United Steles
Environmental Protection
Agency
EPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
CHLORINE, HYPOCHLOROUS ACID AND HYPOCHLORITE ION
DRAFT
ECAO-CIN-D004
lannarv/ 1 QQ4
Prepared for
OFFICE OF DRINKING WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 5268
DRAFT: DO NOT CITE OR QUOTE
NOTICE
This document is a preliminary draft. It has not been formally released by the U.S.
Environmental Protection Agency and should not at this stage be construed to represent
Agency policy. It is being circulated for comments on its technical accuracy and policy
implications.
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2. REPORT DATE
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Drinking
4. TITLE AMD SUBTITLE ^ _
Drinking Water Criteria Document
for Chlorine, Hypochlorous Acid and Hypochlorite
Ion
6. AUTHOR(S)
•Bocttmonrt-
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. EPA
Cincinnati, OH
6. PERFORMING ORGANIZATION
REPORT NUMBER
3. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRE5S(ES)
U.S. EPA
Office of Water
401 M St., SW
Washington, DC 20460
10. SPONSORING/MONITORING
AGENCY REPORT NUMBER
11. SUPPLEMENTARY MOTES
12a. DISTRIBUTION/AVAILABILITY STATEMENT
12b. DISTRIBUTION CODE
13. ABSTRACT (Maximum 200 words)
This document provides the health effects basis to be considered in
establishing the Maximum Contaminant Level Goal. To achieve this ob-
jective, data on pharmacokinetics, human exposure, acute and chronic
toxicity to animals and humans, epidemiology and mechanisms of
toxicity are evaluated. Specific emphasis is placed on literature
providing dose-response information.
IS. SUBJECT TERMS
health effects, chlorine, hypochlorous
acid, hypochlorite ion
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US
16. PRICE CODE
Aok
17. SECURITY CLASSIFICATION
OF REPORT
18. SECURITY CLASSIFICATION
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NSN 7540-01-280-5500 Standard Form 298 (Rev. 2-89)
Prescribed by ANSI Std Z39-18
2y8-1Q2
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DISCLAIMER
This report is an external draft for review purposes only and does not constitute
Agency policy. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended in 1986,
requires the Administrator of the Environmental Protection Agency to publish maximum
contaminant level goals (MCLGs) and promulgate National Primary Drinking Water
Regulations for each contaminant, which, in the judgment of the Administrator, may have
an adverse effect on public health and which is known or anticipated to occur in public
water systems. The MCLG is nonenforceable and is set at a level at which no known or
anticipated adverse health effects in humans occur and which allows for an adequate
margin of safety. Factors considered in setting the MCLG include health effects data and
sources of exposure other than drinking water.
This document provides the health effects basis to be considered in establishing the
MCLG. To achieve this objective, data on pharmacokinetics, human exposure, acute and
chronic toxicity to animals and humans, epidemiology and mechanisms of toxicity are
evaluated. Specific emphasis is placed on literature data providing dose-response
information. Thus, while the literature search and evaluation performed in support of this
document has been comprehensive, only the reports considered most pertinent in the deri-
vation of the MCLG are cited in the document. The comprehensive literature data base
in support of this document includes information published up to 1989; however, more
recent data may have been added during the review process.
When adequate health effects data exist, Health Advisory values for less than
lifetime exposures (1-day, 10-day and longer-term, g10% of an individual's lifetime) are
included in this document. These values are not used in setting the MCLG, but serve as
informal guidance to municipalities and other organizations when emergency spills or
contamination situations occur.
Tudor Davies, Director
Office of Science and
Technology
James Elder, Director
Office of Ground Water
and Drinking Water
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DOCUMENT DEVELOPMENT
Linda R. Papa, Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Scientific Reviewers
Richard Bull
Washington State University
Rullman, Washington
Cynthia Sonich-Mullin
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Rita Schoeny
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Jennifer Orme Zavaleta
Office of Science and Technology
U.S. Environmental Protection Agency
Editorial Reviewers
Erma Durden, B.S.
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Judith Olseri;. B.A.
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Document Preparation
Technical Support Services Staff, Environmental Criteria and Assessment Office*
Cincinnati
iv
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TABLE OF CONTENTS
Page
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES 11-1
ENVIRONMENTAL FATE, TRANSPORT AND DISTRIBUTION II-4
ANALYTICAL METHODS II-7
ENVIRONMENTAL SOURCES, PRODUCTION AND USE 11-12
SUMMARY 11-13
III. TOXICOKINETICS 111-1
ABSORPTION 111-1
DISTRIBUTION III-3
METABOLISM III-5
EXCRETION III-8
SUMMARY III-9
IV. HUMAN EXPOSURE IV-1
To be provided by the Office of Drinking Water]
V. HEALTH EFFECTS IN ANIMALS V-1
INTRODUCTION V-1
ACUTE TOXICITY V-1
Inhalation V-1
Oral V-3
Other Routes V-8
SUBCHRONIC TOXICITY V-8
CHRONIC TOXICITY V-21
TARGET ORGAN TOXICITY V-26
Cardiovascular Effects V-26
nmunoiogical Effects V-29
CARCINOGENICITY V-30
MUTAGENICITY V-34
REPRODUCTIVE AND DEVELOPMENTAL EFFECTS V-38
SUMMARY V-42
v
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TABLE OF CONTENTS (cont.)
VI. HEALTH EFFECTS IN HUMANS VI-1
INTRODUCTION . VI-1
CLINICAL CASE STUDIES VI-2
EPIDEMIOLOGICAL STUDIES VI-9
General Considerations VI-9
Retrospective Studies VI-10
Cross-Sectional Studies VI-23
HIGH RISK SUBPOPULATIONS Vl-26
SUMMARY VI-28
VII. MECHANISMS OF TOXICITY VII-1
SUMMARY VII-6
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
INTRODUCTION VIII-1
NONCARCINOGENIC EFFECTS VIII-7
QUANTIFICATION OF NONCARCINOGENIC EFFECTS V1IH4
Assessment of Acute Exposure Data and Derivation of 1-Day HA . . V1IM4
Assessment of Short-term Exposure Data and Derivation
of 10-Day HA VIIH4
Derivation of Longer-term HA VIIM6
Assessment of Lifetime Exposure and Derivation of DWEL VII-17
CARCINOGENIC EFFECTS VIIH9
QUANTIFICATION OF CARCINOGENIC EFFECTS V1IK1
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS . . . W-21
SPECIAL GROUPS AT RISK Vl^23
IX. REFERENCES IX-1
vi
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LIST OF TABLES
No. Title Page
11-1 Physical and Chemical Properties of Chlorine, Hypochlorous Acid
and Sodium Hypochlorite II-2
II-2 Chlorine Reactions Known to Occur in Aqueous Solution II-5
III-1 Summary of Selected 3SCI Absorption Studies in Male
Sprague-Dawley Rats III-4
V-1 Lethal Concentrations of Chlorine Gas V-2
V-2 Acute Toxicity Studies in Rats V-4
V-3 Subchronic and Chronic Animal Studies Involving Chlorine
Administration in Drinking Water V-9
VI-1 Threshold Levels for Chlorine Gas Inhalation Effects VI-8
VII1-1 Summary of Studies Pertinent to HA Derivation VIII-8
VIII-2 Summary of Studies Pertinent to DWEL Derivation VIII-12
VIII-3 Summary of DWEL and 10-day HA Calculations VIII-20
VIII-4 Existing Guidelines on Human Exposure to Chlorine VIII-22
1
vii
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LIST OF ABBREVIATIONS
ADP Adenosine diphosphate
bw Body weight
DNA Deoxyribonucleic acid
DWEL Drinking water equivalent level
FEL Frank-effect level
GC/MS Gas chromatography/mass spectrometry
Gl Gastrointestinal
HA Health Advisory
HDL High density lipoprotein
HPLC High performance liquid chromatography
LDL Low density lipoprotein
LOAEL Lowest-observed-adverse-effect level
MDR Minimum daily requirement
NOAEL No-observed-adverse-effect level
NOEL No-observed-effect level
PEM Peritoneal exudate macrophage
RfD Reference dose
RNA Ribonucleic acid
SDS Socium dodecyl sulfate
TSH Thyroid stimulating hormone
viii
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I. SUMMARY
Chlorine is a highly reactive element that is widely distributed in the environment.
Elemental chlorine exists as a greenish-yellow gas at 25°C. In water, chlorine reacts
to form hypochlorous acid and hypochlorite ion. At pH 7.4 these two species are in
equimolar concentration; however, as the pH increases, hypochlorite ion will be the
predominant species. Chlorine, hypochlorous acid and hypochlorite ion are the
definitive constituents of "free chlorine" in water. When ammonia or nitrogenous
compounds are present in chlorinated water, chloramines are formed. Chlorine present
in water as chloramines is termed "combined chlorine." "Total chlorine" is, therefore,
the sum of free and combined chlorine. Chlorine is known to react with other organic
material such as humic and fulvic acids to form a wide variety of chlorinated organic
compounds. Each of these chlorine species is important in the disinfection and
treatment of water as a result of their respective oxidation potentials and biocidal
activities.
Chlorine is a common drinking water additive. The occurrence for chlorine,
hypochlorous acid and hypochlorites centers upon their use in drinking water and
wastewater disinfection as well as an intermediate in the manufacture and preparation
of a number of organic products such as antifreeze, rubber, cleaning agents and
pharmaceuticals.
CHLORINE.1
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The measurement and classification of the chlorine species in water can be
performed by several analytical procedures. Appropriate testing procedures for the
detection of chlorine, its oxidants and combined-form species include potentiometry,
amperometry, spectrophotometry, colorimetry and titration. These methods differ in
their sensitivity depending on the form of chlorine (free or combined) being measured;
therefore, selection of the appropriate testing procedure is essential for accurate
estimates of chlorine concentrations.
Chlorine hydrolyzes very rapidly in water with a half-life of 0.005 seconds in
natural waters to yield hypochlorous acid and hypochlorite ion. Because free chlorine
is a strong oxidant, its stability in water is very low and in the presence of light rapidly
undergoes oxidation. Vaporization of molecular chlorine at low concentrations is
insignificant; however, at acidic pH values and high concentrations, vaporization may
become significant.
The toxicokinetics of chlorine, hypochlorous acid and hypochlorite ion in drinking
water are largely unknown due to the fact that these compounds readily react with
biological molecules forming other CI" species. Pharmacokinetic studies have also been
conducted on these chlorinated species; however, these compounds may not be useful
in providing insight in Cl2 toxicokinetics due to significant differences in the oxidative
reactivity of the various compounds.
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Studies in rats have shown that following oral administration of radioactive Cl2,
36CI is rapidly absorbed into the blood. Distribution is the highest in plasma followed
by bone marrow, kidney, testes, lungs, skin and liver. Chlorine is eliminated from the
body primarily through the urine and feces.
Limited information is available regarding the acute effects of chlorine following
oral exposure. Short-term exposure of rats to HOCI or CI in aqueous solution has
resulted in transient decreases in blood glutathione and hypothalamic norepinephrine
levels and liver alterations.
Exposures of >90 days in drinking water of rats, mice, rabbits, guinea pigs,
pigeons and chicks have shown conflicting results. Exposures of >150 mg/L chlorine
in rats, mice, guinea pigs and chicks have shown no adverse effects; however, rabbits
and pigeons exposed to 15 mg/L chlorine experienced increased plasma cholesterol
levels and changes in plasma thyroxine and hydroxyproline levels.
When administered in the diet, chlorine concentrations of 60 mg/kg bw had no
adverse effects on weanling rats; however, exposures of >100 mg/kg bw resulted in
dose-related increases in kidney and liver weights. No significant histopathologic effects
were observed at any dose tested.
A 7-generation study of chlorine in which rats were administered highly
chlorinated drinking water (100 mg/L), resulted in no treatment-related effects on
CHLORINE.1
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lifespan, fertility, growth, hematologic or histopathologic parameters. Long-term
exposure to sodium hypochlorite in drinking water of rats resulted in dose-related
decreases in body and organ weights at doses >13.5 mg/kg bw/day. Chronic exposure
to chlorinated drinking water resulted in no adverse effects in rats or mice at doses up
to 275 mg/L.
Cardiovascular effects have been reported in both pigeons and monkeys
maintained on calcium-deficient or atherogenic diets and exposed to chlorine in their
drinking water. Mice exposed to chlorinated drinking water showed no evidence of
humoral or cell-mediated immune responses.
No evidence of reproductive or developmental effects have been reported. There
is no clear evidence of carcinogenic effects in rats or mice exposed to chlorinated
drinking water. Chlorine, hypochlorous acid or hypochlorite ion have not been shown
to act as either direct carcinogens or initiators of tumorigenesis. Marginal increases in
mononuclear leukemias were reported in female rats; however, these findings were not
dose-related nor supported by similar findings in male rats or male and female mice.
Assessment of the mutagenic potential of chlorine is confounded by the reactive
nature of the chlorine molecule and by the presence of reaction products, which have
been found to be mutagenic. Because of bacteriotoxicity, chlorine or sodium
hypochlorite have given variable results in standard plate incorporation assays. In
mammalian cell systems chlorine has been shown to produce chromosomal aberrations.
CHLORINE.1
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In humans research on the effects of chlorine, hypochlorous acid and
hypochlorites has been overshadowed by the investigation of chlorination by-products
such as chloramines and trihalomethanes. The results of pertinent studies have
indicated that the ingestion of chlorinated drinking water, under most normal
circumstances, does not directly produce toxic effects. Consumption of heavily
chlorinated drinking water (<90 ppm) produces constriction of the throat and irritation
of the membranes of the throat and mouth. Epidemiologic studies that address the
association between chlorinated drinking water supplies and cancer have been limited
by weaknesses in study objectives, design and confounding factors. The results of
these studies do not support or refute a carcinogenic association with exposure to
chlorinated drinking water.
The emphasis of this document is on the derivation of drinking water criteria for
the protection of human health from potential toxicity due to exposure to chlorine,
hypochlorous acid and hypochlorite ion. To that end there is included a discussion of
the physico-chemical and biochemical characteristics of these compounds as relates
to their biological effects. It should be noted, however, that the organic by-products
formed during drinking water disinfection, especially the trihalomethanes, are important
contributors to the overall health risks resulting from water chlorination. Because of the
importance of these compounds, they are discussed in separate U.S EPA documents
on chloramines, trihalomethanes and chlorine dioxide.
CHLORINE.1
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Lack of appropriate data precluded derivation of a 1-day HA for chlorine. It is
recommended that the 10-day HA be adopted as sufficiently protective. A 10-day HA
of 2.5 mg/L for a 10 kg child was derived from a no-adverse-effect level in mice drinking
25 mg/kg free available chlorine in water/day for 33 days.
A 90-day drinking water study was selected for the development of a longer-term
HA. Longer-term HA values of 2.0 mg/L (2000 |ig/L) for a 10 kg child and 6.0 mg/L
(rounded to 6000 ng/L) for a 70 kg adult were derived based on a NOAEL of 16.7
mg/kg bw/day for absence of adverse effects in male rats exposed for 90 days to
chlorinated drinking water. A DWEL of 4.0 mg/L (rounded to 4000 |ig/L) for a 70 kg
adult has been derived based on the NOAEL of 14.4 mg/kg bw/day in female rats
exposed for 2 years to chlorinated drinking water. Caution should be applied in the
interpretation of this health risk assessment in that it does not address the effects of the
chlorinated by-products, especially trihalomethanes, formed during drinking water
disinfection.
CHLORINE.1
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II. PHYSICAL AND CHEMICAL PROPERTIES
The halogen cnlorine (Cl:) is found as a greenish-yellow gas under standard
conditions, or when compressed as a high density amber liquid. The diatomic gas is
characterized by a penetrating and irritating odor. Table 11-1 lists chemical and physical
properties specific to chlorine, hypochlorous acid and sodium hypochlorite.
Chlorine is very reactive and will combine directly with nearly all of the elements
except the rare gases (excluding xenon). The high level of chlorine reactivity is related to
the structure of the chlonne atomic shell in which there are seven electrons; thus, chlorine
will readily accept or share electrons in order to form a more stable electron configuration.
This is reflected in chlorine's functional capacity as an oxidant as a replacement for
hydrogen and hydroxyl groups in organic compounds, as an intermediate in organic
synthesis, and in its role in the saturation of double bonds. Although chlorine usually forms
univalent compounds, it may be found with valences ranging from one to seven.
In pure water, chlorine forms elemental chlorine (Cl2), chloride ion (CI) and
hypochlorous acid (HOCI). As pH increases, hypochlorous acid dissociates to hypochlorite
ion (OCT). The term "free chlorine" (free available chlorine, free residual chlorine) refers
to the concentrations of elemental chlorine, hypochlorous acid and hypochlorite ion that
collectively occur in water. Several factors, including chlorine concentration, pH,
CHLORINE.2
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• TABLE 11-1
Physical and Chemical Properties of Chlorine, Hypochlorous Acid and Sodium
Hypochlorite*
Property
Chlorine
Hypochlorous
Acid
Sodium
Hypochlorite
Chemical formula
Cl2
HOCI
NaOCI
CAS Registry No.
7782-50-5
7790-92-3
7681-52-9
Molecular weight
70.91
52.47
74.45
Boiling point
(25 mm Hg)
-34.05-C
Melting point
-101°C
18eC
Density
Dry gas (0°C/1 atm)
Liquid (0°C/3.65 atm)
(1.4085 mg/L)
3.209-3.214 mg/L
1.468 mg/L
1.4085 mg/L
—
—
Specific gravity
2.482 (0°C)
—
—
Water solubility
7.3 g/L (20°C)
14.6 g/L (0-C)
--
283 g/L (0°C)
Physical form (25°C)
gas
liquid
crystal
Color
greenish-yellow
greenish-yellow
white
Taste threshold (water)
—
—
—
Odor threshold (water)
(air)
0.002 mg/L
0.31 ppm
Conversion factors
1 ppm=2.9 mg/m3
1 mg/m3=0.344 ppm
Residue level (water)
0.2-1.5 mg/L
'Source: Windholz, 1983; NIOSH, 1976, 1984, 1989; Sconce, 1962
CHLORINE.2 II-2 01/20/94
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temperature, exposure to light ana the presence of catalysts or organic.material affect the
stability of free chlorine in aqueous solution. As pH increases above 3.0 and the total
concentration of chlorine decreases below -1000 mg/L, molecular chlorine quantities
diminish, while the hypochiorous acid and hypochlorite ion predominate (NRC, 1980). The
dissociation of hypochiorous acid to hypochlorite ion is largely dependent upon pH and
temperature. The acid is the prevalent form at pH <7.8 and 7 5 for 0° and 20°C,
respectively (Morris. 1978).
Hypochiorous acid is a greenish-yellow liquid whose stability is determined by
reactant concentration. pH. temperature and exposure to light (see Table 11-1).
Hypochiorous acid is the active form of chlorine in treated water and wastewater. When
chlorine reacts with organic compounds in solution, hypochiorous acid acts as an
electrophile in the addition, substitution and oxidation reactions that occur (Morris, 1978).
In water containing significant amounts of ammonia or other nitrogenous compounds,
hypochiorous acid will react to form chloramines. The term "combined chlorine" (combined
available chlorine, combined residual chlorine) refers to the amount of chlorine present as
chloramines. The chloramine hydrolysis constant may favor the reverse reaction such that
hypochiorous acid is produced (at a slower rate) from chloramines in aqueous solution.
This reaction serves as the key mechanism in the chlorination of municipal water supplies,
wastewater and swimming pools.
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Hypochlorite ion is most commonly supplied as a salt. Table 11-1 contains chemical
and physical properties associated with sodium hypochlorite. Calcium hypochlorite and
sodium hypochlorite are used as chlorine sources for the disinfection of drinking water
since hypochlorite solutions are more stable than hypochlorous acid. Their decomposition
may be enhanced by factors such as reactant concentrations, pH, temperature, exposure
to light and ionic strength. Hypochlorous acid is readily formed when hypochlorites
undergo hydrolysis. The utilization of hypochlorites as bleaching agents and disinfectants
is explained by their high oxidation potential and ability to form hypochlorous acid under
favorable conditions.
Environmental Fate. Transport and Distribution
The fate, transport and distribution of chlorine in natural waters is varied and
complex. The major chemical reactions known to occur in aqueous solutions serve as the
primary source of information (Table II-2). Several compilations of thermodynamic,
equilibrium and kinetic data have been made for these reactions. Significant efforts have
been made to integrate water quality data with kinetic and equilibrium data in order to
develop models for predicting the products of the chlorination of natural waters (freshwater
and seawater). Such models will facilitate a more thorough understanding of chlorination,
chlorination products and their ultimate fate and distribution in aqueous solutions {Jolley
and arpenter, 1983a).
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TABLE II-2
Chlorine Reactions Known to Occur in Aqueous Solution*
Reaction Type
Examples
Water
Hydrolysis
CI, + H20 s HOCI + H* + CI"
Ca(OCI)2 + 2 H20 s Ca(OH2 + 2 HOCI
Ionization
HOCIsH* + OCI"
NaOCI - Na+ + OCI"
Ca(OCI)2 - Ca2* + OCI"
Ammonia
Substitution
NH3 + HOCI - NH2CI + H20
Oxidation
2NHCI2 + H20 - N2 + HOCI + 3H+ + 3CI'
Inorganic oxidation
Mn*2 + HOCI + 2H20 - MnO(OH)2 + 3H* + CI"
Disproportionation
30cr - 2cr + cio3-
Decomposition
2HOCI - 2H* + 2CI' + Oj
Organic reactants
Oxidation
RCHO + HOCI - RCOOH + H* + CI"
Addition
RC=CR1 + HOCI - RC(OH)C(CI)R1
Substitution
N-CI bond
RNHj + HOCI - RNHCI + H20
C-CI bond
P.COCH, + 3HOCI - RCOOH + HCCI, + 2H,0
'Source: Jolley and Carpenter, 1983a
CHLORINE.2
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Chlorine hydrolvzes very rapidly in water with a hydrolysis constant ranging from
1.5x10^ at 0°C to 4.0x10"" at 25=C The half-life of chlorine in natural waters is 0.005 s2.
Complete hydrolysis occurs in fresh and wastewaters at pH >6 with the formation of
hypochlorous acid (HOCI) and chloride ion (CI') (Morris, 1978). Hypochlorous acid ionizes
rapidly with a dissociation constant ranging from 1.6x10"8 at 0°C to 3.2x10'8 at 25°C
yielding hydrogen ion (H*)- and hypochlorite ion (OCI"). At pH >5 and 25°C, the
concentration of HOCI and OCI' are essentially equimolar (Morris, 1966). At higher pH
values OCI" becomes the major form of chlorine, while at lower pH values HOCI will
dominate.
Because free chlorine (CU. HOCI and OCI") is a strong oxidant in natural water, its
stability is very low. In the presence of ultraviolet light, free chlorine oxidation of water to
02 proceeds at a significant rate. Half-lives of 8-28 minutes have been measured in
secondary effluents with sunlight. For similar chlorinated effluents without sunlight, a
10-fold greater persistence was measured with a half-life of 1.3-5 hours (Johnson, 1978).
