-------
E
c
*•
tr>
Ul l/J
53
"•
FIGURE 4
STEAM DISILLATE OF COAL (KOH)
HPLC
I I
nil 12 iia!! 14 !
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
TIME (minutes)
Conditions- column: 10 t» Lichrosorb RP-18; program: 50 to 90%. CH3CN in
water, 1.2 ml/min, 30 min (linear program).
15
-------
FIGURE 5
STEAM DISTILLATE OF COAL (NO KOH)
HPLC
il
< 3
CO u-
ss
VI ^
a
0 2 4 6 8 10 12 U 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
TIME (minute.)
Conditions- column: 10 w Lichrosorb RP-18; program: 50 to 90% CH3CN in
water, 1.2 ml/min, 30 min (linear program).
16
-------
Compound
aUanes, al Kyi benzenes
naphthalenes
naphthalenes
c^-naphthalenes
cs-benzene
n-dodecane
cyclododecane
Cg-benzene
Cg-hexahydrophenanthrene?
methylphenanthrene/anthra-
cene
methylf1uoranthene/pyrene
S8
TABLE 2
STEAM DISTILLATES Or COAL
RESULTS OF GC/MS ANALYSIS
HPLC Fraction No.
Mass Spectrum No.
•*
182
156
156
156
156
156
156
170
170
170
170
170
170
170
170
170
170
170
170
184
184
148
170
168
204
212
192
192
216
256
Other
Ions
167
141
141
141
141
141
141
155
155
155
155
155
155
155
155
1b5
155
155
155
169
169
133;iQ5,91
85,71
97,83,69
189,147
197
191
191
201
224,192,160
128,96
Distillate Distillate
with KOH without KOH
F6, F7, F8
F9
F9
F9
F9
F9
F9
F9
F8
F8
F8
F8
F-10
F-10
F-10
F-10
F-10
F-10
F-10
F-10
F9, 10
F9, 10
F-10
F-10
F-10
F-10
F-10
F-10
F-10
F-13
F-14
Distillate"
with KOH
332
260
272
282
287
293
309
283
293
299
303
309
314
322
331
209
212
230
250
341
418
431
Distil
withcu
117
120
125
132
136
141
169
182
*6C conditions—column: 2 mm x 12' with 55! SP-2250 on 100/120 Gas Chrom Q; program: 80-225°C @ 2°/min.
Mass spec: start scan after 2 m1n; scan: 50 to 300 amu ? 7.5 .sec/decade.
bGC conditions—column: 1/8" x 6' with 3% 07-101 on 80/100 Gas Chrom Q; program: Initial hold 1 m1n, 80 to
225°C 9 4°/mtn. Mass spec: start scan after 2 mln; scan: 50 to 300 amu 9 7.5 sec/decade.
17
-------
TABLE 3
REPORTED STUDIES ON THE BIOLOGICAL EFFECTS OF
THE WATER SOLUBLE FRACTION OF PETROLEUM ON FISH
Type of Test Organism(s) Effect Reference
Toxicity, Acute Salmon, trout, cod 24, 96-hr toxicity 127
Sublethal Growth/ Baltic herring
Development
decreased lengths,
malformed larvae
128
Cough Response Salmon
increased cough
rate
129
MFO
Brown trout,
cunner, copelin
AHH introduction
130
Bioaccumulation Fundulus
bioaccumulation of
naphthalenes
46
18
-------
TABLE 4
REPORTED STUDIES ON THE BIOLOGICAL EFFECTS OF
COAL-DERIVED MIXTURES ON AQUATIC ORGANISMS
Test Solution
fly ash leachate
Type of Test Organism Effect
toxicity goldfish Mortality
(embryo larval) redear sunfish (24-72 hr)
coal conversion toxicity
gasifier condensate
fathead
minnow
resorcinol + toxicity Vapkrua
6-methyl quincline
Mortality
(24,48,96 hr)
Mortality
(48 hr)
Reference
131
112
37
19
-------
daphnids were placed into a 250-m£ beaker containing 200 ml 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/I) and 100% mortality during a 96 hr bioassay (25 g coal/£).
Coal distillate caused no significant mortality to adult or newly-
hatched fathead minnows (Table 6) at the highest concentration tested (20%).
Partial mortality of Vaphnio. 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 I 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
-------
TABLE 5
SUMMARY OF BIOLOGICAL TESTING
OF COAL LEACHATE.8
ro
100%
(6.3g/l DDW)
100%
(6.3g/l DDW)
100%
Uncentr if uged
(25g/l DDW)
100%
(6.3g/l DDW)
10-100%
(6.3g/l LW)
100%
(6.3g/l LW)
fathead
minnow
fathead
fathead
minnow
fathead
minnow
fathead
minnow
rainbow
trout
5-15 daya
21jmo
2>s mo
2 mo
*1 yr
1 yr
3 weeks
24 weeks
96 hours
24 weeks
2 or 4 weeks
28 days
Toxicity
Toxicity
Toxicity
Sub lethal
Growth
Bioaccumula-
tion
Sublethal:
Spawning
Sublethal:
AHH Response
Liver Para-
Renewed Static
Renewed Static
Static
Renewed Static
Renewed Static
Renewed Static
15% mortality
not significantly
greater than
controls
No mortality with
centrifuged leachate
100% mortality
Growth rate simi-
lar to that in
Lake Superior
water but onset
of maturity de-
layed . Some
qualitative
differences in
GC analyses of
tissue extracts
36% spawning suc-
cess in leachate
exposures.
90% spawning suc-
cess in control
exposures.
No consistent
differences from
controls
meters
aThe ratio of coal to water and the type of water used to prepare leachates (100Z) are indicated. DDW - distilled
deionlzed water, LW - Lake Superior Water
-------
TABLE 6
SUMMARY OF BIOLOGICAL TESTING
PO
ro
OF COAL DISTILLATE.
Concentration Organism Age at Onset Duration of Exp. Type of Test Bioassay Conditions Response
20, 40, 60%
20%'
(67g/l, DOW)
0.1,1,10,20%
(67g/l, DDW)
1,5,10,207.
(67g/l, DDW)
0.27,
(AOg/1 LW)
Daphnia
fathead
minnow
fathead
minnow
bluegill
rainbow
trout
-24 hr
24-48 hr
9 mo
?
1 yr
48 hr
96 hr
96 hr
24 hr
21 days
Toxicity
Toxicity
Toxicity
Sublethal;
Cough Re-
sponse
Sublethal:
MFO Response
Bioac cumula-
tion
static
static
static ,
recirculating
static
flow-through
Death at high
concentrations
due to low con-
ductivity. Partial
mortality at inter-
mediate concentra-
tions.
No different from
controls
No mortality
No sustained,
elevated cough
rate
P-450 no different
from control
Slight AHH
elevation, D28
No different from
control
The ration of coal to water and the type of water used to prepare distillates (100%) are indicated. DDW= distilled
do Ionized water, LW » Lake Superior Water.
-------
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 (LepomXa macAoc-klnm*,} were exposed
to 1, 5, 10 and 20% concentrations of coal distillate in tanks designed to
monitor opercular movement.'33 Exposure tanks contained 4 £ 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 £ glass aquaria con-
taining 100% leachate (made with Lake Superior water) or Lake Superior water
at 11±1°C 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 ymhos/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 peHgt from
the homogenized liver preparations was analyzed according to Burton.'34 Fif-
teen-minute extractions of the pellet were done at 70° using 3 m£ 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 total volume to
10 m£ with perchloric acid, 200 y£ was then assayed for DNA concentration. The
incubation mixture contained 200 y£ DNA extract, 500 \il 0.5 N perchloric acid,
and 2 m£ 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.
1 35
Protein was determined by the biuret method. Five-hundred y£ samples
of microsomes were combined with 2 rn£ 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 weig-ht of each
liver by the corresponding total fish weight. The values shown in Figure A-8
represent the mean ratios from three fish.
23
-------
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 £ stainless
steel tank using an FMI (Fluid Metering, Inc.) lab pump with stainless steel
fittings and tubing. Flow rates were 27.5 £/hr for the distillate tank and
40 £/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 m£ centrifuge tubes and then evaporated to exactly 3 mi.
Interfering lipids were removed by gel permeation chromatography'36 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 distil late)--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
Vapkrua. 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 VaphnM
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
-------
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
-------
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 Iess137,138 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, XAD resins92,149-151, polyurethane foam!52,153, or
C-18 Corasil143,144,154, followed by analysis of this material with high per-
formance liquid chromatography (HPLC)143,i44,154, gas chromatography (GC)'40,
142,147s anc| gas chromatography/mass spectrometry (GC/MS)142,144. Direct
analysis with fluorescence has also been attempted155.
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 lacking145. 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 reported138,
143,144. However, reverse-phase HPLC alone does not give extensive separation
of various PAH's for accurate identification and quantisation. Therefore, we
have improved upon this procedure15^ 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 CorasilR. 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
-------
The results of the PAH recovery determinations (Table 7) indicate that
sub-micron filters such as the Whatman 6F/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 6F/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 detector157 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
a more 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 somewhat157 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 t 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 Porapak^ 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 y) (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 y C-18 reverse phase column (or a
4.6 x 25£) mm 5 y 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 Tracer 550 chromatograph equipped with
an HNU Systems photoionization detector (model PI-51 max temp =.250°). The
GC inlet was modified by welding a Waters Associates HPLC septum inlet onto
the front of the Tracer septum nut in order to move the location of the sep-
tum (Hewlett-Packard, part number 5080-6721) about 3.5 cm awayfrom the 300°
inlet tube. The PID was connected to the Tracer outlet v-to. a 1/16-inch
27
-------
ro
CD
TABLE 7
DETERMINATION OF PAH RECOVERY FROM AQUEOUS SOLUTIONS OF KNOWN CONCENTRATIONS
USING THE C-18 ADSORPTION—HPLC-GC PROCEDURE
Experiment No.
Coix of PAH in nq/£
# of £ through
C-18 column
# of yg of PAH for
quantitative recovery
Glass fiber filter
Type C-18 packing
% Recovered:
1-Methyl-naphthalene
Fluorene
Dibenzofuran
1-Methyl-4-chl oro-
naphthalene
Phenanthrene
2-Methylanthracene
1-Methylphenanthrene
9-Methy1a n t h ra c en e
Fluoranthene
Pyrene
9-Chlorophenanthrene
1
80
14.5
1.16
None
d
76±3
87±3
81 ±24
87±10
103±21
96±18
65±16
2
80
14.3
1.14
a
d
64±6
101 ±2
64+5
78±13
92±13
101±16
84±28
3
80
14.2
1.14
b
d
87±2
72±11
82±13
87±24
87±20
79±35
4
22
18.2
0.392
b
d
5
80
14.0
1.12
c
d
6
80
12.2
0.978
c
d
7
107
13.8
1.48
c
d
77±11 42±8 72±18 70±20 69±4
105+21
115+18
99±68
81±5
92±55
85±2
61+9
87±22
101±17
67±4
36±10
90+4
87±13
111±12
110±18
76±9
100±11
74±2
86±10
106±12
88+14
106±14
8
600
6.4
3.84
c
d
9
1000
6.2
6.20
None
e
93±16
83±10
89±13
80±8
88±7 86±5
10
965
10.2
9.84
None
e
76+10
95±2
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.
a - S & S 29
b - S & S 30
c - Whatman GF/B
d - Waters Corasil II
e - Waters Porasil B
-------
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 allow 300°
inlet and outlet temperatures with minimum column bleed. SupeltexR 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
•^10-75 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 620-1 and 620-L data
systems.
