IMM20007: Immunotoxicity of Chloramine (CAS No. 10599-90-3) in Female B6C3F1 Mice

Disinfection by-products (DBPs) are contaminants found in drinking water. DBPs are formed as by-products as a result of the chlorination/ozonization process used to purify water to acceptable drinking water standards. The potential effects of drinking water contaminants to affect adversely the immune system is a concern of both the Environmental Protection Agency (EPA) and the National Institute of Environmental Health Sciences (NIEHS). Several drinking water DBPs have been identified and selected for evaluation of their potential effects on the immune system in a joint project between the EPA and the NIEHS.
Chloramine has long been used to provide a disinfecting residual in distribution systems where it is difficult to maintain a free chlorine residual or where DBP formation is of concern. The reaction between chlorine and compounds containing a nitrogen atom with one or more hydrogen atoms attached to it will form chloramine.....

In conclusion, CHL, when administered for 28 days in the drinking water at doses from 2 to 200 ppm, produced minimal toxicological and immunotoxic effects in female B6C3F1 mice. However, Abdel-Rahman et al.1 have reported that the glutathione content in rat blood was decreased significantly after 4 months of monochloramine treatment (0 - 100 mg/l). After 3 months, red blood cell counts and hematocrit were significantly decreased. Hemoglobin concentration and MCHC were also decreased at 10 months of treatment. Thus, exposure of mice to CHL for a longer period may be necessary to insure that CHL does not adversely affect the immune system.

An Ecological Risk Assessment of Inorganic Chloramines in Surface Water

John P. Pasternak, Dwayne R. J. Moore, R. Scott Teed

Inorganic chloramines are formed when chlorine and ammonia are combined in water. These substances are frequently used as a secondary disinfectant for drinking water and are by-products of processes involving the disinfection of wastewaters and the control of biological fouling in cooling water systems. For chloraminate drinking water, the total residual chlorine (TRC) concentration may be almost completely due to monochloramine. Based on 1995 and 1996 survey data, the most significant and prevalent TRC loading to the Canadian environment is from municipal wastewater releases. Drinking water releases are the next most important source of chloramine entry into the Canadian environment, while TRC releases from other sources, such as cooling water, zebra mussel control practices and industrial wastewater, are much less important. A probabilistic water quality model was used to model two wastewater discharges and a cooling water discharge to different freshwater systems. The resulting exposure distributions were then compared with three incipient lethality endpoints, i.e., 50% mortality to the invertebrate Ceriodaphnia dubia and 50% and 20% mortality to juvenile chinook salmon (Oncorhynchus tshawytscha). For each discharge scenario studied, there were moderate to high probabilities of significant adverse effects on aquatic life up to 1.9 km from the effluent sources.
Probabilistic Risk Assessment, Municipal Wastewater Effluents, Ecological Risk Assessment, Inorganic Chloramines.


Project Title: A Rapid Ammonia-Oxidizing Bacteria Measurement Method as an Early-Warning Indicator of Nitrification Episodes

Investigator(s): John (Jay) Regan
Sponsor: American Water Works Association Research Foundation

Environmental Problem Addressed:
Water quality treatment/detection for nitrification episodes.

Research Project Objectives:
This research project aims to improve the reliability of chloramination via the prediction and early detection of nitrification episodes. The specific objectives of the project are to:

   1. Adapt existing molecular-based analysis techniques to provide a protocol that rapidly determines ammonia-oxidizing bacteria concentrations in drinking water samples; and
   2. Evaluate the hypothesis that an increase in ammonia oxidizer numbers in chloraminated systems indicates the pending onset of a nitrification episode, prior to the observed changes in other water quality parameters typically associated with nitrification.


Chloramination offers the drinking water industry a secondary disinfection alternative to chlorination, and its use has increased in the United States over the past decade. Much of this switch to chloramines has been prompted by the increasingly stringent regulations on chlorinated disinfection byproducts and the need to reduce biological regrowth. Chloroamines have been shown to produce lower concentrations of trihalomethanes and haloacetic acids than free chlorine, facilitating the compliance with Stage I Disinfectants and Disinfection By-Products Rule.

Despite these advantages, chloramination has some potential drawbacks. Nitrification episodes are a common operational problem observed in chloraminated distribution systems. While nitrifying bacteria do not pose a direct threat to public health, nitrification episodes are marked by a decline in several water quality parameters that may have public health implications. The growth of nitrifiers, which is promoted by the free ammonia associated with chloramination, is typically accompanied by a deterioration in water quality parameters such as an increase in heterotrophic bacteria, nitrite and nitrate, and a decrease in total chlorine residual.

Once a nitrification episode is underway, increasing chloramine dose is an ineffective means of correcting the problem, requiring utilities to implement alternative control strategies. Therefore, it would be advantageous to detect and respond to the pending onset of a nitrification episode prior to the full establishment of a nitrifying community and the associated water quality impacts.

