ABSTRACT
HEIDI L. VERMEULEN. The Synthesis of Deuterated Bromodichloromethane, Deuterated Chlorodibromomethane, and Mixed Halogenated Acetic Acids. (Under The Direction of Dr. Avram Gold)
The purpose of this research project was to develop procedures for the preparation of isotopically-labeled, mixed halogenated disinfection by-products. Deuterated bromodichloromethane (deuterated BDCM), was synthesized using an aluminum catalyzed reaction. Equal volumes of deuterated chloroform (25 mL) and bromoethane (25 mL) were mixed in a 100 mL round bottom flask. Enough AICI3 was added to the flask such that the reaction mixture turned dark red in color. The reaction was allowed to stir for 46 hours. The product was purified through fractional distillation, in which three fractions were collected. Through GC-MS analysis, the purity of fraction 2 (65 - 72 °C) was calculated to be 93 % and that of fraction 3 (73 - 78 °C) was 85%. For both fractions, BDCM was 99% deuterium enriched. "C NMR and GC-MS analyses indicated that the impurity in both of fractions 2 and 3 was deuterated chlorodibromomethane. This indicated that AICI3 was reacting with the generated deuterated BDCM to yield deuterated chlorodibromomethane. Therefore, by letting this same reaction stir for 7 days it was possible to obtain a significant amount (approximately 5 g) of deuterated chlorodibromomethane. Bromochloro-, chlorodibromo-, and bromodichloroacetic acid were all obtained through a literature synthesis (Zimmer et al., 1990). A new experimental procedure was developed for the synthesis of bromodichloroacetic acid (CBrCl2C00H). Allowing bromodichloromethane to react with 1.6 M n-butyllithium in hexanes, yielded the bromodichloromethide anion. The anion was carboxylated by the addition of dry ice to yield CBrCl2C02"Li"^. The lithium salt was added to water and acidified with concentrated hydrochloric acid. CBrCl2COOH was extracted from solution with methylene chloride. Having demonstrated the feasibility of this reaction, attempts were made to develop a procedure based on Ba^'^COs as a source of CO2. Pilot studies involving BaC03/H2S04 as the CO2 source proved unsuccessful. While using unrealistic quantities of BaCOj (5 g), only trace quantities of bromodichloroacetic acid were obtained. Table of Contents
Page
List of Figures...... i
List of Tables...... ii
Acknowledgements...... iii
L Literature Review...... 1 A. Disinfection...... 1 B. Bromodichloromethane (BDCM)...... 4 1. Chemical and Physical Properties...... 4 2. Occurrence and Prevalence...... 5 3. Metabolism...... 7 4. Toxicity...... 9 a. Hematological Effects...... 10 b. Hepatic Effects...... 10 c. Renal Effects...... 11 5. Carcinogenicity...... 12 C. Halogenated Acetic Acids...... 13 1. Chemical and Physical Properties...... 13 2. Occurrence and Prevalence...... 14 3. Metabolism...... 16 4. Toxicity...... 19 5. Carcinogenicity...... 22
n. Introduction...... 26 in. Experimental Materials and Methods...... 29 A. Instrumentation and Laboratory Materials...... 29 B. Synthesis of Mixed Halogenated Acetic Acids...... 30 1. Bromochloroacetic Acid...... 30 2. Chlorodibromoacetic Acid...... 31 3. Bromodichloroacetic Acid...... 32 a. Literature Synthesis...... 32 b. Synthesis through Lithium Salt Formation...... 33 C. Synthesis of Deuterated Bromodichloromethane...... 34 D. Synthesis of Deuterated Chlorodibromomethane...... 36 IV. Results and Discussion ...... 37 A. Mixed Halogenated Acetic Acids...... 37 B. Deuterated Bromodichloromethane...... 39 1. Lithium Salt Formation...... 39 2. Bromination of Dichloromethide Anion...... 40 Page
3. Hydrogenolysis of Dibromodichloromethane...... 41 4. Aluminum Chloride Catalyzed Reaction...... 42 5. Redistribution of Halogens in a Mixture of Chloroform and Bromoform Under Alkaline Conditions...... 45
V. Conclusions...... 48
VI. References...... 51 List of Figures Figure Page 1 Chemical Structures of THMs...... 58 2 Carbon Centered Oxidation Metabolic Pathway for THMs...... 59 3 Halogen Centered Oxidation Metabolic Pathway for THMs..... 60 4 Reductive Metabolic Pathway for THMs...... 61 5 Reaction Scheme for CHClBrCOOH...... 62 6 Mass Spectrum of CHClBrCOOH...... 63 7 Reaction Scheme for CClBr2C00H...... 64 8 Mass Spectrum of CClBrzCOOH...... 65 9 Reaction Scheme for CClaBrCOOH...... 66 10 Mass Spectrum of CClzBrCOOH...... 67 11 Reaction Scheme for CCl2BrC00H via Lithium Salt Formation...... 68 12 Mass Spectrum of CCl2BrCOOH from Li Salt Reaction...... 69 13 Proposed Reaction Scheme for ^^C-labeled CCl2BrC00H...... 70 14 Reaction Scheme for Deuterated BDCM (AICI3 Reaction)...... 71 15 *^C NMR of Deuterated BDCM Fraction 2...... 72 16 '^C NMR of Deuterated BDCM Fraction 3...... 74 17 GC-MS of Deuterated BDCM Fraction 2...... 76 18 GC-MS of Deuterated BDCM Fraction 3...... 77 19 Reaction Scheme for Deuterated Chlorodibromomethane (CDBM)...... 78 20 "C NMR of Deuterated BDCM Fraction 2 (7 Day AICI3 Catalyzed Reaction)...... 79 21 "C NMR of Deuterated BDCM Fraction 3 (7 Day AICI3 Catalyzed Reaction)...... 81 22 "C NMR of Deuterated BDCM and CDBM Fraction 4 (7 Day AICI3 Catalyzed Reaction)...... 83 23 "C NMR of Deuterated BDCM and CDBM Fraction 5 (7 Day AICI3 Catalyzed Reaction)...... 85 24 Proposed Reaction Scheme for Deuterated BDCM Via Lithium Salt Formation...... 87 25 "C NMR of BDCM From Lithium Salt Reaction...... 88 26 Mass Spectrum of Deuterated BDCM From Lithium Salt Reaction...... 90 27 Proposed Reaction Scheme for Deuterated BDCM Via Bromination of Dichloromethide Anion...... 91 28 Proposed Reaction Scheme for Deuterated BDCM Via Lithium Aluminum Deuteride...... 92 29 Generation of Dichlorocarbenes Under Catalytic Two-Phase Conditions...... 93 30 Reaction Scheme for BDCM Via Mixing Chloroform and Bromoform Under Alkaline Conditions...... 94 List of Tables
Table Page
1 Haloacids Identified as Drinking Water Chlorination By-Products in a Ten-City Survey...... 95 2 Quarterly Median Values for Haloacetic Acids in U.S. Drinking Waters...... 96 3 Cancer Incidence in Mice Exposed to Chloroacetic Acids in Drinking Water...... 97 4 Melting Points of Mixed Halogenated Acetic Acids...... 98 5 Mass Spectral Data For Mixed Halogenated Acetic Acids and Deuterated BDCM...... 99 6 '^C NMR Chemical Shifts...... 100
11 ACKNOWLEDGEMENTS
I would like to sincerely thank Dr. Gold for all of his guidance and support throughout this entire project. He has been an excellent research and academic advisor, and has made my educational experience here at the University of North Carolina a pleasant one. I would like to give special thanks to Dr. Sangaiah. He has been a true inspiration for the past two years. I am indebted to him for his vast knowledge in organic chemistry. I am also very thankful to have shared a laboratory with two very fine scientists, Dr. Jayaraj and soon to be Dr. Rachel Austin. Their insights and support throughout this project have been greatly appreciated. I would also like to thank Dr. Ball and Dr. Christman for being on my research committee. I am very thankful for all of their help and guidance both in my research and in writing my masters technical report. In closing, I would like to express my sincere gratitude to Dr. McKinney and the Environmental Protection Agency (EPA) for their support throughout this project (Grant # CR818552). I am very appreciative for the funding that they have provided for this research project and for my education here at UNC.
lu I. LITERATURE REVIEW
A. DISINFECTION
Disinfection of drinking water was instituted at a time when infectious diseases were the primary focus in public health. From the earliest times, it was clear that water was an important route of transmission for these diseases (Akin et al., 1982). Since 1904, chlorine has been in continuous use as a water disinfectant in the United States and thus has played a vital role in controlling waterbome infectious diseases. It is important to realize that the use of chlorine as a disinfectant began prior to the development of concerns about possible effects of chronic exposure to chlorine and disinfection by-products. Due to the availability of antibiotics to treat water borne diseases, the microbiological hazards of water have faded from the public mind (Bull and Kopfler, 1991). Because of the shifts in the social and political perceptions of risk, toxicological issues concerning disinfection of drinking water have been given greater prominence. The public does need to be reminded however, that the hazards that could arise from the abolition of disinfection would far outweigh any presently proven benefits from reduced toxicological hazards. Nevertheless, it is important to recognize that there are toxicological risks associated with drinking water disinfection. Since chlorine is the most widely used disinfectant, most research to date has evolved around chlorinated disinfection by-
1 products, specifically trihalomethanes (THMs). Chlorine dissolves rapidly in water and establishes an equilibrium with hypochlorous acid (HOCl) and hydrochloric acid (HCl).
CI2 + H2O < = = = = > HOCl + H+ + CI- (pKa = 9.5) (1)
In dilute solutions and at pH levels above 4.0, the equilibrium, as shown in Reaction (1), is displaced to the right and very little molecular chlorine exists in solution. Between pH 6.0 and 8.5, hypochlorous acid dissociates almost completely to form the hypochlorite ion (OCl) (lARC Monographs, 1991).
HOCl <= = = = > OCl- + H+ (2)
At pH levels above 9.0, hypochlorite ions are the dominant species. The total concentration of molecular chlorine, hypochlorous acid and hypochlorite ion is defined as "free available chlorine". Total available chlorine may be defined as the mass equivalent of chlorine contained in all chemical species that contain chlorine in an oxidized state. Combined available chlorine can be defined as the difference between total available chlorine and free available chlorine and represents the amount of chlorine that is in chemical association with various compounds, but that is also capable of disinfecting. Free chlorine species are generally more effective disinfectants than combined chlorine species. An important reaction that often occurs in the chlorination of water that contains bromide ion, is the formation of hypobromous acid from bromide.
HOCl + Br —> HOBr + CI' (3)
Even at low bromide concentrations. Reaction (3) leads to readily detectable levels of brominated organic by-products, (ie. brominated trihalomethanes), due to the reactivity of hypobromous acid. Bromide concentrations in untreated water vary widely across the United States. For example, in nine rivers in various regions of the United States, bromide levels ranged from 10 to 245 ug/L (Amy et ah, 1985). Addition of chlorine to waters containing dissolved organic compounds can result in three possible reactions: (1) addition (2) ionic substitution and (3) oxidation. All of these reactions result in an increase in the oxidation state of the substrate (Pierce, 1978). The amount of organic matter in untreated water varies considerably. Normally, high quality groundwater contains up to 1 mg/L (as organic carbon), river water contains 1-10 mg/L (as organic carbon), and upland water may contain up to 20 mg/L (as organic carbon) which is almost entirely of natural origin (humic substances). The total organic matter present would be roughly double these concentrations (lARC Monographs, 1991). B. BROMODICHLOROMETHANE (BDCM)
The four most commonly studied trihalomethanes (THMs) are: chloroform (CHCI3), bromodichloromethane (CHBrCy, chlorodibromomethane (CHBrjCl), and bromoform (CHBrj). A chemical structure is given for each of these in Figure 1. All of these chemical compounds are known to be chlorination disinfection by¬ products. Of these four, bromodichloromethane (BDCM) will be discussed in detail.
