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', ."r’,“-.2 l. ‘ t ‘- ' i u .1 ._ .H. . ‘ ‘ ' .IA . .451: Ii « .. t . . ‘1 a": “'3', «IL‘ in " "LR-ob 30")»; Mmmuwn aha- f This is to certify that the thesis entitled AN EVALUATION OF THE CHEMODYNAMICS OF FIREMASTER 680 IN THE AQUATIC ENVIRONMENT presented by Ralph Lawrence Bednarz has been accepted towards fulfillment of the requirements for M. S . degree in Limnology 36mg 2% Mia: l Major professor 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution Place in book drop—to I Msuj RETURNING MATERIALS: . ‘ 35/' is ‘ MSU LIBRARIES m ‘ rev“ ' t, fihgclt .',J- from u r 7 '1 RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. AN EVALUATION OF THE CHEMODYNAMICS OF FIREMASTER 680 IN THE AQUATIC ENVIRONMENT by Ralph Lawrence Bednarz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTERS OF SCIENCE Department of Fisheries and Wildlife 1982 ABSTRACT AN EVALUATION OF THE CHEMODYNAMICS 0F FIREMASTER 680 IN THE AQUATIC ENVIRONMENT By Ralph Lawrence Bednarz The primary purpose of this investigation was to develop a base set of data to predict the fate of'Firemaster 680 in the aquatic environment. This compound is an important brominated aromatic flame retardant for use in thermoplastic applications. .A recent contamination incident with Firemaster 680 has been identified in the Raisin River watershed near Adrain, Michigan. Commercial grade Firemaster 680 was identified as 1,2-bis(2,h,6- tribromophenoxy)ethane and is 991 pure. The aqueous solubility was measured as 0.0“:0.01 ug/L. The n-octanol/water partition coefficient was determined to be 1.38 x 107. This value was used to estimate an organitzcarbon normalized sediment distribution coefficient of 5.25 x 106 and a fish bioconcentration factor of 1.58 x 105. Bacteria capable of degrading Firemaster 680 were not found during acclimation, analog enrichment, or cometabolism studies. This study revealed that the degree of adsorption to sediments and suspended matter will most likely dictate the ultimate fate of‘Firemaster 680 in the aquatic environment. To Our Son, Kenton, And Our Forthcoming Child ACKNOWLEDGMENTS I ‘wish to extend my‘ appreciation to Dr. F.M. D'Itri for the opportunity to pursue advanced study and for serving as my major advisor during my graduate studies. I am also indebted to Dr. M.J. Zabik and Dr. D.L. King for their contributions as members of’my guidance committee and for their unique ability as educators. Special thanks to Dr. P.A. D'Itri for her guidance in preparation of this manuscript. Thanks to C.S. Annett for his technical assistance. Sincere thanks to my typist and to J.P. Kilroy for his reprographic work. Very special thanks to my wife, Patricia, for her patience, encouragement, and assistance during the many years of this study. Sincere appreciation to Kenton for tolerating his father's absence from home. I will always be grateful to my parents, Larry and LaVerne Bednarz, for their encouragement and support to pursue my goals. Appreciation is also extended to my relatives and friends for their interest and concern. ii TABLE OF CONTENTS Page LIST m TABLm O O O O O O O O O O O O 0 O O O O O O 0 O O O O 0 iv LIST OF FIGURES O O O O O O O O O O O O O O O O O O O O O O O O 0 v INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O O O O -—I Overview . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Firemaster 680 . . . . . . . . . . . . . . . . . . . . . . . u Environmental Fate Assessment . . . . . . . . . . . . . . . 8 Statement of Objectives . . . . . . . . . . . . . . . . . . 10 MATERIALS AND MHODS O O O O O O O O O O I O O O O O O O O O O O 1 2 Firemaster 680 Chemical Structure and Purity . . . . . . . . 12 Analytical Method . . . . . . . . . . . . . . . . . . . . . 12 Water Solubility Determination . . . . . . . . . . . . . . . 13 n-Octanol/Water Partition Coefficient Determination . . . . 15 Microbial Degradation of Firemaster 680 . . . . . . . . . . 16 RESULTS 0 I O O I O O O O I O O O O O O O O O O O O O O O O O O O 22 Water Solubility Determination . . . . . . . . . . . . . . . 22 n-Octanol/Water Partition Coefficient Determination . . . . 22 Microbial Degradation of Firemaster 680 . . . . . . . . . . 29 DISCU$ION . O O O O O O O O O O O O O O O O O O O O O O O O O O “2 water SOIUbility .- O O O O O O I O O O O O O O O O O O O O O ”2 n-Octanol/Water Partition Coefficient . . . . . . . . . . . N3 Microbial Degradation . . . . . . . . . . . . . . . . . . . H6 SWARY AND CONCLUSIONS 0 O C O O O O O O O O O O O O O O I O O O 50 RE COWENDATIONS O O O 0 C O O O I O O O O O O O O O O O O O O O O 52 APPENDICB O O O O O O O O O O O O O O O O O O O O O O O O O O O 53 Appendix A. Firemaster 680 Toxicity . . . . . . . . . . . . 53 Appendix B. Chemical Characterization of Firemaster 680 . . 59 Appendix C. Analytical Method Development . . . . . . . . . 76 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O O O O O O O 90 iii 10. 11. 12. 13. 1“. 15. 16. 17. LIST OF TABLES Environmental Samples for Bacteria Isolation Firemaster 680 (FM680) Water Solubility Determination HPLC Retention Times and Partition Coefficients for Calibration Standards and Firemaster 680 . . Comparison of Estimated Low Kow Values Using Equation 2 and Reported Log Kow Values . . . . . . . . . . . . Enrichment and Isolation - Spread-Plate Step Enrichment and Isolation - Streak-Plate Step Enrichment and Isolation - Shaker Flask Step Firemaster 680 (FM680) Cometabolism . . . . . Acute Toxicity of Firemaster 680 . . . . . . Comparison of Theoretical and Experimentally Determined Elemental Content of Firemaster 680 (FM680) . . . . . . XAD Resin Extraction Efficiency of Firemaster (FM680) and 2,”,6-tribromophenol (TBP) . . . XAD-2 Extraction Efficiency-FM68O . . . . . . XAD-2 Extraction Efficiency-TBP . . . . . . . XAD-u Extraction Efficiency-FM68O . . . . . . XAD-u Extraction Efficiency-TB? . . . . . . . XAD-2/u Extraction Efficiency-FM68O . . . . . XAD-Z/h Extraction Efficiency-TBP . . . . . . iv 680 Page 17 23 25 27 3O 32 36 38 5a 66 81 82 83 8H 85 86 87 FIGURE 10. 11. 12. 13. 1H. 15. LIST OF FIGURES Chemical Synthesis of 1,2-Bis(2,u,6-tribromophenoxy)ethane Sampling Stations fer Firemaster 680 Degrading Bacteria . Firemaster 680 (FM680) Water Solubility Determination . . Correlation Plot for Log Kow and Log tc of the Test S tandards O O O O O O O O O O O O 0 O O O O O O O O O O 0 Progressive Loss of FM 680 from FM680/g1ucose Growth Medium 0 O C O O O O O O O O O O O O O O O C O C O O O 0 Progressive Loss of FM 680 from FM680 Growth Medium . . . Progressive Loss of FM680 from FM680/DPE Growth Medium . CHN Analyzer Calibration Data Sheet . . . . . . . . . . . CHN Analyzer Data Sheet for Firemaster 680 . . . . . . . Gas Chromatogram - (A) Firemaster 680 (10,000 pg) (B) 1-(2,u,6-tribromophenoxy)-2-bromoethane (a) and 2,“,6- tribromophenol (b) (10 pg) . . . . . . . . . . . . . . . Ultraviolet Absorption Spectrum of Firemaster 680 . . . . Infrared Spectrum of Firemaster 680 . . . . . . . . . . . NMR Spectrum of Firemaster 680 . . . . . . . . . . . . . Graphic Representation of Mass Spectrum of Firemaster 680 XAD-2 Analytical Scheme Standard Calibration Curve for Firemaster 680 (FM680) . . . . . . . . . . . . . . . . . Page 6 18 2M 26 39 H0 in 63 6a 65 67 68 70 71 89 INTRODUCTION ovaavraw Over the past few decades the chemical industry has experienced an unparalleled expansion with the development of many new organic chemicals that have very diverse applications. Approximately four million chemicals are presently known, with about fifty thousand chemicals in everyday use, not including pesticides, pharmaceuticals, and food additives (Maugh, 1978). Under the 0.8. Environmental Protection Agency's Toxic Substance Control Act, the initial Chemical Substances Inventory and Cumulative Supplement lists a total of 55,103 chemicals which are manufactured, imported, or processed for commercial purposes in the United States (U.S. EPA, 1980a). This tremendous increase in chemical development and production has resulted in a substantial burden for the environment. Iliff (1972) has suggested that, worldwide, up to #0 billion pounds of manufactured organic chemicals enter the environment annually. The introduction of synthetic organic chemicals to the environment during the last thirty years has already had a significant impact on the biosphere. A number of catastrophes have resulted from environmental contamination by such chemical agents as mercury, cadmium, polychlorinated biphenyls (PCB), vinyl chloride, chlorinated dioxins, and numerous chlorinated pesticides (e.g., DDT, Kepone, Mirex). A more recent chemical contamination incident was the polybrominated biphenyl (PBB) tragedy which occurred in Michigan (Kay, 1977). This disaster was one of the most extreme l 2 cases of chemical contamination of domestic animals and the human population ever recorded. Polybrominated biphenyls are members of a diverse class of brominated aromatic flame retardants which are currently produced in large quantities (Anonymous, 1977; Anonymous, 1980; Sanders, 1978). Fire safety legisla- tion during the last decade has greatly stimulated. the synthesis, production, and application.of'fire retardant chemicals (Anderson, 1976). The market for brominated aromatic flame retardants in the United States rose from 20 million pounds in 1971 to 115 million pounds in 1973 (Anonymous, 1974). The amounts used in plastics alone in 1976 and 1977 were 22 and 18 million pounds, respectively (Anonymous, 1977). In 1980 the annual consumption of brominated aromatic flame retardants was 32 million pounds, and by 1985 it is expected to reach 55 million pounds (Anonymous, 1980). The potential for environmental contamination has increased along with this rapid increase in production and application. The PBB incident in Michigan is only one of many documented instances of environmental contamination resulting from brominated aromatic flame retardants. The first reported case of environmental pollution from these compounds was in 1975 when residues of pentabromotoluene were feund in sewage sludge from a wastewater treatment plant in Sweden (Mattsson 35 _l., 1975). The source was a factory which used this chemical for flame retardant applications. Since this discovery, several reports have been published on environmental and human contamination resulting from the production and use of brominated aromatic flame retardants (Anderssonland Blomkvist, 1981; DeCarlo, 1979; DiCarlo gt al., 1978; Forba, 1980; Forba gt al., 1980; Hesse and Powers, 1978; Jamieson, 1977; Pellizzari §t_gl., 1978; Powers, 1976). 3 Although brominated aromatic flame retardant production has increased tremendously over the past ten years and numerous incidents of environmental contamination from these compounds have been identified, very little is known about their toxicity and environmental chemistry. Research so far has focused on PBBs. Recent work (Falk gt a;., 1980) has revealed that these chemicals may be more toxic than their chemical relatives, PCBs. Also, limited studies on the environmental transport and transformation characteristics of P888 indicate that they may be as persistent as PCBs (Filonow 92 al., 1976; Jacobs gt al., 1976; Jacobs gt __l., 1978; Matsuo, 1980; Ruzo and Zabik, 1975; Ruzo .e_t__a_l., 1976; Sugiura gtpal., 1978; Zitko, 1977; Zitko and Hutzinger, 1976). Only a few studies have been published on the toxicity and environmental chemistry of other brominated aromatic flame retardants (Hutzinger gt_§l., 1976; Kociba gt 3;” 1975; Liepins and Pearce, 1976; Norris e_t gin 1973a; Norris gt $1,, 1973b; Norris gt al., 1975; Orlando and Thomas, 1975; Zitko and Carson, 1977). These studies indicate a general lack of information on the persistence and fate of brominated aromatic flame retardants in the environment, especially aquatic ecosystems. Our ability to respond objectively to such incidents is limited by the lack of information on this diverse group of'chemical compounds. For this research project environmental fate data were developed on Firemaster 680. This compound was selected for the study because it has been desig- nated to replace Firemaster BP-6 (PBB). Therefore, production volumes and applications are projected to be large. Additionally, a contamination incident involving Firemaster 680 has been identified in a segment of the Raisin River watershed in Michigan, thus necessitating an evaluation of the chemical dynamics of this compound in the aquatic environment. FIREMASTER 680 Firemaster 680 is a non-reactive brominated aromatic flame retardant which is manufactured and marketed by the Velsicol Chemical Corporation, Chicago, Illinois. This flame retardant additive was designed fcr high performance thermoplastic resins such as acrylonitrile-butadiene-styrene terpolymer (ABS), high impact polyester and other polymer systems common in electrical and high temperature applications (Barth, 1979; Nametz and Moore, 1978; Bern, 1978). Firemaster 680 is also widely used in the manufacture of television cabinets and other electrical equipment because it retards flamability without impairing moldability, impact strength, and resistance to ultraviolet light degradation (Velsicol Chemical Company, 1977). This chemical also can be formulated into adhesives, coatings, lattices, and specialty composites (Leitheiser 22 al., 1978; Smith and Shukla, 1978). Chemically, Firemaster 680 is identified either as 1,2-bis(2,u,6- tribromophenoxy)ethane or by the Chemical Abstract Service (CAS) name of 1,1'-[1,2-ethanediy1bis(oxy)]bis[2,”,6-tribromobenzene]. The CAS number is 37853-59-1 or 5976fl-36-2. Structurally, the compound consists of two symetrically brominated phenyl rings attached through a glycol group. The six bromine atoms give this compound a bromine content of 70% by weight and a relatively high molecular weight of 687.67 gm/mole. The known physical data include a melting point range between 223-225°C, a decomposition temperature of approximately 325°C, and a density of 2.58 gm/ml. The compound has moderate solubility in p-xylene, perchloro- ethylene, and boiling dichlorobenzene; a vapor pressure of 0.0019 mm Hg at 170°C; excellent thermal stability; and resistance to ultraviolet light degradation (Michigan Chemical Corporation, 1976). S The synthesis of 1,2-bis(2,u,6-tribromophenoxy)ethane is a two step process (Figure 1). In reaction I the sodium salt of 2,11,6-tribromophenol is formed from 2,u,6-tribromophenol and sodium bicarbonate in a propylene glycol mother liquor which contains 0.51 phenol. The final product is then made (reaction II) by reacting the sodium salt of 2,”,6- tribromophenol with 1,2-dibromoethane in a propylene glycol mother liquor (Heisted, 1977). It is then washed with methanol and water to remove residual sodium bromide and tribromophenol based impurities. The impurities normally associated with this product are 2,”,6-tribromophenol and 1-(2,u,6-tri- bromophenoxy)-2-bromoethane. Additionally, 1-(2,u,6-tribromophenoxy)4 ethanol, 1-(2,h,6-tribromophenoxy)ethylene and numerous 2,”,6-tribromo- phenoxypropylene glycol derivatives may be found as trace impurities. The final product specifications call fer a minimum bromine content of 68.5% which corresponds to a product purity of 98.2% (Heisted, 1977). Velsicol Chemical Corporation has conducted toxicity studies on Firemaster 680, and the results have been submitted to the U.S. Environmental Protection Agency as a substantial risk notice in compliance with Section 8(e) of the Toxic Substances Control Act (U.S. EPA, 1980b). These studies are sunmarized in Appendix A. The results generally indicate that Firemaster 680 is relatively non-toxic to mamals during acute, subacute, and chronic exposures. Some accumulation of Firemaster 680 in fat and other tissues of laboratory test animals were noted, but no signif- icant changes were noted in hematological, biochemical, and urinalysis parameters. The results of an Ames mutagenicity bioassay were negative. The bioconcentration potential of Firemaster 680 in carp (Cyprinus gaggig) was also reported (U.S. EPA, 1980b). The actual test conditions of this study were not specified, but the carp were exposed to nominal ocmnaoAzxoconaoaoonacpuo.z.mvmwmim.P mo mamonucmm Hmoaacso .— ocsmfim 2. 5:33: s N N . as: . .u 13:2. 