United States
Environmental Protection
Agency
Health Effects
Resrarch Laboratory
Research Triangle Park NC 27711
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Research and Development
EPA/600/S1-88/002 Sept. 1988
v>EPA Project Summary
The Stability and Mobility of
Mutagenic Activity from
Wastewater and Sludge in
Agricultural Soils
K. W. Brown and K. C. Donnelly
The objective of this research was to
measure the fate and mobility of organic
mutagens In municipal sludge amended
soil. Initially, seventeen municipal
sewage sludge samples were collected.
The mutagenic potential of 49 separate
fractions extracted from 17 samples of
municipal sewage sludge was determin-
ed using the Salmonella! microsome
assay. None of the fractions extracted
from four of the sludges induced a
positive response In the bioassay.
Twenty-six fractions failed to induce a
doubling of revertant colonies at two
consecutive dose levels, while seven-
teen fractions induced a moderate
mutagenic response (35 to 100 net rever-
tants 10 per milligrams), and six induc-
ed a relatively high mutagenic response
(greater than 100 net revertants per 10
milligrams). The maximum specific ac-
tivity was 187 net revertants per 10 mg
induced by the methanol fraction ex-
tracted from the AD2 sludge with
metabolic activation; while the maximum
weighted activity was 1197 revertants per
gram of sludge induced by the combin-
ed fraction of the SM4 sludge. For the
sludge fractions tested, strain TA98 was
the most sensitive, and the majority of
fractions required metabolic activation to
induce a positive response.
One sludge sample which induced a
strong positive response was selected
for use in a field study. Sludge samples
were collected and applied at three ap-
plication rates to undisturbed soils
enclosed In lysimeters to monitor the
fate and mobility of sludge borne
mutagens in soil. The maximum specific
activity of the sludge amended soil was
416 net revertants per 10 milligrams of
residue induced by the methanol fraction
extracted from the Weswood soil col-
lected 154 days following sludge applica-
tion (totaling 150 Mg/ha). The mutagenic
potential of the sludge amended soil was
observed to decrease slowly with time.
A positive response was still observed
for several of the fractions extracted from
the sludge amended soil collected 207
days after sludge application. The
mutagenic activity per unit weight of soil
decreased to near background levels
over the course of the study for only one
treatment (Padina sand with 50 Mg/ha).
Eight of twenty-seven leachate samples
from the waste amended soils induced
a positive mutagenic response; while
twelve samples induced a weighted ac-
tivity of greater than 100 revertants per
liter. The maximum specific activity of
the acetone extractable organics
recovered from the leachate water was
207 net revertants per 10 milligrams of
residue induced without metabolic ac-
tivation by the leachate water from the
Weswood soil which received two sludge
applications totaling 150 Mg/ha.
None of the extracts from plants grown
on the sludge amended soil induced a
positive response in the bioassay. When
the interactions of the Plant extracts
were evaluated in the presence of
positive controls or the extract of the
sludge, synergistte, antagonistic, and ad-
ditive interactions were observed.
However, the extract of the plants grown
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on the sludge amended soil were
observed to inhibit the mutagenic activi-
ty of the sludge extract.
The results indicate that municipal
sludges may contain a persistent
mutagenic residue. In addition, leaching
of mutagens may be a serious problem
in coarse textured soils, or in areas
which receive high amounts of rainfall.
Uptake of mutagens into plants does not
appear to be a significant problem for the
sludges, soils, plants, and application
rates employed, as none of the extracts
of plants grown on sludge amended soil
induced a positive response.
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
From the time man began to congregate
in urban centers, the safe and economical
disposal of municipal sewage has been a
major concern. Given the present status of
rising fertilizer costs and water shortages,
it is of vital importance to our nation to use
every effort to recycle the nutrients and
water present in municipal sewage.
Large quantities of municipal sewage
sludge are generated each day in the
United States, much of which is ultimately
disposed of by land application. Land treat-
ment is designed to provide for the recycl-
ing of water and nutrients, and the reten-
tion or degradation of organics in the sur-
face layer of soil. Health risks which may
be associated with land application of
sludge include pathogens (e.g., bacteria,
viruses, protozoa and helminths), trace
elements (e.g., cadmium, lead, and zinc),
nitrates and organics. While much informa-
tion exists pertaining to the environmental
fate of specific pathogens, heavy metals,
and nitrate, much less is known about the
fate and composition of organic chemicals
in municipal sewage.
A partial list of organic chemicals which
have been identified in municipal
wastewater or sludge includes a variety of
chemical classes such as chlorinated
solvents, polychlorinated biphenyls (RGBs),
and polycyclic aromatic hydrocarbons.
Many of these chemicals have been found
to induce mutagenic effects in short-term
bioassays.
Care must be taken in the disposal of
municipal sewage to prevent the transmis-
sion of mutagenic compounds to the
human population. In a properly managed
land treatment system, organic mutagens
in a sludge amended soil may be
transformed, degraded or immobilized.
Under conditions of mismanagement or en-
vironmental stress, organic mutagens may
be released from the facility by volatiliza-
tion, leaching into groundwater, runoff into
surface waters, or plant uptake. The present
study was conducted to evaluate the fate
of organic mutagens in a land application
system.
