United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/D-84-001 Jan. 1984
ENVIRONMENTAL
RESEARCH BRIEF
Hazardous Chemicals in Fish
Wisconsin Power Plant Impact Study
John J. Lech and Mark J. Melancon
Introduction
From the operation of a coal-fired power plant, a variety of
organics present in coal might reach the aquatic environ-
ment via leaching from such sources as stored coal, stack
emissions, and ash ponds. The use of petroleum-derived
fuels for the transportation of coal could provide a source
for additional hydrocarbons, and the use of chlorination
procedures on cooling water to retard algal growth could
give rise to chlorinated hydrocarbons. Chemicals which
reach the aquatic environment but which have relatively
low water solubility tend to be taken up by aquatic species
including fish. Thus, the uptake and accumulation by fish of
organics arising from the operation of a coal-fired power
plant might serve as a vector for human exposure to such
organics through the consumption of the contaminated
fish. The present report summarizes studies of the uptake,
disposition, metabolism, and elimination of selected chemi-
cals by fish.
Findings and Conclusions
/. Uptake, Distribution and Elimination of
Naphthalene. 2-Methylnaphthalene and
1,2,4-trichlorobenzene by Fish
Naphthalene and 2-methylnaphthalene were selected for
study because both are among the most water-soluble
components of coal and petroleum and both chemicals are
likely to reach the aquatic environment. 1,2,4-trichloro-
benzene was selected as a representative compound for
chlorination-caused chloro-organics.
The uptake and elimination of these three compounds by
fingerling rainbow trout was studied using a continuous-
flow delivery system in which groups of trout were exposed
to the appropriate 14C-labeled chemical for 4-5 weeks,
followed by a depuration period. The results with naphtha-
lene, 2-methylnaphthalene and trichlorobenzene are pre-
sented in Figures 1, 2, and 3, respectively. The maximum
accumulations attained (muscle 14C/ average water 14C)
were approximately 40 for naphthalene, 160 for 2-meth-
ylnaphthalene and 156 for trichlorobenzene. In additional
experiments with 2-methylnaphthalene, the values were
123 for carp muscle and 403 for bluegill sunfish (whole
body). The t-1/2 of elimination following each of these
exposures is shown in Table 1.
Although these chemicals were accumulated by fish
muscle at 40-160 times the exposure level, they were
generally eliminated rapidly upon termination of the
exposure, with the exception of naphthalene. In earlier
experiments, the elimination of 14C from trout tissues
following short-term (8 hr) exposures to any of these '"C-
chemicals was rapid. Subsequent studies suggested that
the slow elimination of naphthalene metabolites from
muscle tissue following the longer term exposure was
probably responsible for the slower 14C elimination rate.
//. Metabolism of Naphthalene, 2-methylnaphtha-
lene and 1,2,4-trichlorobenzene by Fish
Exposure of fish to these chemicals containing 14C-label
resulted in the appearance of 14C-labeled materials in the
bile of the exposed fish. Thin-layer chromatography indi-
cated that in each case most of the bile 14C resided in high
polarity compounds, suggestive of conjugated metabolites.
By the use of /3-glucuronidase. an enzyme which hydrolyzes
glucuronide conjugates, it was demonstrated that glucuro-
nide conjugates made up a major fraction of these bile
metabolites. Additional studies suggested that other metab-
olites may have arisen from glutathione conjugates.
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Elimination
W 20 30 40
Time (Days)
50
60
Figure
Tissue levels of 14C in trout during a 27-day
exposure to 14C-naphthalene and subsequent
elimination. Each point represents the average of
values from live trout, and the verticl lines
represent the standard error. The average
concentration of 14C-naphthalene in water during
the exposure was 0.023 mg/liter.
• = liver, o = muscle, D = blood, and + = exposure
water.
I
70|-
Exposure
Elimination
++
+** s+
\
20
\ l
\
1 I
30
Time (Days)
\
40
\
50
\
60
Figure 2. Tissue levels of 14C in trout during a 26-day expo-
sure to 14C-2-methylnaphthalene and subsequent
elimination. Each point represents the average of
values from five trout, and the vertical lines
represent the standard error. The average concen-
tration of 14C-2-methylnaphthalene in water
during the exposure was 0.017 mg/liter.
• = liver, o = muscle, D = blood, and + = exposure
water.
70.00!
3.00-r
t
£
"3
I
cu
83 030
c
01
•Q
O
•g 0.70-
0.03
Exposure
Depuration
70
20 30 40
Time (Days)
50 60
70
Figure 3.
Tissue levels of 14C in trout during a 35-day
exposure to 14C-1 ,2,4-trichlorobenzene and
subsequent elimination. Each point represents the
average of values from five trout, and the vertical
lines represent the standard error. The average
concentration of 14C-1 ,2,4-trichlorobenzene in
water during the exposure was 0.018 mg/liter.