1. CHEMICAL AND PHYSICAL PROPERTIES
Bromodichloromethane (BDCM) is a colorless, heavy, nonbumable liquid (Clement Assoc, 1989). It has a density of 1.980 g/mL at 20.4 °C, a boiling point of 90.1 °C, a melting point of -57.1 °C, and a molecular weight of 163.83 g/mol, BDCM is slightly soluble in water (4.5 g/L at 20 °C) (Mabey et al, 1982), and soluble in acetone, ethanol, benzene, chloroform and diethyl ether (Weast, 1989). It has a vapor pressure of 50 mm Hg at 20 °C and its hydrolysis rate at neutral pH and 25 °C is 5.76x10-* per hour (Mabey er fl/., 1982). 2. OCCURRENCE AlSfD PREVALENCE
Most of the BDCM in the environment is formed as a by-product when chlorine is added to drinking water to kill disease-causing organisms. It has been found in concentrations ranging from 1.9 to 183 ug/L (Bull and Kopfler, 1991). In 1980, the EPA estimated that over 800 kkg (Ikkg = 1 metric ton) of BDCM was produced annually through the process of chlorination of drinking water (Clement Assoc, 1989). Studies have shown that ozonation prior to chlorination can enhance formation of BDCM if bromide is present in water (Krasner et al., 1989). Small amounts of BDCM are also made in chemical plants for use in chemical laboratories or as intermediates in making other chemical products. A very small amount (approximately less than 1 % ) is formed by algae in the ocean (Clement Associates,
1989). In the environment, BDCM usually is found as vapor in air or dissolved in water. Because of its relatively high vapor pressure, BDCM evaporates quite easily, so most BDCM that escapes into the environment from chemical facilities, waste sites, or drinking water enters the atmosphere as a gas. BDCM is slowly broken down (approximately 90% a year) in the air via oxidative pathways, with a half-life of about two to three months. Any BDCM that does remain in the water or soil can be broken down slowly by bacteria. BDCM does not adsorb strongly to soils or sediments, and it tends not to bioaccumulate in fish or other animals (Clement Assoc, 1989). The hydrolysis of BDCM in water is very slow, with an estimated rate constant at neutral pH of 5.76x10"* per hour. This corresponds to a half-life of more than 1,000 years. The concentration of BDCM in chlorinated water depends on a number of factors, which include: temperature, pH, bromide ion concentration in the source water, fulvic and humic substance concentration in the water, and the chlorination treatment practices. Bellar et al. (1974) has shown that the amount of BDCM tends to increase as a function of increasing organic content and bromide ion in the source water, BDCM has also been detected in chlorinated swimming pools, where the total
THM concentrations averaged from 120 to 660 ug/L (Beech et al., 1980). In freshwater pools, most of the total THM was chloroform, with BDCM levels ranging from 13 to 34 ug/L. For saltwater pools, bromoform was the principal THM present, and the BDCM concentrations were approximately the same as those BDCM concentrations found in freshwater pools.
Bromodichloromethane is known not to be a common contaminant of food. It occurs only in trace amounts in some samples. Entz et al. performed a market basket study in 1982. Of 39 items tested, BDCM was detected in one dairy sample at 1.2 ppb and in butter at 7 ppb. A study of BDCM in food processing water and processed foods indicated no detectable levels except in ice cream at one processing plant (0.6 to 2.3 ppt) (Uhler and Diachenko, 1987). Soft drinks have also been found to contain BDCM, usually at concentrations of 0.1 to 6 ug/L (Abdel-Rahman, 1982; Entz et al., 1982). It should be noted that cooking foods in water containing BDCM is unlikely to lead to contamination, since BDCM would rapidly volatilize. 3. METABOLISM
BDCM is believed to be metabolized by at least three pathways. These include, microsomal oxidation, microsomal reduction, and conjugation with glutathione (GSH). Most studies to date have centered around analyzing the oxidative metabolism of BDCM. The primary site of oxidation is carbon, by insertion of oxygen in the C-H bond, to give a trihalogenated methanol (CCl2Br-0H) intermediate which spontaneously loses HBr to yield phosgene (COCy (see Figure 2). Studies are currently being conducted to determine the importance of the halogen-centered oxidation of BDCM (see Figure 3).
In studies where oral doses of BDCM have been administered, it has been shown that BDCM was metabolized to CO2 in rats and mice (Mink et al., 1986) and to CO and CO2 in rats (Anders et al, 1978; Mathews et al, 1990).
One of the most recent BDCM metabolism studies was done by Mathews and colleagues (1990). In this study, it was determined that after administering '"^C- labeled BDCM to male Fischer rats, approximately 80-90% of the BDCM was metabolized within 24 hours of dosing, with 70-80% of the administered dose appearing as ^''COj and 3-5% as "CO. Urinary and fecal elimination were considerably less, accounting for only 4-5% and 1-3% of the dose, respectively. The results indicated that there was saturation of metabolism at a dose level of 100 mg/kg, which was indicated by the lower production of CO2. CO2 production dropped to 33% of the total dose administered. This is in comparison to 62% and 66% CO2 production at the 1 and 10 mg/kg doses, respectively. The persistence of BDCM in tissues was relatively low (3-4% of dose). Most of the accumulation (1-3% of dose) was in the liver. The kidney, a known target for BDCM, was the next highest in concentration of '*C-label, particularly the cortical region, which exhibited tissue/blood ratios of 5-8. Like the liver, the cortical region of the kidney contains cytochrome P-450, which is an enzyme that aides in the production of phosgene. As mentioned previously, phosgene is a known intermediate in the metabolism of THM's. This explains why there may be higher concentrations of radiolabeled chemicals in the tissues of these two organs. At a dose of 100 mg/kg/d for 10 days, BDCM was extensively metabolized to CO2. The initial rate of CO2 production from daily doses however, doubled after day 1, and stayed at that rate for the remainder of the 10 day experiment. This suggests that BDCM induces its own metabolism, however no increase in liver weight or toxicity was observed.
Mink et al. (1986) did a study to determine the absorption, distribution and excretion characteristics of four THM's (chloroform, bromodichloromethane, chlorodibromomethane, and bromoform) using '"C-labeled compounds in both mice and rats. The study indicated that there were differences in the distribution and elimination with respect to intercompound/interspecies comparisons. For instance, the mice eliminated 40-81% of the total ^'^C-labeled THM as '*C02 and 5-26% as the unmetabolized parent compound. The rats, on the other hand, eliminated 4-18% of the total '"^C-labeled THM dose as ''*C02, and 41-67% as the unmetabolized parent
8 THM. The data in Mink's study (1986) indicate that BDCM exhibits limited metabolic activation, due to recovery of a high percentage of the dose as the parent compound.
4. TOXICITY
Bromodichloromethane has been shown to produce liver and kidney damage in both mice and rats (Chu et al, 1982; Munson et al, 1982). The effects of BDCM obviously depend on how much is taken into the body. High levels of BDCM can also cause effects in the brain, leading to incoordination and sleepiness. There is also some evidence that suggests that BDCM may be toxic to developing fetuses.
However, this suggestion has not been well studied.
Most estimates of acute oral LD50 values for BDCM in rodents range between 400 and 1000 mg/kg (Chu et al, 1980; Aida et al, 1987). The pathological changes that were observed in the acutely poisoned animals included: fatty infiltration of liver and hemorrhagic lesions in the kidney, adrenals, lung and brain (Bowman et al, 1978).
In a 14 day repeated-dose study in mice, all animals dosed with 150 mg/kg/day died
(NTP, 1987). Males appeared to be slightly more susceptible to the lethal effects of
BDCM compared to females in both mice (Bowman et al, 1978) and rats (Aida et
al, 1987; Chnetal, 1980). a. Hematological Effects Chu et al. (1982) showed that hemoglobin and hematocrit were significantly reduced in male rats following a single dose of 390 mg/kg of BDCM, Exposure in drinking water for 90 days to a dose of 213 mg/kg/day caused no effect on lymphocyte levels in either males or females. Rats fed BDCM in their diets at intake levels of 130 mg/kg/day for 24 months exhibited no hematological changes compared to controls (Tobe et al, 1982).
b. Hepatic Effects Many animal studies have indicated that the liver is susceptible to injury by BDCM. The typical signs include: increased liver weight, pale discoloration, increased levels of hepatic tissue enzymes in serum, decreased levels of secreted hepatic proteins (fibrinogen) in blood, and focal areas of inflammation or degeneration. These effects have been observed at doses of around 1,250 mg/kg and higher in acute (single dose) studies (NTP, 1987). It should also be mentioned that this dose level causes death within two weeks (NTP, 1987),
In subchronic studies (10 to 14 days) in both mice and rats, mild effects on the liver have been observed at doses as low as 37 mg/kg/day (Condie et al., 1983) and 50 mg/kg/day (Ruddick et ah, 1983). The effects include a slight increase in liver weights and microscopic changes that are rated as "minimal". The effects become
10 more pronounced at doses of 125 to 300 mg/kg/day (Condie et al., 1983). Most long-term studies report signs of liver injury in rats or mice at doses of 50 to 200 mg/kg/day (NTP, 1987). These doses are not significantly different from those reported to cause hepatic injury in acute and short-term studies. This suggests that there is a relatively low tendency toward cumulative injury to the liver.
c. Renal Effects
Animal studies indicate that the kidney is also susceptible to injury by BDCM, normally at dose levels similar to those that affect the liver. Munson et al. (1982) performed 14 day studies in which increases in blood urea nitrogen (BUN) in mice dosed with 250 mg/kg/day were observed. Condie et al. (1983) observed a decrease in the uptake of p-aminohippurate (PAH) into kidney slices from mice dosed with 74 to 148 mg/kg/day. Similarly, Ruddick et al. (1983) reported an increased in renal weight in rats dosed with 200 mg/kg/day for 10 days.
In longer-term animal studies, areas of focal necrosis were observed in the proximal tubular epithelium in male mice exposed for 13 weeks to doses of 100 mg/kg/day. Cytomegaly was observed following chronic exposure to 25 mg/kg/day (NTP, 1987). Female mice were somewhat less susceptible than males. In rats, cytomegaly and nephrosis were observed in both males and females at chronic exposure levels of 50 to 100 mg/kg/day (NTP, 1987).
11 5. CARCINOGENICITY
Although no studies with statistically significant conclusions have been done regarding the carcinogenic effects in humans following chronic oral exposure to BDCM, there are several epidemiological studies that indicate that there may be an association between ingestion of chlorinated drinking water (which normally contains
BDCM) and an increased risk of cancer in humans (Cantor et al., 1978; Morris et al., 1992). However, such studies cannot resolve possible effects of one or more of the hundreds of other by-products that are also present in chlorinated drinking water.
Chronic oral animal studies do however provide convincing evidence that BDCM is carcinogenic. In rats, increased frequency of liver tumors was observed in females exposed to 150 mg/kg/day for 180 weeks (Tumasonis et ah, 1985), and kidney tumors were observed in both males and females exposed to 1(X) mg/kg/day (NTP,
1987). The incidence of renal tumors was 13/50 and 15/50 in males and females, respectively. Tumors of the large intestine were also reported in rats, at incidences of 13/50 and 45/50 in males exposed to 50 and 100 mg/kg/day, and at an incidence of 12/47 in females dosed at 100 mg/kg/day. In mice, renal tumors were observed in males dosed with 50 mg/kg/day, and hepatic tumors were observed in females dosed with 75 or 150 mg/kg/day. Increased intestinal tumors were not observed in mice
(NTP, 1987).
12 C. HALOGENATED ACETIC ACmS
Halogenated carboxylic acids are products of the chlorination of fulvic and humic acids. While the haloacetic acids are the most abundantly produced species, a variety of saturated and unsaturated monobasic and dibasic acids containing several carbons have been identified in several laboratory studies as reaction products of chlorine and humic and fulvic acids (Bull and Kopfler, 1991).
Although brominated and mixed halogenated acetic acids have been identified as chlorinated disinfection by-products, little research has been done concerning the metabolism and toxicity of these compounds. Therefore, the following metabolic and toxicological information centers around di- and trichloroacetic acid.
