ 27* 2.85** 2b- .31 B7/ 32- 2.72** ll/ 27- 22- 42/ 28= 1.50 14/ 232. 5 .20 2* .6 5 22/ 2. 3 .39 303?= .37 */ ll/ I*/ .»4 21- 1.10 22/ 29- 6/ 3C« .52 23/ .9 3 9/ 2<». .31 .41 .73 17/ 30?T, .63 , 7e ll/ 27= .41 .20 I)/ ?*•= . 2 4 �X, Table 34 Cni-SQCAKE ITST ,r-F THE DEATH INDEX - PARATHlON 1 DEGREE OF FREEDOM VEHICLE CONTROL WEEK N N HOI PRO DEATH INDEX CHISB 74-01 N N MDI PRG 62.5 MS/KG DEATH INDEX CHISO 7*-01 N N WDI P«5 ?*-01 125. DEATH INDEX CMISQ 250. N >i HEATH • l!l PRO INDEX .? ChlSJ K N "Di P«S DEATH INDEX CHISU MULTIPLE TREATMENT 1 8 23 .5 3 0.00 6 25 .4 2 .5 2 9 27 .33 .4 0 5 22 .23 .32 25 24 .»<•> 12.49** 2 9 27 .33 0.00 11 32 .34 .04 B 23 .5 3 . 4 0 7 23 .30 .01 26 27 .9ft 20.7V** 7ft 7.8*3 ** 3 10 26 .8 3 0.00 7 23 .30 .08 8 20 .0 4 . 4 0 7 ?6 .27 .3-5 JS 3<» 4 4 11 27 .41 0.00 a 25 .32 .13 4 23 .17 2.^1 10 2B .36 .01 * tl .33 .32 25 29 .8 3 0.00 7 25 .8 2 .23 9 .6 3 .02 8 30 .27 .42 10 30 .11 .01 6 8 29 .8 2 0.00 9 28 .32 .01 7 23 • 30 01 10 32 .31 .00 10 27 .37 .22 7 12 29 .41 0.00 12 29 .41 .07 12 21 .7 5 .6 6 il 29 .38 0.00 9 2? •n .12 8 11 32 .34 0.00 11 33 .3 3 .1)3 9 29 .31 .uu 5 30 .17 1.70 10 ^fi .ia .nc 5 11 ** SIGNIFICANT AT PLT 0.01 �Table 33 3F OEAD IMPLANTS PER TOTAL IMPLANTS - PWlTHION 74-ui CONTBOL 74-0\ fc2.i 125. MG/KG 250. .? MULTIPLE TREATMENT 1 S/ 265* .03 IB' £66* .07 ll/ 314« .04 e/ 22*= .04 fc?/ 316= .20** ! / 337= 4 .04 10/ 250* .04 i7/ 23flr .07 77/ 293= .** 2.* 10/ 24fc. .4 0 B/ ?77« .03 97/ 34fl, . 5 2 " 42/ 311« .1* ll/ 266s .4 0 .6 3 Z v : s >• 305* .04 3 16/ ?68x .06 7/ 257. .03 ID/ 28Bs .06 */ 277, .03 5/ 253. . 02*D e S5/ 334. .04 13/ 299* .C4 14/ 2S5» .ns Jl/ 339= .03 2?/ 356s •- *' 323s .03 lu/ 322- .03 9/ 2 ^ 5 * .03 I*/ 331= .* 0 IT/ 273= .6 0 7 l\/ 323« .07 15/ 322= .5 0 23/ 236* .10 22/ 33<» .06 I!/ 30»i = .04 e 1 . 3313' .03 13' 393* .03 9/ 336> .3 0 6/ 355< .21 0*! ll/ 322= .03 * •• 0 SIGNIFICANT AT P LT 0.95 SIGNIFICANT AT P «.? 0.01 DECREASED SEUM COPT&OL �T a b l e 35 CHl-SQCAEUi TEST OF THE FERTILITY INDEX - PARATH ION-METHYL 1 3EGREE OF FREEDOM VEHICLE CONTROL -EEK M N PRG MTU PERT. INDEX CHISU 7»-OS PNG MTO 20 H6/KG FtRT. INDEX CNIbU 74-06 N N PRG MTU' »0 nb/Kb PERT. INDEX CHISU 74-Ci*; N N Prtfc KTD 80 Mlj/Ku TEW N i\ PKG M f D PERT, INCEX CHI SO .2 ' 1.37 29 iNOt* CH1SC* 40 .72 1.37 39 MULTIPLE TREATMENT 1 2 23 27 3 26 4 27 40 .57 0.00 29 40 .72 1.J7 24 40 .60 0.00 29 40 .9 6 0.00 33 40 .82 !.<* 23 40 .57 ,7«? 29 40 .72 .01 27 .9 6 .6 0 40 .5 6 0.00 30 40 .5 7 .4 3 32 40 .0 0 1.57 28 40 .70 .6 0 32 40 .80 1.57 40 .7 6 0.00 Jo 40 .5 9 26 37 .70 .00 £6 39 .67 .03 27 vu .o7 .06 7 39 e.,<i**i 5 29 4Q .2 7 0.00 .»2 »0 .0 8 .£8 2* *0 .6Q >b9 3j 4Q .75 B.CO 30 -»J • * 0.00 6 29 40 .2 7 0.00 33 40 .2 S .5 6 30 40 .75 O'bO 2» 4C .0 6 .9 3 £T 36 .71 .01 7 29 40 .72 0.00 36 40 .0 9 2.VS 32 40 .0 8 .c8 13 40 .2 8 .5 6 27 it, .75 .00 .5 8 .09 36 .72 .28 6 ** I 32 40 .0 6 0.00 SIGNIFICANT AT P LT 0.0! INCREASED ABOVE CONTROL 32 *0 .0 8 .6 0 35 40 .8 6 .7 3 34 40 26 �Table 37 AVEHAGE ISSPLASTS PER PREGNANT FEMALE - PAEATHION-iffill:«J. • ifcK 21) CONTROL 74-05 w.iLTIPLE 40 MG/KG Tt-O'S 80 .2 MG/K3 TRfcATHENT i £6S/ 23*11.52 30i/ 29-10,46* 26a/ 24.11.1? 303/ 29.10.45* 316/ 29=10.90 <J 33' 0. 27.11.3C 3»T/ 3.05 31.2 241/ 2 . 0 »8 31. 323/ 29.11.14 293/ 27=10.85 30s]0.20 3 ( 38' 6. 26*10.31 306/ 331/ 32.10. J4 297/ 28.10.61 348/ 32.10.87 -F 266/ 27« 9 8 .5 * 29S/ 27*10,67 41?.' 38«1«S9 27S/ 26-10.58 2Bi/ 26-10. Bl 5 334/ 29.11.52 39V 32*12.16 30C/ Z4.12.3U 36B/ 30^12.27 35&/ 30=11.87 27.10.11 6 323/ ! 323 / U 381/ * •• 29*11.14 2 . 1 1« 91. 32.11,41 366/ »0)/ 347/ SIGNIFICANT *T P LT 0.05 SIGNIFICANT AT P LT 0,01 334/ 30-11.13 279/ 24*11.62 273/ 36.11.14 357/ 32*11.16 349/ 33.12.09 306/ 27.11.33 32.10.d4* 407/ 3 . 1 63 S1. 402/ 34*11.62 322/ 26*12.38 33*11.39 �Table 33 AVERAGE DEAD IMPLASTS PER PREGNAitT rcMALE - PARATHrON-METHYi. 74-Ob CONTKOL 20 HG/Kli 74-05 »0 Mb/Kb 74-09 80 Mb/KG TtM MULTIPLE TREATMENT 1 8/ 23* 27. .35 9, 29- .31 .JO .! * 10/ 33= 2. 6 .62 8/ 30. .27 *D 16/ 27- .9 5 lb/ 3tl = 5 IS/ 29* .52 23/ a •*/ 2V. .oi 7 21/ 2. 9 e 13/ 32* 2 ll/ 3 16/ 4 8/ 24= 1.U4 * 23- . Jb 16/ .55 62/ 2<i= i | « .* .31 77/ 27 = 2.65** 2ea 1.21 S7/ 32. 2.72** 9/ 26= .35 ll/ 27= •41 12/ 30* .40 22/ 30* .73 .?<> 17/ 27= .3 6 9/ 29= 2<»» 22/ 32- .bS .7 4 b/ 26= . i« *D 32. .72 IT/ £4= .rl 20/ 33= .bl :o.-' 30= .' .i .72 7/ 36. .19 -3 ll/ 32. .34 lex 33* .48 ll/ ?7x .41 .41 ll/ 32= .4 3 18/ 35= .31 12/ 34- .35 ll/ 26= .42 Cn * •• D 25/ SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL 34/ fe/ 24= �Table 39 TEST OF THE DEATH INDEX - PABATHION-METHYL •itrt<. OLE ai.f.1. N N KOI PRO CONTROL DEATH IMOEX CM ISC 74-05 ft N *0i PRO 20 KG/KG C-£ATrt CM I in 74-Ob N h • 1 PR6 0 HG/Ku 40 DEATH iNDtX 7<t-05 * H SO YE* « «/!«-, ft N DCdlf CrUaG CnlSU .? M G /KG •ci P«G UtAT* INDEX CHI 51* NULTIPLE TREATMENT 1 $ 23 .5 3 C.09 t> £9 .S 2 .07 12 24 50 .8 5 9 29 .31 .30 25 29 .6 8 1 . 9 2 4 " 2 5 27 .3 3 0.00 9 33 .? 2 .5 0 b 23 26 .6 0 0 29 .28 .03 26 47 .6 9 20.79** a 33 3 10 26 .8 3 0.5C .27 .. 4' 14 32 »» .02 V .64 25 J2 .8 7 7.85** <• 11 27 .41 f,03 14 38 .37 .00 5 26 19 1.98 I 26 .27 .0 6 6 d7 .38 .32 * il 29 .8 3 0.00 11 32 .4 3 ,1)0 10 24 42 .00 10 30 .33 .01 1C 30 .33 .01 a 2* .28 0»00 11 33 .3 3 .5 0 6 30 27 .05 6 24 .5 2 • 01 10 £7 .37 .22 •41 0.00 6 36 •H 3. ' 4 B 32 25 1.18 13 33 .39 • 01 •i 27 .23 .12 .4 3 0,00 <* 32 .2* .JO 7 35 2u 1.10 & 34 .24 .9 4 1C 36 .6 3 .00 6 7 ; 2 a li •* C? 32 SIGNIFICAST iT f LT 0.01 �T a b l e 40 NUM3E3 OF DEAD [MPLANTS PER TOT^L IMFLWTS - PAH A T H I OS-METHYL «ttK CONTROL. 74-05 20 NG/KG 74-05 «0 KG/KG 74-0 = ItM 80 ,Z MULTIPLE TREATMENT 1 2 N B/ 265s ll/ 305s .03 9/ 301 = .0* 10/ 347. e/ 306. .03 6 25/ , 8 = .3V* id/ 303= .05 62/ 3lt>= .20** .03 B/ 241s .U3 9/ 323, .33 77/ 293» .2&*» .03 22/ 331. .a7 34/ 297s .11 8?/ 343. .5* 2. 9/ 281= .03 !>/ 266= .04 3 16/ 266s .06 4 16/ 288= .6 0 18/ 417= .04 5/ 275= 5 IS/ 334> .04 23/ 389= .06 17/ 300" .06 12/ 3fc8= .03 22/ 356= »C6 6 9/ 323= .03 20/ 366= .05 10/ 334 = .03 6/ 279= .02 17/ 2'3» .06 7 21/ 323= .07 7/ 401 = .02*D ll/ 357= .03 16/ 399s .04 ll/ 306s .34 0 13/ 381= .03 ll/ 347 = .03 18/ 407 = •<J4 12/ 402= .03 11 / 322= -03 J * •* D SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL «u2*D �Table 41 CHL-SQL'ARE TEST O? THE FERTILITY INDEX - QUISTOZ1&E. (PCKS) 1 DEGREE OF FREEDOM :c5C MS.'XG •tf.ti * »« FEBT. P3G ffj INDEX CMIS3 N M PAS HTQ IKOfc* CHtSa 7*-0» N PSS N MTO 2500 MG/KG FERT. INDEX CHISO ?<t-oe H N PRO MTO soco "M; TEM N CrllSQ Mfi/KG N PflG MTO fEBT. SHOP* c*'.sa MULTIPLE TREATMENT g , oc i 2 i f 4 0 . 0 7 .72 0.00 29 ( . j l - l 34 .62 .CO 40 .75 .6 0 31 40 .77 .6 2 29 *0 .? 7 0.00 22 40 .5 5 .7 4 26 40 .5 6 .5 0 27 39 .9 6 .03 30 2 ?6 40 . 65 3 23 »3 .57 O.OC 25 38 .6 6 .27 26 38 ,8 6 ,58 24 40 .72 1.37 32 40 .0 8 3.72 » f 43 .67 0.60 33 33 .87 3.09 30 40 .75 .2* 28 4C .0 7 0.09 27 *0 .7 6 .6 0 5 24 40 .60 0.00 19 39 .50 .4* 28 40 .0 7 .9 4 33 40 .2 8 3.91*1 30 *Q .5 7 1.42 6 2c 38 .63 0.00 33 38 .61 0.00 2 6 40 .5 6 .00 36 40 .90 6-47*1 27 38 .71 .4 2 7 *u 38 .79 0.03 27 38 .71 .26 27 40 .7 6 .8 7 28 40 .0 7 .2 4 27 36 .5 7 .02 31 40 36 .72 .02 e I 27 33 .71 C , 00 40 21 A. 00 SIGHIFICAHT AT F LT 0.05 INCREASED ABOVE COMTSDL JO 3n .T9 .8 2 .7 7 .IS 30 40 .5 7 .02 26 �T a b l f -,2 AVERAGK LMPLAOTS HER PREGSAST FEMALE - JUIYTOZESE (FCNE) CONTROL 74-OH 1350 MS/KG 74-08 2500 74-08 SOOC riu/KG TEW MULTIPLE TREATMENT 349/ 29*12.03 338/ 30 = 11. ,?7 361/ 6 31 = 11 . 5 316/ 29=10.90 303/ 26=11.65 222X 21=10.57 235/ 22=10.68 2S9/ 26= 9 .96* 293/ 27.10.65 3 ?*S/ 23=10.65 296/ 25=11.64 326/ 26=12. -54 «*I 353 / 29=12 .17*1 34B/ 32-10-87 4 309/ 27=11.44 392/ 33=11.88 349/ 30=11.63 317/ 28*11 .32 266X 27= 9.05* 5 27«/ 24=11.42 214/ 19=11. 26 342/ 28=12. <?1 376/ 33.11 . 9 3 356/ 30=11,87 6 302/ 24S12.S8 267/ 23=11.«,! 291/ 26=11.19 ** »00/ 36=11 . 273/ 27=10.11 ** 306/ 27«11.33 322/ 26»12.36"I 1 28:11.39 2 \o 319/ 7 8 346/ 292/ 30=11.53 27=10. 81 26// 3S4/ • S I G N I F I C A N T AT P LT 0.05 «• S I G N I F I C A N T 4T P LT 0.01 I INCREASED ABOVE CONTROL i:- 2T«10.63* 292/ 27=10. «1 30£/ 28=10 .79 30=11.80 336/ 31=10. H4 333/ 30 = 11 .10 �Table ii DEAD IMPLANTS PER P5EGSANT FEMALE - qiiIHIt.-7.ESz ...-Ys "<G/K& 74-08 2500 M6/K6 .2 Mfi/KG TTM 7»-Os MULTIPLE TREATMENT 31" .35 62/ ?<»* 2.1* ** fr/ 26» .23 77/ 2 . 2. 85 ** 7 29» .52 87/ 32» 2.72 ** 10/ 2e= .36* ll/ 27« .41 * .75 lb/ 33= .5 4 2?/ 3. 0 = 71 26= .35 **D 16/ 36s .44*0 I?/ 2. 7 .3 6 !/ & 27= .<-,<) ?0/ 28» .71 ll/ 27* .41 10/ 31= .32 )3/ 30: .43 ll/ 26* .2 4 i 13/ 28* ,*6 ll/ 2S> .38 12/ 30= . tO 2 5/ S6r .31 :a/ 21 = .5V 15/ 22= .8 6 9/ 23* .39 25= .52 7/ 26= .gl 15/ ^ 2/ 27* .07 15/ 33» . 5 * 4 * 7/ 30= .<-3 5 ll/ ». . .6 4 111/ 19. .53 ai/ 28= S SI/ 24s .88 9/ » JO/ 30* 1.00 1 1/ 27s .! * a 5<>/ ?T" i?/ 30 = .S7 ll/ 3 ^ 0 * •• D .70 ti/ 23= SIGNIFICANT AT i» LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELCH COXTROL ^35*0 �Tail? ii CHI-SQ'JARE TEST OF THE DEATH INDEX - QUIST'W.RN* (PCN3) 1 DECJREE OF Frl VEHICLF CONTROL U&E.K N •ni N PRG DEATH INDEX CHISO 74-06 N N • DI PKG 1250 MG/KG DEATH INDEX CHISO 74-03 N N WOI PR'J 74-oa 2SOO MG/KO DEATH INDEX sooo . 3 Mr,/KG N PHG N CiIbQ flEATh INDEX CnlSU N N •DI PKG DEATH INPF* CHJSO MULTIPLE TREATMENT 1 10 28 .3* 0.00 7 29 .24 .44 10 30 .33 .01 •* 31 .2^ .07 25 ^4 .8* 13.27 ** 2 7 26 .27 0.00 10 21 .4B 1.35 9 22 .! * .51 2 26 .08 2.15 26 £T .9ft 24.26** 3 q 23 .9 3 0.00 11 25 .44 .00 6 26 .3 2 .82 11 29 .38 .04 25 32 ."« 7.05** 4 2 27 .07 0.00 12 33 .36 5.44 * 6 3« .20 .97 7 28 .25 i.96 « 27 .3" 3.07 28 .6 4 .1* 13 33 .39 .02 10 JO .31 .00 8 26 .31 3.B6*D 12 36 .33 3.84*D 10 £7 .•*7 2.34 27 .2 5 2.01 10 28 .36 .21 9 £7 .IS .07 9 31 .9 2 .89 12 30 .40 .00 2ft .33 .03 5 9 24 .3fl 0.00 9 19 .47 6 15 24 .63 0.00 6 23 .26 7 8 30 .27 0.00 10 27 .37 .31 a 12 27 .4 4 0.00 13 30 .43 .03 * ** D SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL .12 4.91 *D 13 14 10 �Table 45 .Tfflat; OF DEAD IMPLANTS PER TOTAL IMPLANTS - QUINTiSZiiN? ,.'£ f.«. 74-08 1250 COKTROL 2500 MG/KQ 74-88 ,2 MG/SG TF- 5000 MULTIPLE TREATMENT 1 !3/ 319- .04 ll/ 3*9= .03 12/ 33»> 2 »/ 303- .03 1 . 222= 2' .OS IS/ 235. 3 9/ 245- .04 13' 296- .4 0 7/ 326= 4 2/ 309- .01 IS/ 392- .04** 7/ 349« .03 t,gf 316* 1 .20** 6/ 259- .02 77/ 293= .26** • 1 2 »D ) 15/ 353- .04 B7/ 34«- .* 2* .02 10/ 317- .03* ll/ 266* .4 0* .6 0 16/ 376s .4 0 22/ 3^6* .Oft .03 *D 16/ 400- .* 0 IT/ ?73= .6 0 .6 0 ll/ 2?»» .04 10/ 214- b SI/ 302- .07 fl/26?- 7 30,' 3 «6- .09 ll/ 287- .04 16/ 292* .05 20/ 302= .07 ll/ 306 = .04 .0? I*/ 354 = .5 0 10/ 33«> •03 13/ 333= .04 ll/ 322- • 03 a I?/ 2?2 • SIGNIFICANT AT P LT 0.0«> •* S I G N I F I C A N T AT ° LT 0.01 D DECREASED BELOW COHTXOL .02*0 21/ 342= ll/ 361- S e .OS .4 0 9/ 291 = �Table 46 CHI-SQUARE TEST OF THE FERTILITY INDEX - PHOBATE 1 DEGREE OF FREEDOM • EEK VEHICLE. CONtHUL N N PRO HTO FF.RT. INDEX CHISU 74-04 K N PKb MTU MG/Mi •> FtUT. INDEX CHI SO 74-u* N N PHG MTU 10 Mb/Kb FEBT. INDEX CnlbQ MO/KG 20 N N PHG MTO PERT. IfcDE.lt CHISU N PHli H F U fEKT. INDEX CHISU U) MULTIPLE THEATNENT 1 23 40 .57 0.00 2J 40 .57 .US. 21 40 .52 .t>5 . 4 40 .0 6 o.oo 29 40 .72 1.37 2 27 39 .9 6 0.00 30 40 .75 .10 2J 40 .i>7 .72 29 40 .72 .01 27 J* ,(r,9 .Ob 3 26 40 .5 6 0.00 2b 40 .65 .US 24 40 .60 .liS 31 40 ,77 ."« 32 40 .0 8 1.57 4 27 40 .7 6 0.00 26 40 .65 0. 00 26 40 .65 O.UO 32 40 .0 8 l.OJ 27 40 .*" .06 .72 0.00 #> 40 .3 6 .» !! 27 40 .7 6 .6 0 31 40 .77 .07 30 40 .75 25 40 40 .5 6 .3 4 33 40 .2 8 .S 6 21 3* .71 27 Jb .75 .01' 20 JO .72 .21* b 29 40 b 29 40 .72 O.OU .3 6 .31 26 7 29 40 .72 0.00 30 40 .75 O.UO 29 40 .72 .06 Ji 40 .0 8 B 32 40 .9 8 0.00 26 40 .65 l.»7 29 40 .72 «<B 30 40 .75 1.95 .07 0.00 .01 �Table 47 AVBRAGE IMPLANTS PER PREGNANT FEMALE - PHOIiAT2 7«-0* CCK'SOL 10 NO/Rti .2 TtM 7»-0« MULTIPLE TRLATHtNT 1 ?65/ 5 23-11 . 2 2* I/ 23»i0.te* 221/ 24-11 .21 316/ 29-10.90 30i/ 27-11 .30 326/ 30*10.37 2SS/ 10 .52* 212 .11 . 9 3 0 269/ i 3U7/ 29-10 . 9 5 293/ 27-10.05 3 268/ 2 . .31 6 10 29«/ 26-11.50*1 257/ 2 .10 .71 * 34i/ 31-li .03 3«B/ 32=10.87 * 6 288/ 27.10. 7 2S>1/ 26-11.19 277/ 353/ 32-11 .03 266 / 27- 9 8 .5 S J3*/ 2 .ii .52 * 2?B/ 2b=S1.12 31Z/ 10 6 26- . 5 27.il eb6 6 j2 )V 2 . .1* 9 11 305/ 7 32j/ 29.11 .1* J36/ B .»Bi/ 32- il .91 jib/ AT P I.T 0.05 ** SJG'v^ICANT AT f LT C.C1 1 IHCBEASED ABOVE CONTROL 25.12.20*1 30.11.20 2.:.2 6'21 295/ 3l?/ 33b/ 3 26- li . 5 369/ 31-11 .90 356/ 30-11.87 384/ 33.11 .79 273/ 27.10.11 27.11.33 26-12.3B 10 . 3 * »07/ 35-11 . 3 6 306/ .5 6 J24/ 30*10 .do* 32^/ **- 29- 11 �Table 48 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - PHORATE MG/KG CONTROL r<t-Ut 10 Mti/nG f»-0» 20 Mb/Kb .2 FtM MuLTIfLE THEATHfcNT 1 a/ 23* .35 9/ 23" .39 10/ 21* .*H 3/ 2** .13 6?/ 29= 2.1* ** 2 ll/ 27* ,»1 lt>/ 30* .53 «/ 23* .U 17/ 29* .9 5 77/ 27» 2.85 ** 3 16/ 26* .62 1J/ 26* .50 U/ 24* .SO IS/ 31s .B * 87/ 32- 2.72 ** * 16/ 27s, .59 15/ 26s .SB 26/ 26s l.DO 12/ 32* .6 3 ll/ 27= .41 3 IS/ 29s .52 13/ 25s .S2 ll/ 27* 13/ 31* ,»2 22/ 30* .73 6 9/ 29s .31 13/ 25s .52 7/ 26* .27 7/ 33* .21 30* .43 2i/ 29* .76 7/ 35* 26* .38 26/ 29. .90 ll/ 33* 7 21/ 29* .72 U/ 8 13/ 32s ,»1 10/ • •• D S I G N I F I C A N T AT f LT 0.05 S I 6 N I F I C A H T AT P LT 0.01 DECREASED BELOW CONTROL .! * .37 17/ 27s .63 IS/ 27* .41 ll/ 26* .4? �Table 49 CHI-SQl'ARE TEST OF THE DEATH INDEX - PHORA7E 1 DECREE OF FREOXtt i?Et* ^i.'ilCLE CONTntn. N N »DI Pkb QEATrt INOex V«. ~%K 5 MCi/Kb f. PMC CH;Sw SftDEx CHIiU 74-U* N N • 01 PR6 10 HG/Ku 0£ATH 1NUEX T -.;» 20 N N CH1SU N OE4TM iHliCa .?. HG/KG TEM W<!/KB " N CniSu UEATH INDEX CHI SO HOUTlPLt TREATMENT 25 <:.-> .86 12.49 ** .37 26 ^7 .96 20.7*** .32 .04 25 32 .78 7.b5** 34 .34 .05 8 ^7 .30 .32 1 a 23 .35 w.OO d 23 .J5 .10 7 21 .33 .US 2 24 .00 3.45 2 9 27 .33 U.tO 13 30 ,»3 ,«!S 2 23 .09 3.07 13 2V .45 3 10 « .33 3.00 T s!fe .27 .JS U £4 .50 .,49 10 31 4 11 27 .*k a. 00 11 26 .42 .03 IS) 2b .5tt .»2 11 1 5 il 2<s .38 I . CO Id 25 .48 ,£2 7 2T .26 .46 11 31 ,3S .01 10 JO .33 .01 6 0 2S .28 0.00 10 25 * 40 .46 7 26 .27 .Ob 7 33 .21 .00 10 <*7 .37 .22 7 12 <!9 .41 IJ.CO S 30 .30 .41 15 29 .52 ,*B » 3* .!« 4.66*D 9 e? .33 .12 b,UO 4 26 ,35 .07 14 29 .48 .71 a 30 1C ft, .38 .00 a * ** D ii 3£ .3* SIGNIFICANT AT F Lt 0 0 . 5 SIGNIFICANT AT P LT 0 0 . 1 DECREASED BELOW CONTROL .27 .19 �T a b l p 50 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - PRORATE • EEK MG/Ku CONTROL /4-0* 74-04 10 20 Mb/Ho MG/KQ ICM MULTIPLE. TRtATMtNT .01 *D 62/ 316= .20 ** 17/ 307 = .06 77/ 293= . 6 ** 2 .) 15 IS/ 341 = .04 67/ 348 = .25 ** 26/ <iT7« PU 9 12/ 3S>3= .03 ll/ 266= .0* .05 ll/ 312= .04 13/ 369= .04 Z2/ 356= .06 13/ 3ub = .0* 7/ 29j= ,o2 7. 1-9= .' .02 )7/ 77.1= .6 0 .07 13/ 336= .04 i'i./ 317= .' 0 7/ 407 = .02*0 ll/ 306= .4 0 .3 0 10/ 31b= .03 26/ 33b= . l)B ll/ 324= .03 ll/ 322= .03 .04 10/ 221= .U= 3/ * , . .* lb/ 32b= .05 4/ 2b5= .0^ .Ob 13/ 299= .04 12/ 257= 16/ 288= .06 lb/ 291 = .OS S IS/ 334= .04 13/ 278= 6 9/ 323 = .03 7 21/ 323= 8 13/ 361* 1 8/ 265 = .3 0 2 ll/ 305» .04 3 6. 16/ 2 8 4 • •• D 9/ 241= SIGNIFICANT AT P UT 0.05 SIGNIFICANT AT P UT 0.01 DECREASED BELOW CONTROL �Table 51 DMA REPAIR SYNTHESIS ASSAY OF Monocrotophos (dpm/u« DNA) 4NQO (M) Monocrotophos (M) Sample 1 _£_. tt 65 io-7 io-6 io-8 IO"4 lO-3 46 31 54 70 64 1354 46 46 86 1273 10-5 2 35 42 3 39 39 25 40 36 69 1306 4 34 34 25 40 40 51 975 5 30 26 53 64 64 972 6 20 32 -f 38 46 _.t 32 37 50 92 71 1135 —f 47 1169 SD 7 7 10 7 14 15 168 SE 6 3 5 3 & 6 69 Mean 9* Sample deleted from calculations because of low DNA value. Only five samples used. Cell culture and exporiosontal condj.t_ions T-25 flask cultures of passage 24 WI-38 cells were initiated in medium containing iO% serum. The medium was replaced with medium containing 0.5% sarv.m on day 5 following initiation and subsequently on days 11 and 15. Tho assay was conducted on day 22. Hyriroxyurea (10~*M) preincubation = 1 hour. Compound exposure time = 3 hours. 3 li~TdR added with compound. 3 H-TdR incorporation = 1 uCl/ml (S.A. ==6.7 Ci/mmole), 3 hours. Postincorporation incubation = medium containing TdR, 3/4 hour. Cells were removed with IN NaOH. 1 minute, 70°C. DNA was oxtfflcted by the PCA-hydrolysls procedure and measured following reaction with dtphenylamlne. S«j;aUvu control and compounci solvent -~- 0.5% EtCH. 78 �Table 52 DMA REPAIR SYNTHESIS ASSAY OF Monocrotophoa WITH METABOLIC ACTIVATION (dpm/ng DMA) _Monocrotop_ho8_ OjO DMN (M) 0_ 10-^ 10-J j°Jll_. Sample 1 55 67 43 87 206 2 54 66 48 84 220 3 52 43 52 82 223 54 59 48 84 216 2 14 4 2 9 1 8 2 1 5 Mean SD S E 5 X 10"' Cell £ulture_and experimental conditions T-25 flask cultures of passage 24 WI--38 cells were initiated in medium containing 10<jfe serum. The medium was replaced with medium containing 0.5% serum on day 4 following initiation and subsequently on day 10. The assay was conducted on day 16. Hydroxyurea (10~2M) preincubation - 1 hour. Compound exposure time = 1 hour, with the 9,000 g fraction of a mouse liver homogenaie. 3 H-TdR added with compound. 3 H-TdR incorporation = 1 p,Ci/ml (S.A. =6.7 Ci/mraole), 4 hours. Post.incorporation incubation = medium containing TdR, & hour. Cells were removed with IN NaOH, 10 minutes, 22°C. DNA was extracted by the PCA-hydrolysis procedure and measured following reaction with diphenylamine. Negative control and compound solvent = 0.5^ EtOH. 79 �Table 53 DNA REPAIR SYNTHESIS TESTING OF BROMACIL (dpm/ug DNA) Broroacll (M) _4NOO_(M) -- J>*. IP"7 10~6 10"5 ID-'*1" 10-3t 195 153 212 230 207 201 148 129 212 215 121 248 178 179 231 144 152 204 144 137 200 165 28 12 Sample ! 2 3 4 5 6 Mean SD SE 217 298 218 48 19 156 187 182 251 199 32 13 218 184 220 193 38 15 240 178 209 34 14 Negative control and compound solvent =0.5% DMSO. Slight precipitate observed at 10~3 M and 10™1* M 80 2670 2850 107 2688 2702 80 2438 74 87 50 20 2662 2668 105 134 55 �Table 54 DNA REPAIR SYNTHESIS ASSAY OF BROMACIL WITH METABOLIC ACTIVATION (dpm/pg DNA) Bromacll (M) DMN (M) 0 10~7 10-6 10-5 10-* ID'3 113 102 141 191 112 174 91 102 120 161 400 117 110 102 110 178 397 102 149 131 158 189 82 91 135 218 152 104 167 136 170 96 126 190 145 165 100 119 165 141 SD 41 30 13 37 23 SE 17 12 5 15 9 185 141 133 155 24 10 5 X 10"2 Sample 1 2 3 6 Mean * vample lost. 81 * 529 645 448 484 105 50 �Table 55 DNA REPAIR SYNTHESIS ASSAY OF CAOODYLIC ACID DNA) Cacodylic Acid (If) 4NQO (H) o* io-7 io-3 10-s io-4 1 . at 0 Sample 1 61 41 47 _-* 31 36 1891 2 31 48 27 -_* 27 19 1681 3 32 59 25 63 30 45 2418 4 L8 32 68 29 19 2245 5 25 39 38 __* 23 22 38 1430 35 22 __* 29 28 36 2275 Mean 34 40 34 46 28 32 1990 SD 15 13 10 23 3 11 387 SE 6 5 5 11 1 4 158 6 Negative control and compound solvent =0.5^ DMSO. t Slight lowering of pH at 10~3 M. 4' Sample lost. 82 10-6 �Table 56 DMA REPAIR SYNTHESIS ASSAY OF CAOODYL1C ACID WITH METABOLIC ACTIVATION ONA) Cacodylic Acid (M) DMNjM) 0* io-5 10- « io-3 Sample 1 44 25 21 29 381 2 30 33 22 25 384 3 23 39 28 21 339 Mean 33 32 24 25 368 SD 11 7 4 4 25 SE 6 4 2 2 15 Negative control and compound solvent =0.5$ EtGH. 83 5 X 10~2 �Table 57 DMA REPAIR SYNTHESIS ASSAY OF CAFFAN DMA) Gaptan (M) 8 io- 4MQO(M) fl --12LL io- 10~ 5 10-" IP"5 Sample l 37 43 41 74 81 8 924 2 64 58 53 60 81 6 947 3 76 50 68 52 57 7 1106 4 76 60 73 37 51 5 801 5 63 61 56 50 72 5 760 6 66 44 66 82 65 6 884 Mean 64 51 59 59 68 6 904 SD 14 8 12 16 12 1 122 SE 6 3 5 7 5 Negative control and compound solvent = 0.5$ DN5O. 0.4 50 �Table 58 DNA REPAIR SYNTHESIS ASSAY OF CAPTAN WITH METABOLIC ACTIVATION DNA) )_ io~5 10-* 5**)l.l*?i io-3 5 x io-a Sample 1 30 53 89 7 i 2 40 __t 50 73 5 323 48 71 5 384 35 50 77 6 353 SD 7 2 9 1 43 SE 5 1 5 0.6 31 3 Mean Negative control and compound solvent =0.5$ DMSO, Sample lost. 85 �Table 59 DNA REPAIR SYNTHESIS ASSAY OF CHLOROPYRIFOS (dpm/,ig/DNA) Chloropyrifos 4NQO (M) io-5 JL _10-7_ 115 2801" 282* 2 1 143 " 129 93 110 67 3 98 102 84 60 <t 72 95 64 110 _„* 52 —* 68 5 97 89 99 98 64 37 1208 6 78 - 98 85 86 79 49 1209 Mean 92 103 85 101 64 66 1339 SO 17 15 13 11 9 31 220 SE 7 7 6 5 4 15 98 Sample i 10-6 TO-5 KM __* 6.1 HT3 109 __* Negative control and compound solvent = 0.5^ DMSO. Sample deleted from calculations because of low DNA value. Sample loKt. 1337 —* 1721 1220 �Table 6G DNA REPAIR SYNTHESIS ASSAY OF CHLOHOPYRIFOS WITH METABOLIC ACTIVATION DNA) DMN (M) Chloropyrifos (M) 10- 10-4 10-" 5 X 10~2 Sample 1 72 79 70 52 355 2 75 65 75 71 349 3 55 67 63 79 384 Mean 67 70 69 67 363 SD 10 8 6 14 18 SE 6 5 4 8 11 Negative control and compound solvent =0.5$ DMSO. 87 �Table 61 DEN REPAIR SYNTHESIS ASSAY OF DINOSEB (dpm/^g DNA) Dinoseb (M) 4NQO (M) _°: .10-' 115 101 67 103 106 2 143* 101 68 100 100 3 96 112 54 116 79 1721 4 72 61 58 62 66 1220 5 97 57 63 60 67 1208 6 78 58 73 60 76 1209 Mean 92 82 64 84 82 1339 SO 17 26 7 25 17 220 SE 7 11 3 10 7 98 Sample 1 _L2I± ' ! • 11 •• • _iP±+ Negative control and compound solvent = 0.5$ DMSO. Suggestion of precipitate at 10-" M. Sample deleted from calculations because of low 'DMA value. § Sample lost. 88 HlL. 1337 __ § �Table 62 DMA REPAIR SYNTHESIS ASSAY OF DINQSEB WITH METABOLIC ACTIVATION (dpm/pg MA) - «M»._.n«, 0* Dinoseb (M) , J. _v 1 * . . w- _ _ __ . 