Distilled water was further purified for this work by forcing it through
an 0.45 u 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-£ pressure tank VJM. a 100-y£ 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 acetom'trile-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 injected158, plus the
29
-------
FIGURE 6
TYPICAL GAS CHROMATOGRAM OF
PAH MIXTURE USING PHOTOIONIZATION DETECTOR
u
iu m
33
2 "-
ss
0 2 4 6 8 10
TIME (minutes)
Injection: 1 wl 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
-------
error involved in measurements of the volumes of the HPLC fractions. (See
Appendix 8).
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 mi 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 }flyo]yes removal of the
PAH by extraction!45,159, alkaline digestion/extraction'1^^''^', or headspace
sampling145, followed by clean-up via column chromatography on alumina or
silica'59, reverse-phase or adsorption HPLC' , or gel-permeation chromato-
graphy^O 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/Styrage!R-GC/PID and/or GC/MS sequence. The Styrager 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 (^2-5 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 mi of methylene chlo-
31
-------
ro
TAULE 8
SUfWARY OF ANALYSES OF SPIKED FISH TISSUE SAMPLES
X Recovered
Trial
it
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Grans F1sh
Added
4.3
4.13
5.5
5.8
5.9
4.1
3.1
6.0
7.4
4.6
4.1
2.6
3.4
3.8
3.3
ng PAH
Added
20.0
5.0
5.0
5.0
10.0
5.0
5.0
5.0
10.0
15.0
30.0
5.0
10.0
20.0
40.0
mg PAH
kg fish
4.7
1.2
0.9
0.9
1.7
1.2
1.6
0.8
1.4
3.3
-7.3
1.9
2.9
5.3
12.0
B-Naphthoflavone Dibenzufuran
101+12
64±6
86±18
76±6
85±5
—
—
135±20a
84±7a
—
83t3a
94+18
94±13
9219
72+10
Fluorene
95*12
72±10
83±13
77*5
99*4
—
—
—
—
—
—
—
75 ±18
76:9
57±5
Phenanthrene
—
—
—
—
—
75±2
67*2
—
106+.8
111+9
116*9
105±10
90+2
—
109±12
1-Methyl- 9-Chloro-
phenanthrene phenanthrene Fluoranthene
91+10
57+6
—
101*15
66+9
103+12
102+7
84+12
107+8
112+.9
118+7
100±5
81±2 — 96±6
88t6 — 79+5
80±4
Pyrene
...
—
—
—
...
—
—
—
—
—
—
—
103*11
90*1
...
"Determined by HPLC-UV. All other determinations by GC-PID.
-------
ride for 5 hours. The extract was then concentrated in a Kuderna-Danish
apparatus to ^1.5 mi. The concentrate was injected into the HPLC and frac-
tions containing the PAH material were collected at M8-25 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 quantisation as described in the water analysis section.
33
-------
FIGURE 7
TYPICAL FRACTIONATION OF FISH
TISSUE EXTRACT CONTAINING PAH MATERIAL
USING HPLC-STYRAGEL
tu to
II
S "•
SS
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
-------
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-
ment'O' >I26, 161 ,162 are: a) synthesis by microorganisms^! ,161 and plants
101,161,162; t,) geochemical processes'^6 which result in the formation of
coal'SS and petroleum; c) natural pyrolytic processes such as forest fires
126 , prairie 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 involves free radical re-
actions'66"^6'' and possibly "benzyne" intermediates^6^. Higher temperatures
generally result in formation of less alkylated PAH's126.
The environmental cycle of PAH's has been discussed in several reviews
101,168-170 and a summary has been provided by Suess1^ :
"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
-------
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 oil 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 state168'177, associated with particulate matter16!,168,169,171 and/or
associated with organic matter161'171. Photooxidation101>161,172,173, js
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's176.
The levels of PAH's in the environment have also been discussed in
several reviews101'161'162'168'170'177'178. 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 considerably101 '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
-------
TABLE 9
IDENTIFICATION AND QUANTITATION OF POLYNUCLEAR AROMATIC HYDROCARBONS:
RESULTS FROM SELECTED LITERATURE REPORTS
Source
Remote region of Bolivia
(air paniculate extract)
Antwerp
(air partlculate extract)
Zurich, Switzerland
(airborne partlculates)
Mew York City
(airborne participate
extract)
Hew York City
(airborne partlculates)
College Park, HD
(airborne partlculates)
Oslo, Norway
(airborne partlculates)
Stmcoe, Canada
(airborne partlculates)
Urban air partlculates,
Indiana
Air partlculates near
coke oven plant
Coke oven emissions
Coal fly ash
Compounds
/
AIR
anthracene, phenanthrene
methylanthracene, methyl phenanthrene
fluoranthene
pyrene
benzanthracene, chrysene
benzopyrene, perylene
otners
anthracene, phenanthrene
methyl anthracene, methylphenanthrene
fluoranthene
pyrene
benzanthracene, chrysene
behzpyrene, perylene
dtbenzacrldlne
others
phenanthrene
anthracene
fluoranthene
pyrene
benzo[e]pyrene
benzo[a]pyrene
perylene
1so
-------
TABLE 9 (continued)
So1l--Russli
Soil—Swiss Tcurualn town:
center cf ton
at Mc,h*a/
open country
Sediment In a stream flow-
ing through an oil tank fan
1n Kr.onvllle, TN
River sedtfrent from the
Rnondda rar- Valley, South
Wales (site cf cedi mine
operated In 19th and early
20th centuries!
Lake sediiserts,
Graifenste, Switzerland
River sediment near chem-
ical nanufacturing plant
Marine Sediment,
Buzzards Bay, Mass.
Strut dust
Ash from volcano
SOIL AHO SEOMEnT
be-*:o[a]pyrene
PAH (total)
PAH (total)
PAH (total)
nacnthalene
aUyinaphthalene
anthracene
benz(a)anthracene
berrz(a)pyrere
anthracene
flucranthene
pyrene
2.3-'jenzofluorene
chrysene
3,d-benzpyrene
1,2.3,4-dlbenzanthracene
benzoighiiperylene
phenanthrene
anthracene
fluoranthene
pyrene
benzofelpyrene
benzotajpyrene
perylene
fluorene
phenanthrene
methylphenanthrene
fluoranthene
pyrene
others
phenanthrene
fluoranthene
pyrene
others
aza-arenes
phenanthrene
anthracene
fluoranthene
pyrene
benzofelpyrene
benzotajpyrene
perylene
benzopyrene
0.1-6
110,000
220,000
5.000
7.000
320.000
3,400
120
48
7,800
6.500
S.100
1,800
35,500
7,200
5,400
4,200
340
30
420
380
210
160
40
2.000-10,000
200-25,000
400-20,000
1.000-60,000
600-75,000
S3:
130
120
140
3,500
450
10,000
7,200
3,900
2,700
660
5.4-6.1
191
192
176
182
193
102,194 l
102,194 I
182
164
HATER
Hater 1n i stream flowing
through an oil tank farm
in fcioxvllle, TN
Well fn Ames, IA near site
*t l coal gas plant that
operated until 1970's
T1re manufacturing plan
wastewaters
Thames River
Tap water
Switzerland
Tap water, U.S.
Rain water
Tip water. Athens, GA
naphthalene
aIkylnaphthalenes
anthracene
benz[a]anthracene
benz[a]pyrene
acenaphthylene
1-methylnaphthalene
fflsthyl ir.denes
acenaphthene
benzcthiophene
others
naphthalene
1-methylnapnthalene
phenanthrene
methylphenanthrene
flucranthene
pyrene
others
fluoranthene
pyrene
benzcoyrene
perylene
others
pheianthrene
pyrene
benzo[a]pj'rene
phenanthrene
methylphenanthrene
fluoranthene
pyrene
chrysene
ben?cpyrene
others
naphthalene
dit^fizcfuran
fluorenc
phenanthrene
flucran'.henc
8.0
850
3.3
0.19
0.039
19.3
11.0
18.8
1.7
0.4
100
120
70
60
8
10
0.21
0.52
0.33
0.43
0.01
0.01
0.00002-0.002
0.009
0.032
0.031
0.?55
0.02]
176
92
142
139
140
170
19S
ISt
38
-------
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 mammals61>196-200, it was not until 1963
that this metabolic capability was suggested in fishes201. 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's206"208,
3-methylcholanthrene2°9-2129 2,3-benzanthracene247 and benzo(a)pyrene2'3.
Of particular interest from an environmental standpoint has been the
finding that fishes exposed to petroleum under laboratory and field conditions
show induced MFO activity'98»202-205,207,214> Qne JVJPQ enzyme system, aryl
hydrocarbon hydroxylase (AHH), has been suggested as an environmental monitor
202 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 oil by intraperitoneal
injection. After anaesthetizing in 100 mg/l MS-222 (ethyl m-aminobenzoate
methanesulfonic acid salt), each fish received an 0.2 m£injection 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 yg/kg to 300 mg/kg in dose response experiments.
Controls received 0.2 mi 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 diTutor, at a
39
-------
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/t CaC03, hardness 45.1±0.3 mg/t CaCOq, conductivity 90±3 ymhos/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 (fl) 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 \il/l (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 I 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-lined
stir bars within the reservoirs. Calculated concentrations were 50 mg £"' for
coal and 2 yg £~' 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 £/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% KC1 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% KC1 and
resuspended in 1.15% KC1 by sonicating (Branson Instruments) 3 sec after
gentle homogenizing.
40
-------
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 hydroxylaseJAHH) and aniline hydroxylase (AH) were
assayed by literature methodsIyb'^[. 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 mi) contained 50 yM TES {N-tris (hydroxymethyl) methyl-
2-aminoethanesulfonic acid} buffer, pH 7.50; 0.6 mg NADPH (sigma, Type I);
3 yM MgC^, and 200 y£ microsomal suspension.
The assays were initiated by adding 100 y£ substrate: 100 nM B(a)P in
acetone and 10 yM aniline-HCl. Incubations were carried out in a shaking
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-6 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.
