Effects of Coal on Benzo(a)pyrene Uptake-=

The presence of coal particles reduced the total accumulation of B(a)P
to 20% of that accumulated by rainbow trout exposed to B(a)P without par-
ticulates. Ground coal at a concentration of 50 mg/£ (particle size + 0.125mm)
lowered the exposure concentration of B(a)P from 0.4 ug/£ to below detection
(0.1 ug/2)in filtered water samples. The observed accumulation in the pre-
sence of coal particles indicated that B(a)P adsorbed onto particulates might
be accumulated at gill and/or gut membranes. Uptake from particulates may be
an important route of accumulation in the aquatic environmen} because the
major portion of PAH's are associated with suspended solids a.

Relationship of Mixed Function Oxidase Levels to Concentrations of a
Specific Carcinogen {Benzo(a)pyrene} in Tissue of Rainbow Trout--

The few reported environmental concengrations of B(a)P in fishes have
been low. For example, Pancirov and Brown found only 1.5 ug/kg in menhaden
and <1 ug/kg in flounder and cod fish caught off the New Jersey coast.

We have, however, demonstrated that B(a)P can be readily bioaccumulated
from water by rainbow trout. Although the concentration of B(a)P used in the
water uptake experiment (0.49 + 0.21 ug/1) is high compared to ground water
concentrations (0.0001 ug9/1) 61, it was less than the concen}yations from se-
veral metropolitan raw water supplies in the U.S. (1-2 ugs}), > and down-
river from a petroleum industry in Russia (0.05-3.5 yg/1)°°". The observed
bioconcentration in rainbow trout should be contrasted with a study by Lu 2
who found that B(a)P was rapidly metabolized by mosquitofish (Gambusia) in
water uptake experiments and was appreciably bioaccumulated only by food chain
transfer and in the presence of an MFO inhibitor.

The hitherto unknown relationship between hepatic MFO activity and
B(a)P concentration in tissue has been explored in this investigation. In
Table 12, the B(a)P tissue concentrations from Table 11 were compared to
their respective enzyme levels in Figure 9. Also included were the B(a)P
water exposure data from Table 10. From this study it appears that induced
MFO activity might be predicted in fish having B(a)P tissue burdens exceeding
300 g/kg.

49


FIGURE A-3
GAS CHROMATOGRAPHIC ANALYSIS OF A METHYLENE
CHLORINE EXTRACTION OF COAL LEACHATE®

BLANK

ELD. RESPONSE

LEACHATE

ALU, KESPONSE

oO ry 12 1 24 EQ 360 42 48 34 60 66 72 78 84 90

TIME (minutes)
40ne liter samples of distilled deionized water and centrifuged coal leachate
(6-3 g goal distilled deionized water) were extracted with 50 ml methylene
chloride.

GC conditions-column: 5% SP-2250, 2m x 2mm i.d. glass; injector: 250°C ;
detector: 300°C; carrier flow: 20 ml/min; program: 80-260°C at 4°/min.

113


UV ABSORBANCE AT 254nm
(2.0 FULL SCALE)

FIGURE 4
STEAM DISILLATE OF COAL (KOH)
HPLC

rs
@

Fe Se ae ee TOR SS NT I NT SS SS SO SN OT ONS ST SOOO OT

1
4 6 8B 1 12 4 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
TIME (minutes)

Conditions- column: 10 » Lichrosorb RP-18; program: 50 to 902. CHACN in
water, 1.2 ml/min, 30 min (linear program).

15


tel

APPENDIX F
METHYLATED NAPHTHALENES: MASS SPECTRAL DATA FOR REPRESENTATIVE MONO-, DI-, TRI-, AND
TETRAMETHYLNAPHTHALENES (SYNTHESIZED AND COMMERCIALLY AVAILABLE)

20

°

Sn

slau. rl t ry wll, ani a a a a ha a ll °

160 i150 200 eso
SPECG 58 LS SSLM + SOun STEP MASS*1, IvBvS «© 1%



6EL

THRESH(CM) = @.5 Be
OFFSETCHZ)= 162.5
KF FROCMHZ)= @ 6600
NO. INT. OFFSETCHZ) PPM
1 ai 3380.8 + 3380.0v0
2 23 3372.5 - 3372.500
3 20 3347-5) = 3347.500
4 167 3241.2 + 3241.250
5 7 3219.8 - 3219.050
6 174 3191.2) = 3191.250
7 se 3188.7 - 3188.750
8 176 3167-65) = 3167.50
9 206 3163.9 - 3163.950
i) Ss 3152.9 - 3152.98
ny 6 3150.0 + 315¥.u00
12 108 312661 = 3128.100
3 136 481.1 + 481.149
14 iat 0.8 - 6.600
wee teres veces
si j
i CH, |
i



RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and appiication of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

Environmental Health Effects Research

Environmental! Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

Scientific and Technical Assessment Reports (STAR)
Interagency Energy-Environment Research and Development
“Special” Reports

Miscellaneous Reports

ONOnNRWH +

o

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.

This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.


TABLE A-5

COUGH RESPONSE DATA FOR TWO BLUEGILL SUNFISH
EXPOSED TO COAL DISTILLATE

TIME FRAME CONCENTRATION (%) AVE. COUGHS/MIN.

Tank 1 Tank 2 Fish 1 Fish 2
0800 - 0820 0 0 0.55 0.55
0915 - 0935 1 5 1.10 1.00
1000 - 1020 5 20 1.35 1.05
1300 - 1320 5 20 1.55 0.80
1550 - 1610 20 0.50 0.40
0800 - 0820 5 20 0.45 0.50

109


TABLE 18
AHH ACTIVITY IN FATHEAD MINNOWS EXPOSED TO PAHs®

AHH Activity

Exp Tank Compound
# # (pM 3-OH- BaP) {ma | protein/min)
Day 7 Day 2i

1 dibenzofuran

1 fluorene 0.23+0.02 0.30#0.04
2 9-chlorophenanthrene 0.2120.09 1.0+0.10
1 phenanthrene

2b B-naphthoflavone 1.020.4 1.0+0.5 0.69+0.04
2 phenanthrene 0.3120.02 0.4+0.1 0.44+0.02
1 phenanthrene

9-chlorophenanthrene 0.3740.08 1.5+0.2 1,340.3
2 phenanthrene 0.7+0.3 0.7+0.3 0.5+0.2

dibenzofuran
fluorene
3 phenanthrene 1.50.8
1-methylphenanthrane 1.4#0.1 1.0#0.5
fluoranthene
pyrene

The mean and standard deviation for controls was: 0.53140.213 based on
8 samples, each containing 15, 6-10-week-old fathead minnows.

othe AHH values in this experiment were based on duplicate determinations,
whereas all others were done in triplicate.

58


a i
'H 100 MH, _-—__.___} _! .
econ ae i
SOLVENT. TEMP. c '
TUBE 0.D________mm SPIN RATE. rps
LOCK
SIGNAL___ OR FIELD

OBSERVE cwRE FTO 3 ourO) onFO

OFFSET. 864.6495... Hz
SWEEP/SPECTRAL WIDTH... 20. Hz
SWEEP/ACQUISITION TIME eC
RF FIELD/PULSE WIDTH... .dB/usec
PULSE DELAY... sec SPECTR. AMPL.ww
TIME CONS TAN Tenement SEC
DATA POINTS... vovewewene FILTER. Hz
NO. SCANS/TRANSIENTS..

12345
DECOUPLE cateo “O
NucLEUS__H GYROCODE
OFFSET__51319. Hz RF POWER....19..dB

NOISE BANDWIDTH...
INDOR SWEEP WIDTH
OPERATOR

CHART S-100XLA Gaver
PRINTED IN USA

Hz

142


69

Starting PAH

Phenanthrene

Phenanthrene

Phenanthrene

Phenanthrene

Phenanthrene

1-Methy] phenanthrene

1-Nethylphenanthrene

J-Methy] phenanthrene

Fluoranthene

FTuoranthene

tet,)
mg/t

3.7

26.3

19.3

19.5

20.0

3.1

25.6

3.4
22.0

{PAH]
ng/t

236

233

820

239

VaR

925

994

178

824
239

Reaction
Time, hr

0.5

3.0

3.0

3.0

3.0

0.5

3.0°

3.0

0.5
3.0

pH
6.8

6.0

4

4.2

4.05

6.9

4.0

4.0

6.8
5.9

Table 19 (Continued)

Products
Identified
(% Yield)

Phenanthrene
(77414)

Phenanthrene
(8624)
Monochloro-
phenanthrene

(421)

Monochloro-
phenanthrene

Phenanthrene
(994)
Monochloro-
phenanthrene
3825)
Monochloro-
phenanthrene
(3925)

}-Methy] phenan-
threne

Monochloro-1-
methy] phenan-
threne

Monochloro-1-
methy] phenan-
threne (x8)

Fluoranthene

Fluoranthene
(6343)

Basis
for
Assignment

boc

bic
bic

a,b

bc

boc

Comments

Same GC retention time as com-
pound 3 (Tabte 20).

we 212, 214, Same GC retention
time as compound 3 (Table 20).
Same GC retention time as compound

3 (Table 20).

Same GC retention time as compound
3 (Table 20).

wf = 192.

nt = 226,228, GC retention time
identical to compound 5 (Table 20)

GC retention time is identical to
compound 5 (Table 20).

ut = 202


SECTION 2
CONCLUSIONS

The study of organic leaching from Northern Great Plains coal has shown
that solubilized levels less than 10-50 ng/ of an individual polynuglear
aromatic hydrocarbon (PAH) will be obtained upon equilibration at 25°. More-
over, the predominant compound types observed in the leachate (e.g. the lower
molecular weight PAH's and alkyl PAH's) are not those regarded to be highly
carcinogenic and are present at concentrations comparable to background levels
of PAH's in Lake Superior.

There are also polar material leached from coal, but neither these com-
pounds nor the PAH's exhibited severe adverse effects on aquatic organisms at
the concentrations tested. The lipophilic organic compounds appear to be
associated with the particulate coal fraction of leachate but can be effi-
ciently released through volatilization processes (i.e. steam distillation)
that are possible in a natural setting.

PAH's typically considered carcinogens and MFO inducers in mammalian
systems were found to initiate MFO induction in rainbow trout. Moreover,
for the specific carcinogen benzo(a)pyrene, a minimum concentration of about
300 ug/kg in the tissue of rainbow trout was found to be necessary to induce
MFO production. All the PAH's studied (dibenzofuran, fluorene, phenanthrene,
9-chlorophenanthrene, 8-naphthoflavone, 1-methylphenanthrene, fluoranthene,
and pyrene) were readily bioaccumulated (bioconcentration factor =~ 1000-5000),
but the attainment of a steady state value cannot always be assumed due to
external influences such as the presence in the water or tissue of other com-
pounds or an enhanced level of MFO activity in the exposed fish. The release
or conversion of bioaccumulated PAH's was very rapid, usually occurring in
less than 4 days. In addition a study showed the vulnerability of PAH to
aqueous chlorination under conditions typical of those used for disinfection.
The product types (chloro PAH, chlorohydrins, and quinones) observed during
this investigation were thos? expected not only on mechanistic considerations
but also on reports of products isolated from other oxidation processes.

The development of a rapid and selective technique for the general
synthesis of alkylated PAH's aided the characterization process. The utility
of the synthetic method was demonstrated by the preparation of several poly-
Methyl naphthalenes. The sensitive and versatile high performance liquid
chromatography - gas chromatography system was designed for the analysis of
PAH's in water and tissue with a lower limit of detection of ~ 10-50 ng/2 for
an 18 £& sample and about 100 ug/kg for a 1 g sample, respectively.

In summary, the chemical and biochemical portions of the study resulted


FIGURE A-8
EFFECTS OF COAL LEACHATE ON LIVER PARAMETERS
OF RAINBOW TROUT?

ecsee CONTROL
—— LEACHATE

Liver Wt.

@
SPECIFIC ACTIVITY

Rainbow trout were exposed to coal: leachate or to Lake Superior water for 28
days under renewed static conditions. Three fish per exposure were removed
at each sampling period and after weight determinations, the livers were
pooled and homogenized prior to protein, DNA, and AHH measurements.

DRelative liver weight is expressed as mg liver/g total fish weight.

Cprotein is the total microsomal protein content/g total liver weight (3
pooled livers).

DNA is the DNA content of the 15,000 g pellet/g total liver weight.
faHH activity is expressed as pM 3-hydroxybenzo(a)pyrene/mg protein/min,

d

118


176/7LN7DERL 77205 DICHLOROBENZOFURAN

sun
130392

BASE
22364

> >DATE <7SPECO
PEAK

:  70ev
GC
Varian CHS

300

1%

250

Electron Energy
Inlet System
Instrument

govurzronnascosscosncenene
CacomsuNsEANoddeNSaaunUNDS
EOVORAR OOOO RO = mM MMARROMMMS

popepesiinabser rer sr ttt]

WNNNRNANNNNNN NNN NN NRNRNNNNNNNN
AF NOUR OUTARMOOT MOMONNOROS
COO-OMKMROSM®

Sanson oNuSAMN

MAU UR HMM RAUF TM
= ue

RUMR@eTNHOKMNOMseoKacamuMs
SRR UUMMMMS TT ANDODD

100
90
80

124

_|
IvB-s

e200
STEP HASS*1,

tule

T

150

178 LM.DERL 77205 DICHLOROBEN2ZOFURAN

100

ha dedi ll

70

60

50

40

30

10

0
SPECO


In contrast to 8-naphthoflavone, 9-chlorophenanthrene appeared to show a Sig-
nificant increase in AHH activity (Table 18, exp. #1,3) but bioconcentration
factors for phenanthrene in the presence or absence of this chloro compound
were the same, within experimental error (Table 15, exp. #3, Tanks #1,2). De-
puration rates in this latter experiment were also nearly identical (Table 16,
exp. #3, Tank #1,2).

To summarize, the PAH's studied exhibited significant bioconcentration
factors (1000-5000) but the attainment of a steady state can not always be
assumed due to the presence of other compounds in the water or tissue or the
level of mixed-function oxidase activity in the exposed fish. The release or
metabolism of bioaccumulated PAH was usually rapid (< 4 days) after the fish
were returned to relatively PAH-free water.

64


22

TABLE 6

SUMMARY OF BIOLOGICAL TESTING
OF COAL DISTILLATE.”

Concentration _ Organism ___Age at Onset Duration of Exp. Type of Test Bioassay Conditions Response
20, 40, 60% Daphnia $24 nr 48 he Toxicity static Death at high
concentrations
due to low con-
ductivity. Partial
mortality at inter
mediate concentra-
tions.
20% fathead 24-48 br 96 hr Toxicity static No different from
(67g/1, DDW) minnow controls
0.1,1,10, 20% fathead 9 mo 96 br Toxicity static No mortality
(67g/1, DDW) minnow
1,5, 10, 20% bluegill 2 24 be Sublethal: recirculating No sustained,
(67g/1, DDW) Cough Re- static elevated cough
sponse rate
0.2% rainbow Lyre 21 days sublethal: flow-through P-450 no different
(40g/1 LW) trout MFO Response from control
Bioaccumula- Slight AHH
tion elevation, D28

No different from
control

*tThe ration of coal to water and the type of water used to prepare distillates (100%) are indicated. DDW= distilled

delonized water,

LW = Lake Superior Water.


156.

157.

158.

159.

160.

161.

162.
163.
164.

165.

166.

167.

168.

169.
170.

A.R. Oyler, D. Bodenner, K. Welch, R. Liukkonen, R. Carlson, H. Kopper-
man, and R. Caple. "Determination of Aqueous Chlorination Reaction
Products of Polynuclear Aromatic Hydrocarbons by Reversed Phase High

Performance Liquid Chromatography-Gas Chromatography", Anal. Chem.,
50(7), 837-842 (1978). Y

J. Driscoll, J. Ford, L. Jaramillo, J. Becker, G. Hewitt, J. Marshall,
and F. Onishuk. "Development and Applications of the Photoionization
Detector in Gas Chromatography". Amer. Lab., 10(5) 137-147 (1978).

D. Peters, J. Hayes, and G. Hieftje. “Chemical Separations and
Measurements". W.B. Saunders Company, Philadelphia, Pa. (1974), p. 33.

R.L. Gritz and D.G. Shaw. "A Comparison of Methods for Hydrocarbon

Analysis of Marine Biota". Bull. Environ. Contam. Toxicol., 17 4),
408-415 (1977).

D. Kuehl and E. Leonard. "Isolation of Xenobiotic Chemicals from
Tissue Samples by Gel Permeation Chromatography". Anal. Chem., 50,
182-185 (1978). ~

J. Andelman and M.J. Suess. "Polynuclear Aromatic Hydrocarbons in the
Water Environment". Bull. World Hith. Org., 43, 479-508 (1970).

Reference (54), pp. 70-95.

Reference (1), pp. 2-2 to 2-8.

A. Ilnitsky, V. Mischenko, and L. Shabad. "New Data on Volcanoes as
Natural Sources of Carcinogenic Substances". Cancer Letters, 3, 227-
230 (1977). ~

"Particulate Polycyclic Organic Matter". National Academy of Sciences,
Washington, D.C., 1972, pp. 13-15.

B.D. Crittenden and R. Long. "The Mechanisms of Formation of Poly-_
nuclear Aromatic Compounds in Combustion Systems". In Carcinogenesis--
A Comprehensive Survey, Vol. 1., eds., R. Frendenthal and P. Jones,
Raven Press, New York (1976), pp. 209-223.

J. Schmeltz and D. Hoffmann. "Formation of Polynuclear Aromatic Hydro-
carbons from Combustion of Organic Matter". In Carcinogenesis--A Com-

rehensive Survey, Vol. 1, eds., R. Frendenthal and P, Jones, Raven
Press, New York ti976), pp. 225-239.

J. Borneff. "Fate of Carcinogens in Aquatic Environments". Adv. En-
viron. Sci. Technol., 8, 393-408 (1977).

Reference (165), pp. 36-62.

H. Kraybill. "Global Distribution of Carcinogenic Pollutants in Water".

Ann. N.Y. Acad. Sci., 298, 80-89 (1977).

93


stainless steel tube placed inside of a heated steel block. The purge inlet
of the PID was also connected. Linde pre-purified grade nitrogen, which was
run through a gas filter packed with DrieriteR and molecular sieves (Pierce
Chemical Company, part number 06116.23), was used for the carrier and purge
gases. Injections were made with a one-microliter Hamilton #7001 syringe
fitted with a 7-cm needle. The glass GC column (8 ft x 20 mm ID x &" OD) was
packed in the coil with 1.5% SP-2250/1.95% SP-2401 (methyl phenyl silicone
and fluoropropyl silicone) on 100/120 SupelcoportR (Supelco catalog number
1-1947; maximum temperature rated at 250°) and in the ends with Anakrom flux-
calcined diatomaceous earth (110/120 mesh; Analabs) in order to a} low 300°
inlet and outlet temperatures with minimum column bleed. Supeltex® M-2
column ferrules (Supelco) were used. The following GC conditions were typi-
cally employed: carrier flow rate = 23 ml/min, purge flow = 0, inlet tem-
perature = 300°, oven temperature = 182°, outlet temperature = 305°, auxilia-
ry block temperature = 312°, detector temperature = 250°. On certain
occasions increased reproducibility of detector response could be achieved,
at some sacrifice in sensitivity, by adjusting the detector purge to

v10-75 ml/min. An example of a typical gas chromatograph is provided in
Figure 6.

Integrations of HPLC and GC peaks were carried out with a Hewlett-
Packard 3380S integrator.

The GC-MS work was performed by electron impact at 70 eV on a Varian
MAT CH-5 single focusing instrument equipped with Varian 620-I and 620-L data
systems.

Distilled water was further purified for this work by forcing it through
an 0.45 up Millipore filter and a 7 x 600 mm Bondapak C-18 Porasil B reverse-
phase HPLC column (Waters Associates).

Determination of PAH Recovery from Solutions of Known Concentration--

An aqueous solution of known PAH concentration was prepared by adding,
with stirring, the appropriate amount of an acetonitrile solution of PAH to
“purified" water contained in a 19-2 pressure tank via a 100-ul syringe. The
filter holder assembly and the 7 x 50 mm C-18 column were then attached to
the outlet and the pressure inside the tank was adjusted to maintain a flow
rate of about 20-30 ml/min. If necessary, the column was stored at 5°C
prior to HPLC-GC analysis. The column was then inserted into the HPLC system
directly in front of the reverse-phase column and the following linear sol-
vent program was run: 1:1 acetonitrile-water to 9:1 acetonitrile-water over
a 30-minute period with a total flow rate of 1.2 ml/min. Fractions correspon-
ding to various peaks were collected in glass-stoppered, graduated 5-ml cen-
trifuge tubes. A one-microliter sample from each tube was then immediately
injected into the GC along with 3-8 injections of PAH solutions of various
known concentrations. A computer program was used to calculate a least
squares line (GC peak height or area versus weight injected) for each PAH in
the solutions of known concentration and to calculate from this line the
amount of each PAH that occurred in the HPLC fractions. The results are ta-
bulated in Table 7. The errors given in this table reflect the standard de-
viation in the determination of weight of material injected 58, plus the

29


tS

100
SPECO

158 Ls

CH,

Hy
rid
li ull. aii i i ei IL
150 200 250
AS8LM - 153LN STEP NASS*1, IvB-g

4


absorbance of the resulting HPLC fraction, which contained phenanthrene and
8-naphthoflavone, was determined at 270 and 255 nm. Using Beer's Law plots
for each of the compounds at each of the wavelengths, the concentration of the
phenanthrene and g-naphthoflavone in the water could be calculated. For the
analysis of the B-naphthoflavone in fish tissue, the usual styragle-HPLC frac-
tionates produced a methylene chloride fraction containing the flavone (but
not phenanthrene) which was analyzed by reverse phase HPLC. The conditions
for the latter analysis were 10% to 90% acetonitrile in water in 30 min at

1.5 mé/min total flow with a 5 uw Lichrosorb C-18 column.

Calculation of Bioconcentration Factors--

The calculation of the bioconcentration factors and associated errors
(presented in Table 15) were carried out as follows:

FON(I)
BCF =  MWCN(L) - EBCF(T)

where BCF(I) = bioconcentration factor for day I
FCN(I) = mg of compound per kg fish on day I (Table 16)

MWCN(I) = mean concentration of compound in water up to and including
day I (Table 17)
EBCF(I) = maximum error involved in determination of BCF(I) on day I
n
x WCN.
MWCN(I) = j= J
n

=| (ECN(I) + EFCN(I
EBCF(1) | Gus T) = EMNCN(T)) ~ acr(1)|

where j = water sample number (1, 2, 3....n)
n = number of water samples taken up to and including day I
WCN; = concentration of compound in tank water for j~ sample

EFCN(I error involved in determination of FCN(I) (see Appendix B)
and

n 2 n 2
z {WON (1) } - © WCN.(I)
J=1 J

EMWCN(I) = error in MWCN(I) = Jal

n-1
Determination of Particulate PAH Load--

In order to determine if any PAH material was associated with particu-
late matter (11.4 mg/2) in the fish exposure tank of experiment #3, Tank #3,
the GF/F filter, used in the water analysis apparatus, (see Section 5) was

54


SEL

1do0

sb

°

N
°

THRESH(CM) =
UF FSET CHZ)=
KF FRUCMHZ)=

No.

WANA EONS

INT.
5
14
is
la
153
19
14
178
146
207
176
1s9
172
165
ies
26
27
30
62

1.8
140.9

25.1997

OFFSETCHZ)
351165
3497.8
345767
343063
3246-5
3235-3
3229.8
3e22.8
3219.8
3217.9
311665
3066-5
3062.8
304268
302961
197461
1942.6
191863

68

BE
PPM
139-351
138.805,
137.214
1362127
128.835
128.388
126.140
127.892
127.777
127.698
123.676
121.692
121-543
120.749
126.2068
766341
77.089
75-607
8 .0ue



in defining what would constitute a natural exposure setting, Moreover,
toxicity can be ascribed to the presence of particulate matter (i.e. uncentri-
fuged leachate) and/or low conductivity of the water (i.e. high concentrations
of distillate) rather than to the presence of specific toxic compounds.

25


TABLE 17

BIOCONCENTRATION EXPERIMENTS: SUMMARY OF WATER ANALYSIS
Exp. 1 Exp. 2 Exp. 3
Tank 1 Tank 2 Tank 1 Tank 2 Tank 1 Tank 2 Tank 3

Com- dibenzo fluor- 9-chloro- phenan- g-naphtho- phenan- phenan- 9-chloro- phenan- dibenzo- fluor- phenan- I-methyl- fluor- pyrene
Pound furan ene phenan- threne flavone threne threne phenanthrene furan’ ene. threne phenan” nee
bay threne threne threne

0 3.65: 3.722 0.76 4.08 1.82 2.55¢
0.02 0.01 0.01 0.22 0.26 0.14

1 1.92 0.86:
0.14 0.06
2 0.66 = 2.22 1.828 2.32¢ 2.46 2.282 1.30: 1.192 0.894
0.01 0.24 0.19 0.13 0.22 0.14 0.10 © 0.04 0.09
3 3.152 3.49%
0.03) 0.01
4 0,37¢ 2.74 2,98: 1.99%
0.05 0.21 0.17 0.32
§ 2.40 2.56 1.662 0.91¢ 0.98 0.78
O16 0.18 0.18 «= 0.05 0.07 0.21
6 3.07 1.63 2.67
0.08 0.1 0.23
7 3.13 3.78 2.48 1,832 3.35 3.68 2.99 1.23 1.162 1.192
0.14 0.07 “07 0.20 0.34 © 0.43 0.14 (0.07, 0.06 0.07
8 0.872 2.76 1.60: 2.072
0.02 0.07 0.28
9 3.492 3.95 2.86: 1.76 2.70 2.062 2.172 1.448 1.208 1,062
0.15 0.07 0.13 0.22 0.16 0.08 0.09 «0.08 = 0.15 0.13
10 9.602 2.47
0.08 0.14
n 3.26 1.82 2.99
015 0.12 0.08
2 1.392 1,73 2.49% 1,36 1 1.32
0 0:08 0.09 0.06 += 0.05 0.09
13 2.17 1.46 2.262
018 0.28016
14 2.998 3.28% 1,962 2.28 2,308 1.554 1.57 1.548
0.46 = 0.22 010 0.12 O11 0.01 0.15 0.15
5 0.45 9 3.514 1.712 2.38: 1.23
0.08 0.31 (0.22 0.17 0.08 .
16 3.05: 3.424 3.27% 2.282 2.342 2.198
0.28 0.32 0.70 0.12 © 0.13 0.11
wv 0.602
0.08
18 2.29% 1.66 2.79% 1.228 2.938
0.26 0.18 0.16 0.16 = 0.17
19 2.35: 2.524 2.098 1.44% 1.35 0.954
0:08 = 0.08 0.17 0.37 0.25 0.10
20 2.752 1.462
0.17 0.08
a 2,80 3.13¢
0.50 0.15
22 0.50: = 2.07 = 1.744 2.40¢ 1.38 2.43¢  2.50t 2.34 1.33: 1.472 1.448
0.02 0.10 0.15 0.13 0.11 0.10 0.13 0.13 0.10 = 0.12 0.10
23 2.048 2.23
0.08 on
2% 3.19% 2.78 1.502 2.24 2.062 0.97 2.09: 2.56t 2.422 1.59 1,53 1.588
0.21 0.15 0.07 0.17 0.22 0.12 0.200 0.14 (0.15 0.11 0.16 0.10
25 0.472
0.06
26 1.99%
0.15
a 2.42 1.072 2.512 2.572 1.864 1.492 1.87 1.214
0.14 0.14 0.22 © 0.14 0.18 O11 = 0.13 0.09
2 2,87 3.168 2.01f 1.91 16st 1.148
0.44 0.26 0.08 «0.18 «0.19 0.20
Mean 3.19: 3.45 0.58¢ 2.63 1.82 «2.552 2.53 1.41t 2.34 2.342 2.482 2.202 1.382 1.358 1.188
+ Std 0.26 0.38 0.12 0.83 0.18 0:44 «0.44 0.340.545) 0.48 0.25 0.19 2:20 0.27
Dev.

57


71.

172.

173.

174.

175.

176.

177.

178.
179.

180.

181.

182.

183.

184.

185.

S.E. Herbes. "Partioning of Polycyclic Aromatic Hydrocarbons Between
Dissolved and Particulate Phases in Natural Waters". Water Res., ll,
493-496 (1977). —_

Reference (54), pp. 32-40.

Reference (1), pp. 6-30 to 6-34.

Reference (165), pp. 65-74.

D.F. Barofsky and E. Baum. “Exploratory Field Desorption Mass Analysis
of the Photoconversion of Adsorbed Polycyclic Aromatic Hydrocarbons".
J. Amer. Chem. Soc., 98, 8286-7 (1976).

S. Herbes and L Schwall. "Microbial Transformation of Polycyclic

Aromatic Hydrocarbons in Pristine and Petroleum-Contaminated Sediments".
Applied and Environmental Microbiology, 35(2), 306-316 (1978).

R. Harrison, R. Perry, and R. Willings. “Polynuclear Aromatic Hydro-~
(igve) in Raw, Potable and Waste Waters". Water Res., 9, 331-346
1976). ~

Reference (1), pp. 6-1 to 6-10.

W. Cautreels, K. Van Cauwenberghe, and L. Guzman. "Comparison Between
the Organic Fraction of Suspended Matter at a Background and an Urban
Station". Sci. Tot. Environ., 8, 79-88 (1977).

W. Cautreels and K. Van Cauwenberghe. “Determination of Organic Com-
pounds in Airborne Particulate Matter by Gas Chromatography-Mass
Spectrometry". Atmos. Environ, 10, 447-457 (1976).

W. Cautreels and K. Van Cauwenberghe. "Fast Quantitative Analysis of
Organic Compounds in Airborne Particulate Matter by Gas Chromatography
with Selective Mass Spectrometric Detection". J. Chromatogr., 131,
253-264 (1977). —=

W. Giger and C. Schaffner. "Determination of Polycyclic Aromatic
Hydrocarbons in the Environment by Glass Capillary Gas Chromatography”.
Anal. Chem., 50, 243-249 (1978).

M.W. Dong, D. Locke, and D. Hoffmann. "Characterization of Aza-arenes
in Basic Organic Portion of Suspended Particulate Matter". Environ.
Sci. Technol., 11(6) 612-618 (1977).

M. Dong, D. Locke, and D. Hoffman. "Separation of Aza-arenes by High-
Pressure Liquid Chromatography". J. Chromatogr. Sci., 15, 32-35 (1977)

M. Dong. D. Locke, and E, Ferrand. "High Pressure Liquid Chromatogra-
phic Method for Routine Analysis of Parent Polycyclic Aromatic Hydro-
aoe) in Suspended Particulate Matter". Anal. Chem., 48(2), 368-372
(1976). —

94


FIGURE A-6
GAS CHROMATOGRAPHIC ANALYSIS OF A HEXANE EXTRACT
OF XAD-2 PURIFIED LAKE SUPERIOR WATER®

LAKE SUPERIOR WATER

FLO. RESPONSE

XAD PURIFIED LAKE WATER

FID. RESPONSE

J

o 6 2 8 2 30 36 42 48 54 60 66

TIME (minutes)

3, one liter sample of Lake Superior water was collected before XAD treat-
ment and another one liter sample taken after 13 liters of Lake Superior
water had passed through the 50 ml XAD column. Concentrated hexane extracts
were analyzed using the following GC conditions-column: 5% SP-2250, 2m x 2mm
i.d. glass; injector: 250°C; detector: 300°C; carrier flow: 20 ml/min;
program: 125°C 10 min, 125-200°C at 4°/min.