10-5 10~" 10-3 DMNjM) 5 X 10-a Sample 1 72 93 76 71 355 2 75 81 80 64 349 3 55 51 58 89 384 Mean 67 75 71 74 363 SD 10 22 12 13 18 SE 6 12 7 8 11 Negative control and compound solvent =0,5$ DMSO. 89 �Table 63 DNA REPAIR SYNTHESIS ASSAY OF DSMA (dpm/>g DNA) DSMA j>! (M) io-7 ig-g 1Q-S 10- y io-3 Samp Le I 67 51 79 86 100 62 2 58 54 67 64 155 49 3 44 36 99 64 35 58 4 55 36 400* 45 44 59 5 79 62 74 53 55 68 6 105 32 75 107 89 69 Moan 68 45 79 70 80 61 SD 22 12 12 23 45 7 SE 9 5 5 9 18 3 Negative control and compound solvent = H2O. Sample deleted from calculations because of low DNA value. 90 �Table 64 DNA REPAIR SYNTHESIS ASSAY OF DSMA HTITH METABOLIC ACTIVATION (dPm/|ig DNA) DSMA DMN__(M) (M) io-5 10 -< 10-3 5 X 10" Sample 1 44 28 25 38 381 2 30 36 29 36 384 3 23 21 32 28 339 Mean 33 29 29 34 368 SO 11 8 4 5 25 SE 6 4 2 3 15 Negative control and compound solvent - 0.5$ EtOH. 91 �Table 65 DNA REPAIR SYNTHESIS ASSAY OF FENTHJON (dprn/u-g DMA) ', L Fenthion (M 0* Sample 1 89 2 3 43 107 4 10-6 10-5 4NQO (M) 10-j 10~3t io-s 65 105 154 37 64 2983 337* 34 67 2272 83 85 40 36 2552 62 46 54 63 63 4059 5 61 34 102 31 44 1728 6 94 51 44 —§ 33 1893 Moan 76 64 88 41 51 2583 SO 24 26 44 13 15 857 SE 10 11 18 5 6 350 •a Negative control and compound solvent ~ 0.5$ EtOH. Precipitate observed at 10"3 M. ' Sample deleted from calculations because of low DNA value. § Sample lost. 92 �Table «6 DNA REPAIR SYNTHESIS ASSAY OF FENTHIOH WITH METABOLIC ACTIVATION DNA) Fenthion J.M) _0?. SSLUtiL 10-JL 10~ * 10 ~JL 5 X 10~a Sample 1 55 54 30 51 206 2 54 50 46 64 220 3 52 42 64 63 223 54 48 57 60 216 SD 2 6 10 7 9 SE 1 4 6 4 5 Mean Negative control and compound solvent - O.S^t EtOH. 93 �Table 67 DM REPAIR SYNTHESIS ASSAY OF FOLPET DNA) Folpet (M) 4NQO(M) * Sample 1 37 43 63 45 82 29 924 2 64 58 62 54 58 25 947 3 76 91 108 52 92 31 1106 4 76 83 91 92 65 26 801 5 63 107 72 70 85 31 760 o 66 60 84 104 107 29 884 Mean 64 73 80 71 82 28 904 SO 14 24 18 25 18 2 122 SE 6 10 7 10 7 1 50 * Negative control and compound solvent =0.5% DM80. 94 �Table 63 DNA REPAIR SYNTHESIS ASSAY OF FOLPET WITH METABOLIC ACTIVATION (dpm/yg DNA) DMN JM^ _F°lP_et ($ 0* 1 0~" ID-- ID'3 5 X 10~2 30 49 49 —_ T 40 __t 54 63 82 40 323 58 384 35 52 49 353 SD 7 9 43 SE 5 3 2 98 81 18 10 5 31 Sample 1 2 3 Mean 54 Negative control and compound solvent Sample lost. 95 m 0.5% DMSO �Table 69 DNA REPAIR SYNTHESIS ASSAY OF AZINPHOS-METHYL DNA) Azinophos-raethyl (M) JL! 1^ 2 10-" 102 145 99 99 .115 3 77 4 4NQO (M) io-5 10- « 12± 264* 51 55 804 103 99 105* 39 924 96 71 82 55 33 629 97 125 100 __§ 80 85 34 856 5 85 93 79 57 97 761 6 111 n 72 56 68 35 897 95 108 89 79 63 49 822 12 25 16 15 14 25 87 5 10 7 7 6 10 36 Sample 1 Mean SD SE 10- * Negative control and compound solvent - 0.5% DMSO. t Precipitate observed at .10"3 M. t Sample delated from calculations because of low DNA value. 5 Sample lost. 96 �Table 70 DNA REPAIR SYNTHESIS ASSAY OF AZINPHOS-METHYL WITH METABOLIC ACTIVATION (dpm/yg DNA) E>MN (H^ _0_* -!E1 -.19.7.1 JLSZi 5__x_ur_* --1" Sample 1 30 70 75 42 2 40 66 65 55 323 —f 35 78 41 54 384 71 60 50 353 SO 7 6 18 8 43 SE 5 3 10 4 31 3 Mean itNegative control and compound solvent =0.5^ DMSO. Sample lost. 97 �Table 71 DNA REPAIR SYNTHESIS ASSAY OF MALATHION (dpm/pg DNA) Mala thion (M) 4NQO (M) _o*. 10- lOTi 10-5 10-" 1C! 110 128 124 SE 6 9 4 29 12 90 126 67 113 91 156 107 31 13 33 130 116 127 143 128 144 78 74 119 132 127 112 SD 123 125 106 100 114 138 118 14 Sample 1 2 3 A 5 6 Mean 111 86 133 116 110 111 15 6 * Negative control and compound solvent • 0.5% EtOH. 98 34 23 44 39 40 35 7 3 1943 1626 1538 1264 1737 1651 1626 225 92 �Table 72 DNA REPAIR SYNTHESIS ASSAY OF MALATHION WITH METABOLIC ACTIVATION (dpm/jig-HJNA) Halathion (H) 7o-r DHN (Mj_ 0* io-5 Sample 1 55 48 46 38 206 2 54 49 52 48 220 3 52 62 55 223 54 51 37 41 5 6 9 3 4 5 Mean SD 2 53 8 SE 1 5 1 0™ * Negative control and compound solvent =0.5^ EtOH. 99 5 X IO-2 216 �Table 73 DNA REPAIR SYNTHESIS ASSAY OF METHOMYL DNA) Methomyl (M) 0* io-7 Sample 1 135 2 133 3 165 —f 100 4 72 S 6 io-« 10~s 4MJO (M) io-« ID'3 10"6 116 117 126 88 1442 133 108 116 69 1544 129 97 122 69 1518 98 97 115 109 69 1385 104 85 103 109 70 1423 103 95 93 116 139 130 61 118 94 112 118 71 SD 32 6 17 115 14 f — 1462 9 9 67 SE 13 3 7 6 4 4 30 Mean 95 Negative control and compound solvent =0.5^ DMSO. Sample lost. LOO �Table 7-\ DNA REPAIR SYNTHESIS ASSAY OF METHOMYL WITH METABOLIC ACTIVATION DNA) Methpmyl (M) DMN(M) 0* 10-° 1C-4 ID'3 Sample 1 44 26 22 24 381 2 30 25 20 25 384 3 23 22 26 30 339 Mean 33 25 23 26 368 SO 11 2 3 3 25 SE 6 1 2 2 15 ft Negative control and compound solvent - 0.5$ EtOH. 101 5 X 10-a �Table 75 DMA REPAIR SYNTHESIS ASSAY OF MDNUUON DMA) Monuron (M) _°! Sample 1 135 2 133 3 165 4 io-7 113 __* io-6 4NQO (M) io-5 10-' 86 97 83 48 1442 93 88 72 47 1544 10-3' ^ » ^ W « * H 89 77 81 46 1518 72 129 __* 83 75 82 49 1385 5 104 118 92 77 84 45 6 103 131 110 109 85 38 1423 _J 118 123 92 87 81 46 1462 32 8 9 14 5 4 67 13 4 4 6 2 1 30 Mean 3D SE Negative control and compound solvent -'• 0.5% DMSO. Precipitate observed at 10"3 M. t Sample lost. 102 , �Table 76 DNA REPAIR SYNTHESIS ASSAY OF MONURON WITH METABOLIC ACTIVATION DNA) Monuron (ll) .£ 10-" 10- «_. _12I',. 5 x 10~a -t Sample J. 30 78 81 74 2 40 88 68 74 323 3 ~r ' 92 63 88 384 35 86 71 79 353 SD 7 7 9 8 43 SE 5 4 5 5 31 Mean w Negative control and compound solvent =0.5^ DHSO. Sample lost. 103 �Table 77 DNA REPAIR SYNTHESIS ASSAY OF MSMA (dpm/jig UNA) MSMA < k 4NQO (M) 01 ) 10-8 10-4 io-3 10-" 108 60 68 902 84 93 53 93 52 39 1241 44 114 68 61 66 61 1380 4 55 1 235 " 50 63 66 67 990 5 79 75 75 48 60 1087 8 105 53 66 __* 50 59 75 971 Mean 68 73 77 60 64 62 1095 SD 22 29 23 9 16 12 182 SE 9 13 9 4 7 5 74 0* 10-' Sample 1 2 67 38 58 3 10-" Negative control and compound solvent = H20. Sample deleted from calculations because of low DNA value. Sample lost. 104 �Table 78 DNA REPAIR SYNTHESIS ASSAY OF MSMA WITH METABOLIC ACTIVATION (dpm/^g DNA) MSMA (M) -.0! DMN (M) JL°:L 10~5 io-3 JLJLlfiL1. Sample 1 44 27 21 32 381 2 30 25 35 25 384 3 23 21 34 24 339 Mean 33 24 30 27 368 SD 11 3 8 4 25 SE 6 2 5 3 15 •ii- Negative control and compound solvent =0.5^ EtOH. 105 �Table 79 DNA REPAIR SYNTHESIS ASSAY OF PARATHION (dpm/pg DNA) _Q^ Sample 1 2 3 4 5 6 Mean SD SE iQlL Parathlon (M) IP"6 10-5 123 127 151 221 125 106 100 114 138 118 14 6 129 135 124 137 145 133 7 3 155 135 200 160 210 169 29 12 129 157 137 155 126 154 35 14 4NQO (M) IP"5 io± 10- 102 94 90 104 93 116 102 1626 1538 1264 1737 93 100 10 4 1651 1626 225 92 83 72 93 71 86 13 5 * Negative control and compound solvent - 0.5% EtOH. 106 3 1943 �Table 80 DNA REPAIR SYNTHESIS ASSAY OF PARATHION WITH METABOLIC ACTIVATION DNA) Parathion £M) DMN (H) 0* 10-s 10- « 10-3 Sample 1 44 33 42 20 381 2 30 33 32 23 384 3 23 35 30 30 339 Mean 33 34 35 24 368 SD 11 1 6 5 25 SE 6 1 4 3 15 Negative control and compound solvent -0.5% EtOH. 107 5 x 10- * �Table 81 DNA REPAIR SYNTHESIS ASSAY OF PARATHION-METHYL DNA) Parathion-Methyl (M) . £ 4NQO (M) 10-7 10~8 10~B 10~4 10~3 t 10~5 Sample 1 36 36 33 34 44 40 2 34 38 32 .55 44 23 411* 781 3 8G 49 28 31 53 28 782 4 94 56 27 52 41 29 858 5 53 49 35 43 40 29 1296 6 112* 46 41 44 27 ,1103 Mean 61 .6 4 33 85 50 44 ,2 .8 964 SO 28 8 5 20 .5 6 227 SE 13 3 2 8 2 3 102 T,* Negative control and compound solvent = 0.5% EtOH. Precipitate observed at 10~3 M. * Sample deleted from calculations because of low DNA value. 108 �Table 32 DNA REPAIR SYNTHESIS ASSAY OF PARATHION-METHYL WITH METABOLIC ACTIVATION DNA) Parathion-Methyl (M) •M- DMN (M) 0 10- 10-* 10"" "^ Sample 1 55 44 65 51 206 2 54 54 52 48 220 3 52 57 37 45 223 54 52 51 48 216 SD 2 6 14 3 9 SE 1 4 8 2 5 Mean Negative control and compound solvent =0.5$ EtOH. 109 5 X 10~a �Table 83 DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB) DNA) PCNB (M) j£ Sample 1 2 61 3 32 4 18 5 25 6 35 31 Mean 34 SD 15 SE 6 JO"7 io-« IP-6 10-* 35 43 33 21 27 23 22 21 18 37 44 30 31 23 18 19 30 16 18 21 38 20 27 32 22 27 20 39 20 27 35 9 4 27 23 20 26 5 8 4 6 2 3 2 3 Negative control and compound solvent =0.5$ DM90. Precipitate observed at 10~3 M. 110 . 10"3 �Table 84 DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB) WITH METABOLIC ACTIVATION DNA) 10 5 X 10 Sample 1 2 72 75 95 106 355 75 79 71 72 349 3 55 84 73 49 364 Mean 67 79 80 76 363 3D 10 5 13 29 18 SE 6 3 8 17 11 Negative control and compound solvent = 0.5% DHSO. Ill �Table 85 DNA REPAIR SYNTHESIS ASSAY OF PHORATE DMA) . Phorate (M) io-3t 1' 0" 27 56 411* 17 50 45 781 22 42 55 782 26 107 _.§ 43 43 50 858 53 40 44 52 37 61 1295 112* 56 26 77 39 59 1103 Mean 61 43 52 39 40 55 964 SD 28 13 32 23 8 6 227 SE 13 5 14 9 3 2 102 0* 1 36 58 38 25 2 34 45 44 3 86 31 4 94 5 6 Sample 10-7 J£± 1Q-8 J£± Jf. Negative control and compound solvent = 0.5^ EtCH. Precipitate observed at 10"3 M. * Sample deleted from calculations because of low DNA. value. § Sample lost. 112 �Table 86 DNA REPAIR SYNTHESIS ASSAY OF PHOKATE WITH METABOLIC ACTIVATION DNA) Phoraice. (ll) DMN (M) _wrf_ 5 X 10-' J>! _™L* Sample 1 55 45 43 37 206 2 54 59 41 35 220 3 52 63 39 38 223 54 55 41 37 216 2 10 2 1.4 9 1 6 1 08 . 5 Mean SD SE -12™ Negative control and compound solvent =0.5^ EtCH. 113 �Table 87 DNA REPAIR SYNTHESIS ASSAY OF SIMAZItiE (dptn/ug DHUO Slmazlne (M) 4NQO (M) _ai Sample 1 2 3 4 5 6 Mean SD SE io-7 IP"6 10"5 iEi IP"3 10"5 195 153 212 230 217 298 218 48 19 165 91 131 138 152 113 132 27 11 151 171 152 146 179 213 169 25 10 195 190 312 290 237 305 255 55 22 177 193 253 281 166 161 205 50 20 369 208 233 165 205 t 2670 2850 2688 2702 2435 2662 2668 134 55 * Media control and compound solvent » 0.5% DMSO. t Sample lost. 114- 236 78 32 �Table 38 DNA REPAIR SYNTHESIS ASSAY OF SIMAZINE WITH METABOLIC ACTIVATION g MA) Slmazlne __(M) 5 X 10"* Sample 1 72 57 64 59 355 2 75 58 60 58 349 3 55 64 61 76 384 Mean 67 60 62 64 333 SD 10 4 2 10 18 SE 6 2 1 6 11 Negative control and compound solvent = 0.5^> DHSO. 115 �Table 89 DMA REPAIR SYNTHESIS ASSAY OF TRIFLURALIN DHA) Trlfluralin (M) 4NQO (M) 0* io-7 10-e io-» io-« 10-' 10- • Sample 1 56 42 53 51 71 639 2 68 29 26 _J' 152* 97 123 3 51 78 48 68 158* 112 570 4 45 51 76 89 78 83 894 5 50 47 79 97 56 80 663 6 29 41 59 57 43 53 986 Mean 50 48 58 73 62 87 812 SD 13 17 22 19 28 26 222 SE 5 7 10 9 12 11 91 Negative control and compound solvent =0.5$ EtCH. Sample lost. Sample deleted from calculations because of low DMA value. 116 1125 �Table 90 DNA REPAIR SYNTHESIS ASSAY OF TRIFLURALIN WITH METABOLIC ACTIVATION DNA) Trifluralin (M) 0* DMN (M) 10-' 10-* 10-' 5 X 10~a Sample 1 2 72 67 79 51 355 75 58 55 74 61 .._t 349 3 64 79 Mean 67 66 74 56 363 SD 10 8 9 7 18 SE 6 5 5 5 11 * Negative control and compound solvent =0.5% DM30. Sample lost. 117 384 �Table 91 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM Compound Metabolic Activation yg Compound Added/Plate 95 113 Negative control + Positive control, 4-o-tolylazo-l-toluidine v- oo Mofioerotephos — + _ — + + + + + + + Average Number of Histidine-Positive Revertants /Plate TA1538 TA100 TA1537 TA1535 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 13 13 8 10 7 10 87 101 93 107 97 95 126 9 22 14 23 13 17 14 10 9 9 7 11 10 10 6 183 11 10 13 10 9 7 6 101 89 75 79 71 78 113 16 20 16 14 16 19 18 14 13 12 10 15 9 10 12 12 11 10 11 15 10 o n o ft o •o �Table 91 (continued) Compound Metabolic Activation pig Compound Added/Pla te Negative control Positive controls p-Propiolactone AF-2 2-Anthramlne Bromacll ^ . Average Number of Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA1535 145 154 + - + + + + + + + 50 p,l 0.05 50 22 25 25 24 16 30 756 372 63 338 50 1 5 10 50 100 500 1000 120 129 123 117 16 3 10 4 101 23 17 31 29 40 30 14 24 13 22 16 18 15 6 26 14 13 15 1 9 15 11 1 5 1 0 5 0 1 0 0 500 1000 118 131 157 136 138 145 162 35 28 33 26 30 21 8 21 1 6 21 14 12 20 5 16 20 19 13 15 20 16 �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Cacodylic Acid Average Number, of Histidine-Positive Revertants/Plate TA100 TA1537. TA1535 TA1538 56 72 15 14 12 9 7 15 15 15 15 10 11 11 8 14 15 12 11 15 14 13 10 150 5 11 8 8 9 9 8 18 8 18 12 19 15 13 25 25 1 5 10 50 100 500 1000 48 42 42 39 43 44 44 1 5 10 50 100 500 1000 69 53 64 50 64 54 59 17 15 12 18 22 15 16 17 15 16 15 18 21 14 S? n a ^j p™ HO fr £ �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Captan Average Number of Histidine-Positive Revertants/Plate TA1537 TA100 TA1535 TA1538 72 98 2 2 18 14 7 3 8 25 2 5 0 0 0 0 2 2 2 0 0 1 7 14 16 26 6 22 3 100 211 532 822 820 720 1 5 10 15 25 50 Killing 1 5 10 15 25 50 141 210 285 340 330 704 29 80 76 104 SO Killing 20 60 113 55 71 143 19 22 26 21 46 44 •o ft �Table 91 (continued) Compound Metabolic Activation lig Compound Added/Plate Negative control Average Number of . Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA153S 92 80 18 14 12 16 16 16 Positive control, 4-o-tolylazo-o-toluidine Chloropyrifos 5 10 50 100 500 1000 66 92 65 SB 67 87 79 20 22 14 26 20 17 13 12 15 21 15 17 18 20 15 168 22 28 24 25 22 17 22 1 5 10 50 100 500 1000 67 71 87 77 77 72 77 11 9 11 16 11 13 14 15 15 15 18 14 14 11 14 16 20 13 16 30 22 1 3 M O O O CD �Table 91 (continued) Compound Metabolic Activation Vi g Compound Added/Plate 97 30 Negative control Positive control, 4-o-tolylazo-o-toluidine Dinoseb ro Average Number of Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA.1535 15 15 12 16 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 21 17 • 69 59 57 67 82 91 12 12 17 17 12 7 Killing Killing 79 82 94 83 87 104 Killing 13 14 12 14 13 12 Killing 21 16 18 1.7 16 17 Killing 19 18 17 15 17 10 Killing 15 168 15 14 20 17 15 19 Killing 15 14 15 17 14 7 Killing CD s- �Table 91 (continued) Metabolic Activation yg Compound Added/Plate 56 72 Negative control Positive control, 4-c-tolylazo-o-teluidine DSMA Average Number of - *Histidine-Positive Revertants/Plate TA1538 TA100 TA1537 TA1535 15 14 7 15 12 9 . 25 25 10 250 1 5 10 50 100 500 1000 50 56 51 66 71 53 43 12 10 20 15 16 13 12 7 10 10 11 7 12 7 4 5 3 8 8 7 9 1 5 10 50 100 500 1000 54 85 50 55 50 60 53 22 7 16 17 13 15 14 8 8 8 15 3 5 7 10 8 11 9 5 2 o (A �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Azinphos-methyl Average Number of Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA1538 101 102 26 26 6 3 25 25 13 24 13 78 1 5 10 50 100 500 1000 66 74 74 73 75 107 104 39 22 23 30 49 30 31 1 5 10 50 100 500 1000 76 69 68 81 65 84 119 23 23 24 22 24 :io 24 4 3 2 3 4 2 3 1 2 3 3 2 0 0 10 11 9 11 10 10 13 20 25 28 21 19 24 23 o CD �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-taluidine Fenthion Average Numb en of ;. ^c, -Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA1535 94 80 36 20 10 9 12 12 15 168 25 25 1 5 10 50 100 500 1000 64 97 105 112 100 107 90 31 34 32 36 38 42 32 8 11 15 15 16 10 6 13 17 12 14 14 14 12 1 5 10 50 100 500 1000 114 97 81 90 98 86 89 15 17 9 16 14 22 20 10 12 9 12 7 8 10 12 10 17 21 13 10 15 (D 3 O �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Average 'Number of Histidine-Positlve Revertants/Plate TA100 TA1537 TA1535 TA153S 72 93 Negative control Positive control, 4-o-tolylazo-o-toluidine 1 5 10 25 3 7 25 25 Folpet 19 15 50 100 500 10GO 1 5 10 25 50 100 500 1000 127 150 244 300 550 286 20 35 39 48 111 110 Killing Killing Killing Killing 112 173 241 420 720 532 20 51 79 70 218 216 Killing Killing Killing Killing 7 0 1 0 2 0 0 0 2 1 5 6 10 3 0 0 8 20 6 183 5 8 11 14 7 2 Killing Killing 30 26 35 36 45 48 Killing Silling •<B �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control 39 92 Positive control, 4-e-tolylazo-o-tol.uidine OS 1 5 10 10 10 8 10 25 25 Malathion IsJ Average Number of Hiscidine-Positive Revertants/Plate TA1537 TA100 TA1535 TA1538 7 7 6 183 500 1000 54 48 85 99 81 82 61 8 7 7 8 7 12 10 11 12 10 6 7 5 7 3 3 5 7 5 7 4 1 5 10 50 100 500 1000 65 61 99 92 75 90 66 5 6 9 8 9 6 8 7 7 6 12 10 9 5 4 6 5 4 4 50 100 a 7 gf h0) ft HO a �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Methomyl N> vO Average Number of Kistidine-Positive Revertants/Plate TA1538 TA100 TA1537 TA1535 128 149 18 14 33 20 1 5 10 50 100 500 1000 123 112 98 109 110 119 105 28 35 28 27 34 23 24 1 5 10 50 100 500 1000 145 115 125 129 132 133 122 19 20 16 18 21 17 13 12 10 12 10 13 10 14 17 22 17 206 14 10 18 14 24 26 21 18 18 21 19 20 20 24 15 15 13 20 19 18 14 25 25 �Table 91 (continued) Compound Metabolic Activation Mg Compound Added/Plate Negative control Positive control, 4-o-colylazo-o-toluidine Monuron UJ o Average Number of Hisiidine-Positive Revertants/Plate TA100 TA1535 TA153V TA1538 128 149 17 12 17 15 25 25 17 22 6 177 1 5 10 50 100 500 1000 126 . 98 108 122 114 122 125 .15 24 17 19 19 20 22 15 12 12 11 15 18 14 1 5 10 50 100 500 1000 125 142 119 116 108 104 123 10 13 17 15 15 15 11 15 15 13 19 16 15 15 19 18 22 21 29 29 19 21 15 18 21 19 12 17 1 o 9 �Table 91 (continued) Compound Metabolic Activation li g Compound Added/Plate 56 72 Negative control Positive control, MSMA Average Number of Histidine-Positive Revertants/Plate TA100 TA1537 TA1538 TA1535 4-o-tolylazo-o-toluidine 15 14 12 9 7 15 10 250 25 25 1 5 10 50 100 500 1000 79 69 62 52 41 53 48 1 5 10 50 100 500 1000 79 64 65 67 53 66 68 15 15 14 17 15 17 13 11 15 7 7 12 14 10 7 13 11 11 12 8 12 6 4 6 6 7 5 5 11 8 10 14 8 7. 10 10 10 9 7 8 8 10 �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Positive control, 4-o-toIylazo-o-toluidine Parathior Average Number of Histidine~Fositive Revertants/Plate TAiOO TA1537 TA1535 TA1538 95 114 19 21 6 13 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 94 138 85 98 87 110 107 56 75 69 76 88 105 103 12 12 13 13 15 1314 11 16 14 14 17 15 12 7 7 4 4 4 4 3 12 7 5 6 7 8 6 6 7 6 177 8 7 7 8 6 8 13 15 15 19 8 5 9 12 (B rt HO �Table 91 (continued) Compound Metabolic Activation ug Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Phorate Average Number of Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA1538 96 118 15 17 8 11 25 25 1 5 10 50 100 500 1000 70 65 85 65 72 70 58 17 15 17 17 11 14 14 i 5 10 50 100 500 1000 101 103 79 89 79 59 70 14 11 13 15 15 16 19 8 8 7 5 7 6 7 11 8 10 9 6 6 8 8 11 6 177 16 11 11 8 9 6 9 15 10 12 11 7 11 5 I �Table 91 (continued) Compound Metabolic Activation wg Compound Added/Plate Negative control Positive control"-," 4-o-tolylazo-o-toluldine Siaazine .