Vaporization of molecular chlorine (Clz) is insignificant at low concentrations of
chlorine and neutral pH values. At acidic pH values and high concentrations (pH 2 and
3500 mg/L chlorine) significant amounts of Cl3 may be vaporized into the atmosphere
(White, 1972).
CHLORINE.2
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Free chlorine reacts rapidly with inorganics such as bromide and more slowly with
organic material present in natural and wastewaters. The primary chemical reactions of
chlorine, hypochlorous acid and hypochlorite ion in freshwater and seawater are depicted
in Figure 11-1 (Sugam and Helz. 1980). Free oxidant forms (Cl2, HOCI, OCT, HOBr. OBr),
combined oxidant (chloramines. organic chloramines, bromamines. organic bromamines,
mixed halamines), and reaction products (chloro- and bromoorganics, oxidized organicsT
chlorate and bromate ion. oxidized metal ions, nitrogen and oxygen) participate in, a
complex set of interactions. Factors such as reactant concentrations, pH, temperature,
salinity and exposure to light control the extent of reactions. In freshwaters and
wastewaters (reactions on the right side of Figure 11-1) the concentrations of ammonia,
bromine and chlorine, and the pH serve as important regulators of reaction products of
which monochloramine appears to be the most persistent compound formed. Free
chlorine in natural waters is transformed to chloride, oxidized organics, chlororganics,
oxygen, nitrogen, chlorate, bromate and bromoorganics. The term "chlorine demand"
descnbes the quantity of chlorine required in these reactions. The actual concentrations,
distribution and fate of the products associated with chlorine demand reactions have not
been determined (Jolley and Carpenter, 1983a).
Analytical Methods
The me^iurement of chlorine, hypochlorous acid and hypochlorite ion in drinking
water is complicated by such factors as confusion associated with water chlorination
terminology, selection of appropriate testing procedures, and the sensitivity and accuracy
CHLORINE.2
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cu. OCI
*-CI ORGANIC*
^r/„
^ MHjCI r
M-Br ORGANICS
UBr
HOCI. OCI
MHfirCl
CIO
•#0
CIO
CI-ORGANlCS
0XI0I2E0 OHGANIC3
0r —ORGANICS
OXIDIZED ORGANICS
Seawater Freshwater
FIGURE 11-1
Principal chemical pathways for reaction, degration and environmental fate of free available chlorine in the aquatic
envirnment. Presumed pathways (not yet proven) are delineated by dashed arrows. Compounds formed at different
places in the reaction scheme are designated by superscripts a and b to assist in understanding interrelationships
Halides (C1" and Br) are not depicted but are products of many of the chemical pathways.
-------
of the available instrumentation. Combined chlorine, which is associated with the
presence of ammonia or nitrogenous compound, is often analyzed as free chlorine. Iodine,
hypobromous acid and bromammes may also be detected as free chlorine. The terms
"free oxiaant" and "combined oxidant" are used when referring to these compounds. When
considering both free and combined chlorine, the term "total chlorine" (total available
chlorine, total residual chlorine) is applied (Jolley and Carpenter, 1983b).
Appropriate testing procedures for the detection of chlorine, its oxidants, and
combinea-form species include potentiometry, amperometry, spectrophotometry,
chemiluminescence. colorimetry and titration (Jolley and Carpenter, 1983b). These
methods differ in their sensitivity for detection of the various chlorine compounds found in
water. Potentiometnc methods utilize measurement of the spontaneous electrical potential
generated between two dissimilar ion selective electrodes and can be used to detect total
oxidant formation. Limitations associated with the technique include the logarithmic
(Nernstian) response of the electrode and a poor level of precision as compared with other
techniques (U.S. EPA, 1981). The amperometric method of chlorine detection involves the
monitoring of the current generated from the application of a constant external potential
across indicator and reference electrodes. Amperometric methods can be used to
determine both free and total chlorine concentrations depending upon the composition of
the buffer solution (Johnson, 1978); The cell used in the amperometric method can be
modified for the measurement of available chlorine, hypochlorous acid and hypochlorite
CHLORINE.2
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ion. Continuous monitoring amperometric devices are considered to be fairly accurate,
typically ±1% (NRC. 1 976). but are subject to interference from Mn02 (Johnson, 1978).
Ultraviolet spectrophotometry, while not commonly employed, evaluates the
chemical speciation ana degradation reactions of chlorine and bromine compounds. Total
and free available chionne are measured by this process (Opresko. 1980).
Chemiluminescence involves the oxidation of luminol to azaquinone by
hypochlorous acid or molecular chlorine in aqueous solution and is useful as an indicator
method for very low concentrations of free available chlorine (U.S. EPA, 1981),
Hypochlorite ion levels can be detected at concentrations from 10"4 to 10"65 M.
The two most commonly applied methods for the detection of chlorine or its related
compounds in water are colorimetry and titration. Colorimetry has proven to be more
popular based upon its simplicity. In colorimetric techniques indicator chemicals are
reacted with chlorine compounds to produce colors that can be analyzed by comparison
with standard color scales. Generally, colorimetric methods are subject to interference
from sample color, turbidity, Mn03 and organic contaminants. However, the measurement
of chlorine or oxidant residuals at concentrations sO.1 mg/L as chlorine can be readily
accomplished with coiorimexic indicators (Jolley and Carpenter, 1983b).
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Titrimetry consists of four techniques (lodometry, amperometry, ferrousorxhotolidine
and ferrous-DPD) in which chlorine compounds are reacted with a reducing reagent. The
iodometric method involves the titration of iodine with a standard sodium-thiosulfate
solution and is used in measuring total chlorine concentrations <.1 mg/L (APHA. 1985).
The amperometric method is a more sensitive test used for the determination of free or
combined chlorine by titrating at pH 7 in the absence of iodide by a standard solution of
phenylarsine oxide or sodium arsenite. The third and fourth methods of titrimetry include
the ferrous-orthotolidine and ferrous-DPD techniques. The orthotolidene procedures have
been deleted from Standard Methods due to the low accuracy of this technique (APHA,
1985). The ferrous-DPD technique serves as an operationally simple procedure for deter-
mining free available chlorine, total available chlorine, combined available chlorine and
chloramine levels in water. Titrimetric methods are subject to a variety of interferences.
The reliability or accuracy of any information involving chlorine or chlorine-containing
compounds in drinking water is dependent upon the proper selection, execution and
analysis of results of a particular analytical method. Overall, electrochemical amperometric
methods are recommended for the detection of low concentrations of free or combined
chlorine, while iodometric and DPD-titrimetry and DPD-colorimetry are considered
appropriate for the determination of total chlorine levels in water. It is suggested that
measurement techniques and test conditions be described „: reports involving the analysis
of chlorine levels in water. Pertinent information would include the following: water quality,
analyst expertise, methodology and statistical significance (Jolley and Carpenter, 1983b).
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Environmental Sources. Production and Use
Chlorine exists iargely as chloride in combination with sodium, potassium, calcium
or magnesium in compounds such as salt (NaCI), carnalite (KMgCI3>H20) and sylvanite
(KCI) (Sconce, 1962).
As of January 1, 1986, 25 manufacturers with an estimated total chlorine production
capacity of 26.1 billion pounds were reported for the United States (SRI, 1986), Total
chlorine production for the United States was 20.8 and 21.9 billion pounds for 1986 and
1987, respectively (Reisch. 1988). In addition to domestic production of chlorine, the U.S.
Department of Commerce (1986) estimated chlorine imports to be 576.9 million pounds.
Over 95% of the chlorine manufactured in the United States is formed by the
electrolysis of chloride salts, most commonly sodium chloride (Windholz, 1983). In
addition, chlorine is produced through the oxidation of HCI in the presence of catalytic
metal oxides or oxidation with S03 or HN03, also as a by-product of potassium nitrate
production and as a co-product in the production of potassium hydroxide (U.S. EPA, 1981).
Hypochlorous acid is generated in situ by chlorine hydrolysis. Hypochlorite solutions are
prepared by chlorination of caustic or lime slurry (Wojtowicz, 1978).
Chlorine, hypochlorous acid and hypochlorites are used mainly as intermediates in
the production of organic chemicals. They are required to produce antifreeze, rubber,
pharmaceuticals, home permanents and cleaning agents, construction materials,
CHLORINE.2
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automobiles, food and paper. In addition, they are used as intermediates in the production
of inorganic chemicals, wnich in turn are used for a multitude of processes and products.
Chlorine has also been extensively used in the paper industry as a bleaching agent for
pulp and paper products.
Elemental chlorine and hypochlorites are also utilized in various water treatment
processes. As a result of their oxidizing characteristics and toxicity to microorganisms,
they are used to disinfect drinking water, sewage and wastewater, swimming pool water,
seawater, reservoirs, drainage ditches, in-plant water supplies (food industry) and industrial
cooling waters (Wojtowicz, 1978). The chlorine compounds function to sanitize, control
odors, reduce biochemical oxygen demand, implement chemical precipitation, reduce color
and treat cyanide (Dychdala, 1977).
In the food industry, chlonne and hypochlorites are employed for general sanitation,
and bacteria and odor control. Dairies, wineries, breweries, canneries, beverage bottling
plants and food processing plants use chlorine compounds to disinfect the equipment and
utensils, as well as the ingredients associated with the production of food and beverages
(Dychdala, 1977).
Summary
Chlorine, hypochlorous acid and hypochlorite ion are compounds that are noted for
their high oxidation potentials. Hypochlorous acid and hypochlorite ion are formed when
CHLORINE.2 11-13 01/20/94
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chlorine is added to water. The concentration of each agent in water is a function of
reactant concentrations, pH, temperature, exposure to light and the presence or absence
of catalysts or organic material. As a result of their highly reactive nature, chlonne and its
aqueous dissociation products are involved in the synthesis of many other chemical
species. The measurement of chlorine, hypochlorous acid and hypochlorite concentrations
in water involves the use of potentiometry, amperometry, spectrophotometry,
chemiluminescence, colorimetry and titrimetry. Electrochemical amperometric methods
are recommended for evaluation of low levels of free or combined chlorine; total chlorine
levels in water are best determined through iodometric and DPD-titrimetery and
DPD-chlorimetry. The ultimate fate of hypochlorous acid and hypochlorite in water is
dependent upon environmental conditions. Decomposition products may include chloride,
oxidized organics, oxygen, nitrogen, chlorate and nitrate.
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III. TOXICOKINETICS
The toxicokinetics of chlorine, hypochtorous acid and hypochlorite ion in drinking
water are largely unknown due to the fact that chlorine readily reacts with biological
molecules and is short-lived in organisms. Pharmacokinetic studies of Cl2f HOCI and OCI"
have been performed using radio labeled chlorine compounds. However, because these
molecules are so highly reactive in biological systems, these studies may reflect more the
fate of the chlorine ion (CI") or other reaction products generated than that of the parent
molecules. Chlorine is known to react with purine and pyrimidine bases and amino acids,
but the toxicokinetics of these compounds are not known. Pharmacokinetic studies have
also been conducted on chlorination by-products such as trihalomethanes as well as
alternate disinfectants such as chlorine dioxide (CI02) and its metabolites. Studies using
these compounds may not be useful in providing insight into Cl2 toxicokinetics due to
significant differences in oxidative reactivity of the various compounds.
Absorption
Data pertaining to the absorption of chlorine, hypochtorous acid and hypochlorite
ion in humans and animals are limited. The rates of absorption, the factors influencing
absorption, and the forms in which these compounds are absorbed are among topics that
have not been .jlly addressed. A few recent studies, however, have examined the
absorption of chlorine after oral exposure.
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Abdel-Rahman et al. (1983) investigated the pharmacodynamics of hypochlorous
acid. Radioactive hypochlorous acid (HOJ6CI) was administered by gavage to four fasted
and four nonfasted male Sprague-Dawley rats. Blood samples were taken and analyzed
for 36CI levels at intervals <72 hours in nonfasted rats and <96 hours in fasted animals. A
peak 36CI plasma level was reached in 2 hours in fasted rats versus 4 hours in nonfasted
rats following the oral administration of 0.6 mg (3 mL of 200 mg/L H036Cl) and 0.75 mg (3
mL of 250 mg/L H036CI) H036CI per animal, respectively. Body weights for fasted and
nonfasted rats ranged between 220 and 240 g. Doses of H036CI were calculated (using
mean body weight of 230 g) to be 2.61 and 3.26 mg/kg, respectively. The absorption
half-life was 2.2 hours for both fasted and nonfasted rats. Plasma elimination half-lives
were 44.1 hours in the fasted rats and 88.5 hours in the nonfasted rats. In an earlier study,
Abdel-Rahman et al. (1982b) found an absorption half-life of 36CI in plasma of 4.42±1.31
hours in rats receiving an oral dose of HO^CI in 3 mL of 250 mg/L aqueous solution of 36CI.
Approximately 77% of the initial dose was absorbed within 72 hours. These differences
in the absorption and elimination of 3BCf (derived from H036CI) may reflect the greater
tendency for chlorine., hypochlorous acid and hypochlorites to produce chlorinated organic
by-products in the blood or Gl tract of nonfasted animals (Vogt et al., 1979; Mink et ai.,
1983).
Suh and Abdel-Rahman (1983) studied the kinetics of CI" absorption in four fasted
male Sprague-Dawley rats administered an oral dose of 3 mL of 200 mg/L Na^CI solution
(-1.6 mg/kg bw). The blood samples were collected by heart puncture and ^Cl determined
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by liquid scintillation spectrometry. Levels of 35CI" in the plasma reached a maximum after
t- i
8 hours. The absorption and elimination half-lives from plasma were 19.2 and 51.9 hours,
respectively. The absorption half-life for the chloride was s4 times that observed for any
oxygenated forms (Table 111-1).
Distribution
Two studies by Abdel-Rahman et at. (1982b, 1983) examined the tissue distribution
of orally-administered hypochlorous acid in male Sprague-Dawley rats. In the first study
(Abdel-Rahman et al.. 1982b), four animals were gavaged with 3 mL of 250 mg/L H036CI
(3.26 mg/kg bw). After 72 hours the distribution of H03SCI was found to be highest in the
plasma (0.77% of the initial dose), followed by bone marrow, kidney, testes, lung, skin,
duodenum, spleen, liver and carcass, and was lowest in the ileum (0.14% of the initial
dose).
In the Abdel-Rahman et al. (1983) study four fasted animals received an average
dose of 0.6 mg H036CI (2.61 mg/kg bw). Ninety-six hours after administration, the
percentage of 3SCI was highest in the plasma (1.92 vg/g), followed by whole blood, bone
marrow, testes, skin, kidney, lung, packed blood cells, duodenum, stomach, spleen,
thyroid, thymus, liver, carcass and ileum, and was lowest in fat (0.09 Aig/g).
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TABLE 111-1
Summary of Selected 36CI Absorption Studies in Male Sprague-Dawley Rats
Test
Compound
Dose*
Rats
Fasted/
Nonfasted
' 1/2
(hours)
Plasma
Absorption
t m (hours)
Plasma
Elimination
Plasma
36CI
(hours)
Hours
to
Peak
Reference
H03SC!
3 mL of 200
mg/L (-2.61
mg/kg bw)
Fasted
2.2
44.1
2
Abdel-
Rahman
et al., 1983
H036CI
3 mL of 250
mg/L (-3.26
mg/kg bw)
Nonfasted
2.2
88.5
4
H036CI
3 mL of 250
mg/L (3.26
mg/kg bw)
NR
4.4
77.0
NR
Abdel-
Rahman
et al.,
1982b
Na35CI
3 mL of 200
mg/L (-1.60
mg/kg bw)
Fasted
19.2
51.9
8
Suh and
Abdel-
Rahman,
1983 |
"All materials administered orally.
NR = Not reported
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The subcellular distribution of 36CI in rat liver 24 hours following H03SCI
administration was also analyzed by Abdel-Rahman et al. (1983). They found that for
hypochlorous acid. 75% of the total ;6CI activity of the liver homogenate was recovered in
the cytosoi. 2.5% in the microsomal fraction, 1.5% in the nuclear and <0.1% in the
mitochondrial fraction. Only 4% of the total 3SCI activity in the whole homogenate was
recovered in the trichloroacetic acid precipitate of the homogenate following H036CI
treatment.
Metabolism
Free residual chlorine, as Cl2, OCI" and HOCI, is a strong oxidizing agent that readily
reacts with organic molecules to produce a wide variety of chlorinated compounds;
therefore, it is short-lived in biological systems (Seegert and Bogardus, 1980). This
reactivity in biological systems makes it difficult to study the in vivo metabolism of free
residual chlorine and to separate the effects of parent compounds from those of free
residual metabolites. The majority of data concerning the metabolism of free residual
chlorine is either indirect or derived from in vitro preparations.
Mink et al. (1983) studied the in vivo biotransformation of sodium hypochlorite in
fasted and nonfasted male Sprague-Dawley rats. In these experiments, rats weighing
-400 g were divided into control and treatment groups of six each (three fasted and three
-nonfasted animals). The six treated rats were dosed with 7 ml. of an 8 mg/mL solution of
sodium hypochlorite by gavage (140 mg/kg bw). One hour post-treatment all rats were
CHLORINE.3
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sacrificed and the blood and stomach were removed for analysis by GC/MS. Trichloro-
acetic acid and dichloroacetic acid were detected in the stomach contents of both fasted
and nonfasted rats. This indicated that in vivo formation of these chlorinated acetic acids
was not dependent on NaOCI interactions with organic material in the gut. Trichloroacetic
acid was detected in 2/6 plasma samples from dosed rats, and dichloroacetic acid was
detected in 5/6 plasma samples. The minimum detection limits of dichloroacetic acid (0.3
^g/mL) are lower than trichloroacetic acid (1.3 ^g/mL) by broad spectrum capillary GC/MS
scanning. This could account for the higher incidence of dichloroacetic acid observations.
While chloroform was found in the gut contents of ail of the treated rats, it was found in the
plasma of only one dosed (nonfasted) rat. Dichloroacetonitrile was detected in the gut
contents of 2 of the 3 nonfasted rats. None was present, however, in the gut of fasted rats,
indicating that the presence of food can have an influence on the development of
chlorination by-products. Dichloroacetonitrile was not detected in any plasma samples.
These findings emphasize the high reactivity of chlorine and the difficulty in ascertaining
the in vivo metabolic and toxicokinetic profile of chlorine.
The data of Abdel-Rahman et al. (1983) provide some insight into the terminal
product of chlorine metabolism. They found that 96 hours after rats were orally dosed with
HO^CI, 51% of the total MCI dose was excreted through urinary and intestinal routes. The
major metabolite of H036CI in plasma was chloride ion (81%).
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Baker (1947) and Pereira et at. (1973) studied the in vitro reactions of hypochlorite
and hypochlorous acid, respectively, with protein. Baker (1947) summarized the earlier
work of several authors with the following observations: 1) amino acids and their residues
are attacked by sodium hypochlorite at rates that differ greatly from one acid or residue to
another; 2) groups other than the peptide linkage are attacked; 3) both chlorination and
oxidation occur; 4) active chlorine groups of varying stability are formed; and 5) in acidic
conditions, chlorination predominates over oxidation. Baker (1947) showed that in the
course of protein degradation by NaOCl, -2.9 molecules of hypochlorite were reduced per
amino acid residue attached to protein. Pereira et al. (1973) found that hypochlorous acid
converts several a-amino acids into a mixture of the corresponding nitriles (major product)
and aldehydes (minor product) by oxidative decarboxylation. Chlorination of the ring of
tyrosine was also observed. Cysteine when reacted with HOCI yielded cystine and cysteic
acid as the only identified products. They also found that the amide nitrogen bond of
several dipeptides was resistant to HOCI at room temperature and chlorination of these
compounds yielded the corresponding N, N-dichlorodipeptide.
The reaction of hypochlorous acid with nucleotide bases has been examined fairly
extensively in recent years. Patton et al. (1972) found that one equivalent of hypochlorous
acid reacted with cytosine, 5-chlorocytosine or 5-methylcytosine to yield the corresponding
4-N-chloro derivatives. Reaction of three equivalents of HOCI with cytosine produced two
unstable compounds identified as di- and trichiorocytosine in addition to 4-N-chloro
derivatives. Reaction of cytosine with five equivalents of HOCI produced unstable tri- and
CHLORINE.3
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tetrachioro- derivatives. Hoyano et al. (1973) found both stable and chlorinated products
and labile intermediates formed by the reaction of aqueous HOCI with thymine, uracil and
5-bromouracil. The purine ring system of guanine and adenine was more resistant to HOCI
attack than thymine and uracil, but, with sufficient reaction time, parabanic acid was
produced. Dennis et at. (1978) found reaction of uracil with a 10-fold excess of HOCI
resulted in rapid degradation to trichloroacetic acid, carbon dioxide and nitrogen trichloride.
An estimation of the eventual metabolic fate of such chlorinated pyrimidines in
mammals can be extrapolated from experiments in mice where 5-chlorouracil was added
to the drinking water. The chlonnated base was found to be incorporated into the DNA of
r
tissue samples in the liver and testes (Cumming, 1978). Thus, it is presumed that the
bases are phosphorylated and eventually incorporated into nucleic acids.
9
Excretion
The elimination of 36CI-hypochlorous acid and its metabolites from the body has
been studied by Abdel-Rahman et al. (1983). In this study, the urine, feces and expired
air were collected over 4- and 5-day periods after the administration of 3 mL of a 200 mg/L
solution of H036CI (2.61 mg/kg bw) to four fasted male Sprague-Dawley rats. During the
first 24-hour period, 7.05% of the initial HO^CI dose was excreted in the urine and 7.45%
in the feces. After 96 hours, 36.43% of the administered dose was excreted in the urine
and 14.80% in the feces. In an earlier study (Abdel-Rahman et al., 1982b), a gavage dose
of 3.26 mg/kg bw HO^CI was administered to each of four male Sprague-Dawley rats. It
CHLORINE.3
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was observed that after 72 hours, 76 and 24% of the excreted 36CI residues of H036CI were
found in the urine and feces, respectively. 16C1 compounds were not found in the expired
air from any treated rats throughout the experiment.
Summary
At present, the study of the toxicokinetics of chlorine, hypochlorous acid and
hypochlorite ion has been limited to analyses of the gross movement of 3SCI following oral
administrations of various 36CI-containing compounds. Studies in rats have shown that
following oral administration of radioactive H036CI, 36CI is rapidly absorbed into the blood
with peak concentrations reached in 2 hours for fasted animals and 4 hours for nonfasted.
Absorption half-life was 2.2 hours for both fasted and nonfasted animals. When
administered as CI" (Na36CI) in nonfasted animals, peak plasma concentrations were
reached in 8 hours. Seventy-two to 96 hours after oral administration, the distribution of
36CI was highest in the plasma, followed by bone marrow, kidney, testes, lungs, skin and
liver. Lowest concentrations were found in the ileum and adipose tissue.
