United States
Environmental Protection
Agency
Municipal Environmental
Research Laboratory
Cincinnati OH 45268
V f" . ./t" X
Research and Development
EPA-600/S2-84-116 Sept. 1984
&ER& Project Summary
Evaluation of Urban Runoff and
Combined Sewer Overflow
Mutagenicity
Stuart J. Spiegel, Edwin C. Tifft, Jr., Cornelius B. Murphy, Jr., and Randy
R. Ott
A study was conducted to evaluate
combined sewer overflows and urban
runoff for the presence of chemical
mutagens. The Ames Salmonella/m\-
crosome mutagenicity test was used as
a general biological effects test for the
qualitative detection of mutagens in the
sanitary environment, including rain,
urban runoff, sanitary wastewater,
combined sewer overflows, sewage
treatment plant effluent, and receiving
waters. The Ames test is a relatively
sensitive and simple bacterial test for
detecting chemical mutagens. Its ad-
vantages over long-term animal tests
are speed, ease, and relatively low cost.
The test employs previously mutated
Salmonella typhimurium LT2 bacterial
strains that tend to mutate back to their
natural state when exposed to muta-
genic compounds.
Nineteen samples produced a detect-
able response to one or more of the five
S. typhimurium test strains, with or
without metabolic activation. Nine of
these samples (47%) were of urban
runoff in the project area of metropoli-
tan Syracuse (Onondaga County), New
York, and they produced 17 of 30
detectable responses (57%). Five of the
samples (26%) were from combined
sewer overflows, and they produced 7
of 30 detectable responses (23%).
The results indicated that urban
runoff components that produce a
detectable response in the Ames test
may be diluted or inactivated in combi-
nation with sanitary sewage to form
combined sewage, since fewer responses
were detected in the latter than in urban
runoff.
This Project Summary was developed
by EPA's Municipal Environmental
Research Laboratory, Cincinnati, OH.
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Only since the early 1960's have urban
runoff and combined sewer overflows
(CSO) been identified as pollutant contri-
butors to receiving streams. And not until
nearly 10 years later were urban runoff
and CSO implicated as major sources of
particulates, nutrients, and microbial
loading to urban waterways. In recent
years, almost any potential pollutant to be
found in the urban environment can be
traced to receiving streams via this
pathway.
To decrease surface water pollution
from these sources, the U.S. Environ-
mental Protection Agency (EPA) has
engaged studies and provided funds for
the design and construction of prototype
CSO treatment facilities. These facilities
are primarily designed to use some form
of solids removal followed by disinfection.
Though past studies have characterized
urban runoff and CSO, and identified
constituent pollutants, they have not con-
clusively dealt with the most recent
concerns about the fates and effects of
organic chemicals in these systems.
Potentially toxic, mutagenic, carcinogenic,
or teratogenic chemicals are readily
identifiable in the urban environment, but
little attempt has been made to determine
their concentrations in urban runoff and
-------
Two alternative sites were also chosen m
in case difficulties arose at any time in the
collection of samples from the primary
sites:
(a) Site 004 - a large basin drainage area
characterized by mixed residential
and commercial land use; and
(b) Site 074 - a drainage area with
predominantly commercial and in-
dustrial land use.
In addition, three CSO samples were
collected in Washington, D.C., one at each
of three separate sites. Because of
difficulties in obtaining manual grab
samples at these sites, an automatic
sampler was used. Also, two CSO
samples were taken from a site in
Rochester, New York (Site 007, Maple-
wood Avenue), during two storm events.
The sample collection sites were
chosen using three selection criteria:
1. Uniqueness of drainage area char-
acteristics. Drainage areas with
percent imperviousness, popula-
tion density, size, and land use mix
representative of several others in
the city were evaluated for selec-
tion. In addition, drainage areas
that were unique in the city (i.e.,
predominantly open space, such
as Site 046) were selected. -
2. Ease and safety of sample col lee- fl
tion. Where several sites of similar
physical characteristics could
have been selected, ease of sample
collection was considered to pre-
serve sample integrity. In addition,
both receiving streams traverse
busy urban areas where sample
collection would be performed
through manholes in streets. To
ensure the safety of personnel,
accessibility during high-traffic
periods was considered.