116


SECTION 6
ENVIRONMENTAL IMPLICATIONS OF DISSOLVED
POLYNUCLEAR AROMATIC HYDROCARBONS

UBIQUITY OF POLYCYCLIC AROMATIC HYDROCARBONS

The significance of the chemical and biological studies on specific
PAH's extends well beyond the present discussion because these compounds and
other PAH's can be derived from many sources and are considered ubiquitous.

The origins of polynuclear aromatic hydrocarbons found in the environ-
ment101,126,161,162 are: a) synthesis by microorganisms!01,161 and plants
101,161,162: b) geochemical processes!@6 which result in the formation of
gga 6 and petroleum: c) natural pyrglytic processes such as forest fires

: prairis fires!26, and volcanoes!64; and d) man-produced pyrolytic
processes !62 such as coal-fired electrical generating plants. The latter
man-produced origins are considered to be the most significant! - The pyro-
lytic formation of PAH's from organic matter involyes free radical re-
actions!65-167 and possibly "benzyne" intermediates!6’, Higher temperatures
generally result in formation of less alkylated PAH's 126,

The_ environmental cycle of PAH's has been discussed in several reviews
101,168-170 and a summary has been provided by Suess!01;

"The transport pattern of PAH in the environment appears
to be relatively simple. The background PAH, which are formed
by biosynthesis, are quite static and, obviously, remain in
the plants and microorganisms in which they were formed and,
more generally seen, stay within their own ecosystems be it
the soil, which holds the synthesizing bacteria and the plant
roots, or the lake, river or sea with its aquatic biota. How-
ever, it appears probable that PAH in ground-water are leached
out from the soil.

In contrast, PAH formed by high temperature processes,
whether resulting from natural open burning and volcanic erup-
tions or from man-induced combustion reactions including ground,
sea and air transportation, are all emitted into the atmosphere,
and thus are subject to the same dynamic forces which govern the
Movement, transport and fallout of aerosols generally. Because
a significant portion of PAH, absorbed onto the aerosols, will
decompose by photooxidation while still in the atmosphere, either
stationary or in motion, their fallout at great distances from

35


Table 22. The structures were confirmed by microanalyses, NMR (60 MHZ), IR,
and MS techniques. Pertinent NMR data are listed in Appendix E and represen-
tative MS spectral data in Appendix F. The general scheme discussed here
offers a potential route to certain methylanthracenes and phenanthrenes via
the corresponding epoxyanthracenes and epoxyphenanthrenes. ~~

Q . C)
~~ CHALi CHaLi

Mass Spectra--

Mass spectra of each of these compounds have been obtained and presented
in Appendix F. It should be noted that the mass spectra were run on mixtures
of isomeric products, except in (3) and (9) which are isomer free products.
The diagrams in Appendix F are labeled according to the major product.

Analysis of the mass spectra of these methylated compounds reveals two
possible useful fragmentation trends. As the methyl substitution increases:

(1) The wt yMt-1 ratio, increases, and
(2) The size of the M-15 (loss of a methyl radical from the radical
cation) peak increases.

The radical cation may lose either a methyl radical or the elements of
CoH or C3Hq. A hydrogen atom may also be lost by the M radical cation.
With increasing methylation the loss of C2Hp or C3Hq becomes less pronounced.

Experimental Details
Apparatus--

The 60 MHz NMR spectra were obtained on a Varian EM-360 NMR instrument
with tetramethylsilane used as an internal standard and deuteriochloroform
as the solvent. Mass spectral data were obtained at 70 ev on a Varian CH-5
system by Mr. Douglas Kueh] of the EPA Environmental Research Laboratory,

Duluth.
Microanalyses--

The microanalytical data were obtained by Spang Microanalytical
Laboratory, Ann Arbor, Michigan. See Appendix G.

77


Number

Page
21 Synthesis of Polymethylepoxy Naphthalenes ...........0. 80
22 Synthesis of PolymethyInaphthalenes .............0.04 81
A-1 Properties of Coal Leachate... .. 2.2... - eee ee eee 105
A-2 Effects of Coal Distillate in Daphnia pulicaria. ........ 106
A-3 Fathead Minnow Behavioral Response to Coal Leachate ....... 107
A-4 Effects of Coal Leachate on Spawning Success in Fathead

MinnoWS 2... ee 108

A-5 Cough Response Data for Two Bluegill Sunfish Exposed to Coal
Distillate. 2. ee 109

A-6 Effect of Coal Distillate on Hepatic Mixed Function Oxidase
Parameters in Rainbow Trout 110


FOREWORD

Our nation's fresh waters are vital for all animal and plant life, yet
our diverse uses of water -- for recreation, food, energy, transportation,
and industry -- can physically and chemically alter lakes, river, and streams.
Such alterations threaten terrestrial organisms, as well as. the aquatic ones.
The Environmental Research Laboratory in Duluth, Minnesota, develops methods,
conducts laboratory and field studies, and extrapolates research findings

-- to determine how physical and chemical pollution affects
aquatic life;

-- to assess the effects of ecosystems on pollutants;

-- to predict effects of pollutants on the ecosystems through
use of models; and

-- to measure the rate of uptake and bioaccumulation of pollutants
in aquatic organisms that are consumed by other animals, including
man.

A comprehensive program was designed in 1974 because of the "Energy
Crisis" of 1973 to study the adverse effects on the aquatic environment
being created by new energy sources and technologies. Data being reported
in this grant are a part of the comprehensive program and deal with the
possible effects of coal storage on aquatic ecosystems.

Acute and chronic toxicity tests were conducted using fish which
indicated little if any adverse effect. Bioaccumulation, however, was noted
with several polynuclear aromatic hydrocarbons (PAH) in the range of
1000-5000, and that these PAH's induced a mixed-function oxidase, (MFO)
activity in trout similar to those of other mammalian systems.

These findings will be integrated with other studies to aid elected
officials in making environmentally sound decisions on future energy
developments.

Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory-Duluth

iti


Obl

280
1000
500
250
100

25

THRESH(CM) =

OFFSETCHZ)=

KF FRUCMHZ)=
NO. INT.
1 12
2 16
3 13
4 13
5 is
6 137
7 207
8 10
9 an3
18 21
ivy 95
12 12t
3 17
14 3
is 16
16 3
7 99
18 27
19 a

1.8
141.3
25.1997
OFFSETCHZ)
337263
3368.5
3297.63
324263
321062
319365
3168.9
317866
317261
316767
315363
3laiel
313let
197568
1943-6
191163
483-5
169
08

BC

PPM
133.629
133.676
138.853
128.670
127.392
126.732
126.549
126.140
125.884
125.706
1256136
124.652
124.255
782409
770131
75.849
19.189
0.079

@.000

i
1

i
i
\
|

————



2

TABLE 8
SUMMARY OF ANALYSES OF SPIKED FISH TISSUE SAMPLES

Trial Grans Etsh yg PAM mg PA ; S Recovered thyle —_9-Chloro=
led Added g fish g-Naphthoflavone Dibenzufuran Fluorene Phenanthrene phenanthrene phenanthrene Fluoranthene Pyrene
1 4.3 20.0 4.7 -- 101212 95412 -- -— 91210 - -
2 4.13 5.0 162 _ 6426 72210 - a 5726 - --
3 §.5 5.0 0.9 — 8618 83213 — one = - oo
4 5.8 5.0 0.9 --- 7686 7785 - - 101215 - “
5 5.9 10.0 1.7 _ 8525, 9% 4 — - 669 — one
6 4.1 5.0 1.2 7 --- - 742 103212 - oe oo
7 3.1 5.0 1.6 o -- 6722 10227 _ - -
8 6.0 5.0 0.8 135#208 - -- _ 84212 — _ a
9 7.4 10.0 0 1.4 e479 + = 1068 10728 - - —
10 4.6 15.0 3.3 _ —_ see Wise9 V29 - — —
uN 4.1 30.0 -7.3 833" - = 169 1187 — oo =
12 2.6 5.0 1.9 - 9418 -- 10510 7 o 1005 _--
3 3.4 10.0 2.9 -- 94213 753218 902 B12 — 966 103211
4 3.8 20.0 5.3 ~ 929 769 a 8&6 - 795 9021
15 3.3 40.0 12.0 — 7210 SiS 108 12 804 — _

"determined by HPLC-UV. All other determinations by GC-PID.


St

~
oo
I

Juul

°

CH.

SPECe

Ml

ull atbieaty | , , ,

Ad Lt rp
100 150 200 eso

27 LM STEP MASS+1, IvBvs

= 1%


extracted with a Soxhlet apparatus with acetonitrile. Analysis of the ex-
tract by HPLC indicated that less than 1% of the compounds found in the pre-

column (i.e., dissolved in 2.5 2 of water) was in the extract (i.e., associa-
ted with the particulates in 2.5 2 of water).

Results and Discussion

The results of these experiments are summarized in Tables 15-18 and
Figures 8-14.

The reproducibility within experimental error of bioconcentration factors
for phenanthrene in the absence of other compounds (Table 15: exp. #2, tank
#2 and exp. #3, tank #2) and for all compounds of experiment #3 on day 28
(Table 15: exp. #3 all tanks, day 28, trials #1,2) as well as the similar
lipid content of the fish indicate that the data presented in Table 15 can be
utilized for trend analysis. The moderately large errors associated with the
calculated bioconcentration factors (Table 15) are mainly the result of varia-
tions in the concentration of the compounds in thg water (Table 17). Such
variations are common in experiments of this type’ ae

Bioconcentration factors approached a limiting value in most experi-
ments. However, there are some clear exceptions in which the bioconcentra-
tion factors rose to a maximum value and then declined (experiment #3, tank 3).
In this latter experiment it is possible that increased aryl hydrocarbon
hydroxylase (AHH) enzyme activity (Table 18) may have been involved in the
diminution of bioconcentration factor values for phenanthrene, 1-methy1-
phenanthrene, fluoranthene, and pyrene in the second half of the experiment
(Table 15; experiment #3, tank #3 and Figures 12,14).

Further statements about the relationship between AHH activity and bio-
accumulation are difficult to make. The rather large errors involved, in the
AHH activity measurements using the reported fluorimetric procedure and
the previously mentioned errors in bioconcentration factor values make con-
clusions tenuous. For example, the presence of §-napthoflavone

(5,6-benzoflavone), which has been reported to be an AHH inducer2!0+212,243 |
showed little significant increase in AHH activity (Table 18, exp. #2). How-
ever the fish in the tank containing the flavone (Table 16, exp.-#1) appeared
to release or metabolize accumulated phenanthrene at a significantly faster
rate than the fish in the tank containing only phenanthrene (Table 16, exp.#2).

63


Experimental Details

Procedure for aqueous chlorination reactions !56__

Purified water was placed into a pressure tank and, whilg stirring with
an overhead stirring motor, a solution of sodium hypochlorite was added,
followed by sufficient 0.1 N sulfuric acid to attain the desired pH. The
"free chlorjpg” ggncentration of the reaction mixture was determined iodo-
metrically.“>~? An acetonitrile solution of the PAH was then added via
a 100 u1 syringe. After the desired reaction time, the reaction was quenched
by addition of solid sodium thiosulfate (twice the number of chlorine
equivalents). The tank was then fitted with a 7x50 mm C-18 column and the
quantitative analysis proceeded as described in Section 5. For GC-MS identi-
fication work it was necessary to remove the water from the individual HPLC
fractions and to concentrate them. The water removal was effected by addition
of 0.5 ml of methylene chloride to cause separation of layers, followed by
removal of the aqueous layer with a Pasteur pipette. The organic layer which
remained was dried with sodium sulfate and concentrated under a stream of
nitrogen.

Procedure for preparative-scale chlorination reaction--!96

Chlorinated PAH's required for comparison with the products of the
aqueous-chlorination reactions wspe prepared by the reaction of the parent PAH
with chlorine gas in acetic acid“. Work-up of these reactions consisted of
dilution with water followed by washing with sodium bicarbonate, drying with
magnesium sulfate, and solvent evaporation. The material that remained was
then separated and/or purified by preparative-scale HPLC using two 7x600 mm
columns pagked with either Bondapak C-18 Porasil®B (37-75 u, reverse phase)
or PorasilTMA (37-75 uy, normal phase). The compounds obtained were re-
crystallized and checked for purity by GC-MS (see Table 20 and Appendix C).

72


EPA Implications to the
Aquatic
Environment of
Polynuclear
Aromatic
Hydrocarbons
Liberated from
Northern Great
Plains Coal


224.

225.

226.
227.

228.

229.

230.
231.

232.

233.

234.

235.

236.

237.

238.

L.N. Samoilovich and Y.R. Redkin. "3,4-Benzopyrene Pollution of the
River Sunzha by the Petrochemical Industry in Grozny". Hyg. Sanit.,
33, 165-168 (1968).

P. Lu, R. Metcalf, N. Plummer, and D. Mandel. "The Environmental Fate
of Three Carcinogens: Benzo(a)pyrene, Benzidene, and Vinyl Chloride
Evaluated in Laboratory Model Ecosystems". Arch. Environ. Contam.
Toxicol. 6, 129-142 (1977).

Reference (1), pp. 9-1 to 9-163.

R. Lee, R. Sanerheber, and G. Dobbs. “Uptake, Metabolism, and Dis-
charge of Polycyclic Aromatic Hydrocarbons by Marine Fish". Marine
Biol., 17, 201-208 (1972).

R. Lee, C. Ryan, and M. Neuhauser. "Fate of Petroleum Hydrocarbons
Taken Up from Food and Water by the Blue Crab Collinectes supides".
Marine Biol., 37, 363-370 (1976).

W. Roubal, T. Collier, and D. Malins. "Accumulation and Metabolism of
Carbon-14 Labeled Benzene, Naphthalene, and Anthracene by Young Coho
Salmon". Archiv. Environ. Contam. Toxicol., 5s 513-529 (1977).

Reference (54), pp. 61-72.

J. Bend, M. James, and P. Dansette. "In Vitro Metabolism of Xenobiotics
in Some Marine Animals". Ann. N.Y. Acad. Sci., 298, 505-521 (1977).

Jd. Hamelink and A. Spacie. "Fish and Chemicals: The Process of Accu-
mulation". Ann. Rev. Pharmacol. Toxicol., 17, 1670177 (1977).

J. Chambers and J. Yarbrough. '"Xenobiotic Biotransformation Systems in
Fishes". Comp. Biochem. Physiol., 55C, 77-84 (1976).

D. Malins. "Metabolism of Aromatic Hydrocarbons in Marine Organisms".
Ann. N.Y. Acad. Sci., 298, 482-496 (1977).

E. Brown and T. Sinclair. "Chemical Pollutants in Relation to Diseases
in Fish". Ann. N.Y. Acad. Sci., 298, 535-546 (1977).

E. Brown, J. Haydra, L. Keith, J, Greenspan, and J. Kwapinski. "Fre-
quency of Fish Tumors Found in a Polluted Watershed as Compared to Non-
polluted Canadian Waters". Cancer Res., 33, 189-198 (1973).

A. Mearns and M. Sherwood. "Distribution of Neoplasms and Other
Diseases in Marine Fishes Relative to the Discharge of Waste Water".
Ann. N.Y. Acad. Sci., 298, 210-224 (1977).

T. Matsushima and T. Suyimara. "Experimental Carcinogenesis in Small
Aquarium Fish". Progress in Experimental Tumor Research, 20, 367-379
(1976). ~

98


APPENDIX B

CALCULATIONS AND ERROR TREATMENT FOR ANALYSIS OF
WATER AND FISH TISSUE BY GC/PID

For analysis of each set of unknown solutions a standard curve (y =
a + bx) was generated by the method of least squares!5® using the data
obtained by injecting 1 uz of n solutions of known concentration into the
GC/PID:

x
a

weight in nanograms of compound in injection of known solution i

Y= GC peak height or area observed when X; nanograms were injected.

b = slope of least squares line
a = y-intercept of least squares line
n
zx
; -i
n
n
Zi
jy -4
(zm) = 9
E XsYs) - nxy
>» = Sis] i
(7 a. 22
( qx, )- nx
i=1
( n n
aes Ly, - £ x)
Mert ist!

i inj jon k
The weight of compound x, inal us injection of unknown solut
which produced a GC peak of area or height Vg on the jth injection is
given by:

121


OSL

SPECE

cH,

CH,

sn

_ Try
100
46 LS

a
150
37LN

Perepereperey
250

STEP MASS=1,

tial ial al

Ives

1%

T

300

20

10


DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory,
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or recommendation
for use.

Vi


239.

240.

241.

242.

243.

244.

245.

246.

247.

248.

249.

250.

251.

D. Branson, G. Blau, H. Alexander, and W. Neely. "Bioconcentration of
2,2',4,4'-Tetrachlorobiphenyl in Rainbow Trout as Measured by Accelera-
ted Test". Trans. Am. Fish Soc., (4) 785-792 (1975).

W. Neely, D. Branson, and G. Blau. "Partition Coefficient to Measure
Bioconcentration Potential of Organic Chemicals in Fish". Environ.
Sci. Technol., 8(13) 1113-1115 (1974.

S. Herbes and G. Risi. "Metabolic Alteration and Excretion of Anthra-

Cisse” Daphnia pulex". Bull. Environ, Contam. Toxicol., 19, 147-155

J. Neff and J. Ayderson. "Accumulation, Release, and Distribution of
Benzo(a)pyrene-'"C in the Clam Rangia Cuneata". In Proceedings of
Conference on Prevention and Control of O71 Pollution, San Francisco,
March 25-27, 1975, American Petroleum Institute, Washington, D.C.

A. Boobis, D. Nebert, and J. Felton. "Comparison of B-Naphthoflavone
and 3-Methylcholanthrene as Inducers of Hepatic Cytochromes P-448 and
Aryl Hydrocarbon (Benzo{a}pyrene) Hydroxylase Activity". Molecular
Pharmacology, 13, 259-268 (1977).

B. Dunn. "“Technigues for Determination of Benzo(a)pyrene in Marine
Organisms and Sediments". Environ. Sci. Technol., 10, 1018-1021 (1976).

J. Warner. "Determination of Aliphatic and Aromatic Hydrocarbons in
Marine Organisms". Anal. Chem, 48(3), 578-583 (1976).

H. Guerrero, E. Biehl, and C. Kenner. "High-Pressure Liquid Chromato-
graphy of Benzo(a)pyrene and Benzo(ghi)perylene in 0il-Contaminated
Shellfish". J. Assoc. Off. Analytical Chemists, 59(5), 989-991 (1976).

B. Dunn and D. Young. "Baseline Levels of Benzo(a)pyrene in Southern
California Mussels". Marine Pollution Bulletin, 7, 231-234 (1976).

B. Dunn and H. Stich. "Release of the Carcinogen Benzo(a)pyrene from
Environmentally Contaminated Mussels". Bull. Environ. Contam. Toxicol.,

15(4), 398-401 (1976).

B. Dunn and H. Stich. “Monitoring Procedures for Chemical Carcinogens
in Coastal Waters". J. Fish. Res. Board Can., 33, 2040-2046 (1 976).

H. Bravo, S. Salazar, A. Botello, and E. Mandelli. "Polyaromatic Hydro-
carbons in Oysters from Coastal Lagoons Along the Eastern Coast of the
Gulf of Mexico, Mexico". Bull. Environ. Contam. Toxicol., jg, 171-176

(1978).

G. Veith, D. Kuehl, F. Puglisi, G. Glass, and J. Eaton. "Residues of
PCB's and DDT in the Western Lake Superior Ecosystem". Archiv. Environ.

Contam. Toxicol., 5, 487-499 (1977).

99


daphnids were placed into a 250-mé beaker containing 200 m2 of solution of 20,
40, 60 or 80% coal distillate in Lake Superior water. Controls were prepared
using the corresponding amount of distilled water in Lake Superior water.
The temperature was 18+1°C and the photoperiod was 16:8 (light:dark).

The results (Table 5) indicate no toxic effects of centrifuged leachate
to juvenile or adult fathead minnows. However, uncentrifuged leachate caused
25% mortality during the first 2 weeks of a 24-week exposure (6.3 g or
coal/2£) and 100% mortality during a 96 hr bioassay (25 g coal/Z).

Coal distillate caused no significant mortality to adult or newly-
hatched fathead minnows (Table 6) at the highest concentration tested (20%).
Partial mortality of Daphnia exposed to 20, 40 and 60% coal distillate was
noted in 1 out of 3 bioassays (Table A-2). The consistent mortality observed
at 80% levels was also found at comparable distilled water levels, indicating
that the low ionic strength of the test water was the lethal factor.

Sublethal Effect Studies--

Growth--The growth rates of two-month-old fathead minnows exposed to
coal leachate (distilled deionized water) were compared to fish exposed to
purified Lake Superior water (XAD-2) in a 24-week experiment.* The leachates
for the first two weeks of the experiment were not centrifuged and a 50%
mortality of fish was noted in one leachate tank. Thereafter, centrifuged
leachate was used for the experiment. Exposure water was renewed twice a
week and the 6 & tanks were continuously aerated. Temperatures were main-
tained at 24+1°C and the photoperiod set at 16:8 (light:dark). The fish were
2 months old at the onset of the experiment and the maximum loading attained
at the termination of the experiment was 2.7 g fish biomass per liter of tank
water. The fish were fed 5% of their body water per day in 4 feedings. Food
consumption was measured weekly, while total fish weights were determined
every two weeks. Daily observations were made for signs of male spawning
coloration. Growth curves and maturation data are presented in Figure A-7
and Table A-3 (Appendix A) and indicate a similar growth rate, but an appa-
rent retardation in maturation.

Spawning--The spawning behavior of fathead minnows exposed to coal
leachate (made using Lake Superior water) was compared to fish exposed to
only Lake Superior water in a number of 2-4 week experiments. In each ex-
periment 2 male and 4 female fathead minnows (9 mo - 1 yr ) were exposed

to either water or leachate in a tank containing three spawning substrates
prepared by gluing (Corning black silicone) a layer of sand to the inside of
glass half-cylinders (60cm radius). Eggs were removed and counted daily.
Hatchability in Lake Superior water was determined by incubating groups of
50 eggs in glass jars which were mechanically oscillated. In these experi-
ments the fish were fed frozen brine shrimp and pellets twice a day. The

* Gas chromotograms of centrifuged leachate and purified Lake Superior
water are shown in Figures A-2, A-3 and A-6

20


yield was obtained in this step. The significant NMR data are in Appendix E,
mass spectral data in Appendix F, and microanalytical data in Appendix G.

79


CHALi 8

Orty se | Ory

lwo

The value of this route is the flexibility that can be gained by intro-
ducing methyls in the component parts of the epoxynaphthalene synthesis309,

0
fe)
| Q@— Oh
/ CH),
)

cH
(Cy), (Cis) (CH3),

methyl benzynes methyl furans
(generated in situ)

74


FUTURE UTILIZATION OF COAL

In the future a dramatically increased usage of coal to replace dwindling
supplies of gi ig and gatural gas in the production of energy and chemicals iS
anticipated.‘~7> - However, the exact processes to be employed and the
timetable for their introduction have not yet been determined.

Possibilities for energy production from m goR] | ipcdyce improved methods
gf direst combustion, 23 combustion of gases 55 4 and liquids!5,16,18
oer" derived from 28°84> 2 and direct generat ign oF slectrictts from coal by
magnetohydrogynamics. hgmical feedstocks? such as ammonia,® methanol 8
acetic acid, ethylene yco! 8 et ylene,° benzene-toluene-xy]enes 4 (BTX),
petrochemicals!4, and others!* can also be derived from coal.

ENVIRONMENTAL AND HEALTH CONCERNS

There are considerable concerns about the environmental and health
aspects of increased coal utilization because of the types of compounds that
are associated with coal and its transformation processes. 9, These in-
clude inorganic compounds such as heavy metals, sulfur dioxide, and nitrogen
oxides and organic compounds such as phenols, arylamines, alkanes, mono- and
polycyclic aromatic hydrocarbons and their sulfur and nitrogen heterocyclic
analogs.

Extensive studies on Pheno)s indicate that, in general, these compounds
have a high acute toxicity, 3 Igy potential for bioaccumulation,2© and
are readily degraded by microbes . Arylamines have been found to be
toxic to blue-green algae, and other aquatic organisms, . 0 Alkanes undergo
microbial degradation, a3thy rpgatively non-toxic#4-45 and may be accumu-
lated in aquatic organisms .4

The aromatic tyenicieye Ss, and their heterocyclic analogs have caused the
most concern. Mutagenicity* and carcinogenicit. are associated with
many members of this group of com nce including benzo(a)pyrene (B(a)P),
benzo (aJantpracenes, and chrysenes. Toxicity has also been re jprted for some
members such as 6-methy]quinol ine3 and phenanth ERS The bio-
accumulation of these compounds may be a problem as well B54, 56 (see
Section 6 also).

Metabolic activation o 78 Comp aromatic hydrocarbons (PAH) is neces-
sary for cancer production 2? Complex, inducibl@3»°9 enzyme systems which
contain cytochrome P-45' and which are located in the endoplasmic
reticulum 6! and the nucleus2>6 3 of many mammalian cells (including the cells
of liver, skin, blood, placenta and others in human4 ) convert the PAH's
into metabolites such as phenols, epoxides, alcohols and ketones. Some of
the metabo lites ane known to bind covalently to DNA, 85/56 06-74 RNA,°5 268 and
proteins)» 66-68 bas fon studigs on benzo(a)pyrene 68>72 benzo(a)-
anthracene, 77 quinoline,°’ benzen and other compounds. Epoxides or diol
epoxides are currently ato to be the g “ultimate, carcinogens", 90,69,72-82
but other 3 Bgtabol ites such as quinongs ,© 3 phenols ,&4 and some intermediate
specieso3 such as radical cations®? may also deserve consideration.


vb

TABLE 10.
PAH TISSUE CONCENTRATIONS AND MFO MEASUREMENTS DURING WATER UPTAKE EXPERIMENTS*

PAH Concentration Time p-450° AHH AW
(ug/kg) (day) (nM/mg protein) (pM/mg protein/min) (pM/mg protein/min)
pyr fl
9528 318210 3 0.083 + 2.18 aed
20.43
8247 273214 7 0.107 1.18 -
£0.27
159211 40820 10 0.103 1.74 -
£0.02
281223 1,250238 21 0.071 2.50 ieee
£0.43
i) 0 0.107 1.72 se
(controls, n=6) +0.048 20.80
B(a)P
368424 10 0.238* 3.80* 34,52*
20.018 40.62 48.14
73.124° 19 0.115 1.66 11.47
0.028 20.88 £1.13
9 0.175 1.76 20.01
(controls, n=2) 20.032 40.25 43.21

®The mean water concentrations during the exposures were 3.89 +0.08 ug vw, pyrene; 3.31 £0.08 ug rm, fluoranthene;
and 0.40 +9.21 ug 17), B(a)P. Control values were obtained by taking several (n) 3-fish samples from control tanks
at various times during the experiments. Induced enzyme levels (P <0.05) are indicated by asterisks.

bouring the pyr/fl experiment only one measurement of P-450 was made on microsomes prepared from 3 pooled livers;
duplicates were run during the B(a)P exposure.

“The exposure tank contained 50 mg v1 ground egal (s0.125 mm) {n addition to B(a)P. Water concentrations of B(a)P
determined on filtered samples were <0.1 yg 17!.


60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

W. Baird, C. Chern, and L. Diamond. "Formation of Benzo(a)pyrene-
Glucuronic Acid Conjugates in Hamster Embryo All Cultures". Cancer

Research, 37, 3190-3197 (1977).

C. Yang. "The Organization and Interaction of Monoxygenase Enzymes in
the Microsomal Membrane". Life Sciences, 21, 1047-1058 (1977).

E. Bresnick, J. Vaught, A. Chuang, T. Stoming, D. Bockman, and H.
Mukhtar. "Nuclear Aryl Hydrocarbon Hydroxylase and Interaction of
Polycyclic Hydrocarbons with Nuclear Components". Archiv. Biochem.
Biophys., 181, 257-269 (1977).

J. Pezzuto, M. Lea, and C. Yang. "The Role of Microsomes and Nuclear
Envelope in the Metabolic Activation of Benzo(a)pyrene Leading to
Binding with Nuclear Macromolecules". Cancer Research, 37, 3427-3433
(1977).

0. Pelkonen. "Metabolism of Benzo(a)pyrene in Human Adult and Fetal
Tissues". In Carcinogenesis--A Comprehensive Survey, Vol. 1, R. Fren-
denthal and P. Jones, eds., Raven Press, New York, 1976, pp. 9-23.

T.J. Slaga, S. Butz, S. Thompson, W. Bracken, and A. Viaje. "A Kinetic
Study on the in vitro Covalent Binding of Polycyclic Hydrocarbons to
Nucleic Acids Using Epidermal Homogenates as the Activating System".
Cancer Research, 37, 3126-3131 (1977).

J. Pezzuto, M. Lea, and C. Yang. “Binding of Metabolically Activated
Benzo(a)pyrene to DNA and Histones of Rat Liver, Lung and Regenerating
Liver". Life Sciences, 22, 105-110 (1978).

G. Kahl, E. Klaus, H. Jonen, and R. Kahl. "“Enzymic Control of Irrever-
sible Binding of Metabolically Activated Benzo(a)pyrene in Perfused Rat
Liver by Monooxygenase Activity". Arch. Toxicol., 39, 149-158 (1977).

M. Koreeda, P. Moore, P. Wislocki, W. Levin, A. Conney, H. Yagi, and
D. Jerina. "Binding of Benzo(a)pyrene 7,8-diol-9,10-epoxides to DNA,
RNA and Protein of Mouse Skin Occurs with High Stereoselectivity".
Science, 199, 778-781 (1978).

A. Dipple and J. Nebzydoski. “Evidence for the Involvement of a Diol-
Epoxide in the Binding of 7,12-dimethylbenz(a)anthracene to DNA in Cells
in Culture". Chem.-Biol. Interactions, 20, 17-26 (1978).

W. Lutz and C. Schlatter. "Mechanism of the Carcinogenic Action of
Benzene: Irreversible Binding to Rat Liver DNA". Chem.-Biol. Inter-

actions, 18, 241-245 (1977).

K. Straub, T. Meehan, A. Burlingame, and M. Calvin. “Identification of
the Major Adducts Formed by Reaction of Benzo(a)pyrene Diol Epoxide with
DNA in vitro". Proc. Natl, Acad. Sci. U.S.A., 74(12), (1977).

