INVESTIGATIONS IN FISH CONTROL

73. Formalin: Its Toxicity to Nontarget Aquatic Organisms, 

Persistence, and Counteraction

74. Chlorine: Its Toxicity to Fish and Detoxification of 

Antimycin

75. Malachite Green: Its Toxicity to Aquatic Organisms, 

Persistence, and Removal with Activated Carbon

76. Toxicity of Furanace to Fish, Aquatic Invertebrates, 

and Frog Eggs and Larvae

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE
Investigations in Fish Control, published by the Fish and Wildlife Service, include reports on the 

results of work at the Service's Fish Control Laboratories at La Crosse, Wis., and Warm Springs, 

Ga., and reports of other studies related to that work. Though each report is regarded as a separate 

publication, several may be issued under a single cover, for economy. [See Investigations in Fish 

Control 47-50 (in one cover) for list of issues published prior to 1970.]

35. Toxicology of Thiodan in Several Fish and Aquatic Invertebrates, by Richard A. 

Schoettger. 1970. 31 pp.

(Reports 36 through 38 are in one cover.)

36. A Method for Rating Chemicals for Potency Against Fish and Other Organisms, by Leif L. 

Marking. 1970. 8 pp.

37. Comparative Toxicity of 29 Nitrosalicylanilides and Related Compounds to Eight Species of 

Fish, by Leif L. Marking and Wayne A. Willford. 1970. 11 pp.

38. Toxicity of 33NCS (3'-chloro-3-nitrosalicylanilide) to Freshwater Fish and Sea Lampreys, by 

Leif L. Marking, Everett L. King, Charles R. Walker, and John H. Howell. 1970. 16 pp.

(Reports 39 and 40 are in one cover.)

39. Effects of Antimycin A on Tissue Respiration of Rainbow Trout and Channel Catfish, by 

Richard A. Schoettger and Gerald E. Svendsen. 1970. 10 pp.

40. A Resume on Field Applications of Antimycin A to Control Fish, by Robert E. Lennon and 

Bernard L. Berger. 1970. 19 pp.

(Reports 41 through 43 are in one cover.)

41. Identification of MS-222 Residues in Selected Fish Tissues by Thin Layer Chromatography, by 

John L. Alien, Charles W. Luhning, and Paul D. Harman. 1970. 7 pp.

42. Dynamics of MS-222 in the Blood and Brain of Freshwater Fishes During Anesthesia, by 

Joseph B. Hunn. 1970. 8 pp.

43. Effect of MS-222 on Electrolyte and Water Content in the Brain of Rainbow Trout, by Wayne A. 

Willford. 1970. 7 pp.

44. A Review of Literature on TFM (3-trifluormethyl-4-nitrophenol) as a Lamprey Larvicide, by 

Rosalie A. Schnick. 1972. 31 pp.

(Reports 45 and 46 are in one cover.)

45. Residues of MS-222 in Northern Pike, Muskellunge, and Walleye, by John L. Alien, Charles W. 

Luhning, and Paul D. Harman. 1972. 8 pp.

46. Methods of Estimating the Half-Life of Biological Activity of Toxic Chemicals in Water, by Leif 

L. Marking. 1972. 9 pp.

(Reports 47 through 50 are in one cover.)

47. Preparation and Properties of Quinaldine Sulfate, an Improved Fish Anesthetic, by John L. 

Alien and Joe B. Sills. 1973. 7 pp.

48. Toxicity of Quinaldine Sulfate to Fish, by Leif L. Marking and Verdel K. Dawson. 1973. 8 

pp.

49. The Efficacy of Quinaldine Sulfate as an Anesthetic for Freshwater Fish, by Philip A. 

Gilderhus, Bernard L. Berger, Joe B. Sills, and Paul D. Harman. 1973. 9 pp.

50. Residue of Quinaldine in Ten Species of Fish Following Anesthesia with Quinaldine Sulfate, 

by Joe B. Sills, John L. Alien, Paul D. Harman, and Charles W. Luhning. 1973. 9 pp.

(Reports 51 and 52 are in one cover.)

51. Methods for Simultaneous Determination and Identification of MS-222 and Metabolites in 

Fish Tissues, by Charles W. Luhning. 1973. 10 pp.

52. Residues of MS-222, Benzocaine, and Their Metabolites in Striped Bass Following Anesthesia, 

by Charles W. Luhning. 1973. 11 pp.

Use of trade names does not imply U.S. Government endorsement of commercial products.
INVESTIGATIONS IN FISH CONTROL

73. Formalin: Its Toxicity to Nontarget Aquatic Organisms, 

Persistence, and Counteraction

By Terry D. Bills, Leif L. Marking, and Jack H. Chandler, Jr.

74. Chlorine: Its Toxicity to Fish and Detoxification of 

Antimycin

By Leif L. Marking and Terry D. Bills

75. Malachite Green: Its Toxicity to Aquatic Organisms, 

Persistence, and Removal with Activated Carbon

By Terry D. Bills, Leif L. Marking, and Jack H. Chandler, Jr.

76. Toxicity of Furanace to Fish, Aquatic Invertebrates, and 

Frog Eggs and Larvae

By Leif L. Marking, Terry D. Bills, and Jack H. Chandler, Jr.

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE 

Washington, D.C. 1977

Formalin: Its Toxicity to Nontarget Aquatic Organisms, 

Persistence, and Counteraction

by

Terry D. Bills and Leif L. Marking 

Fish Control Laboratory, La Crosse, Wisconsin

Jack H. Chandler, Jr.

Southeastern Fish Control Laboratory

Warm Springs, Georgia

Abstract

The acute toxicity of formalin to selected fishes and aquatic invertebrates was determined in 

standardized laboratory tests. Fish species exposed were chinook salmon (Oncorhynchus 

tshawytscha), rainbow trout (Salmo gairdneri), Atlantic salmon (S. salar), lake trout 

(Salvelinus namaycush), black bullhead (Ictalurus melas), channel catfish (I. punctatus), green 

sunfish (Lepomis cyanellus), bluegill (L. macrochirus), smallmouth bass (Micropterus 

dolomieui), and largemouth bass (M. salmoides). Invertebrates exposed were freshwater prawn 

(Palaemonetes kadiakensis), seed shrimp (Cypridopsis sp.), Asiatic clam (Corbicula leana), 

snail (Helisoma sp.), and backswimmer (Notonecta sp.). Black bullhead and channel catfish 

were the fish most sensitive to formalin (96-h LC 50's, 62.1 and 65.8 //l/l), and Atlantic salmon 

and green sunfish were the most resistant (96-h LCso for each, 173 //l/l). The TILC50 (lethal 

concentration producing 50% mortality independent of time) for formalin against rainbow 

trout was 72.0 //l/l. Seed shrimp were the most sensitive invertebrates (96-h LC50 , 1.05 //l/l), 

and backswimmers were the most resistant (96-h LC50 ,835 //l/l). The toxicity of formalin was 

unchanged in solutions aged as long as 3 weeks; the biological half-life could not be determined. 

Formalin was not detoxified by oxidation or reduction, and filtration through activated carbon 

did not significantly reduce toxicity.

Formalin is one of the most effective and widely 

used compounds in fish culture for therapeutic and 

prophylactic treatment of fungal infections and 

external parasites of fish and fish eggs. Uses of 

formalin in fish culture were reviewed by Schnick 

(1974). Before about 1967, the registration of 

chemicals used to treat diseases of fish in hatcheries 

was not required. Since then the Food and Drug 

Administration and the Environmental Protection 

Agency have required specific information about 

each chemical and its use pattern before registration. 

Information required for the registration includes 

toxicity to target and nontarget organisms, efficacy, 

residues, metabolites, and means of counteraction 

(Lennon 1967). Standardized tests have been 

developed for generating toxicity information 

necessary for the registration of fishery chemicals 

(Marking 1975).

The purposes of this study were to determine (1) the 

toxicity of formalin to nontarget aquatic organisms, 

(2) the toxicity (safety) of maximum use-pattern 

exposures to formalin, (3) the toxicity of formalin to

selected fishes in extended exposures, (4) the effects 

of certain water characteristics on the toxicity of 

formalin to fish, (5) the persistence of formalin in 

water, and (6) the feasibility of counteracting formalin by oxidation or reduction, or removal from 

water with activated carbon.

Materials and Methods

Stock solutions of commercial grade formalin (37% 

formaldehyde) obtained from North Central 

Chemical Co., La Crosse, Wisconsin, were prepared in 

water (the liquid formulation was measured 

volumetrically and diluted with water). All concentrations listed are based on the formulated product. 

To prepare test solutions of the desired concentrations, we pipetted portions of stock solutions into 

the test vessels, and stirred the resulting mixture to 

ensure homogeneity. In flow-through toxicity tests, 

required amounts of the stock solutions were 

delivered by a solenoid-activated pipetting pump 

(Micromedic Systems Automatic Pipette Model 2500).
Fish species exposed were chinook salmon (Oncorhynchus tshawytscha), rainbow trout (Salmo 

gairdneri), Atlantic salmon (S. salar), lake trout 

(Salvelinus namaycush), black bullhead (Ictalurus 

melas), channel catfish (I. punctatus), green sunfish 

(Lepomis cyanellus), bluegill (L. macrochirus), 

smallmouth bass (Micropterus dolomieui), and 

largemouth bass (M. salmoides). Invertebrates exposed were freshwater prawn (Palaemonetes 

kadiakensis), seed shrimp (Cypridopsis sp.), clam 

(Corbicula leana), snail (Helisoma sp.), and 

backswimmer (Notonecta sp.). The fish were obtained 

from State and Federal hatcheries and maintained in 

the laboratory; invertebrates were either cultured 

outdoors in partly shaded vinyl pools or collected in 

the field. Organisms collected in the field were held 

for 7 days in water identical with that used in the 

toxicity tests, before they were exposed to formalin. 

Fish and invertebrates were acclimated to test 

conditions for 24 h before the addition of formalin. 

Ten or more organisms were exposed at each 

concentration. Mortalities were recorded at 1, 3, and 

6 h the first day and daily thereafter during the 96-h 

exposure period. Fish were regarded as dead when all 

opercular movements ceased and invertebrates when 

they became immobile or failed to respond to physical 

stimuli.

Laboratory toxicity tests were conducted according 

to standard procedures described by Lennon and 

Walker (1964) and the Committee on Methods for 

Toxicity Tests with Aquatic Organisms (1975). Static 

tests were conducted in 2.5- or 15-liter glass jars, 

depending on the size of the test organism involved. 

Flow-through tests were conducted in 45 liters of test 

solution; the solution was replaced four times daily 

through a 1-liter dilution apparatus similar to that 

described by Mount and Brungs (1967).

Temperature was controlled by immersing test 

vessels in a water bath equipped with a chilling unit. 

Reconstituted water (Marking 1969) was used in tests 

with fish and clams and limed spring water (pH 6.5 

±0.1, total hardness 20 mg/1 as CaCOs) in tests with 

the other invertebrates. Chemical buffers were added 

to soft water in tests of the effect of pH (6.5-9.5), as 

recommended by Marking and Dawson (1973). The 

pH's of the test solutions were checked daily and 

adjusted to within ±0.2 pH units. For determination of 

the persistence of formalin, aqueous solutions were 

aged 1, 2, and 3 weeks, after which rainbow trout 

fingerlings were introduced and 96-h LC50 's computed. Deactivation indices were computed from 

these data according to the method of Marking (1972).

In counteraction studies, potassium permanganate 

(KMnO.i) at a concentration of 1 mg/1 and sodium 

thiosulfate (Na2 S2 O3 ) at 10 mg/1 were introduced 

into a series of formalin solutions of selected

concentrations 6 h before the introduction offish. In 

aeration tests, the solutions were aerated with air 

stones for 24 h before fish were added. We compared 

the 96-h LC50's with a reference standard to assess 

changes in toxicity. To determine if formalin could be 

removed from aqueous solutions, we filtered a 

concentrated solution (150 01/1) at a flow rate of 

100 ml/min through a 15-cm column of activated 

charcoal (Darco 20 * 40 mesh). Samples of effluent 

were taken at selected volumes (0-200 ml and 

800-1,000 ml). These samples and a sample of the 

stock solution were bioassayed against rainbow trout 

and the 96-h LC50 's compared.

