v'/EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S1-81-016 Mar. 1981
Project Summary
Assessment of Mutagenic
Potential of Mixtures of
Organic Substances in
Renovated Water
M. W. Neal, L. Mason, D. J. Schwartz, and J. Saxena
This study was conducted to deter-
mine the presence of mutagenic activity
in municipal wastewater and to evalu-
ate the performance of available ad-
vanced wastewater treatment systems
for removing such activity. Further,
the distribution of the described acti-
vity among various classes of chemical
compounds was studied in an attempt
to identify the active fractions. The
study was conducted utilizing rapid//?
vitro bioassays for detection of muta-
genic activity and, therefore, was
expected to provide a preliminary
assessment regarding the mutagenic
hazards linked to the potential potable
use of reclaimed wastewater. The
results of this investigation should be
of value to the Environmental Protec-
tion Agency and others in identifying
the problem areas and in setting priori-
ties for in-depth and more specialized
toxicological studies. Knowing that
certain wastewaters have toxic prop-
erties, treatment systems may be
designed to remove the toxic compo-
nents or sources of such components,
for example certain industries could
provide pretreatment measures.
This Project Summary was develop-
ed by EPA's Health Effects Research
Laboratory, Cincinnati, OH, to an-
nounce 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
A full toxicological investigation of the
potential hazards from the consumption
of renovated wastewater is necessary
before considering it for potable reuse.
Wastewaters may contain hazardous
chemicals for which currently no criteria
exist. Such pollutants may pass through
wastewater treatment plants in tracq
amounts, so an evaluation of the toxic-
ological properties of such effluents is
warranted.
Methods
An investigation was undertaken to
evaluate the performance of selected
advanced wastewater treatment pro-
cesses for removing (introducing) muta-
genic substances and to determine the
distribution of the detected activity
among various classes of chemical
compounds. The study utilized specially
constructed strains of Salmonella typhi-
murium and Saccharomyces cerevisiae
to assess mutagenic activity, and the
mammalian cell — BHK21 Cl 13 (to a
limited extent) to determine transform-
ing activity. In an effort to recover the
wide variety of organic contaminants
present in wastewater and renovated
wastewater, three independent concen-
tration methods were utilized. These
included sorption on polyurethane foam
plugs, sorption on XAD resin, and
solvent extraction. Conditions suitable
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for recovery of organics of polyurethane
foam were: wastewater temperature 62
C, flow rate 250 ml/min, with a sample
volume of 10 I for secondary effluent
and 20 I for intermediate or fully treated
wastewater. The conditions used for
XAD resin were: wastewater temperature
50 C, flow rate 100 ml/min, XAD-4 and
XAD-8 columns (1x10 cm) connected
in series, sample volume 51. For recovery
of organics by liquid-liquid extraction,
15% methylene chloride in hexane (75
ml solvent/1 wastewater) was used.
The crude organic mixtures recovered
from wastewaters were separated into
seven major chemical classes of organic
compounds according to their solubility
under acidic, basic, and neutral condi-
tions, and each class was tested for
mutagenic activity. The classes of com-
pounds isolated were; ether insoluble,
water soluble, basic, amphoteric, strong
acid, weak acid, and neutral. Adsorption
chromatography with silica gel columns
was used to subfractionate the neutral
fraction into aliphatic, aromatic, and
oxygenated fractions.
A mutagenic response by the waste-
water concentrates and separated frac-
tions was observed with the Ames Sal-
monella liquid suspension assay which
involved preincubation of the test mix-
ture and Salmonella tester strain in
liquid suspension prior to plating. Con-
ventional plate incorporation and spot
tests failed to detect mutagens in crude
concentrates and separated fractions.
For detection of low concentrations of
active compounds in unconcentrated
wastewater, the assay was performed
by making filter sterilized test water into
media (base agar layer). This modifica-
tion of the assay permitted incorporation
of up to 10 ml of test wastewater in the
Ames assay (70% v/v). The amino acid
histidine, which interferes with the
Ames assay by artifactually increasing
the number of revertants, was not
detected in wastewaters. The yeast
assay (forward and reverse mutagene-
sis, mitotic cross over, and gene conver-
sion) lacked the sensitivity for detection
of mutagens in wastewaters, wastewater
concentrates, or separated fractions.
Only a marginal response was obtained
with strain S288C (forward mutagene-
sis assay) with some samples. The
assay also lacked the capacity to detect
mutagenic compounds which required
mammalian metabolic activation. The
BHK cell system also proved to be
unsuitable for this study because of the
excessive time requirement and its erratic
behavior.
Physical-Chemical System
The influent of the physical-chemical
AWT process at Piscataway, Maryland,
in unconcentrated form, showed mini-
mal mutagenesis in the base pair sub-
stitution mutants TA100 and TA1535 of S.
typhimurium. The activity was lost by
incorporation of mammalian liver en-
zymes in the assay. Wastewater samples
collected after the liming/recarbonation
steps showed increased activity over
that of the influent suggesting that
these treatment steps were capable of
introducing mutagenic substances.
Alternatively, the liming/recarbonation
steps may have removed toxicants
and/or masking agents, thereby permit-
ting detection of mutagens already
present in the influent. The increased
activity observed with the partially
treated sample was removed by break-
point chlorination and/or carbon filtra-
tion. However, the overall response
with the final effluent was no less than
that shown by the influent wastewaters.
