Studies of the toxic interactions of disinfection by-products.

A large number and variety of compounds are formed in the process of chlorinating drinking water. The classes of compounds formed include trihalomethanes, haloacetic acids, haloacetonitriles, halophenols, and halopropanones. Many of the compounds have been shown to be toxic and are currently being further evaluated by the U.S. Environmental Protection Agency (EPA). One group of the halopropanones found in chlorinated drinking water is the dichloropropanones. The toxicological properties of this group have not been well characterized. In addition, a number of investigators have shown that ketones potentiate the hepatotoxicity of haloalkanes. We conducted a series of studies to explore both the toxicity of the dichloropropanones and their potential interactions with a well-characterized haloalkane, carbon tetrachloride. A variety of toxicological and biochemical endpoints were used to evaluate the toxicity of the dichloropropanones and their interaction with CCl4, including cytochrome P-450 concentration, reduced glutathione levels, pentane generation, serum enzyme activities, and histopathology. Administration of 1,1-dichloropropanone (DCP) resulted in elevated serum enzymes associated with periportal necrosis. Glutathione levels were reduced by the administration of 1,1-DCP; pentane generation was not increased. When 1,1-DCP was given prior to CCl4, the data were consistent with additivity. Administration of 1,3-DCP did not result in elevated serum enzymes, nor was there histopathologic evidence of necrosis. Glutathione levels and pentane generation in the 1,3-DCP-treated groups were the same as those of controls. Inhibition of the toxicologic effects of CCl4 in a dose-related manner was observed when 1,3-DCP was administered prior to CCl4.

Mechanistic aspects of ingested chlorine dioxide on thyroid function: impact of oxidants on iodide metabolism.

Toxicological studies dealing with recent findings of health effects of drinking water disinfectants are reviewed. Experiments with monkeys and rodents indicate that the biological activity of ingested disinfectants is expressed via their chemical interaction with the mucosal epithelia, secretory products, and nutritional contents of the alimentary tract. Evidence exists that a principal partner of this redox interaction is the iodide of nutritional origin that is ubiquitous in the gastrointestinal tract. Thus the observation that subchronic exposure to chlorine dioxide (ClO2) in drinking water decreases serum thyroxine levels in mammalian species can be best explained with changes produced in the chemical form of the bioavailable iodide. Ongoing and previously reported mechanistic studies indicate that oxidizing agents such as chlorine-based disinfectants oxidize the basal iodide content of the gastrointestinal tract. The resulting reactive iodine species readily attaches to organic matter by covalent bonding. Evidence suggests that the extent to which such iodinated organics are formed is proportional to the magnitude of the electromotive force and stoichiometry of the redox couple between iodide and the disinfectant. Because the extent of thyroid uptake of the bioavailable iodide does not decrease during ClO2 ingestion, it seems that ClO2 does not cause iodide deficiency of sufficient magnitude to account for the decrease in hormonogenesis. Absorption of one or more of iodinated molecules, e.g., nutrients, hormones, or cellular constituents of the alimentary tract having thyromimetic or thyroid inhibitory properties, is a better hypothesis for the effects seen.

Subchronic toxicity of chlorine dioxide and related compounds in drinking water in the nonhuman primate.

Subchronic toxicities of ClO2, NaClO2, NaClO3 and NH2Cl were studied in the African Green monkeys (Cercopithecus aethiops). The chemicals were administered in drinking water during 30-60 days subchronic rising dose protocols. The only unexpected and significant toxic effect was elicited by ClO2; this chemical inhibited thyroid metabolism in the animals at a dose of ca. 9.0 mg/kg/day. A statistically significant decrease of serum thyroxine occurred after the fourth week of exposure to 100 mg/l.concentration. The extent of thyroid suppression was dose dependent in each individual monkey, and was reversible after cessation of exposure. NaClO2 and NaClO3 failed to elicit similar effects in doses up to ca. 60 mg/kg/day. Also, NaClO4 or NH2Cl did not cause T-4 suppression in doses of 10 mg/kg/day. The selective thyroid effect of ClO2 was unexplained and it appeared to be paradoxical since ClO2 was rapidly reduced by the oral and gastric secretions to nonoxidizing species (presumably Cl-). No evidence of thyroid effects were detected in the serum of human volunteers who ingested approximately 1 mg/l. of ClO2 in drinking water as a result of routine use in the community water treatment process. Sodium chlorite induced dose-dependent oxidative stress on hematopoesis, causing decreased hemoglobin and red cell count and increased methemoglobin content. At the same time, serum transaminase (SGPT) levels showed significant subclinical elevation. The hematologic effects of NaClO2 rebounded during exposure indicating compensatory hemopoietic activity taking effect during oxidative stress. Sodium chlorate and chloramine did not induce detectable hematologic changes in the animals.

