United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
X
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Research and Development
EPA/600/S1-89/001 Aug. 1989
&ERA Project Summary
Fractionation of
Mutagensfrom Municipal
Sludge and Wastewater
Cherylyn W. Wright, Dorothy L. Stewart, and Walter C. Weimer
There are potential environmental
concerns from the disposal of munic-
ipal wastewater effluents and sewage
treatment plant sludges. This report
summarizes the microbial mutagenic
evaluation and chemical analysis of
13 sewage sludge samples from
various sewage treatment plants
located in Texas and Washington
state. The sewage sludge samples
were air-dried followed by sequential
Soxhlet extraction with three organic
solvents of increasing polarity, i.e.,
pentane, methylene chloride, and
methanol. The organic extracts from
three of the samples were further
fractionated by normal phase high-
pressure liquid chromatography
(HPLC). The obtained extracts and
fractions were bioassayed for mi-
crobial mutagenic response using
the standard histidine reversion
assay with Salmonella typhlmurium
strains TA98 and TA100, both with
and without S9 metabolic activation.
Extracts and fractions were chemical-
ly analyzed by high resolution gas
chromatography (GC) using a variety
of element-specific detectors, gas
chromatography/mass spectrometry
(GC/MS), and (to a lesser extent)
liquid chromatography/mass spectro-
metry (LC/MS).
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research project that Is fully docu-
mented in a separate report of the
same title (see Project Report order-
Ing information at back).
Introduction
Potential genotoxicity of sewage
sludges may be of particular concern
relative to agricultural uses and to land
disposal; likewise, the disposal of treated
wastewater is of considerable environ-
mental concern because of the possible
introduction of mutagens into the receiv-
ing water. The spectrum of mutagens
(and organic compounds in general)
found in the sludges is likely to be
different than that found in treated
wastewater. This is related to the fact that
many of the incoming organic com-
pounds become associated with the
sludge, and the biological treatment and
disinfection processes remove some
compounds and synthesize many more
in the treated wastewater. The focus of
this project was on the separation and
identification of mutagenic constituents in
sewage treatment plant sludges. At the
beginning of the project, ten different
sewage sludges were examined. Six were
from various locations in Texas, and four
were from various locations in Washing-
ton state. The organic constituents of
these sludges were isolated by organic
extraction and were screened for
microbial mutagenicity. All of the extracts
were examined qualitatively by high
resolution gas chromatography (GC)
using a variety of detectors to evaluate
similarities in gross chemical composi-
tion. In addition, selected samples were
analyzed by gas chromatography/mass
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spectrometry (GC/MS). These analyses
were performed in an attempt to identify
the principal components found in each
of the extracts.
After the initial preliminary chemical
characterization of the original ten
sewage sludge extracts, it was decided
that further fractionation would be neces-
sary to separate and identify any muta-
genic components of sewage sludge.
Three new sewage sludge samples from
Texas were provided from the same
location, one was an original sludge,
while the two others had been stored in a
Lysimeter for varying amounts of time.
These sludges were extracted in a similar
manner as the previous ten sludges. The
extracts were then subjected to further
fractionation based on polarity using
normal phase, high-pressure liquid
chromatography (HPLC). The HPLC
fractions were screened for microbial
mutagenic activity as were the above
extracts. Selected fractions were ana-
lyzed by GC/MS and liquid chromatog-
raphy/MS (LC/MS).
Procedure
Samples of all 13 of the sewage
sludges as received were air-dried in a
laminar flow hood at room temperature
until they achieved a constant weight;
percent water compositions were deter-
mined. The total carbon contents of the
air-dried samples were determined by
complete combustion. The organic con-
stituents of the dried sludges were
isolated by sequential Soxhlet extraction
using pentane, methylene chloride, and
methanol solvents. Total amounts extract-
ed by each solvent were calculated after
solvent removal.
The pentane, methylene chloride, and
methanol Soxhlet extracts from three
sewage sludge samples were further
fractionated by normal phase HPLC using
a ternary mobile phase gradient of hex-
ane, methylene chloride, and methanol.
Three fractions were collected from the
pentane extracts, four fractions were
collected from the methylene chloride
extracts, and five fractions were collected
from the methanol extracts. Total
amounts in each fraction were calculated
after solvent removal.
Solutions of the extracts and HPLC
fractions from the sewage sludge sam-
ples were tested for mutagenicity in the
Salmonella histidine reversion assay after
evaluations of solvents for maximized
dissolution. Salmonella typhimurium
tester strains TA98, which screens for
frame shift mutagens, and TA100, which
screens for point mutagens, were select-
ed. Each of the extracts and HPLC
fraction was tested with the standard
plate incorporation method at five or
greater concentration levels of test
extract with Aroclor-induced rat liver
homogenate (S9) metabolic activation.
Two to five concentration levels of each
extract were tested without S9 metabolic
activation. The levels of test extracts and
HPLC fractions ranged from 2 to 1250
p.g/plate. The concentration of sludge
extracts and HPLC fractions were chosen
based on preliminary toxicity testing
which indicated cytotoxicity concentra-
tions at the level of 2000 ug/plate. Also,
the higher concentrations of extracts (i.e.,
1000 ug/plate or greater) consistently
exhibited undissolved sludge particulates
when plated with agar.
Revertant colonies per petri plate were
counted electronically (or by hand if
precipitates were present). An extract or
HPLC fraction was considered to give
positive microbial mutagenic response if
it showed a two-fold increased number or
revertants form background or if there
was a positive linear dose response with
a correlation coefficient of 0.75 or greater
and an intercept on the ordinate axis
within 20% of the negative control for the
day as determined by linear regression
analysis.
