600184019
SRC TR-81-620
WATER DISTRIBUTION SYSTEM AS A POTENTIAL SOURCE
OF MUTAGENS IN DRINKING WATER
Dipak K. Basu
Jeet Saxena
Frederick W. Stoss
Joseph Santodonato
Micheal W. Neal
Syracuse Research Corporation
Center for Chemical Hazard Assessment
Syracuse, New York 13210-4080
Grant No. R 806413
Project Officer
Frederick C. Kopfler
Exposure Evaluation Branch
Health Effects Research Laboratory
Cincinnati, Ohio 45268
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio U5268
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CONTENTS
DISCLAIMER i
FOREWARD ii
ABSTRACT iii
LIST OF FIGURES v
LIST OF TABLES vi
LIST OF ABBREVIATIONS vii
ACKNOWLEDGEMENTS viii
1 . INTRODUCTION 1
2. CONCLUSIONS 3
3. RECOMMENDATION 5
4. LITERATURE REVIEW 7
5. MATERIALS AND METHODS 12
MATERIALS 12
EXPERIMENTAL PROCEDURES 15
6. RESULTS AND DISCUSSION 45
RESULTS OF CHEMICAL ANALYSIS FOR PAH 48
RESULTS OF MUTAGENICITY ASSAYS 68
7. REFERENCES 83
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
dose not signify that the contents necessarily reflect the views and policies of
the U.S. Environmental Protection Agency, nor does it mention trade names or
commercial products constitute endorsement or recommendation for use.
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FOREWARD
11
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ABSTRACT
The primary objective of this study was to examine the changes in polycyclic
aromatic hydrocarbon concentrations 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 U.S. A method developed in our
laboratories was used for the collection and analysis of water samples for
polycyclic aromatic hydrocarbons. The polycyclic aromatic compound benzo(a)-
pyrene and benzo(ghi)perylene were among the most frequently occurring poly-
cyclic aromatic hydrocarbons in raw and treated water and in waters travelling
through the distribution lines. With the exception of Wheeling, WV treated
water, which showed an unusually high total polycyclic aromatic hydrocarbon
concentration of 138.5 ppt, the total polycyclic aromatic hydrocarbon concentra-
tion in all other treated water ranged from 0 to 13.4 ppt. The corresponding
total polycyclic aromatic hydrocarbon concentration in water after their passage
through the distribution pipes varied from 0 to 61.6 ppt. This indicates a small
increase of polycyclic aromatic hydrocarbon concentration in waters as a result
of their passage through the asphalt-lined distribution pipes. The increase in
polycyclic aromatic hydrocarbon concentration was maximum with recently
installed asphalt-lined pipes. Waters from a few distribution lines showed
reduction in polycyclic aromatic hydrocarbon concentrations possibly due to
sorption of PAH on the surface of these distribution pipes.
iii
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Mutagenic activity was also detected in many of the water samples treated,
however, the levels of this activity did not correlate with either the transit of
water through the distribution system or the levels of polycyclic aromatic hydro-
carbon in the water. There was some evidence to indicate that the water
treatment process itself may have contributed to the mutagenicity observed in the
finished water and that compounds responsible for the observed activity were
different from the low molecular weight chlorinated compounds produced during
chlorination. This study also provides evidence that mutagenic activity is less
likely to be found in the raw water supply if this supply is from ground water
rather than surface water.
IV
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LIST OF FIGURES
Number
1 Portable sampling suitcase used to collect organic
contaminates from drinking waters 22
2 Locations and configurations of sampling points
after the passage of potable waters through
distribution pipes 25
3 Experimental Design of Bioassays 33
4 Emission spectra of an unidentified compound that
recurred in several samples 6?
5 Gas chromatograms of Elkhart, IN samples collected
at the four sampling points 69
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LIST OF TABLES
Number
1 Levels of Some PAH in Fossil Fuel Derivatives 9
2 Some Characteristics of the Water Supply System
Used for Sampling 16
3 Distance, Type and Configuration of the Distribution
Pipes in the Supply Systems Used for Sampling 26
4 Results of PAH Analyses of Water Samples 49
5 Detection Limit for Six PAH by Thin Layer and
Capillary Gas Chromatographic Methods 60
6 Water supplies with no detectable mutagenic activity 70
7 Variation in Mutagenicity in Raw and Distribution
Water as Compared with the Finished Water 74
8 Comparison of Mutagenic Activity in Raw Water with
the Type of Raw Source 80
vi
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LIST OF ABBREVIATIONS
BaP = benzo(a)pyrene
BghiP = benzo(ghi)prylene
BjF = benzo(j)fluoranthene
FL = fluoranthene
IP = indeno(1,2,3-cd)pyrene
PAH = polycyclic aromatic hydrocarbons
TLC = thin layer chromatography
U.V. = ultra violet
vii
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ACKNOWLEDGEMENTS
Syracuse Research Corporation wishes to acknowledge the assistance and
cooperation provided by the staff members of all the twenty-four water treatment
plants during our sampling trips. Special acknowlegements are extended to
Mr. Conrad Teufel and Mr. James Morgan for assisting us with the field samplings.
We are also grateful to Drs. David Schwartz and Lawrence Mason for their
assistance with some of the Ames Assay work. The generous guidance and
assistance of the EPA Project Officer, Dr. Frederick C. Kopfler, is also
appreciated.
Cost sharing by the American Petroleum Institute at the level of 5% of the
overall cost is appreciated. Special thanks are extended to API Project
Officers, Dr. David D. Disbennet and Dr. David Chen.
viii
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SECTION 1
INTRODUCTION
The presence of trihalomethanes and other potentially carcinogenic
compounds in drinking waters has caused considerable public concern over the
potential effects on health arising from the consumption of these waters. The
two generally recognized sources of chemical contamination of drinking water are
pollutants present in raw water sources of potable waters and compounds formed
during treatment of raw water, such as chlorination of finished waters. A third
and little investigated source is the possibility of contribution of mutagenic or
carcinogenic substances in drinking waters by the pipes used in the distribution
of potable waters. The level of exposure to chemicals through the consumption of
pipe-leachable compounds in potable waters may be low but of long-term duration.
In the absence of a general agreement that a completely safe exposure level of
biologically active chemicals does exist, it is important to identify the
suspected chemicals and evaluate the possible health effects associated with the
use of potable waters after their passage through the distribution pipes.
German workers have shown that significant amounts of polynuclear aromatic
hydrocarbons (PAH), many of which are suspected or proven carcinogens, are added
in the distribution system (Borneff, 1977). In one case, the concentration of
PAH at the end of the water supply network was about 10 times higher than the
concentration at the treatment site. Increases in the concentration of PAH at
the end of the supply network are also indicated from our preliminary investiga-
1
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tion on three water supplies within the United States; the asphalt-based coating
material used within the pipes was suspected to be the cause of this problem.
Microbial mutagenicity assays utilizing histidine dependent strains of
S. typhimurium (Ames et al., 1973) were also performed on drinking waters
obtained from the distribution system at three selected water supplies. The
results revealed a significant increase in the mutagenic activity upon passage of
the treated water through distribution pipes. Some of the compounds introduced
required mammalian metabolic activation (i.e., S-9 mix), whereas others elicited
mutagenic responses without the activation system.
The present investigation is directed at examining the contribution of
mutagenic compounds from the supply network to selected water supplies within the
United States. The overall mutagenic potential will be assessed by utilizing
non-specific bioassay systems. Furthermore, in view of the potential of the
distribution pipes for addition of PAH, the presence of PAH in these waters will
also be examined.
The study proposed is a screening type investigation and is designed to
provide information concerning the intensity and frequency of the problem with
water distribution processes. The results of this investigation will be helpful
to EPA in setting priorities for in-depth and more specialized work, and in the
epidemiological investigation currently underway at EPA to derive a relation-
ship, if any, between the quality of drinking water and the occurrence of certain
types of cancer.
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SECTION 2
CONCLUSIONS
The primary objective of this study was to investigate the possibility of
enhancement of PAH concentration and mutagenicity of treated water as a result of
its passage through distribution pipes. The results indicate that in 67% of the
total water supplies studied, a measurable increase in PAH concentration in water
does occur as a consequence 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 the older
pipes predominantly leached the less water-soluble PAH. In older pipes this is
probably due to depletion of the more water-soluble PAH from the surface of the
lining. Waters from a few distribution lines showed reductions in PAH concentra-
tion instead of the expected enhancements of PAH concentration. This reversing
trend can be explained by assuming sorption of PAH on the surface of the distri-
bution pipes. Distribution 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 both desorption and sorption effects. In addition to the pre-
selected six PAH identified and quantified in this study, many other compounds
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were detected in the water samples. No attempt was made to identify these
compounds.
The results of bacterial mutagenicity assays (Ames assay) indicated the
presence of low levels of mutagenic activity in many of the waters sampled.
There appeared to be no correlation between the level of biological activity and
the passage of water through distribution pipes. Also there was no apparent
correlation between the type of pipe used and the presence or absence of
mutagenic activity. The specific compounds that produced the observed mutagenic
response were not identified, however, there was no correlation between the PAH
levels of water and the observed mutagenicity. In most cases where mutagenic
activity was observed in water at the treatment plant this activity was the same
or greater than the activity in the raw water. This indicates that the treatment
plant was generally ineffective in removing this low level of mutagenic activity,
and that some of the processes employed in water treatment may actually contri-
bute to the presence of mutagens in the distribution system. In considering the
source of water for potable use, ground waters generally demonstrated less
mutagenic activity as compared to surface waters.
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SECTION 3
RECOMMENDATION
On the basis of our experience with the present project, we recommend the
following areas for further investigations:
(1) Samples that showed mutagenicity should be fractionated to
isolate and identify the component(s) responsible 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 coal tar-based distribution pipes. It is
expected that the leaching may be more dramatic with these pipes
than asphalt based distribution pipes.
(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
should be given one of the primary considerations in the selec-
tion of sampling sites. Recently installed pipes are expected to
provide more dramatic enhancement of PAH concentraton than the
older pipes.
(4) Factors, such as pH, residual chlorine, aggressive index of water
and conditioning of the pipes after application of coatingsall
the factors that may affect the leaching of PAH should be more
thoroughly studied.
