United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S7-81-080 Dec. 1981
Project Summary
Bioassay and Chemical
Analysis for Hazardous
Materials in Residual Oils
T. D. Kaczmarek and A. Zervins
Residual fuel oils have been con-
sidered as an energy source for the
chemically active fluidized-bed (CAFB)
process, designed to produce a low-
sulfur, low-Btu fuel gas suitable for
power-plant utilization in a conven-
tional steam generator. This project
was designed to ascertain the presence
of potentially bio-hazardous substances
in residual fuel oils.
In spite of the length of time residual
fuel oil has been available, there has
been little definitive study of what
would be typical compositions for
such materials (or whether even a
composition can be "typical"). There
has been less effort to assay this
product for bio-hazard, and there has
been no real effort to associate possible
bio-hazard with composition. The very
nature of refinery processes leads to
residual oils which will contain poly-
cyclic materials, nitrogen, oxygen,
and sulfur heteroatoms, and be ap-
pended with diverse functional groups.
All these types of materials would be
suspect as potential carcinogens if
water soluble or present in high enough
concentrations.
In fulfillment of this contract, bio-
assay and chemical analyses were
performed on residual fuel oils. The
bioassay work would have been sim-
plified if the contract wording had
been followed in performing bioassays
only on as-received oils. However,
Westinghouse (in the spirit of the
contract and with project officer ap-
proval) applied the bioassay to par-
ticular oil fractions in an effort to more-
deeply probe mutagenicity and poten-
tial carcinogenicity of this type of
material. It was determined early that
mutagenicity (i.e., the accepted mea-
sure of potential carcinogenicity) was
not a serious concern with the 26 as-
received residual oils.
The analyses sought the maximum
information for material characteriza-
tion compatible within the time-cost
constraints of the contract. The major
difficulties associated with these
chemical analyses were sample com-
plexity, low sample volatility, and lack
of reference data.
This Project Summary was devel-
oped by EPA's Industrial Environmen-
tal 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
This project produced several new,
specific chemical and bioassay questions
which need to be considered and
answered when using fuel oil for power
generation. Of particular relevance to
environmental impact analyses is the
discovered novel fluorescent property of
fractionated sample and its associated
mutagenicity.
The data bank for the report was
derived from the following completed
tasks:
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41 Residual fuel oil samples received.
26 As-received samples bioassayed.
32 As-received samples pre-frac-
tionated by column chromatogra-
phy into seven fractions each.
23 Samples examined by combined
gas chromatography/mass spec-
troscopy/data system as seven
fractions each.
6 Samples bioassayed as seven
fractions each.
1 Sample partitioned into dimethyl-
sulfoxide solubles and insolubles
and then pre-fractioned by column
chromatography into seven frac-
tions each.
1 Sample, partitioned and pre-
fractioned as above, examined by
bioassay and gas chromatogra-
phy/mass spectroscopy/data
system.
Direct insertion mass spectrometry,
ultraviolet fluorescence, infra-red anal-
yses, and elemental composition studies
have been performed within the time-
cost framework.
Bioassay of the as-received fuel oils
required solubilization in DMSO or p-
dioxane to introduce the material into
the bacterial test system. Water solubil-
ity is negligible and whatever part is in
solution does not elicit mutagenic re-
sponse in the Ames test. The two-
solvent-extracted residual oil part was
generally non-mutagenic, although
some oils (19%) exhibited a very weak,
positive test result. Residual fuel oils
can be fractionated by column chroma-
tography so that, within the scope of this
effort, a particular fraction is consistently
mutagenic. Even an oil that is not
mutagenic as-received may be positive
in this specific fraction. As with as-
received oils, this particular fraction will
show varying degrees of mutagenicity.
The most powerful tool, discovered in
this project for isolating mutagenicity of
a residual oil is the dimethylsulfoxide
partitioning of the as-received sample
followed by column chromatography
fractionation of the dimethylsulfoxide
soluble portion. In a single test run, each
of the seven fractions obtained was
mutagenic.
Four major factors affect the analyses
for organic compounds in residual fuel
oil:
1. The sheer complexity of the sample
in terms of the hundreds of com-
pounds it contains.
2. The inconsistent source, treatment,
and post-treatment refinery blend-
ing from sample to sample.
3. The essentially non-volatile nature
of residual fuel oils; this precludes
complete sample analyses by the
most powerful analytical tool,
combined gas chromatography/
mass spectroscopy/data system.