Chlorine, as Cl2, OCI" or HOCI, is a strong oxidizing agent that reacts readily in
biological systems producing a wide variety of chlorinated organic compounds. In vivo
studies in fasted rats have reported that 81% of the total ^Clj excreted after 96 hours was
in the form of chloride ion. In vitro studies have shown that hypochlorite and hypochlorous
acid react with proteins and nucleotide bases to form various chlorinated organic
compounds. Chlorine is eliminated from the body primarily through the urine and feces.
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CHLORINE.3 111-10 01/20/94
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IV. HUMAN EXPOSURE
Text to be provided by the Office of Drinking Water.
CHLORINE.4
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V. HEALTH EFFECTS IN ANIMALS
Introduction
The importance of chlorine, hypochlorous acid and hypochlorites in the treatment
of drinking water is evidenced by the relative decline in the incidence of waterborne
infectious diseases in modern times. Although chlorine has proven effective in its
inactivation of bacterial, viral and protozoal pathogens, concern over its use has been
generated by the discovery that water chlorination may result in the formation of potentially
carcinogenic organic by-products, in particular trihalomethanes (Bellar et al., 1974; Rook,
1974, 1976). Thus, attention has focused upon the utilization of alternative water
disinfectants such as chlorine dioxide, chloramines and ozone (Akin et al., 1982). Most
studies of the effects of chlorine-containing compounds in water have either investigated
these alternative disinfectants or the by-products (for example, trihalomethanes,
chloramines) formed as a result of water chlorination. The extent of research on the effects
of exposure to chlorine and its dissociation products in drinking water on animals and
humans is very limited.
Acute Toxicity
Inhalation. Information on the acute toxicity of chlorine has historically been based
upon the results of inhalation studies. Table V-1 lists lethal concentration levels of chlorine
gas administered by inhalation to various animal species.
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TABLE V-1
Lethal Concentrations of Chlorine Gas*
Species
Effect
Exposure Conditions
Rat
LC50
293 ppm/1 hour
Mouse
lc50
137 ppm/1 hour
Dog
LCl0
800 ppm/30 minutes
Cat
LCl0
660 ppm/4 hours
Rabbit
LCl0
660 ppm/4 hours
Guinea Pig
lcl0
330 ppm/7 hours
Mammals
o
O
i
500 ppm/5 minutes
'Source: NIOSH, 1986
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Withers and Lees (1985) reanalyzed the available acute inhalation toxicity data for
mice, rats and dogs. Using the Litchfield and Wilcoxcn method of probits and adjusting for
a common exposure period of 30 minutes, these authors estimated LCS0 values of 256, 414
and 650 ppm for mice, rats and dogs, respectively.
Several studies have been conducted to evaluate sensory irritation and pulmonary
function in mice, rats and rabbits following acute exposures to chlorine gas (Barrow and
Smith, 1975; Barrow et ai.. 1977; Barrow and Steinhagen, 1982; Chang and Barrow,
1984). RD50 values of 9.3 and 25.4 ppm were reported for mice and rats, respectively,
Pretreatment of rats for <2 weeks at concentrations of 1-10 ppm increased the RD50 values
20-fold (Barrow et al.. 1977; Barrow and Steinhagen, 1982; Chang and Barrow, 1984).
Rabbits exposed to 50-200 ppm chlorine gas for 30 minutes had decreased pulmonary
compliance and edema. Recovery time (0.5 hours to 3 days) was related to the level of
exposure and the extent of damage (Barrow and Smith, 1975).
Oral. An oral LD50 of 850 mg/kg bw was reported in rats ingesting calcium
hypochlorite (NIOSH, 1986). Toxicity data on acute oral exposures to chlorine,
hypochlorous acid and hypochlorites are summarized in Table V-2.
CHLORINE.5
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0
1
J—
o
2
z
m
CJ1
<
TABLE V-2
Acute Toxicity Studies in Rals
No ./Sex of Animals
Studied
Study
Length
Chlorine Exposure
Mode of
Administration
Effects
Reference
4 groups of 6/NR
15
hours
0, 20, 50 or 80 mg equivalent
chlorine in 5 mL NaOCI solution
(0. 100, 250 or 400 mg/kg). pH
not reported
Intragastric
intubation
Amount of CHCI^ in blood,
liver, kidney and fal
increased with increasing
doses of NaOCI
Voglel al, 1979
4/males
2 hours
0, 10, 20 or 40 mg/L HOCI
solution (0. 0 2. 0 4 or 0 8 mg/kg)
drinking water
gavage
Decrease in blood
glutathione .ifter 30 minutes
in (ho 10 of 40 mg/l dose
groups, recovery after 2
hours
Adbel-Rahmen et
al. 1982
4 groups of 4/males
3
hours-7
days
0 or 50 mg equivalent free
chlorine in 5 mL NaOCI solution
(250 mg/kg), pH not reported
intragastric
Intubation
Significant decreases in
hypothalamic norepinephrine
levels and increases in
normetanephrine levels after
3 and 24 hours; recovery to
normal levels after 7 days
Vogt et al, 1982
4 groups of 4/males
2-10
days
0 or 50 mg equivalent free
chlorine in 5 mL NaOCI solution
(0 or 142.9 mg/kg), pH not
reported
Intragastric
Intubation
Morphological and
biochemical liver changes
within 2 days of dosing;
recovery after 10 days
Chang el al, 1981
4 groups of 5/males
9 days
0,40, 200 or 1000 mg/L available
chlorine (0. 33 1,180.5 or 902.4
mg/kg/day), pH not reported
in milk ad
libitum
No significant effect on body
wieght gain or organ weights
Cunningham, 1980
4 groups of
10/females
14 days
0, 80, 400 or 2000 mg/L available
chlorine. (0, 8, 40 or 200
mg/kg/day), pH not reported
milk fed by
gavage
Body weight gain enhanced
at 60 mg/L; kidney
enlargement in 2000 mg/L
group
n5
O NR = Not reported
co
4^
-------
Vogt et al. (1979) administered aqueous sodium hypochlorite by gavage to fasted
Sprague-Dawley rats at 0, 100, 250 or 400 mg/kg bw (0, 20, 50 or 80 mg chlorine).
Animals were sacrificed within 1.5 hours after dosing. Chloroform was measured in blood,
liver, kidney, fat and brain. Chloroform concentration was found to be a function of
increasing doses of sodium hypochlorite in all tissues except the brain, in which chloroform
levels remained constant.
A single dose of 10. 20 or 40 mg/L (0.2, 0.4 or 0.8 mg HOCI/kg bw) was
administered to groups of four male Sprague-Dawley rats (Abdel-Rahman et al., 1984).
Decreases in blood glutathione were observed in the low- and high-dose animals by 30
minutes post administration. Recovery was complete in all groups by 2 hours. All treated
animals also showed an increase in red blood cell osmotic fragility at the earliest time point
(15 minutes); this effect was not observed 1 hour after treatment.
Administration of chlorinated water was found to influence the levels of hypothalamic
norepinephrine in rats (Vogt et al., 1982). Three groups of four male Sprague-Dawley rats
were given a single intragastric dose of 5 ml. sodium hypochlorite solution (250 mg/kg),
containing 50 mg equivalent free chlorine. Animals were killed after 3 hours, 24 hours, or
7 days after dosing. A contio group of four rats was given saline solution 12 hours prior
to sacrificing. Measurement of neurotransmitter levels in the hypothalamus at the different
CHLORINE.5
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time intervals revealed that significant treatment-related decreases had occurred in the
levels of norepinephrine (p<0.05). Hypothalamic norepinephrine had decreased by 25%
at the 3- and 24-hour time intervals. Recovery to the levels found in control animals was
completed in the treated rats after 7 days. Concurrent with the decreased norepinephrine
levels, normetanephrine. a metabolic product of the o-methyl transferase system, was
found to be increased. Normetanephrine levels returned to normal in a pattern that
corresponded (in the opposite direction) to the changes seen in norepinephrine levels.
Histamine and dopamine levels were decreased, but the changes were not statistically
significant.
Chang et al. (1981) reported that intragastric administration of chlorinated water to
rats resulted in the development of a condition resembling "fatty liver" syndrome. Adult
male Sprague-Dawley rats (4/group) were given a single 5 mL dose of sodium hypochlorite
solution which contained 1% (50 mg) equivalent free chlorine (142.9 mg/kg). The animals
were sacrificed at 2, 5 or 10 days after treatment. A control group was given saline
solution and killed after 12 hours. After 2 days, morphological changes occurred in the
livers of treated rats such that the liver acquired a fatty, pale colored appearance. Total
triacylglycerols increased 250% in the dosed rats. The composition of the triacylglycerols
was altered in both liver mitochondria and whole liver homogenate. Analysis of the acyl
groups revealed the presence of small but significant amounts of long chain poly-
unsaturated fatty acids (PUFA). It was noted by the authors that hepatic triacylglycerols
CHLORINE.5
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generally contain only trace levels of PUFA. Recovery from the morphological and
biochemical changes had occurrea in 50% of the rats examined at 5 days ana in all of the
rats examined after 10 days.
Cunningham (1980) reported that weight gain in weanling Wistar specific-
pathogen-free rats and albino guinea pigs was enhanced by administration of water or milk
treated with sodium hypochlorite. Four experiments, two of which entailed short-term
exposure, were performed to aid evaluation of practices within the dairy industry. In the
first, sodium hypochlorite was mixed with cow's milk on a daily basis such that the nominal
concentrations of available chlorine in milk were 0, 40, 200 or 1000 mg/L (0, 36.1, 180.5
or 902.4 mg/kg/day). Five male weanling rats/treatment group were given the milk ad
libitum for 9 days. Body weights were recorded 5 times in 9 days. The animals were given
free access to a commercial rat diet, but no water during the exposure period. Weight gain
was found to be enhanced, though not at statistically significant levels, in rats exposed to
the lower two chlorine concentrations. Organ weights (liver, kidneys, heart and brain) as
a percentage of body weight were unaffected.
Similar results occurred in the second experiment in which female rats (10/group)
were given sodium hypochlorite in milk at nominal doses of 0, 8, 40 or 200 mg/kg bw/day
as available chlorine. Treatment was by twice daily gavage at a rate of 1 mL/10 g bw/day.
Water was available ad libitum. Female rats were weighed 10 times over a 2-week study
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period. Weight gain was determined to be significantly enhanced by comparison with
controls in those animals receiving 8 mg/kg/day (p<0.05). Kidney weights of animals of the
highest dose group were also significantly increased (p<0.01).
Other Routes. Sodium hypochlorite was tested in a screening bioassay wherein
deionized distilled water solutions were injected into fertile hens' eggs. An LDS0 was
determined to be 1650 mg/kg bw. This was only slightly toxic by comparison with other
haiogenated hydrocarbons evaluated in the same assay (Hekmati et at., 1983).
Subchronic Toxicity
Limited data are available regarding the subchronic toxicity of chlorine,
hypochlorous acid and hypochlorite ion. Available studies are summarized in Table V-3.
Cunningham (1980) tested the effects of hypochlorite added to the drinking water
of male weanling rats (10/group) for 6 weeks at exposure levels of 0, 20, 40 or 80 mg/L
available chlorine. The average intake of free residual chlorine was calculated to be 0,4.1,
8.1 and 15.7 mg/kg bw/day for the control, low-, mid- and high-dose groups, respectively.
Enhanced weight gain was observed in all treated animals. Weight gain was significantly
(p<0.05) different from controls for those rats in the 8.1 mg/kg bw/day group only.
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0
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TABLE V-3
Subchronic and Chronic Animal Studies Involving Chlorine Administration in Drinking Water
Species
Number of Animals
Studied
Study
Length
Chlorine Exposure
Effects
Reference
Shaver broiler
chicks
240 females
240 males
2B days
0. 300. 600 and 1200
mg/L available
chlorine. pH not
reported
In 1200 mg/L: significant increase in
mortality, decrease in feed efficiency, heart,
kidney, liver and lestes weight. In 600 mg/L
reduced head weights (in both sexes) and
lestes weight. In 300 mg/L: significantly
lower mean body wieyhts, linear decrease in
waler consumption with increasing chlorine
concentration.
Hulan and Proudfool,
1982
Shaver broiler
chicks
240 females
240 males
28 days
0. 37.5, 75 and 150
mg/L available
chlorine, pH not
reported
No effects on organ weights, water
consumption, feed efficiency or mortality
Mice
10 females
10 males
10 male controls
33 days
0 or 200 mg/L free
available chlorine (0
or 25 mg/kg/day). pH
5.9-62
No adverse affects on weight gain, food or
water consumption, histological or
pathological changes
Blabaum and
Nichols, 1956
Guinea pigs
2 groups of 10
males
5 weeks
0 or 50 mg/L
available chlorine (0
or 13.4 mg/kg/day),
pH not reporled
No significant effects on waler consumption or
body weight gain
Cunningham, 1980
Rats
4 groups of 10
males
6 weeks
0. 20, 40 or 80 mg/L
available chlorine (0,
4.1,8.1 or 15.7
mg/kg/day). pH not
reported
Significant increase in total body weight gain
at 40 mg/L, only at 6 weeks.
Mice
10 males
10 male controls
50 days
0 or 100 ppm free
available chlorine (0
or 12.5 mg/kg/day),
pH 6 2-6.5
No adverse affects on weight gain, food or
water consumption, histological or
pathological changes.
Blabaum and
Nichols, 1956
-------
0
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TABLE V-3 (cont.)
Species
Number of Animals
Studied
Study
Length
Chlorine Exposure
Effects
Reference
Cameau pigeons
groups of 12
3 months
0, 2 or 15 mg/L (0,
10 or 7 5
mg/kg/day). pH 6 5 or
0.5
Increase in plasma cholesterol after 2 and 15
mg/L wilh normal diet and at 15 mg/L with
high cholesterol diet; increase in aortic plaque
size in pigeons fed normal diets and exposed
lo 2 and 15 mg/L chlorine, changes in plasma
thyroxine levtilt, .illcf trcatmunl with 2 and 1li
mg/L chlorine
Revis el al, 1906
New Zealand
rabbits
NR
3 months
0. 0.1 or 15 mg/L (0.
0.01 and 1.6
mg/kg/day)
Increase in hydroxyproline in heart tissue after
15 ing/L Irealment
Revis et al.. 1985
Sprague-Dawley
rats
70 males
70 females
3 months
0,25, 100, 175 or
250 mg/L (0. 2, 7.5.
12.8 or 16 7
mg/kg/day males) (0,
3.5. 12.6 19 5 or 24.9
mg/kg/day females)
No consistent treatment-related adverse
effects.
Daniel et al.. 1990
Carneau pigeons
5 groups of 5
9 months
0. 0.1. 10. 15 or 30
mg/L (0,0.05. 5.0.
7.5 or 15.0
mg/kg/day)
Contractile properties of the heart decreased
after 15 and 30 mg/L; relative heart weight
increased at 15-30 mg/L; endocardial and
myocardial fibrosis more intense at 30 mg/L;
more antherosclerotic plaques al 30 mg/L;
increased hydroxyproline in heart tissue after
0.1 mg/L; decrease in serum thyroxine and its
metabolites after 10, 15 and 30 mg/L
Revis et al., 1985
-------
0
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TABLE V-3 (conl)
Species
Number of
Animals Studied
Study
Length
Chlorine Exposure
Effects
Reference
Sprague-Dawley
rats
groups of 4/males
12 months
0.10.10.0 or 100
mg/L (0, 014. 1.4 or
14 mg/kg bw/day)
Significant decreases in red blood cell count
and hematocrit reversed at 6 months,
increased osmotic fragility 1-100 mg/L only
significant at 1 and 100 mg/L; increased mean
corpuscular hemoglobin
Abdel-Rahman et al ,
1984
F344 rals
groups of 50/
males & females
2 years
0, 0 05 and 1%
males (5.5 and 21 9
mg/kg bw) 0. 0 1 and
0.2% females (0,
15 5 and 54.7 mg/kg
bw)
Significant decreases in body and liver
weights all treated animals, decreased brain
and heart weights m males and salivary gland
and kidney weight in females.
Hasegawa et at , 1986
F344 rats
groups of 70/
males & females
2 years
0, 70. 140. 275 ppm
<0,4.2. 7.2, 13.3
mg/kg bw mates) (0.
4.2. 7.8, 14.4 mg/kg
bw females)
No treatment-related adverse effects.
Decreased water consumption, and body
weights.
NTP, 1990
B6C3F1 mice
groups of 70
males & females
2 years
0, 70. 140. 275 ppm
(0, 7.4,14, 24 males)
(0, 7 6, 14, 24
females)
BDII rats
236/sex NR
7
generations
100 mg/L free
residual chlorine (10
mg/kg/day), pH not
reported
No adverse effects on weight gain, food
consumption, water consumption, fertility,
lifespan, growth pattern, hematology,
histology (liver, spleen, kidney or other
organs).
Druckrey, 1968
2 NR = Nol reported
k5
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-------
Cunningham 11980) also tested male albino guinea pigs to determine whether the
enhancement of weight gain was a specific response in rats. Ten guinea pigs/group were
administered either nonchlorinated water or water containing 50 mg/L (13.4 mg/kg bw/day)
available chlorine ad libitum. At the end of 5 weeks, body weight gain was increased over
controls; however, this increase was not statistically significant. No treated animals, rats
or guinea pigs, were observed to decrease their water consumption or to exhibit any signs
of toxicosis.
Interest in the results of the Cunningham (1980) studies provided the impetus to
determine if similar effects might occur in Shaver Broiler chicks (Hulan and Proudfoot,
1982). Two experiments were conducted in which sodium hypochlorite was added to
drinking water to obtain available chlorine concentrations of 0, 300, 600 and 1200 ppm
(experiment 1), or 0. 37.5, 75.0 and 150 ppm (experiment 2). In each experiment, water
was made available ad libitum for 28 days starting when the chicks were 1 day old. The
480 chicks were randomly assigned so that each treatment was offered to three replicate
units of 20 male and 20 female chicks. Food and water consumption was recorded weekly
for the entire unit of 20 rather than for individual birds. On day 28, two birds from each of
the eight treatment groups were killed and hearts, livers, kidneys and testes were removed
and weighed. Administration of 1200 ppm available chlorine resulted in significantly
(p<0.01) increased mortality (as compared with controls,', "lowered feed efficiency,"
reduced water consumption, and decreased heart, liver, kidney and testes weights. Mean
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body weights and water consumption were significantly (p<0.01) lowered in chicks
receiving 300 ppm or more available chlorine in drinking water. There was some indication
of a growth stimulus at the lower concentrations (37 5-150 ppm) but none of the responses
were statistically significant. The results of this study are difficult to interpret, since the
investigators did not provide information on the causes of death for the chicks at the 1200
ppm HOCI treatment concentration. The decreases in body and organ weight may have
been an indirect effect of the unpalatable taste of the highly chlorinated drinking water.
The study lacked any discussion of quality control procedures as to whether the available
chlorine concentrations were measured or confirmed over the exposure period, or if
precautions were taken to prevent volatilization and photodegradation of residual chlorine.
Also, water consumption data are not adequate for deriving a mg/kg bw dose of chlorine.
Although the Cunningham (1980) and Hulan and Proudfoot (1982) studies indicated
that toxic effects may occur after oral administration of chlorine, studies by Blabaum and
Nichols (1956) did not report any evidence of toxicity. Blabaum and Nichols (1956)
administered chlorine to weanling white mice (strain not specified) obtained from a stock
solution prepared by bubbling chlorine gas through city water. In the first part of the
investigation, a group of 10 male mice were given free access for 50 days to 100 ppm
(12.5 mg/kg/day) available chlorine in water (pH ranged from 6.2-6.5). In the second part
of the xperiment two groups of mice (10 females, 10 males) were given 200 ppm (25
mg/kg/day) free available chlorine in drinking water (pH ranged from 5.9-6.2), ad libitum,
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for 33 days. A single control group of 10 male mice was used for both parts of the
experiment. The control group received city water from Madison, Wisconsin (pH=7.3),
which contained 10 ppm CI. Water supplies were changed every 12 hours, at which time
both the old and new drinking water supplies were analyzed with the idometric method to
determine the total residual chlorine. Chlorine test papers were employed to confirm that
the chlorine content of the dispensed water drops were not appreciably different from that
in the bottle. At the end of the experiments the mice were sacrificed, autopsied, and their
stomachs, intestines, kidneys, livers and spleens were removed for histological
examination. By comparison with controls, the mice receiving 100 and 200 ppm free
available chlorine expenenced no differences in weight gain, growth or water consumption.
No gross or physical abnormalities were found upon autopsy or histologic examination.
A NOAEL of 25 mg/kg/day can be identified in this study.
%
Fisher et al. (1983a) fed weanling rats (9/sex/group) diets containing 0, 1257 or
2506 ppm chlorinated flour for 28 days. TWA chlorine concentrations were 118 and 236
mg/kg bw for males and 125 and 249 mg/kg bw for females for the low- and high-dose
groups, respectively. Body weights, food consumption and organ weights were measured.
Histological examination of the kidneys, livers, hearts and brains was performed. There
was no difference in body weight in males. In the females, there was a significant (p<0.05)
inverse correlation between body weigi. jnd chlorine treatment at 28 days. No significant
differences were seen in absolute organ weights; however, there were significant (p<0.05)
CHLORINE.5
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dose-related increases in kidney weights (males) and liver weights (both sexes) when
organ weights were adjusted for body weight covariance. No significant histopathological
effects were found.
In a study by Kotula et al. (1987), weanling F344 rats (24/sex/group) were fed diets
containing 0. 50, 200 or 600 ppm (0, 5, 20 or 60 mg/kg/day) chlorine for 92 days. Rats
were weighed and food consumption recorded weekly. After 92 days, rats were sacrificed
and histopathologic examination was performed on all major organs (adrenals, brain, liver,
kidneys, lungs, ovaries, heart, thyroid, testes, pancreas, colon and stomach). Rats fed the
chlorine-treated diets did not exhibit any adverse treatment-related effects. Male rats fed
the meat diets with or without chlorine developed moderate fatty livers. This was attributed
to an excess intake of fat in the diet (33% fat). A NOAEL of 60 mg/kg/day can be identified
from this study. 1
Revis et al. (1985) exposed male New Zealand rabbits (5/group) that had been
maintained on calcium-deficient diets (80% minimum daily requirement) and supplemented
*
with 10% lard to drinking water containing 0, 0.1, 10, 15 or 30 ppm (mg/L) chlorine for 3
months. Chlorine doses were estimated to be 0, 0.01, 1.6 and 3.2 mg/kg bw assuming an
average body weight of 3.8 kg and 0.41 LYday water consumption. Analyses of the
hydroxyproline content of the rabbits hearts exposed to 0, 0.01 and 1.6 rng/kg bw chlorine
showed a dose-related increase in the hydroxyprobine concentration. This increase was
CHLORINE.5
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statistically significant (p<0.05) at ihe high dose (1.6 mg/kg) only. No other effects were
reported in the rabbits.