A number of researchers have recognized the benefit that would be realized by the drinking water industry with the establishment of an early warning nitrification indicator. The direct enumeration of ammonia-oxidizing bacteria (AOB) presents a reasonable candidate for such an indicator, as AOB growth may be the catalyst that gives rise to the broader symptoms associated with nitrification outbreaks. The recent development of rapid, culture-independent molecular measurement tools offers the prospect of overcoming the limitations of culture-based AOB enumeration techniques and investigating the direct use of AOB numbers as an early warning indicator. This research project will develop quantitative molecular methods to monitor AOB community development, which would allow a rigorous evaluation of the relationships between nitrifier growth and water quality operational parameters.

Anticipated Results:

The desired outcome of this project is the development of a rapid and sensitive technique for quantifying ammonia-oxidizing bacteria concentrations in drinking water supplies. This will provide a marked improvement over the culture-based technique presently used by the water industry by reducing the turnaround time from at least three weeks to one or two days. This work will also show whether a measurable increase in ammonia oxidizer concentration preceeds the onset of a nitrification episode as determined by other water quality indicators.

Public Health Database Results
Practice Type: Model

Program Name: Drinking Water Nitrification Surveillance Program

Organization: Lee County Health Department, FL
Contact Person: Gary Maier, Professional Engineer Administrator
Phone (239) 939-4245
Fax (239) 939-4038
Email gary_maier@doh.state.fl.us
Web Address: http://www.lee-county.com/healthdept/engi.htm

Nitrification is not addressed by the current Safe Drinking Water Act regulations, but it can significantly deteriorate water quality in a public drinking water distribution system. It is a two-step biological process that first converts ammonia to nitrite, and then converts nitrite to nitrate. The second step generally does not occur or occurs very slowly in drinking water, resulting in a buildup of nitrite. Nitrite can accumulate in the drinking water and exceed the maximum allowable contaminant level.

Nitrite in drinking water poses an acute health concern, causing serious illness and sometimes death in infants less than six months old from methemoglobinemia (blue-baby syndrome). Nitrification can also cause a loss of the disinfectant residual in the water, leading to other bacteriological problems.

The American Water Works Association estimates that nitrification occurs to some degree in two-thirds of the public drinking water systems that practice chloramination disinfection. The number of water systems practicing chloramination is expected to dramatically increase in the near future due to new disinfection by-product regulations.

Lee County’s nitrification surveillance practice easily and rapidly monitors for, detects, and confirms nitrification occurrence in a drinking water distribution system, thus allowing a public water utility to execute timely countermeasures. Although nitrification countermeasures are known and available, an easy, fast, and inexpensive early warning system was sorely lacking. This practice fills that void. The cost to implement this practice is insignificant because the labor is attached to the routine microbiological sampling programs that all public water utilities are already required to perform. The benefits of i this practice include protecting infants under six months old from methemoglobinemia and preventing consequential bacteriological problems.

Responsiveness and Innovation:
Nitrification within a drinking water distribution system poses a public health threat that is not addressed by the current Safe Drinking Water Act regulations. Current regulations require water systems to sample for nitrite and nitrate only at the entry point to a water distribution system. The formation of nitrite or nitrate within the water distribution system will not be detected. The model practice will quickly and easily detect nitrification within a drinking water distribution system and provide an early warning to the water system so that the water system can carry out appropriate countermeasures before the nitrite level exceeds the maximum allowable contaminant level. Although nitrification countermeasures are known and available, an easy, fast, and inexpensive early warning was not in place until the inception of this program.

Compliance with current regulations will not result in detection of nitrification within a water distribution system. The scientific literature contains recommendations that water systems can voluntarily implement to monitor for distribution system nitrification, but these recommendations normally involve using inconvenient, slow, or expensive techniques such as monitoring of heterotrophic bacterial populations throughout the water distribution system or developing an accurate nitrogen mass balance along with specific biological monitoring for ammonia-oxidizing bacteria and nitrite-oxidizing bacteria throughout the water distribution system. Lee County’s program can confirm or refute nitrification occurrence using an inexpensive (less than one dollar) one-minute field test in conjunction with the water system’s standard routine microbiological monitoring.

Agency and Community Roles:
The primary collaborative partners will be the public water utilities that practice chloramination disinfection. The local public health agency role can vary depending on the working relationship it has with the public water utilities. The practice is flexible along a wide continuum of shared roles. At one extreme, the local public health agency could simply share this model practice with the appropriate public water utilities and encourage them to adopt it or something similar. At the other extreme, the local public health agency could develop a working relationship with the public water utilities in which the local public health agency conducts all of the water system’s standard routine microbiological monitoring and implements this practice at the same time. The latter approach is the current one in Lee County. A local public health agency could also adopt some middle ground whereby the agency conducts periodic spot checks while encouraging the water utility to implement the model practice.

Public water utilities normally want to do the right thing to protect the health, safety, and welfare of their customers. The key point is that the local public health agency should work with and educate the utility managers and operators and explain:

    * What nitrification is, why it is important, and where and when it can occur.

    * How to detect and confirm nitrification occurrence.

    * How the utility can prevent and fix nitrification problems.

Once all the facts are on the table, the local public health agency and the utility can decide, on a case-by-case basis, the most efficient and effective way to monitor for nitrification and protect public health. There is ample scientific literature on nitrification (see below, under “replication”).

