ACUTE TOXICITY OF CHLOROFORM TO BLUEGILL
(Lepomis macrochirus), RAINBOW TROUT
(Salmo gairdneri), AND PINK SHRIMP
(Penaeus duorarum).
BIONOMICS

-------
ACUTE TOXICITY OF CHLOROFORM 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
4 01 M STREET, S.W.
WASHINGTON, D.C. 20460

-------
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 chloroform. The acute toxicity of chloroform
to bluegill and rainbow trout in both a soft and a hard
water, and pink shrimp in sea water was estimated during
static bioassays.
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.

-------
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 reagent
grade chloroform (CHCI3), a clear liquid manufactured
by Mallinckrodt Chemical Company (lot #WBRC) t.6sted as
100% active ingredient. Results for all tests were ex-
pressed 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 con-
verting 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
f
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

-------
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 con-
crete 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 CaCC^, 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-

-------
Page four
cellent condition. The shrimp were held in 1100-1 fiber-
glass tanks in constantly flowing filtered (10 micrometers)
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 bottled 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 com-
mercial 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 accli-
mated 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 test conditions of water quality and temperature for at
least seven days prior to testing. Water in the test vessels
was not aerated: In all bioassays, the test compound was
pipeted directly into each jar. Animals were introduced
into the test vessel within 30 minutes after the compound

-------
Page five
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 bioassays was the same
as previously described for holding these fish. The hard
water for these bioassays was prepared by adding 192 mg
of NaHCO^, 12 0 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 bioassay consisted of
filtered (10 micrometers) natural sea water with a salinity
of 25 o/oo and a pH of 8.0 + 0.5. Concentrations of dis-
solved 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) con-
centrations 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, consistingvOf at least
five concentrations evaluated the dose-response relationship

-------
Page six
to a degree allowing the LC50 to be calculated from
the data with optimum accuracy. A control, which con-
sisted of the same dilution water, conditions, procedures,
and organisms, was maintained for each species tested.
RESULTS AND DISCUSSION
The estimated LC50 values and 95% confidence intervals
are presented in Table 1 along with the highest nominal
concentration at which there were no discernible effects
on test animals due to exposure to chloroform. A summary
of observed mortality for each individual' test concentration
at 24, 48 and 96 hours of exposure to chloroform 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. The shrimp exposed to the test concentrations
of 56 and 100 mg/1 became lethargic during the first 24
hours of exposure and were often observed lying on their
sides during the test. However, after 96 hours of exposure,
all nine remaining shrimp in the 56 mg/1 concentration were
upright and appeared in good condition; in the 100 mg/1 con-
centration, five of the seven remaining shrimp were on their

-------
Page seven
sides and two were upright. These effects could have been
the result of volatilization of chloroform, and subsequent
reduced concentrations in test water, over the 96-hour
period. The concentrations of dissolved oxygen, measured
at 0, 24, 48 and 96 hours of exposure, 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 conc-
centrations and controls for the shrimp bioassay.
The LC50 values for those bioassays exposing bluegill in
both soft'and hard water were essentially the same after
96 hours of exposure. The 96-hour value for rainbow trout
in soft water was ca 1.5X that for rainbow trout in hard
water. The LC50's for shrimp at 48 and 96 hours of exposure
were between the LC50 values determined for bluegill and
rainbow trout.

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

-------
Table 1 — Acute toxicity of chloroform to bluegilla (Lepomis
macrochirus) , rainbow trout*3 (Salmo gairdneri) , and
pink shrimp0 (Penaeus duorarum). These data are based
on the results of bioassays conducted at the Aquatic
Toxicology Laboratory and the Marine Research Laboratory
of E G & G, Bionomics, Wareham, Massachusetts and
Pensacola, Florida.
Species/
diluent
LC50(mg active ingredient/1)
24 hour	48 hour 96 hour
No discernible
effect level
at 96 hours
(mg/1)
bluegill/
soft water
185
(155-221)'
123
(107-143)
115
(96-138)
100
bluegill/
hard water
119
(96-148)
100
(72-140)
100
(72-140)
75
rainbow trout/
soft water
67.5
(55.2-82.5)
67.5	66.8
(55.2-82.5) (54.8-81.4)
42.0
rainbow trout/	55.5	43.8	43.8
hard water	(40.7-75.7) (36.1-53.2) (36.1-53.2)
24.0
pink shrimp
134	81.5	81.5
(96.3-187) (62.8-106) (62.8-106)
32.0
Bioassays conducted at 21 + 1.0 C, mean wet weight of bluegill 1.1 g.
)
Bioassays conducted at 12 + 1.0°C, mean wet weight of rainbow trout
1.0 g.
Bioassays conducted at 20 + 1.0°C, rostrum-telson lengths of pink
shrimp 35-50 mm.
95% confidence interval.

-------
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) ex-
posed to chloroform.
Nominal
Species/	concentration	% mortality observed	
diluent	(mg/1)	24 hour 48 hour 96 hour
bluegill/
soft water
bluegill/
hard water
320
240
180
140
120
100
control
320
240
180
140
100
75
control
100
60
80
10
.0
0
0
100
100
100
30
30
0
0
100
100
100
80
90
0
0
100
100
100
100
40
0
0
100
100
100
100
90
0
0
100
100
100
100
40
0
0
rainbow trout/
soft water
100
75
56
42
32
control
100
30
10
0
0
0
100
30
10
0
0
0
100
40
10
0
0
0

-------
Table 2 — Continued.
Species/
diluent
Nominal
concentration
(mg/1)
% mortality observed	
24 hour 48 hour 96 hour
rainbow trout/
hard water
100
75
56
42
32
24
control
100
100
40
0
0
0
0
100
100
70
20
10
0
0
100
100
70
20
10
0
0
pink shrimp
320
180
100
56
32
control
100
90
10
10
0
0
100
100
30
10
0
0
100
100
30
10
0
0

-------
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 chloroform.
Species/
diluent
Nominal
concentration
(mg/1)
Dissolved oxygen
(mg/1 and % of saturation)	
0 hour 24 hour 48 hour 96 hour
bluegill/
soft water
320
240
100
control
7.3	(81)
7.7 (87)
7.4	(82)
7.5(85)
6.8(75) 5.2(57) 4.7(52)
6.6(73) 4.8 (53) 4.2(46)
5.8 (64) 4.7 (52) 4.0(44)
bluegill/
hard water
180
100
75
control
8.0(90)
8.1(91)
7.8(88)
7.7(87)
5.8 (64) 4.2(46) 3.9 (43)
5.2 (56) 4.6 (51) 4.0 (44)
6.3(70) 5.8(64) 4.1(45)
rainbow trout/
soft water
100
75
42
control
9.2(85)	-
9.3(86)	7.7(70)	7.2(66)	6.9(63)
9.0 (82)	7.9 (72)	7.5 (69)	7.1(65)
9.0 (82)	7.8 (71)	6.8 (62)	6.5 (60)
rainbow trout/
hard water
75
56
32
control
9.0 (82)
9.8(90)
9.1(84)
9.7(89)
9.8 (90) 7.4 (67) 6.8 (62)
8.4(78) 6.9(63) 6.1(56)
9.7 (89) 6.6 (60) 6.1 (56)

-------
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/
180
6.8 (89)
5.6(74)

_a

sea water







100
6.7 (87)
6.3 (83)
5.
8(76)
3.0(39)

32
6.8(89)
6.2 (82)
5.
7(75)
4.0(53)

control
6.8 (89)
6.8 (89)
6.
0(79)
3.5 (46)
a
Dissolved oxygen not measured due to 100% mortality.

-------