86


AQUEOUS CHLORINATION OF POLYNUCLEAR AROMATIC HYDROCARBONS

Chlorination is the predominant technique used for water renovation and
disinfection. The process has been applied to wastewater treatment problems,
to the disinfection of drinking water and the preservation of acceptable water
quality through distribution systems, to the solubilization of sludge (a
"superchlorination" process involving large doses of chlorine), to the main-
tenance of hygienic conditions in closed swimming abeas. and to the reduction
of algal and bacterial growth in cooling towers 0-269” The development of
the technology for the effective application of chlorine has been considered
largely responsible for saving thousands of lives that could have been lost
through contracting any of several possible water-borne diseases. In short,

chlorination has developed into what has been refgrned eto as the most valuable
and versatile tool available to the water chemist“”~? :

The possible reaction of Spjorine with materials present in the treated
water has long been recognized“°’, mainly because of the very practical ne-
cessity for using pore gghlorine than was anticipated to meet given standards
of turbidity, BoDe > or fecal coliform bacteria. Environmentally, chlo-
rine and chloramines (as reaction products of amponig amino acids, or other
amines with chlorine) are considered deleterious®®*> 70, and considerable
effort has been directed toward their removal by such processes as reduction
(e.g., S09)" or by adsorption-decomposition (activated charcoal), with the
result that documented examples of incorporation of carbon-bound chlorine under
conditions used in water treatment have been quite limited. Early chemical
investigations were only ipikighedoyhen the chlorination process generated
problems of taste and odor“??? . However, since these initial reports,
there have been documented examples of the incorporation of chlopjpe,agto such
systems as "activated" aromatics, humic acids, and nucleic acids“’>"“".
Typically, the chlorine incorporation into these systems results in decreased
degradabiljty and increased toxicity266-269 , Similar studies involving the
ubiquitous and, in some cases, carcipggenic? polynuclear aromatic hydro-
carbons (PAH's) have not been extensive?’. However, it has been reported, that
bhe_sgqcentration of various PAH's in water is reduced upon chlorination ?

and that a few 9f the resulting products, have been identified: chlo-
rinated naphthalene29! 5293 and Co_genaphthalenes : chlorinated acenaphthas
lenes: 3, 5-chloro-3,4-benzopyrene 92 and 3,4-benzopyrene-5,8-quinone! 99>
as well as arene oxides“’©.

Results
The results of the application of the C-18/HPLC/GC-PID/GC-MS method to
the study of aqueous chlorination reactions of PAH's are provided in Table 19.

Chlorinated PAH standards that were prepared and used in this work are listed
in Table 20. The study demonstrates the vulnerability of PAH's to conversion

65


SECTION 5
GENERAL ANALYTICAL TECHNIQUES DEVELOPED FOR PROJECT

This section describes the general analytical techniques that were develo-
ped during this project for the analysis of polynuclear aromatic hydrocarbons
and their derivatives in water and fish tissue.

WATER ANALYSIS

The determination of PAH present in water at the microgram per liter
level or less!3/,138 has usually been carried out by removal of the PAH
material from water using liquid-liquid extraction techniques!39-142, head-
space sampling techniques!43-145, or adsorption techniques employing materials
such as Tenax GC146-148, Xap resins92,149-151, polyurethane foaml52,153, or
C-18 Corasi1!43,144,154, followed by analysis of this material with high per-
fpymance liquid chromatography (HPLC)143,144,154, gas chromatography (GC)!40,
142,14 » and gas chromatography/mass spectrometry fecvms)¥42.1 4. Direct
analysis with fluorescence has also been attempted 55,

Results and Discussion

For the present work it was decided to use an adsorption technique be-
cause liquid-liquid extraction techniques require the use of large volumes of
organic solvents followed by a tedious and time-consuming concentration step,
and extensive data on the successful application of headspace sampling techni-
ques to analysis of PAH in water is lacking!45. The adsorbent chosen was
C-18 Corasil® because a convenient. procedure for analysis of the adsorbed ma-
terial via C-18 pre-column-coupled reverse-phase HPLC has been reported! 38,
143,144 However, reverse-phase HPLC alone does not give extensive separation
of various PAH's for accurate identification and quantitation. Therefore, we
have improved upon this procedure!56 by the introduction of several modifica-
tions, including the use of a gas chromatographic separation (photoionization
detection).

The procedure begins with forcing an aqueous sample through a glass micro-
fiber filter (necessary for environmental samples) connected in series with a
50 x 7 mm column packed with C-18 Corasil®. The column containing the PAH ma-
terial is then attached to a C-18 micro-packed reverse-phase HPLC set-up and
eluted with an acetonitrile-water gradient. Fractions corresponding to each
UV peak are collected and these aqueous-acetonitrile fractions are then in-
jected directly into a GC equipped with a photoionization detector (PID). The
procedure thus provides compound identification and quantitation data from
both HPLC and GC and is very convenient since no concentration or water re-
moval steps are required unless GC/MS analysis is necessary.

26


TABLE § (continued)

Sott

Sott~-Swtss rcuntatn town:
‘center of tenn
at nighmay
open country
Sediment tn a stream flow
ng throuch an atl tank farm
tn kroxvitle, TH

ssa

River sedicent from the
Rnondda Far- valley, South
Wales (site cf ccai mine
operated tn 19th and early
20th centurtes}

Lake sedinests,
Greifensee, Switzerland

River sediment near chor-
fea} manufacturing plant.

Marine Sediment,
Buzzards Bay, Mass.

Street dust

Ash froa volcano

Water in a strean flowing
through an oi? tank farm
Ta Kooxvtiie, TH

Well fn Aces, IA near site
at a coal gas plant that
operated until 1970's.

Tire manufacturing plan
wastenaters

Thames River

Tap water
Switzerland
Tap water, U.S.
Ratn water

Tap water, Athens, GA

verzolaoyrene

PRH (total
PAH (total

PAH (total)
nacnthalene
alkylnaphthalene
anthracere
bens{a}antoracene
denz(a}pyrere
anttracene
‘flucranthene
pyrene
2,2-denzofluorene
chrysene
3,4-benzpyrene

af
benzo(ght)perylene
phenanth=ene
anthracene
fluoranthene
pyrene
benzofe}oyrene
benzotsfpyrene
peryléne

fuorene
phenanthrene
methy! phenanthrene
Fluoranthene
pyrene

others
phenanthrene
fluoranthene
pyrene

others,

aze-arenes
phenanthrene
anthracene
Fluoranthene
pyrene
denzofe]pyrene
benzolalpyrene
erylene

benzopyrene

naphthatene
alkylnaphtnalenes,
anthracene
denzfaJanthracene
benz{a pyrene

acenaphthyTene
‘emethyinaphthatene
methyl incenes
acenaphthene
benzethtophene
others
naphthalene
Tenethyinapathalene
phenanthrene
ethylphenanthrene
flucranthene
pyrene

others
Fuoranthene
pyrene

Benzcoyrene
perylene

others
phenanthrene
pyrene
benzola}pyrene
phenanthrene
ethyloheranthrene
‘Tuoranthene
pyrene

cheysene
benzepyrene
others
raghthalene
Sitenzofuran
Tuerene
phoranthrene
Flyorantnene

+3 ated benzanthracene

38

2,000-10,000
200-25,000

0.01
0.01

0.00002-0.002

191

192

176

193

102,194}
102,194

182

v6

az

139

i)
vo

195

ist


ZoL

TABLE A-3.  FATHEAD MINNOW BEHAVIORAL RESPONSE TO COAL LEACHATE?

EXPOSURE OBSERVATION OF SPAWNING COLORATION # SPAWNINGS EGGS/SPAWNING FISH/TANK

(week #1) (ave. #1)
Leachate (Tank 1) - - - 19
Leachate (Tank 2) 23 - - 8
Control (Tank 3) 15 - - 18
Control (Tank 4) 16 5 43.6 18

Aouring a 24 week exposure to coal leachate, the onset of male spawning
coloration was noted. Minnows were 2 mo. old at the start of the
experiment. Tank 1 and 2 were leachate exposures and tanks 3 and 4
Lake Superior water exposures.


FIGURE 8
EFFECTS OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHs )
ON MICROSOMAL ENZYMES IN RAINBOW TROUT®

pmol 3-OH Ba}P mg protein-! min?

mol p-ominophenol mg protein! min!

= oe ‘phen om “fluor ehtys — Bta)P

The two bars for each compound are values obtained 3 and 5 days after i.p.
injection of 30 mg/kg PAH. For each compound tested, the three enzyme
assays were done using the microsomal fraction obtained from 2-3 pooled
livers. AHH and AH assays were done in duplicate with the error bars
representing the standard deviation. The mean and standard deviation of
seven control fish samples are indicated by the horizontal lines. a=p< 0.05

abbreviations: naph, 1,2,4-trimethylnaphthalene; 1254, Aroclor 1254; phen,
phenanthrene; pyr, pyrene; fluor, fluoranthene; chrys, chrysene; B(a)P,
benzo(a)pyrene.

43


2027LM/OERL 77204 CHLOROPHENANTHRENE

> >DATEC< 7SPECa

sun
268071

BASE
31176
PEAK

3200

Varian CHS
250

:  70ev
GC

°

Electron Energy
200

Inlet System
Instrument

YQaOCConnnwnpececeosconnnncoocoooDeS
EMFUSNUSKNOOE NT ACSSNGGSmAsHONOvAMFH
ERRARA@SGGOODAATTOSOATANURRR RRO a moe

papeeeasaitainl es pbahebebebier tr tnt

= mete

150

CNN NN
VOUFMERTOFONAERTOMMI N=omma@
RAM VINO TRAUMA DOITEMMOM DARED AD
Quuised ve vaoNUvundMnueNs veKanKsore
rere) ae RM Rae omin

POUUUAVAMD USA M MAF HR OMIT FO =O 01
“= uM

La cihaite

ROOUMIDRDOmMSROUMS DOOROMUORATCURDLOS —=
A UU UUAMMMM MT ANDOOOSDORRARA®

100
eae it BERL 77204 CHLORGPHENANTHRENE

100 -.
$0-,
80.
70:
60:

128

Ivars

STEP NASS=1,

SPECS


MIXED-FUNCTION OXIDASE ACTIVITY IN FISH EXPOSED TO POLYNUCLEAR AROMATIC
HYDROCARBONS

Polynuclear aromatic hydrocarbons and many other foreign compounds are
metabolized by the mixed-function oxidase (MFO) system. Although this group
of enzymes is well characterized in mammals6!,196-200, jt was not until 1963
that this metabolic capability was suggested in fishes20!. As in mammals,
the fish MFO system has been shown to be inducible. Some of the compounds
and mixtures demonstrated to be inducers are: petroleum, PCB's¢06-2'
3-methylcholanthrene209-212, 2,3-benzanthracene24’ and benzo(a)pyrene@ 3,

Of particular interest from an environmental standpoint has been the
finding that fishes exposed to petroteum under laboratory and field conditions
show induced MFO activity!98,202-205,207,214 Qne MEO enzyme system, aryl
hydrocarbon hydroxylase (AHH), has been suggested as an environmental monitor

2 since not only does it respond to the presence of hydrocarbons, but it
activates certain PAH's to carcinogenic metabolites.

The current investigation was designed to examine several PAH's to de-
termine whether MFO induction in rainbow trout is a response to particular
(especially carcinogenic) PAH's or to general PAH exposure and to evaluate
the relationship between the concentration of a recognized carcinogen, benzo-
(a)pyrene {B(A)P}, in tissue and hepatic MFO {AHH, aniline hydroxylase (AH)
and P-450} induction.

EXPERIMENTAL DETAILS
Bioassay Setup--

Injection experiments--Rainbow trout were obtained from the Genoa National
Fish Hatchery, Genoa, Wisconsin, and held in flowing Lake Superior water
for six months prior to use. The fish used in the experiments weighed from
30-50 g and had a total length range of 14-17 cm. All fish sampled were
sexually immature and were one year old at the start of the six-month ex-
perimental period.

Several PAH's were administered in sterile peanut oi] by intraperitoneal
injection. After anaesthetizing in 100 mg/2 MS-222 (ethyl m-aminobenzoate
methanesulfonic acid salt), each fish received an 0.2 mlinjection with a 22-
gauge needle. The standard dose of B(a)P was approximately 30 mg/kg. B(a)P
was given in doses from 3 ug/kg to 300 mg/kg in dose response experiments.
Controls received 0.2 m@ injections of peanut oil.

Experiments were carried out in 55-liter stainless steel tanks supplied
with 10#1°C Lake Superior water by a Mount-Brungs proportional dilutor, at a

39


89

Starting PAH
1-Methyl naphthalene

1-Methylnaphthalene

Fluorene

Fluorene

Fluorene

Fluorene

Anthracene

Anthracene
Anthracene
Anthracene

Phenanthrene

tc1)]
mg/e

24.0
20.4

1.205

18.8
23.5

12.9

2.0

3.2

[PAH]
ng/t

531

336

334

819
773

333

1000

1042
965
552

820

TABLE 19

Summary of Aqueous Chlorination Studies

Reaction
Time, hr

3.0

3.0

0.5

3.0
3.0

3.0

4.0

3.75

0.08

0.5

pH
3.8

4.1

7.0

4.1
3.4

3.35

4.0

44

6.5

7

71

Products
Identified
(% Yield)
Monochloro-1~
methylnaphalene

Monochloro-1-.
methyl naphthalene
(7325)

Fluorene
(7344)
Fluorene

Fluorene
Monochlorofluorene

Fluorene (v5)
Monochlorofluorene
(5224)

Anthracene

3

Anthraquinone
(90211

Anthracene
Anthraquinone
(7849)

Anthraquinone
(61216)

Phenanthrene

Basis
for
Assignment

a,b

bse

boc
a,b,c
a,b,c
a

dsc

a,b,c

a,bsc

a,b,c

Comments
ut = 176,178. GC retention time
po) saenetca) to compound 1. (Table
20).

GC retention time identical to
compound 1 (Table 20).

wt = 166

My = 166

M = 200, 202

GC retention time is identical to
compound 2 (Table 20).

w= 178

wt = 208

No anthraquinone produced according
to HPLC

wt = 178,


Reagents--

Furan and anthranilic acid were obtained from Aldrich Chemical Company,
Inc., Milwaukee, Wisconsin. The 3-methy]l and 5-methylanthranilic acids were
purchased from Pfaltz and Bauer, Inc., Stamford, Connecticut. Methy11ithium
was obtained from Alfa Inorganics, Tne The 2,5-dimethylfuran was synthesized
according to the procedure of Newman?¥?.

General Procedure for the Conversion of the Anthranilic Acids to the 1,2-
dihydro- and 1,4-dimethyl-1,4-epoxynaphthalenes--

In a 250-ml 3-neck round-bottom flask equipped with a reflux condenser,
magnetic stirrer, and pressure equilibrated dropping funnel were placed 30 m
of 1,2-dimethoxyethane, 3 mé of isoamy] nitrate (0.022 mol) and 5 mé of furan
(0.070 mol) or 2.5 dimethyl furan (0.045 mo1)°. The reaction mixture was
heated to reflux (furan, 80°, 2.5 dimethyl] furan, 106°) under a nitrogen
atmosphere. The appropriate anthranilic acid (ca. 0.014 mol) in 30 me of
1,2 dimethoxyethane was added dropwise during 2% to 3 hours. Following 30 min
at reflux the reaction mixture was cooled, made basic with 10% sodium bicarbo-
nate, and extracted with six equal portions of ether and water. The aqueous
layer was re-extracted six times and the combined ether extracts were dried
and decolorized. A short column distillation (1 mm, 70°) produced a pure
sample.

The average yield for this conversion is 40%. MNR data were correct for
the proposed structures. Microanalytical data are listed in Appendix G.

General Procedure for the ADdition of Methyllithium to the 1,4-Epoxynaphtha-
lenes--

The epoxynaphthalene (1, 4, 5, 6, 7, or 8), 0.0039 mol, was dissolved in
50 mé of dry THF under an anhydrous nitrogen atmosphere in a 250-mé 3-nick
round-bottom flask fitted with a reflux condenser and magnetic stirrer.
Methyllithium, 0.045 mol, was added dropwise and the solution was refluxed for
90 min. The reaction mixture was then cooled to room temperature and water
was added to destroy the excess methyllithium. The aqueous layer was
extracted with six 25-m2 portions of ether and the combined extracts were
dried over anhydrous magnesium sulfate. A short path distillation (0.6 mm)
produced a relatively pure alcohol (some thermal dehydration inevitably
occurred) at a typical yield of about 40%. The alcohols were not purified
further but carried directly to the dehydration step.

General Procedure for the Dehydration and Production of the Methylnaphtha-
Tenes--

The partially purified alcohols produced above were dissolved in about
50 mé of anhydrous ethyl ether and a few drops of freshly distilled boron-
trifluoride ether were added. The solution was stirred at room temperature
for no more than 15 min. The ether was washed twice with 10-m2 portions of
10% sodium bicarbonate. The ether layer was dried over anhydrous magnesium
sulfate and, after removal of the solvent, the naphthalene was purified by a
short path distillation (Imm,70°). A pure product in almost a quantitative

78


ride for 5 hours. The extract was then concentrated in a Kuderna-Danish
apparatus to v1.5 m2. The concentrate was injected into the HPLC and frac-
tions containing the PAH material were collected at 118-25 min (flow rate =
4 m/min). One-microliter samples of each of the HPLC fractions were in-
jected into the GC/PID along with injections of solutions of known concen-
tration for quantitation as described in the water analysis section.

33


FIGURE A-9
GAS CHROMATOGRAPHIC ANALYSIS OF
FATHEAD MINNOWS EXPOSED TO COAL LEACHATE

LEACHATE

Na

FLO. RESPONSE

LAKE WATER

FLO. RESPONSE

Oo 5 15 20. 25 30. 95 40 45 50 55 60 65 70 75

TIME (minutes)

arathead minnows were exposed to coal leachate (6.3 g coal/L distilled
deionized water) for 24 weeks prior to extraction. .
brathead minnows were exposed to XAD-2 purified Lake Superior water for 24

weeks prior to extraction. ; _
GC onditions-colunn: SP-2250, 2m x 2mm i.d. glags; injgctor: 250°C; detector

300°C: carrier flow: 20 ml/min; program: 100-235 C at 4 /min.
119


291. J.G. Smith, R.B. McCall and P.K. Chan. "Formation of Polychlorinated

Aromatic Compounds During Aqueous Chlorination". Environ. Pollut., 14
289-296 (1977). rl

292. W. Graef and G. Nothhafft. "Chlorination of Drinking Water and Benzo-

pyrene". Arch. Hyg. Bakteriol, 147, 135-46 (1963); Chem. Abstr., 59,
11102 (1963). — =

293. D.P. Spath, Ph.D. Dissertation. "The Chlorination of Coal Tar Deriva-
tives in Water". University of Cincinnati, Cincinnati, Ohio (1972),
Dissertation Abst. 3106B (1972).

294. Standard Methods of Examination of Water and Wastewater. 14th ed.,
American Public Health Association, American Water Works Association
and WPCF, Washington, D.C., 1976, pp. 316-321.

295. P. de la Mare, G. Cum and M. Johnson. "The Chlorination of 1-methyl-
naphthalene by Molecular Chlorine". J. Chem. Soc., C, 1590 (1967).

296. P. de la Mare and H. Suzuki. "Products of Chlorination of Naphthalene,
1-Methyl naphthalene, 2-Methylnaphthalene, and Several Chloro-methyl-
(ey with Sulphury] Chloride". J. Chem. Soc., C, 1586-1590

1967). ~

297. G. Bearen, P. de la Mare, E. Johnson, and N. Klassen. "The Kinetics
and Mechanisms of ARomatic Halogen Substitution. Part XII. Products
of Chlorination of Fluorene in Acetic Acid". J. Chem. Soc., 988-994
(1962).

298. P. de la Mare, N. Klassen and R. Koenigrberger. "The Kinetics and
Mechanisms of Aromatic Halogen Substitution. Part XI. Chlorination
of Phenanthrene in Acetic Acid". J. Chem. Soc., 5285-5293 (1961).

299. M. Shenbor, M. Samodrigina, and N. Zheltonogora. Khim. Tekhnol, (11)
90-5 (1968); Chem. Abstr., 72, 90139r (1970).

300. M. Shenbar and V. Kirichenko. Khim. Tekhnol., (20) 55-60 (1971);
Chem. Abstr., 76, 24955 (1972).

301. M. Blumer and W.W. Youngblood. "Polycyclic Aromatic Hydrocarbons
in Soils and Recent Sediments". Science, 188, 53 (1975).

302. F.C. Monaster. In Sources, Effects, and Sinks of Hydrocarbons in the
Aquatic Environment. Symposium Proceedings of the American Institute
of Biological Sciences, August 9-11, 1976.

303. J.E. Tomaszewski, W.B. Manning and G.M. Muschik. "A Facile Synthesis
of Benz(a)anthracene-7,12-diones". Tetrahedron Lett., uu, 971 (1977).

103


FIGURE A-2
GAS CHROMATOGRAPHIC ANALYSIS OF A HEXANE EXTRACTION
OF COAL LEACHATE?

BLANK

FLD. RESPONSE

LEACHATE

ELD. RESPONSE

|

0 6 2 18 24 30 36 42 48 54 60 66. 72 76 84 90

TIME (minutes)

8Two liter samples of distilled deionized water and centrifuged coal leachate
(6.3 g coal/L distilled deionized water) were extracted with 100 ml hexane.

GC conditions-column: 5% SP-2250, 2m x 2mm i.d. alass; injector: 250°C;
detector: 300°C; carrier flow: 20 ml/min; program: 80-2600C at 4°/min.

112


ZtL

FISH WEIGHT (g/fish)

FIGURE A-7
GROWTH RATES OF FATHEAD MINNOWS
14p EXPOSED TO COAL LEACHATE AND PURIFIED
LAKE SUPERIOR WATER® o—__» LEACHATE

rab o----- © LAKE WATER

1.0F

ob eer

0.6F a

n at. n L L m
°o 2 4 6 8 10 12 4 16 af: } 20 22 24

TIME (weeks)

*Fathead minnows, 2 months old initially, were exposed to coal leachate (6.3 g coal/L
distilled deionized water) or to Lake Superior water (purified using XAD-2) under renewed
static conditions.


HOnp

SOLVENT_CDC13__ TEMP._Auiba___°C
| TUBE O.0__S mm SPIN RATE. 20_tps

i
|

[tock
SIGNAL__CDClo__ RF FIELOZ3/7.. _.uB

OBSERVE cwE FTO tFO) onFO

SWEEP/SPECTRAL WIDTH...
SWEEP/ACQUISITION TIME
RF FIELO/PULSE WIDTH.........04......dB/usec
PULSE DELAY. one
TIME CONSTANT.
DATA POINTS...
NO. SCANS/TRANSIENTS

OFFSET. 86464 Bae Hz |

sec SPECTR. AMPISXL/2

a a

TT
PPM

a Al
DECOUPLE cated O oh
NUCLEUS______. GYROCODE oL
OFFSET____. .Hz RF POWER

NOISE BANDWIDTH...
INDOR SWEEP WIDOT!
OPERATOR.

CHART S-100XLA (Givarion
PRINTED IN USA

141

Te



StL

100
90
804
704

speco = 27 Ln

eee eee EAT
200



EPA-600/3-79-093
August 1979

IMPLICATIONS TO THE AQUATIC ENVIRONMENT OF POLYNUCLEAR
AROMATIC HYDROCARBONS LIBERATED FROM
NORTHERN GREAT PLAINS COAL

by

Robert M. Carlson, Alan R. Oyler, Ellen Heath Gerhart,
Ronald Caple, Kenneth J. Welch, Herbert L. Kopperman,
Donald Bodenner, and Dale Swanson
Department of Chemistry
University of Minnesota
Duluth, Minnesota 55812

Grant No. R803952-03-1

Project Officer

Douglas W. Kueh]
Physiological Effects of Pollutants Section
Environmental Research Laboratory
Duluth, Minnesota 55804

ENVIRONMENTAL RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804


SEL

THRESH(CM)= 168
OFFSETCHZ)= 140.8
RF FROCMHZ)= 25-1997
NO. INT. OFFSETCHZ)
1 ay 325668
2 a 321561
3 146 3202.9
4 203 319964
Ss 107 3198-3
6 103 3179.9
7 138 3150-7
8 114 3088.7
9 120 3085-8
16 19 1969-6
iB) 19 1937.7
12 21 1906.8
413 84 0.08

el

PPM
129.242
127.589
127.104
126.964
126.604
126.190
125.034
122.572
122.459

786163
76.897
75-639
@ 060



199.

200.

201.

202.

203.

204.

205.

206.

207.

208.

209.

210.

I. Bjérkhem. "Rate Limiting Step in Microsomal Cytochrome P-450 Cata-
lyzed Hydroxylations". Pharmacol. Res., 1(3), 327-348 (1977).

A.Y.H. Lu. "Liver Microsomal Drug-inducing Enzyme System: Functional
Components and Their Properties". Federation Proc., 35(2), 2460-2463
(1976). TT —

J.R. Baker, A. Struempler, and S. Chaykin. "A Comparative Study of
Trimethylamine N-Oxide Biosynthesis". Biochem. Biophys. Acta, 71,
58-64 (1963). ~~

J.F. Payne and W.R. Penrose. "Induction of Aryl Hydrocarbon {Benzo-
(a)pyrene} Hydroxylase in Fish by Petroleum". Bull. Environ. Contam.
Toxicol., 14(1), 112-116 (1975).

K.A. Burns. "Microsomal Mixed Function Oxidases in an Estuarine Fish,
Fundulus heteroclitus, and Their Induction as a Result of Environmental
Contamination”. Comp. Biochem. Physiol, 53B, 443-446 (1976).

B. Kurelec, S. Britvic, M. Rijavec, W. Miller, and R. Zahn. "“Benzo(a)-
pyrene Monooxygenase Induction in Marine Fish--Molecular Response to
Oi] Pollution". Marine Biology, 44, 211-216 (1976).

J. Yarbrough and J. Chambers. "Crude Oi1 Effects on Microsomal Mixed-
Function Oxidase System Components in the Stripped Mullet (Mugil
cephalus)". Life Sci., 21, 1095-1100 (1977).

D.W. Hill, E. Hejtmercik, and B.J. Camp. “Induction of Hepatic Micro-
somal Enzymes by Aroclor’ 1254 in Ictalurus punctatus (Channel Catfish)".
Bull. Environ. Contam. Toxicol., 16(4), 485-502 (1976).

E. Gruger, M. Wekell, P. Numoto, and D. Craddock. “Induction of
Hepatic Aryl Hydrocarbon Hydroxylase in Salmon Exposed to Petroleum
Dissolved in Seawater and to Petroleum and Polychlorinated Biphenyls,
Separate and Together, in Food". Bull. Environ. Contam. Toxicol.,
17(5), 5120520 (1977).

E.H. Gruger, Jr., T. Hruby, and N.L. Karrick. "Sublethal Effects of
Structurally Related Tetrachloro-, Pentachloro-, and Hexachlorobipheny]
a Juvenile Coho Salmon". Environ. Sci. Technol., 10(10), 1033-1037
1976).

J.J. Stegeman and D.J. Sabo. “Aspects of the Effects of Petroleum
Hydrocarbons on Intermediary Metabolism and Xenobiotic Metabolism in
Marine Fish". In Sources, Effects, and Sinks of Hydrocarbons in the

Aquatic Environment, Symposium Proceedings (A.I.B.S.), pp. 423-436
1976).

M. Chevion, J. Stegeman, J. Peisach, and W. Blumberg. "Electron Para-
magnetic Resonance Studies on Hepatic Microsomal Cytochrome P-450 From
a Marine Teleost Fish". Life Sci., 20, 895-900 (1977).

96


9g

Exp Tank
on
h
1
l2
2
N
2
3
3

Compound
( benzofuran
If

Wworene

9-chlorophe-
nanthrene

henanthrene

-naphtho~
Yavone

phenanthrene

hhenanthrene

}-chlorophe-
ranthrene,

phenanthrene

idtbenzofuran
ifluorene
phenanthrene
N-rethy1phe-
jnanthrene

Fluoranthene

pyrene

Day #1
0.96:
0.19

0.938
0.19

0.87
0.05
4.032
0.22

5. 14s
0.53

3.79%
0.07

3.798
0.21

Day #2

1.972
26

1.878
0.22

0.85
0.07

2.508
0.21

<0.05

Ans
0.24

4.528
0.65

5.278
0.48

3.17
0.59

3.06¢
0.67

4.548
0.29
1.508
0.41
1,938
0.25
1.108
0,09

SUMMARY OF FISH ANALYSES (BASED ON TOTAL WET WEIGHT OF FISH)

Concentration of PAH tn

Day #4 Day #7 Day #10

4,002 4.03

0.19 0.34

4.15: 4.45%

0.19 0.26

1.132 1.028

0.08 = 0.06

2.522 3.762 4.4de

0:20 © 0.42 0.37
0.05 <0.16

4.962 5.98 8.658

0.81 0.20 0.58

3.882 4.728

0,32 (0.40

9.162 8.278

1.64 1.01

3.388 3.358

0.24 = (0.39

1.932 2.318

0.39 «(0.18

2.05 3.108

0.40 © 0.32

4.628 4.132

0.06 0.26

1.832 1.46

0.45 (0.18

3.082 4.428

0.65 «(0.18

1.08 1.362

0.18 0,20

Fish:

5.122
0.28

8.69
0.44

3.048
0.37

0.33

2.788
0.24

2.56
0.35

ing PAH/Kg ff
Day #14 Day #18 = Day #21

10.06¢
0,42

sh

TABLE 16

3.378
0.22

3.938
0.32

0.668
0.10

4,908
0.43
<0.18

10.9%
0.73

5.632
0.49

7.508
0.68

$.05¢
0.50

2.738
0.22

3.212
0.22

4.88
0.67

2.358
0.28

4.668
0.42

2.808
0,35

3.472
0.18

3.908
0.22

7.13
0.31

9.46¢
071

7.08¢

3.81t
0.23
3.628
0.17

4.928
0.29

1.708
0.24

2.558
0.62

ot

Day #28
May #25 Day #28 Trial #2 Day #29

2.75%
0.19

<0.42

5.97
0.43

1.92
0.31

4.308
4

1,262
0.07
1,324
0.07

1,552
0.28

O.14t
0.21

0.762
0.16

<0.1

Depuratton
Day #30 Day #31 Day #32 Day 635

<0.1 0.1

<0.1 <1

<0.1

59 <0.7
08

ee
oe

<0.1 <0.1

1,794 <0.1
0.48

<0.1
<0

<01

Wean vg PAK/E
water

3.1920.26

3,A820.38

0.5520.12

2.6320.83

1,820.18

2.58¢0.44

2.5% 0.46

1,418 0.34

2.3420.54

2.3420.51
2,4820.48
2.2020.25
1,382.19
1.3520.20

1.1820.27


éoated stirring bar was introduced and the sample extracted by emulsifying the
contents for 2 hr. The organic layer was dried over NagSOq and concentrated
for GC analysis in a Kuderna-Danish evaporator. The recovery efficiency for
known samples was 95+4%.

Results

Effects of Several Polynuclear Aromatic Hydrocarbons on Mixed-Function
Oxidase Activity--

The effects of several aromatic compounds (injected dose 30 mg/kg) on MFO
parameters indicated that in general it was the higher molecular weight com-
pounds that caused induction (Figure 8). Cytochrome P-450 levels were ele-
vated by injection with Aroclor 1254 (a PCB mixture), pyrene, chrysene, and
B(a)P. The CO difference spectra of induced P-450 did not show the spectral
shift to 448 nm characteristic of mammalian systems. This observation is con-
sistent with other studies on piscine P-450 measurements211,217,233. AHH
activity showed a dramatic 12-fold increase three days after B(a)P administra-
tion. Injected Aroclor 1254 also caused significant hydroxylase induction.
This latter observation is in agreement with previous studies where PCB's in-
food have induced AHH activity in coho salmon207,208 and PCB's in water caused
very high AH and N-demethylase activities in channel catfish206, The re-
sponse of AH to injection of PAH's, although lower in magnitude, was generally
similar to that for AHH. The exception was chrysene which caused significant
AH and P-450 induction but no AHH enhancement.

Rainbow trout were also exposed to three dissolved PAH's under flow-
through conditions. Table 10 shows the enzyme and tissue data after exposure
to pyrene and fluoranthene (pyr/f1, combined) and to B(a)P. The day 21
(pyr/f1) and day 10 B(a)P enzyme assays are of particular interest, Although
the total accumulated pyrene and fluoranthene was over four times that of
accumulated B(a)P, the accumulation of pyr/fl did not initiate P-450 or AHH
induction. In contrast, accumulated B(oYP caused significant induction of
both hydroxylase activities and P-450 content after 10 days.