• = liver, + = muscle, and o = blood.
///. Studies of the Hepatic Microsomal Monooxy-
genase System in Rainbow Trout and Carp
As evidence began to accumulate on the in vivo metabolism
of xenobiotics by fish, studies were initiated to compare the
metabolizing systems in fish to those characterized in
mammalian species. Our studies with liver, a major locus of
xenobiotic metabolism, showed that trout liver responded
to homogenization, differential centrifugation and various
enzyme assays in much the same way as did rat liver.
Because a number of aquatic pollutants such as PAHs and
PCBs are known to induce (increase) hepatic microsomal
monooxygenase (xenobiotic metabolizing) activity in mam-
malian species, the effect of these aquatic pollutants in fish
was examined. The data in Table 2 show the effects of
administration of a PAH, a PCB and phenobarbital to
rainbow trout on two hepatic microsomal enzyme activities.
The data in Table 3 show similar information for carp.
Because chemical aquatic pollutants such as PAHs and
PCBs can increase hepatic microsomal metabolism, such
chemicals might affect the metabolism and disposition of
foreign chemicals by fish in vivo.
IV. Studies on the Effects of Modifiers of Hepatic
Metabolism in Fish on Metabolism and Disposition
of Xenobiotics in Fish
The effect of preadministration of /3-naphthoflavone to
rainbowtrout on the metabolism of naphthalene, 2-methyl-
naphthalene, and trichlorobenzene was examined. The
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Table 1. Elimination Half-Lives of 14C from Fish Exposed to
Aqueous 14C-Naphthalene, 14C-2-Methylnaphtha-
lene or 14C-Trichlorobenzene for Several Weeks
Half-Lives, h
Exposure
Muscle Liver Blood Whole Fish
Trout
Naphthalene
0.017 mg/liter
Trout
2-Methylnaphthalene
0.023 mg/liter
Carp
2-Methylnaphthalene
0.013 mg/liter
Bluegill sunfish
2-Methylnaphthalene
0.013 mg/liter
Trout
1,2,4-Trichlorobenzene
0.018 mg/liter
909
13"
711
37"
1942
8"
36
343 379
211
59"
781
23
<24C
353
32
"When two values are given, the upper value is the early rapid
phase of elimination and the lower value is the later slower
phase. The slope and intercept for the slow phase of elimination
were calculated using the data for days 4-36. The data from days
0-2 were corrected for this, and the resulting values were used to
calculate slope for the rapid phase of elimination from days 0-2.
Muscle '4C decreased by approximately 75% during the first 3
days of depuration.
''Data for slow phase, days 8-73 and for rapid phase, days 0-3.
Whole fish "C decreased by approximately 61% during the first
three days of depuration.
"Data for slow phase, days 1-26. Muscle I4C decreased by
approximately 60% during the first day of depuration.
"Data for slow phase, days 1-36. Muscle '"C decreased by
approximately 88% during the first day of depuration.
results presented in Table 4 show that pretreatment
resulted in substantial increases in metabolites of each
chemical appearing in bile, and a decrease in the amount of
each chemical remaining in muscle.
The effect of administration of an inhibitor, piperonyl
butoxide, on metabolism and elimination of two organic
compounds, pentachloroanisole and di-2-ethylhexylphthal-
ate, was studied. Bile from the pretreated trout contained
only one-third of the amount of pentachlorophenol-gluc-
uronide, the major metabolite of pentachloroanisole, as did
the control trout. In the case of di-2-ethylhexylphthalate,
the pretreatment reduced the level of bile metabolites by
one-half and increased muscle levels of the parent chemi-
cals 3-fold.
V. Possible Use of Fish Bile as an Aid in Monitor-
ing for Aquatic Pollutants
Some aquatic pollutants are of concern because they
collect in fish to levels many times higher than the levels
present in the water. For highly lipophilic pollutants such as
PCBs, monitoring for these chemicals might be easier if fish
flesh, rather than the water, were examined. For other
pollutants which are more readily metabolized, the metabo-
lites may appear in bile at much higher levels than in the
water. The data in Table 5 demonstrates the bile-to-water
ratio for a variety of chemicals. A tetrachlorobiphenyl which
is not readily metabolized was present at only 11 -fold
concentration; phenols, which are readily glucuronidated,
were concentrated 1,000-fold to 10,000-fold, with other
chemicals falling between these two extremes. In the long-
term exposures described earlier, the bile-to-water ratios
were much higher for PAHsbut notfortrichlorobenzeneas
shown in Table 6.