Costs and Expenditures:
The only costs of this practice, over and above the costs of routine required microbiological monitoring, are the cost of the water quality test strips and the cost of occasional laboratory analyses for nitrite and nitrate. The program spends about $100 per year for water quality test strips, and about $100 per year for extra laboratory tests. Furthermore, additional staff time is negligible.

In the community, the health department cultivated a working relationship with most of the public water utilities whereby the health department conducts all of the water system’s standard routine microbiological monitoring. The water utilities reimburse the health department for the cost of this service. It is standard procedure to conduct field tests for pH and total chlorine residual at the same time that routine microbiological water samples are collected.

The health department trained field staff to watch for symptoms of nitrification, such as a sharp, unexpected decrease in total chlorine residual or an unexpected decrease in pH. If such symptoms are observed, then staff conducts a one-minute nitrate/nitrite field test using water quality test strips. If the test strip qualitatively confirms nitrification, then staff immediately notify the water utility so that they can 1) take countermeasures and 2) collect a water sample to obtain a certified laboratory analysis of nitrite and nitrate. The certified laboratory results are used to determine whether public notification is warranted.

The water system partners are very committed, and based on the local experience, they take nitrification very seriously. When nitrification is discovered, the water system partners take prompt and effective corrective action without hesitation. The cost of the practice is so low compared to the public health benefits that they do not foresee any reason to discontinue monitoring for nitrification.

Lessons Learned:
Program staff learned to use the water quality tests strips only for the qualitative purpose of confirming the occurrence of nitrification, and not for determining quantitatively the actual level of nitrite or nitrate in the water. If the water quality test strip confirms the occurrence of nitrification, then a water sample for nitrite is collected and nitrate analyses by a certified laboratory. The certified laboratory result should be used to determine whether a public notice is warranted.

Key Elements for Replication:
Read the available scientific literature on nitrification in water distribution systems to better understand the issue. A succinct publication is available on the web from the American Water Works Association Research Foundation at: http://www.awwarf.org/research/TopicsAndProjects/execSum/710.aspx.

Online 2007 Model Practices Application
For any questions, contact Practices@naccho.org, or 202-783-5550, x232


International Agency for Research on Cancer (IARC) - Summaries & Evaluations


CAS No.: 10599-90-3

5. Summary of Data Reported and Evaluation

5.1        Exposure data
Chloramine, formed by the reaction of ammonia with chlorine, is increasingly being used in the disinfection of drinking-water. Monochloramine, dichloramine and trichloramine are in equilibrium, with monochloramine predominating. Exposure to milligram-per-litre levels occurs through ingestion of chloraminated water. Chloramines are also formed in swimming pools from the reaction of chlorine with nitrogen-containing contaminants, and trichloramine has been measured in swimming-pool air. Chloramine generated in situ is also used as an intermediate in the production of hydrazines, organic amines and other industrial chemicals.

5.2        Human carcinogenicity data
Several studies were identified that analysed risk with respect to one or more measures of exposure to complex mixtures of disinfection by-products that are found in most chlorinated and chloraminated drinking-water. No data specifically on chloramine were available to the Working Group.

5.3        Animal carcinogenicity data
Chloraminated drinking-water (predominantly in the form of monochloramine) was tested for carcinogenicity by oral administration in female and male mice and rats without demonstrating clear evidence of carcinogenic activity. In carcinogen-initiated rats, chloramine generated by ammonium acetate (in feed) and sodium hypochlorite (in drinking-water) promoted stomach cancer.

5.4        Other relevant data
36Cl-Labelled chloramine is readily absorbed after oral administration to rats. About 25% of the administered radioactivity is excreted in the urine over 120 h.

Nitrogen trichloride (trichloroamine) is volatilized from food-processing water disinfected with chloramine and from swimming-pool waters disinfected with chlorine, and reacts with ammonia in water to form chloramine. Upon inhalation, it produces lung irritation and may be a cause of occupational asthma. Ingestion of monochloramine produced no clinical abnormalities in male volunteers at concentrations as high as 15 mg/L. No reproductive or developmental effects have been associated with monochloramine.

Chloramine induced single-strand breaks and loss of DNA-transforming activity and was a weak mutagen in Bacillus subtilis. It was not mutagenic to Salmonella typhimurium. In vitro, chloramine caused double-strand DNA breakage in plasmid pUC18, and DNA fragmentation and DNA double-strand breaks as well as chromatin condensation in rabbit gastric mucosal cells and human stomach cancer cells. Monochloramine did not induce micronuclei, chromosomal aberration, aneuploidy or sperm abnormality in mice in vivo, but induced the formation of micronuclei in erythrocytes of newt larvae in vivo.

5.5 Evaluation
There is inadequate evidence in humans for the carcinogenicity of chloramine.

There is inadequate evidence in experimental animals for the carcinogenicity of monochloramine.

Overall evaluation

Chloramine is not classifiable as to its carcinogenicity to humans (Group 3).

Last updated: 29 September 2004
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