United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-019 Jan 1985
Project Summary
Water Distribution System as a
Potential Source of Mutagens in
Drinking Water
Dipak K. Basu, Jitendra Saxena, Frederick W. Stoss, Joseph Santodonato, and
Michael W. Neal
The primary objectives of this study
were to examine the changes in concen-
tration of six polycyclic aromatic hydro-
carbons (PAHs) and the possibility of
changes in mutagenic potential of
treated waters as a result of leaching
during their passage through commonly
used distribution pipes in the United
States. With the exception of the fin-
ished water from one utility in which the
total concentration of the six PAHs was
138.5 parts per trillion (ppt), the total
PAH concentration in all other treated
water ranged from 0. to 13.4 ppt. The
corresponding total PAH concentration
in water after their passage through the
distribution pipes varied from 0 to 61.6
ppt. This demonstrates that PAH con-
centration in waters can increase as a
result of their passage through the
asphalt-line distribution pipes.
M utagenic activity was also detected
in many of the treated water samples,
however, the levels of this activity did
not correlate with either the transit of
water through the distribution system
or the levels of PAH in the water. There
was some evidence to indicate that the
water treatment process itself may have
contributed to the mutagenicity ob-
served in the finished water and that
compounds responsible for the ob-
served activity were different from the
low molecular weight chlorinated com-
pounds produced during chlorination.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park, NC,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Finished waters from the treatment
sites are transported to the consumers
through a variety of pipelines In the
United States, three kinds of pipes are
commonly used as transmission/distri-
' bution Imesfor transporting treated water
to consumers, cast iron/ductile iron;
asbestos/cement, and steel pipes. Other
materials occasionally used as water
distribution lines are reinforced concrete,
a variety of plastic materials, iron, copper,
and rarely, lead. With the exception of
plastic pipes, coating materials are often
used for the interior of the water pipes to
prevent corrosion or to aid in curing the
pipes. For example, cast iron/ductile iron
pipes are lined sometimes with asphalt,
cement, or asbestos/cement coatings;
concrete pipes with asphalt coating; and
steel pipes with cement or coal-tar coat-
ings. Depending upon the characteristics,
manufacturing conditions, thickness, and
age of the pipe or its lining, some leaching
of materials can be expected to occur as
water passes through the pipes. The
leaching of volatile organic compounds
from different types of water pipes has
also been studied in Europe. Cast iron
pipes coated with polyurethane have been
shown to add volatile contaminants such
as chlorobenzene and aromatic com-
pounds in drinking water.
Both asphalt and coal tar are known to
contain high levels of polycyclic aromatic
hydrocarbons (PAHs), some of which are
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known or suspected carcinogens, al-
though the level of PAH in coal tar may be
1000 to 100,000 times higher than in
asphalt It is possible that the coating of
coal tar-based or asphalt-based paint or
sealant on iron, steel, or concrete pipes
could cause leach ing of small amounts of
PAH into drinking water. The concentra-
tion of any given PAH leached into the
water is dependent on its solubility,
concentration in the coating, contact time,
etc. There are reports in the literature that
undesirably high levels of PAH can result
In a German study, a 10-fold increase in
PAH concentration was reported at the
end of water supply pipes resulting from
the leaching of compounds from the
coating used inside the water pipes. In a
study in Pascagoula, Mississippi, more
than nine PAHs, including fluoranthene
at a concentration of 9.7 /ug/L, were
reported in water stored in a tank freshly
coated with coal-tar pitch. Similarly,
potable w-.'er in Portland, Oregon, ob-
tained from a terminal point of a water
distribution pipe coated with coal tar
showed the presence of PAH.
Alt hough the leaching of coating mate-
rials from the pipes can account for the
presence of many compounds including
PAH, it may not be the only mechanism
which could contribute to the deteriora-
tion of the water quality resulting from
passage of drinking waters through sup-
ply networks. The reaction of residential
chlorine with organics leached from
pipes, oxidation of leached materials, or
microbiological synthesis of compounds
on pipe walls and their subsequent
release into the water may contaminate
the water with compounds not originally
present in the finished water.
The examination of changes in water
quality resulting from distribution pipes
can be accomplished by determining
changes in the levels of specific chemi-
cals Because of their reported presence
m some coating materials used inside the
distribution lines, and because of the
known carcinogenic potential of many of
the compounds in this class, PAHs were
chosen as a class of chemicals to be
monitored in order to determine possible
adverse effects of the distribution system
on water quality. To monitor the effects of
other harmful compounds that may be
leached from the pipes or produced in the
distribution system, an assay was also
employed that was not specific for a
particular class of chemicals. This general
assay was an in vitro microbiological
mutagenicity assay which has the capa-
bility of detecting a large variety of
compounds with genotoxic potential.
