Reproductive effects of alternative disinfectants.

Organohalides formed through the reaction of chlorine and organic compounds in natural and waste waters pose potential health hazards. For this reason, alternative water disinfectants that do not form organohalides are being investigated with great interest. Limited data are available on the health effects, in particular reproductive toxicity effects, of these compounds. In our laboratory, we have examined the reproductive effects of chloramine and chlorine administered by gavage in Long-Evans rats. Animals were treated for a total of 66 to 76 days. Males were treated for 56 days and females for 14 days prior to breeding and throughout the 10-day breeding period. Females were treated throughout gestation and lactation. Following breeding, the males were necropsied and evaluated for sperm parameters and reproductive tract histopathology. Adult females and some pups were necropsied at weaning on postnatal day 21. Other pups were treated postweaning until 28 or 40 days of age. These pups were evaluated for the day of vaginal patency and thyroid hormone levels. No differences were observed between control rats and those rats exposed to up to 5 mg/kg/day chlorine or 10 mg/kg/day chloramine when fertility, viability, litter size, day of eye opening, or day of vaginal patency were evaluated. No alterations in sperm count, sperm direct progressive movement (micron/sec), percent motility, or sperm morphology were observed among adult male rats. In addition, male and female reproductive organ weights were comparable to their respective control groups, and no significant histopathologic changes were observed among chlorine- or chloramine-treated male and female rats.