(5) The effect of chlorination of drinking water on some of the
leached compounds should be investigated. A few recent investi-
gations suggest that the leached compounds are susceptible to the
formation of chlorinated or other 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 biological
activity as a result chlorination.
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(7) The mutagenic activity of trace contaminants in treatment
chemicals used for the production of finished water should be
investigated as a possible source of mutagenic activity in
drinking water.
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SECTION 4
LITERATURE REVIEW
The presence of trihalomethanes and other potentially carcinogenic
compounds in drinking waters has caused considerable public concern over the
potential health effects arising from the consumption of these waters. The two
generally recognized sources of chemical contamination of drinking water are
pollutants present in the raw water sources of potable waters and compounds
formed during treatment of raw water, such as chlorination and ozonation used for
disinfecting water prior to human consumption. A third and little investigated
source of contamination is the possibility that the pipes used in the
distribution of potable waters may contribute mutagenic or carcinogenic
substances to drinking waters.
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 distribution lines for transporting treated water
to consumers: cast iron/ductile iron, abestos/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. Of the total
installed length of approximately 685,000 miles of water distribution lines in
the United States, about 75% consists of cast iron/ductile iron pipes, 13?
consists of asbestos/cement pipes, and 6% consists of steel pipes (Goldfarb
et al., 1979).
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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 sometimes lined with
asphalt, cement, or asbestos/cement coatings; concrete pipes with asphalt
coatings; and steel pipes with cement or coal-tar coatings. 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. McFarren ฃt _al. (1977) have demonstrated that galvanized iron
pipes may leach into drinking water, lead, zinc, and cadmium; asbestos/cement
pipes some asbestos fibers; and PVC pipes some residual vinyl chloride. The
leaching of volatile organic compounds from different types of water pipes has
also been studied in Europe (Eklund at _ง!., 1978). Cast iron pipes coated with
polyurethane have been shown to add volatile contaminants such as chlorobenzene
and aromatic compounds in drinking waters.
Both asphalt and coal-tar are known to contain high levels of polycyclic
aromatic hydrocarbons (PAH), some of which are known or suspected carcinogens.
Table 1 presents the levels of a few PAH in coal-tar and petroleum asphalt as
determined by Radding et _al. (1975).
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Table 1. Levels of Some PAH in Fossil Fuel Derivatives (Radding et al., 1975)
PAH Content in Fossil Fuel Derivatives, u.g/g
PAH Identified
Benzo(a)pyrene
Chrysene
Benzo ( a ) anthracene
Coal-Tar
30,000
up to 2,860
up to 6,980
Coal-Tar Pitch
12,500
up to 10,000
up to 12,500
Petroluem Asphalt
0.1-27.
O.M-34.
up to 34.
It is thus possible that the coating of coal tar-based or asphalt-based
paint or sealant on iron, steel or concrete pipes could cause leaching of small
amounts of PAH into drinking water, determined partly by their solubilities in
water. Borneff (1976) reported a ten-fold increase in PAH concentration at the
end of water supply pipes resulting from the coating used inside the water pipes.
In a study in Pascagoula, Mississippi, more than 9 PAH, including fluoranthene at
concentration as high as 9.7 (ig/1, were reported in water stored in a tank
freshly coated with coal-tar pitch (McClanahan, 1979). Similarly, potable water
obtained from a terminal point of a water distribution pipe coated with coal-tar
showed the presence of PAH. The fluoranthene and phenanthrene concentrations in
this Portland, Oregon, water were reported to be 0.6 u.g/1 and 3-2 u.g/1,
respectively (Roebeck, 1979).
Although the leaching of coating materials 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 deterioration of the water quality resulting from
passage of drinking waters through supply networks. The reaction of residual
chlorine with organics leached from pipes, oxidation of leached materials, or
microbiological synthesis of compounds on pipe walls and their subsequent
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release into the water may cause contamination of the water with compounds not
originally present in the finished waters.
The examination of changes in water quality resulting from distribution
pipes can be accomplished by determining changes in the levels of specific
chemicals. Because of their reported presence in some coating materials used
inside the distribution lines, and because of the known carcinogenic potential of
many of the compounds in this class, PAH 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 capability 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 drinking waters as a result of passage of those waters
through the distribution pipes.
The monitoring of the expected low levels of PAH in drinking waters required
the selection of a method (Basu and Saxena, 19?8a) that was capable of concentra-
ting PAH from large volumes of water. The method must also include a chemical
separation procedure capable of eliminating interfering impurities, and a quan-
tification method sensitive enough to detect small amounts of PAH. A method
developed in a previous study (Basu and Saxena, 19?8b) was found to be suitable
and was used for the monitoring of PAH levels in waters.
10
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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 compounds. Second, a high correlation between positive results in
the Ames assay and positive results in in vivo animal carcinogenicity assays has
been established for a large number of compounds (Ames et al., 1973; McCann
et al., 1975).
11
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SECTION 5
MATERIALS AND METHODS
MATERIALS
PAH Standards
Fluoranthane, Benzo(ghi)perylene, Indeno-(1,2,3-cd)pyrene, and Benzo(a)-
pyrene were obtained from Aldrich Chemical Co. Each compound was at least 99%
pure as verified by GC-FID and TLC-fluorimetric analysis. Benzo(j)- and
Benzo(k)fluoranthene were obtained from Community Bureau of Reference (BCR),
Brussels, Belgium. These were certified reference materials of better than 99%
purity and used as received.
Materials for Capillary Column (WCOT) Chromatographic Analysis
Gases: He (carrier gas) was of ultrahigh purity grade, N, (detector make-up
gas) and H were both prepurified medical grade, Linde Division, Union Carbide
Corp.
Aluminum foil-backed septa(Metasep) were from Altech Associates
WCOT Columns were 30 m x 0.25 mm i.d. and 15 m x 0.25 mm i.d., and both were
coated with SE-51* obtained from Supelco, Inc. Analysis of samples collected
after 8-1-80 were performed with the shorter column. The gas chromatograph with
dual flame ionization detector was a Hewlett-Packard Model 5730A with 18740B
capillary column control accessories, operated in the splitless mode, and used
with a Fisher Recordall Series 5000.
12
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Materials for Thin Layer Chromatography
Acetylated cellulose (40^), aluminum oxide G (type E) , 8 x 8 cm glass
plates, TLC tank, Desaga template, and Desaga-Brinkman Spreader were obtained
from Brinkman Instrument, Inc. Spectrophotofluorimeter with thin film scanner
was from American Instrument Co., and the U.V. Lamp (Mineralight) for visualiza-
tion of TLC spots was from Scientific Products, Inc.
Materials for Radiolabeling Work
14
Radiolabeled 7, 10 C-benzo(a)pyrene was obtained from California
Bionuclear Corp. and a Packard Tri-Carb Scintillation Spectrometer with Teletype
Printer was used for determination of radioactivity. Scintillation fluid
contained 2,5-diphenyloxazole (PPO), 2,2'-phenylene bis-(S-phenyl) oxazole
(POPOP) which was purchased from Packard Instrument Co.
Materials for Sampling
Cyclindrical flexible polyurethane foam tube or flask plugs (45 mm x 45 mm)
with a trade name of "Identi-Plug" were obtained from VWR Scientific Co.
Extender type chromaflex columns of 25 mm i.d. with "0" rings, adapters and
clamps were from Kontes Glass Co. The Masterflex pump, controller and pumpheads
were made by Cole-Parmer Instrument Co., Chicago, and the Haake Model FE
Thermostated Circulator with 1000 W Heater was purchased from VWR Scientific Co.
Chemicals, Reagents and Apparatus for Sample Treatment
All solvents were from Burdick Jackson Lab., Inc. and distilled in glass.
Chromatographic grade Florisil (60-100 mesh), was obtained from Floridin
Co.
13
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The Buchler Rotary Vacuum evaporator with thermo-regulator and immersion
heater was purchased from Scientific Products, Inc. and calibrated tubes
(6.5 ml), with each of the last four-tenth ml calibrated to one-hundredth ml were
purchased from Fisher Scientific Co.
Standard PAH Solution
Stock solutions of each of the six PAH were prepared by dissolving 100 mg in
100 ml benzene in a volumetric flask (1000 ppm).
In order to prepare working PAH standard mixtures for GC analysis, 1 ml each
of the six stock solutions were diluted in a 10 ml volumetric flask and brought
up to the final volume with benzene (100 ppm). Standard PAH mixture for TLC was
prepared by mixing 5 ml of the standard (100 ppm) fluoranthene solution and 1 ml
each of the five other PAH (100 ppm) in a glass stoppered bottle, (final concen-
tration: 50 ppm fluoranthene, 10 ppm others).
Materials for Bioassays
Test organisms utilized in the rautagenicity bioassays were specially
constructed strains of Salmonella typhimurium designated TA98, TA100, TA1535,
TA1537, and TA1538, all supplied by Dr. Bruce Ames (Berkeley, California).
During the course of the study it was necessary to obtain subcultures of
S. typhimurium strains TA100 and TA98 from Dr. Edmond J. LaVoie at the American
Health Foundation. Male Sprague-Dawley rats weighing 150-200 gm each were
obtained from a commerical supplier, Taconic Farms, Inc. (Germantown, NY) and
were used in the preparation of liver homogenates for the S-9 metabolic activa-
tion system.
14
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DIFCO Bacto-Agar and DIFCO Nutrient Broth (Scientific Products) were used
for media preparations. Aroclor 1254 (Monsanto Co.) and U.S.P. corn oil (Fisher
Scientific) were used in the induction of rat liver enzymes. Diagnostic mutagens
routinely used for positive controls were: 4-nitro-o-phenylenediamine (Sigma
Chemical), methylmethane sulfonate (Aldrich Chemical), 9-aminoacridine (Sigma
Chemical), 2-aminoanthracene (Sigma Chemical). The DMSO (dimethyl sulfoxide)
was obtained from Burdick and Jackson Laboratories. All salts, and other
chemicals were of reagent grade.
Unconeentrated water samples were filter sterilized using a 90 mm standing
filter holder (Millipore Corporation), with 90 mm Gelman filters (0.20 um,
0.45 pjn, 1.20 MJH, and glass prefilters). Revertant colonies were counted with a
BioTran II automatic colony counter.