The CO difference spectrum -was measured on a Beckman DB-G spectrophotometer
. Protein was determined using a microbiuret method1-". 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-Qz 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 m£ methylene chloride(pyr/fl) or hexane {B(a)P} was placed into a
2 liter volumetric flask followed by 1900 ml of tank water which had been
filtered through a Gelman glass fiber filter (0.2-10 yM). A IV Teflon-
41
-------
coated stirring bar was introduced and the sample extracted by emulsifying the
contents for 2 hr. The organic layer was dried over ^SCty 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 measurements2!!,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/fl, combined] and to B(a)P. The day 21
(pyr/fl) 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(a)P 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 yg/kg dose on day 5 was elevated. Therefore,
the 300 lag/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 cavi'ty, weight variations
42
-------
FIGURE 8
EFFECTS OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHs)
ON MICROSOMAL ENZYMES IN RAINBOW TROUTa
P-450
0.3O.
naph 1254 phen
fluor ehry« B(a)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
-------
TABLE 10
PAH TISSUE CONCENTRATIONS AND MFO MEASUREMENTS DURING WATER UPTAKE EXPERIMENTS3
PAH Concentration
(ug/kg)
pyjr H
95±8 318±10
82±7 273±14
150+11 408±20
2flli23 1.250+38
.0 0
(controls, n=6)
B(a)P
363±24
73.1±24C
0
(controls, n=2)
Time
(day)
3
7
10
21
10
10
P-450D
(nM/tng protein)
0.083
0.107
0.103
0.071
0.107
±0.048
0.238*
±0.018
0.115
±0.028
0.175
±0.032
AHH
(pH/mg protein/ml n)
•2.18
±0.43
1.18
±0.27
1.74
±0.02
2.50
±0.43
1.72
±0.80
3.80*
±0.62
1.66
±0.88
1.76
±0.25
AH
(pM/mg protein/rain)
...
...
—
...
...
34.52*
±5.14
11.47
±1.13
20.01
±3.21
aThe mean water concentrations during the exposures were 3.89 ±0.08 pg 1 , pyrene; 3.31 ±0.08 yg 1 , fluoranthene;
and 0.10 ±0.21 vg !"', B(a)P. Control values were obtained by taking several (n) 3-flsh samples from control tanks
at various times during the experiments. Induced enzyme levels (P <0.05) are Indicated by asterisks.
During the pyr/fl experiment only one measurement of P-450 was made on microsomes prepared from 3 pooled livers;
duplicates v/ere run during the B(a)P exposure.
cThe exposure tank contained 50 mg I'1 ground coal (sO.125 mm) 1n addition to B(a)P. Water concentrations of B(a)P
determined on filtered samples were <0.1 pg I'1.
-------
among the fish, different rates of metabolism, and injection variations.
In the water uptake experiment (Table 10), exposure of trout to 0.4 yg/1
B(a)P resulted in accumulation of 368 yg/kg B(a)P after ten days. Table 10
also shows the corresponding enzyme data, which demonstrated induction of all
three MFO parameters at that time. Trout containing 73 yg/kg B(a)P showed no
MFO induction after a 10-day exposure to B(a)P plus coal particles (low levels
of B(a)P (<0.1 yg/£)}.
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 yg/kg), B(a)P levels above 300 yg/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^'8. 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 greatly en-
hanced the carcinogenic potency of B(a)P in mice skin tumor studies1^19. Also
some compounds, such as the synthetic 8-naphthoflavone, cause MFO induction
but are not carcinogenic^.
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 yg/kg after 21 days. This may explain the lack of MFO response and
points out the importance of determining bioconcentration factors"1 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
-------
FIGURE 9
DOSE RESPONSE FOR HEPATIC MICROSOMAL ENZYMES
IN RAINBOW TROUT3
Each fish was injected l.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
-------
TABLE 11
TISSUE ANALYSES FOR DOSE-RESPONSE EXPERIMENT3
Approximate Injected Dose B(a)P Concentration In
(vg/kg) Tissue (vg/kg)
Day 3 Day 5
30,000 3260i50 458±31
3,000 320±54 537±65
300 249±33 350±21
30 130±27 140±14
3 <30 <30
aThe B(a)P doses indicated were injected i.p. in peanut oil and tissue concen-
trations measured 3 and 5 days later in 2-3 gutted fish (pooled). The mini-
mum detectable B(a)P level was 30 yg/kg.
47
-------
TABLE 12
RELATIONSHIP BETWEEN B(a)P CONCENTRATION IN RAINBOW TROUT
AND MFO INDUCTION9
P-450 AHH AH
3260±50 (i) + + +
537±65 (i) + + +
458±31 (i) + + +
368±24 (w) + + +
350±21 (i) + - +
320±54 (i) + + +
249±33 (i) - +
140±14 (i) - -
130±27 (i) - - -
73±24 (w) -
<30 (i) - -
aThese 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
-------
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 yg/£ to below detection
(0.1 yg/£)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 environment because the
major portion of PAH's are associated with suspended solids1 .
Relationship of Mixed Function Oxidase Levels to Concentrations of a
Specific Carcinogen (Benzo(a)pyrene} in Tissue of Rainbow Trout--
The few reported environmental concentrations of B(a)P in fishes have
been low. For example, Pancirov and Brown"2 found only 1.5 yg/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 (0.40 + 0.21 yg/1) is high compared to ground water
concentrations (0.0001 yg/1)' , it was less than the concentrations from se-
veral metropolitan raw water supplies in the U.S. (1-2 ygZl)" , 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"5
who found that B(a)P was rapidly metabolized by mosquitofish (GambuAia) 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 yg/kg.
49
-------
BIOACCUMULATION OF POLYNUCLEAR AROMATIC HYDROCARBONS
The accumulation of aromatic hydrocarbons from water^l,226,227 and
food226'228'229 by a variety of aquatic organisms161»2^6,^30 including algae,
plankton, mollusks, worms, clams, and fish (Tables 13 and 14) has generated
some interest23'. Organisms containing accumulated aromatic compounds may be
involved in food-chain biomagnification processes which may result in in-
creased concentration of hydrocarbons per weight of tissue at successively
higher trophic levels, perhaps including humans'0' >'71 »"°»"l >"£. Also
aromatic hydrocarbons, accumulated in higher organisms containing mixed-
function oxidase enzymes, might be transformed by these enzymes into meta-
bolites caoable of producing adverse effects on the organism or its progeny
T30,ZT7,229,23T,233,234 These concerns are apparently justified by the ob-
servation of increased tumor frequency in fish living in polluted water con-
taining PAH (e.g., 10-50 yg/£ benzanthracene235) with respect to fish
living in relatively unpolluted water235'237. Although it is not known if
PAH were involved in the tumor production, it is known that certain PAH are
capable of producing tumors in laboratory fish23".
Aquatic organisms, such as Tubx^ex. 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'30,226,225. in
organisms of the latter type, it has been noted that in some cases the pre-
sence of certain amounts of PAH .and other compounds may lead to increased le-
vels of PAH metabolic activity130'204'205'207'231 (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 .presence of an inhibitor may lower the metabolism and raise the
accumulation2 '^".
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.
*Bioconcentration factor = concentration of compound in tissue
concentration of compound in water
50
-------
TABLE 13
LEVELS OF VARIOUS AROMATIC COMPOUNDS IN AQUATIC ORGANISMS IN THE ENVIRONMENT
RESULTS FROM SELECTED LITERATURE REPORTS
Concentration In
Compound
benzo(a)pyrene
benzo(a)anthracene
benzo(a)pyrene
pyrene
nethylpyrene
fluorene
phenanthrene
methyl phenanthrene
benzo(a)pyrene
tenzo(a)pyrene
benzo(gh1 )perylene
benzo(a)pyrene
benzo(a}pyrene
benzo(a)pyrene
benzo(a)anthrene
benza(a)pyrene
pyrene
nethylpyrene
benzo{a)pyrene
pyrene
nethylpyrene
total PAH
Aroclor 1254
polychlorlnated blphenyls
Organism
mussel
oyster
oyster
oyster
oyster
clan
clam
clam
mussel
shellfish
•ussel
mussel
mussel
crab
menhaden
flounder
flounder
oysters
burbot
lake trout
long-nose
sucker
slimy sculpin
nysld
fish
mussels
Location kg
on creosoted
Pilings
Long Island
Sound
LII. Sound
L. 1. Sound
L. I. Sound
S.California
?Ha1ne
S.California
on pilings
new creosoted
timbers
Vancouver
Rarltan Bay,
New Jersey
Rarltan Bay,
New Jersey
Long Island
Long Island
Gulf of
Mexico
H. Lake
Superior
V
Puget Sound
wet weight)
0.049:0.015
0.003
0.002
0.058
0.011
0.046
0.88
0.56
0.0023
0.016
0.025
0.008
0.045
0.215
0.002
0.003
0.006
0.002
0.002
0.002
0.0005
2-9
1.4i0.4
1.8*1.6
0.9^0.8
0.34
0.085t0.029
0.84
0.21
• ' * J J^ WH^CH^I H C IUII
analyzed in water (ug/kg)
whole organism
whole organism
whole organism
whole organism*
whole organism
whole organism*
whole organism
whole organism
whole organism
whole organism
whole organism
whole organism
whole organism0
whole organism
whole organism
whole organism
whole organism
whole organism
whole organism6 .0008
whole Organism6 .0008
whole organism6 .0008
whole organism6 .0008
whole organism6 .0008
whole organisn6
o luiviiivnki a nun
Factor4 (ttire) Ref
244
222
245
237
246
247
248
249
222
222
222
250
1.75x10*
2.25xlOS 251
l.13xlOS
4.25xl05
l.06xlfl5
252
•Btoconeentratlon factor 3 (concentration of compound 1n fish In ug/kg)/(concentration of compound 1n water In pg/kg).
"shucked
'conposltes
51
-------
Compound
TABLE 14
TABULATION OF LEVELS OF VARIOUS AROMATIC COMPOUNDS IN AQUATIC ORGANISMS IN LABORATORY EXPERIMENTS
RESULTS FROM SELECTED LITERATURE REPORTS
en
ro
anthracene0
naphthalene
naphthalene^
naphthalene j
phenanthgene
chrysene .
benzo(a)pyrane
naphthalene .
1-methyl naphthalene
naphthalene .
1-methyl naphthalenej
2-methylnaphthalene .
di-methylnaphthalene .
tri-methylnaphthalene
benzo(a)pyrene
benzo(a)pyrene£
benzo(a)pyrene
benzo(a)oyrene
biphenyl
Aroclor 1254*
Aroclor 1254
heptachlor
trans-Chlordane
Aroclor 1254e
Aroclor 1254e
heptachloronorbornene
pentachlorophenol
pentachloroanisole
A
p-d1chlorobenzene
2,21,4,4l-tetra-
chlorobiphenyl
?Bioconcentration factor =
Organism
Daphm'a pulex
polychaete
polychaete
clam
clam
clam
clam
sheepshead minnow
sheepshead minnow
clam
clam
clam
clam
clam
clam
mosquito larva
snail
mosquito fish
rainbow trout
cockle
tell in
spot
spot
spot
spot
fathead minnow
rainbow trout
rainbow trout
Cone. In
Organism
(mg/kg)
0.015*
•x-5.5
•V.-4.2
0.43±0.01
2.8±1.1
0.54±0.3
0.45±0.1
60
205
1.9
2.9
3.9
4.1
0.8
7.2
0.0942
5.1523
0.0
40.8
34.5
0.308
0.132
27
448
16
6.5
6.0
1.0
3.2
1.0
85.0
2.3
rainbow trout
rainbow trout
Tissue Analyzed
whole organism
whole-body males
whole-body females
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 orgariism(shucked)
whole organism
whole organism
whole organism
whole organism
whole organism
liver
blood
fat
muscle
liver
blood
fat
muscle
muscle
muscle
H20 Cone.