3. Availability of baseline data. Phy-
sical and chemical analyses of
CSO's in the Syracuse area have
been performed during previous
characterization studies. These
data could serve as a baseline for
this study. This criterion was
considered least important of the
three in site selection.
CSO samples were collected during
four wet-weather events in glass con-
tainers lowered from manholes directly
above the overflow pipe. Sufficient
sample was transferred to fill an amber
glass bottle for Ames testing, a 50-ml
plastic container with nitric acid preser-
vative for TOC and heavy metals analysis,
and a plastic half gallon container for
other chemical/physical analyses. This
sample storage and preservation proce-
dure was followed for all samples. Glass
CSO. Several reasons exist for this
omission. Such studies would require the
analysis of long lists of compounds, as in
priority pollutant analysis, and these lists
are constantly expanding. Technological
difficulties exist in the analysis of such
compounds. The detection limits of
available instrumentation may not be
sufficiently low to detect minute concen-
trations of contaminants at levels that
may still pose a potential health hazard.
The cost of analyzing a single sample
under these conditions is high, and the
cost would be staggering to examine
enough samples to provide statistically
significant data for a long list of compounds.
Furthermore, such determinations would
still hot provide any biohazard risk index;
that is, any measure of the biological
effects these compounds might have in
their environmental medium, particularly
effects on the human system or synergis-
tic or multiplicative effects.
At this time, most treatment systems
for CSO are still in the initial planning or
evaluation phases. Though it is generally
agreed that treatment should consist of
solids removal and disinfection, there has
been little evaluation of the need for
additional levels of treatment. Because of
the highly variable nature and occurrence
of urban runoff and CSO, it is doubtful
that the need for advanced treatment can
be determined solely by characterization
of a list of specific chemicals. Instead,
general information describing the po-
tential biohazard risk of these systems
would be a valuable criterion for deter-
mining the need for advanced treatment.
This study has thus been based on the
premise that urban runoff may substan-
tially contribute substances of potential
risk to the human environment and that a
preliminary analytical survey using the
Ames Salmonella/microsome mutagen-
icity test might outline the presence and
scope of that potential. To this end,
samples were taken from seven sources
of environmental contributors to the
Onondaga County (Syracuse), New York,
CSO system. These sources included:
rain, urban runoff, dry-weather sanitary
flows, and CSO, as well as influent and
effluent from the Metropolitan Syracuse
Treatment Plant (Metro) and samples
from Onondaga Lake, the ultimate recep-
tor of the overflows. Samples were tested
by the Ames test and subjected to
analyses for other chemical parameters,
including total suspended solids (TSS),
total organic carbon (TOC), 5-day bio-
chemical oxygen demand (BOD5), oil and
grease (O&G), and the heavy metals
cadmium (Cd), chromium (Cr), lead (Pb),
and copper (Cu). The results of the Ames
test were compared with the baseline
chemical analyses to determine any
correlations that might help identify the
source of mutagenic activity.
Since inorganics (especially metals)
are removed in the sample extraction
procedure, the primary purpose of heavy
metal analysis is to characterize the
samples and evaluate whether their
concentrations were typical or atypical of
the sample type.
This study was completed in two
phases. Phase I involved the collection
and analysis of 74 samples during 1980.
Based on the results of those analyses,
modifications were made to some of the
procedures. Phase II involved the collec-
tion and analysis of an additional 11
samples in 1981 from those drainage
areas that elicited detectable responses
during Phase I.
Sample Collection
As mentioned earlier, provisions were
made in the original study plan to collect
seven types of samples in Onondaga
County (Syracuse), New York (CSO, dry-
weather sanitary flow, urban runoff,
influent and effluent to Metro, Onondaga
Lake, and rain).
CSO Samples
The project plan required the selection
and collection of one CSO sample during
four wet-weather events from each of six
CSO discharges. The object was to select
six drainage areas to be examined, each
with divergent characteristics of size,
land use, population density, and/or
percent imperviousness. Selection was
made based on previous studies performed
in the Syracuse area to characterized
CSO sites.