We used the method of Litchfield and Wilcoxon 

(1949) to determine LC50 's and 95% confidence 

intervals, and a modification of the method given by 

Green (1965) to compute TILC50 's.

Results

Toxicity of Formalin to Fish

The 96-h LCso's for formalin against nine species of 

fish ranged from 62.1 01/1 for black bullheads to 173 

0 1/1 for green sunfish and Atlantic salmon (Table 1). 

Toxicity of formalin increased with time; for 

bluegills, for example, the 3- and 96-h LCso's were 

2,290 01/1 and 100 01/1, respectively. Ictalurids were 

twice as sensitive to formalin as the centrarchids or 

salmonids. Green sunfish were the most resistant 

centrarchid exposed, followed by largemouth bass, 

smallmouth bass, and bluegills. Atlantic salmon 

were the most resistant salmonid, followed by 

rainbow trout and lake trout (Table 1).

The effects of temperature, water hardness, andpH 

on toxicity were determined by exposing rainbow 

trout, channel catfish, and bluegills to formalin. In 

short exposures, formalin was significantly more 

toxic to these species at the higher temperatures; 

however, at 96 h the differences were insignificant 

except in rainbow trout (Tables 2, 3, and 4). Water 

hardness had no apparent effect on toxicity. For 

rainbow trout and channel catfish, formalin was 

more toxic in waters of pH 9.5 than in waters of pH 

6.5, 7.5, or 8.5 (Tables 2 and 3).

In chronic toxicity tests the TILCso for formalin 

against rainbow trout was 72.0 01/1 as compared 

with LC50 's of 157 01/1 at 24 h and 131 01/1 at 96 h.

Toxicity of 

Formalin to Invertebrates

Invertebrates differed widely in their responses to 

formalin. The 96-h LC50 's ranged from 1.05 01/1 for 

seed shrimp to 835 01/1 for backswimmers; those for 

the bivalve and snail 126 and 93 01/1 were similar
Table 1. Toxicity of formalin to fingerling fish of nine species in standard toxicity tests at 12 C.

Species

Rainbow trout

Atlantic salmon

Lake trout

Black bullhead

Channel catfish

Green sunfish

Bluegill

Smallmouth bass

Largemouth bass

Average 

weight 

(g)

0.63

0.60

0.50

0.75

0.40

0.70

0.50

0.68

1.00

LC50 and 95% confidence interval (//l/l) at

3 h

1230 

957-1581

1410 

1049-1896

495 

430-570

2290 

1804-2907

6 h

655 

580-740

840 

751-939

603 

444-819

232 

178-303

1600 

1165-2198

1030 

928-1140

24 h

300 

237-380

389 

333-455

141

114-174

173 

123-243

122 

102-145

323 

250-417

211 

171-260

222 

171-288

283 

229-350

96 h

118 

99.7-140

173 

149-201

100 

78.2-128

62.1 

50.9-75.8

65.8

58.1-74.5

173 

123-243

100 

80.0-125

136 

90.2-205

143 

129-159

Table 2. Toxicity of formalin to fingerling rainbow trout at selected water temperatures, hardnesses, and

pH's.

Temp

7

12

17

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Hard

Very hard

Soft

Soft

Soft

LCso and 95% confidence interval (//l/l) at

PH

7.5

7.5

7.5

6.6

7.8

8.2

6.5

8.5

9.5

1 h

>3000

2310 

1959-2724

1210 

1015-1443

>2500

1740 

1240-2441

1690 

1070-2670

1730 

1233-2427

1390 

1043-1852

1740 

1240-2441

3 h

1810 

1537-2131

1230 

957-1581

1000 

788-1269

1590 

1070-2364

1740 

1240-2441

1690 

1070-2670

1730 

1233-2427

1150 

917-1442

875 

750-1020

6 h

940 

762-1160

655 

580-740

590 

532-654

729 

657-809

925 

783-1093

910 

769-1077

835 

749-931

645 

523-796

500 

355-704

24 h

349 

292-418

300 

237-380

219 

174-276

230 

181-292

388 

332-454

334 

272-411

321 

231-446

300 

220-408

135 

105-174

96 h

245 

213-282

118 

99.7-140

172 

108-274

171 

122-240

171 

122-240

172 

123-241

100 

78.9-127
Table 3. Toxicity of formalin to channel catfish at selected water temperatures, hardnesses, and pH's.

Temp.

12

17

22

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Hard

Very hard

Soft

Soft

Soft

LCso and 95% confidence interval ( jU.1/1) at

pH

7.5

7.5

7.5

6.6

7.8

8.2

6.5

8.5

9.5

1 h

779 

666-911

600 

523-689

559 

475-658

771 

660-901

1050 

798-1382

872 

703-1081

779 

666-912

630

554-717

559 

474-659

3 h

495 

430-570

350 

299-409

284 

229-351

490 

425-565

450 

360-563

439 

346-557

424 

356-505

455 

365-567

282 

228-349

6 h

232 

178-303

282 

228-349

234 

185-297

490 

425-565

355 

298-422

285 

229-355

282 

228-349

285 

230-353

235 

185-298

24 h

122 

102-145

119 

102-138

100 

83.7-119

99.0 

86.0-114

117 

100-137

111 

94.5-130

118 

103-135

94.0 

84.0-105

63.9

54.7-74.7

96 h

65.8

58.1-74.5

65.5 

58.2-73.7

64.0 

58.6-69.9

69.9 

63.7-76.7

49.0 

43.3-55.5

61.9 

53.9-71.1

62.0 

54.4-70.7

56.5 

51.4-62.1

42.9 

36.2-50.9

Table 4. Toxicity of formalin to fingerling bluegills at selected water temperatures, hardnesses, and pH's.

Temp.

12

17

22

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Hard

Very hard

Soft

Soft

Soft

pH

7.5

7.5

7.5

6.6

7.8

8.2

6.5

8.5

9.5

LCso

3 h

2290 

1804-2907

2300 

1822-2904

1750 

925-3312

1800 

1532-2115

1720 

1458-2029

1740 

1499-2019

2310 

1961-2721

2300 

1822-2904

2300 

1950-2712

and 95% confidence interval ( jU.1/1) at

6 h

1600 

1165-2198

780 

670-908

469 

403-545

1230 

1071-1412

1190 

1027-1379

1310 

1038-1654

2290 

1944-2697

1650 

1401-1943

1055 

883-1260

24 h

211 

171-260

189 

153-234

142 

115-176

369 

315-433

249 

166-373

233 

181-300

335 

284-395

230 

182-290

232 

174-309

96 h

100 

80.0-125

73.5 

63.5-85.0

91.0 

81.2-102

88.4 

75.1-104

106 

84.0-134

117 

101-136

125 

89.1-175

86.2 

72.6-102

100 

72.6-138
to those for fish. The freshwater prawn was intermediate in resistance to formalin, having a 96-h 

LC 50 of 465 11 1/1 (Table 5).

Toxicity of Formalin 

at Use-Pattern Concentrations

Recommended use-pattern concentrations of formalin range as high as 250 /t 1/1 for 1 h in tanks or 

raceways and are 15 to 25 ju. 1/1 for indefinite periods 

in earthen ponds. Exposure to use-pattern concentrations caused no mortality in chinook salmon, 

rainbow trout, Atlantic salmon, lake trout, black 

bullhead, channel catfish, green sunfish, bluegill, 

smallmouth bass, or largemouth bass. The seed 

shrimp was the only invertebrate affected; 99% 

mortality could be expected at a 25-/x 1/1 indefinite 

treatment level.

Persistence of Formalin 

in Aqueous Solutions

The toxicity to rainbow trout fingerlings of formalin solutions that had been aged for 1, 2, and 3 

weeks was not substantially different from that of 

fresh solutions (Table 6).

Formalin solutions were not detoxified by either 

oxidation or reduction. The 96-h LCso's for the 

formalin reference solution, an aerated solution, and 

a solution to which thiosulfate had been added were 

not significantly different. However, the 96-h LC50 for 

the formalin:potassium permanganate solution was

Table 6. Effect of aging on the toxicity to rainbow 

trout of formalin in soft water at 12 C.

Aging 

period

(weeks)

0

1

2

3

96-h LC 50 (fi. 1/1) and 

95% confidence interval

119 

91.3-155

111 

94.5-130

141 

114-174

122 

87.5-170

Deactivation 

index

1.00

0.933

1.18

1.03

60.0 /A 1/1 as compared with 107 /x. 1/1 for the formalin 

reference (Table 7).

When the first and last 200-ml portions of the 

filtrate of a 150- /tl/1 formalin stock solution filtered 

through a 15-cm column of activated carbon were 

bioassayed against rainbow trout along with a 

sample of the stock solution, the 96-h LCso's were 

210 n 1/1 for the first 200-ml sample, 132 /A 1/1 for the 

800- to 1,000-ml sample, and 121 /A 1/1 for the 

reference solution. Although this difference indicates 

some removal of formalin, the removal was insignificant when the relative amounts of formalin and 

carbon involved (1 mg formalin/1 g carbon) are 

considered (Table 8).

Species

Table 5. Toxicity of formalin to selected aquatic invertebrates in soft water at 16 C.

______________LCso and 95% confidence interval (/xl/1) at________

1 h 3 h 6 h 24 h

a Toxicity based on immobility.

b Toxicity based on ability to resist attempts to open valves and respond to tactile stimulus.

c Toxicity based on ability to respond to tactile stimulus.

96 h

Seed shrimp (ostracods) a 

Cypridopsis sp.

Freshwater prawna 

Palaemonetes kadiakensis

Bivalves 

Corbicula sp.

Snailc 

Helisoma sp.

Backswimmerc 

Notonecta sp.

9.00 

6.83-11.9

3525 

3201-3881

6.40 

4.91-8.34

2150 

1948-2373

1340 

953-1883

1.20 

0.664-2.17

1900 

1588-2273

780 

629-967

1.15 

0.690-1.97

1105 

896-1362

800 

638-1003

710 

544-925

4500 

3006-6735

1.05 

0.590-1.87

465

368-588

126 

80.9-196

93.0 

69.5-124

835 

652-1069
Table 7. Toxicity of formalin solutions containing selected oxidizing and reducing agents to fingerling

rainbow trout a -

Chemical Concentration

(mg/1)

96-hLC50 (/il/l)and 

confidence interval

Formalin (reference) 

Formalin:aeratiou " 

Formalin:thiosulfate 

Formalin:KMnO4

10

107

89.9-127

117

90.0-152

99.0 

81.4-120

60.0 

53.8-66.9

Fish were added to the reference, thiosulfate, and KMnO4 solutions 6 h after the chemicals were added. 

Solutions were aerated vigorously for 24 h before addition of fish.

Discussion

Information regarding the toxicity of formalin to 

various aquatic organisms is abundant. However, the 

varied test conditions under which the data were 

developed make comparisons difficult, and some of 

the data are unacceptable for use in the evaluation of 

formalin for registration (Schnick 1974). Usually no 

reference has been made to temperature, pH, 

hardness, or other characteristics of water that 

directly affect toxicity and efficacy of other chemicals 

used in fisheries (Marking and Olson 1975; McKee 

and Wolf 1963).

Schnick (1974) pointed out the wide range of 

sensitivity for different species offish, salmonids and 

centrarchids being the most resistant and ictalurids 

the most sensitive. Data from our study follow this 

pattern. Schnick also stated that, although various

Table 8. Toxicity of selected eluates of a 150- \d/l

formalin stock solution filtered through a 15-cm

column of activated carbon to rainbow trout in soft

water at 12 C.

