ACUTE TOXICITY OF PENTACHLOROPHENOL
TO BLUEGILL (Lepomis macrochirus),
RAINBOW TROUT (SaliuO gair-dneri) , AND
PINK SHRIMP (Penaeug duoraruiti)1.-
BIONOMICS
n

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ACUTE TOXICITY OF PENTACHLOROPHENOL
TO BLUEGILL (Lepomis macrochirus),
RAINBOW TROUT (Saliud gaigdjveri) , AJSFD
PINK SHRIMP (Penae,u°. dupraruftt)'
BY
ROBERT E. BENTLEY
TOM HEITMULLER
BEVIER H. SLEIGHT, III
PATRICK R. PARRISH
ORDER NUMBER: WA-6-99-1414-B
PROJECT OFFICER: MR. WILLIAM FOX
ENVIRONMENTAL PROTECTION AGENCY
CRITERIA BRANCH (WH-535)
ROOM 1013 EAST TOWER
4 01 M STREET, S.W.
WASHINGTON, D.C. 204 60

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INTRODUCTION
The current concern regarding the protection of aquatic
life in surface waters has prompted the evaluation of the
effects of exposure to chemicals on aquatic organisms.
The primary objective of these studies was to provide the
Environmental Protection Agency with information to evaluate
the relative susceptibility of aquatic organisms to acute
exposure to pentachlorophenol. The acute toxicity of
pentachlorophenol to bluegill and rainbow trout in both a
soft and a hard water, and to pink shrimp in sea water was
estimated during static bioassays.
The bioassays with fishes were conducted at the Aquatic
Toxicology Laboratory of EG & G, Bionomics, Wareham,
Massachusetts. The shrimp bioassay was conducted at the
Marine Research Laboratory of E G & G, Bionomics, Pensacola,
Florida.

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Page two
MATERIALS AND METHODS
The methodology for acute toxicity testing of fishes
and shrimp closely followed the recommended bioassay
procedures as described in Standard Methods (APHA, 1971)
except for certain conditions described below.
The chemical evaluated in these bioassays was pentachloro-
phenol (ClgCgOH), a white powder (Baker Grade, Lot #326103)
tested as 100% active ingredient. Results for all tests
were expressed as the median lethal concentration (LC50),
the nominal concentration of the test compound in water
causing 50 percent mortality of test animals. The LC50
value and its 95% confidence interval were calculated by
converting the test concentrations and the corresponding ob-
served percent response to logs and probits, respectively.
These values were then utilized in a least squares regression
analysis, and the LC50 value and its confidence interval were
estimated from the calculated regression equation. .
The animals used in these tests were bluegill (Lepomis
macrochirus), rainbow trout (Salmo gairdneri) and pink
shrimp (Penaeus duorarum). The bluegill were acquired from

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Page three
a commercial fish hatchery in Nebraska, and had a mean wet
V.
weight of 1.1 g and a mean standard length of 37 mm. The
rainbow trout were obtained from a commerical fish farmer
in Washington, and had a mean wet weight of 1.0 g and a
mean standard length of 32 mm. The shrimp were collected
by laboratory personnel from Big Lagoon in Pensacola,
Florida and had rostrum-telson lengths of 35-50 mm.
The bluegill and rainbow trout were held in 1700-1 concrete
raceways which are coated with an epoxy resin paint to
prevent leaching of materials into the water. Flow of
well water (having a temperature of 21 + 1.0°C for the
bluegill, and 12 + 1.0°C for the rainbow trout) into these
raceways was at a minimum flow of 4 1/minute, providing
an adequate rate of turnover for holding these species.
This water had a hardness of 35 mg/1 as CaC03, a pH of
7.1 and a dissolved oxygen concentration of at least 6.0
mg/1 (60% of saturation). These species were maintained in
the laboratory hatchery facilities for at least 30 days
prior to testing. During the 30 day period, mortality was
<2%; no mortality was observed during the 48 hours immediately
prior to testing, and these fish were judged to be in ex-
cellent condition. The shrimp were held in 1100-1 fiber-
glass tanks in constantly flowing filtered (10 micrometers)