Immunotoxicologic evaluation of chlorine-based drinking water disinfectants, sodium hypochlorite and monochloramine.

Male Sprague-Dawley rats were exposed to chlorine-based disinfectants in the drinking water from weaning to 12 weeks of age, at which time they were terminated and assessed for immune competence. Chlorine-based drinking water disinfectants used were sodium hypochlorite (5, 15 and 30 ppm) and monochloramine (9, 19 and 38 ppm). Parameters of immunity measured were spleen and thymus weights, antibody production, delayed-type hypersensitivity (DTH) reactions, natural killer cell (NKC) cytotoxicity, oxidative metabolism response (i.e chemiluminescence-CL) and phagocytosis by macrophages, and production of 2 immunoregulatory cytokines, interleukin 2 (IL2) and prostaglandin E2 (PGE2). Significant (P less than or equal to 0.05) reductions of spleen weight, DTH reactions, and oxidative metabolism by macrophages were observed only in groups of rats exposed to high levels (30 ppm) of sodium hypochlorite, while PGE2 production was elevated. Rats exposed to the higher doses of monochloramine had reduced spleen weights (38 ppm), decreased antibody synthesis (9 and 19 ppm) and augmented PGE2 production (19 and 38 ppm). These results extend the earlier observations of others that macrophage function of laboratory rodents may be impaired by exposure to high concentrations of chlorinated drinking water. Furthermore, the function of other major populations of immunocytes and types of immune responses may also be altered following subchronic exposure to high concentrations of chlorinated drinking water. These types of effects on the immune system are a previously unrecognized potential side-effect of the ubiquitous practice of disinfection of water with chlorine compounds. Alteration of immune function of chlorine-based disinfectant-exposed rats in this study was only evident at relatively high doses, and only selected immune responses were altered. It appears, therefore, that these chlorine-based disinfectants are not particularly strong immunodepressants. However, further studies in different species may be warranted in order to better extrapolate to implications to human health following chronic low-level exposure.

Iodination of nutrients in the presence of chlorine based disinfectants used in drinking water treatment.

Under conditions simulating the gastrointestinal tract chlorine dioxide (ClO2), HOCl, and NH2Cl caused covalent organification of iodide to nutrient biochemicals. The extent of binding seemed to be proportional to the electromotive force (EMF) and stoichiometry of the redox couple between iodide and the oxidant. Almost half of 71 nutrients examined were found to bind reactive iodine. Iodide was found to inhibit the quinoidal chromogen formation from tyrosine and ClO2, demonstrating the preferential generation of reactive iodine in complex organic mixtures. These findings indicate that ingestion of residual disinfectants via drinking water may pose a health risk in terms of in vivo generation of iodinated organics. Structure, formation, and biological activity of these compounds are under study.

Chlorine dioxide water disinfection: a prospective epidemiology study.

An epidemiologic study of 198 persons exposed for 3 months to drinking water disinfected with chlorine dioxide was conducted in a rural village. A control population of 118 nonexposed persons was also studied. Pre-exposure hematologic and serum chemical parameters were compared with test results after 115 days of exposure. Statistical analysis (ANOVA) of the data failed to identify any significant exposure-related effects. This study suggests that future evaluations of chlorine dioxide disinfection should be directed toward populations with potentially increased sensitivity to hemolytic agents.

Toxicology of drinking water disinfection byproducts from nutrients. Rate studies of destruction of polyunsaturated fatty acids in vitro by chlorine-based disinfectants.

