INVESTIGATIONS IN FISH CONTROL

60. Toxicity of the Lampricide

3-Trifluoromethyl-4-nitrophenol 

(TFM) to Nontarget Fish in Static Tests

61. Toxicity of the Lampricide

3-Trifluoromethlyl-4-nitrophenol

(TFM) to Nontarget Fish in Flow-Through Tests

62. Toxicity of the Lampricide

3-Trifluoromethyl-4-nitrophenol

(TFM) to Selected Aquatic Invertebrates 

and Frog Larvae

U

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 0 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.
INVESTIGATIONS IN FISH CONTROL

60. Toxicity of the Lampricide 3-Trifluoromethyl4-nitrophenol (TEM) to Nontarget Fish in 

Static Tests

By Leif L. Marking and Lee E. Olson

61. Toxicity of the Lanopricide 3-Trifluoromethyl4-nitrophenol (TEM) to Nontarget Fish in 

FlowHThrough Tests

By Leif L. Marking, Terry D. Bills, and 

Jack H. Chandler

62. Toxicity of the Lampricide 3-Trif luoromethyl4-nitrophenol (TEM) to Selected Aquatic 

Invertebrates and Frog Larvae

By Jack H. Chandler and Leif L. Marking

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. 1975
IN THIS COVER

Pages

Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol

(IMF) to Nontarget Fish in Static Tests, by Leif L. Marking and

Lee E. Olson ................................... 1-27

Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) 

to Nontarget Fish in Flow-Through Tests, by Leif L. Marking, 

Terry D. Bills, and Jack H. Chandler ................... 1-9

Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) 

to Selected Aquatic Invertebrates and Frog Larvae, by Jack H. 

Chandler and Leif L. Marking ........................ 1-7

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402 - Price $1.10

Stock Number 024-010-00402-9
\

FOREWORD

The lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), has been used 

extensively to control larvae of the sea lamprey (Petromyzon marinus) in 

the Great Lakes. Although the toxicity of TFM to lampreys is well documented, its effects on other organisms are unknown.

The use of any toxicant in the environment raises concern as to the 

safety of nontarget organisms. Since invertebrate and lower vertebrate 

populations provide the forage base for many sport and commercial 

fishes, data on how TFM affects these organisms are vital to any application for registration.

The three papers in this series represent a part of continuing research 

on the effects of TFM on aquatic organisms. The papers report the results of tests on 15 species of nontarget fish, the larvae of 3 species of 

frogs, and 16 species of invertebrates. Reports on the effects of TFM on 

algae, midges, mayflies, and selected other invertebrates were published 

as Nos.56, 57, 58, and 59 of Investigations in Fish Control; a complete review of the literature prior to 1972 related to the use of TFM as a lampricide was published in No. 44.

Fred P. Meyer, Director 

Fish Control Laboratories

iii

INVESTIGATIONS IN FISH CONTROL

6O. Toxicity of the Lampricide

3-Trifluoromethyl-4-nitrophenol 

(TFM) to Nontarget Fish in Static Tests

By Leif L. Marking and Lee E. Olson

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. * 1975
CONTENTS

Page

Abstract. ...................................... 3

Introduction. .................................... 3

Materials and methods ............................. 3

Results. ....................................... 6

Purified TFM.................................. 6

Toxicity to selected species of fish .................. 6

Influences of temperature, water hardness, and pH. ....... 6

Field grade TFM. ............................... 6

Toxicity to selected species of fish .................. 5

Influences of temperature, water hardness, and pH. ....... 7

Reduced TFM.................................. 7

Residual toxicity................................ 10

Comparison of various formulations. .................. n

Discussion ..................................... 12

Conclusions. .................................... 12

Literature cited.................................. 13

Appendix. ...................................... 15
TOXICITY OF THE UMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL 

(TFM) TO NONTARGET FISH IN STATIC TESTS

By Leif L. Marking and Lee E. Olson -1 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is applied to 

tributary streams of the Great Lakes for controlling larvae of the sea 

lamprey (Petromyzon marinus). During treatments for lamprey control, 

cohabiting, nontarget fish are also exposed to TFM. Knowledge of the 

margin of safety for these fish is vitally important to the reduction of undesired effects of field applications. The lampricide is toxic to 15 species 

of coldwater and warmwater nontarget fish; the 96-h LCSO's in static tests 

at 12 C range from 1.39 to 16.2ju 1/1 of field grade TFM (35%). The toxicity of TFM is influenced by temperature, water hardness, and pH. The 

most influential factor is pH. For certain species, more than 50 times as 

much chemical is needed to produce the same effect at pH 9.5 as at pH6.5. 

In laboratory test water, TFM detoxifies slowly; solutions lose little or no 

activity over periods up to 8 wk. The margin of safety (LC01 forfish/LC99 

for lamprey) for rainbow trout (Salmo gairdneri) in minimum lampricidal 

concentrations of TFM is influenced by pH and is greater in water of low 

pH (6.5) than in water of higher pH. Under laboratory conditions at pH 7.5 

and 8.5, a 10% mortality of rainbow trout could be expected in lampricidal 

concentrations of field grade TFM.

INTRODUCTION

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is an effective toxicant against 

larval lampreys (Petromyzon marinus) living 

in tributary streams of the Great Lakes (Applegate et al. 1958). However, additional data 

on the toxicity of TFM to nontarget organisms 

are needed to satisfy regulatory requirements 

for toxicants (Lennon 1967). Previous laboratory and field information regarding the use of 

this lampricide was summarized by Schnick 

(1972).

The present study was designed to determine the toxicity of purified, field grade, and 

reduced TFM to fish in laboratory toxicity 

tests and to determine the influence of water

hardness, pH, and temperature on the toxicity 

of TFM. The residual toxicity of TFM in water 

solutions was determined to evaluate the persistence of the toxicant under aerobic conditions. These data were used to derive the 

margin of safety for nontarget fish.

MATERIALS AND METHODS

The static test procedures used follow 

closely those of Lennon and Walker (1964) and 

Taras (1971). Ten fish were exposed to each 

concentration of TFM in glass jars containing 

15 liters of oxygen-saturated test water. The 

test waters were prepared according to the 

schedule in Table 1 to produce desired water 

hardnesses. In separate studies, the pH of

Present address: Fish Pesticide Research Unit. P.O. Box 936. La Crosse, Wisconsin 54601.
4 Investigations in Fish Control 60: Fish and Wildlife Service

Table 1. Quantities of salts and characteristics of reconstituted waters

Water 

type

Very soft

Soft

Hard

Very hard

Salts added in mg/1

NaHC03

12

48

192

384

CaS04 .2H2 0

7.

30.

120.

240.

5

0

0

0

MgS04

7.

30.

120.

240.

5

0

0

0

KC1

0.

2.

8.

16.

5

0

0

0

6.

7.

7.

8.

pH .

4-6.8

2-7.6

6-8.0

0-8.4

ms/i

Hardness

10-13

40-48

160-180

280-320

as CaC03

Alkalinity

10-13

30-35

110-120

225-245

test waters was controlled with chemical 

buffers (Table 2). The solutions were adjusted 

to the appropriate pH before the test and 

readjusted with chemical buffers at 24-h intervals as necessary to maintain the selected 

pH ± 0.2 units. Test temperatures were regulated by immersing the test jars in constanttemperature water baths.

Table 2. Buffer chemicals used to

produce and maintain various pH's

in soft, reconstituted water

PH

ML Hiliters of solutions for 

15 liters of water

IN NaOH 1M KH2P04 0.5M H3B03

6.0

6.5

7.0

1.3

10.0

19.0

80.0

30.0

30.0 _--

7.5

8.0

8.5

9.0

9.5

10.0

 

19.0 20.0

12.0 11.5

8.8

11.0

16.0

 _

___

---

30.0

20.0

18.0

Field grade TFM and analytical grade TFM 

(99% active ingredient) were obtained from 

Hoescht Chemical Company, Summerville, 

New Jersey.2 Field grade TFM is formulated 

with DMF (N,N-dimethylformamide) and is 

approximately 35% active ingredient, but the 

purity varies slightly between batches. Purified TFM was prepared by Aldrich Chemical 

Company 2 (96% active ingredient). Dr. John 

Lech of the Department of Pharmacology of 

the Medical College of Wisconsin at Milwaukee also prepared purified TFM (94% active 

ingredient) and synthesized reduced TFM 

(RTFM) hydrochloride for these experiments. 

The purified materials were weighed on an 

electrobalance and dissolved in acetone; concentrations are expressed as mg/1. Fieldgrade TFM was measured volumetrically and 

dissolved in water; concentrations are expressed as

Fish weighing 1 to 1.5 g each were obtained 

from Federal hatcheries and maintained according to the standard procedures of the Fish 

Control Laboratory (Hunn et al. 1968). The 

fish were acclimated to the desired water 

chemistries and temperature of each test. 

Mortalities were recorded at 1, 3, and 6 h on 

the first day of exposure and daily thereafter 

for the remainder of the 96-h test.

The methods of Litchfield and Wilcoxon 

(1949) were used in computation of theLCSO's 

(concentrations producing 50% mortality) and

unbuffered soft, reconstituted 

water.