Revis et al. (1986) exposed groups of 12 male White Carneau pigeons to 0, 2 or 15
mg/L chlorine in drinking water at pH 6.5 or 8.5 for 3 months. The birds were maintained
on either a calcium-deficient (80% minimum daily requirement) or a calcium-deficient diet
supplemented with 10% lard and 0.5% cholesterol. At 1-month intervals, blood samples
were collected and analyzed for development of atherosclerosis. The authors reported
significant increases in plasma cholesterol levels as compared with controls after 3 months
of exposure to 1.0 and 7.5 mg/kg/day chlorine at pH 8.5 and a normal cholesterol (but
calcium-deficient) diet. Of birds on the high cholesterol diet, plasma cholesterol was
increased in pigeons exposed to 1.0 mg/kg/day chlorine (pH 6.5 and 8,5), but significant
increases (p<0.05) were only observed in those pigeons given 7.5 mg/kg/day at pH 8.5.
Significant increases in the mean aortic plaque size in pigeons fed the normal diet were
observed only in the group exposed to chlorine at pH 8.5 (2 and 15 mg/L), suggesting a
relationship between mean aortic plaque size and plasma cholesterol levels. In an attempt
to determine if the increase in plasma cholesterol level is associated with thyroid function,
the levels of plasma thyroxine (T4) and its metabolite triiodothyroxine (T3) were quantified.
Significant changes in the levels of these hormones were observed in both the 1.0 and 7.5
mg/kg/day trea+;."ents at pH 8.5. Although the implications of these results may be
complicated by the fact that the experiment utilized calcium-deficient diets, the data have
CHLORINE.5
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identified a potential sensitive subpopulation and suggest possible adverse effects on the
cardiovascular system as a consequence of chlorine exposure.
Daniel et al. (1990) administered chlorine in the drinking water of adult male and
female Sprague-Dawley rats (10/sex/dose) for 90 days. Dose levels were 0, 25, 100, 175
and 250 mg/L which correspond to 0, 2, 7.5, 12.3 and 16.7 mg/kg/day for males and 0, 3.5,
12.6, 19.5 and 24.9 mg/kg/day for females, respectively. Food and water consumption,
and body weight were monitored and detailed hematologic, clinical chemistry and
histopathologic examinations were conducted. There were no treatment-related effects
on survival, body weight or histopathologic lesions for either sex. At the highest dose level
(200 mg/L), there was a 62-64% decrease in water consumption compared with controls.
The authors concluded that the highest dose level (200 mg/L) was a NOAEL.
In a more recent report (Daniel et al., 1991) chlorine was administered in the
drinking water of adult male and female B6C3F1 mice (10/sex/dose) for 90 days. Dose
levels were 0, 12.5, 25, 50, 100 and 200 mg/L which corresponded to 0, 2.7, 5.1, 10.3,
19.8 and 34.4 for males and 0, 2.8, 5.8, 11.7, 21.2 and 39.2 mg/kg/day for females.
Mortality, body weight, food and water consumption, hematology, clinical chemistry, organ
weights and histopathology were monitored. There was no reduction in survival or food
consumption or clinical signs of toxicity or nontrea'ment-related histopathologic lesions.
A concentration-related decrease in water consumption was observed in both males and
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females. Decreases were statistically significant (p<0.05) in females at the highest dose
levels (100 and 200 mg/L). A concurrent decrease in body weight gain was also observed
in both sexes with a significant reduction in males at the two highest dose levels.
Reductions in organ weights and serum enzymes were also observed; however, these
decreases were not consistent in males and females and were not reported at all dose
levels. The authors concluded that these effects were consistent with the decreased water
consumption rather than any specific treatment-related toxicity, especially in the absence
of any histopathologic lesions or signs of clinical toxicity. The 50 mg/L dose was
considered to be a NOAEL by the authors, while the two highest doses of 100 and 200
mg/L were considered mild LOAELs.
Bercz et al. (1990) studied the influence of chlorinated water on the development
of hyperlipidemia in monkeys consuming an atherogenic diet. Rhesus (4 females) and
African Green (5 males and 4 females) monkeys were exposed for 89 weeks to chlorinated
drinking water (0, 5 or 30 ppm) while being maintained on either normal or high cholesterol
- diets. The study design consisted of eight treatment periods. In period I (6 weeks),
animals were maintained on a normal diet and given nonchlorinated drinking water, which
served to establish baseline lipid parameters. During periods ll-V, animals were fed an
atherogenic diet (15% lard and 1% cholesterol) and given drinking water containing 0, 5
or 30 mg/L chlorine. Fasting blood samples were drawn weekly and analyzed fcr total
cholesterol, LDL, HDL and triglycerides. Statistical evaluation of measured responses was
CHLORINE.5
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performed by comparing each animal's median response with that of the previous treat-
i
merit period. The results indicated that exposure to 5 mg/L chlorine had no effect on lipid
metabolism. Exposure to 30 mg/L chlorine (14 weeks) resulted in increased LDL and
decreased HDL levels when compared with previous exposure period. It should be noted,
t
however, that cholesterol levels were initially increased with the introduction of the
atherogenic diet (previous treatment period) and may have reached a plateau during the
following period in which animals were exposed to high chlorine levels (30 mg/L). No effect
was seen on triglyceride levels. After 61 weeks, the animals were returned to a normal diet
and allowed to stabilize for 9 weeks with distilled water. Monkeys exposed to 30 mg/L
chlorine and normal diets showed no alterations in lipid metabolism.
Abdel-Rahman et al. (1984) administered HOCI in drinking water to groups of four
male Sprague-Dawley rats for <12 months at chlorine concentrations of 0, 1, 10 or 100
mg/L (0, 0.14, 1.4 or 14 mg/kg bw/day). Blood samples were collected after 2, 3, 4, 6, 7,
10 and 12 months of treatment and blood osmotic fragility and glutathione (GSH) levels
were determined. Data from this study indicated increased GSH levels at 2 months in 10
and 100 mg/L groups followed by decreased levels at 7 months. After 3 months of
treatment, there were significant decreases in red blood cell count and hematocrit;
however, these effects were reversed after 6 months of treatment. Increased osmotic
fragility was observed in "'I treatment groups; however, this effect was statistically
significant (p<0.05) in only the 1 and 100 mg/L dose groups. The authors concluded that,
CHLORINE.5
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based on these results, there was an increased mean corpuscular hemoglobin
concentration indicative of erythrocyte damage. The results of this study should, however,
be viewed with caution in light of the relatively small sample size involved as well as the
inconsistencies in the dose response and duration of effects.
Revis et al. (1985) also exposed groups of five white male Carneau pigeons
maintained on a calcium-deficient diet (80% MDR) to drinking water containing 0, 0.1, 10,
15 or 30 ppm (mg/L) chlorine (as sodium hypochlorite) for 9 months. Based on an average
body weight of 500 g and water consumption of 250 mL/day, this corresponds to -0, 0.05,
5.0, 7.5 and 15.0 mg/kg/day. In the pigeons, blood pressure, ventricular pressure (left
side) and pressure-time measurements were made as well as body and heart weights.
Morphological studies were also conducted on the heart tissue. Although the diastolic and
systolic blood pressure increased in the pigeons exposed to 5-15 mg/kg/day chlorine, this
change was not statistically significant. Contractile properties of the pigeon hearts
(described as dp/dt, the rate of rise of ventricular pressure) decreased significantly in
pigeons exposed to 7.5 and 15 mg/kg/day chlorine. Heart weight was also significantly
increased (p<0.01) at these dose groups. Endocardial and myocardial fibrosis as well as
atherosclerotic plaques in coronary arteries appeared to increase in incidence and seventy
with increasing doses. An analysis of the hydroxyproline content of the pigeon hearts
showed an increased concentration with increased dosage, Significant at 0.05 mg/kg in the
pigeons. Serum thyroxine (T„) and thyroxine metabolite (T3) were also measured in
CHLORINE.5
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pigeons exposed to cnlorinated water. The authors reported a significant decrease in the
levels of these hormones in pigeons given 5,7.5 and 15.0 mg/kg/day chlorine.
Chronic Toxicity
Druckrey (1968) studied the effects of highly chlorinated drinking water (100 mg/L)
given daily to 7 consecutive generations of BD II rats. Solutions were prepared weekly by
bubbling chlorine gas through tap water (Freiberg, Germany). Chlorine content was
monitored by titration with Na2S203. In order to insure a stable total dietary chlorine
concentration, dry rat chow was cooked with the chlorinated water prior to distribution to
the parental generation. Subsequent generations received chlorine only in the water and
ate a standard diet, resulting in an average daily dose of -10 mg/kg bw chlorine.
Parental animals began treatment at 100 days of age. Repeated matings were
done and rats remained on treatment during pregnancy and lactation. Selected progeny
were separated from their dams at 30-40 days and were designated the subsequent
generation at this time. Animals of the F3 and F4 generations consumed chlorinated water
only until the birth of progeny. Subsequent generations remained on test for the entire
lifespan. Two groups of animals served as controls at the beginning and ending of the
experimental period.
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After a period during which the rats became acclimated to the taste of the
chlorinated water, the test solution was well tolerated. Weight gain among neonates was
somewhat depressed during the first few days of life. By maturity the average body weight
for all generations of test animals was -5-10% greater than that of the untreated rats. Of
236 rats observed, no treatment-related effects were noted on the lifespan, .fertility, growth,
hematological measurements, or histology of liver, spleen, kidney and other organs. The
incidence of malignant tumors in the treated rats was not found to differ from that of control
group rats (see the Carcinogenicity Section). A NOAEL of 10 mg chlorine/kg bw could be
identified from this study.
Hasegawa et al. (1986) studied the potential adverse effects resulting from
long-term exposure to sodium hypochlorite in drinking water. Male and female F344 rats
(50/sex/dose) were given sodium hypochlorite in their drinking water at concentrations of
0.05 and 0.1% for males and 0.1 and 0.2% for females for 104 weeks. Similar numbers
of male and female rats received distilled water. All animals were observed daily and body
weights, mortality and water consumption were measured at regular intervals. All animals
surviving until the termination of the experiment were sacrificed, fasting blood samples
were taken and then autopsied. Complete gross and microscopic examination was also
performed on all moribund rats or animals dying spontaneously during the experiment.
Based on information provided by authors, chlorite (OCI") concentrations of 13.5 and 27.7
mg/kg bw for males and 34.3 and 63.2 mg/kg bw for females were estimated. These
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values, however, appear extremely high and inconsistent with those reported by other
investigators who have seen marked reductions in water consumption at similar or lower
chlorine concentrations.
As requested by the U.S. EPA, NTP conducted a 2-year bioassay to evaluate the
chronic toxicity and carcinogenicity of chlorinated drinking water. In this study F344 rats
(70/sex/dose) and B6C3F1 mice (70/sex/dose) were administered chlorinated drinking
water at 0, 70, 140 or 275 ppm available chlorine (as sodium hypochlorite) for 104 weeks.
Based on body weights and water consumption values reported in the study, these doses
correspond to doses of 4.2, 7.3 and 13.6 mg/kg/day for male rats; 4.2, 7.8 and 14.4
mg/kg/day for female rats; 7.4, 14 0 and 24 mg/kg/day for male mice, and 7.6, 14.2 and
24.2 mg/kg/day for female mice. Interim sacrifices were performed at 14 and 66 weeks
on 10 animals/sex/dose. Body weights, organs weights, histopathology and hematology
food and water consumption were evaluated throughout the study.
In rats, there was a dose-related decrease in water consumption. During the
second year of the study, water consumption was decreased 21% in males and 23% in
females. Mean body weights were slightly lower in high-dose females and in all treated
males. However, these decreases were within 10% of the control animals. There were no
biologically significant differences in organ weights or organ-to-body weight ratios. In
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addition, there were no changes in hematology, and no treatment-related gross or
microscopic lesions observed.
In mice, body weights were 5-8% lower for high-dose males and 5-7% lower in
females. There were no treatment-related significant differences in organ weights or
organ-to-body weight ratios. No alterations were reported in hematologic or gross or
microscopic histopathologic parameters. Although there was a high rate of mortality, the
survival rate was similar for all groups of rats, controls and treated animals. Absolute liver
weights were decreased significantly in male and female treated rats when compared with
controls. Significant decreases in weight, salivary gland and kidney weight were observed
in females. In the males, there was a significant decrease in brain and heart weights.
There was no significant increase in the incidence of gross or histopathologic
nonneoplastic lesion of any type in treated animals. No changes in serum chemistry, food
consumption or water consumption were observed. The biological significance of the
reduced organ and body weights is unclear although the authors concluded that these
effects may be suggestive of chronic toxicity.
Several studies have been done to ascertain possible health effects of chlorine as
an additive in flour (Fisher et al., 1983a,b; Ginocchio et al., 1983). Wistar rats
(60/sex/group) v.-ive fed diets consisting of cake prepared from flour containing 0, 1250 or
2500 ppm CI for 104 weeks (Fisher et al., 1983b). All animals were inspected daily, body
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weights and food intake were recorded weekly. Blood and urine analyses were performed
at 1, 3, 12, 18 and 24 months. Organ weight and histological evaluations were also
performed. Chlorine intake was estimated by the investigators to be 12.8 and 25.3 mg/kg
bw/day for males and 17.0 and 35.0 mg/kg bw/day for females for the low- and high-dose
groups, respectively. No differences in mortality were observed in treated animals when
compared with similarly fed control animals. Dose-related increases in hematological
parameters were seen in both males and females; however, these effects were statistically
significant in the males only (p<0.05), A dose-related statistically significant (p<0.05)
reduction in spleen weight was seen in the females. In addition to these effects,
histological lesions characteristic of the aging process were observed in the liver, kidney
and spleen in both control and treated rats; however, these effects appeared earlier in
treated animals. A LOAEL of 12.8 mg/kg can be identified in this study.
In a similar study, Ginacchio et al. (1983) fed Theiller original strain mice (60/sex)
cakes prepared from chlorinated flour containing 0, 1257 or 2506 ppm chlorine for 70-73
weeks. Body and organ weights, food consumption and mortality rates were measured.
Blood and urine analysis as well as histological evaluations were also conducted.
Estimated chlorine intakes were 143 and 286 mg/kg bw/day for males and 175 and 350
mg/kg bw for females for the low- and high-dose groups, respectively. The results showed
an increase in obesity in bo..i male and female treated mice. In females, there was a
statistically significant (p<0.01) increase in heart and kidney weights and a significant
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ip<0.05) decrease in ovary weight. There was also increased mortality in females
(p<0.001) in both dose groups. A LOAEL of 175 mg/kg bw can be derived from this study.
Target Organ Toxicity
Cardiovascular Effects. A reduction in the weight of the heart occurred in both
sexes of Shaver broiler chicks studied by Hulan and Proudfoot (1982) at available chlorine
concentrations of both 600 and 1200 ppm in drinking water.
As pigeons may provide a useful model for cardiovascular disease, Revis et al.
(1985) used these birds in an investigation of the effect of chlorinated drinking water on the
cardiovascular system. Groups of five male white Carneau pigeons were fed
calcium-deficient diets (80% MDR) and drinking water containing either 0 (deionized
water), 0.1, 10, 15 or 30 mg/L (0, 0.05, 5.0. 7.5 or 15.0 mg/kg/day) chlorine ad libitum for
9 months. After 9 months of exposure, pigeons were cannulated and blood pressure, left
ventricular pressure and pressure-time (dp/dt) were measured. Systolic and diastolic
pressure increased in the range of 8-13 mm Hg and 9-17 mm Hg in pigeons exposed to
5.0-15.0 mg/kg/day chlorine, respectively. These changes in blood pressure werei not
statistically significant. Significant increases in heart weight of 22-49%, however, were
observed in all treatment groups. Body weight of treated pigeons did not change by
comparison with controls. Pigeons given 7.5 or 15 mg/kg/day jhlorine showed a significant
decrease in dp/dt, which is frequently observed in chlorine heart failure secondary to
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cardiac hypertrophy. Morphological changes of the heart such as endocardial and
myocardial fibrosis appeared to increase with higher concentrations. Decreases in T4
levels ranging from 25-45% were measured in pigeons given 10, 15 and 30 mg/L chlorine.
The authors suggest that hypothyroidism may be a disease condition associated with
enlargement of the heart.
In a more recent study, Revis et al. (1986) examined the relationship of chlorine to
plasma cholesterol and thyroid hormone levels in pigeons. Groups of 12 male white
Carneau pigeons (age 3-4 months) were fed altered diets and drinking water containing
either 0 (deionized water), 2 or 15 ppm chlorine ad libitum for 3 months (0, 1.0 or 7.5
mg/kg/day). Diets were either (A) reduced to 0.35% calcium (80% MDR for a pigeon), or
(B) reduced to 0.35% calcium with the addition of 10% lard and 0.5% cholesterol. Treated
drinking water was prepared and changed daily. At 1-month intervals blood samples were
collected and plasma levels of cholesterol and T4 were determined. Following a 3-month
exposure to diet (B) and either deionized water or water containing 15 ppm chlorine (pH
8.5), pigeons were observed to have significant increases in plasma cholesterol. Plasma
T4 levels were significantly decreased in pigeons fed a normal diet (A) or high cholesterol
diet (B) and drinking water containing 2 and 15 ppm chlorine (pH 8.5). Plasma T3 levels
appeared to be increased at the 2 ppm level and decreased at 15 ppm in both diets A and
B. There was no clear dost esponse effect for plasma cholesterol observed in any of the
treatment groups. Thus, factors associated with the effects of chlorine on plasma
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cholesterol were not clearly elucidated by these data. The authors suggest that the
changes in plasma cholesterol may be mediated by products formed when chlorine reacts
with organic matter in the upper Gl tract.
Studies by Bercz et al. (1990) indicated that in monkeys maintained on high
atherogenic diets, the consumption of chlorinated water (0, 5 or 30 ppm chlorine) may
influence the development of hyperlipidemia. Exposure of both male and female African
Green and female rhesus monkeys to 30 mg/L chlorine resulted in increased LDL and
decreased HDL levels. There was also an increase in total serum cholesterol levels in
these animals. It should be noted, however, that when these animals were returned to
normal diets and exposed to similar levels of chlorine (30 mg/L) no change in lipid
metabolism was observed.
Hasegawa et al. (1986) found that long-term exposure of F344 rats (50/sex/dose)
to sodium hypochlorite in their drinking water resulted in statistically significant (p<0.05)
decreases in heart weights of males exposed to chlorine concentrations of *13.5 mg/kg
bw/day. This effect was not seen in females.
In a study in which mice (Theiller original strain) were fed diets of cakes prepared
from flour containing 0, 1257 or 2506 ppm chlorine for < 0-73 weeks, there was a
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statistically significant (p<0.01) increase in heart weight in female mice (Ginacchio et al.,
1983).
Immunological Effects. As part of a study on water disinfectants (Hermann et al.,
1982) 30 outbred CRI-1CD-1 mice received chlorinated drinking water for 120 days.
Control groups drank deionized distilled water (0.1 ppm CI, pH 6.1-6.4) or tap water (0.9
ppm CI. pH 7.0±0.2); experimental groups received hyperchlorinated water (15 or 30 ppm
CI, pH 6.4 or 6.8, respectively), chlorinated/acidified water (15 ppm CI, pH 2.5) or hyper-
acified water (0.1 ppm CI, pH 2.0). Fifteen mice from each group were tested for delayed
hypersensitivity to sheep red blood cell, and 15 were assayed for serum antibody
responses to the same antigen. Five mice/group were not immunized but were measured
for delayed hypersensitivity. Nonimmunized mice were used for assessment of
reticuloendothelial phagocytosis of colloidal carbon. None of the mice drinking chlorinated
waters showed evidence of a statistically significant change in humoral or cell-mediated
immune responses.
Fidler (1977) examined the relationship between drinking water hyperchlorination
and peritoneal exudate macrophage (PEM) functions in female C57BI/6N mice. A control
group received tap water (0.5-1.0 ppm chlorine) and the treatment group received
hypep-u,orinated tap water (numbers of animals not specified). The latter treatment was
prepared by adding sodium hypochlorite to tap water twice a week so that total residual
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chlorine (assayed by the o-toluidine method) was maintained at levels between 25 and 30
ppm. At weekly intervals, five control mice and five treated mice were injected i.p. with 2
mL thioglycolate broth in order to collect PEMs. Beginning with the first week of treatment,
there were significant decreases in the number of PEMs collected from mice given
hyperchlorinated water. Furthermore, during the first 2 weeks of treatment, PEMs from
mice receiving hyperchlorinated water showed significant decreases in in vitro cytotoxicity
against mouse melanoma (B16) and fibrosarcoma (UV-112) target cells, and a complete
absence of tumoricidal activity in the remaining 2 weeks. Hyperchlorination of drinking
water was, therefore, determined to have an adverse effect upon the macrophage defense
mechanism of laboratory mice.
Carcinogenicity
Chlorine, hypochlorous acid and hypochlorite ion have not been shown to act as
direct carcinogens or initiators of tumorigenesis. In the 7-generation toxicity study
conducted by Druckrey (1968), the incidence of malignant tumors in rats consuming
drinking water with a free available chlorine level of 100 mg/L was not different from the
incidence in control rats. Average daily dose was determined to be 10 mg/kg bw/day. Of
a total of 236 individuals from all generations of treated rats, 24 malignant tumors were
found. Among the total of 56 control animals, five tumors considered malignant were
found. The authors considered these cidences to be equivalent. There was, however,
an increase in the number of ileocecal sarcomas in the F2 generation of treated rats.
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These were undifferentiated large-cellular sarcomas originating in the retroperitoneal or
mesenteric lymph glands: there was evidence of some metastasis. Fs and Fs rats
consumed test water throughout their lifespan. Of 49 F5 rats only two ileocecal sarcomas
were observed, and among 63 F5 rats only one was noted. The author concluded that the
increased incidence of ileocecal sarcoma in the F2 rats was not attributed to the chlorine
ingestion, as an increase was not noted in the subsequent generations.
In a more recent study, Hasegawa et al. (1986) studied the carcinogenic potential
of sodium hypochlorite on F344 rats. Groups of 50 male and female rats were given
NaOCI in their drinking water at concentrations of 0.05 and 0.1% for males and 0.1 and
0.2% for females for 104 weeks. Although a variety of tumors developed in all groups
(controls and treated), no dose related increase was seen in either incidence, type or
latency period of tumors for any organ or tissue in either sex. Survival was also similar for
all groups. The most frequent tumors and proliferative lesions found were leukemias,
adenomas of the pituitary, C-cell adenomas of the thyroid gland and adrenal cortical
hyperplasia. These tumors are the most commonly found spontaneous tumors in F344
rats. NTP (1990) evaluated the potential carcinogenicity of chlorinated drinking water in
F334/N rats and B6C3F1 mice (70/sex/dose). Animals were administered drinking water
containing 0, 70, 140 or 275 ppm chlorine (as available atomic chlorine) for 2 years.