Benzo(a)pyrene Tissue Levels and Mixed-Function Oxidase Induction--

The specific effects of the known MFO inducer B(a)P were examined by
comparing B(a)P tissue levels with concomitant MFO measurements in a dose-
response injection experiment and in a water uptake experiment. In the dose-
response study, significant induction of both AHH and AH activities was found
at an injected B(a)P dose of 300 ug/kg and above (Figure 9). The cytochrome
P-450 concentration at the 300 ug/kg dose on day 5 was elevated. Therefore,
the 300 ug/kg dose appears to be an approximation of a minimum effective dose
(MED) for MFO induction in rainbow trout. The AH results should be contrasted
with those of Payne!30 who found no basal or inducible AH activity in rainbow
trout.

Gutted fish from the dose-response experiment were analyzed for B(a)P 3
and 5 days after injection (Table 11). The measured B(a)P concentrations
were generally quite different from the injected dose perhaps because of
different rates of absorption from the peritoneal cavity, weight variations

42


131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

W. Birge. "Aquatic Toxicology of Trace Elements of Coal and Fly Ash".
Presented at Ecological Symposium on Energy and Environmental Stress in
Aquatic Systems, sponsored by Savannah River Ecology Laboratory, SRP

National Environmental Research Park, University of Georgia's Institute

of Ecology and United States Department of Energy, November 2-4, 1977,
Augusta, Georgia.

K.E. Biesinger. "Tentative procedure for Daphnia magna chronic testing
in a standing system". Fed. Register, 40(123), 26902-26905 (1975).

R. Carlson and R. Drummond. “Fish Cough Response--A Method for Evalua-
ting Quality of Treated Complex Effluents". Water Res., 12, 1-6 (1978).

K. Burton. “A Study of the Conditions and Mechanism of the Dipheny]-
amine Reaction for the Calorimetric Estimation of Deoxyribonucleic
Acid". Biochem. J, 62, 315-323 (1956).

K. Honn and W. Chavin. “An Improved Automated Biuret Method for the
Determination of Microgram Protein Determinations". Anal. Biochem.,
68, 230-235 (1975.

G. Veith, D. Kuehl, and J. Rosenthal. “Preparative Method for Gas
Chromatographic/Mass Spectral Analysis of Trace Quantities of Pesti-
cides in Fish Tissue". J. Assoc. Offic. Anal. Chem., 58, 1-5 (1975).

M. Fishman and D. Erdman. "Water Analysis". Anal. Chem., 49, 139R-
158R (1977). _

H. Hertz, W. May, S. Wise, and S. Chesler. "Trace Organic Analysis”.
Anal. Chem., 50(4), 428A-436A (1978).

M. Acheson, R. Harrison, R. Perry, and R. Wellings. "Factors Affecting
the Extraction and Analysis of Polynuclear Aromatic Hydrocarbons in
Water". Water Research, 10, 207-212 (1976).

K. Grob, K. Grob, Jr., and G. Grob. "“Qrganic Substances in Potable
Water and in Its Precursor". J. Chromatogr., 106, 299-315 (1975).

R. Harrison, R. Perry, and R. Wellings. "Effect of Water Chlorination
Upon Levels of Some Polynuclear Aromatic Hydrocarbons in Water".
Environ. Sci. Technol., 10, 1151-1156 (1976).

G.Jungclaus, L. Games, and R. Hites. "Identification of Trace Organic
Compounds in Tire Manufacturing Plant Wastewaters". Anal. Chem.,

48(13) 1894-1896 (1976).

S. Chesler, B. Gump, H. Hertz, W. May, S. Dyszel, and D. Enagonio.
"Trace Hydrocarbon Analysis: The National Bureau of Standards Prince
William Sound-Northeastern Gulf of Alaska Baseline Study". “U.S.
Bureau of Standards Technical Note 889, Jan. 1976.

91


FIGURE 5
STEAM DISTILLATE OF COAL (NO KOH)
HPLC

UV ABSORBANCE AT 254m
(1.0 FULL SCALE)

0 2 4 6 8& 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
TIME (minutes)

Conditions- column: 10 » Lichrosorb RP-18; program: 50 to 90% CH,CN in
water, 1.2 ml/min, 30 min (linear program).

16


FIGURE 2
PURIFIED WATER LEACHATE (FILTERED)
LIQUID CHROMATOGRAMS

KNOWN COMPOUNDS
(400 ng/ compound)
q | |
j
§ |—_ WL
ao
4
a
2
u BLANK
co)
= (3.5L)
£
&
st
wo
N
3 Lo
w
9
Zz
a
c
9
3 LEACHATE
15L
2 (15L)
!
~~
° 10 20 30 40 Py

TIME (minutes)

Conditions-column: 5y Lichrosorh RP-18, 4.6 x 250 mm; program: 59-90% CH3CN
in water, 1.2 ml/min, 30 min (linear program). Compounds in standard-
19.85 min: dibenzofuran; 21.22: fluorene; 22.91: phenanthrene; 26.76:
1-methylphenanthrene, fluoranthene; 27.79: pyrene; 29.72: 9-chlorophenan-
threne. Peak at 24.32 min in leachate experiment was shown to be phthalate
ester contamination.

12


186.

187.

188.

189.

190.

191.

192.

193.

194.

195.

196.

197.

198.

M. Fox and S. Staley. "Determination of Polycyclic Aromatic Hydrocar-
bons in Atmospheric Particulate Matter by High Pressure Liquid Chroma-

tography Coupled with Fluorescence Techniques". Anal. :
992-998 (1976). ‘ Anal. chen.» $87),

A. Bjgrseth. "Analysis of Polycyclic Aromatic Hydrocarbons in Particu-

late Matter by Glass Capillary Gas Chromatography". Anal. Chimica Act.
94, 21-27 (1977). we ae

F. Karasek, D. Denney, K. Chan, and R. Clemet. "Analysis of Complex
Organic Mixtures on Airborne Particulate Matter". Anal. Chem., 50,
82-87 (1978). =

M. Lee, M. Novotny, and K. Bartle. "Gas Chromatography/Mass Spectro-
metric and Nuclear Magnetic Resonance Determination of Polynuclear
Aromatic. Hydrocarbons in Airborne Particulates". Anal. Chem., 48(11),
1566-1572 (1976). —

G. Broddin, L. Vaeck, and K. Van Cauwenberghe. “On the Size Distribu-
tion of Polycyclic Aromatic Hydrocarbon Containing Particles from a
Coke Oven Emission Source". Atmos. Environ., 11, 1061-1064 (1977).

L. Shabad, Y. Cohan, A. Ilnitsky, A. Khesina, N. Shcherbak, and G.
Smirnov. "The Carcinogenic Hydrocarbon Benzo(a)pyrene in the Soil".
J. Nat. Cancer Inst., Al, 1179-1191 (1971).

M. Blumer, W. Blumer, and T. Reich. "Polycyclic Aromatic Hydrocarbons
in Soils of a Mountain Valley: Correlation with Highway Traffic and
Cancer Incidence". Environ. Sci. Technol., 11(12), 1082-1084 (1977).

G. Jungclaus, V. Lopez-Avila, and R. Hites. “Organic Compounds in an
Industrial Wastewater: A Case Study of Their Environmental Impact".
Environ. Sci. Technol., 12(1), 88-96 (1978).

W. Giger and M. Blumer. "Polycyclic Aromatic Hydrocarbons in the
Environment: Isolation and Characterization by Chromatography, Visible,
Ultraviolet, and Mass Spectrometry". Anal. Chem., 46, 1663-1671 (1974).

W. Giger, M. Reinhard, C. Schaffner, and F. Ziicher. In Identification
and Analysis of Organic Pollutants in Water, ed. by L.H. Keith, Ann
Arbor Science, Ann Arbor, Mich., 1976, pp. 440-443.

J.R. Gillette, D.C. Davis and H.A. Sesame. "Cytochrome P-450 and Its
Role in Drug Metabolism”. Ann. Rev. Pharmacol., 12, 57-84 (1972).

R. Kato. "Drug Metabolism Under Pathological and Abnormal Physiological
States in Animals and Man". Xenobiotica, 7, 25-92 (1977).

D.W. Nebert, R.C. Levitt, M.M. Orlando, and J.S. Felton. Effects of
Environmental Chemicals on the Genetic Regulations of Microsomal Enzyme
Systems. Clin. Pharmacol. Rev., 22(5), 640-669, 1977,

95


2S

100
SPECH

118 LS

150
1I8LN +

CH,

CH,

AISLA

Q
eso
STEP MASS*i, IvBvs

12


Compound!

10,

1,

12,

13,

lw

[wo

To!

12!

13'

Microanalytical Data

91

91

91

92.
(82.

APPENDIX G
METHYLATED NAPHTHALENES:

Calculated

aC

92.
(82.

51
46

-76
(87.

92,
(82.

94

26
94

75
(83.

12

+25
(83.

12

26
73

NN

@o @cno on

on

MICROANALYTICAL DATA

2H

67
+55

29
57

74
57

«75
97

-66
-97

74
-09

2

lprecursor alcohol in parentheses.

2

155

Not enough pure material obtained.

Observed

aC

92.
82.

91

82.

90.
83.

91

92.
82.

46
48

82
82.

98

79

76
08

23
83.

10

22
90

2H

~mN

0 09

ony ©WOo MO ©

54
-57)

82
.67)
-20)

-01
+90)

75
91)

76
.05)


Leachate exposure resulted in no consistent liver changes during the 21-
day experiment (Figure A-8). Hepatic DNA content, microsomal protein and
relative liver weight in leachate-exposed fish were similar to values obtained
during lakewater exposure. Although a significant elevation of AHH activity
was noted on day 7 of the experiment, no elevated activity was maintained.

Hepatic Mixed-function Oxidase Activity (coal distillate)--In a related
experiment, rainbow trout were exposed to 0.2% coal distillate for 21 days
under flow-through conditions. Distillate was metered into a 55 2 stainless
steel tank using an FMI (Fluid Metering, Inc.) lab pump with stainless steel
fittings and tubing. Flow rates were 27.5 2/hr for the distillate tank and
40 2/hr for the lake water (control) tank. The trout were kept at 11+1°C
under subdued lighting (16:8). Samples of three fish each were removed on
days 3,7,10,14 and 21. The livers were pooled and assayed for cytochrome
P-450 concentration and AHH activity by methods described in the mixed-function
oxidase discussion (Section 6). The results of this experiment are presented
in Table A-6 and indicate an overall lack of MFO induction. There was, how-
ever, a small but observable elevation in AHH activity on day 21 of the experi-
ment.

Bioaccumulation (coal leachate)--In order to determine if there were any
qualitative differences in bioaccumulation between the fish exposed to coal
leachate and the control fish in the 24-week growth experiment described pre-
viously, extractions from the fish were analyzed by gas chromatography. Fish
from each tank were combined and frozen until analysis. The fish were ground
in a blender with 40 g solvent-washed sodium sulfate and Soxhlet extracted
with methylene chloride for 4 hr. The extracts were evaporated under a hood,
transferred to 15 mé centrifuge tubes and then evaporated to exartly 3 me.
Interfering lipids were removed by gel permeation chromatography 38 with re-
distilled, deaerated methylene chloride as the carrier solvent. After con-
centrating in a Kuderna-Danish evaporator, the sample was injected onto a gas
chromatograph (Figure A-9). Results indicate qualitative and quantitative
differences in bioaccumulated material.

Bioaccumulation (coal distillate)--In order to determine if there were
any qualitative differences in bioaccumulation between fish exposed to coal
distillate and control fish in the 21-day hepatic mixed-function oxidase ex-
periment described previously, the gutted fish were extracted and analyzed by
gas chromatography. The results are presented in Figure A-10 and showed no
major differences in the chromatograms.

Discussion

Biological testing of material liberated from Northern Great Plains coal
indicated no severe adverse effects on fathead minnows, rainbow trout or
Daphnia. Toxicity, growth retardation and MFO induction were not observed in
fish exposed to coal leachate, although a lowered spawning success rate was
noted in fathead minnows. Distillate effects were limited to slight Daphnia
mortality at intermediate concentrations (20, 40, 60%) and a small increase in
hepatic MFO activity in rainbow trout.

However, conclusive statements cannot be made about the impact of leached
and volatilized materials on an actual receiving water due to the difficulty

24


GOL

APPENDIX A

ADDITIONAL TABLES AND FIGURES RELEVANT TO BIOLOGICAL STUDIES ON THE LEACHING AND VOLATILIZATION OF COAL

Dissolved Oxygen*
(mg/1)

Total Alkalinity”

(mg/1 CaCo3)
EDTA Hardness?
(mg/1 CaCo3)
pH
(20-22°C)

Conductivity
(umhos/cm)

Turbidity
(N.T.U.)

PROPERTIES OF COAL LEACHATE

Distilled Deionized

TABLE A-1

Distilled Deionized

Lake Superior Water

Lake Superior Water

Water Water Leachate Leachate
(7 samples) (48 samples) (48 samples) ( 6 samples)
mean range mean range mean range mean range
- - 6.81 3.70-8.50 8.06 6.87-8.60 - -

1.27 1.01-1.52 5.11 3.40-6.70 42.62 41.20-44.60 21.53 17,78-24.75
0.0 - 0.84 0.33-1.60 45.50 4446-46 .43 13.98 2.74-14.41
- 5.55-6.92 7.8° 6.8-8.2 7.8° 7.5-7.9 2.85 —7.22-8.09

1.19 0.73-1.63 27 15-32 92 90-104 70 65-77
- - 4.45 1.1-8.8 0.54 0.16-1.9 2.4 1.3 - 3.2

Reference R-294 azide modification

Reference R-294

“Mode of pH values


CHLOROMETHYLNAPHTHALENE

>>DATE<<7SPECO®

SO/LN/DERL 77182

70ev
GC

Electron Energy:
Inlet System

10

BOROPOMA MANA Maa M a NAMA MOM aaa!

Couns VVNoonOMsNNGEROTSH—WOHOND
ERARRRODDOGCCe ARR UMMMS Se TT RARK

WNXRNNRNNNM
ATURMOUOIS

Qreve ee ee
SPUSUVS MM NS VSONON@aMS@nSNsz@
“4 eM RO WHO aeenae

ETNA -aKnorerOcDOMemucaouraonmurs

ZUTKAGOUID MAUNA = MOOT UORMMORATAT

HUOT Ma PaO OUUUMOURTIRAMMFORD

AM ORUAINNS YON DUM UT A CLAM Emr
-“ ou om

Yovenvamnn sg oman oes aorsnvareoam
AHUNUUUMT TFT FUNDODGOORKR DOO

&

cs

Varian CHS

Instrument

126

200 ese 300

150

59 LM DERL 77182 CHLORONETHLYNAPHTHALENE

1%

I/B/s

STEP MASS=1,

SPECt


TABLE 12
RELATIONSHIP BETWEEN B(a)P CONCENTRATION IN RAINBOW TROUT
AND MFO INDUCTION®

P-450 AHH AH

3260+50 (i) + + +
537465 (4) + + +
458431 (4) + + +
368224 (w) + + +
350421 (i) + - +
320454 (i) + + +
249433 (i) - + -
140214 (i) - - -
130227 (i) - - -
7324 (w) - - .
<30 (i) - - -

@These data were compiled from both the injection experiment [i,
Table 2, Figure 9] and the water uptake experiment (w, Table 1).
Pooled livers were analyzed for MFO activity and the corresponding
gutted fish analyzed for B(a)P. A plus (+) indicates enzymes in

induced state.

48


279.

280.

281.

282.

283.

284.

285.

286.

287.

288.

289.

290.

R.M. Carlson, R.E. Carlson, H.L. Kopperman and R. Caple. “Facile
Incorporation of Chlorine Into Aromatic Systems During Aqueous
Chlorination Processes". Environ. Sci. Technol., 9, 674-675 (1975).

L.H. Keith. "Identification and Analysis of Organic Pollutants in
Water". Ann Arbor Science Publishers, Ann Arbor, Michigan (1976).

R. Harrison, R. Perry and R. Wellings. "Chemical Kinetics of Chlorina-
tion of Some Polynuclear Aromatic Hydrocarbons Under Conditions of
(iosG Treatment Processes". Environ. Sci. Technol., 10, 1156-1160
1976). —

R. Perry and R.M. Harrison. "A Fundamental Study of the Removal of
Polynuclear Aromatic Hydrocarbons from Water During Chlorination".

Prog. Wat. Tech., 9, 103-112 (1977).

M. Reinhard, V. Drevenkar, W. Giger. “Effect of Aqueous Chlorination
on the Aromatic Fraction of Diesel Fuel". J. Chromatogr., 116, 43-
51 (1976). —

S.G. Sforzolini, A. Savino, S. Monarca, M.N. Lollini. “Decontamination
of Water Polluted by Polynuclear Aromatic Hydrocarbons (PAH). I.
Action of Chlorine and Ozone on PAH in Double-Distilled and Deionized
Waters". Ig. Mod., 66, 309-35 (1973).

S.G. Sforzolini, A. Saviano and L. Merletti. “Effect of Chlorine on
Some Polycyclic Aromatic Hydrocarbons. The Destruction of Carcino-
genic Compounds in Water". Boll. Soc. Ital: Biol. Sper., 46, 903-906

J. Borneff. "Elimination of Carcinogenic Polycyclic Aromatic Com-
pounds During Water Purification". Gas-Wasserfach, 110, 29-34 (1969).

S.G. Sforzolini, A. Savino and S. Monarca. "Decontamination of Pollu-
ted Waters by Polynuclear Aromatic Hydrocarbons (PAH). II. Action of
Chlorine and Ozone on PAH in Drinking Water and in River Water".

Ig. Mod., 66, 595-619 (1974).

A.P. Ilmikskii, K.P. Ershova, A. Khesina, L.G. Rozhkova, V.G. Klubkov
and A.A. Korolev. "Stability of Carcinogenic Substances in Water and
(ts Fy ticacy of Methods of Decontamination". Gig. Sanit., 36, 8-12
1971). —

R.D. Gabovich, I.L. Kurennoi and Z.P. Fedorenko. “Effect of Ozone and
Chlorine on 3,4-benzopyrene During the Disinfection of Water". Gig.
Naselennykk Mest. 8, 88-91 (1969), Chem. Abstr., 73, 28613h (is7ay"

S. Krishnan, D.G. Kuhn and G.A. Hamilton. "Direct Oxidation in High
Yield of Some Polycyclic Aromatic Compounds to Arene Oxides Using

Hypochlorite and Phase Transfer Catalysts". J. Amer. Chem. Soc., 99,
8121-8123 (1977). ed

102


to "second-order" products during dilute aqueous chlorination conditions
typical of those encountered during disinfection processes. Moreover, the
increasing reactivity of chlorine with decreasing pH is demonstrated in all
examples examined. In the investigation fluoranthene showed the most in-
teresting behavior of all the compounds studied. In one chlorination experi-
ment it produced a very polar compound which eluted from the reverse-phase
HPLC column in approximately ten minutes less than the time required for the
monochlorofluoranthene standard. However, the GC retention times were identi-
cal and the GC-MS of the product indicated it to be a monochlorofluoranthene.
Presumably, this product is a chlorohydrin which readily loses water to form
a monochloro derivative. In a second experiment under similar conditions,

the elimination of water apparently occurred before the HPLC analysis, since
the product had an HPLC retention time identical to the monochiorofluoranthene
standard. In contrast to the other compounds studied, experiments at pH 4
with phenanthrene and its 1-methyl derivative gave very low total recoveries
for products (40% and .8% respectively). Presumably very polar products

were generated in these reactions which were not concentrated by the 7x50 mm

-18 column.

fluoranthene -H

net substitution -H,0,-4°

net addition

Hoon
H
C1
-H0 cl
(O) “Facile (O)

3-chlorofluoranthene polar chlorohydrin

66


TABLI

E 3

REPORTED STUDIES ON THE BIOLOGICAL EFFECTS OF

THE WATER SOLUBLE FRACTION OF PETROLEUM ON FISH

Type of Test

Organism(s)

Toxicity, Acute

Sublethal Growth/

Development

Cough Response

MFO

Bioaccumulation

Salmon, trout, cod 24, 96-hr toxicity

Baltic herring

Salmon

Brown trout,
cunner, copelin

Fundulus

rate

Effect Reference
127
decreased lengths, 128
malformed larvae
increased cough 129
AHH introduction 130
bioaccumulation of 46

naphthalenes

18


32.

33.

34.

35.

36.

37.

38.
39.

40.

41.

42,

43.
44.

45.

H. Braunstein, E. Copenhaver, and H. Pfuderer, eds. “Environmental ,
Health, and Control Aspects of Coal Conversion: An Information Over-
view". Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (1977),
ORNL/EIS--94/95.

W. Ramsay. "Siting Power Plants". Environ. Sci. Technol., 11(3),
238-243 (1977). —

G. Morgan. "Energy Resource Development: the Monitoring Components".
Environ. Sci. Technol., 12(1) 34-43 (1978).

C. Jahnig and R. Bertrand. "Environmental Aspects of Coal Conversion".
Chemical Engineering Progress, 72, 51-56 (1976).

European Inland Fisheries Advisory Commission. "Water quality criteria
for European freshwater fish: report on monohydric phenols and inland
fisheries". Water Res., 7, 929-941 (1973).

S. Herbes and J. Beauchamp. "Toxic Interaction of Mixtures of Two Coal
Conversion Effluent Components (Resorcinol and ewethy Touino) ine}
to Daphnia magna". Bull. Environ. Contam. Toxicol., 17(1), 25-31 (1977).

Reference (1), pp. 7018 to 7-27.

S. Dagley. “Catabolism of aromatic compounds by microorganisms".
Advances in Microbial Physiology, 6, 1-46 (1971).

S. Herbes, G. Southworth, and C. Gehrs. "Organic Contaminants in
Aqueous Coal Conversion EFfluents: Environmental Consequences and
Research Priorities". Proc. of 10th Annual Conference on Trace Sub-
stances in Environmental Health, University of Missouri, June 7-14, 1976.

J. Batterton, K. Winters, and C. Van Baalen. "“Anilines: Selective
Toxicity to Blue-Green Algae". Science, 199, 1068-1070 (1978).

Y. Miura, M. Okazaki, S. Hamada, S. Murakawa, and R. Yugen. "Assimila-
tion of Liquid Hydrocarbon by Microorganisms. I. Mechanism of Hydro-
carbon Uptake". Biotechnology and Bioengineering, 19, 701-714 (1977).

Reference (1), pp., 7-27 to 7-42.

R. Lee, R. Sauerheber, and A. Benson. "Petroleum Hydrocarbons: Uptake
and Discharge by the Marine Mussel Mytilns edulis". Science, 177,
344-346 (1972). —

J. Neff, J. Anderson, B. Cox, R. Laughlin, S. Rossi, and H. Tatem.
"Effects of Petroleum on Survival, Respiration, and Growth of Marine
Animals". In Sources, Effects, and Sinks of Hydrocarbons in the Aquatic
Environment. Proceedings from the Symposium (A.I.B.S.), 1976, pp.
516-539.

84


AH

AHH
B(a)P
bp

icles
GC-MS
GPC
HPLC
IR
MFO
NMR
PAH

PID
TLC
uv

ABBREVIATIONS

aniline hydroxylase

aryl hydrocarbon hydroxylase
benzo(a)pyrene

boiling point

gas chromatography

gas chromatographic mass spectrometry
gel permeation chromatography

high performance liquid chromatography
infrared

mixed-function oxidase

nuclear magnetic resonance

polynuclear aromatic hydrocarbon
polycyclic aromatic hydrocarbon

photoionization GC detector
thin layer chromatography
ultraviolet

xt


Product identification was accomplished by MS and NMR data and, where
possible, by a preparative scale chlorination in acetic acid (see Table 20)
for comparison with known samples or reported melting points. The MS data is
most useful for determining chlorine content (e.g., monochloro, dichloro,
...etc.) but is less useful in assigning the position(s) of substitution.
Representative MS data are included in Appendix C. This appendix also includes
some incomplete work on the dibenzofuran system, where both monochloro and

dichloro derivatives were prepared, and pyrene, where it appears that tri-
chloropyrene is formed.

The structural assignments of the major PAH derivatives are sometimes
tenuous. With this in mind, we thought it prudent to catalog whereygr
possible the 3¢ NMR spectra of all our chlorinated products. The '~C spectra
(with simultaneous !'H decoupling) is very sensitive to the substitution
pattern (both in chemical shifts and relative intensities). Data of this type,
hopefully, will eventually be used not only for the present structural assign-
ments, but to help identify previously unreported chlorinated PAH's that no
doubt will be detected in these or other studies. It is also possible that
high resolution (100 MHz or greater) ‘H NMR spectra with appropriate spin
decoupling experiments can be invaluable in certain structural assignments.

We have used this approach successfully to help establish the monochlorination
product of phenanthrene as 9-chlorophenanthrene. This was done by examination
of the relative chemical shifts of the three de-shielded protons (at C-1, C-8,
and C-10) upon saturatjgn of the remaining six aromatic,protons located
further upfield. The ‘~C spectral data as well as the ‘H 100 MHz data are
given in Appendix D. For a more detailed interpretation of the fluorene
system the reader is referred to the M.S. thesis of Kenneth Welch, "Coal
Derived PAH's and their Aqueous Chlorination Chemistry", University of
Minnesota, Duluth, 1979.

67


SECTION 7
SYNTHESIS OF METHYLATED NAPHTHALENES

Among the ubiquitous PAH's in the aqueous environment, a large number of
methylated derivatives are included’. The monitoring of these potentially
hazardous compounds have been described by Dr. F.C. Mgpastero of the U.S.
Department of the Interior as "a national imperative"°'*. The monitoring of
even the methylated PAH's is no easy task owing to the complexity of the
existing mixtures and the lack of suitable standards. The need to develop
synthetic routes to methylated PAH's is summarized in a recent report by Dr.

J.B. Tomaszews kj of the Chemical Carcinogenisis Section of the NCI Frederick
Research Center303,

The magnitude of the complexity of this problem can be gained by
examination of the simplest PAH, naphthalene. There are 73 methylated naphtha-
lene derivatives with one to eight methyl groups substituted at the eight
available positions. Of these, only seven are presently commercially
available.

8 1
7 2
6 3
5 4

The present work describes a synthetic design that leads to the selective
synthesis of certain mono-, di-, tri-, tetra-, and pentamethy] naphthalenes.
The synthetic scheme appears to be applicable to the anthracene and penan-
threne nuclei as well.

The new procedure is based on the susceptibility of the readily avai jable
naphthalene oxide system 1 to ring opening reactions with alkyllithiums?4",

The general route is illustrated below for the synthesis of 2-methy naphthalene
(2). This particular naphthalene is commercially available,* but not in the
100% isomeric purity as produced in the scheme.

* Aldrich Chemical Company, Milwaukee, Wisconsin
73


Number

AaRwn —

10
1
12
13
14

A-1

A-3

A-4
A-5
A-6

A-7

FIGURES

Page
Lakewater Leachate, Liquid Chromatographs ........... 10
Purified Water Leachate (Filtered), Liquid Chromatographs ... 12
Purified Water Leachate (Centrifuged), Liquid Chromatographs. . 13
Steam Distillate of Coal (KOH): HPLC ...........0- 15
Steam Distillate of Coal (mo KOH): HPLC. ..........2.4 16
Typical Gas Chromatograph of PAH Mixture Using Photoionization
Detector. ©... . ee ee 30
Typical Fractionation of Fish Tissue Extract Containing PAH
Material Using Styragel - HPLC.,..... be ee eee 34
Effects of Polycyclic Aromatic Hydrocarbons on Microsomal
Enzymes in Rainbow Trout... .... 2... ee eee ee eee 43
Dose Response for Hepatic Microsomal Enzymes in Rainbow Trout . 46
Summary of Dibenzofuran Experiments ............4. 59
Summary of Fluorene Experiments ........-++2-+-ee04 59
Summary of Phenanthrene Experiments... . 2... 2. ee eee 60
Summary of 9-Chlorophenanthrene Experiments .......... 61
Summary of Experiment with Six PAH Compounds. ......... 62
Conductivity Changes During Aqueous Leaching of Coal. ..... W1
Gas Chromatographic Analysis of Hexane Extract of
Coal Leachate . 2 2 1 we ee 112
Gas Chromatographic Analysis of Methylene Chloride Extract
of Coal Leachate. ©. 2 2 1 eee ee ee ee ee et ee ee 113
Isooctane Extraction of Coal Distillate. ..........-, 114
UV Scans of Coal Distillate . 2... ee ee et ee eh ee ee 115
Gas Chromatographic Analysis of Hexane Extract of XAD-Purified
Lake Superior Water... 1 1 1 ee ee ee te et ee 116
Growth Rates of Fathead Minnows Exposed to Coal Leachate and
Lake Superior Water... 1. 1 ee ee 117

vii


265.

266.

267.

268.

269.

270.
27).

272.

273.

274.

275.

276.

277.

278.

John D. Keenan and D.A. Hegemann. "Chlorination and Ozonation in
Water and Wastewater Treatment", Chemosphere, 1, 9-28 (1978).

R.M. Carlson and R. Caple. "Chemical/Biological Implications of Using
Chlorine and Ozone for Disinfection”. U.S. Environmental Protection
Agency, Washington, D.C., EPA Publication #600/3-77-066, (1977).

J.C. Morris. "Formation of Halogenated Organics by the Chlorination

of Water Supplies". U.S. Environmental Protection Agency, Washington,
D.C. EPA Publication #600/1-75-002 (1975.

E.L. Barnhard and G.R. Campbell. "The Effect of Chlorination on
Selected Organic Chemicals". U.S. Environmental Protection Agency,
Washington, D.C., EPA Publication #12020EXG03/72 (1972).

R.M. Carlson and Duane W. Long. "Chlorinated Organics as a Factor in
Reduced Biological Oxygen Demand". J. Environ. Sci. Health, A13(s),
177-186 (1978).

W.A. Brungs. J. Water Poll. Contr. Fed., 45, 2180 (1973).

G.F. Vaughn and J.C. Morris. “Tastes and Odors in Water Supplies".
Sci. Technol., 1, 703 (1967).

M.B. Ettinger and C.C. Ruchhoft. "Stepwise Chlorination on Taste

and Odor Causing Intensity of Some Phenolic Compounds". J. Amer. Water
Works Assoc., 43, 561 (1951).

R.H. Burttschell, A.A. Rosen, F.M. Meddleton and Morris B. Ettinger.
"Chlorine Derivatives of Phenol Causing Taste and Odor". J. Amer.
Water Works Assoc., 51, 205 (1959).

D.L. Shumway and J.R. Palensky. "Impairment of Flavor by Water
Pollutants". U.S. Environmental Protection Agency, Washington, D.C.
20460, EPA-R3-73-010.

R.L. Jolly. "“Chiorination Effects on Organic Constituents from
Domestic Sanitary Sewage Treatment Plants". Oak Ridge National Labora-
tory, Oak Ridge, Tennessee, Publication ORNL-TM-4290 (1973).

J.C. Morris. "Formation of Halogenated Organics by the Chlorination
of Water Supplies". U.S. Environmental Protection Agency, Washington,
D.C. Publication EPA-g00/1-75-002 (1975).

E.L. Barnhard and G.R. Campbell. "The Effect of Chlorination on
Selected Organic Chemicals". U.S. Environmental Protection Agency,
Washington, D.C., Publication EPA-12020 EXG03/72 (1972).