Objectives
1. To evaluate the bacterial mutagenici-
ty of the organic extractable fraction
of municipal sludge from a variety of
different sources.
2. To monitor the mobility and stability of
these mutagens in municipal sludge
amended soil.
3. To assess the potential for plant up-
take of these mutagens from sludge
amended soils.
Procedures
Seventeen municipal sludges were
selected for evaluation to represent a range
of domestic and industrial contributions. In
addition, one treatment plant was selected
from which multiple samples were collected
over a ten month period to provide
preliminary data on the variability of
mutagenic constituents in sludge from a
single source. Sludges were sequentially
extracted using methylene chloride and
methanol, and three fractions (methylene
chloride, methanol and a combined frac-
tion) were tested using the standard
Sa/mone//a/microsome assay.
To evaluate stability, sludge was applied
on three separate occasions at two applica-
tion rates to 18 undisturbed monolith
lysimeters. Soil samples were collected
from the sludge amended soil and control
lysimeters immediately following and 28,56,
116,156,175, and 210 days after sludge ap-
plication. Soil samples were extracted with
methylene chloride and methanol using the
same procedures as were utilized for the
sludge samples.
To evaluate mobility, leachate water was
collected from suction cups installed 75 cm
below the zone of incorporation. The
leachate water was concentrated on a com-
bined XAD2/XAD7 resin, and the adsorb-
ed organics eluted with acetone. The
residue from the leachate water was taken
to dryness and redissolved in acetone for
testing in the bioassay.
Finally, a limited study was conducted in
the greenhouse to evaluate the potential for
translocation of mutagens into alfalfa and
bermudagrass grown on sludge amended
soil. Bioassays were also conducted on
mixtures of the plant extracts with known
mutagens to evaluate potential interactions
of plant constituents with mutagenic
chemicals.
Results
The data from this study and that
reported in the literature indicate that a
tremendous variability can be anticipated
between samples of sludge from different
sources, as well as different samples of
sludge from same source. In the present
study, the specific activity of sludge frac-
tions ranged from below detection to 187
net revertants per 10 mg of residue (approx-
imately five times background); while, the
weighted activity ranged from below detec-
tion to 1197 revertants per gram of sludge.
For comparative purposes, a Weswood soil
and a wood preserving waste induced a
specific activity of 67 and 141 net revertants
per milligram of residue, respectively; and,
a weighted activity of 2 revertants per gram
for the Weswood soil, and 20,070 revertants
per gram for the wood preserving waste
(Brown et a/., 1985 and Donnelly et a/.,
1987). Thus, it appears that bioassays can
serve as an indicator of the mutagenic ac-
tivity of municipal sludges, although
chemical analysis is an important tool to
identify the components of active samples.
The data from this study showed that a
minimum of eight weeks were required for
the weighted activity of the soil to reach a
maximum, and that the residual mutagens
in the soil were persistent, and could still
be detected at appreciable levels as long
as 207 days following waste application.
Three of the treatments, the Padina sand
which received one application of 50 Mg/ha
of sludge and the Weswood soil which
received 50 Mg/ha or 100 Mg/ha of sludge,
appeared to return to a weighted activity
which was approximately equal to the
weighted activity observed in the back-
ground soil samples during this time.
In the present study, appreciable quan-
tities of mutagens were detected in several
of the leachate samples. The specific ac-
tivity of the leachate samples ranged from
below detection to 207 revertants per 10
mg. Nine of twenty-six leachate samples in-
duced a doubling of revertants at the
highest dose tested. Two of the leachate
samples collected from the lysimeters
which received 150 Mg/ha of sludge induc-
ed a positive response. In addition, one of
the samples collected from the Padina sand
which received 50 Mg/ha induced a
positive response. These data indicate that
municipal sludge amended soils can
release mutagens into the soil pore water
(Brown et al., 1984).
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The weighted activity of the leachate
water from the soils best defines the quan-
titative bacterial mutagenicity of the
leachate water. The weighted activities of
the leachate samples collected from the
sludge amended soils ranged from below
detection to 416 revertants per liter. Twelve
of twenty-seven samples induced a
weighted activity of greater than 100 rever-
tants per liter, with four of those samples
inducing a weighted activity of greater than
300 revertants per liter. Thus, the weighted
activity of the leachate samples was often
more than twice that of drinking water, and
occasionally greater than the weighted ac-
tivity of surface water contaminated with a
wood preserving waste.
For the sludges, soils, and plants
employed for this study, there does not ap-
pear to be a significant potential for
transmission of mutagens to the food chain
through plants. The results indicate that at
all dose levels tested, none of the extracts
of plants grown on sludge amended soil in-
duced a positive response in the
Salmonella mutagenicity assay. Although
the results indicate that mutagens were not
detected in the plant extracts, this certain-
ly does not indicate that plants will not ad-
sorb mutagens from sewage sludge
amended soils. The lack of mutagenic
response may be due to either 1) the
mutagens were not taken up by plants at
levels which could be detected by the
bioassays or 2) the mutagens were taken
up by and into the plants, but plant consti-
tuents effectively inhibited the activity of the
translocated mutagens.