EXPERIMENTAL PROCEDURES
Sampling Procedures
Selection of Sampling Sites
The sampling locations for this study represent the largest water supplies
within the 24 selected counties in the United States. The areas these water
supplies serve represent communities with both high and low cancer incidence
rates. In an ongoing epidemiological study, the U.S. Environmental Protection
Agency is making an effort to determine if any correlation exists between the
occurrence of certain types of cancer with certain water quality parameters such
as total organic carbon, halogenated hydrocarbons and asbestos. The present
investigation will provide an additional parameter, PAH, for correlation with
EPA's epidemiological data. Table 2 identifies the raw water source, type of raw
15
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water pollution, treatment provided to generate finished water, and the date of
sampling for the water supplies investigated in this study.
Sampling Procedure
Earlier studies (Basu and Saxena, 1978b; Saxena _et al., 1977a; Saxena
gt al., 1977b) showed that water passing through foam plugs at 62 + 2ฐC at a flow
rate of 250 ฑ 10 ml/min can effectively concentrate PAH from water. Four foam
plugs two in each column and connected in series, were adequate for sampling
20 liters of finished water and 10 liters of polluted raw water. A maximum
volume of 75 liters of finished water and 60 liters of raw water were sampled
with a portable sampling unit assembled in our laboratory from a combination of
off-the-shelf hardware available commercially.
With a minor modification, the sampling unit described in our earlier work
(Basu and Saxena, 1978a) was employed for the collection of water samples in the
field. To facilitate air transportation, the components of the sampling equip-
ment were assembled inside suitcases. The overall unit consisted of the
following five parts: (a) variable water pumping unit consisting of a Masterflex
pump with appropriate pump heads and flow controller, (b) Haake Thermostated
Water Circulator, (c) unit containing the column systems, (d) thermometer, and
(e) flowmeter.
The variable pumping unit allowed water from the sample source to be pumped
to a column system (containing the foam plugs) at a controlled rate of 250 ฑ
10 ml/min. The water prior to reaching the column passes through the stainless
steel coil of a Haake heating bath and was maintained at 62 ฑ 2ฐC. The column
system consisted of two 25 mm i.d. Chromaflex extender-type columns connected in
20
-------
series with the help of tapered glass adaptors and double-bent pyrex tubing. The
temperature of the water passing through the columns was monitored by introducing
a water-trap equipped a thermometer. A Gilmont flowmeter calibrated to measure
the flow rate was placed at the end of the sampling unit. The connections were
made with glass tubing as much as possible and only where necessary with Tygon
tubing.
For convenience of transportation, the variable pumping unit with the flow
controller and pump heads was carried in one suitcase and the rest of the unit
containing the Haake Thermostated Water Circulator, the clamps for the column
system, water trap for the thermometer, and the flowmeter were all fixed in place
in another suitcase. The two suitcases were transported to the sampling site
along with an electric timer, 20 liter Jerrican and other detachable items such
as, extender columns, thermometers, clamps, "0" rings, and connecting tubes. The
purpose of the calibrated 20 liter Jerrican was to collect the effluent from the
sampling unit and thereby keep an exact record of the volume of water passed
through the columns. The timer was used to indicate the running time of the
sampler.
At the sampling location, the unit was assembled as shown in Figure 1.
Water from a 1 liter beaker placed under a continuously running water tap was
pumped through two foam columns under the previously described conditions. Since
the residence time for water in the beaker was very short, the losses of PAH due
to adsorption were assumed to be insignificant. Both foam columns were changed
after a certain volume of water (maximum 25 liters) passed through the columns
and the sampling was continued with fresh foam columns. In case of raw water,
21
-------
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13
-------
depending on the suspended solid content, this maximum volume varied from 10
liters to as high as 20 liters. After sampling the desired volume of water, the
columns were wrapped in aluminum foil, cooled in ice, and transported to the
laboratory. In the laboratory, a major part of the foam eluate obtained by
solvent extraction was used for PAH analysis and a fraction of the remaining
extract was used for determination of mutagenic activity.
In addition to collection of samples by concentration on polyurethane
foams, 2 gallons of unconcentrated water were also collected from each sampling
point for determination of mutagenic activity. Collapsable polyethylene bottles
of one gallon capacity were rinsed three times with the water to be collected
prior to filling the bottles with water. A small air space was left in each
bottle for expansion of water during cooling. The bottles were capped with
plastic screw caps, cooled in ice, and transported to the laboratory via air
freight.
Sample Collection Points at Each Water Supply
At each of the sampling locations, samples were collected at four different
points. These were raw water and finished water, both usually at the water
treatment plant, and two samples of finished water. The finished water was
sampled after passage through different lengths of distribution pipes. Where
possible the two sampling points were chosen at the furthest distance from the
treatment plant which would be consistent with obtaining water which had passed
through two different kinds of distribution pipes. These samples represented the
maximum exposure of the finished water to the two different pipes. In locations
where the distribution system consisted of only one kind of pipe, one sample was
23
-------
taken at the most distant practical point from the treatment plant and repre-
sented maximum exposure to the distribution pipe, while the other sample was
obtained from an intermediate point in the system. The sampling points are
diagrammatically shown in Figure 2.
The two finished water samples representing exposure to the distribution
lines were usually collected from water taps inside motels, private or public
buildings at approximately the desired distances from the treatment plant. To
avoid the collection of contaminants which resulted from leaching plumbing lines
in these buildings (as opposed to contaminants leaching from the distribution
line), running water from well flushed lines was used to obtain samples.
Table 3 describes the type of pipes the finished water passed through before
reaching the sampling points (sample 3 and sample 4 in Figure 2), the distance
the finished waters travelled through these pipes and the configuration of the
distribution system with respect to the treatment plant. It should be pointed
out that the distances given in Table 3 refer to the approximate distances
travelled by the waters through the pipes (the distribution lines were not always
straight and could be circuitous) and not the actual distance of the sampling
point from the treatment plant.
Elution of PAH from Foam Plugs
The PAH were eluted from the foam columns by washing each column with 30 ml
acetone and 125 ml cyclohexane. The details of the elution procedure are given
elsewhere (Saxena et al., 1977a). Several impurities belonging to foams were
also collected during the elution process. With the exception of trace amounts
of fluoranthene and benzo(ghi)perylene, other interfering impurities originating
24
-------
RAW
WATER
SOURCE
TREATMENT
PLANT
TYPE A DISTRIBUTION PIPES
SAMPLE 1
(RAW WATER
AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
SAMPLE 3
(MID-POINT
FINISHED WATER I
1
SAMPLE 4
(FARTHEST POINT
FINISHED WATER)
RAW
WATER
SOURCE
SAMPLE 3
(MID-POINT FINISHED WATER)
SAMPLE 1
(RAW WATER
AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
SAMPLE 4
(FARTHEST POINT
FINISHED WATER)
WATER SUPPLY WITH ONE TYPE(A) OF DISTRIBUTION PIPES
RAW
WATER
SOURCE
TREATMENT
PLANT
f l
SAMPLE 1
(RAW WATER
AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
SAMPLE 3
(FARTHEST POINT OF TYPE A PIPE)
SAMPLE 3
( FARTHEST POINT OF TYPE B PIPE )
RAW \
SOURCE 1
TREATMENT
PLANT
TYPE A
DISTRIBUTION PIPE
TYPE B
DISTRIBUTION PIPE
1
SAMPLE 1
(RAW WATER
AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
SAMPLE 3
(FARTHEST POINT
OF TYPE A PIPE)
1
SAMPLE 4
(FARTHEST POINT OF
TYPE B PIPE)
SAMPLE 3
(FARTHEST POINT
OF TYPE A PIPE)
WATER SUPPLY WITH MORE THAN ONE TYPE (A & B) DISTRIBUTION PIPES
Figure 2. Locations and configurations of sampling points
after the passage of potable waters through
distribution pipes
25
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from the foam plugs could be eliminated by precleaning plugs with benzene or
cyclohexane as detailed by Saxena _et al. (1977a).
PAH Separation and Analysis
PAH Separation
During the concentration of PAH from water, several other water contam-
inants were also concentrated on the foam plugs and some of them coeluted with
PAH during the elution step. These impurities interfered with the analysis of
PAH and a cleanup procedure was found necessary. The first cleanup step
consisted of the solvent partitioning procedure of Hoffman and Wynder (1960) as
modified by Acheson jet al. (1976). However, this procedure alone was inadequate,
and additional cleanup involving liquid chromatography through a short Florisil
column was found to be necessary to further separate the PAH from impurities.
Following cleanup, the extract was concentrated to 0.1 ml by a combination of
evaporation in a rotary evaporator and under a stream of nitrogen gas. The
details of the cleanup and concentration procedure are described elsewhere (Basu
and Saxena, 1978b; Saxena jง_t al_., 1977a).
Addition of Internal Standard for the Determination of Recovery Factor
The loss of PAH during the elution and purification procedure was monitored
14
for each analysis by the addition of an internal standard of C-benzo(a)pyrene
to the foam plugs prior to initiating sample workup. A known amount of radio-
labeled material (quantity usually less than 10 ng) was added to the foam plugs,
PAH eluted, purified and concentrated to 0.1 ml as described in the preceding
section. A 10 u.1 aliquot of the concentrate was assayed for radioactivity by
29
-------
liquid scintillation counting. The recovery factor was determined from the
amount of radioactivity initially added to the foam plugs.
PAH Analysis
The PAH quantified in the present study were fluoranthene, benzo(j)fluora-
nthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene and
benzo(ghi)perylene. The compounds selected can be regarded as representative of
the whole polynuclear family, and examination of their presence in drinking
waters has been recommended by -the World Health Organization (1971). PAH
analysis was performed using two techniques: gas chromatography-FID detection
and thin-layer chromatography-fluorescence detection.