Therefore, the present investigation was
primarily oriented to provide information
on two aspects of water quality: PAH
content and the presence of mutagens in
source waters, and the effect of treatment
and passage of those waters through the
distribution pipes on these measures of
water quality.
The sampling locations for this study
represent the largest water supplies
within 24 selected counties in the United
States. These counties were selected by
the U.S. Environmental Protection Agency
for an epidemiological study of associa-
tion between cancer mortality rates and
drinking water quality. Twelve pairs of
counties were selected with contrasting
(high and low) total mortality rates for
colon, rectum, bladder, and liver cancer in
females during the years 1 968 to 1974.
Each pairof counties was matched on the
basis of five demographic characteristics
(geographic region, total population, per-
cent urbanization, median school years
completed, and percent of work force in
manufacturing industry). Only counties
that were at least 50% urban were
included. The studies reported here, were
conducted in these counties to determine
if the occurrence of PAH or mutagenic
activity correlated with the occurrence of
any of the types of cancer.
The monitoring of the expected low
levels of PAH in drinking water required a
sample collection method that was cap-
able of concentrating PAH from large
volumes of water. A method developed in
a previous study was found to be suitable
and was used; the organic contaminants
are adsorbed onto polyurethane foam
plugs and subsequently desorbed by
elution with an organic solvent. This
method also includes a final chemical
separation procedure (thin layer chroma-
tography) capable of eliminating interfer-
ing impurities and a quantification
method (fluorescence spectrometry) sen-
sitive enough to detect small amounts of
PAH. The PAH detection limit for PAHs by
this method was determined to be better
than with capillary-gas chromatography
with flame ionization detection.
The overall mutagenic potential was
assessed by utilizing the Ames Salmonella
assay system. This assay employs a
mammalian metabolic activation system,
thus allowing the detection of compounds
that are biologically active only after
having been metabolized. The system
was chosen primarily for two reasons.
First, it provides a quick screening method
for testing mutagenic potential of com-
pounds. Second, a high correlation be-
tween positive results in the Ames assay
and positive results in in vivo animal
carcinogenicity assays have been estab-
lished for a large number of compounds.
The scoring of samples for positive or
negative mutagenic activity was inde-
pendently conducted by three individuals.
Objective evaluation criteria (i.e., doubling
of spontaneous reversion rate, and
demonstration of dose-related increase
in revertants) were employed in the
interpretation of all Ames assay results.
However, in certain assays where the
results were not definitive (e.g., more
than a doubling but lack of dose-related
increase, or positive dose-response curve,
but number of revertants slightly less
than twice the spontaneous) a subjective
evaluation was also applied to the data. In
these cases, a consensus of the three
reviewers was obtained before declaring
the result to the positive or negative.
Results and Conclusions
The results indicate that in 67 percent
of the total water supplies studied, a
measurable increase in concentration of
PAH in water does occur as a conse-
quence of treated water travelling through
petroleum asphalt-lined distribution lines.
The increases in PAH concentration,
however, were small and amounted to a
maximum of 61 ppt in water. It was also
determined that the maximum leaching
effect occurred with distribution pipes
that had recently been coated with asphalt
linings. The recently installed asphalt-
coated pipes predominantly added the
more water-soluble PAH, whereas leach-
ates from the older pipes showed higher
ratios of less water-soluble to more
water-soluble PAHs. In older pipes this is
probably due to depletion of the more
water-soluble PAH from the surface of
the coating. Waters from a few distribu-
tion lines showed reductions in PAH
concentration instead of the expected
enhancements of PAH concentration.
This reversing trend can be explained by
assuming sorption of PAH on the surface
of the distribution pipes or chemical
interaction with oxidants in water. Distri-
bution pipes that are depleted of the
leachable PAH may act as sorbents for
these compounds. Thus, depending on
the age of the pipes, it is possible to get
either desorption or sorption effects. In
addition to the pre-selected six PAHs
identified and quantified in this study,
many other compounds were detected in
the water samples. No attempt was made
to identify these compounds.