H.L. Kopperman, R.C. Hallcher, Sr. A. Riehl, R.M. Carlson and R. Caple.
"Aqueous Chlorination of a-Terpineol". Tetrahedron, 1621 (1976).

101


tZ

PAH

1-MethyI naphthalene
Fluorene
Phenanthrene

1-Methyl phenanthrene

Fluoranthene

Footnotes for Table 20

®Temperature = 25°C.

Table 20
Monochloro Products

Produced by Preparative Scale Chlorination Reactions. in Acetic Acid?

Moles PAH:
Moles Clo

1:3
V1
V1

1:1

1:2

Reaction
Time, hr

18

7.3

18

Monochloro Products

Monochloro-1-methyl-
naphthalene

Monochiorofluorene
Monochl orophenanthrene

Monochloro-1-methyl-
phenanthrene
Monochloro-1-methyT=
phenanthrene

Monochloro-
fluoranthene

Product
Reference
Number

1

3

Comments

oj1

wt = 176, 178
0,

Mp293-C

Wes 200,202

Mp = 52-52.5°C

M = 212, 214

oj}
MT = 226, 228

Mp = 87.5-80°C

M” = 226, 228

Mp = 93-99°C
MY = 236, 238

Literature

1-Chloro-4-methyl-
naphthalene (295,296)

2-Chlorgfiuorene,
mp 96.5" (297)

9-Chi orophgnanthrene,
mp 53-53.5 (298)

3-chloroflyoranthenc,
mp 101-102° (299,300)


21.

212.

213.

214.

215.

216.

217.

218.

219.

220.

221.

222.

223.

R.M. Philpot, M.O. James, and J.R. Bend. “Metabolism of Benzo(a)-
pyrene and Other Xenobiotics by Microsomal Mixed-Function Oxidases in
Marine Species". In Sources, Effects, and Sinks of Hydrocarbons in

Peretti Environment, Symposium Proceedings (A.I.B.S.), pp. 184-189

Cc. Statham, C. Elcombe, S. Szyjka, and J. Lech. "Effect of Polycyclic
Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of
Cope) enenatene in Rainbow Trout in vivo". Xenobiotica, 8(2), 65-72

9 . ~~

M.G. Pedersen, W.K. Hershberger, and M.R, Juchau. "Metabolism of
3,4-Benzopyrene in Rainbow Trout (Salmo gairdneri)". Bull. Environ.
Contam. Toxicol., 12(4), 4810486 (1974).

J.F. Payne. "Field Evaluation of Benzopyrene Hydroxylase Induction
a a yonttor for Marine Petroleum Pollution". Science, 191, 945-946
1976). —

Y. Imai, A. Ito, and R. Sato. "Evidence for Biochemically Different
Types of Vesicles in the Hepatic Microsomal Fraction". J. Biochem., 60
4170428 (1966). =

T. Omura and R. Sato. "The Carbon Monoxide-binding Pigment of Liver
Microsomes. I. Evidence for Its Hemoprotein Nature". J. Biol. Chem.,
239, 2370-2377 (1964).

J. Ahokas, 0. Pelkonen, and N. Karki. "Characterization of Benzo(a)-
pyrene Hydroxylase of Trout Liver". Cancer Res., 37, 3737-3743 (1977).

H.E. Christensen and E.J. Fairchild. "Suspected Carcinogens", 2nd
edition. A Subfile of the NIOSH Registry of Toxic Effects of Chemical
Substances, HEW Public. No. (NIOSH) 77-149, 251 pp. (1976).

E. Bingham and H.L. Falk. "The Modifying Effect of Cocarcinogens
on the Threshold Response". Arch. Environ. Health, 19, 779-783 (1969).

L.W. Wattenberg, M.A. Page, and J.L. Leong. “Induction of Increased
Benzopyrene Hydroxylase by Flavones and Related Compounds". Cancer Res.
28, 934-937 (1968).

J. Hamelink. "Current Bioconcentration Test Methods and Theory".
Aquatic Toxicology and Hazard Evaluation, ASTM STP 634, F.L. Mayer and
J. Hamelink, eds., American Society for Testing and Materials, 1977,
pp. 149-161.

R. Panirov and R. Brown. "Polynuclear Aromatic Hydrocarbons in Marine
Tissues". Environ. Sci. Technol., 11(10), 989-992 (1977).

J. Saxena, D.K. Basu, and J. Kozuchowski. "Method Development and .
Monitoring of Polynuclear Aromatic Hydrocarbons in Selected U.S. Waters".
Syracuse Research Corp., EPA Grant #803977

97


1.

12.
13.

REFERENCES

H. Braunstein, E. Copenhaver, and H. Pfuderer, eds. "Environmental,
Health, and Control Aspects of Coal Conversion: An Information Over-
view". Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (1977),
ORNL/EIS-94/95, pp. 2-1 to 2-104.

W. Hook. "Primary Coal--Analytical Needs". Pure Appl. Chem., 49, 1465-
1473 (1977). =

B. Ignasiak and M. Gawlak. "Polymeric Structure of Coal. I. Role of
foes in Constitution of High-rank Vitrinite". Fuel, 56, 216-222
1977). —_

C. Woo, A. D'Silva, V. Fassel, and G. Oestreich. '"Polynuclear Aromatic
Hydrocarbons in Coal--Identification by Their X-ray Excited Optical
Luminescence". Environ. Sci. Technol., 12(2), 173-174 (1978).

R. Tye, A. Horton and I. Rapien. “Benzo(a)pyrene and Other Aromatic
Hydrocarbons Extractable from Bituminous Coal". Am. Ind. Hyg. Assoc. J.,
25-28 (1966).

Reference (1), p. 1-1

G. Atwood. "The Strip-mining of Western Coal". Scientific American,
233(6), 23-29 (1975).

P. Spitz. "Petrochemicals from Coal". CHEMTECH, 295-300 (1977).

Anon. “Increased Use of Coal Deemed Safe Through 1985". Chem. Eng.
News, 22-23, Jan. 20 (1978).

G. Kélling. "Products of Coal (Coke, Tar, Gas) and Their Analysis".
Pure Appl. Chem., 49, 1475-1482 (1977).

E. Osborn. "Coal and the Present Energy Situation". Science, 183,
477-481 (1974). =

Reference (1), pp. 2-62 to 2-83.
D. Davies. "Energy Conservation in the Chemical Industry. Part II.

Economics and Politics of the Return of Coal and Cellulose". Chemistry
and Industry, 771-775 (1975).

82


BIOACCUMULATION OF POLYNUCLEAR AROMATIC HYDROCARBONS

e.accumulation of aromatic hydrocarbons from water!71,226,227 and
Food2ebs 2885288 by a variety of aquatic organisms! 6132265230 including algae,
plankton, mol Jusks, worms, clams, and fish (Tables 13 and 14) has generated
some interest©?!. Organisms containing accumulated aromatic compounds may be
involved in food-chain biomagnification processes which may result in ine
creased concentration of hydrocarbons per weight issue at. successively
higher trophic levels, perhaps including heagetots 171,226,281 258 Also
aromatic hydrocarbons, accumulated in higher organisms containing mixed-
function oxidase enzymes, might be transformed by these enzymes into meta-

i a ucing adverse effects on the organism or its progeny
POb'BPF, SBBOBAP, 945 Prog These concerns are apparently justified by the ob-
servation of increased tumor frequency in fish Jjyins in polluted water con-
taining PAH (e.g., 10-50 ug/2 benzanthyagene 35) with respect to fish
living in relatively unpolluted water¢35-23/, Although it is not known if
PAH were involved in the tumor production, it 38 known that certain PAH are
capable of producing tumors in laboratory fis 8

Aquatic organisms, such as Tubigex worms and snails, which do not con-
tain the enzyme system necessary for metabolism of aromatic compounds, tend to
concentrate these compounds to higher levels (per weight of. tissue) than do
organisms such as fish, which contain the necessary enzymes! 3 2226,225. In
organisms of the latter type, it has been noted that in some cases the pres
sence of certain amounts of P ng ot} ompounds may lead to increased le-
vels of PAH metabolic Be Sa tot 305208 268, SOPCEST (i.e. "MFO induction"). The
relationship between increased enzyme activity and levels of accumulation of
polycyclic aromatic compounds in a particular organism has not been studied
extensively, but it appears that the presence of inducers of mixed-function
oxidases may raise metabolism and lower accumulation of PAH in the organism
and that the, Presgnce of an inhibitor may lower the metabolism and raise the
accumulation? !¢> .

The goal of the present study was to provide data, which are presently
scarce, on the potential for accumulation of various polycyclic aromatic com-
pounds in fish and to determine if any relationship exists between the mixed -
function oxidase activity in the fish and the levels of accumulation.

The experimental determination of the potential for a given compound to
accumulate in an aquatic organism usually involves the derivation of a bio-
concentration factor by direct measurement of the concentration of a com-
pound in the fish and in the water at equilibrium under flow-through conditions.

* . a ae
: . - concentration of compound in tissue

Bioconcentration factor = © 7
concentration of compound in water

50


FIGURE 9
DOSE RESPONSE FOR HEPATIC MICROSOMAL ENZYMES
IN RAINBOW TROUT?

0.40} P-450

i
t
r
|
5
t

each fish was injected i.p. with (B(a)P and the MFO response measured 3 and
5 days later. For each dose the three enzyme assays were done using the
microsomal fraction obtained from 2-3 pooled livers. AHH and AH activities
were measured in duplicate with the error bars representing the standard
deviation. The mean and standard deviation of seven control fish samples
are indicated by the horizontal lines. a = p< 0.05

46


SECTION 4
CHEMICAL AND BIOLOGICAL STUDIES OF

NORTHERN GREAT PLAINS COAL
NORTHERN GREAT PLAINS COAL

Northern Great Plains coal is a subbituminous variety of coal which
contains!22 72-76% carbon (mineral-matter free), 18% oxygen, and 35-50%
volatile matter. A large portion of the volatile matter is probably water.123

Experimental Details

The coal was originally derived from the Decker Mine in Montana and
arrived in Superior, Wisconsin, on October 25, 1975. Sixty pounds of coal
were taken from each of twelve railroad cars and the combined samples were
mixed in a large fiber barrel. The coal was then ground in a Wiley mill and
sieved to isolate particles of various sizes (0.125-0.250 mm) for this work.

LEACHING OF COAL--CHEMICAL STUDIES

Reports on the leaching of organic compounds from coal appear to be
non-existent,9398 although it is known that inorganic materials may be
removed from coal storage piles by this process .96-98

Lakewater Leachate--

The leachate was prepared by stirring 11.1 9 of coal (<0.125 mm) per
liter of Lake Superior water for 48 hr. It was then centrifuged in a Sorvall
(SS-3) continuous-flow centrifuge and 14.23 2 of the centrifugate was forced
through a GF/F filter and a 50x7 mm column of C-18 Corasil as described in
the analytical section (Section 5). Column-coupled reverse-phase HPLC pro-
duced a curve which is compared in Figure 1 with a similar curve obtained

from a 14.48 2 blank of Lake Superior water. The water chemistry analyses are
presented in Table 1.

Purified Water Leachate (Filtered)--

This leachate was prepared by stirring 10 g of coal (<0.125 mm) per liter
of purified water (see Section 5 for preparation) for 48 hr. It was then
placed into a stainless steel pressure can and forced through a Whatman GF/F
filter which was replaced seven times in order to maintain a good flow. The
filtered leachate was then forced through a GF/F filter-pre-column combina-
tion (see Section 5) and analyzed by column-coupled HPLC in the usual manner


IDENTIFICATION AND QUANTITATION OF POLYNUCLEAR AROMATIC HYDROCARBONS:

Source

Remote regton of Bolivia
(air particulate extract)

Antwerp
(air particulate extract)

Zurich, Switzerland
(airborne particulates)

Hew York city
(airborne particulate
extract)

New York City
(airborne particulates)

College Park, MD
{airborne particulates)

Oslo, Norway
(airborne particulates)

Simcoe, Canada
(airborne particulates)

Urban air particulates,
indiana

Ate particulates near
coke oven plant

Coke oven entsstons

Coal fly ash

TABLE 9

Compounds
AIR

anthracene, phenanthrene
methylanthracene, methy] phenanthrene
fluoranthene

pyrene

benzanthracene, chrysene
benzopyrene, perylene

otners

anthracene, phenanthrene
methylanthracene, methylphenanthrene
fluoranthene

pyrene
benzanthracene, chrysene
denzpyrene, perylene
dibenzacridine

others

phenanthrene
anthracene
fluoranthene
pyrene
benzofe]pyrene
benzolalpyrene
pery!
{soquinoline

methyl {soquing} ine
ethyl isoquinol ine
benzo(f){soquinol ine

phenanthrene
anthracene
fluoranthene
pyrene

perylene

others.
fluoranthene
pyrene
benzfaJanthracene
chrysene

benzof e}pyrene
benzolalpyrene
benzoperylene

phenanthrene
fluoranthene
pyrene

pyrene
coronene
others

fluorene
phenanthrene
fluoranthene
yyrene
chrysene
benzofluoranthene
benzpyrene
others

120 PAHS identified

anthracene
phenanthrene
fluoranthene
Pyrene
chrysene
perylene
benzopyrenes
others

fluorene
phenanthrene

anthracene

ethylpheranthrene
methylanthracene

fluoranthene

pyrene

chrysene & triphenylene

benzola jpyrene

coronene

many others

anthracene, 9,10-demethylanthracene
benzo(a)pyrene, benzo(e pyrene
benzo(b)phenanthrene, chysene
fluorene, fluoranthene, perylene
phenanthrene, pyrene, triphenylens

37

RESULTS FROM SELECTED LITERATURE REPORTS

van?

0.037

vg/kg

3,400

12,900
18,000

9,300

Reference

79,180,181

1982

183,184

185

187

188

189

190

m



During PAH bioaccumulation exposures, 14-20 fathead minnows were assayed
for MFO activity. After rinsing with lake water, the fish were minced with
scissors, homogenized, and centrifuged as described above. The microsomes
from these whole fish homogenates were then used for AHH assays.

Enzyme Assays--

Aryl hydrocarbon hydroxy] age, {AHH) and aniline hydroxylase (AH) were
assayed by literature methods!?°> . Aliquots of the microsomal preparation
containing 0.4-2 mg protein were added to incubation vials (without substrate)
and frozen for 24-48 hr until analysis. The incubation mixture for both
assays (total vol 0.9 m2) contained 50 yM TES {N-tris (hydroxymethyl) methy1-
2-aminoethanesulfonic acid} buffer, pH 7.50; 0.6 mg NADPH (sigma, Type I);

3 uM MgClo, and 200 ul microsomal suspension.

The assays were initiated by adding 100 y£ substrate: 100 nM B(a)P in
acetone and 10 uM aniline-HC1. Incubations were carried out ina shakjng
water bath under dimmed lights. The water bath temperature was 28.5°C
and the incubation times were 30 min (AHH) and 20 min (AH). Each sample was
measured in duplicate and blanks were run with each set of assays. Hydroxy-
lated B(a)P was measured at 396 nm excitation and 522 nm emission on an
Aminco-Bowman spectrophotofluorometer calibrated against 3-hydroxy B(a)P (re-
ceived through the courtesy of the National Institutes of Health.) For AH
determinations the p-aminophenol content was measured at 630 nm on a Beckman
DB-G spectrophotometer equipped with a recorder scale expander. Sublimed
p-aminophenol was used as the standard.

With the remaining microsomal preparation, cytochrome P-450 concentration
was determined after diluting with 25% glycerol in 0.1 M TES buffer, pH 7.40.
he CO difference spectrum was measured on a Beckman DB>Gspectrophotometer

Protein was determined using a microbiuret method'’*. Crystalline
bovine serum albumen was used as the standard. On most samples, only one
Measurement was made of P-450 concentration on each sample day with repli-
cates generally within 15%.

Tissue Analyses (PAH Exposures: injection and water uptake) --

After the livers were removed for MFO assays, the fish were gutted
(kidney remaining), and rinsed with Lake Superior water, methanol, and
finally water. Fish from each exposure tank were combined and ground tho-
roughly in a blender. The homogenized fish were stored in 8-0z glass jars
with foil-lined lids at -20°C. Ten grams of tissue from fish exposed to
B(a)P (by injection and in water) were extracted and analyzed as described
under Section 5. Fish exposed to pyrene and fluoranthene were extracted and
analyzed similarly to fish exposed to coal leachate and distillate (see Sec-
tion 4.)

Water Analysis (PAH Exposures of Rainbow Trout)--
100 mé methylene chloride(pyr/fl) or hexane {B(a)P} was placed into a

2 liter volumetric flask followed by 1900 m2 of tank water which had been
filtered through a Gelman glass fiber filter (0.2-10 uM). A 13s" Teflon-

41


OL

{ctp] [PAH]
Starting PAK mg/k ng/k

Fluoranthene 17.7 824

Fluoranthene 23.9 239

Footnotes for Table 19:

Reaction
Time, hr

3.0

3.0

Table 19 (Continued)

pH
4.1

Products
Identified
(% Yield)

Fluoranthene
Fluoranthene
chlorohydrin

Fluoranthene
4243)
Monochloro-

fluoranthene
(321)

Basis
for
Assignment

a,byc

See
comments.

boc

bic

Comments

(Y= 202
Appears to lose water readily to
form a monochloro fluoranthenc.

GC retention time identjcal to com-
pound 6 (Table 20). 236,238.
However, reverse ate | HPLC reten-
tion time was much Tess than com-
pound 6 (Table 20).

GC and HPLC retention times are
identical to compound 6 (Table 20

Mass spectral data. bec retention time was identical to an authentic standard. ‘HPLC retention time was identical to an
authentic standard. “Not corrected for recovery efficiency.


207
DICHLOROFLUORANTHENE

7

SHE LN DERE

>>DATEC 4 7SPE

sun
24

a4

BASE
29644

fF 10

MAPA cera cue

——

Varian CH5

70ev
GC

i ceae SHemeer Sry
Snoneoeeam

voousroacn
sures

TUMG SKS TN
UMM AZIrOme
OT OUOM 25th
susan =

SUMT NR ane
yrrsrrrrrn

OF DORSS Hn OHA e GR CH Ree ee Re
© : epreenrererees: ae :

COUPE ENGL ININMTSOSSHANONTANOOTOMERNATI AT.
SER KARA RE EDS OFT ISO a RR NAUMAME FAR RNOSAOTS

eo a et te oe UU

nmos
MMM oe OCSOREFRUCMORN TOROS ONSSM
ESSTUUS ove OMURU SRNR DO OuAl
SYSh ars manna

PSR OMOMOOUNSUUAMOW AUR Tmo

MOMMOR ET MUMS MOR MOURN UT LIM

Bea cumce De or EUOECLUTNOROEMUMOMOm Eh

HR OO Ree Ur NSARM aM FU USO MEM
- = =u

MAUAZ MON ME SAUMFNIR OT CMM NON AOMUMTNONOAS
c RAR UUUUAUAUMAMM MMMM mm

i

Electron Energy
Inlet System:
Instrument:
WS

} About ti

q
4
wed

704
605
50

132

100 150 200 eso

DERL 77207 DICHLOROFLUORANTHENE

50

1%

IvB/s

STEP NASS?1,

e41 in

SPEC


LE4ZLINZDERL 77176 2-CHLOROFLUORENE

sun
225996

BASE
32736

>>DATEC< “SPEC
PEA

10
C)

rey

o
2 EL
fs) =
i= —_
Oo
So % E
oo E-
no > =
s E
B E-
Ss E
26 .. FE
uw PrP
eS Ss —
Soe =
Bet
ovo
oo wu
- ce
oa oe

gu saqons~asrnamenonaosognsconmene
Scans anddMUsndedsamuunarnamsnesaa

ERARRRGDODOVSAIIOT CaM MMMMODODOLOSS
Ramee UO

Udde ne NNN NNN MN NN NN NM NNANN NNN
QOCMM AIBA TMADMUM ING TUTOTMATONT SA
EAANGARFRGERASS TABOO MS TAMER DOOM
PSU MVSGRGOOORSVHOR CANTO SUOANASS
Aeneid == a Surear =

OOR OT FOOT DVTT OM DNODONA DOUMUUUD OM UT
SOOT OFT RAR APOIO TAOTMMAUNUNDTORNMN UNE
ARR Ss MOTT URDINS DOMURUOR MAOPOTRMT 0

VAT AUSSSOAMAUGAMM UUM AAEM UM]M UG T UT

MAUS OPOSUMOR GOUT VORMTONDUMTNONT OH
Sanaa cendAdMUMAMMMs ser T Ti

Weak ote

ay aaa Ea a a a
eo © © © & B&B 8
7, 8 RF OD HR F mM

104

125

300

1%

eso

IvB/s

STEP MASS=1,

100 150 200

114 LM DERL 77176 2-CHLOROFLUGRENE

50

SPECE


Extensive studies68,71,76 on the metabolism of the carcinogen benzo(a)-
pyrene indicate that the diastereomeric 7,8-diol-9,10-epoxides may be the
"ultimate carcinogens." Covalent bonding of these epoxides with nucleic
acids occurs primarily with the 2-amino group of guanine68,71 (Scheme 1) as
well as the 6-amino group of adenine,/! and the 3-N, 4-amino, or 2-oxo group
of cytosine.7]1 In addition, the reaction of these epoxides with phospho-
triester linkages is known to cause strand scission in DNA68;,86 (Scheme 1).
However, final conclusions about the exact mechanism of PAH produced muta-
genicity and carcinogenicity cannot be made at present. 68

POINTS OF ENVIRONMENTAL ENTRY OF COAL-RELATED ORGANIC COMPOUNDS

PAH's and other coal-related compounds may enter the environment at a
number of stages along the coal utilization sequence from the initial mining
operations to the final waste disposal. Mining operations and proposed
underground gasification techniques8/»88 may introduce. significant amounts of
inorganic??> 0 and organic pollutants including PAH's91,92 into ground and
surface water. Transport of the coal from the mines to the site of utiliza-
tion by proposed slurry pipelines might also be a problem.93,94 Storage,
which may require up to 50 acres at a conversion site,9° may introduce
pollutants into the environment through dusting emission9® and runoff of
particulates and dissolved materials.99-98

Aqueous effluents result from the usage of large quantities of water in
many parts of coal conversion processes including pre-treatment of coal,
gas scrubbing, cooling, and quenching.98 The principal types of organic
materials in these effluents are phenols and nitrogen containing heterocyclic
aromatic compounds but mono- and polycyclic aromatic hydrocarbons may also be
present 30937540599, 160 poryey y y

Airborne emissions from coal combustion and conversion processes are
believed to be the major source of ariborne polycyclic and heterocyclic
aromatic hydrocarbons%9:!01-108 jn the atmosphere (see Table 9). These
emissions have been linked with an increased incidence of human cancer.
Moreover, filtrates of respirable coal fly ash have been shown to be
mutagenic to Salmonella typhimurium. 109

Finally, the products of coal conversion (i.e., gasification, liquifica-
tion, etc.) which may contain varying amounts and types of polycyclic com-
pounds, phenols, arylamjnes, and alkanes may also present environmental and
health hazards. 10s 10051 10-121

PRESENT WORK

The purpose of the present project was to determine the identity,
quantity, and environmental implications of organic compounds liberated from
Northern Great Plains coal during storage at facilities such as the one
located in Superior, Wisconsin. At this facility coal, transported to
Superior by rail from the mines in Montana, is stored in an open-air holding
prior to loading on to large lake freighters.


877LN-DERL 77181 DICHLORONETHYLNAPHTHALENE

>DATE<<-SPECO

s °
E
wo F
x=
2 F
° Fo
5 F
> -
vo = =
on
~ o> e
F
pecceucces F
neooencene >
Reusrnecds 2s
rggexngeds  £
RESRSGeacs «2
E23 :
«$5 EB
on = E
x =
Beez E
=o oe a
agenzecras 3 2 8 -
APS BEMARAS ae
Usenconeen GF SOS +
comm erm ——_
Rmrar corns —_
Roe Ecos ae F
UaaRASenan
Sekok. b heeks bod hel =
gueras 3
onumnonane _af
exummesgas :
7
Qundaunaccans~-ooressesoecoss macone _—
Came TUUURUUSORANODENGOUAAMTUGURDOCSFS=M a
ERARRRARR@DODHDOOHDVMDFT AO Mae UMMMMS FUN —
=
a
uae ene ese ay seg gest eae avg aa sane 8¢ 2563503082 =
wg a tac ge eae SERRATE NG =
CORRE Ep LEE ort aT erate t entre ntti 7

SONU CNUs UNS KCURS HOUMeoUSHuas Cuno
=m sae Ua FU

a
Nin
Hea i

FUNG AR DAM TH ORG VOR DOPUF TUNA TDA RGUEMNDT
BAGH PUGS GANT UUMMOFOARATOMMORAMAUMONMOD|Y
RMFASMFAUSPOMIOD AMARA AUD HOM OMT ANA TOM
Lama ener ae Pana Daren mee MUM emer rm Or incu

MEMS RRO KNORR OUMT NDA. SRAUDEMMTNROMT NON MS &
c aN TTT TONY OOD

127

300

200

156

Tress

STEP NASS<1-

DICHLOROMETHYLNAPHTHALENE

87 LN DERL 77181

SPECS


among the fish, different rates of metabolism, and injection variations.

In the water uptake experiment (Table 10), exposure of trout to 0.4 ug/1
B(a)P resulted in accumulation of 368 g/kg B(a)P after ten days. Table 10
also shows the corresponding enzyme data, which demonstrated induction of all
three m0 parameters ay that time. Trout yp nae’ 73 ug/kg B(a)P showed no

Induction after a 10-day exposure to B(a)P plus coal particles {low levels
of B(a)P (<0.1 yg/2)}. is

To determine whether there was a correspondence between accumulated
B(a)P and MFO induction, the B(a)P analyses from the injection experiment
(Table 2) and from the water uptake experiment (Table 10) were compared to
their corresponding MFO measurements (Figure 9, Table 10). From the combined
results (Table 12), it appears that although there was one exception (i.e., a
lack of AHH induction at 350 ug/kg), B(a)P levels above 300 ug/kg were accom-
panied by induced MFO activity.

Discussion
Mixed- function Oxidase Induction by Various Polycyclic Aromatic Hydrocarbons--

The MFO system in rainbow trout was found to be inducible only by certain
compounds.

Of the compounds tested, chrysene, B(a)P, and Aroclor 1254 are classified
as carcinogenic by NIOSH“'®. The injection experiments showed that P-450 and
the hydroxylases responded to these compounds. The PAH water uptake experi-
ments also indicated a degree of specificity of the MFO system since accumu-
lated B(a)P caused induction, but tissue levels of pyrene and fluoranthene
(combined) did not elicit such a response. This lends some support to the
rationale behind using MFO levels, such as AHH, as indicators of environmental
exposure to carcinogens. The situation is confounded, however, because of
possible synergistic interactions among the wide array of watersoluble organic
compounds. For example, the long chain hydrocarbon n-dodecane great} en-
hanced the carcinogenic potency of B(a)P in mice skin tumor studies Also
some compounds, such as ibs synthetic 8-naphthoflavone, cause MFO induction
but are not carcinogenic 0,

Short term injection experiments have shown to be a rapid means of
screening PAH's for MFO inducibility. However, even with pooled samples,
large variations can occur, and the need for adequate sampling is recognized.
Additionally, the physiologically abrupt absorption of compounds from the
peritoneal cavity may augment the MFO response, For example, injection of
pyrene caused P-450 elevation, yet throughout the 21-day water exposure to
pyrene (with fluoranthene), there was no P-450 induction. The relatively low
bioaccumulation potential of pyrene is demonstrated by the accumulation of
only 281 ug/kg after 21 days. This may explain the lack of MFO rgsponse and
points out the importance of determining bioconcentration factors and de-
puration rates when assessing possible carcinogenic impact. Therefore, it
appears that through the use of i.p. injections for initial screening coupled
with bioaccumulation exposures, discrete compounds and components of complex
mixtures could be tested for relative MFO inducibility in fishes.

45


ABSTRACT

. The effects of leaching processes upon Western Great Plains coal were
Investigated to ascertain the potential impact of the organic components on
aquatic organisms. Acute and chronic toxicity testing indicated no criti-
cally adverse effects, which led to a subsequent detailed study on the
lipophilic fraction containing polynuclear aromatic hydrocarbons (PAH) that
might be anticipated to bioaccumulate. HPLC-GC analysis of coal leachate
indicated that the PAH content was of a comparable concentration to samples
obtained from Lake Superior. GC-MS analysis of the lipophilic materials that
are adsorbed on the coal particulates indicated that they were predominantly
low molecular weight PAH's (i.e. naphthalenes, phenanthrenes, anthracenes,
etc.), alkanes, and heterocycles. Synthetic methodology was developed to
provide standard samples of alkylated PAH's of the type observed during the
analysis.

The biological studies on PAH's were aided by the use of a combined
HPLC-GC analysis procedure (ng/2 detection level) developed specifically for
this program. The biological investigation resulted in obtaining bioaccumu-
lation factors in the range of 1000-5000 for several PAH's and noting that
the PAH's that induced mixed-function oxidase (MFO) activity in rainbow trout
were those that showed similar effects in mammalian systems.

Selected PAH's of various structural types were also shown to be quite
susceptible to chemical transformations during conditions typical to
chlorine disinfection. The chlorination products that were observed during
the course of this investigation were mixtures of chloro- and oxygenated
derivatives.

This report was submitted in fulfillment of Grant # R803952 by the
University of Minnesota, Duluth, under the sponsorship of the Environmental
Protection Agency. Work was completed as of July 1, 1978.

iv


eg

‘ '

‘)

OO”

CH, Os

: eee

—————



BIOCONCENTRATION FACTOR

FIGURE 14
SUMMARY OF EXPERIMENT
WITH SIX PAH COMPOUNDS®

7000-
6000,
o———+ DIBENZOFURAN
e-= He -METHYLPHENANTHRENE
oe ~PYRENE
L o— ——e FLUORENE
5000 PHENANTHRENE
FLUORANTHENE
4000+
3000}
2000}-
1000F
soe te ee gg se tg te eg ge gg
0 2 4 6 8 10 12 14 16 18 20 22 24 26 2a 30 32

TIME (days)

"Table 15, Exp. #3, Tank #3.

62


TABLE 2
STEAM DISTILLATES OF COAL
RESULTS OF GC/MS ANALYSIS

HPLC Fraction No. Mass Spectrum No.
Other Distillate Distillate Distillate? Distillate?
Compound a* Tons with KOH without KOH with KOH without KOH
alkanes, alkylbenzenes F6, F7, F8
Cg-biphenyT 182 167 FQ 332
2 naphthatenes 186 141 F9 260
156 14i Fo 272
156 141 &9 282
156 141 Fg 287
156 141 Fo 293
156 141 Fo 309
3 naphthalenes 170 155 Fa uz
170 155 Fe 120
170 155 Fs 125
170 155 Fe 132
170 155 F-10 283
170 155 F-10 293
70 185 F-10 299
170 185 F-10 303
170 19S F-10 309
170 185 F-10 314
170 155 F-10 322
170 155 F-10 331
Cq-naphthalenes 184 169 F9, 10 136
184 169 F9, 10 141
eg-benzene 148 133;,105,91 F-10 209
n-dodecane 170 85,71 F-10 212
cyclododecane 168 97 ,83,69 F-10 230
Cg-benzene 204 189,147 F-10 250
Co-hexahydrophenanthrene? 212 197 F-10 341
methylphenanthrene/anthra~ 192 191 F-10 48
cene 192 151 F-10 431
methy!fluoranthene/pyrene 216 201 F-13 169
Sg 256 «= 224,192,160 Fa14 182

128,96

6c conditions--column: 2 mmx 12' with 5% SP-2250 on 100/120 Gas Chrom Q; program: 80-225°C @ 2°/min.