Eluate 96-hLC50 (/xl/l)and 

95% confidence interval

Reference a

0 to 200 ml

800 to 1,000 ml

121 

105-140

210 

189-233

132 

111-157

a Toxicity of stock solution before filtration.

chemical characteristics of the water and physical 

condition of the fish appear to influence the toxicity of 

formalin, variations in sensitivity within a species 

may be due to genetic composition.

The effect of water chemistry on the toxicity of 

formalin to fish is somewhat controversial. Birdsong 

and Avault (1971) reported that the toxicity of 

formalin to pompano (Trachinotus carolinus) was not 

affected by different salinity levels. Piper and Smith 

(1973) reported that water chemistry has no effect on 

the toxicity of formalin to fish; however, their data 

were based on questionnaires received from various 

hatcheries rather than on experimental data. Marking et al. (1972) also reported that the toxicity of 

formalin was not affected by water hardness or pH. 

Bills (1974) first demonstrated that formalin was 

more toxic to fish and fish eggs in alkaline than in 

acid water. This conclusion is further supported by 

data from the present study, which show that in soft 

water formalin was more toxic to channel catfish and 

rainbow trout at pH 9.5 than at lower pH's.

Formalin is frequently used at concentrations of 15 

to 25 fi 1/1 for control of parasites on fish in earthen 

ponds. Much information is available on the efficacy 

of formalin as a parasiticide; however, the effects of 

formalin on pond flora and fauna, particularly on 

aquatic invertebrates, have not been determined. 

Schnick (1974) reported few data on the toxicity of 

formalin to invertebrates. Our data show a wide 

range of sensitivities for invertebrates; the 96-h 

LC50 's ranged from 1.05 /*!/! for seed shrimp to 

835 /* 1/1 for backswimmers. Our data also show 

formalin to be persistent under laboratory conditions, 

and at use-pattern concentrations some invertebrates 

could be affected. Present governmental controls on 

the use of chemicals in the environment necessitate
counteraction of persistent compounds after they 

have accomplished their purpose (Dawson 1976); 

however, the two most commonly used techniques for 

removal of such compounds (chemical oxidation/reduction or adsorption on activated carbon) 

failed to neutralize the toxicity of formalin. In fact, 

under oxidative conditions the solutions became 

more toxic.

Although some formalin may be removed by 

activated carbon, the amount is insignificant and the 

technique probably would not be applicable to 

hatchery operations.

Conclusions

1. Black bullheads were the species most sensitive 

to formalin (96-h LC50 's = 62.1 ^1/1).

2. Atlantic salmon and green sunfish were the most 

resistant species (96-h LC50 's = 173 /u.1/1).

3. Lake trout were the most sensitive salmonids and 

bluegills were the most sensitive centrarchids.

4. The toxicity of formalin was not influenced by 

water hardness, but in soft water the chemical 

was more toxic to rainbow trout and channel 

catfish at pH 9.5 than at pH 6.5 or 8.5.

5. Formalin was more toxic to rainbow trout, 

channel catfish, and bluegills in warm than in 

cold water in 3-h exposures, but after 96 h the 

difference continued to be statistically significant only in rainbow trout.

6. Formalin was about twice as toxic in chronic 

exposures as in acute exposures.

7. Seed shrimp were the only organisms exposed 

that were affected by formalin at use-pattern 

concentrations.

8. Seed shrimp were the most sensitive invertebrates and backswimmers the most resistant; the 

96-h LCso's were 1.05 /xl/1 and 835 /JL\/\.

9. The toxicity of formalin solutions persisted after 

3 weeks of aging.

10. Formalin solutions were not detoxified by oxidation or reduction; in fact, they became more toxic 

under oxidative conditions.

11. Vigorous aeration for 24 h did not significantly 

change the toxicity of formalin solutions.

12. Only a small proportion of formalin was removed 

by filtration through activated carbon.

References

Bills, T. D. 1974. Toxicity of formalin, malachite green,

and the mixture to four life stages of rainbow trout. M.S.

Thesis. University of Wisconsin-La Crosse. 40 pp. 

Birdsong, C. L, and J. W. Avault, Jr. 1971. Toxicity of

certain chemicals to juvenile pompano. Prog. Fish-Cult.

33(2):76-80. 

Committee on Methods for Toxicity Tests with Aquatic

Organisms. 1975. Methods for acute toxicity tests

with fish, macroinvertebrates, and amphibians.

Ecological Research Series. EPA [Environ. Prot. Agency]-660/3-75-009. 61 pp. 

Dawson, V. K. 1975. Counteracting chemicals used in

fishery operations: current technology and research.

Pages 32-40 in P. H. Eschmeyer, ed. Rehabilitation of

fish populations with toxicants: a symposium. North

Cent. Div., Am. Fish. Soc., Spec. Publ. No. 4. 

Green, R. H. 1965. Estimation of tolerance over an

indefinite time period. Ecology 46(6):887. 

Lennon, R. E. 1967. Clearance and registration of

chemical tools for fisheries. Prog. Fish-Cult.

29(4):187-193. 

Lennon, R. E., and C. R. Walker. 1964. Laboratories and

methods for screening fish control chemicals. U.S. Fish

Wildl. Serv. Invest. Fish Control 1 (Circ. No. 185). 15 pp. 

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified

method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2):99-113. 

Marking, L. L. 1969. Toxicological assays with fish. Bull.

Wildl. Dis. Assoc. 5:291-294. 

Marking, L. L. 1972. Methods of estimating the half-life

of biological activity of toxic chemicals in water. U.S.

Fish Wildl. Serv. Invest. Fish Control. 46. 9 pp. 

Marking, L. L. 1975. Toxicological protocol for the

development of piscicides. Pages 26-31 in P.H.

Eschmeyer, ed. Rehabilitation of fish populations with

toxicants: a symposium. North Cent. Div., Am. Fish,

Soc., Spec. Publ. No. 4. 

Marking, L. L., and V. K. Dawson. 1973. Toxicity of

quinaldine sulfate to fish. U.S. Fish Wildl. Serv. Invest.

Fish Control 48. 8 pp. 

Marking, L. L., E. L. King, Jr., and J. H. Chandler,

Jr. 1972. Section of toxicology. Pages 3-8 in Quarterly

report of progress for July-September 1972. Fish Control

Laboratory, La Crosse, Wis. 

Marking, L. L., and L. E. Olson. 1975. Toxicity of the

lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to

nontarget fish in static tests. U.S. Fish Wildl. Serv.

Invest. Fish Control 60. 27 pp. 

McKee, J. E., and H. W. Wolf. 1963. Water quality

criteria. Calif. State Water Quality Control Bull. Publ.

No. 3A. 548 pp. 

Mount, D. I.,andW.A.Brungs. 1967. A simplified dosing

apparatus for fish toxicological studies. Water Res.

1:21-29. 

Piper, R. G., and C. E. Smith. 1973. Factors influencing

formalin toxicity in trout. Prog. Fish-Cult. 35(2):78-81. 

Schnick, R. A. 1974. Formalin as a therapeutant in fish

culture. U.S. Fish Wildl. Serv. Lit. Rev. 74-09. Natl. Tech.

Inf. Serv. No. PB-235 4487AS. 145 pp.

Chlorine: Its Toxicity to Fish and Detoxification

of Antimycin

by

Leif L. Marking and Terry D. Bills 

Fish Control Laboratory, La Crosse, Wisconsin

Abstract

The 96-h LC50 's for chlorine ranged from 0.156 mg/1 for channel catfish (Ictalurus 

punctatus) to 1.41 mg/1 for black bullheads (L melas) in standardized laboratory tests. The 

toxicity of chlorine was influenced little by pH, temperature, or water hardness. Chlorine at 0.25 

and 0.5 mg/1 effectively detoxified antimycin. The half-life of biological activity for antimycin 

with chlorine ranged from 1.1 h at pH 6.5 to 1.5 h at pH 9.5. Chlorine readily detoxified 

antimycin at pH 6.5, 7.5, and 8.5, but not at pH 9.5.

Chlorine is used extensively for disinfection of 

municipal water supplies and effluents (Fair et al. 

1948; Chambers 1971) and occasionally for sterilization of fish hatchery water supplies (Hagen 1940; 

Bedell 1971) and culture ponds. Due to the widespread 

use of chlorine, precautions must be considered for 

protecting nontarget organisms such as fish, aquatic 

invertebrates, and plants. Brungs (1973), who summarized acute and chronic effects of residual chlorine 

on some aquatic organisms, recommended that total 

residual chlorine not exceed 0.2 mg/1 for a period of 

2 h/day for the more resistant species of fish. The 

evaluation of toxicity data in the literature is 

complicated by lack of information on the water 

chemistry, temperature, chlorine demand, solubility 

conditions, formation of chlorine complexes, and 

exposure time. Merkens (1958) stated that toxicity of 

chlorine depended on the amount of chlorine that 

remained in solution rather than on the amount 

added. Therefore the exact conditions of toxicity tests 

with aquatic organisms should be reported, and 

standardized procedures followed.

Because chlorine is a strong oxidizing agent, it can 

be used to detoxify chemicals that are subject to 

oxidation reactions. Preliminary studies on the fish 

toxicant antimycin indicated that it is effectively 

detoxified with 0.5 mg/1 of chlorine in soft water at 

pH 7.5 (Dawson and Marking 1974); a concentration 

of 10 /xg/1 of antimycin was decreased to less than 

0.2 Mg/l after 2 h. The preliminary results were 

based on sensitivity bioassays with green sunfish 

(Lepomis cyanellus). Although antimycin is nonpersistent in the aquatic environment (Marking and 

Dawson 1972), additional measures to accelerate its 

detoxification are needed when the possibility exists 

that treated water will enter municipal water 

supplies.

This study was designed to determine the toxicity 

of chlorine to fish and to establish the efficiency of 

chlorine for detoxifying the fish toxicant antimycin. 

Factors influencing toxicity of chemicals in water 

were included in the evaluation.

Materials and Methods

Technical grade antimycin was obtained from 

Ayerst Laboratories, Rouses Point, New York. Stock 

solutions were prepared by dissolving weighed 

portions in acetone and further diluting them in 

aqueous stock solutions just before use. Portions of 

the aqueous stock solutions delivered to the static test 

chambers yielded selected concentrations over a 

range which produced mortality at high concentrations but permitted survival at low concentrations. 

Aqueous stock solutions of chlorine were prepared 

from calcium hypochlorite (commercial grade HTH) 

containing 70% available chlorine. Test concentrations were based on active chlorine.

Procedures for the static toxicity tests followed 

those described by Lennon and Walker (1964), with 

some modifications. Test waters were prepared by 

adding mineral salts to deionized water in prescribed 

proportions to yield total hardness (as mg/1 of 

CaCO3 ) of 12 for very soft water, 44 for soft, 160 for 

hard, and 300 for very hard water (Marking 1969). 

The pH of reconstituted water was altered and 

stabilized by adding chemical buffers to soft water to 

yield pH's of 6.5, 7.5, 8.5, and 9.5 (Marking and 

Dawson 1973). The pH of water of different 

hardnesses was stabilized with sodium bicarbonate. 

The pH's of test solutions were checked daily and 

adjusted when necessary. Soft water was used in

1
standardized tests for the determination of the 

toxicity of chlorine to various species of fish.

Fish were obtained from Federal fish hatcheries 

and maintained by a trained fish culturist (Hunn et 

al. 1968). Ten fish, 2 to 5 cm in total length, were 

exposed to each concentration. Fish loading rates did 

not exceed 1 g per liter of water. Common and 

scientific names for fish used are listed in Table 1.

Mortalities were observed and recorded at 1, 3, and 

6 h during the first day and at least daily thereafter. 

We analyzed the data by the methods of Litchfield 

and Wilcoxon (1949) to obtain LC50 's (concentrations 

calculated to produce 50% mortality) and 95% confidence intervals. Chi-square tests indicated acceptability of all data reported. The half-life of antimycin:chlorine solutions was estimated by plotting 

deactivation indices against time on semilogarithmic 

coordinates (Marking and Dawson 1972).

