Leaching study of polycyclic aromatic hydrocarbon bioavailability from a coal-derived solid product

Chemosohere, Vol.16, Nos.lO-12, P r i n t e d in G r e a t B r i t a i n

pp

2613-2622,

1987

0045-6535/87 $ 3 . 0 0 ÷ .OO P e r g a m o n J o u r n a l s Ltd.

LEACHING STUDY OF YOLYCYCLIC AROMATIC HYDROCARBON BIOAVAILABILITY FROM A COAL-DERIVED SOLID PRODUCT C. Y. Ha, I. B. Rubln, B. A. Tomklns, and W, H. Grlest Analytical Chemistry Division Oak Ridge National L a b o r a t o r y Oak Ridge, Tennessee 37831

ABSTRACT The leaching behavior of selected four- to slx-ring polycyclic aromatic hydrocarbons (PAH) from

a

tumorigenic

their

potential

aqueous

coal-derlved

solid product was

bioavailebilltle$.

solubilities

Equilibrium

determined

using

three

PA~4 concentrations

after less than five days of beaching.

media

generally

YAH selectively

to evaluate exceeded

she

accumulated

in

phospholipld vesicles at individual concentrations

greater than i ~g/g, which is ca. 100-fold

over

results

that

in

bioavailable

the

bulk

aqueous

leachate.

from the solid product,

The

suggest

and that cell membranes

that

can be

the

YAH

are

quite

exposed to considerable

quantities of these YAH over an extended bloassay.

INTRODUCTION In a two-year re= inhalation bloassay of the solid product from the Solvent Refined Coal (SRC-I) coal converstion process, 5 of 27 females and I of 40 males exposed to A9 mg/m ~, for 8& weeks

in

the

case

of

females

carcinoma of the lung (1). 10 mg/m 3 . these

However,

lower exposure

inhalation.

and lO~ weeks

in the case

of males,

exhibited

squamous

cell

Squamous cell carcinoma was not observed in animals exposed to I or

a precursor levels.

lesion,

This

keratlnizing

is an unusually

squamous

eplthelioma,

strong tumorigenic

was

response

observed at for particle

The skin tumorigenicity conferred upon high-boiling crude coal liquids by their

content of polycyclic

aromatic hydrocarbons

(YAH)

of several of these YAM in the SRC Solid (7,8)

(2-6)

and the 12 to 280 ~g/g concentrations

suggest that the YAH content of the SRC Solid

may have contributed t o the responses observed in the inhalation bioassay. It

has

transported subsequently nature,

been into

reported cellular

metabollsed

by

(9-12) membranes

that

YAH

before

membrane-bound

adsorbed

on

biological enzymes.

PAH are likely to partition from particles

inhaled

effects

Because

of

particles

can

be

their

must

first

be

PAH

are

exerted. nonpolar,

lipoph[llc

into the lipid bilayers of cell membranes.

One of the in-vltro models

for the membrane bilayer consists of phospholipid vesicles

Phospholipids

the

are

one

of

major

components

of

lung

surfactants

(13),

and

(9-12).

leaching

of

particulate PAH by lung surfactantl such as phospholiplds can provide useful information on PAH

2613

2614 i)ioavailability.

In thls study, three media, including phospholipid vesicles, were utilized to

assess t h e potential bioavailability of selected PP~ dermal tumorlgens from the SRC S o l i d

EXPERIMENTAL

The

solid

coal-derived

inhalation

bioassay

was

supplied

<'IRDC", Mat~awan, HI). run

no.

235

of

the

when

(the

by

the

SRC

Solid)

sample

In~ernatlonal

which

Research

had

and

been

subjected

Development

to

Corporation

This sample is identified as the hydrotreater unit solid product from

Solvent

micronized to a particle i8)

product

Refined

Coal

plant

at Wilsonville,

AL

(8).

It had been air-

size range which yielded a mass median aerodynamic diameter of 3 ,m

resuspended

in

the

inhalation

chambers

at

IRDC.