&> Average Number *of' Histidine-Positive Revertants/PlaLe TA1537 TA1538 TA100 TA1535 98 106 10 7 8 8 83 72 73 87 85 71 6S 84 90 82 S3 87 89 120 7 5 7 7 8 3 4 15 10 20 10 12 7 11 25 26 22 266 22 20 20 22 17 25 22 9 4 8 9 8 4 2 11 11 16 9 11 16 10 22 22 15 14 15 15 18 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 ( 4 H- �Table 91 (concluded) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Trifluralln Average Number of Histidiae-Positive Revertants./Plate TA1537 TAiOO TA153S TA1535 96 80 15 15 17 20 14 8 18 24 29 23 25 22 18 15 168 15 14 14 16 15 11 15 13 16 18 14 16 13 15 9 9 14 13 15 11 10 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 73 81 74 93 86 76 95 72 80 78 90 81 79 81 11 12 16 19 18 18 13 10 13 14 15 8 12 12 l-l p> �Table 92 RESULTS OF ASSAYS WITH ESCHERICHIA COLI WP2 Compound Metabolic Activation Negative control . -f- Positive control, AF-2 + - yg of Compound Added per Plate Moncrotopbos - • + + + + + + Bromacil — B — B . . ^ — + + + + + + 0.05 0.05 1 10 50 100 500 1000 i 10 50 100 500 1000 1 10 50 10 0 500 1000 1 10 50 100 500 1000 Average Number o± Tryptophan Positive Revertants per Plate 68 73 204 220 89 83 76 77 61 70 90 88 76 73 75 95 65 74 71 70 70 67 71 73 66 70 70 71 �Table 92 (continued) Compound Cacodylic Acid Metabolic Activation - + + + + 1 10 50 100 500 1000 1 Average Number of TryptophanPositive Revertants per Plate m 102 89 82 91 85 95 + - + + + + + + 10 50 100 500 1000 j 5 10 15 25 381 733 1358 1755 50 + Captan yg of Compound Added per Plate 76 79 89 81 85 2600 1 5 10 15 25 50 89 182 423 699 955 1712 124 �Table 92 (continued) of TryptophanMetabolic Activation of Compound per Plate^ Dindseb 1 10 50 100 500 1000 57 57 49 71 52 42 1 10 50 100 500 1000 Chloropyrifos 60 73 53 49 61 49 1 10 50 100 500 1000 64 73 5fr 55 44Toxic 1 10 50 100 500 1000 73 69 63 65 49 Toxic �Table 92 (continued) Compound Metabolic Activation DSMA Average Number of TryptophanPositive Revertants per Plate 1 10 50 100 500 1000 + + + + + •i- Fenthion Mg of Compound Added per Plate 86 81 67 68 68 71 1 10 50 100 500 1000 69 62 67 73 81 79 1 10 50 100 500 1000 59 63 62 56 70 71 1 10 50 100 500 1000 64 50 67 64 64 81 �Table 92 (continued) Compound Folpefc Metabolic Activation -^g of Compound Added per Plate 1 5 10 25 50 100 Azinpbos-methj'l 1 5 10 25 50 100 1 10 50 100 500 1000 1 10 50 100 500 1000 Average Humber'Wf "1'ryp^lbphanPositive Reveftantfl "per Plate 65 162 170 424 720 1260 74 167 202 900 16SO 1880 92 87 83 89 68 88 83 74 87 86 73 79 �Table 92 (continued) Halathion Methomyl Metabolic Activation wg of Compound Added per'Plate - 1 10 50 100 500 1000 62 54 60 60 54 48 + + + + + + Compound 1 10 50 100 500 1000 58 50 55 59 75 64 - 1 10 50 100 500 1000 61 76 83 57 68 71 + + + + + + 1 10 50 100 500 1000 70 81 78 83 63 74 . Average Number of TryptophanPositive Revertants per Plate �Table 92 (continued) Compound Metabolic Activation pg of Compound Added per'Plate Average Number of TryptophanPositive Revertants per Plate Momuron - 1 10 50 100 500 1000 72 68 57 63 65 63 MSMA + + + + + + - 1 10 50 100 500 1000 i 10 50 100 500 1000 60 59 47 71 50 61 55 64 57 76 60 63 + + + + + + 1 10 50 100 500 1000 55 71 73 71 61 72 �Table 92 (continued) Compound Parathion Metabolic Activation ]jg of Compound Added per Plate 1 Average Number of TryptophanPositive Revertants per Plate 10 50 100 500 1000 1 10 50 100 500 1000 Parathion-methyl 71 64 66 70 64 64 69 53 76 57 72 66 1 10 50 100 500 1000 1 10 50 100 500 1000 53 56 60 68 63 52 64 83 60 65 53 71 �Table 92 (continued) Compound Metabolic Activation Average-Number of TryptophaaPositive Revertantsv.per Plate 1 10 50 100 500 1000 Qulntozene (PCNB) + + •s+ Phorate ug of Compound Added per Plate 70 60 57 62 1 10 50 100 500 1000 78 67 54 57 59 62 1 10 50 100 500 1000 63 64 65 49 71 60 1 10 50 100 500 1000 78 86 83 73 90 70 54 :-s �Table 92 (concluded) Compound Simazine Metabolic Activation pg of Compound Added per Plate 1 Average Number of TryptophanPositive Revertauls per Plate 10 50 100 500 1000 1 10 50 ICO SCO 1000 Trifluralin 55 51 73 54 54 53 64 66 72 56 71 83 1 10 50 100 500 1000 75 73 81 86 69 60 1 10 50 100 500 1000 58 63 63 65 70 70 �Table 93 MICROBIAL INHIBITION IN ESGHERICHIA COLT AND BACILLUS SUBTILIS Diameter of Zone of Inhibition (mm) • ... \ • Compound TCg of Compound Added to Disc E. coli W3110 p3478 B. subtilis HI 7 m45 Positive control, "i-plieiiyl-3,-.' diiriethyitriazene 1.0 37 52 40 61 Negative control, chlcracsphenacol 0.03 34.5 34 32 31 Monocrotophos 1 6 . 6 6 6 Bromacil 1.0 6.5 6.5 6.5 6.5 Cacodyiic acid I 6 6 6 6 Cap tar. 0.1 6.5 11 9 19 Chloropyfif os 2.5 6 10 6 11 Dinoseb 1 10 17 8.5 11 DMSA 1 6 6 6 6 Fenthion 1 6 6 6 6 Folpet 0.1 6.5 6.5 7.5 Azinphos-methyl 1 6 6 6 CD 10 6 �Table 93 (concluded) Diameter of Zone of Inhibition (mm) Compound mg of Compound Added to Disc E. coli p3478 W3110 B. subtilis H17 m4 5 Malathion 1 6 6 6 6 Me thorny 1 1 6 6 6 6 Monuron 1 6 6 6 6 MSHA 1 6 6 5 6 Parathion 1 6 6 6 6 Parathion-methyl 1 6 6 6 6 Qulntozene (PCNB) 1 6 6 6 6 Phorate 1 6 6 6 6 Simazins 1 6 6 6 6 Trifluralin 1 6 6 S 6 �Table 94 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - SwSiOCRCTCPEOS Metabolic Compound Percent Concentration (w/v or v/v) Mitotic Recombinants Survivors per 105 Cells/ml per ml Percent of 3 7 Control ,. . (x 10- ) Survivors (x 10- ) EXPERIMENT 1 5.7 100 4.5 7.9 5.8 100 4.5 7.8 0.1 5.8 102 1,650 2,845 0.1 46 . 79 1,435 3,120 5 5.7 10O 44 77.2 5 47 . 81. 30 63.8 91 . 100 8 8.8 86 . 100 9.5 5 6.3 69 26 41.2 5 48 . 56 40 83.3 Negative control Positive control 1,2,3,4-Diepoxybutane Monocrotophos EXPERIMENT 2 Negative control Uonocrotophos 11.0 �Table 95 IN VITRO ASSAYS WITH SACCHAROHYCE3 CEREVISIAE 03 - BROHACIL Compound Metabolic Activation Percent Survivors Percent of Concentration Cells/ml (w/v or v/v) (x 10 •>) Control Mi t otic Recombinants per I0a per ml fx 10 3) Survivors EXPER1HHJT 1 4.8 Negative control 4.7 Positive control 1,2,3.4-Dlepoxybutane 100 100 3 3 71 74 104 94 104 92 745 683 3.4 3.5 005 .0 0.01 0.05 01 .0 05 .0 50 . 4.5 5.0 005 .0 0.01 00 .5 0.10 05 .0 Bromacil 0.04 0.04 4.4 3.9 4.3 3.8 2.4 94 83 91 81 51 5 4.5 4.2 00 .4 00 .4 00 .5 01 .0 0.25 4.4 2.0 42 3 2 3 1 1 6.3 6.4 2191 1951 6.0 4.4 6.0 2.3 10.0 3 1 11.4 7.7 7.0 2.6 3 12.5 10 0 10 0 5 11.1 3 7.1 4.5 4.2 100 10 0 80 7 653 5.7 54 . 5.5 49 . 126 120 122 108 7 5 5 1 4 .9 47 . 48 . 4.9 117 112 114 117 4 5 6 1 3 EXPERIMENT 2 Negative control Positive control 1,2,3,4-Dlepoxybutane Bromacil 0.50 0.05 01 .0 0.25 05 .0 1933 1555 12.3 9.3 9.1 20 . 8. 2 10. 6 12.5 2.0 �Table 96 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CACODYLIC ACID Compound Metabolic Activation . Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10~7 ) Control Mitottic Recombioants per 10a per ml 3 (x ID' ) Survivors EXPERIMENT 1 Negative control ; 74 . 7.5 7.7 10 0 5 5 5.6 76 20 35.7 5 6.3 82 11 17.5 7.1 100 3.5 49 . 6.5 100 3 4.6 5 15.5 217 1,187 766 5 17.5 270 1,159 662 + Cacodylic acid so 10O + 10.1 6.5 EXPERIMENT 2 Negative control + Cacodylic acid + �Table 97 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CAPTAN Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Recombinar.ts per 105 per ml 3 (x lO" ) Survivors EXPERIMENT 1 Negative control 7.1 100 3.5 49 . 6,5 100 3.0 4.6 0.003 60 . 84 205 342 0.0 03 9.1 140 145 159 7.5 100 1.5 2.0 60 . 10 0 4 67 . 10 37 481 85 58 114 + Captan + Wl EXPERIMENT 2 _ Negative control + Captan 003 .0 + 0. 0 03 .7 7 5.1 �Table y8 IN VITRO ASSAYS WITH SAGCHAROMYCES CEREVISIAE D3 - CHLOROPYRIFOS Compound Metabolic Activation Percent Concentration (w/v or v/v) Mitotic Recorabinants Survivors Cells/ml ,per 10s .„ Percent of per ml 7 •* (x 10~ ) Control ",,(* 10 -.».) ^urviVorssf EXPERIMENT 1 7.5 100 1.5 2.0 6.0 100 4 6.7 5 7.7 103 7 9.1 5 8.0 133 10 12.5 6.3 100 1.5 2.4 7.4 10O 3.5 4.7 0.1 3.8 60 1,045 2,750 0.1 5.2 70 903 1,737 5 7.9 125 3 5 7.4 100 10 Negative control + ChloropyrifoB + •-J-- EXPERIMENT 2 Negative control + Positive control, 1,2,3, 4-diepoxybutane + Chloropyrifos + 3.8 13.5 �Table 99 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DINOSE3 Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per ml per 10s (x 10-') Survivors EXPERIMENT 1 - 6.3 100 1.5 2.4 + 7.4 100 3,5 4.7 0.1 3.8 60 1,045 2,750 0.1 5.2 70 903 1,737 0.2 62 . 98 9 14.5 0.2 40 . 54 1 2.5 0.3 Negative control . 3 5 5 0.3 2.4 32 5 5.5 100 2.5 4.5 5.2 100 2.0 3.8 0.1 4.4 80 3 68 . 0.1 40 . 77 4 10.0 02 . 4.1 75 8 19.5 0.2 4.3 83 8 18.6 Positive control, 1, 2 ,3 ,4-diepoxybutane + in in Dinoseb + + 167 20.8 EXPERIMENT 2 _ Negative control + Dinoseb + + �Table 100 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DSMA Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-') Mitotic Recombinan,£s 5 per ml per 10 (x 10j3) Survivors: EXPERIMENT 1 Negative control 7.4 100 7.5 10.1 7.7 100 5 6.5 4.5 1.1 15 0 4.5 5.2 68 0 7.1 100 3.5 4.9 6.5 100 3 4.6 5 4.7 66 3 6.4 5 3.4 52 7 + DSHA + Ul a-. EXPERIMENT 2 _ Negative control + DSMA + 20.6 ! �Table 101 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FENTHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10"7) Control Mitotic Recombinants oer ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 6.3 100 1.5 2.4 7,4 100 3.5 4.7 0.1 3.8 60 0.1 5.2 70 903 5 6.6 105 9 5 7.5 101 5 6.7 7.5 100 1.5 2.0 6.0 100 4 6.7 5 7.S 104 4 5.1 5 7.1 118 6 8.5 + Positive control, 1 , 2 , 3 , 4-d iepoxybutane Ul •vj + Fentbicn + 1,045 2,750 1,737 13.6 EXPERIMENT 2 Negative control + Fenthion + �Table 102 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FOLPET Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Percent of Cells/ml Control (x 10-7) ^MJ-.totic Rjgcombinants | "* ' per ml * "~p~e*" 10s . U io-3) Survivors EXPERIMENT 1 _ 7.5 100 1.5 2.0 60 . 100 4 67 . 003 .0 40 . 53 119 298 0 .0 03 3.8 63 65 171 6.3 100 1.5 2.3 74 . 100 3.5 47 . 0 .0 03 9.5 151 89 94 0 .0 03 9.1 123 82 90 Negative control + Folpet + EXPERIMENT 2 _ Negative control + Folpet + �Table 103 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 -AZINPHOS-METHYL Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Percent of Cells/ml Control (x 1 " ) 0' Mitotic Recombinants per ID5 per ml 3 (x 10- } Survivors EXPERIMENT 1 _ 5.7 100 4.5 7.9 5.8 100 4.5 78 . 0.1 5.8 102 0.1 46 . 4.5 4.5 Negative control + Positive control 1,2,3,4-Diepoxybutane + 79 1,435 3,120 5.3 93 15 28.3 5.8 100 15 25.9 100 8 8.8 8.6 100 S.5 5 Azinphos-methyl 2,845 S.I + 1,650 5.7 63 68 119.3 5 6.2 72 80 129 EXPERIMENT 2 _ Negative control -i- Azinphos-methyl + 11.0 �Table 104 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MALATHION Compound Metabolic Activation Percent Concentration (w/v or -v/v) Survivors Cells/mi Percent of Control (x 10~7 ) Mi tot ic Recorabinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 Negative control • . -. . i \ --. • * •. • Positive control, 1 , 2., 3 , 4-diepoxy butane o* o 5.7 100 4.5 7.9 5.8 10 0 4.5 78 . 0.1 5.8 102 1,65O 2,845 0.1 4.6 79 1,435 3,120 5 7.8 137 11 14.1 5 6.3 109 7 11.1 9.1 100 8 8.8 8.6 100 9.5 5 8.1 89 13 16.0 5 7.6 88 8 10.5 + + Malathion + EXPERIMENT 2 Negative control + Malathion + 11.0 �Table 105 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - METHOMYL Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 6.6 5.8 100 100 0.1 0.1 1.8 27 266 1,478 1.5 29 184 1,227 2.0 5.0 76 4 2.0 2.7 50 0 3.0 Positive control, 1,2,3, 4-diepoxybutane + Negative control 3.7 56 8 21.6 3.0 4.1 76 6 14.6 5.5 100 2.5 4.5 5.2 100 2.0 3.8 3 4.7 85 15 31.9 3 4.4 85 10 22.7 + Methomyl + + 4.5 2.5 6.8 4.6 80 . EXPERIMENT 2 _ Negative control + Methomyl + �Table 106 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MONURON Compound Metabolic Activation Percent Concentration (v/v or v/v) Survivors Cells/ml Percent of Control (x 10~7) Mitotic Recorabinants per 105 per ml Survivors . (x 10-») EXPERIMENT i 66 . 100 45 . 68 . 5.4 100 2.5 4.6 0.1 1.8 27 266 148 ,7 0.1 1.5 29 184 1,227 5 3.5 53 3 8.6 5 3.8 70 1 2.6 5.5 100 2.5 4,5 5.2 10 0 2.0 3.8 5 6.9 125 2 2.9 5 6.2 119 9 14.5 Negative control + Positive control, 1,2,3, 4-diepoxybutane + Monuron + EXPERIMENT 2 Negative control + Monuron + �Table 107 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MSMA Compound Metabolic Activation Percent Concentration >/v or v/v) Survivors Cells/ml Percent of (x 10~7) Control Mitotic Recombinants per ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 7.4 100 7.5 7.7 100 5 6.5 5 4,3 58 1 2.3 5 5.4 70 3 5.6 7.1 100 3.5 4.9 6.5 100 3 4.6 5 4.9 69 10.2 20.8 5 5.8 89 10.4 17.9 + MSMA -f- 10.1 EXPERIMENT 2 Negative control + MSMA + �Table 108 IK VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PARATHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Percent of Cells/ml Control (x 10-') Mitotic Recombinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 _ Negative control 5.7 Positive control, 1 , 2 , 3 , 4-diepoxybutane 4.5 7.9 5.8 + 100 100 4.5 7.8 2,845 3,120 1,650 46 . 79 1,435 6.5 3 4.6 5 5.8 114 100 5 8.6 100 8 88 . 8.6 100 9.5 5 ••! 102 9.1 Parathion 5.8 5 + 0.1 0.1 8.8 96 4 4.5 5 8.2 95 5 6.1 EXPERIMENT 2 _ Negative control + Parathion + 11.0 �Table 109 IN_ VITRO ASSAYS WITH SACCHAROMYCES rEREVISLAE D3 - PARATHION-METHYL Compound Metabolic Activation Percent Concentration {w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Reconibinants per ml per 10s 3 Survivors (x ID' ) EXPERIMENT 1 Negative control 5.7 100 4.5 7.S 5.8 100 4.5 7.8 0.1 5.8 102 1,650 2,845 0.1 46 . 79 1,435 3,120 5 7.7 135 16 20.8 5 5.4 93 15 27.8 - 91 . 100 8 S.8 + 8.6 100 9,5 5 7,4 81 19 25.7 5 7.2 84 25 34.7 + Positive control, 1,2,3, 4-diepoxybutane + Parathion-methyl + EXPERIMENT 2 Negative control Parathion-methyl + 11.0 �Table 110 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - QUINTOZENE (PCNB) \— f Compound Metabolic Activation Percent Concentration '',w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 _ 5.7 100 4.5 7.9 5& . 100 4.5 7.8 0 .1 5.8 102 1,650 2,845 0.1 4.8 79 1,435 3,120 2 3.7 65 3 8.1 2 4.2 72 4 9.5 9.1 100 8 8.8 8.6 100 9.5 1 58 . 64 4 69 . 1 7.0 81 7 10.0 2 68 . 75 3 44 . 2 7.5 87 10 13.3 Negative control + Positive control, 1,2,3, 4-diepoxybutane + Quintozeue (PCNB) + EXPERIMENT 2 Negative control + Quintozene (PCNB) + + 11.0 �Table 111 IK VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PHOKATE Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10~7) Control Mitotic Recombinants per ml per 10s (x ID'3 } Survivors EXPERIMENT 1 9.1 100 8 8.8 100 3.5 11.0 5 8.7 96 9 10.3 5 7.5 87 3 40 . 5.7 100 4.5 7.S 5.8 100 4.5 7.8 5 7.5 132 4 5.3 5 7.2 124 7 9.7 Negative control -!- Phorate + 8.8 EXPERIMENT 2 Negative control + Phorate + �Table 112 _IS VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - SIMAZINE Comoound Metabolic Activation Percent Concentration : w/v or v/v) Survivors Cells/ml Percent of Control (x 10~7 ) Mitotic Recombinants per ml per 10 3 (x 10" ) Survivors EXPERIMENT 1 _ 66 . 100 4.5 6.8 5.4 100 2.5 46 . 0.1 1.8 27 266 1,478 0.1 1.5 29 184 1,227 5 3.8 58 3 7.9 5 2.0 37 1 5.0 5.5 100 2.5 4.5 5.2 100 2 3.8 5 7.0 127 7 10.0 5 7.0 135 4 5.7 Negative control ; Positive control, 1 , 2,3,4-diepoxybutane + + Siraasine + EXPERIMENT 2 _ Negative control + Simazlne + �Table 113 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - TRIFLURALIN Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10"7 ) Control Mitotic Hecombinants per 10* per ml (x 10-3) Survivors EXPERIMENT 1 Negative control 7.5 100 1.5 2.0 6.0 100 4 6.7 5 8.4 112 5 5.9 5 6.0 100 2 3.3 6,3 100 1.5 2.4 7.4 100 3.5 4.7 0.1 3.8 60 1,045 2,750 0.1 5.2 70 903 1,737 5 8.7 138 7 80 . 5 8.4 114 3 3.6 + Trifluralin + EXPERIMENT 2 VO Negative control + Positive control, 1,2,3, 4-diepoxybutane + Trifluralin + �Table 114 III VITRO MUTAGENESIS WITH SAIMONELIA TYPHIMURIUM SUMMARY DATA FOR EPA PESTICIDES Positive Response, +; Negative Response, - Pesticide Hottocrotophos BroAacll Cucodylic Acid Captaa ESMA Fen tb Ion Folpet Malathlon Methonyl MS4A Parathloa Parathlon-Bsethyl tpilntozene (PQtB) Phorate Slaazine Trlfluralln TA100 - Metabolic + Metabolic Activation Activation TA1535 - Metabolic + Metabolic Activation Activation TA1S37 - Metabolic + Metabolic Activation Activation TA1538 - Metabolic + Metabolic Activation Activation �APPENDIX A MUTAGENEST.S STUDIES OF PESTICIDE COMPOUNDS MOUSE HERITABLE TRANSLOCATION TEST CAPTAN 171 �SUMMARY SRI conducted a heritable translocation study of Captan in mice to investigate whether heritable mutagenic events occur when the compound is ingested repeatedly over an extended period. For 8 weeks, adult male mice were administered Captan in their diet; 60 mice received 2500 ppm, and 61 received 5000 ppm. A control group of 60 adult male mice received an untreated diet during this time. A positive control group containing 66 adult male mice was treated as a control group for 4 weeks and then received the known mutagen triethylenemelamine (TEM) Jn the drinking water for 4 weeks. After treatment, all males were bred with two virgin females each to produce an F generation, the males of which were raised to maturity. Selected (200 per group) F males were bred to three virgin females each, and presumptive translocates were rebred to three additional females each. A third breeding was conducted with selected nonbreeder and/or presumptive males. Evaluation of the data on fertility, breeding, and litter size distribution for F. and F generations does not suggest the presence of translocation heterozygotes in control or Captan-treated male mice. Data on dead implants and rebreeding did, however, suggest the presence of translocation heterozygotus in the group treated with 5000 ppm Captan. Meiotic cell preparations of the testcs of the presumptive males were evaluated cytogenetically. Normal meiotlc chromosomes were found in the following numbers of F males derived from the. group specified: 8 of 8 controls, 8 of 8 from the 2500 ppm Captan group, 8 from the 5000 ppm Captan group, and 2 of 2 from the 5000 ppm Captan-treated group derived from traumatized F females. Five of 5 TEM-treated F males and i. of 8 from the. 5000 ppm Captan-treated males showed reciprocal translocations. 172 �The results of this study show that under the experimental procedures employed, Captan at. 5000 ppm In the diet of male mice for 8 consecutive weeks can produce a heritable mutagenic event in F generation male mice. 173 �' INTRODUCTION The EPA is reviewing and evaluating the health hazard of pesticides and of substitute candidate, pesticides according to available data. Additionally, the Agency is obtaining supplemental laboratory data. The objective is to enable the EPA to select those chemicals that are minimally hazardous when used according to labeling restrictions. SRI is participating in this Substitute Chemical Program by investigating the mutagenic potential of selected materials by in vitro and in vivo procedures. Captan has been shown to respond in a positive manner in Salmonella typhimurium, Escherichia coll WP2, Saccharomyces cerevisiae, J£. coll (relative toxicity), Bacillus subtilis, WI-38 unscheduled DNA synthesis (UDS) with metabolic activation, and Drosophlla melanogaster experiments. It was not positive in a mouse dominant-lethal test. Based on positive responses in both Tier I (in vitro test) and Tier II (Drosophila) mutagenic studies, It was recommended that a heritable translocation test (Tier III) in the mouse be conducted to further assess thie mutagenic potential of Captan. In this study, young adult male JCR/SIM mice from a closed, randombred colony were administered Captan in the diet for 8 weeks. After treatment, each male was mated to two virgin females to produce an F generation, the males of which were raised to maturity and bred to three virgin females each. Pregnant females were evaluated against pre- determined selection criteria for Identification of suspect F.J males, which were rebred and evaluated again. Presumptive F.. males were examined cytogenetically. Through this procedure, a heritable mutagenic response can be detected. Potential mutagenic effects were Identified by examination of fetuses during the middle to later stages of gestation. 174 Cytogenetic �examinations were made of meiotic cell preparations of the testes from suspect males for confirmation of findings obtained front the breeding studios. Reported here are the results of the heritable translocatlon study of Captan. 175 �MOUSE HERITABLE TRANSLOCAT10N TEST Human populations frequently are exposed to man-made chemicals, often at barely detectable levels, for extended periods. To evaluate the genetic hazards of such chemicals, a prudent approach is to study them In mammalian systems so as to maximize detection of a mutagcnlc response. The study reported here was such an investigation of Captan for its potential to produce heritable genetic defects. Chemical induction of chromosomal aberrations in the mouse is a valuable and Important experimental aid in understanding the many genetic defects due to chromosomal anomalies in humans. To date, mammalian evaluations of chemically induced chromosomal aberrations have been attempted with the dominant-lethal test and cytogenetic studies of somatic and germinal cells. Although these procedures can provide useful information, they do hot measure heritable genetic effects, the most important mutagenlc occurrences ttiat are permanent and transmissible. A need exists for a method to reliably identify compounds that cause heritable chromosomal aberrations in mammalian systems. The mouse translocatlon procedure appears to be such a system. A well-defined translocation test will demonstrate the fertility of an F male population derived from F males created with a tost agent. Confirmation of a nonbreeder, sterile, or partially sterile response, can be obtained by cytological examination of the germ cells from suspected males. Sterility and partial sterility are closely correlated with the induction of translocation heterozygotes. The procedure used in conducting this translocatlon test was based on experimental techniques described by Leonard and DeKmidt,1 Cattanach et al.,'1 Falconer et al.,J and Generoso.4 We modified this approach, in consultation with government and industry scientists actively engaged In mutagenesls research. 