Results

Toxicity of Chlorine to Fish

The toxicity of chlorine to fish varied with the 

species; 96-h LC 50's ranged from 0.156 mg/1 for 

channel catfish to 1.41 mg/1 for black bullheads 

(Table 1). The three coldwater species (coho salmon 

rainbow trout, and lake trout) were more sensitive 

than the warmwater species (except for channel 

catfish). At 7 or 8 mg/1 of chlorine, mortality 

occurred within 1 h of exposure for all species except 

goldfish, carp, fathead minnows, and black 

bullheads. The LC 5,,'s changed little after 24 h of 

exposure.

In rainbow trout exposed at different temperatures, 

chlorine was more toxic at 17 and 12 C than at 7 C, 

after 1 h of exposure (Table 2). This trend was

Table 1. Toxicity of chlorine (from commercial grade HTH) to 12 species offish in soft, reconstituted water at

12 C.

Species LCso and 95% confidence interval (mg/1) at

1 h 3 h 6 h 24 h 96 h

Coho salmon 

Oncorhynchus kisutch

Rainbow trout 

Salmo gairdneri

Lake trout 

Salvelinus namaycush

Goldfish 

Carassius auratus

Carp 

Cyprinus carpio

Fathead minnow 

Pimephales promelas

White sucker 

Catostomus commersoni

Black bullhead 

Ictalurus melas

Channel catfish 

/. punctatus

Green sunfish 

Lepomis cyanellus

Bluegill 

L. macrochirus

Yellow perch 

Perca flavescens

4.25 

3.43-5.26

0.969 

0.886-1.06

1.19 

0.974-1.45

>7.00

>8.00

>7.00

2.00 

1.65-2.42

>8.00

1.38 

1.06-1.79

9.42 

6.84-13.0

10.8 

6.41-18.2

1.32 

0.994-1.75

0.599 

0.541-0.664

0.640 

0.533-0.769

0.615 

0.554-0.682

3.65 

2.91-4.57

2.43 

1.57-3.77

0.880 

0.830-0.933

>8.00

0.346 

0.273-0.438

3.00 

2.21-4.08

1.32 

0.996-1.75

1.16 

0.842-1.19

0.434 

0.383-0.492

0.550

0.450 

0.371-0.545

2.39 

1.87-3.06

2.83 

2.56-3.13

1.38 

0.964-1.98

0.631 

0.538-0.740

2.21

0.286 

0.231-0.354

1.72 

1.23-2.41

1.11 

0.797-1.55

0.735 

0.658-0.821

0.310 

0.247-0.389

0.236 

0.199-0.280

0.246 

0.177-0.343

1.42 

1.14-1.76

0.825

1.00 

0.843-1.19

0.379 

0.318-0.452

1.41 

1.14-1.74

0.156 

0.106-0.229

1.28 

1.01-1.62

0.569 

0.484-0.669

0.570 

0.489-0.665

0.289 

0.226-0.370

0.172 

0.148-0.200

0.200 

0.147-0.272

1.18 

0.902-1.54

0.800

0.998 

0.841-1.18

0.379 

0.318-0.452

1.41 

1.14-1.74

0.156 

0.106-0.229

1.28 

1.01-1.62

0.555 

0.468-0.658

0.558 

0.474-0.657
Table 2. Toxicity of chlorine (from commercial grade HTH) to rainbow trout at selected temperatures, water

hardnesses, and pH's.

Temp

7

12

17

12

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

PH

7.5

7.5

7.5

8.0

8.0

8.0

8.0

6.5

8.5

9.5

LCso and 95% confidence interval (mg/1) at

1 h

3.00 

2.34-3.85

0.969 

0.886-1.06

0.800 

0.721-0.888

3.00 

2.20-4.10

2.87 

2.12-3.89

2.81 

2.28-3.46

2.75 

2.24-3.37

0.999 

0.871-1.15

3.30 

2.36-4.62

>3.00

3 h

1.11 

0.912-1.35

0.640 

0.533-0.769

0.619 

0.561-0.683

0.900 

0.763-1.06

1.00 

0.859-1.16

1.24 

1.03-1.49

0.900 

0.839-0.965

0.476 

0.436-0.520

1.23 

0.957-1.58

1.65 

1.39-1.96

6 h

0.695 

0.594-0.814

0.550

0.434 

0.348-0.541

0.830 

0.757-0.910

0.815 

0.768-0.865

0.879 

0.806-0.958

0.851 

0.806-0.898

0.405 

0.372-0.441

0.900 

0.833-0.972

1.00 

0.901-1.11

24 h

0.310 

0.224-0.428

0.236 

0.199-0.280

0.320 

0.245-0.419

0.362 

0.270-0.485

0.590

0.510 

0.414-0.628

0.439 

0.327-0.590

0.250 

0.218-0.287

0.350 

0.321-0.382

0.308 

0.274-0.346

96 h

0.141 

0.114-0.174

0.172 

0.148-0.200

0.192 

0.145-0.254

0.200 

0.144-0.278

0.211 

0.168-0.265

0.350

0.228

0.143 

0.115-0.178

0.189 

0.158-0.226

0.200 

0.164-0.244

reversed after 24 h, however, and at 96 h chlorine 

was most toxic in the coldest water probably 

because chlorine is more residual at low than at high 

temperatures.

Water hardness at a constant pH of 8.0 (maintained 

by adding equal amounts of bicarbonate to water at 

each level of hardness) did not affect the toxicity of 

chlorine to rainbow trout (Table 2). Although chlorine 

toxicity was influenced little by pH, the general trend 

was toward decreasing toxicity with increasing pH.

Detoxification of Antimycin

Green sunfish were exposed to antimycin, chlorine, 

and a mixture of antimycin and chlorine to determine 

the detoxification efficiency of chlorine. Detoxification was represented by a deactivation index, which 

is the quotient of the LCso for antimycin and chlorine 

in aged and in fresh solutions. For example, the 

deactivation index, at pH 7.5 after 1 h of interaction 

time, was 11.5/6.90, or 1.67 (Table 3). The time 

required for the deactivation index to reach 2.0 

indicating that the toxicity has been decreased by 

one-half coincides with the half-life of the toxicant. 

The half-life of antimycin in combination with 

chlorine at pH 7.5 was estimated at 1.3 h by plotting 

the deactivation indices against time on

semilogarithmic coordinates (Fig. 1). Similar curves 

prepared for the data at other pH's yielded estimated 

half-lives of 1.1 h at pH 6.5 and 1.5 h at pH's 8.5 and 

9.5.

7

Z3 

o

3 HOURS

123456 

TIME IN HOURS

Fig. 1. Half-life (T'/2) curve for antimycin and 0.5 mg/1 of 

chlorine in soft water at pH 7.5 and 12 C.
Table 3. Toxicity and deactivation of antimycin in static tests at 12 C with green sunfish in water 

containing 0.25 mg/l of chlorine at pH 6.5 and 0.50 mg/l of chlorine at pH's 7.5, 8.5, and 9.5.

Compound, and interaction

time3 of chlorine and

antimycin (h)

96-h LC50 , 95% confidence interval, 

and (in parentheses) deactivation index at

pH 6.5 pH 7.5 pH 8.5 pH 9.5

Chlorine (mg/l) 

Antimycin (/u,g/l)

Antimycin (fj.g/1) 

plus chlorine

0.745 

0.623-0.891

0.107 

0.084-0.136

0.949 

0.837-1.08

0.164 

0.138-0.194

0.840 

0.688-1.03

0.640 

0.539-0.761

0.715 

0.578-0.885

16.2 

14.1-18.6

0 0.779

0.644-0.942

(1.00)

0.5 1.72

1.23-2.41

(2.21)

1.0 1.39

1.03-1.87

(1.78)

2.0 2.87

2.15-3.84

(3.68)

4.0 4.28

3.38-5.41

(5.49)

6.0 8.51

7.36-9.83

(10.9)

8.0 7.60

7.05-8.19

(9.76)

6.90

4.69-10.1

(1.00)

10.7

8.73-13.1

(1.55)

11.5

8.64-15.1

(1.67)

20.0

16.9-23.7

(2.90)

36.0

30.0-43.2

(5.22)

44.8

(6.49)

5.19

4.44-6.07

(1.00)

6.00

4.99-7.22

(1.16)

9.00

7.40-10.9

(1.73)

13.3

(2.56)

15.2

11.6-19.9

(2.93)

25.5

20.6-31.5

(4.91)

34.5

29.9-40.0

(6.65)

4.45

2.93-6.76

(1.00)

7.09

5.47-9.18

(1.59)

8.69

6.86-11.0

(1.95)

7.05

5.29-9.39

(1.58)

15.0

11.3-19.9

(3.37)

23.4

18.5-29.5

(5.26)

28.0

25.4-30.8

(6.29)

a Time antimycin and chlorine were in solution before fish were introduced.

Toxicity of antimycin decreased significantly at 

successively higher pH's; the 96-h LCso's ranged from 

0.107/xg/l at pH 6.5 to 16.2/ig/l at pH 9.5 (Table 3). 

The drop was particularly sharp between pH's 8.5 

and 9.5 as previously observed by Marking and 

Dawson (1972). As in the earlier tests, the toxicity of 

chlorine was affected little by pH.

The toxicity of antimycin decreased significantly 

immediately after chlorine was added to the solution 

at pH 6.5, 7.5, and 8.5 with no interaction time 

(Table 3). For example, at pH 7.5 the 96-h LC50 for 

antimycin was 0.164 fig/1 and that for antimycin 

plus 0.5 mg/l of chlorine was 6.90 /u.g/1. The 

difference was perhaps due to the time required for 

antimycin to produce a lethal effect, commonly called 

the effective exposure time (Gilderhus 1972). 

Therefore the biological measure of concentrations

remaining after aging was delayed by the latent 

response of fish.

Toxicity to fish increased when chlorine was added 

to the antimycin solution at pH 9.5 (Table 3). The 96-h 

LC50 was 16.2 /xg/1 for antimycin alone, and 

4.45 fj. g/1 after the addition of 0.5 mg/l of chlorine. 

Most likely the 0.5 mg/l of chlorine contributed 

toxicity, rather than detoxifying the antimycin, and 

its effect was additive rather than antagonistic 

(Marking and Dawson 1975). As the interaction time 

increased, however, antimycin was detoxified; its 

half-life was estimated to be 1.5 h.

Marking and Dawson (1972) demonstrated that 

antimycin detoxifies in water without the addition of 

chemical detoxifiers (half-lives for antimycin alone 

ranged from 310 h at pH 6.5 to 1.5 h at pH 10.0); 

however, the chemicals greatly increase the rate of
detoxification. Although chlorine detoxifies antimycin much less rapidly than does potassium 

permanganate (Marking and Bills 1975), it destroys 

the biological activity of antimycin within a suitably 

short time.

The detoxification of antimycin without detoxifiers 

followed a first order decay curve; with detoxifiers, 

however, the curve was nonlinear. The nonlinearity 

results from loss of chlorine during the interaction 

time due to reactions such as reduction, volatilization, and adsorption. The initial detoxification rate is 

most important, however, because antimycin is 

generally nontoxic after a time equal to one or two of 

the initial half-life periods.

When chlorine concentrations were monitored for 

96 h to ascertain the dissipation rate during a typical 

toxicity test, the concentrations measured (Taras et 

al. 1971) in solutions containing fish were significantly lower than those measured in solutions without 

fish (Table 4). Little, if any, chlorine remained after 

24 h in the fish assays.

Table 4. Dissipation of chlorine (calcium

hypochlorite, 70% active chlorine) in water without

fish and in water with green sunfish (0.75 g/l) in soft

water at 12 C.

Time after

(h)

0

24

48

72

96

Chlorine (mg/1)

Without fish

0.5

0.525

0.350

0.350

0.325

0.300

1.0

1.075

0.825

0.925

0.975

0.925

With fish

0.5

0.275

0.050

<0.01

<0.01

<0.01

1.0

0.725

0.075

0.025

<0.01

<0.01

Conclusions

1. The 96-h LC50 's for chlorine at pH 7.5 ranged from 

0.156 mg/1 for channel catfish to 1.41 mg/1 for 

black bullheads.