Chemical

and

physical

characterization of this SRC Solid simple is reported elsewhere (7,8). The

following

as

received.

used

chemicals The

and

[7,10-t4C]benzo(a)pyrene

International

Ltd.(Amersham,

Research Labs.

(New York, NY).

L-A-phosphatldylcholine

reagents were obtained from the

UK),

and

the

(29.7

Tris

indlcaced

mCt/mmol)

was

sources

bought

from

(tris[hydroxymethylaminomethane)

Tween 80 (polyoxyethylene sorbltan mono-oleate)

and were Amersham from

Mann

and dipalmi~oyl

(DPPC) were obtained from the Sigma Chemical Company (St. Louis, MO).

PAH standards were purchased from the Aldrich Chemical Company (Milwaukee, WI), Analabs, (North Haven,

CT), and ~he Commieslon of the European Communities

grade methylene chloride, hexane, Labs.

Inc~

Biological

(Huskegan, Co.

Hl).

Eagle's

(Grand Island,

ordered from KC Biological,

acatonitrile,

NY),

Inc.

Minimum

(Brussels,

Belgium).

Inc. UV-

and methanol were all from Burdick and Jackson Essential

while Hank's buffer

Medium

(HEM)

was

from Grand

solution and fetal bovine

Island

serum were

(Lexena, KS).

~e#chinz,~edia Tween 80:

A 0.13% solution of Tween 80 in Trls buffer (0.01 H Trls-amlne, 0.05 M KCI, pH

7.50) was prepared by sonlcation of a known quantity of Tween 80 in a buffer solution for i0 man at room temperature. Minimum Essential Medium (MEM):

A mixture of i0~ (v/v) fetal bovine serum in Eagle's MEM

was prepared by mixing the two freshly-thawed reagents at room temperature. stored

at

-4°C.

A wetting

agent of

2% Tween 80 in Hank's buffer

The MLM/serum was

solution was prepared by

sonication for 10 man at room temperature (iA). Dipalmitoylphosphatldylcholine

(DPPC):

DPPC

vesicles

were

prepared

by

sonication

suspension of DPPC at a concentration of lO mg/ml in a buffer (0.01 M Tris-amine~ pH 7.5)

of a

0 05 M KCI,

The milky suspension was heated for one hr at 50°C (I0,Ii).

te~chin~ of SRC,,Solid The 5RC Solid was leached at a concen=ratlon of approximately 1 mg of solid solution.

The

leaching

medium

shaking or ultrasonicatlon solid

particles

m:xture.

were

gradually

to form a homogenous wetted

added

=o the weighed

suspension.

in the wetting

agent

solid with

flasks

per

ml

to addition

of

of

intermic:enc

In leaching with MEM/serum,

prior

A 1,5 ml volume of wetting agent was used per I00 ml of M~M/serum.

leaching was conducted Er!enmeyer

first

was

the

the MEM/serum Generally,

:he

in the dark with volumes of 50, i00, or 150 ml in I00. 150, or 250 ml

(respectively)

placed

selected to model animal lung conditions.

in a 37°C water bath

shaker.

These

conditions

were

2615

At p r e d e t e r m i n e d

intervals

time

(5 days,

2, 4,

or 6 weeks)

a

50 ml aliquot of the SRC

Solid leaching suspension was removed from the water bath and analyzed. sequential leaching study, on every fifth day of leaching, a clinical

benchtop

centrifuge

(model CL International

supernatant was removed for analysis.

In a separate Tween 80

the suspension was centrifuged wi~h

Equipment

Co.,

Needham,

MA)

and the

Fresh Tween 80 soluclon was added to the precipitate to

continue the leaching. Blanks for all three leaching media were prepared as above (except that the SRC Solid was omitted).

Leaching was run for five days before analysis.

~9~,lection.

Isolation.