Our earlier studies (Saxena et al., 1977a) demonstrated that a packed and
temperature-programmed Dexsil-300 GC column failed to provide adequate resolu-
tion for some of the PAH, resulting in erroneously high values. Therefore, with
the exception of the samples from the first six locations, all samples collected
after 8-1-80 were analyzed by SE-54 wall-coated glass capillary columns. The
details of the operating conditions for this packed column are given elsewhere
(Basu and Saxena, 1978b). The conditions used for the capillary GC analaysis are
given below:
Injection mode: Splitless
Vent delay: 40 seconds
Septum purge flow rate: 3 ml/min
Split vent flow rate: 60 ml/min
He carrier gas linear velocity: 25 cm/sec
Detector gases:
Np make-up gas flow rate: 51 ml/min
N gas flow rate: 39 ml/min
Air flow rate: 300 ml/min
30
-------
Column Programming:
Initial temp.: 160ฐC
Program rate : 8ฐC/min
Final temp. : 270ฐC
Final hold : 65 mins for 30 m column and 45 mins for
the 15 m column
Injection port temp.: 250ฐC
Detector temp. : 350ฐC
Nitrogen was used as the detector make-up gas to enhance the sensitivity of
the detector. The sample injection technique and the special nature of the
injection port septa used have been previously described (Basu and Saxena,
1978b).
Thin-layer chromatography was performed using a slight modification of the
plate coatings of Borneff and Kunte (1969). The plates were developed in two
dimensions. The details of the preparation of TLC plates and the method for
solvent development have been described by Saxena et al. (1977a).
The identification of PAH spots on the TLC plates were made by two methods.
The first method, an approximate one, was based on the R_ values of the spots.
D
The R_ values were defined as the ratio of distance travelled by a specific
D
compound to that of benzo(a)pyrene in the two solvent systems. Since R~ values
were found to be somewhat variable depending on the thickness of coating
material, solvent composition and other undetermined factor(s), the unequivocal
identification of a compound was made by the excitation and emission spectra of
the individual spot directly on the plate at room temperature with an Aminco
thin-film scanner attached to an Aminco-Kiers spectrophotofluorimeter.
For quantification of the spots, the signals of fluorescence emission
intensities of the scanned spot produced by the photomultiplier were transferred
31
-------
(as input) to a strip chart recorder (50 mv full scale deflection). The peaks
were manually integrated for area determination. The in situ scanning
eliminated the losses usually encountered during removal of PAH spots for
fluorescence measurement in solvents, and the interferences arising due to
solvent interactions. The details of the TLC identification and quantification
of the spots are reported elsewhere (Basu and Saxena, 1978b; Saxena jit al.,
1977a).
Assay of Mutagenic Activity
The bioassay employed in the present study utilizes the histidine-requiring
strains of the bacteria Salmonella typhimurium developed by Ames and coworkers
(1975). This In vitro bioassay is presently one of the most widely known and
frequently used test systems to detect the mutagenic effects of chemical
substances. The Ames test has the advantages of speed, ease of testing, low
cost, and the generation of relatively precise results. This assay can detect
point mutations of both the frameshift and base-pair substitution types. The
assay has the capacity to approximate metabolic activation of the test substances
by the incorporation of a microsome-containing subcellular preparation obtained
from rat liver (S-9 mix). A flow diagram of the bioassay procedures used during
the present studies is presented in Figure 3.
The assays were performed in both the presence and absence of a mammalian
metabolic activation (S-9) system. The following is a brief description of the
tester strains used in the present study:
32
-------
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TA1535 - Strain reverted by mutagnes which cause base-pair substitutions
TA1537 - Strain that is reverted by mutagens causing frame shift
suppression in G-C rich regions
TA1538 - Strain reverted by mutagens causing frame shift suppression as
small deletions in G-C base-pair regions
TA98 - Similar to TA1538 above, but possessing a pKM 101 plasmid
containing an ampicillin resistance gene and a "mutator" gene,
which increases sensitivity to mutagens
TA100 - Strain derived from TA1535 and like TA98, possesses a pKM 101
plasmid for ampicillin resistance, and a "mutator" gene
Methods for Routine Bioassays
Strains of S_. typhimurium are routinely cultured in our laboratory, and are
stored as frozen bacterial concentrates at -80ฐC. The following procedures
describe the methods employed for the routine handling and storage of the tester
strains, the preparation of the metabolic activation system, and the preparation
of minimal media/agar plates used in the assay.
Preparation of Cell SuspensionCell suspensions of the Salmonella tester
strains for the present study were prepared using a modification of the Ames
procedure. Instead of growing fresh cultures each time an assay was performed,
large batches of subcultured bacteria were prepared and stored frozen at -80ฐC.
Aliquots of these frozen cells were used directly in the bioassay after thawing
and making the appropriate dilutions. In this manner the cells from a single
subculture could be used for a number of bioassays performed on different days.
The bacteria were grown, harvested, and stored by the following methods.
A subculture of the tester strain was prepared by innoculating 100 ml
sterile nutrient broth (8 gm DIFCO Nutrient Broth, 15 ml Q.2% L-cysteine; in
-------
1000 ml distilled water) with bacteria from a frozen stock culture. The innocu-
lated broth was gently shaken in a water bath at 37ฐC for 16 to 18 hours, followed
by as^eptically harvesting the cells by centrifugation at 5500 rpm for 15
minutes. The spent broth was discarded and the remaining bacteria pellet was
resuspended in sterile nutrient broth supplemented with 2Q% sterile glycerine as
a cryo-protective agent. The cell density of the suspension was in the range of
5 to 9 x 10 cell/ml. The cell suspension was as^eptically transferred in 15 ml
aliquots into vials and stored at -80ฐC until used. This technique eliminated
the necessity for the growing of bacterial strains for each experiment, and also
eliminates problems of sparse "lawns" which may arise with the slower growing
Salmonella strains. This technique leads to an improvement in the
reproducibility of the Ames' assay, and saves time. This procedure yields
reproducible results even when aliquots of cells were used after two months of
storage at -80ฐC.
Preparation of the Mammalian Metabolic Activation System (S-9)Metabolic
activation in the bioassay was achieved by incorporation of the 9000 x g
supernatant of a liver homogenate from rats previously injected with a polychlor-
inated biphenyl (PCB) in order to induce microsomal enzyme activity. The
procedure as reported by Ames et al. (1975) for the preparation of the rat liver
homogenate, the S-9 fraction, and the S-9 cofactor mix was employed without
modification. The agent used for induction of the rat liver enzyme system was a
mixture of polychlorinated biphenyls (PCB) under the trade name Aroclor 1254.
Aroclor dissolved in corn oil (100 mg in 0.5 ml cornoil) was administered as a
single intraperitoneal injection at a dosage of 5 mg/kg. To minimize potential
35
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differences among individual animals, at least three rats were used in the
preparation of the S-9 mix. The rats were allowed 5 days for induction of liver
raicrosomal enzymes. Food and water were provided ad libitum for 4 days at which
time the rats were starved 24 hours prior to being sacraficed. The animals were
decapitated and their livers surgically removed under asceptic conditions. The
rat livers were weighed and minced in cold 0.15 M KCฃ (KC2/g liver = 3:1 by
weight). The minced liver tissue was homogenized using a Polytron tissue
ป ti
homogenizer followed by centrifuge^- of the homogenate at 9000 x g for 15 minutes
at 4ฐC. The supernatant (S-9) was asceptically removed and 7-0 ml aliquots
placed into sterile screw-top glass vials. Aliquots of the S-9 fraction were
frozen and stored at -80ฐC.
The complete S-9 mix (liver supernatant plus cofactors) was prepared by the
following procedure. All ingredients were asceptically combined to a final
volumn of 1 ml:
Rat liver supernatant (S-9) 100 ul
0.4 M MgCS-? 20 ul
1.65 M KC& 20 ul
1.0 M glucose-6-phosphate 5 u-1
1.0 M NADP"1" 40 ul
0.2 M sodium phosphate buffer,
pH = 7.4 500 p,l
Sterile distilled water 315 ul
1000 |il
To insure sterility of the final solution, the homogenate-cofactor mixture was
filter sterilized through a 0.2 urn membrane filter. Fresh S-9 mix was prepared
each day, and kept in an ice bath. Unused portions of S-9 mix were discarded at
the end of the day. Each batch of S-9 was checked for metabolic activation
36
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potential using diagnostic mutagens of known activity, and the S-9 was also
checked for possible bacterial contamination.
Preparation of Minimal Media Plates for Liquid Suspension and Plate
Incorporation AssaysA concentrated (50x) minimal salts solution (Vogel-Bonner
minimal salt concentrate) was prepared by adding the following ingredients in
sequence and stirring with a magnetic stirring apparatus until dissolved:
670 ml distilled water
5 gm MgSOjj (anhydrous)
100 gm citric acid (monohydrate)
500 gm potassium phosphate (dihydrous, anhydrous)
170 gm sodium ammonia phosphate ซ4 M
This solution was not autoclaved, and could be stored at room temperature for
long periods of time.
Minimal glucose agar was prepared by adding in a 2 liter Florence flask:
20 ml 50x Minimal salts concentrate
980 ml distilled water
15 gm DIFCO Bacto-agar if plates were desired
r
I/ The contents of the flask were autoclaved at 250ฐJS and 15 psi for 15 minutes.
After removal from the autoclave, 5 ml of sterile 40$ glucose was asceptically
added to the flask. If plates were desired, 25-28 ml of media were asceptically
transferred to 100 x 15 mm plastic petri dishes (Lab Tex, VWR Scientific
Supplies). Minimal media plates were used for the liquid suspension assays, and
for the "1 ml" dose in the plate incorporation assays.
Preparation of Media/Water PlatesThe following method was used for
preparing Media/Water plates in order to enhance the possibility of detecting low
levels of mutagens in unconcentrated water samples. In a 600 ml prescription
37
-------
bottle, 30 gm DIFCO Bacto-agar was added to 550 ml distilled water.
Simultaneously, 40 ml of 50x minimal salts concentrate was added to a. two liter
Florence flask. For those flasks for which half-strength media was required,
700 ml distilled water was added to the appropriate flask. Both the agar and the
/ \,
salts or salts/water flasks were autoclaved at 250ฐC and 15 psi for 15 minutes.
After autoclaving, 5 ml of sterile 40? glucose was added to each of the flasks.
For those flasks used to prepare plates with the maximum amount of test water,
1400 ml of filter-sterilized test water was added. For the half-strength flasks,
700 ml filter-sterilized test water was added to the appropriate flask. The
contents of all flasks were brought to 50 ฐC in a water bath. The molten sterile
agar was added and mixed in each flask. Into 15 x 100 mm plastic petri dishes,
25 to 28 ml of the media/water was asceptically poured. The final amount of test
water in the full-strength plates was approximately 25 to 28 ml per plate and
12.5 ml per plate in the half-strength plates. These media/water plates were
used to assay unconcentrated water samples in the plate incorporation assays.
