United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600/9-81-009
March 1981
Research and Development
&EPA
A Matrix Approach to
Biological
Investigation of
Synthetic Fuels
-------
EPA 600/9-81-009
March 1981
A MATRIX APPROACH
TO BIOLOGICAL INVESTIGATION OF SYNTHETIC FUELS
Proceedings of a Conference Cosponsored by
the U.S. Environmental Protection Agency and Oak Ridge National Laboratory
through the EPA/DOE Fossil Fuels Research Materials Facility
Research Triangle Park, North Carolina
April 26, 1979
Project Officer
David L. Coffin
Research Advisory and Special Studies Office
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or recom-
mendation for use.
ii
-------
ABSTRACT
Documentation is provided for a conference cosponsored by the U.S.
Environmental Protection Agency and Oak Ridge National Laboratory and held
in Research Triangle Park, North Carolina on April 26, 1979. The general
topic is toxicological assessment of health effects from the rapidly
developing synthetic fuels industry.
In particular, the discussions focus on the Paraho crude shale oil
that was produced by Development Engineering, Inc. (Anvil Points, Colorado)
and refined into diesel and jet fuels by the Standard Oil Company of Ohio.
Summaries of both operations are presented. Also discussed is the collection,
storage, and distribution to toxicologists of sample materials from these
operations by the U.S. Environmental Protection Agency/U.S. Department of
Energy Fossil Fuels Research Materials Facility (Oak Ridge National Laboratory).
Other chapters survey ongoing and planned testing of the Paraho shale oil
materials by investigators from Oak Ridge National Laboratory, Battelle Pacific
Northwest Laboratories, Lawrence Livermore Laboratory, and the U.S. Environ-
mental Protection Agency. The application of microbial, cellular, and whole-
animal bioassays is considered.
iii
-------
CONTENTS
DISCLAIMER ii
ABSTRACT iii
ABBREVIATIONS vii
1. OVERVIEW OF THE FOSSIL FUELS RESEARCH MATERIALS FACILITY 1
D. L. Coffin.
Introduction 1
The Matrix Approach 2
History 2
Outlook 3
2. DISTRIBUTION OF PARAHO OIL SHALE AND SOHIO-REFINED PARAHO
SHALE OIL MATERIALS 5
W. H. Griest and M. R. Guerin
Introduction 5
Collection of Samples 5
Distribution of Samples 8
Return of Data 8
Storage in the Repository 8
Acknowledgments 11
3. RECENT PARAHO OPERATIONS 12
R. N. Heistand and J. B. Jones, Jr.
Introduction 12
Recent Paraho Operations 16
Research 22
Conclusions 26
Acknowledgments 27
References 27
-------
4. REFINING OF SHALE OIL BY SOHIO 28
D. L. Cawein
Introduction 28
SOHIO's Toledo Refinery 29
Special Considerations in Refining Shale Oil 30
Steps in the SOHIO Refining Process 30
5. WORK PLAN FOR SHALE OIL STUDY, BIOLOGY DIVISION,
OAK RIDGE NATIONAL LABORATORY 36
T. K. Rao and J. L. Epler
Introduction 36
Level One: Cellular Bioassays 37
Level Two: Mammalian Toxicity Bioassays 41
Input to Health Effects Assessment 45
References 46
6. SHALE OIL BIOASSAYS AT BATTELLE PACIFIC NORTHWEST LABORATORIES . . 47
R. A. Pelroy
Introduction 47
Samples to be Assayed 47
Fractionation Methods 48
Bioassays to be Applied 49
Summary 49
7. APPLICATION OF A BATTERY OF SHORT-TERM BIOASSAYS FOR
TESTING THE GENETIC TOXICITY OF PARAHO SHALE OIL PRODUCTS 51
F. T. Hatch and H. Timourian
Introduction 51
Available Bioassays 53
Strategy of Application 62
Samples to be Assayed 64
Expected Results 66
Acknowledgment and Disclaimer 67
References 68
8. EVALUATION OF POTENTIAL TOXICITY OF SYNTHETIC FUEL
COMBUSTION PRODUCTS 70
D. L. Coffin and J. L. Huisingh
Introduction 70
Ongoing and Planned Studies 70
Research Needs 71
Reference 72
VI
-------
ABBREVIATIONS
API
aprt
ATPase
BaP
CHO
DFM
DOE
DMA
EMS
EO
EPA
ESB
GC/MS
hgprt
hprt
i.d.
JP
LLL
ONR
ORNL
PAH
RNA
SCE
SOHIO
tk
TLC
American Petroleum Institute
adenine phosphoribosyltransferase
adenosine-triphosphatase
benzo(a)pyrene
Chinese Hamster ovary
Diesel Fuel Marine
Department of Energy
deoxyribonucleic acid
ethyl methane sulfonate
equivalent oil
Environmental Protection Agency
ether-soluble base
gas chromatography/mass spectrometry
hypoxanthine-guanine phosphoribosyltransferase
hypoxanthine phosphoribosyltransferase
inner diameter
Jet Propellant
median lethal dose
Lawrence Livermore Laboratory
Office of Naval Research
Oak Ridge National Laboratory
polycyclic aromatic hydrocarbon
ribonucleic acid
sister chromatid exchange
Standard Oil Company of Ohio
thymidine kinase
thin layer chromatography
via.
-------
1. OVERVIEW OP THE
FOSSIL FUELS RESEARCH MATERIALS FACILITY
D. L. Coffin
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
INTRODUCTION
As everyone knows, there is a shortage of energy, and there will certainly
be a place for alternate synthetic fuels derived from either coal or shale.
Accordingly, there is a great need for rapid application of toxicology in
this developing industry. The U.S. Environmental Protection Agency (EPA)/U.S.
Department of Energy (DOE) Fossil Fuels Research Materials Facility ("Reposi-
tory") was created to involve toxicologists "on the ground floor" in the in-
dustry, before appreciable human exposure occurs through environmental prob-
lems, in plants, or with users. EPA's hope was that, with the synthetic fuels
industry, we will be "ahead of the game" for a change.
A substantial amount of research must be accomplished, and accomplished
quickly, if it is to have any impact. Therefore, EPA welcomes and encourages
the participation of all interested toxicologists; the Repository was created
as a focal point for such collaboration. In the beginning, we participated
in numerous discussions with Lt. Comdr. Leigh Doptis of the U.S. Navy, and we
became especially cognizant of the Navy's needs.
-------
THE MATRIX APPROACH
The term "repository" should not imply a place where chemicals are merely
"put in a closet." The primary function of the Repository is to provide a way
of moving from the technologies to the biologists and back to the technologies,
so that the biologists work with materials relevant to the technologies, that
they communicate with one another, and that they have unified specimens to per-
mit nearly automatic comparison of one biological technique to another. This
matrix approach is important from the standpoint of such developing models as
the Ames assay, because — despite all the ongoing work — we still lack close
comparison between these methods and some of the older, more classical methods
of skin bioassay, etc. The Repository automatically encourages comparison of
this sort, because a matrix of investigators applies different techniques to
identical materials. The function of the Repository is not only to provide
the samples, but also to aid in communication between the investigators and
the technologists and administrative personnel.
HISTORY
The Search for Samples
A fundamental goal in creating the Repository was to encourage toxico-
logical studies of synthetic fuel materials before there was large capital
investment and commercial development. We wanted to become involved at a
point somewhere between the developmental stage (the "bench model" level) and
the commercial drive. We were interested in enterprises of sufficient size
to make testing practical and to provide enough material for complete anal-
ysis. In the case of shale oil, for example, we envisioned an analysis that
would extend from mining all the way through end use of the product, to pro-
vide input on environmental problems (with mining and crushing), worker health
problems (retorting, refining, distribution, and use), and problems associated
with end use (usually, some sort of combustion).
-------
At the beginning, it was difficult to find technologies that would lend
themselves to this type of analysis. We found, in certain instances, that a
technology had become outdated and was therefore unsuited for our purposes.
Alternately, many of the governmental efforts remained at a "bench model"
stage and were therefore equally unsuited to our desired approach (i.e., a
complete analysis).
The Paraho Operation
In view of this situation, the Repository team became quite excited at
the opportunity to examine the recent U.S. Navy/Paraho operation. Paraho re-
presented a chance to obtain sufficient materials for biological testing:
production of ~105 bbl (from our standpoint, a significant quantity) of crude
shale oil was planned. Because the Paraho operation was the best available
approximation of an initial commercial module, then, we concentrated most of
our resources on it. This, we felt, was a sensible approach: the Navy had
identified shale oil as the closest in time to a usable fluid fuel of any of
the alternate sources, and Paraho was (and is) the largest shale oil develop-
ment effort available to us.
To date, the Paraho operation has progressed all the way through the
stage of refining. The products are in the Repository, ready for distribution.
Materials from mining and retorting are already distributed.
OUTLOOK
Subsequent chapters in this volume lay out the plan that has been de-
veloped among individual investigators, show how the efforts of these inves-
tigators interrelate, and indicate how data will be returned to the technol-
ogies. The whole point of the effort, of course, is to help technologies
develop clean, safe fuels. If certain problems cannot be removed, the goal
is to develop methods of occupational hygiene that will prevent injury to
workers.
-------
The scope of this matrix approach extends far beyond the range of EPA's
immediate interests: it bears not only on environmental factors and problems
of the community at large, but also on worker health and product safety. (For
example, we are cooperating with the program of Comdr. Lawrence J. Jenkins of
the Naval Medical Research Institute at Wright-Patterson Air Force Base.) The
Repository will attempt to cover the entire field; the methods and lines of
communication developed for the Paraho operation will serve as a model for
investigation of other fuels.
Historically, the perspective of industrial toxicology has been applied
only after some sort of problem (e.g., illness in workers) has developed. EPA
sees the developing synthetic fuels industry as an unusual and important op-
portunity — indeed, as a challenge — for toxicologists to become involved "on
the ground floor" in assessing potential hazard. Hopefully, the coordinating
efforts of the EPA/DOE Fossil Fuels Research Materials Facility will help to
efficiently and successfully meet this challenge.
-------
2. DISTRIBUTION OF PARAHO OIL SHALE AND
SOHIO-REFINED PARAHO SHALE OIL MATERIALS
W. H. Griest
M. R. Guerin
Analytical Chemistry Division
Oak Ridge National Laboratory
Post Office Box X
Oak Ridge, Tennessee 37830
INTRODUCTION
The function of the U.S. Environmental Protection Agency (EPA)/U.S.
Department of Energy (DOE) Fossil Fuels Research Materials Facility ("Reposi-
tory") is to obtain, catalog, store, and distribute research materials from
synthetic fuels production to qualified health effects investigators. An addi-
tional function is to provide physical/chemical fractionation and characteriza-
tion of high-priority materials for health effects testing. Data from the study
of these materials are returned to the source of the samples. In the case of
the oil shale industry, materials corresponding to both production and refining
of shale oil are now available from the Repository. This paper briefly sum-
marizes the collection, cataloging, and distribution of these materials.
COLLECTION OF SAMPLES
Samples from the retorting of oil shale were collected by a Repository
subcontractor at the Paraho above-ground retorting process demonstration site
at Anvil Points, Colorado, in the fall of 1977. At that time, a 10s-bbl pro-
duction run for the U.S. Navy was in progress. Itemized in Table 2-1, these
-------
TABLE 2-1. REPOSITORY INVENTORY OF OIL SHALE
MATERIALS FROM PARAHO ABOVE-GROUND RETORT
Repository
Sample No.
4204
4206
4209
4205
4207
4208
4211
4212
4213
4203
4202
4201
4210
Date
Received
11-8-77
11-4-77
11-8-77
11-8-77
11-8-77
11-8-77
8-l-78a
8-l-78a
11-8-77
11-8-77
11-8-77
11-8-77
11-4-77
Description
Raw Shale
Airborne Raw Shale (hi-vol)
Raw Shale Particles from Baghouse
Retorted Shale
Airborne Retorted Shale (hi-vol)
Retorted Shale Particles from Baghouse
(collected from screw conveyor)
Retorted Shale Particles from Baghouse
(a) 0-10 um sized fraction
(b) >10 um sized fraction
(c) unsized
Product Oil
Product Water (oil separation)
Process Water (gas line drain)
Thermo-Oxidizer Stack Particles
Quantity
50 gal
21 g
5 gal
25 gal
10 g
5 gal
45 Ib
350 Ib
40 Ib
8 gal
2 gal
2 gal
50 mg
Date of receipt of sized particle fractions from subcontractor.
collected materials correspond to both bulk and airborne samples of raw and
retorted oil shale. The latter has been sized into respirable and nonrespir-
able particle fractions. Samples of the shale oil and of process and product
water also are available.