Bioconc.
(time)
Factor
a
0.02
20
27
71
89
66
52
1000
1000
840
340
480
240
30
30.5
2.5
2.5
2.5
250
250
0.14
0.04
1
40
26
26
26
26
24
24
24
24
1.6±0.2
760 (24 hrs)
275*(20 hrs)
156* (20 hrs)
6.1 (24 hrs)
32.0 (24 hrs)
8.2 (24 hrs)
8.7 (24 hrs)
60* ( 4 hrs)
205*( 4 hrs)
2.3 (24 hrs)
8.5 (24 hrs)
8.1 (24 hrs)
17.1 (24 hrs)
26.7 (2*4 hrs)
236 (24 hrs)
2177 ( 3 days)
37 ( 3 days)
0 ( 3 days)
438±38 (»)
163 (40 days)
138 (40 days)
2,200 (72 hrs)
3,300 (72 hrs)
27,000 (56 days)
11,200 (30 days)
615 (24 hrs)
250 (24 hrs)
231 (24 hrs)
39 (24 hrs)
133 (12 hrs)
47 (12 hrs)
3,542 (12 hrs)
96 (12 hrs)
215±21 (-)
9850+2890 (»)
Reference
241
253
45
254
46
225
240
255
256
257
258
259
259
240
239
(concentration of compound in fish in ug/kg)/(consentration of compound in water in ug/kg)
Value calculated for data in article static, C static flow through renewed static
-------
Other methods for the bioconcentration factor derivation include in-
direct approaches based on pharmacokinetic models221'232'239 or estimates
based on Rartltion coefficients22!,232,240, In studies involving measure-
ments «a»«»i^i,OH- or'^C-labeled compounds are most often employed and meta-
bolites are usually, but not always242, 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 m£/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 I water,
which contained ^10 y£ 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 ^2% 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/6C-PID procedure.
The analysis of the 3-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
-------
absorbance of the resulting HPLC fraction, which contained phenanthrene and
B-naphthoflavone, was determined at 270 and 255 run. Using Beer's Law plots
for each of the compounds at each of the wavelengths, the concentration of the
phenanthrene and 3-naphthoflavone in the water could be calculated. For the
analysis of the 3-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 m£/min total flow with a 5 y 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:
BCF =
FCN(I)
MWCN(L)
- EBCF(I)
where BCF(I)
FCN(I)
MWCN(I)
bioconcentration factor for day I
mg of compound per kg fish on day I (Table 16)
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
I WCN.
MWCN(I) = jfj 1
n
EBCF(I) =
/FCN(I) + EFCN(I) * RrFm
1MWCN(I) - EMWCN(I)} " BCF(I)
where j
n
WCNj
EFCN(I7
and
water sample number (1, 2, 3 n)
number of water samples taken up to and including day I
concentration of compound in tank water for j sample
error involved in determination of FCN(I) (see Appendix B)
(WCN. (I)}
EMWCNtI) = error in MWCN(I) =/^_
V
Determination of Particulate PAH Load--
Z WCN. (I)
= J
n - 1
In order to determine if any PAH material was associated with particu-
late matter (11.4 mg/£) 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
-------
TABLE IS
SUMMARY OF BIOCONCENTRATION FACTORS (BASED ON TOTAL WET WEIGHT OF FISH)
en
en
Exp Tank
f 1 Compound
1
1
dlbenzofuran
fluorene
2 9-chlorophe-
nanthrene
2
•j
phenanthrene
8-naphtho-
flavone
2 phenanthrene
phenanthrene
3
1
B-chlorophe-
rianthrene
2 phenanthrene
3
dlbenzofuran
fluorene
phenanthrene
1 -methyl phe-
nanthrene
fluoranthene
Dvrene
Day 11
260*
50
250*
50
1,140*
80
990*
100
2,000*
300
2,000*
200
4.400*
600
Bioconcentratlon factor
Day IZ Day #4 Day #7
540*
70
500*
60
1,200*
240
800*
700
1 ,700*
200
2,400*
600
6,100*
1,000
1 ,400*
350
1,200*
400
2,000*
300
1 ,200*
400
1 ,600*
300
1 ,200*
250
1,200*
200
1,200*
100
1 ,900*
1,200
800*
700
2.000*
250
1 .600*
900
6.400*
11 ,000
1,500*
200
830*
200
830*
300
2,000*
200
1,400*
500
2,600*
700
1 .200*
400
1,200*
200
1 ,200*
100
1 ,700*
1,000
1 ,300*
900
2,300*
200
1 ,800*
800
5,600*
5,000
1 ,400*
400
860*
300
1,100*
500
2,000*
600
1 ,300*
400
4.000*
600
1 .400*
700
• (wg PAH/kg f1sh)/{u9 PAH/kg
Day #10 Day #14 Day #18
1
3
1
1 ,500*
750
*90*
40
3,300*
400
1
5
2
1
1
2
1
3
2
3
2
1
.100*
150
870*
100
,200*
,500
3,700*
700
,900*
600
.600*
,000
,300*
400
,800*
900
,200*
,000
,100*
600
.100*
700
.200*
800
,300*
,000
water)*
Day #21
1 ,000*
200
1,100*
zoo
1 ,200*
600
2.100*
600
5,100*
2,000
2,000*
600
1 ,200*
500
1 .300*
500
2.200*
600
1 .800*
600
3.600*
1,000
2,600*
1,000
Day 28
Day #25 Trial #1
1.100*
160
1.100*
200
5.000*
2,000
1,900* 2.5001
1.100 1,300
*100* «100±
80 40
4,200* 5.100*
1.000 1,600
Z.OOO*
600
5,100*
2,000
3,100*
1,000
1 .700*
600
1 .800*
800
1 ,900*
500
700*
300
1,500*
500
970*
500
Day 28 MFO
Trial #2 Activity Z L1p1d-± S.D
Mn A O*l C
f* v f • Ox 4*3
YAS 4 1+fl R
• *TJ tf IXw*O
V*. A Qj.1 C
f C> *f * OX I • 3
'
2,800*
750
6.700* YM 4.1*0.5
3,000
3,000* N . , .
1,000 "° 1.3*1.0
1 ,600*
500
1,500*
400
-------
TABLE 16
SUWARY OF FISH ANALYSES (BASED ON TOTAL MET WEIGHT OF FISH)
Exp
cn
Tank
/ Compound
Idlbenzofuran
1
Ifluorene
. 9-chlorophe-
nanthrene
1
shenanthrene
j-naphtho-
flavone
2 phenanthrene
Iphenanthrene
1 f
p-chlorophe-
(nanthrene
2 phenanthrene
3
dtbenzofuran
fluorene
phenanthrene
1-irethyl phe-
nanthrene
fluoranthene
pyrene
Day 11
0.96i
0.19
0.93t
0.19
0.87i
0.05
4.031
0.22
<0,05
5.141
0.53
3.79*
0.07
3.791
0.21
Day 12
1.971
0.26
1.87i
0.22
0.851
0.07
2.50*
0.21
<0.05
4.31!
0.24
4.521
0.65
5.271
0.48
3.171
0.59
3.06*
0.67
4.54*
0.29
1.501
0.41
1.93*
0.25
1.101
0.09
Concentration of PAH
Day »4 Day 17 Day 110
4.00i
0.19
4.151
0.19
1.13i
0.08
2.52!
0.20
4.96!
0.81
3.881
0.32
9.161
1.64
3.381
0.24
1.931
0.39
2.051
0.40
4.621
0.06
1.83!
0.45
3.08*
0.65
1.04!
0.18
4.03i
0.34
4.45±
0.26
1.02t
0.06
3.761 4.44i
0.42 0.31
<0.05 <0.16
5.981 8.651
0.20 0.58
4.72t
0.40
8.27i
1.01
3.351
0.39
2.3U
0.18
3.101
0.32
4.131
0.26
1.46*
0.14
4.42!
0.18
1.361
0.20
In Fish: mg PAH/kg fish
Day 114 Day f!8 Day *21
3.491
0.17
3.16i
0.11
l.Slt
0.13
5.12i
0.28
8.69±
0.44
1
3.141
0.37
4.271
0.32
5.331
0.33
6.751
0.27
2.781
0.24
4.011
0.16
2.561
0.35
3.371
0.22
3.931
0.32
0.66i
0.10
10.061
0.42
5.63i
0.49
7.501
0.68
5.05i
0.50
2.731
0.22
3.21i
0.22
4.88±
0.67
2.351
0.28
4.661
0.42
2.801
0.35
Day »25 Day 128
3.47i
0.18
3.901
0.22
2.70±
0.23
4.901 6.431
0.43 0.22
<0.18 <0.19
10.9% 12.90*
0.73 1.04
5.10 1
0.33
7.231
0.43
7.191
0.29
4.021
0.30
4.381
0.70
-.4.201
0.47
0.961
0.19
2.041
0.29
1.141
0.24
Day »2B Depuration Mean ug PAH/I
Trial 12 Day 129 Day «0 Day *31 Day 13? Day H5 water
7.131
0.31
9.46!
0.71
7.05!
0.52
3.81*
0.23
3.62±
0.17
4.921
0.29
1.7W
0.24
2.55!
0.62
oT
<0.1
<0.1
2.75t 0.59*
0.19 0.08
<0.42
5.97i 1.8%
0.43 0.18
1.92i <0.1
0.31
4.30t 1,791
0.44 0.48
1.611 <0.1
0.27
1.261 <0.1
0.07
1.321 <0.1
0.07
1.651 <0.1
0.28
0.141 <0.1
0.21
0.761 <0.1
0.16
<0.1 <0.1
<0.1 3.19-0.26
<0,1 3.«i0.38
<0.1 0.55±0.12
<0.1 2. 63*0.83
1.82*0.18
0.49*. 2.55*0.44
0.10
<0.1 2.53H0.44
<0.1 1.41i0.34
2.34*0.54
2.34*0.51
2.48i0.48
2.20±0.25
1.38i0.19
1.35i0.20
1. 1810.27
-------
TABLE 17
BIOCONCENTRATION EXPERIMENTS: SUMMARY OF WATER ANALYSIS
Com-
pound
Day
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Mean
* Std
Dev.
Exp.