Preparation of Minimal Top Agar and Minimal-Nutrient Broth A minimal top
agar media was made by adding to a 2 liter Florence flask, the following
ingredients:
20 ml 50x minimal salts concentrate
5 gm NaCl
75 gm DIFCO Bacto-Agar
/ 980 ml distilled water
5
The contents of the flask were autoclaved at 250 ฐp and 15 psi for 15 minutes. The
top agar was stored in sterile prescription bottles, in volumes of 100 ml, at
room temperature until used. Before using the agar was remelted by heating in
38
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boiling water, and 10 ml of a sterile 0.5 mM L-histidine and 0.5 ml biotin stock
solution was added to each bottle. The contents of the bottle were gently mixed
by swirling. The trace amount of histidine incorporated into the top agar will
allow the bacteria, to undergo several divisions, and is necessary for
rautagenesis to be observed.
A minimal-nutrient broth (Vogel-Bonner solution supplemented with glucose)
was used as a pre-incubation medium for the liquid suspension assays. These
assays required a one hour preincubation of bacteria, test organic concentrate,
and S-9 mix where appropriate, before plating onto minimal-media plates. The
broth was prepared by adding 20 ml of the 50x minimal salts concentrate to 980 ml
distilled water and sterilizing this solution in an autoclave at 250ฐF and 15 psi
for 15 minutes. After sterilization, 5 ml of a sterile Wf> glucose solution was
asceptically added. For use in the assay 1.0 ml of the sterile minimal-media
broth was asceptically dispensed into each sterile culture tube before bacteria,
mutagen, or S-9 mix were added.
Preparation of Water Concentrates for Ames TestingOrganic concentrates of
water were received from the analytical laboratory for mutagenicity testing in
20 ml of cyclohexane. The water concentrates represented 10 liters of water from
the raw-water source or 15 liters of water from the treated and distribution
sources. Aliquots of each cyclohexane concentrate were transferred to separate
tubes. The cyclohexane was evaported with nitrogen until dry. Upon complete
drying the residue was resuspended in sterile DMSO to provide a stock solution
from which subsequent dilutions were made. The stock solutions provided the "H"
(high) dose solution for each sample location. For the raw water samples the "H"
39
-------
dose represented 150 ml water per 0.1 ml of the final DMSO solution. Subsequent
raw water dilutions were: "X" (intermediate) dose, 100 ml water per 0.1 ml DMSO,
"M" (medium) dose, 50 ml water per 0.1 ml DMSO, and "L" (low) dose, 25 ml water
per 0.1 ml DMSO. For the treated and both of the distribution water samples the
"H" dose represented 300 ml per 0.1 ml DMSO solution. Subsequent dilutions for
each sample were "X" dose, 200 ml water, "M" dose, 100 ml water, or "L" dose,
50 ml water per 0.1 ml DMSO solution. For each concentration assayed by liquid
suspension 0.1 ml of the appropriate DMSO water concentrate solution was used.
Quality ControlsThe performance of the mutagenicity assays was routinely
examined using diagnostic mutagens as positive controls. Diagnostic test
substances of known mutagenic potential and not requiring prior metabolic
activation, included:
TA98 4-nitro-o-phenylenediamine
TA100 methylmethane sulfonate
TA1535 methylmethane sulfonate
TA1537 9-aminoacridine
TA1538 4-nitro-o-phenylenediamine
The performance of the mutagenicity assays involving metabolic activation was
verified by the use of 2-aminoanthracene as a diagnostic positive control mutagen
for all tester strains in the presence of the S-9 metabolic mix.
Confirming the tester strains' genotypes for the presence of the R-factor
conferring ampicillin resistance in strains TA98 and TA100 was routinely
performed. Amplicillin-containing filter-paper discs containing 10 p.g of
ampicillin were placed in the center of nutrient agar petri dishes overlaid with
each of the desired tester strains. Zones of bacterial growth inhibition after
40
-------
24 hours incubation at 37ฐC were observed, as expected, with tester strains
TA1535, TA1537, and TA1538, but not with strains TA98 or TA100.
To confirm the presence of the tester strains' deep rough mutation (fra),
which permits large molecules to enter the bacteria and inhibit growth, a crystal
violet test was carried out in conjunction with the ampicillin resistance test.
A sterile filter paper disc containing 10 ng crystal violet (10 ul of a 1 mg/ml
solution) is placed onto nutrient-agar plates containing bacteria. After 12 to
24 hours incubation at 37ฐC a clear zone of growth inhibition around the disc
indicates the presence of the deep rough mutation.
Experimental Procedures
Two variations of the standard plate incorporation assay were used to assess
the mutagenic potential of drinking waters. One such procedure was designated as
the "Media water assay," and employed unconcentrated, filter-sterilized drinking
water samples. The second procedure was designated the "liquid suspension
assay," and employed organic concentrates of the drinking water samples. Details
of the two experimental procedures are provided in the following sections.
Media Water Plate Incorporation AssayTo facilitate the detection of low
concentrations of mutagens in unconcentrated drinking water samples, the
bioassay procedure was modified by incorporating filter-sterilized test water
into the base agar. Two different amounts of test water in the media were
obtained by dilution of the test water with an appropriate volume of distilled
water prior to the preparation of the media.
For the actual plating procedure sterile 100 x 13 mm glass tissue culture
tubes containing 3 ml of the top agar and 0.5 mM histidine/0.5 mM biotin solution
41
-------
were supplemented with 0.1 ml of the appropriate bacterial suspension and 0.5 ml
of the S-9 mix (where appropriate). The contents of each tube were overlaid onto
the base agar layer. Suspect mutagens would therefore be present in the base
layer, and test organisms and the S-9 mix would be present in the top agar. This
procedure allowed for incorporation of approximately 25 or 12.5 ml of drinking
water per plate (70% of the total volume for the highest concentration).
A third level of exposure was included in the protocol by adding 1.0 ml of
filter-sterilized drinking water into the agar-histidine/biotin top agar
solution, before supplementation with the appropriate bacterial tester strain
and S-9 mix (where required). This "1 ml" exposure was plated onto standard
minimal-media plates in a manner similar to a plate incorporation assay. Assays
on all the water samples collected from one location were performed at the same
time along with distilled water controls for spontaneous reversion rate and
positive mutagenesis controls. Therefore, three concentrations of 25, 12.5 and
1 ml per assay-plate were used to obtain dose response information.
All assays were performed in quintuplicate. Plates were incubated at 37ฐC
in a Sherer Lab incubator for 96 hours before enumerating the colonies on the
Biotran II automatic colony counter. The results were recorded as number of
revertants counted per plate, and expressed as means (+ standard deviation) of
the quintuplicate determinations. Also, the ratio of experimental ^control was
calculated.
In previous laboratory studies conducted at SRC (Neal et al., 1980) using
known mutagens, it was demonstrated that the same mutagens added to the bottom
agar were detected with lower efficiency than those added to the top agar. For
42
-------
example, benzo(a)pyrene, an extremely hydrophobia molecule, was detected in the
media water assay with 10 times less sensitivity compared to the procedure in
which the mutagen was added to the top agar; however, mutagens which were more
hydrophilic were detected with high efficiency. Thus, although some sensitivity
is lost in detecting very hydrophobia mutagens, the ability to add a larger
quanity of sample justifies the use of this procedure.
Liquid Suspension Assay of Drinking Water ConcentratesThis assay involved
incubation of an organic concentrate of water and bacterial tester strains in
liquid suspension prior to plating on minimal-media. Liquid suspension assays
are particularly valuable with assays that involve complex uncharacterized
mixtures, and where the possibility exists that some of the compounds present, or
a metabolically activated form of the compounds present, may be inactivated by
the agar. All of the positive features of the plate incorporation assay are
retained by the liquid suspension assay, therefore this assay can detect any
compound which is positive in the conventional plate incorporation assay.
DeSerres and Shelby (1979) have recommended the use of the liquid suspension
assay where the results of the standard plate incorporation assay are incon-
clusive .
The liquid suspension assay was used in assessing the mutagenic potential of
drinking water concentrates. The pre-incubation medium for this assay contained
1 ml of minimal media (minus agar) broth, 0.5 ml rat-liver homogenate plus
cofactors (S-9 mix) where required for metabolic activation, 0.1 ml of the
appropriate bacterial tester strain, 4 u-g histidine, 6 ug biotin, and the
designated amounts of the test drinking water concentrate. Identical vehicle
43
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control tubes were made with 0.1 ml of DMSO added. Following a 60 minute
incubation of the mixture at 37ฐC in a water bath, 3 ml of soft agar containing
24 |ig histidine and 36 ug biotin per ml was added to each tube. The contents of
each tube were mixed and overlaid onto minimal glucose agar plates. The plates
were inverted and incubated at 37ฐC for 96 hours. The colonies were counted and
the results expressed as the ratio of the number of revertants colonies per
plates receiving exposure to test waters over the number of spontaneous
revertants on control plates.
Intrepretation of the ResultsIn the plate incorporation assays and the
liquid suspension assays with test water concentrates, a ratio of revertant
colonies (experimental/spontaneous control) of 1.5 to 2.0 was considered to be a
weak mutagenic response, and a ratio of > 2.0 was considered as a positive
mutagenic response. Particularly in samples with low activity, the presence of a
positive dose-response relationship was used to assist in the determination of
whether the sample was clearly mutagenic.
-------
SECTION 6
RESULTS AND DISCUSSION
The leaching of PAH from coal tar-coated potable water storage tanks and
water distribution pipes has been demonstrated by a few investigators (Borneff,
1976; McClanahan, 1979; Roebeck, 1979; Alben, 1980a). However, only six percent
of the total installed potable water distribution pipes in the U.S. consist of
steel pipes that are typically lined with coal tar-based coating materials
(Goldfarb, 1979). Instead, the majority (75%) of water distribution pipes are
made of cast/ductible iron (Goldfarb, 1979). Unlined and cement mortar-lined
cast/ductile iron pipes are typically coated with petroleum asphalt paints,
instead of coal tar, for corrosion inhibiton and as a curing aid for the pipe
linings (Goldfarb, 1979). That these asphalt-based paints contain PAH can be
inferred from the investigation of Radding et al., 1975 (see Section 4). The
leachability of PAH in potable waters travelling through distribution pipes
coated with asphalt-based paints, however, is not well studied. The primary
objective of the present investigation was to examine both the change in PAH
concentration and the pattern of mutagenic activity in treated waters as a result
of their passage through commonly used distribution/transmission pipes.