In late 1978 and early 1979, the Paraho shale oil was refined into diesel
and jet fuels by the Standard Oil Company of Ohio (SOHIO) at its Toledo, Ohio
refinery. SOHIO personnel collected samples of the raw and hydrotreated shale
oils, intermediate materials, and final, finished products in 55-gal stainless
steel drums. The samples were shipped to the Repository by mid-March, 1979.
Table 2-2 lists the Repository inventory of these materials.
Corresponding petroleum-derived jet and diesel fuel products (Table 2-3)
also were obtained directly from Comdr. L. J. Jenkins of Wright-Patterson Air
-------
TABLE 2-2. REPOSITORY INVENTORY OF SOHIO-REFINED AND -SUPPLIED
PARAHO SHALE OIL MATERIALS
Repository
Sample No.
4601
4602
4603
4604
4605
4606
4607
4608
4609
4610
4611
4612
Date of
Sampling
11-03-76
11-29-78
11-21-78
11-22-78
11-27-78
11-21-78
11-30-78
1-24-79
11-27-78
2-21-79
11-28-78
not sup-
plied
Description
Crude Shale Oil
Hydrotreated Shale Oil
Weathered Gas Feedstock
JP-5 Before Treating
(Precursor)
JP-8 Before Treating
(Precursor)
DFM Before Treating
(Precursor)
Hydrotreated Residue
JP-5 Product
JP-8 Product
DFM Product
JP-5 Product
Acid Sludge from DFM
Treatment
SOHIO
Reference No.
CSO-556
C5HTSO-554
WGFS-55
PRE JP5-555
PRE JP8-555
PRE-DFM-555
HTR-555
FIN JP5-554
FIN JP8-554
FIN DFM
FIN JP5-N-2A
AS- 5 2
Quantity
55 gal
55 gal
5 gal
55 gal
55 gal
55 gal
55 gal
55 gal
55 gal
55 gal
1 gal
5 gal
Repository
Sample No.
4613
4614
4615
4616
TABLE 2-3
Date
Received
1-19-79
1-10-79
1-10-79
1-10-79
. REPOSITORY INVENTORY OF PETROLEUM-
DERIVED JET FUELS AND DFM&
Material
JP-4 Product
JP-5 Product
JP-8 Product
DFM Product
Quantity
55 gal
55 gal
55 gal
5 gal
^Materials obtained from Comdr. L. J. Jenkins, Wright-Patterson Air Force Base,
Ohio.
-------
Force Base, Ohio, in early January of 1979. These fuels are "reference" mate-
rials which the Navy is using for comparison with shale-oil-derived fuels, and
they were obtained to allow investigators working with SOHIO-refined Paraho
shale oil materials to use the same "references."
DISTRIBUTION OF SAMPLES
In early May 1979, the SOHIO-refined Paraho shale oil materials and
Wright-Patterson Air Force Base petroleum equivalents were carefully mixed and
aliquoted into clean (methanol-rinsed, dried, and rinsed with sample itself)
amber borosilicate bottles, except where large sample volumes required other
containers. After the headspace was briefly flushed with argon, each container
was sealed with a Teflon-lined cap and labeled. Shipments to the investigators
were made in late May 1979. Table 2-4 shows the distribution of the materials.
Table 2-5 lists the names, addresses, and phone numbers of all investigators.
Requests for additional samples should be directed to L. B. Yeatts (FTS
624-4863; commercial 615-574-4863) or W. H. Griest (FTS 624-4868; commercial
615-574-4868).
RETURN OF DATA
Data returned by the investigators will be compiled and fowarded to the
Navy and other sponsors. SOHIO and Development Engineering, Inc. (Paraho)
have requested that all investigators allow them to review any papers before
publication; this review can be arranged through the Repository.
STORAGE IN THE REPOSITORY
Bulk quantities of the remaining materials are being stored under ambient
conditions in the stainless steel drums until refrigerated storage can be
arranged. Smaller aliquots are being refrigerated in borosilicate glass for
future requests and for stability studies. Determinations of infrared spec-
trum, viscosity, simulated distillation, elemental composition, and major
-------
TABLE 2-4. REPOSITORY DISTRIBUTION OF SOHIO-REFINED PARAHO SHALE OIL MATERIALS AND PETROLEUM EQUIVALENTS
Sample Requirements
Investigator No.: 1 2
(see Table 2-5)
» m
Study: g "S
8. S
3 I
Material £ 3
O 04
Shale Oil
Crude Shale Oil (4601) 2 liters 500 ml
Hydrotreated Shale Oil (4602) ~~2 liters 500 ml
Weathered Gas Feedstock (4603)
JP-5 Precursor (4604)
JP-8 Precursor (4605)
DFM Precursor (4606) 2 liters 500 ml
Hydrotreated Residue (4607) 2 liters 500 ml
JP-5 Product (4608)
JP-8 Product (4609)
DFM Product (4610) 2 liters 500 ml
Acid Sludge (4612)
Petroleum Equivalent
JP-5 Product (4614)
JP-8 Product (4615)
DFM Product (4616) 500 ml
comprehensive
analysis w
Marine Ecosystem &
4
4
4
4
4
4
4
4
4
4
1
liters
liters
liters
liters
liters
liters
liters
liters
liters
liters 32 liters
liter
100 ml
4
4
4
4
4
4
4
4
4
4
4
5
u}
u)
ft
(11 £ W Ul TO
•H rf 'HE >i M
Ul Ul W U) 0>
(U rH O 4J E
(0 £ id >•< *O O
4J O -P O C W U
3 J= 3 * 0 u! J=
so aw 04 u o
liters 100 ml
liters 100 ml
liters 1 liter
liters 50 ml
liters
liters
liters
liters
ml
Acute Oral
Mouse Toxicity to
Drosophila
Mutagenesis u>
100 ml 100
100 ml 100
100
100
100
100
100 ml 100
100
100
100
100
100
100
100
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
-------
TABLE 2-5. INVESTIGATORS WHO HAVE RECEIVED SOHIO-REFINED
PARAHO SHALE OIL MATERIALS AND PETROLEUM EQUIVALENTS
Investigator No.
(see Table 2-4)
Investigator,
Sponsor
Phone
Address
10
11
12
13
Dr. William Barkley
(API)
S. C. Blum
(API)
L. W. Burdett
(API)
Dr. Norman Richards
(EPA)
Dr. Mike Holland
(DOE)
Dr. David Coffin
(Dr. Ronald Bradow)
(EPA)
Dr. J. LI. Epler
(DOE)
Dr. L. M. Holland
(DOE)
Dr. F. T. Hatch
(DOE)
Dr. J. M. Giddings
(DOE)
Comdr. M. J. Cowan
(U.S. Navy)
Dr. H. f. witschi
(DOE)
Dr. S. Zimmering
(DOE)
513-872-5785 Department of Environmental Health
Kettering Laboratory
University of Cincinnati Medical
Center
3223 Eden Avenue
Cincinnati, Ohio 45219
201-474-3303 Exxon Research & Engineering Company
Analytical S Information Division
Post Office Box 121
Linden, New Jersey 07036
714-528-7201 Union Oil Company of California
Union Research Center
Post Office Box 76
Brea, California 92621
FTS-686-9011 U.S. Environmental Protection Agency
Environmental Sciences Research
Laboratory
Sabine Island
Gulf Breeze, Florida 32561
FTS-624-0678 Biology Division
Oak Ridge National Laboratory
Post Office Box X
Oak Ridge, Tennessee 37830
FTS-629-2585 U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
FTS-624-0841 Biology Division
Oak Ridge National Laboratory
Post Office Box X
Oak Ridge, Tennessee 37830
FTS-843-2747 Los Alamos Scientific Laboratory
c/o Receiving Department, SM-30
Los Alamos, New Mexico 87545
FTS-532-5611 Lawrence Livennore Laboratory
Post Office Box 5507
Livermore, California 94550
FTS-624-7337 Environmental Sciences Division
Oak Ridge National Laboratory
Post Office Box X
Oak Ridge, Tennessee 37830
FTS-775-3116 Naval Medical Research Institute
Toxicology Detachment
NMRI/TD
Wright-Patterson Air Force Base
Dayton, Ohio 45433
FTS-624-0801 Biology Division
Oak Ridge National Laboratory
Post Office Box X
Oak- Ridge, Tennessee 37830
401-863-2620 Division of Biology and Medicine
Brown University
Providence, Rhode Island 02912
10
-------
organics (gas chromatographic profile) will be conducted periodically on sam-
ples stored in flint glass, borosilicate glass, and stainless steel under am-
bient and refrigerated conditions.
ACKNOWLEDGMENTS
This research was sponsored by the U.S. Environmental Protection Agency
under Interagency Agreement IAG D-7-0129, under Union Carbide Corporation con-
tract W-7405-ENG-26 with the U.S. Department of Energy.
11
-------
3. RECENT PARAHO OPERATIONS
R. N. Heistand
J. B. Jones, Jr.
Development Engineering, Inc.
Post Office Box A, Anvil Points
Rifle, Colorado 81650
INTRODUCTION
The Paraho Process
The Paraho process is operated at Anvil Points, Colorado by Development
Engineering, Inc., a subsidiary of Paraho Development Corporation. The pro-
cess and retort have been described previously (Pforzheimer 1974).
The retort is a cylindrical, vertical kiln having a refractory-lined
carbon steel shell (Figure 3-1). Near the top of the retort is the off-gas
collector, where the oil mist and gas are removed from the retort. Below the
off-gas collector are three gas/air distributors located at separate levels in
the retort. The bottom gas/air distributor is located in the grate mechanism
at the bottom of the retort. This grate at the bottom and the rotating spreader
at the top are the only moving pieces within the retort.
The Paraho retort can be operated in several modes. Figure 3-2 shows the
Direct Mode, where combustion required to produce the heat for retorting occurs
within the retort. Raw shale enters the top of the retort and is preheated in
the mist formation zone by the gases carrying the oil mist out of the retort.
The preheated shale next enters the retorting zone, where hot gases rising
12
-------
RAW SHALE
ROTATING SPREADER
REFRACTORY
COLLECTORS
DISTRIBUTORS
DISTRIBUTORS
DISTRIBUTORS
MOVING GRATE
SHALE MOVED
THROUGH GRATE
GAS/AIR
GAS/AIR
GAS/AIR
RETORTED SHALE
TO DISPOSAL
Figure 3-1. Paraho retort.
13
-------
RAW IHALE
H
£>.
t
MIST FORMATION ZONE
*
RETORTING ZONE
f
COMBUSTION
ZONE
1 t
i >
RETORTED SHALE
COOLING ZONE
1
*
/ \
RETORT
TOP
DISTRIBUTOR
MID
DISTRBUTOR
OFF ei<; OIL/6AS
IIA^^ wlfc' •"•»
W SEPARATORS
*
PRODUCT OIL
T0
trop AI
M
MID
AIR
AIR
_ BOTTOM COO
SAS /^ *i
RECYCLE BAS
BLOWER
P DILUTION CAS
R
ID DILUTION GAS
BLOWER
LING GAS
NET PRODUCT
GAS
RECYCLE
GAS
r
f
RETORTED SHALE
Figure 3-2. Direct Mode schematic.
-------
from the internal combustion heat the shale to retorting temperature. Upon
retorting, the solid organic kerogen in the shale breaks down to gas, oil, and
coke. The gas and oil are swept upward with the hot gases, and the coke remains
on the retorted shale. As the retorted shale enters the combustion zone, some
of this coke is burned with the air introduced through the gas/air distributor.
Only enough air is introduced to produce sufficient heat for the retorting pro-
cess. Recycle gas is added with the air to ensure even distribution across the
bed and to control the flame temperature. Below the middle distributor, the
retorted shale enters the cooling zone, where it is cooled by recycle gas ris-
ing from the bottom distributor. What the Paraho retort involves, then, are
countercurrent flows: gases rise while shale moves downward under the force
of gravity and as controlled by the grate. These flows produce hot combustion
and retorting zones in the middle with heat exchange at both top and bottom,
so that both gases (and oil mist) and retorted shale are relatively cool upon
leaving the retort.
Another mode of operation of the Paraho retort is the Indirect Mode. In
this case, the air blower is replaced by a heater, and the recycle gas entering
the top and middle distributors is heated externally. No combustion occurs
within the retort; the product gas is not diluted with N2 and carbon dioxide
(CO2) by-products of combustion, and the coke remaining on the retorted shale
is not utilized.
Brief History
Paraho oil shale retorting operations began with the Paraho Oil Shale
Demonstration. That demonstration, a $10-million, 3-year project involving 17
industrial participants, is described elsewhere (Jones 1976, 1977). One of
the project achievements was the refining of 1Q1* bbl of crude shale oil into
military fuels.