3 i a 3 3.. s SN a . a... 2 5:32: 5 .5 ~99 . .3. . ~ 7 as"... . . ~ 5 3 a .5 7 concentrations at 0.3 mg/L or 0.03 mg/L Firemaster 680 for up to 8 weeks. The highest concentration factors were 56.6 for the high exposure group and 113.6 for the low exposure group. These values indicate that Firemaster 680 has little tendency to bioconcentrate. A biodegradation study with 1"’C-labeled Firemaster 680 was also submitted to the Environmental Protection Agency (U.S. EPA, 1980b). Tests of aerobic biodegradability were conducted by exposing 1"‘C-labeled Firemaster 680 to acclimated sewage and garden soil microorganisms in a shake flask system. Microbial utilization of Firemaster 680 was followed by measuring respired 1”002. During an 18 day acclimation period, microorganism derived from sewage and garden soil were exposed to Firemaster 680. The acclimated bacteria were then used as seed organisms and added to microbial media which contained 0.1, 1.0, or 10.0 mg/L of 1tic-labeled Firemaster 680. The results appeared to indicate that 1"'COZ was liberated from universally labeled Firemaster 680 in all test samples throughout the study period. However, degradation was slow and the tests were terminated after 30 weeks. Following an initially accelerated 1”002 liberation, the 1“002 absorption decreased to about 0.001 to 0.008% of the total 1uC-activity in the 1.0 and 10.0 mg/L Firemaster 680 test flasks. These results indicate that Firemaster 680 is relatively nonbiodegradable in the presence of sewage and garden soil microorganisms under the conditions of these tests. The toxicity and environmental fate data on Firemaster 680 reported by Velsicol Chemical Corporation appear to indicate that this product is rather non-toxic and environmentally stable. However, only selective studies were reported and most of these data were generated in one laboratory, that of Velsicol Chemical Corporation, the manufacturer of 8 Firemaster 680. Before the environmental hazard potential of Firemaster 680 can be ascertained, these data must be verified, and additional toxicity and environmental fate studies will have to be conducted. Transport and transformation studies are necessary to evaluate the envi- ronmental fate characteristics and exposure potential of Firemaster 680. ENVIRONMENTAL FATE ASSESSMENT The fate of’a brominated aromatic flame retardant such as Firemaster 680 in the environment is controlled by the transport and transformation processes acting on it. In the aquatic environment, the major transport processes include sorption, volatilization, bio-uptake, and physical transport in the water phase. Transformation processes include hydroly- sis, oxidation, photolysis, reduction, and biodegradation. While the ultimate persistence of a chemical is controlled by the transformation processes, its residence time and actual exposure concentration in a segment of the aquatic environment are controlled by both transport and transformation processes. Environmental.fate and exposure assessment are extremely complex and require precise information in all of the categories identified above. At this time, the state-of-the-art has not progressed to the point where the environmental concentrations of a chemical can be predicted with a high level of accuracy by using only laboratory derived data. The actual environmental concentrations can only be ascertained by extensive monitoring studies. However, environmental fate assessment methods have advanced to the extent that they can be used to predict the interactions of a chemical contaminant with the aquatic environment and to begin to estimate, in a qualitative sense, the concentration of the chemical in specific compartments of the aquatic environment. 9 A variety of methods have been proposed to evaluate the fate of chemicals in the environment (Kimerle at al., 1978; Stern and walker, 1978). These range from predictions based on chemical and physical property relationships to laboratory studies which focus on one or more of the fate processes, microcosm studies which attempt to simulate the natural environment, and field studies to validate the predictions. Recently, the Environmental Protection Agency has proposed standard protocols for environmental fate testing which are mandated under Sections u and 5 of the Toxic Substances Control Act (U.S. EPA, 1980c; U.S. EPA, 1981) and Section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (U.S. EPA, 1980d). Additionally, the Organization for Economic Cooperation and Development (OECD) has published a complete set of environmental fate test guidelines for use by its member countries (OECD, 1981). Chemical environmental fate assessments usually follow a step- sequence (tier) testing scheme. The lowest level generally contains a base set of tests which provide the minimum data for estimating the environmental fate of a chemical while the highest level consists of actual field studies. The minimum data base studies have received much attention over the last few years because they require relatively short periods of time and the least capital expenditure. The base set of chemical fate tests are primarily for the purpose of providing data to determine the dominant transport and transformation processes that the chemical may undergo in natural environments. Results from these studies are used to make deci- sions on whether to move up to the next level of the testing hierarchy. The U.S. Environmental Protection Agency (1981) proposed that the base set of data fbr chemical environmental fate assessment include water solubility, vapor pressure, n-octanol/water partition coefficient, 10 boiling and melting points, density, dissociation constant, particle size distribution, UV and visible spectra, sediment and soil adsorption/ desorption, hydrolysis, biodegradation, and bioaccumulation. Thus, an essential first step in a chemical environmental fate analy- sis is to develop an understanding of the chemical, physical, and toxico- logical properties of the compound being studied. For common, high volume chemicals, this information is usually well known and readily available. For specialty chemicals, such as brominated aromatic flame retardants, basic physical and chemical data may be difficult to obtain and toxicologi- cal information might be non-existent. Some physical and chemical informa- tion is usually available from the manufacturer through product bulletins and advertisements appearing in trade journals. In most cases, however, the investigator has to develop much of the information in the laboratory. STATEMENT OF OBJECTIVES This project was undertaken to assess the impact of Firemaster 680 contamination on the south branch of the Raisin River in Michigan. The overall objective of this investigation was to obtain information to predict the fate of Firemaster 680 in the aquatic environment. The specific objectives were: 1. To characterize Firemaster 680 chemically and to determine the chemical purity of the technical grade product. 2. To develop a method for the analysis of Firemaster 680 in water. 3. To determine the water solubility of Firemaster 680. u. To determine the n-octanol/water partition coefficient of Firemaster 680 and to use this parameter to estimate its 11 potential for sediment partitioning and biological up-take by aquatic organisms. 5. To determine microbial degradation of Firemaster 680. MATERIALS AND METHODS FIREMASTER 680 CHEMICAL STRUCTURE AND PURITY Commercial grade Firemaster 680 (Lot No. 611111-17) obtained from Velsicol Chemical Corporation (Chicago, Illinois) was characterized for chemical structure and purity. Elemental analyses with a Perkin-Elmer Model 2H0 Elemental Analyzer detected 24.35% carbon, 1.15% hydrogen, and 0% nitrogen. With a total bromide determination method (Liggett, 195"; Michigan Chemical Company, 1975), the bromine content was determined to be 68.72%. These data indicate a product purity of approximately 99%. The melting point was sharp, within o.5°c of 225°C, which also indicates a relatively pure product. Gas chromatographic analyses for organic impurities revealed less than 0.1% of 2,4,6-tribromophenol and 1-(2,u,6- tribromophenoxy)-2-bromoethane. The ultraviolet, infrared, nuclear mag- netic resonance and mass spectra clearly identified this product as 1,2- bis(2,A,6-tribromophenoxy)ethane. For a more complete discussion of these methods and results, see Appendix B. ANALYTICAL METHOD An XAD resin analytical scheme was devised to extract low levels of Firemaster 680 and potential metabolities from aqueous media. This method is a modification of a procedure developed by Junk gt al. (1974). The extraction efficiencies for Firemaster 680 and 2,“,6-tribromophenol were 12 13 determined with XAD-2 and XAD-u resins separately and in a mdxture of XAD-Z/XAD-H. Standard solutions of Firemaster 680 and 2,4,6-tribromo- phenol were prepared in diethyl ether and spiked water samples were pre- pared in a concentration range of 0.1 to 2.0 ug/L with deionized water. The spiked water samples were extracted through the XAD resin columns. The sorbed fraction of’Firemaster 680 and 2,4,6-tribromophenol.was eluted with diethyl ether, and the eluate was dried, concentrated and analyzed by gas chromatography. The extraction efficiencies for each resin with Firemaster 680 and 2,“,6-tribromophenol were determined by comparing the gas chromatogram peaks of the XAD resin extract fer each spiked water sample with the gas chromatogram peaks for the theoretically equivalent amounts of the standard ether solutions of Firemaster 680 and 2,4,6-tri- bromophenol. The XAD-Z resin system was the most efficient with recoveries of approximately 50% for Firemaster 680 and 100% for 2,14,6-tribromophenol. A standard calibration curve was developed with the spiked water samples for the concentration range of 0.1 to 2.0 ug/L in order to correct for the lower extraction efficiency for Firemaster 680. See Appendix C for a complete description of these methods and results. WATER SOLUBILITY DETERMINATION The time-dependent equilibrium procedure described by Hague and Schmedding (1975) was used to determine the aqueous solubility of Fire- master 680. Approximately 0.5 mg Firemaster 680 were dissolved in 25 m1 of diethyl ether and swirled onto the wall of a 20 liter glass carboy. The residual ether was evaporated by using a nitrogen stream, resulting in a thin film of Firemaster 680. Nineteen liters of deionized, carbon and membrane filtered water were carefully added to the carboy so as not 14 Firemaster 680 film on its side. A teflon coated magnetic stir bar was added and a magnetic stirrer was used to stir the contents of the carboy. The solution was isolated from the magnetic stirrer by a one inch sheet of styrofoam to prevent temperature gradients caused by heat from the stirrer motor. Finally, the carboy was fitted with a glass siphon tube which had a fine frit that extended below the surface of the liquid. This was used to collect samples for analysis. The solution was sampled through the glass frit-siphon tube at weekly intervals for the first month, monthly for the next two months, and once every three months for a complete year. The solution was stirred at approximately 250 rpm for the first month, and then the stirring was stopped. The carboy was held at room temperature (22 i 2°C) for the entire study. Triplicate 500 ml samples were collected at each sampling time and then were concentrated according to the XAD-2 analytical scheme previously described. The concentrates were analyzed by gas chroma- tography. A Beckmn, model GC-65, gas chromatograph equipped with a non- radioactive electron-capture detector and a Beckman 10-inch linear recorder (Beckman Instruments, Inc., Fullerton, California) were used to obtain the gas chromatography data from which the aqueous solubility was calculated. The test samples and standards were chromatographed on a 0.2 (i.d.) X 183 cm glass column packed with 60/80 mesh Gas Chrom Q coated with 3% silicon OV-1 liquid phase (Applied Science Laboratories, Inc,, State College, Pennsylvania) with a helium carrier gas flow rate of 55 ml/min. The gas chromatograph conditions were as follows: inlet temperature, 290°C; column temperature, 280°C; detector line temperature, 310°C; detector temperature, 360°C. The polarizing voltage, carbon 15 dioxide flow, and bias voltage were set for optimum detector response. With this column and appropriate operating conditions, the retention time for Firemaster 680 was 3.9 minutes, and the minimum detectable quantity was 1.0 pg at 2.5 times the background noise level. N-OCTANOL/WATER PARTITION COEFFICIENT DETERMINATON The neoctanol/water partition coefficient for Firemaster 680 was estimated by using a reverse-phase HPLC method similar to the procedure described by McDuffie (1981). Six chemical standards for which Kow have been reported were used to calibrate the elution time in units of Kow‘ The calibration ndxture included benzene (Burdick and Jackson Labora- tories, Inc., Muskegon, Michigan), 1,fl-dichlorobenzene (Aldrich Chemical Company, Milwaukee, Wisconsin), biphenyl (J. T. Baker Chemical Company, Phillipsburg, New Jersey), phenanthrene (J. T. Chemical Company, Phillips- burg, New Jersey), p,p'-DDE (Aldrich Chemical Companyy Milwaukee, Wiscon- sin), and 2,4,6,2',4',6'-hexachlorobiphenyl (Analabs, Inc., North Haven, Connecticut). Methanol solutions containing 100 mg/L of each standard, except 2,u,6,2',4',6'-hexachlorobiphenyl, were prepared with chromatog- raphy grade methanol. A 75:25 (V/V) methanol-hexane mixture was used to prepare solutions of 2,11,6,2' ,11' ,6'-hexachlorobipheny1 and Firemaster 680 at 100 mg/L concentration. The eluting solvent was 75:25 (V/V) methanol- water which was prepared from chromatography grade methanol and distilled- carbon filtered water, and it was degassed by sonication before use. .The liquid chromatograph consisted of an Altex Model 110 solvent meterimg pump (Altex, Inc., Berkeley, California) and a Hitachi Model 100-110 variable wavelength UV detector (Hitachi, Ltd. , Tokyo, Japan) fitted with an Altex Model 155-00 spectrophotometer flow cell module. The flow cell module had a 1.0 cm path length and a 20 ul cell volume. A l6 0.u6 (i.d.) X 25 cm Zorbax-ODS C18-microparticulate reverse-phase column (DuPont Company, Wilmington, Deleware) was used. The column was held at a constant operating temperature in.a DuPont Model 860 column compartment (DuPont Company, Wilmington, Deleware). Retention times were recorded on a Linear Model 255 recorder (Linear Instruments Corp., Irvine, California). Twenty ul of each standard solution was injected onto the column which was held at a constant operating temperature of 35°C. The solutes were eluted isocratically with the methanol-water solvent mixture which was pumped through the column at 1.0 m1/min at a pressure of approximately 2000 psi. The detector was set at a wavelength of’25fl nm for the calibra— tion standards and 290 nm for Firemaster 680. Absorbance peaks were recorded at a chart speed of 30 cm/hr. The retention times of the calibra- tion standards and Firemaster 680 were determined directly from the recorder chart. MICROBIAL DEGRADATION 0F FIREMASTER 680 A modified version of a technique for isolating PCB degrading bac- teria.was employed to isolate natural populations of’microorganisms which can utilize Firemaster 680 as a carbon and energy source for growth (Kaiser and Wong, 197A). The bacterial cultures were then used in a cometabolism study to check for primary degradation of Firemaster 680 and to identify the degradation products. Enrichment and Isolation Microorganisms were obtained from sediment and water samples which were collected in the vicinity of Adrian, Michigan. These locations were previously exposed to Firemaster 680 and other chemicals in process wastes 17 Table 1. Environmental Samples for Bacteria Isolation Sample Sample Firesmater 680 Code Type Location of Collection Contenta W2 Water WWTPb-influent ND° W3 Water WWTP-return sludge ND wu Water WWTP-effluent ND 82 Sludge WWTP-digested sludge 0.11 mg/kg W5 Water WWTP-sludge disposal bed ND 85 Sludge WWTP-sludge disposal bed 1.2 mg/kg W6 Water WWTP-lagoon NAd S6 Sludge WWTP-lagoon 5.9 mg/kg 37 Sludge WWTP-sludge disposal bed 5.3 mg/kg 37B Sludge WWTP-sludge disposal bed NA 88 Sediment East Side Drain-Anderson 55.0 mg/kg Development Company S9 Sediment East Side Drain-end of storm 1.3 mg/kg sewer S10 Sediment East Side Drain-Academy Road 6.3 mg/kg S11 Sediment East Side Drain-Oakwood Street 1.1 mg/kg S12 Sediment South Branch Raisin River-East 1.3 mg/kg Side Drain S13 Sediment South Branch Raisin River-Howell o.u9 mg/kg Highway aData obtained from Michigan Department of Natural Resources surveys (Boerson, 1979a; Boerson, 1979b; Jackson, 1979) bAdrian, Michigan Wastewater Treatment Plant cNone Detected dNot Analyzed l8 mason—0mm $5.3.»ch owe Loumgocwm no.