As model reactions between unsaturated fats and water disinfectants in the GI tract, relative rates of destruction of seven polyunsaturated fatty acids (L, alpha Ln, gamma Ln, Ara, EPA, DH, and DT) by OCl- and NH2Cl were investigated in vitro. Using millimolar solutions of seven PUFAs combined with various OCl- mole ratios, disappearance of PUFAs was followed by UV spectrophotometry at pH = 9.5 and at 35 degrees C via conjugated hydroperoxydienes at 234 nm. While OCl- rapidly destroyed all PUFAs, NH2Cl was inert. Overall second-order rate constants computed for L at increasing times disclosed that the attack on the cis-CH=CHCH2CH=CH moiety by OCl- does not follow simple second-order kinetics. Using a logit-log transform and second-order polynomial regression analysis of L's disappearance in a stoichiometric ([L] = 1.2 mM; [ClO-] = 2.4 mM) mix, data were analyzed by the time ratio method of Schwemer and Frost. These agreed with a sequential system of at least two irreversible second-order reactions having k1 = 15.6 L.mol-1.s-1 and k2 = 2.6 L.mol-1.s-1. Preliminary GC/MS analysis indicated that the initial product is a mix of chlorohydrin isomers. These undergo second addition of HOCl and/or lose halogens and polymerize. Additional minor products were also C5-C9 mono- and bifunctional carboxylates and mixed acid aldehydes. Studies with mol equiv of Cl- - free 36ClO- allowed estimation of covalent binding of Cl by L at various times, supporting the kinetic findings. For other PUFAs of higher degree unsaturation, the complexity of feasible reactions precluded an analogous approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chlorine dioxide on thyroid function in the African green monkey and the rat.

In a previous study from this laboratory, chlorine dioxide (ClO2) treated drinking water depressed thyroxine (T4) levels in the African green monkey. The present study again demonstrated a decrease in T4 levels in the same species after 4 wk of oral exposure. However, after 8 wk of treatment T4 levels rebounded to above pretreatment levels, coinciding with an increase in thyroid radioiodide uptake. This T4 rebound phenomenon and increased iodide uptake may be due to a compensatory endocrinological mechanism. In rats, T4 levels dropped during the 8-wk ClO2 treatment period in a dose-dependent manner, and no rebound effect was observed. Iodide uptake values in the rat were not affected. It appears that ClO2 may have an effect on thyroid function in both species.

An automated method for the determination of sulfate.

An automated method of sulfate analysis is described, which can detect sulfate concentrations as low as 2.5 mug per ml water. The assay is based on the reaction of sodium rhodizonate and barium forming a colored complex. Sulfate quantitavely interferes with this reaction. The assay is reproducible in the range of 2.5 to 30 mug sulfate per ml water. This method conveniently and accurately measures water soluble sulfate filtered from the air, and is especially useful in assaying samples containing microgram quantities of sulfate from experimental inhalation apparatus.

Subchronic toxicology of humic acid following chlorination in the rat.

A subchronic 90-d study was conducted with chlorinated and nonchlorinated humic acids using male Sprague-Dawley rats. Body weight gain, terminal organ and body weights, food and fluid consumption, clinical chemistries, hematological parameters, and urinalyses were determined for all animals. Selected organs were examined microscopically. Significant findings were confined to those rats given the high dose of chlorinated humic acid (1.0 g/l total organic carbon). The terminal body weight and average weekly body weight gain were significantly lower (p less than 0.05) in the high-dose group as compared to the distilled-water control group. This difference can be partially explained by a 16% lower daily fluid consumption. The average weight of the kidneys was significantly higher in the 1.0-g/l chlorinated humic group as compared to distilled-water controls. Hematological parameters and clinical chemistry values were normal in all treatment groups. The most significant finding was the increased incidence and severity of hematuria in the 1.0-g/l chlorinated humic acid group. A thorough histopathological examination of the entire urinary tract indicated that the most likely cause of the more severe incidences of hematuria in the rats was caused by crystalline deposits in the renal pelvis.

Effects of ClO2 on the absorption and distribution of dietary iodide in the rat.