Based in part upon the achievement of that refining run, the U.S. Office
of Naval Research (ONR), U.S. Department of Navy, and U.S. Department of Energy
15
-------
(DOE) contracted with Paraho to continue research and development of surface
retorting technology. These contracts consisted of the following:
• refurbishment of the facility for limited operation;
• installation of crude shale oil storage tanks;
• continued research and development, including production of up
to 105 bbl of shale oil.
Operations were carried out under ONR contracts until December 31, 1977 and
were continued under a DOE contract into 1978.
During the third quarter of 1978, the crude shale oil produced during
these recent operations (1977-78) was shipped for refining into military
fuels. The shipment was carried out by rail, using a shuttle of 40 jumbo rail
tank cars. This was the largest shipment of crude shale oil in the United
States to date. Refining was carried out at the Toledo refinery of the
Standard Oil Company of Ohio.
During these recent Paraho operations (especially during the last half of
1977) , many researchers and specialists visited the Anvil Points site to per-
form environmental monitoring and to obtain samples for further research.
Samples for the U.S. Environmental Protection Agency (EPA)/DOE Fossil Fuels
Research Materials Facility were taken during this period.
RECENT PARAHO OPERATIONS
Mining
During the recent operations, the Anvil Points mine utilized equipment
that had been employed by Paraho in previous operations. This equipment con-
sisted of a rotary drill jumbo, a mechanical sealer, a roofbolter, a 5-yd3
wheel loader, 50-t haul trucks, a water truck, a grader, air compressors, and
ventilating fans. During the 1977-78 operations, nearly 2 x 105 t of shale
were mined for use by DOE and in the Paraho project. Figure 3-3 illustrates
the advances of the room-and-pillar mine during these recent operations.
16
-------
Ventilation
Duct
1977 Mining
1978 Mining
Adit 1
Figure 3-3. Anvil Points mine.
17
-------
Crushing
The crushing operations used a series of crushers provided by the Navy.
These consisted of a primary jaw crusher, a secondary jaw crusher, and a
!i
tertiary double roll crusher. Other support equipment included a small loader,
storage bins, and screens.
Retort Production
Table 3-1 presents a general overview of retort production during the
recent Paraho operations. Data are from the 8.5-ft i.d. Semi-Works operations.
Shale grade varied from 24.5 to 36.6 gal/t; shale rate varied from 416 to 536
lb/h/ft2. Oil production varied from 140 to 200 bbl/d. This production was
limited by the oil/gas separation equipment. When daily production exceeded
180 bbl/d, oil/gas separation equipment capacity was exceeded, and the oil was
not completely separated from the recycle gas. Gas production averaged 1.5 x
106 stdft3/d, with an average gross heating value of 150 Btu/stdft3. Table 3-1
calculates equivalent oil (EO) of the gas on a basis of 6 x 106 Btu = 1.0 bbl
of oil. On this basis, the gas EO production ranged from 30 to 50 bbl/d.
Products
Tables 3-2 through 3-4 outline properties of the raw shale and of typical
products from the recent operations. Except for a low water content, the
crude shale oil (Table 3-2) is typical of most crude shale oils. Water is
removed from the product oil by simple decantation, in order to meet ONR
specifications. The product gas (Table 3-3) is typical of Direct Mode opera-
tions — high in N£ and CO2. Hydrogen sulfide (H2S) is ~0.3% by vol., or 3000
ppm. The raw and retorted shales (Table 3-4) are typical of Anvil Points opera-
tions. Note that most of the organic carbon present in the raw shale is
removed. Most of the sulfur remains, however.
18
-------
TABLE 3-1. SEMI-WORKS RETORT PRODUCTION0
1977
Jan Apr
May Dec
1978
Jan Sept
Shale
Rate (lb/h/ft2)
Grade (gal/t)
Product Gas
432 486 418 536
24.5 29.7 24.7 36.6
From Jones and Heistand (1979).
416 502
24.5 29.2
Production (1000 stdftVd
GHV (Btu/stdft3)
Equivalent Oil (bbl/d)
Product Oil
Production (bbl/d)
.) 1400
118
30
140
1500
141
35
200
1300
118
30
145
1700
153
40
190
1400
120
35
145
1800
180
50
180
TABLE 3-2. PROPERTIES OF CRUDE SHALE OIL
Parameter
Value
Gravity (°API)
Viscosity (SSU at 130°F)
Viscosity (SSU at 210°F)
Pour point (°F)
Water (% by wgt.)
Sediment (ml/100 g)
Carbon (% by wgt.)
Hydrogen (% by wgt.)
Nitrogen (% by wgt.)
Sulfur (% by wgt.)
21.4
83
48
85
0.3
0.1
84.8
11.4
2.0
0.6
19
-------
TABLE 3-3. PROPERTIES OF PRODUCT GAS
Parameter Value
Dry Gas ( % by vol . )
H
2
N
2
Q
2
CO
CH.
4
CO.
2
C2H4
C H
2 6
C.,
3
C.
4
C..+
5
H.S
2
NH,
3
Total
Water Vapor ( % by vol . )
Heating Value (Btu/stdft3 (dry) )
5.5
61.0
0
2.9
2.4
24.2
0.7
0.6
0.6
0.6
0.6
0.3
0.6
100.0
17.5
145
Operability, Reliability, and Yields
Operability of the Paraho Semi-Works retort was most encouraging. The
on-stream factor, determined by dividing the on-stream hours by the total
hours, exceeded 90% for the recent operations (90.8% for 1977 and 90.7% for
1978). These consistently high -on-stream factors are a measure of the opera-
tional reliability of the Paraho above-ground retorting process.
Another measure of reliability is the long continuous run achieved during
performance of the ONR contracts in the first half of 1977. Although long,
20
-------
TABLE 3-4. PROPERTIES OF RAW AND RETORTED SHALE
Analysis
Fischer Assay
Chemical
Elemental
Parameter
Oil (% by wgt.)
Water (% by wgt. )
Oil (gal/t)
Gas + Loss (% by wgt.)
Mineral CO2 (% by wgt.)
Ash (% by wgt. )
Moisture (% by wgt.)
Carbon ( % by wgt . )
Hydrogen ( % by wgt . )
Nitrogen (% by wgt.)
Sulfur (% by wgt.)
Value :
Raw Shale
10.46
1.07
27.39
1.99
17.54
66.67
0.88
16.65
1.75
0.52
0.74
Value :
Retorted Shale
0.06
0.41
0.16
0.18
15.63
81.44
6.48
0.18
0.23
0.80
continuous operations were not a principal objective, a 105-d continuous run
was achieved. The Semi-Works retort was successfully fired off on January 5,
1977 for the 10-d shakedown operations. Without shutdown, operations were
continued. After the required 1.25 x 104 bbl of crude shale oil had been pro-
duced, research efforts were accelerated to determine maximum oil production.
An oil production level of 200 bbl/d was achieved. This exceeded the limits
of the oil/gas separation, and operations were shut down on April 20, 1977.
Total production of shale oil meeting ONR specifications approached 1.5 x 10
bbl of dry oil. Lost time (downtime) during this continuous operation totaled
4.2 h, most of which was devoted to standby while the raw shale rotary seal was
serviced. Other details of the 105-d continuous operation are presented else-
where (Jones and Heistand 1979).
Retort yields are obtained by comparing retort performance with results
of the Fischer Assay (Heistand 1976). The Fischer Assay is an empirical
laboratory method used to determine the oil potential of shale. By comparing
the energy products of the Paraho Direct Mode operations (oil, gas EO, plus
21
-------
the thermal energy EO supplied as heat from the raw shale feed) with the
products of the Fischer Assay (oil plus gas EO), a retort yield is obtained
(Heistand 1979). Typical retort yields for Paraho Direct Mode operations
average 114% of assay (Table 3-5).
The reliability of these data and of the data presented in the balance of
this report is verified by good closures of material and elemental balances
(Table 3-6). Balances for mean data of ~100% for each of the 2-year periods
are typical of results from individual test days. Normally, the overall weight
balance was 100 ± 1% and the balance for total carbon was 100 ± 4%. These
good balance closures indicate accurate measurements of both process flows and
product composition, and demonstrate that the data are valid and reliable.
RESEARCH
Research studies directed towards improving operational reliability, in-
creasing oil yield and production, and minimizing environmental impacts were
carried out during the 1977-78 operations. Major emphasis was given to re-
torting operations studies, which involved more than 100 24-h test days.
These studies employed both the 2.5-ft i.d. Pilot Plant and the 8.5-ft i.d.
Semi-Works. As noted previously, although both retorts were subjected to
wide variations in operating conditions, the overall operability and yields
remained high.
Three of the research studies merit additional discussion. These are:
shale grade; product gas desulfurization; and introduction of air to the lower
section of the retort. These studies have been conducive to improving oil
production, minimizing environmental impacts, and improving operability.
Shale Grade
)
Eight 24-h test periods were carried out in the Pilot Plant over a 2-week
period using raw shale feed of grades of 26.0, 27.9, 31.4, and 35.2 gal/t
(Table 3-7). Oil production mirrored raw shale grade: production increased
22
-------
TABLE 3-5. MEASURES OF YIELD
Analysis Parameter
Fischer Assay Oil
Gas EO
Total
Paraho Yield Oil
Gas EO
Total
Value
(gal/t)
27.4
2.2
29.6
24.4
6.0
30.4
Percent
of Assay
89.1
103
Residual Carbon Fuel Used
EO 3.3
Overall
(Oil + Gas + Fuel) 33.7
114
TABLE 3-6. TYPICAL MATERIAL AND ELEMENTAL BALANCES
Parameter
Weight
Total Carbon
Total Sulfur
1977
Recovery
(%)
99.8
99.0
103.1
1978
Recovery
(%)
99.9
99.2
100.3
aFrom Jones and Heistand (1979)
23
-------
TABLE 3-7. SHALE GRADE RESEARCH3
Date
1/9-10
1/15-16
1/20-21
1/25-26
Raw Shale
(gal/t)
26.0
27.9
31.4
35.2
Oil
(bbl/d)
JL1.5
16.5
18.3
20.8
Gas EO
(bbl/d)
1.4
1.7
1.5
1.9
Total
(bbl/d)
12.9
18.2
19.8
22.7
From Jones and Heistand (1979).
with increasing grade of shale. Retorting operability was not affected by use
of rich (35.2 gal/t) shale. In order to substantiate this operability with
rich shale, another research study was scheduled for the Pilot Plant. Over a
2-week operation, six 24-h test days were run using rich (36.2 ± 0.2 gal/t)
shale. Retorting operability was not affected, and yields remained high for
the six test days.
Gas Desulfurization
A special off-gas collector, designed to be installed without modifying
the middle distributor, was used to test sulfur removal. Results of this
Pilot Plant research are shown in Table 3-8. Sampling of gas within the retort
indicated that H2S was removed from the gas as it passed through the lower bed.
At the middle distributor, the H2S content of the recycle gas was reduced to
0. As the fraction of product gas taken through the middle off-gas collector
increased, H2S in the combined product gas fell from 0.30% to 0.17% by vol.
Individual samples of the middle product gas stream showed H2S levels to be
as little as 0.05% by vol. or ~0.3 Ib H2S/MM Btu. Removal of product gas from
the middle of the retort did not adversely affect yields or oil production.
Significant quantities of H2S can be removed from the gas in the lower retort,
reducing environmental impacts without affecting operability or product yields.
24
-------
TABLE 3-8. GAS DESULFURIZATION RESEARCH0
Date
3/11
3/18
3/21
3/23
Normal
(stdft3/min)
162
76
36
41
Product Gas
Middle
(stdft3/min)
0
86
132
142
Product Production
H2S
(% by vol.)
0.30
0.22
0.17
0.19
Oil
(bbl/d)
14.4
14.8
12.5
13.9
Gas EO
(bbl/d)
2.2
1.5
1.4
2.1
Total
(bbl/d)
16.6
16.3
13.9
16.0
aFrom Jones and Heistand (1979).
Addition of Air to Bottom Distributor
The effect of adding air to the bottom distributor was studied in the
Semi-Works retort over a 4-week period. Six tests (one to four 24-h periods
each) were carried out in which bottom air was varied from 0 'to 97 stdft /min
(Table 3-9). During a 2-d test period (6/27-28), a blockage in the middle
distributor prevented introduction of sufficient air. Oil production fell;
normally, the retorting operations would have been shut down. Instead, air
was introduced through the bottom distributor, oil production again reached
163 bbl/d, and operations were continued for an additional 2 weeks. Although
this technique can be used to extend operations, it does reduce thermal
efficiency. This ability to shift the air injection to various distributors
without adversely affecting production or operability demonstrates the Paraho
technology's good mechanical design and serves to complement its commercial
feasibility. Good on-stream service factors are enhanced in two ways: First,
i
operations are extended. Secondly, unscheduled shutdowns requiring extensive-
downtime are avoided.