“ mcofiumum Mazda—mm m.m.m.:.n.m wcoaumum msaansmm nu m I _ 1:. o o ,_m .Rfl? u i t... .m onsmaa 19 from the Anderson Development Company. A description of the location and types of samples and their content of’Firemaster 680 is included in Table 1 and Figure 2. Subsamples consisting of approximately 2 g of the sediment or sludge or 100 m1 of water, were placed in 300 m1 Erlenmeyer flasks and fortified with 200 mg Firemaster 680 and 100 ml of‘mineral salt solution. The min- eral salt solution was prepared according to Smith gt 3;. (1977) and it contained: KZHPOu, 1.“ g; KHZPOu, 0.6 8; (NHN)ZS°u: 0.5 3; NaCl, 0.1 g; MgSOu-7H20, 0.1 g; CaClz-ZHZO, 0.02 g; FeSOu, 0.005 g; in 1 liter H20 and 1 ml of trace elements solutions. The trace elements solution contained 0.1 g H3803 and 0.05 g each of CuSOu-st-IZO, MnSOu-HZO, ZnSOn-7H20, NaZMoOu, and CoC12'6H20 per liter of water. The samples were incubated for one week at room temperature on a rotary shaker. This procedure presumably selected for those bacteria that could use Firemaster 680 as the sole carbon and energy source fer growth. After incubation, 0.1 ml aliquots of the enriched samples were plated onto sterile growth medium agar plates with a standard spread plate tech- nique (Rodina, 1972). The growth medium agar consisted of the mineral salt solution, agar, and a carbon source. Three carbon sources were used separately or in combination to prepare four growth medium agar substrates with carbon source contents of 0.1%. These included: Firemaster 680; Firemaster 680 and plate count agar which contained glucose, tryptophane, and yeast extract (Difco Laboratories, Detroit, Michigan); diphenoxye- thane (Aldrich Chemical Company, Milwaukee, Wisconsin); and a 1:1 mixture of Firemaster 680 and diphenoxyethane. Diphenoxyethane is the nonbro- minated analog of Firemaster 680. A growth medium agar without any carbon source was also prepared. 20 The inoculated growth medium agar plates were incubated at room tem- perature for one week and were observed fer bacterial growth. On plates showing good growth, colonies of bacteria were removed and purified by repeated streaking onto sterile growth medium agar plates. This was an attempt to isolate specific bacteria capable of utilizing the individual carbon source. The inoculated plates were incubated at room temperature for 16 days. Four observations for bacterial growth were made during this period. The capability of the purified cultures to use Firemaster 680 directly or indirectly as a cometabolite for growth was further tested by growing them in a mineral salt solution that was fortified with the respective carbon source. Colonies of bacteria were taken from the growth medium agar plates and placed in 300 ml Erlenmeyer flasks which contained 200 ml of the mineral salt solution.with.0.01% Firemaster 680,«diphenoxye- thane, Firemaster 680/diphenoxyethane, or Firemaster 680/glucose. The samples were incubated for thirty days at room temperature on a rotary shaker and each flask was observed daily for bacterial growth. Cometabolism Study A specially designed chamber was used for this study. This system consisted of a 9 liter glass carboy to which was fitted a glass frit and a condenser. The glass frit supplied a constant air flow and facilitated sampling the growth medium in the carboy. The condenser was added to eliminate evaporation of the sample. The growth medium was prepared by adding Firemaster 680 to the basal . salts medium to obtain a final concentration of 5 ug/L. The basal salts medium consisted of a 1:1 dilution of the mineral salts solution with deionized water. Five liters of Firemaster 680 growth medium were added 21 to each of the biodegradation chambers. Besides the Firemaster 680, two of the growth chambers received an additional carbon source. Diphenoxyethane was added to one growth chamber to obtain a final concentration of 5 ug/L. The third growth chamber received glucose at 50 mg/L. Each growth chamber was inoculated with the 89a strain of bacteria which had been obtained in the isolation and enrichment phase. This culture was selected because it appeared to grow better than any other that was isolated on FM680+ agar. The bacteria, which were growing on Firemaster 680 plate count agar slants, were suspended in 25 ml of the basal salts medium and then were added to each biodegradation chamber. Samples consisting of 500 ml of the growth medium were collected from each growth chamber at the time of inoculation (to) and at 1 (t1), 3 (t2). 6 (t3), 12 (tn), 90 (t5), and 153 (t6) days after inoculation. ,A 0.1 m1 aliquot was taken from each one at the time of sampling and transferred to growth medium agar plates which contained the same carbon source as the sample. The inoculated plates were incubated at room temperature for two weeks and observed periodically for bacterial.growth. Each set of samples was analyzed for Firemaster 680 and 2,“,6- tribromophenol according to the XAD_2 analytical scheme described in the analytical methods section. RESULTS WATER SOLUBILITY DETERMINATION The results obtained in this study are tabulated in Table 2 and presented graphically in Figure 3. During the first month, the concentration of Firemaster 680 increased, probably approaching an equilibrium value. However, after the stirring was stopped, the concentration of Firemaster 680 decreased sharply over the next two months, perhaps because aggregates of Firemaster 680 may have been formed during the stirring that gave higher solubility values. This possibility is strengthened by the fact that the error associated with the analyses during the first five sampling periods was much greater than during the last five sampling periods. This large variance could be due to the random capture of microcrystals or aggregates of Firemaster 680 during sampling as discussed by May gt al. (1978). The equilibrium solubility value of o.ou ug/L was reached at 2A weeks and was consistent through the end of the 60 week study period. These data show that the equilibrium solubility, including 95% confidence limits, for' Firemaster 680 in deionized, carbon and membrane-filtered water was 0.0” i 0.01 ug/L. N-OCTANOL/WATER PARTITION COEFFICIENT DETERMINATION The results obtained in this experiment are tabulated in Table 3. The corrected solute retention times (tc) were determined according to the equation: 22 23 Table 2. Firemaster 680 (FM680) Water Solubility Determination Time FM680 Sample (Weeks) (ug/L) to o 1.31 (.21)a t1 1 1.37 (.211) 1:2 2 1.111 (.36) t3 3 1.50 (.28) tn u 1.56 (.59) t5 8 0.98 (.22) t6 12 0.13 (.02) t7 2A 0.0A (.02) t8 36 0.0u (.01) 1:9 as 0.01 (.01) t10 ‘ 60 0.0u (.03) a( ) = standard deviation, N=3 t0 = tR - to (Equation 1) where tR is the solute retention time after injection and to is the solvent retention time. The logarithm, base 10, of tc for each standard was calculated and correlated with the reported log K0w values for these chemicals. The log to values were used instead of log tR to obtain an improved linear correlation as discussed by McDuffie (1981). Figure 11 graphically illustrates this correlation which is best described by the equation: log Kow = 3.53(log tC) + 2.52 (Equation 2) 832.5588 32338 teen: 832$ o8 Samantha .m 8&8 nwzmwzu wt: om mm om me oe mm on mu ow my cu m o 24 _ _ _ _ a _ _ a _ _ A _ 9 T R o . ._ mu 0 / o 2 m6 2 a who a .— a m g .p z w mu w z o u m; o m mg. n z .._ m cofiumsvm Bosh nonmasoamom A_ema ..H “OH 25 .oH Ames. ..wm mm ecoesoannuv am.o muononLmuv >m.: Amps, ..mm mm ecoesoatauv _..m a 0040 mo.m u 0040 00.: 0040 om.m Pm.— 39.9 P0.0 ma.o =m.o 0N.0 00.0 m.mm 0.5— z.FP N.0 :.m 0.m N.: Amocmcmmoav 30M mod on men lessees m.m 000 Loummaocwm m.m moau.a..s..m.e.=.m m.m manuhd m.m mcocnucmconm m.m HzconQHm N.m ocouconocoanOH01:.F m.m econcmm ACHEVOu vcsoqsoo one seennsonaa was nonmesaem soaennnaaeu soc neseaoaceeoo soaeaenna can messy sonnetsem one: .m manna 26 e..— N..~ noncommum pooh on» no 0» mom can 30x mom com uoam coaumaoccoo Uh 604 m.o m.o v.0 N.o o _ _ _ _ o I a N I m 9. my 0 .. v x o 0 nu 1 mm .4 1 my 1 b .z ocsmam 27 It has a correlation coefficient (r) equal to 0.980. With this equation, the estimated log Kow fbr Firemaster 680 was 7.1a. To determine the accuracy of this method of estimating Kow' a comparison was made between the literature log Kb" values reported for the calibration standards and their estimated log Kow values by using equation 2 (Table 1). The mean absolute error was 0.2“ with a standard deviation of 0.17. The error associated with the estimated log Kow for Firemaster 680 could be larger because it was necessary to extend the linear regression line past the log Kow range of the calibration standards. However, if the regression line is linear above this range, then the log Kow for Firemaster 680 is 7.11,: 0.12 with 95% confidence. It is interesting to compare the log Kow value for Firemaster 680 determined by using the HPLC procedure with the calculated log Kow values Table A. Comparison of Estimated Log Kow Values Using Equation 2 and Reported Log Kow Values Estimated Literature Absolute Compound log Kow log Kow Deviation Benzene 2.52 2.11 0.41 1,4-Dichlorobenzene 3.uu 3.39 0.05 Biphenyl 3.72 ".09 0.37 Phenanthrene H.21 4.57 0.36 P,D'-DDE 5.73 5.69 0.0” 2,u,6,2',u',6'-PCB 6.5“ 6.3” 0.20 Mean Absolute Deviation = 0.24 28 derived from equations which incorporate other physical properties. Chiou _L _l- (1977) correlated experimental values of the aqueous solubility and n-octanol/water partition coefficient for 3'4 chemicals. Their regression equation showed: log Kow = 5.00 - 0.67(log 8) (Equation 3) where S = aqueous solubility, in umole/L. When the aqueous solubulity of Firemaster 680, 5.82 x 10'5, umole/L, determined in the previous experiment, was substituted into equation 3, the log Kow for Firemaster 680 was calculated to be 7.8h. Kenaga and Goring (1980) extended this correlation to include 90 chemicals and developed the regression equation: log Kow = 11.158 - 0.800(log 3) (Equation ’4) where S = aqueous solubility, in umole/L. Using this equation and the experimentally determined water solubility, 5.82 x 10""5 umole/L, for Firemaster 680, the log Kow was calculated as 7.55. Banerjee _£._l- (1980) reported that the correlation between water solubility and Kow developed by Chiou gt 3;. (1977) could be invalid for high-melting solids due to the crystal structure of these chemicals. To correct for this error, these authors developed a relationship between the aqueous solubility and n—octanol/water partition coefficient by using data on 27 chemicals which included a melting point correction term. Their regression equation showed: log Kow = 6.5 - 0.89(log S) - 0.015(mp) (Equation 5) where S = aqueous solubility, in umole/L, and mp = melting point, in 0C. By using this equation and the experimentally determined water solubility, 5.82 x 10‘5 umole/L, and melting point 225°C for Firemaster 680, the log Kow was calculated to be 6.89. 29 Mackay and co-workers (1980a) extended the melting point correction approach by including a correction factor based on the fugacity concept. These authors used water solubility values and n-octanol/water partition coefficients for #5 organic: chemicals and. developed the regression equation: ln Kow = 6.79(1 - TM/T) - lnCs - 1n(aovo) (Equation 6) where C8 = aqueous solubility, in mole/m3; TM = triple point; T = system temperature, in °K; v0 = molar volume fcr octanol which is approximately 115 x 10"6 m3/mole; and ac = octanol phase activity coefficient which is estimated as 6H.20 for chemicals with molecular weights greater than 300. With this equation and specific parameters for Firemaster 680, C8 = 5.82 x 10'8 mole/m3, Tm = “98°K, T = 295°K and a0 = 6u.20, the log Kow was calculated as 7.3fl. In summary, the HPLC derived log Kow value for Firemaster 680 agreed well with the log Kow values calculated by using equation 5 or 6 and appeared to be within experimental error of the true log Kow value. MICROBIAL DEGRADATION 0F FIREMASTER 680 Enrichment and Isolation The results obtained from the initial spread-plate inoculation are summarized in Table 5. The growth medium containing Firemaster 680 and plate count agar (FM680+) supported abundant growth for all of the environmental samples that were tested. These results were anticipated because the FM680+ medium contained glucose as one of the carbon sources. Such abundant growth was not found on the agar plates which contained Firemaster 680 (FM680), diphenoxyethane (DPE) or Firemaster 680 and diphenoxyethane (FM680/DPE) as the carbon source. These plates appeared 30 Table 5. Enrichment and Isolation - Spread-Plate Step Carbon Sourcea Sample FM680 FM680+ DPE FM680/DPE Control W2 +/- +++ +/- + +/- W3 + +++ + + + W“ +/- +++ + - +/- 32 - +++ +/- - - W5 - +++ - - - SS - +++ - — - W6 - +++ - — _ S6 +/- +++ +/- +/- +/- 87 +/- +++ +/- +/- +/- 373 - +++ - +/- - $8 - +++ + - - S9 + +++ + +/- +/- S10 +/- +++ + +/- +/- $11 - +++ + - - $12 +/- O + +/- +/- S13 + +++ + +/- +/- aData Key - = No Growth + = Slight Growth +++ = Abundant Growth +/- Possible Growth 0 = NO Plate 31 to support some growth, but the colonies were very small and scarce. The growth medium agar without an added carbon source (Control) also supported marginal growth of bacteria from samples W2, W3, wu, S6, S7, S9, 810, S12, and $13. This low level of bacterial growth could have been sustained by organic substrates which were transfered from the enriched samples when the agar plates were inoculated or they could have resulted from impurities in the agar. With these results, no conclusions could be drawn as to whether or not bacteria capable of utilizing Firemaster 680 and/or diphenoxyethane for growth had been isolated. These results did reveal, however, that Firemaster 680 is not toxic to the bacteria growing on the FM680+ agar plates. The results of the streak-plate isolation step are summarized in Table 6. Basically, this rather complex set of data revealed essentially the same bacterial growth pattern that was found during the spread-plate isolation step. The FM680+ agar supported abundant growth while the FM680, DPE, and FM680/DPE agar supported only a few very slow growing colonies. The Wha, Whe, and 39a strains initially appeared to be growing on Firemaster 680, but the small colonies which occurred after two days of incubation never proliferated during the course of the study. This marginal growth pattern was also observed for strains W2a, W2b, W2c, W2f, Wilb, Wllg, 82d, S7a, 37d, 8%, S10d, Sllc, S13a, and S13b. While the results are ambiguous, it can be reasonably concluded that these cultures are not capable of utilizing Firemaster 680 or diphenoxyethane to support active population growth. The spurious results suggest that these bacteria were using the agar or impurities therein as sources of carbon. This phenomenon has been previously descried by Marshall gt gt. (1960), who found a substantial number of soil microorganisms that were capable 32 1\+ + 1\+ 1\+ owozm +omozm nmm +++++ +++++ ++++ +++ +O®©Zh omgm mmm + 1\+ 1\+ 1 man +ooeza emm 1 1 1 1 3989.; +89; emm 1\+ 1 1 1 amaze +omezm nmm +++++ +++++ ++++ +++ +Ow02m 909+...“ mmm +++++ +++++ ++++ +++ +OQ©Z- ommzm 3:3 + 1\+ 1 1 amaze +omozm ms: 1\+ 1\+ 1\+ 1\+ was +owezm as: + + + + man: 009E +owozm 0:3 1\+ 1 1 1 maa\omoza amaze as: 1 1 1 1 amaze amaze 0:: + + 1\+ 1\+ maaxomoza maa\omeze as: 1 + + + omozm amaze me: 1 1 1 1 maa\omezm +omezm em: 1 1\+ 1\+ 1\+ amaze +owezm em: +++++ +++++ ++++ +++ +0093 0002a om: 1\+ 1\+ 1\+ 1\+ man\omeza mao\omeza em: 1\+ 1\+ 1\+ + amaze amaze am: 1 1 1x1 1 man +omezm mm: + + 1\+ 1\+ maa\omezm +omoza em: 1 1\+ 1\+ 1 amaze +omezm om: +++++ +++++ ++++ +++ +0003".