Tank 1
dlbenzo fluor-
furan ene
3.65± 3.72±
0.02 0.01
3.15+
0.03
3.13±
0.14
3.49±
0.15
2.99±
0.46
3.05±
0.28
3.19*
0.21
2.fi7±
0.44
3.19±
0.26
3.49±
0.01
3.78±
0.07
3.95*
0.07
3.28±
0.22
3.42±
0.32
2.78±
0.15
3.16±
0.26
3.45+
0.38
1 Exp. 2 Exp. 3
Tank 2 Tank 1 Tank 2 Tank 1 Tank 2 Tank 3
9-chloro- phenan- B-naphtho- phenan- phenan- 9-chloro- phenan- dlbenzo- fluor- phenan- 1-methyl- fluor- pyrene
phenan- threne flavone threne threne phenan threne furan ene threne phenan ene
threne threne threne
0.76± 4.08± 1.82± 2.55*
0.01 0.22 0.26 0.14
1.92± 0.86±
0.66±
0.01
0.37±
0.05
'
0.47±
0.02
o.eot
0.08
0.45+
0.08
0.60±
0.08
0.50+
0.02
0.47+
0.06
0.54+
0.12
2.22±
0.24
2.48±
0.17
2.86±
0.13
3.51±
0.31
2.29±
0.26
2.07+
0.10
1.50±
0.07
2.63±
0.83
1.82±
0.19
1.83±
0.20
1.76±
0.22
1.71 +
0.22
1.66+
0.18
1.74+
0.15
2.24±
0.17
1.82+
0.18
2.74±
0.21
2.47±
0.14
3.27±
0.70
2.80±
0.50
2.044
0.08
2.01±
0.08
2.55±
0.44
0.14
2.98±
0.17
3.07±
0.08
2.76±
0.09
3.26*
0.15
2.17±
0.18
2.38±
0.17
2.79*
0.16
2.75+
0.17
2.40+
0.13
2.06±
0.22
2.42+
0.14
1.9U
0.18
2.53±
0.44
0.06
1.99+
0.32
1.63±
0.10
1.60±
0.07
1.82+
0.12
1.46±
0.28
1.23+
0.08
1.22+
0.16
1.46+
0.08
1.38+
0.11
0.97±
0.12
1.07±
0.14
1.68±
0.19
1.41 +
0.34
2.13+
0.14
2.28±
0.10
2.67±
0.23
2.07±
0.25
2.99±
0.08
2.26±
0.16
2.21±
0.17
2.93±
0.17
3.13*
0.15
2.23+
0.11
1.99±
0.15
1.14+
0.20
2.34±
0.54
2.32+
0.13
2.40±
0.16
3.35±
0.34
2.70±
0.16
1.39±
0.08
1.96+
0.10
2.28±
0.12
2.35±
0.08
2.43±
0.10
2.09+
0.20
2.51+
0.22
2.34±
0.51
2.46±
0.22
2.56±
0.18
3.68±
0.43
2.06±
0.08
1.73±
0.08
2.28+
0.12
2.34±
0.13
2.52+
0.08
2.50±
0.13
2.56+
0.14
2.57±
0.14
2.48+
0.48
2.28±
0.14
1.66±
0.18
2.39±
0.14
2.17±
0.09
2.49*
0.09
2.30*
0.11
2.19*
0.11
2.09*
0.17
2.34±
0.13
2.42±
0.15
1.86*
0.14
2.20*
0.25
1.30*
0.10
0.91*
0.05
1.23*
0.07
1.44+
0.04
1.36*
0.06
1.55*
0.11
1.50+
U.ll
1.44±
0.37
1.33*
0.10
1.59*
0.11
1.49±
0.11 -
1.38*
0.19
1.19±
0.04
0.98*
0.07
1.16*
0.06
1.20*
0.15
1.38±
0.05
1.57+
0.15
1.49*
0.10
1.35*
0.25
1.47*
0.12
1.53*
0.16
1.57*
0.13
1.35*
2.20
0.89*
0.09
0.78*
0.21
1.19+
0.07
1.06*
0.13
1.32*
0.09
1.54*
0.15
1.03±
O.Q7
0.95*
0.10
1.44*
0.10
1.58*
0.10
1.21*
0.09
1.18*
0.27
57
-------
Exp
#
Tank
#
1
2
1
2
1
TABLE 18
AHH ACTIVITY IN FATHEAD MINNOWS EXPOSED TO PAHsa
Compound
dibenzofuran
fluorene
9-chlorophenanthrene
phenanthrene
6-naphthoflavone
phenanthrene
phenanthrene
9-chlorophenanthrene
phenanthrene
dibenzofuran
fluorene
phenanthrene
1-methylphenanthrane
f1uoranthene
pyrene
AHH Activity
(pM 3-OH-BaP)/(mg protein/min)
Day 7 Day 14 Day 28
0.23+0.02 0.30±0.04
0.2U0.09 1.0±0.10
1.0±0.4 1.0±0.5
0.3U0.02 0.4±0.1
0.37±0.08 1.5±0.2
0.7±0.3 0.7±0.3
1.5±0.8
1.4±0.1
0.69±0.04
0.44±0.02
1.3±0.3
0.5±0.2
1.0±0.5
The mean and standard deviation for controls was: 0.53U0.213 based on
8 samples, each containing 15, 6-10-week-old fathead minnows.
The AHH values in this experiment were based on duplicate determinations,
whereas all others were done in triplicate.
58
-------
4000
ee
g
< 3000
o
pi
at
ui
u
8
i
2000
1000
FIGURE 10
SUMMARY OF DIBENZOFURAN
EXPERIMENTS3
DIBENZOFURAN
(5PAH'S)b
(FLUORENE)6
0 24 6 8 10 12 14 16 18 20 22 24 26 23 30 32
TIME (days)
8Based on data in Tattle 15. Exp. #3, Tank 13; fluorene, phenanthrene,
1-methylphenanthrene, fluoranthene, and pyrene also present. cExp. #1,
Tank #1; fluorene also present.
FIGURE 11
SUMMARY OF FLUORENE
EXPERIMENTS3
4000
FLUORENE
(5 PAH'S)b
(DIBENZOFURAN)*
• • IT • ; ? - - * r -i.- * =_.-...-_.
24 6 8 10 12 14 16 18 20 22 24 26 28 30 32
TIME (days)
aBased on data in Table 15. Exp. #3, Tank #3; dibenzofuran, phenan-
threne, 1-methylphenanthrene, fluoranthene, and pyrene also present.
CExp. #1, Tank #1; dibenzofuran also present.
59
-------
FIGURE 12
SUMMARY OF PHENANTHRENE
EXPERIMENTS3
<
se.
u
Z
O
7000
6000
5000
z 4000
O
3000
2000
1000
PHENANTHRENE
PHEN ALONE (EXP.2)
—. PHEN ALONE (EXP.3) .
—« PHEN+BETA-NAPHTHOFLAVONE (EXP.2)
. _ PHEN+9-CHLOROPHENANTHRENE (EXP.3)
. PHEN+5PAH'S (EXP.3)
_t • t t
0 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32
TIME (days)
Based on data in Table 15. Exp. #2; no other compounds present.
Exp. #3, Tank #2, no other compounds present. dExp. #3, Tank #1;
9-chlorophenanthrene also present. eExp. #2, Tank #1; B-naphthoflavone
also present. Exp. #3, Tank #3; dibenzofuran, fluorene, 1-methyl-
phenanthrene, fluoranthcne, and pyrene also present.
60
-------
8000
7000
6000
5000
u
z 4000
o
§
t? 3000
U
O
2000
1000
FIGURE 13
SUMMARY OF 9-CHLOROPHENANTHRENE
EXPERIMENTS3
9-CHLOROPHENANTHRENE
024 6 8 10 12 14 16 18 20 22 24 26 28 3O 32
TIME (days)
8Based on data in Table 15. Exp. #3, Tank #1; phenanthrene also
present. cExp. #1, Tank #2; no other compounds present.
61
-------
7000r
FIGURE 14
SUMMARY OF EXPERIMENT
WITH SIX PAH COMPOUNDS2
Ctf
2
u
2
z
o
5
as
»-
ui
U
8
o
03
6000
5000
4*
o
o
0
W
0
O
0
2000
1000
-» DIBENZOFURAN
• 1-METHYLPHENANTHRENE
• PYRENE
. FLUORENE
. PHENANTHRENE
._. FLUORANTHENE
0 24 6 8 10 12 14 16 78 20 22 24 26 28 30 32
TIME (days)
Table 15, Exp. #3, Tank #3.
62
-------
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 I of water) was in the extract (i.e., associa-
ted with the particulates in 2.5 £ 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 the water (Table 17). Such
variations are common in experiments of this type2 .
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-methyl -
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|y° and
the previously mentioned errors in bioconcentration factor values make con-
clusions tenuous. For example, the presence of $-napthoflavone
210 212 243
(5,6-benzoflavone), which has been reported to be an AHH inducer
showed little significant increase in AHH activity (Table 18, exp. #2). How-
ever the fish in the tank containing the flavone (Table 16, exp.-fl) 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
-------
In contrast to g-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
-------
AQUEOUS CHLORINATION OF POLYNUCLEAR AROMATIC HYDROCARBONS
Chiorination 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 areas, and to the reduction
of algal and bacterial growth in cooling towers260"265. 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 refeccecLto as the most valuable
and versatile tool available to the water chemist^ '.
The possible reaction of chlorine with materials present in the treated
water has long been recognized267, mainly because of the very practical ne-
cessity for using more,chlorine than was anticipated to meet given standards
of turbidity, BOD" , or fecal coliform bacteria. Environmentally, chlo-
rine and chloramines (as reaction products of ammonia, amino acids, or other
amines with chlorine) are considered deleterious26'*'2'0, and considerable
effort has been directed toward their removal by such processes as reduction
(e.g., S02)*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 initiatedpwben the chlorination process generated
problems of taste and odor266' . However, since these initial reports,
there have been documented examples of the incorporation of chloripe_igto such
systems as "activated" aromatics, humic acids, and nucleic acids^/b .
Typically, the chlorine incorporation into these systems results in decreased
degradabilitv and increased toxicity266'269. Similar studies involving the
ubiquitous'26 and, in some cases, carcinogenic53 polynuclear aromatic hydro-
carbons (PAH's) have not been extensive54. However, it has been reported that
the concentration of various PAH's in water is reduced upon chlorination141'
£o I ~ ti/O _* «J 4-U -i 4. -i -C,-v.., x\-C -4-U^i v»/-\c"ii"I-f--i*"in n v»rvrli ir- -f-c tia \/Q Kaon iclpntlfl ^d * CHI 0""
and that a few of the resulting products have been identified: chlo-
d naphthalene291'293 and C2_3-naphthalenes28l3s chlorinated acenaphtha-
93, 5-chloro-3,4-benzopyrene292 and S^-benzopyrene-S.S-quinone156'^^
rinat
lenes
as well as arene oxides
Results
The results of the application of the C-18/HPLC/GC-PID/GC-MS method to
?tudv of aoueous chlorination reactions of PAH's are provided in Table 19.
the study of aqueous chlorination reactions of PAH's ^re providedi
Chlorinated PAH standarc
in Table 20. The study
Chlorinated PAH standards that were prepared and used in this work are listed
I. The study demonstrates the vulnerability of PAH's to conversion
65
-------
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 monochlorofluoranthene
standard. In contrast to the other compounds studied, experiments at pH M
with phenanthrene and its 1-methyl derivative gave very low total recoveries
for products (^40% and ^% respectively). Presumably very polar products
were generated in these reactions which were not concentrated by the 7x50 mm
C-18 column.