Interim sacrifices of 10 animals/sex/dose were performed at 15 and 66 .veeks. Food and
water consumption, body weights, organ weights, survival, clinical chemistry, hematology,
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and gross and microscopic histopathology were evaluated. Survival among treated
animals (rats and mice) was similar to controls. In rats there was an increased incidence
of mononuclear cell leukemia in mid- and high-dose females. This increase was
statistically significant (p=0.014) for the mid-dose females only. The incidence of
leukemias in females was 8/50. 7/50, 19/51 and 16/50 for the control, low-, mid- and
high-dose groups, respectively. The proportion of animals with leukemia that died before
the termination of the study (104 weeks) and the mean time to death were comparable for
all dose groups and controls. No leukemia were observed in male rats. Other neoplasms,
renal tubular cell adenomas and squamous cell carcinomas in males and islet cell
adenomas and lymphoid hyperplasia in females were also observed. However, the
occurrence of these neoplasms was sporadic, nondose-related or lacked supportive
evidence in either males or females. These neoplasms, therefore, were not considered
treatment related.
In mice there was no increase in neoplasms that were statistically significant when
compared with controls and that could clearly relate to chlorinated drinking water exposure.
Renal tubular cell neoplasms were observed in two high-dose male mice (one carcinoma
and one adenoma). Additional examination of renal tissue from all male mice (treated and
controls) resulted in the observation of one additional tubular cell carcinoma in a low-dose
male and foca1 hyperplasia in controls and treated animals from all dose groups. Since
there was no dose-related increase in the occurrence of these renal neoplasms and the
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incidence of hyperplasia was comparable in controls and treated mice, it was concluded
that these neoplasms were not treatment related.
Dermal application studies performed with sodium hypochlorite in conjunction with
other chemicals have produced conflicting results. In a study by Hayatsu et al. (1971),
three groups of 40 female ddN mice underwent dermal applications of
4-nitroquinoline-l-oxide alone, commercial sodium hypochlorite solution alone (*10% free
residual chlorine), or a combination of both. Application of sodium hypochlorite solution
alone for a total of 60 treatments in 300 days did not produce malignant or benign tumors
after a total observation time of 450 days. However, when 45 applications of sodium
hypochlorite solution were given after 20 treatments of 4-nitroquinoline-1-oxide (0.05 mg
of 0.25% w/w benzene solution), 9/32 mice developed skin tumors. This may indicate a
possible cocarcinogenic potential of sodium hypochlorite.
A later experiment by Pfeiffer (1978) used a study design similar to that used by
Hayatsu et al. (1971). In this case a 0.22% or 0.44% acetone solution of benzo[a]pyrene
was applied to the depilitated skin of female NMRI mice twice a week for 10 weeks,
providing total doses of 750 ug or 1500 ug/animal. Sodium hypochlorite (1 %) was applied
to groups of 100 mice either before, during, or after treatment with benzo[a]pyrene.
Results indicated that skin tumors in animals adn^.'iistered NaOCI prior to benzo[a]pyrene
treatment were reduced in size and number. The number of carcinomas among the tumors
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observed was decreased by g4Q% in all the treatment groups that received chlorine.
Neither the time to tumor induction nor the mortality rates were affected by chlorine
treatment. Thus, these experiments showed chlorine to reduce rather than to increase the
tumor incidence.
In a more recent study, Robinson et al. (1986) treated the skin of female SENCAR
mice with aqueous solutions of HOCI and NaOCI for 4 days. Animals were sacrificed on
days 1, 2, 3, 4, 5, 8, 10 or 12 following the last treatment. The shaved backs of animals
were treated with 0.1.10,100. 300 or 1000 ppm HOCI or NaOCI. For animals treated with
HOCI there was a dose-related statistically significant (p<0.05) increase in epidermal
thickness. Treatment with NaOCI resulted in increased epidermis only at the 1000 mg/L
dose level.
Mutagenicity
Assessment of the mutagenicity of chlorine is complicated by the reactive nature of
the chlorine molecule. Mutagenicity testing of chlorine is confounded by the presence of
its reaction products, which have been found to be mutagenic.
Sodium hypochlorite has been determined to be directly mutagenic at the histidine
locus in Salmonella typhimurium (Wlodkowski and Rosenkranz, 1975). oodium
hypochlorite gave variable results in standard plate incorporation assays, because of the
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bacteriotoxicity of the test compound. This toxicity was reduced by incubating the bacteria
and hypochlorite in suspension cultures and adding ascorbic acid at various time intervals
to decompose the residual hypochlorite. Under these conditions sodium hypochlorite at
a concentration of 0.28 ^moles/mL was mutagenic for the base substitution mutant
TA153Q but did not revert the frameshift mutation in TA1538.
j
Sweeney and Chek (1985) demonstrated that XAD-4 resin concentrates of free
residual chlorine were mutagenic in the Ames assay. Dose-related increase's in the
number of revertants/L were formed when chlorine concentrations of 0, 0.5, 1.0 and 2.0
ppm were tested without S-9 using Salmonella strain TA100.
In a study by Ishidate et al. (1984), sodium hypochlorite and calcium hypochlorite
were shown to be direct-acting mutagens in the Ames assay using Salmonella typhimurium
TA100 at nonbacteriotoxic concentrations.
Mickey and Holden (1971) reported that chlorine can directly produce chromosome
aberrations in mammalian cells. These authors added chlorine to the culture medium of
three different mammalian tissue culture cell systems at 13 different concentrations ranging
from 2.5-60 ppm. No significant increase in chromosomal aberrations of human
lymphocytes was detected jelow concentrations of -20 ppm. Above this concentration
there was an exposure dependent increase in chromatid and chromosome breaks,
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translocations, dicentric chromosomes and gaps. Chlorine was added to Chinese hamster
lung cells or to Indian muntjac cells 24 hours after subculture, and cells were harvested
and processed 24 hours later. Extensive exposure-dependent chromosomal breakage
occurred at concentrations *20 ppm. E-ndomitosis also increased at concentrations of s20
ppm. Thus, in all three test systems, the higher concentrations of chlorine produced
significant increases in chromosomal aberrations and endomitotic figures over control
values and completely inhibited mitosis at still higher chlorine levels.
Patton et al. (1972) showed that aqueous hypochlorous acid reacts with cytosine
to produce various chlorinated cytosine derivatives. Many investigators have shown that
the chlorination of uracil in water produces chlorinated uracil derivatives, the most
abundant of which is 5-chlorouracil (Dennis et al., 1978). Furthermore, Walton and
Gumming (1976) demonstrated 5-chlorouracil to be mutagenic for bacterial test strains.
A 300-fold increase in Escherichia coii WP-1 mutants was observed after the incubation
of the bacteria with 50 ug/mL. Cumming (1978) found that 1 g/L 5-chlorouracil given to
mice in their drinking water was incorporated into DNA. These authors, however, could
not demonstrate any mutagenic activity in a mouse dominant lethal assay from
5-chlorouracil and concluded that it does not constitute a significant mutagenic hazard to
the human populatton. The* formation'of chlorinated nucleic acids in vivo as a
consequence of drinking water exposure is speculative. 5-Ch!orouracil has, however, been
identified in chlorinated sewage effluents (Cumming, 1978).
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Sodium hypochlorite was assayed for its DNA-damaging potential in the E. colipolA
test. Preferential growth inhibition of the bacterial strain lacking the DNA repair polymerase
was observed in a spot test of sodium hypochlorite (0.01 and 0.006 umoles/spot). This
was indicative of the hypochlorite ability to interact with DNA. The author hypothesized
that the hypochlorite attacked the pyrimidine 5-6 double bond to yield 6-hydroxy-
5-chloro-5,6-dihydropyrimidine (Rosenkranz, 1973).
Calcium hypochlorite, likewise, produced chromosomal aberrations in CHL cells in
the absence of exogenous mammalian metabolic enzymes (Ishidate et al., 1984).
Meier et al. (1985) evaluated the cytogenetic effect of oral administration of
hypochlorite ion or hypochlorous acid to Swiss CD-1 mice. Test solutions were prepared
from a stock of NaOCI by adjusting the pH; at pH 8.5 the predominant chlorine species was
OCI", and at pH 6.5 it was HOCI. Five males and five females (or four each for bone
marrow aberration studies) were administered -1.6, 4.0 or 8.0 mg/kg/day. Dosing was
either for 5 days with sacrifice 6 hours after the last treatment (both assays) or a single
dose followed by sacrifice at 6, 24 or 48 hours postexposure (bone marrow aberrations
only). None of the treatments resulted in significant increases in erythrocyte micronuclei
or chromosome aberrations of bone marrow cells.
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Reproductive and Developmental Effects
Animal studies in general have demonstrated no evidence of reproductive or
teratogenic effects of chlorine. C3H/HEJ and G57B1/6J mice were administered drinking
water which had been treated with sodium hypochlorite and hydrochloric acid (10-13 ppm)
to maintain the water at pH 2.5 over a 6-month trial period (Les, 1968). Control animals
received tap water, which varied in pH from 9.2-9.8, but was usually 9.6. In the treated
animals, the number of mice born and the number weaned/dam were greater than in the
control (p<0.01). The authors concluded that the treatment of C3H/HEJ and C57BL/6J
mice with chlorine and hydrochloric acid had no adverse effects on their reproductive
performance.
McKinney et al. (1976) noted a periodic increase in reproductive failure among CD-1
mice. Mating, number of embryos per fertile female, and embryonic development were
affected. The effect was seasonal and was most severe in the winter. In the absence of
any other observed variations in the animal husbandry, the authors attributed the
reproductive deficiencies to the heavily chlorinated Durham city water consumed by the
mice.
Two attempts were made to repeat the observations reported by McKinney et al.
(1976). Chemoff et al. (1979) fo^nd no significant difference in the reproductive
parameters of CD-1 mice consuming Durham, NC drinking water as compared with the
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control group maintained on distilled water. The animals were maintained on the test or
control water for a 2-week period after which mating was begun. Dams were sacrificed on
day 18 of gestation. Exposure was continued throughout the 8-month course of the study
(December-September). Dams were analyzed for differences in numbers inseminated,
number pregnant, weight gain during gestation, organ weight, and percent resorptions.
Fetuses were examined for skeletal and visceral anomalies as well as mortality, and body
weight. No statistically significant maternal or fetal effects were noted, except for a 28.1%
incidence of supernumerary ribs in the tap water consuming group compared with 21.1%
in the control group (p<0.05).
In a second study, Staples et al. (1979) reported no significant overall influence on
the incidence of malformed fetuses (skeletal or visceral malformations) that could be
attributed to the chlorination of drinking water. The incidence of malformations in the
controls was 8.1% compared with 7.8 in treated animals. Differences were in the opposite
direction from the finding of McKinney et al. (1976). Two significant effects occurred in the
month of January, and one occurred in the month of February. In January, a lower number
of mated females CD-1 became pregnant, and the average number of implants/pregnant
female was lower in the purified water group. In February, the average fetal weight was
lower in the purified water group than in the tap water group. Indeed, the presence of
chlorine in the water seemed to confer a beneficial effect. The authors joncluded that the
results of their study did not support the findings of McKinney et al. (1976), although there
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were complicating factors in the Staples et al. (1979) study. There appears to be no
evidence that would link the process of chlorination at levels consistent with current
practice to any adverse reproductive effects in mammals.
Hulan and Proudfoot (1982) studied the effects of sodium hypochlorite in drinking
water on Shaver broiler chickens. Sodium hypochlorite was added to the drinking water
of chicks (240/sex) at doses of 0. 300, 600 and 1200 ppm. A significant (p<0.01) reduction
was found in the weight of chick testes at dose levels of 600 and 1200 ppm of available
chlorine. However at these higher concentrations there was also a decrease in total body
weight, food and water consumption and an increase in mortality.
Meier et al. (1985) demonstrated that oral administration of a sodium hypochlorite
solution, but not hypochlorous acid, resulted in dose-related increases in the amount of
sperm-head abnormalities in male B6C3F1 mice. Ten animals/group were given 1 mL of
a free residual chlorine solution daily for 5 days. Test solutions were prepared bubbling
Cl2 into a 1 M solution of NaOH and adjusting the pH to either 8.5 (predominant species
OCI") or 6.5 (predominant species HOCI). The solutions were diluted with distilled water
to 200 mg/L, 100 mg/L and 40 mg/L chlorine equivalents (8.0, 4.0 or 1.6 mg/kg bw/day
respectively). The mice were then sacrificed at 1, 3 or 5 weeks after the last dose was
administered. .'•¦¦¦ mice given OCI", significant increases in sperm-head abnormalities were
observed only at the 3-week interval at doses of 1.6 and 4.0 mg/kg bw/day. These results
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were reproduced in retrials of the experiment. HOCI administration at any dose was not
associated with increases in sperm-head abnormalities.
Druckrey (1968) conducted a 7-generation study wherein rats were given drinking
water chlorinated to a concentration of 100 mg free chlorine/L. Animals were mated
repeatedly and continued to drink the test water throughout gestation and lactation. There
was a total of 108 matings among the test group resulting in 80 litters for a total of 609
viable progeny. The average number of viable progeny/litter was 7.9 for test animals and
8.2 for controls. Mortality among neonates of test animals was -20% higher than that
observed for controls. This was attributed to decreased milk consumption as a
consequence of the chlorinated odor of the milk. Microphthalmia of one or both eyes was
noted in 17 treated progeny. This finding was observed irregularly, and it was stated that
this condition has been known to occur spontaneously in BDII rats. Mating of
microphthalmic rats produced normal offspring, indicating that the condition did not arise
from a stable germ cell mutation. Druckrey (1968) concluded that no reproductive effects
could be attributed to chlorine ingestion in this study.
Pilot experiments have been performed to determine the possible teratogenic effects
of disinfectants on the developing rat fetus (Abdel-Rahman et al., 1982a). Six virgin
Sprague-Dawley rats we're administered 0, 1, :0 or 100 mg HOCI/L in drinking water for
2.5 months prior to mating. Animals were maintained on the treated water after pregnancy
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was confirmed (day 0) and killed on day 20. Maternal weight at time of death was not
reported. Incidence of fetal anomalies associated with exposure to hypochlorous acid
solutions were not found to be statistically significant. Mean fetal weight from the 10 and
100 mg/L groups were less than control, but this decrease was not statistically significant.
Neither was there a significant difference in numbers of resorptions between control and
treated groups. Examination of general trends in the study indicated an increase (not
significant) in skeletal anomalies in animals treated with 10 mg HOCI/L. Soft tissue
anomalies for the 100 mg HOCI/L treatment group were increased significantly by
comparison with control. The findings of these experiments were limited by the small
number of study animals. Some of the calculations of anomaly percentages reported in
the paper were incorrect. Furthermore, the rate of both skeletal and soft tissue anomalies
appeared to be higher in the control group than in the low-dose treatment groups.
Summary
The majority of research dealing with acute effects of chlorine exposure has been
in the area of inhalation. Short-term exposure of rats by gavage to HOCI or CI in aqueous
solution (to -250 mg/kg/day) has resulted in transient decreases in blood glutathione and
hypothalamic norepinephrine and reversible morphological and biochemical liver changes.
Exposure by gavage in milk to 200 mg/kg available chlorine/day for 14 days resulted in
kidney enlargement in rats. No effects were observed in rats when as much as d02 mg
Cl/kg/day was consumed ad libitum in milk.
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Shaver broiler cmcks were observed to have decreased body weights and organ
weights (including testesi when maintained for 28 days on water with 1200 mg/L available
chlorine. As there was also a dose-dependent decrease in water consumption by the
birds, the effects noted may have been because of the unpalatability of the water. Longer
term studies in rodents have shown mixed results as a consequence of consumption of
chlorinated drinking water. A 7-generation study wherein BDII rats consumed 10 mg
Cl/day in water produced no evidence of adverse effects. However, exposure of F344 rats
for 2 years resulted in decreased body and organ weights. Additional studies in F344 rats
and B6C3F1 mice observed no treatment-related adverse effects.
In these same studies, no clear evidence of increased treatment-related tumor
incidence was reported for rats or mice. Increases in mononuclear cell leukemias in
female rats and renal tubular cell neoplasms in male mice were reported. These increases
were not considered dose or treatment related. There are conflicting data regarding
cocarcinogenicity of sodium hypochlorite. Commercial sodium hypochlorite applied
dermally to female ddN mice enhanced tumor development initiated by 4-nitroquinoline-1-
oxide. By contrast sodium hypochlorite applied to the skin of female NMRI mice treated
with benzo[a]pyrene reduced the tumor incidence. It should be noted that the data
discussed here do not address the possible carcinogenicity associated with organic
by-products formed durin^ water chlorination.
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Chlorine has been shown to produce chromosomal aberrations in mammaiian ceils.
Sodium hypochlorite both damages DNA and causes base substitution mutations. Neither
hypochlorite ion or hypochlorous acid, however, caused chromosomal aberrations.
t
No reproductive dysfunction was noted in a 7-generation rat study wherein chlorine
was administered in drinking water. Hypochlorite ion, but not hypochlorous acid, produced
dose-related increases in sperm-head abnormalities in male B6C3F1 mice.
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VI. HEALTH EFFECTS IN HUMANS
Introduction
Studies of the relationship between chlorine in drinking water and human health
effects are few. The majority of information about chlorine in regard to adverse health
effects pertains to the inhalation of chlorine gas and is likely of questionable relevance
to assessing exposures from ingestion of chlorine in water. Although these data are
considered in an overview of chlorine, the findings presented here will principally address
the results of exposure to chlorine, hypochlorous acid and hypochlorite ion in drinking
water. Some of the more recent information in this chapter, particularly the clinical
studies and analytical epidemiologic studies, was previously summarized in another
technical report (unpublished) for the Office of Drinking Water (Craun et al., 1989) and
is presented here in its original form.
There is no question that disinfection of drinking water with chlorine has clearly
been a beneficial practice. Waterborne infectious disease has been brought under
control, and while outbreaks still occur in the United States, they are of a different
magnitude and nature than occurred before the widespread practice of disinfection was
instituted (Craun, 1986). The current focus on the possible relationship of this practice
to cancer and cardiovascular disease should be weighed against the clearly positive
effects of disinfection.
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Clinical Case Studies
A few early clinical case reports of the toxic effects of chlorine in drinking water
have been documented (Muegge, 1956). Muegge reported that a group of 150 persons
at a military base consumed water with chlorine levels of 50 ppm (-1.4 mg Cl/kg/day)
during a period of water main disinfection. No adverse health effects were noted.
Another incident of elevated chlorine levels in drinking water occurred during a flood
where residents consumed water containing 50-90 ppm (-1.4 and 2.6 mg Cl/kg/day).
Muegge noted that when chlorine levels exceeded 25 ppm, people refused to drink the
water. In this same article, the author cited an incident in which a group of Army
personnel drank water containing >90 ppm chlorine. This ingestion resulted in
constriction of the throat and irritation of the membranes of the throat and mouth of those
exposed. Muegge's overall conclusion was that human beings have a high tolerance to
highly chlorinated water. It must be noted that this was only an anecdotal case report.
Lubbers et al. (1982) investigated the effects of oral ingestion of chlorine,
chloramine, chlorine dioxide and chlorate administered in drinking water under two clinical
study protocols. Study subjects were healthy male volunteers between 21 and 35 years
of age. Phase I consisted of an increasing dose tolerance analysis in which progressive
doses of chlorine were administered in water as chlorate, chlorine dioxide, chlorite,
chlorine and chloramine to six groups: 10 subjects/compound, given every 3 days for a
total of 18 days, at concentrations of 0.1, 1.0, 5.0, 10.1, 18.0 and 24.0 mg/L (0.001,
0.014, 0.071, 0.143, 0.257 and 0.343 mg/kg, respectively) in a total volume of 1000 ml_.
Phase II consisted of 60 subjects who ingested the various compounds listed above, in
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six groups: 10 subjects/compound at daily concentrations of 5 mg/L in a volume of 500
mL of water for 12 consecutive weeks. In both phases, the sixth group consumed
untreated water. Three participants who were deficient in glucose-6-phosphate
dehydrogenase (G6PD) and considered to be theoretically more susceptible to oxidative
stress were included as a separate group in the second phase. They were given daily
5 mg/L of sodium chlorite only in a volume of 500 mL for 12 consecutive weeks.
Sixty-eight biochemical and physiological parameters were measured in each
participant at multiple time points for each phase in the study. The data were analyzed
using analysis of variance parameters in the different exposure groups but no data were
presented to indicate either the direction or magnitude of change. With so few people per
experimental group and so many significance tests computed, it would be inappropriate
to consider the changes to be a true effect of the exposures. The changes appeared to
be randomly distributed among the participants and unrelated to any particular protocol.
No findings were specific to the three subjects who were deficient in G6PD. It is unclear
why the invesgiators would even attempt any formal statistical testing in a group
comprised of only three individuals. No clinically significant changes in any variables
were noted; all fluctuations were within normal range of measurement. Although the
study was limited by the failure to control variables such as diet and other sources of
drinking water, the fact that there were no overt adverse health effects suggests that
ingestion of chlorine at these levels over a relatively short period of time produces no
toxicity in healthy adult males. The most reasonable interpretation of the noted changes
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is that it likely represents a combination of interindividual variability and random laboratory
error.
In a recent series of controlled clinical studies, Wones et al. (1989, 1991)
investigated the effects of chlorinated drinking water on human lipid metabolism. In the
study 19 healthy males (24-62 years old) were given 0, 2, 5 or 10 ppm chlorine in their
drinking water for a total of 15 weeks. Each subject was allowed to serve as his own
control and was required to drink 1.5 L of water/day. All subjects were maintained on a
high fat, high cholesterol diet throughout the study period and intake was weighed and
measured daily. Total serum cholesterol, lipoproteins, apolipoproteins and thyroxine
levels were monitored. Baseline levels were obtained during the first 4 weeks of the
study in which subjects were given the protocol diet and chlorine-free water, after which
chlorine was added to the drinking water at 2, 5 or 10 ppm for a period of 4 weeks each.
A 3% increase in total serum cholesterol occurred during the treatment period and was
statistically significant (p<0.05) for the 5 and 10 ppm dose groups. Total thyroxine levels
were statistically significantly (p<0.05) increased above baseline at all doses throughout
the study. However, these changes were numerically small, not uniform in all subjects
and were not clinically significant. Because there was no separate comparison group that
did not receive chlorine, it is possible that the small observed increases were due to the
protocol diet or other uncontrolled factors and not to the chlorine.