The physical characteristics of the six
CSO sites that were selected are sum-
marized as follows:
(a) Site 005 - a small drainage area with
a high percentage of impervious
surface (86%) and predominantly
commercial land use (however, few
historical chemical data were avail-
able from previous studies to charac-
terize this area);
(b) Site 019 - a large drainage area with
mixed land use;
(c) Site 027 - the only drainage area that
includes a significant amount of
industrial land use;
(d) Site 037- a drainage area with a high
percentage of impervious surface
and mixed land use;
(e) Site 043 - a large, high-density
residential area; and
(f) Site 046 - the only drainage area that
is primarily open space.
-------
containers were used for the collection
and storage of Ames samples, since it
was unknown what effect plastic would
have on the samples.
Dry-Weather Sanitary Flow
Samples
Sanitary sewage samples were collected
from the combined sewer either before or
after a storm event, as appropriate to the
collection schedule, in the same manner
as the CSO samples.
Urban Runoff Samples
Urban runoff samples were collected
from catchbasins in their corresponding
CSO sites. Where necessary, curbside
runoff was used.
Influent and Affluent to Metro
Six samples were collected from the
Metro plant. These included one set of
influent and corresponding effluent
samples during storm flow, a similar pair
of samples during dry-weather flow, and
two influent samples collected during
separate wet-weather flow periods.
O no n dag a Lake Samples
Samples were collected from the north
and south basins of Onondaga Lake 9 m
from the Lake's surface, the lower level of
the epilimnon.
Rain Samples
Rain samples were collected at the
O'Brien & Gere Syracuse office in 36-in.
diameter stainless steel pans prerinsed
with dichloromethane. These pans were
placed away from buildings, cars, trees,
and pavement to minimize contamination
from extraneous sources. Rain from two
pans was combined until a sufficient
sample was collected for analytical pur-
poses. The pH of this sample was taken
immediately.
Mutagenicity Testing
Phase I
Criteria used in this study for determin-
ing a significant level of mutagenicity
were recommended by EPA. The signifi-
cance for each strain is determined by the
formula
MAR=E-C
where MAR=mutagenic activity ratio
E =the number of induced re-
vertants
C =the number of spontane-
ous revertants on the day
that the E revertants were
induced
c =the historical rate of spon-
taneous reversions in the
testing laboratory.
MAR values were calculated for all
samples with all five test strains, without
and with S-9 liver activation. A negative
MAR results when E is less than C for a
given test strain on a particular day. A
zero value is obtained when E equals C.
And a positive value results when E is
greater than C, although this positive
value may only be a fraction if E is only
slightly greater than C. A sample is
considered positive or detectable if the
MAR is 2.5 or greater.
Table 1 summarizes only those samples
and the particular strain(s) with a MAR of
2.5 or greater. The MAR values presented
are for 100 /ul of sample concentrated
200 times (the highest concentration of
any sample available in this study and
therefore the most likely sample dosage
to induce mutagenic activity), with 500//I
of S-9 reaction mixture and 100//I of the
appropriate test culture. Nineteen sam-
ples induced a detectable response in one
or more of the test strains by this
criterion.
Nine of these 19 positive samples (or
47%) were of urban runoff; seven were
collected from Syracuse and two from
Rochester, New York. Five of the 19
samples (or 26%) were CSO samples —
three from Onondaga County and two
from Washington, D.C.
Nine of the various samples listed in
Table 1 as inducing a positive MAR were
mutagenic in TA1538 yet not in the more
sensitive counterpart, TA98. Some
indications are that this is not an unusual
test result; TA1538 may show greater
sensitivity than TA98 with some chemi-
cals.
No MAR of 2.5 or greater was obtained
from analyses of the following samples:
CSO from Sites 4 and 46; sanitary
wastewater from Sites 37, 43, and 46;
urban runoff from Sites 27 and 46;
influent to the Metropolitan Treatment
Plant; and CSO from Rochester, New
York.
The results of all applied dosages for
samples that produced a detectable MAR
(equal to or greater than 2.5) appear in
Table 2. Three concentrations (2X, 20X,
and 200X) were applied at a dosage of
100//I/plate at each concentration. Nine
of the 19 samples that produced detect-
able responses (a positive MAR) did so
across the range of all three concentra-
tions; six of these responses resulted
from metabolic activation. Four of these
nine samples produced a MAR of 2.5 or
greater for all three concentrations. The
four curves produced from the responses
of these samples are generally horizontal
with little slope, indicating that these
samples did not show an increase in the
mutagenic response with an increase in
the amount of sample applied. Generally,
a response that increases with dosage or
concentration would be expected; but a
response such as that shown by these
samples is not unusual. Only sample
number 95871 exhibited a logarithmic
response curve.
