Document XR13zqXwqKYd6xzyxv3rnGGyw

t I GRANT-IN-AID-PROPOSAL ELIMINATION OP POLYCHLORINATED BIPHENYL RESIDUES PROM SILOS submitted to Monsanto Industrial Chemicals Company St. Louis, Missouri b7 Ohio Agricultural Research and Development Center Wooster, Ohio 0A1396* PCB-ARCH0745557 TABLE OF CONTENTS JUSTIFICATION.................................................................................................. 2 PREVIOUS WORK AND PRESENT OUTLOOK............................................... $ OBJECTIVES.......................................................................................................... 11 TECHNICAL APPROACH.......................................................................................12 Silo Coating Material Experiments....................................... 12 Aroolor Feeding Experiments..................................................... 14 ANALYTICAL PROCEDURES................................................................................17 QUALIFICATIONS.................................................................................................19 Personnel and Responsibilities...............................................19 Current PCB and Related Research..........................................19 Facilities and Equipment..........................................................22 PROPOSED TIMETABLE....................................................................................... 27 PROPOSED BUDGET.............................................................................................. 28 LITERATURE CITED............................................................................................ 29 APPENDIX OM 3965 PCB-ARCH0745558 i i \ ELIMINATION OP POLYCHLORINATED BIPHENYL RESIDUES PROM SILOS lumber of silos throughout Ohio and neighboring ptates are contaminated with polychlorinated biphenyl residues. These residues In turn contaminate the allage and the products produoed by livestock that consume this silage. Since poly ohlorlnated biphenyls are a dntamlnant or human foods, livestock producers with contaminated silos are faced with and/or have suffered unmarketable produce and t In 1971 studies were begun In the Department of Dairy Solence of the Ohio Agricultural Research and Development Center to eliminate polychlorinated biphenyl residues from producing dairy cows and their feed supply. Tho three ap proaches to the problem have been: to Increase the rate of PCB clearance from the animal; to remove the milk fat and associated PCB from non-fat milk components; and to develop ooatlng systems to permanently oover the PCB contamination In the silo. The research Included In this proposal repre sents a continuation of this effort to eliminate these resi dues from livestock produots. oax3``>t- PCB-ARCH0745559 ( : -2JUSTIFICATION Polyohlorlnated biphenyls (PCB's) have recently been detected in livestock foodstuffs and subsequently In neat, eggs, poultry, fish and milk. These compounds, as are all synthetic chlorinate hydrocarbons, are 1/flegal oontarelfiants of human food. The dlsoovery of these compounds has caused considerable oonoern among those associated with animal agrl-culture--an4^ubstantlalT^)!nanclal losses to those who have fJ o ro been dlreetly'icffanted by this oontamlnent. major source) of PCB contamination of milk and meat has b^gu-tfllos which were coated to prevent leakage and erosion of the walls with a material that contained the Aroolor's-* These PCB compounds were not specifically produced for lnoorporatlonintosllo coatings, but were used because they are good (plasticlae^a^------- 'f40 /- Reports of silos contaminated with PCB's have come from a number of mldwestern, mideastern and southern states. The exact number of PCB contaminated silos Is not known, however, 7 It Is suspected that over^OO a 1103 In the Ohlo-Mlchigan area are so contaminated. The contamination In these silos Is a ohronlo problem over many years. Silos coated as many as^l6) years ago with the Aroolor containing coating still contain substantial quantities of the PCB. - Currently ^nlj)^Ee~7>hlo State Department of Agriculture Is conducting a regular testing program to detect and ban the lAroolor Is trademark of Monsanto Co. 0413967 PCB-ARCH0745560 I <( UP .. _ . . -3- shlpment of milk that contains more than 5.0 ppm of Aroolor on a fat basis. However, budget and personnel shortages within this Department have prevented routine testing of all producers. Twelve^jtroduoers in Ohio have had to cease milk shipment until the levels of Aroolor decreased to less than the "actionable level." This problem^mllriJV'doubt affect Increasing numbers of dairymen and cattlemen In the near future. State and federal agencies will attain the capabilities to assay these hydro carbons and more producers will have their milk and meat pro ducts checked for PCB contamination. Furthermore, the Food and Drug Administration has proposed lowering the "administra tive guidelines" and reclassifying them as "temporary toler ances." If this proposed ohange occurs then the current allowable tolerance of 5 ppm for milk on fat basis (equal to 0.2 ppm whole milk basis If milk has 1$ fat) would be reduced by half to 2.5 ppm on fat basis. If this change should occur N then many livestock producers who formerly were below the allow- //^ able toleranoe would be subject to a shipment ban on their ) products. A livestock producer who has a silo contaminated with PCB's is faced with considerable financial loss. Replacement of the basic silo structure will probably cost In excess of $4,000.00. If his milk Is unmarketable he may be losing $6.50 per hundred pounds rejected and contaminated, unmarketable livestock may have been worth $36.00 per hundred pounds. Therefore, it seems oaia^bB PCB-ARCH0745561 ) -llJustifiable that an effort be made to develop low cost pro cedures that will reduce the level of the PCB contamination from silos below that which may cause rejection of the farm produot. PCB-ARCH0745562 PREVIOUS WORK AND PRESENT OUTLOOK Description of the distribution, and some biological effeots of PCB's have been reviewed (7, 10, 12, 13). There Is no need to review or discuss these general aspects of PCB