2. Toxicity of chlorine to fish was influenced little 

bypH.

3. Toxicity of antimycin to green sunfish decreased 

as pH increased; the 96-h LC50 's ranged from 

0.107 fjig/l at pH 6.5 to 16.2 /ig/1 at pH 9.5.

4. Chlorine effectively detoxified antimycin. Halflives of antimycin with chlorine ranged from 

1.1 h at pH 6.5 to 1.5 h at pH 9.5.

References

Bedell, G. S. 1971. Eradicating Ceratomyxa shasta from 

infected water by chlorination and ultraviolet irradiation. Prog. Fish-Cult. 33(l):51-54.

Brungs, W. A. 1973. Effects of residual chlorine on 

aquatic life. J. Water Pollut. Control Fed. 

45(10):2180-2193.

Chambers, C. W. 1971. Chlorination for control of 

bacteria and viruses in treatment plant effluents. J. 

Water Pollut. Control Fed. 43(2):228-241.

Dawson, V. K., and L. L. Marking. 1974. Removal and 

deactivation of antimycin using carbon and chlorine. 

Prog. Fish-Cult. 36(1):19.

Fair, G. N., J. C. Morris, S. L. Chang, I. Well, and R. P. 

Burden. 1948. The behavior of chlorine as a water 

disinfectant. J. Am. Water Works Assoc. 40:1051-1061.

Gilderhus, P. A. 1972. Exposure times necessary for 

antimycin and rotenone to eliminate certain freshwater 

fishes. J. Fish. Res. Board Can. 29(2): 199-202.

Hagen, W., Jr. 1940. Sterilizing hatchery water supplies 

with liquid chlorine and powdered derris root. Prog. FishCult. 48(l):19-24.

Hunn, J. B., R. A. Schoettger, and E. W. 

Whealdon. 1968. Observations on the handling and 

maintenance of bioassay fish. Prog. Fish-Cult. 

30(3): 164-167.

Lennon, R. E., and C. R. Walker. 1964. Laboratories and 

methods for screening fish-control chemicals. U.S. Fish 

Wildl. Serv. Invest. Fish Control 1 (Circ. 185). 15 pp.

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified 

method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2):99-113.

Marking, L. L. 1969. Toxicological assays with fish. Bull. 

Wildl. Dis. Assoc. 5(2):291-294.

Marking, L. L., and T. D. Bills. 1975. Toxicity of 

potassium permanganate to fish and its effectiveness for 

detoxifying antimycin. Trans. Am. Fish. Soc. 

104(3):579-583.

Marking, L. L., and V. K. Dawson. 1972. The half-life of 

biological activity of antimycin determined by fish 

bioassay. Trans. Am. Fish. Soc. 101(1):100-105.

Marking, L. L., and V. K. Dawson. 1973. Toxicity of 

quinaldine sulfate to fish. U.S. Fish Wildl. Serv. Invest. 

Fish Control 48. 8 pp.

Marking, L. L., and V. K. Dawson. 1975. Method for 

assessment of toxicity or efficacy of mixtures of 

chemicals. U.S. Fish Wildl. Serv. Invest. Fish Control 67. 

8pp.

Merkens, J. C. 1958. Studies on the toxicity of chlorine 

and chloramines to rainbow trout. Water Waste Treat. 

7:150-151.

Taras, M. J., A. E. Greenberg, R. D. Hoak, and M. C. Rand, 

editors. 1971. Standard methods for the examination 

of water and wastewater. 13th edition. Am. Public Health 

Assoc., New York, N.Y. 874 pp.

Malachite Green: Its Toxicity to Aquatic Organisms, 

Persistence and Removal with Activated Carbon

by

Terry D. Bills and Leif L. Marking 

Fish Control Laboratory, La Crosse, Wisconsin

Jack H. Chandler, Jr.

Southeastern Fish Control Laboratory

Warm Springs, Georgia

Abstract

The acute toxicity of malachite green was determined in standardized laboratory tests for 

chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), Atlantic salmon 

(Salmo salar), brown trout (S. trutta), rainbow trout (S. gairdneri), brook trout (Salvelinus 

fontinalis), channel catfish (Ictalurus punctatus), largemouth bass (Micropterus salmoides), 

smallmouth bass (M. dolomieui), bluegill (Lepomis macrochirus), snails (Pleurocera sp.), 

Asiatic clams (Corbicula leana), ostracods (Cypridopsis sp.), freshwater prawns (Palaemonetes 

kadiakensis), larval midges (Tony tarsus dissimilis), naiads of mayflies (Callibaetis sp.), adult 

newts (Notophthalmus viridescens), larval leopard frogs (Rana pipiens), and larval toads 

(Bufo sp.). Bluegills were the most sensitive (96-h LC 5(l ,0.0305 mg/1), and coho salmon the 

most resistant (0.383 mg/1). The TILC50 (lethal concentration producing 50% mortality 

independent of time) for rainbow trout was 0.0998 mg/1. The responses of frog and toad larvae 

(96-h LC50 ,0.173 and 0.0680 mg/1) were similar to those offish, whereas adult newts were more 

resistant (1.03 mg/1). The invertebrates exposed were generally more resistant than the fish 

and amphibians; the 96-h LC50 's ranged from 0.510 to 3.45 mg/1, except for the Asiatic clam, 

which was extremely resistant (122 mg/1), and the mayfly naiad, which was very sensitive 

(0.0790 mg/1). The toxicity of malachite green to fish was not affected by water hardness or pH, 

except bluegills, in which toxicity was about half as great at pH 6.5 as at pH 7.5 to 9.5, and was 

increased only slightly by increases in water temperatures. Malachite green was very 

persistent in aqueous solutions; it did not detoxify after 3 weeks of aging in glass containers. 

The chemical is readily absorbed from aqueous solutions (pH 7.5, total hardness 44 mg/1, 

temperature 12 C) by filtration through activated carbon; the capacity was 23.4 mg of 

malachite green per gram of carbon.

Malachite green has been used in fish culture as a 

fungicide and parasiticide for about 40 years. It was 

first used as a dip treatment by Foster and Woodbury 

(1936) to treat fungal infections of four species of trout 

and largemouth bass (Micropterus salmoides). More 

recently it has been used in combination with 

formalin to treat Ichthyophthirius, a serious parasite 

of catfishes (Leteux and Meyer 1972).

Although the use of malachite green as a therapeutant in fish culture has many advantages, it also 

poses various potential problems (Nelson 1974): 

toxicity to fishes (Willford 1967); possible teratogenic 

and mutagenic effects (Lieder 1961; Nelson 1974; T.D. 

Bills and L.L. Marking, in preparation); and stress 

induced during and after the treatment of fry of

certain fishes (Glagoleva and Malikova 1968; Bills 

and Hunn 1976).

Malachite green is not registered for aquatic use by 

either the Food and Drug Administration or Environmental Protection Agency, because information 

required for registration toxicity, efficacy, residues, 

metabolites, and counteraction is incomplete. The 

purpose of the present study was to contribute 

laboratory data on (1) the toxicity of malachite green 

to nontarget aquatic organisms; (2) its toxicity to 

rainbow trout (Salmo gairdneri) and bluegills 

(Lepomis macrochirus) in extended exposures; 

(3) the effects of certain water characteristics on its 

toxicity to fish; (4) its persistence in water; and (5) its 

possible removal from water with activated carbon.
Materials and Methods

Concentrated stock solutions of commercial grade 

zinc-free malachite green (4-[P-(dimethylamino)-otphenylbenzylidene]-2,5-cyclohexadien-l-ylidene dimethyl-ammonium chloride) manufactured by MCB 

Manufacturing Chemists, Norwood, Ohio, were 

prepared by mixing weighed portions with water. To 

prepare test solutions of the desired concentrations, 

we pipetted portions of stock solutions into test 

vessels and stirred the resulting mixture to ensure 

homogeneity. In flow-through toxicity tests, the 

required amounts of the stock solution were delivered 

by a solenoid-activated pipette pump (Micromedic 

Systems Automatic Pipette Model 2500).

Tests were conducted according to the methods 

outlined by the Committee on Methods for Toxicity 

Tests with Aquatic Organisms (1975) and the 

protocol described by Marking (1975). Glass jars of 

3.78 or 18.9 liters were used, depending on the size of 

the test organism. Reconstituted water was used in 

tests with fish (Marking 1969), and limed spring 

water (pH, 7.5 ±0.1; total hardness, 20 mg/1 as 

CaCOs) in the tests with amphibians and invertebrates. Chemical buffers were added to soft 

water to adjust the pH (6.5-9.5), as described by 

Marking and Dawson (1973).

Flow-through tests were conducted in a proportional diluter similar to that of Mount and Brungs 

(1967). Test vessels were 45-liter glass aquariums 

supplied with a flow sufficient to replace the entire 

volume at least four times daily. Carbon-filtered, 

municipal well water (total hardness 300 mg/1, pH 

7.5) was used in the flow-through system. 

Temperature was maintained by immersing test 

vessels in a water bath equipped with a chilling unit.

Fish species exposed were chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), 

Atlantic salmon (Salmo salar), brown trout (S. 

trutta), rainbow trout, brook trout (Salvelinus fontinalis), channel catfish (Ictalurus punctatus), 

largemouth bass, smallmouth bass (Micropterus 

dolomieui), and bluegills. Test fishes weighed 0.5 to 

1.5 g. Other aquatic organisms exposed were snails 

(Pleurocera sp.), Asiatic clams (Corbicula leana), 

ostracods (Cypridopsis sp.), freshwater prawns 

(Palaemonetes kadiakensis), larval midges (Tanytarsus dissimilis), naiads of mayflies (Callibaetis sp.), 

adult newts (Notophthalmus viridescens), larval 

leopard frogs (Rana pipiens), and larval toads (Bufo 

sp.).

In tests for the determination of persistence of 

malachite green, aqueous solutions were aged for 1,2, 

and 3 weeks in glass containers. Rainbow trout were 

introduced concurrently to these and a freshly

prepared reference solution for comparison of mortality. Deactivation indices were computed from 

these data according to the method of Marking (1972).

We used the method of Litchfield and Wilcoxon 

(1949) to determine LC50 's and 95% confidence 

intervals, and a modification of the method published 

by Green (1965) for computing TILC,,,'s (lethal) 

concentration producing 50% mortality independent 

of time).

To determine if malachite green could be removed 

from aqueous solutions (pH 7.5, total hardness 

44 mg/1, temperature 12 C), we filtered a concentrated solution (2 mg/1) at a flow rate of 100 ml/min 

through a glass 2.7 cm ID column containing 15 cm 

(35.5 g dry weight) of activated carbon (Darco 

20 * 40 mesh). Samples were taken periodically and 

concentrations in the effluent determined 

colorimetrically (620 nm). The carbon bed was 

considered saturated when the concentration in the 

effluent reached 10% of that in the original stock 

solution (0.2 mg/1). The capacity of activated carbon 

for the chemical was determined by the following 

formula:

Concentration (mg/1) x

Milligrams of malachite _ liters passed through filter 

green adsorbed per gram Grams of carbon 

of carbon (dry weight)

Results

Toxicity to Fish

Malachite green was toxic to all species of fish 

exposed; LC50 's ranged from 0.0305 to 0.383 mg/1 in 

96-h exposures in soft water at 12 C (Table 1). 

Centrarchids were 1.5 to 3.5 times more sensitive to 

the chemical than the ictalurids and 3 to 7 times more 

sensitive than the salmonids. The bluegill was the 

most sensitive species (96-h LC 50 , 0.0305 mg/1) and 

the coho salmon the most resistant (0.383 mg/1). The 

toxicity of the chemical increased as exposures 

lengthened in all species; for bluegills the LCso was 

6.00 mg/1 at 3 h and 0.0305 mg/1 at 96 h.

