ACUTE TOXICITY OF NICKEL TO BLUEGILL
(Lepoiais macrochirus) , RAINBOW TROUT
(Salmo gairdneri), AND PINK SHRIMP
(Penasus duorarum).
BIONOMICS

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ACUTE TOXICITY OF NICKEL TO BLUEGILL
(Lepomis macrochirus), RAINBOW TROUT
(Salmo gairdneri), AND PINK SHRIMP
(Penaeus duorarum).
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-585)
ROOM 1013 EAST TOWER
401 M STREET, S.W.
WASHINGTON, D.C. 20460

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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 nickel. The acute toxicity of nickel to bluegill
and rainbow trout in both a soft and a hard water, and to
pink shrimp in sea water was estimated during static bio-
assays.
The bioassays with fishes were conducted at the Aquatic
Toxicology Laboratory of E G & 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 nickel, as
nickel chloride (NiC^-Sl^O, 25% nickel), a green granular
substance manufactured by Matheson, Coleman & Bell (lot
#10E17). Results for all tests were expressed as the
median lethal concentration (LC50), the nominal concen-
tration of the test compound in water causing 50 percent
mortality of test animals. The LC50 value and its 95% con-
fidence interval were calculated by converting the test
concentrations and the corresponding observed 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
weight of 1.1 g and a mean standard length of 37 mm. The
rainbow trout were obtained from a commercial 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 rainhow 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 CaCO^, 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 flcwing filtered (10 micrometers)

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Page four
natural sea water. The salinity of this water was 25
parts 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 con-
ditions 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 test conditions of water quality
and temperature for at least seven days prior to testing.
Water in the test vessels was not aerated. The test compound
in the bluegill and rainbow trout bioassays was added to
each jar in a solution of water. In the shrimp bioassays,
the test material was introduced into each jar directly. Animals
were introduced into the test vessels within 30 minutes after

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the compound was added. Ten 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 NaHCO-j, 120 mg of CaSO^, 120 mg of MgSO^, and
8 mg of KC1 per liter of deionized water. The resulting
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 o/oo and a pH of 8.0 + 0.5. Concen-
trations 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

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five concentrations, evaluated the dose-response relation-
ship 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.
RESULTS AND DISCUSSION
The estimated LC50 values (95% confidence intervals) for
nickel and the species tested are presented in Table 1
along with highest nominal concentration tested at which
there were no discernible effects on test animals due to
exposure to nickel. A summary of observed mortality for
each individual test concentration after 24, 48 and 96
hours of exposure to nickel 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.
Those bluegill exposed to nickel in soft water exhibited
excessive mucus production at nominal concentrations ^75.0
mg/1 through 72 hours of exposure. This condition sub-
sequently appeared to subside during the final 24 hours
of exposure. Affected shrimp generally lost equilibrium,
lay on their sides, and died.

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Page seven
The concentrations of dissolved oxygen, measured at 0,
24, 48 and 96 hours, are 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 con-
centrations and controls in the shrimp bioassay.
Water quality appeared to have no effect on the toxicity
of nickel to bluegill or rainbow trout. Bluegill exhibited
similar 96-hour LC50 values in both the soft and hard water
bioassays (62.2 mg/1 and 60.3 mg/1, respectively). Rainbow
trout were also observed to have nearly equal sensitivities
at 96 hours (13.7 mg/1 and 16.3 mg/1 for the soft and hard
water, respectively). The shrimp exhibited less susceptibility
to nickel than either of the other two species (112 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 (LC50) of nickela to bluegill^ (Lepomis
macrochirus), rainbow troutc (Salmo gairdneri) and
pink shrimp^ (Penaeus duorarum). These data are based
on the results of static bioassays conducted at the
Aquatic Toxicology Laboratory of E G & G, Bionomics,
Wareham, Massachusetts or the Marine Research Laboratory
of E G & G, Bionomics, Pensacola, Florida.
LC50 - (mg active ingredient/1)
24 hour	48 hour	96 hours
170.0	115.0	62.2
(130. 0-222. 0)e (87. 0-152.0) (50. 5-76.. 6)
Species/
diluent
bluegill/
soft water
bluegill/
hard water
pink shrimp/
sea water
170.0
(124.0-232.0)
>560
110.0
(81.2-150.0)
51.0
(39.3-66.1)
94.3
(77.2-115.0)
415
(276-624)
60.3
(35.2-103.0)
13.7
(10.4-18.2)
16.3
(11.5-23.2)
112
(76.8-163)
No discernible
effect level
at 96 hours
(mg/1)
42.0
37.0
5.6
4.9
<56.0
rainbow trout/ 196.0
soft water (101.0-383.0)
rainbow trout/ 312.0
hard water (211.0-461.0)
a
Nickel chloride (NiCl2•6H2O), 25% nickel.
b
Bioassays conducted at 21 + 1.0°C, mean wet weight of bluegill, 1.1 g.
c
Bioassays conducted at 12 + 1.0°C, mean wet weight of rainbow trout,
1.0 g.
d
Bioassays conducted at 20 + 1.0°C, rostrum-telson lengths of pink
shrimp, 35-50 mm.
e
95% confidence interval.

