United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-83-016 Jan. 1984
&EPA Project Summary
The Evaluation of the
Mutagenicity of Municipal
Sewage Sludge
Philip K. Hopke and Michael J. Plewa
Samples of five municipal sewage
sludges from Illinois cities have been
subjected to a multiorganism testing
program to determine the presence or
absence of mutagenic activity. Chicago
sludge has been the most extensively
tested using the Salmonella/micro-
somal activation assay, the micronu-
cleus test Tradescantia, the wx locus
assay in Z. mays, and sister chromatid
exchange induction in human lympho-
cytes. Mutagenic activity has been
observed in the sludges from Chicago
and Sauget, Illinois. Sludges from
primarily domestic sewage do not
apparently contain mutagens.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory. Research Triangle Park,
NC, to announce key findings of the
research project that is fully documented
in a separate report of the same title (see
Project Report ordering information at
back).
Introduction
A major problem facing the United
States is the disposal of the sludge
resulting from the treatment of municipal
sewage. There has been a substantial
increase in the number and efficiency of
sewage treatment plants in the past
decade, particularly in suburban and
rural areas, resulting in an increased
amount of sludge requiring disposal. In
addition, previously employed sludge
disposal methods such as ocean dumping
may be modified in the near future. The
incineration of sludge requires expensive
facilities and may result in increased air
pollution An attractive alternative disposal
method is the application of this organic
matter to agricultural lands or its use in
the reclamation of land stripped of its
organic matter by surface mining.
There may, however, be problems
associated with sludge disposal by land
application. Analyses of sludge from the
Calumet and Southwest treatment plants
of the Metropolitan Sanitary District of
Chicago showed that concentrations of
several heavy metals were substantially
enriched over typical soil compositions.
Thus, in 1967, a large demonstration and
research program was initiated to examine
the feasibility of land application of
digested sewage sludge from the Metro-
politan Sanitary District of Chicago waste
water treatment plants. A major objective
was to examine compositional changes in
soil, crops, and water arising from
sludge-amended fields.
In the intervening period, there has
been a great deal of study of the uptake and
accumulation of heavy metals (most
notably zinc and cadmium) in corn, the
effects of these elements on soil bacteria,
and the potential for transmission of
heavy metals through a food chain that
might result in impacts on the general
public health if widespread use was made
of sludge as a soil additive Zinc and
cadmium have been found to accumulate in
corn grain and leaf tissues. High levels of
sludge treatment appear to inhibit the
development of several inbred strains of
corn but do not cause overt symptoms of
metal toxicity. Long-term studies of food
chain and field productivity effects of
sludge are in progress.
Additional contaminants that may be
present in municipal sewage sludge are
organic compounds from industrial
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processes or other anthropogenic activities.
Of potential concern are compounds that
are genetically active and may cause
mutations, birth defects, or cancer. In a
metropolitan area like Chicago there are
major chemical, petrochemical, petro-
leum-refining, and other industries that
utilize organic chemicals in large quanti-
ties. Other activities such as fossil fuel
combustion produce genotoxic compounds
that may find their way into the sanitary
sewer system. Therefore, there exists the
possibility that there are genotoxic
compounds present in the sewage sludge
that may be released intotheenvironment
by leaching into surface or groundwater
or incorporated into the human food
chain through the use of sewage sludge
as a soil additive. In order to examine this
question, samples of sewage sludge were
obtained from several local sewage
treatment plants that treat a variety of
wastes ranging from only household
wastes to combinations of industrial and
domestic wastes. These sludge samples
have been subjected to a multiorgamsm
screening procedure to determine if
mutagenic compounds are present in
sludge and sludge-amended soil.
Conclusions
This study has determined that mut-
agenic activity is present in some
municipal sewage sludges. Sludges from
Champaign, Chicago, Hinsdale, Kankakee,
and Sauget, Illinois were studied. The
Chicago municipal sewage sludge sample
clearly has components that induce a
variety of mutagenic responses. Although
it is not possible to compare the quantita-
tive responses in the various test
organisms employed, the sludge or
sludge extracts did induce a response in
all of the species employed. The superna-
tant liquid after appropriate concentration
was found to be mutagenic using the
Ames Salmonella assay. Acetone, hexane,
and chloroforrrrmethanol extracts of the
pellet contained promutagens that could
be activated by mammalian microsomes
(S-9). Acetone was a more effective
solvent than hexane in the extraction of
the promutagens from the sludge pellet.