Toxicity to 

Other Aquatic Organisms

In 96-h exposures, the LCso's for malachite green to 

frog larvae (0.173 mg/1) and toad larvae 

(0.0680 mg/1) were similar to those for fish (Table 2). 

Adult newts were more resistant than frog or toad 

larvae (96-h LC5 o, 1-03 mg/1), but about equally or 

less resistant than most of the invertebrates exposed. 

Mayfly naiads were the most sensitive invertebrate
Table I. Toxicity of malachite green to finger ling fish in soft water at 12 C.

LCso and 95% confidence interval (mg/1) at

Chinook salmon 

(Oncorhynchus tshawytscha)

Coho salmon 

(O. kisutch)

Atlantic salmon 

(Salmo salar)

Brown trout 

(S. trutta)

Rainbow trout 

(S. gairdneri)

Brook trout 

(Salvelinus fontinalis)

Channel catfish 

(Ictalurus punctatus)

Largemouth bass 

(Micropterus salmoides)

Smallmouth bass 

(M. dolomieui)

Bluegill 

(Lepomis macrochirus)

3 h

1.72 

1.22-2.42

3.56

2.77-4.58

1.73 

1.23-2.43

1.41 

1.14-1.74

3.00 

2.06-4.37

>3.00

_

1.36 

1.09-1.70

6.00

4.41-8.17

6 h

1.38 

1.04-1.82

>3.00

1.09 

0.929-1.28

1.27 

0.991-1.63

0.760 

0.649-0.890

1.44 

1.05-1.98

1.10 

0.904-1.34

_

_

2.19 

1.66-2.89

24 h

0.292 

0.245-0.348

0.569 

0.486-0.662

0.497 

0.415-0.595

0.352 

0.280-0.443

0.360 

0.305-0.425

0.300 

0.259-0.348

0.181 

0.123-0.266

0.282 

0.211-0.376

0.154 

0.117-0.202

0.231 

0.184-0.290

96 h

0.224 

0.209-0.240

0.383 

0.327-0.449

0.283 

0.229-0.350

0.237 

0.209-0.268

0.248 

0.193-0.319

0.220 

0.188-0.257

0.112 

0.0893-0.140

0.0728 

0.0604-0.0877

0.0453 

0.0366-0.0561

0.0305 

0.0218-0.0427

Table 2. Toxicity of malachite green to selected nontarget aquatic organisms in limed water at 16 C.

LCso and 95% confidence interval (mg/1) at

Snail 

(Pleurocera sp.)

Asiatic clam 

(Corbicula leana)

Ostracod 

(Cypridopsis sp.)

Freshwater prawn 

(Palaemonetes kadiakensis)

Midge (larvae) 

(Tanytarsus dissimilis)

Mayfly naiads 

(Callibaetis sp.)

Newts (adult) 

(Notophthalmus uiridiscens)

Leopard frog (larvae) 

(Rana pipiens)

Toad (larvae) 

(Bufo sp.)

6 h

5.85 

4.00-8.57

5.00 

3.13-7.99

5.75 

4.95-6.69

_

1.00 

0.875-1.14

1.70 

1.54-1.87

24 h

_

5.85 

4.29-7.97

9.10 

7.29-11.3

1.00 

0.636-1.57

2.75 

2.07-3.65

3.90 

3.47-4.38

0.380 

0.351-0.412

0.355 

0.235-0.276

96 h

0.720 

0.483-1.07

122 

93.8-159

3.45 

2.49-4.80

1.90 

1.76-2.06

0.510 

0.295-1.10

0.0790 

0.0442-0.141

1.03 

0.672-1.58

0.173 

0.149-0.200

0.0680 

0.0530-0.0860
exposed (96-h LC50 , 0.0790 mg/1), and the Asiatic 

clam was by far the most resistant to the chemical; it 

tolerated concentrations in excess of 100 mg/1. The 

other invertebrates exposed were more resistant than 

fish or amphibians, but less resistant than the Asiatic 

clam. The 96-h LC50 's for these organisms were 

between 0.510 and 3.45 mg/1.

Effects of Temperature,

Water Hardness, and

pH on Toxicity

In short exposures of 3 or 6 h, malachite green was 

more toxic to rainbow trout, channel catfish, and 

bluegills in warm water (17 and 22 C) than in cool 

water (7 and 12 C), but at 96 h the LC 50 's at different 

temperatures were not significantly different, except 

for channel catfish (Tables 3, 4, 5). Neither water 

hardness nor pH influenced the toxicity of the 

chemical to any species except bluegills, in which 

toxicity was about half as great as pH 6.5 as atpH 7.5 

to 9.5.

Chronic Toxicity

Rainbow trout and bluegills were exposed 

simultaneously to the chemical in a flow-through 

toxicity test to determine the TILC50 . Mortality 

increased with time in both species. The 24-h LC50 for 

bluegills was 0.151 mg/1. A TILC5o could not be 

calculated because mortality continued until all 

organisms succumbed at the lowest concentration 

(0.0316 mg/1) after 16 days of exposure. The 24-h 

LC50 was 0.230 mg/1 for rainbow trout, and mortality 

continued through 30 days. A TILC50 of 0.0998 mg/1 

was determined after 36 days of exposure.

Persistence of 

Malachite Green in Water

Bioassays with rainbow trout of aqueous solutions 

of malachite green aged for 1,2, and 3 weeks in glass 

jars indicated no significant loss of activity. The LC50 

was 0.173 mg/1 for the freshly prepared reference 

solution and 0.179 mg/1 for the solution aged for 3 

weeks.

Table 3. Toxicity of malachite green to fingerling rainbow trout at selected temperatures, water hardnesses,

and pH's.

Temp

7

12

17

12

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.5

7.5

7.5

8.0

8.0

8.0

8.0

6.5

8.5

9.5

LC5o and 95% confidence interval (mg/1) at

3 h

>2.00

1.41 

1.14-1.74

1.42 

1.15-1.76

2.00 

1.55-2.58

2.31 

1.72-3.10

2.30 

1.43-3.71

2.35 

1.74-3.17

>2.00

2.60 

1.86-3.63

>2.00

6 h

2.30 

1.71-3.10

0.760 

0.649-0.890

0.567 

0.517-0.621

0.780 

0.726-0.838

0.800 

0.659-0.971

1.40 

1.13-1.73

0.820 

0.701-0.959

1.01 

0.764-1.34

0.980 

0.851-1.13

1.26 

0.978-1.62

24 h

0.400 

0.330-0.486

0.360 

0.305-0.425

0.569 

0.516-0.627

0.362 

0.307-0.426

0.280 

0.226-0.347

0.345 

0.296-0.403

0.280 

0.226-0.347

0.279 

0.207-0.375

0.284 

0.229-0.351

0.367 

0.311-0.434

96 h

0.168 

0.137-0.206

0.248 

0.193-0.319

0.284 

0.229-0.353

0.286 

0.230-0.355

0.234 

0.179-0.305

0.288 

0.233-0.356

0.249 

0.195-0.318

0.280 

0.227-0.345

0.212 

0.172-0.262

0.173 

0.136-0.220
Table 4. Toxicity of malachite green to fingerling channel catfish at selected temperatures, water

hardnesses, and pH's.

Temp

12

17

22

12

12

12

12

12

12

12

Table 5.

Temp

12

17

22

12

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.5

7.5

7.5

8.0

8.0

8.0

8.0

6.5

8.5

9.5

LCso and 95% confidence interval (mg/1 at

6 h

1.10 

0.904-1.34

0.552 

0.499-0.610

0.400 

0.331-0.483

0.600 

0.440-0.818

1.30 

1.01-1.67

1.72 

1.23-2.41

1.71 

1.22-2.40

0.960 

0.717-1.29

0.835 

0.665-1.05

0.519 

0.377-0.714

24 h

0.181 

0.123-0.266

0.222 

0.168-0.293

0.0691 

0.0576-0.0831

0.106 

0.0935-0.120

0.285 

0.232-0.350

0.284 

0.229-0.351

0.286 

0.232-0.353

0.236 

0.181-0.308

0.835 

0.665-1.05

0.191 

0.155-0.236

96 h

0.112 

0.0893-0.140

0.0940 

0.0860-0.103

0.0535 

0.0442-0.0647

0.0750 

0.0555-0.101

0.117 

0.0972-0.140

0.142 

0.115-0.176

0.142 

0.115-0.176

0.0975 

0.0937-0.101

0.237 

0.182-0.309

0.162 

0.135-0.194

Toxicity of malachite green to fingerling bluegill at selected temperatures, hardnesses, and pH's.

Water 

hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

LCso and 95% confidence interval (mg/1) at

3 h

7.5 6.00 

4.41-8.17

7.5 2.17 

1.63-2.88

7.5 0.860 

0.737-1.00

8.0 2.30 

1.72-3.08

8.0 >2.00

8.0 >2.00

8.0 >2.00

6.5 7.43 

5.76-9.59

8.5 4.68 

3.77-5.80

9.5 3.70 

2.81-4.87

6 h

2.19 

1.66-2.89

0.656 

0.584-0.737

0.238 

0.184-0.308

2.00 

1.54-2.59

1.52 

1.15-2.00

1.41 

1.14-1.74

1.42 

1.10-1.83

2.18 

1.64-2.90

2.18 

1.64-2.89

2.20 

1.67-2.89

24 h

0.231 

0.184-0.290

0.0920 

0.0663-0.128

0.0780 

0.0594-0.102

0.117 

0.0967-0.142

0.122 

0.100-0.149

0.141 

0.114-0.174

0.141 

0.114-0.174

0.282 

0.219-0.394

0.123 

0.0955-0.158

0.0810 

0.0562-0.117

96 h

0.0305 

0.0218-0.0427

0.0340 

0.0242-0.0477

0.0308 

0.0221-0.0430

0.0413 

0.0343-0.0497

0.0400 

0.0330-0.0486

0.0450 

0.0384-0.0528

0.0440 

0.0370-0.0523

0.0780 

0.0594-0.102

0.0339 

0.0241-0.0476

0.0340 

0.0242-0.0477
Counteraction with 

Activated Carbon

Aqueous solutions of malachite green (2.0 mg/1) 

were filtered through a bed of activated carbon. In 

three runs the activated carbon adsorbed the 

chemical from 420, 401, and 425 liters of solution 

before the endpoint was reached (0.2 mg/1), an 

average of 23.4 mg of malachite green per gram of 

carbon. Activated carbon thus is an excellent means 

for removing this chemical from water.

References

Bills, T. D., and J. B. Hunn. 1976. Changes in the blood

chemistry of coho salmon exposed to malachite green.

Prog. Fish-Cult. 38(4):2147 216. 

Committee on Methods for Toxicity Tests with Aquatic

Organisms. 1975. Methods for acute toxicity tests

with fish, macroin vertebrates, and amphibians.

Ecological Research Series. EPA [Environ. Prot. Agency]-660/3-75-009. 61 pp. 

Foster, F. J., and L. Woodbury. 1936. The use of

malachite green as a fish fungicide and antiseptic. Prog.

Fish-Cult. !8(4):7-9. 

Glagoleva, T. P., and E. M. Malikova. 1968. The effect of

malachite green on the blood compositon of young Baltic

salmon. Rybn. Khoz. (USSR) 44(5):15-18. (Transl. from

Russian by R. M. Rowland) 

Green, R. H. 1965. Estimation of tolerance over an

indefinite time period. Ecology 46(6):887.

Leteux, F., and F. P. Meyer. 1972. Mixtures of malachite 

green and formalin for controlling Ichthyophthirius and 

other protozoan parasites of fish. Prog. Fish-Cult. 

34(l):21-26.

Lieder, U. 1961. Zur Wirkung des Cancerogens and 

Mutagens Malachitgreen [p-dimethylamino-fuchsondimethylimino-oxalat (sulfat)] auf Mitosen bei Fischen 

und Fischeier. [On the effect of the carcinogen and 

mutagen malachite green on mitosis in fish and fish 

eggs.] Naturwissenschaften 48(ll):437-438.

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified 

method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2):99-113.

Marking, L. L. 1969. Toxicological assays with fish. Bull. 