176 �Materials and Methods Animals Male and female ICR/S1M mice were purchased from Simonsen Laboratories, Cilroy, California. The FQ males were 8 to 10 weeks old. The females used in the breeding phases were 10- to 12-week-old virgin stock. Chemical_ Supply A supply of Captan sufficient for all aspects of the experimental program was received from Battelle CoJumbus Laboratory and F.PA-RTP. Lot number SX-640, Chevron Chemical Company, was used for all treatment periods. The excess material has been placed in storage in case it is needed for future reference. Dosage Selection and Compound Administration SRI and EPA staff selected the two dosage levels of Captan Lo be used in this experimental program. For 8 weeks, Captan was fed in the diet at 2500 and 5000 ppm. An appropriate amount of Captan was dissolved and/or suspended in corn oil. Then the compound-oil concentrate was added at a level of 3% to a finely ground commercial diet (Purina) of known composition. The use of corn oil assured even distribution of Captan and prevented its stratification in an otherwise dry diet. Diets prepared at 2-week intervals were refrigerated at 4°C until fv>d to the animals. The diet was replaced in the feed containers twice weekly to minimize the possibility of compound loss. Body weights and food consumption were recorded weekly during the 8-week exposure period. Reference Control Males in the reference control group were fed the Purina diet with only corn oil added at a level of 3%. These mice were treated In the same manner as those in the compound test groups. Body weights were recorded weekly, as was food consumption. 177 �Positive Control For the positive control group, the known mutagen triethylenemelamlne (TEM) was administered in the drinking water at 0.32 ing/liter for 2 weeks and then at '0.124 mg/liter for 2 weeks. TEM treatment was initiated after the males had been on the control diet for 4 weeks. Body weights and food consumption were recorded weekly. TEM is one of the chemical mutagens that have the demonstrated effect of inducing translocations in the F progeny of F_ treated males. Genetic Tests After 8 weeks of treatment, the males in each treatment group were mated to two adult virgin females each, After 1 week, each female was housed individually and allowed to deliver its litter. The F males were discarded. All litters were raisod to weaning age, at which time the females were discarded. The F males were raised to maturity. At maturity (10 to 12 weeks of age) , 200 I*' males from each experimental group were selected randomly and housed individually. Three adult virgin females were housed with each F male for the first breeding. plugs. They were examined daily for the presence of vaginal These females were sacrificed 14 days after mating, and a uterine analysis was performed for determination of the number of total, live, and dead implants. Males bred to females that produced litters fitting our criteria for presumptive classification as sterile, partially sterile, or nonbreeder were rebred to three new virgin females each. The same evaluation was made for the second breeding. Our criteria for presumptive classification of a male as "partially sterile," "sterile," or "nonbreeder" are: • ^Partia 1J_y_ S, t er II e^jto 1 e each must have 9 or fewer live implants, with at least I. having h or fewer live implants. - _If^only_ 2^f_3._fema.JLjes_ .are pregnant, both must have 9 or fewer live Implants, with 1 having 6 or fewer live implants. 178 �. . L £ . . . ? e ? : ? ? l . l ' ? gl-- : > i . l - ? - .S J . , _ 5 J I . . I a n ! . 6 or fewer live implants. tnis female must have • "Sterile" Male - None of 3 females pregnant—previously identified by presence of vaginal plug. • "Nonbreeder" Male - None of 3 females pregnant—not previously identified by presence of vaginal plug. Any F, male that did not fit one of these descriptions waa considered "normal" and was discarded. For each F male in the control and compound-treated groups suspected of being a translocate or nonbreeder after 2 or 3 breedings, a cytogenetic evaluation was made of meiotic cell preparations of its testes. Five males from the positive control group were also subjected to cytogenetic evaluation. I]yaluat^ipn_qf Breeding n_ata F I males were identified as sterile, partially sterile, or nonbreeders by the methods outlined above. Individual data were totaled to give the number of observed (presumptive) translocaticns per treatment group, using a data base of 600 to 800 females per group. Also, for an accurate review of such findings, the F breeding and litter data were thoroughly evaluated. The various measured evaluated included percentage of pregnancies, average litter size, average number of males and females, average number of males with females having zero to five or more dead Implants, average number of females having zero to five or more dead Implants, percentage of females with plugs, and percentage of pregnancies with and without plugs. Meic tic_.Cell Cytogenetic Studies Cytogenetic examinations were made of the testes of 31 F mice, with the two testes from each mouse being examined separately. 179 The procedures �used for the cytogenetic preparations arc as follows. C0« was used to sacrifice the mice. The testes Were removed, weighed, and placed In an Isotonlc solution of 2.2% sodium citrate. The tunica of each testls was punctured to release the tubules, which were then rolled on a glass plate to release the cell contents into the isotonic solution. The resulting cell suspension was centrifuged at 800 rpm for 5 minutes; the supernatant was removed, and each pellet of cells was resuspended in 5 ml of 1% sodium citrate hypotonic and held at room temperature for 15 minutes. The cells were centrifuged again at 800 rpm for 5 minutes and the supernatant was discarded. The cells were then treated with Carney's fixative (3 parts methyl alcohol and 1 part glacial acetic, acid) to give a total volume of 5 ml, and. immediately centrifuged again at 800 rpm for 5 minutes. This procedure was performed twice, Then the cells, suspended In an appropriate amount of fixative, were dropped onto clean, wet microscope slides and allowed to air-dry. The slides were stained with 2% buffered Ciemsa for 5 minutes. Coverslips were attached with Permount. The slides were coded to preclude bias on the part of the scorers. 180 �RESULTS AND DISCUSSION Table 1 presents the average body weights for mice In the various groups. The body weights of the control, TEM, and 2500 ppm Captan group were within normal limits and comparable throughout the experiment. The 5000 ppm Captan-treated group showed a depressed body weight for 5 weeks before demonstrating a recovery trend during Weeks 6 to 8. This body weight depression appeared to be due to the inability of the male mice to acclimate to such a high level of compound in their diet. Table 2 summarizes the average food consumption by treatment group. Both Captan-treated groups (2500 and 5000 ppm) showed a lower average weekly food intake than did the control arid TEM-treated groups. During the week immediately following (..he 1-week F generation rating, one animal rack holding some females that had been mated with the mice given 5000 ppm Captan was accidentally tipped and some cages were sptiled onto the fllor, resulting in our inability to identify which males had been mated with these females. There were, however, sufficient numbers of F, generation males from those females that were not traumatized to allow us to randomly select 200 F males for use in subsequent F generation breedings. In addition, we held all females that had been traumatized by the tipping of the rack and maintained them throughout the remainder of the study is a separate group. From this separate group we selected 50 F males (at least one. per female retained) for use in the F generation breedings and evaluations. F Generation Information on the breeding performance, litter size, sex distribu- tion, and clinical effects of the Ffi generation should be Included in the evaluation of translocation data, because it may provide valuable reference data. 181 �Table 3 .summarizes the breeding .mcl Litter performance of the F generation. NQ adverse effects were observed in the control and 2500 ppm Captan groups. The two 5000 ppm Captan groups showed a reduced pregnancy ratej the rate for the traumatized females was 29% below that of the controls. Litter sizes for the 5000 ppm groups were slightly below control values. As expected, the TEM group had a reduced pregnancy rate and a litter size 47% below the control level. Table 4 presents litter-size distributions of live young from the F generation mating. Although the distribution patterns for the control and Captan-treated animals were within normal ranges for our strain of mouse, there was evident a definite pattern of decreasing litter size and increasing variance and standard deviations between the different experimental groups. As exnected, the TEM-treated animals showed the classic shift toward smaller Jitters. Figure ] graphically presents the data on the F- generation litter-size distribution. J_i Generation Table 5 summarizes breeding data from the first mating of the F generation male mice. In the females mated with TEM nales and with males from the 5000 ppm Captan group of traumatized F_ mothers, there were 10% fewer females with mating plugs, and an increased percentage of nonpregnant females in comparison with control values; also, these two male groups had an increased percentage of males with no pregnant females. Results from males in the 2500 and 5000 ppm Captan groups were within normal limits for this strain of mouse and comparable with t values from control males. Litter-size distributions of live Implants derived from the first mating of F I generation males are presented in Table 6. Responses of control and Captan groups were within normal limits and readily comparable. The TEM group showed approximately a 13% reduction in litter size. Mean litter sizes were 11.72 for the control group, U.73 for tin1 2500 ppm Captan group, 11,56 for the 5000 ppm Captan group, 11.88 for the 5000 ppm (traumatized F 182. female) group, and �10.24 for the TEM-treatcd group. The data on the F generation littersize distribution are. presented graphically in Figure 2. Tables 7 and 8 summarize the data on dead implants per F, male .and dead implants per femaLe, respectively. The 2500 and 5000 ppm Captan groups showed a slight increase in total dead Implants for both males and females at the 4, 5, and >5 levels when compared with controls. TEM animals showed significant increases in dead implants for both males and females. Table 9 summarizes the breeding results by treatment of those FI males classified as presumptive sterile, partially sterile, or nonbreeders after three breedings. Table 10 identifies these F males individually by number and treatment. Details of the breeding and rebreeding data for presumptive F males are presented in Table 11. In the reference co:itrol group, 24 of the 200 males were considered as presumptive translocates. When rebred, 12 males remained in this classification. A third mating of selected questionable and/or nonbreeder males reduced the number of presumptive males to eight: 1 nonbreeder, 1 presumptive sterile, and 6 partially sterile (3 of which were questionable partially sterile). For the TEM group, 83 of 200 F males were identified as presumptive mutants after the first breeding. When rebred, 53 still met the original criteria. A third breeding reduced this number to 49; 4 continued to be nonbreeders, 14 were presumptive sterile, and 31 were partially sterile (6 of which were questionable partially sterile). In the 2500 ppm Captan group, 30 of 200 F males were identified as presumptive mutants after the first breeding. When rebred, 11 still met the criteria: 5 were nonbreeders, 1 was a presumptive sterile, and 2 were partially sterile (1 cf which was questionable partially sterile). The 5000 ppm Captan group also had 30 of 200 F^ males identified as presumptive mutants after the first breeding. The second mating reduced this number to 9. After a third breeding, 8 males still met the 183 �original criteria: 2 were nonbreeders, 1 was a presumptive sterile, and 5 were partially sterile. For the group of F. males derived from traumatized F females and males treated with 5000 ppm Captan, 12 of 50 FI males were identified as presumptive mutants after the first making. A second breeding reduced this number to 4 and a third breeding further reduced to 2 the number of F males that still met the original criteria; both were partially sterile, with one of them being questionable partially sterile. The data on the F. and F.. generation.?' fertility, breeding, and litter-size distribution'as well as the data on the F generation's dead implants and rebreeding show that Captan tends to induce doserelated effects on the reproductive performance of male mice. The data also suggest the presence of translocation heterozygotes in the 5000 ppm Captan group. Review of the data on dead implants, breeding, and rebreedlng for the F- generation of the TEM-treated group showed, as expected, the potential for the presence of translocation heterozygotes in 24.5% of the FI males. Cytogenetic Studies Table 12 presents the findings from the cytogenetic evaluation of meiotic cell preparations from F males in the Captan groups characterized as nonbreeder, presumptive sterile, or partially sterile. Also, eight control males and 5 of 49 TEM males were evaluated. v, Whenever possible, 25 spermatocytes per testls were scored. The slides were decoded only after all scoring was completed. The results are summarized as follows: • All eight males examined In the control group were cytogenetically normal. • The five TEM males all showed positive reciprocal translocations. • All eight males in the 2500 ppm Captan group were cytogenetically normal. • Seven males in the 5000 ppm Captan group were cytogenetically normal; however, the eighth male (No. 657) showed as a positive reciprocal translocation. 184 �• The two males examined In the 5000 ppm Captan group derived from traumatized F iemales were cytogenetically normal. Discussion Increased use of the translocatlon procedure has revealed that a meaningful relationship exists between the Incidence of dead Implants in F matings and the occurrence of a heritable translocatlon event. Previous experiments at SRI and at Oak Ridge National Laboratories have demonstrated this correlation. The following paragraphs discuss occurrences of dead implants in this study. When females had a total implant count of less than six or when all their implants were identified as dead, we generally considered thin to be a result of first breeding or of some factor other than compound treatment (such as background IncJ-denc.c) and excluded those females from this evaluation. Tables 13 through 17 present the total, dead, and live implantation data for suspect translocates of the control, the TEM group, and the three Captan groups. The control group (Table 13) showed a normal implant distribution, with the exception of one F male (No. 122) for whom the number of implants was high. In the TEM group (Table 14), the expected Increase in dead implants and the resultant decrease in live implants occurred, although the numbers of tot.al implants were generally normal. This pattern occurred during all breeding periods. The 2500 ppm Captan group (Table 15) contained seven males in the first breeding with females having high dead implant counts but normal live litter size, according to the criteria. Also, 3 males showed high dead implant counts during the first breeding, with live implant counts fitting the criteria for partially sterile males. This Increase in dead implant occurrence was not repeated in subsequent breeding, and all FI males were classified as normal after the breeding phases. 185 �(1 •'< -I I*-} Five FI males in the 5000 ppm Captan group (Table 16) showed an increase in dead Implants during the first breeding. In the rebreeding of these males, only male No. 657 continued to show the increase in dead implants and the resultant decrease in live implants. All other FI males in this group had a normal distribution of dead and live implants for all breedings. Table 17 presents the implant data for F. males derived from traumatized females in the 5000 ppm Capcan group. With the exception of an occasional female showing an increase in dead implants, the distribution of total, dead, and live Implants was normal. The numbers of total implantations were generally within normal limits for all experimental groups in all breedings. 186 �REFERENCES 1. A. Leonard and G. H. DeKmidt. Mutation Res. 9, 127 (1970). 2. B. M. Cattanach, C. E. Pollard, and T. H. Isaacson. Mutation Res. J>, 297 (1968). 3. D. S. Falconer, D. M. Slizynskl, and C. Auerbach. 81 (1952). J. Genet. 51, 4. W. M. Generoao. Proc. Second Annual Environmental Mutagcn Soc., p. 9 (March 1971). 187 �Table 1 AVERAGE BODY WEIGHTS IN GRAMS FOR MICE RECEIVING VARIOUS LEVELS OF CAPTAK IN THEIR DIETS Week of Test Initial 1 2 3 4 5 6 7 8 Control TEM Dietary Levels of Captan Cppm diet) 2500 5000 33.8 32.5 33.7 34.8 34.6 34.8 37.3 37.7 39.2 33.4 33.8 33.6 32.9 33.3 31.8 34.6 33.7 31.4 35.4 34.4 32.1 35.8 34.4 31.4 35.9 36.0 33.0 38.1 37.5 35.3 38.9 39.3 35.8 39.8 38.9 36.9 188 �Table ?. AVERAGE FOOD CONSUMPTION FOR MICE RECEIVING VARIOUS LEVELS OF CAPTAN IN THEIR DIETS (Grama of Food Consumed/Mouse/Day) Week of Test 1 2 3 4 5 6 7 8 TEM Control Dietary Levels of Captan (ppm diet) 2500 MOO 4.01 4.05 3.74 3.25 4.82 5.00 4.72 4.38 4.91 5.08 4.90 4.40 5.28 5.33 4.45 5.08 5.55 4.95 5.44 5.36 5.56 4.95 4.75 5.81 6.14 5.63 5.27 5,55 5.74 5.13 4.83 189 4.95 �Table 3 TRANSLOCATION STUDY OF CAPTAN F0 GENERATION MICE SUMMARY OF BREEDING AND LITTER DATA Parameter Control TEM 2500 ppm 5 0 ppm 00 5 0 ppma 00 60 66 60 31 30 120 132 119 61 59 Number pregnant 93 77 91 38 33 Percent pregnant 77.5 58.3 76.5 62.3 55.9 8 5 13.3 16.1 11.68 Number of FQ males Number of F females Number of nonbreeder males 8 19 Percent nonbreeders vO o 13.3 Average live litter size 12.30 6.55 12.16 5.84 3.26 5.68 Average number of males weaned/litter 28.8 6.29 — -11.33 5.25 Group of females accidentally tipped off rack following 1 week of mating with 5000 ppm-treated mates. These traumatized females, and their offspring, most of which could not be identified back to a particular FQ male, were considered as a separate group for the remainder of the study. �Table 4 TRANSLOCATION STUDY OF CAPTAN MOUSE LITTER-SIZE DISTRIBUTION OF LIVE YOUNG DERIVED FROM F GENERATION ADULTS 2500 Captan (ppm diet). _5000. jooof 1 0 0 0 0 2 0 I 0 3 0 3 0 0 0 4 0 8 0 1 5 0 10 0 1 0 0 6 0 15 2 1 1 7 8 I 0 0 1 2 12 8 0 0 9 9 8 3 3 1 3 10 6 2 8 6 3 11 8 7 18 9 24 5 12 3 1 8 5 13 22 0 20 9 8 14 18 1 10 6 1 15 6 0 6 1 3 16 3 0 3 0 0 17 0 0 2 0 0 18 0 0 0 0 0 Mean (p,) 12.30 6.55 12.16 11.68 11.33 3.89 5.84 4.97 5.73 6.09 1.97 2.42 2.23 2.39 2.47 Litter __Si*£__ Control 1 0 2 Variance (o3) Standard Deviation () a TEM Traumatized FQ females. 191 �24 \ I .— •in I I I I I I I I I i TO- Control 22 ___«,.. 2600 pprn Captan —»' • — • « 6000 ppm-Ceptan « • 6000 ppm Captan 20 (Traumatized Females) 18 16 ui 12 10 / ,>\ .•""••' -l5^ 8 10 12 14 LIT TEH SIZE FIGURE. 1 LITTER SIZE DISTRIBUTION FO GENERATION 192 16 IB �Table 5 TRANSLOCATION STUDY OF CAPTAN FL GENERATION MICE SUMMARY OF BREEDING DATA- -FIRST BREEDING Parameter Number of Fl males Number of females Number of mating plugs Percent mating plugs Number pregnant Percent pregnant Number pregnant with plug Percent pregnant with plug Number pregnant without plug Percent pregnant without plug Number not pregnant Percent not pregnant Number not pregnant with plug Percent not pregnant with plug Males with no pregnant females Percent males with no pregnant females a Traumatized Fo females. Control 200 600 450 75 492 82 434 88 58 12 108 18 16 15 13 6.5 TEM 2500 200 600 376 63 383 64 326 85 57 15 217 36 50 23 39 19.5 200 600 442 74 472 79 419 89 53 11 128 21 23 18 14 7.0 Captar. (ppm) 5000 jQOOa 200 600 439 73 477 80 413 87 64 13 123 20 26 21 9 4.5 50 150 95 63 112 75 94 84 18 16 38 25 1 3 7 14.0 �Table 6 TRANSLOCATION STUDY OF CAPTAN MOUSE LITTER SIZE DISTRIBUTION OF LIVE YOUNG DERIVED FROM FI GENERATION ADULTS--FIRST BREEDING Litter Size 1 2 3 4 5 6 7 8 9 10 11 12 13 . 14 15 16 17 18 19 20 Control TEM 2500 5 4 2 4 5 8 • 7 14 35 37 73 80 4 4 0 5 6 4 89 >. 