Aqueous chlorine dioxide (ClO2), an alternative disinfectant for drinking water, was found to decrease gastrointestinal (GI) bioavailability of dietary iodide. It has been previously reported that subchronic exposure to ClO2 decreases thyroxine (T4) levels in nonhuman primates. In this study in vitro experiments with animal feed, isolated rat stomachs, as well as in vivo studies with intact rats, showed that ClO2 in drinking water (at in situ concentrations as low as 2 ppm) oxidizes iodide to its reactive elemental (radical) state, binding it to organic substances present in the GI tract. A single instance of acute exposure to ClO2, however, did not decrease blood iodide levels, or thyroid glandular uptake of iodine.

Ten- and ninety-day toxicity studies of 1,2-dichlorobenzene administered by oral gavage to Sprague-Dawley rats.

Ten- and ninety-day toxicity studies of 1,2-dichlorobenzene (DCB) were conducted in male and female Sprague-Dawley rats to meet the needs of the U.S. Environmental Protection Agency for toxicity data on this chemical for use in their determination of possible health risks related to human exposure. 1,2-Dichlorobenzene was administered at doses of 37.5, 75, 150, and 300 mg/kg/day (10-day), and 25, 100, and 400 mg/kg/day (90-day) in corn oil by oral gavage; control animals received corn oil. At time of sacrifice, gross necropsies were performed and selected tissues were weighed and prepared for histological evaluation. Blood was taken for hematology and clinical chemistries. In the 10-day study, exposure of 300 mg DCB/kg body weight to male rats resulted in a statistically significant decrease in final body weight, organ weights (heart, kidneys, spleen, testes, and thymus), and relative organ weights (spleen and thymus). A significant increase in absolute and relative liver weights was also noted in this dose group. Males also displayed significant increases in water consumption (300 mg/kg group), ALT (300 mg/kg) and leukocyte count (150 and 300 mg/kg). A significant increase in the incidence of hepatocellular necrosis was seen in the 300 mg/kg group of males compared to controls. In the 90-day study, male rats exposed to 400 mg DCB/kg displayed a statistically significant decrease in body weight, organ weight (spleen), and relative organ weight (spleen). The absolute weights of kidney and liver and the relative weights for heart, kidney, liver, lung, brain, and testes were increased significantly for this dose group. The absolute and relative weights of both the kidney and liver were significantly increased in the female 400 mg/kg dose group. The only clinical chemistry parameters statistically different than control were increased ALT (100 and 400 mg/kg groups), BUN and total bilirubin in the male 400 mg/kg group and total bilirubin in the 400 mg/kg female group. Histopathological evaluation showed hepatocellular lesions associated with DCB treatment which included centrolobular degeneration and hypertrophy, and single cell necrosis in male and females receiving 400 mg DCB/kg. The NOAEL observed in this study is 25 mg/kg/day.

Evaluation of the developmental toxicity of sodium nitrite in Long-Evans rats.

Sodium nitrite administered in the drinking water to Long-Evans rats during pregnancy and lactation severely affected erythropoietic development, growth, and mortality in their offspring. Pregnant rats were maintained throughout gestation on 0.5, 1, 2, or 3 g NaNO2/liter. There were no significant differences between treated and control litters at birth. Thereafter, pups of treated dams on 2 and 3 g NaNO2/liter gained less weight, progressively became severely anemic, and began to die by the third week postpartum. By the second week postpartum, hemoglobin levels, RBC counts, and mean corpuscular volumes of these pups were all drastically reduced compared to controls. Blood smears showed marked anisocytosis and hypochromasia. Gross chylous serum lipemia and fatty liver degeneration were noted. Histopathology demonstrated cytoplasmic vacuolization of centrilobular hepatocytes and decreased hematopoiesis in bone marrow and spleen. Administration of 1 g NaNO2/liter resulted in hematological effects but did not affect growth or mortality. NaNO2 (0.5 g/liter) was at or near the no observed effect level. Cross-fostering indicated that treatment during the lactational period was more instrumental in producing lesions than treatment during the gestational period. The data presented are consistent with the lactational induction of severe iron deficiency in the neonate.