— 0002m 0N3 + + 1\+ 1\+ maa\omozm mao\omoza em: 1 1\+ 1\+ 1\+ was man am: 1 + 1\+ 1\+ amaze amaze mm: ms\m_\o oe\op\o as\m\e ms\=\e asses: naw< asses: tame Asaanemv Hanan osmaa1xmenam HannaeH enmas1enesam seesaw lessee mmuasmom seem senHA1samnem 1 soaanaonH can usessoassm .e manna 33 1 1 1 1 mm: +owozm oppm + + 1\+ 1\+ mm0\0002m +0mozm oFPm 1 1 1\+ 1\+ omcxm +0mczm oppm +++++ +++++ ++++ +++ +0002m 00020 mppm 1\+ 1\+ 1 1 man +ommzm poem 1\+ 1\+ 1\+ 1 mm0\owczm +omozm mopm + 1\+ 1 1 mma\0wozm m00\omozm oo—m 1 1 1 1 amass +omozm eopm 1 1\+ 1\+ 1\+ man man no—m +++++ +++++ ++++ +++ +00020 00020 mopm 1\+ 1\+ 1\+ 1\+ man +omozm omm 1\+ 1\+ 1\+ 1\+ mm0\omozm +omozm umm +++++ +++++ ++++ +++ +owozh man 00% + + 1\+ 1\+ was mmn can + + + 1\+ owozm omozm mam + + 1\+ 1\+ mma mma chm 1\+ 1\+ 1\+ 1\+ man +owozm cum 1\+ 1\+ 1\+ 1\+ mma\omozm +omozm new + 1\+ 1\+ 1\+ owozm +owozm mum 1\+ 1\+ 1\+ 1\+ man +omczm 00m 1\+ 1\+ 1\+ 1\+ mm0\omozm +0002m 00m 0 a m a omozm +omozm new +++++ +++++ ++++ +++ +0wezm 0002b mom 1\+ 1\+ 1\+ 1\+ man +omozm omm 1\+ 1\+ 1 1 mma\owozm +omozm omm m~\mp\o os\o_\o ms\m\o ms\=\e asaemz name asses: name Asaansmv Hmcam oumamlxmoeum HoauHCH oumHmlvmoLam oaaamm mmuasmom A.e.esoe0 o oases 34 nuzocu amazon u m suzocu oHnHmmom u 1\+ nuzomu pcmccso< >Lo> u +++++ mesons sewaam + nuzoco oz u 1 how mumnm 1 1 1 1 was gem are + + 1\+ 1\+ maa\omeza omezm nmpm + + 1\+ 1 ocean omozs amam m>\mp\e os\op\o a>\m\o ms\z\e asses: same ashes: nnma Asansumv Hagan esnaa1saenem HannasH esnaa1eaenam seesaw Anomav mmuasnom .Ae.ueoo0 a means 35 of growth in a medium with no source of carbon other than agar. Alexander (1977) referred to these microorganisms as oligocarbophiles. In order to distinguish between oligocarbophiles and bacteria capable of growing on Firemaster 680 or diphenoxyethane, a final enrichment step was conducted with a liquid medium. Twenty flasks were prepared and inoculated as described in Table 7. Growth was only observed in the flask containing the FM680/glucose supplemented mineral salts solution. The FM680, DPE, or FM680/DPE growth medium did not support observable growth during the 30 day study. These results substantiate the former conclu- sions. The microorganisms that were isolated were probably oligocar- bophiles rather than bacteria capable of growing on Firemaster 680 or diphenoxyethane. Thus, it appears that Firemaster 680 is not readily degradable as a sole carbon source or cometabolite in these systems. Cometabolism Study The biodegradation chamber containing FM680/glucose fortified growth medium was the only one that supported visible growth. The bacterial population grew rapidly but died off seven days after inoculation, leaving a bacterial cell floc at the bottom of the growth chamber. No growth was observed in the FM680 or FM680/DPE biodegradation chambers during the entire study. The growth medium agar plate results were consistent with these observations. No colonies were formed on the plates which received samples from the FM680 or FM680/DPE growth chambers. However, substantial growth was observed on the plates that were inoculated with bacteria from the FM680/glucose biodegradation chamber. Gas chromatography analyses for Firemaster 680 and 2,H,6-tribromo- phenol revealed a relatively rapid decrease in the concentration of Firemaster 680 and no 2,11,6-tribromophenol in each growth chamber. These 36 Table 7. Enrichment and Isolation - Shaker Flask Step Shaker-Flask Growth Medium Streak-Plate Final Agar Medium Sample Spread-Plate (Strain) Initial Agar Medium 1) W2b DPE DPE DPE 2) W20 FM680/DPE FM680/DPE FM680/DPE 3) W2f FM680+ FM680/DPE FM680/DPE N) WHa FM680 FM680 FM680 5) wub FM580/DPE FMfiaO/DPE FM680/DPE 6) Hue FM680+ FM680/DPE FM680/DPE 7) WHg FM680+ FM680 FM680 8) 32d FM680+ DPE DPE 9) 55b FM680+ FMOBO FM680 10) 37c FM680+ DPE DPE 11) S70 DPE DPE DPE 12) 89a FM680 FM680 FM680 13) 89a FM680 FM680 FM680/glucose 1“) 39b DPE DPE DPE 15) 39b FM680+ FMOBO/DPE FM680/DPE 16) S10d FM680/DPE FM680/DPE FM680/DPE 17) S10f FM680+ DPE DPE 18) $110 FM680+ FM680/DPE FM680/DPE 19) S13a FM680 FM680 FM680 20) S130 FM680 FM680/DPE FM680/DPE 37 data are tabulated in Table 8 and presented graphically in Figures 5, 6, and 7. The concentration of Firemaster 680 in the FM680/glucose growth chamber decreased exponentially over time which is characteristic of a substrate being metabolized by an actively growing bacterial population. However, the results were similar for the FM680 and FM680/DPE growth chambers which did not support bacterial growth. The concentration of Firemaster 680 in the FM680 growth medium initially increased slightly, but after 3 days it also decreased exponentially with time. The concentration of Firemaster 680 in the FM680/DPE growth chamber followed a similar pattern, but its rate of decline was less than that observed in other growth chambers. The concentration of'Firemaster 680 eventually reached a level which was consistent with its equilibrium solubility in deionized water. These data suggest that a mechanism other than microbial metabolism was responsible for the disapearance of Firemaster 680 from the growth medium. Some process of physical removal, such as volatilization or adsorption, may explain these results. Most likely, Firemaster 680 adsorbed on the glass walls of the biodegradation chambers and thus was removed from solution. In addition, sorption of Firemaster 680 by bacteria could have occurred in the FM680/glucose growth chamber. This would explain the increased rate of loss of Firemaster 680 from that growth medium. No matter which mechanism is responsible for these results, it appears that Firemaster 680 was not cometabolized under the conditions of this study. 38 Table 8. Firemaster 680 (FM680) Cometabolism Time FM680 TB? 1 Initial FM680 Sample (Days) (us/L) (ug/L) Concentration A. FM680/glucose Growth Medium t0 0 1.96 ND8 39.2 t1 1 1.37 ND 27." t2 3 0.84 ND 16.8 t3 6 0.69 ND 13.8 t” 12 0.23 ND “.6 t5 90 0.15 ND 3.0 t6 153 0.03 ND 0.6 B. FM680 Growth Medium t0 0 1.N5 ND 29.0 t1 1 1.88 ND 37.6 t2 3 1.96 ND 39.2 t3 6 1.57 ND 31.” t“ 12 0.18 ND 3.6 t5 90 0.22 ND H.“ t6 153 0.05 ND 1.0 C. FM680/DPE Growth Medium t0 0 2.3” ND ”6.8 t1 1 2.0“ ND 40.8 t2 3 1.98 ND 39.6 t3 6 1.75 ND 35.0 t” 12 1.03 ND 20.6 t5 90 0.02 ND 0.“ t6 153 0.0“ ND 0.8 aND - Not Detected (Limit of Detection 0.01 ug/L) 39 save: £320 onoosawxomozm so...“ 0093 no who; gaunewoam nm>¢00 uzuh 00" Deg 0m" 00d 00 00 0* 0w 0 Flu _ F _ p p _ _ 0 1T 1. 0“ .. HUN .um Age .um HZHI-HCJ 1.1.2000 UGZQUZO—ZCI—HDZ N .m shaman 40 00“ suave: nuzoco ommzm scam owozm ho nmoq o>Hmmocmocm nw>¢00 wank 0v" own 00" _ _ _ 00 _ 00 — 0v 0N e/al 0— ON on 0v ow HZHl-HG—l ILIICDQO UOZQUZl—Ofidfl-HOZ N .0 onsmfim 41 2300: 3380 203093 so...“ 0002a no who; cinema—No.5 .N. 95mg nw>mnu wrap 00— Dew 0N" 00~ 00 00 0% 0w 0 _ _ _ _ o) HZHO-HCJ b.2009 UOZUUZI—KCPHOZ N DISCUSSION WATER SOLUBILITY Of the parameters that affect the fate and transport of organic chemicals in the environment, water solubility is one of the most important (Kenaga and Goring, 1980). In general, chemicals that are water soluble are likely to be more widely distributed by the hydrologic cycle than those which are relatively insoluble. The aqueous solubility of a chemical can have an effect on its adsorption and desorption on soils and sediments, on its bioconcentration potential, and on its potential for volatilizing from aqueous media (Freed e_t gt, 1977). The more insoluble a chemical is, the more likely it is to sorb to soils and sediments and to bioconcentrate in aquatic organisms. Water solubility can also affect the possiblity of transformation via hydrolysis, photolysis, oxidation, reduction, and biodegradation in water. In this experiment, the water solubility of Firemaster 680 was deter- mined to be 0.01-1 ug/L. This value is low when compared with the aqueous solubilities of other known environmental contaminants. For example, HaQue and Schmedding (1975) reported a water solubility of 10.3 ug/L for 2,2'11,5,5'-pentachlorobiphenyl and 0.95 ug/L for 2,2'll,ll' ,5,5'-hexachlo- robiphenyl. The lowest solubility value found for P083 was 0.016 ug/L for decachlorobiphenyl (Weil gt gt., 197A). The highly toxic 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) has a water solubility of 0.2 ug/L (Neely, 1979). For comparison with other brominated aromatic flame 42 43 retardants, technical grade octabromobiphenyl has a water solubility of 20 to 30 ug/L (Norris gt gt., 1974) and the solubility of’Firemaster BP-6 in water is 11 ug/L (Jamieson, 1977). In natural waters, the solubility of Firemaster 680 could be higher than reported here due to dissolved organic matter. A number of studies have shown that the presence of dissolved organic material, such as the naturally occurring humic and fulvic acids in rivers and other surface waters, leads to an increase in the solubility of many organic compounds (Hasset and Anderson, 1979; Matsuda and Schnitzer, 1971; Wershaw gt gt., 1969). However, this probably will not affect the solubility of Firemaster 680 by more than one order of magnitude. Because of the low apparent aqueous solubility of Firemaster 680, the dissolved concentrations in water would be expected to be low. This indicates that Firemaster 680 tends to be associated with sediments, suspended solids, and biota in aquatic environments. N-OCTANOL/WATER PARTITION COEFFICIENT In recent years, the n-octanol/water partition coefficient (Kow) has become a key parameter in studies of the environmental fate of organic chemicals. In particular, the n-octanol/water partition coefficient has become a critical property for predicting water solubility (Banerjee gt _t., 1980; Chiou gt gt., 1977; Kenaga and Goring, 1980; Mackay gt gt., 1980b; Tulp and Hutzinger, 1978), soil sorption (Briggs, 1969; Hassett _t __l., 1980; Karickhoff gt a_l., 1979), bioconcentration in aquatic organisms (Chiou gt gt., 1977; Kenaga and Goring, 1980; Neely gt gt., 1974; Southworth gt gt., 1978; Veith gt gt., 1979), lipophilic storage (Davies e_t_ a_l. , 1975), and biomagnification (Metcalf gt a_l., 1973; Metcalf 44 gt_gt., 1975; Lu gt_gt., 1978; Lu and Metcalf, 1975; Tulp and Hutzinger, 1978). The measured values of n-octanol/water partition coefficients for organic chemicals have been found to be as low as 10'3 and as high as 107 (Hansch and Leo, 1979). In terms of log Kowv this range is from -3 to 7. Estimated values for this parameter have been recorded as high a 15.5 (Tulp and Hutzinger, 1978). However, for most environmental.contaminants that are persistent and tend to accumulate in sediments and aquatic biota, log Kb“ values generally are in the range of 5 to 7. Some examples of these include 2,2',4,5,5'-pentachlorobiphenyl; 2,2',4,4',5,5'-hexa- chlorobiphenyl; p,p'-DDT; and TCDD with log Kb" values of 6.11, 6.72, 6.19, and 6.19, respectively (Chiou gt gt., 1977; Neely, 1979). Sugiura gt gt. (1978) have reported a log Kow value of 7.41 for 3,3',5,5'- tetrabromobiphenyl. In the present study, the log Kow for Firemaster 680 was estimated to be 7.14. For neutral organic chemicals, the degree of sorption to sediments is dominated by their interaction with the organic content of the particulates. Karickhoff (1981) has shown that the organic carbon partition coefficient (Koc) for natural sediments correlates well with the water solubility and n-octanol/water partition coefficients for hydrophobic pollutants. In his study, least squares fitting of the Kow and Koc data gave: 103 Koo = 0.989 log KOw - 0.346 (Equation 7) If the K00 value for the environmental contaminant is known or estimated with equation 7, the partition coefficient for sediment sorption (Kp) can be predicted for a number of sediments where the organic content is known or designated. 45 A log Koc value of 6.72 for Firemaster 680 was calculated by using equation 7 and the experimentally determined log Kow' 7.14. For sediment with 3% organic carbon content, the corresponding Kp value would be 157,500. This indicates a strong tendency for Firemaster 680 to sorb onto suspended particulates and sediment in aquatic ecosystems. The accumulation of organic chemicals in fish and other aquatic organism can also be estimated from correlations between bioconcentration and nsoctanol/water partition coefficients (Baughman and Paris, 1981; Geyer gt gt., 1981; Southworth _et _a_1., 1978; Veith _e_t gt., 1979). For fish, the following equation can be used fer estimating the bioconcentration factor (BCF): log BCF = 0.76 log Kow - 0.23 (Equation 8) This regression equation was derived by Veith e_t gt. (1980) from the results of laboratory experiments by several investigators with a variety of fish species and 84 different organic chemicals. The bioconcentration factor for Firemaster 680 was calculated as 158,500 with equation 8 and 7.14 for the log Kow value. The structure-bioaccumulation relationship has one limitation which has not been thoroughly investigated (Veith, 1982). The equation implies that the bioconcentration factor will increase without bounds as the log Kow increases. However, an increase in the log Kow is often accompanied by an increase in molecular volume which eventually is sufficiently large to inhibit membrane permeability (Lieb and Stein, 1969). A number of studies of the accumulation of polybrominated biphenyls by juvenile Atlantic salmon (§§tgg §§tg§) have shown that penta- and higher bromobiphenyls accumulate in the fish to a lesser extent than similar chlorobiphenyls (Zitko, 1977; Zitko, 1979; Zitko and Hutzinger, 46 1976). Octa- and higher brominated biphenyls were accumulated by the fish to a small extent only when administered in food. Low water solublity, possibly acting in conjunction with low membrane permeability, was considered the main factor fer the lack of accumulation of the highly brominated biphenyls (Zitko, 1979). Sugiura _e_t_ gt. (1978) have shown that the accumulation factors for di-, tri-, and tetra-bromobiphenyls in killifish (Ogyzias latipes) were proportional to n-octanol/water partition coefficients when the coefficients were below 10°, but not when the coefficients were above 10°. The molecular size and steric configuration of Firemaster 680 may inhibit membrane permeability. Thus, the calculated bioconcentration factor for Firemaster 680 may significantly' overestimate its bioaccumulation potential. The study reported by Velsicol Chemical Corporation on the uptake of Firemaster 680 by carp (Cyprinus carpio) revealed a maximum accumulation factor of 56.6 (U.S. EPA, 1980b). These data suggest that direct accumulation of Firemaster 680 from water by fish may not occur to a significant extent. However, further investigation of the bioconcentration and ecological magnification of Firemaster 680 in aquatic organisms is necessary before the bioaccumulation potential of this compound is fully understood. MICROBIAL DEGRADATION The transformation of organic compounds by living organisms is a very important factor for the evaluation of their persistence in the envi- ronment. Degradative processes caused by microorganisms are the most important degradative mechanisms for organic compounds in nature, with respect to both the mass of material transformed and the extent to which 47 it is degraded (Brink, 1981). Biodegradation is often a desirable mechanism fer environmental transformations of organic molecules because the enzymatic degradations generally form the metabolites used for growth or potential energy storage, or simple inorganic molecules (Brink, 1981). Considerable progress has been made in understanding how mdcroor- ganisms degrade synthetic chemicals in the environment. Basically, a man-made compound will be biodegraded if it is susceptible to attack by the enzymes acquired by microbes during the course of evolution (Dagely, 1978). This depends on the ability of the microbial enzymes to accept as substrate compounds having chemical structures similar to those found in nature and the ability of these substrates to induce or derepress the synthesis of necessary enzymes (Dagely, 1978). Numerous metabolic reactions in which microbes degraded a variety of synthetic chemicals have been identified in the laboratory (Alexander, 1981; Chapman, 1972; Chapman, 1979; Kobayshi and Rittmann, 1982). Almost all of the reactions involved in biodegradation can be classified as oxidative, reductive, hydrolytic, or conjugative. Examples of the first three kinds of reactions include beta-oxidation, hydroxylation, hydrolysis, dehalogenation, and decarboxylation. Conjugative reactions such as methylation, acetylation, and dimerization have also been observed in the presence of ndcroorganisms. Reactions take place both in the presence and in the absence of oxygen. Some compounds, such as DDT, are transformed under both aerobic and anaerobic conditions (Meikle, 1972). This diversity of microbial mediated reactions of chemical substances influenced many microbiologists to believe in the theory of microbial infallibility a few years ago (Alexander, 1965). They were convinced that every organic compound was able to sustain microbial growth and would 48 be completely degraded or mineralized. This theory has been proven incorrect