72 32 16 7 1 0 0 9 12 .; , 17 '•'•' - 2 1 11 8 17 25 31 53 61 52 27 21 8 1 1 1 0 5 6 13 35 40 77 75 87 54 36 14 7 2 0 1 Captan (ppm) 5000a 5000 3 0 3 0 1 5 0 1 6 1 8 4 11 3 19 4 17 2 53 16 77 14 85 93 0 0 15 17 14 13 5 1 0 0 0 55 19 11 7 3 Mean (JA) 11.72 10.24 11.73 11.56 11.88 Variance Co*) Standard Deviation 8.13 13.59 8.68 7.40 7.20 2.85 3.69 2.95 2.72 2.68 (CO traumatized Fo females. 19A �120 Con 11 ol 110 — TEM 2500 mm GOOO ppm Capinn 100 — 5000 ppm Capinn (Ti,ium,ili/rd Females) in — 1. ..I._!...L.J....L10 12 LITTEfl SIZE FIGURE 2 LITTER SIZE DISTRIBUTION F, GENERATION 195 — FIRST MATING �Table 7 TRANSLOCATION STUDY OF CAPTAN SUMMARY OF DEAD IMP!ANTS PER Fl MALE Number of Males with Females Having Cap tan Copm) 5000 5000a Control TEM 2500 0 Dead implants 68 37 69 49 20 1 Dead implant 54 36 44 68 11 2 Dead implants 3 Dead implants 30 18 25 16 31 13 36 18 7 2 4 Dead implants 8 9 18 9 0 5 Dead implants 2 6 4 3 2 > 5 Dead implants 7 32 7 8 1 Traumatized FQ females. 196 �Table 8 TRANSLCX;ATION STUDY OF CAPTAN SUMMARY OF DEAD IMPLANTS PER FEMALF. Number of Dead Implants/Female Control TEM 2500 0 320 182 295 28^ 79 1 109 83 113 23 2 39 35 37 130 41 3 18 16 16 9 0 4 11 7 11 2 3 o 0 5 3 0 0 > 5 3 45 2 8 1 492 383 472 477 112 Total Pregnant Females Traumatized Fo female. 197 Cap tan (ppjn) „ 5000 j>QO.pJ 9 �Table 9 TRANSLOCATION STUDY OF CAPTAN SUMMARY OF PRESUMPTIVE TRANSLOCATION T MALES AFTER THREE BREEDINGS Control Total num&er of FI males TEM 2500 200 200 200 Number o f nonbree.der males Number o f presumptive sterile males Number of partially sterile males VO 00 Traumatized Fo females Captan 5000 1 1 6(3?) 4 14 31(6?) 200 5 5000a 50 2 1 1 2(1?) 5 0 0 2(1?) �Table 10 TRANSLOCATION STUDY OF CAPTAN INDIVIDUAL IDENTIFICATION OF PRESUMPTIVE AFTER THREE BREEDINGS MALES Partially Sterile Presumptive Sterile Nonbreeder Contro L 16? 65 122? 164? 189 190 72 220 TEM 215 216 232 235? 238 245 262 264 269 280 281 290 292 299? 300 314 315 327? 344 345 350 359 360? 361 371 375 376 388 390 399? 400? 202 231 251 256 268 273 288 317 321 326 339 343 369 389 220 241 391 396 Treatment (Continued) 199 �Table 10 (Concluded) Treatment Partially Sterile Presumptive Sterile Captan 2500 ppm 480? 526 474 449 479 496 523 583 Captan 5000 ppm 635 657 760 779 780 736 634 733 Captan 5000 ppma 805 837? ^Traumatized Fo females. 200 Nonbreeder �Table 11 TRANSLOCATION STUDY OF CAPTAN BREEDING AND REBREEDING SUMMARY OF PRESUMPTIVE MALES (Live Implants Only) Treatment Control Male No. 11 16 1.7 24 39 43 50 59 65 67 72 86 102 122 129 139 144 152 164 179 189 190 192 200 First Breeding (3 Females) .** 202 215 216 220 226 227 228 229 230 1.3 6 (15)* - (LI) 9 4 0 4 6 3 0 1 7 - Totals TEM Third Breeding (3 Females) Second Breeding 13 Females) _ 0 7 0 .2 9 1 /t (14) - . 6 5 (ID 13 9 0 6 - _ 0* 0 12 10 3 14 0 11 11 9 6 11 _ (12) 1 5 7 ~ 10 8 0 0 12 13 - 9 7 14 9 10 10 0 0 .4 (9) 0 - 0 (9) 2 9 - 0 7 12 12 14 14 11 0 U 2 4 12 14 10 11 11 - 12 _ 2 . 16 14 _ 12 12 4 - 14 13 (9) 0 0 0 0 1 1 0 1 - 12 2 '4 0 0 7 . 11 12 1.0 8 0 ._ 4 - 8 11 13 - (Continued) "0" indicates a plug was observed for a female that was not pregnant. ** •-" indicates a plug was not detected and the female was not pregnant. t,, ( ) indicates all implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. 201 �Table 11 (Continued) Treatment TEM First Breeding (3 Females) Females) Wo, 231 232 233 235 237 2218 239 241 244 245 251 253 254 256 258 262 264 267 268 269 270 273 280 281 283 286 288 290 292 294 299 300 302 314 315 317 318 321 322 ** . 0* 3 8 5 3 4 1 7 9 0 6 8 1 _ _ _ © 0 3 4 0 0 2 3 _ 0 9 © 9 0 5 6 8 8 6 9 0 0 4 0 9 0 0 10 8 5 _ Third Breeding (3 Females) Second Breeding (3 Females) Femalea) 1 0 5 _ 7 6 9 2 8 0 5 3 (18) t (15) 3 0 4 0 - 0 4 0 9 0 0 3 (Continued) 0 8 11 7 10 1 13 0 2 15 7 10 2 - 0 5 6 13 14 ©" - 10 1 0 14 13 13 5 © 13 13 0 0 11 14 0 4 5 © 11 3 0 11 5 9 12 _ 6 8 0 8 4 14 7 8 1 0 0 7 3 10 9 8 13 2 0 0 9 0 11 0 5 3 12 4 5 12 1 0 0 4 0 11 - . 5 2 - 10 0 0 8 11 9 2 15 4 0 11 .- "0" indicates a plug was observed for & female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. "( ) indicates all implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. "®" indicates female was pregnant but had no live implants. 202 �Table 11 (Continued) Male First Breeding Q Females) Treatment TEM 326 327 333 334 339 343 344 34e, 346 349 350 355 356 359 360 361 363 367 369 371 372 375 376 381 382 385 387 388 389 390 391 396 397 399 400 Totals ** 0 0 6 - .* 0 0 10 (10) t 0 1 12 4 9 (15) 4 5 0 4 (16) 3 5 (13) 9 . 3 0 5 4 ) 4 4 .6 .. .. 0 (11) 5 4 4 3 - 4 2 0 0 5 4 12 10 8 9 - () 6 0 13 11 7 2 3 12 4 5 10 5 1 11 10 15 3 5 -. 6 (^ F 0 0 4 4 11 8 0 0 3 © 7 14 5 . 5 9 0 12 12 11 10 0 0 5 3 10 I (15) 7 () P 11 5 7 1 . 3 7 4 3 5 3 11 5 4 0 2 5 0 4 2 - 5 2 „ 0 5 (12) 2 () 9 - _ 0 0 3 6 - Third Breeding Second Breeding 13 Females)} (14) (ID 1.1 12 53 83 49 (Continued) "0" Indicates a plug was observed for a female that was not pregnant. ** , '-" Indicates a plug was not detected and Che female was not pregnant. "( ) indicates all Implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. " ( ) " indicates female was pregnant but had no live implants. ? 203 �Table 11 (Continued) n Treatment Captan 2500 ppm Captan 5000 ppm Male No. 430 432 436 449 450 451 452 455 461 469 472 474 479 480 484 489 496 518 523 526 528 546 561 568 569 582 583 585 588 590 Totals 601 604 620 622 626 627 634 635 First Breeding (3 Females) .* * b 4 (17)' _ _ 9 5 Third Breeding (3 Females^ Second Breeding (3 Females) — " 9 8 12 11 _ 11 8 10 _ 12 12 12 13 10 0 13 10 13 0 0 13 0 11 12 11 . 11 _ 0 11 _ 11 13 10 13 11 9 11 10 _ - 5 . 11 13 13 11 0 _ . 8 10 7 13 12 — 0* 7 9 8 (3) 4 11 7 0 8 - 9 8 7 8 . 8 - 1 0 10 14 10 10 - 9 . - 11 6 . _ - 0 14 12 . 3 () 9 10 0 30 (U) 9 . 8 - (1) . . (13) . 12 (12) 9 () 8 9 () 9 8 - . (13) 11 _ 9 _ 0 0 14 9 11 8 12 9 0 11 9 9 0 (12) 10 - 11 0 10 12 12 6 <B 8 12 _ 14 _ 13 9 15 13 11 U 14 (P1 - (Continued) ** "0" Indicates a plug was observed for a female that was not pregnant. "-" Indicates a plug was not detected and the female was not pregnant. "( ) indicates all Implants were in early stages of development and Impossible " to determine If they were live or dead upon gross observation. " ? " Indicates female was pregnant but bad no live implants. () 204 �Table 11 (Concluded) Treatment Captan 5000 ppra Fl (tale No. 638 646 653 657 660 668 671 672 679 722 732 733 734 736 743 760 761 765 767 769 779 780 First Breeding (3 Females) 10 10 0* 2 0 . 6 4 0 7 9 9 10 8 2 4 805 806 815 816 817 818 822 823 837 84L 846 847 (13)f 5 10 . 0 - 5 10 0 0 1 6 1 8 _ 6 8 (15) 8 (12) 1 5 10 11 14 2 14 9 14 6 10 5 (14) Totals 9 (10) 6 - 12 13 13 1 9 12 11 12 11 9 10 4 12 12 14 3 12 12 11 12 14 1 5 12 12 12 0 0 10 0 13 11 13 13 4 2 30 Totals Captan 5000 ppm 6 -** Third Breeding Second Breeding J 3 FemaJjBs) 10 0 11 9 12 10 - 4 9 7 (11) - . 12 13 9 7 11 6 13 7 13 10 10 14 11 0 8 10 12 10 2 10 10 . 12 - 11 2 13 10 12 12 12 14 4 12 Traumatized ?o females. "0" indicates a plug was observed for ; ** "-" indicates a plug was not detected and the female was not pregnant. "( ) indicates all implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. 205 �Table 12 TRANSLOCATION STUDY OF CAPTAN CYTOGENETIC EVALUATION OF FI MALE MICE Fl Testes Weight (ma) 16 64.6 265 Partially sterile (questionable) Normal 58.5 38.8 68.0 48.7 307 237 232 289 Nonbreeder Partially sterile Normal Normal Normal Normal 164 Control Body Weight (g) 17 65 72 122 Treatment Male No. Classification-Based Upon Breeding Data 55.8 314 Partially sterile Presumptive sterile Partially sterile Cytogenetic Classification (questionable) N> o Normal (questionable) TEM Captan 2500 ppm 189 190 232 262 290 345 361 49.6 47.5 245 222 Partially sterile Partially sterile Normal Normal 54.6 41.4 62.6 54.1 50.4 276 243 222 294 278 Partially Partially Partially Partially Partially Positive reciprocal translocation Positive reciprocal translocation Positive reciprocal translocation Positive reciprocal translocation Positive reciprocal translocation 449 474 479 480 60.2 56.5 65.1 58.7 256 175 287 307 Nonbreeder Presumptive sterile Nonbreeder 496 523 526 583 60.9 50.7 54.1 50.7 242 348 295 297 sterile sterile sterile sterile sterile Partially sterile Normal Normal Normal Normal (questionable) Nonbreeder Nonbreeder Partially sterile Nonbreeder Normal Normal Normal Normal �Table 12 (Concluded) Male No. Body Weight (g) Testes Weight (mg) Classification-Based Upon Breeding Data Capcan 5000 ppm 634 635 657 733 736 760 779 780 57.1 56.0 61.0 62.2 57.9 55.9 54.8 53.0 299 312 231 256 256 298 224 295 Nonbreeder Partially sterile Partially sterile Nonbreeder Presumptive sterile Partially sterile Partially sterile Partially sterile Normal Normal Positive reciprocal translocation Normal Normal Normal Normal Normal Captan 5000 ppma 805 837 70.0 56.9 268 303 Partially sterile Partially sterile (questionable) Normal Normal Treatment ro o Traumatized Fn females. Cytogenetic Classification �Table 13 TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES local F Male Female Dead Live Number Number ^plantations Implantations Implantations First breeding 11 16 1 2 3 1 2 .* * - - 24 39 43 50 59 65 67 72 86 102 122 -.v 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 (13)t . . - 3 17 13 • ? . •• . . - . (13) . - 9 0 . 9* 0 . 4 B 7 0 0 . 0 i 1 4 7 6 0 0 - 4 0 5 . - Nonbreeder Partially sterile (questionable) Nonbreeder Nonbreeder 0 5 0 5 . . . •• (11) If, Initial Clas sj.ficatloi Normal Nonbreeder Partially sterile Presumptive sterile u 1 0 0 . - . . . 7 10 2 0 . (11) 6 2 13 Partially sterile Nonbreeder Nonbreeder Nonbreeder Partially sterile (questionable) Normal "0" indicates a plug was obgtfrVod for a f e m a l e t h a t u.is not pregnant. "-" indii:ar.?s a piu); was not dt-tuctb'd and Che female uis not p r e g n a n t . "()" indicates all Implants vrere in e a r l y stages of development, aric! thus d i f f i c u l t to determine if they were live or dene! upon gross 'observation. 208 �Table 13 (Continued) I'RANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MA1JES F Male Female Total Number Number lmp_l anjt ationa First breeding (concl.) 129 139 IV* 152 16/t 179 189 190 192 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 I 2 3 1 2 3 ! 2 1 I 2 _ ..Contrpljjroujg Dead Live Implant a_ti on s Imp lantBliona ** - . - . - 7 12 9 - - 4 3 0 . .. - 0* 1 0 0 1 0 It 0 0 0 6 0 7 0 ? - - . - 2 3 200 (L4) + - 3 1 - - . . 3 9 9 . . 0 1 0 1 It 6 7 (14) - - Initial Claaslficatio Nonbreeder Nonbreeder Nonbreeder Partially sterile Nonbreedei Nonbreeder Partially sterile Partially sterile Partially sterile (questionable) Nonbreeder Final Classification Second 11 breeding 16 I 2 3 1 2 17 3 1 2 3 13 9 (15) - 0 1 •; - - - - - 13 H (15) - Normal Rebred Rebred h b "0" Indicates a plug was observed for a female Ihut was not pregnnnt. "-" iinlic.ii.es .1 plug was ni<t delected and the fern .Tic was not pregnnnt. " ) incllc.itps .ill implants wrrc in cnrlv stages of development, and thus difficult to (" determine if Llicv were l i v e or dead upon gross observation. See t h i r d b r e e d i n g f o r f i n a l i - L a s s if t e a t ion . 209 NOT REPRODUCIBLE �Table 13 (Continued) TRANSLATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE T F Male Female Total Number Number Implantations Second breeding (cont.) 24 39 43 50 59 65 67 72 66 102 122 129 1 2 3 I 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 139 I 144 2 3 i 2 3 MALES Control Croup Live Dead Implantations Implantations 0* 12 tafr 0 12 11 12 10 12 3 6 2 15 11 0 . (12) + 0 1 0 . 0 0 0 0 3 0 0 0 1 0 0 - - ? - - - 10 1 1 6 16 14 14 2 . 15 •• 9 10 0 7 7 8 1 0 0 0 11 • 0 12 11 12 10 9 3 6 2 14 11 - Final Classification Normal Rebred Rebred h K Normal Normal Partially sterile Normal 0 - (12) 9 5 16 7 7 4 14 9 10 Rebred b Rebred b Normal Normal Rebred b Normal Normal "0" indicates a plug was observed for a female tluit was not pregnant. ;( "-" Indicates a plug wns not detected and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine 11 they were live or dead upon gross observation. See third breeding for final classification. 210 �Table 13 (Continued) TRANSLOCATION STUDY OF C APT AM IMPLANTAIION SUMMARY OF PRESUMPTIVE F MALES _ ._..._ F, "Male" Female " "ToTaL™ Dead Live Numhe r Number Lni£].ant_a,tlon8_ IniE.lantatJ.ons. lEJRL™!:8.1-!??.! -_F. Second breeding (cone] .) 152 164 179 169 190 192 200 Third 16 breeding 17 39 43 72 1 2 3 1 2 3 1 2 3 1 2 1 1 2 3 1 2 3 1 2 3 1 2 1 2 3 1 2 3 1 2 3 1 11 8 14 _*•« (9)t 11 - 1 10 0 1 . 13 ? 1 - () 9 10 - 0* 0 0 0 0 1 0 0 13 12 13 .. _ . 12 12 11 10 - 0 0 0 0 I 1 - 0 - . _ . 0 1 0 0 . - - e . . 0 0 0 Normal Rebred Normal Retired 1 0 0 12 11 13 . _ 12 11 11 10 - Rebred Normal Normal Partially sterile (questionable) Nonbreeder Normal Normal Presumptive sterile 86 3 1 2 3 - - 14 14 5 2 2 1 12 12 4 Normal "0" Indicates J plug was observed for a female tliac wjs not pregnant. "-" indicates a plug was not decocted and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine 1C they were live or dead upon gross observation. 'SRC rhird breeding for final classification. 211 �Table 13 (Concluded) TRANSUOCAXION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F HALES Control Croup Dead Live F. Male Female Total Number Number 1Implantations Implantations Implantations Third breeding (concl.) 129 164 189 190 1 2 3 1 2 3 1 2 3 1 2 3 14 ** . • 0* 1 0 1 1 "0" indicates a plug vas observed 0 . . 0 0 0 0 0 14 . . • 0 1 0 1 1 for a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. 212 Final Classification Normal Partial ly sterile (questionable) Partially sterile Partially sterile �Table 14 THANSLOCATION STUDY OF CAPTAN TMPLANFATION SUMMARY OF PRESUMPTIVE F, MALES __ __TEM Oroujs Total Dead Live F. Male female Number Number jjpg 1 an tail on s. Implantations .lmp.1 ant a First breeding 202 215 216 220 226 227 228 229 230 231 232 233 I 2 3 1 2 3 1 2 3 1 2 3 I 7. 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 2J5 3 1 2 217 3 1 2 3 0* 0 0 kit - . (9)* . 0 11 II - () 9 - 8 0 10 . 0 0 0 0 . .. 7 . . 0 7 9 - 7 1 0 1 - 0 - - - - 12 9 9 5 9 - 10 . - - 15 11 11 7 4 5 b 0 3 12 12 0 0 Q . () 9 . .. .0 4 2 . () 9 7 0 9 . . 0 3 4 I - PisaumptLve sterile Nonbreedcr Partially sterile (questionable) Nonbr eerie r Nonbrceder Partially sterile Partially sterile (questionable) Normal Nor.breeder Presumptive sterile P a r t i a l l y sterile Nonbreeder e 7 Partially s t e r i l e 6 5 9 6 P.irtlally sterile "0" lndic«ti* r < ••' pl"K w.is observed [or n fcmalr tli.u was not prepnant. i. •/: "-" ind itiitr i .1 pliiR u,tq nol di-rortrd and Lhc C entitle w,is not pregnant. "()" i n d i c a t e s ,1.1 i m p l . m l s uorr in e a r l y stnges ol d e v e l o p m e n t , and thus d i f f i c u l t to i l c t c n n i n c i f t h e y were 1 L v o or dead upon gross o b s e r v a t i o n , 213 �Table 14 (ContInucd) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRKSUMPTIVE F. MALES F, Male Female Number Number First breeding (cont.J 238 239 241 244 245 251 253 254 256 258 262 264 267 266 269 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Implantations 14 0* 9 .* * . . 6 0 4 0 0 0 12 12 6 0 0 TEM Group Dead Live Implantations Implantations 11 0 8 . . . . . 6 0 1 3 0 I . . _ 0 0 0 9 2 2 0 0 Initial Classification 0 - - 12 12 15 8 8 . 10 4 10 5 3 . - . - - - Nonbreeder _ Nonbrcedcr . - Nonbreeder 0 0 1 0 0 3 10 4 0 - Partially sterile Presumptive sterile Normal Partially sterile Presumptive sterile 0 2 0 5 3 5 . _ . . . "0" indicates a plug wan observed for a female t;<at WAS not pregnant. t- "-" indicates a plug was not detected and the fe.ua le was not pregnant. 214 Partially sterile Presumptive sterile Partially sterile P rtially sterile Nonbreeder Nonbreeder Nonbreeder �Table 14 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES TEM_Groi;ji Total F, Male Female Number Number Implantations First breeding (cont.) 270 273 280 7.81 283 286 288 290 292 294 299 300 302 314 1 2 3 I 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 0* .* * - 13 1? .1 4 12 12 11 9 . - 0 0 0 9 14 11 14 10 0 8 (18)t 11 11 9 (15) 11 . . - Dead Implantations 0 4 5 1 4 6 3 2 1 . 0 0 0 4 9 7 8 7 0 0 9 2 3 3 1 2 . - Live Implantations Initial Classification 0 . 9 7 2 0 6 9 <! 8 .. 0 0 0 5 5 4 6 3 0 8 (18) 9 8 6 (15) 9 . - Presumptive sterile Nonbreeder Partially sterile Partially acerUe Normal Nonbreeder Presumptive sterile Partially sterile Partially sterile Partially sterile (questionable) Normal Partially sterile (questionable) Normal Nonbreeder "0" Indicates a plug was observed for a female that wan not pregnant. "-" Indicates a plug was not detected and the female was not pregnant. "()" Indicates all Implants were In early stages of development, and thus d i f f i c u l t to determine If they were live or dead upon gross observation. 215 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES TEM Group F. Male Female Number Number First breeding (cont.) 315 317 318 321 322 326 327 333 334 339 343 344 345 34fi 34S 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 Total Dead Live Implantations Implantations Implantations Initial Classification 0* 3 0 0 0 0 0 5 9 10 0 0 1 . 0 - - 0 3 0 0 4 4 3 0 0 9 . . . 0 - 0 0 0 - 5 - 6 . - 0 .* * 9 13 13 0 0 10 . . 0 11 . - 0 0 0 0 10 12 14 10 10 . <!'-) + 0 0 0 0 7 7 8 6 5 . . 7 Partially sterile Presumptive sterile Partially sterile Presumptive sterile Normal Nonbreeder Presumptive sterile Partially sterile Nonbreeder 0 - Presumptive sterile 0 0 0 Presumptive sterile 3 r j - Tart tally sterile 6 4 5 . - Partially sterile Nonbreeder Partially gtcrllo (quest lonable) . (12) "0" indicates a wns observed Cor a female rli.it was not pregnant. ""'',.,, ' • • i •% . i - < : . . . » . . '-" indicates a phiR was not Jctp.ct.ed and the female was not. prcgndht . "()" indicates all implants nere in e a r l y stapes of development, and thus d i f f i c u l t to determine if they were live or dead upon gross observation. 216 �Table 14 (Continued) TRANS I/ICATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE I' F "Male _Njumb;" r Flvst breeding (cont.) Fema ' c Number rc'tdl Im£lantat lona 350 I 2 3 TKM Grou P IV ad Live Impi.int.'itloiis Imp Ian t u t lone 12 11 H _.h, 355 356 359 360 36L 363 36 / 369 371 MALES Initial Classification P a r t i a l l y sterile . - . - Nunbreeder . - I 2 3 4 3 2 - 1 2 3 8 8 6 . - - Nonbrceder ft 8 8 9 /i 8 P.irl t a l l y 3 t « r l IP r, 1 2 3 12 16 1 2 3 (l'j) + - •] • - 1 2 3 12 14 11 8 10 2 4 <i 9 1 2 3 9 - 4 - s - I'artUilly sterile i 2 3 . - . - . - Nonbreudur I 2 3 ()'•'' - 0 - - (15) - - P a r t i a l l y start If (rjur.it ioriahle) P.u-tially sterile 0 - Presumptive s t e r i l e - /, I 2 3 LO 13 - 6 7 - 372 1 2 3 (16) - •> - 375 1 2 3 14 .. - 11 . - 3 - P,irti.al Iv s i e r i l e 1 „! 3 11 0 10 6 0 Part i.illy s t e r i Lr '• 5 0 6 1 - . - 17(> 381 6 - (16) - P a r t i a l l y sterile. P a r t i a l l y sterile (questionable) Nonbret'iler "0" i n d i c a t e s a p l » t r ft-1. - w "'! ohsi-r^cil FIT ,1 ( r m a l c t h a t w j s n o i prf-|;iianL . i nil 11.'Hi", ;i p l u j > , was not d i ' l i ' C tod nnd the f o i i ' i l c was not p r e g n . i n t . j "()" Lii(ltc,ii.cs a l l Linplniun u r r o in ''.'ii-lv 'itajies cl dcvi loju'it'il , <uid thus d i f f i c u l t d c i i ' r m i n o i l i h o v won 1 1 i v o IT .lead upon BIMSS n l i s o r v i i l i n n . 2.1.7 to �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES TEM Group F. Male Female Number Number First breeding (concl . ) 382 . 38$ 387 (13) + (11) _** 1 2 3 1 9 6 () 6 . . 14 9 0* . 11 12 14 . . 3 1 2 3B9 390 391 396 397 399 400 Implantations 1 2 3 2 388 Total 3 1 2 3 I 2 3 I 2 3 1 2 3 I 2 3 1 2 3 1 2 3 . (14) 11 9 (ID 12 9 (12) Dead Implantations Live Implantations 7 7 0 1 (13) (ID 9 5 () 6 7 5 0 .. - . 3 4 0 - 7 3 5 . . Partially sterile (questionable) Partially sterile 4 9 8 . - Initial Classification 11 . ? 6 5 7 10 7 7 - Nonbrecder Partially sterile Nonbrecder Partially sterile Nonbrceder Nonbrceder Partially sterile (questionable) (14) 5 4 (ID 2 2 Partially sterile (questionable) Partially sterile (questionable) (12) Final Classification Second breeding 202 2li I 2 3 1 2 0 0 0 11 1 0 0 0 4 5 0 0 0 Presumptive sterile 7 4 Partially sterile 3 "0" indicates a plug was observed for a female chat was not pregnant . * "-" indicates a plug was not detected an;! the female wns not p "(}" indicates; all implants were In early stages of development, and thus difficult to determine if they were live or dead upon gross observation. 218 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES TKM[..Group F. Male Number Second 'iroedlng '.cont.) 216 220 226 227 228 220 230 231 232 Female Number I 2 1 L 2 1 I 2 3 1 2 J 1 2 3 L 2 3 1 2 3 1 2 3 1 7. 