Mass spec: start scan after 2 min; scan: 50 to 300 amu @ 7.5 sec/decade,

bee conditions--column: 1/8" x 6' with 3% OY-101 on 80/100 Gas Chrom Q; program: initial hold 1 min, 80 to
225°C @ 4°/min. Mass spec: start scan after 2 min; scan: 50 to 300 amu @ 7.5 sec/decade.

W7


FIGURE 12
SUMMARY OF PHENANTHRENE

EXPERIMENTS®
7000;
PHENANTHRENE
6000
o————~ PHEN ALONE (EXP.2)
o-----« PHEN ALONE (EXP.3)
” e————e PHEN +BETA-NAPHTHOFLAVONE (EXP. 2) b
% 5000  -————+ PHEN+9-CHLOROPHENANTHRENE (EXP.3)
fe escent PHEN+5PAH’S (EXP.3)
Vv
<
rs
z 4000}
Q
=
<
=
5 3000}
&
Vv
2
g -
@ 2000- 2 4
ro OO Le een sF
et ee eo - ew
1O00F
Nene
Fe ee Se Se Se SO RS SOUR SN SO SS
o 2 4 6 8 W 12 4 1 18 20 22 24 26 28 30 32
TIME (days)

aBased on data in Table 15. be xp, #25 no other compounds present.

CExp, #3, Tank #2, no other compounds present. Fexp, #3, Tank #1;
9-chlorophenanthrene also present. "Exp. #2, Tank #1; B-naphthoflavone
also present. fexp. #3, Tank #3; dibenzofuran, fluorene, 1-methy1-
phenanthrene, fluoranthene, and pyrene also present.

60


9EL

ocho
1000
500
250

io

25

THRESH(CM) =
OFFSETCHZ)=
RF FROCMHZ)=

NO.

SOUS ON=“Svarvauson=

INT.
19
Ss
5

8

7
123
209
87
17
108

108
28
23

96
136

@.5
139.5
25.1997
OFFSETCHZ)
3654.1
3606-5
354961
353951
324161
3204.8
3202.8
316124
315463
304401
3039-6
302461
197461
194263
191863
92761
2.0

BC

PPM
145.018
1436121
140.843
140 2446
132.589

127.182
127.099
125-458
125-168
120.803
1204624
120 869
78.341
77.081
15.887
36.792
8.080



Other methods for the bioconcentration factor derivation include in-
direct approaches based on pharmacokinetic models22! 232,239 or estimates
based on apt ition coefficients22!,232,240, In studies involving measure-
ments229,229,241, 3y- orl4c-jabeled compounds are most often employed and meta-

bolites are usually, but not always¢42, separated from the parent compound
prior to radioactivity determination.

For the present study the direct measurement approach was employed in
which the concentration of a polycyclic aromatic hydrocarbon in the exposure
water and the fish tissue was measured by HPLC-GC procedures (Section 5) over
a four week uptake period and a one week depuration period. The levels of
activity of the mixed function oxidases in the fish were also monitored.

Experimental Details

Aquaria--

Forty-liter glass tanks were equipped with a Masterflex pump (Model 7015,
Cole-Parmer) to deliver 210 mé/min of Lake Superior water and an FMI (Fluid
Metering, Inc.) laboratory pump to deliver 2.1 m/min of an aqueous methanol
solution of polynuclear aromatic hydrocarbons. The tank volume of 36 2 water,
which contained ~10 u£ methanol per liter of water, was turned over every
3.2 hr. The temperature was maintained at 24+1°C and the photoperiod was
16:8. Tanks were cleaned daily.

Fish--

The fish used for these studies were five- to six-week-old fathead min-
nows which were reared in Lake Superior water at the United State Environmen-
tal Protection Agency Environmental Research Laboratory, Duluth, Minnesota.
They were fed a maintenance diet of 12% of their body weight of #1 pellets
(Zeigler Bros.) and live brine shrimp nauplii daily.

Procedure--

The fish (250-300) were exposed to lake water containing PAH for 28 days
and then to only lake water for an additional five days. Sixteen to twenty
fish samples were removed on various days during the experiment, rinsed with
Lake Superior water, and blotted dry on an absorbent towel. Analysis of the
PAH content of the fish then proceeded as described in the analytical section
(Section 5) using the Styragel-HPLC/GC-PID procedure. Determination of the
mixed-function oxidase (MFO) activity of whole fish homogenates was carried
out on days 7, 14, and 28, using methods described in the mixed- function oxi-
dase discussion (Section 6). One to four liters of tank water was also ana-
lyzed on various days as described in Section 5 using the "pre-column" con-
centration/pre-column coupled-reverse phase HPLC/GC-PID procedure.

The analysis of the B-naphthoflavone in water and fish tissue in the pre-
sence of phenanthrene (experiment #2) required some modifications of the
original analytical techniques because this compound could not be determined
by GC. For the analysis in water, the usual pre-column concentration/pre-
column coupled reverse phase HPLC procedure was employed. The ultraviolet

53


FIGURE 1
LAKE WATER LEACHATE LIQUID CHROMATOGRAMS

KNOWN COMPOUNDS
(240 ng/ compound)
a eee
a
4
3}
na
a
3 LAKE WATER BLANK
uw
° (14.5.1)
=
&
c
t
3
n
&
8
z
Fs
c
3 LAKE WATER LEACHATE
<
s (14.2 L)
)

TIME (minutes)

Conditions-column: 5y Lichrosorb RP-18, 4.6 x 250 mm; program: 59-90% CH3CN in
water, 1.2 ml/min, 30 min (linear program). Compounds in standard-21.86
min: dibenzofuran; 23.23: fluorene; 24.92: phenanthrene; 28.67: 1-methy1-
phenanthrene, fluoranthene; 29.73: pyrene; 31.56: 9-chlorophenanthrene.

10


The results of the PAH recovery determinations (Table 7) indicate that
sub-micron filters such as the Whatman GF/F (effective retention of 0.7 mi-
cron) which are often part of the analysis procedure have little effect on
the recovery of PAH's from water. In contrast, the Whatman GF/B filter
(effective retention of 1.0 micron) did lower the recovery of several PAH's
in the one experiment in which it was used.

The HNU Systems photoionization GC detector!57 used in this work is at
least ten to forty times more sensitive to PAH's (lower limit of detection
0.05 to 0.1 ng) than a flame ionization detector (FID) and, in contrast to
the FID, shows little response to water or acetonitrile. The lack of a sig-
nificant solvent peak allows the use of isothermal GC conditions for many
experiments, which results in greater reproducibility of retention times and
amore stable baseline. This detector, which has a maximum temperature limit
of 250°C, gradually becomes fogged which results in a decrease in sensitivity.
In order to regain maximum sensitivity it is necessary to remove the lamp
for cleaning with an abrasive cleaner provided by the manufacturer. The
linearity and reproducibility varied somewhat!57 as is reflected by the va-
riations in the errors given in Table 7.

EXPERIMENTAL DETAILS
Apparatus--

Stainless steel pressure tanks (Amicon, Model RS20) with internal volume
of 19.5 2 were pressurized up to 125 psi with cylinders of Linde high purity
nitrogen which was pre-purified via an in-line 7 x 50 mm PorapakR QS column.
Stainless steel and brass were used for all fittings and lines.

Stainless steel filter holders (Gelman part number 2220, 47 diameter)
were used to hold Whatman or Schleicher and Schuell (S&S) glass microfiber
filters.

Stainless-steel HPLC columns (7 x 50 mm) were dry-packed with Bondapak
C-18 CorasilR II or PorasilR B (37-50 u) (Waters Associates).

The HPLC apparatus was manufactured by Waters Associates and consisted
of the following items: two M-6000 pumps, a model 660 solvent programmer,
a model U6K injector, a model 440 dual channel UV detector equipped to moni-
tor 254 and 280 nm, and a 3.9 x 100 mm 10 u C-18 reverse phase column (or a
4.6 x 250 mm 5 p C-18 Lichrosorb column manufactured by Altex Associates).

SwagelokR stainless steel quick-connect fittings for 1/16 inch tubing
were used on the pressure tanks, the 7 x 50 mm HPLC columns, and the micro~
packed analytical HPLC column.

The GC apparatus consisted of a Tracor 550 chromatograph equipped with
an HNU Systems photoionization detector (model PI-51 max temp = 2509). The
GC inlet was modified by welding a Waters Associates HPLC septum inlet onto
the front of the Tracor septum nut in order to move the location of the sep-
tum (Hewlett-Packard, part number 5080-6721) about 3.5 cm away’from the 300°
inlet tube. The PID was connected to the Tracor outlet via a 1/16-inch

27


fel

SPECco

Fy

ch,

cH,

| ‘lL |

a i i ll
100
40 LM DERL 77178 UND 1,3-DINETHYLNAPHTHALENE

250

STEP NASS*1,

IvBss



TABLE 11
TISSUE ANALYSES FOR DOSE-RESPONSE EXPERIMENT?

Approximate Injected Dose B(a)P Concentration In
(ug/kg) Tissue (ug/kg)

Day 3 Day 5

30,000 3260250 458231

3,000 320254 537265

300 249433 350421

30 130427 140214

3 <30 <30

athe B(a)P doses indicated were injected i.p. in peanut oi] and tissue concen-
trations measured 3 and 5 days later in 2-3 gutted fish (pooled). The mini-
mum detectable 8(a)P level was 30 ug/kg.

47


FIGURE 3
PURIFIED WATER LEACHATE (CENTRIFUGED) LIQUID CHROMATOGRAMS

KNOWN COMPOUNDS
(200 ng/compound )

a NIN
z
oO
7)
4
a
=)
ne
2
= BLANK (7L)
€
c
3
a
& | pe
Ww
5)
z
Fs
5
g LEACHATE (15.7L)
<
s |i.
=)

T r 1 \ T

o 0 20 2 40 50

TIME (minutes)

Conditions-column: 5y Lichrosorb RP-18, 4.6 x 250 mm; program: 50-90% CH3CN in
water, 1.2 ml/min, 30 min (linear program). Compounds in standard- 20.23
min: dibenzofuran; 21.61: fluorene; 23.31: phenanthrene; 27.20: 1-methy1-
phenanthrene, fluoranthene; 28.28: pyrene; 30.19: 9-chlorophenanthrene.
Peak at 37.38 min in leachate was analyzed by SC/MS but nothing was found.

13


vel

APPENDIX D

CHLORINATED POLYNUCLEAR AROMATIC HYDROCARBONS : 13¢ and Wy (100 MHz) NMR DATA
| THRESH(CM) = 1.8 3¢ Fe ee
500 OFFSET(HZ)= 150.4 Pot | | ! “|
KE FRU CMHZ)= 25.1997 honk oh. | | i
250 (NO- INT. OFFSET CHZ) PPM i H j Z |
1 14 3329.5 132.131 at ‘ i _ 4 t
2 10 3285.8 130.394 ~ : 7
100 3 96 3240-5 128.597 : |
4 98 3198.6 126.934 ie foci
50 5 201 3188.7 126.541 ' i
| 6 112 3092.0 122.706 H j-
25 7 12 0.8 9.000 i
i
|
i

7
|
-|
i
7
i

aa



TABLE 21
SYNTHESIS OF POLYMETHYLEPOXYNAPHTHALENES

1, RBH RSF
4, Ry = Hs Ry =
5, Ry =H, Ry =
6, Ry =H, Ry =
7, Ry = CHy, R
B, Ry = CH, Rp

80

2 =

H, R
H, R
CHe,
CHs,

=H,


FIGURE 7
TYPICAL FRACTIONATION OF FISH
TISSUE EXTRACT CONTAINING PAH MATERIAL
USING HPLC-STYRAGEL

(1.0 FULL SCALE)

UV ABSORBANCE AT 254nm

a |

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

TIME (minutes)

Sample: Methylene chloride extract of twelve fathead minnows (1.2738
gram total). Compounds: fluorene (fractions 1,2; 3.6 mg/kg fish),
dibenzofuran ( fractions 2,3; 3.8 mg/fish), phenanthrene (fractions
2,3; 4.9 mg/kg fish), 1-methylphenanthrene (fractions 2,3; 1.7 mg/kg
fish), fluoranthene (fractions 2,3; 2.6 mg/kg fish), pyrene (fraction
3; 0.69 mg/kg fish).

34


We have developed the procedure to the point where up four methyls may
be introduced on the epoxynaphthalene systems at those positions indicated
by the arrows.

\ i“
Ons

The reaction is illustrated below for the selective synthesis of 1,2,4-
trimethyl naphthalene.

a) Sa" Oy

2,5-dimethyl furan

CHA
i H
CHa 2° . BF 5. (CoHg) 0
oS UO 3 Tho
THF 2)
CH,
(of configuration shown) CH
oa
CH,
2

75


es

TABLE 14

TABULATION OF LEVELS OF VARIOUS AROMATIC COMPOUNDS IN AQUATIC ORGANISMS IN LABORATORY EXPERIMENTS
RESULTS FROM SELECTED LITERATURE REPORTS
H20 Conc. Bioconc. Factor

Compound

anthracene®
naphthalene
naphthalene
naphthalene’
phenanthrene
chrysene d
benzo(a)pyrgne
naphthalene d
J-methyI naphthalene
naphthalene d
J-methyInaphthaleneg
2-methyInaphthalene
di-methyInaphthalene
tri-methylnaphthalene
benzo(a)pyrene
benzo(a)pyrene®
benzo(a)pyrene:
benzo(a)pyrene
bipheny?
Aroclor 1254
Aroclor 1234
heptachlor e
trans-Chlordgne
Aroclor 1254)

Aroclor 1254 e
heptachloronorborgene
pentachlorophenol

f
f

pentachloroanisole®

p-dichlorobenzene®

2,21,4,4!-tetrg-
chlorobipheny]
gioconcentration factor

Value calculated for data in article

Organism

Daphi ulex
polychaete
polychaete
clam

clam

clam

clam
sheepshead minnow
sheepshead minnow
clam

clam

clam

clam

clam

clam

mosquito larva
snail

mosquito fish
rainbow trout
cockle

tellin

spot

spot

spot

spot

fathead minnow
rainbow trout

rainbow trout

rainbow trout

rainbow trout

(concentratign of compoun:
Beatie, Mc 4

Conc. In
Organism
(mg/kg)
0.015*
5.5
x42
0.43+0.01
2.821.1
0.5440.3
0.45+0.1

static

Tissue Analyzed

whole organism
whole-body males
whole-body feniales
whole organism(shucked)
whole organism(shucked)
whole organism(shucked)
whole organism(shucked)
whole organism ?

whole organism(shucked)
whole organism(shucked)
whole organism(shucked)
whole organism(shucked)
whole organism(shucked)
whole organism(shucked)
whole organism

whole organism

whole organism

muscle

whole organism(shucked)
whole organism(shucked)
whole organism

whole organism

whole organism

whole organism

whole organism

liver

blood

fat

muscle

liver

blood

fat

muscle

muscle

muscle

Flow through

* a

(age) (HP®)

0.02 760 (24 hrs)
20 275*(20 hrs)
27 156*(20 hrs)
1 6.1 (24 hrs)
89 32.0 (24 hrs)
66 8.2 (24 hrs)
52 8.7 (24 brs)
1000 60*( 4 hrs)
1000 205*( 4 hrs)
840 2.3 (24 hrs)
340 8.5 (24 hrs)
480 8.1 (24 hrs)
240 17.1 (24 brs)

30 26.7 (24 hrs)

30.5 236 (24 hrs)

2.5 2177 ( 3 days)

2.5 37 (3 days)

2.5 ( 3 days)
438238 (=)

250 163 (40 days)
250 138 (40 days)
0.14 = 2,200 (72 hrs)
0.06 3,300 (72 hrs)

1 27,000 (56 days)

40 11,200 (30 days)

26 615 (24 hrs)

26 250 (24 hrs)

26 231 (24 hrs)

26 39 (24 hrs)

24 133 (12 hrs)

24 47 (12 hrs)

24 3,542 (12 hrs)

24 96 (12 hrs)
1.60.2 215421 (=}

9850#2890 (@)

Reference

241
253

45
254

46

225

240
255

256
257
258
259

259

240
239

in fishin ug/kg)/(congentration of compound in water in yg/kg)
renewed static


The only real synthetic limitation to this route, in addition to the
availability of suitably substituted benzynes and furans, is the production
of 2 isomeric products with the attack of methyllithium on an unsymmetrical
epoxynaphthalene. This is illustrated below for the production of 2,6- and
2,7-dimethylnaphthalenes. This offers a limited separation problem for the
isolation of pure individual isomers but nevertheless is amenable to a GC/MS
analysis.

Ol. 6 = ob

CHLi CHALi
OH chy
CH, CH
CH
H 3
, OG)
H
(CoH, ) iW
BF.,.(C,H,),0 -H,0
3°h2"'5/2 | 2 | -ii,0
CH3 CH, CH,
CH
12 12!
Results

The synthetic scheme discussed above has been applied to the syntheses
of the methylepoxynaphthalenes listed in Table 21. The resulting methy1-
naphthalenes resulting from the interaction with methyllithium are listed in

76


CONDUCTIVITY (wmhos/em)

35r

30-

25F

20r

iF

10

FIGURE A-1
CONDUCTIVITY CHANGES DURING LEACHING
OF coaL?

1. 1 ..

3
25 50 75 too
TIME (hr)

30081 leaching was monitored after adding ground coal (<0.5 mm) to
distilled deionized water at a coal to water ratio of 6.3 9/1.

1


FIGURE A-4
GAS CHROMATOGRAPHIC ANALYSIS OF AN
ISOOCTANE EXTRACT OF COAL DISTILLATE

BLANK

FLD. RESPONSE

COAL DISTILLATE

FLD. RESPONSE

o 5 15 20°25 30—*S SCC OOS OS
TIME (minutes)

A steam distillation of 1500 ml distilled deionized was carried out in a
modified Nielsen-Kruger distillation apparatus for 6 hr.

Das above except that 100 g of coal (<0.250 mm) were added.
GC conditions-column: 5% SP-2250, 2m x 2mm i.d. glass; injector: 250°C;
detector: 300°C; carrier flow: 20 ml/min; program: 80-250°C at 4°/min.

114


l2

TABLE 5

SUMMARY OF BIOLOGICAL TESTING
OF COAL LEACHATE.*

Concentration Organism __Age at Onset Duration of Exp. Type of Test Bioassay Conditons Response
100% fathead 5-15 days 3 weeks Toxicity Renewed Static 15% mortality
(6.3g/1 DDW) minnow not significantly
greater than
controls
100% fathead 235 mo 24 weeks Toxicity Renewed Static No mortality with
(6.3g/1 DDW) centrifuged leachate
100% fathead 235 mo 96 hours Toxicity Static 100% mortality
Uncentrifuged minnow
(25g/1 DDW)
100% fathead 2 mo 24 weeks Sublethal Renewed Static Growth rate simi~
(6.3g/1 DDW) minnow Growth lar to that in
Bioaccumula- Lake Superior
tion water but onset
of maturity de~
layed. Some
qualitative
differences in
GC analyses of
tissue extracts
10-1002 fathead al yr 2 or 4 weeks Sublethal: Renewed Static 36% spawning suc-
(6.3g/1 LW) minnow Spawning cess in leachate
exposures.
90% spawning suc-
cess in control
exposures.
100% rainbow lyr 28 days Sublethal: Renewed Static No consistent
(6.3g/1 LW) trout AHH Response differences from

rhe ratio of coal to water and the type of water used to prepare leachates (100%) are indicated.
deionized water, LW = Lake Superior Water

Liver Para-
meters

controls

DDW = distilled


9tL

CH.

|

106 150 200 250 300
SPEC 43 LM UMD DERL 77177 1,4-DINETHYLNAPHTHALENE STEP MASS*#1, IvB/7S © 1%


TABLES

Number

1

10

i
12

13

14

15

16
7
18
19
20

Water Chemistry Analyses for Leachates.............-
Steam Distillate of Coal: Results of GC/MS Analysis ......

Reported Studies on the Biological Effects of the Water Soluble
Fraction of Petroleum on Fish... 2... 2. eee eee ee ee

Reported Studies on the Biological Effects of Coal-Derived
Mixtures on Aquatic Organisms. . 2... 1 1. ee ee ee ee ee

Summary of Biological Testing of Coal Leachate .........
Summary of Biological Testing of Coal Distillate ........

Determination of PAH Recovery from Aqueous Solutions of Known
Concentrations Using the C-18 Adsorption HPLC-GC Procedure .. .

Summary of Analysis of Spiked Fish Tissue Samples. .......

Identification and Quantitation of Polynuclear Aromatic
Hydrocarbons: Results from Selected Literature Reports. ....

PAH Tissue Concentrations and MFO Measurements During Water
Uptake Experiments ..... ry

Tissue Analyses for Dose-Response Experiment. .........

Relationship Between B(a)P Concentration in Rainbow Trout and
MFO Induction. ©... 6. ee ee ee

Levels of Various Aromatic Compounds in Aquatic Organisms in
the Environment: Results from Selected Literature Reports .. .

Levels of Various Aromatic Compounds in Aquatic Organisms in
Laboratory Experiments: Results from Selected Literature
Reports. ........ tee ne ee ee te ee ee ee

Summary of Bioconcentration Factors (Based on Total Wet Weight
of Fish)... ee

Summary of Fish Analyses (Based on Total Wet Weight of Fish) . .
Summary of Water Analysis. . 2... 6 ee ee ee ee ee ee
AHH Activity In Fathead Minnows Exposed to PAH. ........
Summary of Aqueous Chlorination Studies... ........4.

Monochloro Products Produced by Preparative Scale Chlorination
Reactions in Acetic Acid. 2... 1. ee ee eee ee ee ee

ix

18

19
21
22

28
32

37

44
47

48

51

52

55
56
57
58
68

71


77206 OICHLOROPHENANTHRENE

2077LMN/DERL

>>DATEC</SPECY

10

crmqrlpenfonmpenp] 8

©

=

ro)

<

©

> _

@ S

oo 6

XG >
“oogse
Sener Sy
Senne 3

merern oe
eucuascucucd o£

< Gs

wip

26
<

SOME

s 3

pe cf

ov 6

on-u

-— ‘cc

wes

So maMS NSO ONAN SADTUNGSmAMsrHOSKCSTMOK~S HA
SORRARRRRDOODOETT ODOR AUNUMMAM TT FARRAR Doo
Re tt nt ett me UL

OMA S MUSK OMS Ne
BES ONARGOSRaTMaMMRMMOMMSSNaW MATIN SHS Oe

5a Ka aaa ame mM = UMM a ee
on
iS
e
ROS AMUN OOH A DUOMVANOUMOMGDOSCOUUUNGTTNOTT OS
FOMAe OUT EARMOMUT ONAUKR MMR DODOMMOSUITT OU ON
FOR ROMO OMADUAR OO A DODOOMARMGONTNOF SUMAN OUD
Sande me MO “oo UUMT he
wo
Ou
cm SHUT HON OE OO ee ee eee eT ees runnune
ao SS aM UUUAUMMM MMS TTT TTT TMU S
=o

a

la ro oe

129

oO

150 200 250 300
267 LM DERL 77206 DICHLOROPHENANTHRENE

160

50

1%

eh, I7B7S) «

STEP NASS

SPECa


LEL

J i ' oo
: i \
2500  tHeesnccm= 065 13 : |
OFFSETCHZ)= 14266 Cc Poo . .
1000 RF FkucMHd)= 25.1997 ‘ i 1 '
NO. INT.  UFFSETCHZ) PPM a a A

500: } 8 351767, 139.595 \ i | i
+ 2 1s 3454-6 1372103 rs
3 5 327943 130.138 | be |
250 + 4 163 322661 128.025 '
~ 5 204 32168 127.624 _ 1 .. ~ hoe .
100 6 5 320069 127.025 j '
i 7 168 319363 126.724 i
50 8 160 3064-6 121.618 7 !
9 al BW26604 120.1008
| 10 9 1974.3 76.351 ! : 4 }
25 in 12 194262 77.077 \ : ‘
12 12 190968 15.792 i ! H
43 370 - res = 0.030 |



APPENDIX E
METHYLATED NAPHTHALENES: Yy (60 MHz) DATA

Chemical Shift Data in Delta for the Methy] Groups in the

Compound

10 & 10'

118 11'

12 & 12'

13 & 13!

Polymethylated Naphthalenes

No. of Methyls*

2.50

2.73, 2.65,

2.59

6 Values**

2.56, 2.56, 2.47, 2.43

2.95, 2.95,

2.52, 2.52,

2.57, 2.57,

2.95,

2.52,

2.50,

2.95, 2.81, 2.68, 2.58, 2.45

2.52

2.50, 2.50, 2.50, 2.37, 2.37

* 11 compounds exhibit correct CH3/aromatic ratio by integration.

** Chemical shifts are relative to internal TMS.

143


the source (delayed fallout), will be relatively very
limited. However, where fallout of PAH occurs, it will
contaminate the upper layers of the earth, including
vegetation and forests, as well as rivers, reservoirs

and lakes, and some of them will also reach the oceans.
Runoff and the rivers will carry eventually some of this
fallout to the open seas and oceans. As waste treatment
plants do not remove all PAH, the coastal waters will re-
ceive an additional load from domestic and industrial

waste effluents either directly, or indirectly through

the rivers. Such effluents will also carry PAH coming

with oi] pollution. Some of the PAH, settling on land

and vegetation, are bound to be washed into the soil with
minor amounts eventually reaching ground-water. The open
seas and oceans will also be polluted by PAH from activities
connected with oil transport of tankers, including loading
and unloading, as well as oil spills and accidents at seas."

Polycyclic aromatic hydrocarbons in aquatic systems may be in the dis-
solved statel68,1/77, associated with particulate matter 161 ,168,169,171 and/or
associated with organic matter!61,171. Photooxidation!01,161,172,173, jis
probably one of the principal transformation processes that occurs in water
and atmospheric environments. Particulates may enhance the rate of reaction
173-175, Biological transformation processes_in soil and sediment are also
important, especially for smaller ring PAH's!76,

The levels.of PAH's in the envjronment have also been discussed in
several reviews 01,1 1, T62016851 Ost 78, The results of some recently
published work on the identification and quantitation of PAH in the environ-
ment (tabulated in Table 9) indicate that although levels may vary con-
siderably, PAH material may be found almost anywhere. It has been demonstra-
ted that industrialization has elevated the levels considerably !01,102,162

The present studies that will be discussed are a) the induction of mixed-
function oxidases (MFO) in fish by specific PAH's, b) the bioaccumulation of
PAH in fish and the possible relation between bioaccumulation and MFO induc-
tion, and c) the identification and quantitation of products of aqueous
chlorination reactions of PAH.

36


Number
A-8

A-10

Effect of Coal Leachate on Liver Parameters .......,., 118
Gas Chromatographic Analysis of Fathead Minnows Exposed to

Coal Leachate . 2. 6... ee ee eee 119
Gas Chromatographic Analysis of Rainbow Trout Exposed to 0.2%

Coal Distillate... 1. ee ee 120

viii


FIGURE 6
TYPICAL GAS CHROMATOGRAM OF
PAH MIXTURE USING PHOTOIONIZATION DETECTOR

TOS

UV ABSORBANCE AT 254nm
(1.0 FULL SCALE)

TIME (minutes)

Injection: 1 ul of a 0.2 ng/ul acetonitrile solution of dibenzofuran
(1.08), fluorene (1.29), phenanthrene (2.47), 1-methylphenanthrene
(3.82), 9-chlorophenanthrene (4.67), fluoranthene (6.31), and pyrene
(7.62).

30


TABLE A-6
EFFECT OF COAL DISTILLATE ON HEPATIC MIXED-FUNCTION OXIDASE
PARAMETERS IN RAINBOW TROUT®

Day P-450 (nM/mg protein) AHH (pM/mg protein/min)
Control Distillate Control Distillate

3 0.130 0.108 1.234 0.958
+0.161 40.308
7 0,125 0.097 2.224 1.968
+0.161 40.928
10 0.156 0.033 0.968 1.801
40.315 40.374
14 0.065 0.138 1.305 1.353
+0.041 +0.083

21 0.082 0.089 1.124 2.873
+0.105 +0.351

0.112° 0.093° 1.371% 1.790°
+0.037 +0.038 40.494 40.723
(33%) (41%) (36%) (40%)

Rainbow trout were exposed to 0.2% coal distillate under flow-
through conditions.

bs ignificantly elevated compared to controls.

mean and Standard deviation over the 21 day period.

110


(Section 5). The results are presented in Figure 2 and the water chemistry
analyses are presented in Table 1.

Purified Water Leachate (Centrifuged)--

This leachate was prepared by stirring 10 g of coal (<0.125 mm) per
liter of purified water (see Section 5 for preparation) for 48 hr. It was
then run through a continuous flow centrifuge followed by the usual GF/F--
pre-column--HPLC procedure (Section 5). The results are presented in Figure
3 and the water chemistry analyses are presented in Table 1.

Discussion

The results indicate that aqueous leachates of coal prepared as de-
scribed at ambient temperature contain less than .10-50 ng/f, if any, of
individual polynuclear aromatic hydrocarbons. No PAH compounds were identi-
fied. The results presented in Figures 2 and 3 for purified water leachates,
in contrast to the results in Figure 1 for lake water leachates, indicate the
presence of significant amounts of polar materials (phenols?) not present in
the blanks. Analysis of these polar compounds was not attempted.

VOLATILIZATION FROM COAL--CHEMICAL STUDIES

An open storage coal facility, such as the one in Superior, Wisconsin,
which contains a large amount of high-moisture content coal that is periodi-
cally sprinkled with water to reduce dust liberation, has a high potential
for steam volatilization of organic compounds. In this process, relatively
low molecular weight compounds associated with coal may be volatilized at
temperatures less than 100°c.124 It should be noted that temperatures of
40-80°C above ambient temperature have been recorded at a Yorkshire coal pile
and a number of alkanes and benzenes were found in nearby air. 125

Experimental Details

One hundred grams of coal (<0.125 mm) and 1.5 2 of purified water (see
Section 5) were placed in a distillation apparatus. Additional water was
added during the distillation process until 4 2 of distillate was obtained.
In the second of two experiments, 4 g of KOH was added to the distillation
flask in order to minimize the amount of sulfur and organic acids in the
distillate. The two distillates were analyzed in the usual manner using the
pre-column--HPLC--GC/MS procedure (see Section 5). The HPLC traces obtained
are shown in Figures 4 and 5 and a list of compounds identified on the basis
of their mass spectrum is provided in Table 2.

Discussion

The majority of the products volatilized from the coal by steam distilla-
tion appear to be alkanes, alkylbenzenes, and alkyInaphthalenes. The lack
of unsubstituted PAH's is consistent with the high temperature requirement
for the formation of these compounds. !