contamination here. The following discussion will bo limited to the contamination of milk and silage. When dairy cows were fed Aroolor 1254* or a feed contami nated with this ohlorlnated hydrocarbon, residues were deposited in the body fat and secreted with milk fat (3, 5). This residue may appear In the milk within 12 hours (unpublished results from our laboratory) and when fed continually approached a "steady state" in about 40 to 60 days (1, 5). Pries et al. (5) fed 200 mg of Aroolor 1254 dally to nine cows for 60 days. The average excretion of Aroolor residue between the 40th and 60th day was 42.3 mg per day which accounted for 21# of the Intake. As yet It Is not known If the rate of excretion Into milk is linearly related to the concentration of Aroclor consumed. When Aroolor feeding was discontinued a rapid decline In the residue In milk was observed with about a 50# decline In residue concentration In 15 days (5). Thereafter, the rate of deoline was slower being 1.0 to 1.5# per day (4, 5). Polychlorinated biphenyls have been shown to have a similar rate of elimination from cows as do some pestloldes (3, 4)* especially DDE (6). The rate of dleldrln exoretlon was Increased by feeding phenobarbital or activated carbon (17). Unfortu nately, PCB excretion was not Increased by feeding these compounds 0*13970 PCB-ARCH0745563 tv 1 ______________ -6- (1, 4)- Aooeleration of PCB excretion In cows has not yet been achieved; however, studies are In progress In our laboratory utilising high-fat diets. This approach seems promising since Rusoff et al. (14) aooelerated removal of DDT and dleldrln from contaminated cows by feeding high fat diets. This accel erated removal of the chlorinated hydrocarbons was probably the result of lnoreased turnover of the body adipose tissue. Palmqulst and Conrad (9) have shown that a high fat diet may reduce the turnover time of the body adipose tissue pool by 3% The mechanism by whloh PCB's from Aroolor-contalning coatings contaminate the silage was originally thought to be only meohanioal by flaking off of contaminated ohips of the ooating. It is not uncommon for these chips to contain 5,030 10,000 ppm of Aroolor. However, recent work has shown that the Aroolor is soluble in silage juice (15). The solute may well be aeetlo acid, one of the Important organic acids formed when the silage ferments. Aroolor 1254 has been shown to be soluble in acetic acid (8). The Aroolor found in the silo walls and in the silage does not appear to be different than Aroclor standards when analyzed by gas chromatography (1, 15). This suggests little or no microbial degradation of Aroolor 1254 during the ensiling prooess (2). Skrentry et al. (15) reported that the majority of the Aroolor was detected in the six inches of silage adjaoent to the silo wall. However, the contamination diffused through 0*13971 PCB-ARCH0745564 the silage and was detectable 48 inches from the silo wall. Similar results were obtained in our studies, except no con tamination was deteoted beyond 38 inches from the silo wall (16). In the aforementioned study by Willett (16) the use of certain coating materials to cover Aroolor contamination in a alio and on stave blooka substantially reduoed the Aroclor levels in silage. These studies indicated that the thickness of th coating and the solvent carrier used were important factors Influencing the quantity of Aroclor residue found in the silage. By utilizing the data in these previously discussed studios it appears that the recoating of silos that are contaminated with Aroolor may well be a practical means of reducing the contamination to below the proposed maximal tolerance levels. The following example illustrates how this may be a feasible approaoh. Figure 1 is derived from the data of Willett (16) and shows the mean concentrations of Aroclor 124 that appeared in the silage (dry matter basis) at various distances from the silo wall. Those data are from the ooating test sections of a silo with an 8-foot diameter. Also shown is the approximate per centage of the total silage which Is between the various sampl ing distances from the wall. By utilizing these percentages and the mean concentrations of Aroclor within each distance from the silo wall a weighted mean concentration of Aroclor 0*ilV>7z PCB-ARCH0745565 PPM AROCLOR 1 2 5 k I n S ila g e -8- Peroent of Total Silage Represented FIG. 1. Mean concentrations of Aroclor 1251; that appeared In silage (dry matter basis) at various distances from the silo wall. The lower diagram repre sents the approximate percentage of the total silage between the various sampling distances from the wall. PCB-ARCH0745566 , ( t V. ' ' ______ -9- oan be calculated for the blended silage from the entire silo. This gives us a meaningful figure with whloh to work, since on commercial dairy farms, silo unloaders discharge silage frcan all parts of the silo. For this example we will presume that a cow Is being fed 13.6 kg of silage dally, and the silage Is 33* 3?^ dry matter. r \ I ' ^ Jv We will also presume that the oow Is producing 18.6 kg of milk VV dally which Is the average production of cows In Ohio on UHIA ' testing. Utilizing weighted mean concentration of Aroclor from j* lf ` ' Figure 1, the cow In our example would consume 8.8 mg of Aroclor dally. If 21% of this dose Is excreted In the milk, as has teen reported by Fries et al. (), then I.67 mg or 0.1 ppm would be In the milk dally. Hie estimate of the concentration of Aroclor In milk for this example would be a maximal figure. First, development of a ooatlng system that would reduce the level of contamination of silage adjacent to the silo wall to 2.0 ppm or less seems very possible. Second, the majority of the silos In use today are of larger diameter than 8 feet thus the contaminant would be diluted more. Additional research Is necessary to establish two components of the above example. Further testing of ooatlng materials Is necessary to verify their effectiveness as barriers to the PCB's In the silo walls. Also, further verification of the quantity of the consumed dose of Aroclor that appears In milk is necessary. Particularly the percent of the consumed dose 0<U397 PCB-ARCH0745567 appearing In milk must be established when low levels (1-20 ag per day) of Aroolor are fed. 0413975 PCB-ARCH0745568 ) -11 OBJECTIVES 1. To determine how effectively various coating materials prevent oont8mlnatlon of allage with Aroclor 1254 from previously used silo coatings. 