After

the

the entire

leachates

SRC S o l i d

sample

precipitate

the

leaching

9 f L q l c h e d PAH s u s p e n s i o n was removed from t h e

or a 50 ml portion was

solid

were

isopropanol

and A n a l y s i s

particulate

then

filtered

to reduce

matter.

through

a

its hydrophobic

was further centrifuged

centrifuged The

1 ~m

supernates Teflon

shaking

in a clinical from

filter

characteristics.

water bath,

benchtop

the Tween

which

had

either

centrifuge

to

80 and MEM/serum

been

prewashed

with

The supernate from the DPPC leachate

in a Beckman modal L-2 uluracentrlfuge

(Beckman Instruments,

Berkely,

CA> for i hr at 27,000 rpm (ca. 90,000 G) in a type 50 rotor. Nonpolar

fractions

of

the

leachates

were

collected by pumplng measured volumes

of the

centrifuged and filtered leachate$ through a 4.6 mm ID x 3.7 cm, RP-18 Sphere-lO reverse phase guard column cartridge pressure

of

nonpolar

fraction

(Brownlee Labs,

600 pslg.

The

was

eluted

with

CA) at a flow rate of i ml/mln and

Inc., Santa Clara,

column was an

then washed with an equal volume equal

volume

of

methanol.

The

of water,

methanol

and the

eluate

was

evaporated to dryness in a rotary evaporator at 50°C and transferred with methylene chloride to a tared vial.

The solvent was evaporated again in a scream of nitrogen, and the residue was

we ighed. fraction was separated from the DPPC precipitate.

A nonpolar

from the ultracentrlfugation

precipitate which

had

not

been

precipitate was water

removed

suspended

by

the

in methanol

The top layer of the DPPC

was carefully plpetted from the underlying SRC Solid

low-speed

benchtop

clinical

centrlfugation.

and dried in a rotary evaporator.

The

DPPC

The last traces of

were removed azeotroplcally by evaporating with ethanol and benzene.

The residue was

dissolved in ca. 50 ml of benzene, washed with eight 50 ml volumes of Trls buffer, and combined with

benzene

backwashes

of

the Tris buffer.

The

combined benzene

solution was

dried over

filtered through a flne porosity slntered glass funnel, taken t o dryness, and

calcium chloride, weighed,

A subfractlon enriched with four- to six-ring PAH dermal tumorigens was separated from the nonpolar

fractions

chromatography.

using

semipreparative

scale,

normal

phase

high

performance

A 0.25 ml aliquot of the nonpolar fraction was separated on a 9.4mm ID x 25 cm

Partisil PAC i0 Magnum 9 i0/25 bonded phase column purchased from Whatman, The volume of i0% elu~ion

time

liquid

of

benzo(ghi)perylene

(vol./vol.)

methylene

benz(a)anthracene

and

chlorlde/hexane extendlng

was collected and concentrated

Inc.(Clifton,

eluate s~arting at 4 min before

through

i0

mln

after

the

elution

NJ). the of

to 0.i ml along with a known mass of l,l'-

blnaphthyl internal standard. The

PAM

analyses

were

method of internal standards, determined by

a

performed by capillary

column

gas chromatography

(CC)

using the

and were corrected for any interferences from the leaching media

separate blank sample analysis.

The HP 5880 GC (Hewlwtt-Packard, Pale Alto,

2616 CA) was silica

equipped

with

•

30 m x 0 . 2 5

um ID,

ionization

detector.

column and a f l a m e

programmed Co 180oC a t

2 5 ° C / m i n and t h e n

0.25

to

~n f i l m

280°C a t

The hydrogen carrier gas flow rate was 1.5 ml/min. chromatography-mass

spectroscopic

thickness

DB-5 b o n d e d p h a s e

The column o v e n was h e l d

examination

2°C/min,

and h e l d

at

at

100°C f o r

280aC

fused 3 min,

for

20 min.