Effect of gavage vehicle on hepatotoxicity of carbon tetrachloride in CD-1 mice: corn oil versus Tween-60 aqueous emulsion.

This investigation was conducted to evaluate the effect of gavage vehicles on altering the severity of the subchronic hepatotoxicity of carbon tetrachloride (CCl4). Male and female CD-1 mice were gavaged with 0, 1.2, 12, and 120 mg/kg CCl4 in either a corn oil or 1% Tween-60 vehicle once daily for 5 consecutive days per week for 90 days. The study revealed that the hepatotoxicity was greater in the mid- and high-dose groups of mice that had received CCl4 administered in corn oil. Increases in serum enzyme activities were detected in the mid-dose groups of mice that were gavaged with CCl4 in corn oil. The serum enzyme activities were significantly higher in the high-dose groups of animals in which CCl4 was administered in corn oil. Histopathological findings indicated that hepatocellular changes following the administration of CCl4 at the mid- and high-dose levels were more frequent and more severe when CCl4 was given in corn oil than when it was administered in Tween-60. The experimental findings indicate that the no-observed-adverse-effect level from CCl4 exposure was lowered by an order of magnitude (from 12 to 1.2 mg/kg) and that the hepatotoxicity of CCl4 was enhanced in the high-dose treatment groups when corn oil was employed as the gavage vehicle.

Subchronic toxicity study of 1,1-dichloro-2-propanone in Sprague-Dawley rats.

Groups of 10 male and 10 female Sprague-Dawley rats were administered 1,1-dichloro-2-propanone in corn oil by gavage at 0, 10, 20, 40, or 80 mg/kg/day for 90 consecutive days. Food and water consumption, body and organ weights, organ-to-body weight ratios, hematology, and clinical chemistry parameters were determined. Gross and microscopic pathology examinations also were conducted. No treatment-related mortality was observed during the study; however, liver, forestomach, and kidney toxicity was evident. Liver changes consisted of cytoplasmic alteration, cytomegaly, karyomegaly, and bile duct hyperplasia. These occurred with significance of p < or = 0.05 at or above 10 mg/kg/day in both sexes. The forestomach lesions included hyperkeratosis and epithelial hyperplasia in both sexes at 40 and 80 mg/kg/day, and ulcerations at 80 mg/kg/day. Also, an increased incidence and severity of spontaneously occurring chronic progressive nephropathy was most apparent in high dose males. Increases in organ-to-body weight ratios were noted for the liver and kidneys in females at the highest dose level and in males at the two highest dose levels. Serum enzymes (ALT, AST, and LDH) were increased in females and decreased in males. Based on liver lesions and biochemical changes, it was concluded that there was no experimentally definable NOAEL.

Subacute and subchronic toxicity of ethylene glycol administered in drinking water to Sprague-Dawley rats.

Subacute (10-day) and subchronic (90-day) toxicity studies of ethylene glycol (EG) were conducted in male and female Sprague-Dawley rats to provide the U.S. Environmental Protection Agency's (EPA) Office of Drinking Water with toxicity data for final preparation of a Health Advisory for the chemical. Ethylene glycol was administered in drinking water at concentrations of 0.5, 1.0, 2.0, and 4.0% for both sexes in the 10-day study. Based on a projected consumption rate of 100 ml/kg/day, the respective doses on a mg/kg/day basis would be 554, 1108, 2216, and 4432. These dose levels were also used in the 90-day study for females, but dose levels for the males in the 90-day study were 0.25, 0.5, 1.0, and 2.0% (227, 554, 1108, and 2216 mg/kg/day). At time of sacrifice necropsies were performed and tissues were prepared for histological evaluation. Blood samples were taken for hematology and clinical chemistry determinations. Body weights were measured weekly. Water and food consumption were determined three times weekly. No mortality occurred in the 10-day study. In the 90-day study 8/10 females and 2/10 males in the high dose group died prior to sacrifice. Body weights were suppressed in a dose response fashion for males and females. Hemoglobin, hematocrit, erythrocytes, and leukocytes were all significantly decreased in female rats receiving 4% EG for 10 days. The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.