because many synthetic organic molecules are mineralized very slowly or not at all. They endure for long periods of time in nature because microorganisms are not able to degrade them rapidly, if at all. Thus, they have been referred to as recalcitrant molecules (Alexander, 1981). Once released to the environment, these chemicals can be transported great distances and many of them are susceptible to biomagnification. Examples of recalcitrant chemicals include PCBs, DDT, and alkylbenzene sulfonates. A number of scientists are studying these types of chemicals and are trying to understand the basis for recalcitrance and to predict which molecules may not be subject to microbial transformation, especially mineralization (Alexander, 1981). The results from this study indicated that Firemaster 680 was not readily degraded by microorganisms under the experimental conditions. Some loss of Firemaster 680 from the growth medium was observed in the cometabolism study, but no distinct metabolites were detected. It was assumed that the loss of the chemical was primarily the result of adsorption to the glass walls of the growth chambers and, in the case of the FM680/ glucose growth chamber, adsorption, and/or accumulation by microorganisms in the growth medium. Microorganisms that can metabolize Firemaster 680 were not found in any of the environmental samples collected for this study. These data suggest that Firemaster 680 should be classified as a recalcitrant chemical. Resistance of an organic compound to microbial degradation may be attributable to the structure of the chemical or to one of several ecological parameters such as dissolved oxygen, oxidation-reduction potential, temperature, pH, availability of other compounds, salinity, 49 particulate matter, competing organisms, and concentrations of compounds and organisms (Alexander, 1965; Alexander, 1973; Alexander, 1975; Alex- ander, 1981). For Firemaster 680, its structure and physico-chemical characteristics are key factors influencing its resistance to biodegra- dation. The compound's low water solubility and large n-octanol/water partition coefficient suggests that it may not be readily available to the microorganism for biodegradation. The "bulky" chemical structure of Firemaster 680 could inhibit membrane permeability and the ability of'the compound to reach the reaction site in the microbial cell. Furthermore, the extent and pattern of bromine substitution on the Firemaster 680 molecule would tend to inhibit microbial mediated dehalogenation, hyroxylation, and ring cleavage reactions for this compound. Based on the results of this study, it appears that microbial degradation is not an important transformation process formFiremaster 680 in the aquatic environment. However, further research on the biodegra- dation of Firemaster 680 under anaerobic conditions is required since this chemical will most likely reside in the sediments of aquatic systems. Anaerobic degradative pathways such as reductive dehalogenation are now considered important processes in the biodegradation of certain classes of compounds (Kobayashi and Rittman, 1982). SUMMARY AND CONCLUSIONS Firemaster 680 has become an important brominated aromatic flame retardant for use in thermoplastic applications. Production of this com- pound has increased over the last five years and environmental contamina- tion from this chemical has occurred near a number of production facili- ties. A contaminaton incident with Firemaster 680 has been identified in the Raisin River watershed in the vicinity of Adrian, Michigan. The impact of this pollutant on the aquatic ecosystem has not been assessed. Very little is known about the behavior of’Firemaster 680 in aquatic environments. The primary purpose of this investigation was to develop a base set of data which could be used to predict the water-related environmental fate of Firemaster 680. The aqueous solubility was measured as 0.04:0.01 ug/L under controlled laboratory conditions during a 60 week experiment. The logarithm of the partition coefficient for Firemaster 680 between n-octanol and water was determined to be 7.14:0.42. This value was then used to estimate an organic carbon normalized sediment distribution coefficient of 5.25 x 106 and a fish bioconcentration factor of 1.58 x 105. Bacteria capable of degrading Firemaster 680 were not found during acclimation, analog enrichment, or cometabolism studies. The UV spectrum of Firemaster 680 revealed absorption maxima at 282 and 290 nm which indicates the potential for direct photodegradation in the environment. 50 51 The melting point and density were found to be 225°C and 2.58 g/ml, respectively. The vapor pressure for Firemaster 680 at room temperature is expected to be low. These data may be used for a preliminary assessment 'of the chemodynamics of Firemaster 680 in the aquatic environment. Based on the results of this study, it is apparent that physica- chemical processes will largely determine the persistence of Firemaster 680 in the environment. The degree of adsorption to sediments and other suspended matter will, to a large degree, dictate the ultimate fate of Firemaster 680 in aquatic environments. Adsorption on suspended particulates may lead to a wide translocation of this chemical from its original site of entry, and possibly to accumulation through trophic levels by the action of detritus-feeding organisms. In contrast, sedimentation of these materials may lead to burial and removal of Firemaster 680 from aquatic systems. This association with sediments may also have a negative effect on other fate processes. Compared to sorption, processes such as volatilization, photolysis, and biodegradation appear to be minor components governing the behavior of Firemaster 680 in the aquatic environment. RECOMMENDATIONS In view of the apparent importance of sorption of Firemaster 680 to sediments and suspended matter, further studies should be initiated to determine actual distribution coefficients for a variety of sediment types as a function of particle size distribution. The kinetics of adsorption and desorption for this chemical should be examined in the laboratory. Furthermore, studies should be undertaken to determine the ability of Firemaster 680 to undergo biomagnification through trophic levels. Finally, microbial degradation should be assessed under anaerobic conditions and the role of photodegradation in the fate of Firemaster 680 in aquatic systems should be investigated. The results of this study suggests that an assessment of the impact of Firemaster 680 contamination on the south branch of the Raisin River should focus on sediments, especially in zones of deposition. A monitoring program should be developed for Firemaster 680 in suspended and bottom sediments and an assessment should be made on the effects of Firemaster 680 contamination on the benthic community. Additionally, an aquatic biota monitoring program should be initiated to determine possible biomag- nification and the potential for wildlife and human exposure to this contaminant. As a final note, similar studies on other brominated aromatic flame retardants should be undertaken to determine the environmental contamina- tion potential of this diverse class of chemical substances. 52 APPENDICES APPENDIX A FIREMASTER 680 TOXICITY The Velsicol Chemical Corporation has developed a toxicity profile on Firemaster 680 from acute, subacute, and chronic exposure studies with laboratory animals. Based on the results of these studies, the company claims that Firemaster 680 has a very low order of toxicity (Velsicol Chemical Corporation, 1977). These studies have been reported to the 0.3. Environmental Protection Agency as a substantial risk notice in keeping with Section 8(e) of the Toxic Substances Control Act (U.S. EPA, 1980b). A synopsis of these data are reported below. A number of acute toxicity studies with Firemaster 680 were reported and results from these studies are summarized in Table 9. These data appear to indiciate that this chemical is relatively non-toxic during acute exposure. In addition to the acute studies, results from several subacute studies also were submitted (U.S. EPA, 1980b). A subacute dust inhalation toxicity study was reported with two exposure groups with 5 rats per sex in each group and one control group. The exposure group received micro- nized Firemaster 680 dust at concentrations of 5 or 20 mg/L for a 21-day period at 4 hr/day, 5 days/week. At the end of the 21-day period, hematological, biochemical, and urinalysis values were obtained prior to necropsy; ‘Bromine neutron activation analysis of liver, fat, kidney, 53 54 Table 9. Acute Toxicity of Firemaster 680 Acute Study Test Animal Results Oral Toxicity Rat (male) LD50 = 10,000 mg/kg Rat (female) LD50 = 10,000 mg/kg Beagle Dog (male) LD50 = 10,000 mg/kg Beagle Dog (female) LD50 = 10,000 mg/kg Dermal Toxicity Rabbit (male) LD50 = 12,000 mg/kg Rabbit (female) LD50 = 10,000 mg/kg Skin Irritation Rabbit Non-irritating Eye Irritation Rabbit Non-irritating Inhalation Toxicity Rat (male) L050 = 36.48 mg/L Rat (female) LC50 = 36.48 mg/L lung, and blood samples was conducted at necropsy» 'There were no reported deaths in either the control or treated groups. A slight ocular porphyrin discharge was observed only in the treated groups while a clear nasal or prophyrin discharge, soft feces, and respiratory congestion was observed in animals from each group. All of the animals showed similar growth rates and the food consumption values obtained from rats in the control and treated groups were essentially similar. Results of the study indicated no significant differences between the hematologic, clinical chemistry, or urinalysis values of the control animals and the treated animals. However, very marked increases in bromine content, approximately 2,000 times, were reported in the lungs of treated rats. Increases in bromine from 2 to 3 times were also reported in liver, fat, kidney, and blood samples obtained at necropsy; No gross pathologic lesions or organ weight variations related to exposure to Firemaster 680 were reported. Compound related histopathologic lesions were limited to the lungs of 55 rats from both experimental groups which included small local accumu- lations of foamy alveolar macrophages scattered throughout the lungs. A 28-day dermal toxicity study on albino rabbits also was reported (U.S. EPA, 1980b). In this study, the rabbits received doses of'50, 500, and 5,000 mg/kg of Firemaster 680 applied to the shaven intact or abraded skin far 5 days a week for 4 weeks. Rabbits in the control group and in each of the treatment groups exhibited very slight to slight erythema during the study period. One rabbit dosed at the 5,000 mg/kg/day level was reported to exhibit very slight to moderate erythema. The observed erythema was attributed to the normal saline carrier fluid and not the applications of Firemaster 680. No changes related to the compound were reported in hematological, biochemical, or urinalysis analyses which were obtained at day 14 and 28 of the study period. Nor were any compound related gross pathogenic lesions or variations in organ weight found in any rabbits from the experimental groups. In addition to the inhalation and dermal subacute studies, a 28-day rat feeding study was reported in which male rats were exposed to 100 or 1,000 mg/kg Firemaster 680 (U.S. EPA, 1980b). At the end of the 28-day test period, rats in the test group showed less body weight gain and slightly poorer feeding efficiency when compared with the controls. Organ weight data were interpreted to indicate a definite difference between test and control animals with regard to the size of the organs. In general, all organ weights were decreased in the dosed animals when compared with the controls. Also, Firemaster 680 appeared to accumulate in the fat and remained there for some time. A 90-day chronic oral toxicity study was reported in which albino rats were fed dietary concentrations of 0, 1, 10, or 100 mg/kg of Firemaster 56 680 (U.S. EPA, 1980b). The results of this study indicate that the average body weight gains and average food consumption among test animals were similar to those of the control group. However, the animals receiving the highest dose showed histological liver changes which were reflected by increased alkaline phosphatase in the blood. In addition, the kidney weights of the female rats on the 100 mg/kg diet were significantly lower than those of the control group. Also, the ratio of kidney weight to body weight was significantly greater in male rats on the 10 mg/kg Firemaster 680 diet. No data were presented to show the extent of bromine retention in tissues. A mutagenicity evaluation of Firemaster 680 was also conducted (U.S. EPA, 1980b). This study was performed by using the Ames test on tester strains of Sglmonellg typhimurium and Saccharomches cerevisiae. Tests were conducted both in the absence and presence of the rat liver activation system and the dose range was from 0.25 to 50 ug Firemaster 680 per plate. All results were reported to be negative. In addition to mamalian toxicity studies, results from aquatic toxicity studies also were submitted (U.S EPA, 1980b). The 96-hour TL50's of Firemaster 680 reported for rainbow trout (gating gairdneri) and bluegill sunfish (Lepomis macrochirus) were 1410 mg/L and 1531 mg/L, respectively. In these studies, the test material was suspended in the water by sonication. Thus, the fish were exposed to particles of Firemaster 680 along with that dissolved in solution. A Japanese testing company determined the 48-hour TL50 for Firemaster 680 for orange-red killifish (Oryzias latipes) to be 230 mg/L (U.S. EPA, 1980b). This value is significantly lower than the TL50's reported for bluegills and rainbow trout, although the test compound is still only S7 moderately toxic. The difference in the TL50's might be due to a different method of dissolving Firemaster 680. In this study, dimethyl sulfoxide and castor oil carriers, plus sonication were used to drive Firemaster 680 into solution. In a recent study, the porphyrinogenic potential of some recently marketed fire retardants was determined using a primary tissue culture of chick embryo cells assay (Koster gt gt., 1980). The chemicals that were tested included decabromobiphenyl, decabromobiphenyl oxide, octabro- mobiphenyl.oxide, N,N'-ethylenebistetrabromophthalimide, Firemaster 680, and Firemaster BP-6. Decabromobiphenyl and decabromobiphenyl oxide did not cause porphyria in the chick embryo liver cell culture. Firemaster 680 and N,N'-ethylenebistetrabromophthalimide were found to be slightly porphyrinogenic only after pretreatment of cultures with B-naphthoflavone to induce drug enzymes. Octabromobiphenyl oxide and Firemaster BP-6 were found to be strongly porphyrinogenic, even without pretreatment with B- naphthoflavone. The authors suggest that the nonporphyrinogenic effect of decabromobiphenyl and decabromobiphenyl oxide may be explained by their less planar molecular structure and their resistance to metabolic degradation. These studies suggest that Firemaster 680 is relatively non-toxic to mammals and fish when tested under controlled laboratory conditions. However, the initial signs of Firemaster 680 toxicity are very similar to those found for PBBs. For example, several acute and chronic studies have shown that the initial indicators for PBB toxicity in mammals are weight loss or reduced weight gain (Falk gt gt., 1980). Additionally, increase in liver size is indicative of this toxicant. One study revealed that a dose as low as 50 mg/kg Firemaster BP-6 in the diet of male rats 58 for ten weeks produced an enlarged liver (Harris gt gt., 1978a). Another study showed decreased weight gain and an increase or decrease in the activity of some kidney enzymes in female rats when they were fed 100 mg/kg Firemaster BP-6 for 90 days (McCormack gt gt., 1978). When Firemaster FF-1 (Firemaster BP-6 with 2% calcium trisilicate) was fed to rats at doses of either 30, 100, 300, or 1,000 mg/kg of body weight per day, all of the animals exposed to doses greater than 30 mg/kg died within 73 days with the exception of 621 of the males at the 100 mg/kg dose (Gupta and Moore, 1979). All of these rats demonstrated depressed body weights, anemia, and enlarged livers. Further evaluation of the carcinogenic and teratogenic potential of Firemaster 680 should be made before it is designated as non-toxic. Although the Ames test with Firemaster 680 produced negative results, this compound may still possess genotoxic properties. An Ames study with PBB was also negative (Ball 23.21:: 1980), but several long-term animal studies have revealed carcinogenic activity (Gupta g a_l. , 1981; Kimbrough gt gt., 1978; Kimbrough gt gt., 1981). Also, the reproductive and the teratogenic effects of PBB are well known (Allen, 1978; Aulrich and Ringer, 1979; Beaudoin, 1977; Corbett gt gt., 1975; Durst gt gt., 1978; Ficsor and Wertz, 1976; Harris gt gt., 1978b; Jackson and Halbert, 1974; Preache gt gt., 1976; Mercer gt gt., 1976; Moorehead gt gt., 1977; Wastell gt gt., 1978; Wertz and Ficsor, 1978). APPENDIX B CHEMICAL CHARACTERIZATION OF FIREMASTER 680 The reported chemical structure of Firemaster 680 was confirmed by using various analytical instrumental techniques which included gas chromatography, ultraviolet spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, mass spectrometry, and elemental analysis. The purity of'Firemaster 680 was determined from the elemental analysis, melting point, and trace organic impurity content. EXPERIMENTAL Materials Firemaster 680 was obtained from Velsicol Chemical Corporation (Chicago, Illinois) as part of Lot No. 61114-Fu The 2,4,6-tribromophenol standard was purchased from Aldrich Chemical Company (Milwaukee, Wisconsin) and it was 99-1-1 pure. The 1-(2,4,6-tribromophenoxy)-2- bromoethane standard and sodium biphenyl reagent were obtained from the former Story Chemical Company (Muskegon, Michigan). All other reagents were of analytical or spectral grade. Analytical Methods gpd Instrqgggtg Elemental Analysis. The Firemaster 680 was subjected to elemental analyses to determine the carbon, hydrogen, and bromine content. The carbon and hydrogen content were determined with a Perkin-Elmer model 240 Elemental Analyzer (Perkin-Elmer, Norwalk, Connecticut). 