PAH identifications were verified by 8as

of

representative

samples

under

similar

chromatoaraphic conditions. Recovery conducted

measurements

by

liquid

scintillation

of

carbon-i4

spectroscopy,

scintillation

counter

(Packard

labeled benzo(a)pyrene

Instrument

using

Company,

a model

Downers

added

to

C2425

Packard TrICarb

Grove,

the

IL).

leachates

Briefly.

labeled benzo(a)pyrene was added to the leachates after removal of the SRC Solid.

were

liquld

carbon-l. Recovery of

PAH after collection of the nonpolar fraction and isolation of the PAH-enrlched subfraction was estimated

from

the

radlotracer

recovery.

These

analytical

methods

are described

in decail

materials

co • s s e s s

potential

tumorlgens

from

elsewhere (15,16),

RESULTS AND DISCUSSION In rates

this

and

study,

nonionic

surfactant,

leaching

procedure

e m p l o y e d (17)

to

media.

A tissue

because

It

has

c o a t e d on c o a l u s e d as PAH

~hree

extraction

of

selected

•s

selected

PAH d e r m a l

Tween 80 ( p o l y o x y e c h y l e n e •s well

increase culture been

fly

as

the

the

le•chin8

sorbitan

(l&)

•s

medium.

•

of oil

This

MEMwith f e t a l studies

of

the

the

SRC S o l i d .

has been

mut•genicity

testing

b o v i n e serum) was i n c l u d e d the bioavailabilfCy

have been employed in s t u d i e s

and

of

the

physical

and

the

previously

DPPC, a m a j o r c o m p o n e n t o f p u l m o n a r y s u r f • t r e n t

(9-12)

A

was u s e d t o e v a l u a t e

surf•crane

samples with bacterial

leachin 8 sodium in

DPPC v e s i c l e s

matter

monooleace),

methodoloEy.

medium ( • m i x t u r e o f E a g l e ' s

used

particulate

analytical

compatibility

ash samples.

a leaching

from

media were

effic£encies

(13),

o f PAIt a l s o was

o f membrane u p t a k e

chemical

interactions

of

of

lung

surf•cannas with air pollutants and fly ash (18,19). An evaluation of the efficiency of the analytical procedure was conducted by measurin$ the recovery of carbon-14 The

overall

labeled benzo(a)pyrene

recovery

isolation steps, was

of

the

procedure,

added to the leachate samples of the SRC Solid.

~ncludfn8

contributions

from

ca. 65% with all three of the leaching media.

the

collection

and

The collection efficiency

ransed from 70 to 94%, while the fractlonation efficiency varied from 69 to 92%. the

DPPC

leaching

vesicles

not

were

contalne6 by

removed

in both

ulcracentrifusacion of the supornatant In spite of this split,

precipitate

ultracentrifugation,

(DPPC vesicles) plus

aqueous

and

The PAH from \ suspension (DPPC

phase)

phases

from

from the benchtop centrlfuaatlon of the DPPC leech•re.

r~he overall recovery of the PAH from the DPPC medium was the same as

chose for the other ~wo media.

Althouah this level of recovery is not optimum,

it is within •

range which can be used reasonably ~o correct the measured PAH concentrations. The Solid

is

leaching behavior presented

respectively.

in

of selected

Tables

1-3

for

four the

to slx-rln K PAH dermal Tween

80,

MEM/serum,

tumorlgens

and

DPPC

from the arc

vesicles

media,

Because of the relative ease in its analytical preparation and its low blank.

the Tween 80 surf•canna medium was used most extensively to study PAH leachln 8 behavior. In the conulnuous leachlng experiment, replacement

of

the

Tweet

80 medium.

the SRC Solid was leached continuously and without

Samples

were

taken

for

analysis

at preselected

~ime

intervals to determine the time required to reach leaching equilibrium and the equilibrium PAl{ concentrations.

The

PAH concentratlorm generally appeared to reach an equilibrium before the

2617

II

•

.-4

.