1. CHEMICAL AND PHYSICAL PROPERTIES
Dichloroacetic acid (DCA) (FW = 128.94 g/mol), is a colorless liquid with a pungent odor. It has a boiling point of 193-194 °C and a density of 1.563 g/mL. Its two crystalline forms melt at 9.7 °C and -4 °C. DCA is soluble in water, alcohol, ether, and acetone. (Weast, 1989). Dichloroacetic acid is used as a chemical intermediate and in pharmaceuticals and medicine specifically to treat diabetic or hyperlipoproteinemic patients.
13 Trichloroacetic acid (TCA) (FW = 163.39 g/mol), takes the form of nonflammable, deliquescent colorless crystals. TCA, like DCA has a sharp, pungent odor. The crystals melt at 57.5 °C and boil at 197.5 °C. At 25 °C, 1.2 kg of TCA crystals are soluble in 1 liter of water. Trichloroacetic acid is used: in organic syntheses, as a reagent for detection of albumin, in medicine for the removal of warts, as an astringent, in pharmacy, and in herbicides (National Research Council,
1987).
2. OCCURRENCE AND PREVALENCE
In 1977, Rook identified dichloroacetic acid (DCA) as one of the products obtained from chlorinating a solution of peat-derived humic acid. Quimby et al. in
1980, chlorinated solutions of soil-derived humic and fulvic acids and identified
trichloroacetic acid (TCA) as one of the reaction products.
Miller and Uden (1983) determined that the chlorination pH had only a small
effect on the amount of DCA produced but that when the reaction was carried out at
pH 7 or higher, the amount of TCA formed was drastically reduced.
Legube et al. (1989) found that preozonation of fulvic acid destroyed both DCA
and TCA precursor sites when the fulvic acid was subsequently chlorinated. It was
also discovered that the bicarbonate ion significantly improved organohalide precursor
14 destruction. Thus, Legube et al. (1989) proposed that bicarbonate ions stabilize molecular ozone in water by inhibiting the radical chain reaction, and that molecular ozone reacts more readily with nucleophilic aromatic parts of the fulvic acid than does the hydroxyl radical.
Ozonation of fulvic acid in the presence of bromide ion was found to increase the formation of brominated halo acids, such as bromoacetic acid and dibromoacetic acid
(Legube et ah, 1989). Ozone oxidized the bromide to hypobromite and the bromoacids were formed.
Several studies have been done in recent years to determine the prevalence of halo
acids in finished drinking water. Stevens et al. (1989a, 1989b), compared clarified
Ohio River water to raw, unclarified Ohio River water which was chlorinated at pH
5,7, and 9.4. Clarification of the water prior to chlorination reduced the quantity of
both DC A and TCA formed. The quantities of TCA formed at pH 5 and 7 were
similar, and the amount produced at pH 9.4 was drastically lower at any given
reaction time. The concentration of TCA that was produced by chlorination of the
clarified water at these pH values ranged from 20 to 30 ug/L after 144 hr of reaction
time. The concentrations of DCA were essentially independent of pH, and the
concentrations after 144 hr of reaction time also ranged from 20 to 30 ug/L.
In 1985, Stevens et al. (1990) investigated the occurrence of disinfection by¬
products at 10 utilities in the United States. These 10 utilities represented a wide
geographic area, and included both surface and groundwaters with high and low
organic carbon content. The utilities served both large and small communities. All
15 of the utilities in the investigation used free chlorine at some point. Three of the ten utilities were selected because they were known to have high levels of THMs or because use of high chlorine levels on high TOC waters was expected to maximize formation of chlorination by-products. The haloacids identified in these finished waters are listed in Table 1. Semiquantitative data were obtained for the chloroacetic acids, and only the presence at levels three times the blank level was reported for the others. Chloroacetic acid was reported at levels less than 10 ug/L . Dichloroacetic acid was found in two supplies at less than 10 ug/L, in six supplies at levels between
10 and 100 ug/L, and in two supplies at concentrations greater than 100 ug/L.
Trichloroacetic acid was reported in two supplies at less than 10 ug/L and in four
supplies at levels between 10 and 100 ug/L.
Table 2 gives the quarterly median values for haloacetic acids found in U.S. drinking waters from Spring 1988 through Winter 1989 (Krasner et al., 1989). The
study included 25 utilities across the United States with 10 utilities in California.
3. METABOLISM
Mono, di and trichloroacetic acids have been found to activate the pyruvate
dehydrogenase (PDH) complex in several species, including man, by inhibiting
pyruvate dehydrogenase kinase (Blackshear et al., 1974; Whitehouse et al., 1974;
16 Crabb et al., 1976). This enzyme allows pyruvate dehydrogenase phosphatase to convert inactive phosphorylated PDH to the active form. Of the three chlorinated acetic acids, dichloroacetic acid has been more extensively studied, because of its potential use as a hypoglycemic, hypolactatemic, and hypolipidemic agent. It has been hypothesized that the effects of the PDH complex are at least partially responsible for the large accumulations of glycogen that are seen in the livers of animals treated with DCA and to a lesser extent TCA (Bull et al., 1990). DCA is also known to stimulate gluconeogenesis in fed rats, probably by increasing acetyl-
CoA production by PDH. Acetyl-CoA is an activator of pyruvate carboxylase, an enzyme in gluconeogenesis (Crabb er a/., 1981).
In 1981, Crabb et al. summarized the effects of DCA on metabolic processes. As stated previously, DCA activates PDH in many tissues by inhibiting PDH kinase; similarly, DCA activates myocardial branched-chain alpha-ketoacid dehydrogenase by inhibiting branched-chain alpha-ketoacid dehydrogenase kinase (Paxton and Harris,
1984). In an in vitro preparation, DCA inhibited lactate gluconeogenesis by hepatocytes because of the inhibition by oxalate of pyruvic carboxylase, and stimulated the oxidation of leucine because of the transamination of leucine by glyoxalate. In vivo, DCA was found to decrease blood glucose by restricting the supply of precursors for gluconeogenesis in the liver. This effect is consequent to activation in peripheral tissues of pyruvic dehydrogenase. It was also stated in this review by Crabb et al. (1981) that DCA lowers blood cholesterol in hyperlipidemic patients but no conclusion concerning the mechanism was given.
17 The effects of DCA and TCA have been shown to vary substantially between individuals. In a study done by Enser and Whittington (1983), two doses of 300 mg/kg body weight/d of DCA were administered to obese/hyperglycemic mice and to lean mice. It was found that DCA increased blood glucose in obese mice, which was attributable to a failure to secrete insulin. This increase in blood glucose could also be the reason for the increase in fatty acids in the adipose tissue. In the lean mice,
DCA decreased blood glucose and lactate levels. The numerous studies that have been done on humans, has shown similar results at dose levels ranging from 35 to 50 mg/kg/d (Stacpoole et al., 1983; Curry et ah, 1985).
Stacpoole and associates (1983) administered sodium dichloroacetate (NaDCA) to
13 patients who were hospitalized in an intensive-care unit. All patients had lactic acidosis, defined as a level of lactate in arterial blood of 5 mM/liter or higher. Also, their acidosis had been refractory to sodium bicarbonate treatment. Because two of the patients were inadvertently given sodium bicarbonate after initiation of DCA therapy, which was in violation of the intended protocol, these patients were excluded in the analysis of the results.
The results indicated that arterial plasma lactate decreased in all of the 11 patients, with an average decrease of 29%. The DCA-induced reduction in lactate was significant, being greater than a 20% reduction in 7 of the patients. The arterial alanine levels were normal or elevated prior to treatment and fell an average of 54% in the 7 responding patients. Beta-hydroxybutrate concentrations increased an average of 34% after DCA treatment.
18 The study concluded that although the DCA therapy caused a marked improvement in overall morbidity, only one of their patients survived to leave the hospital. Previously, however, none of their patients with hyperiactatemia of this magnitude had survived. Further, the patients whose hyperiactatemia was reversed by
DCA treatment died because of the failure to reverse their primary, life-threatening illnesses. It was concluded that the DCA stimulated PDH activity, leading to accelerated oxidation of pyruvate, lactate, and alanine, as well as to increased bicarbonate formation and consequently, to a rise in arterial pH.
4. TOXICITY
Katz et al. (1981) administered sodium dichloroacetate daily for 3 months to CD rats and beagle hounds. Ten rats of each sex (131 to 156 g bw) were intubated with an aqueous solution of 125 and 500 mg/kg bw per day, and 15 of each sex were intubated with 0 or 2,000 mg/kg bw per day. Dogs received 0, 50, 75, and 100 mg/kg bw orally as a solid contained in gelatin capsules (4 of each sex at 0 and 100 mg/kg bw, 3 of each sex at 50 and 75 mg/kg bw). In rats, 2,000 mg/kg bw proved lethal and 500 mg/kg bw nonlethal. In dogs, the lethal dosage was 75 mg/kg bw with
50 mg/kg bw being nonlethal. Both species experienced reduced food consumption and body weight gain; hind limb weakness; frequent urination; progressive reduction
19 in erythrocyte counts, hematocrit, and hemoglobin levels; reduced blood levels of glucose, lactate ^d pyruvate; vacuolation of myelinated white tracts in the cerebrum
I and to a lesser extent, in the cerebellum; and degeneration of germinal epithelium of the testes, with syncytial giant cell formation. In addition to these symptoms, rats often had aspermatogenesis, whereas dogs had atrophy of the prostate gland, cystic mucosal hyperplasia in the gall bladder, hemosiderin-laden Kupffer cells, and eye lesions consisting of bilateral lenticular opacities, injected bulbar conjunctivae, and superficial corneal vascularization, with a tendency toward keratoconjunctivitis sicca.
Some animals of each species in this study were not killed at the end of the experiment, but were allowed a 1 month recovery period before being necropsied.
During this time frame, there was some recovery, however, the lenticular opacities and gall bladder anomalies in dogs, the brain lesions in both species, and the aspermatogenesis and loss of testicular germinal epithelium in rats persisted or improved only minimally.
At the time of necropsy, the weight of the livers of the female rats at 500 and
2,000 mg/kg bw, were significantly heavier than those of the controls. Relative liver weights were significantiy increased at all doses among both sexes, in a dose- dependent manner. There were also significant increases in relative weights of kidneys (females at all doses) and adrenals (males at 500 mg/kg bw and females at
2,000 mg/kg bw). Absolute and relative organ weights of intoxicated rats approached I ͣ those of the controls at the end of the month recovery period.
The brain lesions in the dogs were at first thought to be characteristic of edema.
20 However, the lesions persistence in the hounds, which were permitted to recover for
5 weeks, indicated that more than simple edema was involved in the development of the lesions. Moreover, the lesions were not seen in the optic or sciatic nerves.
The high dose of 2,000 mg/kg bw resulted in a 13% mortality rate in both male and female rats. Piloerection, tactile-induced vocalization, low body position, and unthriftiness occurred prior to death in male rats. Female rats that died, first exhibited cachexia and unthriftiness. Dogs, on the other hand were dramatically more sensitive. One of the three females died at a dosage level of 75 mg/kg bw and one of the four males died at 100 mg/kg bw. In contrast to the changes observed after oral administration of DCA, the administration by vein to dogs (up to 100 mg/kg bw for
30 days) was without evidence of testicular, prostate, or central nervous system effects.
Woodward et al. (1941) reported an oral LD50 for TCA in fasted mice to be 4.97 g/kg/d and 3.32 g/kg/d for fasted rats. Both the rats and mice quickly passed into a state of narcosis and within 36 hours had either recovered or died. B6C3F1 mice exposed to chronic levels of TCA resulted in massive accumulation of fat, some increases in cell size, and deposition of large amounts of lipofuscin in the liver.
These effects have been observed at doses as low as 60 mg/kg/d.
21 5. CARCINOGENICITY
DCA and TCA have been tested for carcinogenicity in male B6C3F1 mice
(Herren-Freund et al., 1987). The mice received either a single initiating dose of 2.5 mg/kg ethylnitrosourea or vehicle at 15 d of age. Groups of animals were exposed to
5 g/L and 2 g/L DCA (39 mM and 16 mM) or TCA (31 mM and 12 mM) in drinking water for 61 weeks, starting when the mice were 28 d of age. The test chemicals were neutralized with NaOH, and the control groups were exposed to drinking water containing equivalent sodium as sodium chloride.