59 60 The bromine content was measured by using a total bromide determination method (Liggett, 1954; Michigan Chemical Company, 1975). The sample was decomposed with sodium biphenyl reagent which converted the organic bromine into the inorganic form. Following decomposition, the bromide content was determined by potentiometric titration with a silver_nitrate solution and calculated as bromine. Melting Point. The melting point of Firemaster 680 was determined with a Fisher Melting Point Apparatus (Fisher' Scientific Company, Pittsburgh, Pennsylvania). A small amount of sample was placed between two glass cover slips on the hot plate and heated with a temperature rate of increase of 10 °C/min. I_mpurity Determination. The organic chemical impurities in the Fire- master 680 were determined by electron-capture gas-liquid chromatography. Ether solutions of 2,4,6-tribromophenol and 1-(2,4,6-tribromophenoxy)-2- bromoethane standards at concentrations of 10.0 and 1.0 ug/L and Firemaster 680 at 10.0 mg/L were prepared. These samples were subjected to gas chromatography analyses with a Beckman model GC-65 gas chroma- tograph equipped with a non-radioactive electron-capture detector and a Beckman 10-inch linear recorder (Beckman Instruments, Inc., Fullerton, California). The polarizing voltage, carbon dioxide flow, and bias voltage were set for optimum detector response. A 0.2 (i.d.) x 120 cm glass column packed with 1% SP-124ODA on 100/120 Supelcoport (Supelco, Inc., Bellefonte, Pennsylvania) was used with a helium carrier gas flow rate of 40 ml/min. The inlet, column, detector line, and detector tempera- tures were 290, 170, 310, and 360 °C, respectively. Using the above column and operating conditions, the retention times for 1-(2,4,6-tribro- mophenoxy)-2-bromoethane and 2,4,6-tribromophenol were 2.8 and 6.6 min., 61 respectively; and the minimum detectable quantity was 1.0 pg for each standard at 2.5 times the noise level. Ultraviolet Spectrometry. A Gilford model 2600 microprocessor- controlled ultraviolet-visible spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio) equipped with a Hewlett Packard model HP7225A graphics plotter (Hewlett Packard, San Diego, California) was used to obtain UV absorption spectra of‘Firemaster 680. A 100 mg/L solu- tion of Firemaster 680 in spectrophotometric grade cyclohexane (Aldrich Chemical Company, Milwaukee, Wisconsin) was placed in a 1.0 cm quartz cuvette and scanned through the near UV region between wavelengths of’220 to 340 nm. Infrared Spectrometry. Infrared spectra of Firemaster 680 were obtained with a Perkin-Elmer model 337 Spectrophotometer (Perkin-Elmer, Norwalk, Connecticut). A KBr macropellet containing 1.0% Firemaster 680 was prepared and scanned from 2.5 to 25 microns. Nuclear Magnetic Resonance Spectrogetry. The nuclear magnetic reso- nance characteristics of Firemaster 680 were studied by using a Varian model EM—360 NMR Spectrometer System (Varian Instruments, Palo Alto, Cali- fornia). The NMR spectra were obtained for Firemaster 680 dissolved in carbon tetrachloride with tetramethylsilane (TMS) as an internal standard. The spectra were obtained at an applied field frequency of 60 x 106 cps. Mggg Spectrometry; .A DuPont model 321 Mass Spectrometer (DuPont Company, Wilmington, Deleware) was used with a direct probe inlet system to obtain mass spectra data. Operating conditions for the mass spectrometer were as follows: source temperature, 250°C; ionizing potential, 70 eV; total scan time, 5.1 seconds (m/e 45 to 700); internal mass marker, perfluoro-tributylamine (PFA). 62 RESULTS Elemental Analyses. Analyses of Firemaster 680, Lot. No. 61114-F, for elemental carbon, hydrogen, and nitrogen revealed that this sample contained 24.351 carbon, 1.15% hydrogen, and 0.00% nitrogen. Experimental data and calculations fer these values can be found in Figures 8 and 9. The total.bromine analysis showed that.Firemaster’680, Lot No. 61114- F, contained 68.72% bromine. This value was calculated with the equation: 1 Bromine = (ml AgN03) X (N AgNO3) X 0.079909 X 100 sample weight (8) (Equation 9) Experimental data for this calculation included: volume AgN03 = 2.15 ml; normality AgNO3 = 0.1002 N; sample weight of Firemaster 680 = 0.02505 g. Only one total bromine determination was made because of the limited amount of sodium biphenyl reagent, thus no statistical significance could be given to this value. However, it has been reported that organic bromine can be determined within 0.5% of the true value by this method (Ligget, 1954). A comparison of’ the theoretical and experimentally determined elemental content of this sample of Firemaster 680 is presented in Table 10. The experimentally determined values are in close agreement (approximately 99%) with the theoretical values. Melting_Point. The melting point of Firemaster 680 was determined to be 225°C. The crystals began to melt at 225°C and were completely melted at 226°C. tgpggtty Determination. Gas chromatography analyses of Firemaster 680 for organic impurities showed less than 0.1% of 2,4,6-tribromophenol and 1-(2,4,6-tribromophenoxy)-2-bromoethane. A comparsion of the gas chromatograms obtained from this experiment is presented in Figure 10. 63 CALIBRATION DATA SHEET A. Standard Name: A2 B. Date: 12-6-77 C. Standard Weight: (g323__ ug D. Theoretical Percentages: 10.36% N; 71.09 1 C; 6.71 1 H E. Theoretical Weights: 247.91 ug N; 1701.18 ug C; 160.57 ug H F. Total Signals: N Siggal C Siggal H Siggal Reads (uV) 537 x 4 = 2148 876 x 10 = 8760 653 x 16 :: lguggppression (uV) *'* Setting 3 = 30000 *** - Blank Values (uV) = 210 = 190 = 450 - Zero (uV) = 42 = 1330 = 192 Total Signals (uV) = 1896 = 37240 = 9806 G. Sensitivities: K = Total signal (uV) Theoretical weight (ug) 7.65 uv_ 21.81 uv. 61.07 uv KN (7.6) 313’ c (21.8) E’ K11 (61.1) 17g" Figure 8. CHN Analyzer Calibration Data Sheet 64 A. Sample No.: FM680 B. Sample Name: Firemaster 680 C. Date: 12-6-77 D. Sample Weight: 2033 ug E. Theoretical Percentages: __::__ % N; 24.45 3 C; 1.17 % H F. Theretical Weights: __::__ ug N; 497.1 g C; 23.8 ug H G. Sensitivities: KN = 7.6 8%; KC = 21.8 8%} KH = 61.1 E! N Siggal C Siggal H Siggal Reads (uV) 210 x 1 = 210 234 x 10 = 2340 518 x 4 = 2072 +0 Supression (uV) *** Setting 1 = 10000 **9 - Blank Values (uV) = 210 = 200 = 450 - Zeros (uV) = 50 = 1350 = 188 Total Signals (vV) = 0 = 10790 = 1434 I. Calculated Weights and Percentages: Weight (ug) of N, C, or H in a sample = Total Signal (uV) (uV) Sensitivity K ug Weights 0 ug N; 494.95 ug C; 23.41 ug H Percentage of N, C, or H in a sample = Wei ht x 100 Sample Weight Percentages: 0 % N; 24.35 5 C; 1.15 S H Figure 9. CHN Analyzer Data Sheet for Firemaster 680 65 Relative Response 2.0 4.0 0.0 8.0 Retention time [minutes] Figure 10. Gas Chromatogram - (A) Firemaster 680 (10,000 pg) (B) 1-(2,4,6- tribromophenoxy)-2-brommoethane (a) and 2,4,6-tribromopheno] (b) (10 pg) 66 Table 10. Comparison of Theoretical and Experimentally Deter- mined Elemental Content of Firemaster 680 (FM680) Element Theoretical Content Experimental Content 1 Br (6)a 69.72 68.72 C (14) 24.46 24.35 H (8) 1.17 1.15 0 (2) 4.65 N03 a< ) = Number of atoms of each element in Firemaster 680 bOxygen content was not determined. Ultraviolet Spectrometry. The UV absorption spectrum of Firemaster 680 is presented in Figure 11. The spectrum shows B-band absorptions at max of 282 and 290 nm. This absorption pattern is characteristic of a benzene chromophore with auxochromic group substitution. .0 charac- teristic bathochromic shift (red shift) of the B-bands of the benzene chromophore is seen in the Firemaster 680 spectrum. The UV absorption spectrum of Firemaster 680 is consistent with the reported chemical structure of this product. ggggared Spectrometry. The infrared spectrum of Firemaster 680 is shown in Figure 12. The characteristic absorption bands are as follows: (a) aromatic C-H stretCh, 3060, 3030 cm'1; (b) methylene C-H stretch, 2930, 2860 cm“; (0) aromatic C-H overtone band, 1715 cm‘1; (d) aromatic ring c-c stretch, 1550, 1520, 1u30, 1030, 1010 cm“; (e) asymetric c—o- C stretch, 1252 om"; (f) symmetric C-O-C stretch, 1002 cm‘1; (g) out of plane aromatic ring C-H bend, 860, 745, 737 cm“; (h) out of plane aromatic 67 BB'BYE BB'BZE BB'DIS ‘F 00'708 4 0026?. one Lopmmaonwm mo ssnuooom coHpaLomn< poaofi>mauas 2.5 528 3:5 " 83'092 " BZ'BSZ “3‘77? 20°28? BZ'BZZ lb ID o a as a- Bum—.6 annt.n numo.n ausm.a mama.“ auuv.u Gama." sand." num«.~ aan.N eoneqxosqv .FP mtsmfim owe Lounmamgam no sznuooaw ooLmLh:H .m— ogsmfim :19: 3.3:: 000' camp comp OOON can“ 006» coma 609' d 1 000 00. .3 .3 is. 68 .2 .3 .3 .3 .3 2: .1 06 ISIIQJOIII OR 69 ring C-C bend, 663 cm‘1; (i) C-Br stretch, 569 cm’1. This absorption pattern is indicative of an aryl-alkyl ether with the aryl moiety substituted with bromine. This spectrum also reveals that there were no alcohol, amine, acid, nitrile, ketone, or aldehyde functional groups in the molecule. The infrared absorption spectrum obtained for Firemaster 680 is in very good agreement with the reported structure of'this product. Nuclear’Magnetic Resonance Spectroscopy (NMR). The NMR spectrum of Firemaster 680 is presented in Figure 13. The proton NMR spectrum of Firemaster 680 showed resonance signals from two different protons at 4.45 ppm and 7.70 ppm. Both signals were singlets, which indicates chemically equivalent protons contributing to each resonance signal. Furthermore, the resonance signals intigrate 1 to 1 which indicates equivalent number of protons f0r each signal. The singlets at 4.45 ppm is indicative of a methylene proton adjacent to an oxygen atom, specifically an alkyl-aryl ether group. The singlet at 7.70 ppm is indicative of an aromatic proton, specifically one that had been shifted paramagnetically as a result of the inductive effect of a neighboring electronegative atom such as bromine. This resonance pattern is in agreement with the reported structure of Firemaster 680, with four methylene protons of‘ an alkyl-aryl ether group and four equivalent aromatic protons separated by electronegative bromine atoms. Mass Spectrometry. A graphic representation of the mass spectrum of Firemaster 680 is presented in Figure 14. The molecular ion (M+) was found at m/e 682 and the base peak (B) was at m/e 354. The molecular ion peak was followed by a M+2, M+4, M+6, M+8 pattern which is characteristic of a molecule containing six bromine atoms. 7O tn owe 0338003 .uo 3:30on 65 .mF mesmue $2.3 Eng 71 owe Lounmamnfim mo Esauooam mum: no cofiumpcomopaom canamco .z— onswflm m}. ow: om: 0:: cm: co: owm own oem omm com owm com 5...:_:::_:_::__:._ . lo M d z . _ _ -=- o" tau) 2: I; Amy Iéem a: ram ruemv ll 8 8 Kitsueiul eAIquea I .3. Amy 0? com OJN CNN OON OwH 00H OJH ONH OOH ow 0m 0: lo— 18 tony» Ge :3 1.9» EV ram roost AHV 72 r. r\ E T” r9: Kitsuaqux eAtquea 72 0mm. 005 owm owe OJm omw {a 00m can own. o:m A.U.uCOOv .ap mtsmfim omm . com lean, ( 1 r 8 8 r, § Kitsuequl eAIquea 73 The base peak at m/e 354 was followed by a M+2, M+4, M+6 pattern characteristic of three bromine atoms. This fragment was most likely a tribromophenoxyethylene ion with the structure: i-crcu, m 3!: which was produced by cleavage of the parent compound through the ether bond. The other fragment resulting from this cleavage was the tribromo- phenol cation with the structure: 6 11:1 30: which was fOund at m/e 327 (C). Once again, the M+2, M+4, M+6 pattern was present which indicated three bromine atoms. The next major peak was located at m/e 275 (E) which showed an M+2, M+4 pattern, typical of two bromine atoms. This peak was 79 mass units from the base peak (m/e 354) and resulted from cleavage of a bromine atom from the base peak ion. The resulting fragment had the structure: 0-0I=0l2 [E] III The peak at m/e 250 (F) is typical of the fragment which was produced by cleavage of the ethylene group of fragment E along with hydrogen rearrangement. The characteristic two bromine substituent M+2, M+4 pattern is also apparent here. The structure of this fragment was: 2!: 0 [F] 74 Cleavage of a Br and CO group from compound F resulted in a bromocyclopentadiene cation which was found at m/e 143 (H) and had the structure: I (‘9) Additional major peaks were found at m/e 77 (I) and m/e 65 (J) which are indicative of phenyl and cyclopentadienyl cations, which are typical fragments of aromatic compounds. A number of minor peaks (i.e., D and G), which are consistent with Firemaster 680 fragments, were also found. The mass spectrum fragmentation pattern of Firemaster 680 is in agreement with the reported chemical structure of this product. With these data alone, the chemical structure for Firemaster 680 is best described by the structure: The exact location of the bromine atoms on the aromatic rings could not be determined from the mass spectral data. DISCUSSION The ultraviolet, infrared, nuclear magnetic resonance, and mass spectra of Firemaster 680, Lot No. 61114-F, identified this product as 1,2-bis(2,4,6-tribromophenoxy)ethane. Infrared and mass spectra data revealed that Firemaster 680 consists of a double alkyl-aryl ether connected through the ethane C-C bond with three bromine substitutes on each phenyl ring. The NMR spectrum, showing only two singlets, confirmed that the bromine atoms are placed at positions 2, 4, and 6 on each phenyl 75 ring. From these data, the structure of Firemaster 680 was determined to be: It It If 0-0lz-cl2-0 t 3' It This chemical is structurally symetrical with the axis of symmetry through the C-C bond of the ethylene group. This explains the relatively simple NMR and mass spectra for Firemaster 680. The purity of Lot No. 61114-F of Firemaster 680 appeared to be between 98 and 99%. This sample contained approximately 68.7% organically bond bromine which equates to a purity of 98.6% for Firemaster 680. There were less than 0.1% bromine containing organic impurities in the sample. The melting point was sharp, within 0.5°C, which indicates a relatively pure product. Commercial grade products are not normally as pure as Firemaster 680. However, the chemical reactions in the synthesis of this product are specific and the raw naterials are relatively pure which explains the high purity of the final product. APPENDIX C ANALYTICAL METHOD DEVELOPMENT An XAD macroreticular resin analytical scheme was developed for extracting Firemaster 680 and potential metabolites from aqueous media. The method described here is a modification of an analytical scheme reported by Junk pp El! (1974). The extraction efficiency for Firemaster 680 and 2,4,6-tribromophenol is reported for tests with XAD-2 and XAD-4 alone, and a mixture of