~

¢~,

~

~

0%

~O

~

~

,.,-4

~

~

@,4

~

(',,4

i)1 ~i

cU ~D

40

~'~

~

i .C

O

4.J e RI

oJo Ul ~o G

•

cg 1.4

o o

l,-i

~

~e

4=i

I

"-' 0

B ~%

,,4)

r~.

~

-.I'

O

~'~

u u ¢13 iI~ i1)

~

E

¢11

2618 first time point of 5 days.

exposure experiment (ca.

This is very rapid in comparison with the length of the inhalation

2 years),

and i t

suggests

the potential

for considerable

PAt{ l e a c h i n g

during the b i o a s s a y .

A sequential

leaching

protocol

( T a b l e 1) a l s o

was p e r f o r m e d w i t h

examine the PAH leaching behavior in a quasi-d3mamlc leaching model. for 20 d a y s leached

in which

the leaching medium was changed every 5 days.

in the differen~

steps were similar to those

indicating a considerable is possible result

that

the

depleted. Data Table 2.

The PA~ concentrations

in the continuous

leaching experiment,

"capacity" of the PAH available for leaching from the SRC Solid.

slmlarltles

in

the

concentrations

from PAH in both reaching sa~uratlon.

began to fall off,

t h e Tween 80 medium to

The SRC Solid was leached

beuween

the

By the fourth leaching,

~wo

leaching

It

protocols

the PAH concentrations

suggesting that the PAH in the outer layers of the particles were becoming

It also may indicate a decrease in the kinetics of leaching. for

continuous

leaching

The concentrations

experiments

using

the MEM/serum

Tween 80, but the ratios of the PAH concenuraulons were similar. leaching behavior

two

in the

mixture

are presented

in

of leached PAH were sllghtly lower than those observed with ~he

media was

the decline

The main difference in the

in PAH concentrations

with

Some

time.

decreases in PAH concentrations were apparent at 2 weeks of leaching, but 6 weeks were required for ~he effect

to

become p r o n o u n c e d .

It is possible t h a t

proteins

in the serum were binding

PAH from solution, or that microbial degradation was beginning to occur in this medium.

Table 2. Leaching of PAH from SRC solid with minimum essential medlua/serua mixture

Concentration." uz/~ 2 wk 4 wk 6 wk

PAH

5d

Benz(a)anthracene

2.7

1.7

1.9

0.~

Chrysene

2.7

2.1

2.1

1.3

Benzo(b/J)fluoranthenes

6.7

,.7

4.5

2.2

Benzo(k)fluoranthene

0.32

O.&8

0.64

Benzo(e)pyrene

6.3

5.5

4.5

Benzo(a)pyrene

5.5

4.6

3.0

1.6

Indeno(1,2,3-cd)pyrene

3.3

3.0

1.0

0.88

Dibenz(a,c/a,h)anthracenes

1.7

1.3

0.63

~.8~

Benzo(ghl)perylene

6.2

5.2

3.1

1.6

2.8

" C o r r e c t e d f o r b l a n k and r e c o v e r y , d - d a y s , wk - w e e k s . Single determination for each data point,

PAH leaching behavior with the DPPC vesicle system is shown in Table 3. More difficult The

leaching

centrifugation

media media with

Under these more

to prepare could

not

for analysis. be

the benchtop

severe

completely clinical

conditions,

Consequently, separated

cen=rlfuge,

from

This was a much

the data are not as extensive. the

SRC

Solid

by

and ultracentrlfugation

some of the DPPC vesicles

the was

were precipitated,

sample was b i p h a s i c ,

PAH c o n c e n t r a t i o n s

rpm

required. and it was

extremely tedious to pipette them cleanly from the underlying SRC Solid preciplcate.

this

low

Because

had t o be m e a s u r e d i n b o t h t h e u l t r a c e n t r i f u g a t i o n

2619

,

•

.

,

.~

.

4

.~

c~

0

•

i

0

v •

,

,

°

.

m

~

al

m '~

c o u

~ ~

°

o u

ca 4~

cs. |

.