25
-------
TABLE 3-9. RESEARCH ON ADDITION OF AIR TO BOTTOM DISTRIBUTOR
Date
6/13,15
6/18-21
6/27-28
6/30
7/2
7/3-6
Air
Bottom Distributor
(stdft3/min)
0
50
0
60
88
97
Product Production
Oil
(bbl/d)
160
162
151
161
158
164
Gas EO
(bbl/d)
36
35
37
36
33
32
Total
(bbl/d)
196
197
188
197
191
196
From Jones and Heistand (1979).
CONCLUSIONS
The results of Paraho operations at Anvil Points have been most en-
courgaging. Highlights of these results are:
• Production of >105 bbl of crude shale oil
• Continuous periods of operation of >100 d
• Maintenance of a service factor of >90% for a 2-year period
• Demonstration of the ability to retort rich (>35 gal/t) shale
• Demonstration of the ability to produce low-sulfur (<500 ppm H2S) gas
• Demonstration of the ability to operate until scheduled turnaround
With successful completion of two research projects utilizing the Paraho
retorts, we are ready to proceed towards our next objectives:
• Design, construction, and operation of a full-size Paraho module
• Encouragement of oil shale commercialization
• Maintenance of status as a technology-licensing company
26
-------
ACKNOWLEDGMENTS
The work described in this paper was carried out at the U.S. Department
of Energy's Anvil Points Oil Shale Research Facility located on the Naval
Oil Shale Reserves near Rifle, Colorado.
REFERENCES
Heistand, R. N. 1976. The Fisher Assay: Standard for the oil shale industry.
Energy Sources 2(4):40.
Heistand, R. N. 1979. Product yields. 12th Oil Shale Symposium, Colorado
School of Mines, Golden, Colorado, April 18-20.
Jones, J. B., Jr. 1976. The Paraho oil shale retort. 9th Oil Shale
Symposium, Colorado School of Mines, Golden, Colorado.
Jones, J. B., Jr. 1977. Technical evaluation of the Paraho process.
Eleventh Israel Conference on Mechanical Engineering, Technicon, Haifa,
Israel.
Jones, J. B., Jr., and R. N. Heistand. 1979. Recent Paraho operations. 12th
Oil Shale Symposium, Colorado School of Mines, Golden, Colorado,
April 18-20.
Pforzheimer, H., Jr. 1974. Paraho — New prospects for oil shale. Chem. Eng.
Prog. 70(9):62.
27
-------
4. REFINING OF SHALE OIL BY SOHIO
D. L. Cawein
The Standard Oil Company of Ohio
Post Office Box 696
Toledo, Ohio 43694
INTRODUCTION
Some speakers at this conference have mentioned "energy shortage." There
is no energy shortage; there are tremendous amounts of energy in the forms of
oil shale, tar sand, coal, solar radiation, and so on. The problem is one of
economics — there is not the low-cost energy that we had the luxury of using
in the past.
The Standard Oil Company of Ohio (SOHIO) has been interested in oil shale
as an alternate energy source for some 20 years. At the beginning, we acquired
a reserve position in oil shale and entered into a joint venture to investigate
what appeared to be a very likely first process that could reach an early
stage of commercialization and be economic. After spending, with others, some
15 to 20 million dollars in the middle 1960's, we recognized that the technol-
ogy was relatively close. There were technological problems, but they were
solvable; the primary problem was the economics of the process.
Following this initial investigation, SOHIO continued to expand its
studies and reserve position. At that time, the internal combustion vertical-
shaft kiln that the U.S. Bureau of Mines had first researched many years ago
was being further developed by Development Engineering, Inc. in a consortium
called Paraho. To SOHIO, this appeared the most likely first entry into a
28
-------
commercial oil shale industry. So SOHIO, along with Development Engineering,
Inc., organized a consortium of 17 companies to further the research and
development of that technology.
Paraho really spearheaded the operation. Paraho leased the plant from
the U.S. Navy, worked with the Navy for continued funding (along with funding
from various commercial oil companies, including SOHIO), and won approval for
the 2-year operation to make 105 bbl of raw shale oil. Having made the raw
shale oil, the next step was to convert it to products needed in Navy opera-
tions. Because of our interest in furthering the oil shale industry, SOHIO
submitted to Paraho and the Navy a bid to refine 105 bbl into products similar
to those the Navy was using. SOHIO proposed a two-step plan: (1) a laboratory
pilot program, to assess the feasibility of the process; and (2) processing of
the 105 bbl in commercial equipment. In summary, both steps were successful.
At SOHIO1s Research and Development Laboratory in Cleveland, we were able to
process the raw shale oil into fractions very similar to products from crude
oil. Secondly, in commercial equipment in our refinery, we were able to very
nearly duplicate the laboratory results. This accomplishment, we believe, has
important implications for future efforts. This confirmation of pilot plant
results increases the confidence in future pilot studies.
SOHIO'S TOLEDO REFINERY
The SOHIO refinery in Toledo, Ohio is a medium-sized oil refinery:
1.2 x 105 bbl/d of crude oil are processed. The facility differs from the
average refinery in that we concentrate on maximizing yield of gasoline from
crude oil, due to the specific needs of the Ohio market. We produce ~75% gaso-
line from crude oil, in contrast to a national average of ~50%.
The Toledo facility is a very modern, efficient refining system. The
system includes the first commercial Hydrocracker built in the world (1962).
This Hydrocracker was built to convert 7500 bbl/d of diesel fuel/heating oil
into gasoline. (SOHIO had found the market for gasoline to be growing at a
faster rate than the market for heating oil, putting us out of balance with our
customers' needs.) After construction of this facility in 1962, we eventually
increased throughput to ~104 bbl/d.
29
-------
SOHIO volunteered to make its facility available (on a "break-even"
basis) to the Navy for processing the raw shale oil. It is important to re-
member that the facility was not built for processing oil, but for processing
a much easier-to-handle material. On the other hand, it was probably the best
commercial facility available. Of course, one could design and build a facility
much better suited for the characteristics of shale oil. Thus, we can assume
a difference in the yield and quality of products from this facility vs. prod-
ucts from a commercial unit designed to process shale oil.
SPECIAL CONSIDERATIONS IN REFINING SHALE OIL
Table 4-1 summarizes the characteristics of shale oil vs. typical crude
oil. In general, shale oil is much heavier than even the heaviest crude oil.
Shale oil contains less of the gasoline and kerosene kinds of materials and
more of the heating oil and heavier kinds of materials.
The major problem in refining raw shale oil is the nitrogen content.
The 2% nitrogen is some 10 times higher than the nitrogen content of most
crude oils (see Table 4-1). In refining, most crude oils undergo catalytic
processing, and most currently-used commercial catalysts are subject to
poisoning from nitrogen and the by-product ammonia (NHs). As a result, nitro-
gen is the "bad actor" in shale oil refining.
The sulfur content of raw shale oil is relatively low in comparison to
that of high-sulfur crude oils. Certain metals are substantially higher (in
particular, arsenic and iron). These present problems during processing with
normal kinds of refining equipment.
STEPS IN THE SOHIO REFINING PROCESS
This subsection describes the processing scheme used to refine the 105
bbl. (Actually, 7.8 x 10^ bbl were processed.)
Raw shale oil received from Colorado was accumulated in a large tank over
a period of 3 to 4 months. The oil was heated to settle out water, but Paraho
30
-------
TABLE 4-1. CHARACTERISTICS OF SHALE VS. CRUDE OIL
Typical Paraho
Parameter Shale Oil
Specific Gravity (°API)
Distillation (°F)
Initial Boiling Point
10%
50%
90%
Basic Sediments and Water (% by vol.)
Pour Point (°F)
Sulfur (% by wgt.)
Nitrogen (% by wgt.)
Oxygen (% by wgt.)
Ash (% by wgt.)
Metals (ppm)
Arsenic
Nickel
Iron
Vanadium
20
375
520
790
1015
0.1
85
0.7
2.1
1.4
0.01
10
2
38
1
Typical Crude Oil
Heavy, Sour Light, Sweet
25 35
100 75
250 200
750 550
1050 1000
0.2 0.2
10 -10
2.5 0.3
0.3 0.05
-
-
Nil Nil
25 5
10 5
100 1
31
-------
had done such a good job that there was no water. Prior to the shale oil run,
the hydro-treating facility was shut down for "turnaround," to clean it up and
change the catalysts. Thus, the shale oil was processed with clean facilities
and new catalysts.
As shown in Figure 4-1, the process consisted of hydrotreating , fractiona-
tion of the hydrotreating products, and acid treating for JP-5 and diesel
fuel, the primary products being sought. Tankage was included following
fractionation, because there was only a single acid treater and it was nec-
essary to block through first one product and then -the other. Some of the
lighter material (lighter than JP-5) was returned to the refinery pool, to end
up in gasoline. The heaviest part was sent to the refinery's heavy fuel pool.
The sludge produced in acid treating was sent to a landfill.
We had planned to make small amounts of JP-4 and JP-8, but mechanical
difficulties brought us to the end of the run sooner than expected.
Figure 4-2 outlines the hydrotreating process. First, the raw shale oil
was pumped through a guard filter. After the guard filter, the filtered raw
shale oil was mixed with recycle gas having a high concentration of H2 (~80%
H£, with the remainder light hydrocarbons), heated to ~700°, and passed through
a multi-bed reactor. The multi-bed reactor used a commercially-available hydro-
treating catalyst with a high concentration of nickel and molybdenum. (Any
number of available catalysts could have been used.) At 700°, with the
catalyst, with the H2, and at a pressure of ~1500 Ib/in2g, reasonably good
conversion of sulfur to hydrogen sulfide (H2S) and nitrogen compounds to NH3
was accomplished. A still better job could be achieved in a commercial
operation using higher pressures, better catalyst, and more catalyst. These
improvements would permit higher conversion of nitrogen compounds to
An exothermic reaction took place in the reactor. Interbed cooling was
employed to limit temperatures (deactivation occurs with excessive temperature)
A stream of cool H£-rich gas cooled down the products after the first bed and
after the second bed.
32
-------
"2
\
GASOLINE STOCKS
RAW
SHALE
OIL
U)
w
\
HEAVY FUEL
HYDROTREATING DISTILLATION
H2S04
i
-
r-^
n
JP-5
DFM
\
SLUDGE
ACID TREATING
Figure 4-1. Toledo refinery block flow.
-------
MAKE-UP H,
U)
*»
SHALE OIL
STORAGE
PRODUCT
TO LOADING
CLAY ACID
FILTER SETTLER
H2S04
STRIPPER
WET
GAS
HEAVY
FUEL
PRODUCT
STORAGE
FRACTIONATOR
REBOILER
Figure 4-2. Toledo refinery process flow.
-------
After completion of the hydrotreating reaction in the reactor, the next
step was to separate the products by adding water, which absorbed NH3. The
water was separated from the oil in a high-pressure separator. The sour water
containing NH3 and some of the H2S was sent to a stripper to remove these im-
purities. The excess H2 in the reactor (over and above the stoichiometric re-
quirements of the reactions) was recirculated, replacing the amount that had
been consumed with make-up H2-
The cool, dewatered oil then flowed to a stripper, where any remaining
H2S and some of the lighter hydrocarbons were removed. The product fractionator
was a conventional reboiled and refluxed 50-plate fractionating tower. Four
products were produced: a light gasoline stock (lighter than JP-5), the JP-5
product, the diesel fuel product, and the bottom residual material. The tower
design did not permit maximization of JP-5 and diesel fuel yields.
From storage tanks, the products were pumped through a simple acid-
treating system in which the oil was merely contacted with 93% sulfuric acid
(H2SOi4.) to remove residual nitrogen compounds. This acid treating was thorough
but not very efficient: 5 to 10% of the product ended up as an acid sludge.
Final treating was accomplished in a clay filter using a very fine, natural
kind of clay (Attapulgus) to remove any residual trace amounts of acid or
sludge.
The final task was to transfer the final products to the Navy by tank
car. Preliminary testing indicated all products to be well within Navy
specifications.
35
-------
5. WORK PLAN FOR SHALE OIL STUDY,
BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY
T. K. Rao
J. L. Epler
Biology Division
Oak Ridge National Laboratory
Post Office Box Y
Oak Ridge, Tennessee 37830
INTRODUCTION
The principal focus of the Oak Ridge National Laboratory (ORNL) Shale Oil
Study is the testing of primary effluents and products for potential effects
on man. This portion of the evaluation of Paraho samples concerns questions
of relative toxicities of process materials and refinery products.