10°

“OQ
C

ro

0-1

~O-0-O~

(minor)

Scheme 168+

P-450
ah,

71,77 ,86



CONTENTS

Abbreviations and Symbols... 1... 1... eee ee ee ee ee
Acknowledgments. 2. 2 2 2 6 ewe ee ee ee

Introduction. ©. 6 6 ee ee ee ee

Coal Compositiion. ..... 2... ee eee eee eee
Present Utilization of Coal... . 1... 2.2.2.2.
Future Utilization of Coal... 2... eee ee eee
Environmental and Health Concerns... .........
Points of Entry of Coal-Related Organic Compounds. . . .
Present Work 2. 2. ee ee ee ee

Conclusions ....... tee ee be ee eee

Recommendations . . 2... ee ee ee ee ee

Chemical and Biological Studies of Northern

Great Plains Coal. 2 2. ee ee ee ee
Northern Great Plains Coal... 2... ee eee eee
Leaching of Coal-Chemical Studies. .........4.-
Volatilization from Coal-Chemical Studies. .......

Leaching and Volatilization of Coal-Biological
Studies. 6 6 6 ee ee ee

General Analytical Techniques Developed for Project .....

Water Analysis . 2. 1 1 ee ee ee ee ee ee
Fish-Analysis. . 2. 6 1 ee ee ee ee ee ee ee

Environmental Implications of Dissolved Polynuclear
Aromatic Hydrocarbons

NO wWwWNHnNHa =


ea
b? * aut

_ ve ~ psy?
n-2
n
=f (y; - yy?
i=l
n
= 5 (x, - x)?

number of times unknown solution k was injected

m
Zz Yps
i=l kj

m

weight of the compound (W) in the total volume of unknown solution

xl) + Es,q(M) + xp(E)]

volume of unknown solution k in ue

error in volume determination of unknown solution k. For a solu-
tion of volume 1 to 5 ml this error is 0.05 ml.

122


252.

253.

254.

255.

256.

257.

258.

259.

260.

261.

262.

263.
264.

J. Mowrer, J. Calambokidis, N. Musgrove, D. Brager, M. Beng, S. Herman.
"Polychlorinated Biphenyls in Cottids, Mussels, and Sediment in Southern
Puget Sound, Washington". Bull. Environ. Contam. Toxicol., 18, 588-
594 (1977). ~

S. Rossi and J. Anderson. “Accumulation and Release of Fuel-0i1-Derived
Diaromatic Hydrocarbons by the Polychaete Neanthes Arenaceodentata".
Marine Biol., 39, 51-55 (1977).

J. Anderson, J. Neff, B. Cox, H. Tatem, and G. Hightower. "The Effects
of Oi] on Estuarine Animals: Toxicity, Uptake, and Depuration, Respira-
tion". In Pollution and Physiology of Marine Organisms, F. Vernberg
and W. Vernberg, eds., Academic Press, N.Y., pp. 367-379 (1974).

W. Langsten. “Accumulation of Polychlorinated Biphenyls in the Cockle
Cerastoderma edule and the Tellin Macoma balthica". Marine Biol., 45,

265-272 (1978).

S. Schimmel, J. Patrick, and J. Forester. "Heptachlor: Uptake Depura-
tion, Retention, and Metabolism by Spot". J. Toxicol. Environ. Health,
2, 169-178 (1976).

D. Hansen, P. Parrish, J. Lowe, A. Wilson, and P. Wilson. "Chronic
Toxicity, Uptake, and Retention of Arochlor 1254 in Two Estuarine
Fishes". Bull. Environ. Contam. Toxicol., 6(2), 113-119 (1971).

R.L. Spehar, G.D. Veith, D.L. Defoe, and B.V. Bergstedt. "Toxicity
and Bioaccumulation of Hexachlorocyclopentadiene, Hexachloronorborna-
diene and Heptochloronorbornene in Larval and Early Juvenile Fathead
Minnows, Pimephales promelas". Bull. Environ. Contam. Toxicol., in
press (1978).

A.H. Glickman, C.N. Statham, A. Wu, and J.J. Lech. "Studies on the
Uptake, Metabolism and Disposition of Pentachlorophenol and Penta-
chloroanisole in Rainbow Trout". Toxicol. Appl. Pharmacol., 41, 649-
658 (1977). =

G.C. White. Handbook of Chlorination. Van WNostrand-Reinhold Company,
New York, N.Y., (1972).

American Water Works Association. Water Quality and Treatment. McGraw-
Hill Book Company, New York, N.Y. (1971).

J.W. McCoy. The Chemical Treatment of Cooling Water. Chemical Pub-
lishing Company, New York, N.Y. (1974).

G.V. James. Water Treatment. CRC Press, Cleveland, Ohio (1971).

J. Donald Johnson. Disinfection--Water and Wastewater. Ann Arbor
Science Publishers, Ann Arbor, Michigan (1975).

100


BIOCONCENTRATION FACTOR

BIOCONCENTRATION FACTOR

FIGURE 10
SUMMARY OF DIBENZOFURAN
EXPERIMENTS®

4000
DIBENZOFURAN
3000}-
2000+
(5 PAH's)P

JT xc

1oook T + + ——} (FLUORENE)*
ee

© 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
TIME (days)

Based on data in Table 15. bexp, #3, Tank #3; fluorene, phenanthrene,
1-methylphenanthrene, fluoranthene, and pyrene also present. Exp, fy
Tank #1; fluorene also present.

FIGURE 13
SUMMARY OF FLUORENE
EXPERIMENTSTM

40007 FLUORENE
3000}-

OOF
* (5 PaH's)®
1000} (DIBENZOFURAN)*

Fe ee OS OT SY

© 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
TIME (days)

®Based on data in Table 15. bexp. #3, Tank #3; dibenzofuran, phenan-
threne, 1-methylphenanthrene, fluoranthene, and pyrene also present.
Exp. #1, Tank #1; dibenzofuran also present.

59


LEACHING AND VOLATILIZATION OF COAL - BIOLOGICAL STUDIES

Biological testing of complex mixtures derived from fossil fuels has
generally involved the study of acute and/or chronic toxicity and the investi-
gation of sublethal effects (Table 3 and 4 and references cited therein).
Using this same approach, leachates and steam distillates of Northern Great
Plains coal were assessed for toxic effects on Daphnia pulicaria and fathead
minnows (Pimephales prometas) and sublethal effects {growth, spawning
behavior, cough response, bioaccumulation, and mixed-function oxidase (MFO)
induction} using fathead minnows, sunfish (Lepomés macrochinus), and rainbow
trout (Salmo gaindnert) .

Experimental Details and Results

Preparation of Coal Leachate--

Eighteen liter batches of coal leachate were prepared by stirring 6.3 g
of coal (<0.250 mm)/Z of distilled deionized or Lake Superior water for 72-96
hr. at ~ 20°C. The leachates were usually clarified by continuous-flow cen-
trifugation (Sorvall SS-3) at 12,000 rpm prior to use in the biological tests.
Various properties of the leachate including dissolved oxygen, alkalinity,
hardness, pH, conductivity, and turbidity are recorded in Table A-1 and
Figure A-1. The gas chromatograms of methylene chloride and hexane extracts
of centrifuged leachates are presented in Figures A-2 and A-3.

Preparation of Steam Distillate of Coal--

Coal distillates were prepared for fish experiments by boiling 2.5 of
Lake Superior water with 100 g of coal (<0.25 mm) ina distillation apparatus
and collecting 2 2 of distillate. For Daphnia experiments the amount of coal
was increased to 625 g and distilled deionized water was used. The pH of the
distillates varied between 5.0 and 6.8. The gas chromatogram of an isooctane
extract of a distillate is presented in Figure A-4 and ultraviolet spectra of
a distillate is presented in Figure A-5.

Toxicity Studies--

Coal leachates and distillates were tested for toxicity by exposing fat-
head minnows (Pimephales promelas) in static or renewed static bioassays. Ex-
posure tanks were 10 2 glass aquaria containing 6 2 of test solution. Tempera-
tures were maintained at 251°C and the photoperiod set at 16:8 (light:dark)
in all experiments. The test water was aerated and renewed at 72-96 hr in-
tervals during experiments exceeding 96 hr. Air was passed through a column
of glass wool and charcoal before entering the tanks. In tests longer than
1 week, adults were fed frozen brine shrimp (San Francisco Bay Brand) and # 2
pellets (Zeigler Bros., Inc., Gardners, PAY and juveniles fed live brine
shrimp nauplii at 2-5% of their body weight per day. Newly hatched larvae
were exposed in glass jars (12 x 5 cm) with nylon-screened bottoms which were
suspended in an exposure tank.

Coal distillate was also tegged for toxicity to Daphnia pulicaria accor-
ding to the method of Biesinger, In each experiment five 24-hr -old

14


s
2237LMN/7DERL77164 1-CHLOROPYRENE

>ODATE<<7SPECH

sun

253210

BASE
32736

- 10

: 70ev
GC
Varian CHS

———e - 0

Electron Energy
Inlet System
Instrument

Er uusnacaccrcacdmuccnsnarcadsnan
ERR MOG PSSO mA MN UMFANATATOSOMMMM
Re eect tet nd oo ens OL EN LL

UN MN NNNNNMNNMNNNNKNNRNMMNNNNRNMNN
ONUMOOUTT OF OMMAURT mNRROMRSOOMACONY
CDOOUMAUHNFSROMNG DOT AROMTMAUMOATM
PSeeeneedalln nena taraneralntararaer anes araraman aren arataraiN
Advnacvnnencune-seunnouder sroresor

ae Sd= UN = MUaRmOME

HOARE OROCOMMOSHT HO MONNIEMAURORND

ZOR OSS eR OM RRSOMMFUTTMR TOM TMATM

He SADSM OMAN DOHNUDOMKOUUTODDOR@MO

FUMUOURATUOM AC MUUAUATURONATAOWM
a UE Meet

HAUT NOORORUMT VOTOMNORGAUMT NODGOM
Boe RR RUM MMMM mms

x
c
wi
a

— —

Leber

in

133

—

_—_—

EF

5

EB

aT TTT ETT TTT Sa a

2 o 0 © ¢ e 6
@ Soh F a8

100 150 200 250 300

OERL77164 1-CHLOROPYRENE

50

I/Bes

STEP NASS#1,

223 LN

SPECO


ACKNOWLEDGMENTS

We wish to express our appreciation to the Environmental Research
Laboratory - Duluth (ERLD) for providing space for the bioassay procedures
and to Mr. Douglas Kuehl (ERLD) for facilitating access to GC-MS instrumen-
tation. Moreover, the enthusiasm, versatility, and technical capabilities
of Ms. Pat Kosian, Mr. Robert Liukkonen, Mr. David Ostergren, Mr. Jim

Coffey, Ms. Martha Lewerenz, and Mr. Tim Boettcher contributed significantly
to the success of this project.


273/7LM/7DERL 77203 CHLOROFLUORANTHENE

> DATEC</SPECO

70ev
GC
Varian CHS

Electron Energy
Inlet System:
Instrument:

wovonenencocececcenmecce
Emmuncocsacwucsnarsauonos
ERRAGOG COmMAMAARSEOSOMMMM

popeaonehsisiabeheeeerr er tri tity

Qocveaann ou sucnucigicingedn
Ram Ong

FHOMN VS DMONONUMADR MUR oon

ZOHOFOKMAOVUDDSM DOOR NOUT OO

AR DRMANDOORMUMSSHUUSRONS

OM ar We ae MM OUR
a!

MUMFOTUOROMUTVOROMUMS DROT
= Ree RA NAGUAUUM MAMMA MM

cape a -

Lit lherlprborel

ntl

|

273 LN DERL 77203 CHLOROFLUORANTHENE

In

131

300

250

200

150

100

50

1a

IvB/s

STEP WMASS*1-

SPECS


TABLE 4

REPORTED STUDIES ON THE BIOLOGICAL EFFECTS OF

COAL-DERIVED MIXTURES ON AQUATIC ORGANISMS

Test Solution Type of Test Organism Effect Reference
fly ash leachate toxicity goldfish Mortality 131
(embryo larval) redear sunfish (24-72 hr)
coal conversion toxicity fathead Mortality 112
gasifier condensate minnow (24,48,96 hr)
resorcinol + toxicity Daphnia Mortality 37
6-methyl quinoline (48 hr)

19


TECHNICAL REPORT DATA

(Please read Instructions on the reverse before completing)

1. REPORT NO. 2

EPA-600/3-79-093

3. RECIPIENT'S ACCESSION NO.

4. TITLE AND SUBTITLE
Implications to the Aquatic Environment of Polynuclear

Aromatic Hydrocarbons Liberated from Northern Great
Plains Coal

5. REPORT DATE
August 1979 issuing date

6. PERFORMING ORGANIZATION CODE

7. AUTHOR(S)

Robert M. Carlson, Alan R. Oyler, Ellen H. Gerhart,
Ronald Caple, Kenneth J. Welch, Herbert _L. Kopperman

ig. PERFORMING ORGANIZATION REPORT NO.

9. PERFORMING ORGANIZATION NAME AND ADDRESS f)onald Bodenner &
Dale Swanson

Department of Chemistry

University of Minnesota

Duluth, MN 55812

70, PROGRAM ELEMENT NO.

71, CONTRACT/GRANT NO.

R803952-03-1

12. SPONSORING AGENCY NAME AND ADDRESS

Environmental Research Laboratory - Duluth, MN
Office of Research and Development

U.S. Environmental Protection Agency

Duluth, MN 55804

13, TYPE OF REPORT AND PERIOD COVERED

Final 6-30-75 to 7-1-78

74. SPONSORING AGENCY CODE

EPA/600/03

715. SUPPLEMENTARY NOTES

76, ABSTRACT

bioaccumulate.
concentration to samples obtained from Lake Superior.

etc), alkanes, and heterocycles.

The biological studies on PAH's were aided by the

range of 1000-5000 for several PAH's.

The effects of leaching processes upon Western Great Plains coal was investigated
to ascertain the potential impact of the organic components on aquatic organisms.
Acute and chronic toxicity testing of coal leachate indicated no lipophilic fraction
containing polynuclear aromatic hydrocarbons (PAH) that might be anticipated to
HPLC-GC analysis indicated that the PAH content was of a comparable

GC-MS analysis of the lipophilic

materials that are adsorbed on the coal particulates indicated that they were pre-
dominantly low molecular weight PAH's (i.e., naphthalenes, phenanthrenes, anthracenes,
Synthetic methodology was developed to provide
standard samples of alkylated PAH's of the type observed during the MS analysis.

use of a combined HPLC-GC

analysis procedure (na/1 detection level) developed specifically for this program.
The biological investiaation resulted in obtaining bioaccumulation factors in the

Selected PAH's of various structural types were also shown to be quite susceptible
to "second-order" anthropogenic transformations such as chlorine disinfection.

n7.

KEY WORDS AND DOCUMENT ANALYSIS

a. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Coal Bioassay Fathead minnow PAHs 06/A
Leaching Rainbow trout 06/F
Volatilization Daphnia pulicaria 06/T
Polycyclic Mixed-function oxidase 07/C
Analytical Bioconcentration factor
Chlorination HPLC
Synthesis GC-MS
118, DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES
7 23
. AS sis page) 22. PRICE

RELEASE TO PUBLIC UNCLASSIFIED
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION IS OBSOLETE

156 2 US. Gomme Pr OFFICE 1879 -657-060/5445



85.

86.

87.

88.
89.

90.

91.

92.

93.
94.

95.

96.

97.

98.
99.
100.

E. Cavalieri, R. Roth, and E. Rogan. “Metabolic Activation of Aromatic
Hydrocarbons by One-electron Oxidation in Relation to Tumor Initiation".
In Carcinogenesis--A Comprehensive Survey, Vol. 1, Raven Press, New
York, 1976.

H. Gamper, A. Tung, K. Straub, J. Bartholomew, and M. Calvin. "DNA
Strand Scission by Benzo(a)pyrene Diol Epoxides". Science, 197,
671-674 (1977). —_—

T. Maugh. "Underground Gasification: An Alternate Way to Exploit Coal".
Science, 198, 1132-1134 (1977).

Reference (1), pp. 4-86 to 4-87.

J. Boyer and V. Gleason. "Coal and Coal Mine Drainage". J. Water
Poll. Control Fed., 1163-1172 (1977).

R. Corbett. "Effects of Coal Mining on Ground and Surface Water Quality
Monengalin County, West Virginia". Sci. Tot. Environ., 8, 21-38 (1977).

E.D. John and G. Nickless. "Gas Chromatographic Method for the Analysis
of Major Polynuclear Aromatics in Particulate Matter". J. Chromatogr.,
138, 399-412 (1977).

A. Burnham, G. Calder, J. Fritz, G. Junk, H. Svec, and R. Willis.
"Identification and Estimation of Neutral Organic Contaminants in
Potable Water". Anal. Chem., 44(1), 139-142(1972).

Coal Cleaning Review, 1(1), (1977)

Anon. "Slurry Pipeline Support Gains Momentum". Chem. Eng. News,
56(16), 23-24 (1978).

Reference (1), p. 4-58

W. Anderson and M. Youngstrom. "Coal Pile Leachate--Quantity and Quality
Characteristics". Journal of the Environmental Engineering Division,
1239-1253 (1976).

N. Coward, J. Horton, R. Koch, and R. Morden. "Static Coal Storage--
Biological and Chemical Effects on the Aquatic Environment". EPA
Grant #NERC-R-803937-02-0 (1977).

Reference (1), pp. 4-86 to 4-97.

Reference (1), pp. 4-128 to 4-135.

W. Bertsch, E. Anderson, and G. Holzer, "Characterization of Coal-
derived Fluids by Capillary Column Chromatography-Mass Spectrometry".

J. Chromatogr., 126, 213-224 (1976).


1O37LN7DERL 77179 CHLOROMETHYLPHENANTHRENE

>>DATE<</SPECO

10

T
300

1%

250

IvB7s

w
x
=)
i=
oc
> -
Sok
Ro > E
mnonnnne
mdmnsnoe
Fasauaaa “
wea cu cua Os Od >
o
Los
o &
co ss
wu PP
vec
58
>
pe &
o Vo
oe a
- oc
Bee

DemMrNon
WORRARAR

STEP MASS*1,

QOD COMOMOM AMNION SONOS M MOMMA New mmm
CainsuncaanasncamnnsssunocomnancosucamrnereNes
ERAARRRR@@OODAGDAAOS REE AGTEK UAMFNNOSOIOIRROOS

NNNMNNRNMNNNNNMNNNNMN

LF FOMEMSAUMAUMSOSV OTD

(oz uivaucndccr sueKUNUAR Suu suns eoouR ss Aus
un popeednetaioes a ree nao wor

VOU VOOR RNAUUREHRMRERUMOFRATEROMMONAUUODTUUODOE
ZRMMST OF HH TE DION OMUSPUNIMMNOSOMRNKTFDTNEODIODN
AROMOMT OF ORG HEF OTUSUR TOUR MORMORA GOSS=MNDOHOG
MRT AUHRAM MAT UAT MAME Meee Me SUN LTT Mm med Mm

LAUMFAURDROMURDM AAMT NON DF MUOINFOMFARDOUSTNYROTOMIN.
« AAR UUUUUANUUM MMMM SS TT TAMIR OOS

a

183 LM DERL 77179 CHLORONETHYLPHENANTHRENE

TT TEETETETEP TT

130

SPECS


TABLE 22
SYNTHESIS OF POLYMETHYLNAPHTHALENES

0
Ry RQ CH,

CHzLi_ 0 BF RoR,

Ne aS
THF -H,0

R R

1 R 3
3 CH
Ro

3, Ry =H, Rp =H, R, =H

9, Ry = H, Ro = H, RZ = CHA

10 and 10', Ry H, Ro = CH3, Rg = H

1 and 11', R, H, Ro = CH, Rg = CH3
12 and 12', Ry sl CHa, Ro =H, Ry 2H

13 and 13’, Ry = CH3, Ro = H, Rg = H

81


SECTION 1
INTRODUCTION
COAL COMPOSITION

Coal is a compact material which was produced by metamorphosis of plant
matter over periods of time up to about 400 million years. This metamorphic
process proceeded through a continuous series of alterations! (living plants >
peat + lignite + subbituminous coal + bituminous coal > anthracite) which
resulted in an increase in the relative amount of carbon present (up to 93%
for anthracite) and a decrease in the relative amount of volatile matter. 22
The composition of the coal includes inorganic mineral matter from the sur-
rounding geological formations and organic matter derived from the remains
of the plants.’ The organic matter is a complex mixture of various macromole-
cules which varies according to the stage of metamorphosis'(rank). The
structures of these mglecules are not well understood although some models
have been proposed.!> In addition, polynuclear aromatic hydrocarbons,
including 1,2-benzanthracene, 3,4-benzopyrene, benzo-g,h,i-perylene, perylene,
Phenaptbrene, and 1,2-benzopyrene, have been found to be associated with raw
coal.*?

PRESENT UTILIZATION OF COAL

The estimated coal reserves in the United States amount to about three
trillion tons, which represents about 50% of the total known world coal
reserves and about 80% of the total fossil fuel reserves of this country .6
Western coal, which has a low sulfur content and is frequently close enough
to the surface for strip-mining, constitutes at least 72% of the U.S. coal
reserves.’ The estimated coal reserves of the world are capable of providing
enough energy for 730 years based on a consumption rate equal to that of
1973.8 Current U.S. production is around 700 million tons per year? and the
total world production is around 2000 million tons per year. 0° The major
portion of the coal currently produced is burned directly for the generation
of electricity and for heating. In this country, about 20%'' of our energy
needs are met by coal. Most of the remaining coal production is carbonized $8
produce coke which is utilized in steel production_as_an ore-reducing agent.
The volatile byproducts of coke production includel0,12 benzol (consisting
mainly of benzene and toluene), tar (consisting mainly of polynuclear aromatic
hydrocarbons of bp > 180°C) and gases (consisting mainly of hydrogen and
methane) .


72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

K. Shinohara and P. Ceratt. "Formation of Benzo(a)pyrene--DNA Adducts
in Peripheral Human Lung Tissue". Cancer Letters, 3, 303-309 (1977).
C. Bigger, J. Tomaszewski, and A. Dipple. "Differences Between Products
of Binding of 7,12-Dimethylbenz(a)anthracene to DNA in Mouse Skin and

in Rat Liver Microsomal System". Biochem. Biophys. Res. Comman, 80(1
229-235 (1978). mu sol),

E. Huberman and L. Sachs. "DNA Binding and Its Relationship to Carcino-

genesis By Different Polycyclic Hydrocarbons". Int. J. Cancer, 19,
122-127 (1977). =

R. Scott and G. Girling. "Evolution of Volatile Hydrocarbons from Coal
During Storage". Chem. and Ind., 1570-1571 (1961).

J. Selkirk. “Benzo{atpyrene Carcinogenesis: A Biochemical Selection
Mechanism". J. Toxicol. Environ. Health, 2, 1245-1258 (1977).

R. Lehr and D. Jerina. "Relationships of Quantum Mechanical Calculations,
Relative Mutagenicity of Benzo(a)anthracene Diol Epoxides, and ‘Bay
Region' Concept of Aromatic Hydrocarbon Carcinogenicity". J. Toxicol.
Environ. Health, 2, 1259-1265 (1977).

A. Wood, R. Chang, W. Levin, R. Lehr, M. Schaefer, J. Karle, D. Jerina,
and A. Conney. “Mutagenicity and Cytotoxicity of Benzo(a)anthracene Diol
Epoxides and Tetrahydro-epoxides: Exceptional Activity of the Bay
Region 1,2-epoxides. Proc. Natl. Acad. Sci., 74(7), 2746-2750 (1977).

R. Lehr and D. Jerina. "Metabolic Activations of Polycyclic Hydrocar-
bons". Arch. Toxicol., 39, 1-6 (1977).

D. Thakker, W. Levin, A. Wood, A. Conney, T. Stoming, and D. Jerina.
"Metabolic Formation of 1,9,10-Trihydroxy-9,10-dihydro-3-methy]cholan-
threne: A Potential Proximate Carcinogen from 3-Methylcholanthrene" .
J. Amer. Chem. Soc., 100, 645-647 (1978).

B. Tierney, A. Hewer, C. Walsh, P. Grover, and P. Sims, "The Metabolic
Activation of 7-Methylbenz(a)anthracene in Mouse Skin". Chem. Biol.
Interactions, 18, 179-193 (1977).

D. Thakker, H. Yagi, H. Alsagi, M. Koreeda, A. Lu, W. Levin, A. Wood,

A. Conney, and D. Jerina. “Metabolism of Benzo(a)pyrene. VI. Stereose-
lective Metabolism of Benzo(a)pyrene and Benzo(a)pyrene-7 ,8-dihydrodiol
to Diol Epoxides:. Chem.-Biol. Interactions, 16, 281-300 (1977).

F. Beland and R. Harvey. "Model Reactions of the Quinone Metabolites of
Carcinogenic Hydrocarbons with t-Butylthiol". Progress in Biorganic
Chemistry, 6, 415-419 (1977).

P. Wislocki, R. Chang, A. Wood, W. Levin, H. Yagi, 0. Hernandez, H, Mah,
P. Dansette, D. Jerina, and A. Conney. “High Carcinogenicity of 2-
Hydroxybenzo(a)pyrene on Mouse Skin". Cancer Research, 37, 2608-2611
(1977).

87


144.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

W. May, S. Chesler, S. Cram, B. Gump, H. Hertz, D. Enagonio, and S.
Dyszel. "Chromatographic Analysis of Hydrocarbons in Marine Sediments
and Sea Water". J. Chromatogr. Sci., 13, 535-540 (1975).

S. Chesler, B. Gump, H. Hertz, W. May, and S. Wise. "Determination of
Trace Level Hydrocarbons in Marine Biota". Anal. Chem., 50(6),
805-810 (1978). —

V. Leoni, G.,Puccetti, and A. Grella. "Preliminary Results on the

Use of Tenax” for the Extraction of Pesticides and Polynuclear Aromatic
Hydrocarbons from Surface and Drinking Water for Analytical Purposes".
J. Chromatogr., 106, 119-124 (1975).

G. Junk, J. Richard, M. Grieser, D. Witiak, J. Witiak, M. Arguello, R.
Vick, H. Svec, T. Fritz and G. Calder. "Use of Macroreticular Resins

in the Analysis of Water for Trace Organic Contaminants". J. Chromato-
gr., 99, 745-726 (1974).

R. Vick, d. Richard, H. Svec, and G. Junk. "Problems with Tenax®-¢¢
for Environmental Sampling". Chemosphere, (6), 303-308 (1977).

S. Stephan and J. Smith. "Some Conditions for Use of Macroreticular
Resins in the Quantitative Analysis of Organic Pollutants in Water".
Water Res., 11, 339-342 (1977).

D. Pietrzyk and C. Chu. "“Amberlite XAD Copolymers in Reversed Phase
Gravity Flow and High Pressure Liquid Chromatography". Anal. Chem.,
49(6) 757-764 (1977). -

P. Rossum and R. Webb. "Isolation of Organic Water Pollutants by XAD
Resins and Carbon". J. Chromatogr., 150, 381-392 (1978).

J. Saxena, J. Kozuchowski, and D. Basa. "Monitoring of Polynuclear

Aromatic Hydrocarbons in Water. I. Extraction and Recovery of Benzo-
(a)pyrene with Porous Polyurethane Foam". Environ. Sci. Technol., 11,
682-685 (1977). ~

J. Navratil, R. Sievers, and H. Walton. "Open-Pore Polyurethane
Columns for Collection and Pre-concentration of Polynuclear Aromatic
Hydrocarbons from Water". Anal. Chem., 49, 2260-2263 (1977).

W. May, S. Wasik, and D. Freeman. "Determination of the Aqueous Solu-
bility of Polynuclear Aromatic Hydrocarbons: by a Coupled Column Liquid
Chromatographic Technique". Anal. Chem., 50, 175-9 (1978).

F. Schwarz and S. Wasik. "Fluorescence Measurements of Benzene, Naph-

thalene, Anthracene, Pyrene, Fluoranthene, and Benzo{a}pyrene in
Water". Anal. Chem. ,48, 524-528 (1976).

92


80L

TABLE A-4
EFFECTS OF COAL LEACHATE ON SPAWNING SUCCESS IN FATHEAD MINNOWS

EXPOSURE CONDUCTIVITY TANKS. IN WHICH MEAN # EGGS/

Water g COAL/1 woh /em ANEEKS/TEST TANKS SPAWNING OCCURRED # SPAWNINGS SPAWNING ATCHABILITY, % (¥ EGGS)
100% 6.25 i) 4 4 0 ° 0 0
Leachate (65-77)

100% 6.25 70 2 3 1 2 85 88
Leachate (68-75) (50)
90% 5.0 n 2 2 1 3 19

Leachate (66-78)

75% 47 16 2 3 1 2 162 ry
Leachate (71-83) (100)
50% 2.78 82 2 2 1 3 170 88
Leachate (78-86) (150)
25x 1.39 87 2 3 2 5 69 64
Leachate (85-89) (150)
10x 0.85 a 2 5 2 2 97 al
Leachate (100)
Lake Superior 0 93 2 4 3 7 102 90
Water (92-94) (200)
Lake Superior 0 2 4 4 4 16 70 72
Water (89-92) (500)
28% Distilled 0 B 2 2 2 6 138 69
water in Lake (71-74) (300)

Superior Water


SS

TABLE 15
SUMMARY OF BIOCONCENTRATION FACTORS (BASED ON TOTAL WET WEIGHT OF FISH)

&xp Tank Bfoconcentrattion factor = nah PAH/kg fish)/(ug PAH/kg water)* Day 28 Day 7 KFO
# # Compound Day #1 Day #2 Day #4 Day #7 0 Day #14 Day 1B Day #21 Day #25 Tria? #) Trial #2 Activty % Lipids S.p.
dibenzofuran 260: 540: 1,200, 1,200 1,100 1,000 1,100
u 50 70 "200° “200 150 200 160 No 4.821.5
fluorene 250: «$001,200 1,200 870s 1,100 1,100 “B21.
1 50 60 100 = “100 100 200 260
lg Q-chlorophe- 1,140 1,200: 1,900: 1,700 3,200 1,200 §,000 Yes Aele0.8
nanthrene 80 ‘240 1,200 1,000 1,500 600 2,000 +1204
phenanthrene 990: 800 © 800t 1,300 1,500 1,900: 2,500
; Yoo = 700-700 “900 "750 1300 1,300 Yes 4.B1.5
lb-naphtho- 902 4100 «100s Bal.
2 flavone 40 80 40
2 phenanthrene 2,000: 1,700: 2,000: 2,300: 3,300 3,700 4,200: 5,100 ho 9,820.7
300 '200-« 250200400 700 1,000 = 1,600 +820.
menanthrene 2,000 2,400 1,600 18008 1,900: 2,100 2,000: 2,800
H 600 © "900 "800 600 600 600 780
=chlorophe- 4, ie 6.100% 6,400: 5,600 56002 5,100 5,100: 6,700 «= Y*S 4180.5
anthrene 1,000 11,000 5,000 2,000 2,000 2,000 3,000
2 phenanthrene 1,500 1,400 1,300 2,000 3,100¢ 3,000,
"200 "400 4c0 600 Y000° 100" No 4,321.0
ibenzofuran 1,400: 830: 8608 1,800 1,200 1,700: 1,600
350 200» 300 '900 500 600 500
3 Ftuorene 1,200: 830-1, 100 2,200 1,300 1,800: 1,500¢
400 300 "500 1,000 500 800400
yhenanthrene 2,000: 2,000: 2,000 3,100 2,200 1,900 2.2002 y,
300 200 600 600 600 500 es 4,420.9
N-rethyiphe- 1,202 1,400: 1,300 2,100 1,800: 7002 1.2408
anthrene ‘400 "500 «= "400 700 600 300
IFluoranthene 146008 2,600 4,000 3,200 3,600 1,500 i ate
300 600 300 1,000 500
lbyrene 1,200 1,400 2,300 2,600: Sto: 6008
250 Vioo. 700 1,000 1,000 500 600

calculated from data presented ‘in Tables 16 and 17.

b His z mean concentration of PAH determined from all analyses performed up to and including the day of fish sampling.