2 Use of trade names does not imply U.S. Government endorsement of commercial products.
Marking and Olson: Toxicity of TFM to Nontarget Fish in Static Tests S

Table 3. Toxicity of purified TFM to fingerling fish in 

toxioity tests at 12 C

LC5Q and 95% confidence interval (mg/1) at

Species 

Ih 3 h 6 h 24 h 96 h

.Coho salmon 6.80 5.60 5.60 4.30 2.70 

(Onoorhynchus kisutcfr) 6.24-7.41 4.69-6.69 4.69-6.69 3.78-4.89 2.26-3.22

Chinook salmon 4.00 3.40 3.10 2.24 

(anGorhynchus tshaw- 3.30-4.06 3.06-3.78 """ 2.70-3.55 1.94-2.59 

ytsoha)

Rainbow trout 4.40 3.08 2.92 2.91 1.97 

(Saljno gairdneri) 4.02-4.82 2.87-3.31 2.65-3.22 2.57-3.31 1.78-2.18

Brown trout 7.00 4.93 4.78 3.89 2.63 

(Saljno trutta) 6.33-7.74 4.47-5.43 4.20-,5.44 3.57-4.24 2.35-2.94

Jake trout 2.72 1.93 1.43 1.43 1.40 

(Salvelinus namaycush) 2.30-3.21 1.71-2.18 1.16-1.75 1.16-1.75 1.11-1.77

Northern pike 5.55 1.85 1.85 1.25 0.947 

(Esox lucius) 4.67-6.59 1.25-2.73 1.25-2.730.847-1.84 0.594-1.51

Carp ... 2.10 1.74 1.25 

(Cyprinus carpio) 1.85-2.37 1.43-2.11 1.00-1.56

Channel catfish 5.15 2.38 1.34 1.20 1.00 

(Ictalurus punctatus) 4.26-6.23 2.05-2-76 1.20-1.50 1.03-1.400.803-1.25

Bluegill 12.9 $.90 6.42 6.23 6.23 

(Lepomis maorochirus) 11.4-14.6 8.22-9.64 5.99-6.89 5.50-7.05 5.50-7.05

Sraallraouth bass 11.1 7.96 6.42 6.42 6.30 

(Micropterus dolomieui) 10.2-12.1 7.10-.8.93 5.66-7.28 5.66-7.28 5.63-7.04

Largemouth bass 5.45 3.85 2.19 - 

(Micropterus salmoides) - 5.01-5.93 3.41-4.35 1.82-2.63

Yellow perch 6.20 3.38 2.88 2.51 2.07 

(Perca flavescens) 5.05-7.61 2.63-4.35 2.47-3.35 2.19-2.88 1.69-2.54

Walleye 7.10 3.00 2.05 1.88 1.88 

(Stigostedion vitreum) 4.96-10.1 2.47-3.65 1.84-2.28 1.61-2.19 1.63-2.16

Purity (94$) for tests with coho salmon and (96$) for tests with others.
6 Investigations in Fish Control 60: Fish and Wildlife Service

95% confidence intervals. Regressions were 

drawn and inspected for each set of data. All 

data fulfilled the Chi-square test requirement 

for acceptability.

Deactivation indices were derived lor field 

grade TFM in water at four different pH's. 

Aged solutions of the toxicant were bioassayed 

to determine the biological activity remaining 

after selected time periods. The deactivation 

index was determined by dividing the LC50 of 

aged solutions by the LC50 of unaged solutions 

under corresponding test conditions (Marking 

1972).

RESULTS

Purified TFM 

Toxicity to selected species of fish

Purified TFM is toxic to coldwater and 

warmwater fish in soft water; the 96-h LCSO's 

range from 0.947 to 6.30 mg/1 of TFM (Table 

3). Northern pike and channel catfish were 

most sensitive, and the 96-h LCSO's were not 

significantly different from each other 

(P = 0.05). Smallmouth bass and bluegill are 

the most resistant; LCSO's were significantly 

different from those with other species at all 

comparable exposure periods. Most of the 

other species were sensitive to 1 to 3 mg/1 of 

TFM.

All species responded rapidly to the toxic 

effects of TFM (as shown by the 1-h LCSO's), 

and the toxicity changed little with prolonged 

exposure (the 1-h LCSO's which are only 2 to 5 

times greater than 96-h LCSO's). The LCSO's 

for 24- and 96-h exposures were not significantly different for lake trout, northern pike, 

channel catfish, smallmouth bass, bluegill, 

yellow perch, and walleye.

Influences of temperature, water hardness, 

and pH

The toxicity of purified TFM to fish was 

altered considerably by water quality, and the 

alteration was fairly uniform for different 

species. Test results are presented for rainbow trout in Table 4 and for other species in 

Appendix Tables 1 to 7.

The lampricide was more toxic to rainbow 

trout in warm than in cold water. The 96-h 

LCSO's were significantly different at temperatures of 7, 12, and 17 C. This influence 

was more consistent for coldwater species 

than for warmwater species. The 96-h LCSO's 

for carp, for instance, were not significantly 

different at 12, 17, and 22 C.

Purified TFM was more toxic to rainbow 

trout in soft water (total hardness, 44 

mg/1) than in hard or very hard water (total 

hardness, 170 and 300 mg/1, respectively). 

The respective 96-h LCSO's at 12 C were 

1.97, 5.47, and 9.45 mg/1. The hardness of 

test water influenced the toxicity to other 

species in a similar manner.

The toxicity of purified TFM to fish decreased substantially as the pH of test waters 

increased (Table 4 and Appendix Tables 1 to 

7). The 96-h LCSO's were significantly different for each pH increment for rainbow trout 

as well as for other species. The magnitude 

of change in toxicity can be compared by 

dividing the LC50 value at the lowest pH by 

that at the highest pH. The factors are as 

follows: coho salmon - 45, rainbow trout - 29, 

brown trout - 50, lake trout - 59, carp - 36, 

channel catfish - 23, bluegill - 21, and yellow 

perch - 26. The factor for rainbow trout is 

lower than that for other salmonids because 

the pH ranged only from 6.5 to 9.0, whereas 

for the other species the pH ranged from 6.5 

to 9.5.

Field grade TFM 

Toxicity to selected species of fish

Field grade TFM also was toxic to coldwater and warmwater fishes in soft water 

(Table 5). The 96-h LCSO's for 15 species 

ranged from 1.39 to 16.2ju 1/1 of TFM (35.4%). 

Considering only the active ingredient in the 

formulation, the range was 0.39 to 4.58ju 1/1. 

Field grade TFM thus appeared to be more 

active than purified TFM; however, the difference was slight and may have been due to 

variations in sensitivity among the different 

groups of fish exposed. Among the families of 

fishes represented, centrarchids were the 

most resistant to TFM. Bluegill and green
Marking and Olson: Toxicity of TFM to Nontarget Fish in Static Tests

Table 4. Toxicity of purified TFM (96$) to fingerling rainbow trout at 

selected temperatures, water hardnesses, and pH's

Temp. Water 

(°C) hardness

LC50 and 95$ confidence interval (mg/l) at

1 h

12

12

12

12

3 h 6 h h 96 h

7

12

17

12

Soft

Soft

Soft

Hard

7.5

7.5

7.5

7.8

6.20 

5.61-6.85

4.40 

4.02-4.82

3.10 

2.93-3.28

_«_

4.00 

3.73-4.29

3.08 

2.87-3.31

2.62 

2.33-2.94

9.00

3.00 

2.78-3.24

2.92 

2.65-3.22

2.62 

2.33-2.94

8.43

2.89 

2.64-3.16

2.91 

2.57-3.30

2.05 

1.70-2.47

8.00

2.44 

2.16-2.75

1.97 

1.78-2.18

1.58 

1.35-1.85

5.45

8.41-9.63 7.67-9.26 7.43-8.61 4.74-6.27

Very hard 8.2 18.5 15.0 14.7 13.8 9.45

16.4-20.9 13.9-16.2 13.5-16.0 12.6-15.1 9.12-9.79

Soft 

Soft 

Soft

6.5 2.00 1.42 1.30 1.26 1.10

1.74-2.30 1.28-1.57 1.17-1.45 1.13-1.39 0.744-1.63

8.0 15.4 10.9 10.0 7.75 6.19

14.3-16.5 9.96-11.9 9.39-10.6 6.82-8.80 5.56-6.89

9.0 37.9 32.1 

33.5-42.9 29.5-34.9

sunfish were the most resistant species to 

field grade TFM and smallmouth bass to purified TFM. Channel catfish were the most 

sensitive species to field grade TFM.

Influences of temperature, water hardness, 

and pH

The toxicity of field grade TFM to fish was 

influenced by temperature, water hardness, 

and pH in patterns similar to those observed 

with purified TFM. In general, TFM (35.7%) 

was most toxic to fish in warm, very soft, and 

low pH (6.5) water. Toxicity data for rainbow 

trout are in Table 6, and those for other species are in Appendix Tables 8-13. The greatest influence on the toxicity of TFM (35.7%) 

was from pH. Several 96-h LCSO's were unavailable, but the 24-h exposure produced a 

good approximation of the 96-h results. The 

24-h toxicity of TFM (35.7%) to rainbow trout

decreased by a factor of approximately 10 as 

pH increased from 6.5 to 8.5 and by a factor 

of nearly 100 as pH increased from 6,5 to 9.5 

These factors were much greater than those 

for purified TFM at pH's of 6.5 to 9.0 (Table 

4). Apparently the toxicity change accelerated 

above pH 9.0. Also, the data for most other 

species (Appendix Tables 8-13) showed a significant increase in toxicity from 24- to 96-h 

exposures at the high pH; the LCSO's at pH9.5 

were 31 and 83 times greater than those at 

pH 6.5 for yellow perch and lake trout, respectively.