233 235 237 ?18 239 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 Tot nl Imj)1 an tat Ions Imp lantatlons .* * . 14 5 12 11 a i/i 12 12 15 14 13 11 10 0* 0 0 17 12 15 11 15 7 11 13 . - 2 3 0 7 0 0 0 1 I 0 0 0 0 0 0 0 9 10 10 0 I 1 Live implantations ImpJLanta - 4 Normal a 14 12 11 Normal 14 14 Normal n 11 10 - 8 2 5 11 15 6 1 2 0 . - 3 - 7 13 Normal Normal Norm.il Partially sterile 13 - Normal - Rebred "0" I n d i c a t e s a p l u j ^ was observed for n f o m a l c that was nor p r e g n a n t . "-" i n d i c a t e s a p l u g was not d e t e i t c ' d and clip female was not pregnant. 219 Partially sterile 7 ,'. Si'e clii.nl breeding for f i n a l c l a s s i f L e a l ion. Presumptive sterile 0 8 10 13 0 . . - Normal 0 0 9 12 13 - Rebrodb Normal LU 10 14 n Rebred 12 2 12 11 14 14 14 Final Classification . 4 (> I 1 4 1 2 241 Dead �Table 14 (Continued) TRANSLATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES TEH droop Totnl F. Male Female Number Number Implantations Second breeding (cont.) 244 245 251 253 254 1 2 3 I 2 3 1 2 3 1 2 3 1 2 256 258 262 264 267 268 269 270 273 280 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 I 2 3 1 2 3 Dead Implantation Ltve Implantations 12 13 11 1 0* 7 0 0 G 16 11 .* * 2 0 0 0 0 4 0 0 0 2 1 10 13 11 1 0 3 0 0 0 14 11 13 14 11 . 13 0 B 12 10 9 11 12 14 I 14 _ 0 0 0 - 13 L4 11 13 0 - .. 6 7 - 8 0 11 11 10 0 0 3 8 5 9 6 3 1 L 2 _ . 0 0 0 - - 0 - 5 4 5 0 5 9 13 0 12 _ 6 7 B - - - 3 3 5 8 B 5 "-" Indicates .1 plug was not detected and the female was not pregnant. Sec t h u d breeding fci f i n a l classi.ficdti.ori. 220 Normal Partially sterile Presumptive sterile Normal Normal b Rebred Normal Partially sterile Partially sterile Normal Rebredb Partially Rebred 0 "0" I n d i c a t e s a plug was observed Tor a fe.uale Lli.-it was nut pregnant.. :> Final Classification Rebred sterile b b Partially sterile �T a b l e 14 (Continued) TPANSLCCATION STUDY OF (.A.PTAN IMPLANI'ATrON SUMMARY OF PRESUMPTIVE F, MALES JNk'T.bs.1. •Second breeding (cont . ) 281 (in1???!?..!?. 1 1. 3 1 2 Total i3lP':anJ:1tA(-nilc T.KM_Gruup Dead Live !mP'j?,n.Ln..!LLPnl?. .-!S.P.!-Ji1?.a!:-^yP..5. 11 I? 10 7 11 8 4 1 2 Final C.lagsIficatton Partially sterile 14 3 14 0* .** 286 1 . . .. - - - 28S 2 3 1 0 0 0 0 0 0 0 0 0 6 3 0 7 5 0 (i 11 3 3 3 1 0 1 5 8 1 8 10 2H3 2 290 292 294 3 1 'f. 1 1 "I 3 ;. 2 :i 299 ; 2 300 :i 1 302 2 3 1 2 314 315 :> i i 3 1 2 317 3 i 11 H 0 9 14 11 11 12 12 1/4 321 Retired Presumptive sterile Partial ly sterile P-ITI islly sterile No rma I 9 5 4 10 8 Partially sterile 5 (questionable) 0 1 0 13 7 12 1? 5 11 Part tally sterl le 8 2 I 4 0 0 0 0 0 0 f) 0 0 Partially storlle n 0 0 0 0 =, b 2 9 4 11 0 0 0 i) - 13 q 12 13 n 15 \ 14 9 1 2 3 12 11 Normal No rma 1 0 2 1 0 - 4 9 12 12 L 318 0 0 - n 0 n - - 2 12 15 "0" I n d i c a t e s a plup. was observed for n female thai w.ii, not p r e g n a n t . r.-* "-" indicates i plug uda nut doii'clod and thr female wns not Si.'u third breeding for f i n a l classification. 221 pregnant. Normal Presumptive .sterile Normal Presumptive sterile �Table 14 ( C o n t i n u e d ) TRANSLOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES TEH Group F, Male Nurtb'er Second breeding (cent.) 322 326 327 333 334 339 343 344 345 346 Female Number Total Im plantations Dead Implantations 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 I 2 3 1 11 11 _** 0 0 0 - 2 349 3 1 2 350 3 1 2 355 3 I 2 356 3 1 2 3 0* - Live Imp Ian tat ion a Final Classification 11 11 0 0 (10) Normal Presumptive sterile 13 (10)t 12 0 - 13 •) - 0 0 0 0 Presumptive sterile 0 0 0 0 0 0 Presumptive sterile 0 2 0 - 0 0 0 0 0 0 10 5 10 6 6 2 8 I 0 1 - 11 6 1? 4 12 12 10 11 1? a 0 3 2 0 10 8 0 9 n 0 - 0 0 - IL . (1^) 0 . •> b Rcbred 10 0 No rnifl 1 . - Uebrecl n - 0 5 4 Partially sterile 5 4 4 3 12 11 - Partially aterile Normal 10 8 10 0 0 I 11 0 Normal Partially sterile Normal - 11 . Normal (15) "0" Indicates a plug was observed for a female that was nor pregnant. *i - indicates a plug was not detected and the female was not pregnant. "()" indicates all implants ucrc in early stages of development, and thus difficult, to determine if they were live or dead upon gross observation. Sue third breeding for final c lassification. 222 �Table 14 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE f } I'EM C r o u p Dea.l Live Implantations Implantations F, Mali? Number Second breeding (cont .) Female Number Total Implantations 359 1 2 3 LO 9 16 1 2 3 10 13 _** 1 2 3 13 13 8 I/. K 10 10 360 361 363 367 369 371 372 375 376 381 3H2 J65 387 3KH "0" I 2 3 1 2 3 1 2 3 3 6 9 8 13 - Partially (questionable) 2 0 - 12 14 - Norma 1 f> 6 0 11 11 . - 4 5 11 Normal 10 11 14 1 2 3 11 9 - 1 0 - 1 2 3 5 11 1 l, 11 0 4 0 - 3 0 0 0* - sterile Partially sterile 8 -3 1 2 3 1 2 3 7 3 7 2 0 Final Class.If Icatlc 3 7 0 *) 6 9 1 2 3 MALES - - 5 5 5 10 9 5 7 1 1 11 0 - Partially sterile Robred ' Partially sterile Normal Partially sterile Partially sterile Normal 1 2 - - - - " Rcbrrd 1 2 3 10 17. 7 0 0 0 10 12 7 Normal 1 2 3 I'j 12 b 0 15 - Normal 1 2 J 9 3 R i;> intl i rates <i |il"R uas observed for 3 3 4 a [(.male Lli.-u VMS not pregnant. -.• "-" IndicaiTb n plui' war. not ilererred and the female* was not pregnant. Sec tlilnl brucilinK for f i n a l *. l a s s i fi c a t i o n . 223 b Partial ly sterile �Table 16 ( C o n t i n u e d ) TRANSLOCAIION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRF.SUMPTIVE F MALES TEM Group F Male Female Number Number Second breeding (concl.) 389 1 2 390 391 396 397 399 400 3 I 2 3 1 2 1 2 3 1 2 3 I 2 3 1 2 3 256 1 2 3 I 2 3 1 2 3 1 268 Third breeding 2 3 l 216 220 241 Total Imp lao tat ions bead Implantations .* * 14 12 12 • . 11 0* 0 11 7 9 • Live Implantations Final Classification 2 5 3 ~ 273 b Partially sterile Rebred b . 0 0 0 9 3 0 5 8 1 . 11 Pebrcdb 0 Normal 0 5 2 4 Partially sterile (questionable) 4 5 2 Partially sterile (questionable) 4 . . . Partially sterile . 5 . . - - - - 0 0 0 14 7 4 9 13 3 9 . - (questionable) Nonhrecder Nonbreeder Presumptive sterile 2 270 Rebred **r Presumptive sterile 1 2 9 - 0 - 9 - 1 2 . . „ - - - Normal Presumptive sterile 3 "0" Indicates a plug was observed Cor a fomale tliir was no I pregnant. t; "-" indicoLes a plug was nor. detected and the fc'in.i'.c was not pregnant . Set- third breeding for final classification. 224 �Table 14 (Concluded) TRANSLOCATION STUDY OF CATTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MAIES TEM Croup F. Male Number Third breeding (cone I.) Ftfma IE Number 286 1 2 3 1 2 3 1 327 334 2 369 3 1 2 332 389 391 1 2 J 1 2 1 1 2 396 3 I 2 3 Total Iro plantations 11 .* * 11 (9) t - L'ead Jjnplantnt ion.j I 10 •i - Live il.1Pla.n.?flI_'-5n,?. Final Class if icat Ion 10 1 (9) - 14 12 . - 2 0 . - 12 12 . - 11 10 - 0 f) 0 0 - .- - - - • . - - - - . . - - - Partially sterile (questionable) 11 0* Normal - 10 - Normal Fresumpt ! v sterile Normal Presumptive sterile Nonbrccdcr Nonbreeder "0" indicates a plug was observed for a femulc that was not pregnant. "-" Indicates a plug was not detected and the Ecmalc was not pregnant. " ) indicates all implants were In early stages of development, and tl'-us difficult to (" determine if they were I Ive or dead upon gross ibsrrvation. 225 �Table 15 TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F L MALES F. Ma,le Female Number Number First breeding 430 432" 436 1 2 3 1 2 3 1 2 449 450 451 452 455 461 469 472 474 479 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 480 464 3 1 2 3 I 2 3 Total Implantations 2500 ppm Group Dead Live Implantations Implantations .* * - . - 4 4 (17)t 9 14 7 11 . 9 0* 5 14 11 0 - 10 12 13 . . , _ 9 11 12 . . . (3) f) 11 16 11 9 5 2 3 . - 4 (17) 9 9 5 8 . . 4 0 1 0 7 0 9 10 10 . Nonbreeder Partially sterile (questionable) Partially sterile Nonbreeder 14 4 Initial Classification 0 0 1 2 3 .. .. 1 1 '3 - . . No nn a 1 Norm.! I Normal Nonbreeder Nonbreeder Nonbreeder 8 10 Normal 9 . . - 7 0 • (3) 0 - 7 2 0 14 11 Nonbrecjdef Nonbreeder Partially sterile 4 Normal "0" indicates a plug was observed for a female that was not pregnant. :• - indicates a plug was not detected and the female was not pregnant. "O" Indicates all Implants were l.i early stages of development, and thus difficult to dutprmine if they were live or dead upon gross observation. 226 �Table 15 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES F, Male Female _Nfunibe_r_ Number Firs I breeding (concl .) 489 1 2 3 496 1 i 518 3 I 523 3 I 2 2 526 3 1 2 1 528 546 561 1 2 3 1 2 3 1 2 568 3 1 2 569 582 583 585 588 590 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 1 I 2 3 Total Dead I.«!]p..l_ant_at_i._ons. Implantations )4 13 8 .* * . . - 3 1 2 . . . - Live Initial Classification 11 12 6 Normal - Nonbreeder - Nonbres-der - Nonbi'p.edei 7 7 4 (l)t 0 1 • ) 3 (I) 0* - 0 0 - . . . . - - L2 () 9 (13) . 4 8 () 9 .. . - - - . - 9 I . . - - - 10 11 1 15 (12) 11 0 9 - 1 3 . i 3 0 0 Partially sterile Presumptive sterile Nonbreeder Nonbreeder Partially sterile (questionable) (13) . . 9 10 12 - Nonbreeder Nonbreeder Nonbreedur Normal Partially sterile (questionable) (12) 8 0 Normal 9 "0" indicates <n pluK was observed for a female chat van not pregnant. * - indicates a plug was not decoded and the Eemjlc wus not pregnant. 4- " ) indicates all implants were in early stages of development, and thus difficult Lo (" determine I f they were l l v r or deaf I upon Rrosa observation. 227 �Table 15 (Continued) TRANSLOCAT10N STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES F, Male Female Number Number Second breeding 430 432 436 449 450 1 2 3 1 2 3 1 2 3 1 2 3 1 2 451 452 455 4f.l 469 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 472 474 3 I 2 3 I 2 479 480 3 1 2 3 1 2 3 Total Implantations 2500 ppm Group Live Dead Implantations Implantations Final Classification 12 e 14 12 11 13 12 12 13 .* * 11 13 14 0* 0 11 14 11 13 10 12 12 13 11 11 . . 12 0 12 11 0 (13)t 8 I 12 8 12 11 10 12 11 12 13 .10 13 13 0 0 10 L 0 0 0 0 1 13 11 13 10 12 11 0 0 13 11 9 .. 11 0 0 2 1 1 1 1 0 0 1 0 1 0 0 2 . . I ! 0 11 10 0 - Normal Normal Retired 0 1 t Normal Norm ill Normal Normal Normal 0 Normal Rcbred - b Normal 0 - Normal Rcbred b b - (1J) 9 Rcbred b "0" indicates a plug was observed for a female that was not pregnant. .-* - indicates a plug was not detected and the female VMS not pregnant. "()" Indicates a l l implants were in early stages of development, and thus difficult to determine If they were live or dead upon press observation. Sec third breeding for final classification. 228 �Table 15 (Continued) TRANS LOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES ? 5.QP.. PJ"P . F, M a l e jluinbot Second breeding Fundlc Number 48'. 1 2 3 n 1 2 3 9 13 14 ( con t . ) 489 496 518 523 526 528 546 561 568 569 582 •>B1 505 588 I 2 3 1 2 3 1 2 3 1'Otlll Tmplan in t tons. Dcnd ImjjJ an t a t. limn Live Imp ) an ta tlcna 0 0 2 11 0 12 Normal 0 9 11 14 No rma 1 0* 14 'i 0 - .** . - . - Kchrcd n 15 . . . - I 2 3 No nn a 1 - 0 r 1 2 3 0 11 IJ - n • 1 2 3 > 0 0 0 0 •j b Rcbred b b 0 14 - Normal - Rob red - i . . - - Rcbred 1 2 3 11 11 H 2 0 0 9 11 8 Normal 1 2 3 11 - n - 11 - 0 10 in Normal 1 2 3 7 0 7 !') 2 > 13 8 1 ? 3 - - - Rrbri'd I 2 3 12 11 13 0 12 13 13 Normal 1 2 11 <) - 0 - 9 - Rehriid r> (1 0 - "0" indicate."-, a p l " H was observed 1'or a Crm.ilc 111,11 u is ncl p r r K M ' U i L :.-v< v Rebred" 0 0 0 0 11 1 2 3 . - Final C l a o s i f l c "-" i n d i c a t e s a plug was noi d e l e c t e d and die f o n m l e \im nol p r e g n a n t . hire r h i r i l b r e a d i n g for f i n . ' j l c l a s s i f L c u c i o n . 229 Normal b b �Table 13 (Concluded) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES 2500 ppm Croup F, Male Female Number Number Second breeding (concl.) 590 Third breeding 449 474 479 480 1 2 3 1 2 3 I 2 3 1 2 3 1 2 496 523 526 546 561 583 588 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Dead Implantations Implantations 0* 13 12 .* * . 10 . •• . 2 I 0 9 13 15 11 - 0 2 0 . , . B . . 0 0 0 0 0 0 0 - - - 12 . - - 1 Live Implantations Final Classification 0 11 12 . . Nonbreeder - Presumptive sterile 2 - Nonbreeder . 2 I 0 9 13 15 11 11 - "0" indicates a plug was observed for n fcmnU* chat was not pregnant. ft "-" (nilicat.es .1 p l u g was not detected and the fur.ialo was not pregnant. 230 Normal Pdrtially sterile (questionable) Nonbreeder Nonbreeder Partially sterile Normal Normal Nonbreeder Normal �T a b l e 16 TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F^ Male" Female Number Number First 601 breeding 604 620 622 1 2 3 1 2 3 1 2 3 1 2 626 3 I 2 3 627 634 635 1 2 3 1 ? 3 1 2 3 638 1 2 3 646 653 657 660 1 2 3 1 2 3 1 2 3 1 2 668 3 1 2 3 Total Implantations .* * ( 1 I) + (8) 7 11 14 H . - CM . 9 11 R . •• 10 6 6 12 - 5000 ppin Grou P_ Dead Live IseJ.SPiat.lonS. Jj)J tial Classification . 9 7 0 2 •i 0 '! _ - 0 ) 8 8 . - . Partially sterile (questionable) Normal Normal Partially sterile (questionable) Nonbreeder Normal R . - 0 0 0 2 - Nonbreeder Nonbrocder 10 6 0* 0 ? - 9 7 8 7 0 11 H 7 . (H) (8) 7 9 ') 8 -. . - 1 3 0 - (13) 13 8 MALES 0 1 0 . 1 6 Normal 10 0 (13) - Normal Partially sterile (questionable) 2 5 1 0 10 Partially sterile Normal H . - Partially sterile 6 "0" i n d i c a t e s a plug was observed for a female t l u t u.-is not p r e g n a n t . V: "-" indir.-itcs a plug was not detected an<i rhc female uas not pregnant. t '( ) i.ndical.es all Implants were in early stcigrs of development, and tbus difficult to determine if chpy were live or dead upon gross observation. 231 �Table 16 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE Fj MALES F. Male Female Number Number First breeding (cont . ) 671 672 679 722 732 733 734 736 743 700 7ft 1 765 767 I 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 Total Implantations .* * . 6 0* 12 . 4 . . -. 0 - (15)t 8 5 9 12 12 11 - 5000 ppm Group Dead Live Implantations Implantations _ .. . 6 0 0 0 8 4 . . 4 0 . . . . 0 0 t (15) 1 7 5 0 8 1 9 3 . 9 3 1 10 - 1 8 (12) 8 •t Nonbreeder Nonbreeder Partially sterile Nonbreeder Partially sterile Nonbreeder Nonbreeder Nonbrseder Partially sterile (questionable) Partinlly sterile Normal Normal 10 - 2 3 769 2 3 12 .. - Initial Classification 8 (1?) Normal Partially sterile (questionable) "0" indicates a plug was observed for a female thnr was not pregnant . "-" indicates a plug was not detected and tlic female was not pret-nanL. " ) indicates all implants wen1 In early stages of devulopmont, and thus difficult to (" determine if thoy were live or dead upon Rro.i.9 obsctvation. 232 �T a b l e 16 TRANSLOCATION STUDY OF CAP! AN IMPLANTATION SUMMARY OF PRF.SUMPTIVE F ] MAJ,ES F" Male _Numher_ First breeding i.concl .) 'Female fjumbfii: 779 1 2 3 780 I 2 3 _ __5000_ ppni G r c u p Tor.al Dead Live I m p l a r t a t iona Imp Ian tat iona .Implantations 2 0 1 0 4 1 0 0 0 n* 2 0 1 4 1 ;_ I n i t i a l Classlilcatic P a r t i a l l y sterile P a r t i a l l y sterile Final C l a s s i f i c a t i o n Second breed ing 601 604 620 622 626 627 634 635 <>38 '>46 M3 1 2. 3 I 2 J n 1 0 11 0 Normal 10 0 0 I 9 10 1'. Normal 12 1 2 3 1 2 10 1 2 3 (12) t 12 1 2 3 I 2 3 13 1 2 3 I 2 1 2 J 1 2 J 0 1 Normal 0 0 0 12 Normal 0 (12) 12 Normal 1 10 6 0 No rma 1 - 0 I 9 Rebred " - 1 1 0 Rebred 10 12 11 0 0 0 10 12 11 Nonndl 11 0 0 1 Norm al 10 1I 13 9 n 0 0 0 14 13 10 b Normal n 10 "0" i n d i r a r c s a p l n p w.is obsorvod for J f e m a l e I b a i was n o t p r e g n a n t . !• "-" i n d i i . i t , " ; .1 p l u R was n n t d o t c c t r d find tliv f c m . i l r wds IIOL p r e g n a n t . * " ( ) " i n d i r d t o - j . i l l i m p K n U i wc'rr i n p . i r l y s t a p e s oC i l l v c l o p m c n L , .mil t h u s d i f f i c u l t i l j l v r m l n c L f Liu1)1 were l i v e nr dcjd upon i>ross n ' . ) ? o r v a t ion. Sec r'.urd h r c c d i n n f;n l n u l c \ .i.->s i f ic.ii ion . 233 to �Table 16 ( C o n t i n u e d ) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F , MALES F. ,Mj»lc Number Second breeding (cont . ) Female Number Total Implantations 657 1 2 3 1 ? 3 I 2 3 1 2 3 1 2 3 1 5000 pp<n Group _ Dead Live Implantations Imp! an t a t lo n 9 Final Classlf Lcat ion 7 9 5 14 10 12 9 13 13 14 11 -** .660 668 671 672 679 2 722 732 733 734 736 7'3 , 760 761 765 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 '3 5 2 1 8 1 4 0 1 0 14 9 12 Normal 0 9 12 12 Normal 1 1 0 0 . - - U» 0 7 12 ' ' 0 0 14 11 - - - - 14 7 12 3 - 12 0 - - Normal Normal 12 - Partially sterile 1 - - - 0 0 11 12 _ - Normal Rebred Normal - Re b red 11 12 _ . - - - 14 10 11 0 0 10 1 0* 0 0 1 0 0 0 5 14 1.2 0 0 5 13 11 12 11 9 1 ! 3 0 0 13 11 9 h Normal LO "0" indicates a pliifl was observed for a female that was not pregnant. "-" indicates n plug was not di'toclcd and rhu female was inn. pregnant. 234 Rebred 14 .1 Soc 1'iird breeding for final classi Cication. 1) Normal - 1 b Partially sterile Normal No nn LI 1 �T.ihlo 10 ( C o n e l u l l e d ) IKANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF I'RESUtlPTIVE F MALES SpOOjpm F Male; Number Second brcudlnK (cone I .) 767 ?r,9 Total female Numbc r Implant actions 1 ? 3 I 1 2 0* 4 2 0 0 ll 0 0 0 0 1 0 2 I 0 0 1 . - . 1 . 0 2 ) 771 /HO 2 3 Tlur.1 breeding 634 I 2 3 635 722 •'33 1 2 3 1 2 3 1 2 3 736 Dead Implantations I. 1 2 3 14 13 12 12 13 10 13 . .. - 0 12 13 12 Normal 12 13 10 Normal 0 4 P a r t i a l l y s«ti>rilt! Nonbrccder 1'artlally sterile 12 - - "0" indicates ti plug was observed for a female thai was not pregnant. I: "-" indicates a plup, was not detected and the female was not pregnant. 235 Partially sterile Norma 1 Nonbrecdcr Presumptive sterile �Table 17 fRANS LOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F F Male Female Number Number First breeding 805 806 815 816 8L7 Total Implantations I 2 3 1 2 3 I 2 3 1 2 3 1 2 818 822 823 837 3 I 2 3 1 ? 3 1 2 3 I 2 3 841 84(> 847 1 2 5000 pp.n Group Dead Live Implantations Implantations ** . 6 9 7 - . 0 0 . . 6 9 0 - (10)t (U) . •J - - 10 - 0 . . - . . - - - . - 0 5 - - 1 (14) - •; - Nonbrceder Nonbreeder Partially sterile Partially sterile. (questionable) 10 '.' 0 . - - (10) (U) . . - 6 . - Initial Classification 7 1 2 3 2 3 MALES - - 6 . 5 (14) - Nonbreed?r Normal Nonbrceder Nonbreeder Nonbreoder Nonbreeder Partially sterile Partially sterile (questionable) - Final C l a s a i f i c a t l o n Second breed! n i>. H05 Bebred ' i n d i c . u o s t [V.umiiLl/.c'd F., f e m a l e s . ,v ,v-\ t "0" i n d i c a t e s <i pl'ip, was observed tor a Ornate t l u L was not. "-" indic.'ilc's ,1 plug was n''t detected jnd rht; fomnlc was nol procnjnt. "()" indicate 1 . 1 ; J l l i m p l a n t s wcro in r j r l y suigos o f d e v e l o p m e n t , and thus d i f f i c u l t t o d e t e r m i n e if they were l i v e or dead upon gross o b s e r v a t i o n . 'Sue I ' . i i r d brci-dinp, f o r f i n j l classification. 236 �T a b l e 17 ( C o n t i n u e d ) TRANSLATION STUDY OF CAPI AN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES _ _ _ .5000' ._|jpm (-rpu[)_ _ _ Dead Total Live rmg lap tat ions Implant a t :um:j Implantations Final C l a s s i f i c a t i o n F. Male Niinibe r Second brooding (concl.) Female Number 806 1 2 3 13 14 10 1 0 0 12 14 10 Normal 1 2 ') I'l 11 13 1 0 3 IJ 11 10 Normal I 2 i; * Normal I 2 3 11 n* n 4 0 - 7 0 •• Kobr«-d h 0 0 11 811 816 817 818 822 823 837 841 846 847 1 2 3 805 817 B22 8 1) 2 n 1 0 Normal 12 6 1 2 b 1 2 3 n - 1 2 1 8 1 - - - I1) 2 0 - 13 10 - Normal 0 0 0 10 12 12 Noniia 1 1 10 10 14 Normal I 2 3 1 2 3 1 2 3 Third b reed inn ** 1 2 Kobred'' in in 12 12 ;i in 1/1 . n 0 - 0 0 „ Retired _ 1'artially sterile - 2 7 1I 2 11 Normal in i? i? Normal 1J n 1 2 3 10 12 12 0 I n d i c ' i i l c s tr.jum.it izcd F _' Normal - I 2 3 4 13 - n 0 Inn.ili";. "0" i n d i c a t e s ,1 I ' l n p was n b s c i v e d lor a Connie f i a r vjni MOL prep.n.ini. "-" i n d i c a t e s n pluj; was not d i ' l p c t r d .irnl the female u;is mil p r e g n a n t . Sot t h i r d breeding for l i n j l c la-ii if i r a t i o n . 237 �Table 17 (Concluded) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PKESUMPTIVF, f} MALTS F. Male Female Number Number Third breeding (concl.) 837 1 2 3 Total Implantations -** - 5000a ppm Uroup Dend Live Implantations Implantatlona - - 'Indicates traumntizod F. females. '-" indicates a p l u g was nol. detected and the female was not pregnant. 238 Final Classification Normnl �a a: a. 