Introduction
Chlorine has been the most commonly used disinfectant in the treatment of U.S. water supplies. However, potential health hazards are posed by organohalides formed through the reaction of chlorine with organic compounds present in natural and waste waters (1,2). Trihalomethanes (THM), including chloroform, bromodichloromethane, and dibromochloromethane have been detected in chlorine-treated waters and are the subject of increasing concern regarding potential health effects. Additionally, chloroform has been designated as a carcinogen by the National Cancer Institute (3). The potential reproductive effects of chlorine and chlorine compounds have not yet been addressed fully, despite widespread use and the availability of a significant data base on the general toxicity of chlorine. Druckrey (4) reported no effects on fertility, growth, blood parameters, survival, or the incidence of malignant tumors in seven consecutive generations of rats exposed to up to 100 ppm chlorine. Abdel-Rahman et al. (5) exposed rats to 1 to 100 ppm chlorine for 2.5 months before and throughout gestation. Pregnant rats * were sacrificed on gestation day 20, and fetuses were examined for visceral and skeletal anomalies. No increase in fetal resorptions was observed in any dose group. Abdel-Rahman and co-workers reported a slight increase in skeletal variants among fetuses in the 10 and 100 ppm dose groups and some visceral anomalies in fetuses in the 100 ppm dose group. Unfortunately the number of litters per treatment group was small, so the fetus rather than the litter was used as the unit of comparison. A high percentage of control fetuses were also observed to have skeletal and visceral variants, reducing confidence in the apparent effect in this study.
Monochloramine (NH2CI) is currently used as an alternative to chlorine disinfection. Chloramine disinfection is advantageous because the persistence of chloramine provides a residual disinfectant in the water supply not maintained in chlorine-treated waters (6). Chloramine also provides the taste-and odor-control advantages of chlorine dioxide (C102) use (7). Although NH2C1 has been shown to be a weak mutagen (8), the only reported health effects associated with chloramine are hemolytic crises in long-term dialysis patients (9,10) and in vitro methemoglobin formation and hemolysis in human erythrocytes (10). Abdel-Rahman (5) reported hio adverse effects in rat fetuses exposed in utero to up to 100 ppm NH2Cl. Berez et al. (11) demonstrated a significant decrease in serum thyroxine in African green monkeys exposed to 100 ppm C102, but observed no rats per dose group were gavaged with 0, 1.0, 2.0, or effects of chloramine on thyroid function. The studies 5.0 mg/kg (10.0 mL/kg) aqueous chlorine (HOCI, pH reported here were designed to evaluate reproductive 8.5) for 56 days prior to breeding and throughout the effects of chlorine and NH2Cl in Long-Evans rats. 10-day breeding period. Twenty-four female rats per dose group were given the same dose levels of chlorine Materials and Methods by gavage for 14 days prior to breeding and throughout breeding, gestation, and lactation until the pups were Long-Evans rats, 4-6 weeks of age, were obtained weaned on day 21. Doses chosen were the highest pracfrom Charles River Breeding Laboratories (Portage, ticable considering solution stability and potential gas-MI) and were housed two per cage in polycarbonate tric irritation. Animals were housed one male to two cages with Absorb-Dri bedding. The animal room was females throughout the 10-day breeding period. Femaintained at 69-75°F and 40-60% humidity, with a 12-males were checked for the presence of sperm in a lahr/12 hr light-dark cycle. Animals were given ad libivage smear before 9:30 a.m. each morning. Females tum access to Purina Certified Rodent Chow 5002 and observed to be sperm positive (gestation day 0) were deionized water. Two separate experiments were con-housed individually. The day of parturition was desigducted. In the first study, the effect of aqueous chlorine nated lactation day 0. on reproductive outcome was evaluated. Twelve male Following the breeding period, males were bled by 'Pups used for practice hormone bleeds. Table 3. Summary of male reproductive parameters in Long-Evans rats exposed to aqueous chlorine (X ± SD). 0 mg/kg 1.0 mg/kg 2.0 mg/kg 5.0 mg/kg No. males examined 12 12 11 11 Sperm cone. (X 109/g epidid.) 1.14 ± 0.22 retro-orbital puncture for a complete blood count and from the vena cava for determinations of thyroid hormone levels. They were then sacrificed by pentobarbital overdose, and given a complete gross necropsy. The reproductive tract, including testis, epididymis, prostate, and seminal vesicles, was removed, weighed, and, except for the right cauda epididymis, preserved in neutral buffered formalin for histopathologic evaluation. The right cauda epididymis was weighed and a small sample of seminal fluid was expressed from the cut end ofthe vas deferens onto the edge of a clean scalpel blade. This fluid was mixed with two drops of phosphate buffered saline (PBS) with 0.1% glucose (pH 7.4, 37°C) on a prewarmed microscope slide. The slide was coverslipped and immediately observed using phase-contrast microscopy. The cauda was then finely minced in 10 mL of PBS. All slides, coverslips, solutions, and pipettes were maintained in an incubator at 37°C. A modification of Katz and Overstreet's (12) videomicrography method was used to videotape and evaluate a minimum of ten 10-sec fields at x 160 magnification and ten 10-sec fields at x 400 magnification. An aliquot of the minced cauda sperm suspension was heat-killed and used for determining sperm counts. An 0.5 mL aliquot was heat-killed, stained with aqueous eosin Y, and used to prepare four to six slides Table 6. Summary of male reproductive parameters in Long-Evans rats exposed to aqueous monochloramine (X + SD). per animal for sperm morphology evaluation. The videotapes were later evaluated for percent sperm motility (10 fields) and sperm direct progressive movement (five sperm/field; ten fields). Sperm motility was estimated by two independent observers. Sperm showing any movement were judged motile. The observers' results agreed within 5%. Sperm for progressive movement determinations were randomly selected with the videotape stopped. The tape was advanced, and the distance traveled was measured. The distance traveled on the videoscreen (millimeters) was correlated with the actual distance traveled (micrometers). The elapsed time was determined from a running digital time clock that was videotaped simultaneously when videotaping the sperm. Only morphologically normal sperm were selected for progressive movement determinations. Dams were observed for fertility, length of gestation, body weight gain, and maternal behavior. At necropsy on lactation day 21, dams were bled by retro-orbital puncture for complete blood counts and via the vena cava for hormone analysis, then sacrificed by pentobarbital overdose and given a complete gross necropsy. The reproductive tract was removed, weighed, and preserved for histopathologic evaluation.
Litters were evaluated for viability, litter size, day of eye opening, body weight gain, and gross external abnormalities. The day on which all pups in a litter had both eyes open was designated as the day of eye opening. At necropsy on lactation day 21, 10 pups/sex/dose were bled for complete blood counts and hormone analysis. The reproductive tract was weighed and preserved. Randomly selected pups were retained for observation of the day of vaginal patency and hormone evaluation.
In the second experiment, groups of 12 male and 24 female rats were administered 0, 2.5, 5.0, or 10.0 mg/ kg (10.0 mL/kg) chloramine in deionized water by oral gavage for 66 to 76 days according to the regimen described for the first experiment. Chloramine stock solution was prepared by titrating chlorine stock solution with ammonia. All other methods were the same as that described for the study of chlorine.