To correct for these methodologic deficiencies, a second study was conducted to
determine if drinking water containing 20 ppm chlorine affected lipid orthyroid metabolism
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in healthy women unselected for baseline total cholesterol levels or in healthy men
selected to have baseline total cholesterol levels above the-50th percentile for their age
(Wones et al., 1991). Because the impact of drinking water chlorine on lipid metabolism
was unimpressive in the first study (Wones et al., 1989), the second study was restricted
to men with baseline cholesterol levels above the 50th percentile in order to determine
whether elevated baseline cholesterol might be associated with an increased
responsiveness to chlorinated drinking water. The protocol consisted of a 4-week dietary
stabilization period during which all subjects drank distilled water followed by a 4-week
treatment period during which half of the subjects were assigned randomly to continue
consuming distilled water while the other half were assigned to consume 1.5 L/day of
drinking water containing 20 ppm chlorine. There were 15 men and 15 women in each
group. There were no differences between the chlorine and distilled water groups in any
of the lipid or thyroid parameters. No subgroups (age, race, baseline total cholesterol,
source of pre-study drinking water) were differentially affected by exposure to chlorine.
These findings do not support the concern for chlorine's effect on lipid and/or thyroid
metabolism that was raised in response to the animal studies reviewed in Chapter v.
Acute exposure to chlorine has occurred through the ingestion of household
bleach. This occurs most commonly in children, and the bleach usually consists of 3-6%
solutions of sodium hypochlorite in water with pH values averaging -11.0. The typical
amount of bleach ingested by a child has been estimated to be -4-5 mL. Intake of this
small amount of bleach generally results in irritation of the oropharynx and esophagus,
a burning sensation in the mouth and throat, spontaneous emesis, and in rare instances,
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permanent injury to the esophagus with perforation or stricture formation dependent upon
the pH of the solution (Mack. 1983).
Strange et al. (1951) reported a case in which a 49-year-old male ingested a quart
of liquid bleach containing roughly 5% free available chlorine in the form of sodium
hypochlorite (-6557 mg/kg). Injury to the stomach eventually necessitated a total
gastrectomy. The individual's esophagus appeared to be healthy.
Ingestion of a few teaspoons of bleach proved fatal for an 18-month-old girl (Done,
1961). The bleach solution was apparently aspirated into the trachea where it caused
acute tracheobronchitis.
The list of reported adverse health effects associated with chlorine gas exposure
ranges from bronchitis, asthma and pulmonary edema to headaches, meningitis and heart
disease. Not all of these findings have been confirmed in more than one report (NRC,
1976; WHO, 1982).
Shortly after World War I, it was believed that chlorine gas in small amounts
decreased the incidence of respiratory diseases among exposed workers. Small amounts
of chlorine gas were also used to treat the common cold, influenza and bronchitis (NRC,
1976).
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Adverse health effects related to acute exposure to chlorine gas include pulmonary-
congestion, respiratory failure, pulmonary edema and bronchopneumonia (WHO, 1982).
Table VI-1 indicates the threshold levels of the effects produced by inhaling chlorine gas.
The authors of the WHO (1982) report also stated that there were no indications of
permanent respiratory damage in persons surviving acute exposures to chlorine gas. In
cases of acute, low-level exposure to chlorine gas, complete and rapid recovery occurred
with symptomatic treatment.
Episodes of dermatitis have been linked to exposure to chlorine and hypochlorite
ion (U.S. EPA, 1981). Sodium hypochlorite disinfectants, in particular, have been
determined to be the causal agent 1n the development of occupational allergic reaction
or irritation of the skin. Eun et al. (1984) reported a case of dermatitis in a 46-year-old
male veterinary surgeon who was frequently exposed to an undiluted animal antiseptic
with a sodium hypochlorite concentration of 4-6%. A skin patch test demonstrated the
surgeon's sensitivity to the concentrations of 0.04-0.06% sodium hypochlorite. Hansen
(1983) studied 541 members of a hospital cleaning department and determined that
irritant skin dermatitis was caused by occupational exposure to sodium hypochlorite or
alkaline trisodium phosphate.
A case involving an accidental exposure to sodium hypochlorite occurred while a
61-year-old white female patient with end-stage renal disease was undergoing
hemodialysis (Hoy, 1981). Approximately 2 L of undiluted sodium hypochlorite cleaning
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TABLE VI-1 '
Threshold Levels for Chlorine Gas Inhalation Effects*
Effect
CI Threshold Levels
mg/m3
ppm
Odor perception/irritation
0.06-5.8
0.02-2.0
Perceivable sensory irritation
2.9
1.0
Intolerable sensory irritation
11.6
4.0
Chronaxie/visual adaptation changes
1.5
0.52
Pronounced dyspnea, anxiety, vomiting,
cyanosis, pulmonary edema
87.0-116.0
30.0-40.0
"Source: WHO, 1982
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solution (Chlorox), containing 5.25% sodium hypochlorite and 5% available chlorine, were
added to the dialysis bath. It was estimated that -30 mL (-30 mg/kg) of sodium
hypochlorite solution crossed the dialysis membrane and entered the patient's circulating
volume. The patient experienced massive hemolysis, hyperkalemia, cyanosis and
cardiopulmonary arrest. Serum electrolytes drawn before and after the sodium
hypochlorite exposure indicated a sudden rise and fall in serum sodium, potassium and
chloride concentrations. Blood count, arterial pH and prothrombin time did not change.
The patient recovered in 1 week.
Epidemiologic Studies
General Considerations. Since the early 1970s, a number of epidemiologic studies
have attempted to assess the relationship between drinking water quality and cancer.
These studies differ markedly in both their objectives and design which in turn limits
specific inferences to be drawn from their findings. The studies have evolved from
general descriptions of disease rates in various geographic areas with different drinking
water sources and presumed contaminants to well-designed interviewcase-control studies
with incident cancer cases (Murphy and Craun, 1989).
These studies were never intended to assess whether chlorine itself is responsible
for adverse health effects but rather were designed to test the hypothesis that
tril.Jomethanes or other organic compounds occurring in drinking water as a result of
chlorination are associated with an increased risk of gastrointestinal and urinary cancer.
To date, chlorine itself has not been found to be carcinogenic in animals.
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Exposure measures utilized in these studies have been quite variable and are
determined by both study design and availability of information. Ecologic studies have
used information that is available for larger geographic areas such as proportion of a
population served by different water sources using different treatment practices.
Case-control death certificate studies are limited to the information that is recorded on the
death certificate. Place of usual residence as recorded has been used as a surrogate
variable for presumed exposure to different sources and types of drinking water. Rarely
will there be sufficient information available to assess and control for confounding with
these study designs. This is particularly important when considering the relationship of
low-level, long-term exposure to chronic diseases with long latent periods. Inadequate
control of confounding and exposure misclassification may obscure any small elevations
in risk that truly exist. Many of these limitations, however, do not exist in the most recent
studies of this problem, although there will always be some methodologic problems to
consider in assessing the final reported results (Craun, 1985; Murphy and Craun, 1989).
With these limitations in mind, the following is an overview of the major studies that
have assessed the relationship between exposure to chlorinated drinking water and
several site-specific cancers.
Ecological and Retrospective Studies. The earliest studies of this problem were
ecologic and used a variety of cancer endpoints and surrogate exposure variables. The
detailed results have been summarized and critiqued by many others (Crump and Guess,
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1982; Shy, 1985; Craun, 1985; Craun et al., 1989; Murpny and Craun, 1989) and will not
be presented in this document. Although there are clear limitations to the interpretation
of ecologic studies (Morgenstern, 1982), the studies were instrumental in raising the issue
of a possible hazard associated with a long-accepted public health practice (Craun, 1988)
and provided the impetus for further, better designed studies to address this potential
problem.
The earliest case-control studies of the association between chlorinated drinking
water and cancer involved decedent cancer cases and had only limited information on
individual risk factors and exposures. Odds ratios derived from these data range from
1.13 (Kanarek and Young, 1982) to 1.93 (Alavanja et al., 1978) for rectal cancer, 1.05
(Gottlieb et al., 1981) to 1.61 (Alavanja et al., 1978) for colon cancer and 1.04 (Kanarek
and Young, 1982) to 1.69 (Alavanja et al., 1978) for bladder cancer. These relative risk
estimates, in light of differences in study populations, exposure variables, etc. are not
strong enough to discount the effects of possible confounding factors; neither do the
studies have sufficient statistical power to rule out a relationship between chlorine and its
dissociation products and adverse health effects.
More recent case-control studies have used improved study designs to further
examine the relationship between exposure to chlorinated drinking water and colon and
bladder cancers (Cragle et al., 1985; Cantor et al., 1985, 1987, 1989; Zierleretal., 1986,
1988; Young et al., 1987, 1989).
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Cragle et al. (1985) investigated the relationship between water chlorination and
colon cancer using 200 incident cases of colon cancer from seven hospitals in North
Carolina and 407 hospital-based comparison subjects without evidence of cancer and no
history of familial polyposis, ulcerative colitis, adenomatous polyposis, or any other major
chronic intestinal disorder. Both cases and comparison subjects were required to be
residents of the state for at least 10 years to be included in the study. Comparison
subjects were matched on age, race, gender, vital status and hospital to prevent potential
confounding by these characteristics. Additional information on potential confounders,
including alcohol consumption, genetic risk (number of first-degree relatives with cancer),
diet, geographic region, urbanicity, education and number of pregnancies, was obtained
by either mailed questionnaire or telephone interview. These characteristics were
assessed and statistically controlled in the analysis. Approximately 71% of the eligible
population was included in the study. Water exposures were verified for each address
and categorized as chlorinated and unchlorinated for the analysis. Logistic regression
analysis showed genetic risk, a product term between alcohol consumption and high fat
diet, and an interaction term between age and chlorination to be positively associated with
colon cancer. The association between chlorinated water and colon cancer was found
to be highly dependent upon age. Rate ratios for persons who drank chlorinated water
at their residence for >16 years were consistently higher than those exposed to
chlorinated water <16 years, but a statistically significant association between water
chlorination and colon cancer, controlling for possible confounders, was found only for
those above age 60. For example, 70-79 year olds who drank chlorinated water for >16
years had twice (RR=2.15) the risk of colon cancer compared with 70-79 year olds who
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drank unchlorinated water; in the same age group the risk of colon cancer was about 50%
(RR=1.47) higher in those who drank chlorinated water for <16 years compared with
those who drank unchlorinated water.
Young et al. (1981) in an earlier case-comparison mortality study had reported an
association between colon cancer mortality in Wisconsin and exposure to chlorinated
drinking water estimated by the average daily chlorine dosage 20 years past. The study
included 8029 cancer deaths and 8029 noncancer deaths in white females matched on
county of residence, year of death, and age. Death certificates provided information on
urbanicity, marital status and occupation, and these were considered as potential
confounders. This association was further pursued in an interview study of 347 incident
cases of colon cancer, 611 population-based comparison subjects, and 639 comparison
subjects with cancer of other sites (Young et al., 1987; Kanarek and Young, 1989).
Lifetime residential and water source histories and information on water-drinking habits,
diet, sociodemographics, medical and occupational histories, lifestyle and other factors
were obtained by questionnaire. Data on past THM levels in drinking water were
estimated using a predictive statistical model based on current, quantitative THM levels
and routinely recorded operating data for Wisconsin water supplies. Multivariate
analyses, controlling for variation in water consumption, population size and other factors,
were used to estimate the colon cancer risk for various drinking water related factors.
While this case-comparison study associated colon cancer incidence with use of
chlorinated drinking water for 0-10 years prior to diagnosis, no increased risks were
observed between colon cancer incidence and use of chlorinated drinking water for >10
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years prior to diagnosis nor were estimated THM exposure in Wisconsin associated with
colon cancer incidence. However, current THM levels in these water supplies were
generally low with 98% of samples <100 }jg/L Colon cancer cases were also found to
more frequently consume water from municipal groundwater supplies, which are more
likely to be chlorinated than not chlorinated. Since groundwaters in the United States
have also been shown to be contaminated with synthetic volatile organic compounds, the
possibility of other water exposure was considered. A study that considered exposures
of this population to organic contaminants in groundwater showed higher estimated
relative risks (RR=1.7-2.4) of colon cancer incidence in populations exposed to
tetrachloroethylene, trichloroethylene and 1,1,1 -trichloroethane in municipal groundwaters
(Kanarek and Young, 1989).
Cantor et al. (1985) reported results from a collaborative Environmental Protection
Agency (EPA)-National Cancer Institute (NCI) study of the association between water
chlorination and bladder cancer. Included were a total of 2982 persons (73% of those
eligible to participate) between the ages of 21 and 84 diagnosed with cancer of the
urinary bladder in 1978 and residing in 10 areas of the United States (Connecticut, Iowa,
New Jersey, New Mexico, Utah, and the metropolitan areas of Atlanta, Detroit, New
Orleans, San Francisco, and Seattle) and 5782 population-based comparison subjects,
randomly selected and frequency matched on gender, age and study area. Subjects
were interviewed at home by a trained interviewer, and data were collected for a number
of possible confounders including smoking, occupation, artificial sweetener use, coffee
and tea consumption, and use of hair dyes. A complete residence history was obtained
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to categorize individuals according to water sources and chlorination status on a
year-by-year basis, and information was obtained on use of bottled water and fluid
consumption. Of the 587,568 person-years lived by all residents since 1940, 76% were
at a known water source. Logistic regression analysis was used to control for potential
confounders. Among persons in all study areas combined, risk was not elevated in those
respondents living in areas with chlorinated water supplies for 20, 20-39, 40-59 and 60
or more years. However, it should be noted that this study was originally designed to
determine if saccharin.was a human carcinogen rather than to determine cancer risks
associated with water chlorination. The study areas were not selected to provide the
optimal variability of water sources and treatment, and the statistical power of the study
is less than suggested by the large number of individuals studied, as the five metropolitan
areas were served primarily by chlorinated water supplies. Among the 10 study areas,
participants from the three states with agricultural land use did show evidence of
increased risk for bladder cancer with the number of years at a surface source, but the
number of participants in these areas was small compared with the other areas. It is
curious that another analysis of these same data (Cantor, 1987) reported this trend to be
in the other nonagricultural areas. Among nonsmokers who were never employed in a
high risk occupation (a group otherwise at low risk for bladder cancer) the risk was
elevated among those served by chlorinated surface sources with some evidence of a
duration of exposure-response relationship. However, only in nonsmokers who resided
60 or more years at a residence served by chlorinated water was the risk statistically
significant; in this instance the risk of bladder cancer was more than double (RR=2.3) the
risk among nonsmokers who resided in areas served by unchlorinated water.
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Previous studies have reported an increased risk of bladder cancer associated with
a high total fluid intake but this has not been thoroughly studied. A high volume of total
fluid may affect the bladder by increasing its work load or the types of fluid consumed
may contain constituents that are either carcinogenic or protective. Cantor et al. (1987,
1990) has recently reported a further analysis of the EPA-NCI national bladder cancer
study according to beverage intake level and type of water source/treatment. Among the
white participants, complete information on beverage consumption and cigarette smoking
was available for 5793 males and 1983 females. Risk of bladder cancer was reported
to be associated primarily with the tap water component and to increase with greater
consumption of tap water. After correcting for age, smoking and other potential
confounding characteristics, it was observed that people who reported drinking the most
chlorinated tap water had a bladder cancer risk about 43% higher (RR=1.43) than people
who drank the least. When tap water consumption was analyzed separately for males
and females, however, only among males was the association between water ingestion
and bladder cancer risk statistically significant.
Evaluation of bladder cancer risk by the combined effects of duration of chlorinated
surface water use and tap water intake showed that only among the study participants
who drank chlorinated surface water for 40 or more years did the bladder cancer risk
increase with the higher tap water consumption. A risk gradient with water consumption
was not found among consumers of chlorinated surface water for <40 years or among
long-term consumers of unchlorinated groundwater. Duration of exposure to chlorinated
surface water was associated with bladder cancer risk among women whose tap water
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consumption was above the median. However, the rate ratios were statistically significant
in only one category, females who had resided 60 or more years at a residence served
by chlorinated surface water and whose tap water consumption was above the median.
Evaluation of risk by smoking status revealed that most of the duration effect was seen
in nonsmokers. Among nonsmokers who consumed tap water in amounts above the
median, a risk gradient was apparent only for males. However, a high risk was also seen
for nonsmoking females who consumed less than the median. It is difficult to accept
these findings as indicative of an association between tap water consumption and bladder
cancer because of the random variation found when the results are analyzed separately
by gender; the lack of statistical significance in all but a few categories of water
consumption; and inconsistent high risks among nonsmoking males who consumed more
water than the median and females who consumed less water than the median. In
addition, recently reported results from a population-based incident case-consumption
study in Utah (Slattery, 1988) found neither total fluid intake nor tap water consumption
to be related to bladder cancer after adjustment for cigarette smoking, age, gender,
history of diabetes and bladder infections.
Because chloramination has been shown to produce very low levels of THMs,
epidemiologic studies were conducted in Massachusetts where chlorine and ammonia
have been used since 1938 to disinfect surface water provided to most communities in
tne Boston metropolitan area. A recently completed mortality study (Zierler et al., 198bj
showed a slight increase in bladder cancer in populations in Massachusetts receiving
chlorinated surface water compared with populations receiving chloraminated surface
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water. To address problems in interpretation of the results of this study, a
case-comparison study of 614 individuals who had died of primary bladder cancer and
1074 individuals who had died of other causes was also conducted (Zierler et al., 1988).
Confounding by age, gender, smoking, occupation, and socio-economic status was
controlled by multiple logistic regression.
The largest water utility in the state, the Massachusetts Water Resources Authority
(MWRA), provides water to Boston and to more than 25 additional towns and cities. The
water is of high quality because of restricted access to much of the watershed.
Chloramine disinfection began in 1938, and only recently have chemicals been added for
fluoridation and pH adjustment. The Authority has provided chloraminated water directly
to some 20 cities and towns; an additional 10 cities and towns have purchased untreated
water from the MWRA or use the MWRA water sources and provide chlorine for
disinfection. Other communities using surface water sources of similar quality with
chlorination were also included in the studies. Exposure was defined according to
duration of residence in communities using chlorine or chloramine disinfection.
Individuals who resided from 1938 until the year of diagnosis exclusively in communities
supplying chlorinated drinking water were classified as having lifetime exposure;
individuals residing exclusively since 1938 in communities with chloraminated drinking
water were classified as having no exposure to chlorinated water and lifetime exposure
to chloraminated water. Because all of the subjects died between 1978 and 1984,
lifetime use means that only one type of disinfectant was used in the residential water
supply 40-46 years before death. All individuals not meeting this definition for lifetime
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exposure were included in a separate analysis considering "usual" exposure lo either
disinfectant. Also analyzed separately using the definition for lifetime exposure were data
among the subset of residents in communities using only MWRA water, which is obtained
from a common source and is disinfected with either chlorine or chloramine by different
communities.
A positive association was detected between both usual and lifetime chlorinated
drinking water exposure classification and bladder cancer mortality. The bladder cancer
association was highest for lifetime residents of chlorinated drinking water communities
relative to lifetime residents of chloraminated drinking water communities. Using
lymphatic cancers as the comparison group, the risk of bladder cancer mortality among
lifetime consumers of chlorinated water was almost three times (RR=2.7) the risk of
bladder cancer mortality among lifetime consumers of chloraminated drinking water;
among usual consumers the risk was doubled (RR=2.0). A slightly higher risk (RR=3.5)
was observed when the subset of lifetime residents using MWRA water was analyzed.
When all deaths were used for comparison, the bladder cancer mortality risks were higher
than was observed when only lymphoma deaths were used for comparison. The rationale
for the lymphoma comparison resulted from consideration of possible associations
between disinfected water and other diseases in the comparison group whereas there are
no data to suggest that lymphoma may be associated with disinfected water. Because
the observed bladder cancer risks were consistently lower when the analysis included all
deaths, it is possible that one or more of the causes of death in the comparison group are
related to water chlorination and resulted in the observation of this lower risk. More
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careful consideration should be given to the proper selection of a comparison group for
future epidemiology studies of water.contaminants. For example, use of a comparison
group that includes cardiovascular deaths could dilute the observed magnitude of effect
if cardiovascular disease is associated with chlorinated water.
Although most of the studies reviewed here have looked at colorectal or bladder
cancer risk, one recently published work investigated the risk of pancreatic cancer in
relation to presumed exposure to chlorinated drinking water. IJsselmuiden et al. (1992)
conducted a population-based case-control study in Washington County, Maryland, using
the same population data that was originally ascertained during a private population
census for an earlier cohort study (Wilkins and Comstock, 1981). The cohort study did
not find any association between pancreatic cancer and chlorinated drinking water
(OR=0.80, 95% Cl=0.44-1.52).
The case-control study was conducted to reexamine chlorinated drinking water as
a possible independent risk factor for pancreatic cancer in this population. Cases were
residents reported to the County cancer registry with a first time pancreatic cancer
diagnosis during the period July 1975 through December 1989, and who had been
enumerated in the 1975 census (101 cases). Controls were randomly selected by
computer from the 1975 census population (n=206). Drinking water source, as obtained
during the 1975 census, was the exposure variable used. In univariate analyses,
municipal water as a source of drinking water, increasing age, and not being employed
were significantly associated with increased risk of pancreatic cancer. Multivariate
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analyses that controlled tor confounding variables indicated that the use of municipal,
chlorinated water at home was associated with a significant odds ratio of 2.23 (95%
Cl=1.24-4.10).
A clear interpretation of these findings is hampered by several problems regarding
the assessment of exposure, including the fact that information obtained in 1975 on type
of water and other variables is really cross-sectional and may not truly reflect actual
exposure patterns, and there is no information on the actual amounts of water consumed.
Additionally, different residential criteria were used for the cases and controls. The cases
had to still be residing in the County at the time of their cancer diagnosis to be included
in the study, but the controls may not have been current residents. If controls emigrated
out of the county differentially on the basis of exposure, the odds ratio may be an over-
or underestimate of the true value. Finally, it can not be ruled out that the exposure
variable used for this and other studies — residence served by a particular water
source — is simply a surrogate for some other unidentified factor associated with nonrural
living. The nonspecific relationship of several different causes of death and water source
at home observed in the earlier cohort study (Wilkins and Comstock, 1981) lends some
support to this possibility. Better studies with more valid individual exposure information
are needed to confirm or refute the findings of this and other studies.
Morris et al. (1992) conducted a meta-analysis of 11 epidemiologic studies of cancer
and presumed exposure to chlorinated water and its byproducts. The authors reported
a combined relative risk estimate of 1.21 (95% CI, 1.09-1.34) for bladder cancer and 1.38
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(95% CI, 1.01-1.87) for rectal cancer. Odds ratios for 10 other site-specific cancers
including colon and pancreas were reported to be not significantly elevated. This attempt
to quantitatively summarize the available epidemiologic studies is hindered by the
previously discussed methodologic problems in the individual studies, particularly
inadequate control of important covariates and a lack of individual exposure estimates.
The individual studies have been considered to be inconclusive primarily because of
these resolved methodologic problems, not because of a lack of statistical significance
due to inadequate sample sizes. Additionally, the extreme variability in the composition
of drinking water over time and across geographic areas means it is unlikely that the
exposures across all these studies are really the same and therefore raises a serious
question as to whether a statistical meta-analysis of this body of data is a valid exercise.