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 The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 012 Folder The folder containing the original item. 0101 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 Simmon, Vincent F. Ann D. Mitchell Ted A. Jorgenson Description An account of the resource Corporate Author: Stanford Research Institute, Menlo Park, California Date A point or period of time associated with an event in the lifecycle of the resource 1977-05-01 Title A name given to the resource Evaluation of Selected Pesticides as Chemical Mutagens 'In Vitro' and 'In Vivo' Studies Subject The topic of the resource animal testing pesticide testing https://www.nal.usda.gov/exhibits/speccoll/files/original/8dd45d44d672ef1c545bc2019d64ceab.pdf dbb36089c3a06149ea2ff579fb93a3a3 PDF Text Text Item ID Number: Author Corporate Author 00054 Boush, G.M. University of Wisconsin, Department of Entomology, Madison, Wisconsin Pesticide Degradation By Marine Algae Journal/Book Title Year 1975 Month/Day A ril Color W Number of linages 23 DeSCrlptOU Notes Contract N00014-67-A-01 28-0023, Task No. NR 306-061 P ' Friday, November 17, 2000 Page 54 of 57 �Boush,G.M., et al 1975 Pesticides Degradation by Marine Algae .AD A 008 275 AD-A008 275 PESTICIDE DEGRADATION BY M A R I N E ALGAE G. M. Boush, et al Wisconsin University P r e p a r e df o r : Office of Naval Research 1 April 1975 DISTRIBUTED BY: Hiflimi TttfciHtil hififiiititi ftftfrt V. 1 BEMRTMENT if C8MMERCE �118104 10 Off lev of Haval Research Contract H0001U-67-A-0128-0023 00 o o Task Ho. HB 306-061 PHIAL REPORT Pesticide Degradation by Marine Algae by G. N. Bousb and F. Matsumura University of Wisconsin Department of Entomology Madison, Wisconsin 53706 April 1, 1975 Reproduction in whole or in part is permitted for any purpose of the United States Government Approved for public release: distribution unlimited This research was supported in part by the Office of Haval Research, Haval Biology Program, under Contract Ho. H0001U-67-A-0128-OO23, HR 306-061. Reproduced by NATIONAL TECHNICAL INFORMATION SERVICE US (topulnmt ol Comn«« Sprl.gfM<l. VA. 2JI51 �DOCUMENT CONTROL DATA - R I D l*ll> i <im«l w *nl*n4 wh*r< lh» •rmtmll ttffrl /« «•/««»<«•«) I*. RIPORT f C C U R I T V CLASSIFICATION I. ORIGINATING A C T I V I T Y Department of Entomology University of Wlseouln Madison, Wisconsin 93706 1. HtPMT TITL« Pesticide Degradation by Marine Algae 4. DC«CRIPTIV« NOTCt (Tyf» 01 np*r« and Final report »- »0 TMOMMI rFinf HMwTiiMffi Mail. lm»l MM) George M. Boush and Fumio Matsumura • - RCPOMT OATC If. TOTAL NO. OF FAOU 7». NO. Of KCFI April 1, 197? 0 M. eONTHACT ON CHANT NO ». PHOJ.CT NO. H B HOOO1H-OY-A"0120-0023 M. OMIOINATOITl KCPOHT NUMBCHI*) 306-061 M>. OTHCM HKPOMT NOIII (Any oHm Itwmbfn timt muy b* •• 10. DISTMIC JTION STATKMCNT Standard Distribution List II. SUPPlCMCMTAItV NOTt* 12. SPONSORING M I L I T A R Y A C T I V I T Y Office of Naval Research Rone I AStTRACT Various algae species are tested for their susceptibilities towards chlorinated hydrocarbon insecticides. Deildrin, which is the most frequently found pesticidal contasdnant in the 1)8, and its analogs were found to inhibit the growth of certain of algae species. Anacystis nidulans in particular showed narked susceptibility to endrin dieldrin, ketoendrln sad photodieldrin. This species was also susceptible towards dieldrin metabolites such as metabolite F and 0. Among DDT metabolites DDD (TDE) was found to be the most toxic material) followed by DDE, DDT and FW-152. It has not been mown that DDT should be more toxic to algae. In terms of acute toxiclty phenylmercuri acetate was by far the most alglcidal agent among all pesticidal chemicals tested. This pesticide is toxic to both A. nidulans and A. quadruplicatum at the concentration of 1 ppb. Algae, along with other plankton, are known to bioaccumulate pesticides and thereby play a vital role in the process of food-chain accumulation of these micropollutants Oar studies indicate that the rates of pick-up of pesticides are very rapid. To study the feasibility of constructing a model ecosystem we used algae as a key food chain organism. By this way we could demonstrate that TCDD, the most toxic contaminant of 2, >-T does not really accumulate in the aquatic organisms as compared to DDT. Algae as a whole are not very active in degrading pesticidal chemicals in vivo. They were found to play, however, a key role in the process of environmental alteration of pesticidal residues. The way they participate in such processes was found to be through aynerglstlc actions on photochemical reactions. Algal products, when tested in the form of aqueous extract from dead algal cells, were found to be excellent photosensitizers for DDT and mexacarbate degradation by the sun-light f«ini»-i>t.»rt «»n i«miO 00.^.1473 S/N 0102^)14^600 (PAGE t ) PLATE NO. 21BJ6 Security CUmlTic«tion PWCB SUBJECT TO CHANGE �Ill LINN A • HIV ••no* •OLB *T •»t oblarlmtrt hydrocarbon* ioMMllua quadrupliotiai •icrcfcial tegxmtetion DDT up-tak* t>ceuul»tlon food elmini teniflAl realdue* TCDD •otel •eocystmu i X i • DD (PAGE 2) < BACK » LIMN e LIMN- • N«Ll Security CU»ific«tlofi tt«LI •T �,UA«* /v/ TABLE CT COITOrTS Page or RESEARCH ACCOMPLISHED - r - i I. - Effect* of pesticide* on plankton - — . . — . — . 1 . II. - Effect* of degradation products . . — . — . . 2 . . .. III. > Effects of pesticide •icro-contaainaats -— - 6 nm or -nccraiic/u REPORTS -....17 or ALL PUHLICATICHS ..................i? MUOR ACCCMPLISBffiHTS - - 16 DOCUMENT caiTROiL DATA - R & D 19 KEY WORDS . - - - . _ . . . 20 �-1- SttMAHY OF RESEARCH ACCOMPLISHED I. Studies relating to the affects of pesticides on plankton. It has been suggested that the varying resistance of marine phytoplankton to effects of chlorinated hydrocarbons could have far-reaching effects in terms of phytoplankton population balance. Studies in this laboratory have also shown varied growth inhibition of planktonic bluegreen algae by chlorinated hydrocarbons. TABLE 1 shows the effects of aldrin, dieldrin, and endrin on growth rates (generations per 2U hr.) of Anacystis nidulans (freshwater species) and Agme'nellum quadrupllcatum (marine species).It is noticeable that generally the marine Isolate is more tolerant than the freshwater isolate. This may be due to the influence of the growth medium on the insecticide. The toxiclty of a pesticide in aquatic environments may vary according to the physical characteristics of water. Although much variation is noted in the data, the general trend indicates both algae are tolerant to these insecticides except at concentration* higher than reported in natural waters. Also notable is the sensitivity of A. nidulana to dieldrin, an isomer of TABLE 1 Growth Response of Agmenellum quadruplicate* and Anacystis nidulans to Aldrin*, Dieldrin, and """^ ppb Aldrin A. q u a d r u - A . 0 pllcatum*5 nidulans 950 U75 95 19 0.2 Control 6.2 * 0.7 7.1 * 0.5 6.6 * 1.2 6.8 * 0.3 6.U * i.o 6.6 * 0.5 Dieldrin Endrin A. q u a d r u - A ^ A. q u a d r u - A T plicatum nidulans plicatum nidulans 6.U * O.U 5.8 i 0.9 6.7 * 0.2 6.8 * 0.5 7.1 * O.U 7.2 i 0.3 6.S * O.U 6.0 - 0.8 6.0 - 1.0 6.5 - 0.7 5.3 * 1.2 6.2 i 0.9 3.2*0.8 3.9*1.5 6.9*0.6 7.2*0.9 6.7*1.3 . 6906 .*. 3.5 * 0 9 . U.8 * 1.5 U.9 * 2.2 5.6 * 1.3 *0.3 6.6 * 0.5 2.2 * 0.7 3.2 * 1.0 6.3 * 0.3 6.6 * O.U 7.0 * 0.5 6.6 * O.U Concentrations for aldrin 9 0 U55, 91, 18 and 0.2 ppb. 1, Values reported as number of generations per 2U hours, represents mean of 3 to 5 replicate cultures Aajaenellum quadrupllcatum (strain FR-6), Anacystic nidulans (strain TX20) In preliminary experiments the growth response of these two algae was also tested against phenylaercuric acetate (FHA), an algicide and fungicide once used extensively in 'Industry. The results are summarized in TABLE 2. �-2TABIE 2 Susceptibility of Two Species of Blue-Green Algae Against Fhenylmercuric Acetate 0.10 A. nidulans A. quadruplicatum . 109 Phenylmercuric acetate PPb) 0.75 0.25 0.50 1L.OO 100 109 112to 10 0 8° 7 6* 8 112 1.0 00 0 0 Expressed in % relative growth against controls as 100. Only 3 of U replicates grew during the experiment. *? Only 2 of k replicates grew. Only 3 of 6 replicates grew. Thus results showed A. nldulans to be affected by as little an 0.50 ppb PMA. At this and higher concentrations growth was irregular and vts preceded by lag phases. In view of mercury contamination reported in oceanic environments it was of interest to also consider the toxicity of FHA to A. quadruplieatum. Duplicate cultures in two experiments yielded the growth values as compared to controls (TABLE! 2). Thus it is evident that A. quadruplicatum is more tolerant to FMA than A. nldulans; however, neither organism showed any growth at 10 ppb IMA. ~~ Much research has shown that in addition to growth, beneficial activities of microorganisms can be affected by pesticides as well. Bacteria in soil which convert organic matter to ammonia, and several herbicides have been seen to influence soil nitrification. II. Effect of degradation products. Pesticides, as they may adversely affect microorganisms, involve not only the parent compound, but the intermediate and terminal residues of these compounds as well. Recent investigations have pointed out the potential of certain "terminal" residues to be as Ijxic as the original pesticides. Data from this laboratory alco support this obeservation. Anacystis nidulans and Agmenellum quadruplieatum were grown in media containing microbial degradation products of aldrin, dieldrln, and endrin. The data in TABIE 3 show that A. nldulans continues to be sensitive to photcaldrln and ketoendrin, two metabolites of aldrin and endrln, respectively. Agmenellum quadruplicatum appears resistant to both compounds. However, both organisms show of dieldrin, as shown in TABI£ U. formed microbially, photodieldrin by the action of UV or sunlight. continued sensitivity to metabolites While metabolites P and G are only is also known to form on plant surfaces Hence, it was of interest to assay the �-3- Qrowth Response* of Agmenellum quadruplicatum and Anaeystis nidulans to effects of Metabolites of Aldrin and Kndrin, toB-i»ll Photoaldrtn A. quadruplicatum A. nidulans ppb 950 *75 95 19 0.2 Control Ketoendrin A. quadrupllcatun A. nidulans 6.2 6.U 6.5 5.9 6.0 6.6 7.U - O.U 6.8 ± 0.2 7.3 * 0.3 7.1 * 0.8 7.3 * 0.6 6.6 i 0.5 * 1.0 * O.U * 0.7 ;0.7 * 0.6 t 0.5 5.3 -0.8 6.3 * 0.7 6.U 1 0.7 7.0 i 0.6 7.0 i 0.6 6.8 ± O.U fc.5 * 1.1 3.5 - 0.1 6.0 - 0.8 6.6 ;* 0.2 6.3 - 0.1 6.8 t O.U Conditions as in 1AHUE 1. TABLE Growth Response of Agmenellum quadruplicatum and Anaeystis nidulans to Metabolites of Dieldrin Metabolite F A. A. quadrunidulans ppb 950 475 95 19 02 . Control 5.* I 0.7 5.9 ±0.9 6.1» - 1.2 6 8 - 0.8 . 6.2 ±0.9 3.5 U.8 6.5 7.2 6.7 6.9 *Q.k ±0.3 ± 0.5 ±0.5 ±0.3 ±0.6 Metabolite 0 A. quadruA. plicatum nidulans Photodieldrin A. quadruA. plicatum nldulans M - 1.0 k.Z - l.U 6.U ±0.8 5.9 * 0.6 6.U * 0.9 6.7 * 0.1 6.U i 1.2 6.7 * 0.5 6.5 ± 1.1 6.U ± 0.1 6.2 ±0.9 6.9 * 0.6 5.6 - 1.0 6.9 ±0.5 6.6 ±O.U 6.U ±0.6 7 1 ± 0.2 . 6.2 ± 0.9 u.o Jo.u 5.3 7.2 7.1 71 . 6.9 ±0.8 ± 0.1 ±0.9 ±0.8 ± 0.6 response of other algal species to photodieldrin. TABUS 5 shows that of the algae tested, Hostoc sp. and the green alga Chlorella spporensis appeared only slightly affected by photodieldrin at high concentration, while A. nidulans was most inhibited. Continued growth of A. nidulans in successive cultures in medium plus various levels of photodieldrin did not improve initial growth rates. Other attempts to improve the organisms' tolerance by varying growth conditions and dark incubation intervals were unsuccessful. �ft. TABLE 5 Growth Response of Several Blue-Green Algae* to Photodieldrin*»c Alga* I II III V VI* VII3 Control 2.6 * 0.1 5 0.2 3.5 * 0.2 7.0 * 0.7 - v.i(,\ 6.2 * 0.6}? 3.3 * 0.3* ' 6 5 - I°- 2 /l! 3.6 * 0.5<6> 0.2 ppb 2.7 * 3.6* 6.7* 6.9* 3 3 7.1 * "-I""/^ 3.7 * 0.7<6> - ; 19ppb >..& * 0.2J3J - 950 ppb 95 PPb tf\ 2.7* 3.6* 7.1 - w.«/_\ SilitjSl I:!!?:!?! 3.5 * 0.5<6> k'.O * 2.9 * 0. 3.3 * 0. 5i2 3.2 7.1 3.fc * 0. * 0. * 0. * 0. * I • Anabaena variabilia; II » Hostoc «p. ; III » Anacyitig nidulana; IV Chlorella • _^___^ V * Agpenellum quadruplicatua (strain BS-l); VI » Agaenellua quadruplicatum' ( train PR-6J; VH Coceochloris elabans. a All algae tested here are blue-green species, except C soproensis, a gre* * b Values reported as number generations per 2k hours. ~ e Rubbers in parentheses indicate replicate cultures. d Indicates marine isolate; others are freshwater isolates. �-V ' S"*-^ •'' ,;;,•'*:tBT*1S, ". '!.-:i,"i £"«?£ i«*3. ':r E'-f».V< ;<*--;---;; ^m •BHI |J£J2*g|nlB£gB!|»Dxg ^Sf^^i^HKi^s^i^SiSfff^iHSH9^tMfUffS^HniSSo^3i^^SISKSS^SSS3^S^SSSESS3^Si^ Vjjpirfhi'^H -5ThHl£ 6 Growth Response of Agnenellum quadrupllc '.turn and Anacystis nidulans to DDT and its Metabolite** »» ppb DDT A. quadruplicatua 885 442 88 18 0.2 (13) 12) »0 0.0 5.3 5."» G.2 6.2 6.7 6.3 * 1.0 6 * 0.9 5 * 0.4 3 ± 0.3 ( 4 * 0.4 ( 4 * 0.6 ( 8 791 395 79 8 0.8 0.0 ODD I 1.1 J 1.3 * 0.6 ± 0.4 * 0.6 ± 0.7 791 395 79 8 0.8 0.0 DDE 5.1 5.* 6.3 6.5 6.1 6.1 3.4 5.3 6.7 6.8 7.0 6.k * 0.0 ( * 0.8 ( i 0.1 ( i 0.3 [ * Q.k * 0.7 1[ 6.9 * 0.2 ( 6.7 - 0.0 ( 7.k * 0.2 [ 7.1 * 0.0 7.k * 0.2 6.9 * 0.2 700 350 70 7 0.7 DBA ' 0.0 DBF 6.5 * 0.3 720 6.9 6.6 6.8 6.6 6.9 360 72 7 0.7 0.0 FW 152 ( 4) ( 4) (16) • 0.3 - 0.1 * Q.k * 0.1 * 0.2 8 0.8 0.0 5.8 - l.l 5.8 * 0.8 6.5 * 0.6 <f M "gUSjTaiJl-i-liai 2)c 2c k 2 2) 2) 6) 4.2 * 0.3 5.3 * 0.3 6.2 t 0.3 6.8 ± 0.3 6.4 * 0.3 7.0 4 0.3 ( j^ C 2) 2) 2) 6.2 * 0.1 2 5.9 * 0.2 2) 6.5 * 0.4 2 2) 3) 6.8 ± 0.0 6.0 ± 0.3 2 2 2 2 2 3) 6.4 t o.l 6.2 i 0.0 6.8 * 0.2 7.1 £ 0.»» 6.6 ± 0.1 6.0 ± 0.3 ( 2) 6.8 * O.I* ( 3) 6.6 * 0.3 ( »») •AM «t^«%v^ srn^t**^ •••••ilia •• 5 6.2 * 0.2 4 6.8 J 0.7 4 6.8 t 0.5 16) 5.4 - 2.0 6.5 * 1.5 U 6.5 * 0.4 ( 2) 7.2 * 0.0 ( 2 6.8 * 0.5 ( 7 6.6 i 0.6 i. 6) o o 7) 2 2 3 2 2 2 2 2 3) 6.3 i 0.4 2) 7.2 t 0.2 2) 7.4 * 0.2 ( 2) 7.2 ± 0.1 ( 2) 7.«» * 0.2 ( 2 7.0 * 0.3 ( 3 • Values expressed as number of generations/24 hours. • 16 4.6 * o.O 6.1 - 0.1 4) 6.8 * 0.3 3) 6.6 * 0.* 3) 6.3 * 0.* 4) 821 410 82 A. nidulans *«w* «MMt1 4 t^+^m Growth occurred in only one or two of several replicates. �-6TABUE 6 Incorporates data of several growth-response experiments of A. quadruplicatum and A. nidulans to DDT and five DDT-analogs. Again, although much variation occurred, the data show little growth-rate depression of either alga at concentrations below 100 ppb insecticide. Both species show continued, perhaps greater, sensitivity to the two analogs DDE and ODD, as well as DDT. However, the more polar compounds DDA, DBF, and FW 152 apparently have little effect as seen in these experiments. III. Microbial uptake and accumulation. Toxicants, one taken up by primary producers such as marine algae, can be passed up the food chain to higher trophic levels. In addition, many toxicants can, depending upon the environmental conditions and species involved, be accumulated within the cell to levels many-fold higher than ambient. From the few studies available, accumulation appears to be primarily by inactive surface adsorption. However, Glooschenko found that dividing marine diatom cells in light accumulated 2 °3Hg longer than did non-dividing cells, thus indicating the possibility of some active uptake mechanism. The exotic and demanding minor-element nutritional requirements of many organisms would tend to support active uptake in some instances. We have found that yeast cells of Rhodotorula gracilis rapidly accumulated yii> of the DDT in a 2-ppm aqueous solution! Likewise, another yeast, Torulopsis utilis, took up 9^6 of the DDT in 3 minutes. In TABLE 7, showing the percent radioactivity of the ^C-DDT in the cellular fraction, the control values showed a random distribution of DDT between the medium and the cellular fractions, ranging from 21 to 56£. These results were obtained by centrifuging, decanting, and filtering the aqueous medium, and the distribution of i^C-DDT between the medium and cellular fractions was determined by liquid scintillation counting. In contrast to the erratic control values, the cellular fractions accumulated DDT at a constant rate over the <X)£ level after 3 minutes. An extract of R. gracilis was prepared by sonicating the cells before the additior~of the ^C-DDT. The sonicated pellet was found to accumulate an average of 96£ of the DDT. We also attempted to correlate cellular lipid content with the uptake of DDT. Rhodotorula gracilis when grown on a medium rich in carbohydrates and deficient in nitrogen and phosphorus will produce approximately 6o£ lipids, whereas when not deprived of N and P, lipid production is reduced to approximately UOjt. When cultures were grown under both circumstances, no differences in DDT pick-up were noted. It is apparent that the complexities of pesticidal-microbial in- �-7- terrelationahips warrants continued study. It has been amply demonstrated that members of the microbial world vary widely in their response to pesticides and that several factors may influence the toxicity of pesticides. Likewise, the microbial tolerance of pesticides may be affected by growth conditions, physiological condition of the cells, and various stress factors which might exist in natural populations (e.g., temperature, limited nutrients, competition). For example, growth experiments with A. nidulans established separate tolerances of 1% NaCl and 800 ppb DDT (Batterton, Boush and Matsumura, 1972). However, growth of this alga is severely inhibited in meul^u containing both 1% NaCl and 800 ppb DDT. Figure 1 illustrates relative growth of A. nidulans in various concentrations of DDT r.nd NaCl. The resulting growth pattern indicates the combined stresses of NaCl and DDT significantly changes the tolerance of A. nidulans to either substance. However, in similar experiments with test-tube cultures, growth inhibition was contraindicated when the calcium concentration of the growth medium was increased fivefold. It is particularly interesting to note the similarities in nearly all c the uptake-accumulation studies. First, pick-vp of the toxicant is extremely rapid—varying from a matter of seconds to a few minutes. And secondly, removal of the toxicant from the medium is quite high— usually more than 90jt of the total being removed by the cells (dead or alive), even when ambient levels were many-fold higher than those usually encountered in nature., However, one factor should not be ignored. Few, if any, studies have included competitive adsorptive substrates. Might not DDT, for example, readily aifsorb to organic matter, silica, etc., if available? The apolarity, affinity for lipids, and low water solubility of virtually all of the persistent insecticides make studies in aqueous substrates difficult. Of even greater importance, cau we extrapolate from our work, even to a United degree, to conjecture as to what occurs in nature? It is doubtful if we can overstress the important of microbial accumulation. After more than 25 years of world-wide use and study, the real threat from persistent pesticides is in their unfortunate ability to concentrate with food chains. This would not occur were the toxicants not picked up from low background levels, concentrated in the cell, and finally, stable for considerable periods of time. The results shown in Figure 2 indicate the general susceptibilities of brine shrimp, Artemia salina, to various terminal residues and analogs of DDT. It can be seen that these analogs, though many of them have been regarded as non-insecticidal, are indeed toxic to this species. IV. Effects of chlorinated insecticides on NaCl-tolerance mechanisms. Since Na+, K -ATPases have been known to serve as the enzyme re- �-8sponaible for Na+ and K+ exchanging across many biological membranes, we have decided to study first the effect of DDT on the salinity regulatory mechanism of a blue-green algae (Batterton et al., 1972). The initial experimental results indicate that the susceptibility of a blue-green alga, Anacystis nidulans, against DDT varies greatly under different salt concentrations. At high NaCl concentrations the bluegreen alga becomes extremely sensitive to DDT. It is clear from the result that this fresh water species loses its NaCl-tolerance capability in the presence of a low level of DDT: the level normally would not affect the specie. . A spearate experiment in vitro showed also that DDT was indeed an inhibitor of Na+, X+-ATPases of A. nidulans. blocking all ouabain sensitive ATPase activities. The most important indication that the ATPase is related to the Nad-tolerance mechanism comes from the in vivo finding that Ca , when added externally to the medium, can antagonize the effects of DDT. TABLE 7 Percent Radioactivity of lJ*C-DDT in Yeast Cells Time (minutes ) 17.5 32.5 Culture 2.5 7.5 12.5 Control Torulopsis utilis Rhodotorula gracilis Extract-R. gracilis Protein producing medium-R. gracilis Lipid producing medium-R., gracilis 21 92 97 98 U2 96 98 56 91 97 38 95 98 Average 39 96 97 96 97 96 97 95 98 . 