We propose a parallel, two-level program to expeditiously and cost-
effectively answer these questions. Level One is cellular bioassays. These
assays will accumulate base-line data on typical effluents and emissions and
ascertain how the relative toxicities of major effluents and fractions vary
with changes in process conditions. In addition, biological effects studies
using cellular assays will provide an essential data base for eventual cor-
relation with acute and chronic toxic effects in whole animals.
Level Two consists of mammalian toxicity bioassays. These assays will
involve characterization of the acute, subacute, and chronic toxicities of
primary process precursors and products. As data from the analytical chemistry
and cellular bioassay programs become available, this information will help in
36
-------
determining whether additional evaluation of the process by other materials or
tests is indicated.
The various assay systems and their application to appropriate test
materials or selected active compounds representative of the biohazard present
are divided into two categories: (1) testing that is specifically applicable
to shale oil, and (2) research or validation that is applicable to the ongoing
generic approach of the U.S. Department of Energy (DOE) and other agencies
in health effects studies of synthetic fuel technologies. For effective
evaluation of the facilities and processes, the two approaches must interrelate
and reinforce each another. This paper discusses only the segments necessary
to gather specific, comparative data on shale oil samples.
Since even the cellular bioassays are only predictive (and, at that,
still developmental in nature), this program is offered only as a practical
use of state-of-the-art assays. Considerable basic research must parallel
these screening efforts in order to reflect accurately on the question of
environmental acceptability of various liquefaction and shale oil processes.
LEVEL ONE: CELLULAR BIOASSAYS (J. L. Epler, Principal Investigator)
Relationship to Health Effects Assessment
These tests are intended to function as (1) predictors of profound long-
range health effects such as mutagenesis and/or carcinogenesis, (2) a mechanism
to rapidly isolate and identify hazardous biological agents in complex mix-
tures, and (3) a measure of biological activity correlating base-line data
with changes in process conditions. Since complex mixtures can be fraction-
ated and approached in short-term assays, information reflecting on the actual
compounds responsible for biological effects may be accumulated. Thus, tests
in this category will (4) aid in setting priorities for (a) further validative
testing, (b) testing in whole animals, and (c) more definitive chemical anal-
ysis and monitoring.
Tables 5-1 and 5-2 list the tests to be applied and materials to be tested.
37
-------
TABLE 5-1. BIOASSAYS TO BE APPLIED (LEVEL ONE)
AT OAK RIDGE NATIONAL LABORATORY
Screening Bioassay (all samples and fractions)
Salmonella
Yeast Gene Mutation (selected samples and fractions)
DNA Repair
Cytotoxicity
Teratogenesis
Validative Assays (selected fractions)
Drosophila
Mammalian Cell Gene
Mammalian Cell Chromosomal (CHO)
Mammalian Cell Chromosomal (Leukocyte)
TABLE 5-2. SAMPLES TO BE BIOASSAYED (LEVEL ONE)
AT OAK RIDGE NATIONAL LABORATORY
Shale Oil Materials
Crude Shale Oil
Hydrotreated Shale Oil
Gas Feedstock
JP-4 Precursor
JP-5 Precursor
JP-8 Precursor
DFM Precursor
No. 6 Fuel Oil
JP-4
JP-5 (Final)
JP-8
DFM
Acid Sludge
Petroleum "Equivalents"
JP-4
JP-5
JP-8
DFM
38
-------
Confirmation with a Battery of Tests
Because of the intrinsic limitation of each mutation assay, testing with
only one microbial system has often led to faulty conclusions for pure com-
pounds. To overcome this shortcoming, we will employ short-term mutagenesis
and/or DNA repair assays to comprehensively screen for both mutagenic and
carcinogenic hazards in primary effluents and potential fugitive emissions.
Some segments of the battery of assays will be used only on selected active
compounds as determined by the coupled effort of chemical and initial bio-
logical screens. The actual components will then be characterized as either
highly purified fractions or actual pure chemicals. Thus, feedback to chemical
screening will become a feasible monitoring method.
Selected samples will also be assayed in cultured mammalian cells. Two
major biological end points will be under surveillance: gene mutation and
cytogenetic damage. The decision to apply these important assays will be a
function of the overall toxicity of the sample; conceivably, only pure iso-
lated and identified components will be tested.
With substantial progress, these ongoing EPA-DOE-cosponsored research
activities will benefit from knowledge gained by biological and chemical evalua-
tion of coal liquid test materials. In a reciprocal sense, these studies will
feed back into an overall assessment of the hazards of the materials.
Fractionation Methods
Development of a standardized methodology for biopreparation fractiona-
tion will be approached through comparison of multiple samples (final shale
oil products, crude oils, and precursor products). As shown in Figure 5-1,
this method will involve: (1) removal of volatile materials by distillation,
(2) collection of distillate, (3) acid/ether extraction, (4) alkaline/ether
extraction, and (5) LH-20 chromatography of neutrals via isopropanol and
acetone to yield (a) aliphatic, (b) aromatic, and (c) polyaromatic fractions.
The array of "oil" samples will be evaluated with multiple extractions and
multiple bioassays.
39
-------
Oil
Distill / Evaporate
>'
Residue
HCI / Ether Partition
A *
Aqueous
Basify with NaOH
Back Extract with Ether
Ether
Extract with NaOH
Ether
Aqueous*
Ether
LH-20 / Isopropanol
+ Acetone
Aliphatic
±
Aqueous
Acidify with HCI
Back Extract with Ether
Aromatic
Polyaromatic
= for chemical / biological studies
Aqueou
* = retain until studies complete
Figure 5-1.
Practionation scheme for shale oil study at Oak Ridge National
Laboratory.
40
-------
In previous studies, ether-soluble base (ESB) fractions of several crudes
and aqueous wastes fractionated by a solvent partition method exhibited high
biological activities as measured by the Ames microbial mutagenesis test. The
ESB fractions of various shale oils will be chosen for subfractionation to iso-
late and identify the mutagenically active components.
Expected Results
Fractions showing biological activity will be chemically characterized
(identification and quantitation when required) to determine the possibly
responsible constituents. Further subfractionation will be carried out as
necessary, and eventually we will obtain an estimate of relative mutagenicity
(potential biohazard). By fractionating and subfractionating active samples,
mutagenic activity will be located according to chemical type. (Thus, com-
pounds in most active fractions will require chemical definition.) At this
point, we will test the activity of known and newly identified individual
compounds in the subtractions and attempt to correlate these results with
whole-animal carcinogenicity data. This procedure will provide information on
relative hazard in addition to identification of defined biohazards.
LEVEL TWO: MAMMALIAN TOXICITY BIOASSAYS
To be coordinated with the cellular bioassays, the mammalian toxicity
bioassays will involve characterization of acute, subacute, and chronic
toxicities of a limited number of primary process materials. The samples
tested will be materials for which there is a high probability of direct or
indirect human exposure. Infprmation from the analytical chemistry, area
monitoring, and cellular bioassay programs will guide decisions on whether a
thorough evaluation of the process will require additional materials or tests.
The authors recognize the extreme importance of Level Two and also the
limited opportunity to obtain representative samples. Accordingly, we expect
other EPA/DOE/American Petroleum Institute programs to supplement and consider
alternatives to our studies.
41
-------
Acute Mammalian Toxicity (H. P. Witschi, Principal Investigator)
The following compounds will be tested: (1) retort oil, (2) hydrotreated
product, and (3) No. 6 fuel oil. Testing will include: (1) acute oral median
lethal dose (LDso) in mice, (2) acute skin toxicity in rats, (3) primary skin
and eye irritation in rabbits, and (4) dermal sensitization in guinea pigs.
Acute Oral Toxicity—
Graded doses of the three fractions will be administered by gavage to
young male and female mice. The animals will be observed for 2 weeks after
dosing or until all signs of reversible toxicity subside, whichever occurs
later. The LDso with confidence limits will be calculated.
Acute Dermal Toxicity—
Test agents will be applied to the skin of rats. Except for the different
species, the protocol will be essentially the same as in the acute oral toxi-
city study.
Primary Eye and Skin Irritation-
Test animals will be young albino rabbits. To evaluate eye irritation,
each agent will be placed on the everted lower lid of one eye and the lids
then gently held together. The contralateral eye will remain untreated as a
control. Ocular lesions will be read and graded according to standard proce-
dures during an observation period of up to 14 d.
Primary skin irritation will be evaluated by introducing each test sub-
stance onto clipped skin under a 1-in2 gauze patch. The test substance will
be kept in contact with the skin for 24 h. Signs of irritation will be ob-
served and scored until all irritation subsides.
Dermal Sensitization—
Albino guinea pigs will be sensitized 3 times weekly for 3 weeks by
intradermal injection or topical patch application. Following the 9th sen-
sitizing treatment, the animals will be set aside for 2 weeks and then chal-
lenged by a final injection. Erythema, edema, and. other lesions will be
scored according to standard procedures at 24 and 48 h after each application.
42
-------
Acute, Subacute, and Chronic Dermal Toxicity (J. M. Holland, Principal
Investigator)
These studies will evaluate the skin penetrability, distribution, and
persistence of Paraho shale oil crudes and refined products and establish the
correlation between these parameters and specific activities of the whole mix-
tures , both as skin irritants and as epidermal carcinogens, in vivo.
The following samples will be evaluated: raw crude, hydrotreated crude,
No. 6 fuel oil, and DFM.
In Situ Skin Fluorescence—
We have developed a method using native fluorescence to follow the move-
ment of synthetic crude, oils through intact skin. The method may also be used
to evaluate various barrier creams or cleanup procedures, as well as to quan-
titate differences in bioavailability between materials.
Using this method, we will apply known amounts (per unit area) of raw
crude, hydrotreated crude, and each of the finished products to shaved mouse
skin. At 24-h intervals animals will be killed, the skin excised, and frozen
sections examined to quantitate levels of fluorescing constituents trapped in
situ within sebaceous glands. Once in the sebaceous glands, materials can
escape by only two significant pathways: metabolic clearance or mechanical
excretion onto the skin surface. Our evidence suggests that, after an initial
equilibration period, the sebaceous gland becomes a reservoir for hydrocarbons,
and surface concentrations are maintained as a result of the slow (days) but
constant secretion of sebaceous lipid containing residual fluorescing hydro-
carbons. It is likely that loss from the skin surface is much more dynamic
and is mediated through normal desquamative and mechanical processes. One of
the things we will learn from the assay is whether retention or trapping of
fluorescent materials is greater for some crudes and, if it is, the degree to
which this phenomenon correlates with skin irritation or carcinogenesis or
both. Some available data suggest a positive correlation between trapping/
persistence and toxicity/carcinogenicity, although too few samples have been
tested to allow generalization (Holland et al. 1979d).
43
-------
In Vitro Metabolic Profile—
Our laboratory has developed a simple and efficient method to assess the
capacity of native material to modify the metabolism of marker polycyclic
aromatic hydrocarbon (PAH) compounds in intact skin. To date, studies have
been performed exclusively with labeled benzo(a)pyrene (BaP), but we are
extending our observations to other "off the shelf" marker PAH's, each of
which reflects a particular metabolic pathway.
Using short-term organ cultures, we will compare the various crude,
hydrotreated, and finished products with respect to extent, direction, and
nature of influence on overall rate of BaP metabolism. Assuming funds for
necessary equipment can be obtained, we plan to compare BaP metabolic profiles
obtained in the presence of materials in both mouse and human skin. These
assays may provide information on whether the mixtures contain modifiers of
PAH metabolism. By comparing rates and profiles for a range of whole crudes
as well as the Paraho shale oil and products, we will determine whether signif-
icant metabolic disparities occur and what effect, if any, they have on
biological potency in vivo.
Pulse Skin Carcinogenesis Bioassay—
Previous experience with prototype whole synthetic crude oils has provided
information sufficient for an adequate provisional assessment of relative
carcinogenic potency of related materials. This assessment has been achieved
within a comparatively short period of time and with reduced test material
requirements. We have determined that synthetic crudes differ markedly in
capacity to evoke direct skin irritation. In addition, we have obtained
evidence suggesting that this in vivo cytotoxicity may inhibit expression of
skin neoplasms (Holland et al. 1979d). Because PAH's are also toxic following
metabolic activation (Nebert et al. 1977), it follows that tumors are expressed
only if neoplastic cells are differentially refractory to continued application
of the carcinogen (Farber and Solt 1978) or if carcinogen application is dis-
continuous. Our approach to bioassay of complex and variably cytotoxic
materials strives to maximize the probability of tumor expression while mini-
mizing cytotoxicity and preserving the data's relevance to assessing potential
44
-------
consequences of occupational cutaneous exposure (which would be episodic
rather than continuous).