The sampling locations selected for the study represent the largest water
supplies within 24 selected counties in the U.S. These water supplies serve
areas that represent both high and low overall cancer incidence rates as
established by U.S. EPA epidomiological study (Kopfler, 1977). To avoid bias in
the analysis, however, the relative cancer mortality rates among these areas
45
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remained unknown to us. As shown in Table 3, the samples collected from these
water supplies represent contact with a variety of distribution pipes. The
majority of the distribution lines in these locations consist of unlined or
cement-lined cast/ductile iron pipes. A small number of samples were collected
from concrete, asbestos/cement, cement-lined steel, plastic and copper pipes, or
a combination of these. The exact sampling points for each of the water supplies
tested are shown in Table 3 and Figure 2. The rationale for selecting the
sampling points is given in Section 5.
The presence of a wide variety of PAH, including several animal carcinogens,
in environmental samples is not uncommon. For example, in one study (Kunte,
1979), more than 200 different PAH were detected in an ambient air sample.
Because it is difficult and generally unnecessary to quantify all PAH in a
complex environmental sample, it seems practical to choose a few specific and
representative compounds for water monitoring purposes. The six PAH analyzed in
this study included fluoranthene (PL); benzo(j)fluoranthene (BjF); benzo(k)-
fluoranthene (BkF); benzo(a)pyrene (BaP); indeno(1,2,3-Gd)pyrene (IP); and
benzo(ghi)perylene (BghiP). The World Health Organization (WHO) recommends,
with the exception of benzo(j)fluoranthene, the analysis of these PAH in drinking
water. Benzo(b)fluoranthene, which is recommended for analysis by WHO, was
replaced with its isomer benzo(j)fluoranthene in our analysis because benzo(b)-
fluoranthene was not available to us as a reference standard. This group of six
PAH is regarded by WHO as a reliable indicator of the entire polycyclic family
(WHO, 1971). Except for the ease of fluorescence detection and their frequent
occurrence in environmental samples, the reason for selecting these six
46
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compounds by WHO is further explained (Borneff and Kunte, 1979). In the absence
of other guidelines, we decided to analyze the above six PAH for the following
additional reasons: the six PAH represent compounds varying from strongly
carcinogenic (benzo(a)pyrene) to non-carcinogenic (fluoranthene) (NAS, 1972),
and include compounds that are hard-to-separate; i.e., the isomers benzo(j)-
fluoranthene and benzo(k)fluoranthene. The ability of the analytical method to
separate carcinogenic PAH (for example, benzo(a)pyrene) from its nonmutagenic
isomer (for example, benzo(e)pyrene) can be tested by including the last two
isomers, which are difficult to separate.
PAH represent only one class of biologically active compounds which may be
either deposited in raw water or leached from distribution pipes. In order to
detect the presence of other biologically active compounds of importance to water
quality, an in vitro mutageniciity assay was performed on samples from each
collection site. This assay was performed on the same organic concentrate used
for PAH analyses, and also on the unconcentrated water. The use of unconcen-
trated water allows for detection of biologically active compounds that are not
absorbed by polyurethane foam plugs, as well as compounds which are lost during
the extraction and preliminary chemical clean up phases. The experimental
bioassay data, expressed as revertants per plate, for each sampling site are
tabulated in Appendix A.
The PAH in water samples taken from the different locations were collected
by a polyurethane foam retention method. In a previous study (Basu and Saxena,
1978b), the retention efficiency of polyurethane foams for six PAH in finished
waters was reported to be nearly 90$. Using the foam concentrative technique
-------
described in Section 5, the investigators collected water samples from 24 potable
supplies. Water samples from 5 of the 24 supplies were resampled for the
following reasons: the water samples collected during the first trip to Fremont,
OH; Elkhart, IN; and Fairborn, OH were not analyzed for mutagenic activity, and
the original samples collected from Champaign, IL, and Ft. Myers, FL showed
elevated levels of mutagenic activity. Therefore, water from these five supplies
were resampled primarily for the analysis or confirmation of their mutagenic
activity.
RESULTS OF CHEMICAL ANALYSIS FOR PAH
The results of PAH analyses are given in Table 4. The values given for
fluoranthene and benzo(ghi)perylene in Table 4 have been corrected for the trace
amounts of these PAH contributed by the foam plugs from each lot. The details of
these corrections are given elsewhere (Saxena et al., 1977). Benzo(a)pyrene
values have been corrected for the amount of radiolabelled compound added as an
internal standard. Correction has not been made for the PAH retention efficiency
of the foams, which was assumed to be quantitative. Because the retention
efficiency of PAH from foam plugs will always be lower than 100?, the actual
amount of PAH in waters should be slightly higher than the values shown in the
table. The loss of PAH during solvent extraction of the foams and the subsequent
chemical clean up was determined by the addition of radiolabelled benzo(a)pyrene
as an internal standard. The amount of radiolabelled benzo(a)pyrene added to
each of the samples was such that it provided statistically significant counts
over the background and yet did not interfere with the analysis of low level
benzo(a)pyrene.
48
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The values of the six PAH determined were corrected using the recovery
factor from radiolabelled benzo(a)pyrene. It was assumed that the recovery of
all six PAH was the same as the recovery of the benzo(a)pyrene. The average
recovery of PAH for all finished water was slightly higher (68?) than the average
recovery for the raw waters (63?). This lower recovery in the case of raw waters
is understandable because in some instances the impurities in raw waters, pro-
duced emulsions during the solvent extraction steps.
The final concentrations of the six PAH presented in Table H are based
primarily on TLC-spectrofluorometric values. The GC-FID method in the vast
majority of cases failed to detect PAH because less than 5 u& of the final
concentrate (less than 556 of the total) could be used for quantification, and
because the method has a lower sensitivity than the fluorometric method. In
cases where both values could be obtained, the GC results served as a crosscheck
for the TLC values. In a limited number of cases, the TLC method did not provide
concentration values for one or two PAH compounds in the samples because of
incomplete separation from impurity spot(s). Some of these compounds were
separated and quantified by the capillary GC-FID method. The separability and
sensitivity of the capillary GC-FID method was superior for the early eluting
peaks, such as fluoranthene, benzo(j)fluoranthene and benzo(k)fluoranthene. As
found by other authors (Bjrirseth, 1979), benzo(j)fluoranthene and benzo(k)f-
luoranthene were separated by an SE-5^ capillary column.
The detection limit for the six PAH by both TLC-fluorometric detection
(Saxena et al., 1977) and capillary GC-FID detection is given in Table 5. The
capillary GC-FID detection limit is based on a minimum output response of four
59
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Table 5. Detection Limit for Six PAH by Thin Layer and
Capillary Gas Chromatographic Methods
TLC-fluorometric
PAH
FL
BjF
BkF
BaP
IP
BghiP
Absolute
limit, ng
14.0
7.5
5.0
10.0
10.0
20.0
Limit in 60 1
water (ppt)
2.3
0.1
0.2
0.2
0.2
0.3
Capillary GC-FID
Absolute
limit, ng
6.2
8.3a
10.3
Limit in
water
3.5
6.9a
8.6
60 1
(ppt)
compound not separated by GC
60
-------
times the background values obtained at an output attenuation of 1 x 6H and a
maximum sample loading volume of 2 u& from a total of TOO uA concentrate. The
detection limits for indeno(1,2,3-cd)pyrene and benzo(ghi)perylene by the
GC-method are not shown in Table 5 because neither of these compounds were
detected by the capillary GC-FID method in any of the samples. Nevertheless the
detection limits for these two compounds by the capillary GC method were higher
than those determined by the packed column method. With the slower eluting
peaks, similar results were obtained with other capillary columns by Lao et al.
(1973).
If PAH contaminants are leached from the pipes or their linings, one would
expect enhancement of PAH levels as the treated waters travel through these
pipes. Also, in a distribution system served by one kind of pipe, an enhancement
of PAH level in waters at an intermediate point of the distribution system should
signal a further enhancement of PAH concentration in water at the most distant
point of the distribution system. In general, an increase in PAH level was
observed in waters after their passage through cast/ductile iron and cement-
coated cast/ductile iron pipes. Of the 24 sites monitored for the possibility of
enhancement of PAH concentation by the distribution system, 67% showed increases
in PAH concentrations in waters travelling through at least one sample collection
point in the distribution lines. The remainder of the water supplies showed
reductions in PAH concentrations.
A correlation between the PAH level in water and the distances these waters
passed through the distribution lines was also found. An increase in the
distance usually enhanced the PAH levels in water. Because petroleum asphalt-
61
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based paints and sealants are employed with cement-coated and uncoated
oast/ductile iron pipes, this enhancement of PAH levels may be attributable to
the increased leaching of the pipe linings associated with increased contact time
inside the pipes. The amount of PAH leached into water, however, was small and
found to produce incremental increases in the part per trillion range.
The amount of PAH leached was also found to be related to the age of the
installed pipes. The maximum incremental difference in PAH concentrations of
53.4 ppt, 61.2 ppt, and 42.2 ppt were detected in waters from Fremont, OH;
Elkhart, IN; and Tascaloosa, AL respectively. The Fremont, OH water was
collected from a recently installed cement-coated cast/ductile iron section of
the distribution system. The cast/ductile iron pipes in Tascaloosa, AL were
coated with asphalt-based paints and probably were installed recently. Although
the age of the pipes in Elkhart, IN was not known, the substantive increase in
PAH concentration in water may indicate that a major new section of pipe in the
distribution system had been recently installed. It is interesting to note that
in all three locations, the increase in the concentration of the more water
soluble PAH (Santodonato et al., 1979) accounted exclusively for the substantial
increase in the total PAH concentration in the water. Distribution pipes coated
with PAH-containing materials tend to leach out the more water soluble PAH, such
as flouranthene, at the earlier stages of their use. As the pipes become older,
their surfaces become depleted of the more water soluble PAH and they leach out
less soluble PAH, such as benzo(a)pyrene and benzo(ghi)perylene, in higher
proportions. This age dependence of PAH leaching was also observed by Alben
(1980a) in studies on a coal tar-coated storage tank. Therefore, an increase in
62
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the concentrations of the more water soluble PAH in waters from distribution
lines provided a further indication that they were recently installed.