Reduced TFM

In waters of three different hardnesses, the 

reduced form of purified TFM was considerably less toxic to rainbow trout than the parent 

material. The 96-h LCSO's ranged from 29.0 

mg/l of RTFM in very soft to 48.0 mg/l in 

very hard water (Table 7); however, the
8 Investigations in Fish Control 60: Fish and Wildlife Service

Table 5.--Toxicity of field grade TFM (35.4$) a to fingerling fish in

toxicity tests at 12 C

LC50 and 95% confidence interval (/*!/!) at 

Species _________________________ __ 

1 h 3 h 6 h 24 h 96 h

Chinook salmon 11.5 8.66 7.62 5.98 4.20

(Oncorhynchus 9.66-13.7 7.86-9.54 6.48-8.96 5.09-7.03 3.52-5.02

tschawytscha)

Brown trout 9.63 5.83 4.94 4.53 3.53

(Salmo trutta) 8.46-11.0 5.33-6.38 4.15-5.88 3.86-5.32 3.04-4.09

Rainbow trout 5.83 4.83 4.46 3.83 3.83

(SaJjno gairdneri) 5.36-6.34 4.33-5.39 4.05-4.91 3.31-4.43 3.31-4.43

Lake trout 14.5 4.94 4.52 3.84 2.94

(Salvelinus namaycush) 12.5-16.9 3.81-6.38 3.50-5.82 3.11-4.72 2.64-3.28

Goldfish 38.5 12.7 7.17 5.22 5.00

(Carassius auratus) 29.4-50.4 10.8-15.0 6.50-7.91 4.32-6.30 3.97-6.29

Carp 8.27 4.51 3.35 3.35 3.35

(Cyprinufl carpio) 7.00-9.77

Golden shiner 18.8 

(Notemigonus 18.3-19.3 

(crysoleucas)

Fathead minnow 17.5 

(Pimephales promelas) 15.9-19.3

3.24-6.29 2.37-4.74 2.37-4.74 2.37-4.74

11.4 10.0 8.20 7.62

9.98-13.0 9.01-11.1 7.10-9.48 6.29-9.23

10.5 5.54 4.79 4.79

8.14-13.5 4.65-6.60 4.19-5.47 4.19-5.47

White sucker 10.0 6.50 6.26 4.50 3.95

(Catostomus 8.24-12.1 5.16-8.19 4.94-7.93 3.22-6.28 2.69-5.81 

commersoni)

Black bullhead 13.5 5.50 3.85 2.34 2.41

(Ictalurus melas) 12.2-14.9 4.67-6.48 3.29-4.50 1.89-2.90 2.10-2.77

Channel catfish 11.9 4.54 3.86 2.40 1.39

(Ictalurus punctatus) 10.2-13.9 4.09-5.25 3.30-4.51 2.08-2.77 1.12-1.73

Green sunfish 26.2 16.8 13.1 12.9 9.40

(Lepomis cyanellus) 24.3-28.3 14.9-18.9 11.6-L4.7 11.4-14.6 7.88-11.2

Bluegill 25.4 16.2 16.2 16.2

(Lepomis macrochirus) 21.5-30.0 14.1-18.6 13.6-19.3 13.6-19.3

Largemouth bass . 10.0 6.04 6.04

(MLcropterus salmoides) 5.28-18.9 4.07-8.97 4.07-8.97

Yellow perch 11.4 7.00 5.85 5.80 4.35

(Perca flavescens) 10.0-12.9 6.35-7.71 5.27-6.49 4.98-6.76 3.45-5.48

TFM (35.7$) for chinook salmon, brown trout, lake trout, and rainbow trout,
Marking and Olson: Toxicity of TFM to Nontarget Fish in Static Tests 9

Table 6. Toxioity of field grade TFM (35.7$) to fingerling rainbow trout at 

selected temperatures, hardnesses, and pH's

Temp. Water LC5° and 95 ^ confidence interval (yul/l) at 

(°C) hardness PH

Ih 3h 6h 24 h 96 h

7 Soft 7.5 10.2 6.68 4.78 4.37 3.68

9.16-11.4 5.93-7.52 4.23-5.40 3.95-4.84 3.38-4.01

12 Soft 7.5 5.83 4.83 4.46 3.83 3.83

5.36-6.34 4.33-5.39 4.05-4.91 3.31-4.43 3.31-4.43

17 Soft 7.5 4.10 3.40 3.40 2.79 2.37

3.75-4.48 3.05-3.79 3.05-3.79 2.34-3.33 2.05-2.75

12 Very soft 6.6 3.77 3.27

3.32-4.28 2.85-3.75

12 Hard 7.8 50.3 26.0 19.0 14.1 8.38

43.5-58.2 23.0-29.4 16.7-21.6 12.8-15.5 7.41-9.48

12 Very hard 8.2 88.3 45.9 36.6 27.2 19.0

79.4-98.2 40.5-52.0 33.2-40.4 21.8-34.0 16.8-21.5

12 Soft 6.5 4.12 2.82 2.56 2.52 2.52

3.71-4.57 2.56-3.10 2.17-3.01 2.16-2.94 2.16-2.94

12 Soft 8.5 74.0 42.4 36.7 20.5

65.0-84.2 38.5-46.7 32.1-42.0 

12 Soft 9.5 >300 270 239 230

	228-320 205-278 204-259

Table 7. Toxicity of reduced TFM to fingerling rainbow trout in standard,

reconstituted water at 12 C

Water

hardness

Very soft

Hard

Very hard

LC50 and

24 h

30.0 

24.6-36.6

64.0 

51.7-79.2

52.0 

44.4-60.9

95$ confidence interval

48 h

30.0 

24.6-36.6

60.0 

49.2-73.2

50.0 

43.3-57.7

(mg/1) at

96 h

29.0 

26.2-32.1

49.0 

42.7-56.3

48.0 

41.5-55.5
10 Investigations in Fish Control 60: Fish and Wildlife Service

influence of water hardness was not as great 

as with the parent compound.

Residual Toxicity

Field grade TFM (35.4%) was added to test 

waters and permitted to age for 1 wk before 

rainbow trout were introduced. A comparison 

of the toxicity of aged solutions and unaged 

reference solutions showed that TFM detoxified slowly, if at all, in water solutions (Table 

8). The 96-h deactivation index (LC50 of aged 

solution/LC50 of unaged solution) was 0.91 for 

pH 6.5, 1.03 for pH 7.5, 1.09 for pH 8.0, and 

1.14 for pH 9.0. Although detoxification tended 

to be slightly greater at high pH's, the toxicity of aged and unaged solutions was not significantly different at any pH's.

Additional deactivation studies were carried 

out for longer aging periods to determine the 

rate of detoxification at different pH's. The 

toxicity and deactivation indices of purified 

TFM (96%) were determined at four pH's after 

aging periods up to 8 wkat 12 C (Table 9). The 

indices were near 1.0 for pH's 6.5, 7.5, and 

8.5 but were erratic and did not show significant detoxification of TFM with aging. TFM 

was much less toxic to rainbow trout at pH 9.5 

than at lower pH's. Activity decreased with 

aging, but the decrease again was erratic. 

Although the index at 4 wk of aging (1.95) indicated a considerable decrease in activity, 

the activity remained constant in a series of 

solutions aged for 6 wk. Apparently, TFM was 

detoxified in some instances but not in others. 

Determination of the rate of detoxification

Table 8. Toxicity of field grade TFM (35.4%) to fingerling rainbow trout in 

buffered solutions at 12 C freshly prepared (F) or aged 1 week (A)

Type of 

pn solution

(F)

6.5

(A)

(F)

7.5

(A)

(F)

8.0

(A)

(F)

9.0

(A)

LC50 and 95% confidence interval (/-ti/1)

1 h

3.45 

3.10-3.84

4.00 

3.57-4.49

11.9 

10.3-13.8

19.1 

16.7-21.9

42.0 

36.0-49.0

42.0 

38.4-45.9

 

238 

187-303

3 h

3.00 

2.68-3.35

3.30 

2.87-3.79

8.70 

7.98-9.48

11.6 

9.96-13.5

35.0 

30.6-40.0

33.0 

29.3-37.2

 

142 

121-167

6 h

2.85 

2.59-3.13

3.29 

2.87-3.77

8.70 

7.98-9.48

11.1 

9.39-13.1

32.0 

28.4-36.1

31.0 

27.6-34.8

137 

118-159

24 h

2.85 

2.59-3.13

3.10 

2.75-3.49

6.33 

5.63-7.11

8.58 

7.34-10.0

26.0 

23.6-28.7

23.0 

21.0-25.2

 

117 

99.2-138

at

96 h

2.46 

2.17-2.79

2.24 

2.01-2.50

4.78 

3.97-5.76

4.90 

3.61-6.66

11.0 

10.1-12.0

12.0 

10.4-13.8

39.5 

33.8-46.2

45.0 

38.5-52.5
Marking and Olson: Toxicity of TFM to Nontarget Fish in Static Tests 11