Exposure to both DCA and TCA resulted in an increased incidence of liver cancer and an increased tumor burden (see Table 3). A prior treatment with an initiating dose of ethylnitrosourea was not required for the chloroacetic acids. This suggests that both DCA and TCA appear to be functioning as complete carcinogens in the B6C3F1 mice. On a molar basis, however, DCA appeared to be a more potent hepatocarcinogen than TCA.
In this study (Herren-Freund et al., 1987), no response was observed between the high dose (5 g/L) and the low dose (2 g/L) test groups for either DCA or TCA.
Because water consumption measurements were not made during this study, an average daily intake for the mice in the two experimental groups could not be calculated. Herren-Freund and associates (1987) observed that when the mice were exposed to drinking water containing DCA or TCA, the test mice initially limited their daily intake to almost one-half of the amount consumed by the control animals.
22 However, after a period of 4 to 6 weeks, the treated mice increased their intake to
90% of that of the control mice.
Although both DCA and TCA appeared to function as complete carcinogens in this particular study, their role as initiators, with an ability to directly damage DNA is still not understood. Neither DCA nor TCA possessed mutagenic activity in tests using various Salmonella strains. Meier and Blazak (1985) discovered that both DCA and TCA could induce weak statistically significant increases in sister chromatid exchanges in cultured Chinese hamster ovary cells, but only at high concentrations and in the absence of metabolic activation. When tested in the mouse in vivo, however, neither DCA nor TCA induced bone marrow sister chromatid exchanges or micronucleus formation.
It should also be mentioned that the B6C3F1 mouse is noted for a high incidence of spontaneous liver tumors; therefore, it is quite possible that DCA and TCA might be promoting the outgrowth of spontaneously initiated cells (Reddy, et ah, 1980). Although most data indicates that DCA and TCA have little potential for inducing genotoxicity, much data does suggest that the carcinogenicity of DCA and TCA might be caused by their ability to function as peroxisome proliferators. Several studies indicate that following short-term administration, both DCA and TCA induced peroxisome proliferation in mice and rats (Elcombe, 1985; Everett, 1985 and DeAngelo et ah, 1986). Because many of the chemicals that induce peroxisome proliferation also increase the incidence of hepatocellular cancer in rodents, Reddy et al. (1980) proposed that they constitute a unique class of chemical carcinogens which
23 share other biological properties, including the ability to induce hepatomegaly and hypolipidemia. The mechanism by which peroxisome proliferator compounds induce liver tumors involves the excessive production of hydrogen peroxide and the reactive oxygen species, which results from lipid peroxidative reactions. The accumulation of hydrogen peroxide and the reactive intermediates initiate carcinogenesis by damaging cellular DNA. In DeAngelo's study (1990), the data indicate that peroxisome proliferation is not a mechanism for the carcinogenicity of DCA and TCA. DCA, a potent carcinogen relative to TCA, was the weaker inducer of peroxisome proliferation. In fact, in this study, the cancer incidence and liver tumor burden were the same for both the high dose mice with a moderate elevation of palmitoyl CoA oxidase activity and the low dose mice with a statistically significant lower activity of this marker enzyme in the liver. Both DCA and TCA increased liver weights in exposed B6C3F1 mice, and the degree of enlcu-gement correlated with the carcinogenicity of the acid. Although DCA was a relatively weak inducer of peroxisome activity, the hydrogen peroxide and reactive oxygen intermediates formed could result in large amounts of DNA damage if hepatocytes were turning over. The damage from TCA, which is a relatively strong peroxisome proliferator, would be less if it did not induce a large amount of hyperplasia. However, since little data is available on the mitogenic potential of either DCA or TCA, the role of liver cell hyperplasia in the carcinogenicity of
chlorinated acids is not clear.
24 To date, no studies have been done concerning the toxicity or carcinogenicity of the mixed halo acetic acids, specifically, bromochloroacetic acid, bromodichloroacetic acid, and chlorodibromoacetic acid. One can only hypothesize that the mixed halo acetic acids would react similarly to DCA and TCA.
25 n. INTRODUCTION
Bromochloroacetic acid, dibromochloroacetic acid, and bromodichloroacetic acid, are known to occur in drinking water as a result of chlorination of raw water containing a high bromide ion concentration. When chlorine is added to water containing bromide ion, the bromide is oxidized to hypobromous acid according to
Reaction (3).
HOCl + Br <= = = = > HOBr + CI" (3)
This reaction is the basis for bromine-containing disinfectant by-products.
Particular areas of the United States that are prone to high bromide ion concentrations in their raw water include; Brownsville, Texas; Lexington, Kentucky and a number of coastal cities of Florida. Because these halo acids represent a potential health hazard to the consumers of these waters, several studies have been done in order to synthesize and identify these three mixed halogenated acetic acids for use in investigations of biological effects. (Ireland et al., 1988 and Zimmer et al.,
1990)
Trihalomethanes are a prevalent contaminant of chlorinated drinking water supplies, and have been measured in concentrations ranging from 0.1 to 540 ug/L
(U.S. EPA, 1990). In waters containing bromide ion, the trihalomethane,
26 bromodichloromethane (BDCM) is known to form. BDCM has been known to induce neoplasms in the kidney and large intestine of male and female rats, in the liver of female mice, and in the kidney of male mice (NTP, 1987). Chloroform (CHCI3), the most prevalent THM in chlorinated drinking water and perhaps the most extensively studied THM, appears to be less carcinogenic compared to BDCM (Dunnick et al., 1987). Studies also indicate that BDCM is more acutely toxic than CHCI3 (Chu et ah, 1982). The results of these studies indicate the need for further research on
BDCM.
Current research is being conducted to determine the relationship of the chemical structure of BDCM to metabolism and its relationship to different toxic endpoints, including carcinogenicity. In order to do this, an understanding of the potentially important metabolic pathways is needed. The three major metabolic pathways that are known for BDCM include: oxidation, reduction, and conjugation reactions with glutathione. By using deuterium-labeled BDCM in rodent studies, researchers will be able to study the heavy isotopic effect in bond cleavage. This information will lead to a clearer understanding of the metabolism of this compound by indicating whether C-H bond cleavage is a rate-determining step. In a study done by Pohl and Krishna (1978), on the deuterium isotope effect of chloroform, they found that the cleavage of the C-H bond appeared to be the rate- determining step in the reaction, since deuterium labeled chloroform is biotransformed into phosgene (COCy more slowly than CHCI3. Pohl and Krishna believed that CHCI3 was metabolically activated to phosgene by cytochrome P-450 which is found
27 in the liver microsomes. This reaction was hypothesi7ed to proceed through an oxidative dechlorination mechanism that involved the oxidation of the C-H bond of
CHCI3 to produce the trichloromethanol (CI3C-OH) derivative, which would spontaneously dehydrochlorinate to produce COCI2. CDCI3 also appeared to be less hepatotoxic compared to CHCI3. At a dosage of 2.49 mmole/kg, CDCI3 produced only minor histological changes, and there was no elevation of the level of serum glutamic pyruvic transaminase (SGPT) (which was used to assess hepatic damage). At this same dosage, CHCI3, produced considerable necrosis of the centrolobular region of the liver as well as elevated SGPT values. When the dosage was increased to 4.98 mmole/kg, 85% of the rats treated with CHCI3 died within 24 hours, whereas all of the CDCI3 treated rats survived. This information suggests that a similar pathway of metabolism is responsible for the hepatotoxic properties of chloroform.
28 III. EXPERIMENTAL MATERIALS AND METHODS
A. INSTRUMENTATION AND LABORATORY MATERIALS
'^C nuclear magnetic resonance (NMR) spectra were obtained at the UNC Department of Chemistry on a Varian XL-400 at 100.573 MHz. Deuterated acetone, obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI), was used as the solvent. Mass spectral analyses were performed in the Department of Environmental Sciences and Engineering at UNC on a VG 70S 250SEQ mass spectrometer in the EI mode at 70 eV. GC-MS analyses were performed in the Department of Environmental Sciences and Engineering at UNC in Dr. Rappaport's laboratory. The gas chromatograph was a Hewlett Packard 5890 Series II model and the mass
spectrometer was a Hewlett Packard 5971 Mass Selective Detector. The column was a 30 meter DB-1 from J & W Scientific (Folsom, California). The carrier gas was helium (He), with a flow rate of 1 mL/min. The port temperature was set at 250 °C and the detector temperature was set at 280 °C. The column temperature program
was as follows: 50 °C for 3 minutes, 25 "C/min. to 150 "C. One microliter of a 1 mg/mL sample in hexane was injected into the GC in the splitless mode. All melting points were determined using a Fischer-Johns apparatus and are uncorrected. All of the routine chemicals were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI) or Eastman Kodak (Rochester, NY). All solvents were reagent
29 grade, and were dried over CaH2 or LiAl4 and distilled where indicated.
B. SYNTHESIS OF MIXED HALOGENATED ACETIC ACIDS
A literature synthesis (Zimmer et al., 1990) was used to obtain bromochloroacetic acid, chlorodibromoacetic acid, and bromodichloroacetic acid.
1. BROMOCHLOROACETIC ACID
To a mixture of 0.1 mole of malonic acid and 70 mL of anhydrous ether, cooled in an ice bath to 10 °C, was added 16.0 g (0.1 mole) of bromine. The bromine was added dropwise through an addition funnel over a period of 6 hours. Volatile materials were distilled off, leaving a yellowish solid residue product. This product was filtered and washed with cold chloroform to give bromomalonic acid (see Figure 5 for reaction scheme) which was dissolved in 40 mL anhydrous ether, cooled to 10 °C, and reacted with 0.1 mole of distilled sulfuryl chloride. The distilled sulfuryl chloride was added dropwise to the flask, using an addition funnel, over a fifteen minute period. After the addition was complete, stirring was continued for an
30 additional 30 minutes at room temperature. The solvent and volatile materials were again evaporated using the rotary evaporator. Upon adding petroleum ether, colorless crystals of bromochloromalonic acid formed. This product was heated to 130 °C for decarboxylation. To the resulting brownish liquid, were added 10 mL anhydrous ether. The solution was treated with activated carbon and filtered to yield bromochloroacetic acid. The product was confirmed through melting point determination (Table 4) and through MS EI mode analysis (Figure 6).
2. CHLORODIBROMOACETIC ACID
To a 3 neck 50 mL round bottom flask which was flushed with argon (Ar) gas and equipped with a reflux condenser and addition funnel, were added 12.5 g monochloroacetaldehyde diethyl acetal. Using the addition funnel, 26.5 g (8.5 mL) of bromine were added to the flask. With the use of an oil bath, the reaction flask was heated to 65 °C when bromine started to disappear. The temperature was then increased slowly to 100 °C. The bromine addition took approximately 7 hours. After the addition of bromine, unreacted bromine and ethyl bromide were distilled off at 100 °C. The remaining liquid was treated with 1.0 g of solid calcium carbonate and distilled (120 °C - 140 °C) to give a clear liquid. This liquid was treated with 1.0 g phosphorus pentoxide and distilled again, yielding dibromochloroacetaldehyde (135
31 "C - 140 °C) (see reaction scheme, Figure 7). To 4.0 g of this aldehyde were added 6 mL of red fuming nitric acid. This mixture was heated in a 50 mL round bottom flask equipped with a reflux condenser for two hours. Upon cooling, the reaction mixture was placed in the refrigerator to allow for crystallization. The formation of chlorodibromoacetic acid was confirmed by melting point determination (Table 4) and
MS EI mode analysis (Figure 8).