In our method, groups of animals are exposed either 2 or 3 times weekly
to graded doses of the materials diluted in an appropriate solvent. The
highest dose is one that can be tolerated without frank erosion or ulceration
of the skin. For moderately carcinogenic crudes, >l/3 of the initial popula-
tion will develop tumors within 32 weeks (Holland et al. 1979b, 1979c, 1979d).
By comparison, our C3H mouse has an average tumor latency of 16 weeks at 50 g
BaP 3 times/week (Holland et al. 1979a). At 20 or 30 weeks, exposure is dis-
continued and mice (with and without tumors) are held for an additional 20
weeks to assess the clinical progression of induced neoplasms in the absence
of continued exposure. Following this clinical observation phase, surviving
mice are killed and those with skin tumors examined for signs of metastasis.
All mice (including those that died during the course of the study) are ex-
amined for signs of systemic pathology.
Our resources for tests of this nature are extremely limited. Therefore,
we will evaluate only the raw crude, hydrotreated crude, No. 6 fuel oil, and
DFM. It may be possible to consider the remaining materials in a second cycle
of tests. Our reason for selecting No. 6 fuel oil and DFM for carcinogenicity
tests is that their higher boiling range might be expected to make them the
most carcinogenic of the various products. In other words, if tests of DFM
and No. 6 prove negative, we will be surprised if any of the jet fuels are
later found to be more active.
INPUT TO HEALTH EFFECTS ASSESSMENT
The two-level program described above is designed to provide specific
information on specific process materials. This generic approach, coupled
with chemistry, health effects, and environmental studies, will place syn-
thetic fuel materials into context with other materials and processes for
which data are available. Direct information on potential mutagenicity,
carcinogenicity, and overall toxicity of the process samples will provide
45
-------
perspective with respect to other technologies. Comparative information and
published data on similar materials will permit an ordered estimate of bio-
hazard for each sample. Our team approach will encourage expedient extra-
polation of data on known materials.
The relationship of these screening tests to risk assessment in man
remains to be demonstrated, and is the focus of considerable ongoing research.
By this we do not imply that the concept of screening is at present invalid,
but simply that many tests remain developmental to varying degrees. The shale
oil research by Paraho/Standard Oil Company of Ohio represents a major op-
portunity to demonstrate practical applicability of the "screening approach'1
in toxicity testing.
REFERENCES
Farber, E., and D. Solt. 1978. A new model for the sequential analysis of
chemical carcinogenesis in the liver. In: Slaga, T. J., A. Sivak, and
R. K. Boutwell (eds.), Carcinogenesis —A Comprehensive Survey. Vol. II.
Mechanisms of Tumor Promotion and Cocarcinogenesis, pp. 443-448. New
York, Raven Press.
Holland, J. M., D. G. Gosslee, and N. J. Williams. 1979a. Epidermal carcino-
genicity of bis(2,3 expoxycyclopentyl)ether, 2,2 bis(p-glycidyloxyphenyl)-
propane, and m-phenylenediamine in C3H and C57BL/6 inbred male and female
mice. Cancer Res. 39:1718-1725.
Holland, J. M., R. O. Rahn, L. H. Smith, B. R. Clark, S. S. Chang, and T. J.
Stephens. 1979b. Dosimetry of coal and shale derived crude liquids as
mouse skin carcinogens. J. Occ. Med. 21:614-618.
Holland, J. M., M. S. Whitaker, and L. C. Gipson. 1979c. Chemical and bio-
logical factors influencing the skin carcinogenicity of fossil liquids.
Proceedings of Park City Environmental Health Conference, Park City, Utah,
April 4-7.
Holland, J. M., M. S. Whitaker, and J. W. Wesley. 1979d. Correlation of
fluorescence intensity and carcinogenic potency of synthetic and natural
petroleums in mouse skin. Am. Ind. Hyg. Assoc. 40:496-503.
Nebert, D. W., R. C. Levitt, N. M. Jensen, G. H. Lambert, and J. S. Felton.
1977. Birth defects and aplastic anemia: Differences in polycyclic
hydrocarbon toxicity associated with the Ah locus. Arch. Toxicol.
39:109-132.
46
-------
6. SHALE OIL BIOASSAYS AT BATTELLE PACIFIC NORTHWEST LABORATORIES
R. A. Pelroy
Biology Department
Battelle Pacific Northwest Laboratories
Post Office Box 999
Richland, Washington 99352
INTRODUCTION
Efforts by the Biology Department of Battelle Pacific Northwest Labora-
tories will include in vitro mutagenicity, DNA damage, and cellular toxicity
testing of Paraho shale oil, intermediate process streams, and refined shale
oil products. Chemical fractionation of the complex mixtures will be accom-
plished by solvent extraction and thin layer chomatography (TLC). The in
vitro assays coupled to analysis by gas chromatography/mass spectrometry
(GC/MS) of crude material, solvent extracts, and TLC fractions will be the
main analytical tools for relating activity to chemical species.
SAMPLES TO BE ASSAYED
Our testing regime will examine the unrefined shale oil (starting mate-
rial) , intermediate process streams and by-products, and refined products.
Table 6-1 presents an itemized list of these materials. At present, our De-
partment lacks the resources to investigate airborne matter, raw shale, spent
shale, and similar materials, although we remain interested in such potential
studies.
47
-------
TABLE 6-1. SAMPLES TO BE BIOASSAYED AT BATTELLE PACIFIC NORTHWEST LABORATORIES
Crude Shale Oil
Hydrotreated Shale Oil
No. 6 Fuel Oil
Gasoline Stock
JP-4 Precursor
JP-5 Precursor
JP-8 Precursor
DFM Precursor
JP-4
JP-5
JP-8
DFM
Acid Sludge
Water (retort oil separation)
Water (from stripper)
FRACTIONATION METHODS
Our Department currently employs two fractionation schemes to break down
complex hydrocarbon mixtures into component chemical classes. The scheme
currently used for most of our work is a simple acid-base solvent extraction
procedure that yields acidic, basic, neutral, and polynuclear-aromatic-
hydrocarbon-containing fractions. These fractions are not subdivided (e.g.,
into weak acids, weak bases, etc.) and are thus somewhat less refined than
samples obtained with a "Swain-type" procedure. Our main purification step
occurs with TLC separation of the components in the fractions.
A second fractionation method is currently being applied to shale oil (
and may eventually replace the simple solvent extraction procedure, provided
we can show significant improvements in yields or resolution of biologically
active materials. This scheme is based on Swain-type solvent extraction of
complex mixtures followed by column fractionations similar to those developed
48
-------
at Oak Ridge National Laboratory. We plan to carry out direct comparisons of
the two types of fractionation schemes (i.e., simple solvent extraction versus
solvent extraction-column fractionation) in terms of the materials yielded for
bioassay.
BIOASSAYS TO BE APPLIED
Table 6-2 lists the bioassays that our Department will use to assess the
various samples. The main test system will be the Ames Salmonella assay;
direct comparisons will be made with the other systems listed.
TABLE 6-2. BIOASSAYS TO BE APPLIED AT BATTELLE PACIFIC NORTHWEST LABORATORIES
Test System
Bacteria for mutagenesis Salmonella
(Ames assay)
Mammalian cell cultures CHO cells
for mutagenicity at hgprt
Mammalian cell cultures CHO cells
for SCE
Mammalian cell cultures CHO cells
for toxicity
SUMMARY
By way of summary, Figure 6-1 presents a schematic diagram of our in-
tended protocol for assessing shale oil materials. To reiterate, we will
emphasize a simplified solvent extraction procedure with heavy reliance on
TLC in combination with GC/MS. Our approach will be essentially analytical,
focusing on the structural relationships that may relate mutagenic activity to
chemical species. The Ames assay will serve as the main connecting link be-
tween the chemistry and the biology.
49
-------
SAMPLES
Unrefined shale oil,
intermediate process streams
and by-products, refined products
ASSAYS
Ames, CHO (mutation,
SCE, toxicity)
FRACTIONATION
Solvent extraction to yield acid,
base, neutral (etc.) fractions;
TLC to yield subtractions; GC/MS
analysis of subtractions
Ln
o
Heaviest reliance will be placed on the Ames assay, with direct comparisons to other assays (e.g.,
Ames back mutation vs. CHO SCE assay, etc.).
DGC/MS analysis will be routinely used only on samples yielding positive iii vitro assays.
Figure 6-1. Schematic representation of procedure for assessing shale oil materials at Battelle Pacific
Northwest Laboratories.
-------
7. APPLICATION OF A BATTERY OF SHORT-TERM
BIOASSAYS FOR TESTING THE GENETIC TOXICITY
OF PARAHO SHALE OIL PRODUCTS
F. T. Hatch
H. Timourian
Lawrence Livermore Laboratory
University of California
Biomedical Sciences Division
Post Office Box 5507
Livermore, California 94550
INTRODUCTION
The objective of this project is to determine the relative mutagenicities
and genetic toxicities of crude, hydrotreated, and refined shale oil products
from the Paraho surface retort and to compare the potential health hazards
of these materials with hazards of similar petroleum-derived materials. Applica-
tion of a battery of bioassays consisting of a standard microbial test and in
vitro and in vivo mammalian systems will provide a basis for estimating human
health hazards.
A substantial portion oft the Biomedical Sciences Division program at
Lawrence Livermore Laboratory (LLL) is devoted to integrated application of
cell biology, analytical cytology, and biochemical techniques to problems of
environmental mutagenesis, carcinogenesis, and injury to the reproductive
system. Most of the test systems in the available battery of bioassays (Table
7-1) were developed and validated at LLL. This battery emphasizes mammalian
systems and includes both in vitro (cell culture supplemented with metabolic
activation) and short-term in vivo components.
51
-------
TABLE 7-1. BIOASSAYS AVAILABLE AT LAWRENCE LlVERMORE LABORATORY
in
to
Test System
Bacteria for Salmonella strains
mutagenesis that require
(Ames assay) histidine
Cultures (mammalian
cells) for:
Toxicity CHO and mouse
hepatoma cells
Mutagenicity CHO cells
Chromosome CHO cells
damage
Whole animals for:
Chromosome Mice
damage
Sperm Adult male mice
morphology
Oocyte Newly born
depletion female mice
Average
End Point or Process Time Cost Rangea
Parameter Measured (weeks) (dollars/sample)
Growth of reverse- 1 350-600
mutant colonies in
the absence of
histidine
Growth of cell 2 350-600
colonies in
presence of test
substance
Growth of drug- 8 2500-5000
resistant mutants
in presence of
lethal dose of drugs
Sister chromatid 2 700-1500
exchange in cells
Sister chromatid 2 800-1500
exchange in bone
marrow cells
Abnormal morphology 8 3000-6000
of epididymal sperm
Survival of primary 4 1500-3000
oocytes
Testing
Capability
(samples/
yr/FTE)
150-200
150-200
20-30
100
90
10-15
25-30
Cost includes testing at several doses to give a dose-response curve.
-------
Five types of bioassays are available for the project:
(1) Microbial mutagenesis by detecting revertants to histidine
independence in Salmonella typhimurium (Ames assay)
(2) Mammalian cellular toxicity by measuring differential toxicity
in cultures of mouse hepatoma cells lacking or containing aryl
hydrocarbon hydroxylase and cultures of Chinese hamster ovary
(CHO) cells with and without defects in repair of deoxyribonucleic
acid (DNA)
(3) Mammalian cellular mutagenesis by measuring the frequency of
mutations at multiple gene loci in CHO cells (hprt, aprt, ATPase,
tk)
(4) Mammalian cellular and genetic toxicity to germ cells by
measuring the frequency of induction of abnormal head shape
in sperm and killing of primary oocytes in vivo
(5) Chromosomal j.njury and misrepair by measuring the frequency of
induction of sister chromatid exchange (SCE) in vivo
AVAILABLE BIOASSAYS
Ames Assay
This test determines mutation induction (reversion) in histidine
auxotrophs (histidine requiring strains) of Salmonella typhimurium. The
bacteria are exposed to the shale oil materials; revertants that survive and
form colonies in histidine-free media are counted. The number of revertant
colonies represents a direct measure of induced mutation. Since some mutagens
require enzymatic activation, bacteria are exposed with and without S9, a
preparation of rat liver microsomes (Ames et al. 1975).
53
-------
Mouse Hepatoma Cell Assay
Mouse hepatoma cells permit rapid detection of benzo(a)pyrene (BaP) and
other polycyclic aromatic hydrocarbons in complex mixtures. Two genetic cell
lines, one sensitive and the other resistant to BaP (Hankinson 1979), are
exposed to the shale oil materials. A differential toxicity response provides
a rapid screening method (Figure 7-1). Since the major genetic difference
between the two strains is the ability to activate BaP, this test does not
require addition of a microsomal activating system.