Table 9. Toxicity to rainbow trout of fresh and aged solutions of purified 

TFM (96%) at four pH's in 12 C water. (Deactivation indices

shown in parentheses)

Aging 

period 

(weeks)

6

8

96-h LC50 and 95% confidence 

interval (mg/l) and (deactivation index)

pH 6.5 pH 7.5 pH 8.5

1.20

0.973-1.48 

(1.25)

0.842 

(0.875)

2.32

1.98-2.71 

(1-12)

2.17

1.83-2.57 

(1.04)

1.78 

(0.856)

2.05

1.70-2.47 

(0.986)

2.05

1.71-2.46 

(0.986)

5.81

5.24-6.45 

(1.08)

5.63

5.09-6.22 

(1.04)

4.40 

(0.816)

5.81

5.23-6.46 

(1.08)

pH 9.5

0

0.962

0.850-1.09

(1.00)

2.08

1.73-2.50

(1.00)

5.39

4.84-6.00

(1.00)

40.5

35.8-45.8

(1.00)

71.2

64.9-78.2 

(1.76)

66.3

59.2-74.2 

(1.64)

79.0

69.4-89.9 

(1.95)

51.8

46.8-57.3 

(1.28)

from these erratic indices was not possible 

nor was it feasible to extend the aging periods. The data were sufficient, however, to 

indicate that TFM did not detoxify readily 

under laboratory conditions. Concentrations of 

TFM remaining in aged solutions were confirmed by spectrophotometric analysis (Olson 

and Marking 1973).

Comparison of Various Formulations

Some of the various formulations of TFM 

that have been prepared for laboratory use

and for field applications were tested to determine their activity against rainbow trout 

(Table 10). The high-percentage formulations 

tested were comparable in activity but were 

slightly less active than field grade TFM 

(35.7%). The greater activity of the field grade 

TFM was expected because the formulating 

process overcomes some of the problems associated with solubility. The carrier used in 

the field grade formulation may increase dispersion, reduce particle size, or enhance ionic 

state of the TFM molecule since may carriers 

are used for such purposes.
12 Investigations in Fish Control 60: Fish and Wildlife Service

Table 10. Toxicity of TFM in various formulations to rainbow trout in

soft water at 12 C

Percent 

active ingredient

Analytical

Purified

Purified

Field

(99+)

(96)

(94)

(35.7)

m . 96 -h LC50 and 95$ confidence interval at Toxic ^

unit 

Total formulation

mg/1 1.39 

1.17-1.66

mg/1 1.50 

1.35-1.67

mg/1 1.55 

1.36-1.76

Ml/1 3.38 

2.91-3:92

Active TFM

1.39 

1.17-1.66

1.44 

1.27-1.57

1.46 

1.28-1.65

1.21 

1.03-1.40

DISCUSSION

Data on the toxicity of TFM to nontarget 

fishes helps assess the margin of safety for 

such fish. Because the toxicity of TFM is influenced significantly by water hardness and 

pH, the margin of safety can be determined 

accurately only if the tests for toxicity to 

target and nontarget organisms are done in 

comparable water media. Toxicity data from 

field applications usually cannot be compared 

with laboratory toxicity data because of the 

differences in water quality and the presence 

of other biota. Ideally, the margin of safety 

should be determined by testing field grade 

TFM against target and nontarget organisms 

in the water to be treated.

Efficacious concentrations of field grade 

TFM were determined in standardized laboratory tests by Dawson et al. (In press), who 

used test water identical to that used in our 

toxicity tests. For example, in soft water 

(44 mg/1 total hardness; 12 C), the 24-h LC99 

in jul/1 of TFM (35.7%) against larval lamprey 

was 0.950 at pH 6.5, 3.25 at pH 7.5, and 12.0 

at pH 8.5. In corresponding water quality, the 

24-h LC01 in jul/1 of TFM (35.7%) against 

rainbow trout was 1.60 at pH 6.5, 2.60 at pH 

7.5, and 10.4 at pH 8.5. The margin of safety 

(LC01 for fish/LC99 for lamprey) at the respective pH's was 1.760, 0.800, and 0.866. 

Because values less than 1.0 indicate incomplete survival of the trout, some trout would 

be expected to die at minimum lampricidal 

concentrations at pH 7.5 or 8.5, but not at 

pH 6.5.

If the margin of safety is calculated on the 

basis of LClO's for fish and LC99's for lamprey, the value is near or higher than 1.0 at 

the three pH's. Therefore, a 10% mortality of 

rainbow trout could be expected at pH's 7.5 

and 8.5 under these conditions. Trout in pH6.5 

water are safe (LC10/LC99 = 2.11).

CONCLUSIONS

1. Purified and field grade TFM are toxic to 

coldwater and warmwater fish in brief exposures (1, 3, and 6 h) as well as in 96-h 

exposures. The toxicity increased little 

during prolonged exposures.

2. TFM was generally more toxic to fish at 

higher temperatures, but the trend was not 

consistent for all warmwater species.

3. TFM was more toxic to fish in very soft 

water than in very hard water by a factor 

as great as 10.

4. TFM was considerably more toxic in acid 

than in alkaline water. The factor was 

more than 50 for pH's 6.5 to 9.5 for some 

species.
Marking and Olson: Toxicity of TFM to Nontarget Fish in Static Tests 13

5. Reduced TFM was less toxic than TFM to 

fish, but the toxicity of RTFM was influenced less by water hardness.

6. TFM was very persistent in laboratory 

test waters; activity decreases were small 

or nil for periods up to 8 wk.

7. On the basis of active ingredient, field 

grade TFM appeared to be slightly more 

toxic than purified TFM.

8. The margin of safety for rainbow trout in 

minimum lampricidal concentrations of 

field grade TFM was influenced by pH and 

was greatest at pH 6.5.

LITERATURE CITED

Applegate, V. C0 J. H. Howell, and M. A. Smith. 1958. 

Use of mononitrophenols containing halogens as selective sea lamprey larvicides. Science 127(3294): 

336.338.

Daws on, V. K., K. B. Gumming, and P. A. Gilderhus. 

1975. Laboratory efficacy of 3-trifluoromethyl-4- 

nitrophenol (TFM) as a lampricide. U.S. Fish Wildl. 

Serv., Invest. Fish Control 63.

Hunn, J. B.t 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. 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. 185). 15 pp.

Litchfield, J. T., Jr0 and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. 

J. Pharmacol. Exp. Ther. 96(2):99-113.

Marking, L. L. 1972. Methods of estimating the halflife of biological activity of toxic chemicals in water. 

U.S. Fish Wildl. Serv., Invest. Fish Control 46. 

9pp.

Olson, L. E., and L. L. Marking. 1973. Toxicity of 

TFM (lampricide) to six early life stages of rainbow 

trout (Salmo gairdneri). J. Fish. Res. Board Can. 

30(8):1047-1052.

Schnick, R. A. 1972. A review of literature on TFM 

(3-trifluormethyl-4-nitrophenol) as a lamprey larvicide. U.S. Fish Wildl. Serv., Invest. Fish Control 

44. 31 pp.

Taras, M. J., ed. 1971. Standard methods for the examination of water and wastewater. American Public Health Association, American Water Works 

Association, Water Pollut, Control FecL, New York, 

New York. 874 pp.

APPENDIX

Appendix Table 1. Toxicity of purified TFM (94%) to fingerling coho salmon at se

hardnesses, and pH! s

Temp. water 

v C) hardness

7 Soft

12 Soft

17 Soft

12 Very soft

12 Hard

12 Very hard

12 Soft

12 Soft

12 Soft

pH 

1 h

7.5 9.60 

8.74-10.5

7.5 6.80

6.24-7.41

7.5 3.61

3.35-3.89

6.6 7.40 

6.64-8.24

7.8 16.0

14.7-17.5

8.2 33.0

30.6-35.6

6.5 2.30

2.05-2.58

8.5 18.5

16.2-21.1

9.5

LC50 and 95$ confidence interval 

3 h

6.10 

5.37-6.93

5.60

4.69-6.69

3.47

3.11-3.87

4.60 

4.10-5.16

15.6

14.5-16.8

33.0

30.6-35.6

1.72

1.54-1.92

16.9

15.2-18.7

 

6 h

5.60 

4.69-6.69

5.60

4.69-6.69

3.47

3.11-3.87

4.55 

4.20-4.93

15.0

13.5-16.7

32.0

29.8-34.3

1.69

1.52-1.88

16.3

14.2-18.7

 

2

4.2

3.7

2.3

2.6

1

12.1

2

23.