2. To determine the durability of various coating materials when used in a silo to cover PCB contamination. 3. To determine the concentration of residue from Aroclor 1254 in milk resulting from known concentrations of Aroclor 1254 in the silage. ' PCB-ARCH0745569 ) -12TECHNICAL APPROACH In order to achieve the outlined objectives, four approach es will be used. Three experiments will evaluate coating mater ials for silos and one will determine the concentration of resi due from Aroclor 1254 In milk resulting from known levels of Intake. Criteria for the selection of coating materials to be tested were: The effectiveness of certain types of coatings In the pre liminary study (15), the physical and chemical properties of the coating (thickness, solvents, etc.); and, the cost of the mater ial. Only materials that would cost less than $600,00 for pur chase and application (14' x 50' silo) are used. The levels of Aroclor for the feeding experiment are de signed to approximate trace contamination of silage yet main tain the milk residue at 0.1 ppm and less. Silo Coating Material Experiments. 1. Silo stave block experiments. Cumar is the name of a coating used by several mldwestern silo companies and Is respon sible for most. If not all, of the Aroclor contaminated silos. , This coating contains 70 lb. Aroclor 1254 and 20 lb. Aroclor j 5460 per 47 gallons of paint. It should not be confused with Neville R-1-6A resin that has the same name. / 1 In these studies concrete silo stave blocks will be coated with Cumar. After Ihe Cumar has had adequate time to cure then the various experimental coatings will be applied to cover this 0*13977 PCB-ARCH0745570 t ' ) -13Aroclor containing material. Untreated and Cumar coated blocks will be maintained as controls. The blocks will be placed In polyethylene lined 55 gallon drums, chopped corn or alfalfa will be pressed Into the drums and sealed for four months. During this period the ensiling process will take place In the same manner as In a large silo. Silage samples will be collected at three depths and three distances from the blocks and analyzed.for Aroclor 1254* These block studies provide the opportunity to rapidly and at low cost evaluate the performance of the coatings as barriers to Aroclor contamination of silage. These studies will provide for minimal extraneous contamination and sampling can be very acourate. Unfortunately the stave block experiments do not evaluate the durability of the coating when used In an actual silo. 2. Experimental silo. A silo which had a residue of over 600 ppm of Aroclor In scrapings of the wall will be used as the test structure. This silo was divided Into test sections with Masonite sheets, the walls were wire brushed to provide a sound clean surface and various coatings were applied. See the report by Willett (16) for additional details. . These experiments will be conducted by filling the silo to the top of the test sections with chopped corn or alfalfa and allowing normal ensiling to take place. Pour months after fill ing, the silage will be sampled at specific depths and distances from the wall. These will be analyzed for Aroclor to determine PCB-ARCH0745571 ) -14- the vertical and horizontal distribution of the residue. This silo will be serially filled and sampled. This will test the durability of the various coatings a3 well as determine whether the coating maintains an effective barrier to Aroclor when used continually. 3. Field silos. Some silos that are known to be contami nated with Aroclor will be entirely coated with the materials that appeared most effective In our preliminary study (16). These will bo silos that are In U30 .on commercial dairy farms. Prior to recoating of these silos, samples of silage, silo wall scrapings and milk from the bulk tank will be analyzed for Aro clor. Following filling, biweekly samples of silage will be collected adjacent to the silo wall. On the same day that the silage Is collected samples of milk from the bulk tank will also be collected. All samples will be analyzed for Aroclor. Presently, one silo is on experiment and another will be coated In August 1972. The number of silos that will be Involved In this phase of the research Is not known because the dairyman must volunteer to have his silo used, must stand all costs and assume all risks. Aroclor Feeding Experiments. In order to determine the amount of Aroclor residue that appears In the milk from a known dally Intake, 70 day feeding trials will be conducted. Initially six animals will be allot ted to one of three levels of dally Intake of Aroclor. These Aroclor feeding levels will be U4..O mg, 7.0 mg, and 3.5 mg 0413979 PCB-ARCH0745572 ) ________ -15- daily. If tho results from these animals are highly variable, additional anlraalswlll need to be employed. All animals vrf.ll be on a standard feeding program and will be fed corn silage, alfalfa hay and grain concentrate at the ratio of 1:1:1 on the as fed basis. The Aroclor will be fed onoo-dally by adding a premeasured acetone dilution to the silage. The acetone will be allowed to evaporate prior to feeding. Re fusal (If any) will be weighed and analyzed for Aroclor so that the Intake can be corrected. Milk will be sampled from each animal biweekly and ana lyzed for Aroclor residue. Weekly samples of urine and fece3 will also be collected and analyzed. Data from these animals will be analyzed to determine the percentage of the consumed do3e that appears In the milk. Other analysis will determine: If parallelism occurs between the con sumed dose and levels of residue In the milk; the time required to reach biological equilibrium; and, variability among animals. Levels of Aroclor residue In urine and feces should further sub stantiate the proportions of this hydrocarbon cleared by those excretory pathways. Since unverified reports olalm reproductive failure and calfhood mortality among cows fed Aroclor contaminated silage, accurate records of the reproductive performance of the animals will be kept during the Aroclor feeding periods. Feeding of such low levels of Aroclor are not expected to alter the repro ductive pattern. 