97 In another set of experiments, the brine shrimp, A. salina, was subjected to various chlorinated hydrocarbon insecticides under different salt concentrations (Figure 10). The results clearly indicate that the effects of these chlorinated hydrocarbons are strongest at either extremely low or high salt concentrations. The brine shrimp is noted for its great capabilities of tolerance on different salt concentrations. It is often found in abundance in inland salt lakes (e.g., salt ponds and lakes in Utah) where the salt concentration is so high that no other organism can survive. The loss of salt tolerance mechanisms for this species by the presence of these insecticides is, therefore, quite a surprising phenomenon. The examples illustrate only one aspect of pesticidal pollution. It is important to note, however, that such a finding comes from fundamental knowledge of the chemical interactions with biological materials. �-9AN'ACYST.'S _N!PULANS FIGURE 1. Relative growth of Anacystis nidulane in response to varied DDT and NaCl concentrations. Liquid culture (15 ml) in 60 mm petri dishes inoculated ( 2 , 0 cells/ml) and incubated 72 hr. under 1000 200 ft-c fluorescent lamps at 37° C. After correction for evaporation growth was measured as optical density at 660 run. All O.D. values normalized to 1.0. «* 0.4 a* u> CONCENTRATION xo IN PPM FIGURE 2. Differential toxicities of DDT analogs and metabolites on brine shrimp, Artemia salina; 2k hours exposure at 2l*° C. �" •• ' • > , • • - . -10- It is aJ.so necessary to stress that those stable terminal residues and contaminants would not have been detected from the environments if not for the specific knowledge accumulated through basic researches in the laboratory as to their chemical characteristics and behavior. Factors involved in the interactions of pesticides with variouii ecosystems are numerous and complicated, but it certainly is hoped that there are a number of rate-limiting, key factors that can be analyzed through controlled laboratory experiments. V. Effects of pesticide micro-contaminants: Model ecosystem study. While the problem of pesticidal contamination of the environment is far from beirg solved, considerable useful information has emerged from the research efforts made by many scientists in recent years. First, we now know by experience that the chemicals that cause environmental problems are the ones which are extremely persistent in nature, biologically active, and easily concentrated in biological systems. Compounds which lack any of the above qualifications usually do not play any significant role in pesoicidal pollution no matter how acutely toxic they are. The above analysis becomes more important, when one insiders other aspects of pesticidal pollution. For instance, we are concerned about only biological effects in considering pollution, with particular emphasis on the effects on non-target organisms. In the case of polychlorinated dibenzo-p-dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the question of bioactivity is indisputable, as it is one of the most toxic compounds known to occur as a pesticidal impurity. Its chemical stability is also questionable. Thus the central question of its hazard to the environment must be studied from the viewpoint of bioconcentration in various ecosystems. Published data on environmental fate of chlorodibenzo-p-dioxins are scarce at present. For instance, residues of dioxins were not found in several aquatic animals at detection limits of 0.01-0.01* pg/g. In the study reported herein we have made efforts to measure the degree of bioaccumulatior. of TCDD in relation to well established pesticides by using several model ecosystems. The data are still preliminary, In that several model ecosystems are still being compared for their relative merits in assessing the actual impact of pesticides in nature. 'The data obtained have been, however, useful in assessing the relative tendency of a pesticide in comparison with other pesticides. Materials and Methods—Approximately 100 microbial strains which have previously shown the ability to degrade persistent pesticides were screened for their ability to degrade TCDD. Screening was carried out and the metabolic products were examined by thin-layer chromatography (TLC) by the method of Matsumura and Boush. The pesticides (0.1 umole each) were deposited on 1 g of clean sea sand, which was placed on a *7* TS '*" ''' Urn *" '1 �-11column of sandy loam type soil. Water was then slowly dropped onto the surface of the sand at a rate of approximately 2 rnl/hr. The water and sections of soil were extracted with chloroform. Three groups of invertebrates were used for the pesticide accumulation study: Qatracoda species, Artemis, salina, and Aedes aegypti larvae, and one fish species northern brook silverside, Laludesthes sicculus sicculus. Pour pesticides were selected from representative groups of important compounds: dioxin (TCDD), DDT, /-3HC, and zectran. All compounds were l^-labeled in the benzene rings. Three model ecosystems were used to study bioaccumulation. In model I, the pesticides (5 and 10 pmole) dissolved in a solvent were added directly to water along with the primary food organism, such as algae and yeast, and this mixture was then added to the aquarium containing the invertebrate test organisms. In model II, the pesticides (20 pmole) were deposited on the inner surface of the glass container by evaporating the solvent to form a thin film. The primary food organism were grown in the container for 2U hr. and then transferred along with the culture media to the aquarium contairihg the test invertebrate organism. In model III, the pesticides (5 and 10 praole) were deposited on 1 g of sand and the solvent evaporated to form a thin film on the surface of the sand particles. The sand was added to the test aquarium containing invertebrates and/or fish. In all cases the test organisms were maintained in the aquarium at room temperature (2U° C), except for the fish cultures which were maintained at 12° C. Test organisms were either homogenized in counting solution or carbonized (Model 300 Packard Tri-Carb Oxidizer), and the amount of ^C02 measured. Measurements of the amount of labeled material in the water, primary food organism, and on sand and glass surfaces were made by extracting with chloroform. All studies were short-term (k-7 days), in small volume containers (200 ml). As shown in Table 8, the extent of translocation of TCDD from the sand to the organic soil layer is extremely small. Virtually no TCDD was found to leach out from the column. The mobility of TCDD in soil, therefore, must be considered much less than that of DDT. Thus, the mode of translocation of TCDD in the environment would be limited to movement of soil particles or dust-carried dispersion and biological transfer (but not plant-mediated transfer), particularly in aquatic environments. As for the microbially mediated degradation of TCDD, our current survey indicates that such capabilities are rather rare in nature. Approximately 100 microbial strains in which the ability to degrade persistent pesticides has been previously demonstrated were screened for this purpose. Among them, only five strains showed some ability to de- �-12- grade this compound. We ha e not been able to manipulate cultural conditions to increase the rate of degradation of TCDD in any of the microorganisms so far. In studying the extent of biological transfer of TCDD, three different model systems were devised. In model system I, pesticides in acetone were introduced directly into water along with the primary food organisms. In model system II, pesticides were applied to the inner surface of a glass container, and the primary food organisms were grown in the container for 2k hr. and were transferred to the aquarium. In model III, pesticide-coated sands were placed directly in the aquarium contairAig the test organisms. In the model I experiment (Table 9), DDT behaved quite differently from other pesticides, showing high degrees of affinity to each test organism, in close agreement with the phenomenon actually observed in nature. Although this model system is simple and appears to offer a quick straight-forward answer to the general tendency of pesticidal accumulation by biological systems, it has one weakness, i.e., that one is forced to work above the limit of water solubility of some of the compounds. TCDD for instance was measured at a level 100 times its water solubility. Also the extent of direct pick-up due to partitioning and food intake is uncertain. In the model II experiment, where only the portion of pesticide picked up by the primary food organisms and the media were introduced into the test aquarium, the levels of total pickup were further reduced in the case of TCDD (but not DDT) (Table 10). To circumvent the problem of solubility, the model III system was devised. In this way, only that portion of pesticide that is soluble should be present in water at any time. The results shown in Table 11 indicate that the rate of TCDD pick-up is extremely low in brine shrimp and fish under the experimental conditions. Mosquito larvae, which are bottom feeders, showed a surprising rate of TCDD pick up. The reaction is not at its maximal rate, since further increase in the level of the pesticide apparently increases the pick up by the larvae. Also noted is the difference between the bioconcentration pattern in fish as compared to other invertebrates. y-HHC, in particular, shows high degree of concentration in fish. To study the effects of food consumption, the same test was repeated in the presence or mosquito larvae. As expected, the level of TCDD (Table 12) in the fish increased in the presence of mosquito larvae, which are the best concentrators of TCDD among the organisms tested. On the other hand, the levels of other pesticides did not significantly change, indicating that the route through ingestion of mosquito larvae does not represent the major source of uptake in these pesticides. It is apparent from these data that the reaction of biological concentration is greatly influenced by the external conditions and the design of the experiment, the physical and biological nature of the �,. ,3t;i,,-*,i[*-i*>»^ ',.', "$&•:•" "'' *?• ., *uf. ; '.Tjr • .,!«,. . u. -13organisms, and by chemical characteristics of the pesticides. To facilitate understanding of the role of chemical nature of pesticides in determining the rate of bioconcentration, a comprehensive list has been prepared to illustrate their Important properties (Table 13). It can be seen here that general tendencies of bloaccumulation in invertebrate species follow closely the trend of the partition coefficients. In model II experiments, however, the valuos for TCDD come much lower than expected from this rule. Thus it is likely that water solubility (and solvent solubility) must play an important role where the initial pick-up is the rate-limiting factor. It is apparent that species-specific factors play a much more important role than once suspected. For instance, the pattern of bioaccumulation and concentration in fish is quite different from those in other organisms studies, in that both /"-BUG and zectran snow higher degrees of affinity than DDT and TCDD, respectively. Although the data are not sufficient to permit a definite conclusion, they suggest the possibility that water-soluble pesticides tend to accumulate in fish. TABLE 8 Vertical translocation of pesticides from sand to organic soil.a Pa s t ic ide c on tent, * DDT13 Top sand 0-0.5 cm 0.5-1.0 cm 1.0-1.5 cm 1.5-2.0 cm 2.0-2.5 cm Water 1st 2nd 3rd eluatec 50 ml 50 ml 50 ml Zectran" 90. Ul 7.32 1.0U 0.50 0.26 0.18 65.01 30.75 3.51 0.55 0.26 0.19 0.07 0.06 0.08 0.05 0.06 0.06 0.12 0.08 0.06 0.0*1. 0.02 1*9. U 17.6 29.1 0.09 10 x 1.5 cm glass column Pesticide introduced: 0.1 jamole each (33-8 for DDT, and 22.2 ;ig for Zectran) Water eluted per day, 50 ml for dioxin, 35.5 ;ig �-1UTABLE 9 Bloaccutnulation of pesticides by aquatic invertebrates for model I (pesticides introduced directly into ambient water with the primary food organisms). Test organisms (primary Pesticide food) Original concentration in water, ppb Final concentration found in test organisms, Concentration ppb factor Paphnia (algae) Dioxin DDT Zectran 32. ** 35.8 22.2 1,592 Ijl»,l6>» 1,969 '•9 123U 89 Ostracod .Tfalgae ) Dioxin DDT Zectran 32. k 7,069 50,771 7,265 218 1U18 327 Brine shrimp (yeast) Dioxin DDT /•-BHC Zectran 35.8 22.2 16.2 If 956 12,336 2,688 17.9 1U.7 11.1 121 689 183 1U 155 TABIE 10 BJ©concentration of pesticides by aquatic invertebrates for model II (primary food organisms allowed to pick up pesticide from glass surface and then given to the test organisms). Test organism (primary food) Pestici-ie Original amount, jig (theoretical concentration, ppb) Daphnia (algae) Dioxin DDT Zectran 6.U8 (162) 3.58 (179) 2.22 (111) Ostra :od (algae) Dioxin DDT Zectran 6.U8 (162) 3.58 (179) 2.22 (111) of the test. Final concentration found, ppb Water Test aquarium organisms O.U 22.9 15.1 2.6 50.8 H:.<, 879 U3.123 37,l»99 279 36,391 6,177 Concentration factor* 2,198 1,883 2,U88 107 716 1U2 �TABI£ 11 Bioconcentration of pesticides by aquatic organisms for model III (pesticides introduced into system in the form of rp-iuues on sand). Amount of pesticide Concentration found, ppb Test Water (including food) organisms Test organism Pesticide Brine shrimp Dioxin DDT V-BHC Zectran 1.62 1.79 1.47 1.11 0.1 0.5 5.2 5-0 Dioxin 1.62 O.U5 2.1+0 0.85 1.40 Mosquito larvae PC DDT /-3HC Zectran 3.24 1.79 3.58 1.47 2.9k 1.11 2.22 Fish (silverside) Dioxin DDT y^-SHC Zectran 1.62 1.79 1.47 1.11 6.6 13.1 5.45 10.8 0 2.1 1.8 4.7 157 Concentration factor 1,570 3,092 495 89 6,184 95 18 " 4,150 0 9,222 5,000 16,765 21,571 220 221 0 89 8 2 458 2,904 *?• 218 12,000 14,250 30,200 1,450 2,900 213 —. 1,613 U5 w* �-16'CABLK 12 Two-step bioconcentrution of pesticide by mosquito larvae, and northern brook silverside (model III). Pesticide 1.62 Dioxin DDT /•-BHC Zectran Water (including Hood) 1.3 1.1 1.79 l.U? 1.11 1.9 5 Concentration factor Mosquito Fish larvae 3,700 17,900 690 0 Amount of pesticide jig Concentration found, ppb Mosquito Fish larvae 2.8U6 16,273 708 337 1080 76 383 0 5^ 306 600 15 TABIB 13 Physiocochemical characteristics of dioxin in comparison with other insecticides. Water solubility Solvent solubility Water solubility 0.2 1.2 100 10 ioio 1 Dioxin DDT Zectran /•-BHC 5 ppb ppb ppm ppm Partition coefficient (vs. hexane) 106 10 * 105 100 ,0* 1000 0,0 100* 1,700 Benzene solubility, g/10Q g O.OU7 80 80 - Estimates The data indicate that TCDD is not likely to accumuhte in as many biological systems as DDT. This is likely because of TCDD's low solubility in water and lipids as well as its low partition coefficient in lipids. Since microbial degradation is not expected to be a rcajor factor, the predominant mode of elimination of this compound in the environment is photodecopposition by sunlight. VI. Degradation of pesticides by algae. Three salt water algae, Porphyridium sp., lAinaliella tertiolecta, ard Coccochloris elabans strain Di, were selected and studied for their ability to degrade a number of environmentally important pesticides (2,U-D, 2,U,5-T, mexacarbate, and DDT) and a pesticide contaminant �-17(tetrachlorodihcnzo d i n x i n ) . Pure algae cultures were grown on a dci'initlve raedla under laboratory c o n d i t i o n s . The compounds were studied under the following conditions: (l) growing al|,ao undor 2't hour light, (2) heat killed algae under 2'* hour light, (3) growing algae undor total dark (standard mrdia amended w i t h glucose) and ('0 controls (the medium alone but no algae) under ?.h hour light. The above studies were conducted for 7 days. Three co-npomids, 2,U-D, 2,U,5-T and TODD were reslstent to breakdown under those conditions. Mexn.carba.te, and DDT were readily broken down. Although inexaoarbatc was defended in the presence of light alone, in the presence of algae ( l i v i n g and d e a d ) , over '(0$ of the compound was converted to water soluble materials not fo^nd in controls. These compounds became solvent exl.ractable only after acid hydrolysis. A chloroform soluble metabolite was also dotcc'-ed v/hich was not found in c ntro.ls. This material had an Rf (ethyl ether, hexane, othanol; Y7:?0:3) belween methyl fonaami.do and formamldo mexacarbate derivatives. Tlie degradation of DDT under the above light conditions seem to give a small amount of DDA. In the presence of algae (living and dead), under light conditions, two other compounds were formed. One compound has tentatively been identified as DDE. The other compound using three TT£ systems has been identified as DDOH. Due to the fact that dead algae also forms the metabolites of mexacarbate and DDT it is postulated that a compound is formed by the algae v/hich causes photo-decomposition. The identification of the metabolites and the nature of the photosensitizers are proposed for future study. INDEX OF TECHNICAL REPORTS 1. No Technical Reports have been issued. BIBLIOGRAPHY OF ALL PUBLICATIONS 1. Batterton, J. C . , G. M. Boush and F. Matsuraura. 1972. DDT: Inhibition of sodium chloride tolerance by the blue-green algae Anaeystis nidulans. Science 176: llUl-llU3. 2. I'atsumura, F. and H. J. Benezet. 1973• Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzop-dloxin. Environ. Health Persp. 5: 253-253. 3. Boush, G. M. . Effects of pesticide terminal metabolites on algae and brine shrimp. (In preparation). �Mt~ *J -18M.JOR Varioi'-. alijue species are tested for i.hclr :iii;;<:opUb1 lities towards chlorinated hydrocarbon Insecticides. D i o l d r l n , which in the rnout frequently found pestlcidal contaminant in the Uf5, and Its analogs were found to Inhibit the growth of certain of algae :;pccles. Anacjfstis nidulans In pai-ticular showed marked susceptibility to endrin, die.l.drin, ketixrulrin and p h o t o d i e l d r i n . This species was also susceptible towards d l o l d r i n metabolites r.uch as metabolite F and (}. Among DDT me tab o I. i to s ODD (TDK) was found to be the most toxic material; followed by DDK, DDT and KW-l'p3. It has not been known that ODD should be more toxic to algae. In terms of acute toxicity phenylmercuric acetate was by far the most algicidal agent among all pesticldal chemi c a l s tested. This pesticide is toxic to both A_. nidulans^ and A. !n,a-lnipj l-.ratvrn at the concentrition of 1 ppb. Algae, along w i t h other plankton, are known to bioaccumulate pesticides and thereby play a vital role in the process of food-chain aoetnaulattnn of these mieruponutants. Our studies indicate that the rates of pick-up of p o s t i c i i l o s are very rapid. To study the feasibility of constructing a model censysl<.3i we u;:ed algae as a key food chain or(7xnism. By this way we could dc-nonntrate that 'WJDD, the most toxic contaminant of 2,'^,5-T does not really accumulate in the aquatic organisms as compared to DDT. Algae as a whole are not very active in degrading pesticldal chemicals iji vivo. They were found to play, however, a key role in the process of environmental alteration of pesticidal residues. The way they participate in such processes was found to be through synerfjistic actions on photochemical reactions. Algal products, when tested in the form of aqueous extract from dead algal cells, were fouiid to be excellent photosensitlsers for DDT and mexacarbate degradation by the sun-light (simulated sun lamp). � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 005 Folder The folder containing the original item. 0054 Series The series number of the original item. Series 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 Boush, G.M. F. Matsumura Description An account of the resource Corporate Author: University of Wisconsin, Department of Entomology, Madison, Wisconsin Date A point or period of time associated with an event in the lifecycle of the resource April 1 1975 Title A name given to the resource Pesticide Degradation By Marine Algae Subject The topic of the resource biodegradation dioxin pesticide testing