The use of meta-analytic techniques in the area of environmental epidemiology
requires a great deal of careful consideration particularly with regard to the problems of
exposure assessment and variability. A major problem in the application of meta-analysis
is deciding when a collection of studies are indeed combinable. The decision to combine
the water chlorination studies appears to have been based more on statistical rather than
logical grounds. Instead of encouraging additional studies designed to resolve some of
these important exposure assessment and other methodologic issues, the study of Morris
et al. (1992) may have the unintended opposite effect. Further high quality epidemiologic
research of this issue may be stifled because of the authors' erroneous interpretation that
a clear and significant association between cancer and consumption of chlorinated
drinking water has been demonstrated by the meta-analysis.
CHLORINE.6 VI-22 11/01/93
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Cross-Sectional Studies. Results have recently been reported for a cross-sectional
epidemiologic study of 1520 adult residents, aged 40-70 years, in 46 Wisconsin
communities supplied with chlorinated and unchlorinated drinking water of varying
hardness (Zeighami et al., 1989, 1990). This study was designed to determine whether
differences in calcium or magnesium intake from water and food and chlorination of
drinking water affect serum lipids.
The study communities from central Wisconsin: 1) were small in population size
(300-4000) and not suburbs of larger communities; 2) had not undergone more than a
20% change in population between 1970 and 1980; 3) had been in existence for at least
50 years; 4) obtained water from groundwater sources with no major changes in water
supply characteristics since 1980 and did not artificially soften water. The water for the
communities contained total hardness of either >80 mg/L or >200 mg/L CaC03; 24
communities used chlorine for disinfection and 22 communities did not disinfect. Eligible
residents were identified through state driver's license tapes and contacted by telephone;
an age-gender stratified sampling technique was used to choose a single participant from
each eligible household. Only persons residing in the community for at least the previous
10 years were included and participants were required to spend at least 9 months of each
year in the community. A questionnaire was administered to each participant to obtain
data on occupation, health history, medications, dietary history, water use, water supply,
and other basic demog.aphic information. Water samples were collected from a selected
subset of homes and analyzed for chlorine residual, pH, calcium, magnesium, lead,
CHLORINES
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cadmium, and sodium. Specimens of fasting blood were collected from each participant
and analyzed for total cholesterol, triglycerides, high density and low density lipoproteins.
Among females, serum cholesterol levels were found to be significantly higher in
chlorinated communities than in nonchlorinated communities. Serum cholesterol levels
were also higher for males in chlorinated communities, on the average, but the
differences were smaller and not statistically significant. LDL cholesterol levels followed
a similar pattern to that for total serum cholesterol levels, higher in chlorinated
communities for females, but not different for males. However, for both sexes, HDL
cholesterol levels are nearly identical in chlorinated and nonchlorinated communities and
there were no significant differences found in the HDL7LDL ratios. The implications of
these findings for cardiovascular disease risk are unclear at this time given the
inconsistencies in the data. The possibility exists that the observed association in
females may have resulted from some unknown or undetermined variable in the
chlorinated communities.
The results from a second study, designed to further explore the findings among
female participants in the Wisconsin study (Riley et al., 1992, manuscript submitted for
publication). Participants were 2070 white females, aged 65-93 years who were enrolled
in the Study of Osteoporotic Fractures (University of Pittsburgh Center) and had
completed baseline questionnaires on various demographic and lifestyle factors. Total
serum cholesterol was determined for all participants. Full lipid profiles (total cholesterol,
triglycerides, LDL, total HDL-2, HDL-3, Apo-A-I, and Apo-B), were available from fasting
CHLORINE.6 VI-24 11/01/93
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blood samples for a subset of 821 women. Interviews conducted in 1990 ascertained
residential histories and type of water source used back to 1950 and all reported public
water sources were contacted for verification of disinfectant practices. Private water
sources were presumed to be nonchlorinated. A total of 1896 women reported current
use of public, chlorinated water, 201 reported current use of nonchlorinated springs,
cisterns or wells, and 35 reported having mixed sources of water. Most of the women
had been living in the same home with the same water service for at least 30 years.
Overall, there were no meaningful differences detected in any of the measured serum
lipid levels between women'currently exposed to nonchlorinated water and those exposed
to chlorinated water (246 mg/dL vs. 247 mg/dL, respectively, for total cholesterol). The
data were also stratified by age and person-years of exposure to chlorinated water at
home. There was some suggestion that women with no exposure to chlorine had lower
total cholesterol levels but this was very inconsistent. This finding may be spurious since
there was no trend noted with LDL cholesterol or Apo-B, both of which are known to
correlate with total cholesterol. There was also no association between increasing
duration of exposure to chlorine and HDL cholesterol, Apo-A-I, or triglycerides.
The only notable differences were that women with chlorinated water reported
significantly more cigarette and alcohol consumption than the women with nonchlorinated
drinking water (Riley et al., 1992). This was evident in all age groups and across strata
of duration of exposure. This finding leads support to the possibility that the previously
reported association of chlorinated drinking water and elevated total serum cholesterol
CHLORINE.6 VI-25 11/01/93
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(Zeighami et at., 1990) may have arisen due to incompleted control of lifestyle factors
which were differentially distributed across chlorination exposure groups.
High Risk Subpopulations
At present, there are no definitive studies pointing to specific high risk populations.
The information presented in this section consists of a few clinical cases of asthma linked
with chlorinated water, a question of a possible risk group of persons deficient in G6PD
and a postulated risk to those persons who are already at risk of cardiovascular disease.
Reports of the precipitation of asthma attacks as a result of exposure to chlorinated
water have been published (Watson and Kibler, 1933; Sheldon and Lovell, 1949; Cohen,
1933). Severe asthma attacks in a 53-year-old female subsided when iodized salt was
removed from her diet, though milder symptoms persisted until chlorinated drinking water
was replaced by distilled or spring water. Municipal water supplies in this case contained
0.2-0.4 ppm chlorine. Further investigations revealed that asthmatic symptoms returned
within 8-10 hours after the subject had ingested municipal water, distilled water or spring
water to which sodium hypochlorite had been added. The subject experienced asthmatic
attacks upon a few hours of exposure to air contaminated with chlorinated (4-6 ppm load)
swimming pool water. Asthma attacks were also precipitated by the subject's weekly
washing with a chlorine rinse (NRC, 1976; Sheldon and Lovell, 1949). It must be noted
that this report and others describing a link with asthma are anecdotal case reports
published over 35 years ago. The lack of subsequent reports in the literature may be an
indication that these cases may have been erroneously attributed to chlorine exposure.
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In the study of male college student volunteers by Lubbers et al. (1982), three
participants were noted to have G6PD deficiency. These three were treated as a
separate group for treatment and analysis because, theoretically, they were believed to
be more susceptible to oxidative stress. Although no adverse effects were noted among
these three individuals, a larger sample size is needed to properly address this question.
Another possible high risk group are those persons already at risk of cardiovascular
disease. As discussed in Chapter V of this document, Revis et al. (1985) showed an
increase in cholesterol levels as well as atherosclerotic lesions in pigeons receiving a
calcium-deficient diet after exposure to chlorine in water. However, these findings have
not been reproduced in either animals or humans. The recent study of Wones et al.
(1992), designed to investigate a group at higher risk for cardiovascular disease, does not
support the earlier findings of a chlorine-induced elevation of either total serum
cholesterol or the specific cholesterol subfractions, so it is very questionable as to
whether this is truly a high risk subgroup.
The results from the EPA-NCI bladder cancer study (Cantor, 1985, 1987) indicated
that if there is indeed a causal relationship of long-term consumption of chlorinated
surface with bladder cancer, it may only be occurring in nonsmokers with no known
occupational risks, a group considered to be at otherwise low risk for development of
bladder cancer. This finding should be followed-up in a study specifically designed to
address this risk in a large sample of nonsmokers, as very little is otherwise known about
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bladder cancer risks that are independent of cigarette smoking status (Kabat, 1986;
Slattery, 1988).
Summary
At this time, there are no known long-term adverse human health effects from
exposure to chlorine, hypochlorous acid or hypochlorite ion in drinking water. The
majority of literature on the adverse health effects of chlorine deals with the inhalation of
chlorine gas. Acute exposure to chlorine gas produces pulmonary congestion, respiratory
failure, pulmonary edema and bronchopneumonia. If a person survives the initial
exposure, however, symptomatic treatment leads to rapid and complete recovery.
Consumption of heavily chlorinated drinking water (>90 ppm) produces constriction -
of the throat and irritation of the membranes of the throat and mouth. Levels of 25 ppm
chlorine in drinking water make the water unpalatable. Ingestion of household bleach
(pH-11) produces irritation of the oropharynx and esophagus. Cases of dermatitis have
been linked with the use of sodium hypochlorite disinfectants.
Epidemiologic studies that address the association between chlorinated drinking
water supplies and cancer are of limited use in defining adverse health effects of chlorine,
hypochlorous acid and hypochlorite ion primarily because of differences in study
objectives and relative nonspecificity regarding exposures from disinfected drinking water.
The most sophisticated of these studies have attempted to assess whether there are
adverse health effects associated with by-products created during the disinfection process
CHLORINE.6 VI-28 11/01/93
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and were not designed to specifically look at the disinfectant, i.e., chlorine, itself as a
potential etiologic agent in human disease.
The study of Cantor et al. (1987) has created a lot of concern regarding the risk of
bladder cancer in long-term consumers of chlorinated drinking water. It must not be
forgotten that this is the first study to date that has analyzed water consumption data in
incident bladder cancer cases and population-based controls. Because it stands alone
at this time, substantial confirmation will be required before any interpretation regarding
causality can be made (Deresa et al., 1990). The results of Zeighami et al. (1989,1990)
must be viewed in a similar light.
With the paucity of data pointing to the adverse health effects of chlorine,
hypochlorous acid and hypochlorite ion in drinking water, high risk subpopulations are
very questionably defined based on old case reports, animal studies and theory. They
include asthmatics, persons deficient in G6PD and possibly persons already at risk of
cardiovascular disease.
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VII. MECHANISMS OF TOXICITY
Chlorine is a highly reactive element that readily combines with a variety of organic
compounds and radicals. This property makes it difficult to characterize the toxic
mechanisms of chlorine, hypochlorous acid and hypochlorite ion in mammalian organ
systems. Thus, much of the information on the mechanism of toxicity has been
hypothesized from studies with microorganisms and in vitro experimentation with
macromolecules. Only a limited amount of research has been performed with mammals
to delineate further the mechanisms of toxicity of these compounds.
The mechanism of action for the bactericidal properties of chlorine, hypochlorous
acid and hypochlorites has not been clearly defined. Several theories have suggested that
an Interaction occurs between chlorine and proteins in the cell membrane, which interferes
with normal cell metabolism and results in the destruction of the cell wall (Dychdala, 1977).
Cell wall damage may be the most immediate effect of free available chlorine upon
bacteria. Studies by Friberg (1957) demonstrated that small amounts of chlorine were
effective in disrupting bacterial wall permeability, which led to leakage of the marker, P32,
from nucleoproteins of the bacterial cell. Similar disruptions of the cellular membrane may
explain the acute edematogenic and corrosive effects of chlorine on exposed surfaces in
mammalian tissues.
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In addition to cell wall lesions, key enzymatic reactions have been shown to be
nhibited in bacteria exposed to chlorine. Enzymatic reactions involving carbohydrate
metabolism have received the most attention. Green and Stumpf (1946) postulated that
chlorine irreversibly inhibits the metabolism of glucose in bacteria at the point of oxidation
of triosephosphoric acid to phosDnoglyceric acid. Knox et al. (1948) suggested that the
inhibition of carbohydrate metabolism was a result of chlorine reaction with sulfhydryl
groups of enzymes. This hypothesis has been challenged by more recent experiments
with viruses. In these studies it has been shown that compounds that are reactive toward
sulfhydryl groups are not always lethal. It appears that the inactivation of RNA may be
more critical to the viricidal action of chlorine than is inhibition of sulfhydryl-dependent
I
enzymes (NRC, 1976).
Further evidence that chlorine may act through its toxic effects on enzymes or other
cellular proteins was provided by Baker (1947). In experiments with egg albumin, the
addition of sodium hypochlorite resulted in the denaturation of protein and the formation
of N-chloro-groups. Pereira et al. (1973) demonstrated that by a process of oxidative
decarboxylation, hypochlorous acid converts several amino acids into a mixture of
corresponding nrtriles and aldehydes. Hypochlorous acid also chlorinated the ring of
tyrosine and reacted with cysteine yielding cystine and cysteic acid.
Hypochlorous acid is also able to interact with the nucleotide bases of RNA and
DNA. Patton et al. (1972) demonstrated that hypochlorous acid reacts with cytosine to
CHLORINE.7
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form y-N-chloro derivatives, di-. iri- and tetrachlorocytosine. depending on the
hypochlorous acid:cytosine ra:;o. Hoyano et al. (1973) found that aqueous hypochlorous
acid interacted with purine ana pyrimidine bases forming both labile intermediates and
stable end products. Dennis si al. (1978) reported that uracil reacted with an excess of
hypochlorous acid and formea trichloroacetic acid, carbon dioxide and nitrogen trichloride
as the break-down products. This interaction of hypochlorous acid with nucleotide bases
led the authors to believe that active chlorine might modify or destroy ceilular DNA. This
could result in direct death of ;ne cell, or initiation of a mutagenic event.
Other research with regents has indicated that the components of the plasma
membrane may be a site of toxic effects of chlorine. Vogt et a I. (1979) discussed in vitro
experiments where decreases in membrane-bound (Na* + K") -ATPase activity occurred
in rat cells after application of increasing concentrations of hypochlorite. Alterations in
membrane structure and function were also noted by Sun et al. (1980) when in vitro prepa-
rations of mitochondria from rat liver were exposed to various concentrations of chlorine.
Although mild chlorine treatment (0.9-1.8 ppm) did not result in changes of protein profiles
on SDS gel electrophoresis, mitochondria were observed to lose respiratory control. At
chlorine concentrations of 4-8 ppm, changes were found in the protein profiles and the
ADP-coupling mechanism in the oxidative phosphorylation was lost. At the highest levels
(40-80 ppm) chlorine caused tha loss of membrane proteins and lipids and the inhibition
of mitochondrial respiratory activity. At the lower concentrations, chlorine was thought to
cause changes in the microenvironment of the mitochondrial enzymes, which were part
CHLORINE.7
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of the ADP-coupling mechanism. ; he higher chlorine levels induced more severe
degradation of the membrane structure and function.
Disturbances in the enzymatic processes in liver membranes were hypothesized to
have caused liver changes that occurred in rats after acute administration of chlorinated
water (Chang et al., 1981). Rats were sacrificed 2, 5 or 10 days after a single intragastric
dose (5 ml.) of sodium hypochlorite water containing 1% free residual chlorine; that is, 50
mg of residual chlorine was given to each animal. Morphological and biological changes
occurred largely at the 2-day interval, and recovery was complete within 10 days. Liver
tricylglycerols increased and the acyl group composition of triacylglycerols and
phospholipids was altered in liver mitochondria and whole homogenate. Long-chain
polyunsaturated fatty acid levels increased. These effects suggested that the chlorinated
compounds had altered the lipoprotein secretion system and membrane transport function.
Vogt et al. (1982) determined that the synaptic membrane in the hypothalamus of
rats was a site of effects occurring after administration of chlorinated water. Each rat was
treated with 50 mg of free residual chlorine in 5 mL of a sodium hypochlorite solution and
then sacrificed after 3 hours, 24 hours or 7 days. Hypothalamic norepinephrine was
decreased at the 3- and 24-hour intervals, but recovery occurred within 7 days.
Administration of the chlorinated water was thought to have sffocted either the release,
reabsorption or metabolism of the neurotransmitter. A corresponding increase in the
norepinephrine metabolite, normetanephrine, was found at 3 and 24 hours with a return
CHLORINE.7
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to normal levels after 7 days. I hus, the reabsorption process and other active transport
functions were thought to have been affected.
r
In the studies performed by Vogt et al. (1982) and Chang et al. (1981), the
researchers noted similarities in the effects of sodium hypochlorite administration and the
changes that occur when chlorinated hydrocarbons, such as chloroform and carbon
tetrachloride, are given to animals. Thus, it is possible that the toxic effects at the cell
membrane may be mediated by these chlorinated compounds rather than as a direct result
of chlorine, hypochlorous acid or hypochlorite ion treatment.
As previously mentioned, chlorine, hypochlorous acid and hypochlorite ion are
strong oxidants. The high oxidative potential of these compounds makes them highly
corrosive to the skin and mucous membranes (Done, 1961). Many cases of accidental
ingestion or inhalation of hypochlorite ion have been cited in the medical literature.
Postmortem examinations have revealed focal areas of necrosis, hemorrhage and
superficial erosion of the Gl mucosa and pulmonary epitheleum (Strange et al., 1951;
Done, 1961; Muhlendahl et al., 1978). Thus, the effects of chlorine, hypochlorous acid and
hypochlorites may be a direct result of the high oxidative and corrosive effects of these
compounds upon tissues and cells.
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Summary
The toxic actions of chlorine, hypochlorous acid and hypochlorite ion are many.
Toxicity is dependent on their ability to penetrate cell membranes, their high oxidative
capacity and their ability to interact directly with proteins and nucleotide bases. In addition,
these free residual chlorine compounds readily react with organic material, generating
trihalomethanes or other chlorinated organic compounds, which may also be associated
with toxic effects.
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VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
Introduction
The quantification of toxicological effects of a chemicai consists of separate
assessments of noncarcinogenic and carcinogenic health effects. Chemicals that do not
produce carcinogenic effects are believed to have a threshold dose below which no
adverse, noncarcinogenic health effects occur, while carcinogens are assumed to act
without a threshold.
In the quantification of noncarcinogenic effects, a Reference Dose (RfD), [formerly
termed the Acceptable Daily intake (ADI)] is calculated. The RfD is an estimate (with
uncertainty spanning perhaps an order magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk
of deleterious health effects during a lifetime. The RfD is derived from a no-observed-
adverse-effect level (NOAEL), or lowest-observed-adverse-effect level (LOAEL), identified
from a subchronic or chronic study, and divided by an uncertainty factor(s) times a
modifying factor. The RfD is calculated as follows:
(NOAELor LOAEL) „ fj
RfD = mgtkgfday
[UncertaintyFactor^s) x ModifyingFactot]
Selection of the uncertainty factor to be employed in the calculation of the RfD is based
upon professional judgment, while considering the entire data base, of toxicological effects
for the chemicai. In order to ensure that uncertainty factors are selected and applied in a
CHLORINE.8 VIII-1 01/18/94
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consistent manner, the U.S. EPA employs a modification to the guidelines proposed
by the National Academy of Sciences (NAS. 1977, 1980) as follows:
Standard Uncertainty Factors (UFs)
Use a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor is intended to account for the variation in
sensitivity among the members of the human population. [1 OH]
Use an additional 10-fold factor when extrapolating from valid results
of long-term studies on experimental animals when results of studies
of human exposure are not available or are inadequate. This factor
is intended to account for the uncertainty in extrapolating animal data
to the case of humans. [ 10A]
Use an additional 10-fold factor when extrapolating from .less than
chronic results on experimental animals when there is no useful
long-term human data. This factor is intended to account for the
uncertainty in extrapolating from less than chronic NOAELs to chronic
NOAELs. [10S]
Use an additional 10-fold factor when deriving an RfD from a LOAEL
instead of a NOAEL. This factor is intended to account for the
uncertainty in extrapolating from LOAELs to NOAELs. [10L]
Modifying Factor (MF)
Use professional judgment to determine another uncertainty factor
(MF) that is greater than zero and less than or equal to 10. The
magnitude of the MF depends upon the professional assessment of
scientific uncertainties of the study and data base not explicitly treated
above, e.g., the completeness of the overall data base and the
number of species tested. The default value for the MF is 1.
The uncertainty -actor used for a specific risk assessment is based principally upon
scientific judgment rather than scientific fact and accounts for possible intra- and
interspecies differences. Additional considerations not incorporated in the NAS/ODW
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guidelines for selection of an uncertainty factor include the use of a less than lifetime study
for deriving an RfD, the significance of the adverse health effects and the counterbalancing
of beneficial effects.
From the RfD, a Drinking Water Equivalent Level (DWEL) can be calculated. The
DWEL represents a medium specific (i.e., drinking water) lifetime exposure at which
adverse, noncarcinogenic health effects are not anticipated to occur. The DWEL assumes
100% exposure from drinking water. The DWEL provides the noncarcinogenic health
effects basis for establishing a drinking water standard. For ingestion data, the DWEL is
derived as follows:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 L/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for exposures of shorter
duration (1-day, 10-day and longer-term) are determined. The HA values are used as
informal guidance to municipalities and other organizations when emergency spills or
contamination situations occur. The HAs are calculated using an equation similar to the
RfD and DWEL; however, the NOAELs or LOAELs a.^ identified from acute or subchronic
studies. The HAs are derived as follows:
DWEL
DrinkingWaterVolumein Llday
where:
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(NOAELor LOAEQ x (bw)
HA = = _ maiL
{UF) x ( LIday)
Using the above equation, the following drinking water HAs are developed for
noncarcinogenic effects:
1. 1-day HA for a 10 kg child ingesting 1 L water per day.
2. 10-day HA for a 10 kg child ingesting 1 L water per day.
3. Longer-term HA for a 10 kg child ingesting 1 L water per day.
4. Longer-term HA for a 70 kg adult ingesting 2 L water per day.
The 1-day HA calculated for a 10 kg child assumes a single acute exposure to the
chemical and is generally derived from a study of <7 days duration. The 10-day HA
i
assumes a limited exposure period of 1-2 weeks and is generally derived from a study of
<30 days duration. The longer-term HA is derived for both the 10 kg child and a 70 kg
adult and assumes an exposure period of -7 years (or 10% of an individual's lifetime). The
longer-term HA is generally derived from a study of subchronic duration (exposure for 10%
of animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chemical, based on the
overall weight-of-evidence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from epidemiology studip?
to support a causal association between exposure to the chemical and human
cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of carcinogenicity in
animals with limited (Group B1) or inadequate (Group B2) evidence in humans.
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Group C; Possible Human Carcinogen. Limited evidence of carcinogenicity in
animals in the absence of human data.
Group D; Not Classified as to Human Carcinogenicity, inadequate human and
animal evidence of carcinogenicity or for which no data are available.
Group E: Evidence of Noncarcinoaenicitv for Humans. No evidence of
carcinogenicity in at least two adequate animal tests in different species or in both
adequate epidemiologic and animal studies.
If toxicological evidence leads to the classification of the contaminant as a known,
probable or possible human carcinogen, mathematical models are used to calculate the
estimated excess cancer risk associated with the ingestion of the contaminant in drinking
water. The data used in these estimates usually come from lifetime exposure studies using
animals. In order to predict the riSK for humans from animal data, animal doses must be
converted to equivalent human doses. This conversion includes correction for
noncontinuous exposure, less than lifetime studies and for differences in size. The factor
that compensates for the size difference is the cube root of the ratio of the animal and
human body weights. It is assumed that the average adult human body weight is 70 kg
and that the average water consumption of an adult human is 2 L of water per day.