0*11980 PCB-ARCH0745573 16All experimental animals that die or are killed will he neoropsled and selected tissues analyzed for Aroolor residue accumulation. o* 13981 PCB-ARCH0745574 -17ANALITICAL PROCEDURES The facilities and equipment available for these studies ore discussed In the "Qualifications" section of this proposal and will not be repeated here. The procedure for the extraction of silage Is In the appendix. Milk, urine and feces samples are also conducted by this method with only slight modifications. The extraction procedure (unpublished); consists of an acetone extraction, partitioning against petroleum ether and a clean up on a silica gel-florsil column; and was developed by A. C. Waldron with modifications by L. B. Willett. For silage, this method has provided very repeatable results, low backgrounds and consistent recovery of known standards. The mean background lovol for silage samples has been 0.020 ppm. The mean recovery from standards (between 0.05 and 2.0 ppfti) was 67.45 + 1.31$. The procedure has provided very low background levels (0.009 ppm) and good repeatability for the extraction of milk samples. However, the percent recovery is not constant between 0.05 and 1.0 ppm. Instead there Is a repeatable los3 of about 0.045 ppm from all standards between 0.1 and 1.0 ppm. This ex traction procedure has not proven dependable on standards with a,concentration of 0.05 ppm. For this reason, Aroclor levels will be determined on milk fat samples rather than whole milk. Gas chromatography of samples Is conducted on an F & M 42 63 ' with a "Ml electron capture deteotor. The column Is an 8 foot by l/8 Inch and packed with 4$ DC-200 and b% QF-1 on Gas Chrom Q O'.U'JSZ PCB-ARCH0745575 (' ) ______ -18- (80-100 mesh). The running conditions are: Column 210, detector 300 and Argon-Methane flow l\2 ml per min. A typical standard tracing Is enclosed In the appendix. Quantitation of the sample Is done by disc Integrations and comparison of these to the Integrations of known standards. Care Is taken to maintain sample concentration within the linear range of the detector. For silage samples total inte grations from peak "F" through peak "K" (see appendix) are used, while for milk the Integrations from peak "D" through peak "P" are used. Silage samples are extracted on a wet basis as they come from the silo. The Aroclor concentrations In silage samples are expressed on a dry matter basis to remove variability due to moisture. Dry matters ar6 determined on a portion of the sample by weighing before and after drying at 100 C for lp3 hours. Aroclor levels In milk are reported both on a whole milk and fat basis. Milk fat percent is determined by the TESA method. 0*13983 PCB-ARCH0745576 (' ) -19- QUALIFICATIONS PERSONNEL AND RESPONSIBILITIES. The proposed research program will be conducted by personal of the Ohio Agricultural Research and Development Center, departments of Dairy Science and Entomology (Pesticides Analyti cal Laboratory). The personnel, their departments, fields of speciality and responsibilities to the project are listed below. Biographical resumes are included in the Appendix. Dr. L. B. Willett. Dairy Science, Physiology, and dairy management. Dr. Willett will be the project leader. He will direct and conduct the silo and animal phases of the studies. He will also direct most of the Aroolor analyses. Dr. A. C. Waldron. Entomology. Pesticide and PCB analysis. Dr. Waldron will oonduct a portion of the Aroolor analyses. He will also assist with the interpretation of PCB values obtained. Dr. H. R. Conrad. Dairy Science. Ruminant Nutrition. Dr. Conrad will direct the nutritional aspects of the proposed studies. CURRENT PCB AND RELATED RESEARCH. Presently, studies are being conducted cooperatively between the departments of Dairy Science and Entomology (Pesticides Analytical Laboratory) to develop methods by which dairy systems can be decontaminated of PCB residues which originate from 0*1398* PCB-ARCH0745577 ooatings used to seal silos which contain Aroolor 1254* There are four objectives of the ourrent research. First, tf> study methods of and recommend a procedure for de contaminating silos contaminated with PCB. Second, to Identify dietary agents abetting the removal of PCB from dairy cows. Third, to develop a feasible approach for selectively skimming milk from highly contaminated cows to reduce PCB contamination of the total milk supply to below the actionable level. Fourth, to determine any pathological or toxicological changes and PCB levels In the body tissues of PCB-contaminated animals that die or are killed. Two lines of investigation have been used in the study of silo decontamination. A silo, which Is known to contain a substantial quantity of PCB In Its protective coating. Is used as the major structure for these studies. The silo has been sectioned Into test blocks which were mechanically coated with solvent- and water-based materials that are considered impervious to the Aroclor In the silo walls. Six to eight months after the silo was filled the effectiveness of the cover coatings were determined by analyzing the silage for Aroolor. A copy of the preliminary results Is Included In the Appendix. In other studies concrete silo blocks have been painted with a coating that contains Aroclor 1254* Then the blocks have been recoated with the materials designed to prevent Aroclor migration. These blocks were placed In air tight drums of silage for four months. The silage has been analyzed for Aroclor 0'.13'J8^ PCB-ARCH0745578 ) V I 21- oontamination. Experiments with 10 coating materials have been completed. Further experiments are In progress utilizing those coatings which appear most effective plus new coatings with the properties- ' that seem to form good barriers to PCB contamination. A series of experiments are presently In progress to deter mine the biological behavior of a single oral dose of Aroclor 12%h given to cows fed various dietary regimes. This study involved a comparison of the rate of Aroclor excretion