The data from the literature and from the
present study indicate that the potential of
plant uptake of mutagens is limited. Addi-
tional information from the literature in-
dicates that plant uptake will largely be
governed by a chemical's water solubility,
rate of biodegradation, and the clay con-
tent of the soil. Thus, the major health
threat of plant uptake of mutagens is likely
to be associated with the application of an
unusually toxic sludge, unusually high con-
centrations of sludge, or sludge which had
been weathered which may contain signifi-
cant quantities of water soluble mutagens.
Conclusions
1. The bacterial mutagenicity of
municipal sewage sludges exhibits tremen-
dous variation both between different
sources and from a single source.
2. The specific activity of several sludge
fractions was not appreciably greater than
the solvent control. For the mutagenic
sludges, the maximum specific activity was
188 net revertants per 10 milligrams for the
methanol fraction from the AD2 sludge with
metabolic activation; and, the maximum
weighted activity was 1197 net revertants
per gram of sludge for the combined frac-
tion of the SM4 sludge.
3. There does not appear to be a correla-
tion between the level of mutagenicity in
sludge, and the level of industrial contribu-
tion; however, this correlation may be more
direct if it were possible to base this com-
parison on the specific type of industrial
contribution.
4. Although a limited number of sludge
fractions contained direct acting mutagens,
for the majority of sludge fractions,
mutagenicity was only observed in the
presence of metabolic activation.
5. For the sludges and soils studied, the
specific activity of the sludge amended soil
increased with time following application,
and required approximately six months to
reach a maximum activity before exhibiting
signs of detoxification. In addition, selected
fractions of samples collected as long as
207 days following sludge application con-
tinued to induce a response that was
greater than twice the concurrent solvent
control.
6. For the majority of treatments
evaluated, the weighted activity of the
sludge amended soil was unchanged or
slightly reduced for as long as 207 days
following sludge application. However, for
the fractions extracted from the Padina
sand which received one sludge applica-
tion at a rate of 50 Mg/ha, the weighted ac-
tivity of the sludge amended soil collected
175 days after application was approximate-
ly equal to the weighted activity of the con-
trol soil.
7. Several samples of leachate water from
the sludge amended soil were found to con-
tain appreciable levels of organic
mutagens. Although the chemical consti-
tuents of these samples were not identified,
the results do indicate that mutagenic
chemicals can migrate below the zone of
sludge incorporation.
8. None of the extracts of plant samples
tested induced a mutagenic response.
Although these results suggest that
translocation of mutagens did not occur, in-
hibition of mutagens by chlorophyll in the
plants may have masked the activity of
mutagenic chemicals which were present
in the plant extracts.
Recommendations
1. Although bacterial mutagens were
detected in most of the sludges tested, a
more accurate description of the genotox-
ic potential of the municipal sludges would
be obtained if additional testing were con-
ducted using assays to measure DNA
repair and chromosome damage, and
these results were combined with a
chemical analysis.
2. Short-term bioassays should be utiliz-
ed as a tool for determining the persistence
of mutagens in sludge amended soils.
3. The extraction procedure used for
sludge and soil samples in this study is
useful for environmental monitoring;
however, a more detailed fractionation pro-
cedure would be valuable to isolate the
most active mutagens in the sludge.
4. The rate of sludge application should
be carefully monitored and regulated to pre-
vent sludge born mutagens from leaching
into the subsurface environment.
5. Additional research is needed to
evaluate the interactions of climatic condi-
tions and soil texture, especially as it affects
degradation and leaching of mutagens in
sludge amended soil.
References
Donnelly, K. C., K. W. Brown and D. Kamp-
bell. 1987. Chemical and biological
characterization of hazardous industrial
waste. I. Prokaryotic bioassays and
chemical analysis of a wood-preserving
bottom-sediment waste. Mutat. Res.
180:31-42.
Brown, K. W., K. C. Donnelly, J. C. Thomas
and P. Davol. 1985. Mutagenicity of Three
Agricultural Soils. The Sci. of the Total
Environ 41:173-186.
Brown, K. W., K. C. Donnelly and J. C.
Thomas. 1984. Use of Short-Term
Bioassay to Evaluate Environmental Im-
pact of Land Treatment of Hazardous In-
dustrial Waste. Final Report. Grant No.
R. 80770101. U.S.E.P.A., Robert S. Kerr
Environmental Res. Lab. Ada, OK.
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K. W. Brown andK. C. Donnelly are with Texas A&M University, College Station,
TX 77843.
Kate Smith is the EPA Project Officer (see below).
The complete report, entitled "The Stability and Mobility of Mutagenic Activity
from Wastewater and Sludge in Agricultural Soils," {Order No. PB 88-237
243/AS; Cost: $25.95, 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
United States Center for Environmental Research
Environmental Protection Information
Agency Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S1-88/002
0000329 PS
U S EJIWR PROTECTION ftSEMCT
CHICAGO IĞ- 60604
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