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Table 2 — Concentrations tested and corresponding observed
percentage mortalities for bluegill (Lepomis
macrochirus), rainbow trout (Salmo gairdneri), and
pink shrimp (Penaeus duorarum) exposed to nickel
for 24, 48.and 96 hours.
Species/
diluent
Nominal
concentration
(mg/1)
% mortality observed	
24 hour 48 hour 96 hour
bluegill/
soft water
320
210
140
100
75
56
42
control
100
70
30
0
0
0
0
0
100
100
50
0
10
10
0
0
100
100
100
100
60
60
0
0
bluegill/
hard water
370
240
140
100
65
37
control
100
90
50
0
0
0
0
100
100
90
0
10
0
0
100
100
100
100
50
0
0

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Table 2 — Continued
Species/
diluent
Nominal
concentration
(mg/1)
% mortality observed	
24 hour 48 hour 96 hour
rainbow trout/
soft water
rainbow trout/
hard water
320.0
210.0
100.0
75.0
56.0
42.0
28.0
24.0
16.0
7.5
5.6
control
420.0
320.0
210.0
140.0
100.0
87.0
65.0
49.0
32.0
21.0
14.0
6.5
4.9
100
10
0
0
0
0
0
0
0
0
0
0
100
10
0
0
0
0
0
0
0
0
0
0
0
100
100
100
60
20
20
30
0
0
0
0
0
100
100
100
90
80
10
20
0
0
0
0
0
0
100
100
100
100
100
100
100
60
50
10
0
0
100
100
1C0
100
100
100
100
100
90
50
10
10
0

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Table 2 — Continued.
Species/
diluent
Nominal
concentration
(mg/1)
% mortality observed	
2 4 hour 4 8 hour 96 hour
pink shrimp/
sea water
560
320
180
100
56
control
30
10
0
0
0
0
80
30
0
0
0
0
100
100
50
30
10
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 nickel.
Species/
diluent
Nominal
concentration
(mg/1)
Dissolved oxygen
(mg/1 and % of saturation)	
0 hour 24 hour 4 8 hour 96 hour
bluegill/
soft water
320
210
100
control
8.3(94)
8.2 (92)
8.0 (90)
8.4 (95)
8.2 (92) 7.6 (85)
7.8 (87) 7.2 (80) 6.4 (70)
8.0(90) 6.8(75) 5.4(60)
bluegill/
hard water
370
140
37
control
8.4(95)
8.2 (92)
8.0(90)
8.4(94)
8.2 (92) 6. 9 (77)
7.1 (79) 6.2 (67) 5.1(56)
8.0 (90) 6.6 (73) 5.4 (60)
rainbow trout/
soft water
320.0
210.0
5.6
control
9.2 (85)
9.2(85)
8.2 (75)
8.8 (80)
9.2(85)
6.8(62) 5.6(52) 6.2(57)
8.8 (80) 6.8 (62) 6.1(56)
rainbow trout/
hard water
420.0
210.0
4.9
control
9.2 (85)
9.5 (87)
9.0	(83)
9.1	(84)
9.5(87)
8.2(75) 6.8(62) 5.5(51)
9.2 (85) 8.3 (76) 8.2 (75)

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Table 3 — Continued.
Nominal	Dissolved oxygen
Species/	concentration	(mg/1 and % of saturation)	
diluent	(mg/1)	0 hour 24 hour 48 hour 96 hour
pink shrimp/
sea water
560
320
56
control
6.8	(89)	6.6 (87)	6.7 (88)	-a
6.8(89)	6.8(89)	6.4(84)
6.9	(90)	6.8 (89)	6.1(80)	5.7(75)
6.8 (89)	6.8 (89)	6.0(79)	3.5 (46)
a
Dissolved oxygen not measured due to 100% mortality.

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