XAD-2/XAD—4. EXPERIMENTAL Materials and Apparatus Reggents. Deionized water was freed of organic matter by passing it through a column containing activated charcoal. All of the solvents were analytical grade and were obtained from Burdick and Jackson Laboratories (Muskegon, Michigan). The concentrated HCl (approximately 37%) was obtained from Mallinckrodt (St. Louis, Missouri). The macroreticular resins, XAD-2 and XAD—4, were obtained from Rohm and Haas (Philadelphia, Pennsylvania). The fines ‘were removed by slurrying in methanol and then decanting. The remaining resin beads, predominantly 20-60 mesh, were purified by sequential solvent extractions with methanol, acetonitrile, and diethyl ether in a soxhlet extractor for 76 77 8 hours with each solvent. The purified resins were stored under methanol in glass-stoppered bottles to maintain their purity. Test Solutions. Firemaster 680 and the 2,4,6-tribromophenol used to prepare standard samples and spiked water samples were obtained from Velsicol Chemical Corporation and Aldrich Chemical Company. Standard solutions containing 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1000 ug/L of Firemaster 680 and 2,4,6-tribromophenol were prepared in diethyl ether. Spiked water samples were prepared to contain 0.0, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.6, and 2.0 ug/L of Firemaster 680 and 2,4,6- tribromophenol. Instruments. A Beckman, model GC-65, gas chromatograph equipped with a non-radioactive electron-capture detector and a Beckman 10-inch linear recorder were used to obtain the gas chromatography data. The polarizing voltage, carbon dioxide flow and bias voltage were set for optimum detector response. A 0.2 (i.d.) x 120 cm glass column packed with 1% SP-1240DA on 100/200 Supelcoport (Supelco, Inc., Bellefonte, Pennsylvania) was used with a helium carrier gas flow rate of 60 ml/min and a column temperature of 160°C to analyze the 2,4,6-tribromophenol. For Firemaster 680, a 0.2 (i.d.) x 183 cm glass column packed with 60/80 mesh Gas Chrom Q coated with 3% OV-1 liquid phase (Applied Science Laboratories, Inc., State College, Pennsylvania) was used with a helium carrier gas flow rate of 60 ml/min and a column temperature of 270°C. The inlet, detector line, and detector temperatures were 290, 310, and 360°C, respectively. With these columns and operating conditions, the retention times for Firemaster 680 and 2,4,6-tribromophenol were 4.3 and 2.7 minutes, respectively; and the least detectable quantity was 1.0 pg for each at 2.5 times the noise level. 78 Analytical Procedpge Column Preparation. The apparatus used to remove trace quantities of Firemaster 680 and 2,4,6-tribromophenol from water consisted of a 1.5 (i.d.) x 30 cm glass column which was fitted with a 300 ml sample reservoir and a teflon stopcock. A clean, ether-extracted silanized glass wool plug was inserted near the stopcock. The purified resin was added as a methanol slurry until a resin bed approximately 7.0 cm high was obtained (6.0 g dry resin); then a second silanized glass wool plug was inserted above the resin. The methanol was drained through the stopcock until the level reached the top of the resin bed; then the resin was washed with three 20 ml portions of deionized water. Each portion of the flow was stopped when the liquid level reached the top of the resin bed. An XAD- 2, XAD-4, and 50/50 mixture of XAD-2/XAD-4 column were prepared by using this procedure. §agple Preparatipp, All of the standard solutions were prepared with diethyl ether. All of the spiked water samples were prepared by injecting a calibrated volume of a standard solution of Firemaster 680 and 2,4,6-tribromophenol into a 500 ml volumetric flask containing deionized water. The spiked water samples were then acidified (pH 2.5) by adding 2.5 ml of concentrated HCl. g9;pmp Extraction. The acidified spiked water samples were added to the glass columns containing XAD resin. The sample was allowed to pass through the XAD resin column by gravity flow at a rate of 30 to 50 ml/min. When most of the sample had passed through the column and the liquid level was at the top of the resin, the reservoir walls were carefully washed with a 20 ml portion of deionized water and drained through the column until the level reached the top of the resin bed. This wash was 79 repeated twice, letting the water drain completely only after the last wash. Elution and Regeneration. The reservoir walls were washed with two 10 ml portions of diethyl ether, and each wash was allowed to drain into the XAD resin but not through the column. The column was then capped and the diethyl ether -was allowed to equilibrate with the resin for 10 minutes. Then the cap was removed, the stopcock was opened, and the ether was allowed to flow through the column into a 30 ml screw-top test tube. An additional 5 ml of diethyl ether was added to the column and immediately wasnallowed to flow through the resin into the test tube. The last traces of diethyl ether in the column was eluted with purified air under pressure. The XAD column was regenerated immediately after the ether was eluted. Methanol was added to the column, and the air bubbles were removed by shaking the XAD resin with the methanol. A total of 30 ml of methanol were passed through the column. The stopcock was closed when the methanol reached the top of the resin bed, and the silanized glass wool plug was inserted. An additional 15 ml of methanol was added, and the reservoir was capped with a stopper. The XAD resin column was ready for subsequent analyses without further treatment beyond wetting the resin with deionized water as outlined in the column preparation step. Egyipg. Most of the residual water was removed from the diethyl ether eluate by freezing. After the water was frozen, the diethyl ether eluate was decanted through anhydrous sodium sulfate into a 20 ml graduated glass centrifuge tube. The walls of the screw-top test tube were immediately washed with 1 ml of diethyl ether and the ether was added to the eluate in the centrifuge tube. 80 Concentration of Eluate. In the graduated glass centrifuge tube, the eluate was concentrated by evaporating the diethyl ether with a stream of purified air. The eluate was allowed to concentrate to 1 ml, then the final volume was increased to 2 ml with additional diethyl ether. Separation and Quantification. A 1.0 ul aliquot of each 2.0 ml concentrate was injected into the gas chromatograph with a syringe. When the gas chromatography separation of the Firemaster 680 or 2,4,6- tribromophenol in the concentrates was completed, this process. was immediately repeated with the set of standard ether solutions of Firemaster 680 and 2,4,6-tribromophenol. The concentration range was similar to that expected in the 2.0 ml concentrates, assuming complete recovery of the solute from the water sample. The gas chromatographic conditions were held rigidly constant for both sample and standard during the tests. The chromatogram peaks were integrated by a computer which was interfaced with the gas chromatograph. The peak areas were used to calculate the percentage of the organic solutes that were removed. RESULTS The extraction efficiencies for each resin for Firemaster 680 (FM680) and 2,4,6-tribromophenol (TBP) at each concentration are summarized in Table 11. The experimental data and calculations are presented in Tables 12-17. The extraction efficiency results show that the XAD-2 resin system was most efficient in extracting Firemaster 680 and 2,4,6-tribromophenol at all concentrations tested. The mean recovery for’2,4,6-tribromophenol was slightly greater than 100%, whereas the mean recovery for Firemaster 680 was approximately 50%. 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N.F m.mw m.omm Ap.mvm.nmp 0mm o.P m.om >.Pmp AP.=V:.@oP mmm m.o 5.0» m.mmp Ao.mmvp.:m oom m.o z.m~ o.mmp A=.opvp.m> mup >.o 0.5m m.mor A>._vb.om mm? m.o ..Po m.m: oAm.mV>.Fm mp m.o o.mmp :.mm uAuvm.:m mm _.o nu oz 092 o o.o nAuv «Away Ammg< m>flamHmmv Amgv A4\msv >Lm>oomm omuomficH mma mammamc< ow omuooncH amp cofiumaucmocoo mme ucsoa< Hmsuo< maaamm Lmumz cmxfiqm acsoa< Hmofiamnomne oaaamm Lmumz nmxfiam mmanmocmflofiuum cofiuomnuxm :xm-onso coHuanHHmu vnmvcmum mamsom Hmowuhamc¢ NIQ¢N Aoma owhuwfizm ommzu hzsozc mauuhmmowzh Axum L Azuv _ Axum _ Axum _ any" _ Aum DAUA .umA Azuw Axum n:um Axum nzu¢ .mp shaman (KM—JCO—H>I.IJ CEOZLIJCE LITERATURE CITED LITERATURE CITED Alexander, M. 1965. Biodegradation: Problems of Molecular Recalcitrance and Microbial Infallibility. Adv. Appl. Microbiol.‘1:35-80. Alexander, M. 1973. Nonbiodegradable and Other Recalcitrant Molecules. Biotechnol. Bioeng; 15(3):611-6u7. Alexander, M. 1975. Environmental and Microbiological Problems Arising from Recalcitrant Molecules. Microbial Ecol. g:17-27. Alexander, M. 1977. Introduction 39 Soil Microbiology. John Wiley and Sons, New York. #67 pp. Alexander, M. 1981. Biodegradation of Chemicals of Environmental Concern. Science 211:132—138. Allen, J.R., Lambrecht, L.K., and Barsotti, B.A. 1978. Effects of Polybrominated Biphenyls in Nonhuman Primates. IQ; Amer. Vet. Med. Assoc. Anderson, E.V. 1976. Government Rules: Problems, Opportunities. Chem. Eng; News 5fl(1gl:12-13. Andersson, O. and Blomkvist, G. 1981. Polybrominated Aromatic Pollutants Found in Fish in Sweden. Chemosphere 10(9):1051-1060. Anonymous. 197”. Bromine Outlook Tied to Clean Air Rules. Chem. Eng. News §g:11-12. Anonymous. 1977. Living With the New Regulations: Flame Retardants. Mod. Plast. 54S22:58-60. Anonymous. 1980. Good Growth Forecast for Fire Retardants. Chem. Eng. News 58(H6):26. Aulerich, R.J. and Ringer, R.K. 1979. Toxic Effects of Dietary Polybrominated Biphenyls in Mink. Arch. Environ. Contam. Toxicol. §:fl87- “98. Banerjee, S., Yalkowsky, S.H., and Valvani, 8.0. 1980. Water Solubility and.Octanol/Water'Partition Coefficients of Organics. Limitations of the Solubility-Partition Coefficient Correlation. Environ. Sci. Technol. 13:1227-1229. 90 91 Barth, H.G. 1979. Flame Retardant ABS Resins. Proc. Env. Conf. Flammability Fire Retard., pp. 1-9. Baughman, G.L. and Paris, D.F. 1981. Microbial Bioconcentration of Organic Pollutants from Aquatic Systems--A Critical Review. ggg Crit. Rev. Microbiol. 8:205-228. Beaudoin, A.R. 1977. Teratogenicity of Polybrominated Biphenyls in Rats. Environ. Res. 13:81-86. Boerson, G. 1979a. Soil and water Samples Collected at Adrian WWTP. Michigan Department of Natural Resources Memorandum, March 27, 1979. Boerson, G. 197%. Water Samples and Sludge Samples from the South Branch Raisin River Near Adrian. Michigan Department of Natural Resources Memorandum, April 3, 1979. Briggs, C.G. 1969. Molecular Structure of Herbicides and Their Sorption by Soils. Nature {London} gg3:1288. Brink, R.H. 1981. Biodegradaton of Organic Chemicals in the Environment. In: Environmental Health Chemistry, J.D. McKinney, Ed., pp. 75-100. Ann Arbor Science Publishers, Ann Arbor, MI. Chapman, P.J. 1972. An Outline of Reaction Sequences Used for the Bacterial Degradation of Phenolic Compounds. In: Degradaton 3g Synthetic Organic Molecules _lfl _th_e Bios here, pp. 17-55. National Academy of Sciences, Washington, DC. Chapman, P.J. 1979. Degradation Mechanisms. In: Proceedings of the Worshop: Microbial Degradation of Pollutants in Marine Environments, A.W. Bourquin and P.H. Pritchard, Eds., pp. 28-66. EPA-600/9-79-012. Chiou, C.T., Freed, V.H., Schmedding, D.W., and Kohnert, R.L. 1977. Partition Coefficient and Bioaccumulation of Selected Organic Chemicals. Environ. Sci. Technol. ll:u75-fl78. Corbett, T.H., Beaudoin, A.R., Cornell, R.G., Anver, M.D., Schumacher, R., Endres, J., and Szwabowska, M. 1975. Toxicity of Polybrominated Biphenyls (Firemaster BP-6) in Rodents. Environ. Res. 10:390-396. Dagely, S. 1978. Microbial Catabolism, the Carbon Cycle and Environmental Pollution. Naturwissenschaften §§:85-95. Davies, J .E., Barquet, A., Freed, V.H., Haque, R., Morgade, C., Sonneborn, R.E., and Vaclavek, C. 1975. Human Pesticide Poisonings by a Fat-Soluble Organophosphate Insecticide. Arch. Environ. Health 39:608-613. DeCarlo, V.J. 1979. Studies on Brominated Chemicals in the Environment. DiCarlo, F.J., Seifter, J., and DeCarlo, V.J. 1978. Assessment of the Hazards of Polybrominated Biphenyls. Environ. Health Perspect. g3:351- 365. 92 Durst, H.I., Willett, L.B., Schanbacher, F.L., and Moorehead, P.D. 1978. Effects of PBBs on Cattle. I. Clinical Evaluations and Clinical Chemistry. Environ. Health Perspect. 23:83-9. Falk, H.L., Posner, H.S., Guthrie, J., Jurgelski, W., Bernhein, N.J., Damstra, T., Luster, M., and Vouk, V.B. 1980. The Toxicity of PBBs (BP- 6 or FF-1) in Domestic and Laboratory Animals. Final Report National Institute of Environmental Health Sciences (NIEHS), October 2", 1980. Ficsor, G. and Wertz, G.F. 1976. Polybrominated Biphenyl Nonteratogenic c-Mitosis Synergist in Rat. Mutat. Res. 38:388. Filonow, A.B., Jacobs, L.W., and Mortland, M.M. 1976. Fate of Polybrominated Biphenyls (PBBs) in Soils. Retenton of Hexabromobiphenyl in Four Michigan Soils. J; Agric. Food Chem. gg:1201-120u. Forba, R.W} 1980. Pine River Contamination Survey, St. Louis, Michigan. Final Report, Office of Enforcement, U.S. Environmental Protection Agency. EPA-330/2-80-031. Forba, R.W., Peckham, A.F., and Benson, B.E. 1980. Investigation for Hazardous Waste Contamination at the Velsicol Chemical Corporation Plant Site, St. Louis, Michigan. Final Report, Office of Enforcement, U.S. Environmental Protection Agency. EPA-330/2-80-O32. Freed, V.H., Chiou, C.T., and Haque, R. 1977. Chemodynamics: Transport and Behavior of Chemicals in the Environment--A Problem in Environmental Health. Environ. Health Perspect. 29:55-70. Geyer, H., Viswanathan, R., Freitag,, D.,and Korte, F. 1981. Relationship Between Water Solubility of Organic Chemicals and Their Bioaccumulation by the Alga Chlorella. Chemosphere 19:1301-1307. Gupta, B.N., McConnel, E.E., Harris, M.W., and. Moore, J.A. 1981. Polybrominated Biphenyl Toxicosis in the Rataand Mouse. Toxicol. Appl. Pharmacol. §1:99-118. Gupta, B.N. and Moore, J .A. 1979. Toxicologic Assessment of a Commerical Polybrominated Biphenyl Mixture in the Rat. Amer. g; Vet. Res. 5g:1u58- 1u68. Hansch, C. and Leo, A. 1979. Substituent Constants for Correlation Analysis in Chemistry and Biology. John Wiley and Sons, New York, NY. 339 PP- Haque, R. and Schmedding, D. 1975. A Method of Measuring the Water Solubility of Hydrophobic Chemicals: Solubility of’Five Polychlorinated Biphenyls. Bull. Environ. Contam. Toxicol. 13:13-18. Harris, S.J., Cecil, H.C., and Bitman, J. 1978a. Effects of Feeding a Polybrominated Biphenyl Flame Retardant (Firemaster BP-6) to Male Rats. Bull. Environ. Contam. Toxicol. 12:692-696. 93 Harris, S.J., Cecil, H.C., and Bitman, J. 1978b. Embryotoxic Effects of Polybrominated Biphenyls (PBB) in Rats. Environ. Health Perspect. Hassett, J .P. and Anderson, M.A. 1979. Association of Hydrophobic Organic Compounds with Dissolved Organic Matter in Aquatic Systems. Environ. Sci. Technol. 13:1526-1529. Hassett, J.T., Means, J.C., Banwart, W.L., and Wood, S.G. 1980. Sorption Properties of Sediments and Energy-Related Pollutants. Final Report, U.S. Environmental Protection Agency. EPA-600/3-80-0u1. Heisted, D. 1977. Personal Communication. Hesse, J.L. and Powers, R.A. 1978. Polybrominated Biphenyl (PBB) of'the Pine River, Gratiot and Midland Counties, Michigan. Environ. Health Perspect. 23:19-25. ‘ Horn, W.E. 1978. Flame-Resistant Aromatic Polycarbonate Compositions. Ger. Offen., Patent No. 29211325. Borg-Warner Corp. Hutzinger, 0., Sundstrom, G. and Safe, Sn 1976. Environmental Chemistry of Flame Retardants. Part 1. Introduction and Principles. Chemosphere Iliff, N. 1975. Organic Chemicals in the Environment. Ngw Sci. 53(7812:263-265. Jackson, G. 1979. Curene and Firemaster Sampling from Raisin River Sediments. Michigan Department of Natural Resourcesd Memorandum, July 2, 1979. Jackson, T.F. and Halbert, F.L. 197". A Toxic Syndrome Associated with Feeding of Polybrominated Biphenyls-Contaminated Protein Concentrate to Dairy Cattle. J; Amer. Vet. Med. Assoc. 165:“37-439. Jacobs, L.W., Chou, S.F., and TiedJe, J.M. 1976. Fate of’Polybrominated Biphenyls (PBBs) in Soils. Persistence and Plant Uptake. .J; Agric. Food Chem. 2“(6):1198-1201. Jacobs, L.W., Chou, S.F., and Tiedje, J.M. 1978. Field Concentrations and Persistence of Polybrominated Biphenyls in Soils and Solubility of PBB in Natural Waters. Environ. Health Perspect. 