in milk, urine and feces when the cows were fed high-grain and high-fat diets versus those fed normal diets. It is known that the turnover of fat In cows occurs at a more rapid rate when a high-fat ration is fed compared with the turnover of fat in animals receiving normal amounts of roughages. Conversely, hlgh-grain rations reduce the turnover rate of adipose tissue. The determination of Aroclor that appears in the feces Indicates the percentage of a single three gram Aroclor dose that was absorbed from the gut. Urinary Aroclor levels show the Importance of this excretory route. The Aroclor level in milk Indicates the amount of Aroclor excreted in milk, the rate of excretion, the time required for the milk level of Aroclor to fall below the actionable level (0.2ppm and/or O.lppm) and the Influence of high-fat and high-grain rations on the aforementioned excretion of Aroclor into milk. The results of these studies are not yet available as data analysis has not been completed. PCB-ARCH0745579 -22- An effort has been made to develop a system of management procedures for skimming milk oentrlfugally in order to remove the fat portion of the milk and the PCB contaminants. In this study milk from contaminated cows as well as milk contaminated with known quantities of Aroclor were assayed for levels of PCB. Milk was then subjected to centrifugal separation of the milk fat. The skimmilk and fat portions were then reassayed for PCB levels. The results of this study agree with those of Platonow et gJL. (11) which show thatnearly all of the PCB is associated with the milk fat and can be separated from the skimmilk. Aside from the PCB analyses conducted for the above studies, research at the Pesticides Analytical Laboratory includes: analyses of PCB and organochlorine, organophosphate and carbamate insecticides, and various herbicides in lake and river drainage wator; analyses of milk and feed products for DDT and dieldrin residues; and several projects concerning residues in fruit vegetables and animal tissues. FACILITIES AMD EQUIPMENT. The Ohio Agricultural Research and Development Center is oharged with the responsibility of conducting basic and applied research in agriculture and home economics and their related fields. It also has the responsibility of dissemination of new knowledge for the benefit of all segments of our society, and of training graduate students in cooperation with The Ohio State University Graduate School. oei3<>87 PCB-ARCH0745580 ) -23The O.A.R.D.C. Is a separate legal and budgetary entity and Is governed by Its own Board of Control. The professional staff of the 14 subject matter departments includes about 240 scien tists, including 120 faculty members at The Ohio State University who devote part-time to O.A.R.D.C. research. This effort Is supported by about 18? technicians and research assistants, 29? civil service employees, and 120 temporary seasonal workers. Some ?00 people are employed at Wooster during summer and about 415 during other seasons of the year. One of the newest additions to the O.A.R.D.C. research program Is the Laboratory for Environmental Studies, established In June 1970. The new laboratory coordinates environmental research and Initiates additional studies on pollution and Im provement of the environment. A 70,000 volume research library on campus Is housed In a new trl-purpose building. This facility has the library, a 1000 seat auditorium, and the statistical laboratory. The latter facility Is equipped with IBM 1130 computer and has the necessary supporting accessory equipment. The statistical labora tory has a statistician for assistance In statistical analyses. A photography laboratory, staffed by two experienced photographers, . and print shop services are available to O.A.R.D.C. personnel. Trained veterinarians In the Veterinary Science Department provide animal health services and surgical procedures when necessary. Experimental rations are prepared at the O.A.R.D.C. feed pro cessing facility, having micro and macro mixing capabilities, o*.^988 PCB-ARCH0745581 whioh can provide foodstuffs In a variety of Toms including pellets. The Dairy Science Department has 110 cow milking herd which consists of both Jerseys and Holsteins. The young female animals are reared at the Research Center for experimental purposes and as herd replacements. The dairy herd is used for studies in milk secretion, nutrition and physiology, herd management, and environ mental quality. The North Central Branch of O.A.R.D.C. has a herd of 100 registered Holsteins which are used for nutritional and breeding studies. The dairy farm unit has facilities to handle cows in groups, as individuals or in isolation. Facilities and procedures are routinely used whereby experimental animals are individually fed, milked and urine and feoes collected. Laboratories are available at the dairy unit with facilities to determine dry matter of feed and feces samples as well as facilities for the preparation of samples for storage. These dairy facilities will be used for the proposed research. Experienced technical support is presently employed at the dairy unit to do the feeding, milking, Aroclor administration and sample collection. Cows for the proposed experiment will be obtained from the O.A.R.D.C. herd. Three laboratories in the Dairy Science Department are devoted entirely or in part to the analysis of PCB residues. The PCB extraction laboratory (300 sq. ft.) is'complete with tables, 0*13989 PCB-ARCH0745582 (` ) -25- glasaware, chromatography oolumns, solvent distillation apparatus and storage space. The gas-chromatography laboratory (300 sq. ft.) is equipped with two P and M Model 402 gas-chromatographs, one of whloh Is equipped with Nl^ electron capture (EC) detection. ....... Also available Is a Varlan Aerograph HI PI gas-chromatograph. The F and M 402 with the EC detector Is committed to PCB studies. The analytical chemistry laboratory (1000 sq. ft.) Is also used for some phases of PCB analysis and for -protein, mineral and vitamin A analyses. This laboratory Is also complete with tables and glassware Including: Kjeldahl apparatus; fraction collector; electrophloresls unit; colorimeter; refrigerated centrifuge; auto