4. The lack of signal reference data to
tie the sample components to
known materials.
The organic analyses obtained in this
program suggest that:
1. The most mutagenic components
of a residual fuel oil reside in the
carbazole-type compounds class.
2. The second most mutagenic agents
of a residual fuel oil are associated
with polycyclic ketones such as
anthraquinone and benzanthrone.
Concentration-related toxic effects
appear to prevent these substances
from being bioassayed in bacterial
systems as the most mutagenic
agents.
3. Major mutagenic substances in
residual fuel oils are not associated
with "classical" organic classes,
such as polycyclic aromatics (e.g.,
benzopyrene).
4. The unusual, orange fluorescence
effect under ultraviolet irradiation
of the more mutagenic sample
fractions suggests that this phe-
nomenon should be studied further
for routine bioassay application in
fossil fuel analyses.
The elemental composition of residual
oils is elucidated to some extent in this
report. The nature of inorganic material
occurrence is such that it does not
appear that these materials can be
solubilized as required for performance
of bioassay testing.
Based on the work that has been
performed, it is recommended that:
1. Because there is little analytical
reference data to connect materials
of interest in residual fuel oils to
known compounds, efforts should
be continued to identify mutagens
in residual fuel oils that will add to
the known list of such bio-active
materials.
2. Residual fuel oils should be ex-
amined by second-tier bioassay
such as Syrian Hamster Embryo
cells, Chinese Hamster Ovary,
Sister Chromatid Exchange, or
Mouse Lymphoma System. It ap-
pears that mutagenicity of residual
fuel oils does not principally reside
in the "classical" polycyclic aro-
matic hydrocarbon portion. It is
therefore possible that accepted
"relationships" between the Ames
test and potential carcinogenicity
of residual oil components may
not be reflected in the literature
and may not follow expected
trends.
3. A test plan should be formulated
and carried out to evaluate the
potential of usage/environment
concentration of mutagenic factors,
and to check undesirable environ-
mental alterations which residual
fuel oil can undergo. Any muta-
genicity of an as-received residual
fuel oil studied in this work has
been very weak. Fractions from
mutagenic oil as obtained from
dimethylsulfoxide partitioning/
column chromatography can be
very strong, due to active agent
concentration.
4. Further chemical identification of
mutagenic species should be car-
ried out. This would certainly
include use of the dimethylsulfox-
ide partitioning/column chroma-
tography technique. It would also
include re-evaluation of the well
known "separation by classes"
technique of Fuson and Shriner
which could now be effective since
presumed obstructive and incon-
sequential matrix (i.e., dimethyl-
sulfoxide insolubles) is removed
from the sample. This effort should
also include attempts to upgrade
the column chromatography
method.
5. The feasibility of developing
dimethylsulfoxide partitioning and/
or the phenomenon of ultraviolet
fluorescence as a "field test" for
mutagenicity should be explored.
6. Each of the above recommenda-
tions should be applied for chemi-
cal analysis and genetic toxicology
examination of materials, product,
and waste from other energy con-
version processes such as coal
gasification, coal liquefaction, and
shale oil extraction.
7. The 15 as-received oils not ex-
amined should be bioassayed by
Ames test. The collection of 41
residual fuel oil samples assembled
here is unique and probably could
never be duplicated. Given the
"individuality" of composition of
such samples, all 41 should be
subjected to DMSO partitioning
followed by column chromatogra-
phy. The final fractions should be
bioassayed and examined by or-
ganic chemical analysis.
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8. Any of the above work should be
performed in serial rather than
parallel fashion consisting of:
sample separation, bioassay, and
chemical analysis. Results of a
prior operation should determine
if there will be a subsequent opera-
tion.
In summary, environmental affects of
residual oil use in power generation is
minimal.
T. D. Kaczmarek and A. Zervins are with the Westinghouse Research and
Development Center. 1310 Beulah Road, Pittsburgh. PA 15235.
Samuel L. Rakes is the EPA Project Officer (see below).
The complete report is in two volumes, entitled "Bioassay and Chemical
Analysis for Hazardous Materials in Residual Oils":
Volume 1. {Order No. PB 82-117 078; Cost: $24.00. subject to change)
Volume 2. (Order No. PB 81-190 944; Cost: $34.50, 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 can be contacted at:
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
U.S GOVERNMENT PRINTING OFFICE 1981—559-017/7402
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