Recommendations
1. The rapid elimination of certain organics after short-
term exposure suggests that intermittent brief expo-
sures should not lead to substantial bioaccumulation.
Long-term exposure however will lead to significant
bioaccumulation dependent upon the lipophilicity and
metabolism of a particular chemical. Additional studies
should be done on representative PAHs of greater
lipophilicity and on mixtures of these chemicals.
Table 2. The Effect of Inducers on the Kinetics of Monooxygenation in Rainbow Trout Hepatic Microsomes Following
Intraperitoneal Pretreatment
Ethoxyresorufin-O-deethylase
Pretreatment
Corn oil
1 ml/ kg
Aroclor
150 mg/kg
fi-Naphthoflavone
100 mg/kg
Pentobarbital
65 mg/kg
Vmax
nmol/min/mg
0. 136±0.26"
1.85 ±0.04"
6.06±0.18"
0.088±0.017
control
100
1367
4455
65
Km.
nM
144±6
154±0
125±8
170±0
Ethoxycoumarin- O-deethylase
Vmax
nmol/min/mg
0.1 01 ±O.O10
0.286±0.47a
1. 19±0.28"
0.065±0.017
control
100
283
1178
64
Km.
nM
129±9
57±/b
41 ±4"
105 ±5
^Values are mean ± SE; all values obtained 72 h at, r injection of fish.
^Significantly different from'corn oil control group, P '. 0.05.
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Table 3.
Maximum Induction of Cytochrome P450 and
Mixed-Function Oxidase Activities in Carp Liver
and Kidney Microsomes
Liver Microsomes
Kidney Microsomes
P450
ECOD"
EROD
BaPH
BNF
2.8"
1.6
73.5
234
A 1254
1.9
1.5
84.9
29.6
TCB
0.8
0.8
1.4
BNF
2.3
1.7
A 1254
2.2
61
TCB
1.5
2.3
"Values represent the maximum ratios of P450 content or enzyme
activity of treatment group to control (corn oil) group,
bECOD = ethoxycoumarin-O-deethylase activity; EROD = ethoxy-
resorufin-O-deethylase activity and BaPH = benzofajpyrene
hydroxylase activity.
2. The substantial bioconcentration of metabolites of
PAHs which are carcinogens and of phenols in fish bile,
compared to the levels in exposure water suggest that
use of bile could prove useful in monitoring for certain
pollutants in the aquatic environment.
Bibliography
1. Lech,J.J., M.J.Vodicnik, and C. R. Elcombe. 1982.
Induction of monooxygenase activity in fish. pp.
107-148. In: L V. Weber (ed.). Aquatic Toxicology.
Raven Press, New York, N.Y.
2. Breger, R. K., R. B. Franklin, and J. J. Lech. 1981.
Metabolism of 2-methylnaphthalene to isomeric di-
hydrodiols by hepatic microsomes of rat and rainbow
trout. Drug Metab. Disposit. 9(2):88-93.
3. Vodicnik, M. J., A. H. Glickman, D. E. Rickert, and J.
J. Lech. 1980. Studies on the disposition and
metabolism of pentachloroanisole in female mice.
Toxicol. Appl. Pharmacol. 56: 311 -316.
Table 4. Effect of Pre-Administration of /8-Naphthoflavone on the Disposition and Metabolism of 14C-Labeled Chemicals in
Rainbow Trout
Tissue
Bile'
Muscle*
Liver*
Blood"
Bile"
Muscle"
Liver0
Com Oil
Tissue level of
parent chemical
+ metabolites
(ug/g or ug/ml)
67 2±5. 1
2.25±023
2. 05 ±0.1 2
1 83+0.23
150+24
4.9
108
Metabolites
%
Naphthalane
98
5.1 ±04
8.5±0.5
0 97 ±0.08
2-Methylnaphthalene
96 1233+2O1
2
10
fi-Naphthoflavone
Tissue level of
parent chemical
+ metabolites
(ug/g or ug/ml)
308±21
1,25±0.16
1.72±001
1OO
26
5.0
Metabolites
%
99
12.3±0.9
24.0±1.8
10
40
Blood"
33+02
1.9±0 1
1,2,4-Trich/orobenzene
Bile"
Muscle"
Liverc
Blood"
14.7+0.8
575"
22"
2.01+J0.12
65
08
3.7
87.5±5 5
299"
42"
1 03±0 04
98
2 1
6.2
Groups of eight trout were injected mtrapentoneally with corn oil or a solution of BNF in corn oil(100 mg/ml) at a rate of 1 ml/kg After 48 h,
groups of fish were exposed to one of the above chemicals for 24 h The water levels of the chemicals for control and induced trout were
naphthalene, 0.52 and0.45 mg/liter; 2-methylnaphthalene, 0.28 and0.36 mg/liter, and 1,2,4-trich/orobenzene, 0 20 and0.20 mg/liter.
respectively.