3. BROMODICHLOROACETIC ACID
a. Literature Synthesis To a 100 mL 3 neck round bottom flask, equipped with a reflux condenser and addition funnel, were added 24.0 g (0.128 moles) of dichloroacetaldehyde diethyl acetal. The flask, under argon gas, was placed in an oil bath and heated to 110 "C. Bromine (6.6 mL = 0.128 moles) was added to the flask through an addition funnel over a period of 8 hours. Upon completing the bromine addition, the volatile materials were distilled off. The remaining liquid was cooled to 0 °C and shaken for approximately 1 minute with 25 mL cold concentrated sulfuric acid in a separatory funnel. This mixture was allowed to stand at room temperature for 30 minutes. The bottom layer was collected in a 50 mL Erlenmeyer flask containing 2.5 g of solid calcium carbonate and was left to stand for 3 hours. The mixture was filtered, and
32 the filtrate was distilled. The fraction that was collected between 110 "C and 120 °C was treated with 1.8 g of phosphorus pentoxide and redistilled giving dichlorobromoacetaldehyde (see reaction scheme, Figure 9). This aldehyde was collected at 115 °C - 121 °C. The dichlorobromoacetaldehyde was refluxed with 3 mL red fuming nitric acid for 3 hours at 100 ° C. Upon cooling, the reaction flask was placed in the refrigerator to allow for crystallization. The crystals formed slowly. Bromodichloroacetic acid was confirmed through melting point determination (Table 4) and through MS EI mode analysis (Figure 10).
b. Synthesis Through Lithium Salt Formation To a 3 neck, 250 mL round bottom flask, purged with Ar(g), were added 1 mL bromodichloromethane and 50 mL distilled THF. The flask was placed in a dry ice acetone bath and allowed to stir for 5 minutes. Upon cooling the flask, 8.0 mL of 1.6 M n-butyllithium in hexanes were added to the flask slowly via syringe. This reaction mixture was allowed to stir 1.5 hours. Carbon dioxide was bubbled into the
reaction flask for 30 minutes, using dry ice as the CO2 source (see Figure 11). After 30 minutes, the reaction mixture was poured into a beaker containing a layer of dry ice. Deionized water was added to the lithium salt and then the reaction mixture was acidified with concentrated hydrochloric acid. The solution was saturated with sodium chloride and extracted three times with methylene chloride. The three organic
33 extracts were combined, dried over sodium sulfate and filtered into a 100 mL round bottom flask. A rotary evaporator was used to remove the methylene chloride. The sample was placed in the refrigerator for crystallization to occur. Bromodichloroacetic acid crystals did form and were confirmed by MS EI mode analysis (Figure 12). Several attempts were made to develop a procedure based on Ba^COs as a source of CO2 (see reaction scheme, Figure 13). In pilot studies, BaCOj was treated with concentrated H2SO4 and the evolved CO2 was condensed in a liquid Nj cold trap. The LiCBrClj solution was then added to the trap, which was allowed to warm to -80 °C. Using this procedure, it has been possible only to recover trace quantities of CBrCl2C00H using unrealistic quantities of BaCOj (5 g) to generate CO2. Since it is possible to use no more than 10 mCi (300 - 1000 mg) based on an activity of 20 - 60 mCi/mol, this route did not appear practicable for preparation of the '"C-labeled compound.
C. SYNTHESIS OF DEUTERATED BROMODICHLOROMETHANE
The synthesis of deuterated BDCM was based on the procedure of Dougherty (1929) involving an aluminum chloride catalyzed reaction. To a 100 mL round bottom flask, equipped with a magnetic stir bar, were added 25 mL 99.8% d-
34 chloroform and 25 mL bromoethane (see Figure 14). The reaction flask was placed in an ice bath, while the aluminum chloride was being added. Enough AICI3 was added so that the reaction mixture turned a bright crimson red. A drying tube was used to seal the flask to prevent any moisture from entering. The reaction mixture was allowed to stir for 46 hours. During this time, the reaction mixture remained a bright crimson red color.
The reaction was stopped by adding approximately 15 mL deionized water, followed by dilute HCl. The water layer was removed by the use of a separatory funnel. The organic layer was washed with 30 mL of a 5% NaOH solution, and again the water layer was removed by the use of a separatory funnel. The product was dried over sodium sulfate and collected in a 100 mL round bottom flask. The solution was distilled using a Vigreux column distillation apparatus. Three distillation
fractions were collected; fraction 1: 24-64 °C, fraction 2: 65-72 °C, and fraction 3:
73-78 °C.
By "C NMR analysis, deuterated BDCM was identified in Fractions 2 and 3 (see Figures 15 and 16). Using the relative peak areas given by GC analysis for fractions 2 and 3, the purity of fraction 2 was calculated to be 93% and that of fraction 3 was 85% (see Figures 17 and 18). For both fractions, the bromodichloromethane was
99% deuterium enriched.
35 D. SYNTHESIS OF DEUTERATED CHLORODffiROMOMETHANE
To a 100 mL round bottom flask, equipped with a magnetic stir bar, were added 25 mL 99.8% CDCI3 and 25 mL CjHsBr. While the reaction flask was placed in an ice bath, AICI3 was added to the flask until the reaction mixture turned a dark crimson red (see reaction scheme, Figure 19). A drying tube was used to seal the flask to prevent any moisture from entering the reaction. This solution was then allowed to stir for 7 days. Over this time, the color of the reaction did not dissipate. The reaction was stopped by adding approximately 15 mL of deionized water and a dilute solution of HCl. The organic layer was removed from the water layer by use of a separatory funnel. The organic layer was subsequently washed with 30 mL of a 5% NaOH solution. The organic layer was collected and dried over sodium sulfate and collected in a 100 mL round bottom flask. The solution was distilled using a Vigreux column distillation apparatus. Five fractions were collected: fraction 1: 27 - 67 °C; fraction 2: 68 - 72 °C; fraction 3: 73 - 78 °C; fraction 4: 79 - 84 °C and
fraction 5: 90 °C (under vacuum). '^C NMR analyses (see Figures 20 - 23) indicate that deuterated chlorodibromomethane is present in fractions 4 and 5 by observation of a 1:1:1 triplet peak centered about 36 ppm. Deuterated BDCM is present in all four fractions as
indicated by a 1:1:1 triplet peak centered at 59 ppm.
36 rV. RESULTS AND DISCUSSION
A. MIXED HALOGENATED ACETIC ACIDS
The synthesis of bromochloroacetic acid (CHBrClCOOH), chlorodibromoacetic acid (CBr2ClC00H), and bromodichloroacetic acid (CBrClzCOOH) was obtained using the literature synthesis published by Zimmer et al. (1990). Each of these halo acids was confirmed by melting point determination (see Table 4) and by mass spec analysis (see Figures 6, 8, and 10). The variations between the observed melting points and the literature values most likely reflects the need for recrystallization to remove traces of water. We did not recrystallize any of the three halo acids, all of which are hygoroscopic, thus likely to contain some water. The presence of water will lower the melting points. The mass spectra indicate the correct isotopic clusters at the appropriate molecular weights for each of these acetic acids (172, 174, 176 for bromochloroacetic acid; 250, 252, 254, 256 for chlorodibromoacetic acid; 206, 208,
210, 212 for bromodichloroacetic acid). Due to facile fragmentation, the molecular
ion in each case is relatively weak. Although, it was possible to obtain bromodichloroacetic acid (CBrClzCOOH) using the procedure described by Zimmer et al. (1990), this synthetic approach was not feasible for the preparation of "C-labeled bromodichloroacetic acid because labeled starting compounds were not readily accessible. A synthetic approach was developed, in which 1.6 M n-butyllithium in hexanes was reacted with either
37 CHBrCl2 or CBr2Cl2 and carboxylated to yield the lithium salt of bromodichloroacetic acid (see Figure 11) (Fischer and Kobrich, 1968), As seen in Figure 11, the bromodichloromethide anion is carboxylated by the addition of dry ice to yield CBrCl2C02'Li"^, which is subsequently poured over a layer of dry ice. This procedure generated a significant yield of CBrClzCOOH. The product was confirmed by mass spectra EI mode analysis (see Figure 12). Having demonstrated the feasibility of this reaction, attempts were made to develop a synthetic procedure based on Ba^COj as a source of CO2. In pilot studies, BaCOj was treated with concentrated H2SO4 and the evolved CO2 was condensed in a liquid nitrogen cold trap (see Figure 13). The LiCBrClj solution was then added to the trap, which was allowed to warm to -80 °C. This approach did not yield the significant quantity of product obtained with a large excess of CO2 from dry ice. Only trace quantities of CBrCljCOOH were recovered while using impractically large quantities of BaCOj (5 g) to generate CO2. Since it is not possible to use more than 10 mCi (300 - 1(XK) mg) based on an activity of 20-60 mCi/mol, this route does not appear to be practicable for the synthesis of the ^''C-labeled compound. A second route was proposed that involved the chlorination of commercially available bromoacetic acid (CHjBrCOOH). '"^C-labeled bromoacetic acid is available as either CH2Br"C00H or as *''CH2BrC00H. This synthetic approach could also be used to generate the other mixed halogenated acetic acids. However, to date no pilot studies involving the chlorination of unlabeled bromoacetic acid have been initiated.
38 B. SYNTHESIS OF DEUTERATED BROMODICHLOROMETHANE
1. LITHIUM SALT FORMATION
Because of the feasibility of generating the bromodichloromethide anion, attempts were made to synthesize deuterated bromodichloromethane (see Figure 24) by quenching the anion with D2O.
It was hypothesized that deuterium oxide would exchange rather rapidly with lithium to yield deuterated bromodichloromethane. However, this did not appear to be the case, since no color change was observed when deuterium oxide was added to the reaction flask. The crude sample was analyzed by ''C NMR and MS (see Figures 25 and 26) which indicated the presence of a number of different by-products. Based upon the relative intensities of the isotopic clusters from the mass spectral data, bromodichloromethane, present in trace amounts, was estimated to be 20% deuterium enriched. Attempts to isolate bromodichloromethane by distillation through a packed column were unsuccessful. In order to purify the sample, a preparative GC would
have been needed.
Sherman and Bernstein (1951), describe a similar reaction, in which 0.1 mole of purified halomethane (CHBrCla and CHBraCl) was mixed with 0.1 mole 98% D2O and 0.01 mole sodium deuteroxide in a heavy-walled Pyrex vessel. The contents were outgassed at -78 °C, and the vessel was sealed and placed in an oven at 105 °C for four days. After four days, the reaction vessel was cooled to -195 °C and opened.
39 The yield reported was 43% CBr2ClD and 16% CBrCl2D. Upon recharging the reactor the yields increased to 64% and 36% respectively. They concluded that the rate of exchange of halogens between bromodichloromethane and chlorodibromomethane is more rapid than the rate of hydrolysis.
2. BROMINATION OF DICHLOROMETHroE ANION
Due to the poor yield and our inability to purify CDBrClj through fractional distillation using the approach shown in Figure 24, a similar reaction was proposed that involved brominating the dichloromethide anion (Blumberts, LaMontagne, and
Stevens, 1972).
The reaction involves mixing deuterated methylene chloride with 1.6 M n- butyllithium in hexanes, to yield the deuterated dichloromethide anion. This product is subsequently brominated to give the desired product (see Figure 27). The reaction, as before, was performed at -78 °C in order to stabilize the lithium salt. However, bromine did not react with LiCDClz at -78 °C, presumbaly because, bromine froze out of solution. The reaction was repeated with addition of bromine at 0 °C, and again the bromine remained unreactive. Since the dichloromethide anion would be unstable
at higher temperatures, it was apparent that this approach was not feasible for the
synthesis of deuterated BDCM.