Chlorine
Neither clinical signs of toxicity, hematology changes, nor body weight depression were observed for male or female Long-Evans rats exposed to 0, 1.0, 2.0, or 5.0 mg/kg chlorine in deionized water for up to 76 days. The observed fertility rate varied between 75% and 96%, but did not vary in a dose-dependent manner (Table 1). Litter survival, litter size, and pup weight did not differ among groups (Table 2). Developmental landmarks such as the mean day of eye opening (day 17) and average day of observed vaginal patency (day 33) were comparable across groups. The day of eye opening is a general maturational index, whereas the day of vaginal patency provides information on rate of sexual maturation. Adult male rats exposed to up to 5.0 mg/ kg HOCl showed no adverse reproductive effects (Table  3). Sperm concentration ranged from 1.03 x 109 sperm/ g epididymal tissue (1.0 mg/kg HOCI dose group) to 1.14 x 109 sperm/g epididymal tissue (control group).
The percentage of motile sperm and sperm progressive movement (,um/sec) were not different when compared to controls. The percentage of abnormal sperm morphology was comparable for all groups, ranging from 0.71% (2.0 mg/kg HOCI dose group) to 1.22% (1.0 mg/ kg HOCI dose group). No histopathologic lesions were observed in the reproductive tract of adult male or female rats exposed to 0 to 5.0 mg/kg chlorine.

Chloramine
No clinical signs of toxicity, hematology changes, or evidence of body weight suppression were observed for adult male or female rats dosed with 0, 2.5, 5.0, or 10.0 mg/kg NH2Cl by gavage for up to 73 days. Fertility, fecundity, length of gestation, litter size, and litter survival rates were unchanged by NH2Cl treatment (Table  4). Mean pup weight also was unaltered ( Table 5). The average day of eye opening (day 16) and the average day of observed vaginal patency (day 31.8 to day 32.6) were comparable among groups. No treatment-related adverse reproductive effects were observed in adult male rats treated with up to 10.0 mg/kg NH2CI. These results are presented in Table 6. The sperm concentration ranged from 587.5 x 106 sperm/g epididymal tissue to 644 x 106 sperm/g epididymal tissue. The percentage of abnormal sperm morphology ranged from 0.4 + 0.5% to 2.6 ± 4.7%, but did not vary in a dose-dependent manner. Sperm motility and sperm progressive movement were unaffected by dose exposure. No treatmentrelated histopathologic lesions were observed in the reproductive tracts of male or female rats.

Discussion
There is a dearth of reproductive and developmental toxicity data for chlorine and alternative water disinfectants (e.g., chloramine, chlorine dioxide) and their by-products such as chlorite and chlorate. Evaluations of chlorine and NH2Cl (5) have indicated that these chemicals have little or no teratogenic effect. A slight increase in skeletal variants and soft tissue variations were noted, but the number of dams per group for both chemicals were insufficient to evaluate teratogenicity. Druckrey (4) reported no toxicity associated with exposure of seven consecutive generations of rats to up to 100 ppm chlorine.
Our studies of the potential reproductive toxicity of chlorine and chloramine also indicate no adverse reproductive effects following subchronic administration of these two drinking water disinfectants. Although the percentage of abnormal sperm forms in the 5.0 mg/kg NH2Cl dose group (2.6 + 4.7%) was elevated relative to control males (1.1 + 1.3%), the large standard deviation of the former precluded statistical significance. Abnormal sperm were observed in only two of the 12 rats at 5.0 mg/kg, and no adverse effects were observed among males treated with 10.0 mg/kg NH2Cl. Thus, this result was not considered to be chloramine-induced. This work was supported by EPA Project No. CR810862-02. It has been subject to the Agency's review and has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Thanks are offered for technical assistance to Ms. Crystal Carmon and Ms. Lisa Duty and to Mr. William Kaylor of EPA/HERL for preparing the chlorine stock solutions.