1 2
10
Cl
6 5
fluoranthene -H
net substitution
ffl
Cl
facile
3-chlorofluoranthene
polar chlorohydrin
66
-------
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 wherever
possible the '•'C 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-l, C-8,
and C-10) upon saturation 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.
10 9
67
-------
TABLE 19
Summary of Aqueous ChloHnatlon Studies
oo
Starting PAH
1 -Methyl naphthalene
1- Methyl naphthalene
Fluorene
Fluorene
Fluorene
Fluorene
Anthracene
Anthracene
Anthracene
Anthracene
Phenanthrene
[C12]
mg/4
24.0
20.4
1.205
18.8
23.5
21.3
12.9
0.0
12.4
2.0
3.2
[PAH]
ng/i
531
336
334
819
773
333
1000
1042
965
552
820
Reaction
Time, hr
3.0
3.0
0.5
3.0
3.0
3.0
3.7
4.0
3.75
0.08
0.5
PH
3.8
4.1
7.0
4.1
3.4
3.35
4.0
4.4
6.5
7.1
7.1
Products Basis
Identified A for
(% Yield) Assignment
Monochl oro-1-
methylnaphalene
Monochl oro-1-
methyl naphthalene
(73±5)
Fluorene
(73±4)
Fluorene
Fluorene
Monochl orof 1 uorene
Fluorene (-vS)
Monochl orof 1 uorene
(52±4)
Anthracene
(3)
Anthraqulnone
(90±H)
Anthracene
Anthraquinone
(78±9)
Anthraqulnone
(61±16)
Phenanthrene
a,b
b,c
b,c
a.b.c
a.b.c
a
b,c
b,c
a,b,c
a.b.c
c
b,c
b,c
a.b.c
Comments
M* = 176,178. GC retention time
is Identical to compound 1 (Table
20).
GC retention time identical to
compound 1 (Table 20).
M* = 166
M! = 166
M = 200, 202
GC retention time is Identical to
compound 2 (Table 20).
M* - 178
M* = 208
No anthraquinone produced according
to HPLC
H+ • 178.
-------
o>
to
Table 19 (Continued)
Starting PAH
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
1 -Methyl phenanthrene
1 -Methyl phenanthrene
1 -Methyl phenanthrene
Fluoranthene
FTuoranthene
[C12]
mg/i
3.7
26.3
19.3
19.5
20.0
3.1
21
25.6
3.4
22.0
[PAH]
ng/t
236
233
820
239
na
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
4.1
4.2
4.05
6.9
4.0
4.0
6.8
5.9
Products
Identified.
(2 Y1eld)d
Phenanthrene
(77±14)
Phenanthrene
(86+4)
Monochloro-
phenantnrene
(4±1)
Monochloro-
phenanthrenc
Phenanthrene
(9*4)
Monochloro-
phcnanthrene
(38±5)
Monochloro-
phenanthrene
(39±5)
1 -Methyl phenan-
threne
Monochloro-1-
methyl phenan-
threne
Monochloro-1-
methyl phenan-
threne (-v-O)
Fluoranthene
Fluoranthene
(63±3)
Basis
for
Assignment
b,c
b,c
b,c
a,b
b,c
b.c
b,c
a,b,c
a,b
b,c
a,b,c
b,c
Comments
Same GC retention time as com-
pound 3 (Table 20).
M « 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).
M* • 192.
M* = 226,228. GC retention time
identical to compound 5 (Table 20)
GC retention time 1s identical to
compound 5 (Table 20).
M* » 202
-------
Table 19 (Continued)
Starting PAH
Fluoranthene
[ci2]
mg/A
17.7
[PAH]
ng/A
824
Reaction
Time, hr
3.0
PH
4.1
Fluoranthene
23.9
239
3.0
4.03
Products
Identified
(5! Yield)3
Fluoranthene
Fluoranthene
chlorohydrln
Fluoranthene
(42±3)
Monochloro-
fluoranthene
(32*1)
Basis
for
Assignment
a.b.c
See
comments.
b,c
b,c
Comments
M* = 202
Appears to lose water readily to
form a monochloro fluoranthenc.
GC retention time identical to com-
pound 6 (Table 20). M = 236,238.
However, reverse phase HPLC reten-
tion time was much less, than com-
pound 6 (Table 20).
GC and HPLC retention times are
identical to compound 6 (Table 20
Footnotes for Table 19:
aMass spectral data. !|GC retention time was Identical to an authentic standard. CHPLC retention time was identical to an
authentic standard. Not corrected for recovery efficiency.
-------
PAH
1-Methyl naphthalene
Fluorene
Phenanthrene
1-Methylphenanthrene
Fluoranthene
Table 20
Monochloro Products
Produced by Preparative Scale Chlorination Reactions In Acetic Acid"
Moles PAH:
Moles C12
1:3
1:1
1:1
1:1
1:2
Reaction
Time, hr
18
3
7.3
18
18
r i uuuitl*
Reference
Monochloro Products Number Comments
Monochl oro-1 -methyl -
naphthalene
Monochl orof 1 uorene
Monochl orophenanthrene
Monochl oro-1 -methyl -
phenanthrene
Monochl oro-1 -methyl -
phenanthrene
Monochl oro-
fluoranthenc
1
2
3
4
5
6
0}1
M =
Mp»9:
M =
MD =
M =
OJ1
M*
MD
M
MD
M
176, 178
J°C
200,202
52-52. 5°C
212, 214
226, 228
87.5-88°C
226, 228
93-99°C
236, 238
Literature
l-Chloro-4-methyl-
naphthalene (295,296)
2-Chlorpfl uorene,
mp 96.5° (297)
9-Chlorophgnanthrene,
mp 53-53.5 (298)
3-chlorofluoranthcno,
mp 101-102° (299,300)
Footnotes for Table 20
temperature - 25°C.
-------
Experimental Details
Procedure for aqueous chlorination reactions156--
Purified water was placed into a pressure tank and, while stirring with
an overhead stirring motor, a solution of sodium hypochlorite^bu was added,
followed by sufficient 0.1 N. sulfuric acid to attain the desired pH. The
"free chlorine" concentration of the reaction mixture was determined iodo-
metrically.^0' An acetonitrile solution of the PAH was then added via
a 100 yl 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 M3.5 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.
1 cc.
Procedure for preparative-scale chlorination reaction--
Chlorinated PAH's required for comparison with the products of the
aqueous-chlorination reactions were prepared by the reaction of the parent PAH
with chlorine gas in acetic acid2^^. 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 packed with either Bondapak C-18 PorasilRB (37-75 y, reverse phase)
or PorasilRA (37-75 y, normal phase). The compounds obtained were re-
crystallized and checked for purity by GC-MS (see Table 20 and Appendix C).
72
-------
SECTION 7
SYNTHESIS OF METHYLATED NAPHTHALENES
Among the ubiquitous PAH's in the aqueous environment, a large number of
methylated derivatives are included'31" . The monitoring of these potentially
hazardous compounds have been described by Dr. F.C. Mopastero of the U.S.
Department of the Interior as "a national imperative"302. 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.E. Tomaszewski 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.
The present work describes a synthetic design that leads to the selective
synthesis of certain mono-, di-, tri-, tetra-, and pentamethyl 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 available
naphthalene oxide system 1_ to ring opening reactions with alkyllithiums-™4.
The general route is illustrated below for the synthesis of 2-methylnaphthalene
(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
-------
CH3Li
exo attacK
H20
-H20
The value of this route is the flexibility that can be gained by intro-
ducing methyls in the component parts of the epoxynaphthalene synthesis305.
(CH,)
3'n
and
(CH3)
3'm
m
methyl benzynes methyl furans
(generated J_n_ situ)
74
-------
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.
The reaction is illustrated below for the selective synthesis of 1,2,4-
trimethylnaphthalene.
2,5-dimethylfuran
CH3Li
THF
BF3.(C2H5)20
-H20
(of configuration shown)
75
-------
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 methyl lithium 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.
and
CH3Li
CH,
CH3Li
-H20
Y
-H£0
12
12'
Results
The synthetic scheme discussed above has been applied to the syntheses
of the methylepoxynaphthalenes listed in Table 21. The resulting methyl-
naphthalenes resulting from the interaction with methyl lithium are listed in
76
-------
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 methyl anthracenes and phenanthrenes via
the corresponding epoxyanthracenes and epoxyphenanthrenes.
CH3Li
etc.
CH3Li
etc.
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 M+/M+-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
pHo or C3H4. A hydrogen atom may also be lost by the M radical cation.
ith increasing methylation the loss of ^2 or C3H4 becomes less pronounced.
Wi
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 Kuehl of the EPA Environmental Research Laboratory,
Duluth.
Microanalyses--
The microanalytical data were obtained by Spang Microanalytical
Laboratory, Ann Arbor, Michigan. See Appendix G.
77
-------
Reagents--
Furan and anthranilic acid were obtained from Aldrich Chemical Company,
Inc., Milwaukee, Wisconsin. The 3-methyl and 5-methylanthranilic acids were
purchased from Pfaltz and Bauer, Inc., Stamford, Connecticut. Methyllithium
was obtained from Alfa Inorganics, Inc. The 2,5-dimethylfuran was synthesized
according to the procedure of Newman30".
General Procedure for the Conversion of the Anthranilic Acids to the 1,2-
dihydro- and 1,4-dimethyl-l,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 mi of isoamyl nitrate (0.022 mol) and 5 m£ of furan
(0.070 mol) or 2.5 dimethyl furan (0.045 mol)b. 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 m£ of
1,2 dimethoxyethane was added dropwise during 2h 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 6.
General Procedure for the ADdition of Methyllithium to the 1,4-Epoxynaphtha-
lenes--
The epoxynaphthalene (JU 4^ 5_, 6_, 7_, or 8J, 0.0039 mol, was dissolved in
50 m£ of dry THF under an anhydrous nitrogen atmosphere in a 250-m£ 3-nick
round-bottom flask fitted with a reflux condenser and magnetic stirrer.
Methyl!ithium, 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 methyl!ithium. The aqueous layer was
extracted with six 25-m£ 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.
Genera! Procedure for the Dehydration and Production of the Methylnaphtha-
lenes--
The partially purified alcohols produced above were dissolved in about
50 m£ 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-m£ 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 (1mm,70°). A pure product in almost a quantitative
78
-------
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 6.
79
-------
TABLE 21
SYNTHESIS OF POLYMETHYLEPOXYNAPHTHALENES
and
1, R,
4, R}
5., R]
6, R1
7., R1
8, R
H»
» o
H, R2 = H, R3 * CH3
H»
H,
o> O
H
ni Rp = H» R« = H
qi Rp = Hj KO = C
80
-------
TABLE 22
SYNTHESIS OF POLYMETHYLNAPHTHALENES
CH3L1;
THF '
H20
1.