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Page four
natural sea water. The salinity of this water was 25
part per thousand (o/oo) and the temperature was 20 + 1.0°C.
The static bioassays were conducted in 19.6-1 wide-mouth
soft-glass bottles containing 15 liters of test solution.
Exposure mixtures for the bluegill bioassays were maintained
in water baths at 21 + 1.0°C by immersion coil heaters and
mercury column thermoregulators. Test solutions for the
rainbow trout and shrimp, were maintained in water baths at
12 + 1.0°C and 20 + 1.0°C/ respectively, by use of commercial
refrigeration units. Each species was from the same year
class, and the standard length of the longest fish or shrimp
was no more than two times that of the shortest fish or
shrimp. The bluegill and rainbow trout were acclimated to
test conditions of temperature and water quality over a 96-
hour period prior to testing. These species were not fed
during the 48 hours immediately prior to testing or during
the tests. The shrimp were acclimated to. cest conditions of
water quality and temperature for at least seven days prior
to testing. Water in the test vessesl was not aerated. The
test compound in the bluegill and rainbow trout bioassays
was added to each jar in a solution of reagent-grade acetone.
In the shrimp bioassays, the test material was introduced into
each jar directly. Animals were introduced into the test
vessels within 30 minutes after the compound was added. Ten

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Page five
bluegill or rainbow trout were randomly assigned to each
test vessel. Ten shrimp (2 replicates, 5 animals/vessel)
were exposed to each concentration.
The dilution water used in the fish bioassay was the same
as previously described for holding these fish. The hard
water for these bioassays was prepared by adding 192 mg
of NaHCC>3, 120 mg of CaS04, 120 mg of MgSO^, and 8 mg of
KCl per liter of deionizea water. This water had a pH of
7.6 and a total hardness of 200 mg/1 as CaCO^. The dilution
water for the shrimp bioassays consisted of filtered (10
micrometers) natural sea water with a salinity of 25 c/oo
and a pH of 8.0 + 0.5. Concentrations of dissolved oxygen
were measured with a combination temperature-oxygen probe
and meter in selected concentrations at 0, 24, 48 and 96
hours of exposure.
Two series of concentrations were established within a
bioassay, a series of range-finding (preliminary) concen-
trations and a series of definitive concentrations. The
preliminary test was conducted to determine the approximate
range of concentrations for evaluating the dose-response
relationship. The definitive test, consisting of at least
five concentrations, evaluated the dose-response relationship

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Page six
to a degree allowing the LC50 to be calculated from the
data with optimum accuracy; A control, which consisted of
the same dilution water, conditions, procedures, and organisms,
was maintained for each species tested. A solvent control,
which contained a volume of acetone equivalent to the
greatest amount introduced into any vessel, was also main-
tained for each test.
RESULTS AND DISCUSSION
The estimated LC50 values and 95% confidence intervals for
pentachlorophenol and the species tested are presented in
Table 1 along with the highest nominal concentrations at
which there were no discernible effects on test animals due
to exposure to pentachlorophenol. A summary of observed
mortality for each individual test concentration at 24, 48,
and 96 hours of exposure to pentachlorophenol is also presented
(Table 2). The mortality syndrome among fish from those
concentrations where mortality was observed was similar. Fish
generally became dark and lethargic, lost equilibrium, and
expired. Affected shrimp generally lost equilibrium, lay on
their sides, and died. The concentrations of dissolved
oxygen, measured at 0, 24, 48 and 96 hours of exposure, are

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presented in Table 3. Final pH was 7.0 + 0.5 for all
test concentrations and controls where bluegill and rainbow
trout were exposed in soft water. Comparable pH's for the
test concentrations where bluegill and rainbow trout were
exposed in hard water were 7.5 + 0.5. Final pH was 8.0 +
0.5 for all test concentrations and the control in the
shrimp bioassay.
The LC50 values for those bioassays exposing bluegill and
rainbow trout in both soft and hard water were similar.
The LC50 values for those tests ranged from 0.060 mg/1
(bluegill in.soft water) to 0.092 mg/1 (rainbow trout in
hard water) after 96 hours of exposure. The pink shrimp
were much less susceptible to this compound than were fish,
exhibiting a sensitivity ca 61X less than the fresh water
species (96-hour LC50 = 5.6 mg/1).

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LITERATURE CITED
A.P.H.A. 1971. Standard Methods for the Examination
of Water and Wastewater. 13th Edition, 874 pp.

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Table 1 — Acute toxicity of pentachlorophenol to bluegilla (Lepomis
macrochirus) , rainbow trout'3 (Salmo gairdneri) , and pink
shrimpc (Penaeus duorarum). These data are based on results
of bioassays conducted at the Aquatic Toxicology Laboratory
and the Marine Research Laboratory of E G & G, Bionomics,
Wareham, Massachusetts and Pensacola, Florida.
No discernible
-Species/	LC50 (mg active ingredient/1)	effect level
diluent	24 hour	48 hour	96 hour	(mg/1)
bluegill/ 0.130	0.063	0.060	0.042
^soft water	(0.106-0.158)d(0.052-0.076)	(0.048-0.073)
bluegill/ 0.202	0.116	0.077	0.042
hard water	(0.115-0.354)	(0.071-0.190)	(0.059-0.101)
rainbow trout/ 0.100	0.075	0.075	0.056
soft water	(0.072-0.140)	(0.055-0.107)	(0.055-0.107)
rainbow trout/ 0.207	0.175	0.092	0.042
hard water	(0.123-0.347)	(0.131-0.235)	(0.072-0.118)
pink shrimp 8.2	7.4	5.6	3.2
(6.3-11)	(5.7-9.5)	(4.6-6.9)
a
Bioassays conducted at 21 + 1.0°C, mean wet weight of bluegill, 1.1 g.
b
Bioassays conducted at 12 + 1.0°C, mean wet weight of rainbow trout,
1.0 g.
c
Bioassays conducted at 20 + 1.0°C, rostrum-telson lengths of pink
shrimp35-50 mm.
d
95% confidence interval.