23:1-8. Jamieson, J.W.S. 1977. Polybrominated Biphenyls in the Environment. Economic and Technical Reivew Report, Environmental Protection Service, Environment Canada. EPS-3-EC-77-18. Junk, G.A., Richard, J.J., Brieser, M.D., Witiak, D., Witiak, J.L., Arguello, R., Vick, R., Svec, H.J., Fritz, J.S., and Calder, G.V. 1974. Use of Macroreticular Resins in the Analysis of Water for Trace Organic Contaminants. J; Chromatogr. 22:7fl5-762. 94 Kaiser, K.L.E. and Wong, P.T.S. 197fl~ Bacterial Degradation of Polychlorinated Biphenyls. I. Identification of Some Metabolic Products from AroclorR 12112. Bull. Environ. Contam. Toxicol. 11:291-296. Karickhoff, S.W. 1981. Semi-Empirical Estimation of Sorption of Hydrophobic Pollutants on Natural Sediments and Soils. Chemosphere Karickoff, S.W., Brown, D.S., and Scott, T.A. 1979. Sorption of Hydrophobic Pollutants on Natural Sediments. Water Res. 13:2fl1-2u8. Kay, K. 1977. Polybrominated Biphenyls (PBB) Environmental Contamination in Michigan, 1973-1976. Environ. Res. 12:74-93. Kenaga, E.E. and Goring, G.A.I. 1980. Relationship Between Water Solubility, Soil Sorption, Octanol/Water Partitioning, and Concentration of Chemicals in Biota. In: Amzatic To_xicology, ASTM STP 707, J.G. Eaton, P.R. Parrish, and A.C. Hendricks, Eds., pp. 78-115. American Society for Testing and Materials, Philadelphia, PA. Kimbrough, R.D., Burse, V.W., and Liddle, J.A. 1978. Persistent Liver Lesions in Rats after a Single Oral Dose of Polybrominated Biphenyls (Firemaster FF-1) and Concomitant PBB Tissue Levels. Environ. Health Perspect. 23:265-273. Kimbrough, R.D., Grace, D.F., Korver, M.P., and Burse, V.W. 1981. Induction of'Liver Tumors in Female Sherman Strain Rats by Polybrominated Biphenyls. J1 Natl. Cancer Inst. §§:535-5u2. Kimerle, R.A., Gledhill, W.E., and Levinskas, G.J. 1978. Environmental Safety Assessment of New Materials. In: Estimatigg 1:113 Hazard 915: Chemical Substances 19 Aquatic Life, J. Cairns, Jr., K.L. Dickson, and A.W. Maki, Eds., pp. 132-1u9. American Society for Testing Materials, Philadephia, PA. Kobayashi, H. and Rittmann, B.E. 1982. Microbial Removal of Hazardous Organic Compounds. Environ. Sci. Technol. 16:17OA-183A. Kociba, R.J., Frauson, L.O., Humiston, C.G., Norris,J.M., Wade, C.E., Lisowe, R.W., Quast, J.F., Jersey, G.L., and Jewett, G.L. 1975. Results of a Two Year Dietary Feeding Study with Decabromodiphenyl Oxide (DBDPO) in Rats. ‘11 Fire Flammability/Combust. Toxicol. 2:267-285. Koster, P., Debets, F.M.H., and Strik, J.J.T.W.A. 1980. Porphyrinogenic Action of Fire Retardants. Bull. Environ. Contam. Toxicol. 25:313-315. Leitheiser, R.H., Londrigen, M.E., Akerberg, D.W., and Bozer, K.B. 1978. Fire Retardant Furan Resins. Proc. Env. Conf. Flammability Fire Retard. Lieb, W.R. and Stein, W.D. 1969. Biological Membranes Behave as Non- Porous Polymeric Sheets with Respect to the Diffusion of Non-Electrolytes. Nature 22u:2uo-2u3. 95 Liepins, R. and Pearce, E.M. 1976. Chemistry and Toxicity of Flame Retardants for Plastics. Environ. Health Perspect. 11:55-63. Ligget, L.M. 19511. Determination of Organic Halogen with Sodium Biphenyl Reagent. Anal. Chem. 26:7u8-750. Lu, P.-Y. and Metcalf, R.L. 1975. Environmental Fate and Biodegradability of Benzene Derivatives as Studied in a Model Aquatic Ecosystem. Environ. Health Perspect. 10:269-28u. Lu, P.-Y., Metcalf, R.L., and Carlson, E.M. 1978. Environmental Fate of Five Radiolabeled Coal Conversion By-Products Evaluated in a Laboratory Model Ecosystem. Environ. Health Perspect. 25:201-208. Mackay, D.., Mascarenhas, R., and Shiu, W.Y. 1980a. Aqueous Solubility of Polybrominated Biphenyls. Chemosphere 2:257-264. MacKay, D., Bobra, A., and Shiu, W.Y. 1980b. Relationships Between Aqueous Solubility and Octanol-Water Partition Coefficients. Chemosphere Marshall, K.C., Whiteside, J.S., and Alexander, M. 1960. Problems in the Use of Agar for Enumeratmn of Microorganisms. Soil Sci. Soc. Am; Proc. a:61-620 Matsuda, K. and Schnitzer, M. 1977. Aqueous Solubility of Polynuclear Aromatic Hydrocarbons. 4; Chem. Eng. Data 22:399-H02. Matsuo, M. 1980. The i/o-Characters to Relate Accumulation Factors of Some Halogenated Biphenyls in Fish. Chemosphere 9(1):61-65. Mattsson, P., Norstrom, A., and Rappe, C. 1975. Identificatin of the Flame Retardant Pentabromotoluene in Sewage Sludge. _J_._ Chromatog. 111:209-213. Maugh, T.H. 1978. Chemicals: How Many Are There? Science 199:162. May, W.E., Wasik, S.P., and Freeman, D.H. Determination of the Aqueous Solubility of Polynuclear Aromatic Hydrocarbons by a Coupled Column Liquid Chromatographic Technique. Anal. Chem. 59:175-179. McCormack, K.M., Kluwe, W.M., Sanger, V.L., and Hook, J.B. 1978. Effects of Polybrominated Biphenyls on Kidney Function and Activity of Renal Microsomal Enzymes. Environ. Health Perspect. 23:153-157. McDuffie, B. 1981. Estimation of Octanol/Water Partition Coefficients for Organic Pollultants Using Reverse-Phase HPLC. Chemosphere 19:73-83. Meikle, R.W. 1972. Decomposition: Qualitative Relationships. In: Organic Chemicals in the Soil Environment, Vol. 1, C.A.I. Goring and J.W. Hamaker, Eds. Marcel Dekker, New York. Mercer, H.D., Teske, R.H., Condon, R.J., Furr, A., Meerdink, 0., Buck, W., and Fries, G. 1976. Herd Health Status of Animals Exposed to Polybrominated Biphenyls (PBB). J. Toxicol. Environ. Health 2:335-3fl9. 96 Liepins, R. and Pearce, E.M. 1976. Chemistry and Toxicity of Flame Retardants for Plastics. Environ. Health Perspect. 11:55-63. Ligget, L.M. 19511. Determination of Organic Halogen with Sodium Biphenyl Reagent. Anal. Chem. 26:7u8-750. Lu, P.-Y. and Metcalf, R.L. 1975. Environmental Fate and Biodegradability of Benzene Derivatives as Studied in a Model Aquatic Ecosystem. Environ. Health Perspect. 19:269-28u. Lu, P.-Y., Metcalf, R.L., and Carlson, E.M. 1978. Environmental Fate of Five Radiolabeled Coal Conversion By-Products Evaluated in a Laboratory Model Ecosystem. Environ. Health Perspect. 25:201-208. Mackay, D.., Mascarenhas, R., and Shiu, W.Y. 1980a. Aqueous Solubility of Polybrominated Biphenyls. Chemosphere 9:257-2611. MacKay, D., Bobra, A., and Shiu, W.Y. 1980b. Relationships Between Aqueous Solubility and Octanol-Water Partition Coefficients. Chemosphere I2:7O1=711. Marshall, K.C., Whiteside, J.S., and Alexander, M. 1960. Problems in the Use of Agar for Enumeraton of Microorganisms. Soil Sci. Soc. Am; Proc. 23:61-62. Matsuda, K. and Schnitzer, M. 1977. Aqueous Solubility of Polynuclear Aromatic Hydrocarbons. J1 Chem. Eng. Data gg:399-uoz. Matsuo, M. 1980. The i/o-Characters to Relate Accumulation Factors of Some Halogenated Biphenyls in Fish. Chemosphere 9(1):61-65. Mattsson, P., Norstrom, A., and Rappe, C. 1975. Identificatin of the Flame Retardant Pentabromotoluene in Sewage Sludge. _J_. Chromatog. 111 :209-213. Maugh, T.H. 1978. Chemicals: How Many Are There? Science 199:162. May, W.E., Wasik, S.P., and Freeman, D.H. Determination of the Aqueous Solubility of Polynuclear Aromatic Hydrocarbons by a Coupled Column Liquid Chromatographic Technique. Anal. Chem. 29: 175-179. McCormack, K.M., Kluwe, W.M., Sanger, V.L., and Hook, J.B. 1978. Effects of Polybrominated Biphenyls on Kidney Function and Activity of Renal Microsomal Enzymes. Environ. Health Perspect. 23:153-157. McDuffie, B. 1981. Estimation of Octanol/Water Partition Coefficients for Organic Pollultants Using Reverse-Phase HPLC. Chemosphere 19:73-83. Meikle, R.W. 1972. Decomposition: Qualitative Relationships. In: Organic Chemicals _ig the Soil Environment, Vol. 1, C.A.I. Goring and J .W. Hamaker, Eds. Marcel Dekker, New York. Mercer, H.D., Teske, R.H., Condon, R.J., Furr, A., Meerdink, G., Buck, W., and Fries, G. 1976. Herd Health Status of Animals Exposed to Polybrominated Biphenyls (PBB). L Toxicol. Environ. Health 2:335-3119. 97 Metcalf, R.L., Kapoor, I.P., Lu, P.-Y., Schuth, C.K., and Sherman, P. 1973. Model Ecosystem Studies of the Environmental Fate of Six Organochlorine Pesticides. Environ. Health Perspect. 5:35-uu. Metcalf, R.L., Sanborn, J.R., Lu, P.-Y., and Nye, D. 1975. Laboratory Medel Ecosystem.Studies of the Degradation and Fate of Radiolabeled Tri-, Tetra-, and Perta-Chlorobiphenyl Compared with DDE. Arch. Environ. Contam. Toxicol. 3:151-165. Michigan Chemical Corporation. 1975. Firemaster 680 Total Bromide Determination, Analytical Method Number u60-B. Michigan Chemical Corporation, St. Louis, MI. Michigan Chemical Corporation. 1976. Firemaster 680. Product Bulletin No. FHOHS. Michigan Chemical Corporation, Chicago, IL. Moorhead, P.D., Willett, L.B., Brumm, C.J., and Mercer, H.D. 1977. Pathology of Experimentally Induced Polybrominated Biphenyls Toxicosis in Pregnant Heifers. J1_Amer. Vet. Med. Assoc. 170:307-313. Nametz, R.C. and Moore, P.O. 1978. Flame Retardant Polystyrene Plastic Compositions. Brit. 115 Pat. Appl. Patent No. 20251427. Velsicol Chemical Corporation. Neely, W.B. 1979. Estimating Rate Constants for the Uptake and Clearance of Chemicals by Fish. Environ. Sci. Tech. 13:1506-1510. Neely, W.B., Branson, D.H., and Blau, G.E. 197". Partition Coefficient to Measure Bioconcentration Potential of Organic Chemicals in Fish. Environ. Sci. Tecnol. 8:1113-1115. Norris, J.M., Kociba, R.J., Schwetz, B.A., Rose, J.Q., Humiston, C.G., and Gehring, P.J. 1973a. Toxicological Evaluation of Fire Retardant Chemicals: Decarbromodiphenyl Oxide and Octabromobiphenyl. Pharma- colggist 15S22:226. Norris, J.M., Ehrmantraut, J.W., Gibbons, G.L., Kociba, R.J., Schwetz, B.A., Rose, J.Q., Humiston, C.G., Jewett, G.L., Crummet, W.B., Gehring, P.J. , Tirsell, J.B., and Brosier, J.S. 1973b. Toxicological and Environmental Factors Involved in the Selection of Decabromodiphenyl Oxide as a Fire Retardant Chemical. Appl. Polym, Sci. Symp. 22:195-219. Norris, J.M., Ehrmantraut, J.W., Gibbons, G.L., Kociba, R.J., Schwetz, B.A., Rose, J.Q., Humiston, C.G.,Jewett, G.L., Crummett, W.B., Gehring, P.J., Tirsell, J.B., and Brosier, J.S. 1974. Toxicological and Environmental Factors Involved in the Selection of Decabromodiphenl Oxide as a Fire-Retardant Chemical. 1; Fire FlammabilityZCombust. Toxicol. 1:52-77. Norris, J.M., Kociba, R.J., Schwetz, B.A., Rose, J.Q., Humiston, C.G., Jewett, G.L., Gehring, P.J., and Mailhes, J.B. 1975. Toxicology of Octabromobiphenyl and Decabromodiphenyl Oxide . Environ . Healfl Perspect . 98 OECD. 1981. OECD Guidelines for Testing 9_f_ Chemicals. Organization for Economic Cooperation and Development, Paris, France. Orlando, C.M. and Thomas, D.P. 1975. A New Aromatic Brominated Flame Retardant and Its Application in High Impact Polystyrene. ,1; Fire Flammability/Fire Retard. Chem; 2:183-185. Pellizzari, E.D., Zweidinger, R.A., and Erickson, M.D. 1978. Environmental Monitoring Data Near Industrial Sites: Brominated Chemicals. Part I and II. Final Report, Office of Toxic Substances, U.S. Environmental Protection Agency. EPA-560/6-78-002. Powers, R.A. 1976. Status of Polybrominated Biphenyls (PBB) Contamination of the Pine River, Gratiot and Midland Counties, Michigan, August and September, 1976. Final Report, Toxic Materials Section, Michigan Department of Natural Resources. Preache, M.M., Cagan, 8.2., and Gibson, J.E. 1976. Perinatal Toxicity in Mice Following Maternal Dietary Exposure to Polybrominated Biphenyls. Toxicol. Appl. Pharmecol. 31:171. Rall, D.P., Moore, J.A., and Huff, J.E. 1980. National Toxicology Program. NTP Technical Bulletin 1§32:9-11. Rodina, A.G. 1972. Methods 1g Aquatic Microbiology. University Park Press, Baltimore, MD. ”61 pp. Ruzo, L.O., Sundstrom, G., Hutzinger, 0., and Safe, S. 1976. Photodegradation of'Polybrominated Biphenyls (PBB). 'g1nggric. Food Chem; Ruzo, L.O. and Zabik, M.J. 1975. Polyhalogenated Biphenyls: Photolysis of Hexabromo and Hexachlorobiphenyls in Methanol Solution. Bull. Environ. Contam. Toxicol. 13:181-182. Sanders, H.J. 1978. Flame Retardants. Chem. Eng. News 39:22-36. Smith, J.B., Mabey, W.B., Bohonos, N., Holt, B.R., Lee, S.S., Chou, T.- W., Bomberger, D.C., and M111, T. ‘1977. Environmental Pathways of Selected Chemicals in Freshwater Systems. Part I: Background and Experimental Procedures. Environmental Research Laboratory, Athens, GA. EPA-600/7-77-113. Smith, R.E. and Shukla, G.J. 1978. Flame-Retarded Nonwoven Textile Material. Eur. Pat. Appl. U.S. Patent No. 821111. Velsicol Chemical Corp. Southworth, G.R., Beauchamp, J.J., and Schmieder, P.K. 1978. Bioaccumulation Potential of Polycyclic Aromatic Hydrocarbons in Daphnia pulex. Water Res. 12:973-977. Stern, A.M. and Walker, C.R. 1978. Hazard Assessment of Toxic Substances: Environmental Fate Testing of Organic Chemicals and Ecological Effects Testing. In: Estimating the Hazard 91 Chemical Substances £9 Aquatic Life, J. Cairns, Jr., K.L. Dickson, and A.W. Maki, Eds., pp. 81-131. American Society for Testing Materials, Philadelphia, PA. 99 Sugiura, K., Ito, N., Matsumoto, N., Youzou, M., Murata,K., Tsukakoshi, Y. , and Goto, M. 1978. Accumulation of Polychlorinated and Polybrominated Biphenyls in Fish: Limitation of "Correlation Between Partition Coefficients and Accumulation Factors". Chemosphere 7(92:731-736. Tulp, M. Th. M., and Hutzinger, O. 1978. Some Thoughts on Aqueous Solubilities and Partition Coefficients of PCB, and the Mathematical Correlation Between Bioaccumulation and Physico-Chemical Properties. Chemosphere‘1:8fl9-860. U.S. Environmental Protection Agency. 1980a. Toxic Substances Control Act Chemical Substances Inventory, Cumulaftive Supplemen . Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency. 1980b. Chemical Screening: Initial Evaluations of Substantial Risk Notices, Section 8(e). TSCA Chemical Assessment Series. EPA-560/11-80-008, pp. 29u-299. U.S. Environmental Protection Agency. 1980c. Proposed Environmental Standards; and Proposed Good Laboratory Practice Standards for Physical, Chemicals, Persistence, and Ecological Effect Testing. Fed. Regist. A55227):77332-77365, Friday, November 21, 1980. U.S. Environmental Protection Agency. 1980d. Proposed Guidelines for Registering Pesticides in the United States. Subpart N Chemistry Requirements: Environmental Fate. NOCFR, Part 163. U.S. Environmental Protection Agency. 1981. Agency Policy to Premanufacture Testing of New Chemical Substances and Announcement of Rescheduled Meeting and Extension of Coment on Certain Environmental Test Standards. Fed. Regist. l16(17l:8986-8993, Tuesday, January 27, 1981. Veith, G.D. 1982. State-of-the-Art of Structure Activity Methods Development. Annual Project Sumary, Environmental Research Laboratory, U.S. Environmental Protection Agency, Duluth, MN. EPA-600/S3-81-029. Veith, G.D., Defoe, D.L., and Bergstedt, B.V. 1979. Measuring and Estimating the Bioconcentration Factor of Chemicals in Fish. g_. Fish. Res. Board Can. 36:10110-10118. Veith, G.D., Macek, K.J., Petrocelli, S.R., and Carroll, J. 1980. An Evaluation of Using Partition Coefficients and Water Solubility to Estimate Bioconcentration Factors for Organic Chemicals in Fish. In: Acmatic Toxicology, ASTM SP 707, J.G. Eaton, P.R. Parrish, and A.C. Hendricks, Eds., pp. 116-129. American Society for Testing Materials, Philadelphia, PA. Velsicol Chemical Corporation. .1977. Advertisement, "Announcing a New Low Cost Flame Retardant Firemaster 680." Mod. Plast. §h§92:17. Wastell, M.E., Moody, D.L., and Plog, Jr., J.F. 1978. Effects of Polybrominated Biphenyl on Milk Production, Reproduction, and Health Problems in Holstein Cows. Environ. Health Perspect. 23:99-103. 100 Weil, V.L., Dure, G., and Quentin, K.-E. 197A. Wasserlgslichkeit von Insektiziden .Chlorierten Kohlenwasserstoffen und Polychlorierten Biphenylen im Hinblick auf eine Gewgsserbelastung mit Diesen Stoffen. Wasser Egg Abwasser Forschu g 1:169-175. Wershaw, R.L., Burcar, P.J., and Goldberg, M.C. 1969. Interaction of Pesticides with Natural Organic Material. Environ. Sci. Technol. 3:271- 273. Wertz, G.F. and Ficsor, G. 1978. Cytogenetic and Teratogenic Test of Polybrominated Biphenyls in Rodents. Environ. Health Perspect. 23:129- 132. Zitko, V. 1977. The Accumulation of Polybrominated Biphenyls in Fish. Bull. Environ. Contam. Toxicol. 11:285-292. Zitko, V. 1979. The Fate of Highly Brominated Aromatic Hydrocarbons in Fish. In: Pesticide and zgnobiqtic Metabol;§m 1g Aquatic Organisms, M.A. Khan, J.J. Lech, and J.J. Menn, Eds., pp. 177-182. ACS Symposium Series, No. 99. American Chemical Society. Zitko, V. and Carson, W.G. 1977. Uptake and Excreton of Chlorinated Diphenyl Ethers and Brominated Toluenes by Fish. Chemosphere:293-301. Zitko, V. and Hutzinger, O. 1976. Uptake of Chloro- and Bromobiphenyls, Hexachloro- and Hexabromobenzene by Fish. Bull. Env. Contam. Toxicol.