Chinese Hamster Ovary Cell Toxicity Assays
CHO cells can be used to study cytotoxicity in two ways. In the first
method, a plating efficiency curve is run at a series of exposure doses to
determine 037(M) (the molar dose of agent that kills 63% of the initial cell
population). A review of the literature on mutagenesis in mammalian cell
cultures (Carver et al. 1979b) and extensive data of June Carver (LLL) indicate
a high correlation between induced mutation frequency (per mole per liter
exposure dose) and D^(K) that is applicable for several genetic markers and
several mammalian species (Figure 7-2). Thus, in screening and setting priori-
ties for further testing, this simple and rapid measurement of cytotoxicity is
often predictive of mutagenicity, and any errors will be conservative.
The second method employs one or more mutant strains of CHO cells now
being developed by Larry Thompson (LLL). These mutants are substantially
more sensitive to various classes of mutagens than the "wild"-type strain,
presumably owing to defects in DNA repair mechanisms. Thus, differential
cytotoxicity between "wild" and mutant cells indicates damage to DNA.
Chinese Hamster Ovary Cell Mutagenesis Assays
CHO cells are incubated with the test material alone or with a microSjOmal
activating system. After exposure, the CHO cells are tested for reproductive
ability (by determining plating efficiency) and for specific single step
54
-------
BaP concentration
M9/ml
0
0
0.1
0.3
1.0
3.0
Mouse
hepatoma
cell lines:
Hepa-1
sensitive
Hepa-B6
resistant
Figure 7-1.
Mammalian cellular toxicity assay employing mouse hepatoma cells. Duplicate cultures
containing 2 x lO4 cells/well of either sensitive or resistant cells were exposed to BaP.
After 5 d, sensitive cells were killed and resistant cells grew to confluence at all BaP
concentrations.
-------
104
103
O
c
01
§102
S
*f-
co
I 10°
3
T)
10'
BP/MBA-7br
/—
I ! I S i 1J 1 \ I i ! i 1 t 1i I I i J I 5 i 1 I I
102 103
104 105 106
1
D37(M)
10'
10
8
Figure 7-2.
Results of simple CHO cell toxicity assay of 22 chemical muta-
gens. Cytotoxic potency correlates with mutagenic potency as
assayed at hprt, aprt, and tk loci in five rodent and human in
vitro cell systems. Data for induced mutations are plotted as a
function of the reciprocal of D37(M). See Carver et al. (1979b)
for details.
56
-------
mutations at four different loci (by determining resistance to lethal drugs).
Plating efficiency provides a measure of toxicity and, in some cases, an in-
direct measure of mutagenicity. To determine mutagenicity, cells exposed to
the test material are subsequently cultured in lethal concentrations of a drug
(e.g., azaadenine, azaguanine, fluorodeoxyuridine, or ouabain). Survival and
growth in the presence of the drug are a measure of induction of mutation by
the test material (Figure 7-3). In general, the effects at different markers
are correlated, but recent data (Carver et al. 1979a; Thompson 1979) indicate
that certain mutagen classes may show differential effects at the markers,
so that the multiple marker CHO assay may provide a broader spectrum to detect
mutagens than the commonly used strains. The CHO cell bioassays are also
sensitive to mutation induction by metal ions and their methylation products
(Taylor et al. 1979a, 1979b).
Mammalian Germ Cell Toxicity Assays
Sperm Head Abnormalities-
Four weeks after in vivo exposure to shale oil materials, sperm head
abnormalities may be detected in adult male mice (Figure 7-4). The fractions
of epididymal sperm that are morphologically abnormal are counted by micro-
scopic observation. Isogenic strains of mice are used because the percentage
of abnormal sperm in each strain is constant. The induction of abnormal sperm
morphology by mutagens is well documented (Wyrobek and Bruce 1978) . Recent
studies in industrial populations indicate sensitivity of human spermatogenesis
to pesticides and other chemicals (Wyrobek and Gledhill 1978).
Oocyte Depletion— '
Following in vivo exposure to shale oil materials, oocyte depletion may
be detected in newly born female mice (Figure 7-5). Exposure is either direct
to the newborn by gavage or indirect (in utero) by treatment of the pregnant
mother. Mice are sacrificed 14 d after exposure, ovaries are sectioned, and
numbers of oocytes are counted. The number of oocytes in the ovaries of an
organism is set before birth and normally diminishes at a predictable rate
during lifetime. Oocytes are very sensitive to mutagens, and an accelerated
rate of depletion may indicate genetic damage (Dobson et al. 1978). The median
57
-------
(a) Drug-sensitive CHO cells
(b)
200
.£
_o
o
o
Exposed to test substance for 20 h
Culture cells to allow recovery and
expression of mutant phenotype
"5 100 -
.a
3
z
Test for toxicity
survival and
SCE induction
0.5
Fraction of cells
surviving (1-dose)
0.1
Culture in presence
of drug
Normal cells die;
mutants multiply
and form colonies
Figure 7-3. CHO cell mutagenesis assay. CHO cells exposed to energy effluents
or their fractions are tested for toxicity survival, mutation in-
duction, and SCE. (a) Mutations are detected by cell survival in
the presence of lethal doses of drugs such as azaadenine or aza-
guanine. These drugs are structurally similar to adenine or
guanine, bases that make up DNA and RNA (nucleic acids that carry
genetic information). A mutation in genes that specify the struc-
ture of enzymes using guanine or adenine will prevent the incor-
poration of the drug into DNA and RNA and make the cells drug- '
resistant. (b) The number of azaguanine-resistant mutant cells I
as a function of the mutagen ethyl methane sulfonate (EMS) is
shown by plotting the number of mutant colonies vs. the fraction
of cells that survived the toxic effects of EMS (survival is in-
versely proportional to applied mutagen dose).
58
-------
(a) Inject test substance
(b)
10
Adult male
Q.
CO
o
c
4
weeks
Score for sperm
morphology from epididymis
T I\\ TTTT
Background
number of
abnormalities
i i i
i i i
1 5 10 50
Dose of test substance — mg/kg
(c)
Normal
Abnormal
Figure 7-4.
Assay for morphological abnormalities in sperm of adult male mice.
Induction of abnormal sperm morphology by test substances provides
an indication of genetic damage. (a) Sperm development takes 4
weeks; detrimental effects of the test substances are easily de-
tected by counting abnormal sperm after this period. (b) Plot of
the effect of 3-methyl cholanthrene on the frequency of abnormal
sperm as a function of dose. (e) Typical samples of sperm mor-
phology as seen under the microscope.
59
-------
(a) Inject test substance
(b)
1 2-day-old female
davs
o
Oocyte survival:
count primordial oocytes in
serial sections of dissected ovary
100
75
'E
50
25
0.25 0.8 2.5 8.0 25
Dose of 3-methyl cholanthrene — mg/kg
80
Figure 7-5.
Assay for depletion of oocytes in newly born female mice. The highly sensitive oocytes of
young female animals are killed by most mutagens. (a) Surviving oocytes are counted in
serial sections of the ovary to calculate the LDsg. (b) Oocyte survival after injection of
different doses of 3-methyl cholanthrene.
-------
lethal dose (LDsg) for X-rays is ~5 rad; LD50 values for several polycyclic
aromatic hydrocarbons range from 1 to 20 mg/kg (representing, in the newborn
mouse, a total dose of a few ug).
Sister Chromatid Exchange Assay
SCE's may be assayed in mice after in vivo exposure to shale oil materials.
At 5 to 8 h before exposure to test material, mice are prepared by implanta-
tion of BrdUrd pellets under the skin. Animals are sacrificed 12 to 15 h later
and cells in bone marrow examined and scored for SCE's (Figure 7-6). The SCE's
(exchanges of segments between sister chromatids) are visible in metaphase
chromosomes because BrdUrd incorporated during DNA synthesis stains the new
and old chromatids differently. SCE's indicate that genetic damage and repair
may have taken place. The number of SCE's has been correlated with the number
of mutations in mammalian cell cultures for several mutagens (Carrano et al.
1978). We assume that the relationship that applies in cell cultures will
apply in whole animals (Stetka et al. 1978).
0,h
5to8h
WINDOW
2T 23f
Implant Challenge Inject Sacrifice,
BrdUrd with test Colcemid process
pellet material (40 g) cells
U_ chromosomes
oq.
Figure 7-6. Sister chromatid exchange assay. Induction of SCE's is determined
after in vivo exposures in mice prepared with BrdUrd and later
injected with colcemid to arrest systemic cell division at mitosis.
61
-------
STRATEGY OF APPLICATION
The major biological concern with environmental pollution is widely
believed to be DNA damage resulting from low-dose, often chronic exposure.
Such DNA damage can cause defects in the information content of the genome.
The principal consequences are carcinogenesis in the current generation and
an increase in the load of detrimental mutations in future generations.
Since direct measurement of either of these consequences from the large
variety of agents of concern is too time-consuming and expensive, a variety
of short-term tests was developed to indicate hazard and to aid in setting
priorities for definitive testing. An early concept was the "tier" approach,
in which application of a simple and rapid test was followed, if positive,
by successively more elaborate tests. As time passed, virtually every short-
term test proved fallible or even blind to certain classes of agents.
The currently favored strategy is a "sequential battery" in which two
or more tests (preferably based upon different genetic principles) are applied
at several levels of complexity. Even this approach has drawbacks. For exam-
ple, Purchase et al. (1976) calculated that application of eight tests in which
each has 90% accuracy results in a high probability for identification of all
toxic agents. However, 43% of the truly negative agents would also give a
positive result in one or more tests of the battery; therefore, careful judgment
is required to assess the correct status of some agents. Also, in the sequen-
tial battery approach it is probably wise to carry some random samples of ini-
tially negative agents forward into certain of the more elaborate assays.
An important consideration in comparing bioassays is sensitivity (i.e.,
the minimum concentration of an agent that will give a positive response). In
this respect, the principal experience of the authors' laboratory at LLL has
been with groundwater from wells surrounding the burn zone of an in situ coal
gasification experiment at Hoe Creek, Wyoming. Figure 7-7 shows the sensi- ,
tivities of our assay battery expressed as the reciprocal of parts per million
organic matter in sample required for a positive effect. The sensitivities
62
-------
CTl
Ul
>
!|
c
£
1 5
05
CC
Chinese hamste/ ovary cells
f m
T
133
Mouse hepatoma
.
cells LD5Q
-Salmonella n
. Ames Test
/ *
\
^
/
^
!v!
•:•
jif.
-i
/
^
rrr
1
::;!
>
d.
•:|:
$•
•:•:
:£:
'
/
/
/
/
/
/
/
:::::
;.|.
i?:
;!;:
Juvenile
female
mice
/
',
/
/
/
. — • —
s
-
—
-
ais
Relative sensitivity =
(1/ppm needed to
show effect) X100
Hoe Creek 2 experiment.
Water obtained from
within gasification cavity.
Figure 1-1.
Relative sensitivity of different bioassays to chemical fractions from underground water
after coal gasification.
-------
of the assays appear roughly similar; however, there is some indication that
the two assays for CHO cell toxicity are somewhat more sensitive than the
others (including the Ames assay).
The manpower and time requirements for bioassays are, of course, important
considerations. In general, except-for the Ames assay and the simpler
mammalian tests, the assays require ~0.5 man-month for a complete dose response.
Turnaround times range from 1 week (for the simpler tests) to ~2 months (for
the more elaborate ones).
When the results of a battery of tests are available, there remain the
important tasks of assessing human risk from realistic estimates of exposure
and of comparing relative risks in alternative courses of action. This new
area of genetic toxicology does not yet have a satisfactory theoretical, or
even empirical, framework. The authors' Division at LLL has limited experience
in the area but expects to become fully involved in the future. In particular,
there are strong possibilities for comparison of data from the LLL bioassay
battery with data becoming available from short-term genetic tests applied
directly in humans exposed to industrial agents or medical therapy. The
authors' Division is active in development and validation of some of these
tests, and application will greatly facilitate the development of risk-estima-
tion techniques (Carrano 1979; Wyrobek and Gledhill 1978).
SAMPLES TO BE ASSAYED
Initial plans are to assay crude and hydrotreated shale oil samples.
Additional samples will be tested if preliminary results from other labora-
tories (in particular, the laboratory of J. Epler at ORNL) indicate some of the
refined shale oil products to also be mutagenic. Initial results from muta-
genicity tests will be confirmed in mammalian cell lines and whole-animal tests.