Of the 30 detectable responses (those
from more than one test strain for six of
19 samples), 10 were from Site 004 (in
three samples), and four were from Site
037 (also in three samples). A more
interesting observation is that 17 responses
of the total of 30 (57%) were from nine
runoff samples (47% of the 19 samples
from which detectable responses were
obtained). CSO samples produced seven
responses from five samples, and dry-
weather sanitary samples elicited four
responses from three samples. Thus,
urban stormwater runoff may contribute
potentially mutagenic substances to CSO
and receiving waters. But since mutagen-
ic activity is not as evident in the
respective CSO samples as it is in the
urban runoff, it is probable that the
concentrations of mutagenic substances
in the runoff are diluted below the
detectable limits of this study as the
runoff mixes with sanitary waste to form
CSO.
Phase II
For this verification phase of the study,
an additional 11 samples were collected in
Syracuse in the late summer of 1981.
These samples included two runoff and
two CSO samples from both Sites 004
and 037, two samples of Metro influent,
and a rain sample. One gallon of each
sample was extracted and brought to a
volume of 20 ml with dimethyl sulfoxide
(DMSO), resulting in a concentrate of
189.25 times the original sample. Five
dosages (500, 400, 300, 200, and 100//I)
were applied.
In two instances, the samples did elicit a
detectable response. One sample was
CSO from Site 004, and the other was
Metro influent. The CSO sample (OBG
No. 28524) had a MAR of 5.2 for tester
strain TA 1538 at a dosage of 200 /ul;
higher dosages resulted in a weaker
response. The Metro influent had a MAR
of 6.1 for TA1538 at the highest applied
dosage of 500 /t/l; lower dosages did not
result in a MAR greater than 2.4.
-------
Table 1. Phase I Mutagenicity Results
MAR*
TA98
Sample
Number
94492
95366
95368
95475
95476
95477
94491
95739
94493
94497
95745
95871
95873
95474
95369
94765
94766
94662
94663
City
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
4
4
Source
1
1
1
2
2
2
3
3
3
3
3
3
3
4
7
1
1
3
3
ID
Location
19
27
43
4
19
27
4
4
19
37
37
37
43
2
.
24
34
7
7
CODE
Descriptor
1
1
1
2
2
2
1
1
1
1
1
1
1
2
.
1
1
1
1
Without
S-9
-t
-
-
-
-
-
-
3.0
-
2.7
-
-
.
_
_
-
-
-
-
With
S-9
2.9
.
-
-
-
-
3.3
2.6
-
-
-
-
_
-
-
-
-
MAR MAR MAR MAR
TA100 TA1535 TA1537 TA1538
Without
S-9
2.8
-
-
-
-
.
-
-
-
_
-
6.8
.
_
_
-
76.2
-
-
With Without With Without With Without With
S-9 S-9 S-9 S-9 S-9 S-9 S-9
.
72
9.1
2.7
3.9
11.2 - - - - - 5.1
4.1 2.6 3.4 2.5
4.0 4.8
2.6 - - - -
_
29
2.9 ----- -
10.0
6.1
2.6
4.4
4.2 -
4.5
3.8
*MAR - mutagenic activity ratio.
t - No activity at 2.5 or above.
Table 2. Detectable Responses from Phase I Analyses
Sample
Number
94492
94492
95366
95368
95475
95476
95477
95477
94491
94491
94491
94491
94491
95739
95739
95739
95739
94493
94497
95745
95871
95871
95873
95474
95369
94765
94766
94766
94662
94663
Organism
TA98
TA100
TA 1538
TA 1538
TA 1538
TA 1538
TA100
TA 1538
TA98
TA 1537
TA 1537
TA 1538
TA 1538
TA98
TA98
TA 1537
TA 1537
TA 1535
TA98
TA 1538
TAWO
TA100
TA 1538
TA 1538
TA 1538
TA 1538
TAWO
TA 1537
TA 1538
TA 1538
S-9 Reaction
Mixture fal)
500
-
500
500
500
500
500
500
500
500
-
500
500
-
500
-
-
500
-
500
500
500
500
-
-
-
-
-
200-Xt
2.9
2.8
7.2
9.1
2.7
3.9
11.2
5.1
3.2
4.1
2.6
3.4
2.5
3.0
2.6
4.0
4.8
2.6
2.7
2.9
68.