,

o

~.e.,

ixl ~L

,.-¢ °,.i ,~

~

~.

,

~,~

~-~

...I"

~.~

r.~

c

c

e.-

t.l al

1.1

w

ec.

o ra

0 ~1

m

•

Id

o m

~

u

I~

o

II

c

~ A

U

0

~

•

~1

,=.d w=.t

~

U c

~

o

4; ".-"

m

~-~ 0

~-~ 0

~ 0

-~ 0

0 ~

w eJ

~,

2620

suspension and DPPC

precipitates.

This more complex procedure

for the analysis of the DPPC

leachates limited the analyses ~o time points of 5 days and 6 weeks. suspension

a~ ~wo other ~ime points.

phase

than in the Tween 80 and MEM/serum media.

The solubilities

Concentrations

very similar ~o those for the Tween 80 medium. leacha~e

(from the suspension and preclpi~ate

(corresponding

to DPPC vesicles)

in the vesicles

leacha~e~

PAB

Although wa~er

Individual

(Table

4),

~he

mainly

media.

the

vesicles

concentrated

exceeded

were

enhanced

It

The general Solid,

I #g/g

in the DPPC vesicle

rather

than

in ~he aqueous

phase

suggests

that

cell

can

membranes

which

concentrations

some

PAH~

two

absorb

the bulk dissolved

of the PAH

or

results of these leaching studies suggest that in the inhalation bioassay of the

PA/~ may be quite bioavailable

(49 mg/l

(6 hrs

for

process.

and concentrations

far in excess

water solubilities may accumulate relatively rapidly in cell membranes.

protocol

in the bulk

phase by ca.

constitutes

efficiently

efficiently particlpa~e directly in the PAl4 leachlng/dissolution

the SRC

than

This finding shows that the PAl{ leached from ~he SRC Solid are present

in the vesicles

leaching

in

of the PAH for the DPPC vesicles+

lO0-fold more

in all three leaching media were generally greater =ban in pure

PAH concentrations

orders of magnitude.

the affinity

were ca.

concentrations

the PAB solubilities

of other PAH in the leacha~es were

Comparison of the data calculated for the total results) with those measured for the precipitate

illustrates

The PAH concentrations

Data are included for the

for the larger PAH were greater

per

for the high exposure

day,

5 days

per

week,

group) for

of their

Given the high aerosol

of the SRC Solid and the long exposure

20-24

months)

of

the bioassay,

it

is not

surprising ~hat lung tumors have appeared in the animals.

REFERENCES i.

"Two Year Inhalation Toxicity and Oncogenlcity Study on SRC Solid in Rats," International Research and Development Corporation Report on Study 5000-250 to the U.S. Department of Energy, International Research and Development Corporation, Hattawan, Ml, February 20, 1987.

2.

J. M. Holland,

3.

J.M. Holland, F.W. Larlmer, T.K. Rao, J. L. Epler, C.-h. Ho, M. V. Buchanan, and M. R. Guerln, ~. ADDI, Tox,, ~, 117 (198A).

4~

D. D. Hahlum, C. W. Wright, E. K. Chess, and g. W. Wilson, Cancer Res.,

5

B. W. Wilson, R. A. Pelroy, and D, D, Hahlum, "Chemical Characterization and Genotoxlc Potential Related t o Boiling Point for Fractionally Distilled SRC-I Coal Liquids," PNL-&277, Battelle Pacific Northwest Laboratory, Richland, WA (July, 1982).

6.

C.-h. Ho, A. R, Jones, J. E. Caton, g.H. Grlest, L . E . Smith, and H. R. Guerln, "Upgrading Coal-Derlved Liquids by Means Other Than Hydrotreatment II. Tumorigenicity and Tumorigens," ORNL/ TM-9324, Oak Ridge National Laboratory, Oak Ridge, TN (May, 1985)+

7.