Table 7-2 shows a priority matrix of available assays and materials to be tested.
Funding for application of our bioassay battery to samples of fossil
fuels or effluents remains quite limited. Furthermore, the number of technology
sources competing for our limited manpower and resources is growing rapidly (e.g.,
64
-------
TABLE 7-2. PRIORITY MATRIX FOR BIOASSAYS AT LAWRENCE LIVERMORE LABORATORY
w
Bioassay
Crude Hydrotreated
Ames Assay A/O A/O
Mouse Hepatoma
Cell Line
CHO Cell
Mutagenicity A A
and Toxicity
SCE in Mice A A
Abnormalities
Oocyte
Depletion
DFM
(pre-acid-
treatment)
A/0
B
B
B
B
B
Sample
DFM Various
(final) Jet Fuels
A/0 A/0
B B
B B
B B
B B
B B
Equivalent
Petroleum
Products
A/0
B
B
B
B
B
A = Highest priority.
B = High priority only if positive results are found in other bioassays.
O = May be tested in other laboratories (e.g., J. Epler at ORNL).
-------
Paraho shale fuels, Oxy in situ fuels and effluents, LLL retort samples, RCRA
leachates, and Rio Blanco in situ experiments). To reiterate, the authors
consider the highest current priority to be the issue of hydrotreating shale
oil to significantly lower its toxicity. Accordingly, our Division will assay
selected Paraho samples as appropriate. Particularly for the Navy's concerns,
however, the authors regard the study of combustion effluents from these fuels
as more important than assaying the neat fuels.
EXPECTED RESULTS
Initially, the Ames assay will be used to determine if hydrotreatment,
a necessary step to improve flow and refining characteristics, reduces the
mutagenicity of the crude shale oil. Use of the Ames assay on other refined
products (as performed by LLL or ORNL) will determine relative mutagenicities.
In vitro bioassays using mouse hepatoma and CHO cell lines will confirm
Ames assay results. Previously, matched hepatoma lines (BaP-sensitive and
-resistant) treated with graded doses of crude and hydro-treated Paraho shale
oil in a continuous 5-d exposure responded similarly in dose toxicity.
However, both cell lines were killed at lower concentrations of the crude
sample. For example, growth inhibition at 11 ug/ml with the crude sample was
comparable to inhibition at 94 ug/ml with the hydrotreated sample. These
results indicate that the measured toxicity is not due to BaP-like compounds,
but perhaps to substances causing nonspecific toxicity.
In vivo animal bioassays will test the effects on mutagenicity of dif-
ferent routes of administration, access of agents to germ cells, genetic
capacity for activation, and various dose rates. In a previous study, whole-
animal LDso levels in mice were determined as a preliminary to in vivo tests.
Juvenile mice were injected intraperiotoneally with single doses of either
crude or hydrotreated Paraho shale oil. After 2 weeks, LDso values of
3.8 g/kg body weight (Paraho crude) and 31.7 g/kg body weight (hydrotreated;
product) were determined. The higher toxicity of the crude was emphasized by
the fact that at 13.2 g/kg all animals exposed to crude were dead within 1 week.
66
-------
In contrast, all animals exposed to the same dose of hydrotreated product were
still alive 2 weeks later.
Although the significance of the relative toxicities of crude and hydro-
treated shale oil is not yet clear, note that crude was 8 times more toxic
than hydrotreated in both systems tested (in vitro hepatoma cell lines and
in vivo juvenile mice). This eightfold difference in toxicity is likewise
reflected in the relative carcinogenic activity of the two materials. For
example, in skin painting experiments using crude and hydrotreated Paraho shale
oil, Coomes (1979) reported tumor development in 13% of animals painted with
hydrotreated shale oil and 97% of those painted with crude. Such results con-
tribute to an understanding of the spectrum of toxicity of shale oil products
in bacterial and mammalian systems, and help to predict potential effects on
humans.
In toxicologic studies of synthetic fuels (particularly the work of
J. Epler at ORNL and R. Pelroy at Battelle Pacific Northwest Laboratories),
the basic alkaline pH fraction appears to have the highest potency; this
fraction contains a variety of nitrogeneous heterocyclic aromatic compounds.
In a separate project, the authors recently found that the major mutagenic
activity produced in cooking beef is also in the basic fraction. Although the
genetic toxicology of naphthylamines, azo dyes, and aminobiphenyls has been
studied, many classes of organic bases deserve further investigation.
ACKNOWLEDGMENT AND DISCLAIMER
This work was performed under the auspices of the U.S. Department of
i
Energy by the Lawrence Livermore Laboratory under Contract W-7405-ENG-48.
NOTICE
"This report was prepared as an account of work sponsored by the United States
Government. Neither the United States nor the United States Department of
Energy, nor any of their employees, nor any of their contractors, subcontrac-
tors, or their employees, makes any warranty, express or implied, or assumes
67
-------
any legal liability or responsibility for the accuracy, completeness or use-
fulness of any information, apparatus, product or process disclosed, or
represents that its use would not infringe privately-owned rights."
REFERENCES
Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for detecting
carcinogens and mutagens with the Salmonella/mammalian-microsome
mutagenicity test. Mutat. Res. 31:347-364.
Carrano, A. V. 1979. Sister chromatid exchange: Relation to mutation and
application to human population studies. Abstract, DHEW Subcommittee
on Environmental Mutagenesis, May 7. National Institutes of Health,
Bethesda, Maryland.
Carrano, A. V., L. H. Thompson, P. A. Lindl, and J. L. Minkler. 1978. Sister
chromatid exchange as an indicator of mutagenesis. Nature 271:551-553.
Carver, J. H., G. M. Adair, and D. L. Wandres. 1979a. Mutagenicity testing
in mammalian cells. The development and validation of multiple drug-
resistance markers having practical application for screening potential
mutagens. Preprint UCRL-81703. Lawrence Livermore Laboratory, Livermore,
California.
Carver, J. H., F. T. Hatch, and E. W. Branscomb. 1979b. Estimating maximum
limits to mutagenic potency from cytotoxic potency. Nature 279:154-156.
Coomes, R. M. 1979. Carcinogenic testing of oil shale materials. 12th Oil
Shale Symposium, Colorado School of Mines, Denver, Colorado, April 18-20.
Dobson, R. L., C. G. Koehler, J. S. Felton, T. C. Kwan, B. J. Wuebbles, and
D. C. L. Jones. 1978. Vulnerability of female germ cells in developing
mice and monkeys to tritium, gamma rays, and polycyclic aromatic hydro-
carbons. Develop. Toxicol. of Energy-Related Pollut. CONF-77107,
DOE Symp. Series 47, pp. 1-14.
Hankinson, O. 1979. Single-step selection oif clones of a mouse hepatoma cell
line deficient in aryl hydrocarbon hydroxylase. Proc. Natl. Acad. Sci.
76:373-376.
Purchase, I. F. H., E. Longstaff, J. Ashby, J. A. Styles, D. Anderson, P. A.
Lefevre, and F. R. Westwood. 1976. Evaluation of six short term tests
for detecting organic chemical carcinogens and recommendations for theif
use. Nature 264:624-627. ,
Stetka, D. G., J. L. Minkler, and A. V. Carrano. 1978. Induction of long-
lived chromosome damage, as manifested by sister-chromatid exchange in
lymphocytes of animals exposed to Mitomycin-C. Mutat. Res. 51:383-396.
68
-------
Taylor, R. T. , J. H. Carver, M. L. Hanna, and D. L. Wandres. 1979a. Platinum
induced mutations to 8-azaguanine resistance in Chinese hamster ovary
cells. Mutat. Res. 67:65-80.
Taylor, R. T., J. A. Happe, M. L. Hanna, and R. Wu. 1979b. Platinum
tetrachloride: Mutagenicity and methylation with methylcobalamin. J.
Environ. Sci. Health A14(2):87-109.
Thompson, L. H. 1979. Validation of conditions for efficient detection of
HPRT and APRT mutations in suspension-cultured Chinese hamster ovary
cells. Preprint UCRL-82111. Lawrence Livermore Laboratory, Livermore,
California. Also: Mutat. Res. (in press).
Wyrobek, A. J., and R. Bruce. 1978. The induction of sperm-shape abnormalities
in mice and humans. Chemical Mutagens, Vol. 5 (A. Hollaender, ed.),
pp. 257-285. Plenum Press, New York.
Wyrobek, A. J., and B. L. Gledhill. 1978. Human semen assays for workplace
monitoring. Proc. Workshop on Methodology for Assessing Reproductive
Hazards in the Workplace, NIOSH. Preprint UCRL-81810. Lawrence Livermore
Laboratory, Livermore, California.
69
-------
8. EVALUATION OF POTENTIAL TOXICITY
OF SYNTHETIC FUEL COMBUSTION PRODUCTS
D. L. Coffin
J. L. Huisingh
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
INTRODUCTION
In evaluating the potential health effects of synthetic fuels, it is
necessary to test the fuels at every stage of extraction, refining, trans-
portation, and end use at which a possibility exists of significant direct
or indirect human contact. The production and subsequent refining of a
synthetic crude oil from shale have presented a timely opportunity to examine
combustion products. This report presents a brief overview of current and
proposed studies of combustion products by investigators from EPA's Office
of Research and Development at Research Triangle Park, North Carolina.
ONGOING AND PLANNED STUDIES
Studies are planned of the emissions which result from combusting
synthetic fuel oil in commercial boilers. Comparisons to standard petroleum
fuel oil emissions will seek possible differences in emitted products having
direct toxicity (e.g. carcinogenicity or mutagenicity) or indirect effect
(e.g., contribution of reactive hydrocarbons, nitrogen oxides, or other
precursors to photochemical smog).
70
-------
The mutagens and potential carcinogens associated with diesel combustion
are of concern because of the high particle emission rate as compared to
current gasoline vehicles. The minute particles emitted from diesel combus-
tion consist of elemental carbon with adsorbed organics containing extractable
mutagens (as indicated by Ames testing). Previous studies (Huisingh et al.
1978) showed that petroleum-derived diesel fuel combustion products
(particles) possess significant mutagenic activity as measured by the Ames
test. Furthermore, the characteristics of the fuel were observed to influence
the Ames test results.
Underway is a joint EPA/Department of Transportation project to compare
automotive combustion emissions from shale-oil-derived and petroleum-derived
diesel fuels. This work employs the Diesel Fuel Marine refined by the
Standard Oil Company of Ohio from the Paraho crude produced by Development
Engineering, Inc. This fuel is to be compared for mutagenicity with standard
petroleum-derived Diesel Fuel No. 2 obtained from local sources. As an
analytical reference, these experiments will also include a No. 2 National
Average (a fluid used for vehicle certification). Particles will be collected
from a prototype test vehicle operated on a chassis dynomometer simulating
the actual driving pattern of the Highway Fuel Economy Test cycle. Collection
will be accomplished by filtration on Pollflex filters from a standard
dilution tunnel. The filters will be extracted with dichloromethane for 48 h
by the Soxhlet method and diluted with dimethyl sulfoxide for bioassay in
the Ames test.
RESEARCH NEEDS
The type of information that is obtained in such studies is needed as
quickly as possible in the hope of obviating, during extraction and refining
operations, any problems that may be unique to synthetic fuels. In the
present case, shale is considered a useful source for strait run middle
distillates; as such, shale might prove a valuable substitute for petroleum
for automotive diesel fuel. Information is needed not only on the currently
available refined product (Diesel Fuel Marine) but also on the contribution
71
-------
of mode of extraction and refining to the development of mutagens in the
exhaust. Experiments are required to determine the influence of precursors
in the fuel (regardless of the fuel's source) on the content of mutagenic
compounds in the exhaust. Thus, basic experiments are needed not only on
finished fuels but also on precursor fuels that have not been subjected to
post-distillation treatment and, most importantly, on fuels in which the
hydrocarbons have been deliberately altered in order to delineate the roles
of specific fuel compound classes on mutagen synthesis by the diesel engine.
Such a program, together with improvement of engine combustion efficiency,
can likely lead to significant reduction in the content of potentially
hazardous exhaust products.
REFERENCE
Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada,
J. Bumgarner, F. Duffield, M. Waters, V. F. Simmon, C. Hare,
C. Rodriguez, and L. Snow. 1978. Application of bioassay to the
characterization of diesel particle emissions. Application of Short-
Term Bioassays in the Fractionation and Analysis of Complex Environmental
Mixtures (M. D. Waters, S. Nesnow, J. L. Huisingh, S. S. Sandhu, and
L. Claxton, eds.), pp. 381-418. Plenum Press, New York.
72
« US. GOVERNMENT PRtHnNQ OFFICE: 1W1-757-064/0279
------- |