The results indicate that a moderately
potent agent can be extracted from the
sludge since, with microsomal activation,
the material extracted from only 155/ul of
whole Chicago sludge was able to induce
twice the number of revertant colonies
per plate of Ames strain TA98 compared
to the background negative control
plates.
The Champaign, Hinsdale, and Kankakee
sludges showed a weak response from
2
samples obtained by the further chemical
fractionation of the chloroformimethanol
extracts following activation Only the
Sauget sample demonstrated direct
acting mutagenic activity as well as very
substantial mutagenicity after activation
with mammalian hepatic microsomes.
In this project, the experiments using
the Tradescantia micronucleus test
encompassed the analysis of over 2.0 x
106 meiotic cells that were individually
scored for micronuclei. The project mean
negative control frequency based on 2.Ox
105 tetrads was 3.53MCN/100 tetrads.
The overall positive control (50 mm
maleic hydrazide) frequency based on 4.7
x 104 tetrads was 10.71 MCN/100
tetrads.
The Tradescantia data clearly indicate
that whole or diluted Chicago sludge
induces chromosome aberrations in
meiotic cells. The aqueous phase is much
less mutagenic than whole sludge and it
appears that a major portion of the
mutagenic agents remain in the sludge
solids. The acetone fraction of the
Chicago sludge gave a negative response
in the micronucleus test. When whole
sludge samples from each municipality
except Sauget were evaluated an inter-
esting pattern of response emerged. A
ranking in order of decreasing mutagenic
response is Chicago, Kankakee, Hinsdale
and Champaign. When concentrated,
whole sludge samples are evaluated, the
same ranked order is maintained. How-
ever, the Chicago sludge is a much more
potent mutagen than the Kankakee
sludge samples. A one half dilution of
whole Chicago sludge induced a mean
value of 5.19 MCN/100 tetrads as
compared to a value of 6.30 MCN/100
tetrads for the 13.3x concentration of
Kankakee whole sludge. Neither the
Hinsdale nor Champaign sludge samples
induced a significant frequency of
micronuclei in Tradescantia tetrads. The
Sauget sample has not yet been tested
with this assay. There is an apparent
correlation of the cytogenetic potency of
the sludge sample and the relative
industrialization of the municipality.
In order to provide a mammalian
endpoint, the ability of sludge extracts to
induce sister chromatid exchanges
(SCE's) in human peripheral lymphocytes
has been examined. It is quite difficult to
culture the cells in the presence of the
extracts, and only partial results are
currently available. We have discovered a
direct-acting agent(s) in an acetone
extract of Chicago sludge. A 100 /j\
sample of a 15-fold concentrated sample
of this extract induced a statistically
significant increase in the frequency of
SCE s (pO.01) The control frequency was
0.21 ±0.02 SCE/chromosome while the
sample increased the frequency to 0.32 +
0.02 SCE/chromosome. A positive con-
trol of 5 mM ethyl methane sulfonate
(EMS) induced an SCE/chromosome
frequency of 0.48 ± 0.02. This acetone
extract is not directly mutagenic when
tested with the Salmonella assay but also
showed activation by mammalian S-9.
The results also show that these acetone
extracts are toxic to human lymphocytes
at high concentrations. Further studies
with this assay are in progress.
The in situ tests of sludge-amended soil
using Chicago sludge and the maize wx
locus assay demonstrate that mutagens
are available to the plant when sludge is
used as a soil amendment. Toxic as well
as mutagenic responses were observed.
The laboratory studies indicate that
sludge can induce a substantial number
of mutations at the wx locus in inbred
Early-Early Synthetic as well as reverse
mutation at the wx-90 locus observed in
the field studies. Champaign sludge did
not demonstrate mutagenic activity in the
forward mutation assay with Early-Early
Synthetic. The other sludges were not
tested in this assay because of time
constraints. These studies indicate that
mutagens present in sludge-amended
soil are transported into a crop plant and
can induce genetic damage in germ cells.
However, these studies do not provide
any indication of mutagenicity in the
kernels grown on sludge-amended soil or
transfer of the mutagens from the sludge
to ground or surface waters. However,
these results do suggest the need for
further study of the possible adverse
effects of land application of sludge from
large industrialized cities.
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Philip K. Hopke and MichaelJ. Plewa are with the University of Illinois, Urbana, IL
61801.
Norman E. Kowal is the EPA Project Officer (see below).
The complete report, entitled "The Evaluation of the Mutagenicity of Municipal
Sewage Sludge," (Order No. PB 83-264 077; Cost: $10.00, subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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