analyzer for nitrogen, urea, calcium, phosphorus, blood sugar and protein-bound Iodine; and separate rooms for handling and counting radioactive materials, amino acid analysis and analytical weighing. Walk-ln cooler and freezer space as well as freeze drying equipment are available for preserving samples. The Pesticides Analytical Laboratory, In the Entomology Depart ment, will oonduot a portion of the PCB analyses. This laboratory has been In operation since 1965, and Is under the direction of Dr. A. C. Waldron. In this laboratory a wide range of pesticides, herbicides and PCB's have been studied In water, milk, eggs, plants and animal tissues. The Pesticides Analytical Laboratory is complete with tables and vacuum evaporators, ovens, water baths, balanoes, mixers, grinders and appropriate glassware required for the analysis of PCB residues. Also available is a Barber-Colman Series 5000 0A139<I0 PCB-ARCH0745583 (i ) -26 gas-ohromatograph with electron capture detector and a Beckman nBn spectrophotometer. Additional equipment available to the laboratory include: Dohrmann miorocoulometer detector C-200-A with chloride and sulfur capture detector, P and M gas chromato- graph with EC detector, Beckman DU-2 speotrophotometer with speotral fluorescence attachment, Beckman IR-10 infrared spectro meter and a Kontes Sweep co-distillation apparatus. PCB-ARCH0745584 > 27PROPOSED TIMETABLE If full time technical assistance can be hired by October 1, 1972, the following timetable should be possible: January 1, 1973 - Data available from second silo trial and from second stave block trial. Complete data should be available from first filling of one field silo. Partial data should be available on the Aroclor feeding study, April 1, 1973 - Data available on the third silo trial and the Aroclor feeding study. Partial data should be available on continued used of the field silos and third stave block trial. July 1, 1973 - All data should be available for final analysis unless previously done. October 1, 1973 - All aspects of the outlined research should be completed. . 0*13992 PCB-ARCH0745585 { i ) -28PROPOSED BUDGET Elimination of Polychlorinated Biphenyl Residues from Silos The following Proposed Budget will cover the direct oosts and administrative overhead for the PCB studies described In this proposal. This Proposed Budget Includes the costs that will be Incurred between Ootober 1, 1972 and September 30, 1973. Labor Costs: Technical Assistant (Temporary) 12 month employment Student Labor (Part-time) To do dishwashing, eto $7,296.00 600.00 TOTAL LABOR 7,896.00 Supplies: Solvents for extraction Chromatography reagents Mlso. glassware and reagents Gas-chromatography gas Gas-ohromatography service & supplies 300.00 320.00 260.00 TOTAL SUPPLIES 2,10!|.00 Administrative Overhead: 2,600.00 GRAND TOTAL $12,600.00 0*13993 PCB-ARCH0745586 u ) -29BITERATURE CITED 1. Fries, 0. P. 1972. Polychlorinated biphenyl residues In milk of environmentally and experimentally contaminated cows. Environ. Health Pers., 1: 55. 2. Pries, G. P. 1972. Degradation of chlorinated hydrocarbons under anaerobic conditions. Adv. Chem. Ser. In press. 3. Pries, G. p. and G. S. Marrow, Jr. 1972. Relationship of milk fat and body fat concentrations of the organochlorlne compounds In cows. J. Dairy Scl., 545 796. 4. Pries, G. P., G. S. Marrow, Jr. and C. H. Gordon. 1971. Similarity In behavior of DDE and polychlorinated biphenyl (Aroclor 1254) residues In an environmentally contaminated herd of dairy cows. Abstract. ' J. Dairy Scl., 54* 796. 5. Fries, G. P., G. S. Marrow, Jr., and C. H. Gordon. 1972. Excretion of polychlorinated biphenyl (Aroclor 1254) in milk of cows. J. Agr. Pood Chem. In press. 6. Fries, G. P., G. S. Morrow, Jr., and C. H. Gordon. 1972. Similarity of a polychlorinated biphenyl (Aroclor 1254) and DDE in rate of elimination from cows. Bull. Environ. Cont. Toxicol. In press. 7. Gustafson, C. G. 1970. PCB's - prevalent and persistant. Environ. Scl. & Tech., 4s 814. 8. Monsanto Company. (Undated Report). Aroclor Plasticizers. Tech. Bull. 0/PL-306A. _ 9. Palmquist, D. L. and H. R. Conrad. 1971. Origin of plasma fatty acids in lactatlng cows fed high grain or high fat diets. J. Dairy Scl., 54: 1025. 10. Peakall, D. B. and J. L. Blncer. 1970. Polychlorinated biphenyls: Another long-life widespread chemical in the environment. Bio Science, 20: 958. 11. Platonow, N. S., H. S. Funnell, H. D. Bullock, D. R. Arnott, P. W. Saschenbrecker, and D. G. Grieve. 1971. Pate of polychlorinated biphenyls in dairy products processed from the milk of exposed cows. J. Dairy Scl., 54s 1305. 12. Presett, I., D. J. Jefferies and H. W. Moore. 1970. " Polychlorinated biphenyls in wild birds In Britain and their avian toxicity. Environ. Pollut., 1: 3. PCB-ARCH0745587 ) u 3013. Risebrough, R. W., P. Rleche, S. G. Herman, D. B. Peakall and M. N. Klrvln. 1968. Polychlorinated biphenyls in the global ecosystem. Hature (London), 220: 1098. 14. Rusoff, L. L., A. S. Achacoso, J. H. Gholson, R. Bonner and E. A. Epps, Jr. 1971. Effect of dietary energy levels on acceleration of DDT and dieldrin residue removal in milk of contaminated cows. J. Dairy Scl., 54* 796. Abstract. 15. Skrentny, R. P., R. W. Hemken and H. W. Dorough. 1971. Silo sealants as a source of polychlorobipehnyl (PCB) contamination of animal.feed. Bull. Environ. Contami nation & Toxicol., 6: 409. 16. Willett, L. B. 1972. Barrier coatings to prevent poly chlorinated bipehnyl (PCB) contamination of silage. Ohio Agr. Res. & Develop. Cntr., 59: 28. 17. Wilson, K. A., R. M. Cook and R. S. Query. 1968. Effects of charcoal feeding on dieldrln excretion in ruminants. Fed. Proc., 27: 558. 0413995 PCB-ARCH0745588 APPENDIX 0*l3`,<,b PCB-ARCH0745589 { ) A1 PCB Extraction of Silage 1. Grind sample until largest particles are 0.5 cm In length. 2. Weigh $0 g of the silage sample and place In 500 ml flask. - 3. Extract with 250 ml redistilled Acetone by shaking for 12 hours on shaker. 4. Filter flask contents through Whatman's #1 filter paper Into a 500 ml graduate cylinder. 5. Record the volume of the Acetone extract. 6. Put the entire acetone extract volume in a separatory funnel and add 500 ml of 2% Na^O^, 7. Extract the above phase lx with 200 ml redistilled pet ether (30 - 60 C) and then again lx with 150 ml redistilled pet ether. 