Selected sludge sample extracts were
analyzed by GC using a HP 5880A gas
chromatograph. The extracts were
analyzed using multiple detectors includ-
ing a flame ionization detector (FID), a
nitrogen-phosphorus detector (NPD), an
electron capture detector (ECD), and a
sulfur-specific flame photometric detector
(FPD). The percent of each extract that
was chromatographable was estimated.
Elemental sulfur was quantified in some
of the sludge extracts by calibration of
the FPD with an elemental sulfur stand-
ard. Some unknown nitrogen-containing
components detected in the extracts
were quantified based on the NPD
response of an internal standard,
benzo[b]carbazole.
Selected extracts and HPLC fractions
were analyzed by GC/MS using an HP
5982 or 5987 quadrupole mass spectro-
meter interfaced to an HP 5710 or 5840
gas chromatograph. The mass spectro-
meters was operated in the electron im-
pact mode at 70 e V and were scanned
from 50 to 500 atomic mass units (amu).
Some of the extracts and fractions were
derivatized with diazomethane prior to
analysis.
Two HPLC fractions were analyzed by
LC/MS using an HP 5988A thermospray
system. A binary mobile phase of 95:5
(v:v) 0.1 M ammonium acetate:acetonitrile
and acetonitrile was used. The masj
spectrometer was scanned from 140 \
600 amu. Prior to the analyses by LC/MS
many of the extracts and fractions wer<
analyzed by HPLC using a photodiod<
array UV detector. UV spectra wer<
plotted from responses at wavelengths o
254 nm and 340 nm. Samples were
selected for LC/MS if it was felt there was
adequate response on the UV detector tc
be detected by the mass spectrometer.
Results
The water content of the sludges was
highly variable, ranging from less than
1% water to nearly 85% water. The total
carbon contents of the dried sludges
were in the 22-31% range. The percent
solvent extractable ranged from aboul
2% to 15%. There was a direct linear
correlation between the carbon contents
of the sludges and their percent
extractable. The HPLC chromatograms
showed there were components of
increasing polarity in the extracts from
solvents of increasing polarity.
The sludge extracts and HPLC
fractions were difficult to bioassay mainly
due to the presence of undissolved
sludge particulates when the samples
were plated with agar. Data resulting fro
assays in which the formation
precipitates occurs cannot be meaning-
fully expressed, and they cannot be
useful for comparing the activities of dif-
ferent materials. The microbial mutagenic
responses of all the sludge extracts and
fractions was low, regardless of tester
strain or metabolic activation. All extracts
expressed less than 0.5 revertants per
mg and all fractions expressed less than
1.2 revertants per mg microbial muta-
genic activity. Fewer extracts exhibited
microbial mutagenic activity when tested
with strain TA100 compared to strain
TA98. Some of the mutagenic responses
were direct-acting. The microbial muta-
genic activity of the extracts and fractions
were not clearly separated into any one
extract or fraction.
While there was considerable variability
for the percent chromatographable
amongst the extracts from the sludge
samples, approximately two-thirds of the
pentane-extractable components, one-
third of the methylene chloride-extract-
able components, and 10% of the meth-
anol-extractable components were chro-
matographable under the conditions em-
ployed. Three major components elec-
tron-capturing components were detected
in all the extracts, regardless of origidfl
Elemental sulfur as well as three otheV
sulfur-containing components were de-
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tected in the low parts per thousand
(mg/g) region in some of the sludge
extracts when analyzed by FPD.In ad-
dition, some high molecular weight,
nitrogen-containing components were de-
tected in the parts per thousand region
(mainly in the methanol extracts) in some
of the sludge extracts when analyzed by
NPD.
Generally, the same components were
found in all the different sludge extracts,
regardless of origin. The components did
tend to be extracted preferentially into
different solvents. The major components
of all the sewage sludge extracts and
fractions as identified by GC/MS were
fatty acids having 16 or 18 carbon
molecules. Overlap of these components
between the extracts and in adjacent
fractions was observed. There was also
evidence of the presence of benzenedi-
carboxylic acids in some of the HPLC
fractions. No compounds were identified
by LC/MS due to lack of sensitivity.
Conclusions and
Recommendations
Accurate assessments of mutagenic
activities were difficult due to the
formation of precipitates with the agar
using the standard microbial mutagenicity
assay. Extreme care must be taken in the
evaluation of sewage sludge samples for
mutagenic activity to prevent inaccurate
reporting. Using the methods employed,
the microbial mutagenicity, and hence,
the mutagens of the sewage sludge
samples were not clearly separated. The
overall levels of mutagenicity in all
extracts and fractions were low (less than
1.2 revertants per mg), and no specific
mutagens were identified in any of the
extracts of fractions using GC and
GC/MS techniques.
Due to the extremely low levels of
mutagenicity expressed by the extracts
and fractions of the sewage sludge
samples studied, further studies should
concentrate on samples that exhibit
higher levels of biological activity as
determined by a screening method. Fur-
ther characterization would only be done
as mutagenicity dictated. This may
increase the likelihood of identifying
some mutagens present in sewage
sludge samples. The estimated low
amounts of the organic extracts and
fractions that were amenable to analysis
by GC and GC/MS indicates the need for
other sensitive chemical analyses for
high molecular weight biological and/or
highly polar components to better char-
acterize the sewage sludge.
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Cherylyn W. Wright, Dorothy L Stewart, and Walter C. Weimer are with Battelle
Pacific Northwest Laboratories, Richland, WA 99352.
M. Kate Smith is the EPA Project Officer (see below).
The complete report, entitled "Fractionation of Mutagens from Municipal Sludge
and Wastewater," (Order No. PB 89-161 491/AS; Cost: $15.95, 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
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S1-89/001
000085833 PS
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