40 3. HYDROGENOLYSIS OF DIBROMODICHLOROMETHANE
Pilot studies were started involving the hydrogenation of dibromodichloromethane by lithium aluminum hydride (see Figure 28). Johnson and co-workers (1948) hydrogenated several alkyl halides by lithium aluminum hydride and lithium hydride. Since aluminum hydride reacts with lithium hydride in ether to produce lithium aluminum hydride, it was considered reasonable that alkyl halides could be hydrogenated by means of lithium hydride with only a small amount of lithium aluminum hydride present. Johnson et al. reported that tetrahydrofuran (THF) was an excellent reaction medium because it is a good solvent for the reagent, it is miscible with water and it has a boiling point (65 °C) that allows for convenient separations of products by distillation. Nevertheless, the boiling point of THF still permits the use of a temperature range so that the reactions can be accelerated. The temperature should however be kept below 100 °C, since lithium aluminum hydride may decompose violently above this temperature. Using this synthetic approach, it was evident that lithium aluminum hydride acts as the hydrogen carrier, since no reaction seemed to occur with lithium hydride alone. The use of lithium hydride greatly
reduces the amount of lithium aluminum hydride that is necessary, and minimizes the possibility of the formation of aluminum halide. Johnson and co-workers (1948) also
found that in general, alkyl bromides reacted more readily than alkyl chlorides with lithium aluminum hydride. Primary halides reacted more readily than secondary halides which in turn reacted more readily than tertiary halides. Alicyclic and
41 aromatic halides proved to be very unreactive. Based upon this information, an attempt was made to react CBr2Cl2 with lithium hydride and a minimum amount of lithium aluminum hydride as shown in Figure 28. If this reaction proved to be successful, lithium deuteride (LiD), and lithium aluminum deuteride (LiAlD4) would be used as the deuterium source. "C NMR analysis were performed on the reaction mixture after 1, 4 and 8 hours. In each case, there was no evidence of CHBrClj. Thus, this reaction did not prove feasible for the synthesis of deuterated BDCM.
4. ALUMINUM CHLORmE CATALYZED REACTION
In 1929, Dougherty discussed the catalytic activity of aluminum chloride. Aluminum chloride forms addition compounds with many different types of organic molecules, and in an attempt to explain the mechanism of this Friedel-Crafts reaction, the catalytic activity of the aluminum chloride has been connected to the formation of these complexes. The ability of aluminum chloride to act as a strong lewis acid complexing agent is normally explained electronically on the grounds that the aluminum atom in aluminum chloride has an outer shell consisting of six electrons, and in order to achieve the more stable arrangement of eight, aluminum will share a pair belonging to some other atom or molecule. Unless a rearrangement occurs, this
42 new molecule will be polar. It has been proven that solutions of aluminum halides in alkyl halides, and organic aluminum halide solutions in general, often display considerable conductivity.
Dougherty described the action of aluminum chloride on halogen compounds using the following reactions:
RX + AICI3 < = = = > RX-AICI3 < = = = > R+-(XAlCl3)- (4) R,Xi + AICI3 < = = = > RjXi-AlClj < = = = > R,+-(XiAlCl3)- (5) R+-(XAlCl3)- + Rj-^-CXiAlClj)- < = = > R^-CXiAlCy- + Ri^-CXAIC^" (6) R+-(XiAlCl3)- < = = = > RXi + AICI3 (7) Ri+-(XA1C13)- < = = = > RjX + AICI3 (8)
If either of the new alkyl halides, RjX or RX, are volatile in comparison with RX and RjXi, the reaction RX + RjX, — > RjX + RX, should occur. If all of the alkyl halides are of the same order of volatility, the reaction should proceed to an equilibrium point.
Dougherty (1929) discovered that the reaction between chlorides and bromides in the presence of aluminum chloride, undergo such Friedel-Crafts reactions without appreciable formation of complex by-products and tars. Mixing equal portions of bromoethane and chloroform and adding a small amount of aluminum chloride, bromodichloromethane was synthesized, giving approximately a 35% yield.
This procedure given by Dougherty (1929) was used to synthesize deuterated bromodichloromethane. This compound was prepared by mixing equal volumes of bromoethane and deuterated chloroform, adding enough aluminum chloride such that
the reaction mixture turned dark red in color, and letting the reaction mixture stir for
43 approximately 46 hours (see Figure 14). The yield of deuterated bromodichloromethane calculated for this reaction was approximately 28%, in which the compound was 99% deuterium enriched. These calculations were based upon "C
NMR and GC-MS analysis (Figures 15 - 18). These results also indicated the presence of another deuterated product in the highest boiling distillation fraction (see Figures 16 and 18). The "C NMR indicated the presence of a deuterated trihalomethane at approximately 37 ppm. Based upon literature values of methyl carbon shieldings of chlorobromomethanes, the value given for chlorodibromomethane is 34.6 (Strothers, 1972). Mass spectra data indicated a molecular ion peak at 207,
209, 211 which is consistent for the isotopic masses for deuterated chlorodibromomethane. In fact, Dougherty (1929) notes that a small fraction (b.p. 120-132 ° C) was collected in which he believed to be a mixture of bromoform and chlorodibromomethane. However, due to difficulties in the fractional distillation, he was not able to identify these two compounds with certainty. It was hypothesized that as our desired product (deuterated BDCM) was forming, aluminum chloride was reacting with it to yield deuterated chlorodibromomethane (see Figure 19). Thus, it was assumed that if the reaction was allowed to react long
enough, a significant yield of deuterated chlorodibromomethane could be obtained.
The same reaction was repeated; however instead, of letting the reaction stir for 46 hours, it was allowed to stir for seven days. "C NMR results, as shown in Figures
20-23, do indicate a substantial yield of deuterated chlorodibromomethane
(approximately 5 g) in the highest distillation fraction (fraction 5 = 90 °C under
44 vacuum). Thus, one can conclude that this aluminum catalyzed reaction can be used to synthesize both deuterated bromodichloromethane and deuterated chlorodibromomethane in substantial yields and at 99% deuterium enrichment. The reaction is not only simple to perform but it is also relatively easy to purify the samples through simple fractional distillation. Because of the feasibility of this reaction, "C and ''^C-labeled bromodichloromethane and chlorodibromomethane can be obtained. For both of these syntheses, a preparative GC will be required for purification on a small scale. A GC with a thermal conductivity detector has been obtained from NIH. While waiting for the delivery of 10 g '^CHClj from Cambridge Isotope Inc., the GC is being assembled to perform preparative analysis.
5. REDISTRIBUTION OF HALOGENS IN A MIXTURE OF CHLOROFORM
AND BROMOFORM UNDER ALKALINE CONDITIONS
Fedorynski et al. (1977) report that when dichloro- or dibromocarbene are
generated under catalytic two-phase conditions (CTP) in the presence of
tetraalkylammonium phase transfer reagents, they remain in equilibrium with their carbanionic precursors and thus with the starting haloforms. This phenomenon is due
to the excellent solubility of tetraalkylammonium trihalomethides and halides in
45 haloforms and other nonpolar solvents. When a mixture of chloroform and bromoform is treated with a concentrated sodium hydroxide solution in the presence of triethylbenzylammonium chloride (TEBA) and an alkene, three dihalocyclopropanes form. This result is explained in terms of an equilibrium that exists between dihalocarbenes and trihalomethylanions (see Figure 29). It is this equilibrium that prompted Fedorynski et al. (1977) to test whether or not these conditions could be applied to the redistribution of halogens in a mixture of chloroform and bromoform without trapping the dihalocarbenes. One possible disadvantage of this reaction is the hydrolysis of the haloforms.
Fedorynski et al (1977) successfully synthesized bromodichloromethane and chlorodibromomethane by reacting bromoform and chloroform with concentrated aqueous NaOH and triethylbenzylammonium chloride (TEBA) (see Figure 30). This reaction gave a mixture of products, including tetrahalomethanes. Fedorynski et al. states that the mixture was easily separated via fractional distillation. The yields reported for BDCM and CDBM were 33% and 54% respectively. When the haloforms were regenerated the yields improved to 48% and 72% respectively. Although hydrolysis of the haloforms is a possible drawback to this reaction,
Fedorynski and co-workers reported that hydrolysis in their experiments did not exceed 15%.
Although this synthetic approach is less time consuming (reaction time = 1 hr), it is not practicable for labeled compounds. In order to obtain deuterated BDCM,
deuterated chloroform, deuterated bromoform, NaOD, and D2O would all be
46 required. Because of the number of products this reaction yields, this method is also only applicable on a large scale.
47 V. CONCLUSIONS
Chlorine, is by far the most commonly used chemical for the disinfection of drinking water. The primary purpose of disinfection is to protect the consumer from unnecessary exposures to water borne diseases. However, in the past two decades the discovery of potentially toxic compounds in drinking water as a result of chlorination has raised many concerns. Adding to the problem is the fact that the composition of chlorinated drinking water to which the consumer is exposed varies according to location. Variables that affect the production of possible harmful disinfection by¬ products include, total organic carbon concentrations, pH, ammonium and bromide
ion concentrations and the qualitative composition of the organic matter (IARC,
1991). Because of these possible health risks, current research is focusing on the
metabolism of these compounds, so that proper risk assessments can be made.
Trihalomethanes (THMs) are perhaps one of the most studied classes of disinfection
by-products.
The purpose of this project was to develop a synthesis for deuterated bromodichloromethane, so that it could be used eventually for the study of the heavy
isotopic effect in bond cleavage. This will lead to a clearer understanding of the
metabolism of this compound. The importance of bromodichloromethane is that past
research has shown that this compound is suspected to be more toxic compared to
chloroform. Therefore it is of great importance to understand the metabolism of this
compound in order to develop any sort of hazard/risk assessment. Deuterated
48 bromodichloromethane was synthesized using an aluminum catalyzed reaction as described by Dougherty (1929). The reaction gave a yield of 28% deuterated bromodichloromethane, in which the desired compound was 99% deuterium enriched.
The reaction proved not only to be quite simple, but BDCM could be purified through simple fractional distillation.
This reaction proved to have an additional advantage, in that it was possible to synthesize not only deuterated BDCM but also deuterated chlorodibromomethane. By allowing the reaction mixture to react for one week, it was possible to obtain considerable amounts of both deuterated BDCM and deuterated chlorodibromomethane.
Because of the considerable success and ease of this reaction, the lack of complex by-products and convenient separation of desired products, this synthetic procedure can be used to generate the desired '^C and '''C-radiolabeled compounds in small scale. The only requirement for the small scale reactions is a preparative gas chromatograph for efficient separation. The use of large amounts of '^C and ^''C chloroform is unrealistic because of cost and in the case of '"C, the undersirability of handling large quantities of radioactive compounds. Work is currentiy being done on setting up a preparative GC, while waiting for the delivery of 10 g of "C-labeled chloroform (99% enrichment) from Cambridge Isotope Inc.. Once synthesized, the '^C-labeled bromodichloromethane, will be used in human studies as a measuring device, to determine the amount of '^COj that is expired by the subjects. This will allow insight into how bromodichloromethane is metabolized in the human body.
49 Recently, the finding of mixed halogenated acetic acids in areas where water contains high concentrations of bromide ion has raised a lot of concern. These acids, like THMs represent a potential health hazard to those who consume these waters.
To date most of the toxicological data available on acetic acids has centered around dichloroacetic acid (DCA) and trichloroacetic acid (TCA). Little is known about the mixed bromo-chloroacetic acids.
A procedure was developed for the synthesis of bromodichloroacetic acid that involved the generation of the bromodichloromethide anion which was subsequently carboxylated using dry ice as the CO2 source. The success of this reaction, prompted a pilot synthetic procedure based on Ba'^COj as a source of CO2. BaCO, was treated with concentrated H2SO4 and the evolved CO2 was condensed in a liquid nitrogen cold trap. The bromodichloromethide anion solution was added to the trap, and the reaction mixture was allowed to warm to -80 °C. Unfortunately, the pilot studies proved to be unsuccessful in obtaining significant amount of bromodichloroacetic acid. Only trace quantities were recovered while using impractically large quantities of BaCOj (5 g). Because it is not possible to use more than 10 mCi (300 - 1000 mg, based on an activity of 20-60 mCi/mol), it was concluded that this synthetic route was not applicable to "C-labeled bromodichloroacetic acid. A second route was proposed that involved the chlorination of commercially available bromoacetic acid. ^'*C-labeled bromoacetic acid is available as either
BrCH2*'*C00H or Br*'*CH2COOH. However, to date no pilot studies, involving the
chlorination of unlabeled bromoacetic acid have been initiated.