1.4

1

10.
16 Investigations in Fish Control 60: Fish and Wildlife Service

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Appendix Table 3.--Toxicity of purified TM (96$) to fingerling lake trout at s

water hardnesses, and pH*s

Temp. Water 

( °c) hardness

7 Soft

12 Soft

12 Very soft

12 Hard

12 Very hard

12 Soft

12 Soft

12 Soft

PH 

1 h

7.5 6.02 

4.89-7.41

7.5 2.72

2.30-3.21

6.6 2.35

2.13-2.59

7.8 10.6

9.43-11.9

8.2 18.0

16.5-19.7

6.5 1.81 

1.61-2.04

8.5

9.5 52.0

39.5-68.4

LC50 and 95$ confidence interval 

3 h

2.23 

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1.93

1.71-2.18

1.35

1.21-1.51

6.68

6.14-7.27

12.2

11.6-12.9

0.842 

0.750-0.945

9.42 

8.04-11.0

50.0

38.8-64.5

6 h

1.89 

1.61-2.22

1.43

1.16-1.75

1.19

1.02-1.38

5.93

,5.45-6.45

11.1

10.4-11.8

0.704 

0.593-0.836

8.52 

7.73-9.39

50.0

38.8-64.5

1.5

1.1

1.0

5.3

1

9.8

0.59

7.0

5

38.
18 Investigations in Fish Control 60: Fish and Wildlife Service

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hardnesses, and pH's

Temp. Water 

( C) hardness

12 Soft

17 Soft

22 Soft

12 Very soft

12 Hard

12 Very hard

12 Soft

12 Soft

12 Soft

pH 

1 h

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7.5 3.38 

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6.6

7.8

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3.57-5.62

8.5 22.2

17.7-27.8

9.5 47.2 

39.2-56.8

LC50 and 95% confidence interval 

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2.63-4.35

2.68 

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2.80 

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12.0

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19.5

16.0-22.9

1.63

1.14-2.33

12.0

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44.3 

38.3-51.3

6 h

2.88 

2.47-3.35

2.68 

2.22-3.24

2.80 

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9.00

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15.2

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1.18

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10.4

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44.3 

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2

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Appendix Table 9. Toxicity of field grade THA (35.7%) to fingerling brown t

temperatures, hardnesses, and pH's

Temp. Water 

( C) hardness

7 Soft

12 Soft

17 Soft

12 Very soft

12 Hard

12 Very hard

12 Soft

12 Soft

12 Soft

pH 

1 h

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7.5 9.63

8.46-11.0

7.5 6.53

5.68-7.51

6.6 7.00

5.98-8.20

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55.2-64.8

6.5 4.10

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8.5 80.0

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9.5 > 150

LC50 and 95$ confidence interval 

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4.72

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3.58

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13.8

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39.8

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2.46

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38.8

34.0-44.3

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6 h

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4.94

4.15-5.88

4.72

4.17-5.34

3.42

3.07-3.81

12.8

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35.7

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24 Investigations in Fish Control 60: Fish and Wildlife Service

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mcMocoo °5 <*> ^ *Q Q ONHO 1 1 CM O 1 O

O-^- OtO H CM vOCMCM 

CM»{*>» A A H .

S vo A A in A CM H

m vo to CM in in

I> VO I> tO VO tO

4J TJ

tj Cl

O CQ

CQ A

&J +» h ft *p ft fr» ft ^ Qi **^4

O 0) JOI (U O C5 t/5 ' ^> Si} ^> CQ c/5

CM CM CM CM CM CM 

CM H H H H H

tovO

to

vO

^0

^^

"*

A

8

NT

A

m

ON

S

c8

a

S)

CM
Appendix Table 13.~Toxicity of field grade TBM (35.7%) to fingerling yellow 

temperatures, hardnesses, and pH's

Temp. Water 

( C) hardness

12 Soft

17 Soft

22 Soft

12 Very soft

12 Hard

12 Very hard

12 Soft

12 Soft

12 Soft

i TT pH 

1 h

7.5 14.3

11. 3-18.1

7.5 9.35

8.31-10.5

7.5 8.30

6.89-10.0

6.6 6.30 

5.60-7.08

7.8 45.0

39.1-51.9

8.2 93.0 

81.4-106

6.5 2.68

2.40-3.00

8.5 42.4 

38.3-46.9

9.5 >200

LC50 and 95%

3 h

5.98

5.05-7.09

5.30

4.76-5.90

6.00

5.36-6.72

2.88 

2.26-3.68

17.2

15.0-19.8

47.6 

42.8-53.0

1.70

1.45-1.99

20.0 

17. 3-23; 2

165

147-186

confidence interval 

6 h

5.85

4.85-7.05

5.30

4.76-5.90

6.00

5.36-6.72

2.60 

2.22-3.05

16.9

14.5-19.7

33.3 

30.4-36.4

1.25

0.962-1.62

13.8 

10.3-18.4

109

92.5-128

3.7

4.1

3.3

1.9

1

12

3

27

0.85

8.1

8

71

INVESTIGATIONS IN FISH CONTROL

61. Toxicity of the Lampricide

3-Trjfluoromethyl-4-nitrophenol

(TFM) to Nontarget Fish in Flow-Through Tests

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

United States Department of the Interior

Fish and Wildlife Service

Washington, D. C. * 1975
CONTENTS

Page 

Abstract. ....................................... 3

Introduction. ..................................... 3

Materials and methods .............................. 3

Results. ........................................ 4

Discussion ...................................... 8

Conclusions. ..................................... 8

Literature cited................................... 8
TOXICITY OF THE 

LAMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL

(TFM) TO NONTARGET FISH IN FLOW-THROUGH TESTS

By Leif L. Marking and Terry D. Bills 

Fish Control Laboratory, La Crosse, Wisconsin

and

Jack H. Chandler 

Southeastern Fish Control Laboratory, Warm Springs, Georgia

ABSTRACT

Field grade 3-trifluoromethyl-4-nitrophenol (TFM) was tested for acute 

and chronic toxicity to 11 species of nontarget fish in 4- and 30-day exposures, respectively. The species used were coho salmon (Oncorhynchus 

kisutch), rainbow trout (Salmo gairdneri), brown trout (Salmo trutta), brook 

trout (Salvelinus fontinalis). lake trout (Salvelinus namaycush), goldfish 

(Carassius auratus). golden shiner (Notemigonus crysoleucas). channel 

catfish (Ictalurus punctatus), bluegill (Lepomis macrochirus), red-ear 

sunfish (Lepomis microlophus), and yellow perch (Perca flavescene). The 

96-h LCSO's for the lampricide in flow-through tests ranged from 8.79 

to 32.1 /ul/1 in hard water and from 2.15 to 17.5 /ul/1 in soft water. The 

toxicity of the TFM formulation to two species of salmonids did not 

change significantly (P = 0.05) between 1- and 30-day exposures. The 

results of simultaneous static and flow-through acute toxicity tests with 

channel catfish were not significantly different in two experiments.

INTRODUCTION

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is effective for killing larval 

lampreys (Petromyzon marinus) living in 

tributary streams of the Great Lakes without 

decimating the endemic fish populations (Applegate et al. 1958). The effects of TFM on fish 

have been observed during numerous stream 

applications and in the laboratory (Marking and 

Olson 1975, Dawson et al. (In press), and 

Schnick 1972). The registration of TFM as a 

lampricide has been supported primarily by 

laboratory data developed in static test systems. However, flow-through toxicity tests 

simulate the use pattern of TFM more closely 

than static toxicity tests.

This study was designed to determine acute 

and chronic toxicities of field grade TFM to 

nontarget fish in flow-through toxicity tests. In 

addition, the acute toxicity of TFM was compared in static and flow-through systems.

MATERIALS AND METHODS

Field grade TFM, obtained from American 

Hoechst Chemical Company, Somerville, New 

Jersey,1 was used for these experiments. Because the percentage of active ingredient varies 

from one batch to another, purity is specified.

1 Use of trade names does not imply U.S. Government 

endorsement of commercial products.
Investigations in Fish Control 61: Fish and Wildlife Service

The liquid formulations were measured volumetrically and diluted in water to prepare 

stock solutions, and toxicity values were calculated and reported on a formulation volume 

to volume (/ul/1) basis. The concentration of 

TFM in each test aquarium was determined 

daily by colorimetric analysis (Olson and 

Marking 1973), and the toxicity was calculated 

on the basis of the mean values for the concentrations.

The flow-through toxicity tests were conducted in an apparatus similar to that described 

by Mount and Brungs (1967) but with modifications according to McAllister et al. (1972). The 

apparatus was designed to deliver 1 liter of 

test solution each cycle. Each glass aquarium 

contained 45 liters of test medium. The rate of 

flow was sufficient to replace the entire volume 

of test medium at least four times each day. 

The flow-through units were designed to deliver 

seven successively lower concentrations of the 

toxicant; each concentration was approximately 

25% less than the preceding one. The control 

for each test contained dilution water but no 

toxigant. The temperature of test solutions was 

maintained with a water bath.

Two types of water were used in the flowthrough tests. Reconstituted water, prepared 

according to Marking (1969), was used for 

some 96-h tests and for comparing the toxicity of TFM in static and flow-through tests. 

Charcoal filtered municipal well water was 

used for other 96-h tests and for 30-day exposures. The reconstituted water was soft (total 

hardness of 44 mg/1 and pH of 7.5), whereas 

the well water was hard (total hardness of 300 

mg/1 and pH of 7.7). Procedures for the static 

tests followed those of Lennon and Walker 

(1964).

National Fish Hatcheries furnished the fish 

for these experiments. Fish used in 96-h tests 

were not fed during acclimation before each 

test nor during exposure (Hunn et al. 1968). 

Fish for the 30-day tests were fed dry commercial pellets during acclimation and exposure. The fish ranged in size from 1.1 to 

19.9 g; for tests in which the weight is not 

specified, the fish weighed 2 to 5 g. Observations on survival and mortality were recorded 

daily, and dead fish were removed during each 

observation.

