Metabolism and pharmacokinetics of alternate drinking water disinfectants.

The chlorination of surface waters is known to elevate trihalomethanes; consequently, chlorine dioxide (ClO2) is being considered as an alternative disinfectant. The primary products resulting from ClO2 disinfection of waters are chlorites (ClO2-) and chlorates (ClO3-). Studies in rats revealed that ClO2 is converted to chloride (Cl-), ClO2- and ClO3-. ClO2- and ClO3- are excreted as Cl-, ClO2- and Cl-, ClO2-, ClO3-, respectively. Radioactivity was rapidly absorbed from the gastrointestinal tract following the administration of 36ClO2 orally, and the half-life for the elimination of 36Cl from the rat was 44 hr, corresponding to a rate constant of 0.016/hr. After 72 hr, radioactivity was highest in plasma, followed by kidney, lung, and stomach. 36Cl in plasma reached a peak at 2 hr and 1 hr after oral administration of 36ClO2- and 36ClO3-, respectively. 36Cl excretion was greatest 24 hr after the administration of 36ClO3-, but in the case of 36ClO2-, the excretion probably represented saturation of the biotransformation and excretion pathway. A low activity in packed cells compared to plasma was detected in chlorate ingestion, rather than an even distribution in chlorite treatment. Chloroform determinations in rat blood after one year indicated that chloroform was significantly higher than control in the 100 and 1000 mg/l. ClO2 groups. However, no significant values were observed in the 1 or 10 mg/l. ClO2 and ClO2 metabolites groups. ClO2 and its metabolites are eliminated from the body more rapidly than chlorine, and they do not appear to increase trihalomethane concentrations at low dosages.


Introduction
Chlorination is, and always has been, the most widely used method for disinfection of public water supplies. Concern over its possible adverse effects and those ofits by-products on humans has increased considerably in recent years. The formation of trihalomethanes such as chloroform (CHC13), bromodichloromethane (CHBrC12), dibromochloromethane (CHBr2Cl) and bromoform (CHBr3) during the chlorination process has become evident (1,2).
The United States Environmental Protection Agency conducted a comprehensive survey of 80 water supplies in the U.S. for halogenated organics (3). It was found that the four trihalomethanes CHCl3, CHBrCI2, CHBr2CI, CHBr3-are widespread in chlorinated drinking water in the U. S. and result from chlorination, since these substances were not found in raw waters. The trihalomethanes were not a result of impurities in the chlorine being used but were a result of the reaction of chlorine with precursor. Morris (4) made the point that the number of trihalomethane precursors is extensive and includes ethanol, acetaldehyde and the methyl ketones. Rook (1) and Morris (4) indicated that the bromide ion present in a natural water is rapidly oxidized by aqueous chlorine to hypobromous acid (HOBr), and that this acid reacts with precursor to account for the formation of the brominated species.
Since these trihalomethanes may be a health hazard, chlorine dioxide (CI02) is one of the alternative disinfectants being considered because it prevents the formation of trihalomethanes (5,6). C102 is a more effective oxidizing agent than chlorine and greatly improves the taste of water by avoiding the unpleasant taste associated with chlorination of water (7). The unpleasant taste of chlorine in water is caused partially by the build-up of chlorinated phenols.
Miltner (6) has reported that the primary products resulting from C102 disinfection of surface waters include chlorites (01021 and chlorates (C1030, which appear in concentrations of 50% and 30% of C102 demand, respectively. Toxicity studies demonstrated that rats drinking C102, C102and C103-(Cl compounds) daily for 9 months exhibited depressed red blood cell counts, hemoglobin concentration and packed cell volumes. Also, these C1 compounds in drinking water for three months inhibited the incorporation of 3Hthymidine into nuclei of rat testes (8).
The studies described in this report were conducted to provide information on metabolism and pharmacokinetics of chlorine, chlorine dioxide, chlorite and chlorate in the rat. Also, the effect of C102 in drinking water on the formation of CHC13 in rat blood and various organs was studied.