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57 Figure 1: Chemical Structures of Trihalomethanes (THMs)
CI CI
H — C — CI H •C CI I I CI Br
Chloroform Bromodichloromethane
CI Br I I H —C—Br H — C ~ Br I I Br Br
Chlorodibromomethane Bromoform
58 Figure 2: Carbon Centered Oxidation Metabolic Pathway for THMs.
Xi Xi HXi Y X2-C—H ----^ X2-C-O-H ----^^-^^ C=0 I I / X3 X3 ^3 Phosgene X2 "O2C j^ \^^^_^Cysteine_ V- \_ O + HX^ + HX3 x/"^3 \ 0/
+ H2O \ ------^ C=0 + HX2
t MalonylCoA-*ͣ-• CO2 + HX3
COo^ i'^'^^Biotin PEP t , Fatty Acid Oxaloacetate —-»- Malate — ͣ*- OAA --^-.^ CO2 Synthesis
RS + 2RSH \ ------^ C=0 + HX, + HX, RS /
2 RSH RSSR ^ - ^» CO + HXo + HXo
+ macromolecules ------•«- Binding in Tissues
CHCI3: Xi = X2 = X3 = Cl CHBraCl: Xj = Xj = Br, X3 = CI CHBrClj: X^ = Br, X2 = X3 = CI CHBrj: Xj = X2 = X3 = Br 59 Figure 3: Halogen Centered Oxidation Metabolic Pathway for THMs O o-
I x,o- Xi Xi I +0 r X2-C—H ------^ Xo-C—H -^-Xo-C—H ^ X2-C^—H I I I I X. X, X3 X3
ox, X2.V HXi I c=o —^ X. -c- H I X x,o X3
X2-C^—H Binding to Macromolecules-*- I S-G X3 I + GSH X2-C-H HOX, I X3
OH + H2O ^^ X5-C-H —*- CO + HX3 I X, HoO
NAD^ NADH Formyltetrahydrofolate ^--^ CO2
Purines Thymine Methionine Glycine CHCI3: Xi = X2 = X3 = CI CHBrjCl: X, = X2 = Br. X3 = CI CHBra: Xi=X2 = X3 = Br CHBrCl2: Xj = Br, Xj = X3 = CI 60 Figure 4: Reductive MetaboHc Pathway for THMs.
X, H X, e I -•ͨ Interactions with macromolecules: X2—C — H ------^^-^^ Xo—C I I Lipids X, X3 Proteins Nucleic Acids
^ X2-C—H
X,
CHCI3: Xi=X2 = X3 = Cl CHBriCl: Xj = Xj = Br. X3 = CI CHBrClj: Xj = Br, X2 = X3 = CI CHBrj: Xi=X2 = X3 = Br
61 Figure 5: Synthetic Reaction Scheme for Bromochloroacetic Acid
COOH COOH / Bro HoC H—C —COOH Ether I \ COOH Br
SO2CI2 Ether
COOH COOH Z^ CI—C-H CI — C — COOH I CO, I Br Br
62 Figure 6: Mass Spectrum of Bromochloroacetic Acid
I III11 III 111111iIII 111111111111111111111111111111111111111111111111111iͣ11111.111.llͣ11111i111ͣ11111
o
H o
o
o in H
o
o
H
o H
o
o o 00' 00
o H O
o
O o m' in iiiHiiiiliiiHiiiHiiiHmi[Minniniiiniiiniiii|iiinim[nininniiiniiiniiiHiiini»ii looinotooinotooiooinoinoinomo O) H OEt Br O I I I II 2Bt2 CH CI — C — cC — H CI C I I I Br H OEt HNOo Br I CI — C — COOH I Br 64 File:Vl6l5 Scan:4 Acq:22-0CT-91 18:57:05 +0:09 70SEQ EI+ Function:Magnet Bpl:4131086 TIC:44567808 File Text:VERMEULEN DIBROMOCHLORO ACETIC PROBE 4. 1E6 100 129 L3.9E6 95i L3.7E6 90i L3.5E6 85i L3.3E6 BOi 13.1E6 75i 173 L2.9E6 70i L2.7E6 65J L2.5E6 60J L2.3E6 55J L2. 1B6 50. 79 Ll.9E6 45J L1.7B6 40J 91 Ll.4E6 35 L1.2E6 30J:48 Ll.0E6 25i L8.3E5 20i L6.2E5 15i 63 145 208 L4. 1E5 lOj 107 160 21 252 L2. 1E5 5J J-O.OEO Oi .iiiii I. II. ^ .1 —|.l.l|i,.i|i„i|. 220 240 260^0 2^280 M/Z 60 80 00 12 140 160 i6o 260 Ic Figure 9: Synthetic Reaction Scheme for Bromodichloroacetic Acid CI OEt CI o I I I II Bro CI — C-CH CI C C—H H^ I I I Br H OEt HNO, CI I CI C COOH I Br 66 File.:V2090 Scaa:2i Acq:21-JJ0V-»l 21:16:16 +0:46 7QSZQ, KE+ Puaction:Maga«t Bpl: 3954626 TIC:27511722 Fil* Text :VERiJDBLSN OICHBRACZTXC ACID PROBS xoo 8.3 .Z25.00- 4.0E6 95J L3.8S6 90j L3.6E6 8SJ L3.4S6 U3 SOJ m C L3.2B6 ' (0 TSJ m 13.0E6 70J iz.szs 2 GSJ 0) L2.6E6 0) €0l 01 L2.4E6 SSJ w L2.2B6 m o SOJ rr L2.0S6 c 45J 3 L1.8S6 127 O 40J it) L1.6B6 w 3SJ n L1.4E6 o 3 30j o |1.2S6. ZSJ o L9.9Z5 tr 20j o L7.9E5 63 o 1SJ CJ L5.9BS o ro 10J rf 99 163 L4.0BS H- sJ O L2.0ES > o l,..|ll.ljl lll.i.ljul.ji -oaWi illn: LO. 0E0 t 80 IdO lio' 140 160J 180 200 220Si 240 2^0 M/2 Figure 11: Synthetic Reaction For Bromodichloroacetic Acid Via Lithium Salt Formation. dry ice I CHBrCl2 t or + LiC4H9 ͣ^ LiCBrCl2 + C02(s) CBr2Cl2 J CI CI O DIH2O I II CI — C — COOH CI- c - c —O — Li^ HCl I Br Br 68 t "70SEDM* -TextiTERMEUIEN EW- PrmctdoniMagaet DICEBRJ^CETIC BpI:B14aiS4 ACID TIC:EI 121417888 I0IU 8? Btz : €3 127 •CTJ o u ssi SOJ -4SJ-4S 117 40J t3.3S€- » a H ' 'I'lm ii .in la n> 1-1 3li ii iiii ss lit) 20J is] SI IDJ 71 173 156 207 5J o 0, lM IlJ ib 1 lili 11 iLi. -JJ. €0 60 100 lio 140 160 180 Qi 260 220 240 260 14 Figure 13: Proposed Scheme for C-Labeled Bromodichloroacetic Acid Ba CO3 + H2SO4 CHBrCl2 t or - + LiC4H9 LiCBrCl2 + CO2 CBr2Cl2 J CI CI o I DIH2O CI — C — COOH CI- c - c— O—Li^ HCl I I Br Br 70 Figure 14: Synthetic Reaction Scheme for Deuterated Bromodichloromethane CI H H 1 D- c-ci + H-C-C— C—Br 1 Cl AlCL Cl H H I I D-C—Cl + H-C-CC-C —Cl I I I H H Br 71 OBSERVE CARBON FREQUENCY 100.573 MHZ in SPECTRAL WIDTH 22624 HZ c ACQ. TIME 0.663 SEC (D PULSE WIDTH 39 DEGREES AMBIENT TEMPERATURE cn NO. REPETITIONS 800 GATED DECOUPLING > M H W SPIN RATE 20 HZ n o H DOUBLE PRECISION ACQUISITION w 2 DATA PROCESSING S O W LINE BROADENING 1.0 HZ 0) ft o FT SIZE 32K &) Ml H TOTAL TIME 8.9 MINUTES ͨ< o N (0 (D C Oj n- (0 ?d M (D fu QJ rt «s~ O rt) r^ ft Oi ͣ<*• I-- 0 a o Ol 3 a CM • n s 1 ,^ <-^ hj OJ o CM rt h- ; o 3 CM in N> •iMi« to >»wJw^iH>,||»MM> I f,iVi*|«i'ii^M»»ifW>N^ SPIN RATE 20 HZ n n DOUBLE PRECISION ACQUISITION DATA PROCESSING 3 n » LINE BROADENING 1 . 0 HZ (+ o FT SIZE 32K 0) Ml CM TOTAL TIME 8.9 MINUTES *< o in CD C Qi ft fD ?d i-( fD OJ O) DJ rt — 03 O fD 00 CTl rt Oi . ͣ>!- H- CO Ol O dd ID 00 P O '^ , in • n Ol n CM in O (+ ^ o 3 jll C n H > i«'4w^wr^^vk<*'y'VfM»iS^ft»»M>>>M»i4i | ' ' ' 1 I I I I I ( I I t t [ t 'l 1 J | i I ] 1 | I I 1 I | I I I 1 | I I I I [ 1 I 1 1 j I I TT [ I. I i I 1 I I i. I [ 80 70 60 50 40 3a PPM 20 Figure 17: GC-MS Results for Fraction 2 (Deuterated BDCM) AlCl^ Catalyzed Reaction. TIC: BDCMl.D [Abundance i 2e+07 - 1.5e+07 le+07 1 1 5000000- -0.a,Ju>^-fULJUUul_JL,.^J^JLjuu» .^JuUuJ '"-XJUuoUuJ 'MlX^J.OjUUOi.>^>*-*-/-*-*^JULa,j^JULJ.JLvwJuJLJki-JU*-JLJLJLjU«-A^>-Jl-a_iOL-*-JuX-j^J_*-Jl-.wVJ^AJ-^J.J^.,A--*vjLJUL 1 1 1 1 1 1 1 1 1 1" , , 1 , ͣ , , , 1 ' 0^^--1—1----T----1----1----1----1----1— 12.00 Time -> 4.00 6.00 8.00 10.00 14.00 1 p^bundance 8 6 Scan ,68 (4.353 min): BDCMl.D - CORRUPT | 6000000- 1 5000000 4000000- 3000000 i:JO 2000000- 1000000- f\ 1 1. , 1 31^ 210 246 278 316 353 386 431 461485 518544 | U -" 1 M/Z -> 100IflU^—[-"V^ 150 200 250 300 350 400 450 500 550 | 76 Figure 18: GC-MS Results for Fraction 3 (Deuterated BDCM) AlCl^ Catalyzed Reaction. [Abundance TIC: BDCM2.D 1 ^ 2.5e+07 - 2e+07 - 1.5e+07 - le+07 - 5000000 - v-j»-<-JUUuJ '^-J->-tJOl_n_>_«_l_kJ *-ͣ*—^--'^-ͣ'—*^-*~~'-*—*~J--i-^_JLj_^^ __LJU>_.VwJ>w*_J ^^A_J~-*-^--A->-JUjl^ A-_A—>L_>wi-*-A-*^J'- 1 1 1 1 1 ' ' 1 ' 1 1 1 1 1 1 1 1 ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1 ' ' 1 1 1 1 1 0 -h—1—1—I— 10.00 11.00 rime -> 4.00 5.00 e• .00 7.00 8.00 9.00 12.00 1 Abundance Scan 67 (4.341 min): BDCM2.D - CORRUPT 86 6000000 5000000- 4000000 130 3000000 2000000- 1000000 Ifty 200 26378 305 346 393409 46476_j---—.J---1 506 540r "T" 1—I ͣ"•(ͣͣ I—I ͣͣ I "r—I—I—r^T—1—I—[^—1—I—1—1—]—I—I—1—1—I—1 ] 1 I I 1 I I I I I I I I I ' 1 r- M/Z -> 100 150 200 250 300 350 400 450 500 550 77 Figure 19: Synthetic Scheme for Deuterated Chlorodibromomethane Via an Aluminum Chloride Catalyzed Reaction. CDCI3 + C2H5Br ^^^^^ . C2H5^ + BrAlCl3 + D-C-Cl I Br Br I D C- 01 + CIAICI3 Br 78 o ID CO 01 in o ID ~D OPSETRVE CARSON o ifl n cn C FREQUENCY 100.573 MHZ CM h SPECTRAL WIDTH 22624 HZ (T) ACQ. TIME 0.663 SEC to o PULSE WIDTH 39 DEGREES AMBIENT TEMPERATURE NG REPETITIONS 800 GATED DECOUPLING O O SPIN RATE 20 HZ s DOUBLE PRECISION ACQUISITION n to DATA PROCESSING rt O LINE BROADENING 1.0 HZ Hi M FT SIZE 32K •< O N o TOTAL TIME 8.9 MINUTES O c o ft (D OJ rt O rt H- O CO 3 o o • * CO o -M 0C O H — oo 0) o W rt (D (U o O 3 rt H- O 3 iil^i^mii00(t^^ KiiHiii'inyii m]qtfiwi*m* mmmimim** r i i i | i i i i [ i i i i | i i i i | i i i i | i i i i [ i i i i | i i i i [ i i i i | i i i i [ i i i i | i i i i | i i i i | i i i i [ i i i 'i | i i i i [ i i i i | i i i i I i i i i | i i i i i i 180 150 140 120 100 80 60 40 20 PPM 0 CO o lyJUM(mft'#)n» ^x^w^^n,!