8. Combine both pet ether extracts and wash with 100 ml of 2% NaKCO^. 9. Dry the pet ether on rotary evaporator 42 C to a volume of about 20 ml. 10. Prepare a chromatography column as follows: a. Put glass wool In bottom b. Put In Z\ Inches of Flora11 60/100 mesh (Activated) c. Put In 5 Inches of Silica Gel (Davidson 923) (Activated) d. Put In 1 Inch of NagSOj, (Anhydrous) e. Wash column with 100 ml of pet ether 11. Add sample (In the + 20 ml pet ether) to the column. When the sample Is completely absorbed In the column, elute It with a. 250 ml of 2% Benzene In pet ether - elutes PCB's b. 250 ml of Benzene .12. Dry down on rotary to about 10 ml and transfer Into vials for storage with eluting solvent. 0413997 PCB-ARCH0745590 Biographical Sketch Kame: Willett, Lynn B. Birth Date: 2 Aug. 1944 Title: Ass't. Prof. Nationality: U.S. Sex: Male Birthplace: Colorado Springs, Colo. Education: Institution Location Degree Year Colo. State Univ. Purdue Unlv. Purdue Unlv. Fort Collins Lafayette, Ind. Lafayette, Ind. B.S. M.S. Ph.D. . 1966 1968 1971 Honors: Sigma XI ' Major Research Interest: a. General interests are the physiology and management of dairy cattle. b. Specific Interests are the decontamination of dairy systoms of PCB's, and steroid physiology with respect to environmental adaptation. Research and Professional Experience: 1. Graduate Research Assistant Department of Animal Sciences Purdue University, Lafayette, Ind. 1966-1971 2. Assistant Professor, Research ~ Department of Dairy Science Ohio Agricultural Research and Development Center Woo3ter, Ohio 1971-present Selected References: 1) Willett, L. B. and Albright, J. L. Dairy Management In Larger Herds. J. Dairy Scl., l: 138. 1968. 2) Willett, L. B. Albright, J. L. and Cunningham, M. D. OnceVersus Twice-Daily Feeding of Milk Roplacer to Calves. J. Dairy Scl., 52: 390. 1969. ~3) Willett, L. B., Dunn, T. G., Brown, B. L. and Erb, R. E. Excretion of 4"^*'0-Cortlcosterolds In Urine by an Ovariectomized Bovine. J. Animal Scl., 32: 119. 1971. fr' .J** V X&qpr, Oimw : - ' PCB-ARCH0745591 i 4) Willett, L. B. and Erb, R. E. Short Term Changes In Plasma _ Cortlcolds In Dairy Cattle. J. Animal Scl. (Acoepted), 1971 ` ' ' ' $) Willett, L. B., Brown, B. L, and Erb, R. E. Isolation of Meta bolites of li-^C-Contlcostorolds from Urine of an Ovarleotomlied Heifer. J. Dairy Scl. (` led). 1972. Willett, L. B I 0413999 . V ; '-KWttv. ifla -y-T-.-tTl t rpt*gireiaaMayaiiFKatnet --r-~. -*-** PCB-ARCH0745592 Biographical Sketch Name: Conrad, Harry li. Birth Date: 3 Oct. 1925 Title: Professor Nationality: U.S. Sex: Male Birthplace: Burlington, Ky. Education: Institution Location De_greo Year Univ. of Ky. The Ohio State Univ. The Ohio State Univ. The Ur.iv. of Term. AEC Agr. Res. Program Univ. of Calf. Harvard School of Public Health Lexington Columbus Columbus Knoxville Davis Boston B.S. M.S. Ph.D. Postdoc. Sab. leave Sab. leave 1948 1949 1952 1955 1961 1968 Honors: Sullivan Award - 19J+8 - University of Kentucky (outstanding senior) A.F.M.A. Award - 1959 - Citation by the American Dairy Science Assoc. Borden Award - 1966 - also by the A.D.S.A. for outstanding con tributions to knowledge of dairy cattle nutrition Major Rcsearch Intores_t: a. General research Interest are In nutrition nnd physiology of ruminants. b. Specific interests are nitrogen net aboil sir., factors regulating food intake and the application of tracer techniques in quan titative nutrition. Research and P-ofosslonal Experlence; Professor Conrad is currently leader of three major research pro jects and lecturer In Animal Energetics at the Ohio State University. He has been a member of the Graduate Faculty 3ince 1955. Dr. Conrad has 1? years experience in ruminant research including 16 years with isotope tracer methods. Contributions include studios on radiocalcium metabolism, rumen digestion, nitrogen motabollsr.. blood flow, causes of anemia, radioiodino uptake in cattle, regu lation of feed Intake in rur.inant3, and essential amino acid syn thesis in tho rumen. He is senior author of 60 and coauthor of 51 scientific journal articles and papers. During 1961 he studied tracer methods in animal metabolism with Professors Max Kleiber and A. L. Black of the University of California. In 1968 Dr, Conrad completed a sabbatical year of study on the regulation of nutrient balance in the laboratory of Professor Jean Mayer of the Harvard School of Public Health. O^l'tOOO PCB-ARCH0745593 Selected References: 1) Conrad, H. R., and Hansard, Sam L. Effects of Massive Doses of Vitamin D on Physiological Behavior of Calcium in Cattle. J. Applied Physiology, 10: 90. 1957. 2) Conrad, H. R., Hlbbs, J. W., Pratt, A. D., and Davis, R. R. Nitrogen Metabolism in Dairy Cattle. I, The Influence of Grain and Meadow Crop3 Harvested as Hay. Silage, or Soilage on Efficiency of Nitrogen Utilization. J Dairy Scl., 44: 85. 1961. 3) Conrad, h'. R., Pratt, A. D., and Hlbbs, J. W. Regulation of Feed Intake in Dairy Cows. I. Change in Importance of Physical and Physiological Factors with Increasing Digesti bility. J. Dairy Scl., 47: 54. 1964. 4) Conrad, II. R. , Miles, R. C., andButdorf, Janet. Estimation of Methionine Synthesis in Intact Cows after Administering Sulflde-35s. J. Nutrition, 91: 337. 1967. 5) Conrad, H.R., Hlbbs, J. W., and Pratt, A. D. Effect of Plane of Nutrition and Source of Nitrogen on Kethlonlno Synthesis In cows. J. Nutrition, 91: 343. 1967. Conrad, H. R. 0*1*001 PCB-ARCH0745594 Biographical Sketch Name: V/aldron, Acle C. Birth Date: 4 Feb. 1930 Title: Ext. Specialist Nationality: U.S. Pesticide Chemicals Sex: Male Birthplace: Malad City, Idaho Education: Institution Location Degree Year Idaho State College Brigham Young Univ. The Ohio State Univ. Tho Ohio State Univ. Pocatello Provo, Utah Columbus Columbu s None B.S. M.S. Ph.D 1948. 