&EPA
United States
Environmental Protection
Agency
Robert S. Kerr Environmental Resear
Laboratory
Ada OK 74820
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Research and Development
EPA-600/S2-81-007 Mar. 1981
Project Summary
Mutagenistic Testing of
Industrial Wastes From
Representative Organic
Chemical Industries
Susan Stinnett and John E. Matthews
The general applicability of the Ames
test for screening wastewater samples
was investigated. Application of the
Ames test to raw and treated waste-
waters from representative organic
chemical industries involved the in-
vestigation of several problems: (1)
the feasibility of using the Ames test
to detect mutagens in wastewater
samples, (2) the relative effectiveness
of various waste treatment processes,
(3) the mechanics of establishing an
Ames testing program, and (4) the
economics of using the test in routine
environmental screening.
Samples collected from 14 industrial
sites were analyzed using the Ames
procedure. Results were interpreted
on the basis of relative increases in
revertant colonies on test plates as
compared to control spontaneous re-
version plates. A "positive," sample
consisted of six replicate test plates
with an average count of at least twice
the control value. Of 28 samples
tested, 6 were interpreted as positive
and 22 were interpreted as negative.
This Project Summary was devel-
oped by Robert S. Kerr Environmental
Research Laboratory, Ada. OK, to
announce key findings of the research
project which is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
An increasing concern about the pos-
sible introduction of carcinogens into
the environment has resulted in a
search for a simple, sensitive, and reli-
able method for the detection of these
chemicals. The Ames test, developed by
Bruce Ames, of the University of Califor-
nia, Berkeley, has achieved considerable
attention among industries and govern-
mental agencies interested in routine
screening for potential carcinogens in
water supplies.
The Ames test was originally designed
to determine the ability of a specific
compound to cause mutations. Since
most carcinogens are also mutagens, a
correlation has been made between
positive results in the test and potential
carcinogenicity. Test strains of bacteria,
supplemented with extracts of rat liver
to simulate mammalian metabolism,
respond readily to the presence of most
mutagens in minute quantities. The test
is currently being investigated as a pos-
sible method for detecting mutagens in
industrial effluents.
In conducting this study, the general
applicability of the Ames test to screen-
ing wastewater samples was investi-
gated. The problems involved in pre-
paring samples for testing and in
interpreting the results are markedly
different for environmental samples as
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opposed to pure compounds or extracts.
The test was applied to raw and treated
industrial wastewaters, in line with
several objectives: (1) the determination
of the feasibility of using the Ames test
to detect carcinogens in the environ-
ment, (2) the relative effectiveness of
various waste treatment procedures in
removing carcinogenic substances, (3)
the mechanics of establishing an Ames
testing program, and (4) the economics
of using the test for screening.
Methods
Raw and treated wastewater samples
collected from 14 industrial sites by the
Environmental Protection Agency's
Robert S. Kerr Environmental Research
Laboratory were analyzed using the
Ames procedure. The industrial sites
represented plants involved in petroleum
refining and production of organic
chemicals, pesticides, wood preserva-
tives, rubber, and pharmaceutical
products.
The Ames test was designed to deter-
mine whether a pure compound can
cause bacteria to mutate. The signifi-
cance of this test system lies in the
correlation between the ability to cause
mutations in bacteria and the ability to
cause mutations in mammals, including
humans. Testing chemicals on animals
is an expensive and time consuming
process. Therefore, it is hoped that the
much simpler Ames test can give valid
preliminary information about mutagens.
The test works by inducing mutations
in the areas of the bacterial chromosome
that have already been mutated. To ap-
pear on test plates, histidine-biotin defi-
cient mutants must regain the capacity
to synthesize these compounds. This is
accomplished when the mutagenic
compound reacts with the genetic ma-
terial, reversing the original mutations.