Methods
Synthesis of 36C1 Compounds H36C1 was obtained from New England Nuclear.
Kinetics of Chlorine Dioxide, Chlorite, Chlorate and Chlorine in the Rat Four groups of four male Sprague-Dawley rats (230 ± 20 g) each were used in these experiments. 36C0 compounds were administered by gavage as follows: 3 ml of 100 mg/I. 36C102 (0.7 ,uCi) was administered to each rat, producing a dose of 1.5 mg/kg body weight; 3 ml of 10 mg/ 36CIO2-(0.17,ui) ABDEL-RAHMAN, COURI AND BULL was administered to each rat, producing a dose of 0.15 mg/kg body weight; 3 ml of 5 mg/I. 36CO3-(0.85 ,uCi) was administered to each rat, producing a dose of 0.065 mg/kg body weight; 3 ml of 250 mg/I. H036C1 (0.45 ,uCi) was administered to each rat, producing a dose of 3.26 mg/kg body weight.
Heparinized blood samples were collected at 5, 10, 20, 30 and 60 min, and 2,4,8,24 and 48 hr by orbital sinus puncture. The blood was centrifuged at lOOOg for 15 min to separate the red blood cells from the plasma. Then the radioactivity was counted in a liquid scintillation counter (Beckman LS 7500), and the rate constant and T1/2 of absorption and elimination from plasma were calculated for each compound.

Distribution of Cl Compounds in the Rat
After 72 hr following the administration of 36C1 compounds as described previously, rats were killed by decapitation, and blood was collected in heparinized tubes. Tissue specimens of stomach, testes, lung, kidney, duodenum, ileum, spleen, liver, bone marrow, carcass and skin were prepared for the determination of 36C1 content by liquid scintillation spectrometry as follows: Whole blood and packed cells were prepared according to the method of Mahin et al. (10). Samples of wet tissue weighing up to 200 mg and samples of bone marrow up to 50 mg were placed in individual glass LSC vials with Teflon or polyethylene-lined caps. Then 2 ml Protosol was added to each vial, which was capped tightly to prevent loss of solvent. Samples were solubilized by heating overnight at 5000, then cooled at room temperature; afterwards 0.2 ml of 30% H202 was added for decolorization. The samples were heated at 5000 for 30 min, then cooled, and 15 ml of Aquasol-2 was added.
Sample quench effects were corrected by using the method of channel ratios, and efficiency was greater than 80%.

Metabolism and Excretion Studies
Four groups of four male Sprague-Dawley rats/ group were used in these studies. Each group was administered orally the same concentrations of 36C1 compounds described above. Animals were housed in modified Roth all-glass metabolism chambers for the collection of expired air and fecal and urine samples. The radioactivity of the total 36C1 compounds was measured. The analysis of radioactive metabolites (W-, C102-, C1031 was performed as described in a previous report, (9), and measured as a percentage of the initial dose.

Effect of Chlorine Dioxide, Chlorite and Chlorate on the Formation of Chloroform in Rat Blood and Organs
Male Sprague-Dawley rats (50-170 g) were used in this experiment. The animals were housed in a controlled environment (temperature, 22°C; humidity 50%) with a 12 hr light/dark cycle beginning at 6:00 A.M. Food was available ad libitum. Groups of rats were allowed to drink doubly distilled water containing 10 or 100 mg/l. C102, C102or C103for 20 hr/day, 7 days/week for 12 months. C102 was generated daily and water concentrations determined by the DPD (diethyl-p-phenylenediamine) method of Palin (11).
After one year, rats were killed by decapitation and blood collected in heparinized tubes. Tissue samples of liver, kidney, spleen, brain, and testes were homogenized in isotonic sucrose medium. Tissue homogenate or blood sample (3 ml) was extracted by 5 ml pentane. The pentane layer was then separated by centrifugation at lOOOg for 5 min. A gas chromatograph equipped with an electron capture detector was used for the quantitation of chloroform in each sample, with bromodichloromethane being used as the internal standard. 21

Kinetics of Cl Compounds in the Rat
The rate constant and T1/2 for absorption and elimination from plasma of Cl compounds are shown in Table 1. The rate constant for absorption was highest for 36CI02 (3.77 ± 0.24/hr) followed by 26CI03-, 36C102-, and lowest for HO3CI (0.157±0.001/hr).