The small amount of PAH detected in the water after passage through
cast/ductile iron pipes is not surprising. In a recent investigation, Miller
et al. (1981) measured the concentration of the six PAH in water by circulating
PAH-free water up to 232 hours through 34' x 6" petroleum asphalt-coated ductile
iron pipes. The total PAH concentration in the water after passage through the
pipes did not exceed 10 ppt. The concentrations of the six PAH measured in the
leached waters from the present investigation were of the same order of magnitude
as those determined by Miller et al. (1981).
In certain instances, particularly in waters from Fond Du Lac, WI; Austin,
TX; and Wheeling, WV, marked reductions of as much as 98$ were observed in PAH
concentrations in the waters after distribution. This reduction in PAH concen-
tration cannot be correlated with any specific kind of distribution pipe. While
the distribution pipes in Wheeling, WV consisted of a combination of copper, cast
iron, cement-coated ductile iron and asbestos/cement, the distribution pipes in
Fond Du Lac, WI and Austin, TX consisted of cast iron and cement mortar lined
steel pipes, respectively. A possible explanation for the reduction in PAH
concentration lies in the age of the distribution pipes. Cast iron and cement-
coated cast/ductile iron and steel pipes may be coated with asphalt-based paints
and sealants. Up to a certain age, the asphalt coating might continuously leach
slowly decreasing amounts of PAH in the travelling waters. When the surface of
the pipes becomes depleted of PAH leachables, the bare metal and cement surfaces
may act as a sorbent of PAH as water containing PAH travels through them.
63
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The quality of water as a result of its passage through two other non-
metallic pipes, for example, asbestos/cement (Wichita Falls, TX; Hutchinson, KS;
and Fayetteville, NC) and reinforced concrete (Olympia, WA) , was also examined
during this investigation. As expected, with pipes not coated with PAH leachable
materials the water passing through these distribution lines showed no change in
PAH concentrations other than fluctuation in the concentration of benzo(ghi)-
perylene.
Water quality parameters, such as the pH, hardness, and alkalinity of the
water may also affect the leachability of PAH from the pipe surfaces. It was
shown by McFarren et al. (1977) that the aggressive index of travelling water is
important in determining the leachability of asbestos fibers from the
asbestos/cement pipes. The laboratory studies of a few investigators (Alben,
1980b; Oyler et al., 1978) suggest the possibility of interaction of residual
chlorine in potable waters with the compounds leached from the pipe surfaces.
Chlorinated and other oxidized products were identified as evidence of these
interactions. Because no dramatic leachings of PAH were observed from the
distribution pipes in the present investigation, no attempt was made to study the
effect of water quality parameters on the leachability of PAH from the distribu-
tion pipes.
No data regarding the precision and accuracy of the results can be given
because a single analysis for each sample was performed. The data presented in
Table 4, however, were obtained under the following quality assurance condi-
tions.
64
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(1) The retention efficiencies of the 6 PAH by polyurethane foams were
determined with PAH-spiked water every time a new batch of foams was purchased.
The retention efficiencies were found to exceed 80%.
(2) The recovery of PAH in each sample resulting from foam desorption and
chemical cleanup was determined from the recovery of radiolabelled benzo(a)-
pyrene that was added as an internal standard.
(3) We analyzed external quality control samples received from U.S. EPA,
and the results of these analyses indicated that our quantification methods were
satisfactory.
(4) Whenever there was more than a 10$ variation in the sensitivity of
fluorometric detection (due to change in lamp intensity or photocell response) as
determined by a standard quinine sulfate spot, fresh fluorometric standard
curves were made and used for the determination of unknown sample concentrations.
(5) For samples quantified by the GC-FID method, at least one standard
mixture was injected into the GC following sample injection to ascertain any
changes in relative retention times and sensitivity of detection.
(6) A fresh solvent and reagent blank was run with the samples whenever a
new lot of solvent and/or chemicals was used.
It is evident from Table 4 that with the exception of treated water from
Austin, TX, more than 90% removal of PAH was accomplished by the treatment
processes in the water supplies when the concentration of total PAH in raw water
exceeded 10 ppt. The efficiency of PAH removal by the treatment processes was
less significant when the level of total PAH in raw water was less than 10 ppt.
In fact, in certain cases as in treated water from Augusta; GA, Ft. Myers, FL;
65
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Eau Claire, WI; Fond Du Lac, WI; Hutchinson, KS; Olyrapia, WA; and San Luis
Obispo; CA, the PAH level might have increased as a result of treatment. The
increase may be due to the presence of PAH impurities in treatment chemicals or
due to leaching of the pipes employed to transport the treated water to the tap
(where they were sampled), or both.
We studied the possibility of variations in PAH concentration of surface
waters in samples collected from Fremont, OH and Ft. Myers, FL at two different
times. The raw surface water collected from Fremont, OH during the spring of
1981 showed a large increase of PAH concentration compared to the sample
collected from the same location in the summer of 1977. The raw surface water
collected from Ft. Myers, FL, however, showed no change in PAH concentration.
The observed concentration variation in the Fremont, OH raw water sample might
have been due to seasonal variation of PAH concentration, but this possibility
was not further explored. In the absence of home heating during the winter
months, which represents the largest source of PAH contamination in the environ-
ment (NAS, 1972), the surface water from Ft. Myers would not be expected to show
any seasonal variation in PAH concentration.
In addition to the six PAH identified and quantified by the TLC-fluorometric
and GC-FID methods, a number of unidentified compounds were detected by both
methods. For example, in Wheeling, WV raw water samples, as many as 13
luminescent spots remained unidentified on the TLC plate. Some of the
unidentified spots on the TLC plates showed characteristic absorption and
emission spectra. One such compound with a characteristic emission spectra that
recurred in many water samples is shown in Figure 4. Similarly, the capillary
66
-------
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67
-------
GC-FID method showed the presence of several unidentified compounds in the
chromatograms of many of the water samples. The capillary GC-FID chromatogram
obtained from Elkhark, IN water samples collected from the four sampling points
illustrate the complexity of these chromatograms (Figure 5). These chromato-
grams also qualitatively demonstrate the changes in the water quality as the raw
water is treated to finished water and is finally passed through the distribution
pipes. No attempt was made, however, to identify the unidentified compounds.
RESULTS OF MUTAGENICITY ASSAYS
In most cases, the raw water and finished water (before and after distribu-
tion) 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 contributes to the presence of
rautagens or, on the other hand, whether some pipes adsorb mutagens which had not
previously been removed during treatment. Of the 2U water supplies tested, three
showed no detectable mutagenic activity in either the raw water or water taken
from any of the collection points (Table 6). Six 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 indicates that these values fall within the normal
variation of the Ames assay. This interpretation is more difficult, however, in
the cases of Fayetteville, NC and Tascaloosa, AL a ratio of greater than 1.5 was
68
-------
131
Figure 5. Gas chromatograms of Elkhart, IN samples collected
at the four sampling points
69
-------
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70
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observed at the highest concentration tested, since this could indicate that a
higher dose level would produce a clearly positive response.
The lack of observed rautagenic response in most samples probably results
from the absence of sufficient quantities of mutagens in the sample. There is
also the possibility that some of these samples contain mutagenic compounds and
that the biological activity of these compounds was masked by the presence of
toxic substances in the sample. The interference by toxic materials with the
performance of the Ames assay has been reported by Heartlein et al. (1981) in a
study of the mutagenicity of drinking water in agricultural areas. 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 encountered 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 sample from
Fremont, Ohio where the 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. In the
study of Hearthein et al. (1981) the addition of S-9 mix decreased the toxicity
of the sample to the -feafe- bacteria, however, in the present study the presence of
S-9 mix decreased toxicity in certain cases while in other cases it increased
toxicity. It is apparent from the effect of metabolic activation on bacterial
71
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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 water samples that demonstrated no
mutagenic activity are shown in Table 6. The pipes listed in Table 6 represent
all the various types encountered in this study, ranging from the cast iron to
asbestos/cement. Although many variables are unknown about the pipes such as
age, condition, and the physical properties (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 weak mutagenic response or a mutagenic response in at least one
of the four strains of bacteria tested. For a response to be considered weakly
mutagenic in this study we require that the reversion ratio fall between 1.5 and
2.0 and be accompanied by a positive dose-response trend. A clear mutagenic
response in this study was considered to occur when the reversion ratio 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 demonstrating clear mutagenic potential in this study, the response was
not great, with most ratios being only slightly greater than 2.0.
72
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As a result of the low level of activity, even in samples designated as
showing a positive mutagenic response, it was not possible to have precise
guidelines to interpret the effects of water treatment and type of distribution
system on mutagenic activity. Since the interpretation of these mutagenicity
data required a higher degree of subjective evaluation than for most Ames assay
data, three individuals in our group independently interpreted the data, as
presented in Appendix A. Where differences in interpretation exist a consensus
was obtained and this consensus is presented in Table 7.
The interpretation of these data was based on weight of evidence approach.
A positive mutagenic response was considered more credible if a dose-response
relationship could be demonstrated even when individual values which comprised
this dose-response relationship could be demonstrated even when individual
values which comprised this dose-response were below a ratio of 1.5. The
metabolic activation (S-9 mix) was considered when comparing mutagenic response
among different points in the sampling scheme; e.g., mutagenicity was considered
to be enhanced if distribution point I showed activity in the presence and
absence of S-9 mix, while the finished water showed activity only in the absence
of S-9 mix. It was necessary to do this in order to account for the appearance of
different classes of mutagens in the raw, finished, and distribution system
water. In the same manner, the information from the two types of assays (liquid
suspension and plate incorporation) and all bacterial tester strains was used to
arrive at a final interpretation with the realization that each tester strain is
sensitive to different types of compounds and that each of the two assay systems
might actually be evaluating both qualitatively and quantitatively different
73
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chemical mixtures from the same sampling point. This information was combined to
indicate the change in mutagenic activity in the raw water and water from
distribution points I and II as compared with the mutagenic activity of the
finished water.