10 and 10',
11 and IT,
12 and 12',
13 and 13',
n
«
= H
» H,
H, R2 = CH3,
o
= H
= CH
- ru p = H p - u
— urio» l\o rij i\o n
<3 b 0
— ru o — u D — u
- U13, K2 - H, K3 - H
81
-------
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APPENDIX A
ADDITIONAL TABLES AND FIGURES RELEVANT TO BIOLOGICAL STUDIES ON THE LEACHING AND VOLATILIZATION OF COAL
o
in
Dissolved Oxygen3
(rag/1)
Total Alkalinityfc
(mg/1 CaCo3)
EDTA Hardnessb
(mg/1 CaCo3)
pH
(20-22°C)
Conductivity
(pmhos/cm)
Turbidity
(N.T.U.)
Distilled Deionized
Water
( 7 samples)
mean range
1.27
0.0
1.19
TABLE A-l
PROPERTIES OF COAL LEACHATE
Distilled Deionized
Water Leachate
(48 samples)
mean range
Lake Superior Water
(48 samples)
mean range
Lake Superior Water
Leachate
( 6 samples)
mean range
6.81 3.70-8.50 8.06 6.87-8.60
t
1.01-1.52 5.11 3.40-6.70 42.62 41.20-44.60
21.53 17.78-24.75
0.84 0.33-1.60 45.50 44.46-46.43 13.98 2.74-14.41
5.55-6.92 7.8C 6.8-8.2
0.73-1.63 27
15-32
7.8C
92
4.45 1.1-8.8
7.5-7.9
90-104
0.54 0.16-1.9
70
7.22-8.09
65-77
2.4 1.3 - 3.2
Reference R-294 azide modification
Reference R-294
cMode of pH values
-------
TABLE A-2
EFFECTS OF COAL DISTILLATE ON DAPHNIA PULICARIA9
Coal Distillate. Conductivity Mortality Distilled Water . Conductivity Mortality
Concentration (%) (ymhos/cm) (%) Concentration (%} (vimhos/cm) (%)
0 88
20 72±6
40 56±1
60 42±2
80 24±1
0
1.7
3.3
10.0
58.3
10
25
50
75
80
100
82
70
48
27
19±1
4.7
0
0
0
0
83.3
100
aDaphnia 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.
Coal distillate and distilled water were diluted with Lake Superior water.
-------
TABLE A-3. FATHEAD MINNOW BEHAVIORAL RESPONSE TO COAL LEACHATE1
EXPOSURE OBSERVATION OF SPAWNING COLORATION
(week #1)
# SPAWNINGS EGGS/SPAWNING FISH/TANK
______ (ave. #1)
Leachate (Tank 1)
Leachate (Tank 2)
Control (Tank 3)
Control (Tank 4)
23
15
16
43.6
19
8
18
18
During 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.
-------
TABLE A-4
EFFECTS OF COAL LEACHATE ON SPAWNING SUCCESS IN FATHEAD MINNOWS
EXPOSURE
Water g COAL/1
100X 6.25
Leachate
100% 6.25
Leachate
90% 5.0
Leachate
_, 75% 4.17
o Leachate
Co
50* 2.78
Leachate
25% 1.39
Leachate
10% 0.55
Leachate
Lake Superior 0
Water
Lake Superior 0
Water
25% Distilled 0
water In Lake
Superior Water
CONDUCTIVITY
pmohs/cm
70
(65-77)
70
(65-75)
71
(66-78)
76
(71-83)
82
(78-86)
87
(85-89)
91
93
C92-94)
91
(89-92)
73
(71-74)
WEEKS/TEST
4
2
2
2
2
2
2
2
4
2
ITANKS
4
3
2
3
i
3
5
4
4
2
WANKS IN WHICH
SPAWNING OCCURRED
0
1
1
1
1
2
2
3
4
2
1 SPAWNINGS
0
2
3
2
3
5
2
7
16
6
^SPAWNING*' "ATCHABILITY. % {* EGGS)
0
85
79
162
170
69
97
102
70
138
0
88
(50)
94
(100)
88
(150)
64
(150)
81
(100)
90
(200)
72
(500)
69
(300)
-------
TABLE A-5
COUGH RESPONSE DATA FOR TWO BLUEGILL SUNFISH
EXPOSED TO COAL DISTILLATE
TIME
0800
0915
1000
1300
1550
0800
FRAME
- 0820
- 0935
- 1020
- 1320
- 1610
- 0820
CONCENTRATION (%)
Tank 1 Tank 2
0
1
5
5
5
5
0
5
20
20
20
20
AVE. COUGHS /MI N.
Fish 1 Fish 2
0.55
1.10
1.35
1.55
0.50
0.45
0.55
1.00
1.05
0.80
0.40
0.50
109
-------
Day
TABLE A-6
EFFECT OF COAL DISTILLATE ON HEPATIC MIXED-FUNCTION OXIDASE
PARAMETERS IN RAINBOW TROUT3
P-450 (nM/mg protein) AHH (pM/mg protein/min)
3
7
10
14
21
Control
0.130
0.125
0.156
0.065
0.082
0.112C
±0.037
(33%)
Distillate
0.108
0.097
0.033
0.138
0.089
0.093C
±0.038
(41%)
Control
1.234
±0.161
2.224
±0.161
0.968
±0.315
1.305
±0.041
1.124
±0.105
1.371C
±0.494
(36%)
Distilla
0.958
±0.308
1.968
±0.928
1.801
±0.374
1.353
±0.083
2.873b
±0.351
1.790°
±0.723
(40%)
aRainbow trout were exposed to 0.2% coal distillate under flow-
through conditions.
Significantly elevated compared to controls.
°Mean and Standard deviation over the 21 day period.
110
-------
FIGURE A-l
CONDUCTIVITY CHANGES DURING LEACHING
OF COAL9
25
50
TfME (hr)
75
100
aCoa1 leaching was monitored after adding ground coal (<0.5 mm) to
distilled deionized water at a coal to water ratio of 6.3 g/1.
Ill
-------
FIGURE A-2
GAS CHROMATOGRAPHIC ANALYSIS OF A HEXANE EXTRACTION
OF COAL LEACHATE3
BLANK
LEACHATE
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90
TIME (minutes)
3Two liter samples of distilled deionized water and centrifuqed 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. glass; injector: 250°C;
detector: 300°C; carrier flow: 20 ml/min; program: 80-2600C at 4°/min.
112
-------
FIGURE A-3
GAS CHROMATOGRAPHIC ANALYSIS OF A METHYLENE
CHLORinE EXTRACTION OF COAL LEACHATE3
BLANK
LEACHATE
30 36 42
54 60
72 78 84 9O
TIME (minutes)
aOne liter samples of distilled deionized water and centrifuqed coal leachate
(6.3 g coal/L distilled deionized water) were extracted with 50 ml methylene
chloride.
RC 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
-------
FIGURE A-4
GAS CHROMATOGRAPHIC ANALYSIS OF AN
ISOOCTANE EXTRACT OF COAL DISTILLATE
BLANK
COAL DISTILLATE
0 S 10 15 20 25 30 35 40 45 SO 55 60 65 70 75
TIME (minut.,)
aA steam distillation of 1500 ml distilled deionized was carried out in a
modified Nielsen-Kruger distillation apparatus for 6 hr.
As 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
-------
FIGURE A-5
UV SCANS OF COAL DISTILLATE
250 300
WAVELINOTH (MI)
a. UV spectra of distilled water (DW), Lake Superior water (LW), and coal
distillate (DIST).
b. After obtaining a stable baseline using distilled water, various dilutions
of coal distillate were made using Lake Superior water as the diluent.
c. Coal distillate (pH 5.0) was scanned and then rescanned after raising the
pH to 11.3 with 1 N NaOH.
115
-------
FIGURE A-6
GAS CHROMATOGRAPHIC ANALYSIS OF A HEXANE EXTRACT
OF XAD-2 PURIFIED LAKE SUPERIOR WATER3
LAKE SUPERIOR WATER
XAD PURIFIED LAKE WATER
IS
24
30 36
TIME (mlnul«i)
42 48
54
60
A 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
-------
1.4
1.2
1.0
« 0.8
o>
I 0.6
LU
x
E 0.4
0.2
FIGURE A-7
GROWTH RATES OF FATHEAD MINNOWS
EXPOSED TO COAL LEACHATE AND PURIFIED
LAKE SUPERIOR WATER3
LEACHATE
-• LAKE WATER
10 12 14
TIME (weeks)
16
18
20
22
24-
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.
-------
FIGURE A-8
EFFECTS OF COAL LEACHATE ON LIVER PARAMETERS
OF RAINBOW TROUT3
CONTROL
LEACHATE
20
18
16
14
12
Liver Wt.
14
21
28
01 3
d I
3JO
24
U
14
DNA
0 1 3
28
aRainbow 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.
Relative 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).
dDNA is the DNA content of the 15,000 g pellet/g total liver weight.
eAHH activity is expressed as pM 3-h.ydroxybenzo(a)pyrene/mg protein/min.
118
-------
FIGURE A-9
GAS CHROMATOGRAPHIC ANALYSIS OF
FATHEAD MINNOWS EXPOSED TO COAL LEACHATE
LEACHATE
LAKE WATER
20 25
30 35 40
TIME (minuHi)
45
50
55
60 65
75
aFathead minnows were exposed to coal leachate (6.3 g coal/L distilled
.deionized water) for 24 weeks prior to extraction.
bFathead minnows were exposed to XAD-2 purified Lake Superior water for 24
weeks prior to extraction. . . 0
GC conditions-column: SP-2250, 2m x 2mm n.d. glags; injector: 250 C; detector
300°C; carrier flow: 20 ml/min; program: 100-235 C at 4 /mm.
119
-------
FIGURE A-10
GAS CHROMATOGRAPHIC ANALYSIS OF RAINBOW
TROUT EXPOSED TO 0.2% COAL DISTILLATE
LAKE WATER
DISTILLATE
24
30
36 42 48
TIME (mlnut.s)
54 60
66
Rainbow trout were maintained in flowing Lake Superior water for 21 days
prior to extraction.
Rainbow trout were exposed to 0.2% coal distillate by metering distillate
into flowing Lake Superior water over a 21 day period.
GC conditions-column: 3% OV-101, 2m x 2mm i.d. glass; injector: 250°C;
detector: 300 C; carrier flow: 20 ml/min; program: 80-250 C at 4 /min.
120
-------
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 squares158 using the data
obtained by injecting 1 y«, of n solutions of known concentration into the
GC/PID:
x* = weight in nanograms of compound in injection of known solution i
y.j = 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
E x.
- _
x
n
yi
y -
n
-nx2
, l/?v sx\
a = ri z Y4 - .z,xi)
n\i=l 1 i=l 7
The weight of compound X|< in a 1 va injection of unknown solution k
which produced a GC peak of area or height ykj on the jtn injection is
given by:
121
-------
xk =
where
'xk
2
n-2
bZU
k is:
r = Z (y. - yr
1=1
o n 9
r = z (x, - xT
1=1 n
m = number of times unknown solution k was injected
m
1 yki
; =l£Lli
'k m
The weight of the compound (W) in the total volume of unknown solution
W = xk(M) ± [sxk(M) + xk(E)]
where
M = volume of unknown solution k in y£
E = 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
-------
APPENDIX C
CHLORINATED POLYNUCLEAR AROMATIC HYDROCARBONS:
MASS SPECTRAL DATA
»ORTE«xSPEC»
214/Ltl^DERL 77202 CHLORODIBEH20FURRN
Co
100-?