Distribution of Cl Compounds in the Rat
The distribution of 36CI compounds after 72 hr from the time of oral administration is shown in Table 2. Values are expressed as a percentage of aValues were calculated from the time course of the elimination from rat plasma; n = 4 rats per treatment.  aValues represent the mean ± SE as percentage of the initial dose from four rats per treatment, throughout the 72 hr studied. bNone detected.
the initial dose. The distribution of 36C102 was highest in the kidney, followed by lung, plasma, stomach, ileum, liver, duodenum, spleen and bone marrow, while the distribution of HO36C1 was highest in plasma, followed by bone marrow, kidney, testes, lung, skin, duodenum, spleen, stomach, liver, carcass and ileum. The distribution of 36C102was highest in plasma, followed by stomach, testes, skin, lung, duodenum, kidney, carcass, spleen, ileum, bone marrow and liver. In the case of 36CI03-, plasma was highest, followed by stomach, lung, testes, kidney, skin, duodenum, spleen, ileum, carcass, liver and bone marrow.
In whole blood, when plasma was separated from packed cells, radioactivity was divided more or less 40.14 ± 2.14 3.14 ± 1.0 H036C1 (250 mg/I.) 21.52 ± 2.51 7.09 ± 0.24 aValues represent the mean ± SE as percentage of the initial dose from four rats per treatment, throughout the 72 hr studied. 36Cl was not detected in expired air.

Metabolism of Cl Compounds
The administered 36CI compounds are found as various metabolites in rat urine, as shown in Table  3. After 72 hr from the time of administration, 36C102 and 36CI03-are found in urine as C1, C1O2and C103-whereas 36ClO2-is found as Cl-and C102-. In all three cases, Clis the major metabolite (approximately 87, 84 and 64% of the total radioactivity found in urine for 36CIO2,36CIO2and 36CIO3-, respectively).

Excretion of Cl Compounds
The total of the 36CI compounds recovered in the urine and feces 72 hr after administration was determined for each compound and calculated as the percentage of the initial dose. In the case of 36C102, 75% of the recovered dose was found in the urine, while 25% was found in the feces. For 36CIO2-, 87 and 13% was found in urine and feces, respectively; and for H036CI, 76 and 24% was found in urine and feces, respectively (Table 4). 36C1 compounds were not found in the expired air of any treatment group throughout the 72 hr time period.
Effect of Cl Compounds on the Formation of Chloroform in Rat Blood and Organs Table 5 shows the effects of chronic treatment with chlorine dioxide and its metabolites for one year on the formation of chloroform in rat blood, liver, kidney, spleen, brain and testes. A concentration of 100 mg/l. C102 daily in the drinking water caused an increase in chloroform levels in blood, liver, testes and brain. A concentration of 10 mg/l. C102 increased chloroform levels in rat testes, while levels in blood, liver, kidney, spleen and brain remained unchanged. Treatment of rats with 100 mg/l. C102for one year elevated chloroform levels in the liver and brain, while the levels in blood were not affected. Treatment with 100 mg/l. Cl03-increased liver but not blood chloroform concentrations after one year.

Discussion
From the kinetics study, it is apparent that C102 and its metabolites (C102and C1030 are more rapidly absorbed into the bloodstream than HOC1. The effect is very pronounced when comparing C102 itself with HOCI. Likewise, HOCI is more slowly eliminated from rat plasma than C102 or its metabolites.
The distribution study revealed that although plasma and whole blood contain the highest activity, the bone marrow contained far less in 36C102, 3C102-and 36C103 treatment, while HOCI had a higher activity in bone marrow. The high activity in testes, even after 72 hr, indicates a possible pharmacologic action at this site. Effects of these compounds on the reproductive system are currently in progress in our laboratory.
It is important to indicate that after 72 hr, high amounts of radioactivity were still found in the stomach, duodenum and ileum.
The Cl compounds studied are eliminated mainly by kidney and intestinal routes, since no radioactivity was found in expired air. For H036C1 and 36C102 treatment, about 75% of the recovered dose was found in the urine; in the case of 36CI02-and 36CI03 , about 90% of the recovered dose was in the urine.