The results of bacterial mutagenicity
assays indicated the presence of low
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levels of mutagenic activity in many of the
waters sampled. In general, the raw,
finished, and distributed drinking waters
tested in this investigation demonstrated
no significant or consistent mutagenic
activity in the Ames test system. Thus, it
is not clear whether the leachate from
certain distribution pipes actually con-
tributes to the presence of mutagens or,
on the other hand, whether some pipes
adsorb mutagens which had not previous-
ly been removed during treatment. Of the
24 water supplies tested, two showed no
detectable mutagenic activity in either
the raw water or water taken from any of
the collection points. Five other water
supplies had a number of sampling sites
in which an individual assay point
demonstrated a mutation ratio (number
of revertants in experimental plate divided
by number of revertants in control plates)
of between 1.5 and 2.0 The random
appearance of these weak mutagenic
responses and the concomitant lack of a
dose-related increase in mutation rate
indicate that these values fall within the
normal variation of the Ames assay
When samples produced no mutagenic
response the reason was probably that
there were insufficient quantities of
mutagens present. There was also the
possibility, though, that some of these
samples contained mutagenic compounds
but the biological activity of these com-
pounds was masked by the presence of
toxic substances in the samples. Since
toxicity tests were not performed on any
of the water samples or their concen-
trates, the only detectable evidence of
severe toxicity in our investigation would
be the absence of a bacterial lawn (the
limited growth of histidine-dependent
bacteria allowed by the trace amount of
histidine supplied by the media in the
minimal plate). The absence of a bacterial
lawn did not result from the testing of any
samples in this study. However, slight
bacterial toxicity was occasionally encoun-
tered as indicated by a dose-dependent
decrease in the number of revertants
with some samples. An example of this
toxic effect was observed with the raw
water samplefrom Fremont, Ohio, where
average number of revertants for strain
TA1535 declined from 79.5 for control to
62.0, 50.0, 56.5, and 8.4 as the dose
increased. It is apparent from the variable
effect of metabolic activation on bacterial
toxicity (in some samples S-9 increased
activity while in others it decreased
toxicity) that the chemical composition
and/or amounts of the toxic agents in
individual samples varied extensively
among the different collection locations.
In most samples, the lowest dose tested
appeared to be nontoxic to the bacteria.
The types of pipe associated with the
two water samples that demonstrated no
mutagenic activity were cast iron and
asbestos/cement. Although many vari-
ables are unknown about the pipes such
as age, condition, and the physical prop-
erties (pH, types of solutes) of the water, it
is apparent that this study shows no
consistent relationship between the type
of pipe and the absence of observed
mutagenic activity in the water which has
been transported by these pipes.
Fifteen of the water supplies provided
at least one sampling point which either
produced a mutagenic response or a weak
mutagenic response in at least one of the
five strains of bacteria tested. In this
study a response was considered to be
weakly mutagenic if the reversion ratio
fell between 1.5 and 2.0 and was accom-
panied by a positive dose-response trend.
A clear mutagenic response in this study
was considered to occur when the rever-
sion ratio equaled or exceeded 2.0. It
should be noted that it is not unexpected
to observe mutagenic responses with
environmental samples where the high
dose point has a lower ratio than the
intermediate and low dose point. Such
data are generally indicative of the toxicity
to the tester strain of bacteria used in the
Ames assay. Even in samples demonstra-
ting clear mutagenic potential in this
study, the response was not great, with
most ratios being only slightly greater
than 2.0.
Recommendations
On the basis of the results of the
present project, the investigators recom-
mend the following areas for further
investigations:
1. Samples that showed mutagenicity
should be fractionated to isolate
and identify the component(s) re-
sponsible for the mutagenic effect.
2. Since no dramatic PAH leaching
was observed with the petroleum
asphalt-lined pipes, further study
should be directed towards the
leaching effects from distribution
pipes lined with coal tar-based
products.
3. In the present investigation, the age
of the installed pipes was found to
be one of the most critical factors in
the leachability of PAH from the
pipe linings. In future studies, the
age of pipes, condition of coating,
and contact time should be given
primary considerations in the selec-
tion of sampling sites. Recently
installed pipes are expected to
provide more dramatic enhance-
ment of PAH concentration than
the older pipes.
4. Factors, such as pH, residual chlo-
rine, aggressive index of water, and
conditioning of the pipes after ap-
plication of coat ings (all factors that
may effect the leaching of PAHs),
should be morethoroughly studied.
5. The effect of chlorination of drinking
water on some of the leached
compounds should be investigated.
A few recent studies suggest that
the leached compounds are con-
verted to chlorinated or oxidized
products in the presence of chlorine.
6. The apparent increase in mutagenic
activity resulting from conventional
water treatment processes should
be studied in greater detail This
should include an investigation of
the production of high molecular
weight compounds having biologi-
cal activity as a result of chlorina"-
tion.
7. The mutagenic activity of trace
contaminants in treatment chemi-
cals used for the production of
finished water should be investi-
gated as a possible source of muta-
genic activity in drinking water.
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D. K. Basu, J. Saxena, F. W. Stoss, J. Santodonato, and M. W. Neal are with
Syracuse Research Corporation, Syracuse, NY 13210-4080.
Frederick P. Kopfler is the EPA Project Officer (see below).
The complete report, entitled "Water Distribution System as a Potential Source of
Mutagens in Drinking Water,"(Order No. PB 85-125 474; Cost: $19.00, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
* U S GOVERNMENT PRINTING OFFICE, 1985 — 559-016/7875
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