Wildl. Dis. Assoc. 5:291-294.

Marking, L. L. 1972. Methods of estimating the half-life 

of biological activity of toxic chemicals in water. U.S. 

Fish Wildl. Serv. Invest. Fish Control 46. 9 pp.

Marking, L. L. 1975. Toxicological protocol for the 

development of piscicides. Pages 26-31 in P.H. 

Eschmeyer, ed. Rehabilitation of fish populations with 

toxicants: a symposium. North Cent. Div., Am. Fish. 

Soc., Spec. Publ. No. 4.

Marking, L. L., and V. K. Dawson. 1973. Toxicity of 

quinaldine sulfate to fish. U.S. Fish Wildl. Serv. Invest. 

Fish Control 48. 8 pp.

Mount, D. L, and W. A. Brungs. 1967. A simplified dosing 

apparatus for fish toxicological studies. Water Res. 

1:21-29.

Nelson, N. C. 1974. A review of the literature on the use of 

malachite green in fisheries. U.S. Fish Wildl. Serv. Lit. 

Rev. 74-11. Natl. Tech. Inf. Serv. No. PB-235 4507AS. 

79pp.

Willford, W. A. 1967. Toxicity of 22 therapeutic compounds to six fishes. U.S. Fish Wildl. Serv. Invest. Fish 

Control 18. 10 pp.
Toxicity of Furanace to Fish, Aquatic Invertebrates, 

and Frog Eggs and Larvae

by

Leif L. Marking and Terry D. Bills 

Fish Control Laboratory, La Crosse, Wisconsin

Jack H. Chandler, Jr.

Southeastern Fish Control Laboratory

Warm Springs, Georgia

Abstract

Furanace (6-hydroxymethyl-2[2-(5-nitro-2-furyl)vinyl]pyridine), a nitrofuran originally 

designated as furpirinol, nifurpirinol, or P-7138, is effective against certain bacterial infections 

in fish, especially myxobacteriosis. The toxicity of the drug to fish, frog eggs and larvae, and 

aquatic invertebrates was determined in standardized laboratory toxicity tests and in use 

pattern exposures. Additional tests were conducted in aged solutions of Furanace to determine 

the persistence in water. This research was done to broaden existing toxicity data and to help 

fulfill drug registration requirements. Furanace was not toxic to any test species in use pattern 

exposures of 1 mg/1 for 1 h daily for up to three treatments. In 96-h exposures, LCso's ranged 

from 0.820 to 3.00 mg/1 for six species of fish and from 1.13 to 20 mg/1 for six species of 

invertebrates, and was 0.770 mg/1 for larvae of the leopard frog (Rana pipiens). Toxicity 

increased with elevated temperatures in tests with rainbow trout (Salmo gairdneri), channel 

catfish (Ictalurus punctatus), and green sunfish (Lepomis cyanellus). Increased water 

hardness and pH decreased toxicity to rainbow trout, but did not influence toxicity to channel 

catfish and green sunfish.

Furanace (6-hydroxymethyl-2[2-(5-nitro-2-furyl) 

vinyl]pyridine), a nitrofuran originally designated as 

P-7138, furpirinol, or nifurpirinol, has proved to be 

therapeutic against certain fish diseases. It was 

developed as a fish bactericide by Dainippon Pharmaceutical Co., Ltd. in Japan (Shimizu and Takase 

1967) and was tested further against fish diseases in 

the United States (Amend and Ross 1970; Ross 1972). 

A review of literature on the drug was provided by 

Herman (1974). U.S. studies were conducted with the 

intention of registering the drug for fishery use as 

outlined by Lennon (1967).

Amend and Ross (1970) demonstrated that experimentally induced columnaris disease in coho 

salmon (Oncorhynchus kisutch) can be controlled 

with Furanace, that the drug is readily absorbed and 

eliminated, and that it is nontoxic to juvenile salmon. 

Additional studies by Amend (1972) showed that 

furunculosis in coho salmon was partially controlled 

by 8 or 10 mg/1 of Furanace with two daily 1-h baths, 

but not by feeding the drug because fish refused to eat 

the medicated feed. He also found that one or two 1-h 

treatments of 1 mg/1 of Furanace in static baths 

resulted in excellent control of myxobacteriosis and 

that the drug had low toxicity to fish and posed no 

residue problems 9 days after the last treatment.

We expanded the information on Furanace toxicity 

by (1) including more species of fish and additional 

test conditions, (2) including six species of aquatic 

invertebrates, and frog eggs and larvae, (3) determining the toxicity of use pattern exposures, and 

(4) determining the persistence of Furanace in water. 

The sequence of test procedures and conditions were 

standardized as suggested in the toxicological 

protocol of Marking (1975).

Materials and Methods

Purified Furanace, furnished by Abbott 

Laboratories, North Chicago, 111., was dissolved in dimethylformamide or acetone to prepare stock 

solutions, portions of which were then added to static 

test chambers to yield desired concentrations. Toxicity was calculated on the basis of 100% activity for the 

added quantity of drug.

The static test procedures were those recommended 

by the Committee on Methods for Toxicity Tests with 

Aquatic Organisms (1975). Ten fish were exposed to 

each concentration in glass jars containing 15 liters 

of oxygenated, reconstituted water prepared from 

deionized water (Marking 1969). Test waters were of

1
four different total hardnesses (mg/1 as CaCOs in 

parentheses): very soft (10), soft (44), hard (170), and 

very hard (300). In separate tests, chemically buffered 

solutions (Marking 1975) were used to assess the 

effects of pH on toxicity. Test temperatures were 

regulated by immersing the test jars in constant 

temperature water baths.

Fish weighing 1 to 1.5 g each were obtained from 

National fish hatcheries and maintained according 

to standardized procedures of the Fish Control 

Laboratory (Hunn et al. 1968). Fish were acclimated 

to the test conditions before they were exposed to the 

drug. Mortalities were recorded at 1,3, and 6 h on the 

first day of exposure and daily thereafter for the 

remainder of the test. Scientific names of the fishes 

are listed in Table 1.

Six species of invertebrates (scientific names listed 

later in Table 5) and eggs and larvae of the leopard 

frog (Rana pipiens) were collected from the wild. 

These organisms were exposed to Furanace in a 

manner similar to that for exposing fish except that 

exposures were in 3 liters of limed spring water 

(21 mg/1 of total hardness) in glass jars. Frog eggs (2- 

to 8-cell stage) were placed in concentrations of 

Furanace ranging from 0.01 to 5.0 mg/1 and allowed 

to complete their development. After 7 days in the test 

vessels, the various stages of development were 

assessed. Invertebrates and frog larvae were exposed 

for 96 h to Furanace concentrations ranging from 0.1

exposed to Furanace for 4 and 24 h and then placed in 

floating cages in a pond.

Computations of LC50's (concentrations calculated 

to produce 50% mortality) and 95% confidence 

intervals were computed according to the methods of 

Litchfield and Wilcoxon (1949). All data reported 

fulfilled the chi-square test requirement for acceptability. Lego's were considered significantly 

different when their confidence intervals did not 

overlap.

Residues of Furanace in water were determined by 

Abbott Laboratories, North Chicago, 111., by gas 

chromatography (R.E. Crutcher and J. T. 

Manneback, in preparation).

Results

Toxicity to Fish

The toxicity of Furanace was not significantly 

different in 24-h exposures at 12 C to Atlantic 

salmon, rainbow trout, fathead minnows, and 

channel catfish; the 24-h LC50 's ranged only from 

4.27 to 4.50 mg/1 (Table 1). The 96-h LC50 's for green 

sunfish and the 96-h LC50 for bluegills were 

significantly greater than those for the other species.

Table 2. Toxicity of Furanace to rainbow trout at 

selected water temperatures, hardnesses, and pH's.

tu £j\j ing/ i ui tu i/iic use pattciii uuiiuciitiatiuii ui

1.0 mg/1. Higher concentrations of Furanace were 

not used because such solutions approached saturation and often precipitated. The observation period

was extended to 60 days for a group of clams that were

Table 1. Toxicity of Furanace to selected species of 

fish in soft water at 12 C.

Species

Atlantic salmon

(Salmo salar)

Rainbow trout

(Salmo gairdneri)

Fathead minnow

(Pimephales promelas)

Channel catfish

(Ictalurus punctatus)

Green sunfish

(Lepomis cyanellus)

Bluegill

(Lepomis macrochirus)

LC5o and 95% confidence 

interval (mg/1) at

24 h

4.50

3.84-5.28

4.32

3.64-5.13

4.39

3.96-4.87

4.27

3.40-5.36

6.60

6.16-7.07

96 h

1.41

1.14-1.74

1.00

0.824-1.21

0.820

0.717-0.938

1.07

0.854-1.34

2.48

2.07-2.97

3.00

2.45-3.68

Temp

(°C)

7 

12

17

12

12

12

12

12

12

12

Water 

hardness

Soft 

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.5 

7.5

7.5

8.0

8.0

8.0

8.0

6.5

8.5

9.5

LC5o and 95% confidence 

interval (mg/1) at

24 h

9.60 

8.02-11.5

4.32 

3.64-5.13

3.69 

3.11-4.38

2.45

1

(\f\ Q I f* .yu-o.io 

2.83

2.29-3.50

2.82

2.28-3.99

2.82

2.28-3.49

3.28

2.76-3.90

3.82

3.20-4.55

3.29

2.77-3.90

96 h

1.73 

1.23-2.44

1.00 

0.824-1.21

0.795 

0.661-0.956

0.569

0.450-0.719

0.618

0.490-0.779

0.760

0.615-0.939

1.18

0.913-1.52

0.489

0.432-0.554

0.800

0.696-0.917

1.42

1.05-1.92
Elevated temperatures increased the toxicity of 

Furanace to some species. The LCso's were 

significantly different at the lowest and highest 

water temperatures at which rainbow trout and 

channel catfish were exposed (Tables 2 and 3).

Furanace was significantly less toxic to rainbow 

trout at 96 h in very hard than in soft or very soft 

water (Table 2). However, the toxicity to channel 

catfish and green sunfish at 96 h was not significantly different at the four different water hardnesses 

(Tables 3 and 4).

Toxicity of Furanace to rainbow trout was influenced by pH in 96-h exposures, and the drug was more 

toxic at pH 6.5 than at pH 8.5 and at pH 8.5 than at pH 

9.5 (Table 2). However, toxicity of Furanace at 96 h to 

channel catfish and green sunfish was not affected 

by pH (Tables 3 and 4).

Toxicity of Use 

Pattern Exposures to Fish

The suggested use pattern of Furanace as a fish 

therapeutant is a 1-h exposure daily to a 1-mg/l 

solution for up to 3 consecutive days, as necessary. 

Accordingly, rainbow trout were exposed to concentrations ranging from 0 to 3.0 mg/1 of Furanace for 3 

consecutive days and then observed for 10 additional

days. No mortality occurred and no stress was 

observed during the 13-day period at any of the 

exposure concentrations.

Exposure of leopard frog larvae to concentrations 

of 0.2 to 20 mg/1 of Furanace for 3 consecutive days 

resulted in no mortality after 7 days. Many of the 

larvae were immobilized in the 10- and 20-mg/l 

concentrations during the exposure, but they 

recovered when placed in fresh water. Furanace in 

the use pattern exposure was nontoxic to fish, frog 

larvae, and aquatic invertebrates.

Toxicity to Invertebrates

Invertebrates were more resistant than fish to 

Furanace; the 96-h LC50 's ranged from 1.13 mg/1 for 

snails to more than 20 mg/1 for water fleas, 

freshwater prawns, and backswimmers (Table 5).

Asiatic clams that were exposed for 4 or 24 h and 

placed in floating cages in a pond also survived 

concentrations of Furanace greater than the 

therapeutant treatment level. The 4-h exposure 

produced less mortality than the 24-h exposure. 

Mortality from most of the exposures increased 

during the 60-day holding period. However, there was 

some survival among those exposed to concentrations as high as 20 mg/1.