The toxicity of TFM was calculated according to the statistical procedures of Litchfield 

and Wilcoxon (1949). Toxicity was defined by 

LCSO's (concentrations calculated to produce 

50% mortality) and 95% confidence intervals. 

Chi-square tests were applied to each set of 

data to test for goodness of fit.

RESULTS

Four species of fish were exposed to field 

grade TFM (39.45%) in flow-through toxicity 

tests using soft, reconstituted water at 17 + 1 

C (Table 1). Channel catfish are the most 

sensitive and red-ear sunfish the most resistant; the 96-h LCSO's were 2.15 and 17.5 M 1/1 

of TFM, respectively. The toxicity of the 

lampricide did not change significantly (P= 0.05) 

after 24 h for goldfish, golden shiner, and redear sunfish.

Seven species were exposed to field grade 

TFM (35.7%) in charcoal filtered municipal 

well water at 12 C (Table 2). The 96-h LCSO's 

ranged from 8.79 to 32.1 fil/1 of the formulation. Larger fish of a given species were 

more resistant than smaller ones to the toxicant. For instance, the 96-h LC50 for TFM 

was 10.5 jul/1 against 1.3-g coho salmon and 

29.0 nl/1 against 7.4-g coho salmon and was 

8.79 Ml/1 against 1.3-g rainbow trout and 

13.8 yul/1 against 19.7-g rainbow trout. Lake 

trout (17.0 g) were more resistant than rainbow trout of similar size (96-h LC50= 16.9

Toxicosis was apparent in very short exposures (1 to 6 h) to TFM, and the toxicity did
Marking, Bills, and Chandler; Toxicity of TFM to Nontarget Fish in Flow-Through Tests

Table 1. Toxicity of TFM (39.45$) to fingerling fish in flow-through tests 

with soft, reconstituted water at 17 ± 1 C

Species

Goldfish 

(Carassius auratus)

Golden shiner 

( Notemigonus crysoleucas)

Channel catfish 

(ictalurus punctatus)

Red-ear sunfish 

(Lepomis microlophus)

LC50 and

3 h

7.00 

5.22-9.38

30.0 

25.5-35.3

95% confidence

6 h

8.10 

5.65-11.6

13.2 

9.96-17.5

4.80 

3.82-6.03

 

interval (

24 h

4.85 

3.26-7.01

10.6

8.56-13.1

4.05 

3.11-5.27

17.5 

14.2-21.6

[Ml/1) at

96 h

4.25 

2.86-6.31

8.50 

5.79-12.5

2.15 

1.52-3.03

17.5 

14.2-21.6

not change significantly for many exposure 

time increments (Table 2). In fact, toxicity did 

not change significantly between 6- and 96-h 

exposures for coho salmon (7.4 g), rainbow 

trout, brown trout, brook trout, lake trout, 

and bluegill. Considering only small sizes of 

fish, brook trout were more resistant than 

other salmonids, bluegills, or yellow perch.

Three species of fish were exposed to field 

grade TFM (35.7%) for 30 days in charcoal 

filtered municipal well water at 12 C (Table 3). 

The 30-day LCSO's against coho salmon, brook 

trout, and lake trout ranged from 10.5 to 19.6 

Atl/1 of the formulation. As in shorter exposures, brook trout were most resistant even 

though they were smaller than lake trout. Also, 

toxicity did not change significantly between

10 and 30 days with coho salmon and between 

1 and 30 days with lake trout.

Channel catfish were exposed to TFM in 

simultaneous static and flow-through tests to 

compare the toxicity and to assess the need 

for establishing the toxicity of TFM in flowthrough facilities. Three separate tests showed 

that TFM (39.45%) was uniformly toxic in the 

two types of tests (Table 4). All three tests 

showed that TFM was more toxic in static 

than in flow-through facilities; however, the 

difference was significant only in the second 

trial. Therefore, additional tests in the flowthrough facility with water of different temperature, hardness, and pH are perhaps unnecessary because those characteristics have 

been examined intensively in previous work.
Table 2. Toxicity of TFM (35.7$) to selected fish in 4-day flow-through tests us

municipal well water at 12 C

Species

Coho salmon

(Qncorhynchus kisutch)

Coho salmon

Rainbow trout

(Salmo gairdneri)

Rainbow trout

Brown trout

(Salmo trutta)

Brook trout

(Salvelinus fontinalis)

Lake trout

(Salvelinus namaycush)

Bluegill

( Lepomis macrochirus)

Yellow perch

(Perca flavescens)

Average 

weight

(g)

1.3

7.4

1.3

19.7

2.7

2.2

17.0

1.1

2.6

LC50 and 95$ confidence inte

1 h

14.4

13.2-15.7

34.9 

28.8-42.3

22.0

13.1-36.8

14.8

13.2-16.6

12.9

12.1-13.7

35.4

30.6-41.0

22.3

18.7-26.6

20.6

15.7-27.1

3 h

1/4.2

13.2-15.7

29.9 

25.5-35.0

19.7

12.6-30,8

13.8

12.8-14.9

10.8

10.0-11.7

32.1

28.1-36.7

16.9

15.3-18.7

22.2

19.3-25.2

17.6

13.9-22.3

6 h

14.2

13.2-15.7

29.8 

25.1-35.3

13.2

7.70-22.6

13.8

12.8-14.9

10.8

10.0-11.7

32.1

28.1-36.7

16.9

15.3-18.7

16.1

14.9-17.4

15.1

11.8-19.3
Marking, Bills, and Chandler: Toxicity of TFM to Nontarget Fish in Flow-Through Tests

Table 3. Toxicity of TFM (35.7$) to fish in 30-day flow-through tests using 

charcoal filtered municipal well water at 12 C

Species

Coho salmon

Brook trout

Lake trout

Average 

weight

(g)

1.3

2.2

17.0

LC50 and 95$ confidence

1 day 10 days

12.6 10.5

11.3-14.1 9.30-11.7

32.1 32.1

28.1-36.7 28.8-35.8

16.9 16.9

15.3-18.7 15.3-18.7

interval (

20 days

10.5

9.30-11*7

22.5

19.0-26.6

16.9

15.3-18.7

!/i]/D at

30 days

10.5

9.30-11.7

19.6

15.4-24.9

16.9

15.3-18.7

Table 4. Toxicity of TFM (39.45$) ,to channel catfish in static and 

flow-through tests with soft, reconstituted water at 17 ± 1 C

Type Of LC50 and 95$ confidence interval (/ul/1) at 

assay '

Ih 3h 6h 24 h 96 h

Static 14.2 6.00 4.25 3.55 2.85

13.3-15.2 5.39-6.68 3.89-4.65 3.16-3.99 2.45-3.32

Flow-through 3.50 3.28

	3*17-3187 2.99-3.60

Static 12.5 6.25 3.75 3.15 2.75

11.6-13.5 5.53-7.06 3.07-4.58 2.72-3.64 2.29-3.30

Flow-through 4.85 4.30 3.42

4.57-5.14 3.88-4.77 3.05-3.84

Static 11.8 6.60 4.60 3.48 1.80

10.4-13.3 5.76-7.56 4.01-5.28 2.94-4.12 1.26-2.58

Flow-through 7.00 4.80 4.05 2.15

5.22-9.38 3.82-6.03 3.11-5.27 1.52-3.03
8 Investigations in Fish Control 61: Fish and Wildlife Service

DISCUSSION

The use of the flow-through technique has 

been recommended over the static technique 

for certain kinds of toxicity determinations. 

The flow-through technique is more complicated and expensive than the static technique; 

however, and the use of water with different 

characteristics is not as practical in flowthrough tests as in static tests. Because the 

toxicity of field grade TFM is similar in both 

techniques, the static procedure probably is 

sufficient to estimate the acute toxicity of TFM 

to fish. However, the flow-through technique 

must be used for determining chronic toxicity.

Large fish of a species were more resistant 

than smaller ones. The increase in resistance 

with size is perhaps related to the greater 

ability of larger fish to metabolize TFM. Lech 

and Costrini (1972) demonstrated the formation of TFM glucuronide (reduced TFM) in 

vitro and suspected that the same metabolite 

was formed in vivo in rainbow trout. Other 

studies showed that TFM and reduced TFM 

are excreted in the urine of rats (Lech 1971). 

Thus TFM is apparently readily metabolized 

and excreted. Mature fish probably have more 

effective enzyme systems than do juveniles 

for metabolizing TFM and adjusting to a continuous exposure to the toxicant.

The lampricide kills fish in short exposures 

(1 to 6 h) at concentrations equal to or nearly 

equal to those required in long exposures (4 to 

30 days). In fact, the toxicity did not change 

after 3 h with salmon (7.4 g), rainbow trout 

(19.7 g), brown trout, brook trout, and lake 

trout in 4-day tests (Table 2). The same trend 

occurred in the 30-day trials in which the 

LCSO's were identical for lake trout after 1 

and 30 days of exposure (Table 3). The change 

in toxicity of TFM to brook trout in 1- and 

30-day exposures was significant. However, 

considering the magnitude of change for brook 

trout and tests with other species, fish generally succumb immediately or survive chronic 

exposure by employing enzymatic defenses.