Since this phenomenon occurs with
some regularity in the absence of muta-
gens, the background "spontaneous
reversion" rate must be determined
before and during Ames testing. It is
generally accepted that a given amount
of material is considered to have muta-
genic activity if test plates incorporating
that dose show at least twice as many
colonies as the average number of
spontaneous revertants for the bacteria
used.
Results and Discussion
Results were interpreted on the basis
of relative increases in revertant colonies
on test plates as compared to control
spontaneous reversion plates. For a
sample to be scored positive, six replicate
test plates had to give an average count
of at least twice the control value. Of 28
wastewater samples tested, 6 were
interpreted as positive (4 raw and 2
treated) and 22 were interpreted as
negative.
Positive results were anticipated
because some of the molecules gener-
ated by organic chemical industries
have shown positive results in pure
compound testing. In industrial waste-
waters, however, the exact chemicals
present and their individual concentra-
tions are unknown. It is probable that
some mutagens may be present in a
sample, but in concentrations belowthe
threshold of detection using these
methods.
Some workers attempting to use the
Ames test to screen wastewaters have
approached the concentration problem
by extracting the samples with methylene
chloride. This was not done in this study
because methylene chloride is a known
carcinogen, and handling it increases
the hazard and the time needed in
sample preparation; because it is a
mutagen, any residue of methylene
chloride remaining in the extracts could
alter the results of the testing. By not
using the extraction procedure, these
objections were eliminated while others
were raised. The lack of concentration
in test samples may lead to negative
scores that might be positive if extracts
were tested. Also, it is obvious that only
the soluble phases of the samples are
tested; any mutagens in or on particu-
late matter have been removed prior to
testing.
In reviewing the validity of using the
Ames test as a-screening procedure, it is
important to recognize that a potential
exists for obtaining false results. The
Ames test is a much more complex
bioassay than it appears; with such a
large number of controls necessary,
even the rigid maintenance of quality
control standards allows a margin of
error to remain.
The potentials for false results (posi-
tive or negative) are always present in
this type of bioassay; they cannot be
totally eliminated. Any attempt at mini-
mizing these potentials will detract from
the value of the test as a rapid screening
tool, but will certainly increase the
confidence in the data. It is apparent
that the experimental design used in
this project was suitable only for pre-
liminary screening of samples. It is not
reasonable to classify a sample as
mutagenic or not on the basis of this test
alone.
Alternative bioassays, similar to the
Ames test, have been developed for
detecting mutagenic activity of pure
compounds. These assays use yeasts,
other bacteria, or tissue culture cells as
the test organisms. If the Ames testing
procedure outlined in this project is
coupled with one or more of these
assays, it may prove to be very useful as
a screening technique. Ames testing
should not be used as the only screening
test for determining mutagenic activity
of wastewaters.
Conclusions
It is possible to detect positive muta-
genesis in at least some wastewater
samples with the procedure used in this
project. Because the mutagenic agents
are so dilute in wastewater, negative
results obtained from these tests do not
necessarily indicate the absence of
mutagens. There is also a potential for
obtaining false positives using this pro-
cedure. Therefore, it is not reasonable to
classify a sample as positive or negative
on the basis of this test alone.
While the test does not appear to
require much sophistication, it requires
a large laboratory used exclusively for
Ames testing. The initial expense of
establishing a laboratory for screening
samples by Ames testing will be high.
Once the laboratory is equipped and
staffed with people who have had time
to perfect the necessary technique,
routine testing will be inexpensive
when compared to animal studies.
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Susan Stinnett is with East Central Oklahoma State University, Ada, OK 74820,
and John E. Matthews is with the Robert S. Kerr Environmental Research
Laboratory, Ada. OK 74820.
John E. Matthews is the EPA Project Officer (see below).
The complete report, entitled "Mutagenistic Testing of Industrial Wastes from
Representative Organic Chemical Industries," (Order No. PB 81-155 574;
Cost: $6.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
P.O. Box 1198
Ada, OK 74820
1 U.S. GOVERNMENT PRINTING OFFICE: 1961-757-01Z/7054
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