Table 3. Toxicity of Furanace to channel catfish at selected water temperatures, hardnesses, andpH's.

Temp

12

17

22

12

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.5

7.5

7.5

8.0

8.0

8.0

8.0

6.5

8.5

9.5

LCso and 95% confidence interval (mg/1) at

6 h

32.6

23.4-45.5

I

14.2 

11.5-17.6

27.6 

22.5-33.9

24.6 

20.6-29.4

24.6 

20.6-29.4

22.8 

18.2-28.6

_

~

24 h

6.19 

5.78-6.63

5.25 

4.48-6.15

2.90 

2.41-3.49

2.90 

2.62-3.21

4.00 

3.59-4.46

4.00

3.57-4.48

4.38 

3.96-4.84

8.25 

7.76-8.77

7.59 

7.20-8.01

6.55 

5.95-7.21

96 h

1.72 

1.48-2.00

1.90 

1.55-2.33

1.22 

1.05-1.42

0.945 

0.791-1.13

1.00 

0.821-1.22

0.960

0.784-1.18

0.969 

0.810-1.16

1.82 

1.62-2.05

1.80 

1.60-2.03

1.42 

1.15-1.76
Table 4. Toxicity of Furanace to green sunfish at 

selected water temperatures, hardnesses, and pH's.

Temp

12

17

22

12

12

12

12

12

12

12

Water 

hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.5

7.5

7.5

8.0

8.0

8.0

8.0

6.5

8.5

9.5

LC50 and 95% confidence 

interval (mg/1) at

24 h

6.30 

5.55-7.16

5.88 

5.29-6.54

4.25 

3.84-4.70

7.00 

6.10-8.04

6.28 

5.88-6.71

6.60 

6.16-7.07

7.85 

7.32-8.42

6.00 

5.38-6.69

7.00 

6.71-7.31

4.64 

3.53-6.09

96 h

2.42 

2.04-2.86

2.00 

1.59-2.52

1.42 

0.980-2.04

2.44 

2.06-2.89

2.20 

1.79-2.70

2.48 

2.07-2.97

2.50 

2.20-2.84

1.73 

1.23-2.43

2.00 

1.10-3.63

2.00 

1.08-3.69

Effects of Furanace on 

Frog Eggs and Larvae

Fertilized eggs at the 2- to 8-cell stage were exposed 

to Furanace at concentrations of 0.2 to 20 mg/1 for 

1 h on 3 consecutive days and then held in fresh 

water. After 7 days, there was no apparent effect of 0.2 

to 4.0 mg/1 of Furanace on the development of 

embryos. At concentrations of 6.0 to 20 mg/1, 

however, fewer embryos survived; some developed a 

head and tail and then died, and others appeared to 

develop normally but failed to escape the egg mass.

Frog larvae were considerably more sensitive to 

Furanace than were frog eggs, invertebrates, or fish. 

The 96-h LC50 was 0.770 mg/1 in limed water at 16 C 

(Table 5).

Persistence of Furanace in Water

The persistence of Furanace in water was measured 

biologically by determining the toxicity to rainbow 

trout of solutions that had been aged for periods as 

long as 5 weeks. Additionally, Furanace residues in 

the aged solutions were determined analytically. 

Aging for 5 weeks reduced the toxicity of Furanace 

(i.e., increased the 96-h LC50 ) by about 50% (Table 6). 

The same waters showed a similar loss of drug as 

determined by gas chromatographic analysis. 

Therefore the half-life of biological activity corresponded with the half-life of chemical integrity, i.e., 

about 5 weeks (Table 7).

Table 5. Toxicity of Furanace to selected species of aquatic invertebrates and frog larvae in limed water at

16 C.

Species LCso and 95% confidence interval (mg/1) at

24 h 96 h

Snail 

(Physa spj

Asiatic clam 

(Corbicula leana)

Water flea 

(Daphnia magna)

Amphipods 

(Hyalella azteca)

Freshwater prawn 

(Palaemonetes kadiakensis)

Backswimmer 

(Notonecta spj

Leopard frog (larva) 

(Rana pipiens)

8.00 

6.98-9.17

>20.0

>20.0

16.0 

11.3-22.7

>20.0 

>20.0

6.90 

5.55-8.57

1.13 

0.860-1.47

11.6

8.75-15.4

>20.0

13.6 

9.43-19.6

>20.0

>20.0

0.770 

0.590-1.01
Table 6. Deactivation of Furanace in soft water at 12 C, as determined by changes in 96-h LC50 's of rainbow

trout.

Aging period 96-h LC50 's (mg/l) and 

(weeks) 95% confidence interval

0 0.705 

0.570-0.872

1 0.810 

0.677-0.969

2 0.900 

0.763-1.06

3 0.900 

0.763-1.06

4 1.33 

1.19-1.48

5 1.46 

1.31-1.62

Deactivation 

index a

1.0

1.1

1.3

1.3

1.9

2.1

a Deactivation index =

LCnn of fresh solution

Table 7. Concentrations of Furanace (mg/l) detected by colorimetry and gas chromatography in water

solutions aged for 0 to 5 weeks.

Concentration 

before aging 

(calculated)

1.0 

3.0

1.0 

3.0

1.0 

3.0

1.0 

3.0

1.0 

3.0

1.0 

3.0

Aging 

period 

(weeks)

0 

0

1 

1

2 

2

3 

3

4 

4

5 

5

Concentration after aging

Colorimetric

1.0 

3.11

0.67 

2.89

0.67 

2.89

0.78 

2.22

0.56 

2.56

0.78 

2.50

GC-eCa

1.15 

2.56

0.654

1.82

0.602 

0.807

0.575 

2.52

0.466 

1.74

0.485 

1.58

Gas chromatography-electron capture average of two samples.
Discussion and Conclusions

The toxicity of Furanace was relatively low to fish 

and aquatic invertebrates. Saturated solutions (about 

10-20 mg/1) usually did not cause sufficient mortality to permit the derivation of LC5o's in exposures 

shorter than 24 h. No frog eggs or rainbow trout 

succumbed after use pattern exposures of 1 mg/1 for 

1 h daily for up to three treatments, and none of the 

invertebrates died after a single use pattern treatment. In fact, most fish and invertebrates survived 

96-h exposures to 1 mg/1 of Furanace.

Toxicity of Furanace was influenced by certain 

physical and chemical characteristics of water. 

Toxicity increased with increases in water 

temperatures in tests with rainbow trout and channel 

catfish. Increasing water hardness and pH decreased 

the toxicity of Furanace to rainbow trout but had no 

effect on its toxicity to channel catfish and green 

sunfish.

References

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nifurpirinol in salmonids. U.S. Fish Wildl. Serv. Tech.

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Amend, D. F., and A. J. Ross. 1970. Experimental control

of columnaris disease with a new nitrofuran drug,

P-7138. Prog. Fish-Cult. 32(1): 19-25.

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Organisms. 1975. Methods for acute toxicity tests

with fish, macroin vertebrates, and amphibians.

Ecological Research Series. EPA [Environ. Prot. Agency]-660/3-75-009. 61 pp. 

Herman, R. L. 1974. A review of the literature on the use

of Furanace in fisheries. U.S. Fish Wildl. Serv., Eastern

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Hunn, J. B., R. A. Schoettger, and E. W.

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Marking, L. L. 1969. Toxicological assays with fish. Bull.

Wildl. Dis. Assoc. 5:291-294. 

Marking, L. L. 1975. Toxicological protocol for the

development of piscicides. Pages 26-31 in P.H.

Esehmeyer, ed. Rehabilitation of fish populations with

toxicants: a symposium. North Cent. Div., Am. Fish.

Soc., Spec. Publ. No. 4. 

Ross, A. J. 1972. In vitro studies with nifurpirinol

(P-7138) and bacterial pathogens. Prog. Fish-Cult.

34(1):18-20. 

Shimizu, M., and Y. Takase. 1967. A patent

chemotherapeutic agent against fish diseases: 6-

hydroxymethyl-2[2-(5-nitro-2-furyl) vinyl] pyridine

(P-7138). Bull. Jpn. Soc. Sci. Fish. 33(6):544-554.
(Reports 53 through 55 are in one cover.)

53. Toxicity of Mixtures of Quinaldine Sulf ate and MS-222 to Fish, by Verdel K. Dawson and Leif 

L. Marking. 1973. 11 pp.

54. The Efficacy of Quinaldine Sulfate:MS-222 Mixtures for the Anesthetization of Freshwater 

Fish, by Philip A. Gilderhus, Bernard L. Berger, Joe B. Sills, and Paul D. Harman. 1973. 9 pp.

55. Residues of Quinaldine and MS-222 in Fish Following Anesthesia with Mixtures of Quinaldine 

Sulfate:MS-222, by Joe B. Sills, John L. Alien, Paul D. Harman, and Charles W. 

Luhning. 1973. 12 pp.

(Reports 56 through 59 are in one cover.)

56. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to 10 Species of Algae, by A. 

W. Maki, L. D. Geissel, and H. E. Johnson. 1975. 17 pp.

57. Acute Toxicities of 3-Trifluoromethyl-4-nitrophenol (TFM) and 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) to Larvae of the Midge Chironomus tentans, by J. A. Kawatski, M. M. 

Ledvina, and C. R. Hansen. 1975. 7 pp.

58. Acute Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nymphs of 

Mayflies (Hexagenia sp.), by C. R. Fremling. 1975. 8 pp.

59. Toxicity and Residue Dynamics of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) in 

Aquatic Invertebrates, by H. O. Sanders and D. F. Walsh. 1975. 9 pp.

(Reports 60 through 62 are in one cover.)

60. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nontarget Fish in Static 

Tests, by L. L. Marking and L. E. Olson. 1975. 27 pp.

61. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nontarget Fish in FlowThrough Tests, by L. L. Marking, T. D. Bills, and J. H. Chandler. 1975. 9 pp.

62. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Selected Aquatic 

Invertebrates and Frog Larvae, by J. H. Chandler and L. L. Marking. 1975. 7 pp.

(Reports 63 through 66 are in one cover.)

63. Laboratory Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM) as a Lampricide, by V.K. 

Dawson, K. B. Gumming, and P. A. Gilderhus. 1975. 7 pp.

64. Effects of 3-Trifluoromethyl-4-nitrophenol (TFM) on Developmental Stages of the Sea 

Lamprey, by G. W. Piavis and J. H. Howell. 1975. 4 pp.

65. Accumulation and Loss of Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in Fish Muscle 

Tissue: Laboratory Studies, by J. B. Sills and J. L. Alien. 1975. 5 pp.

66. Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in a Stream Ecosystem after Treatment for 

Control of Sea Lampreys, by P. A. Gilderhus, J. B. Sills, and J. L. Alien. 1975. 5 pp.

67. Method for Assessment of Toxicity or Efficacy of Mixtures of Chemicals, by L. L. Marking and 

V. K. Dawson. 1975. 7 pp.

68. Development and Evaluation of On-site Toxicity Test Procedures for Fishery Investigations, 

by R. M. Burress. 1975. 8 pp.

(Reports 69 and 70 are in one cover.)

69. Toxicity of 3-trifluoromethyl-4-nitrophenol (TFM), 2',5-dichloro-4'-nitrosalicylanilide (Bayer 

73), and a 98:2 Mixture to Fingerlings of Seven Fish Species and to Eggs and Fry of Coho 

Salmon, by T. D. Bills and L. L. Marking. 1976. 9 pp.

70. The Freshwater Mussel (Anodonta sp.) as an Indicator of Environmental Levels of 3- 

trifluoromethyl-4-nitrophenol (TFM), by A. W. Maki and H. E. Johnson. 1976. 5 pp.

71. Field Tests of Isobornyl Thiocyanoacetate (Thanite) for Live Collection of Fishes, by R. M. 

Burress, P.A. Gilderhus, and K. B. Gumming. 1976. 13 pp.

72. Toxicity of Rotenone to Fish in Standardized Laboratory Tests, by L. L. Marking and T. D. 

Bills. 1976. 11 pp.

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