CONCLUSIONS

1. Field grade TFM was toxic to nontarget 

coldwater and warmwater fish in brief exposures (1 to 6 h) as well as in 96-h exposures in flow-through tests.

2. In hard water, the 96-h LCSO's ranged 

from 8.79 to 32.1 /ul/1 of TFM formulation 

and in soft water from 2.15 to 17.5 ytil/1 of 

TFM formulation.

3. Field grade TFM was chronically toxic to 

nontarget fish; however, the toxicity 

changed little between 1- and 30-day exposures.

4. Field grade TFM was more toxic to small 

than to large sizes of fish of the same 

species in 96-h exposures.

5. The toxicity of TFM to fish was greater in 

static tests than in flow-through tests, but 

the difference was not significant in two of 

three experiments.

LITERATURE CITED

Applegate. V. C.. J. H. Howell, and M. A. Smith. 1958e 

Use of mono-nitrophenols containing halogens as 

selective sea lamprey larvicides. Science 127(3294): 

336-338.

Daws on, V. K., K. B. Gumming, and P. A. Gilderhus. 

1975. Laboratory efficacy of 3-trifluoromethyl-4- 

nitrophenol (TFM) as a lampricide. U.S. Fish Wildl. 

Serv.. Invest. Fish Control 63.

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.

Lech, J. J. 1971. Metabolism of 3-trifluoromethyl-4- 

nitrophenol (TFM) in the rat. Toxicol. Appl. Pharmacol. 20:216-226.

Lech, J. J., and N. V. Costrini. 1972. In vitro and in 

vivo metabolism of 3-trifluoromethyl-4-nitrophenol 

(TFM) in rainbow trout. Comp. Gen. Pharmacol. 

3(10):160-166.

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, Bills, and Chandler; Toxicity of TFM to Nontarget Fish in Flow-Through Tests

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

Bull. Wildl. Etts. Assoc. 5:291-294.

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.

McAllister, W. A., Jr., W. L. Mauck, and F. L. Mayer, 

Jr. 1972. A simplified device for metering chemicals in intermittent-flow bioassays. Trans. Am. 

Fish. Soc. 101(3):555-557.

Mount, D. I., and W. A. Brungs. 1967. A simplified 

dosing apparatus for fish toxicology studies. Water 

Res. 1:21-29.

Olson, L. E.t and L. L. Marking. 1973. Toxicity of TFM 

(lampricide) to six early life stages of rainbow trout 

(Salmo gairdneri). J. Fish Res. Board Can. 30(8):1047- 

1052.

Schnick, R, A. 1972. A review of literature on TFM 

(3-trifluoromethyl-4-nitrophenol) as a lamprey 

larvicide. U.S. Fish Wildl. Serv., Invest. Fish Control 

44. 31 pp.

INVESTIGATIONS IN FISH CONTROL

62. Toxlclty of the Lampricide

3-Trifiuoromethyl-4-nitrophenoi

(TFM) to Selected Aquatic Invertebrates

and Frog Larvae

By Jack H. Chandler and Leif L. Marking

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. 1975
CONTENTS

Page 

Abstract ....................................... 3

Introduction ..................................... 3

Materials and methods .............................. 3

Results and discussion .............................. 4

Conclusions. ..................................... 7

Literature cited................................... 7
TOXICITY OF THE UMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL 

(TFM) TO SELECTED AQUATIC INVERTEBRATES AND FROG LARVAE

By Jack H. Chandler 

Southeastern Fish Control Laboratory, Warm Springs, Georgia

and

Leif L. Marking 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) was tested 

against various groups of nontarget aquatic organisms. Invertebrates exposed were flatworms (Catenula sp.), annelids (Tubifex tubifex), daphnids 

(Daphnia magna), seed shrimps (Cypridopsis sp.), glass shrimp (Palaemonetes kadiakensis), mayfly nymphs (Callibaetis sp.), backswimmers 

(Notonecta sp.), mosquito larvae (Culex sp. and Anopheles sp.), bivalve 

mollusks (Corbicula sp., Sphaerium sp., Elliptio sp., and Plectomerus sp.), 

and snails (Physa sp., Helisoma sp., and Pleurocera sp.). Vertebrates exposed to TFM were larvae of gray tree frog (Hyla versicolor), leopard frog 

(Rana pipiens), and bullfrog (Rana catesbeiana). Larvae of tree frogs were 

the most sensitive organism to TFM (96-h LC50 = 1.98 mg/1), and backswimmers were the least sensitive (96-h LC50 = 555 mg/1). Soft-bodied 

invertebrates were less sensitive than snails and bivalve mollusks to TFM. 

The invertebrates tested were not as susceptible as larval lampreys 

(Petromyzon marinus) in similar standardized tests.

INTRODUCTION

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been effective for controlling 

sea lamprey (Petromyzon marinus) in the 

Great Lakes. The lampricide is applied to 

streams in which the larvae live and is more 

toxic to larval lampreys than to other fishes 

(Applegate et al. 1958). Schnick (1972) reviewed the literature on the lampricide and 

summarized the data available that support 

existing registration of this pesticide. Recently completed studies have defined the toxicity of TFM to selected nontarget organisms 

(Marking and Olson 1975; Marking et al. 

1974; Maki et al. 1974; Fremling 1974; 

Kawatski et al. 1974; Sanders and Walsh 1974)

and the efficacy against larval lampreys 

(Dawson et al. In press).

The present study was designed to determine 

the toxicityof TFM to selected aquatic invertebrates and larvae of frogs.

MATERIALS AND METHODS

Field grade TFM (39.45% active ingredient) 

was measured gravimetrically and diluted with 

deionized water to prepare stock solutions for 

static and flow-through toxicity tests. Concentrations were calculated on the basis of the 

formulation used in the field rather than on 

active ingredient.
Investigations in Fish Control 62: Fish and Wildlife Service

Static tests were conducted in small jars 

containing 3 liters of test water or in large 

jars containing 15 liters of water. The jars 

were immersed in a water bath to the level of 

the test fluids; the water bath was equipped 

with a commercial chilling device (Frigid 

Units, Inc.1). At least 10 concentrations and 1 

water control were employed in each test.

Flow-through tests were performed in a 

modified version of the Mount and Brungs 

(1967) apparatus, but a different chemical 

metering device (Chandler et al. 1974) was 

substituted for the conventional form. Five 

concentrations and a control were used in each 

of the flow-through systems. Each of the 5 test 

chambers held 45 liters of diluted toxicant in 

which each successive concentration was approximately 50% less than the previous one. 

The colorimetric method of Olson and Marking (1973) was used periodically to determine 

actual concentrations of toxicant in each of the 

aquaria. A rate of flow was maintained which 

ensured a minimum of three complete replacements of test solutions per day in each of the 

chambers. Test media were cooled with water 

bath equipment similar to that used in static 

tests. Tests were conducted at 16 to 17 C.

Most of the tests were conducted in spring 

water to which lime was added (hereafter 

called limed water) to bring the total hardness 

(as CaCO3) to approximately 20 mg/1. The pH 

of the test waters varied from 6.8 to 7.0. Reconstituted waters routinely used for toxicity 

tests involving fish (Marking 1969) were used 

only in tests with clams, because it appeared 

in initial tests that the soft-bodied invertebrates might have been adversely affected by 

the test media.

Most test organisms were collected in ponds 

and streams. A few were reared outdoors in 

partly shaded, vinyl pools or in the laboratory. All forms collected in the field were 

retained for a minimum of 7 days in waters 

identical with those used in the tests. Only 

vigorous individuals of uniform sizes were 

used in tests. Small or delicate organisms 

were placed in cylindrical cages fabricated 

from Nitex screen. The cages were suspended 

in the test chamber of the flow-through appa1 Use of trade names does not imply U.S. Government 

endorsement of commercial products.

ratus to facilitate observation and to prevent 

loss or damage to organisms by turbulent 

water.

Mortality determinations were made on an 

appropriate hourly or daily basis, and dead 

organisms and detritus were removed after 

each examination. Mortalities were based on 

immobility or lack of response of test organisms to various mechanical stimuli. Snails 

were assumed to be dead when they failed to 

retract the "foot" into the shell, and bivalves 

when they were unable to close their shells.

The statistical procedures of Litchfield and 

Wilcoxon (1949) were used to calculate the 

concentration of toxicant necessary to produce 

50% mortality (LCSO's) and to obtain 95% confidence intervals.

RESULTS AND DISCUSSION

Eight groups of invertebrates (hereafter referred to as "soft-bodied" invertebrates) were 

exposed to TFM in static or flow-through 

tests. Of the eight groups, tubificids (Tubifex 

tubifex) were the most sensitive and flatworms 

ranked next (the 96-h LCSO's were 2.50 and 

11.6 mg/1, respectively, Table 1). The sensitivity of these organisms may be greater in 

laboratory tests than in their natural environment. Tubificids normally live in bottom substrates, whereas those used in our test were 

exposed to TFM in water solutions with no 

substrate. Flatworms (Catenula sp.) were exposed to TFM in hard water (160 mg/1 total 

hardness) and other organisms were exposed 

in soft water (20 mg/1 total hardness). Because TFM is less toxic to invertebrates in 

hard or high pH water (Fremling 1974; Kawatski et al. 1974), the 96-h LC50 for TFM against 

flatworms in soft water would probably be less 

than 11.6 mg/1 as shown in Table 1.