^ »w^i, {mm w>*v'**fii ro *i (D o» us Cl of Deuter talyzedfU R ft 'NJ ͣ* O (D ft Pi d en H- •n N i on (7 Day Reaction). BDCM (Fraction 3) rrrr I I I I | M I I | 1 M I | I 1 I I | I I I I | I 1 I 1 I I 1 i I 11 I I TTTTTT rfTTTTp TTT I'l 1 i 1 i I I 1 1 I1 1 i 1 | i M i 1 180 160 140 120 100 80 60 40 2 0 PPM 0 C h CO to mW^mii^^^w^^^ I I I I I [~~I I i I-? i i I I | I I I I I i I T i i i i i i i i r i i i i I i n i | i i i | i i i i I i i i i | 80 70 60 50 40 30 PPM 20 in en OBSERVE CARBON FREQUENCY 100.573 MHZ SPECTRAL WIDTH 22624 HZ AGO. TIME 0.663 SEC PULSE WIDTH 39 DEGREES AMBIENT TEMPERATURE NO. REPETITIONS 800 GATED DECOUPLING SpIN RATE 20 HZ DOUBLE PRECISION ACQUISITION DATA PROCESSING LINE BROADENING 1 . 0 HZ FT SIZE 32K 00 TOTAL TIME 6.9 MINUTES •-' o ro — 00 m cn co lTJ "J CO i I'l I | I i I i | i i I 1 | I i I i I I I i I I I I | i I I TTTTTTT TTT 1111 i 1111 I [ I 1111111111 I I'l 111111111 111111 111 111111 i 11 180 160 140 120 100 80 50 40 20 PPM 0 00 y^WMftWHtfb^^ ^V^Jim^^^Ww^W^v^^ "•^ͣ'wt.y^4t-^^^M F I I 1 I I I I I i I I i I i I I 1 I I I I i I i rn i i i i i i i i i i i i n i i i i r i i | i i i i I i i i i [ 80 70 60 50 40 30 PPM 20 OBSERVE CARBON FREOU=.NCv 100 5^3 MHZ 5PECTRA'_ WID^H 22t24 HZ ACG. tI\IE 0.663 SEC C,JLCE U1CTH 3? DE3REES AMBIENT ͣ"EMP£RAT'»RE NC RE::,ETITIOiN:S LOO GATED DECOUPLING SPIN tvATE 10 HZ rOUBLE. PRECISION A^QUISITIO.I da"a pr: :Er:iNS LINE SRCaDENING 1.^ HZ FT :iZE 32< 00 U1 "OTAu "; IxlE £ 9 MINUTET h n a Cb 0*fimmmMwmm iHmH**m*^^ 1111111111111111111111 I I | i i i I J I I I I | I I I I I I I M | I I I I I I I I I | I I I I I i I I I | I | I I I 1 | I I I I | I I I I j I i I I |.l I I 1 | I 180 160 140 120 100 80 60 40 20 PPM 0 c n (D > 00 *ftf^M»l4fWM if i i i i i I iiit r~i i i i | i i i i i i r i i i r i i | i i i i I i I I | i 1 I I i i t i i i i i i | i i i i | 80 70 60 50 40 30 PPM 20 Figure 24: Proposed Synthetic Scheme for Deuterated Bromodichloromethane Via Lithium Salt Formation. CHBrCl2 + LiC4H9 ------*- LiCBrCl2 ^^Q „ CDBrCl2 87 OBSERVE CARBON tQ FREQUENCY 100.572 MHZ c n a SPECTRAL WIDTH 22624 HZ eo f^ _ ACQ. TIME 0.663 SEC ,- O) ci PULSE WIDTH 39 DEGREES AMBIENT TEMPERATURE NO. REPETITIONS BOO GATED DECOUPLING J77S SPIN RATE 20 HZ 2 DOUBLE PRECISION ACQUISITION S DATA PROCESSING LINE BROADENING 1 . 0 HZ FT SIZE 32K U) to ^ 00 00 TOTAL TIME 8.9 MINUTES * CO l*MiWrtN*llrt^^ ^>^>#>i^(^^W mmm i.... i CZ-U »a c ^ n (D to CO j ͣ i; i T j i. i •! 1 ^j i i i i j I i i i j I i i i j i i i i i I i i i j i i I i j i i I i j i i i i j i i i i j i ] i T o : 'ͣ70 -. ͣ .= • .. A*-* 3 J 40 30 PPM 20 # Flle:V2753 Scan:10 Acq: 9-APR-92 03:49:53 +0:19 70SEQ E1+ Function:Magnet Bpl:4677526 TIC:72258400 File Text:HEIDI CDCI.2BR EI PROBE 100 57 .4.7E6 c 95i .4.4E6 90i .4.2E6 to 85i .4,0E6 50 2 80J .3.7E6 (D D) 01 0) o cn 75J .3.5E6 (i- H- cn 0 T3 70J .3.3E6 D (D 71 •^ a • rt 651 .3.0E6 t-i c 3 sol .2.8E6 O Ml 55i .2. 6E6 o • (D 50i .2.3E6 C 85 rt (D 45J .2.1E6 n tu rt 40 J .1. 9E6 (D a. 35i 97 .1.6E6 D .1.4E6 o 30j 109 s 25J .1.2E6 2o| .9.4E5 ft .1. 0E5 c 15j 91 il3 3 1 ni 122 143 103 L4.7E5 CO 119 135 0) ¥-^ 167 5i 131 L2.3E5 OJ—iiiiiiiii|i| |i!ltl!UljlHiit'titl|i|l(iiMifliifkhM'»if'^'^^ LI^O.OEO 50 60 70 80 90 lOO 110 120t 130t 140 150 160 170 1^0 190 20020 M/Z Figure 27: Proposed Reaction Scheme for Deuterated Bromodichloromethane Via Bromination of the Dichloromethide Anion. CD2CI2 + LiC.Hg JTHF^ LiCDClo + Bu-D Bro CDBrCl2 + LiBr 91 Figure 28: Proposed Reaction Scheme for Deuterated Bromodichloromethane Via Lithium Aluminum Deuteride. CBr2Cl2 + LiD ^!^^^ * CDBrCl2 + LiBr 92 Figure 29: Generation of Dichlorocarbene Under Catalytic Two-Phase Conditions (Fedorynski et al., 1977). CHCi3„,g. + ^N^^cr^rg. + NaOH CCl," N. + NaClaq / \ + H20,q org CCV/N^ org CCl2org. + )N;^Cl-,rg. 93 Figure 30: Synthesis of BDCM By Reacting Chloroform and Bromoform Under Alkaline Conditions (Fedorynski etal., 1977). CHBr3 + CHCI3 NaOH aq CHBr3 + CHBr2Cl + CHBrCl2 + CHCI3 + CCI4 + CBr4 * Triethylbenzylammonium Chloride (TEBA) phase transfer catalyst 94 Table 1: Haloacids Identified as Drinking Water Chlorination By-Products in a 10-City Survey (Stevens et al, 1990). Haloacid Number of Supplies Sampled Chloroacetic 6 10 Dichloroacetic 6 Trichloroacetic 4 Bromochloroacetic 9 2,2-Dichloropropanoic 2-Chloropropanoic 8 3,3-Dichloropropenoic 5 9 Chlorobutanedioic, isomer 2 2,2-Dichlorobutanoic Chloro-methoxy-dicarboxylic 1 95 Table 2: Quarterly Median Values for Haloacetic Acids in U.S. Drinking Waters (Krasner er a/., 1989). Spring 1988 Summer 1988 Autumn 1988 Winter 1989 Haloacid (ugA^) (ug/L) (ugyl.) (ug/L) Chloroacetic <1.0 1.2 <1.0 1.2 Dichloroacetic 7.3 6.8 6.4 5.0 Trichloroacetic 5.8 5.8 6.0 4.0 Bromoacetic <0.5 <0.5 <0.5 <0.5 Dibromoacetic 0.9 1.5 1.4 1.0 Total Haloacids 18 20 21 13 96 Table 3: Cancer Incidence in Mice Exposed to Chloroacetic Acids in Drinking Water (Herren-Freund et al., 1987), Prior ^ Concentration of 2 Mice With Carcinomas Initiation Chloroacetic Acid Carcinomas (%) per Mouse (No.) 0 - NaCl (22) 0 + NaCl (22) 5 0.05 t 0.05 - 39 mM DCA (26) 81 1.69 - 0.29 1.47 - 0.24 + 39 mM DCA (32) 72 1.17 - 0.22 + 16 mM DCA (29) 66 - 31mMTCA(22) 32 0.50 t 0.17 + 31mMTCA(23) 48 0.57 t 0.21 0.64 - 0.14 + 12mMTCA(33) 48 2.5 ug ethylnitrosourea/g body weight at 15 d of age. 9 Parentheses indicate the number of mice at sacrifice. 97 Table 4: Mixed Halogenated Acetic Acid Melting Points m.p. (lit, values ) °C m.p. (exp. values) °C BrClCHOOH 25 Br2ClCCOOH 108 - 110 97-102 BrCl2CCOOH 78 - 80 69 - 74 98 Table 5: Mass Spectral Data for Mixed Halogenated Acetic Acids and Deuterated BDCM. Compound (M/Z) Nt -COoH M""-Br M" -C02-Br M^"-CI CHClBrCOOH^'^ 172 127 93 49 137 (Figure 6) (3,5.5,2.5) (0.9,0.2,0) (41.5,42.3) (8.8,3,0.5) (0.9,3.9) CClBr2COOH'' 250 205 171 127 215 (Figure 8) (2, 5.9,5,2) (4,9.2,6.8,1.2) (52,73.5,35) (86.9,100,37.9) (6.5,12.1,6) CCl2BrC00H^ 206 161 127 83 171 (Figure 10) (2.5,4,3.2,1.2) (6,9.1,4.8,1.2) (41.5,37,8.5) (100,73.5,16.8) (2,2.5,1) CDCl2Br*' 163 84 128 (Figure 17) (weak) (48.4,100,30.8) (32.4,40.7,9.9) ^ In spectra, M"^-Br and M'^-Cl daughter peaks appear to absorb a hydrogen. Numbers in parentheses represent the relative intensity of peaks at X, X+2, X+4, and X+6 respectively due to chlorine and bromine isotopes. 99 Table 6: ^^C NMR Chemical Shifts (5) Compound 5 (ppm) d-Acetone 29.8 (7) d-Bromodichloromethane 59 (3) d-Chlorodibromomethane 37 (3) d-Chloroform 77 (3) d-Methylene Chloride 53.8 (5) Tetrahydrofuran 67.4 (1) () indicates multiplicity 100