29.
10.
6.1
2.6
4.4
16.2
4.2
4.5
3.8
MAR*
20X
0.5
0.3
3.2
6.0
2.8
4.0
0.1
4.6
0.2
2.5
0.9
2.8
0.7
1.1
0.6
3.2
1.9
-0.1
0.3
-0.7
12.
1.7
2.1
5.6
1.3
0.8
-0.5
-0.5
0.6
0.6
2X
-
1.2
2.3
3.4
4.0
-
5.4
-
-0.7
0.2
-
-
-
0.8
-
-
-
-
1.7
-
6.5
-
-
-
-
-
-
* MAR = mutagenic activity ratio.
t Concentrated 200 times.
-------
Two samples produced toxicity; runoff
from Site 004 (OBG No. 28885) with
TA100 (no activation) and Metro influent
(OBG No. 28888) with TA100 (with and
without activation).
The four aforementioned samples that
produced positive or toxic responses
were re-analyzed for verification. None of
the analyses resulted in a MAR of 2.5 or
greater at any applied dosage.
Conclusions
The following conclusions resulted
from the project:
1. Nineteen samples out of 85 total
samples (22%) may be interpreted to
have induced a detectable mutagenic
response from one or more of the test
strains, with or without metabolic
activation. Nine of these samples
were of urban runoff. Some samples
required a 200-fold concentration for
detection of this activity. Of 30
detectable responses, 17 (57%) were
found in urban runoff, and seven
(23%) were found in CSO.
2. The samples with a detectable
response did not appear to be
correlated to levels of any of the
chemical parameters measured or to
any particular CSO site. The experi-
mental design involved too few
samples and chemical parameters to
obtain correlations between detect-
able mutagenic activity and chemical
characteristics of samples or sample
types (CSO, urban runoff, etc.).
3. Fewer responses were detected in
CSO than in urban runoff. Thus
substances present in urban runoff
that produce a detectable response
in the Ames test may be diluted or
inactivated when the runoff is added
to sanitary sewage to form combined
sewage (CSO).
4. This study indicates the need for a
more comprehensive survey of the
subject. Definitive conclusions are
difficult because of problems en-
counterd in analytical methodology,
sample toxicity to test bacteria, and
the inherent chemical variability of
CSO and urban runoff.
5. The spot test (preliminary screening)
may not be usable as a preliminary
step in analyzing polluted aquatic
samples with the Ames Salmonella/
microsomemutagenicity test because
of sample toxicity at ambient concen-
trations.
6. Dichloromethane (methylene chlo-
ride) may serve as both a disinfec-
tant and an extractant. But if extrac-
tion or concentration is not required
in other aquatic environmental
samples, a more suitable disinfec-
tion procedure may be necessary.
The full report was submitted in
fulfillment of Cooperative Agreement No.
CR-806640 by O'Brien & Gere Engineers,
Inc., and by the Department of Drainage
and Sanitation, Onondaga County, under
the sponsorship of the U.S. Environmental
Protection Agency.
Stuart J. Spiegel. Edwin C. Tifft, Jr., and Cornelius B. Murphy, Jr. are with
O'Brien and Gere Engineers, Inc., Syracuse, NY 13221; and Randy R. Ottis with
the County of Onondaga, Department of Drainage and Sanitation, North
Syracuse, NY 13212.
Richard Field (see below) and Robert Turkeltaub are the EPA Project Officers.
The complete report, entitled "Evaluation of Urban Runoff and Combined Sewer
Overflow Mutagenicity." (Order No. PB 84-211 168; Cost: $14.50, 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, Richard Field, can be contacted at:
Storm and Combined Sewer Program
Municipal Environmental Research Laboratory—Cincinnati
U.S. Environmental Protection Agency
Edison, NJ 08837
*USGPO: 1984-759-102-10671
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
Pb OuO
U 6 E^y/l*
KFbluu >
HkUTtCTlON
IU fr 060^4
------- |