90-f
60-j
70H
60-i
SH
40-
30-i
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10
0
BRSE
SUM
24501 94694
PEHK INT
S 2456
3 2928
6 1434
7 1827
8 1487
9 1900
10 1907
11 1697
12 2509
14 1262
IS 1646
\7 1478
19 2336
20 3517
Z\ 17033
22 163?
23 1738
2? 24501
28 3164
g9 7555
30 1243
jlLiii
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1 0. Q2X 69. 0
11.9SX 69.5
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7. 4SX 75. 1 _-
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» • r 5 A 85.1
6:9sx B?.\ Inlet System: GC
t n o U V A Q t
s! isx 99^0 Instrument: Varian CHS
6. 71X 101. 0
6. 03X 113.0
9. S3X 137. 0
14. 35X 138. 0
69. SIX 139. 1
6. 68X 140. 1
7.09X 167.0
1 00. OOX 202. 0
12. 91X 203. 0
30. 83K 204. 0
5. 072 205. 0
20
1 0
50 100 ISO
SPECO 214 in DERL 77202 CHLORODIBEMHOFORHH
200 250
STEP MflSS=l, I/B'S • 15J
300
-------
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178XLM/OERL 77205 DICHLOROBEH20FURBH
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100-
90-
80-
70-
60-
50-
HO-
30-
20-
10-
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22364 1
PERK INT
1 2158
2 1131
3 2162
7 3852
8 2183
9 1449
14 1674
15 3546
16 2023
17 3362
23 1493
25 1149
30 2680
33 2641
34 1755
39 4723
>IO 6456
47 1641
48 14935
50 3886
58 1281
61 22364
62 11647
63 2989
64 1755
sun
30392
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9. 64X
5. 05X
9. 66X
17. 22X
9. 76X
6. 47X
7. 48X
15. 85X
9. 04X
15. 12X
6. 67X
5. 13X
1 I. 98X
11. 80X
7. 84X
21. 11X
28. 86X
7. 33X
66. 78X
17. 37X
5. 72X
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52. 072
13. 36X
7. 84X
1 • •
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74:4 Inlet System: GC
75.4
es.o Instrument: Varlan CH5
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86. 5
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99. 0
111.0
118. 0
119.0
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STEP rinss'i, I^BXS « ix
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-------
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ro
in
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32736
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2776 8. 47X 83. 0
3519 10. 74X 85. 0
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6566 20. QSX 87. 1
1991 6.. 08X 88. 1
3646 1 1 . 13X 89. 1
3095 9.45X 98.0
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34
36
37
38
42
43
5237 15. 99X 115.1
2076 6. 34X 135. 0
2758 8. 42X 137. 0
2125 6. 49X 138. 0
3963 12. 10X 139. 0
2092 6. 39X 161.1 1
3902 11.91X 162.0 1
44 18892 57.71X 163.0 1
45 13978 42.69X 164.0
46 32736 100. OOX 165. 1 ]
47
9371 28. 62X 166. 1
49 14452 44. 14X 200.0 1
50
51
I
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STEP riflSS'l, IxB'S * IX
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CHLOROMETHYLNAPHTHALENE
Electron Energy:
Inlet System:
Instrument:
70ev
GC
Varian CH5
en
100-^
80
70
60
50
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59'LI1-'DERL 77188
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sun
32736 259891
PEflK INT
I 2224
6 1645
9 6472
13 7311
16 2182
22 2987
23 5128
25 5064
27 10181
29 1914
31 2128
43 6650
45 5278
47 26218
48 2233
49 2309
54 2003
58 2292
60 2729
61 22488
64 4470
65 32736
66 2934
68 5108
74 3578
79 4471
80 23042
81 4774
83 5944
"T"'|""l | 1 ' 1 ' | ' 1
0 200
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6. 79X
5. 02X
19. 77X
22. 33K
6. 66X
9. 12X
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15. 46X
31 . 1 OX
5. 84X
6. 5 OX
20 31X
16. 12X
80. 08X
6. 82X
7. 05X
6. I IX
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8. 33X
68. 69X
13. 65X
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8. 962
15. 60X
1 0. 92X
13. 65X
70. 38X
14. 58X
18. I5X
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MflSS
70. 0
72. 7
73. 8
74. 9
76. 0
86. 1
87. 2
68. 1
89. 2
91.2
99. I
13. 1
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15.3
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26. 1
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39. 2 10
140.2
141.3
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175. 2
176. 2
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250 300
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ro
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1325
3966
1211
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1 063
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1 059
9346
1627
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24. 20X
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45. 84X
7. 98X
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14.. 15X
5. 54 X
30. 7SX
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5. 84 X
9.S6X
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13. 80X
10. 36X
18. OSJi
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5. 33X
20. £6X
21 . 72X
S. S4X
92. 04X
24. 32X
26. 65X
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18. 26X
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"5| 70 112? 5.52X
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-,J'| 73 £371 II.63X
7* ' « 75 6124 30. 04 X
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12 ° 82 10134 49.71X
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174.
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37. 1
|?.6 inlet
88.1
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11 t Instrument:
97. 0
98. 1
99. 1
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11.0
12. 0
13. 0
14.0
15.0
16. 0
25. 0
37. 0.
33. 1
138. 5
139. 1
140.1
149. 0
150. 0
151.1
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ro
CO
100-;
50-
80
70-
60
50-
40-
30-
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w-
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sun
31176 888071
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7
9
10
18
13
16
17
18
30
21
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24
27
30
32
33
34
38
40
50
59
to
61
62
66
67
69
69
70
73
77
78
79
80
INT
1814
8073
12490
2929
2743
1935
2919
SOS?
3629
18437
2393
1707
2113
1653
3536
3898
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1775
5820
17438
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5087
9049
29732
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2236
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25. 892
40. 062
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73. 1
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75. 0
76. 0
77. 0
85. 1
86. 1
87. 1
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88. 1
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93. 0
94. 0
98. 0
99. 0
100.0
106. 0
107. 0
126. 0
149. 1
150. 1
151. 1
152. 1
174. 0
175. 0
176. 0
177. 0
178. 0
186.0
212. 0
213. 0
214. 0
215. 0
77204 CHLOROPHENflMTHREHE
Electron Energy: 70ev
Inlet System: GC
Instrument: Varian CH5
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106 150
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2 0 0 £50
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300
-------
»DHTE«XSPEC» 207/LMXDERL 77206 DICHLOROPHEHflHTHREME
r\j
10
100 ;
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»OftTE«/SPECO
77179 CHLOROtlETH YLPHENRM THREME
CO
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8899
PEHK
1
2
3
4
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7
8
9
1 0
1 1
15
17
18
21
22
23
24
85
26
27
28
29
31
32
36
37
39
40
43
44
47
48
50
52
54
55
56
57
58
59
60
65
66 .
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miJTTnjrmfnt
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0 28019
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2776
7036 2
4382 1
1606
2340
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5742 1
2992 1
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8967 1
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4961 1
1537
5029 1
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3028 1
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61 08 2
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11679 4
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7
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9. 57X
4. 27X
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5. 53X
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APPENDIX D
CHLORINATED POLYNUCLEAR AROMATIC HYDROCARBONS:
iTHKESH(CM)* 1.0 13£ : ]-—••. •—
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3178.6 126.140
3172.1 125.884
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3153.3 125.136
3141.1 124.652
3131.1 124.255
1975.8 78.409
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142
-------
APPENDIX E
METHYLATED NAPHTHALENES: ]H (60 MHz) DATA
Chemical Shift Data in Delta for the Methyl Groups in the
Polymethylated Naphthalenes
Compound No. of Methyls* 5 Values**
3 1 2.50
9 3 2.73, 2.65, 2.59
10 & 10' 4 2.56, 2.56, 2.47, 2.43
11 & IT 8 2.95, 2.95, 2.95, 2.95, 2.81, 2.68, 2.58, 2.45
12 & 12' 4 2.52, 2.52, 2.52, 2.52
13 & 13' 8 2.57, 2.57, 2.50, 2.50, 2.50, 2.50, 2.37, 2.37
* All compounds exhibit correct CH3/aromatic ratio by integration.
** Chemical shifts are relative to internal TMS.
143
-------
APPENDIX F
METHYLATED NAPHTHALENES: MASS SPECTRAL DATA FOR REPRESENTATIVE MONO-, DI-, TRI-, AND
TETRAMETHYLNAPHTHALENES (SYNTHESIZED AND COMMERCIALLY AVAILABLE)
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1 ST
•10
STEP nnss'i, I/B/S
-------
in
CO
too-
90-
80-
70-
60-
50-
HO-
30-
£0-
10-
9-
8PECO
-CH,
100
ISO
gOO 850
STEP MflSS'l, I/B/S •
-------
in
CH.-,
100-n
80-
70-
60-
50-
HO-
100
SPECO 158 US
^
150
1S8LM • 153LM
•It
a 00
250
STEP HflSS-l, I/B/3 •
-------
Compound
APPENDIX G
METHYLATED NAPHTHALENES: MICROANALYTICAL DATA
Microanalytical Data
1
9
10, 10'
11, 11'
12, 12'
13, 13'
Calculated
%C
92.51
(82.46
91.76
(87.94
92.26
(82.94
91.75
(83.12
91.25
(83.12
92.26
(82.73
%H
7.67
7.55
8.29
8.57
7.742
8.57
8.75
8.97
8.66
8.97
7.74
8.09
Observed
%C
92.46
82.48
91.82
82.98
82.79
90.76
83.08
91.23
83.10
92.22
82.90
%H
7.54
7.57)
8.82
8.67)
8.20)
9.01
8.90)
8.75
8.91)
7.76
8.05)
Precursor alcohol in parentheses.
2
Not enough pure material obtained,
155
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
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
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Donald Bodenner
Dale Swanson
10. PROGRAM ELEMENT NO.
Department of Chemistry
University of Minnesota
Duluth, MN 55812
11. 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
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
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
bioaccumulate. HPLC-GC analysis 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 pre-
dominantly 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 MS analysis.
The bioloaical studies on PAH's were aided by the use of a combined HPLC-GC
analysis procedure (ng/1 detection level) developed specifically for this program.
The biological investigation resulted in obtaining bioaccumulation factors in the
range of 1000-5000 for several PAH's.
to
Selected PAH's of various structural types were also shown to be quite susceptible
"second-order" anthropogenic transformations such as chlorine disinfection.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Coal
Leaching
Volatilization
Polycyclic
Analytical
Chlorination
Synthesis
Bioassay
Fathead minnow PAHs
Rainbow trout
Daphnia pulicaria
Mixed-function oxidase
Bioconcentration factor
HPLC
GC-MS
06/A
06/F
06/T
07/C
8, DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
IJNr.LASSIFTFD
21. NO. OF PAGES
168
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
PREVIOUS EDITION IS OBSOLETE
156
» U.S. GOVERNMENT PRINTING OFFICE 1979 -657-060/5445
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