1957 1959 1961 Honors: Sigma .'Cl Gamma Signa Delta Vho's Who in tho Midwest Major P.esearch Interest: Pesticide residues In agricultural crops and livestock, In soils, and In water; and tho safe use of pesticides. Research anri Profes3Ional_Esperlonce: 1. Extension Specialist, Pesticide Chemicals, Ohio Cooperative Extension Service, 1%6-present. Major responsibility supervision of Pesticides Analytical Laboratory concerned with determination of pesticide residues In agricultural crops, livestock, soils, and water. Pesticide Coordinator dissemination of information on pesticide regulations and registrations, pesticide safety, environmental concerns with pesticides and all similar Information. Promotion of the pesticide safety program. 2. Method Development Chomlst, Agricultural Division, American Cyanamid Co., Princeton, New Jersey. 1961-1966. Concerned with tho development of analytical methods for the determination of pesticide residues In plant and animal tissues and In formulations. 3. Research Assistant, Ohio State Experiment Station, O.S.U., Columbus, Ohio 1957-1961. In conjunction with graduate - work. Studied the extraction, separation, and Identifica tion of organic nitrogen and phosphorus compounds In soli oei-io02 PCB-ARCH0745595 organic natter and tho composition of the fluorescent fraction of soil organic natter. 1).. Publications Editor, TJ.S. Army Chemical Corp. Commissioned Officer duties involved writing and revising Chemical Corps field manuals and sub-courses. SelectedReferences: 1) Waldron, A. C. and Mortensen, J. I>. Soil Nitrogen Complexes: III. Distribution and Identification of Nitrogenous Con stituents in Electrophoretic Separates. Soil Scl. 93: 286-293. 1962. 2) Waldron, A. C., Pasarela, N.R., Woolford, M. H. and Ware, J. H. Improved Colorimetric Determination of Phorate Residues in Plant Tissues. J. Agr. & Food Chem. 11: 241-214i. 1963. 3) Pasarela, N. R. and Waldron, A. C. Fluorometric Method for Kicrogran Quantities of Tehylenodlamine Residues in Milk. J. Agr. & Food Chem. 15: 221-226. 1967. U) Waldron, A. C., Kaeser, H. E., Golenan, D. Staubus, J. R. and Niemccyk, D. Heptachlor and Heptachlor Epoxide Residues in Fall Treated Alfalfa and in Milk and Cow Tissues. J. Agr. & Food Chem. 16: 627-631. 1968. 5) Waldron, A. C., Robb, C. J. and Sleesman, J. P. The Determina tion of HCN Residues on Greenhouse Tomatoes. Bui. Envir. Cont. & Toxic. 5: 95-97. 1970. Waldron, A. C. 0414003 PCB-ARCH0745596 "i >i" (m'lOO'i PCB-ARCH0745597 Admin, fi>rm 20 fkiiineit Office . OHIO AGRICULTURAL RESEARCH AND DEVELOPMENT CENTER Grant-in-aid Agreement ARTICLES OF AGREEMENT between the Ohio Agricultural Research and Development Center, Party of the First Part, hereinafter called the Center and ______________________________________ Monsanto Industrial Chemicals Co. 800 N. Lindbergh Boulevard St. Louis, Missouri 63166 Name and Address of Donor Party of the Second Part, hereinafter called the Donor, for a cooperative investigation described as follows: ELIMINATION OF POLYCHLORINATED BIPHENYL RESIDUES FROM SILOS The investigation to be carried out by the Center under the terms and conditions specified herein. Article 1 TIME: This agreement is executed for the following period: October 1, 1972 through September 30, 1973'______________________________________________ ___ with the understanding that it may be extended for additional periods upon the written agreement of both parties. Article 2 CONTROL AND SUPERVISION: The Center will selector designate the personnel and place to un dertake the research and will plan and conduct the work in accordance with the recommendations of person nel best qualified to evaluate the problem and suggest procedures for its solution. 04 1400b PCB-ARCH0745598 o ) Article 3 FACILITIES: The Center will provide competent supervision and will use its facilities and existing equipment so far as possible. It is agreed, however, that apparatus, equipment, and other facilities not available in the laboratories of the Center, and all materials and supplies required in the investigation may be purchased from and charged against the funds for this investigation provided by the Donor and shall be come the property of the Center. Article 4 RECORDS AND PUBLICATIONS: The data obtained from the study will be interpreted by the project personnel and the results released at their discretion in publications of the Center or in scientific jour nals. Such publication shall constitute final release to the Donor and dedication to public use. If for some reason publication is not mode or is unduly delayed a final report of the work will be pro vided to the Donor. The Donor shall not use the name of the Center or Center personnel or any unpublished information or data relating to the project, in any publicity or advertising unless copy is submitted and writ ten approval of the Director of the Center is obtained. Article 5 REPORTS OF DONOR: Authorized representatives of the Donor shall at all times have access to the data secured and results computed from the investigation, subject, however, to the restrictions named in Article 4. The Center may submit appropriate reports of progress to the Donor. Article 6 POSSIBLE PATENTS: It is agreed that all results of experimental work, including patentable dis coveries carried on under the^direction of the scientific staff of the Center, belong to the Center. Article 7 GRANT OF FUNDS: The Donor agrees to pay to the Center the sum of investigation, such payments to be according To the following schedule: ----- for this Date September 1, 1972 $12,500*00 Date Date Date PCB-ARCH0745599 Jt is understood and agreed that the above sum is conditional upon the conduct of the investigation by the Center with due diligence so as to secure the greatest possible progress consistent with the nature of the work. An administrative charge of twenty percent will be deducted from the total grant. The balance of the fund will be administered completely apart from and independent of other funds of the Center. Rec ommendations for expenditure of the donated funds may be made by the project supervisor through the Chairman of the Department involved and approved by the Director. Any unexpended balance is to be re tained by the Center for further research and study. Approved by: Donor By Title 'yu. ____________ Director ' JDhio^Agricultural Researcfvand Development Center Asscxflate Director Oi)J6 Agricultural Research and Development Center Date fcjy- y i~ Date Date 0u*7 PCB-ARCH0745600