W. H. Griest, C. Y. Ma, I. S. Rubln, and B. A. Tomkins, "Physical and Characterization of SRC Toxicology Program Samples", ORNL/TM-10204, Oak Ridge Laboratory, Oak Ridge, TN (November, 1986).

8

B. Z. Drozdowlcz and C. M. Kelly, "Interim Report on the Genetic and Animal Toxicity Testing of SRC-I Products, Intermediates, and Waste Materials, Appendix G," DOE/OR/0305~-50, International Coal Refining Company, Allentown, PA (September, 1983).

9.

J. R, Lakowlcz,

D. R. Seven, and S. C. Riemer, Biochim.

i0.

J. R. Lakowicz,

F. Englund, and A. Hidmark,

D. A. Wolf, and B. R. Clark, Environ. Health P~rsDect.,

38,

149 (1981).

&__qa,5176 (198~).

Chemical National

Bioohvs. Acr~a, 629, 243 (1980).

Ibld., 543, 202,

(1978).

2621

0 if%

,,= u

if3 e..

I1 >

0~

7-,

~

~-,

~

%

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~

~

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2622

iI.

J. R. Lakowicz and J. L. Hylden, Nature, 275, ~ 6

12.

D. R. Bevan, S. C. Riemer, and J. R. Lakowicz, "Transfer of Polynuclear Aromatic Hydrocarbons from Particulate Matter co Membrane Measured by Fluorescence Spectroscopy" in Polvnuclear Aromatic Hydrocarbons, M. Cooke and A. J. Dennis, Eds,, Battelle Press, Columbus, OH (1981),pp. 603-614.

13.

S. A. Rooney, Environ. Health PersD., 55, 205 (1984).

I~.

A. Bjorseth, G. Bather, M. J. Chang, K. L. McNelll, and J. L. Fisher, "Comparative Elution Studies with Vaporor Liquld-Phase l*C-Banzo(a)pyrene Coated Coal Fly Ash", in ~ynuclear Aroma=it Hydrocarbons, M. Cooke and A. J. Dennis, Eds., Battelle Press, Columbus, OH (1984), pp. 189-197.

15.

5. A. Tomkins, W. H. Griest, J. E. Caton, and R. R. Reagan, in Polvnuc~ear Aromatic ~y~rocarbons: Physical and Blolo~ical Chemls~rv, M. Cooke, A . J . Dennis, and G. L. Fisher, Eds., Battelle Press, Columbus, OH (1982), pp. 813-824.

16.

~+ H. Grlest, B. A. Tomklns, R. R. Reagan, and J. ~. Russell,

i7.

C. Y. Ma, C. -h. Ho, R. B. Quincy, M. R. Guerln, T. K. Rao, B. E. Allen, and J. L. Epler. ~ , 118, 15 (1983).

18.

E

S. ~llkins and P. FetCissoff,

19

E

Wllkins, M. G. Wilklns,

20.

B

G. ~hicehouse,

21

W

E. May, S. P. Waslk, and D. H. Freeman, ~ h L _ f J l ~ ,

22.

F

P. Schwartz, J. Chem. Enz. Data, 22, 273 (1977).

23.

C L. Parker and D. I. Dykstra, Eds. " Envlronmen=al Assessment Data Base for Coal Liquefaction Technology: Volume II, Synthoil, H-Coal, and Exxon Donor Solvent Processes", EPA-600/7-78-184B, Envlronmen=al Protection Agency, Washlngton, D.C. (1978), pp. 314-315.

24.

D. Mackay and W. Y. Shlu, J. Chem. En2. Data, 22, 399 (1977).

(Received

in U S A

Fuel, 66, ~08-411 (1987).

J, Environ. Scl. Heal~h, A16, 477 (1981).

and O. Seoudi, ~. Environ. $ci. Health, AIT, 169 (1982).

~L~,~.~,

? June

(1978).

19872

14, 319 (1984).

accepted

20 J u l y

1987)

50, 997 (1978).