FIGURE 13
SUMMARY OF 9-CHLOROPHENANTHRENE
EXPERIMENTS®

sooo; 9-CHLOROPHENANTHRENE

7000/--

6000F

a

5000

4000

3000}- L

2000

BIOCONCENTRATION FACTOR

1000,

ce ek Ss ee Se Se SOO SON SO J a |

2 cz 1.
© 2 4 & 8 10 12 14 18 18 20 22 24 26 28 30 32
TIME (days)

Based on data in Table 15. bexp. #3, Tank #1; phenanthrene also

present. “Exp. #1, Tank #2; no other compounds present.

61


in the development of new analytical methodology, insight into the suscepti-
bility of PAH's toward external transformations involved in such processes aS
chlorine disinfection and metabolism, and the tendency of PAH's to bioaccumu-
late in aquatic organisms.


6tL

1003

SPECe

100
95 LN

cH,

CH

|,

a i i i i il i

OERL 146 1,5-DINETHYLNAPHTHALENE

200

250
STEP MASS=1,

Ive/s

1%

10


101.

102.

103.

104.

105.

106.
107.
108.
109.

110.
1.

12.

113.

114.

115.

M.J. Suess. "The Environmental Load and Cycle of Polycyclic Aromatic
Hydrocarbons". Sci. Tot. Environ., 6, 239-250 (1976).

R. Hites, R. Laflamme, and J. Farrington. "Sedimentary Polycyclic
Aromatic Hydrocarbons: The Historical Record". Science, 198, 829-
830 (1977). —=

G. Miller. "Pollution Research on Dated Sediment Cores from Lake
Constance. III. Historical Evolution of N- and P-compounds--Relation-
ship to the Development of Heavy Metals and Polycyclic Aromatic Hydro-
carbons". Z. Naturforsch., 32c, 920-925 (1977).

G. Miller. "Pollution Research on Dated Sediment Cores from Lake
Constance. II. Historical Evolution of Heavy Metals--Relationship to
the Evolution of Polycyclic Aromatic Hydrocarbons". Z. Naturforsch.,
32c, 913-919 (1977). TO

M. Lee, G. Prado, J. Howard, and R. Hites. “Sources Identification of
Urban Airborne Polycyclic Aromatic Hydrocarbons by Gas Chromatographic
Mass Spectrometry and High Resolution Mass Spectrometry". Biomedical

Mass Spectrometry, 4(3), 182-186 (1977).

Reference (1), pp. 4-120 to 4-123.
Reference (54), pp. 89-96.
Reference (1), p. 9-5.

C. Chrisp, G. Fisher, and J. Lammert. "Mutagenicity of Filtrates from
Respirable Coal Fly Ash". Science, 199, 73-74 (1978).

Reference (1), pp. 4-100 to 4-120.

R.C. Lao, R. Thomas, and J. Monkman. “Computerized Gas Chromatographic-
Mass Spectrometric Analysis of Polycyclic Aromatic Hydrocarbons in
Environmental Samples". J. Chromatogr., 112, 681-700 (1975).

T. Schultz, S. Davis, and J. Dumont. "Toxicity of Coal-Conversion
Gasifier Condensate to the Fathead Minnow". Bull. Environ. Contam.
Toxicol., 19, 237-243 (1978).

P. Buryan, J. Macak, and V. Nabirach. "Investigation of the Composi-
tion of Coal-tar Phenols and Xylenols by Capillary Chromatography".
J. Chromatogr., 148 203-214 (1978).

D. Grant and R. Meiris. "Application of Thin-layer and High-perfor-
mance Liquid Chromatography to the Separation of Polycyclic Aromatic
Hydrocarbons in Bituminous Materials". J. Chromatogr., 142, 339-351
(1977). ~~

N. Goeckner and N. Griest. "Determination of Methyl Chrysenes in a
Coal Liquification Product". Sci. Tot. Environ., 8, 187-193 (1977).

89


tL

Alkalinity
(mg/2 Caco,)

EDTH Hardness
(mg/2 caco,)

pH
(at 20°C)

Conductivity
(umhos/cm)

Turbidity

TABLE 1

WATER CHEMISTRY ANALYSES FOR LEACHATES

LAKE WATER LAKE WATER LEACHATE® PURIFIED WATER PURIFIED WATER LEACHATE
42.49 7.62 1.04 4.12%, 3.08”
43.84 2.90 0 0.07, 0.49°

7.8 8.1 6.3 6.87,6.5°
88 108 4.9 33, 87°
0.6 3.6 0.20 0.70%, 2.25°

3 Leachate prepared using a coal to water ratio of 10 g/%, followed by 48 hr stirring and filtration
through GF-F filters.

> similar to (a) but the leachate was centrifuged.


rate to give a tank turnover time of 2.5 hr. The photoperiod was 16:8
(light:dark). Fish were fed 3% of their body weight per day Zeigler Bros. _
3/16" pellets) during the experiments except on the day they were to be sacri-
ficed. The tanks were siphoned daily.

The water chemistry data during the experiments were: alkalinity 40.8+0.6
mg/2 CaCO3, hardness 45.1+0.3 mg/& CaCO3, conductivity 90+3 yumhos/cm, pH 7.05-
7.79, and dissolved oxygen 92+5% saturation.

Polycyclic aromatic hydrocarbon flow-through exposures of rainbow trout--

Conditions were similar to those described above. Rainbow trout from the
same cohort were exposed to both pyrene (pyr) and fluoranthene (f1) in one ex-
periment and to B(a)P in the other. During each experiment aliquots of a PAH
stock solution were diluted 1:1000 with lake water and added daily to a 20-
liter reservoir. A control reservoir was filled with 0.1% acetone in lake
water. Nominal acetone levels for both control and experimental tanks were
10 ue/2 (ppm).

During the B(a)P exposure, rainbow trout (1% yr old) were exposed to
solubilized B(a)P in water, with and without the addition of coal particles.
Three exposures were run in 55 £ stainless steel tanks containing: coal,
B(a)P, and coal plus B(a)P. Reservoirs were filled daily with B(a)P stock
solutions diluted 1:1000, and ground coal particles ($0.125 mm). The control
(coal alone) reservoir received the same proportion of acetone. Under the
two reservoirs containing coal were magnetic stirrers powering 2" Teflon-J] ined
stir bars within, the reservoirs. Calculated concentrations were 50 mg 27’ for
coal and 2 ug 27! for B(a)P.

FMI (Fluid Metering, Inc.) lab pumps with stainless steel fittings and
tubing were used to meter reservoir solutions into the tanks. Three water
samples were taken during each experiment. Lake Superior water at 10+1°C for
the pyr/fl experiment and 13+1°C for the B(a)P experiment was delivered at a
rate of 25 £/hr (2.2-hr tank turnover time) and 16.7 2/hr (3.3-hr tank turn-
over time) respectively.

Fish for enzyme and tissue analyses were taken on days 3,7,10 and 21 for
the pyr/fl experiment and on day 10 for the B(a)P experiment.

Preparation of Microsomes--

The trout were killed by cervical dislocation and the livers quickly re-
moved and placed in cold 1.15% KC1. Individual livers were blotted and
weighed before homogenizing. Microsomes were then prepared by grinding 2-3
pooled livers in 4 vol 1.15% KCl using a Potter-Elvehjem homogenizer with
Teflon pestle. Homogenates were centrifuged at 15,000 G for 20 min and the
supernatant spun at 100,000 G for one hour on a Beckman L5-50 ultracentri-
fuge (0°C). The microsomal pellet was rinsed three times with 1.15% KC and
resuspended in 1.15% KCl by sonicating (Branson Instruments) 3 sec after
gentle homogenizing.

40


46.

47.

48.

49.

50.

51.
$2.
53.

54.

55.
56.
57,

58.

59.

J. Neff, B. Cox, D. Dixit, and J. Anderson. "Accumulation and Release
of Petroleum-Derived Aromatic Hydrocarbons by Four Species of Marine
Animals". Marine Biology, 38, 279-289 (1976).

J. McCann and B. Ames. "Detection of Carcinogens as Mutagens in the
Salmonella/microsome Test: Assay of 300 Chemicals: Discussion". Proc.

Nat. Acad. Sci., U.S.A., 73, 950-954 (1976).

J. McCann, E. Choi, E. Yamasaki, and B. Ames. "Detection of Carcinogens
as Mutagens in the Salmonella/microsome Test: Assay of 300 Chemicals".
Proc. Nat. Acad. Sci., U.S.A., 72, 5135-5139 (1975).

P. Brookes. "Mutagenicity of Polycyclic Aromatic Hydrocarbons". Muta-
tion Research, 39, 257-284

M. Hollstein, R. Talcott, and E. Wei. "Quinoline: Conversion to a Muta-
fore) Human and Rodent Liver". J. Natl. Cancer Inst., 60, 405-410

Reference (1), pp. 9-62 to 9-63.
Reference (1), pp. 10-16 to 10-23.

R. Frendenthal and P. Jones. '"Carcinogenesis-~A Comprehensive Survey".
Vol. 1, Raven Press, New York, 1976.

S. Radding, T. Mill, C. Gould, P.Liu, H. Johnson, D. Bomberger, and

C. Foju. "The Environmental Fate of Selected Polynuclear Aromatic
Hydrocarbons". United States Environmental Protection Agency, Washing-
ton, D.C. (1976), EPA 560/2-75-009, pp. 48-72.

Reference (1), pp- 9-42 to 9-43.
Reference (1), pp. 9-5 to 9-36.

H. Seliger and J. Harmman. "Chemical Production of Excited States.
Chemi luminescence of Carcinogenic Hydrocarbons Accompanying Their Meta-
bolic Hydroxylation and a Proposal for Common Active Site Geometries for

Hydroxylation"”. J. Phys. Chem., 80(20), 2298-2306 (1976).

C. Heidelberger. "Studies on the Mechanisms of Carcinogenesis by Poly-
cyclic Aromatic Hydrocarbons and Their Derivatives". In Carcinogensis,
Vol. 1. Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and
Carcinogenesis, ed. by R. Frendenthal and P. Jones, Raven Press, New
York, 1976.

I. Kano, J. Gielen, H. Yagi, D. Jerina, and D. Nebert. “Subcellular
Events Occurring During Aryl Hydrocarbon Hydroxylase Induction: No
Requirement for Metabolism of Polycyclic Hydrocarbon Inducer". Molecular

Pharmacology, 13, 1181-1186 (1977).

85


tanks were aerated and the water renewed twice a week. Spawning success
(Table A-4) is defined as the percentage of exposure tanks in which at least
one spawning occurred during the 2-4 week experimental period. There was only
a 36% spawning success in the 22 leachate exposure tanks compared to a 90%
spawning success in 10 control exposure tanks. In spite of the overall lower
spawning success observed during the leachate exposures, spawning ultimately
occurred at all leachate concentrations tested.

Cough Response--Two bluegill sunfish (Lepomis macrochitus) were exposed
to 1, 5, 10 and 20% concentrations of coal distillate in tanks designed to
monitor opercular movement. !33 Exposure tanks contained 4 2 of water, and
fish were exposed under recirculating static conditions. Physiographs were
activated 1 hr before introduction of distillate to obtain baseline data for
each fish. After the test was initiated, 20-min scans were picked at selected
intervals and the frequency of the cough response tabulated. The results of
this experiment are presented in Table A-5. In both tanks the initial eleva-
tion of cough rate rapidly diminished indicating a lack of imminent toxicity.

Hepatic Mixed-function Oxidase Activity (coal leachate)--Rainbow trout
(1 yr old, 10-15 cm) were exposed to 100% coal leachate for 28 days in order
to determine the effect on hepatic mixed-function oxidase activity and other
liver parameters. These fish, which had been held in flowing Lake Superior
water for three months prior to use, were exposed in 40 2 glass aquaria con-
taining 100% leachate (made with Lake Superior water) or Lake Superior water
at 11419C under subdued lighting (16:8). The water in each tank was renewed
every 72 hr and vigorous aeration maintained. The mean conductivity of the
water control tank was 91 wmhos/cm. The fish were fed 1% of their body weight
in two daily feedings. Samples consisting of three fish were removed from
each tank on days 1, 3, 7, 21 and 28. Microsomes, prepared from the livers of
these fish, were analyzed for aryl hydrocarbon hydroxylase activity (AHH) by
methods described in the mixed-function oxidase discussion (Section 6). Hepa-
tic DNA context, microsomal protein, and relative liver weight were also
assayed at each sampling period. The DNA content of the 15,000 g pe}]) t from
the homogenized liver preparations was analyzed according to Burton. Fif-
teen-minute extractions of the pellet were done at 70° using 3 mé 0.5 N per-
chloric acid. During the first extraction, 2 drops of 0.5% sodium dodecyl sul-
fate were added in addition to the acid. After adjusting the tota] volume to
10 mé with perchloric acid, 200 ue was then assayed for DNA concentration. The
incubation mixture contained 200 v2 DNA extract, 500 ul 0.5 N perchloric acid,
and 2 mé freshly prepared diphenylamine reagent. Duplicate tubes plus DNA
standards and a reagent blank were all incubated at 30°C for 10-20-hr and then
read against the blank at 600 nm on a Beckman DB-G spectrophotometer. Calf
thymus DNA (Sigma) was used to prepare standards.

Protein was determined by the biuret method. 135 Five-hundred pl samples
of microsomes were combined with 2 m2 of freshly prepared biuret reagent and
the absorbance at 550 nm was determined. Crystalline bovine serum albumen was
used as the standard.

Relative liver weight was determined by dividing the wet weight of each
liver by the corresponding total fish weight. The values shown in Figure A-8
represent the mean ratios from three fish.

23


APPENDIX C

CHLORINATED POLYNUCLEAR AROMATIC HYDROCARBONS
2147LN/DERL 77202 CHLORODIBENZOFURAN

MASS SPECTRAL DATA

sun
94694

BASE

> DATEC</SPECA
eusor

f 20

: 70 ev
GC
Varian CHS

300

250
= 12

Ivers

el,

Electron Energy
Inlet System
Instrument

COSTUTUONRETHMROOSRAMI
EOORRDOAQGAPOMMMNTOSSOS
Barer uaa

STEP MASS

200

156

WI NRNNNN NNN MNARNNNNAA NAAN

or eension
2a emm

POOFARORRATUVOVAMROSTAM
ZUUMAUDESROOTAMAMMMOONT
AF OrOreeonaorMNnevRnminy

UU aan UMN ZN

LUMORDPOMATURTOMUMROTS
« Soe eR NANOGNAM
7
a

|

I,
214 LM DERL 77202 CHLORODIBENZOFURAN

| ih

ee

[rm eereeerepreererty
co ee © Ss 6
o 7 ®@ 8

123

so4
404
304
204
104

04

50
SPECO


SECTION 3
RECOMMENDATIONS

The results of the study suggest areas for further chemical investiga-
tion before design and siting criteria can be firmly established for coal
storage and handling facilities. These areas of study include the analysis
of the polar portion of the coal leachate and distillate, the determination
of the ability of aquatic organisms to bioaccumulate PAH {20m particulate
matter, the examination of environmental samples of water and sediment for
the presence of the chlorine disinfection products, and the field evaluation
of the impact of the volatilization process.

Field studies of the tissue content of PAH and the levels of MFO acti-
vity in fish would be appropriate. Determination of the relative importance
of uptake of PAH from food, in comparison to uptake from water and particu-
lates, would be useful for the estimation of maximum bioconcentration poten-
tial of PAH in fish. In addition, field studies of the PAH content of
organisms of several trophic levels would illuminate the environmental par-
titioning of PAH within a biological community.


TABLE 13
LEVELS OF VARIOUS AROMATIC COMPOUNDS IN AQUATIC ORGANISMS IN THE ENVIRONMENT
RESULTS FROM SELECTED LITERATURE REPORTS

Concentration tn
organisa (fg compd/ Tissue Concentratfon —_Bloconcentratton

Coapound Organism Location kg wet weight) analyzed in water (ug/kg) Factor® (tice) Ref.
benzo(a)pyrene mussel on creosoted 0.049:0.015 whole organisa” 24
pilings
benzo(a)anthracene oyster ong tstand 0,008 whole organisa?
und
benzo(a)pyrene oyster Lit. Sound 0.002 whole organism? 22
pyrene oyster L. 1. Sound 0.058 whole organisa
nethylpyrene oyster L. 1. Sound 0.011 whole organisa?
flvorene clan 0.046 whole organisa?
phenanthrene clam 0.88 whole organisn® 245
nethylphenanthrene clan 0.56 whole organisa?
benzo(a)pyrene mussel S.California 0.0023 27
benzo(a)pyrene shellfish TNatne 0.016 whole organisa? 246
benzo(ght)perylene 0.025 whole organisa?
benzo(a)pyrene mussel S.californta 0.008 whole organtsa® 287
on pilings
benzo(a)pyrene mussel new creosoted 0.045 whole organisa? 28
tinbers
benzo(a)pyrene mussel Vancouver 0.215 whole organtsa® 29
denzo(a)anthrene crab Rarttan Bay, 0.002 whole organisa 22
New Jersey
benzo(a)pyrene 0.003
pyrene 0.006
natiylpyrene 0.002
benzo(a)pyrene enhaden Raritan Bay, 0.002 whote organism 2
New Jersey
pyrene founder Long Island 0.002 whole organism wz
nethylpyrene flounder Long Island 0.0005 whole organtsa
total PAH oysters Gulf of 2-9 whole organism 250
Hexico
Aroclor 1254 burbot w. Lake 1.4204 whole organtsn® — .0008 175x108
Superior 6
Take trout, . whole organisn® — .0008 2.25x10! 251
Yong-nose . whole organisa® 0008 Ttaxto®
sucker 5
slimy sculpin * 0.34 whole organism’ 0008 425x107
mysid . 0.08520.029 whale organism’ — 0008 1.06x10'
ce 2
polychlorinated biphenyls fish Puget Sound 0.84 whole organtsn 22
mussels 0.21

"gtoconcentratton factor (concentration of compound in fish in ug/kg)/(concentration of conpound tn water tn vg/kg).

Pshucked
“composites

51


FIGURE A-10
GAS CHROMATOGRAPHIC ANALYSIS OF RAINBOW
TROUT EXPOSED TO 0.2% COAL DISTILLATE

LAKE WATER

FLD. RESPONSE

Tl bistutare

FLD. RESPONSE

Me

o 6 2 W 2 % 36 42 46 54 60 66
TIME (minutes)

tJ

3Rainbow trout were maintained in flowing Lake Superior water for 21 days
prior to extraction.

PRainbow trout were exposed to 0.2% coal distillate by metering distillate
into flowing Lake Superior water over a 21 day period.

Gc conditionsscolumn: 3% OV-101, 2m x 2mm i.d. glass; injgctor: 350°C;
detector: 300°C; carrier flow: 20 ml/min; program: 80-250°C at 4°/min.

120


304. R. Caple, G.M.S. Chen and J.D. Nelson. "The Addition of Butyl-
lithiums to Benzonorbornadiene and 1,4-Dihydronaphthalene 1,4-endo-
Oxide". J. Org. Chem., 36, 2874 (1971).

305. L.F. Fieser and M.J. Haddadin. "Isobenzofurane, A Transient Inter-
mediate". Can. J. Chem., 43, 1599 (1965).

306. M.S. Newman, H.M. Dali and W.M. Murg. "Synthesis of 1,4-Dihydro-
1,4-dimethy1-1,4-epoxynaphthalene and Conversion to 1,4-Dimethyl-
1,2,3,4-tetrahydronaphthalene and o-Diacetylbenzene". J. Org. Chem.,
40, 262 (1975).

104


90L

TABLE A-2

EFFECTS OF COAL DISTILLATE ON DAPHNIA PULICARIA®

Coal Distillate, Conductivity Mortality Distilled Water b Conductivity Mortality
Concentration (%) (umhos/cm) (%) Concentration (%) (umhos/cm) (%)
0 88 0 10 82 0
20 7246 1.7 25 70 0
40 56+1 3.3 50 48 0
60 4242 10.0 75 27 0
80 24+1 58.3 80 19+1 83.3
100 4.7 100

Daphnia were exposed to coal distillate (and 80% distilled water) during 3 separate bioassays with
4 replicates per concentration. The conductivity of the exposure water was measured at the
beginning of each bioassay and the mean and standard deviation indicated. The distilled water and
lake water controls were tested in 1 bioassay, with 4 replicates per concentration.

bCoal distillate and distilled water were diluted with Lake Superior water.



Ubiquity of Polycyclic Aromatic Hydrocarbons .........

Mixed-Function Oxidase Activity in Fish Exposed
to Polynuclear Aromatic Hydrocarbons

Bioaccumulation of Polynuclear Aromatic Hydrocarbons

Aqueous Chlorination of Polynuclear Aromatic
Hydrocarbons . 2... 1 ee ee ee te ee

Syntheses of Methylated Naphthalenes .............
References 2. 1 ee ee ee eee
Appendices

A. Additional Tables and Figures Relevant to
Biological Studies on the Leaching and
Volatilization of Coal... 2... eee eee eee

B. Calculations and Error Treatment for Analysis
of Water and Fish Tissue by GC/PID..........
C. Chlorinated Polynuclear Aromatic Hydrocarbons:
Mass Spectral Data... ......2.2.5-50 ee eee
D. Chlorinated Polynuclear Aromatic Hydrocarbons:
"3¢ and 'H (100 MHz) NWR Data...
Methylated Naphthalenes : 'H (60 MHz) Data... ....
Methylated Naphthalenes: Mass Spectral Data. ....
Methylated Naphthalenes: Microanalytical Data

vi


14.

1.
16.
7.

18.

19.
20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.
31.

J. O'Hara, E. Becker, N. Jentry and T. Harding. "Petrochemical Feed-
stocks From Coal". Chemical Engineering Progress, 73, 64-72 (1977).

Reference (1), pp- 1-1 to 1-25.
G. Mills. "Alternate Fuels from Coal". CHEMTECH, 4180423 (1977).

J. Walsh. “Problems of Expanding Coal Production". Science, 184,
336-339 (1974). ~ =

L.E. Swabb, Jr. "Liquid Fuels from Coal: From R & D to an Industry".
Science, 199, 619-222 (1978).

A. Hammond. "An Interim Look at Energy". Science, 199, 607 (1978).

R. Wishart. "Industrial Energy in Transition: A Petrochemical Per-
spective". Science, 199, 614-618 (1978).

K. Schlupp and H. Wien. "Production of 0i1 by Hydrogenation of Coal".
Angew. Chem. Int. Ed., 15(6), 341-346 (1976).

R. Gordon. "The Hobbling of Coal: Policy and Regulatory Uncertainties".
Science, 200, 153-158 (1978).

P. Fennelly, H. Klemm, R. Hall, and D. Durecher. “Coal Burns Cleaner
ina Fluid Bed". Environ. Sci. Technol., 11(3), 244-248 (1977).

N. Cochran. "Oil and Gas from Coal". Scientific American, 234(5),
24-29 (1976). —

D. Luntz, P. Buckingham, S. Kimmel, N. Cochran, and D. Garrett. "Gaso-
line from Coal". Chemical Engineering Progress, 73, 49-54 (1977).

J. O'Hara. "Coal Liquification". Hydrocarbon Processing, 221-226 (1976).
I. Schwager and T. Yen. "Coal-liquification Products from Major Demon-
stration Processes. I. Separation and Analysis". Fuel, 57, 100-104
(1978) =

J. Grey, G..Satton, and M. Zlotnick. "Fuel Conservation and Applied
Research". Science, 200, 135-142 (1978).

A. Hammond, W. Metz, and T. Maugh, II. "Energy and the Future”.
American Association for the Advancement of Science, Washington, D.C.,
1973, pp. 25-28.

Reference (1), pp. 3-1 to 3-94.

Anon. "Mobil Proves Gasoline-from-methanol Process". Chem: Eng. News,
26-28, Jan. 30, 1978.

83


82

TABLE 7
DETERMINATION OF PAH RECOVERY FROM AQUEOUS SOLUTIONS OF KNOWN CONCENTRATIONS
USING THE C-18 ADSORPTION--HPLC-GC PROCEDURE
Experiment_No. 1 2 3 4 5 6 7 8 9 10

Cone of PAH in ng/z 80 80 80 22 80 80 107 600 1000 965
# of 2 through

C-18 column 14.5 14.3 14.2 18.2 14.0 12.2 13.8 6.4 6.2 10.2
# of ug of PAH for

quantitative recovery 1.16 1.14 1.14 0.392 «1.12 0.978 1.48 3.84 6.20 9.84
Glass fiber filter None a b c c c None None
Type C-18 packing d d d d d d e e

% Recovered:

an
ao

1-Methyl-naphthalene_ = -- -- -- -- -- -- -- -- -- 76410
Fluorene 7643 6446 77411 4248 72218 70420 6924 93416 89413 --
Dibenzofuran -- -- -- - -- -- 83410 8048 --
1-Methy]-4-chloro-

naphthalene -- -- -- -- -- “- -- -- -- 9542
henanthrene 87+3 10142 8742 105421 85+2 90+4 -- -- -- --
2-Methylanthracene 81424 = 6445 7211 115#18 = 6149 87413 7442 -- -- --
-Methylphenanthrene 87410 78413 82413 99468 87422 111412 86:10 -- -- --
9-Methylanthracene 103+21 92413 87224 -- 101417 11018 106212) -- -- --
Fluoranthene 96+18 101416 87420 8125 6744 7649 88414 -- -- --
Pyrene 65416 84428 79435 92455 36210 100+11 106214

9-Chlorophenanthrene -- -- -- -- -- -- --

The errors given in this table reflect the standard deviation in the determination of the weight of
material injected into the GC plus the error involved in the measurement of the volumes of the HPLC
fractions.

88+7 865 --

a-$&S$ 29 b-S&S 30 c - Whatman GF/B d - Waters Corasil II e - Waters Porasil B


Stl

eer

SPECO

Mo

|

\

:

ry
°

nr a

: a oe

50

@

us

Lid di
100
eLA +

T

BOLN

Li

rT

repre

mt 0
200
STEP NASS*1, Ivers

1%


FIGURE A-5
UV SCANS OF COAL DISTILLATE

as

a2
8
&

- UV spectra of distilled water (DW), Lake Superior water (LW), and coal

distillate (DIST).

. After obtaining a stable baseline using distilled water, various dilutions
of coal distillate were made using Lake Superior water as the diluent.

- Coal distillate (pH 5.0) was scanned and then rescanned after raising the

pH to 11.3 with 1 N NaOH.
115


116.

117.

118.

119.

120.

121.

122.
123.
124.

125.

126.

127.

128.

129.

130.

J. Schiller and D. Mathiason. "Separation Method for Coal-derived
Solids and Heavy Liquids". Anal. Chem., 49, 1225-1228 (1977).

J. Swansiger, F. Dickson, and H. Best. "Liquid Coal Compositional
Analysis by Mass Spectrometry". Anal. Chem., 46, 730-734 (1974).

R. Pugmire, D. Grant, K. Zilm, L. Anderson, A. Oblad, and R. Wood.
"Carbon-13 Magnetic Resoncance of Coal-derived Liquids". Fuel, 56,
295-301 (1977). =

W. Dark, W. McFadden, and D. Bradford. “Fractionation of Coal Liquids
by HPLC with Structural Characterization by LC-MS". J. Chromatogr.

Sci-, 185 454-460 (1977).

J.E. Schiller. "Nitrogen Compounds in Coal-derived Liquids". Anal.
Chem., 49, 2292-2294 (1977).

J. Schabron, R. Hurtubise, and H. Silver. "Separation of Hydroaromatics
and Polycyclic Aromatic Hydrocarbons and Determination of Tetralin and
oye in Coal-derived Solvents". Anal. Chem., 49, 2253-2260
1977). Oe

Reference (1), p. 2-14.
Reference (1), p. 2-86.

A. Krubsack. "Experimental Organic Chemistry". Allyn and Bacon, Inc.,
Boston (1973), pp. 64-68.

R. Scott and G. Girling. "Evolution of Volatile Hydrocarbons from
Coal During Storage". Chem. Ind., 1570-1571 (1961).

M. Blumer. "Polycyclic Aromatic Compounds in Nature". Sci. Am., 234,
36-45 (1976). OS

S. Rice, J. Short and J. Karinen. "Toxicity of Cook Inlet Crude Oil
and No. 2 Fuel Oi] to Several Alaskan Marine Fishes and Invertebrates".
In Sources, Effects, and Sinks of Hydrocarbons in the Aquatic Environ-
ment. Proceedings of the Symposium (A.1I.B.S.), pp. 395-422 (1976).

0. Linden. "Biological Effects of Oil on Early Development of the
(i978) Herring Clupea harengus membras". Marine Biol., 45, 273-283
1978). =

Jd. Barnett and D. Toews. "Effects of Crude 0i1 and Dispersant,
Oilsperse 43, on Respiration and Coughing Rates in Atlantic Salmon
(Salmo salar)". Can. J. Zool., 56, 307-310 (1978).

J. Payne. "Mixed Function Oxidases in Marine Organisms in Relation
to Petroleum Hydrocarbon Metabolism and Detection". Marine Pollution
Bulletin, 8, 112-116 (1977).

90


error involved in measurements of the volumes of the HPLC fractions. (See
Appendix B).

Analysis of Samples of Unknown Composition--

For analysis of environmental samples, coal leachate samples and other
unknown solutions, the filter holder was fitted with a Whatman GF/F filter
and the analysis proceeded as described above. The filter was replaced as
often as necessary to maintain the flow rate of 15-30 m/min. For GC-MS
identification work it was necessary to remove the water from the individual
HPLC fractions and to concentrate them. The water removal was effected by
addition of ~0.5 mt of methylene chloride to cause separation of layers,
followed by removal of the aqueous layer with a Pasteur pipette. The organic
layer which remained was dried with sodium sulfate and concentrated under a
stream of nitrogen.

FISH ANALYSIS

The analysis of PAH in biological tissues usually 49¥o]¥gs removal of the
PAH by exfractionl 45,159, alkaline digestion/extraction'*»>!94, or headspace
samp] ing! 5, followed by clean-up via column ghroma tography on alumina or
silical59, reverse-phase or adsorption HPLC 45. or gel-permeation chromato-
graphy!©0 with subsequent analysis by GC, GC/MS, ultraviolet and/or fluor-
escence.

Results

For the present work a procedure was developed which employs an ex-
traction-HPLC/Styragel®-Gc/PID and/or GC/MS sequence. The StyragelTM step
(size separation) provided excellent separation of PAH material from biomole-
cules. The resulting PAH fractions were analyzed directly (no concentration
required) via gas chromatography with photoionization detection or concen-
trated for GC/MS analysis as described above. The results of determinations
of recovery efficiencies for the procedure with fish samples spiked with
known amount of PAH prior to the extraction step are provided in Table 8.

Experimental Details
Apparatus--

The HPLC apparatus was manufactured by Waters Associates and consisted
of the following items: an M-6000A pump, U6K injector, 254 nm UV detector
and two 7.8 x 1220 mm 60A Styragel columns connected in series. Methylene
chloride was used at a flow rate of 4.0 m/min. An example of a typical HPLC
fractionation is provided in Figure 7. The gas chromatograph used for this
work was described above.

Procedure--

The fish (12-5 grams) and “15 g of sodium sulfate were ground in a Waring
blender. The material obtained was mixed with an additional 15 g of sodium
sulfate and extracted in a Soxhlet extractor with 170 m2 of methylene chlo-

31