Organisms of intermediate sensitivity (96-h 

LCSO's, 21.3 to 89.0 mg/1) were daphnids, 

seed shrimp, mayfly nymphs, and mosquito 

larvae. The toxicity of TFM to mayfly nymphs 

(Callibaetls sp. - a form that lives in streams) 

exposed in static and in flow-through tests did 

not differ significantly in either case (Table 1). 

The least sensitive species were glass shrimp 

(96-h LC50 =125 mg/1) and backswimmers 

(96-h LC50 = 555 mg/1).
Table 1. Toxicity of TFM (39.45$) to selected aquatic invertebrates in static 

limed water (20 mg/1 total hardness)

Exposed in hard water (160 mg/1 total hardness). 

Flow-through toxicity tests.

Species

Flatworma 

Catenula SD.

Annelid 

Tubifex tubifex

Daphnid 

Daphnia magna

Seed shrimp 

Cypridopsis sp.

Glass shrimp 

Palaemonetes

kadiakensis

Mayfly 

Callibaetis sp.

Callibaetis sp.^

Backswimmer 

Notonecta sp.

Mosquito larvae 

Anopheles sp.

Culex sp.

LC50 and 95$ confidence interval 

1 h

17.4 

13.8-21.9

186 

164-212

117 

80.1-171

372 

214-441

14,250 

9,901-20,510

3 h

44.5 

30.2-66.0

16.3 

11.7-22.6

130 

104-163

60.0' 

40.0-91.1

660 

555-785

182 

157-211

7,900 

5,901-10,576

6 h

35.0 

27.2-45.0

12.5 

7.20-21.6

122 

102-146

60.0 

40.0-91.1

550 

436-694

166 

135-204

2,000 

1,248-3,206

18

7.

47

36

1

60

7
6 Investigations in Fish Control 62; Fish and Wildlife Service

Exposures for 96 h indicated greater toxic- Snails and bivalves which were exposed to 

ity than exposures for shorter periods. In- TFM in soft water at 17+_ 1 C, generally were 

vertebrates reacted differently than fish in more sensitive than soft-bodied invertebrates; 

that respect; changes in the toxicity of TFM to 96-h LCSO's ranged from 2 to 9 mg/1 of TFM 

fish were small or nil in 3- to 96-h exposures for all the species except fingernail clams 

(Marking and Olson 1975). Since TFM is ap- (Table 2). Fingernail clams were more replied over shorter periods (8 to 12 h) to con- sistant than other mollusks, and 96-h LCSO's 

trol lamprey larvae, the values at 24 h of ex- were 16.3 and 15.3 mg/1 in static and flowposure are perhaps more important than 96-h through tests. TFM appeared to be more toxic 

values for estimating the sensitivity of these to snails in static tests than in flow-through 

organisms during lampricidal treatments. tests.

Table 2. Toxicity of TFM (39.4-5%) to snails, bivalves, and frog larvae in 

soft water (4-4- mg/1 total hardness) at 17 ± 1 C (based on ability of organisms to respond to tactile stimulus)

Test 96-h LC50 and 95% confidence interval (mg/1) organism 

In static tests In flow-through tests

Snails

Physa sp. 3.05 4.60

2.35-3.95 3.03-6.97

Helisoma sp. 3.75 4.10

3.03-4.64 2.89-5.82

Pleurocera sp. 3.90 8.65

2.96-5.14 ' 5.51-13.6

Bivalves

Asiatic clam 2.30 4.10 

Corbicula sp. 1. 54-3.43 2.77-6.06

Mussels - 3.65 

Elliptic sp. 2.66-5.00

Plectomerus --- 8. 10

6.77-9.69

Fingernail clam 16.3 15.3 

Sphaerium sp. 10.6-25.0 7.4-2-31.3

Amphibians

Gray tree frog larvae 1.98 

ffyla versicolor 1.77-2.22

Leopard frog larvae 2.76 

Rana pi pi ens 2.4-5-3.11

Bullfrog larvae 3.55 

Rana catesbeiana 2.62-4.82
Chandler and Marking: Toxicity of TFM to Aquatic Invertebrates and Frog Larvae

Frog larvae also were more sensitive than 

soft-bodied invertebrates to TFM. In static 

tests larvae of the gray tree frog were the 

most sensitive (96-h LC50 = 1.98 mg/1), and 

larvae of the bullfrog were most resistant 

(96-h LC50 of 3.55 mg/1). Bullfrog larvae 

were exposed to TFM in flow-through tests 

and the other frog larvae in static tests. There 

is little difference in sensitivity among the 

three species.

Dawson et al. (197-) tested TFM (35.7%) for 

its effectiveness against larval lampreys 

(Petromyzon marinus) in standardized laboratory tests. In soft water (pH = 7.5) at 17 C, 

the 96-h LC50 was 1.60 mg/1 and the 12-h 

LC99 was 2.90 mg/1. Thus larval lampreys 

were much more susceptible than the softbodied invertebrates.

Although the 96-h LC50 values for TFM 

against snails, bivalves, and frog larvae indicated sensitivity for some species at larvicidal concentrations, these organisms would 

be less sensitive in 12-h exposures used to 

treat streams for larval lampreys. Few, if 

any, of these organisms should be affected by 

stream treatments with the lampricide.

CONCLUSIONS

1. The lampricide (39.45%) was toxic to 

aquatic invertebrates in standardized laboratory tests, but the invertebrates were 

not as susceptible as larval sea lampreys 

under similar test conditions.

2. Most of the soft-bodied invertebrates were 

less sensitive than snails and bivalve 

mollusks to TFM.

3. Larvae of gray tree frogs were the most 

sensitive to TFM (96-h LC50 = 1.98 mg/1), 

and backswimmers were the most resistant (96-h LC50 = 555 mg/1).

4. The toxicity of TFM to invertebrates increased in longer exposures (up to 96 h), 

whereas the reported toxicity of TFM to 

fish changes little after 3-h exposures.

5. TFM appeared to be more toxic to snails 

in static than in flow-through tests.

LITERATURE CITED

Applegate, V. C0 J. H. Howell, and M. A. Smith. 1958. 

Use of mononitrophenols containing halogens as selective sea lamprey larvicides. Science 127(3294): 

336-338.

Chandler, J. H., Jr., H. 0. Sanders, and D. F. Walsh.

1974. An improved chemical delivery apparatus for 

use in intermittent-flow bioassays. Bull. Environ. 

Contain. Toxicol. 12(1): 123-128.

Dawson, V. K., K. B. Gumming, and P. A. Gilderhus.

1975. Laboratory efficacy of 3-trifluoromethyl-4- 

nitrophenol (TFM) as a lampricide. U.S. Fish Wildl. 

Serv., Invest. Fish Control 63.

Fremling, C. R. 1975. Acute toxicity of the lampricide 

3-trifluoromethyl-4-nitrophenol (TFM) to nymphs of 

mayflies (Hexagenia sp.)e U.S. Fish Wildl. Serv., 

Invest. Fish Control 58. 8 pp.

Kawatski, J. A., M. M. Ledvina, and C, R. Hansen. 

1975. Acute toxicities of 3-trifluoromethyl-4- 

niti»ophenol (TFM) and 2',5-dichloro-4*-nitrosalicylanilide (Bayer 73) to larvae of the midge 

Chironomus tentans. U.S. Fish Wildl. Serv., Invest. Fish Control 57. 7 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.

Maki, A. A., L. D. Geissel, and H. E. Johnson. 1975. 

Toxicity of the lampricide 3-trifluoromethyl-4- 

nitrophenol (TFM) to 10 species of algae. U.S. Fish 

Wildl. Serv., Invest, Fish Control 56. 17 pp.

Marking, L. L. 1969. lexicological assays with fish. 

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

Marking, L. L., T. D. Bills, and J. H. Chandler. 1975. 

Toxicity of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to nontarget fish in flow-through tests. 

U.S. Fish Wildl. Serv., Invest. Fish Control 61. 9 pp.

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.

Mount, D. U, and W. A. Brungs. 1967. A simplified 

dosing apparatus for fish toxicology studies. Water 

Res. 1:21-29.

Olson, L. E., and L. L. Marking. 1973. Toxicity of 

TFM (lampricide) to six early life stages of rainbow 

trout (Salmo gairdneri). J. Fish. Board Can. 30(8): 

1047-1052.

Sanders, H. 0., and D. F. Walsh. 1975. Toxicity and residue dynamics of the lampricide 3-trifluoromethyl-4- 

nitrophenol (TFM) in aquatic invertebrates, U.S. Fish 

Wild. Serv., Invest. Fish Control 59. 9 pp.

Schnick, R. A. 1972. A review of literature on TFM 

(3-trifluoromethyl-4-nitrophenol) as a lamprey 

larvicide. U.S. Fish Wildl. Serv., Invest, Fish 

Control 44. 31 pp.

4U.S. GOVERNMENT PRINTING OFFICE! 1975-567^72



(Reports 53 through 55 are in one cover.)

53. Toxicity of Mixtures of Quinaldine Sulfate 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. A. 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. 0. Sanders and D. F. Walsh. 1975. 9 pp.

Fish Control Laboratories

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