"Aliquots of blood and bile from each fish were used to determine levels of ' "C. Values are the average + SE. Metabolite determinations
utilized pooled bile samples.
"Each sample consisted of pooled muscle or liver from two fish Thus four samples per group were used to determine tissue ">C levels and
percentage of metabolites Values are average ±S E
"Each sample consisted of pooled muscle or liver from all eight fish in the group
"Tissue weights were not calculated The total parent compound plus metabolites were extracted as given
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Table 5. Biliary Concentration of Xenobiotics by Rainbow Trout
Concentration
inHzO
(mg/liter)
Radioactivity (dpm/ml)
H2O
(0 hours)
Bile"
(24 hours)
Ratio
(bile ">C)/
(H2O "C)
2',5-Dichloro-4'-nitrosa/icy/ani/ide
(Bayer 73; chlorosalicylic acid; nng-UL-14C)
Di-2-ethylhexy/phthalate (DEHP; carboxy/-'4C)
2-Methylnaphthalene (ring UL-'4C)
1 -Naphthyl-N-methylcarbamate
(carbaryl; naphthyl- 1-'4C)
Naphthalene (rmg-UL-'4C)
Pentachlorobiphenyl (POP; ring-UL-'4CJ
2,5,2-,5--Tetrachlorobiphenyl(TCB; ring-UL-">C)
1,1,1 -Trichloro-2,2-bis(p-chlorophenyl)ethane
(p,p'-DDT; ring-UL-'4C)
3-Trifluoromethyl-4-mtrophenyl (TFM; ring-UL-'4C)
0.05
0.5
0.005
025
0.005
0.1
0.5
0.1
0.5
3,010
1,070
310
1,030
305
4.070
3.640
180
2,020
30,500,000
265,000
796,000
975,000
127,000
21,800,000
39,000
22,500
2,150,000
10.100
247
2,570
947
414
5,360
11
124
1,064
"Exposures were made at 12°C for 24 hours.
Table 6. Maximum Concentration of Bile Metabolites Found in Fish During Long-Term Continuous Exposure
Chemical
Naphthalene
2-Methylnaphthalene
1 ,2,4-Trichlorobenzene
Species
trout
trout
carp
trout
Water Level
mg/l
0.017
0.023
0.013
0.018
Bile level, parent
Chemical + Metabolites
mg/ml
0.327
0.434
1.835
0.024
Bile Level
Water Level
19.200
18,900
141,200
1.3
4. Melancon, M. J., and J. J. Lech. 1980. Uptake,
metabolism, and elimination of 14C-labeled 1,2,4-tri-
chlorobenzene in rainbowtroutandcarp J.Toxicol.
Environ. Health 6: 645-658.
5. Melancon, M. J. 1979. Metabolism of phthalate
esters in aquatic species. ACS Symp. Series No 99,
Pesticide and xenobiotic metabolism in aquatic
organisms. M. A. Q. Kahn.J.J. Lech, and J J Menn
(eds.), American Chemical Society
6. Elcombe, C. R., R. B. Franklin, and J.J Lech 1979.
Induction of hepatic microsomal enzymes in rainbow
trout. ACS Symp. Series No. 99, Pesticide and xeno-
biotic metabolism in aquatic organisms. M. A. Q.
Kahn, J. J. Lech, and J. J. Menn (eds.), American
Chemical Society.
7. Gumey, P. D., M. J. Melancon, Jr., J.J. Lech, and R.
E Peterson. 1979 Effects of egg and sperm
maturation and spawning on the distribution and
elimination of a polychlormated biphenyl in rainbow
trout (Salmo gairdneri) Toxicol Appl. Pharmacol.
47:261-272.
10.
Statham, C. N., W. A. Croft, and J. J. Lech 1978.
Uptake, distribution, and effects of carbon tetra-
chlonde in rainbow trout (Salmo gairdneri). Toxicol.
Appl. Pharmacol. 45: 131-140.
Lech, J.J , A. H Glickman, and C. N. Statham. 1978.
Studies on the uptake, disposition and metabolism
of pentachlorophenol and pentachloroanisole in
ra\r\bowtrout(Salmo gairdneri) Pentachlorophenol,
Plenum Publishing Corp , New York, N.Y.
Guiney, P. D., R E Peterson, M J Melancon, Jr.,
and J. J Lech 1 977. The distribution and elimina-
tion of 2,5,2',5'-['4C]-tetrachlorobiphenol in rainbow
trout (Salmo gairdneri) Toxicol Appl. Pharmacol.
39. 329-338.
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