Mutagens were not present as conjugates
in wastewaters since the presence of
the hydrolytic enzyme /3-glucuronidase
in the assay did not result in an increas-
ed mutagenicity. The organic mixtures
recovered from wastewater by the
concentration methods showed muta-
genic activity, but not in the same tester
strains as with unconcentrated waste-
waters. Also, the activity could be
detected only when the indicator organ-
ism was incubated with the concentrates
in liquid suspension prior to the assay.
These findings suggested that the con-
centration methods employed failed to
recover all the active compounds from
wastewaters. The recovered activity of
the purified effluent was found to be
distributed mainly in weak acid, basic,
and aliphatic and aromatic classes of
compounds. The combined activity of all
the separated fractions was greater
than that of the crude concentrate,
suggesting masking of activity in this
complex mixture, either because of
toxicants or masking agents. The chemi-
cal separation scheme used in this
investigation resulted in the uncovering
of some of the activity but not all, since
many of the separated fractions remain-
ed toxic to the test organisms. Only a
small percent of the original organics,
as measured by total organic carbon,
was recovered by the three concentra-
tion methods and the results must
tempered by this fact.
Biological With Physical-
Chemical System
The influent and effluent from Bay
Park AWT Plant, at East Rockaway, New
York, which utilized combined biological
and physical-chemical treatment, show-
ed mutagenic response in the base pair
substitution mutants TA100 and TA1535.
A comparison of the mutagenic response
of AWT influent and effluent revealed
that the AWT method employed at Bay
Park, as was observed with the Piscata-
way treatment, not only failed to remove
certain mutagenic substances but also
added new mutagens to the final efflu-
ent. Unlike the results with unconcen-
trated wastewater, the organic mixtures
recovered from Bay Park wastewater
showed activity in the f rameshift mutant
TA1538. The mutagenic chemical class-
es derived from the influent were basic,
water soluble, and amphoteric. Activity
in these classes was partially reduced
as a result of treatment but activity
appeared in two new fractions: the
weak acid and ether insoluble.
I
Industrial Waste Plant
Wastewaters from the physical-chem-
ical Niagara Falls, New York AWT pro
cess, which consists of 60-70% indus
trial wastes, showed weak to moderate
mutagenicity in base pair substitutior
and frameshift tester strains of S
typhimurium. Mammalian metabolii
activation was required for mutagenesif
in frameshift mutants. The mutagenii
activity was higher in influent waste
water than in partially treated and fina
effluent suggesting removal of mutagen
during treatment. There were, however
mutagens still present in the fina
effluent. The influent wastewaters a
the point samples had undergone partie
treatment including liming/pH adjusl
ment, and the addition of mutagen
during these treatment steps may hav
been responsible for increased muta
genie activity of the influent.
Domestic Wastes
Influent and AWT effluent sample
from the rapid sand filtration process i
Lake George failed to cause mutagem
sis in the Salmonella strains tested, bi
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showed an inhibitory effect on the
spontaneous reversion rate of strain
TA98. Organic mixtures recovered from
these wastewaters were also free of
mutagens. The data revealed that micro-
bial synthesis of mutagens did not occur
following prolonged contact with the
natural delta sand beds. Because of the
absence of mutagens in the influent
wastewater, efficiency of the process
for removing mutagens could not be
assessed. These wastewaters did not
contain any industrial wastes and it is
significant that wastewaters strictly of
domestic origin do not have mutagenic
properties.
ment steps themselves may contribute
mutagens. Further toxicological and
analytical studies are required to
determine the nature of potentially
hazardous compounds present in reno-
vated wastewater.
Sand Filtration System
Information on removal of mutagens
by sand filtration was obtained using
simulated laboratory sand columns
which were dosed with partially treated
primary effluent from the heavily indus-
trialized area of Niagara Falls. The
primary effluent did not show mutagenic
activity in any of the chemical fractions
except for the strong acid fraction. This
was surprising since the wastewaters
from this facility, when tested in uncon-
centrated form, showed high mutagenic
response. After filtration of the waste-
waters, through one half of the sand
column, the strong acid fraction became
less mutagenic, but strong mutagenic
activity appeared in the water soluble
and ether insoluble fraction, and weak
mutagenicity was noted in weak acids,
basic, and amphoteric fractions. On
continued contact with sand, mutagens
of the amphoteric, weak acid, and basic
classes were removed to a nondetectable
level but mutagens of ether insoluble,
water soluble, and strong acid classes
were unaffected. The presence of sev-
eral new classes of mutagens in the
midpoint and final effluent, not detected
in the influent, suggests synthesis of
mutagens during the sand filtration
process. Alternatively, the sand filtration
process may have removed toxicants
and/or masking agents thereby permit-
ting expression of the mutagens origi-
nally present in the influent.
Conclusions
The study shows that the physical-
chemical, biological, and sand filtration
processes studied were unable to com-
pletely remove mutagenic activity from
wastewater. In several instances, treat-
M. W. Neat, L Mason, D. J. Schwartz, and J. Saxena are with the Syracuse
Research Corporation, Syracuse. NY 13210.
Herbert R. Pahren is the EPA Project Officer (see below).
The complete report, entitled "A ssessment of Mutagenic Potential of Mixtures of
Organic Substances in Renovated Water," (Order No. PB 81-153 843; Cost:
$11.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
US GOVERNMENT PRINTING OFFICE 1981-757-012/7011
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