The summary of the data in Tables 6 and 7 indicates in general that the
distribution system neither adds nor substracts from the mutagenicity present in
the finished water, in any consistent manner. The types of pipes used in these
water systems included cast/ductile iron, cement-lined steel and asbestos/cement
pipes. The water systems which indicated either an increase or decrease in
mutagenic activity during distribution represented all the various types of
pipes. Thus, the data suggest that, although in some cases the mutagenic
activity of finished water may be affected by the distribution system, this
effect cannot be simply be attributed solely to the type of pipe used to trans-
port the water.
A further indication of the complexity of the interaction of compounds in
drinking water with the distribution pipes is demonstrated in the two water
supplies which were tested twice. The sample obtained from the first trip to
Champaign was only tested in a limited manner for mutagenic activity, with only a
single concentration of the organic concentrate tested. This limited assay
produced over a five fold increase in the reversion rate for TA98 in the presence
of metabolic activation at the two distribution points. This result contrasts
with the finding of no mutagenic activity in this strain with samples of raw
water and finished water. Upon collecting and assaying a new water sample from
Champaign the total mutagenic response was considerably lower, and this low level
75
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of activity was observed in samples from all collection points. In testing two
independent samples from Ft. Myers similar results were obtained. The first
samples showed a decrease in mutagenic activity in water obtained from the
distribution system while the second indicated that the distribution system had
no effect on mutagenic activity. Since the distribution lines had not changed
during the interval between the sampling trips, other variables must account for
the observed differences.
Some of the variables which may affect the mutagenic activity in drinking
water, and the interaction of this activity with the distribution pipes include
variation in raw water quality, treatment processes in the prepartion of finished
water, and differences in the microbiological population in the pipes. The first
two variables can cause qualitative changes in the trace organic content of the
finished water. If these changes generate different mutagens or promutagens it
might not be unexpected that different types of interaction with the distribution
system may occur. Also, changes in the raw water quality and treatment processes
may alter the pH of the water, the chlorine content, and the amount of disolved
and suspended solids. Any of these changes could affect the ability of the water
to leach material from the distribution pipe, or may affect the biological
activity of leached compounds. Finally, Alexander (197*0 has reported the
synthesis of mutagens by microbiological action, and if this is occurring in the
distribution lines then changes in the microbiological population will cause
changes in the patterns of mutagenicity.
It is difficult from the results of the present study to speculate on
whether any of the above processes could account for the different results
76
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obtained from the two water samples taken from Champaign and Fort Myers.
However, a recent report by Clark and Johnston (1981) describes an extensive
analysis of the mutagenic activity in the drinking water of Champaign. In this
study not only was marked seasonal variation was observed. A correlation was
made between mutagenic activity and the alternating use of choramine and break-
point chlorination in the treatment processes. In a similar manner those changes
in the treatment processes could account for both the quantitative differences in
mutagenic activity and the differences in the patterns of mutagenic activity
observed by us in the two sampling trips to Champaign. There is no information
on what caused the differences in mutagenicity in the Ft. Myers samples except
that during the second trip there was a severe drought condition which resulted
in poor water quality as measured by total organic carbon levels.
PAH, many of which are mutagenic (Santodonato et al., 1979), were detected
in most of the water samples. Although mutagenic PAH were present, the concen-
tration of PAH did not correlate with the level of mutagenic activity. The range
of mutagenic response included ratios of 1.51 to 4.13 for samples which contained
no detectable levels of the six representative PAH. The highest mutagenic
activity was observed in Champaign (first trip) even though the total PAH concen-
tration was only 6.2 ng, whereas the sample from Wheeling had the highest PAH
level, 1586 ng/&, but produced no demonstrable mutagenic activity. It is also
important to note that PAH require metabolic activation before mutagenicity can
be observed in the Ames assay, and a number of water systems showed mutagenic
activity in the absence of metabolic activation. Although the PAH either present
in the raw water or leached from distribution pipes may contribute to some of the
77
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mutagenic response observed, the cannot be the major source of biological
activity in the sample. Also, it is clear that the levels of PAH would be a poor
indicator of the overall mutagenic potential of drinking waters examined in this
study.
The results of this study indicate that the mutagenic compounds present in
drinking water represent a diverse group of contaminants when the water supplies
are compared with each other. As can be determined from an examination of the
mutagenicity data in Appendix A most of the positive results were obtained with
the plate incorporation assay, however, at least some of the positive responses
were observed in the liquid suspension assay. The starting material for the
adsorbed and eluted from polyurethane foam plugs. The compounds adsorbed to
polyurethane foam plugs probably represent relatively hydrophobic compounds,
whereas the unconcentrated water containing both polar and nonpolar compounds,
was assayed only with the plate incorporation technique. The presence of
mutagenic activity in only one assay system is suggestive evidence that the
mutagens are predominantly polar (plate incorporation assay) or nonpolar (liquid
suspension assay) compounds. Some of the water supplies appear to have mutagens
which do not adsorb to polyurethane foam plugs while others have mutagens which
could be concentrated by this technique.
The second indication that the mutagens in drinking water represent a
diverse group of compounds is the varying requirement for metabolic activation
for expression of mutagenicity. Mammalian metabolic activation (i.e., S-9 mix)
is needed to convert pro-mutagens to ultimate mutagens in the Ames assay. As
shown in Table 7 some water supplies displayed mutagenic activity only in the
78
-------
presence of S-9 mix, indicating that only pro-mutagens were present while other
supplies revealed the presence of ultimate mutagens exclusively, as indicated by
activity only in the absence of S-9. The absence in these cases of activity in
the presence of S-9 indicates that mammalian enzymes present in the S-9 mix are
capable of metabolizing the ultimate mutagens to inactive compounds. In samples
where activity is present both with and without metabolic activation it is
impossible to say whether both ultimate and pro-mutagens are present or that the
ultimate mutagens are refractory to mammalian metabolism. The above evidence
suggests that a number of classes of compounds may be responsible for the
mutagenic activity observed in the water supplies tested in this study.
Along with the effect of the distribution system on mutagenic activity of
drinking waters, this study also provides additional information on the
efficiency of removal of mutagens by water treatment, and on the presence of
mutagens in raw waters. One of the earliest reports which described mutagenic
activity in raw water was the study of Pelon et al. (1977). The water tested was
from the Mississippi River which receives wastewater from a large number of
industries. Pelon et al. (1977) used unconcentrated raw water in their study
while in a more recent investigation, Kool et al. (198la) observed mutagenic
activity in organic concentrates from surface waters in the Netherlands. In the
present study 10 of the 24 raw waters tested displayed detectable mutagenic
activity (Table 8). It is of interest to note that most of the locations showing
mutagenic activity received their raw water from surface water sources, while
most of the ground waters tested in this study showed no mutagenic activity. The
presence of pollutants in surface waters is well documented, with the pollution
79
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occurring primarily from intentional discharge, atmospheric fallout, and ground
run off. On the other hand, filtration through sand has long been used to purify
water, and this type of filtration along with biological degradation which would
naturally occur prior to water entering the aquifer probably accounts for the
general lack of activity shown in ground water.
The presence of mutagens in drinking water has also been well documented
(Kool et al., 198lb, 198lc; Loper et al., 1978; Schwartz et al., 1979; Simmon
et al., 1977). As can be seen in Table 7 we found in many cases the water after
treatment was more mutagenic than the raw water. In three of the water systems
we tested the raw water was nonmutagenic while the finished water contained some
mutagenic activity. A number of studies have linked the mutagenic activity in
drinking water to the formation of low molecular weight chlorinated compounds
produced by chlorination (Simmon et al., 1977; Rapson et al., 1980; Brett and
Calverly, 1979; Dolara et al., 1981; Anonymous, 1981). Although chlorination
may be the source of the increased mutagenicity observed in finished water, the
activity probably did not result from chlorinated low molecular weight
compounds, since neither of the assay procedures used in this study are very
effective at the detection of volatile compounds. In addition to chlorination,
mutagens may also be added to finished water through the presence of trace
contaminants in the chemicals used during purification (e.g., lime addition).
Without more extensive investigation of each process employed in the purifica-
tion of drinking waters we studied the specific source of the increase in muta-
genic activity cannot be determined. Table 7 also shows that in two cases the
81
-------
treatment process was effective in the removal of mutagenic activity, while in
six cases the treatment had no effect on the level of mutagens.
Many of the samples tested, including both finished water and distribution
c" '
water,'low levels of mutagenic activity (Appendix A). The implication to human
health of these low levels of mutagenic activity are not fully understood at the
present. This is particularly true in the assessment of mutagenic activity in
complex environmental mixtures in which a large number of interactions may occur.
Supression of mutagenic activity has been observed in other environmental
samples as a result of interactions (Shahn and Fournier, 1978). Although
enhancement of activity has not been reported there have been no systematic
investigations of the performance of the Ames assay with complex environmental
mixtures. The interpretation of mutagenicity is further complicated by the low
levels of activity commonly observed in water samples and the toxicity to the
bacteria of the organic concentrates which prevent the use of high dose levels.
Overall the results obtained with the Ames assay in this study indicate that some
drinking water suplies contain compounds that have potential biological
activity. The presence of biologically active compounds in drinking water
indicate that these water supplies should be first studied in greater detail to
determine the source of this contamination before attempting to assess any public
health; arising from the consumption of this water.
82
-------
SECTION 7
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Environmental Fate of Selected Polynculear Aromatic Hydrocarbons. EPA
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3rd ed., p. 35. Geneva, Switzerland.
86
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APPENDIX A
Mutagenic Activity in Drinking Waters
-------
Key to Tables 1-131:
Metabolic Activation = rat liver S-9 homogenate/extract for enzymatic activa-
tion is present ( + ) or absent (-)
2
Dose: C = control; L = low; M = medium; X = intermediate; H = high
Dose: C = control; 1 ml = 1 ml of filter sterilized water per plate, 1/2 = equal
volumes of media water and distilled water used in preparing agar; Full = only
filter sterilized media water used to prepare agar
*Weak mutagenic response indicated; **Mutagenic response indicated
NA = not applicable; NC = Not calculated; NT = not tested; 3D = standard devia-
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