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Table 2 — Concentrations tested and corresponding observed
percentage mortalities at 24, 48 and 96 hours for
bluegill (Lepomis macrochirus), rainbow trout
(Salmo gairdneri), and pink shrimp (Penaeus duorarum)
exposed to pentachlorophenol.
Species/
diluent
Nominal
concentration
(mg/1)
% mortality observed
24 hour 48 hour 96 hour
bluegill/
soft water
bluegill/
hard water
0.240

100
100
100
0.180

90
100
100
0.140

40
100
100
0.100

30
100
100
0.075

0
80
90
0.056

0
30
70
0.042

0
0
0
control
(acetone)
0
0
0
control

0
0
0
0.240

80
100
100
0.180

50
100
100
0.140

0
80
100
0.075

0
0
10
0.056

0
0
10
0.042

0
0
0
control
(acetone)
0
0
0
control

0
0
0

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Table 2 — Continued.,
Nominal
Species/	concentration	% mortality observed
diluent	(mg/1)	24 hour 48 hour 96 hour
rainbow trout/
soft water
0.180	100
0.140	100
0.100	40
0.075	0
0.056	0
0.042	0
control (acetone) 0
control	0
100
100
100
30
0
0
0
.0
100
100
100
30
0
0
0
0
rainbow trout/
hard water
0.240	90
0.180	10
0.140	0
0.100	0
0.075	0
0.056	0
0.042	0
control (acetone) 0
control	0
100
80
0
0
0
0
0
0
0
100
90
70
60
70
10
0
0
0
pink shrimp/
sea water
32.0
18.0
10.0
5.6
3.2
100
100
30
10
0
control (acetone) 0
control	0
100
100
70
20
0
0
0
100
100
100
30
0
0
0

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Table 3 — Measured concentrations of dissolved oxygen during
96-hour exposures of bluegill (Lepomis macrochirus),
rainbow trout (Salmo gairdneri), and pink shrimp
(Penaeus duorarum) to pentachlorophenol.
Species/
diluent
Nominal
concentration
(mg/1)
Dissolved oxygen
(mg/1 and % of saturation)	
0 hour 24 hour 48 hour 96 hour
bluegill/
soft water
0.240
0.140
0.042
control
8.3 (94)
8.0(90
7.9(88)
8.4(95)
8.0(90)
7.8(87)
7.9(88)
5.2(57) 4.8 (53)
6.2 (67) 5.3(58)
bluegill/
hard water
0.240
0.140
0.042
control
8.2 (92)
8.1(91)
8.0 (90)
8.5(96)
7.2(80)
7.5(83)
7.2 (80)
6.0 (66) 4.7 (52)
6.3(68) 5.8 (64)
rainbow trout/
soft water
0.140
0.075
0.042
control
9.1(84)
9.3 (86)
9.2 (85)
9.5 (88)
7.8 (72) 6.2 (57) 5.4 (50)
9.1(84) 6.6 (60) 4.1(37)
8. 8(81) 7.4 (67) 6.1(56)
rainbow trout/
hard water
0.240
0.140
0. 042
control
9.5(88)
9.5	(88)
9.6	(89)
9.5 (88)
9.2(85)
9.4(87)
9.5(88)
9.7(89)
6.2(57) 4.5(41)
7.2 (66) 4.4 (40)
6.8 (62) 5.0 (46)

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Table 3 — Continued.
Species/
diluent
Nominal
concentration
(mg/1)
Dissolved oxygen
(mg/1 and % of saturation)	
0 hour 24 hour 48 hour 96 hour
pink shrimp/
sea water
10.0
5.6
3.2
control
6.8	(89)	6.4 (84)	5.8 (76)
6.9	(90)	5.9 (78)	4.8(63)	4.5 (59)
6.8 (89)	5.7 (82)	5.0 (66)	3.3 (43)
6.8 (89)	6.2 (82)	5.4(71)	3.1(41)
a
Dissolved oxygen not measured due to 100% mortality.

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