For contaminants with a carcinogenic potential, chemical levels are correlated with a
carcinogenic risk estimate by employing a cancer potency (unit risk) value together wrth
the assumption for lifetime exoosure from ingestion of water. The cancer unit risk is
usually derived from a linearized multistage model with a 95% upper confidence limit
providing a low dose estimate; that is, the true risk to humans, while not identifiable, is not
CHLORINE.8 VIII-5 01/18/94
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likely to exceed the upper limit estimate and, in fact, may be lower. Excess cancer risk
estimates may also be calculated using other models such as the one-hit, Weibull, logit and
probit. There is little basis in the current understanding of the biological mechanisms
involved in cancer to suggest that any one of these models is able to predict risk more
accurately than any other. Because each model is based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of cancer risk rate
levels has an inherent uncertainty that is due to the systematic and random errors in
scientific measurement. In most cases, only studies using experimental animals have
been performed. Thus, there is uncertainty when the data are extrapolated to humans.
When developing cancer risk rate levels, several other areas of uncertainty exist, such as
the incomplete knowledge concerning the health effects of contaminants in drinking water,
the impact of the experimental animal's age, sex and species, the nature of the target
organ system(s) examined and the actual rate of exposure of the internal targets in
experimental animals or humans. Dose-response data usually are available only for high
levels of exposure and not for the lower levels of exposure closer to where a standard may
be set. When there is exposure to more than one contaminant, additional uncertainty
results from a lack of information about possible synergistic or antagonistic effects.
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Noncarcinoqenic Effects
Several acute ano subchronic studies have provided information that may be relevant
to the derivation of short-term HA values. Effect levels associated with the ingestion of
chlorine, hypochlorous acid and hypochlorites have been described in the work of several
authors; the most pertinent studies are summarized in Table VI11-1.
Chang et al. (1981) and Vogt et al. (1982) observed transient morphological and
biochemical changes in the liver and a decrease in the levels of the hypothalamic
neurotransmitter, ncreDinephrine. m Sprague Dawley rats that were administered a single
intragastric free residual chlorine dose equivalent to 50 mg of elemental chlorine.
Recovery to a normal liver appearance and biochemical composition was complete in
animals examined after 10 days (Chang et al., 1981). The norepinephrine levels had
returned to normal 7 days after administration (Vogt et al., 1$82).
Cunningham (1980) reported varying effect levels in male and female Wistar rats
administered ad libitum or gavage sodium hypochlorite (NaOCI) in milk. A NOAEL of 100
mg/L or 902.4 mg/kg/day chlorine was found for rats that were given chlorinated milk (ad
libitum) for 9 days. However, female rats, treated (by gavage) with chlorinated milk for 14
days, were reported to have increased body weight gain when they were given 80 mg/L
available jhlorine. Body weight gain was not affected at higher nominal doses (400 or
2000 mg/L) of available chlorine. Yet, an increase in kidney weight was found in female
rats that received a 2000 mg/L nominal concentration of available chlorine added to milk.
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TABLE Vill i
Summary of Studies Pertinent to HA Derivation
Experimental Animal
Duration of Study
Chlorine Exposoure
Dose
(mg/Kg/day)
Effect
Reference
Sprague-Dawley rats (males)
7 days
50 mg (NaOCI soln; acule)
250'
Decreased norepinephrine levels
Vogt et al.. 1962
Wistear specific-pathogen-free rats
(males)
9 days
1000 mg/L (milk ad lib)
902 4'
No adverse effects reported
Cunningham. I960
Wistar specific-pathogen-free rats
(lemaies)
14 days
2000 mg/L (gavage)
200"
Kidney enlargement
Cunningham. 1980
Sprague-Dawley rats (males)
10 days
50 mg (NaOCl soln. acule)
M2 9*
0 34'
Morphologic and biochemical liver
changes
Chang el al 1981
Humans (males)
18 days
24 mg/L (donkmg water)
No adverse effects reported
Lubbers el al . 1982
White mice (strain n"' identified) male
and female
33 days
200 mg/L (drinking water)
25"
No adverse effects reported
Blabaum and Nichols.
1956 /
Guinea pigs (males) r
5 weeks
50 mg/L (drinking water)
134
No adverse effects reported
Cunningham, 1980
Wistar rats (males)
6 weeks
20. 40 or 80 mg/L (drinking
water)
4 1. a 1 or
15 7
Enhhanced weight gain all
treatment groups, significant (or
40 mg/L only
Cunningham, 1980
Mice (males)
50 days
100 mg/L (drinking water)
12 5
No adverse effects reported
Blabaum and Nichols,.
1956
Sprague-Dawley rats (males and
females)
90 days
25. 100.175 or 250 mg/L
(drinking water)
2.7.5. 12 8
or 16.7 for
males and
3.5, 12.6.
19.5 or 24 9
for females
No adverse effects reported
Daniel et al , 1990
/
C"' *
F344 rats (males and females)
2 years
70, 140, 275 ppm (drinking
waier)
4 2. 7 3.
13 6 for
males and
4 2,7 6.
14.4 for
females
No adverse effects
NTP, 1990
U
'Assuming rat weight ¦- 0 35 kg
"Dose given in paper
'Assuming human weight¦ 70 kg and water consumption in this study = 1 L/day
-------
Rats given 40 mg/Lavailable cnlorine in drinking water experienced significant increase
in weight gain after 6 weeks (Cunningham. 1980). Weight gain, although increased, was
not significantly affected at any other treatment level (0, 20 or 80 mg/L available chlorine)
or study interval (1, 2, 3, 4 or 5 weeks). No effects on body weight gain or water
consumption were noted in male guinea pigs that received 50 mg/L available chlorine in
drinking water for 5 weeks (Cunningham, 1980).
Hulan and Proudfoot (1982) utilized sodium hypochlorite as a source of available
chlorine in the drinking water of broiler chicks in an attempt to duplicate the results of
Cunningham (1980). No effects on mortality or biologic performance were found in chicks
that received available chlorine concentrations of *150 ppm for 28 days. However,
administration of 600 or 1200 ppm chlorine was associated with decreases in mean body
weights, water consumption and kidney, liver, heart and testes weights. At an available
chlorine concentration of 1200 ppm, significant increases in mortality were also found. The
results of the experiments are of limited value for criteria development because water
consumption for individual animals was not monitored, and decreases in body and organ
weights may have been due to reduced water consumption: actual chlorine concentrations
in the drinking water during the test was not well documented.
In the Abdel-Rahman et al. (1984) study, 0, 1, 10 or 100 mg/L of free residual chlorine
was administered daily in the drinking water of male rats (4/group) for 1 year, intermittent
alterations in blood glutathione content, osmotic fragility and blood cell compartment were
CHLORINE.8 VIII-9 01/18/94
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reported. The authors of this study concluded that blood glutathione levels were changed
by exposure to HOC! and that blood cell compartment changes indicated some degree of
damage to erythrocytes. However, these results were not dose or duration related and
fluctuated in direction and statistical significance for both the treatment groups. Therefore,
the results of this study could not be definitely linked to specific adverse effects in the rat
and are not suitable for criteria determination.
In the study performed by Lubbers et al. (1982), 10 male volunteers were administered
increasing doses of residual chlorine (0.1, 1.0, 5.0, 10.0, 18.0, 24.0 mg/L elemental
chlorine) every 3 days over a 8-day period followed by daily ingestion of 5 mg/L chlorine
in drinking water for 12 weeks. No clinically important effects occurred in any of the
individuals as a result of free residual chlorine ingestion in drinking water.
Blabaum and Nichols (1956), conducted a two-part experiment in which weanling white
mice were administered chlorine water at 100 ppm available chlorine (pH ranged from
6.2-6.5, 12.5 mg/kg/day) for 50 days or at 200 ppm free available chlorine (pH ranged from
5.9-6.2, 25 mg/kg/day) for 33 days. A single control group of 10 male mice was used for
both parts of the experiment. By comparison with controls, the rats receiving 100 and 200
ppm free available chlorine experienced no differences in weight gain, growth or water
consumption. No y^ross or physical abnormalities were found upon autopsy or histologic
examination.
CHLORINE.8
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Male and female Sprague-Dawley rats (10/sex/dose) were administered chlorinated
drinking water at 0, 25, 100, 175 ana 250 mg/L for 90 days (Daniel et aL 1990). 'These
dose levels correspond to 0.2.75. 12.8 and 16.7 mg/kg/day for males and 0, 3.5, 12.5,
19.5 and 24.9 mg/kg/day for femaies. Food and water consumption, body weights, organ
weights, clinical chemistry and histopathology were evaluated. No consistent effects on
organ weights or tissue histopathology were observed. Decreases in water consumption
and body weight were reported: however, these effects were not considered to be
biologically significant and treatment-related. A NOAEL of 200 mg/L was identified by the
authors.
Results of assays considered fcr derivation of a DWEL are summarized in Table VIII-2.
Recent studies by Revis et al. f 1985. 1986) and Bercz et al. (1990) have investigated
the possible effects of chlorinated drinking water on the cardiovascular system. Using
male white Canneau pigeons, Revis et al. (1985, 1986) found that exposure of *15 ppm
chlorine in drinking water resulted in increased heart weight and total serum cholesterol
and decreased T3 and Td levels. Studies by Bercz et al. (1990) indicated that in monkeys
exposure to chlorinated drinking water and high atherogenic diets influenced the
development of hyperlipidemia. The lack of adequate control data and the small number
of animals used in these studies precludes their use for quantitative risk assessment.
CHLORINE.8
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TABLE VIII-2
Summary of Studies Pertinent to DWEL Derivation
Experimental
Animal
Exposure
Dose
(mg/kg/day)
Duration of
Study
Reference
F344 rats
(50/group) male
and female
0, 0.05, 0.1
and 0.2%
(drinking
water)3
0, 13.5, 27.7
(males) 0, 34.3,
63.2 females"
104 weeks
Hasegawa
etal., 1986
BDII rats (236),
male and female
100 mg/L
(drinking
water)
10°
Lifetime for 7
generations
Druckery,
1968
F344 rats
(70/group) male
and female
0, 70, 140.
275 ppm
(drinking
water)
0, 4.2, 7.3, 13.6
(males) 0. 4.2,
7.8, 14.4
(females)
104 weeks
NTP, 1990
"Average OCI intake estimation from average body weight and fluid intake given in
study
^Assuming rat weight = 0.35 kg and rat water consumption = 0.035 L/day.
CHLORINE.8
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Druckrey (1968) studied the effects of highly chlorinated drinking water (100 mg/L)
given daily to 7 consecutive generations of BDII rats. Two groups of animals served as
controls.at the beginning ana ending of the experimental period. Weight gain among
neonates was somewhat depressed during the first few days of life, but by maturity the
average body weight for all generations of test animals was 95-10% greater than that of
the untreated rats. Of 236 rats observed, no treatment-related effects were noted on the
life span, tumor incidence, fertility, growth, hematological measurements, or histology of
liver, spleen, kidney and other organs.
Hasegawa et at. (1986) studied the potential adverse effects of long-term exposure to
sodium hypochlorite in drinking water. Male and female F344 rats (50/sex/group) exposed
for 2 years to 0.05-0.2% NaOCI (13.5-63.2 mg/kg bw) experienced significant dose-related
decreases in body weight and absolute linear weights. Dose-related decreases were also
reported in the salivary gland of female rats. Significant decreases were found in the heart
and brain of the high-dose males (0.1%) and in the kidneys of the 0.2% dose group for the
females. Although there was high mortality throughout the study, survival rates were
similar for both control and treated animals.
NTP (1990) conducted a 2-year bioassay using F344 rats (70/sex/dose) in which
chlorinated drinking water was administered at doses of 0, 70, 140 or 275 ppm. Based on
body weight and water consumption values reported in the study, these doses correspond
to concentrations of 0, 4.2, 7.3 or 13.6 mg/kg/day for males and 0, 4.2, 7.8 or 14.4
CHLORINE.8 VIII-13 01/18/94
-------
rmg/kg/day for females. There was a dose-reiated decrease in water consumption for both
males and females. A decrease of 5-8% in body weight was reported for all dose groups.
¦No other nonneoplastic effects were reported in treated animals. A NOAEL of 13.6
mg/kg/day for males and 14 4 mg/kg/day for females was identified.
Quantification of Noncarcinoaenic Effects
Assessment of Acute Exposure Data and Derivation of 1-Day HA. Two studies
have been performed that involvea a single administration of a chlorinated solution to rats.
In both studies, specific morpnological or biochemical effects were noted as a
consequence of the intragastric intubation of rats with a chlorinated solution (containing
free residual chlorine equivalent to 50 mg elemental chlorine). It would be inappropriate
to derive a 1-day HA value from the data found in these two studies in view of the fact that
only a single concentration of chlorine was administered and the measurements taken
were limited to either reversible liver effects (Chang et al., 1981) or neurotransmitter level
changes (Vogt et al., 1982).
In the absence of suitable data to derive a 1-day HA, it is recommended that the 10-day
HA of 2.5 mg/L be used as a conservative estimate of the 1-day HA.
Assessment of Short-term Exposure Data and Derivation of 10-Dav HA. Data
considered for derivation of the 10-day HA for a 10 kg child are presented in Table VIII-1.
CHLORINE.8
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Several animal studies were flawed in their design, performance, description of the
experiment or duration of exposure.
The Lubbers et al. (1982) human study was also not useful, since the dosages appear
to be well below the threshold level of effect.
The NOAEL found in the Blabaum and Nichols (15(56) study was the most suitable
effect level for the development of the 10-day HA values. In this study, 20 mice (10/sex)
received 0 or 200 mg/L free available chlorine for 33 days and a second group of 10 males
received 100 mg/L free available chlorine in drinking water for 50 days. The average
weight of the mice was 0.02 kg, and their water consumption was determined to be 0.0025
L/day. The results indicated that ingestion of 200 mg/L (or 25 mg/kg/day) free available
chlorine did not result in gross lesions, histologic abnormalities or differences in weight gain
or growth as compared with control animals that received normal municipal tap water. A
NOAEL of 25 mg/kg/day was thus defined.
The 10-day HA for a 10 kg child is calculated as follows:
10-aay HA - 25 1° , 2.5 mgll_
1 l/day x 100
wheie:
25 mg/kg/day = NOAEL based on the absence of adverse gross or histologic
effects (Blabaum and Niohote, 1956)
CHLORINE.8
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10 kg
assumed weight of a child
1 L/day
assumed of water consumption by a child
100
uncertainty factor chosen in accordance with U.S. EPA (1988)
guidelines for use of a NOAEL from an animal study.
Derivation of Longer-term HA. The only oral subchronic study that may be
considered is the drinking water study by Daniel et al. (1990). In this study,
Sprague-Dawley rats (10 sex/group) were administered chlorinated drinking water
containing 0, 2, 7.5, 12.8 or 16.7 mg/kg chlorine for 90 days did not exhibit any
treatment-related adverse gross or histologic effects. A NOAEL of 16.7 mg/kg/day for
males was identified in this study.
The longer-term HA for a 10 kg child is calculated as follows:
where:
16.7 mg/kg/day = NOAEL, based on the absence of adverse effects in male rats
exposectto chlorinated drinking water for 90 days (Daniel et al.,
1990)
Longer-term HA =
(16.7 mg/kglday) (10 kg)
100* 1 LIday
= 1.67 mg!L (roundedto 2.0 mgIL)
10 kg
assumed body weight by a child
100
uncertainty factor, chosen in accordance with U.S. EPA (1988)
guidelines for a NOAEL from an animal study
1 L/day
assumed water consumption of a child
CHLORINE.8
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The longer-term HA for a 70 kg adult is calculated as follows:
Longer-term HA = C16-7 mgtkg/day) (70 kg) _ ^ mg/i_ (roundedto 6.0 mgIL)
100 x 2 Llday
where:
16.7 mg/kg/day = NOAEL, based on absence of adverse effects in male rats
exposed to chlorinated drinking water for 90 days (Daniel et al.,
1990)
70 kg = assumed body weight of an adult
100 = uncertainty factor, chosen in accordance with U.S. EPA (1988)
guidelines for use of a NOAEL from an animal study
2 L/day = assumed water consumption by an adult
Assessment of Lifetime Exposure and Derivation of DWEL. There are three
chronic oral studies on the effects of chlorine in drinking water that may be considered for
the derivation of the DWEL, Druckrey (1968), a more recent study by Hasegawa et al.
(1986), and the ffTP (1990) bioassay. In the Druckrey (1^68) study, BDII rats were
exposed for 7 generations to 100 mg/L chlorinated drinking water. The study did not reveal
any effects on fertility, growth, blood chemistry, histopathology of organs or longevity. A
NOAEL of 10 mg/kg/day can be identified in this study.
In the more recent study by Hasegawa et al. (1986) previously discussed, F344 rats
exposed for 2 years at 0, 0.05 or 0.1% chlorinated drinking water for males and 0.1 or
0.2% chlorinated drinking water for females experienced a dose-related reduction in body
weight gain and organ weight at the 0.05% (13.5 mg/kg bw) dose group. A LOAEL of 13.5
CHLORINE.8 VIII-17 01/18/94
-------
mg/kg bw/day has been identified in this study. However, the biological significance of
these effects-is unclear since there was no evidence of gross or microscopic lesions with
increasing doses. Also, there appear to be inconsistencies in the reported doses and
estimated concentrations based on water consumption values. It should also be rioted that
the reported consumption values are inconsistent with those reported by other investigators
who have observed decreased water consumption at these high concentrations.
In the 2-year bioassay conducted by NTP (1990), F344 rats were administered 0, 20,
140 or 275 ppm chlorinated drinking water. No adverse nonneoplastic effects were
observed in males or females at any dose level. Mean body weights were slightly
decreased in all treated males and females at the highest dose level. However, these
decreases were <10% when compared with controls. Water consumption was decreased
21-23% for males and females, respectively. A NOAEL of 13.6 mg/kg/day for males and
14.4 mg/kg/day for females was identified from this study.
Step 1: Determination of the Reference Dose (RAJ)
RfD = 14 4 mglkg bwlday __ g.144 mg/frgfday (roundedto 0.1 mglkglday)
100
/
where:
14.4 mg/kg bw.uay = NOAEL, based on absence of adverse effects in female rats
(NTP, 1990) exposed to chlorinated drinking water for 2 years
100 = uncertainty factor, chosen in accordance with U.S. EPA
guidelines for use of a LOAEL from an animal study
CHLORINE.8
VIII-18
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Step 2: Determination of the Drinking Water Equivalent Level (DWEL)
DWEL =
C0.1 mglkglday) (70 kg)
2 L/day
- 3.5 mgIL (roundedto 4.0 mg/L)*
where:
0.1 mg/kg/day = RfD
70 kg
assumed body weight of an adult
2 L/day
assumed water consumption by an adult
HAs are summarized in Table VIII-3.
'Caution should be applied to the interpretation of this health risk assessment, in that it
does not address the adverse effects associated with chlorinated by-products, especially
trihalomethene.
Carcinpgenic Effect?
Animal data are limited (Druckrey, 1968; Hasegawa et al., 1986; NTP, 1990) for
assessing the carcinogenic potential of chlorine, hypochlorous acid or hypochlorite ion.
The NTP (1990) 2-year bioassay concluded that there was no evidence of carcinogenicity
in male F344 rats and equivocal evidence in females. The increased incidence in
mononuclear cell leukemias in female rats does not support a carcinogenic association
based on the following factors:
1. The increase was slight and not clearly dose-related.
2. No decrease in tumor latency was observed.
3. Incidence in concurrent controls was lower than historical controls.
4. No supporting incidence was observed in n.-ie rats or male or female mice.
CHLORINE.8
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TABLE VIII-3
Summary of DWEL and 10-day HA Calculations
Criteria
Calculated Level
(mg/L)
Reference
1-Day HA (10 kg child)
ND
Blabaum and Nichols, 1956
10-Day HA (10 kg child)
2.5
Blabaum and Nichols, 1956
Longer-term HA (10 kg child)
2.0
Daniel et al., 1990
Longer-term HA (70 kg adult)
6.0
Daniel et al., 1990
DWEL
4.0
NTP, 1990
ND = No suitable data; it is recommended that the 10-day HA be adopted as the 1-day
HA.
CHLORINE.8
VIII-20
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-------
There was no evidence in male or female B6C3F1 mice. The results of these studies
do not indicate that chlorine or its dissociation products are directly carcinogenic to humans
or experimental animals. It should be recognized, however, that these compounds in water
may form organic by-products (for example halomethanes) that may have carcinogenic
potential. Using the U.S. EPA (1986) carcinogen risk assessment guidelines chlorine may
be classified in Group D, not classifiable. This category is for agents with inadequate
animal and human evidence of carcinogenicity.
Quantification of Carcinogenic Effects
Since no definitive carcinogenic effects have been detected for chlorine, hypochlorous
acid or hypochlorite ion, no quantification of effects is appropriate.
Existing Guidelines. Recommendations and Standards
Most of the recommendations for chlorine exposure involve inhalation. Occupational
exposure guidelines are listed in Table VIII-4. The U.S. EPA (1081) recommended an
ambient water quality criterion of 10.0 mg/L chlorine for the protection of human health.
A water quality criterion of 10 ^g/L of residual chlorine has been recommended for the
protection of aquatic life.
CHLORINE.8
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TABLE VIII-4
Existing Guidelines on Human Exposoure to Chlorine3
Type of Guideline5
Exposure Level (ppm)
ACGIH
TLV-air TWA
STEL-air
1
3
OSHA-Standard
Chlorine in air
1
U.S. MSHA
Standard-air TWA
1
Criteria Document:
Occupational exposure to chlorine in air
0.5 for 15 minutes
TLV (West Germany, Switzerland,
Yugoslavia)
0.5
TLV (USSR, most eastern European
countries)
0.3
TLV (most other countries)
1
'Source: ACGIH, 1986
bTLV = Threshold limit value; STEL = short-term exposure lirrit; OSHA = Occupational
Safety and Health Administration
CHLORINE.8
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Special Groups at Risk
While there has been some research on the chemistry of chlorine and its dissociation
products, little attention has been directed to the effects of such chemicals on individuals
or groups within the human population who are potentially at higher risk.
Individuals who are allergic to chlorine products or who are asthmatic may be at high
risk for adverse reactions after inhalation or ingestion of chlorine compounds. Asthmatic
attacks have been reported after consumption of municipal drinking water that contained
0.2-0.4 ppm chlorine (Sheldon and Lovell, 1949). Studies by Lubbers et al. (1983, 1984)
have indicated that individuals with an A-variant form of G6-PD deficiency may also be at
higher risk due to oxidant stress. Newborns, especially those with enzymatic deficiencies,
are also a group to be at increased risk from oxidant-stress or agents (Jones and
McCance, 1949; Ross, 1963).
CHLORINE.8
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